pmid stringlengths 8 8 | pmcid stringlengths 8 11 ⌀ | source stringclasses 2
values | rank int64 1 9.78k | sections unknown | tokens int64 3 46.7k |
|---|---|---|---|---|---|
35497460 | PMC9049231 | pmc | 4,753 | {
"abstract": "Different nanostructured surfaces have bactericidal properties that arise from the interaction between the bacteria and the nanostructured surface. In this study, we focused on the relationship between bacterial motility and bactericidal properties. The motility of Escherichia coli ( E. coli ) was tuned by genetic engineering, and four types of E. coli (wild type (WT), lacking flagella, and flagellated with deficient motility or deficient chemotaxis) were used to evaluate the adhesion and bactericidal properties of nanostructured surfaces. Cicada ( Cryptotympana facialis ) wings and Si nano-pillar array substrates were used as natural and artificial nanostructured surfaces, respectively. Differences in motility and chemotaxis strongly influenced the adhesion behavior and to some extent, the damage to the cell membrane. These results suggest that the bactericidal properties of nanostructured surfaces depend on bacterial motility.",
"conclusion": "4. Conclusion We evaluated the adhesion properties of E. coli strains with different behaviors by deleting genes related to cell motility and adhesion. The number of adhered cells with motility defects were markedly lower than that of WT cells with motility. The motile and non-motile strains preferred to adhere to hydrophobic and hydrophilic surfaces, respectively. From these results, we proposed that not only the presence of flagella but also flagellar motility play a role in the adherence to nanostructured surfaces. Moreover, we found that directed movement toward nutrients accelerated the adhesion of the cell to the nanostructured surface. This motile behavior might be applied to sterile materials by collecting bacterial cells efficiently on the nanostructured surfaces. In addition to cell adhesion, we confirmed that bactericidal speed was also dependent on cell motility. From these results, we proposed two bactericidal mechanisms: that the cell membrane is damaged by contact with the nanostructure after the adhesion, and that an imbalance in cellular development leads to cell crash as well as bactericidal colloids.",
"introduction": "1. Introduction Human beings have developed many kinds of bactericidal materials to overcome infection by microorganisms. Currently, many household articles, such as furniture, sanitary goods, and dishes with antibacterial properties, are commercially available to the public. Most bactericidal materials consist of different chemical substances, such as nanosized metals, 1–3 antibiotic agents 4–6 and antimicrobial compounds. 7–9 These materials have disadvantages such as short stability, cost, and being harmful to human beings. Recently, microorganisms presenting antimicrobial resistance have become a serious threat, causing approximately 700 000 deaths per year. Additionally, it has been estimated that the number of antimicrobial resistance-related deaths will increase to 10 million by 2050. 10 Nanostructure-based bactericidal materials, including Si pillar arrays and polymer pillar arrays, can kill bacteria with antimicrobial resistance because the principle underlying their bactericidal effects is different from that of the bactericidal chemical materials. The bactericidal effect of the nanostructures was discovered by studying natural nanostructured surfaces such as cicada wings, 11–15 dragonfly wings, 12,16,17 and gecko fingers. 18,19 Later it was discovered that artificial nanostructured surfaces composed of inorganic materials, such Si 11,15,20,21 and carbon nanotubes (CNT), 22 and organic materials, 23–26 also have bactericidal properties. The bactericidal activity on a nanostructure originates from its physical instead of from its chemical properties; the cell membranes are stretched by the nanostructured surface, which causes cell break. It is possible to estimate cell death occurrence using commercially available DNA-staining reagents such as SYTO 9 and propidium iodide (PI), which indicate cell membrane damage. 11–19 This technology gives us information about cell membrane damage. It is noted that some population of bacteria inactivated by various methods such as UV and heat treatment to destroy micro-organisms can still survive as injured cells. 27–29 Injured cells are defined as cells exposed to physical and chemical stresses. They are not destroyed after sterilization but their life and death are judged by additional broth condition after the sterilization. Our group reported that the cicada wing surface caused the effusion of intercellular fluid of Escherichia coli ( E. coli ) cells adhered to it. 15 To prove this, we measured the decrease in a fluorescent protein expressed by E. coli , which indicated that the lysis of cells adhering to the nanostructure. The changes in fluorescence intensity were classified into three stages: just trapped in the nanostructure, small effusion, and large effusion. The same phenomenon was observed on an artificial nanostructured surface. From these results, we hypothesized that the cell membrane was damaged after the attachment, which led to intercellular fluid effusion into the environment and finally to cell death. In fact, E. coli trapped in the nanostructure resulted in death. Therefore, we used SYTO 9 and PI as reagents to analyze the cell death in real time. An important step for bactericidal behavior is the adhesion of bacteria to the nanostructured material surface because adhesion is the first step on the nanostructure-based bactericidal material. Our group reported that the number of attached cells depended on presence or absence of nanostructure and the surface wettability. 15 The number of attached cells increased with the contact angle for water (WCA: water contact angle) and that on the nanostructured surface was about twice compared to the flat surface at the same WCA. To develop an application in the field of sanitary engineering, it is important to increase the number of attached cells to improve the bactericidal and antibacterial properties of the nanostructured surface. Thus, we focused on bacterial characteristics that could affect attachment. Flagella play an important role in the adhesion to a surface because they can sense its physicochemical properties. 30 In this study, we evaluated the relationship between attachment to the nanostructured surface and cell damage after attachment using a wild type (WT) E. coli strain and three genetically modified strains with deficit of flagella, deficit of flagellar motility (cells with non-motile flagella), and deficit of chemical sensors (those that lead the cells toward nutrient-rich environments).",
"discussion": "3. Results and discussions 3.1 \n E. coli adhesion to the glass surfaces depends on bacterial behavior We observed the cell movement behavior dependent with E. coli strains and wettability of the flat glass substrate by fluorescence microscopy. WCAs of the glass surface before and after the plasma treatment were 69.5 ± 1.5° and 16.7 ± 1.9°, respectively. The microscopy images of adhered cells on the hydrophobic glass surface were shown in Fig. 2 . We could confirm that cells of RP437 ( Fig. 2(a) ), UU2612 ( Fig. 2(b) ) and RP6894 ( Fig. 2(d) ) had flagella but that of RP437 fliC did not have flagella ( Fig. 2(c) ). In this case, flagella adhered on the surface then they could not move at all (see ESI Movies 1(a)–(d) † ). On the hydrophilic glass surface, body of all types of the cells attached to the surface due to hydrophilic interaction. Then, flagella of RP437 and UU2612 moved (see ESI Movies 2(a) and (b) † ). Flagella of RP6894 did not moved because of its own trait. The cells of RP437 fliC and RP6894 moved randomly due to Brownian motion (data not shown). These results indicate that main contact area of E. coli to solid surface was strongly dependent with wettability of the solid surface, because the body of the E. coli cell is negatively charged and has hydrophilicity due to long glycan chains 37 and flagella have hydrophobicity due to four hydrophobic segments of motor protein MotoA. 38 Fig. 2 Fluorescent microscopy images of Cy3 labelled genetically modified cells attached on the hydrophobic glass surface. Scale bars in each photo show 10 μm. (a) RP437, (b) UU2612, (c) RP437 fliC, (d) RP6894. Enlarged views were inserted in each photo. In this case, scale bars show 5 μm. 3.2 \n E. coli adhesion to the nanostructured surfaces depends on bacterial behavior We monitored the changes in the number of E. coli cells adhered to the nanostructured surface by microscopy. We used cicada wings as a natural nanostructured surface and Si nano-pillar array substrates with hydrophilic and hydrophobic surfaces as artificial nanostructured surfaces. Fig. 3(a) shows the number of adhered cells per unit area 60 s after dropping the suspension of three of E. coli strains on the nanostructured surfaces. Here, each data shows the average with error bars ( N = 3) by counting adhered cells on different view fields. Gray, red and blue bars represent the strains of E. coli strains RP437 (WT), RP437 fliC, and RP6894, respectively. Our results showed that WT strain cells had higher adherence to each sample than the genetically engineered strains. The WCAs on the cicada wings, hydrophobic Si nano-pillar array, and hydrophilic Si nano-pillar array were 140 ± 2.6°, 78.3 ± 1.3° and 23.1 ± 1.0°, respectively, which suggests that the amount of adhered WT cells increased with the WCA. These results agreed well with our previous report showing that the number of adhered cells on the artificial nanostructured surface increased with WCA. 15 Fig. 3 (a) Number of adhered cells per unit area 60 s after dropping the suspension of three E. coli strains on the nanostructured surfaces. Gray, red and blue bars represent E. coli strains, RP437 (WT), RP437 fliC (absence of flagella), and RP6894 (presence of flagella but deficit of motility), respectively. Each data shows the average ( N = 3) with error bars. (b) Number of adhered cells per unit area 60 min after dropping the suspension of two E. coli strains on the nanostructured surfaces. Gray and patterned bars represent E. coli strains, RP437 and UU2612 (absence of chemotaxis), respectively. Each data shows the average ( N = 3) with error bars. The number of adhered cells of the non-motile strains (RP437 fliC and RP6894) was higher in hydrophilic surface. These contrasting results in motility and hydrophobicity might indicate that different cell structures interacted with the sample surface in the different strains. For the motile WT strain, flagella may play an important role in cell adhesion, and they prefer to attach to hydrophobic surfaces. 15,33 In contrast, the interaction between the cell membrane and hydrophilic nanostructures may be important in non-motile strains, and E. coli is a Gram-negative bacteria whose cell membrane is negatively charged and consequently hydrophilic as mentioned above. Among the non-motile strains, RP6894 had flagella, but they could not move. Therefore, in the non-motile strains, the interaction between the cellular membrane and the sample surface might be preferred to that of the flagella and the sample surface. Moreover, the number of adhered cells in the non-motile strains, RP437 fliC and RP6894, was considerably lower than that in the WT strains. These results show that not only having flagella but also flagellar motility is important for the adhesion to the material surface. Next, we evaluated the relation between cell adhesion and cellular behavior in terms of motility, directed or undirected, toward nutrients. We compared the WT strain, which moved toward a nutrient-rich environment using chemical sensors and the UU2612 strain, which had a random movement because its chemical sensors were defective. The hydrophobic surface of the Si nano-pillar array substrate was used for the test. Since the cells of the UU2612 strain took a long time to adhere the surface, we counted the number of adhered cells per unit area 60 min after dropping the suspension. The results showed that the number of adhered WT cells was approximately 5 times that of adhered UU2612 cells ( Fig. 3(b) ), suggesting that directed motility toward nutrients accelerates bacterial adhesion onto the surface. The contribution of motile direction might reflect the fact that WT cells move toward dead cells, which constitute a nutrient source and adhere to the surface of the nanostructure. This directed cell motility called chemotaxis, might be used in the construction of sterile materials by leading bacterial cells efficiently to the nanostructured surfaces. 3.3 Cell membrane damage depended on the E. coli strains As written above, SYTO 9 diffuses into the cell cytoplasm through the cell membrane and stains DNA green. In contrast, PI enters the cell cytoplasm and stains DNA red when the cell membrane is damaged. Therefore, adhered cells without membrane damage were colored green. Fig. 4(a) shows representative fluorescence microscopy images recorded every 5 minutes, and Fig. 4(b) shows the time-dependent active cell ratio of the hydrophobic nano-pillar array substrate derived from Fig. 4(a) , the active cell ratio was calculated using the following equation: Fig. 4 (a) Representative images obtained by the fluoresce microscopy, recorded every 5 minutes after dropping the suspension of three E. coli strains. (b) Time-dependent active cell ratio of the hydrophobic nano-pillar array substrate dependent on the strains. Gray circles, red triangles, and blue squares represent the strain RP437 (WT), RP437 fliC (absence of flagella), and RP6894 (presence of flagella but deficit of motility), respectively. Each data shows the average ( N = 3) with error bars. \n Fig. 4(b) clearly shows that the active cell ratio decreased drastically on WT cells but little on RP437 fliC and RP6894 cells. From these results, we concluded that membrane damage strongly depended on bacterial cell motility. We checked the cell membrane damage on the flat Si surface by using WT cells. The time-dependent active cell ratios on the hydrophilic and hydrophobic surface are plotted on Fig. S1(a). † In addition, the time-dependent active cell ratios with/without nano-pillar array on the hydrophobic surface are displayed on Fig. S1(b). † Active cell ratios of WT cells on the flat surfaces rarely decreased without regard to surface wettability, which means that cell membrane was little damaged on the flat surface. These results confirm that the cell membrane damage was occurred on the nanostructure. In addition, we confirmed that the nano-pillars penetrated the cell after the cell membrane damage test as shown in Fig. S2. † This SEM image explained the cell membrane was damaged and deformed. 3.4 Macroscopic antibacterial properties depended on the E. coli strains As stated in Subsection 3.3, the cell membrane of motile strains was damaged quickly, whereas cells without motility adhered to the nanostructured surface but cell membrane damage progressed slowly. The macroscopic antibacterial properties on each E. coli strains were evaluated in the long-term. This evaluation is important in terms of industrial applications. The results are plotted on Fig. 5 . Circles, triangles, squares and diamonds represent E. coli strains WT (RP437), RP437 fliC, RP6894 and UU2612, respectively. Closed symbols plotted against incubation time show the alive cell ratio obtained on the Si nano-pillar array substrate. Opened symbols show the alive cell ratio after a 24 h incubation obtained on the flat Si substrate, which served as a reference. Fig. 5 Time-dependent alive cell ratio of four E. coli strains evaluated by the macroscopic bactericidal property test. Circles, triangles, squares, and diamonds represent E. coli strains RP437 (WT), RP437 fliC (absence of flagella), RP6894 (presence of flagella but deficit of motility), and UU2612 (absence of chemotaxis), respectively. Closed symbols plotted against incubation time show the alive cell ratio obtained on the Si nano-pillar array substrate. Opened symbols show the alive cell ratio after a 24 h incubation obtained on the flat Si substrate, which served as a reference. The alive cell ratio on the flat surface was over 1% for all strains, showing that the Si flat surface did not have antibacterial properties. In contrast, the alive cell ratio on the nanostructured surface for all strains was below 1% after 24 hours of incubation, showing that the nanostructured surface had antibacterial properties against all strains. The alive cell ratio of WT strain was the lowest at every incubation time and reached approximately 1% after 1 hour of incubation. The alive cell ratio of the RP6894 and RP437 fliC strains decreased gradually and was lower than that of the reference. This behavior is attributed to the motility of the cells because cells without motility adhered to the nanostructure by gravity, it took them a long-time to adhere. The alive cell ratio of the UU2612 strain was lower than that of the RP6894 and RP487 fliC strains before 8 hours. This result could be explained because the UU2612 strain also has the ability to swim toward the nanostructured surfaces. These data indicate that the antibacterial properties of the nanostructured surface depended on cellular motility, in addition to the adhesion properties. Moreover, the alive cell ratio of the UU2612 strain was higher than that of the WT strains before 8 hours. This difference between the strains can be explained by differences in the motile direction as discussed in Subsection 3.2. In our previous study, we proposed the following model of bactericidal effect of the nanostructured surface: a bacterium searches for its favorite hydrophobic surface relying on sensors present on its flagella. Consequently, the flagella are the first cellular structures to make contact with the nanostructure and adhere to it. Then, the flagella get entangled in the nanostructure, but the cell can still move. Then, the cell hits the nanostructure and suffers abrasions that cause the cell cytoplasm to effuse gradually. Over time, the small abrasions grow into major scars that cause the cytoplasm to effuse drastically. Finally, the cell dies. The proposed mechanism agrees with the results of this study in the case of WT strain. Bandara et al. reported that motility after adhesion is a key point leading to cell death. 39 Our results showed that cells without motility died slowly while cells with motility died fast. The results of this study suggest that there are two mechanism of bactericidal nanostructured surfaces, the first depends on bacterial motility as described above, and the second is due to the disruption of the bacterial wall and nanosized bactericidal colloids. 40 The bactericidal speed of the later mechanism is lower than that of the first mechanism because it depends on the speed of cell development. Thus, bactericidal speed depended on bacterial motility."
} | 4,746 |
24810513 | null | s2 | 4,755 | {
"abstract": "The photosynthetic efficiency of C3 plants suffers from the reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) with O2 instead of CO2 , leading to the costly process of photorespiration. Increasing the concentration of CO2 around Rubisco is a strategy used by photosynthetic prokaryotes such as cyanobacteria for more efficient incorporation of inorganic carbon. Engineering the cyanobacterial CO2 -concentrating mechanism, the carboxysome, into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have chosen to explore the possibility of producing β-carboxysomes from Synechococcus elongatus PCC7942, a model freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple β-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts, and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the β-carboxysome are able to assemble in plant chloroplasts into highly organized assemblies resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a 17-amino-acid CcmN peptide to the shell proteins inside chloroplasts. Our experiments establish the feasibility of introducing carboxysomes into chloroplasts for the potential compartmentalization of Rubisco or other proteins."
} | 392 |
37050308 | PMC10096731 | pmc | 4,756 | {
"abstract": "A reliance on fossil fuel has led to the increased emission of greenhouse gases (GHGs). The excessive consumption of raw materials today makes the search for sustainable resources more pressing than ever. Technical lignins are mainly used in low-value applications such as heat and electricity generation. Green enzyme-based modifications of technical lignin have generated a number of functional lignin-based polymers, fillers, coatings, and many other applications and materials. These bio-modified technical lignins often display similar properties in terms of their durability and elasticity as fossil-based materials while also being biodegradable. Therefore, it is possible to replace a wide range of environmentally damaging materials with lignin-based ones. By researching publications from the last 20 years focusing on the latest findings utilizing databases, a comprehensive collection on this topic was crafted. This review summarizes the recent progress made in enzymatically modifying technical lignins utilizing laccases, peroxidases, and lipases. The underlying enzymatic reaction mechanisms and processes are being elucidated and the application possibilities discussed. In addition, the environmental assessment of novel technical lignin-based products as well as the developments, opportunities, and challenges are highlighted.",
"conclusion": "8. Conclusions To tackle public issues such as global warming and pollution, organizations and investors must adjust not only the politics but the socio-economic framework as well. Enhanced global cooperation might boost technical lignin commercialization by channeling funds and facilitating the discovery of innovative routes. In the coming years, research is expected to investigate the usage of lignin in a variety of advanced next-generation applications, as is evident by the current European funding scheme. In our review, we introduced emerging studies on enzymatically modifying technical lignin into mechanical responsive hydrogels coatings, adhesives, and fillers. It is foreseen that more lignin-based smart materials with high performance will be designed and prepared in the future. Moreover, the increasing number of enzymatically modified technical lignins with flexible physicochemical properties allow for more advanced techniques in lignin processing. Lignin-based polymeric materials could be promising alternatives to traditional fossil-based materials. Given the progress of lignin modification chemistry and the development of new processing techniques and techniques for technical lignin purification, lignin is a promising renewable resource for high performance materials. Lignin valorization has emerged as an important research field in the near future.",
"introduction": "1. Introduction The total European pulp production accounts for 4.4% of the sulphite pulp production, which results in 1.7 tons of available pulp. In general, most pulp production sites are in Sweden (31.2%) and Finland (30.2%). In total, 25.3% of the global pulp production is situated in Europe [ 1 , 2 ]. The worldwide production of technical lignin is approximately 100 million tonnes/year, valued at USD 732 million in 2015. It is expected to reach USD 913 million by 2025 with a compound annual growth rate (CAGR) of 2.2% [ 3 ]. Lignin represents the second most abundant source for sustainable aromatic polymers [ 4 , 5 ]. Although the structure of lignin is complex, it can be broken down into three repetitive structural motifs, the so-called monolignols. The complexity of lignin results from the many different linkages possible between the monolignol units as seen in Figure 1 . These molecules are hydroxycinnamic alcohols containing a sidechain formed of three carbons (labelled α, β, and γ) attached to an aromatic ring system (labelled one to six), differing only in the number of methoxy groups attached on the aromatic ring. There is no methoxy group present in p-coumaryl alcohol, coniferyl alcohol has one at position C3, and sinapyl alcohol has two at positions C3 and C5. While in nature, a cocktail of different enzymes (cellulases, cellobiohydrolases, peroxidases, and laccases) works synergistically on the degradation of lignocellulosic biomass, in the industry harsh conditions are required to separate biomass into its single compounds. In the paper industry, the pulping process generates purified cellulose and hemi-cellulose product streams. These are used for the generation of high-quality paper and fine chemicals. Meanwhile, lignin is regarded as a side product and is mainly burned for the re-generation of some of the energy needed during the pulping process. The rising awareness of the value of lignin, because of its structure, and in light of the circular bio economy concept value-added use of this resource is of more interest. Depending on the solvent used during pulping, different processes can be distinguished leading to different types of lignin with varying properties, the so-called technical lignins presented in Figure 2 . The most common industrial processes are the sulfite, kraft, soda. and organosolv pulping [ 6 , 7 , 8 ]. In sulfite pulping, proper-sized wood chips (15–25 mm long) are cooked at high temperatures ranging from 140 to 170 °C at an acidic, neutral, or alkaline pH, depending on the sulfite salt added. The typically used counter ions are Ca 2+ , Na + , Mg 2+ , or NH 4+ . During cooking, the ether bonds within the lignin are hydrolyzed and subsequently sulfonated leading to the solubilization of lignin. The pulp not only contains lignin but also residual cellulose and hemicellulose, as well as some inorganic molecules. Thus, it is filtrated afterwards leading to accumulation of the lignin in the spent liquor. The modified lignin is then called lignosulfonate (LS), which is soluble in water (due to the sulfonation) of relatively high molecular weights (15,000–60,000 Da) when compared to other types of technical lignins and has a low concentration in phenolic groups [ 7 , 9 ]. In the kraft pulping process, wood chips are cooked for several hours at temperatures from 155 to 175 °C in an aqueous solution of NaOH and Na2S, the so-called white liquor. Under these extreme conditions the aromatic ether bonds crack, leading to the dissolution of the lignin and the precipitation of the cellulose and hemicellulose. This is also indicated by the color change of the liquor from white to dark brown or black. This black liquor has a high alkaline pH, typically around 13 to 14, rendering the lignin in its deprotonated form and thus soluble. In the black liquor, not only lignin is accumulated but also cellulose and hemicellulose residues, as well as inorganic compounds. Membrane filtration or acidification are methods applied to purify the kraft lignin (KL) further. In the industrially applied LignoForce process, membrane filtration is used. First, the black liquor is sparged with oxygen until the sulfite concentration is reduced to a specific level. Then, the solution is acidified by the addition of CO 2 until a pH of 10 is reached. Finally, the lignin is separated by filtration and afterwards the filtration cake is washed with diluted sulfuric acid and dried, resulting in a high-quality technical lignin [ 10 , 11 , 12 ]. Another industrially applied purification method is based on acidification and is known as the LignoBoost process. Therein, the black liquor is first acidified with CO 2 until a pH of around 10 is reached, resulting in precipitation of about 75% of lignin. The precipitated lignin is then re-suspended and further purified through the addition of H 2 SO 4 until a pH of 3 is reached. To remove the remaining water-soluble parts of ash, washing with acidified water is necessary [ 10 ]. Upon the acidification of the black liquor, the deprotonated and thus soluble functional groups of lignin become protonated again (depending on their respective pKa values), leading to re-protonation, allowing for the formation of new linkages between the single molecules, leading to an increase in the molecular size, and, finally, to the precipitation [ 13 ]. This low-cost process results in kraft lignins of high purity and yield [ 14 ]. Kraft lignin generally is insoluble in water of low molecular weights (ranging from 200 to 20,000 Da) and has a relatively high concentration of phenolic groups [ 7 , 9 ]. Sulfite and kraft pulping are the industrially most common processes, with the latter being the dominant pulping process today. An example of a commercially available kraft lignin is Indulin AT. However, besides the structural changes of the native lignin molecule due to the harsh reaction conditions, sulfur groups are also incorporated, leading to severe alterations of the lignin molecule. In order to better understand the reaction mechanisms, it is essential to work with native lignin. In search for processes that allow for the isolation of more natural lignin, sulfur-free pulping processes were developed [ 15 ]. In the soda pulping process, mainly non-woody biomass is cooked at temperatures between 160 to 170 °C in the presence of NaOH and anthraquinone. The addition of anthraquinone is optional but leads to a higher lignin yield due to an increased ether bond cleavage. This process results in soda lignin, which is of a low molecular weight (800–3000 Da) and is sulfur-free and, thus, can be considered purer than other technical lignins, such as LS or KL [ 10 , 16 ]. The organosolv pulping process is another sulfur-free process. Various organic solvents, such as ethanol, methanol, acetone or mixtures of them with water, are used to dissolve lignin at high temperatures (100–250 °C) and pressures. The generated organosolv lignin a has high molecular weight (2000–9000 Da), is insoluble in water. and is closer to native lignin than other technical lignins (LS and KL). Organocell (using methanol as solvent) and Alcell (using ethanol as solvent) organosolv lignins are commercially available today [ 17 ]. Further, the hydrotropic delignification utilizing sodium benzoate is researched to obtain technical lignins with beneficial properties [ 18 , 19 ]. However, at the moment, there is no such thing as native lignin, as all technical treatments lead to at least slight modifications, such as condensations, of the lignin structure. Thus, new processes to isolate lignin are constantly developed, such as treatment with hot water, diluted acids, alkaline solutions, ionic liquid. or enzymatic hydrolyses. On a laboratory scale, milled wood lignin is thought to be the one that comes closest to native lignin. Thereby, ball-milled plant material is treated with a dioxane-water mixture as the solvent to extract the lignin from the plant cells [ 7 ]. From a bioeconomy perspective, lignin is currently mainly used to produce bioenergy (electricity and heat) but has recently received attention as a renewable raw material for the production of chemicals and materials to replace petrochemical resources and sometimes also provide technical improvements [ 20 ]. Other examples of interesting applications where lignin can be used to replace conventional materials are displacing urea-formaldehyde in adhesives [ 21 ], bitumen in asphalts [ 22 ], polyacrylonitrile in carbon fibers [ 23 ], and polyol in polyisocyanurate foams [ 20 ] and liquid fuels [ 24 ]. Yet, lignin is largely underexploited for these purposes, although many scientific studies are conducted to forward these fields [ 25 ]. Moreover, lignin can be used in other industrial applications that can benefit from the good surface activity of lignin [ 26 ], such as adsorbents for CO 2 capture [ 27 , 28 ] and catalysts [ 2 , 29 , 30 ]. A number of chemical and physical processes for the improvement of the properties have been developed and reviewed in the past. This review, thus, mainly focus on the recent development of “green” enzyme-based upgrading of lignins. When in nature the monolignols are incorporated into the growing polymer during enzyme-catalyzed lignification, the resulting phenylpropanoid units are called p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). The most common linkages formed between the monolignol units during radical cross-linking are aryl-ether (β- O -4), resinol (β-β), phenylcoumaran (β-5), biphenyl (5-5), and diaryl ether (4- O -5) linkages. Generally, ether linkages are more common than ester or carbon-carbon linkages. Amongst these linkages, the aryl-ether linkages (β- O -4) are the most abundant in native lignin, representing a relative labile linkage, easily cleaved during lignin pretreatment or depolymerization processes [ 4 , 6 ]. Aside from the interunit linkages present between the monolignols, their abundance and distribution also effect lignin reactivity. They vary vastly between different types of biomasses. Softwood lignin mainly contains G-units, in hardwoods both G- and S-units are present, and in grasses and herbaceous plants all three H-, G-, and S-units can be found. The structure of the monolignols also defines the possible structure of the formed polymers. G-units mainly build branched polymers due to the free position at C5, while S-units tend to form linear polymers. Hence, hardwood lignins are imagined as more linear polymers, while softwood lignins are expected to be rather branched [ 17 ]. In general, all technical lignins can be suitable for enzymatic modification or synthesis with the respective pretreatments. Kraft lignin, being readily available and of low cost, would be the best option from an economic and ecological point-of-view. Anyhow, the condensation of the C5 position in the aromatic ring, as well as the γ-elimination of the primary alcohol and the other conformational changes in enzymatic reactions are difficult to perform without extensive pretreatment. In addition, its low water solubility makes it necessary to engineer enzymes able to perform under extreme conditions such as high temperatures and at high pH values [ 31 , 32 , 33 ]. In contrast, lignosulfonates have been found to be more suitable for enzymatic modification. Their hydrophilicity due to the presence of anionic carboxylate groups, anionic sulfonate groups, and phenolic hydroxyl groups is beneficial. Furthermore, their polydisperse nature and wide range of molecular weight offer a multitude of reaction possibilities [ 34 , 35 ]. The lignins gained from soda pulping (also called organosolv lignins) are often described as the most native, and, therefore, often considered as beneficial for enzymatic modification. Studies show that enzymatic reactions are hindered by the low content of hydroxyl groups and the substitution of aromatic rings and steric barriers. Therefore, reactions like demethylations are employed [ 36 , 37 ]. With the latest geopolitical developments, the general availability and pricing of technical linins has vastly changed. Due to high energy prices, the burning of lignin for energy production stays in strong competition to their use as renewable raw material for the production of chemicals and materials to replace petrochemical resources. It is yet to be seen what consequence this has on further research and development in this area."
} | 3,796 |
39814598 | PMC11735340 | pmc | 4,757 | {
"abstract": "Abstract A growing body of theoretical studies and laboratory experiments has focused attention on reciprocal feedbacks between ecological and evolutionary processes. However, uncertainty remains about whether such eco‐evolutionary feedbacks have an important or negligible influence on natural communities. Thus, recent discussions call for field experiments that explore whether selection on phenotypic variation within populations leads to contemporaneous effects on community dynamics. To help fill this gap, in this study, we test the hypothesis that selection on consumer traits in a population of predatory drilling snails can drive eco‐evolutionary dynamics in a rocky intertidal community in California, USA. We first conducted a laboratory selection experiment to raise newly hatched dogwhelks ( Nucella canaliculata ) on four diet treatments encompassing a range of prey species and shell thicknesses. Snails that survived to adulthood under these diet treatments differed in their capacity to drill thick‐shelled mussels. Dogwhelks from these treatment groups were then outplanted to intertidal field cages for 1 year to test whether groups experiencing selection differed in their effects on mussel bed succession. As expected, succession proceeded most rapidly in the reference treatment with dogwhelks excluded. However, successional patterns differed minimally among dogwhelks raised under the different diet treatments. Thus, although our laboratory results suggest that prey can impose selection that leads to rapid adaptation and divergent consumer traits, these feedbacks were not strong enough to result in clear community effects in the field. We propose that a limited range of variation in functional traits within populations, moderate strengths of selection, and a background of substantial abiotic and biotic variation may all act to dampen the potential for strong eco‐evolutionary dynamics in this and many other natural communities.",
"introduction": "INTRODUCTION Increased recognition that evolution can proceed rapidly (Boag & Grant, 1981 ; Reznick & Ghalambor, 2001 ) has resulted in the emerging field of eco‐evolution dynamics. Despite growing attention to eco‐evolutionary feedbacks (Fussmann et al., 2007 ; Hendry, 2017 ; Pelletier et al., 2009 ), it is still unknown whether these feedbacks are consequential in natural communities (Hendry, 2019 ; Schoener, 2011 ). Most studies of eco‐evolutionary dynamics have been conducted in the laboratory or in mesocosms (but see Agrawal et al., 2013 ; Farkas et al., 2013 ; Reznick & Travis, 2019 ), with relatively few experimental tests conducted in nature, particularly at the scale of a community or ecosystem (but see Bassar et al., 2010 ; Palkovacs et al., 2009 ). Field experiments are needed to determine whether eco‐evolutionary feedbacks are swamped or amplified by external factors in more complex, natural environments (Hendry, 2019 ). In addition, existing experimental studies of eco‐evolutionary dynamics have generally compared two or more locally adapted populations that have evolved under spatially divergent selection (Des Roches et al., 2013 ; Farkas et al., 2013 ; Fukano et al., 2022 ). While such studies provide proof of principle that evolutionary processes can influence ecological dynamics, this approach often relies on substantial phenotypic variation that has evolved through divergent selection imposed on separate populations over extended periods (e.g., decades to a century or more). In contrast, there have been increased calls for studying the dynamics of systems where there is no separation in time between the evolutionary and ecological processes under consideration (Bassar et al., 2021 ; Hersch‐Green et al., 2011 ; Tack et al., 2012 ). One promising approach is to explore whether abiotic and biotic variation acting over rapid timescales (e.g., months to years) might impose selection on existing within‐population variation in functional traits that are linked to community dynamics (Hughes et al., 2008 ; Reusch et al., 2005 ; Whitham et al., 2003 ). However, to date, few field studies have tested whether ecological processes might be altered by feedbacks with contemporaneous selection on the phenotypic variation present within a local community (but see Agrawal et al., 2013 ; Carvajal‐Endara et al., 2020 ; Schoener et al., 2017 ). Evaluating the importance of such eco‐evolutionary dynamics will ultimately require a body of field studies that span a range of ecosystems. However, a logical starting point is to seek evidence of eco‐evolutionary dynamics in natural communities where these effects are expected to be particularly strong. Given that predator–prey interactions can be tightly coupled (Thompson, 1999a ), and often lead to strong top‐down control of communities (Menge & Branch, 2001 ), it is not surprising that many studies of eco‐evolutionary dynamics have focused on these interactions (Reznick & Travis, 2019 ; Yoshida et al., 2003 ). One might also expect eco‐evolutionary feedbacks to be strong in communities where a predator has: (1) substantial intrapopulation variation in foraging traits that have a heritable basis and are linked to community dynamics; and (2) a relatively fast generation time and exposure to substantial environmental variation, high mortality, and the potential for strong selection. Study system Intertidal communities have long served as testing grounds for ecological theory (Menge & Branch, 2001 ), in part because species interactions in these systems are often strong and easily manipulated. In the northeast Pacific, dogwhelks ( Nucella spp.) are important rocky intertidal predators that feed by drilling a tiny hole through their barnacle and mussel prey (Carriker, 1981 ). Beds of the mussel Mytilus californianus dominate the mid‐intertidal zone, providing habitat for diverse species and exhibiting a well‐documented sequence of succession following disturbance (Dayton, 1971 ; Paine & Levin, 1981 ). Prior work indicates that Nucella spp. can alter the rate of succession by consuming early colonizing space holders (Berlow, 1997 ; Wootton, 2002 , 2013 ), and perhaps M. californianus later in succession (Sanford et al., 2003 ; Sanford & Worth, 2009 , 2010 ). Nucella spp. produce benthic egg capsules with crawl‐away young that are dependent on small, newly recruited prey (barnacles and/or mussels) that are spatially and temporally variable in their abundance, and potentially their shell properties (e.g., shell thickness). Mortality of juvenile Nucella in the field can reach 90%–99% during the first 2 months of life (Spight, 1975 ). Thus, seasonal and interannual variability in prey and other environmental factors might impose strong selection on Nucella phenotypes during the juvenile phase when mortality is especially high (Spight, 1982 ). Previous work indicates that populations of the channeled dogwhelk, Nucella canaliculata , differ geographically in the length and thickness of M. californianus mussels that can be drilled (Longman & Sanford, in review; Sanford & Worth, 2009 ). Drilling phenotypes in this species persist after two generations in common laboratory conditions, establishing a genetic basis for this variation (Sanford & Worth, 2009 ). The N. canaliculata population on the Bodega Marine Reserve (BMR) in northern California, USA, is particularly interesting as it contains a mix of drilling phenotypes that vary both among and within N. canaliculata families (Sanford & Worth, 2009 ). The processes that generate and maintain this variation are unknown, but might include greater than expected gene flow from nearby populations. Alternatively, BMR is located within a coastal region that has oceanographically driven seasonal and interannual variation in prey recruitment (Morgan et al., 2009 , 2012 ) and mussel shell thickness (Kroeker et al., 2016 ), which could impose temporally fluctuating selection. Collectively, prior work in this system suggests the potential for temporal variation in prey to impose strong selection on within‐population variation in drilling phenotypes in N. canaliculata , a predator known to influence mussel bed succession. Although we focus on pairwise interactions and the potential for prey to impose selection on consumer traits, we acknowledge that selection imposed by multispecies interactions in natural communities is likely far more complex (De Meester et al., 2018 ; Govaert et al., 2021 ; Strauss & Irwin, 2004 ). For example, N. canaliculata partially overlaps in its intertidal distribution and diet with the dogwhelk Nucella ostrina (Wieters & Navarrete, 1998 ). These congeners might compete for prey and thus variation in the density of Nucella ostrina could impose selection on the drilling traits of N. canaliculata . Similarly, Nucella spp. are subject to predation, especially by cancrid crabs, and variation in crab predation may impose selection on the foraging behavior of N. canaliculata (Neylan et al., 2024 ). Nevertheless, the potential for variation in prey to select for drilling traits in Nucella is one of the most plausible pathways for strong eco‐evolutionary feedbacks to occur in this study system. Thus, in this study, we first hypothesized that variation in early‐life diet would select for specific drilling phenotypes in dogwhelks. To test this hypothesis, we conducted a laboratory selection experiment to rear newly hatched dogwhelks on four prey treatments. Second, we hypothesized that the resulting divergence in predator phenotypes would alter the trajectory of mussel bed succession, with an increased frequency of strong drilling phenotypes slowing succession relative to weaker drillers. To test this hypothesis, we outplanted snails from the selection experiment to field enclosures to quantify their effects on community succession over 1 year.",
"discussion": "DISCUSSION Empirical field studies of the community‐level effects of eco‐evolutionary feedbacks are rare. As a result, uncertainty remains about whether such effects are ecologically important or relatively trivial (Hendry, 2017 , 2019 ; Schoener, 2011 ). Our experiments focused on a study system where there were compelling reasons to suspect that eco‐evolutionary dynamics might be important. Previous research had shown that the drilling traits of N. canaliculata varied strongly both across its species range and within the focal population for this study (Sanford & Worth, 2009 , 2010 ). When we raised snails in the laboratory under different prey regimes mimicking the natural seasonal and interannual variation in recruitment of prey at this focal site (Morgan et al., 2009 , 2012 ; Sanford & Worth, 2010 ), mortality was highest in hatchling snails raised on BMR M. californianus , creating the potential for strong selection. Indeed, this treatment showed the highest frequency of strong drillers suggesting that selection can rapidly modify this predator–prey interaction. However, despite well‐established links between dogwhelk predation and succession (Sanford & Worth, 2010 ; Wootton, 2002 ), divergence in drilling traits generated by our laboratory treatments did not lead to clear community‐level effects on succession. This suggests that, at least in this well‐studied system, eco‐evolutionary feedbacks were relatively weak in a natural field setting, perhaps due to the dampening effects of environmental and biotic variation, a restricted range of within‐population phenotypes, and incomplete selection. Eco‐evolutionary feedbacks may often be swamped in the field by the overriding effects of substantial spatial variation in environmental conditions and the complex dynamics of biotic processes. In our study, fine‐scale physical variation, including the vertical tidal gradient, was a primary driver of mussel cover with lower plots progressing more rapidly in succession toward competitive dominance by mussels. Trajectories of mussel bed succession are also dependent on patterns of recruitment of barnacles and mussels, which can vary strongly over small spatial and temporal scales (Berlow, 1997 ). The effects of Nucella predation on succession in a given plot can in turn be strongly affected by these stochastic rates of prey recruitment (Berlow, 1997 ). Rates of succession in mussel bed communities can also be influenced by a range of other species interactions, including both facilitation and competition (Berlow, 1997 ; Navarrete, 1996 ; Wootton, 2002 ). Against this backdrop of substantial environmental variation and the noise‐amplifying effects of complex biotic networks, the influence of intrapopulation variation in drilling traits on rates of succession may be comparatively minor. A second factor that may weaken eco‐evolutionary feedbacks in nature is a limited range of within‐population variation available for selection to act upon. Polymorphism and genetic variation in behavior and other functional traits are common within populations (Smith & Blumstein, 2008 ; Wilson, 1998 ), and selection on this variation can have ecological consequences (Hughes et al., 2008 ; Whitham et al., 2003 ). Within‐population variation can be maintained by fluctuating selection, gene flow from other populations, or genetic drift (Star & Spencer, 2013 ). However, the range of functional variation in ecologically important traits within a population is generally small compared to that found across populations (Tack et al., 2012 ). Theories about eco‐evolutionary dynamics have typically been tested by comparing the effects of locally adapted populations or divergent ecotypes (e.g., Des Roches et al., 2013 ; Farkas et al., 2013 ; Fukano et al., 2022 ). An analogous approach has been common in the field of plant community genetics where plants are collected from distant, diverged populations and their effects on insects are quantified in a common garden environment (Hersch‐Green et al., 2011 ). These approaches often create a mismatch in the spatial scales used to test the relative importance of genetic versus environmental influences on community processes (Tack et al., 2012 ; but see Hughes & Stachowicz, 2004 ; Reusch et al., 2005 ; Tack et al., 2010 ). This mismatch raises concerns that comparing the effects of distant populations in a common environment may inflate genotypic and phenotypic variation and its importance within a community (Bassar et al., 2021 ; Hersch‐Green et al., 2011 ; Tack et al., 2012 ). Nevertheless, few studies of eco‐evolutionary dynamics have assessed whether temporal variation in selection within a population causes contemporaneous genetic/phenotypic changes that ultimately have ecological consequences (Bassar et al., 2021 ; but see Carlson et al., 2011 ; Carvajal‐Endara et al., 2020 ; Schoener et al., 2017 ). In our study system, the range of variation in drilling capacity within our focal population (BMR) is substantially less than that encompassed by geographically separated populations; populations of N. canaliculata from California can drill mussels 3.4 times thicker than populations from Oregon (Longman & Sanford, in review). Previous studies indicate that Nucella spp. are weak drillers in the Pacific Northwest with minimal impacts on M. californianus (Sanford & Worth, 2009 ) and that these snails facilitate the rate of succession by removing early primary space holders (Wootton, 2002 ). In contrast, our study of a California population of N. canaliculata indicated that snails slowed and inhibited succession via their predation on M. californianus , the primary species that dominates later in succession. Had we outplanted snails from populations with highly divergent drilling phenotypes, the California populations would likely have slowed succession whereas Oregon snails may have accelerated succession. This approach would have highlighted the influence of intraspecific phenotypic variation on ecological processes. However, to assess the importance of eco‐evolutionary feedbacks within a community, we prioritized studying the effects of intrapopulation variation to minimize the amount of separation between evolutionary and ecological processes (Bassar et al., 2021 ). Lastly, eco‐evolutionary feedbacks might be weaker than expected in nature if selection on a population is not strong enough to produce extreme divergence in functional traits. The existence of wild populations comprised of phenotypically diverse individuals suggests that past selection, even if relatively strong, has not been strong enough to eliminate variability in traits under more realistic selection regimes (Carlson et al., 2011 ; Grant & Grant, 2002 ). If selection on variation within a population tends to result in moderate shifts in the frequency of different functional traits, the community‐level consequences of this selection may be dampened relative to comparisons of highly divergent ecotypes selected from separate populations (Tack et al., 2012 ). Our selection treatments mimicked natural variation in prey abundance and tested the community consequences of selection on functional variation within a consumer population. Bodega Marine Reserve lies within a strong coastal upwelling region that is known for high levels of seasonal and inter‐annual variability in larval supply and recruitment (Morgan et al., 2012 ; Wing et al., 2003 ). These fluctuating food sources may select for different drilling phenotypes of N. canaliculata within and among years, ultimately maintaining a mix of phenotypes in the population. Although our BMR M. californianus treatment did select for a higher frequency of stronger drillers (Figure 3 ), there were likely still some strong drillers in all of the diet treatments outplanted to the field cages. The maintenance of this variability in consumer traits likely dampens eco‐evolutionary effects in this and other natural communities. The laboratory portion of our experiment relied on artificial selection, which can have unintended phenotypic consequences due to genetic linkage with the focal trait or experimentally imposed conditions (Conner, 2003 ). These inadvertent outcomes can subsequently impact an individual's fitness under natural conditions (Baskett & Waples, 2013 ) and may have influenced the ecological effects of Nucella once they were outplanted to the field. It is also possible that some component of variation in drilling ability in our laboratory treatments arose from phenotypic plasticity rather than selection. In the few cases where plasticity in gastropod feeding structures has been documented, morphological variation appears to be a response to recent diet, as snails completely replace their radula over a span of 3–4 weeks (Padilla, 1998 ). In our study, after dogwhelks were subjected to the selection phase, they were raised on a common diet for 6 months before their drilling abilities were scored (to minimize any effects of plasticity). In addition, previous research has shown that SBR, BMR, and other populations vary in drilling phenotypes after being reared in the laboratory on a common diet ( M. trossulus ) through two generations, thus establishing a genetic basis to this trait variation (Sanford & Worth, 2009 ). The SBR population consists of uniformly strong drillers (Sanford & Worth, 2009 ), and the drilling ability of dogwhelks in this population was unaffected by the early‐life diet treatments. In contrast, the BMR population (comprised of a mix of drilling phenotypes; Sanford & Worth, 2009 ) responded strongly to the diet treatments, consistent with the hypothesis that phenotypic variation was a result of differential mortality and selection. Although the SBR population exhibited no phenotypic plasticity in response to the diet treatments, we cannot completely rule out the possibility that plasticity contributed to phenotypic variation in the BMR population. Additional studies are underway to explore genomic differences underlying variation in drilling traits in N. canaliculata (Longman et al., unpublished data). Overall, we suspect that all three of the factors discussed in this section—environmental and biotic variation, restricted within‐population variation in phenotypes, and relatively modest levels of selection—contributed to weaken eco‐evolutionary feedbacks in this rocky intertidal community. Experimental designs that use divergent populations from distant locations are common yet likely inflate the importance of genotypic/phenotypic variation in functional traits (Bassar et al., 2021 ; Hersch‐Green et al., 2011 ; Tack et al., 2012 ). Although such an experimental design would have been consistent with the approach of many studies of eco‐evolutionary dynamics to date, we agree with recent syntheses highlighting the need to study feedbacks where there is no separation in time between ecological and evolutionary dynamics (Bassar et al., 2021 ). In our view, this distinction lies at the heart of the debate about the relative importance of eco‐evolutionary feedbacks. There is a long history of studies demonstrating that spatially divergent selection on geographically separated populations can lead to local adaptation and altered species interactions, with community‐level impacts (Foster & Endler, 1999 ; Thompson, 1999b ). However, the novel and pressing question for the field of eco‐evolutionary dynamics is whether selection on variation in important functional traits within populations influences the dynamics of natural communities in a consequential way. In our study system, many aspects of natural history, phenotypic variation, selection, and food web structure were aligned to create the potential for strong eco‐evolutionary feedbacks. However, the effects of these feedbacks on community dynamics in the field ultimately proved to be weak. Although similar studies are required across many communities to assess the generality of our results, we suggest that a broad range of factors may often constrain and dampen the strength of eco‐evolutionary dynamics in natural communities."
} | 5,535 |
26645284 | null | s2 | 4,758 | {
"abstract": "Interactions between polymer molecules and inorganic nanoparticles can play a dominant role in nanocomposite material mechanics, yet control of such interfacial interaction dynamics remains a significant challenge particularly in water. This study presents insights on how to engineer hydrogel material mechanics via nanoparticle interface-controlled cross-link dynamics. Inspired by the adhesive chemistry in mussel threads, we have incorporated iron oxide nanoparticles (Fe3O4 NPs) into a catechol-modified polymer network to obtain hydrogels cross-linked via reversible metal-coordination bonds at Fe3O4 NP surfaces. Unique material mechanics result from the supra-molecular cross-link structure dynamics in the gels; in contrast to the previously reported fluid-like dynamics of transient catechol-Fe(3+) cross-links, the catechol-Fe3O4 NP structures provide solid-like yet reversible hydrogel mechanics. The structurally controlled hierarchical mechanics presented here suggest how to develop hydrogels with remote-controlled self-healing dynamics."
} | 263 |
27966672 | PMC5155288 | pmc | 4,759 | {
"abstract": "The order Thermoplasmatales ( Euryarchaeota ) is represented by the most acidophilic organisms known so far that are poorly amenable to cultivation. Earlier culture-independent studies in Iron Mountain (California) pointed at an abundant archaeal group, dubbed ‘G-plasma’. We examined the genomes and physiology of two cultured representatives of a Family Cuniculiplasmataceae, recently isolated from acidic (pH 1–1.5) sites in Spain and UK that are 16S rRNA gene sequence-identical with ‘G-plasma’. Organisms had largest genomes among Thermoplasmatales (1.87–1.94 Mbp), that shared 98.7–98.8% average nucleotide identities between themselves and ‘G-plasma’ and exhibited a high genome conservation even within their genomic islands, despite their remote geographical localisations. Facultatively anaerobic heterotrophs, they possess an ancestral form of A-type terminal oxygen reductase from a distinct parental clade. The lack of complete pathways for biosynthesis of histidine, valine, leucine, isoleucine, lysine and proline pre-determines the reliance on external sources of amino acids and hence the lifestyle of these organisms as scavengers of proteinaceous compounds from surrounding microbial community members. In contrast to earlier metagenomics-based assumptions, isolates were S-layer-deficient, non-motile, non-methylotrophic and devoid of iron-oxidation despite the abundance of methylotrophy substrates and ferrous iron in situ , which underlines the essentiality of experimental validation of bioinformatic predictions.",
"discussion": "Discussion Isolation of previously uncultured microorganisms from the environment remains one of the bottlenecks in microbiology hindering physiological and biochemical studies and demanding a resolution. It is especially important for archaea, the relatively recently discovered Domain, and which embraces a majority of difficult-to-culture organisms. The cultured diversity of archaea is dramatically low: according to the Euzeby LSPN online resource ( http://www.bacterio.net/ ), only some 116 genera and 451 species with validly published names of archaea (of which 55–60% are haloarchaea-related organisms) vs some 2277 genera и 11940 species of cultured and described bacteria are known to-date. The acidophiles of the order Thermoplasmatales are a good example of this status of things, accounting for only six cultured genera published since 1970, despite numerous documentations on the presence of highly diverse Thermoplasmatales -like organisms in low-pH habitats worldwide. The present genomic analysis of new successfully cultured Thermoplasmatales members 13 brought us closer to the understanding of functional diversity within this archaeal group. Interestingly, these archaea represent a unique case for Thermoplasmatales , when organisms from the same species and almost identical genomes from different geographic locations became cultured. Metabolically, Cuniculiplasmataceae resemble other Thermoplasmatales members, however certain discrepancies suggest some variety of their evolutionary trajectories. Cuniculiplasma spp. genomes encode the A1-type heme-copper oxidases forming a distinct clade at the root of A-type reductases and closely branching to the B-type oxygen reductases and are deficient in membrane-integral oxygen reductase subunits III and IV, suggesting that, in contrast with other Thermoplasmatales, they have a more ancient and less energetically efficient B-type enzymes. Cuniculiplasma spp. exhibit largest genomes among Thermoplasmatales seemingly at the expences of genetic loci for heavy metal resistance and defense systems. Scavenger type of nutrition was confirmed as a characteristic trait for Cunicuiplasma spp., which is reflected in their genomic blueprints and physiology, suggesting these organisms feed in situ on proteinaceous compounds derived from primary producing organisms. Based on the reconstructions of metagenomic data, the archaea related to this species previously supposed to be uncultured and associated to ’G-plasma cluster‘ are found in many acidic environments 1 6 . Certain features predicted from the metagenomic assembly “G-plasma” have not been confirmed highlighting the essentiality of cultivation efforts and experimental functional validation of genomic predictions. Almost identical genomes of the two European isolates and their North American sibling and strong conservation within their genomic islands, suggest a massive stabilizing selective pressure in similar acidic environments and/or significant fidelity of DNA repair systems assure their genome stability. Isolation of reference strains and experimental validation of genomic predictions for this archaeal group should be considered in the future as tasks of a highest priority."
} | 1,198 |
35567168 | PMC9099972 | pmc | 4,761 | {
"abstract": "Legumes are usually used as cover crops to improve soil quality due to the biological nitrogen fixation that occurs due to the interaction of legumes and rhizobia. This symbiosis can be used to recover degraded soils using legumes as pioneer plants. In this work, we screened for bacteria that improve the legume–rhizobia interaction in nutrient-poor soils. Fourteen phosphate solubilizer-strains were isolated, showing at least three out of the five tested plant growth promoting properties. Furthermore, cellulase, protease, pectinase, and chitinase activities were detected in three of the isolated strains. Pseudomonas sp. L1, Chryseobacterium soli L2, and Priestia megaterium L3 were selected to inoculate seeds and plants of Medicago sativa using a nutrient-poor soil as substrate under greenhouse conditions. The effects of the three bacteria individually and in consortium showed more vigorous plants with increased numbers of nodules and a higher nitrogen content than non-inoculated plants. Moreover, bacterial inoculation increased plants’ antioxidant activities and improved their development in nutrient-poor soils, suggesting an important role in the stress mechanisms of plants. In conclusion, the selected strains are nodulation-enhancing rhizobacteria that improve leguminous plants growth and nodulation in nutrient-poor soils and could be used by sustainable agriculture to promote plants’ development in degraded soils.",
"conclusion": "5. Conclusions Rhizosphere of Medicago spp. plants in the Piedras river estuary (southwest Spain) contains PGPR with appropriate properties to be used as biofertilizers. Particularly, the strains Pseudomonas sp. L1, Chryseobacterium soli L2, and Priestia megaterium L3 were able to improve M. sativa development and nodulation in a nutrient-poor soil, acting as NER. Although single bacterial inoculation had a positive effect in M. sativa growth under nutrient poverty stress, the combination of the three PGPR showed the best performance, demonstrating that a consortium of bacteria with complementary traits works better than single inoculation. The next step to confirm the positive obtained results with the inoculation with these bacteria should be to perform a trial in the original soil without sterilization in order to observe the results in a more realistic situation. As a final conclusion, legumes inoculated with Pseudomonas sp. L1, Chryseobacterium soli L2, and Priestia megaterium L3 could be used as biological tools for the ecological restoration of degraded soils and to promote sustainable agriculture.",
"introduction": "1. Introduction Legumes are a family of plants ( Fabaceae/Leguminosae ) formed by 765 genera and around 19,500 species [ 1 ]. This family of plants is characterized by the symbiotic relationship with rhizobia, a group of α- and β-proteobacteria including several genera, among others, such as Bradyrhizobium , Ensifer , Mesorhizobium , Rhizobium , and Sinorhizobium [ 2 ]. In this symbiosis, plants offer both a niche and carbon source to the bacteria while the latter provides NH 4 + by its ability to reduce atmospheric N 2 within the nodules [ 3 ]. Thanks to this association, legumes are pioneer plants that can grow and colonize degraded environments, overcoming abiotic stresses (nutrient-poor soils, saline soils, polluted soils, etc.) [ 4 , 5 ] and contributing to enrich degraded soils, with nitrogen improving their quality and fertility [ 6 , 7 ]. For this reason, legumes are used as a transition plant in intercropping to recover soil quality after a crop cultivation [ 8 , 9 ]. Nevertheless, nodulation and the nitrogen fixation effectiveness can be affected by abiotic stress [ 10 , 11 ], which also conditions the rhizobial population present in these kinds of soils [ 11 ]. In the rhizosphere, a higher concentration of bacteria exists around roots due to the exudates of plants which bacteria use as a source of nutrients [ 12 , 13 , 14 ]. Several of these rhizospheric bacteria possess plant growth-promoting (PGP) properties that help plant development and are known as plant-growth-promoting rhizobacteria (PGPR). PGPR assist plants by means of direct and indirect mechanisms such as nutrient (phosphorous, iron, and nitrogen) acquisition, phytohormones production, ethylene level modulation, and the production of lytic enzymes against phytopathogens [ 5 , 15 , 16 ]. Among these, the solubilization of phosphorous, siderophores production and nitrogen fixation are the major beneficial properties involved in nutrients acquisition. Thus, the presence of these properties in the bacteria helps plants to solubilize insoluble phosphates, improve iron uptake with siderophores, and increases the nitrogen content of the plant through nitrogen fixation [ 14 ]. In addition, PGPR can synthesize and produce phytohormones such as auxins (mainly indole-3-acetic acid, IAA), stimulating root elongation and improving nutrient acquisition [ 14 ]. The stress level in plants can also be modulated by PGPR’s amino-cyclopropane carboxylic acid (ACC) deaminase activity, an ethylene’s precursor degrading enzyme [ 14 , 17 ]. Among all PGPR, there is a non-rhizobia group which, in addition to its plant promoting properties, improves the symbiotic relationship between legumes and rhizobia enhancing nodulation, known as nodulation-enhancing rhizobacteria (NER) [ 11 ]. This kind of bacteria helps the plant to form nodules thanks to PGP properties—IAA production and ACC deaminase activity, the main traits implicated in this nodulation improvement [ 11 ]. The aim of this study is to enhance the growth of legumes in soils with nutrient poverty by means of the NER activities in order to recover degraded soils and improve their quality and fertility. For that, we selected soils of the Rio Piedras estuary (Huelva, Spain) as nutrient-poor soil, M. sativa as a model legume, and Ensifer medicae MA11 as the rhizobium to carry out the experiments. The interaction Medicago - Ensifer is well-known [ 18 ], and the strain MA11 was isolated from a degraded environment, showing nodulation capabilities under these kinds of conditions [ 19 ]. To accomplish the main aim, the following objectives were pursued: (i) isolation of rhizobacteria from the rhizosphere of Medicago spp. from the Rio Piedras estuary; (ii) first screening based on the ability to solubilize phosphate; (iii) identification and characterization of the phosphate solubilizing bacteria displaying additional PGP properties and enzymatic activities; (iv) selection of the best isolated PGPR; (v) determination in vitro of the role of the selected PGPR in the germination and nodulation in M. sativa ; (vi) determination of the effect of selected PGPR in the growth and the physiological status of plants of M. sativa grown in soil from the Rio Piedras estuary under greenhouse conditions.",
"discussion": "3. Discussion For decades, natural and human activities have caused negative effects in lands, giving rise to degraded soils characterized by poor quality, a decrease in fertility and nutrients content, and an increase of abiotic stresses such as salinity, heavy metals content, or drought. The presence of abiotic stresses in soils reduces plant biodiversity and creates an imbalance of nutrients [ 20 ]. Nutrient-poor soils produce a loss of microbial biodiversity and fertility and prevent the growth and development of plants, decreasing the ecological value of estuaries [ 21 , 22 ]. In addition, low fertility is one of the main limitations in crop production [ 23 ], so the loss of fertile soils is a worldwide challenge regarding food security [ 24 ]. A possible solution for the ecological restoration of agricultural lands is the use of legumes as cover crops. Legumes are beneficial in agricultural practices because their use reduces greenhouse effect gases, reduces the need for nitrogen based artificial fertilizers, avoids soil erosion, and, most importantly, provides organic compounds and nitrogen, increasing the fertility, nutrient content, and quality of soils [ 25 , 26 , 27 ]. The main goal of this work is the nodulation and development improvement of legumes, particularly M. sativa , in environments with nutrients poverty in order to use them as pioneer crops to recover the quality of degraded soils, promoting a sustainable agriculture. M. sativa was selected, in addition to the benefits that legumes provide to soils, because it is an important forage crop digestible for animals with great yield and high nutrient values [ 28 ]. For nodulation to be effective under stress conditions, proper rhizobial strains with high resistance towards stress must be used. In our case, Ensifer medicae MA11 had previously been shown to effectively nodulate Medicago plants under arsenic stress [ 19 ]. In addition to using a tolerant rhizobial strain, the isolation, characterization, and selection of PGPR and NER from the rhizosphere of Medicago spp. plants growing in a nutrient-poor estuarine soil were performed, since PGPR can assist plant growth through direct and indirect mechanisms [ 14 , 15 , 16 ], and NER can improve legumes nodulation [ 11 ]. Rhizospheric soil collected from the Rio Piedras estuary was mainly composed of sand, making it a light and drain soil. Furthermore, this soil had less than 1% of organic material and a low quantity of nutrients. These characteristics correspond to the definition of nutrient-poor soil [ 29 , 30 , 31 ], so the estuary of Rio Piedras could be considered as a degraded environment. The knowledge of the microbial population of this degraded soil is very important to find PGPR and NER able to promote legumes growth under the scarcity of nutrients [ 32 ]. In this work, 71 cultivable bacteria were isolated and, due to the low level of phosphorus in the soil, a first screening was performed, selecting those bacteria capable of solubilizing phosphate, reducing the selection of isolates to 14. Phosphorus is one of the most limiting factors for plant development, and phosphate solubilizing bacteria make it available to plants through the secretion of phytases, phosphatases, and organic acids into the soil [ 33 ]. The diversity of root-associated bacteria depends on the environment and its stress level [ 34 , 35 , 36 , 37 , 38 ], showing a lower number of microorganism taxa and fewer interactions among taxa in degraded soils with erosion [ 35 , 39 ]. This low diversity was also observed in rhizosphere of Medicago spp. From the Rio Piedras estuary at the genus level, Pseudomonas being the most representative ( Table 2 ). The presence of Pseudomonas as the most represented genus among the cultivable bacteria in this study is not a surprise since it is ubiquitous in soil and has well known genetic, environmental, and physiological adaptability to survive in any environment [ 40 ]. In addition, rhizosphere bacteria belonging to genus Pseudomonas had been isolated from other legumes such as peanut, soybean, and broad bean [ 41 , 42 , 43 , 44 ]. The rest of the genera isolated in this work, namely, Chryseobacterium , Priestia , Bacillus , and Buttiauxella , have been also described as associated to legumes by other authors [ 45 , 46 , 47 , 48 , 49 , 50 ]. In addition to phosphate solubilization, isolated bacteria showed individually several PGP properties ( Figure 1 A). IAA is an auxin, a phytohormone involved in numerous processes in plant development, mainly in root elongation facilitating nutrient absorption by plants [ 51 ]. Moreover, IAA is also involved in nodule formation because it intervenes in the relationship between rhizobia and legume, and the functionality of the nodule gets to delay the senescence by the interaction with the bacteroid inside the nodules [ 11 ]. This property was observed in all isolates, making all of them good candidates to improve the nodulation of legumes growing in degraded estuaries. Siderophores production is another important PGP property in degraded soil with nutrient deficiency because they have affinity for iron, forming a complex that can be assimilated by plants [ 52 , 53 ]. Furthermore, the production of siderophores is also related to biotic control due to competition for Fe with phytopathogens [ 12 ]. A total of 87% of isolated strains in this study were grown in minimal medium without a nitrogen source, indicating that they could fix nitrogen, which would additionally increase the amount of nitrogen fixed within the nodules [ 54 ]. ACC deaminase, detected in half of the rhizobacteria ( Figure 1 A), allows bacteria to modulate the ethylene concentration in plants since this enzyme hydrolyzes the ethylene’s precursor [ 55 , 56 ]. With this modulation, bacteria promote plant growth under stress conditions [ 57 ] and even improve the nodulation and the functionality of the nodules because ethylene is also involved in nodule senescence [ 58 , 59 , 60 ]. Finally, although biofilm formation was the less represented property among isolates, it could help plants to grow in degraded environments since biofilm concentrates bacteria coating roots and facilitates important processes like nitrogen fixation, the mineralization of organic N and P, and nutrients absorption [ 61 ]. The secretion of lytic enzymes such as chitinases, lipases, pectinases, or proteases acting against the wall of phytopathogens are also interesting traits to enhance plant development [ 55 ]. In this respect, only L1, L2, and L3 showed enzymatic activities for protease, pectinase, and chitinase. Moreover, strain L3 also showed cellulase activity, important to degrading the vegetal cell wall and facilitating the rhizobia entry in roots [ 62 , 63 ]. These three strains, Pseudomonas sp. L1, Chryseobacterium soli L2, and Priestia megaterium L3, which also showed five of the six tested PGP properties with high values individually and all of the PGP properties altogether, were selected to continue with plant experiments. The inoculation of M. sativa seeds with selected strains showed that all of them increased the number of germinated seeds individually, although inoculation with the three ( consortium ) showed the highest number of germinated seeds. This positive effect in M. sativa seed germination was also reported in [ 64 ], where Bacillus spp. strains improved the number of germinated seeds. The results concerning plant development and nodulation were similar both in vitro and under greenhouse and nutrient-poor conditions. Inoculated plants showed an increase in both shoots and roots development, the increase in those inoculated with the consortium being the highest one. Supporting these results, several authors verified that the inoculation of M. sativa plants with PGPR improved plant biomass and length in nutrient-poor substrate, both in vitro [ 65 ] and in greenhouse conditions [ 66 ] and even in the presence of other abiotic stresses such as salinity and heavy metals [ 28 , 64 , 67 , 68 ]. In addition to plant development, the nodulation in plants was also higher in inoculated groups, in agreement with the results obtained in other studies where the nodulation in M. sativa was tested under stress conditions [ 69 ]. The fact that co-inoculated plants had a greater number of nodules could be related to the nitrogen content in plants since they also showed higher values of nitrogen [ 28 ]. The positive effect in plant physiology was also observed in photosynthetic parameters since inoculation with the consortium improved the photosynthetic status in plants under nutrient poverty stress. Similarly, in M. sativa plants growing in a substrate with a low quantity of phosphorous, the inoculation with Priestia megaterium, formerly Bacillus megaterium [ 70 ], increased the chlorophyll content [ 66 ], and in M. sativa plants under heavy metals stress, inoculation was involved in higher values of physiological parameters such as the ETR, Fv/Fm, Φ PSII , A N , and g s [ 66 ]. Finally, the stress level in plants growing in nutrient-poor estuarine soils was determined by the measurement of antioxidant enzymatic activities. The inoculation with the consortium of the selected bacteria showed a significant increase in these enzymatic activities. This increase was also observed in other studies with M. sativa plants under different abiotic stresses [ 28 , 64 ], suggesting that bacterial inoculation could elevate the stress response in plants, improving and recovering the plant status in degraded environments. According to the results, the inoculation of M. sativa with the consortium of the three selected strains showed more vigorous plants with more nodules than the single inoculations, suggesting that the consortium had the higher effect on the physiological status and nodulation of alfalfa plants under nutrient-poor soil stress. These positive effects could be due to the presence of protease, pectinase, chitinase, and cellulose activities together. As mentioned above, cellulase activity is involved in the nodulation process [ 62 , 63 ] because it facilitates the entry of the rhizobium in the root. In a similar way, pectinase can also degrade the cell wall and could increase the nodulation when plants are inoculated with the consortium [ 63 ]. In relation to the improvement in the germination, protease could be involved in the mobilization of the protein reserves in seeds during germination. The presence of all of the PGP properties in the consortium could be the reason for the increase in all of the measured parameters. The biofilm formation and the ACC deaminase activity seem to be the main differences that provide the consortium with the stronger effects, improving the acquisition of nutrients and decreasing the ethylene levels in plants in response to the nutrient poverty in soils [ 17 , 61 ]. More studies should be performed in order to elucidate the role of each PGP property in the plant and nodulation improvement."
} | 4,494 |
28587442 | PMC5459778 | pmc | 4,763 | {
"abstract": "The first methanotrophic syntheses of polyhydroxyalkanoates (PHAs) that contain repeating units beyond 3-hydroxybutyrate and 3-hydroxyvalerate are reported. New PHAs synthesized by methanotrophs include poly(3-hydroxybutyrate- co -4-hydroxybutyrate) (P(3HB- co -4HB)), poly(3-hydroxybutyrate- co -5-hydroxyvalerate- co -3-hydroxyvalerate) (P(3HB- co -5HV- co -3HV)), and poly(3-hydroxybutyrate- co -6-hydroxyhexanoate- co -4-hydroxybutyrate) (P(3HB- co -6HHx- co -4HB)). This was achieved from a pure culture of Methylocystis parvus OBBP where the primary substrate is methane and the corresponding ω-hydroxyalkanoate monomers are added as a co-substrate after the cells are subjected to nitrogen-limited conditions. Electronic supplementary material The online version of this article (doi:10.1186/s13568-017-0417-y) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction Polyhydroxyalkanoates (PHAs) are microbial storage polymers accumulated by many different groups of bacteria as an intracellular carbon and energy reserve. PHAs are biodegradable, biocompatible, and renewable bioplastics (Myung et al. 2014 ) that could substitute for petrochemical-derived plastics in many applications. Accumulation of PHAs occurs when bacterial cells grow under conditions where substrates other than the electron donor (typically the carbon source), such as nitrogen or phosphorus, limit growth. Depending upon the carbon co-substrates supplied during this nutrient-limited period, PHAs with different compositions are produced. Over one hundred different carboxylic acid monomers are reported to be incorporated into PHAs, resulting in polymers with a wide range of material properties (Steinbüchel and Gorenflo 1997 ). Among the variety of polymers produced, 4-hydroxybutyrate (4HB) homopolymer or its copolymer are of interest for various biomedical applications (Martin and Williams 2003 ). It is a strong, flexible thermoplastic material that can be processed easily to form scaffolds, heart valves, or cardiovascular tissue supports (Martin and Williams 2003 ). In addition, 4HB polymer is extremely well tolerated in vivo because biological hydrolysis of 4HB homopolymer or copolymer yields 4HB, which is a common metabolite in the human body (Nelson et al. 1981 ). A copolymer of 3-hydroxybutyrate (3HB) and 4HB units is degradable by lipase and PHA depolymerase, in contrast to most PHAs, which cannot be degraded by lipase (Saito and Doi 1994 ; Wu et al. 2009 ). Aside from 4HB, the presence of structurally similar monomer units such as 5-hydroxyvalerate (5HV) and 6-hydroxyhexanoate (6HHx) in PHAs also adds elasticity to the polymer and enhances lipase-mediated degradation of the polymer (Mukai et al. 1993 ). Use of methane (CH 4 ) as a feedstock for PHA production can significantly decrease costs and environmental impacts (Rostkowski et al. 2012 ; Strong et al. 2015 ). Methane is currently widely available as the major component of natural gas and biogas obtained from the anaerobic degradation of organic waste. When CH 4 is the sole feedstock, high molecular weight poly(3-hydroxybutyrate) (P3HB) is the sole PHA product (Wendlandt et al. 2001 ; Pfluger et al. 2011 ; Pieja et al. 2011 ; Myung et al. 2015b , 2016b ). Recently, we reported production of poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBV) copolymer by a methanotrophic enrichment (Myung et al. 2015a ) and a pure culture of obligate Type II methanotrophs (Myung et al. 2016a ) when fed CH 4 as a primary feedstock and valerate as a co-substrate. In general, bacterial enzymes involved in PHA synthesis have broad substrate specificity (Poirier et al. 1995 ). For example, in Alcaligenes eutrophus , the PHA synthase can incorporate 3-hydroxyvalerate (3HV), 4HB, 4-hydroxyvalerate, 5HV, and 4-hydroxyhexanoate into PHAs (Haywood et al. 1989 ; Valentin et al. 1992 , 1994 ). To our knowledge, this same broad specificity for substrates was not known for methanotrophic bacteria. Herein, we report the first methanotrophic synthesis of PHAs that contain repeating units beyond 3HB and 3HV, including poly(3-hydroxybutyrate- co -4-hydroxybutyrate) (P(3HB- co -4HB)), poly(3-hydroxybutyrate- co -5-hydroxyvalerate- co -3-hydroxyvalerate) (P(3HB- co -5HV- co -3HV)), and poly(3-hydroxybutyrate- co -6-hydroxyhexanoate- co -4-hydroxybutyrate) (P(3HB- co -6HHx- co -4HB)). This was achieved by a pure culture of Methylocystis parvus OBBP when the primary substrate is CH 4 and the corresponding ω-hydroxyalkanoate monomers are added as co-substrates.",
"discussion": "Discussion Our group and others have previously reported production of P3HB and PHBV by pure culture methanotrophs using CH 4 and various co-substrates (Cal et al. 2016 ; Flanagan et al. 2016 ; Myung et al. 2016a ). The general rule was that even-numbered carbon co-substrates (e.g. 3HB, crotonate) led to production of P3HB, whereas odd-numbered carbon co-substrates (e.g. propionate, valerate, 2-pentenoate, or pentanol) led to production of PHBV. Depending on the number of carbon atoms, fatty acid substrates are processed via the beta-oxidation pathway into either acetyl-CoA or propionyl-CoA, the precursors of P3HB and PHBV. Addition of ω-hydroxyalkanoate co-substrates resulted in a different outcome. In this case, M. parvus OBBP synthesized a random copolymer containing 3HB and ω-hydroxyalkanoate monomers. While various species of bacteria have synthesized PHAs containing 4HB or 5HV monomers (Poirier et al. 1995 ; Chanprateep et al. 2010 ; Chuah et al. 2013 ), this is the first report of methane-dependent production of PHA copolymers other than PHBV. The outcome of ω-hydroxyalkanoate co-substrate addition was dependent upon the presence and position of the hydroxyl group (Fig. 5 ; Table 1 ). When butyrate or 3HB was added, P3HB was produced; when 4HB was added, the resulting polymer was P(3HB- co -4HB). When valerate was added, the resulting polymer was PHBV, but when 5HV was added, the product was P(3HB- co -5HV- co -3HV). When hexanoate was added, the polymer product was P3HB, but when 6HHx was added, the product was P(3HB- co -6HHx- co -4HB). From these results, we can conclude that the presence of a hydroxyl group and its position play a key role in determining the composition of the PHA produced. When there is a hydroxyl group on the nth carbon (e.g. ω-hydroxyalkanoates), the co-substrate seems to be incorporated directly into the polymer by PHA synthase (PhaC) (Fig. 6 ), and it can also undergo beta-oxidation, as evidenced by formation of 4HB monomer units derived from the 6HHx co-substrate. Fig. 5 Scheme of PHA production using methane and co-substrates. a Without the hydroxyl group and b with the hydroxyl group \n Fig. 6 C 1 -oxidation dependent synthesis of PHAs in serine-cycle methanotrophic bacteria. The bold arrow denotes an acyl-CoA pathway likely activated by ω-hydroxyalkanoate co-substrates \n Thermal stability is critical for polymer melt processing. P3HB has a narrow processing window, melting at 175–180 °C and thermally degrading at ~190 °C. PHA copolymers produced by incorporation of ω-hydroxyalkanoate monomers have significantly lower melting temperatures (T m ), expanding the processing window (Table 2 ). These copolymers also had lower T g values suggesting increased chain mobility compared to P3HB. This would manifest as a decrease in brittleness, an expectation confirmed by the results of mechanical testing summarized below. Key mechanical properties for useful application of bioplastics are Young’s modulus (E), tensile strength (σ t ), and elongation at break (ε b ). P3HB has high E and σ t , but is brittle, with a small ε b . Short chain-length monomers generally confer toughness and high crystallinity, and medium chain-length monomers generally confer elasticity and low crystallinity. Thus, a mixture of the two enables production of PHA that is both tough and elastic, an important combination of properties for many applications. Heterotrophic bacteria have been shown to produce P(3HB- co -4HB) (Doi et al. 1990 ; Nakamura et al. 1992 ; Saito and Doi 1994 ; Chanprateep et al. 2010 ) and P(3HB- co -5HV- co -3HV) (Doi et al. 1987 ; Steinbuchel and Valentin 1995 ), but methanotrophic synthesis of these PHA copolymers has not been reported previously. To the best of our knowledge, this is the first reported microbial synthesis of P(3HB- co -6HHx- co -4HB). Generalizing these results, we envision that control over the structure and concentration of added co-substrates will enable synthesis of copolymers suitable for a broad range of applications (Fig. 4 ). Use of CH 4 as the primary substrate for PHA synthesis is of interest because CH 4 is abundant, cheap, and its use does not adversely impact the food supply, unlike cultivated feedstock (Levett et al. 2016 ). The cost of ω-hydroxyalkanoate co-substrates is high, but can be significantly reduced when lactones are used as the precursor for chemical synthesis of ω-hydroxyalkanoates (see “ Materials and methods ” section). We conclude that provision of methane as primary substrate and addition of ω-hydroxyalkanoate as co-substrates is a promising route for synthesis of polymers with tunable physical properties."
} | 2,319 |
37143804 | PMC10153088 | pmc | 4,767 | {
"abstract": "Cellular transport systems are sophisticated and efficient. Hence, one of the ultimate goals of nanotechnology is to design artificial transport systems rationally. However, the design principle has been elusive, because how motor layout affects motile activity has not been established, partially owing to the difficulty in achieving a precise layout of the motile elements. Here, we employed a DNA origami platform to evaluate the two-dimensional (2D) layout effect of kinesin motor proteins on transporter motility. We succeeded in accelerating the integration speed of the protein of interest (POI) to the DNA origami transporter by up to 700 times by introducing a positively charged poly-lysine tag (Lys-tag) into the POI (kinesin motor protein). This Lys-tag approach allowed us to construct and purify a transporter with high motor density, allowing a precise evaluation on the 2D layout effect. Our single-molecule imaging showed that the densely packed layout of kinesin decreased the run length of the transporter, although its velocity was moderately affected. These results indicate that steric hindrance is a critical parameter to be considered in the design of transport systems.",
"conclusion": "4. Conclusion The finding that a densely-packed layout decreased the run length reinforces the importance of the spatial layout in designing motile systems. The power of the DNA origami approach should reveal the similarities and differences between different motor proteins ( e.g. , kinesin, myosin, and dynein), explaining the differences in periodicity (intermolecular distance) in natural systems, such as muscles (for myosin) and cilia/flagella (for dynein). Moreover, combining theoretical model analysis 29,31,46,47 with our evaluation of 2D layout effects should pave the way for constructing functional artificial transport/motile systems in natural and artificial cells, allowing the investigation of how other factors influence motility. 48",
"introduction": "1. Introduction In natural motile systems, motor proteins are aligned in a specific layout and exhibit power-efficient and reliable movements. 1–7 Mimicking these natural systems to create efficient artificial motile systems is one of the ultimate goals of nanotechnology. The fundamental question of these motile system constructions is how collective motility (team activity) differs from the motile activity of a single molecule and how the individual motor contributes to overall collective movement. 3,8–10 One approach to understanding collective movement is elucidating the effects of key parameters ( e.g. , motor number and layout) on motile activity (such as velocity and run length). Kinesin is a processive motor protein that walks along the microtubules and has been used as a model protein for collective movement. In previous studies, the effect of motor number has been examined; however, the effects of density and layout remain elusive. 11–16 This is because of the (1) heterogeneity of the samples and (2) difficulty in controlling the number and density/layout separately. These drawbacks can be attributed to conventional assays (such as beads and gliding assays), where the motors are generally randomly adsorbed to the transporter, and the distributions of motor number and intermolecular distance are broad. To overcome these limitations, DNA-based assays that allow researchers to design and construct transporters with a defined number and layout of motor molecules have been developed. 17–19 Although these studies have successfully evaluated the effect of one-dimensional (1D) intermolecular distance of motor proteins on motility, the effect of intermolecular distance remains controversial. 20 Therefore, a more systematic examination of intermolecular distances is crucial. DNA origami is a versatile method used to construct custom two- and three-dimensional structures and control precise molecular layouts. 21–28 The molecular layout capability of DNA origami at nanometer resolution could provide a solid platform to elucidate the effect of intermolecular distance precisely. However, the advantages of DNA origami are yet to be completely exploited, partly because of the technical limitations of transporter construction. Previous studies used a hybridization method to integrate motor proteins, 19,29–31 where single-stranded DNA (ssDNA)-bound motor proteins were hybridized to ssDNA handles protruding from the DNA nanostructure. However, a short ssDNA handle, reduces the yield of protein integration. In contrast, long double-stranded DNA (dsDNA) handle makes it difficult to evaluate the intermolecular distance precisely, especially for short-distance ranges. Subsequently, direct and covalent attachment of motor proteins have been reported, 20,32,33 where protein-tag technology, such as SNAP-tag protein, was used to ensure the stable binding of the protein to the DNA nanostructure with a smaller handle. The central problems associated with these covalent binding tags are slow assembly speed and low integration yield. This reduction in integration may be partly owing to the charge repulsion between the protein and DNA nanostructure, where both are negatively charged in neutral buffer conditions [the isoelectric points (pIs) of two major tag proteins, SNAP-tag and HaloTag, are 6.0 and 4.9, respectively]. Therefore, a high molar ratio of the motor protein to the DNA nanostructure is required to compensate for the low integration yield, which makes the removal of the excess motor proteins during the purification step challenging. These contaminating free motor proteins impede the evaluation of transporter activity even when using single-molecule imaging. Moreover, in some cases, a long incubation time with a high protein concentration induces the aggregation of proteins, DNA nanostructures, or protein–DNA nanostructure complexes. Here, we partially solved the problem of a low integration yield by introducing a positively charged peptide tag (poly-lysine tag composed of 5–10 lysine amino acids, Lys-tag) with an up to 700 times higher association rate ( k on ) of SNAP f -tag-fused kinesin. This Lys-tag allowed us to decrease the molar ratio of kinesin to the DNA nanostructure while maintaining high integration efficiency (>86%, where more than half of the transporters have the correct number in the case of the 4-kinesin integrated transporter). We successfully constructed and purified transporters with a high motor density (intermolecular distance of 7 nm, which matched the unit lattice size of the microtubule of 8 nm). Based on this system, we evaluated the effect of the 2D layout of kinesin molecules on artificial transporters. Our results showed that the densely packed layout of kinesin decreased the run length of the transporters, although the speed was similar. These results indicate that steric hindrance is a critical parameter to consider while designing artificial transporters.",
"discussion": "3. Discussion The key to building a sophisticated and efficient motile system is establishing a design principle. It is essential to evaluate the layout effect, because the architecture of motor proteins is integral to natural motile systems, such as muscles, cilia/flagella, and vesicle transporters. In this context, researchers have exploited the advantages of DNA nanostructure platforms to control the number and layout of molecules. However, understanding of the effect of the molecular layout ( e.g. , intermolecular distance) on the motility of transport systems is limited, partly owing to practical obstacles to transporter construction. Here, we introduced a Lys-tag to improve the construction process and successfully evaluated the effect of the 2D molecular layout. We found that the density of the kinesin motor protein affected the run length but only moderately affected the velocity. Both processive (dimer) and non-processive (monomer: catalytic domain only) kinesins support this conclusion, suggesting that steric hindrance control is the key to transporter design. In transporter construction, there are two major steps: (1) integration of motor proteins and (2) removal of excess unbound motor proteins from the transporter complex (purification of the transporter complex). The slow assembly of the protein on the DNA nanostructure hinders the first integration step. The necessity of a high concentration of molecules to drive an efficient reaction and achieve a high yield causes side effects, such as the aggregation of DNA nanostructures (ESI Fig. 5 † ). The second purification step is critical because of the intrinsic characteristics of the DNA nanostructure. The negative charge of the DNA nanostructure decreases the binding rate of the transporter (DNA nanostructure–protein complex) to the rail protein ( e.g. , microtubule) compared to that of the unbound free protein (ESI Fig. 17 † ). Thus, the complete removal of excess unbound motor proteins is critical for the assay. The trade-off between the requirement of a high concentration in the construction step and the desirability of a low concentration in the purification process makes transporter construction difficult. The Lys-tag partially resolved this trade-off and allowed us to decrease kinesin concentration during the integration process while maintaining a high final transporter yield. The lysine number had a strong effect on the acceleration factor of k on : 3 and 700 times for 5 K and 10 K of SNAP f , respectively ( Fig. 2 ). Moreover, the acceleration factor of the Lys-tag differed from those of the SNAP f -tag and HaloTag (700 vs. 10 with 10 K Lys-tag). These results might be attributable to the difference in the estimated charge on the tag protein (−18.8, −9, −5.3, and +4.5 at pH 7.4, for Halo-0 K, Halo-10 K, SNAP f -0 K, and SNAP f -10 K, respectively), as the apparent association rate ( k on ) of the tag protein to the DNA nanostructure apparently depended on the charge (ESI Fig. 10 † ). This hypothesis is supported by the HaloTag mutant results, where replacing surface amino acids with positively charged amino acids improved the k on of the HaloTag protein to the DNA nanostructure. 37 Further optimization of the peptide sequence and/or fusion to the DNA-binding motif should improve the assembly of many proteins on DNA nanostructures, 38,39 where it might also be possible to design the integration process precisely using the difference in k on . We evaluated the 2D layout effect on two key parameters of the transporter (velocity and run length) using a DNA origami platform with a defined number and layout of motor molecules. We found that the layout moderately affected the velocity, whereas the densely packed layout decreased the run length (when comparing transporters with the same motor number). Regarding velocity, the number of kinesins involved in the motile movement of a transporter should range from one to a fully defined number ( e.g. , 4 for a 4-Kin transporter) in our system. Using a gliding assay, it has been reported that the velocity of kinesin's collective movement is independent of motor density. 11 Our results reflected this characteristic. The similar velocities of the transporter with different defined motor numbers support this view ( Fig. 3 ). Another interpretation is that the number involving motile movement is less than the threshold number required to demonstrate the effect of counterforce produced by the microtubule-bound motors that do not produce the effective movement of the transporter while imposing negative interference. 12,40 Future transporter studies with a larger number of motors are required to provide more precise information. For this line of experiments, improvement of the construction yield is the key, 36 including improving the origami design, such as using rationally designed scaffold–staple pairs. 41,42 In the present study, in contrast to velocity, molecular layout affected run length under these conditions. We observed short run lengths with densely packed layouts (when only comparing transporters with the same motor number). The run length was also affected by ionic strength (NaCl concentration; Fig. 3 and ESI Fig. 20 † ), suggesting that electrostatic interactions are essential for transporter activity 43 ). Regarding the motile mechanism, there are two key parameters (on- and off-rates of the motor 15,20 ). Currently, the reason for this remains unknown; however, we speculate that steric hindrance might affect both the on- and off-rates (see ESI † Discussion). The effect of two-dimensional (2D) motor array, i.e. , side-by-side arrangement versus along the axis of the microtubule rail, is an interesting issue regarding the movement of the transporter. Our results suggest that the effect was not strong under our experimental conditions (see ESI Fig. 21 † and 4 for the two- and four-motor systems, respectively). However, our current data do not reveal the exact orientation of the DNA origami transporter relative to that of the microtubule rail. In future studies, further confirmation with qualitative analysis is required with a similar approach to the literature ( 14,15 , i.e. , attaching a marker to observe the individual motor and transporter movement). Currently, this is technically challenging owing to the large size of probes such as quantum dots (QD, 25 nm, 14 ) and gold nanoparticles (40 nm, 44 ) compared to the intermolecular distance between two kinesin molecules (such as a minimum of 7 nm in our study). Future advances in new probes with smaller sizes while providing sufficient photon numbers (such as nano-diamonds 45 ) might overcome these technical issues."
} | 3,410 |
23061026 | PMC3463476 | pmc | 4,769 | {
"abstract": "The widespread exchange of genes between bacteria must have consequences on the global architecture of their genomes, which are being found in the abundant genomic data available today. Most of the expansion of bacterial protein families can be attributed to transfer events, which are positively biased for smaller evolutionary distances between genomes, and more frequent for classes that are larger, when summed over all known bacteria. Moreover, “innovation” events where horizontal transfers carry exogenous evolutionary families appear to be less frequent for larger genomes. This dynamic expansion of evolutionary families is interconnected with the acquisition of new biological functions and thus with the size and distribution of the genes’ functional categories found on a genome. This commentary presents our recent contributions to this line of work and possible future directions."
} | 223 |
36428233 | PMC10078134 | pmc | 4,772 | {
"abstract": "Abstract Plant recruitment interactions (i.e., what recruits under what) shape the composition, diversity, and structure of plant communities. Despite the huge body of knowledge on the mechanisms underlying recruitment interactions among species, we still know little about the structure of the recruitment networks emerging in ecological communities. Modeling and analyzing the community‐level structure of plant recruitment interactions as a complex network can provide relevant information on ecological and evolutionary processes acting both at the species and ecosystem levels. We report a data set containing 143 plant recruitment networks in 23 countries across five continents, including temperate and tropical ecosystems. Each network identifies the species under which another species recruits. All networks report the number of recruits (i.e., individuals) per species. The data set includes >850,000 recruiting individuals involved in 118,411 paired interactions among 3318 vascular plant species across the globe. The cover of canopy species and open ground is also provided. Three sampling protocols were used: (1) The Recruitment Network (RN) protocol (106 networks) focuses on interactions among established plants (“canopy species”) and plants in their early stages of recruitment (“recruit species”). A series of plots was delimited within a locality, and all the individuals recruiting and their canopy species were identified; (2) The paired Canopy‐Open (pCO) protocol (26 networks) consists in locating a potential canopy plant and identifying recruiting individuals under the canopy and in a nearby open space of the same area; (3) The Georeferenced plot (GP) protocol (11 networks) consists in using information from georeferenced individual plants in large plots to infer canopy‐recruit interactions. Some networks incorporate data for both herbs and woody species, whereas others focus exclusively on woody species. The location of each study site, geographical coordinates, country, locality, responsible author, sampling dates, sampling method, and life habits of both canopy and recruit species are provided. This database will allow researchers to test ecological, biogeographical, and evolutionary hypotheses related to plant recruitment interactions. There are no copyright restrictions on the data set; please cite this data paper when using these data in publications."
} | 600 |
34093485 | PMC8170126 | pmc | 4,773 | {
"abstract": "Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of “ Candidatus Methylacidithermus pantelleriae” PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, “ Ca. Methylacidithermus pantelleriae” might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs.",
"conclusion": "Conclusion and Ecological Role “ Ca. Methylacidithermus pantelleriae” PQ17 presents most of the typical characteristics of verrucomicrobial methanotrophs. This microorganism was detected in a thermoacidophilic volcanic environment and its genome predicts it to be an aerobic bacterium able to fix carbon via the CBB cycle. Methane oxidation to methanol may use the methane monooxygenase encoded by the pmoCAB operon and the conversion of methanol could be carried out by the XoxF-type MDH. Contrary to other verrucomicrobial methanotrophs, the genome of strain PQ17 does not encode genes for nitrogen fixation, nor for the oxidation of hydrogen, a common energy substrate for verrucomicrobial methanotrophs. These features, together with phylogenetic analysis, suggest that “ Ca. M. pantelleriae” has evolved differently from other verrucomicrobial methanotrophs. This bacterium probably utilizes exclusively methane or methanol for energy production and provides nitrogen for biomass mainly via nitrate and ammonia and not by fixing N 2 gas.",
"introduction": "Introduction Verrucomicrobial methanotrophs are a group of aerobic bacteria usually found in the acidic soil of geothermal active regions ( Dunfield et al., 2007 ; Pol et al., 2007 ; Islam et al., 2008 ; Sharp et al., 2014 ; van Teeseling et al., 2014 ). Their genomes all encode one or up to three particulate methane monooxygenase enzymes (pMMO) for the conversion of methane to methanol and a XoxF-type methanol dehydrogenase (MDH) to transform methanol to formate. The peculiarity of their XoxF-MDH is the strict dependence on rare earth elements (REEs), which are present in the active site together with the pyrroloquinoline quinone (PQQ) cofactors ( Pol et al., 2014 ). Formate is ultimately converted to CO 2 by a formate dehydrogenase ( Picone and Op den Camp, 2019 ). Inorganic carbon is fixed autotrophically using the Calvin-Benson-Bassham (CBB) cycle rather than the serine- or ribulose monophosphate (RuMP) pathways used by proteobacterial methanotrophs ( Khadem et al., 2011 ; van Teeseling et al., 2014 ). Two verrucomicrobial methanotrophs were shown to be able to grow in the absence of methane when supplied with a mixture of carbon dioxide and hydrogen ( Mohammadi et al., 2017a ; Schmitz et al., 2020 ). Moreover, they are capable of nitrogen fixation and partial denitrification ( Khadem et al., 2010 ; Mohammadi et al., 2017b ). The current classification divides verrucomicrobial methanotrophs into two genera: Methylacidimicrobium , generally mesophilic and extremely acidophilic and the thermophilic but less acidophilic Methylacidiphilum ( Dunfield et al., 2007 ; Pol et al., 2007 ; Islam et al., 2008 ; Sharp et al., 2014 ; van Teeseling et al., 2014 ; Picone et al., 2021 ). Verrucomicrobial methanotrophs were detected in different geothermal ecosystems, including the Favara Grande, a volcanic area on Pantelleria Island, Italy. In particular, pmo -containing bacteria closely related to Methylacidiphilum fumariolicum SolV, were detected in site FAV2 ( Gagliano et al., 2014 ). This site was characterized by pH values of 4–4.5 and temperature ranging from 60°C in the top layer of the soil, to 92°C at 50 cm depth. Ammonia was scarce, whereas high emissions of carbon dioxide (CO 2 ), hydrogen (H 2 ), and methane (CH 4 ) were recorded ( Gagliano et al., 2016 ). A recent metagenomic analysis of site FAV2 revealed a methanotrophic community composed of Proteobacteria and Verrucomicrobia ( Picone et al., 2020 ), supporting the findings of Gagliano et al. (2014) . Two Metagenome Assembled Genomes (MAGs) that belonged to the phylum Verrucomicrobia were retrieved. One of these MAGs (MAG5) was a novel Methylacidimicrobium species ( Picone et al., 2021 ). MAG9, instead, did not classify as Methylacidiphilum or Methylacidimicrobium , indicating that it may represent a novel genus. In this study we determine the phylogeny of this new verrucomicrobial methanotroph species and analyze its genome to predict the metabolic potential.",
"discussion": "Results and Discussion Proteobacterial and Verrucomicrobial Methanotrophs in the Soil of Favara Grande The Favara Grande is an area on Pantelleria Island characterized by hydrothermal activity with gas emissions of CO 2 , H 2 , and CH 4 ( Picone et al., 2020 ). Within the bacterial community, methanotrophs belonging to the Gammaproteobacteria and Verrucomicrobia phyla could be identified through pmoA sequencing ( Gagliano et al., 2014 ). 16S rRNA gene amplicon sequencing analysis, instead, did not detect Verrucomicrobia in the soil of Favara Grande, but potential methanotrophy was mainly attributed to Gammaproteobacteria ( Gagliano et al., 2016 ). Recent Illumina metagenomic sequencing at a much higher resolution could show the presence of five MAGs related to methanotrophs ( Picone et al., 2020 ; Supplementary Figure 1 ). MAG2 resembled a novel gammaproteobacterial Methylobacter species ( Hogendoorn et al., 2021 ) and MAG8 and MAG16 were related to Methylococcus sp. The two remaining MAGs clustered within the phylum Verrucomicrobia. A detailed description of Methylacidimicrobium thermophilum AP8, an isolated representative of MAG5, was recently published ( Picone et al., 2021 ). 16S rRNA analysis of MAG9 revealed a species phylogenetically distant to other known verrucomicrobial methanotrophs. The closest cultured relatives were Methylacidiphilum sp. RTK17 and Methylacidiphilum infernorum V4, that shared only 89.9% 16S rRNA identity to MAG9. This MAG was analyzed in detail. Phylogenetic analysis showed that the 16S rRNA gene of MAG9 clustered in between Methylacidimicrobium and Methylacidiphilum species ( Figure 1 ; Dunfield et al., 2007 ; Pol et al., 2007 ; Op den Camp et al., 2009 ; van Teeseling et al., 2014 ; Schmitz et al., 2021 ). The 16S rRNA and AAI values ( Table 1 and Supplementary Table 1 ) fell below the threshold for species delimitation (95% for AAI and 98.7–99% for 16S rRNA) ( Stackebrandt and Ebers, 2006 ; Thompson et al., 2013 ). Considering the AAI thresholds proposed by Konstantinidis et al. (2017) for uncultivated microorganisms (45–65% for the same family, 65–95% for the same genus and 95–100% for the same species), these results classified MAG9 as representing a new species of a new genus, for which we propose the name “ Candidatus Methylacidithermus pantelleriae” sp. PQ17. This “ Ca. Methylacidithermus” genus is the third genus of methanotrophic Verrucomicrobia within the family Methylacidiphilaceae, that adds to the previously described Methylacidiphilum and Methylacidimicrobium genera ( Op den Camp et al., 2009 ; van Teeseling et al., 2014 ; Schmitz et al., 2021 ). FIGURE 1 16S rRNA gene-based phylogenetic tree of methanotrophic Verrucomicrobia. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.41438253 is shown. The percentage of replicate trees (>50%) in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 17 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1,575 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 ( Kumar et al., 2016 ). TABLE 1 Average amino acid identity (AAI) value comparison between different verrucomicrobial methanotroph species. Species 1 2 3 4 5 6 7 1. “ Ca . Methylacidithermus pantelleriae” PQ17 50.6 50.8 51.0 53.6 53.3 52.7 2. Methylacidiphilum fumariolicum SolV 50.6 75.3 94.2 54.6 54.8 54.6 3. Methylacidiphilum infernorum V4 50.8 75.3 75.4 55.1 55.1 54.6 4. Methylacidiphilum kamchatkensis Kam1 51.0 94.2 75.4 55.1 55.2 54.8 5. Methylacidimicrobium thermophilum AP8 53.6 54.6 55.1 55.1 78.0 78.5 6. Methylacidimicrobium cyclopopanthes 3C 53.3 54.8 55.1 55.2 78.0 79.9 7. Methylacidimicrobium tartarophylax 4AC 52.7 54.6 54.6 54.8 78.5 79.9 Values are expressed in %. In bold, the comparison of strain PQ17 with other verrucomicrobial methanotrophs. Genomic Characterization of “ Ca. Methylacidithermus Pantelleriae” The draft genome of strain PQ17 was analyzed in details to gain a better understanding about its metabolic potential and its role in the geothermal soil of Pantelleria. MAG9 consisted of 71 contigs ranging from 401,379 to 2,075 bp, for a total of 2,466,655 bp, containing 3,127 predicted CDSs and an overall 55.2% GC-content. CheckM analysis revealed a completeness of 98.6, 1.3% contamination and no strain heterogeneity ( Supplementary Figure 2 ). A total of 3,204 genes could be identified, 3,127 of which were protein coding genes and 77 were RNA genes. One 16S and two 23S and 5S rRNA genes were retrieved, indicating that one 16S rRNA copy was probably missing from the draft genome. Functions could be assigned to 2,164 protein coding genes ( Table 2 ). 47.4% of the predicted genes were allocated into Clusters of Orthologous Groups ( Supplementary Table 2 ). TABLE 2 Genome statistics of “ Ca . Methylacidithermus pantelleriae” PQ17. Attribute Value Genome size (bp) 2,466,655 DNA coding (bp) 2,037,457 DNA G + C (%) 55.2% DNA scaffolds 71 Total genes 3,204 Protein coding genes 3,127 RNA genes 77 rRNA genes 5 tRNA genes 65 Pseudo genes 8 Genes in internal clusters – Genes with function prediction 1,231 Genes assigned to COGs 1,482 Genes involved in carbon, nitrogen and sulfur metabolism were analyzed in detail. Their pathways will be described in the upcoming sections and a schematic representation of the metabolism of strain PQ17 can be found in Figure 2 . FIGURE 2 Overview of metabolic pathways in “ Ca. M. pantelleriae.” Colors of enzymes and transporters indicate nitrogen metabolism (green), carbon metabolism (red), sulfate metabolism (purple), phosphate metabolism (blue) and complexes of the respiratory chain (gray). Genes: pmoABC , methane monoxygenase; xoxF , methanol dehydrogenase, xoxG , cytochrome c L ; xoxJ , periplasmic binding protein; folD , bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methylene-tetrahydrofolate cyclohydrolase; fhs , formate-tetrahydrofolate ligase; fdh , formate dehydrogenase; cynT , carbonic anhydrase; frdAB , fumarate reductase; pstABC , phosphate transporter; cysTW/sbp , sulfate transporter; amtB , ammonia transporter; ntrABC , nitrate transporter; nasC , assimilatory nitrate reductase; nasD , assimilatory nitrite reductase; norBC , nitric oxide reductase; cynS , cyanase; cysCDH , adenylyl-sulfate kinase, sulfate adenylyltransferase, phosphoadenosine phosphosulfate reductase; cysJ , sulfite reductase; cysK , cysteine synthase; cysE , serine acetyltransferase; mtoX , methanethiol oxidase. Methanotrophy As a first step in methane oxidation, methane monooxygenases use molecular oxygen to break the energetically strong C-H bond of methane to form methanol ( Sirajuddin and Rosenzweig, 2015 ). So far, two types of this enzyme have been described: a membrane-bound pMMO and a soluble methane monooxygenase (sMMO). One single pmoCAB operon and a pmoD subunit, encoding a copper-binding protein ( Fisher et al., 2018 ), were found in MAG9, whereas no other mmo genes were identified ( Table 3 ). This is in line with previously described verrucomicrobial methanotrophs, although the number of pmo operons seems to be variable ( Op den Camp et al., 2009 ; van Teeseling et al., 2014 ; Erikstad et al., 2019 ; Picone et al., 2020 ; Schmitz et al., 2021 ). The PmoA phylogenetic tree including strain PQ17 ( Supplementary Figure 3 ) supports the phylogeny derived from 16S rRNA gene analysis. TABLE 3 Genes encoding for enzymes involved in methane oxidation pathway, along with their Enzyme Commission (EC) numbers and percentage identity to the most similar homologue. Enzyme Identifier EC number Gene Identity (%) Species Particulate methane monooxygenase MPNT_60061 1.14.18.3 pmoA 62.7 M. infernorum V4 MPNT_60059 pmoB 47.3 M. infernorum V4 MPNT_60062 pmoC 59.2 M. infernorum V4 MPNT_60058 pmoD 35.9 M. infernorum V4 Methanol dehydrogenase MPNT_10387 1.1.99.8 xoxF 74.7 M. fumariolicum SolV Cytochrome C L MPNT_10390 xoxG 41.7 M. fumariolicum SolV Putative periplasmic binding protein MPNT_10389 xoxJ 55.4 M. infernorum V4 Putative TonB-dependent receptor MPNT_80102 cirA 51.4 M. ishizawai ABC transporter ATP-binding protein MPNT_20035 54.5 Verrucomicrobia Tous-C9LFEB Methenyltetrahydrofolate cyclohydrolase/methylenetetrahydrofolate dehydrogenase MPNT_320016 3.5.4.9/1.5.1.5 folD 57.7 M. infernorum V4 Formate-tetrahydrofolate ligase MPNT_330006 6.3.4.3 fhs 56.9 M. marinum Formate dehydrogenase MPNT_130014 1.17.1.9 fdh 77.2 C. sequanensis Formate dehydrogenase MPNT_310008 1.2.1.2 fdsA 77.3 N. kurashikiensis MPNT_310010 fdsB 69.1 M. ishizawai MPNT_310011 fdsC 62.1 Rhizobium MPNT_310006 fdsD 54.8 Burkholderiales bacterium The second step in methane oxidation is the conversion of methanol to formaldehyde or formate. Two types of pyrroloquinoline quinone (PQQ)-containing MDHs are generally found in methanotrophs and methylotrophs: The MxaFI and the XoxF type MDH. Whereas MxaFI depends on calcium for its catalysis, XoxF was found to contain lanthanides in the active site ( Pol et al., 2014 ). Analysis of the MDH sequence of strain PQ17 showed that this enzyme presented a conserved Asp residue required for the coordination of lanthanides in the active site ( Keltjens et al., 2014 ; Pol et al., 2014 ; Good et al., 2020 ). Furthermore, it exhibited 74% amino acid identity to XoxF from Methylacidiphilum fumariolicum SolV, confirming that this protein was a XoxF type and it belonged to group XoxF1 ( Keltjens et al., 2014 ). xoxG and xoxJ genes were also found in the genome of strain PQ17 ( Table 3 ). xoxG encodes a cytochrome C L that functions as electron acceptor for XoxF, whereas xoxJ encodes a periplasmic binding protein that is proposed to be involved in the activation of XoxF and, more specifically, in the insertion of the PQQ cofactor in apo-XoxF ( Zheng et al., 2018 ; Featherston et al., 2019 ; Versantvoort et al., 2019 ). In the Methylacidiphilum species SolV and Kam1 these proteins are exceptionally present as the fusion protein XoxG/J ( Islam et al., 2008 ; Versantvoort et al., 2019 ). Several genes have been proposed as candidates for lanthanide incorporation in bacterial cells ( Ochsner et al., 2019 ). The gene cirA , encoding a TonB-dependent receptor, and a component of the ABC transport system have been identified in other Verrucomicrobia ( Picone et al., 2021 ) and were also shown to be present in the genome of strain PQ17 ( Table 3 ). The lanthanide binding protein lanmodulin described in M. extorquens , instead, could not be found ( Cotruvo et al., 2018 ). XoxF from strain SolV is known to convert methanol to formate in vitro in a four electron process ( Pol et al., 2014 ). In Methylobacterium extorquens AM instead, XoxF generated formaldehyde ( Good et al., 2018 ), which is converted to formate by formaldehyde dehydrogenase. Similarly to strain SolV, no formaldehyde dehydrogenase was detected in “ Ca. M. pantelleriae.” If formaldehyde is produced, different detoxification pathways are known. The tetrahydrofolate pathway was the only pathway identified in strain PQ17. The first step in this cycle is a spontaneous reaction which couples formaldehyde to tetrahydrofolate forming 5,10-methylenetetrahydrofolate. The bifunctional enzyme FolD catalyzes the second and third steps of this cycle, converting 5,10-methylenetetrahydrofolate via 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate. The last reaction step is catalyzed by formate tetrahydrofolate ligase (Fhs), in which formate is produced and tetrahydrofolate is regenerated ( Table 3 and Figure 2 ; Vorholt, 2002 ). Formaldehyde could also be produced by methanethiol oxidase (MtoX, MPNT_180031), an enzyme apparently conserved in verrucomicrobial methanotrophs ( Eyice et al., 2018 ; Picone et al., 2021 ). In the last step of methane oxidation, formate is converted to CO 2 by formate dehydrogenase (FDH). In the bacterial kingdom, a large variety of FDH exist, which are all highly diverse regarding cofactor usage and mechanism ( Hartmann et al., 2015 ). For “ Ca. M. pantelleriae,” two different FDHs were found in the genome, one was cytoplasmic and the other was predicted to be a membrane-bound enzyme complex composed of four subunits ( Table 3 ). Central Carbon Metabolism Methanotrophs assimilate carbon into their metabolism using different pathways. Verrucomicrobia are generally able to fix CO 2 via the Calvin-Benson-Bassham (CBB) cycle ( Khadem et al., 2011 ). This cycle is initiated by the reaction of carbon dioxide with ribulose bisphosphate, which is catalyzed by ribulose bisphosphate carboxylase (RuBisCO) ( Tabita, 2007 ). The small and a large subunit of this enzyme could be identified in the genome, together with two carbonic anhydrases ( Table 4 ). The products of RuBisCO are two molecules of 3-phosphoglycerate (3-PG), which are converted back to ribulose bisphosphate through a series of gluconeogenic and pentose phosphate pathway reactions. Every three CO 2 molecules yield net one molecule of 3-PG, which can be incorporated in central carbon metabolism. TABLE 4 Key enzymes for three major carbon assimilation pathways in methanotrophs. Enzyme Identifier EC number Gene Identity (%) Species Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) a MPNT_100035 4.1.1.39 cbbS 64.4 Gemmatimonadetes bacterium MPNT_100036 cbbL 84.4 M. infernorum V4 RuBisCo-like protein MPNT_20090 47.7 T. mobilis Carbonic anhydrase 1 MPNT_100082 4.2.1.1 mtcA1 76.8 NC10 bacterium Carbonic anhydrase 2 MPNT_210022 4.2.1.1 mtcA2 79.3 Verrucomicrobia bacterium Serine hydroxymethyltransferase b MPNT_220042 2.1.2.1 glyA 60.4 M. infernorum V4 Serine-glyoxylate aminotransferase b MPNT_60063 2.6.1.45 sgaA 68.5 M. kamchatkense Kam1 a CBB cycle. b Serine cycle. And their respective Enzyme Commission (EC) numbers, and percentage identity to the most similar homologue. Beside the CBB cycle, the Serine and the RuMP pathways are other strategies for carbon incorporation in microorganisms ( Chistoserdova, 2011 ). Some genes involved in the Serine pathway could be found in the genome of strain PQ17 ( Table 4 ), but four essential genes were lacking ( hprA EC 1.1.1.29, gckA 2.7.1.165, mtkB EC 6.1.2.9, mcl EC 4.1.3.24). Likewise, two genes required for the RuMP pathway were also absent ( hxlA EC 4.1.2.43, hxlB EC 5.3.1.27). Therefore, it is highly unlikely for “ Ca. M. pantelleriae” to fix carbon using these pathways. All glycolytic, gluconeogenic and pentose phosphate pathway genes could be retrieved from MAG9 ( Supplementary Table 3 ), except for phosphofructokinase (EC 2.7.1.11). The other two genera of verrucomicrobial methanotrophs, Methylacidiphilum and Methylacidimicrobium have genes encoding this protein. All genes for the citric acid cycle were found. Moreover, genes for synthesis and degradation of glycogen were identified. Energy Conservation and Respiration “ Ca . M. pantelleriae” uses O 2 as electron acceptor. Complex I of the respiratory chain ( nuoABCDEFGHIKLMN ) was found in the genome. For complex II, subunits a, b and c of succinate dehydrogenase were found, but subunit d was lacking. Subunits for a canonical complex III could not be retrieved. The verrucomicrobial methanotrophs, including strain PQ17, possess genes encoding an Alternative Complex III, a structurally different protein complex with similar function (MPNT_10279-10285) ( Refojo et al., 2012 ; Schmitz et al., 2021 ). Finally, the electrons are transferred to complex IV and the F 0 F 1 ATP synthase (complex V) generating ATP using the proton motive force ( Supplementary Table 3 ). “ Ca . M. pantelleriae” possesses genes encoding two distinct Complexes IV: aa3-type and a ba3-type. The genomes of the other verrucomicrobial methanotrophs encode for an additional cbb3-type Complex IV ( Schmitz et al., 2021 ). Amino Acid Biosynthesis Pathways for the biosynthesis of 12 amino acids (alanine, isoleucine, leucine, proline, valine, phenylalanine, tyrosine, tryptophan, arginine, lysine, threonine, and cysteine) were completely present in the genome. For histidine, only one gene encoding the biosynthesis protein HisE was absent. The complete pathways for asparagine/aspartate and glutamine/glutamate biosynthesis could not be fully resolved but the enzyme for the conversion of oxaloacetate to aspartate was identified ( aspC MPNT_250010). Genes encoding enzymes for the formation of asparagine, instead, could not be retrieved. Glutamate could be formed from 2-oxoglutarate via glutamate synthase (GltB, MPNT_40080) or from gamma-aminobutyric acid through glutamate decarboxylase (MPNT_510001). Glutamate dehydrogenase (GDH) was not identified, whereas glutamine synthetase and glutamate synthase (GS-GOGAT) were both present ( Supplementary Table 3 ). GDH and GS-GOGAT pathways are also used for ammonia incorporation into biomass. Ammonia incorporation via GS-GOGAT usually happens under low ammonia concentrations ( Tyler, 1978 ; Bellion and Bolbot, 1983 ). The presence of an alanine dehydrogenase (MPNT_50137) in the genome indicates that ammonia could also be incorporated though alanine, starting from pyruvate and NH 4 + . The pathways for glycine and serine biosynthesis are less straightforward. If serine is synthetized from 3-PG, only D-3-phosphoglycerate dehydrogenase/2-oxoglutarate reductase was present ( serA , MPNT_20138), whereas phosphoserine aminotransferase ( serC , EC 2.6.1.52) and phosphoserine phosphatase ( serB , EC 3.1.3.3) were absent. We cannot exclude that these reactions are still performed in vivo , but catalyzed by unknown enzymes. The fragmentation of the genome could also prevent us from retrieving these genes. Assuming that these pathways are actually missing in strain PQ17, serine can still be synthesized in a one-step reaction catalyzed by serine hydroxymethyltransferase ( glyA , MPNT_220042) using 5,10-methylenetetrahydrofolate and glycine. For this to be a feasible strategy, “ Ca . M. pantelleriae” should be able to synthesize its glycine from a different source than serine. As all four subunits of the glycine cleavage system are present in its genome (MPNT_20097, 20098, 20099, 420008), we propose that “ Ca . M. pantelleriae” could use this machinery in reverse to synthesize glycine from ammonia, carbon dioxide and 5,10-methylenetetrahydrofolate ( Kikuchi et al., 2008 ), which has also been described previously for Clostridium acidiurici ( Gariboldi and Drake, 1984 ). Furthermore, glycine can be synthetized from glyoxylate ( agxt2 , MPNT_100077) and from sarcosine ( dauA , MPNT_10078). Nitrogen Metabolism Ammonium from the environment can be imported directly into the cell using either of two AmtB transporters (MPNT_100073, 250005). Alternatively, nitrogen can be obtained by uptake of nitrate via a NrtABC transporter ( Supplementary Table 3 ), followed by reduction to ammonia by NasC and NasD ( Table 5 and Supplementary Table 3 ). Interesting is the presence of the gene cynS , which encodes cyanase, an enzyme converting cyanate and bicarbonate to carbon dioxide and ammonium. Cyanate can act as energy and nitrogen source for nitrifiers ( Palatinszky et al., 2015 ) and its presence has been detected in other verrucomicrobial methanotrophs ( Picone et al., 2021 ). Unlike other verrucomicrobial methanotrophs, genes encoding a nitrogenase enzyme could not be found ( Op den Camp et al., 2009 ; Khadem et al., 2010 ; Schmitz et al., 2021 ). Fixed ammonium is mostly used for biosynthetic purposes, but some ammonium is also converted into hydroxylamine by pmoA , which is a structural homolog of ammonium monooxygenase amoA ( Sirajuddin and Rosenzweig, 2015 ). As hydroxylamine is toxic to the cell, it must be further metabolized into less harmful compounds. However, hydroxylamine oxidoreductase ( hao ), which is present in ammonia oxidizers but also in other methanotrophs such as M. fumariolicum SolV ( Pol et al., 2007 ), could not be retrieved from the MAG. The nitric oxide reductase encoded by norBC ( Table 5 ) was identified, whereas other denitrification genes, like narB (EC 1.7.5.1) and nosZ (EC 1.7.2.5), were not detected. TABLE 5 Genes encoding for enzymes involved in nitrogen metabolism, along with their Enzyme Commission (EC) numbers, and percentage identity to the most similar homologue. Enzyme Identifier EC number Gene Identity (%) Species Assimilatory nitrate reductase MPNT_110017 1.7.1.1 nasC 57.1 P. methylaliphatogenes Nitrite reductase MPNT_110016 1.7.1.4 nasD 46.3 P. methylaliphatogenes Cyanate hydratase MPNT_50182 4.2.1.104 cynS 64.1 K. tusciae Nitric oxide reductase MPNT_410005 1.7.2.5 norB 72.9 O. profundus MPNT_410004 norC 73.4 O. profundus Sulfur and Phosphate Metabolism The primary way to fix sulfur for “ Ca . M. pantelleriae” is to reduce sulfate to biologically available sulfide. For this, sulfate needs to be transported into the cell using the sulfate ABC-transporter sbp/cysTW (MPNT_580001-580004). Subsequently, sulfate can be reduced to sulfite via adenylyl sulfate and 3′-phosphoadenylyl sulfate intermediates by the genes, catalyzed by cysD (MPNT_10354), cysC (MPNT_10355) and cysH (MPNT_20190), respectively. Finally, sulfite can be further reduced to H 2 S by cysJ (MPNT_10061, MPNT_40049) or sir1 (MPNT_20189) and used for cysteine biosynthesis ( cysK (MPNT_110064). Phosphate can be transported directly over the membrane using the ABC-transporter encoded by pstABCS ( Supplementary Table 3 ) and does not require further conversions. The presence of polyphosphate particles has been observed in verrucomicrobial methanotrophs ( van Teeseling et al., 2014 ). Polyphosphate storage is likely in strain PQ17 as genes encoding polyphosphate kinase (MPNT_190035) and exopolyphosphatase (MPNT_40183) were identified."
} | 6,969 |
34808039 | PMC8689693 | pmc | 4,774 | {
"abstract": "Powered by (sun)light\nto oxidize water, cyanobacteria can directly\nconvert atmospheric CO 2 into valuable carbon-based compounds\nand meanwhile release O 2 to the atmosphere. As such, cyanobacteria\nare promising candidates to be developed as microbial cell factories\nfor the production of chemicals. Nevertheless, similar to other microbial\ncell factories, engineered cyanobacteria may suffer from production\ninstability. The alignment of product formation with microbial fitness\nis a valid strategy to tackle this issue. We have described previously\nthe “FRUITS” algorithm for the identification of metabolites\nsuitable to be coupled to growth (i.e., side products in anabolic\nreactions) in the model cyanobacterium Synechocystis . sp PCC6803. However, the list of candidate metabolites identified\nusing this algorithm can be somewhat limiting, due to the inherent\nstructure of metabolic networks. Here, we aim at broadening the spectrum\nof candidate compounds beyond the ones predicted by FRUITS, through\nthe conversion of a growth-coupled metabolite to downstream metabolites\nvia thermodynamically favored conversions. We showcase the feasibility\nof this approach for malate production using fumarate as the growth-coupled\nsubstrate in Synechocystis mutants. A final titer\nof ∼1.2 mM was achieved for malate during photoautotrophic\nbatch cultivations. Under prolonged continuous cultivation, the most\nefficient malate-producing strain can maintain its productivity for\nat least 45 generations, sharply contrasting with other producing Synechocystis strains engineered with classical approaches.\nOur study also opens a new possibility for extending the stable production\nconcept to derivatives of growth-coupled metabolites, increasing the\nlist of suitable target compounds.",
"conclusion": "Conclusions The feasibility of a metabolic engineering strategy\naiming at exploiting\na growth-coupled compound, fumarate, to drive the stable production\nof a downstream metabolite, malate, has been proved in this study.\nBy implementing this strategy, a malate-producing strain of Synechocystis, Δ me Δ mdh , has been engineered. The conversion of fumarate into\nmalate has been further improved by overexpressing heterologous FumC\nin the Δ me Δ mdh strain.\nThe synthesis of an extra amount of this protein did not appear to\ncompromise the fitness of the microorganism and did not result in\na progressive loss in malate productivity due to phenotypic instability.\nThese results indicate that the strategy proposed here in principle\ncould be applied to other compounds deriving from a growth-coupled\nmetabolite that is a side product of anabolism.",
"introduction": "Introduction Cyanobacteria are promising\nmicrobial hosts that can be designed\nas cell factories to enable the development of sustainable production\nprocesses. Energized by (sun)light through their photosynthetic systems,\nengineered cyanobacteria can directly convert inorganic carbon dioxide\ninto a number of chemicals such as alcohols and derived fuels (ethanol,\nbutanol), 1 , 2 chemical building blocks (isoprene, lactate,\npolyhydroxybutyrate), 3 − 5 and fine chemicals. 6 , 7 Classical\nstrain improvement aims to rewire cellular metabolism\nto enhance the production of valuable native compounds or to endow\ncells with the ability to newly produce non-native ones. 8 , 9 However, the diversion of carbon fluxes and energy toward the production\nof compounds of interest implemented by the introduction and/or modification\nof biosynthetic pathways interferes with cellular functionalities\nthat are subject to multilevel (e.g., proteome-, metabolic-, population-,\nand environmental) constraints. 10 − 14 The host cells could therefore experience notable pressure to opportunely\nreallocate resources between biomass precursors and target compound\nformation. 15 − 18 Strong evolutionary pressure favors revertant cells with impaired\nproduction, challenging the feasibility and sustainability of the\nindustrial exploitation of cyanobacteria as cell factories. 19 , 20 Although negative results are seldom shown, the issue of production\ninstability in cyanobacteria engineered to obtain valuable products\nsuch as ethylene, lactic acid, isopropanol, mannitol, and fatty acids\nhas been reported in a handful of reports. 3 , 21 − 23 Developing cyanobacterial-producing strains\nin biotechnological\nsetups requires minimizing the negative impact of instability raised\nby the loss of fitness of engineered strains. Therefore, one approach\nis to make the growth of the engineered strain dependent on the synthesis\nof the compound that is to be produced (i.e., coupling the product\nforming pathway to microbial growth). In this way, the selection acting\non engineered strains during their evolutionary trajectories allows\nproducing strains to outcompete the nonproducing ones. This growth-coupled\nproduction has been implemented computationally using the FRUITS algorithm—that\n“Finds Reactions Usable in Tapping Side-products”—and\nexperimentally validated in Synechocystis sp. PCC6803\n(hereafter, Synechocystis ) engineered to independently\naccumulate either acetate 24 or fumarate. 25 The biotechnologically relevant compounds obtainable\nthrough this approach are defined as the side products of anabolic\npathways. Those side products can be accumulated by disrupting endogenous\nenzymatic reactions responsible for reintroducing them back into metabolism\nwithin a resource-efficient usage framework. In summary, the compounds\nresulting from this approach are growth-coupled since they are obligate\nside products of the formation of essential precursor metabolites.\nHowever, the number of candidates for implementing this growth-coupled\nstrategy is limited to the metabolites that meet such criteria under\nthe conditions for which it is determined using FRUITS. For Synechocystis , using model iJN678 26 constrained to simulate photoautotrophic growth, this is restricted\nto nine metabolites. 24 The metabolic\nstrategy shown in the present study broadens the\naforementioned limitations inherent to the FRUITS approach. Based\non a combination of metabolic modeling and genetic engineering solutions,\nwe show how we could exploit a growth-coupled metabolite previously\nidentified in Synechocystis ( 25 ) to achieve the production of a metabolite positioned immediately\ndownstream of the growth-coupled product itself. Specifically, we\nreport the first stable malate-producing strain of Synechocystis obtained by exploiting the stoichiometrically growth-coupled metabolite\nfumarate as a supply for the accumulation of malate, the direct subproduct\nof fumarate in Synechocystis. Malate is a C4-dicarboxylic\nacid with a wide range of industrial\napplications since it is used as an acidulant in the feed and food\nindustry, 27 building block for bioplastic\nand resin production and a component of drug delivery systems. 28 Given the manifold applications and the increasing\nmarket demand, alternative and sustainable production systems for\nmalate are highly advisable, for instance, in a photoautotrophic host. 29 In this study, we created the markerless\ndeletion Synechocystis mutants with impaired malate\nconsumption pathways and further improved\nthe production by overexpressing the enzyme that converts fumarate\ninto malate, namely, fumarase C (FumC). The rationale behind the latter\nis that cells would then accumulate the target product malate because\n(i) the reactions that consume malate have been deleted and (ii) the\nformation of its direct precursor, fumarate, has been previously shown\nto be a strict requirement for cell growth. 25 Moreover, we validated the stability of the malate production of\nthe engineered strains over prolonged continuous cultivation. The\nstrains obtained by implementing our new metabolic engineering strategy\nrepresent the first report of stable cyanobacterial production of\nmalate via the conversion of the closest upstream growth-coupled compound,\nfumarate. It provides a starting point for the application of this\nnovel engineering strategy to the next stages of industrial exploitation.",
"discussion": "Results\nand Discussion In Silico Simulation for\nMalate Production In Synechocystis under\nphotoautotrophic conditions,\nmalate is exclusively produced from the precursor fumarate. The latter\nis mainly produced as a side product of urea and purine anabolism,\nand it is subsequently recycled via anaplerotic reactions. First,\nthe FumC enzyme catalyzes the conversion of fumarate into malate ( Figure 1 a). Then, malate\nis converted either into oxaloacetate by malate dehydrogenase (Mdh)\nor into pyruvate by the malic enzyme (Me) through oxidative decarboxylation\n( Figure 1 a). It has\npreviously been demonstrated that fumarate can be produced in a growth-coupled\nmanner, as also highlighted by the correlation existing between fumarate\nproductivity and the growth rate of the engineered strain. 25 Considering that for Synechocystis fumarate is a nonessential metabolite that can be produced in a\ngrowth-coupled fashion, it is conceivable that channeling fumarate\ntoward the accumulation of its first derivative metabolite, malate,\nwould not cause major fitness impairments. This hypothesis is supported\nby Flux Variability Analysis (FVA) 30 of\nthe genome-scale metabolic model (GSMM) of Synechocystis iJN678, which predicted that if enzymatic reactions are responsible\nfor the consumption of malate are deleted, either the latter or fumarate\nwould then be accumulated without affecting the growth capabilities.\nIn particular, according to FVA, the single knockout of me or mdh\ngenes would neither enhance malate accumulation nor affect Synechocystis growth ( Tables 1 and 2 ), since one could take\nover the function of each other in simulations. On the other hand,\nthe double deletion of the two target genes would induce the accumulation\nof malate up to a total amount of 0.860 mM gDW –1 , with a small negative effect on cell growth ( Table 2 ). The reaction catalyzed by the FumC is\nreversible and, according to a biochemical thermodynamic analysis\nperformed through eQuilibrator, 31 the conversion\nof fumarate into malate is allowed until the equilibrium of the reaction\nis reached, which corresponds to a molar ratio malate:fumarate of\n4:1 under standard conditions ( Figure 1 b). For this reason, a residual amount of fumarate\nis always to be expected. Figure 1 Malate metabolism in Synechocystis and mutant\nconstruction. (a) Schematic representation of malate-producing and\nconsuming pathways, as reported by the genome-scale metabolic model\n(GSMM). 26 The genes deleted in this study\nare indicated in red, the overexpressed gene is indicated in green.\n(b) Thermodynamic analysis of the reaction catalyzed by the FumC.\nCalculations were obtained using eQuilibrator 31 for the reaction fumarate + H 2 O ↔ malate. The\ndashed lines indicate the equilibrium point of the reaction (Δ G r = 0). (c) PCR confirmation of the strains\nconstructed for markerless deletion of me and mdh . With the primers on each side of the upstream and downstream\nhomologous region (H1 and H2 of ∼1 kb each), a markerless construct\ngave a PCR product of 1.5 and 1.8 kb for Δ me and Δ mdh , respectively. (d) PCR confirmation\nof the fumC overexpression strain. The integration\nof fumC gene in the neutral site slr 0168 gave a PCR product of 4 kb. The genomic DNA of the WT and pBB1\nwere used as negative and positive controls, respectively. Table 1 List of Plasmids and Strains Used\nin This Study plasmids\nand strains description reference pFL-AN BioBrick “T” vector with Avr II and Nhe I on each side ( 42 ) pWD42 Amp r Km r , containing the\nselection cassette ( 43 ) pWD71 pFL-AN derivative, Amp r , containing mdh upstream and downstream homologous regions this study pWD72 pFL-AN derivative, Amp r Km r , containing\nthe selection cassette flanked by mdh upstream and\ndownstream homologous regions this study pWD73 pFL-AN derivative, Amp r , containing me upstream and downstream homologous\nregions this study pWD74 pFL-AN derivative, Amp r Km r , containing\nthe selection cassette flanked by me upstream and\ndownstream homologous regions this study pHKH001 Amp r Km r , integration\nvector at slr 0168 genomic locus ( 3 ) pBB1 pHKH001 derivative, Amp r Km r , fumC (from Escherichia coli ) expressed under the cpcBA promoter this study Synechocystis sp. PCC6803 Synechocystis sp. PCC6803 wild type D. Bhaya WD163 Synechocystis sp. PCC6803 mdh gene knockout\nmutant this study WD169 Synechocystis sp. PCC6803 me gene knockout mutant this study WD170 Synechocystis sp. PCC6803 mdh and me gene knockout mutant this study WD198 Synechocystis sp. PCC6803 fumC overexpressing\nmutant this study WD199 Synechocystis sp. PCC6803 mdh and me gene knockout, fumC overexpressing\nmutant this study SAA023 Synechocystis sp. PCC6803 expressing L- ldh gene from Lactococcus lactis ( 36 ) Table 2 Growth Rate and Product Yields for\nMalate and Fumarate Relative to Biomass Either Predicted by FBA or\nExperimentally Observed during the Exponential Phase in Wild-Type Synechocystis and Derivative Strains Impaired in Malate\nConsumption Reactions a 2 growth\nrate (μ, h –1 ) malate\nyield ( Y p/x , mM gDW –1 ) fumarate\nyield ( Y p/x , mM gDW –1 ) strains model prediction measured model prediction measured model prediction measured wild\ntype 0.052 0.055 ± 0.001 0 0 0 0 Δ me 0.052 0.052 ± 0.000 0 0.067 ± 0.019 0 0.274 ± 0.014 Δ mdh 0.052 0.055 ± 0.000 0 0 0 0 Δ me Δ mdh 0.050 0.050 ± 0.001 0–0.860 0.607 ± 0.091 0–0.860 0.557 ± 0.002 a FVA was\nused for evaluating the\nrobustness of the network and predicting the expected range for malate\nand/or fumarate production. The biomass equation was always used as\nthe primary objective function in all FBA and FVA simulations. The\nexperimental data are referred to the batch cultivation reported in Figure 2 . To experimentally validate the feasibility\nof our approach by introducing\nthe necessary genetic modifications, three markerless deletion mutants\nof Synechocystis as Δ me , Δ mdh , and Δ me Δ mdh were constructed ( Figure 1 c) and further characterized. Malate Production of Δ me , Δ mdh , and Δ me Δ mdh in a Batch Photobioreactor Growth\nand production capabilities\nof engineered and WT strains were monitored by photoautotrophic cultivation\nunder continuous light illumination in the Multi-Cultivator. Δ mdh grew similarly to the WT until the end of the experiment,\nwhile Δ me and Δ me Δ mdh showed a measurably slower growth rate (0.052 and 0.050\nh –1 , respectively) in the exponential phase compared\nto the WT (0.055 h –1 ) ( Figure 2 a and Table 2 ). A similar negative effect on the growth rate due to the disruption\nof the gene encoding for the malic enzyme has been observed before. 32 Comparison of fumarate and malate extracellular\nproduction between the WT and engineered strains allowed assessing\nthe impact of deleting me and mdh genes on the metabolism of Synechocystis . As expected\nfrom previous work investigating the secretion of organic metabolites\nby Synechocystis grown under photoautotrophic conditions, 33 neither fumarate nor malate was detected in\nsignificant amount in the supernatant of WT ( Figure 2 b,c). In contrast to the in silico analysis carried out, which predicted a null malate yield for the\nΔ me strain ( Table 2 ), this mutant was able to accumulate modest\namounts of malate, suggesting that the conversion of malate into oxaloacetate\nby Mdh is less effective than the conversion of malate into pyruvate\nmediated by Me. This result is in partial agreement with 13 C flux analysis showing that during continuous illumination, similar\nto that of our setup, the high levels of ATP generated inhibit the\npyruvate kinase, which converts phosphoenolpyruvate (PEP) into pyruvate\n( Figure 1 a), 34 , 35 inducing cells to convert malate for supplying the necessary pyruvate.\nIn this context, indeed, the higher activity of Me compared to Mdh\ncould be functional to provide pyruvate, thus compensating the bottleneck\nthrough the pyruvate kinase. In accordance with model predictions\n( Table 2 ), both compounds\nwere released in the extracellular broth by Δ me Δ mdh strain, which also showed higher yields\ncompared to Δ me strain ( Figure 2 b,c). For the Δ me Δ mdh strain, the maximum titers of malate and fumarate observed\nwere to 1.2 and 1.0 mM, respectively, after 14 days of batch cultivation\n( Figures 2 b,c and S1 ) and the maximum malate to fumarate molar\nratio was about 1 ( Figure 2 d). Figure 2 Characterization of fumC overexpressing strains\nof Synechocystis compared to the WT and to the double\nknockout Δ me Δ mdh cultivated\nunder continuous light in a photobioreactor. (a) Growth curves and\nextracellular concentration of (b) malate and (c) fumarate of different\nstrains. (d) Profiles of the malate/fumarate molar ratio in Δ me and Δme Δ mdh strains. Values are the mean of at least two biological replicates.\nError bars are standard error in panels (a)–(c) and combined\nstandard uncertainty in panel (d). Concentration values below the\ndetection limit of the analytical techniques used (i.e., 2 μM\nfor malate and 20 μM for fumarate) are reported as zero in panels\n(b) and (c). Malate/fumarate molar ratios are not reported whenever\neither malate or fumarate concentration falls below the detection\nlimit. Overexpression of FumC for Improving Malate\nAccumulation When considering the Δ me Δ mdh strain, which was the mutant with the\nhighest malate production\n( Figures 2 b and S1 ), the presence of a considerable amount of\nfumarate in the culturing broth ( Figure 2 c) suggests that the intracellular conversion\nof fumarate into malate catalyzed by FumC might be limited. The possible\nenzymatic bottleneck related to the low availability of the enzyme\nand/or restricted access to the substrate has been explored by overexpressing fumC . Two strains based on the WT strain and on the background\nof Δ me Δ mdh mutant, i.e.,\nΔ NSI::fumC and Δ me Δ mdh Δ NSI::fumC , have been engineered\nby inserting the fumC gene from E.\ncoli in the chromosomal neutral site slr 0168 ( Figure 1 d).\nThe E. coli fumC gene\noverexpressed is only 59.4% identical to the native one of Synechocystis , which avoids the occurrence of undesired\nrecombination events. The fumC overexpressing\nstrains were cultured photoautotrophically in the Multi-Cultivator\nunder the same conditions as the deletion mutants. The growth curve\nof Δ NSI::fumC was similar to that of the WT,\nwhereas Δ me Δ mdh Δ NSI::fumC showed a slower growth during the exponential\nphase when compared to the Δ NSI::fumC ( Figure 3 a). Conversely, Δ me Δ mdh Δ NSI::fumC grew similarly to the Δ me Δ mdh in the first stages of growth, reaching the steady state earlier\nthan the double mutant ( Figure 3 a). Concerning malate and fumarate extracellular production,\nthe overexpression of fumC alone in Synechocystis was not sufficient to enhance the secretion of detectable quantities\nof neither of the two compounds ( Figure 3 b). On the other hand, Δ me Δ mdh Δ NSI::fumC was\ncapable of releasing in the extracellular environment higher titers\nof malate and lower titers of fumarate compared to the Δ me Δ mdh strain ( Figure 3 b; Figure S1 normalized\nby biomass content), determining a higher molar ratio of malate over\nfumarate in the extracellular broth throughout the entire cultivation\n( Figure 3 c). When analyzing\nthe malate productivity (calculated as the ratio between the variation\nof the malate production and the difference in biomass observed between\ntwo consecutive sampling points), the highest productivity was observed\nduring the exponential phase for both the Δ me Δ mdh Δ NSI::fumC and\nΔ me Δ mdh strains ( Figure 3 d). In this growth\nphase, the malate productivity of the overexpressing triple mutant\nwas significantly higher than that of the double deletion mutant,\nonce more indicating an improved ability of conversion of fumarate\ninto malate in the Δ me Δ mdh Δ NSI::fumC mutant. Figure 3 Characterization of fumC overexpressing strains\nof Synechocystis compared to the double knockout Δme Δ mdh cultivated under continuous\nlight in a photobioreactor. (a) Growth curves and (b) extracellular\nproduction of malate and fumarate of different strains. Variation\nof (c) the malate/fumarate molar ratio and (d) the malate productivity\nof Δme Δ mdh and ΔmeΔmdh Δ NSI::fumC s trains.\nDry weight concentration was calculated from OD 730 measurements\nusing a conversion factor of 148 mg L –1 OD –1 obtained in a similar setup. 44 Values\nare the mean of at least two biological replicates. Error bars are\nstandard errors in panels (a) and (b) and combined standard uncertainty\nin panels (c) and (d). Concentration values below the detection limit\nof the analytical techniques used (i.e., 2 μM for malate and\n20 μM for fumarate) are reported as zero in panel (b). Malate/fumarate\nmolar ratios are not reported whenever either malate or fumarate concentration\nfalls below the detection limit. Testing\nthe Stability of Δ me Δ mdh and Δ me Δ mdh Δ NSI::fumC The engineering approach\nadopted to build the Δ me Δ mdh strain is based on the removal of the two consumption pathways of\nmalate. Cyanobacterial strains engineered adopting an analogue deletion\nmethod have been shown to be stable and productive over dozens of\ngenerations. 24 , 25 Consequently, also the Δ me Δ mdh strain is expected to be phenotypically\nstable. Most importantly, malate production should be stably maintained\ndue to the grounds that its precursor, fumarate, is a growth-coupled\nmetabolite. In the Δ me Δ mdh Δ NSI::fumC strain, more cellular resources\nare involved in the synthesis of an extra amount of heterologous FumC,\nthus potentially introducing an additional protein burden to the cell.\nTherefore, assessing the phenotypic stability of this strain is of\nutmost pertinence. Phenotypic stability of the most productive\nstrains, Δ me Δ mdh and\nΔ me Δ mdh Δ NSI::fumC, was assessed through a serial propagation experiment\nin which cultures were kept in exponential growth (OD 730 between ∼0.6 and 1) over a period of ∼2 months to\nensure a constant selective pressure. 10 As a control strain, we used SAA023, a lactate-producing mutant\nof Synechocystis , which carries L-LDH that converts\npyruvate into l -lactate. 36 SAA023\nhas been shown to be unstable during prolonged culturing since spontaneous\nrevertants with mutations in the ldh cassette become\ndominants after a few generations. 15 The\nproduction instability of this strain is associated with the metabolic\nengineering strategy used that imposes a high trade-off between biomass\ngrowth and product formation because an essential metabolite of the\ncentral metabolism, here pyruvate, is depleted. Throughout our\nlaboratory evolution experiment, each culture was\nmonitored between 42 and 50 generations, depending on the specific\ngrowth rate of the strain. Δ me Δ mdh and Δ me Δ mdh Δ NSI::fumC maintained a similar ability to\naccumulate malate in the extracellular environment until the end of\ntheir serial propagations ( Figure 4 a), albeit with some fluctuations along time. In contrast,\na clear drop in lactate extracellular concentration was recorded for\nthe SAA023 strain ( Figure 4 b). To check whether this decrease in lactate was due to the\npresence of mutations in the ldh cassette, the gene\nencoding for the l -LDH and the upstream promoter sequence\nwere amplified from 15 single colonies (five from each of the three\nbiological replicates) and subjected to Sanger sequencing. Mutations\nin the ldh cassette were found in all of the analyzed\nsequences ( Table S2 and Figure S2 ). Interestingly,\nthe mutations were found only in the coding sequence, directly hampering\nthe correct translation of the l -LDH protein. On the other\nhand, no mutations were found in the fumC cassette\ntested with an analogous approach. Figure 4 Malate and lactate extracellular production\nof different strains\nduring the serial propagation experiment. (a) Malate production normalized\nby the OD 730 of Δ me Δ mdh and Δ me Δ mdh Δ NSI::fumC strains. (b) Lactate production\nnormalized by the OD 730 of SAA023 strain. The values are\nthe mean of three biological replicates, the y error\nbars are combined uncertainties, and the x error\nbars are standard errors. Taken together, these results show the promise of strategies aiming\nat producing compounds (here, malate) derived from growth-coupled\nmetabolites (here, fumarate), rather than from central C-compounds\nnecessary for growth. 24 , 25 The nonappearance of mutations\nin the fumC gene indicates that (i) malate and fumarate,\nat these concentrations, do not pose a significant challenge to be\ntransported out of the cell; (ii) nor is the accumulation of neither\nof them at these levels inhibitory for the cell; and (iii) at least\nin the absence of nutrient limitations, the protein overexpression\nper se does not seem to pose a sufficiently large fitness cost for\nthe cell. Although this finding can be somewhat unexpected a priori,\nit is supported by the fact that no decrease in the growth capability\n( Figure 3 a) and no\nloss in malate production ( Figure 4 a) were recorded for the Δ me Δ mdh Δ NSI::fumC strain."
} | 6,316 |
31974310 | PMC7022211 | pmc | 4,775 | {
"abstract": "Significance Genetic modification of plant cell wall polymers is key to improvement of lignocellulosic biomass for forage, fuel, and renewable chemicals. However, such modifications can often lead to ectopic activation of defense responses and reductions in biomass yield. Here, we show that defense gene induction in transgenic Arabidopsis thaliana with altered lignin content and composition through down-regulation of two different lignin pathway enzymes results from the ectopic expression of a pectin-degrading enzyme in vascular tissue, leading to release of cell wall epitopes that serve as signals for defense gene activation. This knowledge provides a basis for uncoupling lignin modification from ectopic defense gene induction.",
"discussion": "Discussion Lignin Modification Is Associated with Extensive Cell Wall Remodeling. Lignin is an important component of both preexisting and inducible defense responses in plants ( 6 ). Various hypotheses have been put forward to explain why reduction in lignin content often appears to enhance rather than reduce plant disease resistance ( 7 , 8 ); these include antimicrobial activity of lignin pathway intermediates ( 42 ) and release of elicitor-active molecules from incorrectly assembled plant cell walls ( 43 ). The present results provide a mechanistic explanation for the latter hypothesis. Impacts of lignin modification on plant gene expression may be initiated through alterations in cell wall integrity, likely, in part, operating through effects on cross-linking to other cell wall components such as pectin and/or hemicellulose. Although pectin is generally considered in the context of primary cell walls, its importance for secondary wall structure is probably greater than previously realized, as lignin modification can affect the expression of pectin-related genes ( 44 , 45 ), and, conversely, pectin modification affects the expression of lignin pathway genes ( 46 ) and lignin content ( 47 ). It has been proposed that pectin forms a nucleation site for lignification in alfalfa ( 48 ), and partially methylated pectins can interact with lignin polymers composed of coniferyl alcohol to form hydrophobic clusters in vitro, suggesting that activity of pectin methyl esterases might regulate pectin−lignin interactions ( 49 ). Moreover, PGase genes have been shown to exhibit high expression during the differentiation of tracheary elements in Zinnia elegans ( 50 ) or during secondary wall formation in trees such as aspen and poplar ( 51 , 52 ), suggesting that pectin modification might function in cell wall remodeling associated with lignin deposition. Although the exact nature of lignin−pectin interactions in the secondary cell wall is not clear, some studies indirectly support the importance of such interactions in orchestrating cell wall integrity ( 19 , 53 ). This concept is supported further by the ability of C. bescii with a deleted pectinase gene cluster to grow on HCT-RNAi and ccr1 Arabidopsis ; clearly, disruption of pectin is critical for opening the cell wall structure to degradation by other enzymes to release the sugars necessary for bacterial growth. It is not possible to determine which of the many induced cell wall-degrading enzymes is responsible for the overall changes in cell wall integrity in the HCT-RNAi and ccr1 lines; although ADPG1 is the most strongly induced pectin-degrading enzyme and the only one induced in common between the two lines, a number of pectin lyase genes are also induced. In both HCT-RNAi and ccr1 mutant lines, pectins were more easily extractable from cell walls in water, oxalate, or carbonate compared to wild-type cell walls. Increased extractability of pectic backbone epitopes is one of the cell wall remodeling features previously shown in response to abiotic stresses such as low soil moisture availability in stem wood ( 54 ). Arabinogalactan and RG-I are the predominant polysaccharide epitopes in the water extracts of HCT-RNAi and ccr1 cell walls, based on glycome and compositional analysis showing increased levels of monosaccharides that constitute these types of molecules (namely fucose, arabinose, galactose, rhamnose, xylose, and galacturonic acid). However, heteroxylans are also preferentially released from ccr1 cell walls. Lignin Modification Uncovers Latent Cell Wall-Derived Elicitors of Defense Gene Expression. Molecules or epitopes present on cell wall components with the ability to activate defense pathways have been termed DAMPs ( 23 ). To date, they have been shown to be OGs of different sizes originating from pectin, or oligoglucosides ( 55 ). The DAMP concept is, in essence, a restatement of the earlier oligosaccharin hypothesis ( 9 , 12 ) formulated in a series of seminal papers that described plant cell wall structures that elicited plant defenses and/or impacted plant growth and development ( 14 , 15 , 27 , 56 , 57 ). Subsequent studies on oligosaccharins derived from xyloglucans or pectin ( 13 , 16 ) led to the hypotheses that plants possess specific receptors for such molecules that may act to transduce signals from the cell wall during attempted penetration catalyzed by pathogen-derived wall-degrading enzymes, and that the effects of oligosaccharins on growth and development may operate through antagonism of auxin action ( 16 , 58 ). Genetic approaches have been applied to understand oligosaccharin signaling and its potential dual role in defense and development ( 59 , 60 ), but, in most cases, the elicitor molecules investigated have been limited to synthetic homo-OGs, so the extent of the repertoire of DAMPs/oligosaccharins that function naturally in plant defense has remained unclear. Analyses of the HCT-RNAi and ccr1 mutant reported here, along with Arabidopsis plants with loss of function or overexpression of the F5H that serves as the entry point to S lignin biosynthesis ( 29 ), show that different types of lignin modification lead to release of different elicitors that activate different defense response pathways (PR proteins in the present case; genes involved in response to oomycetes or tritrophic interactions with insects in the case of F5H misregulation) ( 29 ). These elicitors, even as crude water-soluble extracts, do not exhibit cross-reactivity for defense gene induction. The pectic framework clearly has the structural complexity to provide such diverse and apparently specific elicitors. Based on the results of ion exchange and size fractionation, the actual elicitor molecules are likely polymorphic, containing epitopes that confer activity along with additional nonactive portions. The elicitor-active components from both HCT-RNAi and ccr1 lines are destroyed by digestion with PGase and arabinan-1,5-α- l -arabinosidase. This suggests that they are derived from RGs. Classical RG-I contains, among other substitutions, linear five-linked arabinan side chains attached to a central polymer consisting of alternating galacturonic acid and rhamnose residues, whereas RG-II contains highly complex side chains consisting of multiple sugar types attached to a linear chain of α-1,4−linked galacturonic acid residues, with a few arabinose units only attached as end-groups ( 61 ). The preference of ADPG1 for apple pectin rather than PGA suggests that the elicitors, or at least their precursors, may contain methylated HG. Consistent with lignin modification being the primary reason for cell wall remodeling and elicitor release, complementation of the ccr1 mutant with a wild-type copy of CCR1 with expression targeted to xylem prevented the induction of PR1 in stems. This suggests that lignifying xylem cells are the origin of the released elicitors, although some lignification is also restored in fibers of the ProSNBE:CCR1 line ( 40 ). ADPG1 Is Required for Release of Elicitors of PR Genes. ADPG1 is highly induced in both HCT-RNAi and ccr1 lines, but is not induced in F5H misregulated lines in which lignin composition but not lignin content is altered ( 29 ). This PGase is the only pectin-modifying enzyme that is induced in both the HCT-RNAi and ccr1 lines, and loss of function of ADPG1 results in reduction of PR gene expression in HCT-RNAi and ccr1 genetic backgrounds and the loss of elicitor activity in extracts from cell walls of HCT-RNAi/ adpg1 plants. However, the observation that water extracts from cell walls of ccr1 adpg1 mutant plants possess elicitor activity only after treatment with RC-ADPG1 suggests that the enzyme has a specific role in elicitor release, and is not itself necessary for the cell wall remodeling that results in solubilization of latent elicitors. ADPG1 is normally expressed in siliques and anthers prior to dehiscence, where it is likely that it degrades pectin to cause cell wall breakage, as its loss of function delays, or, in the case of strong alleles, prevents anther dehiscence ( 41 ). Anther dehiscence is also prevented by loss of function of NST1 in Medicago truncatula ( 62 ), or NST1 and NST2 in Arabidopsis ( 63 ). These NST genes encode NAC family transcription factors that regulate lignin deposition in secondary cell walls ( 63 ). The fact that both lignin and pectin modification impact anther dehiscence is consistent with a role for pectin in a structural complex with lignin. The action of ADPG1 in vivo must be limited, specific, and perhaps localized for it to release elicitor-active molecules without destroying them. Furthermore, induction of ADPG1 does not appear to be a result of the activity of the pectic elicitors released from cell walls of these lines. Thus, it is likely that signaling to induce ADPG1 occurs first, with resulting release of pectic/oligosaccharide elicitors that then activate defense responses. In the model in Fig. 5 , an initial stimulus (perhaps a released cell wall component or a physical change in the wall recognized by receptors in the plasma membrane) activates ADPG1 transcription. Several receptors that monitor the “status” of cell wall components have recently been identified ( 64 ). The ADPG1 enzyme releases oligogalacturonide elicitors from RG-I and/or RG-II, which, either directly or after processing, may be recognized by the wall-associated kinases which have the ability to bind OGs and PGA ( 59 ). This reception results in elevated levels of SA [inferred for Arabidopsis ccr1 mutants and directly demonstrated in previous studies on the ccr1 mutant of M. truncatula ( 65 )] and HCT-down-regulated alfalfa and Arabidopsis lines ( 7 , 66 ) and consequent induction of PR genes. Assuming that the cell culture system used allows elicitor-mediated induction of all genes irrespective of their tissue specificity, induction of genes such as SESA2 and CRA2 in the ccr1 mutant is likely a secondary effect, as these genes are not induced by the released elicitors. Fig. 5. Model for the activation of PR genes in HCT-RNAi and ccr1 Arabidopsis plants. In the proposed model, changes in lignin content in xylem cells of HCT-RNAi or ccr1 Arabidopsis are perceived initially by the cell through activation of plasma membrane-localized cell wall integrity receptors. This results in initiation of a signaling cascade that induces the expression of cell wall remodeling genes, including PECTATE LYASES , XYLOGLUCAN ENDO-TRANSGLYCOSYLASES ( XTH s), and ADGP1 . ADPG1 activity may contribute to solubilization of pectin, but is necessary for release of elicitor fragments, most likely from RG-II. The soluble elicitors activate expression of PR defense response genes through a signaling pathway involving SA ( 66 ). Many of the other transcriptomic changes occurring in the lignin-modified plants, such as the activation of seed-specific genes in stems of ccr1 , may result from secondary effects. The modification of pectin is also, at least in part, responsible for the reduced recalcitrance of the biomass. The suite of cell wall disassembly genes that is induced in the transcriptomes of the HCT-RNAi and ccr1 lines is, in many ways, reminiscent of the genes active in plant abscission zones ( 32 , 34 , 67 ). Interestingly, it has been suggested that PR proteins are part of the proteinaceous cell wall components in the protective layer of abscission zones ( 68 ), and, extrapolating from the present data, ADPG1 may therefore be a component of the signaling that strengthens defenses in the exposed surfaces postabscission, triggered initially by altered lignin−pectin interactions. Plants with modified lignin content and/or composition provide an excellent model system for deciphering the complexity of latent signal molecules sequestered within plant cell walls and characterization of their receptors. Improved approaches for the analysis of plant cell wall-released pectic fractions will facilitate these efforts ( 69 ). Understanding how plants remodel their cell walls as a result of engineered structural perturbations may allow us to better design improved lignocellulosic energy crops by optimizing bioprocessing quality, yield, and stress resistance."
} | 3,274 |
26727898 | null | s2 | 4,776 | {
"abstract": "Both positive and negative interactions among bacteria take place in the environment. We hypothesize that the complexity of the substrate affects the way bacteria interact with greater cooperation in the presence of recalcitrant substrate. We isolated lignocellulolytic bacteria from salt marsh detritus and compared the growth, metabolic activity and enzyme production of pure cultures to those of three-species mixed cultures in lignocellulose and glucose media. Synergistic growth was common in lignocellulose medium containing carboxyl methyl cellulose, xylan and lignin but absent in glucose medium. Bacterial synergism promoted metabolic activity in synergistic mixed cultures but not the maximal growth rate (μ). Bacterial synergism also promoted the production of β-1,4-glucosidase but not the production of cellobiohydrolase or β-1,4-xylosidase. Our results suggest that the chemical complexity of the substrate affects the way bacteria interact. While a complex substrate such as lignocellulose promotes positive interactions and synergistic growth, a labile substrate such as glucose promotes negative interactions and competition. Synergistic interactions among indigenous bacteria are suggested to be important in promoting lignocellulose degradation in the environment."
} | 320 |
37279908 | PMC10337742 | pmc | 4,779 | {
"abstract": "Abstract Deep-sea mining may lead to the release of high concentrations of metals into the surrounding seabed, which can disturb important ecosystem functions provided by microbial communities. Among these, the production of N 2 O and its reduction to N 2 is of great relevance since N 2 O is an important greenhouse gas. Metal impacts on net N 2 O production by deep-sea bacteria are, however, currently unexplored. Here, we evaluated the effects of cadmium (Cd) on net N 2 O production by a deep-sea isolate, Shewanella loihica PV-4. We performed a series of Cd exposure incubations in oxic conditions and determined N 2 O fluxes during induced anoxic conditions, as well as the relative expression of the nitrite reductase gene ( nirK ), preceding N 2 O production, and N 2 O reductase gene ( nosZ ), responsible for N 2 O reduction. Net N 2 O production by S. loihica PV-4 exposed to Cd was strongly inhibited when compared to the control treatment (no metal). Both nirK and nosZ gene expression were inhibited in reactors with Cd, but nirK inhibition was stronger, supporting the lower net N 2 O production observed with Cd. The Cd inhibition of net N 2 O production observed in this study poses the question whether other deep-sea bacteria would undergo the same effects. Future studies should address this question as well as its applicability to complex communities and other physicochemical conditions, which remain to be evaluated.",
"introduction": "Introduction Over the next few decades, deep-sea mining of earth minerals is expected to increase as demand is growing and the technical limitations of mining the deep ocean are being resolved. Over 29 exploration contracts already exist to mine locations in international waters, mostly aiming to extract minerals from manganese nodules, cobalt crusts, or massive sulfide deposits (Cuyvers et al. 2018 , Miller et al. 2018 ). Deep-sea mining may pose environmental risks to benthic life that would otherwise be virtually undisturbed by human activities (Orcutt et al. 2020 ). The operation of heavy machinery on the ocean floor may lead to sediment restructure and mining metallic substrates may release toxic concentrations of metals, which can affect biological processes across various domains of life (Magalhães et al. 2011 , Jordi et al. 2012 , Semedo et al. 2012 , Hauton et al. 2017 ). Cadmium (Cd) is one of the metals present in deep-sea ore deposits that may be released during deep-sea mining operations (Hauton et al. 2017 ). Due to its high toxic potential and to previously reported impacts in microbial metabolic and biogeochemical processes in different environments (Magalhães et al. 2007 , Liu et al. 2016 , Afzal et al. 2019 , Broman et al. 2019 ), it is important to investigate Cd impacts on deep-sea microbial life. Deep-sea microorganisms provide several ecosystem functions of great value to environmental sustainability (Orcutt et al. 2020 ). They are responsible for the majority of nutrient cycling in the deep-sea, methane production/oxidation, nitrous oxide production/reduction, and so forth. Nitrous oxide (N 2 O) is a potent greenhouse gas predicted to have a major impact on the globe's climate over the next 100 years (Ravishankara et al. 2009 , Neubauer and Megonigal 2015 ). The fine balance between its production and consumption in deep waters is of great relevance to the control of greenhouse gas emissions from the ocean, the largest ecosystem on earth (Miller et al. 2018 , Bange et al. 2019 ). Denitrification, the stepwise reduction of nitrate (NO 3 − ) and nitrite (NO 2 − ) to nitric oxide (NO), nitrous oxide (N 2 O), and dinitrogen gas (N 2 ), can be a source or sink of N 2 O due to its modularity (Graf et al. 2014 ). If the enzymes responsible for the steps preceding N 2 O formation, such as nitrite reductase (EC 1.7.1.4), are more abundant or active than the N 2 O reductase enzyme (EC 1.7.2.4.), responsible for N 2 O reduction, denitrification is a source. On the other hand, when N 2 O reductase is more abundant or active, denitrification becomes a sink. Nitrite reductase is encoded by nirS or nirK while N 2 O reductase is encoded by nosZ , with two distinct clades, nosZI and nosZII (Hallin et al. 2018 ). The relative expression of sources ( nirS or nirK ) vs. sinks ( nosZ ) will, ultimately, determine N 2 O fluxes in a particular environment or bacterial culture. While some studies report denitrification as an important pathway for NO 3 − removal in deep-sea sediments (Xu et al. 2022 ) or deep-sea sponge grounds (Rooks et al. 2020 ), denitrification activity and gene expression regulation by deep-sea bacterial isolates is relatively understudied. In this work, we take a closer look at net N 2 O production (resulting from production and consumption) by a deep-sea isolate due to the current lack of evidence about its drivers in the deep-sea as well as their susceptibility to metal pollution. It is reasonable to expect that denitrification and, consequently N 2 O production/reduction can be disturbed by the intensification of deep-sea mining. Increased metal exposure can lead to reduced microbial growth, denitrifying activity, and diversity of denitrifying microorganisms in various environments (Kandeler et al. 1996 , Huang et al. 2008 , Magalhães et al. 2011 , Baptista et al. 2015 ). Even metals that are essential cofactors of microbial enzymes, such as copper in nitrous oxide reductase, can inhibit their reactions when environmental concentrations exceed a certain level (Magalhães et al. 2011 , Glass and Orphan 2012 ). Indirect effects may also be relevant. For instance, metal availability and consequent oxidation in the deep seawater may decrease the pH, analogous to an acid mine drainage in terrestrial environments (Orcutt et al. 2020 ). Lower pH is known to inhibit N 2 O reduction by inhibition of nosZ gene expression or post-transcriptional interference (Liu, Frostegård and Bakken 2014 ; Samad et al. 2016 , Gaimster et al. 2017 ), which may lead to increased N 2 O fluxes from deep-sea communities. Despite the potential risk for increased mobilization and bioavailability of metals in deep-sea sediments related to mining activities, little is known about the impacts of metals on microbial life in the deep-sea. A few studies have addressed the toxic effects of metals in larger organisms in deep-sea conditions (low temperature and high pressure) and reported that the increased pressure and/or lower temperatures affected the toxicity potential of each metal (Hauton et al. 2017 ). For example, when a crustacean was exposed to copper and/or cadmium, the toxicity of copper increased at higher hydrostatic pressure but cadmium toxicity did not change with pressure (Brown et al. 2017 ). The colder temperatures and higher pressures of deep-sea conditions will likely affect the toxicity of metals to any living organism, however, the direction of that effect on microorganisms is currently unknown. In fact, to our knowledge, no studies with these metals have been performed so far with microorganisms isolated from the deep-sea and/or able to tolerate deep-sea conditions. In this research, we attempted to initiate the investigation of metal impacts on denitrification and N 2 O metabolism in deep-sea microorganisms. The specific objective was to investigate the Cd impacts on net N 2 O production from Shewanella loihica PV-4, a deep-sea isolate carrying nirK and nosZI genes (Jones et al. 2013 , Graf et al. 2014 ). To achieve this goal, we performed a series of exposure experiments with dissolved Cd and measured N 2 O concentration over time during induced anoxia. To further understand the potential impacts of the metal on the production and reduction of N 2 O, we measured the relative expression of both nirK and nosZ genes in the same period.",
"discussion": "Results and discussion The physicochemical conditions observed during the experiments were similar in both Cd-treated and control reactors. Temperature was constant at 28ºC while pH varied between 6.9 and 7.9 ( Fig. S2 , Supplementary Material ). It is worth noticing that the largest pH change was observed after sealing the reactors for inducing anoxia. The interruption of gas exchange in the headspace may have led to CO 2 accumulation with the consequent pH decrease in the media. Dissolved Cd in the media was quantified to confirm metal exposure at the target concentration ( Table S2 , Supplementary Material ). Cadmium (Cd) concentrations measured at the beginning of the experiment in metal-treated bioreactors (1.67 ± 0.06 mg/L) was approximate to the target Cd concentration of 1.83 mg/L (0.01 mM). At the end of the experiment, however, Cd concentration dropped to an average of 0.69 ± 0.05 mg/L, less than half of the initial concentration. The sharp decline in dissolved Cd concentration could have been caused by multiple mechanisms that are frequently observed during bacterial incubations with dissolved metals, such as adsorption to bacterial cells (Mullen et al. 1989 ) or sulfide particle precipitation (Janssen et al. 2014 , Ma and Sun 2021 ). Biological strategies that may lead to precipitation can also be activated by bacteria to increase metal resistance, such as biofilm formation (Ma and Sun 2021 ) or the production of external vesicles, that is known to be stimulated upon exposure to high metal concentrations (Lima et al. 2022 ). Shewanella loihica PV-4, for instance, was previously shown to be a strong oxidizer of another transition metal, Mn, in aerobic conditions, with the ability to form metal finely grained particles (Wright et al. 2016 ). The growth curves of S. loihica PV-4 during the exposure experiments are shown in Fig. 1 . The three replicate experiments followed a typical bacterial growth curve with three phases (lag, exponential, and stationary). The beginning of the exponential phase, however, was slightly different between experiments, with experiment C and B starting a few hours earlier than experiment A due to shorter lag phases, especially in experiment C. Besides the earlier beginning of the exponential phase, experiment C also displayed steeper slopes during the exponential phase, hence, higher maximum growth rates ( Table S3 , Supplementary Material ). Due to these differences, the sampling period of each experiment (when anoxia was induced) was adjusted so that the growth phase and biomass (assessed by the OD readings) were similar between experiments. The OD values of each reactor when anoxia were induced was between 0.6 and 0.9 for all reactors in each experiment. Figure 1. Optical density at 600 nm in the bioreactors during the exposure experiments with S. loihica PV-4 in oxic conditions. Each point represents the OD600 measurement from a single reactor at a given time point. The shaded area represents the 95% confidence interval of the locally estimated scatterplot smoothing (solid line). The dotted vertical lines represent the period when anoxia was induced for N 2 O and gene expression measurements. The Cd exposure effects on growth were similar in the three replicate experiments. Despite a slight delay in the start of the exponential phase in Cd-treated reactors, when compared to control, the metal did not inhibit growth of S. loihica PV-4 at the test concentration (0.01 mM). The calculated maximum growth rates were very similar between control and Cd-treated reactors across the three experiments ( Table S3 , Supplementary Material ). This observation was expected, based on our preliminary dose-response experiment, which showed growth inhibition only at the Cd concentration of 0.05 mM or higher ( Fig. S1 , Supplementary Material ). The absence of growth inhibition at 0.01 mM indicates that S . loihica PV-4 may be relatively resistant to Cd, since the same metal concentration in the media can be lethal to other model bacteria, such as Escherichia coli K-12 (Ferianc et al. 1998 ). Nevertheless, it is important to have in mind that S. loihica PV-4 relative resistance to Cd is limited. For instance, S. loihica PV-4 is far more sensitive than some bacteria isolated from Cd-polluted soils, that presented minimum inhibitory concentrations as high as 6 mM (Yu et al. 2021 ), and moderately more sensitive than some photosynthetic purple bacteria (Mohamed Fahmy Gad El-Rab et al. 2006 ) or even human pathogens, such as Campylobacter jejuni (Kaakoush et al. 2008 ). To our knowledge, no other deep-sea isolates have been tested against Cd exposure in growth media, even though it is reasonable to expect that deep-sea bacteria may be more resistant than shallower counterparts since Cd concentrations are known to increase with depth in the ocean water column (Xu and Morel 2013 , Janssen et al. 2014 ). However, the overall susceptibility or resistance of deep-sea bacterial growth to metals is still poorly understood and its further investigation is out of the scope of this work, which focus on the Cd effects on N 2 O metabolism. The N 2 O amounts in control and Cd-treated reactors during anoxia are shown in Fig. 2 . Positive N 2 O fluxes were only detected in control reactors, while Cd-treated reactors consistently had R 2 values < 0.80 for the linear increase over time, hence, null fluxes ( Table S4 , Supplementary Material ). A significant effect of treatment was observed in the calculated N 2 O fluxes (Mann–Whitney-Wilcoxon test, P = 0.017). Taking the three replicate experiments into account, the average N 2 O fluxes were 0.101 ± 0.039 µmoles N 2 O-N/min in control reactors and 0 µmoles N 2 O-N/min in Cd-treated reactors, strongly suggesting that Cd might inhibit net N 2 O production in this deep-sea isolate. Besides the overall difference between control and Cd-treated reactors, we must also note that the N 2 O fluxes in control reactors were considerably variable between experiments, with much lower fluxes in experiment A (especially reactor 2), when compared to experiments B and C ( Table S4 , Supplementary Material ). The lower fluxes observed in experiment A are probably due to the slightly later stage of growth when anoxia was induced (Fig. 1 ). The later stage of exponential growth could represent a lower amount of remaining glucose in the media (electron and C source), which would explain the lower N 2 O fluxes observed in experiment A. Figure 2. Nitrous oxide amount in the bioreactors during the exposure experiments with S. loihica PV-4. Each point represents the N 2 O measurement from a single reactor at a given time point during the anoxic period. The solid line represents the linear regression per reactor and the shaded area represents the 95% confidence interval. Calculated slopes and fluxes are presented in Table S4 . As opposed to Fe or Cu, necessary for the catalytic activity of NO 2 − and N 2 O reductase enzymes and potential direct impact on enzyme activity (Glass and Orphan 2012 , Giannopoulos et al. 2020 ), Cd is not known to be a structural component of these metalloenzymes. Thus, Cd effects on N 2 O production and consumption are expected to be associated with cellular toxic effects, such as effects on cell growth, metabolism, and oxidative stress (Kaakoush et al. 2008 , Behera et al. 2014 , Cheng et al. 2022 ) as well as community effects, such as changes in microbial community structure and diversity in the environment (Yu et al. 2021 , Sun et al. 2022 ). To our knowledge, no previous studies have investigated the effects of Cd on N 2 O metabolism using pure cultures or bacterial isolates. However, a few studies have investigated the same impacts on complex microbial communities. Using estuarine sediments, a previous study has found inhibited N 2 O reduction with consequent increase in net N 2 O production after Cd exposure (Magalhães et al. 2007 ), while others have found Cd stimulation of N 2 O reduction with an increase in N 2 production and no change in net N 2 O production in marine sediments (Broman et al. 2019 ). In metal polluted soils, researchers have found inhibited net N 2 O production, when compared to background soil, although these effects may change over time (Liu et al. 2016 , Afzal et al. 2019 ). The contrasting results may be due to multiple factors, such as different environmental contexts, organic content, dose of exposure, etc. For instance, the Cd effects on total denitrification from wetland sediments was shown to be dose-dependent, with inhibitory effects being observed only at concentrations higher than 500 mg/Kg of sediment, with no effect at 100 mg/Kg (Sakadevan et al. 1999 ). Regarding deep-sea microbial communities, no studies have addressed this question so far, although this is a relevant investigation considering the emergence of deep-sea mining activities and potential release of trace metals. To help unveiling the cellular mechanisms driving the inhibitory effect of Cd on net N 2 O production, we quantified the relative expression of the nirK and nosZ genes by qPCR (Fig. 3 ). The relative expression of both genes was lower in Cd-treated reactors than in control (mean and SEM values per treatment: nirK CTRL = 140 ± 39 , nirK Cd = 20.2 ± 6.0; nosZ CTRL = 10.9 ± 2.2, nosZ Cd = 2.38 ± 0.38), with a significant effect of treatment detected in both genes (2-way ANOVA, P -value ( nirK ) = 0.0444; P -value ( nosZ ) = 0.00393). The inhibition of nirK relative expression, however, was stronger than the inhibition observed for the nosZ gene, resulting in a lower nirK / nosZ ratio in Cd-treated reactors, when compared to control ( nirK/nosZ CTRL = 12.8, nirK/nosZ Cd = 8.49). These results contribute to explain the inhibitory effect of Cd on net N 2 O production, since a lower nirK / nosZ ratio supports a lower production to reduction potential. Recent studies have also reported that Cd decreases the number of nirK and nosZ transcripts in soil and marine sediment communities (Afzal et al. 2019 , Broman et al. 2019 ) as well as decreased abundances of nirK and nosZ genes in soils contaminated with a mixture of trace metals, including Cd (Liu et al. 2016 ). Our study provides additional evidence that Cd exposure may inhibit nirK and nosZ gene expression at the individual strain level. Additionally, our results strongly suggest that this inhibition may be particularly stronger for nirK than for nosZ , with consequences for net N 2 O production. Interestingly, other researchers have found a stronger inhibitory effect of soil metal pollution on nirK gene abundance than nosZ (Liu et al. 2016 ), which suggests that our results may be transposable to complex communities. Figure 3. Relative expression of nirK and nosZ genes in S. loihica PV-4 grown with Cd at 0.01 mM (red) and without the metal (blue). Relative expression was estimated by normalizing gene transcript copy numbers by the average of recA and rpoB transcript copy numbers in each sample. The boxes represent the first and third quartiles, with median value bisecting each box. The whiskers extend to the largest/smallest value, excluding outliers (data beyond 1.5 x interquartile range). In conclusion, we show here that Cd inhibits net N 2 O production in S . loihica PV-4 at levels near the ERL. Furthermore, we found that this inhibition is associated with the decrease in nirK / nosZ relative expression, suggesting that nirK gene expression may be more susceptible than nosZ to Cd exposure. It is important, however, to have in mind that our study was limited to a single bacterial strain. Since these effects have not been investigated in other pure cultures, future research should investigate whether our findings are specific to a few strains or observed across a wide range of taxa. Due to the emergence of deep-sea mining, investigating other deep-sea isolates could be of great environmental relevance. Additionally, the observed impacts of Cd exposure should also be investigated with deep-sea environmental communities as well as in deep-sea physicochemical conditions, such as high hydrostatic pressure and low temperature."
} | 5,040 |
34103505 | PMC8187645 | pmc | 4,780 | {
"abstract": "Bacillus subtilis is a soil bacterium that is competent for natural transformation. Genetically distinct B. subtilis swarms form a boundary upon encounter, resulting in killing of one of the strains. This process is mediated by a fast-evolving kin discrimination (KD) system consisting of cellular attack and defence mechanisms. Here, we show that these swarm antagonisms promote transformation-mediated horizontal gene transfer between strains of low relatedness. Gene transfer between interacting non-kin strains is largely unidirectional, from killed cells of the donor strain to surviving cells of the recipient strain. It is associated with activation of a stress response mediated by sigma factor SigW in the donor cells, and induction of competence in the recipient strain. More closely related strains, which in theory would experience more efficient recombination due to increased sequence homology, do not upregulate transformation upon encounter. This result indicates that social interactions can override mechanistic barriers to horizontal gene transfer. We hypothesize that KD-mediated competence in response to the encounter of distinct neighbouring strains could maximize the probability of efficient incorporation of novel alleles and genes that have proved to function in a genomically and ecologically similar context.",
"introduction": "Introduction The spore-forming bacterium Bacillus subtilis is found in soil- and gut environments and is arguably the best-studied gram-positive model species 1 . B. subtilis swarms over surfaces and has diversified into a vast diversity of strains able to recognise non-kin swarms, resulting in the formation of clear swarm boundaries 2 . Kin discrimination (KD) in B. subtilis is mediated by a rich arsenal of intercellular attack and defence molecules with extensive variation in transcription levels upon encounter of non-kin 3 . KD genes are present in unique combinations in different strains and likely frequently acquired through horizontal gene transfer 3 . The combinatorial nature of the B. subtilis KD system means that genomically divergent strains generally also differ to a greater degree in their carriage of antimicrobial genes. As a result, swarm boundaries between unrelated strains (i.e. non-kin) are very distinct, whereas genomically highly similar strains (i.e. kin) exhibit swarm merging 2 . KD-mediated barriers to swarm-merging result in the territorial sorting of strains according to genetic relatedness during the colonisation of plant roots 2 . However, it is not well-understood whether interference competition is the prime selective force underlying the radiation into many KD types, or whether this mechanism could have other functions. Another explanation for bacterial KD is that it could facilitate horizontal gene transfer between unrelated strains 4 . Recognition and lysis of neighbouring genotypes via the release of effectors by the T6SS secretion system coupled to natural transformation have been demonstrated in the gram-negative species Vibrio cholerae 5 and Acinetobacter baylyi 6 , 7 . In the gram-positive species Streptococcus pneumoniae , bacteriocin release can result in lysis of neighbouring susceptible genotypes and likewise increase transformation-mediated horizontal gene transfer 8 , 9 . B. subtilis is naturally competent 10 , 11 but transformation has mostly been studied in the context of single clones growing in liquid medium in this species (but see refs. 12 – 14 ), precluding the action of social interactions such as swarming found in structured environments. We hypothesised that the antagonisms observed between genetically distinct B. subtilis strains could lead to transformation-mediated recombination. Here, we show that swarm boundary interactions between non-kin B. subtilis strains result in horizontal gene transfer mediated by the cell-envelope stress-response ( sigW ) in the donor strain, whereas interactions between more closely related kin strains that do not form boundaries do not result in increased recombination. Our results demonstrate that competence regulation mediated by social interactions can be more important than sequence homology for successful recombination.",
"discussion": "Discussion Here we demonstrate that kin discrimination promotes horizontal gene transfer in B. subtilis through upregulation of competence in response to cell envelope stress. B. subtilis antimicrobial genes ( sunA , sdpC , sboA , yobL , skf , wapA , srfA ) have been shown to be upregulated at the non-kin boundary 3 . It is possible that antibiotics that affect cell-envelope integrity induce sigW at the boundary which, together with competence development, increases tolerance to antimicrobials and augment the more competent strain 27 . It could also be speculated that sigW activated cells with damaged membranes release stress-related metabolites that upregulate competence in the dominant strain as was documented during inter-species transformation experiments where lysis of E. coli cells and subsequent release of cell metabolites affected the transformation ability of B. subtilis recipient cells 28 . Similarly, it was recently shown that exposure to antibiotics increases cell-to-cell natural transformation in B. subtilis by affecting the donor strain through as yet unknown mechanism 29 . Induction of competence in B. subtilis requires activation of a quorum sensing (QS) system, encoded by the comQXPA operon 30 , 31 , that ensures activation of the competence genes via the induction of ComS 32 , 33 . We and others have shown that B. subtilis genotypes evolved extensive polymorphism in the ComQXPA QS system 15 , 34 – 36 . In liquid competence media, competence can only be induced if two strains express the same pheromone (i.e. both strains belong to the same pherotype) 15 , 34 , 37 . Limited cross-talk and sometimes even inhibition of competence has been observed between different pherotypes 34 , leading to the expectation that transformation between pherotypes is lower than within pherotypes. In contrast, the non-kin strains that engage in transformation in this study all belong to different pherotypes 2 , 15 , for example, PS-216 and other kin strains belong to “pherotype 168”, whereas PS-218 and PS-196 belong to “NAF4 pherotype” 15 , yet still show higher transformation efficiency when paired than do more closely related kin strain pairs belonging to the same pherotype. This discrepancy with earlier studies could potentially result from the fact that different mechanisms could be important governing HGT in structured (agar) environments versus unstructured (broth) environments. Interestingly, we found the rate of horizontal gene transfer to increase with genomic divergence. This result seems counterintuitive at first, as the efficiency of transformation-mediated recombination of more divergent DNA fragments has been shown to decrease log-linearly with increasing sequence dissimilarity in Bacillus 38 – 40 . Social interactions leading to competence development thus can play a more significant role in the efficiency of uptake of foreign DNA than constraints imposed by the recombination machinery in this species. This highlights the importance of considering social interactions in the study of bacteria generally, and in the study of horizontal gene transfer specifically. A variety of (non-mutually exclusive) explanations for the evolutionary benefits of natural transformation have been put forward, one of which is that increased genetic variation through recombination with foreign DNA facilitates adaptation 25 , 26 or that it can aid curing the genome of selfish deleterious elements 41 . The observed increase in recombination between genomically more distant strains is not predicted by the DNA for food hypothesis and poses a problem for the DNA repair hypothesis but would be consistent with the sex hypothesis. The coupling of competence to strain-specific killing could ensure that recombination is not upregulated in (near) clonal swarms (which would not introduce any genetic variation) nor upon encounter of dissimilar species (which would unlikely result in successful recombination). Instead, lysis of distinct but closely related strains occupying the same patch maximises the probability of efficient incorporation of novel alleles and genes that have proved to function in a similar genomic and ecological context 42 . Such non-kin interactions are common in natural populations of B. subtilis : 84% of strain combinations isolated from two microscale soil aggregates were shown to be non-kin 2 . Evolution experiments incorporating ecological realism are needed to shed light on the adaptive benefits of KD-mediated antagonism and transformation."
} | 2,191 |
36044593 | PMC9826165 | pmc | 4,782 | {
"abstract": "Abstract Mixtures of n ‐carboxylic acids ( n ‐CA) as derived from microbial conversion of waste biomass were converted to bio‐fuel using Kolbe electrolysis. While providing full carbon and electron balances, key parameters like electrolysis time, chain length of n ‐CA, and pH were investigated for their influence on reaction efficiency. Electrolysis of n ‐hexanoic acid showed the highest coulombic efficiency (CE) of 58.9±16.4 % ( n =4) for liquid fuel production among individually tested n ‐CA. Duration of the electrolysis was varied within a range of 0.27 to 1.02 faraday equivalents without loss of efficiency. Noteworthy, CE increased to around 70 % by hetero‐coupling when electrolysing n ‐CA mixtures regardless of the applied pH. Thus, 1 L of fuel could be produced from 12.4 mol of n ‐CA mixture using 5.02 kWh (<1 € L −1 ). Thus, a coupling with microbial processes producing n ‐CA mixtures from different organic substrates and waste is more than promising.",
"conclusion": "Conclusion Using a n ‐carboxylic acids mixture as substrate for Kolbe electrolysis results in improved yields of liquid fuels by hetero‐coupling of the derived radicals. A CE fuel of nearly 70 % for the n ‐CA mixture was demonstrated to be 10 % higher than CE fuel when using only C 6 , which is the most suitable single n ‐CA. Thereby, the radicals formed from C 4 and C 8 mainly undergo hetero‐coupling with a selectivity of 21.7±0.9 %, while the radicals of C 6 mainly homo‐couple (S homo =49.7±1.1 %). This performance, jointly with the economic consideration showing an operational expenditure of less than 1 € L −1 , demonstrates that n ‐CA mixtures can be successfully used for the production of drop‐in fuel via Kolbe electrolysis. Further, we shed light on influencing parameters for an efficient formation of Kolbe products that strongly depends on the chain length and the concentration of the n ‐CA in the aqueous reaction solution. There is a delicate balance between the need for the formation of a hydrophobic layer on the electrode surface as well as the need of relative proximity of the formed radicals at the electrode. Specifically, the Kolbe electrolysis enables the upgrading of n ‐CA mixtures, originated from microbial conversions of a variety of substrates such as corn beer, \n [13] \n acid whey, \n [12] \n or other organic waste streams[ \n 30 \n , \n 31 \n , \n 32 \n ] to fuels or fuel additives. In general, the combination of microbial and electrochemical conversion in electrobiorefineries offers the potential to make an important contribution to a circular and bio‐based but also viable economy.",
"introduction": "Introduction In the endeavor of a sustainable and eco‐friendly economy, bio‐based resources have to replace fossil feedstock for the synthesis of chemicals and fuels. For establishing a circular and bio‐based economy, the sector of electric energy harvest and storage needs to be intimately interweaved into the chemical industry. Electrochemistry provides that connecting thread by enabling storage of electric energy in form of chemical energy carriers. Therefore, electro‐organic reactions like cathodic CO 2 reduction[ \n 1 \n , \n 2 \n ] and hydrogenations/hydrodeoxygenations[ \n 3 \n , \n 4 \n ] as well as anodic coupling of biomolecules or decarboxylation[ \n 5 \n , \n 6 \n , \n 7 \n ] have to be brought to the spotlight. One specific reaction holding great promise for establishing electrobiorefineries \n [8] \n is the anodic decarboxylation of n ‐carboxylic acids ( n ‐CA), also known as Kolbe electrolysis. The Kolbe electrolysis, already discovered in the 19th century, \n [9] \n can be performed at ambient temperature and in aqueous solutions, making it environmentally friendly. Till now, mainly the Kolbe electrolysis of short‐chain n ‐CA at low concentrations was studied.[ \n 9 \n , \n 10 \n ] For implementation in electrobiorefineries, highly concentrated n ‐CA with a chain length between 4 and 8 C‐atoms, also called medium‐chain CA (MCCA), in alkaline aqueous solutions need to be electrolysed. \n [11] \n The MCCA in alkaline solutions are gained from the conversion of complex feedstock and waste by biological processes, for example using acid whey \n [12] \n or corn beer. \n [13] \n Using Kolbe electrolysis, MCCA can be converted into mixtures of alkanes (Kolbe products) or oxygenates like alcohols or esters (non‐Kolbe products) \n [14] \n via different reaction pathways leading to hydrocarbon mixtures (see Figure S1). For the electrolysis of different MCCA towards Kolbe products in aqueous solutions on monolithic platinum electrodes, a coulombic efficiency for the products (CE product ) around 50 % and also a yield for the products ( Y \n product ) around 50 % was achieved in batch systems. \n [15] \n Also, electrodes coated with Pt nano‐particles showed good results of CE product between 45–65 % and Y \n product between 35–50 % using n ‐octanoic acid as reactant.[ \n 16 \n , \n 17 \n ] The CE product was reported to be even increased up to 67 % and the Y \n product to 75 % when performing electrolysis in flow reactors. \n [18] \n In a previous study, we provided proof of principle for a whole process line for the production of a hydrocarbon mixture with fuel‐like properties starting from corn beer. \n [13] \n Biologically synthesized MCCA were used as starting material for the Kolbe electrolysis, achieving a CE of up to 80 % and a n ‐CA conversion rate per electrode surface area of 2.1×10 −3 mol cm −2 h −1 . The total carbon efficiency (expressed in chemical oxygen demand equivalents) of the entire process line was 0.5 g fuel g corn beer \n −1 . \n [13] \n \n To gain fuel‐like hydrocarbon mixtures in large quantities and also to exploit other feedstock \n [19] \n that can provide different MCCA mixtures using biological conversion, further electrochemical process engineering is required. Therefore, there is an imperative for assessing and engineering reactor components like the electrode material \n [20] \n as well as process parameters such as pH and supporting electrolyte. \n [21] \n Also, the monitoring of the products and not only the n ‐CA degradation, defined as the amount of electrolytically converted acid, as well as providing energy and carbon balances are of utmost importance for bringing the Kolbe electrolysis to industrial scale. The MCCA produced via biological conversion of biomass usually have to be extracted from the fermentation broth using an organic or aqueous extraction solution. Thus, investigations of process parameters of Kolbe electrolysis can be performed in aqueous solutions using pure n ‐CA. Thereby, also current‐controlled (galvanostatic) and not potential‐controlled operation is necessary to allow scale‐up as well as implementation into industrial processes.[ \n 22 \n , \n 23 \n , \n 24 \n ] Performing the Kolbe electrolysis directly in the fermentation broth would require intensive process engineering and development as well as investigations regarding, for example, possible inhibitors of the electrolysis stemming from the biological process step, side reactions with media components, or the influence of biomass. This study describes the Kolbe electrolysis of single n ‐CAs at high concentrations that are commonly gained from bioconversion using reactor microbiomes ( n ‐butanoic, n ‐hexanoic, and n ‐octanoic acid). The electrolysis of the mixture thereof was also performed since using mixtures is representative for scouting the implementation of a combined biological–electrochemical process. Thereby, the whole product spectrum was monitored and the influence of the pH on the CE is assessed. Here, it is important to mention that n ‐CAs with a chain length longer than 4 C‐atoms are only limitedly soluble in aqueous solutions. Therefore, only neutral and alkaline pH were investigated, allowing also n ‐hexanoic acid and n ‐octanoic acid to be fully dissolved in the aqueous electrolyte. Additionally, the influence of the carbon chain length and pH on the formation of micelles, typically leading to electrode blocking, \n [25] \n was investigated. We show that high yield and selectivity, as well as high CE for the Kolbe electrolysis of different n ‐CA being relevant products of the biological biomass conversion can be reached, especially by hetero‐coupling of the formed radicals when using n ‐CA mixtures.",
"discussion": "Results and Discussion Influence of the degree of conversion We previously performed Kolbe electrolysis of 0.5 m \n n ‐hexanoic acid (C 6 ) at 0.5 faraday equivalents (FE) yielding a CE dimer of around 50 %. \n [20] \n FE reflect the amount of charge required to convert a defined share of the substrate assuming 100 % efficiency and selectivity of the corresponding reaction. Thus, in the case of 0.5 FE the amount of charge that is necessary to convert 50 % of the substrate (here theoretically 52 mmol C 6 and hence 5004 C for 200 mL solution) is used. In this study, using the same conditions, a comparable product spectrum and CE dimer was reached showing excellent reproducibility (see Figure S2). Further, it was of interest, if the CE dimer could be increased by decreasing or increasing the time of electrolysis, meaning using different FE (see Figure S3). This is of particular interest because a variable electrolysis time should allow easier linking of the chemical industry and the energy sector. As Figure S3 shows, the optimum for the electrolysis of C 6 is reached with CE dimer =51.2±14.7 % at 0.43±0.02 FE that was therefore further used in this study. Thereby, the highest Y \n dimer of 76 %, reported by Sanderson et al. that was achieved for the electrolysis of 1.0 m C 6 is comparable to the results for the optimized conditions presented here ( Y \n dimer =68.6±18.6 %, see Table 1 ), especially when considering that a higher substrate concentration is favorable for the formation of the Kolbe product. \n [26] \n Remarkably, in the range of 0.3 to 1.0 FE the Kolbe electrolysis of C 6 also possess a high CE dimer , whereas exceeding 1.0 FE leads to a drastic decrease, because of reaching the limiting n ‐CA concentration (see Section S7).\n Table 1 Selectivity ( S \n dimer ) and yield ( Y \n dimer ) for the production of the Kolbe product (dimer) per converted n ‐CA for C 4 , C 6 , and C 8 as substrate as well as selectivity ( S \n fuel ) and yield ( Y \n fuel ) for the sum of fuel‐like compounds in the organic phase per converted n ‐CA for an artificial n ‐CA mixture as substrate. Carbon balance, CE fuel , and CE overall for the electrolysis of the individual n ‐CA and the artificial acid mixture. [a] \n \n Substrate \n \n Carbon balance [%] \n \n \n Y \n dimer or Y \n fuel [%] \n \n \n S \n dimer or S \n fuel [%] \n \n CE fuel [%] \n \n CE overall [%] \n \n \n n ‐butanoic acid ( n =3) \n \n 42.1±24.5 \n \n 11.3±7.5 \n \n 14.4±3.7 \n \n – \n \n 11.5±5.3 \n \n \n n ‐hexanoic acid ( n =4) \n \n 83.4±15.2 \n \n 68.6±18.6 \n \n 68.4±4.9 \n \n 58.9±16.4 \n \n 73.2±15.9 \n \n \n n ‐octanoic acid ( n =3) \n \n 29.0±9.7* (111.3±10.6) \n \n 24.0±9.0* (92.0±10.4) \n \n 69.9±5.4 \n \n 25.4±3.3 \n \n 35.1±5.5 \n \n mix, pH=7.07±0.25 ( n =3) \n \n 87.2±16.2 \n \n 81.9±16.8 \n \n 78.6±2.0 \n \n 69.5±11.1 \n \n 81.2±12.0 \n \n mix, pH=8.21±0.34 ( n =3) \n \n 86.1±3.7 \n \n 80.1±4.7 \n \n 78.5±1.9 \n \n 67.5±2.2 \n \n 80.3±1.2 \n [a] – indicates that no liquid organic phase was formed. n provides number of replicates and ± represents the 95 % confidence interval. Please note that values marked with * are not representative because of phase separation and low solubility of n ‐CA, which lead to an overestimation of acid consumption in the aqueous phase. Consequently, values given in brackets are based on the acid consumption being calculated from CO 2 and ester production (see Experimental Section: carbon balance, yield, selectivity, and rates of the electrolysis). Wiley‐VCH GmbH Kolbe electrolysis of individual n ‐CA The use of n ‐CA with variable chain length obtained from biological processes is of great interest for its conversion into fuel‐like hydrocarbons using Kolbe electrolysis. Therefore, the optimized experimental conditions for the electrolysis of hexanoic acid (C 6 ) were used as comparison point to study the Kolbe electrolysis of n ‐butanoic (C 4 ) and n ‐octanoic acid (C 8 ). Figure 1 shows the results from the electrolysis of C 4 and C 8 compared to C 6 . Converting all three single acids via Kolbe electrolysis is possible, but there are limitations when using C 4 or C 8 as substrate. The electrolysis of only C 4 shows a low CE acid degradation . This is strongly supported by the low CE CO 2 \nof 9.1±5.1 %, as CE CO 2 \nis directly linked to the conversion of n ‐CA (Figure S1). As shown in Figure 1 , no liquid organic compounds and only gaseous products including the volatile dimer derived from C 4 (i. e., n ‐hexane) were formed during electrolysis of C 4 . In addition to n ‐hexane with CE dimer =3.0±1.9 %, only ΣProp (the sum of propane and propene) with CE ΣProp =8.6±3.4 % and S \n ΣProp =85.5±3.6 % was produced during electrolysis of C 4 , resulting in a ratio between dimerization and disproportionation of 1 : 5.8, meaning disproportionation occurs about 6 times more frequently than dimerization for C 4 . This indicates a mechanism shift towards non‐Kolbe products under the applied conditions using C 4 as substrate. Furthermore, the CE O 2 \nincreased significantly, up to 31.0±14.3 % compared to C 6 (CE O 2 \n=2.0±0.8 %, Figure 1 ). This shows that the main share of the electrons is used for the competitive reaction [i. e., the oxygen evolution reaction (OER)] and not to the electrolysis of n ‐CA. The results for Kolbe electrolysis of C 4 presented here are lower compared to literature values. Levy et al. could achieve a Y \n dimer of 20.5 %, and Lopez‐Ruiz et al. reported a CE acid degradation around 75 % with a carbon selectivity of 35.1 % for n ‐hexane.[ \n 27 \n , \n 28 \n ] However, the experimental setup and conditions of both studies differ from the ones used here that are relevant for integration into electrobiorefinieries. Lopez‐Ruiz et al. performed the electrolysis potentiostatically at 5 V vs. Ag/AgCl, and Levy et al. used a flow‐through system with an n ‐CA concentration of 1.4 m . This may explain the differences, as it is known that both potential and acid concentration are critical parameters and can have a major impact on the efficiency of the Kolbe electrolysis.\n Figure 1 Kolbe electrolysis of 0.5 m \n n ‐butanoic acid (C 4 ), n ‐hexanoic acid (C 6 ), and n ‐octanoic acid (C 8 ) in a two‐chamber electrochemical cell with 150 mA cm −2 up to 0.45 FE at pH=7 (C 4 and C 6 ) or pH=8.6 (C 8 ). CE \n i \n for substrate consumption and the formation of different products of the electrolysis are presented. The shown values are averages of the replicates ( n ) and the error bars represent the 95 % confidence interval. Note: The value for CE acid degradation of C 8 is not representative because of phase separation caused by low solubility and agglomeration of the n ‐CA, leading to an overestimation of acid consumption in the aqueous phase. On the other hand, the use of C 8 acid as substrate requires that the starting pH of the solution has to be increased, here from pH=7 to pH=8.6, because of foam formation during the electrolysis. This effect might be caused by the partly undissolved acid in combination with the formation of gases at the electrodes. In the electrolysis at pH=8.6, the dimer n ‐tetradecane is the main product with CE dimer =16.81±5.48 % at S \n dimer of nearly 70 %. This shows that dimerization is the preferred reaction pathway using C 8 as substrate, which can also be seen by the ratio between dimerization and disproportionation of 1:0.1. The CE O 2 \nfor C 8 (27.8±10.2 %) is comparable to that reached when using C 4 as substrate. This implies that the OER is also not as successfully suppressed over the whole duration of the electrolysis of C 8 as during electrolysis of C 6 (Figure 2 ). This can be explained by the pH dependent behavior of C 8 solutions. Having alkaline pH at the beginning of the experiment, C 8 is fully soluble in the aqueous solution providing the octanoate anion as substrate for the Kolbe electrolysis. But already at neutral or slightly acidic pH, C 8 starts to form agglomerates in aqueous solution at these concentrations. \n [25] \n This limits the availability of the octanoate anion at the electrode surface that is required for the Kolbe electrolysis. During electrolysis of C 8 the pH of the anolyte decreases to 6.9, leading to agglomerate formation and inhibition of the Kolbe electrolysis, as well as a further increase in the OER at reaction times longer than 180 min (Figure 2 ). At that point, the OER is favored over the Kolbe electrolysis explaining the high CE O 2 \nfor C 8 as substrate. This agglomeration behavior of C 8 is dependent on pH and concentration, which is shown in Figure S6. For a 0.5 m C 8 solution the conductivity starts to drop at pH≈8, reaching the minimum conductivity at a pH range from 6.8 to 6.2. On the other hand, a 0.1 m solution shows no conductivity collapse. In comparison, neither 0.5 m C 4 nor 0.5 m C 6 are showing any conductivity collapse due to agglomeration during the conductometric titration (Figure S6). The results presented here for the Kolbe electrolysis of C 8 are comparable to literature values. For a similar electrolyte composition and n ‐CA concentration a CE acid degradation of 57±0.4 % could be achieved. \n [25] \n Yuan et al. reported a CE hydrocarbons around 65 % with an Y \n hydrocarbons around 50 % using electrodes coated with Pt nanoparticles. \n [16] \n Hydrocarbons in this respect are the sum of n ‐tetradecane, n ‐heptane, and n ‐heptene, with n ‐tetradecane and n ‐heptane being produced in equal portions. Thus, the CE hydrocarbons and Y \n hydrocarbons are high, but the use of these kind of tailor‐made specialized electrode materials is not applicable for a technical process and comes with significantly higher costs than the electrodes used here. \n [20] \n \n Figure 2 Oxygen evolution over the duration of the electrolysis for different n ‐CA in a two‐chamber electrochemical cell with 150 mA cm −2 up to 0.45 FE at pH=7 (C 4 and C 6 ) or pH=8.6 (C 8 ). The shown values are averages of the replicates ( n ) and the error bars represent the 95 % confidence interval. The interaction and coverage of the electrode surface with the respective n ‐CA differs depending on the carbon chain length of the n ‐CA (Figure 3 ). Thereby it is of note that the physical‐chemical properties are different for the here used concentrations in comparison to the ideal that is infinitely diluted aqueous solutions. For C 4 , the surface coverage with butanoate anions is relatively low in comparison to C 6 and C 8 , as due to its lower hydrophobicity C 4 has a higher solubility in the aqueous phase. This lower coverage of the electrode surface with butanoate anions results in their lower availability for the first oxidation step of the Kolbe electrolysis yielding the C 3 ‐radical. Thus, insufficient radicals are formed closely enough to allow dimerization with high efficiency. This explains the low CE hexane =3.0±1.9 % for electrolysis of C 4 . In addition, the low hydrophobicity means that the formed radicals do not remain on the electrode surface, preventing the second oxidation step to non‐Kolbe products formed from the oxidation of the carbocation. Instead, the radicals disproportionate forming propane and propene which can be seen in the higher CE ΣProp =8.6±3.4 %. The highest CE O 2 \namong the different single n ‐CA is obtained with C 4 , which can also be seen in Figure 1 . Due to the low hydrophobicity of C 4 , no hydrophobic layer forms on the electrode surface, which would prevent water electrolysis. Thus, water molecules reach the electrode surface and are oxidized, leading to a CE O 2 \n=31.0±14.3 %. This hypothesis is strongly supported by the measured contact angles at the electrode surface. The electrolyte solution without n ‐CA shows a contact angle of θ =65.9±4.9°, indicating slight hydrophobicity of the electrode surface. If C 4 is added to the solution, the contact angle increases even further to θ =72.1±1.9° (see Table S7). This shows that the hydrophilicity even increases, supporting that no hydrophobic layer consisting of butanoate anions forms on the electrode surface.\n Figure 3 Representation of the electrode surface–molecule interaction during the Kolbe electrolysis of different single n ‐CA. (A) Kolbe electrolysis of n ‐butanoic acid (C 4 ). (B) Kolbe electrolysis of n ‐hexanoic acid (C 6 ). (C) Kolbe electrolysis of n ‐octanoic acid (C 8 ). The thickness of the arrows represents the dominating reaction pathways as discussed. In the case of C 6 as substrate for Kolbe electrolysis the coverage of the electrode with hexanoate anions can be considered high, leading also to a high concentration of radicals and therefore an efficient dimerization (CE dimer =51.2±14.7 %). Additionally, because of the higher hydrophobicity of C 6 a hydrophobic layer is formed on the electrode surface which inhibits water oxidation nearly completely (CE O 2 \n=2.0±0.8 %), which can also be seen in Figure 1 . This formation of a hydrophobic layer is strongly supported by the smaller contact angle of the electrolyte solution containing C 6 ( θ =48.7±1.0°, Table S7) at the electrode surface. Apparently, the fast dimerization also prevents disproportionation to shorter‐chain alkanes. Since the dimerization after the first oxidation step is the favored reaction pathway the CE \n i \n for non‐Kolbe products formed from the carbocation after the second oxidation step is low. Overall, this leads to a high Y \n dimer and S \n dimer (see Table 1 ). Using C 8 as substrate, the CE dimer of 16.8±5.5 % is lower than when using C 6 . For C 8 as substrate for the Kolbe electrolysis the coverage of the electrode with octanoate anions can be expected to be at least as high as when using C 6 (see Figure 3 ), which is also very much in line with the low contact angle of C 8 electrolyte solution of θ =23.6±0.3°. This is even lower than for the solution containing C 6 suggesting an even higher attraction of octanoate anions to the electrode surface. However, the overall kinetics seem to be lower leading to a lower concentration of C 7 ‐radicals at the electrode surface and hence a less efficient dimerization. Further, this can be also explained by the larger molecule size of C 8 . As a result, the intermolecular distance of the C 7 ‐radicals is increased, and hence dimerization is less efficient. Therefore, a higher portion of the radicals undergoes disproportionation. Also, with C 8 as substrate a hydrophobic layer is formed on the electrode surface, which inhibits the OER in the beginning of the electrolysis. In line with the measured contact angle, a high attraction of octanoate to the electrode surface is expected. Therefore, a really dense hydrophobic layer can be assumed. This hypothesis is further supported by the increased cell potential ( E \n cell ) and working electrode potential ( E \n WE ) during the electrolysis of C 8 compared to C 4 or C 6 (Figure S8). Due to the dense hydrophobic layer the internal resistance increases, leading to an increasing overpotential. However, with decreasing pH in the solution C 8 starts to form agglomerates, and this hydrophobic layer detaches leading to an increasing oxygen evolution over time (see Figure 2 ). Overall, it becomes clear that C 6 seems the best‐suited single substrate for Kolbe electrolysis among the tested n ‐CA. Portions of C 8 in an expected n ‐CA mixture can also be converted to a potential drop‐in fuel. However, the proportion should be kept sufficiently low to avoid agglomeration of C 8 at higher concentrations. C 4 , on the other hand, can only be electrolysed with low efficiency, and hence no liquid organic phase forms that can be used as drop‐in fuel. Another important point to mention is the relative carbon loss due to decarboxylation during the Kolbe electrolysis (see Figure S1). For C 4 , 1 out of 4 C‐atoms and for C 8 , only 1 of 8 C‐atoms is lost as CO 2 , meaning that depending on the chain length the carbon loss ranges between 12.5 and 25 %. Kolbe electrolysis of a n ‐CA mixture To achieve drop‐in fuel/ fuel additive production from biomass using an electrobiorefinery, Kolbe electrolysis of a mixture of n ‐CA is necessary. Here a n ‐CA mixture that resembles the solution gained by Xu et al. from a two‐stage microbial conversion of acid whey was used for the Kolbe electrolysis. \n [12] \n Additionally, the influence of the pH was investigated to decipher the impact of agglomeration that can be seen for C 8 during single acid electrolysis. Thereby, the pH of 7 was chosen to compare the results of the electrolysis of the n ‐CA mixture with the results of the single acids (see above). The used pH of 8.2 resembles a possible cost‐effective and direct combination with the extraction by pertraction as is described by Xu et al. \n [12] \n \n It can be expected that intermolecular interactions between the different n ‐CA may have a weakening effect on the agglomeration of C 8 in a n ‐CA mixture. This, however, is only the case to a limited extent as reflected by the contact angels of θ =25.1±5.1° and 35.5±11.1° for pH=7 and 8.2, respectively. For C 4 these intermolecular interactions may improve the efficiency of the process, because the C 3 ‐radical, formed from C 4 , can combine with radicals formed from other n ‐CA leading to longer chain alkanes than n ‐hexane, which are less volatile. \n [27] \n \n Figure 4 shows a high CE acid degradation >80 % for both tested pHs. Thereby, in line with the relative molar concentrations the largest share is C 6 conversion (CE acid degradation , C 6 \n≈63 %), followed by the other two n ‐CA with CE acid degradation , C 8 \n≈11.5 % and CE acid degradation , C 4 \n≈8 % (Figure 4 B). As expected, compared to the single acid electrolysis the product spectrum was more diverse because of the greater variety of possible recombinations of the formed radicals. Not only homo‐coupling, meaning the recombination of two radicals of the same chain length, takes place but also hetero‐coupling. Homo‐coupling is the dominating reaction pathway for C 6, meaning that two C 5 ‐radicals form n ‐decane. Since C 6 shows the highest conversion, the highest CE \n i \n among the products is CE decane with above 40 % for both pHs. Noteworthy, the CE decane is lower than can be expected for only homo‐coupling of C 6 , which strongly suggests that n ‐decane formation via recombination of C 3 ‐radicals and C 7 ‐radicals is very unlikely. Compared to the single acid electrolysis the CE for the homo‐coupled dimers from C 4 and C 8 decreases to CE hexane and CE tetradecane below 1 % at both pHs. Instead, the formed radicals from C 4 and C 8 performed preferably hetero‐coupling with the C 5 ‐radical formed from C 6 . This leads to a high CE \n i \n for n ‐octane with around 8 %, produced via combination of C 5 ‐ and C 3 ‐radicals, and n ‐dodecane with around 10 %, gained via combination of C 5 ‐ and C 7 ‐radicals,. The CE \n i \n of all produced organic compounds in the liquid organic phase can be summed up because the combined organic phase is a very likely to serve as drop‐in fuel/fuel additive. In total, an excellent CE fuel =69.5±11.1 % for pH 7 and CE fuel =67.5±2.2 % for pH 8.2 was achieved. This is well above the CE fuel =58.9±16.4 % using only C 6 as substrate. This shows that using a n ‐CA mixture increases the efficiency of fuel production via Kolbe electrolysis compared to using single n ‐CA. Also, the OER is successfully suppressed at both pHs using a n ‐CA mixture as substrate, as CE O 2 \nwas below 1 % for both cases (see Figure 4 ).\n Figure 4 Kolbe electrolysis of a 0.47 m artificial n ‐CA mixture containing n ‐butanoic acid (C 4 ), n ‐hexanoic acid (C 6 ), and n ‐octanoic acid (C 8 ) in a molar ratio of 3 : 8 : 1 in a two‐chamber electrochemical cell with 150 mA cm −2 up to 0.45 FE at two different pH (pH=7 and 8.2). (A) CE \n i \n for total substrate consumption and the formation of different products of the electrolysis are presented. (B) CE \n i \n for the different n ‐CA in total substrate consumption. A detailed product distribution is given in Figure S9. The shown values are averages of the replicates ( n ) and the error bars represent the 95 % confidence interval. Additionally, the selectivity and yield for the production of the respective dimer were calculated (see Table 1 ). C 6 shows the highest combination of yield and selectivity for dimer production with Y \n dimer =68.6±18.6 % and S \n dimer =68.4±4.9 % among the individually tested n ‐CA. S \n dimer =69.9±5.4 % for C 8 is comparable to that of C 6 . The Kolbe electrolysis of C 4 shows only a low yield as well as a low selectivity for dimer formation. This confirms that C 6 is the optimal substrate for Kolbe electrolysis among the individually tested n ‐CA. Furthermore, Table 1 shows that the selectivity and yield for the sum of fuel‐like compounds, using a n ‐CA mixture, resembling the composition gained by Xu et al. from a two‐stage microbial conversion of acid whey, \n [12] \n as substrate for the Kolbe electrolysis, is around 80 %. This demonstrates that most of the products are in the liquid organic phase that could be used as drop‐in fuel and only a minor share are present in the gas phase that may also serve as combustion fuel. A schematic representation of the interaction and coverage of the electrode surface with carboxylic acid molecules as well as the selectivity for the different reaction pathways using a n ‐CA mixture as substrate is shown in Figure 5 . With a selectivity around 70 % dimerization is the preferred pathway. However, it has to be distinguished between homo‐ and hetero‐coupling. Homo‐coupling with S \n homo =49.7±1.1 % mainly takes place for C 5 ‐radicals derived from C 6 as already mentioned before. Thereby, 66.1 % of the converted C 6 molecules undergo homo‐coupling of the C 5 ‐radicals while only 8–10 % of the converted C 4 and C 8 result in homo‐coupled dimers of the resulting radicals. The C 3 ‐radicals and C 7 ‐radicals, on the other hand, preferably perform hetero‐coupling with C 5 ‐radicals resulting in S \n hetero =21.7±0.9 %. Only about 7 % of converted C 6 can be accounted for hetero‐coupled products, but between 24–26 % of the converted C 4 and C 8 . With a similar selectivity to hetero‐coupling also disproportionation takes place, mainly for C 4 and C 6 . The least likely pathway is the 2nd oxidation step to a carbocation, which produces non‐Kolbe products like esters and alcohols. This has only a selectivity of around 10 %. Here, it is important to stress that biological conversions of bio‐based feedstock yield mixtures of n ‐CA and usually not solutions containing single acids. This diversity of substrate can be detrimental for other chemical conversions. This is not the case here. Even further, using a n ‐CA mixture as substrate for the Kolbe electrolysis can overcome the drawbacks of the electrolysis of individual n ‐CA, which as single acids did not form any liquid organic products or show agglomeration, and is therefore a highly elegant way to produce bio‐based fuel.\n Figure 5 (A) Representation of the electrode surface–molecule interaction with the thickness of the arrows representing the dominating reaction pathways and the selectivity, S i \n , for the different reaction pathways. (B) Achieved product selectivity during the Kolbe electrolysis of a n ‐CA mixture. The shown values are averages of the replicates ( n =6, Mix pH 7 and Mix pH 8.2) and the error bars represent the 95 % confidence interval. In addition, the operational expenditures (OPEX) of a process similar to our previous study by Urban et al. without the need of down‐streaming and demonstrating the comparable fuel‐like properties of the gained product including a higher heating value of around 46 MJ kg −1[13] were analysed (see Section S13). The used n ‐CA mixture serves as model for a real n ‐CA mixture (MCCA mixture) derived from bio‐based feedstock or even waste via a biological conversion. \n [12] \n Then, the production costs of fuel additives via Kolbe electrolysis can be simplified as follows: Per conversion of 1 mol of n ‐CA (average for the mixture) from the mixture 80.4 mL of liquid fuel mixture are produced. In order to obtain 1 L fuel, 12.4 mol of n ‐CA have to be converted using 5.02 kWh (see Table S8). Considering the electric energy price, this results in OPEX of 0.53 € (0.59 $) per produced liter of fuel mixture. Although this calculation does not consider agitation costs and other costs for drive peripherals, it conveys one important key message: the costs of fuel produced via Kolbe electrolysis can compete with the costs for traditional petroleum‐based fuel and, in addition, governmental funding for the development and expansion of such processes is desirable. \n [29]"
} | 8,297 |
30042299 | PMC6117655 | pmc | 4,783 | {
"abstract": "Herein, efficient antimicrobial porous surfaces were prepared by breath figures approach from polymer solutions containing low content of block copolymers with high positive charge density. In brief, those block copolymers, which were used as additives, are composed of a polystyrene segment and a large antimicrobial block bearing flexible side chain with 1,3-thiazolium and 1,2,3-triazolium groups, PS 54 - b -PTTBM-M 44 , PS 54 - b -PTTBM-B 44 , having different alkyl groups, methyl or butyl, respectively. The antimicrobial block copolymers were blended with commercial polystyrene in very low proportions, from 3 to 9 wt %, and solubilized in THF. From these solutions, ordered porous films functionalized with antimicrobial cationic copolymers were fabricated, and the influence of alkylating agent and the amount of copolymer in the blend was investigated. Narrow pore size distribution was obtained for all the samples with pore diameters between 5 and 11 µm. The size of the pore decreased as the hydrophilicity of the system increased; thus, either as the content of copolymer was augmented in the blend or as the copolymers were quaternized with methyl iodide. The resulting porous polystyrene surfaces functionalized with low content of antimicrobial copolymers exhibited remarkable antibacterial efficiencies against Gram positive bacteria Staphylococcus aureus , and Candida parapsilosis fungi as microbial models.",
"conclusion": "4. Conclusions In summary, efficient antimicrobial porous coatings were fabricated by the breath figures approach from blends containing very low contents of antimicrobial polymers. Highly active amphiphilic copolymers with a large cationic block bearing a flexible side chain with 1,3-thiazolium and 1,2,3-triazolium groups were used as antimicrobial polymers with high charge density. Due to the high biocidal effectiveness of the copolymers and the controlled roughness of the porous surfaces, the resulting films exhibit high killing efficiency against the studied microorganisms. Thus, we can conclude that this breath figures approach, using only a low content of cationic polymers, allows the formation of surfaces with accessible polycationic chains for killing the microorganisms S. aureus and C. parapsilosis by surface contact.",
"introduction": "1. Introduction Healthcare-associated infections are a major problem nowadays, causing high morbidity and mortality rates and substantial increase in health care costs. These infections are mainly associated with surgery procedures and medical devices such as ventilators or catheters. Prescription of antibiotics is typically used as a prevention method and/or treatment to avoid such transmissions of nosocomial pathogens; however, antibiotic consumption is a primary cause of antibiotic resistance [ 1 ], and consequently, there is an urgent need for alternatives, as well as strategies to prevent healthcare-acquired infections [ 2 ]. Inhibition of bacterial growth on the surfaces of medical equipment and devices is necessary to prevent the transmission of diseases by contact, and one promising approach is the development of antimicrobial coatings. Many of these self-disinfecting coatings are based on impregnating the surfaces with antimicrobial agents including antibiotics, silver compounds, light active species, and antimicrobial polymers such as polycations [ 3 , 4 , 5 , 6 , 7 ]. Other strategies limit the bacterial colonization of surfaces by inducing micro and nano-roughness, which modifies the surface area of contact with the microorganisms [ 8 , 9 , 10 ]. Although in the past the effect of surface topography on bacterial inhibition has received less attention, it is gaining popularity nowadays [ 11 , 12 ]. Many of these works study antifouling effects [ 13 , 14 ] based on superhydrophobic surfaces with reduced surface contact [ 10 , 15 ]. Most of the antifouling surfaces are able to effectively reduce the initial bacterial attachment, but only within a relatively short period. If adhesion occurs, the bacteria rapidly proliferate, leading to the formation of the biofilm. Alternatively, the introduction of roughness can produce the opposite effect, dramatically increasing the contact adhesion area. Thus, combinations of surface roughness with chemical biocidal functionalities can create more effective bactericidal properties than flat surfaces [ 16 , 17 , 18 ]. Nowadays, many techniques are available to create surfaces with finely controlled topography, including lithography approaches and colloidal templates [ 19 , 20 ]. Most of these techniques usually require multiple stages, expensive equipment and prefabricated masks. One of the most versatile and simple methodologies to create polymeric porous surfaces with controlled pore size, and thus tailored roughness, is the so-called breath figures approach [ 21 , 22 , 23 , 24 ]. This method prepares ordered porous films with water droplets as the template. Basically, polymer solution is cast onto a substrate under humid atmosphere. The solvent evaporation induces the condensation of water droplets, which self-organize into a hexagonal array and after solvent evaporation, honeycomb-patterned films are obtained. Although this technique can be used with a diversity of polymers and functionalities [ 25 ], it is limited to polymers soluble in non-polar organic solvents such as CS 2 or chloroform, which are mostly hydrophobic polymers, polymers with polar end groups or some amphiphilic copolymers. An alternative for obtaining porous surfaces functionalized with highly hydrophilic polymers or polyelectrolytes is the use of polymer blends, consisting of incorporating low amount of hydrophilic polymers into a hydrophobic polymeric matrix such as polystyrene [ 26 ]. Due to the formation mechanism of the breath figures, the hydrophilic polymers tend to migrate towards the condensed water droplets, which imply their localization at the surface, on the wall of the pores [ 26 , 27 , 28 ]. In this context, cationic antimicrobial polymers based on quaternary ammonium groups have been incorporated into breath figures films by using blends [ 29 ]. However, only systems based on copolymers with low positive charge density have been prepared due to the difficulty to dissolve them in organic solvents [ 29 , 30 ]. In this work, we prepared porous films by a breath figures approach functionalized with high charge density by the incorporation of antimicrobial cationic polymers bearing two quaternary ammonium groups per monomeric unit. In fact, these structures are based on methacrylic monomers with 1,3-thiazolium and 1,2,3-triazolium side-chain groups, and have demonstrated a broad spectrum of antimicrobial activity in solution [ 31 , 32 ], and also when immobilized onto a surface [ 16 , 33 ]. It is well known that surface positive charge density is an important parameter for defining antimicrobial efficiency [ 33 , 34 ], and the incorporation of polymers with high charge density as blend component will enhance the biocidal activity of the microstructured surfaces, maintaining the physicochemical properties of the resulting coating.",
"discussion": "3. Results and Discussion Porous films were prepared by the breath figures method from blends containing antimicrobial copolymers with high charge density as a minor component or additive. By this approach, bactericidal films on contact with antimicrobial chemical functionalities at the surface and controlled microstructure can be fabricated in a very simple and effective way. In addition, structural parameters, such as pore size and pore density, can be easily controlled by selecting the experimental conditions of humidity, concentration of the solution or the type of polymer [ 22 ]. In fact, the use of polymeric structures with polar moieties, i.e., amphiphilic polymers, favors the formation of ordered porous arrays, because these structures help the stabilization of the condensed water droplets. Herein, amphiphilic copolymers based on a hydrophilic block with two quaternary ammonium moieties per monomeric unit ( Figure 1 ) were added to commercial polystyrene as a modifier and antimicrobial component. THF solutions of these blends were cast onto glass substrates under controlled humidity. It has to be mentioned that the cationic copolymers PS 54 - b -PTTBM-R 44 are not soluble in the common organic solvents typically used in the breath figures approach, such as CS 2 and chloroform. Thus, THF was selected to be more compatible with both components of the blend, although it is known that THF is not an ideal solvent and typically leads to irregular arrays due to its miscibility with water. Nevertheless, THF only allows the solubility of low content of copolymers. For this reason, THF solution was prepared with a polymeric concentration of 30 mg/mL, with PS/PS 54 - b -PTTBM-R 44 ratios of 97/3, 94/6 and 91/9 wt %. Films were prepared from these THF solutions under different humidities: 60%, 70% and 90%. As mentioned above, the relative humidity is a fundamental parameter in the preparation of porous films by the breath figures approach and has a large impact on the morphology of the films. As shown in Figure 1 , when the humidity was set at 60%, only flat surfaces were obtained; thus, in this case, higher humidity was necessary to fabricate porous films. In effect, at higher humidity values, such as 70% and 90%, porous films were found; however, films prepared at 90% show irregular patterns containing large and heterogeneous pores mixed with smaller pores, resulting from the coagulation of the rapidly condensing water droplets during the breath figure process, which leads to a dramatic increase in the droplet size ( Figure 1 c,d) [ 37 ]. On the other hand, when the humidity was set at 70%, more homogeneous patterns were obtained, as shown in Figure 1 b. Thus, the following experiments for the fabrication of antimicrobial surfaces were carried out under this relative humidity. Films were prepared at 70% relative humidity from blend solutions composed of commercial PS and a low amount of the antimicrobial copolymer quaternized with butyl or methyl iodide, PS 54 - b -PTTBM-B 44 or PS 54 - b -PTTBM-M 44 , respectively. It is well known that the alkylating agents affect the efficacy of the antimicrobial polymers based on quaternary ammonium groups, because they modify the hydrophobic/hydrophilic balance [ 31 , 38 ]. Additionally, the alkylating agents would also influence the microstructure of the breath figure films. Figure 2 shows SEM images of the films containing different contents of copolymer prepared at 70% humidity, in which porous films are observed in all cases. Additionally, it is observed from the cross-section image that there is only a single layer with pores. In these SEM images, the influence of the type of copolymer and its concentration on the pore structure and morphology of the porous breath figure films can also be seen. Previous works indicate that, in general, copolymers with large hydrophilic blocks produce poorly ordered structures because the interfacial tension tends to decrease and, consequently, the coalescence of the water droplets increases [ 29 , 37 , 39 ]. However, in this case, relatively ordered porous arrays are obtained for all the blends, using both types of antimicrobial copolymers with large cationic segments as additives. Table 1 shows the quantitative evaluation of the order obtained by using Voronoi polygon construction on low-magnification SEM images. The images were processed and analyzed by the software ImageJ to calculate the conformational entropy, which is compared with the entropy for an ideal hexagonal array (S = 0) and a randomly organized array (S = 1.71) [ 40 ]. The large entropies obtained between 1.17 and 0.86 indicate relatively poorly ordered arrays, although these values are also substantially less than S = 1.71 for random packaging. It has to be mentioned that they are typical values for breath figures made from water-miscible solvent such as THF [ 41 , 42 , 43 ]. Concerning the pore size, highly homogeneous pore diameters can clearly be seen for all the samples in SEM images. When the different films are compared, in general, higher diameters are obtained in films containing the copolymer quaternized with butyl, PS 54 - b -PTTBM-B 44 ( Figure 2 a–c), which is more hydrophobic than PS 54 - b -PTTBM-M 44 quaternized with methyl groups. Nevertheless, the differences are slight, as seen in Table 1 , which summarizes the mean pore sizes of all prepared porous films, determined by measuring at least 100 pores from the SEM images. Additionally, when the concentration of both copolymers incorporated into the films is varied from 3 to 9 wt %, a slight influence is noted in the pore size, which decreases when the content of copolymer increases. In general, we can conclude that as the hydrophilicity of the system is augmented, either by the use of more hydrophilic copolymer or by the use of more percentage of the cationic copolymer, the size of the pores decreases. It is well known in the breath figures approach that amphiphilic structures help to stabilize the water droplets condensed at the surface of the polymeric solution; thus, in polymeric blends, the content of amphiphilic copolymers significantly influences the porous structures [ 22 ]. As the content of copolymer increases in the blend, more droplets can be stabilized and, therefore, more and smaller pores can finally be formed at the surface [ 22 , 44 ]. The surface wettability of the films and, then, their contact with culture media mainly depends on both the chemical functionality and the roughness of the surface. The water contact angle values of obtained films were found to be ~120° for all the samples measured, independent of the copolymer content. However, as the cationic copolymer content increases in the sample, so does the hydrophilicity, and the contact angle should decrease. Therefore, the roughness of the sample, as expected, also contributes to the wettability of the films [ 45 , 46 ]. Table 1 summarizes the Wenzel roughness factor, r f , defined as the ratio between the actual and the projected areas of the surface [ 47 ]. This factor is equal to one for flat surfaces and is greater than one for rough surfaces. It is observed that the roughness of the films increases with the content of the copolymers, and in films containing the copolymer quaternized with methyl iodide. Pore diameter slightly decreases with the content of the cationic copolymer, but at the same time, pore density also increases, which contributes to the augmentation of the roughness. These contrary contributions to wettability, chemical functionality and roughness could be the reason for the similar contact angles values found in the films. Therefore, in principle, microbial contact with the surface would be rather similar for all the samples. The antimicrobial activity of the prepared breath figure films was evaluated against S. aureus Gram-positive bacteria and the fungi C. parapsilosis as model microbes, since they are common pathogens responsible of many nosocomial infections. The shake flask method [ 36 ] was employed to quantify the antimicrobial activity of the films under dynamic contact conditions. Table 2 summarizes the cell killing percentage in microbial medium in contact with the films for 24 h, and then the growth in agar plates for 24 h and 48 h for bacteria and fungi, respectively. The cell killing percentages were expressed with respect to control experiments in which the microbial reduction was null (experiments performed with films prepared from commercial PS, 0 wt % of copolymers, and without any films). It can be seen that all films exhibit high killing efficiency against S. aureus bacteria, with a reduction of more than 99.99% in the culture medium. On the other hand, moderate activity was found against C. parapsilosis fungi, with reduction of up to 90% for contents of copolymer higher than 6%. It is worth mentioning that these films present relatively high antimicrobial activity even with very low content of cationic copolymer; films containing only 6 wt % copolymers can reduce 99.99% of S. aureus and 90% of C. parapsilosis exposure to the films. Thus, these results reveal that the preparation method provides films with enough accessible active groups at the surfaces to kill the microorganisms by surface contact, even when low amounts of copolymer are incorporated in the film. Remarkably, these breath figure films provide better efficiencies than flat films prepared directly from the copolymer solution; that is, 100 wt % of PS 54 - b -PTTBM-B 44 , PS 54 - b -PTTBM-M 44 [ 16 ]. These findings demonstrate the importance of the surface roughness on the antimicrobial activity of contact-active films, which allows the use of very low amounts of antimicrobial component in the coating while maintaining excellent biocidal activity."
} | 4,242 |
35771209 | PMC9541195 | pmc | 4,784 | {
"abstract": "Abstract Spatial synchrony is a ubiquitous and important feature of population dynamics, but many aspects of this phenomenon are not well understood. In particular, it is largely unknown how multiple environmental drivers interact to determine synchrony via Moran effects, and how these impacts vary across spatial and temporal scales. Using new wavelet statistical techniques, we characterised synchrony in populations of giant kelp Macrocystis pyrifera , a widely distributed marine foundation species, and related synchrony to variation in oceanographic conditions across 33 years (1987–2019) and >900 km of coastline in California, USA. We discovered that disturbance (storm‐driven waves) and resources (seawater nutrients)—underpinned by climatic variability—act individually and interactively to produce synchrony in giant kelp across geography and timescales. Our findings demonstrate that understanding and predicting synchrony, and thus the regional stability of populations, relies on resolving the synergistic and antagonistic Moran effects of multiple environmental drivers acting on different timescales.",
"introduction": "INTRODUCTION A fundamental feature of population dynamics is spatial synchrony, the tendency for populations in different locations to exhibit correlated fluctuations over time (Moran, 1953 ). Spatial synchrony (hereafter, ‘synchrony’) is ubiquitous, having been observed in a wide range of taxa and over scales of centimetres to thousands of kilometres (Liebhold et al., 2004 ). Synchrony is important to population dynamics because it influences regional population persistence, stability, and resilience. Local population fluctuations (those of populations in different locations) that are asynchronous compensate for each other, whereas those that are synchronous reinforce each other, increasing population variance at the regional scale. Hence, strong synchrony increases temporal variability of regional abundance, which can reduce stability and increase extinction risk (Descamps et al., 2013 ; Hanski & Woiwod, 1993 ; Heino et al., 1997 ; Ojanen et al., 2013 ). Moreover, these effects can cascade to community dynamics and biodiversity (Cattadori et al., 2005 ; Haynes et al., 2009 ; Kent et al., 2007 ; Satake et al., 2004 ; Walter et al., 2020 ; Walter, Shoemaker, et al., 2021 ). Due to its pervasiveness and significance, understanding the patterns, causes, and consequences of synchrony is a key goal in ecology and its applications in conservation (Earn et al., 2000 ; Tack et al., 2015 ), agriculture (Sheppard et al., 2016 ; Walter et al., 2020 ), forestry (Haynes et al., 2018 ; Peltonen et al., 2002 ), wildlife management (Post & Forchhammer, 2002 , 2004 ), and epidemiology (Earn et al., 1998 ). Despite the importance of synchrony, three major aspects remain poorly understood: (1) Populations may be synchronised to different extents on different timescales (i.e. periods of fluctuations, such as annual or decadal) or during specific, transient periods (Keitt, 2008 ; Vasseur et al., 2014 ; Walter et al., 2020 ; Walter, Hallett, et al., 2021 ), but traditional approaches often ignore or misidentify such temporal complexity (Anderson et al., 2019 , 2021 ; Defriez et al., 2016 ; Desharnais et al., 2018 ; Sheppard et al., 2016 , 2019 ). (2) Synchrony can differ regionally, but most investigations overlook geographical patterns in synchrony and their underlying drivers (Anderson et al., 2019 ; Koenig et al., 2017 ; Walter et al., 2017 , 2022 ). (3) The relative influence of multiple drivers of synchrony and—most importantly for this study—their interactions are still understudied (Ranta et al., 1995 ; Sheppard et al., 2019 ; Walter et al., 2017 ) outside of laboratory experiments with microorganisms (Duncan et al., 2015 ; Fox et al., 2011 ; Thompson et al., 2015 ; Vogwill et al., 2009 ) and a few well‐described real populations (e.g. defoliating moths; Walter et al., 2017 ; Haynes et al., 2019 ). Spatially correlated environmental fluctuations can synchronise populations across space in a phenomenon known as the ‘Moran effect’ (Moran, 1953 ). In theory, many environmental processes can induce Moran effects concurrently, but the separate and combined effects of multiple Moran drivers, as well as whether these differ across spatial and temporal scales, are little studied. Recently, Sheppard et al. ( 2019 ) demonstrated for marine phytoplankton that drivers of synchrony can interact synergistically, producing more synchrony than would be expected through independent additive effects. It remains unknown how widespread and important interactive Moran effects are, or whether antagonistic Moran interactions can dampen synchrony, but it is reasonable to suspect that interactions are common because most species are influenced by multiple environmental drivers, which are often spatially autocorrelated. Resolving these gaps in understanding is urgent in light of accelerating global change. Changes in synchrony are associated with climatic variation (Allstadt et al., 2015 ; Cattadori et al., 2005 ; Hansen et al., 2013 ; Kahilainen et al., 2018 ; Ong et al., 2016 ; Post & Forchhammer, 2002 , 2004 ) and recent studies suggest that some systems are becoming more or less synchronous in association with climate trends (Defriez et al., 2016 ; Di Cecco & Gouhier, 2018 ; Koenig & Liebhold, 2016 ; Ojanen et al., 2013 ). The degree to which climate shifts cause changes in synchrony remains underexplored but is now recognised as likely to be important (Hansen et al., 2020 ; Özkan et al., 2016 ). Changing interactions between Moran drivers have the potential to be a crucial but unrecognised means by which climate change alters population synchrony and stability. Here, we examined spatial and temporal patterns of synchrony in a broadly distributed marine foundation species, giant kelp Macrocystis pyrifera , using a 33‐year spatial time series of canopy biomass spanning >900 km of coastline in California, USA. The outstanding availability of biological and oceanographic datasets in our study region enabled us to overcome the typical challenges to resolving the patterns and drivers of synchrony. We used wavelet techniques to quantify time‐ and timescale‐specific patterns of synchrony, and how these varied geographically. We then applied newly developed multivariate wavelet regression models to investigate how disturbance (storm‐driven waves) and resources (seawater nutrients) act individually and interactively to structure giant kelp synchrony, and whether the importance of these forces varies across timescales and geography. In doing so, we accomplish three goals: (1) quantify the timescale structure of giant kelp synchrony and identify the main causes of synchrony; (2) demonstrate that multiple environmental factors can combine to produce timescale‐specific synchrony via synergistic or antagonistic Moran effects, thereby providing evidence that the new mechanism of interactions between Moran effects is potentially widespread; and (3) show that the influence of individual and interactive synchrony drivers can differ strongly across geography and timescales.",
"discussion": "DISCUSSION Despite the ubiquity and importance of spatial synchrony, resolving how multiple factors interact to determine synchrony across scales in time and space has been a long‐standing challenge (Liebhold et al., 2004 ; Moran, 1953 ). Our study of long‐term giant kelp canopy biomass dynamics across California helps narrow this knowledge gap by supporting three major conclusions about environmentally induced synchrony (the Moran effect): (1) Synchrony differs greatly across timescales and geography commensurate with differences in multiple environmental drivers affecting both population decline and growth, such as disturbance and resources. (2) Substantial interactions occur between Moran drivers, which can be synergistic (producing additional synchrony) or antagonistic (reducing synchrony from what would otherwise be expected). (3) The influence of Moran drivers and their interactions differ strongly across timescales and geographical regions, reinforcing the importance of studying synchrony using timescale‐ and geography‐specific approaches (e.g. Defriez et al., 2016 ; Sheppard et al., 2016 ; Walter et al., 2017 , 2022 ). These findings represent an important advance because, to our knowledge, interactions among Moran drivers of synchrony have been identified only once previously (Sheppard et al., 2019 ). Our results also comprise the first example in which disturbance and resources interact to structure synchrony, and the first empirical evidence that Moran effects can interact antagonistically to produce less synchrony than would be expected through additive effects. How can we intuitively understand the mechanism of interactions between Moran drivers? And how can interactions be synergistic on some timescales and simultaneously antagonistic on others, as we found for giant kelp forests in central California? First, note that large waves have immediate negative effects on kelp biomass, whereas elevated nutrients have positive effects that are delayed by one quarter (Appendix S2 ) because it takes several weeks to months for new kelp recruitment and growth to reach the water surface and achieve densities detectable from satellites (Bell & Siegel, 2022 ; Schiel & Foster, 2015 ). In central California, waves tend to achieve their annual maximum in the winter, whereas nutrients achieve their annual maximum in spring (Bell, Cavanaugh, Reed, & Siegel, 2015 ). Thus, annual wave effects are negative and occur in winter, whereas annual nutrient effects are positive and manifest (due to growth delays) in summer. So, nutrient and wave effects can temporally align and reinforce each other in producing large annual oscillations: large increases in kelp canopy biomass in summer due to replete nutrients can be followed by major crashes in winter due to large waves. In this scenario, positive interactions between wave and nutrient Moran effects on annual timescales (17%; Table 1 ) can occur, whenever years with large waves coincide with years with replete nutrients in other locations, a likely common occurrence because both phenomena are related to oceanographic climate. If a large‐wave year in one location coincides with a high‐nutrient year in another location, both locations will tend to have bigger annual kelp oscillations in that year, accentuating annual synchrony. However, sub‐annual timing delays and seasonal differences become negligible when considering long interannual timescales (4–10 y). On such timescales, large‐wave years and nutrient‐replete years counteract each other, so that whenever large‐wave years coincide with nutrient‐replete years in other locations, antagonistic interactions between Moran effects are observed (−44% in central California; Table 1 ). If a multiannual period of larger‐than‐average waves in one location coincides with a multiannual period of higher‐than‐average nutrients in another location, the interaction between waves and nutrients will tend to reduce kelp canopy biomass in the first location but augment it in the second location, reducing interannual synchrony. Synergistic interactions on annual timescales are not observed in southern California probably because, in that region, variations in waves and nutrients are not as strongly seasonal as in central California and may involve different time lags (Bell, Cavanaugh, Reed, & Siegel, 2015 ). Using a simple model, Sheppard et al. ( 2019 ) further illuminated the mechanisms of interacting Moran effects, showing that the interaction between the effects of two drivers varies in relation to the phase relationship between their effects. However, more analytical modelling is needed to advance a general theory of interacting Moran effects and their effects on synchrony. We hypothesise that interactions between Moran effects are common and thus argue they should be considered when studying climate effects on synchrony. Both our investigation of kelp forest synchrony and a recent study of synchrony in marine phytoplankton (Sheppard et al., 2019 ) revealed important interactions between Moran drivers. In our study, interactions between disturbance and resources (nutrients) amplified or dampened kelp synchrony at certain timescales; in Sheppard et al. ( 2019 ), synergistic interactions between temperature and predators (grazing zooplankton) enhanced phytoplankton synchrony. We suspect that interacting Moran effects are widespread because multiple, interrelated environmental drivers influence most ecosystems. Hence, future research should quantify between‐variable synchrony for environmental drivers, systematically assess the commonness of interactions between Moran effects, and resolve the potential for climate change to alter such interactions to affect population synchrony and stability (Hansen et al., 2020 ). Differences in the geographical setting and time period of prior investigations have contributed to uncertainty about the relative importance of disturbance and resources in structuring kelp forest ecosystems (e.g. Broitman & Kinlan, 2006 ; Dayton et al., 1992 , 1999 ; Parnell et al., 2010 ; Reed et al., 2008 , 2011 , 2016 ). Our large‐scale, long‐term study helps clarify this debate by supporting the idea that waves and nutrients work together to synchronise giant kelp canopy biomass via influences on kelp loss, recovery, and growth, but that the strength of these synchronising forces varies over space and timescale. Large waves cause massive giant kelp mortality and canopy loss (Bell, Cavanaugh, Reed, & Siegel, 2015 ; Graham et al., 1997 ; Reed et al., 2011 ; Young et al., 2016 ), and sustained low nutrients reduce kelp recruitment and growth (Deysher & Dean, 1986 ; Hernández‐Carmona et al., 2001 ; Kopczak et al., 1991 ; Zimmerman & Kremer, 1984 , 1986 ); both processes induce kelp synchrony via the Moran effect because they are spatially autocorrelated (Bell, Cavanaugh, Reed, & Siegel, 2015 ). Our results also build substantially upon earlier results showing that increasing geographical separation leads to exponential decreases in synchrony (Cavanaugh et al., 2013 ) by revealing that giant kelp is more synchronous in central than southern California across all timescales, but particularly at annual timescales. These conclusions reinforce prior work demonstrating broad, consistent seasonality of giant kelp canopy biomass in central California—where seasonal wave disturbance (Bell, Cavanaugh, Reed, & Siegel, 2015 ; Reed et al., 2011 ) and upwelling of nutrient‐rich water (Huyer, 1983 ) are more intense—and a lack of consistent giant kelp seasonality in southern California (Bell, Cavanaugh, & Siegel, 2015 ). Endogenous circannual rhythms related to predictable annual changes in day length (photoperiod) may explain some observed annual synchrony in kelp canopy biomass, but strong geographical differences in the seasonality of kelp biomass (Bell, Cavanaugh, & Siegel, 2015 ) and the consistency of annual fluctuations (this study) suggest that photoperiod per se probably plays a relatively limited role. Like many plants and macroalgae (Jackson, 2009 ; Lüning, 2005 ), giant kelp exhibits annual synchrony in the initiation of reproduction (winter spore production and release; Reed et al., 1997 ). However, pronounced variation in environmental conditions over space and time can reduce the spatial synchrony and annual consistency of recruitment to adult sporophytes (e.g. Dayton et al., 1992 ; Deysher & Dean, 1986 ; Reed et al., 2008 ; Reed & Foster, 1984 ). Further study may clarify the role of photoperiod in structuring synchrony across multiple processes (e.g. reproduction, recruitment, growth) that contribute to annual fluctuations in kelp canopy biomass, and how these differ geographically (Bell & Siegel, 2022 ). Additional research may also reveal how the global generalisability of our results is mediated by giant kelp phenotypic plasticity to diverse oceanographic conditions (Demes et al., 2009 ). For instance, disturbance may play a more limited role in inducing synchrony among wave‐sheltered populations in southern Chile that exhibit an annual life cycle (Graham et al., 2007 ). We found that the North Pacific Gyre Oscillation (NPGO) is a dominant driver of kelp synchrony at interannual timescales, particularly those fluctuations >4 years. NPGO variation corresponds with large‐scale periodic strengthening of coastal nutrient delivery (Di Lorenzo et al., 2008 , 2013 ; Pennington & Chavez, 2018 ). Thus, it is likely that the power of NPGO in predicting giant kelp synchrony at long interannual timescales (and more modestly at short interannual timescales) manifests mechanistically through nitrate variability. These findings reinforce the important relationship between the NPGO and giant kelp canopy biomass (Bell, Cavanaugh, Reed, & Siegel, 2015 ), and help clarify the mechanisms by which NPGO variability structures kelp forest dynamics. However, some caution is warranted in interpretation of the strength of synchrony explained by NPGO and its local manifestations (e.g. nutrients) at long interannual timescales due to the potential for model overfitting with relatively few temporal cycles (i.e. about three decadal cycles in our 33‐year time series). Because the NPGO fluctuates predominantly on roughly decadal timescales (Di Lorenzo et al., 2008 , 2013 ), it is not surprising that it was unrelated to annual kelp synchrony. We did not examine other climate indices, such as the El Niño Southern Oscillation or the Pacific Decadal Oscillation, because they are not highly correlated with overall giant kelp biomass dynamics (Bell et al., 2020 ; Bell, Cavanaugh, Reed, & Siegel, 2015 ; Cavanaugh et al., 2011 ). However, the strongest El Niño events on record (1982–1983; 1997–1998; 2015–2016) were associated with widespread giant kelp declines (Cavanaugh et al., 2019 ; Edwards, 2019 ). More research is needed, but it may be that typical ENSO variability is not a major driver of synchronous increases or decreases in giant kelp, but extreme ENSO events are important. Our models could not explain all observed kelp synchrony on any timescale in either region, but predictive power was relatively modest on annual timescales in southern California and on short interannual timescales in both central and southern California. Nevertheless, the positive contributions to synchrony by waves and nutrients, and their antagonistic relationship on multiyear timescales were detectable in the short interannual timescale band. This supports our conceptual model that kelp synchrony is depressed by the correlated but opposing effects of waves and nutrients. Residual synchrony, not explained by these factors, may be due to variables unmeasured in our study, such as species interactions (herbivory, competition) and propagule dispersal. Herbivory may be an important driver of kelp synchrony because grazing sea urchins can denude entire kelp forests (Dayton et al., 1984 ; Dean et al., 1984 ; Filbee‐Dexter & Scheibling, 2014 ), while synchronous sea urchin mortality can reverse state changes (Ebeling et al., 1985 ; Pearse & Hines, 1979 ). Sea urchin recruitment in California is highly synchronous on sub‐regional scales (15–100 km; Okamoto et al., 2020 ), although it remains unclear whether synchronous urchin recruitment leads to synchronous grazing pressure. On the other hand, urchin grazing intensity can vary greatly over short distances (Harrold & Reed, 1985 ; Rennick et al., 2022 ) and this may reduce the strength of Moran effects (Bell, Cavanaugh, Reed, & Siegel, 2015 ). Synchrony may also be diminished by spatial variation in competition between early life stages of giant kelp and benthic algae that suppress kelp recruitment (Beckley & Edwards, 2021 ; Reed, 1990 ). Further study of potential synchronising species interactions in kelp forests and possible dependency on abiotic factors (e.g. nutrients, waves) may help explain geographical variation in the synchrony of giant kelp recovery following catastrophic events (Cavanaugh et al., 2019 ; Edwards, 2004 ). Dispersal is another widely accepted mechanism of synchrony (Abbott, 2011 ; Duncan et al., 2015 ; Gouhier et al., 2010 ; Luo et al., 2021 ) and we argue above that interacting Moran effects may be widespread. However, interactions between dispersal and Moran drivers remain unknown beyond simple theoretical models (Kendall et al., 2000 ). Giant kelp populations are demographically linked by the passive dispersal of spores by ocean currents, and this process is important to patch colonisation and extinction (Castorani et al., 2015 , 2017 ; Reed et al., 2006 ; Young et al., 2016 ). At scales of hundreds of metres, synchronous recruitment after mortality events can produce cohorts with similar age structure (Dayton et al., 1992 ). Because adult giant kelp sporophytes typically live for 2–3 years (Reed et al., 2008 ; Rosenthal et al., 1974 ), the growth and senescence of these cohorts may cause short‐interannual synchrony of kelp canopy biomass in areas not exposed to annual wave disturbance (Bell & Siegel, 2022 ; Rodriguez et al., 2013 ). On the other hand, the synchronising effect of dispersal may be interrupted by local processes that inhibit recruitment, such as grazing (Dean et al., 1984 ; Leonard, 1994 ), competition (Beckley & Edwards, 2021 ; Reed & Foster, 1984 ), or nutrient limitation (Deysher & Dean, 1986 ; Hernández‐Carmona et al., 2001 ). The extent to which dispersal synchronises giant kelp over large spatial scales, and whether this effect interacts with local or regional drivers, have not been established. Our results suggest additional promising directions for future research. Because giant kelp is a foundation species that exerts a strong influence over productivity and biodiversity in kelp forests (Castorani et al., 2018 , 2021 ; Lamy et al., 2020 ; Miller et al., 2018 ) and sandy beaches (Dugan et al., 2003 ; Schooler et al., 2017 , 2019 ), we hypothesise that synchrony of giant kelp and other foundation species may cascade to the community through species interactions (Lee et al., 2020 ; Morton et al., 2016 ). This possibility may provide a promising avenue for future research because the cascading effects of population synchrony on community and ecosystem dynamics have been explored in very few systems (Cattadori et al., 2005 ; Haynes et al., 2009 ; Kent et al., 2007 ; Satake et al., 2004 ; Turkia et al., 2020 ). Moreover, because synchrony is related to population instability, improved understanding of the patterns and drivers of synchrony in foundation species may help predict how environmental changes influence spatial and temporal ecosystem stability and its ecological services (Kremen, 2005 )."
} | 5,758 |
29467721 | PMC5808251 | pmc | 4,785 | {
"abstract": "The factors leading to changes in the organization of microbial assemblages at fine spatial scales are not well characterized or understood. However, they are expected to guide the succession of community development and function toward specific outcomes that could impact human health and the environment. In this study, we put forward a combined experimental and agent-based modeling framework and use it to interpret unique spatial organization patterns of H1-Type VI secretion system (T6SS) mutants of P . aeruginosa under spatial confinement. We find that key parameters, such as T6SS-mediated cell contact and lysis, spatial localization, relative species abundance, cell density and local concentrations of growth substrates and metabolites are influenced by spatial confinement. The model, written in the accessible programming language NetLogo, can be adapted to a variety of biological systems of interest and used to simulate experiments across a broad parameter space. It was implemented and run in a high-throughput mode by deploying it across multiple CPUs, with each simulation representing an individual well within a high-throughput microwell array experimental platform. The microfluidics and agent-based modeling framework we present in this paper provides an effective means by which to connect experimental studies in microbiology to model development. The work demonstrates progress in coupling experimental results to simulation while also highlighting potential sources of discrepancies between real-world experiments and idealized models.",
"introduction": "Introduction Spatial organization has a strong influence on the development and dynamics of biological systems (Kreft et al., 1998 ; Lardon et al., 2011 ; Halsted et al., 2016 ; Hansen et al., 2016 ; McNally et al., 2017 ; Timm et al., 2017 ). The factors leading to changes in organization of multicellular assemblages at fine spatial scales are not well characterized or understood, however, they are expected to guide the succession of community development and function toward specific outcomes (Liu et al., 2009 ; Cline and Zak, 2015 ; Dini-Andreote et al., 2015 ). The organization of distinct microbial populations can be shaped by physical and chemical processes, and affect important activities such as antibiotic resistance, efficient energy conversion, C and N cycling and quorum sensing (Ginovart et al., 2005 ; Gras et al., 2010 , 2011b ; Sahari et al., 2014 ; Wang and Ma, 2014 ; Koonin and Wolf, 2015 ; Biteen et al., 2016 ). Microbe-microbe interactions can also depend on direct and indirect competition for resources between different community members (Kreft, 2004 ; Hellweger et al., 2008 ; Borenstein et al., 2015 ; McNally et al., 2017 ). The microscale/local transport of essential microbe-derived metabolites and cell-to-cell competition are likely to be strongly influenced by spatial confinement and individual cell behavior in the environment (Lardon et al., 2011 ; Pintelon et al., 2012 ; Vogel et al., 2015 ; McNally et al., 2017 ). Consequently, investigating the complexity of these processes and emergence of unique behaviors requires the combination of experimental and computational tools that can be used to explore the impact of spatial organization, while correlating individual microbial behavior and interactions to specific outcomes (Dini-Andreote et al., 2015 ; Zhu et al., 2015 ; Hansen et al., 2016 ). Cells can compete directly with surrounding species through physical contact, and in more specialized cases, are capable of transferring toxic effector proteins to susceptible cells. The Type VI secretion system (T6SS) is an important example of such a pathway, being responsible for the assembly of a pilus apparatus that can be used to contact neighboring cells and potentially induce cell death (Hood et al., 2010 ; Chou et al., 2012 ; LeRoux et al., 2012 ). Hood et al. ( 2010 ) showed that the H1-T6SS of Pseudomonas aeruginosa is required to direct the injection of toxins from T6SS active cells (T6SS+) into T6SS-susceptible cells (T6SS−) that lack immunity. Other important secretion systems such as H2- and H3-T6SS in P . aeruginosa direct toxins preferentially to eukaryotic cells. However, because the H1-T6SS toxin is preferentially directed toward other bacteria, it is particularly well suited for studies of contact-mediated interactions between neighboring and competing prokaryotes (Mougous et al., 2006 ; Sana et al., 2016 , 2017 ). T6SS interactions in mixed microbial populations also play an important role in the regulation of more complex biological processes and microbial community dynamics (Russell et al., 2014 ; Verster et al., 2017 ). For instance, the T6SS interactions occurring amongst commensal bacteria in the mammalian gut microbiome have been shown to modulate community composition and interactions, as well as provide a mechanism for defending commensal bacteria from invading pathogens (Hecht et al., 2016 ). Furthermore, these T6SS interactions are highly active and prevalent, where > 10 9 T6SS firing events (i.e., predicted pilus injections) min −1 g −1 colonic contents can occur and nearly 25% of human gut microbiota have been shown to encode a T6SS pathway (Wexler et al., 2016 ; Sana et al., 2017 ). Using two-member communities as a model system of T6SS interactions in the laboratory, Borenstein et al. ( 2015 ) demonstrated that established colonies of T6SS− Escherichia coli could survive contact with T6SS+ Vibrio cholerae . Agent-based modeling (ABM) simulations further showed that T6SS− cells could survive T6SS+ attack when placed in situations of nutrient limitation and relatively slow growth rates, and could even outcompete the T6SS+ cells, as long as T6SS− cells were able to establish microcolonies within the mixed community (Borenstein et al., 2015 ). These results demonstrate the importance of spatial confinement and local organization on cell growth and survival. Thus, competition between neighboring microbial cells and spatial confinement are expected to drive changes in cell assemblage and organization (Borenstein et al., 2015 ; Halsted et al., 2016 ; Hansen et al., 2016 ). Numerous advances in our understanding of cell-to-cell behavior and interactions at fine spatial scales have stemmed from the use and development of nano/micro-fabricated platforms (Wang et al., 2013 ; Yamazaki et al., 2014 ; Swennenhuis et al., 2015 ; Xue et al., 2015 ; Hansen et al., 2016 ; Zhang et al., 2016 ; Timm et al., 2017 ; Yeh et al., 2017 ). Timm et al. ( 2017 ) used a microwell array platform to study the contact-mediated T6SS interactions of P . aeruginosa . The microwell array platform enabled high-resolution and high-throughput imaging of mixed T6SS+ and T6SS− cells growing under spatial confinement within microwells, with well diameters ranging from 20 to 100 μm and 5 μm depth. Interpreting the results of these cell-to-cell interactions with simplified analytical models of overall growth within each well becomes challenging and potentially unreliable when trying to capture the complex interactions reflected by spatial organization of microorganisms within the microwells. Alternatively, ABM simulations can capture how changes at the level of individual microbial interactions lead to changes observed at the community and microcolony levels. In conjunction with laboratory experiments ABM simulations can be used to infer and test important growth parameters that impact spatial organization within colonies (Borenstein et al., 2015 ). In this study, we have developed an ABM model around experimental data obtained from a microwell array platform. We use the model to interpret spatial organization patterns of P . aeruginosa mutants growing under spatial confinement. The novelty of our approach relies on the high throughput nature of both the experiment and ABM simulations, which allows investigating how the initial ratio of community member abundances, initial growth location and T6SS interactions affect spatial organization during growth. The model is written in the language NetLogo (Wilensky(1999), NetLogo, http://ccl.northwestern.edu/netlogo/ ; Center for Connected Learning and Computer-Based Modeling, Northwestern University, Evanston, IL) and is linked to a computational framework that permits submitting many calculations in parallel for different initial parameters, where each combination of parameters can be conceptualized to represent a micro-environment of interest. The ABM model has been deployed in the Compute and Data Environment for Science, CADES ( http://cades.ornl.gov/ ), which also stores the relevant experimental data used during fitting routines. We find that key parameters, such as spatial constraints, local concentrations of growth substrates/metabolites and associated rate constants alter the impact of P . aeruginosa Type VI secretion activity on the spatial organization of cells in confined environments.",
"discussion": "Discussion The T6SS of P. aeruginosa is an important biological model for understanding how cell-to-cell contact directs the succession and organization of microbial communities (Robinson et al., 2009 ; Hood et al., 2010 ; Sarris and Scoulica, 2011 ; LeRoux et al., 2012 ; Das et al., 2013 ). As mentioned in the introduction, T6SS interactions play a significant role in the regulation of microbiomes, which has important implications for biomedical and pathogen research, particularly for understanding mammalian gut microbiomes, and also environmental biogeochemistry relevant to native microbial interactions with plants and soil. However, the factors leading to changes in organization of microbial cells at fine spatial scales, driven by T6SS interactions, are not well characterized or understood. Recent laboratory investigations and ABM simulations indicated that established T6SS− colonies of Escherichia coli could persist during cell-to-cell interactions with Vibrio cholerae T6SS+ cells (Borenstein et al., 2015 ). The results of Timm et al. ( 2017 ) further suggested that spatial confinement, as well as T6SS activity between growing effector and susceptible P. aeruginosa mutants, could potentially direct cell organization in micro-colonies and affect the survival of susceptible cells. In the present study, building upon additional analysis of the complete dataset of Timm et al. ( 2017 ), and in combination with ABM simulations, we provide supporting evidence that both spatial confinement and T6SS activity can lead to changes in the organization and persistence of P. aeruginosa . We found that discrete zones of clearing occurred around T6SS− cell assemblages during co-growth with T6SS+ cells in ABM simulations (Figure 1 ; see also Supplementary Movie 2 ). This cell-to-cell organization of T6SS− cells, surrounded by a zone of clearing, is consistent with T6SS-induced cell lysis at the boundary between both P. aeruginosa mutants (Hood et al., 2010 ; Borenstein et al., 2015 ). This zone of clearing provides a mechanism of P. aeruginosa cellular organization, as previously observed in Si-based microwell arrays (Timm et al., 2017 ). We speculate that during growth of both mutant strains, these buffer zones can occur randomly during growth, perhaps forming safe-pockets for susceptible cells to continue growing, and can become more defined as microcolonies of both species expand and interact at their outer boundaries. Fitting of the complete experimental dataset indicated that starting at the apparent peak in cell growth for both strains, a general decay in T6SS+ GFP signal intensities began, while T6SS− m-Cherry intensities subsequently remained more persistent over time (Figure 2 ). Borenstein et al. ( 2015 ) demonstrated that more-established microcolonies of T6SS-susceptible cells can potentially survive T6SS attack, which helps explain the persistence of susceptible P. aeruginosa mutants as deduced from the fluorescence intensities taken from our experimental data. We also observed that T6SS+ cells could outgrow a well once the interior of the well cavity had become nearly filled by growing cells (Supplementary Movie 1 ); this may explain, to some extent, the sharp decay phase generally observed for T6SS+ GFP intensities (Figure 2 ). We found that the initial seeded cell density per well on the experimental platform decreased as a function of increasing well diameter between 15 and 100 μm (Figure 4 ), but cell density did not have an apparent effect on cell organization during different growth simulations, which is consistent with the results of Timm et al. ( 2017 ) that demonstrated microcolony formation across all well sizes between 20 and 100 μm diameters. The correlation between initial cell density after seeding and well size likely reflects the preparation of the experimental microwell platform. For instance, following the experimental cell seeding step (Timm et al., 2017 ): (1) slight drying of the aqueous culture media before contact with the nutrient agarose cover; (2) difficulty rinsing cells from smaller diameter wells during the final water rinse step; or (3) a larger side-wall to floor area ratio per well could have affected initial cell densities such that smaller wells were more densely packed than larger wells, particularly at well edge boundaries. However, qualitatively, we found that spatial organization into distinct T6SS mutant assemblages during experimental and simulated growth was not strongly influenced by cell density or close packing. Future quantitative analysis of assemblage size and spacing for different well sizes may reveal a more defined mechanism. Densely packed cell assemblages have been shown in previous studies to follow similar biological-phase separation where distinct microcolony formation is favored regardless of cell-to-cell density in spatially confined environments (Tolker-Nielsen and Molin, 2000 ; Berk et al., 2011 ; Borenstein et al., 2015 ; Cutler et al., 2015 ; McNally et al., 2017 ). We generally found the impact of well size to be negligible for the size ranges explored in experiments, see Figures 5A,B . Well size did not have a significant impact on overall growth rates per initial cell number or maximum growth rate per initial cell number. This was unexpected. Indeed, in well diameters < 25 μm, competition for resources and cell-to-cell interactions would have been expected to suppress T6SS− growth. Reductions in spatial confinement within larger wells would, in principle, allow T6SS− cells to grow more efficiently with increasing well diameters, reducing the likelihood of encountering T6SS+ because of the lower seeding densities and more available area. In wells of 45 μm diameter and greater, at much lower initial densities (Figure 4 ), the individual T6SS− colonies may have had the potential to develop with less competition and become more established before interacting with the more aggressive mutant strain. In this case, the perimeter of T6SS-interactions around a colony would be overshadowed by the more established interior of each mutant colony. In other words, with larger wells above 40 microns, T6SS-killing should have become secondary to the size of mutant colonies by the time they interact at their edges. Clearly, a more systematic study of micro-colony size and distribution is needed to understand these results. The behavior of T6SS+ cells across well sizes displays variation in the data that makes it difficult to draw specific conclusions about T6SS+ growth as a function of well size. Finally, average a ′, μ′ and τ for each well size vs. the entire well size distribution were also calculated, see Figure 7 ; the results obtained in a mixed population of T6SS+ and T6SS− cells were compared to those obtained in control experiments comprising only one cell type. As seen in Figure 7D , for the control experiments, the average a ′, μ′ and τ of T6SS− cells are practically insensitive to the well size, whereas the same data for T6SS+, Figure 7C , shows variation and an increase at larger well sizes. In mixed populations, the data for T6SS− cells shows a similar trend, Figure 7B , although the error bars are larger, illustrating the interactions with T6SS+ cells. The data for T6SS+ cells, Figure 7A , shows even larger error bars, which is surprising because these cells should not be negatively impacted by the presence of T6SS− strains. As mentioned above, inspection of the data reveals that on some occasions T6SS+ cells can outgrow/leave the well boundaries (Supplementary Movie 1 ). We believe this is one of the primary reasons for the large variation observed in Figures 7A,B . Consequently, whether or not T6SS+ cells outgrow or escape the wells should also affect the growth of T6SS− cells remaining within the same wells. Figure 7 Growth parameters vs. well size. Average a ′ (red), μ′ (blue) and τ (green) growth parameters over all the T6SS+:T6SS– initial ratios across wells of different size. (A) T6SS+ average growth parameters as a function of well size when mutants are grown together. (B) T6SS– average growth parameters as a function of well size when mutants are grown together. Error bars represent the 95% confidence interval for the linear fit demonstrated in Figure 3 . (C,D) Panels show equivalent parameters in monoculture. In this study we have developed an experimental-ABM framework that can be used to interpret unique spatial-organization patterns of P . aeruginosa cells growing under spatial confinement. The ABM model developed here, although qualitative, is capable of showing microcolony formation regardless of initial density, or whether the bacteria prefer to begin growth at the well edges, which is consistent with other recent studies that have examined different microbial species under T6SS interactions. Our model was also capable of reproducing the behavior of a ′, μ′ and τ of T6SS+ cells for a particular well size, and the same was true for T6SS− cells once the aggressiveness level of T6SS+ cells was lowered. As such, this model can be used to extract information regarding aggressiveness levels, amount of available resources, and rate of consumption of nutrients, as well as how all these variables affect the growth of the bacterial colony. Yet, there are some uncertainties that the current model does not take into account. Future work will focus on optimizing the model by identifying the most essential growth parameters and developing a more quantitative description of variables used for running the simulations. Further, the ABM model is capable of investigating 5000 wells in 30 min, and in connection with the microfluidic platform, constitutes a powerful framework to connect microbiological experiments to ABM simulations, while improving the ABM models to more accurately reproduce the experimental observations. Finally, this new microfluidic-ABM framework could be used in the future to predict the types of microcolonies that are likely to develop when different microbial species are mixed, which is expected to advance our understanding of microbial ecology at fine spatial scales, as well as mechanistically describe how microbial succession occurs in nature and shapes environments of interest."
} | 4,831 |
23748443 | PMC3710538 | pmc | 4,787 | {
"abstract": "The mammalian gut ecosystem has significant influence on host physiology 1 – 4 , but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to this ecosystem, such as through antibiotic treatment or diet, are currently interpreted at the level of bacterial phylogeny 5 – 7 . Less is known about the contributions of the abundant population of phage to this ecological network. Here, we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine fecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phage from treated mice afford aerobically cultured naïve microbiota increased resistance. Systems-wide analyses uncover post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome broadly enriches for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress."
} | 416 |
23748443 | PMC3710538 | pmc | 4,787 | {
"abstract": "The mammalian gut ecosystem has significant influence on host physiology 1 – 4 , but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to this ecosystem, such as through antibiotic treatment or diet, are currently interpreted at the level of bacterial phylogeny 5 – 7 . Less is known about the contributions of the abundant population of phage to this ecological network. Here, we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine fecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phage from treated mice afford aerobically cultured naïve microbiota increased resistance. Systems-wide analyses uncover post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome broadly enriches for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress."
} | 416 |
23748443 | PMC3710538 | pmc | 4,788 | {
"abstract": "The mammalian gut ecosystem has significant influence on host physiology 1 – 4 , but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to this ecosystem, such as through antibiotic treatment or diet, are currently interpreted at the level of bacterial phylogeny 5 – 7 . Less is known about the contributions of the abundant population of phage to this ecological network. Here, we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine fecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phage from treated mice afford aerobically cultured naïve microbiota increased resistance. Systems-wide analyses uncover post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome broadly enriches for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress."
} | 416 |
23748443 | PMC3710538 | pmc | 4,788 | {
"abstract": "The mammalian gut ecosystem has significant influence on host physiology 1 – 4 , but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to this ecosystem, such as through antibiotic treatment or diet, are currently interpreted at the level of bacterial phylogeny 5 – 7 . Less is known about the contributions of the abundant population of phage to this ecological network. Here, we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine fecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phage from treated mice afford aerobically cultured naïve microbiota increased resistance. Systems-wide analyses uncover post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome broadly enriches for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress."
} | 416 |
37279263 | PMC10268221 | pmc | 4,791 | {
"abstract": "Communication is a fundamental feature of animal societies and helps their members to solve the challenges they encounter, from exploiting food sources to fighting enemies or finding a new home. Eusocial bees inhabit a wide range of environments and they have evolved a multitude of communication signals that help them exploit resources in their environment efficiently. We highlight recent advances in our understanding of bee communication strategies and discuss how variation in social biology, such as colony size or nesting habits, and ecological conditions are important drivers of variation in communication strategies. Anthropogenic factors, such as habitat conversion, climate change, or the use of agrochemicals, are changing the world bees inhabit, and it is becoming clear that this affects communication both directly and indirectly, for example by affecting food source availability, social interactions among nestmates, and cognitive functions. Whether and how bees adapt their foraging and communication strategies to these changes represents a new frontier in bee behavioral and conservation research."
} | 279 |
38358263 | PMC10936165 | pmc | 4,792 | {
"abstract": "ABSTRACT Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here, we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR-Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55-kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the wild type (WT). This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome-to-phenome relationships. IMPORTANCE Genome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.",
"introduction": "INTRODUCTION Cyanobacteria are the most ancient and abundant oxygenic photosynthetic organisms that are largely responsible for the Earth’s viable environment ( 1 ). These photosynthetic prokaryotes have been identified as potential platforms for sustainable carbon-neutral bioproduction because of their unique ability to harvest sunlight as their sole energy source for converting greenhouse gases (carbon dioxide) into value-added chemicals. This bioprocess in theory is also economically sustainable as it enables free and ubiquitous substrates to enter the bio-economy. In comparison to other photoautotrophs, cyanobacteria have several advantages, and, therefore, efforts are ongoing to understand and develop the cyanobacterial platform as sustainable bio-factories ( 2 – 5 ). However, relatively slower growth and limited knowledge of their genomic traits as compared to heterotrophs such as Escherichia coli or yeast have restricted progress in this direction. Although the recent isolation of a few fast-growing strains has made cyanobacterial-based bioproduction more compelling and tractable than ever, concerted efforts are needed to get their productivity at par with their heterotrophic counterparts ( 6 – 10 ). Although most commonly studied cyanobacterial genomes are generally smaller in size than that of E. coli , they exhibit cryptic metabolic and regulatory features, owed likely to their photosynthetic lifestyle, unique evolutionary history, and adaptation to various unfavorable environmental conditions ( 11 ). The main aim of this study was to test the feasibility of employing the genome reduction strategy as a means to shed excess biological complexity and simplify the genome of a fast-growing cyanobacterium without compromising its desirable traits. The clade Synechococcus elongatus hosts all of the fast-growing cyanobacterial strains identified to date ( 8 ), and a genome minimization approach will be beneficial for unraveling the genome-level function and the overall metabolism of these strains, which in turn will aid their development into cell factories. The goal is not to obtain a truly minimum genome for a photosynthetic organism but rather to identify and remove genes dispensable under bioproduction-relevant conditions (high light and CO 2 ) without compromising growth and productivity. Genome streamlining is a natural evolutionary process of eliminating non-beneficial genes, since a smaller genome reduces the metabolic burden on the cell and improves fitness ( 12 – 14 ). As an engineering strategy, it has been successfully employed in model heterotrophs leading to improved fitness and performance ( 15 – 18 ). A genome reduction of 25% in E. coli led to a 1.6-fold improvement in growth rate and improved recombinant protein production ( 19 , 20 ). Similarly, genome streamlining in a Pseudomonas strain resulted in several appealing traits such as faster growth, increased biomass production, enhanced plasmid stability, and, overall, a more efficient energy metabolism ( 21 , 22 ). These studies indicate that a chassis strain with a streamlined genome avoids the unnecessary burden of replicating and expressing genetic elements that are not useful under production conditions. Reducing this unnecessary genetic burden may ensure that more cellular resources are available for the expression of heterologous pathways. By removing genes of unknown function, it also creates a chassis organism that is more fully understood and more amenable toward genetic engineering and synthetic biology efforts ( 16 ). Despite the favorable outcomes of genome minimization in heterotrophs ( 20 – 23 ), limited efforts have been made to implement such strategies in phototrophs. So far, two reports explore genome streamlining. Removing ~2% (118 kb) of the genome of Anabaena PCC 7120 has been demonstrated using the CRISPR-Cas12a system for targeted deletions, but this study did not explore the effects of these edits on strain performance ( 24 ). Deletion of several large fragments of DNA from the genome of S. elongatus PCC 7942 has been performed, producing a septuple mutant with approximately 3.8% of its genome removed. These mutants were further studied to understand the changes in the transcriptomic profile of the cells resulting from the deletions. The CRISPR-Cas12a-editing tool was used to obtain the targeted, specified, and markerless deletions in this study ( 25 ). Although Cas12a is a dynamic and versatile tool, it does not allow the flexibility to explore and identify unknown stretches of dispensable and indispensable regions in the genome of a strain. Recently, a novel Class I multi-Cas protein was commissioned for large deletions in heterotrophs. The dual helicase and exonuclease activity of Cas3 enabled large simultaneous bidirectional deletions, without the necessity of a repair template ( 26 ). Recently, in one of our studies, we have successfully commissioned an inducible CRISPR-Cas3 system in S. elongatus UTEX 2973 to truncate the light-harvesting antenna structure for maximizing fitness and productivity under specified condition ( 27 ). However, this tool has not been explored for genome streamlining in cyanobacteria. Therefore, it is of interest to investigate this Cas system and exploit its beneficial features for genome minimization of photoautotrophs. In this study, we investigate the effect of systematic reduction of dispensable regions from the genome of Synechococcus 2973, the fastest growing, high light thriving cyanobacterium that exhibits high sucrose production titer ( 6 , 10 ). We first identified five large stretches of dispensable genomic regions in this strain using the MinGenome algorithm ( 28 ) and then commissioned the novel CRISPR-exonuclease system, CRISPR-Cas3 to achieve flexible progressive large deletions. CRISPR-Cas3, besides deleting large regions, enabled identification of non-dispensable genes in Synechococcus 2973, which otherwise were predicted as dispensable. We successfully deleted the optimal stretch of two of the five dispensable regions identified by our in silico analysis. The two large deletions were combined to obtain a strain with genome reduction of 55 kb. The strains with a reduced genome showed improved growth and sucrose productivity. This proof-of-concept study ( Fig. 1 ) demonstrates that systematic minimization of cyanobacterial genomes has the potential to develop these organisms as super-strains that might hold the potential to boost a carbon-neutral bio-economy and mitigates climate issues. Fig 1 Schematics showing the strategy used for systematic genome streamlining in cyanobacteria. A combinatorial approach integrating computational (design) and experimental (test) tools to first identify the dispensable regions in S. elongatus UTEX 2973 and further use novel CRISPR tool to validate the prediction and create a strain with improved fitness and productivity.",
"discussion": "DISCUSSION Genome streamlining is a synthetic biology approach that allows strategic reduction of the genome for attaining a desirable strain phenotype, and this approach has been demonstrated to be successful in heterotrophs. There are two approaches of genome streamlining, bottom-up and top-down ( 33 ). Although most minimized genome studies have employed a bottom-up strategy to create a truly minimal genome from scratch ( 16 , 34 ), this strategy can pose several challenges as it demands a vivid and thorough knowledgebase of all biological processes and interactions and requires efficient synthetic DNA synthesis and assembly tools. Moreover, cyanobacteria being a polyploid organism exponentially enhance the challenge of introducing and maintaining the synthetic chromosome. The other approach is top-down, which involves systemic streamlining of the existing genome based on prior information regarding the core essential genes. Most genome streamlining efforts have relied on traditional and, more recently, the CRISPR-Cas12a/9-mediated genome-editing tools ( 20 , 21 , 24 , 25 ). For strains where the essentiality information is available, the strategy to delete fixed, specified regions of the genome is advantageous as it leaves less room for casualties such as drastic loss of fitness. However, even with prior knowledge, the experimental outcome might not correlate with in silico predictions. Like, in E. coli MG1655, a reduction of 29.7% exhibited severely impaired phenotype, while a 7% reduction showed no retarded phenotype ( 20 ). Therefore, for known and, more importantly, for newly discovered strains, the spontaneity or randomness in the extent of deletion might be the key to obtain a strain with enhanced features, and CRISPR-Cas3-mediated genome editing has the potential ( 26 , 27 ). The RNA-guided Cas3 protein has dual helicase-nuclease activity that allows large, progressive, random deletion of genomic region unless encountered with an essential gene. CRISPR-Cas3 has been demonstrated to be effective in heterotrophs, such as Pseudomonas aeruginosa , where deletion of genomic regions as large as 424 kb with a mean of 92.9 kb and median of 58.2 kb deletion ( 26 ) was observed. Therefore, its use in photoautotrophs for streamlining and other large-scale editing purposes is worth exploring. In this study, we focused on developing a genome streamlining strategy for a non-model cyanobacterium Synechococcus 2973 to obtain a strain with reduced genome and improved fitness. Synechococcus 2973 is the fastest-growing cyanobacterium known so far with a doubling time comparable to heterotroph model strains such as yeast ( 6 , 35 ). This organism has the potential to be developed as the next-generation production host; however, the complexity of the strain poses challenges for major engineering efforts. We attempted to minimize the metabolic burden on this strain by first identifying dispensable genomic regions using the MinGenome algorithm ( 28 ) and then removing them under bioproduction-relevant conditions without compromising growth and productivity ( Fig. 1 ). This iterative integrated approach led to the creation of engineered Synechococcus 2973 strains with minimized genomes exhibiting significant growth advantage ( Fig. 5a ). Our results indicate that the extent of growth advantage is not dependent on the extent of genome reduction but on the set of deleted genes. Our analysis revealed that some genes in R1 are predicted as phage-associated proteins. Although the SG55 strain has the largest range of deletion (55 kb), the growth improvement is more in SG33 (33.5-kb deletion), and this might be due to the deletion of prophage-like genes. A previous study in Vibrio natriegens revealed that the deletion of prophage-containing genomic regions is an effective engineering strategy for improving growth ( 36 ). Since a majority of the genes are annotated as hypothetical, further analysis to decipher the genetic features responsible for the phenotype was not possible. We tested the effect of minimization on productivity by engineering SG55 strain for sucrose overproduction and observed a 22.7% improvement in sucrose titer ( Fig. 5b ) as compared to sucrose-producing WT ( 10 ). This strategy of streamlining the genome for improved growth and phenotype might come at the cost of robustness under non-controlled conditions such as outdoor-like conditions (carbon limited). Under the carbon-limited condition, the engineered strains failed to show the improved phenotype (Fig. S4). This decreased fitness in conditions mimicking the natural environment (limited carbon available) might be a boon as it lessens the risk of their release and the chance to overtake the WT populations. However, in under elevated CO 2 , these strains might be advantageous to utilize more available carbon. A more high-throughput understanding of the phenotype and genotype relationship would provide a better insight into the complexity of the strains. The stochastic nature of the CRISPR-Cas3 system offers the potential to discern the essentiality of genes in the targeted regions and obtain an optimal dispensable stretch while retaining or enhancing strain fitness. This hybrid strategy of combining computational analysis with progressive deletion of genomic regions can generate a library of engineered model and non-model photoautotrophic strains with streamlined genomes, thereby paving the path for developing these remarkable green hosts into predictable bio-systems with the potential to mitigate climate problems and boost bio-economy."
} | 3,755 |
35518690 | PMC9061852 | pmc | 4,795 | {
"abstract": "Three-dimensional graphene based materials with superhydrophobic/superoleophilic attributes are highly desirable for water treatment. The graphene aerogel (GA) was prepared by hydrothermal reaction of the graphene oxide (GO) solution in the presence of dopamine followed by freeze-drying. The subsequent surface modification of GA using fluoroalkylsilane occurred by a vapor–liquid deposition process. The superhydrophobic graphene aerogel (SGA) fabricated from GA exhibits superhydrophobicity and superoleophilicity with the water contact angle of 156.5° and the oil contact angle of 0°. With this property, SGA could selectively adsorb various types of oils/organic solvents from the oil–water mixture. Moreover, the SGA possesses excellent low bulk density (9.6 mg cm −3 ), high absorption capacity (110–230 fold weight gain), and superior adsorption recyclability. With all these desirable features, the SGA is a promising candidate for oil-polluted water remediation.",
"conclusion": "Conclusion In summary, SGA with superhydrophobic and oleophilic properties was prepared from PDA functionalized graphene aerogel via hydrothermal method and subsequent hydrophobic modification using fluoroalkylsilane through vapor–liquid deposition. The modification not only introduces nanoscale roughness on the surface of the graphene, but also decreases the surface tension. The resulting SGA showed a high water contact angle of 156.5° and low water adhesion property. Furthermore, the SGA can selective removal of organic pollutants from water and adsorb a broad variety of oil liquids with enormous adsorption capacities. The superhydrophobic GA is very stable in oils and can be used repeatedly for oil/water separation.",
"introduction": "Introduction Recently, the environmental and ecological damage caused by oil spills and chemical leakage has been an area of great concern around the world. 1–9 Among multifarious methods that use of oil skimmers, chemical dispersants, adsorbents, in situ burning, and microorganisms, 10–14 adsorption is considered as the most economical and efficient choice due to low cost, simple operation, and prevention of secondary pollution. 15 However, most conventional adsorption materials show low oil adsorption capacity and poor selectivity for the oil–water mixture. Thus, the development of novel adsorption materials for the cleanup oil polluted water is of great significance. 16–24 Graphene nanosheets with intrinsic hydrophobic properties are attracting increasing attention in the oil–water separation field. 25 3D graphene-based monoliths, such as aerogels, foams and sponges, not only keep the advantage of the unique structure of graphene sheets, but also possess low density, high porosity, and large surface area. 3D graphene-based materials are a promising candidate of adsorption material for the removal of oil and organic solvents from water. For example, Sun et al. fabricated graphene sponge as the oil adsorption material through a hydrothermal method. 26 Li and Shi prepared graphene aerogel (GA) adsorption materials by the chemical reduction of graphene oxide (GO) dispersion. 27,28 Liu et al. synthesized graphene foam for oil adsorption by the thermal reduction of graphene oxide foam. 29 Qu and Xie manufactured nitrogen doped graphene framework as the adsorbents of oils and organic solvents. 30,31 Losic and Gao prepared graphene/carbon nanotube composite aerogels. 32,33 Our previous study also reported porous graphene foam with good hydrophobicity and oil adsorbability, which could be used for oil/water separation. 34,35 Although the above graphene-based monoliths have high adsorption capacity, they have no selectivity for oil and water, decreasing the separation selectivity and efficiency. 36,37 In order to endow the neat graphene monolith with superhydrophobicity and superoleophilicity so as to further improve its separation efficiency, designing surface structure with high roughness and low surface energy is expected to be a feasible and effective pathway. 38–42 In this work, the superhydrophobic graphene aerogel (SGA) was prepared by polydopamine (PDA) functionalized GA via hydrothermal method and subsequent hydrophobic modification using fluoroalkylsilane through vapor–liquid deposition. Various techniques were employed to study the morphology and surface properties of the materials. The wetting characteristics of SGA monolith were measured to confirm its superhydrophobicity and superoleophilicity. The oily compounds adsorption and oil–water mixture separation properties of monolith were systematically characterized.",
"discussion": "Results and discussion The synthetic routes of SGA are illustrated in Fig. 1 . First, the PDA functionalized graphene hydrogel was prepared by hydrothermal reduction of the mixtures of GO and dopamine. Second, the graphene hydrogel was by freeze-drying to produce GA. Third, the low-energy PFOES layer coated on the GA surface through the reaction of the hydroxyl groups in GA with the alkoxysilane group of PFOES. Finally, the resulting SGA was obtained by using the vapor–liquid deposition method. Different from the frequently used SGA prepared solution-processed coating approaches, vapor–liquid deposition avoid the unmanageable solvent removal process and can maintain the stable shape, initial monolithic volume and structure of the materials. Importantly, the surface of the graphene based material can be modified according to the need of oil–water separation. As a result, a piece of SGA could effortlessly stand on the top of foliage without deforming the supporter at all ( Fig. 1 ). The calculated density of aerogel based on the weight and dimension of sample was 9.6 mg cm −3 , belonged to the range of ultra-low density material. Fig. 1 The fabrication process and the digital image of SGA. The morphology of the surface of GA and SGA was examined by SEM. SEM images of the surface of GA in different magnifications are shown in Fig. 2a and c . As revealed by SEM images of the GA ( Fig. 2a ), the curly graphene nanosheets were randomly cross-linked to form 3D interconnected hierarchical porous network with pore size distributions in the scope of submicrometers to ten micrometers. The self-assembly of graphene nanosheets into the 3D structures could be attributed to the partial overlapping or coalescence of the flexible reduced GO nanosheets via noncovalent interactions, such as hydrogen bonding, and π–π interactions. 44,45 The surface of the graphene was smooth at the magnified scale ( Fig. 2c ). The smooth surface of GA indicated that PDA was uniformly coated on the basal planes of graphene, which might be due to the strong affinity between the dopamine aromatic rings and graphene nanosheets. 46 After the in situ vapor–liquid deposition process, a similar porous morphology with interconnected frameworks was observed for SGA ( Fig. 2b ). However, a compact coating with a random distribution of many nanoscale granules was observed on the graphene surface ( Fig. 2d ), which showed that the accomplishment of covalent interaction on the surface of SGA. The nanoparticle morphology provided the nanoscale roughness to complement the microscale roughness inherent in the graphene aerogel. Therefore, the surface microscale and nanoscale roughness was an essential necessity to realize the superhydrophobicity of SGA. Energy dispersive spectroscopy (EDS) analysis was used to further investigate the chemical composition of aerogels. As shown in Fig. 2e , only peaks of C, O, and N were detected on the GA, and no other impurities could be observed. After the deposition process and hydrophobic modification, new peaks of F and Si could be observed besides C, O, and N for the SGA ( Fig. 2f ). These results show that F and Si element is successful introduction overall the surface of SGA. The 3D porous and hydrophobic structures of SGA are highly desirable for oil and organic solvent adsorption. Fig. 2 Low- and high-magnification SEM images and corresponding EDS analysis of (a, c and e) GA and (b, d and f) SGA. To further verify the formation mechanism of the superhydrophobic surface, XPS measurements were used to compare the chemical composition of the GO, GA, and SGA. The peaks of C1s and O1s were observed in GO, while a new peak of N1s emerged in GA, which should arise from polydopamine on the graphene hydrogel during the process of preparing ( Fig. 3a ). After the modification process, the XPS spectrum of SGA ( Fig. 3a ) had fluorine component originating from SGA, indicating that the covalent functionalization of GA by PFOES successfully occurred. This result was consistent with the results of the EDS analysis. The detailed deconvolutions of the C1s spectra for GO, GA, and SGA were investigated as shown in Fig. 3b . Deconvolution of the C1s signal in GO sample showed a strong peak at high binding energy for the heavily oxygenated carbon species. This result was consistent with the results of the previous GO. 35 After hydrothermal reduction, the intensity of the peak was decreased obviously for the heavily oxygenated carbon species, and the peak related to C \n \n\n<svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>\nCreated by potrace 1.16, written by Peter Selinger 2001-2019\n</metadata><g transform=\"translate(1.000000,15.000000) scale(0.017500,-0.017500)\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>\n\n C/C–C (∼284.6 eV) became dominant ( Fig. 3b ). Furthermore, deconvolutions of the C1s spectrum for SGA appeared new peak associated with the C–F covalent bonds at about 291.9 eV corresponding to C–F bonding ( Fig. 3b ). 47 The new Si2p (102.3 eV) ( Fig. 3c ) and F1s (689.2 eV) ( Fig. 3d ) peak in the XPS spectrum of the SGA confirmed the successful binding of fluoroalkylsilane on the surface of the as prepared SGA. The atomic ratios of carbon, oxygen, nitrogen, silicon, and fluorine measured by XPS in different materials were summarized in Table 1 . After reduction, the content of carbon increased gradually to 81.07% while the oxygen content decreased gradually. This result shows that the majority of oxygen-containing groups are removed and some of them may be from PDA ( Fig. 3b ). GA contained 1.62% of N originating from the PDA, indicating that the PDA successfully deposited on surface of the GA. Additionally, after the hydrophobic modification process, the new element of silicon and fluorine content in the SGA sample was 0.72% and 2.12%, respectively. The above results indicate fluorine and silanes are chemically bound on the SGA surface by the hydrolysis/condensation reaction. Fig. 3 XPS results. (a) XPS wide-scan spectra and (b) C1s high-resolution spectra of the GO, GA, and SGA. (c) Si2p and (d) F1s high-resolution spectra of SGA. Atomic ratio of GO, GA and SGA Sample C (%) O (%) N (%) Si (%) F (%) GO 66.67 33.33 — — — GA 81.07 17.31 1.62 — — SGA 77.34 18.12 1.7 0.72 2.12 The FT-IR spectra of GA and SGA were shown in Fig. 4 . For the spectrum of the GA, the peak appeared at 1445 cm −1 (N–H of amide group shearing vibration) and 1677 cm −1 (N–H in-plane stretching vibration), indicating the presence of amine groups after polydopamine-mediated assembly. After surface functionalization with PFOES, new bands at 1162, 1262, and 1385 cm −1 were rocking vibration peaks of C–F bond, and two bands at 2917 and 2961 cm −1 appeared, showing the stretching of the –CH 2 groups from the alkyl chains assigning to silane moieties of PFOES-GAs. 48 Besides, the bands at 1065 and 1109 cm −1 were attributed to the Si–O–Si and Si–O–C bonds, indicating the successful chemical functionalization of GA by PFOES. 49 Fig. 4 FT-IR spectra of GA and SGA. The wetting behavior of the SGA is shown in Fig. 5 . The dynamic wettability of water and oil on the surface was investigated through the high-speed camera system to record the liquid droplet permeating process. For the water droplet adhesion performance, a water droplet was driven to completely contact with the surface of SGA and then lifted it up. From the corresponding photographs of the water droplet ( Fig. 5a ), no deformation was observed when water droplet left the surface of aerogel. The result confirms the SGA has extremely low water adhesion. Conversely, when oily liquid droplet contacted with SGA, it continuously spread and permeated on the material surface with the contact angles of 0° ( Fig. 5b ). The SGA can adsorb the hexadecane droplet within 5 s. The SGA behaves as a superior oil-adhesion property. The SGA exhibit the superhydrophobicity with a water contact angle of 156.5°, while the oil drop is adsorbed completely by the SGA and no contact angle can be found. Solid surface energy was 76.82 and 0.96 mN m −1 for the GA and SGA, respectively, which was calculated by Owen's two liquid methods. The lower surface energy of the SGA is crucial for get superoleophilic surface in air. The superhydrophobic and superoleophilic wettability of SGA avoids direct contact with water and ensures fast oil permeation and moving during oil–water separation process. Fig. 5 Wetting behavior of (a) water and (b) hexadecane droplet on SGA surface. Owing to the above-mentioned superhydrophobicity, hierarchical structure, and ultra-low density, the aerogels are considered as a promising material for highly efficient oil–water separation and organic solvents adsorption. As a demonstration, the light oil atop water surface ( Fig. 6a ) and heavy oil underwater ( Fig. 6b ) were used as an example to show the adsorption process of SGA. Fig. 6a showed the adsorption performance of SGA to light oil atop water. The SGA could immediately adsorb the hexadecane layer (dyed with oil red) around it and leave transparent regions on water surface when a piece of it contacted with the hexadecane on water. The hexadecane was completely absorbed by SGA within a few seconds because the SGA had a rapid adsorption process for oils and organic solvents on water. Fig. 6b demonstrated that SGA could quickly adsorb heavy oil underwater as well. The droplet of chloroform was immediately sucked into the aerogel once the SGA was immersed into water and started to contact with chloroform. There was no detectable water in the adsorbed aerogel materials. The above results demonstrate the SGA has excellent adsorption selectivity for immiscible oils–water. The adsorption performance of SGA was further evaluated through a series of adsorption experiments for various organic solvents and oils. Compared with GA, SGA has a high adsorption capacity ( Fig. 6c ). It can be seen that adsorption capacity of SGA in the range of 110–230 times higher than its own weight for various oils and organic solvents ( Fig. 6c ). The capacities of SGA for all oily liquids are over 100.0 g g −1 . The SGA showed high adsorption capacity for various oils and organic solvents, which were much higher than most previous activated carbon and polymers, 50,51 and superhydrophobic graphene-based aerogels reported in the previous literatures. 26,52 Furthermore, similar to the previously reported, 33 the adsorption capacity of SGA was approximately proportional to the density of the various solvents. Combined homogeneous internal structure with low surface energy, the adsorption capacity of SGA toward oil and other organic chemicals is greatly improved. Fig. 6 Oil adsorption performance of different aerogels (a) illustration showing the adsorption process of SGA for hexadecane and (b) chloroform; (c) adsorption capacity of SGA and GA for various organic liquids. The recyclability and recoverability are important property for an ideal adsorption material in the actual application of oil cleaning. As shown in Fig. 7 , octane was used as an example to evaluate the recycling potential and recovering of SGA for the oils and organic solvents through a simple sorption-drying cycle. As a result, only a small decline for the adsorption capacity of SGA was observed after ten times adsorption–desorption cycles test. The adsorption capacity was still higher than 70 g g −1 for the used common solvent. This result highlights the excellent recyclability of SGA for oil-adsorption by taking this simple sorption-drying method over ten repeat times. Notably, a slight drop for oil adsorption capacity may be attributed to the residual oil entrained in the pores of the aerogels. 53 These results indicate the good recyclability and recoverability of SGA by a simple sorption-drying cycle method. Fig. 7 Recyclability of SGA for oil adsorption ((a) adsorption recyclability of aerogel over ten times cycles; (b) remained adsorption capacity of sample)."
} | 4,212 |
27345956 | PMC5390555 | pmc | 4,796 | {
"abstract": "Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host’s genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which—by virtue of mitochondria—metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host’s vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny—sex—in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically evolving lineages viable by rescuing the incipient eukaryotic lineage from Muller’s ratchet. The origin of mitochondria was, in this view, the decisive incident that precipitated symbiosis-specific cell biological problems, the solutions to which were the salient features that distinguish eukaryotes from prokaryotes: A nuclear membrane, energetically affordable ATP-dependent protein–protein interactions in the cytosol, and a cell cycle involving reduction division and reciprocal recombination (sex).",
"conclusion": "Conclusion Our proposal has it that eukaryotic sex arose as a necessary replacement for prokaryotic gene transfer mechanisms in a cell that had evolved a nucleus (as a consequence of mitochondrial group II introns) and that was consequently able to undergo the transition to MDCS as a fortuitous byproduct of mitochondrial energetics. The mitochondrion transformed its host, generating a cytosol 1) where protein could be synthesized in amounts no prokaryote had ever experienced, 2) where proteins could interact without interfering either with chromatin or with cell division, and 3) where spontaneous aggregation of structural proteins to higher order structures and ATP-dependent disaggregation became possible. Selection was acting upon the outcome of spontaneous protein interactions—spontaneous chemical processes were thus decisive at this major evolutionary transition. Once it exists, the benefits of sex are manifold. The how and why of how sex came into existence are more challenging. The DNA damage hypothesis ( Hörandl and Hadacek 2013 ) posits that meiosis evolved as a repair response to oxidative damage. Our model would not exclude a role for recombination in repair, since the enzymes involved in meiotic recombination are homologous to prokaryotic repair machineries ( Camerini-Otero and Hsieh 1995 ; Ramesh et al. 2005 ), and because mitochondria (the source of reactive oxygen species in eukaryotes) are present in the cell that evolved sex. Homolog synapsis involving cohesins, members of the SMC protein family (Jeppsson et al. 2014), has been suggested as the key innovation that allowed for the evolution of meiosis from mitosis ( Wilkins and Holliday 2008 ). In our proposal, the chromosomes are initially heterogeneous. Hence until a homogeneous, ancestral eukaryote genome with defined chromosome numbers having haploid and diploid states during an evolved cell cycle and life cycle had emerged, homologous pairing would not have been obviously beneficial. Congruent with that view, many prokaryotes possess clear homologs of the SMC proteins that promote eukaryote chromosome condensation and that lead to pairing of sister chromatids ( Soppa 2001 ; Soppa et al. 2002 ), yet prokaryotes neither condense their chromatin at cell division nor do they possess sister chromatids. A number of major evolutionary transitions in eukaryote evolution involve endosymbiosis: The origin of mitochondria, the origin of plastids, and the origin of major algal groups through secondary endosymbiosis. Each of those endosymbiotic transitions also left a major impact on the genome in the form of gene transfers from organelles to the nucleus ( Martin and Müller 1998 ; Martin et al. 2002 ; Ku et al. 2015 ). But endosymbiosis is not readily accommodated either by mathematics or by the gradualist paradigm of population genetics, which is why mitochondria play no role whatsoever in population genetic approaches to understanding the prokaryote–eukaryote transition ( Lynch and Conery 2003 ; Lynch 2006 ; Lynch and Marinov 2015 ). Indeed, population genetic investigations tend to recognize no difference at all between prokaryotes and eukaryotes, both of which appear to map out along an uninterrupted continuum by the measure of population genetic parameters ( Lynch and Conery 2003 ). That is not a criticism of population genetics, it is a statement about the evolutionary divide separating eukaryotes from prokaryotes. Viewed solely through the looking glass of population genetics and allele frequencies, one would not even be able to tell the difference between a lion and a palm tree, because on the long term, both have sex and diploid genetics. Clearly, population genetics does not tell us everything that we need to know about eukaryote evolution, if we are interested in the physiological and cell biological differences that distinguish eukaryotes from prokaryotes. At the prokaryote to eukaryote transition, many major changes took place: Mitochondria, endomembrane system, nucleus, meiosis, mitosis, and the origin of sex. We have endeavored here to account for those differences in one essay. In doing so we necessarily crossed a border from modern endosymbiotic theory, starting from an archaeaon as the host, to population genetics (sex). We were able to sketch, albeit in broad strokes, a general outline that bridged the evolutionary gap between prokaryotes and eukaryotes and that resulted in a mitochondriate, nucleated, sexually recombining and mitosing cell. Whether population genetic approaches would be able to predict the single origin of plastids or mitochondria during evolution remains an open question. Whether the eukaryotic cell cycle or endomembrane system could be modeled as a population genetic parameter is also an open question. When it comes to modeling the processes underlying the cell biological differences that distinguish eukaryotes from prokaryotes and taking them apart into simpler, conceptually tractable components, endosymbiotic theory works fairly well. Yet endosymbiotic theory is founded in comparative biology and comparative physiology, not in mathematics and statistics, hence as a field it does not interface well with population genetics. Maybe future progress will improve that circumstance. Sex, the cell cycle, chromosome division, and alternation of generations are processes. Inferring the evolutionary origin of processes is arguably more difficult than inferring the origin of structures. Understanding the origin of organelles and structures that distinguish eukaryotes from prokaryotes, for example, the endomembrane system, is important, and a strong case can be made that the origin of mitochondria had the decisive role in endomembrane origin ( Gould et al. 2016 ). A better understanding of eukaryote origin requires understanding the evolution of eukaryotic processes. The syncytial stage has many virtues as an evolutionary intermediate. In terms of structuring the problem of eukaryote process origins, it also helps to break down the complex eukaryotic cell cycle and life cycle ( fig. 1 ) into simpler component processes ( fig. 4 ), and in doing so draws attention to the largely (but not completely: Cavalier-Smith 2010 ) neglected issue of the origin of alternation of generation in eukaryotes. Students of biology have had to learn alternation of generations for over a century, yet without an evolutionary context that could account for the origin of the sexual processes (karyogamy and reduction division) that connect the alternating generations. We have made an effort here to put sex and the alternation of generations into the evolutionary context of modern endosymbiotic theory. Our evolutionary intermediates obtain new gene variants through karyogamy in a syncytium and through archaeal-type spore fusion. The end result of the inference is a population of free-living, unicellular, sexually recombining and mitosing cells that have a cell cycle, archaeal ribosomes in the cytosol, and mitochondria. In recent years, views on the origin of eukaryotes have changed in that 1) the mitochondrion is now recognized to be ancestrally present in the eukaryote common ancestor and in that 2) the host is now considered to have been an archaeon ( Martin and Müller 1998 ; Martin and Koonin 2006 ; Cox et al. 2008 ; Lane and Martin 2010 ; Williams et al. 2013 ; McInerney et al. 2014 ; Raymann et al. 2015 ; Spang et al. 2015 ; Sousa et al. 2016 ; Gould et al. 2016 ). Views on the origin of sex have not changed at the same pace, though there is increasing interest in the possible role(s) of mitochondria in promoting the establishment of meiotic recombination ( Lane 2009 , 2015 ; Speijer et al. 2015 ; Havird et al. 2015 ; Radzvilavicius and Blackstone 2015 ). Here, we have considered the origin of sex on the basis of those newer premises. In contrast to earlier theories, we do not assume that the cell in which sex arose had already evolved a mitotic cell cycle. Rather, we start from a symbiotic association of two prokaryotes, which led to the origin of mitochondria, and consider the evolutionary sequence of events. As the most notable departures from previous theories on the origin of eukaryotes, mitosis or sex, 1) we posit that mitotic division is evolutionarily derived from meiotic division; 2) we place both processes in their natural context of eukaryotic cell cycle origin; 3) we propose a coenocytic eukaryote common ancestor, Coeca, which allowed nuclei harboring defective in chromosome sets to complement each other through mRNA in the cytosol; 4) we suggest that the first form of eukaryotic cell division was budding of meiospores from the coenocyte, a process that selected viable combinations of chromosomes and mitochondria; and 5) we suggest that the ability of meiospores to fuse, a property of archaeal cells, allowed them to undergo homologous meiotic recombination, or sex. It is an observation that sex has been retained in all eukaryotic lineages for over 1.7 Gyr, we suggest that the reason for its retention is the same as the reason for its fixation at eukaryote origins: It saves eukaryotes from extinction through Muller’s ratchet. Our inference orders the origin of major evolutionary innovations in the eukaryotic lineage as follows: Mitochondria, followed by the nucleus and endomembrane system, MDCS, reduction division in a syncytial eukaryote common ancestor, meiospore production, division and fusion leading to a meiotic life cycle and cell cycle (sex), and finally mitosis through bypassing of recombination and reduction division during the life cycle. The syncytial nature of the eukaryote common ancestor is a particularly interesting, and possibly useful, element of our proposal. It is interesting because it decouples the processes of chromosome segregation, ploidy cycling, karyogamy, progeny generation, cell division, and the cell cycle from each other. It is useful because it allows one to consider the evolution of each of those actions as a tendentially independent biological process, which fits well with the diversity, or lack thereof, observed for each of these processes among contemporary eukaryotic groups.",
"introduction": "Gene Transfer, Introns, the Nucleus and Ploidy All cells that have sex have a nucleus. How does the nucleus fit into eukaryote origin? The mitochondrial endosymbiont is more than a source of energy, it is a source of genes, lots of genes, and large scale chromosomal mutations. This process of gene transfer, from the bacterial symbiont to chromosomes of the archaeal host, is called endosymbiotic gene transfer, or EGT ( Martin et al. 1993 ; Timmis et al. 2004 ), it is unidirectional, it still operates today involving insertion of whole organelle genomes hundreds of kilobases in length into nuclear DNA ( Huang et al. 2005 ). The mechanism of DNA integration is nonhomologous end joining ( Hazkani-Covo and Covo 2008 ), the mechanism of DNA release to the host is organelle lysis ( Huang et al. 2004 ), the process of EGT has operated throughout eukaryotic history and is observable as an ongoing process, even during human evolution, with the most recent mitochondrion-to-nucleus transfers dating to the Tschernobyl incident ( Hazkani-Covo et al. 2010 ). Eukaryotes are usually described as descendants of archaea ( Cox et al. 2008 ; Williams et al. 2013 ), but if we look at the whole genome, bacterial genes vastly outnumber archaeal genes in eukaryotes ( Esser et al. 2004 ; Thiergart et al. 2012 ), and genes that trace to the mitochondrion vastly outnumber those that trace to the host ( Ku et al. 2015 ). At the outset, the host has no nucleus, and as long as cell division is not impaired, the symbiosis of prokaryotes is stable, as long as the environment supports growth. The stability of the symbiosis changes however, probably as a consequence of EGT, a mutational mechanism that is specific to the eukaryotic lineage: Gene transfer from symbiont to host carries some fateful hitchhikers—self splicing group II introns. Group II introns are important, and their transition into spliceosomal introns could have precipitated the origin of the nucleus ( Martin and Koonin 2006 ). How so? Group II introns occur in prokaryotic genomes ( Lambowitz and Zimmerly 2011 ), they are mobile, they can spread to many copies per genome ( Lambowitz and Zimmerly 2004 ) and they remove themselves through a self-splicing mechanism that involves the intron-encoded maturase ( Matsuura et al. 1997 ). Their splicing mechanism is similar to that in spliceosomal intron removal ( Lynch and Richardson 2002 ), for which reason they have long been viewed as the precursors of both 1) spliceosomal introns and 2) their cognate snRNAs in the spliceosome ( Sharp 1985 ). The crux of the intron hypothesis for nuclear origin ( Martin and Koonin 2006 ) is that group II introns, which are mobile elements in prokaryotes ( Lambowitz and Zimmerly 2011 ), entered the eukaryotic lineage through gene transfer from the mitochondrial endosymbiont to the archaeal host. In the host’s chromosomes they spread to many sites and underwent the transition to spliceosomal introns, as evidenced by the observation that many introns are located at conserved positions across eukaryotic supergroups ( Rogozin et al. 2003 ) and by the presence of spliceosomes in the last eukaryote common ancestor ( Collins and Penny 2005 ). The transition from group II introns to spliceosomal introns evokes a curious situation: Spliceosomal splicing is slow, on the order of minutes per intron ( Audibert et al. 2002 ), whereas translation in ribosomes is fast, on the order of 10 peptide bonds per second ( Sørensen et al. 1989 ). As the transition to spliceosomal introns set in, the host’s cytosol was still a prokaryotic compartment with cotranscriptional translation. With the origin of bona fide spliceosomes and spliceosomal splicing, nascent transcripts were being translated (ribosomes are fast) before they can be spliced (spliceosomes are slow). Translation of introns leads to defective gene expression at many loci simultaneously (though one essential locus would suffice), a lethal condition for the host unless immediately remedied. The solution to this condition was, we posit, physical separation of the slow process of splicing from the fast process of translation so that the former could go to completion before the latter set in. Separation in cells usually involves membranes, and that is the central tenet of the intron hypothesis: The initial pressure that led to selection for nucleus–cytosol compartmentation (the origin of the nuclear membrane) was the requirement for physical exclusion of active ribosomes from nascent transcripts, to restore gene expression and intron-containing genes ( fig. 2 D ). The primordial nuclear membrane allowed the slow process of splicing to go to completion around the chromosomes, thereby initially allowing distal diffusion, later specific export of processed mRNAs to the cytosol for translation, furthermore precipitating the origin of nonsense-mediated decay, a eukaryote-specific machinery that recognizes and inactivates intron-containing mRNAs in the cytosol ( Martin and Koonin 2006 ) ( fig. 2 D ). The reader might protest that we have specified neither a mechanism nor a source for the vesicles that give rise to the nuclear membrane in the host’s archaeal cytosol. That is the topic of a separate paper ( Gould et al. 2016 ), in which we outline how outer membrane vesicles produced by the mitochondrial endosymbiont in an archaeal host are likely both the physical source and the evolutionary origin of the eukaryotic endomembrane system. A primitive nuclear membrane rescues gene expression, and DNA replication can continue to proceed as long as the cytosol supplies dNTP precursors. But there is no mechanism for chromosome segregation in place. Chromosomes replicate without division, polyploidy, extreme polyploidy in all likelihood ensues, and the symbiosis seems to be headed straight toward a dead end. But mitochondria can make a difference."
} | 4,645 |
23171087 | null | s2 | 4,797 | {
"abstract": "Bacteria utilize multiple regulatory systems to modulate gene expression in response to environmental changes, including two-component signalling systems and partner-switching networks. We recently identified a novel regulatory protein, SypE, that combines features of both signalling systems. SypE contains a central response regulator receiver domain flanked by putative kinase and phosphatase effector domains with similarity to partner-switching proteins. SypE was previously shown to exert dual control over biofilm formation through the opposing activities of its terminal effector domains. Here, we demonstrate that SypE controls biofilms in Vibrio fischeri by regulating the activity of SypA, a STAS (sulphate transporter and anti-sigma antagonist) domain protein. Using biochemical and genetic approaches, we determined that SypE both phosphorylates and dephosphorylates SypA, and that phosphorylation inhibits SypA's activity. Furthermore, we found that biofilm formation and symbiotic colonization required active, unphosphorylated SypA, and thus SypA phosphorylation corresponded with a loss of biofilms and impaired host colonization. Finally, expression of a non-phosphorylatable mutant of SypA suppressed both the biofilm and symbiosis defects of a constitutively inhibitory SypE mutant strain. This study demonstrates that regulation of SypA activity by SypE is a critical mechanism by which V. fischeri controls biofilm development and symbiotic colonization."
} | 369 |
39746973 | PMC11696919 | pmc | 4,798 | {
"abstract": "Thermophilic microbial communities growing in low-oxygen environments often contain early-evolved archaea and bacteria, which hold clues regarding mechanisms of cellular respiration relevant to early life. Here, we conducted replicate metagenomic, metatranscriptomic, microscopic, and geochemical analyses on two hyperthermophilic (82–84 °C) filamentous microbial communities ( Conch and Octopus Springs , Yellowstone National Park, WY) to understand the role of oxygen, sulfur, and arsenic in energy conservation and community composition. We report that hyperthermophiles within the Aquificota ( Thermocrinis ), Pyropristinus ( Caldipriscus ), and Thermoproteota ( Pyrobaculum ) are abundant in both communities; however, higher oxygen results in a greater diversity of aerobic heterotrophs. Metatranscriptomics revealed major shifts in respiratory pathways of keystone chemolithotrophs due to differences in oxygen versus sulfide. Specifically, early-evolved hyperthermophiles express high levels of high-affinity cytochrome bd and CydAA’ oxidases in suboxic sulfidic environments and low-affinity heme Cu oxidases under microaerobic conditions. These energy-conservation mechanisms using cytochrome oxidases in high-temperature, low-oxygen habitats likely played a crucial role in the early evolution of microbial life.",
"introduction": "Introduction Atmospheric levels of oxygen in the Archaean are thought to have been negligible ranging from 10 −5 to 10 −6 of present-day atmospheric levels 1 . Early microbial metabolisms involving methane and other single-carbon compounds may have relied on respiration using sulfate, nitrate, or ferric iron 2 – 5 . Although the rise of atmospheric oxygen during the Great Oxidation Event (GOE) commencing circa 2.5 Gya 6 , 7 caused extensive radiation of aerobic microorganisms and eukaryotes 8 , 9 , the role of oxygen in microbial metabolism prior to the GOE is uncertain. Recent studies on the evolution of oxygen utilizing enzymes by microorganisms 10 provide phylogenetic evidence of microbial oxygen metabolism well before the GOE. These results are consistent with other geochemical studies that indicate the importance of oxygen in ancient biosignatures that predate the GOE, such as microaerobic steroid biosynthesis 11 , transition metal anomalies 12 , or micrometeorite analysis in the Archaean 13 . Numerous oxygen reductases are present and function in hyperthermophilic bacteria and archaea including heme Cu oxidases (HCOs; types 1, 2, and 3), cytochrome bd (ubiquinol) oxidases and the CydAA’ oxidases 14 – 19 . Type 3 (cbb3) HCOs and cytochrome bd ubiquinol oxidases have high affinities for oxygen and can support aerobic growth at nanomolar levels of oxygen 20 even in the presence of high sulfide 21 , 22 . Therefore, thermal habitats containing high sulfide and/or low levels of oxygen are extremely relevant to environments likely common prior to the GOE when atmospheric oxygen concentrations were significantly lower than modern levels 1 , 6 , 7 , 12 . Filamentous communities residing in the outflow channels of alkaline (pH 8-9) chloride geothermal springs rely on attachment in high-velocity (~0.2 m s −1 ) outflow channels to capitalize on the chemical energy available as highly reduced fluids mix with oxygen 21 , 23 – 25 . Variable levels of dissolved sulfide (DS) in different alkaline springs represent natural laboratories for testing hypotheses focused on microbial respiration under low-oxygen conditions at similar pH and temperature. Our objectives were to examine the genomics of chemolithotrophic electron transfer processes responsible for the growth and activity of thermophilic microorganisms in two filamentous ‘streamer’ communities thriving under different levels of DS versus DO. ‘Streamer’ is used here to define microbial communities that contain dominant filamentous members attached to solid substrates in high-velocity currents. Integrated and replicated genomic, transcriptomic, geochemical, and microscopic analyses were performed for nearly 10 years at two alkaline-chloride springs in YNP to elucidate microbial community structure and function under microaerobic (40–50 µM DO) versus sulfidic (~100 µM DS) conditions. Here we show that despite differences in DS and DO, several microbial populations including Thermocrinis , Pyrobaculum and Caldipriscus (a member of the early-evolved Pyropristinus lineage) are abundant in each habitat. However, the primary mechanisms of energy conservation and respiration vary under sulfidic (sub-oxic) versus microaerobic conditions. These results have important implications for the effect of oxygen concentrations on microbial community function and biogeochemical cycling under microaerobic and suboxic conditions, at temperatures often considered too high to support aerobic metabolism.",
"discussion": "Results and Discussion Geochemistry and microbial community composition Two alkaline-chloride geothermal springs ( Conch and Octopus Springs ) with similar geochemical profiles (Table 1 ) were chosen for this study to provide a direct comparison between geothermal habitats with contrasting levels of DS and DO (Figs. 1 and 2 ). Both springs discharge near-boiling water (~90°C at 2500 m) but Conch Spring is highly sulfidic (>120 µM DS) with no detectable oxygen (<1 µM), while Octopus Spring contains ~20 µM DO and low DS (<2-3 µM) at the source. DO concentrations increase quickly in the Octopus Spring outflow channel due to oxygen ingassing, and within several meters, DO concentrations reach 40–50 µM at streamer sampling sites (Fig. 2 ), which is approximately 30% of oxygen saturation at 80 °C and 2500 m (~130 µM). Table 1 Aqueous geochemistry a at sample sites within Conch and Octopus Springs b , Yellowstone National Park (WY, USA) Sampling Date T pH DO c DS c Na + K + Ca 2+ NH 4 + Cl − SO 4 2− Si B As DIC c DOC c δ 13 C-DIC d δ 13 C-DOC d Conch Spring (˚C) µM µM mM mM µM µM mM mM mM mM µM mM µM (‰) (‰) 10/13/2011 84 8.3 bd 70 13.5 0.31 14 0.9 8.3 0.2 3.9 0.4 21.9 3.3 39.2 −2.9 −19.0 10/5/2012 85 9.9 bd 120 13.6 0.3 11 na e 8.4 0.3 3.5 0.3 17.7 3.3 42.4 −2.7 −21.5 10/5/2012 82 9.8 bd 98 13.7 0.32 14 na 8.6 0.3 3.3 0.3 25.5 3.6 47.2 −2.7 −21.3 8/28/2013 85 9 bd 90 14.7 0.36 12 na 8.6 0.2 3.2 0.3 26.0 3.6 44.5 −2.5 −26.4 8/28/2013 82 9 bd 78 14.8 0.39 14 na 8.9 0.2 3.1 0.3 27.7 3.9 44.8 −2.3 −24.8 Octopus Spring 10/13/2011 84 7.7 20 bd 12.3 0.36 13 2.3 6.6 0.2 4.5 0.3 20.1 4.9 51.1 −2.2 −22.6 10/5/2012 84 8.6 25 bd 11.9 0.35 10 na 6.6 0.2 4.1 0.2 19.3 5.3 70.1 −1.8 −22.1 10/5/2012 82 8.6 47 bd 12 0.35 10 na 6.7 0.2 4.2 0.2 19.7 5.3 62.6 −1.8 −21.8 8/28/2013 84 8 32 bd 12.9 0.36 14 na 6.7 0.2 3.8 0.2 20.6 4.9 106.5 −1.9 −26.3 8/28/2013 82 8.2 53 bd 12.5 0.41 15 na 6.8 0.2 3.7 0.2 20.6 4.8 60.0 −1.7 −24.1 a Aqueous geochemistry (<0.2 µm): ICP-OES and IC for major cations and anions (see details in methods for others) b Site latitude/longitude coordinates (standard UTM zone 12, easting/northing): Octopus Spring = 516058/4931215, Conch Spring = 513330/4933689. c DO dissolved oxygen determined using modified Winkler method 56 , 57 (below detection <1 µM); DS dissolved sulfide (below detection <1 µM); DIC dissolved inorganic carbon; DOC dissolved organic carbon. d13 C isotope values of DIC and DOC (‰) used to predict the relative amounts of autotrophy in microbial biomass 35 . e na- not available on these dates; other dates confirm values within 5%; NO 3 -N below detection at 1 µM. Fig. 1 Two filamentous streamer communities at similar pH (8–9) and temperature (82–84 °C) exhibit differences in population structure and function due to geochemical differences in dissolved sulfide (DS) versus oxygen (DO) (DS:DO = ratio of DS to DO). A \n Conch Spring \n B \n Octopus Spring (Lower Geyser Basin, YNP). Scanning electron micrographs show filamentous organisms and extensive extracellular matrix (high-resolution insets) that dominate the physical fabric of both communities. Micrographs were chosen from a large collection of over 30 replicates from 3 different sample years and a minimum of 10 replicate images per year. Fig. 2 Concentrations of DS and DO (μM) in Conch and Octopus Springs . Filamentous communities were sampled down-gradient of spring discharge at transect position B, C (82–84 °C) for metagenomic and transcriptomic analyses (DS was <1 μM in Octopus Spring and DO <1 μM in Conch Spring ). Error bars are standard deviations of 4–6 replicates, where absent, error bars fall within symbol. Source data are provided as a Source Data file. Both streamer communities contain highly abundant populations of Thermocrinis (Aquificota) , Pyrobaculum (Thermoproteota), and Caldipriscus (Pyropristinus) genera (Fig. 3 ). Together, these three keystone populations comprise from ~ 50% to > 90% of the Octopus and Conch microbial communities, respectively. However, the other abundant community members were unique to each habitat type: Conch Spring is less diverse and contains only 3-4 additional microbial populations while Octopus communities contained greater diversity for a total of ~ 15 different populations (Fig. 3 , Table 2 ). Microorganisms present in Conch Spring but not in Octopus included Thermodesulfobacteriaceae, Thermosphaera , and several members of the Desulfurococcales. Conversely, Octopus Spring contained ~10 populations not seen in Conch Spring and these included additional early-evolved bacteria as well as additional archaea. Both habitats also contain low amounts (<1 % abundance) of very similar populations of Thermus aquaticus , Nanopusillus and Acidilobaceae (Table 2 , Supplementary Table 1 ). The strong reproducibility in population abundances in 2011 and 2012 (Supplementary Fig. 1 ) as well as the consistent compositional differences between Conch and Octopus Springs are also evident in tetranucleotide frequency t-SNE plots (Supplementary Figs. 2 – 4 ). Fig. 3 Relative abundances of microbial populations present in Conch and Octopus Springs sampled in 2011 and 2012. Organisms common to both sites include Thermocrinis , Pyrobaculum , and Caldipriscus (Pyropristinus) (hatched). Other populations (clockwise, in order of abundance) include Thermodesulfobacteria and 2 members of the Desulfurococcales at Conch Spring (yellow patterns), and Thermoproauctor , Calescibacterium (Calescamantes), Armatimonadota T1, Calditenuis aerorhuemensis 59 , Acidilobaceae, Armatimonadota T2, Thermoflexus , and several others <2% at Octopus Spring (blue patterns) (Table 2 and Supplementary Table 1 show complete list of phylotypes). Abundances were calculated based on the fraction of mapped reads from random metagenome sequence (CheckM 61 ). Taxonomic references are based on nucleotide identity (>95%) at either the phylum, order, family, or genus/species level. Source data are provided as a Source Data file. Table 2 Metagenome assembled genomes (MAGs) from high-temperature filamentous microbial communities sampled a from the outflow channels (82–84 °C) of Conch and Octopus Springs , Yellowstone National Park (WY, USA) Abund. c Size Numb Largest G + C MAG Name b (%) (kbases) Contigs (kbases) (%) SD d Cov. e SD Comp. f Redund. Heter. tRNA CRISPR Thermocrinis CON-C 37.91 3515 537 301 44.5 2.9 345.0 283.6 97.2 84.7 96.8 79 26 Pyrobaculum CON-C 32.70 4887 661 255 55.0 4.8 131.4 164.7 100.0 7.3 70.0 35 60 Caldipriscus CON-C 21.55 1528 20 429 44.5 1.3 364.1 123.4 78.9 4.1 0.0 47 0 Thermodesulfobacteriaceae CON-C 3.55 1950 159 134 37.4 5.1 58.4 70.3 96.5 1.4 11.1 46 4 Thermosphaera CON-C 3.42 1436 47 203 45.4 4.1 46.6 31.4 98.1 0.0 0.0 30 1 Acidilobaceae CON-C 0.87 1350 298 20 48.2 1.7 21.4 3.6 79.7 2.0 50.0 37 0 Thermocrinis OCT-B 28.97 4467 940 42 44.6 3.0 257.3 198.3 95.4 153.4 97.2 88 28 Pyrobaculum OCT-B 9.84 4169 700 41 57.4 3.3 95.9 94.0 93.8 16.2 68.8 68 42 Caldipriscus OCT-B 6.28 1892 475 17 43.8 2.4 192.3 94.4 78.8 42.2 98.7 62 5 Thermoproauctor OCT-B 22.12 1943 201 39 27.4 2.0 327.9 174.4 93.2 2.5 100.0 45 8 Calescibacterium OCT-B 10.76 4031 543 58 33.7 2.2 85.3 61.6 81.7 67.1 45.9 81 5 Armatimonadota-T1 OCT-B 5.05 4059 464 50 52.3 2.1 51.5 29.5 88.9 2.5 25.0 50 10 Calditenuis aerorheumensis OCT-B 4.03 2022 309 42 56.9 5.6 95.4 83.3 91.8 9.3 85.7 45 10 Acidilobaceae OCT-B 3.93 2295 233 90 45.7 3.8 38.6 30.7 99.6 3.2 60.0 53 12 Armatimonadota-T2 OCT-B 2.54 3066 340 45 58.3 1.6 48.2 16.8 89.4 1.3 20.0 47 2 Thermoflexus OCT-B 1.81 3297 283 77 68.2 1.9 27.6 10.5 91.8 2.0 0.0 51 10 Geoarchaeaceae OCT-B 1.77 1646 96 110 29.3 1.7 43.5 78.1 95.6 1.8 11.1 38 1 Desulfurococcales OCT-B 1.44 2380 245 58 44.2 1.9 19.8 6.4 98.7 5.1 7.1 41 5 Thermus aquaticus OCT-B 0.60 2040 192 53 68.5 1.5 15.6 2.8 98.7 0.9 50.0 48 3 Patescibacteria OCT-B 0.59 875 79 114 27.1 1.8 19.6 16.7 73.6 5.6 0.0 50 0 Nanopusillus OCT-B 0.27 344 97 13 24.1 1.9 9.7 10.7 48.2 0.9 25.0 20 0 a sampled in October 2011 and sequenced at 10% of full lane of Illumina; replicate samples (October 2012) were sequenced using full-lane Illumina (Supplemental Table 1 ). b MAG name reflects taxonomic resolution (closest relative) at phylum, order, family, genus or species level. OCT \n Octopus Spring ; CON \n Conch Spring . MAGs from 2011 carry a ‘-r01’ suffix to separate them from MAGs curated from the 2012 samples (-r02) c Abundance calculations made with CheckM 61 as the percentage of reads mapped to each MAG relative to all mapped reads. d SD is the standard deviation of either G + C content or Coverage ( e Cov.) f Comp. Estimate of genome completeness, redundancy and heterogeneity are based on CheckM 61 . Nanopusillus completeness is low due to the small genome. Phylogenetics of early-evolved thermophiles A bacterial phylogeny based on a standard group of 16 ribosomal proteins (Fig. 4A ) shows that the Pyropristinus lineage is the most deeply rooted bacterial group independent of the Candidate Phyla Radiation (CPR) 26 . The Pyropristinus lineage also includes the WOR-like populations described in the Genome Taxonomy Database 27 (GTDB) (Fig. 4A ). A bacterial phylogeny based on the 16S rRNA sequence (Fig. 4B ) also shows that Caldipriscus and Thermoproauctor represent two separate branches within the Pyropristinus lineage 28 , each branching deeper than the Aquificota and Thermotogota 24 . In summary, both microbial communities contain several early-evolved bacterial lineages including the Pyropristinus, the Aquificota, and the Calescibacteria. Functional traits of these groups may provide insight into the activities of microorganisms thought to have been important in early life 4 , 29 . Fig. 4 Phylogenomics of early-evolved bacteria in Conch and Octopus Springs . Bayesian phylogenetic trees of Bacteria using A a concatenation of 16 ribosomal proteins (2447 residues) or B the 16S rRNA gene (sequences > 1000 bp only). Members of the Pyropristinus lineage include Caldipriscus and Thermoproauctor spp. from circumneutral (pH 7–9) hyperthermal (>75 °C) geothermal springs [OCT Octopus Spring, CON Conch Spring, BCH Bechler, FF Fairy Falls, PS Perpetual Spouter; *16S rRNA clones EM3 and EM19 from Octopus Spring 23 ; ** lineages contain populations from Octopus Spring (e.g., Patescibacteria within Candidate Phyla Radiation 26 (CPR) (2 types, OCT 2012), Thermus aquaticus OCT 2011, 2012, Thermoflexus hugenholtzii OCT 2011, 2012) or Conch Spring (Thermodesulfobacteria); Uncollapsed versions of these phylogenetic trees are provided in Supplementary Figs. 5 and 6 . Geochemical forcing: Impacts on functional genomics The geochemical conditions in Conch and Octopus Springs are linked to differences in the distribution of key functional genes that regulate electron transfer reactions involving arsenic, sulfur, and oxygen. Keystone populations present in these alkaline (pH 8-9) habitats included Thermocrinis , Pyrobaculum and Caldipriscus , and although the metabolic potential of each of these organisms was highly similar between the two habitats, there were several notable exceptions (Fig. 5 ). For example, Pyrobaculum populations in Octopus Spring contained an arsenite oxidase (Aio) and HCO complex, while these genes were completely absent in the Pyrobaculum from Conch Spring . Caldipriscus populations also contained a type 1 HCO but only the population from Conch Spring contained a cytochrome bd ubiquinol oxidase (Cyt bd), known to be a high-affinity oxygen reductase 20 as well as a sulfide:quinone oxidoreductase (Sqr) that is important in the oxidation of sulfide 30 . Thermocrinis , the predominant population in both communities, exhibited similar metabolic potential in each habitat, including the oxidation of thiosulfate (Sox complex) and arsenite (Aio) using oxygen as an electron acceptor (HCO). However, as will be shown, the actual transcription of these genes was remarkably different between sulfidic and microaerobic environments. A broader look at energy-related genes using hierarchal clustering (Supplementary Fig. 7 ) shows similar results, and high reproducibility between each of the phylotypes found in both habitats (i.e. , Thermocrinis , Pyrobaculum , Caldipriscus ). Thermocrinis populations in both environments contained a convincing set of genes necessary for the synthesis and use of flagella (Supplementary Fig. 8 ). Fig. 5 Electron transfer and carboxylation genes in microbial populations from Conch and Octopus Springs . Keystone microbial populations present in both springs ( A ), or populations present only in Conch ( B ) or only in Octopus ( C ) Springs [ sox sulfur oxidation pathway, aio arsenite oxidase, sqr sulfide:quinone oxidoreductase, ttr tetrathionate reductase, psr polysulfide reductase, dsr dissimilatory sulfite/sulfate reductase, hco heme Cu oxidases, cyt bd cytochrome bd ubiquinol oxidase, cyd AA’ archaeal cytochrome oxygen reductase, ccl/ccs citryl-CoA lyase/citryl-coA synthetase, fdh formate dehydrogenase, acc acetyl CoA carboxylase, por pyruvate oxidoreductase]. Dissolved sulfide and oxygen concentrations affect the overall community composition in each geothermal habitat, as well as the metabolic attributes of additional community members (Fig. 5 ). For example, higher sulfide levels in Conch Spring result in lower microbial diversity and select for organisms that are known to prefer suboxic conditions (DO < 1 µM) such as organisms within the Thermodesulfobacteria 31 and Thermosphaera 32 . These populations also contain higher affinity oxygen reductases including the cytochrome bd ubiquinol oxidase (bacteria) and cytochrome AA’ oxidase (archaea) 18 . Moreover, the number of genes potentially involved in the oxidation of sulfide (Sqr), the reduction of sulfur compounds (Ttr, Psr, Dsr) and the reduction of nitrate (NarG) were notably higher in the sulfidic (and suboxic) Conch Spring (Fig. 5B ). The Thermodesulfobacteriaceae in Conch Spring was the only population with a nearly complete set of genes necessary for dissimilatory sulfate reduction 33 . Pyrobaculum populations from both springs contain DsrAB, but lack other major proteins required for sulfate reduction. The complete absence of Thermoproauctor , Calescibacterium , Calditenuis , and other heterotrophs under sulfidic conditions ( Conch Spring ) is likely due to the lack of a high-affinity cytochrome bd ubiquinol oxidase and/or direct toxicity from sulfide. Conversely, the microbial community in Octopus Spring was more diverse, which is attributable to higher levels of DO ranging from 40–50 µM (Fig. 2 ). Specifically, genes required for oxygen respiration using type 1 HCO complexes were present in nearly all the microorganisms from Octopus Spring (Fig. 5C ). Most of the additional organisms in Octopus Spring are heterotrophic, as evinced by the absence of chemolithotrophic markers (e.g., Fig. 5C ) and high gene counts of a diverse suite of carbohydrate degrading enzymes (Supplementary Fig. 9 ). Numerous glycosyltransferases (e.g., families GT4, GT5, GT9 of the CAZy database 34 ), glycoside hydrolases (family GH109), and carbohydrate binding modules (e.g. family CBM50) were observed in Armatimonadota (types 1 and 2), Calescibacteria and Themoflexus populations, respectively. The keystone populations common to both habitats have genes necessary for the fixation of carbon dioxide via either the reverse TCA cycle (primarily Thermocrins , e.g., ccl/ccs = citryl-CoA lyase/citryl-coA synthetase 24 ) or other carboxylases (e.g., acc = acetyl coA carboxylase; por = pyruvate oxidoreductase) (Fig. 5 ). This is consistent with stable-isotope ( 13 C) analyses, which revealed high levels of CO 2 fixation in both communities 35 . In fact, microbial biomass from Conch Spring originates nearly entirely (>90%) from the fixation of CO 2 35 , which correlates with the large fraction of primary autotrophs Thermocrinis , Pyrobaculum , and Caldipriscus (up to 90%, Fig. 3 ). Conversely, the presence of numerous aerobic heterotrophs in Octopus Spring explains 13 C-biomass values that reveal a greater fraction of microbial biomass (up to 40%) acquired via dissolved organic carbon 35 . Consequently, higher oxygen levels in Octopus Spring select for a more diverse, aerobic, and heterotrophic microbial community than present in the highly sulfidic Conch Spring , where DO levels were below detection (<1 µM). Finally, the acquisition of sufficient Fe can be problematic in high pH environments due to the low solubility of ferric iron solid phases. Concentrations of dissolved Fe in Conch and Octopus Springs were below detection using ICP-OES (~1 µM) and numerous phylotypes in these habitats showed genomic capabilities for enhanced Fe transport, siderophore production, as well as Fe gene regulation (Supplementary Figs. 10 and 11 ). No evidence of Fe reduction and/or Fe oxidation existed in these communities, which is consistent with low dissolved Fe as well as the absence of any Fe solid phases. Metatranscriptomics: Energy conservation and respiration Thermocrinis , Pyrobaculum and Caldipriscus MAGs accounted for ~80–90 % of the total transcript sequence mapped to community populations from Conch and Octopus Springs (Fig. 6A ). Metatranscriptomes from Octopus Spring (years 2011 and 2016) were highly similar ( p < 0.01), and the fraction of transcripts mapped to Thermocrinis , Pyrobaculum and Caldipriscus was notably consistent (also see Supplementary Figs. 12 and 13 ). The large number of transcripts mapped to these highly abundant and active populations provided sufficient coverage to evaluate the activity of numerous cellular processes (Supplementary Data 1 – 3 ). Fig. 6 Analysis of metatranscriptomes from Conch (2016) and Octopus (2011, 2016) Spring filamentous streamer communities. A Transcript abundance by major phylotype (Octopus 2011 solid blue, Octopus 2016 hatched blue, Conch 2016 hatched yellow). B Abundance of transcripts mapped to specific functions within Thermocrinis , Pyrobaculum , and Caldiprisc us metagenome assembled genomes (MAGs), expressed as a percent of total transcripts within each MAG. [Armatimona. T1 Armatimonadota type 1, aio arsenite oxidase, sox sulfur oxidation complex, hco heme Cu oxidase complex subunits I, II and III, cyt BC cytochrome bc complex, rhod rhodanese sulfur transferase, sqr sulfide:quinone oxidoreductase, cyt BD cytochrome bd ubiquinol oxidase; cyd AA’ = archaeal cytochrome oxidase, por pyruvate ferrodoxin oxidoreductase complex, hgl hemoglobin. Source data are provided as a Source Data file. The ratio of DS to DO ranges from > 100 in Conch Spring to <0.025 in Octopus Spring , a factor of approximately 4000 times. Transcript abundances of genes involved in energy conservation and electron transport revealed several major shifts in metabolism consistent with this large difference in sulfide: oxygen ratio (Fig. 6B ). Thermocrinis arsenite oxidase (Aio) and sulfur oxidase (Sox) complexes were highly transcribed (4–5% of transcripts) in Octopus Spring yet these same genes were not expressed in Conch Spring . Arsenite oxidases were also highly transcribed (~2% of transcripts) in Pyrobaculum populations in Octopus Spring , yet these transcripts were completely absent in Conch Spring despite nearly identical levels of total soluble arsenic (Table 1 ). Moreover, the type 1 HCOs of Pyrobaculum , along with associated cytochrome bc complexes were only transcribed in Octopus Spring (> 1% of transcripts), which indicates that oxygen is serving as an electron acceptor for the oxidation of arsenite and generation of ATP 36 . The large fraction of transcripts consistently mapped to Thermocrinis and Pyrobaculum AioAB genes indicates the importance of arsenite as a critical energy source for chemolithoautotrophs under non-sulfidic, microaerobic conditions (Fig. 7 ). Arsenite oxidases were previously shown to be expressed in several Aquificales-dominated filamentous communities using reverse transcriptase-PCR 36 , especially in non-sulfidic geothermal channels. Moreover, Thermocrinis ruber was cultivated from Octopus Spring 37 and has been shown to oxidize arsenite aerobically in pure culture 38 . The Sox sulfur oxidation pathway was also highly expressed in Thermocrinis , but only in Octopus Spring where 4–5% of Thermocrinis transcripts were mapped to the Sox genes, including subunits SoxA, SoxB, SoxX, SoxY and SoxZ (Fig. 7 ). No SoxCD was observed in the Thermocrinis Sox complex, which suggests that thiosulfate is partially oxidized to S and sulfate 39 . The Thermocrinis Sox complex was not expressed in Conch Spring where high sulfide: oxygen ratios favored Sqr proteins (Fig. 6 ). Fig. 7 Primary respiratory metabolism and energy generation (percent of total transcripts within each organism) in keystone populations present in both Conch and Octopus Springs. Ratios of DS:DO were nearly 4000 times higher at sample sites in Conch versus Octopus Springs . [Gene names: sqr = sulfide:quinone oxidoreductase, cyt BD = bd-type ubiquinol cytochrome oxidase; cyd AA’ = archaeal cytochrome oxidase; sox = sulfur oxidation complex including subunits sox A, sox B, sox X, sox Y, and sox Z; aio AB = arsenite oxidase large and small subunits; HCO = heme Cu oxidase complex subunits I, II and III; and energy generating ATPase subunits a, b, and c.]. High-affinity oxygen reductases including the cytochrome bd ubiquinol ( Caldipriscus ) and cytochrome CydAA’ oxidases ( Pyrobaculum ) 18 were highly transcribed in Conch Spring , indicating a major shift in energy metabolism yet still dependent on oxygen (Figs. 6 and 7 ). The CydAA’ cytochromes have recently been shown 18 to serve as bona fide oxygen reductases and are important in numerous archaea 17 , 40 . Expression of the high-affinity cytochrome bd ubiquinol and CydAA’ oxidases occurred when DO levels were below detection (<1 µM) and likely in the nanomolar range. The HCOs of Caldipriscus and Thermocrinis were over-expressed in Conch Spring , along with increased levels of the cytochrome bc complex compared to Octopus Spring . Sqr proteins implicated in the oxidation of sulfide 30 were only expressed significantly in Conch Spring (Fig. 7 ), as well as rhodanese sulfur transferase domains and an oxygen-binding hemoglobin gene ( Thermocrinis ). The major shifts in respiration pathways observed for these keystone populations reveal a tight linkage between the availability of oxygen and the metabolic strategies employed for energy conservation. Type IV filaments: Microbial community attachment One of the most noticeable features of high-temperature filamentous streamer communities is the surprising abundance of extracellular structures resembling pili with diameters of ~ 20–25 nm (Fig. 8 , Supplementary Fig. 14 ). Numerous genes important in the synthesis, secretion and function of type IV filament (Tff) machinery 41 – 43 were highly expressed in Thermocrinis populations in both Conch and Octopus Spring (Fig. 8 ). Genes involved in Tff production and activity were found on two different contigs and were observed in numerous metagenomes as well as Thermocrinis ruber , which was isolated from Octopus Spring 37 . Piliation can serve multiple functions including natural transformation through DNA uptake, export of filamentous phages, protein secretion, adhesion, and electron transport 41 – 45 . Fig. 8 Type IV Filament Systems and Field-Emission Scanning Electron Microscopy. Highly expressed type four filament (Tff) systems in Thermocrinis MAGs ( A ) likely explain the extensive network of pili-like structures (~20–25 nm diameter) commonly observed in filamentous streamer communities from Conch Spring ( B ) and Octopus Spring ( C ). [Abbreviations and Definitions: Bechler 2008 = Thermocrinis entry from Bechler Spring, YNP 24 . Arrows in C indicate Tff structures versus cells. [ pil A = Type IV pilus assembly, pil W = Type IV pilus assembly, pul G = Type II secretory pathway, fim T = Type IV fimbrial biogenesis, pil Y = adhesin; nfu A = Fe-S biogenesis, pil Q = pilus secretin, rec J = ssDNA exonuclease, HCO = heme copper oxidase complex subunits I, II, and III. Source data are provided as a Source Data file. Micrographs shown in B and C were chosen from a large collection of over 30 replicates from 3 different sample years and a minimum of 10 replicate images per year. Additional FE-SEM micrographs are provided in Supplementary Fig. 14 . Complete tables of observed transcripts and expression levels are provided in Supplementary Data 1 – 3 ]. Very high transcript levels of pil A (1.3 to 5.8% of transcripts) and nfu A (0.6 to 2.5% of transcripts) in Thermocrinis confirm the microscopic evidence that large amounts of cellular resources and carbon are being directed to the formation and maintenance of an extensive Tff network (Fig. 8 ). PilA is the protein comprising actual major pilin structure 43 and NfuA is an Fe/S assembly protein used in maturation and repair of Fe-S proteins, which is often associated with electron transport and Fe stress 46 , 47 . Genes for the fixation of inorganic carbon (citryl co-A lyase and citryl-CoA synthetase/succinyl-CoA synthetase) 24 were also highly expressed in Thermocrinis (up to 0.1 % of transcripts in Conch Springs ). We have shown using 13 C isotope analyses that autotrophically fixed inorganic carbon (CO 2 ) is a very significant fraction (i.e., 50- > 90 %) of the total biomass carbon in both communities 35 . In fact, the percent of biomass originating from CO 2 is highly correlated with the fraction of keystone populations in each community (e.g., Fig. 3 ). Ultimately, the energy required to reduce significant amounts of inorganic carbon and form extensive Tff systems comes from the oxidation of arsenite and reduced sulfur species (i.e., sulfide, polysulfide(s), and/or thiosulfate). Type IV filament systems are central to several processes important in the colonization and survival of Thermocrinis in turbulent, high-velocity (~0.2 m s −1 ) geothermal outflow channels. Firstly, adhesion to solid substrates is an absolute prerequisite for attachment and growth of filamentous streamer communities (Supplementary Videos 1 and 2 ). PilY, which was transcribed by the ‘Pil’ operon in Thermocrinis (Fig. 8 ) is a known adhesin protein shown to be important in bacterial attachment to surfaces 48 . Secondly, ‘pilin’-like structures can be conductive and promote electron transfer reactions. Evidence obtained here shows that the HCO complex (subunits I, II and III) are located near the nfu A and pil Q and were co-transcribed in Thermocrinis populations from Conch Spring (Fig. 8 ). Finally, the actual secretin ( pil Q) 49 in Thermocrinis was also conserved across numerous metagenome entries and present in Thermocrinis ruber . Early-evolved thermophiles: Relevance to early metabolisms Low-oxygen environments were undoubtedly very important under early-Earth conditions 6 prior to the GOE. Early-evolved thermophiles provide clues for understanding how early microbial lineages may have harnessed low levels of oxygen in extreme environments. Alkaline-chloride filamentous communities contain several major lineages of aerobic thermophilic bacteria including the Aquificota 24 , two representatives of the Pyropristinus lineage ( Caldipriscus and Thermoproauctor 28 ), the Calescibacteria ( Calescibacterium ) as well as two novel aerobic organisms distantly related to members of the Armatimonadota shown in the GTDB 27 . Each of these aerobic lineages, including the Pyropristinus, Aquificota and Calescibacteria (and to a lesser extent, Armatimonadota) occupy deep phylogenetic positions (e.g., Fig. 4 ). The monophyletic HCO genes within these lineages is consistent with the hypothesis that oxidases were important in the early evolutionary history of archaea and bacteria. Protein modeling of the subunit I HCO (Supplementary Fig. 15 ) present in Caldipriscus and Thermoproauctor (Pyropristinus lineage) as well as for Thermocrinis and Pyrobaculum indicate that these proteins are all type 1 HCOs (Supplementary Table 4 ) expected to reduce oxygen and create proton motive force driving the production of ATP 50 . Arsenite oxidase has also been considered an ancient bioenergetic protein, which existed prior to the divergence of archaea and bacteria 51 , 52 . Moreover, arsenite may have served as an electron donor for anoxygenic phototrophy in the early Archaean 53 . Indeed, in the current study, both Thermocrinis and Pyrobaculum (bacteria and archaea, respectively) obtain energy for metabolism using arsenite as an electron donor under low-oxygen conditions. However, high levels of sulfide suppress the oxidation of arsenite, which may be due to a combination of a stronger donor via Sqr proteins and/or oxygen limitation due to high sulfide: oxygen ratios. The discharge of reduced geothermal and/or hydrothermal fluids creates complex mixing environments where turbulence and gas-exchange (e.g., oxygen, sulfide) converge to create optimum habitats for filamentous chemolithoautotrophs 24 , 54 . These same processes were likely critical in hydrothermal environments thought to be important on an early Earth 4 , 29 . The solubility of oxygen in water is approximately 2 times lower at 75–80 °C versus 25 °C. However, oxygen reduction by relevant electron donors is highly exergonic even under nanomolar levels of oxygen 54 , which have been shown to support microbial aerobic respiration using high-affinity oxygen reductases 14 , 18 , 20 , even in the presence of sulfide 21 , 22 . Transcriptomic measurements reported here provided sufficient resolution to understand the in situ physiology of three keystone populations, which were important in both communities but exhibited major metabolic shifts due to changes in sulfide and oxygen concentrations. Type I terminal oxidase complexes were highly expressed under microaerobic conditions and contributed to extensive heterotrophic microbial diversity. Conversely, high-affinity oxygen reductases including both the cytochrome bd ubiquinol oxidases ( Caldipriscus ) and the cytochrome CydAA’ oxidase ( Pyrobaculum ) were highly expressed under sulfidic conditions where DO levels were below detection (<1 µM), and likely in the nanomolar range. Both geothermal habitats have unique relevance to possible geochemical circumstances prior to the GOE and indicate the metabolic resilience of early-evolved thermophiles to low-oxygen conditions."
} | 8,938 |
38074452 | PMC10702926 | pmc | 4,799 | {
"abstract": "Forests are highly productive ecosystems that contribute to biogeochemical cycles of carbon and nitrogen, through which it regulates climate and global change. Forests are also spatially highly heterogeneous ecosystems that comprise a multitude of microbial-mediated reactive interfaces. These are mainly the root–soil interface, litter–soil interface, root–root interface, and plant–atmosphere interface. Each of these interfaces has its own unique characteristics, e.g., specific drivers that affect the microbial abundance, nutrient availability, microbial community, and the dominance of certain microbial taxa. Here, we review the microbial-mediated reactive interfaces in forests, focusing on interrelation and dynamics of fungi and bacteria on a broad temporal scale with ecosystem processes ranging from short-term events (e.g., seasonal changes) to long-term stand development suffering a global climate change (e.g., global warming or nitrogen deposition). We argue that in-depth knowledge of forest microbiology can only be obtained by exploring the complex forest microbiome and its ecosystem functions. Underpinning the basis for individual forest variation would ultimately facilitate the formulation of microbiome-based strategies in the future.",
"introduction": "1 Introduction Forest biomes cover an area of approximately 40.6 million km 2 of the terrestrial surface, encompassing more than 3 trillion trees [ [1] , [2] , [3] ]. Forest ecosystems play an important role in maintaining regional ecosystem services, including food and timber production as well as the reduction of carbon footprints and biogeochemical cycles of elements [ 2 , 4 , 5 ]. Ecological research on forests has accumulated much information, with the focus mainly on temperate and boreal forests [ [6] , [7] , [8] ]. Although an individual tree represents a multitude of habitats that are highly spatially heterogeneous, it comprises several microbial-mediated reactive interfaces (or niches), such as root–soil interface [ 9 ], litter–soil interface [ 10 ], root–root interface [ 11 ], and plant–atmosphere interface [ 12 ]. All these interfaces are interrelated and have crucial implications for forest ecosystem processes and functions [ 13 , 14 ]. In this regard, microorganisms drive niche-based processes with significant influences on plant nutrients, interactions, and competition [ 15 , 16 ]. Rapid changes in soil microbial community affect the responses of a tree distribution and population coexistence to new selection pressures in the environment [ 17 , 18 ], thus modifying the interactions between microbiomes and tree hosts and showing increasing joints regarded as holobionts [ 19 ]. Compared to bacteria, fungi appear to attract more attention in forest ecosystems [ 20 ], and studies have shown that fungi in forests are highly diverse and sensitive to environmental changes in terms of community structure, diversity, and biomass [ 21 , 22 ]. The study of forest microbial communities lags behind that of agricultural plant microbiomes, as the development of a forest ecosystem is a complex systematic process that is affected not only by the soil but also by roots and rhizosphere, litter, and phyllosphere [ 6 ]. Interactions between trees and their associated microbial communities are tremendously complex [ 6 , 13 , 14 ]. The spatial heterogeneity of these interfaces complicates stand-level predictions of ecosystem functions, and all these microbial-mediated interfaces are interrelated by frequent microbe flows (i.e., microbial dispersion and blending). Moreover, most ecosystem processes affect multiple habitats at the same time and cannot be properly understood without considering their functioning as a whole [ 6 ]. Thus, there exists an urgent need to proceed from the “forest microbiome” by integrating the microbiology related to the main reactive interfaces in forest ecological processes, with the aim to address the whole microbiota associated with multiple habitats in the forest of any type. The collective information on microbial status (e.g., the structure and function of microbiota) associated with multiple habitats from certain forest allows for an understanding of the relationships among microbes and between the microbiome and environmental parameters. Metatranscriptomics and high-throughput sequencing approaches help explore microbial communities and their functioning at an unprecedented resolution, and stable isotope labeling and NanoSIMS (nanoscale-secondary-ion mass spectrometry) approaches are powerful tools for tracing the fate of plant residues and nutrients in soil [ 20 , 23 , 24 ]. Here, we review microbial-mediated processes in forests on the scale of individual reactive interfaces and highlight the forest microbiome being substantial for understanding the forest ecosystem functions. We mainly focus on bacterial and fungal communities, for which genomic resources and literature data are available. Despite forest ecosystem processes being highly complex and affected by multiple interrelated interface processes, studies have so far most progressed at the individual interface level. We demonstrate that a comprehensive understanding of forest ecosystem functions can be achieved by considering microbial-mediated processes across different reactive interfaces. We also summarize the current knowledge gaps to help guide future research on this topic."
} | 1,350 |
38079551 | PMC10743459 | pmc | 4,800 | {
"abstract": "Significance Anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) often associate as multicelled consortia in methane seep sediments and carbonates, with a poorly understood preservation potential in the rock record. Here, we provide evidence for microbially enhanced precipitation of an amorphous silica-rich phase on the exterior of ANME-SRB consortia both in methane-rich sediments, carbonates, and a methane-oxidizing laboratory enrichment culture. This consortia-associated mineral phase may represent a newly discovered microbially enhanced biomineralization mechanism, potentially involved in the preservation of fossilized ANME-SRB consortia in ancient methane seep carbonates. Our results expand on the knowledge of microorganisms and metabolisms facilitating silica mineralization, with important implications for the morphological and structural preservation of microbial fossils in deep time on Earth and other planetary bodies.",
"conclusion": "Conclusions Silicates have been found in association with ANME-SRB consortia, but a proof of their direct involvement in silicate biomineralization was lacking. We provide evidence for common occurrence of Si-rich phases in carbonate and sediment-hosted consortia and show precipitation of Si-rich phases with clay-like chemistry and morphology by actively growing ANME-2/SRB consortia in long-term AOM enrichment cultures. Our results suggest a direct role for methanotrophic microbial assemblages in the precipitation and evolution of silicate-bearing mineral structures, a process possibly distinct from observed microbially mediated silicate biomineralization in diverse microorganisms, all of which have poorly understood mechanisms. As the early precipitation of authigenic silicates is generally thought to improve the preservation potential of fossils ( 22 , 81 , 82 ), our results further support the possibility that massive chert precipitation as observed in classic microfossil localities such as the Gunflint ( 83 ) may not be critical for preserving microbial fossils in the rock record. Instead, microscale associations between microorganisms and amorphous Si-rich phases may provide adequate preservation potential, offering a search image in paleo-seep carbonates, and further serve as a geochemical trace fossil in the rock record.",
"discussion": "Discussion We observed and identified Si-rich phases on ANME-SRB consortium exteriors from seep sediments, authigenic carbonates, and sediment-free AOM enrichment cultures that were incubated in artificial seawater media undersaturated with respect to silica. Measurement of [Si] in sediment-free incubation media via ICP-MS precluded abiotic mechanisms of Si enrichment of consortium-attached Si-rich phases, as [Si] was too low to drive either amorphous silica precipitation or Si sorption on preexisting consortium-attached silicates. Additionally, most consortium-attached phases are enriched in Si with respect to detrital silicates in the sediments from which they were sourced, and also compared to known clay mineral compositions, suggesting that these phases are not simply a product of the attachment of clay minerals in sediment. For example, in Si-rich endmember clay compositions such as montmorillonite [(Na,Ca) 0.33 (Al,Mg) 2 (Si 4 O 10 )(OH) 2 ·nH 2 O)], with a 2:1 ratio of tetrahedral to octahedral sheets and where Si occupies all tetrahedral sites, the octahedral cation to Si ratio is 0.5 ( 53 ); however, consortium-attached Si-rich phases had octahedral cation: Si ratios generally <0.5 ( Fig. 3 A ). For comparison, methane seep sediment samples from which consortia were extracted had octahedral cation to Si ratios typically >0.5, consistent with clay minerals ( Fig. 3 A ). These results contrast with a previous report of incrustation of AOM consortia with clay minerals ( 10 ), however, the phases reported in that study also had cation: Si ratios generally <0.5, implying there also existed minerals more enriched in Si compared to known clay minerals. Our mineral stability diagrams ( Fig. 3 B and SI Appendix , Fig. S4 ) showed that the media solution chemistry was undersaturated with respect to quartz (amorphous or crystalline), but not with respect to clay precipitates, notably kaolinite ( Fig. 3 B ). Furthermore, the morphology of our Si-enriched precipitates is not consistent with crystalline clay phases ( Fig. 1 and SI Appendix , Fig. S1 ), but rather with the spheroid-like particles expected for the formation of clays within organic assemblages ( 26 ). However, the Al/Si content is lower than what is found in clay composition. Altogether, our findings led us to propose that the Si-rich phase associated with AOM consortia was likely a result of two types of biological processes: biomineralization of Si-enriched precipitates from solutions undersaturated with respect to silica and the formation of clay precipitates in intimate associations with the organic/cellular assemblages. Microbial precipitation of silica in undersaturated conditions has been previously proposed to be mediated by iron ( 39 ) and magnesium ( 54 , 55 ) in solution. Experimental studies in cyanobacteria have shown that sulfate-rich EPS and an increase in pH promoted by photosynthesis enhance the precipitation of magnesium-rich silica, where magnesium likely acts as a cation bridge between positively charged silicic acid and negative functional groups in the EPS ( 55 ). Although we did document a carbon-rich rim around two ANME-SRB consortia within the carbonate rock ( Figs. 4 B and 5 A ), consistent with the presence of EPS, this mechanism is not congruent with our observation that the silicates attached to ANME-SRB consortia have low Al, Fe, and Mg content (<0.6 % wt). In this study, Si-rich phases were observed in association with the exteriors of ANME-SRB consortia, consistent with a previously published study ( 10 ). This is surprising since cell interiors might be more conducive to silicification in undersaturated conditions, for example, as is observed in the intracellular Si-concentrating mechanisms in diatoms ( 56 ), but it is similar to observations of Si on the surfaces of cyanobacterial cells ( 54 ). The silicification at cell surfaces is thought to be mediated by EPS and magnesium in cyanobacteria ( 55 ), and during the reductive dissolution of Fe 3+ -rich clay minerals by metal-reducing Shewanella oneidensis ( 25 , 57 ), which appear to share common textural characteristics with the amorphous silica phase reported here ( 25 ). Notably, the involvement of AMME-2/SRB consortia in extracellular electron transfer (EET) between partners ( 58 ) and, in some circumstances with iron or manganese oxides (e.g., ref. 59 ), may point to the local generation of conditions favorable for silicification facilitated by EET. Silicate precipitation concomitant with microbial respiration of Fe in clay minerals ( 57 , 60 ) has been proposed to occur through interactions with polyamines ( 60 ). Long-chain polyamines (>7 aminopropyl units) can precipitate silica from silica oligomers in undersaturated conditions ( 61 ), which is mediated by bacteria under natural ( 62 ) and experimental ( 60 ) saturated silica conditions. Microbially mediated silicate precipitation has also been observed in the spore coat of Bacillus subtilis , in this case mediated by a serine- and arginine-rich protein ( 63 ). The zwitterionic nature of this protein conferred by serine and arginine residues is similar to that of silacidins and silaffins, proteins that mediate silica precipitation in diatoms ( 64 , 65 ). To explore potential biochemical mechanisms of Si-enriched mineral precipitation by ANME-SRB consortia, we screened the genomes (MAGs) of diverse ANME and syntrophic SRB assembled from metagenomic datasets ( 14 , 66 , 67 ). Following the approach of previous work targeting silaffin-like proteins in diatom genomes ( 68 ), we performed a search of our genomic database to find candidate proteins from ANME and SRB MAGs in sediment-free cultures with serine- and arginine-rich domains. We also searched our genomic database for homologs of aminopropyl transferases. However, no definitive homologs known to be associated with silicate precipitation were identified. The fact that there are currently several independent pathways for microbially mediated silica precipitation described suggests there are likely additional discoveries of biological mechanisms underlying this process. Different polyamines, such as copolypeptides with high block ratio of lysine and phenylalanine, polyallylamine, polyethylenimine, poly[acrylamide-co-2-(dimethylamino)]ethyl methacrylate or amine terminated dendrimers can act as templates for silica formation in vitro ( 69 ). Additionally, several posttranslational modifications (PTMs) have shown to be essential for the Si-precipitating property of silaffins, such as di- or trimethylation or alkylation of lysine residues with N-methylated oligo-propyleneimine chains, hydroxylation and phosphorylation of all the trimethylated lysine residues at the δ-position, and phosphorylation of all serine hydroxyl groups. The PTMs introduce a significant amount of both positive and negative charges, which render the peptide zwitterionic, a property critical for silica precipitation ( 70 ). These characteristics could be used as targets for searching and identifying potential proteins involved in this process. While the genomic mechanism currently remains elusive, our geochemical measurements ( Fig. 3 B ) and modeling results ( Fig. 3 C ) support the biologically mediated formation of Si-rich precipitates by AOM consortia in undersaturated conditions that is inconsistent with abiotic precipitation. Silicification relies on reactive cell surface ligands that can adsorb silica from solution, implying that cell surface charge may be critical for the initial silicification process ( 13 ). A neutral membrane, such as the one that enhances silica biomineralization in the cyanobacterium Calothrix , would be hydrophobic, thus enhancing the attachment of silica ( 38 ). For negatively charged cells, metal cation bridges (e.g., Fe 3+ , Al 3+ ) might be necessary for silicification since the organic ligands at cell surfaces would be electrostatically repulsed to the negatively charged silica species ( 13 ). Notably, the potential for proton buffering by polymerized silica previously demonstrated in diatoms ( 71 ) may also benefit the ANME-SRB methanotrophic syntrophy, where proton build-up is predicted to occur during direct interspecies electron transfer, as electron transfer is faster than proton diffusion, resulting in local pH gradients ( 72 , 73 ). We suggest that future research focuses on investigating cell or consortia surface charges ( 74 ), and differences in the functional groups between the cell wall and EPS, to gain further insights into the silica precipitation mechanisms used specifically by methanotrophic ANME-SRB consortia. The mode of ANME-SRB biologically mediated silica precipitation of clay-related Si-rich phases described in this study may be important for the preservation of organic carbon in seep carbonates. Early diagenetic silica is observed in fossil seep carbonates spanning the Phanerozoic, where silica appears as fibrous and botryoidal cements replacing aragonite ( 49 , 75 – 79 ). Early silica precipitation in seep carbonates can entomb organics; in one striking example, preservation of Cretaceous chemosynthetic symbiotic tube worm chitin in early silica cements has been documented ( 80 ). The observations that ANME-SRB consortia precipitate Si-rich phases both in laboratory enrichment cultures and what appears to be in situ within seep sediments and authigenic carbonates suggest that early amorphous silica cementation in seep carbonates may also improve the preservation potential of body fossils and associated organics from ANME-SRB consortia in ancient seeps. We propose that future work could use Si-rich rings around carbonaceous domains in fossil seep carbonates as a search image to examine the potential preservation of ANME-SRB consortia preserved in the rock record."
} | 3,038 |
27379068 | PMC4908113 | pmc | 4,802 | {
"abstract": "We investigated the role of arbuscular mycorrhizal fungal (AMF) hyphae in alternation of soil microbial community and Aroclor 1242 dissipation. A two-compartment rhizobox system with double nylon meshes in the central was employed to exclude the influence of Cucurbita pepo L. root exudates on hyphal compartment soil. To assess the quantitative effect of AMF hyphae on soil microbial community, we separated the hyphal compartment soil into four horizontal layers from the central mesh to outer wall (e.g., L1–L4). Soil total PCBs dissipation rates ranged from 35.67% of L4 layer to 57.39% of L1 layer in AMF inoculated treatment, which were significant higher than the 17.31% of the control ( P < 0.05). The dissipation rates of tri-, tetrachlorinated biphenyls as well as the total PCBs were significantly correlated with soil hyphal length ( P < 0.01). Real-time quantitative PCR results indicated that the Rhodococcus -like bphC gene was 2–3 orders of magnitude more than that of Pseudomonas -like bphC gene, and was found responded positively to AMF. Phylogenetic analyses of the 16S rDNA sequenced by the Illumina Miseq sequencing platform indicated that AMF hyphae altered bacterial community compositions. The phylum Betaproteobacteria and Actinobacteria were dominated in the soil, while Burkholderiales and Actinomycetales were dominated at the order level. Taxa from the Comamonadaceae responded positively to AMF and trichlorinated biphenyl dissipation, while taxa from the Oxalobacteraceae and Streptomycetaceae responded negatively to AMF and PCB congener dissipation. Our results suggested that the AMF hyphal exudates as well as the hyphae per se did have quantitative effects on shaping soil microbial community, and could modify the PCBs dissipation processes consequently.",
"conclusion": "Conclusion This study demonstrates the important role of arbuscular mycorrhizal hyphae in degradative efficiency of different PCB congeners. Analysis of bacterial growth, bph gene abundance, and bacterial community composition were also shown to vary with different soil mycorrhizal hyphal biomass. The results strongly supported our previous hypothesis that the mycorrhizal hyphae could accelerate the dissipation of low-chlorinated biphenyls as well as shaping the soil PCB congener profiles via altering bacterial growth and community compositions in the mycorrhizosphere. As the AMF-plant symbiosis is ubiquitous in terrestrial ecosystems, the influence of AMF on PCB congener dissipation is broadly relevant across terrestrial ecosystems for the remediation of PCB contaminated soils. These data improved our understanding of mycorrhizal hyphae-bacteria interactions in PCB dissipation. Further investigation is needed to identify the PCBs degraders in AM fungal hyphosphere soil, and understand the metabolic pathways involved in PCBs degradation.",
"introduction": "Introduction Arbuscular mycorrhizal fungi (AMF) are ubiquitous in the terrestrial ecosystem. It is estimated that 250,000 species of plants worldwide, including many arable crops, are capable of forming the symbiosis with this group of fungi ( Smith and Read, 2008 ). AMF receiving carbon from their host, and in return, delivering nutrients and water back. It has been estimated that in natural ecosystems plants colonized with AMF may invest 10–20% of the photosynthetically fixed carbon in their fungal partners ( Johnson et al., 2002 ), and this significant input of energy and carbon into the soil ecosystem could be crucial to microorganisms associated with the AMF. As the extraradical hyphae of AMF provides a direct pathway for translocation of photosynthetically derived carbon to the soil, the continuous provision of energy-rich compounds, coupled with the large surface area of the hyphae that intact with the surrounding soil environment (hyphosphere) provide important niches for bacterial colonization and growth ( Toljander et al., 2007 ). The AMF hyphae may have both positive ( Johansson et al., 2004 ; Toljander et al., 2007 ) and negative ( Welc et al., 2010 ) effects on microbial growth. Using quantitative real-time PCR method, we also detected significant higher 16S rDNA abundance in both the bulk and the rhizosphere soils of Acaulospora laevis and Glomus mosseae inoculated treatments ( Qin et al., 2014a ). Compared to the quantitative changes in bacterial numbers, more studies have demonstrated shifts in bacterial community occurred in the presence of AMF ( Lioussanne et al., 2010 ; Leigh et al., 2011 ; Nottingham et al., 2013 ; Nuccio et al., 2013 ). However, some studies also found AMF have no discernable effect on the composition of the microbial community present in litter-containing soil ( Hodge et al., 2001 ; Herman et al., 2012 ). Though some bacterial species can utilize the hyphae themselves as substrate ( Toljander et al., 2007 ), it is trusted that the changes in the bacterial community in the hyphosphere were not due to the amount of mycelium per se , suggesting that the qualitative effects (e.g., composition of exudates) of the AMF on the hyphosphere bacteria are more important than the quantitative development of AMF hyphae ( Andrade et al., 1997 ; Johansson et al., 2004 ). Carbohydrates ( Hooker et al., 2007 ; Toljander et al., 2007 ) and citric acid ( Tawaraya et al., 2006 ) were detected in mycorrhizal hyphal exudates. In such a manner, the change of soil microbial biomass and the modification of the soil microbial community could be mainly dependent on quantitative and qualitative changes of hyphal exudates ( Filion et al., 1999 ; Toljander et al., 2007 ; Lioussanne et al., 2010 ). Few studies have explicitly studied how AMF influence the soil bacterial community responsible for PCB degradation. AMF have been proved having great potential on the rhizoremediation of organic pollutants through mycorrhizosphere (the zone influenced by both the root and the mycorrhizal fungus) effect ( Joner and Leyval, 2003 ). Teng et al. (2010) reported that Glomus caledonium and Rhizobium meliloti had a significant synergistic effect on field soil PCBs removal when compared with non-inoculated alfalfa treatment. In our previous study, the dissipation rates of Aroclor 1242, both in bulk and rhizosphere soil, were greatly enhanced by the inoculation of Acaulospora laevis or Glomus mosseae ( Qin et al., 2014a ). The results also demonstrated a significant contribution of Actinobacteria to the PCB congener profiles in the bulk soil, indicating the important role of mycorrhizal extraradical hyphae on shaping bacterial community and PCB congener profile compositions. However, we did not separate the mycorrhizosphere and hyphosphere effects in the study. To our knowledge, no previous studies have investigated the effect of AMF on the hyphosphere soil microbial community mediating PCBs dissipation. The AMF hyphal exudates not only contain low-molecular-weight sugars and organic acids, but also unidentified high-molecular-weight polymeric compounds ( Toljander et al., 2007 ). These compounds are energy-rich, and can stimulate or otherwise affect the growth of hyphosphere soil bacteria ( Toljander et al., 2007 ). There are also the possibilities that some of the exudates have the similar chemical structure to PCBs, and may act as inducers for PCBs degradation ( Singer et al., 2003 ). We investigated how the AM fungus, Glomus mosseae , altered the bacterial communities and PCB congeners dissipation in soil spiked with Aroclor 1242. To accomplish this, we used a two-chamber rhizobox to allow AMF hyphae access to the Aroclor 1242 contaminated soil (Supplementary Figure S1 ). Furthermore, we separated the contaminated soil into four horizontal layers from the central mesh to the outer wall to assess the quantitative effect of mycorrhizal hyphae on soil bacterial community and PCBs dissipation. We hypothesized that different quantity of AMF hyphae results different native bacterial community and PCB congener profile compositions. The results may help us clarify the importance of AM hyphae on soil PCBs dissipation, and promote the field application of AM fungi on soil PCBs bioremediation.",
"discussion": "Results and Discussion AMF Biomass in the Rhizoboxes Zucchini root mycorrhizal colonization rate in the Glomus mosseae M47V inoculated treatment was 52% ± 6% after 40 days of growth, while no mycorrhizal colonized roots were detected in the non-mycorrhizal control, indicating the zucchini could be a good host plant for Glomus mosseae M47V colonization. Soil mycorrhizal hyphal lengths were significant higher ( P < 0.05) in the L1-L3 layers of the AM-inoculated treatment when compared to the L4 layer, and no mycorrhizal hypha was detected in the control ( Table 1 ). Moreover, the hyphal length in L3 layer was lower than that in the L1 and L2 layers, though there were no significant differences. The result proved our hypothesis that the mycorrhizal hyphae can penetrate the double meshes and the hyphal length decreasing with the increasing distance from the meshes. Similar decreasing trend was also found when using PLFA biomarker 16:1ω5c as an indicator of AMF biomass ( Table 1 ). However, AMF PLFA biomarker was also detected in the non-mycorrhizal control. One possible reason is the AMF spores which still existed because the soil we used was not sterilized. Furthermore, the biomarker 16:1ω5c can also be detected in some gram-negative bacteria according to Zelles (1997) , which could lead to the overestimation of soil AMF biomass. Table 1 Soil arbuscular mycorrhizal fungal hyphal length and phospholipid fatty acid (PLFA) biomass in different soil layers under mycorrhiza inoculation treatments and the control (mean ± SD; n = 3). Hyphal length PLFA biomass Control ND 1.09 ± 0.09 ab AM-L1 3.27 ± 0.40 a 1.57 ± 0.28 c AM-L2 3.24 ± 0.26 a 1.26 ± 0.15 bc AM-L3 2.71 ± 0.23 a 0.91 ± 0.10 ab AM-L4 1.77 ± 0.47 b 0.81 ± 0.22 a Different lowercase letters in the same column indicate significant differences between the control and different soil layers by Duncan’s multiple range test at P < 0.05 . Soil PCBs Dissipation After 40 days of incubation, the total PCBs concentration of the rhizobox soil in AMF inoculated treatment was significant lower ( P < 0.05) than that in the control, indicating the AMF hyphae had beneficial effect on soil PCBs dissipation. Soil Aroclor 1242 dissipation rates ranged from 35.67% of L4 layer to 57.39% of L1 layer in AMF inoculated treatment, in which the L1 layer was significant higher ( P < 0.05) than other three layers ( Figure 1 ). Among the four soil layers, the di-, tri-, and tetrachlorinated biphenyls concentrations were always exhibit the highest value in the L4 layer, and there were significant differences between the L4 and L1 layers ( P < 0.05) while no statistical differences were found between the L1 and L2 layers. In general, high overall PCB reduction was observed for tri- and tetrachlorinated biphenyls than for di- and pentachlorinated biphenyls. Pearson’s correlation coefficients indicated that the concentrations of soil tri- ( r 2 = -0.793), tetrachlorinated biphenyls ( r 2 = -0.938) and the total PCBs ( r 2 = -0.923) were significantly and negatively correlated with soil hyphal length ( P < 0.01), while only trichlorinated biphenyl concentration was found negatively correlated with AMF PLFA biomass ( r 2 = -0.605, P < 0.05). AMF remediation of organic pollutants contaminated soils has been well-studied yet, however, with most of them only focused on the whole mycorrhizosphere effect ( Joner and Leyval, 2003 ; Teng et al., 2010 ; Lu et al., 2014 ). To our knowledge, this is the first time to demonstrate the PCBs dissipation in the hyphosphere, and the enhanced dissipation of PCBs could be due to the increased bacterial growth and vitality induced by the external mycelial exudates ( Toljander et al., 2007 ). FIGURE 1 Concentration of PCB homolog groups in different soil layers of AMF-inoculated treatments and the control after 40 days of Cucurbita pepo L. growth . AM-L1 to AM-L4 were four horizontal soil layers from the central mesh to outer wall in the hyphal compartment of AMF-inoculated treatment, respectively. Bars represent standard errors and different letters above bars indicate significant differences, determined by Duncan’s multiple range test at P < 0.05. The mycorrhizal hyphae did not only increase PCB dissipation rate, but also alter the soil PCB congener profiles. The PCB congener profiles of different soil layers in the AMF-inoculated treatment were grouped together, and distinctly separated from the control in PC1 ( Figure 2 ). Among the different soil layers, the L4 layer was distinguished from the other three layers by positive PC1 values. According to our previous study which indicated that different plant root exudates or secondary metabolites resulted in different PCB congener profiles ( Qin et al., 2014b ), we can conclude that mycorrhizal hyphal exudates can play an important role in both degrading PCBs and shaping congener profiles. FIGURE 2 Principal component analysis (PCA) of PCB congener profiles in different soil layers of AMF-inoculated treatments and the control after 40 days of Cucurbita pepo L. growth . Samples were represented by open symbols while PCB congeners by solid asterisks. Numbers beside asterisks represent IUPAC congeners. PCB-Degrading Related Gene Abundance The copy numbers of soil bacterial 16S rDNA gene varied from 1.57 ± 0.35 × 10 9 to 2.44 ± 0.32 × 10 9 g -1 dry soil ( Table 2 ). The 16S abundance in the L1 and L2 layers of AMF-inoculated treatment were higher, but only the abundance in the L1 layer was significant higher than the other two layers and the control ( P < 0.05). The hyphosphere could provide important niches for bacterial growth through transferring photo-assimilates into the soil or rapid turnover of mycorrhizal mycelium, in which the exudation of carbohydrates by living hyphae may enable a more direct and reciprocal interaction between mycorrhizal fungi and other microorganisms ( Staddon et al., 2003 ; Toljander et al., 2007 ). Table 2 Abundances of 16S, bph A, bph C( Rh ), and bph C( Ps ) genes in different soil layers under mycorrhiza inoculation treatments and the control (mean ± SD; n = 3). 16S gene (×10 9 copies g -1 ) bphA gene (×10 4 copies g -1 ) bphC ( Rh ) (×10 5 copies g -1 ) bphC ( Ps ) (×10 3 copies g -1 ) Control 1.59 ± 0.06 a 6.75 ± 0.65 a 7.15 ± 0.19 a 1.59 ± 0.41 a AM-L1 2.44 ± 0.32 b 9.43 ± 1.02 b 9.23 ± 0.58 c 2.16 ± 0.98 a AM-L2 1.89 ± 0.40 ab 7.42 ± 1.15 a 7.84 ± 0.12 ab 1.54 ± 0.50 a AM-L3 1.57 ± 0.35 a 6.78 ± 0.42 a 8.72 ± 0.74 bc 1.13 ± 0.21 a AM-L4 1.57 ± 0.33 a 6.59 ± 1.28 a 7.26 ± 0.47 a 1.08 ± 0.08 a Different lowercase letters in the same column indicate significant differences between the control and different soil layers by Duncan’s multiple range test at P < 0.05 . Several studies have successfully calculated the PCB-degrading bacterial population in various contaminated soils using qPCR technique to target the bphA or bphC genes ( Correa et al., 2010 ; Petrić et al., 2010 ; Lu et al., 2014 ). In the present study, the bphA and bphC genes were detected by qPCR in both AMF-inoculated treatment and the control. The bphA gene population was significant higher in the L1 layer than other soil layers and the control, while no significant difference was observed between the L2-L4 layers and the control ( Table 2 ). The population size of Rhodococcus -like bphC gene [ bphC ( Rh )] was found approximately 2–3 orders of magnitude more than that of Pseudomonas -like bphC gene [ bphC ( Ps )], indicating that the bphC ( Rh ) gene played a key role in Aroclor 1242 dissipation in this spiked soil. The bphC ( Rh ) gene abundance was significant higher in the L1 layer when compared to the control ( Table 2 ), and was found correlated significantly with the soil AMF hyphal length ( r 2 = 0.624, P < 0.05) and PLFA biomass ( r 2 = 0.577, P < 0.05). Evidences have shown that hyphal exudates from Glomus sp. contained low molecular weight (LMW) sugar and organic acids, which could probably be metabolized by bacterial ( Toljander et al., 2007 ). Furthermore, some LMW organic acids, such as salicylate, can act as inducers of PCB degradation by indigenous bacteria ( Singer et al., 2003 ). It can be hypothesized that the mycorrhizal hyphal exudates may contain some LMW organic compounds which could act as inducers of the bphC ( Rh ) gene and elevate the gene expression level. This could partly explain the correlation between the bphC ( Rh ) gene abundance and the hyphal length. However, it seems that the hyphal exudates of Glomus mosseae we used in the present study lack of inducers of bphC ( Ps ) gene. As different AMF or host plant may result in different hyphal exudates, further studies are needed to explore potential inducers for bph gene expression and elucidate their mechanisms. Changes in Microbial Community A total of 67513 sequences were obtained after passing several specific filters ( Yadav et al., 2014 ; Yang et al., 2014 ). The relative abundance of the top 14 taxonomic categories at class level is demonstrated in Supplementary Figure S3 . The taxonomic classes other than these results are categorized as “Others.” Sequences that do not show homology in the NCBI database have been labeled as “Unclassified.” The Betaproteobacteria was largely dominant, representing 12.15–14.26% of the population in all soils. The Actinobacteria was the next dominant taxonomy observed in all the soils, followed by the Alpha - and Gammaproteobacteria . Among the 14 selected classes, the Anaerolineae was found negatively correlated with the concentration of dichlorinated biphenyls ( r 2 = -0.660, P < 0.01) ( Table 3 ). Previous studies have revealed that the phylum Chloroflexi was one of the several most abundant groups associated with Aroclor 1242 degradation ( Mercier et al., 2014 ) or perchloroethene (PCE) dechloronation ( Kittelmann and Friedrich, 2008 ). Mercier et al. (2014) found that Chloroflexi was the second-most abundant phylum shared between three granular activated carbons which were incubated for a month with Aroclor 1242 spiked sediment, and the Anaerolineae class was found dominated in this phylum. The strictly anaerobic genera in Anaerolineae class could ferment carbohydrates and amino acids around the mycorrhizal hyphae, and may also utilize low chlorinated biphenyls such as dichlorinated biphenyls. Aside from the Anaerolineae , the class Alphaproteobacteria was also found negatively correlated with the concentration of tetrachlorinated biphenyls ( r 2 = -0.525, P < 0.05). Most of the Alphaproteobacteria were assigned to the Rhizobiales and Rhodospirillales orders. One possible reason is that certain members of the Rhizobiales or Rhodospirillales have the ability to biotransform highly chlorinated PCBs ( Ahmad et al., 1997 ; Bertin et al., 2011 ). Otherwise, there is also the possibility that mycorrhizal hyphal exudates stimulated the growth of Alphaproteobacteria and enhanced the co-metabolization of tetrachlorinated biphenyls by PCB degraders consequently. Table 3 Pearson’s correlation coefficients relating PCB congener concentrations and the relative abundance of bacterial groups at the class, order, and family level, respectively. Classification level Bacterial group Pearson’s correlation coefficients Class Anaerolineae Di-CBs ( r 2 = -0.660 ∗∗ ) Alphaproteobacteria Tetra-CBs ( r 2 = -0.525 ∗ ) Order Rhizobiales Tetra-CBs ( r 2 = -0.548 ∗ ) Sphingomonadales Di-CBs ( r 2 = 0.694 ∗∗ ) Xanthomonadales Tri-CBs ( r 2 = 0.566 ∗ ) Family Anaerolineaceae Di-CBs ( r 2 = -0.660 ∗∗ ) Comamonadaceae Tri-CBs ( r 2 = -0.516 ∗ ) Oxalobacteraceae Di-CBs ( r 2 = 0.575 ∗ ), Tri-CBs ( r 2 = 0.562 ∗ ), Penta-CBs ( r 2 = 0.526 ∗ ), total PCBs ( r 2 = 0.623 ∗ ) Streptomycetaceae Tri-CBs ( r 2 = 0.545 ∗ ), Penta-CBs ( r 2 = 0.521 ∗ ) ∗ P < 0.05; ∗∗ P < 0.01 . At the order level, Burkholderiales , Actinomycetales , Sphingobacteriales , Bacillales , Xanthomonadales , Rhizobiales are dominated, among which the Burkholderiales is the most abundant. Offre et al. (2008) reported that subgroups such as Comamonadaceae , Oxalobacteraceae and Rubrivivax affiliated to Burkholderiales are preferentially associated with AM. Soil Burkholderiales has been shown to be involved in biological suppression of pathogen, plant-growth promotion, and N-fixation ( Xu et al., 2015 ). Our results were in consistent with the previous studies that species of the genus Burkholderia were abundant both in rRNA and in rDNA clone libraries generated from the polluted soil ( Nogales et al., 2001 ). The Burkholderia xenovorans LB400 has been considered as one model organism to study the aerobic biodegradation of PCBs because of their ability to oxidize wide range of congeners as well as use some congeners as sources of carbon and energy ( Tillmann et al., 2005 ; Parnell et al., 2010 ). Though no correlation was found between the abundance of Burkholderiales and soil PCB congeners concentrations, the Burkholderiales was more abundant in the L1 layer of the AMF-inoculated treatment than other layers and the control ( P < 0.05), indicating mycorrhizal hyphal exudates may benefit the growth of this group of bacteria. The negative correlation between the abundance of Rhizobiales and the concentration of tetrachlorinated biphenyls ( r 2 = -0.548, P < 0.05) were proved ( Table 3 ), which provided strong evidence for our above hypothesis about the relationship between Alphaproteobacteria and tetrachlorinated biphenyls. Among the families classified by the RDP classifier, Chitinophagaceae , Xanthomonadaceae , Cyanobacteria Family XIII, Comamonadaceae and Burkholderiaceae are the most abundant five families. The relative abundance of family Comamonadaceae was negatively correlated with trichlorinated biphenyls ( r 2 = -0.516, P < 0.05) ( Table 3 ). The Comamonadaceae are a physiologically heterogeneous group of bacteria; they are known to consume a broad spectrum of organic carbon compounds that range from simple sugars to complex aromatic compounds ( Kersters et al., 2006 ). Genera in this family such as Acidovorax sp. (formerly Pseudomonas sp.) strain KKS102 and Hydrogenophaga taeniospiralis IA3-A have the well-known ability of PCB- and biphenyl-degrading or cometabolizing ( Lambo and Patel, 2006 ; Ohtsubo et al., 2006 ). It is suggested that members of the Comamonadaceae may be stimulated by the presence of AMF in PCBs contaminated soil. The results was in accordance with Offre et al. (2007) , who found that in the Medicago truncatula rhizosphere, the presence of AMF increased the relative abundance of Comamonadaceae taxa. However, results from Pivato et al. (2009) and Nuccio et al. (2013) indicated that taxa from Comamonadaceae responded negatively to AMF. The mechanisms for these interactions are still unknown, and may result from the direct or indirect manipulation of the community through hyphal exudates ( Toljander et al., 2007 ; Nuccio et al., 2013 ). Similar to the class Anaerolineae , the family Anaerolineaceae was also negatively correlated with the concentration of dichlorinated biphenyls ( r 2 = -0.660, P < 0.01). We also found positive correlations between the relative abundance of family Oxalobacteraceae and di- ( r 2 = 0.575, P < 0.05), tri- ( r 2 = 0.562, P < 0.05), pentachlorinated biphenyls ( r 2 = 0.526, P < 0.05) and the total PCB concentrations ( r 2 = 0.632, P < 0.05), while the relative abundance of family Streptomycetaceae was correlated with tri- ( r 2 = 0.545, P < 0.05) and pentachlorinated biphenyls ( r 2 = 0.521, P < 0.05) ( Table 3 ). Given that these two families were relatively lower in the L1 and L2 layers when compared with the other two layers and the control, we can speculate that the mycorrhizal hyphae may secrete certain inhibitors or compete nutrition with them and resulted in decreased biomass consequently. Principal component analysis showed distinct separation of the microbial community from the control in the PC2 to the microbial community that developed under different soil layers of the AMF-inoculated treatment ( Figure 3 ). Among the four soil layers in the AMF-inoculated treatment, the L1 layer was distinguished from the other three layers by positive PC1 value, while no obvious difference in microbial community was found between each of the three soil layers. The PCA results strongly supported our hypothesis that the existence as well as the abundance of arbuscular mycorrhizal hyphae could play an important role in shaping the mycorrhizosphere bacterial community which responsible for PCB dissipation. FIGURE 3 Principal component analysis of bacterial communities in different soil layers of AMF-inoculated treatments and the control after 40 days of Cucurbita pepo L. growth . AM-L1 to AM-L4 were four horizontal soil layers from the central mesh to outer wall in the hyphal compartment of AMF-inoculated treatment, respectively."
} | 6,312 |
37792894 | PMC10578598 | pmc | 4,805 | {
"abstract": "The horizontal transfer of genes is fundamental for the eco-evolutionary dynamics of microbial communities, such as oceanic plankton, soil, and the human microbiome. In the case of an acquired beneficial gene, classic population genetics would predict a genome-wide selective sweep, whereby the genome spreads clonally within the community and together with the beneficial gene, removing genome diversity. Instead, several sources of metagenomic data show the existence of “gene-specific sweeps”, whereby a beneficial gene spreads across a bacterial community, maintaining genome diversity. Several hypotheses have been proposed to explain this process, including the decreasing gene flow between ecologically distant populations, frequency-dependent selection from linked deleterious allelles, and very high rates of horizontal gene transfer. Here, we propose an additional possible scenario grounded in eco-evolutionary principles. Specifically, we show by a mathematical model and simulations that a metacommunity where species can occupy multiple patches, acting together with a realistic (moderate) HGT rate, helps maintain genome diversity. Assuming a scenario of patches dominated by single species, our model predicts that diversity only decreases moderately upon the arrival of a new beneficial gene, and that losses in diversity can be quickly restored. We explore the generic behaviour of diversity as a function of three key parameters, frequency of insertion of new beneficial genes, migration rates and horizontal transfer rates.Our results provides a testable explanation for how diversity can be maintained by gene-specific sweeps even in the absence of high horizontal gene transfer rates.",
"conclusion": "Conclusion We have shown that the simplified framework of our eco-evolutionary model with an underlying metacommunity structure, can support a “gene sweep” dynamics, without eliminating genome diversity. Instead, gene sweeps can lead to a moderate reduction of diversity even in the absence of diversity-restoring mechanism. Inclusion of a diversity-restoring mechanism (e.g, a neutral biodiversity model in our specific case) can increase the minimal observed diversity. Conversely, for high rates of beneficial mutations, it could lead to a reduction in the maximum diversity. The mechanism by which a metacommunity maintains diversity under a gene sweep is compatible with small HGT rates compared to typical migration time scales. Unlike prior work, our model does not explicitly require additional ingredients such as frequency-dependent selection at the individual level, induced by genome-level processes or by ecological interactions. Most of the limitations of our model come with a trade-off with its simplicity. Specifically, numerous oversimplifications, including the absence of spatial organization, no relationship between migration and HGT rates, and a highly simplistic approach to intra-population dynamics, present intriguing questions for future research endeavours. For example, in our model we considered exclusively neutral non-beneficial mutations, and we did not include deleterious mutations. The presence of such deleterious mutations could potentially diminish overall genetic diversity and introduce the possibility of modifying sweep timescales through linkage effects. Similarly, we did not investigate clonal interference effects. In addition, the model outcomes rely on simple time-scale competition arguments. From this standpoint, our hypothesis is related in spirit to the classic proposition put forward by G. A. Hutchinson [ 44 ] to justify the very high observed microbiological diversity in samples of ocean pythoplankton (which was at odds with the principle of competitive exclusion, according to which the survival of a single species within a population should be privileged). To reconcile diversity with competitive exclusion, Hutchinson argued that if the time scale at which the exclusion principle is enforced were comparable to the time scale over which environmental conditions change significantly, a state of equilibrium would never be reached, and therefore there would be no predominance of a single species. Our focus on a metacommunity is complementary to the approach assumed by the previous study by Niehus and coworkers [ 6 ], which focused on intra-patch diversity of a single population and the role of migration of non-carrier individuals, favouring diversity in moderate amounts. The same study also showed that such migration effects are enhanced in a small metacommunity, made of multiple patches. In our model, all populations are connected, and the within-population dynamics are assumed to be fast and result in a single winner. More precisely, we took the conservative assumptions that (i) migration of carriers (which reduces the diversity) is the dominant process and that (ii) the presence of the beneficial gene on a patch, whether it is carried by a species invading the patch by migration or if is acquired by HGT, is sufficient to guarantee a full sweep of the population. Phenomena akin to those described by Niehus and colleagues would increase the prediction of the residual diversity in our model. Thus, in light of their study, we can consider our estimates as lower bounds for diversity. Apart from these differences, the mechanisms described by our work are conceptually similar to the ones discussed by Niehus and coworkers, in that they are a result of the balance between HGT and migration rates. As noted in ref. [ 6 ], these mechanisms lead to an effective frequency-dependent selection (which in our case acts completely at the level of populations within a metacommunity, not on individuals), as it reproduces the same effect defined by Takeuchi and coworkers [ 23 ]. However, we note that this dependency has a different origin than the processes hypothesized by Takeuchi and coworkers [ 23 ] (which act at the level of an individual within a population). In the scenario assumed by Takeuchi and colleagues, the diversity is favoured by ubiquitous and diverse deleterious loci that are linked to the acquired beneficial gene. In such cases, the diversity of bacterial species should be capped by the number of deleterious linked effects (e.g. phage diversity). If these linked alleles can be quantified in data, they should be linked to residual diversity after a gene sweep. Importantly, even though they are conceptually different, these hypotheses are not mutually exclusive, and possibly can both be detected in data or addressed in controlled experiments. To address negative frequency-dependent selection due to linkage, genomic analysis of microbial communities undergoing gene sweeps should be able to isolate the linked deleterious loci that co-occur with the beneficial gene in each species or strain. In order to test the role of a patchy community in restoring diversity, experiments could induce gene sweeps in a laboratory metacommunity with varying densities of patches. In such a setting, spatial patterns of the frequency spectrum or the genetic diversity, intended as the two-point measures of diversity related to the variance of allele frequency could be compared to the variance of the number of co-existing species in the metacommunity predicted by different models (see S2 Fig ). More specifically, the key observables would be statistics of the observed polymorphisms, as the central point is to identify the presence of selective forces acting on genomic regions other than the one embedding the favoured gene. Additionally, the model predicts how migration effects may affect residual diversity in a gene sweep, and this could be possible controlled in such “laboratory gene sweep” setups. Experimental systems that might allow this are conceivable today [ 28 ], although complex spatio-temporal processes might complicate considerably the experimental scenario compared to the simple, purely conceptual, model proposed here [ 45 , 46 ]. Despite these limitations, future studies of genomic data may be able to differentiate a gene-specific sweep with or without high HGT based on an analysis of additional selective forces in other portions of the genome.",
"introduction": "Introduction Horizontal Gene Transfer (HGT) plays a crucial role in the processes that shape bacterial evolution [ 1 – 5 ]. HGT accelerates the adaptation of bacterial communities to new ecological niches [ 6 , 7 ] and reduces the deleterious effects associated with the accumulation of genetic load by clonal reproduction [ 8 ]. HGT is also a widespread pathway through which pathogenic bacteria acquire resistance to antibiotics [ 7 , 9 ]. According to classical population genetics theories [ 10 ], when the rate of HGT is low, then vertical inheritance is the main mechanism for the expansion of novel genetic variants. In such cases, the evolutionary dynamics is described by clonal evolution. In this case, because of linkage effects, transfer of a highly beneficial gene results in a drastic reduction of the diversity, as a consequence of the clonal expansion of the mutant carrying the gene, whose genome would “sweep” together with the beneficial gene. We can term this process a genome-wide selective sweep. In contrast to this scenario, several lines of metagenomic evidence from communities of phylogenetically related strains [ 1 , 11 , 12 ], support an alternative scenario of gene-specific sweeps, an evolutionary dynamics where a beneficial gene can reach fixation across species or strains, without erasing diversity. Reconciling this scenario with standard population genetic models would require very high recombination rates [ 11 ] compared to standard direct measurements of such rates [ 13 – 15 ]. Hence, the consensus is that more complex mechanisms should be in place [ 1 ]. In the past years, several alternative hypotheses were put forward to reconcile the evidence of gene sweeps with the estimated values of the recombination rate [ 13 – 15 ]. A first mechanism was inspired by the evidence collected by Shapiro and coworkers [ 11 ], and was introduced by Polz and coworkers [ 16 ]. This hypothesis uses the observation that HGT rates between pairs of species decline rapidly, following an exponential pattern, as a function of their genetic distance [ 17 , 18 ], an effect that leads to an effective HGT barrier between populations of different species/strains, provided that the intra-population rate of the genomic changes is faster than the inter-population HGT rate. Therefore diversity could persist in a metacommunity (a group of populations including different species/strains based on spatially separate patches) in presence of selective effects. Moreover, another crucial factor resides in the diversified selection experienced across separate patches, stemming from the inherent environmental heterogeneity. This dynamics could maintain diversity by favouring divergent species or strains in distinct geographical locales. A second mechanism, proposed by Niehus and coworkers [ 6 ], proposes that the combination of HGT rates close to realistic estimates [ 13 – 15 ] and a migration rate smaller than the typical selection rates of beneficial mutations, can lead to the fixation of beneficial genes without the decrease of genome diversity. However, dynamic models based on this second mechanism predict gene-sweep times to be excessively short (ranging from months to years), and would require high HGT rates (based on current estimates of genetic transfer rates) to match the observation that timescales of horizontal gene-sweeps remain extremely short compared with phylogenetic timescales [ 18 – 20 ]. A different hypothesis for gene sweeping might be the so-called “soft sweeps” [ 21 , 22 ], where widespread (e.g. neutral) recombination through HGT can generate a pool of standing variation (in our case promoting the presence of one or more beneficial alleles of a given gene across species) that is sufficient to support the emergence of multiple (interfering) sweeps in parallel upon a change of selective pressure. In this case the gene-specific sweep would consist of multiple parallel sweeps of a gene that previously spread neutrally by HGT onto diverse genetic backgrounds. However, this scenario cannot explain situations where a gene sweep originates from a gene that is not already initially present neutrally in many species. Additionally, this requires high HGT rates, as in the previous explanations. Another mechanisms, put forward by Takeuchi and coworkers in 2015 [ 23 ], involves the linkage of the beneficial sweeping gene with widespread (species-specific) deleterious alleles, which would lead to negative frequency-dependent selection. This mechanism was shown to explain a gene-sweep dynamics in quantitative terms, provided that the basal recombination rate, (the spontaneous rate, not affected by selective pressure), is sufficiently low. The widespread linkage of the beneficial gene with deleterious alleles or more generally the presence of linked loci under negative frequency-dependent selection, does not have a simple explanation, but the authors speculate that it could be the consequence of ecological interactions between bacteria and viral predators [ 23 ], possibly supported by a “Kill the Winner” dynamics [ 24 ]. Interestingly, this mechanism can work with relatively low gene-transfer rates, and actually requires a low basal recombination rate. This is notable because it challenges the intuition (and the requirement of the previous models) that high recombination rates are necessary for gene-specific selective sweeps. Here, we propose a complementary eco-evolutionary mechanism whose key ingredient is a metacommunity structure. Our approach is related to the classic population genetics perspective on selective sweeps in structured environments [ 25 – 27 ], where it is well known that under certain conditions, the effect of a selective sweep on the neutral variation of a subdivided population can be different from naive expectations. Specifically, we assume that the environment is characterized by the presence of multiple patches ( e.g ., nutrient patches as in marine snow [ 28 ]) and that their physical separation is the main limitation to the spread, through genome migration or HGT, of beneficial genes. As we show, a metacommunity structure can preserve diversity during the fixation dynamics of a beneficial gene without requiring high recombination rates.\n\nMetacommunity diversity loss resulting from the introduction of a beneficial gene We next ask how much diversity is lost upon the introduction of a beneficial gene, even in the absence of diversity-maintenance mechanisms. This section assumes only a single beneficial gene, and that no two beneficial mutations can simultaneously sweep at distinct loci. In order to address this question, we focused on the dynamics following the introduction of a beneficial gene that can spread through the metacommunity via both HGT and sweep (gene-specific sweep) and genome-wide sweeps on single patches. The initial diversity was set using the neutral model described in the previous section (hence depends on ν , see Eq 1 ). We considered the limiting situation where no diversity-restoring mechanism was in action during the whole sweep. In other words, we assume that the fixation dynamics of the advantageous gene in the metacommunity is much faster than the equilibration time scale of the neutral biodiversity model. Roughly, if there are N individuals per patch, this limit corresponds to the condition\n ν ≪ 1 M log N and ν ≫ 1 M N , \n (2) \ni.e., where the genome sweep time M log( N ) is much smaller than 1/ ν , but this is in turn much smaller than a metacommunity-wide fixation time of the neutral dynamics, which is order MN (see Methods ). This assumption, which will be relaxed in the next paragraph, is the most conservative scenario (the most adverse in terms of diversity loss) for the introduction of a new beneficial gene in a metacommunity. Under these assumptions, only two processes take place ( Fig 3A ): (i) migration-sweep of a patch by a species carrying the beneficial gene (genome-wide sweep), which leads to a reduction of diversity, and (ii) spread of the beneficial gene by HGT (gene sweep), which does not reduce diversity. Hence, diversity can only decrease in this scenario, and we are interested in the magnitude of the decrease relative to the diversity baseline (see details in the Methods , Eq 7 ). 10.1371/journal.pcbi.1011532.g003 Fig 3 In presence of HGT only, and no diversity-maintenance mechanism, the fixation of a beneficial gene in the metacommunity can lead to a moderate loss of diversity. A . A beneficial gene can spread across patches (i) via migration events (reducing diversity) or (ii) via HGT-sweep (maintaining diversity) The two processes take place at each time step, with rate p m (per patch, per time step) and p h (per patch, per time step) respectively. Here, we assume that no diversity-maintenance mechanism counteracts the diversity loss (this assumption will be relaxed later) B . Simulations of this model show a diversity ( S ( t )) loss from the initial value ( S i ) to a new stable value ( S f ), corresponding to complete invasion of the beneficial gene. Each solid line is a realization, with initial condition of the simulations generated by the neutral model described in Fig 2 ( M = 10000 and ν = 0.02), and with parameters p h = 0.2 and p m = 1 − p h . C-D . Comparison between analytical prediction ( Eq 3 ) and simulated data for the sweep parameter Q = 〈 S f / S i 〉 as a function of the ratio p h / p m (panel C , ν = 0.01), and of the innovation rate of the neutral model generating the initial diversity (panel D , p h = 0.1, p m = 0.9). Simulations performed with M = 10 000. The panels CD show the distribution of diversity S over 100 realizations as a box plot (blue line: mean value, box: inter-quartile range, fences: max and min values). To implement genome-wide migration-sweep events, at each time step, with a rate p m (per patch, per time step), two patches are picked randomly. If the first patch carries the beneficial gene and the second one does not, the species of the second one is replaced by a copy of the first one. Similarly, HGT gene-sweep events occur at each time step with a rate p h (per patch, per time step). In such events, two random patches are selected. If the species of the first one carries the beneficial gene and the second one does not, then the gene is horizontally transferred and spreads into the second patch, without any displacement of species. HGT and migration rates are independent and we consider a fully connected network of communities with uniform rates (the spatial distance between patches is not modelled). This model configuration corresponds to an evolutionary regime where the maintenance of diversity is slow compared to the time scale of fixation dynamics. For this regime we find (see Methods ) that the number of populations (patches) carrying the beneficial gene follows a logistic growth (see Methods , Eq 11 ) and after a time τ fix ≃ 2 M log ( M ) p h + p m steps = 2 log ( M ) p h + p m gen the diversity reaches an absorbing state where all the species in the metacommunity carry the advantageous gene. Mathematically, this evolutionary regime is defined by the condition τ fix ≪ τ eq (see Methods ). The key aspect is the residual value of the diversity after the fixation of the beneficial gene. \n Fig 3B shows an example of the typical dynamics of the diversity after the introduction of the beneficial gene. The initial value of the diversity ( S i ≃ 〈 S 0 〉) decreases after the introduction of the beneficial gene and reaches a new stationary value S f . We quantify the effect of the fixation of the beneficial gene by the “sweep parameter” Q 0 ≡ 〈 S f S 0 〉 . Thus, by definition, a value of Q 0 ≃ 0 corresponds to a scenario of a genome-wide sweep across the metacommunity, while for Q 0 > 0 some diversity is regenerated. We have derived an approximate analytical solution for the model dynamics in this regime (i.e., when τ fix ≪ τ eq , see Methods ), which leads to the following expression for the sweep parameter,\n Q 0 = 1 - log ( 1 - ( 1 - ν ) e - p h p m ) log ( ν ) . \n (3) \n Eq (3) was derived under the assumptions of small ν , small p h , large metapopulation size M ≫ 1 (see Methods ), and is in good agreement with numerical simulations of the model ( Fig 3C and 3D ). These results show that a full genome-sweep dynamics ( Q 0 = 0) can only be reached when p h / p m → 0, i.e., when the HGT rate is completely negligible (e.g., for p h → 0 or p m ≫ p h ) and the “invasion” dynamics of the species with the beneficial gene is the only relevant one. However, as soon as the HGT rate is non-negligible (for any positive value p h / p m > 0), there is more than a single species within the metacommunity after the fixation of the beneficial gene. More specifically, for values of p h / p m ≃ 0.1 and ν = 0.01, we already obtain Q 0 ≃ 0.5, which means that a HGT-sweep rate ten times slower than the typical migration-sweep time is sufficient to regenerate (in the worst-case scenario) half of the diversity within a metacommunity. We note that the selection coefficient for beneficial mutations does not play a role here in the expressions of Eqs 3 and 4 , as we have assumed that any carrier of the beneficial gene will sweep a patch."
} | 5,385 |
34499072 | null | s2 | 4,806 | {
"abstract": "Invention of DNA origami has transformed the fabrication and application of biological nanomaterials. In this review, we discuss DNA origami nanoassemblies according to their four fundamental mechanical properties in response to external forces: elasticity, pliability, plasticity and stability. While elasticity and pliability refer to reversible changes in structures and associated properties, plasticity shows irreversible variation in topologies. The irreversible property is also inherent in the disintegration of DNA nanoassemblies, which is manifested by its mechanical stability. Disparate DNA origami devices in the past decade have exploited the mechanical regimes of pliability, elasticity, and plasticity, among which plasticity has shown its dominating potential in biomechanical and physiochemical applications. On the other hand, the mechanical stability of the DNA origami has been used to understand the mechanics of the assembly and disassembly of DNA nano-devices. At the end of this review, we discuss the challenges and future development of DNA origami nanoassemblies, again, from these fundamental mechanical perspectives."
} | 286 |
39422488 | PMC11559036 | pmc | 4,807 | {
"abstract": "ABSTRACT Myxococcus xanthus uses short-range C-signaling to coordinate multicellular mound formation with sporulation during fruiting body development. A csgA mutant deficient in C-signaling can cheat on wild type (WT) in mixtures and form spores disproportionately, but our understanding of cheating behavior is incomplete. We subjected mixtures of WT and csgA cells at different ratios to co-development and used confocal microscopy and image analysis to quantify the arrangement and morphology of cells. At a ratio of one WT to four csgA cells (1:4), mounds failed to form. At 1:2, only a few mounds and spores formed. At 1:1, mounds formed with a similar number and arrangement of WT and csgA rods early in development, but later the number of csgA spores near the bottom of these nascent fruiting bodies (NFBs) exceeded that of WT. This cheating after mound formation involved csgA forming spores at a greater rate, while WT disappeared at a greater rate, either lysing or exiting NFBs. At 2:1 and 4:1, csgA rods were more abundant than expected throughout the biofilm both before and during mound formation, and cheating continued after mound formation. We conclude that C-signaling restricts cheating behavior by requiring sufficient WT cells in mixtures. Excess cheaters may interfere with positive feedback loops that depend on the cellular arrangement to enhance C-signaling during mound building. Since long-range signaling could not likewise communicate the cellular arrangement, we propose that C-signaling was favored evolutionarily and that other short-range signaling mechanisms provided selective advantages in bacterial biofilm and multicellular animal development. IMPORTANCE Bacteria communicate using both long- and short-range signals. Signaling affects community composition, structure, and function. Adherent communities called biofilms impact medicine, agriculture, industry, and the environment. To facilitate the manipulation of biofilms for societal benefits, a better understanding of short-range signaling is necessary. We investigated the susceptibility of short-range C-signaling to cheating during Myxococcus xanthus biofilm development. A mutant deficient in C-signaling fails to form mounds containing spores (i.e., fruiting bodies) but cheats on C-signaling by wild type in starved cell mixtures and forms spores disproportionately. We found that cheating requires sufficient wild-type cells in the initial mix and can occur both before mound formation and later during the sporulation stage of development. By restricting cheating behavior, short-range C-signaling may have been favored evolutionarily rather than long-range diffusible signaling. Cheating restrictions imposed by short-range signaling may have likewise driven the evolution of multicellularity broadly.",
"introduction": "INTRODUCTION Microbiomes often contain bacteria that adhere to biotic and abiotic surfaces, forming biofilms that affect ecosystems and human health in diverse and important ways ( 1 – 3 ). Within biofilms, bacteria communicate using both long- and short-range signaling mechanisms ( 4 ). Long-range signaling involves the release of diffusible signal molecules from cells and does not require cell–cell contacts ( 5 ). Short-range signaling typically depends on cell-surface-associated protein assemblies that mediate direct cell–cell contact ( 6 ). Both long- and short-range signaling shape the composition, spatial structure, ecology, and evolution of biofilms ( 7 – 9 ). A better understanding of the mechanisms and functions of signaling interactions within biofilms will facilitate their manipulation for societal benefits ( 3 , 10 ) and provide insights into the evolution of multicellularity ( 11 , 12 ). Signaling often promotes cooperation between individuals but exposes the community to exploitation by cheaters, which reduce or eliminate the production of the signal molecule but gain a fitness advantage by responding to the signal molecule produced by cooperators ( 13 ). Cheating is pervasive in microbial communities, and its consequences can be profound (e.g., community collapse), so cooperators evolve to combat cheating ( 8 , 9 , 14 , 15 ). Efforts to manipulate battles between cooperators and cheaters for therapeutic, agricultural, industrial, and environmental applications are gaining traction ( 10 , 16 – 22 ). In this study, we used a biofilm formed by Myxococcus xanthus as a model to investigate cheating on short-range C-signaling. M. xanthus adheres to the bottom of a container and forms a biofilm submerged under a thin layer of liquid ( 23 ). In the absence of nutrients, the cells coordinate their movements to build dome-shaped mounds, which mature into fruiting bodies as some of the rod-shaped cells differentiate into round spores. Other rods lyse or remain outside fruiting bodies as peripheral rods ( 24 , 25 ). During the developmental process, C-signaling coordinates mound formation with spore differentiation ( 26 – 28 ). A csgA mutant deficient in C-signaling fails to build mounds or form spores ( 29 – 31 ). Upon co-development with an equal number of wild-type cells, csgA mutants have been reported to form an approximately equal number of spores as the WT ( 29 , 32 ) or ~100-fold ( 33 ) to ~380-fold ( 34 ) more spores than the WT. An equal number of spores indicates rescue of csgA development by WT C-signaling. A greater number of csgA than WT spores indicates developmental cheating by csgA on WT C-signaling. Clearly, csgA mutants respond to WT C-signaling, but our understanding of the requirements for rescue and cheating behavior is incomplete. Neither are the C-signal production and reception mechanisms completely understood (reviewed in reference 35 ). In one model, starving cells secrete a protease ( 36 , 37 ) that cleaves CsgA to a 17-kDa fragment (p17) at the surface of producer cells ( 38 , 39 ), and responders detect p17 with an unidentified cell-surface receptor. In another model, starving cells synthesize intact 25 kDa CsgA with cardiolipin phospholipase enzymatic activity that releases diacylglycerols from the inner membrane ( 40 ), but how these signal molecules exit producer cells and how responders perceive them are unknown. The two models are not mutually exclusive (i.e., CsgA might be bifunctional). Although gaps remain in our molecular understanding of C-signaling, knowledge continues to grow about the cellular requirements for efficient C-signaling. Early work indicated that C-signaling requires cells to be in close proximity, possibly in contact ( 38 ), and that cell motility and alignment increase C-signaling ( 41 – 43 ). Recently, tracking of csgA mutant cells mixed with a 10,000-fold excess of WT cells revealed complete rescue of csgA participation in mound formation, despite differences from WT in motility behavior (primarily, faster speeds of csgA rods compensated for their weaker bias in the persistent duration of movement toward nascent mounds) ( 44 ). C-signaling also affects the expression of many genes during development ( 32 ), apparently by activating the transcription factor FruA posttranslationally ( 45 , 46 ), although the mechanism is unknown. We recently used confocal microscopy and cell segmentation to visualize and quantify C-signal-dependent gene expression of cells within 5–10 µm of the bottom of NFBs ( 47 ), which mature to a height of ~50 µm. We found that expression in transitioning cells (TCs) (i.e., cells intermediate in morphology between rods and spores) and spores later in development correlated with earlier cell density, alignment of neighboring rods, and tangential orientation of rods, suggesting that the arrangement of cells within NFBs affects the efficiency of C-signaling, which regulates the spatiotemporal patterns of gene expression and cellular differentiation. To investigate the cheating behavior of a csgA mutant deficient in C-signaling, we mixed the mutant with WT cells proficient at C-signaling. We induced mixtures at different ratios to co-develop and used our new methods of visualizing and quantifying the arrangement and morphology of cells near the bottom of the biofilm. Our results show that cheating by csgA on WT C-signaling requires sufficient WT cells in the initial mixture and can occur both before and after the mound-building stage of development. By restricting cheating behavior to specific initial cell ratios, short-range C-signaling may have provided a selective advantage evolutionarily. We discuss how cheating restrictions imposed by short-range signaling may have likewise led to their prevalence in biofilm and animal development.",
"discussion": "DISCUSSION We discovered that mixtures of WT and csgA mutant cells at ratios ranging from 1:4 to 4:1 exhibit dramatically different developmental phenotypes in submerged culture. At 1:4, development failed completely. At 1:2, a few mounds formed, but neither WT nor csgA made many spores, indicating that C-signaling by the WT minority was insufficient to support normal development of the population. In contrast, when WT comprised half or more of the population, mounds formed normally, and cheating occurred. Interestingly, at 1:1, C-signaling by WT efficiently rescued csgA participation in mound building, but cheating did not occur until later, during the sporulation stage of development. Strikingly, at 2:1 or 4:1, csgA cheated on C-signaling by the WT majority both before and after mound formation. Greater survival of csgA than WT accounted for cheating prior to mound building. Cheating during the sporulation stage involved csgA forming spores at a greater rate, while WT disappeared at a greater rate. These new insights into cheating behavior pose important questions for future research. Since cheating by csgA required equal or excess WT in the initial mixture, we conclude that short-range C-signaling severely restricts cheating behavior, which likely favored its evolution rather than long-range diffusible signaling. We propose that cheating restrictions imposed by short-range signaling may have likewise proved advantageous during the evolution of bacterial biofilm and multicellular animal development. Short-range C-signaling restricts cheating behavior Excess csgA mutant cells in mixtures with WT interfered with development. The mixtures initially at 1:2 and 1:4 showed a progressive and dramatic decrease in mound formation compared with the mixtures at ≥1:1 (Fig. S3), suggesting that csgA rods require frequent C-signaling from WT rods to participate in mound building. The inability of csgA rods to produce C-signal presumably impairs two positive feedback loops necessary for mound formation. One loop involves the movement of rods into alignment for enhanced C-signaling ( 41 – 43 ). The other loop involves C-signal-dependent transcription of the act operon ( 51 ), whose products control C-signal production ( 52 ). These positive feedback loops depend on the cellular arrangement to enhance C-signaling during mound building. Long-range diffusible signaling could not likewise communicate the cellular arrangement. Rather, it simply communicates the cell density, which presumably is insufficient to build dome-shaped multicellular mounds that become spore-filled fruiting bodies. In the few mounds that formed at 1:2, very few rods became spores ( Fig. 3B ; Fig. S10), consistent with previous observations supporting that spore formation requires efficient C-signaling ( 26 – 28 , 47 ). Developmental interference by excess csgA mutant cells would limit the invasion of WT populations subject to selection for development. Indeed, a mixture of WT and csgA cells at 99:1 exhibited cheating by csgA during an initial cycle of co-development and co-growth, but csgA persisted as the minority with little change in the population dynamics during four subsequent cycles ( 53 ). Albeit short in duration, this experimental evolution study supports the notion that cheating restriction imposed by short-range C-signaling likely favored its evolution. In contrast, long-range diffusible signaling, such as secreted “public goods,” are highly susceptible to cheating ( 13 , 15 , 54 ). The restriction of csgA cheating to populations with equal or excess WT is reminiscent of the negatively frequency-dependent fitness of two developmental cheaters that evolved from WT clones passaged in liquid culture ( 14 , 55 , 56 ). Further exploration of the frequency-dependent fitness of evolved and defined (e.g., an a sgB mutant defective in diffusible A-signaling) ( 56 ) cheaters co-developed with WT may shed light on the evolutionary implications of different cheating mechanisms. We speculate that a population-level restriction on cheating behavior strongly favored short-range C-signaling evolutionarily rather than long-range diffusible signaling. Interestingly, WT can evolve further restrictions on cheating behavior by a csgA mutant. WT rapidly evolved cheater suppression and selfish policing during 20 cycles of co-development with csgA at 1:1 ( 34 ). Even in the absence of csgA , WT clones that had evolved as motile colonies on nutrient agar and differed from the ancestral WT by no more than 20 mutations frequently also evolved resistance to developmental cheating by csgA ( 57 ). The mechanisms of enhanced cheater resistance remain to be elucidated. Based on our results, we propose that restrictions on cheating behavior were driving forces in the evolution of multicellularity and explain the prevalence of short-range signaling in bacterial biofilm and animal development. Cheating during animal development can cause tumor formation and lead to metastatic cancer ( 13 , 58 ). Defects in short-range signaling mechanisms, such as those involving growth factors ( 59 , 60 ), Hedgehog ( 61 ), Wnt ( 62 ), and Notch ( 63 ), are often associated with carcinogenesis, suggesting that restrictions on cheating behavior by these mechanisms may have selected their use and explain their prevalence in animal development. Short-range signaling is likewise prevalent in bacterial biofilm development ( 6 – 8 ). Numerous examples of kin selection mediated by cell–cell contact-dependent mechanisms restrict cheating in biofilms ( 6 , 8 , 9 ), including in M. xanthus ( 64 – 67 ). The extracellular matrix and environmental forces also restrict cheating by spatially structuring biofilms, thus limiting cell dispersal and diffusion of public goods and signals with potential longer range ( 8 , 9 ). Cheating before mound formation involves greater survival of the csgA mutant in mixtures with excess wild-type cells Excess WT cells in mixtures initially at ≥2:1 allowed cheating by csgA before mound formation owing to greater survival of csgA than WT by 18 h PS ( Fig. 5 ). Greater survival of unmixed developing csgA rods compared with WT has been reported previously ( 46 , 48 – 50 ), suggesting a partial defect in developmental lysis of csgA . The mechanism of developmental lysis is unknown ( 49 , 68 ). Interestingly, we did not observe greater survival of csgA than WT at 18 h in the mixture initially at 1:2, and csgA barely outnumbered WT in the mixture initially at 1:1 ( Fig. 5 ). Taken together, these observations suggest that an initial excess of WT promotes greater csgA survival in mixtures, but equal or less WT inhibits csgA survival ( Fig. 5 ). Perhaps, components of living and/or lysed WT cells exert different concentration-dependent effects on csgA , whose stringent response to starvation differs from that of WT ( 69 ). Further elucidating differences between csgA and WT in the stringent response, lysis, and signaling will be important for deeper understanding of csgA cheating behavior in mixtures prior to mound formation and likely later in development as well. Cheating during mound formation generates spatial differentiation within NFBs. We observed a high proportion of csgA rods at 24 and 30 h PS near the radial center of NFBs formed by mixtures initially at ≥2:1, and a high proportion of csgA spores there later for mixtures initially at ≥1:1 ( Fig. 3B ). Presumably, this pattern forms via differential survival and/or movement. We favor differential survival as the explanation for the high proportion of csgA spores near the radial center of 36-h NFBs formed by mixtures initially at 1:1, since we did not observe a high proportion of csgA rods there earlier. However, our results do not exclude the possibility of differential movement of csgA and WT rods between 30 and 36 h. Neither do our results exclude differential movement of csgA and WT rods as the explanation for the high proportion of csgA near the radial center of 24-h NFBs formed by mixtures initially at ≥2:1. Our confocal microscopy method is currently incompatible with tracking of individual cells within NFBs (see below), but our comparison of the overall level of cheating within 24-h NFBs to proximal and distal locations outside revealed little or no difference ( Fig. 5 ). Moreover, the cheating levels were similar to those in the 18-h biofilms, so a similar ability of csgA and WT rods to move into mounds and persistence there could account for the similar level of cheating within and outside of NFBs. In agreement, the accumulation rates of csgA and WT within mounds were indistinguishable in cell tracking experiments ( 44 , 70 ). The detailed motility behavior of csgA and WT rods differed, but spatial differentiation within the mounds was not reported. The cell tracking method used a small fraction of labeled trackable cells (≤0.04%) and frequent (every 30 s for ~5 h), brief (600 ms) imaging. In contrast, our method is currently incompatible with tracking since we have used 17%–50% labeled cells and infrequent, lengthy imaging (4 z -stacks, ~2.5 min each, 3–6 h apart) ( 47 ). It may be possible to use a cell tracking method ( 44 , 70 ), and the insights about cheating behavior revealed by our study to determine the contributions of differential survival and movement to spatial differentiation within NFBs formed by mixtures of csgA and WT. Cheating after mound formation involves more efficient sporulation of the csgA mutant in mixtures with equal or excess wild-type cells When WT comprised half or more of the population, cheating occurred during the sporulation stage of development ( Fig. 3 and 4A ) and involved csgA forming spores at a greater rate, while WT disappeared at a greater rate ( Fig. 4B ; Fig. S10). Why does csgA form spores more efficiently than WT? There are many possible reasons. The altered stringent response of csgA ( 69 ) may enhance its protein synthesis capacity (relative to WT), improving its sporulation efficiency. The csgA mutant lacks cardiolipin phospholipase activity ( 40 ) and fails to synthesize lipid bodies or undergo cell shortening ( 71 ), so its lipid and membrane metabolism differ from WT, as does its developmental gene expression ( 35 , 46 , 72 ). C-signaling from WT may trigger efficient sporulation of csgA without completely restoring normal metabolism and/or gene expression, perhaps akin to chemically induced sporulation of M. xanthus ( 73 , 74 ). This deserves further investigation. Importantly, our data show that csgA cheating occurs early in the sporulation stage, primarily between 30 and 36 h ( Fig. 3 and 4 ; Fig. S10), much earlier than tested in previous studies ( 33 , 34 , 53 , 56 , 57 ). Our results imply that csgA rods are poised to efficiently transition to spores upon sensing WT C-signaling in NFBs. Why does WT disappear from NFBs at a greater rate than csgA ? Under our conditions of submerged culture development, unmixed WT cells appear to lyse more rapidly than unmixed csgA cells at 18–48 h PS ( 46 ), so it is possible that differential lysis contributes to the greater disappearance of WT than csgA during the spore-forming stage of development ( Fig. 3 and 4 ; Fig. S10). We cannot confidently rule out the possibility that WT rods preferentially exit NFBs after 30 h. There was no evidence of preferential WT exiting to proximal or distal locations at 30 h, but the uncertainty was great ( Fig. 5 ) likely due to low cell numbers, which decline further by 36 h near the edge of NFBs ( Fig. 1, 2A and 3A ) and in samples of the entire biofilm ( 46 ). In summary, we conclude that short-range C-signaling restricts cheating in mixtures with a WT minority, cheating occurs only during the spore-forming stage in mixtures with equal WT and csgA cells, and cheating occurs during both the mound- and spore-forming stages in mixtures with a WT majority."
} | 5,179 |
21567179 | PMC3114082 | pmc | 4,808 | {
"abstract": "Microalgae of numerous heterotrophic genera (obligate or facultative) exhibit considerable metabolic versatility and flexibility but are currently underexploited in the biotechnological manufacturing of known plant-derived compounds, novel high-value biomolecules or enriched biomass. Highly efficient production of microalgal biomass without the need for light is now feasible in inexpensive, well-defined mineral medium, typically supplemented with glucose. Cell densities of more than 100 g l −1 cell dry weight have been achieved with Chlorella , Crypthecodinium and Galdieria species while controlling the addition of organic sources of carbon and energy in fedbatch mode. The ability of microalgae to adapt their metabolism to varying culture conditions provides opportunities to modify, control and thereby maximise the formation of targeted compounds with non-recombinant microalgae. This review outlines the critical aspects of cultivation technology and current best practices in the heterotrophic high-cell-density cultivation of microalgae. The primary topics include (1) the characteristics of microalgae that make them suitable for heterotrophic cultivation, (2) the appropriate chemical composition of mineral growth media, (3) the different strategies for fedbatch cultivations and (4) the principles behind the customisation of biomass composition. The review confirms that, although fundamental knowledge is now available, the development of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of this promising technology.",
"conclusion": "Conclusions and future trends Recent advances in microalgal biotechnology have created opportunities for the efficient production of high-value (natural) compounds with the properties of plant-derived products that provide unique benefits (e.g. plant-like glycosylation) compared to their analogues resulting from chemical synthesis or recombinant microorganisms. The laboratory-scale bioreactor cultivations included within this review provide a first insight into the feasibility of carrying out heterotrophic processes with microalgae at an industrial scale. These processes have, in part, already been commercialised with the biotechnological production of PUFA. (To date, the authors are not aware of any literature on the heterotrophic large-scale fedbatch cultivation of microalgae, in contrast to literature on batch cultivation referred to by Apt and Behrens 1999 ; Behrens 2005 ; Wynn et al. 2005 ). This review confirms that the development of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of the promising microalgae technology. The generic cultivation strategies outlined are based on the experimental data of natural microalgae but, in principle, could also be applied to emerging strains improved by genetic engineering. Fundamental knowledge enabling strain design may be derived from advanced metabolic flux analyses (Xiong et al. 2010b ). A promising new avenue for transgenic microalgae is developing based on the knowledge gained over the past two decades, which includes the complete sequencing of the first microalgal genomes (Leon-Banares et al. 2004 ; Parker et al. 2008 ; Rosenberg et al. 2008 ; Walker et al. 2005a ; Walker et al. 2005b ).",
"introduction": "Introduction Microalgae, a large and heterogeneous group of microscopic algae, are an almost untapped pool of metabolic versatility. As many of the species occurring in nature have not yet been identified and/or physiologically characterised, their potential awaits exploitation in the biotechnological manufacturing of high-value biomolecules or deliberately enriched biomass (Guedes et al. 2011 ; Raja et al. 2008 ; Rosenberg et al. 2008 ; Wijffels 2008 ). The term ‘microalgae’ is typically used in its narrowest sense as a synonym for photoautotrophic, unicellular algae utilising CO 2 and gaining energy from light. Although certain species are obligate photoautotrophs, numerous microorganisms currently classified as microalgae are in fact obligate heterotrophs (Droop 1974 ; Gladue and Maxey 1994 ), and others are capable of both heterotrophic and photoautotrophic metabolism either sequentially or simultaneously (Chojnacka and Marquez-Rocha 2004 ; Droop 1974 ; Gladue and Maxey 1994 ; Lee 2001 ). Heterotrophic cultivation without light and with the controlled addition of an organic source of carbon and energy is similar to procedures established with bacteria or yeasts in multipurpose stirred closed tanks sterilised by heat. To date, only a small number of microalgal species have been cultured heterotrophically in conventional bioreactors (Chen 1996 ; Perez-Garcia et al. 2011 ). The few commercialised processes in which microalgae are grown under heterotrophic conditions are focussed on the manufacture of polyunsaturated fatty acids (PUFA) in 100-m 3 scale (Behrens 2005 ). These biotechnological processes represent a sustainable alternative to the extraction of PUFA from fish oil (Apt and Behrens 1999 ; Barclay et al. 1994 ; Barclay 1992 ; Kyle and Gladue 1991 ; Kyle et al. 1991 ; Mendes et al. 2009 ; Wynn et al. 2005 ). Several other heterotrophic processes that utilise microalgae have been established at laboratory scale to deliberately enrich the biomass with compounds such as pigments and antioxidants (Pulz and Gross 2004 ; Raja et al. 2008 ; Spolaore et al. 2006 ). l -Ascorbic acid (Running et al. 1994 ) and polysaccharides (Ramus 1972 ) are examples of commercially valuable extracellular products obtained from microalgae. Classes of compounds that are found in microalgae and that exhibit desirable properties for treating inflammation, tumours and viral or microbial infections are attracting new interest (Guedes et al. 2011 ). Moreover, research in the rapidly expanding field of biofuels (Wijffels and Barbosa 2010 ) provides a valuable source of fundamental information on the physiology and biochemistry of microalgae, producing high-value compounds (e.g. Brányiková et al. 2010 ; Xiong et al. 2010b ). The growing interest in microalgae, either non-recombinant or with appropriate genetic modification (Potvin and Zhang 2010 ; Specht et al. 2010 ), suggests that heterotrophic microalgal processes offer significant commercial opportunities (Rosenberg et al. 2008 ). In contrast to plants or seaweeds, in which biomass is fairly compact, the harvesting of unicellular microalgae dispersed in natural habitats of microbial consortia is not as straightforward. Low cell densities of several grams per litre are an important cost factor for established production processes with photoautotrophic microalgae in conventional open ponds or photobioreactors (Molina Grima et al. 2003 ). However, cell densities of more than 100 g l −1 cell dry weight, achieved with Chlorella , Crypthecodinium and Galdieria species, highlight the potential of heterotrophic microalgal processes (de Swaaf et al. 2003c ; Doucha and Lívanský 2011 ; Graverholt and Eriksen 2007 ; Wu and Shi 2007 ). Moreover, systematic screening for new compounds is only feasible provided that sufficient quantities of concentrated biomass from axenic (pure) cultures are attainable (Olaizola 2003 ; Wijffels 2008 ). From taxonomic studies, it is acknowledged that microalgae exhibit considerable metabolic plasticity (Trainor 2009 ). In response to their surroundings, particular species can occur in alternative phenotypes, and these can result in the altered formation of metabolites and/or products. Thus, the composition of biomass (or intracellular products) or the production of desired extracellular products is typically affected by culture conditions (Hu 2004 ; Illman et al. 2000 ; Jakobsen et al. 2008 ; Lv et al. 2010 ; Shi et al. 2006 ; Xiong et al. 2010b ; Yongmanitchai and Ward 1991 ). In turn, this large environmental adaptability provides opportunities to modify the production of targeted natural compounds and to control their formation at high titres, yields, productivities and the required quality (purity). However, screening the various (natural) phenotypes under different conditions is a complex, time-consuming task involving a large number of culture variables. The basic principles of systematic screening were established during studies of the species suitable for use in aquaculture hatcheries (Gladue and Maxey 1994 ). Although the opportunities for heterotrophic processes with microalgae have been considered in several review papers (Apt and Behrens 1999 ; Borowitzka 1999 ; Lee 2001 as well as more recently by Eriksen 2008b and Perez-Garcia et al. 2011 ), few cover aspects of cultivation technology in depth (e.g. Chen 1996 ; Chen and Chen 2006 ). In an attempt to address the outstanding issues, this review paper outlines the current best practices in the heterotrophic high-cell-density cultivation of microalgae for the production of biomass or specific products for health and nutraceutical applications. The main topics dealt with include (1) the characteristics of microalgae suitable for heterotrophic cultivation, (2) the appropriate chemical composition of mineral growth media, (3) strategies for high-cell-density cultivation and (4) the principles of customising biomass composition. Thus, the potential and limitations of fedbatch technology are outlined. The generic process strategies described are based on experimental data collected for non-recombinant microalgae and are, in principle, also applicable to emerging strains improved by genetic engineering."
} | 2,403 |
33379675 | PMC7771893 | pmc | 4,809 | {
"abstract": "The development of superhydrophobic metals has found many applications such as self-cleaning, anti-corrosion, anti-icing, and water transportation. Recently, femtosecond laser has been used to create nano/microstructures and wetting property changes. However, for some of the most common metals, such as aluminum, a relatively long aging process is required to obtain stable hydrophobicity. In this work, we introduce a combination of femtosecond laser ablation and heat treatment post-process, without using any harsh chemicals. We turn aluminum superhydrophobic within 30 minutes of heat treatment following femtosecond laser processing, and this is significantly shorter compared to conventional aging process of laser-ablated aluminum. The superhydrophobic surfaces maintain high contact angles greater than 160° and low sliding angles smaller than 5° over two months after the heat treatment. Moreover, the samples exhibit strong superhydrophobicity for various types of liquids (milk, coffee, CuPc, R6G, HCl, NaOH and CuCl 2 ). The samples also show excellent self-healing and anti-corrosion properties. The mechanism for fast wettability conversion time is discussed. Our technique is a rapid process, reproducible, feasible for large-area fabrication, and environment-friendly.",
"conclusion": "5. Conclusion The environment-friendly method of fabricating nano/micro structures by the femtosecond laser ablation with a grid pattern of 100 μm step size, followed by the heat treatment post-process at 200°C for only 30 minutes can produce superhydrophobic aluminum surface in an extremely short time, without using any harsh chemical coatings. The wettability conversion time is reduced incredibly from 30 days to 30 minutes, which is shortened approximately 1440 times. All the samples demonstrated for the stable superhydrophobicity for over five months (high contact angles as 160° and small sliding angles as smaller than 5°). The presented technique demonstrated its reproductivity and feasibility of large-area fabrication, which is advantageous for manufacturing and intended industrial applications. The interaction between femtosecond laser and the metal surface can provide different surface morphology as well as different surface chemistry compared to the case of nanosecond laser, which is important for the wettability conversion from superhydrophilic to superhydrophobic. Moreover, the produced superhydrophobic aluminum surfaces exhibited brilliant properties such as repellence with various types of liquids (milk, coffee, CuPc, R6G, HCl, NaOH, and CuCl 2 ), self-healing effect, and anti-corrosion. The mechanism for wettability conversion, for self-healing property, and for anti-corrosion property were also explained. The studied technique can open a new environment-friendly approach in the production of a superhydrophobic metal surface for various practical applications.",
"introduction": "1. Introduction The harsh and moisture environment can accelerate corrosion for metals. Superhydrophobic surfaces, where water cannot stay on the surface, usually possess anti-corrosion effect. Normally, hydrophobicity is defined when the contact angle of a water droplet on the surface is great than 90°; and superhydrophobicity is defined when the contact angle is greater than 150° with a sliding angle smaller than 10° [ 1 , 2 ]. The superhydrophobic property of lotus leaf is due to nano/microstructures on its surface and the low surface energy [ 3 ]. These two factors can also be applied to other materials to make superhydrophobic surfaces for dealing with corrosion problems. A variety of methods have been used to make nano/microstructures, such as Chemical Vapor Deposition (CVD), electrochemical corrosion, plasma sprayed, and other chemical-based methods [ 4 – 8 ]. However, these techniques are costly and complex. With the development of laser techniques, many researchers have focused on femtosecond laser processing to make nano/microstructures on different materials, which is lower cost and faster fabrication speed than CVD and other chemical-based methods [ 9 , 10 ]. Femtosecond laser processed surfaces also show drastic changes in surface wettability [ 11 – 13 ]. Stable hydrophobicity may be more difficult to obtain for some metals, such as aluminum. Often time, an aging process or chemical coating is applied following laser treatment. For example, aluminum requires 14–30 days to convert from superhydrophilicity to superhydrophobicity [ 14 – 17 ]. Another approach is coating chemicals such as Teflon, lauric acid or other harsh chemicals, on the femtosecond-laser-ablated metal to reduce the surface energy without waiting for any aging time [ 18 – 20 ]. However, this approach requires a few hours to over one day for the coating process. In several techniques, complex coating system or harsh chemicals are required. In our previous researches [ 21 , 22 ], we have tried to combine nanosecond laser and low temperature annealing process to accelerate the wettability conversion time from superhydrophilic to superhydrophobic. However, the required annealing treatment time is at least 6 hours to get the good superhydrophobicity with a high contact angle (greater than 150°) and a low sliding angle (smaller than 10°). Moreover, the self-healing property and anti-corrosion property have not been studied on these researches. In this research, a combination with femtosecond laser ablation and heat treatment post-process is introduced. The femtosecond laser is employed because it has a different interaction mechanism between the laser beam and metal surface, compared to that of the nanosecond laser [ 23 – 25 ], which can affect wettability conversion time. The proposed technique can solve the current limitations of other conventional techniques; and this technique also improves significantly the fabrication time as well as the superhydrophobic property compared to our obtained results in the previous works. First, a femtosecond laser was used to ablate a grid pattern on a flat aluminum surface to create nano/microstructures. Then, the just-after-laser-ablated surfaces were put in a commercial oven at 200 °C for heat treatment post-process. Interestingly, within 30 minutes under heat treatment, the samples converted from superhydrophilic to superhydrophobic, without the usage of any harsh chemicals. The wettability conversion time of femtosecond-laser-ablated aluminum can be shortened significantly from approximately 30 days in the common aging process to 30 minutes in this treatment post-process. The fabrication time is shortened approximately 1440 times. Compared to our previous studies, the wettability conversion time is reduced roughly from 6 hours to 30 minutes, which is shortened 12 times. Moreover, the superhydrophobic property in this technique shows better performance compared to our previous results. All the samples exhibited contact angles greater than 155° and sliding angles smaller than 5°. In this research, the fabricated superhydrophobic aluminum surface showed excellent effects such as stable superhydrophobicity (over two months), varied liquid-repellency, anti-corrosion, and self-healing. Moreover, the proposed technique demonstrated its reproductivity and feasibility of large-area fabrication.",
"discussion": "4. Discussions 4.1 Mechanism for femtosecond laser the wettability conversion When combining femtosecond laser ablation and heat treatment post-process, the morphology and the chemical composition of the aluminum surfaces were changed, which could reveal the mechanism for the fast wettability conversion from superhydrophilicity to superhydrophobicity. Due to the short time scales involved in the ablation of the femtosecond laser, the ablation process can be considered as a direct solid-vapor (or solid-plasma) conversion, which is different from the nanosecond laser ablation process; i.e. the energy first heats the solid target surface to the melting point and then to the vaporization temperature. This is the great advantage of femtosecond lasers compared to nanosecond lasers because of the dissipation of energy or the minimization of the heated affected zone. With the unique solid-vapor conversion, femtosecond laser ablation could create micro/nano structures as well as produce many nanoparticles on the surface, which increased the area that adsorbs organic matter in the air. In addition, the femtosecond laser directly evaporated the aluminum to make lotus-leaf-like structures surface as shown in Fig. 2(a) and Fig. 3 ; whereas, this could not be made by nanosecond laser. The nanosecond laser made burrs, as microstructures with double sharp tent shape, along the ablated lines by melting process [ 23 – 25 ]. L. Jiang et al. found that lotus-leaf-like structures can decrease the content area with the water and increase the superhydrophobic property [ 1 ]. In addition to the change of surface morphology, the chemical composition of the aluminum surface also changed when interacting with femtosecond lasers. When the femtosecond laser beam interacts with the aluminum surface, it can reach a high temperature up to approximately 5447 °C [ 26 ], which can easily oxidize the metal surface [ 27 ]. Aluminum and aluminum oxide are both inherently hydrophilic, which is strongly attracted to water. Therefore, when the surface roughness increased, the femtosecond-laser-ablated aluminum surface exhibited superhydrophilic property. This property was followed by Wenzel’s theory [ 28 ]. Other researchers have found that the femtosecond-laser-ablated aluminum surfaces convert from superhydrophilic to superhydrophobic when putting in the ambient air for a couple of weeks to a couple of months [ 14 – 17 ]. In this research, the femtosecond-laser-ablated samples, which were put in the ambient air for one month since laser ablation (without any heat treatment post-process), also converted from superhydrophilic to near superhydrophobic or superhydrophobic (135°—150°). The reason came from an organic adsorption mechanism. S. Banerjee has found that aluminum oxides, which were made at around 500 °C temperature by evaporation of the initial water layer on the oxides, exist nanoholes. These nanoholes have adsorption capacity, which is always used to made alumina adsorbent balls with nanostructures and microstructures [ 29 ]. Similarly, in this research, the just-after-laser-ablated surface also had many aluminum oxide nanostructures compared to our previous study in nanosecond laser [ 21 , 22 ]. Therefore, the nanostructures acting like the alumina adsorbent may have higher adsorption capacity on the femtosecond laser treated surface compared to nanosecond laser treated one. Moreover, the hydroxyl group (–OH) is also an effective adsorptive site, could adsorb the organic matters such as acetic acids, formic and polymeric hydrocarbons from air moisture [ 30 , 31 ]. Therefore, the organic matter in the ambient air are adsorbed on the femtosecond-laser-ablated surface to reduce the surface energy. The FTIR result on the femtosecond-laser-ablated samples, which were put in the ambient air for one month since laser ablation (without any heat treatment post-process), demonstrated the appearance of hydrophobic (–CH 2 –) bonding, (–CH 3 ) bonding, and (–C = C–) [ 32 ]. The increment of the surface roughness and the appearance of hydrophobic bonding makes the femtosecond-laser-ablated aluminum samples, which were put in the ambient air for one month since laser ablation (without any heat treatment post-process), became superhydrophobic. One month is a relatively long time and is not suitable for any manufacturing purposes. Therefore, by applying the heat treatment post-process for 30 minutes at 200 °C, the femtosecond-laser-ablated surface showed high contact angles greater than 160° and low sliding angles smaller than 5°. The heat treatment post-process only played a role as an acceleration process of the inherent organic adsorption phenomenon on femtosecond-laser-ablated surfaces. After the heat treatment, the chemical compositions changed, compared to the results of the just-after-laser-ablated surfaces. The EDS results demonstrated the incredible increment of carbon content. The FTIR results also showed the clear appearance of (–CH 2 –) bonding and (–CH 3 ) bonding, which were similar to the case of the femtosecond-laser-ablated aluminum samples, which were put in the ambient air for one month since laser ablation (without any heat treatment post-process). However, in the case of heat treatment, hydrophobic (–CH 2 –) bonding and (–CH 3 ) bonding appeared with higher intensity. This agreed with the wettability results (contact angle and sliding angle). The femtosecond-laser-ablated surfaces after one month in the air (without any heat treatment post-process), which existed low intensity of hydrophobic bonding, showed high contact angle (135° – 150°) and no sliding angle; whereas, the heat-treated one (after heat treatment for 30 minutes), which existed high intensity of hydrophobic bonding, showed high contact angle (about 160°) and low sliding angle (smaller than 5°). Therefore, the heat treatment could support the femtosecond-laser-ablated aluminum to get sufficient organic matters to decrease its surface energy to show the superhydrophobic property. This acceleration process can reduce the common wettability conversion time to superhydrophobicity on the femtosecond-laser-ablated aluminum surface from one month to 30 minutes. In summary, the wettability conversion from superhydrophilic to superhydrophobic aluminum surface depended on two main factors: surface roughness (nano/microstructures) and low surface energy of the materials. After femtosecond laser ablation, the aluminum increased its surface roughness with nanostructures and microstructures, without any formation of burrs or debris as same as the case of nanosecond laser ablation. These structures made by femtosecond are more absorbent for the organic matter in the ambient air. The heat treatment post-process accelerated the organic adsorption phenomenon on the femtosecond-laser-ablated aluminum surface to form an organic layer as shown in Fig. 7 . The combination of nano/microstructures and hydrophobic organic layer makes surfaces superhydrophobic. Fig. 7. The mechanism for wettability conversion from superhydrophilicity to superhydrophobicity. 4.2 Reproductivity and feasibility for large-scale fabrication The step size of 100 μm showed the best superhydrophobic performance among the obtained result in this research study. To investigate the reproductivity of this technique (femtosecond laser ablation and heat treatment), ten fabrication times were carried out with the same laser fabrication parameters. As shown in Fig. 8(a) the wettability results of 10-time repetitions were presented and each time includes three aluminum samples. All samples exhibited contact angle as approximately 160° and sliding angles as smaller than 5°. This demonstrated the reliability and reproductivity of the fabrication technique in this research. Moreover, all samples maintained their superhydrophobicity even over five months. Fig. 8. The contact angles and sliding angles of the heat-treated aluminum surface after laser ablation for the 10-time repetition. 5 mm x 5 mm grid pattern area is small for any practical applications. Therefore, an investigation on the feasibility of large-scale fabrication was performed. Several samples were ablated to form the grid pattern by femtosecond laser with larger fabrication areas. Herein, samples with 15 mm x 15 mm (scale-up of 3 times) and samples with 20 mm x 20 mm (scale-up of 4 times) was employed. After laser fabrication, all samples were put in the oven for 30 minutes at 200 °C as same as the heat treatment conditions on the samples with the 5 mm x 5 mm grid pattern area. Interestingly, all samples showed the superhydrophobic property as shown in Figs. 8(b), (c), and (d) . The contact angle and sliding angle of the 20 mm x 20 mm sample were similar to that of the 5 mm x 5 mm sample, as shown in Supplement 1 , Fig. S1. The heat treatment time and the heat treatment temperature were independent of the fabrication area. This can be extremely useful when making a large-scale superhydrophobic aluminum surface for practical applications in the industry because the heat treatment time is short. This fabrication method can also be used to process different common metal surfaces into superhydrophobic showed in Supplement 1 , Fig. S2. Visualization 1 showed the water bouncing on the large-scale heat-treated surface after laser ablation. 4.3 Self-healing property The self-healing property of the obtained superhydrophobic aluminum surface was investigated by using ultrasonic cleaning. When the obtained superhydrophobic samples were cleaned under ultrasonic at 40 °C for 4 hours, they became hydrophobic and hydrophilic in different positions. After ultrasonic cleaning, samples were put in the room condition for 6 hours. Interestingly, they recovered their superhydrophobicity, called as self-healing effect. To reduce six hours of waiting time, other samples after ultrasonic cleaning were put on the hot plate at 100 °C for 2 minutes. They could also recover their superhydrophobicity. In this experiment, ultrasonic cleaning could make the water go inside of the channels between the nano/microstructures because of the vibration [ 33 ]. The air gap between the water and the superhydrophobic surface was removed. The water went through the gap between the nano/microstructures and formed a thin water layer inner the structures. When dropping water on these surfaces during contact angle measurement, the water layer can combine with the dropping water and make samples hydrophobic or hydrophilic. However, when the samples were put in the air for 6 hours, the thin water layer can be naturally evaporated. Therefore, the samples become superhydrophobic again. When samples were put on the hot plate, high temperatures at 100 °C can enhance the evaporation process of the resultant thin water layer from ultrasonic cleaning. The fabricated aluminum surfaces in this research can recover their repellent property after losing the superhydrophobicity, like the self-healing effect as shown in Fig. 9 . Fig. 9. Schematic diagram of self-healing property. 4.4. Anti-corrosion property After putting the samples into the 5 wt % copper (II) chloride solution for 3 minutes, the red copper appeared on the whole surface of the flat surface and the just-after-laser-ablated aluminum surface; whereas the superhydrophobic aluminum surface did not appear the red color on the fabricated area, as shown in Fig. 10 . This could demonstrate that there is no copper formation in this area. The area around this fabricated area of the superhydrophobic surface appeared red copper because this is the flat aluminum surface. It means that the sample after femtosecond laser and heat treatment exists the anti-corrosion property. Additionally, the whole corrosion process on the flat aluminum surface, on the just-after-laser-ablated aluminum surface, and on the superhydrophobic aluminum surface, were presented in Visualization 2 . In the superhydrophobic area, the organic layer on the aluminum surface can protect the surface and it can also make an air barrier which prevents the solution to go inside the surface to contact aluminum. After immersing in the copper (II) chloride solution for 3 minutes, all the surfaces were ultrasonically cleaned. The taken images as shown in Fig. 10 indicated clear damage of the flat surface and the just-after-laser-ablated aluminum surface; whereas the superhydrophobic aluminum surface did not show any damage. The SEM images and the 3D structures, as shown in Supplement 1 , Figs. S3 and S4, respectively, could support well for the anti-corrosion property of the superhydrophobic aluminum surface. While the strong damage of structures on the flat surface and the just-after-laser-ablated aluminum surface was observed clearly; the structures on the superhydrophobic aluminum surface were similar to the superhydrophobic ones before the anti-corrosion testing. Moreover, on the superhydrophobic aluminum surface after immersing into the 5 wt % copper (II) chloride solution, the contact angle almost did not change and the sliding angle increased a bit, approximately to 5°. The mechanism can briefly be explained by the reactions as shown as Eq. ( 1 ), ( 2 ), and ( 3 ). First, Cu 2+ could replace the aluminum on the surface and became copper (Cu), which can be deposited on the surface of the sample. After this reaction, the corroded surface would turn to red because of the appearance of copper on the top of the sample surface. Then, the H + , which had been produced by hydrolyzed of Cu 2+ and Al 3+ , could create hydrogen with the reaction of aluminum. During this reaction, bubbles could be formed on the aluminum surface. The superhydrophobic surface can prevent the corrosive solution to interact with the surface due to the air gap. The anti-corrosion property with 5 wt % copper (II) chloride on the superhydrophobic aluminum surface is demonstrated. (1) 3 C u 2 + + 2 A l → 2 A l 3 + + 3 C u \n (2) C u 2 + + 2 H 2 O → 2 H + + C u ( O H ) 2 \n (3) 6 H + + 2 A l → 2 A l 3 + + 3 H 2 Fig. 10. The pictures of the heat-treated surface after laser ablation, flat aluminum surface, and just after-laser-ablated surface before and after immersing into the copper (II) chloride CuCl 2 for 3 minutes. Moreover, for electrochemical measurements, the corrosion potential (Ecorr) and corrosion current density (Icorr) can be obtained by the extrapolation method in this polarization (Tafel) plots. The Tafel plots are shown in Fig. 11 . The corrosion voltage of the flat surface is -384.118 mV and the one on the superhydrophobic surface is -369.443 mV. As for the corrosion current, the flat surface is 22.849 μA and the superhydrophobic surface is 13.4 μA. The values of the Ecorr, Icorr, βa, and βc derived from Tafel plots showed in Table 2 [ 34 ]. Base on the Icorr, CR (corrosion rate) can be gotten on Eq. ( 4 ) Fig. 11. Tafel plots of the flat aluminum surface and heat-treated surface after laser ablation in 3.5 wt % NaCl solution. \n Table 2. Corrosion Current Density (Icorr), Corrosion Potential (Ecorr), Anodic Slope (βa) and Cathodic Slope (βc), CR and the CIE of the flat aluminum surface and heat-treated surface after laser ablation Sample Icorr (A cm −2 ) Ecorr (mV) βa (mV/ dec) βc (mV/ dec) CR (mm/year) CIE (%) Flat aluminum surface 2.2849 × 10 −5 -384.118 311.2 307.8 0.255021 0 Heat-treated surface after laser ablation 1.34 × 10 −5 -369.443 309.3 310.7 0.149559 41.4 \n (4) CR ( mm / year ) = 3.27 × 10 − 3 × I c o r r × M n d in which M is the relative atomic mass of metal (g/mol), d is the density of the metal (g/cm 3 ) and n is the number of electrons required to oxidize an atom of the element in the corrosion process. The corrosion inhibition efficiency (CIE) can be calculated by Eq. ( 5 ): (5) C I E / % = I c o r r − I ′ c o r r I c o r r × 100 Icorr is the corrosion current of the flat aluminum surface and Icorr is the corrosion of the heat-treated surface after laser ablation [ 35 ]. The heat-treated aluminum surface showed better anti-corrosion property due to its superhydrophobicity. Additionally, the superhydrophobic aluminum surface showed a high contact angle to acid and base, as shown in Supplement 1 , Fig. S5. Therefore, the superhydrophobic aluminum surface using femtosecond laser fabrication and heat treatment had the anti-corrosion property, which can prevent or slow down the corrosion reaction on its surface."
} | 5,927 |
21154995 | null | s2 | 4,810 | {
"abstract": "Many bacteria use quorum sensing (QS) to regulate cell-density dependent phenotypes that play critical roles in the maintenance of their associations with eukaryotic hosts. In Gram-negative bacteria, QS is primarily controlled by N-acylated L-homoserine lactone (AHL) signals and their cognate LuxR-type receptors. AHL-LuxR-type receptor binding regulates the expression of target genes necessary for QS phenotypes. We recently identified a series of non-native AHLs capable of intercepting AHL-LuxR binding in the marine symbiont Vibrio fischeri, and thereby strongly promoting or inhibiting QS in this organism. V. fischeri utilizes N-(3-oxo)-hexanoyl L-HL (OHHL) as its primary QS signal, and OHHL is also used by several other bacterial species for QS. Such signal degeneracy is common among bacteria, and we sought to determine if our non-native LuxR agonists and antagonists, which are active in V. fischeri, would also modulate QS phenotypes in other bacteria that use OHHL. Herein, we report investigations into the activity of a set of synthetic LuxR modulators in the plant pathogen Pectobacterium carotovora subsp. carotovora Ecc71. This pathogen uses OHHL and two closely related LuxR-type receptors, ExpR1 and ExpR2, to control virulence, and we evaluated their responses to synthetic ligands by quantifying virulence factor production. Our results suggest an overall conservation in the activity trends of the ligands between the ExpR receptors in P. carotovora Ecc71 and LuxR in V. fischeri, and indicate that these compounds could be used as tools to study QS in an expanded set of bacteria. Notable differences in activity were apparent for certain compounds, however, and suggest that it might be possible to selectively regulate QS in bacteria that utilize degenerate AHLs."
} | 448 |
35858419 | PMC9335220 | pmc | 4,813 | {
"abstract": "Significance Biofilms are ubiquitous in porous media, including soils and technical applications such as bioremediation and wastewater treatment systems. Biofilms can drastically alter the transport of nutrients and contaminants through porous media by forming preferential flow paths, which are subject to strong intermittency in fluid flow. This intermittency manifests through the opening and closing and spatiotemporal rearrangement of flow paths, and its mechanism has to date remained unresolved. Here, we show that intermittency is driven by the competition between microbial growth that governs the closing of preferential flow paths and biofilm compression and rupture that control their opening.",
"discussion": "Discussion Our work has elucidated the mechanism underlying PFP intermittency in biofilm-bearing porous media and has shown that intermittency is due to the competition between microbial growth and fluid shear stress. Closing of the PFPs is controlled by the nutrient mass flow; in an experiment in which flow contained no nutrients, intermittency ceased completely. The opening of the PFPs, due to detachment of the biofilm and its compression normal to the flow direction, results from the viscoelastic mechanical behavior of the biofilm under shear stress ( 42 ). Using a mathematical model based on the Darcy–Brinkman–Biot formulation, we confirmed that suppressing biofilm growth or decreasing the shear stress exerted by the fluid flow halted PFP intermittency. To compare the dynamics of PFP closing to that of the clogging of individual pores, we characterized individual pore clogging by using the extended Gompertz growth model, which is extensively used to describe both bacterial growth in bulk ( 43 ) and biofilm growth under flow ( 44 ). We found that biofilm growth dynamics appear to be maintained in porous media both at the pore scale and during clogging of the entire system, despite the shear forces to which the biofilm is exposed. When comparing the plateaus of the Monod fits for the clogging of individual pores and for the closing of PFPs, we found that the maximal PFP closing speeds were an order of magnitude smaller than the maximal individual pore-clogging speeds. This is likely explained by differences in the shear rate, which is much higher in PFPs as the porous medium approaches full clogging than in individual pores in the early stages of clogging. The shear rates in a microfluidic device of pore size d = 300 µm at a flow rate Q = 1 mL/h range from 10 1 to 10 3 1/s in the early stage ( Fig. 4 A ), whereas in a PFP, shear rates are estimated to range from 10 4 to 10 6 1/s ( Fig. 2 F ). Once PFPs have formed, most of the fluid flow is channeled through the open paths since very little flow can be accommodated by the biofilm due to its very low permeability ( 13 ). Biofilm sloughing off within porous media leading to the rapid opening of PFPs will affect downstream clogging, as the flow transports chunks of biofilm and individual bacterial cells. Mass transport and the regaining of cell motility can lead to the subsequent spreading of bacteria within a pore network ( 45 , 46 ). The occurrence of detachment is determined by fluid flow, nutrient supply, and the geometry of the habitat but also by the mechanical properties of the biofilm ( 42 , 47 ). The mechanical properties of the biofilm can vary in space and time depending on the depth (normal to the direction of the PFP) into the biofilm. In space, we observed a gradual increase in density (i.e., a decrease of light intensity) deeper into the biofilm. In time, we observed a gradual pressure increase in the successive closing–opening cycles of the PFP. Furthermore, the elastic properties allow the biofilm to withstand disturbances shorter than the elastic relaxation time, while longer stress will lead to nonreversible viscoplastic deformation, sloughing, and detachment ( 48 ). In our experiments, we observed that the biofilm was sloughed off intermittently. This hints at a non-Newtonian behavior: a critical shear stress is required to overcome the yield stress of the biofilm in order to trigger PFP intermittency. These results have an abiotic analogy in cohesive sediment transport, where a critical shear stress is required to induce sloughing ( 49 ). In natural settings, biofilms in sediments can prevent sediment erosion, thanks to the yield stress of the biofilm ( 50 , 51 ). In order to capture biofilm behavior in models of porous media, it is crucial to take the mechanical properties of the biofilm into account. In our study, we modeled the biofilm as a viscoplastic material with a yield stress, which allowed us to reproduce the intermittent PFP behavior. The viscoplastic behavior captures the observed, intermittent sloughing off better than a viscoelastic behavior, which would imply greater flowing of the biofilm rather than detachment. The rheological properties of the biofilm depend on the composition of the extracellular polymeric substances, which, in turn, can vary greatly depending on the microorganisms present, the nutrient availability, and the environmental conditions ( 52 ). The interplay between biofilm rheology and local flow conditions determines biofilm morphology, as recently demonstrated ( 53 ). In particular, our results show that the biofilm’s viscoplastic behavior drives its capability of clogging and forming PFPs in a porous medium, as the rheological descriptors of the biofilm were critical parameters of the mathematical model we developed. We hypothesize that a change in biofilm rheology, for example, due to the presence of a different bacterial species or a drastic shift in environmental conditions, may affect the occurrence of PFP intermittency and the frequency of opening and closing events. Furthermore, fast-growing bacterial communities are more efficient in clogging porous structures and will consequently reduce their access to flowing nutrients so that slow-growing competitors may be favored ( 54 ). Similarly, the occasional sloughing will increase access to nutrients by preventing flow paths from being choked off, thus promoting bacterial growth locally. This implication is counter to the implied consensus in the literature that more robust biofilms or stronger biofilm growth are always beneficial to bacteria growing on surfaces under shear stress and may imply that biofilms can tune their physical properties depending on cell density and access to nutrient supply in order to promote survival. The intermittent opening and closing of the PFPs were reflected in the pressure difference through the system: the closing of the PFPs resulted in an increase in the pressure difference due to a reduction in the bulk hydraulic conductivity of the system, whereas the opening of the PFPs reduced the pressure difference. We observed this behavior for completely clogged porous media; this stands in contrast to bioinduced partial clogging of a porous medium, where the continuous reduction in pressure difference results mainly from biofilm formation in the upstream part of the porous medium ( 2 ). From this, it can be concluded that the observation of a recurring pressure increase and decrease within a porous medium cannot solely be attributed to the breakthrough of a bioinduced plug but can also be due to intermittent PFPs. We demonstrated that a power law with a negative exponent that falls between the Hagen–Poiseuille and Darcy solutions best describes the relation between pressure difference and PFP width. Our systematic study of the dependency of PFP intermittency on fluid flow rate and pore size expands and generalizes the conditions under which PFP intermittency is known to occur ( 28 , 29 ). We showed that intermittency occurs only under certain conditions, at fluid flow rates larger than 0.5 mL/h and pore sizes larger than 75 µm. Whether PFP intermittency occurs can be explained by the impact of fluid shear stresses in combination with the material properties of the biofilm. We observed an increased frequency of opening of PFPs with increasing shear forces, which caused more biofilm detachment ( 19 , 29 ). We observed a stabilization in the frequency of PFP opening over time. We attribute this to a more stable biofilm structure over time because of densification deeper into the biofilm due to compression and the regular removal by shear of the newly formed biofilm closest to the PFP ( Movie S1 ). The PFP closing speed correlated with the nutrient mass flow rate and followed a Monod kinetic, as we also observed for the clogging speed of individual pores. Weak or no PFP intermittency was observed at low flow rates because no PFPs were formed. Instead, complete bioclogging was observed. For the smallest pore size, in contrast, a PFP was formed due to a catastrophic rupturing, but without subsequent intermittent opening and closing ( SI Appendix , Fig. S10 ). This is likely explained by the larger surface for biofilm attachment with a smaller pore size, which allows the biofilm to form a denser and more rigid network. The biofilm can thus withstand higher pressures until it abruptly ruptures and forms a large flow path without intermittency ( SI Appendix , Fig. S10 ). By combining microfluidic experiments and mathematical modeling, we were able to unravel the competing mechanisms driving PFP intermittency and fully characterize PFP behavior under a range of geometric and hydraulic conditions relevant in environmental sciences and industrial technologies such as filters and bioreactors. In our microfluidic devices, the grains are regularly arranged and all pores have the same initial fluid flow conditions. Despite this scenario being a simplification of the irregular nature of soils, it allowed a systemic study of pore-clogging and an unprecedented comparison of different hydrodynamic conditions. In our experiments, the fluid flow rates ranged from 0.2 to 2 mL/h (corresponding to velocities of 16 to 160 m/d), which are comparable to transport velocities of microorganisms in soils ( 55 ) and in bioremediation applications ( 56 ). In addition to determining the effect of irregular grain arrangements on intermittency, it will be interesting to study how intermittency changes when one considers pressure-driven flow, which is common in natural environments, rather than an imposed flow rate as done here. Additionally, we highlight that imposed flow is found in certain technical applications, including biomineralization ( 57 ) and biochemical reactors ( 58 ) and that our experimental fluid flow velocities are of the same order of magnitude as in those applications. The formation and dynamics of PFPs in porous media strongly impact mass transport by creating spontaneous chemical inundations during PFP opening. Each opening results in a rapid change of the velocity distribution in the biofilm–porous medium system, leading to a transient flow system with high variance in residence time and strong mixing of chemicals with resident solutions and hence increasing the efficiency of reactions ( 59 ). PFP intermittency, a phenomenon based on the interplay between hydromechanical and biological processes, can be relevant in natural and industrial systems, with applications ranging from soil and aquifer bioremediation, bioreactors, filtration (design of membranes), and enhanced oil recovery. Our findings can contribute to improving the understanding of natural systems and assist in the design of applications that harness the properties of biofilms."
} | 2,883 |
40021616 | PMC11871289 | pmc | 4,815 | {
"abstract": "Ionic fluidic devices are gaining interest due to their role in enabling self-powered neuromorphic computing systems. In this study, we present an approach that integrates an iontronic fluidic memristive (IFM) device with low input impedance and a triboelectric nanogenerator (TENG) based on ferrofluid (FF), which has high input impedance. By incorporating contact separation electromagnetic (EMG) signals with low input impedance into our FF TENG device, we enhance the FF TENG’s performance by increasing energy harvesting, thereby enabling the autonomous powering of IFM devices for self-powered computing. Further, replicating neuronal activities using artificial iontronic fluidic systems is key to advancing neuromorphic computing. These fluidic devices, composed of soft-matter materials, dynamically adjust their conductance by altering the solution interface. We developed voltage-controlled memristor and memcapacitor memory in polydimethylsiloxane (PDMS) structures, utilising a fluidic interface of FF and polyacrylic acid partial sodium salt (PAA Na + ). The confined ion interactions in this system induce hysteresis in ion transport across various frequencies, resulting in significant ion memory effects. Our IFM successfully replicates diverse electric pulse patterns, making it highly suitable for neuromorphic computing. Furthermore, our system demonstrates synapse-like learning functions, storing and retrieving short-term (STM) and long-term memory (LTM). The fluidic memristor exhibits dynamic synapse-like features, making it a promising candidate for the hardware implementation of neural networks. FF TENG/EMG device adaptability and seamless integration with biological systems enable the development of advanced neuromorphic devices using iontronic fluidic materials, further enhanced by intricate chemical designs for self-powered electronics.",
"conclusion": "Conclusion This study demonstrated a soft, two-terminal iontronic fluidic memristor and memcapacitor using a fluidic FF and PAA Na + interface within a PDMS channel. The iontronic fluidic memristor (IFM) exhibits neuromorphic behaviours observed in biological systems, making it a promising candidate for advanced neuromorphic computing applications. A key innovation of this work is integrating a self-powered mechanism by combining the iontronic fluidic device with FF TENG/EMG. The TENG, characterised by its high input impedance, is effectively combined with the low input impedance of the IFM device. The contact separation EMG combined with FF TENG improves the ferrofluid TENG’s performance, enabling the IFM device’s autonomous powering. This self-powered capability is particularly significant for the development of neuromorphic systems, where traditional power sources can be bulky, limiting the flexibility and adaptability of these devices. The autonomous energy generation and storage provided by the FF TENG/EMG device eliminates the need for external power sources, enhancing the portability and integration of neuromorphic devices in real-world applications. Furthermore, the self-powered nature of these devices contributes to their long-term sustainability and reliability. By harvesting energy from environmental stimuli, such as mechanical movement or bio-signals, the FF TENG/EMG device can continuously power the IFM, supporting its synapse-like adaptive learning and memory functions, including STM and LTM. This capability not only extends the operational lifetime of the device but also aligns with the principles of energy-efficient and sustainable design, which are increasingly important in modern technological applications. The findings from this work suggest a novel paradigm for neuromorphic hardware that leverages self-powered iontronic fluidic materials to create multifunctional structures. These materials can offer new possibilities for adaptive sensing, signal processing, intelligent edge computing, and memory functions in self-powered robotic and neuromorphic systems. This approach represents a significant advancement in developing flexible, autonomous, and energy-efficient neuromorphic devices that can operate independently and adapt to their environment, making them ideal for a wide range of self-powered wearable sensing applications.",
"introduction": "Introduction Neuromorphic computing systems aim to achieve the same density, connectedness, and efficiency as the brain 1 – 6 . To do this, networks of highly parallelised dynamic elements and materials with signal processing and memory capabilities are located in the same place, similar to biological synapses 1 , 7 . The biological synapse is the crucial connection for signal processing in brain-like networks 2 , 7 . The ion–neurotransmitter–ion route at chemical synapses plays a significant role in the intricate neurological processes observed in advanced organisms such as humans 8 – 10 . Comprehending and replicating chemical synapses operational processes is crucial to implementing synaptic intronic devices successfully 11 . Chemical synapses have three main elements: the presynaptic neuron, the synaptic cleft, and the postsynaptic neuron 8 , 10 . During a synaptic event, an electrical ion current is transmitted from the presynaptic neuron 8 , 12 . This current triggers the release of neurotransmitters into the synaptic cleft, which in turn stimulates the postsynaptic neuron 12 . As a result, the signal is transmitted from the presynaptic neuron to the postsynaptic neuron 9 , 10 (as depicted in Fig. 1a ). Fig. 1 Neuromorphic Behavior of Iontronic Fluidic Memristor. a Illustrating the synaptic response between the presynaptic and postsynaptic neurons compared to the ionic memristor interface with future technology. b The primary mechanism schematic manipulates ion and liquid transport in the ionic liquid discrete channel. c The device voltage sweeps at different voltages. d The device voltage sweep of ±4.5 V for 100 endurance cycles. e The step response from 3.3 to 4.5 V with a voltage step of 0.1 V. f The current and voltage response with time domain for continuous positive and negative voltage cycles. The multi-state switching behaviour at dual voltage sweep of g 0 V to 4.5 V to 0 V and h 0 V to −4.5 V to 0 V. i The capacitance response of the device at a bias voltage of 3 V in a frequency range of 3–10 kHz. j Capacitive voltage sweep at ±4.5 V of ionic device in a frequency range of 3–10 kHz Despite this, solid-state devices simulating the electric pulse pattern are utilised for most neuromorphic functions executed thus far. The simulation of a chemical synapse in a solution-based environment uses these solid-state devices 13 – 15 . In an aqueous environment, fluidic-based memristors 16 are regarded as the most advantageous devices for achieving neuromorphic functions due to their remarkable compatibility with biological systems and capacity to imbue neuromorphic devices with an extensive array of capabilities via the incorporation of various chemistries 17 . To optimise the performance of neuromorphic computing by simulating synaptic behaviours in neuronal networks via synaptic iontronic devices, it is vital to have a thorough understanding of the synaptic properties and the process by which the devices replicated these properties 18 (Fig. 1a ). Ion solutions permit the coexistence and navigation of various ion types 19 . Furthermore, ions function as substantial carriers of charge in biological systems as well as fluidic memristors 20 . Therefore, the communication between devices and actual neurons is facilitated by the unparalleled convenience that the fluidic memristor’s biological compatibility affords 21 . Moreover, by incorporating an ionic liquid electrolyte interface into discrete channels, continuous plasticity was successfully attained 22 . Several studies have reported the presence of memcapacitance in confined systems. While a few fluidic memristors have been developed, there’s been limited progress in physically realising memory capacitors. Currently, there’s no realisation of devices exhibiting ideal analogue memcapacitance resulting from molecular-scale geometrical changes in materials 17 , 18 , 21 – 24 . It is critical and highly desirable to conduct independent research on memcapacitive based on iontronic fluidic materials. Exploring alternative memcapacitive materials to overcome these challenges is crucial and highly desirable. Additionally, most observed memcapacitive behaviours are linked with memristive switching, with little attention paid to the memcapacitive mechanism itself 24 – 26 . This lack of focus undermines the rational and effective design and optimisation of memcapacitors in the future. The field of iontronic offers an unprecedented prospect for developing neuro iontronics devices based on memristor and memcapacitor behaviour that emulate the operational capability of the human brain 1 , 16 , 25 . As the development of advanced human-machine interfaces (HMIs) continues to accelerate, there is an increasing demand for devices that are not only energy-efficient but also capable of performing multiple functions, such as sensing, memory storage, and learning 8 , 27 . TENGs are particularly valuable in this context due to their versatility, especially when integrated with synaptic resistive memory devices 27 . These memory devices mimic the behaviour of biological synapses, thereby enabling both memory retention and learning capabilities within the system 27 . By merging the sensing and energy-harvesting functions of TENGs with the memory and learning functionalities of synaptic resistive memory devices, it is possible to create intelligent neuromorphic systems that operate with remarkably low power consumption 27 . TENGs, when integrated with these memory devices, can generate spike-like electrical impulses that closely resemble the action potentials found in biological neural networks 27 . Previous approaches to integrating TENGs with synaptic resistive memory devices have largely focused on systems with high input impedance 27 – 29 . TENGs, naturally possessing high input impedance, are well-suited for powering resistive memories with high input impedance characteristics. However, a significant challenge remains that there is currently no effective method for self-powering low input impedance iontronic fluidic memristor (IFM) devices using TENG/EMG. The incorporation of ferrofluid (FF) presents a novel hybrid solution. In this approach, the TENG’s active layer is engineered to be sensitive to magnetic fields, which enhances the overall performance of the TENG. Additionally, using an electromagnetic coil provides a hybrid approach which reduces the input impedance, thereby enabling the efficient powering of IFM. This innovative combination improves FF TENG/EMG based systems functionality and expands their applicability in next-generation IFM neuromorphic devices for self-powered electronics as compared in Supplementary Tables S1 and S2 . This paper presents an IFM capable of effectively performing various neuromorphic activities. These capabilities include emulating electric pulse patterns and chemical-electric signal transduction. IFM is motivated by biological ion channels, which perform the job of natural memristors by regulating ion flow via spatial confinement. Using a fluidic interface consisting of FF and PAA Na + , we developed and manufactured a PDMS fluidic channel iontronic memory, as shown in Fig. 1a . The mechanism is based on ion migration, electroosmosis, and redox reactions. The ability of iontronic devices to precisely regulate the movement of ions in a fluidic medium enables the creation of memory systems with history-dependent behaviour, mimicking biological synapses. The fluid was contained by PAA Na + in this manner, which allowed for the construction of cation concentration equilibrium and charge balance between the inside and outside of the fluidic medium. This occurred under the stimulation of electric fields, causing a redox reaction that injected Cu 2+ ions, ultimately leading to the formation of history-dependent ion memory. The apparatus demonstrates properties typical of biological synapses, such as spike rate-dependent plasticity (SRDP). In addition, the device has features that characterise memory functions in the brain, such as long-term memory (LTM) and short-term memory (STM). Changes to the weights of the IMF are very reproducible and have been successfully examined for the creation of a convolutional neural network (CNN) with reasonable accuracy in output. Furthermore, by integration of the FF TENG/EMG energy-harvesting system with IFM devices to create intelligent systems that mimic the complex signal transduction and learning behaviours of the brain, offering substantial progress toward energy-efficient, bio-compatible, and high-performance neuromorphic computing platforms. Furthermore, the flexibility and bio-compatibility of IFM devices make them ideal for soft robotics applications, where their ability to mimic synaptic functions can enable adaptive control, sensory feedback, and real-time learning in soft robotic systems, enhancing their responsiveness and versatility in dynamic environments.",
"discussion": "Results and discussion Mechanism of IFM Iontronic devices precisely regulate the movement of cations and anions 16 , 30 , 31 . The fundamental operational concept of iontronic devices depends on the ability to alter their electrical characteristics selectively. The behaviour of electrolytes is the fundamental basis of the physics that enables the existence of fluidic memristors 10 . As illustrated in Fig. 1b , when a voltage signal is applied across a fluidic channel, even a small voltage can generate an enormous electric field \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\vec{E}}_{e}$$\\end{document} E ⃗ e , which induces the cations to drift along the channel. This liquid motion in the presence of an electric field \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\vec{E}}_{e}$$\\end{document} E ⃗ e , is known as electroosmosis. Prolonged application of the sweep voltage would sustain the migration and motion of ions, thereby potentially introducing substantial non-linearities into ionic transport. The fundamental mechanism for discrete channel devices is based on ion migration, concentration polarisation, and redox reactions (Cu 2+ ions form through anodic oxidation at the positive electrode, while Na + ions from the polyacrylic acid migrate toward the negative electrode). In this device, ions with identical surface charges repel one another and are attracted to ions with opposite charges, as described by the electroosmotic flow equation in Fig. 1b . Notably, because the ion selectivity on the FF side is negligible compared to the PAA Na + solution, the electric body force \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\vec{{f}_{e}}$$\\end{document} f e ⃗ is only considered on the PAA Na + side. In contrast to the ions in the bulk region of FF, the predominant factor in transport is the counterions (positive and negative ions) that are attracted to the charges on the wall surface of PAA Na + . During the presence of an applied electric field \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\vec{E}}_{e}$$\\end{document} E ⃗ e on an interface between FF and PAA Na + , the electric body force \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\vec{{f}_{e}}$$\\end{document} f e ⃗ generates an imbalance between co-ions and countries \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$({n}_{+}-{n}_{-})$$\\end{document} ( n + − n − ) , resulting in a net charge. This phenomenon is illustrated in Fig. 1b . The conductance tuning in our discrete channel device will enable neuromorphic computing to be executed due to the electroosmotic flow. It is possible to see a decrease in conductance during electrical body force due to electrode metallisation resulting from the oxidation and reduction reactions. Cu 2+ ions mobility plays a significant part during electroosmotic flow, leading to ion concentration polarisation and electrode metallisation. As a result of the diffusion of concentration gradient flux, the flow of ions reduces, ultimately leading to the formation of a state with high resistance (metallisation process). After the polarity of the voltage is altered, the Cu 2+ ions will travel in the opposite direction, resulting in the conduction of ions becoming weak. During each voltage sweep, this process will be repeated, and the end outcome will be a reduction in conductance due to electrode metallisation. Memristor and memcapacitor Solid-state memristor’s energy consumption and functionality are still not on par with biological neurons that use ions as information carriers 5 . Thus, creating artificial memristors that use ions as information carriers opens new research avenues for scientists to pursue. Among the ionic-based memristors, IFM memristors have advanced quickly in recent years 3 , 32 , 33 . Electronic synapses rely on electrons and holes for conduction, whereas biological synapses primarily use ions for information transit and processing. This is the primary difference between conventional synapses and their counterparts 34 , 35 . Ions have recently shown promise as a candidate for use in identifying synaptic devices due to ion mobility being significantly lower than electron mobility 31 , 36 . Due to their diverse structures, ionic current can provide more information than electronic current because ions have various valences, sizes, and polarizabilities. Figure 1c shows the measured current–voltage ( I – V ) sweep characteristics on different voltage sweeps from ±1 V to ±4.5 V with a voltage step sweep of 0.5 V, which reveals that hysteresis and conductance tuning are visible with only PAA Na + and FF in the discrete channel of IFM. Based on Fig. 1d , we may deduce that when the voltage is positive, an electrical body force will be generated inside the PAA Na + solution and directed from that side of the device to the FF side. The PAA Na + solution is pushed against the FF by this force, and the fluidic channel’s total conductance is improved. This I – V explains (Fig. 1d ) the initially accelerated increase in electroosmotic flow caused by the movement of metallic ions Cu 2+ under applied voltages, as indicated by the increasing slopes of the marked region ①. The device will undergo a high resistance state after a specific voltage due to the ion concentration of polarisation at the anode and cathode marked region ②. This stage of high resistance will increase as a marked region ③. The electrical current drops following the reverse biased voltage, shown by marked region ④. The same procedure will be used for the negative voltage sweep. Starting from an initial voltage sweep in the positive direction. The voltage reaches a sufficient positive value to induce an anodic current, causing oxidation of the copper metallic electrode and forming Cu 2+ cations, which subsequently migrate to the negative electrode. The redox reaction persists until most surfaces of the electrode are oxidised, peaking the anodic current. Subsequently, the current decreases for the remaining forward scan duration until the voltage sweep is reversed. The current decreases until the potential reaches a point where the reduction of Cu 2+ begins, resulting in the formation of Cu metal. The cathodic current peaks at this point, coinciding with a notable reduction in Cu 2+ ion concentration. The cathodic current then decreases from its peak after the ion concentration of polarisation. The negative sweep proceeds like the positive scan. Figure 1e demonstrates the effects of raising the SET-stop voltage from 3.3 V to 4.5 V. It was observed that the SET-stop voltage influences the resistance values in both the LRS and HRS. Notably, multiple switching states can be achieved by incrementally increasing the SET voltage by 0.1 V. To confirm the conductance and shift even further, we performed ten successive triangular positive (+4 V) and negative (−4 V) voltage sweeps with time, as seen in Fig. 1f . For successive positive and negative sweeps, the findings indicated a reduction in the current of the device over time; these features are seen as most advantageous for device use in neuromorphic computing. Setting or reset pulses may restore this adjustable state to its starting point. These results imply that memristor conductance may be gradually adjusted by applying optimum programming voltage pulses on the devices’ presynaptic terminals. Figure 1g, h depict the memristive characteristics of IFM, captured through a series of voltage sweeps. Initially, ten consecutive sweeps were applied from 0 V to +4.5 V to 0 V, followed by ten consecutive sweeps from 0 V to −4.5 V to 0 V. It is noteworthy that the conductance of the device decreased progressively with each sweep, regardless of whether a positive or negative voltage was applied. A particularly interesting observation is that the second sweep almost entirely overlaps with the first, highlighting a distinct change between successive sweeps. Capacitive neural networks offer an alternative physical implementation of resistive-based neural networks that more accurately emulates neural functions and potentially reduces circuit power dissipation due to the conversion of signals from current to voltage 23 , 24 , 37 – 39 . As a result, the memcapacitive characteristics of IFM devices at the intronic fluid interface of FF and PAA Na + are investigated. As the frequency increases from 3 kHz to 10 kHz, the capacitance decreases progressively in the capacitance–frequency (C–F) measurement with a bias voltage of 3 V (Fig. 1i ). Due to the limited time available for ions to traverse the electrical field and generate an electrical double-layer capacitance at high frequencies, high-frequency capacitance is diminished compared to low-frequency capacitance. As frequency increases from 3 kHz to 10 kHz, capacitances decrease from 172.98 to 70.80 nF in the C–F curves. As the biassing frequency increases from 3 kHz to 10 kHz, the capacitance amplitude and the hysteresis loop areas decrease, as illustrated in Fig. 1j . Using memory characteristics, the device can convert between the low and high capacitance states (LCS and HCS). The capacitance of the ON state is significantly greater than that of the OFF state in our experiment. The decrease in capacitance of the HCS with increasing frequency is attributed to the greater likelihood that positive charge (Na + and Cu 2+ ) becomes confined at its interface with the electrode at lower frequencies. Therefore, the optimal frequency should be selected to maximise the capacitive difference between the two phases. Neuromorphic computing Promising synaptic characteristics of iontronic fluidic devices have been shown in the electrochemical responses of the ions, which can replicate the movement of ions in the nervous system, so these characteristics can be used to build synaptic devices. Every process in the brain that involves encoding, transferring, or decoding information begins with the neuron (Fig. 1a ). In biosynapses, an action potential moving through the presynaptic neuron triggers the opening of voltage-gated calcium (Ca 2+ ) channels. This leads to an influx of Ca 2+ ions into the presynaptic terminal. The Ca 2+ ions then bind to synaptic vesicles loaded with neurotransmitters, facilitating their docking at the presynaptic plasma membrane. Once docked, these vesicles fuse with the membrane, releasing neurotransmitters into the synaptic cleft, the space between the presynaptic and postsynaptic neurons. The released neurotransmitters then bind to specific receptors on the postsynaptic plasma membrane, particularly to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. These receptors function as ion channels, and their activation by neurotransmitters leads to the opening of these channels, allowing ions to flow into the postsynaptic neuron. This ion influx, primarily involving sodium (Na + ) and Ca 2+ ions, causes depolarisation of the postsynaptic cell, a critical step in transmitting the signal to the next neuron in the network. It is important to note that, under resting conditions, these ion channels remain closed, maintaining the postsynaptic membrane in an insulating state. This insulating state is disrupted only when neurotransmitters bind to their respective receptors. Upon binding, the conductance of the postsynaptic membrane increases sharply, enabling the ion flow that drives neuronal communication. This precise mechanism ensures that signals are transmitted efficiently and only in response to specific neurotransmitter binding, maintaining the fidelity of neural communication. Spiking signals are how neurons communicate, as shown in Fig. 2a . The relationship between frequency and capacitance in memcapacitance devices is crucial for understanding their behaviour in neuromorphic computing. In our memcapacitance spiking test, we performed capacitor-based neuromorphic computing across a frequency range of 3–10 kHz, with a frequency step of 1 kHz, applying 100 pulses during each test with a pulse width of 1 ms as shown in Fig. 2b . During this frequency sweep, a clear trend of decreasing capacitance was observed. At 3 kHz, the capacitance dropped significantly from 1.88 µF to 532 nF. As the frequency increased to 4 kHz, it decreased from 1.31 µF to 383 nF. This trend continued at 5 kHz, with the capacitance falling from 1 µF to 332 nF, and at 6 kHz, it was reduced from 742 nF to 273 nF. Similar reductions were observed at 7 kHz (from 584 nF to 238 nF), 8 kHz (from 509 nF to 231 nF), 9 kHz (from 409 nF to 216 nF), and finally at 10 kHz, where it dropped from 332 nF to 195 nF. This progressive reduction in capacitance as the frequency increases suggests that higher frequencies compress the range of capacitance variation, likely due to reduced time for charge redistribution within the memcapacitance device. Consequently, the device’s response becomes increasingly nonlinear, posing challenges for stable neuromorphic computing as its behaviour becomes less predictable. This study provides valuable insights for selecting an optimal frequency range that offers a stable response with a more pronounced difference between maximum and minimum capacitance values and a more linear synaptic change. Such an approach is essential for the effective hardware implementation of neural networks, enabling more reliable and consistent performance in neuromorphic systems, which is crucial for developing efficient and scalable hardware for artificial intelligence applications. Based on the frequency test (3 kHz to 10 kHz), we selected 5 kHz to evaluate the stability of the capacitance spiking response during the set and reset processes, as shown in Fig. 2c . At 5 kHz frequency, 100 pulses were applied for both the set and reset operations, with voltages of +3 V and −3 V, respectively, and a pulse width of 1 ms. The choice of 5 kHz was selected by its more stable capacitance response, where the capacitance decreased from 983 nF to 241 nF during the set process and from 978 nF to 237 nF during the reset process. This stability indicates that 5 kHz is an optimal frequency for achieving consistent and reliable performance in capacitance-based neuromorphic computing, as it offers a more predictable and uniform response during both operational phases. Fig. 2 Synaptic Behavior and Training Characteristics of Iontronic Fluidic Memristor. a Illustration of the biosynapse similar to IFM behaviour. b The capacitance spiking response was measured in a frequency range of 3 kHz to 10 kHz with a pulse width of 1 ms. c The stability response of capacitance behaviour at 5 kHz with positive +3 V set and −3 V reset pulses with a pulse width of 1 ms. d The different pulse amplitude responses at 2.0, 2.4, 2.8, 3, 3.5, and 4 V with u;lse 1 ms and pulse interval 1 ms. e Plasticity characteristics with varying pulse intervals and intervals of 100 μs, 500 μs, 1 ms, 6 ms, 10 ms, 30 ms, 60 ms, and 90 ms. f The device training will increase by 50 pulses for each set and reset the level of training of iontronic devices. The transition of the IFM device from STM to LTM was evaluated through three distinct phases: g the first learning and decay process, h the second learning and decay process, and i the third learning and decay process. j Neurosim 2.0 convolution Neural network based on GCC 8. k The device retention and endurance test for 5400 pulses with stable set and reset process. l The output accuracy of CIFAR-10 is based on the weight update information of IFM These signals of IFM can be distinguished by pulse and frequency-modulated nature, referred to as spike rate-dependent plasticity (SRDP). The pulse duty cycle and amplitude were varied to investigate the IFM device’s SRDP in the discrete channel. As shown in Fig. 2d , the current drops from 4.26 mA to 25.8 µA at various voltage amplitudes of 2.0, 2.4, 2.8, 3.0, 3.5, and 4 V with a pulse width of 1 ms and an interval of 1 ms. This indicates a lower electrical body force between the interface of FF and PAA Na + at a lower voltage than a higher voltage. As seen in Fig. 2e , the synaptic weight can be controlled by sequential stimuli of externally applied pulses of varying widths 100 μs, 500 μs, 1 ms, 6 ms, 10 ms, 30 ms, 60 ms, and 90 ms and intervals of 100 μs, 500 μs, 1 ms, 6 ms, 10 ms, 30 ms, 60 ms, and 90 ms corresponds to frequency response (1/(pulse interval + pulse width) in a range of 5 kHz, 1 kHz, 500 Hz, 83.3 Hz, 50 Hz, 16.7 Hz, 8.33 Hz, and 5.67 Hz, respectively. The 10 μs pulse width does not indicate a noticeable drop in current, maintaining a level from 1.081 mA to 526.86 μA. However, when comparing longer pulse widths of 100 μs, 500 μs, 1 ms, 6 ms, 10 ms, 30 ms, and 60 ms, with 90 ms the current changes significantly from 1.081 mA to 125.19 μA with a high I on/off ratio of 8.65. The device has a 90 ms duty interval, which results in significant non-linearity. To achieve high convergence with low non-linearity, a suitable duty cycle is selected of 1 ms with a current change from 1.081 mA to 356.77 μA I on/off ratio of 3.03, which provides good non-linearity value of 2.63. It is possible that using these weight change factors would be beneficial to creating an application that utilises convolutional neural networks (CNN) for data reorganisation. High-frequency operation is crucial for real-time neuromorphic applications, but it introduces several challenges related to the rate of change of current in IFMs. When operating at frequencies such as 5 kHz, the current change rate significantly slows down due to the inability of ions to migrate fast enough within the electric field. The ions require a longer pulse width to migrate and redistribute at this frequency. This extended pulse width reduces the system’s responsiveness, as the ions cannot follow the rapid changes in the applied voltage. This slow reaction time negatively impacts the effectiveness of weight updates during the training of neuromorphic systems, making 5 kHz unsuitable for efficient device operation in these applications. In contrast, at very low frequencies, such as 5.67 Hz, the current change rate is too fast for the device to handle effectively. While the ions may be able to migrate more quickly, the system becomes prone to non-linearity and instability, as the rapid current changes lead to a mismatch between the expected and actual behaviour of the device. The system’s response becomes erratic, and the device fails to maintain consistent performance at such low frequencies, which are unsuitable for real-time neuromorphic tasks. Through our characterisation of the system between 5 kHz and 5.67 Hz, we found that 500 Hz offers the best balance between efficient ion migration and stable device operation. At 500 Hz, the ions can move with sufficient speed to track the applied voltage changes while avoiding the slow response seen at higher frequencies. At 500 Hz, the system also ensures that weight updates occur optimally for training the neuromorphic system. This frequency balances speed and stability, enabling the system to operate effectively without performance degradation at higher frequencies or instability at much lower frequencies. Therefore, 500 Hz is the most suitable frequency for iontronic fluidic memristors in neuromorphic computing, offering the best rate of change of current for real-time applications. Several strategies can be proposed to alleviate the degradation of device performance at high frequencies. First, optimising the electrode materials can help minimise polarisation effects, significantly hindering ion mobility at high frequencies. This can be achieved by selecting materials that facilitate better ion transport and reduce the accumulation of charges at the electrode-electrolyte interface. Second, improving the ionic conductivity of the electrolyte material can enhance ion migration speed, enabling the device to operate more efficiently at higher frequencies. This can be done by engineering the fluidic properties of the electrolyte, such as reducing its viscosity and increasing its ionic conductivity. Third, using advanced pulse-shaping techniques can help manage the pulse width at high frequencies, allowing for better synchronisation between ion movement and voltage changes, thus improving the rate of change of current. Additionally, integrating optimised ionic liquids with specific characteristics that match the operating frequency range of the device can enhance the system’s overall performance. By refining the frequency range, electrode design, and electrolyte materials, these strategies can help maintain stable performance in iontronic fluidic memristors at higher frequencies, ensuring that they meet the demands of real-time neuromorphic computing applications. As illustrated in Fig. 2f , the modification procedure facilitated the long-term storage of analogue weights and was completely reversible. This was accomplished by applying one hundred set spikes (+3 V) and reset spikes (−3 V). Linearity is of critical importance for system-level weight tracing. To gain insight into the learning process, a pulse scheme consisting of 100 sets and reset pulses with a duty cycle of 1 ms (pulse width and interval) was devised. For each subsequent set/reset cycle, 50 pulses were added to the previous synaptic training. The device can undergo additional training at higher current levels, and the ion/off ratio will increase with each set of trials. During the training process, as the number of training pulses increases, the device experiences non-linearity, as marked in red circles in Fig. 2f . The weight update behaviour must be nearly linear to ensure efficient tracing and have a favourable I on/off ratio. This will facilitate the realisation of neural networks at the hardware level with greater precision. This work demonstrates that these devices can simulate fundamental functions in biological synapses. Before proceeding, it was verified that the current could be decreased by increasing the voltage of a specific polarity. The conversion from short-term potentiation (STP) to long-term potentiation (LTP) in the device is achieved by modulating its current or conductivity through pulse stimulation, mirroring the human brain’s transformation of STM LTM, as shown in Fig. 2g–i . This process aligns with the Ebbinghaus Forgetting Curve, which describes the cycle of “Learning-Forgetting-Re-learning-Memorising” as the brain assimilates and retains information. The learning behaviour observed in IFMs is attributed to the electrolyte’s ability to preserve its current state by forming stable ion clusters. This process typically occurs within a few milliseconds, enabling the device to emulate memory retention mechanisms effectively. In the initial training phase, as shown in Fig. 2g , the device’s current decreased from 9.5 mA to 0.31 mA after 100 electrical pulses with a 1 ms pulse width and interval. After removing the stimuli, the device was periodically tested every 500 s using a 1 ms pulse width and a 3 V read voltage to measure current decay, which progressed from 0.31 mA to 0.44 mA over 4000 s, mimicking the brain’s short-term learning behaviour. In the second training phase, depicted in Fig. 2h , the device’s current dropped from 0.48 mA to 0.191 mA after another 100 pulses. During the subsequent 4000-s forgetting period, the current changes from 0.191 mA to 0.25 mA. Finally, in the third training phase (Fig. 2i ), the current decreased from 0.29 mA to 0.15 mA after 100 pulses, and over the same forgetting period, the current exhibited minimal attenuation, rising only slightly from 0.15 mA to 0.178 mA. This diminished decay signifies the successful transition from STM to LTM, demonstrating the device’s ability to emulate human memory consolidation by retaining information more effectively with repeated training. These findings highlight the potential of such devices for neuromorphic applications by mimicking the human brain’s learning and memory mechanisms. Convolutional neural network simulation Neuromorphic computer systems, which mimic the functions of the human brain, show significant potential for overcoming the limitations of traditional implementation of artificial intelligence algorithms 40 , 41 . As a biological medium for computation, the brain fundamentally diverges from the conventional architecture of electron-based computing that relies on digital circuits. The brain employs biological elements such as synapses and neurons, whilst the latter depends on memory blocks and transistors. The advent of ion-based computing can potentially transform the existing electron-based system substantially. Ion-based computing offers several advantages, including reduced energy consumption, exceptional compatibility with brain-computer interfaces, and the capacity to employ ions of varying sizes and valence. Fluidic systems have great potential in promoting the development of highly efficient computers, particularly in simulating biological brain networks. For utilising CNN, the current pursuit device array method employs a parallel read-out analogue embedded Non-Volatile Memory (eNVM)-based pseudo-crossbar. Further details can be found in Supplementary Figs. S1 and S2 . The device was used with neuromorphic functionalities, leading to operational data collection. CNN is used to evaluate the proposed IMF. The CIFAR-10 dataset is used as the input data, and six convolutional layers are used to extract features. Subsequently, the last three ultimately linked layers are used to classify these properties, as shown in Fig. 2j . A parallel-read-out analogue eNVM-based pseudo-crossbar detects devices in an array when using CNN. Initially, an endurance test is undertaken to evaluate the device’s weight, which involves subjecting it to 5400 pulses. The device has constant and dependable endurance repeatability over cycles, indicating that the interface between the high-resistance and low-resistance areas stays mostly intact even after removing the set/reset voltages. This value, shown in Fig. 2k , is essential for training on the chip. Furthermore, the device parameters are determined based on the endurance data, encompassing discrete levels, set and reset voltage, maximum and minimum resistance, non-linearity, cycle-to-cycle fluctuation, and duty cycle. The number of pulses shown in Fig. 2k is the same at 100 for both positive (+3 V) and negative (−3 V) stimulation, with a pulse interval of 1 ms and width of 1 ms. In constructing artificial synapses, it is essential to attain a significant degree of consistency in the electrical conductivity of a device when exposed to a series of set/reset voltage pulses. The device’s low resistance is 10.16 kΩ, while its high resistance is 29.70 kΩ. The R off/on ratio is around 3.42. The non-linearity coefficient for positive pulse data is 2.74, whereas, for negative pulses, it is 3.13. The cycle-to-cycle variance for positive pulses is 0.032, whereas for negative pulses it is 0.038. Based on this data, kernels and synapses in all layers were trained to reduce the difference between the actual and expected output. Figure 2l illustrates the accuracy of the CNN after 200 epochs, with a recorded output of 85% for the real weights. Further simulation of the ideal device was conducted using a linear function to provide a genuine case comparison. Figure 2l illustrates that the most effective device attains a maximum accuracy of around 87%. The results closely correspond to those obtained from the device in real-life and optimal situations. The low cycle-to-cycle variation in the current IFM, as shown in Fig. 2k , may be attributable to its interfacial memristor nature. Based on device structure and switching voltage, STDP, and CNN accuracy, the performance parameters are compared with already reported work in Supplementary Table S1 . Self-powered computing of IFM using FF TENG/EMG The human body’s biological perception system processes and transmits information through action potentials, which are signals generated by neurons that play a vital role in neural networks. As shown in Fig. 3a , sensory neurons are specialised in detecting stimuli, such as environmental changes or object movements, through bioreceptors. These neurons are essential for processing these signals, ensuring the body responds appropriately to external stimuli. Similarly, neuron-like devices that mimic this function in artificial neural networks must be developed. These devices must generate real-time responses to external stimuli and perform basic computations on the incoming signals, as depicted in Fig. 3b . We have proposed an IFM synaptic device, which processes and stores information and adapts their behaviour through weight adjustments due to synaptic plasticity. This is achieved using the integration of IFM devices with FF TENG/EMG has led to significant advancements in neuromorphic computing, particularly in self-powered systems. This work is compared with self-powered neuromorphic computing resistive memory devices in Supplementary Table S1 . This approach eliminates reliance on traditional energy supplies, promoting long-lasting, efficient performance free from conventional power systems’ environmental and operational limitations. These self-powered memory devices allow soft robots to function autonomously without needing bulky, external power sources to power memory computing units. As soft robots demand increasingly sophisticated sensory feedback and adaptive learning, the FF TENG/EMG self-powered IFM system represents a cutting-edge solution, driving forward the development of autonomous, self-powered soft robotics that can be deployed in a wide range of real-world applications. The artificial perception system based on these IFM synaptic devices is advantageous for processing simple, singular data streams. It offers an efficient and responsive model for certain information-processing tasks and the biological systems they are designed to emulate. The FF TEMG/EMG structure is depicted in Fig. 3b . A magnet controls the microstructure of FF by adjusting the magnetic field strength 42 . FF is a liquid containing nanoscale ferromagnetic particles suspended in a carrier fluid, like an organic solvent or water 43 , 44 , as shown in Supplementary Fig. S3 . These particles are coated with surfactants to prevent them from clumping due to van der Waals forces and magnetic interactions. Without a magnetic field, the FF remains non-magnetic. However, when a magnetic field is applied, the ferromagnetic particles align with the field, giving the fluid magnetic properties. In this state, the particles stay well-dispersed, and their arrangement is stable as long as the magnetic field is maintained. When the magnetic field reaches a particular strength, it can overcome the FF surface tension and gravity, leading to normal-field instability, where the fluid’s surface forms folds or peaks due to the interaction between magnetic forces and the fluid’s properties. The FF TENG/EMG working principle (Fig. 3c ) shows that PTFE attracts electrons from the FF due to its high electron affinity, resulting in a negatively charged PTFE film and a positively charged ferrofluid (state I). At this stage, no electromagnetic generation occurs since there are no changes in magnetic flux relative to the coil. When pressure is released, positive charges in the FF drive electrons from the negative electrode attached to PTFE (state II). Simultaneously, the EMG initiates an anticlockwise current I EM in the coil due to reduced magnetic flux. With no pressure applied, equilibrium is reached, and no electrical signal is observed in FF TENG/EMG (state III). Upon pressure application, induced electrons flow back to the FF electrode to balance the potential change on the electrode (stage IV). Additionally, a clockwise current I EM is induced in the coil, attributed to increased magnetic flux. This cyclic process produces an AC signal with alternating positive and negative pulses. Supplementary Figure S4 illustrates the contact electrification between the FF and PTFE using an electron cloud interaction model. In this model, d represents the distance between electron clouds, E1/2 is the potential energy required for electrons to escape, and EA/B is the energy level of electrons in the material atoms. Before contact, electrons are trapped by potential wells, preventing transfer. Upon contact, electron clouds overlap, forming an asymmetric double-well potential, allowing electron transfer between materials. After separation, the transferred electrons remain due to an energy barrier unless external conditions change. Fig. 3 Self-Powered Neuromorphic Computing with Ferrofluid TENG/EMG Integrated with Iontronic Fluidic Memristor. a Illustration of the synapse event triggered by the external stimuli. b The interface circuitry of FF TEMG/EMG and IFM using a double bridge rectifier shows a self-powered system. c Complete working principle of the FF TENG/EMG. d The loading resistance effect on current and voltage of FF TENG/EMG. The e voltage and f current response of FF TENG/EMG at 1 kΩ. g Self-powered response of IFM based on the output of FF TENG/EMG at 1 kΩ input impedance Pursuing an electronic soft fluidic sensor and memory that emulates the human mechanoreceptor system, enabling communication with nerves and facilitating intelligent sensing, opens new possibilities for self-powered computing applications. Notably, FF-TENG and EMG show significant potential in converting mechanical energy into electrical energy, which is advantageous for neuromorphic computing with IFM devices. However, FF-TENGs, due to their high input impedance, cannot generate the desired output signal at the required input impedance. As illustrated in Supplementary Fig. S5 , at a load of 1 kΩ, the system can generate 0.5 V and a current of 30 µA. High impedance can limit the current output, making achieving the desired power levels needed to drive IFM devices difficult. To enhance the performance of the FF-TENG, we introduced an EMG coil with 1200 turns and a coil diameter of 3 cm. The EMG coil, having a low input impedance, can generate a higher current of 1.85 mA and a voltage of 2 V at an input impedance of 1 kΩ, as shown in Supplementary Fig. S6 . This improvement is crucial for providing the necessary power to the IFM devices, enabling them to function properly in neuromorphic computing applications. By combining the outputs of the FF-TENG and EMG generators, we can significantly enhance the overall output performance. The combined FF-TENG/EMG generator output is 2.5 V at 1 kΩ with a current of 1.89 mA, as shown in Fig. 3d . The stability of FF TENG/EMG is analysed for 40,000 cycles, as shown in Supplementary Fig. S7 . This combination not only improves the output performance of the TENG but also strengthens the input stimuli operation for the IFM. The increased voltage and current ensure that the IFM devices receive a steady and sufficient power supply, which is essential for self-powered computing applications. The output voltage and current signals generated by the FF-TENG/EMG are depicted in Fig. 3e, f , providing detailed insights into how this output signal interacts with the IFM, as shown in Fig. 3b . For computing applications, it is essential to consider pulse width, pulse interval, and pulse potential. The pulse interval is 100 ms, with a rectified pulse width of 58.5 ms, the active pulse width of two rectified pulses being 15 ms, each pulse potential of 2.5 V, a Pulse current of 1.89 mA and a pulse power of 4.725 mW. The output of the IFM, based on these input stimuli, shows a computing current ranging from 668 µA to 257 µA over a 9 s stimulus generated by the FF-TENG/EMG at 5 Hz, as shown in Fig. 3g . This study represents the first detailed investigation into developing a self-powered neuromorphic device using IFM technology. The successful integration of FF-TENG and EMG generators with IFM devices opens new possibilities for creating self-powered systems that can operate independently of external power sources. Such systems could be used in a variety of applications. Self-Powered Soft Skins: These could be used in robotics to create sensors that detect pressure, temperature, and other environmental factors, providing robots with a sense of touch. Soft Robotics: The enhanced performance of IFM devices could lead to the development of advanced soft robots capable of complex, adaptive behaviours."
} | 12,804 |
30956539 | PMC6417436 | pmc | 4,816 | {
"abstract": "Biological invasions can have various impacts on the diversity of important microbial mutualists such as mycorrhizal fungi, but few studies have tested whether the effects of invasions on mycorrhizal diversity are consistent across spatial gradients. Furthermore, few of these studies have taken place in tropical ecosystems that experience an inordinate rate of invasions into native habitats. Here, we examined the effects of plant invasions dominated by non-native tree species on the diversity of arbuscular mycorrhizal (AM) fungi in Hawaii. To test the hypothesis that invasions result in consistent changes in AM fungal diversity across spatial gradients relative to native forest habitats, we sampled soil in paired native and invaded sites from three watersheds and used amplicon sequencing to characterize AM fungal communities. Whether our analyses considered phylogenetic relatedness or not, we found that invasions consistently increased the richness of AM fungi. However, AM fungal species composition was not related to invasion status of the vegetation nor local environment, but stratified by watershed. Our results suggest that while invasions can lead to an overall increase in the diversity of microbial mutualists, the effects of plant host identity or geographic structuring potentially outweigh those of invasive species in determining the community membership of AM fungi. Thus, host specificity and spatial factors such as dispersal need to be taken into consideration when examining the effects of biological invasions on symbiotic microbes. Electronic supplementary material The online version of this article (10.1007/s10530-018-1710-7) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction A large number of studies have examined the direct effects of biological invasions on the diversity of invaded communities (Vitousek et al. 1996 ; Sax and Gaines 2008 ; Vilà et al. 2011 ). However, it remains to be seen whether invasion effects aboveground are mirrored in belowground soil microbial communities or how microbial mutualists, such as mycorrhizal fungi, may reduce or exacerbate the negative effects of invasive plant species on native flora (Traveset and Richardson 2006 ; Desprez-Loustau et al. 2007 ; Pringle et al. 2009 ; Lekberg et al. 2013 ; Zubek et al. 2016 ). Because mutualisms underlie ecosystem functioning, productivity and stability (Klironomos et al. 2000 ; Renker et al. 2004 ; van der Putten et al. 2009 ; Wagg et al. 2011 ) and can strongly influence plant invasions (Simberloff and Von Holle 1999 ; Richardson et al. 2000 ; Lekberg et al. 2013 ), the impact of biological invasions on mutualist communities is of both theoretical and practical importance. In circumstances where invaders compete directly with native organisms, most studies and meta-analyses support a loss of local diversity as a direct result of invasions (Fridley et al. 2007 ; Vilà et al. 2011 ; Chase et al. 2015 ). However, rather than invaders having direct effects on mutualists, the effects of invasions on mutualists may be indirect for example, via changes in the density or abundance of host organisms (Simberloff and Von Holle 1999 ). Consequently, conceptual frameworks for invasion processes and outcomes based on the direct interactions of invasive species with their native counterparts cannot necessarily be assumed to apply to mutualistic organisms (Richardson et al. 2000 ; Callaway et al. 2004 ). The symbiosis between plants and mycorrhizal fungi is one of the most widespread mutualisms on earth (Smith and Read 2008 ). The most common type of mycorrhizal fungi are the arbuscular mycorrhizal fungi (subphylum Glomeromycotina, former phylum Glomeromycota, Spatafora et al. 2016 ) which form obligate associations with > 80% of terrestrial plant species, including many invasive plant species (Brundrett 2009 ; Pringle et al. 2009 ). In this mutualism, the host plant passes carbon fixed through photosynthesis on to its associated AM fungi in exchange for increased access to growth-limiting soil nutrients, especially phosphorus (Smith and Read 2008 ). Due to the importance of AM fungi for host plant fitness and their broad associations with a diversity of hosts, they are ideal candidates to examine the effects of invasions on microbial mutualists’ community dynamics. In general, the arbuscular mycorrhizal symbiosis is thought to be relatively non-specific where host plants can benefit from a diversity of geographically or phylogenetically disparate AM fungi and vice versa (Moora et al. 2011 ; Davison et al. 2015 ; Lekberg and Waller 2016 ; but see van der Heijden et al. 1998 ; Vandenkoornhuyse et al. 2003 ; Alguacil et al. 2009 ; Bunn et al. 2015 ). However, symbiont compatibility is only one of many ecological filters that AM fungi must pass through in order to establish. Other factors such as dispersal ability, environmental suitability, and intra-guild biotic interactions may outweigh the relative importance of invasive host compatibility on the ability of AM fungi to establish and persist (Leibold et al. 2004 ; Filotas et al. 2010 ; Pillai et al. 2014 ). Various scenarios have been put forth as to how plant invasions may alter the diversity of mycorrhizal fungi. Previous research has suggested that invasive plants may have positive (Lekberg et al. 2013 ; Chen et al. 2015 ), neutral (Richardson et al. 2000 ; Wolfe et al. 2010 ), or negative (Mummey and Rillig 2006 ; Murat et al. 2008 ) effects on the species richness of mycorrhizal fungi (see Dickie et al. 2017 for a recent review). A decrease in species richness may be the result of introducing non-mycorrhizal hosts or invaders that require fewer fungal partners than native hosts (Richardson et al. 2000 ; Pringle et al. 2009 ; Nuñez and Dickie 2014 ), while no change in species richness may be due to invasive host plants partnering with the extant mycorrhizal community (Richardson et al. 2000 ; Catford et al. 2009 ; Pringle et al. 2009 ; Moora et al. 2011 ; Nuñez and Dickie 2014 ), and increases in richness may be the result of co-invasions of non-native hosts and fungi (Dickie et al. 2010 ; Lekberg et al. 2013 ; Bogar et al. 2015 ), yielding net impacts of plant invasions on mycorrhizal fungal diversity that are difficult to predict (Dickie et al. 2017 ). Previous studies of the effects of plant invasions on AM fungal communities are primarily focused on forbs, grass and shrub invasions (Bunn et al. 2015 ). However, woody species such as trees are also common invaders, especially in the tropics, and the majority of invasive trees also associate with AM fungi (Nuñez and Dickie 2014 ). Prior research on AM fungi and invasions has also been focused on habitats in temperate or Mediterranean climates despite the fact that tropical islands tend to experience a disproportionate rate of plant invasions (Kueffer et al. 2010 ). To date there have been no studies that examine the effects of tree invasions on AM fungal diversity in tropical island ecosystems. To fill this gap, we explore the effects of tree-dominated invasions on AM fungal diversity on the island of Oahu in the Hawaiian archipelago, which is one of the invasion capitals of the world (Vitousek et al. 1996 ). There, three of the most common and pernicious invasive trees are strawberry guava ( Psidium cattleianum Myrtaceae), Christmasberry ( Schinus terebinthifolius Anacardiaceae), and Australian redcedar ( Toona ciliata Meliacae). The first two were introduced to Hawaii between the early nineteenth century and sometime before 1911. Strawberry guava and Christmasberry are spread by birds, and the former is also dispersed by non-native ungulates and rodents. Both trees now form thick monodominant stands that have replaced historically native vegetation (Motooka et al. 2003 ). Australian red cedar was potentially introduced to Hawaii as early as the mid-nineteenth century and later broadly planted as fast-growing timber species and is now considered invasive (Wagner et al. 1999 ). We test the hypothesis that independent of geographic location or host identity these tree invasions have led to similar changes in AM fungal richness, species incidence and phylogenetic community structure. We compare AM fungal richness across spatial gradients in paired native and invaded watersheds. We assess the impacts of invasions on the diversity of AM fungi by comparing diversity measures based on species incidence, with phylogenetic distances metrics. We chose to assess changes in AM fungal richness, species incidence and phylogeny because there are no previous studies that have compared the effects of invasion on all three making it difficult to predict whether invasions will lead to changes in all, some, or none of the above.",
"discussion": "Discussion The successful establishment of species to new environments is a complex process that depends on innumerous factors. For plants with high mycorrhizal dependency, success in new areas is linked to the success of their mutualistic partners. However, in addition to partner compatibility, there are several abiotic and biotic filters underlying the establishment of mycorrhizal plants and fungi (Gladieux et al. 2015 ). We studied the impact of tree-dominated plant invasions on the richness, community composition and phylogenetic structure of AM fungi in Hawaiian soils. Despite the different compositions of native and invasive plant species among plots, we found that invasions consistently increased the richness of AM fungi. Furthermore, we found no indication that AM fungal community composition or richness was related to any of the environmental factors we considered. Rather, AM fungal community composition was stratified by geographic location, in this case, watersheds. This finding was consistent whether we took into consideration the phylogenetic structure of AM fungal communities or not. This study provides new data on the effects of biological invasions in understudied tropical ecosystems that experience inordinate negative effects of invasions. Though we are far from finding any axioms for the effects of plant invasions on mycorrhizal communities, by adding new data from tropics, our results complement those of Lekberg et al. ( 2013 ). Based on OTU incidence data, Lekberg et al. ( 2013 ) found an increase in AM fungal diversity with plant invasions in alpine habitats. They suggest that resource availability and the ability of hosts to supply carbon to AM fungi may be an important driver of AM fungal diversity in their study system. Host supply of resources may also be a key factor affecting AM fungal richness in our system where invasive hosts are known to have greater rates of resource acquisition and often higher demands for resources than natives, especially for growth-limiting elements such as water and light (Cavaleri et al. 2014 ; Kagawa et al. 2009 ; Durand and Goldstein 2001 ). An additional factor leading to an increase in AM fungal richness among invasive plots could be co-invasions of AM fungi with introduced hosts. However, based on our indicator analyses we did not detect clear differences between the AM fungi present in native or invasive plots. The lack of detectable differences in AM fungal community composition among native and invasive sites suggests that if any AM fungi are co-introduced, they are not restricted to sites dominated by non-native plants. While we found little evidence that aboveground invasions systematically reduced or increased the presence of any particular AM fungi OTUs, we did find that subsets of our AM fungi OTUs were strictly found in either native or invasive sites. In these cases, host identity is likely playing a crucial role in determining AM fungal communities. Besides direct host-symbiont interactions there may be indirect effects of other invasive species on AM fungal diversity especially in our invaded plots. There, higher AM fungal diversity may in part be due to the presence of invasive ungulates and other invasive mammals such as rodents that have continually been moving soil around within these areas, but have been excluded from native habitats for greater than a decade. Within native areas, this lack of soil mixing may have led to more heterogeneous AM fungal communities (Wood et al. 2015 ). However, since non-native herbivores often reduce, rather than increase mycorrhizal diversity (Rossow et al. 1997 ) and both native and invasive plots have a similar long-term histories of invasive mammal activity, this effect seems less likely to have led to the increase in AM fungal richness that we observed in the invasive plots. Furthermore, even if invasive mammals were the primary driver of differences in AM fungal richness among habitat types, this is still evidence that biological invasions can have non-neutral and indirect effects on microbial mutualist communities. Based both on OTU incidence and phylogenetic inference we found significant differences in AM fungal community composition among watersheds that were not related to environmental factors or vegetation type. These findings support the idea that rather than invasion status of the vegetation, geographic features such as high ridge tops may be effective barriers for the migration of most AM fungi among watersheds. However, had we targeted roots of specific native and non-native hosts, rather than bulk soil for our analyses, we may have found that invasive hosts versus native ones harbored discrete AM fungal communities. Thus the lack of observed differences in AM fungal community membership within watersheds may be owed to AM fungi dispersing within these geographic boundaries, which is independent of whether they are actively colonizing a host or not. We found that all clades of AM fungi were stratified among watersheds except for clade I of Glomeraceae (Fig. 4 ), which was present among all three watersheds. This clade contains some of the most globally widespread and common AM fungal taxa and some of the few taxa to disperse well by air (Moora et al. 2011 ; Kivlin et al. 2011 ; Egan et al. 2014 ; Davison et al. 2015 ). The fact that these taxa are found throughout our study sites, and that they have arrived and established in Hawaii (the most remote oceanic island archipelago on Earth), lends additional support to their cosmopolitan nature. This result also indicates that the dispersal biology of AM fungi clades likely differ. Dispersal ability of AM fungi has been shown to vary based on species identity (Egan et al. 2014 ), and it would be interesting to test if dispersal traits are conserved at deeper phylogenetic levels. The abundance of Glomeraceae clade I across our study sites and the common pattern of long-tail species (OTU) frequency distributions of environmental microbial communities (Shoemaker et al. 2017 ), also found here (SI Figure S4) helps to explain why the q = 2 confidence intervals overlap (Fig. 2 c). This estimator places more weight on the frequency of abundant species and discounts rare ones (Chao et al. 2014 ), thus it is the least likely of the three Hill numbers and estimators to accurately represent our true OTU diversity. We found that despite significant differences among watersheds in soil nitrogen, elevation and precipitation there is no evidence that the community composition of AM fungi is related to these, or other environmental factors. This finding is in contrast to previous studies where environmental conditions such as temperature and pH were strong predictors of AM fungal diversity (Dumbrell et al. 2010 ; Davison et al. 2015 ). This result is surprising given that Hawaii is renowned for its strong environmental gradients that have led to multiple adaptive radiations (e.g. Baldwin and Sanderson 1998 ; Gillespie 2004 ; Tokita et al. 2017 ). In addition to the relatively greater potential importance of host and geography rather than local environment in determining AM fungal community composition, it is also possible that our sampling sites did not represent strong enough environmental gradients to see a response in AM fungal community composition, that our target locus for sequencing is not variable enough to detect these differences (Bruns and Taylor 2016 ), or that we did not measure the environmental factors of import for determining AM fungal community membership. It is important to highlight that there is no universal way to study AM fungal communities (Hart et al. 2015 ). It is possible that the same experimental set up followed by different methods would yield different outcomes. Hart et al. ( 2015 ) show that factors ranging from preservation methods of soil samples, to choice of genetic marker and subsequent bioinformatics, may influence the results of every study. Both preservation methods and genetic marker choice may lead to biases towards specific AM fungal groups, which makes the comparison across studies challenging. Sample preservation is a crucial step in preserving DNA of AM fungi. Although snap-freezing in liquid nitrogen is probably the most efficient preservation method, it is not possible to use this method in many circumstances, namely when sampling in remote places. In our study, we used oven drying (50–60 °C; Janoušková et al. 2015 ), which has been shown to be efficient at preserving DNA of AM fungi while being the least expensive and simplest preservation method. Also, it has been suggested as one of the best preservation methods of AM fungal DNA (Hart et al. 2015 ). As for our choice of genetic marker, the LSU primers used in this study are known to select against certain AM fungal taxa in the Paraglomerales, Archaeosporales and Diversisporales (Krüger et al. 2009 ). Despite these overarching biases inherent to each specific method, all samples from this study were handled the same. Thus, while we acknowledge that we may not have assessed AM fungal diversity and community composition in their entireties, any methodological bias was equal across all our samples, which makes relative comparisons valid. Furthermore, because we used practices common in other studies of AM fungi our results are extractable and comparable to prior, and future studies. Overall, our results suggest that aboveground invasions can lead to an increase in belowground microbial symbiont richness, but not changes in community membership, and that particular environmental conditions do not always lead to the assembly of certain taxa. Our results also support the diffuse nature of the arbuscular mycorrhizal symbiosis, even under biological invasions. We posit that factors such as host identity and functional traits as well as AM fungal dispersal barriers may play important roles in determining mycorrhizal diversity and deserve further attention. To disentangle the contribution of each to AM fungal community dynamics, more exhaustive studies need to be carried out. Future research that investigates the mycorrhizal community dynamics of invasive plant species in their native habitats relative to their introduced ranges, and are aimed at understanding the mechanisms driving AM fungi species coexistence would be particularly valuable. Lastly, mycorrhizal surveys should be designed to consider temporal and spatial effects, because time since invasion and the geographic scale at which observations of biodiversity are made can affect the perceived impact of biological invasions (Fridley et al. 2007 ; Powell et al. 2013 ; Chase et al. 2015 ). Such integrative approaches are necessary to shed additional light on the causal factors and consequences of biological invasions on microbial symbiont communities and their hosts."
} | 4,937 |
35923404 | PMC9339997 | pmc | 4,817 | {
"abstract": "Natural biodegradation processes hold promises for the conversion of agro-industrial lignocellulosic biomaterials into biofuels and fine chemicals through lignin-degrading enzymes. The high cost and low stability of these enzymes remain a significant challenge to economic lignocellulosic biomass conversion. Wood-degrading microorganisms are a great source for novel enzyme discoveries. In this study, the decomposed wood samples were screened, and a promising γ-proteobacterial strain that naturally secreted a significant amount of laccase enzyme was isolated and identified as Serratia proteamaculans AORB19 based on its phenotypic and genotypic characteristics. The laccase activities in culture medium of strain AORB19 were confirmed both qualitatively and quantitatively. Significant cultural parameters for laccase production under submerged conditions were identified following a one-factor-at-a-time (OFAT) methodology: temperature 30°C, pH 9, yeast extract (2 g/l), Li + , Cu 2+ , Ca 2+ , and Mn 2+ (0.5 mM), and acetone (5%). Under the selected conditions, a 6-fold increase (73.3 U/L) in laccase production was achieved when compared with the initial culturing conditions (12.18 U/L). Furthermore, laccase production was enhanced under alkaline and mesophilic growth conditions in the presence of metal ions and organic solvents. The results of the study suggest the promising potential of the identified strain and its enzymes in the valorization of lignocellulosic wastes. Further optimization of culturing conditions to enhance the AORB19 strain laccase secretion, identification and characterization of the purified enzyme, and heterologous expression of the specific enzyme may lead to practical industrial and environmental applications.",
"conclusion": "Conclusion Taken together, a new bacterial strain Serratia proteamaculans AORB19 has been characterized that stably secreted high-level laccase activity after 24-h culture over an extended culture time. The combination of selected culture conditions resulted in a 6.0-fold increase in laccase production without the addition of any toxic inducers. This is, to our knowledge, the first report on the natural production and parameter selection for the expression of extracellular laccase by a Serratia proteamaculans strain. The isolated bacterial strain preferred weak alkaline pH and mesophilic culture conditions that are desired traits for many industrial applications. This first phase of research focused on identification and characterization of a new bacterial strain and screening of important factors that influence laccase production using the OFAT strategy. Future experiments will be designed to explore the impact of other important factors such as the amount of oxygen in the culture medium on enzyme activity and the interactions among different nutritional and conditional parameters and to identify the optimized conditions by the statistical method—Design of Experiment and Response Surface Methodology. The final optimized process should allow secretion of further increased amount of laccase that confers strain AORB19 greater potential to be either directly used in fermentation process or for laccase enzyme production. In addition, the high-level enzyme expression would expedite the biochemical purification and characterization of the specific AORB19 laccase. A greater understanding of the functionality of this bacterial enzyme will likely lead to its practical application in lignin valorization and other biotechnologies that focus on cost-effective biomass conversion and environmental remediation.",
"introduction": "Introduction Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing oxidoreductases that can oxidize a wide range of phenolic and nonphenolic substrates, including ortho, meta, and para diphenols, specifically lignin, a lignocellulose component, and is employed as an attractive tool for the pre-treatment of biomass and its valorization. They are versatile oxidative biocatalysts that contain copper atoms in their active site and oxidize diverse substrates using only molecular oxygen as the known co-substrate instead of hydrogen peroxide, as in peroxidases (Agrawal et al., 2018 ; Agrawal and Verma, 2020 ). As green biocatalysts, laccases have been exploited for a potential application in broad biotechnological areas, viz., environmental remediation processes, biosensor design, synthesis of fine chemicals, food, cosmetic, and pharmaceutical industries, and synthetic dye decolorization capacity to detoxify a range of noxious and recalcitrant environmental pollutants (Yang et al., 2017 ; Becker and Wittmann, 2019 ; Zerva et al., 2019 ). However, most currently known laccases are difficult to overproduce in heterologous hosts (Kim et al., 2010 ). Abundant and inexpensive laccases are needed if the microbiological treatments are to compete with chemical treatments in industries to which they appeal. Identification and engineering for more efficient and tolerant laccases for industrial applications are an ongoing effort to date. Laccase genes dwell in numerous biologically important taxa including plants, insects, lichen, bacteria, and fungi, with the Basidiomycetes class of fungi being the most important source (Hoegger et al., 2006 ; Arregui et al., 2019 ). It has been reported that microbial laccases are primarily involved in wood decay, lignin decomposition, and detoxification and linked to resistance to different environmental stresses (Arregui et al., 2019 ; Janusz et al., 2020 ). Fungal laccases have been studied in a variety of biotechnological applications due to their high redox potential (Abdel-Hamid et al., 2013 ; Upadhyay et al., 2016 ). However, their application is usually hampered due to the long fermentation periods, acidic pH optima, intolerance to extreme conditions, and difficulty in overproducing in heterologous hosts (Baldrian, 2006 ; Kim et al., 2010 ; Du et al., 2015 ). Meanwhile, small laccases, both three- and two-domain laccases, from bacterial sources have gained attention recently, due to their exceptional attributes such as ability to withstand wide temperature and pH ranges, ease in genetic manipulation, and tremendous stability even when inhibitory agents are present (Mukhopadhyay et al., 2013 ; Chauhan et al., 2017 ; Arregui et al., 2019 ; Gianolini et al., 2020 ; Sharma and Leung, 2021 ). In addition, a short generation time of bacteria makes it easier to scale up laccase production processes on a commercial scale (Brugnari et al., 2021 ; Akram et al., 2022 ). The major bacterial genera that have been reported to produce laccase-like multicopper oxidases include Streptomyces, Bacillus, Meiothermus, Gramella, Geobacillus, Aquisalibacillus, Lysinibacillus, Azospirillum, Rhodococcus, Ochrobactrum, Amycolatopsis, Pseudomonas, Stenotrophomonas, Iodidimonas, Alteromonas, and Nitrosomonas (Chauhan et al., 2017 ; Granja-Travez et al., 2018 ; Arregui et al., 2019 ; Jeon and Park, 2020 ; Yang et al., 2021 ). Bacterial species under the genus Serratia, belonging to the family Enterobacteriaceae, has been identified for its numerous applications in biodegradation (Majumdar et al., 2020 ; An et al., 2021 ; Dabrowska et al., 2021 ). Serratia proteamaculans , a gram-negative, non-pigmented strain under this genus, was first reported to cause a leaf spot disease and is the only identified phytopathogen under the genus Serratia (Paine and Standsfield, 1919 ; Mahlen, 2011 ). It is widely distributed in nature and is frequently isolated from the gut microbiota of insects, including spiders and bark beetles (Bersanetti et al., 2005 ; Mikhailova et al., 2014 ). Serratia proteamaculans has been recently recognized for its capability to produce bio-degradative enzymes (Mehmood et al., 2009 ; Madhuprakash et al., 2015 ; Cano-Ramírez et al., 2016 ) and possess remarkable antagonistic traits against plant pathogens (Wang et al., 2014 ). Even though its ability to synthesize extracellular enzymes such as chitinase, endoglucanase, and protease has been reported (Mikhailova et al., 2014 ; Madhuprakash et al., 2015 ; Cano-Ramírez et al., 2016 ), no research regarding its growth and extracellular laccase production has been reported so far. During an investigation of the cultivable microbial community from decomposed wood on the Ottawa River bank, a large number of bacterial and fungal strains were isolated. Among them, a new strain of Serratia proteamaculans , AORB19, has been isolated and characterized that showed significant laccase activities during the screening process. This study characterized the identified strain AORB19 and investigated its laccase production potential and the important culture parameters for enhanced laccase production under submerged culture conditions.",
"discussion": "Discussion As the quest for economic, novel, and robust enzymes becomes increasingly important for biotechnological applications, more attention has been paid to systematic exploitation of microorganisms through natural biodiversity screenings and optimization of cultural conditions to produce abundant enzymes that are affordable as well as stable and active under desired operational conditions. By screening microorganisms of decayed wood stumps in this study, we have isolated a bacterial strain that naturally secreted a significant amount of laccase enzyme activities ( Figure 5 ). Figure 5 Schematic representation for identification and characterization of laccase producing bacterial strain AORB19. The selected strain was characterized phenotypically based on its morphological, physiological, and biochemical characteristics. Unlike many other species in the genus Serratia (Grimont et al., 1977 ), the identified bacterial strain produced non-pigmented colonies with a characteristic mucoid texture on nutrient agar. The biochemical reactions such as l-arabinose, ornithine and lysine decarboxylase, dulcitol, adonitol, d-arabitol, and d-sorbitol fermentation can aid to diversify different biogroups or species within the genus Serratia (Rafii, 2014 ). The bacterial strain was identified with a high probability score of 99.9% from the MicroScan version 4.4 x databases as Serratia liquifaciens complex which included S. proteamaculans, S. grimesii , and S. liquifaciens . As species-specific identification of Serratia liquifaciens complex was not included in the database, the results were further validated using 16S rRNA gene sequencing. The results of 16S rRNA gene sequencing were comparable with the biochemical characterization, and the species showed 99.9% homology to Serratia proteamaculans . Aromatic compounds formed during the lignin degradation process are lethal to many microorganisms and can negatively affect productivities in the fermentation of lignocellulosic hydrolysates (Henson et al., 2018 ). However, while investigating the lignin-degrading potential, it was found that the isolated strain AORB19 was able to grow well with lignin as a sole carbon source in the culture media and produced copious laccase. In the study by Wang et al. ( 2020 ), it was reported that the cell growth in the lignin-based culture medium is linked to lignin degradation by extracellular enzymes. Thus, Serratia proteamaculans AORB19 possesses an important phenotype of lignin-degrading microorganism in terms of bacterial cell proliferation and laccase production in lignin-based media. Beyond this indication, the bacterial strain also exhibited exceptionally stable laccase activity in the culture medium for a prolonged period, suggesting an inherent tolerance of the strain and its enzymes to end-product accumulation during lignin degradation and metabolism. Microbes produce extracellular enzymes to break down complicated organic materials into usable compounds that can be transported through the cell membrane (Ramin and Allison, 2019 ). The time-course analysis of strain AORB19 revealed extracellular laccase production throughout the growth phase following a short lag phase. During the exponential phase (12–36h), there was a marked increase in laccase production in the growth medium and peaked at the early stationary phase, reaching the maximum by 48 hours of incubation. The time for optimum laccase production varies depending on the microorganism. For Pseudomonas extremorientalis BU118 and Bacillus subtilis MTCC 2414, 24 h and 96 h were reported, respectively (Muthukumarasamy et al., 2015 ; Neifar et al., 2016 ). The temperature and pH of the culture medium have a substantial impact on the strain's cell growth and metabolite synthesis. Although the production of laccase was estimated between pH 5 and 12, the optimal range of pH was about 7–10, which corresponded to the optimal cell growth in the study. It was noticed that the laccase production remained steady throughout the incubation period except minor ups and downs during the stationary phase of incubation. Laccase production from other species of Serratia has been reported around a neutral to alkaline pH range and in mesophilic temperature (Chandra et al., 2012 ). For strain AORB19, the optimal laccase production and cell growth occurred between 27 and 33°C similar to Bacillus sp., 30°C (Sondhi and Saini, 2019 ). As nitrogen is a fundamental component of proteins and nucleic acids, it is widely known that it is one of the most important nutrients for microbial metabolism (Hernández et al., 2015 ). For strain AORB19, yeast extract was the most effective nitrogenous source and was consistent with a previous discovery regarding Bacillus subtilis DS (Kumar et al., 2018 ). In this study, maximum laccase production was noticed while using sucrose as the carbon source consistent with a study by Muthukumarasamy et al. ( 2015 ) for Bacillus subtilis MTCC 2414 strain. Cellulose and fructose significantly inhibited laccase production. While fructose is readily assimilable, cellulose may increase medium viscosity and thus lower oxygen supply and interfere with cell division and metabolic rate (Othman et al., 2018 ). Laccase production of strain AORB19 was enhanced by the organic solvents; acetone and chloroform with acetone exhibited a higher impact. Such inductions by organic solvents have also been reported with Serratia marcescens and fungus GBPI-CDF-03, and the results were in concurrence with prior studies (Dhakar and Pandey, 2013 ; Kaira et al., 2015 ; Wu et al., 2019 ). Our study showed that several metal cations improved strain AORB19 laccase production. Copper was found to induce laccase production in MTCC2414 (Muthukumarasamy et al., 2015 ). At 0.5 mM, Cu 2+ increased laccase production, but at 1 mM, Cu 2+ initially lowered laccase production at 24 h and then increased enzyme production on further incubation. This reversal can be explained by the considerable toxicity of copper at greater concentrations, as well as the restriction of normal metabolic pathways caused by redox cycling of ions. As a result, laccase production could be viewed as a regulating system used by bacteria to survive the toxicity of inorganic materials like metal ions by changing their oxidation status under aerobic conditions (Kaur et al., 2019 ). Laccase synthesis by microorganisms can be influenced by many factors such as chemical and nutritional composition of the media; the optimization approach used may be strain specific. In this study, factors [temperature 30 °C, pH 9, yeast extract (2g/l), Li + , Cu 2+ , Ca 2+ , and Mn 2+ (0.5mM), and acetone (5%)] were used to enhance laccase synthesis using a one-factor-at-a-time strategy, resulting in a 6.0-fold overall increase (73.3 U/L) in laccase yield. However, by employing a statistical approach of culture media optimization using five factors by response surface methodology, a 5.5-fold increase in laccase yield (58 U/L) was reported for Pseudomonas putida LUA15.1 (Verma et al., 2015 ). Similarly, Streptomyces psammoticus MTCC 7334 produced 3-fold increase in laccase using response surface methods (Niladevi et al., 2009 ). Furthermore, when compared with the unoptimized media (2.05 U/ml), a 4-fold increase in laccase production in Bacillus cereus TSS1 (9.03 U/ ml) was attained after altering the media variables using response surface approach (Rajeswari et al., 2015 )."
} | 4,084 |
38643972 | null | s2 | 4,819 | {
"abstract": "Horizontal gene transfer (HGT) is a fundamental process in prokaryotic evolution, contributing significantly to diversification and adaptation. HGT is typically facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages, which often impose fitness costs on their hosts. However, a considerable number of bacterial genes are involved in defence mechanisms that limit the propagation of MGEs, suggesting they may actively restrict HGT. In our study, we investigated whether defence systems limit HGT by examining the relationship between the HGT rate and the presence of 73 defence systems across 12 bacterial species. We discovered that only six defence systems, three of which were different CRISPR-Cas subtypes, were associated with a reduced gene gain rate at the species evolution scale. Hosts of these defence systems tend to have a smaller pangenome size and fewer phage-related genes compared to genomes without these systems. This suggests that these defence mechanisms inhibit HGT by limiting prophage integration. We hypothesize that the restriction of HGT by defence systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and the fitness effect of HGT in bacterial populations."
} | 318 |
38978602 | PMC11230466 | pmc | 4,821 | {
"abstract": "Conducting polymers are of great interest in bioimaging, bio-interfaces, and bioelectronics for their biocompatibility and the unique combination of optical, electrical, and mechanical properties. They are typically prepared outside through traditional organic synthesis and delivered into the biological systems. The ability to call for the polymerization ingredients available inside the living systems to generate conducting polymers in vivo will offer new venues in future biomedical applications. This study is the first report of in vivo synthesis of an n-doped conducting polymer (n-PBDF) within live zebrafish embryos, achieved through whole blood catalyzed polymerization of 3,7-dihydrobenzo[1,2-b:4,5-b′]difuran-2,6-dione (BDF). Prior to this, the efficacy of such a polymerization was rigorously established through a sequence of in vitro experiments involving Hemin, Hemoproteins (Hemoglobin, Myoglobin, and Cytochrome C), red blood cells, and the whole blood. Ultimately, in cellulo formed n-PBDF within cultured primary neurons demonstrated enhanced bio-interfaces and led to more effective light-induced neural activation than the prefabricated polymer. This underscores the potential advantages of synthesizing conducting polymers directly in living systems for biomedical applications.",
"conclusion": "Conclusion The conductive polymer n-PBDF is synthesized in a biological environment with natural enzymes (hemoproteins), and inside live zebrafish embryos. The in cellulo formed-PBDF exhibits enhanced bio-interfaces with primary cultured neurons for effective neural activation. Coupled with its simplicity and superior biocompatibility, this innovative approach of polymerization inside living organisms opens up promising prospects for its application in future biomedical innovations.",
"introduction": "Introduction Conducting polymers (CPs) have gained significant attention in biomedical applications due to their biocompatibility and tunable electronic, optical, and electrochemical properties. 1 , 2 They are not naturally produced in living systems and are introduced externally for various applications such as bioimaging, drug delivery, biosensors, and neural interfaces. 3 – 7 However, these strategies often result in poor bio-integration with soft tissues, creating a gap between in vitro and in vivo device performance and longevity. 8 – 10 To overcome such shortcomings, attempts have been made to synthesize CPs within biological systems, with preliminary work showing that CPs can be safely synthesized in vivo with electrochemical polymerization. 11 Recent work developed the in vivo assembly of CPs directly onto neural membranes, either by genetic engineering to express enzymes that catalyze polymerization 12 – 14 or by using external oxidative enzymes that trigger endogenous metabolites (i.e., H 2 O 2 ) to promote local polymerization in living fish and medicinal leeches. 15 , 16 These methods still present limitations, such as the formation of toxic byproducts from over-expression of oxidative enzymes leading to cell apoptosis. 17 Therefore, it is appealing to assemble CPs in vivo by only using endogenous metabolites to initiate and promote polymerization. Recently, n-doped conducting polymer poly(3,7-dihydrobenzo[1,2- b :4,5- b ’]difuran-2,6-dione) (n-PBDF) was reported, showing features such as high conductivity, and air/water stability, and biocompatibility. 18 , 19 The reaction mechanism involves oxidative polymerization by mild oxidants and reductive doping by water, which could conceivably occur inside living organisms. Here, we discovered that utilizing endogenous enzymatic proteins, such as hemoproteins, can lead to efficacious in vivo polymerization of n-PBDF in aqueous media. We then demonstrated in vivo synthesis of n-PBDF through whole blood catalyzed oxidative polymerization and water-promoted reductive doping. We further verified that it is possible to form n-PBDF in live zebrafish embryos without any casualty, showing excellent biocompatibility. We eventually demonstrated the potential of in cellulo synthesized n-PBDF as photoacoustic transducers with enhanced bio-integration, enabling non-genetic neural stimulation in cultured primary neurons with a submillimeter resolution."
} | 1,064 |
29954335 | PMC6022435 | pmc | 4,824 | {
"abstract": "Background Quorum sensing is a mechanism of cell to cell communication that requires the production and detection of signaling molecules called autoinducers. Although mesophilic bacteria is known to utilize this for synchronization of physiological processes such as bioluminescence, virulence, biofilm formation, motility and cell competency through signaling molecules (acyl homoserine lactones, AI-1; oligopeptides, peptide based system and furanosyl borate diester, AI-2), the phenomenon of quorum sensing in thermophiles is largely unknown. Results In this study, proteomes of 106 thermophilic eubacteria and 21 thermophilic archaea have been investigated for the above three major quorum sensing systems to find the existence of quorum sensing in these thermophiles as there are evidences for the formation of biofilms in hot environments. Our investigation demonstrated that AI-1 system is absent in thermophiles. Further, complete peptide based two component systems for quorum sensing was also not found in any thermophile however the traces for the presence of response regulators for peptide based system were found in some of them. BLASTp search using LuxS (AI-2 synthase) protein sequence of Escherichia coli str. K-12 substr. MG1655 and autoinducer-2 receptors (LuxP of Vibrio harveyi , LsrB of E. coli str. K-12 substr. MG1655 and RbsB of Aggregatibacter actinomycetemcomitans ) as queries revealed that 17 thermophilic bacteria from phyla Deinococcus- Thermus and Firmicutes possess complete AI-2 system (LuxS and LsrB and/or RbsB). Out of 106 thermophilic eubacteria 18 from phyla Deinococcus- Thermus , Proteobacteria and Firmicutes have only LuxS that might function as AI-2 synthesizing protein whereas, 16 are having only LsrB and/or RbsB which may function as AI-2 receptor in biofilms. Conclusions We anticipate that thermophilic bacteria may use elements of LsrB and RbsB operon for AI-2 signal transduction and they may use quorum sensing for purposes like biofilm formation. Nevertheless, thermophiles in which no known quorum sensing system was found may use some unknown mechanisms as the mode of communication. Further information regarding quorum sensing will be explored to develop strategies to disrupt the biofilms of thermophiles. Electronic supplementary material The online version of this article (10.1186/s12866-018-1204-x) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion Thermophiles are one of the major groups of extremophile. They are known to form biofilms at high temperature. Identifying the phenomenon to form biofilms at high temperature may reveal how these microorganisms adapted themselves at such adverse conditions. As mentioned in previous studies, AI-1 and oligopeptides are damaged by hydrolytic cleavage whereas; AI-2 is quiet stable at different pH range and temperatures [ 12 , 44 ]. Therefore AI-2 might be the mode of communication used by thermophilic bacteria. Other unknown quorum sensing systems may be prevalent in thermophiles which have to be explored further. To conclude, our study is an attempt to understand the mechanism of quorum sensing used by thermophiles as information regarding quorum sensing has not been explored in most of the thermophiles. Further this information can be utilized for developing strategies to disrupt the biofilms formed by these thermophiles at high temperature.",
"discussion": "Discussion The way thermophilic bacteria adapt themselves in hot environment has become the topic of interest since 1960’s after the discovery of T. aquaticus . Presence of evidences for quorum sensing in mesophiles demands the investigation of quorum sensing mechanism(s) utilized by thermophiles. Thermophilic bacteria viz. T. thermophilus and thermophilic archaea viz. P. furiosus have been shown to form biofilms [ 45 ]. These thermophiles with other thermophilic biofilm formers are included in this study to suggest that these may interact with each other by quorum sensing. Previous work on the presence of peptide based quorum sensing in T. maritima [ 7 ], temperature dependent formation of AI-2 in T. maritima and archaean P. furiosus [ 11 ] and the expression of LuxS in C. mediatlanticus led us to search for quorum sensing systems present in thermophilic community. In this study, the most prevalent quorum sensing systems were investigated. AI-1 system was not found in thermophiles which is in accordance with the results of Schopf et al. [ 10 ] that suggests the heat labile nature of AI-1 system. However, few thermophilic eubacteria showed traces for the presence of peptide based quorum sensing system (Table 2 ) but the identity with their mesophilic counterparts was very low. Only few members of phyla Firmicutes, Deinococcus-Thermus, Chloroflexi and Thermodesulfobacteria showed some identity with the response regulator AgrA and ComA but none of them showed any good identity with receptor histidine kinases which may suggest the presence of incomplete two component system in thermophilic eubacteria or they do not posseses any peptide based system or there may be some unknown proteins involved in two component peptide based system for thermophiles. Search for MTA/SAH nucleosidase (Pfs), autoinducer-2 synthase (LuxS) and SAH hydrolase in the proteomes of thermophilic bacteria showed that the universal autoinducer-2 type of quorum sensing is the mode of communication. Pfs and SAH hydrolase are important enzymes like LuxS as they play role in the detoxification of SAH [ 20 – 22 ]. The Pfs mutant of Neisseria meningitidis completely stopped the release of AI-2 due to the accumulation of inhibitors such as MTA and toxic SAH [ 23 ]. These results were further supported by the previous work in which Pfs and LuxS inhibitors showed reduction in biofilm formation and did not produce autoinducer-2 [ 24 – 26 ]. Absence of LuxS and presence of Pfs and SAH hydrolase in Fervidobacterium islandicum, F. nodosum, Thermosipho melanesiensis, Thermotoga sp. and an archean Aeropyrum pernix in this work predicts that Pfs and SAH hydrolase play role in the detoxification of SAH and they do not produce AI-2 . Therefore, in these bacteria due to the lack of LuxS no autoinducer-2 is synthesized. Phylogenetic analysis of LuxS in thermophiles and mesophiles did not correlate with their 16S rRNA phylogeny, that indicates LuxS is more conserved in bacteria of different phyla and environment and mesophilic bacteria may have acquired LuxS from thermophiles (Fig. 2 ). Further LuxS from themophilic bacteria of same phylum were evolutionarily closer than from other phyla. This finding was supported by the Perez-Rodriguez et al. [ 12 ] who showed the LuxS gene flow from thermophiles to mesophilic epsilonproteobacteria. This work showed the events of horizontal luxS gene transfer, as certain thermophilic bacteria inhabiting similar environment were having LuxS that was evolutionarily more identical. Anoxybacillus spp . and Geobacilllus spp prevalent in dairy industry, B. anthracis and B. cereus bearing close phenotypic and genotypic resemblance, T. thermocopriae and C. perfringens found in decaying vegetables, M. hydrothermalis and O. profundus , inhabitants of deep sea hydrothermal vent, S. marcescens and E.coli , pathogenic bacteria, S. enterica (human pathogen) and P. rhabdus (insect pathogen) were having more identical LuxS. Further, V. harveyi , S. marcescens and E.coli were nested within thermophilic lineage as Nitratiruptor sp. SB155–2 is known to be phylogenetically related to epsilon-proteobacterial pathogens [ 27 ]. Thus this evolutionarily identical LuxS among proteobacteria may be the result of this relation. Further, this study showed that some thermophilic bacteria viz. M. hydrothermalis , M. taiwanesis, M. cerbereus, M. rufus, O. profundus and T. aquaticus have LuxS but no Pfs. However, except M. hydrothermalis all of them possess SAH hydrolase . This indicates that here the detoxification of S-adenosylhomocysteine (SAH) occurs through SAH hydrolase since Pfs is absent in them. However, except O. profundus all of them lack the known receptors for AI-2 which predicts that they may not be involved in AI-2 type quorum sensing and their LuxS carries out the metabolic process such as the production of homocysteine for converting it back to methionine in AMC. However, the possibility of the presence of some unknown AI-2 receptors in them cannot be ruled out. Multiple sequence alignment of LuxS of thermophilic and mesophilic bacteria showed the presence of invariant HXEEH motif which is in accordance with the work done by Keersmaecker et al. [ 28 ]. Further, our analysis showed that all thermophilic bacteria possess amino acids in the similar positions as in persistant hub which is the characterstic of extremophilic LuxS protein as described by Bhattacharyya and Vishveshwara [ 29 ]. M. silvanus and M. chilarophilus both showed autoinducer-2 synthesizing complete pathway as well as SAH hydrolases. Presence of both pathways in these two thermophiles allows them to recycle methionine more economically which is further supported by the work of Schell et al. [ 30 ] that showed the presence of both of these pathways in Bifidobacterium longum . Except M. silvanus and M. chilarophilus, all other thermophiles lacking complete autoinducer-2 system have SAH hydrolases which indicates that they may have role in detoxification of SAH to homocysteine and adenosine without synthesizing AI-2. Our results are further supported by Winzer et al. [ 31 ] who showed the presence of only SAH hydrolase and no autoinducer-2 synthesizing machinery in Leptospira interrogans. Nevertheless, the possibility of unknown mechanisms to synthesize AI-2 exists. LuxP was not found in any of the thermophilic bacteria, corroborating the previous work showing the LuxP type receptors are only limited to Vibrionales and Oceanospirillales [ 32 , 33 ]. Another well known receptor for autoinducer-2 was found to be LsrB. There are evidences for the presence of LsrB in Enterobacteriaceae and Rhizobiaceae and Bacillaceae families [ 34 ]. Autoinducer-2 ABC transporter which shares identity with LsrB ABC transporter was found in some thermophilic bacteria in this study. M. silvanus, O. profundus, A. geothermalis, C. subterraneus, M. thermoacetica and T. naphthophila were the only thermophiles having autoinducer-2 ABC transporter identical to LsrB. Our results showed that M. silvanus , A. geothermalis and O. profundus possess both Pfs and LuxS as well as LsrB, complete autoinducer-2 quorum sensing circuit which can produce as well as detect autoinducer-2. However, all of these thermophiles posseses RbsB as well. It indicates that they may utilize two receptors for autoinducer-2 detection like in Aggegatibacter actinomycetemcomitans [ 35 ] where LsrB protein competes with the LuxP of Vibrio harveyi during autoinducer-2 bioassay and inhibits the interaction of AI-2 with LuxP. They further showed that LsrB competes with LuxP for the AI-2 produced by V. harveyi while RbsB competes with LuxP for the AI-2 produced by A. actinomycetemcomitans. It may be concluded from our work that C. subterraneus, M. thermoacetica and T. naphthophila possess autoinducer-2 ABC transporter protein which is identical to LsrB ABC transporter but they all lacked LuxS protein which indicates that they might respond to autoinducer-2 produced by other bacteria in biofilms without producing it themselves. This is in accordance with the results of Rezzonico and Duffy [ 32 ] which showed the presence of orphan lsr operon in R. sphaeroides and S.meliloti that lack luxS. It may be possible for bacteria to detect the AI-2 signal even if it does not produce it. Pseudomonas aeruginosa does not contain luxS but in the presence of enzymatically produced AI-2 or in co-culture with Streptococcus mitis , some of its virulence gene promoters were upregulated [ 36 , 37 ]. Similar findings were observed in some mixed biofilm formation [ 38 ]. In previous reports, RbsB has been speculated as another receptor for autoinducer-2 in Borellia burgdorferi and Actinobacillus actinomycetemcomitans [ 35 , 39 ]. Our study found a large number of thermophilic bacteria having D-ribose binding protein identical to RbsB of A. actinomycetemcomitans (Additional file 12 ). Ribose ABC-transporter is distributed ubiquitously among thermophilic bacteria regardless of the prevalence of Pfs and LuxS and these results are in accordance with the previous work [ 32 ]. It may thus work as the autoinducer-2 receptor in thermophilic bacteria lacking LuxP and LsrB. Furthermore, there are certain mesophilic bacteria ( Actinobacillus pleuropneumoniae, Borrelia burgdorferi, Helicobacter pylori, Mycobacterium avium and Streptococcus spp. ) which are known to form biofilms and other quorum sensing phenotypes and produce AI-2 but lack the known LuxP, LsrB and RbsB like receptors [ 40 – 43 ]. Similarly in many thermophiles no known receptor for AI-2 has been found, so unknown alternative receptors might exist for them. Presence of LuxS and autoinducer-2 receptor in thermophiles may explain their role in quorum sensing. Presence of autoinducer-2 receptor but lack of autoinducer-2 synthase in certain thermophiles may explain their interactions with neighbours in biofilms regardless of producing autoinducer-2 synthase but responding to autoinducer-2. Peptide based or other unknown quorum sensing systems may be the mode of communication used by some thermophilic archaea as no autoinducer-1 or autoinducer-2 system is present in them."
} | 3,425 |
26636551 | PMC5029187 | pmc | 4,826 | {
"abstract": "The occurrence of anaerobic oxidation of methane (AOM) and trace methane oxidation (TMO) was investigated in a freshwater natural gas source. Sediment samples were taken and analyzed for potential electron acceptors coupled to AOM. Long-term incubations with 13 C-labeled CH 4 ( 13 CH 4 ) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, 13 C-labeled CO 2 ( 13 CO 2 ) simultaneously increased with methane formation, which is typical for TMO. In the presence of nitrate, neither methane formation nor methane oxidation occurred. Net AOM was measured only with sulfate as electron acceptor. Here, sulfide production occurred simultaneously with 13 CO 2 production and no methanogenesis occurred, excluding TMO as a possible source for 13 CO 2 production from 13 CH 4 . Archaeal 16S rRNA gene analysis showed the highest presence of ANME-2a/b (ANaerobic MEthane oxidizing archaea) and AAA (AOM Associated Archaea) sequences in the incubations with methane and sulfate as compared with only methane addition. Higher abundance of ANME-2a/b in incubations with methane and sulfate as compared with only sulfate addition was shown by qPCR analysis. Bacterial 16S rRNA gene analysis showed the presence of sulfate-reducing bacteria belonging to SEEP-SRB1. This is the first report that explicitly shows that AOM is associated with sulfate reduction in an enrichment culture of ANME-2a/b and AAA methanotrophs and SEEP-SRB1 sulfate reducers from a low-saline environment.",
"introduction": "Introduction Anaerobic methane oxidation (AOM) coupled to sulfate reduction (SR) was first discovered to occur in marine sediments ( Martens and Berner, 1974 ; Reeburgh, 1976 ). The process was found to be catalyzed by communities of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) of the Deltaproteobacteria ( Hinrichs et al. , 1999 ; Boetius et al. , 2000 ; Orphan et al. , 2001a , 2001b ). More recently, AOM was also reported to be coupled to other electron acceptors besides sulfate. In freshwater environments, AOM was coupled to the reduction of nitrate and nitrite ( Raghoebarsing et al. , 2006 ; Ettwig et al. , 2008 , 2009 ; Hu et al. , 2009 ; Deutzmann and Schink 2011 ; Haroon et al. , 2013 ). Microbial methane oxidation with iron and/or manganese reduction was described in marine sediments ( Beal et al. , 2009 ; Riedinger et al. , 2014 ), brackish sediments ( Egger et al. , 2015 ), a terrestrial mud volcano ( Chang et al. , 2012 ) and also in freshwater environments ( Crowe et al. , 2011 ; Sivan et al. , 2011 ; Amos et al. , 2012 ). Recently, humic acids (HAs) were also hypothesized to act as electron acceptor for AOM ( Gupta et al. , 2013 ). AOM coupled to SR in freshwater is likely limited by the low-sulfate concentrations, which are around 10–500 μ M ( Holmer and Storkholm, 2001 ). Sulfate-dependent AOM has been observed in freshwater systems, but the involvement of other electron acceptors could not be excluded. Moreover, the responsible microorganisms were either not analyzed nor conclusively identified ( Grossman et al. , 2002 ; van Breukelen and Griffioen, 2004 ; Eller et al. , 2005 ; Schubert et al. , 2011 ; Segarra et al. , 2015 ). ANME-1-related archaea have been found in a terrestrial subsurface ( Takeuchi et al. , 2011 ), but 13 C-labeled carbon dioxide ( 13 CO 2 ) formation from 13 C-labeled methane ( 13 CH 4 ) also occurred in control incubation where no electron acceptor was added. This was also the case in other incubation studies ( Beal et al. , 2009 ; Sivan et al. , 2011 ; Egger et al. , 2015 ). These observations make it difficult to link ongoing methane oxidation to a particular electron acceptor. Moreover, 13 CO 2 can also be produced during methanogenesis in a process called trace methane oxidation (TMO) ( Zehnder and Brock, 1979 ). TMO was demonstrated to occur in pure cultures of different methanogens ( Zehnder and Brock, 1979 ; Harder, 1997 ; Moran et al. , 2005 , 2007 ), in granular sludge ( Zehnder and Brock 1980 ; Harder 1997 ; Meulepas et al. , 2010 ) and in freshwater and terrestrial environments ( Zehnder and Brock, 1980 ; Blazewicz et al. , 2012 ). Differentiation between AOM and TMO is difficult for several reasons: (a) both processes can produce 13 CO 2 at comparable rates; (b) at elevated methane partial pressure, TMO rates increase ( Zehnder and Brock, 1980 ; Smemo and Yavitt, 2007 ) while methanogenesis is repressed, which favors SR ( Meulepas et al. , 2010 ); and (c) ferrous sulfate addition may result in enhanced TMO rates ( Zehnder and Brock, 1980 ). This means that with elevated 13 CH 4 partial pressure and presence of sulfate, an increase in 13 CO 2 and sulfide production cannot be taken as evidence for sulfate-dependent AOM unless net methane consumption is demonstrated. Moreover, although there is convincing evidence that anaerobic methane oxidizing archaea (ANME) are capable of net AOM, detecting ANME sequences or cells in mixed communities that perform methanogenesis does not prove that AOM takes place, since ANME could perform methanogenesis as well ( Lloyd et al. , 2011 ; Bertram et al. , 2013 ) and as a consequence could perform TMO. In this study we used long-term incubations (>168 days) with samples taken from a freshwater natural gas source with added 13 CH 4 to investigate the occurrence of both TMO during net methanogenesis and AOM. AOM was distinguished from TMO by simultaneous detection of 13 CH 4 , 12 CH 4 (produced during methanogenesis) and 13 CO 2 . We investigated the effect of different electron acceptors that possibly might be involved in AOM. Control incubations without addition of methane were carried out to accurately distinguish between net methane oxidation and net methanogenesis. Archaeal community analysis of long-term incubations with methane and sulfate (CH 4 +SO 4 2− ), sulfate only (SO 4 2− -only), and methane only (CH 4 -only) was performed at 323 days of incubation. Incubations with sulfate and with and without methane were monitored for an extended period of 728 days.",
"discussion": "Results and discussion Trace methane oxidation Methane production was observed in most conditions, but was negligible in the presence of sulfate and did not occur in the presence of nitrate ( Figure 2 ). Methane production in conditions with and without added methane showed a similar pattern, but the amount of methane produced was lower in incubations where methane was added ( Supplementary Figure S1A ). This was probably caused by the increase of TMO due to a higher methane concentration ( Zehnder and Brock, 1980 ; Smemo and Yavitt, 2007 ). Production of 13 CO 2 was apparent in all incubations with 13 CH 4 in the headspace, except in the conditions with nitrate and HAs ( Figure 2 and Supplementary Figure S1B ). Typical for TMO, 13 CO 2 simultaneously increased with methane formation in the conditions with ferrihydrite, ferrihydrite + HAs and the control without electron acceptor ( Figure 2 ). The 13 CO 2 production was not substantially different between ferrihydrite, ferrihydrite + HAs and the control conditions (Wilcoxon rank-sum test, P <0.05; Table 1 ), indicating that TMO was not influenced by the electron acceptors added. When ferrihydrite was added, the 13 CO 2 production continued when all iron was reduced to Fe(II) after 300 days, without addition of HAs ( Supplementary Figure S2 ). Iron reduction did occur faster in incubations with ferrihydrite + HAs than in the incubations with only ferrihydrite. The incubations with 20 g l −1 HAs contained an average of 28.8 (±1.0) m M acid soluble Fe(II) after 300 days of incubation and did not show any detectable 13 CO 2 increase ( Figure 2 , Supplementary Figure S1B and Table 1 ). The HAs batch used contained calcium which could scavenge produced CO 2 to form calcium carbonate. After acidification of the samples, an increase in total CO 2 was observed but the percentage of 13 CO 2 did not increase. It was reported that reduced methane emission after addition of HAs to peat ecosystems could be caused by increased methane oxidation ( Blodau and Deppe, 2012 ). In contrast, here we observed higher methane production after addition of HAs but no methane oxidation. Anaerobic methane oxidation Only in the incubations with sulfate, an increase in 13 CO 2 with no increase in 12 CH 4 was observed ( Figure 2 ). The ratio of methane oxidized per methane produced was only >1 for conditions with sulfate, which is indicative for AOM ( Table 1 ). In previous studies, sulfate addition inhibited methane formation and thus 13 CO 2 production from TMO in freshwater sludge ( Zehnder and Brock, 1980 ; Meulepas et al. , 2010 ) and in freshwater slurries ( Segarra et al. , 2013 ) and only stimulated methane oxidation in brackish water slurries ( Segarra et al. , 2013 ). In our study, the pooled inoculum contained an average of around 2 m M sulfate ( Supplementary Table S3 ). All sulfate was reduced after 41 days of incubation and methanogenesis continued in most conditions, which was accompanied by continued 13 CO 2 production during TMO ( Figure 2 ). Only where sulfate was added, sulfate addition stimulated methane oxidation and repressed methane production, indicating AOM coupled to SR at low salinity. AOM could not be coupled to any other electron acceptor than sulfate. Inductively coupled plasma measurements of all samples prior to mixing showed that only sulfur and iron were present, which in oxidized form could be possible electron acceptors for AOM, whereas the amount of selenium and manganese was not significant ( Supplementary Table S4 ). In incubations with nitrate and humic acids, no 13 CO 2 was produced. Reduction of the electron acceptors sulfate, ferrihydrite and nitrate occurred in all conditions with and without addition of methane ( Table 2 ). The reduction rates of sulfate with and without added methane in the first 168 days were similar (two-tailed t -test with unequal variance, P <0.05), which was probably due to endogenous SR masking sulfate-dependent AOM. After 343 days of incubation, the SR rate in incubations with only sulfate had substantially decreased due to endogenous substrate depletion whereas in conditions with methane and sulfate, there was no difference in SR rates. However, in this time period AOM could not be linked to SR and sulfide production as the abundant green sulfur bacteria Chlorobiaceae ( Supplementary Figure S3 ) could have caused the fluctuations in sulfide levels. Growth and activity of Chlorobiaceae explained the green coloration occurring specifically in incubations amended with sulfate, which derived from the bacteriochlorophyll of green sulfur bacteria ( Gorlenko, 1970 ). Green sulfur bacteria are strictly anaerobic autotrophic sulfide oxidizers and have been found to be active when exposed to very little light ( Beatty et al. , 2005 ; Manske et al. , 2005 ), which could explain activity even in the dark with limited exposure to light during sampling of our incubations. Their activity probably kept the sulfide concentration low. After maintaining complete darkness in the slurries, the 13 CO 2 production continued throughout incubation time and free sulfide was eventually measured. In bottle 1A-2 that showed the highest 13 CO 2 production after 168 days of incubation ( Table 1 ), sulfide production increased simultaneously with 13 CO 2 production during the last period between 343 and 728 days ( Supplementary Figure S4 ). This shows that at long term, net methane oxidation accompanied sulfide production. Microbial community profiling Microbial community profiling was only carried out on triplicates of the conditions CH 4 +SO 4 2− , CH 4 -only, SO 4 2− -only, and the original sediment after 323 days of incubation. For all samples that were analyzed, the highest average percentage of 16S rRNA reads for Archaea clustered within the Methanosarcinaceae, Methanoregulaceae, Methanosaetaceae , Methanobacteriaceae , and the Miscellaneous Crenarchaeota Group (MCG) ( Supplementary Figure S5 ). Archaeal OTUs that showed a significantly higher percentage of reads (Kruskal–Wallis, P <0.05) in condition CH 4 +SO 4 2− , as compared with CH 4 -only ( Figure 3a ) and SO 4 2− -only ( Figure 3b ) make up less than 10% of all reads. In condition CH 4 +SO 4 2− , ANME-2a/b sequences represented 0.16% of all reads and were much more abundant than in the condition CH 4 -only. Higher abundance of ANME-2a/b in conditions CH 4 +SO 4 2− compared with SO 4 2− -only was shown by qPCR analysis ( Supplementary Figure S6 ). This indicates the involvement of ANME-2a/b in AOM coupled to SR, as shown before in marine environments ( Orphan et al. , 2001a ). The ANME-2a/b OTU showed 98% identity with ANME-2a/b from both marine and non-marine environments and do not form a monophyletic cluster with ANME-2a/b found in other low-sulfate environments ( Figure 4 ). A marine enrichment of ANME-2a/b species that share 98% identity was previously shown to be completely inhibited in AOM activity at a salinity of 5‰ ( Meulepas et al. , 2009 ), indicating that the ANME-2a/b detected in this study are adapted to low salinity. A higher percentage of reads was also found for 1 OTU of Methanosarcinales GOM Arc I (OTU 4) in conditions CH 4 +SO 4 2− compared with both CH 4 -only and SO 4 2− -only ( Figure 3 ). This GOM Arc I group was previously named ‘ANME-2d' ( Mills et al. , 2003 ) but was renamed to ‘GOM Arc I' since it was not monophyletic with other ANME-2 subtypes and no AOM activity or aggregation with sulfate reducers had been shown ( Lloyd et al. , 2006 ). Recently, the name ANME-2d was re-adopted for a cluster that harbors ‘ Ca . Methanoperedens nitroreducens', which performed AOM coupled to nitrate reduction ( Haroon et al. , 2013 ). This cluster was previously identified in a nitrate-dependent AOM enrichment ( Raghoebarsing et al. , 2006 ) and was named ‘AOM associated archaea' (AAA) ( Knittel and Boetius, 2009 ). The GOM Arc I related OTU 4 found in this study was 97% identical to ‘ Ca. M. nitroreducens' and was 99% identical to other AAA members that were proposed to be responsible for freshwater AOM coupled to SR in Lago di Cadagno sediments ( Schubert et al. , 2011 ) ( Figure 4 ). The AAA were also found to be abundant in an aquifer where methane and sulfate were present ( Flynn et al. , 2013 ). It was already shown that ‘ Ca . M. nitroreducens' uses the complete reverse methanogenesis pathway and it was suggested that the genes for nitrate reduction were obtained from a bacterial donor ( Haroon et al. , 2013 ). We did not find nitrate-dependent AOM activity, which leaves open the possibility that the AAA in this study could perform AOM coupled to SR. Sulfate addition in the presence of methane also had a positive effect on other GOM Arc I related OTUs ( Figures 3a and 4 ), which makes a contribution of GoM Arc I to AOM activity likely. The higher percentage of reads of Methanolobus in conditions CH 4 +SO 4 2− compared with SO 4 2− -only ( Figure 3b ) implied that methane addition had an effect on Methanolobus abundance. The reason for this effect is unclear, since this genus is known to be able to utilize methylated compounds ( Zhang et al. , 2008 ), but not methane. However, Methanolobus was also found in a marine methane-oxidizing bioreactor ( Girguis et al. , 2003 ). Bacterial diversity was high in all samples, with the highest relative number of reads for all samples clustering with the Deltaproteobacteria ( Syntrophobacteriacaea and Desulfobacteraceae ) and Gammaproteobacteria ( Methylococcaceae ), Bacteroidetes , Chloroflexi, Firmicutes and Chlorobi (family Chlorobiaceae ) ( Supplementary Figure S3 ). Bacterial OTUs that showed a substantially higher percentage of reads (Kruskal–Wallis, P <0.05) in condition CH 4 +SO 4 2− as compared with both CH 4 -only and SO 4 2− -only make up less than 0.5% of all reads ( Figure 5 ). These OTUs clustered with the Desulfobacteraceae, Clostridiales and Planctomycetaceae. The OTUs of Desulfobacteraceae belonged to the Sva0081 sediment group, Desulfobacterium spp. and the SEEP-SRB1 cluster. The latter OTU of SEEP-SRB1 (AB630772) was only found in condition CH 4 +SO 4 2− . However, other SEEP-SRB1 OTUs that were detected did not show a difference in read abundance between the conditions CH 4 +SO 4 2− , CH 4 -only and SO 4 2− -only. The SEEP-SRB1 clade has been detected in several marine AOM-mediating environments ( Orphan et al. , 2001b ; Knittel et al. , 2003 ; Lösekann et al. , 2007 ; Pernthaler et al. , 2008 ; Harrison et al. , 2009 ; Yanagawa et al. , 2011 ; Vigneron et al. , 2013 ) and enrichments ( Jagersma et al. , 2009 ; Zhang et al. , 2011 ). The SEEP-SRB1 OTUs found in this study clustered in undefined subgroups outside the marine SEEP-SRB1 subgroups that were described previously ( Figure 6 ), of which SEEP-SRB1a was identified as the dominant bacterial partner of ANME-2a/b in marine AOM-mediating enrichments ( Schreiber et al. , 2010 ). From the other OTUs that showed a higher percentage of reads in condition CH 4 +SO 4 2− , little is known about their role in AOM coupled to SR. It has been shown before that different SRB besides SEEP-SRB1 belonging to Desulfobacteraceae form consortia with different ANMEs ( Orphan et al. , 2002 ; Vigneron et al. , 2013 ) and even non-SRB were found to aggregate with ANMEs ( Pernthaler et al. , 2008 ). We did not find any sequences related to the NC10 phylum of bacteria, harboring the nitrate-dependent methanotrophic bacterium ‘ Ca . Methylomirabilis oxyfera' ( Ettwig et al. , 2010 ), and we also did not obtain any PCR product using specific primers for this clade (data not shown), which is in line with the lack of AOM coupled to denitrification. AOM at low-sulfate concentrations The sulfate concentration was 0.07 m M in the gas source effluent and about 2 m M in the pooled inoculum ( Supplementary Table S3 ). The measured conductivity and chloride concentration of the gas source effluent and pooled inoculum samples ( Supplementary Table S3 ) indicate a somewhat higher salinity than typical freshwater, but a much lower salinity than typical brackish environments. This could correspond to the historical marine influence of the adjacent lake (Markermeer) that was formed due to dike construction, as described for proximal sites ( van Diggelen et al. , 2014 ). In marine environments, the sulfate:chloride ratio is around 1:19. The sulfate:chloride ratio of the lake surface water was around 1:2.6, with 1.7 m M sulfate and 4.4 m M chloride ( Supplementary Table S3 ). Therefore, marine influences cannot explain the relatively high-sulfate concentrations. The sulfate concentration in deeper layers of the gas source could be even higher than measured in the gas source effluent before AOM took place. In marine systems, AOM rates started to be affected below 2–3 m M sulfate ( Meulepas et al. , 2009 ; Wegener and Boetius, 2009 ) but occurred even below 0.5 m M of sulfate ( Beal et al. , 2011 ; Yoshinaga et al. , 2014 ). In typical freshwater environments, the sulfate concentration is generally lower than 0.5 m M , making AOM-SR feasible but at low rates. AOM in freshwater was recently shown to be a strong methane sink at sulfate concentrations as low as 1.2–0.1 m M ( Segarra et al. , 2015 ). Our finding of AOM activity only in conditions with methane and sulfate, and the enrichment of ANME-2a/b and SEEP-SRB1, suggests that these syntrophic clades are ubiquitously distributed in marine and in low-salinity environments and perform AOM at low-sulfate concentrations."
} | 4,955 |
35547892 | PMC9087907 | pmc | 4,827 | {
"abstract": "Stretchable and flexible photoelectric materials are highly desirable for the development of artificial intelligence products. However, it remains a challenge to fabricate a stable, processable, and cost-efficient material with both high photoelectric sensitivity and remarkable deformability. Herein, a new kind of photoelectric sensitive, highly stretchable and environmentally adaptive materials was developed through in situ synthesis and π–π conjugation design. Specifically, a photoelectric elastomer zinc porphyrin SEBS(Zn-PorSEBS) was synthesized by introducing porphyrin to SEBS chain via a one-pot method. Then, graphene/zinc porphyrin SEBS (G/Zn-PorSEBS) composites were obtained by combing the elastomer with graphene sheets through solution blending. Notably, the resultant flexible composites were capable of capturing light changes with illumination on or off, and the maximum photocurrent density reached 0.13 μA cm −2 . Moreover, the photoelectric composites exhibited a dramatic elongation (more than 1000%) and an excellent tensile strength about 20 MPa. This proposed strategy represents a general approach to manufacture photoelectric and flexible materials.",
"conclusion": "Conclusions In conclusion, a facile and effective strategy is proposed for the synthesis of photoelectrical sensitive and highly stretchable elastomer. Firstly, we developed a stretchable and photoelectric sensitive elastomer by introducing the porphyrins to the SEBS chain with a controllable graft ratio through in situ synthesis. The obtained Zn-PorSEBS elastomer exhibited excellent photoelectricity and stretchability. Then, photoelectric composites G/Zn-PorSEBS with higher stimulated photocurrent were successfully constructed by assembling the as-prepared substrates and nanostructured conductive graphene sheets via π–π conjugation. Without sacrifice its outstanding elasticity of Zn-PorSEBS elastomer, the photoelectric sensitivity and tensile strength of G/Zn-PorSEBS were significantly enhanced. The obtained flexible G/Zn-PorSEBS composites can be used as photoelectric switcher to capture light changes with illumination on or off. These remarkable properties enable the designed composites to be a new generation of promising flexible photoelectric responsive materials for a range of applications such as wearable devices, electronic skin, and optoelectronic sensors.",
"introduction": "Introduction Organic photoelectronic materials have drawn considerable attention due to their wide applications in solar cells, 1,2 photoelectronic sensors, 3,4 and light-emitting diodes, 5,6 etc . Generally, good sensitivity and passable conversion efficiency are highly necessary for a desirable photoelectronic device. However, devices made from traditional photoelectronic materials are always too stiff to stretch, fold or wrinkle, which severely limits their application in flexible electronic devices such as wearable devices, 7 electronic skins, 8 and soft sensors. 9 In recent years, many strategies have been exploited to develop flexible electronics. For instance, Lai et al. reported a stretchable transparent electrode via embedding AgNWs beneath the surface of PDMS. 10–12 Lee et al. prepared a stretchable and patchable strain sensor based on a sandwich-like stacked nanohybrid film. 13 Li et al. developed a stretchable conductor by the electroless plating technique. 14 However, these materials exhibit insensitivity to light, which restricts their application in capturing light changes. Therefore, it is of great significance to develop a flexible material which is sensitive to light to cater the needs of soft photoelectronic electronics. Poly(styrene–ethylene/butylene–styrene) (SEBS) triblock copolymer, which is synthesized through anionic polymerization and hydrogenation using styrene and butadiene as raw materials, is a kind of useful commercial thermoplastic elastomer. 15 It features in microphase separated micromorphology in which polystyrene phases with high modulus are incompatible with soft EB phases. Polystyrene hard block phase which act as a physical crosslinking impart the thermoplastic nature and polybutadiene soft block phase contribute to the high elasticity of SEBS at ambient temperatures. Owing to its soft nature, excellent ageing resistance, weather fastness, hydrolysis and moisture resistance comparing to other thermoplastic elastomers such as polyurethane (TPU) and nylon elastomers (PEBA), SEBS has been widely applied as shock absorbing materials, adhesives, sealants, coatings and cable sheath. 16 Therefore, SEBS is a promising organic optoelectronic substrate that can be used in load bearing, outdoor, corrosive and wet, etc. harsh conditions. In recent years, porphyrins and its derivative have attracted great attention in materials science since it has been awarded the Nobel Prizes in Chemistry several times due to their biological functionalities. 17 Porphyrins are visible light harvesting chromophores that exhibit unique physical, chemical and biological features similar to that of natural chlorophylls. They can act as efficient electron donors in various photoinduced electron transfer processes. 18,19 This feature makes porphyrins extremely useful in combination with other electron acceptors. Based on the interactions with electron acceptors, they could achieve the conversion of optical signal to electrical signal under external stimuli. 20,21 In order to endow the elastomer with photoelectric sensitivity, previous researchers have proposed a method to introduce porphyrins to the polymer chains, which included four steps. 22,23 First of all, a phenyl porphyrin substituted with a reactive group on the outer ring should be synthesized. Then, the substituted porphyrin should be complexed with a metal ion. After that, the surface of the polymer material should be chemically modified to produce corresponding reactive groups. Finally, a porphyrin grafted polymer is obtained through a reaction between these two reactive groups. However, the products of substituted porphyrins are always accompanied with many homologues, and separation of the target product from these homologues with extremely similar structures requires permeation chromatography, which is quite complicated and inefficient. Moreover, the yield of the target product is also not satisfactory. Herein, a novel and effective strategy was proposed to design a stretchable photoelectric composite. The matrix of zinc porphyrin SEBS (Zn-PorSEBS) was developed through four modification steps, including chloromethylation, hydroformylation, porphyrinization and complexation by zinc acetate. The porphyrins are synthesized via a one-pot method, and then dynamically grafted to the SEBS chain, which greatly simplifies the separation processes of the target substituted porphyrins. Furthermore, the graft ratio of porphyrins can be artificially controllable by adjusting the reaction time in this strategy. The resulting Zn-PorSEBS elastomer exhibited good photoelectric sensitivity and remarkable stretchability. A small amount of graphene was added via solution blending method, and a graphene/zinc porphyrin SEBS (G/Zn-PorSEBS) composite can be obtained. The photoelectron transfer efficiency was highly enhanced due to the strong π–π conjugation between the porphyrins and graphene. In addition, without sacrifice its excellent elasticity, the mechanical strength of the composite was significantly improved. Moreover, the composite was extremely flexible to undergo a variety of deformations such as twisting, bending, folding and curling to different shapes. This simple and efficient strategy presented in this work probably open up a new opportunity for design and scalable fabrication of flexible optoelectronic materials, which possesses the advantages of being steady, soft and easy to process. This optoelectronic material could be used to fabricate smart electronic devices such as wearable devices, artificial skins of robots and stretchable electronic sensors.",
"discussion": "Results and discussion Molecular structure analysis The synthesis of PorSEBS mainly involves three steps. At first, the chloromethyl groups were grafted to the SEBS chains at the para position of benzene rings through Blanc chloromethylation. 25 Then chloromethyl groups were oxidized into aldehydes by using DMSO as the oxidants, which follows the Kornblum oxidation mechanism. 26 Finally, the obtained aldehydes were reacted with pyrrole and benzaldehyde under the catalysis of lactic acid. Thus, porphyrin rings were in situ synthesized at the para position of the benzene rings of SEBS. FTIR spectroscopy, which is rather sensitive to the groups change can be used to confirm the chemical structure of the intermediate products in each step, as shown in Fig. 2a . In Fig. 2a , for pure SEBS, the wavenumber region of 1601–1452 cm −1 is assigned to the skeleton vibration of benzene rings. The peaks at 757 cm −1 and 698 cm −1 can be attributed to the monosubstituted benzene rings. The peak at 1378 cm −1 corresponds to the C–H bending vibration of the EB segments. It is noted that, a prominent peak at about 1265 cm −1 which belongs to the C–H bending vibration of CH 2 –Cl groups appears after chloromethylation (Cl-SEBS), 27 indicating that the chloromethyl groups were successfully grafted on the benzene rings. However, the peak at 1265 cm −1 is completely absent after the following Kornblum reaction. Instead, a conspicuous new peak at 1701 cm −1 which probably belongs to the C \n \n\n<svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>\nCreated by potrace 1.16, written by Peter Selinger 2001-2019\n</metadata><g transform=\"translate(1.000000,15.000000) scale(0.017500,-0.017500)\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>\n\n O stretching vibration emerges, confirming the successful synthesis of AlSEBS. At last, for PorSEBS, some new peaks located at 3310 cm −1 (stretching vibration of N–H groups), 1350 cm −1 (skeleton vibration of porphyrin ring), 965 cm −1 (in-plane bending vibration of N–H groups), and 798 cm −1 (skeleton vibration of porphyrin rings) can be clearly observed. 28 Besides, the band at 1701 cm −1 was completely disappeared. These changes can be attributed to the fact that the aldehyde groups on the benzene rings were replaced by porphyrin rings. Fig. 2 (a) FT-IR spectra of SEBS, Cl-SEBS, ALSEBS and PorSEBS. (b–e) 1 H NMR spectra of SEBS, Cl-SEBS, ALSEBS and PorSEBS. (f) XPS spectra of PorSEBS and Zn-PorSEBS. Furthermore, the 1 H NMR spectra of the intermediate products are shown in Fig. 2 , which reflects their precise molecular structures. Compared to that of SEBS ( Fig. 2b ), the spectrum of Cl-SEBS ( Fig. 2c ) shows a new proton peak (H1) at 4.4 ppm, which can be attributed to the p -methylene protons of benzene ring. 27 After Kornblum reaction, it can be clearly seen that a new proton peak (H2) at 9.92 ppm appeared ( Fig. 2d ), which can be assigned to the protons of aldehyde groups in ALSEBS. In the 1 H NMR spectra of PorSEBS ( Fig. 2e ), the resonance signals at 8.6, 8.2 and 7.8 ppm are assigned to the ortho - (H4), meta -(H5) and para -protons (H6) of benzene rings in porphyrin, respectively. Typically, the resonance signals of protons in double bond (H3) and secondary amine (H7) in pyrrole rings are appear at 8.8 and −2.75 ppm, respectively. 28 All these results also clearly demonstrated that pyrrole is successfully grafted to the SEBS chains. XPS measurement was used to confirm the complexation behavior between zinc ion and porphyrins, as shown in Fig. 2f . The peaks at 285.7 eV, 400.8 eV and 533.0 eV are shared by the full spectra of PorSEBS and Zn-PorSEBS, which correspond to C 1s, N 1s and O1s, respectively. In particular, the spectrum of Zn-PorSEBS is characterized by the peak at 494.6 eV which is assigned to Zn (LMM) and the peaks at 1031.0 eV and 1044.8 eV are corresponding to Zn 2p. 29 Furthermore, the surface content of Zn element ( C x ) can be obtained from the XPS spectrum according to the peak area ( A i ) and sensitivity factor ( S i ) of desired element, which is expressed as C x = ( A x / S x )/(∑ A i / S i ). As a result, the C x is calculated to be 0.68 wt%. The above analysis fully demonstrate that the target product Zn-PorSEBS was successfully synthesized. Mechanical behavior Tensile test was employed to evaluate the stretchability and elasticity of the composites. As shown in Fig. 3a , the Zn-PorSEBS elastomer exhibits a dramatic elongation (more than 1200%) and a desirable tensile strength about 15 MPa. Comparing to the Zn-PorSEBS elastomer, the G/Zn-PorSEBS composites shows a significant increase in mechanical strength but just a slight decrease in elongation. Graphene adds only 0.3 wt% and the breaking strength enhanced to 17.5 MPa. When the graphene content was added to 0.9 wt%, the breaking strength was further improved to near 20 MPa, and the elongation was still extremely high over 1000%. This is because the reinforcement due to the π–π conjugation between the porphyrins and the incorporated graphene. 30,31 As shown in Fig. 3b–f , it is worth noting that the composites are not only highly stretchable but also extremely flexible to twisting, bending, folding and curling to different shapes (Movie S1 and S2 † ). These results fully demonstrated that the designed photoelectric composite possesses a remarkable stretchable capability and elasticity, which is critical for its applications in flexible electronic devices. Fig. 3 (a) Typical stress–strain curves of Zn-PorSEBS and G/Zn-PorSEBS with different graphene content. Inset photos showing the high stretchability of the sample. (b–f) Pictures giving the different deformation process of G/Zn-PorSEBS composites. Optoelectronic property The photoelectric response performances of Zn-PorSEBS elastomer with different porphyrin grafting ratio and G/Zn-PorSEBS composites with different graphene content were shown in Fig. 4 . Since the neat SEBS is insensitive to light, so no photocurrent can be detected ( Fig. 3a ). Differently from the neat SEBS, the resultant Zn-PorSEBS elastomer shows strong photoelectric response performance due to the introduction of highly photoconductive porphyrin groups. Under illumination, photon-generated excitons dissociate into free electrons and free holes, resulting in the generation of photocurrents. 32 On the contrary, if suffer from dark or insufficient light, the free electrons and free holes will be blocked by the tunnelling barrier. 33 The photoelectric response spectra shows two well-defined states, including a high-current state under illumination and a low-current state under dark, and with the light turns on and off, the two states alternate accordingly (Movie S3 † ). Fig. 4 Photocurrent switching response at 5 s intervals in a 0.3 V 1 M NaOH aqueous solution under 500 W Xenon lamp illumination. (a) Photocurrent response of neat SEBS and Zn-PorSEBS elastomer with different porphyrin grafting ratio. (b–d) Photocurrent response of Zn-PorSEBS elastomer and G/Zn-PorSEBS with different graphene content at the same porphyrin grafting ratio. (e and f) Photographs giving the light on/light off process. To investigate the influence of porphyrinization degree on the photoelectric response performance of Zn-PorSEBS, three samples with different porphyrin grafting ratio were synthesized. The porphyrinization degree of Zn-PorSEBS( i ), Zn-PorSEBS( ii ) and Zn-PorSEBS( iii ) were 7.2%, 10.9% and 13.6% respectively (Fig. S3–S5 and Table S2 † ). It was found that the photocurrent intensity of Zn-PorSEBS elastomer enhanced with increasing of the porphyrinization degree ( Fig. 4a ). The Zn-PorSEBS( iii ) elastomer achieves a highest photocurrent of about 0.06 μA cm −2 . Moreover, compared to the Zn-PorSEBS elastomer, the G/Zn-PorSEBS composites are more sensitive to the incident light due to the existence of graphene ( Fig. 4b–d ). The photocurrent intensity of G/Zn-PorSEBS composites enhances with increasing the mass fractions of graphene. The 0.9% G/Zn-PorSEBS( iii ) composite shows the maximum photocurrent value of about 0.13 μA cm −2 , which is about 2 times larger than that of Zn-PorSEBS( iii ) elastomer. Previous studies indicated that this enhancement is related to the π–π interaction taking place between electron-abundant aromatic rings and conjugated surfaces of graphene, which enables the electron transfer. 34–36 This interaction can be captured by the UV-vis absorption spectra. As shown in Fig. 5a , Zn-PorSEBS elastomer exhibits an intense absorption band around 420 nm referred to as the Soret or B-band and four weak bands in the range of 500–700 nm named as the Q-bands, which are peculiar to the extended π electron structure of porphyrins. 37 The B-band of G/Zn-PorSEBS composite is observed at 426.6 nm, indicating a red shift existence after the incorporation of graphene, which can be attributed to the conjugation effect between graphene and Zn-PorSEBS. The Soret and Q-bands appearing in the UV-vis spectra both arise from the π–π* transitions of porphyrin ligands, which can be explained by the four frontier orbitals (HOMO and LUMO orbitals). 38,39 In Fig. 5b , an energy diagram of molecular orbitals shows the photoinduced electron transfer process from porphyrin ligands to graphene. Under irradiation of ultraviolet or visible light, porphyrins on the PorSEBS chains are excited, leading to the formation of the electrons and hole carriers. 40 Since the Fermi level of graphene is higher than Zn-PorSEBS ligands, the electrons induced by light are rapidly transferred to graphene, resulting in the electron–hole separation and thereby producing a photocurrent in an external circuit. 41 Moreover, incorporation of porphyrins light absorbing chromophores through a π–π conjugation with the highly conductive 2D graphene nanosheets would constitute an ideal donor–acceptor systems that can convert light signals to electrical signals rapidly and improve the charge-carrier mobilities and thereby mitigate the electron–hole recombination. 36,41 That's the main reason why the photocurrent intensity was remarkably enhanced when the graphene was introduced. Fig. 5 (a) UV-vis spectra of Zn-PorSEBS matrix and G/Zn-PorSEBS composite. (b) Molecular orbital energy diagram of photo-induced electron transfer from porphyrin to graphene. (c) SEM and (d) EDS mapping images of G/Zn-PorSEBS composite. Beyond that, the dispersion of graphene nanosheets and zinc ions are also key factors affecting the photoelectric response properties of G/Zn-PorSEBS composites. It can be seen from the SEM image of the G/Zn-PorSEBS composite ( Fig. 5c ), the graphene nanosheets are uniformly embedded in the PorSEBS matrix. Besides, EDS mapping image ( Fig. 5d ) indicates that the zinc ions disperse homogeneously throughout the matrix phase. No agglomerations can be observed."
} | 4,788 |
25289937 | PMC4188562 | pmc | 4,829 | {
"abstract": "Coral disease is one of the major causes of reef degradation. Dark Spot Syndrome (DSS) was described in the early 1990's as brown or purple amorphous areas of tissue on a coral and has since become one of the most prevalent diseases reported on Caribbean reefs. It has been identified in a number of coral species, but there is debate as to whether it is in fact the same disease in different corals. Further, it is questioned whether these macroscopic signs are in fact diagnostic of an infectious disease at all. The most commonly affected species in the Caribbean is the massive starlet coral Siderastrea siderea . We sampled this species in two locations, Dry Tortugas National Park and Virgin Islands National Park. Tissue biopsies were collected from both healthy colonies and those with dark spot lesions. Microbial-community DNA was extracted from coral samples (mucus, tissue, and skeleton), amplified using bacterial-specific primers, and applied to PhyloChip G3 microarrays to examine the bacterial diversity associated with this coral. Samples were also screened for the presence of a fungal ribotype that has recently been implicated as a causative agent of DSS in another coral species, but the amplifications were unsuccessful. S. siderea samples did not cluster consistently based on health state (i.e., normal versus dark spot). Various bacteria, including Cyanobacteria and Vibrios , were observed to have increased relative abundance in the discolored tissue, but the patterns were not consistent across all DSS samples. Overall, our findings do not support the hypothesis that DSS in S. siderea is linked to a bacterial pathogen or pathogens. This dataset provides the most comprehensive overview to date of the bacterial community associated with the scleractinian coral S. siderea .",
"conclusion": "Conclusions \n S. siderea maintains a geographically-conserved bacterial community with considerable diversity (>600 genera). There is notable overlap between the bacterial community composition of S. siderea and its sister species S. stellata at high levels of taxonomy. Our data do not support the hypothesis that DSS is a bacterial disease. There was neither a single dominant bacterial group observed in all DSS samples to indicate a primary pathogen, nor was there a community shift toward a specific or predictable secondarily opportunistic consortium. Future work should focus on determining if this discoloration indicates the same type of lesion across different coral species via histological analyses. Metagenomics also could be used to address the presence of a particular fungal ribotype.",
"introduction": "Introduction Diseases of reef-building corals are now considered a major cause of global coral reef ecosystem decline [1] , [2] . The past two decades have seen a dramatic increase in the number of reports of coral diseases, particularly in the Caribbean [3] – [5] . Most of these diseases are known or suspected to be microbial in origin [6] . Moreover, microbiology is a key part of coral biology, in the same way that human microbiome studies are revealing microbes to be a critical part of human biology [7] . \n Siderastera siderea , also known as the massive starlet coral, is a common component of Caribbean reefs, occurring from the Gulf of Mexico to South America [8] . However, there has been little attention focused on the bacterial communities associated with this coral, other than one culture-based study [9] , two clone library studies focused specifically on black band lesions [10] , [11] and a recent pyrosequencing study of a white plague-like disease [12] . Dark spot syndrome (DSS) was first reported as a discoloration observed on S. siderea , Stephanocoenia intercepta , Porites astreoides , and Montastraea cavernosa near Columbia in the early 1990's [13] . Dark spot lesions are described as purple, black, or brown discolored areas of tissue that may be circular, elongate, ring-shaped, or occur lining the coral tissue-algal boundary of an older lesion [14] – [17] ( Fig. S1 ). There is some argument in the literature as to whether to define dark spot as a disease (DSD) or a syndrome (DSS) [18] . This tissue discoloration has been linked to both physical [14] and microbiological [19] , [20] causes, leading some to suggest it may be a non-specific stress response [14] , [21] . Unlike coral diseases such as black band or white plague, dark spot lesions rarely cause whole colony mortality and result in relatively low net tissue loss [22] . Further, dark spot lesions have been observed to disappear in as little as a month [21] , [22] . It has been suggested that dark spots may signal different conditions in different coral species [20] , [22] – [24] . Given that breadth of scope, we have opted to use the more comprehensive term ‘dark spot syndrome’ (DSS), but acknowledge that it can be regarded as synonymous with DSD given the recent push to apply a broader medical definition (“any inhibition of normal function”) to coral diseases [18] , [25] . Although DSS has been identified in several species of Caribbean corals, S. siderea is the most frequently affected [14] , [24] , [26] – [29] . Previous coral disease work has shown that bacterial communities shift when their hosts are stressed (even in normally pigmented tissues [30] ). Therefore, we hypothesized that there would be a shift in the bacterial communities of DSS-affected tissues compared to healthy colonies, regardless of whether DSS was due to a general immune response or an infection. Because there appear to be multiple possible etiologies for DSS lesions, we wondered if there would be geographic differences or perhaps multiple clusters of DSS bacterial communities rather than the single diagnostic grouping we observed previously in a white plague-like disease [31] . To address these questions, we collected S. siderea samples from Florida and the Virgin Islands and used PhyloChip G3 DNA microarrays to examine the breadth of taxonomic diversity of the bacterial communities associated with both healthy and DSS-affected colonies. This is the first study to apply molecular techniques to the study of DSS in S. siderea .",
"discussion": "Discussion Opinions on the importance of DSS to reef ecology range from those considering it of limited significance due to the low mortality rates and limited tissue loss [26] to those who feel that the high frequency of occurrence and links to greater susceptibility to subsequent bleaching or disease make it a useful indicator of reef health [14] , [46] . Our initial hypothesis was that we would detect differences in the bacterial communities between healthy and DSS-affected tissues as we had between healthy and white plague-like diseased corals [31] , possibly with a geographic component. While half of the DSS samples formed a cluster at 88% similarity ( Fig. 4 ), this was not statistically significant (healthy versus DSS; ANOSIM Global R = 0.162, p = 0.067), and the remaining DSS samples grouped with healthy corals or alone. We did not detect a difference in overall bacterial diversity nor relative abundance of specific taxa (as in [12] ) between all normally pigmented S. siderea colonies and all those with DSS. Further, there were not substantial geographic differences between the Florida and Virgin Island samples. Most previous microbiological work comparing healthy and DSS-affected S. siderea has been conducted on corals from the southern Caribbean (i.e., Columbia, Venezuela) [9] , [12] , providing an opportunity for biogeographic comparisons to our data from Florida and the Virgin Islands. Bacterial diversity of healthy Siderastrea siderea \n This dataset provides the most comprehensive overview to date of the bacterial community associated with the healthy scleractinian coral S. siderea . Previous work has consisted of a culture-based study of S. siderea mucus [9] and a recent study that employed pyrosequencing [12] . Gil-Agudelo et al. [9] cultured bacteria from S. siderea mucus from both healthy and DSS-affected corals. The coral-associated bacterial isolates were identified by metabolic tests (BIOLOG) rather than 16S rRNA sequencing and consisted entirely of Gram-negative Gammaproteobacteria, which probably reflects selection bias of the GASWA culture medium employed [47] . Most metabolic groups were found in both healthy and DSS corals and corresponded to genera identified in this study (e.g., Vibrio , Klebsiella , Aeromonas ; Table S1 ), most notably those in families Enterobacteriaceae and Vibrionaceae ( Fig. 3 ). Cárdenas et al. [12] used pyrosequencing of the 16S rRNA gene to examine healthy and white plague-affected corals. Their dataset includes 378 OTUs from healthy S. siderea . These were dominated by Proteobacteria (75% of sequences), followed by less than 10% each of Firmicutes, Actinobacteria, and Bacteroidetes (in decreasing order of relative abundance) [12] . This perfectly matches the order of relative abundance of our four top phyla ( Fig. 2 ), suggesting the microarray data adequately reflects relative distributions of prominent community members. The remaining phyla detected by pyrosequencing [12] included low percentages of Chloroflexi, Fusobacteria, Verrucomicrobia, Chlorobi, Tenericutes and Cyanobacteria, all of which we also detected ( Fig. 2 ; note that Fusobacteria and Chlorobi were compiled into the ‘other’category). Phyla detected by the PhyloChip G3 in our S. siderea samples ( Fig. 2 ) that were not found by Cárdenas et al. [12] include Acidobacteria, Gemmatimonadetes, Planctomycetes and Spirochaetes. This may be due to biogeographical differences between the northern and southern Caribbean, but more likely is due to the greater depth of 16S rRNA sequences queried by the microarray [48] . Two recent clone library studies allowed us to compare S. siderea with its sister species from Brazil, S. stellata \n [49] , [50] . From these papers we identified 50 sequences, representing 119 clones, from healthy S. stellata . At the phylum level, the two species looked similar, with healthy S. stellata dominated by Proteobacteria (>80% of sequences), followed by much lower percentages of Bacteroidetes, Cyanobacteria, Actinobacteria, and Verrucomicrobia [49] , [50] . However, the two species were markedly different when examined at the family level. Families in common between the two species were Comamonadaceae, Corynebacteriaceae, Flavobacteriaceae, Rhodobacteraceae, and Rhodospirillaceae, as well as Bradyrhizobiaceae, Brucellaceae, Burkholderiaceae, Moraxellaceae, Propionibacteriaceae, Puniceicoccaceae, and Sphingomonadaceae (the latter group being present in S. siderea but summed under ‘other’ in Fig. 3 ) [49] , [50] . Families present at very low relative abundance in S. stellata but not detected in S. siderea were Cytophagaceae and Ruaniaceae [49] , [50] . While it is likely that some of the variation seen between these two coral species is due to methodological differences between the studies, we expect that some of the bacterial-community differences are genuine indicators of biogeographic and species differentiation. Bacterial diversity of DSS-affected tissues Gil-Agudelo et al. [9] found a group of bacteria metabolically similar to Vibrio carchariae present only in cultures from DSS-affected corals, but subsequent inoculation experiments failed to trigger disease signs. We detected many different Vibrio species using the PhyloChip G3, but V. carchariae was not one of them ( Table S1 ). There were several OTUs affiliated with V. campbellii , V. harveyi , and V. orientalis that had increased relative intensities in many of the Virgin Island samples compared to those from Dry Tortugas ( Fig. S2 ). Perhaps this is an indication that the Virgin Island corals are more stressed (and therefore conducive to hosting higher abundance of this opportunistic taxon). The Virgin Islands National Park hosts an order of magnitude more visitors per year than the Dry Tortugas National Park (e.g., in the 2009 sampling year, VIIS had 415,847 visitors versus 52,011 in DRTO; NPS Visitor Use Statistics https://irma.nps.gov/Stats ). Further, while a large proportion of the VIIS visitors will reach the easily accessible Hawknest Bay, only a small percentage of the visitors to DRTO reach Loggerhead Key since most day trips visit Garden Key alone. The only previous study to employ molecular techniques in examining DSS-affected corals found significant differences between the bacterial communities in healthy versus affected colonies of Stephanocoenia intersepta , both by DGGE and clone library analyses [40] . In contrast, our data did not indicate a significant difference between healthy and DSS-affected corals or between corals from different geographic locations ( Figs. 2 , 3 , 4 ). This could mean that DSS represents a different condition in St. intersepta than in S. siderea , as has been previously suggested due to different lesion morphologies [20] , [23] . It could also be due to the broader taxonomic coverage achieved using the PhyloChip G3 microarray to examine the coral-associated bacterial communities (4,978 OTUs versus 50 clone library sequences). Specifically, Sweet et al. [40] found an increase in Cyanobacteria and Actinobacteria associated with all DSS-affected samples and that the Cyanobacteria were dominated by a species of Oscillatoria . The genus Oscillatoria (reference sequence is Pseudocillatoria coralii ) is represented by two OTUs out of the 4,978 OTUs shared by all of our samples and was detected in half of our DSS samples (DRTOSSD08, VIISSSD06, VIISSSD09, and VIISSSD10) but none of our healthy (i.e., normally pigmented) samples ( Fig. S3 ). In addition, there were two other cyanobacterial OTUs (53258 – Phormidium and 53730 – Leptolyngbya ) that had higher relative intensities in a subset of the DSS samples (VIISSSD06, VIISSSD07, VIISSSD09, VIISSSD10) but were present in both healthy and DSS samples ( Fig. S4 ). All four of these cyanobacterial genera have been associated with black band disease (BBD) [44] , [51] , [52] and so clearly have some capacity to negatively impact corals in certain conditions. Unlike BBD, there was no indication of increased sulfate-reducing or sulfur-oxidizing bacterial populations in our DSS samples. Only two OTUs were unique to DSS and present in more than 50% of (>4) DSS samples in our study: 53656 representing Chroococcidiopsis , a genus of Cyanobacteria in the class Oscillatoriophycideae and OTU 7560 representing an unclassified Vibrio . Sweet et al. [40] also found two Vibrio species that were only present in DSS-tissues, but concluded that, like V. carchariae \n [9] , they were probably not directly involved as pathogens. Further, they also detected Oscillatoria sp. in lower abundance in one of their healthy samples, leading them to conclude that this cyanobacterium may contribute to the dark pigmentation of DSS in St. intersepta but is unlikely to be a pathogenic cause [40] . Our S. siderea data corroborate these conclusions ( Figs. S2 , S3 , S4 ). Another aspect of the St. intersepta study was that different size DSS lesions were sampled and a difference was found in the bacterial communities between small (1–2 cm) and larger (5–10 cm, >10 cm) spots, suggesting a community shift as the lesion aged [40] . Unfortunately, all of our DSS areas would be categorized as small, although many were collected from the edge of a much larger older lesion (i.e., central dead area with algal recruitment; Fig. S1 ). This sort of purpling along the edge of an older lesion (e.g., one caused by black band disease) has been described as Dark Spot Syndrome Type II [14] . Fungi Previous histological work on S. siderea colonies from the Bahamas, Little Cayman, Florida Keys, and Puerto Rico described unidentified endolithic fungal cells associated with dark spot lesions [53] , [54] . Sweet et al.'s [40] recent discovery of a fungal ribotype consistently associated with DSS in St. intersepta that was genetically similar to a fungal plant pathogen ( Rhytisma acerinum ) that causes ‘tar spot’ disease adds some molecular evidence that DSS may be a fungal disease. Unfortunately, we were unable to amplify fungal 18S rRNA from our S. siderea samples due to the ITS primers being overwhelmed by coral 18S rRNA. Further, due to the collection and processing methods we followed, it was not possible to examine any of the samples by histology to look for physical signs of fungal infection. Given the uncertainty of whether DSS is a disease or a non-specific stress response to any physical, chemical, or microbiological insult [55] , [56] , as well as being unclear if these signs represent different conditions in different coral species [20] , it is imperative that future studies combine histology with microbiology to link potential causal agents to a specific pathology at the cellular level [57] . Future studies in the vein of Closek et al. [58] may shed more light on what the coral host is doing during these occurrences, providing greater insight into coral immune responses."
} | 4,307 |
23741364 | PMC3669381 | pmc | 4,831 | {
"abstract": "Social animals can use both social and private information to guide decision making. While social information can be relatively economical to acquire, it can lead to maladaptive information cascades if attention to environmental cues is supplanted by unconditional copying. Ants frequently employ pheromone trails, a form of social information, to guide collective processes, and this can include consensus decisions made when choosing a place to live. In this study, I examine how house-hunting ants balance social and private information when these information sources conflict to different degrees. Social information, in the form of pre-established pheromone trails, strongly influenced the decision process in choices between equivalent nests, and lead to a reduced relocation time. When trails lead to non-preferred types of nest, however, social information had less influence when this preference was weak and no influence when the preference was strong. These results suggest that social information is vetted against private information during the house-hunting process in this species. Private information is favoured in cases of conflict and this may help insure colonies against costly wrong decisions.",
"introduction": "Introduction Being well informed can mean the difference between a good and bad decision. Animals frequently make fitness-critical decisions based on information acquired through individual experience (private information) and via signals or cues from other animals (socially acquired information) [1] . Private information, while generally considered more reliable, can be costly or difficult to acquire [1] , [2] . Social information, on the other hand, can be relatively cheap to obtain [3] , [4] and, under the right circumstances, a more than adequate substitute [5] , [6] . However, social information may also be outdated or unreliable, and a dependence on copying can lead to negative outcomes via information cascades [2] , [7] , [8] . Information cascades arise when individuals copy the behaviour of others without themselves assessing the environmental cues on which the behaviour was based, and can lead to sub-optimal outcomes when the individual copied chooses poorly [2] , [9] . It is thus not surprising that many animals weight social or private information differently depending on the environmental context [10] – [14] . It remains unclear, however, in which context one form of information should be favoured over the other [7] , [11] . Social insects exhibit some of the most sophisticated systems of information exchange, from the complex chemical messages encoded in chemical trails to the dance language of honey bees. These systems perform critical roles in the optimisation of collective processes [15] – [18] . Honey bees, for example, communicate the location of resources to other potential foragers via the waggle dance, varying the number of dance circuits with the quality of the resource [19] . Many other social insects coordinate foraging and colony movements using social information in the form of chemical (pheromone) trails [20] – [22] . Ants in particular rely on pheromone trails to recruit to food sources, and adaptively deploy these chemical signals based on the quality of the target [23] , [24] . However, while both dance language and pheromone trails provide insect colonies with a means to effectively exploit available resources without the need for central control, there are notable differences in the flexibility of the two systems. Individuals interact directly to share information and recruitment is linear in honey bees, a dynamic communication system which enables colonies to switch sites rapidly in the case of resource depletion or new discoveries [15] , [25] . Pheromone trails, on the other hand, are subject to momentum and runaway positive feedback because communication of information is indirect and recruitment is nonlinear [15] , [25] , [26] . As a result, once a trail is established, it may be difficult to switch targets [26] – [28] . This represents a form of information cascade [9] as although individuals continue to make independent assessments, the rapid amplification of initial choices (including poor ones) means that social information can soon overwhelm any dissent arising from private information, leading to potentially sub-optimal outcomes at the group level. In addition to holding an important role in foraging, information exchange is critical to the process of finding a new home. Social insects relocate to a new nest when the present site becomes unsuitable or during the process of colony fission [29] , and this process has been intensively studied in Temnothorax ants and honey bees [30] , [31] . New sites are selected via a process of consensus decision making, in which a small proportion of the colony decide collectively from among candidate sites [32] . Scouts that have visited a suitable site share this information via waggle dances (honey bees) or by leading other scouts to the site via tandem running ( Temnothorax ) and, once a critical number of individuals (‘quorum’) is in favour of a particular site, the process shifts rapidly to that of relocation. A collective response emerges as a product of numerous individual decisions, each of which is made based on the private and social information available to that individual. In this manner, colonies are able to make accurate choices among sites of varying quality while maintaining colony integrity [31] , [32] . Unlike other social insects so far studied (though see [33] ), the small-colony ant Myrmecina nipponica relies on pheromone trails to navigate during house hunting [34] . Trails are laid by scouts that have found a suitable new nest site, leading to the recruitment of other nest-mate scouts and, once an apparently quorum-based threshold is reached, a switch to brood transport [34] . As in other species, scouts do not individually assess all candidate sites, and rely heavily on social information. However, as social information takes the form of a pheromone trail, consensus decisions made when selecting a new nest could be subject to information cascades. The initial sequence of events is decisive in this regard: if scouts first locate an acceptable, but sub-optimal nest and commence laying trails, subsequent scouts may be drawn to the same site over potentially superior sites. As pheromone trail strength increases, its influence over subsequent behaviour increases disproportionately [26] , and the probability that scouts will locate other sites diminishes rapidly. Hence, if the initial sequence of events is sufficiently biased, an acceptable, but sub-optimal nest may eventually be selected. The cost of such wrong decisions during house hunting may be higher than during foraging, because all colony members (including queen and brood) are exposed during relocation and additional costs associated with the construction or modification of a new nest may be incurred. We might therefore expect private and social information to be weighted differently during house hunting. However, while studies have examined the use of conflicting social and private information in the context of ant foraging [35] – [38] , and the possession of prior information is known to influence nest site selection [39] , [40] , no study to date has examined the effect of information conflict during house hunting (though see [41] ). Furthermore, whereas information cascades are thought to explain a wide range of collective behaviours in humans, relatively little attention has been invested in the study of this concept in animal societies [27] , [42] , [43] . In this study, I examine how social and private information contribute to the consensus decision process during house-hunting in the ant M. nipponica , and assess whether the use of pheromone trails in this species can lead to information cascades during nest site selection.",
"discussion": "Discussion House-hunting M. nipponica colonies were strongly influenced by existing pheromone trails in choices between equivalent nests, and able to exploit this social information to reduce relocation time. In contrast, trails had no influence on choice when they lead to strongly non-preferred (dry) nests, while in weak preference trials, a higher proportion of colonies again selected non-preferred nests in the presence of trails. The selection of a new nest site is an emergent consequence of numerous semi-independent individual choices based on social and private information. In the main experiment social information (as represented by a fully established trail) was constant, whereas private information obtained by ants visiting non-preferred nests varied in the degree to which it conflicted with social information, from nil (in controls) to high (in strong preference trials). House hunting ants and bees visiting a given site assess its quality and either decide to accept it and begin recruiting to the site, or otherwise continue searching [31] , [47] – [49] . This decision is independent for each individual in that scouts assess the site for themselves, but dependent on other colony members as scouts are more likely to visit sites already flagged by social information [8] . In this study, when private and social information were not in conflict, social information in the form of established trails influenced nest site selection. Trails recruited ants to the site and, on finding the site suitable, these ants presumably reinforced the trail, eventually giving rise to a consensus response. In circumstances where social information conflicted with private information, however, social information was less effective in influencing the colony level response. In strong preference trials, ants eschewing highly non-preferred (dry) sites were able to establish a competitive trail to the alternative site, eventually leading to an optimal colony level response in all twelve trials despite the established trail. In weak preference trials, a higher proportion of colonies selected the non-preferred nest in the presence of trails, though this effect was somewhat muted relative to that observed in controls. This suggests that social information was in some cases sufficient to bias nest site selection despite contradictory private information, presumably because weakly non-preferred nests (light nests and wide entrance nests) had sufficient support among scouts to give rise to a consensus response before a suitably competitive trail could be developed to the alternative site. That is, in weak preference trials the influence of pre-established social information was sufficient to overcome the low level of dissent arising from private information and influence nest choice. This represents a form of information cascade, as although copying is not unconditional, a sub-optimal group level outcome can arise via positive feedback following initial poor choices (represented here by the established trail). Information cascades are thought to explain a range of rapid, broad-scale emulative responses observed in humans [2] , [9] , and the available evidence suggests this may also be the case in animal societies [27] , [28] , [42] , [43] . The relative cost and reliability of private and social information is thought to regulate the conditional weighting of one over the other [1] , [3] , [4] . The ‘costly information hypothesis’ suggests that social information should be preferred when private information is difficult to obtain [3] , [4] , and is supported by empirical studies in vertebrates [50] , [51] . In house-hunting Myrmecina , the cost of acquiring private information is almost certainly high: scouts move slowly and thus finding a new home takes time, during which scouts risk desiccation, predation, and getting lost, and the colony as a whole is exposed. In addition, at least two other factors suggest social information should be favoured in this context. Firstly, social information in the form of pheromone trails can be considered reliable because it is provided by nest-mates with shared interests and the ephemeral nature of trails means that information is up to date. Secondly, Rendell et al . [5] , [11] suggest that copying should be adaptive provided that the individuals which are copied behave rationally and select the best option. Ants deploy pheromone trails in proportion to the quality of the target both in foraging [24] , [26] and in nest site selection [52] , [53] , and thus the very existence of a trail implies that an adaptive choice has been made. These arguments suggest that social information should be highly valued in relocating M. nipponica and, while this supposition is perhaps reflected in the strong influence of trails on choices between equivalent nests, the reduced influence of trails leading to non-preferred nests suggests that social information is not blindly accepted, but vetted against private information before a decision is made. These data support previous studies indicating that in the event of conflict ants defer to private information [36] , [38] , [54] , [55] . Stroeymeyt et al. \n [41] showed that house hunting Temnothorax ants relied on prior experience (navigational memory) when chemical markings in their laboratory arena were experimentally reversed. This suggests a preference for private information over social information, though ants of this genus rely on tandem-running for recruitment during consensus decision making [48] and the role of chemical markings differs to that in Myrmecina \n [33] . Maintenance of some degree of independence in collective decisions such as this is perhaps not surprising as an individual component to choice is thought to be integral to an effective quorum response [32] , and this vetting process may also buffer colonies against negative information cascades [8] . Studies of ants have also revealed the use of negative feedback mechanisms which can curb potential runaway positive feedback associated with the use of pheromone trails [56] – [58] . Vetting and damping systems such as these may be common in species that employ feedback mechanisms subject to information cascades, particularly when negative outcomes have potentially high fitness consequences. Conditional use of information sources appears to be common in animal learning, and present evidence suggests it is probably widespread both taxonomically (reviews for fish [12] , mammals [59] , and birds [60] ) and in terms of the context in which is it employed [7] , [12] . Ants have been shown to use social and private information conditionally when foraging [10] , [35] , [61] and, combined with the present study, this suggests that at least in ants (i) the use of social and private information is not mutually exclusive and (ii) social information is not blindly accepted but vetted against private information, with deference to the latter in circumstances of conflict. While data presented here can be interpreted to largely support the costly information hypothesis, they suggest that even in situations where copying occurs, asocial learning is maintained and may function as an insurance mechanism against negative information cascades. The extent to which similar mechanisms can be found in other species is at present unknown, and indeed, there is a paucity of empirical experiments investigating the potential for maladaptive decisions arising from the use of social information [7] . Rieucau and Giraldeau [42] demonstrated that nutmeg manikins ( Lonchura punctulata ) could be induced to make maladaptive decisions when strong social information apparently lead them to disregarded personal information in a manner consistent with information cascades. Maladaptive choices connected with the use of social information have also been implicated in fish [62] and young birds [63] . Nonetheless, while information cascades are purported to explain a diverse range of human collective responses such as consumer fads and crowd panic behaviour [9] and have the support of laboratory studies [64] , surprisingly few corresponding investigations have been undertaken in other animals. The growing body of literature on the subject demonstrates the importance of social information in the adaptive behaviour of animals, and future studies should investigate the potential for information cascades to influence conditional information use in other species."
} | 4,129 |
38524498 | PMC10955578 | pmc | 4,833 | {
"abstract": "This review provides a comprehensive and accessible\nliterature\nreview on the integration of nanoparticles into biolubricants to enhance\nwear and friction regulation, thus improving the overall lubricated\nsystem performance. Nanotechnology has significantly impacted various\nindustries, particularly in lubrication. Nanobiolubricants offer promising\navenues for enhancing tribological properties. This review focuses\non oxide nanoparticles, such as zinc oxide (ZnO), aluminum oxide (Al 2 O 3 ), copper oxide (CuO), titanium dioxide (TiO 2 ), zirconium dioxide (ZrO 2 ), and graphene oxide\n(GO) nanoparticles, for their ability to enhance lubricant performance.\nThe impact of nanoparticle concentration on biolubricant properties,\nincluding viscosity, viscosity index, flash point temperature, and\npour point temperature, is analyzed. The review also addresses potential\nobstacles and limitations in nanoparticle incorporation, aiming to\npropose effective strategies for maximizing their benefits. The findings\nunderscore the potential of nanobiolubricants to improve operational\nefficiency and component lifespan. This review aims to provide valuable\ninsights for researchers, engineers, and professionals in exploring\nand leveraging nanotechnology’s potential in the lubrication\nindustry. This review paper explores the basics of tribology along\nwith its significance, green principles, mechanisms, and energy savings\nbecause of friction, wear, and lubrication. Condition monitoring techniques\nare also explored to achieve brief knowledge about maintaining reliability\nand safety of the industrial components. Recent advances in tribology\nincluding superconductivity, biotribology, high-temperature tribology,\ntribological simulation, hybrid polymer composite’s tribology,\nand cryogenic tribology are investigated, which gives a thorough idea\nabout the subject.",
"conclusion": "8 Conclusions This paper presents an\ninclusive review on the use of biolubricants\nalong with oxide nanoparticles. The tribological properties like wear\nscar diameter (WSD), coefficient of friction (COF), viscosity, and\nviscosity index (VI) were analyzed. It can be concluded that incorporation\nof oxide nanoparticles in biolubricants in an optimum quantity is\na good alternative for ameliorating lubrication performance. Also,\nit can be stated from literature that nanobiolubricants have the potential\nto provide better lubricating properties than conventionally used\nmineral-based oils. For example, when coconut oil was investigated\nusing CuO NPs, it was found that wear volume and COF both decrease\nby 37% and 93.75%, respectively. Further, different additives which\nwere traditionally used for enhancement of properties of base oil\nwere being analyzed. For example, papaya leaf extract is a biobased\norganic corrosion inhibitor for copper surface in sulfuric acid. Principles\nof green tribology were being studied, which are helpful for conserving\nenergy through tribological consequences. Various condition monitoring\ntechniques were explored in order to provide brief knowledge on maintenance\nof the machine to avoid unusual breakdowns. Recent advancements in\ntribology were being studied, which shows that there has been rapid\ngrowth in the field of tribology. Superlubricity with diamond-like\ncarbon (DLC) coatings and many 2D materials have a scope of research\nand tend to have lower COF. The following are the noteworthy points\nthat can be concluded from this review paper: The use of biolubricant as a fuel in vehicles is a bit\nchallenging, but it can be efficiently used as gear oil, engine oil,\ncompressor oil, brake oil, etc. in various commercial applications. As the VI of nanobiolubricants gets increased,\nthis\ncould improve the economy as the consumption of fuel gets decreased. On adding nanoparticles into the base lubricant,\nviscosity\ngets improved, which leads to the improvement in wear and frictional\nproperties because there is no direct contact of surface asperities. Biolubricants have the potential to decrease\nthe environmental\nimpact, as these are very easily degradable and emit no harmful pollutants.",
"introduction": "1 Introduction Tribology is basically\na study of the relative motion of interacting\nsurfaces, remarkably their friction, wear, and lubrication. 1 , 2 In many applications, particular oils are used to control and manage\nfriction and wear by lubricating the contact surfaces like bearings,\ngears, seals, etc. 3 − 7 The vegetable oils are favored over mineral oil as a lubricating\nbase oil due to their excessive biodegradability, renewability, and\nlow toxicity. 8 , 9 Biolubricants synthesized from\nvegetable oils are obtained from plant vegetables, fruits, seeds,\nand leaves. These are effectively used in pharmaceutical products,\ncosmetics, soaps, and shampoos. These plant-based bio-oils are composed\nof triglycerides, phytosterols, natural pigments, phospholipids, and\nfatty acids. 10 A lubricant provides a defensive\nfilm between the contacting surfaces to avoid the friction and wear\nto some extent. 11 Figure 1 shows the basic classification of lubricating\noils. Figure 1 Classification of lubricating oil. 12 − 14 Apart from solid and liquid lubricants that are\nbeing used rigorously,\nnow-a-days there are intelligent lubricant materials that are being\nused. Such materials adjust themselves in the changing environment\nand manifest their functions according to the change that occurred. 15 , 16 A good lubricant must contain good thermal stability so that it\ncan resist heat which develops during mitigation of contacting surfaces.\nAlso, it should possess a higher Viscosity Index so the lubricant\nwill affect less with variation of temperature. Besides this, a lubricant\nalso must possess a low freezing point and high boiling point, and\nit should be capable of resisting the oxidation process. 17 Tribological performance is usually associated\nwith lubricity. Lubricity is the formation of a layer of lubricant\non the contacting sliding surface. The thickness of this tribo film\nshould be optimum always. High lubricity lowers friction and energy\nloss by reducing direct contact between surface asperities. 18 , 19 In recent years, there has been a constant hunt for sustainable\nand environmentally friendly solutions for lubrication, and it has\ndriven extensive research into the domain of biolubricants. These\nbiodegradable lubricants which are derived from renewable resources\npossess certain properties, because of which these are considered\nas potential candidates for industrial applications. However, it\nis a key challenge for the researchers to enhance their tribological\nperformance to obtain their full potential. There are several\nstrategies among those, and incorporation of\nmetal oxide nanoparticles (NPs) into the biolubricants has emerged\nas a noteworthy approach. Metal oxide nanoparticles own unique physiochemical\nproperties and high surface area, and eventually they have accumulated\nsignificant attention as additives to enhance the lubricating performance\nof biolubricants. This review paper gives a brief idea of the\ncurrent state of research\non the impact of metal oxide nanoparticles on the tribological properties\nof biolubricants. The fundamental mechanisms underlying the interactions\nbetween nanoparticles and biolubricants were explored along with the\ntribological behavior of metal oxide nanoparticles like zinc oxide,\ntitanium dioxide, silica, etc. in biolubricants."
} | 1,851 |
35528092 | PMC9074719 | pmc | 4,836 | {
"abstract": "A productive and novel method for fabricating stretchable transparent heaters with recognised thermochromic properties using commercially available thermochromic ink (TM-55-blue) and silver nanowire (AgNW)-coated polydimethylsiloxane (PDMS) is proposed. Lower resistance, elevated heat generation, and higher transparencies were the expected essential prerequisites for the fabrication of items such as smart windows and window defrosters. AgNW-coated PDMS (hereafter PH devices) satisfied the essential prerequisites but did not produce sufficient color change. In addition to the appreciable electrical and optical characteristics and mechanical robustness, observable color changes represent a critical factor in effortless temperature monitoring by the heating device. Blending TM-55-blue thermochromic ink with PDMS (PBH device) improves the heating rate and color transformation and promotes the ultralow response time appreciably. More notably, it produces a visible transformation from blue to colorless. Color changes visible to the naked eye, ultralow response time, and heating rate represent valuable features for deploying the PBH devices as window defrosters and in smart window applications.",
"conclusion": "4. Conclusion We have presented a stretchable heater with desirable credentials such as high transparency, favorable photochromic properties, high stability, and rapid heating rate. Comparing our novel PBH device with conventional PH devices evidences the superiority of the novel PBH device. The dependence of nanowire network density on sheet resistance and transmittance was studied and optimized to produce an energy-efficient transparent stretchable heater. In addition, an ultralow response time of 20 s further elevated the utility of the fabricated heating device. The color transition was noticeable even at an extremely low temperature (31 °C), meaning that our PBH device is able to compete with other smart window applications. Considering the heating stability, homogeneity, robustness, and high transparency of our PBH device, it is a promising candidate for use in the design of outdoor displays, defoggers, and smart windows; its use may also be extrapolated for the development of energy-efficient transparent electrodes used in LEDs, OFETs, solar cells, touchscreens, and other such devices.",
"introduction": "1. Introduction Stretchable heaters have represented a field of interest over the past few years due to their applicability in various fields, such as in defrosters, smart windows, thermotherapy pads, smart garments, and personal health care equipment. Researchers have endeavored to fabricate devices with both high transparency and high conductivity. ITO has been conquering arenas relating to equipment such as solar cells, 1 light emitting devices, 2 field emission transistors, 3 photodetectors, 4 supercapacitors, 5 transparent heaters, 6 and gas sensors 7,8 due to its low sheet resistance and high transparency. ITO possesses high brittleness that limits its applicability in flexible and stretchable electronics. It also possesses certain limitations such as slow heating and cooling rates attributable to lower thermal conductivity and limited thermal stability. 9–11 In addition, the increasing cost of the rare element indium incentivizes researchers to search for alternatives to ITO. Carbon-based materials such as carbon nanotubes, 12,13 and graphene 14,15 were utilized as transparent conductive electrodes in the fabrication of transparent heaters. The limitation associated with carbon-based electrodes is their high sheet resistance. Lower sheet resistant heaters can stem the substrate to undergo Joule heating, elevating temperature maximally with low input voltage. Conductive polymer offers superior performance because of its higher conductivity, light weight, and solution processability, for example. Post-treatment of PEDOT PSS, such as acid treatment and chemical reduction, has significant effects in improving thermoelectric properties. 16 This post-treatment hampers the material's applicability on an industrial scale, owing to corrosive treatment, time management, and other troublesome factors. Metallic nanowires have gained attention due to characteristics such as their high conductivity, high transmittance, reduced sheet resistance, robustness, large-scale synthesis, and shapeability. Metal nanowire-based heaters present faster thermal responses even under lower voltages. In particular, AgNWs possess superior properties such as excellent flexibility, stretchability, high conductivity, high transparency, easily tunable nanowire synthesis, and relative stability in terms of oxidation. 17–19 For generating nanowire networks, the spray-coating method possesses remarkable advantages compared with other techniques. Spray coating bestows several benefits such as simplicity, time saving, homogeneity in distribution, applicability to 3D-structured materials, flexible operation, waste minimization of functional inks and low-cost techniques for utilization in large-scale processes. 20–22 Silver nanowire (AgNW) network density can be adjusted by varying the spray-coating duration, which controls the optoelectronic properties of the film. 20,23 Fabricating functional heaters still remains challenging in real-time applications because of flexibility and stretchability restrictions. For smart heaters to be utilized under adverse conditions, the heaters' characteristics must include stretchability, homogeneous heat generation, strain tolerance, low hysteresis, endurance, and repeatability. Hong and colleagues fabricated stretchable and transparent heaters using laser ablation patterning of AgNW on a PDMS substrate, and conferred advantages such as high flexibility, stretchability, and transparency. 24 An and colleagues utilized CuZr metallic glasses in the form of metallic nanotroughs for fabricating stretchable heaters, resulting in considerable stretchability owing to the high elastic limit of CuZr metallic glasses. 25 Huang et al. designed a brilliant flexible thermochromic smart window with good mechanical stability towards various deformations. 26 To date, only a few studies have reported on the inclusion of thermochromic ink onto stretchable heaters. Integration of thermochromic ink may enable better results in fabricating smart heaters, smart windows and window defrosters. More recently, our group Cu/Ag core/shell nanofibrous films exhibiting the ultra-low sheet resistance, improved stability and robustness to find the potential application in flexible electronics. 27 In this study, we fabricated two stretchable heaters, namely a conventional PDMS heating (PH) device and a novel TC-PDMS blended heating (PBH) device by employing a simple spray-coating technique. Fig. 1 and S1 † illustrate the fabrication process of the PBH and PH heating devices. The detailed fabrication processes for the PBH and PH are discussed in the experimental section. PDMS was chosen as a substrate because of its inherent flexibility, elasticity, biocompatibility, and nontoxicity, for example. The addition of a crosslinker expedites the bond formation between PDMS and thermochromic ink (TM-55-blue) to form thermochromic ink incorporated PDMS substrate (TC-PDMS). Thermochromic ink (TM-55-blue) was chosen on the basis of considerations such as its low transition temperature (31 °C), appropriateness toward various substrates, and high heat and UV resistance. Commercially available AgNWs were spray-coated over the TC-PDMS substrate. AgNWs possess considerably lower sheet resistance, which directly reduces the thermal resistance for the effective transduction of electrical energy into heat energy, even at much lower applied potentials. Through this fabrication technique, we integrated both heating and thermochromism into our generated novel PBH device. Through comparing PH devices and PBH devices, it was revealed that the PBH exhibits high heating ability and retains considerable stability under various applied biases. The spray-coating time for AgNWs and AgNW density influence the sheet resistance and transmittance of the fabricated PH and PBH devices. The ultrafast heating rate and the thermochromism exhibited by fabricated PBH devices verify their credible potential in window defrosters and smart window applications. This research will remain instrumental for designing safety-assisting devices, smart windows, and defrosters. Fig. 1 Schematic representing the fabrication of a heater blending polydimethylsiloxane with TM-55-blue thermochromic ink (PBH heater) through a drop-casting process and spray-coating technique (inset figure demonstrates the thermochromic ability of the fabricated PBH device).",
"discussion": "3. Results and discussion 3.1 Surface characteristics of pristine and thermochromic ink-blended PDMS Fig. S2 † depicts the SEM images of pristine PDMS and thermochromic ink-blended PDMS (TC-PDMS). The SEM image represents a smooth surface, which has a predominant function in accommodating AgNWs. Pristine PDMS and TC-PDMS cross-sectional SEM images illustrate the dispersivity of PDMS (Fig. S3a and b † ). A comparison between pristine PDMS with TC-PDMS evinced that dispersivity was elevated to a greater extent in the TC-PDMS substrate (Fig. S3b † ). Emerging dispersivity and homogeneity in the TC-PDMS substrate might have contributed to the smooth surface morphology of the substrate, in accordance with the top-view FE-SEM image (Fig. S2b † ). Pristine PDMS and TC-PDMS substrates were plasma-treated to clean their surfaces, imparting hydrophilicity to the substrates; this is considered to be a promising factor for AgNW spray coating. AgNWs was chosen on the basis of several promising advantages, including excellent intrinsic stretchability, high conductivity, high transparency, large-scale solution processability, and easy tunability. 3.2 Optimization of spray-coating time A silver nanowire suspension solution was used to spray coat the pristine PDMS for three different durations: 15 s (Device A), 30 s (Device B), and 60 s (Device C). Spray coating minimized the waste of functional inks in generating nanowires and made possible the creation of interpenetrating ultralong nanowires. Increasing the spray-coating duration is evidently favorable for the formation of dense nanowire networks. FE-SEM analyses of the prepared pristine PDMS-embedded AgNWs are illustrated in Fig. 2a–c , and these images confirm the expected trend. SEM imaging revealed that longer spraying times result in denser nanowire network formation ( Fig. 2c ). Higher transmittance, lower sheet resistance, and significantly low surface roughness have been achieved by exploiting spray-coating techniques. 14,21,28 Fig. 2 (a–c) Polydimethylsiloxane (PDMS) substrate spray-coated for (a) 15 s, (b) 30 s, and (c) 60 s; (d–f) thermochromic ink-blended PDMS substrate spray-coated for (d) 15 s, (e) 30 s, and (f) 60 s. TC-PDMS substrates were spray-coated with AgNWs for three different durations: 15 s (Device D), 30 s (Device E), and 60 s (Device F), as shown in Fig. 2d–f . The spray-coating method permitted similar surface AgNW embedment on the TC-PDMS substrate (as observed for pristine PDMS). Similar surface observation features strongly corroborate that the TC-PDMS substrate is suitable for fabricating the heating device framework in place of conventional pristine PDMS, which lacks sufficient photochromism. 3.3 Electrical and optical performance of AgNW-coated TC-PDMS We further studied the electrical and optical performances of the AgNW-coated TC-PDMS substrates (Devices D, E, and F), portrayed in Fig. 3 . Data were furnished with sheet resistance, and the transmittance plot demonstrates the utility of the thermochromic heater. The electrical sheet resistance measurements were calculated under the various applied strains (10%, 20%, and 30%) for Devices D, E, and F ( Fig. 3a ). The sheet resistance increases by approximately 80 Ω sq −1 when Device D undergoes tensile straining to 30%. On relaxation, the resistance returns to its initial value without any significant change. Device E was also able to restore the sheet resistance after relaxing from strain. Device E's sheet resistance inclination was relatively low compared with that of Device D: the elevation in sheet resistance was approximately 20 Ω sq −1 . Device F conserved almost the same sheet resistance over the entire 30% elongation. This lowered hysteresis suggests reliable electrical performance by the AgNW-coated TC-PDMS even for a 30% stretched state. Fig. 3 (a) Electrical property and (b) optical transparency of the polydimethylsiloxane (PDMS) based fabricated heater after different spray-coating times, (c) sheet resistance versus strain, and (d) sheet resistance stability of the 15 s spray-coated TC-PDMS. Transmittance variation over the entire visible region on Devices D, E, and F feature in Fig. 3b . Wide and flat transmittance over a broad range is desirable for light-emitting-device and solar-cell-panel fabrication. Device D exhibits higher transmittance compared with Devices E and F. Increasing network density evidently causes a decrease in transmittance. This outcome agrees with other reports. 21,27,29 Device D may be considered the most suitable heating device (compared with Devices E and F) because of its higher transparency and considerably lower sheet resistance. Verifying resistance stability of the device under strain is essential. Sheet resistances for Devices D, E, and F were analyzed under strains of 10%, 20%, and 30% ( Fig. 3c ). The sheet resistance increased from ∼70 Ω sq −1 to ∼170 Ω sq −1 (more than twofold increment). The other two devices, Devices E and F, exhibited comparatively lower sheet resistance. Sheet resistance stability was examined by measuring the sheet resistance for each 20 cycles and is plotted in a graph ( Fig. 3d ). The observed results for sheet resistance portray the stability of the fabricated PBH device. DC voltage was applied to the copper terminals of the fabricated device using the alligator clips through the copper terminals. On applying the potential, transparencies were achieved following the attainment of transition temperature (31 °C). PBH changes from blue to transparent when lower voltages of 5 V are applied ( Fig. 4a ), in accordance with transmittance and absorbance peaks after voltage application ( Fig. 4b–e ). Devices D was initially fabricated with a TC-PDMS blending ratio of 20 mg/3 g (TC ink : PDMS). Devices G and H were also fabricated with different ratios of thermochromic TM 55 blue ink : PDMS (30 mg/3 g and 50 mg/3 g). Fig. S4 † shows photographs of the thermochromic heaters: Device D, Device G, and Device H. The color of the thermochromic heater intensified when the concentration of thermochromic ink in the blended films was increased. Fig. 4 (a) Thermochromic response exhibited by the thermochromic ink and silver nanowire-coated polydimethylsiloxane device after the application of 5 V, (b and c) transmittance and absorbance plot of thermochromic ink-blended polydimethylsiloxane (TC-PDMS) before applying potential of 5 V, and (d and e) transmittance and absorbance plot of TC-PDMS after applying potential of 5 V. Optical properties were measured on the PBH device before ( Fig. 4b and c ) and after the potential was applied ( Fig. 4d and e ) using UV-visible spectroscopy. Absorption and transmittance peaks exhibited by Devices D, G, and H before the application of potential complies with the existence of blue color. When voltage was applied, the absorption was almost zero ( Fig. 4e ), and transmittance was observed to be approximately 90% ( Fig. 4d ) (constant over the entire visible spectrum), which was considered to be the primary criterion for use in applications such as window defrosters and smart windows. The results in Fig. 4d suggest that among Devices D, G, and H, Device D constitutes the best proposal, owing to its suitable transparency (90%). Such visual color changes have been utilized in several applications, including chromogenic sensors and heavy metal detectors, which has been discussed in numerous studies. Our group fabricated such a system to detect heavy metals such as Cu, Hg, and Fe. 30–32 3.4 Heating performance of the fabricated PBH device Devices D, E, and F are of great interest because of their lowered sheet resistances and low hysteresis. Considerable attention has been given by various groups to transparent heaters, and their results rely upon the reduced sheet resistance. We therefore applied 10 V across the copper terminals and monitored the heating performance of Devices D, E, and F. Generally, many studies have analyzed transparent heaters based on the Joule's law of heating. According to Joule's law, heaters' temperatures increase considerably by restricting the sheet resistance. In our case, we follow the same trend as observed in other literatures. As it is evidenced from lowered sheet resistance observed ultimately contributed to the maximum saturation temperature within a shorter response time ( Fig. 5a ). Device F exhibited a maximum temperature of approximately 110 °C which compares favorably with that of Device E (approximately 60 °C) and D (approximately 50 °C). Device D and E exhibited limited heating efficiency because of the internal resistance between the AgNW junctions. Fig. 5 (a and b) PBH device heating performance as a function of spray-coating time, (c) temperature versus time under a constant applied voltage (2–10 V), (d) Device F temperature change versus applied voltage, and (e) IR camera image of Device F on applying 2 V, 5 V, and 10 V biases. The existence of junctions is apparent when the density of AgNWs is increased. On stretching, the cracks developing on 30 s and 60 s spray-coated substrates were visible with the naked eye (Fig. S5 † ) because of the higher AgNW density and the interconnected AgNW ruptures that occur more predominantly. On the other hand, control PDMS exhibited lower heating rate (Fig. S6 † ) whereas TC-PDMS device exhibit relatively higher heating rate because of the following two reasons: the first cause for such increase in heating rate was due to the enhanced compatibility between the TC-PDMS and AgNWs. A comparison between pristine PDMS with TC-PDMS evinced that dispersivity was elevated to a greater extent in the TC-PDMS substrate (Fig. S3b † ). The second cause is that the dispersion facilitates the better AgNWs spray distribution thereby producing the enhanced heating rate. The results in Fig. 5b suggest that the temperature rise on an increase in the potential from 2 V to 5 V was nearly 15 °C for Device F, whereas it was less than 5 °C for Devices E and E. On increasing the voltage to 10 V, we observed a temperature rise of approximately 75 °C for Device F ( Fig. 5d ). Devices E and D exhibited temperature rises of only approximately 40 °C and 25 °C, respectively. The results obtained corroborate that the heat-elevating performance of Device F tended to escalate drastically on biasing the heater to 10 V (three times greater than Device D). 3.5 Highlighted features of PBH devices The versatile nature of PBH devices under applied potential biases was established by studying the thermochromic heater under different applied potential biases ( Fig. 5c ). The response time of a heater is defined as the time required to reach a steady-state temperature when starting from room temperature, and this response time is considered a promising factor in evaluating the heater's performance. Minimizing response time is desirable for fabricating a superior device. The temperature of the PBH device increased abruptly once constant voltages were applied, stabilizing within 20 s. Ultra-low response time is a considerable benefit of our heating device. The ultrafast response of our fabricated heater can be compared with the outcomes reported in other papers. 33–41 An ultralow response time for the photochromic heater color change was illustrated with the help of the ESI Video M1. † This rapid thermochromic transition characteristic furthermore adds value to our fabricated PBH device. We suspect this accelerated response to be related to the thickness of the heating device. Such visual color changes were utilized in several applications such as chromogenic sensors and heavy metal detectors, as discussed in the literature. High transmittance and rapid response are credulous requirements for reliable heaters to be used in defrosters and in smart window applications. In addition, heating inhomogeneity constituted a major problem in thermotherapy and heating applications; we verified heating homogeneity with an IR camera. Fig. 5e depicts the PBH device heating homogeneity in the absence of any localized hotspots. The thermal IR camera image reveals the homogeneity of the temperature distribution on the PBH device. Hotspots present in the heating device may harm users in some wearable heating devices. Apart from 2 V, 5 V, and 10 V, the remaining biased PBH device images feature in Fig. S7. † As our PBH device fulfills all of the necessary criteria mentioned herein, we have made advancements in elucidating the processes of defrosting experiments. 3.6 Applicability of the PBH device The defrosting experiment using PBH devices demonstrates their heating and thermochromic characteristics ( Fig. 6a ). A piece of ice was placed on top of the PBH device and was heated by applying 5 V. The ice completely melted within 55 s and turned hot within 90 s. In addition, color transformation was visually demonstrated with this defrosting experiment. Color transformation starts within 10 s and is completed within 15 s, as seen in the ESI Movie M1. † We designed the PBH device, a stretchable transparent heater with thermochromic properties, such that its credulous characteristics make it suitable for defrosting applications, even with low potential. Heating stability was furthermore tested by switching the device from 0 V to 10 V under various applied strains (0–30%). The PBH devices were allowed to undergo tensile elongation (10–30%) and the temperature was monitored. The heat-generating capability of a PBH device under three different strains was studied, and the results appear in Fig. 6b . The temperature decreases as tensile elongation increases from 0% to 30% because of nanowire junction failures. This temperature deterioration trend complies with results observed in other works. 24,25,37 The heat-retaining capacity of the fabricated device upon applied tensile elongation can be quantified using temperature decay (%). This temperature decay (%) can be calculated using the following formulae: where T initial is the temperature observed upon 0% elongation of the heater device, T elongation is the temperature observed with the heater device subject to various elongations (for example 10%, 20%, and 30%). The results are collectively listed in Tables 1 and S1. † The heat-retaining capacity of both the PH and the PBH device are almost comparable (only slight deviations were observed with 30% stretching). Fig. 7 and ESI Movie M1 † elucidates the credibility of our novel PBH device including heat generation, faster response, thermochromic switching ability along with 30% stretchable features. In addition, our novel PBH device works flawless without causing harm to heat generation and their corresponding color transition for 10 repetitive cycles proving its excellent lifetime applicability (Fig. S8 † ). Therefore, our novel PBH device offers a potential alternative to commercial PDMSs, with additional thermochromic properties having plausible bright pathways for use in temperature monitors, thermotherapy, activating filters, and smart homes. Fig. 6 (a) Silver nanowire-coated thermochromic ink blended polydimethylsiloxane device (PBH) device in the defrosting experiment and (b) PBH device heating performance based on thermochromic ink-blended polydimethylsiloxane under varied strains (0–30%). Temperature decay of silver nanowire-coated thermochromic ink blended polydimethylsiloxane device S. no Voltage (V) Temperature °C (0% stretch) Temperature °C (30% stretch) Temperature decay% (30%) 1 2 31 30 3% 2 5 51 34 33% 3 10 107 66 38% Fig. 7 Schematic representation illustrates the heating, stretching, and thermochromic switching characteristics of the PBH device."
} | 6,106 |
30060189 | PMC6198282 | pmc | 4,837 | {
"abstract": "Abstract The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host’s metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.",
"conclusion": "Conclusion Taken together, our results show that glycolysis, contrary to the textbook view on well-investigated model organisms, not only occurs in the cytosol, but also occurs in the mitochondria. All tested stramenopiles show evidence of the second half of glycolysis taking place in the mitochondria and the cytosol, with the exception of the human pathogen Blastocystis , in which the second half of glycolysis occurs exclusively in the mitochondria. Mitochondrial glycolysis therefor seems to be a common feature of the stramenopiles, despite the considerable metabolic and physiological diversity within this group. Although it remains unclear whether this feature is ancestral or derived, our findings show that the intracellular distribution of even the most basic metabolic pathways is variable between the different groups of eukaryotes.",
"introduction": "Introduction Mitochondria provide the bulk of cellular ATP for eukaryotes via oxidative phosphorylation, also known as cellular respiration ( Müller et al. 2012 ). In addition, mitochondria are essential for the production of iron–sulfur clusters ( Lill et al. 1999 ) and play roles in heme synthesis, in fatty acid and in amino acid metabolism ( Scheffler 2008 ). For cellular respiration, pyruvate is produced in the cytosol via glycolysis and imported into the mitochondrion. The pyruvate is then decarboxylated by a mitochondrial pyruvate dehydrogenase to acetyl-CoA. This acetyl-CoA enters the citric acid cycle, subsequently producing one GTP (or ATP) and precursors for several anabolic pathways. More importantly, the reduction of NAD + to NADH and the production of succinate power the respiratory electron transport chain and subsequently ATP synthesis by the proton gradient driven ATP synthase, which is responsible for the majority of cellular ATP synthesis. Glycolysis, the pathway that produces the pyruvate, is a widespread metabolic pathway that converts the six-carbon sugar glucose via a series of ten reactions into the three-carbon sugar pyruvate. During this conversion, energy is stored (two ATP per glucose) and reducing equivalents are formed (two NADH per glucose). To keep the pathway going, NADH needs to be recycled to NAD + , which can happen in a fermentative process, most commonly leading to the formation of lactic acid or ethanol in the cytosol, or by shuttling of the reducing equivalents to the mitochondrial respiratory electron transport chain, which leads to an increased ATP yield. Glycolysis is present in all known eukaryotes, with the exception of some extremely reduced intracellular parasites ( Keeling et al. 2010 ; Wiredu Boakye et al. 2017 ). Glycolysis is nearly universally present in the cytosol of most eukaryotes and also found in specialized microbodies known as glycosomes, originally found in trypanosomes ( Opperdoes and Borst 1977 ), but more recently found to be perhaps a more general feature of all the euglenozoa ( Morales et al. 2016 ). Two glycolytic enzymes were also found to be targeted to peroxisomes in fungi due to posttranscriptional processes ( Freitag et al. 2012 ). An unusual TPI-GAPDH fusion protein was reported to localise to the mitochondrion of a stramenopile, the diatom Phaeodactylum tricornutum ( Liaud et al. 2000 ). In addition, bioinformatics studies ( Kroth et al. 2008 ; Nakayama et al. 2012 ) have hinted at the possible mitochondrial location of several glycolytic enzymes. Stramenopiles are a large and extremely diverse eukaryotic group of organisms, including phototrophic members such as the multicellular kelps (brown algae) and unicellular microalgae including diatoms. They also include nonphototrophic members such as oomycetes, plant pathogens with an enormous impact on agriculture ( Jiang and Tyler 2012 ) and the human pathogen Blastocystis ( Stensvold and van der Giezen 2018 ). The latter species has an anaerobic lifestyle and lacks many features commonly found in mitochondria ( Müller et al. 2012 ; Gentekaki et al. 2017 ). The stramenopiles evolved by endosymbiotic uptake of a red alga but the question as to whether the nonphotosynthetic members never possessed a plastid, or simply lost it, remains unclear ( Baurain et al. 2010 ; Petersen et al. 2014 ; Derelle et al. 2016 ). Members of the stramenopiles can be found in most ecosystems on earth: in marine and fresh water environments, in soil, and as pathogens of humans, animal and plants. Despite their enormous variety in lifestyle a clear monophyly of this group is undisputed ( Walker et al. 2011 ; Derelle et al. 2016 ). Here, we report that the second half of glycolysis, the C3 part, is targeted to mitochondria in the stramenopiles. This exclusive feature of the stramenopiles might be a synapomorphy of this large group of eukaryotes. Mitochondrial glycolysis only covers the pay-off phase of glycolysis, in which the three carbon sugars are converted to pyruvate, leading to the release of energy and reducing equivalents in the form of ATP and NADH.",
"discussion": "Discussion Eukaryotes evolved from a symbiosis between an archaeal host and a bacterial endosymbiont that became the mitochondrion ( van der Giezen 2011 ; Martin et al. 2015 ). Although many different hypotheses have been posited over the years, they principally boil down to two scenarios. The phagotropic origin of eukaryotes suggests they evolved gradually from a less complex prokaryote and once phagotrophy had evolved, the mitochondrial endosymbiosis was possible (see O'Malley 2010 ). The syntrophic eukaryotic origin suggests the establishment of the mitochondrial endosymbiont was the same event as the origin of eukaryotes (see Martin et al. 2015 ). Arguments have been put forward for and against either scenario and it seems that biochemical/physiological arguments favor a synthrophic origin and cell biological/morphological arguments favor a phagotrophic origin. Both scenarios seem to agree that the host was archaeal and the endosymbiont bacterial ( Martin et al. 2015 ; Roger et al. 2017 ). The subsequent replacement of the host’s gene repertoire encoding metabolic capacity has been explained by endosymbiotic gene transfer (reviewed in Timmis et al. 2004 ) and the resulting chimeric nature of eukaryotes had been noticed earlier ( Rivera et al. 1998 ). The nature of the mitochondrial endosymbiont has long been understood to be alpha-proteobacterial ( Gupta 1995 ) but only recently have studies zoomed in on the more precise affiliations of the archaeal host ( Cox et al. 2008 ; Williams et al. 2013 ; Martin et al. 2015 ; Eme et al. 2017 ; Zaremba-Niedzwiedzka et al. 2017 ). A recent study suggests that mitochondria are perhaps ancestral to alpha-proteobacteria, but does not exclude an alpha-proteobacterial origin ( Martijn et al. 2018 ). A few billion years of independent evolution of the endosymbiont’s lineage and widespread bacterial lateral gene transfer (even predating the mitochondrial symbiosis) can explain that not all eukaryotic metabolic proteins have a clear alpha-proteobacterial evolutionary signal. Despite all this, glycolytic enzymes of eukaryotes do all cluster with bacterial homologues in phylogenetic trees ( supplementary fig. S1 , Supplementary Material online; Martin et al. 1993 ; Martin and Herrmann 1998 ; Esser et al. 2004 ). It is indeed implicit of eukaryotic origin theories ( Martin and Müller 1998 ; Martin et al. 2015 ) that glycolysis was originally acquired from the mitochondrial endosymbiont. It is therefore interesting to consider whether the mitochondrial targeting of glycolytic enzymes in stramenopiles represents an ancestral or a derived state for eukaryotes. The deep branches of the eukaryotic tree are not known with certainty, but there is substantial phylogenomic support for the grouping of stramenopiles with alveolates and rhizarians to form the “SAR” supergroup ( Burki et al. 2008 ). Intriguingly, predicted mitochondrial targeting has been reported for several glycolytic enzymes—including TPI-GAPDH fusion proteins—in members of the cercozoa, a group of rhizarians ( Nakayama et al. 2012 ); the distantly related apusozoan Thecamonas trahens also encodes a TPI-GAPDH fusion protein ( Nakayama et al. 2012 ). Taken together, these data raise the possibility that at least some of the latter steps of glycolysis may have occurred in the mitochondria of the SAR common ancestor ( Nakayama et al. 2012 ). However, these inferences are currently based on a very limited sample of SAR diversity, and testing hypotheses about the localization of glycolysis in early eukaryotes will require both more genomes and more of the experimental characterization that we report here. Evolution of mitochondrial protein targeting was a requirement for the successful integration of the mitochondrial endosymbiont and should have happened at least concomitant with endosymbiotic gene transfer if those gene products had to function in the newly formed organelle (comparison to more recently evolved host/symbiont systems suggests that the first proteins that are targeted to an endosymbiont in fact do not originate from the endosymbiont and that the evolution of protein targeting precedes the direct transfer of endosymbiont genes to the host nucleus [ Nowack 2014 ]). Mitochondrial targeting signals do not conform to a strict consensus sequence and secondary structure is a key factor in their functionality ( Schatz and Dobberstein 1996 ). These presequences form amphipathic alpha helices with alternating hydrophobic and positively charged amino acids ( Allison and Schatz 1986 ; von Heijne 1986 ; Roise et al. 1988 ). Mitochondrial targeting sequences can arise randomly ( Baker and Schatz 1987 ), exist in bacteria ( Lucattini et al. 2004 ) and can be acquired by DNA recombination or exon shuffling ( Wischmann and Schuster 1995 ; Long et al. 1996 ; Kubo et al. 1999 ). The predicted presequences for the stramenopiles are in the size range of known mitochondrial targeting signals ( von Heijne et al. 1989 ) and are also enriched in alanine, leucine, serine and arginine ( Neupert and Herrmann 2007 ). Organellar targeting signals for mitochondrial remnants such as the mitochondrial organelle in Blastocystis tend to be shorter than but not as short as those found for Trichomonas hydrogenosomes ( van der Giezen et al. 2005 ; Garg et al. 2015 ). However, there do seem to be some characteristic features even for these hydrogenosomal presequences with often a leucine at the second position and an arginine two places before the cleavage site ( Bradley et al. 1997 ; van der Giezen et al. 1998 ). It is difficult to conclusively determine the selective advantage, if any, for the retargeting or the conservation of glycolysis to/in stramenopile mitochondria. In Blastocystis , similar to many parasitic eukaryotes ( Mertens 1993 ), two key glycolytic enzymes have been replaced by pyrophosphate using versions. Normally, the reactions catalysed by phosphofructokinase and pyruvate kinase are virtually irreversible. However, the reactions performed by diphosphate-fructose-6-phosphate 1-phosphotransferase and phosphoenolpyruvate synthase (pyruvate, water dikinase) are reversible, due to the smaller free-energy change in the reaction. As Blastocystis is an anaerobe and does not contain normal mitochondrial oxidative phosphorylation ( Stechmann et al. 2008 ; Gentekaki et al. 2017 ), any ATP not invested during glycolysis might be a selective advantage. However, in the absence of these irreversible control points there is a risk of uncontrolled glycolytic oscillations ( Chandra et al. 2011 ). Separating the investment phase from the pay-off phase by the mitochondrial membranes might therefore prevent futile cycling. However, as not all stramenopiles use pyrophosphate enzymes, this cannot be the whole explanation. Similarly to the peculiarity of pyrophosphate utilization in Blastocystis , diatoms also show metabolic peculiarities that are not shared with other organisms ( Gruber and Kroth 2017 ). One such peculiarity is the presence of an Entner–Doudoroff pathway in the mitochondria of P. tricornutum ( Fabris et al. 2012 ). This pathway, like glycolysis, degrades glucose to pyruvate. However, the net ATP yield of the Entner–Doudoroff pathway is lower (one ATP per glucose) and the two reducing equivalents that are formed are one NADH and one NADPH per glucose. The degradation of glyceraldehyde 3-phosphate in the Entner–Doudoroff pathway uses identical reaction steps as the glycolysis. Mitochondrial glycolysis therefore might be a complement of the mitochondrial Entner–Doudoroff pathway in P. tricornutum (and other photosynthetic stramenopiles with an Entner–Doudoroff pathway) ( Fabris et al. 2012 ). However, we did not find evidence for an Entner–Doudoroff pathway in nonphotosynthetic stramenopiles, so again, this explanation might not be valid for all stramenopiles with mitochondrial glycolysis. Glycolysis depends on recycling of the reducing equivalents that are formed in the GAPDH reaction (in which NAD + is reduced to NADH). How NAD+ is regenerated depends on the presence of oxygen. Under anoxic conditions, pyruvate usually is reduced in a fermentation which recovers oxidized NAD + (most commonly lactic acid or ethanol fermentation). Under aerobic conditions, the reducing equivalents are transferred to O 2 in the mitochondrial respiratory electron transport chain. In organisms that operate glycolysis exclusively in the cytosol, NAD + /NADH apparently cannot be transported directly into mitochondria. Instead two shuttle systems, the glycerol phosphate shuttle and the malate-aspartate shuttle, lead to indirect exchange of reducing equivalents between cytosol and mitochondria. To release reducing equivalents directly in the mitochondrial matrix where they can be accepted by the respiratory electron transport chain without the need of a shuttle system seems an elegant solution. Similarly, if the redox shuttle system between cytosol and mitochondrial matrix is absent, it also makes sense that the NADPH generating glucose-6-phosphate-dehydrogenase reaction in the above mentioned Entner–Doudoroff pathway in photosynthetic stramenopiles takes place in the mitochondria. The malate-aspartate shuttle requires a cytosolic malate dehydrogenase (MDH). P. tricornutum does not possess a cytosolic MDH ( Ewe et al. 2018 ), which might also suggest an absence of a malate-aspartate shuttle in this diatom. However, if difficulties in redox shuttling would require the redox reactive steps to occur in the mitochondria, this would not explain mitochondrial glycolysis in Blastocystis , an organism that does not rely on oxidative ATP generation. Furthermore, physiological data suggests that in diatoms, considerable shuttling of reducing equivalents from the plastid to the mitochondria may occur as a measure to prevent the formation of reactive oxygen species at the photosystems when excessive excitation energy is absorbed ( Allen et al. 2008 ; Bailleul et al. 2015 ). These findings, and also the presence of unusual transport proteins for nucleotides ( Ast et al. 2009 ; Chu et al. 2017 ), do not support the hypothesis of a lack of efficient shuttling, but underline the importance of stramenopile mitochondria as electron sinks in the recycling of electron acceptors that are reduced either in the mitochondria (in the mitochondrial pay-off phase of glycolysis or in the above-mentioned Entner–Doudoroff pathway) or in other compartments (in the cytosolic part of glycolysis or in the photosynthetic electron transport chain in the plastids). Recently, Abrahamian et al. (2017) reported similar findings to ours and used GFP-tagged proteins in P. infestans to demonstrate the mitochondrial localization of glycolytic enzymes. They also report the targeting of several steps of a serine anabolic pathway to P. infestans mitochondria and suggested the shared 3-phosphoglycerate intermediate would be the raison d'être for the mitochondrial glycolysis ( Abrahamian et al. 2017 ). All the points discussed above might indeed provide several possible physiological explanations for the observed mitochondrial glycolysis in stramenopiles, but unfortunately do not answer the question whether mitochondrial glycolysis is a primary or secondary state in these groups of eukaryotes. The end-product of glycolysis, pyruvate, is transported into mitochondria via a specific mitochondrial transporter that has only recently been identified ( Herzig et al. 2012 ) and that is absent from the Blastocystis genome ( Gentekaki et al. 2017 ). The translocation of the C3 part of glycolysis into mitochondria would necessitate a novel transporter (presumably for triose phosphates). The identification and characterization of such a transporter would open up new possible drug targets against important pathogens. Examples include Phytophthora infestans , the causative agent of late potato blight, which has a devastating effect on food security, but also fish parasites such as Saprolegnia parasitica and Aphanomyces invadans. Both have serious consequences for aquaculture and the latter causes epizootic ulcerative syndrome, an OIE listed disease ( Jiang and Tyler 2012 ; Stentiford et al. 2014 ). Our recent genome analysis of Blastocystis identified several putative candidate transporters lacking clear homology to nonstramenopile organisms ( Gentekaki et al. 2017 ). Such a unique transporter would not be present in the host (including humans) and could be exploited to prevent, or control, disease outbreaks that currently affect food production while the world population continuous to increase ( FAO 2009 )."
} | 4,807 |
32071817 | PMC7007733 | pmc | 4,839 | {
"abstract": "Background The plant microbiome is one of the key determinants of plant health and metabolite production. The plant microbiome affects the plant’s absorption of nutrient elements, improves plant tolerance to negative environmental factors, increases the accumulation of active components, and alters tissue texture. The microbial community is also important for the accumulation of secondary metabolites by plants. However, there are few studies on the niche differentiation of endophytic microorganisms of plants, especially at different elevations. Methods We investigated the effects of altitude on the community composition of endophytic fungal communities and the differentiation of endophytic microorganisms among different niches in Paris polyphylla Sm. The rhizosphere soil, roots, rhizomes and leaves of wild-type P. polyphylla Sm. at different altitudes were sampled, and the fungal communities of all samples were analyzed by internal transcribed spacer one amplification sequencing. Results The results showed that in rhizosphere soil, the number of operational taxonomic units (OTUs) that could be classified or identified decreased significantly with increasing altitude, whereas in the endosphere of plants, the total number of OTUs was higher at intermediate altitudes than other altitudes. Furthermore, the structural variability in the rhizosphere fungal community was significantly lower than that in the endophytic communities. In addition, our results confirmed the presence of niche differentiation among members of the endophytic microbial community. Finally, we also determined that the predominant genus of mycobiota in the rhizome was Cadophora . This study provides insight into the relationships between the endosphere microbiome and plants and can guide the artificial cultivation of this plant.",
"conclusion": "Conclusions This study revealed the structural variability and niche differentiation in the rhizosphere and endosphere fungal microbiomes of wild Paris plants at different altitudes. The results show that the structural variability in microbiome communities in the rhizosphere soil is lower in wild P. polyphylla Sm. than in endosphere fungal communities. The formation of rhizosphere fungal communities is a stable process, and endophytic colonization is variable. In addition, our data confirm reports of niche differentiation in rhizosphere soil-root microbiome communities. Furthermore, our study not only reveals the relationships and differences in endosphere fungal communities in various plant tissues but also clearly shows relationships between altitude and the endosphere microbiome in plants. With increasing altitude, the diversity of the plant endosphere microbiome first increased and then decreased; this pattern is inconsistent with the relationship generally observed between soil microorganisms and altitude. In addition, the core members of the endophytic microbial communities in the rhizome of P. polyphylla Sm. were successfully identified. The present findings provide a basis for further studies on the interactions between the endosphere microbiome and hosts and can inform efforts involving the artificial planting of this plant.",
"introduction": "Introduction Interactions between microorganisms and plants have become a popular research topic in microbiology and botany, with studies conducted on tree ( Beckers et al., 2017 ; Cregger et al., 2018 ), crop and floriculture microbiomes. Plants and microorganisms have close mutualistic or competitive relationships. In most cases, microorganisms play key roles in the survival and performance of plants ( Hackstein, 2010 ; Hacquard et al., 2015 ). In addition, microorganisms are also important for the regulation of plant immune systems ( Jones & Dangl, 2006 ; Kau et al., 2011 ; Lebeis et al., 2015 ; Lee & Mazmanian, 2010 ) and influence plant metabolism ( Khan et al., 2011a , 2011b ). Microorganisms can influence medicinal plants in different ways, such as by affecting the absorption of nutrient elements, improving tolerance to stress, increasing the accumulation of plant active components, and causing changes in tissue texture. The presence of microorganisms in plants is also affected by the host plant genotype ( Cregger et al., 2018 ). To some extent, microorganisms are specific to their hosts. Even within the same genus, endosphere microbiome communities may vary significantly. The symbiosis between fungi and medicinal plants involves a series of complex processes, such as cell morphological changes, signal recognition, signal transduction, nutrient exchange and gene expression ( Huang, Long & Lam, 2018 ). Endophytic fungi produce the same secondary metabolites as host plants ( Guo, 2016 ; Lu et al., 2018 ; Huang et al., 2007 ; Xue, 2013 ). Some endophytic fungi can directly or indirectly affect the secondary metabolism of medicinal plants ( Yadav, Aggarwal & Singh, 2013 ; Bao et al., 2017 ). In addition, the associated bacterial communities may play important roles in the regulation of the plant immune system ( Kau et al., 2011 ; Lebeis et al., 2015 ). Therefore, endosphere microbiome communities are often called the second or extended genome of the host ( Beckers et al., 2017 ). Recently, the effects of endosphere fungi on the growth, development, environmental stress resistance and secondary metabolite synthesis of medicinal plants have attracted increased concern. Plant–Microbe interactions has received substantial attention in recent years as a subject of scientific and commercial interest ( Turner, James & Poole, 2013 ). Studies have shown that the content of secondary metabolites from the same medicinal plant species can be different depending on their location of cultivation, which could in part be related to different composition in their associated microbes when grown at different sites ( Huang, Long & Lam, 2018 ; Köberl et al., 2013 ). For some microbes, their metabolites could also be involved in modulating the production of bioactive phytometabolites, such as paclitaxel produced by Taxomyces andreanae ( Köberl et al., 2013 ). Paris polyphylla Sm. is a famous medicinal plant belonging to Paris in the Liliaceae family. This plant is found in the tropical and temperate regions of Eurasia and is mainly distributed in Southwest China around altitudes of 900–2,000 m. The rhizome is the medicinal part of the plant. Plant components reach the required effectiveness after 5 years. The chemical components of this plant have hemostatic and anti-inflammatory properties as well as good therapeutic effects in treating snake bites. In China, P. polyphylla Sm. is used as a raw material for many medicines. There are many studies on the chemical composition and application depth of this plant, but the endosphere microbiome is less understood. However, wild P. polyphylla Sm. is very rare. This study focuses on the endosphere microbiome of wild P. polyphylla Sm. The fungal communities of all samples were analyzed by internal transcribed spacer (ITS) one amplification sequencing. We assessed the fungal microbiomes in rhizosphere soil, rhizomes, and leaves of P. polyphylla Sm. at different altitudes; DNA samples were amplified by PCR and analyzed by sequencing. The niche differentiation of the related fungal microbiome was also investigated. These studies will help us understand the correlation between the endosphere microbiome and plants and help guide the artificial cultivation of plants.",
"discussion": "Discussion Quality of the pyrosequencing analysis We used ITS1-1F and ITS1-5F primer mixtures to maximize the phylogenetic coverage of fungi. The high abundance of chloroplast genes can lead to unexpected co-amplification of non-target sequences ( Kabir, Peter & Jennifer, 2016 ; Roesch et al., 2007 ). In this study, the amplified plant chloroplast genes were culled. Remarkably, the singletons in rhizosphere soil and in different plant compartments had been removed before annotation. Due to the structural differences between soil and plants, we were unable to extract high-quality and high quantity DNA from all plant samples using the same DNA extraction kit. To ensure high-quality and high quantity DNA from all studied samples, we chose different pre-processing methods, and extraction kits were used for DNA extraction from rhizosphere soil and plant samples. As a result, the high discrepancy in the number of singletons in the plant parts could be attributable to genuinely rare (singleton) OTUs in the rhizosphere soil ( Table 1 ). In fact, microbiomes in the rhizosphere soil are generally considered the most diverse regions ( Lugtenberg & Kamilova, 2009 ; Coleman-Derr et al., 2016 ). For further analysis, we chose to remove all singletons from the data sets. However, the involvement role of singletons ecological roles functions is largely unknown, this requires need the further study. Endophytic fungal communities in different niches We estimated the richness, evenness and diversity of alpha diversity based on OTUs. We found that the abundances of OTUs in rhizosphere soil and plant compartments (roots, rhizomes and leaves) were clearly different. The results are consistent with the general view of endosphere colonization. The diversity and evenness varied greatly from rhizosphere soil to endosphere. Because of chemotaxis and colonization of the rhizosphere microbiome, rhizosphere microbial communities are rich and diverse ( Huang, Long & Lam, 2018 ; Bulgarelli et al., 2012 ; Hardoim, Van Overbeek & Van Elsas, 2008 ). The rhizosphere soil-root interface acts as a selective barrier for endosphere fungal colonization. High variability of endophytic OTU richness, as depicted by the box plot, could possibly be caused by sporadic and nonuniform colonization of the roots, rhizomes and leaves of Paris ( Gottel et al., 2011 ; Beckers et al., 2017 ). Therefore, our data suggest considerable variation in endophytic colonization as a major reason for the high variability in the box plot. At the genus level, Cadophora and Tetracladium were the dominant microorganisms among the plant mycobiota. Although their OTU abundance was abundances were different, both Cadophora and Tetracladium belong to Ascomycota , which is considered the most likely endophytic taxon to colonize plants ( Guo, 2016 ). Ascomycota is widespread in soil and plants, such as forage, flowers and crops ( Kottke et al., 2008 ; Edwards et al., 2015 ; Hacquard et al., 2015 ). Enrichment and depletion of specific microbiomes within the plant-associated microbiome are initiative processes that depend on active selection of microbial consortia by the plant host and opportunistic colonization of the available ecological niches ( Bulgarelli et al., 2013 ; Mehta & Rosato, 2001 ; Wen et al., 2007 ). Therefore, the colonized fungi are limited to specific fungal species. Our results illustrated that the diversity and evenness decreased from the rhizosphere to the endosphere. Only a limited number of microbes could adapt to the way of life in the plant. As a result, the plant niches consist of specific endosphere communities. Previous studies of other plants have also reported niche differences in the distribution of endogenous microbial communities, such as poplar ( Beckers et al., 2017 ; Cregger et al., 2018 ), which may have an effect on plant metabolism ( Chen et al., 2018 ). Different niches of the plant may be associated with differences in plant metabolism. Studies of the differences in the endogenous microbial community in different niches could provide a basis for future studies of differences in metabolism in different parts of medicinal plants. To compare the structure of the endosphere fungal community in different plant compartments, we clustered all samples utilizing PCoA and hierarchical clustering (Binary-Jaccard) ( Fig. 4 ). At the OTU level, the rhizosphere soil gathered together. However, there was no obvious relationship between the fungal microorganism clusters in different plant tissues. This result was further confirmed by UPGMA hierarchical cluster analysis. P. polyphylla Sm. is a perennial herb. In each growth cycle of P. polyphylla Sm., only the aboveground part of the plant dies, while the rhizome remains alive. This growth pattern may be one reason for the lack of obvious typical mycobiota in different plant compartments. In addition, for microorganisms, the endophytic environment of plants is complex, In contrast to rhizosphere colonization, intricate interplay between endophytic microbe and the host plants innate immune system, it of the host plant is completely different from the soil, which is also an important reason for the difference between the soil and plant microbial communities. In this study, different rarefaction curves ( Fig. 2 ) were obtained from the rhizosphere soil and endosphere samples. Compared with plants, the rhizosphere soil displayed much greater microbial diversity, possibly because the plant provides a relatively stable interior environment, which leads to lower variability in the plant’s internal fungal community. In addition, we found that the OTU numbers in soil were almost three times those in plants. This result is consistent with the widely accepted view that soil contains many microbes. The structure of the endosphere fungal communities varied more markedly than the structure of the rhizosphere community ( Bulgarelli et al., 2012 ; Nallanchakravarthula et al., 2014 ). Soil microbe communities form one of the most abundant microbial ecosystems on Earth ( Coleman-Derr et al., 2016 ; Wu et al., 2005 ). In addition, root exudates and nutrients from mucilage sources also attract countless organisms to gather in the rhizosphere. The microbes in plants need to be strongly competitive to successfully colonize roots ( Hackstein, 2010 ); competitive ability might manifest as the ability to break through plant cells (producing enzymes that degrade cell walls) and the ability to adapt to the innate immunity of plants ( Turner, James & Poole, 2013 ; Compant, Clément & Sessitsch, 2010 ; Nie et al., 2005 ). Differences in the microbiome of plants among different altitudes The content of secondary metabolites from the same medicinal plant species can vary depending on the location of cultivation, which could in part be related to differences in the composition of the associated microbial communities at different sites. According to Köberl et al. (2013) , many of the variations in the quality of traditional herbal medicine may be attributable to changes in the microbial community either in the rhizosphere or in the endophytic compartment of the medicinal plant ( Huang, Long & Lam, 2018 ). The functional characteristics of endophytic communities residing inside the roots of rice have revealed that endophytes may be involved in the metabolic processes of rice ( Vain et al., 2014 ). Salvia miltiorrhiza harbors a distinctive microbiome that is enriched in functions related to secondary metabolism and thus may contribute additional metabolic capabilities beyond those encoded in the genome of the host plant ( Huang, Long & Lam, 2018 ; Chen et al., 2018 ). These observations suggest that the soil and climate at different locales can influence the metabolite content of the medicinal plant. Altitude is one of the most important factors affecting climate, therefore we analyzed the variation in the endophytic fungal communities among different altitudes. We divided the plant distribution into three elevation zones: low, middle and high altitude ( Fig. 5 ). The results showed downward trends of the richness and diversity of soil microorganisms in the rhizosphere with increasing altitude. Previous studies have shown that fungal community composition is influenced by both ecological factors and evolutionary factors. Spatial scale is considered a major factor contributing to differences in fungal diversity ( Kabir, Peter & Jennifer, 2016 ). However, the richness and diversity of endophytic microorganisms do not follow the distribution law of soil microorganisms. In the endophytic environment, the richness and diversity of endophytic microorganisms are the most abundant in the plant endophytic environments, which is similar to the distribution law of plants, rather than following the law of altitude. This phenomenon has rarely been reported in previous studies. Through systematic study of the associated microbiome in medicinal plants, we should be able to clarify the distribute situation of various microbes in plants at different elevations. This information could guide better selection of growing environments for the cultivation of medicinal plants, which in turn may improve the medicinal quality and evaluation standards of medicines that will facilitate their passing more rigorous scientific and commercial evaluations."
} | 4,218 |
35079136 | PMC9038690 | pmc | 4,840 | {
"abstract": "Spatial self-organization is a hallmark of surface-associated microbial communities that is governed by local environmental conditions and further modified by interspecific interactions. Here, we hypothesize that spatial patterns of microbial cell-types can stabilize the composition of cross-feeding microbial communities under fluctuating environmental conditions. We tested this hypothesis by studying the growth and spatial self-organization of microbial co-cultures consisting of two metabolically interacting strains of the bacterium Pseudomonas stutzeri . We inoculated the co-cultures onto agar surfaces and allowed them to expand (i.e. range expansion) while fluctuating environmental conditions that alter the dependency between the two strains. We alternated between anoxic conditions that induce a mutualistic interaction and oxic conditions that induce a competitive interaction. We observed co-occurrence of both strains in rare and highly localized clusters (referred to as “spatial jackpot events”) that persist during environmental fluctuations. To resolve the underlying mechanisms for the emergence of spatial jackpot events, we used a mechanistic agent-based mathematical model that resolves growth and dispersal at the scale relevant to individual cells. While co-culture composition varied with the strength of the mutualistic interaction and across environmental fluctuations, the model provides insights into the formation of spatially resolved substrate landscapes with localized niches that support the co-occurrence of the two strains and secure co-culture function. This study highlights that in addition to spatial patterns that emerge in response to environmental fluctuations, localized spatial jackpot events ensure persistence of strains across dynamic conditions.",
"introduction": "Introduction Microbial communities frequently experience perturbations and spatiotemporal fluctuations in their local environmental conditions [ 1 – 6 ]. Such perturbations and fluctuations can have important effects on community stability [ 7 ] and can modulate inter- and intra-specific cell–cell interactions [ 8 ]. For example, many soil environments experience alternating cycles of wet and dry conditions that can induce changes in community composition by promoting growth during hydrated conditions [ 9 , 10 ] and reducing distances between individual cells that facilitate cell–cell interactions during unsaturated conditions [ 11 ]. In coastal environments, tidal dynamics can modify environmental conditions and consequently impose changes on community composition [ 12 ] and metabolic activity [ 13 ]. On plant leaf surfaces, diurnal fluctuations can modulate resource availability and change the set of available carbon resources, which can again impose changes on community composition and metabolic activity [ 14 ]. Finally, in the human gut, changes in dietary conditions can induce changes in the structure and functioning of the gut microbiome [ 1 ]. Thus, environmental perturbations and fluctuations significantly influence the ecological and evolutionary processes governing community structure and functioning [ 6 , 15 ]. One mechanism by which changes in environmental conditions can affect community structure and functioning is by modulating the types of interactions that occur between different cell-types (e.g., mutualism, commensalism, antagonism, competition, etc.) [ 8 , 16 , 17 ]. In turn, changes in the type of interaction can change how those cell-types arrange themselves across space (referred to hereafter as spatial self-organization) [ 18 – 21 ]. Importantly, spatial self-organization is a determinant of many community-level properties and behaviors [ 22 – 27 ], including the metabolic processes performed by microbial communities [ 8 , 28 , 29 ], the resistance and/or resilience of microbial communities to invasion [ 30 , 31 ] and the evolutionary processes acting on microbial communities [ 32 – 36 ]. In populations growing in an unstructured and steady-state environment, the emergence of rare stochastic events, such as the accumulation of beneficial mutations, are referred to as jackpot events [ 37 ]. However, in spatially structured populations, the persistence of such a mutation during range expansion requires that the mutation emerge in a favorable spatial position that secures its presence at the expansion edge [ 38 ]. This is particularly relevant for sessile growth of microbial colonies, where small populations expand into adjacent unoccupied space and growth is confined to a thin layer of cells at the expansion edge [ 32 ]. In the absence of environmental perturbations, microbial communities undergoing range expansion show a decrease in diversity with only a few lineages persisting at the expansion edge [ 39 – 41 ]. Laboratory and in silico experiments demonstrated that stochastic processes [ 32 , 36 ] and mechanical forces acting between cells [ 42 , 43 ] in combination with initial spatial positioning [ 24 , 36 ] can control the dynamics of diversity loss during sessile microbial range expansion. Further investigations demonstrated the importance of initial spatial positioning when sustained by the local substrate landscape, thus leading to the establishment of successful lineages at the expansion edge [ 44 ]. In other words, the presence of a specific cell-type at the expansion edge may result from stochastic processes that do not require beneficial mutations. We use the term “spatial jackpot events” to emphasize the importance of favorable initial spatial positioning [ 38 ] to position cell-types at the expansion edge while the metabolic strength guarantees their stable position at the expansion edge during environmental perturbations. Although spatial self-organization during range expansion has been frequently studied under steady-state conditions (e.g., stable redox conditions), further attention is required to understand how environmental perturbations and fluctuations affect microbial interactions and spatial self-organization. In this study, we investigated the stability of a cross-feeding microbial co-culture under fluctuating environmental conditions. We hypothesized that temporal fluctuations in environmental conditions that alter the nature of interspecific interactions can lead to irreversible transitions in spatial patterns of cell-types, thus affecting co-culture composition and metabolic functioning. Our hypothesis is based on the following two assumptions: (1) environmental conditions that foster different types of interspecific interactions promote the formation of different patterns of spatial self-organization, and (2) the patterns of spatial self-organization that emerge under one set of environmental conditions can alter co-culture composition, spatial self-organization, and functioning under a different set of environmental conditions. The above assumptions are not met if spatial jackpot events emerge that enable cell-types to maintain a stable position at the expansion edge. We tested this hypothesis with a microbial co-culture that satisfies both of the above-mentioned assumptions. The component strains engage in competition under oxic conditions and mutualistic cross-feeding of the conditionally toxic metabolite nitrite (NO 2 − ) under anoxic conditions (Fig. 1a ). Oxic and anoxic conditions promote the formation of fundamentally different patterns of spatial self-organization [ 19 ] (Fig. 1b, c ), satisfying the first assumption discussed above. In addition, we predict that the patterns of spatial self-organization that emerge under oxic conditions are detrimental to the co-culture as a whole under anoxic conditions, satisfying the second assumption discussed above (Fig. 1d ). Briefly, anoxic conditions result in a dominance of the nitrite-producing strain (referred to as the producer) at the expansion edge [ 19 , 34 , 45 ] (Fig. 1d ). If the environment changes to oxic conditions, the producer will have preferential access to resources supplied via diffusion from the periphery and will increase in abundance relative to the nitrite-consuming strain (referred to as the consumer). If the environment switches back to anoxic conditions, the increased relative abundance of the producer will result in nitrite accumulation. Over a series of anoxic/oxic transitions, we predict a continual increase in the relative abundance of the producer and the potential accumulation of nitrite to toxic concentrations, thus creating detrimental conditions for the co-culture as a whole. We tested this prediction by repeatedly transitioning the environment between anoxic and oxic conditions and quantifying the effects on co-culture composition and local spatial organization at the expansion edge. Fig. 1 Two-strain microbial co-culture used in this study. a The co-culture is composed of two isogenic mutant strains of P. stutzeri that differ in their ability to reduce nitrate (NO 3 − ) and nitrite (NO 2 − ). One strain can reduce nitrate but not nitrite (referred to as the producer; solid blue horizontal lines) whereas the other can reduce nitrite but not nitrate (referred to as the consumer; solid green horizontal line). The two strains also carry either the ecfp blue or egfp green fluorescent protein-encoding gene. Different patterns of spatial self-organization emerge depending on redox conditions. b 1 Anoxic conditions induce a mutualistic interaction and “producer-first expansion”, where the producer expands ahead of the consumer. This is because the consumer cannot grow until the producer begins producing nitrite. b 2 The community is punctuated by individual “consumer-first expansion” patterns that persist to the expansion edge (referred to as spatial jackpot events). c Oxic conditions induce a competitive interaction and “simultaneous expansion” of the two strains, resulting in segregated sectors with interspecific boundaries lying approximately parallel to the expansion direction. The scale bars are 1000 μm. d In a fluctuating environment, the previous range expansion determines the initial spatial positionings of the strains for the subsequent range expansion, and may thus fundamentally alter spatial self-organization. We predict that repeated transitions between anoxic and oxic conditions will result in a gradual decrease in the ratio of consumer-to-producer, thus potentially leading to the accumulation of nitrite to toxic concentrations. This is due to the preferential spatial positioning of the producer at the onset of oxic conditions.",
"discussion": "Discussion In this study, we investigated how fluctuations in environmental conditions that alter interactions between two microbial strains influence the emergence and evolution of spatial self-organization. Using a microbial co-culture consisting of two strains that cross-feed nitrite (NO 2 − ) under anoxic conditions and compete under oxic conditions, we conducted a series of range expansion experiments and complemented experimental observations with insights gained from a mechanistic agent-based model that mimics the experimental conditions. Overall, the emerging patterns of spatial self-organization are consistent with our previous observations of producer-first expansion under anoxic conditions and simultaneous expansion under oxic conditions [ 19 ] (Fig. 1b, c ). They are also consistent with our expectation that repeated transitions between the two environmental conditions should result in increased abundance and dominance of the producer (Fig. 1d ). Contrary to our initial expectation (Fig. 1d ), however, we found that the composition of the co-culture is preserved despite repeated transitions between anoxic and oxic conditions (Figs. 2 – 4 ). We attribute the stability in co-culture composition and spatial self-organization to the emergence of spatial jackpot events that enable the consumer to remain located at the expansion edge under anoxic conditions, and subsequently secure its position after transition to oxic conditions (Fig. 3 ). Thus, spatial jackpot events are an important mechanism that enables stable community composition in the face of environmental fluctuations and perturbations (Fig. 6 ). In essence, spatial jackpot events are a form of local spatial pattern diversity within microbial communities [ 56 ]. Thus, just as genetic diversity can provide compositional and functional stability to microbial communities [ 57 – 59 ], spatial pattern diversity can also contribute toward compositional and functional stability. Why do spatial jackpot events emerge, and what enables their propagation? The term jackpot event has typically been used in relation to genotypic events, where rare mutations can emerge that enable new genotypes to proliferate and persist [ 37 , 38 , 60 ]. In our case, spatial jackpot events emerge from a stochastic process that does not have a genetic basis, as we demonstrated via heritability tests and genome re-sequencing analyses in a previous study [ 56 ]. Spatial patterns can diversify due to local variations in the initial spatial positionings of individual cells, which results in two different patterns of spatial self-organization that emerge simultaneously [ 44 , 56 ]. The dominant pattern is “producer-first expansion”, where the producer expands first and the consumer follows. In this scenario, the expansion edge is occupied by producer cells that rapidly proliferate due to their preferential access to nitrate (NO 3 − ) whereas initially negligible nitrite (NO 2 − ) concentrations result in an exclusion of consumer cells from the expansion edge. The minority pattern is “consumer-first expansion” (referred to here as spatial jackpot events). During the development of a spatial jackpot event, the producer pushes a few consumer cells forward within the expansion area (Fig. 1b ) [ 44 , 56 ]. Our detailed modeling results show evidence for two important mechanisms that facilitate the nucleation of spatial jackpot events (Supplementary Fig. S6 ). First, the inoculated consumer cell needs to persist at the expansion edge via shoving by producer cells (Supplementary Fig. S6a ). Furthermore, the results suggest that there is a stronger sensitivity to local conditions at pH 7.5, where the consumer benefits from a cluster of adjacent consumer cells supported by a background of producer cells in their vicinity that push the consumer cluster toward the expansion edge (Supplementary Fig. S6b, c ). Once sufficient nitrite is available, the consumer cells that remain near the expansion edge gain a localized relative growth rate advantage due to abundant nitrite (in comparison to the diminishing nitrate availability per consumer cell) that results in the persistence of the observed spatial jackpot event (Fig. 5 ). In contrast, at pH 6.5 the local growth rate advantage of the consumer does not require a high number adjacent consumer cells in order to nucleate a spatial jackpot event (Supplementary Fig. S6b, c ). Thus, stochastic processes determine the initial spatial positionings of individuals while deterministic processes then act on those individuals to generate a range of spatial patterns as a function of different relative growth rates and behaviors [ 33 , 36 , 61 – 63 ]. Previous studies that investigated range expansion in microbial communities highlighted that increasing the strength of a positive interaction can slow the loss of diversity under constant redox conditions [ 45 ]. We found that the persistence of the consumer is increased at the expansion edge in the face of environmental perturbations by strengthening the mutualistic interaction. The relative abundance of the two strains and also their intermixing showed comparable outcomes (Fig. 2 ), where the relative abundance and intermixing are both higher at pH 6.5 than at 7.5. How generalizable are our main conclusions? Fluctuating environmental conditions frequently occur in natural systems such as in soils. Redox fluctuations following intermittent rainfall events, where anoxic conditions rapidly develop in saturated soils while oxic conditions prevail in unsaturated soils, expose soil microorganisms to fundamentally different environmental conditions that affect community composition and function [ 17 ]. Thus, our study may be of relevance for understanding the resistance and resilience of soil microbial communities to changes in redox. More generally, the principle that we investigated here may be relevant for any type of environmental perturbation or fluctuation conditional that the two assumptions discussed above are satisfied (i.e., different environmental conditions promote the emergence of different patterns of spatial self-organization and the patterns of spatial self-organization that emerge under one set of environmental conditions are detrimental under other sets of environmental conditions). How widespread are spatial jackpot events likely to occur in nature? We argue that such spatial jackpot events may be typical features of self-organizing microbial communities. When any surface is colonized by microbial cells, individuals will not be distributed uniformly. Instead, colonized surfaces will contain local differences in the initial spatial positioning of individuals. These differences, in turn, can create spatial pattern diversity, where some of the patterns may provide new community-level properties such as resistance or resilience to environmental change. Thus, spatial jackpot events may be widespread and inevitable features of surface-associated microbial communities."
} | 4,395 |
30424340 | PMC6187469 | pmc | 4,842 | {
"abstract": "Triboelectric nanogenerators (TENGs) are used as self-power sources for various types of devices by converting external waves, wind, or other mechanical energies into electric power. However, obtaining a high-output performance is still of major concern for many applications. In this study, to enhance the output performance of polydimethylsiloxane (PDMS)-based TENGs, highly dielectric TiO 2−x nanoparticles (NPs) were embedded as a function of weight ratio. TiO 2−x NPs embedded in PDMS at 5% showed the highest output voltage and current. The improved output performance at 5% is strongly related to the change of oxygen vacancies on the PDMS surface, as well as the increased dielectric constant. Specifically, oxygen vacancies in the oxide nanoparticles are electrically positive charges, which is an important factor that can contribute to the exchange and trapping of electrons when driving a TENG. However, in TiO 2−x NPs containing over 5%, the output performance was significantly degraded because of the increased leakage characteristics of the PDMS layer due to TiO 2−x NPs aggregation, which formed an electron path.",
"conclusion": "4. Conclusions In summary, we demonstrated a facile approach to improve the output performance of TENGs by embedding high dielectric TiO 2−x nanoparticles in the PDMS layer. The output performance of the TiO 2−x NPs embedded (5%) TENG showed the highest output voltage and current of 180 V and 8.15 µA, respectively, which is strongly related to the change of oxygen vacancies on the PDMS surface as well as the increased dielectric constant. Since the oxygen vacancies are positively charged, the increment of oxygen vacancies of the negative tribo-material surface could induce electrons from the top electrode by attraction force. Therefore, the TiO 2−x NPs-embedded PDMS layer has larger oxygen vacancies, which can contribute to the enhanced TENG performance. In contrast, with TiO 2−x NPs embedded samples over 5%, the output voltage and current were drastically decreased to 38.7 V and 0.6 µA, respectively. This significant degradation of output performance is due to variation of the tribo-series, resulting in reduced tribo-electrification effects. In addition, the electron path could be formed due to TiO 2−x NPs clustering, which can move the electrons induced on the surface thereby increasing leakage current. Last, our optimal TENG was also presented as a practical application for wind energy harvesting and could obtain 424 V of the peak output voltage via the proposed windmill.",
"introduction": "1. Introduction Triboelectric nanogenerators (TENGs) have demonstrated the ability to convert surrounding ambient mechanical energy into electricity based on the coupling effects of contact triboelectrification and electrostatic induction. However, the need for a low-cost method and an improvement in output performance remain a challenge in TENG fabrication. A practical application to harvest mechanical energy is also required. In general, the two key factors that significantly affect the output performance of TENGs are the tribo-material and the effective contact area of the friction layers. Another study showed that the surface charge density enhanced the triboelectric material and that the surface morphologies in micro/nanopatterning control the TENGs performance [ 1 , 2 ]. As shown in 2012 by Wang’s group [ 3 ], TENGs operate in four basic modes: vertical contact-separation mode; lateral sliding mode; single electrode mode, and freestanding triboelectric-layer mode. Among these working modes, the vertical contact–separation mode was introduced first and has become the primary mode due to its simple operation, durability, stable and high-output performance. Basically, using the electrostatic charges created on the surfaces of two dissimilar materials when they are brought into contact, the tribo-charges can generate a potential drop when two surfaces are separated by an external force and, thus, drive electron flow between two electrodes connected on both sides of the tribo-material. Recently, TENG development has become very common in many fields, such as energy harvesting, self-powered sensors, self-charging systems, or self-powered wearable electronic devices. TENGs has been investigated to obtain higher electricity output using surface charge density enhancement via ionized injection, which alters the effective contact area between the friction tribo-layers. This improvement is through its surface patterning or through new materials developed using functional group attachment. However, the simple, low-cost, and flexible structure of TENGs through embedded nanoparticles (NPs) is still being considered as a way to increase the capacitance of TENGs. By modifying the inside polymers through filling with nanoparticles and forming pores, many studies have reported the advantages of an embedded high dielectric constant material in polydimethylsiloxane (PDMS), such as BaTiO 3 , SrTiO 3 [ 4 , 5 ], Au, or Ag nanoparticles [ 6 ], for improving TENG capacitance. In addition, the output performance of TENG can be increased by adjusting the distribution depth of the tribo-charges in the friction layer as described by Cui et al. [ 7 ]. C. Wu et al. [ 8 ] also demonstrated that the charge density in the friction layer is enhanced by utilizing a reduced graphene oxide (rGO) acting as electron-trapping sites [ 8 ]. These methods can lead to the higher output performance of TENGs, but are still limited in real applications due to the complexity, high-cost, and lack of both physical characteristic and analysis properties for understanding embedded NP behaviors inside the tribo-polymers layer. Moreover, oxygen vacancies in the oxide nanoparticles are electrically positive charges [ 9 ], which is an important factor that can contribute to the exchange and trapping of electrons when driving a TENG. In previous studies, however, the effect of oxygen vacancies within the tribo-material surface according to the oxide nanoparticle embedded ratio was not studied. In this work, we report a significant improvement of TENG performance by considering TiO 2−x NPs embedded in PDMS as a function of weight ratio. Through this work, the optimal TiO 2−x NPs embedded PDMS is determined, and we investigated the correlation between TENG output behavior and the physical properties of the TiO 2−x NPs-embedded PDMS, such as the surface potential, dielectric properties, oxygen vacancy, and electronic structures. Different TiO 2−x NPs embedded according to PDMS weight ratios from 0 to 30% were chosen for tribo-layer fabrication. The results show a significantly enhanced TENG output upon the use of TiO 2−x NPs embedded PDMS as compared to a pristine PDMS tribo-layer. The optimal TiO 2−x NPs embedded PDMS with a 5% weight ratio exhibited the highest voltage of 180 V and current of 8.15 µA under a 5 N pushing force and 5 Hz pushing frequency. Furthermore, for practical applications, a portable windmill system integrated with our optimal TENGs using a low-cost, simple fabrication process involving plastic bottle waste is proposed.",
"discussion": "3. Results and Discussion The proposed TENG was operated using a simple model of contact–separation, as depicted in Figure 1 a. The TiO 2−x NPs-embedded PDMS layer was laminated on the Al electrode as a negative tribo-material. When the TiO 2−x NPs-embedded PDMS layer comes into contact with the top layer Al electrode (i.e., positive tribo-material) as well, the TiO 2−x NPs-embedded PDMS layer becomes negatively charged while the Al electrode is positively charged because of the different electron affinities. During the release process, potential is created between the two opposite electrostatic charges. The potential across these electrodes leads to an electron flow through the external circuit from the bottom Al electrode to the top to obtain electrical equilibrium. After full separation, the electrodes revert back to their original states, and at last, the tribo-charge distribution reaches electrical equilibrium. Applying a continuous external pushing force to the TENGs, repeated TENG operation can be observed. Since the titanium oxide materials have various physical structures, such as anatase, rutile, and bookite, and their electrical, optical, and physical properties depend on their physical structure [ 10 ], XRD patterns of the TiO 2−x NPs were investigated, as shown in Figure 1 b. The TiO 2−x NPs represent the typical polycrystalline rutile structure with diffraction peaks of (110), (101), (200), (101), (210), (211), (220), (002), (310), (301), and (112), and the calculated particle size was about 20 nm through main XRD peak of (110). [ 11 , 12 ] The energy band diagram of the PDMS layer and TiO 2−x NPs-embedded PDMS layer with 5% and 30% are shown in Figure 1 c. As TiO 2−x NP increased, the work function difference between the two contact materials is reduced from 2.61 eV to 0.72 eV. This implies that the transport of electrons from the top Al electrode can be reduced. Figure 1 d shows the scanning electron microscope (SEM, compact SEM GENESIS-1000, Emcrafts, Korea) images of the PDMS layer and TiO 2−x NPs embedded PDMS layer with 5% and 30%. The surface morphology of these PDMS and TiO 2−x NPs-embedded PDMS layers are uniformly wrinkle-like and similar, except for the higher weight ratio TiO 2−x NPs-embedded PDMS layers. The average roughness of these TiO 2−x NPs-embedded PDMS layers is confirmed by the AFM image, and the mean surface roughness of the PDMS layer and TiO 2−x NPs-embedded PDMS layer with 5% and 30% was 17.8, 22.1, and 19.8 nm, respectively. (Not shown here). Figure 2 a,b show the output voltage and current characteristics of the TENG without/with TiO 2−x NPs as a function of weight ratio (measured under a relative humidity of ~55% and room temperature of 27 °C). As the weight ratio of TiO 2−x NPs increased from 0% (pristine PDMS) to 5%, the output voltage and current increased significantly from 112 V and 3.5 µA to 180 V and 8.15 µA, respectively. However, at weight ratios greater than 5%, the output voltage and current drastically decreased to 38.7 V and 0.6 µA, respectively. Moreover, to examine the effect of the pushing force on the output performance of the TENG device with pristine PDMS, 5% (optimal weight ratio) and 30% TiO 2−x NPs-embedded PDMS, the output voltage and current of the TENG were measured and analyzed at different pushing forces ranging from 5 to 20 N and pushing frequency of 5 Hz ( Figure S2—Supporting Information ). It clearly indicates that the peak voltage and current of the TENG were increased from 48.2 V and 1.6 µA to 91.8 V and 2.9 µA for the pristine TENG, from 64.9 V and 3.8 µA to 126.8 V and 6.1 µA for 5%, and that of 30% TiO 2−x NPs-embedded PDMS based TENG increased from 30.6 V and 0.9 µA to 58.7 V and 2.1 µA by enhancing the applied pushing forces from 5 N to 20 N, respectively. The highest output voltage and current values of 126 V and 6.1 µA were obtained under the externally applied force of 20 N. This result is mainly attributed to the surface potential dramatically changing among these kind of TENGs. This was especially true for the 30% TiO 2−x NPs-embedded PDMS which became a positively charged material, leading to the drop of the TENG output performance, even though a strong applied pushing force. In addition, the trend of the TENG output among pristine, 5%, and 30% is the same as the measurement at 5 N and 5 Hz shown in Figure 2 . It was noted that the surrounding conditions were 75% relative humidity and 32 °C room temperature for the different applied forces measurements. Figure 2 c shows the effect of voltage and current characteristics of the TENG as a function of external load resistance ranging from 0.1 to 1000 MΩ. These measurements were performed under a pushing force and frequency of 5 N and 5 Hz, respectively. As shown in Figure 2 c, the TENG voltage increased by increasing the resistance from 0.1 to 200 MΩ, while the current value followed the opposite trend. Afterward, both the voltage and current became saturated at very high load resistances (i.e., 200–1000 MΩ), and the TENG exhibited the highest voltage and lowest current values ranging from 230.6–260 V and 4.8–2.6 µA. In addition, the average power density ( P avg ) was calculated by the following the equation: (1) P a v g = V I S \nwhere V and I are the output peak voltage and current values of the TENG at various load resistances, respectively, and S is the active area (i.e., 9 cm 2 ) of the TENG. Figure 2 d shows the effect of the TENG output power density as a function of external load resistance. Following Figure 2 d, the P avg values of TENG increased by increasing the load resistance and then decreased at a relatively high load resistance. Specifically, at the moderate load resistance of 40 MΩ, the TENG exhibited a maximum power density of 1.84 W.m −2 , under the pushing force and frequency of 5 N and 5 Hz, respectively. More discussion is provided below regarding the output performance behavior of the TENGs and the PDMS properties according to the TiO 2−x NP embedding ratio. Previous studies show that as the embedded weight ratio of NPs with high dielectric constants increases, the TENG output performance was improved because of the increasing dielectric constant of the tribo-material [ 13 ]. Our results show the same trends as previous studies, as shown in Figure 3 a. In addition, the dielectric constant according to frequency does not change significantly. However, we provide below not only an increase in the dielectric constant of the tribo-material but also suggest an additional mechanism based on the role of oxygen vacancy in TiO 2−x NP embedded PDMS to effectively enhance TENG performance. To investigate changes of chemical bonding state within the PDMS layer according to the amount of embedded TiO 2−x NPs, the oxygen (O) 1 s spectra of PDMS and TiO 2−x NPs 5% embedded PDMS layers were observed via XPS, as shown in Figure 3 b. For the detailed analysis of chemical bonding states, the O 1 s spectra were carefully normalized and de-convoluted with three different Gaussian peaks as indexed with O1, O2, and O3 centered at 530.9 eV, 531.8 eV, and 532.9 eV, respectively. Each of the representatively assigned peaks from a low binding energy is related to the oxygen state in the metal-oxide lattices, the oxygen-deficient state, and chemisorbed or dissociated oxygen states, or OH − impurities, respectively [ 14 , 15 ]. Among them, the relative area of the oxygen-deficient peak (O2) is dramatically increased from 18.80% to 24.63% after TiO 2−x NPs embedding, which is remarkable. Moreover, we measured and de-convoluted O 1 s ’ peak of other films that have different concentration, as shown in Figure S3 . In the result, the oxygen-deficient states were similar regardless of increases of TiO 2−x concentration. This saturation can be observed due to the formation of TiO 2−x clusters in the composite film (see Figure 1 d). The TiO 2−x nanoparticles in a cluster act like a single particle. This means that the number of active oxygen deficient states are as same as a single TiO 2−x nanoparticle. Due to the oxygen vacancies being electrically positively charged, the increment of oxygen vacancies of the negative tribo-material surface could induce electron flow from the top electrode by the attraction force, as shown in Figure 3 c. Therefore, the TiO 2−x NPs-embedded PDMS layer has a larger oxygen-deficient state by oxygen vacancies, which can contribute to an enhanced TENG performance. To theoretically understand the output performance enhancement of the TiO 2−x NPs-embedded TENG, a COMSOL Multiphysics (5.0, COMSOL, Inc.) simulation was conducted. Figure 3 d shows the corresponding results for the triboelectric potential distributions of pristine TENGs and TiO 2−x NPs-embedded TENG, respectively. The details of the COMSOL Multiphysics simulation parameters and equations are provided in our previous study, Reference [ 16 ]. As a result, the COMSOL simulations showed that the 5% TiO 2−x NPs-embedded TENG can exhibit higher performance than the pristine TENG. To elucidate the cause of the significantly decreased output performance of TENG containing over 5% embedding TiO 2−x NPs, the correlation between the electronic structures of the TiO 2−x NPs-embedded PDMS and the energy conversion mechanism of the TENG were investigated. Figure 4 a shows the hybridized molecular orbital structures in the conduction band of the TiO 2−x NPs-embedded PDMS as a function of the weight ratio of TiO 2−x NPs measured by XAS. The normalized intensities of the oxygen K-edge spectra of pristine PDMS and TiO 2−x NPs-embedded PDMS layers directly reflect the unoccupied hybridized states by the transition of electrons from the occupied O 1 s state [ 17 ]. Figure 4 a co-plots the normalized O-K edge XAS spectrum of TiO 2−x NPs (gray lines) for easy comparison with the changes in the TiO 2−x NPs-embedded PDMS layer spectra. In particular, XAS analysis is a surface sensitive analytical method that measures the current flowing on the sample surfaces after incident X-ray exposure. Therefore, this is suitable for analyzing tribo-materials whose output performance changes according to surface conditions. The XAS spectra of TiO 2−x NPs are located in the two strong resonance peaks at 531.5 and 534 eV [ 18 ]. With an increased weight ratio of TiO 2−x NPs from 0 to 30%, the electronic structure of PDMS is clearly changed by the addition of the electronic structure of TiO 2−x NPs. These changes in the tribo-material surface can cause changes in the tribo-series, resulting in reduced tribo-electrification effects. For the experimental demonstration of our claim, KPFM measurements were carried out to detect the surface potential of different surfaces according to the weight ratio of TiO 2−x NPs, as shown in Figure 4 b. As the weight ratio of TiO 2−x NPs increased from 0 to 30%, the average surface potential dramatically changed. Specifically, the TiO 2−x NPs 30% sample became a positively charged material. Moreover, considering the SEM image in Figure 1 d, the electron path can be formed due to NPs clustering in the case of the TENG containing over 5% embedding TiO 2−x NPs. The electron path formed by the NPs cluster can move the electrons induced on the surface and increase the leakage current, as shown in Figure S4 (supporting information) , which may be a reasonable cause to reduce TENG performance above 5% embedding TiO 2−x NPs, as shown in Figure 4 c. To demonstrate this with a practical application, we designed a windmill system to harvest the wind energy utilizing the pristine TENG and optimal TENG devices (TiO 2−x NPs 5% embedded TENG), as shown in Figure 5 a. In the presence of wind flow, the windmill blades are forced to rotate and lead to the rotation of a quad nose cam. Subsequently, the quad nose cam transmits its rotation motion to linear motion on the top plate of the TENG. Resulting from that motion, the TENG operates in the vertical direction via contact and separation mode repeatedly ( Figure 5 b), which produces the TENG electrical output. Moreover, the relation of wind speed and output performance of the windmill-integrated TENG was also analyzed at various wind speeds, as shown in Figure 5 c. The measured voltage is clearly shown in the output performance of both the pristine and optimal TENGs, which show linear enhancement upon increasing the wind speed applied to the windmill blades. The peak voltage values of the windmill attached to pristine TENG at the wind speeds of ~10, 15, 20, 25, 30, and 35 are 70, 92, 114, 130, 182, and 190, and that for the windmill integrated with an optimal TENG are 94, 172, 220, 268, 304, and 304, respectively. The output data in Figure 5 c also show a linear enhancement regarding the number of pushing cycles, i.e., pushing frequency and force on the top plate of the TENG as the wind speed increases. With faster wind speeds, the output performance of the windmill TENG can be enhanced. The results clearly demonstrate that our windmill TENG system can efficiently harvest wind energy."
} | 5,065 |
20890598 | null | s2 | 4,845 | {
"abstract": "Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives."
} | 251 |
27194953 | PMC4853940 | pmc | 4,847 | {
"abstract": "Inorganic storage granules have long been recognized in bacterial and eukaryotic cells but were only recently identified in archaeal cells. Here, we report the cellular organization and chemical compositions of storage granules in the Euryarchaeon , Archaeoglobus fulgidus strain VC16, a hyperthermophilic, anaerobic, and sulfate-reducing microorganism. Dense granules were apparent in A. fulgidus cells imaged by cryo electron microscopy (cryoEM) but not so by negative stain electron microscopy. Cryo electron tomography (cryoET) revealed that each cell contains one to several dense granules located near the cell membrane. Energy dispersive X-ray (EDX) spectroscopy and scanning transmission electron microscopy (STEM) show that, surprisingly, each cell contains not just one but often two types of granules with different elemental compositions. One type, named iron sulfide body (ISB), is composed mainly of the elements iron and sulfur plus copper; and the other one, called polyphosphate body (PPB), is composed of phosphorus and oxygen plus magnesium, calcium, and aluminum. PPBs are likely used for energy storage and/or metal sequestration/detoxification. ISBs could result from the reduction of sulfate to sulfide via anaerobic energy harvesting pathways and may be associated with energy and/or metal storage or detoxification. The exceptional ability of these archaeal cells to sequester different elements may have novel bioengineering applications.",
"conclusion": "5. Conclusion The occurrence, location, size, and compositions of two types of intracellular bodies in the thermophilic archaean Archaeoglobus fulgidus VC16 are demonstrated for the first time. Each is composed of distinct primary and secondary metals and is likely involved in nutrient and/or energy storage. The presence of polyphosphate bodies in the archaea along with bacteria and Eukarya suggests an ancient origin of these structures. Future studies are needed to explore the biogenesis and physiological uses of these inclusion bodies.",
"introduction": "1. Introduction \n Archaeoglobus fulgidus strain VC16 is a hyperthermophilic, sulfur oxide-reducing, anaerobic archaeon. Belonging to the Archaeoglobales division of the Euryarchaeota, the species is commonly found in marine thermal vents, hot springs, and thermophilic oil field waters. The production of thiosulfate as well as hydrogen sulfide has been implicated in oil and gas souring and in oil pipeline corrosion [ 1 , 2 ]. A. fulgidus can produce biofilms in response to stress which may be important for metal detoxification, surface adherence, and nutrient acquisition [ 3 ]. Due to A. fulgidus being hyperthermophilic, A. fulgidus cells are used for metal sequestration in water treatment and serve as a source of high temperature stable enzymes. \n A. fulgidus VC16 is able to grow chemoheterotrophically, thereby reducing sulfate. Initially isolated from marine hydrothermal vents in Italy [ 4 , 5 ], it can utilize a variety of carbon compounds as electron donors for sulfate, as well sulfite and thiosulfate reduction to sulfide [ 6 ]. Some A. fulgidus strains are also capable of chemolithotrophic growth and use hydrogen as an electron donor with oxidized sulfur compounds as electron acceptors [ 7 ]. A. fulgidus VC16 cells are morphologically spherical to irregularly coccoid in shape and some strains may be motile by appendages, possibly by flagella [ 5 , 6 ]. In this study, we employ a combination of cryo electron microscopy (cryoEM), cryo electron tomography (cryoET), and electron dispersive X-ray (EDX) spectroscopy analyses to identify and characterize high-density inclusion bodies (also called granules) distributed within the cytoplasm of A. fulgidus VC16. We show that these structures are of two types which can each reach ~240 nm in diameter. One type is rich in compounds containing phosphorus and oxygen and the other in those containing iron and sulfur: both are typically positioned nearby or on the cell membrane and at opposite sides of the cell when the two types are present. Potential functions of these inclusion bodies include phosphate, iron, and sulfur deposits and energy storage in the form of polyphosphates and iron polysulfides, as well as metal sequestration in response to cell toxicity.",
"discussion": "4. Discussion Intracellular granules have been characterized in bacteria, eukaryotes, and archaea utilizing both energy-filtered TEM and energy dispersive X-ray spectroscopy in order to analyze the elemental composition of the inclusion bodies [ 13 , 20 – 22 ]. Our current study demonstrates the coexistence of two types of granules, PPB and ISB, in the same A. fulgidus VC16 cell located near cell membranes. Notably, the PPBs lack the iron and sulfur elements abundant in the ISBs, while the ISBs lack the phosphate and oxygen abundant in the PPBs. Likewise, the less abundant elements present in the PPBs (magnesium, calcium, and aluminum) are low to absent in the Fe-S bodies and, conversely, the PPBs lack iron. The ratios of the predominant elements seen in PPBs and ISBs are diagnostic: the former exhibits a characteristic oxygen, phosphorus, iron, sulfur ratio of 2 : 1 : 0 : 0, while the latter has an elemental ratio of 0 : 0 : 1 : 2. This elemental analysis also provides a potential approach to high throughput STEM assisted cell screening to characterize inorganic granules in A. fulgidus and in other archaeal and bacterial strains. Further analyses of the variations in granule density and at different stages of development are also now possible. Both types of granules (PPBs and ISBs) within A. fulgidus strain VC16 are positioned in characteristic membrane-adjacent locations (Figures 2 – 4 ). These uniform locations suggest a means to position each granule type within the cell along with the genetic ability to spatially and temporally program granule formation. The precise positioning of granules is also found in a magnetotatic bacterium which produces a magnetosome structure composed of arranged magnetic granules, though there is no evidence for the formation of ISBs or PPBs via invagination of the inner membrane or specific associated proteins, as found with the magnetosome [ 23 , 24 ]. The effect of cell nutrition on A. fulgidus VC16 granule formation was also examined where limiting the carbon or phosphate supply resulted in formation of no PPBs or ISBs per cell. This observation is consistent with the ability of the cell to monitor environmental conditions and control elemental sequestration accordingly. Potential roles of the PPB granules in A. fulgidus were mentioned above based on prior PPB studies in the bacteria and eukaryotes over the past thirty years [ 17 – 19 , 25 ]. Besides roles in phosphate storage and cell energy capture, other PPB functions include roles in chromosome replication, cell division, metal chelation, and metal detoxification. The role(s) of the newly described ISB granules in A. fulgidus are unknown. Besides potential roles in iron or sulfur-based energy storage, ISB granules may also have roles in metal sequestration and/or detoxification by analogy to the PPBs. A. fulgidus species thrive in highly reducing and metal rich environments. Fluids flowing from hydrothermal vents, for example, from black and white smoker vents, are reported to contain dissolved calcium, copper, zinc, iron, manganese, and strontium in the low to high micromolar range [ 26 ]. Nearby sea waters rich in dissolved magnesium, phosphate, and sulfate recirculate within these vent fluids which would supply primary sources of phosphorus, sulfur, and other metal cations. These black and white smokers also have other associated metal precipitates and soluble by-products that may be toxic to nearby microbes. The ecology of these habitats is relatively unstudied. As shown in Figures 5 and 6 , the PPB and ISB granules are positioned in characteristic cell locations nearby or on the cytoplasmic membrane surface. This location suggests that the cell possess a genetic means to initiate development of each granule type in a spatial and temporal context. This membrane proximity would presumably facilitate accumulation of nutrients from the environmental surroundings for chemical storage and utilization as cell reserves. We expect that enzyme machinery to facilitate PPB and ISB formation resides at or nearby the cell membrane. PPBs near cell membrane could coordinate accumulation of phosphate from the cell exterior via high affinity uptake systems (e.g., AF0791, AF1356–1360, and AF1798) with colocalized polyphosphate polymerizing enzymes nearby for granule assembly. The presence of structures or scars visualized along the inner surface of the cytoplasmic membrane supports the notion of associated enzyme machineries (Sup. Figure S1). For example, polysulfur and/or iron depositing enzymes would be associated with the ISB granules. It was previously shown that A. fulgidus cells metabolize sulfate and sulfite as well as thiosulfate [ 6 ], and the pathway intermediates leading to sulfide production could provide potential substrates for granule formation. The genome contains two uptake systems for iron 2 (AF0246 and AF2394) and for iron 3 (AF 04302 and AF1401-1402), a highly unique iron storage ferritin (AF0834), a P-type copper transporter (AF1052), and a copper chaperone (AF0346), plus one sulfate ABC-type system (AF00923). Tests of their annotated roles and means of granule formation await the development of genetic tools. From the A. fulgidus VC16 cryoEM measurements of cell envelope and granule dimensions we can accurately document the cell compartment volumes and surface areas. A spherical cell of one micron in diameter would have an overall cell volume of 0.524 μ m 3 (volume, V = (4/3) πr \n 3 ). Using the following measures of the cell membrane cross section (~37 angstroms thick), the S-layer lattice (~110 angstroms thick), and the periplasmic-like space (~130 angstroms thick) which is sandwiched between and separates the two structures ( Figure 2(a) ), the A. fulgidus volume is partitioned into 84.3% cytoplasm (0.441 μ m 3 ), 2.1% cell membrane (0.011 μ m 3 ), 7.2% cell periplasm (0.038 μ m 3 ), and 6.4% cell S-layer (0.034 μ m 3 ). The intracellular PPB and ISB granules observed in A. fulgidus cells can individually compose up to 1.4% of the cytoplasmic space (~230 nm diameter). Assuming the PPB granule density reported by Toso et al. [ 13 ], a single A. fulgidus PPB would store several-hundred-fold more energy in the form of phosphoanhydride bond energy than contained in the cellular ATP pool. Compared to the cross section of a typical E. coli cell envelope (~29 nm thick including the CM, periplasmic space, and outer membrane (OM)) the analogous A. fulgidus envelope dimensions are remarkably similar (~28 nm thick) [ 27 ]. Here, the S-layer lattice (~11 nm in cross section) replaces the bacterial peptidoglycan and OM layers (~6.9 nm for the E. coli OM) and may contribute to cell rigidity and shape [ 28 ]. The corresponding cross section dimensions of the archaeal and E. coli periplasmic spaces also differ by about 25% (~13 nm in A. fulgidus versus ~16 nm in E. coli ). They provide analogous roles while having remarkably different cell architectures and molecular compositions. Few examples of inorganic storage granules are reported in archaea in contrast to PPB granules in bacteria and eukaryotes [ 17 – 19 ]. We recently described the presence of PPBs in the methanogen Methanospirillum hungatei JF1 [ 13 ] which contained spherical granules of approximately 150 nm in diameter. They were positioned along the central axis of the cell and away from the cell membrane relative to A. fulgidus granules. The M. hungatei PPBs bodies also differed from A. fulgidus PPBs reported in this study, whereby the M. hungatei bodies contain iron plus calcium rather than magnesium, aluminum, copper, and calcium ( Figure 6(c) ). These data establish subclasses of PPBs in archaea which differ in the types of cations accumulated. Additionally, since A. fulgidus VC16 cells can possess both PPBs and ISBs, it is evident that this archaean can somehow discriminate between available cations and selectively incorporate them into their two granule types (e.g., the PPBs lack iron, while the ISBs lack Ca, Al, and Mg). Reports of PPB-like structures in other archaeal genera include several strains of Sulfolobus and Methanosarcina [ 20 , 22 ]. This study is the first to report ISBs in archaea, although formation of polysulfides and polythionates has been described in the phototrophic purple sulfur bacteria (e.g., Chromatiaceae and Ectothiorhodospira species [ 29 , 30 ]), where reduced sulfur compounds are oxidized as electron donors during anoxygenic light energy harvesting. In the chemoautotrophic species Thiobacillus ferrooxidans, “sulfur globules” were reported to contain an inner core of S 7–12 polysulfur plus an outer layer of S 19+ polythionates [ 31 ]. The molecular composition of the A. fulgidus VC16 ISBs is currently unknown. Inspection of negatively stained cell sections of A. fulgidus strain 7324, which is related to A. fulgidus VC16 , reveals the presence of electron dense bodies nearby the cell membranes (Figure 1; [ 1 ]). Although not described by the authors, these dark bodies likely contain polyphosphate (PPBs) and/or Fe-S (ISBs) reported in this study. Future investigations are needed to understand the nutritional, biochemical, and genetic basis for PPB and ISB granule formation in archaea as well as their physiological roles in cell metabolism/detoxification."
} | 3,412 |
30654480 | PMC6356191 | pmc | 4,848 | {
"abstract": "Nanosecond laser ablated metallic surfaces showed initial super-hydrophilicity, and then experienced gradual wettability conversion to super-hydrophobicity with the increase of exposing time to ambient air. Due to the presence of hierarchical structures and change of surface chemistry, the laser-induced Inconel alloy surfaces showed a stable apparent contact angle beyond 150° over 30-day air exposure. The wetting states were proposed to elucidate the initial super-hydrophilicity and the final super-hydrophobicity. The basic fundaments behind the wettability conversion was explored by analyzing surface chemistry using X-ray photoelectron spectroscopy (XPS). The results indicated that the origins of super-hydrophobicity were identified as the increase of carbon content and the dominance of C–C(H) functional group. The C–C(H) bond with excellent nonpolarity derived from the chemisorbed airborne hydrocarbons, which resulted in dramatic reduction of surface-free-energy. This study confirmed that the surface chemistry is not the only factor to determine surface super-hydrophobicity. The laser-induced super-hydrophobicity was attributed to the synergistic effect of surface topography and surface chemical compositions. In this work, the corresponding chemical reaction was particularly described to discuss how the airborne hydrocarbons were attached onto the laser ablated surfaces, which reveals the generation mechanism of air-exposed super-hydrophobic surfaces.",
"conclusion": "4. Conclusions In this study, the super-hydrophobic surfaces with line or grid pattern were successfully finished on IN718 material via nanosecond laser ablation. The as-prepared surfaces initially showed super-hydrophilicity. During the air exposing period, the laser fabricated surfaces experienced gradual wettability conversion to steady super-hydrophobicity. The wettability conversion mechanism was extensively explored based on the analyses of surface morphology and surface chemistry. It can be concluded that after laser treatment, the gradual conversion of APCA related with the chemisorbed airborne contaminants from air moisture. The attached organic matters can introduce nonpolar C–C(H) bond onto the laser-induced surfaces, which can slowly lower surface free energy. In terms of surface morphology and surface roughness, the initial super-hydrophilicity was analyzed based on the Wenzel theory, and the comparison of super-hydrophobicity between the line- and grid-patterned surfaces was also elucidated. Noticeably, this study confirmed that the surface chemistry is by no means the only factor to determine surface super-hydrophobicity. The laser-induced super-hydrophobicity was attributed to the synergistic effect of the surface morphology and the surface chemistry. This research not only benefits academic to better understand the generation of air-exposed super-hydrophobic surfaces by laser, but also further inspire factories to alter surface chemistry and effectively produce durable super-hydrophobic surfaces for engineering applications.",
"introduction": "1. Introduction As a precipitation-hardenable super-alloy, Inconel 718 alloy (IN718) has attracted more attention due to its significant potentials in gas turbines, rocket motors and spacecraft. This material shows perfect high strength, thermal and wear resistance in serious conditions [ 1 , 2 ]. However, IN718 alloy is regarded as a difficult machining material by conventional techniques due to its low material removal rate, high shear strength, and excessive tool wear [ 3 ]. Previous literatures have demonstrated that surface modification with a super-hydrophobic property plays an important role in various applications, for instance, self-cleaning [ 4 , 5 ], anti-bacteria [ 6 ], anti-corrosion [ 7 ], enhanced heat transfer [ 8 ], and drag reduction [ 9 , 10 ]. It is therefore urgent to find alternative method to manufacture IN718 alloy with super-hydrophobic property to expand its value in potential applications. By investigating materials from natural creatures including lotus leaves and butterfly wings, two main factors for achieving super-hydrophobic surfaces are rough micro/nano structures and the low-free-energy coatings [ 11 ]. Up to now, many scholars design functional interface with super-hydrophobicity using the approaches of thermal embossing [ 12 ], sol-gel [ 13 ], chemical etching [ 14 , 15 ], electrodeposition [ 16 , 17 ], chemical vapor deposition [ 18 ], and laser ablation [ 19 , 20 , 21 , 22 , 23 , 24 ]. Among the above-mentioned methods, ultrafast laser ablation is intensively used to produce micro/nano-scaled devices for various applications including micromechanics [ 25 ], optics [ 26 ], optomechanics [ 27 ], and microfluids [ 28 , 29 ]. Particularly, laser process is considered as a facile technique to directly obtain super-hydrophobic surfaces on varieties of materials [ 30 ]. Laser ablation results in stable 3D binary rough structures through precise control, which is necessary to prolong surface durability [ 31 ]. For instance, Sun et al. successfully fabricated the super-hydrophobic silicon wafers with various regular surface patterns employing excimer laser, showing an apparent contact angle of 163 ± 1° [ 32 ]. Kietzig et al. used femtosecond laser to produce microstructures on several pure metallic surfaces. The ablated substrates initially presented super-hydrophilic property. With the aging time exposed to ambient air, the contact angle reached about 160° with very small hysteresis [ 33 ]. In our previous paper, the titanium substrate was ablated by nanosecond laser, the fabricated surface also showed initial super-hydrophilicity. After being exposed to air for approximately 30 days, the laser-induced titanium substrates presented slow wettability conversion to final super-hydrophobic state [ 34 ]. It is worthwhile to mention that there are some differences of the time-dependent contact angle hysteresis between the nanosecond laser-induced surfaces and the femtosecond laser-induced surfaces. For example, Rung et al. showed an increased contact angle hysteresis over time after the femtosecond laser ablation [ 35 ], while Yang et al. presented a decreased contact angle hysteresis after the nanosecond laser ablation [ 36 ]. These findings can enable laser ablation to various applications, such as ink-jet printing, immersion lithography [ 37 ], self-cleaning [ 38 ], and so on. Many previous papers reported that surface topography and surface chemistry contributed to the super-hydrophobicity of the laser ablated surfaces [ 39 , 40 ]. However, the relationship between laser-induced surface topography and surface chemistry was seldom investigated. By exploring the fresh and aged pristine IN718 surfaces as well as the laser ablated IN718 surfaces, we investigated the mechanism of time-dependent apparent contact angle and contact angle hysteresis, and found that the surface chemistry was by no means the single factor to determine laser-induced super-hydrophobicity. In this study, the IN718 substrates were ablated with line or grid pattern by nanosecond laser. The as-prepared surfaces initially presented a super-hydrophilic nature and then reached a steady super-hydrophobic state over 30 days air exposure. Surface morphology was observed by scanning electron microscopy (SEM), indicating that the laser-induced binary rough structures amplified the initial super-hydrophilicity based on the Wenzel theory. The roughness-based explanation was also proposed to elucidate the level of final super-hydrophobicity. The variations of surface chemistry detected using X-ray photoelectron spectroscopy (XPS) implied that the air-exposed wettability conversion could be ascribed to the increase of carbon content and reduction of surface polarity, due to the chemisorbed organic matters from air moisture. Particularly, the fresh and aged flat-IN718 substrates were investigated in this research, which revealed that the laser-induced super-hydrophobicity was attributed to the synergistic effect of surface roughness and chemisorbed organic substances.",
"discussion": "3. Results and Discussion 3.1. Surface Morphology The dashed lines in Figure 1 illustrates the movement of laser pulse, and the pulse distance can be calculated by the equation: d = V / f with f as the repetition rate and V as the scanning speed [ 44 ]. The laser pulse number of 24 per spot can be obtained by the used laser parameters shown in Table 2 . In order to investigate the laser ablation process, we just fabricated a single groove on the IN718 surface as shown in Figure 2 a. The typical SEM images show a distinct μ-channel was formed by the ablation of confined region, which resulted in the localized melting, evaporation and solidification in sequence. Meanwhile, it can be seen from Figure 2 b that many irregular particles with the size from several hundred nanometer to micrometer were created and redeposited on the brim of the formed channel or the pristine substrate. We conjecture that the formation of this nano/micro particles was due to the ejection effect during the laser ablation process [ 45 , 46 ]. Figure 2 c,e represent the SEM images of the line- and grid-patterned surfaces after laser ablation, and their corresponding high-magnified image are shown in Figure 2 d,f, respectively. It is noted that many micro-scaled μ-channels with nano-scaled irregular particles were formed, creating the hierarchical micro/nano structures on the line-patterned IN718 substrate. Obviously, due to the overlap of bidirectional laser scanning, the grid-patterned surface experienced more serious ablation effect, causing all the ablated regions were covered by re-solidified materials. Compared with the line-patterned surface, no virgin IN718 area was observed on the grid-patterned surface. The high magnified SEM image also shows large numbers of submicron or nano particles covered on the laser-induced pillars. The generation of hierarchical topographies was due to laser ablation which can cause an increase temperature on the IN718 interface because of the absorption of laser energy. The μ-channels or μ-pillars combined with many irregular particles generated the hierarchical surface structures, leading to the increase of average surface roughness. As a result, many air pockets can be trapped underneath the water droplet, decreasing the contact area between liquid droplet and the laser-induced surfaces. 3D profiles and average surface roughness ( R a ) of the laser ablated samples with line and grid patterns were further measured using laser confocal microscopy. Figure 3 a demonstrates that the average width ( W ) of the laser ablated single μ-groove was 29 ± 5.8 μm and the average depth ( D ) was 25 ± 3.2 μm. It is clear from cross-sectional image that after laser ablation process, the molten materials were resolidified on the brims of the ablated groove (as shown the red box in Figure 3 a). Therefore, the surface morphology changed significantly after laser ablation process, and as-prepared surfaces became obviously rough. 3D profiles of laser-induced surfaces with line and grid pattern are displayed in the Figure 3 b,c, respectively. It is noted that the grid-patterned surface was much rougher than the line-patterned one, which can be proved by the average surface roughness values ( R a ). The results indicate that the as-prepared surface with line pattern had an average surface roughness value of 18 ± 1.2 μm, and such roughness value on grid-patterned surface was increased to 26 ± 1.2 μm. According to the Wenzel theory, the surface roughness has a close relationship with the apparent contact angle. In the following section, the initial super-hydrophilicity will be extensively discussed in terms of the surface roughness. 3.2. Wettability The line- and grid-patterned surfaces were exposed to ambient air after laser ablation, and surface wetting property was evaluated through measuring the APCAs. The increase of APCA implies the increment of hydrophobicity. The time-dependent APCAs for the air-exposed patterned surfaces are shown in Figure 4 . The fresh pristine IN718 surface (IN-i) presented hydrophilic property since its APCA was only 45.2 ± 0.6°. The aged pristine IN718 surface (IN-ii) also showed a hydrophilic character with the APCA of 49.5 ± 3.5°, almost the same with the fresh polished surface (IN-i), indicating that the surface wettability was not obviously changed even though the polished IN718 surface had been stored in the ambient air for long period. However, the initial as-fabricated IN718 surfaces exhibited super-hydrophilic character immediately after laser ablation. When the liquid contacted the laser-induced hierarchical rough surface, the water droplet could quickly spread out and was almost completely penetrated into the surface channels. It was therefore unable to obtain APCA value. Within 4 days after laser ablation, this super-hydrophilic state on fresh laser-induced IN718 surfaces was particularly pronounced. The initial conversion from original hydrophilic to super-hydrophilic was possibly due to the modification of surface morphology. The amplified hydrophilicity can be described by Wenzel theory in terms of increase of average surface roughness value [ 47 ].\n (1) cos θ w = rcos θ 0 \nwhere θ w denotes the APCA of the laser-induced rough surface. Because of the difficulties to prepare the ideal solid surface, θ 0 is in accordance to the APCA of the fresh polished IN718 surface (i.e., the smooth IN-i surface). r > 1 is the average surface roughness value ( R a ). Equation (1) implies that with the increment of surface roughness, the APCA will decline for a pristine hydrophilic surface. Conversely, the APCA will grow for a pristine hydrophobic surface. In terms of hydrophilic IN718 material, at early stage of wettability evolution, the fresh line- and gird-patterned surface showed ultralow APCA due to the enhancement of average surface roughness. In addition, the oxidation effect during the laser ablation process could result in a layer of surface oxide. This may be another possible reason to explain the occurrence of initial super-hydrophilicity because the produced oxide has a hydrophilic nature, which will be discussed later. It is noted that after 5 days air exposure, APCAs of the two patterned surfaces started to grow. Interestingly, on the Day 5, the line-patterned surface showed a larger APCA comparing with the grid-patterned surface, which resulted from the difference of surface topography. This is because the grid-patterned surface presented no virgin IN718 materials left, leading to a higher surface polarity due to more serious laser ablation effect. On the contrary, some virgin areas presented on the line-patterned surface can slightly balance the surface polarity after the laser ablation. From Day 5 to Day 10, the APCAs witnessed a sharp increase to around 140°. Posteriorly to 15 days, the APCA of line- and grid-patterned surfaces showed slight increase. Over a period of 20 days, the growing trend stopped. The as-prepared surfaces showed relatively steady APCAs in the following days. Over 30 days air exposure, the ablated surfaces showed typical super-hydrophobicity with the relative steady APCA of 152.3 ± 1.2° for line-patterned surface, and 156.8 ± 1.1° for the grid-patterned surface. In addition, the line-patterned surface showed a contact angle hysteresis of 7.6 ± 1.0° (advancing contact angle: 155.2 ± 1.0° and receding contact angle: 147.6 ± 1.0°). The grid-patterned super-hydrophobic surface had a relative smaller contact angle hysteresis of 6.2 ± 1.0° (advancing contact angle: 159.6 ± 1.0° and receding contact angle: 153.4 ± 1.0°). It is revealed that the aged laser-induced samples presented the unique super-hydrophobic property due to the high APCA greater than 150° and low contact angle hysteresis less than 10° [ 48 , 49 ]. Therefore, it is concluded that after laser treatment, the as-prepared surfaces presented initial super-hydrophilicity, then slowly reached super-hydrophobic state in ambient air over 30 days. The wettability conversion mechanism and the difference of stable APCAs between the line- and grid-patterned surfaces will be extensively discussed in Section 3.4 . 3.3. Surface Chemistry During the laser ablation process, surface oxidation reaction was inevitable, resulting in the formation of oxide layer on the laser treated samples. Previous literatures have clearly demonstrated that the formation of hydrophilic oxide layer will produce large number of surface defects. Therefore, the created oxide layer is not wanted because it will considerably increase surface free energy and prolong the interval for the laser-induced surfaces to reach super-hydrophobic property [ 44 ]. In this study, we mainly focus on the insights into the slow wettability conversion on the line- and grid-patterned surfaces after being exposed to the ambient air. It has been reported that growth of surface carbon, which can reduce surface polarity, plays an important role in the air-exposed wettability conversion to super-hydrophobicity [ 36 , 44 ]. Therefore, the mechanism of time-dependent wettability was investigated though analyzing surface chemical compositions of pristine IN-i and IN-ii surfaces, super-hydrophobic IN-iii and IN-iv surfaces using XPS. The XPS spectra and the corresponding surface elemental components are shown in Figure 5 and Table 3 , respectively. The displayed spectra indicate that three main metallic peak signals of Ni 2p, Cr 2p and Fe 2p were presented on these surfaces. Besides, C 1s and O 1s peaks appeared at approximately 285.0 eV and 531.5 eV, respectively. It is noted from Table 3 that the fresh pristine surface (IN-i) possessed over 20% carbon, instead of zero, indicating that the fresh surface was not free of carbon and was deposited by some contaminants containing carbon element. We conjecture that the deposited carbon possibly came from two key sources: the absorbed organic matters before XPS measurement and the residual oil in the XPS vacuum chamber. Compared to the fresh pristine surface (IN-i), the relative carbon content on the as-prepared super-hydrophobic surfaces (IN-iii and IN-iv) showed significant increase after the patterned surfaces were exposed to air over 30 days. Correspondingly, the relative oxygen and metallic elements declined since the total content of carbon, oxygen and metallic elements was 100%. Stunningly, the carbon content on the aged pristine IN718 surface (IN-ii) considerably rose to 61.29% when exposed to ambient air for over one year, which implies that a vast of complex contaminants having large amount of carbon were absorbed on the flat IN718 surface. It is believed that the surface morphology showed no obvious change during the exposure to air condition. Thus, the surface wettability conversion was related with the modification of surface chemistry. The growth of surface carbon content was due to the attachment of airborne hydrocarbon contaminants from air moisture, causing the reduction of the surface free energy. It can be concluded that the surface hydrophobicity was enhanced due to the combination of surface hierarchical structures and the reduction of surface free energy. Therefore, the relative content of absorbed airborne hydrocarbon can be characterized by the amount of surface carbon. The deconvolution of C 1s was performed utilizing the software of CasaXPS in order to further analyze the carbon state and explore the chemisorption mechanism of airborne hydrocarbon. Here, the deconvolution of C 1s spectra presented four contributions as shown in Figure 6 . One contribution located at around 284.7 eV was assigned to hydrocarbon chains or graphitic structure (C–C(H)). The C–O contribution was related with the alcohols/ether, the C=O functional group corresponded to aldehydes/ketones, and the O–C=O bond contributed to carboxyl/ester matters. The binding energies at approximately 289.6, 287.5, and 285.6 eV were corresponding to the functional group of O–C=O, C=O and C–O, respectively. Generally, C–C(H) is considered as the nonpolar bond, contributing to the increase of surface hydrophobicity. Otherwise, ether, carbonyl and carboxyl present hydrophilic property because of high polarity [ 50 ]. Therefore, the relative amount of C–C(H) bond can be used as an indicator to characterize the surface wetting property. In addition, the relative concentrations of C–C(H) bond on the studied surfaces were compared. The results indicate that the fresh pristine surface (IN-i) presented very small amount of C–C(H) content and thus polar sites were the dominance of surface free energy, resulting in the hydrophilic property. However, although the aged flat surface (IN-ii) showed hydrophilic property, it possessed the highest C–C(H) concentration of 87.32% among the four samples. The accumulation of organic contaminants contributed to the large amount of C–C(H) concentration on the aged pristine substrate. When the line- and grid-patterned samples with unique surface texture were stored in ambient air over 30 days, their relative C–C(H) concentrations were both over 70%, which could contribute to the super-hydrophobicity due to the dominance of the nonpolar sites. Interestingly, slight difference of C–C(H) concentration is noticed between the line-patterned surface (70.52%) and grid-pattered surface (78.07%). We conjecture that the higher C–C(H) concentration could account for the larger APCA on the as-prepared super-hydrophobic surfaces. It is reported that there are various organic matters possessing nonpolar short chain molecules, for example, acetic acid, formic and methanol [ 51 ]. Thanks to the carboxylation effect, surface free energy can be continuously decreased due to the gradual chemisorption of nonpolar chains from air moisture. The time-dependent wettability conversion on the as-prepared surfaces can be ascribed to this depolarization effect. This aspect will be extensively discussed in the following section. 3.4. Wettability Conversion Mechanism The formation of a carbonaceous film with nonpolar sites will significantly reduce surface polarity, making the laser-induced surfaces more hydrophobic. The attached airborne contaminants originating from air moisture, such as acetic acids, formic and polymeric hydrocarbons, could introduce short nonpolar molecules and thus decrease surface free energy. In this process, surface hydroxylation is regarded to play a key role in the chemisorption mechanism. Laser ablation can produce many unsaturated metal and oxygen atoms which served as strong Lewis acid and base pairs, respectively [ 52 ]. Once reacting with the dissociated OH − and H + from interfacial water vapor molecules, the Lewis acid and base pairs will be quickly hydroxylated, leading to the reduction of surface free energy due to the weakness of Lewis acid-base sites. However, surface hydroxylation cannot elucidate the slow wettability conversion as the hydroxylation reaction is very fast. On the other hand, the surface with hydroxides present highly adhesive property to water molecules, implying that the hydroxylation effect is not the reasonable explanation to elucidate the gradual wettability conversion. However, the hydroxyls generated in the hydroxylation process can absorb the carboxylates (for instance, acetic acids, formic) and other airborne organic matters (such as polymeric hydrocarbons). Once these organic matters reacted with hydroxyls, their nonpolar chain R containing C–C(H) functional groups can be chemisorbed onto the laser-induced surfaces, leading to the reduction of surface polarity [ 53 ]. The corresponding chemical reaction can be described by the following Equation (2): (2) Ni - OH + RCOOH ( g ) → RCOO - Ni + H 2 O This chemisorption process to depolarize surface energy is relatively slow and therefore can elucidate the step-by-step wettability conversion from initial super-hydrophi licity to final super-hydrophobicity when the laser-induced surfaces were stored in ambient air. The concentration of O–C=O group can further verify this assumption. The results infer that on the IN-i surface, the O–C=O concentration was only 2.39%. However, 30 days later, the relative concentration of O–C=O bond dramatically rose to 5.24% for the line-patterned surface, and to 7.01% for the grid-patterned surface. Although the polar O–C=O functional group was also attached on the laser ablated surfaces, these surfaces were dominated by the nonpolar C–C(H) groups. It is therefore possible for the laser patterned surfaces to show super-hydrophobicity. The results confirm that the condensation reaction is conducive to the gradual super-hydrophobicity due to the absorption of nonpolar molecules, which leads to the slow reduction of surface polarity. Remarkably, although the aged pristine IN718 sample with flat surface structure had the largest carbon content as well as the highest relative C–C(H) concentration, it still presented the hydrophilic property. Thus, except for the modification of surface chemistry, the laser-induced wettability conversion should be explored further based on surface topography. Previous literatures proved that the coating of low-free-energy substance on flat surfaces cannot ensure the super-hydrophobicity [ 54 , 55 ], implying that surface chemical modification is not the only factor to determine surface super-hydrophobicity. Obviously, the difference of the aged pristine sample (IN-ii) and the as-prepared super-hydrophobic surfaces (IN-iii and IN-iv) is the surface roughness. Therefore, the influence of surface roughness on the wettability conversion mechanism should be investigated in detail. In terms of the initial super-hydrophilicity, surface roughness of the laser-induced samples significantly increased due to the formation of μ-channels with micro/nano particles. In this case, the laser ablated surfaces can be modeled by Wenzel state (as shown in Figure 7 a), inferring that with the increase of surface roughness, the contact angle will decline regarding the pristine hydrophilic IN718 surface. Thus, the created hierarchical topographies amplified the effect of surface hydrophilicity, leading to super-hydrophilicity immediately after laser ablation. After stored in air for over 30 days, the line- and gird-patterned surfaces reached super-hydrophobic state due to the chemisorbed nonpolar organic molecules. Many studies utilized following simplified Cassie-Baxter equation to explain the conversion of super-hydrophobicity [ 56 , 57 , 58 , 59 ]: (3) cos θ c = f s ( 1 + cos θ 0 ) − 1 \nwhere θ 0 presents the APCA of the polished surface, and θ c is the APCA of the laser-induced rough surface. f s denotes the portion of solid/liquid area. However, this oversimplified Cassie-Baxter Equation (3) is not suitable to elucidate the wetting state of the aged super-hydrophobic surfaces due to the presence of laser-induced hierarchical topographies. According to the SEM and 3D images of the laser-induced surface, more accurate wetting state can be shown in Figure 7 b. Based on previous papers, it has been both experimentally and theoretically proved that the hierarchical multi-scale topography can enhance water repellency of surfaces [ 60 , 61 ]. This is because many air pockets were generated underneath the water droplet and the air cushion could prevent the liquid from further penetrating into the grooves. In this current study, the grid-patterned surface experienced much more serious laser ablation effect. Therefore, more air would be trapped underneath the water droplet and less solid area was wetted by water, which resulted in a better super-hydrophobicity on the grid-patterned surface. Because of the formed solid-air composite interface with low-surface-energy molecules, the dispensed water droplet could hardly penetrate into the micro/nano structures on the aged super-hydrophobic surfaces."
} | 7,000 |
38559250 | PMC10979890 | pmc | 4,850 | {
"abstract": "Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N -acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa , yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also caused mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR specific inhibitory activities over a range of concentrations below that causing toxicity. Ortho- Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho -vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho -vanillin as a small molecule QS modulator against RhlR.",
"introduction": "INTRODUCTION Many bacteria sense and respond to changes in population density using a gene regulation system called quorum sensing (QS). QS can regulate diverse behaviors including light production in marine bioluminescent bacteria, virulence factor production in plant and animal pathogens, and motility in many soil bacteria ( 1 ). In Proteobacteria, one type of QS system involves N -acyl L-homoserine lactone (AHL) signals (for reviews, see refs. ( 2 , 3 )). AHLs are produced by LuxI-type signal synthases, and detected by LuxR-type signal receptors, which are cytoplasmic transcriptional factors. At low population densities, AHLs are produced at low levels and accumulate in the local environment with increasing population density. The AHLs diffuse in and out of the cell, although active efflux can also contribute to the export of certain long chain AHLs ( 4 ). AHLs bind to the LuxR-type receptor protein and—for most of the known associative-type receptors—when they reach a critical concentration, they cause conformational changes to the protein that enable binding and activation of target gene promoters. AHLs interact with their cognate LuxR protein by making a series of hydrogen-bonding and hydrophobic contacts with residues in the ligand-binding pocket. AHL binding pockets vary structurally among LuxR family members to ensure specific responses to cognate AHLs, which differ in acyl chain structure. Pseudomonas aeruginosa is an opportunistic pathogen that can cause debilitating infections in immunocompromised patients and is difficult to treat due to its multi-drug resistance. P. aeruginosa has two LuxI/R-type systems, LasI/R and RhlI/R. The LasI/R system produces and responds to N- (3-oxo)-dodecanoyl L-homoserine lactone (3OC12-HSL), and the RhlI/R system produces and responds to N- butanoyl L-homoserine lactone (C4-HSL). Upon AHL binding, LasR and RhlR activate distinct and overlapping regulons ( 5 , 6 ). Among those are the genes encoding factors with known roles in virulence, such as the secreted toxins phenazine and hydrogen cyanide, proteases, and biofilm matrix proteins. These systems have been shown to be important for P. aeruginosa virulence in numerous acute animal infection models ( 7 – 11 ). Thus, P. aeruginosa QS has been proposed as an attractive target for the development of novel anti-virulence therapeutics ( 12 ). Over the past 30 years, there has been considerable effort to identify molecules that block QS in P. aeruginosa and other bacteria. These prior studies have identified several promising approaches such as inhibiting LuxI-type synthases ( 13 ), destroying or sequestering AHLs ( 14 ), or inhibiting LuxR-type receptors ( 15 ). The latter strategy has received the most attention to date in P. aeruginosa , with much focus on the LasR receptor, and more recently RhlR, in P. aeruginosa . As a result, several promising molecules have been identified that inhibit these receptors ( 16 – 19 ). These molecules have potencies in the high-nM to mid- to low-μM range. In general, the most potent molecules have been identified as a result of high-throughput screens of small molecule libraries or by making targeted changes to the native AHL or other promising lead compounds via chemical synthesis. In addition to these synthetic agents, there also has been widespread study of readily available molecules that can be re-purposed as QS inhibitors. Many of these compounds are natural products and were initially identified because of their ability to block QS-dependent phenotypes in the native species, not via studies of their ability to target specific QS pathways. These compounds include halogenated furanones ( 20 ), flavonoids such as baicalein ( 21 , 22 ) and several benzaldehydes such as cinnamaldehyde ( 23 – 28 ). Despite the widespread use of these molecules as chemical tools for studies of QS inhibition, relatively little is known of the specificity, potency, and mechanism of action for most of these compounds. New tools to study QS are of considerable interest, as many of the known chemical modulators have limitations, including relatively low potencies, efficacies, solubilities in aqueous media, and/or chemical stabilities. Consequently, re-purposed bioactive agents and readily-available natural products (and analogs) with promising QS inhibitory activities represents a valuable space to search for new chemical probes to study bacterial signaling. In this study, we used E. coli reporters to evaluate the ability of several naturally occurring benzaldehydes and related derivatives to inhibit the P. aeruginosa QS receptors LasR and RhlR. We focused on compounds reported to disrupt QS-dependent phenotypes in P. aeruginosa , such as cinnamaldehyde and salicylic acid, along with several previously unstudied compounds with some structural similarity, such as orsellinaldehyde and ortho -vanillin ( Fig. 1 ). We observed antagonism of the E. coli LasR and RhlR reporters at concentrations in the mid- to low-μM range, with ortho -vanillin showing the most promising effects. The compounds also caused mild reductions in growth and could nonspecifically antagonize a constitutive reporter at higher concentrations; however, at lower concentrations there was a suitable window of activity allowing for LasR and RhlR antagonism without any observable toxicity. In follow-up structure-function studies using LasR mutants, we found that critical AHL-binding residues in LasR were not required for ortho -vanillin to antagonize LasR. However, our results support that ortho-vanillin might specifically interact with RhlR. Together, our results indicate that naturally occurring benzaldehydes could have utility in QS inhibition and motivate future studies to develop this chemical scaffold into small-molecule tools to explore LuxR-type protein function and QS pathways.",
"discussion": "DISCUSSION The contribution of QS to a wide array of phenotypes, including virulence, in P. aeruginosa has attracted significant attention to the identification of QS inhibitors for use as chemical probes and in therapeutic development. Despite considerable work in this area, there are relatively few highly potent and selective QS inhibitors in P. aeruginosa and related proteobacteria. Most of these compounds target LuxR-type receptor proteins, including V-06–018 that antagonizes LasR in P. aeruginosa ( 18 ) and the chlorolactone AHL analog (CL) that antagonizes CviR from Chromobacterium violaceum ( 37 , 38 ). Beyond these classes of synthetic compounds, there are many naturally derived compounds or extracts that have reported activities as QS inhibitors in bacteria. For example, salicylic acid can downregulate production of the QS-controlled virulence factors pyocyanin and elastase and attenuate the ability of P. aeruginosa to infect plants ( 28 ). However, detailed studies to determine the molecular mechanisms by which these natural products elicit their effects on QS are limited. In this study, we evaluate the ability of salicylic acid, cinnamaldehyde, and several related benzaldehyde derivatives, to antagonize the P. aeruginosa LuxR-type receptors LasR and RhlR using heterologous reporters in E. coli . We provide evidence that one of these compounds, namely ortho- vanillin, can specifically antagonize these receptors within a lower range of concentrations in which they are not generally toxic. These results provide a basis to guide the use of these compounds in QS studies and suggest chemical scaffolds to advance for the design of new QS receptor antagonists. The investigations described here indicate that ortho- vanillin can specifically antagonize LasR and that it does so through a non-competitive mechanism ( Fig. 5 ). There are prior reports of other compounds that might inhibit LuxR-type receptors noncompetitively. Halogenated furanones, such as bromofuranone, have been shown to inhibit the Vibrio fischeri LuxR receptor noncompetitively ( 39 ). Inhibition might involve a mechanism of increasing turnover of the receptor protein in the cell ( 39 ), although bromofuranone can also be broadly toxic at inhibitory concentrations ( 40 ). Some flavonoids also have been reported to inhibit LasR noncompetitively, such as baicalein, although in the case of baicalein the mechanism is not known ( 22 ). Our discovery that ortho -vanillin can antagonize LasR noncompetitively adds to this list of noncompetitive antagonists. In the case of RhlR, ortho -vanillin appears to act as a specific, competitive antagonist in the E. coli reporter ( Fig. 7 ). Competitive inhibition is by far the most invoked mechanism for known LuxR-type inhibitors; the crystal structure of chlorolactone (CL) bound to CviR and stabilizing an inactive conformation provides perhaps the most compelling support for this mechanism ( 38 ). There are several other known competitive inhibitors of RhlR, most of which closely resemble its native ligand C4-HSL, and our prior detailed structure-function studies have revealed portions of the molecules that are essential for strong inhibitory activity ( 41 ). With the recently determined crystal structure of RhlR ( 35 ), it is now possible to carry out more detailed studies to better understand RhlR-ligand binding interactions, including with the native ligand C4-HSL. Such studies will be interesting to reveal important insight into the mechanism of RhlR-ligand interactions and advance the design of compounds that can modulate RhlR activity. Our results with E. coli reporters show that ortho- vanillin is more potent against RhlR than LasR. This difference could be due to the relatively small size of this molecule and/or its lack of an acyl tail. The natural ligand of LasR, 3OC12-HSL, has a long 12-carbon acyl tail, whereas the RhlR ligand C4-HSL has a much shorter 4-carbon acyl tail. Prior structure-function studies of LasR and 3OC12-HSL reveal that there are important hydrophobic contacts formed between the long tail of 3OC12-HSL and residues within the LasR binding pocket ( 42 ). These contacts contribute to the strength and specificity of the interaction with LasR. In addition, studies with V-06–018 analogs showed that shorter acyl tails weaken LasR interactions ( 43 ). In turn, we have shown that RhlR is both activated and inhibited by AHLs analogs with shorter tails. ortho- Vanillin largely lacks such a hydrophobic tail ( Fig. 1 ), which might weaken its ability to antagonize LasR, while enhance its ability to engage with RhlR. Our results support the idea that the hydrophobic tails of ligands play a critical role in the specificity and strength of interactions with LuxR proteins. As this competitive activity for ortho -vanillin in RhlR, and its non-competitive activity in LasR, were observed in E. coli reporter systems, additional experiments including in vitro studies will be necessary to provide further clarity into its molecular mechanisms of action and the hypotheses outlined here. The relative simplicity of the ortho -vanillin scaffold suggest straightforward routes to alter its structure and examine impact on potency and specificity, along with reducing any associated toxicity. Overall, these studies illustrate the importance of performing rigorous studies to determine the specificity and function of small molecule QS inhibitors to inform their use as research tools and other applications."
} | 3,290 |
32927721 | PMC7570729 | pmc | 4,851 | {
"abstract": "The swarm intelligence (SI)-based bio-inspired algorithm demonstrates features of heterogeneous individual agents, such as stability, scalability, and adaptability, in distributed and autonomous environments. The said algorithm will be applied to the communication network environment to overcome the limitations of wireless sensor networks (WSNs). Herein, the swarm-intelligence-centric routing algorithm (SICROA) is presented for use in WSNs that aim to leverage the advantages of the ant colony optimization (ACO) algorithm. The proposed routing protocol addresses the problems of the ad hoc on-demand distance vector (AODV) and improves routing performance via collision avoidance, link-quality prediction, and maintenance methods. The proposed method was found to improve network performance by replacing the periodic “Hello” message with an interrupt that facilitates the prediction and detection of link disconnections. Consequently, the overall network performance can be further improved by prescribing appropriate procedures for processing each control message. Therefore, it is inferred that the proposed SI-based approach provides an optimal solution to problems encountered in a complex environment, while operating in a distributed manner and adhering to simple rules of behavior.",
"conclusion": "5. Conclusions The proposed routing protocol overcomes the disadvantages of AODV and improves routing performance via the incorporation of collision avoidance, link-quality prediction, and maintenance techniques that mimic the ACO algorithm. SI can operate reliably in accordance with simple rules of behavior, while providing reliable solutions to given problems in complex environments. In this study, the performance of the routing protocol was improved by replacing the periodic “Hello” message with an interrupt message capable of detecting and predicting link disconnections. As observed, routing performance can be further improved via the addition of processing procedures for each message type. In addition, when the signal strength of a received data packet approaches the residual-energy threshold prior to energy loss, an increase in path-loss tendency can be observed. In contrast, when the signal strength of a received packet falls below the minimum threshold, a pre-warning packet is generated. Simulation results reveal that the proposed method is superior to the existing AODV and DSR routing protocols. The trade-off between the acquisition time of the sensing information and the energy consumed by the nodes is well resolved. In addition to providing a highly reliable and robust path for information transmission, the proposed method improves source-to-destination data latency, thereby reducing the frequency of the link disconnections and unnecessary control packet transmissions within the network. In view of its above-mentioned features and advantages, the proposed biomimetic algorithm is expected to be effectively utilized in large-scale communication networks. This requires sophisticated mathematical modeling of various biological systems, as well as rigorous performance verification based on several system-environment variables. In future, further research is required on techniques to maintain a continuous alternative path in the proposed algorithm and to increase the reliability of the path in one-way links that often occur in wireless network environments. In addition, some path improvements can be made, such as using the results of the proposed method to compare with other methods (e.g., recursive neural networks).",
"introduction": "1. Introduction Swarm intelligence (SI) originates from the collective behavior of life groups [ 1 ]. It is a mechanism that can overcome the limitations of the perceptions of individual agents. SI deals with complex systems, in which individual agents interact with each other with minimal communication with neighboring agents. Owing to this property, SI has been applied to several engineering applications [ 2 , 3 , 4 ]. The SI-based bio-inspired algorithm shows features such as stability, scalability, and adaptability in an environment where many individuals exist, the environment changes dynamically, available resources are restricted, and objects with heterogeneous characteristics remain distributed and autonomous. This is similar to a communication network environment, along with its service requirements [ 5 , 6 , 7 ]. In this study, the ant colony optimization (ACO) algorithm—one of the most effective bio-inspired algorithms used in communication and networking technology—has been employed to address the limitations of wireless sensor networks (WSNs) [ 8 , 9 , 10 , 11 ]. Accordingly, an ACO-based WSN-routing algorithm has been proposed in this paper. The basic idea of the ACO algorithm is to provide the trailing ant with decision results from the leading ant, such that the trailing ant can use this information to identify an optimal solution [ 12 , 13 , 14 ]. The ant system is suitable for use in large dynamic systems, such as WSNs, for two reasons. First, within an ant colony, ants search for routes while exclusively using local information (i.e., the number of pheromones). Therefore, the ACO-algorithm-based system corresponds to a distributed control system used for communication over a wireless sensor network. Secondly, the ACO algorithm adapts well to unpredictable changes in the environment. In WSNs, an ACO-algorithm-based system is designed to reroute data according to the network traffic, thereby improving the performance of the entire transmission network. Therefore, using SI, an algorithm that maximizes the service life of a sensor network, while simultaneously distributing data traffic across the same, can be developed. This paper presents the swarm-intelligence-centric routing algorithm (SICROA) for use in WSNs that aim to leverage the advantages of the ACO algorithm. The proposed algorithm considers each data packet transmitted to the base station as an ant, whereas each packet is considered the residual-energy pheromone of the sensor connected to the corresponding link upon selection of each successive hop in the path. Additionally, SICROA ensures efficient energy utilization by each node, thereby maximizing the sensor network’s lifetime, while also allowing it to adapt to variations in the network environment [ 15 ]. Section 2 discusses the ACO and other applied algorithms that form the basis of the proposed algorithm. Section 3 presents the realization of collision avoidance via use of interrupts, link-quality prediction, and maintenance technologies incorporated within SICROA. Using simulation results, Section 4 compares the handling of the general routing problem by the proposed and other protocols, thereby demonstrating the superiority of the proposed algorithm. Finally, Chapter 5 discusses the simulation results, conclusions, and future work."
} | 1,720 |
35424497 | PMC8978675 | pmc | 4,852 | {
"abstract": "A simple, one-step electrodeposition process was rapidly performed on a metal substrate to fabricate calcium superhydrophobic surfaces in an electrolyte containing calcium chloride (CaCl 2 ), myristic acid (CH 3 (CH 2 ) 12 COOH), and ethanol, which can avoid the intricate post-processing of surface treatment. The morphology and surface chemical compositions of the fabricated superhydrophobic surfaces were systematically examined by means of SEM, XRD, and FTIR, respectively. The results indicate that the deposited surfaces were mainly composed of calcium myristate, which can dramatically lower surface free energy. The shortest process for constructing a superhydrophobic surface is about 0.5 min, and the maximum contact angle of the as-prepared surfaces can reach as high as 166°, showing excellent superhydrophobicity. By adjusting the electrodeposition time, the structure of the cathodic surface transforms from the turfgrass structure, loose flower structures, larger and dense flower structures, secondary flower structures, and then into tertiary or more flower structures. The superhydrophobic surfaces showed excellent rebound performance with a high-speed camera. After a pressing force, their hardness increases, but the superhydrophobic performance is not weakened. Inversely, the bouncing performance is enhanced. This electrodeposition process offers a promising approach for large areas of superhydrophobic surfaces on conductive metals and strongly impacts the dynamics of water droplets.",
"conclusion": "4. Conclusions In conclusion, we have successfully reported a simple one-step electrodeposition process to construct a soft calcium superhydrophobic surface on the cathodic substrate with an electrolyte solution, and its rebound performance was also studied systematically. The needed shortest electrodeposition time can be largely shortened to 0.5 min. A maximum contact angle of 166° and a low rolling angle of less than 3° are achieved on the prepared cathodic copper surface. The cooperation of H 2 bubbles, flower-like structures, and calcium myristate with low surface energy play an important role in adjusting surface wettability. Such unique surface structures can contribute to trapping a large amount of air and showing a superhydrophobic performance. Moreover, this kind of calcium superhydrophobic surface has an excellent bound performance. After the pressing force, the bound performance of the as-prepared surface will be increased. The technique is expected to provide a promising way for the large-scale fabrication of superhydrophobic surfaces and promote the bound performance by a simple pressing force.",
"introduction": "1. Introduction Wettability is one of the most significant and fundamental characteristics of solid materials, and it depends on the surface chemistry and morphology. The chemical compositions of the surface determine the surface energy, which has a significant effect on its wettability. 1–4 There are many superhydrophobic surfaces in nature, such as rice leaves, rose petals, waterfowl feathers, strider legs, spider silk, and especially the famous lotus leaves. 5–7 Superhydrophobic surfaces have attracted wide attention because of their importance in scientific research and engineering. Two main factors are widely accepted common features of such surfaces: micro/nano rough structures and low surface free energy. Various methods and technologies have been employed successfully to prepare artificial superhydrophobic surfaces by controlling these two parameters. Several review articles have been published about different aspects of superhydrophobic surfaces. 8–11 However, most published methods suffer from many constraints such as specialized equipment, harsh conditions, multi-step procedures, and being very expensive and difficult to scale up for large areas in the fabrication process. 12 Recently, a few electrodeposition methods 13 have been widely used to prepare superhydrophobic surfaces on the electrode substrate due to their simplicity in adjusting the deposition parameters, low cost, and ease of scalability, especially applied on a wide range of materials. In 2012, Chen et al. reported 14,15 a new facile method for the electrodeposition of superhydrophobic surfaces. As a standard method, significant progress has been made over several years, including the use of Fe, Co, Ni, Cu, La, etc. , to prepare superhydrophobic surfaces. 16–19 In addition, hydrogen has attracted extensive research interest owing to its environmental friendliness and high energy density. Recently, hydrogen bubbles arising from the electrochemical reduction of H + in the deposition process functioned as the soft template for electrodeposition products. 20–22 Hydrogen bubble templates have advantages compared with hard templates, including low cost, ease of preparation, and facile control and synthesis process. This method was gradually used to prepare superhydrophobic structures. 23 Meantime, calcium, the third most abundant metal in nature, had a decisive advantage over the other metals due to its peculiar coordination chemistry. Although certain research about superhydrophobic surfaces, including calcium elements, has been reported in the literature, 24–26 to our knowledge, few studies have systemically reported about calcium superhydrophobic surfaces including preparation and certain impacting dynamic behaviors of water droplets, and their underlying mechanism is still not entirely clear. In this work, a simple and effective method of the electrodeposition process offers promising approaches for large areas of superhydrophobic surfaces on conductive metals. The calcium superhydrophobic surface with the presence of a flower-like rough structure contributes to excellent bounce performance for water droplets. This study aims to reveal the influence of deposition time on surface morphology as well as wettability and bounce. Both the surface morphology and chemical compositions were carefully analyzed to reveal the formation mechanism of the electrodeposited superhydrophobic surface. The research on the impact of substrate hardness and type on bounce performance was reported systematically. Three metal materials, copper, aluminum, and stainless steel, differing in hardness, were compared to test the impacting velocity of different droplets and the substrate surface—the bounce behavior of water droplets under the influence of electrodeposition time, voltage, and external force. The analysis shows that the as-prepared surfaces are soft superhydrophobic surfaces and become harder after pressing by an external force. While the superhydrophobic surface does not lose its superhydrophobicity, its bounce performance has been enhanced. This method of electrodeposition is straightforward, effective, and low cost, which offers a promising approach for large areas of soft superhydrophobic surfaces on various conductive substrates.",
"discussion": "3. Results and discussion 3.1 Morphology, wettability and chemical compositions of the copper superhydrophobic surfaces The SEM measurement was used to characterize the morphology of the studied calcium surfaces, and the results are shown in Fig. 1 . SEM images of the cathodic copper surface formed in the solution of 0.1 M myristic acid and 0.2 M calcium chloride at a varying time, namely, 0.5 min, 1 min 35 s, 10 min, 15 min, 30 min, and 45 min, are illustrated in Fig. 1(a)–(f) . The representative images are from different samples produced under the same conditions except for the deposition times. As shown, with the electrodeposition in the solution for a short time ( Fig. 1(a) , 0.5 min), there appear a few random small crystallite clusters on the surface, and each cluster is like a loose bud with about a diameter of 2 μm (see the inset of Fig. 1(a) ), and there are many microstructures similar to turfgrass. Prolonging the electrodeposition time to 1 min 35 s, the number of clusters increases and improves the uniformity of surfaces. As shown by the inset of Fig. 1(b) , the high magnification image revealed that the cluster with a size of about 4 μm is formed, and each cluster is similar to that of 0.5 min. However, the difference is larger and denser. As the electrodeposition time is further extended to 10 min, the entire substrate is almost covered by the dense and homogeneous crystallite clusters. Each cluster has a size of about 5 μm. The structure of a single bug is more obvious as the electrodeposition time increases to 15 min, and competitive growth of two or more clusters in the inset of Fig. 1(d) can be seen. With a further increase in the electrodeposition time to 30 min, these clusters evolve into larger flowers, as shown in Fig. 1(e) . Furthermore, when increasing the electrodeposition time up to 45 min, all of the nanostructure assemblies develop into flowers, the heterogeneous structures with particles and flowers are formed, and the single flower has a diameter of about 15 μm (see Fig. 1(f) ), and the upper flower about 5 μm, which is similar to Fig. 1(b) . Fig. 1 SEM images of the cathodic copper surface in 0.1 M myristic acid and 0.2 M calcium chloride solution with a DC voltage of 30 V for different electrodeposition times: (a) 0.5 min; (b) 1 min 35 s; (c) 10 min; (d) 15 min; (e) 30 min; (f) 45 min. In order to further clarify the formation mechanism of the superhydrophobicity, as an example, Fig. 2 shows a schematic illustration of the superhydrophobicity surface growth process. The first step is to create nucleation sites via process reaction growth as shown in Fig. 2(a) . Oriented nanocrystals grow from these nucleation sites ( Fig. 2(b) ), and in the subsequent process steps, new crystals nucleate and grow on the crystals produced in previous stages. In a word, with the electrodeposition time evolution, the structure of the cathodic copper surface transforms from the turfgrass structures, loose flower structures, larger and dense flower structures, secondary flower structures, and then into tertiary or more flower structures (see Fig. 2(c)–(f) ). Fig. 2 Schematic illustration of the superhydrophobicity surface growth process. (a) Creation of nucleation centers similar to the turfgrass on a substrate; (b) growth of small and loose flowers on the substrate; (c) growth of big and dense flowers on small and loose flowers; (d) Ostwald growth of the big and dense flowers on the substrate, typically competition growth and merge growth; (e) secondary flower growth from the Ostwald growth patterns; (f) tertiary or more growth from the flower growth morphology. To understand the influence of electrodeposition time on the wetting properties of the prepared Ca cathodic surfaces, the variation curve of the contact angle with the electrodeposition time is displayed in Fig. 3 . As shown, the contact angle value reaches 156° at a short deposition time (0.5 min), indicating that the surface wettability has already achieved the superhydrophobic state, and strongly demonstrating that this method is quite time saving and highly efficient. 3,27 With the increase of deposition time, contact angles of surfaces show an increasing trend. When the deposition time is about 1 min, the contact angle increases to 160°. When prolonging the electrodeposition time to 3 min, the contact angle is improved to 165°. As the deposition time is further increased from 10 min to 20 min, the contact angle remains at a maximum value of 166°, and the rolling angle is less than 3°. However, further increasing the deposition time to 25 min, the contact angle decreased slightly to 165°, and then the contact angle began to decrease slowly with the electrodeposition time, but still retained good superhydrophobicity. 16 Fig. 3 The variation curve of the contact angle with the electrodeposition time. Apart from the analyses of surface chemical compositions, according to the Cassie–Baxter equation, 28,29 the influence of surface structure on the fabrication mechanism of the superhydrophobic property is also discussed below. 1 cos θ r = f cos θ − (1 − f ) where f is the normalized interfacial area of the solid surface in contact with the liquid droplet, (1 − f ) is that of trapped air among the micro/nano structures, r is the contact angle of micro/nano structure surfaces, and θ is that of the smooth surface. For this research, the contact angle of the smooth surface after modification with myristic acid is only about 109°, while the maximum contact angle value of the electrodeposition surface is about 166°. When these angles are substituted into eqn (1) , f and (1 − f ) can be calculated to be 0.04 and 0.96, respectively. It can be deduced that the air stored in the micro/nano structure surface prevents a water drop from infiltrating the surface, and it plays an important role in improving the wettability. Therefore, the surface exhibits superhydrophobicity within an electrodeposition time of 0.5 min; this technique has the advantages of quickness and simplicity. The chemical compositions of the as-prepared surface were studied by XRD, FTIR and schematic diagram of the reaction progress. In Fig. 4(a) , the typical XRD spectrum for the sample is displayed. As shown, in the small-angle region a set of well-defined diffraction peaks is observed. These diffraction peaks belong to the ( l 00) reflections, indicating that the as-prepared cathodic superhydrophobic surfaces are crystallized and regularly ordered layered structures. 30–33 Fig. 4 (a) The corresponding XRD spectrum; (b) FTIR spectrum of the superhydrophobic surface obtained from the as-fabricated cathodic copper; (c) the schematic diagram in this experiment. FTIR analysis was further used to investigate the absorption feature characteristic of the chemical groups of the products in the range of 4000–500 cm −1 as reported in Fig. 4(b) . In the low-frequency region, the two strong peaks at 1438 and 1578 cm −1 can be assigned, respectively, to the symmetric ( ν s (COO − )) and antisymmetric ( ν as (COO − )) stretching vibrations of the carboxylate group. No typical peak attributable to ν (C \n \n\n<svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>\nCreated by potrace 1.16, written by Peter Selinger 2001-2019\n</metadata><g transform=\"translate(1.000000,15.000000) scale(0.017500,-0.017500)\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>\n\n O) is identified at about 1701 cm −1 , indicating that the obtained sample is not contaminated with myristic acid, 27 but only its metal salt. When the frequency was higher, in the high-frequency region, the absorption peaks at 2850 cm −1 and 2918 cm −1 correspond to symmetric and asymmetric stretching vibrations of methyl groups (C–H), respectively. 34–37 As a consequence, it demonstrated that myristic acid was grafted on the surface through chemical bonding during the formation process, and calcium myristate (Ca[CH 3 (CH 2 ) 12 COO] 2 ) with low surface energy was formed on the cathodic copper surface, which contributed to the superhydrophobicity. In order to clarify the electrodeposition process in this experiment, the schematic diagram in the experiment is illustrated in Fig. 4(c) . The electrodeposition reaction process is explained that when the copper electrodes are immersed in the electrolyte solution with the application of DC voltage, some Ca 2+ ions near the cathode react with myristate (RCOO − ) ions, and the reaction is shifted towards the formation of a carboxylate complex on the cathodic surface. In the meantime, most hydrogen (H + ) ions are ionized from the myristic acid electrolyte and generate H 2 bubbles. These released H 2 bubbles can be considered a soft template and lead to the loose micro/nano structures on the obtained superhydrophobic surface. 15,16,38 Thus, we can infer that H 2 bubbles, electrodeposited micro/nano structures, and calcium myristate with low surface energy play an important role in the transition progress. The reaction equations can be described as follows: 2 Ca 2+ + 2CH 3 (CH 2 ) 12 COOH → Ca[CH 3 (CH 2 ) 12 COO] 2 + 2H + 3 2H + + 2e − → H 2 ↑ 3.2 Effects of various experimental parameters on structures and wettability To study the effects of preparing conditions on the electrodeposited micro/nano structures, only the concentration of calcium chloride was changed, and the others were fixed. As shown in Fig. 5(a) , with increasing the concentration of calcium chloride, the value of the contact angle can reach above 160°. When increasing the concentration to 0.2 M calcium chloride, it can obtain 165°, whereas with the concentration of the calcium chloride, the value of the contact angle will decrease slowly. The reason can be deduced that the surface will become much thicker in the higher concentration of calcium chloride. It also can be seen from the inserted SEM figures that it has many clusters with about 20–30 μm diameters, which is advantageous to store many breaths of air and keep the high superhydrophobicity. Inversely, the much thicker surfaces decrease their contact angle. Fig. 5 (a) The curve between the content of calcium chloride and value of the contact angle; (b) the curve between the value of voltage and value of the contact angle. The morphology and wettability of the surface at varying voltage (5, 15, 30, 45, and 60 V) are shown in Fig. 5(b) . It shows the relation curve of contact angle and voltage value in 0.2 M calcium chloride and 0.1 M myristic acid solution for 5 min. When the voltage is low (5 V), the contact angle of this surface is about 153°. There are many microstructures similar to turfgrass and without flower-like structures. Upon increasing the voltage to 15 V, the contact angle increases to 158°, and its microstructures are still the turfgrass structures. Several loose flower-like structures begin to appear above the turfgrass structures. With a further increase in voltage value to 30 V, some flowers can be seen obviously in the inserted figure, and the corresponding contact angle is enlarged to 165°. It can be explained that the voltage represents the driving force of the reaction. 39 At a low voltage, the amount of hydrogen bubbles was not enough to produce a significant stir of the solution layer near the cathodic surface. Thus, the surface was primarily controlled by diffusion rather than kinetics growth; an increased voltage predominantly can dramatically increase the H 2 bubbles and reaction rate. Thus, a higher voltage has bigger and denser flower-like structures, which induce the increase of the superhydrophobicity. However, when the voltage increases to 45 V, the corresponding contact angle is decreased to 163°. When the voltage is close to 60 V, the corresponding contact angle is decreased to 159°. The reason can be that with increasing the voltage, at first electrodeposition current is 2.8 × 10 2 A m −2 at 45 V, and increases to 4.3 × 10 2 A m −2 at 60 V, which dramatically increases the nucleation rate at the cathodic surface, the clusters become larger and more compact, and result in the surface becoming over thick, and the contact angle decreases naturally. In addition, to further confirm the method with universal application for conductor materials, in this section, the prepared superhydrophobic surface can be extended to other general conductor materials. SEM images of the cathodic surface in 0.2 M calcium chloride and 0.1 M myristic acid solution with a DC voltage of 30 V for a different time on the different substrates are presented in Fig. 6 . Fig. 6(a1) is the stainless steel substrate surface at a low current density of 2.4 × 10 2 A m −2 at 2 min 20 s. There appear many uniform flower-like structures on the surface. The high magnification SEM images in Fig. 6(a2) and (a3) show that the flower-like structure is composed of many compact turfgrass structures, and the diameter of each flower is about 5 μm. When the electrodeposition time is 10 min, it can be seen from Fig. 6(b1)–(b3) that the surface of stainless steel shows many compact flower-like structures similar to that of Fig. 6(a1)–(a3) . Two single flower-like structures with competitive growth forming structures with a size of about 10 μm can be seen in Fig. 6(b3) . When the electrodeposition time is extended to 30 min, a more compact flower structure can be seen in Fig. 6(c1) and (c2) . Furthermore, it is clear from the magnification image in Fig. 6(c3) that the flower-like structures show competitive growth forming tertiary or more flower structures with a size of about 15 μm, similar to those on the copper cathodic surface in Fig. 1(f) . Fig. 6 SEM images of the cathodic surface in 0.2 M calcium chloride and 0.1 M myristic acid solution with a DC voltage of 30 V for different times and substrates. (a1) Low magnification 500× for stainless steel at t = 2 min 20 s; (a2) middle magnification 1000× for stainless steel at t = 2 min 20 s; (a3) high magnification 3000× for stainless steel at t = 2 min 20 s; (b1)–(b3) 500×, 1000×, 3000× for stainless steel at t = 10 min, respectively; (c1)–(c3) 500×, 1000×, 3000× for stainless steel at t = 30 min, respectively; (d1)–(d3) 500×, 1000×, 3000× for aluminum at t = 3 min 40 s, respectively; (e1)–(e3) 500×, 1000×, 3000× for aluminum at t = 10 min, respectively. In addition, a pair of aluminum and stainless steel substrates are replaced under the same electrolyte conditions and can be seen in Fig. 6(d1)–(d3) and (e1)–(e3) , respectively. When the electrodeposition time is 3 min 40 s, many uniform clusters appear on the substrate surface in Fig. 6(d1) , and a 1000 times magnified figure in Fig. 6(d2) shows a competitive growth forming structures with a size of about 10 μm. In Fig. 6(d3) , the flowers are very similar to those in Fig. 6(a3) . When the electrodeposition time is prolonged to 10 min, compact clusters are shown on the substrate surface (see Fig. 6(e1) ), and a 1000 times magnified figure in Fig. 6(e2) shows competitive growth of three or four flowers forming structures with a size of about 15 μm. Fig. 6(e3) shows tertiary or more flower structures. By comparing stainless steel with aluminum and copper for an electrodeposition time of 10 min, these flowers on three kinds of substrates are very similar, and one cluster structure size of the stainless steel surface is the largest. This is mainly because the conductivity and the current density of stainless steel are the highest. 3.3 Impacting behavior of water droplets on the superhydrophobic surface Then, to further explain the superiority of the as-prepared surface, we used a high-speed camera to study the bounce performance of water droplets impacting onto superhydrophobic surfaces. In the following experiment, 1 ml water in a syringe was divided by some number of droplets, and the average volume of every droplet is about 10 μl. When a droplet impacts the superhydrophobic surface, it deforms and stores the kinetic energy, making the droplet recoil later on. 40–44 Fig. 7 shows the evolution process of the droplet bounce shape on the calcium superhydrophobic surface prepared by the electrodeposition of copper, aluminum, and stainless steel for 1 min, respectively. During the bounce process, the inertia forces of the droplet cause it to spread out, and during the spreading the kinetic energy of the droplet is converted into surface energy, which will be used for the retraction and rebound of the droplet. Fig. 7(a)–(d) are the bounce shapes of droplets at different velocities under a copper substrate. For a 10 μl droplet in Fig. 7(a) , at 0 ms, the small droplet indicated is produced by the pinch off when the droplet separates from the syringe needle, and a similar droplet can also be observed in Fig. 7(b)–(f) . A series of images of the droplet at V = 0.626 m s −1 are shown in Fig. 7(a) . It can be seen from the figure that the droplet deformed, a cavity at the center of the droplet was formed, a droplet with an overlapping pancake shape was formed in the spreading stage at 4 ms, some air in the pancakes can be entrapped as the droplet retracts, and most of the air can be squeezed out when it recoiled. However, a small part of the air was trapped at the top of the cavity retracted faster than the bottom. As the retraction continued, an ejection of satellite drops was observed at 9 ms. When t = 17 ms, the droplets pull up again to form an approximately circular droplet. At 31 ms, it deforms again in the air. This phenomenon becomes more obvious as the velocity increases. Fig. 7(b) shows the snapshots of the impacting droplet on the superhydrophobic surface at V = 1.25 m s −1 . It was found that a pinning-like state was observed when t = 9 ms and the droplets were greatly elongated before taking off. This phenomenon is more clear when V = 1.53 m s −1 in Fig. 7(c) ; as shown, the main droplet and four satellite drops rebound taking off the surface at 17 ms. Fig. 7 The evolution process of the droplet bounce shape of the calcium superhydrophobic surface prepared by the electrodeposition of copper, aluminum, and stainless steel for 1 min. (a) V = 0.626 m s −1 for the copper substrate; (b) V = 1.25 m s −1 for the copper substrate; (c) V = 1.53 m s −1 for the copper substrate; (d) V = 1.77 m s −1 for the copper substrate; (e) V = 1.25 m s −1 for the aluminum substrate; (f) V = 1.25 m s −1 for the stainless steel substrate. When the velocity reaches V = 1.77 m s −1 , as shown in Fig. 7(d) , the droplets were torn into a large droplet and two satellite drops at 9 ms. The reason is maybe the larger pinning force and rebound velocity. The main part of the droplet rebounded off again, while the satellite drops were pinned on the surface with Wenzel state at 17 ms. Nevertheless, when the main droplet impacted the surface again, moreover, it was also observed that the bouncing height of the main droplets was not as superior as that of droplets at low velocities. This rule is similar to the results reported in the related literature; 45 that is, the greater the initial velocity, the stronger the bounce performance of the droplet, but the best bounce performance within a certain range. Next, the versatility of the bounce performance was further verified under the aluminum and stainless steel substrates under the same preparation condition with a copper substrate. As shown in Fig. 7(e) and (f) , at 17 ms, two satellite drops can be seen on the aluminum substrate and stainless steel, which means that when the droplets impacted the surface of the substrate, the rebound velocity was greater. These results proved that the metal substrate plays an effective role in promoting the bounce of the droplets. Although many methods and technologies to fabricate superhydrophobic surfaces have been reported, few products have been launched using such surfaces. This is mainly because these surfaces are generally very weak to resist mechanical contact. 46 In real environmental conditions, the destruction of surface structures by external forces can lead to a very fast loss of superhydrophobicity. Thus, we investigated the changes of these samples by SEM structures and bounce performance under a manual pressing force of a thumb by a glass slide. Before the pressing force, when the electrodeposition time was prolonged to 5 min for the copper substrate, it can be seen from the SEM image magnified 1000 times in Fig. 8(a) that some large flower-like structures grow on the surface of the substrate before pressing. Similar to Fig. 7 , at 0 ms, the small droplet indicated is produced as in Fig. 7 . As shown, the impact velocity of V = 1.25 m s −1 on the surface droplets shows that, at 17 ms, a jet is gradually developed, and cut off and a satellite drop is separated. After a short time, the jet leaves the surface. When t = 23 ms, there are two different heights for the drops, and at t = 31 ms, they coalesced into a large droplet with each other, and rebounded off again. Fig. 8 The SEM structure and impact performance of the surface before and after the pressing force of a thumb for 5 min on the electrodeposited copper surface. (a) 1000× SEM for the copper substrate at 5 min, V = 1.25 m s −1 ; (b) 3000× SEM for the copper substrate at 5 min, V = 1.53 m s −1 ; (c) 1000× SEM for the same copper substrate with the pressing force at 5 min, V = 1.25 m s −1 ; (d) 3000× SEM for the same copper substrate with the pressing force at 5 min, V = 1.53 m s −1 . After the superhydrophobicity surface was pressed by the pressing force of the thumb, about 98 N, the corresponding SEM image is shown in Fig. 8(b) . It can be seen that the main part of the displayed flower has formed black, dense structures via applying a pressing force. Under the same size droplet impact and the same velocity V = 1.25 m s −1 , the phenomenon of a jet breakup is no longer the same as that described in Fig. 8(a) . At t = 17 ms, after the separation of the first satellite drop, separation into the second satellite drop has already begun. At t = 23 ms, the large, medium and small drops are clearly distributed at different bounce heights, respectively. At t = 31 ms, the distance between the small droplet and the main droplet is further increased. Thus, we can conclude that the superhydrophobic surface prepared by this method is a soft surface. After being pressed by a pressing force, its hardness increases, but the superhydrophobic performance is not weakened. Inversely, the bouncing performance is enhanced. When magnifying the surface microstructures 3000 times, in Fig. 8(c) , the SEM shows that the structure of each flower is about 5 μm. If the impact velocity increases to 1.53 m s −1 , at 17 ms, it is obvious that with the increase of impact velocity, the number of satellite drops increases. When t = 23 ms, three drops of different sizes and heights are clearly visible. At 68 ms, it can be seen that the rebound height is much higher than that of Fig. 8(a) , which shows that the velocity obviously affects the rebound height of the droplet. On the contrary, under the condition of keeping the water droplet velocity, the impact progress was carried out on the same substrate which is pressed by the pressing force strength, and it was found that its bounce performance was further enhanced in Fig. 8(d) . It can be clearly seen that at 17 ms, three satellite drops are formed, and separation into the fourth satellite drop has already begun. From the beginning, when t = 23 ms, four droplets are formed, and when t = 68 ms, the rebound height becomes higher. The reason can be that the droplet has larger potential energy which makes the droplet have greater kinetic energy during the impact process. Therefore, the rebound velocity increases with the increase of the droplet bounding velocity. The larger the rebound velocity the smaller the radius of the jet column, the thinner the column is, and the easier it is to be cut off. Therefore, the number of satellite drops increases with the bounding velocity and manual pressing progress. \n Fig. 9 shows a comparison diagram of droplet rebound characteristics of superhydrophobic surfaces prepared on different substrates before and after a pressing force by a glass slide. Fig. 9(a) reveals the bouncing result of a copper substrate for 30 min electrodeposition at V = 1.25 m s −1 . It should be noted that 0 ms is also set as the time that the droplet just contacts the superhydrophobic surface. At 9 ms, it forms a jet shape, and the formed jet gradually elongates and thins rather than rapidly breaking up. At a time of 17 ms, droplets just now pinch off the surface, and two satellite drops are successively developed on the tip of the jet. By comparing it with superhydrophobic copper surfaces ( Fig. 7(b) and 8(a) ), it can be observed that the rebound performance of the superhydrophobic surface with an electrodeposition time of 30 min is better than that of the latter. And three satellite drops can be observed appearing at 17 ms after pressing the surface ( Fig. 9(b) ), and we found that the satellite drops fall back to the surface, coalesce with main droplets, and then completely rebound. It proved that rebound performance is better after the pressing force. Fig. 9 The impact performance of the surface before and after the pressing force of the thumb for different substrates. (a) V = 1.25 m s −1 for the copper substrate at 30 min; (b) V = 1.25 m s −1 for the copper substrate with the pressing force at 30 min; (c) V = 1.25 m s −1 for the aluminum substrate at t = 30 min; (d) V = 1.25 m s −1 for the aluminum substrate with the pressing force at 30 min; (e) V = 1.53 m s −1 for the aluminum substrate at 15 min; (f) V = 1.53 m s −1 for the aluminum substrate with the pressing force at 15 min; (g) V = 1.53 m s −1 for the stainless steel substrate at 15 min, overlook views with a tilt angle of about 5°; (h) V = 1.53 m s −1 for the stainless steel substrate with the pressing force at 15 min, overlook views with a tilt angle of about 5°. \n Fig. 9(c) and (d) show the rebound behavior of the water droplets on the aluminum surface for 30 min electrodeposition at V = 1.25 m s −1 , respectively. It can be seen that, at 68 ms, the superhydrophobic surface after the pressing force has a higher rebound height. Fig. 9(e) and (f) show the rebound behavior of water droplets with V = 1.53 m s −1 impacting the aluminum superhydrophobic surface for 15 min electrodeposition. Before pressing, an impacting droplet is deformed during spreading and forms an oval shape. When the maximum extension is reached (9 ms–17 ms), then it resumes back to the spherical shape, and takes off with a jet and small satellite drops similar to that of Fig. 9(a)–(d) . The reason maybe is that the aluminum surface has grown a thicker surface than that of 1 minute, and the prepared surface is a soft surface, so the droplets will collide and bounce at a certain speed. It consumes a part of the momentum, so that its bounce height is lower and the chance of satellite drop formation is greatly reduced. After pressing, as shown in Fig. 9(f) , the droplet bounces up obviously, and the phenomenon is similar to that of Fig. 9(a)–(e) . It is also proved that the pressing force can improve the soft surface superhydrophobicity. In order to further study the rebound behavior, we imaged the rebound behavior of water droplets on the stainless steel superhydrophobic surface with a tilt angle of about 5°. Fig. 9(g) shows the water droplet rebound behavior of stainless steel for 15 min electrodeposition at V = 1.53 m s −1 . It can be seen more clearly that it impacts the surface to form a round cake shape at 4 ms. In the retraction progress, at 10 ms, one satellite drop cannot follow the main droplet's receding motion and thus pinches off on the surface (9–68 ms in Fig. 9(g) ). However, the main droplet bounces at t = 10 ms, and then leaves the small satellite drop pinning on the surface. Fortunately, after the pressing force, the surface can obtain a bounce ability similar to that in Fig. 9(c) . Therefore, we can conclude that within a certain range of droplet impact velocity, the larger the velocity, the stronger the bounce performance. At the same impact velocity, the rebound performance of aluminum and stainless steel is better than that of the copper substrate. The main reason is that the hardness of aluminum and stainless steel is higher than that of stainless steel. The proper increase of the electrodeposition time is advantageous to the rebound performance at the same velocity and the same substrate. More importantly, the superhydrophobic surface that is pressed with a glass slide by the pressing force of the thumb can significantly strengthen the rebound performance of water droplets. The reason is believed to be mainly due to the fact that the flower-like structures on the surface become denser, harder, and consume less energy for the bounce of droplets after the pressing force."
} | 9,080 |
25491472 | null | s2 | 4,853 | {
"abstract": "Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically localized within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, overactivation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events."
} | 351 |
28239550 | PMC5315440 | pmc | 4,855 | {
"abstract": "Mine tailings from copper mines are considered as one of the sources of highly hazardous acid mine drainage (AMD) due to bio-oxidation of its sulfidic constituents. This study was designed to understand microbial community composition and potential for acid generation using samples from mine tailings of Malanjkhand copper project (MCP), India through 16S rRNA gene based amplicon sequencing approach (targeting V4 region). Three tailings samples (T1, T2 and T3) with varied physiochemical properties selected for the study revealed distinct microbial assemblages. Sample (T3) with most extreme nature (pH < 2.0) harbored Proteobacteria , Actinobacteria , Chloroflexi while the samples (T1 and T3) with slightly moderate nature (pH < 4.0 and > 3.0) exhibited abundance of Proteobacteria , Fimicutes , Actinobacteria and/or Nitrospirae . Metagenomic sequences are available under the BioProject ID PRJNA361456."
} | 229 |
28939278 | null | s2 | 4,858 | {
"abstract": "Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs."
} | 392 |
37704622 | PMC10499878 | pmc | 4,860 | {
"abstract": "Bacterial remineralization of algal organic matter fuels algal growth but is rarely quantified. Consequently, we cannot currently predict whether some bacterial taxa may provide more remineralized nutrients to algae than others. Here, we quantified bacterial incorporation of algal-derived complex dissolved organic carbon and nitrogen and algal incorporation of remineralized carbon and nitrogen in fifteen bacterial co-cultures growing with the diatom Phaeodactylum tricornutum at the single-cell level using isotope tracing and nanoSIMS. We found unexpected strain-to-strain and cell-to-cell variability in net carbon and nitrogen incorporation, including non-ubiquitous complex organic nitrogen utilization and remineralization. We used these data to identify three distinct functional guilds of metabolic interactions, which we termed macromolecule remineralizers, macromolecule users, and small-molecule users, the latter exhibiting efficient growth under low carbon availability. The functional guilds were not linked to phylogeny and could not be elucidated strictly from metabolic capacity as predicted by comparative genomics, highlighting the need for direct activity-based measurements in ecological studies of microbial metabolic interactions.",
"introduction": "Introduction Algal-bacterial interactions are fundamental to primary productivity in the oceans and other surface waters, including algal bioenergy and bioproduct production ponds 1 . Studies have shown that bacteria can increase algal productivity by providing vitamins 2 , siderophores 3 , and algal growth hormones 4 . More fundamentally, bacteria can remineralize nutrients, through for example, the deamination of organic nitrogen to ammonium 5 . It is generally assumed that algae-associated bacteria grow on algal-derived organic matter, making these interactions mutualistic or at least commensal (i.e., the bacteria benefit and the algae are unaffected), but few studies have empirically measured the fraction of bacterial carbon (C) and nitrogen (N) that is derived from photosynthetic microalgae. To our knowledge, the reverse process (transfer from bacteria to algae) has never been directly quantified. These microscale exchanges, when integrated over large volumes in outdoor algal cultures and in aquatic ecosystems, have profound consequences for elemental cycling 6 . The last few decades have greatly increased our knowledge of the mechanisms of bacterial C and N recycling in aquatic ecosystems. Algal blooms support a microbial community that progresses over time in response to the changing availability of algal-derived organic matter over the course of weeks 7 and as quickly as within one day 8 . Generally, algal-associated bacteria that recycle algal organic matter have been divided into those that specialize on polymers and macromolecules, dominated by Bacteroidia , and those that specialize in smaller molecules, dominated by Rhodobacterales 9 . In both cases, the primary mechanisms for degradation and uptake involve extracellular enzymes and transporters 10 , and many resources now exist to identify genes involved in such processes, including polysaccharide 11 and protein 12 degradation. Compounds released by bacteria and known to be reincorporated by photoautotrophs for biomass include ammonium 5 , amino acids 13 , and carbon dioxide 14 . However, nutrient remineralization has not been identified as a primary mechanism of algal growth promotion by bacteria 15 , perhaps because in nature, they are often decoupled in time and space 16 . Nonetheless, since different bacteria are known to consume different sources of organic matter, it seems plausible that different bacteria likewise express different rates of nutrient remineralization. Thus, there is a need to measure net exchanges of C and N between algae and bacteria, identify whether these exchanges are correlated to mutualism or physical association and whether the quantity can be predicted by phylogenetic affiliation or genomic content. A practical approach to quantify metabolic activity and exchange is the use of stable isotope incubations. DNA-stable isotope probing (SIP) was developed to identify organisms that incorporate a pure substrate, for example, 13 C-labeled methanol 17 . Further studies have used isotope-labeled extracts from phytoplankton to qualitatively identify bacterial taxa that incorporated these mixed substrates 18 . A more quantitative analysis from such incubations can be carried out at the single-cell level using a nanoSIMS imaging secondary ion mass spectrometer through an approach called nanoSIP 19 . This high-resolution method allows the quantification of isotope incorporation by single cells, including small cells that are attached to one another 20 . For example, nanoSIP has been used with mixed bacterial communities growing with one phototrophic alga to show that different bacterial communities have different effects on algal growth and cell-specific carbon fixation, and in some cases this mutualism is mediated by cell-to-cell attachment 21 . However, working with mixed communities makes it challenging to attribute specific impacts to individual taxa, and is also complicated by bacteria-bacteria interactions that might obscure otherwise recognizable interactions. These inherent challenges therefore required us to develop modified culturing and SIP approaches that enable taxon-specific probing and focused observations of bacterial activity and exchange with their algal partner. Here, we relate the identity, genetic potential, and activity of algal-associated bacteria using isotope tracing to quantify the transfer of complex algal C and N to bacteria and remineralized C and N to the algae. We define ‘remineralization’ in these experiments by measurements of net C and N incorporated by the algal cells. Thus, this does not represent gross remineralization of algal organic matter, but rather what is assimilated by the algal cells as they are actively growing, prior to nutrients becoming depleted. Linked activity and metabolic potential 22 can be useful in order to model complex microbial processes, for example using functional trait approaches 23 . We isolated fifteen representative bacteria from mixed community enrichments previously growing with the diatom Phaeodactylum tricornutum under autotrophic conditions. We reinoculated these bacterial isolates into previously bacteria-free P. tricornutum , allowed these co-cultures to reach equilibrium ratios of algae to bacteria based on metabolic exchange, and tested their impact on algal biomass yield. Then we added chemically extracted isotope-labeled P. tricornutum -derived organic matter to these co-cultures to measure organic C and N incorporation by the bacterial cells as well as remineralization of C or N back to P. tricornutum cells. We further examined bacterial incorporation of nitrate in the presence of glycolate, a simple C source known to be produced by P. tricornutum . In addition to comparative genomics analyses of the 15 strains, we focused on two bacterial isolates representing contrasting phenotypes, linking genotype and phenotype by investigating their most abundantly expressed proteins during growth in algal cultures. All together, our data led us to develop a conceptual framework of bacterial association with microalgae that takes into account nutrient exchange, competition, and metabolism, suggesting that algal-associated bacteria in our experiments can be classified into three major functional classes (or ‘guilds’) that exhibit distinct strategies allowing them to proliferate in high nutrient and high-density algal systems.",
"discussion": "Discussion In this study, we quantified algal DOC and DON uptake and remineralization by bacteria associated with P. tricornutum and used these net flux measurements to statistically categorize bacteria with similar activities into functional categories (‘guilds’, Fig. 4 ), using K-mean clustering (Fig. S 5 ) to partition the strains. One guild, that we call macromolecule remineralizers (identified in blue on Figs. 1 , 2 , and 4 ), incorporated high amounts of complex DON and DOC and remineralized detectable levels of N and C to the algal cells. A second guild, which we call macromolecule users (green), also incorporated both DON and DOC and remineralized detectable C, but not N to the algal cells. Alcanivorax is an exception in this category because it did not incorporate complex algal DON and incorporated some N from nitrate and is more similar to the third guild in that way. The third guild, which we call small-molecule users (black), incorporated complex DOC but not complex DON, likely using smaller DON molecules not captured by the extraction protocol and some N from nitrate; they also appeared to be highly efficient recyclers of lost CO 2 through anapleurotic fixation based on 13 C bicarbonate incorporation with no algal C present. As noted above, these guilds do not map to phylogeny and are not predicted by genome content. In addition, there was no pattern regarding attachment to algal cells or mutualism. Note that these conclusions are specific for P. tricornutum incubated in the laboratory in high nutrient F/2 media with high cell concentrations and may not be applicable to different algal taxa or environmental conditions. Fig. 4 Conceptual figure of bacterial- Phaeodactylum metabolic interactions, based on measurements of C and N incorporation and remineralization by 15 bacterial isolates and remineralization of C and N to (remineralization defined as detectable transfer of remineralized algal-derived DOM to the diatom cells). Bacteria are classified as macromolecule remineralizers (evidence of nutrient feedback to the diatom cells) or users (no detected N incorporation by diatoms), and bacteria that did not incorporate complex organic N are classified as small-molecule users. Underlined genera have shown evidence of mutualism in co-culture. The thickness of the arrows approximates relative fluxes as measured by isotope incorporation. In addition to the strain-to-strain variability in activity, within each strain, we also identified cell-to-cell variability in isotope incorporation, which is commonly found even in clonal populations 29 . This variability may contribute to the ecological success of these bacteria given that they rely on organic matter from algal photosynthesis. The macromolecule remineralizers all exhibited a coefficient of dispersion (CD) for DON incorporation over 30% and the macromolecule users under 30%, suggesting that more heterogeneity in N incorporation is linked to N remineralization. One possible mechanism to explain this pattern is that remineralizers may have less efficient coupling between organic matter hydrolysis and uptake, driven by a subset of the population expressing the hydrolytic enzymes for the benefit of the entire population, including the algal cells. In this way, the algal cells and the non-enzyme-expressing bacteria are cheaters, getting benefits without expanding energy to break down the organic matter. Also, some strains included glycolate incorporators and non-incorporators within the same population. This type of heterogeneity in a clonal population has previously been hypothesized to be a response to resource limitation 30 , suggesting this process could also be at play here. Another study 31 , using an autotrophic clonal cyanobacterium culture, documented cell-to-cell heterogeneity caused by several other mechanisms, including temporal population differentiation in an assimilatory process, intracellular resource allocation and differential turnover rates, and heterogeneity in the uptake of multiple substrates. Here, we can consider several potential mechanisms leading to cell-to-cell variability in isotope incorporation by both bacteria and diatom cells. First, the cultures were in batch mode and not in chemostats and were not synchronized, so both algal and bacterial cells were in different phases of cellular division. We note that chemostat-grown cells can still exhibit cell-to-cell heterogeneity, but potentially less so than batch-mode cells 31 . Second, the cultures were not agitated, and P. tricornutum exists both as a benthic and pelagic organism, often forming aggregates of multiple cells, so resources might be heterogeneously distributed or differentially accessible in the culture tubes. Third, bacterial cells can be either algal-attached or free-living, which could result in differential access to externally added isotope-labeled algal organic matter, again leading to spatial heterogeneity of resources. Even among free-living bacteria, they were simultaneously incorporating the labeled extracellular algal organic matter and unlabeled organic matter being exuded from the algal cells, and the latter should not be homogeneously distributed. Unlike DON, incorporation of DOC and remineralization of C back to algal cells was variable but statistically detectable for all tested co-cultures. Also of note is that although as a population, axenic algal cells were not statistically more enriched than killed controls, some of these cells (18%) were statistically enriched. This suggests a subset of the algal population was expressing some unusual metabolic potential to incorporate its own released DOC, through an uncharacterized mechanism. Further, this brings up the possibility that DOC-remineralizing bacteria weren’t actually remineralizing C back to the algal cells but simply signaling to the algal cells to express this DOC incorporation metabolism. We do not currently have data able to fully reject this hypothesis, although the P. tricornutum proteome collected during co-culture with Rhodophyticola and Marinobacter was not statistically different from axenic cultures, suggesting that algal cells were in a metabolically similar state with or without bacteria. The relationship between bacterial C incorporation and remineralized C incorporated by the algal cells in the corresponding co-culture (Fig. S 6A ), was positive but not strong. It likely reveals the C use efficiency of the strains on complex algal-derived organic carbon, with most taxa falling along a positive relationship: greater bacterial C incorporation corresponds to greater remineralized C. Three exceptions from this trend were Pusillimonas and Marinobacter , with low C remineralization given their C incorporation, and Alcanivorax , with the opposite (high C remineralization given its low C incorporation). This suggests that Pusillimonas and Marinobacter are highly efficient incorporators of complex algal C, and Alcanivorax is a highly inefficient one. It is likely that expanding the number of categories would split Pusillimonas and Alcanivorax into distinct groups, based on the second dimension of the K-means clustering locating them at opposite ends (y-axis in Fig. S 6B ). No information from previous studies with either Alcanivorax or Pusillimonas suggests they are high and low respirers of CO 2 , respectively. However, a future study linking single-cell respiration rates 32 and C remineralization to algal cells could directly test this hypothesis. Algal incorporation of bacterial remineralized organic matter can sometimes lead to the emergence of mutualism: for example, under long-term cultivation with low nutrient concentrations, picocyanobacterial and heterotrophic bacteria exchange organic C for remineralized nutrients 33 . Here, we defined mutualism as increased algal biomass in co-culture compared to axenic cultures at the early stationary growth phase. We did not find that the exchange of N was associated with mutualism: none of the three mutualistic strains showed evidence of N remineralization. Furthermore, the highest remineralizers of DON ( Muricauda , Oceanicaulis , Arenibacter , and Algoriphagus ) were not mutualistic, and two of those strains ( Muricauda and Algoriphagus ) were previously shown to be growth-inhibiting in co-culture with P. tricornutum 25 . In fact, most of the 15 tested strains, including the three mutualists, provided little to no remineralized N to the algal cells, at least from the complex organic N extracted with the protocol used. This suggests that bacterial remineralization of complex algal DON and coupled transfer to the algal cells is not as common as we expected (Fig. S 6D ), at least under the relatively high nitrate concentrations tested here and commonly used in outdoor cultivation of microalgae 34 . Direct bacterial uptake of nitrate (60% of the isolates) was more prevalent than N remineralization to algal cells, although the data suggest that nitrate did not provide full bacterial N requirement for many of the taxa, based on low nitrate N net compared to glycolate C net (Fig. S 7 ). Bacterial nitrate incorporation for biomass is more energetically expensive than ammonium, and our data confirm that the algal-associated bacteria likely did not use nitrate directly for growth. However, it is possible that algal-associated bacteria used nitrogen cycling for energy. Four strains contain the metabolic pathway for dissimilatory nitrate reduction (nitrate to ammonium), three contain genes for denitrification (nitrate to N 2 O; Supplementary Data 3 ), and strains of Marinobacter 35 and Thalassospira 36 have been shown to denitrify. Furthermore, some recent evidence suggests that the same Marinobacter strain used here increased its growth rate in response to the availability of F/2 nutrients, likely nitrate, even in the presence of P. tricornutum 37 . It remains to be seen whether the mechanism is assimilatory or dissimilatory nitrate reduction, as we did not find proteomic evidence of either of these processes. Our data and resulting conceptual framework have implications for a better understanding of C and N cycling in natural and engineered algal-dominated ecosystems. Due to the complexity of bacterial communities and the difficulty in predicting activity from ‘omics data, the bacterial community is generally considered a black box with general terms for remineralization, respiration, and other biogeochemical processes 38 . Our conceptual framework should help to divide this black box into categories with shared traits, which should then help to more accurately model the impact of different microbial communities on elemental cycling. Unfortunately, our analyses suggest that activity cannot be predicted by phylogeny or the presence of metabolic pathways related to central C and N metabolism derived from genomic data. Linking genome content and activity is crucial in order to use genome data in metabolic or biogeochemical models, and future efforts should be aimed at identifying metabolic pathways that are correlated to microbial activity. We suggest that it is ultimately possible to link activity and genomic data, but that this will require identifying the unknown genomic pathways that result in these activity categories (perhaps requiring single-cell RNA or protein expression, and more comprehensive RNA/protein expression). Single-cell heterogeneity is also likely to have genomic underpinnings, and this could be another focus of future targeted research. It remains to be seen whether the activity data resulting in the conceptual framework of bacterial metabolic interactions with P. tricornutum incubated in the laboratory translates to other taxa and natural communities. Nonetheless, Phaeodactylum is a model biofuel crop, shown to produce high amounts of biomass and lipids and can be grown in large volumes with high nutrient concentrations 39 , and our results likely apply at least to this genus. Understanding the role of the different members of the Phaeodactylum -associated bacterial community could be useful for optimal production of algal biomass or intracellular lipids, reduction of excreted waste DOM, and recycling of nutrients after harvest. One strategy might be to optimize the algal microbiome for different phases of algal cultivation to maximize in-situ metabolic exchange during algal growth and removal of DOM from the algal-spent medium after biomass harvest. Microbiome optimization might also be a critical component of algal cultivation efforts that use recycled nutrients from wastewater or other external sources that includes both organic and inorganic nutrients."
} | 5,100 |
23417799 | null | s2 | 4,862 | {
"abstract": "Genome-scale metabolic network reconstructions, assembled from annotated genomes, serve as a platform for integrating data from heterogeneous sources and generating hypotheses for further experimental validation. Implementing constraint-based modeling techniques such as flux balance analysis (FBA) on network reconstructions allows for interrogating metabolism at a systems level, which aids in identifying and rectifying gaps in knowledge. With genome sequences for various organisms from prokaryotes to eukaryotes becoming increasingly available, a significant bottleneck lies in the structural and functional annotation of these sequences. Using topologically based and biologically inspired metabolic network refinement, we can better characterize enzymatic functions present in an organism and link annotation of these functions to candidate transcripts; both steps can be experimentally validated."
} | 226 |
26528266 | PMC4604316 | pmc | 4,863 | {
"abstract": "The bacterial microbiota of plants is diverse, with 1000s of operational taxonomic units (OTUs) associated with any individual plant. In this work, we used phenotypic analysis, comparative genomics, and metabolic models to investigate the differences between 19 sequenced Pseudomonas fluorescens strains. These isolates represent a single OTU and were collected from the rhizosphere and endosphere of Populus deltoides . While no traits were exclusive to either endosphere or rhizosphere P. fluorescens isolates, multiple pathways relevant for plant-bacterial interactions are enriched in endosphere isolate genomes. Further, growth phenotypes such as phosphate solubilization, protease activity, denitrification and root growth promotion are biased toward endosphere isolates. Endosphere isolates have significantly more metabolic pathways for plant signaling compounds and an increased metabolic range that includes utilization of energy rich nucleotides and sugars, consistent with endosphere colonization. Rhizosphere P. fluorescens have fewer pathways representative of plant-bacterial interactions but show metabolic bias toward chemical substrates often found in root exudates. This work reveals the diverse functions that may contribute to colonization of the endosphere by bacteria and are enriched among closely related isolates.",
"introduction": "Introduction In carbon-poor soil environments plant root exudates and fine root turnover provide a rich source of carbon substrates that attract and feed a plethora of soil bacteria (Bais et al., 2006 ). Plant-associated bacteria are diverse, with 50–1000 operational taxonomic units (OTUs) associated with any individual plant (DeAngelis et al., 2009 ; Uroz et al., 2010 ; Gottel et al., 2011 ; Weinert et al., 2011 ; Lundberg et al., 2012 ). While it is clear there is extreme phylogenetic diversity in the bacterial community, the functional diversity of bacteria and their contribution to the overall function of the microbiome is less apparent. The root microbiota is commonly distinguished by two environments: the rhizosphere, the volume of soil directly influenced by the root, and the endosphere, the internal root tissue. The rhizosphere is generated by plant cell death and abscission from growing roots and/or active secretion of root exudate, a mixture of small molecules that can solubilize nutrients in the soil for subsequent uptake by the plant (Kirk et al., 1999 ; Dakora and Phillips, 2002 ). The specific chemical composition of the exudate depends on plant species, nutrient status (Dechassa and Schenk, 2004 ), environmental factors (Raynaud, 2010 ) and root age (Schnepf et al., 2012 ; Dunbabin et al., 2013 ), but generally has been shown to include amino acids and peptides, sugars, and small organic acids (Dakora and Phillips, 2002 ; Dechassa and Schenk, 2004 ; Carvalhais et al., 2011 , 2013 ) that directly influence the microbial community associated with the plant (Glick, 2005 ; Bais et al., 2006 ; Hartmann et al., 2008 ; Stearns et al., 2012 ; Hunter et al., 2014 ; Ludwig-Müller, 2015 ). A relatively small fraction of bacteria that associate with the plant gain access to the internal root endosphere compartment (Compant et al., 2010 ; Gottel et al., 2011 ; Lundberg et al., 2012 ; Bulgarelli et al., 2013 ; Oldroyd, 2013 ; Shakya et al., 2013 ). These bacteria are exposed to a different biochemical environment which can include storage carbohydrates, complex structural polymers, and secondary metabolites such as nucleosides and aromatic compounds. Within the endosphere, bacteria can inhabit multiple environments such as inter- and intracellular spaces that may have a unique biochemical profile (Gaiero et al., 2013 ). Relationships between bacteria and host plants, regardless of whether they are found in the rhizosphere or endosphere, can be mutually beneficial and enhance growth of both organisms. For example, plants in need of phosphorus exude organic acids to release soil-bound phosphates; the soil bacteria consume the organic acids from the plant and further solubilize phosphate in the environment, leading to increased available nutrient pools for both host and microbiome (Rodriguez et al., 2004 ; Vyas and Gulati, 2009 ; Ahemad and Khan, 2010 ). Beneficial bacteria can also induce systemic resistance in host plants to help prevent infection (Weston et al., 2012 ) or may directly inhibit pathogen growth through niche space competition or the production of antibiotics (Pérez-García et al., 2011 ). To thrive in the root microbiome, bacteria must compete with other community members for resources. Investigation of the Populus rhizosphere microbiota by cultivation independent approaches has demonstrated that γ- Proteobacteria , primarily Pseudomonas fluorescens -like strains, are highly abundant and represent one of the dominant bacterial groups in this environment, along with α- Proteobacteria, Acidobacteria , and Actinobacteria (Gottel et al., 2011 ; Shakya et al., 2013 ). The P. fluorescens group includes many plant-associated strains and is genetically diverse (Silby et al., 2009 ; Loper et al., 2012 ), with recent assessments showing the core genome of 2789 genes (CDSs) only contributing ~50% to any individual genome in the group and a large pan-genome of 13,872 genes (Loper et al., 2012 ). Given the genetic diversity of P. fluorescens , it has been proposed that the group represents multiple bacterial species, however the boundaries between these species are often obscure (Silby et al., 2009 ; Loper et al., 2012 ). Pseudomonas species are well-studied for aerobic degradation of aromatic compounds (Stanier and Hayaishi, 1951 ; Díaz et al., 2013 ), a class of molecules that are prevalent in the Populus metabolome. To investigate host-associated bacterial functional diversity rather than diversity driven by phylogeny or geographic location, we isolated diverse bacterial strains from the endosphere and rhizosphere compartments of native Populus deltoides trees in central Tennessee (Gottel et al., 2011 ; Weston et al., 2012 ). The observed diversity in the Pseudomonas fluorescens group (Silby et al., 2009 ; Loper et al., 2012 ) and the prevalence of Pseudomonas in our culture collection motivated us to investigate how genomic diversity and functional plasticity differ in endosphere and rhizosphere isolates collected from a single host plant species. Therefore, we have sequenced the genomes of 19 Pseudomonas fluorescens strains that are classified in the same OTU at 99% similarity by 16S rRNA gene sequencing from both the endosphere and rhizosphere compartments of P. deltoides roots (Brown et al., 2012 ). We screened these strains for functional attributes relevant to interaction with the host plant, including phosphate solubilization, denitrification, and ability to promote Arabidopsis root growth. Using both genomic and phenotypic analysis of the strains, we describe the diversity in these strains and identify attributes that distinguish strains isolated from the endosphere and rhizosphere. This work reveals the functional diversity that can exist within a single bacterial OTU in plant-microbiota systems, highlighting the complex associations between bacteria and their host organism.",
"discussion": "Discussion In this study we compared genome sequences and phenotypes of 19 Pseudomonas fluorescens strains isolated from the Populus deltoides endosphere or rhizosphere. Despite the similar isolation conditions and relative taxonomic closeness of these isolates (99% similarity between 16S rRNA genes), there was significant diversity in the genomes and phenotypes, highlighting the considerable functional diversity that can exist within a single OTU class in the plant microbiome. There were no gene clusters or phenotypic traits that uniquely discriminated between rhizosphere and endosphere isolates, which could be attributed to the: (1) wide range of potential mechanisms for plant-bacteria interactions, (2) misidentification of pathways, (3) actual expression of these pathways on plant, or (4) inability to predict function for all genes. However, within the strains isolated from endosphere or rhizosphere, we observed trends that require further study. In endosphere isolates we observed additional genomic elements dedicated to the metabolism of plant-relevant compounds, e.g., either synthesis or modification of plant hormones or catabolism of nucleosides and sugar acids, carbon-rich and complex molecules, which are more abundant in the endosphere compartment. The most distinguishing plant-relevant phenotypes were production of IAA, antimicrobial compounds and denitrification, all of which were biased toward endosphere isolates. The production of IAA has been observed in numerous plant growth promoting bacteria (Spaepen et al., 2007 ; Santner and Estelle, 2009 ; Gallavotti, 2013 ; Pacifici et al., 2015 ). Phenotype data showed that endosphere isolates could perform more activities relevant to interactions with the plant or competition in the microbiome relative to rhizosphere isolates. That is, the measured activities contribute to overall system function by direct interaction through molecular signaling or by indirect mechanisms due to changes in microbiome composition or nutrient availability. Nearly all isolates showed antimicrobial activity as measured by the ability to inhibit growth of four test organisms, but endosphere isolates generally inhibited a higher proportion of the tested organisms. There is more phylogenetic diversity in the rhizosphere (Bulgarelli et al., 2013 ), suggesting more interspecific competition, and potentially necessitating the ability to inhibit a broader range of organisms, but our activity results did not support this hypothesis. Alternatively, the production of anti-microbial compounds and inhibition of growth within the endosphere can contribute to pathogen resistance (Mazzola et al., 2014 ; De Coninck et al., 2015 ) or biocontrol of the community (Vetsigian et al., 2011 ; Tyc et al., 2015 ), both mechanisms ultimately benefiting the host plant. Four endosphere isolates were capable of denitrification, which has been shown to be a beneficial function for competitive ability for P. fluorescens in the rhizosphere (Ghiglione et al., 2002 ) and for colonization in the endosphere in Ralstonia infections of plants (Dalsing et al., 2015 ), likely due to the growth advantage in micro-aerobic environments in the endosphere due to the ability to use nitrate as an electron acceptor. Endosphere isolates tended to have additional pathways relative to rhizosphere isolates, as indicated by pan-genome analysis, metabolic models, and manual pathway identification. Unexpectedly, we did not observe relatively smaller genome sizes in endosphere isolate indicative of evolution of symbiotic relationships (McCutcheon and Moran, 2011 ). In fact, the endosphere isolates appeared to have relatively larger genome sizes relative to rhizosphere isolates, potentially due to a requirement that endosphere isolates must provide some benefit to the host, while still being able to survive and compete in the soil environment. In rhizosphere isolates we observed genomic biases toward cell structure biosynthesis, cofactor production pathways, and metabolism of amino acids and carboxylic acids, consistent with adaptation to an environment with less nutrient availability. Alternatively, endosphere isolates have access to complex cofactors and are under less pressure to maintain diverse, alternate pathways. For example, tryptophan catabolism via the kynurenine pathway proceeds by converting L-tryptophan into anthranilate, which is processed into catechol before entering the ortho -cleavage pathway (Stanier and Hayaishi, 1951 ; Koushik et al., 1997 ; Kurnasov et al., 2003 ). Anthranilate can also be siphoned into the biosynthesis of nicotinamide adenine dinucleotide (NAD) and quinolones (Farrow and Pesci, 2007 ), potentially important for growth in carbon-poor environments. Another explanation for increased genome size stems from the decreased diversity in the endosphere relative to rhizosphere, such that, the strains that do have access to the endosphere may have to make up for the lack of diversity by performing the anti-microbial duties that are performed by other community members in the rhizosphere. The availability of specific carbon sources is a strong selection for bacterial adaptation. The results of this study show that classes of molecules rather than specific metabolites distinguish endosphere isolates from rhizosphere isolates. Specifically, endosphere isolates were biased toward the catabolism of peptides, sugar acids, nucleosides and monosaccharides, compounds that are expected to be prevalent in the endosphere. One of the highly biased compounds (10/14 endosphere, 0/4 rhizosphere isolates), galacturonic acid, is the monomer found in pectin, a polysaccharide commonly found in plants and reported in Populus roots (Cooke et al., 2005 ; Smith et al., 2011 ). Rhizosphere isolates were biased toward carboxylic and amino acids, substituted monosaccharides and sugar alcohols, compounds potentially prevalent in root exudates. It is unclear how the consumption of plant-produced carbon sources by bacteria directly impacts the host, though carbon source has been shown to dictate Enterobacter gene expression thus serving as a signal for interaction with the host plant (Taghavi et al., 2015 ). It is likely that the definitions of endosphere and rhizosphere in this study are too coarse to attribute to specific phenotypes. Within the endosphere, strains can colonize multiple root tissues and may be localized to the inter- or intra-cellular space within those tissues. The endosphere is not chemically homogeneous and may have specific zones such as root tips, branch points, or structural components that have alternate chemical compositions/environments. Similarly, the rhizosphere is spatially heterogeneous. Energy rich compounds secreted by the root are most likely degraded rapidly by rhizosphere bacteria, while lower energy compounds could persist and diffuse farther from the root, generating a gradient that could impact rhizosphere bacteria. Further, the rhizosphere chemical composition at root hairs is different than the chemical composition at the root tip due to programmed cell death and cell abscission at the root tip during active growth. These spatial heterogeneities define niches to which specific bacteria can adapt. All of these examples would be masked by the current definition of endosphere and rhizosphere. Similar to previous studies of the Pseudomonas fluorescens group (Silby et al., 2009 ; Loper et al., 2012 ), we also observed three clades within our genomes, supporting the segregation of the P. fluorescens group into multiple species. Despite the potential speciation, we observe functional ability (both genomic and phenotypic) correlated with isolation compartment, highlighting potential functional requirements for colonization of the endosphere or rhizosphere environments. The diversity in functions displayed by the isolates in this study suggests that bacteria from a single OTU can fill multiple roles in the microbiome, potentially explaining the poor correlation between host genotype and microbiome as measured at the OTU level (Shakya et al., 2013 )."
} | 3,865 |
17217521 | PMC1780118 | pmc | 4,865 | {
"abstract": "Background Ant colony algorithm has emerged recently as a new meta-heuristic method, which is inspired from the behaviours of real ants for solving NP-hard problems. However, the classical ant colony algorithm also has its defects of stagnation and premature. This paper aims at remedying these problems. Results In this paper, we propose an adaptive ant colony algorithm that simulates the behaviour of biological immune system. The solutions of the problem are much more diversified than traditional ant colony algorithms. Conclusion The proposed method for improving the performance of traditional ant colony algorithm takes into account the polarization of the colonies, and adaptively adjusts the distribution of the solutions obtained by the ants. This makes the solutions more diverse so as to avoid the stagnation and premature phenomena.",
"conclusion": "Conclusion The method for improving the performance of traditional ant colony algorithm presented here was performed on a Pentium PC. The experiment results showed in Fig. 1 and Fig. 2 was carried out by applying the algorithm to symmetric and asymmetric TSP benchmarks provided by TSPLIB. These TSP benchmarks were also tested using the classical ant colony algorithm to compare its performance with our algorithm. In DGAA the probability of ants selecting the vertexes and the increment of pheromone updating are adaptively adjusted according to the solutions of the former iteration of the ants. This makes the system never intensify the pheromone on the best path excessively, while the ants can make their evolutionary search towards the correct directions to get strong \"mountain climb\" capabilities in all directions of the solution space, and obtain optimal solution efficiently. In addition, DGAA takes into account the polarization of the colonies, and adaptively adjusts the distribution of the solutions obtained by the ants. This makes the solutions more diverse so as to avoid the stagnation and premature. Therefore, our algorithm makes a dynamic balance between convergence speed and stagnation, and this also shows that it is suitable for solving large scaled optimization problems.",
"discussion": "Discussions We want to provide a diversity guaranteed ant colony algorithm (DGAA) by adopting the immunogenic methods to ant colony algorithm and simulating the behaviours of biological immune system. This new type of ant colony algorithm uses the immunogenic methods of immune selection, immune memory, immune metabolism, density controlled strategy and isolation niche technique. But how to integrate immune strategies into ant colony, how to verify its performance is still an open problem for us. The ant colony algorithm is mainly composed by an iterative algorithm including generation and verification operation, and the global search often realized by the pheromone feedback of the individuals in the colony, so that each individual can has an evolutionary chance or tendency, but meanwhile it may also be degenerated inevitably, even this degeneration is evident under some circumstances. According to the conception and theory of immunology [ 11 , 12 ], to maintain the eminent performance of the classical algorithm, we make full use of the characteristic of the problems and coalesce the specialty of the immune system to restrain the degeneration in the iteration."
} | 830 |
40269231 | PMC12019164 | pmc | 4,868 | {
"abstract": "Coral reef ecosystems face escalating threats from anthropogenic global climate challenges, leading to frequent bleaching events. A key issue in coral transplantation is the inability of fragments to rapidly grow to sizes that can resist environmental pressures. The observation of accelerated growth during the early stages of coral regeneration provides new insights for addressing this challenge. To investigate the underlying molecular mechanisms, we study the fast-growing stony coral Acropora muricata . Using single-cell RNA sequencing, bulk RNA sequencing, and high-resolution micro-computed tomography, we identify a critical regeneration phase around 2–4 weeks post-injury. Single-cell transcriptome analysis reveals 11 function-specific cell clusters. Pseudotime analysis indicates epidermal cell differentiation into calicoblasts. Bulk RNA-seq results highlight a temporal limitation in coral’s rapid regeneration. Through integrated multi-omics analysis, this study emphasizes the importance of a comprehensive understanding of coral regeneration, providing insights beyond fundamental knowledge and offering potential protective strategies to promote coral growth.",
"introduction": "Introduction Coral reef ecosystems are undergoing a transformative phase with increasingly brief intervals between coral bleaching events, impeding the recovery of mature coral colonies. As of the 1880s, Earth’s surface temperature has surged by nearly 1 °C due to anthropogenic climate warming and El Niño, continuously setting unprecedented records 1 , 2 . Recent global sea surface temperature spikes further accelerate the warming trend, reducing the median return time between severe coral bleaching events to just 6 years by 2016 3 , 4 . This escalating frequency poses a significant threat to coral reefs, vital thermal refuges for diverse marine species 5 . Ominous predictions suggest many reefs may vanish by midcentury 4 . Faced with this challenge, urgent and informed management strategies are crucial to protect coral reefs and restore their compromised structure and ecological functionality. Globally, researchers, conservationists, and environmental managers are actively devising innovative strategies to preserve coral reef ecosystems amidst diverse local and global threats 6 . Genetic and reproductive interventions, like managed selection and breeding, aim to propagate stress-tolerant corals 7 . Techniques such as gamete and larval capture 8 , coral cryopreservation 9 , and genetic manipulation 10 offer novel ways to enhance genetic diversity. Physiological interventions, including pre-exposure 11 and algal symbiont manipulation 12 , tap into corals’ adaptive abilities. Strategies like microbiome manipulation 13 , antibiotic application 14 , and phage therapy 15 target coral microbial communities for improved health and stress resistance. Coral population interventions, like managed relocation 16 , propose shifting corals to favorable environments. Environmental strategies, including shading 17 and seawater carbonate chemistry 18 interventions, suggest innovative ways to mitigate thermal stress and modify seawater chemistry. Despite implemented measures, coral reefs decline unabated, with a 14% loss of coral cover globally between 2009 and 2018, which exceeds the total coral currently found on Australia’s coral reefs 19 . One of the crucial factors is the need for substantial recovery volume to preserve reef structure while mitigating coral losses 20 , based on the premise that growth should not come at the expense of genetic diversity and ecosystem stability. Recognizing this, micro-fragmentation leverages corals’ regenerative capacity, segmenting colonies for transplantation. Research indicates accelerated growth in micro-fragments post-fragmentation 21 , 22 , highlighting its potential efficacy. However, molecular mechanisms remain unclear. Lock et al. 23 proposes that micro-fragmentation in Porites lobata disrupts calcium homeostasis, energizing coral expansion as a wound healing response, contributing to survival. The study hypothesizes enhanced calcification rates for skeletal regeneration, but understanding these molecular processes is limited. To uncover the molecular mechanisms driving stony coral regeneration, we focus on Acropora muricata , a fast-growing, perforate coral crucial for reef structure 24 . As a perforate coral, the tissue and skeleton of A. muricata are highly interconnected, which makes it particularly well-suited for studying coral regeneration. Our study integrated scRNA-seq, bulk RNA-seq, full-length transcriptome sequencing, and miRNA analysis for a holistic view of coral regeneration. Differences between scRNA-seq and bulk RNA-seq data emphasize the complex regulatory processes. The multifaceted approach aimed to elucidate cellular dynamics, gene expression, and communication networks, contributing to coral regeneration understanding.",
"discussion": "Discussion The expeditious regeneration of coral colonies is essential for their survival, as demographic indicators, including growth, fecundity, and mortality, exhibit a pronounced reliance on size 44 , which implies a prioritization of energy allocation. Moreover, coral colonies demonstrate a more extensive level of physiological integration than previously recognized, coordinating energy and resources across the entire colony to support regeneration 45 . Acute mechanical damage, caused by factors such as tropical storms, predation, and fragmentation for transplantation, can lead to increased mortality rates, and even loss of reproductive capacity 46 . Previous research has shown a widespread decline in reproductive output in locations distal to the damaged tissue 45 , suggesting that the regeneration from injury in corals may necessitate extended colony integration, challenging the notion that the energy requirements for this crucial process are solely provided by polyps directly adjacent to the damaged area. Therefore, facilitating the rapid and substantial growth of transplanted corals becomes imperative to ensure their survival and successful integration into the reef ecosystem. Here, our morphological study identified a peak phase in the recovery of A. muricata , occurring approximately 2 − 4 weeks post-injury (Fig. 1 ). Building upon this observation, we established the A. muricata cell atlas using scRNA-seq to characterize the cellular dynamics and molecular processes underlying the regeneration phenomenon (Fig. 2 ). The analysis revealed that immune cells were abundant in both cnidocytes and digestive filament cells, suggesting potential immune-related gene or process involvement. Given cnidocytes’ role in prey capture and anti-predator defense 47 , and digestive filaments’ function in food acquisition 48 , these cell types may exhibit immune functions. Additionally, the findings demonstrated a consistent repertoire of cell types between unwounded and wounded samples, without the generation of novel cell subtypes. This observation may suggest that A. muricata primarily relies on the activation and functional adaptation of existing cell types to meet the demands of tissue repair and biomineralization, rather than forming new cell identities. Such a strategy may reflect an efficient regenerative approach, utilizing established cells for injury repair without invoking additional differentiation pathways. Nevertheless, discernible adjustments in the quantity of specific cell types during regeneration were noted, particularly an increase in epidermal cells and a decrease in calicoblasts, suggesting their specific involvement in the regeneration process as they adjust to meet functional and physiological demands. Building on these findings, we further identified potential molecular markers by analyzing genes that were highly expressed and conserved across different treatments. Previous studies, such as Yoshioka et al. 49 , have identified various symbiosis-related genes in Acropora corals. Our analysis focused on sulfate transporter, saccharopine dehydrogenase, and Ca2 as notable markers, complementing broader efforts on symbiosis-related genes in corals. Sulfate transporters may play a critical role in sulfur metabolism during early coral life stages, supporting essential sulfur processes that facilitate symbiotic interactions 49 . Previous studies have confirmed that the presence of symbiotic algae induces the upregulation of coral sulfate transporter homologs 49 . Similarly, saccharopine dehydrogenase, involved in lysine degradation 50 , was detected exclusively in symbiotic anemones and was absent in aposymbiotic ones 51 , suggesting it may enhance nitrogen transport and cycling within the symbiotic host. Additionally, Ca2 is involved in ion transport and pH homeostasis, playing a crucial role in carbon-concentrating mechanisms essential for symbiont photosynthesis 52 . In cnidocytes, we identified high expression of leucine-rich repeat extensin-like proteins. This observation aligns with Levy et al. (2021), whose data also highlighted elevated expression of these proteins in this specific cell type 37 . Together, these findings unveil a range of potential cell markers that contribute to our understanding of the functional roles and specialized characteristics of diverse cell types within coral organisms. Previous research has highlighted genes containing a conserved BRICHOS domain (PF04089) as being differentially expressed in corals exposed to heat and acidification stress 53 . In our study, we observed that Itm2b , which contains this domain, was specifically highly expressed in seven cell types under the wounded treatment. This suggests that Itm2b may play a critical role in the coral’s response to environmental stress, particularly during regeneration. In addition, epidermal cells exhibited specific upregulation of a group of oxidoreductases during coral regeneration, with the most prominent being AOS-LOX, a pathway present across all cnidarian lineages, which is capable of producing oxylipins, crucial stress mediators that indirectly regulate the expression of defense genes 54 . Previous studies have indicated a significant upregulation of AOS-LOX in tissues near the wound and distal parts of coral colonies in the soft coral Capnella imbricata after injury, reaching peak expression at 1 h and 6 h post-injury, respectively 55 . This suggests that the AOS-LOX pathway serves as a rapidly initiated stress response to acute mechanical damage. However, in our study, by integrating the temporal gene expression patterns of AOS-LOX (Supplementary Data 5 ), we observed significant up- and down-regulation at multiple time points during coral regeneration, particularly showing early-stage upregulation until later stages where expression stabilized. This implies that AOS-LOX may function as an enzyme with prolonged activity throughout the coral regeneration process. Because the activity of the enzyme can be influenced by various factors, despite gene expression is downregulated, previously synthesized enzyme molecules may persist and maintain activity for a certain period. Therefore, the intricate temporal regulation of AOS-LOX suggests its potential as a long-acting enzyme during coral regeneration. Several other oxidoreductases, including CAT, CYBA, and PXDN, also exhibited distinct regulation at certain time points during coral injury. CAT catalyzes the degradation of hydrogen peroxide, scavenging reactive oxygen species (ROS) and promoting cell growth, playing a crucial role in the coral response to external stimuli 56 . CYBA, a subunit of NADPH oxidase (NOX), is involved in ROS production and plays an essential role in the immune system, with upregulation observed in response to coral fragmentation 23 . PXDN, another key immune response gene, has been significantly upregulated in response to stony coral tissue loss disease 57 and heat stress 58 . Therefore, forthcoming studies should incorporate assessments of enzyme activity to holistically comprehend the functional roles of these enzymes in response to coral wounding. In addition to stress-related responses, metabolic processes also appear to be critical during regeneration. Notably, we identified Acsl4 as upregulated in symbiocytes under the wounded treatment. Previous studies suggest that Acsl4 provides essential components for energy storage and nutrient availability in corals, which could be vital for meeting the high energy demands of regeneration 59 . Conversely, the downregulation of certain genes observed during coral regeneration suggests a strategic reallocation of cellular resources, prioritizing functions essential for tissue repair and recovery. Specifically, the suppression of genes related to protein synthesis and mitochondrial ATP production may indicate a temporary reduction in energy-intensive processes, such as protein translation and oxidative phosphorylation. This metabolic adjustment likely reflects the high energy demands of regeneration, as cells divert energy to critical processes like stress response and skeletal reconstruction. Similarly, the downregulation of genes involved in transcriptional regulation and protein folding implies a shift away from growth-related activities, redirecting resources to maintain cellular homeostasis and support recovery. These observations underscore the intricate balance of metabolic priorities during coral regeneration, ensuring efficient recovery while minimizing energy expenditure on non-essential functions. Developmental trajectory analysis disclosed a continuous transformation of epidermal cells into calicoblasts during coral regeneration. Notably, epidermal cells predominantly transformed into a specific subtype of calicoblasts during this process. This observation suggests that the primary types of calicoblasts involved in biomineralization differ between normal coral growth and regeneration, reflecting the dynamic nature of coral cell differentiation and functional adaptation in response to distinct developmental and regenerative demands. The predominance of one calicoblast subtype during regeneration may indicate that this subtype represents a transitional state, tailored to the immediate requirements of the regenerative process. In later stages, this transitional calicoblast subtype could potentially revert to normal developmental patterns and transform into another subtype, as observed during typical coral growth. These findings imply that the two calicoblast subtypes observed in unwounded samples may not represent fundamentally distinct cell types but rather reflect cells at different stages of development. During regeneration, both epidermal cells and calicoblasts in earlier developmental stages show a significant increase in numbers, suggesting an enhanced capacity to differentiate more epidermal cells into calicoblasts. This increase in early-stage calicoblasts may reflect distinct biomineralization potential compared to mature calicoblasts. Supporting this, in the early stages of calicoblast development, a noteworthy upregulation of the Ca2 gene was observed, responsible for supplying essential carbonate ions for calcification 52 . Additionally, there was a significant augmentation in the secretion of skeletal organic matrix proteins, pivotal components of the coral skeleton 60 . Moreover, we also identified other genes potentially involved in coral biomineralization that were specifically upregulated in cluster 1. Notably, PRG4 is a key protein, as existing studies suggest that the biomineralization process begins with the secretion of a proteoglycan matrix 61 , further confirming that this period corresponds to the early stages of calicoblast development. MMP24, belonging to the matrix metalloproteinase gene family, which plays a vital role in balancing and regulating the degradation of ECM proteins 62 , might also participate in coral calcification. In addition, distinct biomineralization-related genes were also found in other clusters. These findings underscore the potential variability of key genes responsible for biomineralization in calicoblasts at different developmental stages, and this variability might be intricately linked to the underlying mechanisms governing coral skeletal formation. These findings are further supported by the results of the cell fate analysis, which revealed distinct gene expression patterns and functional enrichment across the two calicoblast fates identified. Cell fate 1, predominantly composed of calicoblasts from the unwounded treatment, was enriched in genes associated with extracellular matrix remodeling and stabilization. These genes play critical roles in creating and maintaining the extracellular matrix 62 , suggesting that calicoblasts in cell fate 1 may prioritize structural support and maintenance during normal coral growth. In contrast, cell fate 2, which includes the majority of calicoblasts from the wounded treatment and a portion from the unwounded treatment, exhibited enrichment in genes such as Ca2 , galaxin , and Mucin , associated with biomineralization. These expression patterns suggest that calicoblasts in cell fate 2 may play a critical role in matrix deposition and calcification during regeneration. The gene enrichment in processes related to ATP synthesis, transmembrane transport, and cell proliferation suggests that these calicoblasts are energetically and metabolically prepared to support the demands of skeletal repair during regeneration. The distinct gene expression profiles observed in the two calicoblast fates further underscore the variability in cellular functions during coral development and regeneration. Importantly, these results align with the earlier hypothesis that the two calicoblast clusters may represent different stages of development rather than fundamentally distinct cell types. During regeneration, the observed predominance of calicoblasts associated with cell fate 2 could indicate a transitional state tailored to meet the immediate demands of skeletal repair, while calicoblasts associated with cell fate 1 might represent cells engaged in long-term skeletal maintenance. Together, these findings highlight the dynamic nature of coral calicoblasts and their ability to adapt their molecular functions in response to changing physiological needs. This flexibility ensures the effective fulfillment of distinct requirements during normal growth and regeneration, emphasizing the intricate coordination of developmental and regenerative processes in corals. In our study, we observed that some of the upregulated, downregulated, and biomineralization-related DEGs in the wounded treatment of scRNA-seq displayed opposite expression patterns in the bulk RNA-seq data (Cluster 2 in Fig. 6a–c ). This suggests that there is significant cellular heterogeneity in gene expression during the injury response, which is not fully captured by bulk RNA-seq. Since bulk RNA-seq represents an average gene expression across the entire sample 63 , it may obscure the specific expression patterns of smaller cell populations, particularly those that are critical for regeneration but less abundant in the sample. In contrast, single-cell RNA-seq provides a more detailed view, capturing the molecular differences within individual cells, which is essential for understanding complex biological processes like injury repair 64 . This observation underscores the necessity of single-cell RNA-seq technology in studying intricate biological processes such as coral regeneration. By enabling the identification of gene expression variations at the single-cell level, single-cell RNA-seq allows for a more precise understanding of cellular dynamics and molecular mechanisms during the repair process, which might be missed when using bulk RNA-seq alone. Although we observed some gene expression discrepancies between the single-cell and bulk RNA-seq data, the overall temporal gene expression analysis revealed that, after an initial period of dynamic gene activity, the expression patterns stabilize as the regeneration process progresses. This stabilization may signify a transition from the rapid regeneration phase to a state resembling normal growth, as the coral gradually returns to its steady-state physiological processes following the completion of tissue repair and biomineralization. Building upon this, we hypothesize that the enhanced biomineralization observed during the short-term coral repair stems from the generation of a substantial quantity of newly formed calicoblasts endowed with high biomineralization capability. This provides an explanation for the immediate short-term enhanced skeletal deposition observed in coral micro-fragments following initial fragmentation and tissue damage 21 , 22 . However, the precise mechanisms governing the transformation of epidermal cells into calicoblasts remain elusive. Regarding the question of why corals accelerate the deposition of their skeletons in the early stages of regeneration, similar to other organisms, the coral skeleton plays a crucial role as an attachment substrate for the soft and vulnerable polyp. It acts as the primary physical barrier to protect the soft tissue from external environmental influences 65 . Simultaneously, due to its richness in peptide neurotoxins, the coral skeleton provides biochemical protection 66 . Therefore, the observed brief period of rapid growth post-injury serves as a defensive strategy, reinforcing damaged tissues. In our investigation of the regeneration of A. muricata , we primarily focused on understanding the molecular mechanisms underlying rapid skeletal remodeling following coral injury, particularly the cellular and transcriptomic dynamics involved. However, our study did not address the tissue repair and regeneration processes, which are equally crucial in coral regeneration. Existing literature suggests that the first step in coral regeneration is wound healing, a rapid tissue repair process that prevents infection and further tissue loss after injury 67 . Therefore, investigating the mechanisms of tissue repair and regeneration is of paramount importance for a more comprehensive understanding of coral regeneration. Future research could build on our findings by focusing on functional validation of key genes and cellular dynamics involved in both tissue and skeletal regeneration. Moreover, integrating multi-omics approaches, including proteomics and metabolomics, as well as exploring species-specific variations and environmental influences, will help advance the field of coral regeneration research."
} | 5,662 |
39149354 | PMC11326212 | pmc | 4,869 | {
"abstract": "Background: Synthetic microbial communities offer an opportunity to conduct reductionist research in tractable model systems. However, deriving abundances of highly related strains within these communities is currently unreliable. 16S rRNA gene sequencing does not resolve abundance at the strain level, standard methods for analysis of shotgun metagenomic sequencing do not account for ambiguous mapping between closely related strains, and other methods such as quantitative PCR (qPCR) scale poorly and are resource prohibitive for complex communities. We present StrainR2, which utilizes shotgun metagenomic sequencing paired with a k-mer-based normalization strategy to provide high accuracy strain-level abundances for all members of a synthetic community, provided their genomes. Results: Both in silico , and using sequencing data derived from gnotobiotic mice colonized with a synthetic fecal microbiota, StrainR2 resolves strain abundances with greater accuracy than other tools utilizing shotgun metagenomic sequencing reads and can resolve complex mixtures of highly related strains. Through experimental validation and benchmarking, we demonstrate that StrainR2’s accuracy is comparable to that of qPCR on a subset of strains resolved using absolute quantification. Further, it is capable of scaling to communities of hundreds of strains and efficiently utilizes memory being capable of running both on personal computers and high-performance computing nodes. Conclusions: Using shotgun metagenomic sequencing reads is a viable method for determining accurate strain-level abundances in synthetic communities using StrainR2.",
"conclusion": "CONCLUSIONS Through analysis of data both in silico and experimental data, we demonstrate that StrainR2 provides highly accurate strain abundances and prevalences using a fraction of the computational resources of previous approaches. StrainR2 makes shotgun metagenomic sequencing reads a viable tool for accurate strain abundances in synthetic communities without the need for high performance computing. This may eliminate the need for more time consuming or expensive methods to assess strain abundance, such as qPCR to which it provides comparable abundances. StrainR2 is also able to provide abundances in scenarios where designing primers for qPCR would be extremely difficult or impossible, as would be the case for the E. lenta community. StrainR2 is available via GitHub, Bioconda, and as a Docker container.",
"discussion": "RESULTS AND DISCUSSION Throughout analysis, two communities of strains were used to generate reads in silico across 6 abundance distributions that represent various scenarios ( Figure S1 , Tables S1 - S4 ). The first community, sFMT1+Cs, represents a more realistic scenario for a synthetic community where most strains are not very similar but with five of the species having two or more strains each ( Figure 2A , Table S1 ). As in the development of StrainR1, a mock community of 22 E. lenta strains were also used to represent an extreme scenario where the number of unique k-mers would be at a minimum ( Figure 2B ). To evaluate StrainR2’s improvement over using FPKM values in the case of uniformly abundant strains, the coefficient of variation was used as a benchmark, as lower coefficients of variation are closer to a uniform distribution. In the case of the sFMT1+Cs community with uniformly abundant strains, StrainR2’s wpFUKM was able to normalize the reads such that the coefficient of variation was 1.69% as compared to FPKM with a coefficient of variation of 17.44% ( Figure 2A ). In the case of uniformly abundant E. lenta strains, the coefficients of variation were 3.93% and 86.82% for wpFUKM and FPKM, respectively ( Figure 2B ). Using StrainR2, this corresponds to fold change differences of 1.085 and 1.159 between the highest and lowest reported abundances for wpFUKM on sFMT1+Cs and E. lenta strains, respectively. Despite the high similarity between strains, wpFUKM still resembled a uniform distribution unlike FPKM. FPKM tended to underestimate the abundance of strains with higher similarity, whereas wpFUKM remained unbiased. With uniformly abundant E. lenta reads, StrainR2’s wpFUKM best followed a uniform distribution out of all methods tested as determined by coefficients of variation ( Figure S2A ). While median FUKM (mFUKM) is the only abundance estimate provided by StrainR1, StrainR2 is still able to quantitatively improve on this measure, with the coefficient of variation decreasing from 6.22% to 5.53%. Specifically, StrainR2 achieves this by including overlaps between subcontigs, using larger k-mers, and marking k-mers from excluded subcontigs as non-unique. Furthermore, NinjaMap showed the worst performance out of all the methods tested, with several cases of strain abundances being off by more than tenfold ( Figure S2B ). To further assess the accuracy examined the recovery of strain abundances across 6 different distributions ( Figure S1 , Tables S3 and S4 ). Jensen-Shannon divergence between true and predicted abundances was used. All measures of abundance were first converted to be a percentage of the total abundance so that all measures of abundance were comparable, then the Jensen-Shannon divergence was calculated. Resulting values can be between 0 and 1, where 0 represents the least divergence. Across all types of distributions, StrainR2 had a Jensen- Shannon divergence at least two magnitudes smaller than either NinjaMap or FPKM ( Figure 3A ). Across each distribution, StrainR2 consistently provided the most accurate recovery of relative abundances ( Figure 3B ). NinjaMap’s abundance predictions were less accurate than using FPKM in all community distributions, which results from high abundance predictions for the strains with the lowest abundances. Its accuracy degraded significantly when spanning strains with orders of magnitude difference in abundance. Moreover, the E. lenta community decreased the accuracy of all tools, but StrainR2 observed the smallest decrease in accuracy. As a representative example, StrainR2 more closely correlates with true abundances in a log-normal distribution of sFMT1+Cs strains than other methods. StrainR2 had the highest Pearson correlation, with an R 2 =0.9996, whereas NinjaMap and unnormalized FPKM reported correlations of R 2 =0.6098 and R 2 =0.9723, respectively. In the log-normal distribution of E. lenta strains, the R 2 values are decreased to 0.9944 (StrainR2), 0.1497 (FPKM), and 0.0314 (NinjaMap). The scatterplots for other distributions and communities show a similar trend with NinjaMap and FPKM performing far worse with E. lenta strains, whereas StrainR2 maintains high correlations (R 2 >0.9944) ( Figure 3B ). The frequency of log2(fold changes) from the true abundance summed across all 6 distributions for each tool shows that StrainR2 has the largest peak around 0 with few abundance predictions more than two fold different from the true abundance ( Figure 3C ). NinjaMap had the largest amount of predictions at more than a 4 fold change from the true abundance, which mostly arises from low abundance organisms. These could be a result of NinjaMap’s use of reads that map ambiguously, which inflates abundances in the case where most ambiguously mapped reads originate from another organism. StrainR2 has similar such cases for very low abundance strains, but at a significantly reduced rate. StrainR2 can also be used to test the presence or absence of a strain depending on if it outputs zero as an abundance. The rate of false positives and negatives heavily depends on which weighted percentile is used as the final abundance ( Figure S3A ). Using a higher weighted percentile increases false positives, whereas lower weighted percentiles increase false negatives, usually in the case of low abundance organisms. A weighted percentile of 60 was chosen to compare StrainR2’s strain presence or absence predictive ability. F1 scores of StrainR2 and three other tools reveal that StrainR2 predicted presence with the highest accuracy ( Figure S3B ). Only three distributions are shown as these are the only distributions that contained absent strains. These results suggest that StrainR2 performs slightly better than YACHT for predicting the presence or absence of strains with recommended parameters, and shows a large improvement over NinjaMap and FPKM. To further test the presence/absence prediction of these tools, the tools were run on ten replicates of the zero-inflated log-normal community, each with different strain abundances, with StrainR2 still showing the best performance ( Figure S3C ). To assess how StrainR2 scales with synthetic community complexity, the memory in GB and run times of the computationally intensive database generation steps of StrainR2, StrainR1, and NinjaMap were gathered as described in the implementation section on an Ubuntu server with dual Intel Xeon Silver 4214 CPUs and 384 GiB of memory ( Figure 4 ). Run times for StrainR2 grew linearly and remained low for all inputs as compared to StrainR1 ( Figure 4A ) and the memory usage showed similar trends ( Figure 4C ). To test if the trend would hold for highly similar inputs, runs were performed on one through 22 of the E. lenta strains. StrainR2 maintained its low run times, whereas StrainR1 and Ninjamap’s run times grew non-linearly ( Figure 4B ). Memory usage on the E. lenta strains is also shown ( Figure 4D ). StrainR2 run times scale closely with the number of unique k-mers there are in a community, meaning it is unaffected by highly similar communities ( Figure S4 ). To validate function outside of a high-performance computing environment, the sFMT1+Cs community was profiled through a StrainR2 Bioconda installation on a personal computer. The system was running OS X Ventura 13.1 with an Apple M1 Pro processor and 16Gb of RAM. The run times were 1 minute 31 seconds, and 6 minutes 56 seconds for PreProcessR and StrainR respectively for 51.9 million reads (7.8 Gbases) of paired-end NovaSeq 6000 data with 8 threads. This highlights that StrainR2 does not require high performance computing nodes, and is a tangible strategy available to most research groups. While StrainR2 was shown to have the best accuracy in silico , we sought to validate its function using experimental samples and a gold-standard method for strain quantification: qPCR with strain-specific probes. Shotgun metagenomic sequencing data was obtained from 17 fecal samples of gnotobiotic mice colonized with sFMT1 or sFMT1+Cs. As a control, two of the mice were also germ-free. To determine the true abundance of strains, qPCR was performed on samples from the same mice for four of the strains present in sFMT1 (JEB00023, JEB00029, JEB00174, and JEB00254). JEB00023 and JEB00174 were selected as they are both strains of the species Bacteroides uniformis and represent an important use case of StrainR2. JEB00029 was selected as our previous experiments had suggested it could not colonize the mice, while JEB00254 was capable of colonization at low abundances [ 9 ]. To compare abundances from each method, the fold change from the geometric mean of strain abundances within a sample was calculated. StrainR2 maintained a close relationship with the results obtained from qPCR, as well as correctly predicting when strains were absent, as was the case with JEB00029 and the two germ-free mice ( Figure 5A ). NinjaMap showed a weaker correlation with the data from qPCR, and was inconsistent with predicting abundances of strains between samples ( Figure 5B ), with FPKM having similar results ( Figure 5C ). FPKM and NinjaMap both incorrectly assigned abundances to JEB00029 and strains in the germ-free mice, showing that another strength of StrainR2 is more accurate presence/absence prediction, as it was the only tool to agree with qPCR on the absence of strains. Per-strain correlations with each tool are described in Figure 5D . Pearson correlation values for StrainR2, NinjaMap, and FPKM versus copies/g across all strains are R 2 =0.9432, R 2 =0.3139, and R 2 =0.3559, respectively."
} | 3,028 |
26183259 | PMC4518299 | pmc | 4,870 | {
"abstract": "Ocean acidification is predicted to impact ecosystems reliant on calcifying organisms, potentially reducing the socioeconomic benefits these habitats provide. Here we investigate the acclimation potential of stony corals living along a pH gradient caused by a Mediterranean CO 2 vent that serves as a natural long-term experimental setting. We show that in response to reduced skeletal mineralization at lower pH, corals increase their skeletal macroporosity (features >10 μm) in order to maintain constant linear extension rate, an important criterion for reproductive output. At the nanoscale, the coral skeleton's structural features are not altered. However, higher skeletal porosity, and reduced bulk density and stiffness may contribute to reduce population density and increase damage susceptibility under low pH conditions. Based on these observations, the almost universally employed measure of coral biomineralization, the rate of linear extension, might not be a reliable metric for assessing coral health and resilience in a warming and acidifying ocean.",
"discussion": "Discussion Results of the present study complement previous research on B. europaea at this same vent site, which revealed no changes in skeletal calcium carbonate polymorph, organic matrix content, aragonite fibre thickness and skeletal hardness in corals growing along the pH gradient 13 . There was, however, a significant reduction in population density along the pH gradient, decreasing by a factor of 3 with increasing proximity to the vent crater centre (that is, from S1 to S3) 13 . Figure 4 summarizes these results at the ocean, population, macro, micro and nanoscales for B. europaea . At the macroscale, increasing acidity was associated with a reduction in net calcification rate and a parallel increase in skeletal porosity, coupled with a decrease in skeletal bulk density. Linear extension rate and corallite shape (biometry and interseptal volume fraction) did not depend on pH, probably as a result of the compensation of reduced net calcification rate by increased skeletal porosity. At the micro/macroscale, the declining skeletal stiffness with decreasing pH could be coupled to an increased volume fraction of pores having a size comparable to the indentation area (that is, at the border between the micro and macroscales). At the nanoscale, porosity, biomineral hardness and density were not significantly affected by pH. These results, bolstered by qualitative SEM and AFM analyses, suggest that the ‘building blocks' produced by the biomineralization process are substantially unaffected, but the increase in skeletal porosity is both a gain and a loss for the coral. In fact, in an acidic environment, where the net calcification is depressed, enhanced macroporosity keeps linear extension rate constant, potentially meeting functional reproductive needs (for example, the ability to reach critical size at sexual maturity); however, it also reduces the mechanical strength of the skeletons, increasing damage susceptibility, which could result in increased mortality and the observed population density decline 13 . While the results reported here for B. europaea may not be representative of the generalized response of all coral species to OA, they are consistent with field observations made on other reef-building scleractinians. For example, while maintaining constant skeletal linear extension, decreased rates of calcification and losses in bulk skeletal density as a function of reduced aragonite saturation have been observed in Montasraea annularis 17 and Porites astreoides 18 . While low aragonite saturation as a sole driver for the observed reduction in coral calcification has been discussed 19 , our conclusions regarding a balance between reduced net calcification rate and increased macroporosity to maintain constant linear extension can explain the outcomes of those studies 17 18 . In fact bulk density depends both on biomineral density and porosity. Our multi-scale analysis shows that all the skeletal features at the nano and microscales, including biomineral density, are unchanged. The decrease of bulk density with decreasing pH depends on the increase of macroporosity, leaving the linear extension rate constant. Our findings, together with the well-described detrimental effects of heat stress on the scleractinian zooxanthellate coral B. europaea 16 20 21 22 23 , provide several independent and consistent clues regarding the sensitivity of this species to global climate change predicted for the coming decades. Together with results from previous studies 24 , we demonstrate that the almost universally employed measure of coral biomineralization, namely the rate of linear extension, might not be a reliable metric for assessing coral health and resilience in a warming and acidifying ocean. Indeed, although the coral's ability to maintain linear extension rate and gross skeletal morphology under conditions of decreasing oceanic pH could allow it to reach sexual maturity, it could reduce skeletal resistance to environmental challenges, affecting the long-term survivability of the species."
} | 1,283 |
31831795 | PMC6908680 | pmc | 4,872 | {
"abstract": "Microalgal photosynthesis is a promising solar energy conversion process to produce high concentration biomass, which can be utilized in the various fields including bioenergy, food resources, and medicine. In this research, we study the optical design rule for microalgal cultivation systems, to efficiently utilize the solar energy and improve the photosynthesis efficiency. First, an organic luminescent dye of 3,6-Bis(4′-(diphenylamino)-1,1′-biphenyl-4-yl)-2,5-dihexyl-2,5-dihydropyrrolo3,4-c pyrrole -1,4-dione (D1) was coated on a photobioreactor (PBR) for microalgal cultivation. Unlike previous reports, there was no enhancement in the biomass productivities under artificial solar illuminations of 0.2 and 0.6 sun. We analyze the limitations and future design principles of the PBRs using photoluminescence under strong illumination. Second, as a multiple-bandgaps-scheme to maximize the conversion efficiency of solar energy, we propose a dual-energy generator that combines microalgal cultivation with spectrally selective photovoltaic cells (PVs). In the proposed system, the blue and green photons, of which high energy is not efficiently utilized in photosynthesis, are absorbed by a large-bandgap PV, generating electricity with a high open-circuit voltage ( V oc ) in reward for narrowing the absorption spectrum. Then, the unabsorbed red photons are guided into PBR and utilized for photosynthesis with high efficiency. Under an illumination of 7.2 kWh m −2 d −1 , we experimentally verified that our dual-energy generator with C 60 -based PV can simultaneously produce 20.3 g m −2 d −1 of biomass and 220 Wh m −2 d −1 of electricity by utilizing multiple bandgaps in a single system.",
"conclusion": "Conclusions Understanding the solar spectrum allows the design of more efficient energy conversion systems that use sunlight as an input. We applied spectral engineering to achieve enhanced photosynthesis efficiency of microalgae. Despite their great potentials, the classical approaches that use luminescent materials to convert blue photons to red photons have not yielded enhanced biomass productivity, possibly due to the intrinsic low optical efficiencies. As an advanced scheme, a dual-energy generator that combines spectrally selective PVs and photosynthesis was proposed, utilizing multiple bandgaps of fullerene (2.6 eV) and chlorophyll (1.8 eV) with minimum energy losses. The proposed system experimentally achieved 85% of the reference biomass productivity, while simultaneously producing an expected additional electricity of 220 Wh m −2 d −1 under the AM 1.5 G illumination of 7.2 kWh m −2 d −1 .",
"introduction": "Introduction In addition to the electricity generated from solar and wind power, biofuel is an attractive renewable energy source, especially for systems such as transportation, which require liquid forms of energy. The aquatic microalgal biomass is considered to be one of the best-suited feedstocks for this purpose, and it does not require arable land area 1 – 3 . Moreover, a wide range of the potential applications of the microalgal biomass (food, medicine, agriculture, etc.) makes it more promising in the view of marketability. However, to become economically viable for mass production, the fundamental issue of limited biomass yield, although one-order higher than terrestrial plants, must be resolved. As a process to convert sunlight into a utilizable form, photosynthesis has a similarity to the photovoltaic devices (PVs); however, photosynthesis for terrestrial or microalgal biomass production suffers from the limited power conversion efficiency (PCE), approximately one order lower than that of PVs 4 , restricting the productivity per area. In chlorophyll, which is to be considered a semiconductor with a band-gap ( E g ) of 1.78 eV, infrared (IR) photons below the band-gap are not absorbed (53%) and the corresponding maximum electron flux is 1.2 × 10 21 m −2 s −1 under 1 sun, as shown in Fig. 1a . In this case, the maximum power conversion efficiency (PCE) reaches only 27%, and the excess energy of the absorbed visible photons is lost as heat. This PCE is lower than the maximum achievable value of 34% at the optimal band-gap of 1.34 eV, called the Shockley-Queisser limit 5 , 6 . Moreover, in the photosynthesis process depicted in Fig. 1b , through photosystems (PSs) I and II, 48 photons are consumed to produce one molecule of glucose (C 6 H 12 O 6 ) with a chemical energy 29.8 eV. The corresponding maximum photosynthesis efficiency (PE) becomes [(1.2 × 10 21 m −2 s −1 ) × (29.8 eV/48)/(1000 W m −2 )] ~ 12%. In reality, as not all the absorbed photons enter the PSs, it is known that at least 57 photons 7 – 9 are consumed per glucose molecule, and the maximum PE becomes ~10% (assuming no loss during the conversion from glucose into real biomass consisting of diverse molecules). While diverse single-junction PVs, such as GaAs ( E g ~ 1.4 eV), crystalline silicone (c-Si, E g ~ 1.1 eV), and perovskite ( E g ~ 1.5 eV) -based PVs, have achieved high PCEs of more than 70% of their theoretical limits, as shown in Fig. 1a 10 , the PEs of outdoor microalgae cultivation under sunlight are reported to be only ~4% for photobioreactor (PBR) systems 11 – 15 and 3- to 5-fold lower for open pond systems 3 in warm locations, far below their theoretical limit, implying significant opportunity for further technical improvement. It should be noted that some of the previous reports 13 , 14 chose a different definition of PE and did not count IR photons as inputs. Figure 1 Theoretical efficiency. ( a ) Theoretical maximum power conversion efficiency (temperature: 300 K) and electron flux of semiconductors with various bandgaps under AM 1.5 G illumination. ( b ) Spectra of AM 1.5 G and chlorophyll absorption (top) and simplified photosynthesis model (bottom). To improve the low PE and produce more bioenergy, controlling the quality of light is an attractive strategy. Although blue photons have higher energy than red photons, the absorption of blue light is inefficient during photosynthesis because (i) the excessive energy of the blue photons (2.5–3.1 eV) is lost as heat in PS I or II with fixed bandgaps (~1.8 eV), (ii) the quantum efficiency of microalgal photosynthesis is known to be lower for blue photons than for green and red photons 16 – 20 , and (iii) the absorption of those high-energy photons may cause photoinhibition 21 – 23 . Moreover, there have been a few studies 24 – 28 reporting that microalgae cells cultivated under red light contain more lipids, which are used for fuel production, than those under blue light, while further investigation would be required to reveal the mechanism and quantify the relationship. Accordingly, direct exposure of microalgae to the full spectrum appears to be suboptimal for utilizing solar energy and the available land area. For those reasons, there have been attempts to down-convert blue or green photons to red photons by adopting luminescent materials 11 , 27 – 34 . For example, L. Wondraczek et al . reported a >20% enhanced photosynthesis rate by adopting a photoluminescent phosphor 27 . In a similar manner, H. Amrei et al . presented further enhancement of microalgal biomass productivity up to 74% using organic luminescent dye 33 . The improved lipid content of 30–70% using spectral conversion was also demonstrated by Y. Seo et al . with 20–40% enhanced biomass productivity 11 . However, despite these achievements that showed the potential and importance of light quality control of microalgal photosynthesis, unsolved issues remain: (i) the spectral information of the light source has been rarely reported and are difficult to compare, (ii) the spectrum and intensity of the light used in these experiments were much different from sunlight and the results may not be reproducible in outdoor conditions, and (iii) quantitative analysis of their effectiveness and general design rules are lacking. In this study, we investigated the optical strategies for solar spectrum engineering of photosynthesis with a controllable AM 1.5G-simulating light source. For the spectral conversion scheme, the optical losses due to the low quantum yield and geometrical light propagation efficiency of luminescent materials were shown to be limiting factors for using spectral conversion materials. Based on the analysis of the potential and limitations of spectral conversion, we introduce an alternative means of utilizing high-energy blue photons in an add-on device, namely, a dual-energy generator that combines a microalgal photosynthesis system with a high-bandgap photovoltaic (PV) module. This innovative device enables the use of red photons for efficient microalgal photosynthesis and blue photons for high-voltage electricity generation. While there have been a few previous approaches that combined PVs with photosynthesis 35 , 36 , they consisted of a simple combination of two different systems and there were no synergetic effects from the control of light quality. We aimed to achieve the multiple bandgap effect, possibly overcoming the efficiency limit of single semiconductor system. We use a fullerene-based organic photovoltaic cell with a high bandgap, which is best suited for the spectrum separation of sunlight and the utilization of high-energy photons.",
"discussion": "Results and Discussion Spectral conversion: bioreactor adopting luminescent materials While most previous reports on the spectral characteristics of microalgal photosynthesis have been based on light emitting diode (LED)-based environments, the quantity and quality of LED were significantly different from the sun and the results may not be applicable to outdoor conditions. For this reason, adopting the appropriate illumination, which mimics the sun in a controllable manner, is crucial to study the optical behavior of an aquatic photosynthesis system in outdoor conditions. Accordingly, we implemented an artificial AM 1.5 G light source, integrating optical filters with a white metal-halide lamp. As shown in Fig. 2a , despite the sharp fluctuations of the measured spectrum of our light source, the integrated proportions of visible photons (400–500 nm, 500–600 nm, and 600–700 nm) roughly matched the AM 1.5 G spectrum, with an error range of 5–15% 9 . Moreover, periodic boundary conditions (PBCs) were applied to simulate large-scale cultivation using smaller bioreactors with limited illumination area. A reflecting metal (aluminum foil or stainless-steel walls) on the sides can prevent energy efflux from inside to outside and energy influx from outside to inside, as depicted in Fig. 2b . In the optical view, the system is identical to the large-scale reactor, where the influx and efflux of light can be compensated. The illumination area was confined by the aperture to strictly define the input energy and prevent overestimation. Figure 2 Spectral conversion. ( a ) Number of photons per wavelength (lines in A.U.) and their relative portion (dots) for the real AM 1.5 G (black) and artificial solar-simulating light source (red) used in this study. UV, visible, and IR regions are shown in blue, green, and pink colors, respectively. ( b ) Outdoor-simulating experimental set-up with solar-simulating light source, defined illumination area, periodic boundary condition, and back reflector. ( c ) Measured absorbance and photoluminescence spectrum of D1 in the film state. ( d ) The growth curves of microalgae in volume concentrations with and without D1 coating. ( e ) The schematic illustration of optical loss mechanism for dye-integrated bioreactors. Under simulated solar illumination, we examined the change of the algal growth rate by adopting a spectral conversion material. For this experiment, we chose an organic dye, 3,6-Bis(4′-(diphenylamino)-1,1′-biphenyl-4-yl)-2,5-dihexyl-2,5-dihydropyrrolo3,4-c pyrrole -1,4-dione (D1) 37 , as a spectral conversion material, which absorbs photons with wavelengths shorter than 550 nm (i.e. blue and green light) (peak at 495 nm) and emits photons with a photoluminescence (PL) peak at 618 nm (i.e. red light), as shown in Fig. 2c . Such spectral characteristics of optical absorption and luminescence are well-matched with the optical properties of microalgae. While the typical red luminescent dyes show a limited quantum yield of 10–20% in the film state, mainly due to the aggregation and π–π stacking interactions of the molecules in the solid state, D1 was designed to minimize such quenching loss in the film using the phenomenon of aggregation induced emission (AIE) 38 , 39 . The measured quantum yield of D1 was 24.7% in the film state on a quartz plate 40 , and such outstanding optical properties make D1 an ideal candidate for our proposed system. Figures 2d and S1 show the results of the microalgae ( Chlorella sp .) cultivation with and without D1. The light intensity was set to be 0.2 or 0.6 sun with continuous illumination, corresponding to 400 and 1200 μmol m −2 s −1 of visible photons, or 4.8 and 14.4 kWh m −2 d −1 of energy. D1 was coated on the polycarbonate cover of the bioreactors with a volume of 500 ml and illumination area of 56 cm 2 . The results of the duplicate reactors were compared with another duplicate reference reactors without dye coatings, as depicted in the inset of Fig. 2d . From their average values, we found no evidence of improved biomass production from the experiments using spectral converting materials. While the average growth rates were already high for the reference reactors (0.41 and 0.49 g L −1 d −1 ), with the aid of a high-density photon supply (0.2 and 0.6 sun, respectively), the coating of D1 failed to add productivity and yielded even lower growth rates (0.39 and 0.47 g L −1 d −1 , respectively). Our results contradict previous research 11 , 27 – 34 that reported enhancements in the biomass productivity by modifying the quality of incident illumination. We speculate that these discrepancies may be due to our different illumination conditions, that is, simulating sunlight. First, organic dyes tend to degrade easily by photooxidation under strong illumination 41 . We observed that the color of D1 after the cultivation was not as dark as the initial state, suggesting degradation. Second, even with identical systems, the light intensity can produce different results. Typical LED light sources have a photon flux far below 0.1 sun and spectra that poorly match chlorophyll absorption spectra. So, the additional illumination with converted red photons may help to enhance the photosynthesis rate. On the other hand, the artificial sunlight we used was as strong as outdoor illumination and contained a sufficient number of red photons to saturate the photosynthesis. Hence, the photon supply may not be a limiting factor for photosynthesis. In previous research 11 , while up to a 40% enhancement of photosynthesis efficiency was achieved with spectral conversion under 0.05 sun, no enhancement was observed in the same system under 0.15–0.2 sun. In addition to the issues mentioned above, the study of the intrinsic optical limitations of the schemes using luminescent materials would provide further insights into formulating design rules. As depicted in Fig. 2e , the luminescent material absorbs the incident light and re-emits converted photons in dipole form. Subsequently, the emitted photons propagate (i) in a forward direction to the algal solution, (ii) in a backward direction toward the incident light source, and (iii) at the edges guided by total internal reflection (TIR) at the surfaces of the dye and substrate and their interfaces. Assuming an isotropic distribution, the amounts of (i) and (ii) are almost identical and become 1/2 n 2 /2 per each, where n is the refractive index of the emitting material 42 , 43 . Hence, only 4–11% of the emitted photons for n = 1.5–2.5 can be transported to microalgae through (ii), and the photons radiated to (i) escape the system. The other photons are all trapped by TIR, as described in (iii). There is a small chance that the photons will be transported by the re-emission of dye molecules, if the trapped photons are re-absorbed by the dye. In addition to the non-perfect internal quantum yield, such intrinsically limited photon transport efficiency significantly restricts the efficiency of the given architecture. For example, with an internal quantum yield of 25% and light transport efficiency of 11%, a single red photon must be more beneficial than 36 blue and green photons for the photosynthesis process, a seemingly unrealistic requirement, to enhance the overall productivity using this scheme. It should be noted that the reported optical efficiency of red photons is only 10–20% higher than that of blue photons according to their action spectra ( A action ( λ )). There exist several possible, but not perfect, strategies to suppress any losses. If the dye-coated substrate directly touches water without an air gap, TIR occurs less and we can obtain a higher optical transmission to the water side (path (i) above), while keeping the undesired backward transmission (path (ii) above) low. If the emitting material is synthesized to have a low refractive index near that of water ( n = 1.33), as well as a high quantum yield and large Stokes shift, TIR loss can be completely removed, and escape through the backside (~17%) is the only path for losing photons. The use of luminescent materials directly inside the bioreactor in the state of the aqueous solution might be an efficient additional approach to overcome the limitations of film-state dyes. Moreover, control of the dipole orientation and roughening of the film interface can be effective ways to suppress TIR losses. Therefore, while many limitations are present in the current configuration, the development of novel materials and advanced configurations have great potential for improving the biomass productivity of microalgae by modifying the quality of solar illumination. Spectral separation: bioreactor combined with spectrally selective photovoltaic cell Photosynthesis may not be best process for utilizing blue photons. Even with a 100% efficiency scheme to convert blue photons to red photons, the fundamental energy loss of high energy blue photons, of which the excess energy is wasted as heat, is inevitable during photosynthesis because of the fixed bandgap, and limits the PE, as discussed in Fig. 1 . Therefore, instead of changing the color of the light, we used an alternative approach to fully utilize the high-energy blue photons. Figure 3a illustrates our proposed system, named a dual-energy generator . A “spectrally selective PV,” which absorbs only high-energy photons, was installed on one side of a blazed-shape cultivator. In this way, the incident light first impinges on the PVs, and the solar energy can be spectrally split; high-energy (blue) photons are then absorbed by the PV to generate electricity with a high voltage, and low-energy (red) photons are used for biofuel production with the optimal spectral match. The distinctive feature of our approach of combining PVs and photosynthesis is the separation of the spectrum, not the quantity, of incident light with multiple bandgaps. While the theoretical limits of PCE were calculated to be 34% and 27% for semiconductors with bandgaps of 1.34 eV (optimal) and 1.8 eV (chlorophyll), respectively, as shown in Fig. 1a , it is known that the limit increases to ~45% when two different bandgaps exist in a single system and the heat loss of high energy photons is alleviated 44 . Figure 3 Dual-energy generator. ( a ) Dual-energy generator integrating PVs with microalgal photosynthesis. ( b ) Energy band diagram of the C 60 -based PV and its current density ( J )-voltage ( V ) curve. ( c ) Separately measured EQE and absorption of a C 60 -based PV and the optical distance of microalgae cells with arbitrary concentrations. ( d ) Simulated EQE (red) and absorption of the C 60 -based PV (grey) and microalgae cells (green) in the dual generator (inset: photosynthetic rate profile inside the configuration with R max = 0.30 W g −1 ). ( e ) Measured growth curve of microalgae cells in the growth phase for the flat PBR (gray) and dual-energy generator (red). (Inset: Blueprint and photograph of the dual-energy generator used in our experiment). Typically, commercial PV materials, such as c-Si, III-V materials, CdTe, and Cu(In,Ga)Se 2 (CIGS), have relatively low bandgaps (1.0–1.5 eV), sacrificing output voltage to absorb more red and near-infrared photons. However, in the dual-energy generator, the sacrificing the voltage is not necessary since red photons do not need to be absorbed. As a result, the hybrid system produces both electricity that can be directly consumed and biofuels that can be stored, with minimal interference to the productivity of either process. The bioreactor can also act as a supporting frame and coolant for the PV, and the PV absorbs UV light that is harmful to microalgae, thereby synergistically reducing the energy production cost. As a spectrally selective PV, we chose a fullerene (C 60 )-based organic PV consisting of tetraphenyldibenzoperiflanthene (DBP) and C 60 , in a ratio of 1:9, whereby the DBP helps to separate the generated charges in C 60 45 . As shown in Fig. 3b , C60 has a high bandgap of 2.6 eV; thus, it absorbs blue photons with a wavelength shorter than 480 nm. The current density ( J )-voltage ( V ) characteristics of the PV under 1 sun irradiation exhibited an open-circuit voltage ( V oc ) of 0.88 V, which is higher than the V oc s of conventional c-Si PVs (0.6–0.7 V), at the cost of narrowing the absorption band from 300–1100 to 300–500 nm, and a power conversion efficiency (PCE) of 3.00%, which can generate 220 Wh m −2 d −1 under 7.2 kWh m −2 d −1 illumination, corresponding to the typical values for the world’s hottest regions. From an economic perspective, such organic PVs are much cheaper than c-Si PVs 46 , 47 , and an electricity production of 220 Wh m −2 d −1 (at 0.07 USD kWh −1 ) is equivalent to a biomass production of 29 g m −2 d −1 (at 540 USD ton −1 48 ). Covering only ~6% of the total area with this scheme is sufficient to supply electricity for the circulation and dewatering processes of a self-operating cultivation system 48 . If C 60 is replaced by C 70 with a lower bandgap (2 eV) in the same PV structure, green photons are absorbed more by the PVs, and the PCE and electric power production further increase to 7.02% and 510 Wh m −2 d −1 , respectively, at the cost of reducing microalgal production, as shown in Fig. S2 . Therefore, the types of PV materials chosen must be finely tuned to obtain the desired amounts of electricity and biofuel. The measured external quantum efficiency (EQE) and absorption of a C 60 -based PV are shown in Fig. 3c with the optical density of microalgae at an arbitrary concentration, measured separately. The absorption spectra of the device were complementary to the absorption spectrum of microalgae. The device is yellow in color because it reflects red and green light. The EQE of ~20% near 600 nm, which is out of the typical C 60 absorption band, was due to the absorption of DBP. The performance of the C 60 -based PVs and the bioreactor simultaneously installed inside the dual-energy generator was assessed by optical simulations, as shown in Fig. 3d . While the EQE of the PV was calculated by transfer-matrix formalism (TMF), assuming the internal quantum efficiency (IQE) of 90% 46 , 47 , the spatial photosynthesis profile of the microalgae (inset of Fig. 3d ) was obtained by combining Monte-Carlo simulations with optical response models for photosynthesis 49 – 51 , fit to our experimental results, as presented in our previous publication 9 . It should be noted that, because photons directly impinge on the PV side first, the calculated PV characteristics of the dual-generator were similar to the measured values of the “PV-only” device, while the calculated algal absorption was highly influenced by the PV properties. According to the simulation results, more than 80% of the blue photons were absorbed by the PVs on the first impingement, and the non-absorbed red photons (near 700 nm) entered the bioreactor and provided energy for photosynthesis. The biomass productivity calculated from the integration of the photosynthesis profile was 15.1 g m −2 d −1 , whereas it was 10.9 g m −2 d −1 for the C 70 -based PVs (Fig. S2b ), where a biomass of 1 g corresponds to 4.9 Wh from its heat value (4.2 kcal g −1 ). Interestingly, the simulated PCE of the C 60 -based PVs in this system was 3.19%, which is, albeit only slightly, even higher than that of the PV-only system. This PCE enhancement can be attributed to the light-trapping effect 9 , 52 – 54 of this system, allowing the PV to recycle the photons that are unabsorbed and reflected by the bioreactor. We also experimentally implemented the dual-energy generator by combining a bioreactor with C 60 -based PVs, as shown in Figs. 3e and S1 . A C 60 (60 nm)-coated film, which had almost identical optical characteristics to the full PV device, was placed at a titled angle of 30° from the vertical plane. The photons initially pass through the illumination area of 45 cm 2 , and those reflected on the PV enter the bioreactor ( Chlorella vulgaris ) with a cultivation volume of 500 ml. The surfaces other than the illuminated area were covered with aluminum foil to block undesired photon flux. The reference flat bioreactor had a volume of 100 ml and illumination area of 10 cm 2 . Since the reference and our configuration have completely different geometries and illumination areas, the volume productivity is not a suitable figure of merit for comparison. Therefore, we divided the total biomass concentration by the illumination area to obtain the areal biomass productivity. The illumination of 0.6 sun was given with a photoperiod of 12 h:12 h, amounting to a total of 7.2 kWh m 2 d −1 . As shown in Fig. 3e , while the areal biomass productivity for the reference bioreactor was 23.9 g m −2 d −1 (PE = 1.6%), for the dual-energy generator, it was possible to achieve a biomass productivity of 20.3 g m −2 d −1 (PE = 1.4%) with an expected electricity production of 220 Wh m −2 d −1 . From the absorption spectrum, it can be calculated that the PV absorbs visible photons of approximately 550 μmol m −2 s −1 of the total 1200 μmol m −2 s −1 under 0.6 sun illumination. Thus, the dual-energy generator achieved 85% of the reference biomass productivity with only 55% of the photon numbers, additionally generating high voltage electricity from the remaining high energy photons. Such higher utilization efficiency with a smaller number of photons resulted from the improved quality of red photons and diluted quantity of strong illumination. The summation of 20.3 g m −2 d −1 and 220 Wh m −2 d −1 corresponds to the PCE of 4.4% over the entire solar illumination spectrum, which becomes 9.2% if only the visible spectrum is counted as the input. Figure 4a presents the theoretical maximum energy production of the dual-energy generator as a function of the PV band gap under an illumination of 7.2 kWh m −2 d −1 . As the band gap increases, PV PCE (red) decreases and biomass productivity (green) increases; the biomass was assumed to contain an energy of 4.2 kcal g −1 from its measured heat value. For the band gap of 2.6 eV, it is theoretically possible to achieve a PV efficiency of 11.5% and a biomass productivity of 121.2 g m −2 d −1 simultaneously, assuming 57 absorbed photons generate one glucose (and no further loss occurs) in the photosynthesis process, implying that there remains further room for improvement in our experiment. Figure 4 Potential of dual-energy generator. Theoretical maximum PCE (PV) and biomass productivity (containing a bioenergy of 4.2 kcal g −1 or 9.45 kcal g −1 ) of the dual-energy generators as a function of the PV band gap under 7.2 kWh m −2 d −1 , when light first impinges on ( a ) PV (red region in the insets) or ( b ) microalgae (green region in the insets). The absorptions of the PV and algae were assumed to be perfect for the photons below their bandgaps. Genetic engineering of microalgae to accumulate greater lipid content can result in a higher yield of bioenergy production, allowing a larger amount of biofuel to be extracted from the same amount of biomass. However, since the lipid contains a higher density of energy (9.45 kcal g −1 ) than the other components of the microalgae (e.g. proteins, carbohydrates), higher lipid contents correspond to higher energy densities of the biomass. Thus, a smaller amount of biomass is produced from the same amount of energy produced by photosynthesis. The upper bound of the lipid production can be calculated from the extreme case that all the produced bioenergy is converted into lipid (i.e. an immortal algal culture which produces lipids without any side products). With the heat value of 9.45 kcal g −1 , the maximum lipid biomass productivity (blue) is only 44% of the green curve with 4.2 kcal g −1 , as shown in Fig. 4a . This curve indicates the theoretical maximum amount of lipid production under an illumination of 7.2 kWh m −2 d −1 . For example, the maximum lipid productions of 53.9 g m −2 d −1 and 64.8 g m −2 d −1 are possible for systems with PVs ( E g = 2.6 eV) and without PVs, respectively. Figure 4b shows the theoretical maxima for another type of dual-energy generator, which receives light on the bioreactor side first. If the PV has a bandgap lower than 1.8 eV, it can generate electricity with the photons reflected by the bioreactor side (inset). Contrary to the PV-first system in Fig. 4a , this microalgae-first system can be beneficial for achieving maximum biomass productivity. Since the PV absorbs only IR photons over 700 nm, the maximum PCE is lower and the red curve is shifted to left (i.e. lower value) compared to that in Fig. 4a . The c-Si PV, the most popular PV in the present market, might be a suitable choice for this system due to its bandgap of 1.12 eV near the optimal point."
} | 7,557 |
36685370 | PMC9852669 | pmc | 4,873 | {
"abstract": "Many organisms in nature such as beetles and cacti can survive in arid places by their own surface structures that are still able to collect mist. These surfaces have micro-nano structures that maintain a very low adhesion, allowing them to continuously collect and transport water. Here, we used a light curing three dimensional molding process to create a template for a water harvesting system inspired by the back of a beetle, a hydrogel-like beetle back surface for water transport. By changing the curvature structure of the water evacuation channels and altering the hydrophilic and hydrophobic properties of the surface, the designed large-scale artificial water harvesting study was made possible. The results show that if the surface has a proper curvature structure and hydrophobic density, the water collection on the super-impregnated surface is much higher than that on an ordinary hydrophobic surface. Based on this, a new efficient and environmentally friendly water collection scheme is proposed. The data show that the triangular tip structure imitating beetle-backed hydrogel surface collects the highest amount of water with a water weight of 16 g in 2 h. This study offers interesting prospects for designing a new generation of structural materials with a bionic structure distribution for high-efficiency water harvesting. The results of the study are useful for pushing the improvement of environmental-friendly water collection, transport and separation devices. Abbreviations The dorsal shape of the beetle's back is critical for water collection. In this work, while redesigning the shape of the back of the beetle, the method of 3D printing the beetle back template was used to prepare the beetle back made of hydrogel, which greatly improved the water collection performance and has certain engineering application prospects.",
"conclusion": "4 Conclusion In summary, based on the problem of water scarcity in life, we prepared a mock beetle back surface using 3D printing technology and investigated the curvature structure and hydrophilicity on the surface. The three-dimensional convex curvature of the back of the beetle has been optimized, and different structures of the back of the beetle have been designed. The results show that the hydrophilic triangular structure of the hydrogel surface has more robust water collection ability compared with other structures. The key factors include: first, the unique arch-shaped structure of the beetle back plays the role of integrated curvature in the process of fog water collection and transportation; second, the Laplace pressure induced by the surface gradient of the triangular structure confers an effective water collection and transportation system on the beetle back surface; third, the hydrophilic hydrogel surface intensifies the adhesion of small droplets in the air, and the water film formed on the surface accelerates the droplet transport rate, thus enhancing the water collection water collection. The study of the structure on this surface will help us to design new materials and devices to collect water in fog and transport condensate efficiently. the introduction of 3D printing technology enables rapid fabrication and mass production, and may have a role in alleviating water scarcity in desert areas.",
"introduction": "1 Introduction One of global concerns is the shortage of freshwater resources. Some arid regions in Africa, Latin America, and Asia have a severe shortage of freshwater resources for daily production and use [ 1 , 2 , 3 ]. Many efforts have been devoted in the last decades to solve this issue of water stress with various technologies, such as purification technologies of wastewater, desalination technologies of seawater and water harvesting technologies [ 4 , 5 , 6 , 7 , 8 ]. Especially for water harvesting, some plants and animals in desert areas rely on their unique structure, which makes them very good at trapping dew and fog. For example, cacti can effectively trap mist from all directions using spikes and micro-notches on their conical surfaces [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ]; Namibian desert beetles can quickly collect water in humid air using striated bumps on their bodies and hydrophilic and hydrophobic regions on their backs [ 16 , 17 , 18 ]; and tiny periodically arranged spindle bodies on spider silk also make them good at trapping mist [ 19 , 20 , 21 ]. These organisms cleverly exploit surface free energy and Laplace pressure differences as the main driving force for water collection [ 22 , 23 , 24 , 25 , 26 ]. For plants and animals in arid desert regions, mist is a better water resource than precipitation and can also be used as a supplementary water source for humans. Hence, inspired by biological structures water, the biomimic natural fog collection systems has attracted intensive interest and been widely studied to improve the efficiency of fog collection capacity [ 27 , 28 , 29 ] Researchers have designed various fog water collection systems based on differences in surface wettability. Li et al. designed a multi-directional artificial spine surface and sputtered hydrophobic coatings on the surface to accelerate water flow and improve water collection efficiency [ 30 ]. Li et al. based on the bionic structure of multiple curved bodies to trap water and illustrated the mechanism of water trapping in the periplasmic pores of pigweed [ 31 ]. Ju et al. investigated the relationship between conical spine clusters and trichomes on cactus stems for fog collection [ 32 ]. Based on the mechanistic explanation of the above studies, many have used the Laplacian pressure difference induced by shape and wettability gradients to achieve efficient water collection performance [ 16 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ]. Although these reports have artificially fabricated hybrid wettability surfaces based on beetle backs, most of them have focused on water collection efficiency studies of single arrays, whereas the natural beetle back is a complete arch-shaped collection system with amazingly efficient mist water collection capabilities. Artificially preparing controlled arrays of multi-channel hybrid wettability surface systems is challenging. There are few reports about significant improvement of the efficiency of fog water collection by controllable three-dimensional shapes. Although much effort has been made by researchers to design hybrid surfaces with high wettability differences by achieving superhydrophilic clustering on a superhydrophobic background, the actual hydrophilic three-dimensional raised structures of fog collecting surfaces on curved beetle-back surfaces have rarely been studied. For example, some flat surfaces with micro-nano structures have been reported previously, which only resemble beetle backs in microstructure, but do not correspond to the actual situation in macroscopic morphology [ 42 ]. Therefore, it is necessary to construct three-dimensional concave and convex structures on curved geometrical morphological surfaces and to study the effect of their surfaces on fog water collection. Light cured three dimensional molding technology (briefly called as 3D printing) is one of the effective methods for rapid fabrication of complex multi-curvature parts, which has been widely applied in industry, scientific research, and daily life in recent years [ 43 ]. The application of 3D printing technology in bionic material fabrication is a novel direction [ 44 , 45 , 46 , 47 , 48 ]. Here, we designed a water collection surface imitating the back of a desert beetle and changed the wettability of the surface to improve the water collection efficiency. The curvature structures of the imitation beetle back surfaces were investigated and optimized based on aerodynamic theory. The triangular tip structure hydrogel surface of the imitation beetle back shows excellent water collection performance. As an environment-friendly water collection system, the optimized 3D-printed surface of the bionic beetle back shows latent capacity in applications of high-efficiency water collection, transportation of water and separation of oil-water.",
"discussion": "3 Results and discussion A typical beetle dorsal surface adhering to water droplets is shown in Figure 1 A. There are many tiny raised structures on the dorsal surface of the beetle, which are capable of retaining water from the air on the dorsal surface and conducting directional transport to obtain water resources that can sustain life [ 17 ]. Inspired by the water collection principle of beetle dorsum, we exploit light-curing 3D printing technology to prepare bionic beetle dorsal water collection devices and obtained a set of beetle dorsal structures with different curvature structures (as shown in Figure 1 B). The water collection ability of 3D printed beetle backs was investigated in saturated humid air with 90% relative humidity at room temperature. Figure 1 C shows the laboratory homemade fog water collection device, including humidifier, iron holder and glass dish. When the 3D printed beetle back was put in a high humidity environment, water droplets were able to adhere to and gradually cover the entire surface on the curvature structure of the surface, and drip down into the glass dish under the effect of gravity. Figure 1 Inspired by the Namib beetle and biomimetic preparation of the beetle's back. (A) Natural beetle using its back to collect water [ 13 ]. (B) Physical view of the 3D printed beetle back. (C) Schematic diagram of the printed beetle back simulating a water collection device in a high humidity environment. Figure 1 Figure 2 shows the preparation process of imitation beetle-back hydrogel. The beetle back template was printed using 3D printing technology, and then PVA and DMSO were dissolved in deionized water by heating at a certain mass ratio to obtain PVA hydrogel. The hydrogels were poured into the templates and frozen at −10 °C for 10 h to prepare hydrogel beetle backs with different curvature structures. Figure 2 Schematic diagram of the preparation process of hydrogel-like beetle back structure. Figure 2 The ability of the beetle back to trap mist is related to the surface structure and the adhesion of the surface to the droplets, while the Laplace pressure of the surface structure affects the droplet transport rate. As shown in Figure 3 , three surface shapes with different curvatures, rectangular, triangular and circular structures, were designed, and the three structures were compared with convex and concave surfaces (as shown in Figure 3 A, B, C, D, E, F). It is well known that the chemical surface modification and the surface roughness will affect the surface tension and adhesion of water, and hydrophilic surfaces are more favorable for water collection [ 8 , 36 ]. Therefore, we coated the surface of 3D printed structures with a hydrophilic PVA hydrogel to change the hydrophilicity of the original resin surface. As can be seen from Figure 3 J, K, L, the water contact angle of the resin surface used directly for 3D printing was 65°, while the contact angle was reduced to 48° after coating the resin surface with a layer of PVA hydrogel, and the contact angle of the PVA hydrogel surface was only 19°, and the contact angle was approximately equal to 0° after 3s. We have also tested the sliding angle of these hydrophilic surfaces. The results are shown in Figure S5 in the supporting information. Figure 3 3D printed beetle back model. (A–C) 3D printed beetle backs with different structures on convex surfaces; (D–F) 3D printed beetle backs with different structures on concave surfaces; (G–I) Hydrogel beetle backs with different structures; (J–L) Contact angles of the printed resin material, resin coated hydrogel and PVA hydrogel material surfaces. The inset corresponds to a partial enlargement of the beetle back model. Figure 3 As shown in Figure 4 A, the curves of water collection with time from the fog trapping experiments show that the water collection of beetle dorsal for triangular structures on the convex surface is better than that of rectangles similar to that of circles, and the water collection of triangular structures for 2 h is higher than that of rectangles by about 0.5 g. Because the curvature change of triangles and circles is greater than that of rectangles, the fog is more easily collected and transmitted on the surface of the structures. While the water collection of different structures of beetle back on the concave surface is similar to that on the convex surface (as shown in Figure 4 B), the water collection of triangular and circular structures is about 1 g higher than that of rectangles, and the water collection of rectangular structures on the concave surface is 0.37 g lower than that of the convex surface. Because the rectangular structures on the convex surface are more dense, while the size of rectangles on the concave surface is larger. As can be seen in Figure 4 A and B, the triangular structure collects slightly more mass of fog water than the circular structure due to the greater curvature of the tip of the triangle and the smoother tip of the circle, so the Laplace pressure of the triangular structure is greater than that of the circular structure [ 21 ]. As shown in Figure S1, the trend is very stable when test other new samples. While the rectangle has the flattest tip, so it collects the least amount of water. Since the effect on water collection is not significant between the same structures on the convex and concave surfaces, we performed only hydrogel replicas of the convex structures at the center of our hydrogel replica beetle dorsum study. Figure 4 Effects of beetle back shape and hydrophilicity on water-collecting performance. (A) Curves of water collection of beetle back with time for 3D printing different structures on convex surface. (B) Curves of water collection of beetle back with time for 3D printing different structures on concave surface. (C) Curves of water collection of hydrogel beetle back with time for different structures. (D) Variation of water collection of hydrogel beetle back optimal model (Triangle-3) with different number of cycles. Figure 4 The natural beetle back is a patterned surface that collects and then transports water from the hydrophilic region which is highly adhesive to water. We replicated the beetle dorsum with a very hydrophilic PVA hydrogel to obtain a hydrogel replica as in Figure 3 G, H, I. As seen in Figure 4 C, the water collection of the hydrogel replica with triangular structure (Triangle-3) is significantly enhanced to 16.65 g, which is 8 times higher than the previous Triangle-1 and Triangle-2. The water collection of Rectangle-3 is still lower than Triangle-3 and Round-3, confirming the effect of Laplace pressure and curvature on water collection. The water in the mist adheres heavily to the hydrophilic hydrogel and forms a water film on the surface, which accelerates the liquid transfer rate as the water continues to collect, thus enhancing the water collection; whereas the pristine resin surface is less hydrophilic and it is difficult for the water in the air to collect on the back surface of the beetle. Figure 4 D shows that the optimal triangular structure hydrogel beetle back can stabilize the water collection on the 2 h surface above 16 g after 10 times mist water collection, and this structure shows excellent stable water collection performance. The detailed sequential optical images during water collecting process were provided in Figure S6. The movies about water collecting process can be seen in Supplementary Information. Based on the structural characteristics of the beetle back, it's water collection process is divided into three stages: firstly, water in the air is adsorbed onto the hydrophilic bumps on the back (as shown in Figure 5 a); secondly, small water droplets on the bumps continuously polymerize with air and adjacent water droplets to grow into large water droplets; thirdly, large water droplets of a certain size slide down the ridges of the bumps into the bottom transfer channel to slide down into the collection device. As shown in Figure 5 b, for the strongly hydrophilic hydrogel beetle back surface, the water in the mist is adsorbed by the strongly hydrophilic raised surface and gradually grows to form a water film. At the same time, the water film surface attraction will further adsorb water, and when the water film spreads between the hydrophilic bump and the bottom transfer channel, it will be anchored on the surface because the three-phase contact line is too small, and the water film increases in the thickness direction and gradually slides down the transfer channel directionally. The whole process of water collection is through the cycle of adsorption→agglomeration→transfer, thus achieving a directional mist collection effect. The combination of commonly available gel materials and 3D printing technology to prepare a low-cost water collection system has a certain reference value for solving the drought problem in desert areas. Figure 5 Mechanism of water collection on the back of beetles. (A) Model diagram of water transport on the back of a complete beetle, with red arrows showing the direction of transport. (B) Schematic diagram of the cross-section of the beetle's back when collecting water, with the inset showing the water collection process: adsorption→agglomeration→transport. Figure 5 As shown in Figure 6 , we have also compared the collecting efficiency of this beetle back with other structures [ 49 , 50 , 51 , 52 ], although the efficiency of beetle back is a little bit lower than that of spider web, it still provide a biomimic strategy for water collecting. Figure 6 Comparision of the water collection effect between our work and previously reported works by other processing methods. Figure 6"
} | 4,483 |
33941908 | null | s2 | 4,874 | {
"abstract": "Photosynthesis is readily impaired by high light (HL) levels. Photosynthetic organisms have therefore evolved various mechanisms to cope with the problem. Here, we have dramatically enhanced the light tolerance of the cyanobacterium Synechocystis by adaptive laboratory evolution (ALE). By combining repeated mutagenesis and exposure to increasing light intensities, we generated strains that grow under extremely HL intensities. HL tolerance was associated with more than 100 mutations in proteins involved in various cellular functions, including gene expression, photosynthesis and metabolism. Co-evolved mutations were grouped into five haplotypes, and putative epistatic interactions were identified. Two representative mutations, introduced into non-adapted cells, each confer enhanced HL tolerance, but they affect photosynthesis and respiration in different ways. Mutations identified by ALE that allow photosynthetic microorganisms to cope with altered light conditions could be employed in assisted evolution approaches and could strengthen the robustness of photosynthesis in crop plants."
} | 274 |
37314359 | PMC10266292 | pmc | 4,877 | {
"abstract": "Abstract Lignocellulosic biomass is still considered a feasible source of bioethanol production. Saccharomyces cerevisiae can adapt to detoxify lignocellulose-derived inhibitors, including furfural. Tolerance of strain performance has been measured by the extent of the lag phase for cell proliferation following the furfural inhibitor challenge. The purpose of this work was to obtain a tolerant yeast strain against furfural through overexpression of YPR015C using the in vivo homologous recombination method. The physiological observation of the overexpressing yeast strain showed that it was more resistant to furfural than its parental strain. Fluorescence microscopy revealed improved enzyme reductase activity and accumulation of oxygen reactive species due to the harmful effects of furfural inhibitor in contrast to its parental strain. Comparative transcriptomic analysis revealed 79 genes potentially involved in amino acid biosynthesis, oxidative stress, cell wall response, heat shock protein, and mitochondrial-associated protein for the YPR015C overexpressing strain associated with stress responses to furfural at the late stage of lag phase growth. Both up- and down-regulated genes involved in diversified functional categories were accountable for tolerance in yeast to survive and adapt to the furfural stress in a time course study during the lag phase growth. This study enlarges our perceptions comprehensively about the physiological and molecular mechanisms implicated in the YPR015C overexpressing strain’s tolerance under furfural stress. \n Construction illustration of the recombinant plasmid. a) pUG6-TEF1p-YPR015C, b) integration diagram of the recombinant plasmid pUG6-TEF1p-YPR into the chromosomal DNA of Saccharomyces cerevisiae .",
"introduction": "Introduction The production of biofuel and biochemicals using lignocellulose-based techniques has a promising possibility of reducing greenhouse gas emissions, bringing benefits to rural economies, and advancing energy security ( De Bhowmick et al. 2018 ; Patel et al. 2019 ). Due to its highly effective hexose fermentation ability, the Saccharomyces cerevisiae yeast is one of the most well-known host microorganisms in the fermentative food sector. It is also the appropriate microorganism for the generation of biofuels. However, the native S. cerevisiae strain cannot utilize xylose, the second most abundant carbohydrate in lignocellulosic-derived inhibitor hydrolysate ( Ask et al. 2013 ). The recalcitrant nature of lignocellulosic biomass, on the other hand, poses a technological challenge to release fermentable sugars; consequently, a major bottleneck for the production of renewable from them ( De Bhowmick et al. 2018 ; Singh et al. 2018 ). They constrain very high inhibition of microbial proliferation and fermentation processes ( Singh et al. 2018 ; Sankaran et al. 2020 ). Lignocellulosic biomasses are often pretreated before further processing ( Kumar et al. 2020 ; Padmapriya et al. 2021 ). The numerous commonly used pretreatment methods, including alkaline, ammonia fiber expansion, and dilute acid hydrolysis, usually generate various chemical byproducts’ inhibition and inhibitory compounds ( Kumar et al. 2020 ; Padmapriya et al. 2021 ). Furfural is a commonly encountered inhibitor that affects microbial growth by causing intracellular acidification and reducing enzyme activities and protein and ribonucleic acid (RNA) syntheses, breaks in deoxyribonucleic acid (DNA), and accumulating reactive oxygen species ( Ask et al. 2013 ; Moreno et al. 2019 ). Overexpression of target genes in yeast strains from haploid laboratory background has been used to establish molecular-based genetic techniques to improve strain tolerance to lignocellulosic hydrolysate ( Gorsich et al. 2006 ; Park et al. 2011 ). Transcriptomic characterization of overexpressed target genes of laboratory S. cerevisiae strains has demonstrated associated tolerance with improved proliferation under the challenge of furfural inhibitors through NAD(P)H-dependent reductions ( Li et al. 2015 ; Zhao et al. 2015 ). Transcriptomic analysis of overexpressed diploid and haploid yeast strains of S. cerevisiae , known to survive lignocellulose hydrolysates inhibition, revealed elevated levels of expression of SFA 1, ADH 6, and ADH 2 genes ( Zhu et al. 2020 ). Additionally, it has been proven that yeast of S. cerevisiae clones overexpressing ADH 6, ADH7 , and ADH1 genes can endure in the presence of furfural ( Petersson et al. 2006 ; Laadan et al. 2008 ; Liu et al. 2008 ). The stress growth response of the overexpressing yeast strains to furfural was evaluated, and a lag phase length was used to determine the strain tolerance level. It is common for S. cerevisiae strains to undergo a lag phase of decreased cell proliferation after challenges with furfural and HMF inhibitors. However, cell growth and fermentation of the overexpressing yeast strains accelerated compared to its parental strain ( Zhao et al. 2015 ). Due to comparative transcriptomic analysis, our previous study discovered an uncharacterized gene, YPR015C of S. cerevisiae , to be significantly up-regulated under furfural stress condition. Furthermore, BLAST analysis of the protein encoded by YPR015C revealed that it has conserved domains similar to transcription factors from the zinc-finger family. Therefore, we concluded that YPR015C is a putative transcription factor of the zinc-finger family. However, it is unknown whether YPR015C contributes to furfural inhibitor stress tolerance. Herein, the complete fragment of the coding sequence of YPR015C was successfully amplified by PCR. The homologous recombination method was used to establish the YPR015C overexpressing strain. We identified differences in phenotypic traits and transcriptional expression between the YPR015C overexpressing strain and its parental strain that exhibit different stress tolerance capacities under furfural challenge in time course study during the lag phase growth. Our findings provide information about the molecular mechanisms of S. cerevisiae strain tolerance to the lignocellulose-derived furfural inhibitor. This knowledge can be applied to aid engineering efforts to improve tolerant strains.",
"discussion": "Results and Discussion Cell growth response under furfural challenge. Overexpression of RPB4, PRS3 , and ZWF1 exhibited tolerance under the treatment of lignocellulose-derived inhibitors compared to the parental strain ( Cunha et al. 2015 ). Furthermore, a yeast tolerance response to furfural was demonstrated, and the lag phase for cell proliferation in response to the inhibitor challenges was used to evaluate stress tolerance manifested at the genome level ( Liu 2011 ). In this study, the YPR015C overexpressing- and the parental yeast strains were tested in the presence of 35 mM furfural during the lag phase. Cell growth was decreased under furfural challenge, and a lag phase was detected in the YPR015C overexpressing and the parental strains. The YPR015C overexpressing strain recovered more rapidly and displayed a lag phase at about 12 h. However, this lag phase was expanded to 24 h in the parental strain ( Fig. 1 ). Notably, the lag phase duration in the YPR015C overexpressing strain was much shorter and reached the exponential phase more rapidly than that of its parental strain. This strain showed a much shorter lag phase than those previously reported for the YNL134C overexpressing strain (24 h) when challenged by furfural during the lag phase ( Zhao et al. 2015 ; Li et al. 2015 ). Fig. 1. Cell growth of Saccharomyces cerevisiae strains overexpressing YPR015C and BY4742 as measured at OD 600 on a defined synthetic medium containing 35 mM of furfural. The prolonged lag phase indicates the inhibitory effect of furfural on the parental strain. Aldehyde resistance performance. In S. cerevisiae , multiple reductases have activity for reducing furfural ( Heer et al. 2009 ; Wang et al. 2017a ). The co-factors NAD(P)H revealed aldehyde reduction activity in both the YPR015C overexpressing and the parental strain. Under furfural and HMF stress conditions, the YPR015 overexpressing strain performed noticeably better ( Fig. 2 ). NADH co-factor showed that the highest specific activity for furfural reduction was about 9 U/mg, 9.5 U/mg and 8.5 U/mg at 2, 8 and 16 h, respectively ( Fig. 2a ). When NADPH was used as the co-factor, the YPR015C overexpressing strain also showed reduction in furfural enzyme activity at 2.5 U/mg, 4.5 U/mg and 6 U/mg after 2, 8 and 16 h treatments, respectively ( Fig. 2c ). In summary, in vitro enzyme assays indicated that the strain overexpressing YPR015C possessed a NAD(P)H-dependent enzyme activity for furfural reduction. In a recent study, overexpression of YKL071W showed both co-factors NAD(P)H-dependent enzyme activity for furfural reduction in crude and purified protein during lag phase growth ( Heer et al. 2009 ; Wang et al. 2017a ). Similarly, enzyme activity assay in this study indicated that the YPR015C overexpressing strain induced reduction in the two co-factors, NADH and NADPH for furfural and HMF inhibitors, respectively ( Fig. 2 ). Furthermore, overexpression of YPR015C led to a much better specific activity (9.5 U/mg) when compared to other earlier reports on aldehyde reductases, such as in the overexpression of Yll056cwp (0.47 U/mg), Ykl071wp (3.38 U/mg), Ykl107wp (2.56 U/mg), and Ymr152wp (5.05 U/mg) from S. cerevisiae for furfural reduction ( Wang et al. 2017a ; Wang et al. 2017b ; Ouyang et al. 2021 ). Fig. 2. Furfural and HMF reduction of activities in the crude cell extracts of the parental ( BY4742 ) and YPR015C overexpressing strains at 35 mM of furfural. The activities were measured using NADH or NADPH as cofactors. a) NADH and c) NADPH used for furfural reduction, b) and d) for HMF reduction, respectively. The data represent averages of three experiments. * p < 0.05; ** p < 0.01 indicates significant differences. Accumulation of reactive oxygen species. Furfural is one the most well-known lignocellulose-derived inhibitors that cause intracellular oxygen radical species accumulation. The oxidizing environment induced by ROS is known to cause irregularity and inactivity of cell components in S. cerevisiae ( Almeida et al. 2008 ; Allen et al. 2010 ; Liu et al. 2020 ). It has been demonstrated that furfural is a thiol-reactive electrophile that can lead to oxidative stress ( Kim et al. 2013 ; Liu et al. 2020 ) by activating transcription factors that mediate S. cerevisiae ’s response to oxidative stress ( Toone et al. 1999 ). In this study, following 2 h of treatment with 35 mM furfural, 14% and 12% of the cells of the strain overexpressing YPR015C and the parental strain, respectively, exhibited a positive oxygen radical signal ( Fig. 3b ). After 8 h of treatment, 38 and 34% of cells of the overexpressing YPR015C and the parental strain, respectively, showed positive ROS signals. Fig. 3. Accumulation of reactive oxygen species (ROS) caused by furfural. a) Representative images of cells stained with the ROS indicator 2’,7’-dichlorofluorescein diacetate, b) percentage of cells at each concentration of furfural and peroxide that stained positive for ROS by 2’,7’-dichlorofluorescein diacetate after 2, 8, and 16 h. The data represent averages of three experiments with standard error. * p < 0.05; ** p < 0.01 indicates significant differences when at least 100 cells were examined on each bright-field image. Furthermore, positive ROS signals were detected in 84% and 70% of cells of the YPR015C overexpressing strain and the parental strain, respectively, after 16 h of treatment. The YPR015C overexpressing strain and its parental strain showed significant differences in the positive ROS signals displayed in the medium supplemented with the same furfural concentration independent of time. Remarkably, the percentage of yeast cells stained positive for ROS signals increased over the time intervals studied ( Fig. 3b ). We also found that the percentage of cells that accumulate ROS after 16 h decreased to a lower level in the YPR015C overexpressing strain compared to its parental strain after the treatment with 35 mM furfural. This result implied that ROS accumulation peaked after 16 h of furfural treatment, indicating that the percentage of cells containing ROS increased over time. Excessive ROS accumulation at this time may cause damage to DNA, proteins, and lipids ( Rowe et al. 2008 ). Cell wall susceptibility analysis. \n S. cerevisiae cell wall and membrane act as the first-line defense barrier to external toxic stimuli under external factors such as temperature. Cell wall analysis in the furfural treated and untreated cells were used to validate the increased furfural stress tolerance in cells that had encountered cell wall improvement using a lytic enzyme, a β-1,3-glucanase from Arthobacterluteus ( Teixeira et al. 2014 ). After 4 h of lyticase treatment, the cell density of samples cultured with 35 mM furfural for 2 h was absorbed without substantial differences compared to that of its parental strain, while samples treated with 35 mM furfural decreased steadily after supplementation with lyticase in 8 h of culture, lowering to approximately 43.7% ( Fig. 4 ). The YPR015C overexpressing strain treated with 35 mM furfural revealed a rapid decrease in cell density in the lyticase-supplemented medium after 16 h transcriptional level under furfural challenge treatment during the late stage of the lag phase. Interestingly, we discovered one SRX1 extended list of genes involved in the oxidative damage stress response that had increased expression in response to furfural challenge, which had not been reported in earlier studies. In conclusion, our of treatment; this decrease was more significant than the cell density decrease reported at 2 and 8 h after furfural challenges compared to its parental strain. Fig. 4. Changes in cell densities after the treatment with lyticase during 4 h of incubation. Saccharomyces cerevisiae strains were treated for 2, 8, and 16 h (from top to bottom) with 35 mM of furfural, respectively. The mean values of relative optical density are presented with vertical error bars, each representing a single standard deviation (n = 3). The results demonstrated that treatment for 16 h increased the resistance of cells to lyticase, with a maximum effect observed. After 8 hours, cells exposed to 35 mM furfural showed noticeably higher lyticase resistance. However, cells treated for 2 h showed no difference in resistance to lyticase compared with its parental strain. In cell wall susceptibility analysis, we observed that it slowly lowered after lyticase was added to the media after 8 h of furfural treatment, dropping to about 53.5% and 44.9%, respectively ( Fig. 4 ). After 16 h, the samples treated with 35 mM furfural showed a rapid decrease in cell density (31.77%) in the lyticase-supported medium. In comparison to its parental strain, the reduction in cell density in these samples was more significant than the reductions in the samples at 2 h (98.3%), and 8 h (44.9%) following furfural challenge treatment ( Fig. 4 ). Transcriptome sequencing data analysis. The quality statistics of sequencing data are described in Table SII. Consequently, the RNA sequencing data in this work was reliable in consideration of statistics of RNA sequencing data. The correlation coefficients of samples within and between groups were calculated based on the FPKM values of all the genes in each sample, and a heat maps were constructed (Fig. S3). Transcriptome differential expressions. Yeast tolerance to furfural inhibitor stress conditions can be shown at the genome level and is most possibly observed during the lag phase ( Liu et al. 2004 ). It is a common practice to use gene expression responses to environmental stimuli to discover tolerant genes under specified conditions ( Unrean et al. 2018 ). S. cerevisiae was used to study the genes correlated with stress resistance reaction to single and synergetic inhibitors. About 184 consensus genes were differentially expressed towards lignocellulose-derived inhibitor resistance between S-C1 and YC1 strains. Overexpression of SFP 1, and ACE 2 gene in the stress resistant strain S-C1 and YC1 increases bio-ethanol productivity in the existence of furfural ( Chen et al. 2016 ). In this report, we explored how differences in stress tolerance between the YPR015C overexpressing and its parental strain were explained by gene expression changes under 35 mM furfural. Seventy-nine up-regulated and down-regulated key genes implicated in various biochemical processes were recognized during the lag phase of the early (2 h), middle (8 h), and late (16 h) stages under furfural challenge. Currently, we identified genes involved in amino acid biosynthesis, oxidative stress response, cell wall and membrane-related response, heat shock protein response, and mitochondrial-associated proteins for the YPR015C overexpressing strain (Table SIII). Out of the genes identified, two genes are implicated in cellular metabolism; MTD1 and IMD2 had significantly higher transcription levels in the YPR015C overexpressing strain. MTD1 is a cytoplasmic malate dehydrogenase and monophosphate dehydrogenase. It has been shown that furfural causes yeast to produce more reactive oxygen species ( Allen et al. 2010 ). Reactive oxygen species, a reduced form of superoxide anion, and hydroxyl radicals, cause high damages to the cellular components such as lipids proteins, and DNA in S. cerevisiae . The oxidative damage stress response began as a defensive system against reactive oxidants in S. cerevisiae , as in most aerobically developing organisms ( Liu and Moon 2009 ; Liu et al. 2020 ). In previous studies, four genes essential for oxidative stress response to inhibitory compounds were identified in the transcriptome analysis of an industrial yeast strain ( Liu and Ma 2020 ; Liu et al. 2020 ). According to a recent proteomic analysis, Zwf 1 consistently showed a high level of enhanced fold changes in response to furfural and HMF treatment ( Thompson et al. 2016 ; Liu and Ma 2020 ). In other reports, a comprehensive phenotyping study discovered a tolerable mutant with the trait of overexpressing the thioredoxin-encoding TRX 1 gene ( Unrean et al. 2018 ). Recent studies have shown that thioredoxin and glutaredoxin, two distinct classes of small proteins essential for resistance to oxidative stress, are the key elements linked to several transcriptionally active genes involved in both enzymatic and non-enzymatic systems ( Liu and Ma 2020 ). In this work, the YPR015C overexpressing strain showed an enhanced oxidative response with a significantly higher expression activity than its parent strain against the furfural inhibitor challenge. Among the identified genes, three essential genes are included in oxidative stress response. Genes encoding oxidoreductase enzymes are highly expressed, including AHP 1 (thioredoxin peroxidases), SRX 1 (sulfiredoxin), and GPX 2 (phospholipid hydroperoxide glutathione peroxidase). Glutathione and glutaredoxin are implicated in both enzymatic and non-enzymatic defense reactions in the YPR015C overexpressing strain against the challenge of furfural inhibitor. It is clear that the two different classes of small proteins glutaredoxin and thioredoxin, which are required for oxidative stress resistance, were the crucial components related to several transcription-active genes involved in different non-enzymatic and enzymatic systems. The specific enzymatic and non-enzymatic defense response system, centered on glutathione, was distributed with these genes. Previous studies have shown that the expression of a few genes encoding the specific oxidative-stress response, including TRR1, ECM4, GLR1, GRX1 , and CTT1 , increases under the furfural challenge ( Heer et al. 2009 ; Wang et al. 2017a ; Liu et al. 2020 ). However, in this study, these genes are not expressed at the work’s results were consistent and similar to those of early reported studies. Yeast cell wall and membrane-related proteins are involved in biological activities and serve as the first line of defense against external toxic stimuli. The cell wall is made up of several proteins, chitin, β-1,3- and β-1,6-glucans, and other substances that can be cross-linked to create higher-order complexes. Its configuration and degree of cross-linking alter with cell proliferation and in response to different stressors ( Wang et al. 2017a ). The S. cerevisiae cell wall structures were modified to adapt to ethanol stress conditions ( Wang et al. 2017a ). Glycosylphosphatidylinositol-linked cell wall proteins SPI 1, SED 1, and PIR 3, as well as DIT 1 and GIP 1 for forming spore walls, have recently demonstrated a sustained increase in the expression levels following furfural and HMF treatment at various stages ( Liu et al. 2020 ). In another study, a protein that is hypothesized to be involved in the control of cell walls was produced when up-regulated genes were expressed. This protein was a crucial part of yeast cell membranes and has been connected to resistance to furfural inhibitors ( Terashima et al. 2000 ). In this study, 15 genes involved in the yeast cell wall and membrane-related proteins showed significant expressions in the recombinant strain after furfural treatment compared to its parental strain. Among them SED1, PIR 3, CWP 1, YPK2, TIP1, IZH 4, and IZH 2 were upregulated after the furfural challenge. These genes encode proteins related to cell walls with different functions. For instance, PIR 3 is an O-glycosylated, covalently bonded cell wall protein that is necessary for the stability of the cell wall, SED 1 is a major cell wall glycoprotein that is structurally triggered by stress, TIP1 – a major cell wall monoprotein, YPK2 is involved in cell wall integrity signaling pathway, and CWP 1 is a cell wall mannoprotein. Some of the proteins encoded by these genes may not be found in the cell walls, but they encode cell wall-related proteins of various activities. For instance, the SED 1 and PIR 3 genes encode proteins which attach glycosylphosphatidylinositol to the cell wall. In conclusion, our work’s results were consistent and similar to those of early reported studies. Amino acid and fatty acid biosynthesis have been revealed to contribute to the biosynthesis of proteins implicated in stress response and suggested as a component of S. cerevisiae tolerance against lignocellulose-derived inhibitors ( Okazaki et al. 2007 ; Liu et al. 2019 ). Another study found that the Y31-N strain could keep the integrity of cell membrane function to resist furfural stress by controlling the fluidity of the cell membrane, modulating the framework of the palmitic acid and stearic acid in the cells ( Wang et al. 2023 ). In this study, at least seven key important genes are implicated in amino acid biosynthesis and fatty acid metabolism including, ARG 1, ARG 3, IME 2, and LEU 4. The LEU 4, ARG 1 and ARG 3 were mainly involved in the metabolism of amino acids (Table SIII). OLE 1 and FRM 2 genes were involved in fatty acid biosynthesis. The findings from earlier studies reveal that OLE1 , which encodes the Δ-9 fatty acid desaturase, catalyzes the double bonding between carbons 9 and 10 of stearoyl CoA and palmitoyl CoA ( Mcdonough et al. 1992 ). In addition to its activity in fatty acid production, OLE 1 is also essential for the formation and functioning of the mitochondria ( Hermann et al. 1998 ). In this work, the OLE 1 gene showed more than 2.3-fold change and a p -value of less than 0.05 in a time course study at the late stage of the lag phase. Furthermore, in our previous study, we found that phenol tolerance in Candida tropicalis affected the expression of genes implicated in fatty acid degradation. These genes were responsible for its tolerance under the stress of phenol ( Wang et al. 2020 ). Four of the seventy-nine genes identified were heat shock proteins and related to amino acid biosynthesis. Among those with known functions, ARG1 and ARG3 were involved in arginine biosynthesis and metabolism, while IME2 is involved in serine/threonine biosynthesis. The down-regulated genes are primarily involved in ribosome biosynthesis, derivative metabolic processes and amino acid, RNA metabolic processes, and other functional categories. It has recently been discovered that the genes involved in the S. cerevisiae survival process and those involved in the response to heat shock stress do not overlap significantly ( Gibney et al. 2013 ). Members of the HSP 70 and Hsp 110 families including SSA 4, SSE 2, SSE 2, and SSA 4, were significantly repressed, as shown in Table SIII. These repressed genes served to fold proteins either directly or indirectly. Other genes coding for heat shock protein, stress-responsive protein, chaperone, and co-chaperone were repressed in the YPR015C overexpressing strain to counteract furfural stress damage to proteins such as HSP 10, HSP 30, HSP 78, and HSP 15. HSP 12 is involved in the maintenance and organization of the plasma membrane. Other genes mainly engaged in the essential categories of cell wall stability, cell wall protein ( PST 1, SPI 1), spore wall assembly ( LDS 2), ARO9 for aromatic aminotransferase, RTC 3 RNA metabolism, and PHM 8 for lysophosphatidic acid hydrolysis were repressed. From this study, we concluded that the accumulation of overexpressed transcriptional genes further shortened the lag phase in response to furfural stress to a certain extent. Regarding the specific activity of aldehyde reductase, the overexpressed YPR015C improves the aldehyde reductase activity dependent on the NADH and NADPH cofactor. The results of comparative transcriptomic profiling identified genes associated with the cell wall and membrane-related biosynthesis, cellular detoxification, and oxidative stress response. The findings of this research report contribute to our knowledge of the adaptation and tolerance mechanisms of yeast, which will aid in improving yeast tolerance to stress."
} | 6,586 |
30971733 | PMC6458172 | pmc | 4,879 | {
"abstract": "We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum , but less polysaccharides and lignin in the cell wall. S. spontaneum , and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum , and one of the CADs isolated ( CAD B) had higher expression in S . officinarum . The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene.",
"conclusion": "Conclusions The set of data obtained here enabled the association of patterns to better understand the process of lignin deposition in four Saccharum species. The differences between the species studied became evident, whether in relation to structural and non-structural carbohydrates or in the quantity and type of lignin. The data enabled the coherent separation of the two species that have been identified as energy canes, S. spontaneum and S. robustum , which accumulate more fiber, from the other two, which accumulate more sucrose. Moreover, the first two species contain more insoluble lignin, the lowest S/G ratios, greater abundance of intermonomeric linkages (lignin oligomers), and lower percentages of saccharification. Gene expression analysis of the lignin biosynthesis pathway genes in S. officinarum and S. spontaneum showed that in general the later species has higher expression in culm tissues especially in mature culms. Surprisingly the sequences of the identified genes showed high conservation in the four Saccharum species including the commercial hybrids. This feature is desirable for the genetic manipulation of energy cane, since knowledge has already been gained with low lignin commercial varieties of sugarcane 39 , 44 , 92 , 93 . It has been show in other grasses that lignin biosynthesis has a complex regulation by transcription factors, which can activate or repress the expression of the several genes of the route 93 – 98 . However, to our knowledge, this is the first report describing that lignin genes are highly conserved among species of the same genus and, consequently, the differences they have regarding the polymer content and composition can be only fully understood after gaining knowledge on the sequencing of the regulatory regions of each gene or at least of a set of genes.",
"introduction": "Introduction Bioethanol can be produced from starch and sucrose (first-generation ethanol – 1GE) but also from lignocellulosic biomass (second-generation ethanol – 2GE). For the production of 2GE the sugars used in the fermentation are from the depolymerization of the carbohydrates present in the cell wall, cellulose and hemicellulose 1 , 2 . Depending on the plant species, tissue wall material can represent between 40% and 80% of the plant biomass 3 , 4 . Grasses with C4 metabolism, especially those belonging to the subfamily Panicoideae, such as sugarcane ( Saccharum spp.), sorghum ( Sorghum bicolor ), species of Miscanthus , and Panicum virgatum , represent plants with the greatest potential for 2GE production due to their large capacity for carbon fixation and biomass accumulation 5 . Lignocellulosic biomass used in 2GE production is composed of cellulose, hemicellulose, and lignin, which are arranged in a chemically ordered manner in the wall. Cellulose is organized into crystalline microfibrils that are embedded in a matrix of hemicellulose, which is covalently linked to the complex structure of lignin. In 2GE production the chemical bonds between wall polymers must be broken to release sugars for downstream fermentation processes. Usually, a chemical pretreatment is needed to allow the access of enzymes to the wall polysaccharides 6 . One of the main difficulties in accessibility to the polysaccharides is the presence of lignin, which is highly resistant to degradation due to a diversity of low reactivity linkages, making this phenolic the main polymer responsible for the cell wall recalcitrance 7 – 9 . In addition, pretreatments can release lignin residues that can inhibit the fermentation process. Lignin is a complex heteropolymer formed by oxidative combinatorial coupling of three alcohols that are synthesized in the cytoplasm of plant cells: p-coumaryl, coniferyl, and sinapyl alcohol. These alcohols differ in their degree of methoxylation 10 and are transported from the cytoplasm to the apoplast, where they are oxidized by peroxidases and/or laccases into radicals, that are then incorporated by random radical reactions into the preformed polymer 11 . After the incorporation, the monolignol residues are called p -hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively, and their proportion in the lignin structure varies significantly between the type of plant cells, tissues, and species 12 , 13 . Lignin present in gymnosperms consists of G units and small amounts of H units, whereas in angiosperms they are composed of G units, S units, and only trace amounts of H units. In monocotyledons, both S units and G units are presented at similar levels and the amount of H units is higher than in dicotyledons 14 . The S/G ratio and the inter-monomeric linkages in the lignin polymer are important characteristics to predict the degree and nature of the condensation of the polymer and, consequently, about plant biomass recalcitrance 15 . In addition, the complexity of the lignin structure and its recalcitrance can be affected by other phenylpropanoids that can be incorporated into the polymer structure to different levels 16 . For example, a recent structural characterization of cell walls of several monocotyledons showed that the flavonoid tricin is part of native lignin 17 , 18 , and this monomer may act in the formation of a nucleation site for the beginning of lignin biosynthesis 18 – 20 . Several species of plants have been genetically modified to change the content and composition of lignin, and the degree of modification depends on the responsible gene and on the position of the encoded enzyme in the biosynthetic pathway 21 , 22 . In general, changes in the expressions of C3H , HCT , or 4CL lead to quantitative changes in the levels of lignin, while the regulation of F5H and COMT leads to changes in the S/G ratio and, consequently, in the type of lignin 7 , 23 , 24 . The recent identification of another lignin biosynthesis enzyme, Caffeoyl Shikimate Esterase (CSE), adds another step in this metabolic pathway that can be manipulated 25 , 26 . Indeed, transgenic poplar plants silenced for CSE showed reduced lignin content, altered S and G composition, and increased saccharification yields 27 . Many of the studies on the biosynthetic pathway of lignin monomers were conducted in some dicotyledons (e.g. Alfalfa and Populus ) and model plants such as Arabidopsis thaliana and Nicotiana tabacum 28 , in which a high degree of conservation was observed. The information obtained with these plants has been applied in studies of monocotyledons used for 2GE production 28 – 30 , but studies with monocotyledons are still proportionally smaller in number. The study of lignin biosynthesis in sugarcane has been conducted recently in a systematic manner 13 , 31 – 36 and transgenic plants of sugarcane silenced for COMT and CAD 37 – 39 were produced. The genus Saccharum comprises more than 10 species 40 and the term sugarcane is generally used to define complex hybrids originated from the species S. officinarum and S. spontaneum , which appear to have contributed with 90% and 10%, respectively, to its genotype 41 . Sugarcane is a C4 grass, which is highly efficient in the production of photoassimilates and biomass accumulation 42 , in addition to storing up to 18% of sucrose (wet basis) in its culms 43 . The sucrose-rich syrup obtained by crushing the culms is used in the alcoholic fermentation and production of 1GE 2 , 44 . The residual biomass called “bagasse” – composed primarily of cellulose (39%), hemicellulose (25%), and lignin (23%) – has huge potential for 2GE production 45 – 47 . However, the use of sugarcane bagasse to produce 2GE has several technical hurdles, among them the recalcitrance of the lignocellulosic material mainly due to the presence of lignin, which drastically decreases the efficiency of saccharification yield for downstream fermentation 33 . A new type of cane, called energy cane, with lower accumulation of sucrose in the stem and richer in fiber has been considered for 2GE production 48 . The term energy cane has been used generically for the species S. spontaneum as well as for its hybrids with commercial varieties of sugarcane. In addition to its application in biofuel production (first and second generation ethanol) energy cane can be burned to generate electricity 42 because of its high lignin content and the greater heating value of this polymer 49 . To date, a systematic study related to lignin biosynthesis and cell wall biochemistry has only been conducted on S. officinarum , but not on any other Saccharum species 33 . Some species of the genus have different sucrose and fiber contents, such as S. spontaneum , S. officinarum , S. robustum , and S. barberi . S. officinarum and S. spontaneum differ in sucrose and fiber content whereby the first accumulates more sucrose but has a lower fiber content. S. officinarum is the only species within the genus Saccharum whose chromosome number is not variable between individuals 50 and it is believed that it originates from S. robustum . On the other hand, S. spontaneum is a complex, highly polymorphic species, and the most primitive of the species of the genus Saccharum . The high genetic variability of this species has been used in genetic breeding programs seeking to develop commercial varieties with potential for biomass production 51 . Abundant molecular evidence indicates that S. spontaneum is genetically very different compared to the other species of Saccharum 52 , 53 . Similarly to S. spontaneum plants of S. robustum have culms that are rich in fiber and poor in sucrose, and although the plants are vigorous, they are susceptible to abiotic and biotic stresses 54 . Although S. robustum has potential to be used in breeding programs because its vigor, its use has been restricted to Hawaii 51 . Apparently, the species S. barberi originated from the natural hybridization of S. officinarum with S. spontaneum 55 . This species has been cultivated and has moderate content of sucrose, displaying resistance to stresses and high content of fibers in relation to S. officinarum . Currently, there is little interest in using S. barberi in breeding programs, mainly due to the difficulty of flowering and flower sterility. Because of differing fiber content and the potential for E2G production of these species, this study aims at investigating the cell wall components, the content and type of lignin, as well as to determine and evaluate the relative expression of the genes related to lignin biosynthesis in S. spontaneum, S. officinarum, S. robustum , and S. barberi . Such information may help not only in a better understanding of the accumulation of lignin within the genus Saccharum but also provide useful information for the adoption of these species for 2GE production.",
"discussion": "Discussion Sugarcane has the capacity of storing soluble, readily fermentable sugars (mostly sucrose) up to 18% of the fresh mass in the stalk 2 , 58 . The large accumulation of sucrose occurs in the maturation of the culms. Energy cane accumulates half or less sucrose than sugarcane and much of the fixed carbon is shuttled to structural polysaccharides such as cellulose and hemicelluloses 59 . By comparing the mature internodes between the Saccharum species studied, the lowest values for cellulose, hemicellulose, and pectin were found in the species S. officinarum , and the highest values were found in S. spontaneum (Fig. 1 ). The opposite was observed for sucrose, the primary soluble sugar in mature culms (Fig. 2 ). With some variation, S. barberi had closer levels to those of S. officinarum , while S. robustum was closer to S. spontaneum . This inverse relationship appears to be reflected in the wall monosaccharide composition evaluated by 2D-HSQC NMR spectroscopy. S. officinarum and S. barberi biomass harbor a higher xylose content, while S. spontaneum and S. robustum a higher glucose content (Fig. 7D ) reflecting the competing sinks for these carbohydrates, hemicellulose and cellulose, respectively 60 , 61 . Interestingly, while the cellulose content remained the same in new and mature culms of S. barberi and S. officinarum , it increased in the other two species. This behavior is opposite to the sucrose levels, that is, the disaccharide increases with maturation in the culms of S. barberi and S. officinarum , but remains practically the same in S. robustum and S. spontaneum . On the other hand, the comparison of reducing sugar contents in new and mature culms shows a much greater variation for S. barberi and S. officinarum , suggesting that reducing sugars in these species are directed towards sucrose synthesis, whereas in the other two species towards structural polysaccharides, in particular cellulose 62 . Similar to Panicum virgatum 63 , 64 , Brachypodium distachyon 60 , 65 , and Zea mays 66 , 67 , during the development of the internodes in S. spontaneum and S. robustum there was higher accumulation of carbon as unsoluble polysaccharides (cellulose, hemicellulose and pectin) in the cell wall, than the soluble sucrose in the parenchymal cell. While the starch content was reduced during the maturation of the culms in S. officinarum , S. robustum , and S. barberi , it increased notably in S. spontaneum as also visually observed in the histochemical analyses. Starch granules were detected in the fundamental parenchyma of mature internodes of S. spontaneum .The presence of starch in S. spontaneum had been reported previously 59 , where 215 clones related to the genera Saccharum , Erianthus , and Miscanthus were analyzed. While S. robustum was the species with only traces of starch, S. spontaneum harbors the highest content. It has been suggested that the accumulation of starch in mature internodes of this species could be due to its capacity for tillering and high metabolic activity and as a strategy to cope with biotic and abiotic stresses 68 . Lignin is the second largest biopolymer present in the cell walls of grasses 69 . Although it is essential for plant growth and development, lignin is the main factor responsible for the recalcitrance to processing of plant biomass in 2GE, including sugarcane 33 . Lignin content in the Saccharum species was determined using the Klason method, which distinguishes the soluble and insoluble fractions together providing a total estimate of lignin 70 . Regarding internode age a negative correlation was observed between these two types of Klason lignin, indicating greater amount of soluble Klason lignin (monomers and oligomers precursors of insoluble lignin polymers) in young internodes, and insoluble lignin in mature internodes. This is not surprising as lignification of the wall is still underway in young internodes. However, most of the lignin biosynthetic genes analyzed had a lower expression in young culms suggesting that the larger amount of soluble lignin in these tissues would be correlated to the polymerization process and not with monolignol production. In the culm, the rind contains a high percentage of densely packed vascular bundles and is a metabolically active region with high peroxidase activity, therefore polymerizing and thus accumulating lignin 31 , 33 . When comparing the insoluble lignin content in mature internodes of the four species, S. spontaneum (20%) and S. robustum (18%) contain higher values than S. barberi (16%) and S. officinarum (14.5%). This difference was also observed in the histochemical analyses with phloroglucinol-HCl. Compared with S. officinarum and S. barberi , the rinds of mature internodes of S. spontaneum and S. robustum have higher density of vascular bundles and the walls of cellular elements such as hypodermis, epidermis, sclerenchyma and vascular fibers seem thicker and more lignified, contributing significantly to the higher content of this polymer. A general analysis of the expression of lignin biosynthesis pathway genes in the tissues of the culms displays a higher expression in S. spontaneum compared to S. officinarum , and a higher expression in tissues (rind and pith) of internode 5 compared with internode 3, supporting the higher insoluble lignin content in S. spontaneum and in mature tissues of the stalk. These gene expression differences, however, varied slightly depending on the species and tissue, for example, C4H in S. spontaneum , C3H in pith of the two internodes, CAD A and CAD B in rind and pith of S. officinarum , CCoAOMT A in rind of S. officinarum , and HCT in pith of S. spontaneum . The nature of inter-monomeric linkages between lignin oligomers and their modifications can be exploited for the production of more degradable lignins 15 , 71 , 72 enabling greater efficiency in fermentation process using cell wall sugars for 2GE production. The linkages 8-O-4 (β aryl ether) are the most common and are characterized as those of easiest cleavage. Lignins rich in G units have more recalcitrant linkages, such as 8-5 (phenylcoumarins), 5-5 (resinols), and 5-O-4, while S lignins are less interlinked and less recalcitrant to hydrolysis 15 , 73 . Overall, the analyses of the profiles of oligomers obtained by UPLC/MS from the four species studied identified 11 structures, between aldehydes, monomers, dimers, and trimers (Table 1 ). The distribution of these structures allowed a clear distinction between the internodes of the Saccharum species, and there was higher frequency of lignin oligomers in mature internodes than in young internodes. On the other hand, the highest amount of soluble phenols in all species were found in young culms, with markedly higher quantities in S. robustum and S. spontaneum compared with the other two species. Large quantities of free phenols, such as hydroxynnamic acids and chlorogenic acids, are found in tissues in lignification 10 , 16 , 25 . Also mature internodes of S. robustum and S. spontaneum the highest frequency and diversity of lignin oligomers (dimers and trimers) were found. Morreel et al . 74 commented that the various lignin oligomers in tissues that undergo extensive lignification are derived from the availability of monolignols that are coupled under oxidative conditions for cell wall lignification, justifying the correlation between lignin content and frequency of oligomers. The 8-O-4 linkage was the most common type of lignin linkage (Fig. 7F ). According to Santos et al . 75 , this type of linkage is dominant in lignins of grasses, corresponding to 60% of the total. Other works such as those presented by Bottcher et al . 33 and Kiyota et al . 15 also corroborate these results. It was also observed that G units were found more frequently than S units in oligomers of the four Saccharum species. We could not find H units, although the lignin of grasses is characterized by having more of these units than the lignin of dicotyledons 76 . The non-detection of H units in the Saccharum species could be explained by the fact that these units occur essentially as free terminal, inert phenolic groups, and their incorporation prevent the growth of the lignin polymer. Due to their high oxidative potential they are insoluble in ethyl acetate, which was the solvent used in the extraction of the oligomers 76 . Linkages containing only G units in this study, such as the dimers m/z = 357 [G(8-5)G] and m/z = 375 [G(8-O-4)G] and the trimer m/z = 553 [G(8-O-4)G(8-5)G], were identified notably in mature internodes of S. spontaneum and S. robustum . This result might explain why mature internodes of these species showed a lower S/G ratio than mature internodes of S. officinarum and S. barberi . The structures corresponding to the dimers m/z = 387 [S(8-5)G] and [S(8-8)G] and the trimer m/z = 583 [G(8-O-4)S(8-5)G] were identified in all internodes, which suggests that these structures are conserved and fulfil an important role in the growth and development. Sinapyl alcohol (S) ( m/z = 209) was found more frequently in young internodes of the species S. officinarum and S. barberi . Young internodes of S. officinarum and S. barberi have a higher soluble Klason lignin content than the other Saccharum species, which could be correlated with an increased frequency of the structure m/z = 209 as soluble Klason lignins are primarily composed of S units 77 . In comparison to the studies of Bottcher et al . 33 and Kiyota et al . 15 , only two structures m/z = 357 [G(8-5)G] and m/z = 583 [G(8-O-4)S(8-5)G] were always present indicating that they are conserved among the species of the genus Saccharum . In sugarcane hybrids it has been observed that the S/G ratio increases with the development of the stalk 33 . The same was observed in other grasses such as Festuca arundinacea 78 , Zea mays 66 , and Panicum virgatum 79 . However, such a ratio increase was not observed here, with the S/G ratio being higher in young internodes of S. barberi and S. robustum and equal for the other two species. Local growth conditions may have affected the S/G ratio, but in the case of the S. barberi and S. robustum the low values might be due to the amount of one of the monomers being higher in the pith or rind. We did not separate rind and pith for S/G analysis but based on the histochemical analysis the G amount (stained yellow, see Fig. 8 ) was elevated in the pith compared to the rind. Lignin composition (S/G ratio) affects the yield of saccharification 7 since tissues rich in S are more susceptible to hydrolysis than those rich in G 80 . We found no significant difference in saccharification in young internodes of the four species studied, which is not unexpected, since the lignification process has not been completed based on the content of soluble and insoluble lignin, oligomers, and phenols. However, it is interesting to note that in young tissues there seems to be no relationship between saccharification yield and S/G ratio, since S. barberi and S. robustum have higher S/G ratio, but saccharification yield is equal. However, mature internodes of S. spontaneum and S. robustum with lower S/G ratio resulted in a lower yield of saccharification. Therefore, higher yield of saccharification is related to S/G ratio, but only in tissues whose maturity has been reached and, thus, where the secondary cell wall formation process has been completed. F5H and COMT are thought to be the determinant enzymes in defining S unit content in plants 38 , 81 . In P. radiata , the joint action of the two activities led to an increase of S units, with the increase being smaller when only F5H was overexpressed 81 . In sugarcane, the reduction in the expression of COMT and F5H using RNAi led to different situations 38 . While plants with partial silenced F5H did not show a reduction in lignin content, one of the lines had a reduced S/G ratio with a concomitant increased saccharification yield. One of the mutants of COMT displayed a reduction in lignin content and improvement in saccharification yield. One of the mutants of COMT exhibited a reduction in the S/G ratio. Our data do not indicate a direct relationship between the expression of COMT and F5H and the S/G ratio. Using S. spontaneum as an example, this species had a similar S/G ratio between young and mature internodes; however, the expression of COMT and F5H was a little higher in pith of mature internodes but equal to the rind of young and mature internodes. On the other hand, the expression of F5H was much higher in mature tissues. A similar situation was also observed in S. officinarum , but with lower expression values. It cannot be ruled out that other hitherto unidentified isoforms of COMT and F5H are involved in lignin biosynthesis in these two species, but it is noteworthy that Bottcher et al . 33 isolated only one COMT and one F5H in sugarcane, and its sequences have a high homology with the sequences isolated in the four species studied. Another factor that has been recognized as negatively affecting plant biomass processing into 2GE is the degree of O -acetylation of cell wall polymers, since acetate, when released during pretreatment represents a powerful inhibitor of fermenting microorganisms 82 . O -acetylation of hemicelluloses also reduced enzymatic hydrolysis due to steric hinderance of the acetate 83 . Therefore, reducing the content of O -acetyl groups in biomasses with bioenergetic potential is desirable 84 . The main hemicelluloses in grasses are xylans 3 and their degree of O -acetylation may vary according to plant species, type of tissue and organ, and state of development 85 . Xylan acetylation occurs more frequently in position O-3 (up to 30%) and less frequently in O-2 (up to 25%), but acetylation in both positions has been reported 85 . In the Saccharum species studied here, it was found that the total percentage of acetylation (36.9–39.9%) was similar to values found in other grass biomasses 86 . On the other hand, acetylation in position O-3 was predominant (21.8–24.7%) with respect to the substitution O-2 (11.8–13.0%) and to O-2/O-3 (1.47–3.47%). Analyses by 2D-HSQC NMR spectroscopy showed that S. robustum and S. spontaneum were the species that presented the lowest percentages of acetylation in position O-3 (21.8% and 22.9%, respectively) and total acetylation (36.9–37.4%, respectively). However, the hypothesis that biomass with a reduced percentage of acetylesters results in higher saccharification yields 87 could not be supported here. S. officinarum and S. barberi , with a higher degree of acetylation than S. spontaneum and S. robustum , exhibited a higher yield of saccharification. Since it is known that in secondary walls xylans are closely associated with cellulose 88 , a lower percentage of acetyl groups in S. spontaneum and S. robustum could lead to an even tighter association of xylan with cellulose adding to recalcitrance in these species, and limiting the yield of saccharification 83 . The strategy used in this study to identify genes involved in the lignin biosynthetic pathway in the four Saccharum species involved the amplification of fragments produced in RT-PCR reactions using primers designed from conserved regions of gene sequences of sugarcane and of several other close species. Therefore, such primers are likely to amplify sequences of closely related genes encoding similar enzymatic activities. There is a possibility that not all genes of a gene family are amplified. However, the isolated genes represent the highest expressed genes in the tissues is high. Taking into account that the four species studied presented distinct genetic characteristics, it was surprising to observe that the isolated sequences are highly similar among the species and very close in sequence to the ones identified by Bottcher et al . 33 in sugarcane. Such similarities could be explained not only by the evolution of the lignin biosynthetic pathway in terrestrial plants but also by the origin of the genus Saccharum and of the commercial cultivars of sugarcane. The parental genomes of S. officinarum (80–90%) and S. spontaneum (10–20%) contributed to sugarcane hybrids including to some extent recombinant chromosomes 89 . Additionally, the lignin biosynthetic pathway is very conserved between plants and modifications in this pathway generate similar phenotypes between monocotyledons and dicotyledons. The approaches to manipulate lignin in alfalfa 7 can be transferred to other species such as switchgrass and sugarcane 29 , 37 . Genes related to sugar accumulation in sugarcane culms arose through differential expression of other regulators suggesting a specific epigenetic control. PAL is highly conserved between plants and seems to precede the divergence of dicotyledons and monocotyledons 90 . Genes related to transcriptional activation are highly conserved in grasses 91 . An example is the gene SND1 which activates several transcription factors: SND3, MYB46, MYB83, MYB85, and MYB105; apparently very conserved during evolution 91 ."
} | 7,358 |
39634420 | PMC11616513 | pmc | 4,880 | {
"abstract": "Sediment plays a pivotal role in deep-sea ecosystems by providing habitats for a diverse range of microorganisms and facilitates the cycling processes of carbon, sulfur and nitrogen. Beyond the normal seafloor (NS), distinctive geographical features such as cold seeps (CS) and hydrothermal vent (HV) are recognized as life oases harboring highly diverse microbial communities. A global atlas of microorganisms can reveal the notable association between geological processes and microbial colonization. However, a comprehensive understanding of the systematic comparison of microbial communities in sediments across various deep-sea regions worldwide and their contributions to Earth's elemental cycles remains limited. Analyzing metagenomic data from 163 deep-sea sediment samples across 73 locations worldwide revealed that microbial communities in CS sediments exhibited the highest richness and diversity, followed by HV sediments, with NS sediments showing the lowest diversity. The NS sediments were predominantly inhabited by Nitrosopumilaceae , a type of ammonia-oxidizing archaea (AOA). In contrast, CSs and HVs were dominated by ANME-1 , a family of anaerobic methane-oxidizing archaea (ANME), and Desulfofervidaceae , a family of sulfate-reducing bacteria (SRB), respectively. Microbial networks were established for each ecosystem to analyze the relationships and interactions among different microorganisms. Additionally, we analyzed the metabolic patterns of microbial communities in different deep-sea sediments. Despite variations in carbon fixation pathways in ecosystems with different oxygen concentrations, carbon metabolism remains the predominant biogeochemical cycle in deep-sea sediments. Benthic ecosystems exhibit distinct microbial potentials for sulfate reduction, both assimilatory and dissimilatory sulfate reduction (ASR and DSR), in response to different environmental conditions. The presence of nitrogen-fixing microorganisms in CS sediments may influence the global nitrogen balance. In this study, the significant differences in the taxonomic composition and functional potential of microbial communities inhabiting various deep-sea environments were investigated. Our findings emphasize the importance of conducting comparative studies on ecosystems to reveal the complex interrelationships between marine sediments and global biogeochemical cycles.",
"conclusion": "4 Conclusions In conclusion, our research utilized deep-sea sediment metagenomic data collected from worldwide locations to study microbial diversity and biogeochemical processes across different benthic ecosystems in the deep sea. The analysis of the microbial genomic catalog allowed us to identify the global dispersal and dominant microbial phylotypes present in different deep-sea sedimentary ecosystems. Specifically, the NS sediments are primarily composed of AOA, whereas ANME and SRB predominate in CS and HV sediments, respectively. Understanding microbial interaction patterns provides insights into the intricate ecological dynamics within these benthic ecosystems. Notably, the complexity of the biogeochemistry of HV sediments is underscored by the limited observed microbial interactions in this environment. Furthermore, the metabolic potentials of microbial communities in deep-sea sediments play diverse and important roles in biogeochemical cycles, including carbon fixation, sulfur compound reduction and oxidation, nitrogen fixation, and greenhouse gas production. The ecological functions of these metabolic pathways differ among habitats. Further examination revealed that the 3DC/4-HB and 3-HP/4-HB pathways of carbon fixation, along with the assimilatory sulfate-reducing pathway, were more abundant in NS sediments. In contrast, the rTCA and WL pathways of carbon fixation and the dissimilatory sulfate-reducing pathway were dominant in CS and HV sediments. Additionally, the presence of nitrogen-fixing organisms in CS sediments may significantly influence global nitrogen balance, alongside methane oxidation. These distinct pathways are integral to marine biogeochemical cycles. Overall, this research highlights the importance of microbial communities in deep-sea sediments for the biogeochemical cycles and ecological dynamics of different benthic ecosystems. Understanding the specific metabolic pathways and interactions within these communities is crucial for comprehending the complex processes occurring in the deep sea.",
"introduction": "1 Introduction The deep-sea benthic ecosystem plays a crucial role in maintaining the overall health and functioning of marine environments [ [1] , [2] , [3] ]. Despite the harsh conditions, deep-sea sediments host diverse and unique communities of organisms that have adapted to survive in these challenging environments [ 4 , 5 ]. In addition to normal seafloor ecosystems, cold seeps and hydrothermal vents are two distinct ecosystems [ [6] , [7] , [8] ]. Cold seeps are characterized by fluid emissions containing hydrocarbons and hydrogen sulfide, with temperatures akin to seawater [ [8] , [9] , [10] , [11] ]. Within cold seep ecosystems, there are primarily two types of microorganisms, anaerobic methane-oxidizing and sulfate-reducing microorganisms, that play crucial roles in the degradation and recycling of organic matter, contributing to overall carbon and energy cycling in these ecosystems [ 10 , [12] , [13] , [14] , [15] , [16] ]. In contrast to cold seeps, hydrothermal vents typically contain water rich in sulfide and metals, with temperatures ranging from 2 °C to 400 °C [ 17 ]. Hydrothermal vent ecosystems are predominantly situated at spreading centers on the seafloor, arising from hydrothermal vent within the Earth's crust [ 7 ]. Chemosynthetic bacteria and archaea constitute the primary trophic level within hydrothermal vent ecosystems, serving as the cornerstone of these unique ecosystems by converting inorganic carbon into organic biomass through chemosynthesis [ 18 , 19 ]. Previous research has demonstrated that hydrothermal vent and cold seep habitats exhibit similar community structures, yet they also harbor habitat-specific species [ 20 , 21 ]. Moreover, the most notable disparities in communities across different seafloor ecosystems occur within hydrothermal vent habitats and cold seep habitats, and the significant presence of endemic species can be found in both ecosystems [ 22 ]. For instance, at the phylum taxonomic level, both hydrothermal vents and cold seeps contain a considerable proportion of common phyla, including Chloroflexi , Proteobacteria , Crenarchaeota and Thaumarchaeota , irrespective of whether bacteria or archaea are considered [ [23] , [24] , [25] , [26] ]. However, at more detailed taxonomic level, such as the genus and species levels, the number of shared genera and species is limited. This may be related to variations in the fluid composition of the two ecosystems, in conjunction with differences in their discharge volumes and rates of organic deposition [ 20 , 27 , 28 ]. These differences in chemical compositions not only influence the distribution of microbial communities capable of utilizing specific compounds but also affect their adhesion and metabolic activities [ 20 ]. Although deep-sea hydrothermal vents and cold seeps are all sunlight-independent and chemosynthetic ecosystems, microbial metabolism in hydrothermal vent and cold seep ecosystems exhibits significant differences. In cold seep ecosystems, microbial metabolism predominantly hinges on the anaerobic oxidation of methane [ 29 ]. Conversely, in hydrothermal ecosystems, microbial metabolism is more reliant on the oxidation of inorganic substances such as hydrogen sulfide and hydrogen [ 30 ]. Within cold seep ecosystems, ANME archaea can transform methane into inorganic carbon and also generate organic carbon, such as acetic acid. This provides a carbon source for heterotrophic microbes within the ecosystem, significantly impacting deep-sea carbon cycling and global climate change [ 29 ]. Within hydrothermal vent ecosystems, the sulfide cycle is one of the most prominent biogeochemical cycles. Microbes in hydrothermal vent ecosystems primarily derive energy through the oxidation of sulfides and reduction of metals [ 31 ]. For example, some hyperthermophiles like Pyrococcus yayanosii utilize hydrogen sulfide and oxygen for metabolism under high temperature and pressure conditions [ 32 ]. Additionally, existing literature has reported a series of other biogeochemical element cycles in these ecosystems, such as the rTCA cycle and nitrogen fixation pathways in cold seeps, and the nitrogen cycle in hydrothermal vents [ [33] , [34] , [35] ]. Despite the burgeoning interest in the biogeochemical processes within cold seep and hydrothermal vent ecosystems, the majority of research has been confined to localized sites or limited regional samples, and focusing on specific metabolic pathways. To date, there is a notable absence of extensive global comparative analyses that amalgamate data from various cold seep and hydrothermal vent habitats. Such comparative studies are essential for elucidating the broader ecological and biogeochemical implications of these unique marine environments. In this study, we gathered a total of 163 metagenomic datasets from normal ecosystems, cold seeps, and hydrothermal vents across the globe. After assembling and binning, we constructed a catalog consisting of 3048 species-level genome bins (SGBs). The primary objective of this analysis was to investigate the composition, diversity, and metabolic potential of microbial communities within the three ecosystems. Additionally, we established correlations between microbial taxa and metabolic potential while predicting the metabolic patterns of microbial communities in different geographic regions of deep-sea sediments. Specifically, we analyzed metagenomes from three distinct ecosystems to address the following questions: (i) identify the predominant and frequently occurring microbial groups in deep-sea sediments; (ii) evaluate the influence of environmental factors on the organization principles of microbial community structure and function in deep-sea sediments; (iii) discover distinctive indicators in various ecosystems; and (iv) explore potential variations in the contribution of microbial community metabolism to biogeochemical cycling across different ecosystems.",
"discussion": "3 Discussion Previously, microbial communities in marine sediments were classified as aerobic or anaerobic ecosystems based on the analysis of microbial diversity in 299 sediment samples collected from various locations worldwide [ 36 ]. Another study categorized marine sediments according to their environmental characteristics by comparing the microbial diversity among shallow marine sediments, cold seeps, and hydrothermal vent habitats [ 22 ]. Cold seep communities were found to have moderate microbial richness and unique bacterial and archaeal groups that are widely distributed, compared to other marine ecosystems [ 22 ]. While these studies have provided valuable insights, they have certain limitations, primarily due to their reliance on amplifying 16S rRNA gene sequences. However, the emergence of high-throughput sequencing technologies, such as metagenomic sequencing, has addressed these limitations by providing information on potential metabolic functions in addition to microbial community composition [ 37 ]. In contrast to these prior researches, our study not only offers a systematic comparison of deep-sea sediments based on geographical attributes but also employs metagenomic data to precisely determine the correlation between microbial composition and ecological functions. This approach sheds light on the complex interactions within these environments. Based on the taxonomic profiles of 3048 SGBs, our analysis revealed that only five phyla demonstrated global dispersion. Although this discovery was unexpected, it could be explained by sampling strategies or sequencing depth. Despite their limited representation, these phyla collectively accounted for an average relative abundance of 35.21 % and 30.72 % in the CS and HV ecosystems, respectively. Additionally, these taxa constituted more than half of the microbial communities in the NS ( Fig. S4 ). It is suggested that the majority of microbial phylotypes are infrequent, with only a limited number being prevalent, while many of these infrequent phylotypes show a widespread distribution throughout marine sediments. Moreover, ANME and SRB were identified as the highly abundant populations within CS and HV, respectively. A previous study revealed that the dominant microorganisms in sediments at a depth of 390 m in Storfjordrenna seeps was ANME/Seep-SRB1 [ 38 ], which aligns with our findings. It can be speculated that microbial phylotypes and particular habitats are inevitably related. However, further investigations are needed to determine the ecological roles and functions of these highly abundant populations and their interactions with the less abundant phylotypes in marine sediments. Furthermore, we observed significant differences in microbial community diversity among the sediments within the NS, CS, and HV environments. The distinct community compositions, as indicated by inter-sample similarities in SGB composition, suggest a greater level of diversity among marine sediment environments. This elevated diversity is likely influenced primarily by local geochemistry, specifically by the concentrations of methane, sulfide, nitrate, and other substances, rather than by the geological environment or random diffusion events [ [39] , [40] , [41] ]. Notably, our findings align with recent studies that have reported the highest microbial species diversity at cold seep sites [ 22 , 33 , 42 , 43 ]. This could be attributed to the local heterogeneity of the sampling infiltration system, which potentially affects the availability of ecological niches for microbial colonizers. An unexpected finding was the microbial communities within the HV ecosystem exhibit a lower degree of network connectivity in comparison to those in the NS and CS ecosystems. Prior research has shown that, in contrast to cold seeps, hydrothermal vents support a greater variety and abundance of metal ions and hydrogen gas, which can serve as sources of nutrition for many microorganisms [ 44 ]. Despite the absence of metal ions and physicochemical parameters such as hydrogen, we propose that this outcome may be ascribed to a more varied growth strategy of microorganisms in nutrient-abundant hydrothermal vent ecosystems, rather than solely depending on microbial interactions. The biogeochemical cycle in marine sediments and the control of organic matter as a dynamic repository are driven by intricate interactions between microbial communities and geochemical processes [ 45 ]. Previous studies have indicated that the cycling of active substances in sediments involving microbes includes carbon fixation, sulfate reduction and sulfide oxidation, nitrogen fixation, and ammonia oxidation, among others [ 21 , [46] , [47] , [48] ]. Another study further revealed that microorganisms residing in deep-sea sediments have diverse potential roles in the biogeochemical cycle, which are highly related to their habitats [ 49 ]. Although the metabolic potential of most carbon fixation pathways was similar, the WL pathway was found to be enriched in the CS and HV sediment microbiota. Furthermore, our findings suggest that while sulfate reduction is the predominant sulfur metabolism pathway in deep-sea sediments, different habitats exhibit distinct preferences for dissimilatory and assimilatory pathways. Additionally, our study indicates a discrepancy in the metabolic potential for the greenhouse gas N 2 O production and utilization within the NS sediments, highlighting an accumulation of N 2 O-producing capabilities without a corresponding increase in N 2 O-consuming pathways. This could be a significant factor in climate change, particularly considering the vast expansion of normal deep-sea beds. Further investigations into the relationship between the metabolic preference of the deep-sea microbial community and its adaptation to specific habitats could significantly contribute to our understanding of the ecological importance of the deep-sea microbiota. We discovered that the CS sediment had a greater abundance of nifH than the NS and HV sediments. These findings support previous research conducted by Dong et al., who analyzed global metagenomic data from 11 cold seep sites and identified a wide range of nitrogen-rich organisms with diverse phylogenetic and metabolic decomposition patterns [ 33 ]. These nitrogen-rich organisms in CS sediments likely play a crucial role in maintaining the global nitrogen balance. Furthermore, our study revealed that despite differences in microbial community composition, genome size and physiological characteristics, microorganisms in diverse ecosystems exhibit similar metabolic capabilities [ 50 ]. This observation is consistent with our findings that functional microorganism diversity varies across habitats, but all of these microorganisms play pivotal roles in the cycling of carbon, nitrogen, and sulfur. However, we also observed that microorganisms in different environments have distinct metabolic contributions when faced with significant geochemical disparities."
} | 4,364 |
39021578 | PMC11249780 | pmc | 4,881 | {
"abstract": "The potential mining of deep-sea polymetallic nodules\nhas been\ngaining increasing attention due to their enrichment in metals essential\nfor a low-carbon future. To date, there have been few scientific studies\nconcerning the geochemical consequences of dewatered mining waste\ndischarge into the pelagic water column, which can inform best practices\nin future mining operations. Here, we report the results of laboratory\nincubation experiments that simulate mining discharge into anoxic\nwaters such as those that overlie potential mining sites in the North\nPacific Ocean. We find that manganese nodules are reductively dissolved,\nwith an apparent activation energy of 42.8 kJ mol –1 , leading to the release of associated metals in the order manganese\n> nickel > copper > cobalt > cadmium > lead. The composition\nof trace\nmetals released during the incubation allows us to estimate a likely\ntrace metal budget from the simulated dewatering waste plume. These\nestimates suggest that released cobalt and copper are the most enriched\ntrace metals within the plume, up to ∼15 times more elevated\nthan the background seawater. High copper concentrations can be toxic\nto marine organisms. Future work on metal toxicity to mesopelagic\ncommunities could help us better understand the ecological effects\nof these fluxes of trace metals.",
"conclusion": "Conclusions This work demonstrates the potential release\nof many heavy metals\ndue to the reductive dissolution of Mn nodules if the deep-sea mining\nwastewater were to be discharged into the ODZ. As interest in Mn nodule\ndeep-sea mining continues to increase, it is vital that we understand\nthe influence of mining dewatering waste to trace metal biogeochemical\ncycling, especially for potentially toxic dissolved Cu, and its potential\nimpact on the mesopelagic ecosystem. Based on our incubation results,\nwe expect to see a slower release of metals due to nonreductive dissolution\nprocesses if released outside of the ODZ, which is critical information\nfor choosing the depth of dewatered mining waste discharge. However,\nregardless of the discharge pipe depth, Mn nodule reductive dissolution\nremains important as a result of inevitable surface spills that will\nsettle MnO 2 particles through the ODZ layer. Overall, more\nwork on the release of trace metals into oxygen-rich waters above\nand below the ODZs is needed in addition to studies of putative metal\ntoxicity to mesopelagic communities in order to gauge ecological effects\nof these fluxes.",
"introduction": "Introduction Marine polymetallic nodules in certain\nabyssal regions, also known\nas manganese (Mn) nodules, have attracted interest over several decades\nfrom mining companies due to their enrichment of metals such as cobalt\n(Co), nickel (Ni), copper (Cu), and rare earth elements (REEs). 1 − 3 These nodules grow up to 20 cm in their longest dimension and are\nformed by the slow growth of Mn and iron (Fe) oxides (dominantly δ-MnO 2 , vernadite, and todorokite) around a nucleus at a rate of\nseveral millimeters to centimeters per million years. 4 − 6 A significant increase in demand for minerals and metals,\nsuch\nas lithium (Li), Cu, Co, Ni, and REEs, is predicted in order to build\nthe batteries required for the clean energy transition to a low-carbon\nfuture. 7 − 10 As the proposed demand for these metals is likely to double by 2060, 11 , 12 deep-sea mining of Mn nodules is an emergent industry that may provide\nadditional mineral resources beyond the growth of land-based mining\nto meet this proposed demand. Mn nodules are widely distributed\nin the global ocean. 13 One of the most\nextensive deposits of Mn nodules\nis the Clarion-Clipperton Zone (CCZ) in the Eastern Tropical North\nPacific Ocean, an area between Hawaii and Mexico at ∼8–15°\nN. The CCZ is located beneath one of the world’s most extensive\noxygen-deficient zones (ODZs), where waters between 200–800\nm are generally lower than 20 μM ( Figure 1 ) of oxygen and reach anoxic conditions in\nsome areas. As interests in mining escalate, the International Seabed\nAuthority (ISA) has issued exploration contracts to 17 countries and\ncommercial entities in the CCZ ( https://www.isa.org.jm/exploration-contracts ). To date, no mining, meaning commercial exploitation, of polymetallic\nnodules has taken place, either in the CCZ or elsewhere. The ISA is\ncurrently developing exploitation regulations for deep-sea mining,\nfor which environmental regulations will include governing waste discharge. 14 , 15 In June 2021, the “two-year rule” at the ISA was triggered\nby the Republic of Nauru to complete exploitation regulations by July\n2023. This date has now passed, but how that provision will be interpreted\nremains to be seen. 16 Relevant scientific\nstudies in the near term will be integral to the development of these\nregulations. Figure 1 Map of the Clarion-Clipperton Zone (CCZ) in the Northeast\nPacific\nOcean. Blue and vermilion boxes are exploration and reserved areas,\nrespectively. Light gray squares are areas of particular environmental\ninterest (APEIs) designated by the ISA. The bluish-green diamonds\nare the U.S. GEOTRACES GP15 cruise track, and the location of Station\n22, where the filtered seawater used for the incubation study was\ncollected, is labeled. The location of Site A where the Mn nodules\nused in this study were collected is marked by using a star. The thick\nblack contour lines are dissolved oxygen concentrations of 10 and\n20 μM at the 500 m isobath, which were retrieved from the World\nOcean Atlas 2018, 32 illustrating the horizontal\nextent of the oxygen deficient zones (ODZs). Shapefiles are downloaded\nfrom the International Seabed Authority Web site ( https://www.isa.org.jm/minerals/maps ). Environmental research to date has focused mostly\non the impacts\nof nodule removal on the benthic environment and assessment of the\nresilience of benthic fauna and microbial ecosystems in the mining\nzones. 17 − 22 In proposed mining operation workplans to date, the waste from shipboard\ndewatering of polymetallic nodules is delivered back to the water\ncolumn. 23 Importantly, the depth of waste\ndischarge during potential future deep-sea mining operations, which\ncould contain both crushed Mn nodules and deep-sea sediments, remains\nan open and controversial question. Naturally occurring manganese\noxides are reductively dissolved in the ODZs of the Pacific Ocean,\nas evidenced by observations of low particulate Mn (<0.01 nM),\nhigh dissolved Mn (>1 nM) concentrations, 24 − 28 and measurements of trace element redox couples. 29 , 30 Based on these studies, we hypothesize that discharge of waste mining\nslurry containing crushed Mn nodule particles into an ODZ at mesopelagic\ndepths will lead to reductive dissolution of discharged Mn oxides\nand the associated mobilization of heavy metals originally sorbed\nand incorporated into the Mn mineral phase of the nodules. This potential\nflux of reductively mobilized dissolved metals into ODZs is largely\noverlooked in the current literature of the environmental impacts\nof deep-sea mining, 14 , 31 and the magnitude of these fluxes\nis central to the question of which depth dewatered mining waste should\nbe discharged to the ocean. In this study, we conducted incubation\nexperiments to evaluate\nthe potential mobilization of heavy metals from the reductive dissolution\nof Mn nodules during mining waste discharge into the ODZ overlying\nthe CCZ. Trace metal clean sampling and analytical procedures enabled\nus to detect nanomolar concentration changes of dissolved trace metals\nassociated with Mn nodule reduction and estimate the accumulation\nrates of Mn and other trace metals in the dissolved phase. This work\noffers one of the first geochemical perspectives of mining waste discharge\nin the mesopelagic ocean, evaluates a potential budget of metal release\nfrom the discharge plume, and explores the fate of these metals within\nthe ODZ (dilution, sinking, complexation, etc.) to inform decisions\nrelated to future mining discharge operations.",
"discussion": "Results and Discussion In our experimental design ( Table S1; Figure S1 ), we tested for the reduction of crushed Mn nodules (hereafter\nMnO 2 ) and release of associated metals in the presence\nand absence of an added labile DOC source (acetate) and at two incubation\ntemperatures, for a total of six groups: 64 μM MnO 2 at room temperature (22 °C) (Group 1, G1: +Mn–Ac–T)\nand at 42 °C (Group 2, G2: +Mn–Ac+T); 64 μM MnO 2 + 500 μM acetate at room temperature (Group 3, G3:\n+Mn+Ac–T) and at 42 °C (Group 4, G4: +Mn+Ac+T); and 500\nμM acetate only at room temperature (Group 5, G5: –Mn+Ac–T)\nand at 42 °C (Group 6, G6: –Mn+Ac+T) (no MnO 2 controls). Characterization of CCZ Mn Nodules Used for the Incubation The specific surface area of the Mn nodules was 153.3 ± 0.7\nm 2 g –1 . This value is lower than the\nsurface area of biogenic Mn oxides produced by the bacterium Leptothrix discophora (224 m 2 g –1 ) 39 but comparable to previously reported\nspecific surface area measurements of CCZ Mn nodules; ∼120\nm 2 g –1. 40 Crushed\nCCZ nodules in this study contain 32.1 wt % of Mn, 9.3 wt % of Fe,\n1.1 × 10 –3 wt % of cadmium (Cd), 0.3 wt % of\nCo, 1.1 wt % of Cu, 1.5 wt % of Ni, and 6.5 × 10 –2 wt % of lead (Pb), which are consistent with compiled major and\nminor chemical compositions of CCZ nodules. 13 , 41 , 42 X-ray diffraction (XRD) results show that\nthe starting material consisted mainly of 10 Å turbostratic phyllomanganates.\nMinor quartz detrital material is also apparent ( Figure S2 ). Changes of Dissolved Trace Metals with Time during the Incubation In groups G1–G4 with added MnO 2 , dissolved Mn\n(dMn) concentrations increased with time, at first linearly, in support\nof the method of initial rates most similar to open system conditions\nrecorded for MnO 2 reductive dissolution rates in previous\nstudies. 43 The increase was larger at \nhigher temperature (G2 or G4) and in groups with higher acetate (G3\nor G4) ( Figure 2 ).\nThe change of dMn from Day 0 to Day 1, however, was similar for groups\nwith or without acetate: at 22 °C, the increase of dMn concentrations\nfrom Day 0 to 1 was 2.5 nM in the presence of acetate and 2.7 nM without\nacetate; at 42 °C, dMn increased by 18.4 nM and 11.2 nM with\nand without acetate, respectively. Given the similar and small increase\nin dMn between Days 0 and 1 regardless of acetate presence, we hypothesize\nthat the nonreductive dissolution of Mn oxides or desorption of Mn 2+ dominates in the first day ( Figure 2 ). Figure 2 Dissolved trace metal concentrations (unit:\nnM) change with time\nduring the incubation. (a–f) Trace metal concentrations (Mn,\nCd, Co, Cu, Ni, Pb) in +MnO 2 only groups with no acetate\nat 22 °C (G1: +Mn–Ac–T) and at 42 °C (G2:\n+Mn–Ac+T); (g–l): trace metal concentrations (Mn, Cd,\nCo, Cu, Ni, Pb) in +MnO 2 +acetate groups (G3: +Mn+Ac–T;\nG4: +Mn+Ac+T). Different groups (G1, G2, G3, or G4) are illustrated\nwith different colored lines. Note that concentration scales change\nfor each element between the two treatment groupings, the top and\nbottom rows. Error bars are standard deviations from duplicates. Weighted\nModel I regression is used to fit dissolved Mn concentrations during\nDays 0–4 for G1 and G2 and during Days 4–15 for G3 and\nG4, visualized as thick black and purple dashed lines, respectively.\nInsets in (g, i) are the expanded plots in the first 2 days of incubation\nfor dissolved Mn and labile Co. We also monitored a competing electron acceptor\nto MnO 2 , nitrate plus nitrite (NO x ), since the\nreduction of nitrate coupled to the oxidation of organic matter has\nsimilar Gibbs free energy to the reduction of MnO 2 , 44 and nitrate is of much higher concentrations\nin ODZs than particulate Mn. 27 , 45 The relatively unchanged\nNO x concentrations from all groups at\nDays 0 and 1 further support the nonreductive release of dMn at the\nbeginning of the incubation ( Supporting Information Text & Figure S3 ). After Day 1, NO x concentrations were quickly depleted in the presence of acetate\n(G4 or G3: +Mn+Ac±T) but decreased more slowly in the absence\nof acetate (G2 or G1: +Mn–Ac±T), suggesting that acetate\nwas used as the electron donor in the reduction of NO x ( Figure S3 ). Similarly,\nthe much higher rate of dMn accumulation in the presence of acetate\nafter Day 1 suggests that acetate also acted as the electron donor\nin the reduction of MnO 2 . The slower accumulation of dMn\nin the absence of acetate (G2 or G1) after Day 1 suggests nonreductive\ndissolution or desorption processes occurring throughout the incubation\nand/or slower rates of reductive dissolution using the more recalcitrant\nbackground DOC as the electron donor. For simplicity, we will refer\nto processes in groups G2 or G1 (+Mn–Ac±T) as nonreductive. Dissolved Mn 2+ is known to strongly adsorb onto Mn oxide\nsurfaces, 46 but an overall net accumulation\nof dMn in all groups during the beginning of the incubation indicates\nthe dominance of Mn dissolution and excess of desorption over adsorption.\nConcentrations of dMn then plateaued and decreased toward the end\nof the incubation, which can be explained by a gradual dominance of\nsorption of dissolved Mn 2+ onto remaining Mn nodules in\nthe closed system of our experimental setup. Secondary adsorption\nand related processes are less important in an open system such as\nthe ocean where dissolved Mn 2+ cannot easily accumulate. 47 Similar to dMn, dissolved Cd (dCd), dissolved\nCu (dCu), and dissolved\nNi (dNi) concentrations increased with time in all groups ( Figure 2 ) and were enhanced\nat higher temperatures (G4 or G2) and with the addition of acetate\n(G4 or G3). Dissolved labile Co (dlCo) decreased with time in the\nabsence of acetate (G2 or G1: +Mn–Ac±T), possibly as a\nresult of readsorption of released dlCo back onto remaining Mn nodule\nparticles due to its high affinity for Mn oxides. 48 − 50 A similar decrease\nin dlCo, about 0.1–0.3 nM, occurred initially between Day 0\nand Day 1 in the presence of acetate (G4 or G3: +Mn+Ac±T), when\nnonreductive release of Mn dominated, but switched to net dlCo ingrowth\nafter Day 1 once reductive dissolution of Mn nodules increased. Dissolved\nZn and Fe concentrations are not discussed due to potential contamination\nissues ( Supporting Information Text and Figure S4 ). Dissolved lead (dPb) is also known to strongly scavenge\nonto Mn\noxides. 39 , 51 , 52 Unlike that\nof dlCo, the accelerated accumulation of dMn caused by the addition\nof acetate did not lead to a similar accumulation of dPb. Dissolved\nPb concentrations decreased with time in all groups, implying that\nreadsorption of dPb was faster than its release from any reductive\ndissolution of Mn nodules. The contrasting behavior between dlCo and\ndPb observed in G3 and G4 (+Mn+Ac) is consistent with laboratory reductive\ndissolution experiments using soil Mn nodules 53 and with studies that show a higher readsorption rate of dPb onto\nMn oxides than dlCo. 51 , 54 − 56 Overall, the\nextent of dissolved trace metal accumulation in the incubation bottles\nwas Mn > Cu > Ni > Cd > Co ≈ Pb in the absence\nof acetate (G2\nor G1: +Mn–Ac±T) and Mn > Ni > Cu > Co > Cd\n> Pb in the\npresence of acetate (G4 or G3: +Mn+Ac±T). Rates and Metal Accumulation from Nodule Reduction during Incubation Reductive dissolution of Mn oxides by organics under anoxic conditions\nhas previously been demonstrated to be a surface- rather than transport-controlled\nreaction. 43 , 57 , 58 We quantify\ndMn release from Mn nodules by calculating the slopes of the initial\ndMn concentration increase with time ( Figure 2 ). Similar methods have been used to characterize\nMn reduction rates under anoxic conditions using either synthesized\nMn oxides 43 , 59 or marine sediments. 60 , 61 As expected, higher concentrations of dissolved Mn 2+ accumulated\nfrom reductive dissolution of Mn nodules in G3 and G4 (+Mn+Ac) than\nfrom nonreductive dissolution processes in G1 and G2 (+Mn–Ac).\nInitial dissolved Mn accumulation rates from the linear (“open\nsystem”) portion at the beginning of the incubation (Day 0–4\nfor G1 and G2; Day 4–15 for G3 and G4) were 2.1 ± 0.3\nnM day –1 for G1 (+Mn–Ac–T), 5.8 ±\n0.4 nM day –1 for G2 (+Mn–Ac+T), 11.4 ±\n9.9 nM day –1 for G3 (+Mn+Ac–T), and 34.5\n± 3.3 nM day –1 for G4 (+Mn+Ac+T) ( Figure 2 ). The trace\nmetal release in different groups, as defined by the ratios between\nthe accumulation of dissolved trace metals and dMn (dTMs/dMn), was\ncalculated to quantitatively evaluate which metals were more/less\naccumulated than one might expect based on congruent (stoichiometric)\nnodule dissolution ( Figure 3 ; Table S2 ). In the absence of\nacetate (G1 and G2: +Mn–Ac), estimated dTM/dMn ratios and their\nstandard errors were (3.8 ± 0.9) × 10 –3 mol Cd/mol Mn, (2.5 ± 0.4) × 10 –1 mol\nCu/mol Mn, and (1.7 ± 0.2) × 10 –1 mol\nNi/mol Mn. In the presence of acetate (G3 and G4: +Mn+Ac), estimated\ndTM/dMn ratios were (4.3 ± 0.6) × 10 –4 mol Cd/mol Mn, (2.5 ± 0.2) × 10 –3 mol\nlCo/mol Mn, (3.6 ± 0.4) × 10 –2 mol Cu/mol\nMn, and (7.2 ± 0.5) × 10 –2 mol Ni/mol\nMn. Figure 3 Relationships between dissolved trace metals and dissolved Mn (unit:\nnM). (a–e) +Mn–Ac groups (G1 and G2); (f–j) +Mn+Ac\ngroups (G3 and G4); (a, f) dissolved Cd (dCd); (b, g) dissolved labile\nCo (dlCo); (c, h) dissolved Cu (dCu); (d, i) dissolved Ni (dNi); (e,\nj) dissolved Pb (dPb). Robust regression is used in the fit to minimize\nthe effect of outliers on the linear relationships. Note that only\ndata after Day 1 in G3 and G4 (+Mn+Ac) are included in the regression,\nsince different processes likely occur before and after Day 1. Only\nregression fits with p values smaller than 0.05 are\nshown. Again, note that concentration scales change for each element\nbetween the two treatment groupings. The bulk molar ratios of crushed Mn nodules (unit:\nmol/mol) used\nfor the incubation were 1.7 × 10 –5 for (Cd/Mn) nodule , 8.5 × 10 –3 for (Co/Mn) nodule , 3.0 × 10 –2 for (Cu/Mn) nodule ,\nand 4.2 × 10 –2 for (Ni/Mn) nodule ( Table S2 ). These trace metal-to-Mn bulk\nmolar ratios in nodules (TMs/Mn) nodule were similar to\nthe dTMs/dMn ratios in solution in G3 and G4 (+Mn+Ac) and were about\none magnitude lower than the dTMs/dMn ratios in G1 and G2 (+Mn–Ac)\n( Figure 4 ). The order\nof magnitude higher dTMs/dMn ratios in the nonreductive G1 and G2\n(+Mn–Ac) experiments show that some dTMs nonreductively dissolve\nor desorb from the MnO 2 surface upon meeting seawater,\nwhich is relevant to mining waste discharge at any depth (not just\nODZs), as it is independent of reductive conditions. In contrast,\nthe overall extent and similarity between dTMs/dMn ratios and bulk\nMn nodule composition in the presence of acetate (G3 and G4), especially\ndCu/dMn and dNi/dMn, confirm that a source of acetate appears to facilitate\nthe reduction of the Mn nodule under anoxic conditions and release\ntrace metals that are structurally incorporated in the MnO 2 octahedral layers. Figure 4 Bar plots of the ratios between trace metals and Mn (unit:\nmol/mol)\nof dissolved phases in different incubation groups (dTMs/dMn) compared\nwith concentrations measured in solid bulk nodules used for the incubation\n(TMs/Mn) nodule . Error bars are standard errors of the dTMs/dMn\nslopes in G1 and G2 (+Mn–Ac) and in G3 and G4 (+Mn+Ac). Inset\nis the expanded bar plot of Cd and Co. The ratio of dlCo/dMn in G1\nand G2 is not determined due to strong readsorption of dlCo onto Mn\noxide surfaces. The relative abundance of Cu and Ni released is\naffected by the\npresence or absence of acetate: more Ni than Cu was released during\nreductive dissolution in G3 and G4 (+Mn+Ac), but the opposite was\ntrue during nonreductive dissolution in G1 and G2 (+Mn–Ac).\nThis may be due to the structural incorporation into phyllomanganate\nlayers being more favorable for Ni (∼10–45% incorporated\ninto layer vacancy sites) 62 , 63 than Cu (∼0–20%\noccupying vacancy sites) 64 , 65 at pH 7–8, whereas\nthe desorption from the interlayer between MnO 6 octahedral\nlayers and layer edge sites is likely to favor Cu over Ni. The\ndCd/dMn ratio in the presence of acetate (G3 and G4: +Mn+Ac)\nwas ∼25 times higher than (Cd/Mn) nodule of 1.7 ×\n10 –5 for bulk Mn nodules, whereas the dlCo/dMn ratio\nwas ∼3.5 times lower than (Co/Mn) nodule of 8.5 ×\n10 –3 ( Figure 4 ; Table S2 ). Differences between\ndTMs/dMn measured in the incubation seawater in the presence of acetate\nand the (TMs/Mn) nodule of bulk Mn nodules suggest that\nreductive dissolution is not the only important process during the\nincubation. Readsorption of released dTMs back onto remaining Mn nodules\nis also relevant, and its relative magnitude varies between different\ntrace metals ( Figure S5 ): a higher readsorption\nthan that of dMn leads to a smaller accumulated dTMs/dMn than (TMs/Mn) nodule of bulk Mn nodules assuming congruent dissolution. For\nexample, the dlCo/dMn ratio is smaller than the bulk (Co/Mn) nodule ratio because dlCo readsorbs more than dMn to the Mn nodule surface,\nbut the dCd/dMn ratio is larger than its nodule stoichiometry because\nthere is less readsorption for dCd. Therefore, our incubation experiment\ndemonstrates that the sorption affinity of dissolved metals onto Mn\nnodules follows a general order of Cd < Ni < Cu ≈ Mn\n< Co < Pb, which is consistent with previous sorption experiments 54 , 66 − 68 and the thermodynamic stability of surface complexation. 69 Consideration of Relevance to Dewatering Operations There are three aspects to consider, temperature, MnO 2 concentrations, and microbial abundances, before extrapolating our\nincubation experiments to a potential mining waste plume scenario. First, the temperature effect of the reductive dissolution of Mn\noxides by various organic compounds has previously been modeled using\nthe Arrhenius equation between the temperature range of 5 to 40 °C. 57 , 70 − 72 We can estimate Mn reduction rates at the temperature\nof ODZ seawater at 200 m overlying the CCZ (approximately 11 °C) 73 based on rates from our incubation experiments\nusing the Arrhenius equation, with potential error quantified for\nour results and also taking into account previous analyses for similar\nsystems ( Supporting Information Text ).\nWe model the kinetics of MnO 2 reduction as a second-order\nreaction 74 with respect to concentrations\nof MnO 2 and DOC for groups G3 and G4 (+Mn+Ac) where MnO 2 reduction occurred. 1 where and are the change of Mn oxides and dissolved\nMn concentrations with time, respectively, k is the\nsecond-order rate constant (unit: L mol –1 day –1 ), and [MnO 2 ] and [DOC] are concentrations\nof MnO 2 and DOC, respectively. The incubation of Mn nodules\nis conducted with excess concentrations of DOC (acetate + background\nDOC ≈ 550 μM) and Mn nodules ([MnO 2 ] = 64\nμM). The rate of Mn oxide reduction, therefore, can be treated\nas pseudozero order with respect to the initial concentration of Mn\noxides and DOC in the bottle as follows: 2 where k ’\nis the pseudozero-order rate constant (unit: mol L –1 day –1 ). The apparent activation energy E a of the reaction can be estimated using two\ntemperatures (22 and 42 °C) based on the Arrhenius equation: 3 4 where A is\na pre-exponential factor (unit: mol L –1 s –1 ), E a is the activation energy (unit:\nJ mol –1 ), R is the gas constant\n(8.314 J mol –1 K –1 ), T is the temperature (unit: K), and subscripts 1 and 2 represent\nthe parameter (rate constant or temperature) at two different temperatures. Using dMn accumulation rates calculated from our incubations in\nthe presence of acetate (G3: k ’ = 11.4 nM\nday –1 at 22 °C; G4: k ’\n= 34.5 nM day –1 at 42 °C), the apparent activation\nenergy E a is estimated as 42.8 kJ mol –1 . This E a falls within\nthe range of 17.4–60 kJ mol –1 found in previous\nkinetic studies of the reductive dissolution of Mn oxides, 57 , 70 − 72 consistent with a surface-controlled reaction. 47 We can then derive a dMn accumulation rate of\n5.8 nM day –1 for an ODZ temperature of 11 °C\nas the pseudozero-order rate constant k ’ ( eq 4 ). Second, we compared\nconcentrations of MnO 2 used in our\nincubation to the dewatering waste plume to confirm that they are\ncomparable after accounting for turbulent mixing upon waste discharge.\nTo estimate dewatering plume composition, we utilize the composition\nof a sediment plume created by mobilization of CCZ sediments from\nthe nodule miner with and without added crushed Mn nodule material.\nWe use the density for a commercial sediment plume in the CCZ of 8\ng L –1 . 75 The lower bound\nof particulate Mn concentrations in the waste plume is 24 mg L –1 (436.4 μM), which assumes crushed Mn nodules\nare absent from the waste plume and the plume is entirely composed\nof surface CCZ sediments without nodules that are characterized by\nabout 0.3 wt % of leachable particulate Mn. 76 As an upper bound, we assume that the discharged waste sediment\nconsists of entirely crushed Mn nodules that are about 30 wt % Mn,\nequivalent to 2400 mg L –1 or 43.6 mM of particulate\nMn. A recent field study discharging CCZ sediments into the subsurface\nNortheast Pacific Ocean demonstrated that the descending waste plume\nis diluted 100–1000 times upon discharge by ambient seawater\nvia strong turbulent mixing as the dynamic plume\nand then laterally advected by ocean currents as the ambient plume. 75 Such dilution decreases the\nparticulate Mn concentration and sets the initial particulate Mn concentrations\nat 0.4–436.4 μM in the ambient plume.\nTherefore, the MnO 2 concentration used in our incubation\nexperiment, 64 μM particulate Mn, falls within the range of\nthe initial MnO 2 concentration of the ambient waste plume, which suggests that both zero-order kinetics and the\nrelative rates that we found may be applicable. Third, it remains\nunclear how relevant our estimated abiotic dMn\naccumulation rates are compared to in situ Mn reduction\nrates within the ODZ that are mediated by microbial processes. To\nthe best of our knowledge, there have not been any direct field measurements\nof MnO 2 reduction or dMn accumulation rates within any\nODZs in the water column. To estimate in situ MnO 2 reduction\nrates, we assume that previous observations from the eastern tropical\nPacific that show a decrease in Mn oxide concentrations in ODZs are\nbecause of reductive dissolution. 24 , 27 , 28 We can use observations of the decrease of Mn oxides\nwith depth from the U.S. GEOTRACES Eastern Pacific Zonal Transect\n(GP16) in the Peruvian ODZ 77 to estimate\na pseudo-first-order rate constant of Mn reduction of (1.6–8.4)\n× 10 –3 day –1 , using the sinking\ntime scale for a Mn oxide particle according to Stokes’ Law 78 ( Supporting Information Text ). Using this in situ rate constant for\nMn reduction from the GP16 cruise to approximate the ambient plume under anoxic conditions in the CCZ, we estimate MnO 2 reduction rates within the ambient plume as 0.7–3686\nnM day –1 given MnO 2 concentrations of\n0.4–436 μM. The dMn accumulation rate of 5.8 nM day –1 at 11 °C derived from our abiotic Mn nodule\nreduction incubation experiments is at the low end but comparable\nto our calculated in situ MnO 2 reduction\nrates that might occur if dewatered mining waste was discharged into\nODZs (0.7–3686 nM day –1 ). Note that the dMn\naccumulation rates in our incubations include strong readsorption\nof Mn 2+ onto Mn oxide surfaces 46 , 60 and can be up to 10 times lower than MnO 2 reduction rates\nas shown in anoxic incubations of marine sediments in the East Sea 61 and the Norwegian Trough. 60 If we consider the effects of strong readsorption onto\nMn oxides in the waste plume, the dMn accumulation rate within the\ninitial ambient plume would be 0.07–368.6\nnM day –1 . Thus, the dMn accumulation rate from our\nincubation may serve as a representative rate, which is very similar\nto the estimated geometric mean of 5.0 nM day –1 within\nthe projected waste plume should it be discharged to the ODZ. Estimations of Trace Metal Budgets within the Waste Plume We hypothesize that the trace metal compositions (dTMs/dMn) observed\nduring our experiments are applicable to potential future waste plumes,\nwhich would include direct dewatered waste discharge as well as any\naccidental spills from either the riser pipe or the ship into surface\nwaters, which would settle through the ODZs ( Figure 5 ). Insights into trace metal budgets during\nmining dewatering operations, therefore, can be gained by extrapolating\nour experimental results to a waste discharge scenario. We use the\ndMn accumulation rate from our incubation, representative of rates\nwithin the initial ambient plume, to estimate the\noverall budget of dissolved Mn and other trace metals that may result\nfrom discharging a mining waste plume within the ODZ. Figure 5 Schematic of the trace\nmetal inputs in a potential deep-sea mining\ndewatering waste plume and accidental spills from both the riser pipe\nand the mining platform. The section plot is dissolved oxygen concentrations\n(unit: μmol kg –1 ) along 12° N. Dissolved\noxygen data are retrieved from the World Ocean Atlas 2018 annual climatology, 32 and the gray contours are oxygen concentrations\nof 30 μmol kg –1 . The most enriched trace metals,\nin comparison to the background seawater concentrations, that are\nreleased from the reductive dissolution of Mn nodules are shown as\nwhite, curved arrows. Our incubation results suggest that Pb likely readsorbs\nonto remaining\nMn surfaces after reductive dissolution of Mn oxides, whereas other\ntrace metals (Cd, Co, Cu, and Ni) are released into the water column\nby both nonreductive but much more so by reductive dissolution processes\n( Figure 2 ). Trace metal\naccumulation rates in a hypothetical waste plume are estimated using\nthe dMn accumulation rate (5.8 nM day –1 ) and trace\nmetal composition during the reductive dissolution of Mn nodules ( Figure 3 f–i). Estimated\naccumulation rates in the ODZ seawater, therefore, are 2.5 ×\n10 –3 nM day –1 for Cd, 1.4 ×\n10 –2 nM day –1 for Co, 2.1 ×\n10 –1 nM day –1 for Cu, and 4.2\n× 10 –1 nM day –1 for Ni. How significant are these accumulation rates of trace metals in\nODZ seawater? Are released trace metal concentrations from the waste\nplume much higher than the background level? The upper bound of concentrations\nof trace metals in seawater that encounter the waste plume occurs\nin the absence of further dilution within the ambient plume. During the ∼100 days of sinking required to pass through\nthe 600 m-thick ODZ ( Supporting Information Text ), the amount of dMn resulting from a single mining waste plume is\nestimated as 1.0 nM (55.0 ng L –1 of Mn) distributed\nuniformly across the ODZ. The most extreme case of continuous waste\ndischarge, in which a plume of MnO 2 particles is reductively\nreduced across the entire thickness of the ODZ, would lead to 580\nnM dMn (equivalent to 31.9 μg L –1 of Mn) released\nduring the sinking time scale under steady state. The estimated upper\nlimit of plume-sourced dMn (1–580 nM) exceeds background dMn\nconcentrations of 0.5–1.0 nM in the ODZ of the Eastern Tropical\nPacific Ocean close to the CCZ by 2 orders of magnitude. 79 Likewise, the input of other dTMs into the mesopelagic\nocean of the CCZ would be 4.3 × 10 –4 to 2.5\n× 10 –1 nM for Cd (4.8 × 10 –2 to 2.8 × 10 1 ng L –1 ), 2.5 ×\n10 –3 to 1.5 × 10 0 nM for Co (1.5\n× 10 –1 to 8.6 × 10 1 ng L –1 ), 3.6 × 10 –2 to 2.1 ×\n10 1 nM for Cu (2.3 × 10 0 to 1.4 ×\n10 3 ng L –1 ), and 7.3 × 10 –2 to 4.2 × 10 1 nM for Ni (4.3 × 10 0 to 2.5 × 10 3 ng L –1 ) ( Table S3 ). The background concentrations of dCd,\ndlCo, dCu, and dNi at the mesopelagic depths of ODZ in the Eastern\nTropical Pacific Ocean are about 0.1–1.1 nM, 0.03–0.1\nnM, 0.6–1.6 nM, and 3–8 nM, respectively. 79 These results suggest that Co or Cu could be\nthe most enriched trace metal within the waste plume compared to the\nbackground seawater concentrations. Concentrations of dlCo, dCu, and\ndNi associated with Mn oxide reduction within the waste plume can\nbe 0.02–15.0, 0.02–13.1, and 0.009–5.2 times\nthe highest concentrations found in the mesopelagic, respectively,\nwhereas dCd concentrations would be diluted by the mining waste discharge\n( Figure 5 ; Table S3 ). Metal release during nonreductive\ndissolution of MnO 2 , such as into water depths outside\nof the ODZ, would be lower and have a slightly different elemental\nprofile (see data from G1 and G2 experiments). The possible\nenrichment of dCu in the waste plume may be a concern\ngiven its strong toxicity to some phytoplankton. 80 − 82 Cu is strongly\ncomplexed by organic ligands in the ocean, 83 and the toxicity of Cu mostly manifests in its inorganic form. Paul\net al. 84 detected high concentrations of\nexcess Cu-binding ligands (up to ∼200 nM) in bottom waters\nand deep-sea pore waters of CCZ sediments, and they argued that excess\nligands could chelate some of the Cu through organic complexation\nand reduce its toxicity to benthic fauna during future mining operations.\nConcentrations of excess organic Cu-binding ligands within the ODZ\nare up to 2 nM in the Eastern Tropical South Pacific, 85 which can only bind part of the Cu 2+ we project\nto be released from the waste plume at mesopelagic depths. How the\nremainder of Cu (up to 18 nM) from the waste discharge plume would\ninfluence the mesopelagic ecosystem remains unclear. For context,\n18 nM of dCu is higher than typical concentrations of Cu in the open\nocean (maximum ∼4–5 nM near the bottom) 79 but falls on the lower end of dCu concentrations in pelagic\nsediment pore waters in the Pacific. 84 , 86 Two aspects\nof added Cu from the waste discharge at mesopelagic depths, beyond\ncomplexation by excess organic ligands, that could supplement the\nfindings from our study are (1) The toxicity threshold of Cu for mesopelagic\necosystems (e.g., zooplankton and fishes) is unknown. Such thresholds\nvary between different plankton communities, 80 , 87 and better baseline studies of Cu ecotoxicology are needed for mesopelagic\ncommunities. (2) The behavioral response of mesopelagic communities\nto added Cu is also unknown. Increased ligand production has been\nreported for both cyanobacteria and eukaryotic phytoplankton in response\nto the addition of high levels of Cu. 82 , 88 It remains\nunclear whether mesopelagic microbial communities would have similar\nresponses, and this also requires a future investigation. This\nstudy provides a geochemical perspective of the influence\nof mining based on current proposals and offers a feasible metal release\nbudget if the mining wastewater were to be discharged into the ODZ.\nImportantly, the trace metal budgets presented here are estimated\nusing a dMn accumulation rate derived from incubation experiments.\nThis rate is likely more representative of dMn accumulation rates\nin the early phase of the waste plume, before any further dilution\noccurs during plume advection (dilution factors up to 40,000–400,000), 75 thereby serving as an upper bound for the waste\ndischarge scenario. The estimated trace metal budget may also change\ndue to its sensitivity to factors such as the choice of dMn accumulation\nrates and the concentrations and compositions of the sediments in\nthe waste plume ( Supporting Information Text ). Future studies on in situ dMn accumulation rates during the transport\nof the waste plume would help better constrain trace metal budgets\nduring mining dewatering operations."
} | 8,852 |
39902321 | PMC11789664 | pmc | 4,883 | {
"abstract": "Background Soil microorganisms are crucial for plant growth, and both plants and their associated rhizosphere microbes are impacted by changes in soil moisture. Inoculation with beneficial fungi can improve bacterial community structure and soil parameters. Aim Under drought stress conditions, the effects of inoculation with Morchella on the physicochemical properties, enzyme activity, and bacterial community structure of the rhizosphere soil of Poa pratensis were studied. Methods High-throughput sequencing was employed to study rhizosphere soil bacterial communities in both Morchella -inoculated and uninoculated Poa pratensis rhizosphere soil subjected to moderate (50% soil moisture) and severe (30% soil moisture) drought stress, as well as under normal water conditions (70% soil moisture). Results Morchella inoculation significantly increased the alkaline nitrogen (AN) and available phosphorus (AP) contents, protease activity (PA), and alkaline phosphatase activity (APA) of Poa pratensis rhizosphere soil. Both Morchella inoculation and drought stress significantly altered the abundance and diversity of the P. pratensis rhizosphere community. The Chao1, Shannon, and Pielou diversity indices decreased with increasing drought stress. The effect of Morchella inoculation was improved under moderate drought stress and unstressed conditions. In addition, Morchella inoculation may help to stabilize the rhizosphere bacterial community under various levels of soil moisture.",
"conclusion": "Conclusions In this study, we conducted an indoor pot experiment to study the effects of Morchella inoculation and drought stress on the P. pratensis rhizosphere community. Morchella inoculation significantly increased the contents of AN and AP and the activities of PA and APA, and decreased the content of AK and the activity of CAT. Both Morchella inoculation and drought stress significantly altered the abundance and diversity of the P. pratensis rhizosphere community. Sphingomonas , RB41 , AKYG587 , Bacillus , Flavobacterium , Pseudomonas , Pir4 , and 10 other genera were the dominant bacterial genera across different treatments. The Chao1, Shannon, and Pielou diversity indices decreased with increasing drought stress. According to PCoA and PLS-DA analyses, Morchella inoculation and water stress both affected bacterial community structure. The effect of Morchella inoculation was improved under moderate drought stress and unstressed conditions. In Morchella -inoculated samples, the abundances of only a few soil bacteria were significantly influenced by moisture levels.",
"introduction": "Introduction Plant growth and developmental processes not only shape plant health but are also essential to the survival of soil microorganisms ( De Vries et al., 2020 ). These soil-level physical, biological, and chemical processes make up the “plant-soil-microbe” trinity ( Das et al., 2022 ). Furthermore, rhizospheric microorganisms strongly influence agricultural production, plant growth, plant drought resistance, and plant disease resistance ( Wang et al., 2019a ; Ahmad et al., 2022 ; Wang et al., 2022 ; Lau & Lennon, 2012 ; Niu et al., 2017 ; Zhang et al., 2021 ). Morel mushrooms ( Morchella spp.) have culinary, therapeutic, nutritional, scientific, and commercial significance ( Du & Yang, 2021 ; Sambyal & Singh, 2021 ). Humans began collecting and consuming wild morel mushrooms in the 19 th century and later transitioned to cultivating them artificially ( Du & Yang, 2021 ; Liu et al., 2022a ). In China, large-scale Morchella farming began around the turn of the century, and there are more than 8,000 acres of Morchella farms today ( Du & Yang, 2021 ; He et al., 2018 ). Morchella cultivation is primarily soil-based and has raised many questions regarding how Morchella cultivation affects soil chemistry, physical structure, and ecology, as well as nearby plant communities. At present, there are relatively few reports on the impact of morel inoculation on plants. In 2021, a study found that intercropping Morchella with peach trees significantly enhanced the physical structure, fertility, and enzymatic activity of the soil, and increased peach yields ( Song et al., 2021 ). The bulk of the soil microbiome associated with Morchella production is reported to be composed of Pedobacter , Pseudomonas , Stenotrophomonas, Flavobacterium ( Benucci et al., 2019 ). Under continuous cropping regimes, an increased richness and diversity of soil microbiome was observed, but the abundance of Lactobacillus and Bacillus exhibited a notable decrease. The reduction in Morchella fruit body yield has been attributed to the enrichment of pathogenic fungi and acidified soil, which has aggravated over the years ( Yue et al., 2024 ). Kentucky bluegrass ( Poa pratensis L.) is a widespread perennial grass that exhibits superior drought tolerance, hardiness, and regeneration qualities, and is commonly used as a turf grass and for animal feed ( Kim et al., 2022 ; Kumar et al., 2022 ). However, little is known regarding the effects of Morchella inoculation and drought stress on the P. pratensis rhizosphere soil’s physicochemical characteristics, enzymatic activity, and bacterial community structure. There have been no reports on the interaction and symbiosis between Morchella and Poa pratensis L. However, during our investigation and research, we found that when collecting Morchella in the wild, Poa pratensis L. often appears near it. Therefore, in response to this discovery, we conducted the following research to verify whether Morchella inoculation has an impact on the growth of Poa pratensis L. Here, we conducted an indoor pot experiment to study how Morchella inoculation treatment and drought stress affects the P. pratensis rhizosphere. We utilized DNA amplicon sequencing of the 16S rRNA gene to study the rhizosphere soil bacterial community. The results presented here will be of great significance for both Morchella and P. pratensis farmers to improve the health of the crops, maximize yields, and ensure operational sustainability.",
"discussion": "Discussion Previous studies have shown that inoculating plants with fungi could yield significant growth-promoting effects. For instance, it is found that inoculation of arbuscular mycorrhiza not only significantly increases the plant height, ear length, ear weight, straw yield, and grain yield of barley, but also improves the plant’s ability to absorb nitrogen, phosphorus, and potassium elements ( Masrahi et al., 2023 ). A soil fungus, JF27 ( Aspergillus terreus JF27), isolated from the rhizosphere of chili peppers, can significantly increase tomato yield and quality, while also enhancing the tomato’s resistance to pathogens ( Yoo et al., 2018 ). The isolation of endophytic fungi in Bromus tectorum revealed that Morchella species could infect plant roots in a non-mycorrhizal manner, demonstrating a mutualistic relationship with this fire-tolerant plant ( Baynes et al., 2012 ). A Morchella crassipes strain collected from post-fire Populus simonii stands exhibited root colonization, significantly enhanced drought tolerance, increased biomass, and improved resistance to Fusarium in sweet corn ( Yu et al., 2016 ). In the current study, the introduction of Morchella resulted in changes in soil nutrients and enzyme activities. Specifically, it led to an increase in AN and AP content, as well as PA and APA activity. Additionally, a decrease in AK content and CAT activity was also observed. This is in agreement with a previous study that found intercropping Morchella with peach trees significantly enhanced soil fertility and structure, including increasing total N, total P, and effective P contents ( Song et al., 2021 ). Changes in soil texture resulting from Morchella inoculation may be the primary driver of these improved soil physicochemical properties. Fungal amendments have been shown to promote soil aggregation and induce changes in soil hydrological properties. Formation of aggregates was found to be positively correlated with fungal biomass, through which soil particles were linked by hyphae ( Angulo et al., 2024 ). The tight relationship among soil texture, nutrient availability, and root exudates may serve as the underlying mechanism responsible for the alterations observed in soil chemical properties and the rhizosphere microbiome after Morchella inoculation of P. pratensis plants ( Adeniji et al., 2024 ). Species richness (Chao1 index) was higher in the Morchella inoculation treatments than in the uninoculated treatments, although the result was not statistically significant. In addition, Chao1 was lower in severely water stressed (30% soil moisture) treatments compared to moderate water stressed (50%) treatments and unstressed (70%). Similarly, in Morchella -inoculated treatments, both the Shannon and Pielou indices decreased as soil moisture decreased. The PCoA analysis of β-diversity showed that Morchella -inoculated P. pratensis rhizosphere soil samples were more evenly distributed along the PCo1 and PCo2 axes, while the uninoculated soil samples were concentrated along PCo1. Overall, the impact of Morchella -inoculation on the α-diversity of rhizosphere bacteria is not significant. This phenomenon is also observed in other studies, with some bacteria increasing in abundance and others decreasing, while the overall change (α-diversity) remains minimal ( Duan et al., 2021 ; Xiong et al., 2014 ). Drought can have a significant impact on rhizosphere microorganisms because changes in soil moisture alter nutritional characteristics, enzyme activity, and bacterial community composition ( Tian et al., 2018 ; Brockett, Prescott & Grayston, 2012 ). The significant impact of drought stress on α-diversity was confirmed in both the Morchella -inoculated and Morchella -uninoculated treatment groups in our study. Across all treatments, the ten most abundant bacterial genera were Sphingomonas , RB41 , AKYG587 , Bacillus , Flavobacterium , Pseudomonas , Pir4 lineage , Pirellula , JGI 0001001-H03 , and Stenotrophobacter . Morchella inoculation tended to decrease the abundance of Bacillus while increasing the abundance of Pseudomonas and JGI 0001001-H03 . In addition, Morchella inoculation may have helped to stabilize the rhizosphere bacterial community under various levels of soil moisture. Even under severe drought stress (30% soil moisture), the abundances of Pseudomonas , Luteolibacter , Pseudarthrobacter , and Arenimonas were substantially higher in X30 than in K30. Under moderate drought (50% soil moisture), Morchella inoculation increased the relative abundances of JGI 0001001-H03 , Stenotrophomonas , Pseudarthrobacter , and Phyllobacterium . Under unstressed conditions, Morchella inoculation increased the relative abundances of JGI 0001001-H03 , Ohtaekwangia , Gemmata , and Pseudarthrobacter . Under both mild drought stress and normal moisture conditions, JGI 0001001-H03 tends to be more prevalent in Morchella -inoculated soils than in uninoculated soils. The introduction of fungi, particularly growth-promoting species, notably influences the structure and composition of the bacterial community within the rhizosphere. For instance, inoculation of Trichoderma harzianum T-63 significantly increased the relative abundance of Pseudomonas , Flavobacterium , and Arthrobacter in the rhizosphere soil of alfafa plants ( Zhang et al., 2019 ). Moreover, Trichoderma -bacteria co-inoculations (usually with Pseudomonas and Bacillus ) often exhibits synergistic effects on plant growth promotion and stress resistance ( Poveda & Eugui, 2022 ). It remains to be further verified whether the changes in the rhizosphere bacterial community caused by the introduction of Morchella are associated with the enhanced drought resistance of P. pratensis . The use of bacterial biofertilizers and inoculants has been shown to significantly decrease the abundance of soil pathogens and increase the abundance of beneficial microbes such as Flavobacterium , Pseudomonas , and Agrobacterium ( Fuentes-Ramirez & Caballero-Mellado, 2006 ; Qin et al., 2017 ; Tao et al., 2020 ; Wang et al., 2019b ). In particular, Pseudomonas has been found to produce growth-promoting hormones, solubilize P, increase soil inorganic nutrients, promote plant growth, and manage plant diseases ( Schillaci et al., 2022 ; Garcia-Salamanca et al., 2013 ; Preston, 2004 ; Hu et al., 2016 ). Furthermore, Pseudomonas induces systemic resistance (ISR) in plants by secreting certain organic compounds and participating in the breakdown of soil macromolecular organophosphorus into small fractions of phosphate esters ( Zhu et al., 2021 ; Olanrewaju, Glick & Babalola, 2017 ; Iavicoli et al., 2003 ; Schuhegger et al., 2006 ). In another study, it was found that the mycorrhizal fungus Suillus luteus tended to increase the abundance of Pseudomonas , resulting in improved soil physicochemical properties, including soil organic matter and total N ( Zhou et al., 2022 ). Fungi can alleviate both abiotic and biotic stresses in plants, such as enhancing drought resistance, enhancing tolerance to high and low temperatures, enhancing salt and acid tolerance, alleviating the toxicity of heavy metals to plants, and improving plant disease and insect resistance ( Zhang, Gu & Duan, 2018 ). Fungal inoculation can promote the transformation of organic matter and accelerate the degradation of nitrogen and phosphorus compounds in soil, providing sufficient nitrogen and phosphorus for plant growth and development ( Wei et al., 2021 ). Notable differences in the mean abundances of RB41 , Nitrospira , and Pseudomonas were observed between soil moisture levels in uninoculated samples. Conversely, in Morchella -inoculated samples, the only notable difference was that of AKYG587 in X30, the abundance of which was significantly lower in X30 than in X50 and X70. Recently, it has been reported that RB41 significantly affects how effectively soil organisms like Acidophilus utilize nutrients. In addition, the addition of fermented food waste to the soil improved RB41 colonization, and RB41 favorably enhanced the nutritional level of the soil ( Meng et al., 2022 ; Liu et al., 2022b ). Nitrospira participates in the nitrogen cycle by converting nitrite into plant-available nitrate. The abundance of Nitrospira in the rhizosphere can be significantly increased, especially under intercropping practices ( Chen et al., 2018 ; Tang et al., 2020 ). The presence of Nitrospira may indirectly enhance plant drought resistance by increasing nitrogen transformation. Recently, a survey of oil palm soil with a low incidence of soil disease showed that it contained higher relative abundances of several beneficial bacterial taxa, including AKYG587 and Calditrichaeota ( Goh et al., 2020 ). In other studies, the relative abundance of AKYG587 increased dramatically after Pseudomonas or Bacillus biocontrol agents were introduced to the soil, and soil health and plant biomass was also significantly improved ( He et al., 2019 ; Zhao et al., 2019 ). Correlation analysis indicated that four environmental variables (AN, AP, APA, and PA) were strongly associated with Morchella inoculation. It was shown that the activities of soil catalase, sucrase, cellulase, and urease are significantly improved by interplanting Morchella and peach trees ( Song et al., 2021 ). Fungal secretions can significantly improve soil microbial structure, enzymatic activity, and N accumulation ( Wang et al., 2014 ; Vázquez et al., 2000 ; Zhang et al., 2024 ). The relative abundance of Bacillus was significantly positively correlated with AK content and CAT activity. A significant positive correlation was found between the relative abundance of Pseudomonas and the contents of AN and AP as well as the activities of APA and PA. As one of the earliest utilized PGPR, Bacillus has been manifested to be effective in promoting the release of soil N and P, enhancing root activity and plant growth, and to accumulate nutrients in the aboveground tissues ( Radhakrishnan, Hashem & Abd_Allah, 2017 ; Liu et al., 2017a ). Pseudomonas and other beneficial bacterial species are relatively abundant in the Morchella rhizosphere soil ( Benucci et al., 2019 ; Pion et al., 2013 ; Liu et al., 2017b )."
} | 4,127 |
36160374 | PMC9488016 | pmc | 4,884 | {
"abstract": "Lack of suitable electron donors or acceptors is in many cases the key reason for pollutants to persist in the environment. Externally supplementation of electron donors or acceptors is often difficult to control and/or involves chemical additions with limited lifespan, residue formation or other adverse side effects. Microbial electrochemistry has evolved very fast in the past years – this field relates to the study of electrochemical interactions between microorganisms and solid-state electron donors or acceptors. Current can be supplied in such so-called bioelectrochemical systems (BESs) at low voltage to provide or extract electrons in a very precise manner. A plethora of metabolisms can be linked to electrical current now, from metals reductions to denitrification and dechlorination. In this perspective, we provide an overview of the emerging applications of BES and derived technologies towards the bioremediation field and outline how this approach can be game changing."
} | 247 |
38431514 | PMC11310912 | pmc | 4,885 | {
"abstract": "Highlights The time is right for targeted engineering of electroactive microbes for electrobioproduction. Current mixed culture microbial electrosynthesis (MES) and microbial electromethanogenesis are limited to a few low-value products. The product spectrum of MES will greatly benefit from engineered microbial pure cultures, especially with regard to high-value products. To quickly advance MES, standardized, off-the-shelf electrobioreactors are urgently needed.",
"conclusion": "Concluding remarks and future perspectives It is time to usher in a new era of electrobiotechnology – the use of flexible and tunable microbial catalysts for the tailored electrosynthesis of high-value compounds in an industrial environment. Opening this new chapter requires a more creative and flexible design of electrosynthetic pathways to generate high-value specialties and commodities with highest efficiency. To achieve this, the generation of highly specific production strains through genetic engineering will be required. Production strains can be based on pure culture isolates with native electroactivity (e.g., through high-throughput screening of promising habitats such as hydrothermal vents) or via engineered electroactivity of biotech workhorses such as E. coli . Simple-to-use and standardized off-the-shelf electrobioreactors will be crucial to enable widespread use and testing of pure cultures catalysts in application-relevant scales and environments, and will be key to transferring this effort to processes. These electrobioreactors need to be as convenient and intuitively operated as those used in other areas of biotechnology. Going beyond these immediate tasks, strategies can be extended to optimize the overall microbial or process performance (see Outstanding questions ). For example, some disadvantages of pure culture applications can be overcome by using defined cocultures. The benefits of collaborative biocatalysts extend from sharing metabolic tasks in the generation of a target product to alleviation of oxidative stress by consuming oxygen and producing reduced metabolites such as H 2 S. New advances in spectroscopic online monitoring enable insight into the individual performance of coculture members and can unlock options for informed control of the biocatalysts to optimize MES performance. For process intensification, for instance, the combination of different microbial catalysts for both targeted anodic and cathodic reactions needs to be investigated more deeply. From an energetic and process-engineering perspective, creating value at both the anode and cathode of an electrochemical reactor is of utmost importance. This can be achieved, for instance by combining cathodic MES from CO 2 with anodic electrosynthesis of hydrocarbons from organic acids. The challenges are not trivial, but with more reliable and reproducible methodology and standardized platforms, as well as reporting of progress, groundbreaking scientific success is certain to follow. Outstanding questions How can we go beyond proof-of-principle? Most current examples of engineered pure culture MES systems target model products without consideration of the economic driving forces. To go from academic exercise to biotechnological application, target products with high economic viability need to be defined and made accessible via MES. The chicken versus egg problem: how can a good biocatalyst be evaluated without proper infrastructure? At this point, the lack of off-the-shelf electrobioreactors greatly limits the degrees of freedom for the larger academic as well as the early-stage industrial biotech field to explore MES. With standardized reactors, testing and benchmarking of new MES concepts will become feasible. Application of redox mediators: can we afford to add external mediators? One great challenge for expanding MES concepts is the lack of electroactivity in established biotech hosts and the metabolic inflexibility of native electroactive microbes. As a solution, the application of externally added redox mediators has long been suggested. However, despite the additional costs of the addition and especially recovery of the mediator during downstream processing, this approach only makes sense if the reaction benefits from pure electron balancing (discharge or uptake). If the MES reaction needs to be wired into the metabolism of the host cell, the addition of external mediators alone will not be sufficient. Can metabolic and technical hurdles be overcome by using defined cocultures? For many detailed aspects of MES function, the strategy of defined cocultivation might provide a valid option to overcome performance and compatibility challenges: combining an electroactive specialist that provides a simple fuel product with a bioproduction specialist; combining an oxygen-sensitive MES specialist with an oxygen scavenger; or combining a cathodic MES specialist with an anodic MES specialist for 200% cell MES (synthesis at both electrodes). Alt-text: Outstanding questions"
} | 1,250 |
35368716 | PMC8958488 | pmc | 4,888 | {
"abstract": "As global ocean temperatures continue to rise, severe declines in coral reef health and diversity are reported on a global scale. Recovery of coral reefs relies on reproduction and increased rates of successful recruitment, which can vary tremendously across coral species. We investigated the effects of increased temperatures in the environment of parental colonies on larval production, size, settlement and survival, in the heat-resistant coral Leptastrea purpurea in Guam . Thanks to two tank experiments (eleven and four weeks, respectively) conducted over two consecutive years we found that larvae released by heat-treated parents (30 °C) were significantly smaller in size but greater in number, had normal settlement behavior and increased post-settlement survival rates compared to those released by control parent colonies (28 °C). We conclude that changes in the environment of parental L. purpurea colonies trigger an anticipatory maternal effect which leads to the release of preconditioned larvae with an increased chance of survival. Supplementary Information The online version contains supplementary material available at 10.1007/s00338-022-02241-y.",
"introduction": "Introduction Increases in sea surface temperature have led to severe declines in coral reef health and diversity on a global scale (Spalding and Brown 2015 ; Hughes et al. 2018 ; Smale et al. 2019 ). Between 2013 and 2017, consecutive worldwide mass coral bleaching events struck reefs around the island of Guam and resulted in losses of up to 60% in coral cover (Raymundo et al. 2019 ). Coral bleaching can have various consequences for corals and their surrounding ecosystems in both the short and long term. During and shortly after bleaching, corals often experience shifts in Symbiodiniaceae composition, compromised photosynthetic efficiency, reduced skeletal growth, and are more susceptible to disease and predation (Lesser 2006 ; Baird et al. 2009 ). In the long term, corals can exhibit increased risk of partial and full mortality as well as decreased fecundity and growth (Ward et al. 2002 ). Additionally, a coral depleted reef may be overtaken by algae and experience shifts in fish community composition potentially reducing fishery productivity (Graham et al. 2007 ). Recovery of coral reefs, including the rebuilding of depleted adult populations and maintaining resilience in the face of increasing environmental pressures, relies heavily on reproduction and increased rates of successful recruitment (Holbrook et al. 2018 ). However, coral reproduction, settlement and recruitment can all be affected by thermal stress to various extents depending on the species. For example, increased temperatures may lead to a decrease in larval survivorship and settlement in spawning corals, particularly in Acroporids (Hughes et al. 2019 ), as well as in the brooding coral Favia fragum (Randall and Szmant 2009 ). Interestingly, Cyphastrea japonica , Favites and Acropora millepora species show increased larval development coupled with increased larval mortality rates at higher temperatures (Figueiredo et al. 2014 ). In Porites astreoides, elevated seawater temperature can affect larval motility or reduce photosynthesis and energy supplies, ultimately causing an increase in larval mortality (Edmunds et al. 2001 ). Elevated temperatures can also shorten the pelagic larval phase and skew larval settlement from their preferred to a less suitable substrate in Stylophora pistillata (Putnam et al. 2008 ). Few studies have identified examples of trans-generational acclimation to stressful environmental conditions through anticipatory parental or maternal effects in corals (Putnam and Gates 2015 ). This rapid-response mechanism assumes that parents or mothers shift the allocation of resources to the offspring when brooded in a stressful environment to increase offspring fitness (Crean and Marshall 2009 ). Corals that can pass on greater heat tolerance to their offspring may adapt better to the predicted environmental conditions of the future and dominate future reefs. Given the variation in corals’ response to heat stress, broadening the number of studied scleractinian species is essential to refine our understanding of coral reproduction under climate change, and better predict recovery patterns after disturbance events (McLachlan et al. 2020 ). Here, we focused on Leptastrea purpurea , an encrusting coral that forms small colonies and is commonly found throughout the Indo-Pacific region (Veron 2000 ; Arrigoni et al. 2020 ). In Guam, L. purpurea is found in abundance on back-reefs (Nietzer et al. 2018 ). Leptastrea purpurea is a brooding coral, producing and releasing larvae daily, which makes it ideal for reproduction experiments (Nietzer et al. 2018 ). Like most other corals, L. purpurea larvae settle preferably on crustose coralline algae (CCA) and larval metamorphosis is induced in the presence of the CCA Hydrolithon reinboldii (Nietzer et al. 2018 ). Dispersal abilities of L. purpurea larvae are unknown and could vary from a couple hundred to thousands of meters. In fact, larvae of the brooding coral P. astreoides , can travel distances up to 1,900 km in favorable current conditions (Serrano et al. 2016 ), while the larvae of brooding Heliopora coerulea can only disperse throughout their natal reef, within 350 m of parent populations (Harii et al. 2002 ). Leptastrea purpurea is relatively resistant to thermal stress and there is anecdotal evidence of a surge in larval production in response to sea surface temperature increase (van Woesik et al. 2011 ; Nietzer et al. 2018 ). Over the course of four years of survey during recurrent bleaching events (2013–2017) on Guam, L. purpurea was recorded to experience bleaching, but showed no mortality despite significant losses in other coral species. In addition, 2017 surveys recorded new recruitment and growth of L. purpurea colonies (Raymundo et al. 2019 ). In the present study, we investigate the effects of parental heat treatment on larval production and fitness (quantity, size, settlement and recruit survival rates) in order to better understand the effects of increased seawater temperature on L. purpurea reproduction. On small islands such as Guam, understanding coral reproduction is vital to predicting future coral assemblages after disturbances and restoring healthy ecosystems.",
"discussion": "Results and discussion Heat-treated parents release more but smaller larvae Leptastrea purpurea is known to be a bleaching-resistant species (Bahr et al. 2016 ; Raymundo et al. 2019 ) and both our experiments confirmed that adult colonies can sustain medium to long-term heat stress without showing external signs of bleaching. In both experiments, the average tissue color of heat-stressed colonies remained as dark as the average tissue color of the control colonies (Fig. 2 , Supplementary Table 1). Fig. 2 Average coral tissue color of heated and control parental colonies for Experiment 1 ( a ) and Experiment 2 ( b ). Color was measured by visual comparison to values on a CoralWatch Coral Health card (Siebeck et al., 2006 ), where 6 is the darkest hue and 0 corresponds to white or bleached tissue. None of the comparisons between control and heat-treated colonies are significant The reproductive consequences of heat stress in corals usually include decrease in gametogenesis, egg size, fecundity (the number of oocytes per polyp), and spawning (release of gametes or larvae; Szmant and Gassman 1990 ; Mendes and Woodley 2002 ; Cox 2007 ). However, over the course of both experiments, heat-treated colonies released significantly more larvae than the controls (p < 0.001; Table 1 , Fig. 3 a, b, Supplementary Figure 1; Supplementary Table 2). Noticeably, throughout both experiments the number of larvae released in both treatments decreased with time (Table 1 , Supplementary Figure 2) most likely caused by a combination of handling-induced stress and life in captivity, as reported previously (Nietzer et al. 2018 ). During Experiment 1, heat-treated colonies released a total of 234 larvae (3.7 larvae/day on average), whereas the control colonies only released 115 larvae (1.8 larvae/day) over 64 days of collection. During Experiment 2, the heat-treated colonies released a total of 55 larvae (3.7 larvae/day), while the control colonies only released 25 larvae (1.7 larvae/day), over 15 days of collection. We noticed that the increased time between larval collections in Experiment 2 (8 days vs. 3 days in Experiment 1), which could allow some larvae to either escape or settle in the tank prior to collection day, did not affect the total number of larvae collected across the two experiments. Only a few other species of brooding corals such as S. pistillata , Pocillopora damicornis or Echinopora lamellosa are known to release larvae when temperatures are within their upper survival threshold (Fan and Dai 1999 ; Edmunds et al. 2011 ; Crowder et al. 2014 , 2017 ). These studies showed that elevated temperatures accelerate gametogenesis and the timing of planulae release in coral species that follow a reproductive cycle (e.g., lunar, monthly or seasonal, etc.; Crowder et al. 2014 ). Here, we show that elevated temperatures can also influence planulation in species that release larvae in a non-seasonal and non-cyclic manner, like L. purpurea (Nietzer et al. 2018 ). In the context of climate change, understanding how elevated temperatures affect reproduction in various coral species is essential to better predict patterns of reproductive success and colonization. Table 1 Summary of data collected in Experiments 1 and 2 Nb Larvae Collected Average Larvae Size Total Nb Larvae Settled Total CCA Plastic Experiment 1 Control 115 / / / / Heat treated 234 / / / / Experiment 2 Control Batch 1 15 0.7 (SE = 0.05) 15 8 7 Control Batch 2 6 0.49 (SE = 0.02) 6 4 2 Control Batch 3 4 0.59 (SE = 0.08) 4 4 0 Control Overall 25 0.64 (SE = 0.04) 25 16 9 Heat-treated Batch 1 21 0.55 (SE = 0.03) 20 10 10 Heat-treated Batch 2 18 0.52 (SE = 0.04) 18 15 3 Heat-treated Batch 3 16 0.54 (SE = 0.04) 14 13 1 Heat-treated Overall 55 0.54 (SE = 0.02) 52 38 14 Fig. 3 Larval counts and sizes. Average larval count by treatment and experiment. a when collected once every 3 days during Experiment 1, b when collected once every week during Experiment 2. c Average size of larvae released by control and heat-stressed colonies of Leptastrea purpurea during Experiment 2 The average size of larvae released by heat-treated colonies over all collection batches was significantly smaller than that of the larvae obtained from the control colonies (p < 0.05; Table 1 , Fig. 3 c, Supplementary Table 3). Average sizes per batch were also smaller for larvae released by heat-treated colonies compared to controls but this difference was only significant in the first batche (which is also the batch that produced the largest number of larvae; Table 1 , Supplementary Figure 2 and Table 4). Under stressful environmental conditions organisms may produce smaller eggs or offspring; this phenomenon has been documented in several marine invertebrates including polychaetes, bryozoans, the brooding coral P. damicornis and other broadcast spawning corals (summarized in Putnam and Gates 2015 ). There is also evidence that larvae released early within the spawning cycle of the brooders P. damicornis , P. asteroides and Seriatopora caliendrum are smaller in size than larvae released later (Putnam et al. 2010 ; Cumbo et al. 2012 ; de Putron et al. 2017 ; but see Zhang et al. 2019 ). This observation combined with the small larvae released by heat-treated L. purpurea colonies, could be another indication that elevated temperatures accelerate either gametogenesis or the timing of larval release in this species. Smaller larvae have lower lipid content, higher respiration rates and larger energy demands which leads to an accelerated development (Harii et al. 2002 ; Edmunds et al. 2011 ; Putnam and Gates 2015 ). Our results suggest that in response to a potentially stressful environment, L. purpurea colonies modulate their larval number and size most likely to increase settlement success. The phenotypic plasticity observed in larval number and size in L. purpurea could be attributed to an anticipatory maternal effect (Marshall and Uller 2007 ). In response to a change in the environment, anticipatory maternal effect allows mothers to increase offspring fitness with regards to the predicted new environment; thus, increasing offspring’s chance of survival (Marshall and Uller 2007 ). In our context this means that the smaller and more numerous larvae produced by L. purpurea colonies in elevated temperatures should fare better in warmer water as well, which we did not test for in the present study. However, this could explain why the number of recruits of L. purpurea increased after the Guam 2017 bleaching event caused by increased seawater temperatures (Raymundo et al. 2019 ). In the context of global climate change, corals that are capable of anticipatory maternal effects have a clear advantage over other species (Crean and Marshall 2009 ). Further studies aiming at exploring this effect in stress-resistant corals species will help us better predict the future composition of coral reefs. Parental heat-treatment does not affect larval settlement but increases survival of recruits Despite being smaller in size, the larvae released by heat-treated parental colonies settled at similar rates as larvae released by control colonies (63.6% and 68% after 72 h, respectively, p = 0.05; Table 1 , Fig. 4 a). When the settlement assays were interrupted due to the covid19 pandemic, 94.5% of the larvae released by heated colonies had settled versus 100% for the control larvae (p = 0.9; Fig. 4 a). Similarly, substrate preference was not affected by the parental treatment. Within both treatment groups, larvae significantly preferred to settle on CCA over the plastic wells (p < 0.05; Fig. 4 b, 4c; Supplementary Table 6). Overall, 64% and 73% of control and treated larvae settled on CCA, respectively. Moreover, larvae produced by heat-treated parents settled on CCA over plastic at the same rate than larvae produced by control parents (p > 0,05; Fig. 4 b; Supplementary Table 5). Elevated temperatures can cause larval developmental aberrations or larvae that do not properly settle or mature into adulthood after settling ( Diploria strigosa Bassim and Sammarco 2003 ; Fungia scutaria Schnitzler et al. 2012 ). The similarity in settlement rates and substrate choice between the two larval types under ambient temperatures highlights the fact that increased temperatures during reproduction of L. purpurea do not have detrimental effects on larval settlement in this species. This remarkable observation most likely contributes to explaining why this species was reported to be mildly affected by recent and recurrent thermal anomalies (Raymundo et al. 2019 ). Fig. 4 Larval settlement (Experiment 2). a Percent settlement of larvae released by heat-stressed and control parental colonies, after 72 h. and overall, b Preferred settlement substrate of larvae released by heat-treated and control parental colonies, c Photograph of an 11-day old recruit on CCA Interestingly, batch 1 larvae released by heat-treated colonies tended to have a significantly higher survival rate post-settlement than controls when observed under ambient seawater temperatures (p < 0.0001, Fig. 5 , Supplementary Table 7). The differences in survival rate between larvae released by heat-treated and control parents were, however, not significant for batches 2 and 3 (p = 0.25 and p = 0.99, respectively). We suspect that the absence of significance between treatments in batches 2 and 3 is due to the combined reduced amount of time we could run the survival experiment (3 weeks and 2 weeks, respectively) and the reduced number of larvae produced by both batches that made it to the survival experiment (24 and 18, respectively) compared to batch 1 (which ran for 4 weeks with 35 larvae total). Brooded embryos and larvae may be able to acclimate to the environmental conditions their parents experienced during their development within the parental colony, a phenomenon otherwise called preconditioning (Putnam and Gates 2015 ). Our experiment showed that despite being preconditioned to high temperatures, larvae produced by heat-treated parents are not at any significant disadvantage compared to control larvae when exposed to a different and less stressful environment than the one in which they were initially released in. In the longest survival experiment (i.e., 4 weeks), recruits from heated-treated parents actually displayed a significant advantage compared to controls, which raises the question of whether these preconditioned recruits might in fact have a higher fitness regardless of the thermal environment they found themselves in. There is evidence that some corals modify their endosymbiotic Symbiodiniaceae community in response to thermal stress and transfer these modifications to their offspring, increasing the offspring’s chance to acclimate to their environment (Quigley et al. 2019 ). Additional research should be conducted to verify if L. purpurea has this ability and whether the increased survival rate of offspring released by heat-treated parents is a trend that sustains itself through time and in elevated temperatures. Nevertheless, our research seems to suggest that parental effect (whether anticipated maternal effect or preconditioning) may contribute to rapid transgenerational changes in L. purpurea. Fig. 5 Larval survival (Experiment 2). Kaplan–Meier survival curves of Leptastrea purpurea recruits produced by control (black) and heated (gray) parental colonies Overall, our results provide empirical evidence that three to eleven weeks of increased seawater temperature did not have detrimental effects on the reproduction of adult L. purpurea corals or on larval settlement and survival. Instead, elevated temperature increased reproduction and survival of recruits. We also did not observe any signs of bleaching in adult L. purpurea corals. These notable results help explain the observed absence of significant mortality rates and rapid increase in numbers of recruits reported for L. purpurea colonies after the widespread bleaching event of 2017 that caused a 30% to 60% decrease in coral cover on Guam (Raymundo et al. 2019 ). It is important to note that although we collected corals of about the same size (all between 8 and 10 cm 2 ), we did not specifically control for surface area (number of polyps capable of releasing larvae) or genotype when distributing corals in the experimental tanks. As a result, some of the observed difference between treatments could be attributed to coral size or other factors such as genotype. Nonetheless, our findings reveal that adult L. purpurea colonies can unequivocally display plasticity in their reproduction strategy and that these changes can happen very rapidly. Whether this plasticity is beneficial over the long term (for adults, larvae, and recruits) still needs to be confirmed with long-term experiments. Furthermore, understanding and characterizing levels of reproductive plasticity across various coral species is a pressing question because plasticity can determine species chances to survive in an ever and fast changing environment (Via et al. 1995 ). Despite having a large geographical distribution spanning the whole Indo-Pacific, L. purpurea are not major reef-builders. Instead, they are encrusting corals capable of growing on fragments of dead coral skeletons or other hard and bare substrates. As seawater temperatures continue to rise, the need to increase the scope of studied coral species beyond the more commonly studied reef-builders (e.g., Acroporids, Poritids or Pocilloporids) becomes more pressing, as less common, and smaller species might dominate future reefs."
} | 5,034 |
35136559 | PMC8809449 | pmc | 4,890 | {
"abstract": "Abstract Kelp forests are in decline across much of their range due to place‐specific combinations of local and global stressors. Declines in kelp abundance can lead to cascading losses of biodiversity and productivity with far‐reaching ecological and socioeconomic consequences. The Salish Sea is a hotspot of kelp diversity where many species of kelp provide critical habitat and food for commercially, ecologically, and culturally important fish and invertebrate species. However, like other regions, kelp forests in much of the Salish Sea are in rapid decline. Data gaps and limited long‐term monitoring have hampered attempts to identify and manage for specific drivers of decline, despite the documented urgency to protect these important habitats. To address these knowledge gaps, we gathered a focus group of experts on kelp in the Salish Sea to identify perceived direct and indirect stressors facing kelp forests. We then conducted a comprehensive literature review of peer‐reviewed studies from the Salish Sea and temperate coastal ecosystems worldwide to assess the level of support for the pathways identified by the experts, and we identified knowledge gaps to prioritize future research. Our results revealed major research gaps within the Salish Sea and highlighted the potential to use expert knowledge for making informed decisions in the region. We found high support for the pathways in the global literature, with variable consensus on the relationship between stressors and responses across studies, confirming the influence of local ecological, oceanographic, and anthropogenic contexts and threshold effects on stressor–response relationships. Finally, we prioritized areas for future research in the Salish Sea. This study demonstrates the value expert opinion has to inform management decisions. These methods are readily adaptable to other ecosystem management contexts, and the results of this case study can be immediately applied to kelp management.",
"conclusion": "5 CONCLUSION Our use of expert opinion and a structured literature review resulted in a comprehensive framework to support management decision‐making despite a paucity of local data. Ultimately, management outcomes will depend on a number of external factors but by utilizing multiple, informed lines of evidence to inform management decision making one greatly increases the chances of a positive outcome. The complexities of modern anthropogenic stressors on nearshore environments require a diverse suite of approaches to identify relevant pathways and to prioritize knowledge gaps for additional quantitative research. By gathering a focus group of relevant experts on the Salish Sea, we were able to rapidly diagram the multiple stressor pathways that are likely contributing to regional kelp decline and use this diagram to inform a systematic literature survey that was then used to identify critical knowledge gaps to direct future research efforts. This targeted, multistage approach allowed us to resolve complex linkages that otherwise would have been missed by using only a single approach. The results inform future research directions while also providing a tool managers can use in the absence of regional quantitative data. Kelps provide important habitat in the Salish Sea, and the loss of this habitat will likely have cascading impacts on other fish, invertebrate, and mammal species that are part of nearshore food webs and the humans that rely upon them. The approach developed here can be extended to other ecosystem‐based management decision‐making processes where quantitative data are lacking, and expert opinion can be incorporated in a more standardized way by linking directly to a conceptual model of the system. Managing and restoring threatened ecosystems such as the Salish Sea, which are under increasing pressure from both the influences of climate change and human intervention, will require us to draw upon both qualitative and quantitative data and expert opinions from many different sources in order to best manage these complex and dynamic ecosystems.",
"introduction": "1 INTRODUCTION Coastal marine ecosystems are experiencing unprecedented changes due to climate variability and other human activities (e.g., vessel traffic, upland and nearshore development, and alterations of trophic structure), posing a significant challenge for resource managers and decision makers (Crain et al., 2009 ; Harley et al., 2006 ; Hewitt et al., 2016 ). Species found in shallow coastal environments can be especially vulnerable to the cumulative effects of human modifications to the environment, despite adaptations to disturbance often observed in variable nearshore regions (Crain et al., 2008 ; Jordan et al., 2009 ; Peterson & Lowe, 2009 ; Thrush et al., 2021 ). These coastal environments often provide critical habitat for ecologically, economically, and culturally important species; therefore, effective management to assure the sustainability of these habitats and the ecosystem services they provide is paramount (Erlandson et al., 2015 ). Kelp forests are among these important coastal ecosystems that provide critical ecosystem services (e.g., carbon sequestration, primary productivity, erosion control) and habitat for important life stages of fishes, invertebrates, and marine mammals (Calloway et al., 2020 ; Duggins et al., 1989 ; Krause‐Jensen & Duarte, 2016 ; Teagle et al., 2017 ). In recent decades, kelp forest ecosystems have suffered widespread declines across much of their range (Filbee‐Dexter & Wernberg, 2018 ; Krumhansl et al., 2016 ; Smale, 2020 ; Wernberg et al., 2019 ). The drivers of these declines differ by place and include climate change‐amplified marine heatwaves, eutrophication, altered trophic structures, and shoreline development, among other anthropogenic stressors (Bischof et al., 2019 ; Halpern et al., 2019 ; Rogers‐Bennett & Catton, 2019 ; Smale, 2020 ; Figure 1 ). These drivers can affect multiple life‐history stages of kelps and may interact to reduce growth, reproduction, and survival of individual kelps and their populations. The impacts of these stressors may also depend on the strength and timing of the impacts and the functional role of different kelp species in the ecosystem: While some species float toward the surface and create upright, buoyant canopies, others remain close to the benthos. Additionally, kelps have a biphasic life history composed of micro‐ and macroscopic stages, each of which may respond differently to stressors (Figure 2a ). Regardless of which functional groups make up a given kelp forest, the macroscopic stages create complex, three‐dimensional habitats that form the structural and energetic bases for an abundance of life (Teagle et al., 2017 ). Declines in kelp populations can therefore have large and cascading impacts on ecological and human communities (Graham, 2004 ; Shaffer et al., 2020 ). FIGURE 1 Stressors impacting nearshore kelp forest ecosystems. Figure art by Su Kim FIGURE 2 (a) Bull kelp life cycle, and (b) the proportion of studies identified by stressor and life stage (green represents zoospore, orange—gametophyte, pink—juvenile sporophyte, and blue—adult sporophyte). Numbers in each pie chart indicate the number of studies found A region of particularly high kelp species diversity is the Salish Sea (Druehl, 1970 ), a fjordal system of inland waterways straddling Washington State (U.S.) and British Columbia (Canada). There have been 21 species of kelp identified within this region, with the bull kelp ( Nereocystis luetkeana ) as the primary floating canopy‐forming species, while the majority of species lie within a few meters of the bottom. Most kelps in this region grow as small forests along a narrow depth band near the shore where they are exposed to large seasonal swings in temperature and salinity. These kelp forests provide critical habitat for threatened or endangered fish and invertebrate species, including Pacific salmon ( Oncorhynchus spp.), rockfish ( Sebastes spp.), herring ( Clupea pallasii ), and abalone ( Haliotis kamtschatkana ) (NMFS, 2005 , 2014 ). Recently quantified declines in the extent of kelp forests in Puget Sound raised concerns regarding the availability of critical habitat for these threatened species which motivated the creation of the Puget Sound Kelp Conservation and Recovery Plan (Berry et al., 2021 ; Calloway et al., 2020 ). Although the drivers of the declines remain unclear, they are likely the result of cumulative effects from multiple natural and human stressors on the system such as increasing sea surface temperatures and incidences of marine heatwaves (Iwabuchi & Gosselin, 2019 ; Masson & Cummins, 2007 ), changes to watersheds and nearshore terrestrial environments (Hansen et al., 2013 ), and changes to marine ecological communities (Pietsch & Orr, 2015 ; Zier & Gaydos, 2016 ). Mapping efforts in other regions of the Salish Sea found kelp population trends were stable or slightly declining, suggesting that stressor intensity and impact varies across basins (Pfister et al., 2018 ; Schroeder et al., 2020 ), but differences in the spatial and temporal scales of these studies make comparisons difficult. The level of data required to quantitatively model the cumulative impacts of multiple stressors on ecosystems such as kelp forests can rapidly surpass available resources (Foley et al., 2017 ). To overcome this challenge, expert knowledge is increasingly being used as a valuable data source in modeling ecosystem processes, answering management questions, and forecasting the impacts of disturbance. For example, Reum et al. ( 2019 ) used diverse expert and stakeholder input to assess management options to rebuild a collapsing fishery in the presence of ongoing climate change; and Stier et al. ( 2017a , 2017b ) quantified how perceptions of food webs based around Pacific herring differed among scientific, local, and traditional knowledge experts. Expert knowledge is an especially valuable data source when modeling complex systems with interacting stressors for which there is little experimental or observational data to build purely quantitative models (McBride & Burgman, 2012 ). When used in conjunction with quantitative approaches, expert knowledge can guide future research so that limited available resources can focus on the most critical data needs. In addition to modeling complex ecological processes in data‐poor systems, this approach builds communication among stakeholders and increases transparency in decision‐making processes. This is critical because increased stakeholder participation in management decisions promotes support for management actions and successful implementation, as was seen in the design and implementation of marine protected areas in California (Fletcher et al., 2014 ). One way to organize conceptual and empirical understandings of complex coastal ecosystems is the DPSIR (Drivers–Pressures–State–Impact–Response) framework (Lewison et al., 2016 ). The DPSIR framework links ultimate and proximate causes to changes in state variables and allows resource managers to assess the relative impacts and responses of potential management strategies (Turner, 2000 ). The main components of the model are as follows: (1) Drivers—human activities with an environmental effect (indirect stressors); (2) Pressures—direct positive and negative effects of the Drivers on the environment (direct stressors); (3) State—the condition of the environment; (4) Impact—the effect of the Pressures, measured as the change in State; and (5) Response—policies, interventions, or management priorities adopted to improve the State (Kristensen, 2004 ). A major strength of the DPSIR methodology is its flexibility, which allows for the use of quantitative data, when available, or expert opinions in the absence of quantitative data. The DPSIR framework has been used to organize understandings, identify research needs, and support management decisions in a number of complex social–ecological systems (Lewison et al., 2016 ), including recent applications to global microplastic pollution (Miranda et al., 2020 ), fisheries management in Kenya (Dzoga et al., 2020 ), and ecotourism in Thailand (Suursaar & Kornpiphat, 2021 ). In an effort to fill existing knowledge gaps for Salish Sea kelp ecosystems to inform management decision‐making, we undertook a multistep process. First, we brought together a group of diverse experts from academic institutions and federal, regional, and Indigenous governments in Washington and British Columbia to map the direct and indirect stressors believed to be contributing to kelp decline in the Salish Sea. We used a modified DPSIR framework to organize how experts identified direct and indirect stressors on kelp populations. We then conducted a comprehensive literature review of each stressor identified by the experts, focusing on both regional research in the Salish Sea and related work in global temperate marine ecosystems. In the course of the literature review, we identified research gaps and limitations in local data to guide and prioritize future research efforts. The development of these linkages and the information from the literature review could help drive subsequent semiquantitative analyses, such as qualitative network models or Bayesian belief networks, that evaluate how important each direct and indirect linkage is between Drivers, Pressures, and the State of kelp populations (Hollarsmith et al., 2021 ). By combining both expert opinion and a comprehensive and structured literature review, we were able to create a robust analysis to inform management despite local data gaps.",
"discussion": "4 DISCUSSION In management scenarios where data are limited, it is common to elicit the advice and opinions of regional experts to provide the best available science for the management decision‐making process, particularly when related to questions concerning how ecosystems or habitats may respond to natural and anthropogenic pressures (Donlan et al., 2010 ; Martin et al., 2012 ; Ryder et al., 2010 ; Turner, 2010 ). Here, we invited researchers and resource managers to develop an inclusive conceptual model of the pressures, and ultimately human activities, that affect the status and trends (State) of kelp in the Salish Sea. This work was motivated by disturbing disappearances of bull kelp forests in Puget Sound, and the paucity of local quantitative information to explain this decline (Berry et al., 2021 ). Losses of these and other kelp forests in the Salish Sea could negatively impact the availability of nearshore habitat to commercially and ecologically significant species (Teagle et al., 2017 ), while also reducing the productivity of nearshore environments (Duggins et al., 1989 ). Consequently, management actions that facilitate the recovery and conservation of kelp forests in the Salish Sea would increase the provisioning of ecosystem services and ensure the long‐term functioning and productivity of coastal ecosystems. It is important to note that another set of individuals from a greater diversity of the general public, including individual citizens, local stakeholders, and more representation from tribes and First Nations may have developed different models (Reid et al., 2020 ; Ressurreição et al., 2012 ; Rosellon‐Druker et al., 2019 ; Stier et al., 2017 ). The combination of this focus group's conceptual model and the relatively consistent support for these pathways found in the literature review suggests expert perceptions of the system are a good starting point for understanding the dynamics important to informing the decision‐making process for conservation and management of kelp in the Salish Sea. The validation of this conceptual model, in addition to quantifying the strength of directionality in relationships, may provide the foundation for predicting anthropogenic impacts on kelp forests in the Salish Sea using semiquantitative and quantitative modeling techniques that could give further insight into the relative importance of each linkage on kelp forest persistence (Hollarsmith et al., 2021 ). However, further inclusion of regional stakeholders and the general public in participatory processes related to this conceptual model and specific management actions will ensure other nodes of the social–ecological system are accounted for in the decision‐making process (Dietz, 2013 ; Stier et al., 2017 ). Overall, we found considerable support in the literature for a majority of the Driver–Pressure–State pathways identified in the conceptual model developed by the expert‐based focus group. However, the vast majority of supporting studies were based on research performed outside of the Salish Sea region, 87% for Driver‐to‐Pressure pathways and 96% for Pressure‐to‐State pathways. The Salish Sea is an oceanographically diverse and complex set of inland waterways with estuarine‐style circulation patterns that leads to net seaward flow of brackish surface layers and net landward flow of deep, dense oceanic waters (Alford & MacCready, 2014 ; Babson et al., 2006 ; Masson, 2002 ). There are numerous sills that constrict and alter geomorphological and oceanographic processes that isolate specific regions at various temporal and spatial scales. These characteristics may impose environmental conditions for kelp that are dissimilar from other coastal kelp habitats where much of our mechanistic understanding of these Driver–Pressure–State relationships have been studied. The lack of data to support these relationships directly may limit the specificity of advice for the conservation and management of kelp in this region. Notably, however, we found generally high consensus in directional relationships between the Salish Sea and global literature, so the results from the global literature may be a good approximation of processes in the Salish Sea. While we found multiple studies to support the impacts of expert‐identified Pressures on various kelp species, these studies were not evenly distributed across the stages that comprise the complex life cycle of kelp. The vast majority of studies focused on the adult sporophyte stage of kelp, which is the stage that provides the most three‐dimensional habitat structure and organic carbon to the kelp forest ecosystem. However, the earlier microscopic life stages may be an important and largely invisible bottleneck in the kelp reproductive cycle (Hollarsmith et al., 2020 ; Muth et al., 2019 ). For pathways that had studies on multiple life‐history stages, there was a high degree of consensus about the direction of the impact, with the exception of water clarity, which was largely positively related to sporophyte performance metrics. The only study investigating other life stages found that for populations from turbid areas, water clarity did not impact gametophytes (Gerard, 1990 ). Generally, this suggests that results for one life‐history stage may be able to be cautiously extrapolated to other stages; however, more research on environmental impacts to spore, gametophyte, and microscopic sporophyte stages is warranted. This literature review was designed to evaluate the pathways identified in the focus group's conceptual model, not to seek out any missing pathways; however, during our keyword searches, we did identify four driver‐to‐pressure pathways and two pressure‐to‐kelp pathways that did not fit into the expert‐identified pathways and that may need to be considered going forward. First, the human activity of net‐pen aquaculture was identified in the broader temperate coast literature searches as increasing nearshore contamination, benthic sedimentation, and nutrients; and decreasing water clarity (Claudet & Fraschetti, 2010 ; Feng et al., 2004 ; Lalonde & Ernst, 2012 ; Wang et al., 2020 ). Second, invasive algal species, included under the category of human impacts to trophic structures, may enhance benthic sedimentation rates (Bulleri et al., 2010 ). Third, temperature has been found to be positively related to epiphyte growth (Werner et al., 2016 ); and fourth, shoreline development can alter nearshore substrate (Dethier et al., 2016 ). We also found that viral disease can negatively impact kelp growth and survival (Beattie et al., 2018 ), which may not be currently affecting kelp in this region but could represent a future threat, considering viral outbreaks have recently affected other taxa in the region (Hewson et al., 2014 ). Another omission was the direct impact of water motion, currents, and wave action on kelp performance, which was included as a mediator between drivers and pressures in the initial diagram. We encountered evidence that suggests it has a direct impact on kelp (Berry et al., 2021 ; Kregting et al., 2016 ; Millar et al., 2020 ; Peteiro & Freire, 2013 ; Starko et al., 2019 ). There are likely other direct or indirect pathways not identified by the expert‐based conceptual model that the literature search also did not capture as the perceptions, knowledge, and biases of experts can vary widely, even within the narrow demographic range of ‘kelp experts’ used in this study (Drescher et al., 2013 ; Martin et al., 2012 ; Stier et al., 2017 ). 4.1 Research priorities for the Salish Sea While studies from the global literature may serve as effective approximations of processes in the Salish Sea, the extreme paucity of literature on pressures impacting floating and nonfloating kelp species in the region indicates an urgent need for research to inform local resource management decisions for kelp conservation and recovery. Situated in a temperate rainforest and composed of deep fjords and large glacial‐fed estuaries, the oceanography of the Salish Sea is distinct from many of the other regions represented in our global temperate literature search. The estuarine environment is unusual for kelp, with periodic or seasonal changes in salinity, temperature, turbidity, and other water column parameters that are often much larger than observed in open coast environments where most kelps are found (MacCready et al., 2021 ). Research has shown that kelps can exhibit population‐level differences in response to environmental stress (Buschmann et al., 2004 ; Flukes et al., 2015 ; Hollarsmith et al., 2020 ; King et al., 2019 ), and recent population genetic work on bull kelp in the Salish Sea revealed distinct genetic clusters that aligned with oceanographic currents, geographic and benthic features, and environmental variables (Gierke, 2019 ). Evidence for genetic structure further supports the need for more research on Salish Sea kelp populations to more accurately understand current and future changes in kelp extent across the different basins. Human actions that are managed at the local level, such as nearshore and upland development and regional fisheries, are some of the Drivers that most need research in the Salish Sea to support management decision making. Historic fisheries and other human activities in the Salish Sea region depleted a number of species, including Pacific cod ( Gadus macrocephalus ), Pacific hake ( Merluccius productus ), rockfish ( Sebastes spp.), and walleye pollock ( G . chalcogrammus ) (Essington et al., 2021 ; Gustafson et al., 2000 ; Harvey et al., 2012 ; Palsson et al., 2009 ; Williams et al., 2010 ). Of note, rockfish populations have declined by an estimated 70% over the past 40 years (Drake et al., 2010 ; Tolimieri et al., 2017 ). In the same time period, pinniped populations have increased dramatically after the passage of the Marine Mammal Protection Act in 1972 (Jeffries et al., 2003 ; Johannessen & McCarter, 2010 ). These species, among others, occupy mid‐ to top‐trophic levels, and they likely play an important role in the Salish Sea ecosystem by maintaining healthy linkages with its trophic systems. For instance, various rockfish species have been found to feed on kelp crabs and other invertebrates that eat kelp in Puget Sound (Washington et al., 1978 ). The decline of rockfish and other fish that eat or impact grazer populations may be contributing to the decline of kelp (Calloway et al., 2020 ). However, we found very limited literature regarding trophic changes impacting kelp within the study area, indicating a large gap in the primary literature. Given the ubiquity of the trophic cascade impacts to kelp worldwide, it is likely this dearth of research represents a data gap for the region and would be worth further investigation. Similarly, research of the more potentially acute conditions in the Salish Sea related to human activity, such as contaminants, impacts of vessel traffic, water quality changes, and nearshore and upland development are warranted. Watersheds that drain into the Salish Sea are extensively logged (Hansen et al., 2013 ), human populations in the region are increasing rapidly (OFM, 2020 ), and the timing and magnitude of delivery of fresh water is changing as climate change results in more rain than snow and glaciers rapidly recede (Mote & Salathé, 2010 ; Riedel & Larrabee, 2011 ). At the same time, stronger environmental protection legislation has improved water and air quality and reduced historic contaminant and pollutant levels, though emerging pollutants remain a concern (EPA, 2021 ). Despite these substantial changes to hydrology and environmental quality in the region, we found very few studies that explicitly address how these changes impact the marine environment. Of note, research of these factors in the Salish Sea should account for the regional diversity of environmental conditions that naturally affect water retention times, temperature regimes, and consequences of changing contaminant identities, concentrations, and distributions throughout the region."
} | 6,466 |
34938590 | PMC8647914 | pmc | 4,891 | {
"abstract": "Abstract Community and invasion ecology have mostly grown independently. There is substantial overlap in the processes captured by different models in the two fields, and various frameworks have been developed to reduce this redundancy and synthesize information content. Despite broad recognition that community and invasion ecology are interconnected, a process‐based framework synthesizing models across these two fields is lacking. Here we review 65 representative community and invasion models and propose a common framework articulated around six processes (dispersal, drift, abiotic interactions, within‐guild interactions, cross‐guild interactions, and genetic changes). The framework is designed to synthesize the content of the two fields, provide a general perspective on their development, and enable their comparison. The application of this framework and of a novel method based on network theory reveals some lack of coherence between the two fields, despite some historical similarities. Community ecology models are characterized by combinations of multiple processes, likely reflecting the search for an overarching theory to explain community assembly and structure, drawing predominantly on interaction processes, but also accounting largely for the other processes. In contrast, most models in invasion ecology invoke fewer processes and focus more on interactions between introduced species and their novel biotic and abiotic environment. The historical dominance of interaction processes and their independent developments in the two fields is also reflected in the lower level of coherence for models involving interactions, compared to models involving dispersal, drift, and genetic changes. It appears that community ecology, with a longer history than invasion ecology, has transitioned from the search for single explanations for patterns observed in nature to investigate how processes may interact mechanistically, thereby generating and testing hypotheses. Our framework paves the way for a similar transition in invasion ecology, to better capture the dynamics of multiple alien species introduced in complex communities. Reciprocally, applying insights from invasion to community ecology will help us understand and predict the future of ecological communities in the Anthropocene, in which human activities are weakening species’ natural boundaries. Ultimately, the successful integration of the two fields could advance a predictive ecology that is urgently required in a rapidly changing world.",
"conclusion": "Conclusion We have presented a mechanistic framework to classify both community and invasion models, using combinations of six different processes: dispersal, drift, abiotic interactions, within‐guild interactions, cross‐guild interactions, and genetic changes. Characterizing models according to these processes allowed us to avoid biases and gaps from overly focusing on specific processes. The classification of representative models from the two fields following this framework and their comparison using a novel method based on network theory has helped not only to provide a synthesis of representative models in the two fields, but also to identify differences and overlaps between them. This enables us to identify where there may be scope to increase coherence both within (as Catford et al. 2009 , Vellend 2016 ) and across these fields in the future. In particular, it shows that concepts in invasion ecology tend to focus on the identification of specific processes, whereas community ecology has transitioned to explore how different combinations of multiple processes can provide a more mechanistic understanding of a whole suite of patterns. We hope that the bridge developed in this paper will help to advance both fields concurrently following a process‐based approach generating hypotheses to be validated experimentally. Using perspectives from one field to investigate questions in the other may create an integrative perspective in ecology that is still lacking (Rosindell et al. 2015 , Courchamp et al. 2017 , Pearson et al. 2018 ), advancing a more predictive ecology that is sorely needed in a rapidly changing world.",
"introduction": "Introduction The fields of community and invasion ecology have traditionally had different, but interrelated scopes. Community ecology aims primarily to explain how multiple species can coexist. Its scope encompasses the origin, evolution, maintenance, and dynamics of biodiversity within communities in diverse environments (Vellend 2016 , Leibold and Chase 2017 ). Invasion ecology, on the other hand, focuses on species introduced to novel environments by humans (termed alien species) and asks questions relating to how populations of alien species spread and interact with other species in these environments. Invasion ecology has a strong applied focus and has grown largely from concepts in population ecology; most early studies of invasions focused on understanding and controlling particular invasive species with major impacts. Except for studies of enemies or mutualists of alien species, invasion ecology has largely progressed independently from community ecology, at least until the last decade or two (Hui and Richardson 2017 ). Despite their largely separate historical trajectories, it is now accepted that community and invasion ecology are not independent from each other: Once a species is introduced to a novel environment, it interacts with the local community and forms part of the network of interacting species (Hui and Richardson 2019 a \n ). In addition, communities are often invaded by multiple alien species which, once established, can become impossible to control and, in some cases, become permanent members of the landscape, creating novel ecosystems (Hobbs et al. 2014 ). The traditional perspective of single alien species interacting only with specific native species, or with the abiotic environment, clearly does not capture the complexity of multiple alien species interacting with each other, with multiple native species, and with abiotic factors in a spatially heterogeneous environment. Consequently, community ecology has repeatedly been proposed as a crucial framework for invasion ecology (Shea and Chesson 2002 , MacDougall et al. 2009 , Pearson et al. 2018 ). Correlative studies and meta‐analyses bridging both perspectives (e.g., Gaertner et al. 2009 , Gallien and Carboni 2017 ) have shown that invasion ecology can benefit from insights that have accrued in community ecology regarding the coexistence of multiple species competing for limited resources and space, and the effects of disturbance and stochasticity on species persistence and coexistence. We will show here how merging insights from the two fields, through a mechanistic framework, creates a much‐needed integrative perspective in ecology, which will ultimately allow us to achieve accrued predictive power about the success or failure of biological invasions, but also to forecast changes in the structure of communities invaded by multiple alien species. Reciprocally, biological invasions can be seen as a kind of perturbation to native communities. Invasions have been framed as biogeographical assays, providing unique opportunities to uncover the mechanisms that structure communities (Cadotte et al. 2006 , Rouget et al. 2015 ). Biological invasions have also been shown to trigger regime shifts, altering multiple facets of ecological communities such that their new structures are hard, or impossible, to reverse (Gaertner et al. 2014 ). Biological invasions therefore have the potential to revolutionize our view of ecological communities and meta‐communities, from a closed system with coexisting species to an open system with a high rate of multi‐species propagule exchange through permeable boundaries and co‐evolving components (Frost et al. 2019 , King and Howeth 2019 , McGrannachan and McGeoch 2019 , Hui and Richardson 2019 a \n , \n b \n ). Applying insights from invasion biology to community ecology will help us better understand and predict the future of ecological communities in the Anthropocene, in which human activities are weakening species’ natural boundaries. Despite the clear interplay between the two fields, community and invasion ecology have developed their own sets of models, theories, and hypotheses. Community ecology tends to seek an overarching and universal theory of the assembly and maintenance of biodiversity, and heated debates arise when different models appear to contradict each other. This is exemplified by arguments around Hubbell’s ( 2001 ) Unified Neutral Theory of Biodiversity, which contradicts the well‐established niche theory (see Clark 2012 and Rosindell et al. 2012 for contrasting perspectives). The effect of spatial scale on community patterns further complicates the study of ecological communities (Chase et al. 2018 ), as does the fact that local ecological communities interact with each other within meta‐communities via propagule exchange between locations with different environmental conditions (Leibold and Chase 2017 ). Ecological communities are therefore complex and involve dynamic interactions among many organisms, each with their own traits and functions for the maintenance of biodiversity. To reduce complexity and redundancies in community ecology, Vellend ( 2016 ) proposed a conceptual framework based on four high‐level processes (dispersal, selection, speciation, and drift) that, he argued, described the fundamental dimensions of community ecology, thereby bringing coherence to the field. Rather than searching for overarching models, most work in invasion ecology seeks to explain or predict how species perform in a recipient ecosystem outside of their native ranges and the impacts of such biological incursions (see also Catford et al. 2009 , Jeschke and Heger 2018 ). Many of the models and hypotheses that have emerged in recent decades are nonetheless interrelated, and understanding how they relate to each other is not straightforward (Enders et al. 2018 ). Frameworks have therefore been proposed to structure the models and hypotheses of invasion biology, thereby contributing to the development of overarching theories. Catford et al. ( 2009 ), in particular, proposed classifying invasion models and hypotheses according to the combination of three key components: propagule pressure, the abiotic characteristics of the receiving ecosystem, and the biotic characteristics of the recipient community and of the alien species. Although emerging independently, this process‐based classification of invasion models and hypotheses maps onto the concept of dispersal, environmental, and biotic filters used to explain community assembly (Stokes and Archer 2010 ) and shares many similarities with Vellend’s ( 2016 ) conceptual framework of high‐level processes for community ecology. A mechanistic (process‐based) framework unifying community and invasion ecology is yet to emerge. This is highlighted by the lack of a general model to predict spread and impacts of alien species and the response of recipient communities (Courchamp et al. 2017 ). Here, we collate and extend process‐based conceptual frameworks from both community and invasion ecology to better capture their interplay (Catford et al. 2009 , Vellend 2016 ). We propose a set of processes that can be applied across community (including metacommunity) and invasion models (Tables 1 , 2 ), which we use to examine, characterize, compare, and synthesize a representative set of existing models at local and regional scales (given the lack of consensus in ecology about what qualifies as a theory, see Marquet et al. 2014 , or even a hypothesis, see Murray 2004 , we will use “community model” and “invasion model” as overarching terms for simplicity and coherence through the article). Based on the resulting process characterization, we also match community and invasion models and analyze the results using a novel method based on network theory, to complete the conceptual picture of the two fields and identify alignments and gaps. We see this as a crucial first step toward a synthesis enabling both fields to maximize benefits from one another, therefore providing novel perspectives to improve the ability to address interrelated issues in community and invasion ecology in the current context of global changes, and to move toward predictive models supporting robust management actions for nature conservation and invasion control in a holistic fashion. Table 1 Community models and their classification as process‐ or pattern‐based (expanding on Vellend 2016 ). ID Name Description Reference(s) Classification C1 Adaptive dynamics (AD) Mutation limited evolution of phenotypic traits driven by ecological interactions determines the structure of a community. Fussmann et al. ( 2007 ) Process C2 Bottom‐up regulation (BUR) Community composition is driven by resources (lower trophic levels). Oksanen et al. 1981 , Matson and Hunter ( 1992 ) Process C3 Colonization‐competition trade‐off / patch dynamics (CCT/PD) Good colonizers (dispersers) are bad competitors and reciprocally. Levins and Culver ( 1971 ) Process C4 Community Assembly Phase Space (CAPS) The combination of neutral and niche processes can generate structures that lie outside of the neutral‐niche continuum due to feedbacks. Latombe et al. ( 2015 ) Process C5 Competitive exclusion principle (CE) Two species competing for the exact same resource cannot coexist because one will inevitably have a slight advantage. Gause ( 1934 ) Process C6 Ecosystem engineering (EE) Community structure is influenced by severe effects of one species on the abiotic environment. Jones et al. ( 1994 ) Process C7 Enemy‐mediated coexistence (EMC) Enemies (predators, pathogens, etc.) have a larger effect on the most abundant species; that is, negative density dependence. Holt et al. ( 1994 ) Process C8 Equalizing/stabilizing criteria (ESC) Coexistence between species is permitted by (i) a reduction in fitness difference and (ii) niche differentiation between species. Chesson ( 2000a ) Process C9 Facilitation‐based theory (FBT) Community structure is explained by positive interactions between species, which promotes coexistence. Bruno et al. ( 2003 ) Process C10 Genetic feedback (GF) Natural selection enables a species with poor interaction ability to change its interaction mechanism and to recover. Pimentel ( 1968 ) Process C11 Hump‐shaped diversity‐productivity hypothesis (HSDPH) Low and high productivity generate stress and competitive exclusion, which reduces diversity, while constraints are relaxed at intermediate productivity. Grime ( 1973 ) Process C12 Intermediate disturbance hypothesis (IDH) Intermediate disturbance decrease competition and therefore the dominance of strong competitors. Grime ( 1973 ), Connell ( 1978 ) Process C13 Intransitive competition (IC) Each species is competitively superior to some and inferior to others, similar to rock‐paper‐scissors. Gilpin ( 1975 ) Process C14 Janzen‐Connell effects (JC) Species‐specific enemies accumulate around adult trees, preventing local regeneration of that species. Connell ( 1970 ), Janzen ( 1970 ) Process C15 Mass effect (ME) Colonization from occupied sites enables a species to survive in a site with unfavorable environment. Holyoak et al. ( 2005 ), Leibold and Chase ( 2017 ) Process \n C16 \n \n Maximum Entropy Theory of Ecology (METE) \n \n Community patterns are generated by maximizing information entropy under constraints on area (A), species richness (S), species abundance (N), and total metabolic rate of the individuals (E)–ASNE model . \n Harte ( \n \n 2011 \n \n ) \n \n Statistical property \n \n C17 \n \n Multiple stable equilibria (MSE) \n \n Positive feedbacks and perturbation/stochasticity can lead the community to switch between different equilibria . Scheffer ( 2009 ) \n Pattern (Process) \n C18 Neutral theory (NeT) All species are equivalent from a per capita perspective and species coexistence emerges from immigration and speciation. Hubbell ( 2001 ) Process C19 Neutral‐niche continuum (NNC) Communities have structures that lie between the structures generated by pure neutral (no interactions) and pure niche (only interactions) processes. Gravel et al. ( 2006 ) Process C20 Niche theory (NiT) Umbrella term for models based on interaction processes, biotic or abiotic. Chase and Leibold ( 2003 ) Process \n C21 \n \n Priority effect (PE) \n \n Initial colonists of a given site inhibit or facilitate the establishment of other species, for different possible reasons . \n Fukami ( \n \n 2010 \n , \n 2015 \n \n ) \n \n Pattern \n C22 R* theory (R*) When dealing with multiple resources, species with the lowest R* (lowest level of resources at which it can persist) outcompete other species. Tilman ( 1982 ) Process C23 Relative nonlinearity of competition (RNC) Interactions with resources fluctuates temporally due to the impact on resource levels by the species, resulting in non‐linear fitness responses to resource levels. Armstrong and McGehee ( 1980 ) Process C24 Spatial storage effect (SSE) Species have different niches and can persist where the environment is not optimal (e.g., through seed banks). In addition, per capita intraspecific competition is greatest at high abundance, and interspecific competition is greatest at low abundance. Chesson ( 2000b ) Process C25 Species pool hypothesis (SPH) Local community diversity is limited by the regional species pool, which is determined by regional and historical interactions, dispersal, speciation, and drift processes. Taylor et al. ( 1990 ) Process C26 Species sorting (SS) Species differ in their fitness in different abiotic environments (similar to niche theory but abiotic only). Holyoak et al. ( 2005 ), Leibold and Chase ( 2017 ) Process C27 Species‐energy theory (SET) Species richness is driven by a trade‐off between immigration from a global species pool and local extinction, which is driven by available energy (similar to TIB with energy instead of area). Wright ( 1983 ) Process C28 Stochastic niche theory (SN) Niche theory incorporating drift and propagule pressure. Tilman ( 2004 ) Process C29 Succession theory (ST) Umbrella term for community dynamics, for example, after disturbance, incorporating all processes but speciation. Pickett et al. ( 1987 ) Process C30 Temporal storage effect (TS) Species have different niches and can persist when the environment is not optimal (e.g., through seed banks). In addition, per capita intraspecific competition is greatest at high abundance, and interspecific competition is greatest at low abundance. Chesson ( 2000b ) Process C31 Theory of island biogeography (TIB) Species richness is driven by a trade‐off between immigration from a global species pool and local extinction, which is driven by area. MacArthur and Wilson ( 1967 ) Process C32 Top‐down regulation (TDR) Community composition is driven by predators (higher trophic levels). Matson and Hunter ( 1992 ) Process Notes Italics denotes models that are not process based under the strict characterization. The multiple stable equilibria models are considered to be pattern‐based under a strict characterization scheme, and process‐based under the inclusive characterization only, as indicated between parenthesis. John Wiley & Sons, Ltd Table 2 Invasion models and their classification as process‐ or pattern‐based (adapted from Catford et al. 2009 and Enders et al. 2018 ). ID Name Description Reference(s) Classification I1 Adaptation (A) The invasion success of alien species depends on their pre‐introduction adaptation to the conditions in the exotic range. Alien species that are related to native species are more successful in this adaptation. Duncan and Williams ( 2002 ) Process \n I2 \n \n Biotic acceptance aka “the rich get richer” (BA) \n \n Ecosystems with more native species are more invaded. This can be due to multiple processes . \n Stohlgren et al. ( \n \n 1998 \n \n ) \n \n Pattern (Process) \n I3 Biotic indirect effects (BID) Combinations of cross‐guild and potentially abiotic processes can lead to indirect biotic interactions between species of the same guild. Callaway et al. ( 2004 ) Process \n I4 \n \n Biotic resistance aka diversity‐invasibility hypothesis (BR) \n \n Ecosystems with high richness get less invaded than ecosystems with lower richness. This can be due to multiple processes . \n Elton ( \n \n 1958 \n \n ) , Levine and D’Antonio ( \n \n 1999 \n \n ) \n \n Pattern (Process) \n I5 Darwin’s naturalization (DN) The invasion success of alien species is higher in areas with few phylogenetically close species than in areas with many phylogenetically close species. Darwin ( 1859 ) Process \n I6 \n \n Disturbance (DS) \n \n The invasion success of alien species is higher in highly disturbed than in relatively undisturbed ecosystems . \n Elton ( \n \n 1958 \n \n ) , Hobbs and Huenneke ( \n \n 1992 \n \n ) \n \n Pattern (Process) \n I7 Dynamic equilibrium (DEM) The establishment of an alien species depends on natural fluctuations of the ecosystem, which influences the competition of local species. Huston ( 1979 ) Process I8 Empty niche (EN) The presence of empty niches increases the likelihood of alien species with adequate niches to invade. MacArthur ( 1970 ) Process I9 Enemy inversion (EI) Introduced enemies of alien species are less harmful for them in the exotic than the native range, due to altered biotic and abiotic conditions. Colautti et al. ( 2004 ) Process I10 Enemy of my enemy (EE) Introduced enemies of an alien species are more harmful to the native than to the alien species, giving the alien species a competitive advantage. Eppinga et al. ( 2006 ) Process I11 Enemy reduction (ERD) Enemies are less frequent in the introduced range, resulting in being less harmful. Similar to enemy inversion but due to population abundance than to actual predation mechanism. Colautti et al. ( 2004 ) Process I12 Enemy release (ER) Enemies are absent in the introduced range, resulting in fitness improvement for the alien species. Keane and Crawley ( 2002 ) Process I13 Environmental heterogeneity (EVH) A highly heterogeneous environment provides more niche therefore more invasion opportunities (similar to the empty niche for the abiotic environment). Melbourne et al. ( 2007 ) Process I14 Evolution of increased competitive ability (EICA) Release from natural enemies leads alien species to allocate more energy in growth and/or reproduction (this re‐allocation is due to genetic changes), resulting in a competitive advantage. Blossey and Notzold ( 1995 ) Process I15 Global competition (GC)–equivalent to Sampling (SP) A large number of different alien species is more successful than a small number because there is more chance than at least one of them will outcompete native species due to interaction processes. Crawley et al. ( 1999 ), Alpert ( 2006 ) Process I16 Habitat filtering (HF) The invasion success of alien species whose niche fits the abiotic environment in the introduced area is high. Darwin ( 1859 ), Melbourne et al. ( 2007 ) Process \n I17 \n \n Human commensalism (HC) \n \n Species living in close proximity to humans are more successful in invading new areas than other species . Jeschke and Strayer ( 2006 ) \n Pattern \n \n I18 \n \n Ideal weed (IW) \n \n The invasion success of an alien species is determined by its specific traits, such as life‐history traits . \n Baker ( \n \n 1965 \n \n ) , Rejmánek and Richardson ( \n \n 1996 \n \n ) \n \n Trait‐based \n I19 Increased resource availability (IRA) High resource availability increases the invasion success of alien species. Sher and Hyatt ( 1999 ) Process I20 Increased susceptibility (IS) High genetic diversity increases the chance to defend against enemies, and therefore to invade novel environments. Colautti et al. ( 2004 ) Process \n I21 \n \n Invasional meltdown (IM) \n \n The presence of alien species in an ecosystem increases the probability of invasion by additional species . \n Simberloff and Von Holle ( \n \n 1999 \n \n ) , Sax et al. ( \n \n 2007 \n \n ) \n \n Pattern (Process) \n \n I22 \n \n Island susceptibility hypothesis (ISH) \n \n Islands are more susceptive to biological invasions than are mainland . \n Jeschke ( \n \n 2008 \n \n ) , Moser et al. ( \n \n 2018 \n \n ) \n \n Pattern (Process) \n I23 Limiting similarity (LS) The invasion success of alien species is high if their niche highly differs from that of native species, and it is low if they are similar to that of native species. MacArthur and Levins ( 1967 ) Process I24 Missed mutualisms (MM) / co‐introduction The absence of mutualist species in the introduced environment decreases the probability of invasion by an alien species. Richardson et al. ( 2000 ), Colautti et al. ( 2004 ), Mitchell et al. ( 2006 ) Process I25 New associations (NAS) Alien and native species can have novel positive or negative interactions, therefore influencing the probability of alien species to establish. Colautti et al. ( 2004 ) Process I26 Novel weapons (NW) Alien species possessing a trait that is new to native species and affects them negatively gives alien species a competitive advantage. Callaway and Ridenour ( 2004 ) Process I27 Opportunity windows (OW; fluctuating resources) Like the empty niche, but niche availability fluctuates spatially and temporally and alien species can only invade at specific places and times. Johnstone ( 1986 ) Process \n I28 \n \n Phenotypic plasticity (PH) \n \n The ability of an alien species to change its phenotype to increase its fitness in a novel environment increases the probability to invade such environment . \n Baker ( \n \n 1965 \n \n ) , Richards et al. ( \n \n 2006 \n \n ) \n \n Trait‐based \n I29 Propagule pressure (PP) High propagule pressure increases the chance of an alien species to invade through sheer numbers. Lockwood et al. ( 2005 ) Process \n I30 \n \n Reckless invader (RI) \n \n The invasion performance of an alien species can vary, rapidly increasing its population shortly after introduction followed by a decrease in population and potentially extinction due to various reasons . \n Simberloff and Gibbons ( \n \n 2004 \n \n ) \n \n Pattern \n I31 Resource‐enemy release (RER) Similar to the enemy release hypothesis, but assumes that invasion success is then maximized when resources are high. Blumenthal ( 2006 ) Process I32 Specialist‐generalist (SG) Enemies present in the introduced range must be specialist, and therefore less likely to affect alien species with which they have not coevolved, whereas mutualists should be generalists, to benefit alien species. Callaway et al. ( 2004 ) Process \n I33 \n \n Tens rule (TEN) \n \n At every step of the invasion process, about 10% of alien species progress to the next step (Introduced, Established, Invasive) . \n Williamson ( \n \n 1996 \n \n ) , Williamson and Brown ( \n \n 1986 \n \n ) , Jeschke and Pyšek ( \n \n 2018 \n \n ) \n \n Pattern \n Notes Italics denotes models that are not process based. Some models are considered to be pattern‐based under the strict characterization scheme, but process‐based under the inclusive characterization only. These models are classified as process‐based between parenthesis. John Wiley & Sons, Ltd",
"discussion": "Discussion Differences between the processes addressed by invasion and community ecology Characterizing and matching invasion and community models according to their underlying processes using our framework highlights important differences in research focus in the two fields. About a quarter of the invasion models considered rely on the classification of invasion patterns under the strict characterization. Process‐based invasion ecology models also appear to more often consider the role of single mechanisms in isolation, as shown by the low number of processes in combinations. The smaller number of multi‐process models in invasion ecology (Fig. 2b ) is consistent with the search for case‐specific explanations of biological invasions integrating information about species biology and ecosystem characteristics, that is, invasion syndromes (Kueffer et al. 2013 , Perkins and Nowak 2013 , Novoa et al. 2020 ). Invasion ecology indeed often relies on observational approaches (see Fig. 17.3 in Jeschke and Heger 2018 ) allowing only limited control on the conditions of invasion. These approaches are therefore designed to investigate specific processes (see Jeschke and Heger 2018 for a synthesis of support or rejection of different models based on such approaches in the literature). In contrast, the larger number of processes used in combination in the community models reflects the fact that community ecology has strived for a more overarching, mechanistic perspective that emphasizes how the interplay of multiple processes can address a wide range of questions on the generation, dynamics, maintenance, and evolution of communities over a wide range of temporal and spatial scales (Gravel et al. 2006 , Latombe et al. 2015 , Vellend 2016 , Leibold and Chase 2017 ). Differences in the number of processes considered by community and invasion models can be explained by the different level of emphasis on interaction processes in the two fields. The predominance of interaction processes (especially cross‐guild interactions) in the list of invasion models has led us to identify a number of overlapping models, and therefore a highly skewed distribution of processes across invasion models (Fig. 3b ), which could be a source of ambiguities (Latombe et al. 2019 ). For example, the enemy inversion (EI), the enemy of my enemy (EE), the enemy reduction (ERD), and the enemy release (ER) models (I9‐I12) are all variations of the same cross‐guild interaction process (although such models can be further distinguished and related to each other using a hierarchy of hypotheses; Jeschke et al. 2012 , Jeschke and Heger 2018 ). Although the distribution of processes is less skewed for community models (Fig. 3a ), interaction processes are also the focus of a number of community models, which can be explained by the fact that interaction processes also dominated community ecology for decades (e.g., reviewed in Leibold 1995 , Chase and Leibold 2003 ). Within‐guild processes are nonetheless predominant, indicating a focus on horizontal communities, consistent with Vellend’s ( 2016 ) original framework. Consistently, the biggest modules in the bi‐adjacency matrix (Fig. 4 ) contain community and invasion models based predominantly on combinations of all interaction processes. Due to the high degree of attention they received historically, it is not surprising that interaction processes have been combined in many different ways, resulting in a limited degree of coherence between models based on these processes within and across the two fields. More recent depictions of community models (e.g., SN, Tilman 1994 ; NNC, Gravel et al. 2006 ; CAPS, Latombe et al. 2015 ), however, spurred on by Hubbell’s ( 2001 ) neutral theory which emphasized the role of dispersal and stochasticity, provide a more balanced perspective, and recognize the interplay of multiple processes, rather than considering independent processes in isolation (Vellend 2016 , Leibold and Chase 2017 ). In contrast, few invasion models consider post‐introduction processes other than interaction processes, resulting in fewer combinations of processes overall. Most invasion studies on dispersal focus on the human‐mediated introduction/invasion pathways (e.g., Wilson et al. 2009 ). However, it has also been shown that different dispersal kernels (e.g., Hui et al. 2012 ), and especially the presence or absence of long‐distance dispersal (Berthouly‐Salazar et al. 2013 ), are crucial for determining the range expansion of alien species in novel environments (Kot et al. 1996 , McGeoch and Latombe 2016 ). Given the importance of feedback between dispersal and interactions for explaining community assembly (Latombe et al. 2015 ), and the role of spatial and temporal correlations of stochasticity in population size and growth in boosting invasion performance (Cuddington and Hastings 2016 , Hui et al. 2017 ), combinations of neutral and interaction processes will likely reveal unexpected trajectories for both the invaders and the structure of the recipient community, even for single‐species invasions. This also applies to the combination of these processes with genetic changes, as rapid evolutionary changes in introduced species have been shown to be quite commonly associated with invasion success (Whitney and Gabler 2008 ). Lawton ( 1996 ) wrote with reference to patterns in community ecology: “Too often, ecologists seem obsessed with finding a single explanation for some process or pattern of interest.” Community ecology has, however, recently transitioned toward a more comprehensive perspective that embraces the interplay between multiple processes. There are several possible reasons why invasion ecology often considers the role of specific processes in isolation to explain biological invasions. First, invasion models tend to have a narrower scope (exploring factors that mediate survival and establishment of a particular introduced species in a novel environment; Pysek et al. 2020 ) compared to community models (whose scope range from the generation and dynamics to the maintenance and evolution of communities). More importantly, invasion ecology has only started to develop as a field more recently (Vaz et al. 2017 ). Searches on Web of Science with the keywords “community ecology” and “invasion ecology” as topics return articles dating back to 1914 and 1986, respectively. It is therefore possible that the lists of models used here, which have similar lengths, may overlook overlaps between community models that may have existed when the field was younger. This list also likely underestimates the number of early community models focusing on the identification of patterns, such as the mathematical formulation of species‐area relationships (Connor and McCoy 1979 ) or species abundance distributions (Williamson and Gaston 2005 ). This is actually good news for invasion ecology, as it would indicate that the field can benefit from the long history of community models to develop further from a mechanistic perspective and produce a coherent synergy between the two fields, as we elucidate below. Toward a stronger synergy between invasion and community ecology Fitting the process‐based framework presented here to existing models, theories, and hypotheses is useful to obtain a much‐needed coherent and synthetic picture and an overarching view of community and invasion ecology. Because of the small number of processes considered simultaneously by invasion models, we argue that we should move toward emphasizing a process‐based invasion ecology, to complement the experimental search for specific reasons to explain successful biological invasion events. Attempts to reconcile community and invasion ecology have often focused on specific interaction processes between one alien species and a native community. Shea and Chesson ( 2002 ) introduced the concept of niche opportunity, which encompasses the different interaction processes of our framework. In their this framework, niche opportunities allow an invading population to have a positive growth rate through access to resources or decrease in natural enemies. MacDougall et al. ( 2009 ) extended this concept by building on the perspective of equalizing vs stabilizing mechanisms as proposed by Chesson ( 2000 a \n ). Wolkovich and Cleland ( 2011 ) showed how phenology can also provide niche opportunities. Pearson et al. ( 2018 ) further incorporated dispersal processes by building on the similarity between the dispersal, abiotic, and biotic ecological filters from community ecology (e.g., Stokes and Archer 2010 ) and invasion ecology (Catford et al. 2009 ). Although each of these frameworks has included several of the six processes described in this paper, they were considered either separately, or additively, not in a truly interactive fashion considering feedbacks and complex outcomes, as explored by community ecology and highlighted by our framework. A truly mechanistic perspective of biological invasions would follow the direction taken by more recent community ecology models (e.g., Gravel et al. 2006 , Latombe et al. 2015 , Leibold and Chase 2017 ) by exploring how different combinations and feedbacks between the processes described in this framework generate different community and invasion patterns. It would then be possible to generate hypotheses that can be systematically tested through experiments or field observations. This approach would enable invasion and community ecology to advance simultaneously. This would help encourage further research on multi‐species interactions in invasion ecology. Such a whole system approach will enable us to achieve a more complete picture of biological invasions (Gurevitch et al. 2011 ), to understand and potentially predict the fate of invaded communities, including the trajectories leading to regime shifts (Gaertner et al. 2014 ) and the dynamics of thresholds between historical, hybrid, and novel ecosystems (Hobbs et al. 2014 ). This will in turn contribute to improve our understanding of community assembly and structure. To develop such a mechanistic, process‐based approach to invasion ecology, future work should clarify the relationship between process‐ and pattern‐based invasion models. This framework should establish the relationship between invasion patterns and different combinations of processes. Patterns generated by the same sets of processes could then also be related to each other (Appendix S2 : Fig. S2). This would also enable us to clearly define nestedness and partial overlap between models, as both metrics are defined based on process similarity. Using this approach will also enable us to remove potential ambiguities when pairing community ecology and invasion models. We acknowledge that our six‐process framework may evolve to capture more accurately the specificities of the models. This is why it was designed in a hierarchical fashion from Vellend’s ( 2016 ) initial four high‐level processes, which already captured the essence of the relevant processes. For example, we have not characterized biotic interactions as positive (mutualistic) or negative (antagonistic), although both kinds have been argued to be important drivers of species assembly and coexistence in community ecology, and of invasion success in invasion biology (e.g., Francis and Read 1995 , Christian 2001 , Colautti et al. 2004 , Traveset and Richardson 2020 ). Other mechanisms, such as frequency dependence, which can apply to different processes and are integral parts of some models (e.g., priority effects), could also be considered in parallel to this framework. While restricting our framework to six processes allowed for generality and a broad, synthetic perspective across community and invasion ecology, considering additional processes may reveal complex and unexpected behaviors in modeled invaded communities. Finally, it is important to explicitly incorporate spatial and temporal scales when expanding this process‐based framework. The processes defined here, as those on which they are based (Catford et al. 2009 , Vellend 2016 ), are not restricted to any particular scale. Rather, as scale is important to detect ecological patterns such as changes in species richness and turnover over space and time (Chase et al. 2018 ), it may change the perspective on the importance of each process at play (Chase 2014 , Viana and Chase 2019 ). For example, competition may only be detected at fine spatial scales (e.g., between adjacent fruiting plants), whereas cross‐guild interactions with frugivorous birds dispersing seeds would occur at a much larger scale. Environmental heterogeneity also varies across scales, changing our perception of the importance of related processes. The relevant scales therefore depend on how the involved taxa perceive and are affected by, the different processes over specific spatial and temporal scales (Theoharides and Dukes 2007 , McGill 2010 ). This process‐based framework, like those on which it is based, can therefore offer a bridge between multiple scales (Vellend 2016 )."
} | 10,090 |
31223337 | PMC6570963 | pmc | 4,894 | {
"abstract": "Background Methane is the primary component of natural gas and biogas. The huge abundance of methane makes it a promising alternative carbon source for industrial biotechnology. Herein, we report diamine compound, putrescine, production from methane by an industrially promising methanotroph Methylomicrobium alcaliphilum 20Z. Results We conducted adaptive evolution to improve putrescine tolerance of M. alcaliphilum 20Z because putrescine highly inhibits the cell growth. The evolved strain 20ZE was able to grow in the presence of 400 mM of putrescine dihydrochloride. The expression of linear pathway ornithine decarboxylase genes from Escherichia coli and Methylosinus trichosporium OB3b allowed the engineered strain to produce putrescine. A higher putrescine titer of 12.44 mg/L was obtained in the strain 20ZE-pACO with ornithine decarboxylase from M. trichosporium OB3b. For elimination of the putrescine utilization pathway, spermidine synthase (MEALZ_3408) was knocked out, resulting in no spermidine formation in the strain 20ZES1-pACO with a putrescine titer of 18.43 mg/L. Next, a genome-scale metabolic model was applied to identify gene knockout strategies. Acetate kinase (MEALZ_2853) and subsequently lactate dehydrogenase (MEALZ_0534) were selected as knockout targets, and the deletion of these genes resulted in an improvement of the putrescine titer to 26.69 mg/L. Furthermore, the putrescine titer was improved to 39.04 mg/L by overexpression of key genes in the ornithine biosynthesis pathway under control of the pTac promoter. Finally, suitable nitrogen sources for growth of M. alcaliphilum 20Z and putrescine production were optimized with the supplement of 2 mM ammonium chloride to nitrate mineral salt medium, and this led to the production of 98.08 mg/L putrescine, almost eightfold higher than that from the initial strain. Transcriptome analysis of the engineered strains showed upregulation of most genes involved in methane assimilation, citric acid cycle, and ammonia assimilation in ammonia nitrate mineral salt medium, compared to nitrate mineral salt medium. Conclusions The engineered M. alcaliphilum 20ZE4-pACO strain was able to produce putrescine up to 98.08 mg/L, almost eightfold higher than the initial strain. This study represents the bioconversion of methane to putrescine—a high value-added diamine compound. Electronic supplementary material The online version of this article (10.1186/s13068-019-1490-z) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion The obligate methanotrophic bacterium M. alcaliphilum 20Z has become an attractive microbial platform for methane bioconversion to a value-added product due to the development of genetic tools and new insights into methane assimilation and the central metabolic pathway. In this study, we applied a genome-scale metabolic model to identify the metabolic engineering targets, inactivated the putrescine utilization pathway in M. alcaliphilum 20Z, overexpressed the ornithine biosynthesis pathway, and optimized the nitrogen source for putrescine production. The engineered M. alcaliphilum 20ZE4A-pACO strain produced putrescine up to 98.08 ± 2.86 mg/L in simple flask culture. To the best of our knowledge, this work represents the first biotechnological application for producing putrescine from methane.",
"discussion": "Discussion Methanotrophs with the ability of using methane as the sole carbon and energy source are becoming promising strains for methane bioconversion. M. alcaliphilum 20Z, a haloalkali-tolerant methanotroph, is an industrially promising biocatalyst for the conversion of methane to value-added products due to a well-characterized central metabolic pathway, rapid growth rates, high cell density cultivation, and genetic tools [ 37 ]. In addition, M. alcaliphilum 20Z possesses an efficient recycling pathway for the production of ornithine-derived product, compared to the linear ornithine biosynthesis pathway of type II methanotrophs, which forms acetate as the bypass product. In this study, we employed M. alcaliphilum 20Z for the conversion of methane to an important industrial chemical, putrescine. However, compared to engineered E. coli , M. alcaliphilum 20Z was very sensitive to putrescine. E. coli can grow well in the presence of 250 mM putrescine dihydrochloride (22 g/L putrescine), and the growth rate significantly decreased in the presence of 500 mM putrescine dihydrochloride (44 g/L putrescine) [ 6 ]. The effect of putrescine on the growth of C. glutamicum was also reported. C. glutamicum was able to grow in the presence of up to 500 mM putrescine dihydrochloride, but the growth rate was reduced (34%) at 740 mM putrescine dihydrochloride [ 9 ]. Therefore, we have conducted the adaptive evolution to enhance putrescine tolerance of M. alcaliphilum 20Z. The evolved strain was able to grow in the presence of 400 mM putrescine dihydrochloride. We constructed an IncP-based broad host-range plasmid for expression of ornithine decarboxylase from a well-known source, E. coli, and from a type II methanotroph, M. trichosporium OB3b, driven by a tac promoter. The expression of ornithine decarboxylase from M. trichosporium OB3b showed higher activities at an alkali pH in a cultivation medium of M. alcaliphilum 20Z. In addition, we inactivated the putrescine utilization pathway to accumulate putrescine in M. alcaliphilum 20Z. M. alcaliphilum 20Z possesses five genes predicted to be associated with spermidine synthase. However, the knockout of one spermidine gene MEALZ_3408 completely blocked spermidine formation in the mutant strain (data not shown). Gene MEALZ_3304 could be associated with spermine synthase, which converts spermidine to spermine. We also found that a small amount of putrescine accumulation in the culture medium promoted the growth of M. alcaliphilum 20Z. A genome-scale metabolic model of M. alcaliphilum 20Z and its application have been recently published [ 14 ]. By employing the genome-scale metabolic model, we present genetic engineering strategies to obtain the engineered M. alcaliphilum 20Z strains where putrescine production increased or growth was coupled. The ACKr gene was identified as a potential target for increase in putrescine production. With ACKr knockout, strain 20ZE3-pACO was able to produce 26.69 ± 1.86 mg/L of putrescine. For further improvement of putrescine production, we introduced key genes in the ornithine biosynthesis pathway-argDJ by integration. These genes were driven by tac promoter into the genome of M. alcaliphilum 20Z, which led to an improvement of 21.08% with a maximized titer of 39.04 ± 1.35 mg/L. We further examined the effects of different nitrogen sources on the growth and putrescine production of the wild-type and engineered M. alcaliphilum 20ZE4A-pACO strain. Generally, M. alcaliphilum 20Z has been cultured with nitrate as the sole nitrogen source. Due to the incompatibility of ammonia oxidation and methane oxidation, methane monooxygenases (which lack substrate specificity) oxidized ammonia to hydroxylamine that inhibits methane oxidation [ 35 , 36 ]. Nitrate assimilation is an energetically expensive process. However, the assimilation of ammonia via glutamate dehydrogenase, which was employed in energy-limited environment, could be favorable for a glutamate-derived product like putrescine. M. alcaliphilum 20Z was unable to grow with ammonia as the sole nitrogen source. We found that the growth of M. alcaliphilum 20Z was promoted and putrescine production was also significantly improved with up to 2 mM of ammonium chloride supplement. A maximized titer of 98.08 ± 2.86 mg/L putrescine with a productivity of 2.9 nmol/gDCW/h was obtained in a simple flask culture. Transcriptome analysis of the engineered strains cultured on mineral salt medium with different nitrogen sources showed upregulation of most genes involved in methane assimilation, TCA cycle, and ammonia assimilation in ammonia nitrate mineral salt medium compared to nitrate mineral salt medium. Despite the incompatibility of ammonia with methane, ammonia could be used as a potential nitrogen source for growth of M. alcaliphilum 20Z and production of glutamate-related metabolites. The maximum titer of putrescine obtained in this study was much lower than sugar-based production [ 6 , 9 ]. The experimental putrescine yield was 0.0276 g/g-CH 4 , much lower than the yield predicted by Optgene (0.408 g/g-CH 4 ), but the experimental specific growth rate of the engineered strain (0.048 h −1 ) in NMS was higher than the maximum specific growth rate predicted by Optgene (0.032 h −1 ). The difference in growth rates between simulated data and experimental data was due to the adaptation to putrescine by engineered strain. To enhance the productivity of putrescine from methane, the remaining obstacles of methane bioconversion including methane mass transfer limitation and methanotrophic methane oxidation efficiency [ 5 , 38 , 39 ] should be solved. In addition to the conventional limitations of methane bioconversion (such as low methane mass transfer, lack of genetic tools, and low carbon conversion efficiency), further studies need to be investigated for engineering methane monooxygenase to allow M. alcaliphilum 20Z to grow in ammonia, which can reduce the energy consumption by nitrate assimilation."
} | 2,355 |
34337805 | PMC7611957 | pmc | 4,896 | {
"abstract": "Abstract The design of dynamic, reconfigurable devices is crucial for the bottom‐up construction of artificial biological systems. DNA can be used as an engineering material for the de‐novo design of such dynamic devices. A self‐assembled DNA origami switch is presented that uses the transition from double‐ to single‐stranded DNA and vice versa to create and annihilate an entropic force that drives a reversible conformational change inside the switch. It is distinctively demonstrated that a DNA single‐strand that is extended with 0.34 nm per nucleotide – the extension this very strand has in the double‐stranded configuration – exerts a contractive force on its ends leading to large‐scale motion. The operation of this type of switch is demonstrated via transmission electron microscopy, DNA‐PAINT super‐resolution microscopy and darkfield microscopy. The work illustrates the intricate and sometimes counter‐intuitive forces that act in nanoscale physical systems that operate in fluids.",
"conclusion": "3 Conclusion Here we presented a nanoscale, fully operational DNA origami switch. The controllable and reversible reconfiguration of the switch is driven by the transition from dsDNA to ssDNA and vice versa. The contractive force responsible for the switching is the result of merely a change in elasticity upon transition from dsDNA to ssDNA. This simple mechanism of dynamic reconfiguration is hardcoded directly into the building material of the switch itself and it has the potential to become an integral component for the development of synthetic biological machineries that mimic essential cellular behaviors such as membrane transformation and sculpting. Another potential application of our sequence‐specific signal transduction mechanism will be biosensing where pathogenic RNA or DNA sequences trigger mechanical changes that are easily detectable on the single‐structure level.",
"introduction": "1 Introduction Due to its predictable Watson‐Crick base‐pairing, DNA has been used successfully over the last decades as an engineering material for the bottom‐up self‐assembly of well‐defined structures and devices on the nanoscale. [ \n \n 1 \n , \n 2 \n , \n 3 \n \n ] In particular, DNA origami, [ \n \n 4 \n , \n 5 \n , \n 6 \n , \n 7 \n \n ] in which a long single‐stranded DNA (ssDNA) scaffold is self‐assembled into 2D and 3D predefined shapes with a set of specifically designed short oligonucleotide staple‐strands, allows building unprecedented complex and functional nanostructures with high yields. [ \n \n 8 \n \n ] DNA also adapts to mechanically stressed conformations, enabling the realization of curved, twisted, and bent DNA origami structures. [ \n \n 9 \n , \n 10 \n , \n 11 \n \n ] By using the entropic spring behavior of ssDNA, the construction of prestressed tensegrity, [ \n \n 12 \n \n ] bent, [ \n \n 13 \n , \n 14 \n \n ] and force clamping [ \n \n 15 \n \n ] DNA structures has been reported. In related approaches, complementing the ssDNA gap regions in DNA origami trusses with the help of DNA polymerases resulted in unidirectional transitions from bent to straight trusses, [ \n \n 16 \n \n ] or the formation of a rigidified tetrahedron through RecA protein filament assembly on ssDNA sections of a DNA origami tripod structure. [ \n \n 17 \n \n ] \n Aside from static nanoarchitectures, DNA nanotechnology also enables the construction of dynamic and autonomous switches. [ \n \n 18 \n \n ] The operation of these dynamic switches can be divided into two main categories: first, operation via molecular interaction and second, operation via external stimuli. The main molecular interactions employed to control motion on the nanoscale are DNA hybridization (mainly toehold‐mediated strand displacement) and base stacking. Examples of such motion controlled by molecular interactions include reconfigurable plasmonic devices, [ \n \n 19 \n \n ] hinges, [ \n \n 20 \n , \n 21 \n \n ] tweezers, [ \n \n 18 \n , \n 22 \n \n ] rotary devices, [ \n \n 23 \n , \n 24 \n , \n 25 \n , \n 26 \n \n ] walkers, [ \n \n 27 \n \n ] drug carriers [ \n \n 28 \n , \n 29 \n \n ] and robots sorting molecules or nanoparticles. [ \n \n 30 \n , \n 31 \n \n ] Other molecular interactions as driving mechanisms include target molecule binding [ \n \n 32 \n , \n 33 \n \n ] and aptamer [ \n \n 28 \n , \n 29 \n \n ] as well as nucleosome interactions. [ \n \n 34 \n \n ] Operation via any molecular interaction, which includes all mechanism described above, has the advantage of controllable molecular recognition and specificity. However, the operation speed is limited by diffusion and interaction kinetics of the molecules and thus often quite slow. Notably, several approaches have been developed to increase the response speed of dynamic DNA devices. On the other hand, external stimuli such as light, [ \n \n 35 \n , \n 36 \n \n ] temperature, [ \n \n 37 \n \n ] ions, [ \n \n 11 \n , \n 23 \n \n ] pH, [ \n \n 38 \n , \n 39 \n , \n 40 \n \n ] and electric fields [ \n \n 21 \n , \n 41 \n \n ] often enable much faster operation up to an increase in speed by many orders of magnitude. [ \n \n 41 \n \n ] For example, Karna et al. used the reversible, pH‐dependent formation of i‐motifs between adjacent nanostructure domains to facilitate the actuation of a coiled DNA nanospring that in turn impacts the motility of cultured cells via integrin coupling. [ \n \n 40 \n \n ] Any of these stimuli which we here termed external, however, have the limitation of acting globally and they lack the specificity molecular recognitions can offer. We here developed a molecular interaction‐based mechanism for the actuation of a DNA origami switch that performs against intuition to some extend: removing one of the two strands of a region of double‐stranded DNA (dsDNA) inside a DNA structure leaves a section of single‐stranded DNA (ssDNA) of the same length. This remaining single strand is now much floppier as the persistence length of ssDNA is ≈1 nm compared to ≈50 nm of dsDNA. This floppiness, however, does not lead to increased flexibility and extension but, on the contrary, to a substantial contraction of the region in question. This spring‐like behavior results primarily from the entropic properties of a polymer in solution that can theoretically be described using, for example, the modified freely jointed chain model (mFJC) [ \n \n 42 \n \n ] or the worm‐like chain model (WLC). [ \n \n 43 \n \n ] While this transition from dsDNA to ssDNA in our experiments is driven by strand‐displacement and thus suffers the same lack of reaction speed as previously described mechanisms, it offers great simplicity.",
"discussion": "2 Results and Discussion We designed a 140 nm long DNA origami switch composed of three rectangular blocks linked together by in total six parallel interconnected DNA double helices at the bottom ( Scheme \n \n 1 \n ). Four dsDNA helices (2 × 2) at the top, each formed by hybridization of four staple strands to the scaffold strand, interconnect the blocks and span the 87‐nucleotide (nt) ‐long (≈30 nm) gaps between the blocks. Each dsDNA bridge has four identical seven nt‐long toehold domains (violet) protruding from the 3’ ends of the staple strands (the design details and the list of oligonucleotides can be found in Figures S1 – S3 and Table S2 , Supporting Information). The transition from dsDNA to ssDNA occurs when an excess amount of fuel strands (complementary to the staple strands in the dsDNA bridge helices) are added. The strand displacement reaction [ \n \n 22 \n , \n 44 \n \n ] is initiated via the toehold domain and dislocates the staple strands from the scaffold, producing unreactive dsDNA waste. The reaction rate here is dependent on several parameters, including toehold length, [ \n \n 45 \n \n ] overall strand length, concentration of added fuel strands, temperature and buffer conditions. [ \n \n 46 \n \n ] In our system, with a 7 nt long toehold, a 22 nt long duplex length and a concentration of 100 × 10 −9 \n m of the added fuel strand, the estimated response time for closing of the switch is on the order of seconds to minutes. To ensure efficient switching we thus incubated our switches for several hours. After the staple strand is removed, the entropic force of the remaining ssDNA now pulls the rectangular blocks toward each other at their upper part while bending the layer of double helices at the bottom of the DNA origami, facilitating a motion and change of state. As a rule of thumb one stretch of ssDNA that is extended to the length of its double‐stranded counterpart exerts a contractile force of ≈5.5 pN on its ends, irrespective of its total length. In our design, pairs of DNA duplexes bridge the upper half of the origami structure resulting in a pulling force of about 11 pN acting on each of the three blocks (see Figure S2 and Text S1 in the Supporting Information for a detailed description). While we avoided notorious hairpins within the m13mp18 scaffold for the choice of our spring regions, we were ad hoc not able to find four regions that completely lack secondary structures. Thermodynamic analysis of the four regions enabled by the NUPACK suite [ \n \n 47 \n \n ] reveals possible formations of short hairpin stems of up to 5 base pairs (bps) (Figure S4 , Supporting Information). Such hairpins will be transient but may add residual forces to the springs. [ \n \n 48 \n \n ] In the next step, the addition of an excess of the sequences that have been removed before leads to these staple strands hybridizing again to the ssDNA scaffold, realizing the reverse transition (from ssDNA to dsDNA). This re‐opening of the switch is again expected to occur on the minute time scale, again we incubated the samples for hours. The switching between the open and closed states can thus be controlled through a series of dissociation and hybridization steps. Scheme 1 Schematic overview of the DNA origami switch. Close view of the dsDNA‐to‐ssDNA transition that leads to the reversible change between the open and closed state. The DNA origami switch was self‐assembled in the open state in a one‐pot reaction by thermal annealing. Correct assembly of the switches was confirmed by agarose gel electrophoresis and transmission electron microscopy (TEM) ( Figure \n \n 1 \n and Figure S5 : Supporting Information). After folding and purification, an excess amount of fuel strands was added to the solution containing the samples to mediate the strand displacement and thus switch the device from the open to closed, U‐shaped state as apparent from the TEM micrographs shown in Figure 1B and Figures S6 – S8 (Supporting Information). We measured the end‐to‐end distance of the switch in both states. The histogram of the end‐to‐end distance followed an asymmetric distribution skewed slightly toward smaller distances than designed for the open state and larger distances for the closed state (Figure S9 , Supporting Information). Both distributions were approximated with a lognormal fit and the median end‐to‐end distance was calculated to be 130 nm in the open state and 33 nm in the closed state. To test the reversibility of our system, we further added an excess of staple strands complementary to the ssDNA scaffold region of the closed switches, leading to re‐opening (Figure 1C and Figure S8 : Supporting Information). The distribution of the end‐to‐end distance after re‐opening was almost identical to the distribution of the initial open state with a median end‐to‐end distance of 130 nm (Figure S9 : Supporting Information). It has to be noted, however, that this “perfect” result is partly an effect of TEM imaging being performed on dried samples. Structures that are slightly bent in solution will stretch out to full length when adsorbing with their bottom‐ or topsides on the TEM grid. Figure 1 TEM micrographs of the DNA origami switch in the A) open, B) closed, and C) re‐opened states. Scale bar: 100 nm. To demonstrate the consecutive switching of individual DNA origami structures and to obtain information about configurational variability in solution, we employed multiplexed DNA‐PAINT super‐resolution microscopy. [ \n \n 49 \n , \n 50 \n \n ] First, we immobilized the structures (in the open state) via biotin‐modified DNA strands extending from the bottom under the central block to a BSA‐biotin coated glass surface (linked by streptavidin, Figure \n \n 2 A ). In order to visualize the state of the DNA origami with DNA‐PAINT, we added docking sites on each end of the structure. In the first imaging round, we visualized the open state configuration. The measured center‐to‐center distance of the localization cluster of about 130 nm is in good agreement with the end‐to‐end distance measured in TEM micrographs (Figure 2B , cyan). To switch the DNA origami structure to its closed configuration, we incubated the origami with the fuel strands while being mounted on the microscope. The second round of DNA‐PAINT imaging depicts the closed state (Figure 2B , magenta). Next, we switched the structure back to the open state and performed a third round of imaging (Figure 2B , yellow). In the re‐opened state, the measured distances between both ends were in some instances reduced compared to the initial open state. This can be explained by incomplete transitions back to the double‐stranded form (e.g., one or several of the 16 required staple strands missing). Although the structure did not always switch back to the full ≈130 nm distance, the experiment demonstrates the repeated switching capability between the two states of individual switches followed over time (nine different, exemplary overlays of the three imaging rounds are shown in Figure 2C , a set of 186 individual switches together with a distance analysis is shown in Figures S10 – S13 , Supporting Information). Figure 2 Observing switching of individual structures via DNA‐PAINT. A) Surface immobilization of the DNA origami switch on a glass substrate via biotin‐streptavidin conjugation. DNA origami structures carry DNA‐PAINT binding sites at both ends. B) DNA‐PAINT super‐resolution imaging to visualize different states of individual switches. Cyan represents the open state, magenta the closed state and yellow the re‐opened state. Scale bar: 25 nm. C) Overlay of the three consecutive imaging rounds of nine different switches. Colocalization of at least two of the three channels (cyan, magenta, and yellow) results in white spots. Scale bar: 100 nm. Next, we tested whether the structure can still perform controlled switching when it carries large cargos. We attached 50 nm gold nanoparticles (AuNPs) to both ends of the structure ( Figure \n \n 3 A ). AuNPs were functionalized with thiolated poly‐T oligonucleotides complementary to extensions on both ends of the switch structure. To attach the AuNPs to the designated position on the DNA origami, the switches with open states were mixed with functionalized AuNPs and then separated from excess AuNPs as well as aggregates by agarose gel electrophoresis (Figure S14 , Supporting Information). Successful attachment of AuNPs as well as switching from open to closed to re‐opened state was verified by TEM (Figure 3A and Figures S15 – S17 : Supporting Information). Figure 3 AuNP attachment to the DNA origami switches. A) Schematics showing the reversible switching between open and closed states of DNA structures carrying two AuNPs on opposing ends (left) and TEM micrographs of a representative structure for each state (right). Scale bar: 50 nm. B) Histograms of the interparticle distances (surface‐to‐surface) for the open state (top) and the closed state (bottom). Lognormal fits are drawn as a solid black line and the median as a dashed line; median open = 102 nm; median closed = 15 nm; n is the number of analyzed single nanoswitches. C) Darkfield images (insets) and scattering spectra of exemplary single switches in the open state (top) and closed state (bottom). The proximity of the AuNPs in the closed state leads to plasmon coupling and hence a red‐shift of the scattered light peak. D) Density plot (Kernel density estimation) of the RGB pixel intensity distribution in darkfield microscopy images of all analyzed single particles for the open state (top) and closed state (bottom). N is the number of analyzed single switches. Furthermore, we analyzed the interparticle distance between two attached AuNPs for the open and closed states of > 100 switches in TEM micrographs (Figure 3B ). We again approximated both distributions with a lognormal fit and the median interparticle distance was calculated to be 102 nm in the open state and 15 nm in the closed state. Similar to the distribution of the end‐to‐end distance from the bare switches (Figure S9 , Supporting Information), the distance distribution is asymmetric, especially in the closed state. Another method to monitor the interparticle distance is to record the scattered light from the AuNPs in darkfield microscopy. The individual 50 nm AuNPs used here exhibit plasmon resonance in the visible range (≈550 nm) and the coupling of plasmons [ \n \n 51 \n \n ] from particles in close proximity results in a shift of the scattered light toward longer wavelengths. [ \n \n 52 \n \n ] This plasmon coupling is highly distance dependent [ \n \n 51 \n \n ] and indeed we observed discernible red‐shifts in darkfield microscopy images upon switching of the structures from the open to the closed state. Figure 3C shows two exemplary images together with scattering spectra: the open structure carrying two separated AuNPs appears green whereas the two AuNPs in direct proximity on the closed device bring about orange spots as well as a pronounced red‐shift in the spectrum. We also analyzed all individual structures in our darkfield microscopy images via the pixel intensity of each spot in the three channels of the RGB color camera (images of all individual structures are shown in Figure S18 , Supporting Information). This intensity distribution of the three channels is shown in Figure 3D . As expected, we observed unimodal intensity distributions for the open structures in all three channels. However, for the closed switches we observed bimodal intensity distributions in both the red and the green channel with an additional peak of higher intensity in red and correspondingly a new peak of lower intensity in green. This attests that the AuNPs of a large fraction of the switched structures came close enough to enable plasmon coupling strong enough to be discernible with the RGB color camera. At the same time, we did not expect to see efficient coupling at interparticle distances greater than ≈15 nm. Since approximately half of the closed switches exhibit a larger interparticle distance (median closed = 15 nm, Figure 1B ), the observed bimodal intensity distributions in the red and green channels of the closed switches are in good agreement with our TEM‐based measurements of the interparticle distance. Note, however, that in contrast to PAINT imaging both TEM and darkfield measurements were performed with dried samples which potentially leads to particles being pushed closer together due to drying and surface tension effects."
} | 4,766 |
34020712 | PMC8138999 | pmc | 4,897 | {
"abstract": "Background Beginning in the last century, coral reefs have suffered the consequences of anthropogenic activities, including oil contamination. Chemical remediation methods, such as dispersants, can cause substantial harm to corals and reduce their resilience to stressors. To evaluate the impacts of oil contamination and find potential alternative solutions to chemical dispersants, we conducted a mesocosm experiment with the fire coral Millepora alcicornis , which is sensitive to environmental changes. We exposed M . alcicornis to a realistic oil-spill scenario in which we applied an innovative multi-domain bioremediator consortium (bacteria, filamentous fungi, and yeast) and a chemical dispersant (Corexit® 9500, one of the most widely used dispersants), to assess the effects on host health and host-associated microbial communities. Results The selected multi-domain microbial consortium helped to mitigate the impacts of the oil, substantially degrading the polycyclic aromatic and n-alkane fractions and maintaining the physiological integrity of the corals. Exposure to Corexit 9500 negatively impacted the host physiology and altered the coral-associated microbial community. After exposure, the abundances of certain bacterial genera such as Rugeria and Roseovarius increased, as previously reported in stressed or diseased corals. We also identified several bioindicators of Corexit 9500 in the microbiome. The impact of Corexit 9500 on the coral health and microbial community was far greater than oil alone, killing corals after only 4 days of exposure in the flow-through system. In the treatments with Corexit 9500, the action of the bioremediator consortium could not be observed directly because of the extreme toxicity of the dispersant to M . alcicornis and its associated microbiome. Conclusions Our results emphasize the importance of investigating the host-associated microbiome in order to detect and mitigate the effects of oil contamination on corals and the potential role of microbial mitigation and bioindicators as conservation tools. Chemical dispersants were far more damaging to corals and their associated microbiome than oil, and should not be used close to coral reefs. This study can aid in decision-making to minimize the negative effects of oil and dispersants on coral reefs. \n Video abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-021-01041-w.",
"conclusion": "Conclusions Our study concluded that the chemical dispersant Corexit 9500 was far more toxic to M . alcicornis than the oil itself, in a flow-through experiment simulating realistic conditions. This study can help companies and governmental agencies in their decision-making about the use of chemical or biological remediation, since we showed that BMC-BC minimizes the negative oil effects without being toxic to the coral. This is also the first study to explore the effects of Corexit 9500 on the microbiome of calcifying cnidarians. Our results showed that Corexit 9500 caused a significant shift in the bacterial community associated with the hydrocoral M . alcicornis . In addition, this study is a proof-of-concept that multi-domain BMC-BCs consortia can be used to mitigate the impacts of oil on coral reefs and adjacent areas. The results emphasize the importance of investigating the host-associated microbiome to protect corals from anthropogenic impacts, as well as the possibility of using beneficial microbes as a tool for conservation purposes.",
"introduction": "Introduction Coral reefs are especially sensitive to environmental changes [ 1 ], which is becoming apparent as reefs experience increasing mass-bleaching events worldwide [ 2 ]. Corals “bleach” when they expel the microalgae living in their cells, without which the host cannot maintain a minimal energy input and will die if conditions are not stabilized [ 3 , 4 ]. Although climate change is presumed to be the main reason for coral bleaching and the disappearance of modern reefs [ 2 , 5 , 6 ], other factors such as poor water quality and pollution [ 7 – 11 ] can also cause bleaching and damage to coral cells. Oil spills occur worldwide in marine environments [ 12 – 15 ]. Exposure to chronic oil contamination can impair biological functions in corals, including reproduction and recruitment [ 16 ]. Chemical dispersants consist of a mixture of emulsifiers and solvents able to break oil into smaller droplets [ 17 – 19 ]. Previous studies have reported substantial declines in the health of corals in response to short-term exposure (0–96 h) to dispersants, and more severe impacts in response to oil-dispersant mixtures [ 20 ]. Among chemical dispersants, Corexit® products are the most commonly used worldwide, applied in some of the largest oil spills and cleanup operations. Oil and dispersants may also disturb the symbioses between corals and a diverse range of associated microorganisms (i.e., viruses, dinoflagellates, archaea, bacteria, and fungi) that are essential for host homeostasis [ 21 – 23 ]. Except for microalgae, symbiotic interactions between corals and other microbial-associated groups are only beginning to be revealed, but studies suggest that they play roles in nutrient cycling [ 24 , 25 26 ], antibiotic production [ 27 ], UV-damage protection [ 28 ], the production of photosynthate in the skeleton [ 29 ], and coral tissues [ 30 ]. Marine host-associated microbes are therefore key drivers of the structuring and functioning of ecosystems [ 31 ], and as either single strains or microbial consortia show potential applications as probiotics in conservation endeavors. Neutralization of toxic compounds is an important probiotic trait, with several potential applications for corals, as some coral-associated microbes can serve as oil-bioremediation agents. For example, Santos et al. [ 32 ] manipulated bacterial strains to protect corals against oil impacts by developing an oil-degrading bacteria consortium isolated from the coral Mussismilia harttii [ 32 ]. The authors assessed hydrocarbon degradation through the culture medium, with crude oil as the sole carbon source. Based on the success of this bioremediation study, a strategy for the manipulation of coral microbes was later proposed [ 21 ] and validated [ 33 ], which used “beneficial microorganisms for corals” (BMCs) to increase overall coral fitness through specific mechanisms. This new research field of coral probiotics opened several possibilities for mitigating threatening impacts on corals, including impacts from oil industry activities. Although past BMC experiments have used only bacteria to defend against pathogen and temperature stress [ 33 ], previous research has shown that specific hydrocarbon fractions can be more effectively degraded using a multi-domain coculture of bacteria and fungi [ 34 ]. Therefore, our main objectives were to (1) develop an environmentally friendly oil-mitigation alternative to chemical dispersants, i.e., bioremediation, through a multi-domain consortium (putative BMC-bioremediator consortium or pBMC-BC) composed of filamentous fungi, yeasts, and bacteria; (2) evaluate the effects of oil, dispersants, and pBMC-BC on corals; (3) investigate host-associated bacteria to evaluate their response to treatments, and identify microbial indicators for each treatment, thereby increasing our knowledge of potential targets for further surveys.",
"discussion": "Discussion Water pollution is one of the three main causes of reef loss globally [ 23 ]. Local management to minimize stressors can increase the ability of corals to cope with global impacts by reducing the synergistic effects caused by several stressors [ 48 , 49 ]. To this end, the United Nations recently emphasized the need to reduce marine pollution and protect and restore coral ecosystems in the “Global Goals for Sustainable Development” [ 50 ]. Recently, a committee of the National Academies of Sciences, Engineering, and Medicine reviewed possible local and global interventions to increase the resilience of coral reefs [ 51 ]. Among these interventions, the manipulation of beneficial microorganisms [ 21 ] and the development of pollution remediation approaches were listed as possible strategies to help coral persistence. Bioremediation methods have advantages compared with other oil cleanup techniques, which include sustainability, lower costs, and applicability across different ecosystems with minimal impacts [ 32 , 52 , 53 ]. The use of oil-degrading bacteria to remediate oil contamination may have benefits in addition to the degradation of compounds. For instance, Santos et al. [ 32 ] successfully minimized the toxicity of oil to the coral Mussismilia harttii with a bacterial probiotic consortium. Probiotics were initially defined as live microbes that can benefit human health [ 54 ]. This definition was later extended to include any host system, including corals [ 55 ]. One of the benefits provided by microbial probiotics is neutralization of toxic compounds [ 56 , 57 ], protecting the hosts against their harmful effects. Therefore, the use of coral-associated microbes to mitigate oil contamination and its consequent impact on coral health can be considered a probiotic approach. However, uptake of a specific inoculated oil-degrading strain by corals is not crucial for defining it as a probiotic, since oil is often degraded in the surrounding water. Our results showed that, although the pBMC-BC consortium members could not be detected in the coral-microbiome assays, inoculation of the consortium was able to mitigate the negative physiological effects observed from the application of oWSF, as indicated by our indirect proxy ( F v / F m rates) and visible physiological responses (death and bleaching). The use of coral-associated microbial consortia has proven to benefit coral health in the presence of oil [ 32 ], marine pathogens, and increased temperatures [ 33 ]. The application of the multi-domain consortium resulted in degradation of n -alkanes and significant decrease of PAH hydrocarbon fractions. A specific strain of microorganism is usually unable to degrade several different hydrocarbon fractions of oil; rather, hydrocarbon degradation is more efficient when there is a set of microorganisms that degrade certain components [ 58 ]. This study provides evidence that a multi-domain consortium isolated from the coral microbiome was efficient in degrading different oil fractions. Furthermore, the detected oil degradation was associated with improved direct and indirect coral health metrics at the last sampling time of the experiment, compared to samples without the pBMC-BC inoculation. The corals were, however, severely affected by Corexit 9500, in spite of the application of the beneficial consortium or the concomitant exposure to oil. Measurements of F v / F m revealed a separation into two main groups, those containing the dispersant and those without it. This information, together with photodocumentation of dead, bleached, or damaged tissue in the presence of Corexit 9500, showed that the dispersant damaged the animals shortly after application in the experimental conditions (Fig. 2 a, b). In addition, this study demonstrated that exposure to Corexit 9500 caused a significant change in the associated bacteria community of calcifying cnidarians, which occurred in parallel with a negative impact on the host physiology. This new information on the effect of Corexit on the associated bacterial community of a marine calcifying organism adds to the list of known harmful effects of chemical dispersants on the physiology of several species from different ecosystems [ 20 , 59 , 60 ]. In corals, the damage ranges from obvious effects such as bleaching and tissue necrosis [ 20 , 61 ] to more subtle consequences such as inhibition of fertilization and larval metamorphosis [ 62 ], both of which affect species perpetuation. Here, we observed these effects in a realistic, open-system experiment, and also revealed one more “invisible” impact that directly affects coral health: the effect of Corexit 9500 on the associated microbiome. Over 200 microbial genera have been reported as able to facultatively degrade petroleum hydrocarbons [ 63 ]. Among these, the genera Roseovarius and Erythrobacter increased in abundance in the presence of dispersant. However, the presence of the dispersant also reduced the abundance of some other oil-degrading bacteria, such as Thalossospira and Hyphomonas [ 64 – 66 ] (Fig. 5 ). This last genus, Hyphomonas , was also found to be a potential bioindicator of the presence of oil (Fig. 6 ). Different oil-degrading bacteria occurred in both the presence and absence of Corexit 9500, making it unclear whether the dispersant is affecting the capacity of the microbial population to remediate oil under the tested circumstances. In previous studies, chemical dispersants not only proved ineffective in promoting oil degradation but also retarded biodegradation [ 67 ]. Microorganisms have been used as bioindicators of different pollutants in marine ecosystems [ 32 , 68 ]. The presence of dispersants also increased the number of bacteria that were found to be related to diseased and stressed corals. For instance, the genus Ruegeria , previously reported as associated with diseased [ 69 ] and stressed [ 70 ] corals, increased in the treatments containing dispersants. Additionally, members of the genus Roseovarius , which are also associated with diseased [ 71 – 73 ] and stressed [ 70 ] corals, increased in the presence of dispersant over time (Figs. 5 , 6 and S 7 ). Other bioindicators of dispersants include Shimia , Thalassobius , Erythrobacter , and Desulfovibrio , all found to be related to diseased and stressed corals [ 70 , 72 , 74 – 76 ]. Taken together, these results suggest that disruption of the beneficial interactions of the associated microbial community could weaken the host, through an increase of commensal and opportunistic microbes, or as an immediate consequence of exposure to the dispersant. At the family level, an OTU closely related to a member of Flavobacteriaceae was one of the dispersant bioindicators. Our data agree with the findings of McFarlin et al. [ 77 ], which showed that the family Flavobacteriaceae was enriched in the presence of Corexit, and therefore an indicator of Corexit [ 77 ]. Members of this family include well-known opportunistic and pathogenic species [ 78 , 79 ] and are often overabundant in corals exposed to several stress factors [ 80 , 81 ]. Through our results and reports in the literature, we can predict that Flavobacteriaceae may have been one of the groups of microorganisms involved in the initial process of dysbiosis leading to the death of the coral. Although not classified as bioindicators, Vibrio OTUs increased in relative abundance in the presence of Corexit 9500 over time, which can be explained by the ability of some Vibrio species to metabolize dispersants [ 82 ]. Additionally, several species of Vibrio are pathogenic and opportunistic bacteria with many different groups of hosts, attacking humans, plants, and corals, among others [ 83 ]. They have many lysogenic islands that can be transferred horizontally intra- and inter-specifically [ 84 , 85 ], and their virulence can increase in stress conditions, such as a temperature increase [ 86 ]. In corals, Vibrio species are associated with several diseases [ 87 – 89 ]. We also observed an increase in members of the genus Vibrio after the exposure to Corexit 9500; these were the most abundant isolates in the presence of the dispersant [ 90 ]. These results suggest that chemical dispersants may affect coral health not only through their toxicity itself, but may also increase the abundance of opportunistic or pathogenic bacteria (i.e., members of the genus Vibrio ), which may cause dysbiosis and disease. Despite the fact that most of the bioindicators of Corexit 9500 have been described as opportunistic pathogens, one dispersant bioindicator, a member of the genus Labrenzia , has been previously reported as showing potential beneficial characteristics for corals by producing antimicrobial compounds [ 91 ]. The genome analysis of a Labrenzia strain associated with coral revealed 4 halo acid dehalogenase-encoding genes and one haloalkane dehalogenase-encoding gene, which can be used to degrade a broad range of aromatic halogens, haloalcohols, and halo acids [ 92 ]. Identification of members of this genus as dispersant bioindicators may be useful in further development of BMCs specifically selected to protect corals against Corexit 9500. Species of this genus are both potential BMCs and oil degraders, which makes them candidates for future experiments on cleanup of petroleum contamination close to coral reefs. On the other hand, bacteria previously correlated with healthy corals were also found to be bioindicators of the absence of dispersants, meaning that they were severely affected by the presence of Corexit 9500. Examples are a member of the genus Thalassospira , previously correlated with healthy coral hosts [ 93 ], and potentially involved with the phosphorus cycle [ 94 ] , Parvularcula , also associated with healthy corals [ 95 ], as well as the genus Inquilinus , reported as important for heat tolerance in corals [ 96 ]. The well-known coral symbiont Endozoicomonas was also negatively affected by the presence of chemical dispersants. Members of this genus have been frequently associated with healthy corals [ 97 – 99 ] , and the different strain genomes revealed functional adaptation and plasticity [ 100 ], suggesting that the relationship between this bacterial genus and the host is important to the adaptation and survival of the holobiont. This study addressed the impact of oil and Corexit 9500 on the coral physiology and microbiome, as well as the development of a bioremediation strategy that avoids the use of chemical dispersants in reef areas. As expected, the presence of Corexit 9500 impacted both the physiology and microbiome of the host shortly after application. In contrast, even though exposure to oil also impacted the coral health and physiology, it did not significantly change the microbiome structure. This result suggests that the natural microbiome of corals may be resilient to oil contamination up to a certain level, even when physiological parameters on the host side are affected. Previous research has shown that some resident coral-associated bacteria have the ability to degrade oil using it as a carbon source [ 101 ]. When confronted with an oil spill, oil-degrading bacteria can increase in abundance, as seen in deep-sea coral reefs impacted by the Deepwater Horizon oil spill [ 102 ]. The association with oil-degrading bacteria may be exploited as an important adaptation tool for the coral holobiont in areas experiencing oil spills, as it may increase the survivability of exposed corals [ 103 ]. Thus, administration of the multi-domain pBMC-MC consortium may further contribute to this adaptive response by increasing the abundance of oil-degrading bacteria in the holobiont. Indeed, the multi-domain pBMC-BC consortium was able to protect the corals from the negative effects of oil exposure, by increasing oil degradation and consequently improving host health, as measured by the Fv/Fm indirect health proxy and morphological traits. These results support the hypothesis that the bioremediation consortium could assist not only in degrading the oil in the water but also in maintaining the resilience of the natural coral microbiome against occasional oil spills. Thus, application of a multi-domain biodegrading consortium as an oil-spill response technique could be a useful alternative to dispersants, since it could provide two advantages: (1) filling the niche with probiotics that can prevent pathogenic organisms from colonizing coral reefs, and (2) helping to reduce hydrocarbon concentrations and their potential impacts on corals. As our data showed, physiological improvements to coral health can be achieved via a multi-domain consortium without causing major changes to the coral microbiome. It may be that the chosen microbial consortia consisted of microbes that are part of the rare biosphere which can perform critical functions, such as degrading oil without having high relative abundances, which has also been shown to occur in coastal seawater samples [ 104 , 105 ]. Inoculation of probiotics can also contribute to the establishment and succession of other beneficial microbes, as demonstrated through the use of pre- and probiotics in humans [ 106 , 107 ]. As the field of environmental probiotic research continues to grow, these rare taxa may be key in understanding how best to implement coral probiotics in the field without causing long-term changes to reef microbial communities. Another possible reason for the low abundance of the consortium members is that, although the members do not increase in abundance in the holobiont tissue, they may have increased in abundance in the surrounding water. However, this hypothesis is merely speculative. Further studies should also include the characterization of microbial abundance in the surrounding water using metagenomics and metatranscriptomics approaches, along with more-detailed analysis and quantification of hydrocarbons. Through these approaches, we can address yet-unanswered questions, allowing us to better understand the molecular mechanisms and ecological principles underpinning the beneficial effects of these microbial consortia on the holobiont. In view of the correlation between inoculation and improved coral health, in the future we will refer to them as BMC-BCs and not pBMC-BC. Currently, there are no known negative effects of readministering native beneficial bacteria back into a coral reef system to combat stress conditions. Nevertheless, many things remain to be learned about environmental probiotics and their application in natural systems. For instance, various obstacles must be overcome to make BMCs applicable and effective at large scale. Among these are consortium large-scale production and optimization; bioproduct maintenance during storage; delivery alternatives compatible with the actual conditions of offshore application; and logistical concerns, some of them extensively discussed by Peixoto and colleagues [ 49 ]. These challenges will be further addressed based on the results of ongoing studies. Ideally, before applying these developed technologies in the field, long-term experiments using realistic mesocosm systems, such as the present one, would be used to test their efficiency and map any potential risks. However, as time is increasingly short, urgent interventions must be put into practice, and the use of Beneficial Microorganisms for Corals (BMCs) is considered an extremely promising alternative. The persistence of coral reefs depends on many changes that are needed in the near future. The scientific community and environmental organizations must try to minimize the local and global impacts that affect reef survival. Coral reefs in the South Atlantic, considered major reef refuges [ 108 ], are currently experiencing unprecedented impacts, resulting in mass die-offs in this area [ 109 ]. A recent mass-mortality event affected about 90% of the fire coral M . alcicornis at one site [ 109 ]. Investigation of the M . alcicornis microbiome and selection of probiotics that can help to mitigate the effects of oil spills and other stressors can contribute to the protection of this important, and now potentially threatened, reef builder in the South Atlantic. This study examined the response of the coral microbiome to exposure to a chemical dispersant, furthering the understanding of ecological interactions—such as symbiosis and pathogenicity—between the host and its associated microbes under adverse stress conditions. Innovative actions in environmentally friendly strategies to mitigate marine oil pollution without causing side effects are insufficient [ 15 ] but are still needed. Our results and other studies in this field can contribute immensely to inform local actions to protect coral reefs in the Anthropocene, such as the mass die-offs caused by global change."
} | 6,096 |
37485419 | null | s2 | 4,898 | {
"abstract": "Hyperaccumulators' ability to take up large quantities of harmful heavy metals from contaminated soils and store them in their foliage makes them promising organisms for bioremediation. Here we demonstrate that some ecotypes of the zinc hyperaccumulator "
} | 63 |
33009437 | PMC7532197 | pmc | 4,899 | {
"abstract": "The increasing demand for high-density data storage leads to an increasing interest in novel memory concepts with high scalability and the opportunity of storing multiple bits in one cell. A promising candidate is the redox-based resistive switch repositing the information in form of different resistance states. For reliable programming, the underlying physical parameters need to be understood. We reveal that the programmable resistance states are linked to internal series resistances and the fundamental nonlinear switching kinetics. The switching kinetics of \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 -based cells was investigated in a wide range over 15 orders of magnitude from 10 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^5$$\\end{document} 5 s to 250 ps. The capacitive charging time of our device limits the direct observation of the set time below 770 ps, however, we found indication for an intrinsic switching speed of 10 ps at a stimulus of 3 V. On all time scales, multi-bit data storage capabilities were demonstrated. The elucidated link between fundamental material properties and multi-bit data storage paves the way for designing resistive switches for memory and neuromorphic applications.",
"introduction": "Introduction The class of redox-based resistive switching devices (ReRAM) based on the valence change mechanism (VCM) is a potential type for future non-volatile memory 1 , 2 , and computation-in-memory applications 3 – 7 . A typical VCM cell consists of a resistively switching oxide layer sandwiched between a high work function metal electrode such as Pt and a low work function metal, e. g. Ta. Among the numerous resistively switching oxides, \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 is a promising material in terms of endurance 8 , scalability 9 , switching speed 10 , and multilevel switching capability 11 . Before the VCM cell can be switched repetitively between a high resistive state (HRS) and a low resistive state (LRS), an electroforming step is required. For this, a voltage is applied to the cell and the oxide thin film is locally reduced by extraction of oxygen resulting in a highly-conducting, oxygen-deficient filamentary region 1 , 12 . The resistive switching effect has been attributed to a movement of mobile donors such as oxygen vacancies or cation interstitials, and a subsequent change in the filament composition leading to a valence change in the cation sublattice 1 , 13 – 15 . As the switching mechanism is dominated by the drift of ions, the switching operation is inherently bipolar. One voltage polarity is needed to set the cell from HRS to LRS, whereas the opposite voltage polarity is required to reset the device from LRS to HRS. Typically, an abrupt set transition is observed, whereas the reset transition is gradual 16 – 18 . The abrupt SET transition is a result of the local Joule heating in the filamentary region. As the device is initially in the HRS, only a small current flows through the device at the beginning of an electrical stimuli. This small current, however, increases the local temperature, which in turn increases the electrical conductivity. This results in a thermal runaway leading to an abrupt SET transition 19 , 20 . During the RESET Joule heating also occurs, and the oxygen vacancies drift away from the active electrode (high work function metal). The resulting depletion of oxygen vacancies at the active electrode decreases the electrical conductivity. Additionally, a diffusion current of oxygen vacancies toward the active electrode sets in. Both effects lead to the gradual RESET of VCM devices 17 . The capability of multilevel operation, i. e. storing multiple bits per cell, enhances the storage density 21 – 23 . In that case, the programming process is stopped at a specific intermediate resistive state (IRS) and is controlled either by the applied voltage during the gradual reset of the cell 17 , 21 , 24 or by a current compliance during the set operation 24 , 25 . For neuromorphic applications, the feature of multilevel switching is essential 26 , 27 . In order to meet the needs for future non-volatile memories, the so-called voltage-time-dilemma has to be overcome 1 . This corresponds to an extremely nonlinear switching kinetics of the ReRAM cell characterized by a low-voltage read-out operation over a long period up to ten years and a fast write process in the nanosecond regime or below by applying a voltage that is about ten times higher than the read voltage. While several groups have studied the switching kinetics of ReRAMs in certain limited ranges as compiled in 28 , an investigation over the complete dynamic range has not been demonstrated yet. To cover the full time-domain, the measurements have to be extended to the sub-nanosecond regime, too. Resistive switching in the sub-nanosecond regime has been qualitatively demonstrated for VCM cells based on \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {HfO}_2$$\\end{document} HfO 2 29 , \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 10 , \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {SiO}_2$$\\end{document} SiO 2 30 , and AlN 31 . The switching event, however, could not be resolved in these studies and the reproducibility of the switching on a single cell was rather low. Here, we present a comprehensive study of the switching kinetics of \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 -based VCM cells from 250 ps to up to 10 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^5$$\\end{document} 5 s by the means of an optimized coplanar waveguide (CPW) device structure and the use of multiple measurement setups. This enables us to resolve the switching time over 15 orders of magnitude at the same VCM cell. The work is exclusively focused on the set process. The reset kinetic is also topic of the authors’ current work and will be published separately. Furthermore, we demonstrate highly reproducible multilevel programming performed by varying the amplitude and length of the pulse. The data analysis reveals that the programmed LRS is linked to the inherent nonlinear VCM switching kinetics and an internal series resistance. Based on this finding, we discuss design rules for optimizing the multilevel programming capability of VCM cells integrated with a passive selector.",
"discussion": "Discussion By the combination of the results of different time regimes, the strong dependence of the set switching time on the pulse amplitude can be illustrated over 15 orders of magnitude (Fig. 4 ). Each red colored data point represents a resistive switching event from the HRS to a state of higher conductance, which may be either the LRS or one of the IRS. To the best of the authors’ knowledge it is the first time that such a high dynamic range of the switching kinetics including the picosecond regime is presented. Figure 4 Non-linear switching kinetics of a \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 CPW cell with \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A_1$$\\end{document} A 1 by the means of a \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{\\mathrm{set}}(V_p)$$\\end{document} t set ( V p ) plot over approximately 15 decades at the time scale (dark red and light red circles), and corresponding fit to Eq. ( 3 ) for \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$V<1.4$$\\end{document} V < 1.4 V (red line). Furthermore, \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{p}(V_{\\mathrm{min}})$$\\end{document} t p ( V min ) is plotted for each \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_p$$\\end{document} t p of Fig. 3 a (blue squares) and fitted according to Eq. ( 3 ) (blue line). The measurement limit is given by the RC time (770 ps) of the device. In the voltage range \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V| < 1.4$$\\end{document} | V | < 1.4 V (Fig. 4 ), the experimental data show a very strong nonlinearity following the empirical relation 3 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} t_{\\mathrm{set}} = t_0 \\displaystyle \\exp \\left( \\frac{\\kappa }{|V_p|-V_0}\\right) \\end{aligned}$$\\end{document} t set = t 0 exp κ | V p | - V 0 with the fit parameters \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\kappa =11.2$$\\end{document} κ = 11.2 V and \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$V_0=0.162$$\\end{document} V 0 = 0.162 V. The parameter \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_0 = 1.19\\times 10^{-13}$$\\end{document} t 0 = 1.19 × 10 - 13 s is equivalent to a wavenumber \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\tilde{\\nu }}=280\\,\\mathrm{cm}^{-1}$$\\end{document} ν ~ = 280 cm - 1 for amorphous \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 , which was found for the deformation modes of the Ta−O−Ta and Ta \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\equiv$$\\end{document} ≡ O bonds by infrared absorption spectroscopy 43 . Based on the suggestion that the electric-thermally activated migration of oxygen vacancies in \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 thin films is the responsible switching mechanism, the phonon vibrations represent the lower limit of the switching time for high voltages \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$V_p\\rightarrow \\infty$$\\end{document} V p → ∞ . The same behavior was theoretically found in our previous study in which the oxygen vacancy movement was described by the Mott–Gurney Law 44 . In the voltage range \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V| > 1.4$$\\end{document} | V | > 1.4 V, which corresponds to shorter pulses, the measured behavior in Fig. 4 deviates from the expected one and the course of data points flattens towards slower switching times with increasing pulse amplitude. This is due to the fact of the non-neglectable RC time of the device. The equivalent circuit shown in Fig. 1 c–d comprises the series resistance \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R_{\\mathrm{S}}$$\\end{document} R S , the capacitances between the CPW electrodes and the ground planes \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$C_{\\mathrm{S}}$$\\end{document} C S , the capacitance of the ReRAM cell \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$C_{\\mathrm{cell}}$$\\end{document} C cell and the time-dependent cell resistance \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R_{\\mathrm{cell}}(t)$$\\end{document} R cell ( t ) . The time-invariant capacitances are determined with impedance measurements at 1 MHz for a cell area \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A_1$$\\end{document} A 1 and amount to \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$C_{\\mathrm{S}} = 10.6$$\\end{document} C S = 10.6 pF and to \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$C_{\\mathrm{cell}} =4.6$$\\end{document} C cell = 4.6 pF. Using the fit parameter \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R_{\\mathrm{S}} = 167$$\\end{document} R S = 167 \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Omega$$\\end{document} Ω for the series of 10 ns pulses of Fig. 3 a results in \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{RC} = 770$$\\end{document} t RC = 770 ps. The switching time is, consequently, not limited at 250 ps by internal physical processes, such as the migration of oxygen vacancies 19 , but by the capacitive charging of the cell. It was already shown in 10 that faster SET times down to 105 ps are possible in \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 devices, which coincides also with the limit of their setup. Based on these facts, we believe that faster SET times down to tens of picoseconds are realizable in ReRAM devices. Thus, the measured data pairs ( \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V_p|,t_{\\mathrm{set}}$$\\end{document} | V p | , t set ) represent an upper limit of the set time at a given pulse height. An improvement of the measurement accuracy could be possible by the means of RC reduction by decreasing the cell capacitance area. However, such an approach may run into a more pronounced impedance mismatch causing a stronger damping of the transmitted signal and a worse temporal resolution. In an ideal case ( \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{RC} \\rightarrow 0$$\\end{document} t RC → 0 ), the extrapolated behavior in Fig. 4 indicates an internal switching speed of about 10 ps for \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V| \\approx 3$$\\end{document} | V | ≈ 3 V. The blue colored data pairs \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{p}(V_{\\mathrm{min}})$$\\end{document} t p ( V min ) in Fig. 4 illustrate the relation between pulse width and minimum voltage taken from Fig. 3 a. In fact, these data points behave similarly to \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{\\mathrm{set}}(V_p)$$\\end{document} t set ( V p ) and can be fitted in a similar way via Eq. ( 3 ) with the parameter set \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_0 = 1.10\\times 10^{-13}$$\\end{document} t 0 = 1.10 × 10 - 13 s, \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\kappa =10.3$$\\end{document} κ = 10.3 V, and \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$V_0=0.124$$\\end{document} V 0 = 0.124 V. The resulting curve lies slightly below the switching kinetics data. For a given \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$V_{\\mathrm{min}}$$\\end{document} V min , the corresponding \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{p}$$\\end{document} t p represents the moment, at which the switching does not occur anymore. Otherwise, for a given \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{p}$$\\end{document} t p , the corresponding \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$V_{\\mathrm{min}}$$\\end{document} V min marks the voltage at which the transition halts. This analysis reveals the link between programmable resistance states and the intrinsic switching kinetics of the ReRAM cell. According to Eq. ( 2 ) and assuming an invariant internal series resistance, the programmed resistance at a specific pulse width is determined by the applied voltage and the minimum voltage. Pulse width and minimum voltage, however, are not independent of each other due to the switching kinetics. If the kinetics is strongly non-linear as it is indicated by the steep slope in the log( t )- V -diagram of Fig. 4 for \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V|_{\\mathrm{min}} < 1$$\\end{document} | V | min < 1 V and \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{p}>100$$\\end{document} t p > 100 ns, the minimum voltage is almost constant for all \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{p}$$\\end{document} t p and the programmed resistance predominantly depends on the pulse voltage amplitude. For a weak non-linearity, i. e. a flat slope \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$d\\log (t)/dV$$\\end{document} d log ( t ) / d V , an additional dependence of R on the time scale is present because of the sensitivity of \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V|_{\\mathrm{min}}$$\\end{document} | V | min to \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$t_{p}$$\\end{document} t p . From this point of view, a highly nonlinear switching kinetics will be beneficial in terms of variability, which is permanently of major interest for resistive switching cells 45 . As pointed out in 36 , 37 , the voltage divider effect caused by an external resistance improves the variability and the device endurance. For multilevel programming, however, a slight voltage variation close to \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V|_{\\mathrm{min}}$$\\end{document} | V | min could evoke a larger (not acceptable) resistance variability. Thus, the pulse amplitude \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V_p|$$\\end{document} | V p | should be sufficient higher than \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$|V|_{\\mathrm{min}}$$\\end{document} | V | min . As the series resistance is linear, the resistances programmed with different voltages lie close to each other. In a big array these different resistance states might be indistinguishable considering cell-to-cell variability 46 . A potential strategy to overcome this problem is the use of a nonlinear series resistance. Without additional elements, the multilevel programming of our devices is only feasible for the set operation. As already explained above, the reset is an abrupt transition from the LRS into the HRS due to the voltage divider effect. For neuromorphic applications, however, it is desirable to program different resistances during the set as well as during the reset operation. An suitable approach is the reduction of the voltage divider effect to emphasize the intrinsic gradual reset transition, e. g. by introducing a selector element with an asymmetric I - V characteristics 47 , 48 . For the set mode, this selector should limit the current and define the programmed resistance. For the reset mode, the selector should be highly conducting, so that the applied voltage would drop completely over the actual resistively switching element and the intrinsic gradual reset transition appears. In this way, multilevel programming capabilities could be achieved for both voltage polarities. In this work, the multilevel resistive switching of \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {Ta}_2 \\hbox {O}_5$$\\end{document} Ta 2 O 5 cells at pulse lengths down to 250 ps was presented. For nanosecond pulses the monitoring of transient currents enables us to resolve the set switching event, and to find a clear dependence between the applied voltage and the resulting switching time. In combination with long pulse experiments, the non-linearity of the switching kinetics over 15 orders of magnitudes was demonstrated. For pulse lengths longer several ns the migration of oxygen vacancies is the limiting parameter, for shorter pulses the RC time of the set-up restricts the switching speed. Nevertheless, the over-all behavior implies the overcome of the voltage time dilemma, which is essential for the use of any resistive two-terminal devices. The multilevel capability together with the high intrinsic switching time of a single bit, which was estimated with 10 ps at 3 V without any parasitic effects, provides the option to store multiple bits per cell in a time regime down to 100 ps, which is significantly faster than writing times of state-of-the-art memory devices."
} | 7,903 |
37095301 | PMC10284849 | pmc | 4,900 | {
"abstract": "Bacteria commonly face attacks from other strains using the type VI secretion system (T6SS), which acts like a molecular speargun to stab and intoxicate competitors. Here we show how bacteria can work together to collectively defend themselves against these attacks. This project began with an outreach activity: while developing an online computer game of bacterial warfare, we noticed that one strategist (“Slimy”) that made extracellular polymeric substances (EPS) was able to resist attacks from another strategist that employed the T6SS (“Stabby”). This observation motivated us to model this scenario more formally, using dedicated agent-based simulations. The model predicts that EPS production can serve as a collective defence mechanism, which protects both producing cells and neighbouring cells that do not make EPS. We then tested our model with a synthetic community that contains a T6SS-wielding attacker ( Acinetobacter baylyi ), and two T6SS-sensitive target strains ( Escherichia coli ) that either secrete EPS, or not. As predicted by our modelling, we find that the production of EPS leads to collective protection against T6SS attacks, where EPS producers protect each other and nearby non-producers. We identify two processes that explain this protection: EPS sharing between cells and a second general mechanism whereby groups of resistant cells shield susceptible cells, which we call “flank protection”. Our work shows how EPS-producing bacteria can work together to defend themselves from the type VI secretion system.",
"introduction": "Introduction Bacteria commonly live in densely populated, multispecies communities where they must compete for limited space and nutrients [ 1 – 3 ]. Life in these environments has driven the evolution of an array of bacterial weapon systems, used to inhibit or kill competing microorganisms [ 4 , 5 ]. One of the most widespread and sophisticated of these weapons is the type VI secretion system (T6SS), a contractile nanomachine that fires a needle into neighbouring bacteria to deliver toxic effector proteins [ 6 ]. Possession of a T6SS can confer a strong ecological advantage to bacterial cells [ 7 – 10 ]. Conversely, being on the receiving end of a T6SS attack can be lethal. Defending against the T6SS, however, presents a unique set of problems for targeted cells, as—unlike many other weapon systems—the T6SS does not rely on the target cell’s surface receptors or transport systems. Instead, it uses mechanical force to pierce the target cell’s membrane and deliver its toxic cargo, which limits the potential range of mechanisms that can be employed in defence [ 11 ]. In the face of these challenges, here we show how bacteria can work together to protect themselves against T6SS attacks. Individual-level defence mechanisms are known, including immunity proteins, capsule formation, target modification, envelope stress responses, and the removal of susceptibility factors [ 12 – 16 ]. However, some of the most powerful defences in bacteria come about when bacteria act as a collective, allowing groups of cells to take advantage of their large numbers [ 17 – 22 ]. This project began when we serendipitously identified a potential collective defence mechanism against the T6SS while developing a computer game, as part of an outreach activity. The game— Gut Wars [ 23 ]—uses an agent-based model developed by our group for research [ 17 , 24 – 26 ] to illustrate different bacterial warfare strategies in the human gut microbiome, and is freely available online for outreach at http://www.oum.ox.ac.uk/bacterialworld/gutwars/ . Running the game repeatedly, we noticed that “Slimy” strategists, which secrete extracellular polymeric substances (EPS), were particularly resistant against “Stabby” competitors armed with the T6SS. Secretion of EPS is widespread among bacteria and plays a key role in the formation of biofilms [ 27 , 28 ]. From observation of the game, we hypothesised that EPS has the potential to protect both the cell that produces it and others around it, thereby functioning as a powerful collective defence mechanism that had thus far been unappreciated. There is evidence that EPS has the potential to be physically resistant to T6SSs [ 12 – 15 ]. Moreover, biofilm formation—involving EPS secretion—is sometimes a response to bacterial competition via T6SSs [ 29 ]. We therefore decided to investigate whether EPS allows bacteria to work collectively in the face of T6SS attacks. Here, we develop agent-based models of competitions between T6SS attackers and EPS-producing target cells. As suggested by the computer game, these analyses predict that secreting EPS is a collective defence mechanism in which producer cells both protect each other, and also cross-protect nearby non-producing cells. We test our predictions using a synthetic community, in which T6SS-wielding Acinetobacter baylyi compete with Escherichia coli strains secreting free (i.e., non-membrane-bound) EPS in amounts comparable to clinical E. coli isolates. These experiments revealed that EPS can indeed serve as a powerful collective defence against T6SS attacks. We identify two processes by which this collective defence can occur: (i) EPS sharing between cells and (ii) what we call “flank protection”, whereby groups of resistant cells shield non-producers without the need for EPS sharing. The latter process appears to be a very general route to collective defence, because it is predicted to occur whatever the underlying mechanism of protection against the T6SS.",
"discussion": "Discussion The production of EPS is a defining property of bacterial biofilms, where the majority of bacteria are thought to spend their lives [ 28 ]. A key challenge for bacteria living in biofilms is the potential for exclusion by competing strains and species [ 4 ]. Our modelling shows how EPS production can help with this challenge by allowing bacteria to work together and protect themselves against armed competitors. Moreover, an experimental test of our model revealed that EPS dramatically increases E. coli survival in competition with T6SS-wielding A. baylyi . Here, colanic acid is secreted into the extracellular environment and we find evidence that it has the potential to protect cells other than the producer. Secretion of colanic acid is widespread in Enterobacteriaceae such as E. coli , Klebsiella , Salmonella , and Enterobacter [ 53 , 54 ], where it plays an important role in biofilm formation [ 49 , 50 , 55 ], and confers protection against various environmental stressors such as desiccation, osmotic stress, and oxidative stress [ 56 , 57 ]. Moreover, elevated EPS production (mucoidy) readily evolves in E. coli and other species in response to predation by phages [ 58 – 60 ], and membrane-anchored capsules can protect pathogenic bacteria from attacks by the immune system [ 61 – 63 ]. EPS-mediated protection from T6SS attacks could, therefore, evolve as a by-product of adaptation to other types of stressors, as well as a direct response to T6SS attacks itself. Both our modelling and experiments showed that EPS non-producing cells are able to survive T6SS attacks better when co-cultured with EPS producers. Our initial hypothesis was that this cross-protection stemmed from the sharing of loose EPS between EPS producers and non-producers. Unexpectedly, however, our simulations identified a second cross-protection mechanism, whereby groups of T6SS-resistant producers reduced the exposure of non-producers to attackers (flank protection). We turned to image analysis of our experimental system to examine these possibilities, which suggested that both flank protection and EPS sharing are important in practice. The role we find for EPS secretion implies that secreted EPS can shield non-producing cells, at a range spanning multiple cell lengths, in a manner not seen in the model. This finding does not necessarily imply that EPS has evolved to be shared with non-producers, however. Cross-protection of nearby non-producing cells can arise as a by-product of natural selection for sharing between cells of the same EPS-producing genotype. Indeed, social evolution theory predicts that the benefits from shared traits will typically fall on the producing genotype, if they are to be evolutionarily stable [ 33 , 64 , 65 ]. Such high-relatedness conditions are common during biofilm growth [ 64 ]. Overall, our findings suggest that EPS production will frequently result in collective resistance to T6SS attacks, including in cases where EPS is not shared between cells [ 15 ]. Flank protection, in particular, has the potential to be highly general: it is predicted to emerge whenever sensitive cell groups interface with resistant cell groups, without assuming anything about the underlying resistance mechanism. Flank protection, therefore, has the potential to be mediated by other mechanisms of T6SS resistance, such as envelope stress responses [ 13 , 16 ], or the production of immunity proteins [ 14 ]. Indeed, we hypothesise that flank protection has the potential to occur against any contact-dependent weapon, including against the T4SS, T7SS, and Cdi (contact-dependent growth inhibition) systems [ 4 ], adding further complexity to interbacterial interactions. Such effects underline the potential for third parties to weaken interactions between bacterial strains in diverse communities. This idea is known from the study of bacterial cooperation, whereby the presence of other species can limit the potential for cooperating strains to work together (“social insulation”) [ 65 , 66 ]. As such, flank protection is an example of a higher-order interaction [ 67 ] that extends beyond simple spatial segregation known to occur between two warring strains [ 68 ] and highlights the need to consider community context in interbacterial aggression. T6SS-wielding bacteria are increasingly being considered as potential biotherapeutics, capable of delivering toxins into pathogenic bacteria [ 69 , 70 ]. Our work cautions that many species have the potential to resist such attacks due to the widespread ability of bacteria to work as collectives. Consistent with this, bacteria are already known to display collective defences against antibiotics [ 17 , 19 , 22 ], predators [ 71 ] and phage [ 20 ], and biofilm formation continues to be a major issue in industry and medicine [ 72 – 75 ]. Strategies that seek to remove problem strains, therefore, need to consider the ability of bacteria to work both as individuals and as groups."
} | 2,634 |
32765469 | PMC7379126 | pmc | 4,901 | {
"abstract": "Cyanobacteria are the oldest photosynthetic microorganisms with good environmental adaptability. They are ubiquitous in light-exposed habitats on Earth. In recent years, cyanobacteria have become an ideal platform for producing biofuels and biochemicals from solar energy and carbon dioxide. Alka(e)nes are the main constituents of gasoline, diesel, and jet fuels. Alka(e)ne biosynthesis pathways are present in all sequenced cyanobacteria. Most cyanobacteria biosynthesize long chain alka(e)nes via acyl-acyl-carrier proteins reductase (AAR) and aldehyde-deformylating oxygenase (ADO). Alka(e)nes can be biodegraded by a variety of cyanobacteria, which lack a β-oxidation pathway. However, the mechanisms of alka(e)ne biodegradation in cyanobacteria remain elusive. In this study, a cyanobacterial alka(e)ne biodegradation pathway was uncovered by in vitro enzyme assays. Under high light, alka(e)nes in the membrane can be converted into alcohols and aldehydes by ADO, and aldehyde dehydrogenase (ALDH) can then convert the aldehydes into fatty acids to maintain lipid homeostasis in cyanobacteria. As highly reduced molecules, alka(e)nes could serve as electron donors to further reduce partially reduced reactive oxygen species (ROS) in cyanobacteria under high light. Alka(e)ne biodegradation may serve as an emergency mechanism for responding to the oxidative stress generated by excess light exposure. This study will shed new light on the roles of alka(e)ne metabolism in cyanobacteria. It is important to reduce the content of ROS by optimization of cultivation and genetic engineering for efficient alka(e)ne biosynthesis in cyanobacteria.",
"introduction": "Introduction Cyanobacteria are the oldest organisms that perform oxygenic photosynthesis similar to what is seen in higher plants. Ancient cyanobacteria played an important role in changing the Earth’s atmosphere from an anoxic to oxic atmosphere. They are ubiquitous in light-exposed habitats on Earth, including extreme environments such as deserts, rocks, salt lakes, hot springs, and polar regions. During their evolution, cyanobacteria have acquired diverse survival abilities that enable them to acclimate to the stress conditions. Light serves as an energy source for cyanobacteria and plays an essential role in photosynthesis. Cyanobacteria are often exposed to light of different intensities in their natural habitats. High light intensities may destabilize the balance between energy supply and consumption. The excess energy could cause partial reduction of oxygen to generate reactive oxygen species (ROS), such as singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 - ⋅ ) and hydroxyl radical (OH⋅), which would lead to oxidative stress ( Latifi et al., 2009 ; Muramatsu and Hihara, 2012 ). All cyanobacteria in light-exposed habitats are frequently subjected to ROS, as they are inevitably generated due to the fluctuations in light intensity. Acclimation mechanisms to ROS include the synthesis of antioxidant enzymes such as superoxide dismutase ( Herbert et al., 1992 ; Inupakutika et al., 2016 ) catalases ( Tichy and Vermaas, 1999 ; Mironov et al., 2019 ) and peroxidases ( Yamamoto et al., 1999 ; Mironov et al., 2019 ) and the enhanced production of antioxidants such as carotenoids ( Zhu et al., 2010 ; Magdaong and Blankenship, 2018 ), tocopherol ( Maeda et al., 2005 ; Sakuragi et al., 2006 ; Mene-Saffrane, 2018 ), and glutathione ( Cameron and Pakrasi, 2010 ) which can scavenge ROS. Since the ROS inevitably damage the membrane lipids of cyanobacteria, there must be some synergetic systems involved in the maintenance of lipid and redox homeostasis in cyanobacteria. There are three membrane systems, namely, outer membranes, plasma (inner) membranes, and thylakoid membranes, in cyanobacteria. The photosynthesis reactions occur in the thylakoid membranes ( Anderson, 1986 ; Mullineaux, 2014 ). The plasma and outer membranes facilitate the transport of ions and cofactors across the membranes by porins and permeases ( Laudenbach and Grossman, 1991 ). Therefore, membrane lipids are pivotal for the photosynthesis of cyanobacteria. Lipid peroxidation damage can be caused by ROS, which can react with biologically important molecules such as lipids, DNA, and proteins. Alka(e)nes are highly reduced and stable molecules. A wide variety of organisms, such as some plants, insects, and microorganisms, can biosynthesize alka(e)nes ( Howard and Blomquist, 2005 ; Schirmer et al., 2010 ; Bernard and Joubes, 2013 ). Alka(e)nes in plants can form a cuticle, acting as a protective barrier against biotic and abiotic stresses ( Bernard and Joubes, 2013 ). Insects can synthesize alka(e)nes and secrete them onto the cuticles as a waterproof barrier and as a signaling pheromone. Alka(e)nes in eukaryotic microalgae can act as storage intermediates in the biosynthesis of epoxides and other lipids ( Metzger and Casadevall, 1989 ). The alka(e)nes in cyanobacteria can modulate the cyclic electron flow to facilitate cell growth under cold stress ( Berla et al., 2015 ). Alka(e)nes can accumulate in the cytoplasmic and thylakoid membranes to facilitate curvature and promote membrane flexibility potentially ( Lea-Smith et al., 2016 ). Alka(e)ne metabolism is ubiquitous in cyanobacteria, regardless of habitat or morphology ( Klahn et al., 2014 ). Schirmer et al. (2010) found that most cyanobacteria biosynthesize long-chain alka(e)nes (C 15 and C 17 ) via acyl-acyl-carrier proteins (ACP) reductase (AAR) and aldehyde-deformylating oxygenase (ADO). A variety of cyanobacteria, such as Synechocystis strain UNIGA, Nostoc punctiforme , and Spirulina platensis ( Al-Hasan et al., 1998 ; Abed and Koster, 2005 ; El-Sheekh and Hamouda, 2014 ) are capable of degrading alka(e)nes. However, the alka(e)ne biodegradation pathways in cyanobacteria have not been identified. Previous results have shown that ADO can incorporate an oxygen atom into the alkane to generate alcohol and aldehyde, which indicates that ADO has alkane monooxygenase activity and could catalyze the key reaction of alka(e)ne biodegradation in cyanobacteria ( Aukema et al., 2013 ). In addition, an aldehyde dehydrogenase (ALDH, EC 1.2.1.5) encoded by slr0091 in Synechocystis sp. PCC 6803 (hereafter Syn6803) can convert aldehydes to the corresponding fatty acids. It was shown that the transcript level of ado increased notably, while transcript level of aar remained unchanged in Syn6803 under high light ( Mitschke et al., 2011 ). The transcript level of aldh in Syn6803 also showed significant increase under high light and oxidative stress by real-time RT-PCR and transcriptomic analysis, respectively ( Trautmann et al., 2013 ; Hernandez-Prieto et al., 2016 ). The ubiquity of alka(e)ne metabolism has been observed in many photosynthetic organisms; however, the specific roles of alka(e)nes in cyanobacteria remain elusive. In this study, using in vitro enzymatic assays, we characterized a cyanobacterial ADO-ALDH alka(e)ne biodegradation pathway. Genetic and physiological analyses showed that alka(e)nes can serve as both fatty acid and electron sinks for maintaining lipid and redox homeostasis in cyanobacteria under oxidative stress. This study sheds new light on the relationship between alka(e)ne biodegradation and the ROS caused by high light.",
"discussion": "Discussion ADO Plays a Pivotal Role in Alka(e)ne Biodegradation in Cyanobacteria Aldehyde-deformylating oxygenase is a versatile catalyst in alka(e)ne metabolism in cyanobacteria. ADO, the key enzyme in alka(e)ne biosynthesis, can also catalyze the key reaction in alka(e)ne biodegradation. The enzymes involved in alka(e)ne biosynthesis and biodegradation are generally different ( Wang and Shao, 2013 ; Herman and Zhang, 2016 ). Cyanobacteria, which are evolutionarily ancient organisms, have relatively small genomes and few encoding genes. Therefore, cyanobacterial enzymes may play versatile physiological and metabolic roles. The ado – aar locus and the adjacent region consisting in Syn6803 show an extremely complex organization. Unexpectedly, ado and aar do not constitute an operon but are expressed independently and follow different regulatory patterns ( Klahn et al., 2014 ). The expression data suggest that ADO may play an additional role in a different functional context ( Mitschke et al., 2011 ). Aldehyde-deformylating oxygenase belongs to the nonheme diiron family of oxygenases exemplified by soluble methane monooxygenase (sMMO). The crystal structure of ADO is very similar to that of the α subunit of the MMO hydroxylase component (MMOH). The active sites of both ADO and MMO are housed within an antiparallel 4-helix bundle in which the two iron atoms are each coordinated to a histidine and two carboxylate ligands ( Marsh and Waugh, 2013 ). Notably, MMOH can oxidize a wide range of substrates, including C 1 –C 8 alka(e)nes ( Colby et al., 1977 ; Green and Dalton, 1989 ). Methane monooxygenase is much more complicated than ADO. sMMO consists of three components, a hydroxylase (MMOH), which contains the active site, a reductase (MMOR), and a regulatory protein (MMOB) ( Lee et al., 2013 ). MMOH consists of three subunits (α, β, and γ) that form an α2β2γ2 homodimer and the nonheme diiron active site that catalyzes the hydroxylation of methane and other substrates. MMOR plays key roles in transferring reducing power to the active site of MMOH. MMOB is known to regulate the activity of MMOH ( Gassner and Lippard, 1999 ; Walters et al., 1999 ). The diiron center in ADO cycles between different iron valence states. The presence of ROS is conducive to the generation of high-valent metal ions ( Wang et al., 2016 ). ADO can form a high-valent Fe IV Fe IV intermediate similar to compound Q of the MMOH under oxidative stress. The Fe IV Fe IV species could activate oxygen and promote the hydroxylation of the initially formed alka(e)ne by a radical mechanism ( Aukema et al., 2013 ). The radicals are formed by the interaction of organic molecules with metal ions. Radical reactions in biological systems take place at the active sites of enzymes containing metal ions ( Rajakovich et al., 2015 ). Aldehyde-deformylating oxygenase also requires oxygen and reducing power for activity, but proteins analogous to MMOR and MMOB have not yet been identified in cyanobacteria. Hydrogen peroxide (H 2 O 2 ) can activate sMMO to catalyze the oxidation of methane and other substrates. MMOR, MMOB, O 2 , and NADH are generally required for the catalysis but can be substituted by H 2 O 2 , which can act as both the O 2 and electron sources for the reaction ( Jiang et al., 1993 ). H 2 O 2 is produced as an intermediate in O 2 reduction by cyanobacteria under oxidative stress ( Telor et al., 1986 ). Alka(e)ne Metabolism Is a Versatile System for Modulating Lipid and Redox Homeostasis in Cyanobacteria As photosynthetic organisms, cyanobacteria are often exposed to fluctuant light intensities in their natural habitat. From night to noon, the outdoor light intensity can range from near 0 to over 2000 μmol photons m –2 s –1 . In cyanobacteria, the generation of ROS byproducts by light-driven photosynthetic electron transport is inevitable, especially when the rate of electron transport exceeds that of electron consumption. ROS can cause severe oxidative damage to cellular components. Cyanobacteria have evolved various acclimatory mechanisms to maintain the balance between electron transport and consumption and to protect cells under stress conditions ( Latifi et al., 2009 ). Antioxidant systems in cyanobacteria can effectively cope with enhanced ROS caused by high light. Excess alka(e)nes would not cause significant cyanobacterial cell damage, while accumulated aldehydes may increase cytotoxicity and result in cell bleaching. Alka(e)nes localize to the cyanobacterial membranes, predominantly to thylakoid membranes ( Lea-Smith et al., 2016 ). Thylakoids are membrane-bound structures embedded in the cytoplasm of cyanobacteria ( Liberton et al., 2013 ). Alka(e)nes localized in the thylakoid membrane may serve as an emergency system to scavenge ROS and boost lipid turnover in situ . Several bacterial strains can degrade membrane-bound alkanes by soluble cytochrome P450 monooxygenases ( Maier et al., 2001 ; Van Beilen et al., 2006 ). In cyanobacteria, H 2 O 2 is inevitably generated and then reduced to hydroxyl radicals via the Fenton reaction under oxidative stress. The production of hydroxyl radicals is enhanced in the presence of transition metals. Hydroxyl radicals are highly reactive, and are thus very dangerous to the organism ( Sies, 1993 ). Unlike superoxide, which can be dismutated to O 2 or H 2 O 2 by superoxide dismutase, the hydroxyl radical cannot be scavenged by enzymatic catalysis ( Reiter et al., 1995 ). Alka(e)nes are highly reduced molecules, and excess electrons can be stored as alka(e)nes. The alka(e)ne-deficient Synechocystis mutant exhibited enhanced cyclic electron flow ( Berla et al., 2015 ), which indicates that alka(e)nes could serve as electron sinks to decrease electron leak to O 2 . Under oxidative stress, alka(e)nes can act as electron donors to reduce partially reduced ROS. In addition, alka(e)nes can be oxidized to fatty acids. Intracellular alka(e)nes remain at low but constant concentrations in cyanobacteria, which implies ongoing alka(e)ne biosynthesis and biodegradation to maintain homeostasis throughout periodic changes in light intensity. Alka(e)nes are chemically rather inert and must be activated before they can be metabolized. In most aerobic alka(e)ne degradation pathways, the alka(e)ne is oxidized to the corresponding primary alcohol by alka(e)ne monooxygenases/hydroxylases. The primary alcohol is further oxidized to the corresponding aldehyde and fatty acid. The fatty acid can be conjugated to coenzyme A (CoA) to form a fatty acyl-CoA, which is further processed by β-oxidation to generate acetyl-CoA. It has been proven that cyanobacteria lack a β-oxidation pathway ( Taylor, 2012 ). As photoautotrophic organisms, cyanobacteria do not need to use alka(e)nes as sources of carbon and energy. The correlation between oxidative stress and alka(e)ne degradation provides insight into the physiological roles of alka(e)ne metabolism in cyanobacteria. Alka(e)nes serve as a fatty acid and electron sink for maintaining lipid and redox homeostasis in cyanobacteria. Under oxidative stress, alka(e)nes can be converted to fatty acids by ADO and ALDH to regenerate the acyl chains of damaged lipids and scavenge ROS to maintain redox homeostasis in cyanobacteria ( Figure 5 ). FIGURE 5 Proposed model of lipid homeostasis via alka(e)ne biodegradation in cyanobacteria under high light. ROS accumulate significantly under high light, which leads to membrane lipid damage (represented as the red lines close to “Damage”) and changes in the iron valence in the ADO active center to form the Fe IV superoxo species. Then, the ADO with Fe IV superoxo species will convert the alka(e)nes (represented as green sphere) to the corresponding aldehydes, which will be catalytically converted to fatty acids by ALDH. The resulting fatty acids will support membrane lipid homeostasis under oxidative stress. Highly reduced alka(e)nes can serve as electron sinks for maintaining redox homeostasis in cyanobacteria. Under high light, alka(e)nes can act as electron donors to reduce the partially reduced ROS. In cyanobacteria, the transfer of excess electrons to oxygen can generate partially reduced ROS and lead to photodamage of cellular components under oxidative stress caused by high light. Alka(e)nes in cyanobacteria can be oxidized to aldehydes by ADO under high light. Subsequently, the highly reactive aldehyde intermediates can be converted to fatty acids. The fatty acids originating from alka(e)ne biodegradation can then be used for lipid turnover under oxidative stress. As electron sinks, alka(e)nes could simultaneously provide electrons to reduce ROS. Oxidative stress was verified as a critical obstacle to alka(e)ne biosynthesis in cyanobacteria. It is beneficial for the alka(e)ne biosynthesis in cyanobacteria to avoid high light irradiation. Improving the capacity of cyanobacteria to scavenge ROS by genetic engineering of the antioxidant systems will also contribute to alka(e)ne biosynthesis. This study will guide approaches and strategies for efficient alka(e)ne biosynthesis in cyanobacteria."
} | 4,127 |
39612478 | PMC11705919 | pmc | 4,904 | {
"abstract": "ABSTRACT The rhizospheres of plants and soil microorganisms are intricately interconnected. Tea trees are cultivated extensively on the karst plateau of Guizhou Province, China; however, the understanding of the interactions among fungal communities, community taxa, and diseases impacting tea tree in the soil rhizosphere is limited. Our aim is to offer insights for the advancement of modern agriculture in ecologically fragile karst tea gardens, as well as microbiomics concepts for green and sustainable environmental development. This study utilized the internal transcribed spacer high-throughput sequencing technology to explore the symbiotic relationship between rhizosphere fungi and plant disease feedback in multiple tea estates across the Guizhou Plateau. The ecological preferences and environmental thresholds of fungi were investigated via environmental variables. Furthermore, a correlation was established between different taxa and individual soil functions. Research has indicated that tea leaf blight disrupts symbiotic connections among fungal groups. For various taxa, we found that numerous taxa consistently maintained core positions within the community, whereas rare taxa were able to stabilize due to a high proportion of positive effects. Additionally, abundant taxa presented a wider range of environmental feedback, whereas the rare taxon diversity presented a stronger positive association with the soil Z score. This study contributes to our understanding of the importance of rare taxa in plant rhizosphere soil processes. Emphasis should be placed on the role of rare taxa in pest and disease control within green agriculture while also strengthening systematic development and biogeographical research related to rare taxa in this region. IMPORTANCE In this study, based on internal transcribed spacer high-throughput sequencing, fungal communities in the rhizosphere soil of tea trees and their interactions with the environment in karst areas were reported, and the symbiotic relationships of different fungal taxa and their feedback to the environment were described in detail by using the knowledge of microbial ecology. On this basis, it was found that tea tree diseases affect the symbiotic relationships of fungal taxa. At the same time, we found that rare taxa have stronger cooperative relationships in response to environmental changes and explored their participation in soil processes based on fungal trait sets. This study will provide basic data for the development of modern agriculture in tea gardens and theoretical basis for the sustainable prevention and control of tea tree diseases.",
"conclusion": "Conclusions Our study mainly focused on disease-mediated fungal changes in the rhizosphere soil of tea trees in karst tea garden ecosystems, as well as the interaction mechanisms of different fungal taxa and their responses to the environment. Our results provide reliable evidence that tea tree diseases increase fungal species richness but decrease fungal diversity, and that the distance between samples is significantly different. The results also revealed that the abundance of AT is closely related to its core position in the community, while the positive relationship between RT enables it to be stable in the community rather than being input from the external environment, which helps us focus on RT in maintaining plant root health. Importantly, RT has a more linear relationship with environmental variables, which also means that it plays a positive role in soil processes and their interactions with plants, especially in karst areas where carbon and nitrogen limitations are more prominent. In this study, we particularly emphasized the symbiotic relationship between RT and its response to the environment. The study of rhizosphere soil in tea gardens in karst areas via a large-scale geographic analysis will contribute to understanding the biogeographic pattern of RT and its relationship with the environment as a reference for the refined management of modern agriculture in karst areas.",
"introduction": "INTRODUCTION The population characteristics of ecosystems often include a high concentration of abundant taxa (AT) groups, a large number of rare taxa (RT), and low niche occupancy rates ( 1 ), which are widespread from the macro population to the micro world ( 2 , 3 ). Previous studies have shown that identifying the diverse distribution patterns, relationships, and functional properties of AT and RT in various natural ecosystems ( 4 , 5 ) and social phenomena ( 6 ) is always possible. Therefore, exploring the interaction between AT and RT and their involvement in environmental processes can help researchers understand microbially driven ecological processes and functions. Microbial communities participate in soil processes and geochemical cycles ( 7 ) and are essential for maintaining ecosystem stability ( 8 ). With constant advancements in the microbial sequencing technology, studies on the interaction mechanisms between distinct microbial groups and their coupling with the environment have become popular. Recent studies have shown that bacterial diversity and specific bacterial groups are key driving factors for soil multifunctionality in temperate arid and semiarid mountain ecosystems ( 9 ). The interaction between pathogenic and mycorrhizal fungi in soil networks can explain the coexistence of aboveground and underground biological communities in forest ecosystems ( 10 ). In eukaryotes, fungi are very different from bacteria in prokaryotes, such as filamentous fungi that grow on hyphae and are larger in size ( 11 ). Certain types of fungi in the soil infiltrate plant tissues through plant roots or spread at the spore level, forming extensive symbiotic relationships with host plants ( 12 , 13 ). Moreover, fungi play a fundamental ecological role in mediating plant mineral nutrition and alleviating nutrient limitations in other organisms and constitute a key group in ecological restoration research in ecologically fragile areas. The high physiological and morphological plasticity of fungi can improve the availability of soil nutrients and the absorption of crop nutrients along environmental gradients ( 14 ). However, there is limited research on the rich and rare taxa in agricultural ecosystems in ecologically fragile areas, such as karst areas, and little is known about the interaction mechanisms of rare taxa in soil fungal communities and their interactions with abiotic factors. Tea is the oldest and most popular caffeinated beverage worldwide, with tremendous economic, therapeutic, and cultural significance ( 15 ). The Guizhou Plateau has a long history of tea tree ( Camellia sinensis ) cultivation ( 16 , 17 ), with unique karst landforms, diverse climates, and abundant precipitation, which effectively protect tea tree resources in this region ( 18 ). Colletotrichum camelliae is a phytopathogenic fungus that causes brown blight in tea trees ( 19 ). This disease results in significant production and economic losses to the yield of some sensitive cultivated tea varieties ( 20 ). Currently, there are few studies on the rhizosphere fungi of tea gardens in karst areas affected by disease, and it is not clear how different taxa of fungi interact with each other and how they participate in environmental processes. It is understood that disease-induced changes in plant performance can trigger a series of indirect changes in the rhizosphere environment, significantly affecting the composition and assembly mechanism of the rhizosphere microbial community ( 21 ). Owing to the low ecological carrying capacity of karst regions ( 22 ), the transformation of traditional agriculture to a modern agricultural development model is particularly urgent ( 23 ), and green and sustainable agricultural measures need to be further optimized. Therefore, theoretical studies on the diversity of rhizosphere fungi in tea trees and their interactions are particularly important. This study aimed to examine (i) the composition and diversity of rhizosphere fungal communities under the influence of tea brown blight disease, (ii) the occupancy and co-occurrence relationships of different fungal taxa, (iii) the ecological preferences of abundant and rare taxa groups and their response to the environment, and (iv) the relationships between different fungal taxa and individual soil functions. The research results provide support for the geographical distribution of soil microorganisms in tea gardens in karst areas and for green prevention and control in the future.",
"discussion": "DISCUSSION Tea tree diseases lead to the emergence of indicator species in different taxa of rhizosphere fungi Soil is an essential biological matrix in nature, and plant root exudates that are released into the soil can affect soil properties. Rhizosphere and ectorhizosphere soils were the first to respond to this phenomenon because it was proved that root exudates from plants can enhance the abundance of beneficial taxa for specific plant species, thereby affecting the composition and function of soil microorganisms ( 24 ). This study revealed that the available nutrient content was affected by tea tree diseases. The AP and AK contents of healthy samples were slightly lower than those of diseased samples (Table S1) possibly because tea tree diseases cause poor absorption of nutrients that cannot be utilized. Moreover, certain pathogenic microorganisms can also consume nutrients in the soil, leading to nutrient imbalance. This phenomenon has also been observed in previous studies ( 25 ). Importantly, unhealthy tea tree soil frequently becomes acidified (Table S1), which directly changes the soil environment and leads to changes in the beneficial microbial population ( 26 ). These changes are not conducive to the reproduction of beneficial microorganisms but accelerate the emergence of other harmful fungi ( 27 , 28 ). This indicates the importance of management methods based on modern agriculture as a basic concept by utilizing ecological principles and methods. Consequently, the stability of ecosystems and the sustainability of agricultural production can be achieved. Organic fertilizers, pesticides, and biological control methods can be used to improve the soil quality, protect the ecological environment, promote biodiversity, and enhance the quality of agricultural products, further reflecting the irreplaceability of soil as a biological substrate. Ascomycota and Basidiomycota were the main phyla of fungi belonging to GSP (Fig. S1 to S3), indicating that fungal taxa have a certain degree of stability at relatively high levels and are not affected by tea tree diseases. In GSP, compared with that in IT, the proportion of Ascomycota in AT generally decreased, whereas that of Basidiomycota generally increased. Some members of the phylum Basidiomycota form mycorrhizal fungi in symbiosis with plants ( 29 ), which is beneficial for crop cultivation. Some basidiomycetes can cause diseases in forests and garden plants ( 30 ), resulting in economic losses. The abundance‒occupancy relationship is a critical indicator for studying fungal community relationships as a core or host-specific group. A greater abundance of AT had a wider distribution, whereas a lower occupancy rate of RT implied a greater elimination risk ( Fig. 1 ). Previous studies support this viewpoint ( 31 ). In addition, OPLS-DA demonstrated (Fig. S5 to S6) that tea tree diseases have a significant effect on fungal communities, and AT screening revealed that the genus Penicillium was significantly present in the affected samples (M). Penicillium belongs to the heterotrophic aerobic type, which can cause plant Penicillium disease, causing the formation of large areas of disease spots in plants and plant death in severe cases ( 32 ). In RT, two families, namely, Bulleribasidiaceae and Phyllosticaceae, were present in the diseased sample (M), and Phyllosticaceae can cause plant leaf blight ( 33 ). Compared with nonfungi, fungi have relatively high symbiotic rates Microbial network relationships are driven by multiple factors, such as crossfeeding, legacy effects, and environmental filtering ( 34 ). Microorganisms often exist in symbiotic forms that are conducive to the construction of complex ecological networks ( 35 ). Our results revealed a relatively positive correlation in the RT (95.23%) network, and the significance test of the topological structure revealed that AT still played a core role in the community (Table S2; Fig. 2 ). Previous studies on the network relationships of soil microbial communities in the Hexi Corridor region of China have shown that most of the network links within the taxa are positively correlated ( 36 ), which is consistent with our findings. RT is crucial for the construction of microbial networks and serves as an indicator of the evolution of soil processes and vegetation succession ( 4 , 5 ). In this study, we found that the internal connections between the RT taxa were the closest and mostly positive ( Fig. 3a and b ). Studies have shown that rare taxa have important ecological functions, including element cycling, pollutant degradation, and host health ( 37 ). There were fewer connections between AT and RT, and the proportion of negative correlations was greater than that between the other groups ( Table 2 ). RT was located at the center of the network and tended to cooperate with the intermediate taxa (IT) ( Fig. 3a and b ). The reasons for their lower cooperation with AT are partly their abundance‒occupancy relationships ( Fig. 1 ) and partly the key role that they may be playing in enhancing the stress resistance of fungal communities, as well as maintaining their structure and stability ( 38 ). The key role of RT is self-evident. Generally speaking, abundant and rare taxa exhibit different responses to environmental changes, while rare taxa are more sensitive ( 39 ). Studies have shown that RT with flexible and diverse taxa can improve the selection efficiency of key taxa rather than relying solely on the input of new microbial taxa under environmental interference ( 40 ). Notably, tea leaf blight increased the negative correlation between species of different taxa in the fungal network, and the topological structure became loose (Table S3). This means that the resource competition between taxa caused by plant diseases intensifies, thereby disrupting community stability. Environmental response of abundant and rare taxa Soil microbes have significant ecological functions in the soil nutrient cycle and plant mineral nutrition ( 41 ). However, because of their susceptibility to environmental changes, external environmental changes frequently result in changes in variety and community structure ( 42 , 43 ). Here, we discuss the ecological preferences of soil microorganisms for the external environment. First, we observed that, compared with RT (19.75%) (Table S5), AT (28.68%) exhibited a broader ecological preference for environmental variables. This can also explain why, compared with RT, AT has greater adaptability to environmental changes and can effectively utilize a wider range of resources. Research on other agricultural ecosystems supports this view ( 31 ). Interestingly, among the top 25 OTUs in terms of the relative abundances of AT and RT, the response of RT to the environmental variables was slightly greater than that of AT ( Fig. 4 ). This phenomenon can be explained by the efficient selection of RT, which can stabilize it in the community and increase its occupancy rate, rather than by continuously updating RT through environmental intervention. In addition, the response traits of the AP and AK preferences in fungal communities are relatively conserved (Table S5; Fig. 4 ), and previous studies have shown that root exudates can affect soil available nutrients during plant growth stages ( 44 ). Therefore, revealing the ecological preferences of fungal communities for available nutrients can be used to evaluate the effects of soil feedback on plant growth. Soil microorganisms affect the transformation and supply of nitrogen and other nutrients. An environmental threshold analysis revealed more negatively responsive species along the pH gradient in AT compared with positively responsive species ( Fig. 5 and 6 ). This is due to the fact that tea trees are well-suited for acidic soil, and environments with excessively high or low pH levels hinder nutrient absorption ( 45 , 46 ). A reasonable pH (4.0–4.8) is beneficial for improving disease resistance. This study focuses on the relationships between GSP and the individual soil functions of different environmental variables ( Fig. 7 and 8 ). Standardized Z-scores for the environmental parameters revealed that individual soil functions and fungal α diversity differed surprisingly across taxa, as supported by similar studies on bacteria and fungi ( 9 , 47 ). There are several possible explanations for this phenomenon. First, the linear relationship between fungal community diversity and individual soil functions depends on the proportion of positive and negative species that respond to environmental gradients. Our ordinary least squares fitting trend was consistent with the environmental threshold analysis, which explains this issue. Second, as the relative abundance and occupancy of species increase, certain species with specific functions in relatively high-abundance taxa (AT and IT) may play a reduced role in the community, and the active cooperation of RT gradually increases the community stability. Another explanation is that AT and plant roots compete for scarce nutrients, especially in karst areas where nitrogen and phosphorus limitations were more prominent in our study. This hypothesis is supported by previous research ( 48 ). In addition, this phenomenon indicates that the relationships between different fungal taxa and individual soil functions manifest as differences in the ecological niche complementarity and stochastic processes of community ecology ( 49 ). From the perspectives of fungal involvement in co-occurring network construction and soil ecological functions, the interaction between rare fungal groups is crucial for determining the community composition and maintaining the ecosystem multifunctionality ( 50 ). Our research has found that species within rare taxa have more positive interactions, indicating the importance of species interactions within rare fungal subpopulations in supporting ecosystem function and stability ( 51 ). In addition, the cooperation of rare taxa may play a crucial role in their survival in tea garden soil (acidic). Most fungi characterized by hyphal growth are interconnected and form a network through hyphae, providing timely feedback on environmental changes, which helps share resources and coordinate microbial activities ( 52 ). Therefore, closely monitoring the cooperation between rare fungal groups can provide solutions for environmental disturbances, including plant diseases and extreme weather events, and enhance the resilience of microbial communities, even soil quality ( 53 ). Conclusions Our study mainly focused on disease-mediated fungal changes in the rhizosphere soil of tea trees in karst tea garden ecosystems, as well as the interaction mechanisms of different fungal taxa and their responses to the environment. Our results provide reliable evidence that tea tree diseases increase fungal species richness but decrease fungal diversity, and that the distance between samples is significantly different. The results also revealed that the abundance of AT is closely related to its core position in the community, while the positive relationship between RT enables it to be stable in the community rather than being input from the external environment, which helps us focus on RT in maintaining plant root health. Importantly, RT has a more linear relationship with environmental variables, which also means that it plays a positive role in soil processes and their interactions with plants, especially in karst areas where carbon and nitrogen limitations are more prominent. In this study, we particularly emphasized the symbiotic relationship between RT and its response to the environment. The study of rhizosphere soil in tea gardens in karst areas via a large-scale geographic analysis will contribute to understanding the biogeographic pattern of RT and its relationship with the environment as a reference for the refined management of modern agriculture in karst areas."
} | 5,139 |
35476526 | PMC9170161 | pmc | 4,905 | {
"abstract": "Significance Bacteria must respond quickly to environmental changes to survive. One way bacteria can respond to environmental stress is by undergoing a lifestyle transition from individual, free-swimming cells to a surface-associated community called a biofilm characterized by aggregative growth. The opportunistic pathogen Pseudomonas aeruginosa uses the Wsp chemosensory system to sense an unknown surface-associated cue. Here we show that the Wsp system senses cell envelope stress, specifically conditions that promote unfolded or misregulated periplasmic and inner membrane proteins. This work provides direct evidence that cell envelope stress is an important feature of surface sensing in P. aeruginosa .",
"discussion": "Discussion In this study, we present evidence that the Wsp system is stimulated by a wide range of compounds with diverse structures capable of perturbing the cell envelope. Our data suggest that unfolded proteins in the periplasm and inner membrane serve as a major stimulus of Wsp activity ( Figs. 3 and 4 ). Furthermore, reconstitution of the Wsp system in E. coli suggests that it is sufficient for sensing environmental stimuli, such as surface attachment ( Fig. 2 ). Finally, our data also suggest that cell envelope stress is an important feature of surface sensing in P. aeruginosa . One lingering question is how the Wsp system can sense such diverse cues. Previous work demonstrated that the periplasmic ligand binding domain of WspA is dispensable for surface sensing ( 18 ). Therefore, it is unlikely that the ligand binding domain of WspA is directly binding to a ligand in cells treated with these compounds. One possible explanation is the environmental changes that destabilize periplasmic and inner membrane proteins also induce structural changes in WspA which, in turn, activates the system. This phenomenon has been suggested for other methyl-accepting chemotaxis proteins. For example, pH, phenol, and osmotic stress all alter the structure of E. coli chemotaxis receptors and impact the chemotactic output (swimming direction) ( 64 – 66 ). It is possible that a similar mechanism is inducing Wsp signaling. The absence of DsbA activates Wsp. Since WspA has no cysteine residues, it is unlikely that DsbA activity is directly responsible for Wsp function and folding. Therefore, WspA structure and signaling could be influenced by periplasmic redox changes and increases in periplasmic hydrophobicity due to high levels of unfolded proteins and exposed cysteines in a dsbA mutant. Understanding exactly how WspA senses cell envelope stress is an ongoing area of research. The role of the Wsp system in the cell envelope stress-response network in P. aeruginosa remains to be defined. P. aeruginosa has at least two putative cell envelope stress-response systems, AmgRS and AlgU, which are predicted to be akin to Cpx and σ E , respectively ( 67 – 69 ). In E. coli , the Cpx and σ E responses are capable of sensing unfolded, aggregated proteins in the periplasm ( 41 , 42 , 70 ). Despite the similarities between P. aeruginosa and E. coli systems, it is currently unknown if/how AmgRS and AlgU respond to cell envelope stress. Our data revealed that Wsp does not perfectly mimic any E. coli stress-response system ( Fig. 4 A and Table 2 ). Wsp activators have broad effects on the cell envelope ( Table 2 ), and Wsp strongly responds to unfolded periplasmic proteins. The P. aeruginosa cell envelope stress response is likely an interconnected network, much like E. coli , but the degree of overlap between the Wsp, AmgRS, and AlgU pathways, the signals they respond to, and the functions they regulate are currently unclear. Here we show that the Wsp system is activated when unfolded periplasmic proteins are present. We also demonstrate that upon surface attachment, periplasmic proteins lose functionality ( Fig. 4 D ). While this indicates that surface contact and attachment generate cell envelope stress, it is unclear if the Wsp system solely responds to cell envelope stress or can respond to additional surface-induced cues. It is possible the surface sensing and cell envelope stress are two separate pathways that are sensed via the Wsp system and cause increased biofilm formation. Cell envelope stress has been postulated to be a feature of surface sensing in other species. Most of the evidence is indirect. In E. coli , the Cpx system is activated when E. coli contacts a surface and Cpx activation induces dsbA expression ( 33 ) and increases c-di-GMP levels via DgcZ ( 36 , 71 ), hinting that surface attachment might initially result in unfolded proteins. Loss of DsbA also induces biofilm formation in Pseudomonas putida and Salmonella Typhimurium ( 72 , 73 ). Additionally, the E. coli Rcs cell envelope stress response regulates the production of exopolysaccharides, and an rcsC mutant is defective in biofilm development ( 74 ). This suggests that the cell envelope stress and periplasmic protein integrity is linked to the biofilm mode of growth in multiple species. When considering the other surface-sensing mechanism in P. aeruginosa , the Pil-Chp chemosensory system, it’s clear that the cell can respond to different types of surface-associated signals. The nature of the Pil-Chp signal has been a focus of recent research and is linked to the type IV pilus. An output of both Wsp and Pil-Chp is cyclic-di-GMP production. It should be noted that cAMP is an output of the Pil-Chp response as well, so the two systems are not completely redundant in their output. The presence of these two systems begs the question: Will a surface always stimulate both systems? If not, what are the different situations where one system provides the predominant surface sensing response? In summary, our findings emphasize that P. aeruginosa senses surface association through diverse cues. Many of the previously described surface-sensing systems, like Pil-Chp, depend upon an extracellular appendage ( 3 , 4 , 6 , 75 , 76 ). Here, we show that cell envelope stress is one consequence of surface association and that P. aeruginosa can use the Wsp system to sense surface-induced cell envelope stress. Accounting for the different mechanisms by which bacteria sense surface and the heterogeneity in the responses will be critical for understanding the initial steps of biofilm formation and, ultimately, our ability to prevent them from forming."
} | 1,610 |
37637320 | PMC10449417 | pmc | 4,907 | {
"abstract": "Abstract Individual differences in behavior have large consequences for the way in which ecology impacts fitness. Individuals differ in how they explore their environment and how exploratory behavior benefits them. In group-living animals, behavioral heterogeneity can be beneficial because different individuals perform different tasks. For example, exploratory individuals may discover new food sources and recruit group members to exploit the food, while less exploratory individuals forgo the risks of exploration. Here we ask how individual variation in exploratory behavior affects the ability of Argentine ant Linepithema humile colonies to (1) locate novel food sources, (2) exploit known food resources, and (3) respond to disruptions while foraging. To address these questions, we conducted field experiments on L. humile foraging trails in which we manipulated food availability near and at the foraging trails and disrupted the foraging trails. We sampled individuals based on their response to the perturbations in the field and tested their exploratory behavior in the lab. We found that exploratory individuals benefit the colony by locating novel foods and increasing resource exploitation, but they do not play an important role in the recovery of a foraging trail after disruption. Thus, the benefits of behavioral heterogeneity to the group, specifically in exploratory behavior, differ across ecological contexts.",
"discussion": "Discussion We found that exploratory individuals are allocated to where they are most needed in the context of recruitment to new food and changes to existing food resources, but not in response to disturbance. Individuals that veered off the main foraging trail were more exploratory than individuals that kept to the trail ( Figure 2A ) regardless of the distance of the food bait from the foraging trail ( Figure 2B ). Furthermore, the proportion of exploratory individuals on the foraging trail increased in response to adding food to the foraging trail—the trail rate decreased over time ( Figure 3A ) and the average exploratory behavior of sampled individuals persisted ( Figure 3B ). Lastly, we did not find evidence that an ant’s response to a disturbance on a foraging trail is related to its exploratory behavior ( Figure 4 ). Colonies of L. humile seem to allocate exploratory individuals to the discovery of new food sources. Ants that were collected from off the trail were more exploratory than those that were collected from an established foraging trail ( Figure 2A ). This result is especially compelling because ants sampled from the main foraging trail likely included both individuals that would not veer off the trail as well as individuals that might deviate from the trail later. If we had a way to distinguish the two types of individuals when sampling from the main trail and collect only ants that conform to the foraging trail, we might have seen a larger effect size. Contrary to our predictions, we found that the distance an ant veered off the trail did not relate to its exploratory behavior ( Figure 2B ). Individuals that veer off from the main foraging trail can form new recruitment trails to novel foods ( Flanagan et al. 2013 ). Our work shows that behavioral differences among individuals may determine which ants instigate these new trails—the ones that are most exploratory. Previous work showed that exploratory behavior in L. humile is persistent for at least a few days and is linked to the expression of the Lhfor gene ( Page et al. 2018 ). Thus, in the timeframe of our experiments, exploratory behavior was likely a persistent trait. Future work might uncover further proximate mechanisms that underlie exploratory behavior in L. humile workers and determine the duration of its persistence. While it might seem surprising that there was no relationship between how far an ant veered off from the trail and its exploratory behavior, it is possible that high exploratory behavior is simply a switch for leaving an established trail. However, if it is not a switch, examining more and farther bait distances will reveal the relationship between exploratory behavior and bait distance. Linking lab quantification of exploration to the ecological meaning of the behavior ( Mouchet and Dingemanse 2021 ) is important for our understanding of individual variation in exploration. Exploratory behavior was linked to the exploitation of a known food source. Our experimental manipulation of adding food to the trail led to an increase in the proportion of exploratory individuals. The trail rate decreased while the exploratory behavior of individuals sampled from the trail did not change after adding food. Thus, the proportion of exploratory behavior increased. It is possible that because exploratory behavior is often linked with recruitment behavior in social insects ( Lemanski et al. 2019 ), adding a food source to the foraging trail required the recruitment of new ants to the added food. In contrast, in established trails that are not manipulated (like those examined by Page et al. [2018 ]), the proportion of exploratory behavior is at a steady state that is linked to the amount of food the trail leads to. Future studies may examine whether supplementing low-use trails results in a more prominent increase in the proportion of recruiting exploratory individuals relative to the increase in exploration we found on high-use trails. Finally, we did not find a relationship between exploratory behavior and response to disruptions while foraging. Ants that crossed a disturbed area did not differ significantly in their exploratory behavior from ants that did not cross a disturbed area ( Figure 4 ). It is possible that behaviors other than exploration relate to the propensity of an individual to cross a disturbed area. For example, Verbeek et al. (1994) showed that moving into a novel environment is related with boldness/risk taking in great tits. While previous work has linked exploratory behavior with risk taking ( Verbeek et al. 1994 ; Fraser et al. 2001 ; Wilson and Godin 2009 ), we did not find such a link. It is possible that our perturbation did not reflect naturally caused disturbances. Future work could examine different ways for quantifying risk-taking behavior in the field and lab to determine whether “risky” individuals are allocated to particular tasks, just like exploratory individuals. Understanding the ecological consequences of individual differences in behavior on the collective behavior of social animals may bring us closer to understanding the causes of these consistent individual differences. Our work shows that exploratory individuals are allocated to where they can best facilitate the collective foraging of ant colonies. It is possible that individuals that exhibit other behavioral types (like boldness or risk taking) are allocated differentially to where those behaviors are most beneficial for the colony. Uncovering when certain individuals are allocated to particular tasks and which behavioral types facilitate different collective behaviors is fundamental for understanding social organization. More broadly, our work highlights the importance of considering both ecological and social contexts when examining different types of behaviors."
} | 1,823 |
36556886 | PMC9786020 | pmc | 4,909 | {
"abstract": "ITO/WO x /TaN and ITO/WO x /AlO x /TaN memory cells were fabricated as a neuromorphic device that is compatible with CMOS. They are suitable for the information age, which requires a large amount of data as next-generation memory. The device with a thin AlO x layer deposited by atomic layer deposition (ALD) has different electrical characteristics from the device without an AlO x layer. The low current is achieved by inserting an ultra-thin AlO x layer between the switching layer and the bottom electrode due to the tunneling barrier effect. Moreover, the short-term memory characteristics in bilayer devices are enhanced. The WO x /AlO x device returns to the HRS without a separate reset process or energy consumption. The amount of gradual current reduction could be controlled by interval time. In addition, it is possible to maintain LRS for a longer time by forming it to implement long-term memory.",
"conclusion": "4. Conclusions Herein, we studied the ITO/WO x /AlO x /TaN device in which an ultra-thin film of AlO x was inserted between WO x and TaN in the ITO/WO x /TaN device. First, STEM and EDS mapping analyses were used to confirm the components of the device. Comparing the I-V and forming curves for the two devices, the WO x /AlO x -based device had lower energy consumption and higher endurance. The current is suppressed due to AlO x with a large band gap, so low-current switching is achieved by the tunneling barrier. This low current improves the short-term characteristics of the WO x -based device. In the pulse mode, the increased current returns to a high resistance state over time, and its relaxation time varies depending on the amplitude. This relaxation time also can be controlled by adjusting the interval between the read pulses. Thus, the implementation of flexible potentiation and depression is possible without an additional reset process using AlO x .",
"introduction": "1. Introduction The conventional digital computing system based on the von Neumann architecture has significantly developed in the past few decades, leading humans into the information age. Shortly, with the development of technologies such as artificial intelligence (AI) and the Internet of Things (IoT), the amount of data and information used in computing systems will exponentially increase [ 1 , 2 ]. However, the computing system based on the von Neumann structure is not suitable for handling large amounts of data in the Big Data era because of the structure that separates the computing part and the memory part in the computing system and the high-power consumption [ 3 , 4 ]. Thus, new memory devices should be developed to replace conventional memory to enhance the computing system. A neuromorphic system imitates the human brain or a biological system [ 5 , 6 , 7 , 8 ]. Unlike von Neumann’s architecture, a neuromorphic computing system consumes low energy by parallel processing similar to the connections that link neurons and synapses in parallel [ 9 , 10 , 11 ]. It is necessary to understand and imitate biological synapses for the implementation of a neuromorphic system. The human brain has two types of synaptic plasticity to maintain memory: short-term plasticity (STP) and long-term plasticity (LTP). It is possible to emulate bio-synaptic simulation in the RRAM devices by the input pulse repetition, pulse amplitude, and interval time [ 12 , 13 ]. This is very similar to the biological system in which the pre-synapse releases the synaptic transmitters, travels across the pre-synaptic transmission, passes through the post-synapse, and finally enters the post-synapse. The memristor is an electron memory device that can simulate artificial synapses [ 14 , 15 , 16 ]. Among several categories, resistance change-based new memory types, such as magnetic random-access memory (MRAM), ferroelectric random-access memory (FRAM), phase-change random-access memory (PRAM), and resistive random-access memory (RRAM), have been studied [ 17 , 18 , 19 , 20 ]. The mechanism of the MRAM device is based on the manipulation of the magnetization state through the spin-transfer torque effect. The FRAM causes the transition of a ferroelectric material by the voltage bias to lead to a change in the conductivity. The switching of the PRAM device is based on the change in the crystallinity of phase-change materials [ 21 , 22 , 23 ]. The RRAM device consists of a simple metal-insulator-metal (MIM) structure with a lot of materials including metal oxides and metal nitrides [ 24 ]. Among them, metal oxides such as HfO x , TaO x , and WO x are promising due to their superior memory performances. It maintains a low resistance state (LRS) in a specific voltage range and a high resistance state (HRS) in a different voltage range. The process of switching from HRS to LRS in the RRAM cell by applying the turn-on voltage is called “set”, and the process of switching from LRS to HRS by applying the turn-off voltage is called “reset” [ 25 ]. The set process is based on the phenomenon in which conducting filaments (CF) form inside the insulator or switching layer between the top electrode (TE) and bottom electrode (BE). In contrast, the reset process is based on a phenomenon in which the CF disappears. When applying extremely high voltages on the RRAM device, excessive CF can be formed, and the device no longer serves as a memory. Compliance current (CC) should be applied during the set process. If the CC is set up before performing a set switching, the current does not increase above a threshold level. It does not prevent a permanent breakdown of the device but also allows the CF to be resized. A larger CC produces larger filaments during the switching, and hence larger voltages are required to destroy and reconfigure the filaments. Short-term memory (STM) is a temporary potentiation/depression of neural connections. Unlike long-term memory, which lasts in the range of hours to years, an STM typically lasts for seconds to tens of minutes. Owing to the volatility of the memristors, short-term plasticity can be implemented in various memristors [ 26 ]. In addition, STP can be converted to LTP through iterative experience, which is related to stimulus in a synaptic structure. In this study, we fabricated the synaptic behaviors of WO x /AlO x -based RRAM devices for neuromorphic systems. ITO as TE and TaN as BE are compatible with the complementary metal-oxide-semiconductor (CMOS) fabrication process. The combination of a conductive oxide metal (ITO) and a metal (TaN) can control the WO x and AlO x layer for resistive switching by oxygen exchange. The control device was prepared without AlO x , and the device with a ~2 nm thickness between the WO x layer and TaN layer to make a difference in electrical characteristics by the introduction of an ultra-thin high-k film. Previous studies have reported the introduction of an ultra-thin high-k film, for example, HfO 2 and AlO x films [ 27 , 28 , 29 ]. However, there are limited studies on the bi-layer of AlO x and metal-oxide for STM. This study reports the effects of an AlO x layer on STM and its advantages as a neuromorphic device.",
"discussion": "3. Results and Discussion Figure 1 a shows the device stacking and fabrication process. The image shows the device prepared by sample cutting using FIB in the vertical direction from ITO to TaN. The TEM image shows a cross-sectional image of D2. The difference between the target thickness (~100 nm thick ITO, ~50 nm WO x , ~2 nm AlO x , and ~100 nm TaN) and deposited thickness is approximately ±5 nm when compared with the TEM image for each layer. Energy dispersive X-ray spectroscopy (EDS) was performed to analyze the components of D2. Figure 1 c shows the EDS mapping for each element with different colors. For confirmation, as shown in Figure 1 d, the atomic line scan was focused on the AlO x area located between WO x and TaN layers. At ~12 nm on the x -axis, the Ta (tan line) and the N (black line) indicate approximately 34% and 38% in the BE layer, respectively. Metal-TaN approximately contains a 1:1 ratio of Ta:N. Therefore, a metallic TaN layer was deposited [ 30 ]. The O ratio (red line) increases in the positive x -direction going from BE to the AlO x layer; this is because the TaN layer is easily oxidized [ 31 ]. Near 12–15 nm on the x -axis, a ~2 nm AlO x layer is observed with a much higher O ratio than the Al ratio [ 32 ]. The WO x layer, another switching layer from 15 nm on the x -axis, is presented as the W (turquoise line) and the O (red line). The atomic ratio of W and O is approximately 25% and 60%, respectively. In the reported RRAM paper, the WO x layer deposited by tungsten-reactive sputtering also contains 20–30% tungsten and 60–70% oxygen [ 33 ]. Overall, there is more oxygen than tungsten in the WO x layer. The electrical characteristics, including the resistive switching of the RRAM device, were analyzed by I-V curves, as shown in Figure 2 a,b. D1 (w/o AlO x ) and D2 (w/o AlO x ) were prepared to investigate the effect of 2-nm thin AlO x film on resistive switching and synaptic characteristics. The tunneling effect can occur since the AlO x film is ultra-thin; however, when a relatively high voltage such as the forming voltage is applied, the device with the AlO x layer undergoes a soft breakdown, and the tunneling effect is no longer considered. The tunneling effect will be discussed in detail later. The switching after filament formation inside the switching layers is shown in Figure 2 . A forming voltage (9 V) was applied to TE with a CC of 3 mA, which was used to prevent breakdown, inducing filament formation. Both D1 and D2 show similar switching curves after the forming process. This indicates that the switching characteristic of the WO x layer is more dominant after the filament is generated inside the very thin AlO x layer during the conducting filament formation process. Although D1 and D2 have the same thickness for the WO x layer, some differences such as set and reset voltage and forming I-V curve are observed, owing to the ultra-thin AlO x layer. The reset voltage, which is the maximum voltage, allows the switching operation. When −2.1 V is applied to D2, a stable reset process occurs within 100 cycles, whereas D1 requires a higher voltage of −3 V. The set voltage, where the current becomes 3 mA which is the same CC with the forming process, was plotted in Figure 2 c. D1 (1.6 to 2.35 V) has a relatively wider distribution than D2 (1.45 to 1.95 V) during the 100 cycles. This indicates that D2 has a narrower distribution and better uniformity than D1. Figure 2 f shows the endurance of HRS and LRS at the read voltage of 0.1 V for 1000 cycles. D1 maintains the LRS and HRS for 1000 cycles, except for changes in the early and later switching. D2 also maintains HRS and LRS, but the window that is smaller than D1 gradually grows during switching. I-V curves confirmed that the difference in resistive switching depends on the ultra-thin AlO x layer after the forming process. The thin AlO x layer can affect the filament in the thick WO x layer. The forming curves are limited by CC of 3 mA for the cell-to-cell of D1 and D2 in Figure 2 c,d, respectively. In the voltage sweep region (from −1 V to 1 V) in these two curves, the current of D1 increases with the voltage without significant suppression, whereas the current of D2 does not increase in a particular voltage region. The energy band gap (E g ) of AlO x and WO x are 8–8.8 eV and 3.0–3.2 eV, respectively [ 34 , 35 , 36 ]. Many oxygen vacancies exist inside the WO x layer because WO x was deposited by physical vapor deposition (PVD). The AlO x grown by ALD, which is a layer-by-layer process, has small defects such as oxygen vacancies inside the grown layer. Therefore, to form a filament inside AlO x , a higher voltage than WO x is commonly required, and it appears as the current-suppressed area in the forming curve. It can also be identified by the inserted forming voltage. The negative forming voltage of D1 (D2) is −5.45 to −4.05 V (−9.2 to −3.95 V) and the positive forming voltage is 3.9 to 6.45 V (10.85 to 13.25 V). The minimum voltage of D1 is smaller than that of D2 for soft breakdown to work when switching is possible as RRAM. Even before the forming process, switching characteristics significantly vary based on the AlO x layer. When a small voltage (<3.9 V) is applied to D1 without AlO x , it does not change from HRS to LRS. Above 3.9 V, the soft breakdown in the WO x layer may or may not occur. On the contrary, when ~6 V is applied to D2 with AlO x , as shown in the insert figure of Figure 3 a, the set switching from HRS to LRS occurs. Compared with D1, which undergoes non-switching at a small voltage, D2 is distinguishable between HRS and LRS. In addition, the short-term effect of returning to HRS is apparent. To analyze short-term memory characteristics, D2 was set to an interval time of 90 s between 5.0 and 5.5 V applied voltages, which is enough time to return to the HRS. The HRS/LRS ratio is well maintained regardless of the voltage amplitude, and the shift from HRS to LRS is not significant. The degree of shift is shown in Figure 3 b. The processing steps including voltage are the same as those in Figure 3 a, but the interval time is set differently to 0, 30, 60, 90, 120, and 150 s. As a result, the I LRS /I HRS fluctuates the most by 2.03 to 7.2 at 0 s and fluctuates the least by 2.85 to 3.21 at 90 s. The short-term memory effects can be observed in the LRS in a small voltage regime for D2, unlike D1. This behavior probably occurs due to the ~2 nm AlO x layer, which acts as a tunnel barrier. In D2 with an ultra-thin layer, the switching mechanism can be explained as a tunneling effect: Fowler–Nordheim tunneling (FNT) or direct tunneling (DT) [ 37 , 38 , 39 ]. The relationship equation between tunneling current (I) and voltage (V) is as follows: (1) I ∝ V e x p ( − 2 d 2 m * Φ B h ) : D T ( V < V t r a n s ) \n (2) I ∝ V 2 e x p ( − 4 d 2 m * Φ B 3 3 h e V ) : F N T ( V > V t r a n s ) , \nwhere d is the insulator thickness; m * is the effective mass of an electron; Φ B is the barrier height; h is Planck’s constant; and V t r a n s is the transition voltage from DT to FNT. DT is dominant at a very small voltage (< V t r a n s ). DT is not affected by the bias voltage but is influenced by the thickness of the switching layer (~2 nm AlO x ), as indicated by Equation (1). FNT appearing at a relatively high voltage (> V t r a n s ) is more influenced by voltage than thickness, as indicated by Equation (2). Therefore, in Figure 3 c, the dominant current flows by a tunneling effect. D2 can convert the memory state with a small voltage even though there is no forming process inside the switching layer. In Figure 3 d, when a small positive bias is applied to TE in D1, the electrons reach the BE by the formation of defects such as oxygen vacancies inside the WO x layer; however, the oxygen vacancies do not affect the conducting filament in the device. D2 has an AlO x layer located between the WO x and TaN layers; thus, when a small bias positive is applied, the current flow is improved as a result of the oxygen vacancies in the WO x moving toward AlO x . However, when the bias is removed, oxygen vacancies at the interface of WO x and AlO x are randomly dispersed returning eventually to the HRS [ 40 ]. Additionally, retention and pulse measurements showing the short-term characteristics of devices were performed. The retention data of D1 show how long WO x can maintain the LRS after the forming/reset/set process ( Figure 4 a). D2 identified whether the LRS can be maintained for a long time after the set process without forming the process in Figure 4 b. As a result of reading at 0.1 V for 10,000 s in the LRS immediately after the set process, the resistance of D1 increased by 50% from the initial low resistance after 345 s: from 1.683 kΩ to 2.535 kΩ. At this point, the resistance of D2 increased by 177.47% from 10.1 MΩ in the initial LRS to 28.1 MΩ. The fact that the LRS of two devices rises indicates returning to the HRS over time. Although forming bias and reset/set bias are applied to D1, it does not have good retention that preserves LRS or the “1” memory state. The weak retention is largely due to recombination between the oxygen and oxygen vacancies. The resistance of D2 significantly increases compared with that of D1 during the same time. The participating oxygen vacancies are less because the current caused by tunneling is due to the ultra-thin dielectric as well as the WO x internal defects [ 41 ]. Therefore, by adding the AlOx layer, the operating current of the device can be lowered to improve the short-term memory characteristics. To study the plasticity for performing the synapse function, the relaxation time was measured by following a read pulse after one pulse that increases the current of the device. In Figure 4 c, after the current of the device is increased by one pulse with an amplitude of 4 V and a pulse width of 1 ms, the total time for the current by a read pulse of 0.5 V to return gradually to the HRS is approximately 0.51 ms. As shown in the DC retention result, even though only the read is applied without a pulse that increases the current, the current by the pulse is also recovered spontaneously. Synaptic plasticity is a fundamental concept in biological systems. The changes in the weight of synapses can be emulated by the increase or decrease in the conductance in RRAM, for example, STP/LTP and potentiation/depression. First, the ratio at which the current recovers to its initial state was measured with the pulse amplitude. The larger the amplitude of the pulse, the lower the recovery speed. As illustrated in Figure 4 d, at 2 V, the current does not increase significantly, and hence, the relaxation time is also short. On the contrary, as the voltage amplitude increases to 4 V or 8 V, the current visibly increases, which results in a longer relaxation time. In the end, the stronger the stimulus, the greater the LTP than the STP, which is similar to a strong impression in the human brain or biological systems. However, when a significantly large amplitude is applied to the device, D2 eventually becomes forming and works in the same state as D1. Applying several pulses to D2, which is a short-term device with no switching failure to imitate synaptic potentiation and depression, suggests its suitability as a neuromorphic device. Figure 5 b shows a time-dependent pulse schematic of potentiation/depression. The black line represents the synaptic potentiation, and the red line represents the synaptic depression with an amplitude of 2 V, a width of 5 ms, and a read of 0.8 V. The conductance increases with a pair of read and positive pulses, but it decreases to only the read pulse without a negative pulse, using short-term characteristics. The depression is controlled by adjusting the interval time from reading to 0.1, 0.5, and 1 s. As a result, as shown in Figure 5 a, the conductance that performs the synaptic weight sufficiently potentiates and depresses differently depending on the interval time. When the degree of depression is fitted with an exponential decay equation, as shown in Figure 5 c, the factors that determine the slope are 1.00 × 10 − 7 (0.1 s), 8.59 × 10 − 6 (0.5 s), and 6.54 × 10 − 4 (1 s), respectively. Therefore, the reduction in conductance also increases as the time between the reads increases. Signals are transmitted by a neurotransmitter between pre-synapse and post-synapse. As it is transmitted, the weight of the synapse changes as it is activated. The TE performs presynaptic works and the BE performs postsynaptic works controlling the conductivity (weight) of the device. The conductivity change of the device is defined as an excitatory postsynaptic current (EPSC). As shown in Figure 6 a, the EPSC is implemented by applying a pulse width of 0.1 s and an amplitude of 2 V to TE. The black line indicates the pulse schematic, and the red time indicates the reacted EPSC. The conductive filament in bulk generated by the first pulse is then enhanced by the next pulse. As 10 pulses are input, in Figure 6 b, the EPSC is the same as accumulated. Comparing the first pulse with the 10th pulse, the EPSC gain (I 10 /I 1 ) improves from 1.06 to 1.12 when the pulse width (fixed 2 V) lengthens from 100 ms to 500 ms. On the other hand, as the amplitude (fixed 100 ms) enlarges from 1 V to 5 V, the EPSC gain decreases from 1.18 to 1.00. In a large amplitude, the conductivity significantly accelerates by the first pulse, so the 10th conductivity is insignificant. The EPSC of D2 with the short-term characteristic slowly decays to a steady state when the stimulus is removed. Therefore, the change in that magnitude is different by the interval time of the pulses. This effect is paired-pulse facilitation (PPF). The PPF is defined by Equation (3) which indicates how the current is triggered by the paired pulse.\n (3) P P F ( % ) = 100 * ( I 2 − I 1 ) / I 1 Here, I 1 denotes a current increased by the first stimulus, and I 2 denotes a current increased by the second stimulus. Figure 6 c show the PPF index by varying the interval time from 1 to 2000 ms for D2. During a very short interval time, the second current increased relatively, but as the interval time is longer, the increase gradually decreased. As a result, the decaying conductivity is controlled by adjusting the interval time due to the characteristics of recovering to the steady state of D2."
} | 5,408 |
30160973 | PMC6291803 | pmc | 4,910 | {
"abstract": "The\ncontrolled immobilization of biomolecules onto surfaces is\nrelevant in biosensing and cell biological research. Spatial control\nis achieved by surface-tethering molecules in micro- or nanoscale\npatterns. Yet, there is an increasing demand for temporal control\nover how long biomolecular cargo stays immobilized until released\ninto the medium. Here, we present a DNA hybridization-based approach\nto reversibly anchor biomolecular cargo onto micropatterned surfaces.\nCargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary,\nsurface-tethered strand. The cargo is released from the substrate\nby the addition of an oligonucleotide that disrupts the duplex interaction\nvia toehold-mediated strand displacement. The unbound tether strand\ncan be reloaded. The generic strategy is implemented with small-molecule\nor protein cargo, varying DNA sequences, and multiple surface patterning\nroutes. The approach may be used as a tool in biological research\nto switch membrane proteins from a locally fixed to a free state,\nor in biosensing to shed biomolecular receptors to regenerate the\nsensor surface.",
"conclusion": "Conclusions In\nthis report, we have described a generic route to temporarily\nimmobilize small-molecule and protein cargo via DNA hybridization\nonto micropatterned surfaces. Releasing cargo can be tuned via the\nwell-understood toehold-mediated strand displacement to control the\nextent of release. Furthermore, only readily available components\nsuch as DNA oligonucleotides are used. The approach can therefore\nbe easily adopted by other researchers. In future experiments, the\npredictability of DNA hybridization could help tune the duration of\nthe release step such as by shortening the DNA duplex. As a further\nadvantage, the sequence-specificity of DNA interaction could be exploited\nto anchor different cargo to different surface areas. Examples include\nantibodies or natural ligands directly conjugated to the C-DNA but\nalso DNA aptamers. Furthermore, cargo-freed surfaces can be reloaded\nwith biomolecular cargo. The rebinding efficiency is, however, lower\nand probably allows for the loading of cargo for no more than 2–3\ntimes even though the contrast in the micropatterns is not impaired\nby the reloading. When applied to biological experiments, DNA’s\nnegatively charged nature may bias the interaction with cells, but\nthe effect could be minimized by altering the salt concentration of\nthe buffer. Similarly, adhesion of cells to the patterns can be enhanced\nby supplementing surface-passivating BSA with fibronectin. 38 In conclusion, we expect that DNA-mediated release\nof protein cargo will enable exciting research in cell biology.",
"introduction": "Introduction Anchoring bioactive\nmolecules onto surface micro- or nanopatterns\nis important in sensing as well as for biophysical and cell biological\nresearch. 1 − 13 Spatially immobilized bioactive small-molecules or proteins can\nminiaturize biosensing or biophysical assays, such as to increase\nin microarray format the throughput of investigation, and minimize\nsample consumption. In biology, microclusters of biomolecular receptors\ncan mimic cell–cell contact by binding cognate cellular membrane\nproteins and thereby probe how the proteins’ distribution in\ncells and function is influenced by their defined localization. 1 , 2 Reversible anchoring of biomolecules onto surfaces is of increasing\ninterest. In cell biological research, controllable release of receptors\ncan switch membrane proteins from a fixed to an unbound state and\nthereby elucidate cell adhesion, 14 cell\nmigration, 15 signaling, 16 − 18 protein–protein\ninteractions, 19 or lateral diffusion of\nmembrane proteins. 1 , 20 Outside cell biology, the controllable\nshedding of protein-coated surfaces may be used to regenerate biomaterial\nactivity. Generating arrays for controllable release can utilize\nclassical\npatterning routes in which biochemically adhesive surface-patches\nare top-down fabricated and then linked to biomolecular cargo. 21 − 24 However, controllable release requires cleavable linkers that respond\nto an external trigger by severing the bond between cargo and surface.\nSpecialized photolytic linkers have been developed. 25 Yet, avoiding intense light can be beneficial in cell biology\nto minimize cytotoxicity. Of similar advantage would be a chemically\nsimple route with readily available components. Here we use\ndeoxyribose nucleic acid (DNA) association and dissociation\nas a means to spatiotemporally control the reversible attachment of\nbiomolecules onto surfaces. DNA-directed immobilization of molecular\ncargo was pioneered by the Niemeyer group 26 and further developed for use in biosensing and biomedical diagnostics,\nand for fundamental studies in biology and medicine. 27 − 29 Our approach for controlled immobilization and release relies on\ncompetitive hybridization/dehybridization 30 as shown in Figure 1 A. Anchor strand A-DNA ( Figure 1 A, black) is bound to the substrate surface. Figure 1 (A) Schematic\noverview of DNA-mediated binding, release, and rebinding\nof molecular cargo to a surface. Steps: capturing of biotin-tagged\nanchor strand A-DNA to surface-bound streptavidin (gray) followed\nby hybridization of cargo-strand C-DNA, release of cargo (yellow circle)\nby toehold-mediated strand displacement with release strand R-DNA,\nrebinding of C-DNA to A-DNA. (B) Schematic overview of the microstructured\nsurface featuring a dense poly(ethylene glycol) (PEG) film on a glass\nslide. The PEG film is microstructured and features biotin–PEG\npatches that bind streptavidin protein. Cargo strand C-DNA ( Figure 1 A, red) hybridizes to A-DNA thereby forming a short\nduplex\nwith a single-stranded overhang ( Figure 1 A, capturing). The terminus of C-DNA carries\nmolecular cargo ( Figure 1 , yellow circle) which is tethered via the duplex to the anchoring\nsite. However, the cargo can be released from the surface by the addition\nof release strand R-DNA (blue)( Figure 1 A). R-DNA is complementary to the entire length of\nC-DNA, thereby leading via toehold-mediated strand displacement 30 − 32 to a long nontethered duplex ( Figure 1 A, release). The liberated anchor strand can be reloaded\nwith C-DNA ( Figure 1 A, rebinding). The approach is related to the previous use of strand\ndisplacement for release of DNA-bound cargo from DNA-assembled supramolecular\nprotein conjugates 33 and the release of\nDNA-bound cells from solid substrates by means of restriction endonucleases. 34 We implement our approach of triggered\nrelease of molecular cargo\nwith a micropatterned substrate, relevant for many applications including\ncell biology. Our approach is demonstrated with patterns produced\nby photolithography and by microcontact printing. Photolithography\nhas the advantage of being automatable, whereas microcontact printing\nis cheaper and more flexible in its applications. 35 We expect that the combination of DNA-mediated controllable\nimmobilization on micropatterns will enable new research in cell biology\nsuch as on the formation of the immunological synapse. 36",
"discussion": "Results and Discussion Generation of Micropatterned\nSurfaces The substrate\nsurfaces for our DNA-based release strategy featured a homogeneous\npoly(ethylene glycol) (PEG) film grafted to a glass slide ( Figure 1 B). The dense layer\nof end-tethered PEG chains fulfilled two functions. It avoided the\nnonspecific adhesion of DNA and protein. In addition, part of the\nlayer’s polymer chains carried the biotin tag to form a grid-like\nmicropattern. The biotin bioaffinity pattern served to bind streptavidin\n( Figure 1 B) and thereby\nanchor biomolecular cargo onto the surface. The biochemically\npatterned substrate surfaces were generated using a method shown in Figure S1 by (i) grafting PEG diamine (MW 600\nD) onto epoxy-functionalized glass surfaces. The quality of the PEG\nlayer was confirmed with atomic force microscopy ( Figure S2 ). (ii) The free amine end of the PEG chains was\nthen modified with biotin using activated ester chemistry. The pattern\nof biotinylated vs nonbiotinylated areas was attained by photolithography.\nTherefore, the biotin–PEG film was (iii) embedded within a\nlayer of positive photoresist. The resist was (iv) illuminated with\nUV light and a grid-mask featuring round holes of 3 μm diameter\nseparated by a distance of 3 μm. (v) Illuminated photoresist\nwas removed using photolithographic developer solvent, followed by\nplasma-etching to oxidatively breakdown the now no-longer photoresist-embedded\nbiotin-PEG within the round features. Incubation with others organic\nsolvents stripped off the nonilluminated photoresist to yield a grid-patterned\nbiotin-PEG surface. The generation of micropatterns of round holes\nwithin the biotin-PEG layer was demonstrated by AFM analysis ( Figure S2 ). (vi) Exposed glass surface within\nthe round holes was backfilled by grafting with nonbiotinylated methoxy-PEG-silane\nthereby yielding the micropattern featuring the round features of\nnonbiotinylated PEG surrounded by biotin-PEG ( Figure 1 B). The functionality of the biotin-micropatterned\nPEG surface was\ndemonstrated by adding fluorophore-labeled streptavidin protein. It\nwas expected that the protein would bind via specific biomolecular\nrecognition to the biotin-grid pattern but not to the protein-repelling\nPEG discs without biotin. Indeed, fluorescence microscopy visualized\nthe expected grid-like pattern of bound Cy5-tagged streptavidin ( Figure S3 ). The contrast between biotin and nonbiotin,\nas judged by the fluorescence signals, was 0.98 ± 0.02. The contrast\nwas calculated by using the formula: contrast = ( F max – F min )/( F max – BG) whereby F max and F min are fluorescence counts\nin the bright, Cy5-streptavidin-coated biotin-PEG areas and in the\ndim, nonbiotin-PEG areas of the pattern, respectively. BG refers to\nbackground which is the glass surface that had not been exposed to\nCy5-streptavidin. Reversible Anchoring of Small-Molecule Cargo After\nvalidating the functionality of the biotin-patterns, we applied them\nfor our reversible anchoring approach. Therefore, the biotin-patterns\nwere decorated with A-DNA. This was achieved by first coating onto\nthe biotin pattern unlabeled streptavidin ( Figure 1 B) and then binding biotinylated A-DNA ( Figure 1 A, capturing). The\nmolecular interaction of biotin and streptavidin is known to be of\nhigh affinity and very reproducible. The successful decoration of\nthe grid patterns with A-DNA was demonstrated by hybridizing fluorophore\nCy3-labeled capture oligonucleotide C-DNA. The latter oligonucleotide\ncomprises the full complementary sequence of A-DNA but carries a single-stranded\n5′ extension. Fluorescence microscopic analysis ( Figure 2 A) shows that hybridization\nvia a short duplex was successful leading to a clear fluorescence\ngrid-like pattern of C-DNA signal that extends over hundreds of micrometers.\nThe contrast between bound and nonbound areas was 0.52 ± 0.04. Figure 2 Fluorescence\nmicroscopy confirms the DNA-mediated binding and release\nof small-molecule cargo anchored onto microstructured DNA-A surfaces.\nSchematic overview and fluorescence microscopic images of microstructured\nsurfaces (A) after incubation with C-DNA labeled with small-molecule\nCy3 fluorophore, (B) after toehold-mediated strand displacement with\nrelease R-DNA, and (C) after rehybridization of fluorescent labeled\nC-DNA. The line profiles of fluorescent microscopic images along the\nwhite lines of the microscopic images are shown to the right. Image\nsize: 96 μm × 96 μm. We next probed whether targeted release of cargo C-DNA can\nbe achieved\nwith competitive hybridization/dehybridization upon addition of R-DNA\n( Figure 1 A, release).\nIn model experiments, DNA release was first demonstrated in solution\nusing read-out with agarose gel electrophoresis, rather than on the\nglass surface ( Figure S4 ). Releasing cargo-carrying\nC-DNA by R-DNA was also successful on the surface as shown in fluorescence\nmicroscopic analysis ( Figure 2 B). The low fluorescence levels and the virtual absence of\nthe grid pattern indicate the almost complete removal of C-DNA, as\ncompared to the pattern before the addition of R-DNA. Quantitative\nanalysis of the fluorescence signal determined a 300-fold drop of\nDNA coverage ( Figure S5 ). This suggests\nthat R-DNA displaced anchoring A-DNA in the short DNA duplex thereby\nforming a new long DNA duplex between R-DNA and C-DNA ( Figure 1 A, release). Successful release\nand concomitant liberation of anchor strand into a single-stranded\nform was also demonstrated by reloading the freed anchor A-DNA with\nanother charge of fluorophore-labeled C-DNA ( Figure 1 A, rebinding). The microscopic image showed\nagain the grid-pattern ( Figure 2 C). However, the total amount of fluorescence was about 30%\nlower than in the first round of binding ( Figure S5 ). Several controls confirmed the specificity of the triggered\nrelease ( Figure S6 ). Release conditions\nsuch as incubation time with R-DNA were also optimized to achieve\ncomplete release ( Figure S6 ). Reversible\nAnchoring of Protein Cargo The DNA-mediated\nrelease principle was next implemented for protein-based molecular\ncargo ( Figure 3 ). As\na model protein, streptavidin was used. The protein was bound via\nbiotinylated cargo-DNA to the surface ( Figure 3 ). We used streptavidin as cargo as well\nas for obtaining the A-DNA micropatterns given the high affinity and\nhighly reproducible nature of the biotin–streptavidin interaction.\nPossible consequences of the double use of streptavidin, such as nonspecific\nbinding of cargo streptavidin to any residual biotin-PEG was avoided\nby the high-quality of the streptavidin micropatterns 37 ( Figures S6 and S9 ). Figure 3 Schematic overview\nof DNA strand-mediated binding, release, and\nrebinding of protein-based molecular cargo. Steps: capturing of biotin-tagged\nanchor strand A-DNA to surface-bound streptavidin (gray) followed\nby hybridization of conjugate C-DNA and streptavidin; release of protein\ncargo by toehold-mediated strand displacement with release strand\nR-DNA; rebinding of C-DNA onto A-DNA. The protein–DNA conjugate was obtained by mixing Atto550-labeled\nstreptavidin to the biotinylated cargo-DNA at a molar ratio of 1:4.\nRatios ranging from 2:1 to 1:20 were also prepared and analyzed\nvia gel electrophoresis ( Figure S7 ). The\nprotein–DNA conjugate was successfully hybridized onto the\nanchor-DNA modified surface ( Figure 3 , capturing), as shown by fluorescence microscopy analysis\n( Figure 4 A for a streptavidin:\nDNA ratio of 1:4; Figure S8 for a ratio\nof 1:10). Figure 4 Fluorescence microscopic analysis of DNA-mediated binding, release,\nand rebinding of streptavidin–DNA conjugates at microstructured\nA-DNA surfaces. Schematic overview and fluorescence microscopic images\nof microstructured surfaces (A) after incubation with C-DNA-streptavidin\nconjugates labeled with Cy3, (B) after toehold-mediated strand displacement\nwith release R-DNA, and (C) after rehybridization of C-DNA-streptavidin\nconjugates. The molar ratio for C-DNA and streptavidin was 4:1. The\nline profiles of fluorescent microscopic images along the white lines\nof the microscopic images are shown to the right. Image size: 96 μm\n× 96 μm. Microscopic analysis\n( Figure 4 B, Figure S8 ) also confirmed\nthat the surface-tethered protein cargo could be released from the\nsurface by adding R-DNA ( Figure 3 , release). As implied by the experimental results,\nrelease DNA led to the competitive dehybridization of C-DNA from A-DNA,\nand concomitant hybridization of cargo to release strand to form the\nlong duplex ( Figure 3 , release). Single-stranded A-DNA could be reloaded with cargo\nby adding fresh\nAtto550-streptavidin-tagged C-DNA ( Figure 3 , rebinding), as indicated by the grid-like\nfluorescence pattern ( Figure 4 C, Figure S8 ). The extent of loading\nwas, however, lower than for the first capturing ( Figure S9 ). Quantitative analysis of fluorescence brightness\nyielded 1450 ± 150 counts compared to 1800 ± 170 counts\nfor the first hybridization (for a streptavidin/DNA ratio of 1:4).\nFor a streptavidin/DNA ratio of 1:10, the corresponding counts were\n903 ± 126 and 1080 ± 110, respectively. Universality\nof the Anchoring Principle To demonstrate\nits universality, the principle of DNA-mediated anchoring of molecular\ncargo was extended to a non-PEG micropatterned surface. An additional\naim was to probe whether the orientation and sequence design of DNA\nstrands can be altered without affecting the release efficiency. The\nredesigned DNA duplexes and DNA strands are schematically shown in Figure 5 . As a first difference,\nA′-DNA is longer than C′-DNA; the opposite was the case\nin the previous design. Furthermore, R′-DNA binds to the free\ndistal 3′ terminus of the A′-DNA. In the previous design,\nR-DNA binds to the 5′ which is close to streptavidin. Finally,\nR′-DNA hybridizes to the A′-DNA to form the long duplex,\nthereby displacing C′-DNA. A consequence of duplex formation\nbetween anchor and release strand is that A′-DNA cannot be\nreloaded with C′-DNA. We tested the new DNA release design\nwith the fluorophore-labeled cargo strand. Figure 5 Schematic overview of\nDNA strand-mediated binding and release of\nsmall-molecule cargo from microstructured A′-DNA surfaces.\nSteps: Biotinylated A′-DNA is bound to surface-bound streptavidin\n(gray) and hybridizes C′-DNA carrying small-molecule cargo\n(yellow circle, fluorophore); release of cargo by toehold-mediated\nstrand displacement with release strand R′-DNA. The biotin\nand the fluorophore tags are attached to the 5′ termini of\nthe DNA strands. The micropatterned non-PEG\nsubstrate surface is shown in Figure S10 . It features streptavidin-coated patches\nthat are surrounded by a nonadsorptive layer of BSA protein. Both\nproteins are directly linked to the epoxy-coated glass surface. To\nmicropattern the surface with streptavidin and BSA, microcontact printing\nwas used ( Figure S10 ). A microstructured\nstamp composed of polydimethylsiloxane (PDMS) was first “inked”\nwith a solution of streptavidin. After removing streptavidin that\ndid not adhere to the PDMS surface, the stamp was inverted and placed\nonto the glass slide. Consequently, protein adherent to the elevations\nof the stamp were transferred onto the glass surface. Residual areas\nof the slide not coated with streptavidin were covered with BSA by\nadsorption from solution. The functionality of the micropatterned\nsurfaces with the new DNA\nduplex design was tested using fluorescence microscopy read-out ( Figure 6 ). The streptavidin\nsurfaces specifically bound biotinylated A′-DNA ( Figure 5 , capturing) because subsequent\nhybridization of fluorophore-labeled C′-DNA yielded micropatterns\nwith the expected shape and dimensions ( Figure 6 A). The contrast between bound and nonbound\nareas was 0.94 ± 0.08. Figure 6 Fluorescence microscopy images of streptavidin\nmicropatterned surfaces\nafter incubation with (A) A′-DNA and Atto488-labeled C′-DNA,\nand (B) after toehold-mediated strand displacement with R′-DNA.\nCorresponding line profiles of fluorescence microscopy images are\nshown on the right. Image size: 96 μm × 96 μm. Releasing cargo-carrying C′-DNA\nby R′-DNA was also\nsuccessful on the surface as shown in fluorescence microscopic analysis\n( Figure 6 B). After\nincubation with release-DNA the contrast between bound and nonbound\nareas reduced to 0.23 ± 0.03."
} | 4,853 |
39829850 | PMC11741383 | pmc | 4,911 | {
"abstract": "In this era of rapid global change, factors influencing the stability of ecosystems and their functions have come into the spotlight. For decades the relationship between stability and complexity has been investigated in modeled and empirical systems, yet results remain largely context dependent. To overcome this we leverage a multiscale inventory of fungi and bacteria ranging from single sites along an environmental gradient, to habitats inclusive of land, sea and stream, to an entire watershed. We use networks to assess the relationship between microbiome complexity and robustness and identify fundamental principles of stability. We demonstrate that while some facets of complexity are positively associated with robustness, others are not. Beyond positive biodiversity x robustness relationships we find that the number of “gatekeeper” species or those that are highly connected and central within their networks, and the proportion of predicted negative interactions are universal indicators of robust microbiomes. With the potential promise of microbiome engineering to address global challenges ranging from human to ecosystem health we identify properties of microbiomes for future experimental studies that may enhance their stability. We emphasize that features beyond biodiversity and additional characteristics beyond stability such as adaptability should be considered in these efforts.",
"introduction": "Introduction Stability, or the ability of biological communities to maintain their functions in the face of change, is paramount for the persistence of ecosystem services upon which all life on our planet relies. For over 70 years ecologists have debated the relationship between stability and ecosystem complexity (the biodiversity of an ecosystem and the interactions therein 1 , 2 ). Counter to previous paradigms put forth by Odum (1953) 3 , Elton (1958) 4 and others, supporting a positive complexity x stability relationship, May’s 1972 5 seminal paper proposed that more complex communities should be less stable. Among ecologists a resounding critique of May’s work was that these mathematical models did not represent real world systems. In response, food webs emerged as model natural study systems to determine the principles of stability 6 . However, even from these decades-long efforts, food web ecologists have yet to agree on the relationship between stability and complexity. Some propose complexity, inclusive of factors such as richness 7 , trophic interactions 8 and phylogenetic diversity within and among guilds 9 , among others, is a fundamental property of stability as it buffers against extinction cascades; while others propose the opposite, that complexity potentially reduces the proportion of strong interactions among species leaving food webs more susceptible to collapse 1 , 2 , 10 . More recently it has been predicted that other facets of complexity such as dominantly competitive, or other negative interactions among species should enhance stability by diffusing the spread of perturbations, while others find that instead, mutualisms are stabilizing 2 . Part of the incongruence among studies may be that the definition of complexity varies among studies, and study systems vary in their innate complexity. Therefore, identifying inherently complex systems and measuring multiple facets of complexity to examine stability x complexity interactions may help reconcile some of these differences. Microbiomes offer this opportunity as they are some of the most complex biological communities on earth often involving interactions among thousands of taxa and spanning the spectrum of biotic interactions and inhabiting basically every organism and environment on the planet 11 . While microbiomes may not always partition into discrete guilds like food webs, properties affecting their stability and robustness may be similar 12 , 13 . In ecological communities macro-organisms engage in complex relationships with each other ranging from positive (e.g., mutualism, commensalism) to negative (e.g., parasitism, amensalism), which together influence community composition and the health of hosts and ecosystems 14 . Microorganisms are no different 15 , taking part in intricate webs of interactions with other microbes, hosts, and the environment that sustain the metabolic and biogeochemical backdrop against which life persists 16 , 17 . Enhancing microbiome stability has recently come under the spotlight as an aspirational goal for management and engineering efforts, to encourage microbiomes to successfully establish and maintain their functions 18 , 19 . Stability is also considered a key factor for microbiomes to resist or remain resilient against disturbances such as climate change, changes in host diet, or antibiotic treatments that can profoundly affect diversity and community composition and lead to alternative stable states which may, or may not be desirable 20 , 21 . Stability is a property influenced by many interacting factors including community resistance and resilience to disturbance, as well as how a community responds to species losses 22 . Extinction cascades or, the degree to which the loss of one species impacts the loss of others in the same system, generates variation in community robustness, which is an important measure of stability 23 . In the case of microbiomes, extinction cascades can not only lead to a loss of microbial biodiversity, but also potentially profoundly affect host and ecosystem function 13 . Currently, it is unclear whether there are universal principles that govern microbiome stability, or whether certain microbiomes are more robust than others. While some hosts and environments harbor specific microbes, many microbes traverse these boundaries and microbiomes in general have a tendency towards nestedness 24 . Therefore, assessing the guiding principles of microbiome stability demands an ecosystem-scale approach. Unlike many macro-organismal food webs, it is challenging to directly observe phenomena such as extinction cascades, competition, or keystone species in microbiomes due to their complex, ephemeral, and microscopic nature. Methods to overcome these challenges include computational tools such as co-occurrence networks built from targeted or untargeted metagenomic data 25 , which are a practical lens to assess various components of stability including robustness. In these networks adopted from graph theory, nodes represent taxa and edges represent statistically significant occurrence or abundance associations between them, either positive or negative. An important caveat for these computational methods is that they generate predictions of biologically meaningful interactions, which need to be validated. However, the power of network methods lies in their ability to embrace the often otherwise intractable diversity of microbiomes to generate strong scalable hypotheses, which can then be tested through more reductionist approaches. Furthermore, co-occurrence patterns (e.g. presence/absence) form the fundamentals of community assembly regardless of whether members directly interact or not 26 . Similar to stable food webs, stable networks should be robust to node removal 27 , meaning their structures resist rapid collapse when nodes are removed. However, not all nodes are “created equal” and similar to the concept of keystone species, the removal of highly connected and central nodes within a network should lead to more rapid collapse 28 . Other node-specific or global network properties related to complexity should also impact robustness. For example, node richness and the ratio of edges to nodes should be positively correlated with robustness if there is a positive complexity x stability relationship. Modularity, which measures the partitioning of species into distinct and highly connected sub-communities, should also have a positive relationship with network stability where higher modularity should contain the effects of disturbance to specific modules rather than impacting the entire network 29 , a concept reminiscent of what May 5 referred to as “blocks.” Another is connectance or the number of realized edges in a network among all the possible ones. Higher degrees of connectance within a network should result in greater robustness due to more paths among nodes damping the effect of changes in any one node’s persistence on the persistence of others, similar to the concepts derived from MacArthur’s 1955 30 models of population and community stability. However, whether these, or other network properties universally increase microbiome robustness has yet to be established. We broadly define microbiome robustness as the relative ability to maintain network structure in the face of node removal. We predict that the sequential removal of highly central and connected nodes from networks will have the greatest negative effect on robustness relative to the removal of less central and less connected nodes, with the effect of random node removal intermediate between these two extremes. We also predict that network robustness will be positively correlated with multiple measures of complexity including richness, connectance, edge to node ratio, predicted negative interactions, modularity and phylogenetic diversity. We define a universal feature of microbiome robustness as a property that consistently predicts robust microbiomes across networks regardless of the spatial scale they represent. To determine whether there are universal properties of robust microbial networks requires a tractable, yet diverse study system, and so far studies are confined to single systems and sample types (mostly soil), and often single domains of microbial life (mostly bacteria), limiting their generalizability 31 . We address this by capturing free-living and host-associated microbiome diversity across a remarkably steep environmental gradient including connected marine, freshwater stream, and terrestrial habitats in a spatially compact and experimentally tractable watershed in Waimea, Oʻahu, USA ( Figure S1 ). Our model ridge-to-reef Hawaiian ecosystem spans an entire hydrologic cycle and four Köppen climate types, thus our study system plausibly reflects microbial diversity and dynamics at much broader geographic scales 24 .",
"discussion": "Discussion The stability of networks ranging from ecosystems to the internet to neuron pathways in the brain, or in this case, microbiomes, is affected by numerous properties of these systems inclusive of resistance and resilience against disturbance 8 , 35 , the ability to “rewire” interactions 36 , as well as their robustness or the ability to maintain structure and function in the face of loss such as brain damage or species extinctions 37 . The perceived importance of microbiome stability largely stems from studies of human and other organisms’ health where a dysbiotic, or unstable microbiome is considered a disease indicator 13 . However, disruption is natural in any system, therefore, defining the properties that maintain function despite disturbances are key. Network tools have their limitations for inferring specific functions, but there is mounting evidence that network complexity is often linked to stability in real world systems 1 . As demonstrated here, complexity cannot be defined by any one property and some are better predictors of robustness than others. From our assessment of 33 networks spanning microbiomes inhabiting a range of spatial scales, environments and habitats, universal principles of microbiome robustness have emerged. In particular, robust networks were characterized by the maintenance of taxa that are highly connected and central within their co-occurrence networks, especially interkingdom networks with relatively higher proportions of predicted negative interactions. The role of these highly connected and central taxa in maintaining network architecture has parallels to keystone species in food webs, where their extinction has drastic impacts on communities and their functions 38 . While it is difficult to predict from taxa-based co-occurrence networks what functions of the microbiome might be compromised by keystone species’ extinctions, it is clear from our results that the diversity and composition of both fungal and bacterial communities would change significantly. For example, across the whole watershed, removing <10% of the bacteria and fungi with the highest betweenness centrality values led to a loss of >40% of all nodes, and similar patterns were observed across networks at all spatial scales ( Fig. 2 ). While taxa with high betweenness centrality have similar roles in maintaining network structure, their identities among networks were not the same despite significant overlap in microbial community composition across hosts, habitats and the watershed ( Fig. S17 ). Therefore, the shared specific properties of these taxa that encourage robustness deserves further investigation. Interkingdom interactions generally increased robustness, but this may again be a product of node-based properties such as node betweenness centrality and node degree, which were always significantly higher in interkingdom networks than single domains. However, betweenness centrality may be a stronger determinant of stability than node degree alone as previous studies have shown that only these nodes act as bridges connecting other highly central nodes, and their removal decreases network function 39 . Higher interkingdom node degree and betweenness centrality may be owed to fungi acting as connectors between modules in multi-kingdom assemblages 40 , possibly through the provision of physical niche space for bacterial colonization and dispersal 41 , or via metabolites that bacteria may exploit in nutrient-limited environments 42 . Therefore, despite fungal networks alone being least robust, the presence of fungi led to overall increased network stability. We found interkingdom networks followed by bacteria and then fungi, to consistently harbor more predicted negative interactions, as well as a strong positive relationship between robustness and the proportion of negative edges in a given network. Whereas positive interactions have the potential to catalyze the mutual downfall of coupled species 13 , the prevalence of predicted negative interactions among more robust microbiome networks may be due to competition, predator-prey interactions, parasites or pathogens diffusing the effects of disturbances 43 while keeping populations of detrimental species in check. For example, food web models put forth by Gross et al. 10 found predator diversity to be a stabilizing factor by keeping prey populations under control. A similar result was also found in empirical food web research, where low predator-prey ratios tended to stabilize soil food webs 44 . Certain lineages of bacteria achieve this by suppressing pathogenic fungi through competitive root colonization, antifungal metabolite synthesis, or other biocontrol activities 45 . Pathogenic microbes themselves may also stabilize communities by promoting selected taxa and limiting the colonization of other microbes 46 . In our networks, fungal nodes assigned to Candida albicans always formed negative edges with Weissella , a genus of lactic acid bacteria with known antifungal activities that specifically inhibits C. albicans biofilm formation 47 . Weissella spp. also suppress pathogenic bacteria such as those in the genus Acinetobacter , and this negative link was also present in our networks 48 . We set out to assess not only the effect of targeted and untargeted (random) node removal on microbiome robustness, but also the relationship between robustness and various measures of complexity. We use a definition of complexity, node richness and their edges, that parallels the ecological definition of species diversity and their interactions 1 . We find that while node (taxa) richness alone has a positive relationship with robustness, other additional measures of complexity are equally, if not more important for predicting stable microbiomes. Specifically, two related indices, connectance or the proportion of realized predicted interactions relative to all possible ones, and observed edge to node ratio. Both properties have previously been shown to be important for the stability of food webs 6 , social networks and cells 37 , but here we find they are also strong predictors of microbiome stability, even when accounting for the effect of richness on these relationships. So, while much emphasis has been placed on the importance of biodiversity for maintaining function, we suggest that additional consideration of interaction type (positive or negative) and interaction frequency is warranted. Despite their stabilizing effect on bacterial networks, fungal networks alone were universally the least robust and defined by their high modularity, many positive edges and low node degree. High modularity is a network property that has repeatedly been associated with stability, purportedly due to the inability of disturbances to radiate beyond individual modules 14 , 31 . However, in the case of our robustness analyses which measured the remaining size of the largest network component (module) after node removal relative to its starting size, rapid module collapse may be due to the extinction of specific keystone taxa connecting multiple network components. This suggests that fungi connect sub-networks and potentially facilitate connectivity and resource sharing to a greater extent than bacteria 40 , but this increased communicability may be conferred at the expense of network stability 49 . Fungal networks were also composed primarily of positive edges (≥95%; Fig. 3 ), which could potentially explain their low robustness and predicted vulnerability to extinction cascades. Although cooperative mechanisms may be beneficial to the fitness of individual hosts, positive interactions are thought to destabilize ecological networks as perturbations can spread more rapidly when species are tightly linked in positive feedback loops 13 , 50 . For this reason, the loss of any one fungal species causes a more rapid deterioration of the network. We assessed the complexity x stability relationship for a wide range of microbiomes found across land, sea and stream and inhabiting hosts ranging from birds to bugs to plants. While much prior attention has been placed on the value of biodiversity, specifically species diversity, in maintaining stable communities and their functions 2 , we find that additional aspects of complexity such as the frequency and type of interactions among species are equally if not more important predictors of robustness. Also, networks inclusive of the least robust microorganism networks, in this case fungi, generally increased the overall stability of bacterial networks, indicating that interkingdom co-occurrences are another important and often overlooked component of complexity that can positively affect stability. While stability may promote long-term coexistence of species 18 , 19 , other examples of stability in nature include less-favorable ecosystem states such as biological invasions 20 and gut microbiota dysbiosis following antibiotic treatment 21 . Therefore, in the context of microbiome engineering it is critical to consider the properties of the reference system, whether it be a healthy gut, a productive agricultural field or an ecosystem, that are important to emulate, which may, or may not include stable microbial communities or stable functions of the microbiome. Indeed, enhancing the ability of microbiomes to acclimate or adapt rather than just persist may be an equally important aspirational trait for microbiome engineering and one that is only recently beginning to receive attention 51 . Future experiments assessing these principles are encouraged as this watershed-wide model study system has now provided clear testable hypotheses for the fundamental building blocks of microbiome stability."
} | 5,015 |
26477321 | PMC4609964 | pmc | 4,912 | {
"abstract": "Lignin, an aromatic polymer of phenylpropane units joined predominantly by β- O -4 linkages, is the second most abundant biomass component on Earth. Despite the continuous discharge of terrestrially produced lignin into marine environments, few studies have examined lignin degradation by marine microorganisms. Here, we screened marine isolates for β- O -4 cleavage activity and determined the genes responsible for this enzymatic activity in one positive isolate. Novosphingobium sp. strain MBES04 converted all four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGGE), a structural mimic of lignin, to guaiacylhydroxypropanone as an end metabolite in three steps involving six enzymes, including a newly identified Nu -class glutathione-S-transferase (GST). In silico searches of the strain MBES04 genome revealed that four GGGE-metabolizing GST genes were arranged in a cluster. Transcriptome analysis demonstrated that the lignin model compounds GGGE and (2-methoxyphenoxy)hydroxypropiovanillone (MPHPV) enhanced the expression of genes in involved in energy metabolism, including aromatic-monomer assimilation, and evoked defense responses typically expressed upon exposure to toxic compounds. The findings from this study provide insight into previously unidentified bacterial enzymatic systems and the physiological acclimation of microbes associated with the biological transformation of lignin-containing materials in marine environments.",
"discussion": "Results and Discussion Identification of a marine microorganism capable of cleaving the β- O -4 linkage of a dimeric lignin model compound We previously isolated several deep-sea bacteria capable of metabolizing lignin-derived aromatic monomers 27 . Here, we screened the isolates for strains capable of cleaving the β -O- 4 ether linkage of the dimeric lignin model compound GGGE ( Fig. 1 , compound 1) and identified an isolate from sunken wood, Novosphingobium sp. strain MBES04, which metabolized GGGE into two end-products, guaiacylhydroxylpropanone (GHP; Fig. 1 , compound 3) and guaiacol ( Fig. 1 , compound 4). GGGE metabolism by strain MBES04 was quantitatively examined during a 5-day culture in basal medium. The detection of a transient intermediate metabolite ( Fig. 1 , compound 2) indicated that GGGE was oxidized to MPHPV prior to cleavage of the β -O- 4 ether linkage. GHP and guaiacol were produced as end products from MPHPV and accumulated in the culture medium at more than 60% of the estimated maximum yield. Metabolism of lignin-related aromatic monomers, dimers, and a crude extract from milled wood by strain MBES04 Strain MBES04 grew on a wide range of aromatic monomers and esters, including synaptic acid, ferulic acid, caffeic acid, 4-hydroxybenzoic acid, syringic acid, vanillic acid, vanillin, benzoate, protocatechuic acid, and chlorogenic acid, as well as hexoses and pentoses that are commonly distributed in terrestrial plants. Notably, however, strain MBES04 was not capable of growth using either GGGE or MPHPV as the sole carbon source in a minimal salt medium. To examine the metabolism of natural wood components by strain MBES04, a water-soluble fraction of dioxan extract (designated herein as WDM) of milled wood, Quercus myrsinifolia , was added to cultures of strain MBES04. After a 48-h incubation, metabolites in the culture supernatant were identified by reversed-phase LC/MS ( Figure S1 ), which revealed that GHP and 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone (syringyl hydroxyl propanone; SHP), a methoxylated derivative of GHP, were the two predominant metabolites among a number of unidentified compounds. This finding is consistent with a study by Lancefield et al. 29 , who reported that GHP and SHP were specifically produced by the chemoselective breakdown of lignin model dimers, lignin-like synthetic polymers and milled wood lignin by the oxidation of Cα hydroxyl moieties followed by the reductive cleavage of β- O -4 ether linkages. To assess the capability of strain MBES04 to depolymerize and/or modify natural lignin outside of cells, the extracellular activities of oxidative enzymes, including oxidases and peroxidases, which are known to function as bacterial lignin-modifying enzymes 30 , were measured in WDM-supplemented cultures once daily for 3 days using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,6-dimethoxyphenol as substrates with/without a divalent metal salt (FeSO 4 , CuSO 4 , and MnSO 4 ) and with/without H 2 O 2 . However, the oxidative enzymatic activities in WDM-supplemented cultures were below the detection limit of the assay. This result suggests that oxidative enzymes have limited involvement in the depolymerization and modification of polymeric lignin outside of cells, even though the strain MBES04 genome contains putative genes encoding four catalase-peroxidases (GAM03180, GAM05894, GAM05893, and GAM04190) and two multicopper oxidases (laccases) (GAM04037 and GAM03576) ( Table S1 ). Based on these findings, strain MBES04 appears to cleave the β- O -4 ether linkages present in partially depolymerized, low-molecular-weight lignin using intracellular enzymes. Identification of the genes involved in GGGE metabolism According to an earlier study of Sphingobium sp. strain SYK-6 20 , GGGE metabolism mediated by SDRs and GSTs produces the intermediates MPHPV, GHP, and guaiacol, which were also produced from GGGE by strain MBES04. Among the 58 genes in the strain MBES04 genome 28 that showed similarity to reported SDRs of Sphingomonadaceae family members and encoded short-chain alcohol dehydrogenases, 6 candidate genes were selected based on similarities to the 4 SDR genes reported to function as Cα-dehydrogenases (accession numbers: NC_015976/Gene ID; BAK65539, BAK68041, BAK68265, and BAK68263) in strain SYK-6 20 21 and expressed as His-tagged proteins in E. coli ( Table S2 ). The recombinant SDRs were purified and assessed for their ability to dehydrogenate the Cα position of GGGE. Only two recombinant SDRs (SDR3 and SDR5; Figure S2a ), encoded by the genes GAM05523 and GAM05547, respectively, exhibited dehydrogenase activity in the presence of nicotinamide adenine dinucleotide (NAD). SDR3 selectively acted on α( R )-substrate, whereas SDR5 was selective for α( S )-substrate ( Fig. 2 ). A total of 18 genes in the strain MBES04 genome were predicted to encode GST family proteins. BLASTP analyses with sequences of reported β-etherases (BAK65541, BAK65540, and BAK67935) and a glutathione lyase (BAK65542) detected three putative GST genes that may be involved in GGGE metabolism. The three identified GST genes (GST4–6; GAM05530, GAM05531, and GAM05532) were grouped in a cluster in the same orientation. In addition, a fourth putative GST gene (GST3; GAM05529) was found upstream of the three GST genes, but in the opposite orientation. These four GST genes were expressed in E. coli as His-tagged proteins ( Table S2 ), which were then purified ( Figure S2b ) and assessed for enzymatic activity. Two recombinant GSTs (GST4 and GST5) catalyzed the cleavage of β -O- 4 ether linkages in MPHPV using glutathione as a cofactor to produce glutathione conjugates of GHP ( Fig. 3 ). GST4 selectively eliminated the ether-linked moiety of the β( S )-enantiomer ( Fig. 4a ), whereas GST5 reacted exclusively with the β ( R )-enantiomer ( Fig. 4b ). Neither GST4 nor GST5 cleaved the β -O- 4 ether linkages in GGGE. GST3 and GST6 did not catalyze cleavage of the β -O- 4 ether linkages both in GGGE and MPHPV but the removal of glutathione from both glutathione conjugates of GHP produced by GST4 and GST5 under these reaction conditions ( Fig. 4a,b ). Based on these results, it was determined that GST4/GST5 and GST3/GST6 function as β-etherases and β-thioetherases, respectively. Notably, GST6 showed markedly lower activity toward the glutathione conjugate produced by GST5 when present at low enzyme concentrations, indicating that the two glutathione conjugates produced by GST4/GST5 have different configurations. Recently, Gall et al. 31 determined the configuration of glutathione-conjugated intermediates and demonstrated that all of the enzymatic reactions were strictly stereospecific. To date, no enzyme capable of reacting efficiently with the β( S )-glutathione conjugate produced by the enzymes LigE and LigP, which are encoded by BAK65541 and BAK67935, respectively, has been identified. Therefore, it has been suggested that a racemase-like or other enzyme with different stereospecificity from LigG (a β( S )-stereospecific β-thioetherase, BAK65542) for glutathione removal, or other metabolic pathway for the cellular utilization of glutathione conjugates, are functional in strain SYK-6 cells 31 . In the present study, GST3 displayed no apparent preference for either epimer of the glutathione conjugate substrate. Thus, this is the first report of an enzyme capable of directly removing glutathione from a glutathione conjugate in both the β( R ) and β( S ) configurations. Based on structural modeling deduced from amino acid sequences of GST3/GST6 using the Swiss model workspace 32 , GST6 is predicted to be a member of the GST omega class, as expected, whereas GST3 is proposed to be a member of the Nu -class. YghU and YfcG from E. coli 33 and Ure2p from the wood-degrading fungus Phanerochaete chrysosporium 34 are partially characterized members of the Nu -class within the GST family. YghU and YfcG exhibit distinct disulfide bond oxide-reductase activities (scheme 1) and little or no GSH transferase activity towards typical electrophilic substrates 33 35 36 . Nu -class GSTs are atypical in that they bind two molecules of GSH in each active site. Ure2p from P. chrysosporium is able to efficiently deglutathionylate GS-phenacylacetophenone and interacts in vitro with an omega class GST. The present finding that a novel GST belonging to the Nu -class catalyzes the reductive removal of glutathione from glutathione conjugates (scheme 2) and has no preference for the glutathione adducts produced by β( S ) and β( R ) specific-etherases provides new insight into microbial lignin metabolism. The protein structure of the GST enzyme will be solved in future work. scheme 1: E*2GSH + R-S-S-R ↔ E*GSSG + 2RSH scheme 2: E*GSH + R-SG ↔ E*GSSG + RH A possible pathway of GGGE metabolism in strain MBES04, including the responsible enzymes and required cofactors, is presented in Fig. 5 . However, this proposed pathway remains speculative and requires corroboration by gene disruption experiments. Biochemical characterization of SDRs and GSTs To compare the catalytic properties of the SDRs and GSTs identified in this study to closely related enzymes showing the same catalytic activity, the purified SDR and GST enzymes from strain MBES04 were biochemically characterized. The pH and temperature optima for the SDR (SDR3 and SDR5) and GST (GST4 and GST5) activities were determined using GGGE and MPHPV, respectively, as substrates ( Table 1 ). The optimal temperatures for GST4 and GST5 activities were higher than the reported β-etheraes ( Figure S3b ). The specific activities and kinetic parameters of SDR3 and SDR5 were also measured under the optimal reaction conditions using GGGE and veratryllglycerol-β-guaiacyl ether (VGGE), which is a non-phenolic derivative of GGGE, as substrates ( Table 1 ). The catalytic efficiency (specificity constant) ( k cat / K m ) of SDR3 and SDR5 for GGGE were 1.6 × 10 1 and 8.6 × 10 3 min −1 mM −1 , respectively, values that are one order of magnitude lower and higher, respectively, than that of LigD of strain SYK-6 37 . The specific activities and kinetic parameters of GST4 and GST5 were determined using MPHPV and β-guaiacyl-α-veratrylglycerone (GVG) as substrates ( Table 1 , Table S3 ). GVG is a non-phenolic derivative of MPHPV and has been used for determination of the specific activities of β-etherases 24 . The specific activities of GST4 and GST5 for GVG were 3.7 × 10 2 and 1.7 × 10 2 mU/mg, respectively. These specific activities were within the range of those reported in a previous study, in which the specific activities ranged from 1.0 × 10 2 to 6.8 × 10 3 mU/mg for the reported enzymes encoded by the members of sphingomonads 24 ( Table S3 ). The GST activities of GST3, GST4, GST5, and GST6 were also screened using commercially available nucleophilic substrates that are widely used in conventional GST assays 35 36 . However, no GST activity towards any of the tested compounds was detected, with the exception of the activity of GST3 towards 1-chloro-2,4-dinitrobenzene (CDNB) (675.0 ± 16.3 mU/mg). These results indicate that the substrate specificity of GST3 is broader than that of GST4–6. Distribution of SDR and GST homologs involved in GGGE metabolism BLASTP searches using the deduced amino acid sequences of the two SDR and four GST genes identified in strain MBES04 as queries against the NCBI nr protein database, which covers non-redundant GenBank CDS translations, RefSeq, PDB, SwissProt, PIR, PRF, excluding those in PAT, TSA, and env_nr (http://www.ncbi.nlm.nih.gov/), was performed to find homologs reported to have β-etherase or β-thioetherase activity 20 , 23 , 24 . A phylogenetic tree was constructed from alignments of the 15 most similar amino acid sequences to SDR3, SDR5, GST3, GST4, GST5, and GST6 ( Figure S4 ). GST4 and GST5 clustered together with LigEs and LigFs, respectively, which are known to function as β( R )- and β( S )-stereospecific etherases, respectively. GST6 was classified together with LigG, a β( S )-stereospecific β-thioetherase belonging to the omega-class of the GST family. The enzymes characterized in the present and previous studies (highlighted by boxes in Figure S4 ) that belonged to the same clade, also shared the same substrate specificities. In contrast, GST3 was assigned to an uncharacterized branch of GST family proteins, indicating that this enzyme belongs to a new class within the GST family that targets lignin model dimers. BLASTP searches using the deduced amino acid sequences of the four GST genes identified in strain MBES04 as queries showed that the putative GST proteins with homology to GST3 or GST6 were widely distributed among members of the α-, β-, and γ-proteobacteria classes. However, only a small number of homologous proteins to GST4 and GST5 were found. Proteins with similarity to GST4 (E value < e-50) were found exclusively in the Sphingomonadaceae family and were annotated as LigF/GST proteins, whereas proteins with homology to GST5 were found among members of α- and δ-proteobacteria and were predominantly annotated as β-1,3-glucanases. Among members of the Sphingomonadaceae family, 15 complete and 69 draft genome sequences are currently available. Here, the distribution of GGGE metabolic genes in this family was investigated using BLASTP and BLASTX searches against predicted coding sequences and nucleotide sequences, respectively ( Table S4 ). At least one complete set of GGGE-converting enzymes, consisting of Cα-dehydrogenases, β-etherases, and β-thioetherases, was found to be essential for GGGE metabolism in this family. A total of 9 strains were identified as candidates with GGGE metabolic activity and included 7 strains that were isolated from water-logged environments, such as rivers, lakes, sludge, subsurface sediment/water, and seawater, whereas the other 2 strains were obtained from a decomposing plant and the rhizosphere. The organization of the GGGE metabolizing-gene homologs was investigated in the 9 identified Sphingomonadaceae strains ( Table S5 ). The tandem arrangement of SDR3 and SDR5 homologs was detected in 6 strains, and multiple GSTs were found in neighboring loci in 4 strains. Two strains, SYK-6 and a marine isolate, Novosphingobium sp. PP1Y, possessed a complete set of GGGE metabolizing-gene homologs encoding enzymes capable of cleaving the β -O- 4 linkage of lignin model dimers, which were comprised of four stereoisomers with two chiral carbon centers. As was observed in strain MBES04, the four GGGE-metabolizing GST genes were clustered together in strain PP1Y, which was also found to share gene synteny with strain MBES04 ( Fig. 6 ). The tight clustering of all four GGGE-metabolizing GST genes in the two marine isolates, strain MBES04 and PP1Y, suggests that these genes may confer an evolutionary advantage in the marine environment and/or be coordinately regulated and expressed. To elucidate the origin, evolution and diversity of the genes involved in β -O- 4 reductive cleavage and gain a better understanding of the evolutionary processes controlling the assembly of the corresponding enzymes involved in lignin metabolism, pan-genomic studies and experimental evidence at the protein level are needed. Effect of lignin model dimers on global gene expression qPCR quantification of GGGE-metabolizing gene expression, which ranged from 10 −5 to 10 −6 fold of that of the 16 S rRNA gene, confirmed the low level expression of these genes, and no apparent differences between the control and GGGE-added conditions were detected ( Figure S5 ). Whole-genome transcriptional profiling in the early exponential phase was conducted to detect the response of strain MBES04 to the lignin-related compounds GGGE and MPHPV ( Table 2 , Table S6 , Figure S8 ). A total of 28 and 51 genes were upregulated in response to medium supplemented with GGGE and MPHPV, respectively, and included 12 common genes between these two groups. In particular, the expression of genes involved in glycerol metabolism for biosynthesis of the cellular membrane was clearly increased. The gene expression analysis also revealed that 5 and 15 genes were down-regulated in strain MBES04 in response to GGGE and MPHPV, respectively. In response to GGGE alone, expression of genes involved in energy metabolism, including aromatic monomers such as toluene and benzoate, and fatty acid degradation were enhanced. The upregulation of the energy metabolism gene expression may promote the growth of strain MBES04 on lignin-derived aromatic compounds immediately upon exposure to plant materials, including lignin-derived aromatic compounds. In addition, the elevated expression of several stress-response genes, such as the transcriptional regulators PadR and CopG, and multidrug transporters was observed. PadR and CopG are involved in stress responses to various aromatic compounds 38 39 and plasmid replication 40 . Elevated expression of multiple drug transporters is indicative of enhanced stress responses against diverse antimicrobial agents 41 . These responses of strain MBES04 are consistent with the coordinated regulation of the microbial gene expression associated with lignin transformation, as predicted from the analysis of lignin-transforming bacterial scaffolds 16 . In cells exposed to MPHPV, increased expression of a greater number of stress-response genes and a few genes involved in energy metabolism were detected compared to GGGE-supplemented conditions. Specifically, in response to MPHPV, the gene encoding the flagellar basal body-associated protein involved in chemotaxis 42 was enhanced, and several cytochrome C proteins involved in the respiratory chain for energy production were repressed. Energy production may be reduced by repression of cytochrome proteins involved in the respiratory chain, a response that has been shown to lead to cellular dormancy 43 . Such dormancy is referred to as a “bed-hedging” strategy, and is employed by many microorganisms to sustain viability in unfavorable environmental conditions 44 . Thus, the response of strain MBES04 to the lignin model dimers MPHPV and GGGE may be a survival strategy for utilizing the abundant TerrOC discharged into the ocean. The findings from this study provide insight into previously unidentified bacterial enzymatic systems and the physiological acclimation of microbes associated with the biological transformation of TerrOC containing lignin in marine environments."
} | 5,009 |
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