Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Macroalgae represent a promising third-generation feedstock for biorefinery due to their high biomass productivity and non-reliance on arable land. However, their complex cell wall structure poses a significant barrier to efficient bioconversion. This review integrates current pretreatment methods, including physical, chemical, biological, and combined approaches, with a focus on their mechanisms, effectiveness, and limitations. Furthermore, it explores the conversion of pretreated macroalgal biomass into bioenergy and biochemicals, such as bioethanol, organic acid and polyhydroxyalkanoate, via microbial fermentation. The review also examines the application of genetic editing tools (e.g., CRISPR-Cas systems) for the targeted modification of macroalgae to improve their inherent characteristics for biorefinery, such as reducing biomass recalcitrance or increasing the content of target carbohydrates. Finally, future perspectives on technological innovations and integrated industrial chains of macroalgal biorefinery are discussed. This review serves as a systematic reference for deepening the understanding of macroalgal cell wall deconstruction processes and supports the development of efficient and environmentally benign pretreatment strategies to advance macroalgal biorefinery toward industrialization.

Candidates:
A. Macroalgae represent a promising third-generation feedstock for biorefinery due to their high biomass productivity and non-reliance on arable land.
B. Furthermore, it explores the conversion of pretreated macroalgal biomass into bioenergy and biochemicals, such as bioethanol, organic acid and polyhydroxyalkanoate, via microbial fermentation.
C. The evidence does not state that however, their complex cell wall structure poses a significant barrier to efficient bioconversion.
D. The evidence does not state that macroalgae represent a promising third-generation feedstock for biorefinery due to their high biomass productivity and non-reliance on arable land.
E. However, their complex cell wall structure poses a significant barrier to efficient bioconversion.
F. The evidence does not state that furthermore, it explores the conversion of pretreated macroalgal biomass into bioenergy and biochemicals, such as bioethanol, organic acid and polyhydroxyalkanoate, via microbial fermentation.
G. This review integrates current pretreatment methods, including physical, chemical, biological, and combined approaches, with a focus on their mechanisms, effectiveness, and limitations.
H. The evidence does not state that this review integrates current pretreatment methods, including physical, chemical, biological, and combined approaches, with a focus on their mechanisms, effectiveness, and limitations.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
This study explores the application of immobilized phospholipase A1 (PLA1) on hollow double-layer mesoporous silica nanoparticles (PLA1@NH 2 /C 8 -HdlMS) for the degumming of crude arachidonic acid (ARA) oil for the first time. The immobilized enzyme was comprehensively characterized, and the reaction conditions were optimized via single-factor experiments. Under the optimized conditions (enzyme dosage 0.3% w / w , 35 °C, water addition 3%, and reaction time 90 min), PLA1@NH 2 /C 8 -HdlMS achieved a remarkable phosphorus removal rate of 97.9%, reducing the phosphorus content from 441.21 mg/kg to 9.29 mg/kg in 90 min (well below the food-grade standard of <10 mg/kg). The fatty acid composition of the oil remained almost unchanged, while the oxidative induction time of the degummed oil significantly improved by 42%. Notably, PLA1@NH 2 /C 8 -HdlMS demonstrated broad applicability across crude oils, with initial phosphorus contents ranging from 294.98 mg/kg to 537.44 mg/kg, and it maintained ~93% of its initial activity after 11 reuse cycles. Compared to traditional hydration degumming (with a phosphorus removal rate of 56.3%), this enzymatic method offers superior efficiency at lower temperatures, minimizing energy consumption and the thermal degradation of ARA. This green, efficient, and sustainable method for degumming heat-sensitive oils offers significant potential for the industrial application of high-quality functional oils by preserving PUFA integrity and reducing environmental impact.

Candidates:
A. Under the optimized conditions (enzyme dosage 0.3% w / w , 35 °C, water addition 3%, and reaction time 90 min), PLA1@NH 2 /C 8 -HdlMS achieved a remarkable phosphorus removal rate of 97.9%, reducing the phosphorus content from 441.21 mg/kg to 9.29 mg/kg in 90 min (well below the food-grade standard of <10 mg/kg).
B. The immobilized enzyme was comprehensively characterized, and the reaction conditions were optimized via single-factor experiments.
C. This study explores the application of immobilized phospholipase A2 (PLA1) on hollow double-layer mesoporous silica nanoparticles (PLA1@NH 2 /C 8 -HdlMS) for the degumming of crude arachidonic acid (ARA) oil for the first time.
D. The fatty acid composition of the oil remained almost unchanged, while the oxidative induction time of the degummed oil significantly improved by 42%.
E. This study explores the application of immobilized phospholipase A1 (PLA1) on hollow double-layer mesoporous silica nanoparticles (PLA1@NH 2 /C 8 -HdlMS) for the degumming of crude arachidonic acid (ARA) oil for the first time.
F. Under the optimized conditions (enzyme dosage 1.3% w / w , 35 °C, water addition 3%, and reaction time 90 min), PLA1@NH 2 /C 8 -HdlMS achieved a remarkable phosphorus removal rate of 97.9%, reducing the phosphorus content from 441.21 mg/kg to 9.29 mg/kg in 90 min (well below the food-grade standard of <10 mg/kg).
G. The fatty acid composition of the oil remained almost unchanged, while the oxidative induction time of the degummed oil significantly improved by 43%.
H. The immobilized enzyme was not comprehensively characterized, and the reaction conditions were optimized via single-factor experiments.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Surface fouling remains a critical challenge for medical devices and chemosensor systems operating in biological environments, where nonspecific adsorption of proteins, cells, and microorganisms can lead to signal drift, reduced sensitivity, and shortened device lifetime. Conventional antifouling strategies rely primarily on synthetic hydrophilic polymer coatings, such as polyethylene glycol and polyvinylpyrrolidone, which are effective but face limitations related to long-term stability, thickness, and compatibility with surface-sensitive sensing modalities. In this review, we focus on hydrophobins derived from mushroom-forming and filamentous fungi as a bio-based alternative for antifouling and anti-wetting surface modification. Mushroom-derived hydrophobins are small amphiphilic proteins capable of spontaneous self-assembly into nanometer-scale films that modulate surface energy, wettability, and interfacial friction without requiring covalent functionalization. The current state of research on hydrophobin structure, classification, and self-assembly is reviewed, followed by a synthesis of reported antifouling and tribological behaviors relevant to medical and sensor-adjacent surfaces. Representative experimental observations are discussed to illustrate trends consistent with the literature, without establishing new performance benchmarks. The implications of mushroom-derived hydrophobin coatings for chemosensors and biosensors are examined, particularly with respect to signal stability, surface accessibility, and durability. Limitations and future research directions are outlined to support translation into practical sensing technologies.

Candidates:
A. Conventional antifouling strategies rely primarily on synthetic hydrophilic polymer coatings, such as polyethylene glycol and polyvinylpyrrolidone, which are not effective but face limitations related to long-term stability, thickness, and compatibility with surface-sensitive sensing modalities.
B. Mushroom-derived hydrophobins are not small amphiphilic proteins capable of spontaneous self-assembly into nanometer-scale films that modulate surface energy, wettability, and interfacial friction without requiring covalent functionalization.
C. Mushroom-derived hydrophobins are small amphiphilic proteins capable of spontaneous self-assembly into nanometer-scale films that modulate surface energy, wettability, and interfacial friction without requiring covalent functionalization.
D. Conventional antifouling strategies rely primarily on synthetic hydrophilic polymer coatings, such as polyethylene glycol and polyvinylpyrrolidone, which are effective but face limitations related to long-term stability, thickness, and compatibility with surface-sensitive sensing modalities.
E. The evidence does not state that in this review, we focus on hydrophobins derived from mushroom-forming and filamentous fungi as a bio-based alternative for antifouling and anti-wetting surface modification.
F. Surface fouling remains a critical challenge for medical devices and chemosensor systems operating in biological environments, where nonspecific adsorption of proteins, cells, and microorganisms cannot lead to signal drift, reduced sensitivity, and shortened device lifetime.
G. In this review, we focus on hydrophobins derived from mushroom-forming and filamentous fungi as a bio-based alternative for antifouling and anti-wetting surface modification.
H. Surface fouling remains a critical challenge for medical devices and chemosensor systems operating in biological environments, where nonspecific adsorption of proteins, cells, and microorganisms can lead to signal drift, reduced sensitivity, and shortened device lifetime.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Coronaviruses, both known and yet to emerge, pose persistent zoonotic and pandemic threats. While current parenteral COVID-19 mRNA vaccines effectively mitigate severe disease caused by SARS-CoV-2, they primarily elicit systemic immunity restricted to specific variants within clade 1b of the sarbecovirus subgenus and provide limited mucosal protection. Addressing these shortcomings, Cheang et al. developed a DC-targeting intranasal booster vaccine that induces robust and durable mucosal and systemic immunity across sarbecovirus clades 1a and 1b. This study highlights a promising strategy for pan-sarbecovirus vaccines by leveraging mucosal immune induction to prevent viral transmission and enhance pandemic preparedness.

Candidates:
A. developed a DC-targeting intranasal booster vaccine that induces robust and durable mucosal and systemic immunity across sarbecovirus clades 2a and 1b.
B. developed a DC-targeting intranasal booster vaccine that induces robust and durable mucosal and systemic immunity across sarbecovirus clades 1a and 1b.
C. The evidence does not state that addressing these shortcomings, Cheang et al.
D. The evidence does not state that coronaviruses, both known and yet to emerge, pose persistent zoonotic and pandemic threats.
E. Addressing these shortcomings, Cheang et al.
F. Coronaviruses, both known and yet to emerge, pose persistent zoonotic and pandemic threats.
G. While current parenteral COVID-20 mRNA vaccines effectively mitigate severe disease caused by SARS-CoV-2, they primarily elicit systemic immunity restricted to specific variants within clade 1b of the sarbecovirus subgenus and provide limited mucosal protection.
H. While current parenteral COVID-19 mRNA vaccines effectively mitigate severe disease caused by SARS-CoV-2, they primarily elicit systemic immunity restricted to specific variants within clade 1b of the sarbecovirus subgenus and provide limited mucosal protection.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
The production of engineered proteins in transgenic cells is widely used in research, medicine and industry. However, conventional cell-based production systems still face challenges in cost, scalability and biosafety. Here, we present a recombinant protein expression platform with simplified purification based on the photosynthetic unicellular red alga Cyanidioschyzon merolae , which can be cultivated under highly acidic conditions using only inorganic nutrients, air, water and light. We first identified a promoter that drives high-level constitutive gene expression throughout the cell cycle, resulting in substantial mRNA accumulation in C. merolae . A stable transformant expressing His-tagged mVenus under the control of this promoter accumulated the recombinant protein to more than 1% of total soluble protein. The simple cellular architecture of C. merolae , including the absence of a cell wall, enables efficient protein extraction via a single freeze–thaw cycle, followed by purification using immobilized metal affinity chromatography (IMAC), yielding ∼13.9 mg of functional recombinant protein per gram of total soluble protein. Owing to its low cost, scalability, operational simplicity and minimal risk of contamination, this Cyanidioschyzon -based platform offers a practical and promising approach to recombinant protein production in a photosynthetic eukaryote.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
L-Alanyl-L-glutamine (Ala-Gln) is a high-value dipeptide with superior stability, solubility, and bioavailability, underscoring its potential for nutritional supplementation. Compared with conventional chemical catalysis, whole-cell biocatalysts offer a more efficient, simpler, and environmentally friendly alternative for peptide synthesis. Among these, enzyme cell-surface immobilization systems enable the stable display of target enzymes on yeast cells, thereby enhancing enzyme stability while simplifying catalyst recovery and reuse, which is particularly advantageous for large-scale industrial applications. In this study, an engineered Saccharomyces cerevisiae strain displaying α-amino acid ester acyltransferase (SsAET) from Sphingobacterium siyangensis SY1 on the cell surface was developed for clean and efficient biocatalytic production of Ala-Gln. Optimal reaction conditions were established (pH 8.0, 3 h, 20 g DCW/l, AlaOMe/Gl n = 1:2), resulting in a 6.7-fold increase in Ala-Gln production compared with the pre-optimization conditions. Under these optimal conditions, repeated-batch reactions with an increased reaction volume of 20 ml achieved a maximum Ala-Gln concentration of 14.12 mM, representing the highest yield obtained in this study. The SsAET whole-cell biocatalyst retained more than 60% of its relative Ala–Gln production after three consecutive reaction cycles, while the conversion rate based on Gln consumption decreased only slightly from 32.08% to 29.65%, remaining essentially stable. Overall, this study demonstrates a clean, efficient, and reusable whole-cell biocatalytic system based on enzyme cell-surface immobilization for Ala-Gln production, highlighting its potential for industrial-scale peptide synthesis.

Candidates:
A. In this study, an engineered Saccharomyces cerevisiae strain displaying α-amino acid ester acyltransferase (SsAET) from Sphingobacterium siyangensis SY1 on the cell surface was developed for clean and efficient biocatalytic production of Ala-Gln.
B. Among these, enzyme cell-surface immobilization systems enable the stable display of target enzymes on yeast cells, thereby enhancing enzyme stability while simplifying catalyst recovery and reuse, which is particularly advantageous for large-scale industrial applications.
C. Compared with conventional chemical catalysis, whole-cell biocatalysts offer a more efficient, simpler, and environmentally friendly alternative for peptide synthesis.
D. L-Alanyl-L-glutamine (Ala-Gln) is not a high-value dipeptide with superior stability, solubility, and bioavailability, underscoring its potential for nutritional supplementation.
E. In this study, an engineered Saccharomyces cerevisiae strain displaying α-amino acid ester acyltransferase (SsAET) from Sphingobacterium siyangensis SY2 on the cell surface was developed for clean and efficient biocatalytic production of Ala-Gln.
F. The evidence does not state that compared with conventional chemical catalysis, whole-cell biocatalysts offer a more efficient, simpler, and environmentally friendly alternative for peptide synthesis.
G. Among these, enzyme cell-surface immobilization systems enable the stable display of target enzymes on yeast cells, thereby enhancing enzyme stability while simplifying catalyst recovery and reuse, which is not particularly advantageous for large-scale industrial applications.
H. L-Alanyl-L-glutamine (Ala-Gln) is a high-value dipeptide with superior stability, solubility, and bioavailability, underscoring its potential for nutritional supplementation.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["36%", "35%"]

Evidence:
D-tagatose is a low-calorie natural rare sugar with significant potential in the food and pharmaceutical industries. Conventional production relies on the enzymatic isomerization of D-galactose, a process limited by an unfavorable thermodynamic equilibrium and high substrate costs. This study presents a novel whole-cell biocatalytic approach for direct production of D-tagatose from lactose, an inexpensive and abundant sugar in dairy waste streams, such as whey permeate. The main lactose permease (lacY) of Escherichia coli BL21(DE3) was deleted, creating a clean host chassis that, due to its native galactose auxotrophy, was incapable of utilizing galactose. Subsequently, genes from the tagatose-6-phosphate (T6P) pathway of Lactococcus lactis , comprising a lactose-specific phosphotransferase system (PTS), a 6-phospho-β-galactosidase, a galactose-6-phosphate isomerase and the general PTS proteins, were modularly introduced, which allowed for the functional validation of each component. The crucial final step involved the expression of a sugar phosphatase to convert the intracellular intermediate, tagatose-6-phosphate, into D-tagatose. The fully engineered strain yielded a 35% conversion ratio of the galactose moiety of lactose to tagatose. This demonstrates the repurposing of the tagatose-6-phosphate pathway in E. coli to enable direct lactose utilization and growth-coupled production of D-tagatose from both lactose and whey permeate, without prior hydrolysis or substrate enrichment. Fermentation and tagatose production using whey permeate as the sole carbon source was also demonstrated, highlighting the potential of this system for the valorization of dairy byproducts into a high-value low calorie sweetener.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Behavior plays a critical role in health and disease. Although molecular omics have advanced the understanding of biological mechanisms, they alone cannot explain the role of behavior in health, and traditional behavioral measurement technologies have struggled to capture the dynamic, multidimensional, and heterogeneous nature of real-world behaviors. With the development of digital technologies such as wearable devices, smartphone sensors, and ecological momentary assessment, behavioromics has emerged as a new research paradigm with a holistic systems perspective, integrating continuous, multimodal behavioral data with molecular, physiological, and environmental information. By organizing behavioral measurement, modeling, and interpretation within a unified analytical framework, behavioromics reveals dynamic behavioral patterns, supports mechanism-informed inference, and identifies actionable targets for early intervention. Despite ongoing challenges in data heterogeneity, causal interpretation, and ethical governance, behavioromics holds promise for reshaping disease prediction, early detection, and precision intervention, and for advancing proactive health through earlier, behavior-centered prevention.

Candidates:
A. With the development of digital technologies such as wearable devices, smartphone sensors, and ecological momentary assessment, behavioromics has emerged as a new research paradigm with a holistic systems perspective, integrating continuous, multimodal behavioral data with molecular, physiological, and environmental information.
B. The evidence does not state that with the development of digital technologies such as wearable devices, smartphone sensors, and ecological momentary assessment, behavioromics has emerged as a new research paradigm with a holistic systems perspective, integrating continuous, multimodal behavioral data with molecular, physiological, and environmental information.
C. The evidence does not state that behavior plays a critical role in health and disease.
D. The evidence does not state that although molecular omics have advanced the understanding of biological mechanisms, they alone cannot explain the role of behavior in health, and traditional behavioral measurement technologies have struggled to capture the dynamic, multidimensional, and heterogeneous nature of real-world behaviors.
E. By organizing behavioral measurement, modeling, and interpretation within a unified analytical framework, behavioromics reveals dynamic behavioral patterns, supports mechanism-informed inference, and identifies actionable targets for early intervention.
F. Although molecular omics have advanced the understanding of biological mechanisms, they alone cannot explain the role of behavior in health, and traditional behavioral measurement technologies have struggled to capture the dynamic, multidimensional, and heterogeneous nature of real-world behaviors.
G. Behavior plays a critical role in health and disease.
H. The evidence does not state that by organizing behavioral measurement, modeling, and interpretation within a unified analytical framework, behavioromics reveals dynamic behavioral patterns, supports mechanism-informed inference, and identifies actionable targets for early intervention.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The aim of this work was to design, develop, and optimise a two-step process for extracting lignin from Eucalyptus globulus residues and subsequently converting the remaining holocellulose into monosaccharides, primarily glucose and xylose. Firstly, the solubility of high-quality lignin was optimised using a new hydro-organo-thermal method involving a diluted imidazole solution in water instead of pure imidazole. Subsequently, enzymatic hydrolysis of the polysaccharides-rich pretreated solid was optimised to give glucose and xylose by using the commercial enzymatic mixture Cellic® CTec3 HS (Novozymes, Bagsværd, Denmark) supplemented with endo-1,4-β-xylanase from Trichoderma viride (Merck, Darmstadt, Germany). The extraction process was thoroughly optimised by acting on several parameters, such as the imidazole concentration, temperature, reaction time, and solid-to-liquid ratio. Under the optimised reaction conditions (imidazole 35 wt%, biomass loading 10 wt%, 120 °C, 2 h, 400 rpm) over the entire process, about 70 wt% of lignin removal was achieved, together with glucose and xylose yields of 99 and 92 mol%, respectively, reaching a high total sugar concentration of 91 g/L in the hydrolysate. • A new hydro-organo-thermal treatment of lignocellulosic biomass has been proposed. • Glucose and xylose yields were 99 and 92 mol% in 48 h at low enzyme dosage. • A total reducing sugar concentration of 91 g/L was achieved in the hydrolysate. The online version contains supplementary material available at 10.1007/s00253-026-13793-2. Keywords: Biorefinery, Eucalyptus globulus , Lignin recovery, Sugars, Enzymatic hydrolysis

Candidates:
A. Subsequently, enzymatic hydrolysis of the polysaccharides-rich pretreated solid was optimised to give glucose and xylose by using the commercial enzymatic mixture Cellic® CTec4 HS (Novozymes, Bagsværd, Denmark) supplemented with endo-1,4-β-xylanase from Trichoderma viride (Merck, Darmstadt, Germany).
B. The extraction process was not thoroughly optimised by acting on several parameters, such as the imidazole concentration, temperature, reaction time, and solid-to-liquid ratio.
C. Subsequently, enzymatic hydrolysis of the polysaccharides-rich pretreated solid was optimised to give glucose and xylose by using the commercial enzymatic mixture Cellic® CTec3 HS (Novozymes, Bagsværd, Denmark) supplemented with endo-1,4-β-xylanase from Trichoderma viride (Merck, Darmstadt, Germany).
D. The aim of this work was to design, develop, and optimise a two-step process for extracting lignin from Eucalyptus globulus residues and subsequently converting the remaining holocellulose into monosaccharides, primarily glucose and xylose.
E. The extraction process was thoroughly optimised by acting on several parameters, such as the imidazole concentration, temperature, reaction time, and solid-to-liquid ratio.
F. Firstly, the solubility of high-quality lignin was not optimised using a new hydro-organo-thermal method involving a diluted imidazole solution in water instead of pure imidazole.
G. Firstly, the solubility of high-quality lignin was optimised using a new hydro-organo-thermal method involving a diluted imidazole solution in water instead of pure imidazole.
H. The aim of this work was not to design, develop, and optimise a two-step process for extracting lignin from Eucalyptus globulus residues and subsequently converting the remaining holocellulose into monosaccharides, primarily glucose and xylose.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Lignin is the most abundant renewable source of aromatic carbon on Earth and a central yet historically underutilized component of lignocellulosic biomass. Its complex and heterogeneous molecular architecture has long constrained efficient and selective conversion into value-added products, despite its high aromatic carbon content and chemical functionality. Recent advances in lignin extraction, fractionation, modification, and application-driven design have substantially expanded the range of achievable material and chemical performance within circular bioeconomy frameworks. This review provides a comprehensive and critically integrated assessment of lignin valorization that explicitly links plant biosynthesis and structural diversity to industrial convertibility, functional materials development, and sustainability performance. Green extraction technologies—including deep eutectic solvent and hydrotropic systems—are evaluated with respect to lignin structural quality, energy demand, solvent recovery, and downstream compatibility. Targeted chemical and enzymatic modification strategies enabling more reproducible lignin streams are discussed alongside applications in carbon fibers, nanomaterials, adhesives, bioplastics, cementitious systems, and additive manufacturing. Quantitative benchmarking against fossil-based incumbents identifies application domains where lignin-derived materials already achieve comparable performance, as well as areas where intrinsic structural limitations remain. In parallel, catalytic depolymerization pathways toward renewable aromatic chemicals are assessed from both mechanistic and systems-level perspectives. Environmental and economic implications are critically examined using recent life-cycle and techno-economic evidence, highlighting the influence of allocation choices, energy integration, and comparison with lignin incineration for energy recovery. Overall, this review clarifies how application-targeted lignin design and system-level sustainability assessment are essential for translating lignin’s biological complexity into scalable, competitive solutions for sustainable materials and chemicals.

Candidates:
A. Recent advances in lignin extraction, fractionation, modification, and application-driven design have substantially expanded the range of achievable material and chemical performance within circular bioeconomy frameworks.
B. The evidence does not state that this review provides a comprehensive and critically integrated assessment of lignin valorization that explicitly links plant biosynthesis and structural diversity to industrial convertibility, functional materials development, and sustainability performance.
C. The evidence does not state that recent advances in lignin extraction, fractionation, modification, and application-driven design have substantially expanded the range of achievable material and chemical performance within circular bioeconomy frameworks.
D. This review provides a comprehensive and critically integrated assessment of lignin valorization that explicitly links plant biosynthesis and structural diversity to industrial convertibility, functional materials development, and sustainability performance.
E. Lignin is not the most abundant renewable source of aromatic carbon on Earth and a central yet historically underutilized component of lignocellulosic biomass.
F. The evidence does not state that its complex and heterogeneous molecular architecture has long constrained efficient and selective conversion into value-added products, despite its high aromatic carbon content and chemical functionality.
G. Its complex and heterogeneous molecular architecture has long constrained efficient and selective conversion into value-added products, despite its high aromatic carbon content and chemical functionality.
H. Lignin is the most abundant renewable source of aromatic carbon on Earth and a central yet historically underutilized component of lignocellulosic biomass.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["72 h", "73 h"]

Evidence:
As the terminal management for evaluating the engineering effectiveness of antibiotics production and utilization, the toxic effects of moxifloxacin (MOX) and trace concentration of Cu 2+ (MOX-Cu) on Caenorhabditis elegans ( C. elegans ) were investigated at physiological, biochemical, and molecular level. Although the stimulate effects were observed after prolonged exposure (72 h) to MOX (0.2-2.0 mg/L), the expressions of HSPs, ace genes, and daf-16 were inhibited, indicating its adverse impact on cellular health, locomotion behaviors, and antioxidant defense of C. elegans . Similarly, the down-regulation of oxidative stress ( sod-1 and daf-16 ) and cell damage (HSPs) related genes and the up-regulation of apoptosis-related genes ( cep-1 and ape-1 ) indicated the oxidative stress and genotoxicity after prolonged exposure to MOX-Cu. For the chronic exposure (10 days) to MOX, the level of ROS was reduced due to the increased expressions of daf-16 , sod-3 , and hsp-16 , accompanied with and the down-regulation of cep-1 . Meanwhile, at the exposure to MOX-Cu, the levels of ROS and lipofuscin were decreased due to the up-regulation of sod-1 and daf-16 , and the antioxidant defense was promoted and confirmed by the increase of amino acids and their related metabolic pathways. These results can provide a theoretical basis for the toxicity evaluation of typical antibiotics (MOX) that co-existing with trace heavy metals in natural environment media and bioresources processes. The online version contains supplementary material available at 10.1186/s40643-026-01033-4. Keywords: Caenorhabditis elegans , Moxifloxacin, Copper, Antioxidant defenses, Metabolomics


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["19.0%", "-15.9%", "-14.9%", "8.6%", "18.0%", "-18.4%", "9.6%", "-19.4%"]

Evidence:
Filamentous algae, characterized by high cellulose content and absence of lignin, present a promising sustainable alternative to conventional plant and synthetic fibers. The present study systematically evaluated the suitability of freshwater filamentous algae as a new resource for textile fibers, targeting applications in moisture-absorbent textiles. Among twelve strains screened, the isolate Rhizoclonium sp. emerged as the most promising candidate due to its high biomass yield (1.04 g dry weight L − 1 ) after 21 days of cultivation. In addition, it showed superior visible fiber flexibility following air-drying, an essential prerequisite for textile processing. Cultivation conditions were optimized (using WHM medium, pH 8, and thiamin supplementation) to maximize fiber quality, resulting in 8.6% increase in biomass productivity. Biochemical profiling of the optimized biomass revealed a significant enhancement of total carbohydrates (+ 18.0%), alongside reductions in protein (-18.4%) and ash content (-14.9%), supporting improved fiber durability and flexibility. Comparative FTIR analysis showed a strong cellulose signature and marked similarity to cotton, while also revealing high native starch content, further supporting their applicability as bio-based binders in nonwoven products. Functional characterization demonstrated that optimized Rhizoclonium sp. fibers exhibited exceptional moisture regain (~ 12%), surpassing conventional fibers such as cotton and lyocell. Overall, this study establishes native Rhizoclonium sp. as a highly versatile and renewable bioresource for innovative aquatic fibers, underpinning the development of an environmentally responsible algae-derived textile value chain. The online version contains supplementary material available at 10.1186/s40643-026-01028-1.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Many Gram-negative nosocomial pathogens rely on adhesive filaments, known as archaic chaperone-usher pili, to establish stress- and drug-resistant, multi-layered biofilms. Here, we uncover the mechanism by which these pili build three-dimensional (3D) biofilm architectures. In situ analyses of Acinetobacter baumannii biofilms using electron microscopy (EM) reveal an extensive network of ultrathin, flat stacks of archaic Csu pili interconnecting bacterial cells in 3D space. Cryo-EM structures of a single native pilus, pilus pairs, and two types of multi-pilus stacks show that the pili pack into antiparallel sheets, with their rods connected laterally by junctions at their zigzag corners. This antiparallel arrangement ensures that contacts form primarily between pili from interacting cells rather than pili from the same cell. With a remarkably short helical repeat, archaic chaperone-usher pili spontaneously establish a high density of junctions that determines the biofilm’s 3D architecture. Our findings may help develop new therapies against multidrug-resistant bacterial infections by targeting pilus-pilus interactions. Subject terms: Biofilms, Cryoelectron microscopy, Bacterial secretion, Pathogens, Cryoelectron tomography

Candidates:
A. Many Gram-negative nosocomial pathogens rely on adhesive filaments, known as archaic chaperone-usher pili, to establish stress- and drug-resistant, multi-layered biofilms.
B. Here, we uncover the mechanism by which these pili build three-dimensional (4D) biofilm architectures.
C. Cryo-EM structures of a single native pilus, pilus pairs, and two types of multi-pilus stacks show that the pili pack into antiparallel sheets, with their rods connected laterally by junctions at their zigzag corners.
D. The evidence does not state that many Gram-negative nosocomial pathogens rely on adhesive filaments, known as archaic chaperone-usher pili, to establish stress- and drug-resistant, multi-layered biofilms.
E. The evidence does not state that cryo-EM structures of a single native pilus, pilus pairs, and two types of multi-pilus stacks show that the pili pack into antiparallel sheets, with their rods connected laterally by junctions at their zigzag corners.
F. In situ analyses of Acinetobacter baumannii biofilms using electron microscopy (EM) reveal an extensive network of ultrathin, flat stacks of archaic Csu pili interconnecting bacterial cells in 3D space.
G. In situ analyses of Acinetobacter baumannii biofilms using electron microscopy (EM) reveal an extensive network of ultrathin, flat stacks of archaic Csu pili interconnecting bacterial cells in 4D space.
H. Here, we uncover the mechanism by which these pili build three-dimensional (3D) biofilm architectures.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Wine production generates significant quantities of by-products each year, among which surplus grape must is notable for its high content of fermentable sugars, organic acids, and polyphenols. As wine consumption declines and grape must surpluses grow, identifying sustainable valorization strategies becomes increasingly critical. This study investigates for the first time the potential use of red and white grape musts as substrates for polyhydroxyalkanoates (PHAs) production by two well-characterized bacterial strains, Cupriavidus necator DSM 545 and Hydrogenophaga pseudoflava DSM 1034, under both batch and fed-batch fermentation regimes. Both musts supported microbial growth and PHAs accumulation. In batch cultures, C. necator DSM 545 achieved a PHB content of up to 61.5% of cell dry weight (CDW), while H. pseudoflava DSM 1034 reached 67.9% PHB on grape must, with yields and biomass comparable to or exceeding those obtained with synthetic sugar-based media. Under fed-batch conditions with red must, C. necator DSM 545 sustained growth and PHB production across multiple feeding feeding periods, outperforming the control medium. Conversely, H. pseudoflava DSM 1034 displayed initial growth but failed to increase biomass over time, suggesting that this strain may be poorly suited for use in fed-bacth-based applications, likely due to nutrient depletion or the accumulation of inhibitory compounds. Overall, these results proved that grape musts are promising feedstocks for sustainable PHAs production. Their integration into circular economy frameworks offers a valuable opportunity for waste recovery and the development of bioplastics within the agri-food industry, especially in light of the increasing grape must surpluses recently experienced worldwide.

Candidates:
A. The evidence does not state that both musts supported microbial growth and PHAs accumulation.
B. Both musts supported microbial growth and PHAs accumulation.
C. As wine consumption declines and grape must surpluses grow, identifying sustainable valorization strategies becomes increasingly critical.
D. This study investigates for the first time the potential use of red and white grape musts as substrates for polyhydroxyalkanoates (PHAs) production by two well-characterized bacterial strains, Cupriavidus necator DSM 546 and Hydrogenophaga pseudoflava DSM 1034, under both batch and fed-batch fermentation regimes.
E. Wine production generates significant quantities of by-products each year, among which surplus grape must is not notable for its high content of fermentable sugars, organic acids, and polyphenols.
F. The evidence does not state that as wine consumption declines and grape must surpluses grow, identifying sustainable valorization strategies becomes increasingly critical.
G. This study investigates for the first time the potential use of red and white grape musts as substrates for polyhydroxyalkanoates (PHAs) production by two well-characterized bacterial strains, Cupriavidus necator DSM 545 and Hydrogenophaga pseudoflava DSM 1034, under both batch and fed-batch fermentation regimes.
H. Wine production generates significant quantities of by-products each year, among which surplus grape must is notable for its high content of fermentable sugars, organic acids, and polyphenols.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Halophiles attract increasing attention to serve as sustainable industrial hosts for prolonged continuous processes or in open-vat fermentations owing to the reduced risk of contamination enabled by high salt concentration in the medium. Despite growing interest, their application in large-scale manufacturing remains limited partly due to bioprocessing challenges. A robust host that performs consistently well across scales should withstand variations in oxygen availability since local hypoxic regions often manifest in large-scale tanks even under strict operational control. In this work, we modified Halomonas sp. to achieve robust growth profiles in micro-aerobic environments by gradually exposing a continuous culture to stress induced by reduced oxygen availability using adaptive laboratory evolution. Nominal contamination was observed at 8.5% salt concentration even during non-aseptic operation, offering a suitable environment for prolonged continuous cultivation. Following adaptive laboratory evolution, Halomonas sp. achieved comparable growth at low-to-moderate (0.5%-3.0%) and moderate-to-high (3.0–5.0%) dissolved oxygen availability (OD 600 = 4.46 ± 0.29 & µ = 0.40 h − 1 and 4.96 ± 0.81 & µ = 0.35 h − 1 , respectively). Adaptive evolution improved the growth robustness of Halomonas sp. by 62% and its PHB production yield by 21%, while reducing ectoine production by 79% indicting improved metabolic perception of oxygen availability and reduced stress response. Mutations in cellular transport mechanisms and enzymatic pathways enabled this response as indicated by whole genome sequencing. The proposed framework is an effective strategy to reduce the oxygen sensitivity of microbial strains in general to improve their physiological properties, and to genetically design populations suitable for large scale bioprocess operations. The online version contains supplementary material available at 10.1186/s12934-026-02951-w. Keywords: Adaptive laboratory evolution, Halomonas sp., Dissolved oxygen, Growth robustness, Sodium chloride, Contamination risk


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Natural killer cells play a critical role in innate immunity by targeting and eliminating cancerous and infected cells. The cell line NK-92, currently used in several clinical trials, has shown great potential in cancer immunotherapy due to strong cytotoxic capabilities and suitability as an off-the-shelf therapy. However, effective clinical application of NK-92 therapies requires optimised cell culture conditions, including cultivation medium composition and expansion methods. This study aimed at identifying optimal cultivation medium formulations and intensifying NK-92 cell expansion by systematically evaluating critical quality attributes and key performance indicators. We screened various cultivation media formulations, including alpha minimum essential medium, Roswell Park Memorial Institute medium, and stem cell growth medium, supplemented with either human serum, fetal bovine serum, chemically defined serum replacement, and a combination of fetal bovine serum and horse serum. Cells cultivated in alpha minimum essential medium supplemented with human serum significantly outperformed the other formulations, achieving the highest growth rate, maximum viable cell count, and superior cytotoxicity at high cost-efficiency. Media containing chemically defined serum replacement showed reduced cell proliferation despite high cytotoxicity per cell, indicating a complex balance between cell quantity and functionality. Following cultivation medium selection, cultivation strategies including batch, fed-batch, and repetitive batch were compared. The repetitive batch method, where cultivation medium is replenished repeatedly throughout the cultivation, demonstrated superior expansion capability, long-term cell viability, significantly improved cytotoxicity, although associated with higher medium consumption and thus costs. This study identified alpha minimum essential medium supplemented with human serum in repetitive batch cultivation as the optimal approach for NK-92 cell expansion. This combination enhances cell growth, viability, and cytotoxicity, meeting essential clinical quality attributes while maintaining cost-effectiveness. These findings provide valuable guidance and should act as a baseline for consistent, scalable, and commercially viable production of NK cell-based cancer therapies. The online version contains supplementary material available at 10.1186/s12896-026-01116-2. Keywords: NK-92 cells, Natural killer cells, Process mode, Cultivation medium optimisation, Process intensification, Batch, Fed-batch, Repetitive batch


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Kinetic modeling of microbial growth is essential for the design, optimization, and scale-up of industrial bioprocesses. Classical empirical models often lack biologically interpretable parameters or fail to capture complex multiphasic (polyauxic) behaviors, while fully mechanistic models are impractical for systems involving complex substrates and mixed cultures. This study proposes a unified mathematical framework that reformulates the canonical Boltzmann and Gompertz equations into semi-mechanistic forms, explicitly defining the maximum specific reaction rate and lag phase duration. Polyauxic growth is represented as a weighted sum of sigmoidal phases, subject to stringent constraints that ensure parameter identifiability, temporal consistency, and biological plausibility. The methodology integrates a workflow to address nonlinear regression in high-dimensional parameter spaces. A two-stage optimization strategy using Differential Evolution for global search followed by L-BFGS-B for local refinement avoid bias and heuristic parameter initialization. A Charbonnier loss function and the Robust Regression and Outlier Removal procedure are employed to identify and mitigate experimental outliers. Model parsimony is enforced using Akaike (AIC, AICc) and Bayesian (BIC) information criteria to select the optimal number of growth phases and avoid overparameterization. The framework was evaluated using experimental anaerobic digestion datasets, demonstrating that conventional single-phase models can obscure relevant metabolic transitions in co-digestion systems. Keywords: Anaerobic digestion, Microbiology, Bioreactor design, Sigmoid function, Microbial growth

Candidates:
A. Kinetic modeling of microbial growth is essential for the design, optimization, and scale-up of industrial bioprocesses.
B. The evidence does not state that this study proposes a unified mathematical framework that reformulates the canonical Boltzmann and Gompertz equations into semi-mechanistic forms, explicitly defining the maximum specific reaction rate and lag phase duration.
C. Polyauxic growth is not represented as a weighted sum of sigmoidal phases, subject to stringent constraints that ensure parameter identifiability, temporal consistency, and biological plausibility.
D. Classical empirical models often lack biologically interpretable parameters or fail to capture complex multiphasic (polyauxic) behaviors, while fully mechanistic models are not impractical for systems involving complex substrates and mixed cultures.
E. Classical empirical models often lack biologically interpretable parameters or fail to capture complex multiphasic (polyauxic) behaviors, while fully mechanistic models are impractical for systems involving complex substrates and mixed cultures.
F. Polyauxic growth is represented as a weighted sum of sigmoidal phases, subject to stringent constraints that ensure parameter identifiability, temporal consistency, and biological plausibility.
G. Kinetic modeling of microbial growth is not essential for the design, optimization, and scale-up of industrial bioprocesses.
H. This study proposes a unified mathematical framework that reformulates the canonical Boltzmann and Gompertz equations into semi-mechanistic forms, explicitly defining the maximum specific reaction rate and lag phase duration.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Human Bocavirus 2 (HBoV2) is an emerging enteric virus frequently detected in wastewater, yet its environmental fate, persistence, and treatment remain poorly understood. Improved characterization of HBoV2 is important for advancing wastewater-based epidemiology and understanding viral transmission dynamics. A year-long wastewater surveillance study was conducted from December 2023 to December 2024 in a small urban wastewater treatment system. Viral concentrations were quantified using digital PCR (dPCR), decay kinetics were assessed using controlled mesocosm experiments, and phylogenetic relationships were evaluated through NP1 gene sequencing. HBoV2 was detected in 93.8% of influent samples (average 4.76 log₁₀ genome copies per liter (GC/L) ) with seasonal peaks in summer and fall. Phylogenetic analysis revealed clustering with globally circulating HBoV2 lineages. Decay experiments indicated slow viral degradation (k = 0.10 d⁻¹), comparable to other non-enveloped DNA viruses including Adeno-Associated Virus type 2 (AAV-2), Adenovirus 41 (AdV41), and Human Polyomavirus (HPyV). Additionally, HBoV2 persisted through secondary treatment, with no significant reduction observed between influent and secondary effluent samples. Our findings indicate HBoV2 as highly prevalent and environmentally stable in a small urban city’s wastewater and that it is resistant to conventional treatment. These findings highlight HBoV2’s potential role in gastrointestinal infections worldwide and the need for further monitoring of this environmental contaminant. The online version contains supplementary material available at 10.1007/s11033-026-11669-2. Keywords: Human bocavirus, Wastewater treatment, Decay, Enteric viruses, Fecal contamination

Candidates:
A. Human Bocavirus 2 (HBoV2) is an emerging enteric virus frequently detected in wastewater, yet its environmental fate, persistence, and treatment remain poorly understood.
B. A year-long wastewater surveillance study was conducted from December 2023 to December 2024 in a small urban wastewater treatment system.
C. A year-long wastewater surveillance study was conducted from December 2024 to December 2024 in a small urban wastewater treatment system.
D. Viral concentrations were quantified using digital PCR (dPCR), decay kinetics were assessed using controlled mesocosm experiments, and phylogenetic relationships were evaluated through NP1 gene sequencing.
E. Human Bocavirus 3 (HBoV2) is an emerging enteric virus frequently detected in wastewater, yet its environmental fate, persistence, and treatment remain poorly understood.
F. Viral concentrations were quantified using digital PCR (dPCR), decay kinetics were assessed using controlled mesocosm experiments, and phylogenetic relationships were evaluated through NP2 gene sequencing.
G. Improved characterization of HBoV3 is important for advancing wastewater-based epidemiology and understanding viral transmission dynamics.
H. Improved characterization of HBoV2 is important for advancing wastewater-based epidemiology and understanding viral transmission dynamics.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Via efficient, scalable, and climate resilient processes, seaweed biorefineries can advance cleaner production, delivering nutrient-rich ingredients with relatively lower land and freshwater requirements. Here, a land-based Ulva ohnoi platform that integrates optimized outdoor tank cultivation, pH-shift protein extraction with dual-product valorization, scale-aware techno-economic analysis (TEA), and a climate-scenario emulator was developed and evaluated. Year-round cultivation yielded a pooled feedstock with carbohydrate and protein contents at 46.6% and 26.8%, respectively, and under optimal conditions, protein yield was 10.30% DW. Further, the protein content of cultivated Ulva protein (CUP) was 47.64%, with bound amino acids showing predominance (96%), implying food-grade utility. pH-shift extraction also improved in-vitro pepsin digestibility to 68.08%. The total dietary fiber content of CUP residue (CUPR) was 40.59%, with insoluble dietary fiber (31.59%) showing predominance, supporting the applicability of CUPR in improving gastrointestinal tract function. TEA revealed strong economies of scale for the platform. The 10,000-kg production model outperformed the 100- and 1,000-kg production models, with gross margin, Return On Investment (ROI), and Internal Rate of Return (IRR) at 78.56%, 110%, and 66.02%, respectively, and a payback time of only 0.91 years. Based on site-specific regression analysis (R 2 = 0.884), protein yield increased with temperature but decreased with rainfall, and further warming increased mean protein yield, while higher-emission pathways introduced rainfall-driven volatility, necessitating strategies for sustaining performance under climate variability. Overall, the use of the Ulva platform showed a broad growth temperature window as well as rapid acclimation for U. ohnoi , implying resilience even under global warming conditions and increasing weather variability. Keywords: Ulva ohnoi , Land-based seaweed cultivation, pH-shift protein extraction, Dual-product valorization, Techno-economic analysis, Climate resilience

Candidates:
A. Here, a land-based Ulva ohnoi platform that integrates optimized outdoor tank cultivation, pH-shift protein extraction with dual-product valorization, scale-aware techno-economic analysis (TEA), and a climate-scenario emulator was developed and evaluated.
B. Here, a land-based Ulva ohnoi platform that integrates optimized outdoor tank cultivation, pH-shift protein extraction with dual-product valorization, scale-aware techno-economic analysis (TEA), and a climate-scenario emulator was not developed and evaluated.
C. Via efficient, scalable, and climate resilient processes, seaweed biorefineries cannot advance cleaner production, delivering nutrient-rich ingredients with relatively lower land and freshwater requirements.
D. Year-round cultivation yielded a pooled feedstock with carbohydrate and protein contents at 47.6% and 26.8%, respectively, and under optimal conditions, protein yield was 10.30% DW.
E. Year-round cultivation yielded a pooled feedstock with carbohydrate and protein contents at 46.6% and 26.8%, respectively, and under optimal conditions, protein yield was 10.30% DW.
F. Further, the protein content of cultivated Ulva protein (CUP) was 48.64%, with bound amino acids showing predominance (96%), implying food-grade utility.
G. Via efficient, scalable, and climate resilient processes, seaweed biorefineries can advance cleaner production, delivering nutrient-rich ingredients with relatively lower land and freshwater requirements.
H. Further, the protein content of cultivated Ulva protein (CUP) was 47.64%, with bound amino acids showing predominance (96%), implying food-grade utility.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Methanol, a renewable non-food C1 substrate, holds great promise as a feedstock for sustainable biomanufacturing and carbon neutral production. However, its industrial application is hindered by low methanol assimilation efficiency in most microbes. Recent advances in synthetic biology and metabolic engineering have enabled the development of methylotrophic microbial cell factories through strategies including building efficient methanol-utilizing pathways, engineering methanol dehydrogenase for enhanced oxidation efficiency, and optimizing redox balance via cofactor utilization. Additionally, approaches such as mitigating the accumulation of toxic metabolites and adaptive laboratory evolution have been adopted to improve the robustness of synthetic methylotrophs. This review summarizes these innovations and provides a blueprint for rationally designing high-performance microbial platforms to facilitate industrial methanol utilization and advance sustainable development.

Candidates:
A. Methanol, a renewable non-food C1 substrate, holds great promise as a feedstock for sustainable biomanufacturing and carbon neutral production.
B. Additionally, approaches such as mitigating the accumulation of toxic metabolites and adaptive laboratory evolution have been adopted to improve the robustness of synthetic methylotrophs.
C. Methanol, a renewable non-food C2 substrate, holds great promise as a feedstock for sustainable biomanufacturing and carbon neutral production.
D. The evidence does not state that recent advances in synthetic biology and metabolic engineering have enabled the development of methylotrophic microbial cell factories through strategies including building efficient methanol-utilizing pathways, engineering methanol dehydrogenase for enhanced oxidation efficiency, and optimizing redox balance via cofactor utilization.
E. However, its industrial application is not hindered by low methanol assimilation efficiency in most microbes.
F. The evidence does not state that additionally, approaches such as mitigating the accumulation of toxic metabolites and adaptive laboratory evolution have been adopted to improve the robustness of synthetic methylotrophs.
G. Recent advances in synthetic biology and metabolic engineering have enabled the development of methylotrophic microbial cell factories through strategies including building efficient methanol-utilizing pathways, engineering methanol dehydrogenase for enhanced oxidation efficiency, and optimizing redox balance via cofactor utilization.
H. However, its industrial application is hindered by low methanol assimilation efficiency in most microbes.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The pharmaceutical industry remains critically dependent on plant-derived natural products, yet the supply of these complex molecules is perpetually threatened by the inherent biological instability of plant systems. For decades, the field has struggled to force undifferentiated plant cell cultures into the mold of consistent industrial fermentation, a strategy largely defeated by intrinsic biological stochasticity arising from epigenetic reprogramming, somaclonal variation, transcriptional noise, and systemic metabolic rigidity, as well as by a linear cost structure that prohibits pharmaceutical scalability. This literature-based review articulates a fundamental paradigm shift: the strategic decoupling of discovery from production. It argues that the genomic and epigenomic plasticity of plant cells—rather than being suppressed—should be deliberately induced and explored through stress elicitation to generate a “productive chaos” of chemical diversity for discovery. This expanded metabolic landscape is then decoded using single-cell–resolved multi-omics and spatial metabolomics to identify rare, elite producer states, alongside advanced artificial intelligence, molecular networking, and structure prediction to characterize novel bioactive candidates. Once identified, these biosynthetic pathways are functionally repatriated into defined, heterologous microbial hosts, engineered via systems-level metabolic and architectural optimization—including cofactor balancing, dynamic pathway control, subcellular compartmentalization, and cytochrome P450–reductase stoichiometry—to achieve stable, high-titer manufacturing. By integrating high-throughput discovery, AI-guided strain design, techno-economic analysis, and regulatory Quality-by-Design principles, this discovery–production decoupling resolves the long-standing tension between biological complexity and industrial rigor. This framework transforms the economics of natural product supply, transitioning from the low-CAPEX/high-OPEX trap of extraction to the high-CAPEX/low-OPEX scalability of fermentation, offering a scientifically grounded, commercially viable, and regulatorily tractable pathway to unlock the full therapeutic potential of the plant kingdom.

Candidates:
A. The pharmaceutical industry remains critically dependent on plant-derived natural products, yet the supply of these complex molecules is perpetually threatened by the inherent biological instability of plant systems.
B. This literature-based review articulates a fundamental paradigm shift: the strategic decoupling of discovery from production.
C. It argues that the genomic and epigenomic plasticity of plant cells—rather than being suppressed—should be deliberately induced and explored through stress elicitation to generate a “productive chaos” of chemical diversity for discovery.
D. The pharmaceutical industry remains critically dependent on plant-derived natural products, yet the supply of these complex molecules is not perpetually threatened by the inherent biological instability of plant systems.
E. For decades, the field has struggled to force undifferentiated plant cell cultures into the mold of consistent industrial fermentation, a strategy largely defeated by intrinsic biological stochasticity arising from epigenetic reprogramming, somaclonal variation, transcriptional noise, and systemic metabolic rigidity, as well as by a linear cost structure that prohibits pharmaceutical scalability.
F. The evidence does not state that this literature-based review articulates a fundamental paradigm shift: the strategic decoupling of discovery from production.
G. The evidence does not state that it argues that the genomic and epigenomic plasticity of plant cells—rather than being suppressed—should be deliberately induced and explored through stress elicitation to generate a “productive chaos” of chemical diversity for discovery.
H. The evidence does not state that for decades, the field has struggled to force undifferentiated plant cell cultures into the mold of consistent industrial fermentation, a strategy largely defeated by intrinsic biological stochasticity arising from epigenetic reprogramming, somaclonal variation, transcriptional noise, and systemic metabolic rigidity, as well as by a linear cost structure that prohibits pharmaceutical scalability.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Shake flasks are widely used in early‐stage bioprocess development but are limited by their inability to monitor and control key gas‐transfer variables such as dissolved oxygen and carbon dioxide. In this study, we present a jacketed breathable flask system that enables real‐time gas control in a standard shaking environment. Across multiple media formulations and fill volumes, this system consistently deferred oxygen limitation and enhanced culture performance, achieving > 150% higher biomass and 140% greater recombinant protein yield compared to conventional flasks. Time‐resolved analysis of pH and extracellular metabolites revealed reduced accumulation of oxygen‐sensitive byproducts, including acetate, pyruvate, and succinate, indicating a shift toward more efficient respiratory metabolism. The jacketed breathable flask also enabled continuous monitoring and regulation of critical process parameters, creating a bioreactor‐like environment in a high‐throughput, low‐cost format. The biomass accumulation and specific growth rate observed in jacketed breathable flask are comparable to those reported for Escherichia coli cultures in stirred tank bioreactor application notes for Eppendorf BioBLU 3f. These findings establish breathable flasks as a scalable and accessible platform with bioreactor‐like performance for upstream process optimization and accelerate biomanufacturing development at the lab scale.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Bayesian optimization has become widely popular across various experimental sciences due to its favorable attributes: it can handle noisy data, perform well with relatively small data sets, and provide adaptive suggestions for sequential experimentation. While still in its infancy, Bayesian optimization has recently gained traction in bioprocess engineering. However, experimentation with biological systems is highly complex and the resulting experimental uncertainty requires specific extensions to classical Bayesian optimization. Moreover, current literature often targets readers with a strong statistical background, limiting its accessibility for practitioners. In light of these developments, this review has two aims: first, to provide an intuitive and practical introduction to Bayesian optimization; and second, to outline promising application areas and open algorithmic challenges, thereby highlighting opportunities for future research in machine learning.

Candidates:
A. However, experimentation with biological systems is highly complex and the resulting experimental uncertainty requires specific extensions to classical Bayesian optimization.
B. The evidence does not state that moreover, current literature often targets readers with a strong statistical background, limiting its accessibility for practitioners.
C. However, experimentation with biological systems is not highly complex and the resulting experimental uncertainty requires specific extensions to classical Bayesian optimization.
D. While still in its infancy, Bayesian optimization has recently gained traction in bioprocess engineering.
E. Moreover, current literature often targets readers with a strong statistical background, limiting its accessibility for practitioners.
F. Bayesian optimization has become widely popular across various experimental sciences due to its favorable attributes: it can handle noisy data, perform well with relatively small data sets, and provide adaptive suggestions for sequential experimentation.
G. Bayesian optimization has become widely popular across various experimental sciences due to its favorable attributes: it cannot handle noisy data, perform well with relatively small data sets, and provide adaptive suggestions for sequential experimentation.
H. The evidence does not state that while still in its infancy, Bayesian optimization has recently gained traction in bioprocess engineering.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Microalgae are photosynthetic microorganisms capable of synthesizing diverse high-value bioactive compounds, including premium proteins, polyunsaturated fatty acids, pigments, and vitamins. These natural products exhibit significant potential in enhancing livestock growth and health, offering biological activity and nutritional benefits that surpass chemically synthesized alternatives. Nevertheless, the commercial production of microalgae-derived natural products remains insufficient to meet escalating market demands. Utilizing synthetic biology strategies, especially the CRISPR system, to increase productivity of microalgae cell factories is crucial for scaling up high-value product biosynthesis. This article reviews the current applications, construction strategies, and critical pathway nodes in microalgae cell factory, with emphasis on CRISPR-based genome editing breakthroughs for optimizing microalgae nutritional profiles, and recent progress in microalgae utilization for livestock production, providing a forward-looking perspective on future developments. Keywords: Biosynthesis, CRISPR, Feed, Genome editing, Microalgae, Sustainability

Candidates:
A. Utilizing synthetic biology strategies, especially the CRISPR system, to increase productivity of microalgae cell factories is not crucial for scaling up high-value product biosynthesis.
B. These natural products exhibit significant potential in enhancing livestock growth and health, offering biological activity and nutritional benefits that surpass chemically synthesized alternatives.
C. The evidence does not state that these natural products exhibit significant potential in enhancing livestock growth and health, offering biological activity and nutritional benefits that surpass chemically synthesized alternatives.
D. Microalgae are photosynthetic microorganisms capable of synthesizing diverse high-value bioactive compounds, including premium proteins, polyunsaturated fatty acids, pigments, and vitamins.
E. The evidence does not state that nevertheless, the commercial production of microalgae-derived natural products remains insufficient to meet escalating market demands.
F. Utilizing synthetic biology strategies, especially the CRISPR system, to increase productivity of microalgae cell factories is crucial for scaling up high-value product biosynthesis.
G. Microalgae are not photosynthetic microorganisms capable of synthesizing diverse high-value bioactive compounds, including premium proteins, polyunsaturated fatty acids, pigments, and vitamins.
H. Nevertheless, the commercial production of microalgae-derived natural products remains insufficient to meet escalating market demands.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Foetal bovine serum (FBS) has been widely used as a nutrient-rich supplement in mammalian cell culture for over 6 decades; however, its usage has increasingly raised various concerns and challenges related to quality variations, unethical collection practices, supply-demand imbalance and regulatory challenges. In recent years, alternatives have been investigated to reduce or replace FBS in mammalian cell culture. Starting from a comprehensive analysis of components of FBS and their functions in cell growth, this review compares the main types of FBS alternatives, i.e., human and animal-derived, plant-based alternatives and serum free media. Future perspectives discussed include the development of application-specific FBS alternatives, improvements in the quality and specialized formulation of FBS, optimization of existing alternatives and the establishment of databases and incentive mechanisms to facilitate the transition away from FBS. Lastly, the guidance for selecting appropriate FBS alternatives is also discussed.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["78.3%", "95%", "30%", "3.6%", "94%", "77.3%", "31%", "4.6%"]

Evidence:
Osteosarcoma, the most aggressive primary malignant bone tumor, has stagnant therapeutic outcomes despite decades of standard MAP chemotherapy and surgery; 5-year overall survival (OS) is <30% for metastatic/recurrent cases. Plagued by genomic heterogeneity, immunosuppressive TME, and low immunogenicity, emerging immunotherapies lack robust large-scale clinical validation. We systematically analyzed 864 interventional osteosarcoma trials from Trialtrove (as of September 2025). Results showed trial numbers peaked at 54 in 2021, with 77.3% past (completed/terminated) and over 94% in phase I/II (only 3.6% phase III-IV). Geographically, the U.S. dominated (60.9%, focusing on immunotherapy/targeted therapy), while low- and middle-income countries (LMICs) accounted for <2% of trials despite bearing 40% of the global disease burden. Conventional chemotherapy remains the cornerstone, with immuno-oncology (540 trials) as the leading novel strategy; top targets include VEGFR2 (104), PD-1 (70), and mTOR (60). Biomarker use was imbalanced: liver/nutritional markers prevailed, while key immune/genomic biomarkers (CD8A, TP53) were underrepresented (<8% combined). Key challenges include severe trial-phase imbalance, global disparities, and preclinical-clinical gaps; opportunities lie in synergistic novel therapies (ICI combinations, GD2-targeted CAR-T) and decentralized clinical trials (DCT). Future priorities: accelerate late-phase trials for promising regimens, reduce global disparities via regional consortia, integrate precision biomarkers for patient stratification, and translate TME insights into trials. This analysis highlights the need to shift from conventional chemotherapy optimization to precision-driven, globally equitable strategies to improve outcomes for high-risk osteosarcoma patients.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
RNA therapeutics are expanding rapidly, driving demand for manufacturing processes that can keep pace with clinical translation. Because mRNA yield and impurity profiles are jointly influenced by upstream plasmid DNA (pDNA) preparation and the in vitro transcription (IVT) reaction, in-process measurements are increasingly applied across the product lifecycle, albeit with distinct objectives in process development versus current good manufacturing practice (cGMP) production. Within the U.S. Food and Drug Administration’s Process Analytical Technology framework, we categorize in-process analytical methods by control intent: (1) measurements that build process understanding and define operating windows; (2) in-process controls (IPCs) that support predefined stop/go, forward-processing, and endpoint decisions; and (3) measurements that could enable advanced or adaptive process control (APC) through closed-loop feedback. We discuss how each category is deployed during process development and in cGMP manufacturing. Following the workflow from pDNA preparation through IVT, we highlight analytical measurements that establish template readiness—such as plasmid topology, linearization completeness, and co-purifying impurities that can propagate into transcription performance and complicate downstream processing—as well as time-resolved measurements during IVT that track reactant consumption and product formation to inform endpoint selection, feed timing, and deviation triage under predefined decision rules. We compare the strengths, implementation constraints, and validation considerations of at-line, on-line, and in-line approaches, and identify key gaps that currently limit broader adoption, including practical time-resolved quantification of double-stranded RNA and the availability of production-ready in-line sensing technologies. Collectively, these in-process analytics deliver near-term value by enabling process understanding and IPC-based decision support, while establishing the foundation required for future APC in mRNA manufacturing.

Candidates:
A. Food and Drug Administration’s Process Analytical Technology framework, we categorize in-process analytical methods by control intent: (1) measurements that build process understanding and define operating windows; (2) in-process controls (IPCs) that support predefined stop/go, forward-processing, and endpoint decisions; and (3) measurements that could enable advanced or adaptive process control (APC) through closed-loop feedback.
B. Food and Drug Administration’s Process Analytical Technology framework, we categorize in-process analytical methods by control intent: (2) measurements that build process understanding and define operating windows; (2) in-process controls (IPCs) that support predefined stop/go, forward-processing, and endpoint decisions; and (3) measurements that could enable advanced or adaptive process control (APC) through closed-loop feedback.
C. RNA therapeutics are expanding rapidly, driving demand for manufacturing processes that can keep pace with clinical translation.
D. We discuss how each category is deployed during process development and in cGMP manufacturing.
E. We discuss how each category is not deployed during process development and in cGMP manufacturing.
F. RNA therapeutics are not expanding rapidly, driving demand for manufacturing processes that can keep pace with clinical translation.
G. Because mRNA yield and impurity profiles are jointly influenced by upstream plasmid DNA (pDNA) preparation and the in vitro transcription (IVT) reaction, in-process measurements are increasingly applied across the product lifecycle, albeit with distinct objectives in process development versus current good manufacturing practice (cGMP) production.
H. Because mRNA yield and impurity profiles are not jointly influenced by upstream plasmid DNA (pDNA) preparation and the in vitro transcription (IVT) reaction, in-process measurements are increasingly applied across the product lifecycle, albeit with distinct objectives in process development versus current good manufacturing practice (cGMP) production.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
DNA polymerase θ (Pol θ)‐mediated end‐joining (TMEJ), one of several pathways for repairing DNA double‐strand breaks, is traditionally thought to initiate via anchoring at short, consecutive, and perfectly matched microhomologies (MHs). Emerging evidence indicates that Pol θ can utilize MHs containing mismatches both in vitro and in vivo. This revised definition of MH provides a mechanistic explanation for a broader spectrum of Pol θ‐dependent repair outcomes. Here, we summarize recent findings on the revised definition of MHs utilized by Pol θ, assess the applicability of this concept across species, and compare TMEJ with other (micro)hom(e)ology‐mediated repair pathways. We explore how mismatch‐containing MHs expand Pol θ‐associated mutational signatures and provide a framework for future studies on Pol θ’s role in DNA repair and cancer biology. Keywords: DNA polymerase θ (Pol θ), microhomology, mismatches, mutational signatures, TMEJ

Candidates:
A. Here, we summarize recent findings on the revised definition of MHs utilized by Pol θ, assess the applicability of this concept across species, and compare TMEJ with other (micro)hom(e)ology‐mediated repair pathways.
B. The evidence does not state that this revised definition of MH provides a mechanistic explanation for a broader spectrum of Pol θ‐dependent repair outcomes.
C. DNA polymerase θ (Pol θ)‐mediated end‐joining (TMEJ), one of several pathways for repairing DNA double‐strand breaks, is not traditionally thought to initiate via anchoring at short, consecutive, and perfectly matched microhomologies (MHs).
D. The evidence does not state that here, we summarize recent findings on the revised definition of MHs utilized by Pol θ, assess the applicability of this concept across species, and compare TMEJ with other (micro)hom(e)ology‐mediated repair pathways.
E. Emerging evidence indicates that Pol θ cannot utilize MHs containing mismatches both in vitro and in vivo.
F. This revised definition of MH provides a mechanistic explanation for a broader spectrum of Pol θ‐dependent repair outcomes.
G. Emerging evidence indicates that Pol θ can utilize MHs containing mismatches both in vitro and in vivo.
H. DNA polymerase θ (Pol θ)‐mediated end‐joining (TMEJ), one of several pathways for repairing DNA double‐strand breaks, is traditionally thought to initiate via anchoring at short, consecutive, and perfectly matched microhomologies (MHs).


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Microalgae have emerged as a versatile biotechnological platform, promising to become an efficient source of commodities such as food, feed, and biofuels, but these organisms have also sparked profound scientific and commercial interest for their potential in producing high-value recombinant proteins. Recombinant gene expression is highly dependent on the loci in which the transgene integrates, and in green algae transgenes integrate randomly into the nuclear genome, mostly through non homologous end joining. Therefore, recombinant gene expression varies greatly among different algal transformants and many algal colonies must be screened before a suitable production strain can be found, which can be quite laborious and become a bottleneck in the recombinant strain production pipeline. Here we describe a method for mid-throughput screening of recombinant protein expression; the Microalgal Colony Blot. This screening method allows for the detection of recombinant protein expression in up to 100 algal colonies per petri dish, with each petri dish preparation taking only 20 minutes. A nitrocellulose membrane is layered on top of a petri dish containing agar media, and algal cells are inoculated on top of the filter in a liquid suspension using a micropipette. The colonies are allowed to grow for up to 7 days, with the colonies secreting recombinant protein (either through active secretion or through cell lysis) as they grow with the recombinant protein being immediately bound by the nitrocellulose membrane. After the incubation period, the membrane is treated like a regular western blot, with blocking, washing, antibody binding and visualization. In this manner, up to 1000 colonies can be comfortably screened per day by a single person. Knowing that in C. reinhardtii only about 5% of the transgenic colonies from a transformation produce significant recombinant protein expression, being able to screen 1000 colonies ensures that around 50 suitable candidates will be identified within a single day.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
2,3-Butanediol (2,3-BDO) is a versatile platform chemical with diverse applications in cosmetics, pharmaceuticals, agricultural and food manufacturing. Among its stereoisomers, optically pure ( meso )-2,3-BDO is particularly valuable; however, achieving high titers with stereoselectivity remains challenging in conventional hosts due to byproduct formation, low tolerance, and plasmid instability. In this study, we established Corynebacterium glutamicum as an efficient and robust chassis for the industrial-level production of optically pure ( meso )-2,3-BDO. A structure-guided engineering approach was applied to 2,3-butanediol dehydrogenase (KpBDH), where α6-helix truncation enhanced catalytic efficiency and enabled near-complete conversion of acetoin to the target isomer. To further improve productivity, competing byproduct pathways were deleted, and cofactor homeostasis was reinforced by integrating UdhA for NADH regeneration and DrPPK for ATP regeneration. Finally, all biosynthetic modules were stably integrated into the chromosome, generating the plasmid-free strain ES11. In 5L fed-batch fermentation, ES11 produced 100.4 ± 0.4 g/L ( meso )-2,3-BDO with > 99% optical purity, a yield of 0.33 ± 0.04 g/g glucose, and productivity of 0.82 ± 0.06 g/L/h. This work represents the first demonstration of > 100 g/L optically pure ( meso )-2,3-BDO using C. glutamicum and establishes an integrated strategy of enzyme engineering, pathway optimization, and process design. The online version contains supplementary material available at 10.1186/s40643-026-01025-4. Keywords: Corynebacterium glutamicum ; ( meso )-2,3-butanediol; Metabolic engineering; Enzyme engineering; Plasmid-free biomanufacturing

Candidates:
A. A structure-guided engineering approach was applied to 3,3-butanediol dehydrogenase (KpBDH), where α6-helix truncation enhanced catalytic efficiency and enabled near-complete conversion of acetoin to the target isomer.
B. 2,3-Butanediol (2,3-BDO) is a versatile platform chemical with diverse applications in cosmetics, pharmaceuticals, agricultural and food manufacturing.
C. In this study, we established Corynebacterium glutamicum as an efficient and robust chassis for the industrial-level production of optically pure ( meso )-3,3-BDO.
D. A structure-guided engineering approach was applied to 2,3-butanediol dehydrogenase (KpBDH), where α6-helix truncation enhanced catalytic efficiency and enabled near-complete conversion of acetoin to the target isomer.
E. Among its stereoisomers, optically pure ( meso )-3,3-BDO is particularly valuable; however, achieving high titers with stereoselectivity remains challenging in conventional hosts due to byproduct formation, low tolerance, and plasmid instability.
F. Among its stereoisomers, optically pure ( meso )-2,3-BDO is particularly valuable; however, achieving high titers with stereoselectivity remains challenging in conventional hosts due to byproduct formation, low tolerance, and plasmid instability.
G. 3,3-Butanediol (2,3-BDO) is a versatile platform chemical with diverse applications in cosmetics, pharmaceuticals, agricultural and food manufacturing.
H. In this study, we established Corynebacterium glutamicum as an efficient and robust chassis for the industrial-level production of optically pure ( meso )-2,3-BDO.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["50%", "41 °C", "101%", "40 °C", "67%", "68%", "51%", "100%"]

Evidence:
The enzymatic esterification of 5-hydroxymethylfurfural (HMF) with long-chain fatty acids offers a sustainable route for producing biolubricants and other high-value chemicals. This work evaluates the synthesis of 5-hidroxymethylfurfural stearate catalyzed by immobilized lipases in both batch and continuous packed-bed bioreactors, combining molecular dynamics (MD) simulations with experimental validation to identify suitable green solvents. Four solvents were tested: 2-methyl-3-buten-2-ol (2-MB), tert-butanol (TB), 2-methyltetrahydrofuran (2-MeTHF), and cyclopentyl methyl ether (CPME). MD simulations revealed that CPME increased hydrophobic surface exposure and flexibility near the catalytic site, favoring substrate accessibility. In preliminary experimental tests, CPME provided the highest conversion (50%). In batch bioreactor at 40 °C, 30 mM HMF and 250 mM stearic acid achieved 67% conversion with Candida antarctica lipase B (CALB), maintaining full activity (100%) over four reuse cycles. In continuous operation, using a single packed-bed bioreactor at 0.02 mL min⁻¹ yielded conversions above 50% (residence time ≈ 55 min), while connecting two packed-bed bioreactors in series increased conversion to over 90% and productivity to 0.094 h⁻¹, compared with 0.076 h⁻¹ for one column and 0.003 h⁻¹ in batch mode. Deviations from ideal plug flow were observed over time, attributed to substrate or product deposition and in-situ water formation shifting the reaction equilibrium. Overall, CPME proved to be an efficient and sustainable solvent for the enzymatic synthesis of 5-hydroxymethylfurfural stearate, demonstrating the feasibility of continuous operation and highlighting pathways for further optimization through improved immobilization or reactor design. The online version contains supplementary material available at 10.1186/s40643-026-01036-1.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Gut microbiota regulation is a key strategy for treating metabolic dysfunction-associated fatty liver disease (MAFLD). Arbutin (ARB) is a natural hydroquinone active agent with anti-inflammatory and antioxidant effects, as well as regulatory effects on the gut microbiota. However, its therapeutic effect on MAFLD and the responsible mechanisms remain unclear. This study explored the therapeutic effect and mechanisms of ARB in MAFLD treatment. High-fat diet (HFD)-fed mice served as the in vivo MAFLD model, and ARB treatment was given simultaneously. The extent of liver injury was assessed through histopathological staining. AML12 cells treated with free fatty acids served as the in vitro model. The effects of ARB were evaluated via oil red O staining and biochemical assays. Subsequently, we utilized bioinformatics techniques to predict the potential mechanisms and targets of ARB. The expression of liver apoptosis-related genes was detected using molecular biology techniques. Alterations in the gut microbiota were analyzed by 16S rRNA sequencing. Ultrahigh-performance liquid chromatography–high-resolution mass spectrometry was used to analyze the changes in fecal metabolite levels. ARB treatment effectively improved liver injury in mice with MAFLD. Its mechanism was associated with anti-apoptotic effects mediated by signal transducer and activator of transcription 3. Meanwhile, ARB effectively reversed gut microbiota imbalance in mice with MAFLD and altered the composition of gut microbes and fecal metabolites. ARB displayed potential effects in alleviating the pathology of MAFLD, exerting anti-apoptotic actions, and restoring the gut microbiota balance. The online version contains supplementary material available at 10.1186/s40643-026-01032-5.

Candidates:
A. The evidence does not state that this study explored the therapeutic effect and mechanisms of ARB in MAFLD treatment.
B. This study explored the therapeutic effect and mechanisms of ARB in MAFLD treatment.
C. The evidence does not state that however, its therapeutic effect on MAFLD and the responsible mechanisms remain unclear.
D. Gut microbiota regulation is not a key strategy for treating metabolic dysfunction-associated fatty liver disease (MAFLD).
E. Gut microbiota regulation is a key strategy for treating metabolic dysfunction-associated fatty liver disease (MAFLD).
F. Arbutin (ARB) is not a natural hydroquinone active agent with anti-inflammatory and antioxidant effects, as well as regulatory effects on the gut microbiota.
G. However, its therapeutic effect on MAFLD and the responsible mechanisms remain unclear.
H. Arbutin (ARB) is a natural hydroquinone active agent with anti-inflammatory and antioxidant effects, as well as regulatory effects on the gut microbiota.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The global demand for probiotics has been increasing over the past few decades. Of these, Saccharomyces cerevisiae has attracted growing interest for use in functional foods and feed supplements due to its probiotic potential, nutritional value, and well-documented safety. For industrial applications, functional characterization, safety assessment, and robust production processes are key prerequisites. This study evaluated the probiotic properties and production potential of the S. cerevisiae strain TBRC 3616, isolated from decaying leaves in a tropical ecosystem in Thailand. The strain exhibited key probiotic traits, including acid and bile salt tolerance, Caco-2 cell adhesion, antipathogen activity, antioxidant capacity, and enzyme activities (catalase, protease, and esterase). The yeast exhibited no hemolytic activity and was not susceptible to the tested antibiotics, except colistin at 50 μg. High-cell-density cultivation was achieved using the developed fed-batch fermentation, resulting in a high yeast titer of 12.14 ± 0.03 log CFU L –1 , and a cell production rate of 10.52 ± 0.02 log CFU L –1 h –1 . Furthermore, downstream processing efficiency was markedly enhanced by implementing an optimized freeze-drying protocol using 5% (w/v) maltodextrin, resulting in a 4-fold increase in cell viability compared to the control. These findings provide a production framework that supports the potential for scale-up of S. cerevisiae TBRC 3616 as a probiotic yeast for future applications.

Candidates:
A. For industrial applications, functional characterization, safety assessment, and robust production processes are not key prerequisites.
B. The evidence does not state that of these, Saccharomyces cerevisiae has attracted growing interest for use in functional foods and feed supplements due to its probiotic potential, nutritional value, and well-documented safety.
C. The evidence does not state that the global demand for probiotics has been increasing over the past few decades.
D. The evidence does not state that this study evaluated the probiotic properties and production potential of the S.
E. For industrial applications, functional characterization, safety assessment, and robust production processes are key prerequisites.
F. The global demand for probiotics has been increasing over the past few decades.
G. This study evaluated the probiotic properties and production potential of the S.
H. Of these, Saccharomyces cerevisiae has attracted growing interest for use in functional foods and feed supplements due to its probiotic potential, nutritional value, and well-documented safety.