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:
Today, PHB and its copolymers—potential plastic substitutes—are produced by fermenting sugar, which is not scalable to the volumes of plastic consumption. PHB from CH 4 can offer a sustainable process route, with CH 4 potentially produced from a variety of waste biomass streams through anaerobic digestion, gasification, and methanation. The high molar mass (M w ) of PHB is a key determinant of its mechanical properties, and strain, culture conditions and downstream processing influence it. In this work, the strain Methylocystis sp. GB 25 (DSMZ 7674) was grown on natural gas as the sole carbon and energy source and air (1:1) in a loop reactor with 350 L active fermentation volume, at 35 °C and ambient pressure. After two days of continuous growth, the bacteria were limited in P and N for 1, 2, and 2.5 days to determine the optimal conditions for PHB accumulation and the highest Mw as the target. The biomass was then centrifuged and spray-dried. For downstream processing, chloroform solvent extraction and selected enzymatic treatment were deployed, yielding ~40% PHB from the biomass. The PHB obtained by solvent extraction exhibited high average weight molar masses of M w ~1.1–1.5 × 10 6 g mol −1 . The highest M w was obtained after one day of limitation, whereas enzyme treatment resulted in partially degraded PHB. Cold chloroform maceration, interesting due to energy savings, did not achieve sufficient extraction efficiency because it was unable to extract high-molar-mass PHB fractions. The extracted PHB has a high molar mass, more than double that of standard commercial PHB, and was characterized by DSC, which showed a high degree of crystallinity of up to 70% with a melting temperature of close to 180 °C. Mechanical tensile properties measurements, as well as dynamic mechanical thermal analysis (DMTA), were performed. Degradation of the PHB by enzymes was also determined. Methanotrophic PHB is a promising bioplastics material. The high M w can limit and delay polymer degradation in practical processing steps, making the material more versatile and robust.

Candidates:
A. The high molar mass (M w ) of PHB is not a key determinant of its mechanical properties, and strain, culture conditions and downstream processing influence it.
B. Today, PHB and its copolymers—potential plastic substitutes—are produced by fermenting sugar, which is not scalable to the volumes of plastic consumption.
C. In this work, the strain Methylocystis sp.
D. PHB from CH 5 can offer a sustainable process route, with CH 4 potentially produced from a variety of waste biomass streams through anaerobic digestion, gasification, and methanation.
E. PHB from CH 4 can offer a sustainable process route, with CH 4 potentially produced from a variety of waste biomass streams through anaerobic digestion, gasification, and methanation.
F. The high molar mass (M w ) of PHB is a key determinant of its mechanical properties, and strain, culture conditions and downstream processing influence it.
G. Today, PHB and its copolymers—potential plastic substitutes—are produced by fermenting sugar, which is not not scalable to the volumes of plastic consumption.
H. The evidence does not state that in this work, the strain Methylocystis sp.


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: ["5H", "5 L", "4H", "6 L"]

Evidence:
Scalable moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen without heterologous P4H expression, offering a sustainable and vegan alternative to conventional collagens derived from animals. Collagens are structural proteins of the extracellular matrix essential for skin elasticity and integrity. They are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via protoplast transformation. Immunodetection identified collagen-producing lines, and mass spectrometry validated the product and detected prolyl-hydroxylation on 23 sites. The presence of this important post-translational modification underscores the high biomimetic quality of the product. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions, including CO 2 supplementation, pH, and light intensity, were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide in the culture supernatant after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens for cosmetic formulations. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements. The online version contains supplementary material available at 10.1007/s00299-026-03727-7.


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:
Carotenoids and apocarotenoids constitute a structurally and functionally sundry class of isoprenoids whose significance extends from photosynthetic light capture and photoprotection to phytohormone signaling, flavor and aroma formation, and emerging biomedical applications. While recent appraisals have emphasized quantitative advances in microbial production, this mini-review adopts a pathway module-centric perspective. We examine each biosynthetic stage from precursor supply, condensation to geranylgeranyl diphosphate (GGPP), phytoene synthesis, desaturation/isomerization, cyclization, hydroxylation, ketolation, epoxidation, and oxidative cleavage, highlighting novel enzymatic variants, mutagenesis studies, fusion strategies, and compartmentalization approaches that impart metabolic control. Special emphasis is placed on recently discovered and engineered enzymes, as well as synthetic biology tools. This review integrates diverse enzyme sources, host ranges across plants, fungi, algae, yeasts, and bacteria, as well as pathway modularity, to provide an updated review of recent literature. We conclude by outlining future directions that highlight gaps and potential areas for future work. This focused synthesis aims to equip researchers with a hierarchical understanding of the pathways and strategies to advance carotenoid and apocarotenoid biosynthesis.

Candidates:
A. The evidence does not state that we examine each biosynthetic stage from precursor supply, condensation to geranylgeranyl diphosphate (GGPP), phytoene synthesis, desaturation/isomerization, cyclization, hydroxylation, ketolation, epoxidation, and oxidative cleavage, highlighting novel enzymatic variants, mutagenesis studies, fusion strategies, and compartmentalization approaches that impart metabolic control.
B. While recent appraisals have emphasized quantitative advances in microbial production, this mini-review adopts a pathway module-centric perspective.
C. We examine each biosynthetic stage from precursor supply, condensation to geranylgeranyl diphosphate (GGPP), phytoene synthesis, desaturation/isomerization, cyclization, hydroxylation, ketolation, epoxidation, and oxidative cleavage, highlighting novel enzymatic variants, mutagenesis studies, fusion strategies, and compartmentalization approaches that impart metabolic control.
D. Carotenoids and apocarotenoids constitute a structurally and functionally sundry class of isoprenoids whose significance extends from photosynthetic light capture and photoprotection to phytohormone signaling, flavor and aroma formation, and emerging biomedical applications.
E. The evidence does not state that carotenoids and apocarotenoids constitute a structurally and functionally sundry class of isoprenoids whose significance extends from photosynthetic light capture and photoprotection to phytohormone signaling, flavor and aroma formation, and emerging biomedical applications.
F. The evidence does not state that while recent appraisals have emphasized quantitative advances in microbial production, this mini-review adopts a pathway module-centric perspective.
G. Special emphasis is not placed on recently discovered and engineered enzymes, as well as synthetic biology tools.
H. Special emphasis is placed on recently discovered and engineered enzymes, as well as synthetic biology tools.


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:
Different yeast species, including Ogataea polymorpha , are often used as hosts for recombinant protein production. One of the most important factors limiting such applications is yeast-specific modifications of glycoside chains attached to secretory proteins. This problem can potentially be solved by the identification and inactivation of genes responsible for these modifications. Previously we demonstrated that the exceptional resistance of O. polymorpha to vanadate depends on the ABV1 gene responsible for the mannosylphosphorylation of protein glycoside chain in the Golgi apparatus. Here we show that mutations altering protein glycosylation in the secretory pathway can be selected in the abv1Δ mutant by screening for vanadate resistance. For one such mutant, we identified the responsible gene, which encodes a putative α-1,2-mannosyltransferase. To ensure the absence of phosphomannosylation, both O. polymorpha genes, ABV1 and MNN4 , which encode mannosylphosphate transferase homologs, were inactivated. Some vanadate resistant mutants generated in this strain showed defects in N -glycosylation of a recombinant glycoprotein. This demonstrates that the effects of N -glycosylation on vanadate resistance in O. polymorpha are not mediated by phosphomannosylation per se and that identification of certain genes responsible for N -glycosylation in this yeast can be performed via selection of vanadate resistant clones.

Candidates:
A. One of the most important factors limiting such applications is not yeast-specific modifications of glycoside chains attached to secretory proteins.
B. The evidence does not state that previously we demonstrated that the exceptional resistance of O.
C. Different yeast species, including Ogataea polymorpha , are often used as hosts for recombinant protein production.
D. This problem can potentially be solved by the identification and inactivation of genes responsible for these modifications.
E. Previously we demonstrated that the exceptional resistance of O.
F. This problem cannot potentially be solved by the identification and inactivation of genes responsible for these modifications.
G. One of the most important factors limiting such applications is yeast-specific modifications of glycoside chains attached to secretory proteins.
H. Different yeast species, including Ogataea polymorpha , are not often used as hosts for recombinant protein production.


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: ["31%", "30%", "87%", "81%", "88%", "7%", "6%", "80%"]

Evidence:
CAR-T cell therapy is leading the way in the field of cancer cell immunotherapies due to its high success rates. However, the manufacturing of CAR-T cells remains complex and expensive. T-cell enrichment from patient apheresis starting material is a key step in the manufacture but cellular impurities interfere with the ex vivo transduction of T-cells and their proliferation. Current enrichment methods including magnetic bead selection suffer from various limitations. We report here a bead-less T-cell enrichment process through a two-stage procedure based on inertial microfluidics. Using apheresis like starting material samples from healthy donors, the dual-stage process showed an efficient 87% (SD ± 6%) enrichment and 80% (SD ± 30%) recovery of T-cells. Validation of the process with ovarian cancer samples resulted in a T-cell purity 70% (SD ± 10%) from a starting purity of 48% (SD ± 6%) at a 64% (SD ± 4%) T-cell recovery. The two-stage inertial microfluidic process was also shown to have no detectable effect on the proliferation of the cells.


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:
Recombinant protein expression can be a limiting step in the production of protein reagents for drug discovery and other biotechnology applications. We introduce RP3Net (Recombinant Protein Production Prediction Network), an AI model of small-scale heterologous soluble protein expression in Escherichia coli . RP3Net utilizes the most recent protein and genomic foundational models. A curated dataset of internal experimental results from AstraZeneca and publicly available data from the Structural Genomics Consortium was used for training, validation and testing of RP3Net. RP3Net achieves an increase in area under the receiver operator curve (AUROC) of 0.15, compared to a baseline model. When experimentally validated on an independent, prospective, manually selected set of 97 constructs, RP3Net outperformed currently available models, with an AUROC of 0.83, delivering accurate predictions in 77% of the cases, and correctly identifying successfully expressing constructs in 92% of cases. The model, along with installation and running instructions, is available under an MIT licence at https://github.com/RP3Net/RP3Net , DOI 10.5281/zenodo.17243498.

Candidates:
A. A curated dataset of internal experimental results from AstraZeneca and publicly available data from the Structural Genomics Consortium was used for training, validation and testing of RP4Net.
B. RP4Net utilizes the most recent protein and genomic foundational models.
C. RP3Net utilizes the most recent protein and genomic foundational models.
D. Recombinant protein expression cannot be a limiting step in the production of protein reagents for drug discovery and other biotechnology applications.
E. Recombinant protein expression can be a limiting step in the production of protein reagents for drug discovery and other biotechnology applications.
F. We introduce RP4Net (Recombinant Protein Production Prediction Network), an AI model of small-scale heterologous soluble protein expression in Escherichia coli .
G. A curated dataset of internal experimental results from AstraZeneca and publicly available data from the Structural Genomics Consortium was used for training, validation and testing of RP3Net.
H. We introduce RP3Net (Recombinant Protein Production Prediction Network), an AI model of small-scale heterologous soluble protein expression in Escherichia coli .


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: ["3.5 µg/mL", "2.5 µg/mL"]

Evidence:
Saccharomyces cerevisiae is an established production host for therapeutic proteins; many of those are small proteins such as insulin or glucagon-like peptide-1 (GLP-1) analogs. Contrastingly, proteins of higher molecular weight, foremost antibodies, did not reach the market due, among other factors, to limiting productivity. Here we addressed the loss of product to protein degradation through a combination of genetic engineering of the host and medium optimization. We screened target genes that either directly or indirectly can lead to proteolytic degradation. We identified four deletions that are beneficial for expression: PEP1 and VPS30 , which both can channel proteins to the vacuole for degradation; MON2 , which can lead to the re-uptake of secreted proteins; and ALG3 , which can affect the permeability of the cell wall. In parallel, we developed a small-scale fed-batch cultivation system for 24-well deep well plate cultivations and using an amino acid-rich medium. To stabilize secreted proteins, we screened chemical chaperones and osmolytes. We fortified the medium with arginine, 4-phenylbutyrate (4-PBA), and Tween-20. Using the engineered yeast strain, which features VPS30 , PEP1 , and ALG3 deletions, and the small-scale fed-batch system, we obtained 2.5 µg/mL of a secreted chimeric fusion of a nanobody to the crystallizable fragment (Fc) of a human immunoglobulin. Instrumental to the increase in the final titer were the reduced losses. This was achieved by a combination of complementary measures: improving diffusion through the cell wall, achieved through genetic engineering, and reducing losses to proteolytic degradation through medium optimization and genetic engineering. Moreover, we showed that the engineered strain and cultivation set-up are suitable for the production of different antibodies. • Chemical chaperones and amino acid-rich medium increased secreted protein titers. • Medium and host engineering are instrumental for improving productivity. • Small-scale cultivation system enables production levels suitable for characterization. The online version contains supplementary material available at 10.1007/s00253-025-13700-1. Keywords: Saccharomyces cerevisiae , Chemical chaperones, Small-scale production system, Medium optimization, Recombinant antibody production, Chimeric nanobody-Fc fusion protein


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:
Gas fermentation offers a sustainable alternative for valorizing climate-active gases and industrial off-gases. Currently, these gases require energy-intensive purification steps before they can be used in chemical processes such as Fischer–Tropsch synthesis. In gas fermentation, anaerobic bacteria produce acetate from industrial off-gases. Compared to chemical processes, the anaerobic bacteria offer greater tolerance to varying gas concentrations and impurities. One major product of these anaerobic valorization processes is acetate, which can be used as a co-substrate in a variety of biological processes. This study evaluates Corynebacterium glutamicum and Ustilago maydis in benchtop cultivations using 10–20% (v/v) sterile-filtered acetate-rich supernatants from Acetobacterium woodii fermentation to produce L-lysine and triglycerides. Partial substitution of glucose with these supernatants supported robust growth and required no additional purification beyond sterile filtration. C. glutamicum achieved a L-lysine concentration of 3.5 ± 0.27 g∙L −1 and exhibited a diauxic growth pattern on glucose and acetate. In U. maydis , supernatant addition shortened the lag phase by approximately 2 h but reduced triglyceride yields modestly due to higher nitrogen availability. Optimizing the nitrogen-to-carbon ratio in benchtop fermentations resulted in a triglyceride concentration of 12.75 ± 1.17 g∙L −1 , demonstrating the feasibility of this approach. Collectively, the results demonstrate a viable method for replacing a portion of refined glucose with acetate-rich supernatants, thereby enabling a cost-efficient integration of anaerobic gas valorization with aerobic biomanufacturing. The online version contains supplementary material available at 10.1186/s13068-025-02732-4.

Candidates:
A. The evidence does not state that in gas fermentation, anaerobic bacteria produce acetate from industrial off-gases.
B. Gas fermentation offers a sustainable alternative for valorizing climate-active gases and industrial off-gases.
C. The evidence does not state that compared to chemical processes, the anaerobic bacteria offer greater tolerance to varying gas concentrations and impurities.
D. In gas fermentation, anaerobic bacteria produce acetate from industrial off-gases.
E. Compared to chemical processes, the anaerobic bacteria offer greater tolerance to varying gas concentrations and impurities.
F. The evidence does not state that gas fermentation offers a sustainable alternative for valorizing climate-active gases and industrial off-gases.
G. Currently, these gases require energy-intensive purification steps before they can be used in chemical processes such as Fischer–Tropsch synthesis.
H. Currently, these gases require energy-intensive purification steps before they cannot be used in chemical processes such as Fischer–Tropsch synthesis.


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:
Komagataella pastoris is extensively used as a microbial cell factory for the production of recombinant proteins and high‐value compounds. However, tightly controlled promoter systems responsive to safe and economical inducers are required for precise metabolic and pathway engineering in this yeast species. Cumate‐inducible promoters are an ideal choice due to the safety and low cost of cumate. In this study, we systematically optimised the insertion sites of the CuO operator sequence within the strong promoter P GCW14 to isolate a high‐activity variant that we designated as P GCWCuO03 . To fine‐tune the expression of the repressor protein CymR, we developed a truncated promoter of P GAP , designated as P GAP200 . Based on the optimal promoter P GCWCuO03 and the CymR expression unit, we constructed a robust CymR/CuO‐mediated cumate‐inducible promoter, designated as P gc , in K. pastoris . P gc demonstrated outstanding induction properties, resulting in an approximately 11‐fold increase in target protein production following induction. Promoter substitution assays validated the effectiveness of P gc in temporal gene expression control, highlighting the significant potential of this promoter for both basic research and industrial bioprocessing applications in synthetic biology and biotechnology in K. pastoris .

Candidates:
A. However, tightly controlled promoter systems responsive to safe and economical inducers are required for precise metabolic and pathway engineering in this yeast species.
B. Cumate‐inducible promoters are not an ideal choice due to the safety and low cost of cumate.
C. Komagataella pastoris is not extensively used as a microbial cell factory for the production of recombinant proteins and high‐value compounds.
D. Cumate‐inducible promoters are an ideal choice due to the safety and low cost of cumate.
E. Komagataella pastoris is extensively used as a microbial cell factory for the production of recombinant proteins and high‐value compounds.
F. However, tightly controlled promoter systems responsive to safe and economical inducers are not required for precise metabolic and pathway engineering in this yeast species.
G. In this study, we systematically optimised the insertion sites of the CuO operator sequence within the strong promoter P GCW14 to isolate a high‐activity variant that we designated as P GCWCuO03 .
H. In this study, we systematically optimised the insertion sites of the CuO operator sequence within the strong promoter P GCW15 to isolate a high‐activity variant that we designated as P GCWCuO03 .


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:
Precise quantification of recombinant proteins is essential for assessing and comparing expression efficiency and optimizing production processes. Fluorescent proteins have emerged as powerful tools for real-time monitoring of gene expression and protein tracking. However, standardized and validated methods for their quantification, particularly for the widely used green fluorescent protein, remain limited. To date, no universally adopted protocol has emerged. This study presents a high-throughput method for the quantification of recombinantly produced Emerald Green Fluorescent Protein (EmGFP) based on direct fluorescence measurements of the cell suspension while quantifying and integrating potential effects of signal attenuation. The workflow uses solely standard laboratory equipment, ensuring broad accessibility and easy implementation. Moreover, in-house EmGFP standard preparation and quantification is described. The method was validated according to FDA guidelines “Analytical Procedures and Methods Validation for Drugs and Biologics,” addressing the requirements of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and recovery rate. Investigation was conducted using Escherichia coli BL21 cells expressing EmGFP, widely available sodium fluorescein as a chemical standard, commercial GFP, and an in-house EmGFP standard. A robust correlation (linear fitting, R 2 0.96) of the EmGFP concentration and relative fluorescence units (RFU) was established, enabling efficient and high-throughput fluorescence quantification using a standardized workflow in a microtiter-based format suitable for the application in comparative studies across different expression constructs, conditions, and scales. By enabling absolute quantification of fluorescent proteins, this method supports both real-time bioprocess optimization and broader applications in protein production research. The online version contains supplementary material available at 10.1007/s00253-026-13734-z.

Candidates:
A. Precise quantification of recombinant proteins is essential for assessing and comparing expression efficiency and optimizing production processes.
B. To date, no universally adopted protocol has emerged.
C. The evidence does not state that to date, no universally adopted protocol has emerged.
D. The evidence does not state that however, standardized and validated methods for their quantification, particularly for the widely used green fluorescent protein, remain limited.
E. Precise quantification of recombinant proteins is not essential for assessing and comparing expression efficiency and optimizing production processes.
F. Fluorescent proteins have emerged as powerful tools for real-time monitoring of gene expression and protein tracking.
G. The evidence does not state that fluorescent proteins have emerged as powerful tools for real-time monitoring of gene expression and protein tracking.
H. However, standardized and validated methods for their quantification, particularly for the widely used green fluorescent protein, remain limited.


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:
Microbial fermentation is an established technology that is becoming increasingly used to produce key food components. Among the various microorganisms used, yeasts play crucial roles due to their efficiency in synthesizing a wide range of industrially important compounds. The growing demand for sustainable, locally sourced, and animal-free food ingredients has increased the focus on yeast biomass and its derivatives. These yeast-based products, such as food emulsifiers, are a promising next-generation of food components, offering advantages like a low risk of allergenicity. Yeast biomass-based fractions have been effectively used as emulsifiers in various food products including in dairy, meat, bakery, meat alternatives, mayonnaises and salad dressing, with effective properties demonstrated in a range of oil-in-water, water-in-oil, and Pickering emulsion models. Both whole cell biomass and yeast cell fractions such as the yeast cell wall, mannoproteins, glucans, exopolysaccharides and other yeast-derived compounds have been demonstrated to function as effective emulsifiers. An increasingly large number of yeasts, beyond just Saccharomyces cerevisiae , have been studied as potential sources of these emulsifiers with the extraction and purification methods employed depending on the specific emulsifier targeted, the required purity, and the intended application. Efficient, cost-effective, and sustainable processes are key to enabling industrial-scale production of these emulsifiers, as such this article reviews the potential yeast-derived food emulsifiers, lists the various yeast species investigated to date, examines the extraction and purification methods, and highlights the potential food applications of these yeast-derived emulsifiers.

Candidates:
A. Microbial fermentation is an established technology that is becoming increasingly used to produce key food components.
B. The evidence does not state that among the various microorganisms used, yeasts play crucial roles due to their efficiency in synthesizing a wide range of industrially important compounds.
C. Among the various microorganisms used, yeasts play crucial roles due to their efficiency in synthesizing a wide range of industrially important compounds.
D. These yeast-based products, such as food emulsifiers, are a promising next-generation of food components, offering advantages like a low risk of allergenicity.
E. The evidence does not state that the growing demand for sustainable, locally sourced, and animal-free food ingredients has increased the focus on yeast biomass and its derivatives.
F. The growing demand for sustainable, locally sourced, and animal-free food ingredients has increased the focus on yeast biomass and its derivatives.
G. Microbial fermentation is not an established technology that is becoming increasingly used to produce key food components.
H. These yeast-based products, such as food emulsifiers, are not a promising next-generation of food components, offering advantages like a low risk of allergenicity.


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:
Billions of clinical ECGs exist only as paper scans, making them unusable for modern automated diagnostics. We introduce a fully automated, modular framework that converts scanned or photographed ECGs into digital signals, suitable for both clinical and research applications. The framework is validated on 37,191 ECG images with 1596 collected at Akershus University Hospital, where the algorithm obtains a mean signal-to-noise ratio of 19.65 dB on scanned papers with common artifacts. It is further evaluated on the Emory Paper Digitization ECG Dataset, comprising 35,595 images, including images with perspective distortion, wrinkles, and stains. The model improves on the state-of-the-art in all subcategories. The full software is released as open-source, promoting reproducibility and further development. We hope the software will contribute to unlocking retrospective ECG archives and democratize access to AI-driven diagnostics.

Candidates:
A. It is further evaluated on the Emory Paper Digitization ECG Dataset, comprising 36,595 images, including images with perspective distortion, wrinkles, and stains.
B. Billions of clinical ECGs exist only as paper scans, making them unusable for modern automated diagnostics.
C. The evidence does not state that billions of clinical ECGs exist only as paper scans, making them unusable for modern automated diagnostics.
D. The framework is validated on 38,191 ECG images with 1596 collected at Akershus University Hospital, where the algorithm obtains a mean signal-to-noise ratio of 19.65 dB on scanned papers with common artifacts.
E. It is further evaluated on the Emory Paper Digitization ECG Dataset, comprising 35,595 images, including images with perspective distortion, wrinkles, and stains.
F. We introduce a fully automated, modular framework that converts scanned or photographed ECGs into digital signals, suitable for both clinical and research applications.
G. The framework is validated on 37,191 ECG images with 1596 collected at Akershus University Hospital, where the algorithm obtains a mean signal-to-noise ratio of 19.65 dB on scanned papers with common artifacts.
H. The evidence does not state that we introduce a fully automated, modular framework that converts scanned or photographed ECGs into digital signals, suitable for both clinical and research applications.


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:
Bovine mastitis is a major disease affecting dairy cow health and milk production, and current treatment options still have important limitations. Previous studies have shown that a protein called cyclophilin A is closely associated with inflammatory responses during mastitis, but further research requires a stable and reliable source of this protein. In this study, we established a method for the stable expression of bovine cyclophilin A in mammalian cells and demonstrated that these cells can continuously express the target protein. This work provides a solid technical foundation for future studies on the role of this protein in bovine mastitis and for the development of related diagnostic or intervention approaches, and it may contribute to improving the prevention and control of bovine mastitis.

Candidates:
A. Previous studies have shown that a protein called cyclophilin A is closely associated with inflammatory responses during mastitis, but further research requires a stable and reliable source of this protein.
B. Bovine mastitis is a major disease affecting dairy cow health and milk production, and current treatment options still have important limitations.
C. In this study, we established a method for the stable expression of bovine cyclophilin A in mammalian cells and demonstrated that these cells can continuously express the target protein.
D. The evidence does not state that this work provides a solid technical foundation for future studies on the role of this protein in bovine mastitis and for the development of related diagnostic or intervention approaches, and it may contribute to improving the prevention and control of bovine mastitis.
E. Bovine mastitis is not a major disease affecting dairy cow health and milk production, and current treatment options still have important limitations.
F. Previous studies have shown that a protein called cyclophilin A is not closely associated with inflammatory responses during mastitis, but further research requires a stable and reliable source of this protein.
G. This work provides a solid technical foundation for future studies on the role of this protein in bovine mastitis and for the development of related diagnostic or intervention approaches, and it may contribute to improving the prevention and control of bovine mastitis.
H. In this study, we established a method for the stable expression of bovine cyclophilin A in mammalian cells and demonstrated that these cells cannot continuously express the target protein.


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:
Microbial biosurfactants have emerged as natural and sustainable alternatives to synthetic surfactants used in the food industry, due to the growing demand for biodegradable and safe ingredients. Produced by bacteria, fungi, and yeasts, these compounds exhibit important physicochemical properties, such as emulsifying capacity, surface tension reduction, foam stabilization, and favorable interaction with different food matrices. In addition to their technological function, they exhibit relevant biological activities, including antioxidant and antimicrobial action, which contribute to the control of lipid oxidation and microbiological deterioration. These characteristics make biosurfactants attractive for applications in emulsions, fermented beverages, aerated products, probiotic systems, and bioactive packaging. The objective of this work is to provide a narrative literature review that integrates recent advances in the production, functionality, safety, sustainability, and application perspectives of biosurfactants in the food sector. In the field of production, biotechnological advances have made it possible to overcome historical limitations such as high cost and low yield. Strategies such as the use of agro-industrial waste, metabolic engineering, microbial co-cultures, continuous fermentations, and in situ removal techniques have increased efficiency and reduced environmental impacts. Despite the advances, significant challenges remain. Future prospects and advances tend to facilitate industrial adoption and consolidate biosurfactants as strategic ingredients for the development of more sustainable, functional, and technologically advanced foods.

Candidates:
A. In addition to their technological function, they exhibit relevant biological activities, including antioxidant and antimicrobial action, which contribute to the control of lipid oxidation and microbiological deterioration.
B. The evidence does not state that in addition to their technological function, they exhibit relevant biological activities, including antioxidant and antimicrobial action, which contribute to the control of lipid oxidation and microbiological deterioration.
C. Microbial biosurfactants have emerged as natural and sustainable alternatives to synthetic surfactants used in the food industry, due to the growing demand for biodegradable and safe ingredients.
D. The evidence does not state that these characteristics make biosurfactants attractive for applications in emulsions, fermented beverages, aerated products, probiotic systems, and bioactive packaging.
E. Produced by bacteria, fungi, and yeasts, these compounds exhibit important physicochemical properties, such as emulsifying capacity, surface tension reduction, foam stabilization, and favorable interaction with different food matrices.
F. The evidence does not state that produced by bacteria, fungi, and yeasts, these compounds exhibit important physicochemical properties, such as emulsifying capacity, surface tension reduction, foam stabilization, and favorable interaction with different food matrices.
G. The evidence does not state that microbial biosurfactants have emerged as natural and sustainable alternatives to synthetic surfactants used in the food industry, due to the growing demand for biodegradable and safe ingredients.
H. These characteristics make biosurfactants attractive for applications in emulsions, fermented beverages, aerated products, probiotic systems, and bioactive packaging.


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:
Centella asiatica has emerged as a strategic biomass for the sustainable production of high-value biochemicals at the interface of traditional medicine and modern biotechnology. This review consolidates the current knowledge on its phytochemical diversity, emphasizing triterpenoid saponins—asiaticoside, madecassoside, asiatic acid, and madecassic acid—as core bioactive molecules relevant to pharmaceutical, dermatological, nutraceutical, and functional-ingredient applications. Advances in green extraction technologies, including ultrasound-assisted, microwave-assisted, ohmic-heating, and supercritical CO 2 systems, have demonstrated superior efficiency in recovering high-purity biochemicals while significantly reducing solvent use, energy demand, and environmental impact compared with conventional methods. Complementary analytical and standardization platforms, such as HPLC, UPLC, and GC–MS, enable rigorous quality control across the entire value chain, supporting the development of reproducible and regulatory-compliant biochemical extracts. From a biomass valorization and biorefinery perspective, C. asiatica offers multiple metabolite streams that align with circular economy and field-to-market sustainability principles. Key challenges remain, including agronomic variability, scaling up green extraction, and supply chain resilience. However, emerging solutions, such as Good Agricultural and Collection Practices (GACP) guided cultivation, plant tissue culture, metabolic engineering, and integrated biorefinery frameworks, show strong potential for establishing a reliable and environmentally responsible production system. Collectively, C. asiatica represents a model species for sustainable biochemical production, combining scientific efficacy with industrial, economic, and ecological relevance.

Candidates:
A. Centella asiatica has emerged as a strategic biomass for the sustainable production of high-value biochemicals at the interface of traditional medicine and modern biotechnology.
B. Complementary analytical and standardization platforms, such as HPLC, UPLC, and GC–MS, enable rigorous quality control across the entire value chain, supporting the development of reproducible and regulatory-compliant biochemical extracts.
C. The evidence does not state that this review consolidates the current knowledge on its phytochemical diversity, emphasizing triterpenoid saponins—asiaticoside, madecassoside, asiatic acid, and madecassic acid—as core bioactive molecules relevant to pharmaceutical, dermatological, nutraceutical, and functional-ingredient applications.
D. The evidence does not state that centella asiatica has emerged as a strategic biomass for the sustainable production of high-value biochemicals at the interface of traditional medicine and modern biotechnology.
E. This review consolidates the current knowledge on its phytochemical diversity, emphasizing triterpenoid saponins—asiaticoside, madecassoside, asiatic acid, and madecassic acid—as core bioactive molecules relevant to pharmaceutical, dermatological, nutraceutical, and functional-ingredient applications.
F. The evidence does not state that complementary analytical and standardization platforms, such as HPLC, UPLC, and GC–MS, enable rigorous quality control across the entire value chain, supporting the development of reproducible and regulatory-compliant biochemical extracts.
G. Advances in green extraction technologies, including ultrasound-assisted, microwave-assisted, ohmic-heating, and supercritical CO 3 systems, have demonstrated superior efficiency in recovering high-purity biochemicals while significantly reducing solvent use, energy demand, and environmental impact compared with conventional methods.
H. Advances in green extraction technologies, including ultrasound-assisted, microwave-assisted, ohmic-heating, and supercritical CO 2 systems, have demonstrated superior efficiency in recovering high-purity biochemicals while significantly reducing solvent use, energy demand, and environmental impact compared with conventional methods.


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:
Monoacylglycerols (MAGs) are significant intermediate byproducts in the hydrolysis of oils and fats. The accumulation of MAGs not only reduces the quality and purity of the final products in biodiesel production and edible oil refining but also poses challenges for downstream separation processes. Therefore, the development of efficient biocatalysts for the specific MAG conversion is of great industrial importance. The lipase from Aspergillus oryzae (AOL) has shown potential for lipid modification; however, the wild-type enzyme (WT) suffers from poor solubility, tendency to aggregate, and low specific activity towards MAGs in aqueous systems, which severely restricts its practical application. In this study, a combinatorial protein engineering strategy was employed to overcome these limitations. We integrated fusion protein technology with rational design to enhance both the functional expression and catalytic efficiency of AOL. Firstly, the superfolder green fluorescent protein (sfGFP) was fused to the N-terminus of AOL. The results indicated that the sfGFP fusion tag significantly improved the solubility and stability of the enzyme, preventing the formation of inclusion bodies. The fusion protein sfGFP-AOL exhibited a MAG conversion rate of approximately 65%, confirming the positive impact of the fusion tag on enzyme developability. To further boost catalytic performance, site-directed mutagenesis was performed based on structural analysis. Among the variants, the mutant sfGFP-Y92Q emerged as the most potent candidate. In the MAG conversion, sfGFP-Y92Q achieved a conversion rate of 98%, which was not only significantly higher than that of sfGFP-AOL but also outperformed the widely used commercial immobilized lipase, Novozym 435 (~54%). Structural modeling and docking analysis revealed that the Y92Q mutation optimized the geometry of the active site. The substitution of Tyrosine with Glutamine at position 92 likely enlarged the substrate-binding pocket and altered the local electrostatic environment, thereby relieving steric hindrance and facilitating the access of the bulky MAG substrate to the catalytic center. In conclusion, this work demonstrates that the synergistic application of sfGFP fusion and rational point mutation (Y92Q) can dramatically transform the catalytic properties of AOL. The engineered sfGFP-Y92Q variant serves as a robust and highly efficient biocatalyst for MAG degradation. Its superior performance compared to commercial standards suggests immense potential for cost-effective applications in the bio-manufacturing of high-purity fatty acids and biodiesel, offering a greener alternative to traditional chemical processes.

Candidates:
A. Therefore, the development of efficient biocatalysts for the specific MAG conversion is not of great industrial importance.
B. The evidence does not state that the lipase from Aspergillus oryzae (AOL) has shown potential for lipid modification; however, the wild-type enzyme (WT) suffers from poor solubility, tendency to aggregate, and low specific activity towards MAGs in aqueous systems, which severely restricts its practical application.
C. Therefore, the development of efficient biocatalysts for the specific MAG conversion is of great industrial importance.
D. Monoacylglycerols (MAGs) are significant intermediate byproducts in the hydrolysis of oils and fats.
E. The evidence does not state that the accumulation of MAGs not only reduces the quality and purity of the final products in biodiesel production and edible oil refining but also poses challenges for downstream separation processes.
F. Monoacylglycerols (MAGs) are not significant intermediate byproducts in the hydrolysis of oils and fats.
G. The lipase from Aspergillus oryzae (AOL) has shown potential for lipid modification; however, the wild-type enzyme (WT) suffers from poor solubility, tendency to aggregate, and low specific activity towards MAGs in aqueous systems, which severely restricts its practical application.
H. The accumulation of MAGs not only reduces the quality and purity of the final products in biodiesel production and edible oil refining but also poses challenges for downstream separation processes.


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:
Detergent-compatible lipases are increasingly valued for their ability to remove stains under low-temperature and environmentally friendly washing conditions. Their industrial applicability depends on achieving high enzyme production, cost-effective purification, and stability within detergent formulations. Here, we report the purification and characterization of a highly active extracellular lipase from Streptomyces sp. AU-153 (1543 U/mL, p -NPP assay). A simplified aqueous two-phase system (ATPS) of poly(ethylene glycol) and sodium chloride achieved 8-fold purification with a recovery of 272.7%. The purified enzyme exhibited optimal activity at pH 8.0 and 40 °C, maintained stability across pH 7–11, and retained substantial activity up to 60 °C. Activity was enhanced by Ca 2+ , Mg 2+ , and β-mercaptoethanol, whereas PMSF inhibited activity. The lipase remained stable in various commercial detergents and in the presence of surfactants, oxidizing agents, and boron compounds. It also showed affinity toward sunflower and thermally degraded olive oils. Low-temperature washing assays confirmed its effectiveness in oil stain removal. To our knowledge, ATPS-based purification and washing performance of Streptomyces lipases have each been reported only once, and this study is the first to integrate both approaches for the same enzyme. Moreover, Streptomyces sp. AU-153 displayed one of the highest native extracellular lipase activities documented for the genus, while the ATPS protocol achieved one of the highest recoveries reported for microbial lipases. These findings establish strain AU-153 as a promising natural source of detergent-compatible lipases and highlight its potential for enzyme-based washing applications.

Candidates:
A. Here, we report the purification and characterization of a highly active extracellular lipase from Streptomyces sp.
B. A simplified aqueous two-phase system (ATPS) of poly(ethylene glycol) and sodium chloride achieved 8-fold purification with a recovery of 272.7%.
C. Detergent-compatible lipases are increasingly valued for their ability to remove stains under low-temperature and environmentally friendly washing conditions.
D. Their industrial applicability depends on achieving high enzyme production, cost-effective purification, and stability within detergent formulations.
E. The evidence does not state that here, we report the purification and characterization of a highly active extracellular lipase from Streptomyces sp.
F. A simplified aqueous two-phase system (ATPS) of poly(ethylene glycol) and sodium chloride achieved 9-fold purification with a recovery of 272.7%.
G. The evidence does not state that their industrial applicability depends on achieving high enzyme production, cost-effective purification, and stability within detergent formulations.
H. Detergent-compatible lipases are not increasingly valued for their ability to remove stains under low-temperature and environmentally friendly washing conditions.


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:
Extracellular vesicles (EVs) are heterogeneous, lipid bilayer-enclosed vesicles secreted by cells. Research on EVs dates back to the 1940s, and the term “exosomes” - a major subtype of EVs - was coined in 1981 to describe small membrane vesicles shed from cells. However, it is only in the past two decades that research in this area has expanded rapidly. By transferring functional biomolecules, EVs play a pivotal role in intercellular communication and regulate a wide range of cellular functions under both physiological and pathological conditions. Owing to their high biocompatibility, capacity to protect encapsulated cargo from degradation, and ability to cross biological barriers, EVs also show great promise as biomarkers and drug-delivery systems. Following the first, albeit unintentional, isolation of EVs in 1946, the 80th anniversary of EV research is now approaching. In this review, we trace the history of EV research and summarize key advances in the field. We also discuss current challenges and future prospects in this rapidly evolving area.

Candidates:
A. By transferring functional biomolecules, EVs play a pivotal role in intercellular communication and regulate a wide range of cellular functions under both physiological and pathological conditions.
B. Extracellular vesicles (EVs) are heterogeneous, lipid bilayer-enclosed vesicles secreted by cells.
C. Extracellular vesicles (EVs) are not heterogeneous, lipid bilayer-enclosed vesicles secreted by cells.
D. However, it is not only in the past two decades that research in this area has expanded rapidly.
E. Research on EVs dates back to the 1941s, and the term “exosomes” - a major subtype of EVs - was coined in 1981 to describe small membrane vesicles shed from cells.
F. However, it is only in the past two decades that research in this area has expanded rapidly.
G. The evidence does not state that by transferring functional biomolecules, EVs play a pivotal role in intercellular communication and regulate a wide range of cellular functions under both physiological and pathological conditions.
H. Research on EVs dates back to the 1940s, and the term “exosomes” - a major subtype of EVs - was coined in 1981 to describe small membrane vesicles shed from cells.


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: ["4.97 g/L", "2.28 g/L", "9.26 g/L", "3.28 g/L", "5.96 g/L", "3.97 g/L", "10.26 g/L", "4.96 g/L"]

Evidence:
Caldimonas thermodepolymerans DSM 15344, a moderately thermophilic bacterium, has emerged as a promising candidate for next-generation industrial biotechnology (NGIB) due to its ability to utilize lignocellulose-derived sugars for polyhydroxyalkanoate (PHA) production. This study assesses its metabolic potential by evaluating the utilization of various plant-derived sugars and their mixtures, with a focus on xylose, glucose, and cellobiose. The results indicate that C. thermodepolymerans exhibits a strong preference for xylose (3.97 g/L PHB) over glucose (2.28 g/L PHB) but demonstrates even greater efficiency in metabolizing cellobiose (4.96 g/L PHB). However, extracellular hydrolysis of cellobiose leads to glucose accumulation, which constrains overall productivity. Our findings suggest that the primary limitation in glucose metabolism is inefficient glucose transport rather than intracellular catabolism. To address this bottleneck, the glf glucose facilitator gene from the mesophilic bacterium Zymomonas mobilis was introduced into C. thermodepolymerans , enhancing its glucose utilization capacity. The engineered strain (Cald_GLF3) exhibited significantly improved PHA productivity, particularly when cultivated on sugar mixtures containing cellobiose. Despite being grown at suboptimal temperatures due to the thermal instability of Glf from Z. mobilis , Cald_GLF3 outperformed the wild-type strain, achieving notably high PHA yields when cultivated with cellobiose as the sole carbon source (9.26 g/L PHB). These findings highlight the critical role of glucose transport in the metabolism of C. thermodepolymerans and suggest that targeted engineering can further enhance its biotechnological potential. This study establishes C. thermodepolymerans as a promising thermophilic chassis for PHA production from lignocellulosic sugars, contributing to sustainable biopolymer synthesis. C. thermodepolymerans DSM 15344 produces PHA from lignocellulose-derived sugars Xylose and cellobiose are preferred substrates, while glucose is poorly utilized Deficient glucose transport in DSM 15344 restored by Zymomonas mobilis glf gene Keywords: Caldimonas thermodepolymerans , Polyhydroxyalkanoates, Thermophiles, Sugar metabolism, Glucose transporters, Lignocelluloses


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:
Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. In addition, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium . The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV’s scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa -infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for the development of effective alternatives to traditional antibiotics in the fight against this resilient pathogen.

Candidates:
A. The evidence does not state that aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity.
B. Pseudomonas aeruginosa is not a major human opportunistic pathogen associated with a high incidence of multi-drug resistance.
C. Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance.
D. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity.
E. The evidence does not state that in this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P.
F. aeruginosa Type 4 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV.
G. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV.
H. In this study, we employed single B cell sorting from cystic fibrosis patients to isolate human monoclonal antibodies (mAbs) targeting proteins from the P.


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:
M9 minimal media and its enhanced variants (M9 + and M9++) are widely used for recombinant protein expression in Escherichia coli, particularly for isotopic labeling required in structural biology techniques such as NMR spectroscopy. This study investigates how different compositions of M9-based media (M9, M9+, and M9++) influence bacterial growth, metabolic stress, and central carbon metabolism during recombinant expression of the protein. Using 1D ¹H NMR spectroscopy and multivariate statistical analysis, we observed distinct media-dependent metabolic shifts. Standard M9 exhibited limited bacterial growth and heightened stress-related fermentation, indicated by high ethanol and acetate levels. In contrast, M9 + significantly increased biomass but promoted pronounced overflow metabolism. M9 + + presented intermediate biomass levels and markedly reduced overflow metabolites, favoring biosynthesis pathways, notably increasing valine, acetoin, and formate concentrations. These findings suggest that further optimization of glucose concentration, nitrogen sources, and phosphate buffering could significantly improve the metabolic balance of M9++, creating an enhanced medium tailored for efficient, high-quality recombinant protein expression and isotopic labeling in E. coli . Keywords: Recombinant protein expression, M9 minimal media, Metabolomics, NMR spectroscopy, Isotopic labeling

Candidates:
A. This study investigates how different compositions of M9-based media (M9, M9+, and M9++) influence bacterial growth, metabolic stress, and central carbon metabolism during recombinant expression of the protein.
B. M10 minimal media and its enhanced variants (M9 + and M9++) are widely used for recombinant protein expression in Escherichia coli, particularly for isotopic labeling required in structural biology techniques such as NMR spectroscopy.
C. Using 1D ¹H NMR spectroscopy and multivariate statistical analysis, we observed distinct media-dependent metabolic shifts.
D. Using 2D ¹H NMR spectroscopy and multivariate statistical analysis, we observed distinct media-dependent metabolic shifts.
E. Standard M9 exhibited limited bacterial growth and heightened stress-related fermentation, indicated by high ethanol and acetate levels.
F. Standard M10 exhibited limited bacterial growth and heightened stress-related fermentation, indicated by high ethanol and acetate levels.
G. M9 minimal media and its enhanced variants (M9 + and M9++) are widely used for recombinant protein expression in Escherichia coli, particularly for isotopic labeling required in structural biology techniques such as NMR spectroscopy.
H. This study investigates how different compositions of M10-based media (M9, M9+, and M9++) influence bacterial growth, metabolic stress, and central carbon metabolism during recombinant expression of the protein.


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:
Radiation enteritis is a common complication in patients undergoing abdominal radiotherapy. Current management strategies face significant limitations: clinical agents like amifostine are hindered by systemic side effects and demanding administration; direct supplementation with radioprotective metabolites such as propionate suffers from low bioavailability and transient action; and conventional probiotics lack targeted therapeutic output. To address these challenges, we engineered Escherichia coli Nissle 1917 to function as a living therapeutic that continuously produces and delivers propionate directly in the gut. This propionate-engineered probiotic achieved a production yield of 181.33 ± 4.27 mg/L in vitro. In a mouse model of abdominal irradiation, this engineered bacterium alleviated radiation-induced intestinal damage by continuously releasing propionate and enhancing intestinal epithelial barrier function. Multi-omics analysis revealed that the engineered bacterium could restore intestinal microbiota homeostasis, enhancing the abundance of advantageous bacteria with radioprotective properties (e.g., Dubosiella , Akkermansia ). Moreover, it modulated intestinal microbiota metabolism, influencing the metabolism of ascorbic acid, aldoses, and other metabolites. Additionally, it protected the intestinal mucosal barrier from radiation-induced damage, which was associated with the modulation of the SOCS1/JAK2/STAT3 signaling pathway. This study introduces a novel biological therapy to mitigate the side effects of radiotherapy and could open new avenues for preventing and treating radiation-induced intestinal injury. The online version contains supplementary material available at 10.1186/s40643-026-01020-9.

Candidates:
A. Current management strategies face significant limitations: clinical agents like amifostine are not hindered by systemic side effects and demanding administration; direct supplementation with radioprotective metabolites such as propionate suffers from low bioavailability and transient action; and conventional probiotics lack targeted therapeutic output.
B. To address these challenges, we engineered Escherichia coli Nissle 1918 to function as a living therapeutic that continuously produces and delivers propionate directly in the gut.
C. This propionate-engineered probiotic achieved a production yield of 182.33 ± 4.27 mg/L in vitro.
D. This propionate-engineered probiotic achieved a production yield of 181.33 ± 4.27 mg/L in vitro.
E. Current management strategies face significant limitations: clinical agents like amifostine are hindered by systemic side effects and demanding administration; direct supplementation with radioprotective metabolites such as propionate suffers from low bioavailability and transient action; and conventional probiotics lack targeted therapeutic output.
F. Radiation enteritis is a common complication in patients undergoing abdominal radiotherapy.
G. To address these challenges, we engineered Escherichia coli Nissle 1917 to function as a living therapeutic that continuously produces and delivers propionate directly in the gut.
H. Radiation enteritis is not a common complication in patients undergoing abdominal radiotherapy.


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: ["49 h", "48 h"]

Evidence:
Tyrosinase is a binuclear copper oxidase central to melanogenesis and food browning and is a major target for depigmenting and anti-browning agents. Here we evaluate Altenusin, a fungal carboxy-biphenyl polyketide, as a tyrosinase-inhibitor scaffold by combining structure-based screening, enhanced fermentation and mechanistic enzymology. Docking against the mushroom tyrosinase Agaricus bisporus PPO 3 (AbPPO 3 ) highlighted Altenusin as a presumed dicopper-site binder, and genome mining of the producer strain revealed a polyketide synthase gene cluster consistent with its biosynthesis. Fermentation optimization and bioreactor transfer increased Altenusin titers up to 0.254 ± 0.022 g L −1 . In vitro , Altenusin inhibited in a substrate-dependent manner, with IC 50 values of 0.381 ± 0.002 mM ( l -tyrosine) and 0.162 ± 0.023 mM ( l -DOPA); kinetic analysis indicated competitive monophenolase inhibition and mixed-type diphenolase inhibition. Altenusin also showed strong radical-scavenging and copper-reducing activity, moderate Cu 2+ chelation and a narrow cytotoxicity window in HepG2 cells (48 h, CC 50 : 0.093 mM). Overall, these data define Altenusin as a biotechnologically tractable starting point for fungal carboxy-biphenyl inhibitor discovery.


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:
Over the past two decades, Meyerozyma caribbica has been identified as a metabolically versatile and ecologically adaptable yeast with significant relevance to biotechnology, agriculture, environmental remediation, and food applications. Since its formal description in 2005, this species has demonstrated the ability to grow on a wide range of substrates and under various stress conditions, facilitating the production of valuable bioproducts such as ethanol, xylitol, arabitol, and volatile aroma compounds. Multiple strains efficiently ferment lignocellulosic hydrolysates, tolerate inhibitory compounds, and remain active at elevated temperatures, which supports their application in integrated biorefineries. In addition to its fermentative capabilities, M. caribbica serves as an effective biocontrol agent through the production of antifungal metabolites, hydrolytic enzymes, mycoparasitism, nutrient competition, and the induction of plant defense responses. Environmental functions include the degradation of dyes, hydrocarbons, and organochlorine pesticides, as well as metal biosorption and the mitigation of oxidative stress in plants. There is also increasing interest in its potential as a probiotic and as a starter culture that can modulate sensory attributes in fermented foods. This review synthesizes 20 years of research on M. caribbica , focusing on its roles in bioproduct production, plant disease management, bioremediation, and probiotic or food-related applications. Keywords: biodegradation, non-conventional yeasts, biocontrol, probiotics, xylitol

Candidates:
A. Since its formal description in 2006, this species has demonstrated the ability to grow on a wide range of substrates and under various stress conditions, facilitating the production of valuable bioproducts such as ethanol, xylitol, arabitol, and volatile aroma compounds.
B. Over the past two decades, Meyerozyma caribbica has been identified as a metabolically versatile and ecologically adaptable yeast with significant relevance to biotechnology, agriculture, environmental remediation, and food applications.
C. The evidence does not state that multiple strains efficiently ferment lignocellulosic hydrolysates, tolerate inhibitory compounds, and remain active at elevated temperatures, which supports their application in integrated biorefineries.
D. In addition to its fermentative capabilities, M.
E. Since its formal description in 2005, this species has demonstrated the ability to grow on a wide range of substrates and under various stress conditions, facilitating the production of valuable bioproducts such as ethanol, xylitol, arabitol, and volatile aroma compounds.
F. The evidence does not state that over the past two decades, Meyerozyma caribbica has been identified as a metabolically versatile and ecologically adaptable yeast with significant relevance to biotechnology, agriculture, environmental remediation, and food applications.
G. Multiple strains efficiently ferment lignocellulosic hydrolysates, tolerate inhibitory compounds, and remain active at elevated temperatures, which supports their application in integrated biorefineries.
H. The evidence does not state that in addition to its fermentative capabilities, M.


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:
Methylococcus species utilize methane as the sole carbon and energy source, converting it into biomass and other metabolic end products. Owing to this metabolic capacity, they hold particular promise in industrial C1 biotechnology, especially for the production of protein-rich feed. However, the industrial cultivation of Methylococcus -based consortia on methane is inherently nonsterile, exposing the process to potential biological risks that may compromise the stability, duration and productivity of cultivation. One of the most critical threats is bacteriophage infection, whose triggers for rapid phage-mediated lysis and resulting economic losses remain incompletely understood. Elucidating these processes is paramount for devising strategies to mitigate or prevent detrimental outcomes. In this investigation, nine publicly accessible genomes of Methylococcus species were examined, culminating in the identification of eleven prophage sequences distributed variably among the genomes. Sequence annotations revealed that nine prophages are potentially functional and intact, whereas the rest carry incomplete gene sets indicative of nonviability. Phylogenetic analyses corroborated the substantial diversity of prophages, which formed distinct clusters related to γ-proteobacteria phages. Furthermore, comparative genomic analyses demonstrated a high degree of structural conservation despite the presence of rearrangements. The annotation of the CRISPR‒Cas systems provided insights into additional dimensions of phage‒bacteria interactions. Examination of prophage integration sites did not reveal any disruption of metabolic gene structures, thus suggesting minimal risk of deleterious phenotypic outcomes. These findings considerably advance the current understanding of the genetic diversity and biological properties of prophages infecting Methylococcus species, underscoring the importance of holistic approaches for the detection and analysis of these elements. Our findings underscore the need for routine prophage monitoring in industrial methanotrophic consortia, with the pipeline established here serving as a foundational framework for future refinement and industrial adaptation. The online version contains supplementary material available at 10.1186/s13068-026-02738-6. Keywords: Bacteriophages, Prophages, Methane, Methylococcus , Gaprin, Bioinformatics, Genomic analysis, CRISPR‒Cas, Comparative genomics, Biotechnology, Industrial bioconversion


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:
High-throughput yeast engineering is being transformed by biofoundries that integrate automation, artificial intelligence (AI), and standardized workflows. This review examines how these facilities accelerate strain development through the Design-Build-Test-Learn (DBTL) cycle, with advances in genome editing, phenotypic screening, and predictive modelling. It highlights Australia’s involvement through the Australian Genome Foundry, Idea-BIO, and the CSIRO Biofoundiry and explores global efforts to overcome reproducibility and standardization challenges. Despite progress, key barriers remain, including protocol variability and integration of AI tools. We also highlight the opportunity for a shift toward autonomous, self-optimizing ‘self-driving labs’ that transition from DBTL to Design-Build-Deploy cycles. The future of yeast engineering depends not only on technological innovation, but also on the harmonization of international standards, data governance, and ethical safeguards. If fully realized, the convergence of robotics, AI, and synthetic biology will redefine yeast engineering, leading to step changes in strain performance for a variety of important products, thus enabling economic and sustainable biomanufacturing at scale. Keywords: biofoundry, synthetic biology, engineering biology, machine learning

Candidates:
A. Despite progress, key barriers remain, including protocol variability and integration of AI tools.
B. High-throughput yeast engineering is being transformed by biofoundries that integrate automation, artificial intelligence (AI), and standardized workflows.
C. This review examines how these facilities accelerate strain development through the Design-Build-Test-Learn (DBTL) cycle, with advances in genome editing, phenotypic screening, and predictive modelling.
D. High-throughput yeast engineering is not being transformed by biofoundries that integrate automation, artificial intelligence (AI), and standardized workflows.
E. The evidence does not state that it highlights Australia’s involvement through the Australian Genome Foundry, Idea-BIO, and the CSIRO Biofoundiry and explores global efforts to overcome reproducibility and standardization challenges.
F. The evidence does not state that this review examines how these facilities accelerate strain development through the Design-Build-Test-Learn (DBTL) cycle, with advances in genome editing, phenotypic screening, and predictive modelling.
G. It highlights Australia’s involvement through the Australian Genome Foundry, Idea-BIO, and the CSIRO Biofoundiry and explores global efforts to overcome reproducibility and standardization challenges.
H. The evidence does not state that despite progress, key barriers remain, including protocol variability and integration of AI tools.


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:
Recombinant protein expression in mycobacteria faces two major challenges: limited regulatory tools for inducible expression and inefficient secretion of heterologous products. In this study, we developed plasmid-based systems that enable translationally gated secretion in Mycobacterium smegmatis , coupling riboswitch-mediated translational control with efficient extracellular export. The platform integrates the M. tuberculosis antigen 85A promoter and signal peptide for constitutive secretion combined with synthetic riboswitches for inducible translational regulation. We tested two theophylline-responsive riboswitches (riboE and riboE+) and a temperature-sensitive variant (riboU9) by using mCherry as a reporter. Fluorescence assays, RT-PCR, and Western blotting confirmed efficient secretion and strict translational control. The theophylline-inducible systems exhibited a dose-dependent response with maximal expression at 2 mM inducer, while the riboU9 construct showed a clean ON/OFF phenotype triggered by temperature shift. In all cases, transcripts were detected irrespective of induction, confirming regulation at the translational rather than transcriptional level. Secretion was highly efficient, with 10–20 fold higher protein levels in extracellular versus intracellular fractions. Induction during early- and mid-log phases yielded maximal protein, whereas late-log induction reduced output by ∼50%. Together, these results define translationally gated secretion as a new control layer in mycobacterial protein production. This modular platform expands the genetic toolkit available for Mycobacterium research, providing new opportunities for the study of antigens and virulence factors from slow-growing pathogens and offering potential applications in structural biology, vaccine development, and drug target validation.


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:
Plastic waste, especially from packaging, poses major recycling challenges due to the presence of mixed polymers, which often result in inconsistent blends that are unsuitable for reuse in food-grade applications. Chemical recycling, particularly alkaline hydrolysis, offers a promising solution in the case of chemically reactive polymers, such as polyesters, with poly(ethylene terephthalate) (PET) being one of the dominant plastics suitable for both mechanical and chemical recycling. Mechanical recycling is currently used for the largest part of PET recycling, due to the fact that turning the polymer back into its monomeric building blocks requires catalysts, elevated temperatures, or prolonged reaction times. This study presents a recently developed Heated High-Ethanol Alkaline Aqueous (HHeAA) process that enables efficient, catalyst-free PET hydrolysis under milder conditions. Nearly complete hydrolysis was achieved within just 20 min at 90 °C using a loading of 0.624 g of NaOH/g of PET. The process was successfully scaled up with commercial PET bottles, achieving full hydrolysis while significantly reducing the liquid-to-solid ratio from 20 to just 5 L/kg. These results highlight the industrial potential of the HHeAA method as a more sustainable and energy-efficient alternative for PET recycling and chemical reuse and in turn reduced environmental impact.

Candidates:
A. The evidence does not state that chemical recycling, particularly alkaline hydrolysis, offers a promising solution in the case of chemically reactive polymers, such as polyesters, with poly(ethylene terephthalate) (PET) being one of the dominant plastics suitable for both mechanical and chemical recycling.
B. Mechanical recycling is currently used for the largest part of PET recycling, due to the fact that turning the polymer back into its monomeric building blocks requires catalysts, elevated temperatures, or prolonged reaction times.
C. The evidence does not state that this study presents a recently developed Heated High-Ethanol Alkaline Aqueous (HHeAA) process that enables efficient, catalyst-free PET hydrolysis under milder conditions.
D. Chemical recycling, particularly alkaline hydrolysis, offers a promising solution in the case of chemically reactive polymers, such as polyesters, with poly(ethylene terephthalate) (PET) being one of the dominant plastics suitable for both mechanical and chemical recycling.
E. Plastic waste, especially from packaging, poses major recycling challenges due to the presence of mixed polymers, which often result in inconsistent blends that are unsuitable for reuse in food-grade applications.
F. This study presents a recently developed Heated High-Ethanol Alkaline Aqueous (HHeAA) process that enables efficient, catalyst-free PET hydrolysis under milder conditions.
G. Plastic waste, especially from packaging, poses major recycling challenges due to the presence of mixed polymers, which often result in inconsistent blends that are not unsuitable for reuse in food-grade applications.
H. Mechanical recycling is not currently used for the largest part of PET recycling, due to the fact that turning the polymer back into its monomeric building blocks requires catalysts, elevated temperatures, or prolonged reaction times.


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 growing demand for sustainable alternatives to fossil-based chemicals has increased interest in platform chemicals derived from renewable biomass sources, such as malic acid. This C4 dicarboxylic acid is valued for its diverse application potential in food, pharmaceuticals, and bioplastics. Sustainable platform chemicals remain commercially uncompetitive primarily due to high production costs driven by high substrate costs. Microbial production using more cost-effective feedstocks like sugar beet molasses shows promise. However, it faces challenges from high osmolality, growth inhibitors, and predetermined substrate composition during fermentation, as well as elevated pigmentation that complicates downstream processing. Moreover, the separation techniques typically used for highly polar carboxylic acids face considerable yield limitations due to the high solubility of malic acid and its salts. This study developed an all-encompassing production process for malic acid from untreated sugar beet molasses. Fermentative malic acid production with Ustilago trichophora was investigated in batch, fed-batch, and pulsed batch in shake flask scale, followed by a scale-up into 150 L pilot scale. A total of 15.7 kg malic acid was produced in a repeated pulsed batch with membrane-based cell retention with a titer of 108 g/L, a yield of 0.50 g/g, and a space–time yield of 0.66 g/L/h (max. 1.1 g/L/h). In addition, the byproduct succinic acid was detected in concentrations of up to 22.9 g/L. In the subsequent downstream processing, activated carbons were used for two-stage product capture, solvent change, and decolorization, followed by crystallization of the products malic acid and succinic acid. Based on experimental results, an Aspen Plus model was developed to estimate the overall process yields of 0.43 g malic acid (98% purity) and 0.10 g succinic acid per gram sucrose equivalent. A techno-economic analysis suggests production costs within the range of current market prices. Agricultural residue streams are often proposed as cost-effective alternatives for fermentative platform chemical production, although the challenges addressed hamper the direct transfer of process strategies from established organic acid production. By presenting a holistic approach explicitly tailored to malic acid production from untreated molasses, this work demonstrates the techno-economic feasibility of the developed process at a meaningful scale. The online version contains supplementary material available at 10.1186/s13068-026-02736-8.


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:
Yarrowia lipolytica is an emerging host for producing acetyl-CoA– and malonyl-CoA–derived chemicals. However, most processes rely on yeast nitrogen base (YNB), a historical formulation with poorly controlled trace metal content. This variability impairs metabolic performance, limits reproducibility, and complicates process transfer. Commercial YNB batches differed markedly, causing 1.5–2-fold variation in growth and docosahexaenoic acid (DHA) production. We developed a malonyl-CoA–responsive flaviolin reporter strain and combined it with a structured Design of Experiments (DoE) workflow to systematically re-engineer YNB mineral composition. Dissection of all 20 YNB components revealed that vitamins are dispensable under the tested conditions, whereas a small subset of salts and trace elements - particularly ZnSO 4 , FeCl 3 , KH 2 PO 4 , MgSO 4 , CaCl 2 , and CuSO 4 - dominantly shape precursor availability and product formation. One-factor-at-a-time (OFAT), factorial, steepest ascent, and central composite designs converged in an optimized synthetic mineral medium assembled entirely from individual salts and trace metals. This formulation increased flaviolin titers to 1.41 ± 0.08 g L -1 , a more-than threefold improvement over commercial YNB, while ensuring high reproducibility. Key mineral interventions also translated to complex pathways: omission of ZnSO 4 increased PUFA titers by 7.6-fold (docosapentaenoic acid, DPA) and 58-fold (eicosapentaenoic acid, EPA) and enhanced DHA formation in independent production strains. The defined formulation substantially reduces cost and eliminates batch-to-batch variability inherent to commercial YNB powders. Our results establish mineral balancing as a major yet underused lever for improving acetyl-CoA– and malonyl-CoA–derived production in Y. lipolytica and demonstrate a generalizable, model-guided workflow for creating simplified, reproducible, and cost-efficient synthetic media for non-conventional yeast cell factories. The online version contains supplementary material available at 10.1186/s12934-026-02939-6. Keywords: Yarrowia lipolytica, Medium engineering, Yeast nitrogen base, Design of experiments, Flaviolin reporter, acetyl-CoA and malonyl-CoA metabolism, Omega-3 fatty acids, Zinc and iron homeostasis

Candidates:
A. The evidence does not state that this variability impairs metabolic performance, limits reproducibility, and complicates process transfer.
B. Commercial YNB batches differed markedly, causing 1.5–2-fold variation in growth and docosahexaenoic acid (DHA) production.
C. Commercial YNB batches differed markedly, causing 2.5–2-fold variation in growth and docosahexaenoic acid (DHA) production.
D. Yarrowia lipolytica is not an emerging host for producing acetyl-CoA– and malonyl-CoA–derived chemicals.
E. The evidence does not state that however, most processes rely on yeast nitrogen base (YNB), a historical formulation with poorly controlled trace metal content.
F. This variability impairs metabolic performance, limits reproducibility, and complicates process transfer.
G. However, most processes rely on yeast nitrogen base (YNB), a historical formulation with poorly controlled trace metal content.
H. Yarrowia lipolytica is an emerging host for producing acetyl-CoA– and malonyl-CoA–derived chemicals.


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:
Optimising continuous phototrophic cultivation remains a major challenge for scalable, energy-efficient cyanobacterial bioprocesses. Here, we combine controlled photophysiology, long-term continuous experimentation, multi-parameter analysis, and batch-derived Monod kinetic modelling to define a precise operational window for Synechocystis sp. PCC 6803 under flat-plate photobioreactor (FP-PBR) illumination. Using a fully calibrated FP-PBR platform, we first quantified intrinsic growth limits ( µ max = 0.081–0.118 day −1 ) across low, moderate, and high irradiance regimes, establishing the illumination-driven growth ceilings that constrain downstream continuous operation. Guided by these kinetic boundaries, continuous cultivation demonstrated that productive steady-state growth emerges only within a narrow regime governed by light intensity (500–700 µmol photons m −2 s −1 ), temperature (32–34 °C), and dilution rate (0.12–0.14 day −1 ). Single-parameter and 3D interaction analyses revealed strong coupling between photonic supply, thermal sensitivity, and hydraulic residence time, while multi-factor modelling captured these nonlinear constraints and accurately predicted washout boundaries. Translating these insights into sustainability metrics, the optimised regime supports 0.07–0.125 g L −1 day −1 of biomass productivity, equivalent to 8.4–15.0 g biomass day −1 and 176–315 kJ day −1 of chemical energy in a 120 L mini-pilot system. Stoichiometric analysis indicates this corresponds to 15.6–27.6 g CO 2 day −1 sequestered, demonstrating measurable environmental benefit even at a small scale. Together, these results provide a mechanistically grounded, kinetically constrained framework for designing inherently efficient, low-waste, and model-predictive cyanobacterial photobioprocesses aligned with green chemistry and future carbon-neutral manufacturing.

Candidates:
A. The evidence does not state that optimising continuous phototrophic cultivation remains a major challenge for scalable, energy-efficient cyanobacterial bioprocesses.
B. Using a fully calibrated FP-PBR platform, we first quantified intrinsic growth limits ( µ max = 0.081–0.118 day −1 ) across low, moderate, and high irradiance regimes, establishing the illumination-driven growth ceilings that constrain downstream continuous operation.
C. The evidence does not state that here, we combine controlled photophysiology, long-term continuous experimentation, multi-parameter analysis, and batch-derived Monod kinetic modelling to define a precise operational window for Synechocystis sp.
D. Optimising continuous phototrophic cultivation remains a major challenge for scalable, energy-efficient cyanobacterial bioprocesses.
E. Using a fully calibrated FP-PBR platform, we first quantified intrinsic growth limits ( µ max = 1.081–0.118 day −1 ) across low, moderate, and high irradiance regimes, establishing the illumination-driven growth ceilings that constrain downstream continuous operation.
F. Here, we combine controlled photophysiology, long-term continuous experimentation, multi-parameter analysis, and batch-derived Monod kinetic modelling to define a precise operational window for Synechocystis sp.
G. PCC 6803 under flat-plate photobioreactor (FP-PBR) illumination.
H. PCC 6804 under flat-plate photobioreactor (FP-PBR) illumination.


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:
Hyaluronic acid (HA) is a glycosaminoglycan with a wide range of biological functions that depend on its molecular weight (MW). Recently, there has been an increasing interest in producing HA at particular MWs for various cosmetic and biomedical applications. HA is traditionally produced by extraction or microbial fermentation, which is then subjected to chemical or enzymatic treatments to customize the MW. On the other hand, direct microbial synthesis at desired MWs has considerable advantages over conventional techniques. The present study introduces a combinatorial approach using four critical variables which influence the molecular weight of HA (MW HA ): (1) Expression of HA synthases from different Streptococcus species ( S. parauberis , S. uberis , S. zooepidemicus , and S. pyogenes ) which intrinsically produce different MW HA ; (2) Supply of HA precursors by varying heterlogous gene expression in the HA-precursor pathways ( hasAB vs. hasABE ); (3) Re-routing of metabolic fluxes by deletion of the lactate dehydrogenase ( ldh ) gene; and (4) Varying the initial glucose concentration in batch fermentation. Recombinant Lactococcus lactis strains expressing HA synthase genes taken from diverse Streptococcal sp. were found to produce varying MW HA under otherwise identical genetic and bioreactor conditions. The HA synthases sourced from S. uberis and S. parauberis synthesized higher MW HA , whereas those from S. pyogenes produced lower MW HA . In silico analysis of the HA synthase sequences indicated that differences in the transmembrane regions among the various isoforms are the probable cause of variations in MW HA . Compared to their wild-type counterparts, ldh -knockout L. lactis strains showed a noticeable increase in MW HA due to a substantial increase in HA precursor levels. Further, the co-expression of hasE in addition to hasAB , considerably increased MW HA due to a better balance of the intracellular HA-precursor ratios. This multiplexing approach, involving simultaneous manipulation of the above factors, allowed us to produce HA with tailored MW HA over a broad range from 0.2 to 2.6 MDa. Our technology eliminates the need for enzymatic desizing or post-processing of HA to achieve the desired MW HA . In summary, this multiplexing approach enables one-pot synthesis of desired MW HA , opening up new avenues for producing customized HA. The online version contains supplementary material available at 10.1186/s12934-026-02945-8.

Candidates:
A. HA is not traditionally produced by extraction or microbial fermentation, which is then subjected to chemical or enzymatic treatments to customize the MW.
B. On the other hand, direct microbial synthesis at desired MWs has considerable advantages over conventional techniques.
C. Hyaluronic acid (HA) is a glycosaminoglycan with a wide range of biological functions that depend on its molecular weight (MW).
D. The evidence does not state that on the other hand, direct microbial synthesis at desired MWs has considerable advantages over conventional techniques.
E. HA is traditionally produced by extraction or microbial fermentation, which is then subjected to chemical or enzymatic treatments to customize the MW.
F. The evidence does not state that recently, there has been an increasing interest in producing HA at particular MWs for various cosmetic and biomedical applications.
G. Hyaluronic acid (HA) is not a glycosaminoglycan with a wide range of biological functions that depend on its molecular weight (MW).
H. Recently, there has been an increasing interest in producing HA at particular MWs for various cosmetic and biomedical applications.