Bioresource Technology

Raffinose family oligosaccharides (RFOs) are abundant in side streams from food and feed production from legumes, and the transition to plant-based diets increases the volume of such side streams. RFOs in the diet tend to have negative impacts on the consumers gut (e.g., nausea, bloating, diarrhoea), and in many ways, RFOs are comparable to lactose as a side stream from the dairy industry and symptoms associated with lactose intolerance. On the contrary, galactooligosaccharides (GOS) are recognized as prebiotics, and in this study we used a {beta}-galactosidase from Niallia circulans to produce potential prebiotics from RFOs (acceptors) and lactose (donor), which we hypothesized to have a lower fermentability than unmodified RFOs. The transglycosylation reactions resulted in RFO-based -{beta}-GOS, with NMR characterization showing ({beta}1-4) galactosylations on the non-reducing galactose end of RFOs as the major product. In reactions with RFOs, the characteristics were comparable to reactions with lactose alone and the new -{beta}-GOS products made up the largest fraction (by weight). A screening of 11 relevant gut and food microbe strains revealed that the gut commensal Bacteroides ovatus metabolised these modified oligosaccharides for growth whereas other strains grew only after adaption and others did not use them at all. This implies that mixed-linkage -{beta}-GOS are less fermentable by some microbes compared to raffinose, while other (beneficial) bacteria can still ferment them. The enzymatic synthesis established here is an interesting approach to upgrade abundant food side streams towards new prebiotics in a world where functional foods and food waste reduction receive increasing attention. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/731070v1_ufig1.gif" ALT="Figure 1000"> View larger version (22K): org.highwire.dtl.DTLVardef@18e0e62org.highwire.dtl.DTLVardef@1525b4borg.highwire.dtl.DTLVardef@1e7be88org.highwire.dtl.DTLVardef@18df278_HPS_FORMAT_FIGEXP M_FIG C_FIG

Extremophilic red microalgae are promising platforms for sustainable biotechnology, combining robust growth under selective thermoacidophilic conditions with production of thermostable phycobiliproteins and carbon-rich biomass. However, reactor-dependent effects on growth, product formation and biomass composition remain insufficiently resolved. Here, we systematically evaluated the extremophilic red microalga Cyanidioschyzon merolae across cultivation scales and reactor formats and benchmarked its performance against the well-established Galdieria javensis and Limnospira platensis. In small-scale multi-cultivator photobioreactors and microfluidic growth chambers, C. merolae showed superior growth, reaching a maximum growth rate of 0.034 {+/-} 0.001 h-1 and 8.3 {+/-} 0.3 g l-1 cell dry weight. Microfluidic cultivation enabled growth analysis at single-cell resolution and matched growth rates obtained in photobioreactors. To identify scalable production strategies, C. merolae was further cultivated in a flat-panel photobioreactor and a custom-designed internally illuminated photobioreactor. The custom-designed photobioreactor delivered the highest biomass concentration and productivity, yielding 11.5 {+/-} 0.6 g l-1 cell dry weight and 1.07 {+/-} 0.06 g l-1 d-1, and comparable yields with regard to R-phycocyanin and R-allophycocyanin. Biomass analysis revealed substantial carbon and nitrogen contents, starch accumulation up to > 20 % of cell dry weight, and fatty acids dominated by palmitic, linoleic and oleic acids. Despite its reduced cell wall fraction, C. merolae contained structurally diverse, cultivation-dependent polysaccharides. These results establish C. merolae as a versatile chassis for thermostable pigment production and renewable feedstock generation, highlighting photobioreactor design as a key determinant of productivity and biomass quality.

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

Given the current global environmental challenges, waste biomass is an attractive renewable resource for circular economies. Gasification of biomass yields syngas (CO, CO2, and H2) that is a suitable feedstock for gas fermentation in biomanufacturing of fuels and chemicals using acetogen microbes. While it is generally known that syngas composition influences both acetogen growth and process performance, we are lacking a consistent dataset quantifying these effects under controlled fermentation conditions. Here, we mapped the metabolic response of the model-acetogen Clostridium autoethanogenum to seven synthetic syngas mixtures during exponential batch growth in bioreactor fermentations. Notably, distinct gas compositions resulted in different fermentation profiles, affecting both growth and metabolite production. Maximum specific growth rates ranged within 0.05 0.13 h-1, with slower growth for low-CO mixtures. While acetate and ethanol production yields varied between 20-133 and 76-353 mmol per gram dry cell weight, respectively, minor production of 2,3-butanediol was detected. All syngas mixtures supported co-utilization of CO and H2, though gas uptake stoichiometry only moderately correlated with syngas content. Importantly, gas uptake stoichiometry strongly influenced carbon partitioning, with higher relative H2 uptake reducing CO2 loss or even realizing CO2 fixation together with increasing carbon flow towards metabolites. Interestingly, higher syngas H2 content favored ethanol and 2,3-butanediol production, while higher H2:CO uptake ratios increased total flux through the Wood-Ljungdahl pathway rather than selectively favoring reduced by-products. Our results are valuable for a better understanding of syngas composition effects on the acetogen biocatalyst and for process engineering towards optimizing gas fermentation performance. HighlightsO_LISyngas composition affects acetogen growth, gas uptake, and carbon distribution C_LIO_LIHigher H2:CO uptake ratios increase carbon flow through the Wood-Ljungdahl pathway C_LIO_LIHigher relative H2 uptake reduces CO2 loss and increases metabolite production C_LI

BackgroundMicrobial chain-elongation by Clostridium kluyveri using the products (acetate and ethanol) derived from the electrocatalytic CO2 reduction reaction (CO2RR) represents a unique sustainable strategy for producing C4-C6 chemicals from CO2. However, direct integration of electrocatalytic effluents with anaerobic bioprocesses is often impeded by the physiological incompatibility between electrocatalytic product streams and microbial metabolism. Specifically, CO2RR effluents commonly contain formate, which cannot be utilized by C. kluyveri for chain elongation and therefore reduces the overall carbon efficiency of CO2 conversion to C4-C6 chemicals. Moreover, both formate and the elevated phosphate concentrations typical of electrochemical reaction solutions may inhibit microbial growth. ResultsWe show that formate at concentrations of up to 50 mM did not inhibit the growth of or the chain elongation by C. kluyveri. Based on this finding, we developed a modular two-step bioprocess. In the first step, the acetogen Clostridium ljungdahlii converts formate in CO2RR product mixtures into acetate, thereby generating additional substrates for second-step C. kluyveri-driven chain elongation, thus increasing the CO2RR carbon-conversion efficiency to C- C6 chemicals. To address the issue of C. ljungdahliis inhibition by high phosphate concentrations in electrocatalytic solutions, we explored the use of C. ljungdahlii biofilms for the first, i.e. the formate-conversion, step. C. ljungdahlii biofilms exhibit tolerance to concentrated electrolytes, enabling the conversion of up to 50 mM formate in CO2RR solutions. ConclusionsThe demonstrated two-step process constitutes the basis for the development of a robust and carbon-efficient biological process for the scalable upgrading of C1-C2 CO2RR products into higher-value C4-C6 chemicals.

Microbial metabolism can be represented as an energy-conserving process (catabolism) and a biomass-forming reaction (anabolism). Anabolism is traditionally measured through the turbidity of the culture, while catabolism is often assessed by the substrates consumed or the products formed. Standard measurements of biomass and products are intrusive and disrupt cultivation and headspace composition, potentially masking important analytical parameters and interactions. Online pressure and backscatter were combined in small-scale closed batch vials to obtain undisturbed real-time measurements of catabolic and anabolic rates, enabling mapping of metabolic phases throughout an entire batch cultivation cycle. The method identified discrete metabolic phases in yeast cultivation and thermophilic syngas fermentation. In nutrient-rich yeast cultivation, five metabolic phases were characterized, covering growth-associated and non-growth-associated gas formation. In a mixed community syngas fermentation, estimates of catabolic and anabolic rates distinguished early biomass increase from minimal net pressure change from two later gas-driven phases. An initial phase with a higher growth rate, linked to carboxydotrophy, followed by a phase with slightly lower growth and increased gas consumption, corresponding to hydrogenotrophic acetogenesis. The study demonstrates that a simple, affordable experimental setup with online pressure and backscatter measurements can be used to visualize phase-plane mapping of microbial metabolism. An additional advantage is the ability to detect sequential metabolic cascades in mixed microbial communities, which is not possible with gas-sparging bioreactor studies. Using a single simple batch culture, growth and maintenance data can be obtained, even when growth is low or absent, thereby yielding parameters applicable to phenotypic characterization and dynamic metabolic modelling.

The pulp and paper industry produces large volumes of condensed kraft lignin, which is challenging to convert to single chemical products. For this purpose, tandem chemical depolymerization and bioconversion to a single atom-efficient product is a potentially promising strategy. In this study, we conducted copper-catalyzed oxidative depolymerization using pine-derived kraft lignin to generate multiple bioavailable aromatic monomers at a yield of 4.5 weight% (wt%; g monomers per g lignin) from both C--O and C--C bond cleavage, followed by counter-current extraction with a 52 wt% monomer recovery. This resulted in an oxidized lignin product containing vanillin, vanillate, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, 5-formylvanillin, 5-carboxyvanillin, 5-carboxyvanillate, acetovanillone, and vanillyl glyoxylate. Based on this stream composition, we engineered the industrially relevant soil bacterium Pseudomonas putida KT2440 to catabolize the latter five compounds via overexpression of ten heterologous genes (acvABCDEFSYK-6, vceABSYK-6, ligW2SYK-6, and mdlCPP). We combined these engineered pathways with previously reported strategies for muconate production from G- and H-type monomers to generate P. putida KMM428, which utilized 93.6 {+/-} 0.2 mol% of the quantified aromatic monomers in a depolymerized kraft lignin mixture, and produced muconate at a yield of 99 {+/-} 3 mol%, on a quantified monomer basis. Together, this work increases the theoretical carbon conversion efficiency of this process by 37.6 {+/-} 0.1 mol% through incorporation of three {beta}-5 cleavage products, in addition to traditional G-type monomers.

Cassava and cocoyam are major dietary staples in sub-Saharan Africa, commonly processed by natural fermentation before consumption. Although fermentation reduces antinutritional compounds and improves food quality, its molecular effects remain poorly characterized. We used untargeted mass spectrometry-based metabolomics with a computational annotation pipeline to compare fermentation-induced molecular remodeling in the two tubers, which showed distinct responses. In cassava, 718 of 773 significant features (92.9%) were depleted, indicating a predominantly catabolic process. In cocoyam, the response was more balanced, with 385 of 1,013 features (38.0%) enriched, including di- and tripeptides consistent with proteolytic processing. Class analysis, molecular networking, and pathway enrichment revealed tuber-specific signatures: cassava was dominated by purine metabolism, whereas cocoyam showed stronger enrichment of amino acid pathways. Cyanogenic glycoside-related features were depleted, consistent with detoxification. Biotransformation prediction also suggested putative fermentation products absent from current databases, highlighting the under-characterized chemistry of these tubers.

Microalgae and cyanobacteria have emerged as promising resources for sustainable agriculture; however, integrated methodologies for the rational design of crop-specific agricultural formulations remain scarce. This study proposes an integrated framework that combines biomass production, species characterization, nutrient characterization, mixture design, nutrient profile estimation, biological validation, and statistical optimization for the rational development of agricultural formulations based on microalgae and cyanobacteria. As a proof of concept, the proposed framework was applied to formulate consortia composed of C. vulgaris, Scenedesmus sp., and A. platensis using common bean (Phaseolus vulgaris L., ecotype Sangre Toro) and rainfed rice (Oryza sativa L., cv. Fedearroz 2020) as model crops. The experimentally determined nutrient composition of the individual biomasses was integrated into a simplex-lattice mixture design coupled with response surface methodology and desirability analysis to identify crop-specific optimal formulations and estimate their nutrient profiles. The cubic model provided the best fit (P < 0.05), showing high predictive performance and a non-significant lack of fit. The optimal bean formulation consisted of 31.6% C. vulgaris and 68.4% Scenedesmus sp., whereas the optimal rice formulation comprised 62.3% A. platensis and 37.7% C. vulgaris, demonstrating distinct crop- specific responses. The optimized bean formulation exhibited higher estimated concentrations of calcium, phosphorus, iron, and zinc, whereas the rice formulation showed higher estimated potassium and Kjeldahl nitrogen contents. These findings demonstrate the feasibility of developing crop-specific microalgal formulations and highlight that different crops may require distinct formulations rather than a universal approach. The proposed approach offers a reproducible, integrated framework for the development and optimization of next-generation agricultural formulations for sustainable crop production.

Global demand for platform chemicals and biomaterials urges us to seek sustainable strategies along with waste valorization to produce lactic acid (LA) sustainably. The study has designed a one-pot fermentation strategy by employing in-house produced ligninolytic and saccharifying enzymes on rice straw along with a consortium of hexose and pentose sugar co-fermenting microorganisms. Biological pretreatment with in-house ligninolytic enzyme was selected for the one-pot strategy from a comparison study of chemical and enzymatic pretreatment of rice straw. In this study, simultaneous pretreatment and saccharification of rice straw followed by LA fermentation by Lactobacillus casei- Lactobacillus rhamnosus system (35.58{+/-}0.29 g/L) was found out to be more efficient than Lactobacillus casei-Lactobacillus pentosus system (29.80{+/-}0.92 g/L). Thus, the L. casei- L. rhamnosus system (CR system) was selected and was further statistically optimized by response surface methodology (RSM) to yield 64.96 g/L of LA. The fermentation broth was decolorized and purified by ion exchange chromatography to yield 85.56% pure LA with 84.95% optical purity. The one-pot fermentation strategy has reduced the number of unit operations involved to synthesize LA from rice straw without compromising the yield and purity through a greener route. The use of in-house enzymes and consortium of lactic acid producing bacteria in one-pot presents a strategic approach to sustainable LA production. The biological enroute and the minimum use of chemicals during upstream, fermentation, and downstream processing adds to the carbon credit of the process. HighlightsO_LILactic acid was produced from rice straw using one-pot co-fermentation strategy C_LIO_LIUpstream processing employed in-house enzymes from fungal solid-state fermentation C_LIO_LIThe process addresses the underutilization of pentose sugars after saccharification C_LIO_LIA consortium LAB produced 64.96 g/L LA with 0.855 g/L.h productivity C_LIO_LIDownstream processing yielded LA with 85.56% purity and 84.95% optical purity C_LI

Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.

Caldimonas thermodepolymerans is a thermophilic polyhydroxyalkanoate (PHA)-producing bacterium with strong potential for sustainable bioplastic production. Besides serving as intracellular carbon and energy storage compounds, PHAs are increasingly associated with bacterial stress resistance and cellular robustness. This study aimed to investigate the physiological and transcriptomic response of C. thermodepolymerans to osmotic stress induced by elevated NaCl concentrations. Growth analysis demonstrated tolerance up to a supplementation of 2% NaCl, while moderate salt concentrations enhanced PHA accumulation, reaching 65% cell dry weight at 1.5% NaCl supplementation. To better understand the bacterial response to osmotic stress, RNA sequencing was performed under sublethal salt stress conditions. Differential expression analysis revealed major changes in genes related to osmoprotection, trehalose metabolism and type VI secretion systems, whereas motility and chemotaxis genes were strongly repressed. Phenotypic assays confirmed increased biofilm formation and reduced swarming motility under salt-induced osmotic stress. Although canonical PHA biosynthesis genes were not significantly differentially expressed, increased polymer accumulation suggests other underlying mechanisms linked to osmoadaptation. Together, these findings demonstrate that osmotic stress induces metabolic, physiological and regulatory responses in C. thermodepolymerans, highlighting the importance of PHA in stress adaptation besides its industrial applicability.

Managing plastic pollution is challenging because current physical and chemical recycling methods are inefficient and environmentally intensive. Biological recycling approaches have been framed as sustainable alternatives but are challenging to optimize due to a lack of process analytical technologies that provide real time data on microbial plastic metabolism. In this study we used Piscinibacter sakaiensis 201-F6, a model bacterium with a well-studied polyethylene terephthalate (PET) metabolism, to validate non-destructive Raman spectroscopy methods to monitor plastic biodegradation by tracking metabolite production. Cells were grown on PET and known metabolites stemming from PET metabolism. Raman spectroscopy was used alongside destructive mass spectrometry techniques to monitor PET metabolite production and uptake under different growth conditions. Although cells grew effectively using PET, Raman spectroscopy did not detect the known PET metabolite terephthalic acid during growth assays. Instead, Raman detected isophthalic acid (IPA), a metabolite not previously associated with PET metabolism whose identity was confirmed with LC-HRMS. Raman spectroscopy was also used alongside thermoanalytical techniques to predict the biodegradability of PET at different crystallinities through the release of IPA. This study frames Raman spectroscopy as a promising tool to study metabolic pathways for plastic recycling and optimize their application in situ.

Power-to-Gas technologies are emerging as a key strategy to integrate surplus renewable electricity into energy systems, through the conversion of green hydrogen into methane. However, the practical implementation of biological in situ biomethanation is still constrained by operational and design requirements that are incompatible with most existing anaerobic digestion infrastructures. This study demonstrates a stable and efficient mesophilic (37{degrees}C) in situ biomethanation process driven by substrate-induced microbial selection rather than relying on continuous hydrogen supply. Anaerobic digesters co-digesting sewage sludge from a wastewater treatment plant with lipid-rich greases recovered from dairy wastewater developed a pre-adapted hydrogenotrophic consortium capable of effective CO2-H2 conversion under mesophilic conditions. Long-term operation confirmed the robustness and persistence of this microbial structure. Upon H2 addition, methane concentrations up to 82 % were achieved under atmospheric pressure, without biogas recirculation, with hydrogen-to-methane conversion efficiencies up to 90% and methane productivities of 1.64 NLCH4.L-1d-1. 16SrRNA-based microbial community analysis revealed that dairy grease co-digestion selectively enriched hydrogenotrophic methanogens, particularly Methanospirillum, together with syntrophic fatty-acid-degrading bacteria such as Syntrophomonas, promoting efficient interspecies hydrogen transfer. Importantly, the lipid co-substrate enabled the establishment and long-term stability of the hydrogenotrophic pathway independently of hydrogen availability, mitigating challenges associated with intermittent renewable energy supply. Overall, these findings challenge the common reliance on thermophilic conditions, continuous hydrogen input, pressurization, and gas recirculation in in situ biomethanation, demonstrating that substrate-driven microbial selection can replace conventional engineering requirements such as thermophilic operation or reactor modifications, providing a simpler and scalable strategy for mesophilic in situ biomethanation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/731101v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c62086org.highwire.dtl.DTLVardef@1813ed6org.highwire.dtl.DTLVardef@4462bcorg.highwire.dtl.DTLVardef@1ae2cfb_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG Highlights- Lipid-assisted co-digestion promotes stable biogas and biomethane production - Dairy wastewater greases enable mesophilic in situ biomethanation - An enriched hydrogenotrophic methanogenic consortium yields >82% CH4 - 70-90% H2-to-CH4 conversion efficiency under mesophilic, unpressurized conditions - Substrate-driven microbial selection enables in situ biomethanation in WWTP digesters

Bacterial cellulose (BC) is a biopolymer that comes from natural sources. It has high purity, crystallinity, mechanical strength, and biocompatibility, which makes it suitable for various biomedical and industrial uses. Unlike plant cellulose, BC does not contain lignin or hemicellulose. This results in better material quality and simpler processing. However, producing BC on a large scale is limited by high production costs, expensive culture media, and dependence on a few bacterial strains. It is essential to find cost-effective production methods and alternative microbial sources to expand its commercial use. This study looked at the cellulose-producing ability of Acetobacter diazotrophicus, a safe and relatively unexplored bacterium, under different growth conditions. We compared bacterial growth and cellulose production using Hestrin-Schramm (HS) medium, the standard for BC production, and LB supplemented with glucose (LB+Glucose), which we explored as a more affordable option. We analyzed growth rates, inoculum age, and pH levels to find the best conditions for cellulose production. We observed faster bacterial growth in HS medium, with a doubling time of 3.906 hours, compared to 6.241 hours in LB+Glucose medium. Cellulose production was greatly affected by inoculum age, with successful synthesis from cultures that were agitated for 36 to 40 hours. The highest cellulose yield was at pH 6.0 in HS medium (4.6 mg/mL) and at pH 5.5 in LB+Glucose medium (3.6 mg/mL). FTIR analysis confirmed the presence of characteristic functional groups of bacterial cellulose. These results suggest that Acetobacter diazotrophicus is a promising and cost-effective option for producing bacterial cellulose and highlight the importance of medium composition, inoculum age, and pH for optimizing production.

Microbial activity is often inferred from cell density measurements; however, biomass formation is merely an indirect, cumulative result of metabolism, known as anabolism. Microbial activity is more accurately indicated by energy conservation or catabolism. This is especially true under low or no-growth conditions, where anabolism remains constant, and shifts in catabolic fluxes go unnoticed with biomass measurements alone. In anaerobic and gas-based metabolic processes, net gas exchange is linked to energy conservation, and catabolism can then be quantified through headspace measurements. We introduce a sealed-vial, non-invasive workflow that uses high-resolution headspace pressure measurements to estimate gas exchange rates and catabolic reactions, enabling real-time visualisation of metabolic shifts throughout an entire batch cultivation cycle. The method was applied to carbon monoxide (CO) fermentations of three Clostridium autoethanogenum strains (JA1-1, LAbrini, and LAbrini_mut) cultivated in serum bottles. Two of them were indistinguishable by OD-derived max. Pressure-derived gas uptake rates resolved multiple exponential phases of gas consumption and identified specific shifts in the metabolism, consistent with transitions from mixotrophic to autotrophic growth. Small but significant differences in terminal headspace pressure were detected, providing an experimentally accessible end-state parameter for phenotypic characterisation that would be obscured by routine intrusive headspace sampling. Finally, pressure-derived catabolic rates further enabled estimates of relative product formation during the main autotrophic phase. The strains were successfully characterised and distinguished by identifying several exponential phases of gas consumption and their rates, as well as differences in the final absolute pressure threshold. This provided phenotypic characterisation and insights not obtainable from OD measurements alone. The work establishes a practical framework for catabolism-resolved microbial characterisation in sealed batch vials through high-resolution online pressure (gas exchange). The assumption that pressure measurements correlate with CO2 and catabolic rates is sensitive to solubility/buffering and temperature/vapour effects, but these limitations are addressable through controls and complementary analytics.

Food production is a significant contributor to global greenhouse gas emissions and deforestation, exacerbated by substantial food waste. Converting food waste into yeast protein offers a sustainable solution to enhance food security and contribute to a circular economy. However, due to the diverse and variable nature of food waste substrates, numerous experimental trials are required to optimise the preprocessing steps, yeast strain selection, nutrient addition, and fermentation conditions. This study presents a hybrid modelling approach where data-driven machine learning is used to predict microbial growth kinetics from process parameters. The hybrid model was trained on a comprehensive dataset consisting of 963 fermentation experiments from 55 publications, enabling transfer learning across 46 yeast strains and 79 food waste substrates. The hybrid modelling method was integrated with Bayesian optimisation, a sequential strategy to optimise expensive-to-evaluate functions, to efficiently maximise yeast biomass growth from different food waste substrates. The utility of the hybrid model was evaluated using five test datasets selected from previous literature and was shown to facilitate an average reduction of 66% in the number of experimental trials required to identify optimal fermentation conditions compared to without using the hybrid model. This proved that the transfer of knowledge between yeast strains and food wastes improved the optimisation efficiency of real, previously published datasets compared to traditional optimisation methods. The novelty and contributions of this study include the collation of the extensive dataset, provided as supplementary material; and the demonstration that transfer learning by training the hybrid model on this heterogeneous dataset can improve the optimisation efficiency for yeast biomass growth on new strains and substrates.

Past few decades witnessed a boom in pharmaceutical and bioproduct industry with the help of bioprocess technology. Industrially important bioproducts can be produced in large scale for commercialization with the help of fermenters. Here in, pharmaceutically valuable bioproduct melanin, synthesized from Pseudomonas stutzeri strain BTC109 by using two different sized bioreactors. Under controlled conditions the bacteria were allowed to synthesis melanin nanoparticles. The important parameters to be monitored here are pH, dissolved oxygen, agitation, aeration, melanin production and cell biomass concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/737634v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d92fe5org.highwire.dtl.DTLVardef@d76c07org.highwire.dtl.DTLVardef@f5516forg.highwire.dtl.DTLVardef@1b5864b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPharmaceutical and bioproduct development industries witnessed a shoot up due to bioprocess technology. C_LIO_LIIndustrially important bioproduct like melanin can be produced in large scale with the help of industrial fermentation technology. C_LIO_LIThe product thus obtained was found to be nano sized and it can be commercialized. C_LI

This study investigates electro-fermentation candida Tropicalis SY005 to address fermentation kinetics limitation during grape must fermentation. In comparison with non-stimulated control sample, EF substantially enhanced sugar depletion, TSS drop by day 3 and generated a strongly reduced state (ORP -100 to -143mV). The oxidation-reduction shift enhanced cellular NAD+ regeneration, reducing total fermentation duration from 264 h to 72 h. GC-MS analysis showed pronounced major characteristic volatile compound confirming substantial metabolic pathway shifts in flavor of glycolytic flux. Moreover moderate electric field promoted cellular membrane electropermeabilization substantially promoting bioactive extraction.

Biological ethanol production is important for the circular carbon economy and makes up 73% of the U.S. biological fuels market. Previously, we produced ethanol by reversing methanogenesis and capturing methane by cloning methyl-coenzyme M reductase (Mcr) from an unculturable population of anaerobic methanotrophic archaea; this process was predicated on the generation of the intermediate acetate and its conversion by the methanogenic host to ethanol. Moreover, methanogens are generally thought to be detrimental for converting acetate to ethanol and are usually intentionally inhibited. Here, we demonstrate that direct growth on acetate as the sole carbon and energy source by the methanogen Methanosarcina acetivorans C2A results in 40% of the metabolized acetate becoming ethanol and that there is 430% more ethanol produced, compared to growth on methane via Mcr. In addition, we found growth on methanol results primarily in methane generation and low levels of ethanol. Therefore, acetate may be readily converted by the methanogen M. acetivorans to ethanol at high yields.