Applied Microbiology and Biotechnology

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

Cellobiose is an important disaccharide used in food, health, and biorefinery applications, but its efficient enzymatic production from crystalline cellulose remains challenging. In this study, crystalline cellulose was dissolved in ionic liquids and regenerated by dilution, and subsequently hydrolyzed at 80{degrees}C using a highly thermostable cellobiohydrolase, HmCel6A-3SNP. The enzyme retained activity in the presence of low concentrations of ionic liquids. Among the pretreatment conditions tested, cellulose treated with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) showed the highest enzymatic digestibility. After washing to remove residual ionic liquid, the reaction produced reducing sugars at levels 1.5-fold higher than those obtained in the presence of 10% [Bmim]Cl, with cellobiose accounting for approximately 96% of the products. Under the optimized conditions, the hydrolysis yield reached [~]36% after 48 hr. Structural analyses using birefringence imaging, electron microscopy, and Fourier transform infrared spectroscopy indicated that higher-order structural changes in regenerated cellulose strongly influence enzymatic accessibility. These results demonstrate the potential of combining ionic-liquid pretreatment with thermostable enzymes for selective cellobiose production from cellulose.

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

A challenge in using plant biomass is its highly recalcitrant nature, which makes it economically infeasible to utilize. In natural environments, various microbes, including bacteria and fungi, are reported to decompose plant cell wall materials such as cellulose and hemicellulose, and there may be undescribed microbes that contribute to the degradation of plant biomass. We focused on isolating novel plant biomass-degrading bacteria and screened more than 100 isolates from the Tomakomai experimental forest in Hokkaido, Japan. Among them, one novel Bacillus species was chosen for whole-genome sequencing. Comparative genomics and a carbon source utilization assay indicated that the isolate belongs to a subspecies of Bacillus subtilis, which we named B. sp. TTS1. Glucose, cellobiose, xylose, xylan, mannose, or mannan was used as the sole carbon source in the minimum medium, and the growth of this bacterium was determined. Furthermore, a proteomic analysis of B. sp. TTS1 was performed using culture supernatants from various polysaccharide-containing media. In the present study, several key enzymes involved in plant biomass degradation were identified, namely {beta}-1,4-mannanase and xylanase, and they were highly enriched in all tested polysaccharides.

Hydrogenophilus thermoluteolus TH-1 is a thermophilic hydrogen-oxidizing bacterium capable of producing poly(3-hydroxybutyrate) (PHB) from CO2. To redirect carbon flux for producing other useful biomaterials, we disrupted the acetoacetyl-CoA reductase genes (phaB1 and phaB2), which are central to the primary PHB synthesis pathway. Unexpectedly, the resulting {Delta}phaB1B2 mutant still accumulated PHB under autotrophic conditions, reaching approximately 25-35 % of the wild-type level. Furthermore, PHB accumulation in the mutant was significantly restored when fatty acids (butyrate and oleate) were used as carbon sources, whereas acetate and malate resulted in reduced accumulation. These results suggest the existence of a PhaB-independent PHB synthesis pathway. We propose that intermediates from the {beta}-oxidation of fatty acids are converted to (R)-3-hydroxybutyryl-CoA, bypassing the disrupted PhaB enzymes. Additionally, the basal PHB production from non-fatty acid sources implies the involvement of a reverse {beta}-oxidation pathway. This study highlights the metabolic versatility of strain TH-1 for future metabolic engineering.

Poly(ethylene terephthalate) (PET) is a highly recalcitrant polyester plastic whose resistance to degradation has contributed to widespread environmental accumulation. Enzymatic PET depolymerization has emerged as a promising bioremediation strategy, but PET hydrolysis remains challenging due to the insoluble and semi-crystalline nature of PET and the poor thermostability of many PET hydrolases at elevated temperatures. Here, several electrostatically supercharged PET binding modules (PBM) were fused to a PET-hydrolyzing Cutinase Catalytic Domain (CD) from the thermophilic microbe Thermobifida fusca to investigate how engineered PBM surface charge influences PET hydrolysis behavior. All PBM designs were derived from a native T. fusca family-2a carbohydrate binding module (CBM) as starting template. Since PET exhibited a substantially negative zeta potential, and accordingly, all positively supercharged PBMs displayed the strongest PET binding interactions in pull-down binding assays. However, stronger PET binding did not translate to improved hydrolysis activity for the fusion constructs. Instead, a slightly negatively charged PBM-CD fusion (D2 construct) exhibited activity comparable to the Cutinase CD on finely milled PET powder while showing substantially improved activity on intact PET discs, suggesting potential advantages for depolymerization of minimally processed PET feedstocks. Thermostability analysis identified an approximately 10 {degrees}C increase in melting temperature for the D2 fusion construct, corresponding to enhanced catalytic persistence and a shifted optimal hydrolysis temperature. Consequently, this construct exhibited an approximately 2-fold increase in long-term hydrolysis activity on milled PET and up to a 10-fold increase on intact PET discs, even at high solids loadings, compared to the native Cutinase CD. Collectively, these findings demonstrate that thermostability, rather than adsorption to PET alone, is a dominant factor governing functional persistence of PET hydrolases.

Saccharomyces cerevisiae is an excellent microbial platform for sustainable production of next generation biofuels such as the branched chain higher alcohols (BCHAs) isobutanol and isopentanol. A cytosolic pathway for BCHA production is generated from expression of prokaryotic orthologs of branched-chain amino acid (BCAA) enzymes acetolactate synthase (ALS), mutant NADH-dependent ketol-acid reductoisomerase (KARIP2D1-A1), and dihydroxy-acid dehydratase (DHAD). The potential for this pathway has been hindered by the availability of iron-sulfur clusters, particularly the 2Fe-2S cluster, required for DHAD to function in the cytosol. ILV3, the endogenous yeast DHAD located in the mitochondria, can be deleted to create a valine auxotroph. In this study we use bioinformatics, heterologous gene library synthesis, and a valine complementation assay to find prokaryotic iron-sulfur cluster biosynthetic gene clusters (BGC) and accessory genes that aid DHAD function in the yeast cytosol. This work presents, to our knowledge, the first functional BGC that enhances the cytosolic activity of prokaryotic DHADs in S. cerevisiae. The SUF BGC from Bacillus subtilis combined with a ferritin-like protein (FTNB) from Escherichia coli and the Lactococcus lactis DHAD enhanced the production of BCHAs. Combined expression gave an average isobutanol titer of 412mg/L, 1.8-fold greater than L. lactis DHAD expressed alone. This work establishes a blueprint for better biofuel production by improving iron-sulfur cluster dependent enzyme activity in the yeast cytosol.

Insects offer promising opportunities for organic waste bioconversion; however, they cannot efficiently degrade synthetic polymers such as polyethylene terephthalate (PET). Here, we generated transgenic Drosophila melanogaster lines to express in vitro-evolved variants of two PET-degrading enzymes with distinct biochemical properties: an engineered Ideonella sakaiensis PETase variant (TS-{Delta}IsPET) and a leaf-branch compost cutinase variant (TA-{Delta}LCC). Both enzymes, fused to a Drosophila gut-derived secretory signal, were produced and secreted by both Drosophila cultured S2R+ cells and transgenic larvae. Both enzymes were glycosylated upon secretion, a post-translational modification that did not abolish their catalytic activity. Notably, TA-{Delta}LCC displayed [~]6-fold higher esterase activity than TS-{Delta}IsPET in larval extracts and TA-{Delta}LCC-containing extracts depolymerised PET nanoparticles in vitro under enzyme-favourable conditions. Transgenic flies showed normal development, fertility and survival. Morphological and biochemical analysis confirmed that TA-{Delta}LCC expression did not alter midgut structure and function. Together, these results establish Drosophila melanogaster as a model for functional expression and comparative evaluation of engineered PET-degrading enzymes and identify TA-{Delta}LCC as a promising candidate for exploitation in insect species relevant to plastic contaminated waste bioconversion. HighlightsO_LITransgenic D. melanogaster enables in vivo study of engineered PET enzymes C_LIO_LIEngineered TS-{Delta}IsPET and TA-{Delta}LCC are functional in larval extracts C_LIO_LITA-{Delta}LCC was selected for PET nanoparticle assays due to higher pNPA activity C_LIO_LID. melanogaster model enables comparative evaluation of PET-degrading enzymes C_LI

Engineering the biosynthesis of fully biodegradable polyhydroxyalkanoates (PHAs) from non-food and renewable feedstocks like lignocellulose is becoming an attractive strategy for sustainable biomanufacturing. However, the efficiency and diversity of PHA synthesis from lignocellulosic hydrolysate (LH), mainly containing glucose and xylose, still remains challenge. Here, Halomonas TD, a cost-effective PHA-producing chassis, was developed to utilize glucose and xylose (or LH) for effective production of poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by engineering the phosphoketolase pathway-dependent xylose metabolism in the genome, yielding 50.1 g L-1 PHB and 42.6 g L-1 P(3HB-co- 11.4 mol% 4HB) under fed-batch condition. Subsequently, the introduction of Weimberg pathway was found be able to synthesize terpolymer consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV), namely [P(3HB-co-4HB-co-3HV)], from glucose and xylose only due to the isoenzyme activity of keto-acid decarboxylase encoded by kivD, which converts xylose-derived intermediate 2,5-dioxopentanoate and -ketoglutarate into butanedial (4HB synthesis) and 2-ketobutyrate (3HV synthesis), respectively. Finally, a tailored-made xylose-induced system was constructed to achieve exquisite xylose transmembrane transportation control for improved synthesis of terpolymer P(3HB-co-4.5 mol% 4HB-co- 3.0 mol% 3HV), reaching to 6.3 g L-1 under shake-flask condition, together with the co-expression of fine-tuned phosphoketolase and Weimberg pathways. This study provides a feasible and sustainable alternative for lignocellulosic resources valorization powered by the engineered Halomonas TD capable of efficient xylose utilization and diverse PHAs synthesis.

BackgroundSuccinic acid (SA) is a four-carbon dicarboxylic acid of considerable industrial relevance, with applications spanning the food, chemical, and pharmaceutical sectors. The remarkable acid tolerance of the yeast Yarrowia lipolytica makes it a promising microbial cell factory for SA production. Numerous metabolic engineering strategies have focused on disrupting genes encoding the succinate dehydrogenase (SDH) complex to enhance SA accumulation. However, such a modification is associated with impaired growth and the accumulation of by-products, notably acetic acid (AA). ResultsTo improve growth capacity, SA productivity, and reduce AA formation in Y. lipolytica SDH5-deficient strains (Sdh5{Delta}), carbon flux from glycolysis was partially redirected toward the pentose phosphate pathway by overexpression of the native genes encoding glucose-6-phosphate dehydrogenase (ZWF1) and 6-phosphogluconate dehydrogenase (GND1), thereby enhancing NADPH generation. The resulting strain was further engineered to increase NADH availability for the mitochondrial electron transport chain by overexpressing genes encoding either a mutated NADPH-dependent malate dehydrogenase (TfMdh) from Thermus flavus or the soluble transhydrogenase (EcSthA) from Escherichia coli, enabling indirect conversion of NADPH to NADH. This strategy resulted in 2-fold and 2.2-fold increase in SA productivity and titre, respectively, compared to the Sdh5{Delta}-ALE strain during bioreactor cultivation on glucose-based media. Moreover, AA accumulation was reduced 1.2-fold, while growth rates were significantly improved. ConclusionsThe proposed engineering strategies, especially heterologous expression of EcSthA, partly alleviated energy limitations in Y. lipolytica Sdh5{Delta} strain, resulting in improved SA productivity and growth performance.

In many tropical countries, fermentation of palm sap into palm wine is an important fermented beverage contributing to local economies, tradition, and culture. Traditionally made in villages and families, palm sap is not inoculated with starter cultures and fermentation commences spontaneously. It is therefore possible that fermentation is influenced by multiple ecological factors, which affect microbial dynamics and thus flavour outcomes. Here, we studied microbial communities during fermentation of palm sap from three different palm tree species (palmyra, coconut, and sugar palm) on the island of Bali, Indonesia in both the wet and dry seasons. Our results suggest that season of collection has a strong influence on microbial dynamics and succession, and these changes positively correlate to metabolite concentration. The change of the season from the dry to wet season led to the loss of microbial diversity with lower richness in the dry season. The dominance of Saccharomyces cerevisiae was not affected by season and fermentation time and was dominant in all samples. Potential spoilage species, such as Candida tropicalis were negatively correlated to ester production and more abundant in the dry season. As microbial species varied in incidence and thus biochemical activity, the chemical groups of esters from their metabolism related to the change of season and fermentation time, while volatile compounds and small molecules were highly discriminated by season in the resultant wines. Ethyl octanoate was consistently different across all variables through comparison by three-way ANOVA and is proposed as a biomarker of seasonal variation in palm sap fermentation. These findings improve our understanding of microbial dynamics in palm sap fermentation, revealing flavour differentiation within season and suggests that strategies for microbial management, product development and quality assurance will elevate this traditional product into the future.

Taro (Colocasia esculenta (L.) Schott) is an important vegetable and food crop in China, but in recent years, soft rot disease has frequently occurred during its cultivation and production. This disease damages the underground corms and petiole bases of taro, causing decay in the affected parts and emitting a foul odor, leading to wilting and lodging of the entire plant. This has resulted in significant economic losses to taro production in China, along with food safety issues and ecological problems caused by excessive pesticide use, making it urgent to find a green and efficient control method. Due to its specificity and environmental safety, phage therapy exhibits advantages that chemical pesticides cannot match, representing a promising alternative to chemical pesticides for controlling pathogenic bacteria. In the preliminary work of this study, a bacterial strain was isolated from taro soft rot in Shaoguan, Guangdong, and initially identified as Pectobacterium colocasium ZXC0623. Using this strain as the host bacterium, a Pectobacterium phage was screened and named QJphage. We analyzed its physicochemical properties and obtained its biological characteristics, including optimal titer, optimal infection latency period, optimal infection multiplicity, optimal storage solvent, and resistance to ultraviolet light, pH, and chloroform. Through homologous alignment analysis, eight tail fiber proteins encoded in the QJphage genome were predicted as putative receptor-binding proteins (RBPs). To validate this prediction, the corresponding genes were cloned downstream of the egfp gene via homologous recombination, and the resulting recombinant plasmids were transformed into a prokaryotic host to express EGFP-tagged tail fiber fusion proteins. Fluorescence detection and confocal laser scanning microscopy confirmed that the protein encoded by ORF04 functions as the RBP. Furthermore, lipopolysaccharide (LPS) was knocked out in the host strain P. colocasium ZXC0623. Both{Delta} LPS1 and{Delta} LPS2 mutants formed smaller plaques compared to the wild-type strain, and the{Delta} LPS1 mutant additionally exhibited a significant reduction in plaque number, indicating that LPS serves as a receptor involved in QJphage adsorption. Finally, transcriptomic analysis during the latent period of infection focused on 20 genes predicted to be associated with phage-host receptor binding and anti-phage immune systems. The results revealed that pilin proteins act as potential reversible adsorption receptors for QJphage, while the host strain ZXC0623 also possesses a diverse repertoire of anti-phage defense systems. Collectively, QJphage exhibits stable physicochemical properties, a well-defined LPS-dependent infection mechanism, and a host with diverse defense systems, providing a foundation for the control of taro soft rot and future phage-related research. ImportancePhage therapy has emerged as a highly effective biocontrol strategy against Pectobacterium, with its specificity making it particularly valuable. A critical aspect of this approach is the identification of phage receptors. The initial step in the phage life cycle involves adsorption to the bacterial host, beginning with reversible contact followed by irreversible binding between phage receptor-binding proteins and specific bacterial surface receptors. Potential receptors include glycolipids in the Gram-negative outer membrane, capsular polysaccharides, and various membrane proteins or appendages. In this study, we first characterized the physicochemical properties of the isolated QJphage. Through integrated transcriptomic and whole-genome analyses, we demonstrated that the LPS of Pectobacterium specifically interact with the tail fiber proteins of QJphage. This research provides the first evidence revealing the molecular mechanism of interaction between Pectobacterium and its phage, establishing a foundation for developing phage-based control strategies against soft rot diseases.

The purification of industrial enzymes typically relies on costly, multi-step chromatographic protocols. To address this, we developed a novel platform termed Coated Bacterial Enzymes (CBEs), which enables one-step purification and immobilization of recombinant proteins fused to the SlpA cell wall binding domain. As a proof of concept, we used a {beta}-galactosidase from Bifidobacterium bifidum of dairy relevance. The chimeric enzyme BbgII-SlpA was expressed in Escherichia coli and captured from crude lysate onto glutaraldehyde-inactivated Bacillus subtilis cells via SlpA domain. Binding was characterized by a dissociation constant (Kd) of 16.2 {micro}M and maximum binding capacity (Bmax) of 144 {micro}mol/g. The resulting CBE biocatalyst exhibited optimal activity at pH 6.0 for ONPG and lactose, with a broader pH profile than the free enzyme. Optimal temperatures were 60 {degrees}C for ONPG and 50 {degrees}C for lactose, and CBE retained >80% activity after 390 min at 45 {degrees}C, compared to 20% for the free enzyme. Catalytic efficiencies (kcat/Km) were 2.62 x106 M-1{middle dot}s-1 for ONPG and 4.40 x102 M-1{middle dot}s-1 for lactose. Moreover, CBE showed improved tolerance to cations such as Ca2+ and Fe2+. These results suggest that the CBE platform offers a cost-effective alternative for producing high-purity, immobilized enzymes for diverse industrial bioprocesses.

Engineering the ethanologenic Gram-negative bacterium Z. mobilis for the secretion of hydrolytic enzymes is a key step towards establishing a biofuel cell factory that uses complex waste material as feedstock. Secretion strategies in Z. mobilis have exclusively relied on signal peptides, which limit protein transport to the periplasm. To achieve single-step secretion across the Z. mobilis double-layered membrane, we sought to identify a native Type I Secretion System (T1SS) tag for fusion to proteins of interest. While a T1SS operon had been identified in the Z. mobilis genome, its native cargo had remained unknown and the use of T1SS secretion tags had so far been unexplored. Here, bioinformatic analysis identified the Major Intrinsic Protein (MIP) as a putative T1SS cargo, and its role validated through fusion of C-terminal sequences of two lengths (61 and 141 amino acids) to a heterologous {beta}-galactosidase from Bacteroides thetaiotaomicron, expressed in Z. mobilis. The 141 amino acid tag, including two RTX domains, resulted in significantly higher secretion efficiency than the 61 amino acid tag lacking RTX repeats, consistent with the established role of RTX domains in preventing premature cytoplasmic folding, thus improving secretion. As extracellular secretion of hydrolytic enzymes has remained a major bottleneck in the development of Z. mobilis as a sustainable cell factory, the identification of a native T1SS secretion tag directly addresses this limitation, introducing a novel tool for enzyme delivery.

The increasing accumulation of polyethylene terephthalate (PET) waste has prompted the development of sustainable biotechnological strategies for its degradation and valorisation. This study presents an integrated approach combining enzymatic PET depolymerization by Yarrowia lipolytica, engineered to express and secrete the cutinase HiC and the lipase CalB, with the microbial valorization of PET-derived monomers, terephthalic acid (TPA) and ethylene glycol (EG), by Rhodococcus jostii RHA1. Y. lipolytica was further engineered for xylose metabolism, enabling enzyme production from low-cost lignocellulose-derived substrates. Enzymatic assays with HiC and CalB crudes effectively hydrolysate PET to TPA and EG, demonstrating functional enzymatic activity without purification steps. In addition, R. jostii RHA1 was able to use as substrate the released monomers and accumulated intracellular lipids. Overall, this work demonstrates the feasibility of coupling the production of PET degrading enzymes and microbial lipid, using an abundant monosaccharide, with the assimilation of the PET degradation products to also produce microbial lipids. This modular system provides a promising framework for the sustainable upcycling of plastic waste into value-added bioproducts within a circular economy.

As the United Kingdom (UK) targets net-zero emissions by 2050, anaerobic digestion (AD) has become a cornerstone of renewable energy infrastructure. However, mathematical models, such as the Anaerobic Digestion Model No. 1 (ADM1), often struggle with high-solids agricultural feedstocks because they rely on Chemical Oxygen Demand (COD), a metric that introduces significant experimental error. To overcome this, this study applies an established mass-based ADM1 framework tailored for the co-digestion of maize silage and cow manure sourced from a UK AD site. This study uses a parallel reactor framework, using two identical laboratory-scale reactors to physically replicate the dynamic conditions of the full-scale site. A Global Sensitivity Analysis was first conducted, identifying biomass decay and carbohydrate breakdown rates as the most influential factors affecting system stability and model accuracy. The model was calibrated using data from the first reactor and then tested against an independent second reactor subjected to significant organic loading stress. Results show high predictive capabilities, with the model achieving a R2 of 0.81 for biogas production during calibration. The model maintained high predictive accuracy during the validation test of the second physical twin, achieving an R2 of 0.85, proving that the framework is robust and not overfitted to a single dataset. While predicting rapid fluctuations in pH and alkalinity remains challenging, the mass-based approach effectively forecasts gas yields and process stability. This methodology provides a reliable foundation for robust process modelling, offering a scalable tool for the UK biogas sector to optimise AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/721061v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@92c7e2org.highwire.dtl.DTLVardef@80d723org.highwire.dtl.DTLVardef@ac3d24org.highwire.dtl.DTLVardef@1e21a51_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biorefinery applications, yet efficient enzymatic saccharification remains challenging due to the recalcitrant structure of plant cell walls. This study presents a comprehensive comparative analysis of enzymatic activities, saccharification performance, and secretome composition of six fungal species cultivated under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using untreated flax shives as substrate. While SmF yielded approximately 4-fold higher total protein concentrations (0.38 {+/-} 0.13 g.L-1 vs. 0.08 {+/-} 0.02 g.L-1), SSF-derived enzymes demonstrated superior specific enzymatic activities, particularly for endo-xylanase and endo-cellulase, resulting in more efficient biomass saccharification. Proteomics analysis revealed distinct secretome profiles between fermentation modes, with SSF showing higher proportions of polysaccharide metabolism proteins (71.0%) compared to SmF (49.3%), while SmF exhibited greater enzyme diversity including more lytic polysaccharide monooxygenases (LPMOs) and auxiliary activity enzymes. Trichoderma species consistently demonstrated the highest saccharification efficiency, with glucose yields reaching 2.37 mM under SSF conditions. A Scheffe simplex-lattice mixture design comprising 65 enzyme cocktail combinations revealed significant synergistic interactions between several cocktails, with the binary mixture of Trichoderma 2SA21 and P. chrysogenum achieving 54% synergy - in terms of higher sugar release above expectations - and the highest total monosaccharide release (1.80 mM). These findings provide practical guidance for developing cost-effective enzyme cocktails for lignocellulosic biorefinery applications, emphasizing the importance of fermentation mode selection and strategic strain combination over enzyme supplementation complexity. The methodology established here, combining systematic screening, comparative proteomics, and statistical mixture design, offers a robust framework for optimizing fungal enzyme systems across diverse biomass substrates. BULLET POINTSSuperior enzymatic activity (xylanase, cellulase) and saccharification in solid-state fermentation Superior total protein content and diversity in submerged fermentation Specific enzyme cocktails combination can lead to synergistic effects, justifying a combinatorial approach GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/729743v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1f3704dorg.highwire.dtl.DTLVardef@151e4bforg.highwire.dtl.DTLVardef@180ced7org.highwire.dtl.DTLVardef@18bb42f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.

Fermentation is a long-standing tradition on the Indian subcontinent, shaped by local cultures and resources. Regional ingredients create a variety of fermented foods rich in probiotics. Lactiplantibacillus pentosus, found in foods like olives, grains, and vegetables, is a key strain. We studied the genome of L. pentosus from traditional fermented rice in Himachal Pradesh, India, and compared it to 95 global genomes. Our focus was on antimicrobial compounds, especially bacteriocins, which improve food safety. KEGG analysis revealed important metabolic proteins, while BPGA showed genome sizes of 3.4-4 Mb and GC content of 45.5-46%. The Indian L. pentosus strain (krglsrbmofpi2) uniquely contains three bacteriocins: Pentoplantaricin-EF, Pentobovicin, and Pentopediocin. Docking studies assessed their antiviral potential. Pentoplantaricin-EF showed the strongest binding to the prefusion 2019-nCoV spike glycoprotein (PDB 6VSB), while Pentobovicin had the highest affinity for the Omicron spike protein (PDB 7T9J) and the Hepatitis E virus E2 domain (PDB 3RKC).

Black Soldier Fly larvae (BSFL, Hermetia illucens) are highly effective for the bioconversion of food waste. However, their rearing process often produces substantial ammonia emissions, which are malodorous and environmentally concerning. We investigated the co-cultivation of BSFL with the sulfur-oxidizing bacterium Thiobacillus thioparus as a strategy to mitigate ammonia release. Importantly, under conditions where ammonia emissions were significantly reduced, neither larval growth nor bacterial viability was negatively affected. Furthermore, even when the initial bacterial inoculum was reduced to 3.3*105 CFU/g-food wastes, the bacterium rapidly recovered to functional levels and effectively controlled ammonia emissions. This indicates the absence of harmful interaction or nutrient competition between BSFL and T. thioparus. These findings suggest an efficient method for controlling ammonia in large-scale BSFL waste treatment. By reducing the required bacterial inoculum, this approach enables scalable microbial co-culturing with environmental and production benefits.