Applied Microbiology and Biotechnology

Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) and other microorganisms have attracted considerable interest due to their structural diversity and physicochemical properties, which makes them valuable across various industrial applications. To achieve high cell densities and maximize EPS yields, microorganisms are typically cultivated in nutrient-rich media containing yeast extract. However, yeast extract may contain high molecular weight polysaccharides that are not metabolized by the bacteria. This can lead to an overestimation of EPS yields and contamination of the bacterial EPS, potentially resulting in misinterpretation of their structure and biological activity. In this study, we demonstrate the presence of high molecular weight -mannan and {beta}-glucan in yeast extract in EPS isolates using both ultrafiltration and the commonly used trichloroacetic acid/ethanol (TCA/EtOH) precipitation method. These polysaccharides were characterized by size-exclusion chromatography, high-performance anion-exchange chromatography, and nuclear magnetic resonance spectroscopy. Their abundances were estimated to range from 10 to 50 mg/L in MRS medium, depending on the supplier of the yeast extract. The main contaminant identified was yeast -mannan. By cultivating L. rhamnosus GG (ATCC 53103) and L. pentosus KW1 and isolating their respective EPS, we illustrate how these yeast extract contaminants affect the structural interpretation of the EPS and that the contaminants can be completely removed by ultrafiltration of the growth medium prior to bacterial cultivation. In conclusion, we emphasize the necessity of stringent controls in the production and purification of microbial EPS, with particular attention to the chemical purity of medium constituents.

Probiotic-based encapsulation offers unique advantages over purified enzymes, such as increased protection from thermal-, pH-, and protease-mediated degradation, for oral therapeutic delivery applications. However, one of the major disadvantages of whole-cell systems is lower reaction rate due to substrate-product transport limitations imposed by the cell membrane and/or wall. In this work, we explore the potential of different lactic acid bacteria (LAB) - Lacticaseibacillus rhamnosus GG (LGG), Lactococcus lactis (Ll), and Lactiplantibacillus plantarum (Lp) - as expression hosts for recombinant Anabaena variabilis phenylalanine ammonia-lyase (AvPAL*). AvPAL* is used as a therapeutic to treat Phenylketonuria (PKU), a rare autosomal recessive metabolic disorder. Among the three species tested, LGG showed the highest PAL activity followed by L. lactis. Next, we attempted to overcome mass transfer limitation in whole-cell biocatalysts in two ways - expression of heterologous transporters and treatment with different chemical surfactants. Engineered strains expressing heterologous transporters exhibited approximately 3-4-fold increased PAL activity, while chemical treatment did not improve reaction rates. This work highlights the challenges and advances in realizing the potential of LAB as biotherapeutics. Impact StatementOral delivery of phenylalanine ammonia-lyase (PAL) using engineered probiotics is a promising therapeutic strategy to treat Phenylketonuria (PKU). Although PAL expression has been reported in probiotic strains of Limosilactobacillus reuteri, Lactococcus lactis, and E. coli, a systematic comparison of lactic acid bacteria (LAB) is underexplored. This study explores the potential of multiple LAB as hosts for PAL expression and investigates strategies to improve whole cell enzymatic activity. The findings from this study provide a foundation for implementing LAB-based delivery of PAL and indicate an important step towards development of probiotic platform for PKU management.

Proteins are an abundant extracellular polymeric substance (EPS) of activated sludge from wastewater. Hot alkalinization is implemented industrially to recover the EPS from sludge. We sought to assess the feasibility of protein recovery from alkali EPS. We detected a low abundance of bacterial proteins in the EPS. Human keratin was highly abundant and could also be recovered. Keratin was observed as a dominant and integral component of the activated sludge flocs, and it was thus not an extraction artifact. Alkali extraction promoted Maillard reaction between proteins and sugars and removed recoverable peptide signatures. Subsequent chemical modification, along with denaturation, impaired protein binding to ion exchange resins, making bacterial proteins inaccessible to isolation. Keratin has high resistance to Maillard reaction under extraction conditions and thus persists in the EPS. While Maillard modifies bacterial proteins, the resultant product, and possibly even keratin itself, are valuable recoverable byproducts from activated sludge. SynopsisThe different sensitivities of proteins to Maillard reaction determines which proteins dominate alkaline extracellular polymeric substance (EPS) extract from activated sludge, with keratin dominating in alkaline EPS and an integral activated sludge component.

Cultivated meat has undeniable potential to address some of the current detrimental impacts of animal farming, while addressing food security worldwide. However, one of the main challenges in cultivated meat production is manufacturing cost. The main contributor to cost is the culture media which comprises expensive components such as growth factors and animal-derived proteins. This study investigated alternative, food grade, high protein extracts as serum replacements in serum-free media formulations. The extracts were chosen to represent various sustainable sources of proteins: marine (spirulina e.g. cyanobacterium), plant (faba bean) and insect (mealworm flour). Different processing methods and different solvents were investigated for production of cell culture-compatible extracts which were then tested with mouse myoblasts (C2C12) and primary porcine myosatellites (pMyoSCs). A serum-free medium formulation containing 2.6% v/v spirulina extract was found to support long term growth of C2C12 cells for [~]10 population doublings compared to only [~]2 in the control. The processing steps were optimized, showing that a glycerine solution was best for free amino acid and protein yield (4950 {micro}M total free amino acids, 11.45 mg/mL protein concentration). This solution had a positive effect on C2C12 cells, increasing their growth by up to 20% when added to the B8 medium. However, this benefit did not translate to pMyoSCs, which showed no significant growth increases in short-term screening. This work demonstrates a method for converting food grade protein powders into effective culture media supplements and highlights the potential of spirulina-based extracts for the use in cultivated meat. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/702276v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@206658org.highwire.dtl.DTLVardef@11f28ceorg.highwire.dtl.DTLVardef@b00fd6org.highwire.dtl.DTLVardef@dfaaf4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Created in BioRender. Gordon-Petrovskii, W. (2025) https://BioRender.com/by7khs1 C_FIG

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY(R) VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

Granulation is a complex microbial-aggregation process essential for forming aerobic granular sludge (AGS) and other microbial granules used in wastewater treatment. However, the biological mechanisms that drive granule formation remain poorly understood. Cyclic-di-GMP (c-di-GMP) is a well-established second messenger that regulates biofilm formation, suggesting it may be used to enhance microbial granulation. Mycobacterium smegmatis, a nonpathogenic model bacterium for Mycobacterium tuberculosis, naturally forms granules. Because M. smegmatis carries a single c-di-GMP modulating gene, dcpA, that encodes an enzyme with both diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities, it offers a unique opportunity to examine the role of c-di-GMP in granulation. Here, we generated and studied two engineered M. smegmatis strains overexpressing dcpA or dcpA{Delta}EAL, the latter of which is defective in PDE activity. Using these engineered strains, we examined different forms of biofilm growth, cell morphology, plastic surface adhesion, granulation, and settleability. Results of sludge volume index and microscopy indicated that the aggregates of M. smegmatis were granules rather than flocs, and the settleability of the granules was particularly robust when the cells were grown in a carbon rich medium known to promote granulation. Engineered strains sustained stable granulation more effectively than the wildtype under low concentration Tween-80 treatment, which was used to induce dispersion. These results suggest that overproduction of DcpA and thus the modulated level of intracellular c-di-GMP enhances granulation and promotes granule persistence in M. smegmatis. Our study further demonstrates that M. smegmatis is a useful model for elucidating biological mechanisms underlying granulation, which could be leveraged to improve granular technologies for wastewater treatment.

Postharvest losses and rapid nutrient degradation due to fruit spoilage necessitate alternative preservation methods. Wine production presents a viable approach to minimizing fruit waste while retaining essential nutrients. In this study, mixed fruit wines (watermelon, banana, and pineapple) were produced using Saccharomyces cerevisiae isolated from palm wine as a starter culture. After secondary fermentation, the wines maintained an acidic pH range (2.29{+/-}0.1 to 3.25{+/-}0.2), a stable fermentation temperature (26.50{+/-}1.1{degrees}C to 27.00{+/-}1.1{degrees}C), specific gravity values of 1.021{+/-}0.02 kg/L and 1.027{+/-}0.03 kg/L, and total acidity levels of 1.57{+/-}0.2% and 2.11{+/-}0.1% for Wines A and B, respectively. The final alcohol content was 8.40{+/-}2.9% in Wine A and 9.84{+/-}3.6% in Wine B. Proximate analysis demonstrated the retention of key nutrients post-clarification and maturation, and sensory evaluation indicated a higher consumer preference for Wine B (P>0.05). These findings highlight the potential of indigenous S. cerevisiae strains from palm win for efficient wine fermentation and support the utilization of mixed fruits as a sustainable raw material for value-added wine production. This approach not only mitigates fruit wastage but also provides an economic avenue for enhancing fruit utilization.

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

Consumption of sprouted seeds, such as alfalfa sprouts, has increased in recent years due to their perceived health benefits. However, these food products have repeatedly been associated with outbreaks of foodborne pathogens, including Salmonella enterica serovars. An S. enterica serovar Choleraesuis strain previously isolated from melon fruit internal tissues was selected as a model to explore plant-pathogen interactions on alfalfa sprouts. Using this strain, we generated a barcoded transposon mutant library comprising approximately 33,000 unique insertions. This library and a collection of individual insertion mutants derived from it were used to identify genetic mechanisms contributing to the fitness of this S. Choleraesuis strain on alfalfa sprouts. The library was screened on sprouts during cold storage at 8{degrees}C. Negative selection for mutants with insertions in eda, fabF, lpp1_2, pnp, stpA, SCHChr_03621 and two intergenic regions were identified. Competition experiments between individual insertion mutants and the wild type confirmed the phenotype of three genes: eda, coding for a keto-hydroxyglutarate-aldolase/keto-deoxy-phosphogluconate aldolase involved in the Entner-Doudoroff pathway, mnmG, encoding the glucose-inhibited division protein, and fabF, involved in fatty acid biogenesis. This study offers a genome-wide perspective on the genes enabling a plant-associated Salmonella strain to persist on alfalfa sprouts. We highlight factors that are critical not only for persistence throughout the entire cold-storage period under conditions that closely simulate real shelf-life conditions in this high-risk food matrix.

The transcription factor Pho4 is crucial for the response to phosphate starvation in many fungi, and it has been linked to tolerance to alkalinization of the medium and to pathogenicity. It is widely accepted that it is encoded by a single gene. However, the industrially relevant yeast Komagataella phaffii might contain two Pho4-encoding genes (PAS_chr1-1_0265 and PAS_chr2-1_0177, designated here PHO4(A) and PHO4(B), respectively), which have never been functionally characterized. The phenotypic analysis of single and double mutants suggests that Pho4(B) plays a major role in the adaptation to Pi scarcity. While single mutants exhibited limited and non-overlapping phenotypic defects, the pho4(A) pho4(B) strain was sensitive to multiple types of stress, including phosphate starvation and alkaline pH. Transcriptomic analysis confirms that Pho4(B) is crucial for the transcriptional response to phosphate starvation, including induction of typical gene markers (PHO5, PHO89, VTC1, etc.). However, by using a GFP reporter we found that PHO4(A) also participates in the induction of PHO89 under high pH stress. Expression of both PHO4(A) and PHO4(B) in S. cerevisiae complemented the pho4 mutation under phosphate limitation by restoring growth, expression of the Pho84 transporter and secreted phosphatase activity. These results indicate that both transcription factors display partially overlapping functions, responding differently to diverse stimuli, and that together they constitute a key component in the adaptation to a variety of stresses. Therefore, K. phaffii is an exceptional example among fungi that encodes two Pho4 functional transcription factors.

Black rice oligosaccharides (BO) were extracted with 80% aqueous ethanol (v/v) and purified by charcoal-celite chromatography followed by dialysis using a 500 Da molecular weight cut-off (MWCO) membrane, yielding an oligosaccharide fraction with a degree of polymerisation (DP) between three and eight (DP3-DP8; 2.87 {+/-} 0.29% w/w). MALDI-TOF MS showed sodium adduct ions from m/z 527 to 1330, and GC-MS analysis of hydrolysed samples identified glucose and galactose as the major monomers, while ketosyl residues were detected in the intact fraction by selective staining and are most plausibly attributed to fructosyl units based on cereal origin and DP distribution. BO showed high resistance to simulated salivary, gastric, and pancreatic digestion (only 2.08 {+/-} 0.51%, 0.34 {+/-} 0.03%, and 4.29 {+/-} 0.73% hydrolysis, respectively) with approximately 93% remaining carbohydrate available for fermentation. All Lactobacillus strains showed positive prebiotic activity scores, with the highest response observed for Lactobacillus rhamnosus (1.165 {+/-} 0.255) and Lactobacillus plantarum (0.980 {+/-} 0.163). Fermentation produced metabolically relevant short-chain fatty acids (SCFA), mainly acetate (34.82 {+/-} 2.08 mM), as well as strain-dependent propionate and butyrate levels. BO greatly promoted probiotic biofilm formation, with biomass reaching 391.33 {+/-} 26.08% and viable cell counts of 9.01 {+/-} 0.70 log CFU/mL relative to the control. Collectively, the results indicate that BO represents a digestion-resistant, hexose-based oligosaccharide series that is selectively utilised by probiotic lactobacilli, promotes SCFA production and enhances biofilm development. To our knowledge, this work is the first to combine structural profiling with in vitro functional evaluation of a purified, low-DP oligosaccharide fraction obtained from black rice. HighlightsO_LIPurified oligosaccharides (DP3-DP8) were obtained from black rice using charcoal-celite chromatography followed by dialysis. C_LIO_LIStructural analysis confirmed that the oligosaccharides were hexose-based and composed mainly of glucose and galactose. C_LIO_LIBlack rice oligosaccharides exhibited higher resistance to simulated gastric and intestinal digestion compared with starch. C_LIO_LIPositive prebiotic activity scores were observed due to selective utilisation by probiotic Lactobacillus strains. C_LIO_LIFermentation of black rice oligosaccharides significantly increased short-chain fatty acid production. C_LIO_LIPurified oligosaccharides enhanced probiotic biofilm formation, indicating improved colonisation potential. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/705216v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@9a5915org.highwire.dtl.DTLVardef@14eac7aorg.highwire.dtl.DTLVardef@1db8baorg.highwire.dtl.DTLVardef@14ad50a_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionThe use of Saccharomyces cerevisiae to ferment alcoholic beverages is an ancient tradition, with genetic evidence indicating origins in Neolithic Asia, although the domestication process of the species is not fully understood. Kveik is a group of traditional yeasts used in farmhouse brewing in western Norway preserved through generations of rural brewing practice. While recent studies have highlighted the distinctiveness of kveik, its precise phylogenetic position, genetic diversity, and domestication history remain unclear. ResultsWe performed whole-genome sequencing on 62 samples representing 25 unique Norwegian strains selected using cultural heritage criteria, and generated telomere-to-telomere (T2T) assemblies for representative isolates. Phylogenomic and population genetic analyses reveal that kveik forms a paraphyletic and early diverging group with respect to other domesticated S. cerevisiae strains. Most strains exhibit low within-strain diversity, strong geographic clustering, and little evidence of gene-flow or admixture. Mitochondrial genomes and Ty1 retrotransposon profiles corroborate this distinct lineage history. We further show that previously reported signals of gene flow between kveik and Asian fermentation strains are likely artifacts caused by population structure and selection. Divergence time estimates suggest that the common ancestor of beer, kveik, and other liquid-phase fermenting strains originated from ancestral populations 4,000 to 8,000 years ago. ConclusionKveik yeasts represent a relic of early S. cerevisiae domestication, shaped by ancient human practices, migrations, and the spread of agriculture. Our genomic resource sheds light on yeast evolution and domestication. They likely comprise some of the oldest domesticated lineages in continuous use until today, connecting endangered intangible cultural heritage to an early genetic origin.

Yarrowia lipolytica is a yeast of industrial interest exhibiting remarkable lipolytic and proteolytic capacities, with a high potential for white biotechnology applications. This yeast can be isolated from a wide range of natural, polluted or anthropogenic environments, including various food products. The present study aims to increase the data on Y. lipolytica phenotypic diversity by evaluating the growth parameters and secreted enzymatic activities of 28 wild-type Y lipolytica (and Yarrowia sp.) strains isolated from various environments across 10 countries. These data could facilitate the selection of appropriate strains for specific research purposes, particularly when wild-type strains are prioritized over genetically engineered ones, like for food-related applications. Notably, strain SWJ-1b exhibited an outstanding combination of favourable characteristics, with optimum (or near) performances for both growth and enzymatic parameters.

Propionic acid (HPr) accumulation is a major indicator of anaerobic digestion (AD) dysfunction, yet the relative contributions of acidity, undissociated HPr, and propionate ions (Pr-) to process inhibition remain poorly understood. We investigated these effects in mesophilic batch AD microcosms fed with municipal sewage sludge, using a comparative design involving HPr, sodium propionate (NaPr), NaCl, and HCl treatments across two series of experiments. While 20 mM HPr caused a 22% reduction in the maximal methane production rate, 81 mM HPr led to complete inhibition, with the initial pH dropping to 5.1. By contrast, 81 mM NaPr reduced methane production rate by only 40%, and 81 mM NaCl caused no inhibition, demonstrating that acidity is the dominant inhibitory factor, with Pr- exerting a secondary concentration-dependent effect. 16S rRNA gene amplicon sequencing revealed strong, compound-specific shifts in microbial community composition, affecting key functional groups including syntrophs and methanogenic archaea. The proportion of methanogens dropped from 2-3% in control reactors to less than 0.2% under 81 mM HPr, consistent with the observed methane production inhibition. Under HPr81, over 100 ASVs were differentially abundant compared to controls, a pattern largely shared with HCl-treated reactors, further confirming the predominant role of acidity. The number of differentially abundant ASVs was negatively correlated with methane production rates (R{superscript 2} = 0.97), underscoring the link between community reshaping and process impairment. These results provide a unifying framework for propionate inhibition in AD and suggest that microbial community profiling could serve as an early warning tool for process imbalance detection.

Poly({varepsilon}-caprolactone) (PCL) is a well-known biodegradable polyester and is among the few polyesters susceptible to degradation in marine environments; however, marine-derived PCL-degrading enzymes remain poorly characterized. Here, we searched for PCL-degrading enzymes from the marine bacterium Alloacanivorax gelatiniphagus JCM 18425 using a genome-based approach. Five candidate genes were predicted, and one encoded protein, designated Ag0826, was identified as a PCL depolymerase. Recombinant Ag0826 was expressed, purified, and biochemically characterized. The enzyme exhibited optimal activity at 35-40{degrees}C and pH 8.0, although it showed limited thermal stability. Substrate specificity was compared with that of leaf-branch compost cutinase (LCC), a well-characterized poly(ethylene terephthalate) (PET) hydrolase, using various polyesters. Both enzymes exhibited largely overlapping substrate ranges with respect to the presence or absence of monomer conversion activity across the tested substrates. Ag0826 slightly degraded PET to terephthalic acid, indicating potential PET-hydrolyzing activity; its conversion rate, however, was substantially lower than that of LCC, suggesting that Ag0826 exhibits a substrate preference differing from LCC. Phylogenetic analysis based on amino acid sequences revealed that Ag0826 formed a separate clade from LCC and IsPETase (from Ideonella sakaiensis). At a broader level, Ag0826 was positioned near HaloPETase1 (from Halopseudomonas pachastrellae), which has been proposed as a Type III PET hydrolase; in contrast, residues corresponding to the substrate-binding subsites were similar but not identical between the two enzymes. These results suggest that Ag0826 broadly belongs to the group of known PET hydrolases, yet it exhibits a partially distinct sequence profile even within this enzyme family.

Porcine Bacteroides thetaiotaomicron LYH5 demonstrated in vitro antimicrobial activity, suggesting probiotic potential. Due to poor gastric juice tolerance, LYH5 was encapsulated via extrusion using sodium alginate (SA) and gellan gum. Box-Behnken design optimization yielded optimal parameters: SA 1.5%, gellan gum 0.4%, CaCl2 0.9%, bacteria:glue ratio 1:4, achieving an encapsulation rate of 84.22{+/-}0.17%. Its effect on weaned piglet intestinal health was evaluated using 78 piglets (7.69{+/-}0.52 kg) randomly assigned to 4 groups for 40 days: CON (control), T (basal diet + LYH5 live bacteria, 1x10{superscript 1} CFU/mL), TJ (basal diet + LYH5 microcapsules, 1x10{superscript 1} CFU/mL, J (basal diet + empty capsules). The results of this experiment showed that compared with the control group, LYH5 microcapsule can improve the intestinal barrier function without affecting the growth performance of piglets, and provide ideas and references for the development of human next-generation probiotics (NGP). IMPORTANCEThis study addresses the key bottleneck of poor gastric acid tolerance of probiotics via microencapsulation and provides a practical reference for the development of human next-generation probiotics.

The gut microbiota plays an essential role in the conversion of anthocyanins and (epi-)catechins into smaller phenolic acids, which are then absorbed into the blood stream. The phenolic composition of a commercial bilberry extract and grape seed extract was assessed, as well as a formulation extract containing a combination of both extracts. The extracts were subjected to an in vitro salivary, gastric and intestinal digestion environment, based on the INFOGEST Model. The solid fraction end-product of the combined extract from the in vitro digestion was further fermented with faecal samples from six healthy donors, for 72 hours, to assess phenolic acid metabolism, short-chain fatty acid formation and changes in microbial composition. During the in vitro digestion, flavonoid content in all three extract samples (bilberry, grape seed and the formulation extracts) decreased significantly. In the process of anthocyanin and flavonoid digestion, smaller phenolic acid compounds such as benzoic acid, cinnamic acid and mandelic acid increased in bilberry, grape seed and formulation extract samples. All faecal donors harboured unique microbiota compositions, however all faecal microbiota were able to fully convert catechin/epicatechin, the most prominent flavonoids in the formulation extract sample, into smaller phenolic metabolites (phenylacetic, phenylpropionic and benzoic acids) within 24 hours. Using 16S rRNA gene amplicon sequencing, Anaerobutyricum and Enterocloster spp. were correlated with catechin/epicatechin metabolism in the fermentation procedure, however, in single bacterial strain fermentation experiments with the formulation extract or catechin standard, these bacteria were not capable of metabolising flavonoids. HighlightsO_LIFaecal microbiota converted (epi-)catechin to phenolic metabolites within 24 h. C_LIO_LI(Epi-)catechin correlated negatively with Anaerobutyricum and Enterocloster spp. C_LIO_LIFaecal bacterial cultures did not show (epi-)catechin metabolism capacity. C_LI

The dynamics and impact of microbial contaminants in industrial sugarcane bioethanol production in Brazil were investigated through a two-year metagenomic study across two biorefineries. Shotgun metagenomic sequencing revealed that temporal shifts in the contaminant microbiome dynamics within production seasons were more pronounced than inter-annual or inter-mill variations. While Saccharomyces spp. dominated, bacterial communities, primarily within the Firmicutes phylum and dominated by the genera Lactobacillus, Limosilactobacillus, and Bacillus, exhibited dynamic changes. Correlation analyses with industrial process parameters revealed a complex interplay: lower Lactobacillus levels in one mill were associated with increased ethanol yield, whereas higher levels in another mill correlated with reduced yeast viability and increased flocculation. The presence of Limosilactobacillus was linked to decreased yeast viability, whereas Bacillus showed potential for inhibiting both Lactobacillus and Limosilactobacillus. These findings highlight the nuanced and species-specific impacts of bacterial contaminants on bioethanol production, underscoring the need for strain-level functional studies and targeted interventions to optimize fermentation efficiency and stability in industrial settings.

Functional dairy products are increasingly recognized for their ability to provide both essential nutrition and additional health benefits. This study aimed to develop and evaluate a synbiotic yogurt enriched with Lactobacillus plantarum as a probiotic and Stevia rebaudiana extract (1% w/v) as a prebiotic source. Thirteen lactic acid bacteria (LAB) strains were isolated from fermented dairy and vegetable samples and evaluated for probiotic potential through tests for acid and bile tolerance, hydrophobicity, aggregation abilities, and pathogen co-aggregation. Isolate PG1 (Lactobacillus plantarum) demonstrated the highest prebiotic growth stimulation index (49%) in the presence of stevia extract and was selected for yogurt formulation. Yogurt samples were prepared and stored at 4{degrees}C for 10 days. Physicochemical properties (pH, titratable acidity, and protein content), microbiological viability, total phenolic and flavonoid content, antioxidant activity (DPPH assay), and sensory attributes were monitored. The synbiotic yogurt (St-Y) showed enhanced functional properties, with a total phenolic content of 16.67 {micro}g GAE/g, a flavonoid content of 6.28 {micro}g QE/g, and 57.84% antioxidant activity. Additionally, it showed improved protein content and superior sensory scores compared to control samples. These findings suggest that S. rebaudiana fortified probiotic yogurt can serve as a nutritious, antioxidant-rich, and sensory-acceptable functional dairy product.

Microbial fuel cell offers a promising approach to improve wastewater quality and generate bioenergy from dark fermented effluents. In this study, the use of dark-fermented palm oil mill effluent as an electron donor for bioelectricity generation was investigated using a double-chambered microbial fuel cell (MFC). The MFCs were operated at room temperature (29 {+/-} 2{degrees}C), anode electrolytes adjusted to pH 7, and a chemical catholyte as the oxidizing agent. The maximum power {+/-} 8.07 mW/m2 and 155.16 {+/-} 12.88 mA/m2, respectively, were generated from the MFCs inoculated with sludge, which was 5.9 times higher than control without inoculum. Microbial community analysis revealed the enrichment of fermentative and electrogenic representative taxa from the phyla Bacillota, Bacteroidota and Pseudomonadota on the anode electrodes. Optimizations of the running conditions were carried out, suggesting the optimum parameters of 0.5 k{Omega} external resistance, anolyte initial pH 9, and 75% DFPOME substrate concentration. Operation under the optimized conditions increased current production, wastewater treatment, and Coulombic efficiency compared to the non-optimized conditions. Multiple configurations were also evaluated, showing higher cumulative voltage, power, and current densities with the stacked MFC connections, compared to single MFC units. Parallel circuit connection produced higher power and current density than serial connection. This study demonstrated the feasibility of MFC as a promising downstream treatment for biohydrogen production processes, towards higher treatment efficiency and resource recovery.