Mannose constitutes a principal component in numerous biomass hemicelluloses; however, its utilization by the cellulose-producing bacterium Komagataeibacter xylinus is inefficient. Consequently, during biorefinery and the transformation of biomass rich in mannose for the production of the high-value biological product, bacterial cellulose (BC), the biomass components cannot be fully utilized, leading to resource wastage. Moreover, inappropriate discharge of the fermentation effluent may also result in environmental pollution. This study addressed the underutilization of mannose in spent coffee grounds (SCGs) by introducing a fusion vector expressing mannokinase and phosphomannose isomerase into K. xylinus ATCC 23770, thereby enhancing mannose utilization in this strain model. SCG was pretreated with concentrated sulfuric acid to disrupt crystalline structures, followed by enzymatic hydrolysis to extract saccharides. When culturing in SCG hydrolysate, BC pellicle-derived inoculum achieved 1.2-fold higher BC yield than broth inoculum, attributed to BC matrix protection. The engineered strain K. xylinus CGMCC 31806 demonstrated superior mannose assimilation, yielding 1.5-fold more BC than wild type in simulated SCG medium. In authentic SCG medium without extra nitrogen addition, BC production reached 3.80 g/L, a 1.2- to 2.5-fold increase. The obtained BC exhibited enhanced mechanical properties with 40-84% higher tensile strength and 20-75% increased Young's modulus. This study validates a dual strategy combining agro-waste valorization and targeted strain modification, establishing a collaborative optimization framework linking biomass feedstocks, microbial engineering, and bioprocessing for cost-effective BC production. Future research would utilize existing commercial high-yield strains to replicate the expression strategy of this fusion protein, in order to achieve truly efficient industrial production.IMPORTANCEThe inefficient utilization of mannose, a major hemicellulose component prevalent in biomass by Komagataeibacter xylinus represents a significant bottleneck in the sustainable, cost-effective bioproduction of bacterial cellulose (BC). This underutilization would lead to wasted resources and potential environmental pollution from fermentation effluents. The study directly addresses this critical challenge by pioneering a synergistic "Feedstock-Strain-Process" integration strategy in advancing BC industrialization. We demonstrate the significant impact of engineering K. xylinus for enhanced mannose assimilation coupled with optimized biorefinery of agro-industrial waste, taking mannose-rich spent coffee grounds, as the representative raw material. This integrated approach not only valorizes abundant agricultural waste streams significantly boosting BC yields under nutrient-limited conditions but also generates BC with superior mechanical properties. The study establishes an actionable blueprint for advancing BC industrialization through collaborative optimization of biomass feedstocks, targeted microbial engineering, and process innovation, aligning with the goals of the circular bioeconomy and sustainable material production.

When the heterofermentative lactic acid bacterium Weissella cibaria JCM12495 was cultivated anaerobically in medium supplemented with 3-hydroxy-4-methoxycinnamic acid (ferulic acid), all the added ferulic acid was converted to 3-(4-hydroxy-3-methoxyphenyl)propionic acid (HMPA, dihydroferulic acid) via reduction of the propenoic acid side chain. Complete conversion to HMPA was achieved within 16 h, even at an initial ferulic acid concentration of 10 mM. Concurrently, the production of typical heterolactic fermentation products, such as lactic acid and ethanol, was suppressed, while acetic acid formation was increased. Transcriptomic analysis revealed that only two genes were significantly induced by ferulic acid addition, and these genes form an operon in the genome. We individually expressed these two genes in Escherichia coli and assessed their enzymatic activities in vitro against ferulic acid by identifying reaction products using liquid chromatography quadrupole time-of-flight mass spectrometry. When ferulic acid and acetyl-CoA were added to a reaction mixture containing the first expressed protein, feruloyl-CoA was detected as the product. Subsequently, when the generated feruloyl-CoA was incubated with the second expressed protein in the presence of FAD and NADH, the formation of HMPA was confirmed. These results demonstrate that the two genes encode a ferulate CoA-transferase (FarA), which catalyzes the CoA ligation of ferulic acid, and a feruloyl-CoA reductase (FarB), which reduces the propenoic acid side chain of feruloyl-CoA to produce HMPA. Our findings reveal an anaerobic respiratory pathway coupled to heterolactic fermentation, in which ferulic acid acts as an electron acceptor, thereby influencing carbohydrate utilization during heterolactic fermentation.IMPORTANCEProbiotics and lactic acid bacteria (LAB) in fermented foods are of significant interest due to their health-promoting effects. This study shows that Weissella cibaria, a common LAB found in plant-derived fermented foods, uses ferulic acid, a compound bound to dietary fibers such as arabinoxylans, as an external electron acceptor for anaerobic redox balancing. This metabolic strategy is associated with redox regulation during heterolactic fermentation and leads to the production of 3-(4-hydroxy-3-methoxyphenyl)propionic acid, a metabolite that has been reported to have health benefits in humans. These findings reveal an unrecognized link between LAB metabolism, dietary polyphenol conversion, and potential probiotic functions, providing new insight into how gut-associated microbes contribute to host health.

This study presents the probiotic strain Pediococcus pentosaceus SMM847 as a promising therapeutic agent for two clinically significant conditions: hyperuricemia and acute alcohol intoxication. The strain mediates uric acid reduction through modulation of purine catabolism pathways. It is particularly noteworthy that SMM847 exhibits ethanol-catabolizing activity, attenuates alcohol-elicited systemic metabolic dysregulation, and exerts hepatoprotection without intrinsic toxicity. The ability of this single bacterial entity to simultaneously target dual pathological pathways-uric acid metabolism and alcohol-induced metabolic disruption-offers a novel and translatable approach for the development of functional foods or pharmaceutical interventions aimed at promoting integrated metabolic and hepatic health.

Marine environments are important reservoirs of bacteria capable of producing bioactive secondary metabolites; however, many promising antimicrobial producers identified during initial screening fail to retain activity during fermentation. This study demonstrates that pH stability during fermentation, rather than pH value alone, is a key determinant of sustained antibacterial metabolite production in marine sediment-derived Bacillus species. By linking isolation conditions, fermentation physiology, and bioactivity outcomes, the findings provide practical guidance for improving the reliability of marine bioprospecting and antimicrobial discovery pipelines. These insights are particularly relevant for efforts to recover stable antimicrobial producers from complex environmental systems.

Pythium myriotylum is the causal agent of root rot and wilt disease, which can cause significant damage to lettuce in hydroponic systems. Root rot can be challenging to treat with traditional methods once it develops, often resulting in the destruction of the entire crop. Reused nutrient solutions have been reported to harbor microorganisms that may affect disease suppression. Examining how bacterial communities in recycled nutrient solutions change and trigger plant-defense genes may contribute to the reduction of Pythium root rot and provide chemical-free and cost-effective alternatives for soilless cultivation systems. Future studies focusing on specific microorganisms and their bioactive compounds will be essential for advancing biological control methods in hydroponic crop systems.

Polycyclic aromatic hydrocarbons (PAHs) are widespread pollutants and especially difficult to degrade under anoxic conditions. The non-substituted three-ring PAH phenanthrene is activated through carboxylation to 2-phenanthroic acid which is then ligated to Co-enzyme A to produce 2-phenanthroyl-CoA, followed by step-wise reduction of the aromatic rings. The first reduction reaction is catalyzed by 2-phenanthroyl-CoA reductase producing dihydro-2-phenanthroyl-CoA. In this study, we elucidated the second reductase enzyme in the pathway, catalyzing the reduction of dihydro-2-phenanthroyl-CoA to hexahydro-2-phenanthroyl-CoA. Dihydro-2-phenanthroyl-CoA reductase (AprC) was heterologously overproduced in Escherichia coli and purified and characterized. AprC has a specific activity of 15.8 ± 0.3 nmol min-1 mg-1 and an apparent Km value of 59.9 ± 3.8 nM at pH 7.5. Dithionite-reduced methyl viologen is the preferred in vitro electron donor for the four electron-reduction reaction, but it also functions with NADH but not with NADPH. The intermediate appearance of tetrahydro-2-phenanthroyl-CoA during the reduction to hexahydro-2-phenanthroyl-CoA indicates that AprC catalyzes two consecutive two-electron reduction steps. AprC is a monomeric enzyme with a molecular mass of ≈73 kDa and contains one FMN, one FAD, and one 4Fe-4S cluster, indicating that the reductase belongs to the type III aryl-CoA reductases of the old-yellow enzyme family. The high amino acid sequence similarity between dihydro-2-phenanthroyl-CoA reductase and 2-naphthoyl-CoA reductase, as well as the similar cofactor coordination in both enzymes, suggested that dihydro-2-phenanthroyl-CoA reductase follows the same biochemical principle as naphthalene ring reduction by 2-naphthoyl-CoA reductase. Quantum chemical calculation indicated that 5,6,7,8,9,10-hexahydro-2-phenanthroyl-CoA (isomer 6a) is the most stable product of AprC out of eight possible isomers, although the real isomer is not known. The reductase also performed a two-electron reduction of chemically synthesized 9,10-dihydro-2-phenanthroyl-CoA to tetrahydro-2-phenanthroyl-CoA but at much lower reaction rates indicating that the 9,10-dihydro-2-phenanthroyl-CoA isomer is not the natural substrate of the enzyme and, thus, also not the real product of 2-phenanthroyl-CoA reduction by 2-phenanthroyl-CoA reductase AprB. Since 9,10-dihydro-2-phenanthroyl-CoA has two non-conjugated benzene rings, this also indicates that, surprisingly, type III aryl-CoA reductases can reduce benzene ring structures without additional ATP requirement.IMPORTANCEPolycyclic aromatic hydrocarbons (PAHs) are highly toxic, persistent pollutants found in the environment. The anaerobic degradation of larger three-ring PAHs like phenanthrene is still poorly understood. Here, we show that after activation to a CoA-ester, the resonance energy of the aromatic ring system of phenanthrene is overcome by consecutive two-electron reduction steps catalyzed by ATP-independent type III aryl-CoA reductases belonging to the old-yellow enzyme family. This finding contributes to our understanding of the anaerobic degradation of PAHs with three or more rings.

Avibacterium paragallinarum is the etiological agent of infectious coryza, a significant respiratory ailment associated with growth inhibition and diminished egg production in poultry. Given the increasing incidence of antibiotic resistance in bacteria, there is an imperative need for the development of genetically engineered vaccines; however, the lack of effective tools for genetic manipulation in Av. paragallinarum has limited the scope of related research. Herein, we investigated the employment of the gene pheSm as a counterselectable marker for the screening of marker-free mutants of Av. paragallinarum. We report the successful and precise deletion of multiple genes in Av. paragallinarum and an instance of the effective seamless integration of the exogenous gene lpxE using this method. This study presents a valuable genetic tool for investigating the virulence genes and associated mechanisms of pathogenicity of this bacterium, as well as aiding the development of genetically engineered vaccines.

Ultraviolet radiation (UVR) is an environmental stressor to the skin and its associated microbiota. Many commensal bacteria produce tryptophan-derived metabolites that modulate epidermal barrier function through aryl hydrocarbon receptor (AhR) signaling. However, the effects of UVR on these metabolic outputs and the subsequent effects on skin barrier function remain unclear. This study examined these interactions. Individual skin commensal bacteria were irradiated with 37.5 mJ/cm² or 150 mJ/cm² of solar-simulated radiation (SSR). Cell-free supernatants (CFSNs) were collected and analyzed for tryptophan metabolites (using LC-MS), untargeted metabolites (using GC-MS), and AhR agonist activity (via a reporter assay). The effects on barrier function were assessed by measuring transepithelial electrical resistance (TEER) of CFSN-treated keratinocyte cultures. Time-course expression of tryptophan-related genes after irradiation was measured by qPCR. Unirradiated bacterial metabolomes were species-specific. Following irradiation, the abundance of metabolites generated by the indole pathway generally increased after 37.5 mJ but decreased following 150 mJ. Untargeted analyses revealed several decreases in amino and organic acid production after high-dose SSR, while 37.5 mJ resulted in fewer changes. Low-dose SSR upregulated genes involved in tryptophan metabolism (ipdC, ALDH) and synthesis (trpE). AhR agonism increased in 8/11 organisms following irradiation, statistically correlating with increased levels of indole-pathway metabolites (indole-3-acetamide, tryptophol, indole-3-carboxaldehyde, and tryptamine). Keratinocytes treated with irradiated Staphylococcus hominis, Micrococcus luteus, and Staphylococcus capitis CFSNs showed enhanced TEER, concurrent with increased AhR activation; inhibition of the AhR removed this effect. UVR significantly alters the metabolomes of skin commensal bacteria, with knock-on effects for AhR signaling and barrier integrity, potentially influencing the skin's response to UVR.IMPORTANCEThe skin and its commensal bacteria are regularly exposed to ultraviolet radiation (UVR). Many skin bacteria generate tryptophan-derived metabolites that influence host physiology through activation of the aryl hydrocarbon receptor (AhR), a key regulator of barrier integrity and stress responses. However, the impact of UVR on the metabolic activity of skin commensals and the downstream consequences for epidermal barrier function remains poorly understood. Here, we show that UVR significantly alters the metabolic outputs of diverse skin commensals, shifting production of indole-pathway metabolites and modulating AhR agonist activity. These changes translated into measurable effects on keratinocyte barrier function, including enhanced transepithelial electrical resistance following UVR exposure. Our findings reveal a previously underappreciated role for bacterial photometabolism in shaping epidermal barrier regulation and host responses to UVR.

In composting, humification is critical for final compost quality and effectiveness and involves small molecules polymerizing into humic substances (HS). Microbial metabolism, as the core process of composting, drives this transformation. However, the full relationship between microbial metabolic processes and humification-related polymerization remains unclear in current research. This review first examines humification polymerization mechanisms from the perspective of precursors through functional group reactions and radical oxidative coupling. Then, it systematically elucidates the central role of microbial extracellular and intracellular metabolisms in driving the humification process. On the one hand, the effects of hydrolases and oxidoreductases of extracellular metabolism on the polymerization are elaborated in detail. On the other hand, it delves into the dual contributions of intracellular metabolism: catabolism supplies essential precursors and active energy for polymerization reactions through substance degradation and oxidative phosphorylation, while anabolism is directly involved in the biosynthesis of humus precursors. This work aims to provide a comprehensive theoretical framework for understanding the microbial driving mechanisms behind humification.

In the context of drought and increasingly severe water shortages, agriculture faces a major challenge in continuing to feed a growing global population. Irrigation of crops with treated municipal wastewater can contribute to meeting agriculture's water needs. However, the use of treated wastewater carries numerous public health risks due to the presence of various human and animal pathogens including spore-forming Clostridium species, such as Clostridioides difficile and Clostridium perfringens. Antibiotic resistance in these important pathogens is an emerging issue. Spores of these bacteria are extremely persistent, and they can persist for years in a wide range of environments. Their presence in treated wastewater could lead to contamination of soil and of plant-based foods grown for human or animal consumption. Within this context, the present review addresses the pathogenicity of C. difficile and C. perfringens and their potential role in the dissemination of antimicrobial resistance in the context of wastewater reuse for agriculture.