Abstract The phosphotransferase system (PTS) is a conserved mechanism in most bacteria, responsible for carbohydrate transport and regulation. This system plays a key role in carbon catabolite repression (CCR), a regulatory process that prioritizes the metabolism of preferred sugars. However, PTS and CCR have received little attention in fermentations of oligomeric lignocellulose-derived carbohydrates. Thermoanaerobacterium thermosaccharolyticum, a thermophilic gram-positive anaerobic bacterium capable of fermenting a diversity of simple and complex carbohydrates, is a promising candidate for lignocellulosic ethanol production. This study investigated the role of PTS and CCR by deleting the ptsH gene in T. thermosaccharolyticum. Monomeric sugar utilization experiments revealed that deletion of ptsH resulted in the loss of selective sugar utilization by effectively disrupting CCR. Assays with 2-deoxyglucose and cyclic adenosine monophosphate (cAMP) confirmed that ptsH is involved in the T. thermosaccharolyticum CCR response and is cAMP dependent. Its deletion alleviates catabolite repression, especially for cellobiose. Remarkably, fermentation of hemicellulose-derived carbohydrates from sugarcane bagasse spent broth was not affected by deletion of the ptsH gene, indicating that CCR has minimal impact under lignocellulosic carbohydrate substrate conditions. This research provides new insights into sugar catabolism regulation in T. thermosaccharolyticum, and for the first time demonstrates that CCR is not important when fermenting lignocellulose-derived sugars.
Malodorous emissions from rural sanitation systems pose a direct environmental health concern. This study developed a composite microbial deodorant (CMD) using five bacterial strains isolated from activated sludge, combined in an optimal 1:1:1:1:1 ratio determined by orthogonal testing, to mitigate emissions of NH3 and H2S-key hazardous and odorous pollutants. In laboratory simulations, the CMD reduced NH3 and H2S by 82.1% and 86.1%, respectively. A pivotal 6-month field evaluation in three common types of rural household toilets demonstrated sustained, real-world efficacy, with peak removal rates of 79.43% for NH3 and 80.83% for H2S. The intervention also conserved nitrogen, reducing total nitrogen loss by 14.8%. Metagenomic analysis revealed the mechanistic basis: the CMD reshaped microbial metabolic pathways by upregulating genes for nitrification (hao) and sulfur oxidation (sqr), while suppressing reductive genes (nif, aprA), thus curbing emissions at source. This work provides robust field evidence that an in situ microbial strategy can effectively and sustainably improve household air quality in rural settings, offering a practical, low-cost, and eco-friendly solution for in situ odor control in rural sanitation- a critical step for sustaining the health benefits of the ''Toilet Revolution'' and improving rural living environments.One-Sentence Summary A composite microbial deodorant effectively reduces ammonia and hydrogen sulfide emissions and improves air quality in rural household toilets through 6-month field validation, providing an eco-friendly and low-cost odor control solution for rural sanitation.
Thermostable enzymes are central to high-temperature industrial processes because they combine catalytic efficiency with structural resilience under demanding conditions. Among thermophilic bacteria, Geobacillus has emerged as a prominent source of industrially relevant enzymes, particularly hydrolases such as proteases, lipases, and carbohydrate-active enzymes. Although many individual enzymes have been characterized in detail, comparative synthesis across catalytic classes within the genus remains limited. In this review, we integrate biochemical data and structural evidence to explore whether thermostability in Geobacillus reflects isolated enzyme-specific traits or recurring functional patterns across enzyme classes. Reported T-max and pH(max) values from purified and partially purified hydrolases were systematically compiled to assess class-level tendencies. Temperature optima cluster within a relatively narrow thermophilic range across enzyme groups, supporting high-temperature activity as a common feature within the genus. In contrast, pH optima display clearer functional differentiation, with proteases and lipases consistently shifted toward alkaline values relative to carbohydrate-active hydrolases. Recurrent stabilizing elements, including reinforced hydrophobic cores, expanded electrostatic interaction networks, and a balanced rigidity-flexibility profile, are observed across multiple enzyme classes, indicating shared structural trends rather than independent enzyme-specific variation. By situating catalytic optima within a comparative framework and relating them to shared structural determinants, this review provides a comparative interpretation of patterns observed across Geobacillus-derived enzymes, linking structure, catalysis, and functional adaptation.
Large-scale untargeted bacterial metabolomics studies are often challenging to interpret, particularly for natural product discovery. Complex raw mass spectrometry data contains media components, biotransformation products, workup contaminants, and in-source fragments, making it difficult to resolve true biosynthesized bacterial products from background features. Incorporating stable isotope labels into untargeted workflows enables the selective detection of actively biosynthesized compounds, drastically reducing candidate mass spectrometry features to highlight novel or robustly expressed metabolites. Recently, our lab introduced IsoAnalyst, a parallel stable isotope labeling platform that links secondary metabolites to biosynthetic gene clusters by determining the incorporation rates of a panel of isotopically labeled building blocks. In this study we applied the IsoAnalyst protocol to a library of 114 Burkholderiales strains with the goals of profiling the specialized metabolite chemistry of this bacterial order and connecting detected natural products to their cognate BGCs based on their labeling profiles. While our strain-matching protocol, which evaluates correlations between detected metabolites and BiG-SCAPE-defined gene cluster families, did not yield confident BGC assignments, it successfully highlighted widely distributed metabolites with distinct labeling profiles. We targeted a set of these molecules for isolation, revealing two classes of compounds derived from primary biomolecule metabolism that are implicated in cellular stress responses. The first class of molecules were identified as lysophosphatidyl ethanolamine analogues while the second was characterized via NMR spectroscopy and mass spectrometry as methyl-esterified 3-hydroxybutyrate oligomers (HB6-OMe, HB7-OMe, and HB8-OMe), representing higher-degree polymer units than previously reported.
Twenty disinfectant products were evaluated for sporicidal activity using an optimized method. Bacillus subtilis ATCC 6051 was sporulated in Difco sporulation medium for 3 days. The spores were purified using density centrifugation and heat treatment and then prepared for sporicidal efficacy assessment. The quality of the purified spores was verified using sodium hypochlorite solutions with free chlorine concentrations of 1,500 and 5,000 ppm. The active ingredients in disinfectant products included chlorine, hydrogen peroxide/peracetic acid, aldehydes, quaternary ammonium compounds, and phenols. Of the 20 products tested, 6 met the effectiveness criterion of greater than a 5 log₁₀ reduction after exposure of B. subtilis spores for the label-recommended contact time. Among the 11 products claiming sporicidal activity, 4 met the established criterion. Among the five categories of active ingredients evaluated, some chlorine-based and hydrogen peroxide/peracetic acid-based products demonstrated significantly higher sporicidal activity than products in the other three categories. Overall, products that did not meet the study criteria exhibited less than a 1 log₁₀ reduction. When these underperforming sporicidal products were tested with an extended contact time of 10 min, one additional product met the effectiveness criterion. Subsequent quality assessments using freshly prepared, laboratory-made products containing identical active ingredients demonstrated improved sporicidal efficacy compared with the original test items, indicating that defects or degradation of active ingredients may have occurred in some of the tested commercial disinfectant products. One sentence summary Evaluation of 20 disinfectants against Bacillus subtilis spores using optimized methods.
