Applied Microbiology and Biotechnology最新文献

This study presents Bacillus subtilis T7 as the first known strain of B. subtilis capable of simultaneous lignin depolymerization and direct hydrogen production—a dual metabolic capability not previously reported in this species. B. subtilis T7 demonstrated 63.38% lignin degradation and 56.53% Azure B decolorization over seven days, with HPLC detection of aromatic intermediates—ferulic acid (0.85 mg/L) and vanillin (0.666 mg/L)—confirming active lignin catabolism. Agar-based assays revealed robust hydrolytic enzyme activities, including proteases (23.3 mm), cellulases (24.5 mm), amylases (14.2 mm), and xylanases (11.6 mm), surpassing those of many reported Bacillus strains. Whole-genome analysis confirmed a cascade of carbohydrate-active enzymes (CAZymes) including AA10 (lytic polysaccharide monooxygenases), AA3 (oxidoreductases), AA6 (quinone reductases), and CE1 (acetyl xylan esterases). These enzymes are associated with enhanced cellulolytic and xylanolytic activities, as well as increased lignin degradation. Batch fermentation experiments demonstrated that B. subtilis T7 produced hydrogen yields ranging from 0.53 to 1.41 mol H₂/mol substrate across various feedstocks, including xylose, glucose, carboxymethyl cellulose (CMC), starch, and untreated food waste. Xylose exhibited the highest volumetric productivity, achieving 274 mL H₂/g volatile solids, along with the most rapid production kinetics, indicating efficient metabolic utilization of this pentose sugar. In contrast, untreated food waste yielded the maximum molar hydrogen output of 1.41 mol H₂/mol substrate, attributable to its heterogeneous carbohydrate composition and lower average molecular weight, which likely enhanced enzymatic accessibility and substrate solubilization. These findings indicate that B. subtilis T7 encodes a functional [FeFe]-hydrogenase operon, along with an expanded repertoire of oxidative CAZymes, enabling it to bioprocess waste biomass into hydrogen without the need for syntrophic partners.

• B. subtilis T7 show dual potential for lignin degradation and hydrogen production.

• The genome contains CAZymes (AA10, AA3, AA6, CE1) responsible for lignocellulose deconstruction.

• The strain T7 produced a hydrogen yield of 1.411 mol H₂/mol substrate from xylose.

Spread of antimicrobial resistance and lack of new antibiotics have brought attention to alternative strategies of combating pathogenic bacteria. One of these strategies takes advantage of the bacteriolytic activity of peptidoglycan hydrolases. The enzymes allow efficient elimination of pathogenic bacteria while preserving the natural microflora. Such enzymes must meet specific criteria of activity, stability, and safety to become efficient enzybiotics. In our previous work (10.1128/spectrum.03546-23), we have created three chimeric enzymes and demonstrated their high efficacy in the elimination of Enterococcus faecalis and Staphylococcus aureus. In this work, we investigated and addressed issues related to the stability and safety of these enzymes. To improve the stability, we engineered the linkers and optimized storage conditions. Moreover, we demonstrated that such enzymes do not have any cytotoxic effects on eukaryotic cells, Danio rerio or Galleria mellonella. We also investigated the prevalence of resistance development, a particularly important feature for new antimicrobials. In conclusion, we here propose efficient, safe, and stable chimeric enzybiotics to eliminate E. faecalis and S. aureus.

• Optimized linker design enhances enzyme stability.

• Generated chimeric lysins do not display cytotoxicity.

• Chimeras with minimal risk of resistance development were selected.

The current study investigated 17 bacterial strains for their ability to synthesize gold nanoparticles (AuNPs) from the aryldiazonium gold(III) salt (DS-AuCl₄). The study aims to investigate the ability of bacterial cell biomass in the stationary phase of growth to synthesize AuNPs at 28 °C and 37 °C. Eleven bacterial strains were isolated from soil and identified using the VITEK® 2 system and 16S rRNA sequencing. An additional six strains were obtained from the American Type Culture Collection (ATCC). The investigated Gram-positive and Gram-negative bacterial strains successfully produced anisotropic AuNPs at a cell density of 2.0 McFarland (6.0 × 10⁸ CFU/mL). Nanoparticle formation was faster when samples were incubated at 37 °C than at 28 °C across all bacterial strains. The results of UV-vis spectroscopy confirmed the presence of AuNPs, with peaks observed centered at 550 nm. High-resolution transmission electron microscopy (HR-TEM) revealed a variety of morphologies, including spheres, rods, triangles, pentagons, hexagons, irregular shapes, and flower-like structures. Gram-positive and Gram-negative bacteria synthesized AuNPs of sizes 38.7 ± 26.0 and 34.0 ± 18.6 nm, respectively. Lattice-spacing analysis confirmed the formation of metallic AuNPs. Energy-dispersed X-ray spectroscopy (EDS) validated the presence of gold in the samples, and X-ray photoelectron spectroscopy (XPS) confirmed the elemental composition of AuNPs at 84.0 eV. These nanoparticles have potential applications in cancer therapy and diagnosis, antibacterial treatments, and drug delivery.

• The AuNPs were synthesized using various bacterial strains

• The gold precursor is aryldiazonium gold(III) salt

• Various anisotropic morphologies were obtained

Created in BioRender. Ahmady, I. (2025)

Limited research has explored the relationship between microbiota characteristics in both the oral cavity and the intestine, and the prevalence of obesity or overweight status in preschool-aged children. In this study, we collected saliva, oral mucosal plaque, supragingival dental plaque, and fecal samples from 47 preschool children aged 3 to 6 with obesity/overweight, along with 34 age-matched normal-weight controls. These samples were subjected to 16S rRNA gene sequencing for microbial analysis. We found that the overweight/obesity group exhibited a lower richness and diversity of fecal microbiota compared to the control group. Distinct niche-specific clustering was observed across different oral niches. In the overweight/obesity group, five genera showed significantly higher abundance, while six genera showed significantly lower levels in saliva samples. In mucosal samples, five genera exhibited increased abundance, whereas two genera had reduced levels in dental plaque samples. In fecal samples, two genera displayed significantly higher abundance, while eight genera showed lower levels. These distinct microbial taxa were also correlated with clinical parameters, including body mass index (BMI) and skin-fold thickness (SFT). Furthermore, correlation analysis showed that the oral bacteria were significantly negatively correlated with fecal bacteria, which was markedly attenuated in children with overweight/obesity. Overall, our study identifies distinct microbial signatures in the oral cavity and intestine of preschool children with overweight/obesity, compared to those with normal weight. These findings offer new insights into the potential role of microbial dysbiosis in the development of childhood obesity, providing a foundation for future strategies aimed at preventing and intervening in childhood obesity and overweight.

• Distinct niche-specific clustering was observed between different oral niches.

• Shifts of abundance observed in obese children were correlated with BMI and SFT.

• Negative correlations between oral and fecal genera were weaker in overweight/obese children.

Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/L_culture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/L_culture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/L_culture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics.

• Site-specific PGB integration enabled by genome sequencing of B. subtilis 007.

• Antibiotic-free and secretory PGB production with an optimized B. subtilis host.

• Increased PGB production reaching 9.5 µkat/L_culture.

Since plastics pose the greatest threat to humanity, it is essential to find an economic and sustainable solution to combat environmental pollution. In this study, the ability of polyhydroxyalkanoates (PHA) production by the halophilic bacterium Halovirbrio sp. HP20-59 in the presence of different carbon sources was examined. The strain showed a selective substrate preference, with the highest PHA production (reaching up to 73% of cell dry weight) in the presence of galactose, while fructose, arabinose, glycerol and xylose resulted in lower accumulation. Phylogenetic analysis based on the 16S rRNA gene sequence and whole-genome sequencing confirmed the HP20-59 strain as a novel species within the Oceanospirillales order. Draft genome showed a size of 4,165,370 bp with a GC content of 55.1% and a complete set of pha genes. The comparative analysis of the phaC gene identified a 638 amino acid-long class I poly(R)-hydroxyalkanoic acid synthase, showing 91% similarity to Halovibrio variabilis and 89% similarity to species within the Vreelandella genus, suggesting a possible horizontal gene transfer of the pha gene cluster. These findings highlight the unique genetic and metabolic characteristics of Halovibrio sp. HP20-59, making it a promising candidate for industrial PHA production and a valuable resource for research on sustainable biopolymers.

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a critical challenge in clinical settings because of its resistance to conventional antibiotics. This study investigated the antibacterial potential of silica-coated silver nanoparticles (SiO₂@AgNPs) against MDR P. aeruginosa and explored their synergistic interactions with selected antibiotics. A total of 450 pus samples were processed for bacterial isolation, and P. aeruginosa was identified using standard microbiological methods. MDR strains were confirmed using MIC-based VITEK antimicrobial susceptibility testing and RT-PCR for resistance genes. The antibacterial activity of the SiO₂@AgNPs was assessed using the microbroth dilution method. A checkerboard assay was conducted against MDR isolates to determine the synergy between SiO₂@AgNPs and ciprofloxacin, meropenem, and ceftazidime-avibactam. The synthesized nanoparticles were characterized using transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analysis. Of the 450 pus samples, 100 P. aeruginosa isolates were identified, of which 13 were classified as MDR P. aeruginosa. SiO₂@AgNPs exhibited effective antibacterial activity, with an MIC of 500 µg/mL against MDR P. aeruginosa. Checkerboard assays demonstrated strong synergy with meropenem and ceftazidime-avibactam (FICI = 0.375) and partial synergy with ciprofloxacin (FICI = 0.625–1.0625). TEM revealed spherical particles with an average size of 10 nm, FTIR confirmed SiO₂ functional groups, and XRD revealed crystalline silver nanoparticles within an amorphous silica matrix. These findings indicate that SiO₂@AgNPs possess potent antibacterial activity against MDR P. aeruginosa and can enhance the efficacy of certain antibiotics, highlighting their potential in combination therapy against resistant strains.

● Silica-coated silver nanoparticles effectively inhibited MDR P. aeruginosa.

● SiO₂@AgNPs enhance the efficacy of meropenem and ceftazidime-avibactam.

● Nanoparticle-antibiotic combinations may offer new strategies for treating resistant infections.

Nutritional supplements such as trace element-enriched yeasts are becoming increasingly popular to overcome the worldwide problem of zinc (Zn) deficiency. Unlike selenium-enriched yeast, which is already authorized in the European Union, Zn-enriched yeasts (ZnY) have not yet been approved for food purposes in the European Union, as their evaluation is still ongoing, demanding more comprehensive data regarding the Zn species present in ZnY. This study screens ten different industrial yeast strains regarding their Zn-enrichment quota, with further characterization of selected strains using spectroscopic and proteomic approaches. Microfermentation experiments on the industrial yeasts showed Zn levels spanning 0.06–51 pg/cell. Large-scale fermentation in bioreactors was carried out with two strains excelling in either biomass or Zn accumulation. A combination of inductively coupled plasma mass spectrometry (ICP-MS) and various spectroscopic methods confirmed the Zn enrichment, while suggesting that fractions of the Zn accumulated on the cell surface, with simultaneously high values of phosphorus being present. Speciation via X-ray absorption spectroscopy (XAS) analyses revealed that Zn species are transformed and Zn is coordinated to P-O-ligands and to amino acid ligands in both strains. Proteomic analysis showed that ZnY cells moved from a Zap1-governed Zn balance to an intracellular excess response, implying cellular Zn uptake. This study demonstrates that, in a Zn-excess medium, industrial yeast strains exhibit variability in Zn-accumulation capacity, cellular Zn-localization, and regulatory responses involving the expression of Zn-binding proteins. The presented findings contribute to optimizing industrial fermentation processes for producing Zn-rich yeast biomass and enhance the understanding of Zn regulation in yeast, aiding in the approval of Zn-enriched yeasts for supplements and novel food applications.

• Zn enrichment in yeasts is strongly time and strain dependent

• Zn proteome changes under Zn excess suggest that Zn is partly internalized in the yeast cells

• Beside proteins, phosphorous compounds seem to be Zn-binding ligands in Zn-enriched yeast