Applied Microbiology and Biotechnology最新文献

Recent studies on yeast-insect associations demonstrated that social wasps of the genera Polistes and Vespula act as a reservoir for the conservation of yeasts and as vectors capable of transferring such yeasts on the grapes. This work aimed to assess yeast species associated with social wasps and to obtain new strains to be used as bio-protection agents in winemaking. The wine yeast communities present on the exoskeleton, intestine of social wasps, and on the surface of grapes sampled in the vineyards of three Tuscan wineries were determined. Regardless of the wasp species, yeasts were mostly associated with female workers and found mainly in their intestine (up to 7 × 10⁵ CFU/mL). The identification revealed 20 species belonging to 10 genera; Aureobasidium pullulans and Metschnikowia pulcherrima were isolated from wasps of all the wineries, the latter occurring at the highest frequency. Strain-level characterization highlighted that three strains present on grapes were also present in the gut of wasps from the same vineyard. All the isolated M. pulcherrima strains underwent in vitro tests to select the most suitable for use as bio-protective cultures. Three strains showed good inhibitory activity against Kloeckera apiculata and Brettanomyces bruxellensis; hence, they were selected for bio-protection trials on artificially contaminated grapes. Results highlighted the reduction of non-Saccharomyces populations, suggesting the effectiveness of M. pulcherrima as a biocontrol agent. The study confirmed the role of social wasps as yeast vectors in the vineyard and as a reservoir of yeast strains to be exploited for biotechnological applications in oenology.

• Social wasps can carry yeast strains that are also present on the surface of grapes

• Metschnikowia pulcherrima was found within the wasp gut as the prevalent yeast species

• M. pulcherrima proved to be a suitable biocontrol agent on grapes

Extremophilic environments are rich reservoirs for discovering microorganisms with vast biotechnological potential. Among these, microalgae stand out for their pivotal role in sustainable wastewater treatment and nutrient recycling. This study introduces Coelastrella thermophile D14, a microalga isolated from a solar panel, identified through morphological studies and genomic sequencing. The genus Coelastrella has been characterized and classified as highly productive strains valuable for biofuel and bioproduct generation as well as for their ability to produce significant amounts of carotenoids. Experiments revealed the extraordinary resilience of this strain to prolonged desiccation and high-strength piggery wastewater. Notably, D14 cultivated in 10% pig effluent exhibited biostimulant properties, achieving a germination index 23% higher than the control on Lepidium sativum. In a groundbreaking development, we have successfully established an Agrobacterium-mediated transformation protocol for C. thermophila D14, optimizing key parameters for effective T-DNA transfer. This marks a pioneering achievement within the genus Coelastrella. These findings highlight the significant potential of D14 as a robust platform for future biotechnological applications, opening new opportunities for innovative solutions, especially in environmental protection and sustainable agriculture.

• First microalga from solar panel biofilm: Coelastrella sp. D14 isolated and characterized.

• Strain D14 tolerates prolonged desiccation and grows well in piggery wastewater.

• Stable Agrobacterium-mediated transformation enables future metabolic engineering.

Coenzyme Q10 biosynthesis in Escherichia coli is constrained by kinetic mismatches between precursor synthesis and methylation, alongside bioenergetic uncoupling. We implemented an optogenetic phase-control strategy integrating dynamic light induction, ribosome binding site (RBS) engineering, and real-time membrane potential (ΔΨ) feedback. Temporal coordination of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and UbiG methyltransferase (UbiG) via a 6-h phase delay reduced methylglyoxal shunt flux by 41 ± 3% (p < 0.01) through enhanced precursor channeling. Membrane hyperpolarization to − 90 ± 2 mV (relative to − 70 mV in controls) triggered voltage-gated UbiG membrane localization (62 ± 3%) and ATP-driven S-adenosylmethionine regeneration, increasing methylation efficiency 2.3-fold. Multivariate modeling identified ΔΨ and acetate as critical control parameters, enabling optimized fermentation (dissolved oxygen (DO) 15–20%, pH 6.7–6.9). The engineered strain achieved 0.63 ± 0.07 g/L CoQ10 in 5-L bioreactors—a 4.3-fold improvement over the static control strain (0.15 ± 0.02 g/L)—with 82.5% carbon efficiency and 25.8% glycerol-to-product yield. This work establishes bioenergetically coupled temporal control as a scalable paradigm for membrane-bound isoprenoid biomanufacturing.

• Phase-driven enzyme synchronization via optogenetics resolves kinetic mismatch.

• Membrane hyperpolarization gates enzyme localization and ATP regeneration.

• Model-integrated bioenergetic-process control enhances CoQ10 production efficiency.

Kluyveromyces marxianus has high biotechnological potential, particularly because it can utilize a wide range of substrates and grow faster than other yeasts. It has also attracted interest as a prospective probiotic; however, this application is still not well researched. Two K. marxianus strains with promising biotechnological properties and the proven ability to efficiently produce 2-phenylethanol (2-PE) and ethanol were examined in this study to evaluate their potential as probiotics. Yeasts WUT216 and WUT240 exhibited similar survival rates in a simulated human digestive system as the reference K. marxianus strain B0399 (DiarYeast®, LongLife, Milano, Italia). Additionally, the WUT yeasts revealed a high degree of adhesion to cancer and normal cell lines in vitro. Importantly, the WUT strains did not exhibit undesirable characteristics, such as mucolytic and hemolytic activities, and toxic effects on the Caco-2 cell lines in vitro and on Galleria mellonella larvae in vivo. Furthermore, K. marxianus strains displayed attractive probiotic properties, particularly high antioxidant potential, antifungal activity, and significant β-glucan content. This study also showed that the WUT yeasts are sensitive to the commercial antifungals, fluconazole, amphotericin B, and geneticin G418. Overall, the WUT240 and WUT216 exhibit similar probiotic properties to those of B0399. Thus, the WUT yeasts are not only promising producers of 2-PE and ethanol but also attractive probiotic candidates. Additionally, discrepancies in literature reports regarding the analyses conducted were noted, particularly in adhesion and autoaggregation tests.

• K. marxianus strains combine probiotic with its biotechnological potential

• WUT yeasts may positively affect the host (antioxidants, β-glucan, antimicrobials)

• Methodological differences hinder the comparison of potential probiotics

Antinutritional factors present in complete feeds markedly diminish digestive and absorptive efficiency in animals, thereby affecting growth performance and resulting in economic losses. Liquid fermentation technology has been demonstrated to be an effective method of reducing antinutritional factors and enhancing the nutritional value of complete feeds. However, there is a lack of systematic research on the liquid fermentation and the screening of bacterial strains for use. In the present study, Bacillus subtilis and Lactobacillus plantarum characterized by excellent low-temperature tolerance, great enzyme activity, strong bacteriostatic capacity, and exceptional acid production, were evaluated for their suitability in a two-stage (aerobic followed by anaerobic) liquid fermentation process of complete pig feed. The results demonstrated that soybean antigenic protein and crude fiber underwent significant degradation, while crude protein and acid-soluble protein content exhibited significant increases in the feed following two-stage fermentation. Additionally, the accumulation of biogenic amines was inhibited to ensure the palatability of the feed. Furthermore, two-stage fermentation significantly enhanced the antioxidant and enzymatic activity of the feed. High-throughput 16S rRNA sequencing revealed an increased relative abundance of beneficial bacteria and a decreased abundance of pathogenic bacteria after fermentation. This study corroborated that a two-stage fermentation process could enhance the nutritional value, safety, and probiotic functionality of animal feeds. This finding provides a theoretical foundation for the development of functional fermented feeds and provides the necessary technical support for the practical application of liquid fermentation feeds. KEY POINTS: • Developed a novel low-temperature two-stage liquid fermentation feed strategy using Bacillus subtilis 3-16 and Lactobacillus plantarum E5 • Significantly degraded antinutritional factors and biogenic amines, while increasing the crude protein, acid-soluble protein, enzymatic activity, and antioxidant capacity of complete pig feed • Promoted beneficial microbiota dominance (e.g., Lactobacillus plantarum and Bacillus subtilis, while reducing pathogenic bacteria (e.g., Escherichia coli and Staphylococcus aureus).

The study evaluated the antimicrobial efficacy of plasma-activated water (PAW), generated using a plasma bubble reactor, against Escherichia coli O157:H7 and Staphylococcus aureus in both planktonic and biofilm states. The physicochemical properties of PAW, including pH, electrical conductivity, and reactive oxygen and nitrogen species concentrations, were analysed immediately after production and after 24 and 48 h of storage at 4 °C. Additionally, the impact of organic load on PAW's antibacterial activity and its cytotoxic effects on human keratinocytes were investigated. To assess its stability, PAW’s antimicrobial activity after storage was also evaluated in the presence and absence of organic matter. PAW treatment resulted in a significant reduction in bacterial counts, achieving inactivation below the detection limit (1 log CFU/mL) within 20 min for both planktonic and biofilm states. However, the presence of organic matter significantly impaired PAW’s antibacterial efficacy, with higher organic loads leading to significantly diminished bacterial inactivation. PAW stored for 24 h maintained strong antimicrobial activity, which declined after 48 h; the presence of organic matter further reduced its efficacy across all time points. Importantly, PAW’s exposure did not induce cytotoxic effects on human keratinocytes at treatment durations of up to 30 min. These findings highlight the potential of PAW as a sustainable disinfection strategy, demonstrating robust antimicrobial activity against Gram-negative and Gram-positive foodborne pathogens while maintaining biocompatibility. Further research is required to enhance PAW’s efficacy in complex environments with organic contamination to enhance its practical applications in agri-food settings.

• PAW effectively inactivated E. coli and S. aureus in planktonic and biofilm states.

• PAW antimicrobial activity is reduced in the presence of organic matter.

• PAW showed minimal cytotoxic effects on human keratinocytes.

We investigated the mechanism underlying the coexistence of the phenol-degrading bacteria Cupriavidus sp. strain P-10 and Comamonas thiooxydans strain R2 using a chemostat culture supplemented with phenol as the sole carbon and energy source. Quantitative PCR (qPCR) targeting the genes encoding the large subunit of phenol hydroxylase dmpN and aphN for strains P-10 and R2, respectively, revealed the coexistence of the two strains even under phenol-limiting conditions. According to the reverse transcription qPCR targeting dmpN, aphN, catechol 2,3-dioxygenase genes (dmpB and aphB), and catechol 1,2-dioxygenase gene (catA) of strain P-10, both strains transcribed these genes, suggesting that phenol was metabolized individually. However, mathematical simulations demonstrated that the two strains never coexisted because competitive exclusion occurred at any phenol sharing ratio. The simulation revealed that strain P-10 outcompeted strain R2 when it occupied more than 64% of the phenol. These results suggest that phenol sharing, with fluctuations in the ratio within an appropriate range, is indispensable for coexistence under resource-limiting conditions. The phenol concentration in the chemostat remained spatially heterogeneous for 15 s. If coexistence was accomplished through the phenol-sharing, either strain P-10 or R2 could invade another single-strain chemostat culture using phenol, even when inoculated at a significantly lower cell density than the resident strain. Strains P-10 and R2 successfully invaded and coexisted with other strains in the chemostat culture. These results suggest that fluctuation of the limiting substrate sharing ratio enabled stable coexistence of bacterial strains under competitive conditions.

• Phenol concentration was spatiotemporally heterogeneous in the chemostat culture

Virus-like particles (VLPs), which closely mimic the structural and antigenic properties of native foot-and-mouth disease (FMD) virions while lacking infectious genetic material, provide a more economical solution by replacing inactivated viruses in liquid-phase blocking ELISA (LPBE) testing for FMD serodiagnosis and vaccine evaluation. In this study, a novel LPBE was developed by replacing inactivated antigens with serotype O foot-and-mouth disease virus (FMDV) VLPs expressed in Escherichia coli. Based on evaluation of sensitivity and specificity, its correlation with virus neutralization test (VNT) results in immunized animal sera, as well as the feasibility and broad applicability for clinical serum sample detection, was further analyzed. Validation showed that VLPs-LPBE achieved high specificity (98.75%) and sensitivity (97.02%). Notably, in quantifying VLP-induced antibodies, the correlation with VNT results (R² = 0.6809, P < 0.0001) was significantly stronger than that of commercial-LPBE. Blind evaluation of 853 clinical serum samples from vaccinated subjects demonstrated excellent agreement between VLPs-LPBE and conventional LPBE, with a kappa coefficient (κ) of 0.896, an intraclass correlation coefficient (ICC) of 0.904, and the concordance rate of 95.8%. These results confirm the clinical reliability and applicability for serological monitoring. In conclusion, VLPs-LPBE provides a safe, economical, and reliable alternative method for the evaluation of FMD vaccines. It eliminates the biosafety risks associated with the handling of inactivated viruses while maintaining compatibility with existing monitoring workflows.

• VLPs-based ELISA safely replaces inactivated virus, reducing costs while maintaining diagnostic accuracy.

• Stronger correlation with neutralization tests enhances VLPs-vaccine evaluation reliability vs. commercial kits.

• High clinical concordance validates workflow compatibility for large-scale serological monitoring.