Glutamate waste liquid is the waste produced in the production process of glutamic acid, with low pH, high ammonium, and high sulfate. The waste liquid contains glutamic acid and can be used as a raw material to produce poly-γ-glutamic acid (γ-PGA), achieving the recycling of waste liquid. [Objective] To investigate the inhibitory effect of glutamate waste liquid on γ-PGA synthesis, we used Bacillus subtilis KH2 to synthesize γ-PGA and evaluated the inhibitory effect of glutamate waste liquid on the synthesis of γ-PGA. [Methods] Comparative transcriptomics was employed to excavate the key genes and inhibitory factors involved in γ-PGA synthesis, and key gene overexpression and knockout were conducted to identify the inhibitory factors. Fermentation experiments were then performed for verification. [Results] The glutamate waste liquid as the substrate for production of γ-PGA by fermentation showed significant inhibitory effects. A total of 1 819 significantly differentially expressed genes were identified, including 952 genes with significantly up-regulated expression and 867 genes with significantly down-regulated expression. The transcript levels of 10 genes (alsS, pgsA, gltT, budA, fumC, ptsG, racE, opuAB, acoC, and rocG) involved in γ-PGA synthesis of B. subtilis KH2 changed significantly during primary fermentation and glutamate waste liquid fermentation. Eight down-regulated genes (alsS, pgsA, gltT, budA, fumC, ptsG, racE, and opuAB) were overexpressed, which increased the production of γ-PGA by 91.20%, 120.77%, 137.50%, 36.44%, 40.85%, 104.58%, 65.67%, and 69.72%, respectively. The overexpression of pgsA, gltT, ptsG, racE, and opuAB increased glutamic acid utilization by 11.57%, 35.53%, 12.83%, 21.43%, and 14.80%, respectively. The overexpression of alsS, budA, and fumC had no obvious improving effect on the utilization of glutamic acid. The knockout of two up-regulated genes (acoC and rocG) had little effect on γ-PGA production and glutamic acid utilization. [Conclusion] The downregulation of ptsG, gltT, racE, pgsA, and fumC in waste liquid fermentation has significant effects on substrate utilization, glutamic acid configuration conversion and polymerization, and TCA cycle, which reduces the synthesis efficiency of γ-PGA. This study reveals the inhibitory mechanism of glutamate waste liquid in γ-PGA synthesis and provides a sustainable biotechnology for the production of value-added biopolymers from industrial waste liquid.

[Objective] To investigate the in vitro uric acid degradation performance and physiological and biochemical characteristics of Limosilactobacillus fermentum H3260 isolated from human feces and examine the effects of this strain on the serum uric acid level and gut microbiota in the mouse model of hyperuricemia, providing scientific evidence for the development of functional food for the prevention and treatment of hyperuricemia. [Methods] HPLC and the uric acid production assay were employed to determine the abilities of the target strain to degrade uric acid, adenosine, and nucleosides and to inhibit xanthine oxidase. The probiotic characteristics of the strain were evaluated by antimicrobial sensitivity tests and in vitro tolerance tests. The uric acid-lowering effect of L. fermentum H3260 was verified by in vivo experiments. [Results] L. fermentum H3260 was screened out. The strain exhibited degradation rates of (86.84±0.03)% for uric acid, (60.84±2.21)% for adenine, and (100.00±0.00)% for nucleosides, along with an inhibition rate of 22.48% for xanthine oxidase. The strain was sensitive to seven common antibiotics, including erythromycin, ceftriaxone, penicillin G, and chloramphenicol. After treatment in 0.3% bile salt for 2.5 h, the bacterial count remained above 1.00×10⁶ CFU/mL. Animal experiments showed that the strain significantly reduced uric acid, creatinine, and blood urea nitrogen in hyperuricemic mice and regulate the gut microbiota to alleviate hyperuricemia. [Conclusion] We successfully screened out a strain L. fermentum H3260 capable of efficiently degrading uric acid, adenosine, and nucleosides. The strain exhibited good physiological and biochemical characteristics in vitro and significantly improved hyperuricemia-related indicators and regulated the gut microbiota in vivo, showing potential as an elite strain for the prevention and treatment of hyperuricemia.

The puparium of Hermetia illucens is rich in chitin and protein, while efficient and environmentally friendly utilization methods remain to be developed. [Objective] To isolate chitinolytic bacteria from the puparium pile of H. illucens and explore their potential in puparium biotransformation. [Methods] Strains were isolated by the plate screening method and identified by 16S rRNA gene sequencing. The 3,5-dinitrosalicylic acid (DNS) method was employed to determine the chitinase activity. Whole genome sequencing by PacBio HiFi was conducted to elucidate the degradation mechanism. The application potential of the strain was explored by puparium fermentation experiments. [Results] Among the seven isolated strains, Bacillus cereus BSF-CH1 showed the highest chitinase activity, reaching a maximum chitinase activity of 0.48 U/mL on the second day of fermentation. The genome of BSF-CH1 contained three chitinase genes, seven chitin deacetylase genes, and four chitodextrinase genes. Puparium biotransformation experiments showed that BSF-CH1 could degrade 47.2% of puparium mass within 7 days, with degradation rates of 64.6% and 59.1% for chitin and protein, respectively. [Conclusion] This study reports an efficient chitinolytic bacterium isolated from the puparium of H. illucens, providing new insights into the puparium biotransformation and having important implications for promoting the sustainable development of the H. illucens industry.

[Objective] To design and express a recombinant protein rMKIBV incorporating confirmed antigenic epitopes of infectious bronchitis virus (IBV) as a vaccine to provide comprehensive protection. Additionally, it explores the potential of polyclonal yolk antibodies (IgY) harvested from laying hens immunized with the rMKIBV vaccine in the prevention and control of IBV. [Methods] The antigenic epitope sequences of IBV, obtained from online databases, were compared with sequences of representative IBV strains from GenBank. Flexible peptides were designed to link all antigenic peptides. The constructed amino acid sequence was analyzed, reverse-translated, codon-optimized, and then inserted into the pET-28a(+) cloning vector. The recombinant vector was introduced into Escherichia coli for expression. The purified, desalted, and endotoxin-removed rMKIBV protein was used as a vaccine to immunize animals for investigation of its immunogenicity and ability to stimulate specific IgY production in laying hens. [Results] The retrieved IBV antigenic epitope sequences showed high similarity with the published N and S protein sequences of 22 representative IBV strains. The predicted isoelectric point and molecular weight of rMKIBV were 10.25 and 63.39 kDa, respectively. The secondary structure of rMKIBV included a high proportion of random coils, which suggested strong antigenicity. High-purity rMKIBV was obtained from E. coli transformed with the recombinant plasmid pET-28a-mkibv. This protein specifically bound to anti-His-tag antibodies, N protein antibodies, and S protein antibodies. The mice immunized with this protein showed increases in the spleen index (P<0.05), elevations in the levels of serum-specific IgG antibodies (P<0.01) and IFN-γ (P<0.05), and no significant change in the IL-2 level. Immunized laying hens successfully produced IgY in egg yolks, with specific IgY antibody levels significantly increasing. Moreover, the IgY antibody titer gradually rose after immunization, reaching the peak after about 50 days and then gradually declining to reach a stable level. [Conclusion] We successfully constructed and expressed the recombinant protein rMKIBV. The protein demonstrated good immunogenicity, stimulating specific antibody production in both mice and laying hens. Notably, the IgY extracted from the yolks of immunized laying hens offers a novel approach to IBV prevention and control. These findings hold significant scientific and practical value for the development of vaccines against IBV.

Root-knot nematodes(Meloidogyne spp.) are widely distributed and highly destructive, causing substantial economic losses in agricultural production. Biocontrol has been considered as an effective measure for managing these pathogens. [Objective] To explore efficient and eco-friendly biocontrol resources for controlling root-knot nematodes. [Methods] Bacillus strains were isolated from soil via the serial dilution method. Strains with strong nematicidal activity against Meloidogyne incognita second-stage juveniles (J2) were screened by in vitro bioassays. The selected strains were identified based on morphological, physiological, and biochemical characteristics, as well as molecular biological evidence. The biocontrol potential of these strains was further evaluated by egg hatching inhibition assays, phosphorus/potassium solubilization tests, enzymatic activity profiling, and antagonistic spectrum analysis. Additionally, pot and greenhouse experiments were conducted to validate the biocontrol efficacy of strains. [Results] Among 189 bacterial strains isolated from 16 soil samples, strains Sneb2550 and Sneb2556 demonstrated strong nematicidal activity against M. incognita J2, inducing the corrected mortality rates of 95.64% and 95.36%, respectively, after 24 h. Based on morphological features, physiological and biochemical characteristics, and 16S rRNA gene and gyrB sequences, strains Sneb2550 and Sneb2556 were identified as Bacillus proteolyticus and B. amyloliquefaciens, respectively. Functional characterization showed that strain Sneb2550 produced protease and inhibited Fusarium asiaticum, Trichothecium roseum,and Alternaria solani. Strain Sneb2556 produced protease and amylase, solubilized phosphate, and suppressed F. asiaticum, Aternaria alternata, Botryosphaeria dothidea, T. roseum, Colletotrichum gloeosporioides, and A. solani. Moreover, both strains did not adversely affect cucumber seed germination and significantly promoted the radicle growth. Under pot conditions, Bacillus Sneb2550 and Sneb2556 significantly reduced root galls formation, with the reduction rates of 56.02% and 50.19%, respectively, while promoting plant growth. Field experiments showed that root irrigation with Bacillus Sneb2550 and Sneb2556 effectively controlled cucumber root-knot nematodes, with the control effects of 60.90% and 52.63%, respectively, while promoting plant growth. [Conclusion] Bacillus Sneb2550 and Sneb2556 effectively controlled cucumber root-knot nematodes and promoted plant growth, providing new potential resources for the biocontrol of root-knot nematodes.

[Objective] Adipic acid is a key monomer for plastics such as nylon 66 and poly (butylene adipate-co-terephthalate) (PBAT), with a vast market potential. This study aims to explore the optimal expression levels of genes in the biosynthetic pathway of adipic acid. [Methods] We regulated the expression levels of genes in the adipic acid synthesis pathway by randomly combining gradient-strength constitutive promoters. The high-throughput screening based on an adipic acid biosensor was conducted to select the strain with the optimal combination. Subsequently, the fermentation media, carbon sources, metal ions, and precursor substance addition amounts were optimized. [Results] After screening, the optimal strain Escherichia coli MG1655 Δ8-D47 was obtained, with an adipic acid yield of 431.32 mg/L. After fermentation condition optimization, the yield of adipic acid in a shake flask reached 550.34 mg/L, which represented a 134% increase compared with that of the control strain Z1. [Conclusion] Metabolic pathway imbalance in microbial synthesis of adipic acid is the main factor limiting the increase in yield.

Streptococcus suis is a major pathogen in pigs and also a zoonotic pathogen. This bacterium has numerous serotypes, among which S. suis serotype 4 (SS4) is known to infect humans and poses a threat to public health due to its potential high pathogenicity. [Objective] To develop a multiplex PCR method based on specific virulence-associated genes of SS4 virulent strains to achieve precise identification of such strains. [Methods] Based on previous research results, four target genes specific to SS4 virulent strains—sly, igdE, tran, and sao—were selected, and the SS4 serotype-specific gene wzy was used as an internal reference gene to design a pentaplex PCR method. After optimization of the multiplex PCR amplification system, specificity and sensitivity tests were conducted. This method was then employed to detect newly isolated SS4 strains. Additionally, zebrafish virulence assays were performed to validate the accuracy of this method. [Results] The multiplex PCR method specifically amplified the target genes, effectively distinguishing SS4 virulent strains from lowly virulent strains. The method exhibited high sensitivity, with a minimum detection limit of 4.1×10² CFU or 12.5 pg of genomic DNA. Specificity validation confirmed that this method accurately identified SS4 virulent strains. This method was then employed to examine six clinically isolated SS4 strains. Three strains were identified as virulent, showing high pathogenicity in zebrafish and causing a mortality rate of 60.00%-86.67%. The other three strains were identified as lowly virulent strains, exhibiting low pathogenicity in zebrafish and causing a mortality rate of 0-6.67%. [Conclusion] A multiplex PCR method based on virulence-associated genes of S. suis was successfully developed, enabling accurate and sensitive identification of SS4 virulent strains. This method provides technical support for the early diagnosis and effective prevention and control of S. suis infections.

[Objective] To investigate the structural characteristics of microbial consortia in different concentrations of petroleum hydrocarbons, cultivate efficient petroleum hydrocarbon-degrading microbial consortia, and mine the strain resources capable of degrading petroleum hydrocarbons. [Methods] We used 0# diesel as the sole carbon source to domesticate oil-contaminated soil samples through five successive generations by gradually increasing the 0# diesel concentration. The structural changes of microbial consortia were uncovered by 16S rRNA gene amplicon sequencing. The strains with petroleum hydrocarbon-degrading potential were isolated and purified via dilution plating and streaking. Finally, the improved 2,6-dichlorophenol indophenol (DCPIP) cultivation system was employed to identify efficient degrading strains. [Results] During domestication, when the concentration of 0# diesel was raised to 7 000 mg/L, the relative abundance of petroleum hydrocarbon-degrading bacteria including Bacteroidota and Bacillota significantly increased. A total of 58 bacterial strains belonging to 25 genera, 22 families of 4 phyla were isolated, including 31 (53.45%) strains of Pseudomonadota, 13 (22.41%) strains of Actinomycetota, 11 (18.97%) strains of Bacillota, and 3 (5.17%) strains of Bacteroidota. From the isolated strains, 18 petroleum hydrocarbon-degrading strains were screened out. [Conclusion] Through gradient domestication, seven natural microbial consortia were successfully enriched, achieving over 70% degradation of petroleum hydrocarbons at 7 000 mg/L of 0# diesel. Amplicon sequencing revealed that varying 0# diesel concentrations altered the microbial consortium structure. Additionally, 18 strains capable of using 0# diesel as the sole carbon source were identified, providing potential microbial resources for the bioremediation of oil-contaminated soil.

Viruses are the most abundant biological entities on Earth and widely exist in various ecosystems. They play a role in regulating microbial community composition, influencing microbial evolution, participating in element biogeochemical cycles, and even causing plant and animal diseases. Soil is an important reservoir of viruses. Due to the limitations of soil heterogeneity, adsorption of soil colloids, and lack of research and analysis methods, the research on soil viruses lags far behind that on viruses in marine or other aquatic ecosystems. In recent years, the rapid development of molecular biology and the gradual improvement of ecological theory have promoted the research on soil viruses, researchers gradually paid attention to the function of viruses in soil ecosystem. In this paper, we summarized the research status and progress of soil viruses in terms of the types, research methods, and ecological functions. On this basis, the future development trends and key research directions of soil viruses were prospected in this paper. This review can deepen people’s understanding of soil viruses and provide scientific reference for the research on soil viruses.

Antimicrobial susceptibility testing (AST) is of great significance for the detection of antibiotic residues, as well as the prevention and treatment of bacterial diseases. Conventional methods of AST are time-consuming and labor-intensive due to the processes of bacterial culture and colony counting. ATP bioluminescence has been widely employed to assess bacterial contamination in food and healthcare and appears to be a rapid and sensitive alternative to conventional methods of AST. This review illustrates the influencing factors of ATP bioluminescence-based AST in detail. It mainly focuses on how these factors influence the detection performance of the method, giving insights into AST in clinical, environmental, and food analyses.