Abstract:Anaerobic digestion (AD) of biomass waste combined with waste activated sludge (WAS),which is characterized by great stability,low energy consumption,and biogas production,can effectively reduce the volume and improve the dehydration of WAS and thus has been widely applied in methane production.However,the AD process is susceptible to external factors such as microplastics (MPs) or nanoplastics (NPs),which can lead to reduced efficiency or even collapse of AD.The AD system needs the interdependence and interaction of the microbial community to keep stable operation in a dynamic equilibrium state,in which phages play a key role.Phages can not only regulate the structure of the microbial community in the sludge and direct the energy flow but also attach to MPs and NPs with bacteria and archaea for transmission.Nevertheless,the effects of MPs and NPs on such a process were underestimated in previous studies.In this paper,we summarize the research progress in the effects of different types and sizes of MPs and NPs on AD systems,with focus on the ecological connections among microbial communities,especially bacteria,archaea,and phages,in anaerobic systems.Furthermore,we put forward novel viewpoints about the effects of MPs and NPs on microbial communities and make an outlook on the future research directions in this field.

Abstract:Pseudorabies virus (PRV) is a member of the genus Varicellovirus in the Herpesviridae family.It primarily causes pseudorabies characterized by reproductive failure in sows,and neurological and respiratory symptoms in piglets,posing a significant threat to pig production.Vaccination is the most important measure to prevent PRV in pigs.However,due to variations of the virus and its latent infection characteristics,the effectiveness of traditional vaccines is compromised.Consequently,there is an urgent need for new drug preparations to assist vaccine immunization.It has been found that natural plant polysaccharides and small molecules such as flavonoids,phenols,and acids can inhibit PRV infection either by directly blocking the viral infection process or by regulating the immune response.In addition,host antiviral protein type I interferon and its downstream interferon-stimulated genes have significant inhibitory effects on PRV infection.Host defense peptides,including antimicrobial peptides and defensins,also show good inhibitory effects on PRV infection.Interestingly,researchers have recently found that extracts and metabolites from bacteria and fungi also exhibit anti-PRV effects,and it is expected that these bacteria and fungi and their products could be applied for the prevention and treatment of viral diseases in the future.This study focused on the recent research progress of natural bioactive molecules against PRV infection,aiming to provide important references for the research and development of anti-PRV infection drugs.

Abstract:Parkinson's disease is a common neurodegenerative disorder that seriously threatens the health of middle-aged and elderly individuals.However,the pathogenesis of Parkinson's disease is not fully understood.Recent studies have shown that gut microbiota plays an important role in the occurrence and development of Parkinson's disease.Gut microbiota and its metabolites influence the intestinal mucosal barrier,neuroinflammation,endocrine system,and other aspects through the microbiota-gut-brain axis,thereby participating in the occurrence and development of Parkinson's disease.Gut microbiota can be regulated by various methods such as probiotic supplementation,fecal microbiota transplantation,dietary adjustments,and traditional Chinese medicine interventions,being an important target for the prevention and treatment of Parkinson's disease.This article reviews the possible mechanisms of gut microbiota being involved in the occurrence of Parkinson's disease and further discusses the current status of prevention and treatment of gut microbiota dysbiosis.

Abstract:In recent years,antimicrobial peptides (AMPs) are considered alternatives to antibiotics and have received increasing attention.AMPs have a broad antibacterial spectrum and extensive sources and are not prone to drug resistance.At present,AMPs are mainly produced with three methods:extraction from natural sources,chemical synthesis,and microbial expression via genetic engineering.The application of the former two methods is limited due to their complicated processes,high costs,and low yields.Microbial expression via genetic engineering is more economical,scientific,and effective than the above two methods.This article introduces and compares the various expression systems and summarizes the strategies for increasing the heterologous expression levels,with a view to providing theoretical support for large-scale production of AMPs with low costs.

Abstract:Pullulan is an exopolysaccharide produced by Aureobasidium spp.Despite its widespread biotechnological applications,the mechanisms underlying the biosynthesis and regulation of pullulan remain to be studied.In recent years,researchers have employed molecular biological techniques to elucidate the molecular mechanisms of pullulan synthesis and regulation.The transmembrane protein AmAgs2 is identified as a key enzyme for the synthesis of pullulan,and the cAMP-protein kinase A (cAMP-PKA),target of rapamycin 1(TORC1),high osmotic glycerol 1(HOG1),and sucrose nonfermentable 1(Snf1) signaling pathways are involved in the regulation of pullulan synthesis.We reviewed the research progress in this field,aiming to give insights into the research on the synthesis and regulation mechanisms of fungal extracellular polysaccharide and provide theoretical support for building cell factories with high yields of pullulan.

Abstract:Innate immunity constitutes the first line of immune defense.As a part of the body?'s innate immunity,the antimicrobial peptide LL-37 plays a crucial role in maintaining homeostasis,with two primary functions:direct antimicrobial activity and immunomodulation.Currently,research on LL-37 mainly focuses on its function,while its induced expression in vivo has rarely been studied.However,understanding the induced expression of LL-37 is essential for delving into the body?'s immune defense mechanisms.Therefore,this paper briefly reviewed the research progress on the synthesis and functions of LL-37,the factors inducing its secretion,and the regulatory pathways involved in its induced expression.The aim is to offer new insights into the regulatory pathways and immune defense functions of LL-37 in the human body.

Abstract:The pneumococcal vaccines play an indispensable role in defending against various invasive and non-invasive diseases caused by Streptococcus pneumoniae.The capsular polysaccharide is a key antigen,while the impurity residues in the capsular polysaccharide cause side effects of pneumococcal polysaccharide vaccines and conjugate vaccines.As the global demand for high-valent pneumococcal vaccines covering a wide range of serotypes keeps increasing,it is urgent and crucial to develop efficient and scalable production technologies for high-purity capsular polysaccharide antigens.The safety and quality attributes of capsular polysaccharide are influenced by multiple factors encompassing the selection of pathogen strains,formulation of culture media,control of fermentation processes,and purification technologies.Following the concept of Quality by Design (QbD) and the process development path,this article systematically summarizes the knowledge and experience throughout the entire process ranging from upstream strain screening and optimization,refined fermentation process design,to downstream purification,aiming to provide comprehensive and in-depth theoretical guidance and practical strategies for building and improving the production system of capsular polysaccharide.

Abstract:Bacterial biofilms are aggregates surrounded by extracellular polymeric substances produced by bacteria,exhibiting significant resistance to antimicrobials and host immune defense mechanisms.As a result,they become a crucial factor in the recalcitrance of bacterial infections.Phages,as a class of viruses capable of specifically infecting and lysing bacteria,have demonstrated immense potential in the prevention and treatment of biofilm-associated infections.This review summarizes the efficacy,in vitro and in vivo research methods,and mechanisms of phages,phage endolysins,and phage-antibiotic combinations in combating biofilm-associated infections.Furthermore,it discusses the prospects and potential obstacles of phages in this field,aiming to give insights into the development of more effective therapeutic agents.

Abstract:Sugarcane smut caused by Sporisorium scitamineum is one of the major diseases affecting the development of China’s sugar industry,and biocontrol is currently the most efficient and safe means,which necessitates the screening of antagonists with strong environmental adaptability and inhibitory effects.[Objective] To isolate and identify the strains with good antagonistic effects against S.scitamineum from soil and provide high-quality biocontrol strain resources for the efficient prevention and control of sugarcane smut.[Methods]The antagonists were isolated by the plate confrontation assay,and their taxonomic status was determined by morphological observation,physiological and biochemical tests,and 16S rRNA gene sequencing.The pot and field experiments were conducted to study the inhibition effects of the antagonists on sugarcane smut.[Results] Three strains of bacteria with significant antagonistic effects were obtained.Strains GB-3 and GH16-3 were identified as Bacillus velezensis and GH16-8 as B.amyloliquefaciens,with the inhibition zone diameters of (30.00±1.07),(44.00±1.21),and (18.00±0.89) mm and the inhibition rates of 16.12%,31.92%,and 5.91%,respectively.The inhibition effects of the three strains against sugarcane smut were 74.33%,76.57%,and 69.07% in pots and 20.08%,55.59%,and 50.08% in fields,respectively.All the three strains had phosphorus-solubilizing ability,tolerance to extreme salt-alkaline environments,and inhibitory effects on a variety of phytopathogenic bacteria and fungi,with the indole-3-acetic acid (IAA) yields of 2.12,1.30,and 1.22 mg/L,respectively.The application of the three strains increased the sugarcane plant height by 28.25%,17.09%,and 23.31%,respectively.[Conclusion] Strain GH16-3 has strong environmental adaptability,growth-promoting effect,and prevention effect against sugarcane smut,demonstrating a promising application prospect.

Abstract:[Objective] Long-term and excessive application of chemical fertilizers leads to soil degradation and an imbalanced microbial community structure in soil.The combination of organic active substances with chemical fertilizers is considered an important approach for controlling soil degradation and maintaining microbial community stability.[Methods] Metagenomics was employed to study the effects of combining the bioactive substance γ-polyglutamic acid (γ-PGA) with chemical fertilizer on soil microbial community and function in cotton fields.Four fertilization groups were designed:chemical fertilizer (NK),chemical fertilizer combined with γ-PGA aqueous solution (YT),chemical fertilizer combined with γ-PGA granules (GT),and no fertilizer (CK).[Results] GT and YT groups outperformed NK and CK groups in terms of cotton growth and soil nutrient content.The combination of γ-PGA with chemical fertilizer significantly increased the microbial abundance and diversity in soil,while chemical fertilizer alone did not improve soil microbial diversity.In addition,the application of γ-PGA changed soil microbial community composition.Compared with the NK group,YT and GT groups showed a 9.70%–12.72% decrease in the relative abundance of Proteobacteria and 13.33%–20.90% and 8.09%–13.01% increases in the relative abundance of Bacteroidetes and Actinobacteria,respectively.In addition,the relative abundance of Rhizophagus(a genus of mycorrhiza fungi) increased by 19.71% in the YT group.The functional gene analysis showed that GT and YT significantly increased the abundance of functional genes related to amino acid biosynthesis,secondary metabolite biosynthesis,and ABC transporters.[Conclusion] The application of γ-PGA has the potential of improving soil microbial diversity and ecosystem stability in Xinjiang cotton fields.

Abstract:Blastocystis sp.is a widespread unicellular protozoan mainly parasitizing the intestines of humans and animals.This parasite has rich genetic diversity,with 42 subtypes (ST1–ST17,ST21,ST23–ST46) reported,of which ST1,ST2,and ST3 are prevalent.However,the pathogenicity levels and the pathogenicity differences among different subtypes have been controversial.[Objective] To determine the pathogenicity of ST1,ST2,and ST3 and explore whether there are differences in the pathogenicity between the three subtypes of Blastocystis sp.[Methods] Blastocystis sp.strains were isolated from fresh human and macaque faeces and identified by morphological observation,18S rRNA gene amplification and sequencing,and cluster analysis.After BALB/c mice were infected with strains of ST1,ST2,and ST3,the distribution characteristics of the parasites in vivo were observed.In addition,the clinical signs,weight gain,feed conversion rate,and mortality of the infected mice were recorded to compare the pathogenicity of the three subtypes.Finally,the pathological changes in the intestines of mice were observed.[Results] Three subtypes of Blastocystis sp.,ST1,ST2 and ST3,were successfully isolated and identified.Animal challenge results showed that the parasites predominantly colonized the intestine and negatively affected the health of mice.A high dose of ST3 resulted in death in mice,while ST1,ST2,and low doses of ST3 did not affect the survival of mice.Blastocystis sp.damaged the intestinal tissue of challenged mice.[Conclusion]Blastocystis sp.causes harm to the host by damaging the intestine.The pathogenicity varies among different subtypes and ST3 has stronger pathogenicity.This study lays a foundation for further studying the pathogenicity mechanism and provides data for assessing the threat ofBlastocystis sp.to public health.

Abstract:[Objective]To systematically analyze the enzymatic properties of transglutaminase (TGase) from Streptomyces mobaraensis CGMCC 4.1851(strain XM4) and subsequently develop a high-yielding strain by engineering for achieving efficient expression of TGase in Streptomyces with reduced fermentation duration and enhanced production efficiency.[Methods]The pH of the fermentation broth and TGase activity were measured to assess the fermentation characteristics of strain XM4.TGase from XM4 was purified by alcohol precipitation combined with ion-exchange chromatography.The reaction conditions (pH,temperature,metal ions) were optimized for the enzyme,and the enzymatic kinetics were tested.The catalytic efficiency was evaluated by casein cross-linking experiments.Subsequently,genetic engineering was employed to enhance the modified strain through heterologous expression and replacement of the ribosome-binding site (RBS),followed by measurement of TGase production.[Results]TGase from strain XM4 exhibited good activity and stability within the range of pH 4.0–11.0,with the highest activity at 50℃ and pH 10.0.The modification realized efficient expression of TGase in S.mobaraensis,inceasing the production by 103.3% compared with the original strain and reducing the fermentation time to 24 h.[Conclusion]TGase from strain XM4 demonstrates excellent acid-base tolerance and thermal stability,demonstrating broad application prospects in the food industry,especially dairy processing.Additionally,the engineered strain enables efficient production of TGase,providing new options for the industrial production and application of TGase.

Abstract:[Objective] Bovine bones contain a large amount of beneficial nutrients such as proteins,mineral salts,and vitamins for human health.With the calcium-to-phosphorus ratio meeting the optimal absorption ratio for the human body,bovine bones serve as a valuable nutrient source.[Methods] lactic acid bacteria with high yields of l-lactic acid were screened by the calcium-dissolving ring method and the performance of strains in l-lactic acid production was then determined via fermentation of bovine bone meal.Metabolomics was employed to explore the differential metabolites and main pathways during the fermentation.[Results] One strain LP15,identified as Lactobacillus plantarum,was isolated from the liquid sample in the production of sour bamboo shoots for its strong ability to produce l-lactic acid.After fermentation of bone meal for 96 h,the strain produced 3.89 g/L l-lactic acid and 69.82 mg/100 mL free calcium,which represented a 32.6-fold increase compared with that in the control group.The metabolomic analysis revealed ABC transporters,metabolic pathway,and biosynthesis of secondary metabolites as the most important differential metabolic pathways during the fermentation of bovine bone meal.Meanwhile,a huge number of physiological active substances,such as cyclocreatine and 2-hydroxy-2-methylbutyric acid,were detected in the fermented bone meal.[Conclusion] Fermentation of bovine bone meal by L.plantarum LP15 is an effective strategy to promote calcium dissolution and produce beneficial active substances.This study lays a practical foundation for developing health food products by microbial fermentation of bovine bone meal.

Abstract:[Objective] To mine the key enzyme genes associated with spinosad synthesis and the biosynthetic gene clusters (BGCs) in Saccharopolyspora spinosa at different developmental stages by transcriptomics,thus laying the groundwork for the construction of high-yield strains.[Methods]The transcriptomes of S.spinosa during the logarithmic phase (T2-48 h) and the stationary phase (T6-144 h) were compared.The results from qRT-PCR and transcriptome sequencing were mutually validated.Gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) enrichment were performed for the differentially expressed genes (DEGs).Central carbon metabolism analysis was performed.[Results] The transcriptome sequencing of S.spinosa revealed 2 542 DEGs,including 1 188 genes with significantly up-regulated expression and 1 354 genes with significantly down-regulated expression.GO annotation indicated that the DEGs were primarily involved in carboxylic acid metabolic process,oxoacid metabolic process,organic acid metabolic process,and amino acid metabolic process.KEGG enrichment analysis demonstrated DEGs were mainly involved in pathways such as glycine,serine,and threonine metabolism,oxidative phosphorylation,and arginine biosynthesis.Further analysis identified seven genes related to spinosad biosynthesis.Among them,accB,Pfk,G6PD,and dsdA showed significantly up-regulated expression,while GAPDH,aceE,DLAT involved in the consumption of spinosad precursors,as well as genes in the TCA cycle and arginine biosynthesis,exhibited significantly down-regulated expression.The results of qRT-PCR were consistent with the trends observed in transcriptome sequencing,which revealed 12 upregulated BGCs:BGC2(43 846 bp),BGC4(18 330 bp),BGC9(20 501 bp),BGC18(62 621 bp),BGC22(19 626 bp),BGC25(42 896 bp),BGC26(40 086 bp),BGC28(39 392 bp),BGC30(20 282 bp),BGC31(53 657 bp),BGC34(20 787 bp),and BGC35(40 232 bp).[Conclusion]This study elucidated DEGs in S.spinosa at different developmental stages through transcriptome analysis,and analyzed the biosynthetic pathways and BGCs of spinosad.These findings pave the way for optimizing the spinosad biosynthetic pathways and genetically modifying S.spinosa to enhance the spinosad production in subsequent studies.

Abstract:[Objective]Within the framework of global climate governance and considering the pivotal role of the Yellow River basin in China’s ecological progress,this study focused on the Inner Mongolia segment of the Yellow River in July 2023.[Methods]We collected the surface sediment,water,and gas samples,with the aim of investigating the dissolved nitrous oxide (N₂O) concentration in the water,the N₂O emission flux (FN₂O) at the water-air interface,and the microbial community composition and diversity in surface sediments.[Results] The results revealed that the dissolved nitrous oxide (N₂O) concentration in the water of this segment varied between 0.547 8 mg/m³ and 0.598 2 mg/m³,with an average of 0.574 1 mg/m³.The FN₂O at the water-air interface ranged from−3.645 3 mg/(m²·d) to 4.392 5 mg/(m²·d),averaging 1.086 1 mg/(m²·d),which suggested this area was a net source of atmospheric N₂O.FN₂O showed a significantly positive correlation with pH and a significantly negative correlation with potential.The surface sediments harbored 7 784 operational taxonomic units (OTUs),with Proteobacteria (average abundance of 35.13%) being dominant.Ammonia-oxidizing archaea (AOA),a category of nitrifying bacteria,presented low abundance,with 116 OTUs among which unclassified_d__Unclassified(average abundance of 31.69%) was the dominant genus.For denitrifying bacteria,3 660 OTUs were identified,with unclassified_k__norank_d__Bacteria(average abundance of 63.12%) being the dominant genus.In view of the scarcity of N₂O data for the Inner Mongolia section of the Yellow River,the findings of this study enrich the N₂O data repository of the Yellow River.[Conclusion] This study not only augments our understanding of the microbial community structure and functionality in sediments but also supports the conservation and purification of the Yellow River.

Abstract:[Objective] Human norovirus (HuNoV) is one of the most common pathogens causing acute gastroenteritis in humans worldwide.Currently,there are no approved vaccines to prevent this disease.This study aimed to prepare virus-like particles (VLPs) of HuNoV in the flashBAC baculovirus expression system,laying a foundation for the development of vaccines against HuNoV.[Methods]After codon optimization,the full-length gene sequence of the VP1 protein of HuNoV GⅡ.4 was synthesized and cloned into the baculovirus pBacPAK9 transfer vector to obtain the recombinant plasmid pBacPAK9-8his-VP1.After enzyme digestion and sequencing,the recombinant plasmid was co-transfected with the linear baculovirus plasmid (Bacmid) into SF9 cells to obtain a recombinant baculovirus carrying the VP1 gene.Hi-Five (HF) cells were infected by the recombinant baculovirus for protein expression,and the expression of VP1 was analyzed by SDS-PAGE and Western blotting.VP1 was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blotting.The purity of VP1 was examined by high performance liquid chromatography (HPLC).The VLPs were observed by transmission electron microscopy.[Results] A transfer plasmid pBacPAK9-8his-VP1 was constructed,and the VP1 protein,with a molecular weight of approximately 58 kDa,was mainly expressed in the cytoplasm of HF cells.The HPLC results showed that the purity of VP1 was over 99%.The VLPs with a regular shape,uniform sizes,and diameters of 30–40 nm were observed by transmission electron microscopy.[Conclusion] The VLPs of HuNoV GⅡ.4 were prepared with a baculovirus expression system,laying a foundation for the development of HuNoV vaccines.

Abstract:[Objective]Multipurpose bioorganic fertilizers contribute to the sustainable development of the Carya cathayensis Sarg.industry.This work aims to explore the resources of phosphorus (P)-mobilizing bacteria (PMBs) with plant growth-promoting effects from the rhizosphere soil of C.cathayensisSarg.[Methods]PMBs were isolated with the dilution-plate coating method and identified based on 16S rRNA gene homology.Moreover,plate and liquid culture tests were conducted to determine their biological functions.[Results]A total of 34 strains of PMBs were isolated from the rhizosphere soil of C.cathayensisSarg.These PMBs belonged to 10 genera of four phyla:Bacillota,Proteobacteria,Actinobacteriota,and Gracilicutes.Among them,Bacillus(12 strains),Burkholderia(9 strains),and Pseudomonas(5 strains) were the dominant genera,with the strains accounting for 76.47% of the total isolated PMBs.After inoculation of PMBs,the content of soluble P produced by PMBs was 7.01–49.97,3.61–27.11,4.56–342.82,27.71–544.53,and 3.28–27.17 mg/L in the culture media with AlPO₄,FePO₄,Ca₃(PO₄)₂,sodium phytate,and lecithin as the sole P source,respectively.Twenty-three strains were capable of simultaneously mobilize insoluble inorganic and organic P components.Additionally,20,7,23,12,10,14,and 13 strains of PMBs could produce indole-3-acetic acid (IAA),siderophores,extracellular protease,β-1,3-glucanase,cellulase,phosphatase,and lipase,respectively,among which strains S3-6L and S3-22L exhibited five biological functions.[Conclusion] The PMBs identified in this study possess high P mobilization capability and multiple biological functions,enriching the resources of PMB strains and laying a foundation for the development of efficient,green,and composite microbial fertilizers for C.cathayensisSarg.

Abstract:[Objective] To study the role of napF3 in Thermoanaerobacter tengcongensis at different temperatures.[Methods]We constructed ΔnapF3 from T.tengcongensis by homologous recombination and observed the growth of ΔnapF3 at 50℃,60℃,75℃,and 80℃.Transcriptome sequencing was employed to identify the differentially expressed genes (DEGs) between ΔnapF3 and the wild type (WT) at 75℃.real-time PCR was conducted to measure the transcriptional levels of 13 genes and 3 sRNAs in WT and ΔnapF3 at 50℃,60℃,75℃,and 80℃.[Results] ΔnapF3 was successfully constructed,and it showcased suspended growth at 50℃ and 80℃ and slow growth at 60℃ and 75℃.A total of 899 DEGs between WT and ΔnapF3 at 75℃ were identified,including 363 genes with up-regulated expression and 536 genes with down-regulated expression.These DEGs were mainly involved in the biosynthesis of valine,leucine and isoleucine,ABC transporters,two-component system,fatty acid synthesis,thiamine metabolism and other pathways.The transcriptional levels of 13 genes and 3 sRNAs related to the thermophilic mechanism changed under specific temperatures.[Conclusion] napF3 plays a role in the thermophilic adaptation of T.tengcongensis.

Abstract:[Objective] To investigate the enzymatic properties and straw-degrading effect of a recombinant xylanase rRuXyn024.[Methods] We cloned RuXyn024 from the rumen of beef cattle and used bioinformatics tools for detailed sequence analysis.The expression vector pET-RuXyn024 was constructed and transformed into Escherichia coli BL21(DE3) for heterologous expression of RuXyn024.Furthermore,the enzymatic properties and straw-degrading effect of rRuXyn024 were examined.[Results] RuXyn024 was composed of 358 amino acid residues and had a molecular weight of approximately 40 kDa,belonging to the GH 10 family.The optimal pH and temperature of rRuXyn024 were pH 7.0 and 40℃,respectively.The relative activity of RuXyn024 at pH 6.0−9.0 and 30−70℃ remained above 60% and 70%,respectively.With xylan from wheat straw as the substrate,rRuXyn024 showcased the K_m and V_max of 18.8 g/L and 82.6µg/min,respectively.The activity of rRuXyn024 was inhibited by Mg²⁺,Zn²⁺,Cu²⁺,Ni²⁺,EDTA,and SDS at 1 mmol/L and 5 mmol/L,as well as 5 mmol/L Ca²⁺ and β-mercaptoethanol.Cu²⁺ and β-mercaptoethanol at 5 mmol/L nearly inactivated the enzyme.Mn²⁺ at 1 mmol/L and 5 mmol/L increased the activity of rRuXyn024 by 46.9% and 35.8%,respectively.The degradation of xylan from wheat straw by rRuXyn024 produced oligosaccharides,including xylotriose and xylobiose.rRuXyn024 could degrade maize straw,rice straw,soybean straw,and rapeseed straw,with the strongest degrading effect on maize straw.[Conclusion] rRuXyn024 exhibits tolerance to broad scopes of pH and temperature and significant potential for improving the utilization of straw by ruminants.

Abstract:The aquaculture industry has rapidly expanded in recent years in China,whereas it faces the challenge brought by bacterial diseases.Antibacterial agents have been the primary tools to combat these diseases.However,prolonged and haphazard usage of antibacterial agents in aquaculture has exacerbated antimicrobial resistance and led to severe antimicrobial residues.Considering these challenges,scholars worldwide have been exploring natural alternatives,such as Chinese herbal medicines.Among them,Pithecellobium clypearia stands out due to its antibacterial,antiviral,and anti-inflammatory properties,coupled with its safety and lack of antimicrobial resistance.Nonetheless,the potential of P.clypearia in the prevention and control of aquatic diseases remains underexplored.[Objective]This study evaluated the in vitro inhibitory activity of P.clypearia aqueous extract against aquatic pathogenic bacteria,including an artificially induced antimicrobial-resistant strain.Additionally,we investigated changes in bacterial cell membrane permeability and observed cellular alterations by transmission electron microscopy to elucidate the mechanism of the extract.Our findings are expected to pave the way for developing P.clypearia as an environmentally friendly antibacterial agent,reducing antibacterial agent dependency,and mitigating pathogen resistance in aquaculture.[Methods] We employed the microbroth method to assess the antimicrobial resistance of 107 pathogen strains attacking aquatic animals and analyzed the inhibitory activity of P.clypearia aqueous extract against aquatic pathogenic bacteria,including an artificially induced antimicrobial-resistant bacterial strain.Furthermore,we determined the extracellular K⁺ content and ultrastructural changes in Streptococcus agalactiae and Vibrio parahaemolyticus after treatment with the extract.[Results]The resistance rate of 107 pathogen strains to sulfonamides was as high as 67.29%,and 46.73% of the strains showed multidrug resistance,among which Aeromonas sp.showed the most severe resistance.The aqueous extract (12.50 mg/mL) of P.clypearia exerted inhibitory effects on all the pathogenic bacteria,especially on Aeromonas sp.with the minimum inhibitory concentration (MIC) as low as 0.39 mg/mL.The MICs of the extract were similar for the strains belonging to the same genus but with different antimicrobial resistance characteristics.The aqueous extract of P.clypearia showed stronger inhibitory effect on the artificially induced enrofloxacin-resistant strain of A.schubertii than on the original strain,with the MICs of 0.78 mg/mL on the original strain and 0.20 mg/mL on the resistant strain.In addition,the treatment with P.clypearia aqueous extract significantly increased the extracellular K⁺ concentration,leading to damage to the bacterial cell membrane structure,leakage of intracellular contents,and vacuolation of cytoplasm,which suggested that P.clypearia exerted the antibacterial effect by destroying the bacterial membrane structure.[Conclusion]P.clypearia exerts in vitro inhibitory effects on aquatic pathogenic bacteria and demonstrates great potential for further research and development in the prevention and treatment of bacterial diseases in aquatic animals.The antibacterial mechanism of P.clypearia appears to involve disrupting bacterial cell membranes.The application of P.clypearia in aquaculture promises to reduce antibiotic dependency and pathogen resistance,paving the way for a healthy and sustainable aquaculture industry.

Abstract:[Objective] To clarify the domestication characteristics and resistant mechanism of wild watermelon plants and provide a theoretical basis and technical support for building a new evaluation system for watermelon breeding and developing beneficial microorganisms,we compared the endophytic microbial community structure in roots between wild and cultivated watermelon varieties.[Methods] High-throughput sequencing was employed to reveal the community structures of endophytic microorganisms (bacteria and fungi) in the roots of wild and cultivated watermelon varieties.[Results] The phyla and genera of endophytic bacteria and fungi in the roots were significantly different between wild and cultivated watermelon varieties.The roots of wild watermelon varieties had significantly higher relative abundance of Nocardioides and Microbacteriumthan those of cultivated watermelon varieties.Actinoplanes,Mycobacterium,Lechevalieria,Amycolatopsis,Bradyrhizobium,and Rhodococcus were the special dominant endophytic bacterial genera in the roots of wild watermelon varieties.The relative abundance of unclassified_o__Chaetothyriale in the roots of wild watermelon varieties was significantly higher than that in cultivated watermelon varieties.unclassified__o_Chaetothyriales,Preussia,and unclassified_f__Microascaceae were the dominant endophytic fungi specific to wild watermelon varieties.[Conclusion] The beneficial bacteria,such as Nocardioides,Microbacterium,andRhodococcus,which were capable of fixing nitrogen,solubilizing phosphorus,secreting siderophores,and producing bioactive substances and antibiotics,and the growth hormone-producing fungi,such as Preussia,were lost in the roots of cultivated watermelon varieties after the domestication of wild watermelon.Therefore,it can be concluded that parts of endophytic microorganisms in the roots disappear during the domestication process of watermelon varieties,which is a major reason for the weak resistance of cultivated watermelon varieties to stresses.In addition,the bacterial genera such as Nocardioides,Microbacterium,and Rhodococcus and the fungal genus Preussia can be taken as the candidate microbial resources for enhancing watermelon resistance.

Abstract:[objective]Pseudomonas as one of the dominant spoilage bacteria highly form biofilms in chilled meat products and processing environment when contaminating single or mixed with other species.This study aims to investigate the antibiofilm properties of the cell-free supernatants (CFSs) of three Bacillus species isolated from fermented food and rice seeds on Pseudomonas lundensis(PL) or and Acinetobacter johnsonii(AJ) as mono-or dual-species.[Methods] Biofilm biomass,extracellular polymeric substances (EPSs),and biofilm structure were measured by crystal violet staining,spectrophotometry,confocal laser scanning microscopy (CLSM),respectively,as well as transcription of biofilm-related genes determined by qPCR.[Results]The CFSs of Bacillus amyloliquefaciens ZG08,B.velezensis B5,and B.subtilis YB11 inhibited the biofilm formation of PL and AJ without affecting their growth.The treatment with 50% CFSs of ZG08 and B5 decreased the cell viability of two biofilms by 12.73%–21.04%,which was higher than that of YB11(0.15%–4.38%).The inhibition rates of 50% CFSs of the three strains were 59.75%–79.59% against the PL biofilm and 63.62%–78.57% against the biofilm of PL+AJ,in which the CFS of YB11 had weaker activity.The content of exopolysaccharides and exoprotein in the two biofilms treated with these CFSs were reduced by 53.77%–73.30% and 54.84%–62.38%,respectively.The treatment with the three CFSs also reduced the adhesive cells,loosened biofilm structures,and thinned their thickness by 57.63%–74.49% and 60.43%–64.64%,respectively.Moreover,the CFSs of ZG08 and B5 effectively eradicated by 41.77%–69.79% against the mature biofilms of PL and PL+AJ,compared to weak activity of YB11.In addition,the antibiofilm activities of the three CFSs were stable under four enzyme digestion and heating conditions.Compared with the control,the CFSs of ZG08 and B5 significantly down-regulated the expression of six biofilm-related genes,lapA,alg44,pelG,luxR,wspR,and rpoS.[Conclusion] The CFSs of ZG08 and B5 have strong antibiofilm activities against PL and AJ as mono-or dual-species.

Abstract:[Objective]Certain strains ofLigilactobacillussalivariuspossess excellent probiotic properties and strong potential for application.This study characterized strain ATCC 11741 in terms of the growth characteristics,stress tolerance,and adhesion capacity,aiming to provide a theoretical basis for the utilization of L.salivarius. [Methods]L.salivarius ATCC 11741’s accuracy was verified through morphological observations and 16S rRNA gene sequencing;its growth characteristics were investigated by tracking growth and acid production curves;additionally,a range of tolerance tests,including those with acid,alkali,bile salts,hyperosmolarity and temperature,were conducted to evaluate its tolerance;lastly,self-aggregation and hydrophobicity experiments were used to indirectly determine its adhesion.[Results] The growth curve of L.salivarius ATCC 11741 was formed like a “S”.The strain reached its highest acid production between 2–7 h,stabilizing at pH 4.3 after 14 h.After being cultured in the MRS medium at pH 2.0 for 4 h,the strain had a survival rate of 50.48% and grew well in the pH 4.0–11.0 range.Following 2 h of incubation in the MRS medium with 0.10% bile salt,the strain’s survival rate was 94.440 0%.In the MRS medium with 6% NaCl,the strain continued to proliferate.The growth of strain ATCC 11741 was significantly affected by temperature,being promoted at 30–42℃ and inhibited at 20℃ and 50℃.At the 5 h mark,the strain’s hydrophobicity (measured by the hydrocarbon compound adhesion method) was 44.5% and its self-aggregation rate was 41.4%.[Conclusion] L.salivarius ATCC 11741 is a strain with fast growth,strong tolerance to acids,alkali,and salts,moderate tolerance to bile salt,a wide range of suitable temperatures,and strong adhesion capacity.

Abstract:[Objective] Soft rot is one of the major diseases affecting the yield and quality of konjac.This study screened a strain with antagonistic effect on Pectinobacterium aroidearum from the rhizosphere soil of konjac,aiming to provide germplasm resources for the biocontrol of soft rot in konjac.[Methods] An antagonistic strain was screened by the plate confrontation method,and its antagonistic effects on pathogenic fungi were measured.The control effect of GZA12 on soft rot in konjac was examined by the inoculation in konjac corm tissue,pot experiment,and root irrigation.The growth-promoting effect of this strain was tested indoors and preliminarily verified by tomato pot experiments.[Results] A strain GZA12 with antagonistic effect was screened out and identified as Bacillus velezensis.This strain showed the inhibition zone diameter of 21.33 mm against P.aroidearum and the inhibition rates of 58.16%,47.30%,and 54.53% against Botryosphaeria dothidea,Fusarium oxysporum,and F.solani,respectively.Inoculation of GZA12 in konjac corm tissue decreased the disease index by 26.67%,33.33%,and 40.00%,respectively,compared with the inoculation of B.dothidea alone.In the pot experiment,the treatment with GZA12 suspension decreased the disease index by 22.85% compared with the control group and reached the control effect of 53.31%.The results from the root irrigation experiment showed that compared with water irrigation,irrigation with GZA12 fermentation broth reduced the disease index by 4.89% and reached the control effect of 21.57%.Strain GZA12 had the ability to fix nitrogen,solubilize phosphorus,and produce siderophores and indole-3-acetic acid (IAA).Inoculation with GZA12 suspension promoted the growth of tomato seedlings in a concentration-dependent manner.[Conclusion] Strain GZA12 can inhibit the pathogen causing soft rot and promote the growth of konjac,demonstrating the potential for further development and utilization.

Abstract:Porcine epidemic diarrhea virus (PEDV) is an enterovirus that can cause severe diarrhea and dehydration.The widespread epidemic of PEDV has caused huge economic losses to the pig breeding industry,which,however,lacks effective means for prevention and treatment.Nsp8 is an important non-structural protein involved in the replication of PEDV,while the host proteins interacting with Nsp8 remains unclear.[Objective] To screen the host proteins interacting with PEDV Nsp8 and explore the effects of the host proteins on the replication of PEDV,so as to provide a theoretical basis for discovering new key functional receptors or therapeutic targets of PEDV.[Methods] The eukaryotic expression plasmid of PEDV Nsp8 was successfully constructed with the eukaryotic expression vector pcDNA3.1(+).The host proteins interacting with PEDV Nsp8 were screened by co-immunoprecipitation,mass spectrometry,and laser confocal microscopy.The effects of the host proteins on PEDV replication were explored by overexpression and knockdown in LLC-PK cells.[Results] Thirty-six potential host proteins interacting with Nsp8 were screened by mass spectrometry,and the interaction between heat shock protein member 8(HSPA8) and Nsp8 was verified.The overexpression of HSPA8 in LLC-PK cells inhibited the overexpression of Nsp8 in a dose-dependent manner.Meanwhile,it significantly inhibited the replication of PEDV in a dose-dependent manner at the protein and transcriptional levels.Interfering with endogenous HSPA8 expression significantly promoted the replication of PEDV.The 50% tissue culture infectious dose (TCID₅₀) and indirect immunofluorescence further proved that HSPA8 inhibited PEDV replication.[Conclusion] This study screened out the host protein HSPA8 interacting with PEDV Nsp8 and proved that HSPA8 could significantly inhibit PEDV replication,which provided a new idea for the design of HSPA8-targeted drugs for the prevention or treatment of PEDV.

Abstract:[Objective]We isolated the aerobic bacteria capable of effectively degrading polylactic acid (PLA) and characterized the bacterial growth and degradation,aiming to lay a theoretical foundation for the bioremediation of PLA contaminated environment.[Methods]The degrading bacterium was identified by 16S rRNA gene sequencing.Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were employed to analyze the morphological and chemical changes of PLA films before and after degradation.[Results]A strain of Bacillussp.JA-4 was screened from activated sludge,and it caused the PLA weight loss of 10.6% after 30 days.The weight loss of PLA reached 5.6% after incubation with the strain at an inoculation amount of 20%,pH 8.0,and 30℃ for 7 days.Gelatin significantly enhanced the biodegradation of PLA.In the presence of 3% gelatin,the weight loss of PLA reached 23.1% after 10 days of degradation,and the degradation rate was greatly increased.FTIR results indicated that Bacillussp.JA-4 degraded PLA by hydrolyzing the ester bonds.[Conclusion]This study enriched the microbial resources for the biodegradation of PLA and provided technical support for the effective degradation of PLA waste in the environment.

Abstract:The widely used trypsin is mainly extracted from animal pancreas,which has the disadvantages of limited raw materials,high costs,and low purity.In addition,the autolysis of trypsin affects its stability in storage and application.[Objective] To obtain an anti-autolysis recombinant trypsin by heterologous expression and methylation.[Methods]We employed gene recombination to realize the heterologous expression of porcine trypsinogen in Pichia pastoris.Furthermore,we conducted single factor experiments to investigate and optimize the temperature,pH,and time of enzyme activation and improved the anti-autolysis performance of the recombinant trypsin by methylation.[Results]The engineered strain ofP.pastoris expressing trypsinogen was successfully constructed.Under the trypsin concentration of10 mg/mL,methylation reagent addition of 30 μL,and reaction time of 3 h,the methylated trypsin showed the activity loss of only 22% and the relative activity of 79% after autolysis for 6 h,which was about 3.4 times higher than that of the control,suggesting that the anti-autolysis performance of the recombinant trypsin was greatly improved.[Conclusion] This study successfully produced a novel anti-autolysis recombinant trypsin by heterologous expression and methylation,which can improve the production and application of trypsin in China.

Abstract:[Objective] To screen microbial strains with high production of exopolysaccharides (EPS) and provide strain resources for the development of soil improvement agents.[Methods] The string test with a LB-aniline blue plate was employed to qualitatively screen the strains of plant growth-promoting rhizobacteria (PGPR) with EPS production.After fermentation with each strain in four media,the EPS content in the fermentation liquid was determined by low temperature alcohol precipitation and the sulfate-anthranone colorimetric method,on the basis of which the PGPR strain with high EPS production was screened out.The fermentation conditions of the strain screened out were optimized by orthogonal test with EPS content in fermentation liquid as the indicator.The influence of the fermentation liquid of strain F1 on the content of macro-aggregates in sandy loam soil was analyzed by the petri dish culture experiment.[Results] Eight EPS-producing PGPR strains were primarily screened out,among which strain F1 had the highest EPS production.PDA was the best medium for F1 to produce EPS,with the EPS content of 867.54 μg/mL.Based on morphological,physiological and biochemical characteristics,phylogenetic analysis based on 16S rRNA gene sequence,and also average nucleotide identity analysis,F1 was identified as a strain of Bacillus megaterium.The optimal culture conditions for F1 to produce EPS were 28℃ and 180 r/min for 24 h,under which the EPS yield reached1123.39 μg/mL.After F1 was incubated in sandy loam soil for 40 days,the content of water-stable macro-aggregates with the grain diameter>0.25 mm in the soil increased by4.44 times compared with that of the control.[Conclusion] Strain F1 with high EPS production can promote the formation of water-stable macro-aggregates in sandy loam soil.The optimal conditions for F1 to produce of EPS was incubation in PDA at 28℃ and 180 r/min for 24 h.

Abstract:[Objective] To explore the effects of different concentrations of chlortetracycline on the characteristics of microbial communities involved in inorganic phosphorus (Pi) dissolution and organic phosphorus (Po) mineralization in the soil applied with organic fertilizer,focusing on soil P transformation and availability.[Methods] The purple soil collected from Tongnan District of Chongqing was used for a pot experiment with the addition of chicken manure as the organic fertilizer.Three chlortetracycline treatments (No-CTC,Low-CTC,and High-CTC) were designed with the addition levels of 0.0,0.1,and 4.0 mg/kg,respectively.The soil samples were collected on days 7(D7) and 30(D30) after pepper (‘Xinxiang 8’) was planted.Real-time qPCR and Illumina MiSeq high-throughput sequencing were employed to analyze the community characteristics of the bacteria carrying the key genes (pqqC and phoD) of Pi dissolution and Po mineralization,respectively.Furthermore,the sequencing results and biologically based P (BBP) fractionation were employed to examine the effects of CTC addition on soil P transformation.[Results] High-CTC increased the content of Citrate-P and Enzyme-P by 8.2% and 44.0%,respectively,compared with No-CTC on D7.Low-CTC and High-CTC increased the content of Enzyme-P by 44.0% and 65.6%,respectively,compared with No-CTC on D30.The addition of CTC suppressed alkaline phosphatase (ALP) activity and affected the community structures of pqqC and phoD-harboring bacteria in the soil.The Mantel test results showed that Citrate-P was significantly associated with the dominant pqqC-carrying taxa Pseudomonas,Geodermatophilus,and Saccharothrix on D7.The dominant phoD-carrying taxa Bradyrhizobium,Ensifer,and Skermanella exhibited notable correlations with Enzyme-P on D7,and such correlations weakened over time.The average degree of the community network of the bacteria carrying pqqC increased in the Low-CTC treatment and decreased in the High-CTC treatment on D7.The average degree of this network decreased in High-CTC and Low-CTC treatments on D30.The average degree of the community network of the bacteria carrying phoD decreased with the increase in CTC addition on D7,while this trend was opposite on D30.[Conclusion] The addition of CTC significantly affected soil Enzyme-P by regulating the community structure ofpqqC- and phoD-carrying bacteria as well as acid phosphatase (ACP) and ALP activities,thereby affecting the P forms and availability in the soil.This study contributes to a deeper understanding of alterations in microbial communities associated with P cycling in the soil-plant system contaminated by CTC.Moreover,it lays a scientific foundation for enhancing nutrient utilization efficiency in the soil applied with antibiotics.