Abstract:[Background] Indigoidine is a nontoxic blue pigment produced by microorganisms. [Objective] Comparison of the stability of heterologous expressed indigoidine in Escherichia coli and commercial indigo. [Methods] Recombinant E. coli DH5α/p28s strain was constructed to produce indigoidine. The effects of light, UV, pH, temperature, oxidant, reductant, food additive, metal ion on the stability of the two pigments were investigated. [Results] When N,N-dimethylformamide (DMF) was used as solvent, both indigoidine and indigo were sensitive to visible light and UV. The colors of the two pigments were stable at pH 1.0?11.0, but higher pH had a great destructive effect. The tolerance of indigoidine to Vc reduction was stronger than that of indigo. Both maximal absorption values were reduced at a certain degree by oxidant. Indigoidine showed distinguished better thermal stability than indigo below 75 °C, but both were bleached at higher temperature. The food additive citric acid and benzoic acid had significant protective effect on the stability of indigoidine and indigo respectively. Ca2+ and Mg2+ showed certain color-keeping effect on both pigments, while Na+, K+ and Li+ had no overall destruction impact, but Zn2+, Al3+, Cu2+, Fe2+ and Fe3+ exhibited notable destruction on both. [Conclusion] Indigoidine exhibits much better overall stability than indigo, suggesting a broader prospect of development and application.

Abstract:[Background] Biological aerated filter performs well on organic matter and ammonia nitrogen removal, whereas exhibits bad on phosphorus removal. [Objective] To improve the phosphorus removal efficiency of the BAF system. [Methods] The experimental group was an A/O filter, followed by a limestone-filling filter column. The control group was an identical one only without limestone filter. The treatment efficiency of reflux BAF system in experimental group and control group was compared and analyzed. Additionally, based on MPN method and high-throughput sequencing technology, the nitrifying bacteria quantity and microbial community structure in aerobic column were also investigated. [Results] Comparative tests showed that, compared with the BAF system without reflux, the removal efficiency of the reflux BAF system for COD, NH4+-N, TN, and TP increased by 3.16%, 41.21%, 40.62% and 18.93%, respectively. The removal efficiency of COD, NH4+-N, TN and TP in the experimental group increased by 1.75%, 2.3%, 2.2% and 23.1%, respectively. According to the results of high-throughput sequencing, the abundance of Proteobacteria, Actinobacteria, Firmicutes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Xanthomonadaceae in the aerobic filter column of the experimental group increased by 25.2%, 4.8%, 5.5%, 7.4%, 7.3%, 12.0% and 6.6%, respectively, compared with the control group. Meanwhile, the amount of different species between the experimental group and the control group at the genus level was 170. [Conclusion] The removal efficiency of TN and TP could be significantly improved by the reflux of limestone filter column. Calcium ions could improve the removal efficiency of phosphorus by chemical precipitation, and enhance the effect of biological phosphorus removal by improving the structure of bacteria in aerobic column.

Abstract:[Background] Trichoderma asperellum agent has been widely used in biological control and secondary bioremediation of soil secondary salinization, while less is known about the salt-tolerant mechanism and salt accumulation potential of this strain under stress of salinization. [Objective] This research aimed to reveal the physiological response of Trichoderma asperellum CTCCSJ-W-SBW10264 (T264) to Na+-induced oxidative stress and the Na+ adsorption and accumulation characteristics of this strain. [Methods] Gradient concentrations of Na+ in broth were designed as stress factor in fermentation experiment of strain T264, then mycelia samples at different culture stages were collected for the measurement of cell oxidative damage indices including H2O2 content and malondialdehyde (MDA) level together with the activity of cellular antioxidant related enzymes. [Results] Result of Na+ stress cultivation experiment indicated that T264 could survive with Na+ concentration of 1.22 mol/L, and the growth of T264 was not significantly inhibited with Na+ concentration lower than 0.25 mol/L. The results of study on oxidative damage and oxidative damage responses revealed that the higher Na+ concentration lead to the higher cellular oxidation level (MDA content). Moreover, with the accumulation of MDA and H2O2 in hyphae of T264, the activity of antioxidative damage enzymes in cells was also increased significantly. After Na+ treatment for 24 h, the activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) were respectively highest under the Na+ stress of 0.5, 1.0 and 1.22 mol/L, respectively, which were 36.66, 3.34 and 233.3 U/mg. The experimental results of Na+ adsorption and accumulation characteristics showed that hyphae of T264 had a strong adsorption capacity for Na+. After being cultured in the 0.05 mol/L Na+ environment for 72 h, the surface Na+ adsorption capacity of mycelia was 1 347.6 mg/g, and the internal Na+ accumulation capacity of mycelia was 218.6 mg/g. The remove rate of Na+ in the culture medium reached 32% through surface adsorption and internal accumulation of T264 mycelia. [Conclusion] This experiment showed that the antioxidant damage-related enzymes of T264 play an important role in its resistance to Na+ stress, and the strain T264 has a strong adaptability to high concentration of Na+ and an efficient adsorption and accumulation effect of Na+ in the environment.

Abstract:[Background] Pseudomonas chlororaphis HT66 is a plant growth-promoting rhizobacterium (PGPR) with biocontrol safety and high yield of phenazine-1-carboxamide (PCN). It has broad application prospects in biological control, ecological agriculture and sustainable agriculture. SuhB, a small non-coding RNA, involved in the metabolic regulation of multiple processes in cells. [Objective] This study explored the effect of suhB gene on the biocontrol ability of in P. chlororaphis HT66. [Methods] we constructed the suhB deletion mutant HT66ΔsuhB by homologous recombination method and the suhB complemented mutant HT66ΔsuhB-pBBR-suhB by plasmid. They were used to explore the effect of suhB gene on strain growth, biofilm formation, swarming motility and PCN synthesis. [Results] The mutant HT66ΔsuhB grew slowly, the plateau period delayed by 12 h and its biomass decreased to 61.6% of the wild type. The maximum yield of mutant strain in KMB medium can reach 109.5 mg/g (per DCW), which was 2.1 times that of wild type. The biofilm formation increased significantly, which was 1.8 times that of the wild type. However, the swarming motility of HT66ΔsuhB was defective. On the swarm plate, the movement radius of the wild strain was 21 mm, while the movement radius of the mutant strain was 9.7 mm. The suhB gene complemented mutant is similar to wild type in the above aspects. Compared with the wild type, there is no difference on the expression of phzI at the overall level in mutant HT66ΔsuhB. On the contrary, the expression of phzR increased significantly, which is 3.1 times that of the wild type. In addition, the expression of phzAp at the transcriptional level is 1.8 times that of the wild type. [Conclusion] The regulation of suhB gene in P. chlororaphis HT66 participates in the growth, biofilm formation, swarming motility and PCN synthesis. This research provides a theoretical basis for the metabolic transformation and biocontrol applications of P. chlororaphis HT66.

Abstract:[Background] Aflatoxin B1 (aflatoxin B1, AFB1) is a highly toxic and widespread mycotoxin. At present, there is no effective method to control its pollution. [Objective] To discover efficient AFB1 degrading bacteria and explore its degradation characteristics, enzymatic properties of an AFB1 degrading strain (HAI2) from mangrove sludge samples were analyzed. [Methods] Using the structural analogue of AFB1 as the sole carbon source, a highly efficient AFB1 degrading strain was screened out, then 16S rRNA gene sequencing technology was used to identify the strain species, and HPLC was used to analyze the strain’s degradation characteristics to AFB1. [Results] 16S rRNA gene sequence of HAI2 shares 99.85% homology with Pseudomonas putida (NR 113651.1). The main component of degradation AFB1 was extracellular protein. According to the analysis of enzymatic properties, the optimum pH value of HAI2-AFB1 degrading enzyme was 7.0 and the optimum temperature was 37 °C. Fe2+, Ca2+, and Zn2+ could inhibit the degradation rate of AFB1, and Cu2+ and Mn2+ could increase the degradation rate of AFB1. The degradation rate of 100 ng/mL AFB1 by HAI2 supernatant was 71.52% for 24 h. Besides, P. putida HAI2 could inhibit AFB1 synthesis in corn infected by Aspergillus flavus, and the content could be reduced by 63.46%. [Conclusion] P. putida HAI2 had a high ability to degrade AFB1, and its main degradation active substance was extracellular protein, with a large application prospect in the food and feed industry.

Abstract:[Background] Apple tree canker is one of the destructive diseases that caused by Valsa mali in many apple production areas of China with the characteristics of widespread distribution, serious damage, and was difficulty in controlling. Also, the disease of apple tree canker that has been severely restricted the development of apple industry. Therefore, it is urgent to develop an effective measure in control of apple tree canker disease. [Objective] To determine the taxonomic position of antagonistic Z-12A strain that has been isolated from the rhizosphere soil of apple trees in Jingning, Gansu province; and evaluate the biocontrol potential of Z-12A against V. mali in vitro. [Methods] The species of the Z-12A strain and the biological characteristics were identified and determined by the combination of morphological characteristics and ITS sequence analysis, and the plate growth rate method, respectively. the inhibitory efficiency of Z-12A against the pathogens growth was evaluated by dual culture and colony diameter assay, and the detached apple twigs method in vitro. the biosafety on apple branches and mice was determined using the Z-12A strain and its fermented broth, respectively. [Results] The strain of Z-12A was identified as Penicillium egyptiacums using the morphological characteristics and ITS sequence analysis. SDYA and GA were screened as the optimum media for the Z-12A strain hypha growth and spores production, respectively. Xylose and KNO3 were screened as the optimum carbon and nitrogen sources, respectively. The optimum pH and temperatures for hypha growth were at 5.0 and at 25 °C, and 7.0 and 10 °C for spores production. The inhibitory rates of Z-12A strain against V. mali was 88.71% and 61.07% using the plate confrontation and fermentation broth, respectively. Microscopic observation showed that the strain of Z-12A can make the mycelium of V. mali deformity and cell protoplast exosmosis. Additionally, the control efficacy of Z-12A fermentation broth was 66.69% after application in apple twigs in vitro, and also was safe for apple branches and mice after application. [Conclusion] These results indicate that the strain of Z-12A has strong inhibitory activity against V. mali, and exhibited significant bio-control potential in vitro. Thus, our results provide a new bio-control resource in controlling apple tree canker disease in future.

Abstract:[Background] Red yeast rice is an edible rice koji fermented by Monascus on rice, and possess the cholesterol inhibitor monacolin K. However, commercially available red yeast rice has low levels of acid form monacolin K and generally exhibits red color, which limit its applicationl. Monascus sp. var. white 3001-18 has the advantages of not producing pigments and citrinin, but producing high levels of acid form monacolin K. [Objective] To study the effect of micronutrients on the production of monacolin K and the proportion of acid form, and also their effects on synthesis-related genes expression. [Methods] Adding different micronutrients to the solid-state fermentation medium to increase the biomass of Monascus, monacolin K production and the content of acid form. We analyzed the expression of genes related to monacolin K synthesis. [Results] The production of monacolin K improved by 10.63%, reached up to 17.90 mg/g by adding 0.1% sodium acetate. Moreover, the proportion of acid form monacolin K increased from 76.08% to 90.51% by adding 0.015% niacin. RT-qPCR analysis showed that sodium acetate increased the production of monacolin K by promoting the expression of mokA, mokB and mokC genes related to the synthesis of monacolin K. Niacin up-regulated the transcription of mokF, mokH and mokI genes, resulting in faster transport and higher proportion of acid form monacolin K. [Conclusion] Micronutrients can promote the synthesis of monacolin K by increasing the expression of synthesis-related genes. The study provides a certain theoretical basis for the application research of high producing acid form monacolin K.

Abstract:[Background] Salmonella enteritis is an important zoonotic pathogenic bacterium that is transmitted essentially by poultry meat products and eggs. [Objective] In order to confirm pathogen information in a breeder farm where the poultry was suspected to be infected by Salmonella in Yunnan province. [Methods] we collected liver tissues from infected chicken in sterile environment to isolate and culture bacteria, then performed drug sensitive test and pathogenicity test, as well as identification of virulence genes for obtained strain. [Results] The isolated bacterium was identified to be Salmonella enteritidis and named SSYN001. Its antigen structural formula consists of the O antigen 1(+), 9(+), 12(+), H antigen gm(+) and [1,7](+). Drug resistance assay suggests that this strain was resistant to penicillin, bactrim, doxycycline and tetracycline, but sensitive to 8 antibiotics, include amoxicillin, ampicillin, gentamicin, tobramycin and ceftazidime. By detecting its resistance gene, we found that the strain contains tetA, a tetracycline resistance gene. The pathogenicity test shows the lethality of this strain for chicks, layer chicken and mice was 40%, 80% and 100%, respectively; eventually, we found this bacterium has 15 virulence genes consisted of spvB, spiA, pagC, msgA, invA, sipB, prgH, spaN, tolC, iroN, sitC, lpfC, sifA, sopB and orgA. [Conclusion] The present study provides new data of the pathogenicity of laying hen’s Salmonella enteritidis in Yunnan province, with implication for public health on detection of foodborne zoonotic Salmonella.

Abstract:[Background] Methicillin resistant Staphylococcus aureus (MRSA) is a multidrug-resistant zoonotic pathogen that often causes many diseases such as cow mastitis. Bacterial biofilm-formation is one of the important drug-resistant mechanisms of MRSA. It has been found that the polysaccharide intercellular adhesion (PIA) regulated by ica operon, which mediates the biofilm-formation by promoting the adhesion and aggregation of MRSA, and the co-expression of the icaA and icaD genes can significantly increase the activity of N-acetylglucan transferase in S. aureus. However, it is unclear that the effect of the icaA/D protein on the biofilm-formation and drug-resistance of MRSA. [Objective] To study the relationship among icaA/D gene, biofilm-formation and drug-resistance of MRSA and provide a scientific basis for finding new drug targets. [Methods] MRSA M5 isolated from mastitis milk, which exhibited a multidrug-resistant and strong biofilm-forming ability, was used to construct the icaA/D genes deletion strain by homologous recombination technology. The biofilm-forming ability and process of the MRSA wild strain and icaA/D genes deletion strain were assessed by FITC-ConA staining combined with laser confocal microscopy. Finally, the minimum inhibitory concentrations (MICs) of 14 antimicrobial agents to wild strain and icaA/D genes deletion strain were detected by broth microdilution method. [Results] The icaA/D genes deletion strain was successfully constructed. Through observation under a laser confocal microscopy, it was found that the wild strain formed a thick layer of mature biofilm at 16 h. Subsequently, the formed biofilm began to dissociate until 120 h; In contrast, a thin biofilm was formed by icaA/D genes deletion strain at 16 h after culture, and completely dissociated at 48 h. Compared with the wild strain, the MICs of 10 tested antimicrobial agents to icaA/D genes deletion strain were decreased, and the drug sensitivity of icaA/D genes deletion strain to 8 tested antimicrobial agents changed from resistance or intermediation to intermediation or sensitivity. [Conclusion] icaA/D gene deletion can significantly reduce the biofilm-formation ability and drug-resistance of MRSA.

Abstract:[Background] 2-phenylethanol is a high-grade flavoring additive with rosy scent, and widely used in perfume, cosmetics, food and medicine. At present, the synthesis of 2-phenylethanol by engineering bacteria has a good prospect. We isolated an Enterobacter sp. CGMCC 5087 that can synthesize 2-phenylethanol through phenylpyruvate pathway. However, the growth of the bacterium and 2-phenylethanol yield are affected by environmental stress. As a stationary phase Sigma factor and a major regulator in response to stress, RpoS plays an important role in resistant to environmental stress. [Objective] In order to elucidate the role of rpoS gene in Enterobacter sp. CGMCC 5087 under various environmental stresses. [Methods] Using CRISPR gene editing technology to knockout the rpoS gene, and the complementary strain was constructed by introducing the plasmid containing rpoS gene into ΔrpoS strain. The growth of rpoS gene-deficient strain ΔrpoS, the wild-type strain and complementary strain ΔrpoS(rpoS) under high osmotic pressure, high temperature, low pH and oxidative stress environments was detected and statistically analyzed. [Results] Deletion of rpoS gene significantly reduced the growth of Enterobacter sp. CGMCC 5087 strain. Under the stress of 5% NaCl and pH 5.0, deletion of rpoS gene significantly reduced the tolerance of Enterobacter sp. CGMCC 5087. At 42 °C, the deletion of rpoS gene resulted in a significant decrease in the tolerance of Enterobacter sp. CGMCC 5087 in logarithmic phase, and increased during the decline phase. Under the condition of 1 mmol/L H2O2 oxidation stress, the deletion of rpoS gene led to the prolongation of the delay period of Enterobacter sp. CGMCC 5087, and the tolerance of the mutant strain ΔrpoS was significantly higher than that of the wild type. [Conclusion] In Enterobacter sp. CGMCC 5087, RpoS plays an important role in resisting to various environmental stresses, and the response to environmental stresses varies with the different growth periods of bacteria. The study provides a basis for further understanding of the biological characteristics of Enterobacter sp. CGMCC 5087 and the mechanism of RpoS in the synthesis of 2-phenylethanol of Enterobacter sp. CGMCC 5087.

Abstract:[Background] Daptomycin is an important antibiotic isolated from Streptomyces roseospoous NRRL 11379 and is clinically used to treat infections. The long fermentation period of the daptomycin producer restricts its productivity. Hence, a daptomycin heterologous expression strain Streptomyces coelicolor K10 that can significantly shorten the fermentation period, was constructed in our previous work. Unfortunately, the very low titer of daptomycin in S. coelicolor K10 hampers its use in further studies. [Objective] The fermentation medium of S. coelicolor K10 was optimized by response surface methodology to improve daptomycin production. [Methods] Plackett-Burman experiment, the steepest ascent method, and response surface methodology were used to optimize the fermentation medium of S. coelicolor K10; and the experiment data were analyzed using Design Expert 8.0. [Results] The main influential factors were dextrin, yeast extract, and casein; and their optimal concentrations were 25.11, 2.20, and 2.00 g/L, respectively. Under the optimal condition, the production of daptomycin could reach 15.30 mg/L, about 2.17 times higher than that in the original medium. [Conclusion] This study increases the daptomycin titer of S. coelicolor K10 considerably and provides a reliable basis for the follow-up studies of daptomycin.

Abstract:[Background] Bacteriocin is a group of antibacterial-peptide substance produced by bacteria, with an inhibition and killing effect on various pathogenic bacteria. Insect, as the most abundant animal groups, is one of animals containing the most abundant and diverse intestinal microbial community in the world. Insect intestines may contain a rich bacteriocin-producing bacteria resource. [Objective] Sampling Dendrolimus punctatus larvae as experimental materials, its intestinal bacteria were isolated, and these bacteria were further cultured and identified. Bacteriostatic bacteria with obvious inhibitory effect on typical pathogens were obtained. Bacteriostatic substance was preliminarily speculated, and bacteriocin-producing bacteria with high bacteriostatic activity were investigated to further enrich resources of the bacteriocin-producing bacteria. [Methods] The bacteria were isolated by traditional-pure culture method, activity of the bacteriostatic substance was detected by using Oxford cup method, and bacteriocin-producing species were determined by combining morphological characteristics and 16S rRNA gene sequences of colony; interference of organic acid and hydrogen peroxide was further eliminated, and the bacteriostatic substance was preliminarily demonstrated with protein-like properties. [Results] Thirteen strains of the bacteriocin-producing bacteria were screened from intestine of the D. punctatus larvae, which belong to genera Bacillus, Staphylococcus, Psychrobacter and Enterobacter. Results of bacteriostatic experiments showed that these 13 strains have a certain degree of inhibitory effects on two species of typically pathogenic indicator bacteria, Staphylococcus aureus and Escherichia coli. In particular, bacteriocin produced by Bacillus amyloliquefaciens MW-1 maintained a good antibacterial activity after treating with different temperature and pH conditions, and an optimum bacteriostatic activity was obtained under pH 7.0 and 37 °C. [Conclusion] Many species of the bacteriocins-producing bacteria in the intestine of the D. punctatus larvae present antibacterial effects on important pathogens. The strain MW-1 with high bacteriostatic activity for pathogen shows potential to replace antibiotic, bacteriocin of which exhibited a good stability under heat, acids and alkaline treatments.

Abstract:[Background] Peptidoglycan (PG) is an important component of the bacterial cell wall. The type VI secretion system (T6SS) can secrete effectors with peptidoglycan hydrolase activities into a neighbor bacterial cell to kill the recipient. However, the enzymatic functions of these effectors have not been fully characterized due to the technical challenges in PG analysis. [Objective] We aim to establish an analytical method using liquid chromatography and mass spectrometry to qualitatively determine the PG-hydrolyzing activities of two Vibrio cholerae effectors TseH and VgrG3. [Methods] The antibacterial effects of TseH and VgrG3 were determined by survival assays and microscopy analysis when ectopically expressed in Escherichia coli. Peptidoglycan was purified from E. coli, and morphologically characterized by transmission electron microscopy (TEM). The ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS) was used to identify the products of peptidoglycan from digestion by TseH or VgrG3. [Results] TEM micrograph showed that the purified peptidoglycan is translucent. Using UPLC-TOFMS to analyze VgrG3-treated PG, we identified three products including disaccharide dipeptide (Di), disaccharide tripeptide (Tri), and disaccharide tetrapeptide (Tetra). [Conclusion] VgrG3, rather than TseH, can digest the β(1-4) covalent bond between N-acetylglucosamine and N-acetylmuramic acid. The established method could facilitate the characterization of other cell-wall targeting antibacterial effectors and chemical compounds.

Abstract:[Background] Extracellular vesicles are lipid bound vesicles harboring specific subsets of bioactive proteins, lipids, nucleic acids, and metabolites, secreted by all living cells into the extracellular space. They have been named as membrane vesicles (MVs) in Gram-positive bacteria. Recently, vesicle production is described in Listeria monocytogenes, a Gram-positive intracellular foodborne pathogen that causes listeriosis in human as well as in animals. However, the knowledge about the impact of L. monocytogenes-derived MVs on host health and disease is still limited. [Objective] To establish and improve the isolation method to extract MVs from L. monocytogenes, and to explore its roles of biological activities. [Methods] Two methods, the ultrafiltration concentration method and Optiprep density gradient centrifugation method, were used to extract MVs from the wild strain EGDe and its virulence mutants (EGDe?prfA, EGDe?prfA+pERL3-prfA*) and compared. The impact of different virulent L. monocytogenes-derived membrane vesicles on the bacterial biofilm formation and pathogenesis to the insect infection model was investigated. [Results] Comparing with the ultrafiltration concentration method, the MVs isolated by the Optiprep density gradient centrifugation have got higher yield and presented more uniform and clear image under transmission electron microscope, but it’s relatively complicated and took a long time; All different virulent L. monocytogenes strains can secrete membrane vesicles with 20?200 nm in diameter, and no obvious differences in their shapes and sizes. However, these vesicles inhibited biofilm formation of L. monocytogenes, harbored a certain hemolytic activity, reduced the pupation and survival rate of the Helicoverpa armigera larvae, and even caused them death. The toxicity of MVs to the larvae showed a clear link with the virulence of strains they originated. [Conclusion] L. monocytogenes-derived membrane vesicles have got versatile roles, and likely participate in bacteria-bacteria and bacteria-host interactions. These results may be of great significance for the further study of the formation and function of Gram-positive bacteria-derived MVs and the pathogenic mechanism of L. monocytogenes.

Abstract:[Background] Bacteria exist in the form of complex microbial communities in the environment, and the competition among bacteria is an important way for bacteria to survive. Salmonella typhimurium is an important zoonotic pathogen that causes gastrointestinal diseases. It needs to interact with other bacteria in water, food or host intestine to gain survival advantage. [Objective] This study intends to construct a transposon insertion mutant library of Salmonella typhimurium by transposon technology, and to screen the mutant library according to the bacterial competitiveness, thus to explore the relevant mechanism of bacterial competition. [Methods] The EZ-Tn5?Tnp Transposome? kit was used to obtain a mutant library of Salmonella typhimurium containing 1 323 mutants. The competition of the mutants and wild type strain with Escherichia coli JM109 and MG1655 were screened. Then the flanking sequences were identified by reverse PCR, the insertion sites were determined. [Results] This study screened out 13 mutant strains with significant differences in bacterial competition. Among them, 2 mutant strains showed enhanced competitiveness. The inserted mutation genes were polB and flhd. Eleven bacterial mutants with decreased competitiveness were inserted in fstJ, rfbG, recC, rfaI, rfaG, rfbC, udha, plsc, mdh, res and ackA. [Conclusion] Virulence factors, the integrity of bacterial membrane, the normal bacterial metabolic capacity and their natural immunity and the appropriate modification of DNA are all closely related to the ability of bacteria to participate in competition. This study laid the foundation to further explore the specific factors affecting the ability of bacterial competition.

Abstract:In recent years, with the increasing attention to odor pollution, the research on the control of malodorous gas has become a hot field. Compared with physical deodorization and chemical deodorization, microbial deodorization has many advantages, such as low input cost, simple equipment and less secondary pollution. Microbial deodorization has been widely in livestock farms, landfill, and sewage-treatment plant. Four main microbial deodorization techniques, including biological washing, biofiltration, biotrickling filtration and microbial agent addition, are currently used. However, each of these techniques has its own advantages and disadvantages. The processes and mechanisms of microbial deodorization have been intensively studied but the knowledge on them is still limited. Future research in microbial deodorization might focus on: 1) screening microorganisms with high-efficiency deodorizing performance; 2) investigating the spatial and temporal dynamics of microbial community in the deodorization process and the metabolism and degradation kinetics of odorous substances; 3) improving techniques of microbial deodorization.

Abstract:Human gut microbiota is closely related to human health and diseases, so that the modeling and analysis of its metagenomic data is of great significance for scientific research and social application in the field of disease prediction and diagnosis. In this paper, we comprehensively assessed the tools of human gut microbiome data analysis, the principles and processes of prediction algorithms, as well as some typical application cases from the perspective of big data analysis and machine learning. It aims to promote the development of analysis technology for gut microbiome and explore effective approaches for gut microbiome analysis combined with machine learning algorithms. Furthermore, it can also provide reference for the development of new diagnosis and treatment methods based on gut microbiome data.

Abstract:Currently, the prevalence of disorders of glucose metabolism is soaring seriously, especially diabetes. Polysaccharides of edible and medicinal fungi are focused since they exhibit significant benefits against diabetes. However, the mechanisms of them against diabetes are not reviewed well. In this paper, we reviewed the hypoglycemic mechanisms of polysaccharides of edible and medicinal fungi against glucose disorders, taking protein tyrosine phosphatase-1B (PTP-1B) inhibition, insulin pathway regulation, glucose metabolism promotion and gluconeogenesis inhibition, anti-oxidation and anti-inflammation and regulation of intestinal microbiota as examples. However, glucose disorders were not only due to the mutations of key genes or the dysfunctions of key targets, but also caused by multiple abnormalities in overall metabolism. Thus, metabolomics emerged as a new tool for unveiling the mechanisms of polysaccharides of edible and medicinal fungi, since its priorities of probing the systematic changes. Based on it, it was found that polysaccharides of edible and medicinal fungi may ameliorate gluconeogenesis and insulin resistance by modulating amino acid metabolism, relieve cytotoxicity, oxidative stress and inflammation by regulating fatty acid metabolism, maintain serum glucose and microbiota homeostasis by regulating bile acids and ameliorate nephropathy in diabetes by modulating nucleotide metabolism. Combining the considerations of overall mechanisms and key targets may benefit the drug developing of polysaccharides against glucose disorders.

Abstract:The regulator of capsule synthesis (Rcs) system is a non-orthodox two-component regulatory system found in many members of Enterobacteriaceae. This system consists of three core proteins (the transmembrane sensor kinase RcsC, the transmembrane protein RcsD and the response regulator RcsB) and multiple accessory proteins. Rcs system can integrate environmental signals, regulate gene expression and alter the physiological behavior of bacteria. Defining the environmental response mechanism of Rcs system in Enterobacteria has been a major focus in several recent studies. Here, the role of Rcs system in signal perception and conduction, and the target genes regulated by Rcs system and their life phenomena are introduced. This review will highlight the current understanding of the biological role of Rcs system, as well as to provide a theoretical basis for the development of new strategies for bacterial control, prevention and treatment.

Abstract:Aerobic composting is a physiological and biochemical process dominated by microorganisms and is widely used for the recycle-aimed treatment of organic solid wastes. By microbial fermentation, organic wastes can be mineralized, humified, detoxified, and finally converted to organic fertilizers. However, traditional methods of aerobic composting have several disadvantages, including long fermentation period, nutrient losses, and emission of odor and greenhouse gases. Addition of microbial agents during composting process can effectively overcome these disadvantages and improve the quality of composted fertilizers. In this review, the main process of aerobic composting and the major factors influencing aerobic composting were briefly described. The recent proceedings in microbial composition and community succession during aerobic composting, and the application and effects of microbial additives in composting were summarized. In addition, problems in the application of microbial additives were analyzed and possible solutions were discussed.

Abstract:Vaccination has been considered as the most effective method to prevent the further spread of the current corona virus disease 2019 (COVID-19). Nowadays, many research teams world wide have applied different technologies for the research on the vaccines of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This article summarizes the research on multiple aspects, including epitope analysis of SARS-CoV-2, research progress of vaccines and potential risk of vaccination. It discusses the difficulties in the development and application of vaccines in the meantime of evaluating the safety and effectiveness of the vaccines in the respective research routes.

Abstract:Myxobacteriales belongs to Deltaproteobacteria, which is an important group of pharmaceutical-producing microorganisms. However, the isolation and purification of myxobacteria are difficult, which severely limits the exploration and utilization of myxobacteria resources. Myxobacteria are microbial predators, which produce a variety of extracellular hydrolases, such as amylase, protease, chitinase, cellulase, phosphatase, protease etc., to lyse other microorganisms or decompose cellulose as nutrients source. At present, the isolation and purification technology of myxobacteria are mainly based on predator-prey interaction or cellulose induction. Hereby, extracellular hydrolase of myxobacteria is the material basis for studying its isolation and cultivation methods. However, the hydrolases produced by myxobacteria have been less concerned. In this paper, the types, properties and functions of the hydrolases produced by myxobacteria are summarized, which would provide reference sense for the related researches on the isolation and cultivation techniques. Moreover, the exploitation and utilization of myxobacteria resources were also discussed.

Abstract:Foodborne viruses have become an important pathogen causing food safety incidents worldwide, which poses a severe challenge to the continuous development of new detection technologies. The application of PCR in the pathogen detection has promoted the comprehensive understanding of foodborne viruses. In recent years, nucleic acid thermostatic detection technologies have been developed rapidly, including loop-mediated isothermal amplification, recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, etc. They showed obvious technical advantages in resisting complex matrix interference, low equipment requirements and on-site real-time detection, which become a hot direction in the foodborne virus detection. Therefore, this paper reviews the principles, applications, advantages and disadvantages of nucleic acid thermostatic detection technologies for foodborne viruses in recent years, and prospects the development direction in future.

Abstract:In recent years due to some new diseases and the emergence of bacterial resistance, the higher screening repetition rate of secondary metabolites from microorganisms, some metabolic genes not expressed in the laboratory conditions, it is necessary to find new microbial strains and activate genes of microbial metabolites. Marine animals harbor rich symbiotic microbial communities, which can produce many compounds with biological activities, and are potential medicinal resources. This paper reviews the research on the co-culture of marine microorganisms associated with marine animals (ascidians, sponges, corals and sea anemones) in recent years, including the selection of co-culture strains, the conditions of co-culture, the influence of quorum sensing and signal molecules on co-culture strains, and examples of co-culture among different kinds of microorganisms. Compared with single culture, the kinds of secondary metabolites or the biological activity of secondary metabolites or the productions of secondary metabolites are increased in co-culture. Co-culture is an important approach for discovering the active natural products from marine microorganisms associated with marine animals, and a new idea for the development of marine drugs.

Abstract:Klebsiella pneumoniae is one of the common pathogens in nosocomial and community-acquired infections. The clinical specimen isolation rate of Klebsiella pneumoniae is only behind the Escherichia coli in Gram-negative bacteria. Klebsiella pneumoniaes are divided into classic Klebsiella pneumoniae and hypervirulent Klebsiella pneumonia according to the differences characteristics in virulence. Hypervirulent Klebsiella pneumoniae is the main pathogen of suppurative liver abscess, and its infection can cause many endogenous metastases, including eye, lung and central nervous systems. Hypervirulent Klebsiella pneumoniae is also associated with primary extrahepatic infections, including bacteremia, pneumonia, and soft tissue infections. It is noteworthy that in addition to causing severe infection in patients, hypervirulent strains resistant to carbapenem have emerged in recent years, which will bring more challenges in clinical diagnosis and treatment. In this paper, the epidemic status, the virulence factors (including capsular, iron carrier system and virulence genes), and the current status of drug resistance of hypervirulent Klebsiella pneumoniae are reviewed.

Abstract:In recent years, virtual simulation technology has been widely used in experimental teaching and has made remarkable achievements. Due to the characteristics of Medical Microbiology experimental courses, it is particularly suitable for virtual simulation technology application. In a sense, virtual simulation technology reconstructs the experimental teaching system of Medical Microbiology. This study focused on the limitation of the current experimental courses of Medical Microbiology, emphasized the application of virtual simulation technology on the useful practice and exploration of strengthening biosafety education, reconstructing teaching contents and innovating teaching methods. Compared with the traditional methods, this program has greatly enriched the interior of teaching. It could promote comprehensive experimental ability and establish a sense of professional responsibility for patients, which improved the teaching quality significantly. Consequently, it will be good for the construction of virtual simulation experiment and the reform of other basic medical experimental teaching by providing this useful experience.

Abstract:Microbiology is a discipline on the sciences of microbial life and the technologies of microbial application. In order to deeply grasp microbiological knowledge, proficiently master experimental skills and clearly understand social hot topics in limited class hours for the students with different academic backgrounds and different development visions, the curriculum reform of Microbiology has been carried out since 2013 in Zhejiang University. The results showed that the teaching quality was significantly improved by means of changing educational philosophy, integrating curriculum ideology, strengthening practice skills and emphasizing evaluation orientation.

Abstract:COVID-19 is threatening the public health in China and oversea in the spring of 2020. During fighting against COVID-19, course teaching of Virus Biology for bachelor students in the school of life sciences has been adjusted to the new situation in the following aspects, aiming to help students establish scientific and rational viewpoints on the pandemic situation: Besides applying online teaching mode instead of offline mode, teaching contents on virology, epidemiology and public health were strengthened further. Besides the special speech on COVID-19 in the beginning of the new semester, a 5-min talk on COVID-19 has been presented for whole semester. These measurements during teaching of Virus Biology helped students understand scientifically and rationally this pandemic caused by virus infection and spreading. These teaching adjustments were necessary to inspire more interest in virology amongst students, epidemiology and public health, and will be improved with the change of this epidemic situation in China and oversea.

Abstract:The Molecular Enzyme Engineering course is a professional discipline combing the theory and practice. It locates the core position in curriculum provision of bioengineering major postgraduate students and plays an important role in cultivation of professional talents. In order to cater the objective command of professional postgraduate degree development, enhance the innovation of case teaching pattern, promote the practical teaching effect and further improve the postgraduate students’ enthusiasm towards the course as well as cultivate the practical innovation capability, our teaching team investigated the innovation and exploration on the teaching pattern, teaching method as well as teaching technologies for this course by introducing the virtual simulate technology, carrying on the case teaching pattern attempts and utilizing the “Internet plus” technology. In addition, we have obtained some preliminary results and experience for other peers’ reference.

Abstract:[Background] Actinobacteria are a treasure trove of natural products, and 70% of the natural antibiotics currently used in clinics are derived from secondary metabolites of actinomycetes. With the increasing resistance of bacteria to traditional antibiotics, how to efficiently screen new active actinomycete resources from natural habitats and discover new antibiotics has become an important challenge for microbiologists. [Objective] However, screening active actinomycetes by traditional methods not only takes time and effort, consumes large amounts of reagent consumables, but also has a very limited screening throughput, making it difficult to analyze the complex microbial community in natural samples as a whole. This study proposes a new strategy based on micro-well plate droplet array culture, which can screen antibacterial Actinobacteria with high throughput. The study analyzed the culture characteristics and screening conditions of model actinomycetes in micro-droplets, which laid the foundation for the establishment of an ultra-high-throughput bioactive Actinobacteria screening platform based on droplet array technology. [Methods] Our new solution is to use interfacial micropipetting (IMP) technology to miniaturize the traditional multi-well high-throughput screening system to 1 μL level, and generate microliter culture droplet arrays in oil-filled micro-well plates (96-well plates), each microdroplet encapsulates an actinobacterial spore or hyphae. After a short period of cultivation, the actinobacteria complete mycelial differentiation and secretion of secondary metabolites in the microdroplets. At this time, the indicator bacteria with fluorescent markers were added through the second step of IMP and the fusion of the droplets, the active target strains were located through the antagonistic screening of the whole bacteria, and the activity spectrum was converted into quantitative fluorescence values. [Results] the model actinobacteria test was found that the mycelium can reach the optimal culture state in the microdroplets and accumulate enough biomass and metabolites, which have obvious inhibitory effects on the fluorescent indicator bacteria. [Conclusion] By establishing the above-mentioned high-throughput screening technology based on microplate droplet arrays, we can quickly screen strains with antibacterial activity from the single-cell level, which significantly saves screening costs and improves screening throughput, which provides a starting point for the discovery and study of novel functional natural products.

Abstract:[Background] Haemophilus parasuis (HPS) is the pathogen of Gl?sser’s disease. Antibiotic therapy and vaccination are not obvious for the prevention and control of the disease, it is particularly important to establish a rapid and accurate antibody detection method. [Objective] Using the expression and purified transferrin-binding protein (TbpA) of HPS to establish an indirect enzyme-linked immuno sorbent assay (ELISA) method for detecting HPS antibodies. [Methods] The tbpA gene of HPS was cloned and connected pET-SUMO prokaryotic expression vector. After identification by PCR, double enzyme digestion and sequencing, the positive recombinant plasmid was transformed into the receptor bacteria Escherichia coli Rosetta(DE3), and the expression was induced by IPTG, and the expression products were identified by SDS-PAGE and Western Blot. Using purified TbpA as coating antigen, the indirect ELISA method for detecting HPS antibody was established through the optimization of a series of reaction conditions, and its clinical application and evaluation were carried out. [Results] The optimum reaction conditions of this method were as follows: 5 μg/mL, 4 °C overnight concentration of coated antigen; 5% skim milk powder sealed at 37 °C for 2 h; The dilution of serum was 1:1 600, 37 °C incubation 45 min; The dilution of HRP was 1:5 000, 37 °C action 30 min; The optimal reaction time of TMB was 5 min. This method can specifically detect the HPS antibody. The positive serum is still positive after 1:12 800 dilution, but no cross-reactivity with other porcine pathogen positive serum. the coefficient of variation between in the batch and batches all less than 6%. The total coincidence rates of this method were 90.00%, 86.67% and 90.00% compared with the commercial ELISA kit and Western Blot, whole bacterial for indirect ELISA, among which the positive coincidence rates were 90.38%, 88.46%, 92.00%, and the negative coincidence rates were 87.50%, 75.00% and 80.00%, respectively. The positive rate of immune antibody was 80%, and the positive rate of infection antibody was 19.50%. [Conclusion] This study based on the recombinant TbpA established indirect ELISA detection method for HPS antibodies with good specificity, sensitivity and repeatability, as well as the reliability of clinical applications, provides a technical means for HPS immune surveillance and epidemiological investigation.