Abstract:[Background] The filamentous fungus Trichoderma reesei is a eukaryotic microorganism and smaller than any other multicellular, which is widely used in industry, and T. reesei QM9414 were one of the most abundant cellulase producing mutant. [Objective] In order to study cellulase activities and expression regulation in T. reesei, we constructed siRNA interfered vectors and overexpressed vectors to inhibit and enhance HKMT expression in T. reesei QM9414, respectively. [Methods] The siRNA interference fragment was designed according to the hkmt gene sequence of T. reesei and the overexpressed hkmt gene fragment was obtained by reverse transcription. The two fragments were cloned into the constitutive expression vector and the two vectors were transformed the protoplasm of T. reesei QM9414. The hyphae growth of the recombinant strains was observed through a fluorescence microscope. In addition, cellulase activities (CMC enzyme activity and filter paper enzyme activity) of recombinant strains were detected and related gene expressions were also detected by quantitative PCR. [Results] The mycelial morphology of the interfered and the overexpressed recombinant strains were not significantly different from the original strain. Quantitative PCR results showed that the interfered vector and the overexpressed vector can silence and promote the expression of hkmt gene, respectively. The enzyme activities of FPA and CMC in the interfered recombinant strains were increased by an average of 2.5 times compared with the original strain. In addition, the expression levels of cellulase-related genes and activators in interfered recombinant strains increased. However, the recombinant strains for overexpressing HKMT showed the opposite patterns in the genes’ expression levels and enzymatic activities. [Conclusion] These findings indicate that HKMT in T. reesei is involved in the negative regulation of cellulase expression and production.

Abstract:[Background] Bio-butanol is a powerful liquid fuel. The products of butanol fermentation (acetone-butanol-ethanol fermentation, ABE fermentation) are the mixture of butanol, acetone and ethanol, weight ratio of the primary product—butanol over the major by-product—acetone (B/A ratio) is about 2.0. [Objective] Butyric acid is an important precursor for butanol synthesis in ABE fermentation. Using butyric acid/glucose as co-substrate would efficiently produce butanol featured with high B/A ratio and improve the quality of ABE fermentation products. [Methods] In a 7-L anaerobic fermentor, the supernatant originated from butyrate fermentation (BFS) with corn starch/waste Pichia pastoris as the raw materials was directly mixed with glucose solution as the ABE fermentation medium, then BFS and concentrated glucose solution were fed during the fermentation as required. [Results] Compared with traditional ABE fermentation using 150 g/L corn, butanol concentration could be maintained at an equivalently high level of 12.7?12.8 g/L, B/A ratio largely increased from 2.0 to 4.4?5.0, butanol yield to total carbon source also increased from 0.32?0.34 to 0.39?0.41 (mole base), butyrate/glucose consumption ratio reached high levels between 37% and 53%. The ABE fermentation benefited from the efficient utilization of butyrate, oligosaccharides, amino acids, etc. in the BFS and the enhanced NADH utilization efficiency. [Conclusion] The proposed fermentation strategy could save raw materials and operating costs of ABE fermentations, largely reduce the residual oligosaccharides concentrations in BFS, increase diversity and flexibility of the fermentation processes based on variations of marketing requirement and products prices, which have reasonably high economic and environmental effects.

Abstract:[Background] Adaptive laboratory evolution is one of directed evolution methods to improve strain fitness. It improves the tolerance to toxic products or rapidly utilizes specific substrates. Due to the rather low mutation rate of native cells caused by the mismatch repair mechanism in the genome, adaptive laboratory evolution usually takes a long period to screen desired evolved strains and more likely to fail because of microbial contamination during a long process. [Objective] To develop controllable hypermutable cells of Bacillus subtilis. [Methods] A marker-free mutation delivery system is used to replace the mismatch repair system operon MutSL with a rigorous xylose-inducible promoter. [Results] The mutation rates of controllable hypermutable B. subtilis cells could be regulated by the concentration of inducer (xylose). The phenethyl alcohol tolerance experiment indicated that the hypermutable B. subtilis cells had better tolerance than the original strain, suggesting that it is more easily to obtain the mutant with desired trait. The adaptive evolution experiment of growth inhibition by substrate crude glycerol showed that the evolved strain showed the shorter lag time, higher biomass, and greater specific growth rate than the starting strain. [Conclusion] The hypermutable B. subtilis cells had better evolvability and adaptability, which could have a wide range of applications.

Abstract:[Background] Phoebe bournei (Hemsl.) Yang is a precious timber species. Clarifying the characteristics of rhizosphere soil microorganism of P. bournei is helpful for scientific management of P. bournei plantations. [Objective] To understand the changes of arbuscular mycorrhiza community in rhizosphere soil of P. bournei (Hemsl.) Yang plantation with forest age after the afforestation. [Methods] The diversity and composition of arbuscular mycorrhiza fungal communities in the rhizosphere soil of P. bournei plantations at different ages were evaluated by high-throughput Illumina Miseq sequencing. Then, we analyzed response characteristics of soil fugal communities in the rhizosphere soil of P. bournei plantations to changing soil nutrient after afforestation. [Results] In total, 342 555 effective sequences and 253 AMF-OTUs were obtained from the rhizosphere soils of 3 forest ages of P. bournei, belonging to 14 genus, 9 families, 5 orders, 1 class, 1 phylum. In terms of alpha diversities, both Chao1 and Shannon indices of AMF community increased with the plantation age of P. bournei, but there was no significant difference among different plantation ages. There was significant difference of β diversity in arbuscular mycorrhiza fungal community composition among different plantation ages. According to the Bray-Curtis dissimilarity analysis, the similarity of AMF community composition was related to plantation ages. The Spearman correlation analysis between species richness and soil factors indicated that the majority soil factors affecting the abundance of arbuscular mycorrhiza fungal communities were pH, total potassium (K), nitrate nitrogen (NO3–-N) and ammonium nitrogen (NH4+-N). Ammonium was significantly correlated with the abundance of dominant arbuscular mycorrhiza fungal molecular virtual species Glomus-Franke-A1-VTX00076 and Glomus-Franke-A1-VTX00269. [Conclusion] Our findings reveal the relationship between soil microbial diversity and the ecosystem of Phoebe bournei (Hemsl.) Yang plantations and provide reference for understanding the relationships between biodiversity and ecosystem functions.

Abstract:[Background] Phosphorus is the main nutrient that causes eutrophication in water. Biological phosphorus removal has the characteristics of low cost, high efficiency and wide application range, and has become a hot spot in the field of water treatment research in recent years. Although some phosphorus-accumulating bacteria have been screened, their phosphorus removal efficiency is not high, the phosphorus removal conditions of the phosphorus-accumulating bacteria need to be optimized. Immobilization and recycling of the phosphorus-accumulating bacteria are urgently needed to be studied. [Objective] Isolation, screen and identify high-efficiency phosphorus-accumulating strains, optimize the environmental conditions for phosphorus removal, explore the impact of adsorbent materials on the phosphorus removal of the bacteria, and provide a theoretical basis for the development and utilization of phosphorus-accumulating bacteria. [Methods] High-efficiency strains were obtained by routine isolation and screening of bacteria, and the strains were identified through morphological observation, physiological and biochemical experiments and 16S rRNA gene sequence analysis; Combined with single-factor experiments, Box-Behnken design and response surface analysis to optimize phosphorus removal conditions; The immobilization effect of the material was evaluated by measuring the adsorption effect of sponge, non-woven fabric and polyurethane foam on phosphorus-accumulating bacteria. [Results] A high-efficiency phosphorous-accumulating bacterium P49 was isolated and screened from the wastewater of a phosphate mine in Lianyungang city, which was identified as Bacillus amyloliquefaciens; The optimal conditions after optimization were pH 6.8, temperature was 31 °C, equipment the liquid volume was 30.2%. Under this condition, the phosphorus removal rate of P49 can reach 80.43%; The application effect of polyurethane foam was better than that of sponge and non-woven fabric. [Conclusion] Bacillus amyloliquefaciens P49 had a good phosphorus removal effect and provides microbial resources for biological phosphorus removal. Adsorption with polyurethane foam can achieve the immobilization and recovery of the strain.

Abstract:[Background] Whether in the nature or in experimental products, huntite is rare carbonate mineral. [Objective] Explore the cause of the unexpected discovery of the huntite during the culture experiments, on the basis of comprehensive analysis of the condition of all experiments related to huntite, to further explore the formation of the minerals. [Methods] A series of cultural experiments in the medium with Curvibacter sp. strain HJ-1 and initial Mg/Ca ratio of 5.0 were carried out for 30 days. During the incubation, cell density, pH, concentration of Ca2+ and Mg2+ in the medium were determined according to the scheduled time. Mineral type was identified by X-ray diffraction (XRD) and the morphology was observed by scanning electron microscopy (SEM). [Results] The pH value of the medium with strain HJ-1 increased, while the concentrations of Ca2+ and Mg2+ decreased gradually with incubation time. For control experiments (CK) without bacteria, the above three indicators were stable. The precipitation of huntite began to form on the 8th day and continued to the end of the experiment (30th day), while any minerals were not formed in the CK system. XRD patterns indicated that the number of crystal planes of huntite is increasing gradually. This means that the crystallinity of huntite tends to be better with incubation time. [Conclusion] The culture with Mg/Ca ratio of ≥2.0, the total concentration of calcium and magnesium ions of 0.03?0.07 mol/L, saturation index above 1.95 and presence of microorganisms is favorable for the formation of huntite.

Abstract:[Background] Marine microorganism is a treasure pool of natural medicine resources. Marine microorganisms produced many natural products that are different from those produced by terrestrial microorganisms. Mangroves grow in tidal flats at the junction of land and ocean, it is a special ecosystem transitioning from land to ocean, and may contains abundant microbial resources and potentially a large number of metabolites with novel structures. [Objective] This study takes sea mud and sea water from the rhizosphere of mangroves in Xinglin Bay, Xiamen as the research object, to explore the diversity of culturable marine fungi and the antibacterial and antifungal activities of the extracts, to provide strain resources for the discovery of new drug lead compounds. [Methods] After pretreatment, the samples were coated on GPY, PDA, Martin and MEA media respectively, and were cultured upside down at 28 °C. Single colonies were selected and inoculated in PDA media to yield fungal strains. The antimicrobial activity of the extracts of the isolated fungi were screened by disk diffusion test and the biodiversity of the crude extracts were evaluated by high performance liquid chromatography, and four active fungi were identified by colony morphology and rDNA ITS sequence analysis. [Results] A total of 71 fungi were isolated, 49 of which showed bioactivity against Staphylococcus aureus, 6 against Candida albicans and 2 against Escherichia coli. HS5-MEA-4 and HS6-MEA-10 were identified as Aspergillus terreus, HS5-GPY-7 and HS6-GPY-15 were identified as Aspergillus aculeatus and Aspergillus templicola, respectively. [Conclusion] The activity characteristics of fungal extracts from the marine sedimentary environment of Xinglin Bay, Xiamen were preliminarily revealed, which provides resources for the subsequent biodiversity of antibacterial and antifungal secondary metabolites.

Abstract:[Background] As a new type of fuel cell resource, microbial fuel cell (MFC) can be used in sewage treatment field to maximize resources while generating electricity. [Objective] A culturable microorganism isolated from MFC was used to study its electrical performance and to characterize the flocculation ability, heavy metal tolerance and the possibility of phenol degradation in sewage treatment of Stenotrophomonas sp. which provides a theoretical basis for expanding the resources pool of electricigens. [Methods] An electrogenic bacterial strain named EFS1was isolated from the anode of MFC using a WO3 nano-probe. The anode electrode was observed by cyclic voltammetry combined with scanning electron microscope, and the polarization curve and power density curve were measured by changing the external resistance. Finally, the flocculation, heavy metal tolerance and phenol degradation of the strain were determined. [Results] The strain was identified as Stenotrophomonas acidaminiphila through the evidences of 16S rRNA, morphology, physiology and biochemistry. The strain EFS1 had a stable cycle of electricity production, the maximum periodic voltage was 300 mV, and the power density was up to 56.25 mW/m2, scanning electron microscopy found that the strain had direct contact with electrodes and secreted electron mediators to transfer electrons; the internal resistance of MFC was about 1 000 Ω. The flocculation rate of the strain could reach 70% under aerobic conditions and 80% in an anaerobic environment with electron acceptor. The strain also had good tolerance to Cd2+, Cu2+, Mn2+ and phenol degradation performance, in which the phenol degradation rate reached 100% at 48 h and 2–4 mg/L. [Conclusion] This study verified that the electrogenic bacteria strain EFS1 had flocculation ability, heavy metal tolerance, and the possibility of phenol degradation and provide theoretical base for the development of electricity producing bacteria and sewage treatment.

Abstract:[Background] There are abundant bacterial resources in the cave environment. The isolation and cultivation of bacteria in the cave helps for the understanding bacterial diversity, as well as the exploration and utilization of bacterial resources. [Objective] To isolate the bacteria by using different media, and to explore the diversity and interactions of cultivable bacteria on the stalactite surface in cave. [Methods] Totally, 11 kinds of media were used to isolate and purify the bacteria, then the taxonomic position of the isolates was preliminarily determined by analysis of their 16S rRNA gene sequence. Additionally, bipartite packet was used to analyze the interaction among the culturable bacteria under R environment. [Results] A total of 206 strains of bacteria were isolated from the samples. These bacteria belonged to 45 species of 4 genera and 25 genera in community with the Shannon index of 4.78 and the Simpson index of 0.95. Proteobacteria and Bacillus are the dominant phylum (47.09%) and genus (29.61%), respectively. The inorganic oligotrophic media were helpful for the isolation of bacteria from stalactite sediments in the cave. Based on genus level-sampling site network analysis shows that the distribution of culturable bacteria has a non-random and significant nestedness community. Bacillus, Devosia, Alcaligenes, Arthrobacter, and Brevibacterium with more effective cooperation and closeness in community are highly dependent on other bacteria, and are the key groups in the community. [Conclusion] There are abundant bacterial resources on the surface of stalactite sediments in Zhijin cave. The relative abundance and network analysis of different groups should be combined to determine the role of bacteria in communities.

Abstract:[Background] Restriction endonuclease Mlu I is a commonly used tool in molecular biology. However, its three-dimensional structure has not been determined. [Objective] Mlu I gene was cloned and expressed in Escherichia coli. Mlu I and its seleno-derivative proteins were purified, and the crystallization conditions were studied. [Methods] A recombinant expression vector pET28b-Mlu I was constructed and expressed inductively in E. coli BL21(DE3)pLysS. Affinity and gel filtration chromatography were used to purify Mlu I and Se-Mlu I proteins. Mass spectrometry, circular dichroism and enzyme activity analysis were carried out to characterize these proteins. Sitting drop method was used to screen the crystallization conditions. [Results] The recombinant expression vector pET28b-Mlu I was successfully constructed and the purified Mlu I and Se-Mlu I proteins with purity suitable for crystallization were obtained. All 8 methionines were successfully replaced with se-methionines in the Se-Mlu I protein determined by mass spectrometry. The circular dichroism and enzyme activity analysis confirmed that se-methionines replacement had no significant effect on the activity and structure of the Mlu I protein. Preliminary crystallization study and subsequent X-ray diffraction showed that the needle-like crystal of Mlu I formed under one condition diffracted to a resolution of 0.32 nm. [Conclusion] The construction of Mlu I and Se-Mlu I protein purification system and study of crystallization conditions layed the foundation for further analysis of the three-dimensional structure of Mlu I, revealing the molecular mechanism of Mlu I, and directed evolutionary modification of Mlu I.

Abstract:[Background] The characteristic peak at 420?425 nm was frequently observed in anoxygenic phototrophic bacteria and often assigned to carotenoid component(s). However, the absorption peak at 423 nm in Rhodobacter azotoformans R7 did not exhibit the characteristic absorption of carotenoid(s). [Objective] To elucidate the formation mechanism of the 423 nm absorption peak in strain R7. [Methods] Analyses by UV-VIS spectrophotometry, thin layer chromatography, high performance liquid chromatography, mass spectrometry, ultracentrifugation and ion exchange chromatography were conducted to investigate the formation origin of the 423 nm peak. [Results] A characteristic absorption maximum at approximately 423 nm was displayed in the in vivo absorption spectrum of strain R7 cultured in glutamate medium, while it was shifted to approximately 415 nm in the absorption spectrum of pigment extract. However, the cell growth and the amounts of bacteriochlorophyll (BChl) and carotenoid (Car) of strain R7 were significantly reduced by addition of glutamate, compared to those with the supplement of yeast. Pigment composition analysis showed that the 415 nm peak was only characterized by magnesium protoporphyrin IX monomethylester (MPE). MPE could locate on the intracytoplasmic membrane with an absorption peak at 423 nm. These results indicated that the 423 nm absorption peak was caused by MPE accumulation. The analyses of pigment protein complex (PPC) showed that three components of PPCs were detected in both yeast and glutamate cultures, however, only the absorption spectra of peripheral light harvesting complex 2 (LH2) and the reaction center (RC) of glutamate cultures gave the 423 nm characteristic peak. Collectively, strain R7 was capable of produce two different types of LH2; MPE locating on either LH2 or RC could form the characteristic peak of 423 nm. [Conclusion] The characteristic peak at 423 nm in strain R7 originated from MPE accumulation rather than carotenoid. The accumulated MPE inside cells could bind to LH2 and RC and locate inside the intracytoplasmic membrane. It is difficult to obtain a large amount of MPEs as it is a biosynthetic intermediate of BChl. Strain R7 was capable of accumulating MPE, therefore, the further study for biosynthesis regulation of MPE contributes to elucidate the novel mechanism of photooxidation and photoprotection in photosynthesis.

Abstract:[Background] Ferulic acid and its analogues, accumulated in soil, are harmful to crop growth. It is an effective solution to decompose these substances by microorganisms. [Objective] A strain that could degrade ferulic acid efficiently was isolated from the soil. The ferulic acid degradation efficiency of the strain was evaluated for further application of the strain. [Methods] The strain was screened by the special culture containing high concentration of ferulic acid. The partial 16S rRNA gene sequences were used to identify the strain. The ferulic acid degradation characters of the strain were detected by HPLC. [Results] The strain was Arthrobacter sp. and named J6. High degradation efficiency was obtained at ≤1.20% saline stress (i.e., 12 g/L NaCl), pH 5.0?9.0, temperature at 20?40 °C, with/without glucose and with/without yeast. [Conclusion] strain J6 had good application potential in bioremediation of ferulic acid.

Abstract:[Background] In Northeast China, a major maize (Zea mays L.) production region, maize yield is limited severely by insufficient soil potassium supply. Potassium-solubilizing bacteria can mobilize insoluble soil potassium, improve soil potassium availability, and promote maize growth. [Objective] To identify efficient potassium-solubilizing bacteria from maize rhizosphere in black soil, verify their growth-promoting effects under potassium-deficient field condition, so as to provide elite bacteria resource for producing microbial potassium fertilizers adapted to the local environment. [Methods] The bacteria isolated from maize rhizosphere were screened on potassium-solubilizing medium. The taxonomy of the selected potassium-solubilizing bacteria was identified by 16S rRNA gene sequencing. The ecological adaptability (acid and alkali resistance, salt resistance, drought tolerance and pesticide resistance) of the selected bacteria were determined by culturing them on diverse physiological and biochemical mediums. A two-year field inoculation experiment was conducted to verify their effects on maize growth and yield under the condition of potassium deficiency. [Results] Three efficient potassium-solubilizing bacteria, MZ4, KM1 and KM2, were selected. MZ4 and KM2 were identified as Bacillus sp., and KM1 was identified as Brevibacilluse sp.. All the three strains can tolerate drought stress, acid and alkali stress, pesticide (imidacloprid), fungicide (azoxystrobin), and salt stress to a certain degree. In the field without potassium application, the inoculation of MZ4, KM1 and KM2 increased plant height, shoot biomass, leaf area index and chlorophyll content of maize at the jointing and flowering stages. Inoculation with MZ4 and KM2 significantly promoted grain yield by 9.65%?11.50%. [Conclusion] The efficient potassium-solubilizing bacteria which adapt to the black soil in Northeast China were identified. These bacteria can be used as elite germplasm resource for producing microbial potassium fertilizer and analyzing the mechanism underlying efficient potassium solubilization by microorganism.

Abstract:[Background] Sorghum anthracnose in Heilongjiang province is one of the main sorghum diseases, which seriously affects the yield and quality of sorghum. [Objective] To definitize the pathogen species of sorghum anthracnose in Heilongjiang province. [Methods] The pure culture of pathogenic fungi causing sorghum anthracnose was obtained by tissue separation and single spore purification. The isolated pathogenic fungi were confirmed by Koch’s rule. The morphological characteristics of colony, sporulation structure, morphology and size of conidia, were used to identify the pathogen combined with rDNA ITS sequence characteristics. [Results] Three isolates D13, H4 and Z24 isolated from different regions were pathogenic fungi of sorghum anthracnose by Koch’s rule. Morphological observation showed that the colony growth rate of 3 strains on PDA medium (28 °C) was 7.3?12.3 mm/d, the hyphae was white, grayish white to grayish brown, and the colony was light yellow to orange. Setae erected and its base distended, were brown or dark brown, with 3?5 grids. Conidiophore generates directly from hypha, colorless and eseptate, short, erected or slightly curved, unbranched. Conidia are unicellular, sickle, acuminate to both ends, smooth, colorless, sometimes with an oil ball. The appressorium produced from the apex of the hyphae or mycelium, the apex is dilated, with color of brown or dark brown, subglobose, ovoid, elliptic or spindle-shaped and its margin was smooth, lobes, more lobes or deep lobes. The rDNA ITS sequence accession numbers obtained from isolated D13, H4 and Z24 were MW040055, MW040057 and MW040056, respectively. The phylogenetic tree based on rDNA ITS sequence found that the D13, H4 and Z24 of the isolates were together with Colletotrichum sublineola. [Conclusion] The pathogen causing sorghum anthracnose in Heilongjiang province is C. sublineola.

Abstract:[Background] Daqing is located in Songnen plain, which land is heavily salinized. Saline-alkali soil contains a large number of saline-alkali-resistant strains, some of which have special functions of promoting growth and can be used as beneficial bacteria to promote plant growth and improve plant saline-alkali-resistance. [Objective] In order to solve the problem of land salinization effectively and increase the yield of grain crops. [Methods] The saline-alkali soil collected in Daqing area as an experiment material was used to separate the saline-alkali tolerant bacteria under pH 9.0, 50 mmol/L NaHCO3, the growth promoting function of the strain was screened by functional medium, and the yield of corresponding substance was measured accordingly. The strains with complete promoting function and better promoting effect was selected, using analyses of morphology, physiological biochemistry, 16S rRNA gene sequence to identify the strains, so as to determine their taxonomic status. The growth promoting strains were tested on physiological indicators such as germination rate, root length, bud length, rooting number and root length of seeds of red adzuki bean to verify the growth promoting effect under pH 8.5 and 15 mmol/L NaHCO3. [Results] The strain 29 was Zobellella, and the others were Halomonas. All the five strains had the functions of nitrogen fixation and produced indole-3-acetic acid (IAA) and ACC deaminase. The strain 34 and strain 23 had the highest yield of IAA and ACC deaminase, reaching 17.66 mg/L and 0.31 U/mg respectively. In addition, the strain 23 was capable of producing siderophore, which content was 3.19%. The strains 29 and 41 had the ability of organophosphorus-dissolving and the phosphorus solution capacity is 20.05 mg/L and 34.61 mg/L. The results of plant growth promotion test showed that the germination rate and root length of red adzuki bean seeds were increased respectively by 41.67% and 32.63% after the seeds were treated with inoculation under saline-alkali stress. After inoculation, the root length and root number of seedlings increased by 129% and 160%, respectively, compared with saline-alkali stress control. [Conclusion] The five saline-alkali tolerance and growth-promoting bacteria could alleviate the inhibition of saline-alkali stress on the growth of red adzuki bean and promote the growth of its root system. This provides an excellent strain for the development of saline-alkali tolerant and growth-promoting microbial fertilizer.

Abstract:[Background] Short-chain fatty acids (SCFAs) have a wide range of physiological activities and biological effects, such as providing energy, regulating nutrient metabolism, inhibiting endogenous cholesterol synthesis, and so on. [Objective] The model of Caco-2 cell absorption SCFAs was established, which was used to research the effect of Lactobacillus on intestinal uptake of SCFAs. [Methods] The integrity and stability of Caco-2 cell model was evaluated by transepithelial electrical resistance (TEER), cell ultrastructure, apparent permeability coefficients of phenol red (Papp) and cell proliferation toxicity test. The content of propionic acid and butyric acid in the model was determined by gas chromatography mass spectrometry (GC-MS) before and after inoculating with Lactobacillus. [Results] The TEER of Caco-2 cell monolayer was 1 290.73 Ω·cm2 on the 11th day, 1 319.31 Ω·cm2 on the 15th day, and the Papp was less than 1×10?6 cm/s, the cells were closely connected and covered with a layer of microvilli perpendicular to the cell surface. The survival rates of Caco-2 cells were 99.03% and 91.42% after treating with 1 mmol/L propionic acid and 1 mmol/L butyric acid for 3 hours respectively. The propionic acid content of Caco-2 cells in the model inoculated with Lactobacillus plantarum P54, P58, P67, P97, P123 and P198 was higher than non-inoculate significantly (P<0.05), and the butyric acid content of Caco-2 cells in the model inoculated with Lactobacillus fermentum F146 and Lactobacillus plantarum P1 was higher than non-inoculate significantly (P<0.05). [Conclusion] The Lactobacillus strains used in this study can promote the absorption of propionic acid or butyric acid by Caco-2 cells in the model.

Abstract:[Background] A fungus producing a neutral protease was isolated from Daqu of Beidacang liquor and identified as Fusarium oxysporim M1 by morphology and molecular biology in our preliminary work. Neutral proteases are important enzyme preparations which have been widely used in industrial production, such as food, medicine, leather, feed, chemical and waste treatment owing to its mild reaction conditions and high catalytic rate. [Objective] In order to apply the neutral protease from Fusarium oxysporim M1 to related industrial production, it is very necessary to purify the protease and study its enzymatic characteristics. [Methods] The protease was purified through ammonium sulfate fractionation, hydrophobic and ion exchange chromatography in this paper, and then its molecular weight was determined by SDS-PAGE electrophoresis. At same time, the thermal stability and acid-base adaptability of the enzyme were also studied. [Results] The protease purification fold and yield were 26.1 and 7.9% by different chromatography steps. The molecular weight of the purified enzyme was 62 kD. The optimum temperature and pH value were 40 °C and 7.0 respectively. The protease belongs to a neutral protease which is sensitive to acid but strong tolerance to alkali. However, it is interesting that the enzyme have strong heat resistance, but is not inhibited by EDTA. [Conclusion] Therefore, the protease from Fusarium oxysporim M1 can be used as a biological catalyst used in different industrial applications in the future.

Abstract:[Background] rabbit hemorrhagic disease virus 2 (RHDV2) was found in April 2020 in China, which seriously threatened the rabbit breeding industry and ecological balance. Besides, basic researches on the etiology and genetic characteristics of RHDV2 are still lacking in China. [Objective] To isolate and identify RHDV2 strain, and to conduct whole gene sequencing and genetic evolution analysis of the isolated strain. [Methods] We planned to conduct pathological dissection, RT-qPCR detection, and animal regression test to isolate and identify the RHDV2 strain in a rabbit farm suspected to have died from RHDV2 infection. [Results] Further whole gene sequencing and genetic evolution analysis were performed. The necropsies of dead rabbits showed hemorrhage and enlargement of all parenchymal organs, especially the heart, lung and liver. RHDV2 was confirmed by RT-qPCR, and there was no mixed infection with other pathogens. We found the infected rabbits could cause similar lesions. The isolates were named SCCN03, and the gene sequences is 7 464 bp in length, which was about 99.21% consistent with the reference strain (GenBank accession number: MN901451.1). By comparing the amino acid sequence of the reference strain, many missense mutations occurred in the amino acid sequence of the non-structural proteins and structural proteins in the isolated strain, among which several missense mutations of the non-structural proteins P16 and structural proteins may be related to the variation of the virus strain. The evolutionary tree showed that SCCN03 belonged to GI.2 genotype. [Conclusion] One strain of RHDV2 was isolated and identified, and its gene sequence was obtained, which enriched the whole genetic data of RHDV2 and laid a foundation for subsequent studies on virulence of RHDV and related vaccine development.

Abstract:[Background] As a normal intestinal flora of human and animal, Enterococcus faecalis was also a conditional pathogenic bacterium, which could infect human and animal. [Objective] To analyze the pathogen of which cause the death of the chicks in chicken farm, and choose the effective drug. [Methods] The pathogen was isolated from clinical sample based on the clinical symptoms and pathological examination. Then, the physiological and biochemical characteristics, 16S rRNA gene sequence, pathogenicity and drug resistance were conducted to identify the pathogen. Drug therapy was performed to screen the effective drug towards the pathogen. [Results] The clinical symptoms of lethargy, paralysis and ataxia in sick chick could be observed. Swelling spleen, yellow, swelling, brittle and fragile liver with a small amount of bleeding points, thickened and bleeding intestinal mucosa, and mild cerebral edema could be observed by pathological examination. A gram-positive coccus, named CJ517 after purification and culture, was isolated from liver tissue. It was identified as Enterococcus faecalis according to physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The pathogenicity test showed that the mortality rate of CJ517 on mice was 66.67%. It is sensitive to cefotaxime, fosfomycin, butyramecana, while as resistant to doxycycline, kanamycin, neomycin and florfenicol. The disease was relieved after treating with sensitive drug combined immunopotentiator. [Conclusion] The result could provide reference for diagnosis and treatment of disease caused by the infection of Enterococcus faecalis.

Abstract:[Background] Microalga Desmodesmus sp. QL96 was isolated from Tibet Plateau, China. We had previously found that the microalga was able to grow at both 4 °C and 25 °C, and produced 71.68% (W/W) cellular proteins which showed antioxidant activities. [Objective] Herein, an antioxidant protein in Desmodesmus sp. QL96 cells was purified and its structure was analyzed. [Methods] Purification of the antioxidant protein was performed through chromatography. The antioxidant activity of the purified protein was determined using chemiluminescence assays and in vitro cell cultures. The structure of the purified protein was then analyzed using mass spectrometery. [Results] The antioxidant protein took up 11.40% (W/W) of the dry microalgal cells. The purified protein demonstrated scavenging capacity to free radicals of OH?, DPPH, ABTS and H2O2 (over 60% scavenged). In vitro cell cultures using liver carcinoma cell HepG2 demonstrated that the Desmodesmus sp. QL96 antioxidant protein could reduce oxidative damage to the cells. The amino acid sequence of the protein determined using a mass spectrometery showed that the molecular weight of the antioxidant protein was 44.8 kD with a pI of 5.79. The homologous rate of the protein with the known proteins in NCBI was below 59%. [Conclusion] Microalga Desmodesmus sp. QL96 produces a previously uncharacterized protein which demonstrates antioxidant activity. The transcript of the protein will be analyzed to verify the genetic sequence similarity in the future, as well as its potential function and industry applications.

Abstract:[Background] Radioresistant microorganisms are important type of extreme microbial resources, which are of great significance for studying radiation-resistance mechanism and environmental protection. [Objective] To analyze the genetic background of Micrococcus luteus V017 and the transcriptome response to radiation through genome and transcriptome. [Methods] The full genome of strain V017 was sequenced and its unique gene sequence was revealed by comparative genome analysis. Further, transcriptome response of strain V017 to gamma ray radiation was performed, and then followed by functional analysis of their differentially expressed genes. [Results] The whole genome of V017 was obtained through de novo assembly. The genome size was 2 527 399 bp with GC content of 72.9%. Comparative genome analysis revealed many mobile element sequences unique in V017 genome, which may be helpful for it to adapt to the extreme irradiation environment. Transcriptome analysis showed that the basic metabolic pathways such as fatty acid and tryptophan metabolic pathways were significantly down-regulated. By contrary, expression level of the DNA damage repair pathways was significantly up-regulated. Especially, homologous recombination repair may play a major role in response to irradiation. [Conclusion] The genomic and transcriptome data analysis implied that the specific mobile sequences of strain V017 may help to adapt to the radiation environment through modulation of DNA rearrangement. Alternation of DNA damage repair and basic metabolic pathways in response to radiation stress may be an important reason for the moderate radiation resistance of strain V017. These studies laid the foundation for further understanding the adaptation and resistance mechanisms of radioresistant microorganisms, as well as the utilization of its microbial resources.

Abstract:[Background] The abuse of antibiotics leads to the increase and spread of drug-resistant pathogens, which has become an important factor threatening people’s health. Therefore, it is particularly important to explore new antibiotics. New species are likely to produce new bioactive substances, especially rare actinomycetes which are rarely isolated by conventional methods. [Objective] To confirm classification of two novel species of Microbispora and predict the secondary metabolic gene clusters by various molecular biological technologies, which will lay a foundation for the discovery of the medicinal actinobacteria producing novel active substances. [Methods] Taxonomic positions of isolates were preliminarily determined by the 16S rRNA gene sequence analysis. The whole genomes were sequenced and annotated by Illumina genome analyzer. On this basis, a whole-genome-based phylogenomic tree was constructed, and the ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization) values were calculated, so as to determine the taxonomic status of novel species. Based on gene annotation, COG (clusters of orthologous genes), KEGG (Kyoto Encyclopedia of Genes and Genomes), and secondary metabolic gene cluster prediction on antiSMASH were analyzed. Antimicrobial activity was tested by cylinder plate method. [Results] The ANI and dDDH values between strain H10836 and 8 species of the genus Microbispora were 85.3%?92.1% and 33.0%?44.5%, respectively. Meanwhile, the ANI and dDDH values between strain H11081 and 8 species of the genus Microbispora were 85.2%?92.1% and 31.3%?44.5%, respectively. Therefore, all the ANI and dDDH values are well below the cut-off point recommended for delineating species. In addition, a whole-genome-based phylogenomic tree showed that strains H10836 and H11081 formed an independent monophyletic branch within the genus Microbispora. Therefore, these results of phylogenetic analysis support the conclusion that strains H10836 and H11081 could be considered to represent two potential novel species of the genus Microbispora. Furthermore, there were many kinds of biosynthetic gene clusters in the genomes of both strains H10836 and H11081 by antiSMASH analysis, and the similarities with known antibiotic synthetic gene clusters were low. The metabolites of two strains exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). [Conclusion] Strains H10836 and H11081 are two novel species with antibacterial activity, so it is worth mining their novel active natural products. The results of this experiment provide reference for further research and application of the strains.

Abstract:[Background] Currently, with the in-depth study of the yeast and the completion of the whole genome and mitochondrial gene sequence determination, Yarrowia lipolytica is popular for using in experimental research and industrial production. However, compared with conventional yeasts, the lack of effective genetic transformation system is also difficulties in gene regulation for Y. lipolytica. At the same time, the chromosome ploidy of yeast will also affect the transformation. When the haploid cells are as receptors for functional gene modification, it can avoid the effect of interaction between alleles. Solve the problem of incomplete gene knockout in polyploid cells. [Objective] The mutant strain P12 of Y. lipolytica was used as the research object. The haploid protease was isolated by different methods, with the aim of establishing a method of preparing haploid strains of Y. lipolytica. [Methods] Y. lipolytica strain was induced to produce ascospores by solid McClary sporulation medium with the culture condition at 30 °C for 7?14 d and liquid McClary sporulation medium with the culture condition at 200 r/min for 2?4 d, respectively. Then the ascospore cell wall was lysed with 2% snail enzyme at 33 °C for 3 h. Finally, the haploid cells were screened and identified by staining microscopy and PCR. [Results] The growth rate of Y. lipolytica was faster in liquid sporulation medium, and the quality of sporulation was better in solid sporulation medium. In the same field of view, the number of spores in the liquid sporulation medium was about 3.7 times higher than in the solid sporulation medium. Moreover, six strains of Y. lipolytica P12 type B haploid were obtained through preliminary exploration and screening. [Conclusion] This research laid the foundation for the subsequent continued operations of genetic engineering.

Abstract:[Background] Cytotoxin associated gene A protein (CagA) is one of the most important effectors of Helicobacter pylori, and its polymorphism is suggested to be involved in the development and progression of gastric carcinoma. [Objective] To compare the structural differences of CagA as an H. pylori clinical isolate, and explore its effects on the morphology and function of gastric epithelial cells. [Methods] A total of 27 sequences of CagA were analyzed to reveal the differences in composition and amino acids variation. Five H. pylori strains containing different CagA sequences were used to infect low malignant gastric epithelial AGS cells for 6 hours at a multiplicity of infection (MOI) of 30. Then the cell morphology was observed by the microscope and the expression of polarity-regulating kinase 1b (PAR1b) was determined by the Western Blot. Finally, the concentration of interleukin-8 (IL-8) in the medium was detected using enzyme-linked immunosorbent assay (ELISA). [Results] It was found that there were differences in the structure and composition of amino acid of CagA. In particular, the EPIYA motifs of Western strains was detected with more variation, and the cell morphology of the strain with the intact CagA gene changed significantly after infection. Compared with the control group, the expression of PAR1b in the AGS cells infected with Western strains of NCTC 11639 and 26695 increased significantly, while expression of PAR1b in the cells infected with East Asian strain GZ7 decreased significantly, and both of the differences were statistically significant (P<0.05). However, there was no significant difference in the expression of PAR1b in the AGS cells infected with H. pylori GZ15 and H. pylori GZ7/ΔcagA. The concentration of IL-8 in the experimental group was higher than that in the control group for East Asia and Western strains (P<0.05). Furthermore, the potential of East Asia strains to promote IL-8 secretion was greater than that of Western strains. [Conclusion] H. pylori containing different CagA performs different biological functions. The East Asian strains can inhibit the expression of PAR1b and have a more vital ability to promote the secretion of IL-8. In addition, the changes in cell morphology caused by H. pylori depend on the integrity of the cagA.

Abstract:Plant hormones is one of trace endogenous compounds, which probably are the universal “languages” among microalgae cells. The screening and identification of plant hormones play a significant role in understanding the quorum sensing and intercellular communication mechanisms of microalgae cells. However, these hormones are usually in ultra-trace levels, with various species, complicate properties, and coexist with complex matrices. Thus, it is still very challenging to develop an effective method to identify the plant hormones, which includes verifying extraction procedures, improving detection sensitivity, reducing matrix effects and distinguishing isomers. In this work, we reviewed the present research status of plant hormones extraction and detection, especially focused on the methods and strategies on preparing plant hormones from various samples. The advantages and disadvantages of these pretreatment methods were systematically elaborated, from aspects of solid phase extraction, liquid extraction, magnetic solid phase extraction, liquid-liquid microextraction, etc. The detection methods, such as liquid chromatography, liquid chromatography tandem mass spectrometry, capillary electrophoresis, etc., were also compared. Then, we look forward to the prospects of tandem solid phase extraction method on extracting trace amounts of plant hormones from algae slurry. We considered that UPLC-LTQ-Orbitrap-MS method would be a promising technology for plant hormones detection. We aimed to provide some method references for the study of inter-algal microbial ecology.

Abstract:Petroleum-based plastics will cause pollution and affect human health after entering the environment. Therefore, looking for alternatives to petroleum-based plastics has become the trend of future development. Bioplastics have attracted much attention in recent years because of their good biodegradability and safety; in particular, poly-β-hydroxybutyrate (PHB), one of the bioplastics, has become an important source for the production of bioplastics. Photosynthetic bacteria (PSB) is an important raw material for PHB production. PSB can use the cheap carbon source in wastewater as a substrate to accumulate PHB, which can realize the recycling of wastewater, and its application prospects are broad. This paper systematically summarizes the PSB strains that can produce PHB, the synthesis pathway of PHB in the bacteria, the factors affecting the accumulation of PHB by PSB, and the research status of purple non-sulfur bacteria (pnsb) accumulating PHB by wastewater. The prospect of the engineering application of pnsb wastewater recycling technology for PHB production from wastewater is put forward, aiming to provide new ideas and reference for solving the pollution of petroleum based plastics and wastewater recycling.

Abstract:In many countries around the world, it is necessary to upgrade municipal wastewater treatment plants (WWTPs) to meet the more stringent discharge limits of nutrients. However, with the decreased total dissolved nitrogen (TDN) in the effluent, the proportion of dissolved organic nitrogen (DON) in TDN is increasing, which potentially contributes to the synthesis of more nitrogenous disinfection byproducts (N-DBP) and the eutrophication of receiving water bodies. As a result, studies on WWTP DON have been increasing in recent years, and this article systematically reviews its characteristics, transformation and ecological consequences. At present, up to 70% of the influent DON can be removed by coagulation, precipitation, disinfection and other combined physical and chemical processes. However, metabolic activities in the biological treatment units generate new DON, including small molecular amino acids and polyamines, which have a relatively high algal bioavailability. We propose a more comprehensive ASM3-DON model, involving six major processes associated with DON biotransformation, including endogenous respiration, cell growth, and reuse of soluble microbial products. The model can be used to predict wastewater effluent DON concentration with increased accuracy. Future research on wastewater DON should focus on developing more rapid and accurate quantification methods, unraveling its formation and transformation mechanisms, as well as developing more efficient removal techniques to reduce effluent DON.

Abstract:As a fecal bacterial indicator of seawater and an opportunistic bacterium, Enterococcus is used for water quality monitoring and closely related to the public health. In this paper, the physiological and distribution characteristics of Enterococcus in the seawater are described at first. Then, decay mechanism of Enterococcus was introduced under the impact of the key environmental factors and biological factors, and decay kinetics model of Enterococcus in cellular and molecular level are summarized. Finally, the research direction of Enterococcus in seawater is addressed. By establishing the Enterococcus concentration prediction model, early warning of the risk of pathogenic microorganism infection in bathing beaches and other recreational waters will be feasible.

Abstract:Jiuqu starter serves as the saccharifying and fermenting agent for Chinese baijiu production, and plays an important role in determing the quality and flavor characteristics of baijiu. The quality of Jiuqu is associated with the compositions of its microbial communities and enzymes. Studies on Jiuqu microbiome would reveal rich scientific connotation and have extensive application prospects. Jiuqu microbiome presents a high microbial diversity and has abundant enzymes. Meanwhile, Jiuqu microbiome is correlated with the manufacturing environmental factors. Based on profiles of Jiuqu microbiome, researchers try to enhance functional microbes to develop intensified Jiuqu or develop pure culture, to improve the quality and brewing function of Jiuqu. In this review, we summarized advances in traditional Jiuqu microbiome features, Jiuqu microbiome theory development and application, and research direction of Jiuqu microbiome in recent years.

Abstract:Arbuscular mycorrhizal fungi are important components of soil microbial community, which have a variety of beneficial effects on plants and soil. In order to further understand this common underground symbiotic microorganism and investigate its effect on sulfur uptake and utilization of plant, we reviewed the latest research progress. In this paper, the molecular regulation mechanism of plant sulfur nutrition improved by arbuscular mycorrhizal fungi was emphatically analyzed and the factors influencing the sulfur metabolism of mycorrhizal plant were also summarized. Finally, some problems that still exist in this research direction and the focus of future research were also proposed.

Abstract:Ectomycorrhizal occupies an important position in the forest symbiosis system, and plays a role in improving the resistance of plants. In the field of mycorrhizology, the research on the interaction between ectomycorrhizal and abiotic stress is far less than other types of mycorrhizal. In particular, it lacks comprehensive summary comments. This article summarizes the research in the past 5 years, and elaborates the relationship between mycorrhizal symbiotes and plant resistance under abiotic stress (drought, cold, high temperature, saline-alkali, heavy metals and toxic substances); it elaborates that other factors and ectotrophic mycorrhiza have a kind of ability that they can cooperate to improve the plant resistance to abiotic stress; it elaborates how exogenous mycorrhiza alleviates abiotic stress by physiological and genetic means. This paper combines three aspects of research on contaminated soil remediation, functional protein expression, and microbial ecosystems. This paper analyzes current research hotspots and existing deficiencies, and looks forward to future research directions to provide useful ideas for forest ecological restoration and mycorrhizal research.

Abstract:Many kinds of microorganisms with several robust functions are widely distributed and abundant in the natural environment, playing critical roles in human health and safety, ecological stability and species evolution. Although microbial cultivation technologies have been developed for more than one hundred years, by far, succeeded isolated microorganisms only account for 0.1%?1.0% of the total microorganisms in the planet due to plenty of limitation factors. Abundant microbial resources in the natural environments remain to be explored and applied. Therefore, it is particularly necessary to understand the restricting elements for cultivation of microbes and elucidate mechanisms, develop novel and effective methods of microbial isolation and culture. This paper analyzes the constraint factors for the growth of typical environmental microorganisms, then shows findings on the improvements of culture media and conditions. Subsequently, applications of novel techniques in microbial isolation such as in situ cultivation, co-culture, microfluidics cultivation and cell-sorting are reviewed, finally, the present bottleneck problems constraining uncultured microbes are discussed. The integration of multiple technologies, microbial interactions, metabolic pathway and mediation mechanisms, resuscitation mechanisms should be considered in the near future. Hopefully, it can provide a basic reference for the development and utilization of microbial resources.

Abstract:Iron is a key element that influences microbial growth and metabolism by binding to proteins and acting as a catalyst, redox or regulator. The growth metabolism of anaerobic ammonium-oxidizing (ANAMMOX) bacteria is highly dependent on iron, especially iron-containing proteins. The anaerobic lifestyle of ANAMMOX bacteria and the presence of anammoxosome make their pattern of iron metabolism different from that of other microorganisms. Clarification of the pattern of iron uptake metabolism of ANAMMOX bacteria could provide the basis for obtaining their pure cultures and facilitate their application in the environmental field. Here, we combine existing views on iron uptake, utilization, and metabolism with genomic information and limited biochemical and physiological data of ANAMMOX bacteria to propose possible iron utilization pathways of ANAMMOX bacteria for subsequent physiochemical and biochemical studies.

Abstract:Milk is known as “white blood” due to its nutrients. It is becoming more and more important for the traceability of pathogenic bacteria, as the public’s demand for the causes of foodborne diseases increases. The traceability technology could help to understand the structure of pathogenic microorganisms and their diversity origin and evolution, that provides important scientific basis for pathogenic microorganism inspection, epidemic monitoring, prevention and control. Methods such as spectral analysis, mass spectrometry, molecular methods, and whole genome sequencing have played important roles in the traceability technology of pathogenic microorganisms in milk. This paper summarizes the principles and applications of traceability techniques commonly used to track pathogenic microorganisms in milk and milk products.

Abstract:Biofilm dispersal is a programmed response of bacteria in the late development of biofilm to signal changes such as nutrients, low concentration of nitric oxide, D-amino acid, autoinducing peptide (AIP), acyl homoserine lactone (AHL), and adenosine triphosphate (ATP), which is conducive to the bacteria to break away from the harsh internal environment of biofilm to find new colonization sites. In addition, the transient antibiotic resistance of bacteria caused by biofilm returns to normal levels during dispersion, which helps to treat refractory biofilm-related diseases caused by pathogenic bacteria. At present, the research on biofilm dispersion is at an initial stage. In this paper, we hope that by summarizing the phenomenon, signaling molecules and regulatory mechanisms of biofilm dispersion, we can better understand the important significance of bacterial biofilm dispersion for the prevention and control of pathogenic microorganisms and the application of beneficial microorganisms.

Abstract:To fully explore the ideological and political elements in Microbiology Course, and give full play to the important role of professional courses in cultivating talents, this paper introduces the design concept and specific cases of ideological and political education in the course of Microbiology, constructs the knowledge of microbiology as the main body with the development of history, celebrity stories, inspirational allusions, daily life as material resources, and integrates professional knowledge and ideological and political education. In addition, we should guide students to set up dialectical thinking, correct students’ scientific attitude of seeking truth and reality, enhance students’ sense of social responsibility, and realize the innovative education mode “all staff education, the whole process education, all-round education”.

Abstract:In the “student-centered” teaching reform, the main issues of concern are student satisfaction and student performance. The massive open online courses (MOOC) of Medical Microbiology of Beihua University is a high-quality online course in Jilin province. In order to fulfill the advantages of MOOC and reflect the “student-centered” teaching philosophy better, the Department of Medical Microbiology has adopted the blended teaching format of flipped classroom (FC) based on MOOC to carry out a classroom teaching reform. For the first time, the teaching team compared in detail the teaching effect of the blended teaching model of MOOC&FC with the traditional teaching model in Medical Microbiology and conducted a survey on the students’ satisfaction with the blended teaching reform. Independent sample t-test results of the experimental group and the traditional group showed that the blended MOOC&FC teaching method did not affect students’ exam scores or the students’ performance under the two teaching models, and the effectiveness of the two teaching patterns was the same. The statistical results showed that the capability of the experimental group of students to analyze and solve problems improved significantly. The survey feedback indicates that students are more content with the improvement of self-learning ability and teamwork awareness in the MOOC&FC blended teaching, and prefer the flexibility and initiative of MOOC&FC course format.

Abstract:Based on the needs of students of different majors in ethnic colleges/universities for knowledge of microbiology, this paper aims at reforming and practicing of Microbiology course teaching in the aspects of teaching content, teaching methods, teaching resources, etc. We have established a system of “four characteristics” microbiology teaching content, which combined systematization, pertinence, frontierness and effectiveness. Meanwhile, we have created a “three combination” microbiology teaching manner: theory and practice combination, in-class and out-of-class combination, and teaching and scientific research combination. Besides, a “student-centered” self-learning platform for microbiology has been built. These methods will strengthen the cultivation of students’ innovation ability, improve the quality of teaching.