Abstract:[Background] Glucosamine (GlcN) and its derivative N-acetylglucosamine (GlcNAc) are important precursors for the synthesis of glycosaminoglycans, which have a wide range of applications in the fields of medicine, cosmetics, and healthcare products. The traditional production methods of GlcN and GlcNAc have many drawbacks, such as environmental pollution, raw material limit, and unsuitability for people with seafood allergies. Therefore, the microbial fermentation for producing GlcN and GlcNAc has attracted increasing attention. [Objective] To explore the molecular modification for producing GlcNAc by microbial fermentation and optimize the fermentation conditions for increasing the production. [Methods] Firstly, the glmS from Escherichia coli MG1655 and gna1 from Saccharomyces cerevisiae were co-expressed in E. coli MG1655 by the pTrcHisA vector. CRISPR/Cas9 was then employed to knock out the genes responsible for GlcN and GlcNAc transport and metabolism to improve the production of GlcNAc. Finally, the fermentation conditions were optimized to further increase the production of GlcNAc. [Results] The RY-5 strain was constructed by the molecular modification. The production of GlcNAc by RY-5 reached 2.36 g/L after shake flask fermentation for 20 h, which was 29 times that by RY-1 strain. After optimization of the fermentation conditions, the production of GlcNAc reached 7.74 g/L, which increased by 2.3 times compared with that before optimization. [Conclusion] This study successfully developed a recombinant E. coli strain producing GlcN and GlcNAc, which launched a foundation for the industrial production of GlcN and GlcNAc by microbial fermentation.

Abstract:[Background] With low toxicity, good biocompatibility, and biodegradability, biosurfactants are excellent substitutes for chemical surfactants. Most of the available biosurfactant-producing bacteria are mesophilic and it is of great significance to mine efficient biosurfactant-producing strains from cryogenic environments such as the Antarctic. [Objective] To screen psychrotolerant bacteria producing surfactants from the Antarctic soil samples and analyze the chemical structures and properties of the purified surfactants. [Methods] The biosurfactant-producing bacteria from the soil samples in Fildes Peninsula, Antarctic were screened by the oil spreading method. A strain with white solid particles on the colony surface was obtained and identified based on the morphology and the phylogenetic analysis of the 16S rRNA gene sequence. The components in the product were separated by high performance liquid chromatography (HPLC) and identified by nuclear magnetic resonance (NMR). The fermentation medium of the strain was optimized by single factor experiment and response surface design. The emulsifying ability of the product and the diesel oil-degrading ability of the strain were evaluated. [Results] Pedobacter sp. GW9-17 with high surfactant production was screened out. The optimal fermentation medium contained (g/L): soluble starch 18.0, tryptone 9.0, C₃H₃NaO₃ 4.4, K₂HPO₄ 3.6 and MgSO₄ 1.2, with pH value 7.0±0.2. The concentration of the product reached (3.0±0.5) g/L after the strain was incubated in the optimal medium at 28 ℃ and 180 r/min for 7 days with an inoculum amount of 10% (volume fraction). The main component of the surfactant was flavolipid-9U,9U, and the methanol-aqueous solution (the volume ratio is 1:1) of the surfactant crude extract had good emulsifying ability for liquid paraffin oil. In the medium with 5% diesel oil, the strain GW9-17 showed the diesel oil-degrading rates of 40.1% and 57.3% at 4 ℃ and 28 ℃, respectively. [Conclusion] A psychrotolerant strain, Pedobacter sp. GW9-17, efficiently producing the surfactant with a low molecular weight was isolated from the Antarctic soil. The product demonstrated the potential to degrade alkane pollutants and remediate the low-temperature environment of oil pollution.

Abstract:[Background] Diseases caused by aquatic pathogens continue to break out, and finding safe and effective alternatives to antibiotics is an urgent need. Probiotics are often influenced by interests and profit-making, whereas not enough attention has been paid to their safety evaluation. [Objective] To find green and safe probiotics with multiple bacteriostatic activities based on the phenotypic and genetic characteristics of Bacillus subtilis in China’s mariculture system. [Methods] Taking 37 B. subtilis isolated from China’s mariculture system from 2009 to 2021 as the research object, this study used K-B method to detect the resistance of B. subtilis to different antibiotics. The amylase, protease, and hemolytic capacity of B. subtilis were determined by medium plate method. Polymerase chain reaction (PCR) was used to detect the risk of carrying genes related to hemolysis in B. subtilis. The Oxford cup method was used to determine its bacteriostatic effects on six pathogens, including Vibrio parahaemolyticus, Vibrio algaelyticus, Edwardsiella tarda, Vibrio harveyi, Pseudoalteromonas sp., and Photobacterium damselae. The safety of candidate probiotic B. subtilis was evaluated. [Results] The results of drug susceptibility test showed that 37 isolates of B. subtilis showed strong resistance to trimethoprim, pipemidic acid, and streptomycin, moderate resistance to sulfadiazine, low resistance to cefotaxime, ciprofloxacin, and sulbactam, and complete sensitivity to clarithromycin, norfloxacin, florfenicol, flumequine, cotrimoxazole, and tetracycline. The test results of protease and amylase activity showed that 37 isolates of B. subtilis hydrolyzed casein and starch to varying degrees. The hemolytic test results showed that 4/37 isolates of B. subtilis had hemolytic phenomenon, while 8 hemolysis-related genes were detected in 37 isolates of B. subtilis. The analysis of the correlation between hemolytic phenotype and detection gene showed that there was no direct correlation between the strain that produced hemolysis and its hemolytic gene carrier. The analysis of bacteriostatic experiments showed that all isolates of B. subtilis had inhibitory effect on 2 or more pathogenic bacteria, and 2 isolates (strains Bs4 and Bs7) had good bacteriostatic effects on 6 pathogenic bacteria. Safety experiments on Litopenarus vanmamei showed that strain Bs4 had high safety against L. vanmamei, and the survival rate of 7 d was 100%. [Conclusion] Through the comparative analysis of the physiological metabolic phenotype, genetic characteristics, and pathogen antagonism characteristics of B. subtilis isolates, it is revealed that B. subtilis has diversified phenotypes and genetic characteristics in China’s mariculture system. An ecologically safe and probiotic B. subtilis with multiple bacteriostatic activities is screened out, which provides a theoretical basis and technical support for the prevention and control of aquaculture diseases, the development of bacteriostatic microecological preparations, and the healthy and green development of aquaculture industry.

Abstract:[Background] The special habitats of Lop Nur and Wensu Canyon in Xinjiang of China, with their special geographical location, extreme surrounding natural ecological environment, and less influence of human activities, promote their unique high-quality microbial resources. [Objective] To explore the effect of adding antibiotics on the selective isolation and culture of microorganisms and optimize the selective isolation and culture of microorganisms in special habitats of Xinjiang to reduce the repeated isolation of strains. In addition, more microorganisms tolerant to such antibiotics could be selectively cultured to discover more bioactive products. [Methods] With Gauze’s No.1 medium as the foundation medium, different concentrations of β-lactam antibiotic amoxicillin, aminoglycoside antibiotic kanamycin, ansa macrolide antibiotic rifampin, quinolone antibiotic norfloxacin, aminool antibiotic chloramphenicol, and aminoglycoside antibiotic streptomycin were added. Microorganisms from soil samples of Lop Nur and Wensu Canyon in Xinjiang were isolated. [Results] The species and number of microorganisms isolated by norfloxacin were the highest, followed by kanamycin, amoxicillin, rifampicin, and chloramphenicol. Most antibiotics were best separated by 1/10 MIC and 1/15 MIC concentration. Nineteen strains were isolated and distributed in 8 genera. Among them, 6 strains belonged to Bacillus, 5 strains belonged to Streptomyces, 3 strains belonged to Paenibacillus, 1 strain belonged to Rhizobium, 1 strain belonged to Metabacillus, 1 strain belonged to Promicromonospora, 1 strain belonged to Pontibacter, and 1 strain belonged to Nonomuraea. Five new species were isolated. Seventy-three strains were isolated from the soil samples of Wensu Canyon and distributed in 27 genera. Among them, 33 strains belonged to Streptomyces, 1 strain belonged to Sphingomonas, 2 strains belonged to Pseudoxanthomonas, 5 strains belonged to Pseudonocardia, 1 strain belonged to Pseudarthrobacter, 2 strains belonged to Promicromonospora, 1 strain belonged to Priestia, 1 strain belonged to Plantactinospora, 1 strain belonged to Paracoccus, 2 strains belonged to Paenibacillus, 2 strains belonged to Paenarthrobacter, 1 strain belonged to Nocardia, 1 strain belonged to Micromonospora, 1 strain belonged to Methylorubrum, 1 strain belonged to Methylobacterium, 5 strains belonged to Metabacillus, 1 strain belonged to Lentzea, 2 strains belonged to Kribbella, 1 strain belonged to Isoptericola, 1 strain belonged to Ensifer, 1 strain belonged to Bosea, 1 strain belonged to Arthrobacter, 2 strains belonged to Agromyces, 1 strain belonged to Agrococcus, 1 strain belonged to Actinokineospora, 1 strain belonged to Actinoalloteichus, and 1 strain belonged to Bacillus. Fourteen new species were isolated. [Conclusion] The addition of norfloxacin and kanamycin is beneficial to the selective isolation and culture of microorganisms and the discovery of new strains. This study provides guidance for the selective isolation and culture of rare microorganisms in special environments.

Abstract:[Background] Heavy metal pollution in water is a serious environmental problem posing a severe threat to human health. Using microbial adsorbents to remediate heavy metal-contaminated water is an efficient and eco-friendly method. Sphingopyxis is capable of removing heavy metal pollution, while little is known about the mechanism of the removal of cadmium from water by Sphingopyxis. [Objective] To reveal the cadmiumadsorption efficacy and mechanisms of Sphingopyxis sp. YF1 isolated from water. [Methods] The adsorption of Cd²⁺ by live and dead cells of strain YF1 under different pH, contact time, and initial concentrations of Cd²⁺ were analyzed. Kinetic and isothermal models were fitted to investigate the cadmium adsorption characteristics of this strain. Scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM-EDS) were employed to observe the accumulation of cadmium on the surface of live and dead cells. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were employed to identify the functional groups involved in the adsorption of Cd²⁺by YF1 cells, so as to elucidate the adsorption mechanism. [Results] With the rise in pH, cadmium adsorption of both live and dead cells increased within the range of pH 3.0–5.0 and did not change greatly within the range of pH 5.0–7.0. The adsorption mainly occurred in the first 10 min, and then the adsorption rate gradually decreased. The process of Cd²⁺ adsorption by live and dead cells was more in line with the pseudo-second-order kinetic model, which suggested YF1 mainly adopted chemisorption. The adsorption of Cd²⁺ by both live and dead cells was better fitted by the Langmuir model, indicating the adsorption of Cd²⁺ by YF1 was homogeneous. The maximal adsorption capacity of Cd²⁺ by live and dead cells reached 36.20 mg/g and 62.98 mg/g, respectively. Cd(II) was deposited on the surface of both live and dead cells after adsorption, and -OH, C-(O,N), and -NO₂ groups were involved in the adsorption. [Conclusion] Sphingopyxis sp. YF1 has high Cd²⁺ removal ability and is promising in the removal of Cd²⁺ from water.

Abstract:[Background] As an aldehyde pollutant, acetaldehyde widely exists in production and life. Compared with physical and chemical methods, biodegradation with great advantages has become a research hotspot. [Objective] To provide experimental resources for the microbial degradation of acetaldehyde, we screened out an acetaldehyde-degrading strain and optimized the fermentation conditions for degrading acetaldehyde. [Methods] After enrichment culture and acetaldehyde-degrading experiment, a strain with high acetaldehyde-degrading ability was obtained. Single factor tests (carbon source, nitrogen source, metal ions, temperature, rotational speed, inoculation amount, and initial pH) and multi-factor interaction experiments (Plackett-Burman design, steepest ascent design, and Box-Behnken design) were carried out to study the effects of culture medium components and fermentation conditions on the degradation of acetaldehyde by the strain. The growth curve and acetaldehyde degradation curve of the strain were established. [Results] A strain LT-2 with high acetaldehyde-degrading ability was obtained and identified as Bacillus velezensis LT-2. The optimum parameters for degrading acetaldehyde by B. velezensis LT-2 were fermentation in the medium composed of 30 g/L sucrose, 0.6 g/L nutrient broth, and 0.12 mol/L potassium chloride at 28 ℃, initial pH 7.5, inoculation size of 6%, and 200 r/min. Under the optimal conditions, B. velezensis LT-2 can grew in the medium with 1 g/L acetaldehyde and showed the 22 h acetaldehyde degradation rate of 89.77%±2.33%, which was 3.58 times of that before optimization. [Conclusion] B. velezensis LT-2 was effective in degrading acetaldehyde. This study provides experimental and practical references for the biological treatment of acetaldehyde-containing industrial wastewater.

Abstract:[Background] Endophytes associated with halophytes have a wide range of activities, such as promoting plant growth, improving stress resistance, fixing nitrogen, and degrading toxic compounds. [Objective] To deeply understand the diversity, community structure, and potential roles of endophytic fungi of halophytes in the arid land. [Methods] Amplicon sequencing was performed for the endophytic fungi in two halophytes (Anabasis eriopoda and A. truncata) growing on the coast of the western Aral Sea in Uzbekistan. [Results] A total of 166 amplicon sequencing variants (ASVs) were obtained, representing 49 genera of 4 phyla, among which the dominant taxa were Neocamarosporium, Botryosphaeria, Alternaria and their higher taxa. The diversity and community composition of endophytic fungi showed significant differences between the two halophytes, which harbored potential novel taxa. Functional prediction by PICRUSt2 and FUNGuild demonstrated that the endophytic fungi in the two halophytes exhibited diverse roles and trophic modes, being host specific. [Conclusion] Our results suggest that the endophytic fungi in halophytes have high diversity and potential resource values and remain to be studied.

Abstract:[Background] Chinese scholars have reported 60 species of Helvella, while there is still a lack of systematic reports on the distribution of Helvella in Shanxi Province, China. [Objective] To investigate the species and geographical distribution of Helvella in Shanxi Province. [Methods] We collected Helvella species and detailed their morphology. After DNA extraction, we sequenced the heat shock protein (hsp) and ribosome large subunit (LSU) of each species and constructed phylogenetic trees based on Bayesian inference and maximum likelihood method. [Results] The three species were identified as H. capucinoides, H. danica, and H. pubescens, respectively. Their morphology was basically consistent with the description of the type specimens. The phylogenetic trees built based on hsp and LSU supported the identification results based on morphological characteristics. [Conclusion] In review of the Helvella species reported hitherto, we confirmed that these three species are new records in China.

Abstract:[Background] Escherichia coli synthesizes porphyrins through the C5 pathway. 5-aminolevulinic acid (5-ALA) is an important precursor for the synthesis of porphyrins through the C5 pathway. Heme is formed by the chelation of an iron to protoporphyrin IX (PPIX). However, it is still not clear how the secretion of 5-ALA and porphyrin affects the accumulation and conversion of porphyrin to heme. [Objective] To construct E. coli without rhtA and tolC, which encode 5-ALA and porphyrin secretion proteins, respectively, to accumulate porphyrins. Iron was added exogenously, and the ferrochelatase gene hemH and efeB involved in iron uptake were over-expressed to promote the conversion of porphyrins to heme. [Methods] The rhtA and tolC of E. coli BL21(DE3) were knocked out by Red homologous recombination, and different concentrations of FeSO₄ and Fe₂(SO₄)₃ were supplemented. Meanwhile, the recombinant plasmid pEHE for overexpressing hemH and efeB was constructed. The content of porphyrin and heme was analyzed to evaluate the conversion of porphyrins to heme. [Results] The removal of rhtA and tolC did not affect the strain growth significantly. As compared with wild-type strain WT, the porphyrin content of knockout strain WT-RT increased, and the synthesis of heme increased slightly. When 100 μmol/L Fe²⁺ was added exogenously, the heme content in WT-RT strain was 29.44 μmol/g-DCW. When 25 μmol/L Fe³⁺ was added exogenously, the heme content in WT-RT reached 38.22 μmol/g-DCW, which was 1.78 times compared with that in WT. The heme content in efeB-overexpressed strain RT-pEE decreased significantly, while that increased significantly in RT-pEHE strain with over-expressed efeB and hemH. [Conclusion] The deletion of tolC and rhtA leads to the accumulation of porphyrins. The addition of Fe²⁺ and Fe³⁺ at appropriate amount and the co-expression of hemH and efeB can promote the conversion of PPIX to heme. The results provide a new strategy for producing heme by recombinant E. coli.

Abstract:[Background] Glycoside hydrolase family 13 (GH13), known as the largest α-amylase family, does not contain galactosidases. [Objective] To identify a protein BgalPg from the marine bacterium Photobacterium gaetbulicola. [Methods] The family of BgalPg was identified by conserved motif analysis and a phylogenetic analysis. The BgalPg gene was cloned and expressed in Escherichia coli. The enzymatic properties of purified BgalPg were determined. [Results] BgalPg possessing the conserved motifs of GH13 family is a new member of the GH13_38 subfamily, although it showed low sequence identity with characterized α-amylase family proteins. BgalPg demonstrated no catalytic activity against any substrates of the α-amylase family, while it had the activities towards pNP-β-d-galactopyranoside [pNP-β-Gal, (2.8±0.4) U/mg] and oNP-β-d-galactopyranoside [oNP-β-Gal, (1.4±0.3) U/mg]. Moreover, the enzyme hydrolyzed lactose [(0.40±0.01) U/mg], which represented a typical β-galactosidase activity. BgalPg exhibited high stability in pH 7.0–8.5 and the half-life time of 1.5 h at 60 ℃. [Conclusion] This is the first time that an enzyme of GH13 family was biochemically defined as a novel β-galactosidase.

Abstract:[Background] Zearalenone (ZEN) is non-steroidal estrogenic mycotoxin contaminating a variety of grain crops. ZEN can cause serious health problems in livestock and humans through the food chain, leading to great economic losses in the food industry and livestock farming. [Objective] To optimize the conditions for ZEN degradation and evaluate the degradation performance of a ZEN-degrading bacterial strain isolated from microecological preparations, and then study the influence of the strain on the content of phytic acid and vitamins in feed. [Methods] A ZEN-degrading bacterial strain was isolated from microecological preparations. The cytotoxicity and estrogenic activity of ZEN-degrading products were determined by Cell Counting Kit-8 (CCK-8). The phytic acid content in feed before and after detoxification was determined by ferric chloride colorimetry. HPLC was employed to determine the detoxification effect of the isolate in culture medium and feed and the vitamin content in feed before and after solid state fermentation. [Results] One bacterial isolate, Bacillus velezensis PA26-7, was obtained from microecological preparations, which efficiently degraded ZEN by secreting extracellular enzymes. PA26-7 degraded ZEN at the initial pH 4.0–8.0 and the incubation temperature of 25–60 ℃. The cytotoxicity and estrogenic activity of the degradation products were weaker than those of ZEN. The solid state fermentation with PA26-7 for 72 h decreased the content of ZEN by 66.2%–96.8%, decreased the content of phytic acid by 8.40%–32.26%, and increased the content of vitamin B2, vitamin C, and folic acid in the feed samples including soybean meal, bran, and moldy finished feed for chicken. [Conclusion] B. velezensis PA26-7 can be used as a biodetoxification strain for ZEN in feed. The solid-state fermentation with B. velezensis PA26-7 can effectively remove phytic acid in the feed and produce a variety of vitamins, which is conducive to improving the nutritional structure of feed.

Abstract:[Background] The contradiction between edible fungi and forest is increasingly prominent, and the agricultural residues are rich in resources, which can be used as the main substrate for the cultivation of edible fungi. [Objective] To screen out the agricultural residue formula suitable for the growth of Auricularia auricula mycelia by using common agricultural residues. [Methods] Six substrates, including soybean straw, rape straw, corn straw, peanut straw, wheat straw, and sawdust, were used as raw materials in this study, and the formula was designed using the simplex-lattice method. The effects of different substrate interactions on the mycelial growth rate, mycelial growth index, laccase activity, polyphenol oxidase activity, and cellulase activity of A. auricula were analyzed. [Results] Among the substrates of agricultural residues, soybean straw was the most suitable for the growth of A. auricula mycelia, followed by rape straw. The combination of the three main ingredients optimized the substrate ratio which was most suitable for the growth of A. auricula mycelia. [Conclusion] This study has finally optimized an agricultural residue formula suitable for the growth of A. auricula mycelia (49.4% sawdust, 16.4% rape straw, 12.2% soybean straw, 20% wheat bran, 1% sugar, and 1% CaSO₄). This study provides a theoretical basis for the cultivation of A. auricula with grass instead of wood.

Abstract:[Background] Soil salinization has become an increasingly serious problem worldwide, affecting not only crop yield but also the physical and chemical properties of soil. It can inhibit seed germination and hinder the normal growth and the water and nutrient uptake of crops, thereby reducing crop yield. [Objective] Maize growth is limited in saline soil. We studied the effects of mixed soaking with sesbania seed endophytes and sesbania gum on the germination of maize seeds exposed to medium and high concentrations of salt, aiming to provide technical support for improving maize growth in saline soil. [Methods] The LB liquid medium was used to determine the salt tolerance of the sesbania seed endophytic bacterium Bacillus velezensis ZH60. Maize seeds were soaked with 1% sesbania gum, ZH60 suspension (OD₆₀₀=0.8), and their mixture for 3 h. After natural drying, the seeds were cultured on 0.8% agar plates containing 0, 100, and 200 mmol/L NaCl, respectively. The germination rate, root length, and bud length of maize were measured. The maize seedlings at the two-leaf stage were transplanted to the pots filled with vermiculite and irrigated with fluorescence-labeled ZH60 suspension. The maize roots were collected after 1, 5, 11, 17, and 25 days, and the colonization of endophytic bacteria in maize roots was determined by plate colony counting. The colonization of ZH60 in maize roots on day 28 was observed under a scanning confocal microscope. [Results] The strain ZH60 can tolerate 11% NaCl. Compared with the control group, the mixed soaking of maize seeds with 1% sesbania gum, ZH60 suspension, and their mixture increased the germination potential by 28%, 22%, and 30%, the bud length by 158%, 163%, and 150%, and the root length by 36.8%, 21.4%, and 42.9%, respectively, under medium and high concentrations of NaCl. ZH60 could colonize maize roots, and the colonization increased from 2×10⁴ CFU/g on day 1 to 2.5×10⁴ CFU/g on day 25. [Conclusion] The endophytic bacterium B. velezensis ZH60 has high salt tolerance. The germination potential, root length, and bud length of maize are increased by mixed seed soaking with ZH60 and sesbania gum. B. velezensis ZH60 can colonize the roots of maize, which provides strain resources for improving germination and rooting of maize in saline soil.

Abstract:[Background] Sheath blight, caused by Rhizoctonia solani, is one of the major devastated rice diseases in the world, while little is known about the pathogenic mechanism of the pathogen. [Objective] To identify more virulence genes from R. solani and provide a theoretical basis for the control of sheath blight. [Methods] The full-length sequence of RsPG5 was obtained by 3'-RACE, and the structure and biological properties of the deduced protein were predicted by ExPASy online. The pathogenic function of RsPG5 was then determined. [Results] RsPG5 harbored seven exons and six introns, with the coding region of 1 263 bp, which encoded 420 amino acid residues. RsPG5, one member of the glucoside hydrolase family 28, contained a signal peptide of 15 residues and NTD, DD, GHG and RF(I)K domains conserved in the polygalacturonases from fungi. The secondary structure of the deduced protein contained 4 disulfide bonds, α-helix, β-sheet, and random coil, which arranged according to right-handed helix and formed a cleft that was responsible for the enzyme activity. RsPG5 was a stable, water-soluble, exocrine protein localized in cell wall, vacuole, and mitochondria. The eukaryotic expression products of RsPG5 had the polygalacturonase activity to hydrolyze pectin and destroy the sheath cells of rice. Distinct brown necrotic spots appeared 72 h after the expression products were inoculated in the rice sheathes by a needle. The expression level of RsPG5 was up-regulated in the infection course of R. solani. [Conclusion] RsPG5 is a typical polygalacturonase and a major pathogenic factor of R. solani.

Abstract:[Background] The abuse of antibiotics leads to the increase in drug resistance of common pathogenic bacteria in the intestinal tract of yaks. While probiotics have a promising application against drug-resistant bacteria.[Objective] To obtain yak probiotics with excellent probiotic properties is the purpose. [Methods] Twenty yak fecal samples were isolated and purified on MRS medium containing 0.5% CaCO₃, and strains with bacterial inhibitory activity were screened out by the Oxford cup method using Escherichia coli and Staphylococcus aureus as indicator bacteria. After acid and hydrogen peroxide were discharged, the bacteriocin-producing strains were screened out by acid and heat resistance test and protease sensitivity test, and identified by morphology and 16S rRNA sequence analysis. The probiotic properties were analyzed by in vitro bacteriostatic test against diarrhea pathogenic such as E. coli and Salmonella spp., measuring self-aggregation ability and hydrophobicity, tolerance to simulated gastrointestinal juice test, and antibiotic sensitivity test. [Results] Eleven strains of calcium soluble circles were isolated from 20 yak fecal samples, six of which had significant inhibitory effects on E. coli and S. aureus. Two strains of bacteriocin-producing lactic acid bacteria (SC6 and SC9) were obtained by re-screening, both of which were identified as Enterococcus faecium. The strain SC9 had obvious bacteriostatic effect on the bacteria of yak diarrhea, with good tolerance and intestinal adhesion ability in the simulated gastric and intestinal fluid, and it was sensitive to five commonly used antibiotics. [Conclusion] E. faecium SC9 has certain stress resistance and the potential as a probiotic.

Abstract:[Background] The small colony variants (SCVs) of bacteria are subpopulations with small colonies, slow growth, and atypical phenotypic characteristics, leading to recurrent infection. Little is known about the food-borne SCVs of Salmonella in China. [Objective] To provide experimental data for the prevention and control of food-borne Salmonella and the guarantee of animal food safety. [Methods] The SCV was induced by the addition of aminoglycoside antibiotics in the medium of a Salmonella strain isolated from sheep bile. The colony morphology, growth, biochemical characteristics, auxotrophy, antibiotic resistance, resistance genes, virulence genes, and biofilm formation ability of the wild type and SCV were determined and compared. [Results] A heme-dependent Salmonella SCV was obtained after induction with kanamycin. Compared with the wild type, the SCV showed a growth rate decrease of 84%, the inability of using citrate, a hemolysis ability increase of 40%, increased tolerance to sulfonamides and aminoglycosides, a biofilm formation reduction of 45%, and a motility decrease of 78%. [Conclusion] The biological characteristics of the Salmonella SCV were significantly different from those of the wild type, which made it difficult to identify the SCVs of Salmonella. The changes of the pathogenicity and drug resistance of SCVs may pose great challenges to the prevention and control of salmonellosis, and the mechanism remains to be studied.

Abstract:[Background] Antibiotic resistance is arguably the biggest current threat to global health and animal husbandry development. As an emerging solution, bacteriophage can specifically infect and kill bacteria, thus becoming a candidate substitute of antibiotics to enable healthy development of the animal husbandry. [Objective] To study the molecular biological characteristics of the cashmere goat-associated Escherichia coli virulent bacteriophage φPTK, and use mouse model to examine the effect of φPTK in the prevention and treatment of E. coli infections, so as to provide a new strategy for effective control of E. coli infections in cashmere goats.[Methods] The φPTK was concentrated with polyethylene glycol (PEG) 8000-sodium chloride (NaCl) and then the morphological structure was observed by transmission electron microscopy. The nucleic acid of φPTK was extracted with phenol-chloroform method, and the whole genome was sequenced by Illumina HiSeq. Mauve was employed for comparative genomics analysis, and the polygenetic tree was plotted by MEGA. Finally, the effect of φPTK in the prevention and treatment of E. coli infection in mice was analyzed.[Results] The φPTK had isometric head (90 nm diameter) and long contractile tail (about 112 nm in length and 18 nm in diameter). The genome was 169 688 bp with GC content of 37.72% and 264 open reading frames (ORFs). It had the holin-lysin lysis system, anti-holin and lysis inhibition accessory protein, and no virulence-associated and antibiotic resistance genes. The comparative genome analysis indicated it was a novel lytic cashmere goat-associated E. coli bacteriophage. φPTK was applied before and after E. coli infection in mice for prevention and treatment. The result showed that all mice in the positive control group without φPTK died, and the survival rate of mice in the prevention and treatment groups reached 80% and 60%, respectively. [Conclusion] The virulent cashmere goat-associated E. coli phage φPTK (Myoviridae, Caudovirale) is effective in the prevention and treatment of E. coli infections. This study lays a basis for the development of bacteriophage agents for biological control of colibacillosis in cashmere goats.

Abstract:[Background] The bioactive substances in batryticated silkworms (Bombyx mori larvae infected by the entomopathogenic fungus Beauveria bassiana) are widely used in medicine, healthcare, and cosmetic industries. At present, most of strains used in the production of batryticated silkworms are the spore powder from naturally dead batryticated silkworms without purification, and there is no fixed application concentration, which make it difficult to ensure the mortality of silkworms. It is an important direction in the research and development for industrial production of batryticated silkworm to improve the pathogenicity of B. bassiana strains and screen out highly pathogenic strains with excellent biological characteristics. [Objective] To screen out highly pathogenic strains by ultraviolet-microwave compound mutagenesis and provide elite strains for the factory production of batryticated silkworms. [Methods] A strain of B. bassiana was isolated with the spore dilution method from naturally infected domestic silkworms in Shanxi province. The strain was then mutagenized by UV-microwave combination, and the spore production and pathogenicity of the strain were compared before and after mutagenesis. [Results] The original strain obtained by isolation was identified as B. bassiana and named B. bassiana Bb1003. Considering the mortality and positive mutation rate, we optimized the mutagenesis conditions as UV (15 W) irradiation for 30 min combined with microwave (800 W, rated microwave frequency of 2 450 MHz) treatment for 60 s. Six mutated strains, including UMCM1, UMCM2, UMCM3, UMCM4, UMCM5, and UMCM6, were obtained after screening. UMCM2 caused an infection rate of 97.64% for silkworm, produced spores 2.48 times those of the original strain, and had significantly higher pathogenic capacity for silkworm than the original strain. [Conclusion] A highly pathogenic strain was produced by ultraviolet-microwave compound mutagenesis, which laid a foundation for the mass production of batryticated silkworms.

Abstract:[Background] Newcastle disease (ND), one of the Class II infectious diseases induced by Newcastle disease virus (NDV), causes huge economic loss to the poultry industry. Thus, early and accurate screening of NDV is crucial for the prevention of ND outbreak. [Objective] To develop a rapid method for the detection of Newcastle disease virus (NDV) based on the reverse transcription loop-mediated isothermal amplification combined with a TaqMan probe (RT-TaqMan-LAMP). [Methods] The specific primer pairs and TaqMan probes were designed according to the NDV F gene sequence, and the reaction conditions were optimized with the recombinant plasmid pMD-NDV-F as a positive standard to verify the specificity, sensitivity, and reproducibility of the method. With 70 samples, the method was compared with the RT-qPCR method recommended in the national standard (GB/T 16550—2020). [Results] The optimal reaction conditions are as follows: 61 ℃, 60 min, 1.6 μmol/L (FIP/BIP), 0.2μmol/L (F3/B3), 0.8 μmol/L (LF/LB), and 0.2 μmol/L (GTP). The lowest detection limit was 1.651×10² copies/μL, and the sensitivity was 100 folds that of LAMP. No non-specific amplification was observed, and no cross-reactivity with Avian influenza virus (AIV), Mycoplasma gallisepticum (MG), Mycoplasma bursalis (MS), infectious bursal disease virus (IBDV), and herpes simplex virus (HSV) was detected. The intra- and inter-batch coefficients of variation (CV) were＜3%. The method detected one more positive sample than RT-qPCR in 70 clinical samples and the coincidence rate was 98.57% after two retests. [Conclusion] The NDV RT-TaqMan-LAMP method is highly specific and sensitive, with high repeatability, which can effectively avoid non-specific amplification and can be used for accurate detection of NDV and epidemic prevention.

Abstract:[Background] The fowl cholera caused by Pasteurella multocida causes great harm to the production, while the existing culture medium has the problem of low bacterial density. [Objective] To develop the medium for the preparation of a P. multocida vaccine with high antigenic activity. [Methods] Single factor experiment, Plackett-Burman design, and response surface methodology were employed to optimize the medium composition of P. multocida. Next, the immunogenicity of the bacteria in different fermentation phases was determined. Finally, the bacteria cultured in this medium were used to prepare the vaccine, the protective effect of which was evaluated by an animal challenge test. [Results] When the developed medium was used for the culture of P. multocida, the maximum viable count reached 1.84×10¹⁰ CFU/mL in 6 h, which was 2.6 times that of the control medium. The antigenic activity of the fermented product was the highest in the stationary phase. Challenge test showed that the vaccine prepared with this culture medium well resisted the infection of P. multocida. [Conclusion] We developed the medium for preparing a P. multocida vaccine with high antigenic activity, laying a foundation for vaccine production.

Abstract:[Background] The widespread presence of Riemerella anatipestifer in poultry farms causes contagious serositis in ducklings, which seriously endangers the development of the poultry industry.[Objective] To improve the fermentation level and antigenic activity of R. anatipestifer and provide technical guidance for the development of R. anatipestifer inactivated vaccines. [Methods] The single factor test and response surface methodology were employed to optimize the vaccine medium for R. anatipestifer. The antigenic activity of R. anatipestifer was determined at different time points of fermentation, and the inactivated vaccine was prepared at the time point with the highest antigenic activity. The immune effect of the vaccine was evaluated by animal immunization test. [Results] The viable cell count of R. anatipestifer in the developed vaccine medium reached 4.68×10¹⁰ CFU/mL, which was 2.29 times higher than that of the commercial medium for R. anatipestifer. The antigenic activity of the strain peaked after 12 h of fermentation. The inactivated vaccine prepared at this time point induced significantly higher antibody level in mice than the commercial inactivated vaccines, with 100% protection from virus attack. [Conclusion] The medium developed in this study had excellent enrichment effect and can be used for the production of antigens for R. anatipestifer inactivated vaccine. The bacteria can be collected when the antigenic activity of the selected strain reaches the highest level during the vaccine production.

Abstract:[Background] Campylobacter is a common group of foodborne pathogens that can cause gastroenteritis in the world. It is increasingly resistant to clinically important antibiotics and poses a serious threat to food safety and public health. [Objective] To investigate the resistance phenotype and genes of a Campylobacter coli strain carrying both optrA and cfrC, the whole genome characteristics of the strain, the distribution of virulence genes, and the gene environments of optrA and cfrC. [Methods] Agar dilution method was employed to determine the minimal inhibitory concentrations (MIC). Whole genome sequencing (WGS) was carried out to sequence the DNA of the strain. [Results] The strain was highly resistant to tetracycline, clindamycin, azithromycin, florfenicol, and linezolid, and sensitive to ciprofloxacin and gentamicin. WGS identified a circular DNA with a size of 1 436 486 bp (GC content of 31.63%), which carried 12 resistance genes involving four major categories of antibiotics. All of the 12 genes were detected on the chromosome, most of which were aminoglycoside resistance genes. A total of 83 virulence genes involved in adherence, invasion and motility were identified, and most of them were associated with motility. The gene islands GIs002 and GIs003 contained resistance genes. CfrC was located on the GIs002, linked with aph(3')-Ⅲa and flanked by two transposons. OptrA was located on chromosome and connected to the insertion sequence Integrase/IS607 family at upstream and downstream sides. Transposon and insertion sequence could mediate the horizontal transfer of resistance genes. [Conclusion] The genome information, resistance genes, and virulence genes of a multiresistant C. coli strain were analyzed by WGS. The mobile genetic elements (transposase and insertion sequence) played an important role in the transmission of antibiotic resistance of C. coli. The findings provide basic information for risk assessment of antibiotic resistance of Campylobacter.

Abstract:[Background] The sulfide in the produced water of offshore oilfield often exceeds the limit, which affects the viscosity of the polymer-containing injection water. The efficiency of biodesulfurization is poor due to the poor adaptability of conventional mesophilic sulfide-removing bacteria to the high temperature of the produced water after oil removal. [Objective] To analyze the changes of microbial consortium structure in the process of offshore produced water treatment and acclimate a thermophilic bacterial consortium for desulfurization. [Methods] Water samples were collected from Haisan Station of Shengli Oilfield, and 16S rRNA gene sequencing was performed to reveal the structure of the bacterial consortium. The acclimation was conducted in the inorganic medium at different temperatures (55, 60, and 65 °C) for multiple rounds, and atmospheric and room temperature plasma (ARTP) was employed to obtain the efficient thermophilic desulfurizing bacteria. Metagenome sequencing was employed to study the composition of the enriched bacterial consortium and then the desulfurizing ability of the consortium was determined. [Results] The produced water samples contained abundant thermophilic bacteria and sulfate-reducing bacteria, such as Thermodesulfovibrio, Pseudothermotoga, Thermolithobacter, Fervidobacterium, Thermovenabulales, and Pseudomonas. Hydrogenophilus became the most dominant bacteria in the effluent from the nitrogen gas-floated oil removal. The relative abundance of Hydrogenophilus in the effluent water of the central platform and the polymer injection platform was 76% and 84%, respectively. After the enrichment and acclimation with increasing temperature, Thermus became predominant, with the relative abundance of 89.4%, in addition to a few members of Hydrogenophilus. The efficiency of desulfurization was further improved by ARTP. The acclimated bacterial consortium could rapidly desulfurize the liquid containing 8.88 mg/L sulfide at 65 °C, with a removal rate of 100% and a removal speed up to 0.49 mg/(L·h). [Conclusion] An efficient desulfurizing bacterial consortium adapted to 65 ℃ was obtained, which improved the desulfurization of polymer-containing injection water. This study would be helpful for the development of high-temperature oil fields.

Abstract:[Background] Biofilm formation is the major pathogenic factor of Staphylococcus epidermidis. The two-component regulatory system (TCS) composed of VraSR and SrrAB is involved in the growth, biofilm formation and the other biological phenotypes of bacteria. The deletion of vraSR leads to thinner cell wall and decreased biofilm formation, and the deletion of srrAB results in lagged growth before the stationary phase and decreased biofilm formation of S. epidermidis 1457 (SE1457). Both VraSR and SrrAB modulate biofilm formation in an ica-dependent manner. The ica operon is a key regulatory element of biofilm formation in S. epidermidis, which is composed of four genes (icaADBC) and a transcriptional repressor (icaR). [Objective] To explore the synergistic regulation of TCS-VraSR and SrrAB on the growth and biofilm formation of S. epidermidis, so as to lay a foundation for the prevention and control of persistent infection caused by S. epidermidis. [Methods] The recombinant plasmid pKOR1-ΔvraSR was constructed, modified by E. coli DC10B, and transformed into ΔsrrAB. The vraSR gene was deleted from the genome of ΔsrrAB by homologous recombination. The suspected ΔvraSR-srrAB was verified by PCR, RT-PCR, and sequencing. The growth, biofilm formation, and drug susceptibility of ΔvraSR-srrAB were examined. [Results] ΔvraSR-srrAB was successfully constructed. Compared with SE1457, ΔvraSR, and ΔsrrAB, ΔvraSR-srrAB exhibited retarded growth, increased drug susceptibility, and decreased biofilm formation. The RT-qPCR showed that the deletion of vraSR or srrAB down-regulated the transcriptional level of icaA by 13%–17% and up-regulated that of icaR by 5–9 folds compared with that in SE1457. The deletion of both vraSR and srrAB down-regulated the transcriptional level of icaA by 6% and up-regulated that of icaR by 14 folds compared with that in SE1457. [Conclusion] VraSR and SrrAB may cooperatively regulate the biofilm formation of S. epidermidis through the ica pathway, and the mechanism of VraSR and SrrAB in modulating the responses to stress remains to be studied.

Abstract:[Background] Major depressive disorder (MDD) is a common major mental disorder and most MDD patients have gastrointestinal (GI) symptoms. However, little is known about the occurrence mechanisms of GI symptoms in MDD. [Objective] To explore the gut microbiota composition and its correlations with inflammation markers and GI symptoms in the patients with first-episode MDD, providing a theoretical basis for the treatment of MDD. [Methods] The participants included 91 first-episode, drug-naive MDD patients and 105 healthy controls (HCs). The 16S rRNA gene sequencing and bioinformatics tools were employed to reveal the composition of fecal microbiota. The levels of high-sensitivity C-reactive protein (hs-CRP), interleukin-1 beta (IL-1β), IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) in the peripheral blood were measured via enzyme-linked immunosorbent assay (ELISA). Gastrointestinal symptom rating scale (GSRS) and Hamilton depression scale (HAMD) were used to evaluate the severity of GI symptoms and depression symptoms, respectively. [Results] All the MDD patients were accompanied by GI symptoms, and the incidence of anorexia, early satiety, nausea, and vomiting was higher than 70%. Compared with HCs, MDD patients had elevated level of hs-CRP and showed different alpha diversity and beta diversity of gut microbiota (P＜0.05). Linear discriminant analysis effect size (LEfSe) showed that MDD patients had higher relative abundance of Geodermatophilus, Alloscardovia, Bifidobacterium, Blautia, Leptothrix, Rubrivivax, Massilia, Haemophilus, Candidatus Xiphinematobacter, and Chthoniobacter and lower relative abundance of Bacteroides, Parabacteroides, SMB53, Anaerostipes, Clostridium, Lachnospira, Roseburia, Faecalibacterium, Ruminococcus, Dialister, Phascolarctobacterium, and Sutterella. Furthermore, the correlation analysis revealed that the relative abundance of Roseburia, Sutterella, and Parabacteroides were negatively correlated with hs-CRP, total score of HAMD-17, and the total score and some item scores of GSRS (P＜0.05). [Conclusion] This study demonstrates that compared with HCs, MDD patients showed elevated hs-CRP. The altered gut microbiota is closely associated with hs-CRP and depression and GI symptoms in MDD patients.

Abstract:Natural products with remarkable biological activities from Streptomyces are important sources for drug discovery. The advancing sequencing technology has revealed the biosynthetic potential of Streptomyces. Most biosynthetic gene clusters (BGCs) in Streptomyces are at low expression levels or even in silence under routine laboratory conditions, which hinders the discovery of natural products. In situ activation and heterologous expression are effective ways for discovering natural products in Streptomyces, in which promoters as the “switch” of gene expression play a key role. Therefore, the study of promoters can promote the activation of BGCs for the mining of new natural products. We introduce Streptomyces promoters in terms of the structures, mining, design, and applications in natural product discovery. This review is expected to provide new insights and methodological references for the optimization of biosynthesis pathways and the discovery of new bioactive substances in Streptomyces.

Abstract:Tricodesmium, a genus of nitrogen-fixing cyanobacteria, is the most abundant nitrogen-fixing microorganisms in the ocean. They contribute about 42% of marine biological nitrogen fixation and provide a new source of nitrogen to marine ecosystems, driving marine primary productivity and biogeochemical cycles. As a major contributor of new nitrogen in the ocean, Tricodesmium is a group of filamentous nitrogen-fixing cyanobacteria that do not produce heterocysts. Because nitrogenase, the key enzyme of biological nitrogen fixation, is sensitive to oxygen, nitrogen-fixing cyanobacteria usually produce heterocysts or fix nitrogen at night to avoid the inhibitory effects of oxygen on nitrogen-fixing enzymes. The recent studies have discovered that Tricodesmium has a unique nitrogen fixation system, which enables the same filament to complete photosynthesis and nitrogen fixation simultaneously during the daytime and has a complex regulatory mechanism. We review the recent progress in the nitrogen fixation strategy of Tricodesmium and introduce the sophisticated regulatory mechanism between biological nitrogen fixation and photosynthesis. This review helps to deenpen our understanding of the nitrogen fixation mechanism of microorganisms, especially marine cyanobacteria.

Abstract:Vibrio parahaemolyticus, the causative agent of vibriosis in aquatic animals, is a foodborne pathogen responsible for fatal diseases such as gastroenteritis, septicemia, and necrotizing fasciitis in humans via the ingestion of contaminated seafood, posing a great threat to both aquaculture and public health security. The abuse and over-use of antibiotics have caused drug residues in aquatic products and bacterial resistance. It is therefore urgent to develop safe and effective antibiotic substitutes. As a bacterial virus, phage possesses many advantages such as host specificity, easy availability, easy preservation, and high efficiency, which attracted increasing attention in the prevention and control of diseases in aquaculture and in the field of food safety. This paper reviewed the studies of vibriosis in aquatic animals and its bacteriophage therapy, aiming to provide a theoretical basis and data support for the application of V. parahaemolyticus phage in the biocontrol of diseases in aquaculture.

Abstract:Enteroviruses belonging to the family Picornaviridae include poliovirus and other major human pathogens and have become one of the major threats to global health. Innate immunity is an important host defense against viral infection. Diverse enteroviruses have evolved multiple ways to evade immune recognition or inactive the innate immune system. We reviewed the recent studies about the mechanisms of enteroviruses regulating innate immune response and systematically introduced the molecular characteristics of enterovirus evasion of interferon-dependent and interferon-independent antiviral innate immune defense, hoping to provide a reference for deciphering enterovirus pathogenesis and developing targeted antiviral drugs.

Abstract:Porphyromonas gingivalis, the main pathogen of periodontitis, can produce various virulence factors. P. gingivalis and its virulence factors not only destroy periodontal tissue but also spread to the whole body and affect the occurrence and development of various systemic diseases, including Alzheimer’s disease (AD). The outer membrane vesicles of P. gingivalis contain a large number of virulence factors of the species, and they are small in size and more easily distributed to remote tissues and organs. Recent studies have found that the outer membrane vesicles may play an important role in inducing neuroinflammation and promoting the occurrence and development of AD. However, the specific mechanism is still unclear. This article reviews the occurrence and regulation of P. gingivalis outer membrane vesicles, the main virulence factors in the vesicles, and their relationship with AD, which is expected to clarify the biological mechanisms of periodontitis and AD.

Abstract:Diabetic kidney disease (DKD) is a serious metabolic disease caused by diabetes mellitus. In high glucose, DKD causes chronic inflammation and oxidative stress in the kidney, destroys the physiological structure of the kidney, and leads to renal interstitial fibrosis. Numerous studies have shown that intestinal microbiome influences the body’s metabolism and health. This review summarized the latest research findings on the relevance of the intestinal microbiota to DKD, which aimed to elucidate the effects of the intestinal microbiota on the prevention and treatment of DKD. In addition, the association between intestinal barrier and intestinal microbiome metabolism with DKD was established, and the related mechanisms of the intestinal microbiome against DKD in recent studies were summarized. What’s more, the feasibility of supplementing prebiotics and probiotics as well as fecal transplantation in the treatment of DKD was discussed. By combing relevant content, this review provides certain theoretical references and data support for the treatment of DKD.

Abstract:Phosphorus is a vital element in biomolecules and one of the main limiting nutrients for primary production of terrestrial ecosystems. Increasing global food demand and modern agricultural consumption of phosphate fertilizers have led to excessive inputs of phosphate to intensively managed fields, causing increased soil phosphate loss and continued eutrophication of surface waters. Phosphate solubilizing microorganisms (PSMs) are considered as eco-friendly manure that can enhance agricultural productivity and play an important role in improving soil fertility. It is crucial to increase the availability of soil phosphorus that comprehensively and in-depth comprehend the function of PSMs and their role in soil biochemical transformation of phosphorus. This paper systematically reviewed species and distribution diversity of PSMs, functional genes mainly involved in microbial phosphorus cycling process, processes of PSMs involving in soil phosphorus biogeochemical cycling, and the reaction mechanism behind these processes, to better understand the ability of PSMs and make full of them in the future.

Abstract:Aspergillus fumigatus is a saprophytic fungus with worldwide distribution and one of the three most common opportunistic pathogenic fungi infecting human in clinical practice. It is the main pathogen of invasive aspergillosis. A. fumigatus can produce two types of melanins: dihydroxynaphthalene melanin (DHN-melanin) and pyomelanin. We introduce the latest progress in the melanins of A. fumigatus in terms of the production pathways, functions, and interactions with the host immune system. This review aims to deepen our understanding of the pathophysiological features of A. fumigatus and provide a theoretical basis for the rapid diagnosis of A. fumigatus infection and the development of antifungal drugs.

Abstract:Microbiology is a basic course for all the majors of biological sciences and a discipline with strong practical and research properties. The development of the theory and the innovation of biotechnology promote and complement each other. With the advancing of science and technology and the emerging of research achievements, the teaching contents of Microbiology must keep pace with the times and present the latest achievements of the frontiers. Taking the knowledge and taxonomic evolution of actinomycetes as an example, we expound the content innovation of the chapter of actinomycetes in the course of Microbiology. This paper aims to facilitate the establishment of a more reasonable teaching system for Microbiology, so as to improve the teaching and lay a foundation for training a group of high-quality talents in biological sciences.

Abstract:The online-offline blending teaching, a new teaching mode combining the advantages of online teaching and offline teaching, adapts to the background of “Internet plus” and the undergraduate education reform in China. Since the establishment of the online course Environmental Engineering Microbiology in Jiangnan University, deep learning-oriented online-offline blending teaching and flipped classroom has been performed for three continuous years. The exploration and practice indicated that the reorganization of course content, the integration of teaching resources, the combination of diverse teaching methods, and the reform of examination way in this teaching mode benefited the cultivation of students’ abilities of self-study, problem solving, team cooperation and communication as well as international horizon. In this paper, we summarized and reviewed the blending teaching and flipped classroom in this course, hoping to provide valuable references for the reform of teaching mode in specialized courses of environmental science and engineering in colleges and universities of China.