Abstract:[Background] The keratinase KerZ1 could efficiently degrade keratin at the optimum temperature of 60℃. However, the poor activity below the optimum temperature makes the enzyme difficult to be applied in industrial production. [Objective] To improve the low-temperature activity of KerZ1. [Methods] Based on homologous alignment and analysis of folding free energy, site-directed mutagenesis was employed for KerZ1, and then the enzymatic properties of the mutants were characterized. [Results] The introduction of T210S, N211S, or T212G into the flexible loop region (loop 13) increased the low-temperature activity of KerZ1. Subsequently, the compound mutant T210S/N211S/T212G was constructed, which showed the activity 45.68% and 85.74% higher than that of KerZ1 at moderate (40℃) and low (20℃) temperatures, respectively. Meanwhile, the half-life (t_1/2) at 60℃ decreased by only 11.52%, indicating that the low-temperature activity of T210S/N211S/T212G was improved without serious loss of thermostability. [Conclusion] The reduction of hydrogen bonds and surface hydrophobicity may be the main reasons for the increased flexibility of the keratinase molecule, which enhances the low-temperature activity of T210S/N211S/T212G. In summary, this study modifies the keratinase to improve its low-temperature activity and lays a foundation for the practical application of this enzyme.

Abstract:[Background] With the development of industry and agriculture, excessive ammonia nitrogen caused by sewage discharge has gradually become an important issue in water pollution, and ammonia nitrogen removal has become one of research hotspots. In previous studies, it has been reported that nitrifying bacteria were able to convert ammonia nitrogen to nitrate nitrogen, resulting in the reduction of ammonia nitrogen concentration in water. Thus the ammonia nitrogen elimination in polluted water by biological methods mainly depended on nitrifying bacteria. Currently there are more studies on heterotrophic nitrifying bacteria than autotrophic ones. However, the removal efficiency of the existing heterotrophic nitrifying bacteria for ammonia nitrogen in oligotrophic rivers is not ideal. In particular, the bioremediation in situ by nitrifying consortia from oligotrophic rivers has virtually not been reported. [Objective] We aim to enrich and characterize nitrifying consortia from oligotrophic rivers, and explore the potential of the ammonia nitrogen elimination in an oligotrophic river by in situ isolated consortia. [Methods] The mineral media for ammonia-oxidizing bacteria and nitrite-oxidizing bacteria are used to isolate ammonia-oxidizing and nitrite-oxidizing bacterial consortia from rivers. The microbial diversities of two consortia were identified according to sequences of V3-V4 region of 16S rRNA genes. The microscopic counting method was used to detect the growth of the consortia. Two consortia were inoculated into the artificial sewage together with aerating at room temperature, and the depletion of ammonia nitrogen was detected. Two consortia were together introduced into the oligotrophic river in situ, and the depletion of ammonia nitrogen was also detected. [Results] In this experiment, an ammonia-oxidizing bacterial consortium and a nitrite-oxidizing bacterial consortium were enriched and designated AOB1 and NOB1, respectively. In AOB1 and NOB1, 99.28% and 99.64% strains belong to Proteobacteria, respectively. Furthermore, the relatively dominant genera in AOB1 are Aeromonas (73.00%) and Delftia (9.17%). The relatively dominant species in NOB1 are Aeromonas (36.66%) and Pseudomonas (30.82%). After 2-3 d incubation in the mineral media, the biomasses of AOB1 and NOB1 reached to 5.23×10¹⁰ cells/L and 3.63×10¹⁰ cells/L, respectively. When AOB1 and NOB1 were introduced into artificial sewage with low organic matter (glucose concentration being 0.04 g/L), the depletion of ammonia nitrogen was 95.26% after 7 d, without accumulation of nitrite nitrogen. When AOB1 and NOB1 were introduced into water from an oligotrophic river (Taizhou city, Zhejiang province) with aerating (areation rate being 5 L/min) in the lab, and the depletion of ammonia nitrogen was 94.04% after 10 d. AOB1 and NOB1 were also together introduced into a 700-meter water area of the above river, and sewage was still discharged continuously into this river during the testing period. After 16 d, 49.19% ammonia nitrogen in the above water area were removed.[Conclusion] This study has enriched an ammonia-oxidizing bacterial consortium and a nitrite-oxidizing bacterial consortium from oligotrophic rivers, which can together remove ammonia nitrogen in artificial sewage and oligotrophic river water. This study provides microbial resources and a feasibility of using microbes to eliminate ammonia nitrogen pollution in urban oligotrophic rivers. It also has a potential application in implementing the desired goal of "The Beautiful Countryside".

Abstract:[Background] The sulfur-containing odor emitted from municipal sludge composting could not only pollute the surrounding environment but also reduce the compost qualities. Biological desulfurization technology is highly efficient and without secondary pollutions. However, recent studies have been focused on the mesophilic strains, which are usually inactivated in thermophilic composting environments. Studies on the thermotolerant sulfide-oxidizing bacteria are scarce, and the desulfurization performance of the thermotolerant bacteria awaits further study. [Objective] To provide a theoretical basis for the biological application of composting desulfurization, a thermotolerant sulfide-oxidizing strain was isolated and identified, and the environmental conditions of the sulfide oxidation process were optimized. [Methods] The selective medium containing sulfide was used to screen and purify the thermophilic strain from sludge compost materials. The strain was identified by its morphological characteristics, physiological and biochemical analysis, and 16S rRNA gene sequence analysis. Single-factor and orthogonal experiments were used to optimize the fermentation conditions, and we also used the Logistic model to fit the growth kinetics curve of the strain. [Results] A thermophilic sulfide oxidizing strain LYH-1 was isolated and identified as Paenibacillus ehimensis; the GenBank accession number was MW659161. The optimized sulfide oxidation conditions were 50℃ of the temperature, pH 7.5, and 5% of the inoculation amount, the sulfate production amount reached 86.89 mg/L, and the production rate was 36.20%. The optimized growth conditions were 50℃ of the temperature, pH 8.0, and 5% of the inoculation amount, and the OD₄₂₀ reached 0.520; under these conditions, the growth kinetics were fitted to obtain the maximum specific growth rate at 0.304 2 h^-1. [Conclusion] Paenibacillus ehimensis LYH-1 has strong sulfide oxidation and environmental resistance abilities. It provides new bacterial resources for biological desulfurization and theoretical support for sulfur-containing odor control during the high-temperature period of sludge composting.

Abstract:[Background] Microalgae are resistant to heavy metals and have high adsorption rates, which can serve as an excellent biosorbent. [Objective] to explore the influences of environmental factors on the adsorption of Cd²⁺ and Cr³⁺ by Chlorella vulgaris. [Methods] Using CdCl₂·2H₂O and CrCl₃·7H₂O to provide heavy metal ions, we explored the influences of environmental factors such as the concentrations of heavy metal ions, pH, and temperature on the adsorption of Cd²⁺ and Cr³⁺ by C. vulgaris. [Results] At 30℃, pH 5.5, Cd²⁺ concentration of 0.4 mg/L, Cr³⁺ concentration of 4.0 mg/L, and C. vulgaris biomass of 0.59 g/L, the adsorption rates of Cd²⁺ and Cr³⁺ by C. vulgaris reached the maximums of 84.5% and 75.2%, respectively. The adsorption rate of C.vulgaris to Cd²⁺ was greater than that to Cr³⁺. [Conclusion] The adsorption of C. vulgaris to Cd²⁺ and Cr³⁺ is affected by environmental factors and C. vulgaris has certain preference to different heavy metals.

Abstract:[Background] A bacterium strain producing α-L-rhamnosidase was screened and identified as Enterococcus avium by molecular biological methods in the preliminary work. α-L-rhamnosidase can specifically cut terminal rhamnose from natural flavonoid compounds, which has great development prospect and application value in food production, pharmaceutical processing and chemical industry. [Objective] The α-L-rhamnosidase gene from E. avium was cloned and expressed, and the enzymatic properties of the recombinant protein were further studied. [Methods] Based on the putative α-L-rhamnosidase gene sequence in the genome of Enterococcus avium strain 352, specific primers were designed to amplify its coding sequence. Recombinant expression plasmid was constructed using pET-28a(+) as vector and the recombinant protein was expressed in Escherichia coli BL21(DE3) competent cells. The recombinant protein was purified by nickel affinity chromatography, and the enzymatic properties were determined using pNPR as a substrate. [Results] The molecular weight of the fusion protein EaRha1 is about 130 kDa. The optimal pH of EaRha1 is 7.0, the optimal temperature is 50℃, EaRha1 is stable at pH 5.0-8.0 and can maintain higher enzyme activity below 40℃. Metal ions can promote or inhibit EaRha1 in different degrees. Methanol has inhibitory effect on EaRha1, and the inhibitory effect increases with the increase of methanol concentration. The kinetic characteristic constants K_m and V_max of EaRha1 were 0.35 mmol/L and 4.2 μmol/(mg·min) (R²=0.999) respectively. The recombinant EaRha1 could catalyze the hydrolysis of neohesperidin, naringin and rutin. [Conclusion] In this study, the hydrolysis characteristics of the protein to flavonoids was determined by studying the enzymatic properties of recombinant protein EaRha1, which laid a theoretical foundation for the biotransformation of flavonoids.

Abstract:[Background] Soybean hulls as a crude feed have high digestibility, but their fiber content is high, and contains a variety of anti-nutritional factors such as urease and antigen protein, which limits its application in monogastric animal feed. [Objective] The aims of this study to reduce the content of fiber and anti-nutritional factors in soybean hulls by combination of bacteria fermentation and enzymatic hydrolysis. The packaging treatment of soybean hulls by using this method was conducted in the laboratory to evaluate the number of molds and the nutritional value and quality of soybean hulls for the foundation of future industrial application of soybean hulls. [Methods] The experiment was divided into four groups:control group (soybean hulls without fermentation and enzymatic hydrolysis group), strain fermentation group (Lactobacillus, Bacillus, and Saccharomyces mixture fermentation), enzymatic hydrolysis group (cellulase and xylanase enzymatic hydrolysis), and enzymatic hydrolysis + strain fermentation group (Lactobacillus, Bacillus and Saccharomyces compound fermentation + cellulase and xylanase enzymatic hydrolysis). The optimal time for packaging treatment was selected. [Results] Compared with the control group, the pH value, reducing sugar content, urease activity, globulin, and β-conglycinin of the fermentation group were decreased (P<0.05). The reducing sugar, crude protein, and true protein content in the enzymatic hydrolysis group was increased (P<0.05), while the neutral detergent fiber and acid detergent fiber were decreased (P<0.05). The pH value, the contents of neutral detergent fiber, and acid detergent fiber were decreased (P<0.05), while the reducing sugar content, crude protein, and true protein contents were increased (P<0.05) in the bacteria fermentation and enzymatic hydrolysis group. Compared with the fermentation group, lactic acid concentration, and viable bacteria number were higher, while pH value, urease activity, globulin, and β-conglycinin were decreased in the bacteria fermentation and enzymatic hydrolysis group (P<0.05). Compared with the enzyme hydrolysis group, the reducing sugar concentration, the neutral detergent fiber, acid detergent fiber, and hemicellulose in the bacteria fermentation and enzymatic hydrolysis group were decreased (P<0.05). In the bag experiment, the pH value of the enzyme fermentation group was 4.87 at the optimal fermentation time of 5 d, which was lower than that of the other groups (P<0.05). Compared with the fermentation group, the reducing sugar content, crude protein, and true protein in the bacteria fermentation and enzymatic hydrolysis group were increased (P<0.05), while the urease activity, globulin, β-conglycinin, neutral detergent fiber, acid detergent fiber and, hemicellulose in the bacteria fermentation and enzymatic hydrolysis group were decreased (P<0.05). Compared with the enzymatic hydrolysis group, the reducing sugar content, neutral detergent fiber, acid detergent fiber and, hemicellulose in the bacteria fermentation and enzymatic hydrolysis group were decreased (P<0.05). The content of mold in the bacterial enzyme fermentation group was only 0.89 lg(CFU/mL), which was lower than that in the other groups (P<0.05). [Conclusion] The combination of bacteria and enzymes can increase the crude protein and true protein content, reduce the content of fiber and anti-nutritional factors, inhibit the production of mold in soybean hulls, and finally improve the nutritional value and quality of soybean hulls.

Abstract:[Background] Aspergillus niger, a typical filamentous fungus, can produce valuable metabolites, such as enzymes, organic acids, antibiotics, and is widely used in food fermentation, environment protection and so on. Its physiological characters including fungal metabolism and productivity are closely associated with fungal morphologies. However, the relationship between fungal morphologies and the biosorption of dyes and heavy metals is little known. [Objective] To study the pellet-formation factors of A. niger RAF106 and its application in the biosorption of crystal violet. [Methods] The dynamic processes of pellet formation, the effects of the initial pH, temperature, spore inoculum level, agitation frequency, and different carbon and nitrogen sources on pellet formation, and the effects of different morphologies on the biosorption of crystal violet were investigated in tea-derived A. niger RAF106 when incubated in the medium of potato dextrose broth (PDB). [Results] Both conidia and hypha can aggregate and then form pellets in A. niger RAF106. The diameters of pellets were inversely proportional to the initial pH values ranging from 4.0 to 10.0 and spore inoculum levels ranging from 5×10⁴ to 5×10⁶ but had nothing to do with agitation frequency. Moreover, the diameters increased with the increase of culture temperature ranging from 25℃ to 35℃ but decreased with the increase of culture temperature ranging from 35℃ to 40℃, and pellets can not be formed at 45℃. Additionally, the diameters of pellets in the group of glucose are the biggest among the tested groups treated with different carbon sources, but the addition of exogenous nitrogen sources inhibited the diameters. In addition, the fungal biomass was not affected by tested initial pH, temperature, and agitation frequency but positively correlated with spore inoculum levels, and affected by different carbon and nitrogen sources. Furthermore, more crystal violet can be absorbed by smaller pellets than that in bigger pellets, and hypha was the most effective one. [Conclusion] The diameters of pellets are affected by temperature, initial pH, inoculum levels as well as carbon and nitrogen source but not shaker speed. The adsorption of crystal violet depends on fungal morphologies in A. niger RAF106. These results will be helpful to control the fungal morphologies and optimize the wastewater treatment system associated with A. niger.

Abstract:[Background] As an important biocontrol actinomyces, Streptomyces caeruleatusWMF106 exhibits good antagonistic effect on the pathogen of walnut blight, Xanthomonas campestris pv. campestris. To promote the preparation and application of antimicrobial agent in the control of the disease, it is necessary to improve the biomass, antimicrobial activity and stability of antimicrobial active substance for strain WMF106. [Objective] The present study aimed to explore an optimal fermentation condition and evaluate the stability and field control effect of antimicrobial active substance for Streptomyces strain WMF106. This study will be benefit for formulating large-scale fermentation procedure and development of bioagent.[Methods] Single factor experiment and orthogonal tests were used to optimize the fermentation medium and conditions. The effects of pH, temperature, light, ultraviolet, storage temperature on the stability of active substances and the polarity nature was determined by the single factor method respectively. To evaluate the results of optimization, the methods of field trials on leaves and fruits were used. [Results] The optimum composition of the fermentation medium was yeast extract 2.0 g/L, octmeal 20.0 g/L, NaCl 0.5 g/L, K₂HPO₄ 0.5 g/L MgSO₄· 7H₂O 0.5 g/L. The optimized fermentation condition was initial pH 6.0, inoculation amount 8%, medium volume 150 mL/500 mL, fermentation time 9 d, fermentation temperature 28 against the disease that infected leaves and fruits were more than 65.33% and 61.21%, respectively.[Conclusion] The optimal fermentation medium and conditions were obtained. Moreover, it is also concluded that the fermentation broth of strain WMF106 not only possess the great stability, but also have better field control efficacy. Therefore, the strain WMF106 may be considered as potential biocontrol agent against walnut blight.

Abstract:[Background] Muskmelon under protected cultivation faces soil deterioration, serious diseases, and the decline of muskmelon yield and quality as a result of continuous cropping and excessive chemical fertilizer and pesticides application. [Objective] This paper aims to clarify the effect of continuous cropping span of muskmelon on soil chemical properties, enzyme activity, and microbial diversity.[Methods] Topsoil samples were collected from the greenhouses after 1 year, 5 years, and 10 years of continuous muskmelon cropping. Soil chemical properties, enzyme activity, culturable microorganism count, and microbial diversity were determined with conventional methods, enzyme assay kits, dilution-plate method, and Illumina Miseq, respectively.[Results] Continuous cropping increased soil total nitrogen, available nitrogen, available phosphorus, and pH. The total nitrogen, available nitrogen, available phosphorus, and pH in soil after 5 years of continuous cropping were 1.04%, 0.56%, 6.00%, and 4.45% higher than those in soil after one year of planting, respectively, and the corresponding figures for soil after 10 years of continuous cropping were 7.25%, 6.86%, 17.75%, and 5.60%, respectively. However, soil available potassium was reduced significantly with the continuous cropping and the reduction reached 12.68% in soil after 10 years of continuous cropping. Continuous cropping significantly decreased the activity of soil urease, alkaline phosphatase, and sucrase, and the decrease was 2.79%, 41.05%, and 38.81% for soil under 5 years of continuous cropping, respectively, and 23.43%, 42.30% and 66.21% for soil under 10 years of continuous cropping, respectively. However, the activity of catalase reduced first and then rose with the continuous cropping. The counts of bacteria and actinomyces decreased significantly with the continuous cropping. To be specific, the number of bacteria, actinomyces, and fungi was 6.02%, 29.32%, and 32.50% smaller in soil after 5 years of continuous cropping, respectively, and 58.65%, 53.17%, and 33.75% smaller in soil after 10 years of continuous cropping, respectively, than that in soil after one year of planting. Moreover, bacterial diversity was lowered, as manifested by the reduction of the dominant Proteobacteria and Bacteroidetes and the beneficial Rhizobium, Pseudomonas, and Bacillus. The abundance of fungi was increased and the evenness was reduced. Specifically, the abundance of Ascomycota rose. Redundancy analysis indicated that soil total nitrogen, available nitrogen, available phosphorus, and pH and soil Sphingomonas, Gemmatimonas, and Mycothermus were in positive correlation with continuous cropping span. [Conclusion] Continuous cropping of muskmelon resulted in deterioration of soil chemical properties and microbial environment. The study provided a reference for the remediation of soil under continuous muskmelon cropping and sustainable development of muskmelon industry.

Abstract:[Background] Potato early blight caused by Alternaria solani is generally considered to be the second major potato disease in potato production, which occurs in various potato production areas. Serious disease will lead to large-scale production reduction and potato block decay during storage, causing huge economic losses to potato production. [Objective] In order to clarify the effect of AsSlt2 gene on the cell wall integrity of Alternaria solani. [Methods] Congo red, sodium dodecyl sulfate (SDS) and cell wall degrading enzymes were applied to evaluate the cell wall integrity for the mutant strain, and calculate the relative growth inhibition rate. RT-qPCR was used to analyze the transcription of cell wall-related genes. The chitin content and extracellular enzyme activity in the cell wall of ΔAsSlt2 were further detected. [Results] the tolerance of ΔAsSlt2 to cell wall stress factors SDS, Congo red and cell wall degradation enzymes were weaker than that of wild and complementation strain. The protoplast release increased significantly after adding cell wall degrading enzyme. Furthermore, it was found that ΔAsSlt2 was more sensitive to exogenous peroxide stress. The extracellular peroxidase and laccase activity of ΔAsSlt2 mutant were significantly reduced, the chitin content in the cell wall of ΔAsSlt2 mutant were decreased. The expression of laccase related genes and chitin synthase related genes in ΔAsSlt2 mutant were detected by RT-qPCR. [Conclusion] The AsSlt2 plays an important role in cell wall integrity and tolerance to external stress in A. solani.

Abstract:[Background] Cotton Fusarium wilt has gradually become a major disease threatening the development of Gossypium barbadense cotton industry, but the related functional genes of cotton Fusarium wilt are not very clear. [Objective] In this study, a mutant library of F. oxysporum marked by green fluorescent protein (GFP) was constructed to screen mutants with random insertion of T-DNA, which could provide materials for the screening and research of functional genes of F. oxysporum. library of F. oxysporum marked by GFP was constructed.[Methods] The mutant library of GFP-labeled F. oxysporum was constructed by Agrobacterium tumefaciens mediated transformation (ATMT), and the T-DNA insertion and transformation stability were detected and analyzed. The colony morphology, growth rate, sporulation, germination rate, T-DNA insertion copy number and pathogenicity of randomly selected mutants were analyzed, so as to screen mutants with obvious variation and stability. [Results] 1 600 GFP-labeled F. oxysporum transformants were obtained by using the optimized ATMT mediated system. The transformants were transferred to PDA medium without hygromycin B for 7 generations and then transferred to the medium with hygromycin B. The transformants could still grow normally, indicating that hygromycin gene Hyg was successfully inserted into the wild-type genome and stably inherited. Finally, 17 mutants with different colony phenotypes were screened, including slow growth type, dark purple mycelium type, light purple mycelium type, and light yellow mycelium type. Compared with the wild type, the pathogenicity of mutant A-1 increased by 21.98%, the sporulation of mutant A-1 increased by 103.54%, and the sporulation of mutant C-6 and C-7 decreased by 61.90%. [Conclusion] The mutant library of cotton Fusarium wilt marked by GFP mediated by Agrobacteriumwas constructed, and the mutants with changes in colony morphology, growth rate, sporulation and pathogenicity were screened and analyzed, which laid a foundation for further study of functional genes related to cotton fusarium wilt.

Abstract:[Background] Elicitin is small molecule compound secreted by the Oomycetes Phytophthora and Pythium that can induce immune response in the host. [Objective] Identify the elicitin gene family of Pythium porphyrae and analyze its structural features and possible mechanisms of action during the infection. [Methods] Screening the genome of Pythium porphyrae NBRC33253 for members of elicitin gene family using homologous alignment method. Analysis of the physicochemical properties and phylogeny of elicitin family using bioinformatics tools, combined with transcriptomic data and GO functional annotation to discuss possible mechanism of action during the infection. [Results] Twenty-two elicitin gene family members were identified in the Pythium porphyrae genome. 17 elicitin genes were extracellular secretory proteins, four were localized at the plasma membrane and one was anchored to the Golgi apparatus. The elicitin genes are simple and conserved in structure, containing 1-2 CDS sequences, with the number of amino acids ranging from 114 to 2 100 aa and the isoelectric point ranging from 3.61 to 9.88. The phylogenetic analysis revealed an expansion of elicitin family in Pythium porphyrae NBRC33253. Expression analysis showed that six elicitin genes was up-regulated and seven elicitin genes down-regulated after infection, indicating Pythium porphyrae elicitin gene presumably possessed multiple biological functions. As annotated by GO function to cellulose binding elicitor lectin (CBEL) and modulation by symbiont of host defense-related programmed cell death. [Conclusion] The elicitin gene family of Pythium porphyrae is structurally conserved and belongs to the ELL (elicitin-like) subfamily. Due to the annotation, elicitins in NBRC33253 showed several kinds of function, including cellulose binding excitor lectin (CBEL) which could accelerate programmed cell death in host cells by binding cellulose, attaching to the host surface, performing protein kinase activity, triggering host MAPK signaling pathway-mediated immune responses, and promoting HR cell death. This study provides theoretical basis for further elucidation of the pathogenic mechanism of Phythium porphyrae and genetic breeding for disease resistance traits in Pyropia.

Abstract:[Background] Biosurfactants produced by microorganisms have many characteristics, such as low toxicity, high efficiency and biodegradability, which can alleviate the environmental problems caused by chemical surfactants. Therefore, the screening of high-yielding and safe biosurfactant- producing strains has attracted more attention. [Objective] To screen biosurfactant-producing strains for medicine and food use from pickled vegetable. [Methods] A biosurfactant-producing strain obtained from pickled vegetables by the method of drop collapse and oil displaced circle, and this strain was identified by morphological observation, physiological and biochemical identification, rDNA ITS sequence analysis. The composition and functional properties of biosurfactants were identified and determined by the method of droplet collapse diameter, emulsification activity and chemical analysis.[Results] A biosurfactant-producing strain, Pichia manshurica YM-7, was screened from household pickled vegetables in Dianjiang District, Chongqing. The biosurfactant increased the droplet collapse diameter by 27.5%, and decreased surface tension of phosphate buffered saline by 49.21% (from 71.87 mN/m to 36.50 mN/m). The emulsification index of the biosurfactant extracts reached 63.0% after being mixed with vegetable oil in equal proportions at 8 days (the control droped to 0 after being placed for 6 days). [Conclusion] P. manshurica YM-7, an excellent biosurfactant-producing strain, was successfully screened from the pickled vegetable, and its biosurfactant has good emulsifying properties for certain application prospect in food, medicine and cosmetics industy.

Abstract:[Background] As a kind of tetracycline, oxytetracycline (OTC) is used widely in pharmacy making and animal breeding. Abuse and residues of antibiotics can easily lead to drug resistance of probiotics in the environment, and all kinds of probiotics can transfer drug-resistant genes to pathogenic bacteria after entering the body through food. The current screening methods for drug-resistant strains are time-consuming, and the results of the minimum inhibitory concentration determination may be biased, and the results do not directly show the antibacterial mechanism and drug resistance mechanism. [Objective] In this study, surface enhanced Raman spectroscopy (SERS) technology was used to explore the interaction between oxytetracycline and Lactobacillus plantarum, aiming to provide a theoretical basis for the rapid screening of microbial drug-resistant strains and provide ideas and methods for the study of antimicrobial mechanism and drug-resistant mechanism.[Methods] In this experiment, a kind of solid phase substrate with good and stable SERS enhanced effect was obtained after assembling silver nanorods and quartz. After adding oxytetracycline at different concentrations, Lactobacillus planatum was incubated for 30 min, 60 min and 90 min respectively. The assembled solid substrate was used to measure the SERS signal of the bacteria cells. The changes of the peak location and peak intensity of Raman spectrum could reflect the differences in the cell components of Lactobacillus planatum under different oxytetracycline concentrations and action time. The effect of oxytetracycline on the growth of Lactobacillus plantarum can be further inferred.[Results] The results showed that 0.5×MIC oxytetracycline mainly caused the decrease of SERS spectrum intensity. 1.0×MIC oxytetracycline at 90 min may lead to the destruction of the bacterial cell wall, the SERS spectra in, 1612 cm^-1and 1630 cm^-1 appear two peaks represent the tyrosine and amideⅠ. The cell wall was damaged by 2.0×MIC oxytetracycline at 60 min. [Conclusion] Raman spectroscopy detection can reflect the changes of bacterial cell state and intracellular and extracellular components, which is of great significance for the study of antimicrobial agents on bacteria and the mechanism of drug resistance, and also provides a good technical basis for the rapid screening of microbial drug-resistant strains.

Abstract:[Background] amylase hydrolyses starch and has a wide range of applications in the food fermentation industry, including starch-based sugar, white wine, yellow wine, beer, and vinegar. [Objective] In order to provide excellent amylase resources, an amylase-producing Bacillus strain was screened from high-temperature Jiuqu, and its classification and identification were carried out, and the fermentation conditions of amylase production were optimized. [Methods] The Bacillus strain was identified by colony morphology, Gram staining, and 16S rRNA gene sequence analysis. The effects of medium composition (carbon source, nitrogen source, and metal ions) and fermentation conditions (temperature, pH, and inoculum size) on amylase production was evaluated by single factor experiment, Plackett-Burman experiment, steepest climb experiment, and Box-Behnken design experiment. The optimum medium and fermentation conditions were applied to amylase production using starch hydrolysis. [Results] A high amylase producing strain LT-2 was obtained and identified as Bacillus velezensis LT-2. The optimum fermentation parameters for amylase production by B. velezensis LT-2 are maltose 8.00 g/L, soy protein isolate 8.69 g/L, ammonium chloride 3.00 g/L, calcium chloride 13.94 mmol/L, initial fermentation pH 7.5, temperature 34.08℃, and inoculum size 6%. B. velezensis LT-2 had an extracellular amylase activity of 972.88 U/mL in 33 h incubation under optimal fermentation parameters, which was 5.01 times higher than that before optimization; it had an amylase production of (1 198.40±4.56) U/mL in 51 h incubation in a 5 L fermenter, which was 1.23 times higher than in shake flask level and 6.17 times higher than in shake flask level (before optimization). The amylase produced by B. velezensis LT-2 had a good starch hydrolysis ability, and the hydrolysis rate was 6.17×10^-5 g/(U·h). [Conclusion] B. velezensis LT-2 has promising applications in amylase secretion and starch hydrolysis, which provides theoretical support for the improvement of saccharification capacity and liquefaction power of Jiuqu.

Abstract:[Background] Yeasts play an important role during the wine fermentation. In China, most wineries use active dry yeast (ADY) to make wine during industrial fermentations. However, there are few reports on the influence of ADY on indigenous yeast resources and on the competitive relationship between them. [Objective] The purpose of this study was to compare the implantation and persistence of commercial ADY during industrial wine productions using different grape varieties, and to reveal the genetic diversity of different Saccharomyces cerevisiae genotypes. [Methods] In this study, Cabernet Sauvignon, Marselan, Dunkelfelder and Petit Verdot grew in Eastern Foot of Helan Mountain in Ningxia were inoculated with commercial yeast SP. Samples were collected at 1 d, 3 d and 5 d after the inoculation, and 20 S. cerevisiae isolates were collected in each period. Then the genetic diversity of S. cerevisiae was analyzed by Interdelta fingerprinting and microsatellite molecular markers. [Results] For the 240 S. cerevisiae isolates, Interdelta showed 24 genotypes. SP showed 4 genotypes of SP1-SP4, and indigenous yeast showed 20 genotypes of Type1-Type20. For 9 microsatellite loci selected, the above 24 genotypes were further divided into 15 different genotypes by the microsatellite method. SP showed 3 genotypes (SP-1-SP-3), and the indigenous yeast showed 12 genotypes (L-1-L-12). In Cabernet Sauvignon wine fermentation, SP existed in the whole fermentation process and was the dominant strain on 1 d, 3 d and 5 d, while in Marselan wine fermentation, SP was only detected and dominated on 5 d, while the indigenous strains dominated on 1 d and 3 d. Interdelta analysis and SSR analysis showed that SP was not isolated in the whole process of Dunkelfelder wine fermentation. In the fermentation of Petit Verdot wine, SP was the dominant strain on 1 d and 3 d. [Conclusion] In industrial wine fermentations inoculated with commercial yeast SP, the fermentation was completed by both indigenous strains and commercial ADY. Moreover, they competed with each other in the same fermentation and showed a dynamic succession of different strains. The dominant position of SP in wine fermentation was not guaranteed. The genotypes of indigenous S. cerevisiae strains in the industrial wine fermentations were rich. And the degree of variability was high. Compared with SSR, Interdelta fingerprinting showed a higher distinguishing ability for S. cerevisiae strains.

Abstract:[Background] In recent years, the clinical isolation rate of Streptococcus suis serotype 4 (SS4) increased gradually, but systematic studies on SS4 were scarce.[Objective] In this research, we aimed to study on etiological characteristics of 19 isolates of SS4 in China, 2018. [Methods] 2 strains of Streptococcus suis serotype 2 (SS2) used as reference, 19 strains of SS4 were researched by cultural characteristics and morphological observation, biochemical test, animal pathogenicity test, virulence gene detection, determination of biofilm formation ability and multilocus sequence typing. [Results] Compared with 2 strains of SS2, 19 strains of SS4 had a larger colony diameter, the same hemolytic characteristics and microscopy morphology and incompletely same reaction to lactose, raffinose, synanthrin and mushroom sugar. All the strains of SS4 could caused meningitis and death in mice, and the LD₅₀ of the 7 most virulent strains of SS4 and 2 strains of SS2 were in the order of 10⁷-10⁸CFU. There were 2 to 6 virulence genes and 5 virulence genotypes in SS4, and epf+mrp+sly+gapdh+fbps+orf2+ was the dominant (36.8%, 7/19). All the strains of SS4 had the ability of biofilm formation, weak (1+) mainly (89.5%, 17/19), and the reticular biofilm structure was observed under scanning electron microscope. There were 3 ST types in SS4, of which ST94 were prevalent (89.5%, 17/19), while ST1158 and ST1224 were new found.[Conclusion] The culture and morphological characteristics of 19 strains of SS4 were uniform, the biochemical characteristics diverse, the virulence genotypes diverse, the biofilm formation ability generally owned, with strong pathogenicity to mice and genetic diversity.

Abstract:[Background] Avian pathogenic Escherichia coli (APEC) can cause avian acute or subacute infection, E. coli type III secretion system 2 (ETT2) is a new type III secretion system found in recent years. The effect of virulence gene yqeH on APEC pathogenicity in APEC is unclear. [Objective] To explore the role of yqeH in the pathogenesis of APEC, so as to lay a foundation for further research on the pathogenesis of ETT2. [Methods] The yqeH deletion strain ΔyqeH and its complementary strain CΔyqeH were constructed by Red homologous recombination technology. The effects of yqeH on the biological function of APEC were analyzed by motility test, biofilm formation ability, stress resistance tests and serum resistance test. The effect of yqeH on host infection of APEC was investigated by cell adhesion and invasion test, pathogenicity test and inflammatory factor expression level detected by fluorescence quantitative PCR.[Results] The deletion strain ΔyqeH and the complementary strain CΔyqeH were successfully constructed. Compared with the wild APEC81 strain, ΔyqeH strain showed lower biofilm formation ability and movement ability, lower tolerance to acid, alkali, osmotic pressure and oxidative shock, lower serum resistance and lower pathogenicity. The adhesion and invasion ability of ΔyqeH to the epithelial cells of chicken trachea mucosa were significantly decreased. Compared with wild strain APEC81, the transcription level of inflammatory factors in chicken tracheal epithelial cells infected by ΔyqeH significantly reduced. [Conclusion] yqeH can regulate biofilm formation, motility, stress resistance, adhesion and invasion ability, inflammatory factor expression, and serum resistance of APEC, thereby regulate the pathogenicity of APEC.

Abstract:[Background] The long-term massive and irrational use of antibiotics has led to increasing multi-drug resistance of extraintestinal pathogenic Escherichia coli (ExPEC) isolated from pigs. [Objective] To investigate the drug resistance of ExPEC isolated from pigs and its correlation with drug resistance genes and class I integrons. [Methods] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 54 strains of ExPEC isolated from pigs against 22 antimicrobial drugs were determined by micro-broth dilution method. The drug resistance genes were identified based on the results of drug sensitivity tests, and the distribution of drug resistance genes and class I integrons on chromosomal DNA and plasmid DNA were detected by PCR. [Results] The 54 strains of porcine ExPEC were highly resistant to penicillin, florfenicol, ampicillin and amoxicillin, of which 52 strains of porcine ExPEC were highly resistant to trimethoprim and cotrimoxazole, and their MIC values were greater than 256 μg/mL, no MBC value; and MICs to cefazolin, tetracycline, cefadroxil, daikonin, and streptomycin were 1-256 μg/mL with MBC values of 8, 16, 32, 64, 128, and 256 μg/mL, all tolerating more than 11 antimicrobial drugs, of which 17 were predominantly tolerated, accounting for 18.52%, with 47 drug resistance spectrums. We identified 20 drug resistance genes in 7 major categories, 19 drug resistance genes in chromosomal DNA except aph(3')-IIa gene were detected, with the highest detection rate of aadA1 and tetA, and all strains carried more than 5 drug resistance genes; 16 drug resistance genes in plasmid DNA except tetB, tetC, SHV and qnrA genes were detected, with the highest detection rate of floR and parC, and all strains carried more than 5 drug resistance genes. The positive detection rates of class I integrons in chromosomal DNA and plasmid DNA were 96.3% and 98.15%, respectively, and all positive strains were multi-drug resistant strains. [Conclusion] ExPEC isolated from pigs has serious multi-drug resistance, the drug resistance profiles are complex and diverse, a very high distribution of resistance genes and class I integrons in chromosomal DNA and plasmid DNA, and a correlation between the development of resistance and the presence of resistance genes, which in turn increases the risk of horizontal transfer of resistance genes.

Abstract:[Background] Bacteriocin is a kind of protein or polypeptide substances with antibacterial effect produced during the growth of microorganisms, which can effectively inhibit or kill for various foodborne pathogens. Sinocyclocheilus grahami is one of specific fish species in Dianchi, Yunnan. S. grahami survive in harsh ecological environment with long-term in Dianchi, intestines of which might contain a large amount of bacteriocin-producing microorganism resources. [Objective] The bacteriocin-producing strains were screened from the intestinal tract of the S. grahami in Dianchi and the antibacterial properties as well as mechanisms of the bacteriocin-producing strains were explored. [Methods] The intestinal bacteria of S. grahami in Dianchi were isolated and identified. The antibacterial effect of the bacteriocin-producing strains was screened by the Oxford cup double-layer method. The antibacterial properties including enzyme sensitivity, acid-base and high temperature tolerance, determination of the minimum inhibitory concentration (MIC) and antibacterial spectrum of the best strains were determined. The antibacterial mechanism of the bacteriocin was explored through cell membrane permeability, XTT (2,3-bis(2-methoxy-4-nitro-5-sulfonyl)-2h-tetrazolium-5-carboxanilide) experiments, and scanning electron microscopy analysis. [Results] A total of 5 strains of bacteriocin-producing bacteria were screened from the intestinal tract of the S. grahami in Dianchi, and they all belonged to the Bacillus and Lactobacillus. Among them, the antibacterial activity of bacteriocin (LSP01) produced by strain S01 was the best, inhibition zone of which reached to (29.21±0.25) mm. The produced bacteriocin was sensitive to a variety of proteases and maintained more than 60% antibacterial activity after treatment at 100℃ and pH 10.0. LSP01 was also presented a good antibacterial effect against other 12 Gram-positive and Gram-negative pathogenic bacteria, e.g. Escherichia coli, Shigella flexneri, and Staphylococcus aureus etc. In addition, the MIC of LSP01 against Aeromonas hydrophila was 11.06 μg/mL. After treatment with LSP01, the permeability of the cell membrane of A. hydrophila was increased, the metabolic activity of the cells was decreased, and the cell structure was ruptured, resulting in the death of A. hydrophila. [Conclusion] The bacteriocin resources in the intestines of S. graham in Dianchi are abundant, especially the bacteriocin (LSP01) produced by strain S01 has the characteristics of high antibacterial activity, good stability, and broad antibacterial spectrum, which can destroy the cell structure of A. hydrophila, reduce metabolic activity and cell membrane permeability, suggesting potential for alternative antibiotics.

Abstract:[Background] The new antimicrobial agents based on the quorum sensing system control was rapidly developed due to the multiple drug resistance caused by traditional antimicrobial agents. [Objective] To study the effect of Forsythia suspensa extract (FSE) on quorum sensing system in Aeromonas hydrophila and provide useful information for bacteria control. [Methods] The minimal inhibitory concentration (MIC) of FSE against Chromobacterium violaceum CV026 and A. hydrophila was determined by multiple dilution method, and the effects of FSE on the growth, swarming motility and protease activity of A. hydrophila were determined by micro-method. The primary components of the extracts were analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The possible inhibiting mechanism of FSE on the quorum sensing system of A. hydrophila was explored by molecular docking. [Results] The MICs of FSE against C. violaceum 026 and A. hydrophila were both 16.00 mg/mL. Under sub-MIC concentration, and the production of violacein was significantly inhibited by the FSE, with the maximum inhibition rate of 56.30%. The swarming mobility and protease activity of A. hydrophila was reduced by 85.70% and 35.40% with 8.00 mg/mL FSE treatment, respectively. The major components of the FSE included forsythia, forsythioside, caffeic acid and chlorogenic acid, etc. The molecular docking results indicated that caffeic acid and chlorogenic acid could competitively bind with LasR receptor protein to inhibit the quorum sensing system of A. hydrophila. [Conclusion] FSE could be used as a potential quorum sensing inhibitor in the aquaculture and biological control area.

Abstract:[Background] Acinetobacter baumannii is one of the most important pathogens causing clinical infection. Its resistance level to carbapenem antibiotics is inceasing year by year. Using genome sequencing technology to analyze the distribution and epidemiological characteristics of A. baumannii can promote the effective prevention and treatment of clinical infection.[Objective] To study the clinical distribution and genomic epidemiological characteristics of 200 carbapenem-resistant Acinetobacter baumannii (CRAB) strains detected in Cangzhou Central Hospital in 2018, aiming to provide a theoretical basis for the prevention of nosocomial infections and anti-infective treatment. [Methods] The susceptibility of 200 CRAB strains isolated from different clinical department to 16 antimicrobial agents was detected by bacterial identification and drug sensitivity analyzer; PCR amplification was used to detect the carbapenemase gene; multi-locus sequence typing (MLST) was performed to detect strains' sequence type; genomic epidemiological analysis revealed transmission relationships among strains from different department. [Results] CRAB strains was mainly distributed in emergency ICU (47.0%), respiratory department (19.5%) and critical medicine department (12.0%). The CRAB strains showed high resistant level to imipenem, meropenem, ampicillin/sulbactam, ticacillin/clavulanic acid, gentamicin, piperacillin/tazobactam, levofloxacin, ciprofloxacin, amikacin and the third and fourth generation cephalosporins (cephalosporin, ceftriaxone and cefepime) and moderate resistance to sulfamethoxazole. Most strains were still sensitive to polymyxin B, minocycline and cefoperazone/sulbactam. The PCR results of antibiotic resistance genes showed that 180 strains (90.0%) of CRAB strains carried OXA-23 gene, 19 strains (9.5%) carried OXA-24 gene, and only 1 strain carried OXA-58 gene. The results of MLST showed that except for the OXA-58 positive strain, the others were all belonged to international clonal group CC2. Genomic epidemiology analysis showed that the strains in this hospital could be divided into four groups A, B, C and D. Group D was the main epidemic group, and it was distributed in different disease areas. [Conclusions] The multidrug resistance of CRAB strain in our hospital is severe, mainly were CC2 strains carrying OXA-23 gene, and there may be infection transmission among different ward areas. The environmental cleaning work of key departments should be strengthened, the technical operation rules of medical staff should be strictly standardized, and carbapenem antibiotics should be used cautiously to reduce infection and prevent further increase of drug resistance rate.

Abstract:Atopic dermatitis (AD) is a recurrent, chronic and inflammatory cutaneous disease with a clear genetic predisposition, and its incidence rate is increasing year by year. The main pathogenesis of AD is skin microdysbiosis and allergen leakage caused by hereditary or acquired cutaneous barrier damage, which activate the corresponding inflammatory reaction, resulting in the vicious circle of "barrier damage-inflammatory reaction". The traditional treatment of AD is corticosteroid and immunosuppressants. Recent studies have revealed that probiotics are effective in the treatment of AD. Probiotics can reshape the gut-skin axis by altering the skin or intestinal microbiota and repair of the skin barrier, resulting in stopping the cycle of AD progression and restoring healthy skin homeostasis.

Abstract:Keratinophilic fungi are those species that can degrade keratin and utilize keratin materials as the only nutrient source most of which belong to Onygenales, Eurotiales and Hypocreales. Since these fungi are widely distributed in various environment or niches, and have many varieties and nutritional diversity, more and more methods and techniques have been developed for investigation and characterization of these species. In order to promote the understanding of the current status and research shortage of keratinophilic fungi, this paper defines the keratinophilic fungi, organizes their taxa, introduces three main research methods, summarizes their applications in feed, leather, medicine and agriculture, and as bioindicators. Finally, we summarized research directions of keratinophilic fungi in the future work.

Abstract:Adaptive laboratory evolution is a method of screening mutant strains with specific phenotypes by long-term domestication under certain selection pressure. In recent years, this method has been widely used to screen industrial production strains with excellent characteristics through specific evolutionary conditions and screening strategies, such as specific phenotypic screening, efficient use of substrates, target product synthesis, and growth characteristics optimization. In this review, the typical examples and research progress of adaptive laboratory evolution for the breeding of industrial production strains were summarized, the existing problems and solutions were discussed. The prospect of this technology was also forecasted.

Abstract:Biological treatment, with the characteristic of low cost and slightly environmental impact, has become a main removal pathway of environmental organic pollutants. The disadvantage is that the treatment time is long, especially for refractory organic pollutants, which may need several decades or even hundreds of years. Extracellular reactive oxygen species (EROS), with the ability of promoting biodegradation of refractory organic pollutants, were oxygen-containing active group generated from the metabolism of microorganisms. Recently, researches related to the effect of EROS on biodegradation of refractory organic pollutants increased sharply. However, no relative review has been reported yet. In this paper, the latest research achievements on the production of EROS and its promotion on biodegradation of refractory organic pollutants have been summarized, mainly from the aspects of introduction of EROS, producing microbes, generation mechanism, promotion on the degradation of refractory organic pollutants, future direction and challenges, aiming at providing a guidance for the following researchers.

Abstract:A white-rot basidiomycete Ganoderma spp. is a rare edible and medicinal fungus, which is rich in a variety of bioactive components. Liquid fermentation technology has the advantages of short production cycle, high efficiency, high yield and stable quality, which is an important way to develop and utilize Ganoderma resources. In recent years, great progress has been made in the development and application of liquid fermentation of mycelia of Ganoderma species. In this paper, the main active components and medicinal effects of liquid fermentation products of Ganoderma spp., the optimization of liquid fermentation process and the application of fermentation products were reviewed, and the future of this field was prospected.

Abstract:Phenazine is a class of nitrogen-containing heterocyclic compounds. It is usually a secondary metabolite produced by Pseudomonas or Streptomyces. Phenazine has antibacterial activity, and phenazine also has anti-insect and anti-tumor activity. It is not only widely used in agriculture, but also widely used in medical field. But the synthesis of phenazine is very complex, because it is regulated by a multi-cascade network. In this article, the author will summarize the regulatory mechanism of two-component signal transduction system on phenazine biosynthesis in Pseudomonas. This paper elucidates the effect of small RNA on phenazine biosynthesis under the control of each two-component signal transduction system, and the effect of quorum sensing on phenazine biosynthesis under the control of each two-component signal transduction system. The purpose of this study is to provide a reference for the further study of the cascade network of phenazine synthesis regulated by Pseudomonas.

Abstract:Extracellular vesicles (EV) are particles with natural nanostructure, which are composed of biomolecules such as protein and nucleic acid wrapped in a lipid bilayer. EV is a means of cell-free communication between cells and affect cell-to-cell communication by transmitting large quantities of biomolecules, including genetic information. In addition, EV has been associated with many phenomena, such as immune regulation, intercellular competition, horizontal gene transfer, and pathogenicity. EVs secreted by Gram-positive bacteria carry a wide variety of compounds that play important roles in bacterial competition, survival, invasion, antibiotic resistance and infection. Currently, the studies on bacterial EVs mainly focused on Gram-negative bacteria, and few reports on Gram-positive bacteria in which the EVs produced by Staphylococcus aureus and Staphylococcus epidermidis are the most frequently investigated. In this review, the chemical composition, influencing factors, functions and clinical application of Staphylococcal EVs are summarized.

Abstract:Rhamnolipids, the biosurfactants extensively studied, are widely used in enhanced oil recovery, environmental pollution remediation, agriculture, etc. However, compared with chemical surfactants, rhamnolipids has low biosynthetic yield, which leads to the high production cost and limits its large-scale application. Therefore, it is of great significance to study the optimization strategies for the high production of rhamnolipids. This paper briefs the biosynthesis of rhamnolipids and its influencing factors, reviews the recent advances in optimization strategies, including screening and improvement of high-yielding microbial strains, heterologous biosynthesis, biosynthetic pathway regulation, and fermentation optimization, for high production of rhamnolipids, and analyzes the advantages and disadvantages of these strategies. Finally, we put forward insights and prospects for the current research on the high production of rhamnolipids.

Abstract:Marine Microbiology Experiment, a core and basic course of marine science major in colleges and universities, plays an important role in the training of marine science talents. The ideological and political construction in Marine Microbiology Experiment course is very important for college education. This article introduces the overall thinking and practical exploration of "curriculum ideological and political education" in Marine Microbiology Experiment course from the following aspects:the mining and construction of curriculum contains ideological elements, teaching idea, teaching goal, teaching method, examination evaluation system, teaching reflection and so on. In such a way, ideological and political education was integrated into the teaching of Marine Microbiology Experiment course to realize synergy effect.

Abstract:Environmental Ecological Engineering is a new major and its basic teaching and curriculum design have yet to be explored and improved. The course of Environmental Microbiology is compulsory for Environmental Ecological Engineering. How to make the teaching content and format of the course adapt to the overall needs of the major to be discussed. Based on the characteristics and advantages of our university and college, the authors propose to employ Environmental Microbiology course as a carrier to cultivate the innovation ability of undergraduates majoring in environmental and ecological engineering. The course incorporates marine characteristics and ecological points, combining theoretical teaching in class, student lectures, extracurricular study, and experimental skills training. This research can provide a reference for teachers who set up Environmental Microbiology in the same major, and can also give tips for the cultivation of innovation ability of undergraduates majoring in Environmental Ecological Engineering in other universities.

Abstract:Industrial Microbiology, a core course of life sciences, features high applicability. In order to achieve the training of applied talent in local universities, the teaching team actively combined with the development of local industrial characteristics to reform the applied teachers training, reorganize the teaching content, construct the online and offline blended and social practice teaching model, and establish a formative learning evaluation system. The practice indicated that the reformed curriculum teaching could stimulate students' internal learning motivation, effectively improved students' application ability and comprehensive quality, and finally achieved good effects.