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    26 September 2019, Volume 35 Issue 9
    Advances on Properties,Production,Purification and Immobilization of Fungal Laccase
    WU Yi, MA Hong-fei, CAO Yong-jia, SI Jing, CUI Bao-kai
    2019, 35(9):  1-10.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0614
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    Fungal laccase is a kind of polyphenol oxidase with excellent properties. It can oxidize large amounts of substrates such as phenolic and aromatic amine compounds,coupled with a concomitant reduction of molecular oxygen to water and other final products. Owing to the up-mentioned characteristics,fungal laccase is regarded as a green catalyst for environmental demand,and thus has great application potential in various fields,including pulp bleaching,environmental protection,biological detection,and organic synthesis. In this paper,current advances on biological properties,production,purification,and immobilization of fungal laccase were introduced and summarized,and its future development was prospected.
    Effect of Surfactant on Cellulase Hydrolysis and Its Mechanism
    ZHANG Jia-shun, GAO Li-li, MA Jiang-shan, LIU Gao-qiang
    2019, 35(9):  11-20.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0158
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    With the increasing tension of energy crisis and environmental problems,it is becoming growingly important to seek an environmentally friendly,sustainable and adequate energy alternative. The research on fermentation of lignocellulose biomass to produce biofuels and other biomaterials has attracted more and more attention. Enzymatic degradation of lignocellulose is a hot topic in biomass conversion and utilization. Surfactant(SF)can significantly improve the enzymatic hydrolysis efficiency and conversion of cellulose,but it is still affected by many factors. Here we reviewed the research status of SF on enhancing the hydrolysis ability of cellulase,and summarized the types and concentration of SF,the source and composition of cellulase,the influence of substrate structure,and hydrolysis conditions on the effectiveness of SF. Further,we analyzed the mechanism of SF enhancing cellulosic enzyme efficiency and conversion rate based on the adsorption properties of enzymes,the stability and activity of enzyme,and the surface structure of substrate. In view of the current research situation,we prospected the future research directions,that is,further exploring the role of molecular interaction among SF,cellulase and substrates in stabilizing cellulase activity,changing the adsorption properties of enzymes,and the surface properties of substrates,aiming to provide a theoretical reference for the enzymatic transformation of lignocellulose biomass.
    Research Progress on Effect of Fertilization on Soil Properties and Microbiome
    LIU Ji-ai, SHU Ai-ping, LIU Guang-rong, LI Zu-zhang, LIU Zeng-bing, GAO Zheng
    2019, 35(9):  21-28.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0579
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    This paper mainly introduces the detection techniques of soil microbiome,reviews the effects of different fertilization treatments on soil properties(physical properties,nutrients,and enzyme activities)and soil microbiome,and summarizes the responses of soil microbial abundance,diversity and community structure and composition to fertilization. Then the paper also briefly overviews the shortcomings in the study of soil microbiome affected by fertilization at present and prospects the future research trends,aiming at providing a basis for rational fertilization of agricultural production.
    Research Progresses on Insecticide Resistance Mediated by Symbiotic Bacteria
    DUAN Ru-xin, MENG Lei, WANG Ning-xin
    2019, 35(9):  29-30.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0488
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    China is one of the first countries to use pesticide for the prevention and control of crop pests,also is a big country producing and using pesticides. With widely applying pesticides in agriculture and forestry production,the resistance of insects to pesticides is increasing,which has become an important limiting factor in the sustainable development of agriculture and environmental protection. Insect resistance refers to the ability of insects to tolerate doses that kill most individuals in a normal population and this develops in their populations. At present,over 600 pests have developed resistances to different kinds of pesticides,and the growth rate of their resistances is on the rise. Studies have found that insect symbiotic bacteria are related to insect resistance. Among these insects,there is the largest number of resistant insect species in Diptera and Lepidoptera. This paper reviews insect resistance mechanisms,symbiosis groups related to insect resistance and related research progress. With the rapid development of molecular biology technology,the functional research of insect symbiotic bacteria will have an obvious breakthrough. Resistance management mediated by insect symbiotic bacteria is of significance in environmental protection and also provides new ideas for biological control,and lays a research foundation for studying insect-microbial interaction.
    Advances in Research and Application of Microbial Source Tracking Technology in Aquatic Environment
    XU You-fen, LI Zong, LIU Ru-yin, YU Zhi-sheng, ZHANG Hong-xun, HE Wei, LI Ye
    2019, 35(9):  35-44.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0343
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    One of the main environmental pollution problems caused by livestock and poultry farming is the fecal pollution to surface water. It is critical to identify the source of fecal pollution associated with livestock for the efficient control of the fecal pollution to water system. Microbial source tracking(MST)is a kind of technique that may locate pollution sources according to the specific relationship between source-indicating microorganisms and hosts. A variety of intestinal microorganisms have been reported to be used as indicators for source tracking of fecal contamination in surface water environments. Based on their phenotypic or genotypic characteristics,various detection methods have been developed,including culture-dependent methods and culture-independent molecular methods. MST can be classified as single-source-indicator methods and multi-source-indicator methods,specifically including bacterial and viral indicator methods,as well as multi-source-indicating DNA microarray and high-throughput sequencing methods. Different types of source-indicating microorganisms and their related detection methods are reviewed in this paper,including their principles and pros and cons,and the issues and challenges while using MST are also discussed. In addition,the application of MST in total maximum daily load(TMDL)promoted by U.S. Environmental Protection Agency(EPA)is briefly summarized,and the development trend of MST is also prospected,aiming at promoting the application of MST in the monitoring of water quality in China.
    Research Progress of Low-cost Method of Synthetizing Polyhydroxyalkanoates(PHAs)
    QIU Shi-zheng, LI Jia-yi, YANG Jing-chen, LIU Chang-li
    2019, 35(9):  45-52.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0398
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    Polyhydroxyalkanoates(PHAs)is a natural macromolecule biomaterial synthesized in microorganisms. It can be used as a reserve material of microbial carbon source under excessive carbon sources and the condition of insufficient nitrogen and phosphorus. PHA is the best substitute for traditional petrochemical plastics because of its fine biocompatibility,biodegradability and thermal processing properties. With the development of PHA industrialization,4 generations of commercially produced PHA products(PHB,PHBV,PHBHx,and P3HB4HB)have been used in medicine,industry,agriculture,chemical industry and other fields,and become one of the most active research hotspots in the field of biomaterials. However,the issues of high cost of fermentation substrates and sterilization,low production efficiency and low product performance have not been effectively solved,thus it is difficult to maintain a greater competitiveness to petroleum-based plastics,which makes it particularly important to explore a low-cost method of synthesizing PHA. The main types and characteristics of PHAs and the hot fields of its industrialization are introduced. The research and development advances in low-cost synthetic PHA are reviewed,i.e.,scientists around the world have adopted strategies of constructing low-cost metabolic pathways,transforming production strains,using cheap fermentation substrates and improving fermentation process. It is aimed to provide a theoretical basis for PHA to replace petrochemical plastics in a low-cost and large-scale way at an early date.
    Research Progress on Sulfate-reducing Bacteria Using Gas as Electron Donor
    ZHU Chuan-jing, LI Liu-qing, HUANG Jian-hong, TIAN Sen-lin, HU Xue-wei
    2019, 35(9):  53-60.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0592
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    Sulfate-reducing bacteria(SRB)demonstrate significant advantages in the treatment of sulfate-containing wastewater,but their metabolic processes require the support of electron donors. Ordinary organic electron donors(such as lactate,ethanol,etc.)cannot be completely converted by SRB in a short time,leading to high residual COD in effluent,which needs further treatment. The gases(H2,CO2,and CO)as the electron donors in SRB metabolic process have received continuous attentions duo to their advantages of large electron supply per unit mass and low residual organic concentration in effluent. This review focuses on the reaction mechanism,influencing factors(environmental factor,gas mass transfer,sulfur-related component,and donor competition),gas characteristics,and metabolic transformation of SRB while using gas as electron donor,and put forward to some problems that need to be solved in the application of biochemical treatment engineering,such as the mass transfer efficiency of insoluble gas,inhibition and removal of toxic gas(CO),high biological proliferation,maintenance of metabolic activity,etc.,aiming at providing corresponding foundation for subsequent research.
    Biological Improvement Technology for Civil Construction Sites and Building Materials:A Review
    HAO Da-cheng, LU Lan-lan
    2019, 35(9):  61-69.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0512
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    Various aspects of construction biotechnology,especially biological improvement technology for civil construction sites and building materials,have formed a new interdisciplinary field involving environmental and industrial microbiology,biogeochemistry,as well as the application of biotechnology in geotechnical and civil/environmental engineering. This paper summarizes the progress of research and application of microbial technology improvement in civil engineering sites and building materials,and discusses the biogeochemical processes related to construction. Bioaggregation can be used to control wind erosion and dust emission. Biocrusting,bioclogging and soil biocementation have different mechanisms and use modes. Other novel ground improvement methods,such as biodesaturation of water-saturated cohesionless soil,bioencapsulation of soft soil and bioimmobilization of soil pollutants,have their own unique mechanisms and advantages. At present,the most microbial geotechnical engineering research is still in the laboratory stage,and more efforts are needed to transform scientific concepts into feasible technologies. Therefore,this article aims to summarize the research and application progress of microbial technology improvement in civil construction sites and materials,to discuss the microbial processes related to construction that relevant researchers and engineers need to understand,and to briefly discuss the design principles and precautions of various field applications.
    Reducing Cr(VI)from Water System via Immobilized Trichoderma hamatum Spores by Embedding Method
    WANG Wen-jing, LU Song-lin, LIU Xue, LI Si-yu, HAO Xue-meng, LI Yuan-yuan, ZHANG Jie
    2019, 35(9):  70-74.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0331
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    This paper aims to explore a novel method of reducing hexavalent chromium by immobilized microorganisms and to provide a new idea for the further research. Spore-encapsulated microspheres PVA-SA and PVA-SA-PDA with Trichoderma hamatum BSL01 were prepared by embedding method. The performance variation of PVA-SA and PVA-SA-PDA microspheres,such as the reduction efficiency of Cr(VI)and the recycling times,were further studied. The results showed that the reduction efficiency of Cr(VI)by PVA-SA immobilized pellets was 93.4%,and it could be reused 18 cycles. The reduction efficiency of PVA-SA-PDA pellets was 97.2%,and it could be reused 23 cycles. Cr(VI)with a concentration of 50 mg/L was completely reduced using PVA-SA and PVA-SA-PDA in 3 d from the 8th cycle,which was nearly twice as that of free bacteria in reducing Cr(VI). It is the first time that PDA medium and fungal spores are prepared into nucleocapsid microspheres that are recycled more times and have fine results.
    Immobilization of Marine Urease and Its Utilization in the Treatment of Urea Wastewater
    LIU Chang-rong, ZHANG Feng-li, LI Zhi-yong
    2019, 35(9):  75-82.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0254
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    The objective is to develop an appropriate immobilization strategy for marine alkaline urease and to provide technical assessments for urea wastewater treatment via the immobilized urease. The enzyme was immobilized by 3 kinds of materials,and the immobilization effects,enzyme properties were examined. The immobilized urease was loaded in a column for urea wastewater treatment and the operating parameters were optimized. Urease immobilized by alginate-CaCl2 was the best with an immobilization rate reaching 61.47%. Comparing to the free urease with optimal temperature 55℃ and pH 8.5,the immobilized urease increased to 70℃,and pH 9.5,respectively,and sustained over 90% activity after incubating at 35-85℃ for 30 min or at pH 7-10.5 for 6 h. Furthermore,its half-life exceeded 3 months when stored at 4℃. The treatment apparatus achieved a flow rate of 19 mL/min at 70℃ with an initial urea decomposition rate of 90.25%,and the efficiency remained constant for 35 days. In conclusion,the optimal temperature and pH,the thermostability and the alkali resistance of the marine urease are much improved after being immobilized by alginate-CaCl2. The urea wastewater treatment applying the immobilized marine urease represents a considerable alternative technique besides chemical method.
    Optimization of Chromium(VI)Removal by Mixture of Bacteria-microalgae and Determination of Chromium(VI)Reductase Activities
    YANG Sheng-nan, LIU Na, SONG Dong-hui
    2019, 35(9):  83-92.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0322
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    In order to efficiently remove Cr(VI)from industry wastewater,two strains of bacteria that remarkably reduced Cr(VI)were isolated from the wastewater in a dyeing factory of Tianjin. The effect of removing Cr(VI)by the mixture of bacteria-microalgae system and the optimized conditions were studied,as well as the activity of Cr(VI)reductase was determined. 16S rRNA gene sequence alignment was used to identify the bacterial isolates,and Placket-Burman,the Steepest Ascent,Box-Behnken experimental design and response surface methodology were conducted to analyze the interactions among each factor. In addition,the Cr(VI)reductase activities of two bacteria were determined. The result showed that two strains of Cr(VI)reducing bacteria belonged to the genus Serratia and the genus Delftia. The optimal conditions for removing Cr(VI)by the mixture of bacteria-microalgae were as follows:the temperature was 29.74℃,the Cu2+ concentration was 27.65 mg/L,the glucose content was 2.41%(W/V),the sodium pyruvate content was 2%(W/V),the inoculum was 7%,and the pH was 7.0,and no NaCl,respectively. Under the optimized conditions,the removal rate of Cr(VI)by the mixture of bacteria-microalgae reached 97.89% in 24 h. The optimal temperature of the Cr(VI)reductase for two strains S-3 and D-7 was 30℃. The optimal pH of the Cr(VI)reductase for the strain S-3 was 6.0,and the kinetic constant of the enzymatic reaction was Km=86.94 μmol/L,Vmax=2.71 µmol/(L·min). The optimal pH of the Cr(VI)reductase for the strain D-7 was 7.0,and the kinetic constant of the enzymatic reaction was Km=103.18 μmol/L,Vmax=3.38 µmol/(L·min). In conclusion,the optimized mixture of bacteria-microalgae may not only increase the removal rate of Cr(VI),but also shorten the treatment time,and it has a promising application prospect in the treatment of chromium-containing wastewater.
    Screening of a Bacterial Strain Efficiently Degrading Feather Waste and Optimization of Its Expression Condition
    ZHANG Yu-wen, YUAN Hang, YU Jiang-yue, MA Xiao-xiao, SHI Chao-shuo, LI Yu
    2019, 35(9):  93-98.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0370
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    This work is objected to screen and construct a strain of efficiently degrading feather keratin for increasing the secretory expression of keratinase. Soil samples were collected from pigs and sheep pens of five different regions in China,and milk medium was used in preliminary screening,then feathers as the sole carbon and nitrogen source were used to rescreen strain producing keratinase. The strain was classified and determined based on the observed morphology and 16S rDNA sequence Analysis. In order to further improve the fermentation activity of the strain,5 signal peptide sequences(KerK,YoaW,DacB,NprE,and SacB)were screened and optimized,and recombinant plasmid was constructed one the basis of pWB980,which was transformed into Bacillus subtilis WB600 to express keratinase. The strain that efficiently degraded feather was identified as Bacillus sp. At 37℃ and after 48 h fermentation,the keratinase activity of the initial strain M was 21.98 U/mL. The recombinant strain R3-DacB containing signal peptide DacB were obtained via screening and optimizing the signal peptides,and its keratinase activity reached the highest by 226.34 U/mL,which was 10 folds of that by the initial strain M. In conclusion,the recombinant strain R3-DacB has promising effect on the degradation of feather waste,which is of great significance to the industrial production of keratinase.
    Microbial Diversity and Community Structure Characteristics of Yam Rhizosphere Soil at Different Development Periods
    KANG Jie, ZHANG Shu-yan, HAN Tao, SUN Zhi-mei
    2019, 35(9):  99-106.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1041
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    This work is to study the dynamic change law of microbial diversity of soil samples at different reproductive periods from yam rhizosphere. By adopting the method of high-throughput sequencing technology,we studied the soil microbial community structure changes of yam root zone in seedling,flowering and harvest stage. Results showed that the diversity and abundance of bacteria in different growth period did not have significant variation,while there was more apparent variation for fungi,and the variation increased gradually from the start growing season to harvest,reached the highest degree of diversity index and abundance in harvest stage. Proteobacteria and Acidobacteria were the dominant bacteria at phylum level,which had over 50% proportion in different periods. Actinomucor and Mortierella were the dominant fungi at genus level,which accounted for 15% proportion in different periods. Combined with the obvious changes of other advantage bacterial group in different growth stages,it can be seen that these flora are closely related to the growth and development of yam.
    Screening and Identification of Biocontrol Bacillus sp. Against Astragalus Root Rot and Its Pot Experiment
    ZHAO Xiao-xia, NIU Shi-quan, WEN Na, SU Feng-feng
    2019, 35(9):  107-111.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0329
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    Astragalus root rot is one of the diseases that seriously affect the yield of astragalus. In order to obtain one bacillus strain with strong antagonistic activity against the pathogen of astragalus root rot,the pathogenic bacterium Fusarium solaniun KR997532 was used as the target strain,the antagonistic bacillus strains of the plant pathogens preserved in our laboratory were screened and rescreened,one bacillus strain 7-2-1-6 with strong antagonistic biological activity was obtained. Strain 7-2-1-6 was identified as Bacillus subtilis subsp. Spizizenii based on the 16S rDNA sequence analysis and the morphological characteristics and physiological and biochemical characteristics of strain 7-2-1-6. The bio-control effect of strain 7-2-1-6 achieved about 68.80% to astragalus root rot in pot experiment.
    Influence of Anthracene on Laccase Activity and Transcriptional Expression Profiles of Ganoderma lucidum
    HU Chu-xiao, LEI Shan-yu, QIN Yan-ping, ZHAO Yi-jin, XIANG Quan-ju
    2019, 35(9):  112-117.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0260
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    This work is to investigate the effects of polycyclic aromatic hydrocarbons(PAHs)on laccase activity and transcriptional expression of different Ganoderma lucidum strains. Three G. lucidum(Meizhi,Rongbao No.1 and Chuanzhi)strains were used as experimental materials. After 5 days of static culture in the dark,a final concentration of 1.0 mg/L anthracene was added. Then the supernatant was collected for the determination of laccase activity after treatment for 1 h,3h,6 h,9 h,and 24 h. Further the mycelium samples were used for RNA extraction,and the effect of anthracene on laccase activity and transcriptional expression of laccase genes was analyzed. The results showed that the effects of anthracene on the laccase activity and transcriptional expression levels of 3 strains differed. Among the 3 strains of G. lucidum,Chuanzhi presented the highest laccase activity,followed by Meizhi,and he lowest activity for Rongbao No. 1. The laccase activity of Rongbao No.1 increased with the prolongation of treatment time,and was inhibited in the early stage of culture,while promoted in the later stage. The change trend of Meizhi’s laccase activity was similar to Rongbao No.1’s,and the change range was small. Enzyme activity of Chuanzhi was strongly inhibited by anthracene and differed insignificantly over time. Under the treatment of anthracene,little variations were observed in the transcript expression levels of Meizhi laccase gene,and most of the genes demonstrated peak transcriptional expression after 9 h treatment. The expression levels of Rongbao No. 1 laccase genes were similar to that of Meizhi. Under short-term treatment,the transcriptional expression level of Chuanzhi laccase genes were significantly up-regulated,and then down-regulated as the treatment time prolonged. In sum,the laccase activities and laccase genes expression levels of 3 strains of G. lucidum varied in response to anthracene;among them,Chuanzhi was the most sensitive,followed by Rongbao No. 1,and Meizhi had the smallest change and thus the strongest tolerance.
    Screening aoattacin Gene from Amiota okadai Based on Transcriptome Sequencing,Expression of It in Insect Cells and Antibacterial Activity Identification
    CAI Juan, LIU Liu, WANG Ling-jun, CAO Jian-ping, ZHENG Ming-hui, LIU Hui
    2019, 35(9):  118-124.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0157
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    By analyzing the transcriptome data of Amiota okadai,an attacin homologous unigene named aoattacin was obtained,which was expressed in insect cell Sf9 and its antibacterial activity was detected. The antibacterial peptide aoattacin gene was obtained by transcriptome analysis. The antibacterial peptide aoattacin gene was obtained by transcriptome analysis,and the sequence of the whole gene was synthesized to construct the recombinant pFast-bac1-aoattacin vector. The recombinant vector was transfected into DH10Bac cells to obtain recombinant baculovirus,which was transfected into insect Sf9 cells. To obtain the P1 virus and obtain the P2 virus by amplification,and detect the expression of the gene in Sf9 cells. Protein expression was detected by SDS-PAGE and Western Blot,and the antibacterial activity of the expressed product was determined by the minimum inhibitory concentration. The results showed that a high-expressed aoattacin gene screening from transcriptome data was successfully expressed in insect cell Sf9,and Aoattacin demonstrated antibacterial activity against many kinds of bacteria in experiments,such as both Gram-positive bacteria and Gram-negative bacteria,which may be a new broad-spectrum antimicrobial peptide.
    Study on the Degradation of PAHs by Bacillus thuringiensis
    LUO Xiao-fang, CHEN Li-hua, XIA Miao-miao, XU Shu-juan, XIONG Mei
    2019, 35(9):  125-133.  doi:10.13560/j.cnki.biotech.bull.1985.2018-0879
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    In order to study the performance of Bacillus thuringiensis degrading anthracene,pyrene and fluorene at different temperatures,pH and qualities of PAHs,a single factor experiment and a response surface analysis were combined,Soxhlet extraction method was applied to determine the degradation absorbance value using PAHs as the sole carbon source and activated carbon as an adsorption carrier. The experimental results were analyzed via Box-Behnken design in software Design Expert 8.0.6.1. The results showed that the interaction between temperature and pH presented significant effects on the degradation of anthracene and pyrene,and the interaction between temperature and PAHs quality significantly affected the degradation of fluorene. In the optimized experiments,the predicted degradation rates of anthracene,pyrene,and fluorene under respective optimized conditions were 92.13%,87.08%,and 83.56% respectively,the relative error between the predicted value and the actual value 91.60%,86.62%,and 82.95% were ±0.58%,±0.53%,and ±0.73%.The relative error of the three optimization models were all within 2%,indicating that the three experimental models could be used to analyze and predict the degradation effect of B. thuringiensis for anthracene,pyrene,and fluorene.
    Improvement of Catalytic Activity of Amylase from Bacillus amyloliquefaciens and Its High Expression in Bacillus subtilis
    QIU Jin, HUANG Huo-qing, YAO Bin, LUO Hui-ying
    2019, 35(9):  134-143.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0234
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    α-Amylase is a kind of enzymes that catalyzes the random internal hydrolysis of α-1,4 glycosidic linkages in starch chain into dextrin,oligosaccharides and monosaccharides. The enzyme is widely used in bread making and washing detergent etc. Modifying amylase at the molecular level to improve the hydrolysis activity and catalytic efficiency has been the goal for many years and has important research significance. In this study,based on BAMYA,a widely used neutral amylase from Bacillus amyloliquefaciens,the mutation sites(L473K/K474H/N475K)at the C-terminal similar substrate binding region(CBM)were rationally designed as the site-directed mutation was aimed to increase its hydrolysis ability,and its efficient expression in Bacillus subtilis was achieved. The optimal temperature and pH of wild BAMYA were 55℃and 6.0,respectively and the specific activity was 6 835 U/mg. The optimal temperature of mutant BAMYA-L473K/K474H/N475K was 60℃,5℃ higher than that of wild one,while no changes in pH. The specific activity of the mutant was 10 148 U/mg,and it increased by 48% compared to the wild one. The Km and catalytic efficiency of wild BAMYA and mutant BAMYA-L473K/K474H/N475K were 3.58 mg/mL and 2.21 mg/mL,1 577 mL/(s·mg)and 4 760 mL/(s·mg),respectively. The catalytic efficiency increased over two times than that of wild one. In sum,the application of the mutant in industry will effectively improve the efficiency of hydrolyzing starch and reduce the application cost.
    Isolation and Identification of Massilia sp. B260 and Its Effect on Seedling Raising
    GUO Yu-ze, DING Xue-min, YAO Lan, XU De-min, ZHAO Yu-jie, FENG Fu-ying, MENG Jian-yu
    2019, 35(9):  144-149.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0683
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    Massilia strain is widely distributed in the rhizosphere of plants;however,there is still a lack of research on its effect on promoting seedling. To analyze the effect of Massilia strain B260 on promoting seedling growth is aimed to provide theoretical basis for its practical application. Coating and scribing was used to isolate the rhizosphere bacteria. The homology analysis of 16S rRNA gene sequence was based to preliminarily classify and identify the strains. The selective medium was used to analyze the characters and abilities of strains fixing nitrogen,dissolving phosphorus phytic acid and inorganic phosphorus,producing ACC deaminase,iron carrier and IAA. Two groups of treatments(including pB260 of adding strain B260 in the matrix,CK without adding B260)were set up. The seedling growth characters of tomato,cucumber,licorice root and astragalus under 2 treatments in hole plate seedling raising were compared and analyzed. The 16S rRNA of strain B260 had the highest similarity(99.85%)with Massilia plicata 76T,which was preliminarily identified as Massilia. B260 demonstrated the plant growth-promoting character including nitrogen fixation,phosphorus release,hormone production,iron carrier production and ACC deaminase production,among which nitrogen fixation and inorganic phosphorus decomposition were the strongest. Compared with CK,pB260 increased the germination rate,plant height,root length,dry weight and fresh weight of tomato,cucumber,licorice and astragalus,especially root length increased by 6.71%,5.41%,12.28% and 15.79%,respectively. The B260 strain is Massilia,which has traits of plant growth-promoting,and can be used as seedling substrate.
    Analysis of Diversity of Malachite Green-degrading Microbial Community and Degradation Characteristics of Efficiently-degrading Bacteria
    WANG Ya-ni, SONG Jin-long, HAN Gang, MU Ying-chun, JIANG Xu, WANG Jin-yao, RUAN Zhi-yong, LI Le
    2019, 35(9):  150-155.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0135
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    Using triphenylmethane dye malachite green as the sole carbon source,the enriched culture degrading malachite green was obtained by continuous passage domestication. High-throughput sequencing was applied to analyze the microbial community change of the enriched culture,and the main microbial information related to degradation was acquired,and further an efficient strain degrading malachite green was isolated and purified. This strain degraded 91.2% of 50 mg/L malachite green within 7 days. The strain was initially identified as a Pseudomonas,and named as Pseudomonas veronii JW3-6.The optimized condition of degradation by this strain were temperature 30℃,pH 7,and inoculation concentration 1×107 CFU/mL.

    Degradation Characteristics of Catechol and Sodium Benzoate by a Petroleum-degrading Bacterium
    LIU Na, LIU Zhi-min, SONG Dong-hui
    2019, 35(9):  156-164.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0578
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    Catechol and benzoic acid are two common intermediate products in the metabolism of benzene compounds,and are also common environmental pollutants in industry wastewater. In order to efficiently remove benzene compounds from industry wastewater,the petroleum-degrading strain Acinetobacter sp. Tust-DM21 capable of effectively degrading aromatic compounds were used to investigate the degradation of catechol and sodium benzoate. The metabolic process of sodium benzoate was studied by the crude enzyme characteristics of catechol 1,2-dioxygenase(C12O)and the enzyme activity of benzoate 1,2-dioxygenase. In the inorganic salt medium without carbon source,the strain Tust-DM21 was able to grow with catechol and sodium benzoate as the sole carbon source,respectively. The characteristics of the Tust-DM21 degrading these 2 benzene compounds were studied by controlling the dosage of inoculated bacteria,substrate concentration,pH and temperature. Also the optimal pH,temperature and kinetic parameters of C12O and the enzymatic activity of benzoic acid 1,2-dioxygenase were analyzed. As results,the maximum tolerated concentration(MTC)of the strain Tust-DM21 on catechol was 700 mg/L;the optimal conditions for the strain to degrade 600 mg/L of catechol were:5% of inoculum,pH 6.0 and temperature 35℃,respectively;under the optimal condition,the bacteria degraded 92% of catechol. The MTC of the strain on sodium benzoate was 4 500 mg/L. The optimal conditions for the strain to degrade 1 500 mg/L of sodium benzoate were:3% of inoculum,pH 8.0 and temperature 35℃,respectively;under the optimal condition,the bacteria degraded 95% of sodium benzoate. The optimal conditions for the enzymatic reaction of C12O were as follows:pH 8.0 and temperature 35℃;the kinetic parameters of C12O were as follows:Km=25.68 mol/L,Vmax=0.131 mol/(mL·min). The enzyme activity of benzoic acid 1,2-dioxygenase was 3.53 U. In conclusion,the strain Tust-DM21 has a strong capability of degrading catechol and sodium benzoate and has a broad prospective application.

    Isolation and Identification of Cellulose Degrading Microorganisms in Composting Process
    LI Lin-chao, ZHANG Chao, DONG Qing, GUO Cheng, ZHOU Bo, GAO Zheng
    2019, 35(9):  165-171.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0581
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    In order to solve the problem of slow degradation of straw in aerobic compost,strains that efficiently degrade cellulose were screened from corn straw compost. The cellulolytic bacteria in the corn stover compost were screened by the plate dilution method combined with the Congo red staining method,and the re-screening was conducted by the filter paper disintegration test. Two actinomycetes C31 and C37 with high filter paper disintegration ability were screened and identified as Streptomyces drozdowiczii and Streptomyces corchorusii. Strain GD16 was identified as Paenibacillus pabuli. At the same time,the enzyme activities of three strains were quantitatively measured. The strain C31 had the highest cellulase activity and filter paper enzyme activity,which were 4.8 U/mL and 3 U/mL,respectively. The strain GD16 had the highest xylanase activity,and reached 23 U/mL. The results showed that strain C31,C37 and GD16 all had cellulase activity,filter paper enzyme activity and xylanase activity.
    Identification of a Lignin Degrading Bacterium and Its Degrading Characteristics
    HU Xiao-feng, ZHANG Duo-duo, ZHOU Yun-heng, WEI Ya-hong, CHEN Shao-lin
    2019, 35(9):  172-177.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0207
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    In order to enrich the germplasm resources of the high-quality lignin degrading strains,alkali lignin was used as the sole growth carbon source and a bleaching/dyeing method with two kinds of lignin analogues Azure-B and Remazol Brilliant Blue was subsequently used to isolate and screen a bacterium with ability to degrade lignin from dropped leaves-covered soil. 16S rRNA gene sequence analysis was to identify this bacterium,ultraviolet spectrophotometry was applied to determine the activities and their variation trends of lignin peroxidase (LiP),manganese peroxidase(MnP),and laccase(Lac),and the scanning electron microscopy(SEM)was used to observe their morphological changes during degradation. A functional bacterium was successfully isolated and designated as Erwinia sp. QL-Z3. The QL-Z3 grew to stable phase after 48 h incubation in liquid alkali lignin medium,OD600 reached 0.31. The degradation rate of lignin reached 14.23% after 72 h incubation. The enzymatic activities related to lignin degradation reached to peak values at 48 h and 60 h:Mnp reached 263.83 U/L after 60h fermentation,and Lip and Lac reached 104.11 U/L and 77 U/L respectively after 48 h fermentation. SEM results demonstrated that the strain depolymerized the lignin sample,destroyed the lignin structure and broke it into small fragments. This work systematically reveals that the strain Erwinia sp. QL-Z3 has an effective lignin degradation performance,and it can be a potential candidate for lignin biodegradation.
    Screening and Degradation Characteristics of a Strain Degrading Polyacrylamide
    LIU Yu-cheng, QIU Lian, LIANG Jing-jing, LI Ling-li, MA Li-li
    2019, 35(9):  178-183.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0292
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    A bacterial strain degrading polyacrylamide was screened from the sludge containing polyacrylamide(PAM). The bacterium can grow with polyacrylamide as the sole carbon source and nitrogen source. The degradation rate of PAM by the strain(inoculation 5%)reached 43% within 48 h when the initial concentration of PAM was 1.2 g/L,the temperature was 35℃,and the pH was 7.0. The strain was identified as Klebsiella sp.,named PCX,after physiological,biochemical and 16S rDNA sequence analysis. The molecular weight of the degraded product of PAM(molecular weight:3×103 kD)was 0.619 kD. Combined with the results from infrared spectroscopy,the amide group on the molecular chain was hydrolyzed to carboxyl group and the side chain was degraded. The results of acute biological toxicity analysis showed that the EC50 was 106 mg/L,indicating that the degraded products had no toxic effect on aquatic organisms.
    Isolation of a p-Nitrophenol-Degrading Bacterium and Investigation of Its Degrading Mechanism
    REN Lei, LIU Bin, LIN Zhong, ZHEN Zhen, LIU Yue-lian, HU Han-qiao, YAN Yan-chun
    2019, 35(9):  184-193.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0286
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    This work is designed to isolate marine microbes that can efficiently degrade p-nitrophenol and to systematically elucidate related mechanism. The sole carbon source method was employed for the enrichment,domestication and isolation of marine microbes degrading p-nitrophenol. The adaptability of isolated strains to different environmental factors was investigated by single factor experiment. The metabolic pathway of p-nitrophenol was deduced by mass spectrometry analysis of metabolic intermediates. Genes involved in the metabolism of p-nitrophenol were obtained by gene cloning. One marine bacterium RL-JY1,which efficiently degraded p-nitrophenol,was isolated. 100 mg/L p-nitrophenol was completely degraded by the strain RL-JY1 within 72 h. Strain RL-JY1 was identified as Pseudomonas putida via the analysis of morphological,physiological and biochemical characteristics and 16S rRNA gene sequence. The degradation rates of 100 mg/L p-nitrophenol were 97.2%,100% and 100% when RL-JY1 was incubated under 20,30 and 40℃,respectively. The degradation rates of 100 mg/L p-nitrophenol were 64.7%,100%,97.2% and 84.2% within 72 h when the initial pH was 6.0,7.0,8.0 and 9.0,respectively. When NaCl concentration was under 0-8%(W/V),all the degradation rates of 100 mg/L p-nitrophenol within 72 h were 100%. When NaCl concentration was 10% and 12%,the degradation rates of 100 mg/L p-nitrophenol within 72 h were 81.3% and 50.6%,respectively. The metabolic pathway of p-nitrophenol in strain RL-JY1 was deduced via metabolic intermediates identification through mass spectrometry analysis,which was a typical 1,2,4-benzenetriol pathway. Gene pnpABC,involved in the transformation of p-nitrophenol to maleacetic acid,was cloned from the genome of strain RL-JY1 according to known p-nitrophenol degrading related genes. The sequence alignments indicated that the obtained pnpABC gene showed high similarity(all above 99%)with the sequences reported in P. putida DLL-E4. In conclusion,p-Nitrophenol degrading bacterium RL-JY1 was identified as P. putida,and showed promising tolerance to environmental temperature,pH and salinity. p-Nitrophenol was utilized via 1,2,4-benzenetriol pathway by strain RL-JY1. p-Nitrophenol degrading related genes pnpABC were identified in the genome of strain RL-JY1.
    Identification and Denitrification Characters of a High-temperature-resistant Nitrite-denitrifying Bacterium
    LI Yu-qi, MA Pei-yu, LIU Han, WANG Ling-zhi, GAO Ren-ling, LI Hui-juan
    2019, 35(9):  194-201.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0130
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    Due to the toxic effect of nitrite,the screening and denitrification characteristics of denitrifying bacteria suitable for removing high-concentration nitrite-nitrogen are of great significance. Enrichment culturing,preliminary screening with plates and secondary screening with flasks were used to screen denitrifying bacteria from aquaculture wastewater,and a strain Y7 was identified through the physiological and biochemical characterization and the 16S rRNA gene sequence analysis. The effects of carbon sources,nitrite concentration,pH,liquid volume,inoculum size,inoculum age and temperature to the removal rate of nitrite-nitrogen by the strain Y7 were further investigated for increasing the efficacy of removing nitrite-nitrogen. As results,strain Y7 was identified as a member of the genus Bacillus and hence designated Bacillus subtilis Y7. Results of single-factor experiment showed that strain Y7 degraded nitrite from 1 100 mg/L to zero after fermentation for 24 hours at temperature 30℃ and 170 r/min,under the conditions of the glycerol as carbon source,the pH value of the medium being 7.0,the liquid volume being 100 mL in 250 mL triangular flask and the inoculum size being 10%(OD600=1.5). In conclusion,strain Y7 tolerates up to 1 100 mg/L nitrite,and the high temperature of 50℃ does not affect its degradation efficiency to the nitrite-nitrogen. Therefore,strain Y7 has great potential in the treatment of high-temperature industrial wastewater containing high concentration of nitrite and summer aquaculture wastewater.
    Isolation and Identification of an Efficient Aerobic Denitrifying Bacterium
    LI Wen-fu, DU Liu-bing, LIU Si-ying, WENG Mei-sui, SHU Hu, CHEN Qiong-hua
    2019, 35(9):  202-209.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0339
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    New types of aerobic denitrifying bacteria were isolated from the culture pond of the Xilang Pearl River Aquaculture Institute in Guangzhou,aiming at laying the foundation for the development of a new biological nitrogen removal process. Three different screening media were used to isolate the aerobic denitrifying bacteria,and the growth curve and denitrification performance of fermentation in a sole nitrogen source medium were determined. Then morphology and 16S rRNA gene sequence analysis were employed to identify the strains and a phylogenetic tree was constructed. As result,a high-performance aerobic denitrifying strain BB-17 was isolated. When the strain was cultured at 28℃,180 r/min in a sole nitrogen source medium,the logarithmic phase appeared at 6-24 h and the removal rate of ammonia nitrogen was up to 93.92% at 18 h,that of nitrate nitrogen reached 66.11% at 48 h,and that of nitrite nitrogen reached 99.10% at 48 h. BB-17 was a Gram-negative Brevibacterium,the results of 16S rDNA sequence analysis presented 98.41% homology with Pseudomonas vancouverensis(AJ011507),and BB-17 was identified as Pseudomonas and named P. sp. BB17.
    Screening of Aerobic Denitrifying Bacteria Based on Loop-mediated Isothermal Amplification and Denitrification Performance
    LI Qiu-fen, LIU Qi-chen, XU Ai-ling, ZHANG Yan, SONG Zhi-wen
    2019, 35(9):  210-217.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0555
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    In order to quickly and accurately screen aerobic denitrifying bacterial strains,a specific software Primer Explorer V4 was applied to design the LAMP(loop-mediated isothermal amplification technology)primers for the gene nirS,nirK and nosZ. The strains were firstly screened by the LAMP system after accumulation and isolation on suitable medium in aerobic condition,and further screened by PCR and gas-producing detection. Then,the denitrification performance of the selected strains was detected. The results indicated that 7 denitrifying bacteria strains containing genes nirS or nirK gene and nosZ,including KFDX1,KFDX2,KFDX3,KFDX4,KWDX1,FJ3-1 and FJ3-2,were firstly screened,after reacting for 60 min at 60℃ in LAMP system and GeneFinderTM staining direct observation. The result of PCR was consistent with the screening result of LAMP,and finally,6 strains produced gas after gas-generating detection. Finally,5 aerobic denitrifying strains producing N2,i.e.,KFDX1,KFDX2,KFDX3,KWDX1,and FJ3-2 were screed out. The results of denitrifying capacity test indicated that,the removal rates of NO2-N and NO3-N by the 5 selected strains were over 99% and 90% after 24 h,respectively. The maximum removal rates were up to 4.9 mg/(L·h)and 4.03 mg/(L·h). The result of this study is of significance for improving the screening method of denitrifying bacterial strains and promoting its application in wastewater treatment.
    Isolation,Screening and Identification of Active Endophytic Fungi from Yili Wild Walnut
    RANG Feng-ju, REN Yan-li, ZHANG Wei, OUYANG Yan
    2019, 35(9):  218-223.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0443
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    The objective of this work is to screen the endophytic fungi with strongly antibacterial and antioxidant activities from Yili wild walnut and then identify them. We used tissue separation method to isolate the endophytic fungi from wild walnut,and the plate confrontation to conduct the experiment on their antibacterial activities as well as their DPPH radical scavenging capacity. Finally,the strains were identified. As results,(1)49 strains of endophytic fungi were isolated. The most isolates came from the stem,then from the roots,and none from the kernels.(2)Strain YHT-4 and YHT-6 presented significant antibacterial effects and the inhibitory index was > 10,and YHT-4 inhibited pathogenic fungus with diameter > 1.5 cm.(3)The strain YHT-4 demonstrated a stronger ability to scavenge DPPH free radicals which was equivalent to Vc.(4)The strain YHT-4 was molecularly identified as Fusarium tricinctum based on morphology and molecular science. In conclusion,the isolated strain F. tricinctum sp. YHT-4 had strong antibacterial and anti-oxidant activities,and it can he used as potential sources for drug development in further study.
    Cultivable Myxobacteria and Their Antibiotic Activities in the Hulun Buir Area of Inner Mongolia
    WANG Xue-han, MA Qiang, TIAN Yuan, HU Jing, LIU Hui-rong
    2019, 35(9):  224-233.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0210
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    Total 20 soil samples were collected from Hulun Buir,Inner Mongolia and their environmental parameters were measured. Myxobacteria were isolated from 20 soil samples and then purified and identified by Escherichia coli-inducing method,the rabbit dung pellets-inducing method and filter paper-inducting method. Then the antibiotic activities of the myxobacterial strains were determined by the plate confrontation method,and the results were analyzed by statistical methods. The results showed that the soil nutrient content was unbalanced in Hulun Buir,the soil samples were generally acidic,and the soil water content was low. A total of 41 strains of myxobacteria were obtained,belonging to Myxococcus,Corallococcus,Cystobacter and Archangium;and the dominant one was Myxococcus. The strains showed rich diversity in black soil and grassland. There was no significant correlation between the strains and environmental factors. All the strains presented activity against E. coli and Phytophthora infestans,3 strains inhibited Staphylococcus aureus,but they show no inhibitory effect on Bacillus subtilis,Saccharomyces cerevisiae,Sclerotinia sclerotiorum,Rhizoctonia solanikuhn,and Fusarium oxysporum. In conclusion,the cultivable myxobacteria are abundant in Hulun Buir,and all of them demonstrated obvious activity against P. infestans,providing basic data for the research and development of myxobacteria resources in Hulun Buir,Inner Mongolia.
    Recombinant Expression and Immunogenicity Analysis of the Porin Protein OmpF of Aeromonas hydrophila
    GAO Yun-shan, LIU Dan-dan, XU Jun-lin, SANG Yu-nong, LIANG Xia-xia, LIU Jian-xin, WANG Wen-bin
    2019, 35(9):  234-243.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0628
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    Outer membrane protein(Omp)F in Aeromonas spp. is β-barrel like porin,and involves in the adaption of osmotic pressure and is highly conservative in this common pathogen for human and fish,thus poses a promising research value as immune-detection target. The A. hydrophila OmpF fragment published in GenBank was synthesized,and inserted into plasmid pET-28a(+). The recombinant plasmid was transformed into Escherichia coli BL21(DE3)and induced by isopropy-β-D-thiogalactoside(IPTG). The molecular size of the expressed protein was about 40 kD,which mainly expressed as inclusion bodies after optimization of inducing temperatures and IPTG concentrations. BALB/c mice were immunized with purified OmpF inclusion bodies. The results of ELISA showed that the immunized mice serum cross-reacted with 10 of 11 boiled and deactivated strains of Aeromonas spp.,the serum titers were around 1∶81 000. However,the mice serum did not react with Vibrio cholerae,Bacillus licheniformis,V. parahaemolyticus,V. vulnificus,and V. anguillarum. The above results indicate that the protein OmpF is immunogenic,conserved and was promising in developing Aeromonas cross-reactive antibodies.
    Cloning and Expression of Outer Membrane Protein OprM from Aeromonas hydrophila and the Evaluation of Its Immunoprotective Effect
    MAO Ran-ran, LI Xiao-yan, WU Yao, ZHANG Li-shan, LIN Zhen-ping, LIN Xiang-min
    2019, 35(9):  244-248.  doi:10.13560/j.cnki.biotech.bull.1985.2019-0319
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    Aeromonas hydrophila is a common conditional pathogen. Controlling the damage of this bacterium in aquaculture industry is an urgent problem to be solved. Hereby the purified protein of A. hydrophila ATCC 7966 OprM was obtained by molecular biology methods such as cloning and expression. After intramuscular injection of the purified protein to zebrafish,the transcription level of immune-related genes was detected and the immune response was explored. The results showed that most of the immune-related genes were up-regulated,indicating that OprM protein evoked a considerable immune responses in zebrafish. Further velogenic strain A. hydrophila LP-2 was used to challenge the immunized zebrafish,and it was revealed that the relative immune protection rate after the injection of OprM protein reached 89.47%,the above results indicate outer membrane protein OprM is highly immunoprotective against zebrafish and is a potential vaccine candidate for A. hydrophila.
    Effects of Acid Mine Drainage on the Abundance of Functional Genes Involved in Nitrogen Cycle in Soil Profiles
    LI Hui ZHA, Jian-jun, SUN Qing-ye
    2019, 35(9):  249-256.  doi:10.13560/j.cnki.biotech.bull.1985.2018-1094
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    Nitrogen(N)cycle in soil,which is mainly mediated by microorganisms,has always been being hotspots in environmental soil science and soil microbiology. Soil contamination significantly affects the course of N-cycle. The discharge of acid mining drainage(AMD)with low pH and containing high contents of salt and heavy metals generally leads to serious contamination of farmland soils. However,AMD’s influence on N-cycle in soil is rarely reported. A rice paddy contaminated by AMD in Tongling,Anhui province,was chosen as the research site. Based on the technique of molecular biology,the microbial biomass and the abundance of functional genes involved in N-cycle in soil profiles were detected,and main factors affecting the abundance of functional genes in soil profiles were analyzed. The results showed that the biomass of bacteria and archaea decreased with the increase of profile depth in the AMD-contaminated paddy soil. The abundance of functional genes involved in N-cycle was the highest in topsoil(0-10 cm). In the studied soil profiles,the abundance of amoA-AOA gene was greater than that of amoA-AOB,and the abundance of nirK gene was greater than that of nirS gene,thus amoA-AOA and nirK were the dominated functional genes of ammonia oxidation and denitrification in the AMD-contaminated soil profile. Compared with uncontaminated soil,the abundance of nifH gene in the AMD-contaminated paddy soil profile significantly reduced,while the abundance of nirS gene significantly increased. pH was significantly and negatively correlated with the abundance of nirS,amoA-AOA and HZO genes. The iron content was extremely and negatively correlated with the biomass of bacteria and archaea and the abundance of nosZ,amoA-AOA,and HZO genes. In conclusion,there is a close relationship between the abundance of functional genes and both the profile depth and the physic-chemical characters of the rice paddy AMD-contaminated soil.
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    2019, 35(9):  257-257. 
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    2019, 35(9):  258-258. 
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