北极海洋沉积物反硝化细菌的多样性分析
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摘要
中国北方地区冬季污水的生物处理存在着处理不彻底、效率低和出水水质难以达标等难题。根据菌落形态特征,利用反硝化细菌培养基对26个站位的北极海洋沉积物进行富集培养,且纯化菌株,获得了克隆到16S rRNA基因有效序列的菌株达59株;基于16S r RNA基因的相似性分析与系统发育研究结果表明,分离的菌株属于细菌域,门、纲、目、科、属、种的数量分别是3、4、8、13、16和23,γ-Proteobacteria占比例最大(57.63%);得到1株菌的16S rRNA基因序列的相似性最高为96.44%,可能为新种。大部分菌株(95%)的温度和耐NaCl浓度的生长范围分别为0～30℃和0～6.0%,能利用多种碳源生长,并且具有产胞外水解酶的能力;利用简并引物从菌株Pseudomonas sp. C22-DN-54、Pseudomonas sp. P13-DN-7、Marinobacter sp. P12-DN-1和Pseudomonas sp. E24-DN-52克隆到了亚硝酸盐还原酶基因(nir S)。该研究将为北方寒冷地区的低温污水生物处理提供新的菌种资源。
In the northern areas of China,biotreatment techniques of wastewater at low temperatures have some shortcomings,such as not thorough treatment,low efficiency and difficulties in achieving emission standards in winter.On the basis of morphological characters,a total of 59 isolates with valid 16S rRNA gene sequences were obtained from the 26 Arctic Ocean sediments by denitrifying screening medium.Molecular identification and phylogenetic analysis on the basis of 16S rRNA gene sequences showed that these isolates belonged to 3 phyla,4 classes,8 orders,13 families,16 genera and 23 species,respectively in the bacteria domain;among which,γ-Proteobacteria accounted for the majority(57.63%).One isolate having16S rRNA gene similarity 96.44% might be novel species.The growth temperature and NaCl tolerance range of most representative strains was 0～30 ℃ and 0～90%,respectively.These isolates could grow by utilizing many kinds of carbon sources and produce various extracellular enzymes.Nitrite reductase gene(nir S),the key enzyme gene in the denitrification,was cloned from strain Pseudomonas sp.C22-DN-54,Pseudomonas sp.P13-DN-7,Marinobacter sp.P12-DN-1 and Pseudomonas sp.E24-DN-52 respectively by the degenerate primers.The research will provide new microbial resources for the sewage treatment at low temperature in cold northern areas.
In the northern areas of China,biotreatment techniques of wastewater at low temperatures have some shortcomings,such as not thorough treatment,low efficiency and difficulties in achieving emission standards in winter.On the basis of morphological characters,a total of 59 isolates with valid 16S rRNA gene sequences were obtained from the 26 Arctic Ocean sediments by denitrifying screening medium.Molecular identification and phylogenetic analysis on the basis of 16S rRNA gene sequences showed that these isolates belonged to 3 phyla,4 classes,8 orders,13 families,16 genera and 23 species,respectively in the bacteria domain;among which,γ-Proteobacteria accounted for the majority(57.63%).One isolate having16S rRNA gene similarity 96.44% might be novel species.The growth temperature and NaCl tolerance range of most representative strains was 0～30 ℃ and 0～90%,respectively.These isolates could grow by utilizing many kinds of carbon sources and produce various extracellular enzymes.Nitrite reductase gene(nir S),the key enzyme gene in the denitrification,was cloned from strain Pseudomonas sp.C22-DN-54,Pseudomonas sp.P13-DN-7,Marinobacter sp.P12-DN-1 and Pseudomonas sp.E24-DN-52 respectively by the degenerate primers.The research will provide new microbial resources for the sewage treatment at low temperature in cold northern areas.
引文
[1]Galloway J N,Townsend A R,Erisman J W,et al.Transformation of the nitrogen cycle:recent trends,questions,and potential solutions,transformation of the nitrogen cycle:recent trends,questions,and potential solutions[J].Science,2008,320(5878):889-892.
[2]韩永和,章文贤,庄志刚,等.耐盐好氧反硝化菌A_13菌株的分离鉴定及其反硝化特性[J].微生物学报,2013,53(1):47-58.Han Yonghe,Zhang Wenxian,Zhuang Zhigang,et al.Isolation of salt tolerant aerobic denitrifying bacterium A_13 and its denitrification characteristics[J].Journal of Microbiology,2013,53(1):47-58.
[3]张玉芹,刘开启,王革,等.反硝化细菌的筛选及培养条件的研究[J].农业环境科学学报,2005,24(1):165-168.Zhang Yuqin,Liu Kaiqi,Wang Ge,et al.Screening of denitrifying bacteria and the studies on culture conditions[J].Journal of Agro-environment Science,2005,24(1):165-168.
[4]郭岩,周雪媚,杨茂华,等.高效嗜碱好氧反硝化细菌的筛选及其脱硝特性[J].过程工程学报,2013,13(4):687-692.Guo Yan,Zhou Xuemei,Yang Maohua,et al.Screening of highly efficient basophilic aerobic denitrifying bacteria and its denitrification characteristics[J].Process Engineering Journal,2013,13(4):687-692.
[5]Zeng Q W.Screening of denitrobacteria and preliminary study about its denitrification properties[J].Journal of Huazhong Agricultural University,2008.27(5):616-620.
[6]桑军强,王占生.低温条件下生物陶粒反应器运行特性研究[J].环境科学,2003,24(2):112-115.Sang Junqiang,Wang Zhansheng.Study on operation characteristics of biological ceramsite reactor at low temperature[J].Environmental Science,2003,24(2):112-115.
[7]Liao B Q,Allen D G,Droppo I G,et al.Surface properties of sludge and their role in bioflocculation and settleability[J].Water Research,2001,35(2):339-350.
[8]陈皓文,高爱国.中国极地微生物学调查研究进展[J].极地研究,2005,17(4):299-307.Chen Haowen,Gao Aiguo.Research progress on polar microbiology in China[J].Polar Research,2005,17(4):299-307.
[9]Beman J M.Activity,abundance,and diversity of nitrifying Archaea and denitrifying bacteria in sediments of a subtropical estuary:Bahía del Tóbari,Mexico[J].Estuaries&Coasts,2014,37(6):1343-1352.
[10]Wallenstein M D,Hall E K.A trait-based framework for predicting when and where microbial adaptation to climate change will affect ecosystem functioning[J].Biogeochemistry,2012,109(1/2/3):35-47.
[11]Graham E B,Wieder W R,Leff J W,et al.Do we need to understand microbial communities to predict ecosystem function?A comparison of statistical models of nitrogen cycling processes[J].Soil Biology&Biochemistry,2014,68(1):279-282.
[12]Rysgaard S,Glud R N,Risgaardpetersen N,et al.Denitrification and anammox activity in Arctic marine sediments[J].Limnology and Oceanography,2004,49(5):1493-1502.
[13]张海舟,庄燕培,朱庆梅,等.西北太平洋楚科奇海沉积物-水界面营养盐输送通量估算[J].海洋学报,2015,37(11):155-164.Zhang Haizhou,Zhuang Yanpei,Zhu Qingmei,et al.Estimation of nutrient delivery fluxes at the sediment-water interface of the Chukchi Sea in the Western North Pacific[J].Oceanography,2015,37(11):155-164.
[14]Bo T,Dalsgaard T.Production of N2through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments[J].Applied&Environmental Microbiology,2002,68(3):1312-1318.
[15]Prakasam T B S,Loehr R C.Microbial nitrification and denitrification in concentrated wastes[J].Water Research,1972,6(7):859-869.
[16]Huang S,Chen C,Wu Q,et al.Distribution of typical denitrifying functional genes and diversity of the nirS-encoding bacterial community related to environmental characteristics of river sediments[J].Biogeosciences Discussions,2011,8(3):5251-5280.
[17]Dang H,Wang C,Li J,et al.Diversity and distribution of sediment nirS-encoding bacterial assemblages in response to environmental gradients in the eutrophied Jiaozhou Bay,China[J].Microbial Ecology,2009,58(1):161-169.
[18]李晓亮,张良,张培玉,等.北极海洋沉积物低温硝化细菌的筛选、多样性与生长特性[J].应用与环境生物学报,2017,23(2):294-300.Li Xiaoliang,Zhang Liang,Zhang Peiyu,et al.Screening,diversity and growth characteristics of cold-adapted nitrifying bacteria in Arctic ocean sediments[J].Journal of Application and Environmental Biology,2017,23(2):294-300.
[19]Throb?ck I N,Enwall K,Jarvis A,et al.Reassessing PCRprimers targeting nirS,nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J].FEMS Microbiology Ecology,2004,49(3):401-417.
[20]许学伟,吴敏,黄伟达.阿尔金山盐湖极端嗜盐古生菌的分离及16S rRNA序列分析[J].浙江大学学报,2004,38(2):508-511.Xu Xuewei,Wu Min,Huang Weida.Isolation of arctic halophilic archaea in the salt lake of Altyn Mountain and sequence analysis of 16S rRNA[J].Journal of Zhejiang University,2004,38(2):508-511.
[21]李小义,王丽萍,杜雅萍,等.好氧反硝化微生物多样性及其反硝化功能初步研究[J].氨基酸和生物资源,2016,38(2):37-45.Li Xiaoyi,Wang Liping,Du Yaping,et al.Study on the diversity of aerobic denitrifying microorganisms and their denitrification function[J].Amino Acids and Biological Resources,2016,38(2):37-45.
[22]孟婷,杨宏.高效反硝化细菌的快速培养及群落结构多样性分析[J].环境科学,2017,38(9):3816-3822.Meng Ting,Yang Hong.High-spped culture of efficient denitrifying bacteria and analysis of community structure diversity[J].Environmental Science,2017,38(9):3816-3822.
[23]张甲耀,宋碧玉,陈兰洲,等.环境微生物学上[M].武汉:武汉大学出版社,2008.Zhang Jiayao,Song Biyu,Chen Lanzhou,et al.Environmental Microbiology[M].Wuhan:Wuhan University Press,2008.
[24]蔡昌凤,梁磊.高效好氧反硝化细菌的筛选及脱氮特性的研究[J].环境科学与技术,2011,34(1):41-44.Cai Changfeng,Liang Lei.Screening of efficient aerobic denitrifying bacteria and study on denitrification characteristics[J].Environmental Science and Technology,2011,34(1):41-44.
[25]邵晴,余晓斌.好氧反硝化细菌的筛选及反硝化特性研究[J].生物技术,2008,18(3):63-65.Shao Qing,Yu Xiaobin.Screening of aerobic denitrifying bacteria and study on denitrification characteristics[J].Biotechnology,2008,18(3):63-65.
[26]Han S I,Kim J O,Lee Y R,et al.Denitratimonas tolerans,gen.nov.sp.nov.,a denitrifying bacterium isolated from a bioreactor for tannery wastewater treatment[J].Antonie Van Leeuwenhoek,2016,109(6):785-792.
[27]Zheng H,Liu Y,Sun G,et al.Denitrification characteristics of a marine origin psychrophilic aerobic denitrifying bacterium[J].Journal of Environmental Sciences,2011,23(11):1888-1893.
[28]Braker G,Schwarz J,Conrad R.Influence of temperature on the composition and activity of denitrifying soil communities[J].FEMS Microbiology Ecology,2010,73(1):134-148.
[29]Srinandan C S,Shah M,Patel B,et al.Assessment of denitrifying bacterial composition in activated sludge[J].Bioresource Technology,2011,102(20):9481-9489.
[30]Lipsewers Y A,Hopmans E C,et al.Abundance and diversity of denitrifying and anammox bacteria in seasonally hypoxic and sulfidic sediments of the saline Lake Grevelingen[J].Frontiers in Microbiology,2016,11(7):1-15.
[2]韩永和,章文贤,庄志刚,等.耐盐好氧反硝化菌A_13菌株的分离鉴定及其反硝化特性[J].微生物学报,2013,53(1):47-58.Han Yonghe,Zhang Wenxian,Zhuang Zhigang,et al.Isolation of salt tolerant aerobic denitrifying bacterium A_13 and its denitrification characteristics[J].Journal of Microbiology,2013,53(1):47-58.
[3]张玉芹,刘开启,王革,等.反硝化细菌的筛选及培养条件的研究[J].农业环境科学学报,2005,24(1):165-168.Zhang Yuqin,Liu Kaiqi,Wang Ge,et al.Screening of denitrifying bacteria and the studies on culture conditions[J].Journal of Agro-environment Science,2005,24(1):165-168.
[4]郭岩,周雪媚,杨茂华,等.高效嗜碱好氧反硝化细菌的筛选及其脱硝特性[J].过程工程学报,2013,13(4):687-692.Guo Yan,Zhou Xuemei,Yang Maohua,et al.Screening of highly efficient basophilic aerobic denitrifying bacteria and its denitrification characteristics[J].Process Engineering Journal,2013,13(4):687-692.
[5]Zeng Q W.Screening of denitrobacteria and preliminary study about its denitrification properties[J].Journal of Huazhong Agricultural University,2008.27(5):616-620.
[6]桑军强,王占生.低温条件下生物陶粒反应器运行特性研究[J].环境科学,2003,24(2):112-115.Sang Junqiang,Wang Zhansheng.Study on operation characteristics of biological ceramsite reactor at low temperature[J].Environmental Science,2003,24(2):112-115.
[7]Liao B Q,Allen D G,Droppo I G,et al.Surface properties of sludge and their role in bioflocculation and settleability[J].Water Research,2001,35(2):339-350.
[8]陈皓文,高爱国.中国极地微生物学调查研究进展[J].极地研究,2005,17(4):299-307.Chen Haowen,Gao Aiguo.Research progress on polar microbiology in China[J].Polar Research,2005,17(4):299-307.
[9]Beman J M.Activity,abundance,and diversity of nitrifying Archaea and denitrifying bacteria in sediments of a subtropical estuary:Bahía del Tóbari,Mexico[J].Estuaries&Coasts,2014,37(6):1343-1352.
[10]Wallenstein M D,Hall E K.A trait-based framework for predicting when and where microbial adaptation to climate change will affect ecosystem functioning[J].Biogeochemistry,2012,109(1/2/3):35-47.
[11]Graham E B,Wieder W R,Leff J W,et al.Do we need to understand microbial communities to predict ecosystem function?A comparison of statistical models of nitrogen cycling processes[J].Soil Biology&Biochemistry,2014,68(1):279-282.
[12]Rysgaard S,Glud R N,Risgaardpetersen N,et al.Denitrification and anammox activity in Arctic marine sediments[J].Limnology and Oceanography,2004,49(5):1493-1502.
[13]张海舟,庄燕培,朱庆梅,等.西北太平洋楚科奇海沉积物-水界面营养盐输送通量估算[J].海洋学报,2015,37(11):155-164.Zhang Haizhou,Zhuang Yanpei,Zhu Qingmei,et al.Estimation of nutrient delivery fluxes at the sediment-water interface of the Chukchi Sea in the Western North Pacific[J].Oceanography,2015,37(11):155-164.
[14]Bo T,Dalsgaard T.Production of N2through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments[J].Applied&Environmental Microbiology,2002,68(3):1312-1318.
[15]Prakasam T B S,Loehr R C.Microbial nitrification and denitrification in concentrated wastes[J].Water Research,1972,6(7):859-869.
[16]Huang S,Chen C,Wu Q,et al.Distribution of typical denitrifying functional genes and diversity of the nirS-encoding bacterial community related to environmental characteristics of river sediments[J].Biogeosciences Discussions,2011,8(3):5251-5280.
[17]Dang H,Wang C,Li J,et al.Diversity and distribution of sediment nirS-encoding bacterial assemblages in response to environmental gradients in the eutrophied Jiaozhou Bay,China[J].Microbial Ecology,2009,58(1):161-169.
[18]李晓亮,张良,张培玉,等.北极海洋沉积物低温硝化细菌的筛选、多样性与生长特性[J].应用与环境生物学报,2017,23(2):294-300.Li Xiaoliang,Zhang Liang,Zhang Peiyu,et al.Screening,diversity and growth characteristics of cold-adapted nitrifying bacteria in Arctic ocean sediments[J].Journal of Application and Environmental Biology,2017,23(2):294-300.
[19]Throb?ck I N,Enwall K,Jarvis A,et al.Reassessing PCRprimers targeting nirS,nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J].FEMS Microbiology Ecology,2004,49(3):401-417.
[20]许学伟,吴敏,黄伟达.阿尔金山盐湖极端嗜盐古生菌的分离及16S rRNA序列分析[J].浙江大学学报,2004,38(2):508-511.Xu Xuewei,Wu Min,Huang Weida.Isolation of arctic halophilic archaea in the salt lake of Altyn Mountain and sequence analysis of 16S rRNA[J].Journal of Zhejiang University,2004,38(2):508-511.
[21]李小义,王丽萍,杜雅萍,等.好氧反硝化微生物多样性及其反硝化功能初步研究[J].氨基酸和生物资源,2016,38(2):37-45.Li Xiaoyi,Wang Liping,Du Yaping,et al.Study on the diversity of aerobic denitrifying microorganisms and their denitrification function[J].Amino Acids and Biological Resources,2016,38(2):37-45.
[22]孟婷,杨宏.高效反硝化细菌的快速培养及群落结构多样性分析[J].环境科学,2017,38(9):3816-3822.Meng Ting,Yang Hong.High-spped culture of efficient denitrifying bacteria and analysis of community structure diversity[J].Environmental Science,2017,38(9):3816-3822.
[23]张甲耀,宋碧玉,陈兰洲,等.环境微生物学上[M].武汉:武汉大学出版社,2008.Zhang Jiayao,Song Biyu,Chen Lanzhou,et al.Environmental Microbiology[M].Wuhan:Wuhan University Press,2008.
[24]蔡昌凤,梁磊.高效好氧反硝化细菌的筛选及脱氮特性的研究[J].环境科学与技术,2011,34(1):41-44.Cai Changfeng,Liang Lei.Screening of efficient aerobic denitrifying bacteria and study on denitrification characteristics[J].Environmental Science and Technology,2011,34(1):41-44.
[25]邵晴,余晓斌.好氧反硝化细菌的筛选及反硝化特性研究[J].生物技术,2008,18(3):63-65.Shao Qing,Yu Xiaobin.Screening of aerobic denitrifying bacteria and study on denitrification characteristics[J].Biotechnology,2008,18(3):63-65.
[26]Han S I,Kim J O,Lee Y R,et al.Denitratimonas tolerans,gen.nov.sp.nov.,a denitrifying bacterium isolated from a bioreactor for tannery wastewater treatment[J].Antonie Van Leeuwenhoek,2016,109(6):785-792.
[27]Zheng H,Liu Y,Sun G,et al.Denitrification characteristics of a marine origin psychrophilic aerobic denitrifying bacterium[J].Journal of Environmental Sciences,2011,23(11):1888-1893.
[28]Braker G,Schwarz J,Conrad R.Influence of temperature on the composition and activity of denitrifying soil communities[J].FEMS Microbiology Ecology,2010,73(1):134-148.
[29]Srinandan C S,Shah M,Patel B,et al.Assessment of denitrifying bacterial composition in activated sludge[J].Bioresource Technology,2011,102(20):9481-9489.
[30]Lipsewers Y A,Hopmans E C,et al.Abundance and diversity of denitrifying and anammox bacteria in seasonally hypoxic and sulfidic sediments of the saline Lake Grevelingen[J].Frontiers in Microbiology,2016,11(7):1-15.
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