滇池沉积物中反硝化细菌及产甲烷菌的分子鉴别研究
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摘要
【目的】为了解滇池沉积物中反硝化细菌及产甲烷菌的种类组成。【方法】采用引物NirS1F/NirS6R和Me1/Me2分别对沉积物总DNA进行PCR扩增,得到1000 bp的反硝化细菌和760 bp的产甲烷菌特异性片段。【结果】证实了滇池沉积物中存在反硝化细菌和产甲烷菌,且推测产甲烷菌的丰度高于反硝化细菌。2个克隆文库分别获得阳性克隆子55和99个,利用Mothur软件将序列分别划分为10和5个OUT,说明尽管反硝化细菌的丰度低于产甲烷菌,但物种多样性高于产甲烷菌。运用BLASTN程序在GenBank数据库中进行序列比对,发现55个反硝化细菌阳性克隆子可分为10个种类,OTU10类序列与细菌Dechloromonas aromatica的相似性达86%,是反硝化细菌中的优势类群;99个产甲烷菌阳性克隆子可分为5个种类,OTU5类序列与甲酸甲烷规则菌(Methanoregula formicicum)相似性达86%,该类群是反硝化细菌中的优势类群。【结论】滇池沉积物中的反硝化细菌与产甲烷菌多为潜在的未被培养的新种。
【Objective】The study aims to understand the species composition of denitrifying bacteria and methanogens in Dianchi sediments.【Method】The total DNA of sediments was amplified by PCR using primers NirS1F/NirS6R and Me1/Me2 respectively,A 1000 bp denitrifying bacterium and a 760 bp methanogenic bacterial specific fragment were obtained. 【Result】The experiments confirmed that there were denitrifying bacteria and methanogens in the sediments of Dianchi,the abundance of methanogens bacteria was higher than that of denitrifying bacteria. 55 clones and 99 clones were obtained of 2 clone libraries. Although the abundance of denitrifying bacteria was lower than that of methanogens,the species diversity was higher than that of methanogens bacteria. Using BLASTN sequence alignment in GenBank database,we found 55 positive clones of denitrifying bacteria could be divided into 10 species. The similarity of OTU10 sequence to Dechloromonas aromatica was 86 %,which was the dominant group in denitrifying bacteria. 99 methanogens positive clones could be divided into 5 species. 【Conclusion】The similarity between OTU5 sequence and Methanoregula formicicum was 86 %,which was the dominant group in denitrifying bacteria. The denitrifying bacteria and methanogenic bacteria in sediments of Dianchi were mostly new species that were not cultured.
【Objective】The study aims to understand the species composition of denitrifying bacteria and methanogens in Dianchi sediments.【Method】The total DNA of sediments was amplified by PCR using primers NirS1F/NirS6R and Me1/Me2 respectively,A 1000 bp denitrifying bacterium and a 760 bp methanogenic bacterial specific fragment were obtained. 【Result】The experiments confirmed that there were denitrifying bacteria and methanogens in the sediments of Dianchi,the abundance of methanogens bacteria was higher than that of denitrifying bacteria. 55 clones and 99 clones were obtained of 2 clone libraries. Although the abundance of denitrifying bacteria was lower than that of methanogens,the species diversity was higher than that of methanogens bacteria. Using BLASTN sequence alignment in GenBank database,we found 55 positive clones of denitrifying bacteria could be divided into 10 species. The similarity of OTU10 sequence to Dechloromonas aromatica was 86 %,which was the dominant group in denitrifying bacteria. 99 methanogens positive clones could be divided into 5 species. 【Conclusion】The similarity between OTU5 sequence and Methanoregula formicicum was 86 %,which was the dominant group in denitrifying bacteria. The denitrifying bacteria and methanogenic bacteria in sediments of Dianchi were mostly new species that were not cultured.
引文
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[19]洪璇,洪有为,陈仲巍,等.九龙江河口区nir S型反硝化细菌多样性及系统发育学分析[J].微生物学通报,2015,42(9):1639-1650.
[20]方晶晶,马传明,刘存富.反硝化细菌研究进展[J].环境科学与技术,2010(s1):206-210.
[21]马凯.产甲烷菌的分子系统学和生态功能研究[D].中国科学院微生物研究所,2006.
[22]曾志华.闽江河口区盐度梯度下潮汐沼泽湿地产甲烷菌群落结构及对环境因子的响应[D].福建师范大学,2014.
[23]Banning N,Brock F,Fry J C,et al.Investigation of the methanogen population structure and activity in a brackish lake sediment[J].Environmental Microbiology,2005,7(7):947.
[24]Juottonen H,Galand P E,YrjalaK.Detection of methanogenic Archaea in peat:comparison of PCR primers targeting the mcr A gene[J].Research in Microbiology,2006,157(10):914.
[2]周炜,张岳芳,朱普平,等.种植制度对长江下游稻田温室气体排放的影响[J].江苏农业学报,2017,33(2):35-41.
[3]王琛瑞,黄国宏,梁战备,等.大气甲烷的源和汇与土壤氧化(吸收)甲烷研究进展[J].应用生态学报,2002,13(12):1707-1712.
[4]陈泽斌,夏体渊,王定康,等.滇池沉积物中厌氧氨氧化细菌分布的时空差异[J].水生生物学报,2016,40(2):425-430.
[5]周丽,付智丹,杜青,等.减量施氮对玉米/大豆套作系统中作物氮素吸收及土壤氨氧化与反硝化细菌多样性的影响[J].中国农业科学,2017,50(6):1076-1087.
[6]杨秀清,吴瑞薇,韩作颖,等.基于mcr A基因的沁水盆地煤层气田产甲烷菌群与途径分析[J].微生物学通报,2017,44(4):795-806.
[7]严立恩,陈志荣,王晶晶,等.南极乔治王岛陆地土壤和潮间带放线菌多样性及其抑菌活性研究[J].中国海洋药物,2016,35(1):19-28.
[8]尹利方,任禛,夏体渊,等.应用克隆文库构建法研究枇杷根系AMF多样性[J].西南农业学报,2017,30(5):1009-1015.
[9]尹利方,陈泽斌,夏体渊,等.一株铁皮石斛组织培养污染内生细菌的分离与鉴定[J].西南农业学报,2016,29(8):1982-1986.
[10]You S J.Identification of Denitrifying Bacteria Diversity in an Activated Sludge System by using Nitrite Reductase Genes[J].Biotechnology Letters,2005,27(19):1477-1482.
[11]王媛媛.磷酸盐对水稻土中异化铁还原菌丰度和群落结构的影响[D].西北农林科技大学,2014.
[12]蒙小俊,李海波,曹宏斌,等.焦化废水活性污泥细菌菌群结构分析[J].环境科学,2016,37(10):3923-3930.
[13]Ru Y L,Fang H H P.Hydrogen production characteristics of photoheterotrophic Rubrivivax gelatinosus,L31[J].International Journal of Hydrogen Energy,2008,33(3):974-980.
[14]徐爱玲,任杰,宋志文,等.污水处理厂尾水细菌群落结构分析[J].环境科学,2014(9):3473-3479.
[15]Joulian C,Patel B K C,Ollivier B,et al.Methanobacterium oryzae sp.nov.a novel methanogenic rod isolated from a Philippines ricefield.[J].International Journal of Systematic&Evolutionary Microbiology,2000,50 Pt 2(2):525-528.
[16]穆维娜,李玉成,武超,等.秸秆蓝藻混合发酵产沼气过程中微生物群落分析[J].微生物学杂志,2017,37(1):70-77.
[17]Brauer S L,Cadillo-Quiroz H,Ward R J,et al.Methanoregula boonei gen.nov.sp.nov.an acidiphilic methanogen isolated from an acidic peat bog[J].International Journal of Systematic&Evolutionary Microbiology,2011,61(Pt1):45.
[18]穆剑,匡丽,高迎新,等.采油废水厌氧处理系统的微生物群落特征[J].环境工程学报,2014,8(3):807-814.
[19]洪璇,洪有为,陈仲巍,等.九龙江河口区nir S型反硝化细菌多样性及系统发育学分析[J].微生物学通报,2015,42(9):1639-1650.
[20]方晶晶,马传明,刘存富.反硝化细菌研究进展[J].环境科学与技术,2010(s1):206-210.
[21]马凯.产甲烷菌的分子系统学和生态功能研究[D].中国科学院微生物研究所,2006.
[22]曾志华.闽江河口区盐度梯度下潮汐沼泽湿地产甲烷菌群落结构及对环境因子的响应[D].福建师范大学,2014.
[23]Banning N,Brock F,Fry J C,et al.Investigation of the methanogen population structure and activity in a brackish lake sediment[J].Environmental Microbiology,2005,7(7):947.
[24]Juottonen H,Galand P E,YrjalaK.Detection of methanogenic Archaea in peat:comparison of PCR primers targeting the mcr A gene[J].Research in Microbiology,2006,157(10):914.