应用分子生物学与同位素示踪技术研究厌氧氨氧化菌活性及功效  

Determination of activity and contribution of anammox bacteria to N_2 production by molecular biology and isotope analyses

作　　者：叶磊[1,2] 祝贵兵[1] 伦中财[3] 王朝旭[1,2] 王衫允[1] 冯晓娟[1] 尹澄清[1] 

机构地区：[1]中国科学院生态环境研究中心环境水质学国家重点实验室,北京100085 [2]中国科学院研究生院,北京100049 [3]北京京城中水有限责任公司,北京100164

出　　处：《环境科学学报》2011年第6期1206-1211,共6页Acta Scientiae Circumstantiae

基　　金：国家自然科学基金项目(No.21077119);国家科技重大专项(No.2008ZX07209-006-02,2008ZX07101-006-08)~~

摘　　要：厌氧氨氧化是一个新发现的氮循环途径,与反硝化作用都产生氮气,但准确衡量厌氧氨氧化活性及功效的相关方法还是个难题.本研究联合应用分子生物学与同位素示踪技术,以北京某污水处理厂的活性污泥为实验样品,研究其中厌氧氨氧化菌的存在、活性和功效.厌氧氨氧化菌的分子生物学分析是通过16S rRNA进行,选择两步引物分别为pla46-630 r和Amx368 f-Amx820 r进行巢式PCR扩增,并对16S rRNA进行后续基因处理.同位素示踪技术是通过15N标记,对反应体系进行3种处理:①仅加15NH+4;②加15NH+4和14NO-3;③加14NH+4和15NO-3,模拟实验在19℃的厌氧密闭容器中进行,3种处理条件下产物氮气的组成通过气相色谱-同位素比值质谱仪进行分析,通过数据计算获得厌氧氨氧化和反硝化反应的速率.16S rRNA研究结果表明被试活性污泥样品中存在污水处理工艺中常见的厌氧氨氧化菌种类——Candidatus Brocadia.同位素示踪和培养实验的测定结果表明,被试活性污泥样品的厌氧氨氧化和反硝化反应在19℃的速率分别为1.25 nmo.lg-1.h-1和12.16nmo.lg-1.h-1,厌氧氨氧化反应对N2产生量的贡献为9.32%.Anaerobic-ammonia oxidation（ANAMMOX） is a newly found pathway in the nitrogen cycle to produce gaseous N2.However,there are difficulties in quantitative determination of anammox activity and contribution.In this study,we developed a new way to combine molecular biology and isotope analyses to solve that problem.Activated sludge from a wastewater treatment plant in Beijing was sampled,and anammox activity and its contribution to nitrogen removal were analyzed.Molecular analyses of 16S rRNA using nested PCR amplification were followed with other molecule analyses.Primer pairs of pla46f-630r and Amx368f-Amx820r were used in nested PCR amplification.Isotope analyses were based on 15N labeled ammonium and nitrate.Three treatments（① 15NH＋4 only;② 15NH＋4 and 14NO-3;③ 14NH＋4 and 15NO-3） were prepared and experiments were conducted in hermetic anaerobic containers at 19℃.Outcome gaseous N2 was delivered to a gas chromatograph-isotope ratio mass spectrometer for separation of 28N2、29N2 and 30N2.Then the rates of anammox and denitrification activities were calculated.The results showed that one kind of anammox bacteria（Candidatus Brocadia） existed in the activated sludge;the rates of anammox and denitrification at 19℃ were 1.25 nmol·g-1·h-1 and 12.16 nmol·g-1·h-1 separately.Anammox contributed 9.32% of the total N2 production.Most of the N2 production was contributed by denitrification.

关 键 词：厌氧氨氧化 活性 15N同位素 16S RRNA 

分 类 号：X703.1[环境科学与工程—环境工程]