重庆市河水中粪源微生物污染特征及源解析
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摘要
以重庆主要河流水体作为研究对象,使用传统培养技术评估了细菌总数、粪链球菌、肠球菌、脆弱拟杆菌、总大肠菌群及粪大肠菌群的污染水平;同时以拟杆菌作为特异性指示菌,选取人源性粪便专一指示菌引物(HF183)和猪源性粪便专一指示菌引物(Pig-2-Bac)进行源追踪.微生物培养结果表明:重庆市主要河流在春季有15.4%的研究断面未达到Ⅲ类水质,秋季有61.5%的研究断面未达到Ⅲ类水质.在春季,主城区河流主要受人类粪便污染,区县河流主要受动物粪便污染;在秋季,主城区和区县河流都主要受人类粪便污染.指示微生物指标间Pearson相关性分析结果表明粪链球菌、粪大肠菌群、肠球菌两两之间有显著相关性,肠球菌与脆弱拟杆菌有显著相关性.猪源性拟杆菌特异性生物标记Pig-2-Bac和人源拟杆菌特异性生物标记HF183对人和动物粪便污染区分成功率达100%;用这两种特异性引物对春季水样DNA进行扩增,发现13个采样点均未受到猪源粪便污染,唐家沱、朝天门、鸡冠石、合川受到人源粪便污染.
In this study, 13 water samples were collected from the main stem rivers in Chongqing city. The numbers of total bacteria,Streptococcus faecalis, enterococci, Bacteroides fragillis, total coliforms and Fecal coliforms were counted using traditional culturing techniques. The bacteroides were selected as specific indicator bacteria to identify the specific fecal pollution sources by using human-specific bacteroides maker(HF183) and pig-specific bacteroides maker(Pig-2-Bac). The results showed that 15.4% of the main stem river reaches in Chongqing did not reach category Ⅲ water quality in spring, and 61.5% did not reach category Ⅲ water quality in autumn. In spring, the rivers in the main urban areas were mainly polluted by human excrement, and those in the rural areas were mainly polluted by animal manure. In autumn, the main stem river reaches and the rivers in the rural areaswere polluted by human excrement. Pearson correlation analysis of indicator microorganisms showed that there were strong correlation between any two indicator of fecal streptococcus, fecal coliform and enterococci, and there was a significant correlation between enterococci and Bacteroides fragilis. The verification tests showed that pig source-specific biomarkers, pig-2-bac and human-specific markers,HF183, showed good specificity to pig manure and human feces samples, respectively. Using these two specific biomarkers to amplify DNA in water samples, it was found that in the spring all the 13 sampling sites were not contaminated by pig feces; four samples including Tangjiatuo, Chaotianmen, Jiguanshi and Hechuan were polluted by human feces.
In this study, 13 water samples were collected from the main stem rivers in Chongqing city. The numbers of total bacteria,Streptococcus faecalis, enterococci, Bacteroides fragillis, total coliforms and Fecal coliforms were counted using traditional culturing techniques. The bacteroides were selected as specific indicator bacteria to identify the specific fecal pollution sources by using human-specific bacteroides maker(HF183) and pig-specific bacteroides maker(Pig-2-Bac). The results showed that 15.4% of the main stem river reaches in Chongqing did not reach category Ⅲ water quality in spring, and 61.5% did not reach category Ⅲ water quality in autumn. In spring, the rivers in the main urban areas were mainly polluted by human excrement, and those in the rural areas were mainly polluted by animal manure. In autumn, the main stem river reaches and the rivers in the rural areaswere polluted by human excrement. Pearson correlation analysis of indicator microorganisms showed that there were strong correlation between any two indicator of fecal streptococcus, fecal coliform and enterococci, and there was a significant correlation between enterococci and Bacteroides fragilis. The verification tests showed that pig source-specific biomarkers, pig-2-bac and human-specific markers,HF183, showed good specificity to pig manure and human feces samples, respectively. Using these two specific biomarkers to amplify DNA in water samples, it was found that in the spring all the 13 sampling sites were not contaminated by pig feces; four samples including Tangjiatuo, Chaotianmen, Jiguanshi and Hechuan were polluted by human feces.
引文
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[20]王江权,康敉,郑祥,等.海河流域典型河流粪源性指示微生物的污染特征及其时空分布[J].环境科学学报,2017,37(1):138-145.Wang Jiangquan,Kang Mi,Zheng Xiang,et al.Occurrence and temporal-spatial distribution of fecal indicator microorganisms in three rivers of the Haihe River Basin[J].Acta Scientiae Circumstantiae,2017,37(1):138-145.
[21]宫月华.长江及某些大型湖泊、水库地表水细菌指标的研究与分析[D].武汉:华中科技大学,2011.Gong Yuehua.The research and analysis of bacteriology index in the yangtze river water and some big lakes and reservoirs[D].Wuhan:Huazhong University of Science And Technology,2011.
[22]国家环境保护总局.水和废水监测分析方法.[M].4版.中国环境科学出版社,2002.National Environmental Protection Agency.Water and exhausted water monitoring analysis method[M].4th Edition.China Environmental Science Press,2002.
[23]何道领,王华平,王震.重庆市畜禽养殖粪污现状与思考[J].安徽农业科学,2014,(20):6627-6628.He Daoling,Wang Huaping,Wang Zhen.Current situation and thinking of manure pollution in livestock and poultry breeding in Chongqing[J].Journal of Anhui Agricultural Sciences,2014,(20):6627-6628.
[24]李庆康,吴雷,刘海琴,等.我国集约化畜禽养殖场粪便处理利用现状及展望[J].农业环境科学学报,2000,19(4):251-254.Li Qingkang,Wu Lei,Liu Haiqin,et al.The Status and Outlook of Treatment on Excreta from Intensive Animal Farming in China[J].Agro-Environmental Protection,2000,19(4):251-254.
[25]周媛媛,殷捷,杨志敏,等.重庆市畜禽粪污的区域分布及其水环境响应特征分析[J].中国生态农业学报,2016,24(6):811-818.Zhou Yuanyuan,Yin Jie,Yang Zhimin,et al.Regional distribution of livestock manure and response characteristics of water environment in Chongqing[J].Chinese Journal of Eco-Agriculture,2016,24(6):811-818.
[2]US EPA O.Recreational Water Quality Criteria[S].2012.
[3]WHO.Addendum to the WHO Guidelines for Safe Recreational Water Environments,Volume 1,Coastal and Fresh Waters.[R].Addendum to the Who Guidelines for Safe Recreational Water Environments,2009.
[4]EU,2006.Directive 2006/7/EC of the European Parliament and of the Council of 16February 2006 Concerning the Management of Bathing Water Quality and Repealing Directive 76/160/EEC[S].Official Journal of the European Union L64,pp:37-51.
[5]GB3838-2002地表水环境质量标准[S].GB3838-2002 Standard of surface water environment quality[S].
[6]Hagedorn C,Robinson S L,Filtz J R,et al.Determining sources of fecal pollution in a rural Virginia watershed with antibiotic resistance patterns in fecal streptococci[J].Applied and Environmental Microbiology,1999,65(12):5522-5531.
[7]Bernhard A E,Field K G.A PCR Assay To Discriminate Human and Ruminant Feces on the Basis of Host Differences in BacteroidesPrevotella,Genes Encoding 16S rRNA[J].Applied and Environmental Microbiology,2000,66(10):4571-4.
[8]Layton A,Mckay L,Dan W,et al.Development of Bacteroides 16SrRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total,Human,and Bovine Fecal Pollution in Water[J].Applied and Environmental Microbiology,2006,72(6):4214-4224.
[9]Schill W B,Mathes M V.Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial cytochrome b targets[J].Environmental Science and Technology,2008,42(14):5229.
[10]Somnark P,Chyerochana N,Mongkolsuk S,et al.Performance evaluation of Bacteroidales genetic markers for human and animal microbial source tracking in tropical agricultural watersheds.[J].Environmental Pollution,2018,236:100-110.
[11]Seurinck S,Defoirdt T,Verstraete W,et al.Detection and quantification of the human-specific HF183 Bacteroides 16S rRNAgenetic marker with real-time PCR for assessment of human faecal pollution in freshwater.[J].Environmental Microbiology,2010,7(2):249-259.
[12]Mieszkin S,Furet J P,Corthier G,et al.Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific Bacteroidales 16S rRNA genetic markers.[J].Applied&Environmental Microbiology,2009,75(10):3045-3054.
[13]Haugland R A,Varma M,Sivaganesan M,et al.Evaluation of genetic markers from the 16S rRNA gene V2region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR.[J].Systematic and Applied Microbiology,2010,33(6):348-357.
[14]罗荣祥.长江嘉陵江交汇段营养限制因子生态幅研究[D].重庆:重庆大学,2006.Luo Rongxiang.The study on Ecological Amplitude of Nutrition Restricting Factors in Jialing River Intersection of Yangtze River[D].Chongqing:Chongqing University,2006.
[15]国家环保总局.水质总大肠菌群和粪大肠菌群的测定纸片快速法[S].2015.National Environmental Protection Agency.Water quality Determination of total coliforms and fecal coliforms Paper strip method[S].2015.
[16]Converse R R,Blackwood A D,Kirs M,et al.Rapid QPCR-based assay for fecal Bacteroides spp.as a tool for assessing fecal contamination in recreational waters[J].Water Research,2009,43(19):4828-37.
[17]马小雪,杨阳,Randy A Dehlgren,等.应用拟杆菌作为水体粪源污染指示菌的研究进展[J].黑龙江农业科学,2011,(10):150-154.MA Xiao xue,YANG Yang,Randy A.Dehlgren,et al.Review and Current Perspective on Using Bacteroides as A Fecal Source Indicator in Polluted Waterbodies[J].Heilongjiang Agricultural Science,2011,(10):150-154.
[18]吕平毓,米武娟.三峡水库蓄水前后重庆段整体水质变化分析[J].人民长江,2011,42(7):28-32.LU Pingyu,MI Wujuan.Analysis on general variation of water quality in Chongqing reach after impoundment of Three Gorges Reservoir[J].Yangtze River,2011,42(7):28-32.
[19]段逸凡.重庆市老龙洞地下河流域岩溶地下水粪便指示菌环境指示意义的研究[D].重庆:西南大学,2015.Duan Yifan.The study of environmental implications of faecal microbes Laolongdong groundwater basin Chongqing[D].Chongqing:Southwest University,2015.
[20]王江权,康敉,郑祥,等.海河流域典型河流粪源性指示微生物的污染特征及其时空分布[J].环境科学学报,2017,37(1):138-145.Wang Jiangquan,Kang Mi,Zheng Xiang,et al.Occurrence and temporal-spatial distribution of fecal indicator microorganisms in three rivers of the Haihe River Basin[J].Acta Scientiae Circumstantiae,2017,37(1):138-145.
[21]宫月华.长江及某些大型湖泊、水库地表水细菌指标的研究与分析[D].武汉:华中科技大学,2011.Gong Yuehua.The research and analysis of bacteriology index in the yangtze river water and some big lakes and reservoirs[D].Wuhan:Huazhong University of Science And Technology,2011.
[22]国家环境保护总局.水和废水监测分析方法.[M].4版.中国环境科学出版社,2002.National Environmental Protection Agency.Water and exhausted water monitoring analysis method[M].4th Edition.China Environmental Science Press,2002.
[23]何道领,王华平,王震.重庆市畜禽养殖粪污现状与思考[J].安徽农业科学,2014,(20):6627-6628.He Daoling,Wang Huaping,Wang Zhen.Current situation and thinking of manure pollution in livestock and poultry breeding in Chongqing[J].Journal of Anhui Agricultural Sciences,2014,(20):6627-6628.
[24]李庆康,吴雷,刘海琴,等.我国集约化畜禽养殖场粪便处理利用现状及展望[J].农业环境科学学报,2000,19(4):251-254.Li Qingkang,Wu Lei,Liu Haiqin,et al.The Status and Outlook of Treatment on Excreta from Intensive Animal Farming in China[J].Agro-Environmental Protection,2000,19(4):251-254.
[25]周媛媛,殷捷,杨志敏,等.重庆市畜禽粪污的区域分布及其水环境响应特征分析[J].中国生态农业学报,2016,24(6):811-818.Zhou Yuanyuan,Yin Jie,Yang Zhimin,et al.Regional distribution of livestock manure and response characteristics of water environment in Chongqing[J].Chinese Journal of Eco-Agriculture,2016,24(6):811-818.
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