基于PCR-DGGE和拟杆菌(Bacteroides)16S rRNA的岩溶地下水粪便污染来源示踪研究:以重庆南山老龙洞地下河系统为例

Tracing the Fecal Contamination Sources Based on Bacteroides 16S rRNA PCR-DGGE in Karst Groundwater: Taking Laolongdong Underground River System,Nanshan,Chongqing as an Example

岩溶地下水的微生物污染日益严重,其来源的研究得到国际学术界的广泛关注.本研究以重庆南山老龙洞岩溶地下河系统为对象,采用滤膜法监测地下水中的总细菌、大肠杆菌、粪大肠菌及粪链球菌等微生物指标,以拟杆菌(Bacteriodes)为指示细菌,采用PCR-DGGE示踪地下水中大肠杆菌/粪大肠杆菌的来源.结果表明,老龙洞地下河流域各类细菌含量严重超标,总细菌数为10~2.9×10~7CFU·m L^(-1),大肠菌群总数达4.3~4.0×10~5CFU·m L^(-1),其中粪大肠菌群(FC)和粪链球菌(FS)分别最高达到1.1×10~6CFU·(100 m L)^(-1)、1.1×10~5CFU·(100 m L)^(-1);FC/FS多数为2以上,暗示流域地下水受人类粪便影响为主.地下水样和粪便样品的拟杆菌群落的PCR-DGGE比对分析表明地下水与人粪之间相似性为7.1%~69.1%,其中地下河出口处达到69.1%.地下水与猪粪之间相似性为1.1%~53.4%,地下河出口处仅为1.5%.因此,人类粪便为地下河污染的主要来源,猪粪污染为动物粪便污染的一部分,还存在其他动物粪便污染来源.此外,PCR-DGGE产物切胶测序发现大部分Bacteroides为人类肠道或粪便来源的细菌.

Microbial contamination in karst groundwater continually increases and tracing the source researches has become a hot topic for international researchers. In this study,Laolongdong underground river at Nanshan,Chongqing was chosen as an example to adopt filter membrane methods to monitor the fecal microbial contaminations including the total bacterial concentration（ TB）,the total E. coli concentration（ TE）,the total fecal coliform（ FC） and the total fecal Streptocoocci（ FS）. Bacteriodes was used as an indicator and PCR-DGGE analysis was used to trace fecal contamination sources in karst groundwater. The results suggested that groundwater in this area was seriously polluted by microbes from feces. The concentrations of microbial parameters exceeded limited levels greatly and the total bacterial amounts ranged 10-2. 9 × 10^7CFU·m L^-1,the concentrations of E. coli were between 4. 3-4. 0 × 10^5CFU·m L^-1,the max concentration of FC was 1. 1 × 10^6CFU·（ 100 m L）^-1and the max concentration of FS was 1. 1 × 10^5CFU·（ 100 m L）^-1. The FC/FS ratios were mostly over 2 which suggested that the main fecal source in groundwater was human feces. In addition,PCR-DGGE contrastive analysis of Bacteroides communities showed that the similarities between groundwater samples and human feces were in range of 7. 1%-69. 1%,and the similarity of the groundwater sample from Laolongdong underground river outlet was 69. 1%. Bacteroides community similarities between groundwater samples and swine feces were in range of 1. 1%-53. 4%,and the similarity of Laolongdong underground river outlet was merely 1. 5%. The similarity data implied that groundwater contamination resulted mainly from human feces,swine feces contamination composed part of animals＇ fecal contamination,and other animals＇ feces participated too. Furthermore,sequencing results of PCR-DGGE products revealed that most Bacteroides in groundwater originated from human intestinal tract and human feces.