Quantification And Diversity Analysis Of Dehalogenic Bacteria In 1,1,1-Trichloroethane(TCA) Contaminated Groundwater And Its Enriched Cultures

TCA which has carcinogenicity and teratogenicity, is one of the most common organic pollutants in our country and has caused serious damage to human health and ecological environment so the study of TCA pollution treatment is essential. The concentration of TCA, 1,1-dichloroethane(DCA) and chloroethane(CA) of 6 samples from groundwater in a TCA contaminated site was determined using gas chromatography. The existence of DCAand(or) CA in the 6 samples may associate with TCA biodegradation. Real-time qPCR results demonstrated the ratio of Dehalobacter spp. to the total bacteria varied in each sample while sample W6 ranked first. PCR products amplified by combining 16 S rRNA gene universal primer 27 f and Dehalobacter group-specific primer DHB647 r were used to construct six Dehalobacter group-specific clone libraries, respectively. Phylogenetic tree was constructed based on the sequences from the 6 clone libraries and their nearest neighbors from GenBank. 41 sequences were obtained from these 6 clone libraries and all their nearest sequences were affiliated to Dehalobacter. These sequences were clustered into 7 operational taxonomic units(OTUs) at a threshold of 99% similarity. OTU1(24 sequences) and OTU2(8 sequences) are the most dominant OTUs in all and constitute 78.0% of all the sequences. OTU1 and OTU2 both have a similarity of 98% with the TCA reductively dechlorinating strain Dehalobacter sp. str. TCA1. Three other OTUs(OTU3, OTU5, OTU6) only has 95%-96% similarity with their nearest 16 S rRNA gene sequences in GenBank and might be novel species. This study demonstrated the existence of Dehalobacter in all 6 samples with a relatively high diversity and these Dehalobacter bacteria might be responsible for the biodegradation of TCA in the groundwater of sampling sites.The degradation of the four TCA contaminated groundwater samples W1,W2,W4,W6 was explored. The original groundwater was transferred into serum bottles respectively and were cultured at 25℃, shaked at 110r/min in the dark for 9 weeks with the addition of VB12 and yeast extract. There are two groups of cultures, one group is referred as the EA group with the addition of mixed extra electron donor, the other group is referred as the NE group without extra electron donor addition. The rate of degradation of chlorohydrocarbon in each sample was measured using Headspace-GC-FID method, showing that in aforementioned conditions, the rate of degradation of chlorohydrocarbon of each sample in the EA group is higher than the corresponding samples in the NE group. Real-time qPCR of Dehalobacter analysis showed that after cultivation of 9 months, the ratio of Dehalobacter to the total bacteria decreased to no more than 1/20 times of the original in each sample. PCR-DGGE results showed that the preponderant bands were weakened in each sample and new preponderant bands appeared in both EA and NE group after cultivation. The microflora varied significantly after cultivation. The addition of growth factor and extra electron donor, cultivation condition changes including temperature change all could be the reason of this microflora change.