Introduction
Chloroethenes, such as tetrachloroethylene (PCE) and trichloroethylene (TCE) are one of the contaminants in soil and groundwater which is a significant problem domestically and internationally. Without extracting these contaminants from the ground, the in-situ treatment which directly degrades contaminants in soil or groundwater is now closely watched as an economical remediation technique. Especially, the in-situ bioremediation which degrades contaminants in-situ by activities of microbes is a safer and more economical technique than the physicochemical one and is being developed for practical application.
This paper describes that anaerobic bioremediation which cleans up soil and groundwater contaminated by chloroethene such as TCE in-situ by anaerobic bacteria.

1. Degradative mechanism
Anaerobic bioremediation is a remediation technique which injects organics, which become electron donors, together with nutrient salts such as nitrogen and phosphorus into groundwater and degrades contaminants by anaerobic bacteria. It is known that PCE and TCE are dechlorinated reductively by anaerobic bacteria and degraded into dichloroethylene (DCE), vinyl chloride (VC), ethylene and likewise, ethane (Fig. 1). Various types of anaerobic bacteria can degrade not only PCE and TCE but also cis-1,2-DCE. Its speed of degradation is relatively fast. On the other hand, by using cis-1,2- DCE and VC as an electron acceptor, Dehalococcoides bacteria is the only anaerobic bacteria which can completely degrade ethylene and ethane. It has been reported domestically and internationally that, in contaminated sites where Dehalococcoides bacteria is not present, TCE can only be transformed into cis-1,2-DCE but not dechlorinated completely because degradation stops even if a nutritional supplement is injected. In the use of anaerobic bioremediation, the confirmation of the presence of Dehalococcoides bacteria in the objective site is necessary in advance.

Figure-1　The proposed pathways for the degradation of chloroethenes by anaerobic bacteria

2. Determination of Dehalococcoides bacteria
The determination of 16S rDNA of Dehalococcoides bacteria by Real-Time PCR was examined. Consequently, the quantitative analysis of 16S rDNA of Dehalococcoides bacteria in groundwater can be performed with a wide range of 101-107 copies/mL and a high sensitivity of 10 copies/mL with a low determination limit. Fig.2 shows that the increase of 16S rDNA of Dehalococcoides bacteria in groundwater was observed by using this real-time PCR technique, as chlorinated ethane is being degraded with the vial degradation test of TCE using contaminated groundwater.

Figure-2　Degradation of cis-1,2-Dichloeoethene by Dehalococcoides sp

3. Practical example of anaerobic bioremediation
A practical example is introduced that groundwater contaminated by chlorinated ethene was purified to a level better than the environmental standards by applying anaerobic bioremediation to a real contaminated site. The objective site is composed of clay mixed with silt of GL 0m to -6m, sand of GL-6m to -11m and silt of GL-11m to -12m. Groundwater is present in the sand layer of GL-6m to 11m, which makes up the primary aquifer. This primary aquifer is contaminated by cis-1,2-DCE of 0.1 - 0.8 mg/L.
A schematic diagram of the anaerobic in situ bioremediation field test is shown in Fig. 3. Three wells as extraction and injection wells (Ex-3,4,5 and In-3,4,5) were installed. The aquifer of 4m X 4m X 5m surrounded by these wells was made the objective area for remediation (hereinafter called the objective area). The supply of nutritional supplement, which is made up of the mixture of extracted groundwater with ethanol and nutrient salt (ammonia and phosphoric acid), into groundwater was done by injecting it into the injection wells (In-3,4,5).
Performance data of the anaerobic in situ bioremediation field test is shown in Fig. 4. The concentration of cis-1,2-DCE in the objective area decreases gradually and almost all the objective areas were purified to a level better than the environmental standards on the 224th day after the remediation started. Also, 2 or 3 digits higher numbers of DNA of Dahalococcoides bacteria in the objective area were observed by determination of Real-Time PCR. Therefore, it was also found in an actual contaminated site that Dahalococcoides bacteria is very effective in the degradation of cis-1,2-DCE.

Figure-3 　Schematic diagram of the anaerobic in situ bioremediation field test

Figure-4　Performance data of the anaerobic in situ bioremediation field test

Conclusion
This paper reports that anaerobic bioremediation can effectively clean up groundwater contaminated by chloroethenes such as PCE and TCE. In this remediation technique, the presence of Dahalococcoides bacteria in the objective site which involves greatly in the degradation of cis-1,2-DCE or later transformations must be confirmed in advance. Monitoring bacteria concentration in the groundwater after the addition of nutritional supplement, controlling the frequency, and quantity and timing of an injection of nutritional supplement based on the data are all important factors.