• Journal of environmental and Sanitary engineering
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• v.23 no.2
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• pp.1-18
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• 2008

The tetrachloroethylene (PCE) dehalogenase of Clostridium bifermentans DPH-1 (a halorespiring organism) was purified, cloned, and sequenced. This enzyme is a homodimer with a molecular mass of ca. 70 kDa and exhibits dehalogenation of dichloroethylene isomers along with PCE and trichloroethylene (TCE). Broad range of substrate specificity for chlorinated aliphatic compounds (PCE, TCE, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropene, and 1,1,2-trichloroethane) for this enzyme was also observed. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. A partial sequence (81 bp) of the encoding gene for PCE dehalogenase was amplified and sequenced with degenerateprimers designed from the N-terminal sequence (27 amino acid residues). Southern analysis of C. bifermentans genomic DNA using the polymerase chain reaction product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase. So, C. bifermentans could play some important role in the initial breakdown of PCE and other chlorinated aliphatic compounds in sites contaminated with mixtures of halogenated substances.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.1
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• pp.40-46
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• 2005

Clostridium bifermentans strain DPH-1 has already been found to dechlorinate perchloroethylene (PCE) to cis-dichloroethylene (cis-DCE) via trichloroethylene (TCE). In this study, our investigation on different culture conditions of this DPH-1 strain was extended to find a more efficient and cost effective growth medium composition for this DPH-1 strain in bioremediation practices. Temperature dependency of strain DPH-1 showed that the growth starting time and PCE degradation at $15^{\circ}C$ was very slow compared to that of $30^{\circ}C$, but complete PCE degradation occurred in both cases. For the proper utilization of strain DPH-1 in more cost effective bioremediation practices, a simpler composition of an effective media was studied. One component of the culture medium, yeast extract, had been substituted by molasses, which served as a good source of electron donor. The DPH-1 strain in the medium containing molasses, in the presence of $K_{2}HPO_4\;and\;KH_{2}PO_4$, showed identical bacterial multiplication (0.135 mg protein $mL^{-1}h^{-1}$) and PCE degradation rates ($0.38\;{\mu}M/h$) to those of the yeast extract containing medium.

• Journal of Environmental Health Sciences
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• v.36 no.4
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• pp.323-336
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• 2010

We evaluated the properties of a fixed-bed column reactor for high-concentration tetrachloroethylene (PCE) removal. The anaerobic bacterium Clostridium bifermentans DPH-1 was able to dechlorinate PCE to cis-1,2-dichloroethylene (cDCE) via trichloroethylene (TCE) at high rates in the monoculture biofilm of an upflow fixed-bed column reactor. The first-order reaction rate of C. bifermentans DPH-1 was relatively high at $0.006\;mg\;protein^{-1}{\cdot}l{\cdot}h^{-1}$, and comparable to rates obtained by others. When we gradually raised the influent PCE concentration from $30\;{\mu}M$ to $905\;{\mu}M$, the degree of PCE dechlorination rose to over 99% during the operation period of 2,000 h. In order to maintain efficiency of transformation of PCE in this reactor system, more than 6 h hydraulic retention time (HRT) is required. The maximum volumetric dechlorination rate of PCE was determined to be $1,100\;{\mu}mol{\cdot}d^{-1}l$ of reactor $volume^{-1}$, which is relatively high compared to rates reported previously. The results of this study indicate that the PCE removal performance of this fixed-bed reactor immobilized mono-culture is comparable to that of a fixed-bed reactor mixture culture system. Furthermore, our system has the major advantage of a rapid (5 days) start-up time for the reactor. The flow characteristics of this reactor are intermediate between those of the plug-flow and complete-mix systems. Biotransformation of PCE into innocuous compounds is desirable; however, unfortunately cDCE, which is itself toxic, was the main product of PCE dechlorination in this reactor system. In order to establish a system for complete detoxification of PCE, co-immobilization of C. bifermentans DPH-1 with other bacteria that degrade cDCE aerobically or anaerobically to ethene or ethane may be effective.

• Journal of Environmental Health Sciences
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• v.26 no.2
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• pp.14-21
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• 2000

DEAE-Toyopearl 650S, Superdex pg-75, Poros HQ, Superdex H200의 각종 칼러크로마토그래피를 이용하여 C.bifermentans DPH-1균주로부터 테트라클로로에틸렌(PCE) 분해 효소를 정제했다. 이 PCE 분해효소 (PCE dehalogenase)는 PCE를 환원적 탈염소화 반응에 의해 시스디클로로에딜렌 (cis-1,2-dichloroethylene)에 전환 가능하여, 이 때의 Vmax 및 Km 치는 각각 73 nmol/h.mg protein, 12$\mu$M이었다. 본 PCE dehalogenase의 겔여과 분자량 Maker Kit를 이용한 분석결과(70kDa)는 SDS-PAGE에 나타난 분자량(35kDa)의 약 2배인 것으로 확인되었다. 따라서 본 효소는 분자량 70kDa의 이량체(Homo dimer)인 것으로 추정되었다. 본 효소의 최적온도 및 pH는 각각 35$^{\circ}C$ 및 8.0 이었다. 또한 본 효소는 PCE외의 트리클로로에틸렌, 디클로로에틸렌 이성체, 1,2-디클로로에템, 1,2-디클로로프로판, 1,1,2-트리클로로에탄에 대하여도 활성을 타나내었다. N-말단 아미노산 분석결과에서도 본 효소는 현재 알려진 PCE dehalogenase와 그 배열이 전혀 다른 것으로 나타나 각종 유기염소 화합물의 분해능을 보유한 신종의 PCE 분해효소인 것이 확인되었다.