Three strains to degrade chlorophenols were isolated through a selective enrichment procedure with 2,4,6-trichlorophenol (TCP) and 4-chlorophenol (4-CP). On the basis of their morphological and phenotypic characteristics, TCP degrading strain was identified as Pseudomonas solanacearum TCP114, and two 4-CP degrading strains were done as Pseudomonas testosteroni CPW301 and Arthrobacter ureafaciens CPR706, respectively.
A 4-CP degrading bacterium, Pseudomonas testosteroni CPW301, dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites. Resting cell experiments and enzyme assays of the cell extract indicated that both 4-CP and phenol were degraded via the same meta-cleavage pathway. When phenol was added as a secondary substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol enhanced the 4-CP degradation rate greatly because of the increased cell mass and 4-CP degradation activity.
Another 4-CP degrading strain, Arthrobacter ureafaciens CPR706, produced hydroquinone as transient intermediate of 4-CP degradation. This result indicated that the chloro-substituent of 4-CP was eliminated to form hydroquinone, which was demonstrated to be oxidized by enzyme extracts from 4-CP grown cells. CPR706 exhibited much higher tolerance for 4-CP than CPW301, as indicated by the maximum degradable concentration (1.6 mM for CPR706 and 0.8 mM for CPW301). The average 4-CP degradation rate by CPR706 was 13 fold higher than that by CPW301 due to the better specific degradation rate and cell yield of CPR706 (0.32 g cell/g 4-CP) than those of CPW 301. CPR706 was demonstrated to degrade other para-substituted phenols through hydroquinone.
The degradability of one component by a pure culture was strongly affected by the presence of other compounds in the medium. For example, when all three components (TCP, 4-CP, and phenol) were present in the medium, a pure culture of CPR706 and CPW301 could not degrade any of the components present. This restriction on the degradability could be overcome by employing a defined mixed culture of the two strains. The mixed culture could degrade all three components in the mixture through co-operative activity. It was also demonstrated that the mixed culture could be immobilized by using calcium alginate for the semi-continuous degradation of the three component mixture. The immobilization could not only accelerate the degradation rate, but also reuse the cell mass several times without losing the cells' degrading capabilities.
본 연구에서는 3종의 염화페놀 [2,4,6-trichlorophenol (TCP), 4-chlorophenol(4-CP)] 분해균주를 분리하였다. 형태적, 생리 및 생화학적 특징을 근거로 하여 TCP 분해균주는 슈도모나스 솔라나세룸 (Pseudomonas solanacearum TCP114) 그리고 두 4-CP 분해균주는 슈도모나스 테스토스테로니 (Pseudomonas testosteroni CPW301)와 아트로박트 유리아페시언스 (Arthrobacter ureafaciens CPR706)로 각각 동정되었다.
휴식세포실험과 (Resting cell experiments)과 효소활성측정으로 슈도모나스 테스토스테로니는 메타 분해경로로 페놀과 4-CP를 분해함이 밝혀졌다. 이 균은 페놀이 2차 탄소원으로 첨가될때 4-CP 분해효율이 크게증가 하였다. 이결과는 첨가된 페놀이 세포량과 4-CP 분해활성을 증가시시켜 주었기 때문이다.
다른 4-CP 분해균주 아트로박트 유리아페시언스 4-CP 분해도중 중간대사산물로 히이드로퀴논 (hydroquinone)을 생산하였다. 이결과는 4-CP의 염화기가 방향족환이 열리기 이전에 제거됨을 암시한다. 아트로박트 유리아페시언스는 슈도모나스 테스토스테로니보다 4-CP에 보다 강한 내성과 분해속도를 보였다.
순수배양에의한 단일성분의 각균주의 분해능은 타성분의 존재로 강하게 영향을 받았다. 예로 TCP와 4-CP 그리고 페놀이 함께존재할때 슈도모나스 테스토스테로니 와 아트로박트 유리아페시언스의 순수배양은 이들중 어떤 성분도 분해하지 못하였다. 이러한 제한은 혼합배양의 도입으로 해결되어졌다. 즉 혼합배양은 세성분 모두를 상호활성을 통해 분해할 수가 있었다. 칼슘알지네이트 (calcium alginate) 로 고정한 혼합배양으로 이들 세성분의 분해속도는 크게 증가하였다. 칼슘알지네이트고정은 세포체의 재사용을 분해활성의 소실없이 가능케 하였다.