Tetrachloroethene (PCE) and trichloroethene (TCE) are common groundwater contaminants that also impact river flats, especially near urban and industrial areas. However, very little is known about dechlorinating microbial communities in river flats. Titanium pyrosequencing, 16S rRNA gene clone libraries, and dechlorinator-targeted PCR characterize the reductive dechlorinating activities and populations in river sediments collected from Wonju River in South Korea. Four times enrichment process were performed to obtain a more enriched dechlorinating bacteria community, and the final enrichment culture was studied in detail. 2-Bromoethanesulfonic acid was added in B2 but not in B3. The chlorinated ethenes were detected by gas chromatography with a HP-5 column. The dechlorinating mechanism was different between the two enrichment cultures B2 and B3. In the microcosms of B2, PCE dechlorination to TCE began within 10 days, and nearly 100% of the initial amount of PCE was converted to TCE after 12 days. In addition, cis-1,2-Dichloroethene (cis-DCE) was observed after TCE transformed. In B3, PCE was almost directly transformed to cis-DCE with no detectable TCE formation. cis-DCE was observed as the dechlorination end product in both of the enrichment cultures. Pyrosequencing of bacterial 16S rRNA genes revealed that Desulfovibrio populations were predominant in B2 and that Desulfotomaculum populations were predominant in B3. Other abundant groups included Geobacter, Treponema, Denitrobacterium, Tepidimicrobium, Zunongwangia in the B2 and B3 enrichment cultures. The results suggest that BES accounts for the different pathways of these two enrichment cultures, which were from the same originator. Moreover, titanium pyrosequencing provides more detailed insight into the community structure dynamics of dechlorinating microcosms than conventional 16S rRNA gene sequencing. A total of seventeen strains were isolated from B2 and B3. Two of seventeen belonged to Desulfovibrio and fifteen belonged to Sedimentibacter.
During a study of the 4-chlorophenol degradation enrichment culture, tree novel bacterial strains designated as strain 3C5-12T, strain 3C6-41, and strain 2C6-43T were isolated from activated sludge from industrial wastewater treatment in Daegu, South Korea and were taxonomically classified. Strains 3C5-12T and 3C6-41 were gram-reaction-negative, nonspore-forming, aerobic Alphaproteobacteria. Their taxonomic positions were investigated in a polyphasic study. On the basis of 16S rRNA gene sequence similarity, the closest phylogenetic relatives were Rhizobium daejeonense KCTC 12121T (3C5-12T: 99.4%, 3C6-41: 99.4%), Rhizobium selenitireducens LMG 24075T (3C5-12T: 97.3%, 3C6-41: 97.4%), Rhizobium herbae CCBAU83022T (3C5-12T: 97.0%, 3C6-41: 97.1%) and Rhizobium giardinii KACC 10720T (3C5-12T: 96.9%, 3C6-41: 97.0%). The G+C contents 3C5-12T and 3C6-41 were 58.02 % and 63.45 %, respectively. Both showed C16:0 and C18:1 ω7c as the major cellular fatty acids, which were also the major fatty acids for the closest strains Rhizobium daejeonensis and Rhizobium selenitireducens. A nifH gene encoding denitrogenase reductase was detected in 3C5-12T, but not in 3C6-41. Two aromatic compounds ring cleavage enzymes of catechol 1, 2-dioxygenase and 2,3-dioxygenase were detected in 3C6-41, but for 3C5-12T, only 2,3-dioxygenase was positive. The results of physiological and biochemical tests as well as the low DNA-DNA hybridization values also enabled strain 3C5-12T and 3C6-41 to be differentiated genotypically and phenotypically from the recognized Rhizobium species. Though strains 3C5-12T and 3C6-41 shared a high 16S rRNA gene sequence similarity of 99.9%, the nifH gene and 1,2-dioxygenase and 2,3-dioxygenase genes support the differences between these two strains.
Strain 2C6-43 was a gram-positive, rod-shaped, aerobic bacterial strain. A comparative 16S rRNA gene sequence analysis showed that strain 2C6-43T is related most closely to Georgenia soli CC-NMPT-T3T (98.8%), Georgenia muralis 1A-CT (97.6 %), Georgenia thermotolerance TT02-04T (96.8 %), Georgenia ruanii YIM 004T (96.6 %) and Georgenia halophila YIM 93316T (96.0%). The G + C content of the genomic DNA was 66.2 mol%. Cell-wall sugars were rhamnose, ribose and galactose, and the menaquinone MK-8 (H4) was detected as the predominant quinone. The polar lipid contained diphosphatidylglycerol, phosphatidylinositol-mannoside, phosphatidylinositol and phosphatidylglycerol. The peptidoglycan was composed of L-Glu, D-Ala, L-Ala and L-Lys. The aromatic compound ring cleavage enzyme of catechol 1,2-dioxygenase was detected, but 2,3-dioxygenase was not detected in 2C6-43T. The fatty acid profile with anteiso-C15:0, iso-C15:0, and anteiso-C15:1 A as the major components supported the affiliation of strain 2C6-43T to the genus Georgenia. Strain 2C6-43T could be clearly differentiated from its phylogenetic neighbors on the basis of several phenotypic, genotypic and chemotaxonomic features.
Tetrachlroethene (PCE) 와 trichloroethene (TCE)는 지하수 오염물질로 하천 특히는 공업단지 주변지역의 오염이 심각한 상태이다. 그러나 이런 탈염소 미생물 군집에 대해서는 많이 알려져 있지 않다. Titanium pyrosequencing, 16S rRNA gene clone libraries 그리고 dechlorinator-targeted PCR을 진행하여 원주천의 하천 침전물의 reductive dechlorinating activity를 알아보았다. 총 네번의 농화배양을 진행하였고 마지막 농화배양을 중점적으로 연구하였다. B2의 배양액에는 2-bromoethanesulfonic acid 를 첨가하였지만, B3 배양액에는 첨가하지 않았다. Chlorinated ethene은 gas chromatography로 측정하였고 컬럼은 HP-5로 하였다. 탈염소 메커니즘은 B2와 B3에서 상이하였는데, B2는 PCE를 TCE를 거쳐 cis-DCE로 전환하였지만 B3는 본 연구에서는 TCE를 발견하지 못하였고 직접 cis-DCE로 전환하였다. 박테리아 16S rRNA genes의 Pyrosequencing 결과에서 Desulfovibrio 속이 B2에서 가장 우점종으로 나온 반면 B3에서는 Desulfotomaculum 속이 우점종으로 발견되었다. 이 외에도 많이 발견된 속은 Geobacter, Treponema, Denitrobacterium, Tepidimicrobium, Zunongwangia 등이 있었다. 이러한 결과는 아마 배양액의 BES에 기인된다고 생각하고 있다. 이 두 농화 배양액에서 총 17 개의 strain을 분리하였는데 그중 두 strain은 Desulfrovibrio 속에 속하였고 나머지 15개 strain은 Sedimentibacter 속에 속하였다.
4-Chlorophenol 생분해 연구과정에서 세 균주를 분리 및 동정을 진행하였다. 이 세균주는 각각 3C5-12T 와 3C6-41 그리고 2C6-43T로 대구공단의 폐수 활성슬러지에서 분리하였다. 3C5-12T and 3C6-41은 그람음성균, 호기성 포자를 생성하지 않는 Alphaproteobacteria에 속한 균주들이었다. 16S rRNA 염기서열에 의하면 가장 가까운 균주들은 각각 Rhizobium daejeonense KCTC 12121T (3C5-12T: 99.4%, 3C6-41: 99.4%), Rhizobium selenitireducens LMG 24075T (3C5-12T: 97.3%, 3C6-41: 97.4%), Rhizobium herbae CCBAU83022T (3C5-12T: 97.0%, 3C6-41: 97.1%) 그리고 Rhizobium giardinii KACC 10720T (3C5-12T: 96.9%, 3C6-41: 97.0%)로 높은 유사성을 보였다. G+C content는 3C5-12T 는 58.02 %, 3C6-41는 63.45 %이었으며 두 균주가 모두 C16:0 와 C18:1 ω7c를 메이저 지방산으로 존재하고 있었다. 탈질반응을 일으킬 수 있는 유전자인 nifH 유전자는 3C5-12T에서는 발견되었지만 3C6-41에서는 발견되지 않았다. 방향족화합물질 분해효소인 catechol 1,2-dioxygenase 와 2,3-dioxygenase 연구결과 3C6-41은 두 유전자 모두 함유하고 있었지만, 3C5-12는 2,3-dioxygenase에서 양성을 보였다. 위의 결과와 낮은 수치의 DNA-DNA hybridization 실험결과를 종합하여 보면, 3C5-12T 와 3C6-41는 같은 속 다른 종들과 표현형적, 유전학적으로 구분된다는 것을 보여주었다.
2C6-43T 균주는 그람양성, 호기성 간균이었다. 16S rRNA 유전자의 유사성 비교결과 이 균주는 Georgenia soli CC-NMPT-T3T (98.8%), Georgenia muralis 1A-CT (97.6 %), Georgenia thermotolerance TT02-04T (96.8 %), Georgenia ruanii YIM 004T (96.6 %) 그리고 Georgenia halophila YIM 93316T (96.0%)와 가갖 가까운 것으로 나타내었다. G + C content는 66.2 mol %로 측정되었다. 세포벽 당분석 결과 이 균주는 rhamnose, ribose 및 galactose를 함유하고 있었고 menaquinone MK-8 (H4)가 가장 많은 quinonㄷ으로 검출되었다. Polar lipid는 cerol, phosphatidylinositol-mannoside, phosphatidylinositol 및 phosphatidylglycerol을 함유하고 있었고 peptidoglycan은 L-Glu, D-Ala, L-Ala 및 L-Lys을 함유하고 있었다. 방향족화합물질 분해효소인 catechol 1,2-dioxygenase 와 2,3-dioxygenase 연구결과 2C6-43T는 1,2-dioxygenase 에서만 양성을 나타냈었다. 지방산은 anteiso-C15:0, iso-C15:0, 그리고 anteiso-C15:1 A를 메이저로 함유하고 있었다. 이런한 결과는 2C6-43T 가 Georgenia속에 속하지만, 기타 균주와 서로 다른 신종균주임을 제시하여준다.