Molecular analysis for monitoring of Nocardia sp. H17-1 on bioremediation of crude oil-contaminated soil

Abstract

Bioremediation is a technology that utilizes the metabolic potential of microorganisms to clean up contaminated environments. Especially, the addition of microorganisms that have degradative activity into environment (bioaugmentation) is useful. It is difficult that the fate of introduced bacteria be monitored in order to prove its contribution to pollutant degradation and to assess its influence on the polluted sites. This study was investigated the degradation of aliphatic and aromatic hydrocarbons by Nocardia sp. H17-1 KCTC8705P, a crude oil-degrading bacterium, and the detection and enumeration of the quantitiy and activity of introduced bacterium, Nocardia sp. H17-1 using the molecular techniques during bioremediation of crude oil-contaminated soil. The biodegradation of aliphatic and aromatic hydrocarbons, separated from Arabian light crude oil by Nocardia sp. H17-1, a crude oil-degrading bacterium, was investigated during 6 days. At the end of the experiment, the aliphatic and aromatic hydrocarbons were degraded 99.0 ± 0.1% and 23.8 ± 0.8%, respectively, while the resins and asphaltenes were not degraded. Nocardia sp. H17-1 degraded C_(10)-C_(24) n-alkanes, yet hardly degraded the aromatic fraction and grew poorly in the minimal medium containing aromatic hydrocarbons. Detection of the catabolic genes involved in the hydrocarbon degradation using a PCR indicated that H17-1 possessed the alkB gene for n-alkane biodegradation and catA gene for the ortho-cleavage of aromatic hydrocarbons, however, it did not contain the xylE gene for the meta-cleavage of aromatic hydrocarbons and nar gene for the degradation of naphthalene and PAHs. As such, the investigation of the genes involved in the biodegradation of hydrocarbons supported the low degradation activity and growth of H17-1 on the aromatic fractions of the crude oil. The effects of environmental parameters on the degradation and mineralization of crude oil by Nocardia sp. H17-1 were investigated over 50-days. First, three soils of loamy sand (LS), sand (S) and combusted loamy sand (CLS) artificially contaminated with crude oil (50 g/kg) were inoculated with Nocardia sp. H17-1. The degradation efficiency of total petroleum hydrocarbon (TPH) in sand was the highest at 76% among the three soils. TPH degradation rate constant (k_(TPH)) in loamy sand, sand and combusted loamy sand were 0.027 d^(-1), 0.063 d^(-1) and 0.016d^(-1), respectively. In contrast, the total amount of CO_(2) evolved was the highest at 146.1 mmol in loamy sand. CO2 evolution rate constant (k_(CO2)) in loamy sand, sand and combusted loamy sand were 0.051 d^(-1), 0.070 d^(-1) and 0.027 d^(-1), respectively. Second, for the investigation the effect of initial oil concentration, the soil was artificially contaminated with 10, 50 or 100 g of Arabian light oil /kg, respectively, and inoculated with 106 cells/g. After 50 days, Nocardia sp. H17-1 removed 78, 94 and 53% of the each initial concentration, respectively. Also, it produced 1.35, 4.21, and 5.91 mmol of CO_(2) per g of soil, respectively. TPH was reduced in proportion to the initial concentrations while CO_(2) production was increased according to increasing oil contaminations. The growth of Nocardia sp. H17-1 was remarkably inhibited when it was inoculated into soil containing 100 g of oil/ kg. Finally, to evaluate the effect of the inoculum size, the soil was artificially contaminated with 50 g of Arabian light oil/ kg, and inoculated with 3×10^(6), 5×10^(7), 2×10^(8) cells/g, respectively. After 50 days, the extent of TPH was remained with similar all treatment but k_(TPH) was increased according to increasing inoculum size. To monitor the Nocardia sp. H17-1 in the environment and to evaluate its in situ petroleum-bioremediation potential, a species-specific primer was constructed based on the 16S rDNA sequence of the bacterium. Two forward primers and two reverse primers were designed and tested against both closely and distantly related environmental isolates. Nocardia sp. H17-1 was detected by all the primers designed. Primer sets NH169F-NH972R and NH575F-NH972R could be used to detect 50 fg of template DNA and 1.2×10^(4) CFU/g of sandy soil. These two PCR primer sets successfully detected the H17-1 strain in the oil-contaminated soil samples containing heterogeneous DNA. A procedure of quantitative competitive polymerase chain reaction (QC-PCR) using species-specific primers based on 16S rDNA sequences was performed for detection and quantitation of Nocardia sp. H17-1 introduced during bioremediation of oil-contaminated soils. Also, change of the catabolic activity was investigated using the alkane monooxygenase gene fragments, alkB4, during the same time. QC-PCR allowed detecting 60 pg 16S rDNA of Nocardia sp. H17-1 and 6 pg alkB4 DNA. We tried to detect the H17-1 population using QC-PCR in crude oil contaminated soil. At the beginning of the study, the concentration of TPH in soil with H17-1 was 10.11 g/kg. After 120 days, it was reduced to 1.36 g/kg, indicating a removal of 86.5% of TPH. Not only the 16S rDNA but also the alkB4 gene of Nocardia sp. H17-1 dramatically increased while petroleum hydrocarbons were rapidly degraded. These results showed a potential for bioaugmentation of Nocarida sp. H17-1 and the use of QC-PCR as a tool for the monitoring of the bacteria introduced into the bioremediation sites, independent of cultivation. To compare the microbial activity and diversity with different treatments during bioremediation of crude oil-contaminated soil, it was accomplished in five different treatments, an indigenous microorganisms (OI), nutrient (ON), biosurfactant (OM), inoculation (OH), and biosurfactant plus inoculation plus nutrient (OHMN) to compare degradation rate and microbial communities. The extent of remaining hydrocarbons was similar at all treatment, but k_(TPH) was the highest in OHMN treatment. Bacterial communities and alkane monooxygenase gene fragment, alkB2, were compared by denaturing gradient gel electrophoresis (DGGE). There were no significant differences between the diversities of OI and OM DGGE band pattern, but k_(TPH) in OM was 10 times higher than OI. In ON, a prominent band which was not appeared in other treatment was showed during the experimental periods. DGGE analysis of OH and OHMN treatment revealed a simple, dynamic dominant population structure throughout the experiment. Also, DGGE analysis of alkane monooxygenase gene fragment, alkB2, was performed to monitor the shift in the numerically dominant hydrocarbon-degrading bacteria during bioremediation. Sequence analysis on the alkB2 gene fragments from the DGGE bands implied that the biostimulation treatment caused a shift of potential dominant hydrocarbon-degrading bacteria. The phytotoxic effects of crude oil and oil components on the plant growth of red bean (Phaseolus nipponesis OWH1) and corn (Zea mays) was investigated. In addition, the beneficial effects of bioremediation with Nocardia sp. H17-1 were also determined. It was found that crude oil-contaminated soil (≥30,000 mg/kg) was phytotoxic to corn and red beans. In contrast, obvious phytotoxicity was not observed in soils contaminated with 0-1000 mg/kg of aliphatic hydrocarbons such as decane (C_(10)) and eicosane (C_(20)). Phytotoxicity was observed in soils contaminated with 10-1000 mg/kg of the polyaromatic hydrocarbons (PAHs) such as naphthalene, phenanthrene and pyrene. It was observed that phytotoxicity increased with the number of aromatic rings, and that corn was more sensitive than red beans to PAH-contaminated soil. Bioremediation with Nocardia sp. H17-1 reduced phytotoxicity more in corn than in red bean, suggesting that this microbial species might degrade PAHs to some degree.;생물학적 정화기술이란 미생물을 이용하여 오염물질을 제거하는 방법이다. 특히, 오염물 분해 활성을 가지는 미생물의 첨가는 매우 유용한 방법이다. 그러나 오염지에서 첨가된 미생물의 거동과 활성을 조사하는 것은 어렵다. 본 연구는 원유오염토양의 생물학적 복원과정 동안 접종된 H17-1의 정량과 원유분해능을 분자생물학적 방법을 통해 조사하였다. 원유오염토양에서 분리한 Nocardia sp. H17-1의 Arabian light oil과 diesel에 함유된 aliphatic과 aromatic hydrocarbon의 생분해 정도를 조사하였다. 6일 배양 후 원유에 함유된 aliphatic과 aromatic hydrocarbon fraction은 각각 초기 농도의 99.0±0.1%와 23.8±0.8%를 분해하였다. Diesel에 함유된 aliphatic과 aromatic hydrocarbon fraction은 각각 초기 농도의 75.3±2.3%와 24.2±3.5%를 분해하였다. Nocardia sp. H17-1은 C10-C24 n-alkane을 분해할 수 있으나, aromatic hydrocarbon의 분해능은 매우 낮았다. Hydrocarbon 분해에 관련된 유전자를 PCR로 증폭한 결과, H17-1은 alkane 분해에 관련된 유전자인 alkB gene과 aromatic hydrocarbon의 ortho-pathway에 관여하는 catA gene을 가지고 있는 것으로 조사되었다. 한편, aromatic hydrocarbon의 meta-cleavage에 관여하는 xylE와 naphthalene dioxygenase에 관여하는 nar gene은 검출되지 않았다. 이것은 aromatic hydrocarbon fraction의 낮은 분해능을 설명할 수 있다. 오염토양에 유류 분해능을 가진 Nocardia sp. H17-1의 접종시 고려되어야 할 인자 중 soil types, 초기 오염농도, 초기접종농도가 탄화수소 분해와 균주의 생육에 미치는 영향을 조사하였다. Soil types이 H17-1의 탄화수소 분해에 미치는 영향을 조사하기 위해 5 g Arabian light oil/kg of soil이 오염된 loamy sand (유기물 함량 5.8%), sand (유기물 함량 0.9%), combusted loamy sand (유기물 함량 0.9%)를 대상으로 50일간 실험을 실시하였다. 분해 속도 상수 (k)는 sand에서 가장 높았으나, CO2 생성속도 상수 또한 sand에서 가장 높은 값을 나타내었다. H17-1은 실험 50일 동안 초기 오염농도 10, 50, 100 g Arabian light oil/kg of soil에 대해 각각 78.5%, 94.3%, 53.2%의 탄화수소를 제거하였으며, 오염농도가 높을수록 분해속도 상수 (k)는 낮아졌다. CO2의 생성량 또한 오염농도가 높을수록 증가하였으나, 100 g/kg of soil의 오염농도에서는 균의 생육이 저해를 받는 것으로 나타났다. H17-1의 초기 접종농도에 의한 영향은 균의 접종량에 따라 최종 남은 TPH의 양은 큰 차이를 나타내지 않았으나, 분해속도 상수 (k)는 균의 접종량이 늘어남에 따라 크게 증가되었으며, CO_(2)의 생성량 또한 균의 접종농도에 따라 크게 증가하였다. 원유로 오염된 토양의 생물학적 복원과정 동안 접종된 Nocardia sp. H17-1 균주를 확인하기 위하여 16S rDNA sequence에 기초하여 균주 특이적 primer를 제작하였다. 14 균주의 16S rDNA sequences 비교를 통해 제작된 4개의 primer set는 H17-1 균주를 특이적으로 검출할 수 있었다. 특히 NH169F-NH972R과 NH575F-NH972R의 primer set는 50 fg의 DNA와 1.2×104 cfu/g of soil의 균체 농도까지 민감하게 검출할 수 있었다. 이 두 primer set는 원유로 오염된 토양의 bioremediation 과정 동안 접종된 H17-1 균주의 특이적 검출을 가능하게 하였으며, 이는 사용된 primer set에 의해 증폭된 PCR 산물을 제한효소 (Eco RΙ)로 절단한 결과와 PCR-DGGE 결과로부터 제작된 primer set의 H17-1 균주 특이성을 검증하였다. 원유오염토양의 생물학적 복원과정 동안 접종된 H17-1의 정량과 H17-1 alkB gene을 이용한 catabolic activity를 quantitative competitive polymerase chain reaction (QC-PCR)법으로 조사하였다. QC-PCR법은 Nocardia sp. H17-1에서 60 pg의 16S rDNA와 6 pg의 alkB DNA의 검출을 가능케 하였다. 초기 TPH 농도가 10.11 g/kg인 토양에 H17-1을 적용한 결과, 120일 후 TPH의 농도는 1.36 g/kg으로 86.5%가 감소되었다. 이 기간 동안 H17-1은 16S rDNA 뿐만 아니라 alkB gene 또한 급격하게 증가되었다. 이러한 결과는 현장에서 H17-1에 의한 bioremediation의 가능성을 뒷받침해주며, 또한 QC-PCR법은 bioremediation시 토양에 도입된 균주 뿐만 아니라 토착미생물의 탐색에 유용하게 이용될 수 있다. 다양한 처리 방법에 의한 원유오염토양의 bioremediation 과정 동안 각 처리구에서의 microbial population 과 catabolic activity를 비교하였다. 토착미생물 처리구 (OI), 영양원 첨가구 (ON), 생물계면활성제 첨가구 (OM), Nocardia sp. H17-1 첨가구 (OH)와 생물계면활성제+H17-1+영양원 첨가구 (OHMN)에서 분해활성과 미생물군집을 비교한 결과, 잔존 TPH 양은 모든 처리구에서 비슷하였으나, TPH 분해 속도 상수 (k)는 OHMN 처리구에서 가장 높았다. 16S rDNA에 의한 미생물 군집과 alkane monooxygenase gene fragment (alkB)는 PCR-DGGE에 의해 분석되었다. OI 와 OM 처리구에서 16S rDNA DGGE band pattern은 비슷하였으나, 분해속도 상수 (k)는 OM 처리구가 OI 처리구보다 10배 정도 더 컸다. ON 처리구에서는 다른 처리구에서는 발견되지 않는 band가 실험이 종료될 때까지 관찰되었다. DGGE OH와 OHMN 처리구는 다른 처리구보다 단순한 band pattern을 나타내었으나, 접종된 H17-1이 우점하고 있는 것으로 관찰되었다. Alkane monooxygenase gene fragment (alkB)의 DGGE 양상은 bioremediation 과정 동안 전체 미생물 군집 중 일부의 hydrocarbon-degrading bacteria에 의해서만 진행됨이 관찰되었다. H17-1에 의한 Bioremediation 효과를 검증하게 위해 팥과 옥수수를 이용한 식물독성을 측정하였다. 10 g/kg 의 Crude oil contaminated soil은 콩과 옥수수에 매우 toxic 하였다. decane (C_(10))와 eicosane (C_(20))와 같은 aliphatic hydrocarbons은 0-1,000 mg/kg의 농도로 오염된 토양에서 식물독성을 나타내지 않았으나, naphthalene, phenanthrene, pyrene 같은 PAHs는 10-1,000 mg/kg의 농도로 오염된 토양에서 식물독성을 나타내었다. 특히 aromatic hydrocarbon은 ring 수가 증가할수록 독성이 증가하였으며, 팥보다는 옥수수가 민감하였다. Nocardia sp. H17-1로 bioremediation된 토양은 H17-1이 처리되지 않은 토양에 비해 식물독성이 많이 회복되었으며, 팥보다 옥수수에서 식물독성이 감소되었다.