유류 및 중금속 오염토양의 rhizoremediation

Abstract

본 연구에서는 장기간 유류로 오염된 토양에서 자생한 6 종의 식물(돌피(Echinochloa crus-galli, K1), 산괭이사초(Carex leiorhyncha, K2), 닭의장풀(Commelina communis, H1), 흰여뀌(Persicaria lapathifolia, H2), 통보리사초(Carex kobomugi, S1), 쇠뜨기(Equisetum arvense, S2))에서 근권토양과 식물뿌리를 채취하여 근권세균을 분리하였다. 분리한 근권세균을 대상으로 식물 성장 촉진 능력(plant growth promoting ability, PGPA)을 조사하고, PGPA가 있는 균주 중 분류된 균주 중 유류 분해능이 우수한 Gordonia sp. S2RP-17와 유류오염 토양의 정화에 효율이 우수한 옥수수(Zea mays)를 동시에 이용하여 유류오염 토양의 rhizoremediation을 수행하고자 하였다. 또한, 유류와 중금속으로 함께 오염된 토양에 Gordonia sp. S2RP-17, 중금속 내성 균주인 그리고 Serratia sp. SY5, 그리고 옥수수를 동시에 적용하여 오염 토양의 rhizoremediation에 관해 연구하였고, 유류 오염 토양으로부터 분리한 비근권세균인 Gordonia sp. SD8의 bioremediation과 비소 오염 토양의 식물상 복원에 관해 연구하였다. 유류오염 토양으로부터 총 374개의 근권미생물을 분리하였고, 분리한 근권세균을 대상으로 Indole acetic acid(IAA) 생산, 1-aminocyclopropane-1-carboxylic acid(ACC) deaminase 활성, 그리고 siderophores의 합성능을 조사하하였다. IAA 생산능은 86개의 균주가 가지고 있었고(23.0%), ACC deaminase 활성은 168개 균주(44.9%), 그리고 siderophore(s) 합성능은 213개의 균주(57.0%)가 가지고 있었다. PGPA를 가지고 있는 근권세균은 RP(82%)에서 분리한 근권세균이 RS(75%)에서 분리한 근권세균보다 많았다. PGPA를 가진 세균들을 대상으로 3 종류 PGPA 사이의 상관관계를 분석한 결과, IAA 생산능을 가진 세균과 ACC deaminase 활성을 가진 세균 비율 사이에는 음의 상관관계를나타냈다(-0.656, p < 0.05). 또한, 식물의 종류 및 서식지, 3가지 PGPA에 따라 PGPR의 비율을 주성분 분석(principal component analysis, PCA)을 한 결과, PGPR의 분포는 식물의 서식지(RS와RP)에 따라 영향을 받음을 알 수 있었다. 근권미생물 중 ACCd와 siderophore 합성능을 가진 Gordonia sp. S2RP-17 균주의 비성장속도는 0.54 d-1 이였고 최대분해속도가 2434 mg L-1 d-1 이었다. 또한, S2RP-17는 디젤로 오염된 토양에서 옥수수를 재배하였을 때 옥수수의 줄기와 뿌리를 각각 15와 20% 증가 시켰고, 생체량은 각각 29%, 28% 향상 시켰다. 옥수수만 단독으로 존재하는 유류 오염 토양과 옥수수와 S2RP-17이 함께 존재하는 오염 토양의 TPH제거 효율을 비교한 결과, TPH제거 효율이 9% 향상 되었다. 비근권세균 중 siderophore 합성능을 가진 Gordonia sp. SD8 선별하여 유류 분해 특성을 조사한 결과 비성장속도는 .673±0.05 h-1, 대최분해속도는 1900.67±39.27 mg L-1 d-1 있었다. 또한, 디젤로 오염된 토양을 정화하는데 있어 SD8 균주의 유용성을 평가한 결과, 실험을 진행하는 7주 동안 TPH의 농도가 97%가 감소 하였고, Gordonia sp. SD8과 옥수수를 함께 이용하여 유류오염 제거 효율을 알아본 결과, TPH농도가 63.5 mg/kg, SD8 균주와 옥수수가 함께 존재하는 근권토양은 0.0 mg/kg으로 식물과 디젤 분해 균주가 함께 존재할 때 디젤 오염 토양의 정화 효율이 증가하는 것을 알 수 있었다. 유류를 분해할 수 있으며 식물성장 촉진 능력을 가지고 있는 근권세균인 Gordonia sp. S2RP-17과 중금속에 내성을 가지며 동시에 식물 성장 촉진 능력을 가지고 있는 Serratia sp. SY5 균주가 유류와 중금속으로 오염된 토양에서의 옥수수 성장과 오염물질 제거에 미치는 영향에 대해 연구한 결과, 옥수수 줄기와 잎 길이에서는 근권세균의 접종 효과가 없었지만, 잎의 수는 근권세균을 접종한 조건이 우수하였다. 토양에 잔류하고 있는 유류는 근권미생물에 의해 TPH제거가 더욱 향상 되었고, 토양의 잔류 중금속은 함량은 초기 농도에 비해 Pb가 가장 많이 감소하였고, 식물의 체내에서는 줄기 보다는 뿌리에 더 많은 양의 중금속을 축적하였다. 독성이 강한 중금속으로 알려져 있는 비소를 과축적할 수 있는 식물로 오이 선별하였고 오이의 성장과 오이의 비소 축적에 비소 화학종이 미치는 영향을 알아본 결과, As(III), As(V), 그리고 DMA 중 As(V)가 오이의 성장을 가장 크게 저해하였고, 비소 축적율도 가장 높았다. 따라서, Gordonia sp. S2RP-17균주와 Gordonia sp. SD8 균주는 옥수수와 함께 유류 및 중금속 오염 토양의 rhizoremediation에 유용한 자원으로 이용될 것이며, 오이는 비소 오염토양의 remediation에 유용하게 사용될 것으로 기대된다.;In this study, Three hundred and seventy-four rhizobacteria were isolated from the rhizosphere soil (RS) or rhizoplane (RP) of Echinochloa crus-galli, Carex leiorhyncha, Commelina communis, Persicaria lapathifolia, Carex kobomugi, and Equisetum arvense, grown in contaminated soil with petroleum and heavy metals. The isolates were screened for plant growth-promoting potential (PGPA), Rhizoremedition efficiency was evaluated by using a plant growth-promoting rhizobacterium (PGPR) with maize (Zea mays) at a diesel-contaminated soil. Also, diesel and heavy metal contaminated soil was treated with rhizobateria Gordonia sp. S2RP-17, Serratia sp. SY5, and maize. And we isolated Gordonia sp. SD8 from diesel contaminated soil and measured bioremediation ability by SD8. Rhizobacteria were isolated from the rhizosphere soil (RS) or rhizoplane (RP), grown in contaminated soil with petroleum and heavy metals. The isolates were screened for plant growth-promoting potential (PGPP), including indole acetic acid (IAA) productivity, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, and siderophore(s) synthesis ability. IAA production was detected in 86 isolates (23.0%), ACC deaminase activity in 168 isolates (44.9%), and siderophore(s) synthesis in 213 isolates (57.0%). Among the rhizobateria showing PGPP, 162 rhizobacteria had multiple traits showing more than two types of PGPP. The PGPP-having rhizobateria were more abundant in the RP (82%) samples than the RS (75%). There was a negative correlation (-0.656, p < 0.05) between the IAA-producers and the ACC deaminase producers. Clustering analysis by principal component analysis showed that RP was the most important factor influencing ecological distribution and physiological characterization of PGPP-having rhizobateria. To develop phytoremediation technique, this study was performed by using a plant growth promoting rhizobacterium (PGPR) with maize in diesel-contaminated soil column (mesocosm). At first, we successfully isolated a PGPR, Gordonia sp. S2RP-17, which had ACC dearminase activity, and siderophore synthesizing activity. Its maximum specific growth rate was 0.54±0.12 d-1, TPH maximum degradation rate was 24342±124.4 mg L-1 d-1. As positive plant growth indicators, the S2RP-17 promoted plant growth including leaf, stem and root parts. The TPH removal efficiencies were 10% by natural attenuation, 84.5% by plant effect and 94.4% by the combination effect of plant and PGPR, indicating the addition of S2RP-17 could enhance remediation efficiency. Bioremediation is a remediation technique using bacteria to remove contaminants from soils. At first, we successfully isolated a diesel degrading bacteria, Gordonia sp. SD8, which had the specific growth rate of 0.673±0.05 d-1, TPH maximum degradation rate of 1727.33±145.22 mg L-1 d-1. In this study, we evaluated diesel degradation by Gordonia sp SD8 and residual TPH concentrations was measured at the once a week during 7 weeks of study period. The results show that Gordonia sp SD8 caused a decrease of TPH concentration in soil (< 96 %) for 7 weeks. In this study, we studied the influence of Gordonia sp. S2RP-17, rhizobacteria, which can resolve oils and promotes plant growth and Serratia sp. SY5, which has metal-resistant as well as the ability to promote plant growth on the growth of Zea mays on oil- and heavy metal-contaminated soil and the removal efficiency of contaminants. We investigated the effect of rhizobacteria on the Zea mays growth by measuring its length of stems and leaves and the number of leaves for 51 days. As a result, we found that rhizobacteria inoculation was effective on the increase of the number of leaves, but not on the growth of the length of stems and leaves. Also, The concentration of oil remaining in soil is 48.9 ± 80.6 mg/kg when planting Zea mays with rhizobacteria (DHM+P+B); and 209.8 ± 108.3 mg/kg when only planting Zea mays in contaminated soil (DHM+P), meaning that TPH is removed by Zea mays and rhizobacteria. In terms of residual heavy metal in soil, the Pb is reduced the most significantly from the initial concentration. Heavy metals are accumulated more in root than in stem of plants. It is well known that phytoremediation is remediation technique using plants to remove contaminants from soils. In this study, we measured arsenic(As) tolerance in 4 plants; corn(Zea mays), wheat(Triticum aestivum), cucumber(Cucumis sativus) and sorghum(Sorghum bicolor), and evaluated these plant’s phytoremediation-possibility. First of all, we measured seed germination ratio in at 200 mg/L of As concentration, seed germination of corn, sorghum, wheat and cucumber was 40%, 13%, 6% and 87% respectively, so cucumber has the highest tolerance among the tested species. Arsenic translocation factor(TF) of cucumber was 1.9±0.9 and bioaccumulation factor(BF) was 21.1±8.4 As the results, cucumber shows arsenic hyperaccumulator and phytoremediation possibility in arsenic contaminated soils. It was evaluated the effect of arsenic (As) species such as As(III), As(V) and dimethylarsinic acid (DMA) on the As accumulation in cucumber (Cucumis sativus) as well as its growth at soil mesocosm. its growth was remarkably inhibited by the inorganic As (As(III) and As(V)) rather than the organic As (DMA). Irrespective of As species, the As concentrations accumulated in Cucumis sativus were increased with increasing the As concentration at soil. The values of As bioaccumulation factor from soil into the tissue of Cucumis sativus were 17.5-35.4 for As(III), 29.3-42.7 for As(V), and 17.6-25.7 for DMA. In addition, the As translocation factor from its root to its shoot were 0.025-0.031, 0.018-0.032 and 0.014-0.026 for As(III), As(V) and DMA, respectively. In conclusion, Cucumis sativus mainly accumulated As in its root rather than in its shoot, and easily accumulated the inorganic As than the organic As from soil into its tissue. Therefore, Gordonia sp. S2RP-17 and Gordonia sp. SD8 stimulated plant development in the soil contaminated with diesel and hearvy metals, enhacing the efficiency of rhizoremediation. Also, cucmber has a possibility to remediate arsenic contaminated soil.