Bioremediation of phenanthrene contaminated soil by rhamnolipid biosurfactants was studied. Preparation of biosurfactants, biosurfactant-enhanced soil washing, and the mineralization of contaminant in washing solution were the main attempts of this work.
In the preparation of biosurfactants, a glycolipid type of biosurfactants was obtained from a strain which had been isolated from soil. The cell was identified as Pseudomonas aeruginosa from taxonomic characteristics and was designated as YPJ80. Thin layer chromatography and deoxyhexose detection tests were done to verify the type of biosurfactant. Steps for the recovery was investigated by comparing the yield of glycolipid from ultrafiltration, adsorption, and solvent extraction. Further spectroscopic analysis such as IR, $^1H$-NMR, $^{13}C-NMR$ and FAB-MS was done and the structure of the biosurfactant was verified to be rhamnolipid type Ⅰ and Ⅲ. Critical micelle concentration of the surfactant was observed to be 50 ppm and the minimum surface tension was 30.1 mN/m. As an emulsifier, rhamnolipids showed superior activity to emulsan in the emulsification of crude Arabian light oil.
In soil washing, effect of rhamnolipid addition was investigated in terms of enhancement of solubilizing power of the washing solution and the increment in the rate of phenanthrene transfer into washing solution. Significant enhancement upto 50 folds was observed in the phenanthrene solubility over that of control by the addition of 2 g/L rhamnolipids. Further enhancement was observed in the presence of cations such as $Na^+$ and $Mg^{++}$. Upto 10 times higher molar solubility ratio was obtained with 1.0 mM $MgCl_2$ when compared with that of cation free rhamnolipid. The coefficient ($κ_L$) of the phenanthrene dissolution rate was increased from 1.8 to 107.2 by the addition of 2 g/L rhamnolipids in the solution. From the uncertainty analysis of the batch equilibrium of the soil washing process, surfactant concentration was verified to be the most affecting parameter on residual concentration of contaminant after washing.
As a successive step of soil washing, mineralization of phenanthrene dissolved in rhamnolipid solution was investigated by monitoring the evolution of radioactivity-labeled $^{14}CO_2$. The presence of rhamnolipids in the solution did not affect the biodegradation of phenanthrene by Pseudomonas putida CRE7. Effect of non-aqueous phase liquid was, on the contrary, controversial. Enhancement and inhibition was observed for high initial concentration (500 ppm) and for low initial concentration (50 ppm) of phenanthrene. Bacterial adhesion to hydrocarbon assay, cell association study, and distribution of the carbon substrate were followed to elucidate the mechanism.
생물계면활성제를 이용하여 phenanthrene으로 오염된 토양을 정화하는 연구가 진행되었다. 우선, 유화활성이 뛰어난 생물계면활성제를 자연으로부터 분리, 생산하는 과정을 확립한 후, 이를 이용하여 이단계 추출 및 처리방식을 통해 오염토양을 처리할 수 있었다.
유화활성이 우수한 생물계면활성제를 생산하는 미생물을 탐색하였으며 균주동정을 통해 Pseudomonas aeruginosa YPJ80 이라는 미생물을 확보하였다. Thin layer chromatography와 deoxyhexose 검출실험을 통해 당지질계의 생물계면활성제임을 알 수 있었으며, crude Arabian light oil에 대해 emulsan보다 뛰어난 유화활성을 보였다. 이의 순수분리를 위해 한외여과, 흡착, 용매추출 등의 분리방법 들을 비교하였다. 순수분리된 계면활성제의 구조분석을 위해 IR, $^1H$-NMR, $^{13}C$-NMR, FAB-MS 등을 이용한 결과, rhamnolipid Ⅰ과 Ⅲ 형태 임을 알 수 있었다. YPJ80으로부터 생산된 rhamnolipid의 임계미셀농도 (CMC)는 50 ppm 이었으며, 최저 표면장력은 30.1 mN/m 이었다.
토양세척에 있어서 계면활성제의 주요 역할은 오염물질인 phenanthrene 의 용해도 개선 및 용해속도의 증가였다. 2 g/L rhamnolipid 용액의 경우, phenanthrene의 용해도가 수용액 자체의 용해도에 비해 50배 정도 증대되었다. 또한 $Na^+$ 나 $Mg^{++}$ 등의 양이온이 존재하는 경우, 용해도는 더욱 증가되었으며, 1.0 mM $MgCl_2$ 의 경우, 10배의 증가가 이루어졌다. 용해속도에 있어서도 2 g/L rhamnolipid 용액의 경우, 전달계수 값이 1.8에서 107.2로 크게 증가하였다. Batch equilibrium model을 이용하여 토양세척공정을 모사하였다. 불확실성분석을 통해 공정의 주요변수 중에서 계면활성제의 농도가 공정의 안정성에 가장 큰 영향을 미치는 변수로 파악되었다.
토양세척 후처리 공정으로서 세척액 내의 phenanthrene의 처리는, radio-labeled phenanthrene 의 완전분해 산물인 $^{14}CO_2$의 radioactivity를 파악함으로써 진행되었다. 용액중의 rhamnolipid의 존재는 생물학적 분해에 별다른 영향을 주지않아 안정적인 사용이 가능한 것으로 파악되었다. 또한 오염물과 함께 존재할 것으로 여겨지는 NAPL은 phenanthrene의 초기 농도에 따라 증가 내지는 저해의 역할을 하였다. 이의 기작을 이해하기위해 BATH assay, cell association, phenanthrene의 분배에 관한 연구가 진행되었다.
