Rhodococcus erythropois 對碳氫化合物生物降解與其細胞聚集現象之研究

Abstract

在碳氫化合物生物復育研究中，如何加速微生物攝取碳源的效率向來是各界所關注的焦點。本研究從具有烷類降解能力之混合菌株TN-4中分離出Rhodococcus erythropolis NTU-1，在降解實驗過程中發現，NTU-1能夠利用其特有的疏水性細胞表面形成特殊的細胞聚集現象，將碳氫化合物完整包覆於細胞顆粒中，並可藉由物理分離方式達到迅速移除大量碳氫化合物的效果，這項發現對於生物復育研究領域提供了一個新的方向與策略。因此，NTU-1在各種碳氫化合物環境下的生長能力以及細胞聚集現象發生的原因即為本研究最主要的探討項目。 研究結果顯示，在濃度1000~3000 ppmv烷類為單一碳源條件下，NTU-1能夠降解約600~700 ppmv的烷類，並且在培養至第40小時左右形成大小0.1~2 cm的細胞結塊顆粒將殘餘烷類包覆，利用濾網將細胞顆粒與培養基進行初步分離後，即可使培養基中的烷類移除量達到95%以上。細胞結塊顆粒是以沒有規則及方向性的NTU-1堆疊而成，外觀所呈現的形態、大小以及顏色，會與細胞密度以及被包覆烷類量有密切的關聯，而細胞顆粒的重量佔了整體細胞乾重的80~90%以上，每單位重量的NTU-1最高可緊密包覆約7.8倍重的烷類。此外，NTU-1能夠在濃度3000 ppmv以下的正六烷、正八烷、四氯化碳、酚、苯、甲苯以及二甲苯等環境中生長，降解能力範圍也能夠涵蓋碳數C6~C32的烷類。 利用疏水性載玻片所進行的實驗中發現，NTU-1會在形成細胞結塊顆粒的前6小時開始貼附於疏水性載玻片上，而且細胞貼附位置與烷類的分布區域重合，這表示聚集現象的發生是由於NTU-1以烷類做為細胞之間的連結物質，再逐漸以一層層堆疊的方式形成細胞結塊顆粒。另一方面，NTU-1能夠在正十六烷降解過程中釋放界面活性劑，培養基表面張力在70小時的培養過程中會下降至60 mN/m。此外，結塊顆粒的細胞表面11種脂肪酸(C12~C24)含量經過正十六烷與異十九烷培養後大幅增加3~22倍不等，脂肪酸組成與碳源分子結構有關，這導致細胞具有更強烈的疏水性質也使NTU-1包覆烷類更加容易；但若使用NB、酚及正八烷為單一碳源培養時，細胞表面脂肪酸含量不會增加，細胞亦無法產生聚集現象。

Bioremediation is a common method for removal of petroleum hydrocarbon contaminations. In this study, a strain isolated from alkane-degrading mixed culture TN-4 obtained from petroleum contaminated soil was identified as Rhodococcus erythropolis NTU-1. When degrading alkanes, R. erythropolis NTU-1 owned hydrophobic cell surface, tended to flocculate and trapped most residual alkanes in the medium. The separation of oil-bacteria floccules from culture medium facilitated the removal of alkanes and consequently enhanced the efficiency of bioremediation. Therefore, the major purpose of this research was to investigate the issues regarding biodegradability and bioflocculation of the strain NTU-1. In batch cultivation, the biodegradation process was accompanied by the formation of biofloccules with size ranging from 0.1 to 2 cm in diameter. The morphology of biofloccules depended on the cell density and the amount of residual alkanes in culture medium. About 7.8-fold weight of alkanes could be trapped per unit weight of NTU-1. Approximately 95% of 1000 to 3000 ppmv of linear and branched alkanes could be efficiently removed within 40-68 hr with the aid of biofloccules. Therefore, this effective approach could be considered as a promising bioremediation strategy. Noteworthily, NTU-1 could not only degrade alkanes but has the ability to utilize broad range of hydrocarbons including toluene, phenol, benzene, xylene, carbon tetrachloride and other alkanes (C6-C32) as sole carbon source. Results illustrated that NTU-1 could attach to hydrophobic slide in the presence of alkanes, suggesting that NTU-1 might possess a hydrophobic cell surface which is an important driving force in bioflocculation. Besides, production of biosurfactant was observed during the microbial degradation of n-hexadecane with a minimum surface tension value of 60 mN/m, which helps in the adherence of the cells to oil droplets and subsequent biodegradation. When grown on long-chain alkanes, there were at least 11 different major growth-associated fatty acids produced, with carbon chain length ranging from C12 to C24, and cell surface hydrophobicity was enhanced via fatty acids accumulation at the cell surface. The relative amount of these fatty acids was 3-22 folds higher in alkane-grown cell compared to inoculum. In addition, the composition and relative amount of fatty acids depended upon the hydrocarbon chain length and structure supplied as a substrate.