| 摘要: | 烷基苯酚聚氧乙基醇為一被廣泛使用的非離子型界面活性劑，其一旦排放至環境中，通常會生成烷基苯酚(Alkylphenol)如壬基苯酚(nonylphenol, NP)、辛基苯酚(octylphenol, OP)等為環境中不易分解，並具有環境荷爾蒙假性雌激素效應的污染有機物，對人體的內分泌與健康具有潛在的威脅。 鑑於目前對於環境中烷基苯酚之生物分解的瞭解仍非常有限，本研究從長期施用農藥的農田土篩選出兩株可生長於辛基苯酚為唯一碳源之細菌，經過BioLog法、脂肪酸圖譜與16S rDNA序列等三種鑑定方法及明膠水解活性測試，其中之一菌株為Pseudomonas nitroreducens OP1，另一菌株為Pseudomonas sp. OP2。此兩菌株皆能以0.001~0.01%的辛基苯酚為唯一生長碳源，其中，以0.005%辛基苯酚培養時，菌株OP1與OP2之比生長率分別為0.17 d-1及0.31 d-1，且培養16天後，菌株OP1與OP2各能去除82%及46%的辛基苯酚，此顯示兩菌株確實能以辛基苯酚為唯一碳源並去除之。此外，兩株分解菌以辛基苯酚為催化基質時，會分別表現5.3及4.4 nmole/min的耗氧活性，以辛基鄰苯二酚為基質時，也有6.1及3.0 nmole/min的耗氧活性，又進一步以西方墨點法分析，兩株細胞粗萃液中皆不具有類似鄰苯二酚2,3-加氧酵素(catechol 2,3-dioxygenase, C23O)的酵素，此顯示此兩菌株在以辛基苯酚為唯一碳源培養時，並不會產生meta-cleavage之開環酵素，但是否存在ortho-cleavage的開環酵素則需進一步研究。 為進一步探討所分離之辛基苯酚分解菌株應用於土壤復育之潛力，本研究另以土壤縮模(microcosm)模擬受污染的土壤環境，除探討以界面活性劑分解菌P. nitroreducens TX1和P. putida TX2分別搭配菌株Pseudomonas sp. OP2添加於含有辛基苯酚聚氧乙基醇的縮模中，對於辛基苯酚聚氧乙基醇及其代謝物辛基苯酚的去除情形，以及添加菌株Pseudomonas sp. OP2於僅含有辛基苯酚的土壤縮模的去除效果外，並利用16S rDNA片段於變性梯度膠體電泳中之指紋，分析上述土壤縮模中微生物菌群之變化及其與污染物降解之關連。其中，菌株P. nitroreducens TX1已證實能分解辛基苯酚聚氧乙基醇，並以辛基苯酚為最終產物，另一菌株P. putida TX2則對於辛基苯酚聚氧乙基醇及辛基苯酚皆具有分解能力。本實驗將各組縮模之土壤混合後，計選殖出24株菌為選殖庫 (Clone library)，其中，所含菌群多屬γ-proteobacteria，而選殖菌群內Alcaligenes faecalis (AF155147)、Stenotrophomonas maltophilia (AJ293470)、Aeromonas sp. (AB076858)等三個菌種則曾於文獻中被發表具有烷基苯酚化合物之分解能力。菌群分析結果顯示，三株添加菌株對於縮模內原生菌群均不會造成顯著的影響，其中，原生菌群Herbaspirillum sp. Chnp3-5、Phyllobacterium myrsinace、Brucellaceae bacterium、Stenotrophomonas maltophilia和Aeromonas hydropila為含有辛基苯酚聚氧乙基醇之縮模中的優勢菌，另外，當菌株P. nitroreducens TX1或P. putida TX2分別添加於土壤縮模後，利用液相層析儀分析90天後土壤中辛基苯酚聚氧乙基醇之含量，皆較只含原生菌之縮模提高1.5倍去除能力，亦能利用縮模內辛基苯酚聚氧乙基醇生長成為優勢菌。此外，土壤原生菌中之Aeromonas hydropila、 Pseudomonas sp YG-1、Alcaligenes faecalis為含有辛基苯酚之縮模中的優勢菌，而菌株Pseudomonas sp.OP2添加於縮模後，於四個月操作時間內能一直為優勢菌，且與只含原生菌之縮模比較，約可於90天內提高15-20%之辛基苯酚的移除效果。整體而言，雖然原生菌已有部分的OPEOn降解能力，但藉由添加菌株P. putida TX2及Pseudomonas sp. OP2之組合，可幾乎完全去除外加之辛基苯酚聚氧乙基醇，並對於辛基苯酚也有25%之去除能力，為最具潛力應用於清除辛基苯酚環境污染之菌株。 Alkylphenol polyethoxylates (APEOn) were an extensively-used non-ionic surfactants. When these componds are discharged into natural environment, alkylphenol such as nonylphenol and octylphenol are often found to be as accumulates metabolites. However, alkylphenol is more recalcitrant than APEOn and has been demonstrated as an environmental hormone with estrogenic-like activity. However, only a few studies were reported on the biodegradation of alkylphenol. This study isolated two octylphenol-degrading bacteria from topsoil of farm, which was frequently sprayed by pesticides and surfactant. They were respectively identified as Pseudomonas nitroreducens OP1 and Pseudomonas sp. OP2 by BioLog breathprint, 16S rDNA sequence analysis, fatty acid fingerprint and negative for gelatin hydrolysis activity. Both of them were able to grow on octylphenol (0.001~0.01%) as sole carbon source. The strains OP1 and OP2 showed a specific growth rate of 0.17d-1 and 0.31 d-1 as 0.005% octylphenol as sole carbon source, respectively. After 16 days of cultivation, the strains OP1 and OP2 were able to remove 82% and 46% of octylphenol, respectively. Both strains OP1 and OP2 further revealed an oxygen uptake activity of 5.3 and 4.4 nmole/min respectively using octylphenol as the transformation substrate with 1.5 ml cell suspension of OD=0.3. They also showed 6.1 and 3.0 nmole/min of oxygen uptake rate when octylcatechol as used as a substrate. In addition, Western blotting and C23O dioxygenase activity assay also showed that no catechol 2,3-dioxygenase-like enzyme for the cleavage of aromatic ring was detected in both strains OP1 and OP2 grown on octylphenol as the sole carbon source. The study further investigated the bioremediation of octylphenol polyethoxylates (OPEOn) and its primary metabolite, OP in soil microcosms with bacteria exogenously added. Two surfactant-degrading bacteria, P. nitroreducens TX1 and P. putida TX2 were respectively added with Pseudomonas sp. OP2 in different OPEOn-contained microcosms to understand the removal of octylphenol polyethoxylates and octylphenol. The strain TX1 was able to degrade OPEOn and formed octylphenol, while the strain TX2 revealed activities to degrade both of OPEOn and OP. The strain Pseudomonas sp. OP2 was further added into an octylphenol-contained microcosm and the variation of octylphenol was then analyzed. The bacterial communities were analyzed by 16S based denaturing gradient gel electrophoresis (DGGE) of rRNA genes by 24 bacterial strains, which were totally cloned from soils in all microcosms. Among these endogenous bacteria, Alcaligenes faecalis (AF155147)、Stenotrophomonas maltophilia (AJ293470)、Aeromonas sp (AB076858) have been found as alkylphenol-degrading bacteria, in precious study. The analysis of bacteria communities indicated that no obvious effect was observed in the endogenous bacteria community when strains OP2, TX1 and TX2 were exogenously added in these microcosms. When P. nitroreducens TX1 and P. putida TX2 were exogenously added in the OPEOn-contained microcosms, both of them were able to enhance the OPEOn removal by 1.5 fold and also found as the dominant bacterial as well as endogenous bacteria such as Herbaspirillum sp. Chnp3-5、Phyllobacterium myrsinace、Brucellaceae bacterium、Stenotrophomonas maltophilia and Aeromonas hydropila. Moreover, the exogenous strain Pseudomonas sp. OP2 was demonstrated to be able to extra remove 15-20% of OP in soil microcosms, and was also the dominant bacteria as well as endogenous strains Aeromonas hydropila、 Pseudomonas sp YG-1 and Alcaligenes faecalis within four months of operation periods. The study showed that endogenous bacteria was able to remove partial OPEOn in microcosms. However, the removal of OPEOn and its metabolite OP were obviously enhanced when strain P. putida TX2 and Pseudomonas sp. OP2 was exogenously added in soil microcosms. Almost all of OPEOn and 25% of OP was able to be removed respectively. These indicated that these two strains, showed the most potential in the application of OP bioremediation. |
