| 摘要: | 烷基苯酚 (Alkylphenol) 與烷基苯酚聚氧乙烯醇 (alkylphenol polyethoxylates, APEOn) 皆被用在塑膠、塗料、殺蟲劑及清潔劑的製造，並且烷基苯酚為APEOn在環境中代謝的產物，自然情況下不易分解，對環境生物造成威脅。壬基苯酚 (Nonylphenol, NP) 與辛基苯酚 (octylphenol, OP) 是烷基苯酚之中最常見的兩個商品。其中，辛基苯酚的雌激素活性是壬基苯酚的十至二十倍，本研究使用辛基苯酚為唯一生長碳源之菌株為模式，探討細菌在環境中生物分解所扮演的角色與機制。 先前本實驗室已由宜蘭縣員山鄉長期施灑農藥與肥料之稻田表土、所製作之土壤縮模 (microcosm) 及辛基苯酚藥罐中，共篩選出17株能以辛基苯酚為唯一生長碳源之菌株，以95種不同碳源組合圖譜 (BioLog方法)、脂肪酸甲酯圖譜分析方法及 16S rDNA序列比對作菌種鑑定。鑑定結果皆為革蘭氏陰性菌，分屬於α、β及γ-proteobacteria，其中 Pseudomonas 屬佔12株，其餘菌株包括 Alcaligenes sp., Shinella sp., Inquilinus sp.及 Methylobacterium radiotolerans。文獻中，具有降解烷基苯酚類化合物能力的菌株大多為 α-proteobacteria以及 γ-proteobacteria，本研究則選出在 β-proteobacteria中生長速率較佳，並可以利用辛基苯酚的類似碳源辛基苯酚聚氧乙烯醇 (octylphenol polyethoxylates)、辛基苯酚單氧乙烯醇 (octylphenol monoethoxylates)、辛基苯酚雙氧乙烯醇 (octylphenol diethoxylates)、酚 (phenol) 以及辛基鄰苯二酚 (octylcatechol) 作唯一碳源生長之菌株Alcaligenes sp. SH542，進行辛基苯酚轉化測定，以探討α、β及γ-proteobacteria對辛基苯酚之代謝途徑 。以辛基苯酚為Alcaligenes sp. SH542唯一生長碳源之模式，在不同時間長度的培養下，對菌液萃取濃縮，以高效能液相層析質譜儀分析辛基苯酚及相關代謝產物。可發現隨著培養時間增加，18天後，94.2%的辛基苯酚被去除。分析Alcaligenes sp. SH542的代謝產物，在細菌生長對數期時，1,2,4-trihydroxybenzene逐漸出現，並在生長對數遲滯期逐漸被分解，此時，另一代謝產物hydroquinone產生累積，然而在培養22天後，辛基苯酚與其代謝物 hydroquinone 與 1,2,4-trihydroxybenzene 皆被完全代謝。經由實驗室成員進行基因序列分析，Alcaligenes sp. SH542具有 multiple component phenol hydroxylase，以耗氧活性偵測儀可發現該菌株在加入辛基苯酚與其代謝產物 hydroquinone與 1,2,4-trihydroxybenzene時，皆有耗氧增加的情況，因此推測耗氧酵素參與在辛基苯酚的代謝過程。 經由代謝產物分析，我們推測 Alcaligenes sp. SH542 降解辛基苯酚途徑為，經由type II ipso substitution 後，產生 ipsohydroxylation中間物，中央斷裂而產生 hydroquinone，hydroquinone 再經加氧酵素轉化成 1,2,4-trihydroxybenzene。 Alkylphenols and alkylphenol polyethoxylates (APEOn) are used in the formulation and production of mainly plastics, paints, pesticides and detergents, and they are omnipresent in the environment. Nonylphenol and octylphenol are the primary members in the alkylphenol, and are also the major metabolites from APEOn. The prior studies have demonstrated that octylphenol is 10 to 20 folds higher in the estrogenic effect than nonylphenol. In this study, 4-t-octylphenol was used as the sole carbon source for the isolation of octylphenol-degrading bacteria in Taiwan. Seventeen octylphenol-degrading bacteria were isolated from paddy farm topsoil, soil microcosm and 4-t-octylphenol reagent bottle by previous works. The topsoil had been intermittently polluted by agricultural surfactants. These bacteria were identified by 16S rDNA gene sequencing, BioLog substrate profiling and fatty acid methyl ester fingerprinting. All the bacterial strains belong to α, β and γ-proteobacteria. In the past, most of the akylphenol degrading bacteria were found to belong to α and γ-proteobacteria. In this article, we propose the octylphenol degrading mechanism of the soil microcosm isolated Alcaligenes sp. SH542. which is belongs to β-proteobacteria. For the substrate utilization, strain SH542 can grow on MSB plate containing OPEOn, OPEO1, OPEO2, octylcatechol and phenol as sole carbon source. For a time-course study of octylphenol degradation, 100% of octylphenol was removed after 22 days of incubation. In metabolite analysis, the data shows that the 1,2,4-trihydroxybenzene increase coincides with the obvious decrease of octylphenol in bacteria log phase at the first four days. After 12 days of incubation, the intermediate hydroquinone was found to accumulate and was further completely degraded after 22 days. For analysis of genes which related to degrade aromatic compounds, our lab member Tuan et al. (2010) shows that Alcaligenes sp. SH542 has multiple component phenol hydroxylase gene. We assumed that oxygen is involved in the degradation of octylphenol. Strain SH542 shows the oxygen was uptaked during octylphenol degradation, the oxygen also was uptaked in MSB medium with hydroquinone and 1,2,4-trihydroxybenzene as sole carbon sources. In conclusion, octylphenol degradation by Alcaligenes sp. SH542 begins with the transformation of octylphenol to its ipsohydroxylated intermediate by type II ipso substitution. After central cleavage, hydroquinone was formed and further transferred to 1,2,4-trihydroxybenzene. No degradation products from 1,2,4-trihydroxybenzene were detected. |
