以硫酸還原菌厭氧生物降解 Fluorene 與 Phenanthrene 之研究

Abstract

多環芳香烴 (polycyclic aromatic hydrocarbons, PAHs) 廣泛地分佈在環境中。由於它們具有毒性、頑抗性、致癌性及致突變性，使得 PAHs 的去除/生物降解是相當重要的研究議題。在這些 PAHs 當中，三環的 fluorene (FLU) 及 phenanthrene (PHE) 是污染場址中最為常見且大量存在的污染物。許多的研究已證實 FLU 及 PHE 可在硫酸還原條件下或是被硫酸還原菌 (sulfate-reducing bacteria, SRB) 降解。然而，在硫酸還原環境下所產生的代謝產物、代謝途徑以及最適化生物降解條件仍不十分明確。為了進一步取得 FLU 與 PHE 代謝的降解動力學參數、代謝機制、最適化操作條件以及代謝產物與主要基質之間的影響等資訊，本研究以 SRB 為優勢菌種 (87 ± 6%) 進行一系列水相的批次實驗。

在生物降解能力 (biodegradability study) 研究中，主要是探討 SRB 對 FLU (5 mg/L) 與 PHE (5 mg/L) 個別存在時以及混合在一起時 (每個化合物添加濃度為 5 mg/L) 的降解能力。實驗結果顯示經過 21 天的反應後，SRB 可降解 88% 的 FLU 與 66% 的 PHE。然而，當 FLU 與 PHE 同時存在時，生物降解效率則呈現下降的現象。綜觀所有實驗可發現硫酸鹽濃度的下降伴隨著微生物的成長以及 FLU 與 PHE 的生物降解，顯示 SRB 是分解此兩種化合物主要的微生物族群。此外，由氣相層析質譜儀鑑定的結果可確認酚 (phenol) 為 FLU 的代謝中間產物；2-甲基-5-羥基苯甲醛 (2-methyl-5-hydroxybenzaldehyde)、1-丙烯基苯 (1-propenyl-benzene)、對-甲酚 (p-cresol) 以及酚為 PHE 的代謝中間產物。由這些所鑑定的代謝產物可推論水合 (hydration)、水解 (hydrolysis) 、去氫作用 (dehydrogenation) 與去碳酸基作用 (decarboxylation) 為 FLU 與 PHE 生物降解的代謝機制，並且提出以 SRB 分解 FLU 及 PHE 的新代謝途徑。

在最適化研究 (optimization study) 中，是以可旋轉性中央合成設計法 (rotatable central composite design, RCCD) 進行實驗設計。PHE 濃度的設計範圍為 2 至 50 mg/L；硫酸鹽濃度的設計範圍為 480 至 3360 mg/L；微生物濃度的設計範圍為 5 至 50 mg/L。實驗結果指出微生物濃度是最重要的影響因子，其次是硫酸鹽濃度與 PHE 的濃度。本研究利用望想函數法 (desirability functions methodology, DFM) 找出 PHE 的最大比去除率 (maximum specific PHE removal rate, Rs)。在實驗設計的範圍內，當初始的 PHE、硫酸鹽以及微生物濃度分別設定為18.5、841 與 50 mg/L 時可得到最大的 Rs (9.0 mg/g VSS-d)。最適化實驗中所觀察到的最大 Rs 較過去相關研究來得大，證明本研究所馴養的 SRB 對於 PHE 有較佳的生物降解能力。隨後，在此最適化條件下進行驗證實驗 (confirmation experiment)，其結果相當符合 DFM 所估計的最大 Rs。此外，吸附實驗 (Adsorption experiment) 的結果顯示 PHE 的生物吸附與其初始添加濃度成正比。當 PHE 被添加至菌液時，大量的 PHE 會立即被吸附在微生物上，而此被吸附的 PHE 可進一步被 SRB 降解。

最後，在代謝產物生物降解研究 (metabolite biodegradation study) 中，分別進行酚與對-甲酚生物降解實驗 (添加濃度為 5 與 10 mg/L) 以確定 SRB 是否可利用它們作為碳源及能量來源。另一個實驗則是同時添加酚、對-甲酚以及 PHE (每個化合物添加濃度為 5 mg/L) 以探討基質間的交互影響，此外，也評估酚與對-甲酚的存在與累積對於降解 PHE 的影響。實驗結果顯示對-甲酚可被快速地分解且沒有遲滯期。添加 5 mg/L 對-甲酚的實驗組在 21 天內有 88% 的降解；添加 10 mg/L 對-甲酚的實驗組有 65% 的降解。然而，酚則沒有明顯的降解。同時存在酚、對-甲酚以及 PHE 的實驗組結果顯示酚與對-甲酚的降解較添加單一化合物實驗組來得高。但是酚與對-甲酚的存在則會稍微地抑制 PHE 的降解。

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed over the environment. Due to their toxicity, recalcitrance, carcinogenicity and mutagenicity, the removal/degradation of PAHs is one of the most important research issues. Among the PAHs, three-ring PAHs i.e., fluorene (FLU) and phenanthrene (PHE), are the most frequent and abundant pollutants found in the contaminated sites. FLU and PHE degradations are reported under sulfate-reducing conditions or by sulfate-reducing bacteria (SRB). However, the metabolites produced, metabolic pathway(s) and the optimal biodegradation conditions under sulfate-reducing environment are not well understood. In order to obtain and understand the FLU and PHE metabolisms such as degradation kinetics, metabolic mechanisms, optimal operating conditions and the interactions between metabolites and parent compound, a series of aqueous batch experiments were conducted using a sulfate reducing bacterial enrichment culture (87 ± 6%).

In the biodegradability study, batch experiments were conducted with FLU (5 mg/L), PHE (5 mg/L) and a mixture of the two (5 mg/L each). After 21 d of incubation, 88% of FLU and 65% of PHE were degraded by SRB. However, a decrease in biodegradation efficiency was observed in the presence of both FLU and PHE. Throughout the study, sulfate reduction was coupled with biomass growth and PAH biodegradation indicating that SRB were the major group of microorganisms responsible for the degradation of FLU and PHE. Using the GC-MSD analysis, phenol was identified as the metabolite of FLU and 2-methyl-5-hydroxybenzaldehyde, 1-propenyl-benzene, p-cresol and phenol were identified as the metabolites of PHE. These metabolites infer that hydration, hydrolysis, dehydrogenation and decarboxylation are the mechanisms responsible for the biodegradation of FLU and PHE. Based on the observations, novel metabolic pathways of FLU and PHE by the enriched SRB were proposed.

In the optimization study, batch biodegradation experiments were designed using the rotatable central composite design with five levels. The designed concentrations were 2-50 mg/L for PHE, 480-3360 mg/L for sulfate, and 5-50 mg/L for initial biomass. Experimental results indicated that the biomass concentration was the most significant factor, followed by the sulfate and PHE concentrations. In the present study, the desirability functions methodology (DFM) was applied to find out the maximum specific PHE removal rate (Rs). A maximum Rs of 9.0 mg/g VSS-d was estimated when the initial PHE, sulfate and biomass concentrations were 18.5, 841 and 50 mg/L, respectively. The Rs observed in the optimization experiments was higher than the values reported in the previous studies demonstrating the superiority of the enriched SRB culture for the biodegradation of PHE. Subsequently, a set of confirmation experiments were performed under the optimal PHE, sulfate and biomass concentrations i.e., 18.5, 841 and 50 mg/L, respectively; the results matched well with the Rs estimated using DFM. In addition, the results of adsorption experiments exhibited that the adsorption of PHE on biomass was proportional to the initial concentration of PHE. When PHE was added in the biotic system, large quantity of PHE was adsorbed in the biomass instantly and the sorbed PHE was further degraded by the SRB.

Finally, in the metabolite biodegradation study, the ability of sulfate-reducing enrichment culture for the utilization of phenol and p-cresol (5 and 10 mg/L) as carbon and energy sources was examined. In another experiment, the phenol and p-cresol were incubated simultaneously in the enrichment culture with PHE (5 mg/L each) to study the substrate interactions during the biodegradation process and to assess the potential effect of presence and accumulation of phenol and p-cresol on PHE degradation. Experimental results demonstrated that p-cresol was rapidly degraded without a lag phase. About 88% and 65% p-cresol degradations were reached within 21 d for the experiment at 5 mg/L and 10 mg/L of p-cresol, respectively. However, no significant degradation of phenol was recorded. The outcomes of PHE biodegradation experiments in the presence of phenol and p-cresol show that the degradation of phenol or p-cresol is higher than their degradations in the single compound system i.e., system added with only phenol or p-cresol. Nevertheless, the presence of both phenol and p-cresol in the system slightly inhibited the degradation of PHE.