| 摘要: | 土壤環境中多環芳香烴化合物 (PAH) 之生物復育經常利用界面活性劑淋洗技術。過程中化學傳輸行為與生物分解會同時發生，此時生物分解PAH之作用受界面活性劑之影響而發生促進或抑制現象。本研究選用不含土壤有機質黏土-鈣蒙特石與含有機質(1.883 %)土壤-台中土，及Triton X-100與Brij 35兩種非離子界面活性劑，利用自PAH污染土壤中篩選之微生物與分解PAH之體外酵素laccase分解含不同界面活性劑之水溶液系統與土壤/水系統中之PAH，探討PAH在不同界面活性劑-土壤組合之傳輸行為對生物分解的影響，及分解過程土壤中與溶液中微生物族群之結構與生理生化反應，並探討可能之代謝途徑。 水溶液系統中，PAH之分解速率受到PAH結構之影響，系統中存在之界面活性劑亦可被微生物所利用，成為微生物的碳源。PAH與界面活性劑兩分子間之分子內聚能 (cohesive energy) 可用於解釋PAH與界面活性劑之生物分解。兩分子之內聚能愈接近，代表兩者間之分子鍵結形式相同者愈多，系統中微生物分泌之體外酵素於分解PAH與界面活性劑過程可相互周轉利用，PAH與界面活性劑之分解速率因此會加快。在土壤/水系統中，因受鈣蒙特石顆粒對微生物之干擾作用及PAH與界面活性劑於台中土發生之分佈作用影響，其微生物分解速率大多較水溶液系統慢。當PAH-界面活性劑-土壤/水系統之組合不同時，PAH與界面活性劑之分解速率也不同，並且系統中所含之界面活性劑濃度與結構不同也會造成影響。在微生物族群數量與結構方面，水溶液系統或土壤/水系統之自由態總生菌數都有增加之趨勢，其中假單胞菌屬 (Pseudomonas sp.) 為分解過程之優勢菌，不過所增加之數量受到系統不同PAH種類與土壤性質之影響。Domain Bacteria，phylum/subclass之 α-, β-, γ-Proteobacteria族群於分解過程佔有極高比例，且不受到添加界面活性劑之影響。Brevundimonas (Pseudomonas) diminuta, Caulobacter sp.、Mycoplana bullata, Burkholderia sp.、Pseudomonas aeruginosa等特定族群於水溶液系統或土壤/水系統之自由態佔有高比例，用於負責分解PAH。此外，分解過程之族群生理圖譜 (community-level physiological profiling, CLPP)、代謝潛能與對Biolog之碳源利用率、API ZYM水解酵素反應、代謝途徑等也因組合不同而發生改變。 胞外酵素laccase分解PAH方面，實驗結果顯示，水溶液系統含有Triton X-100與Brij 35之溶液中laccase皆促進PAH的分解作用，又因微胞比單體為更有效之分佈介質，使得含微胞相時的溶液能促使生物分解速率加快。另外由於Brij 35碳氫鏈較Triton X-100碳氫鏈長許多，形成之微胞結構較大，造成較大的立體結構障礙，故含微胞之Triton X-100溶液對PAH分解速率大於Brij 35。鈣蒙特石/水系統中兩種界面活性劑仍具促進分解作用，且酵素可直接利用溶於水溶性微胞與吸附性微胞之PAH，使得當平衡溶液含界面活性劑微胞時之分解速率大於平衡溶液含單體者。分別比較鈣蒙特石/水系統中附著態與自由態分解情形，因PAH與酵素在自由態之質傳速率大於附著態，使得分解行為在自由態中較明顯。進一步將酵素固定在鈣蒙特石上，分解不同界面活性劑溶液的PAH結果顯示，添加Triton X-100有促進分解作用，但添加Brij 35卻有抑制分解之現象，推測原因為鈣蒙特石對Brij 35之吸附量較TX-100少，PAH多進入溶液中微胞，反而阻礙固定化酵素之作用。 Combined bioremediation with surfactant flushing system is believed to be an important process to remove polycyclic aromatic hydrocarbon compounds (PAH) in soil/water systems. Many chemical reactions were generated in the integrated process so as to affect biodegradation. The PAH’s hydrophobicity results in these compounds being strongly sorbed onto soils. The use of surfactants may change the sorption behavior of PAH in soil environment. The aim of this study was to evaluate the fate impacts on PAH biodegradation in soil/water systems with nonionic surfactants. The ability of PAH-biodegraders and fungal laccase in degrading PAHs, naphthalene and phenanthrene, was analyzed. The nonionic surfactants tested were Triton X-100 and Brij 35. Soils selected were composed of a clay (Ca-montmorillonite) and a natural soil (Taichung soil). Bacterial community and physiological profile of free-state and attached microorganisms were determined during biodegradation. A comparative study was also carried out by application of free and immobilized laccases in the soil-water system. In aqueous, PAHs were mineralized completely but the biodegrading rate was affected by the structure complexity of PAH. PAH-biodegrading bacteria enabled to utilize nonionic surfactant as carbon source in systems. The influence of surfactant additives on PAH biodegradation was successfully evaluated using chemical molecular interaction method, based on the theory of cohesive energy density (CED). Results from this study suggested, PAH have a relatively higher CED value because aromatics compounds with labile π are more polarized to prompt molecular attractions by the induced dipole force. Under different PAH-surfactant compositions, similar CED values are related to facilitate their intermolecular attractions through π-π electron interactions to represent a similar biodegradation pattern. Extracellular enzymatic activity measurements revealed that when induced enzymes targeted same molecular bonding on PAH and surfactant, rapid PAH degradation rate was observed. In soil/water systems, PAH biodegradation was influenced by the composition of PAH-surfactant-soil/water systems. Particle size of Ca-montmorillonite and the partition of surfactant on Taichung soil played an important role. Rate of biodegradation also was found to be affected by the distribution of PAH in the monomer or micelle surfactant bulk. For bacterial number and diversity, Pseudomonas sp. was dominant during biodegradation although their numbers were affected by PAHs and the soil composition. α-, β-, γ-Proteobacteria of Domain Bacteria had a high percentage. Especially Brevundimonas (Pseudomonas) diminuta, Caulobacter sp., Mycoplana bullata, Burkholderia sp., Pseudomonas aeruginosa took advantage in aqueous or free state in soil/water systems. Moreover, community-level physiological profiling (CLPP), carbon degradation potential, Biolog carbon utilization, API ZYM enzymatic activities even metabolic pathway were alternative in different PAH-surfactant-soil/water systems. Addition of Triton X-100 and Brij 35 enhanced the biodegradation of aqueous PAH by free laccase. When micelles existed in water systems, PAH biodegradation was greater than that of below critical micelle concentration (CMC). The same results were also found in the Ca-montmorillonite-water system. The phenomena can be ascribed to more amount of PAH partition on into micelles than that of monomers. Micellar phase were to provide microorganisms for extra phase to biodegrade PAH effectively. To compare PAH biodegradation in difference phase, the rate in the aqueous phase was higher than that in the soil phase. On immobilized laccase systems, an inhibited biodegradation in the presence of Brij 35 was observed, and an opposite effect presented in the presence of Triton X-100. Different phenomenon in bioavailability may correlate with smaller Brij 35 sorption on Ca-montmorillonite. PAH was easily into the aqueous Brij 35 micelles, instead of interceptions with immobilized laccase. |
