本研究以甲苯為生長基質，誘導純菌Pseudomonas putida F1產生甲苯雙氧加氧酵素 (Toluene dioxygenase)，同時量測菌體的生長特性與甲苯分解動力，並就菌體附著於纖維床(Fibrous-bed)的特性進行研究，此外，將附著P. putida F1菌體之纖維床，應用於生物濾床技術與共代謝生物反應器(Cometabolism bioreactor)，並分別探討氣相甲苯污染物的祛除效果與共代謝分解液相三氯乙烯之特性。 實驗結果顯示，藉由供給氣態甲苯的方式，進行菌體培養，P. putida F1可持續獲得碳源，且不會發生高濃度甲苯毒性抑制的現象。此外，纖維床附著菌體的機制包括棉纖維的截留、棉纖維與菌體間的氫鍵作用及胞外聚合物的生成，且附著的位置主要為棉纖維非孔隙的部份。而值得注意地，在本研究中，菌體在纖維床上具有良好的附著能力，附著菌體量可達95000g VSS/m3，且80%以上的比例為胞外聚合物。此外，以生物滴濾床與浸沈式生物濾床操作，當進流甲苯負荷分別在70 g/m3/h與150 g/m3/h以下時，兩者操作方式均能維持90%以上的甲苯祛除率，且浸沈式生物濾床的操作尚具有避免阻塞的功能。 纖維床生物反應器以批次操作的方式，分批共代謝祛除液相三氯乙烯，當起始三氯乙烯濃度介於2.4~47.6 mg/l之間時，由於纖維床具有較高的菌體附著量，三氯乙烯的分解作用能符合擬一階反應動力，且溶氧濃度介於1~10 mg/l時，分解三氯乙烯之擬一階速率常數值，會隨溶氧濃度增加而提高。在操作的過程中，若每30min添加0.005% (w/v)的過氧化氫，即能持續維持8-9 mg/l的溶氧濃度，在4 h的操作時程內，三氯乙烯的祛除率可達98%。另外，纖維床上P. putida F1生物膜共代謝分解三氯乙烯的轉化容量(Transformation capacity)達0.26 mg/mg，且在18h的操作時程內，三氯乙烯祛除率僅由98%略微降低至93%，故毒性代謝產物對於纖維床上菌體的抑制作用應可忽略。由於三氯乙烯與甲苯雙氧加氧酵素之間具有較佳的親和力，因此，甲苯對於三氯乙烯所產生的競爭性抑制作用並不顯著，當起始甲苯濃度在95 mg/l以下時，於4 h的操作時程內，三氯乙烯祛除率仍能夠維持在90%以上。 This study was aimed to develop a novel fibrous-bed bioreactor (FBR) for removing toluene and trichloroethylene (TCE). The attached specificity of Pseudomonas putida F1 was investigated for the fibrous-bed when toluene was the carbon source. In addition, the FBR were operated at the biofiltration processes to evaluate the performance of removing toluene vapor. The FBR was also used as the co-metabolism bioreactor to study the co-metabolic degradation of TCE in contaminated solution. Results indicated that the culture of cells with the supply of toluene vapor was an acceptable way to obtain high specific-growth rate because the carbon source was supplied continuously without toxic inhibition of high concentration toluene. Moreover, the study proposed that the attached site of biomass was primarily on the surface of cloth fiber. The biomass could attach into the fibrous-bed by means of the entrapment of cloth fiber, hydrogen bonds between the biomass and fibrous matrix, and generation of extracellular polymeric substance (EPS) matrix. Additionally, the fibrous-bed exhibited superior ability for cells attachment. The attached biomass approached 95000 g VSS/m3, and over 80% of biomass was the EPS. The removal of toluene vapor was over 90% when FBR was operated at a trickling filter and the inlet loading was below 70 g/m3/h. However, when FBR was operated in the mode of submerged biofilter, the removal of toluene was greater than 90% when inlet loading was below 150 g/m3/h. Also, the operation of submerged biofilter avoided the problem of clogging. Furthermore, the FBR was operated in the mode of the sequential batch. Attached biomass of fibrous-bed utilized toluene to induce toluene dioxygenase (TDO) and co-metabolized TCE. Because the FBR has attached great amount of biomass, the degradation of TCE followed the first order rate equation even the TCE concentrations was as high as between 2.4 mg/l and 47.6 mg/l. This rate constant also increase with the increase of dissolved oxygen in the range of 1.0 and 10 mg/l. Adding 0.005%(w/v) of hydrogen peroxide per 30 min was an efficient way to sustain 8-9 mg/l of dissolved oxygen in this reactor. The removal of TCE was 98% within 4 h of operation period. Moreover, the transformation capacity of TCE co-metabolism was 0.26 mg/mg by P. putida F1 biofilm. The inhibition of toxic metabolite was neglected within 18 h of operation period because the removal of TCE decreased somewhat from 98% to 93%. Also, the competitive inhibition of toluene degradation was limited on co-metabolism of TCE. The removal of TCE was still over 90% within 4 h of operation period when the initial toluene concentration was below 95 mg/l.
