在第3章的研究中，應用改進式的系統化生物處理方法來整治受石油碳氫化物污染之土方，此全面性的系統化生物復育技術，將生物優植和生物刺激結合分子生物的微陣列生物晶片技術，以達到同步進行土壤監測與整治復育的過程。本研究藉由土壤翻堆的操作和再植種生物製劑(廚餘堆肥)的策略，以提高生物復育效率，達到土壤中的總石油碳氫化合物之降解。本研究包含受燃料油(Fuel oil)及柴油(Diesel)污染，兩種不同的土方來源，其一為KH-92來自一受石油碳氫化合物長期污染的高黏質土壤(Clay loam)；其二為KH-20來自另一受石油碳氫化合物近期污染的高砂質土壤(Sandy loam)。經過50天的第二階段生物復育後，兩個不同土方試驗中的N系列(Non-turning over machine，未經連續翻堆，僅以小山貓機(bulldozer)進行土方推進操作)和T系列(Turning over machine，以耕耘機(Rotavator)進行連續翻堆操作)之生物土堆，其TPHC10-C40的污染降解率分別約65％和76％，證明T系列土堆具有較好的翻堆降解成果。最後，經過198天的兩階段生物復育後，總土方KH-92與KH-20，其TPHC10-C40的污染降解率皆能達到約90% (起始濃度5,718 mg/kg-dry soil)。當在生物復育的第二階段，進行生物優植和生物刺激時，微生物濃度從約105增加到106.5 CFU/g-dry soil，同時TPHC10-C40降解率亦與微生物成長，呈現相同的增加趨勢。藉由微陣列生物晶片對受污染土壤中的微生物進行分析後，其多樣性的表現為：土壤原生菌中的主要族群是Gordonia alkanivorans，Gordonia desulfuricans，Aspergillus fumigates，Aspergillus flavus，Pseudomonas boydii及Candida tropicalis。在生物復育的過程中，兩個不同的生物反應土堆試驗中，額外進行再植種的生物優植試驗組，其呈現的微生物為Acinetobacter sp.，Pseudomonas aeruginosa及Aspergillus niger，皆為已知對於石油碳氫化合物具有高度降解能力之菌群。實場的生物整治結果呈現，經生物優植試驗的生物土堆，其殘留的TPHC10-C40濃度已降低到小於500 mg/kg-dry soil。
在第4章中，主要目的為研究土壤有機質（SOM）含量程度對總石油碳氫化物之生物降解的影響。此研究的批次試驗是用2％或10％的土壤有機質分別添加到不同的柴油或燃料油污染土壤批次中。本研究除了針對TPH C10-C40 (柴油或燃料油)降解效率進行探討外，亦結合寡核苷酸微陣列晶片技術(Microarray)和末端修飾限制片段長度多型性分析(T-RFLP)等分子生物技術，對具有降解石油碳氫化物能力的微生物群落，進行詳細的分析。此研究的整個試驗過程中，四個批次的試驗中，經過了140天的反應期間，碳氫化物的濃度TPH C10-C40從10,000減至1,849-4,352 mg/kg-dry soil。在較高的SOM含量的批次試驗中，能夠觀察到較高的生物降解效率和反應動力常數。本研究亦進行碳氫化合物的三種成分分析，在批次試驗的柴油組(B1與B3)，於試驗反應的後期，檢測到相對較低的Resin和Aromatic等片段。10％SOM的試驗中(B3與B4)，其細菌和真菌的生長量比起2％SOM的試驗(B1與B2)高出約10 CFU / g至102 CFU / g之程度。並且在批次試驗中，其TPH降解率達到最低時，該批次試驗的真菌量亦為最低。利用ITS微陣列技術檢測出優植入的土壤微生物並證明其存活。藉由分生技術所呈現的微生物之生長趨勢證明，隨著反應時間的進行，不同種類的污染油品(柴油與燃料油)會影響微生物族群的多樣性。在不同的批次試驗中，微陣列晶片和T-RFLP的分析結果，皆顯示Gordonia alkanivorans，Gordonia desulfuricans和Rhodococcus erythoropolis是所有試驗中的優勢細菌；另外，在真菌族群中，Fusarium oxysporum與Aspergillus versicolor則是所有試驗中的優勢微生物。利用T-RFLP的數據進行非度量多維度分析(NMS)的統計結果說明，微生物族群的動態會受到不同階段的TPH降解趨勢所影響。
在第5章中，研究目的為比較各種不同比例的柴油與生質柴油混合的化合物所模擬的土壤污染試驗，以及利用生物復育技術，將7種證實具有柴油降解能力的細菌進行生物優植至模擬的污染土壤中。本研究提出的復育方法證實，對於土壤污染的試驗，達到80％-99％的污染物降解。對照組試驗（CT）中，其TPHd降解效率（％）和動力參數（k）的結果顯示，比生物優植試驗（BA）相似或更高。與BA相比，含20％和50％生質柴油的批次試驗(CT-20與CT-50)，結果顯示，CT試驗中的TPHd降解率高於BA試驗的10％。因此，說明TPHd的降解率藉由生質柴油含量的增加而提高。例如，本研究證明，在100％生質柴油污染的土壤中(CT-B100 and BA-B100)，TPHd的降解率達到了99％。在不同程度的試驗中皆顯示，生質柴油的增加亦會使總異營菌總量，從107提高到109 CFU / g-dry soil。分生檢測的結果顯示，Gordonia alkanovorans 與Gordonia desulfuricans為原生族群中的優勢菌。Pseudomonas aeruginosa則為整個復育過程中，能夠耐受環境並優勢存活的微生物。在研究的初期中，細菌生長的趨勢顯示，其微生物剛植入的階段才有效果。在進行土壤復育之前，如果能利用合適的分生技術，來鑑別出土壤中具有降解能力的微生物，將更有助於降低整治的成本，並決定最佳的復育策略。

An improved systematic bioremediation method combine bioaugmentation, biostimulation and a biomonitoring with biomolecular microarray chip, which was developed as an integrated bioremediation technology. It was applied to treat total petroleum hydrocarbon (TPH) in soil by using the landfarming operation with a reseeding strategy to enhance the bioremediation efficiency. After 50 days of the 2nd phase bioremediation process, N- and T-series fuel oils (TPHC10–C40) were degraded up to about 65% and 76%, respectively. Finally, the overall period (198 days), KH92 and KH-20 biopiles exhibit approximately 90% TPHC10–C40 removal. When bioaugmentation and biostimulation were applied in the second phase of bioremediation, the microbial concentration increased from approximately 105 to 106.5 CFU/g dry soil along with an increase in TPH biodegradation. Analysis of the microbial diversity in the contaminated soils by microarray biochips revealed that Gordonia alkanivorans, Gordonia desulfuricans, Aspergillus fumigates, Aspergillus flavus, Pseudomonas boydii and Candida tropicalis were the predominant groups in indigenous consortia; the groups in the augmented consortia were Acinetobacter sp., Pseudomonas aeruginosa and Aspergillus niger in both series of biopiles during bioremediation. Field experimental results showed that the residual TPH concentration in the complex biopile was reduced to less than 500mg TPH/kg dry soil.
In chapter 4, The purpose of this study is to investigate the effect of soil organic matter (SOM) content levels on the biodegradation of total petroleum hydrocarbons (TPH). Batch experiments were conducted with soils with 2% or 10% organic matter that had been contaminated by diesel or fuel oil. In addition to the TPH (diesel or fuel oil) degradation efficiency, a comprehensive investigation was conducted on the TPH-degrading microbial community using molecular tools including oligonucleotide microarray technique and terminal restriction fragment length polymorphism analysis (T-RFLP). TPH was reduced from 10,000 mg/kg to 1,849-4,352 mg/kg dry weight soil. Higher biodegradation efficiencies and kinetic rate constants were observed in higher SOM contents. Hydrocarbon fractional analyses were conducted to explain the optimal operation with relatively low resin and aromatic fractions detected at the end of the remediation. The bacterial and fungal counts in the 10% SOM were approximately 10 CFU/g to 102 CFU/g above those in the 2% SOM, and the lowest fungal level was found when the least TPH degradability was measured. The internal transcribed spacer microarray identified the microorganisms that were introduced and proved their survival. The associated growth pattern confirmed that different kinds of contamination oils affected the microbial community diversity over time. Both the microarray and T-RFLP profiles indicated that Gordonia alkanivorans, Gordonia desulfuricans, and Rhodococcus erythoropolis were the dominant bacteria, while Fusarium oxysporum and Aspergillus versicolor were the dominant fungi. The T-RFLP-derived nonmetric multidimensional scaling concluded that the dynamics of the microbial communities were impacted by the TPH degradation stages.
In chapter 5, the objective is to investigate various percentages of diesel-biodiesel mixtures caused contamination and remediation approaches, including bioaugmentation with seven proved diesel-degrading bacterial species. Degradation of 80%-99% was achieved with the proposed remediation approach. The TPHd degradation efficiency (%) and rates (k) in the control batch (CT) found either similar or superior to the bioaugmentation batch (BA). The batches with 20% and 50% biodiesel achieved 10%improvement of TPHd degradation in CT over BA. The TPHd degradation was enhanced by the increase of biodiesel. For example, degradations of 99% were achieved in the soil polluted with 100% biodiesel. The increase of the biodiesel enlarged the total heterotrophic bacterial counts from 107to 109CFU/g dry soil. The molecular data concluded Gordonia alkanovorans and Gordonia desulfuricans were most dominant in the indigenous community. Pseudomonas aeruginosa was a strong survivor last for the entire remediation. The growth patterns of bacteria indicated the introduced species were useful only in the beginning. Using the recommended molecular tools to identify the useful bacteria prior to a remediation project would be helpful in reducing the cost and determine a wise remediation strategy.

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