| Abstract: | 酚在工業上是常見的污染之一，其苯環結構穩定需要外力或者為生物作用，才能將其開環，成為長鏈之結構以便後續分解程序，生物降解因較具經濟上的優勢，故可選用作為土壤酚污染整治最佳選擇之一。本實驗自加油站排水溝周遭收集的汙泥或土壤，以批次培養方式，篩選出耐高濃度之酚降解菌E1、E3、E7、G2、G4、H1、H3、H4，B1和B3，並對篩選出來的純化菌株進行革蘭氏染色，及鄰苯二酚加氧酶活性測試。結果顯示10株菌株具有鄰苯二酚1,2-二加氧酶活性，經過16S rDNA定序比對，E1為Bacillus sp.NRRL B-149911相似度94%，E3為Bacillus sp.NRRL B-149911相似度95%，G2為Pseudmonas stutzeri A11501相似度99%，H1為Staphylococcus warneri L37603相似度98%，B1為Geobazillus sp.相似度為92%，B1為Bacillus pumilus ATCC7061相似度98%，E7、G4、H3和H4都為Rhodococcus opacus B4相似度94%。利用NCBI及MEGA軟體分析，將菌種製作成親緣樹狀圖，由16S rDNA親緣樹狀圖中顯示菌種分成兩大族群。
再者，選取五株降解效率較好之實驗菌株接種於模擬污染土壤中，進行批次生物降解實驗，監測實驗過程酚濃度之變化，並以平板計數法測定酚降解菌之菌數，同時進行土壤DNA萃取，經引子341GC+534r做PCR反應，以變性梯度膠體電泳分析，藉此追蹤實驗過程中菌株之消長變化。實驗發現接種試驗菌株於模擬污染土壤中均具有良好降解效果，且在3號土壤(粘板岩沖積壤土)中有較好的降解效果。應用變性梯度膠體電泳分析，菌株指紋圖譜亮帶隨著降解時間增加而增加，間接表示菌落數的增加，顯示變性梯度膠體電泳分析比傳統培養方式更具敏感。
Phenol is a common pollutant with a stable benzene ring from industries, it impacts on the aquatic ecosystem and environmental balance extremely. Biodegradation is a very economic technique leading to the cleavage of benzene ring, which is the best choice of the phenol contaminated soil remediation. The phenol high-tolerance degraders were isolated from collected sludge and soil from petroleum station, the isolated strains in this study including E1, E3, E7, G2, G4, H1, H3, H4, B1 and B3. The physiological properties of tested strains were analyzed by Gram stain, activity of catechol dioxygenase. The results showed that all tested strains owned the activity of catechol 1,2-dioxygenase (C12O). The E1 and E3 were showed 94 and 95 % probability of Bacillus sp. (NRRL B-149911), respectively. H1 was showed 98% probability of Staphylococcus warneri L37603, B1 was showed 92% probability of Geobazillus sp., B3 was showed 98% probability of Bacillus pumilus; E7, G4, H3 and H4 were showed 94% probability of Rhodococcus opacus. Phylogenetic analysis of the test strains was identified by 16S rDNA gene fragment from NCBI database and MEGA software. The result indicated that the test strains are clustered in 2 subgroups from the phylogenic tree constructed by 16 S rDNA.
Inoculation of five effective degraders to the test soil suspensions spiked with phenol was carried out by batch culture. The phenol concentration and the number of phenol degrader were monitored in the experimental period, soil DNA was extracted and amplified by primer 341GC+534r of PCR for DGGE analysis at the same time to understand the growth variation of test strains. The results showed that the effective biodegradations inoculated by test strains were performed in the polluted soils. Tracing soil bacterial community by DGGE-PCR technology in the experimental period was observed that inoculated effective strains became dominant population, and this result was similar to the colony forming unit (CFU) was counted by plate-count methods. The denaturing gradient gel electrophoresis (DGGE) is considered one of the most sensitive scanning techniques for soil community. |
