荧光原位杂交技术对土壤石油降解菌的检测

Detection of soil petroleum-degrading bacteria with fluorescence in situ hybridization technique

从辽河油田样品中筛选出一株高效石油降解菌,经鉴定为地衣芽孢杆菌。针对其16SrRNA设计寡核苷酸探针。荧光原位杂交(FISH)技术利用寡核苷酸探针检测特定细胞内的互补核苷酸序列。通过对纯菌和泥浆中地衣芽孢杆菌的FISH进行优化,得到泥浆中地衣芽孢杆菌的荧光原位杂交实验条件:样品固定时间17h,杂交温度46℃,杂交时间3h,杂交液中去离子甲酰胺浓度35%,冲洗缓冲液中与去离子甲酰胺对应的NaCl的浓度88mmol.L-1。运用上述FISH技术监测生物泥浆反应器中地衣芽孢杆菌量的变化,并与泥浆中含油率的变化进行比较,二者的变化情况符合微生物降解石油的趋势,为监测含油污泥中微生物的变化提供了一种可行的技术。

A bacterial strain with high efficiency of degrading petroleum was screened from Liaohe oil field, and identified as Bacillus licheniformis. Oligonueleotide probe was specifically designed for the 16S rRNA of B. lieheniformis, and fluorescence in situ hybridization （FISH） technique was applied for the microbial deteetion in bioslurry. The optimal FISH test conditions for the B. licheniformis in bioslurry were fixation time 17 h, hybridization temperature 46℃, formamide concentration in hybridization buffer 35% , and NaCl concentration in washing buffer 88 mmol · L^-1. The quantitative changes of B. licheniformis in bioslurry reactor were monitored by FISH on different sampling days, and the changes of oil concentration in bioslurry were measured on the same sampling days. The two test results represented good consistency with the trend of petroleum biodegradation, suggesting the feasibility of using FISH technique for monitoring the changes of microorganisms in oil-containing sludge.