Study On Biosurfactant-Producing Bacteria From Soils In The Qinghai-Tibet Plateau And Their Ability For Petroleum Degrading

The petroleum spill has caused serious environmental risks and enormous economic losses.The bioremediation using microbes has been shown to be the most efficient way for the removal of petroleum pollution in environments.Therefore,searching noval petroleum degrading-microbes is necessary and significant to copy with oil contamination.In present study,I isolated 34 bacterial strains from the soils in the Qinghai-Tibet plateau.These strains can secrete biosurfactants in the medium adding 2%of petroleum.The analysis of 16S rRNA gene sequences was used for identification of the isolates.The gravimetric method was used to measure the petroleum degradation rate of the isolates.Colorimetric and ultrasonic methods were used for assaying the emulsifiability and emulsion stability of the biosurfactants produced by the isolates.The types of biosurfactants produced by the isolates were analysed using thin layer chromatograph?TLC?.The petroleum-degrading traits and profiles of the isolates were determined using a GC-MS method.Furthermore,some factors that affect the oil degradation rate by the isolates were explored.The main results obtained from the study are as follows.1.I isolated 34 bacterial strains from the soils in the Qinghai-Tibet plateau.These strains can produce biosurfactants in the medium adding 2%of petroleum as sole carbon source.The results of 16S rRNA gene sequences aligment showed that the isolates are assigned to Acinetobacter,Bacillus,Pseudomonas,Arthrobacter,Lysinibacillus,and Rhodococcus.2.The main kinds of biosurfactants produced by the 34 isolates are glycolipids and phospholipids.The emulsion of biosurfactants is type of O/W,which has superior emulsion stability and oil solubilization.Of the 34 strains,14 strains could degraded more than 50%of petroleum,in the medium containing 2%petroleum as sole carbon source,and cultured at 20?and 180 rpm for 7 days.Of these strains,the bacteria belong to genera Acinetobacter,Pseudomonas,and Lysinibacillus possess higher ability to degrade petroleum.For example,Lysinibacillus fusiformis 23-1 degraded 57%of the petroleum in medium.The results from GC-MS assay showed that this strain exhibited different degrading performance to the component of petroleum.The degrading rate of C₁₂-C₂₈ straight-chain saturated n-alkanes was higher than 99%,the degrading rate of C₁₁-C₂₃ branched paraffin was approximately 85%,and the degrading rate of shorter-chain n-alkane(shorter than C₁₂)and longer-chain n-alkane(longer than C₂₈)was relative low.Another strain D.maris Y35 had a superior degrading performance to C₁₄,C₁₆ alkanes and cyclohexanes,and weaker degrading ability to other alkanes and arenes.We further investigated the factors that affect petroleum degradation of strains L.fusiformis 23-1,L.fusiformis 11-4,A.calcoaceticus YF11-2,and A.calcoaceticus SY03134,and the result revealed the optimal conditions for petroleum degrading,i.e.,initial pH 7.5,culture temperature 25?,and 8 days for incubation.3.The functional genes related to petroleum biodegradation in the genomes of the isolates were detected using PCR method.The results showed that most of the isolates possess alkane hydroxylase gene alkB,flavin containing monooxygenase gene almA,naphthalene dioxygenase gene ndoB,and 2,3-catechol dioxygenase gene xylE.Some strains have cytochrome P450 gene,catechol 1,2-dioxygenase gene C12O,and catechol 2,3-dioxygenase gene C23O in their genomes.4.The expression levels of the functional genes related to petroleum biodegradation of L.fusiformis 23-1 and D.maris Y35 were detected using Q-PCR technology.C₁₈-C₃₆ alkanes significantly up-regulated the expression of alkB of L.fusiformis 23-1 52-fold after 5 h of addition.C₈-C₂₀ alkanes significantly up-regulated the expression of almA of L.fusiformis23-1 15-fold after 5 h of addition.However,C₁₄-C₃₆ alkanes down-regulated the expression of ndoB after 5 h of addition.Furthermore,C₈-C₂₀ alkanes significantly up-regulated the expression of alkB of D.maris Y35 10-fold after 5 h of addition.C₁₀-C₁₄ alkanes significantly up-regulated the expression of ndoB of D.maris Y35 1-fold after 5 h of addition.The other alkanes have no significant effect on almA expression of D.maris Y35,with the exception of C₁₆ alkane.These results might indicate that the different mechanisms of oil degradation are evolved in bacteria to copy with hydrocarbons in environment.