Screening And Characterization Of Cold-adapted Petroleum Degrading Bacteria For Petroleum Degradation

According to the main problem in bioremediation of oil polluted soil,low biodegration of oil,this study was intended to screen cold-adapted bacteria for crude oil degrading,to explore construction of artificial microfloras for rapid oil degradation in reactors,to monitor the change of CO₂ and O₂ concentrations online in reactors for evaluation of oil degradation effect,to explore and optimize the parameters and conditions for remediation of oil-contaminated soil in situ.Seven bacterial strains of Gram-negative bacteria,which could grow with crude oil as sole carbon source at 15?,were screened from the oil-contaminated soils by enrichment at low temperatures,and identified as Arthrobacter sp.JLH 001,Acinetobacter baumannii JLH002,Pseudomonas fragi JLH 003,Ochrobactrum sp.JLH 004,Acinetobacter calcoaceticus JLH 005,Arthrobacter sp.JLH 006,Rahnella sp.JLH 007 respectively,according to the 16S rDNA sequence results.Three of isolated strains grew well at 15?and 25?than at 37?,the other fours grew with little difference at three temperatures.So,the growth temperature characteristics of seven strains show that the seven crude oil degrading bacterial strains are all cold-adapted bacteria.The degradation characteristics of cold-adapted bacteria with hexadecane,cyclohexane and phenanthrene as type compounds of petroleum hydrocarbons were analyzed.At 15?,seven bacteria showed little difference in the degradation rate of cyclohexane after 7 days,with about 55%.JLH 001,JLH 002 and JLH 006 showed strong degradation abilities for hexadecane,more than 20%degradation rate.JLH 003 showed 40%degradation rate of phenanthrene,and except for JLH 004 and JLH 007,the other four bacteria showed high degradation rate of phenanthrene,more than 25%.At 15?,the culture surface tension of JLH001,JLH 003,JLH 004 and JLH 006 were decreased significantly after 15 days,and this indicated that the four strains showed high biosurfactant production.Meanwhile,there were no significant effects on their growth in crude oil mediums after rhamnolipid added.However,the culture surface tension of JLH 002,JLH 005 and JLH 007 showed little change,and their growth was significantly promoted in crude oil mediums after rhamnolipid added,and this suggested that their abilities of producing biosurfactant were poor.Based on degradation characteristics of seven cold-adapted strains for crude oil,six groups of microfloras for crude oil degrading were constructed.When six microfloras cultured with crude oil mediums in 5 L fermentor,the change of CO₂ and O₂ concentrations in the degradation process were monitored online by a gas analyzer to evaluate crude oil degradation abilities of microfloras.The most efficient microflora CST 111,consisted of Arthrobacter sp.JLH 001,Acinetobacter baumannii JLH 002,Pseudomonas fragi JLH 003and Arthrobacter sp.JLH 006,showed maximal O₂ consumption and CO₂ production after 48h and completely degrading 1%crude oil after 72 h at 15?,and when the temperature was imcreased to 25?or 300 mg/L rhamnolipid added,the crude oil degradation by this microflora was accelerated.In conclusion,7 oil-degrading cold-adapted bacteria were screened and identified.After analysis of degradation characteristics and optimization of degrading parameters,the petroleum-degrading cold-adapted microfloras were set up.The microfloras could efficiently degrade crude oil in reactor and could be applied for bioremediation of oil-contaminated soil in situ.In other words,bioremediation of oil-contaminated soil in situ could be performed efficiently and rapidly by the addition of oil-degrading cold-adapted microfloras.