Study On Composting Remediation Of Oil Contaminated Soil In Daqing Oil Field And Structure Of The Microbial Community

Composting serves as an important technique in the remediation of oil contaminated soil.Microorganisms make a major contribution to degradation of petroleum hydrocarbon contaminants during composting.Unfortunately,the indigenous microorganisms in oil are so rich and versatile under different ecological conditions that we've had limited success in the investigation of the microbial community structure in composting(especially in enhanced composting)and mechanisms of the degradation of petroleum hydrocarbons by enhanced composting.On the basis that studies were conducted on screening of microbial flora for effective hydrocarbon degradation and bacteria for producing rhamnolipid surfactants from contaminated soil in Daqing Oilfield,this paper further explored the remediation of contaminated soil in Daqing Oilfield by the composting supplemented with nothing,surfactants or mixed flora.In this paper,the high-throughput 16 s r DNA sequencing technique for bacterial identification was used to monitor microbial community structure during composting and investigate the association between hydrocarbon degradation and microbial flora;and a functional gene Alk B(alkane monooxygenase)clone library was constructed to analyze the versatility and structural changes of degradation microbial flora in different phases of composing;fluorescence quantitative PCR of Alk B gene was carried out to study the population of flora capable of degrading major petroleum compounds,the dynamic characteristics and associations during the hydrocarbon degradation process.The research can not only elucidate the mechanism of microbial ecology in the oil degradation by composting,but also provide the basis for improving the efficiency of remediation of the environment polluted by petroleum.The major research results are presented below:Section one was directed towards screening a heat-intolerant hydrocarbon-degrading bacterial group named wt-3 from the oil contaminated soil in Daqing Oilfield and using 16 S r DNA to determine the dominant flora to be the genus Bacillus,Pseudomonas,Rhodococcus,Pseudoxanthomonas and Lysinibacillus.During the composting with wt-3,the highest degradation rate was 75.3% under the optimum degradation conditions such as the temperature(45°C),initial p H(7),inoculation amount(3%),rotational speed of the shaker(160 rpm)and concentration of oil in the medium(2%).The analysis of wt-3 by column chromatography showed that the appropriate degradation rate reached 83%,50%,25% and 30% respectively,indicating that the mixed flora could effectively degrade petroleum contaminants.In the second section,the high-yielding surfactant strain,wk-4,was screened from oil contaminated soil in Daqing and determined as Pseudomonas aeruginosa by the physiological and biochemical identification,and 16 s r DNA sequencing technique.The optimal fermentation conditions for producing surfactants were as follows: soybean oil as the carbon source(10 g/L),ammonium chloride as the nitrogen source(5 g/L)and the fermentation time(120 h).The yield of the glycolipid surfactant reached 0.81 g/L,at a relatively higher level than previously reported in the literature.Thin layer chromatography analysis of the active surfactants identified glycolipids which was then purified by silica gel column chromatography and collected to be a mixture of mono-rhamnolipid and di-rhamnolipid.Therhamnolipids had a critical micelle concentration of 50 mg/L,and a tolerance to salt concentration below 6%,p H range of 3.0-12.0 and temperature of 20°C-100°C,which could render them to be suitable for composting research.A significantly higher degradation rate was observed with rhamnolipids at a concentration of 3 CMC in the in-situ remediation of petroleum contaminated soil,up to 67.4%.In the third section,the three composting methods had an effective impact on the degradation of total petroleum hydrocarbons(TPH),among which the degradation rate of the two enhanced compostingwas higher than that of natural composting.The rate for natural composting,composting supplemented with surfactants,with mixed flora reached 69%,88% and 80% respectively towards the end of 42-day composting period,and the half-lives of petroleum degradation were 23 d,14 d and 18 d,respectively.The population of bacteria in all stages produced by the enhanced composing clearly outnumbered that by the natural composting,up to 9 orders of magnitude rather than 8 by the natural one.In addition,the thermophilic phase lasted for over 11 days in the enhanced composting but only 7 days in the naturalcomposting,illustrating that the two enhanced composting processes could improve the metabolism and growth of the microorganisms to enhance the reaction efficiency.Determination of TPH also revealed that the succession of degradation of TPH varied with different composting processes and the highest rate of degradation occurred in the curing phase in natural composting,in the cooling phase in composting added with surfactants and in the thermophilic phase in composting added with mixed florarespectively.The petroleum degradation processes were all consistent with the first order reaction kinetic equation.GC-FID analysis of fractions of TPH showed that the two kinds of enhanced composting processes could degrade different dominant compounds of crude oil;and the surfactant was more favorable for the degradation of alkanes while the heat-intolerant oil degrading bacteria could better accelerate the degradation of aromatic hydrocarbons.In section 4,high-throughput 16 s r DNA sequencing for bacterial identification demonstrated a uniform trend in the richness and diversity of microbial flora which was the least in the thermophilic phase and grew on entering into cooling and curing phase,and higher in the enhanced composting than that in natural composting.In the cooling and curing phase,the Simpson's Index of diversity in the stack added with surfactants was 5.22 and 5.48 respectively and that in the stack with mixed flora was 5.10 and 5.26 respectively,higher than 4.98 and 5.12 in the natural stack.The cluster analysis unveiled a significant difference in the bacterial community structure during the thermophilic and cooling phase but no difference during the curing phase in different composting processes.The high-throughput sequencing showed the bacteria mainly from 28 phyla,among which the 5 major bacterial phyla were Proteobacteria,Firmicutes,Bacteroidetes,Chloroflexi and Actinobacteria.At the class level,they belonged to 21 classes,among which 9 major classes were Bacilli,Actinobacteria,?-Proteobacteria,?-Proteobacteria,Anaerolineae,Cytophagia,Flavobacteriia and Clostridia.On the genus level,a statistical analysis of the genera in abundance lower than 40 presented that 12 genera are associated with petroleum hydrocarbon degradation.The bacteria with high relative abundances in natural composting were Rhodococcus sp.,Bacillus sp.and Steroidobacter sp..Based on the significant analysis,the degradation rate of TPH was significantly positively correlated with Rhodococcus sp.and Steroidobacter sp.(P<0.05);the bacteria with high relative abundances in the compost supplemented with surfactants were Pseudomonas sp.,Flavobacterium sp.,Pusillimonas sp.,and Mycobacteriu sp.,the degradation ratewas significantly positively correlated with Pseudomonas sp.and Flavobacterium sp.(P<0.05).As for the compost supplemented with mixed flora,the bacteria with high relative abundances were Pseudomonas sp.,Bacillus sp.,Lysinibacillus sp.And Gordonia sp.,and the degradation rate was significantly positively correlated with Pseudomonas sp.(P<0.05)but significantly negatively with Gordonia sp.(P <0.05).In section 5,construction of Alk B gene clone library for composting samples achieved a total of 466 sequences and 12 OPFs by analysis.The evaluation of ?-diversity of Alk B showed that the addition ofrhamnolipid surfactants into the stacks resulted in higher diversity in the flora than in the natural composting.The major OPFs altered in the different types of composting.In the natural composting,and the one supplemented with surfactants and the one with mixed flora,the major OPFs were OPF6(similar sequence Rhodococcus erythropolis Alk B)and OPF2(Rhodococcus qin-gshengii BKS20-40 Alk B),OPF12(Flavobacteriales bacterium ALC-1 Alk B)and OPF8(Pseudomonas aeruginosa Alk B),and OPF5(Pseudomonas aeruginosa Alk B)respectively,which were significantly positively correlated with the genera detected by high-throughput 16 s r DNA sequencing technique during the degradation of petroleum hydrocarbons.It demonstrated the bacteria displayed a major role in the oil degradation by composting.According to the quantitative fluorescence PCR assay for the analysis of the Alk B gene from the composting samples,it was observed that the Alk B gene copy number didn't reached its peak at the same time in different composts and varied greatly in different phases,but the variance was coherent with the occurrence of the highest peak in oil degradation,indicating that the quantitative analysis of the Alk B gene could reflect the effect of the degradation of oil in a way.