Diversity,Biological Characteristics And Degradation Mechanism At Low Temperature Of Oil-Degrading Bacteria Isolated From The Oil-Contaminated Desert Soil

The hazards and effects on environments and living organisms of oil contamination are becoming serious problems during the oil exploitation and applications,which threat human health.Biodegradation of the oil contaminates with microorganism is one of the most effective and economical methods without the second pollution.The thesis is aimed to analyze the bacteria diversities of the oil-contaminated desert soils in northwest China,isolate high-efficient oil-degrading bacteria,study their biological characteristics,and degradation mechanism.The isolated oil-degrading bacteria were used to deal with the oily wastewater.The main results are enclosed as follows.Illumina Miseq high-throughput sequencing method was used to explore the bacteria diversities of the oil-contaminated desert soils.The results indicated that bacteria showed great diversities in the oil-contaminated desert soils.The bacteria belong to thirty three phyla,forty eight classes,seventy eight orders,one hundre d and seventy-nine families,and four hundred and seventy-one genera.The six predominant bacteria phyla were Firmicutes,Proteobacteria,Bacteroidetes,Actinobacteria,Chloroflexi and Fusobacteria.The predominant bacteria genera were Bacillus,Lactococcus,Oceanobacillus,Enterococcus,Citrobacter,Paenibacillus,Streptococcus,Prevotella,Pseudomonas,Dietzia,Nocardioides,Arthrobacter,Streptomyces,Neisseria,Veillonella,Exiguobacterium,Leptotrichia,Haemophilus,Fusobacterium,Rothia and Acinetobacter,including the most of the common oil-degradation genera.Thirty seven oil-degrading bacteria strains were isolated from the oil-contaminated soils by enrichment culture and spread plate methods.The morphology and 16 S r RNA gene sequence analysis were used to identify the isolates.The results indicated that the isolated bacteria strains belonged to twenty one genera and thirty four species of the phyla Actinobacteria,Gammaproteobacteria,Betaproteobacteria,Bacilli and Alphaproteobacteria.The six predominant bacterial genera were found to be Pseudomonas,Rhodococcus,Micrococcus,Stenotrophomonas,Achromobacte and Staphylococcus,which account for 51.35% of the total isolates.The thirty six strains demonstrated good adaptability to crude oil and grow well in medium using crude oil as the sole carbon and energy source.After being cultured for seven days in medium with an oil concentration of 1500 mg/L,the oil-degrading rates of eight strains were not less than 30.55%.The rates of eleven strains were measured to be between 10.05% and 28.37% where as the rates of eighteen strains were not more than 8.05%.The specific PCR primers were designed according to the related degradation genes of the common oil-degradation bacteria.The PCR detection results showed that the twenty five strains contained alkane monooxygenase genes,six strains had aromatic dioxygenase genes,six strains existed biphenyl dioxygenase genes,four strains contained naphthalene dioxygenase genes,while three strains contain toluene dioxygenase genes.Catechol dioxygenase genes were detected in two strains.Two alkane monooxygenase and one ring-hydroxylating dioxygenase genes were further cloned and analyzed.The biological characteristics and the oil-degradation abilities of four oil-degrading strains including KB1,2182,JC3-47 and 1217 were analyzed.They were identified to be Rhodococcus erythropolis,Rhodococcus equi,Rhodococcus qingshengii and Pseudomonas aeruginosa by morphological,physiological and biochemical characteristics,and 16 S r DNA sequence analysis respectively.The strains KB1,218 2 and JC3-47 grew well at 10⁵0oC,with the initial p H of 3⁹ and the Na Cl concentration of 0⁵%.The optimal temperature for bacterial growth was 35 oC,35oC and 30 oC respectively.KB1 and 2182 could grow at p H 2 and 9.0% of Na Cl.And the strain 1217 could grow even at 5⁶5oC,with the initial p H of 2¹0 and the Na Cl concentration of 0⁹%,the optimal temperature,p H and the Na Cl concentration for 121 7 growth was 35 oC,p H 9 and 0% respectively.The bacteria grew well in broth containing different organic substrates as sole carbon source,such as n-dodecane,n-octadecane,benzene,methylbenzene,xylene and naphthaline.These isolates could effectively degrade medium-and long-chain alkane components of crude oil.KB1,2182 and 1217 grew well in broth containing n-hexadecane.KB1 and JC3-47 could grow in broth containing pyrene.1217 could grow in broth containing catechol.The bacteria produced biosurfactants and decreased the surface tension of the culture broth.They also showed adhesion activities to n-hexadecane.The growing characteristics and the oil-degradation abilities of strain KB1 at low temperature were further studied.The results showed that KB1 grew well at 10 oC in medium with an alkane-mixture concentration of 10500 mg/L.The bacteria counts reached its maximum at the fifth day.GC-MS analysis showed that the total alkane degradation efficiency was 64.55%.with the degradation efficiencies of n-octane,n-decane,n-dodecane,n-tetradecane,n-hexadecane,n-octadecane,n-docosane,n-tetracosane and n-octacosane being 29.27%,46.25%,89.13%,77.59%,70.35%,55.16%,57.38%,67.18% and 82.64%,respectively.Transcriptomes of Rhodococcus erythropolis KB1 cultured in the medium with alkanes-mixture as the sole carbon and enegy soures at 10 oC and 30 oC were analyzed.The results showed that the number of differentially expressed genes was 2957,with the number of up-regulated genes being 1584,while down-regulated genes being 1373.When the Log2 ratio> 5,the 99 genes were up-regulated,and 119 genes were down-regulated,33 of the genes exist in different cell components,108 with molecular function,95 participated in the biological process.Further study indicated that there were 201 differentially expressed genes in the degradation proess of fatty acid,aromatic compounds,methane,naphthalene,xylene,ethylbenzene,toluene and polycyclic aromatic hydrocarbon based on the analysis of Gene ontology and KEGG,and the number of the up-regulated genes was 135,while down-regulated genes were 66.When the Log2 ratio> 5 as the base,the 13 genes of fatty acids,aromatics compounds,methane,naphthalene and ethylbenzene were up-regulated,and only 3 genes were down-regulated.The oil degrading efficiency of the oily wastewater was studied by addition of Rhodococcus erythropolis KB1 with a sequencing batch reactor activated sludge?SBR?process at different temperatures.After 20 days treatment,The effluent concentrations of COD of the control group and the experimental group with KB1 at the temperature of 10 oC and 30 oC were 58.50?49.32?55.68 and 45.73 mg/L respectively,the concentrations of NH4+-N were 7.78?5.52?7.64 and 6.01 mg/L respectively,and the the concentrations of TP were 0.187?0.164?0.232 and 0.196 mg/L respectively,which reached the direct discharge requirements of petroleum refining industrial pollutants?GB 31570-2015?.The remaining oil-concentration and removal efficiency of experiment group with KB1 was 4.27 mg/L and 97.40% at 10 oC respectively,the oil-concentration and removal efficiency of the control group were 18.75 mg/L and 88.58% respectively.The oil-concentration and removal efficiency of experiment group with KB1 was 3.17 mg/L and 98.07% at 30 oC,the oil-concentration and removal efficiency of the control group were 15.38 mg/L and 90.63%.Which indicated that oil-degrading bacteria have obvious promoting effect for oil degradation of the oily wastewater,and still have obvious removal effects under the condition of low temperature.The modified and unmodified peanut shells were applied to adsorb heavy metal ions(Pb²⁺,Cu²⁺ and Zn²⁺)of wastewater.The results showed that the modified peanut shells had more obvious adsorption efficiency than unmodified peanut shells.The adsorption efficiency increased gradually with the p H,reaction duration,init ial ion concentration and adsorbent dosage within a certain range.The adsorption of heavy metal ions by unmodified peanut shells performed well under acidic conditions,while the best results for the modified peanut shells were obtained under alkaline con ditions.The adsorption rate reached about 88% of the maximum adsorption rate after treatment for a 15 min reaction and tended to balance after 50 min.The dynamic behaviors of both the modified and unmodified peanut shells well fit the Lagergren?II?order chemical reaction kinetics model.