| In this study,several samples were collected respectively from several great oil fields of the northwest and northeast China.The effects of different environmental factors,soil type,nutrient and petroleum hydrocarbon content on indigenous microbial community structure and diversity were investigated.The core microbial communities under different environment and the main driving factors of their formation were illustrates.A strain KB 1,which was later confirmed to be Rhodococcus erythropolis and could degrade petroleum hydrocarbon efficiently at low temperature,was isolated from typical sandy soil in Yumen Oilfield.The species and classification of strain KB 1 were determined by multiphase taxonomic method and molecular biology technology.At the same time,the whole genome sequencing technology was introduced to illustrate the genetic basis of degradation of petroleum hydrocarbon compounds at low temperature.Based on combination of transcriptomics,metagenomics and metabonomics,the potential key genes and metabolic pathways for degrading various types of petroleum hydrocarbon compounds were explored,which laid a foundation for further study on the transcriptional regulation mechanism of degrading petroleum hydrocarbon.On this basis,the physiological and ecological responses of Medicago sativa L.to petroleum hydrocarbons in soil contaminated with different concentrations of petroleum hydrocarbons in the presence of KB1 bacterial agent were analyzed by laboratory pot cultivation technique.The microecological response mechanism of oil hydrocarbon degradation by KB1-Medicago sativa L.combined remediation system was elucidated from multidimensional perspective,including soil physicochemical properties,enzyme activities,plant physiological and biochemical characteristics and plant root microbial community.Specific research results were as follows:1.High-throughput sequencing technology was used to investigate the bacterial community structure and diversity in Fushun Oilfield(FS),Qingyang Changqing Oilfield(QY)and Yumen Oilfield(YM)under the combined influence of different soil types(black soil,loess and sandy soil),pollution degrees and environmental factors.The results showed that 16 samples collected from the three regions appeared strong heterogeneity in soil nutrients and enzyme activities,in detail,the bacteria belonged to 239 phyla,508 classes,810 orders,1417 families,2048 genera.Alpha diversity analysis showed that the bacterial abundance in the three regions was QY>FS>YM.The core bacterial communities of different regions also showed significant differences.However,under the stress of petroleum hydrocarbon pollutants,the bacterial communities of different samples showed a certain crossover at phylum and genus level.In general,Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria etc.are the dominant phyla which have wide degradation spectra of petroleum hydrocarbons.Pseudomonas,Bacillus,Nocardioides,Oceanobacillus,Sphingomonas,Alkanindiges and Streptomyces are the main functional degrading bacteria of petroleum hydrocarbon pollutants at genus level.On the other hand,the results of Beta diversity analysis indicated that spatial distance limitation and random variation of repeated samples may be the main factors leading to the differences in microbial community structure.The partial least squares path model(PLS-PM)further analyzed the correlation between each factor and bacterial community structure and diversity,indicating that there was a correlation among soil bacterial community abundance,altitude,annual rainfall and longitude.With the increase of annual rainfall,the bacterial community abundance showed an rising trend.However,the diversity of bacterial community decreased with the increase of altitude,that is,Alpha diversity of microbial community and the content of petroleum hydrocarbons in petroleum hydrocarbon contaminated soil in different regions were indirectly controlled by altitude factor that could affect both soil physicochemical factors and air temperature.2.The sandy soil of Yumen Oilfield in Gansu Province has poor nutrients,low bacterial abundance and diversity,and the most difficult environmental remediation.A strain named KB1 with low temperature resistance,high salt resistance and high degradation rate of petroleum hydrocarbon was isolated from the sandy soil of Yumen Oilfield by streak plate method,and this strain was identified by multiphase taxonomic method.The results showed that strain KB1 is a Gram-positive,aerobic microorganism with short rod-shaped or rod-shaped.Characteristic amino acid composition include meso-DAP(diaminoheptanoic acid),Ala(alanine)and Glu(glutamic acid),Gly(glycine)and Asp(aspartic acid).Characteristic sugar composition of whole cell hydrolysates include arabinose,galactose,mannose and ribose.The main polar lipids include diphosphatidyl glycerol,phosphatidylethanol amine,phosphatidylinositol,phosphatidylinositol mannoside,and an unknown phosphatidyl polar lipid.The non-polar lipids are mainly composed of methylnaphthoquinones.Compared with the database,the strain belongs to a species of Rhodococcus.Phylogenetic tree was further constructed by 16S rDNA sequence and gyrB sequence,and the strain was named Rhodococcus erythropolis KB1 from the evolutionary branch.3.The plant growth promoting activity and organic metabolism ability of KB1 strain under different temperature,pH and salt concentration were studied.The results showed that environmental conditions had a significant effect on the biological activity of KB1(P<0.001).Under the condition of 30?,pH 7.0 and 1%NaCl,the capacity of KB1 strain chelated siderophore reached at 72.43%±0.20%,the production of IAA was approximately 10.50±0.67 mg/L,the formation of biofilm was approximately 0.81±0.02,nitrogen-fixing efficiency was approximately 0.36%±0.01%,the production of betaine was approximately 0.973±0.013 mg/(L·g),ACC deaminase activity and laccase activity were 17.07±0.72 U/mg and 9.33±0.31 U/mg,respectively.This tested strain still appeared good biological activity at low temperature or in the presence of high concentration of salts.When naphthalene was used as substrate,the dehydrogenase activity of KB1 strain reached 178±5.21 mg/(L·h),and the degradation rate of long-chain alkane was more than 85%.Moreover,KB1 strain could grow well under crude oil stress,and the maximum degradation rate could reach at 64.47%±0.015%at 30?,while the maximum degradation rate could still reach more than 50%at low temperature.These results indicate that KB1 strain has strong environmental adaptability and is expected to be applied in the remediation of petroleum hydrocarbon pollution.4.The genetic basis of the main biological characteristics of KB1 strain,such as high degradation efficiency of petroleum hydrocarbon,good cold tolerance and plant growth promotion ability,was revealed from a multidimensional perspective by combination of genome,transcriptome,pan-genome and metabolome techniques.The results of PacBio single molecule length sequencing showed that the genetic material of KB1 strain mainly consists of 1 chromosome and 3 plasmids with a total of 6837 genes.There are about 731 genes encoding mono-oxygenase,dioxygenase,aryl alcohol dehydrogenase and other petroleum hydrocarbon degradation related genes,among which 82 genes play key roles in the degradation pathway of petroleum hydrocarbon components,such as xylene,chlorcyclohexane,chlorobenzene and naphthalene.Based on AntiSMASH,it was predicted that KB1 strain contains 18 secondary metabolite biosynthesis gene clusters including NPRS,Ectoine and RiPP-like,which suggested that KB1 strain can produce siderophores at the genetic level and has favorable adaptabilities to salt stress,drying,high temperature and low temperature.In addition,KB1 strain may produce RiPP-like secondary metabolites such as branch chain fatty acids.Comparative transcriptomic analysis of petroleum hydrocarbon degradation under different temperature conditions suggested that under low temperature,KB1 strain can generate compensatory energy or change the proportion of membrane lipid components to protect itself from cold by up-regulating the expression levels of genes related to ATP synthesis,oxidative phosphorylation and fatty acid metabolism(FC>8),and the temperature difference do not cause significant reversal of the expression intensity of genes related to degradation of petroleum hydrocarbon components.Pan-genome analysis illustrated that compared with R.erythropolis X5?R.equi ATCC33707 and R.ruber YC-YT1 strains,genes specific to KB1 mainly perform various functions including enzyme catalysis,ATP synthesis,oxidative phosphorylation,stress,fatty acid metabolism and biofilm system shaping.The results of bacterial metabonomics of petroleum hydrocarbon degradation under different temperature conditions revealed that,compared with 10?,the treatment at 16? can ensure low temperature stress system of KB1 strain play a better role.However,with the increase of temperature difference,the upregulation of expression of phosphatidylethanolamine increase in both quantity and intensity.And the upregulation of expression of phosphatidylethanolamine is the main metabolic pattern in response to low temperature,which was consistent with the widely reported phosphatidylethanolamine as an important functional membrane lipid component.5.Soil ecological effects of high,medium,low concentration and unpolluted petroleum hydrocarbon degradation in Medicago sativa L.(CK group)and PB group supplemented with exogenous KB1 strain were compared and analyzed by laboratory pot cultivation technique,respectively.The results displayed that in CK group,the contents of soil organic matter,total nitrogen,total phosphorus and the activities of sucrase,protease and petroleum hydrocarbon concentration showed a positive correlation(P<0.05),the contents of available nitrogen,available phosphorus,total potassium and available potassium and petroleum hydrocarbon concentration showed a negative correlation(P<0.05),and high concentration of petroleum hydrocarbon inhibits the activities of soil catalase and urease.With the growth and development of Medicago sativa L.,the contents of soil organic matter,total nitrogen,available nitrogen,total phosphorus,total potassium,available potassium and protease activity increased(P<0.05),while available phosphorus and pH decreased(P<0.05),sucrase activity remained constant,and the activities of catalase and urease decreased(P<0.05)in petroleum hydrocarbon contaminated soil.In detail,high concentration of petroleum hydrocarbon seriously affects soil nutrients and enzyme activities.Medicago sativa L.plant remediation technology can significantly increase the contents of soil organic matter,nitrogen,potassium and protease activities.In PB group,introduction of KB1 exogenous bacterial agent can effectively degrade petroleum hydrocarbon in the soil,increase the contents of soil organic matter,total nitrogen,available nitrogen,total phosphorus,available phosphorus,total potassium and available potassium,and increase the activities of soil proteinase,sucrase,and catalase(P<0.01),suggesting that KB1 strain can significantly improve soil nutrients and assist the healthy growth of Medicago sativa L.On the other hand,it can play a pioneer role in the degradation process of petroleum hydrocarbon by increasing catalase activity.6.The physiological effects of different tissues at different developmental stage of Medicago sativa L.(CK group)and PB group supplemented with exogenous KB1 strain grown in high,medium,low concentration and unpolluted petroleum hydrocarbon environment were compared and analyzed by laboratory pot technique,respectively.The results showed that compared with the unpolluted group,petroleum hydrocarbons significantly affected the membrane lipid,photosynthetic efficiency,non-enzymatic antioxidant and antioxidant oxidase system activities of Medicago sativa L.,and inhibited the development of Medicago sativa L.roots and normal growth of plants,especially in the high concentration group.In CK group,with the growth of Medicago sativa L.,the content of malondialdehyde decreased significantly,the contents of proline,soluble protein and chlorophyll increased continuously,the activity of antioxidant enzymes showed an increasing trend,and the agronomic indexes such as Medicago sativa L.plant height and root length also increased continuously(P<0.05),indicating that Medicago sativa L.can grow normally in soil contaminant with high concentration of petroleum hydrocarbon(7500 mg/kg).Compared with CK group,exogenous KB1 supplementation effectively reduced the malondi aldehyde content in Medicago sativa L.,improved the photosynthetic efficiency,and significantly improved the agronomic characteristics of Medicago sativa L.in different concentrations of petroleum hydrocarbon contaminated soil,such as root length,root diameter,leaf number and soluble sugar content in roots.The change trend of other physiological effects was similar to CK group.Furthermore,the synergistic effect of KB1 can significantly alleviate the damage of high concentration of petroleum hydrocarbons to plant roots and membrane lipids,and the damage caused by reactive oxygen species under stress.7.The effect of addition of exogenous KB1 strain on the structure of Medicago sativa L.root microecosystem at flowering stage was investigated.The results exhibited that the top 50 dominant bacterial abundance in Medicago sativa L.root belonged to 8 phyla,10 classes,22 families and 35 genera,among which 25 strains were Proteobacteria(68.5%),4 strains were Bacteroidetes(11.4%)and 2 strains were Actinobacteria(5.7%),and the other strains belonged to Epsilonbacteraeota(2.8%),Deinococcus-thermus(2.8%),Verrucomicrobia,2.8%),Deferribacteres(2.8%)and Firmicutes(2.8%),respectively.In the uncontaminated group,the addition of KB1 significantly increased the number of OTUs in Medicago sativa L.roots,and then with the increase of petroleum hydrocarbon concentration,both of the number of OTUs of rhizosphere bacteria in Medicago sativa L.and the number of OTUs of specific bacteria showed a trend of first decreasing and then increasing.It was deduced that KB1 strain could change the bacterial community structure of Medicago sativa L.root,revealing the pioneer role of KB1 strain in the process of combined remediation.To be specific,the changes of bacterial community composition and abundance in Medicago sativa L.roots can be reshaped through bacterial degradation of petroleum hydrocarbons of different concentrations,improvement of soil ecological characteristics,change of plant physiological characteristics and secretion of endogenous signal molecules,regulation of soil ecology,plant physiology and composition of root exudates. |
PDF Full Text Request