Respond Of Microbial Community And Key Functional Gene To Petroleum Hydrocarbons In Contamined Soil Of Shale Gas Mining Areas

Microbial remediation of petroleum hydrocarbons contaminated soils has been extensively studied,however,the response law of indigenous microbe to petroleum hydrocarbons in contaminated soils remain to be uncovered.Organic matter is completely degraded into CO₂ and H₂O by microbial aerobic metabolism.If a large amount of CO₂ released into the atmosphere,the"greenhouse effect"will aggravate.Therefore,it is of great ecological value to study the response law of carbon fixation genes during microbial remediation.The soil pollution simulation experiment was undertaken for 63 days,using the clear soil and oil-based drilling fluid in the shale gas mining area of Fuling District.We analyzed the natural attenuation law of petroleum hydrocarbons in soil at three contaminated contents:low-concentration contamination(C_TPH=3523±159 mg/kg,4%),medium-concentration contamination(C_TPH=8715±820mg/kg,9%),and high-concentration contamination(C_TPH=22031±118 mg/kg,2.2%).The dynamic variations of the diversity and composition of soil microbial community in the culturing period were studied.And the impact of petroleum hydrocarbons removal on carbon fixation potential was analyzed by quantifying the abundance of key functional gene for carbon fixation.The major research results are presented below:(1)After cultivation for 63 days,the removal efficiency of petroleum hydrocarbons in high-concentration groups(45.33±3.90%)was significantly lower than those in low-and medium-concentration groups(54.79±0.75%?58.96±4.11%).The lowest removal amount of petroleum hydrocarbons components in each group was>C21?C34components,and the highest removal amount in low-,medium-and high-concentration groups were>C16?C21,>C12?C16,>C12?C16 components.During the culturing period,the largest removal rate of TPH among the low-,medium-and high-concentration groups existed in early stage of culturing(1-5d),and then decreased.The content of petroleum hydrocarbons tended to stabilize during culturing,and it took longer in medium-and high-concentration groups than in low-concentration groups;(2)During the culturing period,the richness and evenness of soil microbial community in the shale gas mining area decreased significantly after a slight rise in the first 5 days,but richness and evenness finally maintained at a low level in medium-and high-concentration groups,showing a great effect on soil microbial community;wherea eventually recovered to the pre-pollution level in low-concentration groups.The contamination degree of petroleum hydrocarbons selectively affected the soil microbial diversity.The TPH under medium-concentration contamination was positively correlated with soil microbial community richness(R²=0.746,P<0.05),and the content of petroleum hydrocarbons under low-concentration contamination was positively correlated with soil microbial community evenness(R²=0.657,P<0.05);(3)There was a hysteresis phenomenon on the soil microbial community composition in the shale gas mining area in the first 5 days,then the soil microbial community composition changed significantly,with a tansfomation from the gram-positive bacteria to gram-negative bacteria.The impact under medium-concentration contamination on community composition was greater than that of low-concentration contamination.Soil microbial community composition was similar between medium-and high-concentration group,while differed from that under low-concentration contamination.There was no significant correlation between TPH and community composition during the culturing period(R²=0.0736,P>0.05);(4)The bacteria from 48 genera of 1 phylum were senstive to petroleum hydrocarbons,such as Sphingorhabdus,Candidatus,Polycyclovorans,Sphingopyxis.The soil microorganisms from 190 genera of 18 phyla may tolerant petroleum hydrocarbons contamination,such as Brukholderia?Alanaindiges?Nevskia?Acidothermus?Ralstonia?Deylla,most of which can degrade petroleum hydrocarbons.The biomarkers in low-,medium-and high-concentration groups were screened out,that can provide a basis for applying the remediation strategies,bioenhancement and biostimulation,to oil-based drilling fluids contaminated soil of shale gas mining areas.(5)Under low-concentration contamination of petroleum hydrocarbons,the cbbL gene copies for carbon fixation recovered to the pre-pollution level in later culturing period(49-63d)and tended to be stable;while those under medium-and high-concentration contamination showed a significant increase in different stages in late culture(20-63d),up to 651.56%.The interactive effect(the initial contamination degree of petroleum hydrocarbons*cultivation time impact)the abundance of cbbL gene significantly(P<0.01).There was no significant correlation between soil cbbL gene copies and petroleum hydrocarbons removal contents in soils contaminated by low-,medium-and high-concentation petroleum hydrocarbons(R²<0,P>0.05).