Bacterial Community Structure Shaped By Light-hydrocarbon Microseep And Potential Indicators Of Underlying Oil And Gas

Light hydrocarbons accumulated in subsurface soil or sediment by long-term microseepage could favor the anomalous growth of indigenous hydrocarbon-oxidizing microorganisms.Studies of the ecological characteristics of bacteria and the light-hydrocarbon oxidizing genes in these ecosystems could provide reliable bioindicators for underlying petroleum reservoirs.Here,Illumina MiSeq sequencing of the 16S rRNA gene was conducted to unveil the bacterial community structures in soil samples obtained from four typical oil fields and one gas field at different locations in China.Of all the light hydrocarbons(C1?C4),butane originated only from oil and gas deposits,thus the anomalous enrichment of butane-oxidizing bacteria in subsurface soil could serve as accurate indicators of petroleum reservoirs.Incubation with 12C-butane were performed to determine the composition of butane-utilizing bacteria in light-hydrocarbon microseepage ecosystems and further confirmation was conducted by DNA-stable isotope probing(DNA-SIP)with 13C-labeled butane.Soluble di-iron monooxygenases(SDIMO)gene clone libraries were constructed to characterize bacterial communities involved in light-hydrocarbon oxidation.The main results were as follows:1.The results showed remarkably higher bacterial diversity in next-to-well samples(OS)compared with background samples(NS).The bacterial diversity in OS samples was mainly affected by moisture,nitrogen and phosphorus.The alpha diversity of samples in Puguang gas field(OPG)and the background samples(NPG)increased significantly after incubated with butane.Bacterial community structures was mainly affected by environmental factors,while the geographic location and the concentration of total solvent extractable matter(TSEM)showed non-significant effect,which was notably different from oil-contaminated environments.2.Proteobacteria was much more abundant in OS samples compared with the NS samples,especially for the Gammaproteobacteria.The oil and gas fields significantly favored the growth of Mycobacterium and Nocardia;other genera,such as Pseudonocardia and lamia,were also enriched in most of the oil and gas fields.The relative abundance of Mycobacterium,Pseudonocardia,Rubrivivax and Hydrogenophaga increased notably in all the samples after butane cultivation.Besides,Rhodococcus,Nocardioides and Polaromonas in Jianghan oil field(OJH)and the background samples(NJH)occupied larger proportions after incubated with butane;and the relative abundance of Variovorax and Alcanivorax increased both in OPG and NPG.3.The composition of butane-utilizing bacteria was further identified by DNA-SIP with 13C-labeled butane.The results showed the uptake of 13C butane by Nocardioides and Pseudonocardia in OPG samples,Mycobacterium was still dominant although non-significant label was found;13C butane was assimilated by Mycobacterium and Nocardioides in NPG samples.Many other genera were also found enriched by butane,such as Variovorax and Sphingomonas,which were not previously thought to contribute to butane oxidation.4.Results of SDIMO gene clone libraries indicated relatively higher diversity in oil and gas field,suggested that SDIMO gene diversity might be used as a potential indicator of underlying oil and gas reservoirs.The clones of original samples were mainly related to SDIMO and ThmA coding genes from uncultured bacterium.Interestingly,clones related to PrmA coding genes in Mycobacterium sp.TY-6 increased greatly in all these samples,and those related to Mmo coding genes in Mycobacterium brisbanense and Pseudonocardia acaciae were more abundant in Puguang samples(both OPG and NPG)after incubated with butane.In short,the relatively higher diversity of bacterial community and SDIMO genes could be used as subsidiary indicators for underlying petroleum reservoirs.Our results highlighted the relatively universal potential of Mycobacterium and Pseudonocardia as indicators of underlying oil and gas deposits,and Nocardioides might be adopted as an indicator at certain area.This work contributes to a broader perspective on the bacterial community structures shaped by long-term light-hydrocarbon microseepage and proposes potential indicators,providing an additional resource for the improvement of microbial prospecting of oil and gas.