Efficacies Of Bacteriophage And Biocides Against Microbial Souring In An Oilfield

During the process of oil exploitation,injecting water into underground oil reservoir to maintain formation pressure is a fundamental technical practice to improve oil recovery.However,the sulfate and other sulfur-containing compounds that enter the reservoir with the injected water may promote the growth and reproduction of microorganisms,producing high amounts of hydrogen sulfide,known as microbial souring.Microbial souring leads to a variety of production and safety problems to oilfield development,which need to be effectively solved.Considering the existing physical and/or chemical methods are insufficient,it is of great value to deeply understand the microbial process of microbial souring and develop efficient prevention and control technologies.In this study,the high-throughput DNA sequencing method combined with Hungate rolling-tube culturing method were employed to probe the microorganisms in the production water from the wells of a high-temperature oilfield in northeast China.Moreover,double-layered plate method was used to isolate lytic phages targeting hydrogen sulfide-producing bacteria(SPB).Finally,the efficacies of phages and biocides were evaluated to inhibit these SPB.The results of high-throughput sequencing showed that the abundant phyla in the produced water samples were Proteobacteria,Bacteroidetes and Thermotogota;the dominant classes were Gamma Proteobacteria,Bacteroidia and Alpha Proteobacteria.The relative abundance of SPB in produced water was very low(0.66%),among which Dethiosulfatibacter and Thermovirga were detected in all samples.Three pure strains of SPB,designated DG8,DG22 and DG24,were isolated by Hungate rolling tube method.The analyses of 16S r RNA gene sequences indicated that DG8,DG22,and DG24 shared the highest similarity with Lactobacillus crispatus(DSM 20584;99.9%),Acetomicrobium thermoterrenum(DSM13490;99.3%)and Pseudothermotoga Lettingae(DSM 5069;100%),respectively.Strain DG8 grows optimally at 65?,p H 8-10,and with the salinity of 0.7%-1%.The optimum temperature of strain DG22 was 60?,the optimum p H was 8-9,and the optimum salinity was 0.7%.The optimal temperature of strain DG24 was 60?,the optimal p H was 8?9,and the optimal salinity was 0.7%?1%.Brominitol,tetramethylphosphorous sulfate(THPS),and glutaraldehyde could effectively inhibit the hydrogen sulfide production by these three SPBs,and the working concentrations was as low as 10mg/L,70 mg/L,and 10mg/L,respectively.The lytic phage against strain DG8 was successfully enriched.This mixed phage culture enriched for 12 days could inhibit DG8 for at least 10 days,but when surviving cells from infected DG8 batch cultures were inoculated into new cultures and reinfected,the inhibition lasted 4 days,indicating immunity of DG8 to the phages occurred.The use of the combination of lytic phage with bronopol,glutaraldehyde and THPS,respectively,showed significant higher inhibition than that of sole phage or biocide.In addition,when phage was first supplemented to decrease the number of DG8,and THPS was dosed 60 hours later,the minimal amount of H₂S was observed,suggesting that the dosing strategy of phage and biocide may have influence on the inhibition effect.This study enriches the scientific understanding of microbial diversity in this specific oilfield,especially in microbial diversity that may cause microbial souring.The isolated hydrogen sulfide-producing bacteria,lytic phages and its combination of effective biocides present important alternatives to the prevention of microbial souring in this oilfield,and provide a new direction for the control of oilfield souring.