Enhanced Bioremediation Of Hydraulic Fracturing Flowback And Produced Water Using An Indigenous Biosurfactant-producing Strain Acinetobacter Sp.Y2

In this study,a biosurfactant-producing strain Y2 was isolated from hydraulic fracturing flowback and produced water(HF-FPW)which was collected from the ma 18 well area of Karamay,Xinjiang Autonomous Region.Strain Y2 was identified as Acinetobacter sp.through 16 S rRNA gene sequencing.The optimization of conditions for biosurfactant production was carried out by using single-factor and orthogonal experiments.The optimal fermentation conditions were determined as follows: 2% olive oil,2 g/L ammonium sulfate,a fermentation temperature of30 °C,a salinity of 10 g/L,and an initial pH value of 7.Y2 was cultured under the optimal conditions and the biosurfactant was extracted using ethyl acetate.The extracted biosurfactant was identified as non-ionic surfactant.The surface activity test showed that the biosurfactant had a critical micelle concentration of 187.5 mg/L with a corresponding surface tension of 30.2 mN/m.The biosurfactant showed a strong tolerance against wide range of pH(2?12),temperatures(4?100 °C),and salinity(0?100 g/L).Through thin layer chromatography,Fourier transform infrared spectroscopy,and gas chromatography–mass spectrometry analysis,the biosurfactant produced by Y2 was characterized as lipopeptide.Subsequently,the Y2 was inoculated into HF-FPW for bioaugmentation.The results showed that the growth and activity of microorganisms in the HF-FPW were greatly improved.Meanwhile,it significantly(P<0.05)promoted the removal of chemical oxygen demand(reduction from 6646.7 mg/L to 1546.7 mg/L in 7 d),and the degradation of n-alkanes(reduction from 2635.4 mg/L to 159.7 mg/L)and polycyclic aromatic hydrocarbons(reduction from 918.6?g/L to 209.6 ?g/L).Results of high-throughput sequencing showed that the microbial diversity and richness of the bioaugmentation treatment decreased significantly.Due to the addition of Y2 in bioaugmentation treatment,the microbial diversity at the genus level decreased dramatically.The Acinetobacter sp.dominated the community,and the relative abundances of some petroleum hydrocarbon degrading genera such as Pseudomonas sp.and Rhizobium sp.increased,indicating that the inoculation of indigenous biosurfactant-producing bacteria enriched the degrading population and enhanced the degradation performance of the bacterial community.Furthermore,studies on the degradation performance and functional genes of Y2 were carried out.The 2,6-DCPIP test showed that Y2 had a strong ability to degrade wide range of substrate.The genomic and plasmid DNA of Y2 were further extracted,form which partial fragments of both alkane monooxygenase AlkB gene and cytochrome P450 gene were amplified,indicating that Y2 contained homologous genes of AlkB and P450.The results of real-time quantitative PCR suggested that the inoculation of indigenous biosurfactant-producing bacteria enhanced the expression level of degradation-related genes of microorganisms in the HF-FPW.Canonical correlation analysis results revealed that Y2 directly involved in the petroleum hydrocarbon degradation and played a vital role in the process.This study not only provides a theoretical and feasible basis for the bioremediation of HF-FPW,and deepens the understanding of the bioremediation mechanism of wastewater,but also contributes significantly to the treatment of oil-related pollution and the sustainable development of oil and gas industry in China.