Investigation Of Petroleum Hydrocarbon Pollution In Zhoushan Sea Area And The Application Of Related Oil-Degrading Microorganisms

This paper analyzed the pollution status of petroleum hydrocarbons in the seawater and sediment of Zhoushan sea area which were surrounded by ship-repairing plant,oil storage and transportation base,as well as passenger and cargo terminals.The results evidenced that the denser distribution of shipyards,docks,oil storage and transportation bases around the sampling point,the higher concentration of petroleum hydrocarbons were detected in the samples.The concentration of alkanes and PAHs in intertidal sediments was higher than that in other similar sea areas in China.The results indicate that the sea area near the ship-repairing plant and docks are subject to different levels of petroleum hydrocarbon pollution.To examine the degradation performance of microorganisms on petroleum pollutants originated from ships,microbial consortia were enriched in the laboratory.Then,the degradation of seawater which was polluted by various ship oil and ship clearing oil sludge was simulated in laboratory.The results showed that almost all of the alkane and PAHs in the ship oil and ship clearing sludge had been degraded compared with the control,indicating that the microbial consortia has potential to bioremediate marine oil pollution which was caused by ship oil and ship clearing oil sludge.To learn the ability of microbial consortia on the remediation of petroleum hydrocarbon pollution in different types of coastal zone,the biodegradation of ship clearing oil-sludge in the subtidal,intertidal and supratidal zones was simulated in the lab.The results showed that petroleum degradation rate of the mixed substrate supratidal zone was the highest(65%).The muddy intertidal zone has the highest rate of petroleum degradation rate(85%),and the sandy tidal zone has the highest petroleum degradation rate(86%).To further study the petroleum hydrocarbon degradation strains,functional bacteria were further isolated.In total,9 bacteria strains were isolated.Among them,the degradation rate of n-hexadecane by Leclercia sp.B45 and Halomonas sp.A2 were 91%,and 64%,respectively.Both strain A2 and B45 produced surfactants during growth.The alkB gene of Leclercia sp.B45 was first amplified using degenerate primers,and then the optimal degradation concentration of alkane by Leclercia sp.B45 was determined by GC-MS.Real-time quantitative PCR(RT-qPCR)was performedto analyze the transcriptional pattern of alkB gene.The results showed that the transcription level of alkB gene in Leclercia sp.B45 was up-regulated when induced by C10,C16,and C28,which provided new information for the expansion of the substrate regulation range of alkB gene.Combined with other results in this section,it can be speculated that the up-regulation of alkB gene expression is associated with the efficient use of low-concentration carbon sources by B45.The Halomonas sp.A2 is halophilic.The results showed that Halomonas sp.A2 could degrade C10,C16,and C28.The seven-day degradation rate could reach 71%.The results of qRT-PCR indicated that the cytochrome P450-5 monooxygenase and mono-1 monooxygenase genes in Halomonas sp.A2 may be involved in the n-decane metabolism in this strain.The petroleum hydrocarbon degrading performance of high-salinity oil-field wastewater was examined in the microbial contact oxidation pond of Liujiu district wastewater treatment plant,Xinjiang Heavy Oil Company.The high-throughput sequencing was used to compare the biofilm community structure in three different depths and levels of oxidation ponds.During the operation,the total petroleum hydrocarbon,sulfide,ammonia,COD,BODs and pH of the effluent reached the Integrated Wastewater Discharge Standard(GB8978-1996).The average removal rate of COD,BODs,petroleum,ammonia,and sulfide was 65.4%,46.6%,54.9%,79.2%,and 62.1%,respectively.