Bioremediation Of Crude Oil-contaminated Soil By Microorganisms Immobilized On Modified Biofuel Ash

Crude oil spillage happens during the processes of petroleum exploration,transportation,storage and refining.Spilled petroleum enters surface water,groundwater and soil,it attaches to plant roots and gets absorbed by the plants,and through a food web ultimately reaches humans,affecting human health?soil-plant-animal-human?.Crude oil contamination is a worldwide environmental problem.Many physical and chemical methods had been developed to remediate petroleum-contaminated soil.However,most of those methods are expensive and have high energy demands,causing secondary contamination.Bioremediation has been regarded as a cost-effective method that can be easily implemented and is environmentally friendly,although it is usually time-consuming compared with other physical or chemical remediation methods.A method to improve the activity of introduced microbes is to immobilize them on the microorganism immobilization carrier?MIC?.MIC can shield the introduced microbes from indigenous bacterial competition,provide protective space and improve enzymatic activities,meanwhile achieve the goal of high petroleum biodegradation rate.Biofuel ash?BFA?is produced by burning of biomass in biomass power plants,with porous structure,bigger specific surface area and the ability of improving soil physical and chemical properties.The modified BFA can be used for MICs,which can simultaneously improve the microbial degradation ability and realize waste recycling.The mixed strains of bacteria,named M3 was isolated from oil-contaminated soil of Xinjiang Province.There were both Gram-positive bacteria and Gram-negative bacteria.The degradation ability of M3 for petroleum contaminated solution?1 g/L?was 73.3%,higher than any other single petroleum degraded bacteria.The biofuel ash between 100-and 40-mesh sieve was added to the modification solution in a ratio of 1:1?m/v?.After natural drying,it was used to immobilize M3.The cell concentration in modified-biofuel ash reached 10¹² CFU/g BFA,which was 3-4 orders of magnitude higher than other modification methods refered in previous studies.The degradation rate for immobilized M3 in oil contaminated soil,whose initial petroleum concentration was 52800mg/kg,reached 51.92%.The most easily degraded component was saturated hydrocarbon,followed by aromatics and resins.The residual content of n-alkanes?C7-C40?was determined by GC-FID.The short chain alkanes were most susceptible to degrade.The degradation rate of C17-C19 reached 70.6%;Immobilized M3 could degraded C28 and C31-C34,which could not be degraded by free cells.Immobilized degrading bacteria enhanced degradation rate of crude oil by increasing enzymatic activities,microbial respiration rate and carbon sources utilization.The enzymatic activities such as dehydrogenase and polyphenol oxidase were higher than free cells significantly,which reached 110.3 ug·d^-1·g^-1 and 163.2 mg·d^-1·g^-1 in IC1.The basic respiration rate of immobilized M3 was also higher obviously in than free cell.The biggest respiration rate in IC1 was 1.92 mgCO₂-C g^-124h^-1.Immobilized technology could improve the biodiversity of the degrading bacteria and the Shannon evenness under extreme conditions.The Shannon Weiner index and Shannon evenness index of IC1?FC1?S+IC?S+FC were 2.71±0.02?1.92±0.05?1.23±0.07?0.44±0.06 and 0.79±0.002?0.56±0.001?0.35±0.005?0.13±0.001,respectively.There were no obvious differences between IC1 and FC1,however,the differences between S+IC and S+FC were significant.