Characteristics Of Halotolerant Heavy Oil-degrading Bacteria And Its Application On Bioremediation Of Oil-spilled Shoreline

Oil exploration,development and transportation activities are intensive in marine and coastal environment where are potentially threated by oilspills.The increasing development,processing and utilization increase the probability of heavy oil leaks.The heavy oil contains high-concentration resin and asphaltenes and is featured with refractory polycyclic aromatic hydrocarbons(PAHs),causing difficult remediation for heavy oil-spilled shoreline.In this study,the halotolerant heavy oil-degrading bacteria consortium was constructed based on the characteristics of heavy oil and shoreline environment.The consortium was utilized in bioremediation of intertidal zone and bioremediation coupled phytoremediation of saline soil,and the remediation conditions,degradation characteristics and mechanisms were investigated.(1)Three halotolerant heavy oil-degrading bacteria,Achromobacter xylosoxidans?Ochrobactrum anthropic and Sphingobacterium mizutaii,were isolated from the heavy oil-spilled shoreline at Dalian,Northeast China,based on investigation of degradation rate,reaction kinetics and salt tolerance.The latter was first reported in the petroleum pollution remediation.The heavy oil was emulsified during degradation of the three bacteria which all could utilize N-hexadecane and PAHs(Naphthalene,phenanthrene,anthracene,fluoranthene,pyrene and chrysenes)as a single carbon source,and the salt tolerance was up to 10 %.The consortium XG-349,which was constructed by the three bacteria,showed high degradation rate of heavy oil(60%)in comparison with sole bacteria(40%~50%).The degradation followed a first order kinetics,the reaction rate constant was 0.0842 d-1 and degradation half-life was 8.2 day.The optimized conditions of degradation were salinity at 3.5 %,pH value at 8.0 and 25??(2)The cylinder simulation equipment for ntertidal sediments was constructed to carry out the laboratory-scale bioremediation study for heavy oil-spilled shoreline.The consortium XG-349 in heavy oil-spilled sediments was rapidly proliferated after a short adaptation;the number of microorganisms and enzyme activity were significantly increased.However,the deterioration of consortium XG-349 occurred due to the deficient of nutrients and washing of seawater.The removal of total petroleum hydrocarbons(TPHs)was only 38.2±2.2% in 30 days.The addition of soluble nutrients(Urea+KH2PO4;SN)favored the early-stage remediation.The removal of TPHs in 30 days slightly increased to 41.4±1.5% owing to rapid loss of nutrients.No significant improvement was obtained in the long-term remediation.The slow-release fertilizer(SRF)Osmocote remained a high concentration of nitrogen and phosphorus under washing and dilution of seawater.The number of microorganisms and enzyme activity kept a high level during the whole remediation.Consequently,the removal of TPHs in 30 days was increased to 51.3±2.0%.(3)The simulation system for intertidal environments was constructed to carry out the pilot-scale bioremediation study for heavy oil-spilled shoreline.The removal of TPHs in XG-349 remediation(BioA)was 42.8±2.7% which was higher than 22.8±3.8% of natural attenuation(NA)in 30 days.BioA+SN slightly promoted the removal of TPHs to 48.2±2.4%.However,the removal for BioA+SRF was increased to 57.6±3.4% due to long degradation activity.The BioA,BioA+SN and BioA+SRF all enhanced removal of total N-alkanes.Among these,BioA+SRF improved degradation of >C20 long-chain N-alkanes.BioA showed high removal of total PAHs(74.0±4.7%)relative to NA(59.3±1.7%).The degradation of high molecular weight(HMW)PAHs including C2-pyrene,C3-pyrene,Benzo[e]pyrene and perylene was significantly improved in BioA.The appropriate nutritional conditions of BioA+SN and BioA+SRF benefited the degradation of biodegradable C2-Fluorene and Phenanthrene,increasing the removal of total PAHs(81.0±3.5% and 85.5±4.2%,respectively).The degradation of HMW PAHs was not improved in BioA+SN and BioA+SRF owing to high condense degree,strong hydrophobicity and alkyl side chains induced steric effect of HMW PAHs.(4)Testuca arundinacea was screened to apply to phytoremediation(PhytoR)and bioremediation coupled phytoremediation(PhytoR+BioA)due to high salt tolerance and TPHs removal.The BioA(58.9±2.6%),PhytoR(52.6±2.3%)and PhytoR+BioA(67.35±2.7%)showed high TPHs removals in pot experiment of saline soil remediation.The large number of microorganisms and high enzyme activity guaranteed high degradation rate of PhytoR+BioA.The removal of total PAHs in BioA,PhytoR and PhytoR+BioA was 78.4±4.9%,92.5±5.1% and 94.8±5.8%,respectively.The rhizosphere effect of PhytoR+BioA provided the degradability to total PAHs and HMW PAHs.The removal of TPHs in field experiment of BioA,PhytoR and PhytoR+BioA was up to 47.2±2.6%,42.2±2.6% and 56.9±2.7%,respectively.The results demonstrated the successful application of Consortium XG-349,and provided a beneficial reference for furture remediation of heavy oil-spilled shoreline.