Construction Of Microbial Degradation Strategy And Rational Guidance To Remediate The Petroleum Hydrocarbon Contaminated-soil

With the continuous improvement of human awareness of ecological and environmental protection,the management of petroleum hydrocarbon pollution has become a focal issue.At present,bioremediation technology has shown great application potential in the remediation of petroleum hydrocarbon contaminated sites,but petroleum hydrocarbons are toxic and hydrophobic,leading to bioremediation technology showing long degradation cycle and low efficiency.Therefore,exploring and establishing an efficient,environmentally friendly,and low-consumption bioremediation strategy is of great significance for the remediation of contaminated sites.In this study,microbial remediation strategy was constructed and optimized to remedy petroleum hydrocarbon contaminated soil,including paraffins(C₁₃-C²¹),polycyclic aromatic hydrocarbons(naphthalene and phenanthrene).The optimized degradation strategy was further applied to evaluate the corresponding remediation characteristics of petroleum hydrocarbon-contaminated soil,which enable to achieve the soil environmental quality-Risk control standard for soil contaminated of development land(trial)(GB 36600-2018).The detailed results will be demonstrated in the following three aspects:(1)Enrichment of petroleum hydrocarbon degrading bacteria and the corresponding pressure response of high concentration petroleum hydrocarbonThe activated sludge was first domesticated by gradual increasing petroleum hydrocarbon content(from 1000 mg/L to 20000 mg/L)in the airlift reactor.The results showed that the degradation efficiency of microbial community in high concentration petroleum hydrocarbon(over 10000 mg/L)was significant lower than these in low concentration petroleum hydrocarbon(below 7000 mg/L)in the airlift reactor(reaction liquid volume 1 L,temperature 30°C,aeration volume 2.0 L/L·h,activated sludge inoculation volume 10 m L).Meanwhile,the short chain alkanes(C₁₃-C₁₆)were priority utilized by microorganisms.Specifically,with the increase of petroleum hydrocarbon concentration,the diversity of microbial community was decreased,and the dominant flora was trend to stable.The microbial community function under high concentration of petroleum hydrocarbon(10000 mg/L and 20000 mg/L)was compared by metagenomics,which suggested that the relative abundance of microbial community functions of environmental adaptation,membrane transport,replication and repair were increased.However,the digestive system,circulatory system,and endocrine system of the microbial community were significantly disturbed by high concentration petroleum hydrocarbon,and eventually caused the decrease of petroleum hydrocarbon degradation rate by the activated sludge.Additionally,the application of activated sludge was limited by redundant microbial community,complex interactions,and uncertain contents.Therefore,the single bacteria or mixed flora would be further isolated from activated sludge to enhance the microbial degrading efficiency for the high concentrations of petroleum hydrocarbons.(2)Optimization of petroleum hydrocarbon degradation strategy and functional characteristics analysis of the hydrocarbons metabolism by microbial floraFrom the above activated sludge(after acclimated)with gradual domestication,six petroleum hydrocarbon degraders were isolated and identified,such as D-1(Microbacterium),D-2(Kocuria marina),D-4(Kocuria rosea),D-5(Microbacterium),D-6(Staphylococcus capitis)and D-7(Bacillus odysseyi).Then,the degradation experiments of petroleum hydrocarbon(5000 mg/L,10000 mg/L,and15000 mg/L of paraffins and polycyclic aromatic hydrocarbons)were further performed using the above identified strains respectively.The highest degradation rate of petroleum hydrocarbon was appeared in 5000 mg/L after 14 d.However,with the increasing of petroleum hydrocarbon concentration(over 10000 mg/L),both of the biomass and degradation rate of single bacteria decreased significantly.To improve the degradation rate of petroleum hydrocarbon with high concentration(10000 mg/L petroleum hydrocarbon),mixed flora(two strains)was further constructed to promote the hydrocarbon metabolism of the microorganism combination.Under the degradation conditions of D-4+D-6(reaction solution volume80 ml,temperature 30°C,rotating speed 200 r/min,initial p H=7.0,bacterial inoculation ratio 1:1),the highest petroleum hydrocarbon degradation rate was observed(reach to 72.09±7.14%)among the all mixed flora.Thereinto,the residual contents of paraffins and polycyclic aromatic hydrocarbons were 1618.31±116.85mg/L and 1172.69±95.81 mg/L at 14 d of degradation,respectively.Furtherly,the strategy of the surfactant was evaluated by exogenous feeding experiments to enhance the degradation rate of mixed flora.Under the condition of Tween80 addition,D-4+D-7 displayed the highest efficiency(81.58±6.29%)to degrade 10000 mg/L petroleum hydrocarbon.According to the results of comparative macrogenomics,the relative abundance of global overmaps,amino acid metabolism,and carbohydrate metabolism function modules was increased in the mixed flora(D-4+D-7).In the combination of D-5+D-7 with rhamnolipid addition,the relative abundances of several functional modules exhibited the obvious enhancement,such as the transport,absorption and degradation of petroleum hydrocarbon.Meanwhile,the gene abundance of alk B and nah was also significant enchanced by adding rhamnolipid.Finally,the degradation rate of total petroleum hydrocarbon could reach to 88.35±7.58%(14 d)in the group of D-5+D-7(rhamnolipid addition).And the residual contents of paraffins and polycyclic aromatic hydrocarbons were745.23±66.05 mg/L and 419.77±36.49 mg/L in above degradation system,respectively.After the comparative analysis,it was also found that the petroleum hydrocarbon degradation strategy(combination of rhamnolipid and D-5+D-7)constructed in this work has the highest efficiency,which can be used to rationally remediate petroleum hydrocarbon contaminated soil.(3)Bioremediation characteristics of petroleum hydrocarbon contaminated soilIn the remediation conditions of petroleum hydrocarbon-contaminated soil(reactor 60.0 L,total petroleum hydrocarbon contents 9316.41±625.87 mg/kg,temperature 20°C-25°C,water content 25%-30%),the bacterial flora diversity,petroleum hydrocarbon degrader number(total petroleum hydrocarbon,alkane,polycyclic aromatic hydrocarbons),and soil enzyme activity(catalase,lipase,dehydrogenase,phosphatase,fluorescein diacetate hydrolase)was significant enhanced using bio-stimulation(added nitrogen and phosphorus)plus bio-augmentation(added D-5 and D-7)plus SER(added rhamnolipid).After 126 d of remediation,the total petroleum hydrocarbon contents of contaminated soil was decreased to 2558.29±138.59 mg/kg,which was lower than the level of soil environmental quality-risk control standard of class II land in soil contaminated of development land(trial)(GB 36600-2018).