Investigation About The Response Of Soil Methanogensis To Polycyclic Aromatic Hydrocarbons And Its Contribution To Soil Phenanthrene Elimination

Polycyclic aromatic hydrocarbons（PAHs）are a class of typical pollutants with a strong"three-factor"effect,which are widely distributed in various environments,such as water,atmosphere,sedimentand soil.In recent years,many studies have paid attentions to the response of soil microorganisms to PAHs,and confirmed the sensitive responses of soil microbial community to PAHs and the closely relationship between soil microbial community and PAHs degradation.It is well known that PAHs can be degraded by microbes under both aerobic and anaerobic conditions,and the latter is more economic and energy-saving than the former.The degradation of PAHs under mthanogensisis considered to be an ideal pathway because PAHs can be converted into methane（CH4）gas that can be used as a clean energy source.Presently,the degradation of PAHs under methanogensis has been confirmed or inveatigated in many natural habitats with anaerobic environments,such as sediments,sewage,sludge.Soil is the largest PAHs sink and CH₄ source in the terrestrial ecosystem,but few studies had reported the PAHs degradation under methanogensis in soil environment.In the soil environment,both the response of methanogensis to PAHs and the potential role of methanogensis to PAHs elimination are still unknown.Therefore,in this study,soil samples were collected from the Fuling shale gas field,and then the relations among the soil proterties,methane production activity,and prokaryotic microbial community were investigated.Subsequently,through an inner-room incubation experiment,a typical PAHs with 3 rings（phenanthrene）was spiked into the clean upland and paddy soils,respectively,and the responses of soil methanogensis and bacterial community to phenanthrene were studied.Finally,in a microcosm incubation experiment with the paddy soil with strong MPA,a specific methanogenic inhibitor（2-bromoethane sulfonate sodium,BES）was used to selectively inhibit the MPA in the soil contaminated by phenanthrene,and then the potential contribution of methanogensis to phenanthrene elimination and its microbial mechanism were investigated.The main results are as follows:（1）Soil methane generation mainly driven by soil prokaryotic microbes can be coupled with the degradation of polycyclic aromatic hydrocarbons（PAHs）;however,the relationship between prokaryotic community structure and methane production activity in soil with the potential risk of PAHs contamination is seldom reported.In this study,3 soil samples（CS-1 to CS-3）in the area nearby an exploratory gas well and 5soil samples（DC-1 to DC-5）in a drill cutting dump area were obtained from the Fuling shale gas field（Chongqing City,China）.Then,the prokaryotic community structure was examined by Illumina Miseq sequencing,and the linkage between soil methane production activity（MPA）and prokaryotic community composition was analyzed.The results indicated that 2 samples（DC-4 and DC-5）collected from the drill cutting dump area had significantly higher MPA than the other samples,and a significant and positive relationship（r=0.44,P<0.05）was found between soil MPA and soil organic matter（OM）content.The prokaryotic community composition in the sample（DC-5）with the highest MPA was different from those in the other samples,and soil OM and MPR were the major factors significantly correlated with the prokaryotic community structure in this soil.The samples（DC-4 and DC-5）with higher MPA had a higher relative abundance of Archaea and different archaeal community structures from the other samples,and the MPA was the sole factor significantly correlated with the archaeal genus composition in this soil.Therefore,both the prokaryotic and archaeal community structures are essential in the determination of soil MPA,and the bacterial genus of Saccharibacteria and the archaeal genus of Methanolobus might be the key contributors for methane generation in this soil from the shale gas field.（2）Through a microcosm incubation experiment,the responses of soil methane production activity（MPA）and microbial community to phenanthrene pollution were studied.There were 2 groups of treatments conducted with the upland and paddy soils,and each group contained 4 sub-treatments with different concentrations of phenanthrene(0,10,50,100 mg·kg^-1).A total of 8 treatments（upland group:DCK,DPL,DPM,and DPH;paddy group:PCK,PPL,PPM,and PPH）were incubated at room temperature for 120 days,and soil samples were taken at day 3,30,60,and 120for the detection of soil MPA,denitrification enzyme activity（DEA）,phenanthrene removal rate（PDR）,and mcrA（Methyl coenzyme M reductase gene）abundance,and microbial community structure（only day 120）.The results showed that,during the incubation,the PDR in both the upland and paddy soil groups increased with time,and the PDR of dry land group was significantly higher than that of the paddy group.Although the MPA and DEA of the paddy group were much higher than those of the upland group,the MPA and DEA in both group showed no obvious responses to to the phenanthrene pollution.The abundance of mcrA gene in paddy soil with medium and high concentrations（PPM and PPH）were significantly higher than that without phenanthrene（PCK）,indicated that the abundance of mcrA gene in paddy soil had a significant response to phenanthrene pollution.The bacterial diversity of the paddy group were significantly lower than that of the upland group,while the microbial diversity in each group was not sensitive to the phenanthrene pollution.The OTU-based community structure of upland group had significant response to phenanthrene pollution,while paddy group showed no responses to to the phenanthrene pollution.The community structure of prokaryotic microorganisms based on phylum level in upland and paddy group showed no responses to to the phenanthrene pollution.（3）As the result above indicated that the MPA in both upland and paddy soil was not sensitive to phenanthrene,the hypothesis that soil methanogensis could participate in the phenenthrene metabolism in soil should be theoretically fesible.In this study,a specific methanogenic inhibitor（2-bromoethanesulfonate,BES）was used to inhibit soil methanogenic activity,and six paddy soil treatments were conducted for clean soil（CK）,clean soil amended with low(ML,BES:0.5 m M·kg^-1)and high(MH,BES:1.5m M·kg^-1)concentration of BES,phenanthrene polluted soil(P,phenanthrene:50mg×kg^-1),phenanthrene polluted soil amended with low(PML,phenanthrene:50mg×kg^-1,BES:0.5 m M·kg^-1)and high(PMH,phenanthrene:50 mg×kg^-1,BES:1.5m M·kg^-1)concentration of BES.After incubation at room temperature in dark for 60days,these treatments were divided into two groups（with and without phenanthrene pollution）for the analysis of methane production activity（MPA）,phenanthrene degradation rate（PDR）,and bacterial and methanogenic ecological characteristics includinga-diversity,community structure,and co-occurrence.The results indicated that the BES amended treatments（ML,MH,PML,and PMH）had significantly lower MPA than those no BES treatments（CK and P）,and high BES concentration(1.5m M·kg^-1)inhibited 72.01%and 60.00%of MPA in the without and with phenanthrene groups,respectively.For the phenanthrene polluted group,the PDR in the no BES treatment（P）was significantly higher（P<0.05）than that in the high BES amended treatment（PMH）,and a significantly positive relationship（R²=0.80,P<0.01）was detected between the soil MPA and PDR.This result clarified the potential role of methanogensis to PAHs elimination in paddy fields.The amendment of BES significantly lower the abundance of mcrA gene,and a significantly positive relationship（R²=0.006,P<0.05）was detected between the soil MPA and the abundance of mcrA gene,further confirmed the specific inhibition of BES on methanogens.The amendment of BES significantly changed the bacterial and methanogenic community structures in both groups,and it was the microbial community structure but not thea-diversity was significantly correlated with soil MPA in both groups and PDR in the phenanthrene polluted group.The result of net-work analysis revealed that BES decentralized the co-occurrence of bacterial genera but intensified the co-occurrence of methanogens,indicating that microbial co-occurrence should be an ecological parameter closely related to soil MPA and pollutants metabolism.The correlation analysis between bacterial genera and methanogenic species in the phenanthrene polluted group revealed that some key bacterial genera,such as Bacteroidetes-vadin HA17,Gemmatimonas,and Sporomusaceae should be responsible for the phenanthrene degradation and CH₄ production in this paddy soil.The above results confirmed the potential role of methanogensis to PAHs elimination in paddy fields.Meanwhile,it was studied that the microbial community structure（especially the bacterial）and the co-occurrence of microbial networks were key indicators that affected the soil methanogensis and phenanthrene metabolism.