Degradation Of Soil Pyrene And Its Effect On Denitrification Process Under Anoxic Condition

Soil has been considered as the most important natural reservoir for polycyclic aromatic hydrocarbons?PAHs?.PAHs derived from anthropogenic activities such as incineration,combustion,automobile exhaust,and industrial production can enter the soil through direct or indirect ways,threatening the soil ecosystem and endangering human health.As a complex eco-system,a large amount of micro-aerobic or anaerobic micro-environment may generally exist in soil,and PAHs in soil is usually adsorbed by soil particles and enwrapped by soil colloids.So the role of anaerobic metabolism in the removal of soil PAHs cannot be ignored.It has previously been confrmed that PAHs could be degraded by soil microbes coupling with denitrifcation,but the relationship between soil denitrifers and PAHs metabolism under anaerobic condition is still unclear.Here,through an anoxic soil incubation experiment,the effect of nitrate on soil pyerne anaerobic degradation,denitrification activity,abundances of denitrification concerned genes and the structure of nirS-type bacterial community was investigated.Subsequently,the effects of different electron acceptors?nitrate and sulfate?on pyrene anaerobic degradation and denitrification process were further investigated under anaerobic soil condition.Finally,the Illumina Miseq Sequencing was used to analyze the effects of nitrate and sulfate on the bacterial community structure in those pyerne contaminated soil treatment under anaerobic condition.The objective was to investigate the relationship between soil denitrification process and the elimination of PAHs under anaerobic condition,which might provide some useful information for bioremediation of PAHs contaminated soil.The main results were as follows:1.Here,to comparatively investigate the effect of nitrate on soil pyrene degradation and denitrification under anaerobic and micro-aerobic conditions,two groups?anaerobic and micro-aerobic groups?of treatments were conducted,and each group contained four treatments with two nitrate concentrations(0mg·kg^-1,N₀;5mg·kg^-1,N₅)and two pyrene concentrations(0mg·kg^-1,P₀;100mg·kg^-1,P₁₀₀),denoted as N₀P₀,N₅P₀,N₀P₁₀₀,and N₅P₁₀₀.Those two groups of treatments were incubated in darkness at 25 ^oC for 45 days under anaerobic and micro-aerobic condition.The pyrene content,soil denitrifcation enzyme activity?DEA?and abundances of denitrifcation concerned genes?narG,periplasmic nitrate reductase gene;nirS,cd1-nitrite reductase gene;nirK,copper-containing nitrite reductase gene?were measured at day 3,14,28,and 45.The results indicated that the final removal rate of pyrene in the N₅P₁₀₀ treatments?29.09%,34.32%?were significantly higher than the N₀P₁₀₀ treatments?22.93%,26.66%?under anaerobic and micro-aerobic conditions?P<0.05?.The final removal rate of pyrene under micro-aerobic conditions was significantly higher than that under anaerobic conditions?P<0.05?.The addition of nitrate significantly promoted the final removal rate of pyrene under both incubation conditions.The DEA of all treatments were gradually increased during incubation.Under anaerobic and micro-aerobic conditions,the DEA of treatments amended with pyrene(N₀P₁₀₀,N₅P₁₀₀)were lower than these of the no pyrene amended treatment?N₀P₀,N₅P₀??P<0.05?.The DEA of treatments(N₀P₁₀₀,N₅P₁₀₀)under micro-aerobic condition were significantly higher than that under anaerobic condition?P<0.05?.It can be seen that the addition of pyrene significantly inhibited the soil DEA.Under anaerobic conditions,the addition of pyrene showed the inhibitory effect on the abundances of narG and nirK genes,but revealled promotive effect on the abundance of nirS gene.While under micro-aerobic condition,nitrate and pyrene could promotive the abundance of narG and nirS genes,but had no significant effect on the abundance of nirK gene.The result of Illumina Miseq Sequencing indicated that the nirS-type denitrifying bacterial OTUs and diversity index?Chao1,Shannon?in the treatments amended with pyrene(N₀P₁₀₀,N₅P₁₀₀)were lower than these of the no pyrene amended treatment?N₀P₀,N₅P₀?.The diversity of treatments under micro-oxygen condition was significantly higher than that under anaerobic condition.Based on the phylum,there was a difference in bacterial community structure between anaerobic with micro-aerobic condition.The relative abundance of the Proteobacteria in the no pyrene amended treatments?N₀P₀,N₅P₀?under micro-aerobic condition were higher than that under anaerobic condition,while the abundance of the Proteobacteria in the pyrene amended treatments(N₀P₁₀₀,N₅P₁₀₀)were lower than that under anaerobic condition.Moreover,Rubrivivax and Ralstonia were found to be enriched in the pyrene-added treatment under anaerobic and micro-aerobic condition,indicating that these two nirS-type denitrifying bacteria might be important for the soil pyrene degradation under anoxic condition.2.The results of section 1 showed that the addition of nitrate could significantly improve the final removal rate of pyrene.Therefore,in this section,the co-effect of two representative electronic acceptors?nitrate and sulfate?on anaerobic pyrene degradation,DEA,and abundances of denitrifcation concerning genes in pyrene(60mg·kg^-1)contaminated soil was further studied.Two groups of treatments without?N₀?and with(N₂₀)nitrate(20mg·kg^-1)amendment were conducted,and each group contained three treatments with different sulfate concentrations(0,10,and 30mg·kg^-1,denoted as S₀,S₁₀,and S₃₀ respectively).Thus,a total of six treatments(N₀S₀,N₀S₁₀,N₀S₃₀,N₂₀S₀,N₂₀S₁₀,N₂₀S₃₀)was incubated at 25^oC in darkness for 45days,and soil samples were collected at day3,14,28 and 45 for the measurments of those items same to part 1.The results indicated that the removal rate of pyrene in the treatment amended with nitrate(N₂₀S₀)was significantly higher than that of N₀S₀ treatment?P<0.05?,while the removal rate in the treatment amended with sulfate(N₀S₃₀)treatment was significantly lower than that of N₀S₀ treatment?P<0.05?.The removal rate of pyrene in treatments(N₂₀S₁₀,N₂₀S₃₀)were significantly higher than that of other treatments?P<0.05?.So the addition of nitrate significantly increased the removal rate of pyrene,which was consistent with the result of part 1.Although single amendment of sulfate significantly inhibited the removal rate,simultaneous addition of nitrate and sulfate significantly improved the removal rate of pyrene.The DEA of all treatments showed a tendency of initial increase and following decrease during the incubation.At day 28,N₂₀S₀ and N₂₀S₁₀ treatments were significantly higher than N₀S₀ treatment?P<0.05?,and they were significantly higher than those of N₀S₁₀ and N₀S₃₀ treatments?P<0.05?.Hence,sole addition of nitrate and sulfate could significantly enhance and inhibit soil DEA,respectively.The quantitative PCR results showed that the addition of nitrate significantly promoted the abundance of narG and nirK genes,but the addition of sulfate significantly inhibited that.3.In order to further understand the effect of nitrate and sulfate on contaminated soil bacterial community structure under anaerobic conditions,treatments at day 3 and 45 in the previous experiment were selected for following Illumina MiSeq sequencing.The result showed that the bacterial OTUs and diversity index?Chao1,Shannon?in the treatments amended with nitrate or sulfate(N₂₀S₀,N₀S₁₀,N₀S₃₀,N₂₀S₁₀,N₂₀S₃₀)were lower than that in N₀P₀ treatment.In the initial stage of cultivation,different treatments had similar community structure.In the later stage of cultivation,the relative abundance of Proteobacteria in all treatments was higher than that in N₀S₀ treatment.Compared with N₀S₀ treatment,the treatment amended with nitrate(N₂₀S₀)has lower abundance of Firmicutes and Actinobacteria,the treatments single amended with sulfate(N₀S₁₀,N₀S₃₀)had lower abundance of Chloroflexi and the treatments simultaneous addition nitrate and sulfate(N₂₀S₁₀,N₂₀S₃₀)had lower abundance of Firmicutes and Chloroflexi.Based on genus,the treatments without sulfate addition(N₀S₀ and N₂₀S₀)had similar bacterial community structure,which was obviously different from these in the sulfate amended treatments(N₀S₁₀,N₀S₃₀,N₂₀S₃₀).Further analysis revealed that the the treatments amended with sulfate(N₀S₁₀,N₀S₃₀,N₂₀S₁₀,N₂₀S₃₀)had higher abundance of Methylophilaceae,implying the strongly selective effect of sulfate on this bacterial genus.Thus,under anaerobic condition,nitrate might have no actute effect on soil bacterial community structure,but sulfate could induce the shifts in soil bacterial community structure.