| PAHs(polycyclic aromatic hydrocarbons)are aromatic hydrocarbons with two or more fused benzene rings in straight-lined,angled or group formations.PAHs could pose a serious threat to the environment and human health due to their intensive carcinogenicity,teratogenicity,and mutagenicity.Microbial remediation has been considered as the main pathway for soil PAHs removal for its high efficiency,low cost,and without secondary pollution.With high oxidation-reduction potential,nitrate is not only the important nitrogen source for soil microbes,but also the terminal electron acceptor for soil denitrifiers.Soil denitrifiers are widely distributioned in almost 70 genus,and it has previously been confirmed that soil PAHs could be degraded coupling with the dinitrification processdriven by soil denitrifiers.Presently,the response of soil denitrifiers to PAHs pollution was seldom reported.Therefore,9 soil samples listed as JH-1 to JH-9 were collected from the JiangHan oilfield with a history of more than 50 years in this study.To investigate the relationship between soil PAHs pollution and community structure of soil denitrifiers,the basic properties and PAHs content were determined,and using the functional genes(narG: nitrate reductase;nirK: Cu-nitrite reductase gene;nirS: cd1-nitrite reductase gene)involved in denitrification as biomarkers,the abundance and community structure of soil denitrifiers were detected by quantitative-PCR and T-RFLP(terminal-restriction fragment length polymorphism)combined with clone library,respectively.Subsequently,the response of activity and abundance of soil denitrifiers to a representative PAHs(pyrene)contamination was further confirmed by an anaerobic microcosm incubation experiment,and the effects of pyrene and nitrate amendments on soil bacterial community structure was studied using Illumina miseq sequencing.The objective was to preliminary investigate the response of soil denitrifiers to PAHs contamination,which might be helpful for deeper understanding of the interaction between PAHs pollution and soil denitrification process.The main results were as follows:1)The Results of direct survey for the natural soil in the Jianghan oilfield showed that the content of PAHs in the soil samples varied in the range of 0.21~8.10 mg·kg-1,which indicates that the soil in this region was not seriously contaminated by PAHs.Meanwhile,the quantitative-PCR analysis showed that the highest abundance of narG in the soil samples was also found in the highest PAHs content(JH-4),and the correlation analysis also showed a positively correlation between the copy numbers of narG and soil PAHs content(R2 = 0.58,P < 0.05).In contrast,the lowest copy numbers of nirK and nirS both were detected in the JH-4 with the highest PAHs content,and the correlation analysis showed a negative correlation between the copy numbers of them and soil PAHs content(nirK: R2 = 0.54,P < 0.05;nirS: R2 = 0.58,P < 0.05).Furthermore,the result of T-RFLP indicated that the nirK community structures in different soil samples varied significantly,which was obviously unique in the sample(JH-4)with the highest PAHs content.The subsequent RDA(Redundancy analysis)also demonstrated that soil PAHs content as well as the available nitrogen and phosphorus was one of the most important factors affecting the nirK community structure in this oilfield soil.Compared with nirK,few variation was shown about the nirS community structure among the soil samples.However,the abundance of nirS-harboring pseudomonas had a remarkably positive relation with the soil PAHs content,which indicated that pseudomonas with strong ability to degrade organic pollutants,might be an essential driver for PAHs degradation via denitrification process in this oilfield soil.2)To further investigate the response of activity and abundance of soil denitrifiers to PAHs stress,according to the result of section 1,a relative clean sample(JH-9)was selected,and an anaerobic soil microcosm incubation experiment with pyrene(a typical 4 ring PAHs,often used as indicator or mode molecules of PAHs biodegradation)amendment was conducted.In this experiment,two groups of soil treatments with(N30)and without(N0)nitrate(30 mg·kg-1)amendment were conducted,and each group contained three treatments with different pyrene concentrations(0,30,and 60 mg·kg-1 denoted as P0,P30,and P60,respectively).Thus,a total of six soil treatments presented by N0P0,N0P30,N0P60,N30P0,N30P30,and N30P60 were incubated under anaerobic condition for 45 days.Soil samples were collected at day 3,14,28,and 45 for the measures of PAHs,N2 O,CO2 and quantitative-PCR.The results showed that the final degradation rate of pyrene was in the range of 26%~43% at the end of incubation(day 45),and the higher degradation rates were found in the treatments amended with higher pyrene concentration.However,it seemed that nitrate amendment had no significant effect on the degradation of pyrene under this anaerobic condition.In addition,obvious production of N2 O in all treatments were only detected at the early stage of incubation(day 3),ranged from 122.82~379.22 μg·kg-1·h-1,and nitrate amendment had a significantly promote for the production rate of N2 O.Furthermore,no significant difference wasfound about the production rate of CO2 among those treatments at day 3,7 and 14,but which were obviously higher in the nitrate added treatments than these in the no nitrate added treatments at day45.Additionally,the results of quantitative-PCR indicated that the abundance of those denitrification concerning genes all showed a tendency of initial decrease and following increase during the incubation,and both nitrate and pyrene had no obvious effect on the abundances of these functional genes.3)To investigate the co-effect of nitrate and pyrene on soil bacterial community structure under anaerobic condition,four representative treatments(N0P0,N0P60,N30P0,N30P60)at day 45 in the previous microcosm experiment were selected for the following Illumina miseq sequencing.The results showed that bacterial diversity index(Chao1,shannon)in the treatments added pyrene or nitrate were higher than that in N0P0.The analysis of bacteria community structure based on the phylum showed that the dominant bacteria are Actinobacteria,Bacteroidetes,Chloroflexi,Firmicutes and Proteobacteria in soil samples.Furthermore,the treatments without nitrate addition(N0P0,N0P60)had similar bacterial composition,which was obviously different from these in the nitrate amended treatments(N30P0,N30P60).In detail,the nitrate added treatments had relative higher abundance of Actinobacteria and lower abundance of Proteobacteria than the treatments without nitrate amendment.In addition,those four soil treatments had similar dominant bacterial genus in both Actinobacteria and Proteobacteria,however the ratios of the dominant bacterial genus in the nitrate amendment treatments(N30P0,N30P60)were different from the no nitrate added treatments(N0P0,N0P60).Thus,under anaerobic condition,pyrene might have no acute effect on soil bacterial community structure,but nitrate could induce the shifts in soil bacterial community structure. |
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