Study On The Coupling Mechanism Between Denitrification And Low Molecular Weight PAHs Metabolism

Polycyclic aromatic hydrocarbons（PAHs）,a class of hydrocarbon compounds,consisting of two or more benzene rings in straight-lined,angled or group formations,are mostly derived from oil production,combustion of fossil fuels,coal processing,etc.PAHs can be transmitted and migrated through the food chain,and gradually enriched in animals and plants due to their characteristics of mutagenicity,carcinogenicity,and bioaccumulation,which should pose a great threat to human health and the ecological environment.Compared with the aerobic degradation of PAHs,although the degradation rate under anaerobic conditions is lower,the role of anaerobic degradation of PAHs can not be ignored in some environments with large amounts of micro-aerobic or anaerobic micro-environments,such as sediments,water,and soil.Denitrification is widespread in terrestrial and aquatic ecosystems,and its coupling with anaerobic metabolism of PAHs has been confirmed.However,the mechanism of denitrification coupling with degradation of PAHs is still unclear,and the role of denitrification in the attenuation of PAHs in soil is still unsure.Therefore,to investgate the coupling mechanism between denitrification and PAHs metabolism,in this study,two bacterial consortiums with abilities to degradate naphthalene and phenanthrene under denitrification were enriched from the soil with potential risk of PAHs pollution,respectively.After a 9-day incubation,thedegradation rate of naphthalene or phenanthrene and the abundances of denitrification concerned genes（narG:periplasmic nitrate reeducates gene;nirS:cd₁-nitrite reductase gene）in each enriched consortium were detected,and the bacterial community composition in each enriched culture was analyzed by Illumina MiSeq Sequencing.Subsequently,the relationship between naphthalene metabolism and denitrification process in the naphthalene degradating consortium was further studied by continuous detection of the dynamic changes in naphthalene concentration,contents of denitrification-related substances（NO₃^-,NO₂^-,N₂O,N₂）,and narG and nirS abundances during the incubation.Finally,for the possible inhibiting effects of nitrification inhibitors（NIs）on soil denitrification activity,a representative NI（DMPP）was used in the following indoor soil incubation experiment to preliminarily investigate the potential influence of denitrification on soil phenanthrene elimination.The main conclusions are as follows:1)The denitrification-degrading bacterial consortiums of naphthalene and phenanthrene were enriched respectively,and the maximum degradation rates of naphthalene and phenanthrene were57.4%and 45.18%.In naphthalene and phenanthrene-degrading bacterial consortiums,the narG gene abundance was significantly lower and higher than nirS,respectively（p<0.05）,indicating that nirS-type denitrifying bacteria with nitrite reduction ability and narG-type denitrifying bacteria with nitrate reduction ability are dominant in the denitrification degradation process of naphthalene and phenanthrene respectively.The results of Illumina MiSeq sequencing showed that Pseudomonas bebong to Proteobacterial phylum played a leading role in the denitrification degradation of naphthalene and phenanthrene.In addition,other bacteria such as Methylophilus,Blastomonas and Azoarcus also participated in the denitrification metabolism of naphthalene.Methylophilus and Microbacteria might play a certain role in the metabolism of phenanthrene.2)The above-mentioned naphthalene-degrading bacteria consortiums were used as a subject to continuously analyze the dynamic changes of denitrification-related substances（NO₃^-,NO₂^-,N₂O,N₂）concentrations and the abundance of narG and nirS during the metabolism of naphthalene in the microbial consortiums.The results were as follows.Under anaerobic condition,the naphthalene removal rate of this enriched consortium was 49.11%within 9 days.Relative higher naphthalene degradation rates were found both at the beginning（day 1-3）and the end（day 7-9）of incubation,and which were significantly higher than that at the middle（day 3-7）incubation stage（P<0.05）.The content of nitrate decreased during the whole culture period,while the content of nitrite increased at the initial incubation（day 1-3）and followed by a rapid decrease from day 3 to 9.Furthermore,obvious productions of N₂O[3.39μg·（L·h）^-1]and N₂[8.97μg·（L·h）^-1]were only measured at the end of incubation（day 7-9）.Thus,it could be seen that both the nitrite accumulation and gas production stages were accompanied by relative higher naphthalene degradation rates,which might indicate that both the initial step（nitrate reducing stage）and the following gas producing steps（nitrite reducing stage）of denitrification could be tightly related to the anaerobic naphthalene degradation.The abundances of both narG and nirS increased during the incubation,indicating the continuous growth of denitrifiers in the enriched consortium along with the incubation period.In summary,this study illustrated that both nitrate reducing stage and gas producing steps of denitrification could be coupled with the anaerobic naphthalene degradation,which might be helpful for deeper investigation about the coupling mechanism between denitrification and anaerobic PAHs degradation.3)Recently,it has been reported that nitrification inhibitors could directly or indirectly inhibit soil denitrification enzyme activity.Thus,in this section,the relationship between denitrification process and PAHs elimination in soil was investigated through an indoor soil incubation experiment（60 days）with addition of representative NI-DMPP.Two kinds of incubation conditions（dry and water-logged）were conducted,and each condition contained five treatments,which were control（CK）,adding phenanthrene（F）,adding DMPP and phenanthrene（DF）,adding urea and phenanthrene（UF）,adding urea,DMPP and phenanthrene（UDF）.The soil ammonium and nitrate contents,phenanthrene content（60 days）,potential nitrification rate（PNR）,denitrification enzyme activity（DEA）,and abundances of soil ammonia-oxidizers（ammonia-oxidizing archaea,AOA;ammonia-oxidizing bacteria,AOB）and denitrification concerned genes（narG,nirS,nirK）were detected at day 7,14,28,and 60.The result indicated that under the conditions of dryland and flooded,there were a large accumulation of NH₄⁺in phenanthrene-contaminated soils with urea and DMPP,but higher levels of NO₃^-were found in the phenanthrene-contaminated soils where only urea were added.At the same time,potential nitrification rate also showed that the addition of DMPP could significantly inhibit the ammonia-oxidizing activity in phenanthrene-contaminated soils with addition of urea and DMPP.This showed that only by providing a nitrogen source,DMPP could better inhibit ammoxidation by inhibiting the activity of ammonia-oxidizing microorganisms.Denitrification enzyme activity was not inhibited under both incubation conditions,probably because the effect of DMPP on denitrification enzyme activity was also affected by other factors.In the result of functional gene abundances,it was found that the gene abundances of AOB in phenanthrene-contaminated soils with addition of urea and DMPP were significantly lower than that in soils containing only urea,but there was no significant difference about the abundances of AOA.This showed that the soil ammonia oxidation under the two conditions were mainly affected by AOB.The addition of DMPP to phenanthrene-contaminated soils containing urea had little effect on denitrification enzyme activity,but it had significant inhibition to denitrification concerned genes under flooded conditions.The addition of DMPP had no significant effect on the removal of phenanthrene in soil,which showed this study could not directly prove the potential influence of denitrification on soil phenanthrene elimination.