Partitioning Characterization Of Nitrogen-transformation Pathways And The Related Microbial Mechanism From A Malodorous River

In order to mitigate the exacerbated contamination of urban waterbody and improve the aquatic environments,many endeavors have been made in treating and remediating the malodorous rivers throughout China.There was a sharp decrease of contaminants after the implementation of engineering projects,while the problem of nitrogen overloading in aquatic system has become prominent.Projects targeting malodorous waterbody remediation have put most emphasis on ammonium(NH4+-N)loss via nitrification instead of nitrogen(N)abatement,which lead to the limited total nitrogen(TN)removal.To find a solution for nitrogen overload,based on regulations of aeration,carbon/nitrogen(C/N)ratio increase and calcium nitrate addition,this research is intended to propose partitioning characterization of N-transformation pathways including ammonium oxidization,denitrification,anaerobic ammonium oxidization(Anammox),dissimilatory nitrate reduction to ammonium(DNRA)with stable isotope tracing technique and mass balance analysis,and to investigate the importance and contribution of each N-transformation process during N-cycling at the sediment-overlying water interface in the malodorous river.The mechanism in terms of the microbial diversity and functional gene abundance is also analysed via high-throughput pyrosequencing and real-time PCR analysis,respectively.This research may help to provide basic theory and referential significance for control of endogenous N release from sediments and the improvement of remediation projects for urban malodorous rivers.The main results of this study are as follow:(1)Based on the inhibition performance assessment of ammonium oxidizing bacteria(AOB)inhibitors including ampicillin,DCD,ATU,acetylene,chloroform and acetylene,ATU was screened as the optimal inhibitor for inhibition of nitrification process in the malodorous river,and the optimized inhibitory dosage for ATU was 25 ?mol·L-1.The inhibition of ATU on nitrification was mainly embodied by its capability of reducing the abundance of AOB-related genus Nitrosospira and inhibiting the activity of AOB.(2)Results indicated that among the controls of aeration,C/N ratio increase and calcium nitrate addition,monotonous aeration(Run 3)was the most effective regulation in terms of NH4+-N reduction.For instance,a sharp decrease of NH4+-N to 2.07±0.24 mg·L-1 was observed for Run 3 by the 18th day.Additionally,combinational regulations of aeration and C/N ratio increase(Run 5,7)were unfavorable to NH4+-N removal.Lag phases of 10?15 days were both presented for Run 5 and Run 7 as NH4+-N level decreased to 2 mg·L-1.In contrast,the combinational regulations favored the rapid removal of TN.Observations showed that level of TN decreased to below 2 mg·L-1 by the 34th?38th day for Run5 and Run7,respectively,shortening 22?26 days as compared to that of the monotonous aeration regulation.No difference was found in terms of NH4+-N and TN removal for Run 5(C/N 10)and Run 7(C/N 20),suggesting that carbon source was not the limiting factor for TN removal as C/N ratio level reached to 10.Calcium nitrate added to the sediment was unfavorable to NH4+-N and TN abatement as suggested by Run 9.Lag phases were observed for all operating conditions in period of NH4+-N and TN reduced to the legislative threshold of Class V of National Surface Water Quality Standard(2 mg·L-1)after addition of nitrification inhibitor.(3)Under aerated conditions,AOB predominated in the malodorous river,contributing 62.63?78.28%ammonium oxidation as compared to 21.72?37.37%for AOA.Additionally,results suggested that denitrification was the dominant pathway of nitrate removal in this malodorous river with the denitrification efficiency of 3.61?mol N·kg-1·h-1,accounting for 83.26?92.46%of total nitrate loss.Measured Anammox rates ranged 0.27?0.48 ?mol N kg-1· h-1,contributing 7.54?16.74%total nitrate reduction.DNRA efficiency was hardly detected.(4)For operating conditions with different C/N ratio levels,AOB took the predominance over AOA in the malodorous river,contributing 62.63?78.28%ammonium oxidation as compared to 21.72?37.37%for AOA,and contribution of AO A was hardly affected by C/N ratio level.Denitrification was the dominant pathway of nitrate removal as revealed by all operating conditions,contributing 64.58?89.17%total nitrate reduction as compared to 10.83?35.42%for Anammox.However,Run7 was an exception which presented equivalent contribution of denitrification and Anammox.Particularly,DNRA efficiency was hardly detected except for Run 5 which presented 5.18?7.45%contribution of DNRA for total nitrate reduction.(5)Contribution of AOA to ammonium oxidation presented an increase with the addition of calcium nitrate to the sediment,accounting up to 24.37?50.26%.Increase of denitri?fication and Anammox rate was observed after calcium nitrate was added.While denitrification was still the primary pathway of total nitrate reduction,accounting for 79.56?85.52%and 14.48?20.44%for Anammox.DNRA efficiency was hardly detected.(6)Bacteria within phylum Proteobacteria,Firmicutes and Chloroflexi predominated in the bacterial communities in sediment from this malodorous river,and marked decrease were presented for abundance of Firmicutes-like bacteria under different environmental controls,while bacteria affiliated to Proteobacteria were most affected by the addition of calcium nitrate;The dominant archaea communities are affiliated to MCG,Euryarchaeota,Crenarchaeota and Thaumarchaeota,and there was a remarkable increase of the predominant bacterial communities for all operating conditions as compared to the initial,whereas little difference was observed for them as affected by various environmental regulations.(7)Molecular biology analysis based on amoA suggested that AOB exhibited a significantly higher abundance than AOA in sediment sample from each condition,with the copy number ranging 3.71×105?1.13×106 copies/g dry sediment,almost 4?9 times as large as AOA numbers.Addition of nitrification inhibitor had adverse influence on AOB belonging to genus Nitrosomonas and Nitrosospira,while no obvious effects were observed for AOA.In addition,DO level was the primary environmental variable responsible for the difference among AOA community structure in various sediment samples.(8)For denitrifiers,markedly higher abundance of nirS was observed than that ofnarG,norB and nosZ,with the copy number ranging 5.39×10?5.47×108 copies/g dry sediment.The dominant denitrifying bacteria communities in the sediment were Pseudomonas,Sulfuritaleaand Dechloromonas.Nitrate was confirmed to be the limiting factor affecting the microbial community composition of denitrifiers among all the environmental controls.There was a sharp increase of bacteria affiliated to Thiobacillus after addition of calcium nitrate to the sediment,becoming the predominant genus.It was postulated that the process of sulfur-dependent autotrophic denitrification may be promoted with calcium nitrate addition.Nitrification inhibitor had no influence on functional gene targeted denitrifying bacteria except for narG-denitrifiers.(9)Candidatus_Jettenia-and Candidatus_Kuenenia-like microorganisms were the dominant Anammox bacteria in all sediment samples.Abundance and composition of Anammox bacterial community were greatly affected by calcium nitrate addition,with its abundance ranging from 2.06×104?8.79×104 copies/g dry sediment to 2.66×105?1.95.×106 copies/g dry sediment.And there was a remarkable increase of relative abundance of Cadidatus_Jettenia-like Anammox bacteria,accounting up to 73.30?73.83%.No variations were observed for Anammox bacteria in sediment samples from other operating conditions.(10)Increase of C/N ratio may stimulate growth of DNRA bacteria.Abundance of DNRA bacteria ranged 3.45×106?9.19×106 copies/g dry sediment in all sediment samples from different conditions.DNRA bacteria within the genus Desulfovibrio predominated in the sediment with the relative abundance ranging 0.7%?1.4%,and bacteria affiliated to spiece Desulfovibrio vulgaris and Desulfovibrio_termitidis were the dominant DNRA bacteria,while they might have low activity or related functional enzymes fail to transcript or express.