The Coupling Mechanism Of Sulfur Autotrophic Denitrification And Anaerobic Ammonia Oxidation In Urban River

The urban river after the remediation of black and odor still existed the common difficulty of high nitrogen loading.Sulfur is a vital element of the river ecosystem,and its impact on nitrogen transformation processes should not be ignored in the rivers.It is worth investigating how the traditionally chemoautotrophic processes such as anaerobic ammonium oxidation（anammox）and sulfur autotrophic denitrification（SAD）participate in the nitrogen transformation pathways.Based on the investigation of current status of the river after remediation of black and odor,this study jointly uses high-throughput sequencing,quantitative polymerase chain reaction（q PCR）,metagenomics and metatranscriptomic techniques to systematically evaluate the potential coupling relationship between SAD and anammox under different external controls.What’s more,the simulated river system was made to further explore the coupling relationship between SAD and anammox under different external controls（include calcium nitrate injection,intermittent aeration,sodium thiosulfate addition and pyrite addition）,analyze the related microbial mechanism and determine the relatively optimized strategy.This study can hopefully improve the basic theory of hydrosphere microbes driving the nitrogen and sulfur cycle mechanism,and help establish technical support to address the challenges associated with high nitrogen level in urban polluted river after eliminating the black and odor.The main results of this study are as follows:（1）After remediation of black and odor,the DO content of the urban river was higher（3.26-6.78 mg/L）,and the p H value was 7.32-7.54;the concentrations of COD_Mnand COD_Cr were 4.80-8.82 mg/L and 33.90-42.33 mg/L,respectively;the major form of nitrogen in overlying water was nitrate,and the concentrations of TN and TP were2.01-4.27 mg/L and 0.15-0.41 mg/L,respectively,which were much higher than the limitation of eutrophication;and the sulfate content was higher while the sulfide content was lower in the overlying water.In addition,ammonium was the major form of nitrogen in pore water,and TN concentration was 26.60-72.33 mg/L.The concentration of AVS（Acid volatile sulfur）in sediment was 2356.15-4042.54 mg/kg.After the remediation of black and odor,the microorganisms in sediment of urban rivers were dominated by the functional genus of nitrogen conversion,sulfur conversion,and degradation of organic matter,mainly including sulfur oxidation related genus Thiobacillus,denitrification related genus norank＿f＿Steroidobacteraceae,Dechloromonas and norank＿f＿Anaerolineaceae as well as organic matter degradation related genus norank＿f＿Bacteroidetes＿vadin HA17 and norank＿f＿norank＿o＿SBR103.In addition,the q PCR results showed that the amount of anammox bacteria hzs B in river sediment was 4.64ⅹ10⁴-1.76ⅹ10⁶ copies/g dry sediment.（2）The NO₃^--N/AVS ratio of 0.4 is the optimal dosage for the coupling of SAD and anammox with addition of calcium nitrate,and the removal efficiency of TN can reach more than 90%.During the stable phase,the concentration of ammonium was14.10±0.10 mg/L.The results indicated that the dosage ratio of calcium nitrate to AVS in this system helped to stimulate the occurrence of sulfur autotrophic partial denitrification which can provide the substrate（nitrate）for anammox,thereby promoting the sulfur autotrophic partial denitrification coupled with anammox for nitrogen removal.Microbial analysis showed that the relative abundances of sulfur autotrophic denitrification related genus Thiobacillus and Sulfurimonas as well as anammox related genus OLB14 showed a trend of simultaneous and significant increase.The macrotranscriptome sequencing results also showed that the denitrifying desulfurization bacteria Thiobacillus and Sulfurimonas,and the anammox bacteria Candidatus Brocadia and Candidatus Jettenia had active expressions in the system with relative abundances of 0.12%,0.06%,1.78%and 0.07%,respectively,further illustrating the existence of SAD and anammox in the system.In addition,the high quantities of sulfur oxidation functional genes sox B,denitrification functional genes nar G and nir S,and anammox functional gene hzs B in this system indicated that the system enhances the activity of sulfur oxidation,denitrification,and anammox microorganisms.The results of metagenomic sequencing also showed that the relative abundance of genes related to denitrification and sulfur oxidation processes increased significantly in the system with NO₃^--N/AVS ratio of 0.4,especially the higher relative abundance of the membrane-bound nitrate reductase gene nar GHI（0.187%）,which confirmed that the strong sulfur autotrophic partial denitrification process in the system.（3）The coupling of sulfur autotrophic denitrification and anammox exists in the systems with typical sulfur-iron minerals.However,the coupling of the two is relatively weak compared with our previous research due to the influence of the release of endogenous ammonium and low nitrate concentration in the sediments.It was found that the common sulfur autotrophic denitrifying bacteria Thiobacillus was the dominant genus in the system based on the high-throughput sequencing analysis.The results of metatranscriptomic sequencing showed that the relatively few of Thiobacillus had the ability of active expressions,and the abundances of Thiobacillus in the sodium thiosulfate group and pyrite group were 0.020%and 0.009%,respectively.It was also found that the anammox bacteria Candidatus Scalindua with active expression existed in these two systems,and the relative abundances of it were 0.008%and 0.014%,respectively.In addition,q PCR results indicated that the abundance ratios of the functional genes hzs B and nir S increased significantly while sox B and nar G changed slightly.The metagenomic sequencing results also found that the expression of these functional genes,but the relative abundance of Nar GHI gene in the system was higher than that of nir S and sox B,and the Hzs gene was not detected.These inconsistent results indicate that microbial technology has certain limitations,which should be combined with other approaches such as isotopes trace to analyze the coupling comprehensively.（4）Different external controls have different effects on the decrease of concentrations of ammonium and TN in the simulated river system.Among them,intermittent aeration was conducive to the rapid decrease of ammonium level in the overlying water（7-9 d）,but this regulation had a relatively poor effect on the decrease of TN concentration（79-82 d）.It took pyrite group 11-13 d to achieve ammonium concentration below 2 mg/L,and 67-70 d to achieve TN concentration below 2 mg/L.Overall,addition of sodium thiosulfate was beneficial to the rapid removal of ammonium and TN in the overlying water（9-11 d and 55-58 d,respectively）,which was a relatively optimal control.The optimal dosage of sodium thiosulfate was S₂O₃^2--S/NO₃^--N=1（molar ratio）.Although calcium nitrate injection was beneficial to the rapid decrease of ammonium of the overlying water in river simulated system（less than 1mg/L）,the effect of it on TN removal is not as good as the potential experiment.This difference is mainly due to the differences of DO content and the method of calcium nitrate injection.It is suggested that the calcium nitrate should be injected into the sludge as deeper as possible in the actual project to make it contact better with the anaerobic system of the bottom sludge,thereby strengthening the removal of endogenous nitrogen.Different external controls have different effects on the facilitation of the coupling of SAD and anammox in river simulated systems.This study found that a relatively strong coupling process of sulfur autotrophic partial denitrification and anammox in the system with sodium thiosulfate.The detected related bacterial genus in the sodium thiosulfate system included Thiobacillus,Sulfurimonas and Denitratisoma,and the relative abundances of them are 2.17%,0.007%and 0.50%,respectively.In addition,the q PCR results also confirmed the strong activity of sulfur oxidation,denitrification and anammox microbes in the sodium thiosulfate system,and the abundance ratios of the corresponding functional genes sox B,nar G,nir S and hzs B were 0.14%,0.05%,5.92% and 6.14%,respectively.