Sulfur Autotrophic Denitrification Coupled With Anaerobic Ammonia Oxidation For Enhanced Denitrification

Many industrial enterprises produce wastewater containing large amounts of nitrogenous pollutants such as nitrate,nitrite and ammonia nitrogen,so the process to explore how to remove various nitrogen pollutants from wastewater simultaneously has received a lot of attention.Sulfur autotrophic denitrification process is a biochemical reaction process carried out by sulfur oxidizing bacteria（SOB）under anoxic or anaerobic conditions using sulfur-containing compounds as electron donors and nitrate or nitrite as electron acceptors,and has been widely used in the field of biological nitrogen removal.Anaerobic ammonia oxidation process,as a new type of wastewater denitrification process,is a chemoautotrophic process in which anaerobic ammonia oxidizing bacteria use ammonia nitrogen as electron donor and nitrite as electron acceptor to produce nitrate and nitrogen gas under anaerobic conditions.Coupling the above two processes can simultaneously have the advantages of low sludge production,no need to inject organic carbon source and reduce the production of residual nitrogen compounds in the wastewater,and also improve the total nitrogen removal rate during wastewater treatment.In this study,a SAD-AMX autotrophic denitrification system was successfully constructed by adding anaerobic ammonia oxidation（Anammox）sludge to a sulfur autotrophic denitrification（SAD）reactor with sodium thiosulfate as the electron donor.The nitrogen removal performance of the coupled system during start-up and steady operation was investigated.The coupled system achieved efficient and steady operation with a maximum total nitrogen removal rate of 94.6%at a temperature of（36±1）°C and a total influent nitrogen load of 0.8 kg·（m³·d）^-1.The influence of S/N(ratio of influent S₂O₃^2--S to NO₃^--N)on the denitrification effect was investigated,and the optimal operating parameters were determined.Among them,the coupled system was able to maintain the best denitrification efficiency when the influent S/N was in the range of 1.6to 2.2.The contribution of anaerobic ammonia oxidation and sulfur autotrophic denitrification pathways to total nitrogen removal was stable at about 96.2%and 3.8%,respectively,and anaerobic ammonia oxidation dominated in the coupled system.The sludge activity after long-term operation was tested by batch experiments,and the results showed that both sulfur autotrophic denitrification bacteria and anaerobic ammonia oxidation bacteria were able to maintain high activity,and both were synergistic cooperation with complementary substrates in the coupled system.After the coupled autotrophic denitrification system was started and operated stably,it was used to treat metal heat treatment wastewater.Two ways were used to treat the wastewater:（1）adding more sodium thiosulfate to the wastewater to remove the excess nitrite through the sulfur autotrophic denitrification pathway;（2）adding ammonium chloride to the wastewater to bring the NO₂^--N/NH₄⁺-N in the wastewater to the theoretical value of the anaerobic ammonia oxidation reaction,and removing the excess nitrite through the anaerobic ammonia oxidation pathway.The results showed that both treatment methods were able to treat the wastewater efficiently,with the highest total nitrogen removal rates of 98.25%and 98.85%,respectively.The average nitrogen removal contribution of anaerobic ammonia oxidation was 67.1%,which was always higher than that of sulfur autotrophic denitrification at 32.9%,and anaerobic ammonia oxidation dominated the nitrogen removal.Microbial diversity and its structural changes in the coupled denitrification system were investigated by high-throughput sequencing techniques.The community structure at the microbial phylum taxonomic level was dominated by Proteobacteria with relative abundances of 21.74%,16.62%,39.7%and 37.37%,respectively.Other common phyla included Planctomycetes and Chloroflexi.The highest relative abundance of anaerobic ammonia-oxidizing bacteria,Candidatus Brocadia,belonging to Planctomycetes,was found in the community structure at the taxonomic level of microbial genera,reaching0%,20.06%,14.07%and 13.85%,respectively.Thiobacillus,the most common genus of sulfur autotrophic denitrifying bacteria,was found in sludge samples S and A were not found,and the relative abundance in SA1 and SA2 was 4.28%and 5.49%,respectively.Metabolic pathway analysis using the KEGG database showed that most of the metabolic pathways in the community remained stable,especially some central metabolic pathways.The relative abundance of both Nitrogen metabolism and Sulfur metabolism increased,effectively enhancing the denitrification performance of the system.The increase of metabolic pathways such as flagellar assembly was helpful to improve the stability of the system.The gene expression abundance of denitrification metabolic pathway was low in inoculated sludge S and A,and the gene expression of denitrification metabolic pathway was gradually increased in SA1 and SA2.The gene expression abundance of hzs A/B/C in the sludge samples SA1 and SA2 was the highest in the nitrogen cycle network,and the anaerobic ammonia oxidation reaction occupied the absolute dominance in the coupled system.