Construction And Denitrification Mechanism Of Extracellular Electron Transfer-dependent Anammox System

Anaerobic ammonia oxidation(Anammox)for the removal of nitrogen from wastewater has the advantages of low energy consumption,low cost,and low sludge production.This process requires the participation of ammonia and nitrite nitrogen,however,nitrogen contaminants in municipal wastewater and many industrial wastewaters are usually present as ammonia nitrogen.Nitrite needs to be produced in other treatment processes.Therefore,the anaerobic ammonia oxidation process needs to be coupled with other processes to generate nitrite to participate in the reaction when treating nitrogen-containing wastewater.In this paper,an anaerobic ammonia oxidation denitrification system with graphene oxide and microbial electrolytic cell anode as extracellular electron acceptors was constructed to investigate the nitrogen metabolism pathway and electron transfer mechanism of the extracellular electron transfer anaerobic ammonia oxidation system.The main findings of this study are as follows:(1)The extracellular electron transfer anaerobic ammonia oxidation system was constructed by replacing nitrite with graphene oxide and the Microbial electrolysis cell’s anode using the ability of extracellular respiration of Anammox bacteria,and the effective removal of ammonia nitrogen under nitrite-free condition was achieved with the efficiency of 44.0%and 85.2%,respectively.The long-term operation of an extracellular electron transfer anaerobic ammonia oxidation system in a continuous flow reactor has demonstrated the feasibility of its engineering application,with ammonia nitrogen removal rates up to 66.7%.(2)The functional microorganisms involved in the extracellular electron transfer type anaerobic ammonia oxidation system were clarified.Metagenomic analysis revealed that its microbial composition was essentially the same during the alteration of extracellular electron acceptors to run the anaerobic ammonia oxidation process;metatranscriptome studies revealed that planctomycetes,which are only about 20%,were found to be more than 80%of the metatranscriptome annotations by metatranscriptome studies,indicating that Anammox bacteria played a major role in the experimental microbial community.(3)The nitrogen metabolism pathway of the extracellular electron transfer anaerobic ammonia oxidation system was clarified.Metagenomic and transcriptomic studies revealed that during the anaerobic ammonia oxidation process run with the microbial electrolytic cell anode as the electron acceptor,the gene hzsABC,encoding hydrazine synthase,was significantly upregulated,accelerating the conversion of ammonia to hydrazine through the catalytic conversion of hydrazine synthase,and hydrazine was converted to N2 by hydrazine dehydrogenase;RT-qPCR experiments verified the metabolic process.(4)The electron transfer pathway of the extracellular electron transfer anaerobic ammonia oxidation system was clarified.The analysis of microbial extracellular polymers revealed that the anaerobic ammonia oxidation process with the anode of the microbial electrolytic cell as the electron acceptor secreted more proteins to media ate more efficient extracellular electron transfer process;the three-dimensional fluorescence spectroscopy revealed that the fluorescence intensity of humic acid-like substances related to the extracellular electron transfer ability was higher;the metatranscriptome results showed that this system mainly transferred electrons to the extracellular area through cytochrome c and secreted electron shuttles,which were accompanied by ammonia conversion and hydrogen ion migration;the proposed electron transfer process was verified by RT-qPCR experiments,and the extracellular electron transfer process dominated by cytochrome was successfully inhibited by adding electron transfer inhibitor in respiratory chain blocking experiments.(5)The nitrogen metabolism equation of the extracellular electron transfer type anaerobic ammonia oxidation system was proposed.Based on the proposed denitrification mechanism,a possible nitrogen metabolism equation was proposed with reference to the amount of carbon and nitrogen assimilation by conventional Anammox bacteria.