Nitrate-nitrogen pertains to the nitrogen component of the overall nitrate present in a given sample in order to reduce nitrate nitrogen pollution in water, nitrate nitrogen removal methods based on iron-carbon micro-electrolysis have become a key research focus. The process and mechanism of nitrate nitrogen removal by microbial coupling was comprehensively explored in a novel iron-carbon micro-electrolysis (ICME) system. In order to establish the transformation pathway of nitrate nitrogen in water, the transformation paths of nitrate nitrogen in water before and after coupling microorganisms in three groups of continuous flow reaction devices, namely sponge iron (s-Fe0), sponge iron + biochar (s-Fe0/BC) and sponge iron + biochar + manganese sand (s-Fe0/BC/MS), were studied. The morphology and composition changes of sponge iron were analyzed by means of characterization, and the microbial population changes in the three groups were analyzed by high-throughput sequencing. Results showed that the nitrate conversion rate in the s-Fe0, s-Fe0/BC and s-Fe0/BC/MS systems reached 99.48%, 99.57% and 99.36%, respectively, with corresponding ammonia nitrogen generation, rates of 3.77%, 9.34% and 11.24% and nitrogen generation rates of 95.71%, 90.23% and 88.12%. Scanning electron microscopy imaging showed that in the s-Fe0/BC and s-Fe0/BC/MS systems the surface of sponge iron was highly corroded, with granular substances in the corrosion product clusters. X-ray photoelectron spectroscopy analysis found that the relative contents of Fe2O3 in the surface oxides of sponge iron after microbial coupling were 38.02% and 71.27% in the s-Fe0/BC and s-Fe0/BC/MS systems, while the relative Fe3O4 contents were 61.98% and 28.72%, respectively. Microbial high-throughput sequencing analysis revealed that the Chao and Ace index values in the s-Fe0 system were 871.89 and 880.78, while in the s-Fe0/BC system they were 1012.05 and 1017.29, and in the s-Fe0/BC/MS system were 1241.09 and 1198.29, respectively. The relative proportion of Thauera in the s-Fe0, s-Fe0/BC, and s-Fe0/BC/MS systems was 16.76%,14.25% and 10.01%, while the proportion of Acetoanaerobium was 15.36%, 13.27% and 11.11%, and the proportion of Chloroflexi was 0%, 1.11% and 2.18%, respectively. Furthermore, FAPROTAX function annotation found that the expression levels of chemoheterotrophs in the s-Fe0, s-Fe0/BC and s-Fe0/BC/MS systems were 43 316 OTU, 37 289 OTU and 34 205 OTU, while nitrate respiration expression levels were 16 230 OTU, 15 483 OTU and 9149 OTU, with nitrogen respiration expression levels of 16 328 OTU, 15 493 OTU and 9154 OTU, respectively. These findings suggest that nitrate is converted into nitrogen gas and ammonia nitrogen through the actions of the coupled system of sponge iron/biochar/manganese sand and microorganisms. The catalytic effect of MnO2 promotes the conversion of Fe2+ to Fe3+, generating more electrons, allowing denitrifying bacteria to reduce more nitrate nitrogen, effectively coupling the manganese-catalyzed ICME reaction and microbial denitrification. The micro-electrolysis system and the addition of manganese sand enhanced biodiversity within the s-Fe0/BC/MS system. The heterotrophic bacteria Thauera and Acetoanaerobium were the dominant microorganisms in all three systems, although the micro-electrolysis system with added manganese sand significantly reduced the proportion of facultative bacteria Thauera and Acetoanaerobium and promoted the growth of autotrophic Chloroflexi bacteria. The ecological functions of the three systems were mainly nitrate respiration and nitrogen respiration. By comparing the expression levels of nitrate respiration and nitrogen respiration in s-Fe0/BC and s-Fe0/BC/MS systems, it can be seen that the addition of manganese sand reduced microbial activity.
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