Optimization of mixing ratio for co-calcined pyrite@biochar mixotrophic system treating aquaculture wastewater

Some aquaculture wastewaters may contain high levels of ammonium and nitrate but low content of organics. The dissolved ammonium can be effectively removed via simultaneous nitrification and denitrification (SND), while the nitrate can be polished via mixotrophic denitrification. The co-calcined pyrite@biochar might be a potent substrate for both SND and mixotrophic denitrification, but the volume ratio of pyrite to biochar on their performance has been scarcely reported. In this study, the co-calcined pyrite@biochar was used as the reaction matrix, and the effect of the pyrite-to-biochar volume ratio on their performance was assessed. The best overall performance within the tested range was achieved at a pyrite-to-biochar ratio of 1:7, resulting in removal efficiency of 62.45% for total nitrogen (TN), 67.47% for chemical oxygen demand (COD), and 80.60% for PO₄ 3− -P. Microbial community analysis revealed that Proteobacteria dominated at this ratio (R4), with a sulfate-reducing bacterium, Rhodanobacter (4.11%–12.31%) emerging as the dominant genus. Broad cooperative microbial interactions existed in the coupled system with Thiobacillus facilitating sulfur reduction and denitrification, and Geothrix contributing to ammonium removal via Feammox. Further gene analysis unraveled significantly elevated abundances of key genes such as narG , soxB , and dsrA in R4, promoting multi-pathway nitrogen removal and suppressing nitrite accumulation. In contrast, systems with 1:0 and 0:1 ratios exhibited lower microbial diversity, reduced gene expression, and poorer stability. This study demonstrates that the pyrite-to-biochar mixing ratio heavily influenced nutrient removal in the coupled system. It also provides some reference for the preparation of the novel substrate for biofilm reactors targeting the sustainable treatment of aquaculture effluent.