Metabolic insights into the enhanced nitrogen removal of anammox by montmorillonite at reduced temperature

Abstract Anammox is a low-cost and high-efficiency nitrogen removal technology. However, anammox bacteria are sensitive to realistic temperature variations of municipal wastewater, limiting their practical application in municipal wastewater treatment. In this study, to protect anammox bacteria against variable reduced temperatures, cheap and abundant clay montmorillonite (MMT) particles were used as anammox biomass carriers. Two 5-L sequencing batch reactors with MMT (RB) and without MMT (RA) were operated for 100 days. When the temperature decreased from 37 °C to 10 °C, under a nitrogen loading rate of 0.36 kg-N m−3 d−1, the total nitrogen removal rate was 0.21 kg-N m−3 d−1 in RB, but only 0.025 kg-N m−3 d−1 in RA, demonstrating the improvement of anammox performance at reduced temperatures by MMT. The 16S rRNA gene sequencing analysis verified that MMT promoted the growth of Candidatus Brocadia, resulting in 11% more abundance in RB than RA at 10 °C. Metabolomics analysis revealed MMT promoted the growth of anammox bacteria at reduced temperatures by stimulating metabolic pathways related to bacterial assimilation. In particular, MMT with excellent NH4+ adsorption capacity elevated L-Glutamine and L-Glutamate contents, subsequently stimulating the downstream metabolic pathways, including amino acid biosynthesis, purine and pyrimidine metabolism, and cryo-protectant synthesis. This study provides metabolic insights into how MMT was able to enhance nitrogen removal by anammox at reduced temperatures, which is meaningful towards overcoming the current limitations of the practical application of anammox in treating municipal wastewater.