Impact of chemical oxygen demand to nitrogen ratio on ANAMMOX bacterial growth in an up-flow anaerobic sludge blanket reactor

While several studies have investigated the effect of varying carbon-to-nitrogen (C/N) ratios on the ANAMMOX performance, there is still a research gap in illustrating the shift in 16S rRNA gene copy number and functional microbial population during operation. Hence, this study focuses on utilizing a reference gene and target functional genes to demonstrate the synergetic interaction between ANAMMOX, ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB), using an up-flow anaerobic sludge blanket (UASB) under different C/N conditions. It was demonstrated that elevating the C/N ratio from 1.0 to 2.0 reduced the COD and NH4⁺-N removal efficiencies from 80.12 to 48.62% and from 88.99 to 72.59%, respectively. Based on the qPCR evaluation, at the C/N ratio of 1.5, the abundance of ANAMMOX, AOB, <i>Nitrobacter</i>, and <i>Nitrospira</i> was 2.52 × 10⁶, 82, 5.39 × 10³, and 12.98 × 10³ copies/μL, respectively. However, with the further increase of C/N ratio to 2.0, their abundance was reduced to 1.09 × 10⁶, 46, 0.98 × 10³, and 3.47 × 10³ copies/μL, respectively. The expression of <i>hzo</i> gene encoding for hydrazine dehydrogenase was 169-folds at C/N = 1 and almost inhibited at C/N = 2. The results of microbial population structure using 16S rRNA reverse transcriptase (RT)-qPCR technique depicted a competition between ANAMMOX and heterotrophic bacteria for the available substrate at higher C/N ratios.