Impact of chemical oxygen demand to nitrogen ratio on anammox bacterial growth in an up-flow anaerobic sludge blanket reactor
Authors:   , Msimango Sandile Simiso 
Published: June 2023

The anaerobic oxidation of ammonium (ANAMMOX) process has been suggested as an economical and innovative means of removing nitrogen from wastewater. Nevertheless, very few studies have evaluated the effect of the chemical oxygen demand (COD) to nitrogen (N) ratio on bacterial communities in an ANAMMOX-mediated system. Heterotrophic bacteria can readily outcompete the slow-growing ANAMMOX bacteria in the presence of organic carbon. This study examined the effect of the organic carbon to nitrogen (C/N) ratio on the performance of ANAMMOX in an upflow sludge blanket reactor using synthetic wastewater as the feedstock. Two UASB reactors (UASB-A and UASB-B) were seeded with biomass from a labscale ANAMMOX reactor and operated for a period of 593 days. Both reactors were operated using similar operational conditions during the enrichment phase (0-400 days). Thereafter, the addition of organic carbon in the medium altered the C/N ratio of one of the reactors (UASBB). During this period, UASB-A served as a control reactor. A CN ratio of 1.0, 1.5, and 2.0 was achieved in the UASB B reactor by increasing the organic carbon concentration every 60 days. The reactors were analyzed at three-day intervals per week for nitrogen and COD removal efficiency. The quantitative PCR method was used to detect the dominant N-removing organisms within both reactors at different phases. In addition, cDNA quantification or reverse transcriptase qPCR (RT-qPCR) was also conducted to determine the dominant and active nitrifying communities. The results indicated that when the C/N ratio is 1.0, almost complete removal of NH4 + -N is observed (92%), and nitrogen removal efficiency (NRE) is approximately 82%. The ratios of ΔNO2 - /ΔNH4 + and ΔNO3 - /ΔNH4 + ratios during this phase (C/N=1) fluctuated from >1.25 to <1.6 and from >0.35 to <0.45 <0.11 to >1.6, respectively, which was within the range of the expected ANAMMOX stoichiometric ratio. In addition, when the C/N ratio was increased from 1 to 1.5, NRE rose from 82 to 88%. However, a decrease of NRE to 83% was observed when the C/N ratio was further increased to 2. The quantitative PCR results showed an increase in total bacteria from 1.4 × 106 copies/µL to 2.3× 106 copies/µL, and 2.4× 106 copies/µL as the ratio of C/N increased from 1.0 to 1.5 and thereafter to 2, respectively. ANAMMOX bacteria showed an increase from 16 × 103 copies/µL to 6.5× 10 4 copies/µL, and 2.06 × 105 copies/µL when the C/N ratio was increased from 1 to 1.5, and 2, respectively. The cDNA analysis further showed an increase of ANAMMOX bacteria transcript abundance from 4.6 × 104 copies/µL to 2.52× 106 copies/µL with an increase in C/N ratio to 1.5. Subsequently, a decrease in ANAMMOX bacteria transcript abundance to 1.09 × 106 copies/µL was observed when the C/N ratio was further increased to 2. The expression of the hzo gene encoding for hydrazine dehydrogenase (HDH), which catalyses the oxidization of the unique ANAMMOX intermediate hydrazine to N2 was 169 folds of expression, which was very high at C/N=1, but showed a decrease to 39 folds expression at C/N=1.5. Almost complete inhibition of hzo gene was observed when the C/N ratio was further increased to 2. Based on chemical analysis, it was further confirmed that the decrease of both ANAMMOX and AOB abundance at a higher C/N ratio caused an increase in effluent NH4 + -N concentrations. In conclusion, the study has shown that a higher C/N ratio could significantly affect the overall nitrogen removal rate and the activity of the diverse microbial populations, more specifically the ANAMMOX bacterial activity.