Polishing Tertiary Effluent Nitrogen Via The Synergy Between Methanol-Driven Partial Denitrification and Anaerobic Ammonia Oxidation In Moving Bed Biofilm Reactors

Introduction
Tertiary moving bed biofilm reactors (MBBRs) have been used as a polishing process for biological nitrogen removal (BNR) to meet low effluent TN concentration, e.g.,< 3 mg TN/L. In these applications, supplemental carbon is often needed to drive nitrate removal. Partial nitrification/denitrification coupled with anaerobic ammonia oxidation (PdNA, PANDA) process can result in capacity, chemical and energy savings throughout the secondary and tertiary processes at treatment facilities if properly applied in tertiary MBBR processes. This is because anammox is an autotrophic process can convert ammonium and nitrite to dinitrogen without external carbon while aeration requirements can be reduced because only a fraction of ammonium needs to be oxidized (Xu et al. 2020). Partial denitrification has indeed emerged as a promising approach for stimulating stable NO2-N accumulation because of the challenge in suppressing nitrite oxidizing bacteria (NOB) under the low mainstream temperature and nitrogen level (Zhang et al. 2019). Briefly, the mechanisms behind PD can be attributed to: i) imbalanced activities of NO3-N and NO2-N reductases, which can be controlled by carbon source types and dosages and HRT; and ii) selection and enrichment of denitratating community. This work documents results from piloting efforts to evaluating the feasibility of using methanol driven PdNA/PANDA to reduce carbon and aeration demand at the Fairfax County Noman M. Cole Pollution Control Plant (NCPCP) and achieve TN limits less than 3 mg/L. The objectives of this study are to: i) verify the feasibility of performing nitrogen removal in the tertiary effluent via the PdNA/PANDA with methanol as external carbon source; ii) evaluate the system performance and the effluent quality under the diurnal and seasonal influent fluctuation; iii) determine the optimal operating conditions (carbon dosages and frequencies, etc.); and iv) make recommendations for full-scale operation.
Materials and methods Reactor setup:
A MBBR treatment train dosed with methanol was operated on site at Noman M. Cole Jr., Pollution Control Plant (NCPCP) (Figure 1a). Briefly, two 14 L working volume anoxic MBBRs filled with K1 media at a volumetric fraction of 45% were connected in sequence and then followed by a 8 L working volume reaeration reactor filled with K1 media at a volumetric fraction of 32% to simulate the full-scale NCPCP MBBR train used in NCPCP. K1 media with established methylotrophs were collected from the same full-scale MBBR train (Figures 1a and b). The designed HRTs of each anoxic and aeration cell in Figures 1a and b were 19.4 min and 10.3 min, respectively. Realtime feed-forward control: Real time NO3-N, NO2-N, NH4+-N, and dissolved oxygen (DO) data of tertiary effluent from Chemscan real-time analyzer and DO probe (Figure 1b) were sent to Spyder (python 3.8) in a PC. A programmable pump receiving signals from the PC can precisely dose chemicals to the systems according to the following controller formulas: NH4+-N feed = NO3-Ninf or a setpoint (1) CODfeed = (CODfeed/NO3-Ninf)setpoint × (NO3-Ninf â# ’ NO3-Neff, setpoint) + (CODfeed/DOinf)setpoint ×DOinf (2) Activity Testing: In-situ batch tests for AMX activity were conducted by dosing ammonium chloride at 10 mg NH4+-N /L and sodium nitrite at 12 mg NO2-N /L after isolating reactors and suspending COD dosing for one hours prior to the batch testing. NO3-N, NO2-N, NH4+-N, and sCOD were measured with Hach tubes in 0.45μm filtered grab samples of reactors at various intervals of time ranging from 5 to 30 minutes for a period of 2.5 hours. qPCR analysis: Quantitative polymerase chain reaction (qPCR) was performed to quantify the copy numbers of total bacterial 16S ribosomal RNA (rRNA) gene, the 16S rRNA of Candidatus Kuenenia and hzsB gene on a QuantStudio 3 thermocycler (Applied Biosystems, Waltham, MA). Each 20-μL qPCR reaction mixture contained 10 μL of 2× SYBRâ„¢ Green PCR Master Mix with ROX (Life Technologies, Carlsbad, CA), 1 μL each of forward and reverse primers (0.2 μM), 7 μL molecular-grade water (Sigma-Aldrich, St. Louis, MO) and 1 μL DNA template. The thermal cycling conditions for qPCR amplification were as follows: 95 ℃ for 2 min, 40 cycles at 95 ℃ for 15 s, 60 ℃ for 15 s, 72 ℃ for 20 s, melt-curve for 45 s (95 ℃ for 15 s, 60 ℃ for 15 s and 95 ℃ for 15 s). All qPCR runs had an efficiency between 90% and 110% with an R2 > 0.95. Each gene was quantified in duplicate with a standard curve and negative control (with water as template). Results were reported as absolute abundance (copies per gram dry weight). Results and discussion PD performance: As can be seen from Figure 2a, after the adjustment of carbon dosing by decreasing NO3-N setpoint and increasing COD/N ratios, NO3-N concentration in Cell B dropped to around 1 mg/L after 180-day operation. Meanwhile, NO2-N residual (0.7 ± 0.2 mg/L) and stable PD efficiency (43.7 ± 4.8%) were achieved in Cell A (Figure 2b). Le et al. (2019) reported that methanol-driven PD was only observed in batch test but not in continuous flow reactors. Conversely, in our study, stable PD was achieved after 6-month operation which indicates that culture acclimation might have played an important role, and methanol-driven PD actually can be achieved in MBBRs fed with low nitrogen concentrations. This is in line with the findings reported by Campolong (2019) who also found that methanol had a lower PD efficiency than glycerol and acetate during the startup, and that the process efficiency gradually improved over time as the community acclimated (Figure 2b) AMX activity and its contribution to total inorganic nitrogen (TIN) removal: Given the stable PD performance, strong AMX activity has been observed in the in-situ batch test (Figure 3a), which is in line with the abundance of Anammox 16S rRNA and hzsB measured by qPCR in Figure 3b. As a result, between 1.5 and 2.0 mg/L anoxic NH4+-N removal was observed and low effluent TIN concentrations (< 3 mg/L) were achieved in MBBR pilot after 6-month operation. AMX were estimated to contribute 43.4 ± 5.5% of the TIN removed (Figure 2c). This work is one of the few where PdNA/PANDA studies were performed in secondary or tertiary processes where NO3-N concentration was well below 10 mg/L (Du et al. 2019, You et al. 2020). Figure 2c shows that methanol can be used as a suitable carbon source for PdNA/PANDA in a low nitrogen environment. Carbon savings: The ΔsCOD/ΔTIN profile in Figure 4 showed that the pilot reactor consumed 41.4% less COD for the removal of a unit TIN as compared to the full-scale MBBR operated in the same plant. Hence, methanol can be used as a more cost-effective and eco-friendly carbon source for full-scale MBBRs applied for polishing the effluent TIN.