Batch studies for evaluation of N and P removal pathways

Since DPAOs can utilize nitrate and nitrite under anoxic conditions and oxygen under aerobic condition in the absence of NOX-N, it is important to evaluate their relative contribution in the PNDPR system. Batch studies 1, 2, and 3 were conducted in order to find the dominant electron acceptor in the PNDPR system while batch study 4 was conducted in order to find any contribution of GAOs in the denitrifying phosphorus removal in the PNDPR system.

3.5.1. Batch study 1: Nitrite accumulation at low DO condition without COD addition

A batch study was conducted to investigate the major nitrification product in the PNDPR reactor operated at low DO condition (batch study#1). The biomass was subjected to low DO (0.2-0.3 mg/L) without COD addition in the absence of any anaerobic period. Ammonium was completely oxidized (AUR 8.5 mg/g-VSS.hr) to nitrite and nitrate with a nitrite accumulation ratio of 82% (Fig.5.4a).

Figure 5.421 Evaluation of N & P removal pathways: (a) nitrification at low DO without COD addition, (b) aerobic P-uptake by DPAOs at low DO, (c) anoxic phosphorus uptake profile at pH 7.5 with anaerobic phosphorus release at different pH of 6.2 & 7.8

This clearly indicates that nitrite instead of nitrate was the major nitrification product in PNDPR system and denitrifying phosphorus removal took place via nitrite pathway. In addition, the biomass was still able to achieve approximately 12% SND indicating PHA left over from the previous cycle were used for denitrification.

3.5.2. Batch study 2: Comparison of phosphorus uptake using nitrate versus nitrite as electron acceptors

The comparison of phosphorus removal with nitrate and nitrite as electron acceptor is summarized in Table 5.1. There is no significant difference in COD removal as majority of the COD was removed during the anaerobic stage.

Table 5.120 Stoichiometry and kinetics of DPAOs using nitrate or nitrite as an electron acceptor Electron acceptor COD removal,% N- removal ,% P- Removal,% Average P- uptake rate (mg- P/g- VSS.hr) Average N- reduction rate (mg- N/g- VSS.hr) ΔP/ΔN ΔCOD/ΔN Carbon saving for SNDPR, % Nitrate 94 38 99 14 3.6 3.9 8.5 - Nitrite 95 85 99 13 7.3 1.8 4 53%

Nitrogen removal efficiency was significantly higher (85%) with nitrite as compared to nitrate (38%). Sp. denitrification rates (SDNR) for nitrite and nitrate were found to be 7.3 ,and 3.6 mg-N/g-VSS.hr, respectively. For pre-anoxic zone treating domestic wastewater, depending on the type and amount of readily biodegradable carbon, SDNR may range from 1.7-17.5 mg NO3-N/ g-VSS.hr, and 3- 27 mg NO2-N/ g- VSS.hr (Lee & Yun, 2014, Metcalf & Eddy, 2014, Peng & Zhu,2006). For post anoxic denitrification where substrate for denitrification is provided by endogenous decay, SDNR may vary from 0.63-2.5 mg NO3-N / g-VSS.hr, 0.9-4 mg NO2-N / g-VSS.hr (USEPA, 2007; Metcalf and Eddy, 2014, Yan et al., 2019). In a typical anaerobic-low DO aerobic process, denitrification via ordinary denitrifiers is comparable to post anoxic process since majority of the biodegradable carbon is utilized in the anaerobic zone. Comparing the abovementioned post anoxic denitrification rates, DPAOs will outperform ordinary denitrifiers in the absence of readily biodegradable exogenous carbon and contribute to simultaneous N and P removal. Comparing the nitrate versus nitrite reduction rates, nitrite denitrification via DPAOs is significantly faster (7.3 mg NO2-N / g-VSS.hr) than post anoxic nitrite denitrification (0.9-4 mg NO2-N / g-VSS.hr) by ordinary denitrifiers. Moreover, carbon demand for denitrification is 53% less via nitrite pathway (Table 5.1). This clearly signifies the benefit of nitrite pathway over nitrate both in terms of DPAOs outcompeting ordinary denitrifiers and carbon savings. In this study, sp. P-uptake rate/ sp. denitrification rate for nitrite and nitrate were 1.8, and 3.9, respectively. The reported typical values for sp. P-uptake rate/SDNR for DPAOs are 1 to

2 and 4 to 6 for nitrite and nitrate, respectively (H. Lee & Yun, 2014; Y. Z. Peng et al., 2011). Ratio of COD utilized to nitrogen reduced was 4 with nitrite and 8.5 with nitrate as electron acceptor. The batch study clearly showed that DPAOs acclimatized at low DO are capable of using both nitrate and nitrite as electron acceptors; albeit, with a reduced efficiency for nitrate. The stoichiometric information from this batch study clearly shows that there will be a significant (~53%) carbon savings when nitrite is utilized as electron acceptor compared to nitrate in simultaneous nitrogen/phosphorus EBPR systems. Thus, achieving nitrite shunt is particularly important for treating carbon limited wastewater for simultaneous N and P removal.

3.5.3. Batch study 3: Kinetics of aerobic P-uptake at low DO condition using oxygen as electron acceptor

Carvhelhaira et al. (2014) reported that aerobic P-uptake rate for Accumulibacter PAOs decreases by about 20% at a DO level of 0.6 mg/L compared to DO 4 mg/L . P- uptake rate decreased in the DO range of 0.1 to 0.6 mg/L by about 70% compared to a DO of 4 mg/L (Carvalheira et al.,2014). However, the impact of low DO on DPAOs for aerobic P-uptake has not been reported. Fig.5.4b shows the aerobic P-uptake kinetics of DPAOs culture at a DO level of 0.3±0.05 mg/L. The sp. P-uptake rate was found to be 19 mg/g.VSS.hr which is within the typical values (6-21 mg/g.VSS.hr) for aerobic P- uptake rate by Accumulibacter PAOs at high DO condition. The high sp. P-uptake rate at low DO reflects non-Accumulibacter DPAOs dominance in the PNDPR reactor.

3.5.4. Batch study 4: Anoxic P-uptake at different pH conditions: role of DGAOs in PNDPR reactor

In order to investigate the role of DGAOs (if any) on the denitrifying phosphorus removal in the PNDPR reactor, two batch studies were conducted at different pH scenarios: (1) Anaerobic (pH 6.2)/Anoxic (pH 7.5), and (2) Anaerobic (pH 7.8)/Anoxic (7.5). According to Filipe et al. (2001), at or above pH of 7.25, PAOs uptake acetate faster than GAOs. Therefore, if the DPAOs are not capable of nitrate reduction and rely on DGAOs for nitrate to nitrite conversation, a lower anoxic P-uptake will occur when

at 6.2. This is due to the fact that at high anaerobic pH (7.8) DPAOs will accumulate VFAs faster, resulting in less carbon available for DGAOs. Similarly, at low pH, DGAOs will have a competitive advantage for carbon storage, perform higher nitrate to nitrite reduction, and facilitate P-uptake by DPAOs (Rubio-Rincón et al., 2017). Fig. 54c shows the P-release and P-uptake characteristics of biomass at different pH. It can be observed from Fig.5.4c that at both pH, acetate was completely consumed. The P-release rate was significantly lower at pH 6.2 (22 mg-P/g-VSS.hr) compared to the pH 7.8 (48 mg-P/g- VSS.hr). However, this does not imply a DGAOs-DPAOs competition for carbon because the P-uptake rate for anaerobic pH 7.8 (13 mg/g-VSS.hr) is significantly higher than anaerobic pH 6.2 (7 mg/g-VSS.hr). In addition, the nitrate reduction was also 2 times higher at pH 7.8 than pH 6.2. Tayà et al. (2013) reported the DGAO culture could not denitrify nitrite, and Zeng et al. (2003)also reported NO3-N reduction is much faster than NO2-N reduction for DGAOs, hence DPAOs get a kinetic advantage over DGAOs for utilizing nitrite. However, in our system at pH 6.2 both nitrate and phosphorus reduction were impacted. If DGAOs were present, a decrease P-reduction but not NO3-N reduction at low pH would have been observed.

Therefore, it is highly likely that DGAOs have been washed out from the PNDPR reactor due to partial nitrification at high NAR (82%). Furthermore, as discussed in section 3.7, most commonly found DGAOs species, such as Competibacter phosphatis was not detected and Defluvicoccus & Propionivibrio accounted for less than 0.1% in the microbial analysis. This further confirms that the DPAOs in the PNDPR reactor are capable of denitrifying directly from nitrate (without denitrification by DGAOs) in addition to nitrite. The lower P-release/acetate uptake at pH 6.2 is primarily due to lack of hydrolysis of poly-p since less energy is required for acetate transportation at low pH(Smolders et al.,1995). Therefore, a low pH anaerobic condition can impact the P- release, synthesis of PHA, and subsequent anoxic P-uptake by DPAOs due to lack of PHA as evidenced in this study.

3.6 Contribution of nitrifiers, DPAOs, and various electron acceptors to overall