Impact of mixing intensity on dissolved oxygen half- velocity constants in a sidestream deammonification environment

A partial nitritation/anammox (PN/A) process was operated at two different mixing intensities to quantify the extent to which diffusional limitations impact process rates. At a steady-state operation, the total inorganic nitrogen removal efficiency in the bench-scale sequencing batch reactors was found to increase as mixing intensity decreased (62 and 84% for average velocity gradient (G) values of 15 and 5.3 s , respectively). The half-velocity constants (KO2 ) with respect to bulk-phase dissolved oxygen (DO) concentration for ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) organisms were estimated on the basis of nitrogen removal rates that were observed in activity tests. The activity tests were conducted over a range of bulk-phase DO concentrations. The best-fit KAOB O2 values were estimated to be 0.68± 0.34 and 0.54± 0.56 mg O2/L for G values of 15 and 5.3 s , respectively. The AOB values were not statistically different (p1⁄4 0.19) between mixing conditions which were consistent with AOB dominating the surface of granules. The best-fit Kanammox O2 values were estimated to be 0.13± 0.09 and 0.55± 0.40 mg O2/L for G values of 15 and 5.3 s , respectively, and were statistically different (p 1⁄4 3:9 × 10 10). The results demonstrated that mixing conditions should be considered when designing PN/A processes and provide quantitative results that can be employed to improve models of these processes. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wqrj.2019.009 ://iwaponline.com/wqrj/article-pdf/55/2/145/709393/wqrjc0550145.pdf Biao Xie Wayne J. Parker (corresponding author) Department of Civil and Environmental Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada E-mail: wjparker@uwaterloo.ca Chao Jin Department of Systems Design Engineering, University of Waterloo, Waterloo, Ontario, Canada This article has been made Open Access thanks to the kind support of CAWQ/ACQE (https://www. cawq.ca).


INTRODUCTION
There is a growing interest in the use of short-cut nitrogen removal processes that remove nitrogen from wastewater streams with fewer resources (energy and carbon) than traditional technologies (Kartal et al. ). Lackner et al. The successful operation of PN/A processes typically depends on establishing conditions (including low DO concentrations) that result in the wash-out of nitrite-oxidizing bacteria (NOB) from the process. The DO profile is critical to successful operations and is typically controlled by setting the duration of aerated and unaerated periods and the target DO concentration while aerating (Lackner et al. ).
The successful operation of PN/A processes typically requires the implementation of sophisticated pH and DO control on aeration (Wett ; Wett et al. ). There is, however, a lack of consensus with regard to the target DO concentration that should be employed during aeration. Historically, the models have assumed that all organisms are directly exposed to the bulk liquid substrate concentrations, and the half-velocity coefficients (referred to as intrinsic) have been considered to be constant. However, recent studies have shown that the values of substrate half-velocity constants should be considered as variables (Arnaldos et

METHODOLOGY Experimental plan
A bench-scale sequencing batch reactor (SBR) was operated to generate data that were subsequently employed to assess the impact of mixing intensity on the rates of substrate utilization. It was operated for an extended period of time at two different mixing intensities to facilitate an assessment of the impact of mixing intensity on the performance of the reactor and the K AOB The goal was to conduct activity tests with the granule morphology that was established through the long-term operation at each mixing intensity. Given the considerable period of time required to complete each phase, resource limitations resulted in the testing of two mixing conditions that spanned a range of intensities. The results of the study facilitated an assessment of which half-velocity parameters were sensitive to mixing intensity. Additional research would be required to develop quantitative relationships between mixing intensity and half-velocity constant values.

Test apparatus
The SBR had an effective volume of 8.0 L, was heated to maintain a temperature of 35 C, and received diffused air Loveland, CO, USA) of the SBR contents were continuously monitored and reported to the data acquisition module. The DO probe was calibrated regularly using the air method that was recommended by the operator manual. The aeration control algorithm is subsequently described.
The SBR was fed with a synthetic centrate to maintain a constant influent composition throughout the study. The centrate (Table 1)

SBR operations
The SBR aeration strategy was adopted from literature

Batch activity tests
The batch activity tests were conducted upon the com- Upon the completion of the anoxic phase, aerobic con-

Statistical methods
Multi-response regression was used to calibrate the DO halfvelocity constants in the bioprocess model (Ni et al. ) that was adopted in this study. Two-sample t-tests (significance level of 0.05) were used to determine whether the DO half-velocity constants differed between the two mixing intensities.

RESULTS AND DISCUSSION
The testing was divided into two phases where the mixer was operated at different speeds and mixing intensities (G 150 rpm and G 75 rpm , respectively). Achieving steady state with respect to the activity of the biomass in the reactor was deemed to be important in terms of increasing the accuracy of the subsequent batch activity testing and the confidence in the estimation of the K O2 . The SBR was therefore operated until steady state was achieved prior to the batch activity tests. Steady-state conditions were assessed based on the presence of stable effluent concentrations of NH þ 4 , NO À 2 , and NO À 3 over time (Figure 1). Steady state was deemed to be achieved for days 81-165 and 199-228 in Phases 1 and 2, respectively. The average (±std dev) MLVSS concentrations at steady state were 1.2 ± 0.35 and 1.9 ± 0.09 g/L in Phases 1 and 2, respectively.
Comparing Phases 1 and 2, the average effluent NH þ 4 concentration in Phase 2 decreased to approximately 33% of that observed in Phase 1. The effluent concentrations of NO À 2 were negligible throughout the steady-state periods, while the average effluent NO À 3 concentration decreased only slightly in Phase 2 as compared to Phase 1. The total inorganic nitrogen (TIN) removal efficiency was calculated for the steady-state periods with values of 62 and 84% for Phases 1 and 2, respectively.
The results indicate that the largest contribution to the increased TIN removal with reduced mixing intensity was the increased removal of NH þ 4 which could be attributed to increase the activity of either AOB or anammox. It was hypothesized that the AOB activity would increase with mixing intensity since it would depend on the availability of both NH þ 4 and oxygen to the organisms and both of these would increase with improved mass transfer. By contrast, the anammox activity is reduced in the presence of DO and therefore may increase under conditions of reduced mass transfer. It was not possible to confirm these hypotheses with the data obtained from the continuous operation of the SBR, and the subsequently described batch testing and model application were conducted to investigate the impact of mixing on AOB and anammox activity in more depth.
The activity tests were conducted with the SBR operating in the batch mode and with known initial concentrations of NH þ 4 , NO À 2 , and NO À 3 . Each consisted of anoxic and aerobic periods with the DO concentration in the latter period differing between tests. Trends in substrate concentrations with time were employed to determine substrate utilization rates that were subsequently used to estimate the K AOB The concentrations of NH þ 4 , NO À 2 , and NO À 3 at G 150 rpm that were observed in a test that was conducted in Phase 1, when the DO concentration in the aerobic portion of the test was 0.46 mg O 2 /L, are shown in Figure 2 and are representatives of those obtained under other test conditions. From Figure 2, it can be seen that the concentration of NH þ 4 initially increased and then decreased linearly in both the anoxic and aerobic conditions. The early increase in the NH þ 4 concentrations was attributed to the time required to dissolve and mix the dry substrates that were added to the reactor. Only the concentration profiles obtained after 15 min were used for further analysis of conversion rates.
The trends in substrate responses in the batch tests were consistent with the metabolisms that were anticipated to be active in the different aeration conditions. Under anoxic conditions, NH þ 4 and NO À 2 concentrations decreased and NO À 3 increased over time. These responses were observed in all of the anoxic tests and were attributed to the activity  responses that were generated in the aerobic batch tests. A positive value for the rate was interpreted to indicate that the production rate of the species was greater than the consumption rate, while a negative value indicated that the production rate was less than the consumption rate.
The NH þ 4 , NO À 2 , and NO À 3 conversion rates under anoxic conditions at both G 75 rpm and G 150 rpm were similar.
The consistency in responses between the different mixing   conditions implied that diffusional limitations were not impacting on the anoxic process rates. The lack of apparent diffusional limitations under these conditions suggested that the rates of anammox consumption were being limited by some other factors such as growth processes; therefore, diffusion was less important.
As DO concentration increased, the NH þ 4 conversion rates became more negative and subsequently plateaued at elevated DO values (Figure 3). The highest NH þ 4 consumption rates occurred when the DO concentration was the highest as would be expected as the oxygen availability to AOB became non-limiting at high DO values. By contrast, the NO À 2 net conversion rates increased and then plateaued as DO increased in the aerobic tests ( Figure 3). The results indicate that when the DO concentration was less than or equal to 0.78 mg O 2 /L at G 75 rpm or 0.46 mg O 2 /L at G 150 rpm , NO À 2 was consumed with the rate of consumption greater than the rate of production, and at higher DO concentrations, NO À 2 was produced with the rate of consumption less than the rate of production. Although the trends in net NO À 2 conversion rates were similar at G 75 rpm and G 150 rpm , the DO concentrations at which the net NO À 2 conversion rate equaled to zero were different. At G 75 rpm , the DO concentration that the net NO À 2 conversion rate equaled to zero was between 0.78 and 1. The net NO À 3 conversion rates were relatively small relative to the other N species, were always positive, and decreased slightly as DO increased. The decrease of NO À 3 conversion rates was most apparent when the DO concentration was greater than 4.0 mg O 2 /L as most NH þ 4 was oxidized to NO À 2 . The decrease in NO À 3 conversion rates as DO increased confirmed the low activity of NOB in the system. When DO was lower than 1.0 mg O 2 /L, some NO À 3 was produced and this was attributed to anammox bacteria; when DO was higher than 4.0 mg O 2 /L, anammox bacteria stopped producing NO À 3 . The results obtained from the activity testing provided information on the net processes that were active in the tests. However, it was not possible to directly estimate half-velocity coefficients from the data. A model (Ni et  increased as the mixing speed decreased in Phase 2. Thus, the anammox bacteria experienced less DO inhibition at G 75 rpm as compared to G 150 rpm ; thereby, the TIN removal efficiency was higher at G 75 rpm . The results of this study provide process engineers with quantitative information that can be employed to establish mixing conditions for granular PN/A processes that could increase TIN removal performance. Furthermore, the K AOB O 2 and K anammox O2 values developed in this study can be employed to improve models of PN/A processes that quantitatively reflect the impact of mixing conditions on biological process rates.

CONCLUSIONS
In this study, the effects of mixing intensity on a PN/A process were characterized. The steady-state operation was achieved after 83 days of operation at 15 s À1 with an average total nitrogen removal of 62%, while after 22 days of operation at 5.3 s À1 with an average of total nitrogen removal of 84%. Activity test results showed that as DO increased at both G values, the rates of ammonium consumption increased and then plateaued; the rate of nitrite production increased and then plateaued; and the rate of nitrate