Accepted Manuscript Combination of upflow anaerobic sludge blanket (UASB) reactor and partial nitritation/anammox moving bed biofilm reactor (MBBR) for municipal wastewater treatment Andriy Malovanyy, Jingjing Yang, Jozef Trela, Elzbieta Plaza PII: DOI: Reference:

S0960-8524(15)00002-4 http://dx.doi.org/10.1016/j.biortech.2014.12.101 BITE 14423

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

8 November 2014 29 December 2014 30 December 2014

Please cite this article as: Malovanyy, A., Yang, J., Trela, J., Plaza, E., Combination of upflow anaerobic sludge blanket (UASB) reactor and partial nitritation/anammox moving bed biofilm reactor (MBBR) for municipal wastewater treatment, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2014.12.101

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Combination of upflow anaerobic sludge blanket (UASB) reactor and partial nitritation/anammox moving bed biofilm reactor (MBBR) for municipal wastewater treatment

Andriy Malovanyy1 *, Jingjing Yang1,2, Jozef Trela1,2, Elzbieta Plaza1 1

Department of Sustainable Development, Environmental Science and Engineering,

Royal Institute of Technology (KTH), Sweden Teknikringen 76, 100-44, Stockholm, Sweden, e-mails: [email protected], [email protected], [email protected], [email protected] 2

IVL Swedish Environmental Institute Valhallavägen 81, 114 28 Stockholm, Sweden.

* Corresponding Author: Andriy Malovanyy Address: Royal Institute of Technology (KTH), Department of Sustainable Development, Environmental Science and Engineering. Teknikringen 76, 100-44, Stockholm, Sweden. Tel. +4687908690 E-mail: [email protected]

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ABSTRACT

In this study the combination of an upflow anaerobic sludge blanket (UASB) reactor and a deammonification moving bed biofilm reactor (MBBR) for mainstream wastewater treatment was tested. The competition between aerobic ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) was studied during a 5 months period of transition from reject water to mainstream wastewater followed by a 16 months period of mainstream wastewater treatment. The decrease of influent ammonium concentration led to a wash-out of suspended biomass which had a major contribution to nitrite production. Influence of a dissolved oxygen concentration and a transient anoxia mechanism of NOB suppression were studied. It was shown that anoxic phase duration has no effect on NOB metabolism recovery and oxygen diffusion rather than affinities of AOB and NOB to oxygen determine the rate of nitrogen conversion in a biofilm system. Anammox activity remained on the level comparable to reject water treatment systems. Keywords: anammox; deammonification; mainstream; AOB; NOB; MBBR 1.

Introduction

The trend for developing future wastewater treatment is to consider wastewater as a resource trying to recover as much energy as possible in the form of biogas while satisfying requirements for nutrient removal. In such systems a first treatment step can be based on organic matter removal by precipitation and coagulation/flocculation (Guida et al., 2007), use of high-rate activated sludge process for maximum removal of organics in a form of sludge (Versprille et al., 1985) or anaerobic wastewater treatment in, for example, an upflow anaerobic sludge blanket (UASB) reactor (Mahmoud et al., 2004). The effluent from the organic matter removal step has a low chemical oxygen 2

demand (COD)/N ratio, and, therefore, it is advantageous to apply a partial nitritation/anammox (deammonification) process for nitrogen removal from pretreated municipal wastewater for maximizing a treatment economy. The main challenges for mainstream nitrogen removal by deammonification process are anammox bacteria retention in biomass and suppression of nitrite oxidizing bacteria (NOB) growth. Until now there are presented a number of studies where effective nitrogen removal in an anammox reactor was successfully maintained in similar to mainstream conditions (Du et al., 2014; Hendrickx et al., 2012; Lotti et al., 2014). There are a limited number of studies where the performance of one stage deammonification at near-to-mainstream conditions was reported. Full scale treatment of a high rate activated sludge (HRAS) effluent in an activated sludge process bioaugmented with the ammonium oxidizing bacteria (AOB) and the anammox bacteria from side-stream line was demonstrated in Wett et al. (2013). Performance of a reactor with granular biomass treating municipal wastewater after HRAS step at 19 °C is reported in Lotti et al. (2013). During the periods of stable reactor operation the average nitrogen removal efficiency of 29 % was observed, reaching to 48.8 % during the periods of the best performance. In Malamis et al. (2013), the combination of an UASB reactor for organic matter removal and a deammonification sequenced batch reactor (SBR), inoculated with sludge from a DEMON plant, was used for synthetic wastewater treatment at 30 °C. The system was operated for 98 days and stable nitrogen removal (due to anammox and denitrifiers) was maintained during the last 60 days. The study focused only on the anammox process and, therefore, nitrate production by NOB and overall efficiency was not reported. Performance of an rotating biological contactor (RBC) during a 360 days period of synthetic wastewater treatment with temperature 3

decreasing from 30 to 14 °C is reported in De Clippeleir et al. (2013). Nitrogen removal efficiencies between 54% (at 29 °C) and 34% (at 17 °C) were obtained. After reaching mainstream temperature the reactor was operated for 80 days with a COD/N ratio between 0-2 during different phases and the average efficiencies of 23-45% were maintained. Conversion of anammox reactor treating high strength synthetic wastewater to one stage deammonification reactor treating synthetic wastewater with the ammonium concentration of 70 mg N/L at temperature 12 °C was demonstrated in Hu et al. (2013). After the conversion to mainstream conditions the reactor was operated for 158 days with efficiency of over 90% and no nitrate in the effluent was detected. This is the only study where no NOB growth in a one stage deammonification reactor treating low-strength wastewater at low temperatures was observed. To summarize, significant progress in understanding one stage deammonification systems, operated at mainstream conditions, have been done. However, most of the studies cover a period of less than one year and only in De Clippeleir et al. (2013) operation at a low influent nitrogen concentration during 440 days was reported. Actual pre-treated municipal wastewater with unadjusted content was used only in Lotti et al. (2013) and Wett et al. (2013). No studies on mainstream wastewater treatment in a one stage deammonification moving bed biofilm reactor (MBBR) were reported, to this end. There is no consensus in the literature if AOB or NOB have higher affinity to dissolved oxygen (DO) concentration (Blackburne et al., 2008; Wett et al., 2013; Wiesmann, 1994). Moreover, the impact of oxygen diffusion in a biofilm on AOB-NOB competition is not sufficiently studied. Application of intermittent aeration for NOB out-selection is often applied and different explanations of the NOB suppression mechanism are suggested (Al-Omari et al., 2013; Kornaros et al., 2010). 4

The aim of this study was to test the mainstream wastewater treatment system, based on a combination of an UASB reactor and a deammonification MBBR. The process stability and microbial community in the deammonification MBBR was evaluated during a period of transition from reject water to mainstream wastewater treatment and a long-term period of feeding with the UASB reactor effluent. Interactions between AOB, NOB and anammox bacteria in a biofilm and suspended biomass of the MBBR were studied. Strategies of NOB suppression were investigated in short-term batch experiments and tested in continuous operation mode. 2. Methods 2.1.

Experimental setup

The treatment scheme consisted of two reactors – a UASB reactor for removal of organic matter and a deammonification MBBR for nitrogen removal. The UASB reactor had a working volume of 6.2 m3 and was fed with primary settled municipal wastewater of the Henriksdal wastewater treatment plant (WWTP), Stockholm, at a constant rate of 24 m3/d. The influent wastewater had in average 100 mg COD/L and total nitrogen (TN) concentration of 42 mg/L. Temperature in the UASB reactor was maintained at 20 °C. The MBBR had a volume of 200 L and had a 40% filling with Kaldnes K1 carriers with a biofilm of nitrification and anammox bacteria (corresponds to a 40 m2 of biofilm area). Before this study the MBBR was used in a 3-year research on reject water treatment (Winkler et al., 2012; Yang et al., 2013). A content of the reactor was mixed by a mechanical stirrer at 100 rpm and the temperature was kept at 25°C. The MBBR was equipped with on-line sensors for measuring DO concentration, pH, oxidationreduction potential (ORP) and conductivity. DO concentration was maintained in the 5

reactor on a desired level by means of a controller regulating the aeration intensity. Either continuous or intermittent aeration was applied. The effluent from the MBBR was passing through a settler, where suspended biomass settled and could be used for activity batch tests. The study was divided into two periods. During period I the deammonification reactor was fed with a mixture of anaerobic digestion reject water and an effluent of the UASB reactor filtrated through a textile filter with a 20 µm pore size (influent content in table 1). Concentration of ammonium was decreased step-wise by changing a volumetric ratio of the UASB reactor effluent to reject water. During period II the deammonification reactor was fed with only UASB effluent (influent content in table 2) and different aeration strategies were studied. 2.2.

Activity tests

Determination of specific anammox activity (SAA) was done using the methodology described in Dapena-Mora et al. (2007). The methodology is based on measurement of the nitrogen gas pressure which is increased with the course of the anammox reaction. Activity of aerobic bacteria in biofilm and suspended sludge were determined by oxygen uptake rate (OUR) tests using the methodology described in Gut et al. (2005). Activity of heterotrophic denitrifies was determined by analyzing nitrate concentration in a liquid medium during the course of denitrification. 2.3. Transient anoxia batch tests To investigate the mechanism of NOB suppression by transient anoxia, attached biomass (in the biofilm) was subjected to the conditions of a DO limitation and a limitation of both DO and nitrite. For the first type of tests 5.34 mM phosphate buffer, supplemented with NaHCO3 and NaNO2 to the final concentrations of 10 mM and 15 6

mg NO2-N/L respectively, was aerated to DO concentration higher than 6 mg/L. Biomass was introduced to the medium and the DO concentration was measured (Hach HQd40 DO meter) until anoxic conditions were reached. The biomass was kept in the absence of oxygen for periods of different duration, after which the DO concentration was increased fast to 2 mg/L by changing a part of the liquid with the oxygen-saturated buffer with substrate and the decrease of the DO concentration was measured again. For the second type of tests the phosphate buffer was supplemented with an alkalinity (10mM) and ammonium source (NH4Cl, 50 mg N/L) and after introduction of biomass the DO concentration decrease was measured. Both AOB and NOB were active during this phase of the test. After the DO depletion all the remaining nitrite was removed by the anammox bacteria within 5 min, which was confirmed by chemical analyses. After an anoxic period, a NO2- source and n-allylthiourea (the specific inhibitor of AOB as reported in Gut et al (2005)) were added to the final concentrations of 15 mg NO2-N/L and 12 mg/L, respectively, the DO concentration was increased to 2 mg/L and oxygen consumption by the NOB was measured. In both types of test anoxic periods between 20 min and 15 h were tested. The rate of oxygen consumption was calculated by determining the slope of DO decrease. 2.4. Batch tests on DO concentration influence on AOB and NOB rate The AOB and NOB rates were determined based on oxygen consumption. OUR tests were done with thoroughly washed biofilm carriers utilizing 5.34 mM phosphate buffer, supplemented with NaHCO3 to the concentration of 10 mM. Separate tests were done with addition of only ammonium, only nitrite or both substrates to concentrations of 50 mg NH4-N/L and 15 mg NO2-N/L respectively. No inhibitors were added during the tests and the DO concentration change from 6 to 0 mg/L was measured. 7

2.5.

Analytical methods

Analyses of nitrogen forms, COD and alkalinity were done using Dr. Lange cuvette tests (LCK303, LCK341, LCK340, LCK314, LCK362, LCK 238). All samples were filtrated through a 0.45µm filter prior to analyses. Biomass in the MBBR was determined according to Standard Methods (APHA 1998). 3.

Results and discussion

3.1. UASB reactor performance Since municipal wastewater treatment in UASB reactors is well-studied and applied in a full scale, the research was focused n nitrogen removal. However, the change of COD and TN concentrations in the influent to and the effluent from the UASB reactor was monitored during the whole operation period. The biogas production was 175 L/d (with methane content of 52%), which corresponds to 7.3 L per 1 m3 of treated wastewater. The TN concentration in the outflow remained on the same level as in the influent and the average effluent COD concentration was 61 mg/L. 3.2.Transition to mainstream 3.2.1. Nitrogen conversion routes The ammonium concentration in the influent to the MBBR was changed by a step-wise increase of the ratio between the UASB reactor effluent and reject water (Table 1). During the first three periods (Ia-Ic) the hydraulic retention time (HRT) was gradually decreased to the level of 0.6-0.8 days (which is the usual retention time in the biological step of Henriksdal WWTP (Stockholm Vatten, 2014)), and was maintained on that level until the end of operation period. The nitrogen loading rate (NLR) decreased from 1.79 to 0.21 g N/(m2·d) during periods Ia-Ig with decrease of influent ammonium

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concentration. The initial period of the MBBR operation (Ia), when treating undiluted reject water, was characterized by a high efficiency of nitrogen removal (in average 86%), a high removal rate (1.6 g N/(m2·d)), an HRT of 2.5 days and a production of

nitrate close to the stoichiometry of anammox reaction (Fig. 1A). During periods Ib-Ie the capacity of the system for nitrogen removal decreased considerably. Since it was hard to balance the nitrogen loading with the air supply under these conditions, there were occasions of under-aeration (in periods Ib and Ic) and overaeration (in periods Id and Ie) which led to a drop of the average nitrogen removal efficiency to 60-65%. Starting from period If, when ammonium concentration in the influent was decreased to 108 mg N/L in average, considerable nitrate accumulation

(relative to the removed ammonium) was observed (Fig. 1A). While in the first five periods the observed nitrate production was close to, or even lower than the stoichiometric values in deammonification process, in periods If and Ig 30-70% of all the oxidized ammonium was converted to nitrate by NOB, which resulted in a low efficiency (43% in average). During period IIa, when the MBBR was fed with the nonspiked UASB effluent, nitrate production continued to grow. Due to this, a nitrogen removal efficiency of only 35% was observed in this period and 53% of all the transformed ammonium was converted to nitrate, in average. 3.2.2. Bacterial activity Anammox activity in the biofilm remained stable during the first five transition periods

(Fig. 1B), even though the removal rate decreased three-fold. In the following periods, when ammonium concentration in the influent was further reduced, the SAA decreased to values of 1.2-1.5 gN/(m2·d). Activity of heterotrophic denitrifiers in biofilm was stable at 0.32±0.14 gN/(m2·d) during the whole transition period. Anammox and 9

heterotrophic activities in suspended sludge corresponded to only 0.5% and 0.9 % of the total respective capacities in the reactor. OUR results for biofilm (Fig. 1C) showed that the activity of heterotrophic bacteria and NOB remained in the range of 0.5-1.5 gO2/(m2·d) for the whole transition period. The activity of AOB remained fairly constant in the range of 3.5-5.5 gO2/(m2·d) during periods Ia-If, but decreased to 2.5 gO2/(m2·d) during period Ig. Oxygen uptake by suspended sludge during the periods Ia-If was mostly limited to AOB activity, since the activities of NOB and heterotrophs were lower than the detection limit. However, the AOB activity of sludge decreased 3-fold from 6.5 to 2 g O2/(m2·d) during periods Ia-If. Starting from period Ig, an increase of the heterotrophic activity to 1 g O2/(m2·d) and the NOB activity to 0.5 g O2/(m2·d) was observed. The dependence of OUR on DO concentration for attached and suspended biomass was determined with the tests similar to those discussed in chapter 3.3.3 and the nitrogen fluxes, which could be expected at the DO concentrations maintained in the reactor,

were calculated from the OUR results (Fig. 1D). Even though the mixed liquor volatile suspended sludge (MLVSS) concentration during the reject water treatment (period Ia) was less than 200 mg/L, suspended biomass was highly active with OUR of 6.5 g O2/(g VSS d). Therefore, the capacity of AOB in suspended sludge for ammonium oxidation at DO concentration higher than 5 mg/L was the same as the capacity of AOB in biofilm at the same conditions. Moreover, slower diffusion of oxygen into the biofilm depth, comparing to the diffusion into flocks of suspended biomass, resulted in approximately 75 % of all the aerobic ammonium oxidation by the AOB of suspended sludge at DO concentration set-point (0.8 mg/L) during period Ia. To the contrast, more than 99% of the anammox activity was in biofilm. These results agree with the results 10

reported by Veuillet et al. (2014), where spatial distribution of activities was also found in a deammonification MBBR treating reject water. With the decrease of ammonium concentration in the influent, the MLVSS concentration decreased sharply as a result of lower sludge production per 1 L of treated wastewater and shorter HRT. Because most of the suspended sludge was composed of AOB, the decreased concentration of suspended sludge caused decrease in the ammonium oxidizing capacity of the reactor. Moreover, lower suspended biomass concentration made biofilm responsible for a bigger share of ammonium oxidation. Suspended sludge was responsible for only 33% of ammonium oxidation in period Ic, 9% in period If and 6% in period IIa. This low impact of suspended biomass on the conversion rates means that bacteria grew mostly in biofilm and the suspended biomass originated from the biofilm detachment. The detachment of older biomass from biofilm explains the decrease of suspended biomass activity, observed during periods Ia-IIa. Since the pilot scale MBBR was operated without suspended biomass retention, mean cell residence time (MCRT) for suspended biomass was equal to the HRT. MCRT of attached biomass was much longer and allowed NOB to remain in biofilm even after few years of reject water treatment. The decrease of suspended biomass concentration during the transition to mainstream led to lower AOB abundance in the reactor. Moreover, the drop in AOB activity of attached biomass during period Ig further decreased the AOB/NOB ratio and led to high nitrate production starting from period If. The anammox bacteria capacity was almost equal to the sum of the capacities of AOB

present in suspended and attached biomass in period Ia (Fig. 1D). During the following experimental periods it was always higher than the AOB capacity and the nitrogen load. Big difference between the anammox capacity and the NRR, observed in periods Ib-Ig 11

is considered to be the main cause of the SAA decrease. Anammox bacteria suffered from insufficient substrate availability and part of them died-off. 3.3. Long-term mainstream operation The goal with long-term mainstream operation of the deammonification reactor was to investigate the stability of biomass in time and ensure that anammox bacteria can be sustained in a biofilm in considerable amount and not be out-competed by NOB and heterotrophic denitrifiers. Since oxygen limitation is one of the main factors for controlling AOB-NOB competition, different modes of aeration were tested during the 16 months of the reactor operation with mainstream wastewater as an influent. Moreover, since nitrate accumulation was considered as the main obstacle for reaching efficient nitrogen removal based on the results of period I, competition of AOB and NOB in biofilm was studied by different batch tests. 3.3.1. Process performance at different aeration strategies After transition to mainstream wastewater the reactor was operated with continuous aeration (period IIa). Since the nitrogen removal efficiency was rather low (Table 2), it was decided to change the aeration to an intermittent mode with a 45 min aerated and a 15 min non-aerated phase and decrease the DO set-point to 0.3 mg/L (period IIb). However, the nitrogen removal efficiency deteriorated further and was 19% in average in this period. After 132 days of operation the aeration mode was changed back to continuous (period IIc). The rate of N removal increased to the same values as in period IIa. The efficiency of N removal increased comparing to period IIb but was lower than in period IIa, where also continuous aeration was applied. This can be explained by different bacterial composition of biofilm. In period IIa anammox activity was similar to

the activities observed during reject water treatment (Fig. 2A). Moreover, the AOB 12

activity (in terms of oxygen consumption) was around 3 times higher than the NOB

activity (Fig 2B). This was likely due to the impact of previous operation with more concentrated wastewater. The results indicated that the duration of aerated phase was too long comparing to the duration of anoxic phase in period IIb. Therefore, aeration was changed back to an intermittent mode with longer anoxic phase duration (30 min) (period IId). In this mode the efficiency of N removal returned to 35%. Higher loading was applied which resulted in higher aerobic activity and higher removal rates. Decrease of the aeration phase duration to 30 min in period IIe did not considerably change the efficiency and led to a minor decrease of the nitrogen removal rate (NRR). Finally, a short aeration cycle with a 15 min aerated and a 15 min non-aerated phase was applied (period IIf) and compared with the cycle with twice longer phases duration, as will be discussed later. The highest average treatment efficiency was obtained with this aeration strategy. 3.3.2. Transient anoxia mechanism of NOB suppression Intermittent aeration is used as a NOB suppression tool in reject water treatment systems (Lackner et al., 2014), in mainstream wastewater treatment with deammonification process (Wett et al., 2013) and in nitritation/denitritation systems (Ge et al., 2014; Kornaros et al., 2010). Based on these studies two potential explanations of the lag-phase in NOB activity in the beginning of the aeration phase are the absence of one or both of the substrates (nitrite and oxygen) and inactivation of metabolic mechanisms during the long period of anoxia. To investigate these hypotheses, transient anoxia was studied using attached biomass in the conditions of DO limitation and limitation of both DO and nitrite. The rates of

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oxygen consumption at different DO concentrations were compared before and after

periods of DO limitation (Fig. 3A). The change of part of the liquid and stabilization of DO measurement took 2-3 min, and therefore, such short NOB activity delays could not be detected. The observed process rates just after DO stabilization were the same as in the reference test without the anoxic period irrespective of the anoxic phase duration. Therefore, the lag phase in NOB activity was not detected even after a long period of DO limitation. There was also observed no difference in NOB activity after periods of both nitrite and DO limitation, irrespective of the period duration (lines 2, 4 and 6 on

Fig. 3B). The decrease of activity comparing to reference tests (lines 1, 3 and 5 on Fig.

3B) is due to AOB inactivation by the specific inhibitor. These experiments showed no influence of oxygen or nitrite starvation period duration on NOB activity recovery and the conclusions are contradictory to what was shown in Kornaros et al. (2010), where NOB lag phase in the beginning of aerated phase was observed and the delay in the recovery of NOB activity was found to be a function of anoxic phase duration. An in-depth study of NOB lag phase after a period of anoxia (Gilbert et al., 2014) revealed that the delay in NOB activity to a great extend depends on a reactor operation strategy and, consequently, species distribution within biomass. The delays of up to 13 min were observed for the nitritation-anammox suspended biomass originating from the reactors, operated at high DO set-point. However, the delays of only up to 6 min were observed for the biomass from the reactors operated at low DO concentration, even after anoxic periods of 60 min. Moreover, part of this delay could be explained by slow oxygenation of mixed liquor and low nitrite concentration in the beginning of aerated phase. The tests done during period IIe in our study, when DO set-point of 0.6 was applied, showed that the delay due to inactivation of metabolic 14

mechanisms could be maximum of 2-3 min. The DO concentration in biofilm was even lower, so the biomass can be considered as adapted to low DO conditions. Therefore, these results support the conclusions of Gilbert et al. (2014) that NOB, adapted to low DO concentration, exhibit low suppression by long anoxic period. Even if inactivation of NOB in anoxic phase was not detected, intermittent aeration is beneficial for a one stage deammonification and for nitritation-denitritation systems. During the anoxic phase residual nitrite is removed by anammox bacteria or denitrifiers to the levels significantly lower than the half-saturation concentration and NOB are suppressed due to substrate limitation in the beginning of the aerated phase. In this case the choice of anoxic phase duration should be based on the time required for complete nitrite removal. 3.3.3. Influence of DO concentration on AOB-NOB competition Until recently, it was recognized that AOB have a higher affinity to oxygen than NOB and the typical values from literature indicated half saturation coefficients (Ko) of 0.3 mg O2/L for AOB and 1.1 mg O2/L for NOB (Wiesmann, 1994). Based on these values, operation of a reactor at low DO concentration can benefit in decreasing an NOB growth rate and nitrate accumulation. However, in Wett et al. (2013), investigation of biomass originating from bench-scale deammonification reactors and a full-scale WWTP showed that NOB have a higher affinity to oxygen than AOB with Ko in ranges of 0.35-0.4 and 0.06-0.16 for AOB and NOB respectively. In this research the influence of DO concentration on AOB and NOB competition in a biofilm was investigated utilizing OUR tests (see chapter 2.4). When only nitrite was

supplied (line 1 on Fig. 3C), dependence of NOB rate on DO concentration could be

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obtained. The maximum activity was reached at DO concentration of approximately 3.5 mg/L which is much higher than in other studies (1.2 mg/L in Regmi et al. (2014), 0.7 in Wett et al. (2013)). This is attributed by the impact of DO diffusion into biofilm, which is the rate limiting step. When only ammonium was added as a substrate (line 3 on Fig 3C), it was observed that with increase of DO concentration until 2.5 mg/L OUR changed almost linearly while further increase of DO concentration led to a minor increase of the rate. In this test most of the oxygen was consumed by AOB, since only the nitrite, produced by AOB, was available. Since the activity of NOB in biofilm was comparable with the activity of AOB, and oxygen diffusion is the rate limiting step, it can be assumed that all the produced nitrite was utilized by NOB and then the rate of only AOB can be calculated

(line 4 on Fig. 3C). This assumption has a weakness that the consumption of the produced nitrite by anammox bacteria (especially in low DO concentration range) is not

taken into account. However, since the obtained rate profile (line 4 on Fig. 3C) coincide

with the profile for NOB (line 1 on Fig. 3C) there is a strong support to conclude that in the studied biofilm system the influence of DO on rates of AOB and NOB is similar and is mainly attributed to oxygen diffusion in biofilm and not the affinities of different bacteria groups to oxygen. This also is supported by the results of OUR test with both nitrite and ammonium addition (line 2 on Fig. 3C). The obtained rate was lower than the sum of AOB and NOB rates even at DO concentration higher than 6 mg/L and was similar to the rates of only AOB and only NOB below DO concentration of 1 mg/L. Taking into account these results and those presented in the previous chapter, in order to provide preference for AOB over NOB in the one-step deammonification system, which relies only on biofilm activity, it is preferential to have as low average NO2 16

concentration during aeration phase as possible (short aeration phase) and as short anoxic phase as needed to consume all the NO2-, produced during the aerated phase. DO concentration does not have a strong direct impact on AOB-NOB competition for oxygen for the studied biofilm system. However, with lower DO concentration it is easier for anammox bacteria to compete with NOB for nitrite since an increase in DO concentration leads to an increase of NOB activity and can lead to inactivation of anammox bacteria by oxygen. 3.3.4. Utilizing transient anoxia in pilot-scale MBBR The conclusions of the batch tests are confirmed by the results of continuous pilot plant operation. Comparing the last four periods of the reactor operation, where the same DO set-point but different aerated and non-aerated phase durations were applied, the lowest removal efficiency was achieved when continuous aeration was used (period IIc). The introduction of a 30 min non-aerated phase (in period IId) improved the reactor performance and there was no significant difference observed between using 45 min and 30 min aerated phase. When the change of nitrogen form concentrations during one aeration cycle in period

IIe was studied (Fig. 4A) it was observed that the nitrite concentration increased to 0.065 mg NO2-N/L during first 15 min of aeration and reached 0.09 mg NO2-N/L in the end of the aeration phase (corresponds to 0.017 µg HNO2-N/L at temperature and pH in the reactor). This value is low comparing to the ammonium concentration in the reactor but is close to the half-saturation constant for NOB, which is reported to be 0.032 µg HNO2-N/L (Wiesmann, 1994). Moreover, it was observed that almost all the nitrite was consumed during the first 15 min of the anoxic phase which indicates that almost no nitrogen conversion occurred in the following 15 min. A minor decrease of ammonium 17

and an increase of nitrate concentrations were observed in the aerated phase and the opposite change during the anoxic phase.

Analysis of the nitrogen forms change in period IIf (Fig. 4B) showed that the nitrite concentration increased to only 0.04 mg NO2-N/L in the end of the aeration phase and the average nitrite concentration during the aerobic phase was twice lower than for the aeration strategy with a 30 min phase duration. The duration of the anoxic phase was sufficiently long to decrease nitrite to the concentrations below the detection limit. Therefore, for the studied system it is preferential to use intermittent aeration with 15 min phases duration to limit NOB activity. This conclusion is supported by the results of the continuous reactor operation which showed that the average efficiency of nitrogen removal increased from 36% to 40% after the change to the shorter phase duration. 3.3.5. Influence of nitrogen concentration on AOB-NOB competition If high nitrogen removal efficiency is achieved in mainstream completely mixed reactor it means that the ammonium concentration and pH will be low (if no pH adjustment is applied). The lower the ammonium concentration is, the higher preference for NOB over AOB is, due to limitation of AOB activity by lower substrate availability. This effect was observed in the deammonification MBBR during continuous operation. From the data of chemical analyses it was observed that the nitrate production, relative to ammonium removal, was correlated with residual ammonium concentration in the

reactor (Fig. 5). Operating the reactor with residual ammonium concentration of around 5 mg NH4-N/L, the highest removal efficiencies could be reached.

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Based on these observations, the hypothesis was proposed that process can be operated in a plug-flow or an SBR and by this increase of the average ammonium concentration in a reactor can be achieved facilitating the AOB-over-NOB preference. Therefore, the batch test with operation of the pilot reactor in a sequencing batch mode was performed. 75% of the reactor liquid was changed to wastewater, the inflow was stopped and the aeration in an intermittent mode was started with phases duration of 15 min. Total duration of the cycle was the same as HRT in continuous operation mode. Results of nitrogen species development during the cycle (Fig. 4C) confirmed this hypothesis. It was observed that during the first 5 h of aeration, nitrate production amounted to 45% of the removed ammonium, while in the following 5.5 h it was 65% and in the last 4.5 h it was 81%. Therefore, only a minor part of nitrogen was removed in the last hours of the cycle and almost all the removed ammonium was further oxidized to nitrate. The nitrogen removal efficiency in the whole cycle was 43%, which is higher than the efficiency in continuous mode of pilot plant operation on the day of the experiment (37%) and the average efficiency the during long time operation of the reactor with the same aeration strategy (40%). This test confirmed that for NOB suppression operation in a sequencing or plug-flow mode is preferential. However, the drawback of such an operation mode is that not only ammonium concentration, but also the concentrations of the possible toxic compounds are higher in the beginning of the cycle in an SBR or close to the start of a plug-flow reactor. This approach could be used in two stage deammonification, but application of it for a one stage process was considered by the authors to have too high risk of irreversible inhibition and loss of anammox activity. Therefore, it was not tested in a continuous operation mode.

19

3.3.6. Biomass stability The main reason for the long-term municipal wastewater treatment in the deammonification reactor was to investigate if the set-up will allow retaining the anammox culture, when concentration of ammonium will be decreased, or the bacteria will be wash-out from the reactor. After 16 months of the system operation it can be concluded that high anammox activity in one stage deammonification reactor treating municipal wastewater at 25 °C can be retained. Even though the SAA decreased 2.5 times when the inflow was gradually changed to mainstream wastewater, after that the activity remained stable at the level of 1.2-1.5 g N/(m2·d) during 14 months. This activity is approximately 5 times higher than the applied nitrogen load and is comparable with the activity of biomass in the full-scale reject water treatment line of the Himmärfjärden WWTP (Plaza et al., 2011). The heterotrophic activity to the end of period II decreased to 0.11 g N/(m2·d) and, therefore constituted only to 8% of the total anoxic activity of the biomass. To this end, this is the first reported study where stable performance of a deammonification reactor was maintained during such a long period. Activities of AOB and NOB in biofilm changed considerably during periods IIa-IIc but were rather stable during the following three periods. The effort to completely outcompete NOB from biofilm by application of intermittent aeration was not successful. The average ratios between AOB and NOB activity in periods IId-IIf were 1.0, 1.3 and 1.4 respectively which can be correlated with the increase of average efficiencies in the respective periods. Activity of all the groups of microorganisms in suspended sludge did not have a considerable influence on the reactor performance because of low concentration of suspended sludge. However, it was observed that the proportion of AOB/NOB activity (expressed as oxygen consumption) in the suspended sludge was 20

always higher than in the biofilm. In periods IIc-IIf the average ratio was 2.9, which is more than twice higher than for the attached biomass. As discussed previously, the suspended biomass was originating mostly from the biofilm detachment. Therefore, it suggests that AOB grew on the outer edge of biofilm while NOB grew somewhat deeper and, therefore, were more protected from wash-out. One of the possible solutions for avoiding wash-out of AOB from the reactor is to separate the suspended biomass from the effluent (e.g. by sedimentation) and recirculate it back to the reactor, i.e. operate the system in integrated fixed film activated sludge (IFAS) mode.

To summarize, with a proper control of the deammonification process high anammox

activity can be maintained leading to removal efficiencies of approximately 40 % when

treating wastewater at 25 °C. Such temperatures are common for municipal wastewater

in hot climate areas or for wastewater leaving an anaerobic treatment step, such as a

UASB reactor tested in this study. However, if the process is to be applied for treatment

of wastewater having lower temperature, it is likely that lower efficiency would be

reached. It is well established that NOB has lower activation energy than AOB

(Wiesmann 1994) leading to higher growth rate of NOB than of AOB. The competition

between NOB and anammox bacteria at low temperature conditions is studied little.

Therefore, more research is needed on the operation strategies for controlling the NOB

suppression at low temperatures. 4.

Conclusions

The one stage deammonification reactor, fed with mainstream wastewater after pretreatment in the UASB reactor, showed high biomass stability during 21 month of operation, which indicated that anammox bacteria wash-out could be avoided. Intermittent aeration with a short aerated phase and an anoxic phase, long enough for all 21

the produced nitrite consumption, was shown in batch tests and continuous system operation to be beneficial for AOB-NOB competition. However, out-competing NOB and avoiding high nitrate production was not easy to obtain due to stronger detachment of AOB from biofilm and wash-out from the system. 5.

Acknowledgement

Financial support for the research by Swedish Governmental Agency for Innovative Systems (Vinnova), Swedish Water Development (SVU), Swedish Environmental Research Institute (IVL) and the Royal Institute of Technology (KTH) is greatly appreciated. We gratefully acknowledge the scholarships provided by Swedish Institute to Andriy Malovanyy and by Lars Erik Lundbergs foundation to Jingjing Yang for their PhD studies. The authors would like to thank Mariusz Rajkowski and Paulina Wit for their valuable input in the pilot plant operation. The experimental work was performed at Hammarby Sjöstadsverk (Swedish Water Innovation Center). 6.

Supplementary information

In supplementary data, a scheme with the used experimental set-up and a graph

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26

Figure captions: Fig. 1. Performance of the deammonification MBBR during transition to mainstream (period I): A – ammonium conversion routes (solid line shows the level of NO3-N, which would have been accumulated if all the removed nitrogen (top part) followed partial deammonification stoichiometry); B – anammox bacteria and heterotrophic denitrifiers activity in biofilm; C – aerobic bateria activity in biofilm; D – comparison of potential capacity and applied nitrogen loading (AOB capacity is calculated by multiplication of ܰ‫ܪ‬ସା →ܱܰଶି flux by 2.32/1.32). Fig. 2. Bacterial activity during mainstream wastewater treatment (period II): A – anammox bacteria and heterotrophic denitrifiers in biofilm; B – aerobic bateria in biofilm. Fig. 3. Batch tests on DO influence on AOB-NOB competition: A – transient anoxia tests with only DO limitation; B – transient anoxia tests with NO2-N and DO limitation; C – tests on separate influence of DO on AOB and NOB. Fig. 4. Competition between AOB and NOB at different pilot reactor operation modes: A – continuous loading and intermittent aeration with 30 min + 30 min phases duration; B – continuous loading and intermittent aeration with 15 min + 15 min phases duration; C –sequenced batch mode and intermittent aeration with 15 min + 15 min phases duration. Fig. 5. Influence of substrate concentration of AOB on process performance (data from the periods IId-IIf).

27

Table captions: Table 1. Performance of the deammonification MBBR during transition to mainstream conditions (period I). Table 2. Performance of the deammonification MBBR during treatment of UASB effluent (period II).

28

Figure 1.

29

Figure 2.

30

Figure 3.

31

Figure 4.

32

Figure 5.

33

Table 1 Period Days of operation

Ia 1-40

Ib

Ic

Id

Ie

If

41-54 55-70 71-84 85-98 99-126

Ig

IIa

127- 158-220

157

UASB : RW ratio1

0:100 50:50 70:30 80:20 85:15

90:10

95:5

100:0

884

463

252

194

146

108

67

31

COD (mg/L)

404

194

95

84

72

56

56

37

Alkalinity (mmol/L)

71

38

21

17

14

10

8

5

TN (mg/L)

141

259

154

87

60

68

46

40

NH4-N (mg/L)

51

143

80

30

39

23

16

7

NO2-N (mg/L)

4.5

3.7

2.6

1.8

1.3

0.4

0.3

0.1

NO3-N (mg/L)

66

25

21

34

18

38

22

14

COD (mg/L)

275

148

94

70

61

48

37

24

Alkalinity (mmol/L)

9.9

12.2

12.6

4.6

5.0

3.2

2.1

2.1

MLVSS (mg/L)

178

82

31.3

28.2

23.5

18.9

23.4

13.4

Biofilm VS (g/m2)

28.7

30.0

30.0

30.8

31.9

28.2

27.6

21.4

DO (mg/L)

0.80

1.14

0.84

0.91

1.36

0.90

0.62

0.50

pH

7.34

7.62

7.23

6.84

6.80

6.54

6.66

6.66

33

53

96

154

124

124

104

134

1.79

1.74

1.22

1.21

0.84

0.69

0.44

0.20

86

63

59

66

63

44

42

35

NRR (g N/(m2·d))

1.60

1.07

0.70

0.84

0.54

0.34

0.19

0.06

HRT (days)

2.45

1.33

1.06

0.81

0.95

0.78

0.76

0.77

outflow

inflow

NH4-N (mg/L)

ORP (mV)

NLR (g N/(m2·d))

Efficiency (%)

1

Volumetric ratio between UASB reactor effluent and reject water

Table 2 Period

IIa

IIb

IIc

IId

IIe

IIf

34

Days of operation

158-220

221-353

354-451

354-539

540-591

592-640

Cont.

45+15

Cont.

45+30

30+30

15+15

(0.5)

(0.3)

(0.6)

(0.6)

(0.6)

(0.6)

NH4-N (mg/L)

31

29

39

41

39

34

COD (mg/L)

37

67

64

60

61

55

Alkalinity (mmol/L)

4.7

4.8

5.8

5.7

5.4

5.4

NH4-N (mg/L)

7.1

9.1

7.4

13.2

19

10

NO2-N (mg/L)

0.13

0.18

0.17

0.06

0.3

0.1

NO3-N (mg/L)

13.7

14.1

21.7

13.3

5.8

12.5

COD (mg/L)

24

44

33.4

34.3

31

28

Alkalinity (mmol/L)

2.1

3.2

2.2

2.4

3.3

2.7

DO (mg/L)

0.5

0.35

0.6

0.47

0.36

0.43

pH

6.66

6.64

6.89

6.92

7.10

6.88

ORP (mV)

134

134

185

85

30

89

NLR (g N/(m2·d))

0.20

0.21

0.26

0.36

0.32

0.27

35

19

26

35

36

40

NRR (g N/(m2·d))

0.06

0.04

0.06

0.13

0.11

0.10

HRT (h)

18.5

16.8

17.4

13.8

14.5

17.0

outflow

inflow

Aeration mode1

Efficiency (%)

1

Cont. stands for continuous aeration; XX+XX stands for duration of aerated and non-aerated

phases respectively; DO concentration set-point is indicated in brackets.

35

•

Transition from reject water to mainstream led to decrease of aerobic capacity

•

Mainstream deammonification operated during 16 months at 25 °C

•

Intermittent aeration with phases duration of 15 min gave the best performance

•

AOB/NOB activity ratio is twice higher for suspended than for attached biomass

•

Anammox activity, comparable to reject water systems was maintained

36