Abstract

Nitrous oxide (N2O) is one of the gases with the greatest impact in the atmosphere due to its persistence and significant contribution to the greenhouse effect. This study provides an insight into the dynamics of N2O production in wastewater nitrogen removal systems. A 10 L sequencing batch reactor containing enriched anammox biomass was subjected to different operational conditions, i.e., temperature, feed time, NO2/NH4+ ratio and the initial concentrations of NH4+ and NO2. Tests showed no significant differences in maximum N2O production when the system was operated with a shorter feed time and no increase in the operating temperature. A higher N2O production was observed when the initial NO2/NH4+ ratio increased from 1.3 to 1.7 and 1.9. The highest initial concentration of NO2 was linked to an increase in residual N2O at the end of the batch cycle, probably due to heterotrophic denitrifying metabolism.

INTRODUCTION

Nitrous oxide (N2O), a powerful greenhouse gas, persists in the atmosphere for more than 120 years and has a global warming potential 298 times greater than carbon dioxide (CO2) (IPCC 2013). Due to its polluting potential, N2O has attracted attention from the scientific community, which has called for efforts to identify and reduce the sources of this gas. Among the sources are wastewater treatment plants (WWTPs): N2O is an intermediate product involved in the metabolic pathways of nitrogen removal bioprocesses. Thus, there is a growing concern about the dynamics of the production and emission of N2O in WWTPs, which are attempting to establish more sustainable units and minimize energy consumption, greenhouse gas emissions and final sludge production (Okabe et al. 2011).

The anaerobic ammonium oxidation process (anammox) is a low-cost metabolic pathway based on the oxidation of ammonia (NH4+) to nitrogen gas (N2) using nitrite (NO2) as the final electron acceptor. In WWTPs, this process is usually preceded by a partial nitrification reactor, to achieve partial oxidation of influent ammonia to nitrite (Law et al. 2011; Castro-Barros et al. 2015). The combined process can also occur in a single reactor using the deammonification process, known as completely autotrophic nitrogen removal over nitrite (the Canon process). It is only established in the presence of a mixotrophic community dominated by anammox and ammonia-oxidizing bacteria (AOB), by adopting specific operational strategies that allow the inhibition of nitrite-oxidizing bacteria (NOB) (Third et al. 2001; Zhang et al. 2012).

Because it is completely autotrophic, the Cannon process does not depend on the influent organic matter, which is essential for heterotrophic denitrification in conventional treatment units. In addition, as the complete conversion of nitrogenous compounds to N2 can be achieved in a single reactor operated under low aeration conditions, the demands for space and energy are reduced (Third et al. 2001). However, the deammonification process can produce N2O (Kampschreur et al. 2009; Schreiber et al. 2012), which has not been reported as an intermediate or end product of the anammox reaction, although another gaseous intermediate (nitric oxide; NO) has been found in experiments using a pure culture of anammox bacteria (Speth et al. 2016). However, nitrifying microorganisms (specifically AOB) and heterotrophic denitrifiers (HET), which coexist with anammox bacteria in deammonification systems, have been identified as contributors to N2O production in biological nitrogen removal systems (Schreiber et al. 2012; Speth et al. 2016).

The heterotrophic community may be retained in deammonification systems by using soluble microbial products (SMPs) for their growth, even if the wastewater fed does not contain organic carbon (Liu et al. 2016). These heterotrophs play an important role in deammonification systems because they act to reduce the nitrate (NO3) produced by the anammox metabolism and protect the anammox bacteria from the inhibitory effect of the dissolved oxygen (DO) (Lotti et al. 2014). Anammox bacteria are generally reported as slow growing organisms, therefore one of the major challenges related to practical applications of deammonification is to ensure an adequate solid retention time for establishing this community.

In this context, the present study verified the dynamics of N2O production in a sequencing batch reactor (SBR) containing anammox enriched granular sludge under different operating conditions, i.e., temperature, feed time, influent NO2/NH4+ ratio and initial concentrations of NH4+ and NO2.

MATERIALS AND METHODS

Bioreactor set-up and experimental procedure

The experiments were conducted in a 10 L Plexiglass SBR equipped with a bath jacket (Julabo GmbH, Germany) for temperature control. Stirring, maintained at 200 rpm, was ensured by a mechanical stirrer (Heidolph, Germany, model RZR 2021) with an impeller with inclined blades (diameter 5 cm). When required, the DO input, initially set to vary between 0.08 and 0.6 mgO2·L−1, was performed by an aeration system which included an air pump (Mistral, model 200), a magnetic valve and a DO probe (Endress + Hauser, Switzerland, model COS22D). The whole system was operated by a programmable logic controller (PLC). pH was measured using a pH meter (Endress + Hauser, Switzerland, model CPS471D), connected to the PLC. pH and DO were measured every 30 seconds. The reactor feed, effluent discharge and the HCl and NaOH dosages were performed using peristaltic pumps (Cole-Parmer, Portugal, model 77200-60).

During the tests, the SBR was operated in eight daily cycles of 180 min. Each cycle was divided into four phases: filling (30 min), mixing or reaction (120 min), sedimentation (20 min) and discarding (10 min). The volume exchange rate, which is defined as the ratio between the volume of medium withdrawn at the end of the cycle and the total working volume, was 75%, once 2.5 L of medium had been added and withdrawn during one cycle. The initial values of the operating cycle phases were based on previous studies (Sobotka et al. 2016; Yin, Z. et al. 2016; Lu et al. 2018). Ten batch tests were carried out to elucidate the dynamics of N2O production by anammox enriched granular sludge under different operating conditions. The parameters evaluated were: feed time (6 min and 30 min), operating temperature (30 °C and 36 °C), the NO2-N/NH4+-N ratio (1.3, 1.7 and 1.9), variation of influent NO2-N and NH4+-N concentrations (with the NO2-N/NH4+-N ratio at 1.3) and the system's response under two extreme conditions: absence of NH4+-N and absence of NO2-N. Each condition tested was maintained for at least eight cycles, in which similar performances were observed. One of the monitored cycles was used to evaluate the results.

During the whole experiment, the reactor was fed with synthetic medium, as described by Dapena-Mora et al. (2004). NH4+-N and NO2-N concentrations in the medium were varied using ammonium sulfate ((NH4)2SO4) and sodium nitrate (NaNO2) were used as the sources of ammonium and nitrite, respectively. The operational conditions tested are shown in Table 1.

Table 1

Initial characteristics of the experiments performed in terms of substrates, temperature, period time, pH, DO and biomass

Test number Synthetic medium
 
Temp (°C) F.T. (min) pH DO (mg·L−1VSS (mg. L−1
NO2-N /NH4+-N ratio NH4+-N (mgN. L−1NO2-N (mgN. L−1
T1 1.3 100 130 33.6 7.4–7.8 <0.17 3,217 
T2 1.3 100 130 33.2 30 7.4–7.8 <0.16 3,217 
T3 1.3 140 182 29.7 30 7.5–7.8 <0.16 2,339 
T4 1.3 140 182 36.1 30 7.5–7.8 <0.15 2,339 
T5 1.3 200 260 30.1 30 7.5–7.8 <0.16 3,217 
T6 1.7 200 340 30.0 30 7.5–7.8 <0.12 3,217 
T7 1.9 180 340 30.0 30 7.5–7.8 <0.14 3,217 
T8 1.3 200 260 30.1 30 7.5–7.8 <0.16 3,217 
T9 – 80 30.1 30 7.6–7.8 <0.34 3,217 
T10 – 80 30.3 30 7.6–7.7 0.3–0.8 3,217 
Test number Synthetic medium
 
Temp (°C) F.T. (min) pH DO (mg·L−1VSS (mg. L−1
NO2-N /NH4+-N ratio NH4+-N (mgN. L−1NO2-N (mgN. L−1
T1 1.3 100 130 33.6 7.4–7.8 <0.17 3,217 
T2 1.3 100 130 33.2 30 7.4–7.8 <0.16 3,217 
T3 1.3 140 182 29.7 30 7.5–7.8 <0.16 2,339 
T4 1.3 140 182 36.1 30 7.5–7.8 <0.15 2,339 
T5 1.3 200 260 30.1 30 7.5–7.8 <0.16 3,217 
T6 1.7 200 340 30.0 30 7.5–7.8 <0.12 3,217 
T7 1.9 180 340 30.0 30 7.5–7.8 <0.14 3,217 
T8 1.3 200 260 30.1 30 7.5–7.8 <0.16 3,217 
T9 – 80 30.1 30 7.6–7.8 <0.34 3,217 
T10 – 80 30.3 30 7.6–7.7 0.3–0.8 3,217 

F.T., feed time; VSS, volatile suspended solids.

Each test was planned to have operational conditions that favored certain biochemical pathways to the detriment of others. In this context, it was possible to compare the results of the tests by relating them to the biochemical pathways and possible microbial groups involved. The average of biomass concentration (in terms of volatile suspended solids; VSS) used in each test was 3.217 gVSS·L−1.

Between each test the SBR was adjusted back to experimental condition T1 (Table 1) for at least 12 hours or until all NO2, NH4+ and N2O had been consumed or the reactor presented a stable N2O concentration profile.

Origin and characteristics of the anammox-enriched granular sludge

Prior to the start of the tests, granular anammox biomass was collected from a full-scale sidestream treatment system in Zurich (Switzerland), and acclimatized in an SBR reactor for 18 months. Phylogenetic analysis performed by Sobotka et al. (2016) revealed the coexistence of all groups involved in the nitrogen removal cycle, including anammox bacteria (genus Candidatus Brocadia 42.9%), heterotrophic bacteria (26.8%), AOB (0.10%) and NOB (0.12%). Approximately 30% belonged to unclassified bacteria groups or were not responsible for the biochemical reactions considered in this study.

Analytical methods

During the tests, a liquid sample was taken at intervals of 10–15 min during the filling and mixing phases. The samples were filtered through an MFV-3 glass fiber filter (porosity 1.2 µm and diameter 47.0 mm). The concentrations of the nitrogenous species (NH4+, NO3 and NO2) were then analyzed in the samples filtered using analytical cuvette tests (LCK 303, LCK340, LCK342, Hach Lange GmbH, Germany). The absorbance readings were carried out using the spectrophotometer Benchtop DR3900 (Hach Lange GmbH, Germany).

N2O concentrations in the liquid phase were monitored online every 15 seconds using a Clark-type microsensor model N2O-R and a model 6276 multimeter (Unisense A/S, Denmark). The measurement range was 0–2.0 mg·L−1, the detection limit was 0.0004 mg·L−1 and the agitation sensitivity was less than 2%. The VSS analyses followed the gravimetric method 2540 E in Standard Methods for the Examination of Water and Wastewater (APHA 2012). Temperature, pH and DO were continuously monitored by both Endress + Hauser pH (model CPS471D) and DO (COS22D) probes.

RESULTS AND DISCUSSION

Effect of feed time on N2O production

Tests T1 and T2 were conducted to verify the effect of varying the feed time (6 min and 30 min) on N2O production in the SBR. As shown in Figure 1(b), the strategy of increasing feed time from 6 (Test T1) to 30 min (Test T2) favored the presence of lower concentrations of NO2 and NH4+ for the first 15 min. In the literature, higher values of ammonia and nitrite half saturation coefficients (KNH4 and KNO2) were found for heterotrophs or AOB (in oxygen limited conditions), compared with anammox (Law et al. 2012; Niu et al. 2016; Lu et al. 2018). Thus, at low substrate concentrations, anammox metabolism may be favored due to their higher affinity for the substrate and, consequently, since anammox does not have N2O as an metabolic intermediary, low N2O production was expected (Figure 1). However, the increase in feed time appeared not to favor the decrease in peak N2O production. Instead of what was predicted, the maximum N2O production reached values close to 0.900 mgN·L−1 in both tests and no significant difference was observed between Tests T1 and T2 in terms of the peak value of N2O production (Figure 1).

Figure 1

Effect of the variation of the feed time of the biological reactor (Tests T1 and T2) on nitrogen removal (a and b) and N2O production (c and d) in the liquid medium during the second cycle of the period monitored (e and f).

Figure 1

Effect of the variation of the feed time of the biological reactor (Tests T1 and T2) on nitrogen removal (a and b) and N2O production (c and d) in the liquid medium during the second cycle of the period monitored (e and f).

However, the results of the N2O concentration profile during the 2 hours of the tests (Figure 2), showed that adopting a longer feed time lead to lower N2O concentrations in the bulk liquid at most of the intervals analyzed. Only in the range between 21 min and 45 min were N2O concentrations higher in Test T2. Nitrogen removal efficiencies were similar (88%) in both cases. The VSS within the SBR did not vary significantly over the course of the experiment and the effect of their concentration on the effluent were negligible. This result was the same in all the conditions tested.

Figure 2

Comparison between production of and decrease in N2O during Test T1 (6 min of feeding) and Test T2 (30 min of feeding).

Figure 2

Comparison between production of and decrease in N2O during Test T1 (6 min of feeding) and Test T2 (30 min of feeding).

Temperature influence

As shown in Figure 3, in the case of N2O production, the increase in the operating temperature from 30°C (Test T3) to 36 °C (Test T4) produced no significant effects. The maximum production of N2O when the reactor was operated at 30 °C was 0.312 mgN·L−1 and it occurred 36 min after the test began. Under the operating temperature of 36 °C, the SBR had the peak N2O production – 0.300 mgN·L−1 – 34 min after the start of the test (Figure 3).

Figure 3

Effect of the temperature on the N2O profile in Tests T3 and T4 during a monitoring period of 20 cycles.

Figure 3

Effect of the temperature on the N2O profile in Tests T3 and T4 during a monitoring period of 20 cycles.

The ratio of N2O production to the nitrogen load rate (NLR) was 1.04% and the ratio of N2O production to the nitrogen removal rate (NRR) for both tests was 1.05%. These values are within the range reported by Ali et al. (2016): 0.1% to 0.6% for anammox reactors, 0.8% to 6.1% for partial nitrification reactors and 0.1% to 3.0% for single-stage nitrification-anammox reactors.

The only significant difference between Tests T3 and T4 was in terms of NO2-N and NH4+-N consumption rates. In this case, the adjustment of these compounds showed that operation at 36 °C led to a higher nitrogen consumption rate (1.47 gN·gVSS−1·d−1) versus 1.07 gN·gVSS−1·d−1 for the reactor operated under 30 °C. The specific NRR values were 0.49 and 0.72 gN·gSSV−1·d−1 for the reactor at 30 °C and 36 °C, respectively. Sobotka et al. (2016) also observed the increase in the specific removal rate and the anammox activity with increasing temperature, up to a limit of 40 °C, when using an enriched anammox granular biomass.

Effects of the NLR increase by the change in the NO2-N and NH4+-N affluent concentrations

In Tests T3 and T5 (Figure 4), the peak of N2O production changed in response to NO2-N and NH4+-N concentrations. The maximum N2O concentration in the liquid phase increased from 0.312 to 0.441 mgN·L−1 when the NO2-N concentration increased from 182 to 260 mgN·L−1 and the NH4+-N concentration increased from 140 to 200 mgN·L−1. These results indicate a positive correlation between N2O production and NO2-N and NH4+-N concentrations. Similarly, the results showed an increase in the maximum production factor (i.e., the N2O produced per N consumed) from 1.03% to 1.45%. These results agree with previous observations of the deammonification process (Ali et al. 2016).

Figure 4

Effects of the NLR increase by the variation in NO2-N and NH4+-N concentrations from 182 mg NO2-N·L−1 and 140 mgNH4+-N·L−1 (Test T3) to 260 mgNO2-N·L−1 and 200 mgNH4+-N·L−1 (Test T5), under the same NO2-N/NH4+-N ratio (1.3) in: nitrogen removal (a and b) and N2O concentration profile (c and d) in the liquid medium during the monitored period (e and f).

Figure 4

Effects of the NLR increase by the variation in NO2-N and NH4+-N concentrations from 182 mg NO2-N·L−1 and 140 mgNH4+-N·L−1 (Test T3) to 260 mgNO2-N·L−1 and 200 mgNH4+-N·L−1 (Test T5), under the same NO2-N/NH4+-N ratio (1.3) in: nitrogen removal (a and b) and N2O concentration profile (c and d) in the liquid medium during the monitored period (e and f).

The increased N2O production was expected, as high substrate concentrations tend to favor the species with the highest specific growth (Lu et al. 2018). Although heterotrophic bacteria most likely had the autotrophic SMPs as a source of organic matter (Liu et al. 2016), this NLR increase probably favored the heterotrophic denitrifying bacteria in comparison with anammox. A similar result was also observed by Domingo-Félez et al. (2017).

Effect of the NO2-N/NH4+-N ratio on N2O production

For tests T5, T6 and T7, a progressive increase in the NO2-N/NH4+-N ratio was provided. The first test was conducted with a ratio of 1.3 (Test T5) and for subsequent tests the NO2-N/NH4+-N ratio was increased to 1.7 (Test T6) and 1.9 (Test T7).

An overall assessment of the data presented in Figure 5 highlights a clear correlation between the NO2-N excess and N2O accumulation at the end of the reaction phase. By maintaining the NO2-N/NH4+-N ratio of 1.3, N2O production was observed, until its maximum, followed by decreases (Figure 5(d)–5(f)). On the other hand, when higher NO2-N/NH4+-N ratios were applied (Tests T6 and T7), the decrease in N2O production ceased once the anammox reaction appeared to be finished due to the lack of NH4+, approximately 1.5 hours after the beginning of the test. From this point onwards, it is likely that heterotrophic denitrification by nitrite reduction played the dominant role in N2O production.

Figure 5

Effects of the NO2-N/NH4+-N ratio variation from 1.3 (Test T5) to 1.7 (Test T6) and 1.9 (Test T7) on nitrogen removal (a–c) and N2O profile (d–f) during the period monitored (g).

Figure 5

Effects of the NO2-N/NH4+-N ratio variation from 1.3 (Test T5) to 1.7 (Test T6) and 1.9 (Test T7) on nitrogen removal (a–c) and N2O profile (d–f) during the period monitored (g).

Test T5 gave the lowest value of N2O (0.441 mg·L−1). When the NO2-N/NH4+-N ratio was increased to 1.7 in Test T6, and subsequently to 1.9 in Test T7, the N2O reached its maximum concentration (0.600 mg·L−1) (Figure 5(e) and 5(f)). Although the T6 and T7 tests produced the same peak value of N2O, the N2O concentration remaining at the end of the reaction phase was different. A higher NO2-N/NH4+-N ratio led to a higher final N2O concentration. The percentage of N2O consumed from the peak value until the end of the cycle decreased from 100% (Test T5) to 54% (Test T6) and 19% in Test T7. So the N2O concentration remaining in the bulking liquid was higher when the NO2 concentration was increased. The final N2O concentration values observed in Tests T5, T6 and T7 were 0, 0.270 and 0.484 mgN·L−1, respectively.

The higher rate of N2O reduction compared with nitrite and nitrate reduction leads to the conclusion that in anaerobic/anoxic environments, the N2O produced can be completely reduced to N2 under favorable conditions (Kampschreur et al. 2009). In the present study, however, there was no input of organic matter. With this in mind, heterotrophic denitrification probably occurred by using SMPs as electron donor sources (LIU et al. 2016), which may have been insufficient to reduce all available N2O.

A positive correlation between the concentration of nitrite and N2O production has been reported in other studies and NO2 is commonly considered to be one of the key parameters in high N2O emissions (Tallec et al. 2006; Kampschreur et al. 2009, 2008; Kim et al. 2010; Ali et al. 2016). Most of these studies, however, were performed with biomass from an activated sludge treatment system under nitrification or denitrification conditions with variable DO concentrations. Consequently, the authors generally attribute N2O production to AOB activity by autotrophic denitrification or by ammonia oxidation via hydroxylamine (NH2OH). In this study, tests were performed with low DO concentrations (less than 0.16 mgO2·L−1). Thus, the predominant mechanisms tended to be autotrophic denitrification or heterotrophic denitrification. However, for Test T6 and Test T7, there was probably a predominance of heterotrophic denitrification, since after 1.5 hours of the tests, there was no more NH4+ in the liquid medium (Figure 5(e) and 5(f)).

After Test T7, in which the NO2-N/NH4+-N ratio applied was 1.9, the SBR was again operated under an initial NO2-N/NH4+-N ratio of 1.3. The subsequent cycles were monitored in order to verify a possible decrease in accumulated N2O levels and the process recovery until total consumption of the remaining N2O (Figure 6). Test T8 gave the highest N2O value of all the proposed tests (1.24 mgN·L−1). The cycle began with a concentration of 0.385 mgN·L−1 and the net N2O production was 0.855 mgN·L−1 (Figure 6(a)). This value is higher compared to other tests, which is probably due to the nitrite remaining from the previous cycle. As shown in Figure 6(c), a NO2-N accumulation of 9.93 mgN·L−1 was observed at the beginning of Test T8. At the end of the reaction phase, 100% of the produced N2O had been consumed and the final N2O concentration was similar (0.370 mgN·L−1), at the beginning and end of the test (Figure 6(e)).

Figure 6

N2O concentration profiles (d and e) in the subsequent cycle of Test T7 (a) whose NO2-N/NH4+-N ratio was reduced from (b) 1.9 (Test T7) to (c) 1.3 (Test T8).

Figure 6

N2O concentration profiles (d and e) in the subsequent cycle of Test T7 (a) whose NO2-N/NH4+-N ratio was reduced from (b) 1.9 (Test T7) to (c) 1.3 (Test T8).

This result endorses all previous observed results where all N2O produced was consumed by the end of the reaction phase when the SBR was operated under the NO2-N/NH4+-N ratio of 1.3. After the reaction phase in Test T8, however, the N2O declined until the beginning of the feeding of the next cycle. So over the whole cycle N2O consumption was higher than production. This result was repeated in the subsequent cycles of Test T8 until no N2O was observed at the end of a cycle and the concentration of N2O in the bulk liquid was stable (Figure 6(a)).

Identification of the dominant N2O production processes (heterotrophic vs autotrophic)

During Test T9 (Figure 7) operational conditions favoring the growth of heterotrophic denitrifiers were adopted: 80 mgN·L−1 of NO2, no addition of NH4+ and DO maintained below 0.1 mgO2·L−1. Consequently, there was no substrate for the anammox or AOB and not enough DO for nitrification by NOB. Thus, the N2O production could be attributed exclusively to heterotrophic denitrification. Higher levels of DO, approximately 0.4 mgN·L−1, were present in the feed phase, probably due to agitation.

Figure 7

Effect of the absence of NH4+ (a and c) or NO2 (b and d) and DO concentration (e and f) on N2O production and nitrogen removal.

Figure 7

Effect of the absence of NH4+ (a and c) or NO2 (b and d) and DO concentration (e and f) on N2O production and nitrogen removal.

The nitrogen removal efficiency observed in Test 8 was 49.38% and 2.24% of the NRR was converted to N2O at a production rate of 0.66 mgN·gSSV−1·h−1. In a study on the emission of N2O in low DO conditions Jia et al. (2013) used 1 L SBR in the process of simultaneous nitrification and denitrification for nitrogen removal. By adding NO2 and using nitrification inhibitors, the authors favored heterotrophic denitrification and observed an N2O production rate of 0.15 mgN·gSSV−1·h−1, 4.5 times lower than the value we found in the present study.

Figure 7(c) shows the observed increase in N2O production throughout Test T9. Confirming the results obtained in previous tests (T6 and T7), the presence of NO2 resulted in N2O accumulation at the end of the cycle (0.221 mgN·L−1). This result highlights that NO2 and heterotrophic denitrification may be closely related to the accumulation and emission of N2O. Okabe et al. (2011) also related N2O production in anammox granular biomass to heterotrophic denitrification and to nitrite accumulation. In an anoxic medium, the authors did not observe nitrous oxide emissions when the granular biomass was given a synthetic medium containing NO2, NH4+ and an inhibitor for AOB and HET (Okabe et al. 2011). On the other hand, production was considerable when biomass from the same source was given a medium with only NO2.

In Test T10 (Figure 7(b) and 7(d)), aeration was provided to maintain the DO close to 0.6 mgO2·L−1 (Figure 7(f)) in the presence of NH4+ only. Those conditions could favor AOB activity to the detriment of HET and anammox bacteria. These metabolisms were inhibited due to the absence of NO2. Accordingly, N2O production could be attributed to oxidation of NH4+ via NH2OH.

The results showed maximum production (0.157 mgN·L−1) at 15 min, even during the feed phase, which corresponds to 1.38% of the oxidized ammonia in that time. Wunderlin et al. (2012) observed this production factor in the range of 1.3% to 3.8% using an inoculum from a pilot plant of activated sludge in an SBR with a DO concentration of 0.6 mg O2·L−1. During the reaction phase, 89% of N2O produced was consumed, probably used as a substrate by HET, or emitted from the liquid phase into the atmosphere. The nitrogen removal efficiency observed during the test was only 6.5%, 7.5 times lower compared to the previous test (T9). This result can be attributed to inhibition by DO concentration and the absence of NO2. Yin, X. et al. (2016) and Yin, Z. et al. (2016) also observed a reduction in the nitrogen removal efficiency (approximately 50%) when an SBR with enriched anammox biomass operated under anoxic conditions, initially operated with DO concentrations between 0.6 to 1.0 mgO2·L−1.

The net N2O production in Test T10 represented only 0.9% of the value obtained at the end of T9. These results indicated that heterotrophic denitrification had a higher impact on net N2O production than NH4+ oxidation via NH2OH. The inhibition of HET, and the inhibition of N2O production via nitrite autotrophic denitrification by AOB, was responsible for the reduction of more than 99% of the N2O production. This result was as expected, since, as an intermediary of ammonia oxidation and nitrate reduction, significant NH2OH accumulation is energetically unfavorable (Casciotti et al. 2003).

CONCLUSIONS

N2O production was influenced by the feed time, NLR and NO2N/NH4+-N ratio. The operating temperature did not influence the production of N2O. The results highlighted nitrite as a key parameter for N2O production and accumulation. When nitrite was not added to the medium, there was a reduction of more than 99% of N2O production. However, the addition of higher concentrations of nitrite in the feed medium, by increasing the NO2-N/NH4+-N ratio, resulted in NO2 accumulation and consequent N2O accumulation at the end of the cycle.

As long as nitrite is not added in excess, the anammox reactor studied presents no N2O residue at the end of the cycle period studied. SBR operation maintaining the NO2-N/NH4+-N ratio lower than 1.3 was effective for minimizing N2O production and no N2O accumulation was observed in this study. This result can be used to control N2O production in deammonification systems with granular anammox sludge in WWTPs, while still maintaining high NRR and efficiency.

AUTHOR DISCLOSURE STATEMENT

The authors declare that they do not have any conflicts of interest and no competing financial interest.

ACKNOWLEDGEMENTS

This study has been financially supported by the Polish National Center for Research and Development under the project ‘Reduction of N2O emissions from wastewater treatment plants – measurements, modeling and process optimization (RENEMO)’ (WPN/7/2013, Polish-German sustainability call) and by the Coordination for the Improvement of Higher Education Personnel (CAPES). During this study, Tiago Duarte Santos Pereira and Xi Lu were visiting researchers at Gdansk University of Technology under the project ‘CARbon BALAncing for nutrient control in wastewater treatment (CARBALA)’, People Maria Curie Actions (FP7-PEOPLE-2011-IRSES). Eduardo C. Pires has a grant from the National Council for Scientific and Technological Development (CNPq, Brazil, Grant no. 307042/2014-6).

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