Chemical energy can be recovered from municipal wastewater as biogas through anaerobic treatment. Effluent from direct anaerobic wastewater treatment at low temperatures, however, still contains ammonium and considerable amounts of dissolved methane. After nitritation, methane can be used as electron donor for denitrification by the anaerobic bacterium ‘Candidatus Methylomirabilis oxyfera’. It was shown that in the presence of 0.7% O2, denitrifying methanotrophic activity slightly increased and returned to its original level after oxygen had been removed. At 1.1% O2, methane consumption rate increased 118%, nitrite consumption rate increased 58%. After removal of oxygen, methane consumption rate fully recovered, and nitrite consumption rate returned to 88%. Therefore, traces of oxygen that bacteria are likely to be exposed to in wastewater treatment are not expected to negatively affect the denitrifying methanotrophic process. 2.0% O2 inhibited denitrifying activity. Nitrite consumption rate decreased 60% and did not recover after removal of oxygen. No clear effect on methane consumption was observed. Further studies should evaluate if intermittent addition of oxygen results in increased growth rates of the slow-growing ‘Candidatus Methylomirabilis oxyfera’.

Municipal wastewater is generally treated in activated sludge processes. In these processes, chemical energy is lost when organic matter is oxidized to carbon dioxide. Through anaerobic treatment, chemical energy can be recovered in the form of biogas instead. Effluent from anaerobic treatment contains nitrogenous compounds, mainly ammonium, and, at low temperatures, considerable amounts of dissolved methane. Nitrogen should be removed from the effluent because it causes eutrophication. Methane should be removed to prevent it from escaping to the atmosphere because it has a high global warming potential. Using methane for nitrogen removal would solve both problems.

For conventional nitrogen removal using sequential nitrification and heterotrophic denitrification, an electron donor is required. The effluent from low-temperature anaerobic municipal wastewater treatment contains insufficient organic matter to sustain heterotrophic denitrification. Addition of an external carbon source is expensive and unsustainable (Thalasso et al. 1997; Modin et al. 2007; Zhu et al. 2010). Methane has been considered a cheap and readily available alternative (Thalasso et al. 1997; Houbron et al. 1999; Modin et al. 2007).

In recent years, microorganisms have been discovered that, under anoxic conditions, directly couple denitrification to methane oxidation (Raghoebarsing et al. 2006; Haroon et al. 2013). The denitrifying methanotrophic archaea ‘Candidatus Methanoperedens nitroreducens’ reduce nitrate to nitrite whilst performing reverse methanogenesis (Haroon et al. 2013). Denitrifying methanotrophic bacteria related to ‘Candidatus Methylomirabilis oxyfera’ reduce nitrite to nitrogen gas whilst oxidizing methane (Hu et al. 2011; Luesken et al. 2011; Kampman et al. 2012). After nitritation of the effluent from anaerobic treatment, these bacteria can be applied to remove both nitrite and methane (Luesken et al. 2011; Kampman et al. 2012). M. oxyfera converts nitrite to nitric oxide, which is then converted to nitrogen gas and oxygen by an unidentified nitric oxide dismutase. Thus, even though these bacteria exist under anoxic conditions, they produce and consume their own oxygen. The oxygen is used for oxidation of methane to methanol which is subsequently oxidized to carbon dioxide (Ettwig et al. 2010). The bacteria consume methane and nitrite in a ratio close to the theoretical ratio of 0.375 (Hu et al. 2011; Luesken et al. 2011; Kampman et al. 2012). At this ratio, 21 mg CH4/L would be required to sustain complete denitrification of 50 mg NO2-N/L (typical nitrogen concentration of anaerobic effluent, assuming its presence as nitrite). This represents only a quarter of the methane produced during anaerobic treatment of municipal wastewater (Mahmoud et al. 2004), meaning this process allows for higher energy recovery than aerobic methane oxidation coupled to heterotrophic denitrification (Modin et al. 2007). If not all dissolved methane is utilized, the remainder could be removed aerobically in the following nitritation process required to oxidize ammonium prior to denitrification (Kampman et al. 2012).

Despite the highly exergonic reaction (ΔG0′−928 kJ/mol CH4, Raghoebarsing et al. 2006) performed by denitrifying methanotrophic bacteria, they grow slowly. M. oxyfera-like bacteria have a doubling time of 1–2 months (Kampman et al. 2012,  2014). The doubling time of bacteria employing the same methane oxidation pathway (ΔG0′ −818 kJ/mol CH4, Thauer & Shima 2008), such as Methylomonas capsulatus, can be as short as 13 h (Foster & Davis 1966). The doubling time of heterotrophic denitrifiers typically is 5 h (Tchobanoglous et al. 2003). Rates of oxygen-dependent reactions in M. oxyfera-like bacteria, such as methane oxidation and energy conserving reactions catalyzed by terminal oxygen reductases, might be increased by addition of oxygen (Luesken et al. 2012). Short term exposure to air did not result in reduced activity of M. oxyfera-like bacteria (Ettwig et al. 2009). Moreover, most M. oxyfera-like bacteria have been enriched from the oxic/anoxic interface of freshwater sediments (Raghoebarsing et al. 2006; Kampman et al. 2012; Zhu et al. 2012) or from wastewater treatment sludge exposed to alternating oxic and anoxic conditions (Luesken et al. 2011; Kampman et al. 2014). However, Luesken et al. (2012) showed irreversible inhibition of denitrifying methanotrophic bacteria upon exposure to oxygen partial pressures of 2% and 8%, without full recovery within 8 h after oxygen exposure. Most genes involved in denitrification and also genes involved in vital cellular functions were significantly downregulated (Luesken et al. 2012).

Possibly, the oxygen concentrations applied in the experiments by Luesken et al. (2012) were too high. The effect of lower oxygen concentrations and if M. oxyfera-like bacteria can use external oxygen to oxidize methane still needs to be studied (Shen et al. 2015; Zui et al. 2015). In wastewater treatment denitrifying methanotrophic bacteria might be exposed to traces of oxygen, remaining from the nitritation process required to produce nitrite (Kampman et al. 2012). Therefore, in the present study the effect of 0.7% (0.35 mg dissolved O2/L), 1.1% (0.49 mg dissolved O2/L), and 2.0% (1.0 mg dissolved O2/L), on denitrifying activity was tested. The results and consequences for enhancing enrichment rate of denitrifying methanotrophic bacteria and their application in wastewater treatment are discussed.

Experimental setup batch tests

The effect of oxygen partial pressures of 0.7% (0.35 mg dissolved O2/L), 1.1% (0.49 mg/L) and 2.0% (1.0 mg/L) on denitrifying methanotrophic activity was tested in 0.6 L bottles. All oxygen concentrations and also controls without oxygen were tested in duplicate. Biomass enriched in M. oxyfera-like bacteria (Kampman et al. 2012) was first siphoned to 1 L bottles under constant flushing with nitrogen gas. Then, in an anaerobic chamber the test bottles were filled with 250 mL reactor sample, 7.5 mL 209 g 3-(N-morpholino)propanesulfonic acid (MOPS)/L (pH 7.0, final concentration 6.28 g MOPS/L) and 0.6 mL 14 g NO2-N/L (final concentration 32 mg NO2-N/L). Hereafter, the bottles were flushed with methane for 10 min, pressurizing the bottles to 1.4 bar, and after 3 h equilibration time, volumetric nitrite and methane consumption rates were determined. After 2–3 days, pure oxygen (3.0 mL, 5.0 mL and 10 mL to achieve final concentrations of 0.7%, 1.0% and 2.0% respectively) was added. The bottles were shaken vigorously, after which activity measurements were resumed immediately. After 22 h, oxygen was removed: the head space of the bottles was replaced with nitrogen (five cycles of vacuum and purge) and flushed with CH4. After 3 h equilibration time, activity was measured for 3–4 more days to establish recovery of the denitrifying activity. During the whole test, the bottles were incubated at 30 °C and mixed by magnetic stirring (100 rpm). Oxygen concentration in the bottles was measured online every minute (PSt3 non-invasive oxygen sensors and Oxy-4 trace oxygen meter, PreSens). Biogas composition and nitrite concentration were measured 2–3 times per day (see Analyses section). Nitrite was added to the bottles at a concentration of 28 mg NO2-N/L every time its concentration was close to zero. For calculations and data representation, the concentration at the moment of addition was corrected for the measured concentration just before addition.

Reactor activity test

For a period of 11 days, four reactors enriched in M. oxyfera-like bacteria were exposed to oxygen. Three membrane bioreactors (MBRs; described by Kampman et al. 2014) and one sequencing fed-batch reactor (SFBR; described by Kampman et al. 2012) inoculated with biomass originating from environments with fluctuating oxygen concentrations had been operated for more than two years prior to this activity test. Biomass was present both in suspension and as biofilm. The reactors were fed with a mineral medium. 10% (v/v) 0.2 μm filtered effluent from aerobic wastewater treatment was added to the medium supplied to two of the MBRs and the SFBR in order to supply potential growth factors (Kampman et al. 2012). Oxygen was supplied in a mixture with methane (92% CH4, 8.0% O2) at a flow rate of 5.1 mL/min, instead of a mixture of 95% CH4 and 5% CO2. After these 11 days, the reactors were flushed for 3 days with CH4/CO2 (95/5%) at an increased flow rate of 20 mL/min to rapidly remove remaining oxygen. After these 3 days, the CH4/CO2 gas flow rate was set to 5 mL/min. The effect of this period of oxygen exposure was evaluated from long-term reactor monitoring. To assess the effect of oxygen on the microbial composition immediately after exposure to oxygen, the reactors were sampled for fluorescence in situ hybridization (FISH; described in the following section).

Analyses

Gas composition (O2, N2, CH4 and CO2) was measured by gas chromatography, using 0.5 mL samples. To monitor reactor performance, nitrite and nitrate concentrations were estimated 3–7 times per week using test strips (Merckoquant®, Merck Chemicals). For more accurate process monitoring, nitrite and nitrate concentrations were measured by ion chromatography. These methods were described in detail by Kampman et al. (2012). Volumetric activity was determined rather than specific activity because biomass was scarce and it was undesirable to sacrifice biomass for analysis of biomass concentration. Moreover, in the reactors, biomass was present both in suspension and as biofilm and therefore the amount of biomass would have been difficult to determine. FISH was performed as described by Ettwig et al. (2008). A hybridization buffer with 20% formamide was used and samples were stored at −18 °C. To target bacteria affiliated with the ‘NC10’ phylum probes S-*-DBACT-0193-a-A-18 (DBACT193) and S-*-DBACT-1027-a-A-18 (DBACT1027) (Raghoebarsing et al. 2006) were used. S-Sc-aProt-0968-a-A-18 (ALF968; Neef 1997) and L-C-gProt- 1027-a-A-17 (GAM42a; Manz et al. 1992) were applied to detect respectively Alpha- and Gammaproteobacteria. EUB mix was used to target almost all bacteria (Daims et al. 1999) and DAPI was used to stain all DNA.

Effect of 0.7, 1.1 and 2.0% oxygen on denitrifying methanotrophic activity

All the batch tests, the results of which will be described in more detail below, demonstrated denitrifying methanotrophic activity before, during and after exposure to 0.7% (0.35 mg/L), 1.1% (0.49 mg/L), as illustrated by Figure 1, and 2.0% (1.0 mg/L) O2. The results indicated that the highest consumption rates of methane and nitrite, along with higher production rates of nitrogen gas could be achieved in the presence of 1.1% O2. 0.7% O2 hardly affected activity. In the presence of 2.0% O2, the denitrifying activity decreased and did not recover the first three days after oxygen was removed. In the controls without oxygen, a constant denitrification rate of 27 mg NO2-N/L·d was observed for the duration of the tests.

Figure 1

Results from an activity test with 1.1% O2 and at 30 °C. Figure (a) shows the total amount of methane (♦) and oxygen (—), figure (b) shows the total amount of nitrogen gas (▪) and nitrite (□) in time. The grey blocks indicate the aerobic period; 1.1% O2 was added at the start of this period. Methane and nitrite consumption rate and nitrogen gas production rate slightly increased in presence of oxygen.

Figure 1

Results from an activity test with 1.1% O2 and at 30 °C. Figure (a) shows the total amount of methane (♦) and oxygen (—), figure (b) shows the total amount of nitrogen gas (▪) and nitrite (□) in time. The grey blocks indicate the aerobic period; 1.1% O2 was added at the start of this period. Methane and nitrite consumption rate and nitrogen gas production rate slightly increased in presence of oxygen.

Comparison of initial activity in test bottles and SFBR

Biomass from the SFBR was used for the activity tests. At the start of the tests, the volumetric nitrite consumption rate in the bottles was 22–27 mg NO2-N/L·d. Conversion rates were linear, i.e. independent of substrate or product concentration. The average conversion ratios of CH4: NO2: N2 were close to the stoichiometric ratio of 3: 8: 4 (Table 1). The consumption rates were somewhat higher than the 19 mg NO2-N/L·d measured in the reactor at the time the sludge was sampled. Differences in rates between the individual test bottles and between the bottles and the reactor could be due to different mixing conditions or different biomass concentrations. The latter were not determined since measuring volatile suspended solids could not be done without sacrificing a substantial part of the biomass.

Table 1

Methane, nitrite and oxygen consumption rates and nitrogen gas production rates in controls; tests with 0.7, 1.1 and 2.0% O2 in the headspace in the first anoxic period, before oxygen exposure; during oxygen exposure; and in the second anoxic period, after oxygen exposure

Before oxygen exposure
During oxygen exposure
 After oxygen exposure
 CH4NO2N2CH4NO2N2O2pO2CH4NO2N2
mg/L·dmg N/L·dmg N/L·dmg/L·dmg N/L·dmg N/L·dmg/L·d%mg/L·dmg N/L·dmg N/L·d
Control 
 average 14 27 32         
 A 12 27 31         
 B 15 27 33         
0.7% O2 
 average 11 26 24 12 29 27 25 0.15 12 28 24 
 A 11 27 22 14 28 28 28 0.13 11 27 23 
 B 11 26 26 10 29 26 22 0.17 12 29 25 
1.1% O2 
 average 9.0 24 26 20 38 33 29 0.29 9.0 21 22 
 A 9.6 23 28 19 40 31 27 0.37 9.3 15 21 
 B 8.3 25 25 20 37 35 31 0.21 9.3 28 23 
2.0% O2 
 average 7.0 24 20 7.0 10 12 42 1.0 5.0 10 10 
 A 6.1 22 19 5.3 7.8 7.8 39 0.89 2.4 8.2 5.9 
 B 7.6 26 21 9.2 12 16 45 1.1 7.5 12 14 
Before oxygen exposure
During oxygen exposure
 After oxygen exposure
 CH4NO2N2CH4NO2N2O2pO2CH4NO2N2
mg/L·dmg N/L·dmg N/L·dmg/L·dmg N/L·dmg N/L·dmg/L·d%mg/L·dmg N/L·dmg N/L·d
Control 
 average 14 27 32         
 A 12 27 31         
 B 15 27 33         
0.7% O2 
 average 11 26 24 12 29 27 25 0.15 12 28 24 
 A 11 27 22 14 28 28 28 0.13 11 27 23 
 B 11 26 26 10 29 26 22 0.17 12 29 25 
1.1% O2 
 average 9.0 24 26 20 38 33 29 0.29 9.0 21 22 
 A 9.6 23 28 19 40 31 27 0.37 9.3 15 21 
 B 8.3 25 25 20 37 35 31 0.21 9.3 28 23 
2.0% O2 
 average 7.0 24 20 7.0 10 12 42 1.0 5.0 10 10 
 A 6.1 22 19 5.3 7.8 7.8 39 0.89 2.4 8.2 5.9 
 B 7.6 26 21 9.2 12 16 45 1.1 7.5 12 14 

A and B indicate the duplicates. Oxygen consumption started after a lag phase of 7 h. pO2 indicates the oxygen concentration at the end of the aerobic period.

Development of oxygen concentration in the test bottles

Biomass in the batch tests was exposed to oxygen for 22 h. (Facultative) aerobic bacteria were present in the enrichment culture: in all batch tests, after a lag time of 7 h, oxygen consumption started (Figure 2). Oxygen may have been consumed by nitrifying bacteria as well, although no nitrate production was observed. Nitrate could have been removed by denitrification without accumulating in the system. Oxygen remained present throughout all tests. After 22 h, oxygen was still present at an average of 0.15% (0.08 mg/L) in the tests started with 0.7% O2, 0.29% (0.14 mg/L) in the tests started with 1.1% O2 and 0.96% (0.47 mg/L) in the tests started with 2.0% O2. These results indicated that higher initial oxygen concentrations resulted in higher oxygen consumption rates (Table 1).

Figure 2

Relative conversion rates in activity tests with 0.7, 1.1 and 2.0% O2. Conversion rates were calculated relative to the conversion rate in the first anoxic period (100%), which is not shown in the graphs.

Figure 2

Relative conversion rates in activity tests with 0.7, 1.1 and 2.0% O2. Conversion rates were calculated relative to the conversion rate in the first anoxic period (100%), which is not shown in the graphs.

0.7% Oxygen

In the presence of 0.7% O2, the average methane, nitrite and nitrogen gas conversion rates increased to respectively 113%, 107% and 111% of the rates in the anoxic period (Figure 2, Table 1). After oxygen was removed, the average methane, nitrite and nitrogen gas conversion rates rate were 104%, 99% and 95% of the rate before oxygen addition, respectively. Both in the presence of and after removal of oxygen, conversion ratios were close to the theoretical ratio of 3 CH4: 8 NO2: 4 N2. Thus, only a minor increase of conversion rates was observed in presence of 0.7% O2 and, when oxygen was removed, rates returned to equal rates as before oxygen exposure.

1.1% Oxygen

Addition of 1.1% O2 increased all conversion rates (Figures 1 and 2, Table 1). The average methane consumption rate increased to 218% of the rate before oxygen addition; nitrite consumption rate increased to 158% and nitrogen gas production rate increased to 125%. The average CH4:NO2:N2 ratio was 3:6.7:2.9. The stoichiometry shifted towards methane. Possibly, the increased methane consumption was due to aerobic methane oxidation. Based on a stoichiometric ratio for O2:CH4 of 2:1, aerobic methane oxidizers alone could, however, only have been responsible for a maximum of 7.3 mg CH4/L·d. Methane consumption rates increased with 11 mg/L·d. This means that, in addition to aerobic methanotrophic activity, also the activity of denitrifying methanotrophic bacteria might have increased. This is supported by the observed increase in nitrite consumption rates and nitrogen gas production rates. Nitrite may have partially been removed by aerobic methane oxidation coupled to denitrification. However, according to the above stoichiometry, not all nitrite consumed was converted to nitrogen gas. It is unclear to what compounds, besides nitrogen gas, the nitrite was converted. M. oxyfera-like bacteria bypass nitrous oxide as an intermediate in denitrification (Ettwig et al. 2010). After oxygen was removed, the methane consumption rate returned to the rate in the first anoxic period. On average, nitrite consumption rate returned to 88% (in one bottle 71%, in the other bottle 105%) and nitrogen gas production rate returned to 81% of the rate in the first anoxic period. The average CH4:NO2:N2 ratio was 3:7.8:4.1, indicating that denitrification coupled to anaerobic methane oxidation was the dominant process. The different response in the duplicates with respect to the nitrite consumption rate could not be explained.

2.0% Oxygen

Although 1.1% O2 enhanced denitrifying methanotrophic activity, a concentration of 2.0% O2 resulted in a decrease in most conversion rates (Figure 2, Table 1). Methane consumption rate was 101% of the rate before exposure. However, denitrifying activity decreased: nitrite consumption rate and nitrogen gas production rate were decreased by 60% and 42%, respectively. Luesken et al. (2012) observed a 25% lower methane consumption rate and a 57% lower nitrite consumption rate at the same oxygen concentration. The latter is in good agreement with the present results. Similar to the tests at 1.1% O2 and as observed by Luesken et al. (2012), at 2.0% O2 the stoichiometry shifted towards aerobic methane oxidation. This may be due to inhibition of the denitrification process in the presence of oxygen. Moreover, denitrifying methanotrophic bacteria may prefer oxygen as an electron acceptor (Luesken et al. 2012). Possibly also activity of aerobic methanotrophs, if present, increased due to increased oxygen concentrations. The CH4:NO2:N2 ratio was 3:4.7:2.9. The discrepancy between nitrite consumption and nitrogen gas production could not be explained. Possibly other nitrogenous compounds, such as nitrogen incorporated in cell debris or ammonium added with the influent (4 mg NH4+-N/L) were converted to nitrogen gas by nitrification–denitrification. After removing oxygen, the methane consumption rate in bottle A was only 38% of the initial rate, in bottle B 95%. Nitrite consumption and nitrogen gas production amounted to 36–46% (respectively bottle A and B) and 29–64% (respectively bottle A and B) of the initial activity, respectively. It is unclear how methane consumption recovered in bottle B, while consumption of nitrite and nitrogen gas did not. Similar to the experiments at 1.1% O2, the stoichiometry shifted towards methane oxidation: the ratio CH4:NO2:N2 was 3:5.7:3.1; in bottle A the ratio was 3:12:4.2. The difference between the bottles cannot be explained.

Contribution of aerobic microorganisms to increased conversion rates

If aerobic methanotrophs were responsible for the increased methane consumption rate, at 2.0% O2 rates similar to or higher than at 0.7 or 1.1% O2 would be expected. However, whereas at 0.7 and 1.1% O2 the consumption rates increased, at 2.0% O2 the methane consumption rate was similar to the rate before oxygen addition (Figure 2). This supports the hypothesis that denitrifying methanotrophic bacteria exhibit increased activity in the presence of 1.1% O2. After exposure to 2.0% O2 and, remarkably, also after exposure to 1.1% O2, denitrifying methanotrophic activity did not completely recover. Irreversible inhibition by 2% oxygen was also reported by Luesken et al. (2012).

Reactors fed with 8% oxygen

Nitrite consumption in the presence of oxygen

For a period of 11 days, all reactors were fed with 92% CH4 and 8% O2 at a flow rate of 5.1 mL/min. Oxygen was consumed in the reactors. Reduced concentrations of 0.6% (MBR2 and SFBR), 0.9% (MBR1) and 1.2% (MBR3) O2 were measured. Batch tests showed that at 1.1% O2 the nitrite consumption rates doubled. If this also happened in the reactors it remained unnoticed due to the inaccuracy of the test strips used to follow reactor performance on a day-to-day basis.

Oxygen sensitivity of denitrifying methanotrophs and the role of the side populations in oxygen removal

All enrichment cultures of denitrifying methanotrophic bacteria described in literature, contained more than 20% unidentified bacteria. Luesken et al. (2012) showed an increased expression of genes involved in methane oxidation of several aerobic methanotrophic bacteria upon oxygen exposure. These bacteria belonged to the classes of Alpha- and Gammaproteobacteria, to which, respectively, type I and type II methanotrophs belong. Also in the enrichment cultures exposed to oxygen Alpha- and Gammaproteobacteria were present before and directly after (Figure 3) oxygen exposure. From the observed oxygen removal and the presence of proteobacteria in all reactors, it is hypothesized that the presence of a side population, consisting of e.g. aerobic methanotrophic bacteria and aerobic heterotrophic organisms, able to consume oxygen, may be important for reducing the effect of oxygen on denitrifying methanotrophic bacteria.

Figure 3

Fluorescence in situ hybridization of biomass. (a) Shows biomass from MBR1 after hybridization with probes EUB mix (probes EUB338 I–III; Cy5; dark blue), detecting nearly all eubacteria; DBACT193 (Flu; green) specific for ‘NC10’ bacteria; and ALF968 (Cy3; red) specific for Alphaproteobacteria. Due to co-hybridization with the specific and general probes, the M. oxyfera-like bacteria appear turquoise and the Alphaproteobacteria appear pink. The scale bar indicates 10 μm. (b) Shows biomass from SFBR after hybridization with probes EUB mix (probes EUB338 I–III; Cy5; dark blue), detecting nearly all eubacteria; GAM42a (Flu; green) specific for Alphaproteobacteria; and DBACT193 (Cy3; red) specific for ‘NC10’ bacteria. Due to co-hybridization with the specific and general probes, the M. oxyfera-like bacteria appear pink and the Gammaproteobacteria appear turquoise. The scale bar indicates 20 μm. Please refer to the online version of this paper to see this figure in color: http://dx.doi.org/10.2166/wst.2018.219.

Figure 3

Fluorescence in situ hybridization of biomass. (a) Shows biomass from MBR1 after hybridization with probes EUB mix (probes EUB338 I–III; Cy5; dark blue), detecting nearly all eubacteria; DBACT193 (Flu; green) specific for ‘NC10’ bacteria; and ALF968 (Cy3; red) specific for Alphaproteobacteria. Due to co-hybridization with the specific and general probes, the M. oxyfera-like bacteria appear turquoise and the Alphaproteobacteria appear pink. The scale bar indicates 10 μm. (b) Shows biomass from SFBR after hybridization with probes EUB mix (probes EUB338 I–III; Cy5; dark blue), detecting nearly all eubacteria; GAM42a (Flu; green) specific for Alphaproteobacteria; and DBACT193 (Cy3; red) specific for ‘NC10’ bacteria. Due to co-hybridization with the specific and general probes, the M. oxyfera-like bacteria appear pink and the Gammaproteobacteria appear turquoise. The scale bar indicates 20 μm. Please refer to the online version of this paper to see this figure in color: http://dx.doi.org/10.2166/wst.2018.219.

Effect of oxygen on nitrite consumption rates during prolonged reactor operation

After re-establishing anoxic conditions, tests were performed to check if denitrification still occurred. Significant denitrifying methanotrophic activity was still present, showing that oxygen had not irreversibly inhibited the bacteria. Hereafter, reactor operation was continued.

Long-term behavior of each reactor after the exposure to oxygen was different. Despite a low oxygen concentration of 0.6% O2 in MBR2, biomass seemed negatively affected by exposure to oxygen (Figure 4(a)): the same nitrite loading rate of 11 mg NO2-N/L·d was applied before and during oxygen exposure. However, at the same loading rate, 3 weeks after oxygen exposure, rapid nitrite accumulation occurred. The nitrite loading rate in MBR2 therefore had to be decreased from 11 to 3 mg NO2-N/L·d to match the consumption rate. Hereafter, slow recovery took place and 2 months after the oxygen exposure, a volumetric nitrite consumption rate fluctuating between 5–6 mg NO2-N/L·d was reached, at which it remained for another two months. In MBR1, the decrease (data not shown) and in MBR3 (Figure 4(b)) the increase in volumetric nitrite consumption rates that had already started before exposure to oxygen continued. The rate in SFBR was not affected and the consumption rate remained around 19 mg NO2-N/L·d (data not shown).

Figure 4

Volumetric nitrite loading rate in (a) MBR2 (at 20 °C) and (b) MBR3 (at 30 °C) in time. The grey bar indicates an 11 d period of oxygen exposure. 8% O2 was fed to the reactors, at the end of the aerobic period 0.6% O2 (MBR2) and 1.2% O2 (MBR3) were measured in the reactors. A loading rate of 0 mg NO2-N/L·d was set in the event of too high nitrite accumulation. In all other cases, nitrite consumption rates were well represented by the nitrite loading rate.

Figure 4

Volumetric nitrite loading rate in (a) MBR2 (at 20 °C) and (b) MBR3 (at 30 °C) in time. The grey bar indicates an 11 d period of oxygen exposure. 8% O2 was fed to the reactors, at the end of the aerobic period 0.6% O2 (MBR2) and 1.2% O2 (MBR3) were measured in the reactors. A loading rate of 0 mg NO2-N/L·d was set in the event of too high nitrite accumulation. In all other cases, nitrite consumption rates were well represented by the nitrite loading rate.

Implications

The present study shows that denitrifying methanotrophic bacteria are not inhibited by low oxygen concentrations of 0.7 and 1.1% O2. Addition of low concentrations of oxygen might even offer opportunities to accelerate the enrichment of the slow-growing denitrifying methanotrophic bacteria. This is hypothesized since at the here observed increased methane and nitrite consumption rates at 1.1% O2, it is likely that also the biomass growth rates increased. This is important for full-scale applications, were denitrifying methanotrophic bacteria are likely to be exposed to traces of oxygen, in case of calamities or remaining from a nitritation process (Kampman et al. 2012) are not anticipated to result in a collapse of a system with denitrifying methanotrophic bacteria. Moreover, although low growth rates do not have to be a problem for full-scale applications, first enough sludge is required to start a plant (Shen et al. 2012). If growth rates can be increased, e.g. by applying a system with intermittent micro-aerobic and anaerobic periods, startup times of reactors with denitrifying methanotrophic bacteria may be reduced. Long term oxygen addition to continuous reactors with denitrifying methanotrophic activity is needed to investigate this.

  • Contrary to previous findings, oxygen did not necessarily inhibit denitrifying methanotrophic activity.

  • Batch tests showed that low oxygen concentrations, resulting either from increased denitrifying methanotrophic activity or from an external oxygen source, could both stimulate (1.1% O2) or inhibit (2.0% O2) denitrifying methanotrophic bacteria.

  • After exposure to oxygen for 11 d (0.6–1.2%) three out of four denitrifying methanotrophic cultures do not seem influenced by the presence of oxygen. In another culture, denitrification rates decreased with 75%.

  • Traces of oxygen that bacteria are likely to be exposed to in wastewater treatment are not expected to negatively affect the denitrification process and might even result in increased activity. Further studies should evaluate if long-term addition of oxygen results in increased growth rates.

This study was financed by Technology Foundation STW (STW project 07736), The Netherlands. STW was not involved in the research or the writing of this article. The authors would like to thank Francisca Luesken (Department of Microbiology, Radboud University, Nijmegen, The Netherlands) for her assistance with fluorescence in situ hybridization.

Daims
,
H.
,
Brühl
,
A.
,
Amann
,
R.
,
Schleifer
,
K.-H.
&
Wagner
,
M.
1999
The domainspecific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set
.
Systematic and Applied Microbiology
22
(
3
),
434
444
.
Ettwig
,
K. F.
,
Shima
,
S.
,
Van de Pas-Schoonen
,
K. T.
,
Kahnt
,
J.
,
Medema
,
M. H.
,
Op den Camp
,
H. J. M.
,
Jetten
,
M. S. M.
&
Strous
,
M.
2008
Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea
.
Environmental Microbiology
10
(
11
),
3164
3173
.
Ettwig
,
K. F.
,
Van Alen
,
T.
,
Van de Pas-Schoonen
,
K. T.
,
Jetten
,
M. S. M.
&
Strous
,
M.
2009
Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum
.
Applied and Environmental Microbiology
75
(
11
),
3656
3662
.
Ettwig
,
K. F.
,
Butler
,
M. K.
,
Le Paslier
,
D.
,
Pelletier
,
E.
,
Mangenot
,
S.
,
Kuypers
,
M. M. M.
,
Schreiber
,
F.
,
Dutilh
,
B. E.
,
Zedelius
,
J.
,
De Beer
,
D.
,
Gloerich
,
J.
,
Wessels
,
H. J. C. T.
,
Van Alen
,
T.
,
Luesken
,
F.
,
Wu
,
M. L.
,
Van de Pas-Schoonen
,
K. T.
,
Op den Camp
,
H. J. M.
,
Janssen-Megens
,
E. M.
,
Francoijs
,
K.-J.
,
Stunnenberg
,
H.
,
Weissenbach
,
J.
,
Jetten
,
M. S. M.
&
Strous
,
M.
2010
Nitrite-driven anaerobic methane oxidation by oxygenic bacteria
.
Nature
464
(
7288
),
543
548
.
Foster
,
J. W.
&
Davis
,
R. H.
1966
A methane-dependent coccus with notes of classification and nomenclature of obligate methane-utilizing bacteria
.
Journal of Bacteriology
91
(
5
),
1924
1931
.
Haroon
,
M. F.
,
Hu
,
S.
,
Shi
,
Y.
,
Imelfort
,
M.
,
Keller
,
J.
,
Hugenholtz
,
P.
,
Yuan
,
Z.
&
Tyson
,
G. W.
2013
Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage
.
Nature
500
(
7464
),
567
570
.
Houbron
,
E.
,
Torrijos
,
M.
&
Capdeville
,
B.
1999
An alternative use of biogas applied at the water denitrification
.
Water Science and Technology
40
(
8
),
115
122
.
Hu
,
S.
,
Zeng
,
R. J.
,
Keller
,
J.
,
Lant
,
P. A.
&
Yuan
,
Z.
2011
Effect of nitrate and nitrite on the selection of microorganisms in the denitrifying anaerobic methane oxidation process
.
Environmental Microbiology Reports
3
(
3
),
315
319
.
Kampman
,
C.
,
Hendrickx
,
T. L. G.
,
Luesken
,
F. A.
,
Van Alen
,
T. A.
,
Op den Camp
,
H. J. M.
,
Jetten
,
M. S. M.
,
Zeeman
,
G.
,
Buisman
,
C. J. N.
&
Temmink
,
H.
2012
Enrichment of denitrifying methanotrophic bacteria for application after direct low temperature anaerobic sewage treatment
.
Journal of Hazardous Materials
227
,
164
171
.
Kampman
,
C.
,
Temmink
,
H.
,
Hendrickx
,
T. L. G.
,
Zeeman
,
G.
&
Buisman
,
C. J. N.
2014
Enrichment of denitrifying methanotrophic bacteria from municipal wastewater sludge in a membrane bioreactor at 20°C
.
Journal of Hazardous Materials
274
,
428
435
.
Luesken
,
F. A.
,
Van Alen
,
T. A.
,
Van der Biezen
,
E.
,
Frijters
,
C.
,
Toonen
,
G.
,
Kampman
,
C.
,
Hendrickx
,
T. L. G.
,
Zeeman
,
G.
,
Temmink
,
H.
,
Strous
,
M.
,
Op den Camp
,
H. J. M.
&
Jetten
,
M. S. M.
2011
Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge
.
Applied Microbiology and Biotechnology
92
(
4
),
845
854
.
Luesken
,
F. A.
,
Wu
,
M. L.
,
Op den Camp
,
H. J. M.
,
Keltjens
,
J. T.
,
Stunnenberg
,
H.
,
Francoijs
,
K.-J.
,
Strous
,
M.
&
Jetten
,
M. S. M.
2012
Effect of oxygen on the anaerobic methanotroph ‘Candidatus Methylomirabilis oxyfera’: kinetic and transcriptional analysis
.
Environmental Microbiology
14
(
4
),
1024
1034
.
Mahmoud
,
N.
,
Zeeman
,
G.
,
Gijzen
,
H.
&
Lettinga
,
G.
2004
Anaerobic sewage treatment in a one-stage UASB reactor and a combined UASB-digester system
.
Water Research
38
(
9
),
2347
2357
.
Manz
,
W.
,
Amann
,
R.
,
Ludwig
,
W.
,
Wagner
,
M.
&
Schleifer
,
K. H.
1992
Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria – problems and solutions
.
Systematic and Applied Microbiology
15
(
4
),
593
600
.
Modin
,
O.
,
Fukushi
,
K.
&
Yamamoto
,
K.
2007
Denitrification with methane as external carbon source
.
Water Research
41
(
12
),
2726
2738
.
Neef
,
A.
1997
Anwendung der In-situ-Einzelzell-Identifizierung von Bakterien zur Populationsanalyse in komplexen mikrobiellen Biozönosen
.
PhD Thesis
,
Technische Universität München
,
Germany
.
Raghoebarsing
,
A. A.
,
Pol
,
A.
,
Van de Pas-Schoonen
,
K. T.
,
Smolders
,
A. J. P.
,
Ettwig
,
K. F.
,
Rijpstra
,
W. I. C.
,
Schouten
,
S.
,
Sinninghe Damsté
,
J. S.
,
Op den Camp
,
H. J. M.
,
Jetten
,
M. S. M.
&
Strous
,
M.
2006
A microbial consortium couples anaerobic methane oxidation to denitrification
.
Nature
440
(
7086
),
918
921
.
Shen
,
L.
,
He
,
Z.
,
Zhu
,
Q.
,
Chen
,
D.
,
Lou
,
L.
,
Xu
,
X.
,
Zheng
,
P.
&
Hu
,
B.
2012
Microbiology, ecology and application of the nitrite-dependent anaerobic methane oxidation process
.
Frontiers in Microbiology
3
(
269
),
1
5
.
Tchobanoglous
,
G.
,
Burton
,
F. L.
&
Stensel
,
H. D.
2003
Wastewater Engineering: Treatment and Reuse
.
McGraw-Hill
,
Boston
,
MA, USA
.
Thalasso
,
F.
,
Vallecillo
,
A.
,
García-Encina
,
P.
&
Fdz-Polanco
,
F.
1997
The use of methane as a sole carbon source for wastewater denitrification
.
Water Research
31
(
1
),
55
60
.
Thauer
,
R. K.
&
Shima
,
S.
2008
Methane as fuel for anaerobic microorganisms
.
Annals of the New York Academy of Sciences
1125
,
158
170
.
Zhu
,
S.
,
Nàcher
,
C.
,
Pellicer
,
I.
,
Merkey
,
B.
,
Zhou
,
Q.
,
Xia
,
S.
,
Yang
,
D.
,
Sun
,
J.
&
Smets
,
B. F.
2010
Effective biological nitrogen removal treatment processes for domestic wastewaters with low C/N ratios: a review
.
Environmental Engineering Science
27
(
2
),
111
126
.
Zhu
,
B.
,
Van Dijk
,
G.
,
Fritz
,
C.
,
Smolders
,
A. J. P.
,
Pol
,
A.
,
Jetten
,
M. S. M.
&
Ettwig
,
K. F.
2012
Anaerobic oxidization of methane in a minerotrophic peatland: enrichment of nitrite-dependent methane-oxidizing bacteria
.
Applied and Environmental Microbiology
78
(
24
),
8657
8665
.
Zui
,
M.
,
Ma
,
A.
,
Qi
,
H.
,
Zhuang
,
X.
&
Zhuang
,
G.
2015
Anaerobic oxidation of methane: an ‘active’ microbial process
.
Microbiology Open
4
(
1
),
1
11
.