Simultaneous Anaerobic and Aerobic Ammonia and Methane Oxidation under Oxygen Limitation Conditions

Abstract

Methane and ammonia have to be removed from wastewater treatment effluent in order to discharge it to receiving water bodies. A potential solution for this is a combination of simultaneous ammonia and methane oxidation by anaerobic ammonia oxidation (anammox) bacteria and nitrite/nitrate-dependent anaerobic methane oxidation (N-damo) microorganisms. When applied, these microorganisms will be exposed to oxygen, but little is known about the effect of a low concentration of oxygen on a culture containing these microorganisms. In this study, a stable coculture containing anammox and N-damo microorganisms in a laboratory scale bioreactor was established under oxygen limitation. Membrane inlet mass spectrometry (MIMS) was used to directly measure the in situ simultaneous activity of N-damo, anammox, and aerobic ammonia-oxidizing microorganisms. In addition, batch tests revealed that the bioreactor also harbored aerobic methanotrophs and anaerobic methanogens. Together with fluorescence in situ hybridization (FISH) analysis and metagenomics, these results indicate that the combination of N-damo and anammox activity under the continuous supply of limiting oxygen concentrations is feasible and can be implemented for the removal of methane and ammonia from anaerobic digester effluents. IMPORTANCE Nitrogen in wastewater leads to eutrophication of the receiving water bodies, and methane is a potent greenhouse gas; it is therefore important that these are removed from wastewater. A potential solution for the simultaneous removal of nitrogenous compounds and methane is the application of a combination of nitrite/nitrate-dependent methane oxidation (N-damo) and anaerobic ammonia oxidation (annamox). In order to do so, it is important to investigate the effect of oxygen on these two anaerobic processes. In this study, we investigate the effect of a continuous oxygen supply on the activity of an anaerobic methane- and ammonia-oxidizing coculture. The findings presented in this study are important for the potential application of these two microbial processes in wastewater treatment.

Keywords: ammonia oxidation; anaerobic methane oxidation; anammox; methane oxidation; nitrification; oxygen exposure; wastewater treatment.

FIG 1

Consumption of ammonium (diamonds), nitrite (circles), and nitrate (triangles) by the reactor. Directly after the addition of oxygen and the increase of the ammonium concentration in the medium, nitrate was produced and is plotted here as a negative consumption. The start of the addition of oxygen is indicated with the dotted line.

FIG 2

Representative fluorescence in situ hybridization (FISH) pictures of the enrichment culture under anaerobic (A and B) and oxygen-limited (C to H) conditions. Bars, 20 μm. (A and B) Samples taken from an N-damo/anammox coculture before oxygen addition. In red, anammox bacteria are visible (Cy3, AMX820), and archaea are visualized in blue (Cy5, ARCH915). Methylomirabilis spp. constitute the majority of the biomass and are visible in green (FLUOS, DAMO1027). (C) Sample taken 292 days after oxygen addition. The percentage of Methanoperedens-like archaea (red, DARCH641) and anammox bacteria (blue, AMX820) increased, while the relative abundance of Methylomirabilis spp. (green, DAMO1027) decreased. (D) Sample taken 732 days after oxygen addition. The percentage of Methylomirabilis spp. (green, DAMO1027b) decreased further. Methanoperedens-like archaea (red, DARCH641), and anammox bacteria (blue, AMX820) became the dominant microorganisms in the culture. (E) Sample of granules sampled 800 days after the start of oxygen-limited culturing conditions. Anammox bacteria (green, AMX820) and archaea (blue, ARCH915) are the dominant microorganisms in the granules. Methylomirabilis spp. (red, DAMO1027) could not be detected. (F) Sample of flocculent biomass taken 800 days after the start of oxygen-limited culturing conditions. Archaea (blue, ARCH915) are nearly absent in the flocculent, while anammox biomass (green, AMX820) is less dense than in the granules. Like in the granules, no Methylomirabilis spp. (red, DAMO1027) was detected in the flocs. (G) Sample of flocculent biomass taken 800 days after the start of oxygen-limited culturing conditions. Next to anammox bacteria, visible in yellow/orange (Cy3, PLA46; Fluos, AMX820), other bacteria present in the flocculent biomass are visible in blue (EUBmix). (H) Negative control.

FIG 3

Labeled dinitrogen gas production by the culture measured using membrane inlet mass spectrometry (MIMS) when the culture was fed medium containing ¹⁵N-labeled ammonium (A), ¹⁵N-labeled nitrite (B), or ¹⁵N-labeled nitrate (C). The reactor was running normally, and sequencing batch reactor (SBR) cycles are indicated with gray bars. Time points at which medium containing ¹⁵N-labeled substrates was connected and disconnected are indicated by the dotted lines and symbols (+ and −). Production of both ²⁹N₂ (black) and ³⁰N₂ (gray) was measured and plotted as ratio of total nitrogen gas (^28+29+30N₂).

FIG 4

Potential ammonium and methane oxidation by the culture. (A) In the presence of ammonium and oxygen, nitrite (circles) was produced by the culture. (B) In the presence of oxygen, methane (closed symbols) was rapidly consumed by the culture; methane was not consumed when oxygen was not present (open symbols). After 2.5 h, additional methane was added (indicated by the dotted line).

FIG 5

Production of methane in batch incubations with and without 25 mM 2‐bromoethanesulfonate (BES). BES was added after 3 h of incubation (indicated by the dotted line).