Methane-dependent Denitrification Research Articles

Revisiting methane-dependent denitrification

Methane-dependent denitrification links the global nitrogen and methane cycles. Since its initial discovery in 2006, this process has been understood to involve a division of labor between an archaeal group and a bacterial group, which sequentially perform nitrate and nitrite reduction, respectively. Yao et al. have now revised this paradigm by identifying a Methylomirabilis bacterium capable of performing methane-dependent complete denitrification on its own.

Methane-dependent complete denitrification by a single Methylomirabilis bacterium.

Methane-dependent nitrate and nitrite removal in anoxic environments is thought to rely on syntrophy between ANME-2d archaea and bacteria in the genus 'Candidatus Methylomirabilis'. Here we enriched and purified a single Methylomirabilis from paddy soil fed with nitrate and methane, which is capable of coupling methane oxidation to nitrate reduction via nitrite to dinitrogen independently. Isotope labelling showed that this bacterium we name 'Ca. Methylomirabilis sinica' stoichiometrically performed methane-dependent complete nitrate reduction to dinitrogen gas. Multi-omics analyses collectively demonstrated that 'M. sinica' actively expressed a well-established pathway for this process, especially including nitrate reductase Nap. Furthermore, 'M. sinica' exhibited a higher nitrate affinity than most denitrifiers, implying its competitive fitness under oligotrophic nitrogen-limited conditions. Our findings revise the paradigm of methane-dependent denitrification performed by two organisms, and the widespread presence of 'M. sinica' in public databases suggests that the coupling of methane oxidation and complete denitrification in single cells substantially contributes to global methane and nitrogen budgets.

Combining biological denitrification and electricity generation in methane-powered microbial fuel cells

Methane has been demonstrated to be a feasible substrate for electricity generation in microbial fuel cells (MFCs) and denitrifying anaerobic methane oxidation (DAMO). However, these two processes were evaluated separately in previous studies and it has remained unknown whether methane is able to simultaneously drive these processes. Here we investigated the co-occurrence and performance of these two processes in the anodic chamber of MFCs. The results showed that methane successfully fueled both electrogenesis and denitrification. Importantly, the maximum nitrate removal rate was significantly enhanced from (1.4 ± 0.8) to (18.4 ± 1.2) mg N/(L·day) by an electrogenic process. In the presence of DAMO, the MFCs achieved a maximum voltage of 610 mV and a maximum power density of 143 ± 12 mW/m2. Electrochemical analyses demonstrated that some redox substances (e.g. riboflavin) were likely involved in electrogenesis and also in the denitrification process. High-throughput sequencing indicated that the methanogen Methanobacterium, a close relative of Methanobacterium espanolae, catalyzed methane oxidation and cooperated with both exoelectrogens and denitrifiers (e.g., Azoarcus). This work provides an effective strategy for improving DAMO in methane-powered MFCs, and suggests that methanogens and denitrifiers may jointly be able to provide an alternative to archaeal DAMO for methane-dependent denitrification.

Rapid enrichment of denitrifying methanotrophs in a series hollow-fiber membrane biofilm reactor

Denitrifying anaerobic methane oxidation (DAMO) process uses methane as electron donor to reduce nitrate/nitrite to dinitrogen, which is a potentially efficient, low-cost and clean biological nitrogen removal technology. However, slow microbial growth rate severely limits the application of this promising process. In this study, a series hollow-fiber membrane biofilm reactor (HfMBR) was operated for 90 days to achieve rapid enrichment of these denitrifying methanotrophs. Finally, the highest relative abundance of denitrifying methanotrophic archaea and bacteria (DAMO archaea and bacteria) reached 47.5% and 11.3%, respectively. And the average abundance of DAMO archaea and bacteria increased 92.9 and 136.6 times respectively during the 90-day enrichment. High growth rate of DAMO archaea with a doubling time of 11.6 days was achieved in the second HfMBR according to quantitative PCR results. The results implied that dissolved oxygen would inhibit the growth of DAMO archaea, but the series HfMBR could effectively counteract this unfavorable factor. This work provided theoretical guidance for the rapid enrichment of denitrifying methanotrophs and contributed to the application of methane-dependent denitrification process.

Magnetite-mediated electrically connected community for shortening startup of methane-dependent denitrification in a membrane biofilm reactor

Denitrifying anaerobic methane oxidation (DAMO) not only mitigates methane emission from wastewater treatment, but compensates for the deficiency of current nitrogen removal process. However, the doubling time of DAMO microbes is usually more than two weeks, resulting in a long-period startup of a methane-dependent denitrification reactor especially with anaerobic sludge as inoculum. The study presented here found that, in the presence of magnetite, the nitrate removal rate reached 17.6 ± 0.4 mgNO3−-N/L/d after the 120-day experiments, which was approximately 2.4 folds higher than that in the absence of magnetite (7.3 ± 0.1 mgNO3−-N/L/d). The nitrate removal rate in the presence of magnetite reached 7–8 mgNO3−-N/L/d at day 80–85, which shortened the startup periods of approximately 35–40 days. Microbial morphology based on scanning electron microscope and transmission electron microscope showed that, the surface of cells was deposited by magnetite and they might be connected via magnetite. Further investigations via cyclic voltammetry and electrochemical impedance spectroscopy showed that magnetite mediated an electrically connected community with a higher capacitance and lower resistance than that in the absence of magnetite. Microbial community analysis showed that, in the presence of magnetite, the potential DAMO bacteria (Methylacidimicrobium and Methylotetracoccus species), methanogens (Methanothrix species) as well as nitrate-reducing bacteria (Bellilinea and Dechloromonas species) were enriched. They might participate in direct interspecies electron transfer (DIET) via magnetite substituting for c-type cytochromes as the electrical connection components, since methane oxidation in the presence of magnetite was insensitive to the external addition of potential intermediates, such as hydrogen.

Mass transfer affects reactor performance, microbial morphology, and community succession in the methane-dependent denitrification and anaerobic ammonium oxidation co-culture

Denitrifying anaerobic methane oxidation (DAMO) combining anaerobic ammonium oxidation (Anammox) process is a novel nitrogen removal technology. However, the roles of methane transfer (gas phase) and nitrogen transfer (liquid phase) in the heterogeneous process remain unclear. In this study, granular DAMO and Anammox co-cultures were inoculated from a hollow-fiber membrane bioreactor into a sequence batch reactor (SBR). Since the methane transfer became limited in SBR, the nitrate removal rate first decreased and then increased to 10 mg/(L∙day), while the ammonium removal rate did not recover and was around 2 mg/(L∙day). The activity of DAMO archaea and Anammox bacteria decreased noticeably. Furthermore, granular aggregates dispersed into small granules and ultimately became flocs with poor settleability in SBR. The content of extracellular polymeric substances decreased, especially that of proteins and humics. DAMO archaea decreased by 94.6% and Anammox bacteria decreased by 72%. In summary, the limitation of methane transfer affected DAMO and Anammox processes more notably than nitrogen transfer, resulting in lower nitrogen removal, granule disruption, and microbial community succession.

Long-term nitrate removal through methane-dependent denitrification microorganisms in sequencing batch reactors fed with only nitrate and methane

Denitrifying anaerobic methane oxidation (damo) bioprocesses can remove nitrate using methane as the electron donor, which gains great concern due to the current stringent discharge standard of nitrogen in wastewater treatment plants. To obtain an engineering acceptable nitrogen removal rate (NRR) and demonstrate the long-term stable ability of damo system under conditions of nitrate and methane, two sequencing batch reactors (SBRs) fed with only nitrate and methane were operated for more than 600 days at 30 °C. The NRR of 21.91 ± 0.73 mg NO3−-N L−1 day−1 was obtained which is, to the best of our knowledge, the highest rate observed in the literatures under such conditions. The temperature was found to significantly affect the system performance. Furthermore, the microbial community was analyzed by using real-time PCR technique. The results showed that the microbial consortium contained damo archaea and bacteria. These two microbes cooperated to maintain the long-term stability. And the number of damo archaea was higher than that of damo bacteria with the ratio of 1.77. By using methane as the electron donor, damo archaea reduced nitrate to nitrite coupled to methane oxidation and damo bacteria reduce the generated nitrite to nitrogen gas. The first step of nitrate to nitrite taken by damo archaea might be the limiting step of this cooperation system. SBR could be a suitable reactor configuration to enrich slow-growing microbes like damo culture. These results demonstrated the potential application of damo processes for nitrogen removal of wastewater containing low C/N ratios.

Effect of methane partial pressure on the performance of a membrane biofilm reactor coupling methane-dependent denitrification and anammox

Complete nitrogen removal has recently been demonstrated by integrating anaerobic ammonium oxidation (anammox) and denitrifying anaerobic methane oxidation (DAMO) processes. In this work, the effect of methane partial pressure on the performance of a membrane biofilm reactor (MBfR) consisting of DAMO and anammox microorganisms was evaluated. The activities of DAMO archaea and DAMO bacteria in the biofilm increased significantly with increased methane partial pressure, from 367 ± 9 and 58 ± 22 mg-N L−1d−1 to 580 ± 12 and 222 ± 22 mg-N L−1d−1, respectively, while the activity of anammox bacteria only increased slightly, when the methane partial pressure was elevated from 0.24 to 1.39 atm in the short-term batch tests. The results were supported by a long-term (seven weeks) continuous test, when the methane partial pressure was dropped from 1.39 to 0.78 atm. The methane utilization efficiency was always above 96% during both short-term and long-term tests. Taken together, nitrogen removal rate (especially the nitrate reduction rate by DAMO archaea) and methane utilization efficiency could be maintained at high levels in a broad range of methane partial pressure (0.24–1.39 atm in this study). In addition, a previously established DAMO/anammox biofilm model was used to analyze the experimental data. The observed impacts of methane partial pressure on biofilm activity were well explained by the modeling results. These results suggest that methane partial pressure can potentially be used as a manipulated variable to control reaction rates, ultimately to maintain high nitrogen removal efficiency, according to nitrogen loading rate.

The content of trace element iron is a key factor for competition between anaerobic ammonium oxidation and methane-dependent denitrification processes

Coupling of anaerobic ammonium oxidation (Anammox) with denitrifying anaerobic methane oxidation (DAMO) is a sustainable pathway for nitrogen removal and reducing methane emissions from wastewater treatment processes. However, studies on the competitive relation between Anammox bacteria and DAMO bacteria are limited. Here, we investigated the effects of variations in the contents of trace element iron on Anammox and DAMO microorganisms. The short-term results indicated that optimal concentrations of iron, which obviously stimulated the activity of Amammox bacteria, DAMO bacteria and DAMO archaea, were 80, 20, and 80 μM, respectively. The activity of Amammox bacteria increased more significant than DAMO bacteria with increasing contents of trace element iron. After long-term incubation with high content of trace element iron of 160 μM in the medium, Candidatus Brocadia (Amammox bacteria) outcompeted Candidatus Methylomirabilis oxyfera (DAMO bacteria), and ANME-2d (DAMO archaea) remarkably increased in number and dominated the co-culture systems (64.5%). Meanwhile, with further addition of iron, the removal rate of ammonium and nitrate increased by 13.6 and 9.2 times, respectively, when compared with that noted in the control. As far as we know, this study is the first to explore the important role of trace element iron contents in the competition between Anammox bacteria and DAMO bacteria and further enrichment of DAMO archaea by regulating the contents of trace element iron.

Methane dependent denitrification- from ecosystem to laboratory-scale enrichment for engineering applications

Managing nitrogen and carbon cycles in engineered and natural ecosystems is an environmental challenge. In this manuscript, we report a process which connects these two cycles with immense ecological and engineering significance. Sediments, collected from Jordan River in Salt Lake City, Utah were used as seed to start a laboratory-scale denitrification coupled to anaerobic methane oxidation (n-DAMO) reactor fed with methane (CH4) and nitrite (NO2−). Methane (CH4)-dependent denitrification in sediments of a nutrient-impaired river was found to be in the range of 40 nmol kg−1 d−1 to 70 nmol kg−1 d−1. Post 19 months of operation of the lab scale reactor, the n-DAMO reactor achieved nitrite removal rate of 2.88 mmol L−1 d−1. Enrichment of n-DAMO prokaryotes was evident from the increase in 16S rRNA gene copy number of bacteria belonging to the NC10 phylum in the reactor, corroborating with increase in the oxidation rates of CH4 coupled with NO2−-N removal from 21 μM to 190 μM of CH4 d−1. Based on stable isotope experiments by other researchers, nitric oxide dismutase (nod) functional gene was hypothesized to be responsible for splitting nitric oxide to nitrogen and oxygen and this internally generated oxygen is utilized by n-DAMO prokaryotes to oxidize methane gas. Primers targeting the unique nitric oxide dismutase (nod) gene were developed and tested on the enrichment culture for the first time. This revealed that n-DAMO organisms are closely related yet distinct from, the M. oxyfera which had been enriched in earlier studies. The results emphasize tremendous future promise to use these novel organisms for wastewater treatment purposes, especially to take advantage of the dissolved methane present in anaerobic digester effluents.

Achieving complete nitrogen removal by coupling nitritation-anammox and methane-dependent denitrification: A model-based study.

The discovery of denitrifying anaerobic methane oxidation (DAMO) processes enables the complete nitrogen removal from wastewater by utilizing the methane produced on site from anaerobic digesters. This model-based study investigated the mechanisms and operational window for efficient nitrogen removal by coupling nitritation-anaerobic ammonium oxidation (Anammox) and methane-dependent denitrification in membrane biofilm reactors (MBfRs). A mathematical model was applied to describe the microbial interactions among Anammox bacteria, DAMO archaea, and DAMO bacteria. The model sufficiently described the batch experimental data from an MBfR containing an Anammox-DAMO biofilm with different feeding nitrogen compositions, which confirmed the validity of the model. The effects of process parameters on the system performance and microbial community structure could therefore be reliably evaluated. The impacts of nitritation produced NO2(-)/NH4(+) ratio, methane supply, biofilm thickness and total nitrogen (TN) surface loading were comprehensively investigated with the model. Results showed that the optimum NO2(-)/NH4(+) ratio produced from nitritation for the Anammox-DAMO biofilm system was around 1.0 in order to achieve the maximum TN removal (over 99.0%), independent on TN surface loading. The corresponding optimal methane supply increased while the associated methane utilization efficiency decreased with the increase of TN surface loading. The cooperation between DAMO organisms and Anammox bacteria played the key role in the TN removal. Based on these results, the proof-of-concept feasibility of a single-stage MBfR coupling nitritation-Anammox-DAMO for complete nitrogen removal was also tested through integrating the model with ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) processes whilst controlling the dissolved oxygen (DO) concentration in the simulated system. The maximum TN removal was found to be achieved at the bulk DO concentration of around 0.17 g m(-3) under the simulation conditions, with the AOB, Anammox bacteria and DAMO organisms coexisting in the biofilm.

Nitrogen Removal from Wastewater by Coupling Anammox and Methane-Dependent Denitrification in a Membrane Biofilm Reactor

This work demonstrates, for the first time, the feasibility of nitrogen removal by using the synergy of anammox and denitrifying anaerobic methane oxidation (DAMO) microorganisms in a membrane biofilm reactor (MBfR). The reactor was fed with synthetic wastewater containing nitrate and ammonium. Methane was delivered from the interior of hollow fibres in the MBfR to the biofilm that grew on the fiber's outer wall. After 24 months of operation, the system achieved a nitrate and an ammonium removal rate of about 190 mgN L(-1) d(-1) (or 86 mgN m(-2) d(-1), with m(2) referring to biofilm surface area) and 60 mgN L(-1) d(-1) (27 mgN m(-2) d(-1)), respectively. No nitrite accumulation was observed. Fluorescence in situ hybridization (FISH) analysis indicated that DAMO bacteria (20-30%), DAMO archaea (20-30%) and anammox bacteria (20-30%) jointly dominated the microbial community. Based on the known metabolism of these microorganisms, mass balance, and isotope studies, we hypothesize that DAMO archaea converted nitrate, both externally fed and produced by anammox, to nitrite, with methane as the electron donor. Anammox and DAMO bacteria jointly removed the nitrite produced, with ammonium and methane as the electron donor, respectively. The process could potentially be used for anaerobic nitrogen removal from wastewater streams containing ammonium and nitrate/nitrite.

Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing

Methane is used as an alternative carbon source in the denitrification of wastewater lacking organic carbon sources because it is nontoxic and may be efficiently produced by anaerobic biological processes. Methane-dependent denitrification (MDD) in the presence of oxygen requires the co-occurrence of methanotrophy and denitrification. Activated sludge was incubated with 13C-labeled methane in either a nitrate-containing medium or a nitrate-free medium. Then, bacterial and methanotrophic populations were analyzed by cloning analysis and terminal restriction fragment length polymorphism analysis targeting 16S rRNA gene and cloning analysis targeting pmoA genes. DNA-based stable-isotope probing (DNA-SIP) analysis of the 16S rRNA gene revealed an association of the Methylococcaceae and the Hyphomicrobiaceae in a MDD ecosystem. Furthermore, supplementation of nitrate stimulated methane consumption and the activity of methanotrophic populations (i.e. the stimulation of uncultivated relatives of distinct groups of the Methylococcaceae). In particular, uncultured type-X methanotrophs of Gammaproteobacteria were dominant when nitrate was added, i.e. in the MDD incubations. On the other hand, most methanotrophs (types I, II, and X methanotrophs) were found to have been labeled with 13C under nitrate-free conditions. This DNA-SIP study identifies key bacterial populations involved in a MDD ecosystem.

06/01054 Methane-dependent denitrification by a semi-partitioned reactor supplied separately with methane and oxygen: Waki, M. et al. Bioresource Technology, 2005, 96, (8), 921–927.

06/01054 Methane-dependent denitrification by a semi-partitioned reactor supplied separately with methane and oxygen: Waki, M. et al. Bioresource Technology, 2005, 96, (8), 921–927.

Methane-dependent denitrification by a semi-partitioned reactor supplied separately with methane and oxygen

Methane (CH 4) can be used as an alternative carbon source for denitrification with added oxygen (O 2). However, the off-gas of denitrification reactors using a CH 4–O 2 mixture contains unused CH 4 and O 2 in proportions that make it unusable for fuel, carry explosion risks, and, if released into the atmosphere, contribute to the greenhouse effect. This study tested a novel reactor with a partition dividing the headspace completely and extending partly into the liquid layer. When CH 4 and O 2 were supplied separately to the liquid layer on opposite sides of the partition, the methane-dependent denitrification (MDD) activity was similar to that when the two gases were supplied as a mixture. In reactors with separate gas supplies, the off-gas from the CH 4 supply side was high in CH 4 and low in O 2, and was usable for fuel, and that from the O 2 supply side was very low in CH 4, and might be released into the atmosphere. MDD activity increased with the O 2 supply rate, and separate discharge of CH 4 and O 2 was maintained. The concentration of dissolved methane in the effluent was decreased by lowering the CH 4/O 2 supply ratio to 1.0 and drawing the effluent from the O 2 supply side. This novel reactor enhances the safety of MDD, allows reuse of methane as fuel, and reduces methane leakage to the atmosphere.

Microbiological activities contributing to nitrogen removal with methane: effects of methyl fluoride and tungstate

When methane (CH 4) and O 2 are present, nitrogen can be removed from wastewater that does not contain other organic carbon sources. In this study, microbial activities during methane-dependent denitrification (MDD) were investigated by adding inhibitors of methane-oxidation and denitrification. Sludge susceptible to MDD showed methane oxidation activity in the presence of CH 4 and O 2, and denitrification activity with methanol and acetate under anoxic conditions. Methyl fluoride (CH 3F) is known to inhibit methane oxidation. When CH 3F was present, MDD did not occur, perhaps because methane oxidation was inhibited. Tungstate (WO 4 2−), a known inhibitor of nitrate reduction, also lowered denitrification activity in the sludge, and partly inhibited methane oxidation. When WO 4 2− was added to the medium, MDD almost ceased, perhaps because of a synergic inhibitory effect on denitrification and methane oxidation. These results show that both methane oxidation and denitrification contribute to MDD.

Effects of CH4 and O2 Concentrations on Methane-dependent Denitrification

Effects of CH4 and O2 Concentrations on Methane-dependent Denitrification

Effects of nitrite and ammonium on methane-dependent denitrification.

For effective application of methane-dependent denitrification (MDD) in the treatment of wastewater containing NO(2)(-) or NH(4)(+), the effect of these inorganic nitrogen compounds on MDD activity needs to be clarified. The MDD activity of sludge acclimatized with CH(4) and O(2) was determined with mineral media of different nitrogen-compound compositions in the presence of 0.21 atm CH(4) and 0.20 atm O(2). Incubations with media containing only NO(2)(-) or two of the three inorganic nitrogen compounds (NO(3)(-)+NO(2)(-), NO(2)(-)+NH(4)(+) or NH(4)(+)+NO(3)(-)) resulted in MDD activity equal to or higher than that with media containing only NO(3)(-). However, there was no MDD activity in media containing NO(2)(-) at 10 degrees C, probably because of serious inhibition of NO(2)(-) on methane oxidation. MDD occurred in media containing only NH(4)(+), although the total nitrogen removal efficiency was very low. These results show that NO(2)(-) and NH(4)(+), in the presence of NO(x)(-), do not inhibit but rather promote MDD. Consequently, NH(4)(+) does not need to be completely oxidized to NO(3)(-) in the nitrification reactor before MDD. However, under psychrophilic conditions, NO(2)(-) seriously inhibited MDD. Therefore, the nitrification reactor must not discharge effluent containing NO(2)(-) under psychrophilic conditions.