Shihu Hu

Anaerobic oxidation of methane coupled to nitrate reduction in a novel archaeal lineage

Anaerobic oxidation of methane (AOM) is critical for controlling the flux of methane from anoxic environments. AOM coupled to iron, manganese and sulphate reduction have been demonstrated in consortia containing anaerobic methanotrophic (ANME) archaea. More recently it has been shown that the bacterium Candidatus ‘Methylomirabilis oxyfera’ can couple AOM to nitrite reduction through an intra-aerobic methane oxidation pathway. Bioreactors capable of AOM coupled to denitrification have resulted in the enrichment of ‘M. oxyfera’ and a novel ANME lineage, ANME-2d. However, as ‘M. oxyfera’ can independently couple AOM to denitrification, the role of ANME-2d in the process is unresolved. Here, a bioreactor fed with nitrate, ammonium and methane was dominated by a single ANME-2d population performing nitrate-driven AOM. Metagenomic, single-cell genomic and metatranscriptomic analyses combined with bioreactor performance and 13C- and 15N-labelling experiments show that ANME-2d is capable of independent AOM through reverse methanogenesis using nitrate as the terminal electron acceptor. Comparative analyses reveal that the genes for nitrate reduction were transferred laterally from a bacterial donor, suggesting selection for this novel process within ANME-2d. Nitrite produced by ANME-2d is reduced to dinitrogen gas through a syntrophic relationship with an anaerobic ammonium-oxidizing bacterium, effectively outcompeting ‘M. oxyfera’ in the system. We propose the name Candidatus ‘Methanoperedens nitroreducens’ for the ANME-2d population and the family Candidatus ‘Methanoperedenaceae’ for the ANME-2d lineage. We predict that ‘M. nitroreducens’ and other members of the ‘Methanoperedenaceae’ have an important role in linking the global carbon and nitrogen cycles in anoxic environments.

Enrichment of denitrifying anaerobic methane oxidizing microorganisms

The microorganisms responsible for anaerobic oxidation of methane (AOM) coupled to denitrification have not been clearly elucidated. Three recent publications suggested it can be achieved by a denitrifying bacterium with or without the involvement of anaerobic methanotrophic archaea. A key factor limiting the progress in this research field is the shortage of enrichment cultures performing denitrifying anaerobic methane oxidation (DAMO). In this study, DAMO cultures were enriched from mixed inoculum including sediment from a freshwater lake, anaerobic digester sludge and return activated sludge from a sewage treatment plant. Two reactors, operated at 35°C and at 22°C, respectively, showed simultaneous methane oxidation and nitrate reduction after several months of operation. Analysis of 16S rRNA gene clone libraries from the 35°C enrichment showed the presence of an archaeon closely related to other DAMO archaea and a dominated bacterium belonging to the yet uncultivated NC10 phylum. This culture preferred nitrite to nitrate as the electron acceptor. The present study suggests that the archaea are rather methanotrophs than methanogens. The highest denitrification rate achieved was 2.35 mmol NO3 (-) -N gVSS(-1)  day(-1) . The culture enriched at 22°C contained the same NC10 bacterium observed in the culture enriched at 35°C but no archaea.

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 fibers 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.

Reducing sewer corrosion through integrated urban water management

Sourcing corrosive sewer sulfides Sewer systems are corroding at an alarming rate, costing governments billions of dollars to replace. Differences among water treatment systems make it difficult to track down the source of corrosive sulfide responsible for this damage. Pikaar et al. performed an extensive industry survey and sampling campaign across Australia (see the Perspective by Rauch and Kleidorfer). Aluminum sulfate added as a coagulant during drinking water treatment was the primary culprit in corroding sewer systems. Modifying this common treatment strategy to include sulfate-free coagulants could dramatically reduce sewer corrosion across the globe. Science, this issue p. 812; see also p. 734 Decreasing sulfate added during drinking water treatment can prevent corrosion of sewers caused by wastewater. [Also see Perspective by Rauch and Kleidorfer] Sewer systems are among the most critical infrastructure assets for modern urban societies and provide essential human health protection. Sulfide-induced concrete sewer corrosion costs billions of dollars annually and has been identified as a main cause of global sewer deterioration. We performed a 2-year sampling campaign in South East Queensland (Australia), an extensive industry survey across Australia, and a comprehensive model-based scenario analysis of the various sources of sulfide. Aluminum sulfate addition during drinking water production contributes substantially to the sulfate load in sewage and indirectly serves as the primary source of sulfide. This unintended consequence of urban water management structures could be avoided by switching to sulfate-free coagulants, with no or only marginal additional expenses compared with the large potential savings in sewer corrosion costs.

Effect of nitrate and nitrite on the selection of microorganisms in the denitrifying anaerobic methane oxidation process

Two cultures were inoculated with sludges taken from a parent culture containing archaea distantly related to anaerobic methanotrophic archaea (ANME) and bacteria related to Candidatus Methylomirabilis oxyfera, both of which have previously been found in cultures performing denitrifying anaerobic methane oxidation process. The cultures were fed with nitrate and nitrite, respectively, along with methane. The nitrate-fed culture, Culture B, showed a stable microbial community composition and denitrifying anaerobic methane oxidation activity. In contrast, the nitrite-fed culture, Culture A, achieved a substantial increase in the nitrite consumption rate, from 1.1 to 7.3 mmol NO2 (-) -N (g VSS)(-1) day(-1) in 90 days. Concurrent with this activity increase, the archaeal population in Culture A decreased with time, and became undetectable after 100 days, while bacteria related to M. oxyfera increasingly dominated the culture. This observation suggests that the bacteria related to M. oxyfera are more competitive for nitrite reduction compared with the archaea related to ANME. This study showed that nitrate or nitrite feeding has a significant impact on the ecology and activities of microorganisms in the denitrifying anaerobic methane oxidation process. This study also revealed that nitrite overloading may have a toxic effect on the bacteria related to M. oxyfera.

Modeling of Simultaneous Anaerobic Methane and Ammonium Oxidation in a Membrane Biofilm Reactor

Nitrogen removal by using the synergy of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) microorganisms in a membrane biofilm reactor (MBfR) has previously been demonstrated experimentally. In this work, a mathematical model is developed to describe the simultaneous anaerobic methane and ammonium oxidation by DAMO and Anammox microorganisms in an MBfR for the first time. In this model, DAMO archaea convert nitrate, both externally fed and/or produced by Anammox, to nitrite, with methane as the electron donor. Anammox and DAMO bacteria jointly remove the nitrite fed/produced, with ammonium and methane as the electron donor, respectively. The model is successfully calibrated and validated using the long-term (over 400 days) dynamic experimental data from the MBfR, as well as two independent batch tests at different operational stages of the MBfR. The model satisfactorily describes the methane oxidation and nitrogen conversion data from the system. Modeling results show the concentration gradients of methane and nitrogen would cause stratification of the biofilm, where Anammox bacteria mainly grow in the biofilm layer close to the bulk liquid and DAMO organisms attach close to the membrane surface. The low surface methane loadings result in a low fraction of DAMO microorganisms, but the high surface methane loadings would lead to overgrowth of DAMO bacteria, which would compete with Anammox for nitrite and decrease the fraction of Anammox bacteria. The results suggest an optimal methane supply under the given condition should be applied not only to benefit the nitrogen removal but also to avoid potential methane emissions.

Nitrate reduction by denitrifying anaerobic methane oxidizing microorganisms can reach a practically useful rate

Methane in biogas has been proposed to be an electron donor to facilitate complete nitrogen removal using denitrifying anaerobic methane oxidizing (DAMO) microorganisms in an anaerobic ammonium oxidation (anammox) reactor, by reducing the nitrate produced. However, the slow growth and the low activity of DAMO microorganisms cast a serious doubt about the practical usefulness of such a process. In this study, a previously established lab-scale membrane biofilm reactor (MBfR), with biofilms consisting of a coculture of DAMO and anammox microorganisms, was operated to answer if the DAMO reactor can achieve a nitrate reduction rate that can potentially be applied for wastewater treatment. Through progressively increasing nitrate and ammonium loading rates to the reactor, a nitrate removal rate of 684 ± 10 mg-N L(-1) d(-1) was achieved after 453 days of operation. This rate is, to our knowledge, by far the highest reported for DAMO reactors, and far exceeds what is predicted to be required for nitrate removal in a sidestream (5.6-135 mg-N L(-1) d(-1)) or mainstream anammox reactor (3.2-124 mg-N L(-1) d(-1)). Mass balance analysis showed that the nitrite produced by nitrate reduction was jointly reduced by anammox bacteria at a rate of 354 ± 3 mg-N L(-1) d(-1), accompanied by an ammonium removal rate of 268 ± 2 mg-N L(-1) d(-1), and DAMO bacteria at a rate of 330 ± 9 mg-N L(-1) d(-1). This study shows that the nitrate reduction rate achieved by the DAMO process can be high enough for removing nitrate produced by anammox process, which would enable complete nitrogen removal from wastewater.

Stratified Microbial Structure and Activity in Sulfide- and Methane- Producing Anaerobic Sewer Biofilms

ABSTRACT Simultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 μm, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescence in situ hybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 μm and 700 μm, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera, Desulfobulbus, Desulfomicrobium, Desulfovibrio, Desulfatiferula, and Desulforegula, while about 90% of the MA population belonged to the genus Methanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates.

A laboratory investigation of interactions between denitrifying anaerobic methane oxidation (DAMO) and anammox processes in anoxic environments

This study investigates interactions between recently identified denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (anammox) processes in controlled anoxic laboratory reactors. Two reactors were seeded with the same inocula containing DAMO organisms Candidatus Methanoperedens nitroreducens and Candidatus Methylomirabilis oxyfera, and anammox organism Candidatus Kuenenia stuttgartiensis. Both were fed with ammonium and methane, but one was also fed with nitrate and the other with nitrite, providing anoxic environments with different electron acceptors. After steady state reached in several months, the DAMO process became solely/primarily responsible for nitrate reduction while the anammox process became solely responsible for nitrite reduction in both reactors. 16S rRNA gene amplicon sequencing showed that the nitrate-driven DAMO organism M. nitroreducens dominated both the nitrate-fed (~70%) and the nitrite-fed (~26%) reactors, while the nitrite-driven DAMO organism M. oxyfera disappeared in both communities. The elimination of M. oxyfera from both reactors was likely the results of this organism being outcompeted by anammox bacteria for nitrite. K. stuttgartiensis was detected at relatively low levels (1–3%) in both reactors.

Modeling the pH effect on sulfidogenesis in anaerobic sewer biofilm

Wastewater pH affects the transfer of H2S from liquid to gas and hence impacts on the extent of odour and corrosion. Sewage pH may change significantly in sewer networks due to, for example, the fermentation of organic compounds, particularly when the hydraulic retention time is long, the discharge of trade wastes that are organic-rich or with extreme pH, and the intentional addition of alkali to raise the sewage pH. Such a variation of pH is expected to affect sulfate reduction by sewer biofilms. In this work, experimental studies were carried out to reveal the detailed effects of pH on sulfidogenesis by anaerobic sewer biofilms developed in a laboratory sewer reactor fed with real sewage, and the potential causes for the inhibitory effects observed under both high and low pH conditions. The data clearly showed that the sewer biofilm had the highest sulfate reduction rate at around neutral pH (6.5-7.5), and the activity decreased when pH was higher or lower. The data also suggested that the inhibiting effect at a higher pH is likely due to the inhibitory effects of free ammonia, while at a lower pH it is more likely due to the pH itself. An inhibition model formulated based on the above hypotheses was able to adequately describe the measured sulfide production rates under different pH levels and different free ammonia concentrations, as well as data previously reported in literature. The calibrated model was used to simulate sulfide production in a sewer network receiving alkali dosage. The model adequately predicted sulfide production in the network, which could not be achieved with a model not considering the pH effect.