Project description:Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2-) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. 15N-labeling experiments revealed that NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH4+ oxidation with electrode as electron acceptor. Complete NH4+ oxidation to N2 without accumulation of NO2- and NO3- was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen.
Project description:We examined nitrate-dependent Fe(2+) oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of "Candidatus Brocadia sinica" anaerobically oxidized Fe(2+) and reduced NO3(-) to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein(-1) min(-1), respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of "Ca. Brocadia sinica" (10 to 75 nmol NH4(+) mg protein(-1) min(-1)). A (15)N tracer experiment demonstrated that coupling of nitrate-dependent Fe(2+) oxidation and the anammox reaction was responsible for producing nitrogen gas from NO3(-) by "Ca. Brocadia sinica." The activities of nitrate-dependent Fe(2+) oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO3(-) ± SD of "Ca. Brocadia sinica" was determined to be 51 ± 21 ?M. Nitrate-dependent Fe(2+) oxidation was further demonstrated by another anammox bacterium, "Candidatus Scalindua sp.," whose rates of Fe(2+) oxidation and NO3(-) reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein(-1) min(-1), respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe(2+) oxidation and the anammox reaction decreased the molar ratios of consumed NO2(-) to consumed NH4(+) (?NO2(-)/?NH4(+)) and produced NO3(-) to consumed NH4(+) (?NO3(-)/?NH4(+)). These reactions are preferable to the application of anammox processes for wastewater treatment.
Project description:Enrichment cultures of anaerobic ammonium oxidation (anammox) bacteria as planktonic cell suspensions are essential for studying their ecophysiology and biochemistry, while their cultivation is still laborious. The present study aimed to cultivate two phylogenetically distinct anammox bacteria, "Candidatus Brocadia sinica" and "Ca. Scalindua sp." in the form of planktonic cells using membrane bioreactors (MBRs). The MBRs were continuously operated for more than 250 d with nitrogen loading rates of 0.48-1.02 and 0.004-0.09 kgN m(-3) d(-1) for "Ca. Brocadia sinica" and "Ca. Scalindua sp.", respectively. Planktonic anammox bacterial cells were successfully enriched (>90%) in the MBRs, which was confirmed by fluorescence in-situ hybridization and 16S rRNA gene sequencing analysis. The decay rate and half-saturation constant for NO2(-) of "Ca. Brocadia sinica" were determined to be 0.0029-0.0081 d(-1) and 0.47 mgN L(-1), respectively, using enriched planktonic cells. The present study demonstrated that MBR enables the culture of planktonic anammox bacterial cells, which are suitable for studying their ecophysiology and biochemistry.
Project description:Natural abundance of stable nitrogen (N) and oxygen (O) isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects (<sup>15</sup>? and <sup>18</sup>?) associated with the respective nitrogen transformation processes must be known. However, the N and O isotope effects of anaerobic ammonium oxidation (anammox), one of the major fixed N sinks and NO<sub>3</sub><sup>-</sup> producers, are not well known. Here, we report the dual N and O isotope effects associated with anammox by three different anammox bacteria including "Ca. Scalindua japonica", a putative marine species, which were measured in continuous enrichment culture experiments. All three anammox species yielded similar N isotope effects of NH<sub>4</sub><sup>+</sup> oxidation to N<sub>2</sub> (<sup>15</sup>?<sub>NH4?N2</sub>) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO<sub>2</sub><sup>-</sup> oxidation to NO<sub>3</sub><sup>-</sup> (<sup>15</sup>?<sub>NO2?NO3</sub>?=?-45.3‰ to -30.1‰). In contrast, <sup>15</sup>?<sub>NO2?N2</sub> (NO<sub>2</sub><sup>-</sup> reduction to N<sub>2</sub>) were significantly different among three species, which is probably because individual anammox bacteria species might possess different types of nitrite reductase. We also report the combined O isotope effects for NO<sub>2</sub><sup>-</sup> oxidation (<sup>18</sup>E<sub>NO2?NO3</sub>) by anammox bacteria. These obtained dual N and O isotopic effects could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO<sub>2</sub><sup>-</sup> reoxidation (N recycling) in various natural environments.
Project description:The application of anaerobic ammonium oxidation (Anammox) process is often limited by the slow growth rate of Anammox bacteria. As the essential substrate element that required for culturing Anammox sludge, Fe (II) is expected to affect Anammox bacterial growth. This work systematically studied the effects of Fe (II) addition on Anammox activity based on the kinetic analysis of specific growth rate using data from batch tests with an enriched Anammox sludge at different dosing levels. Results clearly demonstrated that appropriate Fe (II) dosing (i.e., 0.09?mM) significantly enhanced the specific Anammox growth rate up to 0.172?d(-1) compared to 0.118?d(-1) at regular Fe (II) level (0.03?mM). The relationship between Fe (II) concentration and specific Anammox growth rate was found to be well described by typical substrate inhibition kinetics, which was integrated into currently well-established Anammox model to describe the enhanced Anammox growth with Fe (II) addition. The validity of the integrated Anammox model was verified using long-term experimental data from three independent Anammox reactors with different Fe (II) dosing levels. This Fe (II)-based approach could be potentially implemented to enhance the process rate for possible mainstream application of Anammox technology, in order for an energy autarchic wastewater treatment.
Project description:Anaerobic ammonium oxidation (anammox), the biochemical process oxidizing ammonium into dinitrogen gas using nitrite as an electron acceptor, has only been recognized for its significant role in the global nitrogen cycle not long ago, and its ubiquitous distribution in a wide range of environments has changed our knowledge about the contributors to the global nitrogen cycle. Currently, several groups of methods are used in detection of anammox bacteria based on their physiological and biochemical characteristics, cellular chemical composition, and both 16S rRNA gene and selective functional genes as biomarkers, including hydrazine oxidoreductase and nitrite reductase encoding genes hzo and nirS, respectively. Results from these methods coupling with advances in quantitative PCR, reverse transcription of mRNA genes and stable isotope labeling have improved our understanding on the distribution, diversity, and activity of anammox bacteria in different environments both natural and engineered ones. In this review, we summarize these methods used in detection of anammox bacteria from various environments, highlight the strengths and weakness of these methods, and also discuss the new development potentials on the existing and new techniques in the future.
Project description:Nitrite-dependent anaerobic oxidation of methane (n-damo) and ammonium (anammox) are two recently discovered processes in the nitrogen cycle that are catalyzed by n-damo bacteria, including "Candidatus Methylomirabilis oxyfera," and anammox bacteria, respectively. The feasibility of coculturing anammox and n-damo bacteria is important for implementation in wastewater treatment systems that contain substantial amounts of both methane and ammonium. Here we tested this possible coexistence experimentally. To obtain such a coculture, ammonium was fed to a stable enrichment culture of n-damo bacteria that still contained some residual anammox bacteria. The ammonium supplied to the reactor was consumed rapidly and could be gradually increased from 1 to 20 mM/day. The enriched coculture was monitored by fluorescence in situ hybridization and 16S rRNA and pmoA gene clone libraries and activity measurements. After 161 days, a coculture with about equal amounts of n-damo and anammox bacteria was established that converted nitrite at a rate of 0.1 kg-N/m(3)/day (17.2 mmol day(-1)). This indicated that the application of such a coculture for nitrogen removal may be feasible in the near future.
Project description: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.
Project description:Anaerobic ammonium oxidation (anammox) has been proven to be an important nitrogen removal process in terrestrial ecosystems, particularly paddy soils. However, the contribution of anammox in acidic red soils to nitrogen loss has not been well-documented to date. Here, we investigated the activity, abundance, and distribution of anammox bacteria in red soils collected from nine provinces of Southern China. High-throughput sequencing analysis showed that Candidatus Brocadia dominates the anammox bacterial community (93.03% of sequence reads). Quantification of the hydrazine synthase gene (hzsB) and anammox 16S rRNA gene indicated that the abundance of anammox bacteria ranged from 6.20 × 106 to 1.81 × 109 and 4.81 × 106 to 4.54 × 108 copies per gram of dry weight, respectively. Contributions to nitrogen removal by anammox were measured by a 15N isotope-pairing assay. Anammox rates in red soil ranged from 0.01 to 0.59 nmol N g-1 h-1, contributing 16.67-53.27% to N2 production in the studied area, and the total amount of removed nitrogen by anammox was estimated at 2.33 Tg N per year in the natural red soils of southern China. Pearson correlation analyses revealed that the distribution of anammox bacteria significantly correlated with the concentration of nitrate and pH, whereas the abundance and activity of anammox bacteria were significantly influenced by the nitrate and total nitrogen concentrations. Our findings demonstrate that Candidatus Brocadia dominates anammox bacterial communities in acidic red soils and plays an important role in nitrogen loss of the red soil in Southern China.