Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) was recently discovered and shown to be mediated by "Candidatus Methylomirabilis oxyfera" (M. oxyfera). Here, evidence for n-damo in three different freshwater wetlands located in southeastern China was obtained using stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. Stable isotope experiments confirmed the occurrence of n-damo in the examined wetlands, and the potential n-damo rates ranged from 0.31 to 5.43 nmol CO2 per gram of dry soil per day at different depths of soil cores. A combined analysis of 16S rRNA and particulate methane monooxygenase genes demonstrated that M. oxyfera-like bacteria were mainly present in the deep soil with a maximum abundance of 3.2 × 10(7) gene copies per gram of dry soil. It is estimated that ∼0.51 g of CH4 m(-2) per year could be linked to the n-damo process in the examined wetlands based on the measured potential n-damo rates. This study presents previously unidentified confirmation that the n-damo process is a previously overlooked microbial methane sink in wetlands, and n-damo has the potential to be a globally important methane sink due to increasing nitrogen pollution.

Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in coastal environments.

In the current study, we investigated nitrite-dependent anaerobic methane oxidation (N-DAMO) as a potential methane sink in the Hangzhou Bay and the adjacent Zhoushan sea area. The potential activity of the N-DAMO process was primarily observed in Hangzhou Bay by means of (13)C-labeling experiments, whereas very low or no potential N-DAMO activity could be detected in the Zhoushan sea area. The measured potential N-DAMO rates ranged from 0.2 to 1.3 nmol (13)CO2 g(-1) (dry sediment) day(-1), and the N-DAMO potentially contributed 2.0-9.4 % to the total microbial methane oxidation in the examined sediments. This indicated that the N-DAMO process may be an alternative pathway in the coastal methane cycle. Phylogenetic analyses confirmed the presence of Candidatus Methylomirabilis oxyfera-like bacteria in all the examined sediments, while the group A members (the dominant bacteria responsible for N-DAMO) were found mainly in Hangzhou Bay. Quantitative PCR showed that the 16S rRNA gene abundance of Candidatus M. oxyfera-like bacteria varied from 5.4 × 10(6) to 5.0 × 10(7) copies g(-1) (dry sediment), with a higher abundance observed in Hangzhou Bay. In addition, the overlying water NO3 (-) concentration and salinity were identified as the most important factors influencing the abundance and potential activity of Candidatus M. oxyfera-like bacteria in the examined sediments. This study showed the evidence of N-DAMO in coastal environments and indicated the importance of N-DAMO as a potential methane sink in coastal environments.

Vertical distribution of nitrite-dependent anaerobic methane-oxidising bacteria in natural freshwater wetland soils.

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered process that is catalysed by "Candidatus Methylomirabilis oxyfera". In the present study, the vertical distribution (0-10, 20-30, 50-60 and 90-100 cm) of M. oxyfera-like bacteria was investigated in Xiazhuhu wetland, the largest natural wetland on the southern Yangtze River (China). Phylogenetic analyses showed that group A of M. oxyfera-like bacteria and pmoA genes occurred primarily at depths of 50-60 and 90-100 cm. Quantitative PCR further confirmed the presence of M. oxyfera-like bacteria in soil cores from different depths, with the highest abundance of 5.1 × 10(7) copies g(-1) dry soil at depth of 50-60 cm. Stable isotope experiments demonstrated that the n-damo process occurred primarily at depths of 50-60 and 90-100 cm, with the potential rates ranging from 0.2 to 14.5 nmol CO2 g(-1) dry soil d(-1). It was estimated that the methane flux may increase by approximately 2.7-4.3% in the examined wetland in the absence of n-damo. This study shows that the deep wetland soils (50-60 and 90-100 cm) are the preferred habitats for M. oxyfera-like bacteria. The study also highlights the potential importance of these bacteria in the methane and nitrogen cycles in deep wetland soils.

Evidence for the cooccurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in a flooded paddy field.

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the microbial nitrogen cycle. In the present study, we provide direct evidence for the cooccurrence of the anammox and n-damo processes in a flooded paddy field in southeastern China. Stable isotope experiments showed that the potential anammox rates ranged from 5.6 to 22.7 nmol N2 g(-1) (dry weight) day(-1) and the potential n-damo rates varied from 0.2 to 2.1 nmol CO2 g(-1) (dry weight) day(-1) in different layers of soil cores. Quantitative PCR showed that the abundance of anammox bacteria ranged from 1.0 × 10(5) to 2.0 × 10(6) copies g(-1) (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 10(5) to 6.1 × 10(6) copies g(-1) (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene sequences showed that anammox bacteria affiliated with "Candidatus Brocadia" and "Candidatus Kuenenia" and n-damo bacteria related to "Candidatus Methylomirabilis oxyfera" were present in the soil cores. It is estimated that a total loss of 50.7 g N m(-2) per year could be linked to the anammox process, which is at intermediate levels for the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in wetland soils. In addition, it is estimated that a total of 0.14 g CH4 m(-2) per year could be oxidized via the n-damo process, while this rate is at the lower end of the aerobic methane oxidation rates reported in wetland soils.

Distribution and diversity of nitrite-dependent anaerobic methane-oxidising bacteria in the sediments of the Qiantang River.

Nitrite-dependent anaerobic methane oxidation (n-damo) process was reported to be mediated by "Candidatus Methylomirabilis oxyfera", which belongs to the candidate phylum NC10. M. oxyfera-like bacteria have been detected in lake ecosystems, while their distribution, diversity and abundance in river ecosystems have not been well studied. In this study, both the 16S rRNA and the pmoA molecular biomarkers confirmed the presence of diverse NC10 phylum bacteria related to M. oxyfera in a river ecosystem-the Qiantang River, Zhejiang Province (China). Phylogenetic analysis of 16S rRNA genes demonstrated that the recovered M. oxyfera-like sequences could be grouped into several distinct clusters that exhibited 89.8% to 98.9% identity to the M. oxyfera 16S rRNA gene. Similarly, several different clusters of pmoA gene sequences were observed, and these clusters displayed 85.1-95.4% sequence identity to the pmoA gene of M. oxyfera. Quantitative PCR showed that the abundance of M. oxyfera-like bacteria varied from 1.32 ± 0.16 × 10(6) to 1.03 ± 0.12 × 10(7) copies g (dry weight)(-1). Correlation analysis demonstrated that the total inorganic nitrogen content, the ammonium content and the organic content of the sediment were important factors affecting the distribution of M. oxyfera-like bacterial groups in the examined sediments. This study demonstrated the distribution of diverse M. oxyfera-like bacteria and their correlation with environmental factors in Qiantang River sediments.

Transient and long-term effects of bicarbonate on the ANAMMOX process.

In this study, the effects of both transient and long-term inorganic carbon (IC) addition on the anaerobic ammonium oxidation (ANAMMOX) process under pseudo-steady-state and substrate inhibitions were analyzed using reactor performance and measures of sludge activity. Compared with the nitrogen removal rate (NRR) of 3.42 kg N m(-3) day(-1) in the control bioreactor (ICDR) without IC, the peak NRR reached 21.0 kg N m(-3) day(-1) in the reactor (ICAR) with sufficient IC added. It was revealed that the long-term addition of bicarbonate significantly enhanced the performance of the ANAMMOX reactor. The optimum HCO3 (-)/TN ratio was considered to be 1.20, which is lower than that in normal conditions. The IC concentration affected biomass activity, and the transient addition or removal of IC to differing sludge media caused a significant loss of activity. Sufficient addition of IC alleviated the inhibition of excess substrates, while the inhibition was aggravated by the IC limitation. The half-maximal (50 %) inhibitory concentrations of substrate for the sludge were 295 mg L(-1) NO2 (-)-N and 361 mg L(-1) NH4 (+)-N with 120 mg L(-1) of fixed HCO3 (-) and 346 mg L(-1) NO2 (-)-N and 456 mg L(-1) NH4 (+)-N with unlimited IC, respectively. Changing the HCO3 (-)/TN (in milligrams per milligram) ratio resulted in the variation of ANAMMOX stoichiometric ratios. Sludge characterization parameters in the ICDR, including biomass, extracellular polymeric substances, heme C, and so on, were lower than those in ICAR. Filamentous bacteria and spherical bacteria were also observed in the reactor with limited IC.

Broad distribution of diverse anaerobic ammonium-oxidizing bacteria in chinese agricultural soils.

Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed "Candidatus Brocadia," "Candidatus Kuenenia," "Candidatus Anammoxoglobus," and "Candidatus Jettenia" in the collected soils, with "Candidatus Brocadia" being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 10(4) ± 0.42 × 10(4) to 3.69 × 10(6) ± 0.25 × 10(6) copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.

Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria.

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.

Anaerobic ammonium oxidation (anammox) in different natural ecosystems.

Anammox (anaerobic ammonium oxidation), which is a reaction that oxidizes ammonium to dinitrogen gas using nitrite as the electron acceptor under anoxic conditions, was an important discovery in the nitrogen cycle. The reaction is mediated by a specialized group of planctomycete-like bacteria that were first discovered in man-made ecosystems. Subsequently, many studies have reported on the ubiquitous distribution of anammox bacteria in various natural habitats, including anoxic marine sediments and water columns, freshwater sediments and water columns, terrestrial ecosystems and some special ecosystems, such as petroleum reservoirs. Previous studies have estimated that the anammox process is responsible for 50% of the marine nitrogen loss. Recently, the anammox process was reported to account for 9-40% and 4-37% of the nitrogen loss in inland lakes and agricultural soils respectively. These findings indicate the great potential for the anammox process to occur in freshwater and terrestrial ecosystems. The distribution of different anammox bacteria and their contribution to nitrogen loss have been described in different natural habitats, demonstrating that the anammox process is strongly influenced by the local environmental conditions. The present mini-review summarizes the current knowledge of the ecological distribution of anammox bacteria, their contribution to nitrogen loss in various natural ecosystems and the effects of major influential factors on the anammox process.

New anaerobic, ammonium-oxidizing community enriched from peat soil.

Anaerobic ammonium-oxidizing (anammox) bacteria have been recognized as an important sink for fixed nitrogen and are detected in many natural environments. However, their presence in terrestrial ecosystems has long been overlooked, and their contribution to the nitrogen cycling in natural and agricultural soils is currently unknown. Here we describe the enrichment and characterization of anammox bacteria from a nitrogen-loaded peat soil. After 8 months of incubation with the natural surface water of the sampling site and increasing ammonium and nitrite concentrations, anammox cells constituted 40 to 50% of the enrichment culture. The two dominant anammox phylotypes were affiliated with "Candidatus Jettenia asiatica" and "Candidatus Brocadia fulgida." The enrichment culture converted NH(4)(+) and NO(2)(-) to N(2) with the previously reported stoichiometry (1:1.27) and had a maximum specific anaerobic ammonium oxidation rate of 0.94 mmol NH(4)(+)·g (dry weight)(-1)·h(-1) at pH 7.1 and 32°C. The diagnostic anammox-specific lipids were detected at a concentration of 650 ng·g (dry weight)(-1), and pentyl-[3]-ladderane was the most abundant ladderane lipid.

Microbial and physicochemical characteristics of compact anaerobic ammonium-oxidizing granules in an upflow anaerobic sludge blanket reactor.

Anaerobic ammonium oxidation (anammox) is a promising new process to treat high-strength nitrogenous wastewater. Due to the low growth rate of anaerobic ammonium-oxidizing bacteria, efficient biomass retention is essential for reactor operation. Therefore, we studied the settling ability and community composition of the anaerobic ammonium-oxidizing granules, which were cultivated in an upflow anaerobic sludge blanket (UASB) reactor seeded with aerobic granules. With this seed, the start-up period was less than 160 days at a NH(4)(+)-N removal efficiency of 94% and a loading rate of 0.064 kg N per kg volatile suspended solids per day. The formed granules were bright red and had a high settling velocity (41 to 79 m h(-1)). Cells and extracellular polymeric substances were evenly distributed over the anaerobic ammonium-oxidizing granules. The high percentage of anaerobic ammonium-oxidizing bacteria in the granules could be visualized by fluorescent in situ hybridization and electron microscopy. The copy numbers of 16S rRNA genes of anaerobic ammonium-oxidizing bacteria in the granules were determined to be 4.6 x 10(8) copies ml(-1). The results of this study could be used for a better design, shorter start-up time, and more stable operation of anammox systems for the treatment of nitrogen-rich wastewaters.

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Intercellular electron transfer (IET) refers to the process within which electrons being indirectly or directly transferred to the exterior of cells and eventually delivered to the electron acceptors around cells. IET widely exists in nature, especially when electron acceptor are deficient. IET can be divided into two categories: indirect IET and direct IET. Indirect IET (intercellular substrate transfer) always occurs with electron transfer of hydrogen, formate, and other metabolites. Direct intracellular electron transfer is achieved by the coupling of intracellular and extracellular electron transfer. IET boosts the activity of cellular substrate metabolism and expands the acting space of cells. Moreover, IET generates electric current which provides driving-power for energy sharing between bacteria and transformation of extracellular material (such as heavy metals and humus). There is no doubt that IET has physiological and ecological significance. This review summarized recent advances, making a systematic analysis of the process, characteristics, mechanism and eco-physiological significance of IET.

Quantitative comparison of stability of ANAMMOX process in different reactor configurations.

A new stability evaluating system for ANAMMOX comprising three instability indices i.e. coefficient of variation ratio, coefficient of range ratio and coefficient of regression function derivative was established. Three lab-scale ANAMMOX reactors viz upflow anaerobic sludge blanket (UASB) reactor, upflow stationary fixed film (USFF) reactor and anaerobic sequencing batch reactor (ASBR) were compared for their stability based on the established criterion against the hydraulic and substrate concentration shocks. The results showed that all ANAMMOX reactors under investigation were more tolerant to the hydraulic shock than substrate concentration shock. The UASB reactor was the most stable reactor configuration towards substrate concentration shock, followed by the USFF reactor and ASBR. However, the ASBR proved the most tolerant to hydraulic shock, followed by the UASB reactor and USFF reactor. In terms of stability, UASB reactor was more suitable configuration compared with USFF reactor. The instability indices proved to be effective and explicit for the evaluation of ANAMMOX systems.

Physical and chemical characteristics of PM2.5 and its toxicity to human bronchial cells BEAS-2B in the winter and summer.

With the increasing occurrence of haze during the summer, the physicochemical characteristics and toxicity differences in PM2.5 in different seasons are of great concern. Hangzhou is located in an area that has a subtropical monsoon climate where the humidity is very high during both the summer and winter. However, there are limited studies on the seasonal differences in PM2.5 in these weather conditions. In this test, PM2.5 samples were collected in the winter and summer, the morphology and chemical composition of PM2.5 were analyzed, the toxicity of PM2.5 to human bronchial cells BEAS-2B was compared, and the correlation between PM2.5 toxicity and the chemical composition was discussed. The results showed that during both the winter and summer, the main compounds in the PM2.5 samples were water-soluble ions, particularly SO42-, NO3-, and NH4+, followed by organic components, while heavy metals were present at lower levels. The higher the mass concentration of PM2.5, the greater its impact on cell viability and ROS levels. However, when the mass concentration of PM2.5 was similar, the water extraction from the summer samples showed a greater impact on BEAS-2B than that from the winter samples. The cytotoxicity of PM2.5 was closely associated with heavy metals and organic pollutants but less related to water-soluble ions.

Osmotic stress on nitrification in an airlift bioreactor.

The effect of osmotic pressure on nitrification was studied in a lab-scale internal-loop airlift-nitrifying reactor. The reactor slowly adapted to the escalating osmotic pressure during 270 days operation. The conditions were reversed to the initial stage upon full inhibition of the process. Keeping influent ammonium concentration constant at 420 mg N L(-1) and hydraulic retention time at 20.7h, with gradual increase in osmotic pressure from 4.3 to 18.8x10(5) Pa by adding sodium sulphate, the ammonium removal efficiencies of the nitrifying bioreactor were maintained at 93-100%. Further increase in osmotic pressure up to 19.2x10(5) Pa resulted in drop of the ammonium conversion to 69.2%. The osmotic pressure caused abrupt inhibition of nitrification without any alarm and the critical osmotic pressure value causing inhibition remained between 18.8 and 19.2x10(5) Pa. Nitrite oxidizers were found more sensitive to osmotic stress as compared with ammonia oxidizers, leading to nitrite accumulation up to 61.7% in the reactor. The performance of bioreactor recovered gradually upon lowering the osmotic pressure. Scanning and transmission electron microscopy indicated that osmotic stress resulted in simplification of the nitrifying bacterial populations in the activated sludge as the cellular size reduced; the inner membrane became thinner and some unknown inclusions appeared within the cells. The microbial morphology and cellular structure restored upon relieving the osmotic pressure. Addition of potassium relieved the effect of osmotic pressure upon nitrification. Results demonstrate that the nitrifying reactor possesses the potential to treat ammonium-rich brines after acclimatization.

Anoxic biodegradation of dimethyl phthalate (DMP) by activated sludge cultures under nitrate-reducing conditions.

Worldwide extensive use of plasticized plastics has resulted in phthalates pollution in different environment. Nitrates from industry and agriculture are also widely disseminated in the soils, natural waters and wastewaters. Dimethyl phthalate (DMP) biodegradation by activated sludge cultures under nitrate-reducing conditions was investigated. Under one optimized condition, DMP was biodegraded from 102.20 mg/L to undetectable level in 56 h under anoxic conditions and its reaction fitted well with the first-order kinetics. Using the high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS) analysis, mono-methyl phthalate (MMP) and phthalic acid (PA) were detected as the major intermediates of DMP biodegradation. When combined with the determination of chemical oxygen demand (COD(Cr)) removal capacity and pH, DMP was found to be mineralized completely under anoxic conditions. The biodegradation pathway was proposed as DMP -->MMP-->PA-->...-->CO2 + H2O. The molar ratio ofDMP to nitrate consumed was found to be 9.0:1, which agrees well with the theoretical stoichiometric values of DMP biodegradation by nitrate-reducing bacteria. The results of the non-linear simulation showed that the optimum pH and temperature for the degradation were 7.56 and 31.4 degrees C, respectively.

[Isolation, identification and characterization of nitrate-reducing and sulfide-oxidizing bacteria].

Bacterial strains CB and CS were isolated from the Anoxic Sulfide Oxidizing (ASO) reactor working under steady-state with sulfide and nitrate as electron donor and acceptor, respectively. Based on electron microscopy, physiological test and 16S rDNA phylogenetic sequence analysis, the isolate CB was very close to Bacillus pseudofirmus and the isolate CS was very close to Bacillus hemicellulosilytus and Bacillus halodurans. The 16S rDNA sequences of both isolates were submitted to GenBank, NCBI and accession numbers EF542806 and EF542807 were allotted for isolates CB and CS, respectively. According to Biolog carbon source utilization test, the isolate CB is weak to use the carbon sources, while the isolate CB can use many carbon sources. Both the isolate CB and the isolate CS are able to use nitrate for sulfide oxidation. The isolate CS has greater capability to oxidize sulfide with nitrate as electron acceptor.

[Identification of ammonia oxidation Streptomyces strain A2 and study of its autotrophic ammonium oxidation characteristics].

Streptomyces strain A2 was isolated from a nitrification reactor. According to the characteristics of morphology, cultivation, physiology, (G + C)mol% content, 16S rDNA sequence and DNA-DNA hybridization. it was identified as Streptomyces bikiniensis. Strain A2 could heterotrophically grow on YD medium and could also autotrophically grow on inorganic medium. The heterotrophical growth rate (0.39mg/L.d) was higher than the autotrophical growth rate (0.22mg/L.d). During heterotrophical growth ammonia was mainly assimilated. During autotrophical growth, however, one part of ammonia was assimilated and other part of ammonia was converted into nitrite. When grown on the inorganic medium, the maximum ammonium oxidation rate reached at ammonium concentration of 118mgN/L. The optimal pH for growth and ammonia oxidation was 9.36 and 9.29, respectively. The optimal temperature for growth and ammonia oxidation was 31 degrees C and 40.6 degrees C, respectively. A high concentration of dissolved oxygen was good for growth and ammonia oxidation, and growth was more sensitive to dissolved oxygen change than to ammonia oxidation.

Start-up strategies of UASB reactor for treatment of pharmaceutical wastewater.

Two start-up strategies of upflow anaerobic sludge blanket (UASB) reactor for treatment of pharmaceutical wastewater were investigated. The results showed that both of them were workable. Compared with the strategy that started up the reactor directly using chloromycetin wastewater, the strategy that started up the reactor first using mixed wastewater and then using chloromycetin wastewater could save time by 23%. When the latter strategy was adopted the development of sludge activity fluctuated more largely and its final activity was lower, but the sludge grew faster in the course of start-up.

Start-up of anaerobic ammonia oxidation bioreactor with nitrifying activated sludge.

The anaerobic ammonia oxidation(Anammox) bioreactor was successfully started up with the nitrifying activated sludge. After anaerobically operated for 105 d, the bioreactor reached a good performance with removal percentage of both ammonia and nitrite higher than 95% and volumetric total nitrogen removal as high as 149.55 mmol/( L.d). The soft padding made an important contribution to the high efficiency and stability because it held a large amount of biomass in the bioreactor.