Detection and Quantification of Candidatus Methanoperedens-Like Archaea in Freshwater Wetland Soils.

Candidatus Methanoperedens-like archaea, which can use multiple electron acceptors (nitrate, iron, manganese, and sulfate) for anaerobic methane oxidation, could play an important role in reducing methane emissions from freshwater wetlands. Currently, very little is known about the distribution and community composition of Methanoperedens-like archaea in freshwater wetlands, particularly based on their alpha subunit of methyl-coenzyme M reductase (mcrA) genes. Here, the community composition, diversity, and abundance of Methanoperedens-like archaea were investigated in a freshwater wetland through high-throughput sequencing and quantitative PCR on their mcrA genes. A large number of Methanoperedens-like mcrA gene sequences (119,250) were recovered, and a total of 31 operational taxonomic units (OTUs) were generated based on 95% sequence similarity cut-off. The majority of Methanoperedens-like sequences can be grouped into three distinct clusters that were closely associated with the known Methanoperedens species which can couple anaerobic methane oxidation to nitrate or iron reduction. The community composition of Methanoperedens-like archaea differed significantly among different sampling sites, and their mcrA gene abundance was 1.49 × 106 ~ 4.62 × 106 copies g-1 dry soil in the examined wetland. In addition, the community composition of Methanoperedens-like archaea was significantly affected by the soil water content, and the archaeal abundance was significantly positively correlated with the water content. Our results suggest that the mcrA gene is a good biomarker for detection and quantification of Methanoperedens-like archaea, and provide new insights into the distribution and environmental regulation of these archaea in freshwater wetlands.

Effect of gradual increase of atmospheric CO2 concentration on nitrification potential and communities of ammonia oxidizers in paddy fields.

Currently, the influence of elevated atmospheric CO2 concentration (eCO2) on ammonia oxidation to nitrite, the rate-limiting step of nitrification in paddy soil, is poorly known. Previous studies that simulate the effect of eCO2 on nitrification are primarily based on an abrupt increase of atmospheric CO2 concentration. However, paddy ecosystems are experiencing a gradual increase of CO2 concentration. To better understand how the nitrification potential, abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) respond to eCO2 in paddy ecosystems, a field experiment was conducted using the following two treatments: a gradual increase of CO2 (EC, increase of 40 ppm per year until 200 ppm above ambient) and ambient CO2 (CK). The results demonstrated that the EC treatment significantly (P < 0.05) stimulated the soil potential nitrification rate (PNR) at the jointing and milky stages, which increased by 127.83% and 27.35%, respectively, compared with CK. Furthermore, the EC treatment significantly (P < 0.05) stimulated the AOA and AOB abundance by 56.60% and 133.84%, respectively, at the jointing stage. Correlation analysis showed that the PNR correlated well with the abundance of AOB (R2 = 0.7389, P < 0.001). In addition, the EC treatment significantly (P < 0.05) altered the community structure of AOB, while it had little effect on that of AOA. A significant difference in the proportion of Nitrosospira was observed between CO2 treatments. In conclusion, the gradual increase of CO2 positively influenced the PNR and abundance of ammonia oxidizers, and AOB could be more important than AOA in nitrification under eCO2.

Role and regulation of anaerobic methane oxidation catalyzed by NC10 bacteria and ANME-2d archaea in various ecosystems.

Freshwater wetlands, paddy fields, inland aquatic ecosystems and coastal wetlands are recognized as important sources of atmospheric methane (CH4). Currently, increasing evidence shows the potential importance of the anaerobic oxidation of methane (AOM) mediated by NC10 bacteria and a novel cluster of anaerobic methanotrophic archaea (ANME)-ANME-2d in mitigating CH4 emissions from different ecosystems. To better understand the role of NC10 bacteria and ANME-2d archaea in CH4 emission reduction, the current review systematically summarizes different AOM processes and the functional microorganisms involved in freshwater wetlands, paddy fields, inland aquatic ecosystems and coastal wetlands. NC10 bacteria are widely present in these ecosystems, and the nitrite-dependent AOM is identified as an important CH4 sink and induces nitrogen loss. Nitrite- and nitrate-dependent AOM co-occur in the environment, and they are mainly affected by soil/sediment inorganic nitrogen and organic carbon contents. Furthermore, salinity is another key factor regulating the two AOM processes in coastal wetlands. In addition, ANME-2d archaea have the great potential to couple AOM to the reduction of iron (III), manganese (IV), sulfate, and even humics in different ecosystems. However, the study on the environmental distribution of ANME-2d archaea and their role in CH4 mitigation in environments is insufficient. In this study, we propose several directions for future research on the different AOM processes and respective functional microorganisms.

[Effect of gradual increase of atmospheric CO2 concentration on nitrate-dependent anaerobic methane oxidation in paddy soils]. / 大气CO2æµ“åº¦ç¼“å¢žå¯¹ç¨»ç”°ç¡é ¸ç›åž‹ç”²çƒ·åŽŒæ°§æ°§åŒ–è¿‡ç¨‹çš„å½±å“.

Nitrate-dependent anaerobic oxidation of methane (AOM) is a new pathway to reduce methane emissions from paddy ecosystems. The elevated atmospheric CO2 concentration can affect methane emissions from paddy ecosystems, but its impact on the process of nitrate-dependent AOM is poorly known. Based on the automatic CO2 control platform with open top chambers and the 13CH4 stable isotope experiments, the responses of the activity of nitrate-dependent AOM, abundance and community composition of Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like archaea to the gradual increase of CO2 concentration were investigated in paddy fields. We set up two CO2 concentration treatments, including an ambient CO2 and a gradual increase of CO2(increase of 40 µL·L-1 per year above ambient CO2 concentration until 160 µL·L-1). The results showed the nitrate-dependent AOM rate of 0.7-11.3 nmol CO2·g-1·d-1 in the studied paddy fields, and quantitative PCR showed the abundance of M. nitroreducens-like archaeal mcrA genes of 2.2×106-8.5×106 copies·g-1. Compared to the ambient CO2 treatment, the slow elevated CO2 treatment enhanced the nitrate-dependent AOM rate and stimulated the abundance of M. nitroreducens-like archaea, particularly in 5-10 cm soil layer. The gradual increased CO2 concentration treatment did not change the community composition of M. nitroreducens-like archaea, but significantly decreased their diversity. The soil organic carbon content was an important factor influencing the nitrate-dependent AOM process. Overall, our results showed that the gradual increase of CO2 concentration could promote the nitrate-dependent AOM, suggesting its positive role in mitigating methane emissions from paddy ecosystems under future climate change.

Potential role of nitrite-dependent anaerobic methane oxidation in methane consumption and nitrogen removal in Chinese paddy fields.

Nitrite-dependent anaerobic methane oxidation (n-damo), catalyzed by bacteria closely related to Candidatus Methylomirabilis oxyfera, links the global carbon and nitrogen cycles. Currently, the contribution of n-damo in controlling methane emissions and nitrogen removal, and the key regulatory factors of this process in Chinese paddy fields are poorly known. Here, soil samples from 20 paddy fields located in different climate zones across China were collected to examine the n-damo activity and bacterial communities. The n-damo activity and bacterial abundance varied from 1.05 to 5.97 nmol CH4 g-1 (dry soil) d-1 and 2.59 × 105 to 2.50 × 107 copies g-1 dry soil, respectively. Based on the n-damo activity, it was estimated that approximately 0.91 Tg CH4 and 2.17 Tg N could be consumed annually via n-damo in Chinese paddy soils. The spatial variations in n-damo activity and community structure of n-damo bacteria were significantly (p < 0.05) affected by the soil ammonium content, labile organic carbon content and pH. Furthermore, significant differences in n-damo activity, bacterial abundance and community composition were observed among different climate zones. The n-damo activity was found to be positively correlated with the mean annual air temperature. Taken together, our results demonstrated the potential importance of n-damo in both methane consumption and nitrogen removal in Chinese paddy soils, and this process was regulated by local soil and climatic factors.

[Spatial and temporal characteristics of community structure of nitrite-dependent anaerobic methane-oxidizing bacteria in paddy soil]. / ç¨»ç”°åœŸå£¤äºšç¡é ¸ç›åž‹ç”²çƒ·åŽŒæ°§æ°§åŒ–èŒç¾¤è½ç»“æž„çš„æ—¶ç©ºç‰¹å¾.

Paddy fields are one of the most important methane sources, which have great impacts on climate change. The nitrite-dependent anaerobic methane oxidation, by NC10 phylum bacteria-Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria, is a new process regulating methane emission from paddy fields. However, little is known about the spatial and temporal variations of M. oxyfera-like bacterial communities and the regulating factors in paddy soils. We investigated the community composition, diversity, and abundance of M. oxyfera-like bacteria in 0-40 cm depth of paddy soils at key growth stages of rice, including tillering, jointing, flowering, and milky stages. Results of high-throughput sequencing showed that community composition of M. oxyfera-like bacteria differed significantly among different soil layers, while no significant variation was observed among different rice growth stages. The diversity of M. oxyfera-like bacteria increased with soil depth. Real-time quantitative PCR showed that the 16S rRNA gene abundance of M. oxyfera-like bacteria ranged from 5.73×106 to 2.56×107 copies·g-1 (dry weight), with the highest gene abundance in the 10-20 cm layer. Further, the abundance of these bacteria showed a decreasing trend with rice growth. Soil organic carbon content and soil pH were correlated with the M. oxyfera-like bacterial community structures and abundance. In all, our results suggested a certain degree of heterogeneity of spatial and temporal distribution of M. oxyfera-like bacterial communities in paddy soils, which was largely influenced by soil organic carbon and soil pH.

Response of nitrite-dependent anaerobic methanotrophs to elevated atmospheric CO2 concentration in paddy fields.

Nitrite-dependent anaerobic methane oxidation (n-damo) catalyzed by Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria is a new pathway for the regulation of methane emissions from paddy fields. Elevated atmospheric CO2 concentrations (e[CO2]) can indirectly affect the structure and function of microbial communities. However, the response of M. oxyfera-like bacteria to e[CO2] is currently unknown. Here, we investigated the effect of e[CO2] (ambient CO2 + 200 ppm) on community composition, abundance, and activity of M. oxyfera-like bacteria at different depths (0-5, 5-10, and 10-20 cm) in paddy fields across multiple rice growth stages (tillering, jointing, and flowering). High-throughput sequencing showed that e[CO2] had no significant effect on the community composition of M. oxyfera-like bacteria. However, quantitative PCR suggested that the 16S rRNA gene abundance of M. oxyfera-like bacteria increased significantly in soil under e[CO2], particularly at the tillering stage. Furthermore, 13CH4 tracer experiments showed potential n-damo activity of 0.31-8.91 nmol CO2 g-1 (dry soil) d-1. E[CO2] significantly stimulated n-damo activity, especially at the jointing and flowering stages. The n-damo activity and abundance of M. oxyfera-like bacteria increased by an average of 90.9% and 50.0%, respectively, under e[CO2]. Correlation analysis showed that the increase in soil dissolved organic carbon content caused by e[CO2] had significant effects on the activity and abundance of M. oxyfera-like bacteria. Overall, this study provides the first evidence for a positive response of M. oxyfera-like bacteria to e[CO2], which may help reduce methane emissions from paddy fields under future climate change conditions.

Different responses of ammonia-oxidizing archaea and bacteria in paddy soils to elevated CO2 concentration.

The elevated atmospheric CO2 concentration is well known to have an important effect on soil nutrient cycling. Ammonia oxidation, mediated by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), is the rate-limiting step in soil nitrification, which controls the availability of two key soil nutrients (ammonium and nitrate) for crops. Until now, how the AOA and AOB communities in paddy soils respond to elevated CO2 remains largely unknown. Here, we examined the communities of AOA and AOB and nitrification potential at both surface (0-5 cm) and subsurface (5-10 cm) soil layers of paddy fields under three different CO2 treatments, including CK (ambient CO2 concentration), LT (CK + 160 ppm of CO2) and HT (CK + 200 ppm of CO2). The elevated CO2 was found to have a greater impact on the community structure of AOB than that of AOA in surface soils as revealed by high-throughput sequencing of their amoA genes. However, no obvious variation of AOA or AOB communities was observed in subsurface soils among different CO2 treatments. The abundance of AOA and AOB, and nitrification potential were significantly increased in surface soils under elevated CO2. The variation of AOB abundance correlated well with the variation of nitrification potential. The soil water content and dissolved organic carbon content had important impacts on the dynamic of AOB communities and nitrification potential. Overall, our results showed different responses of AOA and AOB communities to elevated CO2 in paddy ecosystems, and AOB were more sensitive to the rising CO2 concentration.

Different responses of nitrite- and nitrate-dependent anaerobic methanotrophs to increasing nitrogen loading in a freshwater reservoir.

Nitrite (NO2-)- and nitrate (NO3-)-dependent anaerobic oxidation of methane (AOM) are two new additions in microbial methane cycle, which potentially act as important methane sinks in freshwater aquatic systems. Here, we investigated spatial variations of community composition, abundance and potential activity of NO2-- and NO3--dependent anaerobic methanotrophs in the sediment of Jiulonghu Reservoir (Zhejiang Province, China), a freshwater reservoir having a gradient of increasing nitrogen loading from upstream to downstream regions. High-throughput sequencing of total bacterial and archaeal 16S rRNA genes showed the cooccurrence of Candidatus Methylomirabilis oxyfera (M. oxyfera)-like and Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like anaerobic methanotrophs in the examined reservoir sediments. The community structures of these methanotrophs differed substantially between the sediments of upstream and downstream regions. Quantitative PCR suggested higher M. oxyfera-like bacterial abundance in the downstream (8.6 × 107 to 2.8 × 108 copies g-1 dry sediment) than upstream sediments (2.4 × 107 to 3.5 × 107 copies g-1 dry sediment), but there was no obvious difference in M. nitroreducens-like archaeal abundance between these sediments (3.7 × 105 to 4.8 × 105 copies g-1 dry sediment). The 13CH4 tracer experiments suggested the occurrence of NO2-- and NO3--dependent AOM activities, and their rates were 4.7-14.1 and 0.8-2.6 nmol CO2 g-1 (dry sediment) d-1, respectively. Further, the rates of NO2--dependent AOM in downstream sediment were significantly higher than those in upstream sediment. The NO3- concentration was the key factor affecting the spatial variations of abundance and activity of NO2--dependent anaerobic methanotrophs. Overall, our results showed different responses of NO2-- and NO3--dependent anaerobic methanotrophs to increasing nitrogen loading in a freshwater reservoir.

Active pathways of anaerobic methane oxidation across contrasting riverbeds.

Anaerobic oxidation of methane (AOM) reduces methane emissions from marine ecosystems but we know little about AOM in rivers, whose role in the global carbon cycle is increasingly recognized. We measured AOM potentials driven by different electron acceptors, including nitrite, nitrate, sulfate, and ferric iron, and identified microorganisms involved across contrasting riverbeds. AOM activity was confined to the more reduced, sandy riverbeds, whereas no activity was measured in the less reduced, gravel riverbeds where there were few anaerobic methanotrophs. Nitrite-dependent and nitrate-dependent AOM occurred in all sandy riverbeds, with the maximum rates of 61.0 and 20.0 nmol CO2 g-1 (dry sediment) d-1, respectively, while sulfate-dependent and ferric iron-dependent AOM occurred only where methane concentration was highest and the diversity of AOM pathways greatest. Diverse Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria and Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like archaea were detected in the sandy riverbeds (16S rRNA gene abundance of 9.3 × 105 to 1.5 × 107 and 2.1 × 104 to 2.5 × 105 copies g-1 dry sediment, respectively) but no other known anaerobic methanotrophs. Further, we found M. oxyfera-like bacteria and M. nitroreducens-like archaea to be actively involved in nitrite- and nitrate/ferric iron-dependent AOM, respectively. Hence, we demonstrate multiple pathways of AOM in relation to methane, though the activities of M. oxyfera-like bacteria and M. nitroreducens-like archaea are dominant.

Cooccurrence and potential role of nitrite- and nitrate-dependent methanotrophs in freshwater marsh sediments.

Nitrite- and nitrate-dependent anaerobic methane oxidation are mediated by the NC10 bacteria closely related to "Candidatus Methylomirabilis oxyfera" (M. oxyfera) and the ANME-2d archaea closely related to "Candidatus Methanoperedens nitroreducens" (M. nitroreducens), respectively. Here, we investigated the occurrence and activity of both M. oxyfera-like bacteria and M. nitroreducens-like archaea in the sediment of freshwater marshes in Eastern China. The presence of diverse M. oxyfera-like bacteria (>87% identity to M. oxyfera) and M. nitroreducens-like archaea (>96% identity to M. nitroreducens) was confirmed by using Illumina-based total bacterial and archaeal 16S rRNA gene sequencing, respectively. The recovered M. oxyfera-like bacterial sequences accounted for 1.6-4.3% of the total bacterial 16S rRNA pool, and M. nitroreducens-like archaeal sequences accounted for 0.2-1.8% of the total archaeal 16S rRNA pool. The detected numbers of OTUs of the 16S rRNA genes of M. oxyfera-like bacteria and M. nitroreducens-like archaea were 78 and 72, respectively, based on 3% sequence difference. Quantitative PCR showed that the 16S rRNA gene abundance of M. oxyfera-like bacteria (6.1 × 106-3.2 × 107 copies g-1 sediment) was 2-4 orders of magnitude higher than that of M. nitroreducens-like archaea (1.4 × 103-3.2 × 104 copies g-1 sediment). Stable isotope experiments showed that the addition of both nitrite and nitrate stimulated the anaerobic methane oxidation, while the stimulation by nitrite is more significant than nitrate. Our results provide the first evidence that the M. oxyfera-like bacteria play a more important role than the M. nitroreducens-like archaea in methane cycling in wetland systems.

Potential role of anammox in nitrogen removal in a freshwater reservoir, Jiulonghu Reservoir (China).

Currently, the nitrogen removal potential of anaerobic ammonium oxidation (anammox) and its regulating factors in reservoir systems remain uncertain. Here, we provided the molecular and isotopic evidence for anammox in the freshwater sediment of Jiulonghu Reservoir that is located in Quzhou, Zhejiang Province, China. Diverse 16S rRNA gene sequences related to Candidatus Kuenenia and Candidatus Brocadia were detected by using high-throughput (Illumina MiSeq) sequencing of total bacterial 16S rRNA genes, and the Candidatus Brocadia was the most frequently detected anammox bacterial genus. The anammox bacterial abundance was determined based on quantitative PCR on hzsA (the alpha subunit of the hydrazine synthase) genes and varied from 3.1 × 105 to 1.1 × 106 copies g-1 dry sediment. Homogenized sediments were further incubated with 15NO3- amendments to measure the potential anammox rates and determine the contribution of this process to dinitrogen gas (N2) production. The potential rates of anammox ranged between 8.1 and 30.8 nmol N2 g-1 dry sediment day-1, and anammox accounted for 7.7-20.5% of total N2 production in sediment. Higher levels of anammox bacterial diversity, abundance, and activity were observed in the downstream with greater human disturbance than those in the upstream with less human disturbance. Correlation analyses suggested that the inorganic nitrogen level in sediment could be a key factor for the anammox bacterial abundance and activity. The results showed that the nitrogen removal via anammox may not be negligible in the examined reservoir and indicated that human activities could influence the anammox process in reservoir systems.

Comparison of community structures of Candidatus Methylomirabilis oxyfera-like bacteria of NC10 phylum in different freshwater habitats.

Methane oxidation coupled to nitrite reduction is mediated by 'Candidatus Methylomirabilis oxyfera' (M. oxyfera), which belongs to the NC10 phylum. In this study, the community composition and diversity of M. oxyfera-like bacteria of NC10 phylum were examined and compared in four different freshwater habitats, including reservoir sediments (RS), pond sediments (PS), wetland sediments (WS) and paddy soils (PAS), by using Illumina-based 16S rRNA gene sequencing. The recovered NC10-related sequences accounted for 0.4-2.5% of the 16S rRNA pool in the examined habitats, and the highest percentage was found in WS. The diversity of NC10 bacteria were the highest in RS, medium in WS, and lowest in PS and PAS. The observed number of OTUs (operational taxonomic unit; at 3% cut-off) were 97, 46, 61 and 40, respectively, in RS, PS, WS and PAS. A heterogeneous distribution of NC10 bacterial communities was observed in the examined habitats, though group B members were the dominant bacteria in each habitat. The copy numbers of NC10 bacterial 16S rRNA genes ranged between 5.8 × 10(6) and 3.2 × 10(7) copies g(-1) sediment/soil in the examined habitats. These results are helpful for a systematic understanding of NC10 bacterial communities in different types of freshwater habitats.

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.

Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater.

Evidence for anaerobic ammonium oxidation process in freshwater sediments of aquaculture ponds.

The anaerobic ammonium oxidation (anammox) process, which can simultaneously remove ammonium and nitrite, both toxic to aquatic animals, can be very important to the aquaculture industry. Here, the presence and activity of anammox bacteria in the sediments of four different freshwater aquaculture ponds were investigated by using Illumina-based 16S rRNA gene sequencing, quantitative PCR assays and (15)N stable isotope measurements. Different genera of anammox bacteria were detected in the examined pond sediments, including Candidatus Brocadia, Candidatus Kuenenia and Candidatus Anammoxoglobus, with Candidatus Brocadia being the dominant anammox genus. Quantitative PCR of hydrazine synthase genes showed that the abundance of anammox bacteria ranged from 5.6 × 10(4) to 2.1 × 10(5) copies g(-1) sediment in the examined ponds. The potential anammox rates ranged between 3.7 and 19.4 nmol N2 g(-1) sediment day(-1), and the potential denitrification rates varied from 107.1 to 300.3 nmol N2 g(-1) sediment day(-1). The anammox process contributed 1.2-15.3% to sediment dinitrogen gas production, while the remainder would be due to denitrification. It is estimated that a total loss of 2.1-10.9 g N m(-2) per year could be attributed to the anammox process in the examined ponds, suggesting that this process could contribute to nitrogen removal in freshwater aquaculture ponds.

Distribution and activity of anaerobic ammonium-oxidising bacteria in natural freshwater wetland soils.

Anaerobic ammonium oxidation (anammox) process plays a significant role in the marine nitrogen cycle. However, the quantitative importance of this process in nitrogen removal in wetland systems, particularly in natural freshwater wetlands, is still not determined. In the present study, we provided the evidence of the distribution and activity of anammox bacteria in a natural freshwater wetland, located in southeastern China, by using (15)N stable isotope measurements, quantitative PCR assays and 16S rRNA gene clone library analysis. The potential anammox rates measured in this wetland system ranged between 2.5 and 25.5 nmol N2 g(-1) soil day(-1), and up to 20% soil dinitrogen gas production could be attributed to the anammox process. Phylogenetic analysis of 16S rRNA genes showed that anammox bacteria related to Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus and two novel anammox clusters coexisted in the collected soil cores, with Candidatus Brocadia and Candidatus Kuenenia being the dominant anammox genera. Quantitative PCR of hydrazine synthase genes showed that the abundance of anammox bacteria varied from 2.3 × 10(5) to 2.2 × 10(6) copies g(-1) soil in the examined soil cores. Correlation analyses suggested that the soil ammonium concentration had significant influence on the activity of anammox bacteria. On the basis of (15)N tracing technology, it is estimated that a total loss of 31.1 g N m(-2) per year could be linked the anammox process in the examined wetland.

[Research progress on microbial properties of nitrite-dependent anaerobic methane-oxidising bacteria].

Nitrite-dependent anaerobic methane oxidation (N-DAMO) is a recently discovered process that constitutes a unique association between the two major global elements essential for life, carbon and nitrogen. This process is one of the most important discoveries in the fields of environmental science and microbiology. The discovery of N-DAMO process supplements biogeochemical cycles of carbon and nitrogen in nature, extends microbial diversity and urges development of novel simultaneous carbon and nitrogen removal process. The N-DAMO process is mediated by the bacterium " Candidatus Methylomirabilis oxyfera" (M. oxyfera), which belongs to the candidate phylum NC10. Currently, a series of breakthroughs have been made in the research of M. oxyfera. The properties of M. oxyfera morphology, chemical composition, enrichment culture, physiology and biochemistry, and ecology have been revealed. Most importantly, the special ultrastructure (star-like) of the cell shape and unique chemical composition (10MeC(16,1Δ7)) of M. oxyfera have been revealed. In addition, a new intra-aerobic metabolism (the fourth biological pathway to produce oxygen) was discovered in M. oxyfera. It was observed that M. oxyfera bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen gas and oxygen, which was then used to oxidise methane. The present review summarises various aspects of microbiological properties of M. oxyfera.

Occurrence and importance of anaerobic ammonium-oxidising bacteria in vegetable soils.

The quantitative importance of anaerobic ammonium oxidation (anammox) has been described in paddy fields, while the presence and importance of anammox in subsurface soil from vegetable fields have not been determined yet. Here, we investigated the occurrence and activity of anammox bacteria in five different types of vegetable fields located in Jiangsu Province, China. Stable isotope experiments confirmed the anammox activity in the examined soils, with the potential rates of 2.1 and 23.2 nmol N2 g(-1) dry soil day(-1), and the anammox accounted for 5.9-20.5% of total soil dinitrogen gas production. It is estimated that a total loss of 7.1-78.2 g N m(-2) year(-1) could be linked to the anammox process in the examined vegetable fields. Phylogenetic analyses showed that multiple co-occurring anammox genera were present in the examined soils, including Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus and Candidatus Jettenia, and Candidatus Brocadia appeared to be the most common anammox genus. Quantitative PCR further confirmed the presence of anammox bacteria in the examined soils, with the abundance varying from 2.8 × 10(5) to 3.0 × 10(6) copies g(-1) dry soil. Correlation analyses suggested that the soil ammonium concentration had significant influence on the activity and abundance of anammox bacteria in the examined soils. The results of our study showed the presence of diverse anammox bacteria and indicated that the anammox process could serve as an important nitrogen loss pathway in vegetable fields.

Distribution and environmental significance of nitrite-dependent anaerobic methane-oxidising bacteria in natural ecosystems.

Nitrite-dependent anaerobic methane oxidation (N-DAMO) is a recently discovered process that is performed by "Candidatus Methylomirabilis oxyfera" (M. oxyfera). This process constitutes a unique association between the two major global elements essential to life, carbon and nitrogen, and may act as an important and overlooked sink of the greenhouse gas methane. In recent years, more and more studies have reported the distribution of M. oxyfera-like bacteria and the occurrence of N-DAMO process in different natural ecosystems, including freshwater lakes, rivers, wetlands and marine ecosystems. Previous studies have estimated that a total of 2%-6% of current worldwide methane flux in wetlands could be consumed via the N-DAMO process. These findings indicate that N-DAMO is indeed a previously overlooked methane sink in natural ecosystems. Given the worldwide increase in anthropogenic nitrogen pollution, the N-DAMO process as a methane sink in reducing global warming could become more important in the future. The present mini-review summarises the current knowledge of the ecological distribution of M. oxyfera-like bacteria and the potential importance of the N-DAMO process in reducing methane emissions in various natural ecosystems. The potential influence of environmental factors on the N-DAMO process is also discussed.