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.

Effects of abrupt and gradual increase of atmospheric CO2 concentration on methanotrophs in paddy fields.

The simulation of abrupt atmospheric CO2 increase is a common way to examine the response of soil methanotrophs to future climate change. However, atmosphere is undergoing a gradual CO2 increase, and it is unknown whether the previously reported response of methanotrophs to abrupt CO2 increase can well represent their response to the gradual increase. To improve the understanding of the effect of elevated CO2 (eCO2) on methanotrophs in paddy ecosystems, the methane oxidation potential and communities of methanotrophs were examined via open top chambers under the three following CO2 treatments: an ambient CO2 concentration (AC); an abrupt CO2 increase by 200 ppm above AC (AI); a gradual CO2 increase by 40 ppm each year until 200 ppm above AC (GI). Relative to AC treatment, AI and GI treatments significantly (p < 0.05) increased the methane oxidation rate by 43.8% and 36.7%, respectively, during rice growth period. Furthermore, the abundance of pmoA genes was significantly (p < 0.05) increased by 62.4% and 32.5%, respectively, under AI and GI treatments. However, there were no significant variations in oxidation rate or gene abundance between the two eCO2 treatments. In addition, no obvious change of overall community composition of methanotrophs was observed among treatments, while the proportions of Methylosarcina and Methylocystis significantly (p < 0.05) changed. Taken together, our results indicate similar response of methanotrophs to abrupt and gradual CO2 increase, although the magnitude of response under gradual increase was smaller and the abrupt increase may somewhat overestimate the response.

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.

Response of potential activity, abundance and community composition of nitrite-dependent anaerobic methanotrophs to long-term fertilization in paddy soils.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) catalysed by Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria is a novel pathway in regulating methane (CH4 ) emissions from paddy fields. Nitrogen fertilization is essential to improve rice yields and soil fertility; however, its effect on the n-damo process is largely unknown. Here, the potential n-damo activity, abundance and community composition of M. oxyfera-like bacteria were investigated in paddy fields under three long-term (32 years) fertilization treatments, i.e. unfertilized control (CK), chemical fertilization (NPK) and straw incorporation with chemical fertilization (SNPK). Relative to the CK, both NPK and SNPK treatments significantly (p < 0.05) increased the potential n-damo activity (88%-110%) and the abundance (52%-105%) of M. oxyfera-like bacteria. The variation of soil organic carbon (OrgC) content and inorganic nitrogen content caused by the input of chemical fertilizers and straw returning were identified as the key factors affecting the potential n-damo activity and the abundance of M. oxyfera-like bacteria. However, the community composition and diversity of M. oxyfera-like bacteria did not change significantly by the input of fertilizers. Overall, our results provide the first evidence that long-term fertilization greatly stimulates the n-damo process, indicating its active role in controlling CH4 emissions from paddy fields.

[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.

[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.

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.

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 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.