Lidong Shen

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

Significance Given the current pressing need to more fully understand the methane cycle on Earth, in particular, unidentified sinks for methane, identifying and quantifying novel sinks for methane is fundamental importance. Here, we provide previously unidentified direct evidence for the nitrite-dependent anaerobic methane oxidation (n-damo) process as a previously overlooked microbial methane sink in wetlands by stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. It is estimated that n-damo could consume 4.1–6.1 Tg of CH4 m−2 per year in wetlands under anaerobic conditions, which is roughly 2–6% of current worldwide CH4 flux estimates for wetlands. Given the worldwide increase in nitrogen pollution, this methane sink may become more important in the future. 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 × 107 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.

Distribution and diversity of anaerobic ammonium‐oxidizing bacteria in the sediments of the Qiantang River

Anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. However, little is known about the distribution, diversity and abundance of anammox bacteria in inland river ecosystems. Here, we found the presence of diverse anammox bacteria in a freshwater river - the Qiantang River, Zhejiang Province (China). The phylogenetic analysis of 16S rRNA genes showed that Brocadia genus, Kuenenia genus, Scalindua genus and three new anammox bacterial clusters could be detected together in Qiantang River sediments, suggesting a higher anammox bacterial diversity in the Qiantang River ecosystem than in open ocean environments where only Scalindua genus was detected. Brocadia and Kuenenia appeared to be the most common anammox bacterial genera in the Qiantang River. Redundancy analysis showed that the sediment organic carbon (OrgC) content had significant influence on the distribution of anammox bacteria in Qiantang River sediments. Pearson correlation analyses showed that OrgC content significantly influenced the anammox bacterial diversity. The results of real-time quantitative PCR showed spatial variations of anammox bacterial abundances which were highly correlated with the sediment total inorganic nitrogen content. These results demonstrated the distribution of diverse anammox bacteria and the influences of environmental factors on anammox bacterial communities in Qiantang River sediments.

Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

ABSTRACT 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 × 104 ± 0.42 × 104 to 3.69 × 106 ± 0.25 × 106 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.

Spatial distribution and factors shaping the niche segregation of ammonia-oxidizing microorganisms in the Qiantang River, China.

ABSTRACT Ammonia oxidation is performed by both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, the current knowledge of the distribution, diversity, and relative abundance of these two microbial groups in freshwater sediments is insufficient. We examined the spatial distribution and analyzed the possible factors leading to the niche segregation of AOA and AOB in the sediments of the Qiantang River, using clone library construction and quantitative PCR for both archaeal and bacterial amoA genes. pH and NH4 +-N content had a significant effect on AOA abundance and AOA operational taxonomy unit (OTU) numbers. pH and organic carbon content influenced the ratio of AOA/AOB OTU numbers significantly. The influence of these factors showed an obvious spatial trend along the Qiantang River. This result suggested that AOA may contribute more than AOB to the upstream reaches of the Qiantang River, where the pH is lower and the organic carbon and NH4 +-N contents are higher, but AOB were the principal driver of nitrification downstream, where the opposite environmental conditions were present.

Evidence for the Cooccurrence of Nitrite-Dependent Anaerobic Ammonium and Methane Oxidation Processes in a Flooded Paddy Field

ABSTRACT 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 × 105 to 2.0 × 106 copies g−1 (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 × 105 to 6.1 × 106 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.

Enrichment of anammox bacteria from three sludge sources for the startup of monosodium glutamate industrial wastewater treatment system

Three activated sludges from a landfill leachate treatment plant (S1), a municipal sewage treatment plant (S2) and a monosodium glutamate (MSG) wastewater treatment plant (S3) were used as inocula to enrich anaerobic ammonium oxidation (anammox) bacteria for the startup of MSG industrial wastewater treatment system. After 360 days of cultivation using MSG wastewater, obvious anammox activity was observed in all three cultures. The maximum specific anammox activities of cultures S1, S2 and S3 were 0.11 kg N kg(-1) VSS day(-1), 0.09 kg N kg(-1) VSS day(-1) and 0.16 kg N kg(-1) VSS day(-1), respectively. Brownish-red anammox granules having diameters in the range of 0.2-1.0mm were visible in cultures S1 and S2, and large red granules having diameters in the range of 0.5-2.5mm were formed in culture S3 after 420 days of cultivation. Phylogenetic analysis of 16S rRNA genes showed that Kuenenia organisms were the dominant anammox species in all three cultures. The copy numbers of 16S rRNA genes of anammox bacteria in cultures S1, S2 and S3 were 6.8 × 10(7) copies mL(-1), 9.4 × 10(7) copies mL(-1) and 7.5 × 10(8) copies mL(-1), respectively. The results of this study demonstrated that anammox cultivation from conventional activated sludges was highly possible using MSG wastewater. Thus the anammox process has possibility of applying to the nitrogen removal from MSG wastewater.

Microbiology, ecology, and application of the nitrite-dependent anaerobic methane oxidation process

Nitrite-dependent anaerobic methane oxidation (n-damo), which couples the anaerobic oxidation of methane to denitrification, is a recently discovered process mediated by “Candidatus Methylomirabilis oxyfera.” M. oxyfera is affiliated with the “NC10” phylum, a phylum having no members in pure culture. Based on the isotopic labeling experiments, it is hypothesized that M. oxyfera has an unusual intra-aerobic pathway for the production of oxygen via the dismutation of nitric oxide into dinitrogen gas and oxygen. In addition, the bacterial species has a unique ultrastructure that is distinct from that of other previously described microorganisms. M. oxyfera-like sequences have been recovered from different natural habitats, suggesting that the n-damo process potentially contributes to global carbon and nitrogen cycles. The n-damo process is a process that can reduce the greenhouse effect, as methane is more effective in heat-trapping than carbon dioxide. The n-damo process, which uses methane instead of organic matter to drive denitrification, is also an economical nitrogen removal process because methane is a relatively inexpensive electron donor. This mini-review summarizes the peculiar microbiology of M. oxyfera and discusses the potential ecological importance and engineering application of the n-damo process.

Enrichment of an anammox bacterial community from a flooded paddy soil.

This study describes the enrichment of anammox bacteria in a column simulating oxygen limited flooded paddy soils, which are important man-made ecosystems that receive substantial amounts of fixed nitrogen. The upper 50 cm of the paddy soil, containing a high amount of ammonium [1.6-10.4 mmol N kg (dry weight)(-1)], was selected as the inoculum for anammox enrichment. After 18 months of incubation with freshwater from the paddy soil ecosystem, the enrichment culture consumed approximately 4 mmol ammonium l(-1) day(-1) and 5 mmol nitrite l(-1) day(-1). The maximum specific anammox activity of the culture was 35.7 μmol N g (dry weight)(-1) h(-1). Fluorescence in situ hybridization indicated that anammox cells constituted 50% ± 10% of the enrichment culture. The phylogenetic analyses of 16S rRNA and the diagnostic hydrazine synthase (hzsA) genes showed that two dominant anammox species were enriched from paddy soil. The enriched Candidatus Anammoxoglobus-like organisms showed a 16S rRNA gene similarity of 97.5-99.2% to Candidatus Anammoxoglobus propionicus and the Candidatus Jettenia-like organisms showed 92.1-93.1% 16S rRNA gene identity to Candidatus Jettenia asiatica. Real-time quantitative PCR of hzsA gene suggested that up to 10(10) copies g (dry weight)(-1) of soil anammox bacteria were present in the enrichment culture.

The Influence of Intense Chemical Pollution on the Community Composition, Diversity and Abundance of Anammox Bacteria in the Jiaojiang Estuary (China)

Continuous chemical pollution is one of the most serious environmental problems in the Jiaojiang Estuary of the East Sea (China). This chemical pollution has significantly changed the estuarine environmental conditions and may have profoundly influenced the distribution of anammox bacterial communities in this estuary. Here, we investigated the influence of chemical pollution on the community composition, diversity and abundance of anammox bacteria in Jiaojiang estuarine sediments. Phylogenetic analysis of 16S rRNA genes showed that the majority of anammox bacterial sequences retrieved from the estuarine intertidal sediments were associated with Kuenenia. In contrast, different anammox communities composed of Brocadia, Kuenenia, Scalindua and Jettenia were found in the estuarine subtidal sediments. Redundancy analysis (RDA) indicated that the sediment nitrobenzene and organic content had significant impacts on the distribution of anammox communities in the intertidal sediments. Pearson correlation analysis showed that the diversity of anammox bacteria in the intertidal sediments was positively correlated with the organic content. In contrast, RDA results showed that the nitrobenzene content, NO3 − concentration and salinity significantly influenced the distribution of anammox communities in the subtidal sediments. The diversity and relative abundance of anammox bacteria in the subtidal sediments were positively correlated with NO3 − concentration.

pH levels drive bacterial community structure in sediments of the Qiantang River as determined by 454 pyrosequencing

The Qiantang River is a typical freshwater ecosystem that acts as an irreplaceable water source in Zhejiang Province in southeastern China. However, the effects of environmental factors on the bacterial community of this freshwater ecosystem have not been determined. In this study, seven sediment samples were collected along the river. Their bacterial communities were identified using 454 high-throughput sequencing, and the primary environmental factors responsible for shaping the community structure were analyzed. The number of bacterial operational taxonomic units (OTUs) ranged from 2637 to 3933. Using a linear-regression analysis, the OTU numbers were significantly positively correlated with pH (r = 0.832, p < 0.05) and negatively correlated with nitrate concentration (r = −0.805, p < 0.05). A redundancy analysis (RDA) was also performed to test the relationship between the environmental factors and bacterial community composition. The results indicated that pH (p < 0.05) and nitrate concentration (p < 0.05) were the most significant factors that determined the community distribution of sediment bacteria.