Sha Geng

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.

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.

Mdodeling a nitrite-dependent anaerobic methane oxidation process: Parameters identification and model evaluation

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered process that is intermediated by n-damo bacteria that oxidize methane with nitrite to generate nitrogen gas. In this work, a kinetic model based on Monod type kinetics and diffusion-reaction model was developed to describe the bioprocess. Some key kinetic parameters needed in the model were obtained from a series of batch activity tests and a sequencing batch reactor (SBR) operation over 100 days. The growth rate, decay rate, methane affinity constant, nitrite affinity constant and inhibition constant were 0.0277±0.0022 d(-1), 0.00216±0.00010 d(-1), 0.092±0.005 mmol L(-1), 0.91±0.09 mmol L(-1) and 4.1±0.5 mmol L(-1) for n-damo bacteria at 30 °C, respectively. The results showed that the model could simulate actual performance of the SBR in the first 76 days, that methane was not a limiting factor at atmospheric pressure for its high affinity, and that the optimum nitrite concentration was 1.92 mmol L(-1).

Anaerobic Oxidation of Methane Coupled to Nitrite Reduction by Halophilic Marine NC10 Bacteria

ABSTRACT Anaerobic oxidation of methane (AOM) coupled to nitrite reduction is a novel AOM process that is mediated by denitrifying methanotrophs. To date, enrichments of these denitrifying methanotrophs have been confined to freshwater systems; however, the recent findings of 16S rRNA and pmoA gene sequences in marine sediments suggest a possible occurrence of AOM coupled to nitrite reduction in marine systems. In this research, a marine denitrifying methanotrophic culture was obtained after 20 months of enrichment. Activity testing and quantitative PCR (qPCR) analysis were then conducted and showed that the methane oxidation activity and the number of NC10 bacteria increased correlatively during the enrichment period. 16S rRNA gene sequencing indicated that only bacteria in group A of the NC10 phylum were enriched and responsible for the resulting methane oxidation activity, although a diverse community of NC10 bacteria was harbored in the inoculum. Fluorescence in situ hybridization showed that NC10 bacteria were dominant in the enrichment culture after 20 months. The effect of salinity on the marine denitrifying methanotrophic culture was investigated, and the apparent optimal salinity was 20.5‰, which suggested that halophilic bacterial AOM coupled to nitrite reduction was obtained. Moreover, the apparent substrate affinity coefficients of the halophilic denitrifying methanotrophs were determined to be 9.8 ± 2.2 μM for methane and 8.7 ± 1.5 μM for nitrite.

Improvement of the trace metal composition of medium for nitrite-dependent anaerobic methane oxidation bacteria: Iron (II) and copper (II) make a difference

Nitrite-dependent anaerobic methane oxidation (n-damo) is a potential bioprocess for treating nitrogen-containing wastewater. This process uses methane, an inexpensive and nontoxic end-product of anaerobic digestion, as an external electron donor. However, the low turnover rate and slow growth rate of n-damo functional bacteria limit the practical application of this process. In the present study, the short- and long-term effects of variations in trace metal concentrations on n-damo bacteria were investigated, and the concentrations of trace metal elements of medium were improved. The results were subsequently verified by a group of long-term inoculations (90 days) and were applied in a sequencing batch reactor (SBR) (84 days). The results indicated that iron (Fe(II)) and copper (Cu(II)) (20 and 10 μmol L(-1), respectively) significantly stimulated the activity and the growth of n-damo bacteria, whereas other trace metal elements, including zinc (Zn), molybdenum (Mo), cobalt (Co), manganese (Mn), and nickel (Ni), had no significant effect on n-damo bacteria in the tested concentration ranges. Interestingly, fluorescence in situ hybridization (FISH) showed that a large number of dense, large aggregates (10-50 μm) of n-damo bacteria were formed by cell adhesion in the SBR reactor after using the improved medium, and to our knowledge this is the first discovery of large aggregates of n-damo bacteria.