Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field.

The nitrate-dependent denitrifying anaerobic methane oxidation (DAMO) process, which is metabolized together by anaerobic methanotrophic archaea and NC10 phylum bacteria, is expected to be important for the global carbon and nitrogen cycles. However, there are little studies about the existence of this process and the functional microbes in environments. Therefore, the coexistence of DAMO archaea and bacteria in a paddy field was evaluated in this study. Next-generation sequencing showed that the two orders, Methanosarcinales and Nitrospirales, to which DAMO archaea and DAMO bacteria belong, were detected in the four soil samples. Then the in vitro experiments demonstrated both of nitrite- and nitrate-dependent DAMO activities, which confirmed the coexistence of DAMO archaea and DAMO bacteria. It was the first report about the coexistence of DAMO archaea and bacteria in a paddy field. Furthermore, anammox bacteria were detected in two of the four samples. The in vitro experiments did not show anammox activity in the initial period but showed low anammox activity after 20 days' enrichment. These results implicated that anammox bacteria may coexist with DAMO microorganisms in this field, but at a very low percentage.

Biochar modulates intracellular electron transfer for nitrate reduction in denitrifying anaerobic methane oxidizing archaea.

Denitrifying anaerobic methane oxidizing (DAMO) archaea plays a significant role in simultaneously nitrogen removal and methane mitigation, yet its limited metabolic activity hinders engineering applications. This study employed biochar to explore its potential for enhancing the metabolic activity and nitrate reduction capacity of DAMO microorganisms. Sawdust biochar (7 g/L) was found to increase the nitrate reduction rate by 2.85 times, although it did not affect the nitrite reduction rate individually. Scanning electron microscopy (SEM) and fluorescence excitation-emission matrix (EEM) analyses revealed that biochar promoted microbial aggregation, and stimulated the secretion of extracellular polymeric substances (EPS). Moreover, biochar bolstered the redox capacity and conductivity of the biofilm, notably enhancing the activity of the electron transfer system by 1.65 times. Key genes involved in intracellular electron transport (Hdr, MHC, Rnf) and membrane transport proteins (BBP, ABC, NDH) of archaea were significantly up-regulated. These findings suggest that biochar regulates electrons generated by reverse methanogenesis to the membrane for nitrate reduction.

Fundamentals and potential environmental significance of denitrifying anaerobic methane oxidizing archaea.

Many properties of denitrifying anaerobic methane oxidation (DAMO) bacteria have been explored since their first discovery, while DAMO archaea have attracted less attention. Since nitrate is more abundant than nitrite not only in wastewater but also in the natural environment, in depth investigations of the nitrate-DAMO process should be conducted to determine its environmental significance in the global carbon and nitrogen cycles. This review summarizes the status of research on DAMO archaea and the catalyzed nitrate-dependent anaerobic methane oxidation, including such aspects as laboratory enrichment, environmental distribution, and metabolic mechanism. It is shown that appropriate inocula and enrichment parameters are important for the culture enrichment and thus the subsequent DAMO activity, but there are still relatively few studies on the environmental distribution and physiological metabolism of DAMO archaea. Finally, some hypotheses and directions for future research on DAMO archaea, anaerobic methanotrophic archaea, and even anaerobically metabolizing archaea are also discussed.

Long-term nitrate removal through methane-dependent denitrification microorganisms in sequencing batch reactors fed with only nitrate and methane.

Denitrifying anaerobic methane oxidation (damo) bioprocesses can remove nitrate using methane as the electron donor, which gains great concern due to the current stringent discharge standard of nitrogen in wastewater treatment plants. To obtain an engineering acceptable nitrogen removal rate (NRR) and demonstrate the long-term stable ability of damo system under conditions of nitrate and methane, two sequencing batch reactors (SBRs) fed with only nitrate and methane were operated for more than 600 days at 30 °C. The NRR of 21.91 ± 0.73 mg NO3--N L-1 day-1 was obtained which is, to the best of our knowledge, the highest rate observed in the literatures under such conditions. The temperature was found to significantly affect the system performance. Furthermore, the microbial community was analyzed by using real-time PCR technique. The results showed that the microbial consortium contained damo archaea and bacteria. These two microbes cooperated to maintain the long-term stability. And the number of damo archaea was higher than that of damo bacteria with the ratio of 1.77. By using methane as the electron donor, damo archaea reduced nitrate to nitrite coupled to methane oxidation and damo bacteria reduce the generated nitrite to nitrogen gas. The first step of nitrate to nitrite taken by damo archaea might be the limiting step of this cooperation system. SBR could be a suitable reactor configuration to enrich slow-growing microbes like damo culture. These results demonstrated the potential application of damo processes for nitrogen removal of wastewater containing low C/N ratios.

Design and evaluation of universal 16S rRNA gene primers for high-throughput sequencing to simultaneously detect DAMO microbes and anammox bacteria.

To develop universal 16S rRNA gene primers for high-throughput sequencing for the simultaneous detection of denitrifying anaerobic methane oxidation (DAMO) archaea, DAMO bacteria, and anaerobic ammonium oxidation (anammox) bacteria, four published primer sets (PS2-PS5) were modified. The overall coverage of the four primer pairs was evaluated in silico with the Silva SSU r119 dataset. Based on the virtual evaluation, the two best primer pairs (PS4 and PS5) were selected for further verification. Illumina MiSeq sequencing of a freshwater sediment and a culture from a DAMO-anammox reactor using these two primer pairs revealed that PS5 (341b4F-806R) was the most promising universal primer pair. This pair of primers detected both archaea and bacteria with less bias than PS4. Furthermore, an anaerobic fermentation culture and a wastewater treatment plant culture were used to verify the accuracy of PS5. More importantly, it detected DAMO archaea, DAMO bacteria, and anammox bacteria simultaneously with no false positives appeared. This universal 16S rRNA gene primer pair extends the existing molecular tools for studying the community structures and distributions of DAMO microbes and their potential interactions with anammox bacteria in different environments.