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 CH 4 m −2 per year in wetlands under anaerobic conditions, which is roughly 2–6% of current worldwide CH 4 flux estimates for wetlands. Given the worldwide increase in nitrogen pollution, this methane sink may become more important in the future.

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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 N 2 g ؊1 (dry weight) day ؊1 and the potential n-damo rates varied from 0.2 to 2.1 nmol CO 2 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 ؋ 10 5 to 2.0 ؋ 10 6 copies g ؊1 (dry weight) in different layers of soil cores and the abundance of n-damo bacteria varied from 3.8 ؋ 10 5 to 6.1 ؋ 10 6 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 CH 4 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.

Nitrite-dependent anaerobic methane oxidation (n-damo) process was reported to be mediated by "Candidatus Methylomirabilis oxyfera", which belongs to the candidate phylum NC10. M. oxyfera-like bacteria have been detected in lake ecosystems, while their distribution, diversity and abundance in river ecosystems have not been well studied. In this study, both the 16S rRNA and the pmoA molecular biomarkers confirmed the presence of diverse NC10 phylum bacteria related to M. oxyfera in a river ecosystem-the Qiantang River, Zhejiang Province (China). Phylogenetic analysis of 16S rRNA genes demonstrated that the recovered M. oxyfera-like sequences could be grouped into several distinct clusters that exhibited 89.8% to 98.9% identity to the M. oxyfera 16S rRNA gene. Similarly, several different clusters of pmoA gene sequences were observed, and these clusters displayed 85.1-95.4% sequence identity to the pmoA gene of M. oxyfera. Quantitative PCR showed that the abundance of M. oxyfera-like bacteria varied from 1.32 ± 0.16 × 10(6) to 1.03 ± 0.12 × 10(7) copies g (dry weight)(-1). Correlation analysis demonstrated that the total inorganic nitrogen content, the ammonium content and the organic content of the sediment were important factors affecting the distribution of M. oxyfera-like bacterial groups in the examined sediments. This study demonstrated the distribution of diverse M. oxyfera-like bacteria and their correlation with environmental factors in Qiantang River sediments.

Although the current literature has recorded many reports of identifying components from herbal preparations, all of them were largely limited to target components. This paper provides a novel and generally applicable approach to identifying nontarget components from herbal preparations, based on the use of liquid chromatography ion trap time-of-flight mass spectrometry (LC/MS-IT-TOF). A simple program was originally developed for searching the common diagnostic ions from all experimentally generated ions. The components sharing the exact same ions (mass error < 5 mDa) were classified into a family. All families were then connected into a coherent network by the bridging components that are present in two or more families. With the benefit from such a network, it is feasible to sequentially characterize the structures of all diagnostic ions once a single component has been de novo identified. The structures of the diagnostic ions could then be used as "a priori" information for selecting the exact candidates containing the substructures of the corresponding diagnostic ions from the primary database hits. This strategy enables a nearly 7-fold narrowing of the database hits and thus substantially enhances the analytical efficiency and sharpness. With the use of such an approach, 43 out of 53 components incorporated into the network have been successfully identified from the test herbal preparation. For the rest, components failed to be identified using this approach; a complementary approach to screening by sequential loss of specific chemical groups, proposed from the accurate mass differences between fragments, was established to narrow the database hits. All of the 87 peaks detected have been successfully identified by combining the use of both approaches except failed to differentiate some isomers. The presently developed approach and methodology would be useful for the identifications of complicated nontarget components from various complex mixtures such as herbal preparations, biological, and environmental samples.