Molecular Ecology Of Methanogens And Methanotrophs In Wetlands Of The Yangtze River Estuary

Understanding the role of estuarine wetlands, the highly productive environments, in the global cycling of methane is critical for elucidating the global greenhouse gas budget. Although net methane emissions from these important environments have been frequently reported, only few investigations have been undertaken to uncover the identity and abundance of methane cycling microbial communities. Hence, upon involving field measurements and laboratory activities; next generation sequencing (GS FLX Titanium) and quantitative real-time PCR (qPCR), we investigated the molecular ecology of methanogens and methanotrophs in the intertidal mudflat (devoid of vegetation) and salt marsh sediments (vegetated) of Dongtan, Chongming Island, China. In addition to the methyl coenzyme M reductase (mcrA) and particulate methane monooxygenase (pmoA), dissimilatory sulfate reductase B (dsrB) and the16S rRNA genes of bacteria and archaea were included in the investigation to have a comprehensive understanding of methane cycling microbes.In the intertidal mudflat sediments, most of the methanogenic genera including some unclassified members such as Zoige cluster (ZC-I) and anaerobic methanotrophic archaea (ANME) were detected. However, Methanosarcinales and Methanomicrobiales were the most dominant methanogens within the entire depth profile down to100cm. At each sampling point, the abundance of methanogens, as revealed from qPCR of the mcrA gene, were significantly increased with depth. Compared with sulfate-reducing microorganisms (SRM), the abundances of methanogens were found relatively high, suggesting the importance of methanogenesis in the sediments. Diversity indices showed that the diversity of methanogens were much lower in the deeper sediments. Thus, the higher abundances but lower diversities of methanogens in the deeper sediments might suggest the depth-wise enrichment of specific methanogen communities. Indeed, the proportions of Methanomicrobiales, the hydrogenotrophic methanogens that can easily be outcompeted by sulfate-reducing microbes, were relatively higher in the deeper sediments where sulfate levels were relatively low. The proportions of Methanosarcinales, methanogens with members that are nutritionally dynamic, were slightly favored in the near-surface sediments.The vegetated part of Dongtan (salt marsh) was dominated by two plant species; Phragmites australis and Spartina alterniflora. P. australis is recognized as native to the area whereas the latter is exotic that was intentionally introduced to increase land formation. Currently, P. australis is being aggressively replaced by S. alterniflora. We detected higher methane flux rates in the S. alterniflora than P. australis stands, which support the previous reports stating S. alterniflora invasion increases methane flux rates. This might be associated with substrate stimulation of methanogens as total carbon and nitrogen contents of sediments detected in the S. alterniflora stands were greater than P. australis. Consistent with this, the abundances of methanogens and SRM were greater in the S. alterniflora-impacted than P. australis stands, which is likely associated with the substrate stimulation of these microbes in the S. alterniflora vegetated soils. S. alterniflora invasion influenced the diversity of methanogens and SRM differently. The diversity of methanogens was much lower in the S. alterniflora than P. australis stands, depicting the selective pressure of S. alterniflora on methanogen communities. This is consistent with the increase in the proportion of Methanomicrobiales and the reduction of Methanococcales in the S. alterniflora stands. Unlike methanogens, S. alterniflora invasion did not show significant effect on the diversity of SRM.Methane flux is the balance between its production and oxidation. Hence, besides methane producing microbes, it is worthy to understand the identity and abundance of methane consuming microbial communities. Our pmoA-based survey of methanotrophs in the mudflat and salt marsh sediments showed that the distributions of aerobic methanotrophs between the mudflat and salt marsh sediments were variable. Using the conventional pmoA gene PCR primers (A189f/Mb661r), it was difficult to amplify methanotrophs from the mudflat sediments. By contrast, methanotrophs from salt marsh sediments were easily amplified. The inability to amplify aerobic methanotrophs in the mudflat sediments could be associated with the absence of vegetation cover which is believed to provide oxygen to sediments through its root systems. However, we detected large numbers of methane producing microbes which were consistent with net methane fluxes reported previously from this mudflat. Hence, the absence of methanotrophs in the oxic zones of the mudflat sediments is highly unlikely. Indeed, a highly degenerated PCR primer that was designed to amplify both ammonia monooxygenase (amoA) and methane monooxygenase (pmoA) genes revealed the presence of abundant Crenothrix related microbes. Crenothrix species are aerobic methanotrophs with unusual pmoA. These filamentous bacteria have been detected so far from aquatic environments; however, unexpectedly it was abundant in the mudflat sediments of Dongtan. As it was an interesting finding, further experiments are ongoing to unravel the clear identity and physiology of these bacteria. Once we knew that Crenothrix related methanotrophs were abundant in the mudflat and could not be amplified the usual pmoA primer set, we involved two pmoA primer sets; A189f/Mb661r (the usual pmoA primer) and A414f/Creno-616r(Crenothrix specific primer designed in this study) to amplify aerobic methanotrophs from the salt marsh sediments. Results indicated that Crenothrix-related pmoA genes are still present in the salt marsh, suggesting their wide spread distribution. Moreover, the abundance of Crenothrix and the inability to amplify the pmoA genes using A189f/Mb661r suggests the likely underestimation of methanotroph diversity and their role in mitigating atmospheric methane emission. Besides Crenothrix, both type-I and type-Ⅱ methanotrophs were detected in the salt marsh sediments, but Methylosarcina, Methylomonas and Methylocystis were the most dominant genera. S. alterniflora invasion favored the abundance of methanotrophs with almost insignificant influence on their diversity.In conclusion, the current study uncovered, for the first time, the identity and abundance of methanogenic and methanotrophic communities from the estuarine wetland sediments of Dongtan. Moreover, this study demonstrated the significant influences of the aggressively invading S. alterniflora on the structure of methane cycling microbes at this important wetland.