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Linking Methanogen Community Structure to Methane Production Rates in Peatlands
Maria Bañuelos University of Redlands University of Oregon NSF REU Site Program Mentor: Steve McAllister P.I.: Brendan Bohannan
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Methane potent greenhouse gas
emitted from variety of natural and human influenced sources Holds ~25x more infrared radiation than carbon dioxide per unit weight important energy source potent greenhouse gas methane more powerfull greenhouse gas than carbon dioxide -better at trapping heat -but also important energy source because its a primary constituent of natural gas -because its a source of energy, projects to control or utilize methane emissions can be economically and environmentally beneficial Figure courtesy of wiki.nanjing-school.com
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What are Methanogens? group of Archaea with a distinct and unique function produce methane Two distinct biochemical pathways phylogenetically distributed within the methanogens hydrogenotrophic methanogenesis -CO2 + H2 acetoclastic methanogenesis - CH3COOH
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Methanogenesis pathways are taxonomically distinct
14CO2 + 4 H2 → 14CH4 + 2H2O CH3COOH → CH4 + CO2 Euryarchaeota 5 groups of methanogens and 4 of them are hydrogenotrophic yet acetoclastic is globally prevalent pathway Courtesy of Steve McAllister
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Core Question Does methanogen community composition affect methane production rates in peatland soils? Hypothesis: apart from environmental factors, methanogen community structure also has an effect on methane production rates
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Sites Northern Peatlands in Michigan Hold ~33% of terrestrial carbon
Significant methane flux Hold a variety of communities and functional characteristics
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ombrotrophic minerotrophic
Northern peatlands: a variety of communities and functional characteristics low neutral pH precipitation groundwater hydrology hydrogenotrophic acetoclastic methane pathway ombrotrophic minerotrophic Courtesy of Steve McAllister
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Sites Bog Poor Fen Intermediate Fen Cedar Swamp Rich Fen ombrotrophic
PF IF CS RF Bog Poor Fen Intermediate Fen Cedar Swamp Rich Fen minerotrophic courtesy of Steve McAllister
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Experimental Strategy
B1 B2 PF IF CS RF Quantifying methanogenesis pathways Community Structure Analysis: extract and sequence methanogen DNA from all sites extract and sequence methanogen mRNA from all sites courtesy of Steve McAllister
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Sampling CH3COOH → CH4 + CO2 14CO2 + 4 H2 → 14CH4 +2H2O
5 events (two in 2009, three in 2010) 5 replicate samples from each site Quantifying Methanogenesis Pathways: B1 B2 PF IF CS RF 14CO2 + 4 H2 → 14CH4 +2H2O CH3COOH → CH4 + CO2 Samples incubated with 14C-labeled bicarbonate tracer Addition of radioactive substrate allows quantification of each pathway courtesy of Steve McAllister
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Methanogenesis Pathways Summary
acetoclastic hydrogenotrophic Methane Produced (umol/d/g dry soil) Acetoclastic methanogenesis varied with gradient position Hydrogenotrophic methanogenesis varied greatly between years Acetoclastic methanogenesis varied with gradient position but not with years or growing season huge variation for hydrogenotrophic pathway between 2009 and 2010, becoming dominant for almost every site in (2010 unusually wet)
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mcrA as a Molecular Marker
functional gene unique and ubiquitous to methanogens codes for enzyme that catalyzes the final step in methanogenesis methyl coenzyme- M reductase Figure courtesy of Ermler et al. 1997 only one copy per genome
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DNA Extractions for each site we extracted DNA from three of the five cores To verify DNA extraction was successful we ran a PCR reaction in an attempt to amplify mcrA mcrA ~ 450 bp in order to sequence the DNA we need to extract it first and this is what i've been working on this summer Ive been preparing .troubleshooting -concentrating DNA -DNA concentration -MgCl 450 bp CS3 PF1 PF2 PF3 PF1 PF2 PF3
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Results 78 of 90 samples successfully produced and mcrA amplicon
12 of the samples were "problematic" -bog samples in order to sequence the DNA we need to extract it first and this is what i've been working on this summer Ive been preparing .troubleshooting -concentrating DNA -DNA concentration -MgCl 450 bp CS3 PF1 PF2 PF3 PF1 PF2 PF3
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Future Directions: Describing a Community
describe the overall community composition of methanogens in each of our sites describe the active methanogen community composition in each of our sites high throughput sequencing DNA Primer F Primer R High throughput pyro sequnecing mRNA cDNA reverse transcriptase mRNA
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Acknowledgments Steve McAllister Brendan Bohannan Scott Bridgham
Qusheng Jin Bohannan Lab Bridgham Lab Peter O'Day Adam Unger NSF University of Oregon
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