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

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 www.what-is-global-warming.com 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

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

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

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

Sites Northern Peatlands in Michigan Hold ~33% of terrestrial carbon Significant methane flux Hold a variety of communities and functional characteristics

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

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

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

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

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 - (2010 unusually wet)

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

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

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

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

Acknowledgments Steve McAllister Brendan Bohannan Scott Bridgham Qusheng Jin Bohannan Lab Bridgham Lab Peter O'Day Adam Unger NSF University of Oregon