Microbial Acetogenesis Lindsay Rollick, Gerrit Voordouw

1. O2 Fe3+ CO2 Microbial AcetogenesisLindsay Rollick, GerritVoordouw CO2 SO42- Mn4+ NO3- Nitrate Reduction Methanogenesis Iron Reduction Acetogenesis Sulfate Reduction Manganese Reduction Aerobic Respiration Fe2+ S2- CH3COOH Mn2+ CH4 CO2 N2 Microbial Metabolism: What’s for Dinner? Microbes tend to be grouped by lifestyle: Energy Metabolism Methanogens make methane Acetogensmake acetic acid 4H2+ CO2→ CH4 + 2H2O 4H2+ 2CO2 → CH3COOH + 2H2O Lowest energy yield Highest energy yield Oil field microbes Unconventional Conventional Anaerobes Aerobes Why do we care? Early Results Model for Potential Acetogenesis Biotechnology • Acetogenesis consumes 2 CO2 = carbon storage • Acetogenesis could be a useful biotechnology in unconventional oil fields • To understand how to control methanogenesis. Methane is a worse greenhouse gas than CO2! No Added CaCO3 or HCO3- Introduction Acetogens and methanogens live at the lowest energy levels and compete for H2 and CO2. Who wins? Thermodynamics: Methanogens Methanogenesis (Hydrogenotrophic) ΔG`0 = -135 kj/mol1 AcetogenesisΔG`0 = -104.6 kj/mol1 (free energy) B Figure 2. A) Mean acetic acid and mean methane in millimolar for low and regular nutrient medium. B) Mean start and final pH for low and regular nutrient medium. All bottles were performed in triplicate. No bicarbonate or carbonate mineral was added. A But: Over 200 species of acetogens have been identified 1 - Some grow in anti-methanogenic conditions or have higher substrate diversity How do they compete under methanogenic conditions? No added bicarbonate led to poor pH buffering of the solution which inhibited microbial growth. Acetogens and methanogens are acid-intolerant below pH 62. Current Results Table 1. Summary of results for experiments. Methane and acetic acid are averages of 3 replicates and % change is calculated based on comparable control. Promising cultures shown are high-lighted in yellow. Added Solid CaCO3 Nutrient Optimization Methods Objectives Microbes: complex sample from Medicine Hat oil field subsurface waters Anaerobic Minimal salts medium: No O2, or other electron acceptors: only acetogensand methanogens can grow = methanogenic conditions Compare with regular version with a low nutrient version: No added nitrogen, phosphate, trace metals or tungstate-selenite Excess 80%H2/20%CO2HeadspaceConsumed gas replenished Doubling Nitrogen Regular Nutrients: ↑ Acetic acid (+39%) ↑ Methane (+18.2%) Low Nutrients: ↓ Acetic acid (+49%) ↓ Methane (-83.8%) (relative to low nutrients) • Observe competition between methanogenicarchea and acetogenic bacteria under controlled conditions. • Find factors to optimize growth of acetogens over methanogens. A C Adding CaCO3 - buffered pH - ↑ biofilm growth Regular Nutrients ↑ Methane (+39%) ↑ Acetic acid (+53.5%) Subculture Low Nutrients ↓ Methane (-85.5%) ↑ Acetic acid (+60.8%) A Trace Metals Removing: ↓ Acetic acid (-38%) ≈Methane (+5.9%) Conclusions • Nutrient levels other than energy substrate can influence the balance between acetogenesis and methanogenesis. • Low nutrients in subculture and adding CaCO3 promoted acetogenesisand decreased methanogenesis. • Acetogenesisis promoted by greater nitrogen and trace metal availability. • Microbial growth can occur in the presence of CaCO3 which can act as a pH buffer for acid-intolerant microbes. Figure 1. A serum bottle experiment containing added solid CaCO3. All experiments are done in triplicate. Incubation is done at 300C. Doubling: ↑ Acetic acid (+29%) ≈ Methane (-3.5%) Subculture - account for inoculum nutrients and transport shock Analysis - Methane production was tracked with gas chromatography (GC-FID), acetic acid productionwas tracked with liquid chromatography (HPLC), pH with a pH meter B B Figure 3. Mean acetic acid and mean methane in millimolar of A) primary culture and B) of subculture. C) Mean start and final pH for low and regular nutrient medium. All bottles were performed in triplicate. Change is measured against controls. Figure 4. Mean acetic acid and mean methane in millimolar of A) medium with doubled nitrogen, B) doubled nitrogen and with no tracemetals and doubled trace metals. Changes are measured against controls. Varying phosphate and salts had no discerning difference (not shown). References Acknowledgements • Drake, H.L., Kusel, K. and Matthies, C. 2002. Ecological consequences of the phylogenetic and physiological diversities of acetogens. Antonie van Leeuwenhoek. 81:203-213. • Nathoo, S., Folarin, Y., and Voordouw, G. (2012). Potential of microbial formation of acetic acid from hydrogen and carbon dioxide for permeability modification in carbonate reservoirs. World Heavy Oil Congress. Aberdeen, UK, Paper WHOC-12 • Müller, V. 2003. Energy conservation in acetogenic bacteria. Applied Environmental Microbiology.69: 6345–6353. I’d like to thank my supervisor Dr. GerritVoordouw for giving me this project and all of the lab members of the Voordouw and Gieg lab for their help and support. I thank the University of Calgary, the Natural Science Research Council of Canada and Suncor Ltd. for financial support and Baker Hughes for providing the water samples used forsource microbes in this research.