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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. 1. Drake, H.L., Kusel, K. and Matthies, C. 2002. Ecological consequences of the phylogenetic and physiological diversities of acetogens. Antonie van Leeuwen hoek. 81:203-213. 2. 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 3. Müller, V. 2003. Energy conservation in acetogenic bacteria. Applied Environmental Microbiology. 69: 6345–6353. Early Results No Added CaCO 3 or HCO 3 - Current Results Experiment Methane (mM) % Change Acetic Acid (mM) % Change Early Reg. 6.72 N/A 4.01 N/A Early Low 0.259 N/A 2.19 N/A CaCO 3 Reg. 11.01 +39.0 8.63 +53.5 CaCO 3 Low 4.62 +94.4 2.49 +12.0 Subculture Reg. 12.34 +10.8 2.19 -74.6 Subculture Low 2.49 -85.5 6.35 +60.8 2XN Reg. 13.47 +18.2 15.67 +44.9 2XN Low 0.748 -83.8 4.84 +48.6 No TM 14.32 +5.9 11.33 -38.3 2XTM 13.01 -3.5 22.08 +29.0 1) Nutrient levels other than energy substrate can influence the balance between acetogenesis and methanogenesis. 2) Low nutrients in subculture and adding CaCO 3 promoted acetogenesis and decreased methanogenesis. 3) Acetogenesis is promoted by greater nitrogen and trace metal availability. 4) Microbial growth can occur in the presence of CaCO 3 which can act as a pH buffer for acid- intolerant microbes. Microbial Acetogenesis Lindsay Rollick, Gerrit Voordouw Microbial Metabolism: What’s for Dinner? Introduction Aerobic Respiration O 2 CO 2 Nitrate Reduction NO 3 - N 2 Manganese Reduction Mn 4+ Mn 2+ Iron Reduction Fe 3+ Fe 2+ Methanogenesis CO 2 CH 4 Sulfate Reduction SO 4 2- S 2- Acetogenesis CO 2 CH 3 COOH Highest energy yield Lowest energy yield Microbes tend to be grouped by lifestyle: Energy Metabolism Methanogens make methane Acetogens make acetic acid 4H 2 + CO 2 → CH 4 + 2H 2 O 4H 2 + 2CO 2 → CH 3 COOH + 2H 2 O Aerobes Anaerobes Acetogens and methanogens live at the lowest energy levels and compete for H 2 and CO 2. Who wins? Thermodynamics: Methanogens Methanogenesis (Hydrogenotrophic) ΔG` 0 = -135 kj/mol 1 Acetogenesis ΔG` 0 = -104.6 kj/mol 1 (free energy) 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? Objectives 1) Observe competition between methanogenic archea and acetogenic bacteria under controlled conditions. 2) Find factors to optimize growth of acetogens over methanogens. Methods Microbes: complex sample from Medicine Hat oil field subsurface waters Anaerobic Minimal salts medium: No O 2 , or other electron acceptors: only acetogens and 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%H 2 /20%CO 2 Headspace Consumed gas replenished Subculture - account for inoculum nutrients and transport shock Analysis - Methane production was tracked with gas chromatography (GC-FID), acetic acid production was tracked with liquid chromatography (HPLC), pH with a pH meter Why do we care? 1) Acetogenesis consumes 2 CO 2 = carbon storage 2) Acetogenesis could be a useful biotechnology in unconventional oil fields 3) To understand how to control methanogenesis. Methane is a worse greenhouse gas than CO 2 ! Figure 1. A serum bottle experiment containing added solid CaCO 3 . All experiments are done in triplicate. Incubation is done at 30 0 C. Methane Acetic Acid 0 5 10 15 Low Regular Mean Concentration (mM) Medium Nutrient Type 5 6 7 8 Low Regular Maen pH Nutrient Type Start pH Final pH 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 B No added bicarbonate led to poor pH buffering of the solution which inhibited microbial growth. Acetogens and methanogens are acid-intolerant below pH 6 2 . Added Solid CaCO 3 Methane Acetic Acid 0 5 10 15 Low Regular Mean Concentration (mM) Medium Nutrient Type Methane Acetic Acid 0 5 10 15 Low Regular Mean Concentration (mM) Medium Nutrient Type A B 6 6.5 7 7.5 8 Low Regular Mean pH Nutrient Type Start pH Final pH C 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. Adding CaCO 3 - buffered pH - ↑ biofilm growth Regular Nutrients ↑ Methane (+39%) ↑ Acetic acid (+53.5%) Subculture Low Nutrients ↓ Methane (-85.5%) ↑ Acetic acid (+60.8%) Nutrient Optimization Methane Acetic Acid 0 5 10 15 20 Low Regular Mean Concentration (mM) Medium Nutrient Type 2X Nitrogen A Methane Acetic Acid 0 5 10 15 20 25 No TM 2XTM Mean Concentration (mM) Medium Nutrient Type Variations in Trace Metals B Doubling Nitrogen Regular Nutrients: ↑ Acetic acid (+39%) ↑ Methane (+18.2%) Low Nutrients: ↓ Acetic acid (+49%) ↓ Methane (-83.8%) (relative to low nutrients) Trace Metals Removing: ↓ Acetic acid (-38%) ≈ Methane (+5.9%) Doubling: ↑ Acetic acid (+29%) ≈ Methane (-3.5%) Varying phosphate and salts had no discerning difference (not shown). Conclusions 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. References Acknowledgements I’d like to thank my supervisor Dr. Gerrit Voordouw 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 for source microbes in this research. Oil field microbes Conventional Unconventional Model for Potential Acetogenesis Biotechnology
