1. Microbial Acetogenesis Lindsay Rollick, Gerrit Voordouw
6/6/ :25 AM
Microbial Acetogenesis Lindsay Rollick, Gerrit Voordouw
Microbial Metabolism: What’s for Dinner?
Microbes tend to be grouped by lifestyle: Energy Metabolism
Methanogens make methane Acetogens make acetic acid
4H2 + CO2 → CH4 + 2H2O 4H2 + 2CO2 → CH3COOH + 2H2O
Lowest energy yield
Highest energy yield
Conventional
Oil field microbes
Unconventional
Anaerobes
Nitrate Reduction
NO3- N2
Manganese Reduction
Mn4+
Mn2+
Iron Reduction
Fe3+
Fe2+
Methanogenesis
CO2
CH4
Sulfate Reduction
SO42-
S2-
Acetogenesis
CH3COOH
Aerobes
Aerobic Respiration O2
CO2
Why do we care?
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!
© 2007 Microsoft Corporation. All rights reserved. Microsoft, Windows, Windows Vista and other product names are or may be registered trademarks and/or trademarks in the U.S. and/or other countries.
The information herein is for informational purposes only and represents the current view of Microsoft Corporation as of the date of this presentation. Because Microsoft must respond to changing market conditions, it should not be interpreted to be a commitment on the part of Microsoft, and Microsoft cannot guarantee the accuracy of any information provided after the date of this presentation. MICROSOFT MAKES NO WARRANTIES, EXPRESS, IMPLIED OR STATUTORY, AS TO THE INFORMATION IN THIS PRESENTATION.
Early Results
Model for Potential Acetogenesis Biotechnology
No Added CaCO3 or HCO3-
Introduction
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
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
Acetogenesis ΔG`0 = kj/mol1 (free energy)
Objectives
Observe competition between methanogenic archea and acetogenic bacteria under controlled conditions.
Find factors to optimize growth of acetogens over methanogens.
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
Experiment
Methane (mM)
% Change
Acetic Acid (mM)
Early Reg.
6.72
N/A
4.01
Early Low
0.259
2.19
CaCO3 Reg.
11.01
+39.0
8.63
+53.5
CaCO3 Low
4.62
+94.4
2.49
+12.0
Subculture Reg.
12.34
+10.8
-74.6
Subculture Low
-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
Figure 1. A serum bottle experiment containing added solid CaCO3. All experiments are done in triplicate. Incubation is done at 300C.
Microbes: complex sample from Medicine Hat oil field subsurface waters
Anaerobic Minimal salts medium:
No O2, 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%H2/20%CO2 Headspace Consumed gas replenished A B
Doubling Nitrogen
Regular Nutrients:
↑ Acetic acid (+39%)
↑ Methane (+18.2%)
Low Nutrients:
↓ Acetic acid (+49%)
↓ Methane (-83.8%)
(relative to low nutrients) 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 acetogenesis and decreased methanogenesis.
Acetogenesis is 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.
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 production was tracked with liquid chromatography (HPLC), pH with a pH meter 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 Ecological consequences of the phylogenetic and physiological diversities of acetogens. Antonie van Leeuwen hoek. 81:
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 Energy conservation in acetogenic bacteria. Applied Environmental Microbiology. 69: 6345–6353.
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.