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 CaCO3 or HCO3

-

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

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 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 CaCO3 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 CaCO3 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 O2

CO2

Nitrate Reduction

NO3-

N2

Manganese Reduction

Mn4+

Mn2+

Iron Reduction

Fe3+

Fe2+

Methanogenesis

CO2

CH4

Sulfate Reduction

SO42-

S2-

Acetogenesis

CO2

CH3COOH

Highest energy yield Lowest energy yield Microbes tend to be grouped by lifestyle: Energy Metabolism Methanogens make methane Acetogens make acetic acid 4H2 + CO2 → CH4 + 2H2O 4H2 + 2CO2 → CH3COOH + 2H2O

Aer

obes

Ana

erob

es

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 = -104.6 kj/mol1 (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 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

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 CO2 = 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 CO2!

Figure 1. A serum bottle experiment containing added solid CaCO3. All experiments are done in triplicate. Incubation is done at 300C.

Methane Acetic Acid 0

5

10

15

Low Regular Mea

n C

once

ntra

tion

(mM

)

Medium Nutrient Type

5 6 7 8

Low Regular

Mae

n 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 62.

Added Solid CaCO3

Methane Acetic Acid 0

5

10

15

Low Regular

Mea

n C

once

ntra

tion

(mM

)

Medium Nutrient Type

Methane Acetic Acid 0

5

10

15

Low Regular M

ean

Con

cent

ratio

n (m

M)

Medium Nutrient Type

A

B

6

6.5

7

7.5

8

Low Regular

Mea

n 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 CaCO3

- 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

Mea

n C

once

ntra

tion

(mM

)

Medium Nutrient Type

2X Nitrogen

A

Methane Acetic Acid 0

5 10 15 20 25

No TM 2XTM

Mea

n C

once

ntra

tion

(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