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 ResultsNo Added CaCO3 or HCO3

-

Current ResultsExperiment Methan

e (mM)%

ChangeAcetic Acid (mM)

% Change

Early Reg. 6.72 N/A 4.01 N/AEarly Low 0.259 N/A 2.19 N/A

CaCO3 Reg. 11.01 +39.0 8.63 +53.5CaCO3 Low 4.62 +94.4 2.49 +12.0Subculture

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 +44.92XN Low 0.748 -83.8 4.84 +48.6No TM 14.32 +5.9 11.33 -38.32XTM 13.01 -3.5 22.08 +29.01) 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 AcetogenesisLindsay Rollick, Gerrit Voordouw

Microbial Metabolism: What’s for Dinner?

Introduction

Aerobic RespirationO2

CO2

Nitrate ReductionNO3

-

N2

Manganese ReductionMn4+

Mn2+

Iron ReductionFe3+

Fe2+

MethanogenesisCO2

CH4

Sulfate ReductionSO4

2-

S2-

AcetogenesisCO2

CH3COOH

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

robe

s

Anae

rob

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?

Objectives1) Observe competition between methanogenic

archea and acetogenic bacteria under controlled conditions.

2) Find factors to optimize growth of acetogens over methanogens.

MethodsMicrobes: complex sample from Medicine Hat oil field subsurface watersAnaerobic Minimal salts medium:No O2, or other electron acceptors: only acetogens and methanogens can grow = methanogenic conditionsCompare with regular version with a low nutrient version:No added nitrogen, phosphate, trace metals or tungstate-seleniteExcess 80%H2/20%CO2 Headspace Consumed gas replenished

Subculture - account for inoculum nutrients and transport shockAnalysis - 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

storage2) Acetogenesis could be a useful biotechnology in

unconventional oil fields3) 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.

0

5

10

15

Methane

Medium Nutrient Type

Mea

n C

once

ntra

tion

(mM

)

Low Regular BESA5

6

7

8 Start pH Final pH

Nutrient Type

Mae

n 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

Low Regular BESA

0

5

10

15

MethaneAcetic Acid

Medium Nutrient Type

Mea

n C

once

ntra

tion

(mM

)

Low Regular BESA

0

5

10

15

MethaneAcetic Acid

Medium Nutrient Type

Mea

n C

once

ntra

tion

(mM

)

A

B

6

6.5

7

7.5

8 Start pHFinal pH

Nutrient Type

Mea

n 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 growthRegular Nutrients↑ Methane (+39%) ↑ Acetic acid (+53.5%)Subculture Low Nutrients↓ Methane (-85.5%)↑ Acetic acid (+60.8%)

Nutrient Optimization

Low Regular

0

5

10

15

20

MethaneAcetic Acid

2X Nitrogen

Medium Nutrient Type

Mea

n C

once

ntra

tion

(mM

)

A

No TM 2XTM

05

10152025

MethaneAcetic Acid

Variations in Trace Metals

Medium Nutrient Type

Mea

n C

once

ntra

tion

(mM

)

B

Doubling Nitrogen Regular Nutrients: ↑ Acetic acid (+39%) ↑ Methane (+18.2%)Low Nutrients: ↓ Acetic acid (+49%) ↓ Methane (-83.8%)(relative to low nutrients)

Trace MetalsRemoving:↓ 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 AcknowledgementsI’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 microbesConventional

Unconventional

Model for Potential

Acetogenesis

Biotechnology