Butanol Production

from Clostridia
Fermentation
David L. Hanson
Chem 4101
December 9, 2011

Background Information
-For over 40 years, the worlds butanol supply has been
produced industrially via microbial fermentation. Butanol is
produced as a fermentation product by bacteria; known as,
solventogenic Clostridia, when cultured on glucose-rich
media containing acetic acid and butyric acid.
-Recently, butanol has been gaining attention as a possible
alternative to petroleum-based gasoline. Much effort is
currently being made to reduce production costs to make
butanol an economically viable option.

Acetic Acid
MW: 60.05
bp: 117-118

Butyric
Acid
MW: 88.11
bp: 162 C

Butanol
MW: 74.12
bp: 116 118

Problem: Currently, butanol production is a

discontinuous process. The acetic acid and
butyric acid are added at the start of
fermentation. Butanol is produced until the
supply of carboxylic acids have been
exhausted. The carboxylic acids are then
reintroduced to restart the process. This
discontinuity adds to the high cost of
production.
Hypothesis: By determining the individual
rates at which the carboxylic acids are
utilized by the Clostridia, and continuously
adding the acids at these determined rates
butanol production can be made to be a
continuous process.

Studies Needed to Test

Hypothesis
1. Determine the specific ratio of acetic
acid/butyric acid that will yield the highest
concentration of butanol produced.
2. During fermentation take samples of the
media at different time points and measure
the concentration of acetic and butyric acid
present to determine the rates at which they
are utilized
3. Continuously pump acetic/butyric acid into
media at determined rate and measure
butanol concentration over time to determine
if production is stable and continuous.

Requirements for Analytical

Method
1) Must be able to separate multiple analytes.
- High Selectivity
- High Resolution
2) Must be able to detect small changes in
amounts of analyte.
- High Sensitivity
3) Must be able to detect broad ranges in analyte
amount.
- Large Linear Response Range
4) Must be able to quantitate reproducibly.
- High Precision
-High Accuracy

Possible Separation
Techniques
Method Type
Advantages
Disadvantages
Reverse Phase HPLC

Limited sample
processing required.
Separate wide
variety of
compounds.
Existing
technology/expertise.

Only separation
method is retention.
May require
complicated mobile
phase buffers.
To improve
resolution analysis
time must be
increased (minutes).

Capillary Zone
Electrophoresis

Very fast analysis

times (seconds).

Analytes will need to

be modified to carry
charge.
Developing
technology/expertise.

Gas Chromatography

High selectivity due

to separate by boiling
point and retention.
Analysis time fast
(seconds to minutes) .

Require processing
of sample to remove
non-volatilizable
matrix components

Possible Detection
Techniques
Method Type

Advantages

Disadvantages

UV-Vis Absorbance

Ease of use.
Non-destructive.

Need
instrumentation
capable of measuring
multiple wavelengths.
Difficulty detecting
low concentrations.
Affected by flow rate.

Mass Spectrometry

Unlimited list of
compounds capable
of detecting.

Quantitation
difficult, requires
radio-labeled
isotopes.
Destructive.

Flame Ionization

Very good for

hydrocarbon
detection.
Ease of use.
High

Limited list of
compounds capable
of detecting.
Non-selective.
Destructive.

GC-FID
Precision and Accuracy: FID is
mass sensitive and not affected
by changes in flow rate. Giving
good reproducibility.

Diagram of GC. Courtesy: Manzi, A.

Selectivity: GC increases by separating

based on retention and boiling point
Resolution: Can be controlled in GC
controlling the oven temperature.

Diagram of FID. Courtesy: University of

Experiment Sample Prep

1.

2.

3.

Filtration: Solids and cellular

debris will be removed by
using a vacuum filter with a
(0.2m pore size) SFCA
membrane (Cat No. 161-0020,
NALGENE Lab ware).
Evaporation: Glucose and
residual salts will be removed
from filtered solution by
evaporation/condensation
using a rotary evaporator (Cat
No. 8024701, IKA). Heating
temperature ~165C.
Storage: Solution collected in
the condensation vessel will
be transferred to 50mL
centrifuge tubes (Cat No.
89039-656, VWR) and stored
at room temperature until
analysis.

Experiment GC Method
Method Parameters
Injection Temperature: 212 C
Oven Temperature Gradient: 110-175 C
Carrier Gas: Helium
Column Parameters
Type: HP-INNOwax column (Agilent Technologies Part#29091N133LTM)
Stationary Phase: bonded polyethylene glycol (high polarity)
Particle Size: 0.25m
Length: 30m
Diameter: 0.25mm
Stability: >1800C
Controls
Acetic Acid: Sigma-Aldrich Cat# 320099 - ACS reagent, 99.7%
Butyric Acid: Sigma Aldrich Cat# B103500 - 99%
Butanol: Sigma Aldrich Cat# 360465 - ACS reagent, 99.4%

Results and Conclusion

Results
Predicted Elution Order
1: Acetic Acid
2: Butanol
3: Butyric Acid
Conclusion
The use of controls to build a standard curve for each analyte
allows for quantitation. By using a GC-FID method each analyte
may be separated and quantified from various sampling times.
Plotting the concentration of each analyte versus the sampling
times allows to calculate the individual rates that acetic and
butyric acid are consumed. Additionally, method can be used to
monitor butanol production.
Future Work
Experiment with other techniques (CE, HPLC) to determine if a
method can be developed that does not require extensive
sample prep.

References
Agilent Technology. HP-INNOwax Columns. http://www.chem.agilent.com/enUS/products/columns-supplies/gc-gc-mscolumns/jwhpinnowax/Pages/default.aspx (accessed Nov 10, 2011)
2.
IKA Technology. http://www.ika.com/ (accessed Nov 3, 2011)
3.
Lee, S., Cho, M., Park, C., Chung, Y., Kim, J., Sang, B., et al. (2008). Continuous
butanol production using suspended and immobilized Clostridium beijerinckii
NCIMB 8052 with supplementary butyrate. Energy & Fuels, 22(5), 3459-3464.
4.
Manzi, A. (2001). Total Compositional Analysis by HighPerformance Liquid
Chromatography or GasLiquid Chromatography. Current Protocols in
Molecular Biology, [http://www.currentprotocols.com/protocol/mb1719a]
5.
NALGENE Labware. http://www.nalgenelabware.com/ (accessed Nov 3, 2011)
6.
Schmid, R.D., Pocket Guide to Biotechnology and Genetic Engineering; WileyVCH: Weinheim, Germany, 2003.
7.
Sigma-Aldrich. http://www.sigmaaldrich.com/united-states.html (accessed Nov
3, 2011)
8.
Skoog , D, et al. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole:
Belmont, CA, 2007.
9.
University of Adelaide: Department of Chemistry. Flame Ionization Detector
(FID). http://www.chemistry.adelaide.edu.au/external/soc-rel/content/fid.htm
(accessed Dec 6,2011)
10. VWR. https://www.vwrsp.com/index.cgi (accessed Nov 3, 2011)
1.