Butanol Production From Clostridia Fermentation

Butanol Production from
Clostridia Fermentation
David L. Hanson
Chem 4101
December 9, 2011
Background Information
Acetic Acid
MW: 60.05
bp: 117-118
°
C
Butyric Acid
MW: 88.11
bp: 162
°
C
Butanol
MW: 74.12
bp: 116 – 118
°
C
-
For over 40 years, the world’s 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.
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) .
• Existing
technology/expertise
•
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 sensitivity(pg/sec).
•
Large linear response
range (~10
7
)
•
Limited list of compounds

capable of detecting.
•Non-selective.
•
Destructive.
GC-FID
Diagram of GC. Courtesy: Manzi, A.
Diagram of FID. Courtesy: University of Adelaide
Selectivity: GC increases by separating based on
retention and boiling point
Resolution: Can be controlled in GC controlling the
oven temperature.
Precision and Accuracy: FID is mass
sensitive and not affected by changes in
flow rate. Giving good reproducibility.
Experiment – Sample Prep
1. Filtration: Solids and cellular
debris will be removed by using a
vacuum filter with a (0.2μm pore
size) SFCA membrane (Cat No. 161-
0020, NALGENE Lab ware).
2. Evaporation: Glucose and residual
salts will be removed from filtered
solution by
evaporation/condensation using a
rotary evaporator (Cat No.
8024701, IKA). Heating
temperature ~165°C.
3. 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#29091N-133LTM)
Stationary Phase: bonded polyethylene glycol (high polarity)
Particle Size: 0.25µm
Length: 30m
Diameter: 0.25mm
Stability: >1800°C
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
1. Agilent Technology. HP-INNOwax Columns. />US/products/columns-supplies/gc-gc-mscolumns/jwhp-innowax/Pages/default.aspx
(accessed Nov 10, 2011)
2. IKA Technology. (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 High-Performance Liquid
Chromatography or Gas-Liquid Chromatography. Current Protocols in Molecular Biology,
[ />5. NALGENE Labware. (accessed Nov 3, 2011)
6. Schmid, R.D., Pocket Guide to Biotechnology and Genetic Engineering; Wiley-VCH:
Weinheim, Germany, 2003.
7. Sigma-Aldrich. (accessed Nov 3, 2011)
8. Skoog , D, et al. Principles of Instrumental Analysis, 6
th
ed.; Brooks/Cole: Belmont, CA, 2007.
9. University of Adelaide: Department of Chemistry. Flame Ionization Detector (FID).
(accessed Dec
6,2011)
10. VWR. (accessed Nov 3, 2011)