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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
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
Gas Chromatography
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
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.
2.
3.
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%
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.