Butanol Production From Clostridia 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.. The acetic

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 (~107)

• Limited list of compounds capable of detecting

•Non-selective

• Destructive

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

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.

http://www.chem.agilent.com/en-US/products/columns-supplies/gc-gc-mscolumns/jwhp-innowax/Pages/default.aspx

(accessed Nov 10, 2011)

production using suspended and immobilized Clostridium beijerinckii NCIMB 8052 with supplementary butyrate Energy & Fuels, 22(5), 3459-3464.

Chromatography or Gas-Liquid Chromatography Current Protocols in Molecular Biology,

[http://www.currentprotocols.com/protocol/mb1719a]

Weinheim, Germany, 2003.

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/fid.htm (accessed Dec 6,2011)