Butanol Production from Clostridia Fermentation

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

2. Background Information • 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. Butyric Acid MW: 88.11 bp: 162 °C Acetic Acid MW: 60.05 bp: 117-118 °C Butanol MW: 74.12 bp: 116 – 118 °C

3. 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.

4. 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.

5. Requirements for Analytical Method • 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

6. Possible Separation Techniques

7. Possible Detection Techniques

8. 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 Adelaide

9. Experiment – Sample Prep • 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). • 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. • 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.

10. 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%

11. 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.

12. References • Agilent Technology. HP-INNOwax Columns. http://www.chem.agilent.com/en-US/products/columns-supplies/gc-gc-mscolumns/jwhp-innowax/Pages/default.aspx (accessed Nov 10, 2011) • IKA Technology. http://www.ika.com/ (accessed Nov 3, 2011) • 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. • Manzi, A. (2001). Total Compositional Analysis by High‐Performance Liquid Chromatography or Gas‐Liquid Chromatography. Current Protocols in Molecular Biology, [http://www.currentprotocols.com/protocol/mb1719a] • NALGENE Labware. http://www.nalgenelabware.com/ (accessed Nov 3, 2011) • Schmid, R.D., Pocket Guide to Biotechnology and Genetic Engineering; Wiley-VCH: Weinheim, Germany, 2003. • Sigma-Aldrich. http://www.sigmaaldrich.com/united-states.html (accessed Nov 3, 2011) • Skoog , D, et al. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole: Belmont, CA, 2007. • 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) • VWR. https://www.vwrsp.com/index.cgi (accessed Nov 3, 2011)