1. FELC Discussion Group November 12, 2013 Effects of Short Chain Fatty Acids on Yeast Metabolism Moderators: Courtney Ireland (Corn Plus) & Asfia Qureshi Ph.D. (Verenium)

2. FELC Discussion Group Nov 2013 Discussion Norms Please speak kindly and professionally to each other Everyone is welcome to offer ideas and suggestions to the topic Do not belittle each other Learn from each other

3. Short Chain Fatty Acids are C1-C5 What is a short chain fatty acid (SCFA)? Usually C1-C5 Includes formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid Have individual pKa values to indicate what proportion is in the dissociated (COO - ) and undissociated (COOH) form at any pH.

4. Short Chain Fatty Acids Have Specific pKas In alkaline media (pH >7) COO - form will be prominent SCFApKa Formic acid3.77 Acetic acid4.76 Propionic acid4.87 Isobutyric acid4.86 Butyric acid4.82 Isovaleric acid4.98 Valeric acid4.82

5. Short Chain Fatty Acids Interfere in Yeast Metabolism Why are SCFA relevant? Seminal paper from 60 years ago describes the effects of acetate and other short chain fatty acids on yeast metabolism During early phases of glucose fermentation, phosphate is taken up by starved, mature yeast cells. Phosphate accumulation (and thus yeast growth and fermentation) is inhibited by C2, C4, C8 (concentration and pH dependent)

6. Effects of Short Chain Fatty Acids on Yeast Metabolism Short chain fatty acids influence the fermentative activity of yeast cells High concentrations inhibit Low concentrations enhance Increasing chain length of fatty acid increases inhibitory action What are the actions of fatty acids? Bactericidal activity Inhibit growth rate “Glycogen sparring” action in yeast cells

7. A pH Increase Can Circumvent Their Effects How do they work? By binding to enzymes and other proteins in the cell membrane How to overcome this? If SCFA are coming out of methanator, increase the pH at prop and ferm – this will shift FA to their dissociated forms which cannot enter yeast cell to cause inhibition Wash the cells/mash (hexane/ether extraction will remove FA – then analyze by GC)

8. SCFAs are Connected to Fusel Oil Inhibition SCFA inhibition is connected to fusel inhibition in yeasts Fusel oils are By-products of ethanol production process mix of volatile organic acids, higher alcohols, aldehydes, ketones, fatty acids, esters toxic to yeast growth and fermentation usually removed using a rectifier distillation column

9. FELC Discussion Group Nov 2013 Thank you for a great discussion!