Fusel Alcohols in Fuel Ethanol Fermentation Matt Hawkins

Fusel Alcohols in Fuel Ethanol Fermentation Matt Hawkins Lallemand Biofuels & Distilled Spirits

Overview • Fusel Alcohols – Structures and Formation • Fusels in Corn Mash Fermentation – Fusels Produced in Fermentation – Factors Influencing Fusel Formation • Fusels in Liquefaction – Measurement and Impact • Dealing with Fusel Alcohols Title Area

Fusel Alcohol Structures Tert-amyl Alcohol Isopropanol Tert-Butyl Alcohol Iso-amyl Alcohol 1 -Propanol 3, 2, or 1 -Pentanol Isobutanol Title Area

General Fermentation Title Area

Fusel Formation Hazelwood et al, 2008 Appl. Envir. Title Area Micro.

Measurement of Fusels - HPLC Isopropanol 1 -Propanol Tert-amyl Tert-Butyl Title Area Isobutanol 2 -Pentanol 3 -Pentanol 1 -Pentanol Isoamyl

Fusels in Corn Fermentation Title Area Hirose et al. 1962 Agr. Biol. Chem.

Fusels Produced in Fermentation (plant) Title Area Isopropanol tert-butyl Average 0 0 Min 0 0 Max 0 0 1 -propanol 36. 78 30. 73 43. 15 Isobutanol tert-amyl 49. 13 0 39. 07 0 54. 33 0 3 -pentanol 0 0 0 2 -pentanol Isoamyl 0 402. 23 0 300. 80 0 479. 95 1 -pentanol Total 0 488. 14 0 388. 53 0 570. 95

Fusel Production in Fermentation (plant) Fusel Production During Fermentation Fusel Concentration (ppm) 250 200 Tert-Butyl 1 -propanol Isobutanol Isoamyl 150 100 50 0 Liq 10 Hr 18 Hr 25 Hr 32 Hr 39 Hr 45 Hr 50 Hr Drop BW Title Area

Fusels Produced in Fermentation (lab) Isopropanol tert-butyl 1 -propanol Isobutanol tert-amyl 3 -pentanol 2 -pentanol Isoamyl 1 -pentanol Total Title Area Mash A 0 0 183. 28 122. 43 0 0 0 362. 86 0 668. 57 Mash B 0 0 61. 20 76. 61 0 0 0 341. 54 0 479. 35 Mash C 0 0 43. 90 82. 00 0 307. 40 0 433. 30

Fusel Concentration (ppm) Impact of Urea Dose 500 Affect of Urea Dose on Fusel Oil Production 400 300 Total Fusels 200 Isobutanol 1 -propanol Isoamyl 100 0 Title Area Insufficient Minimal Urea Dose Excess

Fusel Concentration (ppm) Impact of Protease Dose 500 Affect of Protease Usage on Fusel Oil Production 400 300 Total Fusels 1 -propanol Isobutanol Isoamyl 200 100 0 Title Area None 10 ppm 30 ppm Protease Dose 100 ppm

Impact of Fermentation Temperature Fusel Cocentration (ppm) Affect of Increasing Temperature on Fusel Oil Production 500 400 300 Total Fusels 1 -propanol isobutanol isoamyl 200 100 0 31 Title Area 32 33 34 35 Temperature (°C) 36 37

Impact of Mash Solids Fusel Concentration (ppm) Affect of Increasing Mash Solids on Fusel Oil Production 600 500 400 Total Fusels 1 -propanol Isobutanol Isoamyl 300 200 100 0 30. 3% Title Area 31. 3% 32. 3% 33. 3% Percent Solids of Mash 34. 3%

Removal of Fusel Oils Tert-amyl Alcohol BP: 214°F (101°C) Isopropanol BP: 181°F (82°C) Iso-amyl Alcohol BP: 268°F (131°C) 1 -Propanol BP: 206°F (97°C) Isobutanol BP: 226°F (108°C) Title Area Tert-Butyl Alcohol BP: 180°F (82°C) Ethanol BP: 173°F (78°C)

Removing Fusel Oils Lower Temperatures Higher Temperatures Title Area Source: Ethanol Producer Magazine

Fusels in Liq Mash Isopropanol tert-butyl-alcohol 1 -propanol Isobutanol tert-amyl-alcohol Avg 5. 18 Min 5. 18 Max 5. 18 Count 1 30. 26 52. 86 73. 60 3. 38 22. 83 73. 60 122. 58 130. 05 73. 60 8 8 1 84. 30 11. 92 184. 76 16 0 0 192. 58 34. 72 932. 64 27 0 3 -pentanol 2 -pentanol Isoamyl alcohol 1 -pentanol Title Area

Testing Fusels in the Lab Fusel Species Isoamyl Alcohol Tert-amyl Alcohol 1 -Propanol Tert-Butyl Alcohol Isobutanol Title Area Concentrations tested (ppm) 35 85 135 235 435 835 1235 0 50 100 200 400 800 1200 69 119 169 269 469 869 1269 0 50 100 200 400 800 1200 Observed Max Min 933 34 185 12 130 23 123 3 74 74

120% Effect of Fusel Addition on Dextrose Concentration (48 hr) 80% Isoamyl 40% Tert-amyl 0% 1 -Propanol Tert-Butyl Fusel Addition Title Area m pp 00 12 pm 0 p 80 pm 0 p 40 pm 0 p 20 0 p pm Isobutanol 10 m pp 50 pm -40% 0 p Dextrose Relative to Control Impact of Fusel Oils on Fermentation

Impact of Fusel Oils of Fermentation 5% 0% Isoamyl -5% Tert-amyl 1 -Propanol -10% Tert-Butyl Fusel Addition Title Area m pp 00 12 pm 0 p 80 pm 0 p 40 pm 0 p 20 pm 0 p 10 pp 50 0 p m -15% pm Ethanol Relative to Control Effect of Fusel Addition on Ethanol Production (48 hr) Isobutanol

Impact of Fermentation Conditions on Fusel Inhibition Effect of Temperature and Isoamyl Alcohol on Ethanol Production (48 hr) Ethanol (%w/v) 14. 0 13. 4 32. 2 12. 8 33. 3 34. 4 12. 2 11. 6 0 Title Area 50 200 400 Isoamyl Alcohol Dose (ppm) 800

Impact of Fermentation Conditions on Fusel Inhibition Effect of Temperature and Isoamyl Alcohol on Ethanol Production (48 hr) 1. 00% Relative Ethanol 0. 00% -1. 00% -2. 00% 32. 2 -3. 00% 33. 3 -4. 00% 34. 4 -5. 00% -6. 00% 0 Title Area 50 200 400 Isoamyl Alcohol Dose (ppm) 800

Impact of Fermentation Conditions on Fusel Inhibition 13. 5 Effect of Mash Solids and Isoamyl Alcohol on Ethanol Production (48 hr) Relative Ethanol 13. 3 13. 1 12. 9 32. 47% Solids 12. 7 33. 70% Solids 12. 5 34. 57% Solids 12. 3 12. 1 0 Title Area 50 200 400 Isoamyl Alcohol Dose (ppm) 800

Impact of Fermentation Conditions on Fusel Inhibition Effect of Mash Solids and Isoamyl Alcohol on Ethanol Production (48 hr) 0. 0% Relative Ethanol -1. 0% -2. 0% -3. 0% 32. 47% Solids -4. 0% 33. 70% Solids -5. 0% 34. 57% Solids -6. 0% -7. 0% 0 Title Area 50 200 400 Isoamyl Alcohol Dose (ppm) 800

Impact of Fermentation Conditions on Fusel Inhibition Effect of Urea Dose and Isoamyl Contamination on Dextrose Consumption 2. 5 Dextrose (%w/v) 2. 0 1. 5 450 ppm Urea 1. 0 650 ppm urea 850 ppm Urea 0. 5 0. 0 0 Title Area 50 200 400 800 Isoamyl Dose (ppm) 800 + P

Impact of Fermentation Conditions on Fusel Inhibition Effect of Protease Use on Ethanol Production in Isoamyl Contaminated Corn Mash 13. 8 13. 7 Ethanol (%w/v) 0 ppm 13. 6 50 ppm 200 ppm 13. 5 400 ppm 13. 4 800 ppm 13. 3 800 ppm +P 13. 2 Drop Ethanol Production Title Area

Impact of Fermentation Conditions on Fusel Inhibition 14 Effect of Propagation Nutrient Addition on Ethanol Production in Fusel Contaminated Fermentations Ethanol (%w/v) +2. 7% 12 10 Control +3. 8% Nutrient 8 6 24 Title Area Time (h) 48

Summary • Fusel oils produced during normal fermentation – Predominately Isoamyl alcohol, Isobutanol, and 1 propanol – Others can be observed as well – High variance in concentration • Providing insufficient nitrogen increases isoamyl alcohol formation – Protease addition marginally reduced fusel production • Fusels are observed in liquefaction samples – Isoamyl was observed as the most inhibitory to yeast – Others can be problematic in combination with isoamyl • Addition of supplemental nutrition can help with fusel issues; as can reduced fermentation temperatures and mash solids Title Area