Advanced FAN nutrition, proteases, and yeast Have we thought through all the possibilities and pitfalls?

Transcription

1 Advanced FAN nutrition, proteases, and yeast Have we thought through all the possibilities and pitfalls? 2016 Fuel Ethanol Laboratory Conference Omaha, Nebraska Dr. Dennis Bayrock Director R&D Fermentation Research Phibro Ethanol Performance Group A Division of Phibro Animal Health Corporation

2 Phibro Animal Health Corporation A Global Manufacturer and Marketer of Animal Health & Nutrition and Performance Products Supported by more than 1,200 employees worldwide More than 450 product registrations in 50 countries across all continents 2,500+ customers in a wide variety of end-use markets Experienced sales, marketing, and technical organization with more than 50 PhD s, DVM s, chemists, and technicians on staff State-of-the-art laboratories and technical centers

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4 Process sequence at ethanol plant Grind/Liquefaction Propagation/Fermentation Centrifugation/Drying Distillation (Adapted from Madson and Monseaux,1990)

5 Protein sources at an ethanol plant (Adapted from Madson and Monseaux,1990)

6 Protein sources at an ethanol plant Corn (Adapted from Madson and Monseaux,1990)

7 Protein sources at an ethanol plant Corn Yeast (Adapted from Madson and Monseaux,1990)

8 Protein sources at an ethanol plant Enzyme (AA) Corn Yeast Enzyme (GA) (Adapted from Madson and Monseaux,1990)

9 Protein sources at an ethanol plant Enzyme (AA) Corn Yeast Enzyme (GA) Bacteria (Adapted from Madson and Monseaux,1990)

10 Protein content in Corn (Lorenz and Kulp, Handbook of Cereal Science and Technology)

11 Protein content in Corn (Lorenz and Kulp, Handbook of Cereal Science and Technology)

12 Protein content in Yeast Elemental composition1 Composition of the cell2

13 Protein content in Yeast Elemental composition1 Composition of the cell2 Up to 59% of the yeast mass is protein! Largest amount of a biochemical in yeast!

14 Protein content in a fermentor From mash (corn) Calculations (plant example) Mash Dry Solids (%DM) Fermentor 33 Fermentor volume (gal) Fermentor volume (L) 750,000 2,838,750 Corn in fermentor (lbs DM) Corn in fermentor (kg DM) 2,021, ,822 Protein content in corn (%DM) Protein content in fermentor (lbs): Protein content in fermentor (kg): ,316 99,934

15 Protein content in a fermentor From propagator transfer of yeast Calculations (plant example) Amount of yeast added to prop (lbs) Amount of yeast added to prop (kg) Volume of propagator transferred (gal) 15,000 Protein content in yeast (%DM) Propagator Growth of yeast Fermentor 59 Yeast cell count at drop (cells/ml) Yeast cell count at drop (cells/gal) 2.50E E+11 Number of cells transferred (cells) 1.42E+16 1 g ADY = 2.2E10 CFU/g All cells are viable (1 CFU= 1 cell) Amount of cells transferred (g) Amount of cells transferred (kg) Amount of protein transferred (kg) 645,

16 Protein content in a fermentor From fermentor production of yeast Calculations (plant example) Maximum yeast multiplication in fermentor 10x From Propagator: Amount of cells transferred (g) Amount of cells transferred (kg) Fermentor 645, Amount of yeast produced in fermentor (g) 6,451,705 Amount of yeast produced in fermentor (kg) 6,452 Protein content in yeast (%DM) 59 Growth of yeast Amount of yeast protein produced (kg) 3,807

17 Protein content in a fermentor From fermentor bacterial contamination Calculations (plant example) Fermentor Fermentor volume (gal) Fermentor volume (L) 750,000 2,838,750 Bacterial contamination (CFU/ml) 1.00E+07 Number of bacteria (CFU) 2.84E+16 1 g LAB bacteria = 3.4E11 CFU/g All cells are viable (1 CFU= 1 cell) Amount of bacteria (g) Amount of bacteria (kg) 83, Protein content in bacteria (%DM) 65 Bacterial protein content in fermentor (kg) 54

18 Protein content in a fermentor From enzyme additions Grind/Liquefaction Calculations (plant example) Protein content in AA/GA (%) 95 Alpha amylase density (g/cc) Glucoamylase density (g/cc) Alpha amylase dose rate (%w/w) Glucoamlyse dose rate (%w/w) Corn in fermentor (lbs) Corn in fermentor (kg) Propagation/Fermentation ,021, ,822 Alpha amylse Amount of alpha amylase (lb) (kg) Volume of alpha amylase (gal) (L) Glucoamylase Amount of glucoamylase (lb) (kg) Volume of glucoamylase (gal) (L) Total amount of enzymes (lb) (kg) 1, Amount of protein (lb) 1,114 Amount of protein (kg) 505

19 Protein sources at an ethanol plant Protein summary for one fermentor Enzyme (AA) Corn Yeast Enzyme (GA) Protein source Corn Propagator yeast transfer Fermentor yeast production Enzyme additions Bacterial contamination Bacteria Propagator yeast transfer kg %Total 99, Fermentor yeast production kg %Total 99, ,807 Fermentor Enzyme Bacterial additions contamination kg %Total kg %Total 99, ,

20 Proteases Rapidly becoming standard addition to fermentors in fuel ethanol industry. Commercially viable products from enzyme manufacturers for fuel ethanol since ~2000. Products continue to be released. Testing at fuel ethanol plants began significantly in 2005

21 Why add protease? 1. Aids in breakdown of corn kernel Protein is a major component within the binding matrix of corn Distribution of major components in corn and some corn processing by-products a Component Whole Kernel (%) Dry weight of components (%) Endosperm Starch Protein Oil Ash Others* Water Germ Pericarp Tip Cap CGF CGM DDGS ** - a CGF, corn gluten feed; CGM, corn gluten meal; DDGS, distillers dried grains with solubles. Data sources: Watson and Yahl (1967); Reiners et al. (1973); Anonymous (1982, 1997, 1999); Neumann and Wall (1984); Watson and Ramstad (1987); Singh and Cheryan (1998). * By difference. Includes fiber, nonprotein nitrogen, pentosans, phytic acid, soluble sugars, xanthophylls. ** Also includes glycerol, organic acids and other byproducts of ethanol fermentation.

22 Why add protease? 2. Increased Oil yield Corn oil yield increases up to 11% over normal operation Energy input reductions Ethanol yield Increases Distribution of major components in corn and some corn processing by-products a Component Whole Kernel (%) Dry weight of components (%) Endosperm Starch Protein Oil Ash Others* Water Germ Pericarp Tip Cap CGF CGM DDGS ** - a CGF, corn gluten feed; CGM, corn gluten meal; DDGS, distillers dried grains with solubles. Data sources: Watson and Yahl (1967); Reiners et al. (1973); Anonymous (1982, 1997, 1999); Neumann and Wall (1984); Watson and Ramstad (1987); Singh and Cheryan (1998). * By difference. Includes fiber, nonprotein nitrogen, pentosans, phytic acid, soluble sugars, xanthophylls. ** Also includes glycerol, organic acids and other byproducts of ethanol fermentation.

23 Why add protease? 3. Provides additional FAN (Free Amino Nitrogen) for yeast nutrition Corn protein H2N ( Protease )x COOH x = 0 to 200,000 Amino Acid Nitrogen (as amino nitrogen)

24 Proteases Addition of proteases to fuel ethanol fermentations is not novel Proposed and researched since 1994 to aid in yeast nutrition (Alison and Ingledew, 1994)

25 Potential issues with protease use Issue #1 Timing of FAN

26 Timing of FAN nutrition How much FAN Nitrogen do we need for fermentation? Why do we target 300 ppm FAN? Free Amino Nitrogen or FAN Anaerobically grown yeast 37% to 42% protein. Amino nitrogen-containing chemical that the yeast can biologically use Yeast is ~6% Nitrogen. Cell density peak in fermentation ~ 250 X 106 cells/ml. 1 g yeast = 5 X 1010 cells FAN is Different than %N (composition) Not all N containing chemicals can be used by yeast 250 X 106 / 5 X 1010 = = 0.5% yeast mass * 0.06 = Nitrogen * 1,000,000 = 300 ppm FAN Nitrogen needed (Minimum) - e.g. Proteins in mash cannot be used directly by yeast

27 Number of yeast Timing of FAN nutrition Fermentor Time (h)

28 Timing of FAN nutrition Number of yeast A λ Fermentor Time (h) Maximum growth rate of yeast between 6 and 24 hours into fermentation Highest demand for proper nutrition and FAN

29 Timing of FAN nutrition Number of yeast A λ Fermentor Time (h) Amino nitrogen available for yeast to use (Definition of Free Amino Nitrogen or FAN) With traditional FAN sources (urea/ammonia) and procedures, FAN is available to the yeast as soon as it is added and can last for the duration of fermentation

30 Timing of FAN nutrition Number of yeast A λ Fermentor Time (h) Proteins? Amino nitrogen available for yeast to use (Definition of Free Amino Nitrogen or FAN) With proteases conversion of proteins to amino acids by proteases requires time - FAN calculations require understanding catalytic rate of enzyme (unpublished) When is the 300 ppm minimum FAN reached for yeast?

31 Timing of FAN nutrition Number of yeast A λ Fermentor Time (h) Proteins? Amino nitrogen available for yeast to use (Definition of Free Amino Nitrogen or FAN) Solutions seem easy (obvious): 1. Add more (increase amount of enzyme) 2. Add earlier than the fermentor (increase contact time)

32 Potential issues with protease use Issue #2 Reduction in protein quantity/quality in DDGS

33 Reduction in protein quantity/quality in DDGS Can we use proteases to supply all of yeast FAN nutrition and still get the benefits of better breakdown of corn as well as better corn oil yield? Is there a risk to DDGS protein content or composition?

34 Reduction in protein quantity/quality in DDGS Can we use proteases to supply all of yeast FAN nutrition and still get the benefits of better breakdown of corn as well as better corn oil yield? Is there a risk to DDGS protein content or composition? = Typical urea dose to fermentors = 4000 lbs (urea can be utilized by yeast for nitrogen) O H2N-C-NH2 urea H2N ( x = 0 to 200,000 Fermentor Protease Protein )x COOH X( ) amino acids

35 Reduction in protein quantity/quality in DDGS Is there a risk to DDGS protein content or protein composition? Fermentor Amino Acid Composition (%) Molecular weight (g/mol) Composition (weighted) Alanine Arginine Aspartic acid Cystine Glutaminc acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Sum Sum Mean molecular weight (g/mol)

36 Reduction in protein quantity/quality in DDGS Is there a risk to DDGS protein content or protein composition? Calculations Fermentor volume (gal) Fermentor volume (L) Dry urea dose (lbs) Dry urea dose (kg) Calculated FAN content for yeast (ppm) Fermentor 750,000 2,838, , Molecular weight of urea (g/mol) Average molecular weight amino acid (g/mol) Amino acid mass equivalent to urea dose (lbs) Amino acid mass equivalent to urea dose (kg) 8,786 3,985 Protein mass required (lbs) Protein mass required (kg) Assumption: 1 protein molecule 8,786 3,985 Total protein amount in fermentor (lbs) Total protein amount in fermentor (kg) 220,316 99,934

37 Reduction in protein quantity/quality in DDGS Is there a risk to DDGS protein content or protein composition? Calculations (cont) Protein mass required (lbs) Protein mass required (kg) Assumption: 1 protein molecule Total protein amount in fermentor (lbs) Total protein amount in fermentor (kg) Fermentor Corn protein required to be hydrolyzed (%) 8,786 3, ,316 99,

38 Reduction in protein quantity/quality in DDGS Is there a risk to DDGS protein content or protein composition? Calculations (cont) Protein mass required (lbs) Protein mass required (kg) Assumption: 1 protein molecule Total protein amount in fermentor (lbs) Total protein amount in fermentor (kg) Fermentor Corn protein required to be hydrolyzed (%) 8,786 3, ,316 99, Typical fermentor has enough protein that (when hydrolyzed) can supply all FAN nutritional requirements of yeast.

39 Reduction in protein quantity/quality in DDGS Is there a risk to DDGS protein content or protein composition? Not all of the amino acids that the yeast accumulates from the mash is funneled into proteins (misconception in industry) Amino Acids from media/mash X 100% Protein Uptake

40 Reduction in protein quantity/quality in DDGS Is there a risk to DDGS protein content or protein composition? Not all of the amino acids that the yeast accumulates from the mash is funneled into proteins (misconception in industry) Amino Acids from media/mash X 100% Protein Uptake Once the fermentable carbohydrate in the fermentor is depleted (~50 hours), the yeast will have no choice but to: 1. Use amino acids for energy production 2. Use amino acids as a C source for metabolism 3. Produce more fusels as a byproduct of energy production

41 Potential issues with protease use Issue #3 Fusels

42 Fusels Are there any differences for the yeast in fermenting amino acids?

43 Fusels Are there any differences for the yeast in fermenting amino acids? - Amino acids within yeast cell converted by Ehrlich pathway to fusel oils, aldehydes, and acids (known for 100 years). - Availability of free amino acids (not FAN) within yeast is required Amino Acids from media/mash Protein H2N ( )x COOH Proteases Uptake Amino Acids - Saccharomyces cerevisiae has internal proteases but does not produce proteases that can be exported from cell.

44 Fusels Are there any differences for the yeast in fermenting amino acids? Ehrlich pathway 1. Transamination Amino Acids α keto acid 2. Decarboxylation 2 oxoglutarate glutamate CO2 'fusel aldehyde' + Reduction NAD+ NADH, H NAD+ 'fusel acid' Oxidation NADH, H+ 'fusel alcohol' ATP Export Production of α keto acid point of no return for yeast and fusels Diffusion ATP 'fusel acid' 'fusel alcohol' Flavor/aroma/volatile compounds 44 Air Cleaner Process Cleaner Fuel Cleaner

45 Fusels 45 Air Cleaner Process Cleaner Fuel Cleaner

46 Fusels Ehrlich pathway 46 Air Cleaner Process Cleaner Fuel Cleaner

47 Fusels Are there any differences for the yeast in fermenting amino acids? Amyl alcohol Isomers of C5H12O - Fusel compounds have different boiling points. - What is potential carryover to DDGS from fermentation (up to 100 ppm)? - Insufficient research in tracing these yeastbiochemicals to DDGS. (Wikipedia)

48 Fusels Are there any differences for the yeast in fermenting amino acids? Yeast Growth Conversions 2.1g/l 0.21 g/100ml 0.21% w/v Ethanol concentration Yeast Death (Okolo et al, 1990) Isoamyl alcohol more toxic to yeast than ethanol by ~15 fold

49 Fusels Are there any differences for the yeast in fermenting amino acids? Fusel oil inhibition of yeast confirmed at multiple ethanol plants Symptoms at operating plant - Plant inconsistently passing 2-4% sugars to still. - Ethanol yield decreased by 1% w/v absolute. Normal production between 15-17%, plant was now producing 14%. - Lactic/acetic acids max delta 0.1%w/v and 0.1%w/v for plant in fermentation. - Fermentors stalled at ~40h. Enzyme conversion still proceeding as glucose levels remain >2% at drop. Not an enzyme issue. - Glycerol levels below 1.7% w/v together with lower ethanol indicates that yeasts are either metabolically inhibited (assuming correct crop of yeast), or not enough yeast entering system.

50 Potential issues with protease use Issue #4 Loss in Ethanol yield

51 Loss in Ethanol Yield Maillard reaction - L.C. Maillard in 1913 investigated the reactions occurring between amino acids and sugars at elevated temperatures. - Amino nitrogen chemical groups react with reducing sugars to form condensation products H2N-CH2-COOH Glycine (amino acid) Maltose (cyclic) Maltose (open-chain)

52 Loss in Ethanol Yield Maillard reaction - L.C. Maillard in 1913 investigated the reactions occurring between amino acids and sugars at elevated temperatures. - Amino nitrogen chemical groups react with reducing sugars to form condensation products H2N-CH2-COOH Glycine (amino acid) Maltose (cyclic) Maltose (with reducing end) NH-CH2-COOH N-subsituted glycosylamine

53 Loss in Ethanol Yield Maillard reaction s of different compounds (permutations) possible between sugars and amino acids - Primary cause for darkening of mash during liquefaction, distillation, evaporation, and drying Is Color the Only or Best Indicator of DDGS Quality? Jerry Shurson, June Maillard reaction accelerated by 500x at temperatures >160 F. - Reaction chemically driven by concentration of sugars and amino-containing chemicals.

54 Loss in Ethanol Yield Maillard reaction products - Are inhibitory to yeast. As little as 1% formation (i.e. 1% darkening of mash) can inhibit yeast fermentation by 10%. - No standard test to quantitate concentration (other than by color) - Cannot be metabolized by yeast. Represents a loss of sugar (and FAN nutrition) for the yeast to ferment to ethanol. - Can be metabolized by many species of bacteria. Provides a second source of nutrients for bacterial contamination when starch sources are exhausted.

55 Loss in Ethanol Yield During liquefaction (oversimplified) Corn protein 1 H2N Protease ( x = 0 to 200,000 )x COOH 200,000

56 Loss in Ethanol Yield During liquefaction (oversimplified) Corn protein 1 Protease ( H2N )x x = 0 to 200,000 Corn starch 1 Amylose/Amylopectin ( COOH 200,000 Alpha Amylase Glucoamylase 30,000 DP3,DP2,DP1 )x x = 0 to 30,000

57 Loss in Ethanol Yield During liquefaction (oversimplified) 160 F 4 hours 200,000 30,000

58 Loss in Ethanol Yield During liquefaction (oversimplified) 160 F 4 hours 200,000 30,000 Since 2014, incidents involving ethanol plants that experience darkening of liquefied/saccharified mash has increased significantly Accounting for all other factors of poorer yeast performance (thorough forensic analysis), ~10% of these plants experience ethanol yield loss (up to 0.2% w/v), poorer yeast propagations/fermentations, and darker DDGS

59 Thank you for your attention! Questions?