1. Lecture 10: Problem Fermentations

2. Reading Assignment: Text, Chapter 4, pages 168- 181

3. This lecture will cover the principle types of fermentation problems that can arise during the alcoholic fermentation: Stuck fermentations and off- character production

4. Problem Fermentations Slow (sluggish) fermentation Stuck (incomplete, arrested) fermentation Off-character production –Hydrogen sulfide –Sulfur volatiles –Acetic acid –Undesired Esters

5. Stuck and Sluggish Fermentations

6. Characterized by failure of yeast to consume sugar Multiple causes Difficult to treat Leads to reduced wine quality

7. The main challenge of slow and arrested fermentations is that they are not recognizable until after fermentation rate has changed. At this point it may be too late to change the adaptive response of the yeast.

8. GOAL: To be able to distinguish a normal from a problem fermentation as soon as possible.

9. Fermentation Profile 1 2 3 4 5 1: lag time; 2: max fermentation rate; 3: transition point; 4: post-transition fermentation rate; 5: overall time to dryness Brix Time

10. Fermentation Profile Lag time –Duration? Maximum fermentation rate –Rate value? –Duration? Transition point –At what Brix level? –How sharp? Post-transition fermentation rate –Value relative to max fermentation rate? –Length of time? –Brix/ethanol/nitrogen level at which it occurs? Overall time to dryness

11. Fermentation Capacity Is a Function of: Yeast Biomass Concentration Fermentative Ability of Individual Cells

12. Causes of Stuck/Sluggish Fermentations Nutrient limitation

13. Nutrient Limitation: Nitrogen Nitrogen: most often limiting Amino acids –Can be degraded as N source via transamination –Can be interconverted with related amino acids –Can be used as that amino acid Ammonia –Mobilized by direct amination

14. Transamination Glutamate + X  -ketoglutarate + N-X Glutamine + X’ glutamate + N-X’ Alanine + X’’ pyruvate + N-X’’ Where “X” is an intermediate in amino acid/ nucleotide biosynthesis, and “N-X” is an amino acid or nucleotide base.

15. Amination NH 4 +  -ketoglutarate glutamate NH 4 + glutamate glutamine

16. Preference for Nitrogen Sources How readily can it be converted to NH 4, glutamate or glutamine? Expense of utilization (ATP, cofactor, oxygen requirement) Toxicity of C-skeleton What else is available?

17. Amino Acid Transport H+ Amino acid H+ ATP ADP Amino acid H+ TransporterATPase Pump

18. Factors Affecting Nitrogen Compound Utilization and Preference pH –Transport is coupled to H + ion movements Ethanol –Inhibits amino acid transporter function (80%  at 5% ethanol for the general amino acid permease) –Increases passive proton flux Other N compounds –Competition for uptake –Nitrogen repression –Induction Yeast strain differences

19. Sources of Nutrients Grape Nutrient additions (winemaker) –Diammonium phosphate –Yeast extracts –Yeast “ghosts” –Proprietary yeast nutrient mix Yeast autolysis

20. Causes of Stuck/Sluggish Fermentations Nutrient limitation Ionic imbalance

21. Ionic Imbalance Ratio of K + :H + Must be at least 25:1 Needs to be adjusted early in fermentation Probably important in building an ethanol tolerant membrane

22. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Substrate inhibition

23. Substrate Inhibition Transporters with a high substrate affinity can get “jammed” at high substrate concentrations FG F GG F

24. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity

25. Ethanol Toxicity Plasma membrane is the most ethanol-sensitive cell structure: Composition: Protein 50% Lipid 40% Other 10% Functions: Permeability barrier Regulation of uptake Mediates response to environment Maintains electrochemical gradients Mediates cell-cell interactions

26. Ethanol Toxicity Impact of ethanol Perturbs membrane structure at protein:lipid interface Leads to increased “passive proton flux” and acidification of cytoplasm Inhibits protein activity Affects membrane “fluidity”

27. Membrane Fluidity Is Required for Transport G G G

28. Ethanol Toxicity Adaptation of membrane requires: Increasing content of sterols Increasing relative content of proteins Increasing level of desaturation (number of double bonds) in fatty acid side chains Modification of phospholipid head groups?

29. Ergosterol HO

30. Fatty Acid Saturation Saturated Unsaturated

31. Phospholipid Head Groups FA PO 4 OH HO FA PO 4 CH 2 CH OOC NH 2 FA PO 4 CH 2 NH 2 FA PO 4 CH 2 N -CH 3 CH 3 H 3 C- + Phoshpatidyl- Inositol Serine Ethanolamine Choline

32. Ethanol Toxicity Sterol and fatty acid desaturation are Oxygen-requiring processes New protein synthesis requires nitrogen be available Phospholipid head group synthesis requires cofactors (S-adenosyl- methionine) be available

33. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Presence of toxic substances

34. Presence of Toxic Substances Toxins may arise from the metabolic activity of other microbes Toxins may arise from metabolic activity of Saccharomyces Toxins may have arisen in vineyard, but are not inhibitory until ethanol has accumulated

35. The Most Common Toxins Acetic acid Higher organic acids (C 2 – C 4 ) Medium chain fatty acids/fatty acid esters Acetaldehyde Fungicide/Pesticide residues Higher alcohols Higher aldehydes Killer factors Sulfur dioxide

36. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Presence of toxic substances Poor adaptation of strain

37. Poor Adaptation of Strain Strain may not display ethanol tolerance Strain may have high nitrogen/vitamin requirements Strain may be a poor fermentor, but capable of dominating the fermentation Temperature effects

38. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Presence of toxic substances Poor adaptation of strain Low pH

39. pH pH is reduced by metabolism of Saccharomyces Low pH musts (below pH 3.0) may drop to an inhibitory level (pH 2.7) Dependent upon K + concentration

40. Causes of Stuck/Sluggish Fermentations Nutrient limitation Nutrient imbalance Substrate inhibition Ethanol toxicity Presence of toxic substances Poor adaptation of strain Low pH Temperature shock

41. Temperature Shock Super-cooling/heating of tank due to equipment failure High temperature fermentations becoming too warm due to yeast metabolism

42. The factors leading to arrest of fermentation are interacting. Limitation for nutrients enhances the toxicity of ethanol as does high temperature and the presence of other toxic substances.

43. Off-Character Production

44. The Saccharomyces Off-Characters Volatile sulfur compounds

45. Volatile Sulfur Compounds Hydrogen Sulfide: H 2 S Methanethiol: CH 3 -SH Ethanethiol: C 2 H 5 -SH Dimethyl sulfide: CH 3 -S-CH 3 Dimethyl disulfide: CH 3 -S-S-CH 3 Diethyl sulfide: C 2 H 5 -S-C 2 H 5 Diethyl disulfide: C 2 H 5 -S-S-C 2 H 5

46. Sources of Sulfur Compounds Sulfate reduction pathway Degradation of sulfur containing amino acids Inorganic sulfur –Non-enzymatic –Requires reducing conditions established by yeast Degradation of S-containing pesticides/fungicides

47. Hydrogen Sulfide Formation Due to nitrogen limitation Sulfate reduction regulated by nitrogen availability Lack of nitrogenous reduced sulfur acceptors leads to excessive production of reduced sulfate and release as H 2 S Strain variation

48. Higher Sulfides Come from degradation of sulfur containing amino acids From reaction of reduced sulfur intermediates with other cellular metabolites? Formed chemically due to reduced conditions?

49. Current Understanding of H 2 S Formation Nitrogen levels not well-correlated with H 2 S formation, but generally see increased H 2 S at lower nitrogen Under complex genetic control Tremendous strain variation in H 2 S production

50. Factors Impacting H 2 S Formation Level of total nitrogen Level of methionine relative to total nitrogen Fermentation rate Use of SO 2 Vitamin deficiency Presence of metal ions Inorganic sulfur in vineyard Use of pesticides/fungicides Strain genetic background

51. Timing of Formation of H 2 S Brix Time H2SH2S

52. Timing of Formation of H 2 S Early (first 2-4 days): due to N imbalance Late (end of fermentation): due to autolysis, degradation of S-containing compounds H 2 S produced early can be driven off by carbon dioxide during active phase of fermentation

53. Sulfate Reduction Pathway SUL1, SUL2 SO 4 Adenylylsulfate Phosphoadenylylsulfate Sulfite Sulfide Cysteine Cystathionine Homocysteine Methionine MET3 MET14 MET16 (1,8,20,22) MET10 (1,5?,8,20) MET17/25/15 MET6CYS4CYS3

54. Regulation of the Sulfate Reduction Pathway Methionine (SAM) Repression Cysteine Inhibition of Inducer Production (O-acetyl serine) General Amino Acid Control Sub-Pathway Controls

55. Homocysteine Methionine S- Adenosylmethionine S- Adenosylhomocysteine Met - tRNA

56. Cysteine γ-Glutamylcysteine Glutathione RD Glutathione OX Cys-tRNA

57. Methionine Repression Antagonized by: Threonine Serine Aspartate Glycine Glutamate Histidine Lysine

58. Methionine Repression is Augmented by: Leucine

59. The Amino Acids of General Amino Acid Control Lysine Histidine Arginine Leucine Valine Serine Phenylalanine Tryptophan Proline Methionine

60. The Saccharomyces Off-Characters Volatile sulfur compounds Acetic Acid

61. Acetic Acid Production by Saccharomyces Levels made by Saccharomyces are generally low, below threshold of detection Strain differences in amount formed Derived from: –Fatty acid biosynthesis/degradation –Amino acid degradation

62. The Saccharomyces Off-Characters Volatile sulfur compounds Acetic Acid Higher Alcohols –Fusel oils –Phenethyl alcohol

63. Higher Alcohols (  C2) Also called “fusel oils” Formed during amino acid degradation R R R HCNH C=O HC=O COOH COOH RCHOH RCOOH Deamination Decarboxylation Reduction Oxidation Amino Acid Alcohol Acid

64. Phenethyl Alcohol H2CH2C CH 2 OH Generic “floral” May be too intense for some wines

65. The Saccharomyces Off-Characters Volatile sulfur compounds Acetic Acid Higher Alcohols Acetaldehyde/Higher Aldehydes

66. Aldehyde Production Acetaldehyde from glycolysis –Released when conversion to ethanol is blocked –Released as SO 2 adjunct Higher aldehydes from amino acid degradation –Released when formation of higher alcohols is blocked

67. The Saccharomyces Off-Characters Volatile sulfur compounds Acetic Acid Higher Alcohols Acetaldehyde/Higher Aldehydes Unwanted Esters –Fatty acid metabolism –Amino acid metabolism Phenethyl Acetate

68. Unwanted Esters Esters form from the reaction of an alcohol and an acyl-CoA molecule O O R 1 -OH + R 2 -C  SCoA R 1 -O-C-R 2

69. Source of Esters Most common ester is ethyl acetate made from the reaction of ethanol with acetyl-CoA Esters can derive from amino acid degradation and reaction of acids with ethanol or of alcohols with acetyl-CoA Esters can derive from fatty acid metabolism

70. Phenethyl Acetate Degradation product of phenyalanine Characteristic “rose oil” odor May be too pungent CH 2 -CH 2 O-C-COOH O

71. The Saccharomyces Off-Characters Volatile sulfur compounds Acetic Acid Higher Alcohols Acetaldehyde/Higher Aldehydes Unwanted Esters Vinyl Phenols

72. OH H CH COOH OH H CH CH 2 OH H CH 2 CH 3 Decarboxylase Vinyl Phenol Reductase

73. Vinyl Phenols Responsible for sweaty, horsy, stable,pharmaceutical off aromas Usually formed by Brettanomyces Saccharomyces possesses the enzymes needed to make vinyl phenols and there are reports that it will make them under certain conditions

74. Moral: Yeast needs are simple, but it can be challenging to keep them happy.