Wine Fermentation — Depth
Malolactic fermentation · Brettanomyces · Reductive faults · Oxidation · Volatile acidity · Temperature control
Verified: OIV Paris · AWRI Urrbrae SA · INRAE Paris · Boulton et al. 1999 · Swiegers et al. 2005 AJGWR · EC Reg 606/2009
Wine fermentation science encompasses two sequential processes: primary alcoholic fermentation (Saccharomyces cerevisiae consuming glucose and fructose to produce ethanol, CO₂, and a constellation of flavour congeners) and secondary malolactic fermentation (the lactic acid bacterium Oenococcus oeni decarboxylating malic acid to lactic acid, reducing acidity and producing diacetyl). Beyond these intentional transformations, wine fermentation is subject to microbial spoilage — principally Brettanomyces bruxellensis producing phenolic off-compounds — and chemical faults including reductive sulphur compounds (H₂S, methanethiol, DMS) arising from inadequate yeast nutrition, and oxidative faults (acetaldehyde) from uncontrolled oxygen exposure. The winemaker's mastery of temperature, nutrition, microbial inoculation, SO₂ management, and oxygen exclusion determines whether these secondary processes become assets or defects.
Malolactic Fermentation (MLF)
Malolactic fermentation is not a true fermentation in the biochemical sense — no yeast is involved, and no alcohol is produced. It is a decarboxylation reaction catalysed by the enzyme malolactic enzyme (malic enzyme, EC 1.1.1.38) produced by lactic acid bacteria, principally Oenococcus oeni. The net result is the conversion of the sharp, diprotic malic acid (two ionisable protons; pKa 3.46 and 5.10) into the softer, monoprotic lactic acid (one ionisable proton; pKa 3.86), with the release of CO₂. This reduces total acidity by approximately 1–3 g/L (expressed as tartaric acid equivalents) and raises pH by 0.1–0.3 units — a significant change given that wine pH ranges typically from 3.0 to 4.0.
Oenococcus oeni — The MLF Organism
Oenococcus oeni is the primary organism responsible for MLF in commercial winemaking. It is extraordinarily well-adapted to the wine environment — tolerant of ethanol concentrations up to 13–14% v/v, low pH (viable at pH 3.0–3.5, though optimal at 4.2–4.5), free SO₂ up to approximately 10 mg/L, and nutrient scarcity. O. oeni is a homofermentative lactic acid bacterium (LAB) of the family Leuconostocaceae; it is gram-positive, non-motile, non-sporeforming, and grows as pairs or short chains. Other LAB — including Lactobacillus spp. and Pediococcus spp. — can also conduct MLF but are less desirable: Lactobacillus strains may produce mouse-taint compounds (acetyltetrahydropyridines, threshold ~10 µg/L) in certain conditions, and Pediococcus can produce "ropy" (viscous) wines via exopolysaccharide production.
Diacetyl — The Butter Compound
The most flavour-significant by-product of MLF is diacetyl (2,3-butanedione, CH₃–CO–CO–CH₃). Diacetyl is produced by O. oeni from citric acid metabolism via α-acetolactate and pyruvate. Its sensory threshold in wine is approximately 0.2 mg/L, above which a distinctly buttery or butterscotch character is perceived. Concentrations during and immediately after MLF can reach 3–10 mg/L; with bottle age and reductive conditions, diacetyl is biologically reduced to acetoin (less odoriferous, threshold ~150 mg/L) and then to 2,3-butanediol (essentially flavour-neutral, threshold >1,000 mg/L). The diacetyl character therefore diminishes with bottle ageing.
Inoculated vs Spontaneous MLF
MLF may proceed spontaneously via indigenous O. oeni and other LAB populations resident in the winery's microbiome, or it may be induced by inoculation with a commercial freeze-dried O. oeni culture (e.g. Lallemand Enoferm Alpha, Chr. Hansen VP41). Spontaneous MLF typically occurs 1–3 months after alcoholic fermentation, once yeast-derived antimicrobial compounds (SO₂, medium-chain fatty acids C8–C12 which inhibit LAB, ethanol) have dissipated sufficiently. The risks of spontaneous MLF include unpredictable timing, incomplete conversion (residual malic acid remaining), and the potential dominance of undesirable LAB strains producing off-compounds including mouse taint, acrolein (from glycerol), or excessive volatile acidity.
Inoculated MLF using selected O. oeni strains offers faster, more predictable completion (typically 2–4 weeks at 18–22°C), reduced risk of off-flavour production, and easier management of the fermentation window. Co-inoculation — adding selected O. oeni simultaneously with or immediately after S. cerevisiae inoculation — has become common in warm-climate winemaking where extended post-AF waiting periods risk acetic acid bacteria (Acetobacter, Gluconobacter) growth and volatile acidity formation. AWRI research has demonstrated co-inoculation efficacy in Australian conditions; OIV Resolution OIV-OENO 328-2009 officially recognises selected MLF starter cultures.
MLF Decision — When and Why
| Wine Style | MLF Typical? | Rationale | Notable Exceptions |
|---|---|---|---|
| Red Bordeaux, Rhône, Barossa Shiraz | Always (100%) | Softens tannin perception, reduces harsh malic acidity, adds body; diacetyl at manageable levels in full-bodied reds | Some minimal-intervention producers skip or prevent for freshness |
| White Burgundy (Chardonnay) | Usually (80–100%) | Reduces sharp malic acidity; intentional diacetyl contribution (butter/cream); lees-aged wines metabolise diacetyl to manageable levels | Chablis: historically no MLF for mineral tension; now winery-specific; Mâcon: usually yes |
| Champagne base wines | Winery-specific | Larger houses (Moët) often block MLF for consistent acidity across vintages; grower-Champagnes often encourage for complexity | — |
| Alsace Riesling, German Riesling | Usually not | Malic acidity contributes essential backbone and aging potential; MLF would create flat, over-soft character | Occasionally in warm vintages when natural acidity is very low |
| Sauvignon Blanc (NZ, Sancerre) | Usually not | Preservation of thiols (3-MH, 3-MHA) responsible for characteristic passionfruit/grapefruit; MLF conditions reduce thiol expression | Some Fumé Blanc styles use partial MLF |
| Rosé | Usually not | Freshness and crispness are style requirements; MLF adds unwanted body and reduces fruit brightness | Provence exceptions in very high-acid years |
Brettanomyces bruxellensis ("Brett")
Brettanomyces bruxellensis (anamorph: Dekkera bruxellensis) is a spoilage yeast that is simultaneously one of the most commercially damaging and most debated organisms in fine wine production. It produces a suite of volatile phenols — principally 4-ethylphenol (4-EP), 4-ethylguaiacol (4-EG), and isovaleric acid — that at high concentrations render wine unacceptable to most consumers, but at sub-threshold or near-threshold concentrations contribute complexity valued by some enthusiasts and critics. The controversy over Brett is genuine and unresolved: concentrations perceived as "barnyard character" by some are "complexity" by others.
Biosynthetic Pathway: Vinylphenols to Ethylphenols
Brett's phenolic off-compounds arise from the successive decarboxylation and reduction of hydroxycinnamic acids naturally present in grape skins and wood-extracted phenolics in barrel-aged wines. The pathway proceeds: Ferulic acid → 4-vinylguaiacol (4-VG) → 4-ethylguaiacol (4-EG) via the enzyme cinnamate decarboxylase (Pad1p), then vinyl phenol reductase (Vph1p). Similarly: p-Coumaric acid → 4-vinylphenol (4-VP) → 4-ethylphenol (4-EP). The key enzymes — Pad1p (phenylacrylic acid decarboxylase) and Vph1p (vinyl phenol reductase) — are expressed constitutively in wine strains of B. bruxellensis. Wine contains substantial precursor substrates: ferulic acid 5–30 mg/L; p-coumaric acid 5–15 mg/L.
Brett Ecology: Growth Conditions in Winery
Brett thrives in red wine barrels, particularly used French oak barrels where the porous wood structure harbours persistent populations that survive sulphur dioxide sanitisation. Key risk factors for Brett proliferation include: residual fermentable sugar (glucose + fructose >100 mg/L post-fermentation creates a substrate for Brett growth); low free SO₂ (free SO₂ below 0.5 mg/L molecular — corresponding to approximately 12–25 mg/L free SO₂ depending on wine pH — fails to inhibit Brett growth); elevated cellar temperatures (Brett growth optimal at 24–28°C; slowed but not halted at 15°C); and long barrel maturation with infrequent topping and monitoring. Molecular SO₂ is the biologically active antimicrobial form: molecular SO₂ = free SO₂ × [1 + 10^(pH−pKa)]⁻¹ where pKa(SO₂) = 1.81. At pH 3.5, a free SO₂ of 30 mg/L yields approximately 0.8 mg/L molecular SO₂; at pH 3.8, the same 30 mg/L free SO₂ yields only 0.4 mg/L molecular — explaining why high-pH red wines are more Brett-susceptible.
Brett: Fault vs Complexity?
The Pomerol–St-Émilion debate is well-documented in the wine literature. Some classic Pomerol (notably Château Le Pin, Vieux Château Certan) and Châteauneuf-du-Pape estates have historically produced wines with detectable Brett character (50–300 µg/L 4-EP range) that received high critical scores, and some importers and critics explicitly describe "Brett complexity." The scientific consensus (AWRI; OIV; Boulton et al. 1999) is that Brett is a fault organism; the commercial reality is that its sensory impact is concentration-dependent and consumer-preference-dependent. AWRI's position (awri.com.au) is unequivocal: 4-EP/4-EG above threshold is a wine fault, irrespective of historical acceptance.
Reductive Faults: H₂S, Methanethiol, DMS
Reductive notes in wine — the family of sulphur-derived off-aromas including struck flint, rotten egg, rubber, cooked cabbage, and garlic — represent one of winemaking's most complex sensory and chemical challenges. The compounds share a sulphur atom but differ fundamentally in origin, volatility, threshold, and winemaker remediation options. Understanding the specific compound is essential for targeted correction.
Hydrogen Sulphide (H₂S) — "Rotten Egg"
H₂S (hydrogen sulphide, thioacid) is produced during primary alcoholic fermentation by S. cerevisiae as a by-product of sulphur amino acid biosynthesis (cysteine, methionine) and by reduction of inorganic sulphur compounds — elemental sulphur (S⁰) from late vineyard sulphur applications, sulphates (SO₄²⁻), and sulphites (SO₃²⁻). The central pathway is sulphur assimilation: SO₄²⁻ is reduced via the sulphate reduction sequence to H₂S; if yeast nitrogen demand is satisfied and the H₂S is incorporated into cysteine, no net H₂S is released. Under nitrogen-deficient conditions — a common winery occurrence — the pathway backs up and H₂S is released into the fermenting must at concentrations well above its detection threshold of 1 µg/L. H₂S is the most volatile of the sulphur compounds (bp −60°C) and dissipates readily: splash racking, vigorous aeration, or addition of copper sulphate (CuSO₄, at 0.2–0.5 mg/L Cu²⁺ — which precipitates CuS) are standard remediation tools.
The "Struck Flint" Note in Chablis
Chablis and certain other cold-climate Chardonnays are often described as having a "struck flint," "gunflint" (French: pierre à fusil), or "minerality" note. The attribution of this character to terroir, to chalk or Kimmeridgian limestone soils, or to simple sulphur chemistry has been extensively debated. The consensus emerging from multiple analytical studies — including AWRI research and work by Tominaga, Murat, and Dubourdieu's group at ISVV Bordeaux — is that the struck flint note is primarily chemical rather than geological: it arises from a combination of dimethyl sulphide (DMS) at sub-threshold concentrations (10–25 µg/L range, below perception threshold but modulating other aromas — a known matrix effect), methanethiol at near-threshold concentrations, and certain volatile thiol compounds (notably 3-methylthio-1-propanol, methional) formed during fermentation under cool conditions and from sur lie ageing. The Kimmeridgian marl hypothesis — that the unique fossil-bearing soil directly imparts a flint character to the wine — is not supported by analytical evidence; the soils do not contain volatile sulphur compounds that could be taken up by vines. Cool fermentation temperatures (10–13°C for Chablis) enhance retention of volatile compounds.
Prevention: Yeast Nutrition Management
The most effective management of H₂S and reductive fault prevention is proactive yeast nutrition management during primary alcoholic fermentation. Diammonium phosphate (DAP) and organic yeast nutrients (yeast hulls, yeast autolysates) provide assimilable nitrogen (YAN — yeast assimilable nitrogen, the sum of free amino nitrogen [FAN] and ammonium ions). Target YAN depends on must sugar concentration: generally 150–250 mg N/L for musts of 22–26 °Brix. Nutrient addition timing matters: DAP additions at inoculation and at approximately ⅓ sugar depletion (avoiding late additions above 5 °Brix remaining, which can leave residual nutrients and encourage late Brett growth). AWRI's "Go-Ferm + Fermaid O + Fermaid K" protocol provides sequential organic then inorganic nitrogen to minimise sulphur stress across the full fermentation arc.
Oxidation and Volatile Acidity
Acetaldehyde — The Primary Oxidation Compound
Acetaldehyde (ethanal, CH₃CHO) is wine's most abundant aldehyde and its principal oxidation product. It forms by the oxidation of ethanol (enzyme-catalysed by alcohol oxidase in microbial systems; abiotic oxidation via metal-catalysed reactions in the glass/tank). Acetaldehyde is also a normal fermentation intermediate — S. cerevisiae produces large quantities during fermentation via pyruvate decarboxylase, immediately reducing it to ethanol via alcohol dehydrogenase (ADH). In finished wine, acetaldehyde concentrations should be below 75–100 mg/L for "fresh" wines; concentrations above this produce a characteristic oxidised, bruised apple or flat, stale character.
In fino and manzanilla Sherry, acetaldehyde at 300–400 mg/L (produced by the flor yeast S. cerevisiae var. beticus surface film) is a desirable, defining characteristic — the almendrado (almond) and camomile note that distinguishes these wines. This illustrates the concentration-dependence of "fault" vs "character": acetaldehyde below 75 mg/L is unremarkable; 100–200 mg/L is an oxidative fault in most contexts; 300+ mg/L under a flor cap is a treasured stylistic feature.
SO₂ and Acetaldehyde Binding
Free sulphur dioxide reacts reversibly with acetaldehyde to form 1-hydroxyethane-1-sulphonate (acetaldehyde-bisulphite addition complex) — chemically "binding" the acetaldehyde. This is the primary mechanism by which SO₂ reduces oxidative character: each mole of acetaldehyde requires one mole of bisulphite SO₂ for binding. The "SO₂ binding capacity" of a must is substantially determined by its acetaldehyde content (plus keto-acids, glucose, gluconic acid from Botrytis-affected musts). Winemakers calculate "bound SO₂" when measuring total SO₂ — total SO₂ = free SO₂ + bound SO₂. EU maximum total SO₂: 150 mg/L (dry red); 200 mg/L (dry white/rosé); 300 mg/L (sweet wines); 400 mg/L (Sauternes, Trockenbeerenauslese, Eiswein) under EC Reg 606/2009.
Volatile Acidity (VA)
Volatile acidity (VA) is the fraction of wine acidity that is steam-distillable — primarily acetic acid (CH₃COOH, ethanoic acid), with minor contributions from formic, propionic, and butyric acids and from ethyl acetate. Acetic acid is a normal fermentation metabolite produced in small quantities by S. cerevisiae via acetaldehyde oxidation (the reverse of ethanol synthesis from acetaldehyde). Unacceptable VA levels result from acetic acid bacteria (Acetobacter spp., Gluconobacter spp.) oxidising ethanol to acetaldehyde and then to acetic acid in the presence of oxygen, or from LAB (particularly heterofermentative strains) producing acetic acid as a co-product of hexose fermentation.
Normal finished wine VA: 0.3–0.6 g/L acetic acid for dry wines. Botrytis-affected wines (Sauternes, Tokaji, TBA) may contain 0.8–1.0 g/L intrinsically, as Botrytis produces acetic acid directly. Historic vintages and naturally sweet wines (some Mosel Auslesen, certain orange wines) may approach or slightly exceed the EU dry wine limit while being stylistically accepted by the market. OIV recommends analysis by steam distillation (Cazenave–Fernandez method) or enzymatic assay (L-D lactic acid/acetic acid kits).
| Wine Category | Max VA (g/L acetic acid) | Typical "Healthy" Range |
|---|---|---|
| Dry red wines | 1.20 g/L | 0.3–0.6 g/L |
| Dry white and rosé wines | 1.08 g/L | 0.2–0.5 g/L |
| Wines with residual sugar ≥5 g/L | Higher limits; see Regulation | 0.4–0.8 g/L |
| Sauternes / Beerenauslese / TBA | Exception: 1.80 g/L | 0.7–1.2 g/L (Botrytis-derived) |
| Ethyl acetate sensory threshold | — | ~160–200 mg/L (nail varnish note) |
Temperature Control in Fermentation
Fermentation temperature is the winemaker's most powerful single variable for determining wine style. Temperature affects three independent dimensions simultaneously: (1) yeast kinetics — warmer temperatures accelerate fermentation rate, risking stuck fermentation at extremes; (2) aromatic retention — cooler temperatures reduce evaporative loss of volatile esters and terpenes during fermentation; and (3) phenolic extraction in red winemaking — warmer temperatures increase anthocyanin and tannin extraction from grape skins into the fermenting must.
Cool White Wine Fermentation (10–15°C)
White and aromatic grape varieties fermented at 10–15°C (commercial range 12–15°C; Sauvignon Blanc sometimes 10–12°C) produce wines with markedly higher concentrations of ester aromatics — principally isoamyl acetate (banana, ≤5 mg/L at cool vs ≤2 mg/L at warm), ethyl hexanoate (apple, pear), and ethyl octanoate — retained because low temperature suppresses evaporative stripping of volatile compounds by CO₂. Thiol precursor conversion is also temperature-sensitive: the β-lyase enzymes responsible for liberating 3-mercaptohexanol (3-MH) and 3-mercaptohexyl acetate (3-MHA) from cysteine-conjugated precursors are more active at 14–18°C than above 20°C (Dubourdieu group, ISVV Bordeaux, 2002). For Riesling, cool fermentation (10–13°C) is essential for preserving linalool, geraniol, and other monoterpene floral compounds.
The tradeoff: at 10–12°C, S. cerevisiae fermentation slows to 2–4 weeks (vs 5–7 days at 25°C), increasing the risk of stuck fermentation if yeast health is poor, and extending tank occupancy. Cold fermentation is commercially feasible only with temperature-controlled stainless steel tanks (jacketed or internal coil refrigeration). In pre-refrigeration traditional winemaking, white wines fermented in cool underground cellars at ambient temperatures of 10–14°C (Chablis in autumn, German Mosel cellars) were effectively conducting cool fermentation by default.
Warm Red Wine Fermentation (22–32°C)
Red wine fermentation operates at substantially higher temperatures for a fundamentally different reason: phenolic extraction. Anthocyanins (the colour compounds in red grapes, concentrated in berry skins, not pulp) extract faster at elevated temperatures — peak extraction of monomeric anthocyanins occurs in the first 3–5 days of fermentation at 25–28°C. Tannins (proanthocyanidins, condensed tannins from skins and seeds) co-extract with anthocyanins and bind them into more stable pigmented polymers over time; at cool fermentation temperatures (below 20°C), tannin extraction is inadequate and wines lack structure. The standard commercial range for premium red fermentation is 24–28°C, with "cold soak" (pre-fermentation skin contact at 5–10°C for 24–72 hours) used increasingly to selectively extract anthocyanins before fermentation begins — without extracting astringent seed tannins which are less temperature-sensitive.
The upper boundary for red fermentation temperature is approximately 32–34°C: above this, S. cerevisiae begins to experience heat stress (expression of heat shock proteins HSP26, HSP42 at 34°C; loss of fermentative capacity above 37°C; cell death at 40°C). For high-alcohol regions (Barossa Valley at harvest Brix 14–15°, Châteauneuf-du-Pape in warm vintages), fermentation exotherm management is critical — must entering at 25°C can self-heat to 36–38°C during peak fermentation without active cooling, risking stuck fermentation at 40% sugar depletion.
| Temperature Range | Application | Primary Effect | Key Tradeoffs |
|---|---|---|---|
| 5–10°C | Cold soak (pre-fermentation skin contact) | Selective anthocyanin extraction before yeast activity; colour without astringency | Risk of native yeast/LAB activity; requires SO₂ or dry ice |
| 10–13°C | Sparkling base wines, high-acid whites | Aromatic retention; very slow fermentation rate | Long tank time; stuck fermentation risk |
| 13–16°C | Sauvignon Blanc, Riesling, Pinot Gris | Thiol preservation; ester retention; floral monoterpene preservation | Moderate tank time; requires refrigeration |
| 16–20°C | Chardonnay (unoaked/semi-oaked), Viognier | Balance of aromatic retention and fermentation efficiency | — |
| 20–24°C | Light reds (Pinot Noir, Gamay), rosé | Moderate extraction; fruity style; lower tannin | Limited skin contact recommended |
| 24–28°C | Premium red wine (Cabernet, Syrah, Nebbiolo) | Optimal anthocyanin and tannin extraction; colour stability; structure | Fermentation exotherm monitoring required; cooling capacity needed |
| 28–32°C | High-colour warm-climate reds (Barossa Shiraz); brief peak | Maximum phenolic and colour extraction; full body | Yeast stress risk; acetic acid bacteria risk above 30°C; careful monitoring essential |
| >34°C | Danger zone | Yeast heat stress; stuck fermentation risk | Active cooling mandatory; HSP proteins expressed; volatile acidity risk escalates |
Carbonic Maceration: Temperature as Biochemical Switch
Beaujolais Nouveau and many Gamay wines use carbonic maceration (whole-cluster fermentation under CO₂ atmosphere), where intracellular fermentation enzymes within intact grape berries (not yeast) convert sugars to ethanol at 30–32°C within the berry cells — before the berries rupture and conventional yeast fermentation begins. Temperature is critical: the intracellular enzymes responsible (particularly malic acid catabolism to ethanol and CO₂) are most active at 30–32°C; at cooler temperatures (below 20°C) the carbonic maceration reactions are incomplete and the wine lacks the classic banana/kirsch/bubblegum character (principally from methyl anthranilate, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, and isoamyl acetate produced intracellularly). OIV Resolution OIV-OENO 33-1997 formally defines carbonic maceration as a permitted winemaking practice.