Brettanomyces in enology: physiology and mechanisms of spoilage.

Why Brettanomyces is still an unresolved issue

Although Brettanomyces bruxellensis has been the subject of intensive scientific research for more than twenty years, it remains one of the most feared microorganisms in the cellar. Its biological plasticity makes each situation a case in itself: the same enological practice can produce completely different results depending on the strain present, the chemical profile of the wine and the production stage considered.

The spread of the problem has increased globally over the past fifteen years. In Italy, until the late 2000s, Brettanomyces was not considered a management priority in wineries; today, the situation has changed radically, with documented contaminations even in young wines and in areas traditionally not exposed to the issue. The main enabling factor is climate change: the increase in the average pH of grapes — as a consequence of later harvests and higher sugar levels — has made many wines structurally more favourable to the development of Brettanomyces. At a pH above 3.5, the yeast multiplies effectively, while sulfur dioxide progressively loses its active molecular fraction, drastically reducing its antimicrobial power at the same added dose.

Physiology of Brettanomyces: similarities and differences with Saccharomyces

From a taxonomic point of view, the term Brettanomyces bruxellensis and its synonym Dekkera bruxellensis refer to the same species: Dekkera is the ascosporogenous form, while Brettanomyces is the non-sporulating form. In wine, the non-sporulating form is almost exclusively isolated, as enological conditions do not allow sporulation. Scientific literature uses both names interchangeably.

Brettanomyces shares some fundamental characteristics with Saccharomyces cerevisiae: facultative anaerobic metabolism, fermentative capacity, ethanol tolerance — higher than that of many non-Saccharomyces yeasts, with documented growth up to alcohol levels of 13–14% vol — and sulfite resistance. This clearly distinguishes it from most wild yeasts. However, the differences are equally significant: Brettanomyces grows slowly in the presence of high sugar concentrations — actively fermenting must is not its optimal habitat —, assimilates carbon sources other than simple sugars, including ethanol and glycerol, and is perfectly adapted to surviving in the presence of oxygen, effectively colonising the wooden surfaces of barrels and winery equipment.

Its genome, sequenced in 2011, contains around 3,000 genes, of which 2,600 are orthologous to those of Saccharomyces cerevisiae. Among the functionally characterised genes are those encoding phenylacrylate decarboxylase (PAD) and vinylphenol reductase (VPR) — the two key enzymes involved in the production of volatile phenols — as well as a set of stress-response genes that differ significantly from their Saccharomyces homologues, explaining the greater resilience of Brett in the wine environment.

The enzymatic pathway of volatile phenols

PAD and vinylphenols

The first step in biosynthesis is catalysed by phenylacrylate decarboxylase (PAD), which converts the hydroxycinnamic acids present in wine — p-coumaric acid into 4-vinylphenol, and ferulic acid into 4-vinylguaiacol. It is important to emphasise that PAD is not exclusive to Brettanomyces: indigenous strains of Saccharomyces cerevisiae with a POF+ character (Phenolic Off-Flavour positive) also possess it and can produce vinylphenols during fermentation. However, vinylphenols have relatively high sensory perception thresholds and do not, in themselves, cause a significant organoleptic deviation.

VPR: the exclusive marker of Brettanomyces

Vinylphenol reductase (VPR) converts vinylphenols into ethylphenols: 4-vinylphenol into 4-ethylphenol, and 4-vinylguaiacol into 4-ethylguaiacol. These compounds have much lower sensory perception thresholds — in the range of 140–440 μg/L for 4-ethylphenol — and are responsible for the characteristic stable, leather, wet animal and smoky odours associated with the Brett fault. VPR is currently considered an exclusive functional marker of Brettanomyces: no comparable activity has been identified in any other enologically relevant microorganism.

The expression and activity of VPR vary considerably from strain to strain, which explains why not all contaminations produce the same level of sensory spoilage. A study by Lorenza Conterno showed that approximately 1 in 6 strains of Brettanomyces does not produce volatile phenols in perceptible quantities, despite possessing the genes to do so: gene expression, rather than the mere presence of the gene, is the discriminating factor in strain-specific pathogenicity.

The complete sensory profile: beyond ethylphenols

The spoilage profile produced by Brettanomyces is more complex than ethylphenols alone. Under conditions of oxygen exposure, Brett produces significant amounts of acetic acid, as its metabolism shifts towards an oxidative pathway in the presence of O₂; under normal winery management conditions, production is more limited but may contribute to unexpected increases in volatile acidity. Tetrahydropyridines (THP), responsible for so-called mouse taint, are another class of compounds associated with Brett, although recent studies — including a 2023 analysis — have shown that this fault emerges more frequently in the presence of microbial consortia that include Lactobacillus spp. and wild strains of Oenococcus oeni. Brett also contributes to the production of biogenic amines — histamine and tyramine — with both organoleptic and toxicological relevance.

This composite scenario means that not all animal-like or fermentative off-flavours detected in a wine should automatically be attributed to Brettanomyces: an integrated analytical approach — microbial counts, ethylphenol quantification and identification of the microbial consortia present — is necessary for a correct diagnosis.

Ecology and population dynamics in the winery

The question of the primary origin of Brettanomyces — vineyard or winery — has long been debated. Recent research has shown, through genetic characterisation, that strains isolated from grapes and those resident in the winery do not show systematic genotypic differences: the same strain can colonise both environments indiscriminately. The first contamination may come from the grapes, but in subsequent years the two sources add up.

The highest-risk moment in the production chain occurs in the time window between the end of alcoholic fermentation and the start of malolactic fermentation: sugars are depleted or nearly depleted, ethanol is present, Saccharomyces is losing vitality, and the pH has not yet been stabilised with SO₂. With as little as 300 mg/L of residual sugars, Brett can begin to multiply effectively. Its ability to form biofilms in wood — with documented penetration of up to 6 mm into the stave — and to colonise fittings, hoses and equipment makes it a persistent resident in the winery.

Strain-dependent variability: the central issue

The most relevant characteristic from an applied perspective is the extraordinary intraspecific variability of Brettanomyces. Strains differ in almost all enologically relevant parameters. Tolerance to molecular SO₂ varies significantly: Australian studies have shown strains capable of growing with 0.8 mg/L of molecular SO₂ in an aqueous medium, and others inhibited at as little as 0.4 mg/L; the presence of ethanol further modifies this framework in a strain-specific manner. The balance between PAD and VPR activity is equally variable: some strains mainly accumulate vinylphenols, while others convert almost every intermediate into ethylphenol almost instantly, making them much more dangerous at the same microbial load.

A particularly significant article analysed the growth of five Brettanomyces strains in more than 50 red wines grouped according to their chemical profile. In “permissive” wines — high pH and low free SO₂ — all strains multiplied in a similar way. In “restrictive” wines, however, strain-dependent differences emerged very clearly: only one strain maintained the same aggressive growth dynamics, while the other four showed radically different kinetics. The chemical profile of the wine interacts with the genetic characteristics of the strain in a way that cannot be predicted using standard analytical parameters alone.

This variability has a direct practical consequence: the same management protocol, applied in different vintages or in different wineries with wines of a similar profile, can lead to opposite results simply because the strain present is different. This is why, fifteen years after the sequencing of the Brett genome, there is still no universal and definitive strategy.

The viable but non-culturable state (VBNC)

Among the most insidious characteristics of Brettanomyces is its ability to enter a state known as VBNC (Viable But Non-Culturable), first demonstrated for this yeast by Hervé Alexandre’s group. In this state, cells reduce their volume, slow their metabolism to basal levels and lose the ability to form colonies on Petri dishes: they are not detectable using conventional microbiological methods, but remain viable and able to resume multiplication as soon as the stress pressure ceases. In an emblematic experiment, cells treated with SO₂ and undetectable for 11 consecutive days reappeared and proliferated after SO₂ removal by stripping.

The practical implications are significant: a negative plate count does not guarantee the absence of viable Brettanomyces. The VBNC state makes conventional microbiological monitoring potentially misleading and suggests the adoption of complementary techniques — quantitative PCR and flow cytometry with viability dyes — capable of detecting cells regardless of their culturability status. The extent of the VBNC response also varies among strains, adding a further level of complexity to risk management.

Conclusions

Brettanomyces bruxellensis is a microorganism that, through its association with the wine environment, has developed an effective set of adaptations: alternative carbon sources, biofilms in wood, the VBNC state, and strain-dependent resistance to SO₂. Understanding these mechanisms is not an academic exercise, but an essential prerequisite for building rational control strategies. The second article in this series addresses the tools currently available — from pre-fermentation bioprotection to chitosan, from molecular SO₂ management to future prospects in applied research — with the aim of providing enologists with an up-to-date, scientifically grounded operational framework.