What's in the Wine Microbiome?

The thousand species of yeast, bacteria, and filamentous fungi that are abundant in the soil, on the vine and the fruit, and even in the cellar form communities known as microbiomes. These organisms are present at various stages in the vineyard and throughout the winemaking process. Substantial yeast and bacterial biodiversity exist at different times and interact throughout the entire process.¹

Many researchers have sought to define the influences of numerous bacteria and fungi on the chemical and sensory properties of wine. While some grape/wine microorganisms can have a negative impact on wine quality, such as the bacteria Acetobacter spp. and the yeast Brettanomyces spp., some microorganisms are beneficial and can enhance sensory complexity of wines.² Potentially, these post-harvest microorganisms could be used as an early predictor of wine chemical composition. Several researchers have shown that grape and wine microorganisms exhibit regional patterns that correlate with wine chemical composition, suggesting that it may influence terroir.³ Other factors that contribute to regional microbial patterns include soil type, climate, topography, viticultural practices, and winemaking techniques.⁴

In this article, we will discuss the changing microbiome throughout the fermentation process, techniques to characterize the microbiome, and finally, the importance of interactions among organisms.

Grapes and Must

Yeast

Grapes are the main natural reservoir of native yeasts. Researchers have reported 93 different yeast species belonging to 30 different genera that were isolated from 49 different grape varieties growing in 22 countries. Renouf et al. (2007) identified 47 yeast species belonging to 22 different genera on fruit.⁵ These yeasts were isolated from the surface of grape berries of six different varieties. Several species of non-Saccharomyces yeast are commercially available as coinocula with Saccharomyces yeasts, which can increase the sensory complexity of wines.

CFU stands for "colony forming unit" and is a measure of viable bacterial or fungal cells present when grown in culture. Although large numbers of yeast species are identified on grapes, the actual cell populations are low. Yeast populations on immature grapes range from 10^1 to 10^3 CFU per gram but increase to 10^3 to 10^6 at harvest time.^6,7 Saccharomyces cerevisiae is infrequently found on clean fruit (compared to other species), but fruit that is damaged may increase its growth.⁸ Some researchers have reported isolation of Brettanomyces on both healthy and sour-rot infected grapes.⁹

The growth of yeasts on the berry surface may be related to the increased surface area of each berry and to the availability of nutrients. During maturation, the berries increase in size; more nutrients are available on the surface of the berries, and sugar concentration increases. The decrease in acidity may also stimulate growth.¹⁰

Several studies report that yeast diversity is dependent on both climatic and micro-climatic conditions. In the vineyard, the microclimate is the climate from the soil upward into the vine canopy, and that microclimate has a significant impact on wine quality. For example, some studies reported higher yeast counts for vintages with high rainfall, which is probably due to substantial fungal proliferation.¹⁰

The health status of berries can also affect the diversity of yeasts. For example, Botrytis cinerea can penetrate the surface and release nutrients, possibly altering the microorganisms on the grape surface.¹¹

Bacteria

In order to minimize the faults caused by LAB and AAB genera, winemakers need to understand the metabolic requirements of these two different bacteria. Control of their growth in a wine depends on how the winemaker manages the oxygen in that wine. LAB genera are classified as a facultative anaerobe as they grow either in the presence or absence of oxygen. In the presence of oxygen, they grow more robustly and express their faults, but it should be noted that they still grow in the absence of oxygen. AAB genera are obligate aerobes, which means they require oxygen to grow. However, they don't die without oxygen—they only lie in wait for oxygen to become available.

A review by Barata et al. (2012a) lists over 50 bacterial species that have been identified on grape berries.⁸ The species isolated mostly belong to two groups: gram-positive lactic acid bacteria (LAB) and gram-negative acetic acid bacteria (AAB). These organisms are separated into two groups based on their different staining reactions, which is the result of differences in cell wall composition. Other organisms recovered include Bacillus spp. and Enterococcus spp. LAB include organisms such as Lactobacillus and Pediococcus as well as Oenococcus. While lactic acid bacteria are the typical agents of malolactic fermentation, Oenococcus oeni has seldom been isolated from the vineyard. The AAB are strictly aerobic bacteria and include Acetobacter and Gluconobacter spp.⁸

Analysis of grape berry bacterial microorganisms revealed changes in the size and structure of the population during berry ripening, with levels rising gradually and reaching their highest level when berries were overripe. Research has found that as the grapes reach maturity, gram-negative communities such as Acetobacter decline, whereas gram-positive communities such as LAB genera increase.¹² A study by Martins et al. (2012) found that at harvest time, averages of the different microbial populations were around 103 CFU per berry for gram-negative aerobic or anaerobic bacteria and 104 CFU per berry for gram-positive bacteria.

AAB are often detected on healthy grapes.⁷ AAB populations are stimulated by berry damage and grow to around 10⁶ CFU per berry on rotten grapes.¹³ Typically, winemaking conditions result in loss of these strictly aerobic bacteria, although they have also been found to survive in the absence of oxygen, but this is usually not the case.¹⁴ Since some AAB may survive the fermentation; they can be implicated in wine spoilage downstream. The most serious consequence of AAB spoilage is the production of high levels of acetic acid.

Other organisms, generally considered as spoilage organisms, can also grow on grapes, including filamentous fungi such as Aspergillus and Penicillium, and may greatly influence the sensory quality of wine through the production of mycotoxins or off-flavors.¹⁵ Downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator), and gray mold (Botrytis cinerea) are also capable of producing off-flavors.¹⁶

Alcoholic and Malolactic Fermentation

Yeast

Once grapes are crushed, non-Saccharomyces yeasts multiply and reach peak populations in the early stages of alcoholic fermentation. Populations of non-Saccharomyces may be as high as 106 to 108 CFU/ml, depending on production conditions.¹⁷ Hanseniaspora/Kloeckera are normally the dominant native yeasts present on grapes at harvest, but their activity may be limited to pre-fermentation and early stages of alcoholic fermentation and may decrease as alcohol concentrations increase. Saccharomyces normally completes the alcoholic fermentation (AF). However, fermentations conducted at temperatures less than 15–20°C may decrease the sensitivity of these species to ethanol. If these species equal S. cerevisiae as the predominant species at the end of fermentation, they may have an impact on wine flavor either positively or negatively.¹⁸

Other yeasts that may be isolated from grapes and must include species of Brettanomyces, Dekkera (the sporulating counterpart of Brettanomyces), Candida, Pichia, Hansenula, and Torulopsis.¹⁹ Diseased and damaged grapes harbor significantly more spoilage yeasts that can negatively affect the fermentation. These yeasts can metabolize the sugar in the grapes, which contain 160 to 240 g/L, mainly in the form of glucose and fructose. In addition, the spoilage yeast species can result in strong off-flavors and may be tolerant to sulfur dioxide.

Bacteria

Like yeasts, lactic acid bacteria (LAB) are also found in the AF. LAB isolated from musts and wines include L. brevis, O. oeni, and Pediococcus. The typical spoilage times for LAB are during stuck fermentations and in finished wines containing low SO₂ and residual malic acid or sugar.

In addition to the production of acetic acid through the metabolism of citric acid as well as glucose, LAB can result in several other faults, such as mousiness, geranium taint, and ropiness. Mousey taint is an aftertaste. It is not volatile at wine pH, but when mixed with the neutral pH of saliva, it becomes apparent. The taste is described as mouse urine and rancid nuts. This taint is usually the result of LAB activity but can also be caused by Brettanomyces. Geranium taint is caused by the metabolism of sorbic acid by LAB. Sorbic acid is a yeast inhibitor added to prevent refermentation in the bottle. Although generally effective as a fermentative yeast inhibitor, sorbic acid shows little inhibition of LAB, AAB, or film yeasts.²⁰

Some strains of LAB, such as Oenococcus oeni, are beneficial. This bacterium is involved in the decarboxylation of malic acid to lactic acid during malolactic fermentation (MLF). This reaction increases pH and results in a "softer" mouthfeel. Diacetyl is also produced, which creates a "buttery" character. One to four mg/L of diacetyl is considered desirable—depending on wine style—while high concentrations (more than 5–7 mg/L) are considered a spoilage characteristic.

In addition to the sensory implications, acetic acid and products of LAB metabolism can act as inhibitors to Saccharomyces. This may cause a delay in the onset of fermentation or may result in a stuck fermentation later on. A sluggish fermentation should never be inoculated with malolactic bacteria. The bacteria can metabolize glucose and fructose to acetic acid, increasing VA by 1 g/L or more.

Bacteria in the acetic acid bacteria group (AAB) include Acetobacter and Gluconobacter. They use ethanol (and glucose) aerobically to form acetic acid. Of the two, Acetobacter is the more commonly encountered. Acetobacter can grow in barreled or bottled wines and use small amounts of oxygen absorbed during clarification and maturation. The most serious consequence of spoilage by AAB is the production of high levels of acetic acid (volatile acidity), as previously mentioned.²⁰

Where fruit deterioration has not occurred, and alcoholic fermentation begins quickly, populations of AAB decline to less than 100 CFU/ml. Gluconobacter is unable to survive the alcoholic environment of wine, even when aerated.²¹ In slowly fermenting or stuck AF, where carbon dioxide levels may be insufficient to prevent oxygen uptake, AAB may be able to grow.

Post-Fermentation

Yeast

During the aging of wines, several different yeasts and bacteria grow, some of which are capable of causing spoilage. The distribution of yeast species in cellar-aging wines includes Dekkera/Brettanomyces, film yeast such as Candida and Zygosaccharomyces.

The most common form of yeast spoilage is due to Brettanomyces bruxellensis. This yeast produces volatile phenols and acetic acid. Examples of flaws include aromas described as "medicinal" in white wines and "leather" or "horse sweat" in red wines. Other aroma descriptors include barnyard, wet dog, tar, tobacco, creosote, plastic, and band-aid.

Brettanomyces can infect red wine 6–10 months after barreling and can spoil bottled wines as well. It can also be transmitted by fruit flies. It may grow on the disaccharide cellobiose, a by-product of toasting in barrel production. Control of this yeast is difficult because it is tolerant to sulfur dioxide.²⁰

Bacteria

The typical spoilage times for LAB include finished wines with low SO₂ and residual malic or sugar. In addition to the production of acetic acid through the metabolism of citric acid as well as glucose, LAB can result in several other faults, discussed previously. AAB growth may be encouraged by autolysis of wine yeasts and O. oeni due to an increased nutrient supply. Please refer to the previous section for discussion of AAB growth implications.

Characterizing the Microbiome

Traditional microbiological methods involving isolation in or on nutritive media can lead to mixed results. Microbes constituting less than 1% of the total population cannot be detected, and these methods may fail to detect viable but not culturable (VBNC) organisms.²² Microorganisms in the VBNC state fail to grow on microbiological media yet still display low levels of metabolic activity. The development of molecular methods such as DNA/RNA amplification allows a more complete and comprehensive view of microbial diversity.⁵

Various researchers utilize the amplification of bacterial 16S rRNA [the internal transcribed spacer (ITS) region is amplified for fungi] to detect the organisms present on grapes and in wine. By amplifying this highly conserved region of the genome, the polymerase chain reaction (PCR) amplifies a few sequences to generate millions of copies that enable identification of these strains, allowing detection and quantification. Each organism has a signature sequence that permits a rapid image of the population of the microbial population at a certain stage.²³

Microbial Interactions

Many factors affect the microbial ecology of wine production, with the chemical composition of the juice and fermentation processes being most important. Where mixtures of different species of yeast and bacteria exist, there is the possibility that interactions will occur between microorganisms.

The first significant interactions between microorganisms occur on the surface of the grapes in the vineyard. Interactions continue throughout AF by yeast²⁴ and the malolactic fermentation by LAB.²⁵

Early growth of yeasts in grape juice can result in a decrease of available nutrients, and, as a consequence, the wine cannot support additional microbial growth. In addition, such growth produces a variety of metabolites, some of which may be toxic to other organisms. The inhibitory effects of ethanol and short-chain fatty acids on some microorganisms are well documented.^26,27 Carbon dioxide production and purging of the juice/wine can limit exposure to oxygen, limiting the growth of aerobic species such as AAB.

Some species may produce inhibitory peptides, proteins, or glycoprotein and enzymes that destroy other species by lysis, a process by which the cell is destroyed due to rupture of the cell wall or membrane. However, there are also mechanisms which lead to enhanced microbial growth. The large amount of yeast biomass produced during fermentation will die and autolyze, releasing amino acids and vitamins. These may encourage the growth of other species later in production.²⁷ In addition, this biomass may function as a bio-adsorbent to remove toxic substances (e.g., metal ions, grape phenols). Proteolytic and pectolytic yeast species may hydrolyze the proteins and pectins in juice to produce substrates (the material upon which an enzyme acts), resulting in possible growth of other species.²⁸

Damage to the skin and surface of grapes increases the availability of nutrients for microbial growth and encourages an increased population (>10⁶ CFU/g) and diversity of yeasts. These yeasts are required to co-exist with other organisms, such as filamentous fungi, acetic acid bacteria, and lactic acid bacteria, which will also grow on damaged fruit.²²

One example of yeast-bacteria interaction is malolactic fermentation. Growth of O. oeni during this fermentation decreases wine acidity by transforming l-malic acid into l-lactic acid. Wine flavor and complexity are achieved through production of additional metabolites. Microbiological stability of the wine is also achieved by removal of residual nutrients.²⁷

Many factors affect the growth of O. oeni in wines and the progression of the malolactic fermentation. Among these, yeast–bacterial interactions can be very important. Research indicates that the strain/s of S. cerevisiae responsible for the alcoholic fermentation can inhibit the growth of O. oeni and thus the malolactic fermentation.¹⁹ The relationship is very much strain-dependent at both the yeast and bacteria levels.

Yeast-yeast interactions, as with others, can be negative or positive. Ethanol produced by S. cerevisiae is the major compound affecting the variety of yeasts during fermentation.²³ Most indigenous yeasts do not survive above 3–10% (v/v) ethanol concentration. However, some non-Saccharomyces yeasts can survive until the end of AF due to their high resistance to alcohol. Examples include Torulaspora delbrueckii, Zygosaccharomyces bailii, and Pichia spp.¹⁸

One of the most well-known examples of negative interaction involves the growth of one strain that is restricted by the coexistence of another and by metabolite secretions. The most extreme example is the killer phenomenon. This involves the production of specific extracellular proteins and glycol proteins by certain yeast strains (killer yeasts) that kill other strains that are more sensitive.²⁹ This phenomenon contributes to the presence of various yeast strains throughout fermentation.

Non-Saccharomyces yeast strains have been shown to have antimicrobial activity against other non-Saccharomyces yeasts. They also expressed antimicrobial action against undesired spoilage yeasts including Brettanomyces/Dekkera.

Commercial strains of non-Saccharomyces wine yeasts are available. Typically, the non-Saccharomyces yeast is inoculated, then followed by a Saccharomyces strain. This allows the winemaker to mimic "wild" fermentations in a controlled setting.

Conclusions

Wine is a product of many varied interactions between yeast, fungi, and bacteria. These relationships start in the vineyard and continue throughout the winemaking and storage process. These relationships can have either a positive or negative influence on wine quality.

Increased control of natural fermentations or fermentation by multistarters requires a better understanding of interaction mechanisms. It is established that when two yeasts co-ferment, it impacts the aromatic profile. Research in the field of the wine microbiome could have a positive impact on production practices. While some strains may produce off-flavors, a program of selection and evaluation could result in the identification of those strains with desirable flavor attributes.

Research has demonstrated that understanding the microbiome could potentially provide tools to winemakers to improve wine characteristics or lessen the incidence of problem fermentations. Such information could be practical for predicting the suitability of potential vineyards or for preventing microbiological issues in abnormal vintages.

There may be several promising applications for grapevine and wine-fermentation management, with the opportunity to develop tailored strategies for improving grape and wine quality of individual varieties. Customized fermentation management strategies could improve product outcomes through moderating sulfite additions, temperature control, oxygen limitation, inoculation, or cold maceration to promote or suppress individual populations based on the microbial composition of a given grape variety.³⁰ This potentially powerful way to manage the winegrowing process may be viewed as a manipulation of terroir, but it also offers an exciting area for future research.

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