The types of microorganisms that may give rise to microbiological instabilities in wine are listed below:
- Fermentative yeasts, including genera such as:
- Acetic acid bacteria, including genera such as:
These organisms may be tentatively identified by their physical properties, such as cell shape and size (morphology) and by the effects they have on the wine.
In modern winemaking it is common practice to innoculate using ‘cultured yeast’. The yeast strains used in this practice are normally Saccharomyces cerevisiae or Saccharomyces bayanus. Saccharomyces sp. can be a problem in bottled wine containing residual sugar, where even a small number of cells in a bottle can referment, causing cloudiness and eventually sediment, and a yeasty aroma and flavour. Wines containing more than 10 g/L of residual sugar may explode if refermentation takes place.
Morphologically, Saccharomyces sp. yeast are spherical to ovoid in shape (approximately 6 – 8 µm in diameter or approximate dimensions of 6 x 7 µm). See photograph.
Brettanomyces sp. is the non-sexual, non-sporulating form of Dekkera sp., which is the sexual, sporulating form of the yeast. Brettanomyces is generally associated with barrel-aged red wines, although the Institute has observed the effects of this yeast in white wine. Wood cooperage is difficult to clean and Brettanomyces can populate the cracks and crevices within the staves, where they are relatively protected from concentrations of sulfur dioxide that might otherwise inhibit their growth. Because of the nature of wooden barrels, there is no sure way to sterilise a barrel once Brettanomyces is established. It is best to reduce the risk of infection in the first place by paying attention to general cleanliness in the cellar, frequent sanitation of equipment, maintaining adequate SO2 concentrations (ideally 0.8 mg/L molecular SO2), and pH adjustment.
The organoleptic effects of Brettanomyces/Dekkera on wine have been described as medicinal, bandaid (a compound called 4-ethyl phenol), barnyard-like, horsy, wet wool, sweaty saddle and mousy.
Brettanomyces/Dekkera sp. yeast may exhibit variable cell morphology, however, the cells generally appear rectangular or elongated (2 – 10 x 5 – 45 µm) when isolated directly from wine. They have also been described as boat-shaped, ogival (having one arch-shaped end), cigar-shaped and apiculate (lemon-shaped). To see a photograph of Dekkera bruxellensis cells, click here.
Kloeckera sp. is the non-sexual, non-sporulating form of Hanseniaspora sp., which is the sexual, sporulating form of the yeast. These fermentative yeast are often the dominant species found on the grapes at harvest , and occur in abundance in unsulfited must/juice during the early stages of fermentation. Both Kloeckera and Hanseniaspora are capable of producing high concentrations of acetic acid and ethyl acetate during the early stages of fermentation.
Morphologically, Kloeckera and Hanseniaspora are apiculate (lemon-shaped) or ovoid to elongate, with approximate dimensions of (1.5 – 5.0) x (2.5 – 11.5) µm.
Zygosaccharomyces, like Saccharomyces, is most often a problem in bottled wine containing residual sugar. Zygosaccharomyces is able to grow at sugar concentrations of greater than 70% v/v and often originates from grape juice concentrate, which can be added to wine at bottling to provide sweetness. Zygosaccharomyces is also tolerant of high ethanol concentrations, low pH, sorbic and benzoic acids and sulfur dioxide, so the best way to avoid problems with this organism is to sterile filter the wine at bottling.
The odours and flavours produced by Zygosaccharomyces are generally described as being wine-like, so the main problem with this yeast is the formation of a haze or deposit after bottling.
Morphologically, Zygosaccharomyces cells are ovoid, ellipsoidal or cylindrical, (3.5 – 7.0) x (5.5 – 14.0) µm, in pairs, short chains or clusters. Zygosaccharomyces tend to appear club-shaped or dumbbell-shaped before sporulating.
A photograph of a yeast suspected to be Schizosaccharomyces sp. is shown here.
Small but significant populations of surface yeasts, such as Candida, Pichia and Hansenula are present on mature, sound grapes, and have also been isolated from winery equipment that comes in contact with grape juice and wine. The growth of surface, or film yeasts is reduced during fermentation because they prefer oxidative conditions, however, significant growth may occur during the early stages of fermentation. The growth of film yeasts becomes more pronounced during storage in vessels which are not completely filled, where they grow as a wrinkled film or scum on the surface of wine exposed to air. Surface yeasts can survive fermentation and under oxidative conditions form acetaldehyde, acetic acid and ethyl acetate, by utilising substrates such as ethanol, glycerol and organic acids.
Film yeasts do not usually cause problems in bottled wine, since they require oxygen for their metabolism, however, they can grow for a short period of time after bottling, until any oxygen present is used up. The yeast will then settle to form a deposit, and although extensive growth is required to impart a negative effect on wine aroma, the presence of a sediment will reduce the marketability of the wine, especially white wine bottled in clear glass.
Since they grow oxidatively, surface yeasts are best controlled by keeping tanks and barrels full and sealed. Control is assisted by maintaining low pH, high SO2 concentration and low temperature, however, removal of surface yeasts requires filtration.
Candida typically grows as a chalky-white film on the surface of low alcohol wines, producing acetic acid and other oxidised end-products from growth on ethanol and organic acids. Concentrations of ethyl acetate ranging from 220?730 mg/L have been reported for Candida krusei.
Morphologically, Candida cells are ovoid to long and cylindrical, with approximate dimensions (5-10) x (2-4) µm, and occur singly, in pairs, short chains and small clusters.
A photograph of a surface yeast isolated from wine suspected to be Candida sp. is shown here.
Pichia grows as a heavy, chalky-white ‘bubbly’ film on the surface of wine. Most species are inhibited by alcohol concentrations of about 10% v/v, however, growth may be found in wines of up to 13% v/v alcohol, depending on temperature.
Pichia cells appear as short ellipsoids to cylindrical-shaped rods when actively growing.
Hansenula sp yeast are capable of producing large amounts of acetic acid and ethyl acetate. In addition, the species Hansenula anomala is capable of utilising malic acid which can result in substantial decrease in titratable acidity and increase in pH.
Hansenula anomala appear as ovoid to oblong-shaped, approximately (2-4) x (2-6) µm, and occur singly, in pairs, or in small clusters. Hansenula sp. reproduce by asexual budding, and often cells do not separate, which results in the formation of pseudomycelium.
Lactic acid bacteria (LAB) are defined as those bacteria which produce lactic acid from fermentation of glucose. However, winemakers are more concerned with the ability of certain LAB to perform the so-called ‘malolactic fermentation’, whereby L-(-)-malic acid is decarboxylated to form L-(+)-lactic acid. Lactic acid bacteria have been isolated from grapes and vine leaves (although in low numbers) and from winery equipment.
Malolactic fermentation (MLF) is desirable in premium red wines and some white wines, however, spoilage may occur if MLF proceeds in the bottle, in which case dissolved carbon dioxide, haze and sediment result. If this occurs, the wine will require decanting, filtering and rebottling. It should be noted that wines that have completed MLF still contain nutrients (such as pentose sugars, organic acids and glycerol) that can support significant bacterial growth, and that completion of MLF before bottling merely reduces the risk of bacterial activity after bottling.
The genera of LAB involved in deposits and hazes in bottled wine are Oenococcus, Lactobacillus and Pediococcus, and these are discussed briefly below.
Generally, Oenococcus oeni, (formerly known as Leuconostoc oenos), is the species of LAB preferred by winemakers to conduct the MLF, and many inoculate their wines with commercial strains of this organism. Oenococcus oeni is most suitable for conducting the MLF because it ferments relatively rapidly and produces few off-odours.
Oenococcus oeni are small coccoid bacteria (diameter approximately 0.5 – 1 µm) which are readily identified by their characteristic ?chain of pearls? configuration (Shown here).
Bacteria of this genus are capable of conducting the MLF, but can also cause spoilage, particularly in the presence of a significant concentration of residual sugar. Lactobacillus sp. can also cause spoilage in dry wines which have completed malolactic fermentation, by metabolism of other constituents including pentose sugars, organic acids and glycerol. In addition, Lactobacillus sp. can produce acetic acid under aerobic conditions, so the conditions under which the wine is stored will determine the nature and extent of any spoilage caused by this microorganism.
Lactobacillus are thin, rod-shaped bacteria and can occur as single cells, pairs or short chains (see picture).Generally, their dimensions are approximately (0.5 – 1) x (1-2) µm, although some species such as L. hilgardii and L. fructivorans, which are important in the spoilage of high alcohol dessert wine, occur as very long rods resembling fibres.
Pediococcus sp. are often slow fermenters and can leave wine with a dirty, unpleasant vegetative smell. Since Pediococcus utilise residual malic acid slowly, they are often responsible for the completion of post-bottling MLF. The precise spherical cell morphology (diameter approximately 1 – 1.5 µm) of the pediococci makes them easily identifiable. Furthermore, they usually appear as diplococci (two cells joined together) or tetrads (four cells joined together), helping to confirm the classification. (See photograph).
Acetic acid bacteria belong to the family Acetobacteriaceae, commonly known as vinegar bacteria, and are often responsible for the vinegary spoilage of wines through the production of acetaldehyde and acetic acid from ethanol (and glucose). Acetic acid bacteria are generally described as being obligate aerobes (Boulton et al. 1996), and even short periods of exposure of the wine to oxygen, which occurs during operations such as racking and transfering, are enough to encourage significant growth of these bacteria (Joyeux et al 1984).
The two genera of acetic acid bacteria that are important to the wine industry are Acetobacter and Gluconobacter. Gluconobacter are normally isolated from grapes and must since they have a preference for sugar (Drysdale and Fleet 1989) and appear to be intolerant of high concentrations of ethanol (Joyeux et al. 1984). Acetobacter species are more tolerant of ethanol and may survive fermentation to have an unfavourable effect on the quality of wines (Toit and Pretorius 2000).
Generally, Gluconobacter predominate on sound, unspoiled grapes whilst Acetobacter species dominate on damaged, spoiled grapes and those infected with Botrytis cinerea. As might be expected, the population of acetic acid bacteria present in freshly crushed must is related to those populations present on the grape at harvest (Drysdale and Fleet 1988).
Because of their aerobic nature, acetic acid bacteria generally grow on the surface of wine and appear as a translucent film. Therefore, the best way of controlling these bacteria is to maintain anaerobic conditions by using inert gases in the headspace of tanks, and ensuring tight bunging and frequent topping of barrels. In addition to maintaining anaerobic conditions, SO2 levels should be checked frequently and maintained at 0.8 mg/L molecular SO2 (Boulton et al. 1996). It should be noted that research has suggested that some strains of acetic acid bacteria have the ability to survive in low numbers in barrels until oxygen becomes available (Drysdale and Fleet 1989).
In wine, acetic acid bacteria generally appear as small, fat rods, sometimes elongated, approximately (1-2) x (2-5) µm.
Bacteria of the genus Bacillus are aerobic and produce endospores. Fugelsang (1997) indicates that the bacilli are primarily soil-borne organisms that are secondarily found in water supplies. There have been infrequent reports of Bacillus sp. growing in wine and, in one case, brandy (Murrel and Rankine 1979). Kunkee (1996) and Boulton et al. (1996) reported that growth of Bacillus in bottled wine did not affect the palate or olfactory properties, and that the problem with the bacteria was limited to sediment/haze formation. As the bacteria are obligate aerobes, the anaerobic conditions of bottled wine probably limit the spoilage damage (Boulton et al 1996). However, for wines stored in tanks or barrels, there is the potential for increased oxygen tension and resulting spoilage. The best preventative and control measures are the same as those given for the acetic acid bacteria, above.
As indicated above, Bacillus produce endospores, which are very resistant structures. Boulton et al. (1996) indicate the endospores produced by Bacillus sp. are resistant to 70°C for 10 minutes and 95% ethanol for 20 minutes. They also indicate that the relative susceptibility to wine preservatives and sterilants is unknown.