attribute for sweet late-harvest wines), development of surface film on the wine (positive for some sherry styles but a negative attribute for many other wines), enhancement of a wine's color or certain varietal characteristics by enzymes in the yeast cells and other metabolic products produced by the yeast, foaming and flocculation tendencies, yeasticidal properties (a trait known as "killer yeast") and tolerance for nutritional deficiencies in a must that may lead to a stuck fermentation. re-hydrating freeze dried yeast cultures preparing a yeast starter culture and gradually cooling the culture down to the must temperature by adding some wine. pure culture yeasts that are grown in a lab are often freeze dried and packaged for commercial use. prior to their addition into must, these yeasts need to be re-hydrated in "starter cultures" that must be carefully monitored (particularly in regards to temperature) to ensure that the yeast cells are not killed off by cold shock. ideally winemakers want to add enough inoculum to have a viable cell population density of 5 million cells per milliliter. the exact amount of freeze-dried culture varies by manufacturer and strain of yeast but it is often around 1 gram per gallon (or 25 grams per 100 liters). wines that could have potentially problematic fermentation (such as high sugar level late harvest or botryized wines) may have more yeast added. similarly, re-hydration procedures will also vary depending on the manufacturer and winery. yeast is often inoculated in a volume of water or grape must that is 5–10 times the weight of the dry yeast. this liquid is often brought to temperature of 40 °c (104 °f) prior to the introduction of the yeast (though some yeast strains may need temperatures below 38 °c (100 °f)) to allow the cells to disperse easily rather than clump and sink to the bottom of the container. the heat activation also allows the cells to quickly reestablish their membrane barrier before soluble cytoplasmic components escape the cell. re-hydration at lower temperatures can greatly reduce the viability of the yeast with up to 60% cell death if the yeast is re-hydrated at 15 °c (60 °f). the culture is then stirred and aerated to incorporate oxygen into the culture which the yeast uses in the synthesis of needed survival factors. the temperature of the starter culture is then slowly reduced, often by the graduated addition of must to get within 5–10 °c (9–18 °f) of the must that the culture will be added to. this is done to avoid the sudden cold shock that the yeast cells may experience if the starter culture was added directly to the must itself which can kill up to 60% of the culture. additionally, surviving cells exposed to cold shock tend to see an increase in hydrogen sulfide production. nutritional needs of wine yeast diammonium phosphate (or dap) is a common additive that provides two necessary nutrients for yeast to have a healthy and sustained fermentation – nitrogen and phosphate. in order to successfully complete a fermentation with minimum to no negative attributes being added to the wine, yeast needs to have the full assortment of its nutritional needs met. these include not only an available energy source (carbon in the form of sugars such as glucose) and yeast assimilable nitrogen (ammonia and amino acids or yan) but also minerals (such as magnesium) and vitamins (such as thiamin and riboflavin) that serve as important growth and survival factors. among the other nutritional needs of wine yeast: phosphate – used for the production of nucleic acids, phospholipids (an important component of the cell membrane) and atp (adenosine triphosphate which the cell uses for transferring energy for metabolism). potassium – important for the uptake and utilization of phosphate biotin – involved in the synthesis of proteins, fatty acids and nucleic acids. pantothenic acid – involved in the metabolism of sugars and lipids. a deficiency of this vitamin could lead into increase hydrogen sulfide production with off-aromas in the resulting wine. nicotinic acid – involved in the synthesis of nicotinamide adenine dinucleotide (nad+), a co-enzyme that is important in maintaining the redox balance of the cell as well as in the process of ethanol fermentation itself. inositol – involved with the secondary messenger molecules that facilitate cell division. trace amounts of calcium, chlorine, copper, iron, manganese and zinc for healthy cell function. one traditional way of providing nutrients for the yeast is the ripasso method where the leftover grape skins and pomace (pictured) from a previous fermentation is added to a newly fermenting wine. many of these nutrients are available in the must and skins of the grapes themselves but sometimes are supplemented by winemakers with additions such as diammonium phosphate (dap), freeze-dried micro-nutrients (such as go-ferm and ferm-k) and even the remnant of dead or extracted yeast cells such that the fermenting yeast can break down to mine for available nitrogen and nutrients. one historical winemaking tradition that is still practiced in some italian wine regions is the ripasso method of adding the leftover pomace from the pressing of other wines into a newly fermenting batch of wine as an additional food source for the yeast. saccharomyces cerevisiae can assimilate nitrogen from both inorganic (ammonia and ammonium) and organic forms (amino acids, particularly arginine). as yeast cells die, enzymes within the cells begin autolyzing by breaking down the cell, including the amino acids. this autolysis of the cell provides an available nitrogen source for the still-fermenting and viable yeast cells. however, this autolysis can also release sulfur-link compounds (such as the breakdown of amino acid cysteine) which can combine with other molecules and react with alcohol to create volatile thiols that can contribute to a "stinky fermentation" or later development into various wine faults. the role of oxygen yeasts are facultative anaerobes meaning that they can exist in both the presence and absence of oxygen. while fermentation is traditionally thought of as an anaerobic process done in the absence of oxygen, early exposure of the yeast to oxygen can be a vital component in the successful completion of that fermentation. this is because oxygen is important in the synthesis of cell "survival factors" such as ergosterol and lanosterol. these sterols are important in maintaining the selective permeability of the yeast cell membrane which becomes critical as the yeast becomes exposed to increasing osmotic pressure and levels of alcohol in the wine. as a waste product of its own metabolism, alcohol is actually very toxic to yeast cells. yeast with weak survival factors and lacking sterols may succumb to these conditions before fermenting a wine to complete dryness, leaving a stuck fermentation. cultured yeasts that are freeze-dried and available for inoculation of wine must are deliberately grown in commercial labs in high oxygen/low sugar conditions that favor the development of these survival factors. one of the reasons that some winemakers prefer using inoculated yeast is the predictability of fermentation due to the high level of survival factors that cultured yeast are assured of having without necessarily needing to expose the wine to additional levels of oxygen. winemakers using "ambient" yeasts that are resident in their winery may not have this same assurance of survival factors and may need to compensate with other winemaking techniques. wild non-saccharomyces yeasts often need a much greater exposure to oxygen in order to build up survival factors which is why many of these yeasts are often found living oxidatively as "film yeast" on the surface of wines in tanks or barrels. wine faults related to yeast film yeast like candida (pictured) and pichia can cover the surface of a wine with a film layer that not only consumed most of the free sulfur dioxide available to protect the wine but also produces high levels of acetic acid that will contribute to volatile acidity in a wine. either directly or indirectly, wine yeast can be a culprit behind a wide variety of wine faults. these can include the presence of "off flavors" and aromas that can be the by-product of some "wild yeast" fermentation such as those by species within the genera of kloeckera and candida. even the common wine yeast saccharomyces cerevisiae can be behind some wine faults with some strains of the yeast known to produce higher than ideal levels of acetic acid, acetaldehyde and volatile sulfur compounds such as thiols. also any yeast can have a low tolerance to nutritional deficiencies, temperature fluctuation or extremes and excessive or low sugar levels that may lead to a stuck fermentation. in the presence of oxygen several species of candida and pichia can create a film surface on top of the wine in the tank of barrel. allowed to go unchecked, these yeasts can rapidly deplete the available free sulfur compounds that keeps a wine protected from oxidation and other microbial attack. the presence of these yeasts is often identified by elevated levels of volatile acidity, particularly acetic acid. some strains of pichia will metabolize acetic acid (as well as ethyl acetate and isoamyl acetate that may also be produced) with the side-effect of substantially decreasing the titratable acidity and shifting the ph of wine upwards to levels that make the wine prone to attack by other spoilage microbes. commonly called "film yeast", these yeasts are distinguished from the flor sherry yeast that are usually welcomed by winemakers in producing the delicate fino-style wines. growth of many unfavorable wild yeasts is generally slowed at lower cellar temperatures, so many winemakers who wish to inhibit the activities of these yeasts before the more favorable saccharomyces yeast kick in, will often chill their must, such as the practice of "cold soaking" the must during a pre-fermentation maceration at temperatures between 4–15 °c (39–50 °f). though some species, such as brettanomyces, will not be inhibited and may even thrive during an extended period of cold soaking. brettanomyces while some wine regions view the influence of brettanomyces on the wine, in limited amounts, as added complexity, many winemakers view the presence of brettanomyces species such as brettanomyces bruxellensis (pictured) in their wineries as a negative influence that needs to be controlled. the wine yeast brettanomyces (or "brett") produces very distinctive aroma compounds, 4-ethylphenol (4-ep) and 4-ethylguaiacol (4-eg), that can have a wine being described as smelling like a "barnyard", "wet saddle" or "band-aid". to some winemakers and with some wine styles (such as pinot noir from burgundy), a limited amount of these compounds could be considered a positive attribute that adds to the complexity of wine. to other winemakers and with other wine styles (such as riesling from the mosel), the presence of any brett will be considered a fault. fruit flies are common vector in the transfer of brettanomyces between tanks and even nearby wineries. as a fermentation yeast, brettanomyces can usually ferment a wine up to 10–11% alcohol levels before they die out. sometimes brettanomyces already present in a wine that has been inoculated with saccharomyces cerevisiae will out compete the saccharomyces strain for nutrients and even inhibit it due to the high levels of acetic acid, decanoic acid and octanoic acid that many strains of brettanomyces can produce. once brett is in a winery, it is very difficult to control even with strict hygiene and the discarding of barrels and equipment that has previously come into contact with "bretty" wine. this is because many species of brettanomyces can use a wide variety of carbon sources in wine and grape must, including ethanol, for metabolism. additionally, brett can produce a wide range of by-products that could influence the wine beyond just the 4-ep and 4-eg compounds previously discussed. many of these compounds, such as the "footprints" of the 4-ep and 4-eg, will still remain in the wine even after yeast cells die and are removed by racking and sterile filtration.