Vineyard Impacts on Flora
Fermentation management begins in the vineyard. This is especially true for native flora fermentations but even inoculated fermentations can be influenced by the nature of the organisms present on the grapes. Grapes, leaves, bark, even the trellis system and irrigation systems support microbial flora in vineyards. The type of microbes best adapted to the localized environment of the plant will dominate that surface, but rarely is a single species of organism able to completely dominate an environmental niche. In fact the opposite is most often true – a community of metabolically diverse organisms is found co- localized in the same habitat. This diversity stabilizes the population against changing environmental conditions and may alternately favor blooms of certain species as conditions warrant. Changing the berry surface flora can therefore be difficult if not impossible as organisms become highly adapted to their environment and difficult to completely displace. With respect to subsequent fermentation many of these organisms are inconsequential, but others may have a profound impact on the fermentation and the flora of the fermentation. If native flora fermentations are to be conducted then it is important to “farm” the optimal flora on the surface of the fruit pre-harvest. There are several viticultural factors that impact grape flora.
There are grape variety-specific factors that can impact the types and numbers of organisms at harvest. The principal factor affecting microbial loads on the surface of the grape is the amount of nutrient seepage from the fruit to its surface. In addition some varietals may make antimicrobial compounds excreted from the surface that may limit the growth of some classes of organisms, or favor the growth of beneficial organisms over undesired or damaging ones. The organisms themselves can produce inhibitory substances so in supporting a beneficial population the plant may gain secondarily if that population has the ability to inhibit invasive organisms. Several studies have shown that grape flora yeast are able to inhibit Botrytis and block invasion of the fruit. The converse is also true – once Botrytis becomes established it is able to make compounds that inhibit the beneficial flora. Which population wins the battle depends upon which population is favored by the nutrients available from the fruit on its surface.
In general, nutrient availability favors the bacteria and molds. Bacteria and molds are both able to produce large amounts of biomass faster and with less organic substrates needed than the yeasts. The yeasts are specialized organisms that do well in more nutrient rich environments, like crushed fruit, than with limiting nutrients. If there is seepage of high sugar contents this favors yeast but this usually only occurs if there is some damage to the berry or cluster. Organisms generally are adapted to specific types of energy sources.
The tendencies of berries to seep nutrients varies by the variety. Some berries detach from the rachis more readily than others leading to greater leakage of components. Fruit with thicker walls are less likely to leak internal components. Varieties that create tightly packed bunches with obvious deformity and damage to fruit will also have higher sugar leakage rates on the fruit surface. If the pulp does not contain any inhibitory phenolic compounds but only growth nutrients then those organism more readily adapted to sugar will be favored. If other berry components are also being released, such as phenolic compounds, or polyphenol oxidase, these components may inhibit sensitive organisms and favor the resistant ones. Although only limited studies have been performed, those that have suggest that as sugar seeps from the fruit there is a change in populations to favoring the lactic acid bacteria (Lactobacillus) and the must non-Saccharomycesyeasts (Hanseniaspora, Metschnikowia, Candida) over the microbes present during earlier stages of maturation.
Site-specific factors can also impact the flora of the grape surfaces at all stages of ripening. Climatic conditions influence fruit composition, but also three other important secondary effects on grape flora: nature and type of insect vectors present in the vineyard, the localized temperature of the surface of the fruit, and the relative humidity of the surface of the fruit or within the cluster. Soil composition affects the nature and concentrations of soil microbes and, depending upon vineyard practices, these organisms may be transferred to the surface of the fruit, generally as transient species, but they may be there at the point of harvest.
Of the climatic factors the two most important are temperature and humidity. Some microbes are more heat tolerant than others, with the bacteria being the most heat tolerant of the microbial kingdom. Warm berry surfaces tend to favor larger populations of bacteria. Humidity also exerts a strong impact. All organisms have a range of available water under which growth is permissive. Above this range (too much water) cells have difficulty maintaining the integrity of the membranes and preventing osmosis from bringing in so much water the pressure causes the membranes to rupture. The relative strength if the cell wall is important here as well. If the available water is too low the cells will lose water and not have sufficient water to keep cellular components hydrated and to foster molecular movement within the cell. Shriveling of the cell can occur, which may be fatal. Many microbes can form spores or other resistant bodies that can withstand extremes of water availability and temperature and under these conditions become dormant but are not eliminated from the population. As with plants, some microbes are more resistant to water excesses or deficiencies than others. Many of the molds require a narrow window of humidity to sustain population growth. Molds can grow quickly as filaments and cover a wide territory but they are very sensitive to dehydration as a consequence. Localized humidity of the clusters is important as well as this can foster mold growth. Molds produce asexual spores that can be airborne and spread the colony over great distances so even a localized infection can become a problem for the entire vineyard if conditions quickly become favorable, such as a late rain. Microbes are much more resistant to cool temperatures so temperature decreases at night or late in the season often may be inhibitory to growth but not lethal to a well-established population on the surface of the fruit.
Another site factor that can have an impact is wind. If the vineyard is in a very windy area, humidity will likely always remain low, but significant wind may impact berry structure leading to small berries with thickened cell walls. This in turn may impact seepage and the flora present on the surface of the fruit.
The microbial flora of the grape surface can be influenced by the season indirectly via impacts on climate and insect and animal vectors resident in or visiting the vineyard. Very few studies have looked at seasonal impacts on resident flora directly. However, the impacts on insects as discussed below may be dramatic across seasons. If climate tends to vary dramatically over the growing season there will be a strong seasonal influence due to climactic variation.
The microbes of the soil environment can be transferred to the fruit during any normal vineyard operation that disturbs soil and creates dust. Often these microbes are highly adapted to the soil environment and unable to displace the microbes of the fruit surface. However most of the soil microbes are spore formers so they can persist on the grape surfaces and may be transferred to the winery without actually needing to have grown on the surface of the fruit. Soli fungi, filamentous bacteria and bacilli have been isolated from winery surfaces and from musts suggesting that they were viable and present at the time of harvest. Depending upon which specific species are present these microbes may or may not cause problems in the production of the wine. In general, they are rarely a problem during fermentation but may contaminate wooden or other surfaces and make metabolites that may gain entry to the wine. Any soil amendment that contains microbes, which is virtually all of them, can be a source of species that may eventually be on the fruit at harvest. There are periodic reports of isolations of intestinal flora from musts or grapes and this most likely reflects a transient transfer from a fertilizer preparation.
The amount of water and nutrients available to the vine impacts the nutrient content of the fruit which can impact the organisms present on the surface of the fruit. Foliar applications of nutrients can have dramatic and direct impacts on grape surface flora. In general addition of inorganic nitrogen to the grape flora ecosystem favors the growth of the organisms that can more readily use these nutrients. Any organism that can create biomass more quickly will have the advantage as more biomass means greater percentage consumption by that species of the available nutrient. Although less well studied in grape, foliar applications in other fruit systems favors blooms of the bacteria and can sometimes be used to control the spread of fungal diseases by encouraging the bacterial inhibition of fungal growth. Of course it is important to remember that fungi can make copious amounts of asexual spores that can be spread throughout the vineyard if aerosols on grape surfaces are created by an operation.
In addition to the variety, vineyard farming practices may also impact grape flora. An open canopy that allows good airflow over and around the clusters will likely reduce humidity and limit growth of molds. Sun exposure leads to ultra violet light (UV) exposure. UV is DNA-damaging and many microbes have developed strategies to prevent the damage from occurring or to identify and repair any such damage. However, sensitivity to UV varies across the microbial world and some organisms, such as Saccharomyces are highly UV-sensitive while other wild yeasts are quite resistant. In addition, the grape surface cells themselves may be damaged by UV exposure, and sun burning of the fruit can occur. This leads to the formation of patches on the surface of the berry that are thought to contain different types of phenolic compounds and less or different nutrients which may impact the organisms living on these areas. UV-exposed surfaces may therefore support a different type of flora than those that are not.
The use of antifungal agents in the vineyard will obviously have an impact on the flora present, depending upon the selectivity of the agent used. Because yeast are members of the same kingdom as the fungi some antifungals can inhibit fermentation by impacting the yeast. Some antifungal compounds derive from bacterial sources and if not sufficiently purified these preparations may contain anti-bacterial substances as well. If fermentations are going to be inoculated with Saccharomyces and the contributions of wild flora minimized, use of such compounds in the vineyard may be low risk. However if a native flora fermentation is desired, manipulation of the native flora via use of antifungals may have deleterious effects on the fermentation.
As the grapes mature the surface of the grape also changes. Prior to veraison the surface of the grape tends to be populated by microbes highly adapted to the nutrients available on the surface, which are generally low in sugar. Sugar content on the surface increases as ripening continues, favoring the appearance of sugar-utilizing organisms. It is at this point that yeasts found in musts first start to appear in higher numbers. As the berry continues to mature leakage may become more pronounced, depending upon the variety. At some point the fruit stops accumulating sugar and further increases in sugar are due to net water loss from the fruit. Water loss from the fruit can decrease the available water on the surface of the fruit and disadvantage those microbes with high water availability requirements, such as the molds. Investigations of the changes in microbial flora with extended hang time have shown an increase in the relative proportions of Lactobacillus. The increase in the numbers of these organisms combined with the generally higher pH of the juice made from such fruit almost guarantees an initial bloom of these organism during the early stage of fermentation before Saccharomyces has become established.
Insects can be divided into two classes those that are native residents of the vineyard (for example, leaf hoppers) and those that are visitors to the vineyard, such as flies, bees and wasps. Native insect residents can move organisms from one sector of the vineyard to another. If there is rot or disease in one section they can spread that disease by alternately visiting healthy and diseased fruit. Removal of diseased clusters and dropping them to the vineyard floor might not prevent such insect-driven transfer if the insects still alternately visit the dropped clusters and those remaining on the vine.
Visitor insects are those that may transiently appear in a vineyard but do not spend their entire life-cycles in the vineyard. Such insects may be a source of microbes from non-vineyard origins. In general, insect-vectored microorganisms must be able to displace berry surface residents to have an impact on wine fermentation. Microbes adapted to green or leafy surfaces, to bark or to soil are generally not able to dominate fruit surface flora. The main vineyard insect visitors that can impact fruit flora are those that are attracted to fruit, visiting other fruity plants or that cause damage to the fruit. Fruit flies have been shown to transmit yeasts from one type of fruit to another and often these yeasts can be found as part of the recipient fruit flora. Wild yeasts have been identified from fruit flies and they are often attracted to yeast metabolites such as ethyl acetate, produced by the non-Saccharomyces berry flora. The type of other agricultural activities located in proximity to the vineyard can have an impact on grape flora, especially if the harvest of that commodity precedes that of the grapes. Yeasts indigenous to tomato surfaces are often found in adjacent vineyards following the tomato harvest as those vineyards are inundated with the flies from the tomato operation. Bees and wasps can also transmit fruit flora from one type of fruit to the next and can transfer wild Saccharomyces strains. Wasps can damage the berries leading to seepage of berry contents to coat the rest of the cluster which also seems to boost yeast populations. These insects are not neutral vectors and can transfer microbes to grape surfaces that are capable of colonizing those surfaces. Whether this colonization is beneficial to the fermentation or not depends upon the actual organisms transferred and displaced by this process.
Fungal molds can be benign residents of grape surfaces or can be invasive such as Botrytis. Invasive molds will have a dramatic impact on the organisms present on the surface of the fruit. The grape responds to mold infection and attack by producing antimicrobial components. Molds in turn produce toxic compounds designed to block the response of the plant and to kill plant tissue. Other organisms are often the collateral damage of this battle between the invasive species and the host plant. Organisms that are not sensitive to the inhibitors being produced may also be influenced by the disruption of plant cell integrity and the release of nutrients that accompanies the invasion by the mold. Bacteria and yeast may grow in the damaged fruit leading to high populations at harvest. Other molds may also bloom at the same time. These molds may contribute to the damage to the fruit and are opportunistic pathogens, that is, not able of initiating an attack on the fruit but able to participate once the defenses of the plant have been breached. In many cases undesired acetic and lactic acid bacteria will bloom in damaged clusters, organisms that can dominate the early fermentation and may persist during the fermentation, depending upon the pH. This can have a dramatic impact on the yeast’s ability to both dominate and complete the fermentation.
Herbivores can cause mechanical damage to fruit as well leading to release of fruit components to the surface of the clusters and blooms of bacteria and yeasts. There are some intestinal flora that have been isolated on occasion from musts or fruit surfaces and these may arise from animal feeding in the vineyard, but this has not been conclusively shown. However any visitor to the vineyard has the opportunity to introduce new microorganisms to the ecosystem.
There are four main vineyard factors that will affect fermentation and therefore should factor into management strategies:
- Nutritional and chemical composition of the fruit
- Level and type of berry/cluster damage
- Invasion by fruit-visiting insects pre-harvest
- Use of antimicrobial compounds too close to harvest
Nutritional and chemical composition of the fruit: The nitrogen, sulfate, phosphate and micronutrient level of the juice at the beginning of fermentation is obviously critical to the fermentation. In general, the higher the nutrient content the faster and more complete the fermentation. However, it is important to note that if the juice is too rich in such nutrients very rapid fermentations may raise the temperature to an inhibitory level and that in the presence of ethanol temperature tolerance is reduced. Similarly the high temperature shock forces a cell membrane adaptation to temperature that then diminishes the cell’s capacity to tolerate high ethanol concentrations. Refrigeration or tank cooling can mitigate this to a certain extent, but excessive nutrient addition should be avoided. It is important to know if there are any deficiencies associated with a particular vineyard so that fermentations can be augmented, but excessive nutrient additions should be avoided.
Another important compositional factor is pH. Yeast are low-pH tolerant, with some able to grow even at pH 2.5. The bacteria are less pH tolerant with many inhibited below pH 3.5. Higher pH juices can support the growth of a wider array of microorganisms and sulfite is less effective at higher pH values as an antimicrobial agent. The pH of the juice will dictate the types and numbers of organisms that can be found in the wine. Tolerance of low pH is influenced by the ethanol concentration as well as by the cation composition of the juice. If the pH is too low and there is a deficiency of ions such as potassium, ethanol tolerance will be impacted and fermentation could arrest or be sluggish.
Level and type of berry/cluster damage: A low percentage of damaged clusters can have a dramatic impact on the microbial flora of fermentation. Damaged clusters can develop high population numbers of acetic and lactic acid bacteria, on the order of 109 cells/mL. In an uninoculated fermentation yeast pick up from the winery is between 102 – 103 cells/ml and, if inoculating, cells are present at roughly 106 cells/mL. If inoculating, one bad cluster per 1000 healthy cluster will yield equivalent numbers of yeast from the inoculation and bacteria from the damaged clusters. If conducting a native flora fermentation, the dilution of the clusters would have to be one to one million healthy clusters to get the bacterial numbers in the same range as the yeast. A small faction of highly damaged clusters can have a strong effect on fermentation flora.
Other factors then become important with respect to giving the yeast an edge to become dominant. If the pH is low enough the yeast will have the advantage. If a native flora fermentation is desired, adjusting the pH to below 3.5 may be of benefit. Also, since the yeast in general do better at lower temperatures, keeping the temperature low at this point may also advantage the yeast, but keep in mind that if there are high numbers of non-Saccharomyces yeasts they may also be advantaged by lower temperatures. The temperature needs to be warm enough to give the advantage to Saccharomyces but not so warm as to advantage the bacteria. Temperature should be in the general range of 15-22°C to promote growth ofSaccharomyces.
Nothing substitutes for looking at the fermentation under a microscope to determine what types of organisms are present. This is beneficial before the addition of nutrients to make sure the correct population is being fed. Some winemakers will use the appearance of bubbles (carbon dioxide) as evidence the fermentation has started. This is clearly evidence that something is going on but does not necessarily mean that Saccharomyces is now dominant. It depends upon the condition of the fruit. For sound fruit this is a safe assumption – that the appearance of carbon dioxide indicates Saccharomyces has dominated, but for unsound fruit this is not the case.
Invasion by fruit-visiting insects pre-harvest: Insects that are attracted to a wide array of fruit can have an impact on the yeast flora at the onset of fermentation. For this to occur a significant number of insects must descend upon the vineyard pre-harvest. This will occur if there is an adjacent agricultural operation that is harvested prior to grape harvest. The insects then migrate to the vineyard and can carry enough yeast and bacteria from the donor fruit to impact the flora of the grape. Whether or not this impacts the yeast during fermentation depends upon the organisms transferred by the insects, inoculation and nutrient supplementation practices, and use of antimicrobial agents at the onset of fermentation.
Use of antimicrobial compounds too close to harvest: On occasion the prevention of cluster rots may mean the proactive use of antimicrobial agents, particularly fungicides, in the vineyard. Since Saccharomyces is also a member of the fungi, some of these compounds may also inhibit Saccharomyces. For most agents the impacts on Saccharomyces are well-known and the vineyard manager and winemaker can ask to see the data for any new agent that might be developed. The effect on both fermentation and aroma profile should be known before using any agent near the harvest date.