21st Century Guidebook to Fungi, SECOND EDITION, by David Moore, Geoffrey D. Robson and Anthony P. J. Trinci

Table of Contents

1. Chapter 16: Fungi as pathogens of animals, including man
2. Chapter 17: Whole organism biotechnology
   1. Fungal fermentations in submerged liquid cultures
   2. Culturing fungi
   3. Oxygen demand and supply
   4. Fermenter engineering
   5. Fungal growth in liquid cultures
   6. Fermenter growth kinetics
   7. Growth yield
   8. Stationary phase
   9. Growth as pellets
   10. Beyond the batch culture
   11. Chemostats and turbidostats
   12. Uses of submerged fermentations
   13. Alcoholic fermentations
   14. Citric acid biotechnology
   15. Penicillin and other pharmaceuticals
   16. Enzymes for fabric conditioning and processing, and food processing
   17. Steroids and use of fungi to make chemical transformations
   18. The Quorn fermentation and evolution in fermenters
   19. Production of spores and other inocula
   20. Natural digestive fermentations in herbivores
   21. Solid state fermentations
   22. Digestion of lignocellulosic residues
   23. Bread; the other side of the alcoholic fermentation equation
   24. Cheese and salami manufacture
   25. Soy sauce, tempeh and other food products
   26. Chapter 17 References and further reading
   27. Buy a PDF of Chapter 17
3. Chapter 18: Molecular biotechnology

17.13 Alcoholic fermentations

The most important fermentation worldwide, of course, is the one that is used to make alcoholic drinks (and see the discussion of fuel ethanol in Section 17.22; CLICK HERE to view now). It’s a simple process; starting with something that’s rich in sugar, like fruit juices, honey, or cereal grains or roots. Then add yeast (or rely on the yeasts already in the starting material, which is a common procedure in traditional wine fermentation) and allow the mixture to ferment. The final liquid may contain up to 16% alcohol, depending upon the yeast and the process (Walker et al., 2017).

Fermentation is such a simple process that alcohol has been incorporated into the way of life of every civilisation, even the most primitive. The earliest records occur in ancient Egyptian murals and tomb ornaments depicting bread, beer and wine making. Yeast cells can be clearly seen by scanning electron microscopy in beer remains in a tomb pottery vessel from the village where workmen lived who built the tombs in the Valleys of the Kings and Queens, dated 1550 to 1307 BC (Samuel, 1996). From the biological point of view it is a remarkable aspect of yeast physiology that the organism responsible for fermentation is invariably the yeast called Saccharomyces cerevisiae (not surprisingly known as Brewer’s yeast) or some closely related variant. Other research has identified the presence of barley, with other components of a beer recipe, in 5,000-year-old archaeological materials from China (Wang et al., 2016).

The chemistry of the ethanol fermentation is summarised in this equation:

C₆H₁₂O₆ → 2 C₂H₅OH + 2 CO₂

This equation is telling you that one hexose molecule is converted into two ethanol molecules and two molecules of carbon dioxide. Many yeast species can carry out this fermentation, but only in an anaerobic environment; in the presence of oxygen, complete oxidative respiration of sugar to carbon dioxide and water occurs.

However, Saccharomyces cerevisiae, and its distant relative Schizosaccharomyces pombe, both preferentially use fermentation even in the presence of oxygen and will yield ethanol even under aerobic conditions given appropriate nutrients.

• Yeasts used for making ales tend to generate froth and grow on the top of the fermenting liquor, and such top fermenting yeasts are Saccharomyces cerevisiae itself.
• Lager yeasts do their fermenting at the bottom of the tank and they belong to the closely related species Saccharomyces carlsbergensis.

In summary, ales, beers and lagers are generally made from malted barley; other cereal grains are used for specialist beers or in places where barley is not available (Briggs et al., 2004; Hutkins, 2006; Montet & Ray, 2015).

The process starts by encouraging the cereal grains to germinate. In two to four days the sprouting seeds start the digestion of their stored starch, producing soluble sugars. Then the sprouted grain is killed by slow heating and mashed into hot water with other cereals like maize, wheat or rice. Finally, the sweet mix is boiled with hops to add bitter flavours to the beer. The boiled liquid is cooled and passed to the fermentation vessels for fermenting.

The four main ingredients of beer are:

• water; dissolved salts in the water influence the character of the final beer by affecting extraction of fermentable sugars from the malted grain and in turn affects the way the yeast behaves during fermentation. Total salts in the water in Pilsen (a city in western Bohemia in the Czech Republic) known as the place of origin of Pilsener lager, amounts to around 30 parts per million (ppm); whereas in Burton-upon-Trent in the U.K., which is the home of English Pale Ale, the local hard water has a salt content of 1220 ppm. It was calcium sulphate in the local water in Burton-upon-Trent that helped to create the pale ale style of beer; the calcium increases the efficiency of extraction of sugars from the malted grain, whilst the sulphate enhances the bitterness of the hops. In addition, the divalent calcium ions form salt bridges between suspended polypeptides and polysaccharides and this aggregation helps to clarify the brew.
• malt; which is germinating barley. Starches in the grain are converted into sugars as the seed germinates. When this process is stopped by drying at an optimal point in the process, the grain will contain some sugars plus a quantity of enzymes to aid the further extraction of fermentable sugars. The process of mashing (see below) makes use of these enzymes to do this.
• hops; the flowers of the hop vine contain resins, which provide bitterness to the final product  but take at least 15 minutes boiling to extract, and oils, which provide aroma but evaporate quickly if boiled. The typically English India Pale Ale was first brewed in England and exported for the British troops in India during the late 1700s. More hops were added to the recipe and the natural preservatives in hops enable this ale to travel well over the long sea voyage to the British colonies in India. British Bitter also contains more hops but combined with pale malts to differentiate this ale from other brews. 
• yeast; some beers make use of wild yeasts but most modern brewers prefer to control the yeast culture. The top foam is skimmed from the current fermentation and used to start the next beer ferment. The sugar content of the liquid is monitored throughout the fermentation and the process is stopped when the desired alcohol strength is reached.

Preparation for the fermentation starts by crushing the malted grain; this is intended to split the grains into smaller pieces to increase their surface area, separating the husks from the grain kernels. The resulting grist (made up of grain which is said to be cracked) is better able to release the sugars and enzymes.

Mashing is the process of combining the grist with water at 67ºC to stimulate the enzymes in the malt to convert starches to sugars. Mashing is done in a vessel called a mash tun and is common to all fermentation processes so the grain is typically malted barley for beers and malt whisky but will be mixed with, or even replaced by, other grains such as corn, sorghum, rye or wheat to prepare for other liquors.

The mash is mixed and sparged for 1.5 to 2 h to maximise the extraction. Finally the liquid extracted from the mashing process (now called wort) is pumped into a large boiler known as a copper and brought to the boil, at which point the first batch of hops is added. These hops provide the bitter taste; volatiles that might contribute to final aroma will be boiled off at this stage. The wort is boiled for 1.5 to 2 h, then heating is stopped and aroma hops are added (a process called dry hopping).

After a short mixing period, the hot wort is decanted or filtered, rapidly cooled and transferred to the fermentation vessel at 17ºC. The yeast is added (or pitched) and fermentation starts and is clearly evident in a few hours. After a week to three weeks, the fresh (or 'green') beer is run off into conditioning tanks cooled below 10ºC. After conditioning for a week to several months, the beer is often filtered and the bright beer is ready for serving or packaging.

Wine making is now also a global industry, although Italy, France and Spain still together account for half the world’s production, which was estimated at 259 million hectolitres (mhl) in 2016 (source: website of the International Organisation of Vine and Wine at http://www.oiv.int/en/). In that year, production in Italy was 48.8 mhl, France 41.9 mhl, and Spain 37.8 mhl. For comparison, production of 22.5 mhl was recorded in the United States, and Australian production was 12.5 mhl. In fact, world production fell by 5% compared to 2015, after a succession of climatic events: severe frosts early in the season, followed later in the year by heatwaves and wildfires, drastically reduced yields in France, Spain and Italy.

The classic wine grape has the scientific name Vitis vinifera. The important cultivars include Sauvignon (red Bordeaux); Pinot Noir (the main red Burgundy grape); Riesling and Silvaner (for German white wines); Barbera and Freisa (northern Italian wines); and Palomero, the main sherry grape. The whole of the black grape is crushed to make red wine; it’s the grape skin pigment that makes it red. Black or white grapes can be used to make white wines but only the pressed juice is used, and extraction of skin pigments is avoided (Jackson, 2008). 

Of course, the quality of the wine depends on the grape used, on production techniques, and on fine points like the type of soil type and what the growing season was like. Grape juice is obtained by crushing grapes and usually contains 7 to 23% pulp, skins, stems and seeds; in the wine industry this grape juice is referred to as must.

The most controlled fermentation of the must is achieved with an elliptically-shaped variant of S. cerevisiae called Saccharomyces ellipsoideus, which may come from natural sources (the grapes or the preparation machinery) but is more usually added as a starter yeast culture made up in grape juice.

Traditional wine makers in Europe prefer to use the ambient (or wild) yeasts that occur on the grapes themselves (the bloom or blush of the grape) because it is a characteristic of the region. Common genera of such ambient yeasts include Candida, Pichia, Klöckera (Hanseniaspora), Metschnikowiaceae and Zygosaccharomyces. High quality and uniquely flavoured wines can be produced this way, but wild yeasts are often unpredictable and may cause spoilage.

The crucial feature for most winemaking is the controlled fermentation by that one specifically isolated and cultured yeast, S. ellipsoideus. Even this exists as several hundred different strains with fermentation characteristics that contribute to the diversity of wine, even when the same grape variety is used.

Fermentation may be carried out in stainless steel tanks, in traditional open wooden vats or wine barrels and can last from 5 to 14 days for primary fermentation and potentially another 5 to 10 days secondary fermentation (Hutkins, 2006; Montet & Ray, 2015). The latter, perhaps, inside the wine bottle as in the production of many sparkling wines.

After the yeast fermentation, quality wines take one to four years to age in wooden casks. For some wines a bacterial fermentation is encouraged during aging to mellow the taste by reducing acidity. To make sparkling wines, sugar, a little tannin and a special strain of S. ellipsoideus that can form granular sediment, are added when the wine is bottled.

A secondary fermentation in the bottle produces carbon dioxide and results in formation of an unstable compound with alcohol (called ethyl pyrocarbonatene) which gives the characteristic lingering sparkle of naturally-produced sparkling wines (compared with the brief sparkle of those that have only had carbon dioxide injected into them under pressure).

Fortified wines, which have had up to 20% brandy added to them, include sherry. This is made from a particular grape and a secondary growth of yeasts in the maturing vats creates the characteristic sherry flavour compounds. Vermouths are wines flavoured with herbs, like wormwood, and with extra alcohol added. Port is a red wine in which the primary fermentation was stopped by adding alcohol or brandy while some sugar still remained. The special flavour of Madeira results from a heat treatment of the fermented wine before extra alcohol is added.

Updated July, 2019