Fermentation Chemistry
In our journey through the science of brewing, we’ve reached the area of greatest mystery — what happens inside our fermenters once we add the yeast to our carefully-crafted wort. Most homebrewers will be happy to leave the work up to the yeast and simply give them the tools they need to get on with the job of making our beer for us. To be honest, a lot of brewpub and microbrewers approach the issue in the same way. Still, a good understanding of the biochemical pathways responsible for fermentation, and of course flavor formation, is useful to any brewers who wish to control their beer’s consistency and quality.
Several large changes occur in the wort during fermentation, namely the reduction in concentration of fermentable sugars, an increase in temperature and a drop in pH. In addition to these changes, the number of yeast cells in suspension increases dramatically, as does ethanol concentration. Along with an increase in alcohol, some other flavor compounds — such as higher alcohols (fusel alcohols), esters, aldehydes and vicinal diketones — also increase.
The reduction in concentration of sugars is due to the uptake of small carbohydrates — primarily glucose, maltose and maltotriose — by the yeast. These are then used for energy and to produce the building blocks needed for cell growth. The disappearance of these carbohydrates is easy to measure with the use of a hydrometer. As the concentration of sugars decreases, the specific gravity of the wort likewise decreases. The rapid drop in pH is due to acid formation and consumption of buffers in the wort, primarily phosphates and amino acids. This lowering of the pH will lead to greater microbial stability of the beer because a lower pH tends to inhibit bacterial growth.
The temperature increase during the main part of fermentation is primarily due to the generation of heat during the breakdown of carbohydrates. The temperature will drop back down once the fermentation slows. In commercial breweries, it is usual to remove this excess heat by cooling the beer during fermentation.
Fusel alcohols and esters are components that are formed as by-products of the yeast metabolism. These and others, such as vicinal diketones (including diacetyl), will be discussed in more detail during the forthcoming article on flavor biochemistry.
Wort Composition
Properly made wort is a rich medium containing all of the nutrients the yeast will require for survival, growth and fermentation. It will contain fermentable carbohydrates, amino acids (sometimes referred to as free amino nitrogen or FAN), chemical ions (such as phosphorus, sulfur, calcium, magnesium, zinc and copper), the vitamin biotin and finally, oxygen. It is important to recognize that the yeast cell’s purpose is not fermentation, but rather self-preservation through growth and multiplication (budding). The yeast cell uses the nutrients in the wort to supply it with the energy and building blocks it will require to grow more cells.
Yeast will take up certain constituents of the wort preferentially to others. With regard to carbohydrates, the yeast will take up glucose and fructose, the simpler six carbon sugars (or monosaccharides) first. Then it will move on to maltose (a disaccharide) and finally maltotriose (a trisaccharide). Sucrose is enzymatically degraded, by an enzyme (invertase) inside the cell wall, to glucose and fructose. The yeast also has a preference for the order in which amino acids are taken up. Amino acids are used to build proteins inside the cell and, since enzymes are proteins, without amino acids cell functions are impossible. A normal all-malt wort has more amino acids than are needed for yeast growth. A wort made from up to 50% low-protein adjuncts (such as rice, corn or sugars) contains fewer amino acids, but there are still usually sufficient amino acids for a healthy fermentation. Yeast bring amino acids into the cell in a certain order. Some aren’t brought into the cell until 24 hours after the growth cycle begins. Still, yeast needs the complete range of amino acids to produce the proteins right from the start. The implication of this fact is that yeast are able to manufacture amino acids internally. This results in a number of biochemical pathways operating that create a number of beer flavors in turn.
Pitching Rate
One of the parameters that brewers can influence directly is pitching rate. Most flavors developed during fermentation are in some way linked to yeast growth. The degree to which yeast will grow in wort is influenced by the amount of yeast you put in. The general guideline used by commercial brewers is that the correct pitching rate is 1×106 live cells/mL/degrees Plato. Very strong beers sometimes require a higher pitching rate than normal since high-gravity worts absorb oxygen less readily.
Wort Aeration
It is important to provide the yeast with an adequate supply of dissolved oxygen in order for normal growth to occur. Yeast use molecular oxygen not to respire but to produce materials it requires in the production of new cell-membrane material. A frequent error that crops up in the homebrewing literature is referring to the initial period of fermentation, when yeast are taking up oxygen, as respiration.
Respiration is carried out by some organisms in the presence of oxygen and is a highly efficient method of collecting large amounts of energy from simple sugars. In brewing yeast cells, however, the glucose content of the wort and intracellular glucose levels are always high enough so that the less energy-efficient fermentation pathway is in operation.
Immediately prior to fermentation is the only time in the brewing process when oxygen is intentionally added. If a yeast has a limiting requirement for a certain level of dissolved oxygen in solution, then aeration can have a profound effect on yeast performance. Many English ale yeasts work best in open fermenters and when brewers attempt to use them in closed fermenters, they run into difficulties.
Very strong worts are difficult to aerate sufficiently, which is part of the reason they are so hard to ferment. It is also why the yeast they produce is unsuitable for use in subsequent fermentations. There is no need to worry about over-aeration of the wort, since yeast will take up most of the oxygen very rapidly and any excess is scrubbed from the wort in the first few hours with no adverse effect. In contrast, inadequate aeration can lead to a whole range of problems including: defective initial fermentation, longer fermentation time, fermentations sensitive to cooling, stalled fermentations, defective secondary fermentation (poor removal of green beer flavors) and beer quality problems. Also be aware that poor aeration can lead to yeast health problems in subsequent repitchings, when the yeast is reclaimed and used again.
Progress of Fermentation
If the fermentation is being carried out in an open or clear glass fermenter, then it is possible to monitor the fermentation by observing the qualities of the various foam heads that form on the surface of the fermenting beer, in combination with measurements of temperature and specific gravity. In a closed vessel, progress can only be monitored by measuring the fall in gravity and the rise in temperature.
An ale fermentation carried out using a yeast that rises to the top of a vessel after fermentation has finished follows a distinct sequence of stages. All are recognizable by their appearance. The first foam head will appear 8–12 hours into the fermentation and will be white with a lacing of brown in it. A second “fluffy” head will start to appear after about 18 hours and the brown solids in the first head will be pushed to the side of the vessel and form a ring around the fermenter. As the gravity reaches 1.014–1.010, the main crop of yeast will rise to the surface and be thick and golden colored. This will remain for only a few hours before falling back in, so must be collected at the correct time. Some brewers collect it by skimming this layer off, leaving behind two to three inches of yeast on top which will then darken and form a protective crust over the beer. Others will drop the beer out from under this layer and reclaim the yeast from the bottom of the vessel.
In the case of lager fermentations, the visual clues are similar. In the initial phase, the young beer becomes covered by a white layer of fine bubble foam. The fermentation has begun. Next you will see the low krausen phase when the fine bubble foam becomes deeper and has brown caps. The foam cover should look as uniform as possible and be creamy. After a couple of days, high krausen begins when the fermentation has entered its most intensive phase. The ridges or crests in the foam become higher and the bubbles coarser. After about five days the krausen collapses, the fermentation becomes less vigorous and the high crests slowly collapse. The foam looks browner. The foam continues to collapse until it forms a loose brown layer over the surface that is removed before transfer to maturation to prevent it mixing with the beer.
Due to poor temperature control, these phases often occur more rapidly as a fermentation progresses quickly. I’ve encountered a few homebrewers who swear their yeast is defective when in fact they pitched too much yeast and allowed the fermentation to get too warm.
Yeast Metabolism
Once inside the cell, carbohydrates such as glucose, maltose and maltotriose are broken down by enzymes to produce carbon dioxide, ethanol and energy.
C6H12O6 > 2C2H5OH + 2CO2 + energy
Some of that energy is captured by the yeast cell in the form of adenosine tri-phosphate (ATP), so that it can be used to continue cell growth. The energy that is not captured is released to the environment, and causes the increase in temperature seen in brewery fermentations.
The main pathway for the metabolism of carbohydrates used by yeast is called the Embden-Meyerhof-Parnas (EMP) pathway, also referred to as glycolysis. This is essentially the breakdown of glucose to produce pyruvate. Pyruvate is the starting point for a number of other pathways, although by far the most common path taken is the breakdown of pyruvate to acetaldehyde and hence ethanol and CO2 (i.e. fermentation). Pyruvate is a branching off point for many biochemical pathways including the manufacture of amino acids (needed for proteins and enzymes), lipids (needed for membranes), nucleic acids (needed for genetic material) and flavor compounds such as esters, vicinal diketones, and higher alcohols.
Glycogen
As yeast cells approach the end of the fermentation, they begin to use the carbohydrates assimilated from the wort to store energy inside the cell. They build the complex carbohydrate glycogen inside the cell. It is this glycogen that will allow it to survive the more austere times that are to come as it waits, in the fridge and outside the wort, for the next time you brew. Glycogen is a polysaccharide made up of glucose molecules with alpha 1-4 links and alpha 1-6 linked side chains, and it resembles amylopectin. Amylopectin is a complex polymer made up of glucose molecules linked together in chains with side chains interspersed at intervals throughout. Thus it is multiple-branched and a compact way for a plant to store glucose in a small space.
Towards the end of fermentation, yeast cells begin to synthesize these compounds to use as an energy source during storage. Glycogen can be enzymically broken down to glucose, which enters the EMP pathway and produces small amounts of ATP. This supplies energy to the cell to maintain metabolic processes until extracellular glucose is again available. Glycogen is also the primary source of glucose for sterol (a cell membrane component) synthesis during the lag phase of fermentation. Cells with depleted glycogen reserves are unlikely to perform normally in fermentations.
Much has been written about the various conditions that influence beer flavor during a fermentation, and the way in which they can be influenced on the large scale. As homebrewers our involvement usually only goes as far as providing a measured amount of a certain yeast strain with an appropriate home, some food to eat and a controlled environment, and hoping that it stays happy. The majority of the time this is exactly what it does.
Temperature control is crucial to flavor generation. Good early wort aeration and correct pitching rate will help with consistency. Ensuring the yeast is free of bacteria and wild yeast will aid in guarenteeing long term flavor stability, as will avoiding oxygen ingress late in the fermentation. Good removal or settling of yeast will aid in clarifying the beer and also ensure that the correct amount of yeast remains in suspension for maturation.
