The Science of Yeast

Recently one of the brewers at Sierra Nevada, talking over a couple of beers, proposed that yeast may be the dominant organism on the planet. The basis for his hypothesis was his observation that many of the brightest human beings in the world seem to devote their lives to caring for yeast and making sure they’re happy. While the scientist’s motives may be ulterior, I think a good argument can be made to support his idea.

Yeast is almost certainly the most important ingredient in beer. Although water, malt and hops are crucial, it is yeast that turns wort into beer. Although the yeast itself is usually not in evidence in finished commercial beers (with some notable exceptions), it is almost always found in homebrewed beers. In addition, the flavors and aromas generated by the yeast are key sensory qualities in beer. Alcohol, higher alcohols, esters, ketones and aldehydes are all products of fermentation. Many brewers go to great expense to accommodate difficult yeast strains to achieve their desired flavor profile, including Anheuser-Busch, with its beechwood aging and specially designed fermenters.

In nature, there are about 350 species of yeasts covering a wide range of functions. Most live on fruits, flowers or other places where simple sugars are found in abundance. Of all the species, there are only a few we are concerned with in brewing.

Brewer’s yeast is not found in nature. It is a domesticated microorganism. Unfortunately yeast, like all biological systems, is inherently difficult to control. A five-gallon homebrew fermentation consists of hundreds of billions of living cells carrying out uncountable trillions of biochemical reactions every second, with the outcome of many affecting the outcomes of many others. Commercial brewers, who harvest their yeast and re-use it repeatedly to save money, have the issue of yeast maintenance and care. In contrast, homebrewers have the luxury of purchasing a “ready to use” yeast that can be used once and thrown away. This certainly simplifies life for the homebrewer and allows him to concentrate on producing a wide range of interestingly diverse products.

Yeast Classification

There are three types of cells on earth: eubacteria, archaebacteria and eukaryotes. Eubacteria and archaebacteria — formerly lumped together and called prokaryotes — are smaller than eukaryotes and have fewer internal cellular stuctures. The bacterial species that contaminate beer, including Pediococcus, Lactobaccilus and Acetobacter, are eubacteria. In contrast, all yeasts are eukaryotes.

Eukaryotes include protists, plants, fungi and animals (including humans). The average eukaryotic cell is about 10 times the size of the average bacterial cell and eukaryotic cells have much more interior structure. Most notably, eukaryotic cells have a nucleus while other types of cells do not.

Yeast are fungi, members of the fungal division Ascomycota. There are about 40 genera of yeast and brewer’s yeast belongs to the genus called Saccharomyces.

Yeast Structure
A mature brewing yeast cell is 8–14 micrometers in diameter, and weighs about 40 pg (40 x 10-12 g). Yeast have a structure that is typical of eukaryotic cells and is thus, in many ways, similar to human cells. Eukaryotic cells have many subunits, called organelles, that help it function.

Cell Wall
The outermost layer of the yeast cell is the cell wall, which gives the cell its shape. The yeast cell wall is composed mainly of beta-glucans (40%) and alpha-mannans (40%), which are carbohydrates. It also contains some proteins (8%), lipids (7%) and mineral constituents (12%). The cell wall makes up about 30% of the total cell mass (dry weight). The outer surface of the cell wall has a high amount of phosphate and carboxyl groupings, giving it an overall negative electrical charge at the pH of beer.

In addition to its contribution to cell shape, the cell wall has several other functions. The cell wall acts as a physical barrier for the yeast cell. It also acts as an osmotic barrier, preventing the cell from drying out or bursting and increasing the cell’s tolerance to extreme concentrations of ethanol and sugar. The cell wall also determines the flocculation properties of yeast strains, the degree to which the cells stick together at the end of fermentation. This also determines whether the yeast is top- or bottom- cropping. A yeast’s flocculation characteristics are based on the types of protein it has embedded in its cell wall.

The cell wall also surrounds several enzymes that are excreted from the cell. The most important of these is invertase, which can convert sucrose to glucose and fructose for yeast metabolism. Another enzyme found associated with the cell wall of some yeasts is melibiase, which can degrade the simple sugar melibiose. While not key in beer fermentation, its presence or absence is important in the identification of yeast cells.

Cell Membrane
Inside the cell wall is a secondary protective layer for the cell, called the cell membrane. You can loosely think of the cell membrane as a balloon inflated against a cage made of the cell wall. The cell membrane is a sheet consisting of two layers of phospholipids (relatively large, polar molecules) with proteins floating in it. Many of the cell membrane proteins span the membrane. The two layers of the phospholipid bilayer are arranged so that the outside has an affinity for water and the inside has an aversion to water.

The proteins embedded in the membrane add to the structural integrity of the membrane and many are used for the transport of material across the membrane. Material attaches to the proteins and is carried across the membrane. The cell membrane not only surrounds the cytoplasm inside the cell, but also connects with the other membranous organs inside the cell, such as the Golgi apparatus.

The cell membrane is involved in the uptake of nutrients for the yeast cell, and the proteins in the cell membrane facilitate this transport. The cell membrane must be fluid to allow transport of these nutrients. (In a future article on fermentation biochemistry, I will explain why.) Aeration of the yeast or wort at the beginning of fermentation is key to obtaining membrane fluidity. It enables the growing yeast cell to synthesize necessary molecules, such as unsaturated fatty acids and sterols.

There are three types of transportation that can occur across the cell membrane.

Passive Diffusion: Passive diffusion is the simplest form of transport into the cell and requires no energy from the cell. Molecules simply pass directly through the cell membrane, or through a pore consisting of a protein bridging the lipid bilayer. The rate of diffusion depends on the charge of the molecule (electrical charge impedes diffusion), its size (smaller molecules diffuse faster than larger ones) and lipid solubility (highly lipid-soluble molecules diffuse faster, since they can penetrate the hydrophobic portion of the lipid bilayer). The net movement is down the concentration gradient for the molecule; in other words, molecules move from regions where they are more concentrated to regions where they are less concentrated. Examples of molecules that can penetrate the cell by passive diffusion are: water, calcium ions (Ca2+), sodium ions (Na+) and potassium ions (K+).

Facilitated Diffusion: Facilitated diffusion is similar to passive diffusion, but requires a permease (carrier protein) to allow diffusion. This allows control of the diffusion, but the diffusion is still down the concentration gradient. In general, facilitated diffusion is faster than simple diffusion, and no energy output by the cell is required. The permeases are specific to the structure of the transported molecules, and they are controlled depending on the needs of the cell. Molecules that enter the cell by facilitated diffusion include: glucose, fructose, maltose, maltotriose, clorine ions (Cl-), and carbonate ions (HCO3-).

Active Transport: Active transport is the only mechanism for moving molecules into the cell against a concentration gradient. It is critical to the health of the yeast cell, since there are many nutrients that must be “hoarded” at a higher concentration than they exist outside the cell. Active transport also uses carrier proteins to transport molecules across the membrane, but they require energy to allow this transport. The transport proteins help move specific molecules, or groups of molecules, across the  cell membrane. Molecules that enter the cell by active transport include amino acids (at differing rates) and calcium ions (Ca2+).

Mitochondria are organelles that are roughly the size of small bacteria. They move around inside the cell and replicate semi-autonomously. They have a small, circular genome (like bacteria) whose DNA sequences are very similar to modern-day purple bacteria. (This, along with much other evidence, has convinced scientists that mitochondria are descended from ancient free-living purple bacteria that came to live inside ancestral eukaryotic cells!)

In most eukaryotic cells, the mitochondria are the site of the electron transport system, a key component of respiration (the aerobic process that releases energy from sugar). For this reason, many biology texts refer to mitochondria as the “powerhouses of the cell.” Some homebrew texts refer to the “respiration phase” of fermentation, proposing that early in the fermentation, yeast use oxygen to respire. They quote the Pasteur effect, which states that if oxygen is present, organisms will respire, rather than ferment. However, the Crabtree effect overrides this.  The Crabtree effect is the observation that, if the glucose concentration is above 0.4%, yeast will ferment, rather than respire, even if oxygen is available to the cell.

During fermentation, glucose is always present at greater than 0.4%,  so brewing yeast are not able to respire. As a consequence, their mitochondria are small and serve little function. If brewing yeast are exposed to oxygen in low glucose conditions, they will overcome the Crabtree effect and their mitochondria will proliferate within six to eight hours. This fact is used to grow yeast rapidly in commercial yeast propogators.

The nucleus is an organelle surrounded by two membranes that resides in roughly the middle of the cell. In a normal yeast cell, the nucleus is approximately 1.5 micrometers in diameter. The nucleus’s double membrane is covered in pores, which allow molecules to migrate into and out of the nucleus. The nucleus is the location of the cell’s genetic material, deoxyribonucleic acid (DNA). Yeast cells have 16 chromosomes and its genome size is approximately 1.3 x 107 base pairs — in other words, there are about 13,000,000 “letters” in its genetic code. Laboratory species of Saccharomyces usually have one copy of each chromosome, except when they undergo sexual reproduction and form a cell with two copies of each chromosome. Brewing strains of Saccharomyces carry approximately three copies of each chromosome, but the number for each chromosome varies. Some strains may have more copies of certain chromosomes.

Endoplasmic Reticulum
The Endoplasmic Reticulum (ER) is a membranous structure found throughout the cytoplasm. It is a maze of tubes or sacs that channel materials around the cell. There are two kinds of ER, smooth ER and rough ER. Rough ER is covered with ribosomes, the organelles responsible for the production of proteins. Smooth ER lacks ribosomes. Many metabolic sequences take place in the smooth ER. Smooth ER is also the site of most lipid synthesis, including the synthesis of triglycerides, phospholipids and steroids, all of which are important to cell membrane components. The rough ER makes many proteins, especially glyocoproteins (proteins with carbohydrates attached),  for export outside the cell.

Golgi Apparatus
The Golgi apparatus is an organelle that looks like a stack of folded membranes. The Golgi apparatus accepts proteins — including glycoproteins and lipoproteins — made in the ER. They enter one end of the Golgi apparatus, are modified within it, and are excreted on the other end. Excreted molecules are contained in vessicles, “bubbles” of membrane that pinch off the Golgi apparatus. These vessicles bind with the cell membrane and dump their contents outside the cell.

The Vacuole
Brewing yeast typically contain only one vacuole, a storage area for enzymes and nutrients. The vacuole is bounded by a single membrane, and typically contains several dense granules of phosphate material that will be used during fermentation. The vacuole contains hydrolytic enzymes that are used to recycle large molecules from the cell (RNA and protein for example). If the cell is subjected to conditions that destroy the vacuole (i.e. high temperature or pH), the vacuole will release its contents and the hydrolytic enzymes will destroy the cell (autolysis).

Yeast Life Cycle
Brewer’s yeast reproduces by budding, and the budding process leaves behind a bud scar on the mother cell. Once a yeast cell has too many of these scars, it will lose the ability to reproduce (a normal yeast cell can bud up to 50 times). During normal fermentation, the yeast will bud several times, resulting in a three- to five-fold increase in the population, as each daughter cell will bud several times as well.

Yeast Qualities
There are several qualities that brewers look for in their yeast. These include: flavor and aroma qualities, sedimentation and flocculation, attenuation,  head size, mutation rate and the consistency of crop.

Flavor and Aroma
Flavor and aroma are often regarded as the most important characteristic of a yeast, as far as the brewer is concerned. In commercial breweries that use only one yeast strain, the flavor and aroma of all of the beers will exhibit the “house character” of that yeast. Many homebrewers will take this approach and develop techniques of reclaiming and re-using their own yeast time and time again. Many commercial brewers also use multiple strains to develop distinct characters in their specialty beers. And as I mentioned earlier, many brewers feel that the flavor and aroma qualities of a particular strain of yeast are so important that they will tailor their process to the idiosyncracies of that particular strain.

Flocculation is the aggregation (grouping) of cells into masses at the end of fermentation. Some yeast flocculate and settle to the top, while others settle to the bottom of the tank. Most modern yeasts can be forced to settle to the bottom of a fermenter, but there are several tenacious top fermenters (such as Bavarian Weiss strains) that can only be harvested from the top of the fermenter. Most of these strains require open fermentations. Flocculation also describes how quickly and densely the yeast tend to drop out of the wort. A yeast that settles too quickly will tend to leave fermentable sugars in the beer, and may also be unable to adequately remove off-flavors during maturation. A poorly flocculating yeast will cause problems in filtering or fining the beer, since it stays in suspension. Flocculation may also affect the flavor characteristics produced by the yeast. The size of the head formed by the yeast while fermenting is important when deciding the size of vessel required for fermentation. Yeast typically produced a head that rises from 15–25% above the level of the wort in the tank, and fermenters should be sized accordingly.

Mutation Rate
Mutation rate describes the genetic stability of the yeast strain. Mutations such as respiratory deficiency cause problems in flavor and aroma characteristics and in flocculation. So-called “petite” mutants are known for their propensity to leave diacetyl in the finished beer (but are not the first place to look if your beer contains diacetyl). Although mutation rate might seem to be the determining factor in the amount of re-pitchings a yeast strain can withstand over time, the bacterial load is more often the determining factor.

Dried or Liquid Yeast

For the commercial brewer, liquid yeast gives more options, but is typically harder to deal with since it must be grown up from a small sample, or even a slant. The pitching rate is more difficult to determine, and you must brew fairly often to keep the yeast viable in storage between brews, or invest in a yeast propagation system. But for the homebrewer, a wide array of liquid yeast are available that are simple and convenient to use.  They are grown on nutrient-rich media in the presence of oxygen and are incrementally fed low concentrations of glucose.

Dried yeast is easier to use, since all it requires is hydrating and dumping the yeast into the fermenter. Although it is easier to control the pitching quantity (simply the weight of yeast added), there are less choices of variety of dried yeasts.

Lager vs. Ale Yeast

Strictly speaking, yeast that can metabolize melibiose are classified as Saccharomyces uvarum, or lager yeast. Brewing yeasts that lack this ability are classified as Saccharomyces cerevisiae, or ale yeast. In recent years, it has been found that the distinction between these yeast on a taxonomic level does not correspond well with the actual performance of the yeast in brewery fermentations. A yeast may have an ale yeast taxonomy (it does not metabolize melibiose), but it may perform like a lager yeast in the brewery (it bottom ferments at low temperatures). Some brewers report that their ale strain routinely is more attenuative than their lager strain. Most brewers are under the impression that lager yeast makes drier beers, but this is not always the case.

Top fermenters vs. bottom yeasts can be determined by cell wall composition and can be tested by placing them in a clean test tube with H2O and shaking. Those that make a “skin” at the top are probably top fermenters. Saccharomyces cerevisiae can ferment glucose, sucrose and maltose, S. uvarum can ferment all including melibiose, and S. diastaticus can ferment all of the primary sugars plus dextrins. The last strain mentioned, S. diastaticus, produces considerable quantities of a buttery off-flavor known as diacetyl and is considered a wild yeast. Since it can ferment carbohydrates the others cannot, it is a big risk in bottle- conditioned beers. It can over-attenuate them and cause bottles to explode.

As a living system, yeast metabolism is even more complex than the enzymatic changes in the mash. Do not expect to be able to completely control the activity of the yeast, since we cannot hope to control all the factors and changing one thing generally affects many others. Watch for patterns that develop in yeast performance, and pay attention to the many factors that might affect them. The amount the yeast grows, the time it takes to fully ferment the wort and the size of the head in the fermenter are all clues to yeast performance. Fermentation flavor and aroma are clues that all is going well, so develop your senses. The variables controlling yeast activity and flavor development are innumerable, and the best you can hope to do is shepherd it in the right direction.

Issue: September 2002