Yeast Micronutrient and Growth

Factor Requirements
Novozymes North America Technical Service - Bioenergy

Yeasts require several micronutrients for optimum growth and fermentation performance at quanti-
ties typically between 0.1 to 100 mM. The requirements are dependent on the yeast strain, fermen-
tation conditions, and interactions with other components. Except for zinc, grain and corn based
mash are not usually deficient in micronutrients. It is important to remember that changes in the
fermentation substrate can cause large differences in micronutrient content. Bioavailability of es-
sential micronutrients can dramatically influence the yeast fermentation performance. Because nu-
trient deficiencies will impact yeast health and nutrition, as well as fermentation, nutrient supple-
ments are offered to “even out” periods of nutrient deficiency and make fermentation conditions
more consistent.

Maintaining yeast health and viability is critical for fermentation. Improper yeast nutrition can result
in weak cells, slowed growth, poor stress resistance, and stuck or sluggish fermentations. Growing
cells produce alcohol 33X’s faster than non-growing cells, and growth is limited by lack of essential
nutrients and ethanol toxicity. Yeast cells require a wide range of nutrients for growth and meta-
bolic functions, such as metabolic regulation and fermentative alcohol production. Since many of
the micronutrients are essential in enzymatic and structural components, an imbalance in micronu-
trients is often reflected in metabolic patterns and growth characteristics. Potassium, magnesium,
calcium, iron, zinc, and manganese are required at concentrations between 0.1 and 1 mM. Cobalt,
boron, cadmium, chromium, copper, iodine, molybdenum, nickel and vanadium are required be-
tween 0.1 and 100 mM. Minerals such as silver, arsenic, barium, mercury, lithium, nickel, osmium,
lead, selenium and tellurium can be inhibitory when concentrations exceed 0.1 mM. Availability of
some of the micronutrients is dependent on pH and the presence of chelating agents (e.g. succinic,
malic, and citric acids). The concentration of other ions can also affect availability.

Magnesium (Mg2+)
Magnesium (Mg2+) is the most abundant intracellular divalent cation in yeast and makes up approxi-
mately 0.3% of the yeast cell dry weight. The requirement for Mg2+ is typically around 100 ppm, and
usually it is adequately provided by the raw materials and water used in the fermentation process.
Mg2+ at 500 ppm has shown to help protect against high temperature, ethanol, and osmotic pressure
stresses. Although yeast cells can tolerate higher levels of Mg2+, cell growth has been shown to be
completely inhibited at 1 M.

Magnesium plays a multifaceted role in yeast cell physiology at the cytological, biochemical and bio-
physical levels. It is an essential cofactor for over 300 enzymes involved in many metabolic and bio-
energetic pathways. It is essential for numerous structural and metabolic functions and cannot be
replaced by other cations. Mg2+ is absolutely required for DNA and ATP synthesis, and it stimulates
the syntheses of essential fatty acids, alleviates the inhibitory effects of heavy metals, regulates cel-
lular ionic levels, activates membrane ATPase, alleviates the toxic effects of heavy metals (Cd, Co,
Cu, and Al), and maintains membrane integrity and permeability. Changes in intracellular Mg2+ can
dramatically influence enzyme activity because Mg2+ maintains the tertiary structure of proteins and
the general structural integrity of cells and organelles, especially under stressed conditions.

Mg2+ is necessary for the activation of several glycolytic enzymes and if limited the conversion of
sugar to alcohol may be suppressed leading to slow or incomplete fermentations. Mg2+ can promote
fermentation of high gravity mashes because it is involved in stress tolerance. It can counteract
yeast stress response caused by temperature, osmotic pressure, oxygen free radicals, and heavy
metals. Under extreme temperature conditions, Mg2+ maintains cell viability when cells are shocked
by preventing the syntheses of heat-shock proteins. Under oxidative stress, it counteracts the stress
caused by reactive oxygen species by neutralizing O2 free radicals. Ethanol increased yeast cell per-
meability to Mg2+, thereby increasing the tolerance to otherwise toxic levels of ethanol. Increasing
Mg2+ bioavailability, either by extracellular supplements or preconditioning, has been shown to im-
prove fermentation performance and increase stress-resistance in Saccharomyces cerevisiae.

Potassium (K+)
Potassium (K+) is an absolute requirement for yeast growth. Yeast cellular K+ content is generally 1-
2% of the dry weight of the cell, and the nutritional requirement is typically 160 ppm. Fermentation
rates have been shown to decrease when K+ concentration is between 4 to 10 mM. The start of cell
growth inhibition can be observed with K+ levels >10 mM, and total growth inhibition occurs at 2 M.

Yeasts require K+ as an essential cofactor for a wide variety of enzymes and synthesis of ribonucleic
acids. K+ is the most prevalent cation in the yeast cytoplasm. It is a central component in the regula-
tion of divalent cation transport and essential for the uptake of other nutrients like phosphate
(H2PO4-). Transport of K+ into the yeast cell is associated with the transport of H+ ions out of the cell,
thereby helping to maintain the cells pH. K+ is involved in osmoregulation and enzyme activity, as
well as involved in improving stress and ethanol tolerance during fermentation.

Calcium (Ca2+)
There are conflicting views on the levels of calcium (Ca2+) required for yeast growth, but approxi-
mately 180 ppm Ca2+ is recommended. Ca2+ makes up approximately 0.5 g/kg dry weight of Saccha-
romyces. In excess amounts, Ca2+ can be inhibitory for yeast. Inhibition of amino acid uptake can
occur above 1mM, and growth inhibition occurs above 25 mM. Ca2+ can interfere with the uptake of
Mg2+ and can block essential Mg2+dependent metabolic processes. A 10:1 ratio of Ca2+/Mg2+ will pre-
vent yeast growth, and 3:1 increases yeast lag phase due to Ca2+ antagonism of essential Mg2+-de-
pendent functions.

Even though Ca2+ might not be a requirement for cell growth, there is some evidence that it may
stimulate cell growth. Ca2+ plays a key role in metabolic responses, cell membrane stabilization, bud-
ding, and the synthesis of cell wall proteins. It is involved in membrane structure and function, im-
portant for flocculation, maintains membrane permeability under adverse conditions, and could im-
prove stress and ethanol tolerance during fermentation.

Manganese (Mn2+)
Manganese (Mn2+) is an essential cofactor required for yeast growth and metabolism, and may stim-
ulate fermentation. Mn2+ makes up 0.03 g/kg dry weight of Saccharomyces, and approximately 0.22
ppm Mn2+ is required to maintain proper yeast health. Concentrations of Mn2+ >10 mM can inhibit

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cell growth. At trace levels Mn2+ acts as an intracellular regulator for key enzymes. It also helps to
regulate the effects of Zn2+, and stimulates the synthesis of proteins and biosynthesis of thiamin.

Copper (Cu2+)
Copper (Cu2+) is an essential nutrient for yeast and is a cofactor for several enzymes at very low lev-
els. Cu2+ makes up 0.05 g/kg dry weight of Saccharomyces. Cu2+ is an essential micronutrient for
yeast respiratory pigments at concentrations around 0.1 ppm, but it can start to inhibit yeast cell
growth at 1 ppm, and at 15,000 ppm cell growth completely ceases. Different yeast strains have dif-
ferent sensitivity to Cu2+.

Iron (Fe2+)
Small amounts of iron (Fe2+) is needed as an essential nutrient for yeast and as a cofactor in several
enzymes, haem-proteins, and cytochromes. Fe2+ makes up approximately 0.1 g/kg dry weight of
Saccharomyces. Yeast typically require 0.17 ppm of Fe2+, which is usually abundantly provided in
mash. Fe2+ can be inhibitory at levels above 0.6 ppm, but does not promote cell death as much as
Cd2+, Cu2+, and Zn2+. Excess can lower activity of malate, pyruvate, and succinate dihydrogenases.

Zinc (Zn2+)
Zinc (Zn2+) is an essential cofactor that is rapidly taken up by the yeast cell. Zn2+ makes up approxi-
mately 0.12 g/kg dry weight of Saccharomyces, and 0.5 ppm is essential for yeast growth and main-
taining health. Some yeast may require more Zn2+ than occurs in mash and yeast supplements may
be needed. It is important to supply during propagation to promote cell growth to adequate num-
bers needed for fermentation. At higher concentrations, Zn2+ can suppress metabolic pathways that
aid in detoxifying yeast, which disrupt membrane structure resulting in intracellular components
leaking out of the cell, activate internal degradative enzymes, and activate cell lysis.

Zn2+ at a concentration of 0.3 to 1 ppm is required for alcohol dehydrogenase (ADH). It is the activa-
tor for drives the final reaction involved in ethanol fermentation, and no other cofactor can substi-
tute. Zn2+ is also essential for yeast cell growth and replication. Insufficient Zn2+ will prevent Saccha-
romyces cerevisiae budding, reduce cell count, and result in slowed fermentation. Less glycogen and
trehalose will be produced, which can cause yeast to be more sensitive to stressors. During fermen-
tation Zn2+ helps increase protein content of fermenting cells, and stimulates the uptake of maltose
and maltotriose. Zn2+ increases the tolerance of yeast to acetic acid, ethanol, heat shock, osmotic
stress, oxidative stress, and salt stress.

Growth factors
Growth factors are specific components that are required for yeast health and performance, but are
not synthesized by the yeast directly. Yeast growth factor requirements can vary widely depending
on process and environmental conditions. The typical required growth factors include vitamins, pu-
rines and pyrimidines, nucleosides and nucleotides, amino acids, fatty acids, sterols and other mis-
cellaneous compounds can vary widely.

Vitamins are important growth factors for yeast. The principal function is enzymatic and they gener-
ally act either as co-enzymes or precursors for fully active enzymes, serving a catalytic function in
yeast metabolism. Essential vitamins are required for maximum fermentation rates and strain de-
velopment. Yeasts vary widely in their need for vitamins, and the need may be different depending
on if the yeast is actively growing or alcoholic fermentation is occurring. A corn or grain mash should
contain sufficient amounts of vitamins, but supplements can be added directly to address any defi-
ciency. Deficiencies in vitamins can result in fermentation problems.

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Most strains require 6 ppb biotin for carbohydrate, lipid and leucine metabolism. Biotin is also spe-
cifically required for urea catabolism, resulting in 2 ammonia and 2 CO2 molecules.

Many types of yeast require calcium pantothenate, Coenzyme A, for several metabolic reactions.
Thiamine-pyrophosphate, Thiamin (B1), is needed for many reactions, including some related to fer-
mentation and amino acid biosynthesis. Inositol is required for structural membrane phospholipids.
Niacin (B4) is required as a coenzyme (NAD+, NADP+) for oxidation/reduction reactions under anaer-
obic conditions. Pyrodoxine (B6), pyridoxal and pyridoxamine are needed for amino acid metabo-
lism. Riboflavin (B2) is used as a coenzyme (FMN, FAD) under anaerobic conditions in oxidation/re-
duction reactions.

References
1. Barnett, J. A. (2003) A history of research on yeasts 5: the fermentation pathway. Yeast.
20:509-543.
2. Ingledew, W. M. Chapter 9: (2009) Yeasts: Physiology, nutrition and ethanol production (The
Alcohol Textbook 5th edition). 101-114.
3. Snyder, C, and M. Ingledew. (2009) Nutrition in fermentation. Biofuels Business. 54-56.

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