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Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi and are generally recognised as being unicellular. The diversity of yeast is shown by their presence in both divisions Ascomycota and Basidiomycota, phylums into which they are classified based on their sexual characteristics. They reproduce vegetatively and are not enclosed in a fruiting body. (Boekhout & Kurtzman, 1996). Lower taxonomic categories are based on morphological, physiological and genetic characteristics. The aim of yeast taxonomists is to classify yeasts to species level while the identification of sub-species or strains is the work of yeast technologists. (Lachance, 1987). Identification and classification of yeasts is very important in the biotechnology industry. For example, it is essential to be able to distinguish between wild type strains and cultured strains in an industrial process. This can be demonstrated in the brewing industry, for example, where the presence of wild yeasts may create undesirable flavours in the product. To date, around 1500 species of yeast have been identified, representing only a fraction of the complete yeast biodiversity on earth. It is estimated that only 1% of all yeast species have currently been described. (Kurtzman et al. 2006). At this rate, it would take mycologists several hundred years to document all new species thought to exist. It is important for yeast biologists not only to appreciate this great biodiversity but to develop ways to preserve remaining species, especially those that have potential use in biotechnology. There is also a huge gap in the knowledge relating to known species of yeasts. For example, approximately 50% of the 6000 genes in Saccharomyces cerevisiae are of unknown function. (Oliver, 1996). Yeasts are found in many areas of the natural environment, dominating fungal diversity in oceans. (Bass et al. 2007). Yeasts are chemoorganotrophic, meaning that they require fixed, organic forms of carbon for growth. There are many sources of carbon available to yeasts in the natural environment such as simple sugars, organic acids, hydrocarbons and alcohols. Different yeasts are able to utilise different carbon sources, which ultimately determines their particular habitat. They can be found inhabiting the surface of plants, surface and intestinal tract of animals, and soil to name a few. Some can also be found in man-made environments. For example, S. cerevisiae is almost the only yeast species found colonising surfaces in wineries. (Martini, 1993). Candida albicans is found in hospitals and in some cases, accounts for over 80% of all hospital-derived infections. (Schaberg et al. 1991). Yeasts are probably the world's oldest domesticated organisms. They have been used to produce alcohol and to leaven bread for thousands of years. The brewing of beer is considered the world's first biotechnology process. Modern yeast technology can now be used to produce many different biopharmaceutical products for preventing and treating a variety of human diseases. S. cerevisiae has been extensively studied and is considered a model eukaryotic organism, as it has contributed significantly to biological knowledge in the areas of environmental technologies, biomedical research, food/chemical industries and healthcare industries. While the majority of yeasts are beneficial to humans, there are some negative aspects associated with them. For example, some humans occasionally have a pathogenic relationship with dietary yeasts, leading to various intestinal disorders. Some yeasts are also responsible for food spoilage.
Yeast nutrition is concerned with how yeasts transport water and essential organic and inorganic nutrients from their growth medium into the cell. Yeast nutrition also refers to the use of nutrients for cell metabolism. The understanding of these nutritional requirements is essential for successful cultivation of yeasts in the laboratory and for the optimisation of industrial fermentation processes. Yeast is comprised of the elemental building blocks: carbon, hydrogen, oxygen, nitrogen, phosphorus and sulphur. These form macromolecules such as proteins, polysaccharides, nucleic acids and lipids. Inorganic and trace elements include potassium and magnesium. Macronutrients need to be at the mM level and micronutrients need to be at the µM level for yeast to acquire their essential nutrients from their growth environment. Table 2.1 summarises the main elemental requirements of yeasts.
Table 2.: Summary of elemental requirements of yeasts
Structural element. Catabolism provides energy
Protons from acids
Transmembrane proton-motive force. Intracellular acidic pH of 5-6
Respiration and fatty acid synthesis
Ammonium salts, urea, amino acids
Structure of proteins and enzymes
Energy transfer, nucleic acid and membrane structure
Ionic balance and enzyme activity
Enzyme activity, cell and organelle structure
Amino acids and vitamins
Hemeproteins and cytochromes
As yeasts are chemoorganotrophic organisms, they obtain their carbon and energy from fixed, organic sources. Sugars are the most common source. Glucose is the most widely utilised sugar by yeast. Glucose is generally not available freely in nature for yeast to use. It is usually polymerised in cellulose, starch and other polysaccharides. Glucose is normally added to laboratory culture media for growing yeast. It is not used a lot in industrial fermentations as many cheaper sugar substrates such as maltose, sucrose, fructose, xylose and lactose are available. Glucose can act as an inhibitor of assimilation of other sugars in some yeast strains.
S. cerevisiae has quite a limited number of sugars that can be used as substrates for growth of biomass or fermentation. These are glucose, fructose, mannose, galactose, sucrose and maltose. Ethanol (a product of S. cerevisiae fermentation) and acetate can act as respiratory substrates in S. cerevisiae. Some yeasts (approx 5%) are not strictly chemoorganotrophs and derive their carbon by carbon dioxide fixation.
S. cerevisiae is a glucose sensitive yeast and utilises it in several different ways. The availability of glucose and oxygen are the two major environmental factors that regulate respiration and fermentation in yeast cells. The Pasteur effect states that, under anaerobic conditions, glycolysis proceeds faster than it does under aerobic conditions. This has been observed when glucose concentrations are low (below 5 mM in S. cerevisiae). The presence of oxygen means the Pasteur effect is no longer operable in S. cerevisiae cells. Respiration accounts for only 3-20% of sugar catabolised in growing cultures of S. cerevisiae, compared with 25-100% in resting cells. (Lagunas et al. 1982). Irrespective of oxygen availability, fermentation is the main route of sugar metabolism in actively growing cells of S. cerevisiae.
If the concentration of glucose in the medium is high, the Crabtree effect operates in S. cerevisiae cells. The Crabtree effect states that, even under aerobic conditions, fermentation predominates over respiration. The repression of the synthesis of respiratory enzymes is known as catabolite repression. These enzymes are not synthesised due to repression of the genes by high glucose concentration. As glucose levels decline, cells gradually become de-repressed, causing respiratory enzymes to be synthesised yet again. The S. cerevisiae cells then metabolise the accumulated ethanol produced by fermentation, as it is a carbon substrate.
The regulation of respiration and fermentation is highly important for many industrial processes which use yeast. The optimisation of respiration is important in the production of yeast biomass, for example, in the food industry. The optimisation of fermentation is important in the production of many alcoholic beverages. For the production of baker's yeast using molasses as a substrate, the sugar level must be controlled by using a fed-batch reactor in order to avoid the Crabtree effect.
As free glucose is quite scarce in the natural environment, yeasts must be able to metabolise non-hexose carbon sources such as pentoses, alcohols, hydrocarbons and organic acids. Free glucose is also scarce in many yeast cultivation media such as malt and molasses. These disaccharides (maltose, sucrose etc.) are hydrolyses into their monosaccharides and can then proceed to the glycolytic pathway. The metabolism of ethanol by yeasts under aerobic growth is very efficient.
Elemental hydrogen is present in many macromolecules of yeast cells and is also available from carbohydrates. Hydrogen ions (protons) are very important for maintaining intracellular and extracellular pH, both of which have a dramatic influence on the growth and metabolism of yeast cells. A pH value of 6.0 has been shown to be the optimal value for yeast biomass growth while a pH value of 5.0 has been reported for optimal fermentation, both using glucose as a substrate. (Bull et al. 1976). Yeasts can grow quite well the culture medium is initially between pH 4-6. Many yeasts can, however, grow over a pH range of 2-8. Yeasts generally grow at a lower pH than most bacteria and therefore inhabit different ecological niches. This also allows them to act as spoilage microorganisms in acidic foods such as citrus fruits. The majority of yeasts do not grow well at alkaline pH. As yeast biomass increases, their culture medium becomes acidified due to a number of factors including ion uptake, proton secretion, organic acid secretion and production of carbon dioxide. The production of ethanol is very sensitive to changes in the pH of the medium. In growing cells, the intracellular pH is regulated within a very narrow range. This value is pH 5.25 in S. cerevisiae. (Cimprich et al. 1995).
Oxygen is a critically important growth factor as many yeasts are unable to grow in the absence of oxygen. The absence of oxygen in the growth medium allows yeast cells to ferment the carbon substrate producing ethanol. A yeast biomass can only grow in the presence of a sufficient amount of oxygen. This is because oxygen provides a substrate for respiratory enzymes during aerobic growth. Different yeasts have varying requirements for molecular oxygen. Pure oxygen at high pressure can in fact inhibit yeast cell growth. (Bull et al. 1976). The rapid growth of yeast biomass is highly dependent on oxygen supply to the bioreactor. Oxygen enters the culture medium as it is soluble in aqueous solution. Bioreactors have been adapted in order to increase the oxygen absorption rate, KLac, by yeast cells, where KL is the rate of oxygen passage from the atmosphere through the liquid interface and into solution, a is the area of the interface and c is the oxygen concentration in the medium. On a laboratory scale, the presence of baffled indentations in conical flasks can greatly increase KLac values and ultimately improve yeast growth.
Nitrogen accounts for approximately 10% of the dry weight of yeast cells. Yeasts are unable to fix atmospheric nitrogen and therefore simple inorganic nitrogen sources such as ammonium salts are commonly used in culture media. The most common salt used is ammonium sulphate as it also provides a source of sulphur. Some organic nitrogen sources include amino acids, peptides, purines, pyrimidines and amines. Industrial fermentations use malt wort as a substrate which is composed of mixtures of amino acids. (Busturia & Lagunas, 1986) showed that depriving S. cerevisiae cells of nitrogen leads to inactivation of the sugar transport systems which in turn reduces the fermentation rate, but does not alter the rate of aerobic respiration. Both organic and inorganic nitrogen is used for the structural and functional nitrogenous compounds in the cells.
Sulphur is required by yeasts mainly for the biosynthesis of sulphur-containing amino acids. Sulphur comprises approximately 0.3 % of yeast cell dry weight. Sulphur sources can be in the form of sulphate, sulphite, thiosulphate, methionine and glutathione. Methionine is the most effectively used amino acid in yeast nutrition. Nearly all yeast species can synthesise sulphur amino acids from sulphate.
Phosphorus is essential for all yeasts as it is present in phospholipids and nucleic acids. The phosphate content of yeast cells accounts for approximately 3-5% of dry weight. The presence of inorganic phosphate is responsible for the negative charge of the yeast cytoplasm. The most common sources of phosphorus in culture media come from orthophosphates (H2PO4-) and condensed inorganic phosphate. Orthophosphate acts as a substrate and effector of many enzymes, including enzymes involved in energy transfer. Yeasts can effectively store phosphate in organelles. A 110-fold higher concentration of phosphate is found in the vacuole compared with the cytoplasm. (Okorokov et al. 1980).
Growth factors are organic compounds required in very low concentrations for specific catalytic or structural roles in yeast. They are not used as energy sources. Yeast growth factors include: vitamins (for metabolic functions as components of coenzymes), purines and pyrimidines, nucleosides and nucleotides, amino acids, fatty acids, sterols and other miscellaneous compounds such as polyamines, choline and meso-inositol. Yeast is said to have a growth factor requirement when it cannot synthesise a particular factor which inhibits a key metabolic process and growth without its addition to the culture medium. S. cerevisiae requires biotin (a cofactor in carboxylase-catalysed reactions), panthothenic acid (for acetylation reactions), inositol and thiamine (for decarboxylation reactions).
Yeast cells are quite easy to grow due to their relatively simple nutritional requirements. For laboratory cultivation of yeasts, there are many synthetic and complex media commercially available. Some yeast strains require a specific growth media. Malt extract broth (beer wort) is a traditionally used complex medium for the rapid growth of yeast cells. This can be prepared in solid form by adding 1-2% (w/v) agar in order to grow cells on a Petri dish. Another complex medium is yeast extract which is a product of the breakdown of yeast structural and storage macromolecules. It is usually supplemented with peptone and glucose and is commonly used in the short-term maintenance of laboratory strains. Other complex media include Sabouraud's medium (glucose and mycological peptone) for medically important yeasts and Wallerstein Laboratories Nutrient for brewing yeasts. An example of a synthetic medium is Yeast Nitrogen Base (by Difco), to which a carbon source must be added, usually to a final concentration of 1% (w/v). Yeast Carbon Base is also available, to which a nitrogen source must be added. Certain selective media contain antibacterial, antifungal and anti-yeast agents in order to select against other microorganisms from samples. Cycloheximide is an antibiotic which also inhibits some yeasts and is sometimes used to detect the presence of wild yeast strains in brewing yeast. For the growth or fermentation of yeast cells on an industrial level, several types of agriculturally based complex media are used such as molasses, malt wort, wine must and cheese whey. It is important to note that some media used for fermentation may not be suitable for the growth of yeast biomass. For example, the high concentration of sugar in molasses promotes fermentation of the medium by yeast cells and is not suitable for aerobic growth. A glucose concentration of approximately 0.2M is considered sufficient for aerobic growth of yeast biomass. Some of the industrial fermentation media mentioned above have low levels of compounds which can be toxic to yeast.
The growth if yeast is concerned with how yeasts transport and assimilate nutrients and how these nutrients are utilised in order for the cell to increase in mass and eventually divide. Yeast cells can divide by budding, fission or filamentation. S. cerevisiae divides by budding. The control of yeast cell populations in liquid culture is important for the performance of industrial processes which use yeasts. The growth of yeast on solid surfaces is also relevant in industry and medicine. Yeast cells can be grown in batch, fed-batch, continuous, phased and immobilised culture systems.
Growth of Yeast Biomass in Batch Mode
When yeast cells are inoculated into a suitable liquid nutrient medium and incubated under optimal growth conditions, a typical batch growth curve results when the population of cells is plotted against time as shown in Figure 3.1.
Figure 3.: Typical yeast growth curve showing the phases of growth of a yeast in batch culture, µmax is the maximum specific growth rate and tD is the doubling time.
The lag phase is a period of zero growth where the specific growth rate, µ = 0. The lag phase occurs when the inoculums cells are subjected to a change in nutrient medium or physical conditions such as temperature. Lag time is not only dependent on growth conditions but also on the density of the inoculum and its growth history. This phase represents the time required for yeast cells to adapt to their new physical and chemical environment.
Yeast cells enter an acceleration phase before exponential growth. In this phase, the cells have started to actively divide. The rate of increase (dx/dt) in yeast biomass (x) with time (t) during this phase is expressed as:
The exponential phase is indicated by logarithmic cell doubling and a constant maximum specific growth rate (µmax). The value of µmax is dependent upon the species of yeast and growth conditions. If growth conditions are optimal and cells double logarithmically, then:
When integrated, this gives the equation:
(Where x0 is the initial cell mass)
The final equation above is the fundamental equation for exponential batch growth. The doubling time (tD) of a culture can be calculated by knowing µmax by the equation:
The exponential growth phase in batch culture is finite but can be extended by using the fed-batch culture method, which involves the addition of substrate in line with yeast growth. Some yeasts can enter a second stage of exponential growth called diauxie. This may occur when yeasts are exposed to two carbon substrates which are metabolised in sequence. For example, S. cerevisiae grows diauxically when aerobically grown cells exhaust glucose. Other enzymes are then used to metabolise ethanol. The exponential phase lasts for a relatively short time due to essential nutrient exhaustion and an increase in metabolites that inhibit cell growth.
Cell growth is retarded in the deceleration phase. There is a much lower specific growth rate than µmax at this stage. When yeast growth is limited by the concentration of one substrate (S), the relationship between µ and S can be defined by the Monod equation:
Where KS (the saturation constant) is the value of S which limits the growth rate to ½ µmax.
In the stationary phase, yeast biomass remains constant and the specific growth rate returns to zero. If yeast cells remain in the stationary phase long enough they may die. This can have an effect on the growth and survival of remaining viable cells. Cell death is exponential and is expressed as:
Where k is the death rate.
The stationary phase allows yeast cells to survive for long periods of time without the addition of nutrients. Some characteristics shown by yeast cells in stationary phase include: slow metabolic rate with low rates of protein synthesis, increased thermal and heat-shock resistance, no cell division and development of thick cells walls resistant to lytic enzymes. Other factors which cause yeast cells to enter stationary phase include: toxic metabolites (ethanol), low pH, high carbon dioxide concentration, variation in oxygen levels and increased temperature.
Growth of Yeast Biomass in Continuous Mode
In a continuous culture, yeast cells can be grown for long periods of time without entering the lag or stationary phases. This involves the continuous addition of fresh culture medium to the vessel while simultaneously removing a volume of exhausted medium containing yeast cells. The vessels used in continuous mode are referred to as chemostats. The change in yeast biomass per unit time is the difference between the growth rate and the removal rate of cells:
Where D is the dilution rate or flow rate per unit volume (h-1).
Once continuous growth is achieved, the growth rate remains constant and a steady state cell concentration is maintained such that dx/dt = 0. Therefore µ = D and yeast growth is determined by the dilution rate. This is achieved by changing the rate at which nutrients are delivered to the chemostat.
Yeast cells can also be grown in continuous mode by using a turbidostat rather than a chemostat. In this system, cell growth is not limited by one nutrient. The maximum growth rate is maintained by monitoring the level of biomass by optical density. Fresh medium is added when yeast biomass exceeds a set optical density value.
Physical Requirements for Yeast Growth
Most yeasts grow well in warm, moist, sugary, acidic and aerobic environments. Some may be more adapted to extreme physical or chemical conditions, such as certain spoilage yeasts.
Temperature is one of the most important physical characteristics influencing the growth of yeast. Most yeast strains grow quite well between 20-30°C, characterising them fundamentally as mesophiles. However some yeasts grow optimally at low temperatures (5-18°C) making them psychrophiles, such as Leucosporidium spp. Some also grow optimally above 20°C and are known as thermophiles, such as Kluyveromyces marxianus. Most yeasts used in biotechnology are mesophilic. If temperature is increases beyond optimal growth levels, cells become damaged and the number of viable cells decreases. Yeast cells also show a heat-shock response when exposed to high temperatures, protecting the cell from damage.
Yeasts require water in high concentrations for growth and metabolism. All substrates and enzymes are in aqueous solution so water is needed for any type of enzyme activity to occur. One of the most severe stresses on yeast is dehydration, commonly carried out on yeast biomass in industry to produce dried baker's yeast
Most yeasts grow well between pH 4-6 but nearly all species are able to survive from pH 2-8. Yeast growth is inhibited more by organic acids such as acetic acid rather than mineral acids such as hydrochloric acid. This is because organic acids are undissociated and lower intracellular pH.
Many factors need to be considered in optimising a culture medium for the growth of yeast. Assuming the S. cerevisiae species is used, it is necessary to consider all elemental requirements, physical and chemical conditions required for optimal growth. One of the key aspects of optimising the media involves having the carbon substrate at the correct level in order to produce a yeasty biomass and avoid fermentation of the medium to alcohol and carbon dioxide. Many commercially available cultivation media can be optimised by the supplementation of growth factors ensuring maximum biomass growth. In addition to the correct substrate concentration, the physical and chemical factors inside the bioreactor play a fundamental role in the growth of yeast cells. Optimal values for temperature and pH must be determined in order to ensure the viability of yeast cells. This combination of the correct nutritional requirements, physical factors and chemical conditions ensure the rapid production of large quantities of yeast cell biomass.