Vegetative reproduction by fission is characteristicIn this article we will discuss about the yeast:- 1. Origin of Yeast 2. Reproduction in Yeast 3. Life Cycles 4. Growth Requirements.
Origin of Yeast:
What is a yeast? The origin of the word in many languages relates primarily to its ability to ferment. The English “yeast” and the Dutch “gist” are derived from the Greek term zestos which means boiled, a reference to the bubbling foam caused by the evolution of carbon dioxide. The German “hefe” and the French “levure” both have their origins in verbs meaning “to raise,” again referring to the bubbling foam.
Industrial terms such as “cultured, true, wild, top, or bottom yeasts” have little meaning from the botanical point of view. In fact, even from an industrial viewpoint they are confusing, for yeast considered as a cultivated organism in one industry, for example- brewery yeast, may well be considered to be wild yeast by bakers. From a botanical point of view, yeasts are recognized to be a heterogeneous group.
Botanists of the 19th century generally accepted the idea that yeasts belong to the plant kingdom. At present, many taxonomists are in agreement with the arrangement proposed by Ainsworth in which the fungi are treated as a separate kingdom and the yeasts are included in the division Eumycota.
Yeasts lack chlorophyll and are unable to manufacture by photosynthesis from inorganic substrates the organic compounds required for growth, as do higher plants, algae, and even some bacteria. Hence they must lead a saprophytic, or in a few instances a parasitic, lifestyle. Yeasts possess rather rigid, thick cell walls, have a well-organized nucleus with a nuclear membrane (eukaryotic), and have no motile stages.
The ability to form sexual spores within an ascus or produce them externally on a basidium places most yeasts in the subdivisions Ascomycotina and Basidiomycotina, respectively. Those yeasts in which a perfect (sexual) stage is not known are grouped together in the subdivision Deuteromycotina.
While the definition of yeasts varies somewhat according to author, they are generally defined as fungi which, in a stage of their life cycle, occur as single cells, reproducing commonly by budding or less frequently by fission. Generally organisms with plurinucleate cells or those producing black pigments or producing asexual spores borne on distinct aerial structures are excluded. The distinction of yeasts from related mycelial fungal forms is highly subjective, resulting in a number of transitional forms between yeasts and the more typical higher fungi.
Taxonomic consideration of the yeasts relies heavily on morphological characteristics for genera. However, differentiation of species, because of the limitations of morphological criteria, relies very heavily upon physiological characteristics. Identification tests are standardized to enable investigators in different laboratories to compare their cultures with standard descriptions of type species and varieties.
Reproduction in Yeast:
Cellular Asexual Reproduction and Morphology:
The early investigators described yeasts as being round to oval in appearance and noted that they divided by budding to form daughter cells. This description agrees with that of many types of yeast. However, among the yeasts, the cell shapes and the means by which they reproduce are quite varied.
The yeast thallus (vegetative body) in its simplest form is a single cell or perhaps one with a bud still attached as exemplified by Saccharomyces cerevisiae. Here the first bud is usually detached when the mother cell initiates a second bud. There are yeasts, however, in which the buds will remain attached so that the mother cell and first daughter cell may produce additional buds. In such instances, this will result in small to large, clusters or chains of cells.
Similar to chain formation, a pseudomycelium (false mycelium) may be formed when, instead of the bud breaking away from the mother cell at maturity, it elongates and continues to bud in turn. In this manner chains of cells are formed, which in appearance resemble true mycelium (where cells are separated by a cross-wall or septum) but differ in the manner in which new cells arise (budding).
Pseudomycelia vary in their complexity from very primitive, in which the numbers of cells are limited and where there is little or no differentiation among these cells, to other forms where cells comprising the main chain are rather elongated and the buds arise in clusters on the shoulder of these elongated stem cells. The side buds in turn may remain spherical, ovoidal, or may also elongate giving rise to further branching and complexity of form.
While a particular yeast species may produce a characteristic pseudomycelial form, it is more commonly observed that several types of pseudomycelia are found within a single species. Consequently, while the type of pseudomycelium formed by yeast is of little value in classification, the ability or inability to form a pseudomycelium is used.
Budding, represents the most common method of vegetative reproduction. Except for the species of a few genera, buds usually arise on the shoulders and at the ends of the long axis of the cells. This type of budding, referred to as “multilateral budding,” is characteristic of Saccharomyces and most other ascomycetous yeasts.
In the case of spherical cells, buds do not appear to be oriented to any particular area of the cell surface. In multilateral budding, only one bud is produced from a particular site on the cell. This is in contrast to some yeasts of basidiomycetous origin, which, while reproducing by budding, apparently produce a number of buds at the same site, although more than a single budding site on the cell surface has been observed.
Special cases of budding are polarly oriented Hanseniaspora and Saccharomycodes species bud repeatedly at one site on 6ach of the tips of the cells. This type of budding causes the vegetative cell to assume a lemon shape and the yeasts are known as apiculates. The genus Pityrosporum is characterized by vegetative cells reproducing by repeated unipolar budding on a broad base. In one genus, Trigonopsis, yeast cells have a triangular shape with budding restricted to the 3 apices.
Vegetative reproduction by fission is characteristic of 2 genera, Endomyces and Schizosaccharomyces. In these yeasts, reproduction is carried out by the formation of a cross-wall (septum) without a constriction of the original cell wall. When the process is complete, the new cell wall divides into 2 individual walls and the newly formed vegetative cells separate from each other. Endomyces also produces true mycelium and by most yeast taxonomists is not considered yeast but rather a “yeast-related” genus. Its imperfect form is Geotrichum, the so-called “machinery mold.”
There are a few yeasts in which asexual reproduction is intermediate between typical budding and fission. This so-called “bud- fission” results from a type of budding in which the base of the bud is very broad, somewhat like a bowling pin. Separation of the daughter cell from the mother is by the formation of a septum across the broad neck.
Saccharomycodes and Nadsonia are genera exhibiting this type of reproduction. Fundamentally, bud-fission differs from typical budding only in the size of the septum. In budding it is so small that it appears that the bud is “pinched off” rather than distinctly separated by a septum.
Budding and Fission:
Some yeasts reproduce vegetatively by both budding and fission. Species of Trichosporon usually grow as mycelial strands (hyphae) by cross-wall formation. The strands can undergo disarticulation into individual vegetative cells called arthrospores, which, upon germination again, produce mycelium. Budding cells may also arise on the mycelial strands. In certain species of Candida and in Saccharomycopsis (Endomycopsis), hyphae with cross-walls and budding cells are found. However, in these genera the hyphae do not disarticulate into arthrospores.
A special type of mycelium is formed by some of the basidiomycetous yeasts (Leucosporidium, Rhodosporidium, and Sporidiobolus) in which clamp connections are formed between adjoining cells during the dikaryotic stage of their special life cycle. The clamp connection is a specialized mechanism which assures that 1 pair of compatible nuclei resides in each cell formed.
Spheroidal, globose, ovoidal, elongated, and cylindrical are descriptive terms for the general shape of many vegetative yeast cells. There are, however, certain yeasts which have highly characteristic cell shapes. The apiculate, due to the bipolar mode of budding, is characteristic of the species of Hanseniaspora and its imperfect genus Kloeckera, yeasts which are commonly isolated from the early stages of spontaneous fermentations or spoilage of fruits and similar raw materials.
Yeasts of the genus Dekkera (imperfect = Brettanomyces) produce ogival cells. These are rather elongated cells rounded at one pole and somewhat pointed at the other. These yeasts are used in the production of ale in Europe and are also found in bottled wines and soft drinks as spoilage yeasts. The unipolar mode of asexual reproduction results in a characteristic “flask-like” cell shape characteristic of the genus Pityrosporum and is associated with skin disorders of warm-blooded animals.
Tripolar budding results from the unique triangularly-shaped cells of Trigonopsis, originally isolated from beer in a Munich brewery and subsequently from grapes in South Africa. Also characteristic are the highly curved cells formed by Cryptococcus cereanus, yeast found present in the fermenting juices of certain rotting cacti.
One must realize that the assignment of a particular cell shape as characteristic of a particular genus or species does not imply that every cell in a population will be that shape. It is true, however, that at some period in the ontogenetic development of yeast cells, the yeast cell will assume that form.
Apiculate yeast, for example, begins its ontogenetic development as a small oval bud and will develop an apiculate shape only after separation from the mother cell and subsequent development of buds at the two cell poles. Upon aging, these cells assume a variety of shapes, most common of which would be an irregularly elongated cell.
The actual size of the individual yeast cells varies considerably and for a particular culture may be quite uniform, while in other species extreme heterogeneity in both size and shape is observed. For example- some yeast cells may be 2-3 μm in length, whereas others may attain lengths of 20-50 μm. Width of the cells is normally not as variable and measures normally between 1 and 10 μm.
While a certain amount of variation is normal and is to be expected within a yeast culture, the cultural conditions and, age of cells can exert a great deal of influence on the culture’s morphological properties. Again, as stated before, descriptions of the various yeast species are based on results obtained with quite standardized conditions and media.
Microscopy has contributed a great amount to our knowledge of yeast cytology. Obviously, direct observation with a light microscope provided our first details. Staining techniques have helped to determine the location of specific cell components or surface areas. Transmission Electron Microscopy (TEM) of ultra-thin sections of yeast cells or of carbon-platinum replicas of freeze-fractured cells has revealed internal details at high magnifications.
Lastly, by use of the Scanning Electron Microscope (SEM) details of external vegetative cell and spore structures have been observed. This method has been particularly useful in gaining information on the topography of vegetative cells and sexual spores, as well as morphological details of asexual and sexual reproduction.
Because various yeast species may show significant differences in the ultrastructure and cellular organization, generalizations must be recognized as such. As with much of our earlier information on other aspects of yeast, Saccharomyces cerevisiae or bakers’ yeast has been the subject of many of the cytological investigations. In the past two decades, using sophisticated equipment and techniques, investigators have used species belonging to some different genera to bring out particular structures lacking in other genera, all of which have multiplied our knowledge tremendously.
It has also been shown that significant changes in cellular structures may be brought about by the cultural conditions under which the culture is grown prior to examination. The shape of the cell, absence or presence of vacuoles, inclusion bodies and lipid globules, as well as mitochondrial development and the extent of capsular polysaccharide formation can be strongly modified by the growing conditions and age of a culture.
Structure and Function of Cellular Components:
The structure and function of cellular components will be given here. These include the cell wall (including extracellular capsule material in some species), the plasma- lemma or cytoplasmic membrane, nucleus, mitochondria, ribosomes and micro-bodies, vacuoles, lipid globules, volutin or polyphosphate bodies, endoplasmic reticulum, and the cytoplasmic matrix itself.
With a regular light microscope the cell wall is observed as a distinct outline of the cell but does not reveal distinct features, appearing smooth and sometimes having slight irregularities but no other details. The wall is fairly rigid and is responsible for the particular shape which a yeast cell possesses. With the employment of electron microscopy and techniques by which isolated cell walls can be prepared, details of the physical characteristics have increased.
In multilaterally budding yeasts, e.g., Saccharomyces cerevisiae, the most obvious structures to be found on a cell wall are the bud scars. The single birth scar resulting on the bud when separated from its mother is not particularly conspicuous but expands with the growth of the bud into a mature cell.
Electron micrographs show that the birth scar area is similar to the rest of the cell wall area in that bud scars from the cell giving rise to buds can be located on the birth scar region of the cell itself. In the ascomycetous yeasts, the bud scars are quite characteristic in appearance, having a raised circular brim surrounding a depressed area of about 3 m2.
Since successive buds are never formed at the same site in the ascomycetous yeasts, the number of bud scars on a cell is indicative of the reproductive capacity of the vegetative cell. Obviously, the number of buds formed per cell is limited. In a normal population which has reached the stationary phase of growth, most of the cells have either no bud scars or from 1 to 6 scars, while a very small fraction of the cell population will have as many as 12-15 bud scars. The limitation of the maximum number of scars on the oldest cells of the population is probably due to the exhaustion of a particular nutrient or to the crowding of cells in a particular medium.
Studies to determine the maximum number of buds that a cell can produce have shown that when successive buds are removed by the aid of a micromanipulator and when cell division is not limited by exhaustion of nutrients or crowding of cells, Saccharomyces cerevisiae can produce as few as 9 and as many as 43 buds per cell. The average number under these conditions was 24.
Since under ideal conditions the yeast cell may duplicate itself within 1½ to 2 hr when vigorously growing, the reproductive capacity of these cells can be imagined. It has been found, however, as cells age, the generation time for a particular medium and set of conditions is extended and finally becomes as long as 6 hr per generation. In physical appearance, the cell wall of old cells becomes somewhat wrinkled in appearance in contrast to a young vigorous cell which has a smooth, turgid appearance except for the birth and bud scar structures.
Birth scars in the case of bipolar budding yeasts are only observed in the young, un-budded daughter cell. Yeasts which have this mode of reproduction have bud scars which are superimposed on each other and with repeated budding are characterized in appearance by a series of ring-like ridges on the polar extensions of the cells. Thus, the more buds a cell has produced, the longer the polar extensions become.
In yeasts of basidiomycetous origin, bud formation repeated at the same site often occurs. In such instances, the walls of successive buds arise each time under the original cell wall, giving rise to concentric collars which are particularly noticeable in TEM of ultra-thin sections cut longitudinally through the scar area.
One bud scar is commonly observed, although 2 or more are not uncommon. In appearance the bud scar is different from that of the multilaterally budding yeasts in that the circular ridge is obscured and the center area either less depressed or not at all, giving rise to a pad-like scar, structure.
Vegetative cells of the genus Schizosaccharomyces, which reproduces only by fission, exhibit scar rings at the point where the cross-walls divide after septum formation. The septum area elongates for the production of a new cell and a new cross-wall is formed at a point located distally from the original scar ring.
The filamentous, ascomycetous yeasts which grow by elongation of the hyphal tip do not show exterior scars. However, ultra-thin sections disclose that the cross-walls formed in these organisms are not normally solid plugs, but rather contain 1 to a number of pores, thus permitting protoplasmic continuity among all of the cells.
Chemical analysis and enzymatic degradation studies have led to our present understanding of the chemical composition of the cell wall of yeast. Again, most of the Work has been done with species of Saccharornyces which we will first discuss and then contrast with information available about other species and-genera.
First, it is believed that the cell wall is composed of 3 layers which are not necessarily distinct from each other. First are an outer layer of glycoprotein which consists mainly of a phosphorylated mannan, then a middle layer of alkali-soluble μ-glucan, and finally an inner layer of alkali-insoluble μ-glucan.
While analyses of cell walls often show variable amounts of lipid materials, their precise location is unknown and in fact may actually represent the lipid content of plasmalemmae not removed during cell breakage and cell wall purification. In addition to the yeast phosphorylated mannose and glucose polymers, a small amount (approximately 1%) of chitin, a linear polymer of N-acetylglucosamine, is present.
Chitin of bakers’ yeast is presently known to be restricted to the area of the bud scars. When the-bud has fully developed it is first separated from the mother cell by a primary cross-wall of chitin which is subsequently covered with glucan and mannan prior to the separation of the mature bud from the mother.
In the yeast-like genus Endomyces and in species of Nadsonia, Rhodotorula, Cryptococcus, and Sporobolomyces, recorded chitin contents are much higher, so it may well be an actual cell wall component. In contrast, in species of Schizosaceharomyces analyzed, the cell walls apparently do not contain chitin.
Capsular materials, while not strictly a component of the cell wall, are produced extracellularly by representatives of several yeast genera. Capsular materials are generally classified as phos-phomannans, μ-linked mannans, neteropoly saccharides (contain more than one type of sugar), and finally a number of hydrophobic substances belonging to the sphingolipid type compounds.
Phosphomannans are produced extracellularly by certain species of Han-senula, Pichia, and Pachysolen. These viscous polymers are water soluble and form a sticky layer on the surface of the cells. They contain only D-mannose and phosphate, which is linked as a diester. The molar ratio of mannose to phosphate is characteristic of the particular species producing the capsular material, although it is known that this ratio can be affected by cultural conditions of growth.
For example- in media in which the phosphate levels are suboptimal the mannose phosphate ratio increases appreciably. In several species of the genus Rhodotorula and in Torulopsis ingeniosa, the chemical composition of the capsule material is a linear or slightly branched mannan in which the linkages between mannose units alternate between β -1,3 and β -1,4 linkages.
Heteropolysaccharides are identified as the capsular material of species of Cryptococcus. Since the antigenic properties of the capsular material of the pathogenic yeast Cryptococcus neoformans are of medical interest, the greatest amount of attention has been focused here. Mannose, xylose, and D-glucuronic acid account for the components of the heteropolysaccharide.
Minor components which have been reported include D-galactose and acetyl groups. Other heteropolysaccharides have been identified from specific yeasts. Lipomyces lipofer produces a heteropolysaccharide consisting of D-mannose and D-glucuronic acid in a ratio of 2:1; Candida bogorensis contains glucuronic acid, fucose, rhamnose, mannose, and galactose.
A different group of compounds, tetraacetyl phytosphingosine and triacetyl dihydrosphingosine, which are complex hydrophobic compounds, appear to be responsible, at least in part, for the tendency of some yeasts (in particular, Hansenula ciferrii) to form surface growth in liquid medium.
Proteinaceous hair-like structures termed fimbriae have been reported on the cell surface of several basidiomycetous and ascomycetous yeasts. They range from 5 to 7 nm in diameter and from approximately 10 μm in the basidiomycetous yeasts to only 0.1 μm in length in ascomycetous yeast. It has been proposed from work with strains of Saccharomyces that these hairs like structures may be involved in cellular flocculation.
Plasmalemma (Cytoplasmic Membrane):
This membrane is between the cell wall layers and the cytoplasm and functions in the selective transport of nutrients from the medium into the cell and conversely protects the cell from the loss of low molecular weight compounds from the cytoplasm. During the growth of the cell it also represents the structure upon which the cell wall components are deposited.
Physically the plasmalemma shows numerous deep invaginations. The outer surface is composed of particles which contain mannan-protein, and these particles may be involved in the formation of the mannan-protein complex of the cell wall. The plasma-lemma is a so-called unit membrane that is a structure about 8 nm thick in which 2 electron-dense layers are separated by an electron-transparent layer.
It is believed that the electron-dense layers represent proteins and the middle electron-transparent layer is lipids and phospholipids. The central layer is believed by investigators to consist of nonpolar groups while the borders adjacent are believed to contain proteins involved in the entry and exit of solutes and in some enzymatic action.
For example- magnesium dependent ATPase, active at neutral pH, is located in this area and is believed to play a role in energy dependent transport of certain soluble into the cell. Some carbohydrate in the form of mannan has also been identified in the central layer.
The ground substance or matrix in which various yeast structures such as the nucleus, vacuoles, etc., are located also contains large quantities of polyphosphates, glycolytic enzymes, ribosomes, reserve glycogen, and, in some yeasts, the reserve sugar trehalose.
Some of the polyphosphates are highly polymerized and are a reservoir of high-energy phosphate utilizable in various metabolic processes such as sugar transport, synthesis of cell wall polysaccharides, etc. Glycogen, one of the main carbohydrate storage products, is a highly polymerized glucose molecule which accumulates primarily in a cell’s stationary stage of growth when nitrogen is limiting and glucose is still available.
Commercial bakers’ yeast may have 12% glycogen on a dry weight basis. The other storage carbohydrate, trehalose, is a non-reducing disaccharide which is present from trace amounts to as high as 16% of the dry weight, depending upon the stage and condition of growth.
It is believed that the cytoplasmic matrix generally contains most hydro- lytic enzymes, the enzymes of the glycolytic cycle, and also those of the pentose cycle. It must be recognized that it is difficult to determine which enzymes are truly soluble components in the cytoplasmic matrix and which ones are inadvertently detached from a cell structure by experimental study procedures.
While there is no general agreement among researchers that yeast nuclei have condensed chromosomes as is found in more advanced forms of life, genetic studies have shown 17 independently segregating groups of centromere-associated genes in cultures of diploid Saccharomyces species.
This number probably represents the chromosome complement of the haploid stage. There is evidence that the number of chromosomes may vary with the species of yeast since Hansenula holstii has been shown genetically to contain 3 chromosomes while H. anomala has only 2 chromosomes.
While normally not directly observable by light microscopy, the nucleus of some yeast can be seen by phase contrast when cells are grown on a medium containing 18-20% gelatin. The nucleus is composed of a nucleolus (an optically dense, crescent-shaped part) and a second more translucent portion which contains the chromatin material, about 90% of the cell’s deoxyribonucleic acid (DNA), some RNA, and some polyphosphate compounds. Preparations stained with an acid fuchsin or iron hematoxylin will show the nucleolus, whereas aceto-orcein and Giemsa solution preferentially stain the chromatin material.
When viewed by TEM, ultrathin sections and freeze-fractured preparations of non-budding cells show the nucleus to be more or less spheroidal. It is enveloped by a pair of unit membranes having numerous circular pores approximately 85 nm in diameter. The pores appear to be filled with small granules which may represent ribosomal subunits.
The nuclear envelope remains intact during cell division of ascomycetous yeasts, where mitosis results by elongation and constriction of the nucleus which normally takes place in the neck of the bud. Both the nucleolus and the chromatin-containing portion of the nucleus divide and are incorporated into the new daughter nuclei.
In basidiomycetous yeasts, however, mitosis is not intranuclear. The chromatin-containing portion of the nucleus appears to move into the bud before cell division, while the nucleolus remains in the mother cell, and, in contrast to the ascomycetous yeasts, there is a partial breakdown of the nuclear envelope, the chromatin material divides in the bud, and the nucleolus in the mother cell appears to disintegrate.
A nuclear envelope reforms around the daughter nuclei after chromatin division along with newly formed nucleoli. One of the daughter nuclei moves back to the mother cell and cell division is completed. Under optimum conditions of growth, this sequence-takes place in about 1½ hr.
The vacuoles are normally spherical in appearance, -more transparent to a light beam than surrounding cytoplasmic material, and vary greatly in size. In actively dividing cells, there are usually numerous small vacuoles, whereas in older cells the number is reduced, often to large vacuole. These cytoplasmic structures are particularly conspicuous in the stationary phase of growth when the cells are observed by phase contrast.
Transmission electron microscopy shows the vacuole to be surrounded by a single unit membrane, and when specimens are freeze-fractured, both the inner and outer surfaces of the yeast membranes appear to be covered with particles
A study of the vacuolar contents has disclosed the presence of a number of hydrolytic enzymes including ribonuclease, esterase, and several proteases. It has also been found that a large fraction of the free amino acid pool of the yeast cell is stored in the vacuoles, as well as substantial concentrations of polymerized orthophosphate (volutin or polymetaphosphate).
Certain purines or their derivatives of low solubility cause the formation of conspicuous crystals which are easily observed in the vacuoles of some yeast and are referred to as dancing bodies due to active Brownian movement. It is thought that the autolysis of yeast cells begins with the breakdown of vacuoles under adverse conditions and the release of enzymes which then can attack cytoplasmic substrates.
A double membrane system similar to the Endoplasmic Reticulum (ER) of higher plants and animal cells has been shown to be present in yeast by TEM examination of ultra-thin sections and freeze- etched preparations. This system appears to be in close association with the plasma-lemma and in some preparations to be actually connected to the outer nuclear membrane. Polyribosomes, which are centers of protein synthesis, are believed to be associated with the endoplasmic reticulum. The ER has also been implicated in the initiation of bud formation.
The primary function of mitochondria is that of oxidative energy conversion for the cell. Mitochondria are known to contain DNA, RNA, RNA-polymerase, and a number of the respiratory enzymes participating in the TCA cycle and electron transport systems. Under conditions of fermentation or under aerobic conditions where 5-10% glucose is present, the mitochondria degenerate to so-called promitochondria.
These structures have poorly developed cristae, no longer synthesize cytochromes aa3 and b. and the cell can no longer respire. Respiration is quickly regained by placing the cells in a medium containing a non-fermentable substrate such as glycerol and by aerating the culture.
The distribution of mitochondria in the cytoplasm seems to vary since in ultra-thin sections of cells of Saccharomyces they are found to be concentrated close to the plasma-lemma, whereas in Rhodotorula, non-fermentative yeast, they are randomly distributed throughout the cytoplasm.
During budding, the mitochondria elongate and divide and are distributed between mother and daughter cells. The mitochondria have an inner membrane with numerous cristae which extend into the mitochondrial bodies and a fairly regular outer membrane. The membrane system has large amounts of lipids, phospholipids, and ergosterol.
Yeasts have globules of lipid material stainable with Sudan Black or Sudan Red. While most yeast cells contain a small amount of these lipid materials, some yeasts, particularly when grown in a medium with a limiting nitrogen supply, can accumulate up to 50% of the dry weight as lipids.
Lipomyces starkeyi, Metschnikowia pulcherrima, and Rhodotorula glutinis are notable fat-producing yeasts. The fat globules are highly refractile when observed by light microscopy. Electron microscopy has not revealed a membrane structure surrounding the globules.
Sexual reproduction constitutes a phase of the life cycle of the yeast, that is, an alternation of the haploid condition (1n set of chromosomes) and the diploid condition (2n). The events leading to the formation of sexual spores in yeasts are of great biological significance because they enable a yeast species to exploit fully the evolutionary processes such as hybridization selection, genetic recombination, and mutation, all of which are well known in higher forms of life and which can be exploited for the benefit of mankind. Since all of the yeasts are eukaryotic, the principles of reduction division or meiosis, which the diploid nucleus undergoes, are basically similar to those in higher forms of life.
Briefly, the fusion of 2 haploid nuclei results in a diploid nucleus which, through meiosis, is again reduced to the haploid number. Although yeast chromosomes are too small to be seen under the microscope, genetic evidence and the analogy with higher eukaryotes suggest that 2 members of each chromosomal pair have homologous genetic material.
During meiosis, the homologous chromosomes become tightly paired, and then form a 4-strand structure or tetrad which eventually results in the formation of 4 haploid nuclei each carrying 1 chromatid of the original tetrad. It is important that one realize that the haploid nuclei formed in this manner are not necessarily identical to those of the 2 cells, or nuclei, which gave rise to the original diploid.
Genetic recombination, which occurs by the random assortment of different chromosomes and breakage of chromatids during the formation of the 4-stranded tetrad, and results in exchange of part of the chromatid (recombination), has been termed crossing over and has been demonstrated in yeast by geneticists.
Four ascospores per ascus are common; however, there are species which characteristically form 1 or 2 spores per ascus. Even when 4 spores is the usual number, asci containing 1, 2, or 3 spores may be observed. In such cases the “extra” nuclei in the ascus are not incorporated and often disintegrate.
In the few yeasts normally containing more than 4 spores per ascus, the tetrad nuclei usually undergo one more mitotic division so 8 spores may be formed in each ascus as in Schizosaccharomyces octosporus. In the case of additional supernumerary mitoses, multispored asci characteristic of some yeast, such as Kluyveromyces polysporus, will result.
The process of spore delimitation within the cell has been studied in great detail in Saccharomyces cerevisiae. The original nuclear membrane of the diploid nucleus remains intact during the various stages of reduction division and results in a 4-lobed structure into which the 4 sets of chromatids are separated.
Around each of the lobes a double membrane (prospore or fore-spore wall) is formed. The prospore wall thickens, causing the original nuclear membrane to break up into 4 individual portions. Part of the cytoplasm of the original cell becomes incorporated into each spore by the developing spore wall but a portion of the cytoplasmic matrix remains in the ascus and is referred to as epiplasm.
This process, termed sporulation by “free cell formation,” distinguishes it from spore formation by the appearance of cleavage planes (partitioning) characteristic of certain other fungi. The wall of the original cell, now an ascus, may be rapidly digested by endogenous enzymes and the spores liberated.
In many types of yeast, however, such as Saccharomyces cerevisiae, the cell wall does not auto digest, and the spores are liberated only after germination, swelling, and mechanical rupture of the cell wall. The sexual spores, whether released by mechanical forces or autolysis, can germinate, thus initiating another sexual cycle.
It should be emphasized that many yeasts require special growing conditions or sporulation conditions to pass through the sexual cycle, and if the specific conditions are not met, yeast can continue to propagate indefinitely in the vegetative form. An excellent example is yeast isolated in 1934 as imperfect yeast which was not induced to sporulate until the proper conditions were known in 1970, a period of 36 years under laboratory culture without evidence of sporulation. Since definitive life cycles for basidiomycetous yeast were not known until the late 1960s, most of our information is derived from those yeasts which form ascospores as a sexual means of reproduction.
Various yeast species, depending upon the strain, are found to exhibit homothallism, heterothallism, and parasexuality.
Homothallic yeasts are those which are capable of self- fertilization, that is, a single spore or cell is capable of carrying out a complete life cycle. Such yeasts are known in both the ascomycetous and the basidiomycetous yeasts.
Four mechanisms of heterothallism are known at the present time, the simplest being the biallelic, bipolar, sexual compatibility system in which species require mating types for sexual conjugation; the determinant for sex is located at 1 locus on a chromosome. The 2 compatible sex alleles, resulting in the 2 mating types, are generally termed a and α. Those yeasts which form ascospores and which are heterothallic have this system of compatibility.
In the basidiomycetous yeasts, however, 3 compatibility systems are known. Some species have a biallelic, bipolar, compatibility system similar to that found in the ascospore forming yeasts.
The second system has the sex determinant at 1 locus but there are 3 or more alleles This system, known as multiallelic-bipolar, results in 3 or more mating types, for example- A1, A2, and A3, etc. In this system, matings which will be fertile result from all paired combinations which involve different alleles such as A1 x A2, A1 x A3, and A2 x A3.
The third and last system is termed tetrapolar and involves 2 unlinked loci and 2 allelic pairs. This system has 4 mating types, A1B1, A1B2, A2B and A2B2. Compatibility must be satisfied at both loci before fertile matings can occur- thus the genetic expression for the diploid would be A1A2B1B2. Matings between other types are not capable of completing the life cycle.
Growth can occur but it is usually abortive and sporulation does not occur. Examples of this are particularly noticed when mating crosses are made and only locus A or B is satisfied, in which instance the mycelium formed is unhealthy and does not form clamp connections, which are indicative of compatible paired nuclei, nor does it form teliospores from which the sexual spores are formed.
While the occurrence of heterothallism in other fungi has been known for quite some time, the occurrence in yeasts was recognized only in 1943. It actually occurs quite commonly among the various groups of yeasts and its importance cannot be overemphasized.
Due in part to the classic method of isolation for obtaining pure cultures, where one selects single well-isolated colonies on an isolation medium, many species have been described as lacking a sexual phase when in fact the isolation has been of only 1 mating type Thus the recognition of heterothallism has aided in the proper taxonomic designation of many of these organisms when the isolate has been mixed with compatible strains of a known species and the mating results in sporulation.
Further recognition should be made of the fact that the mating reaction itself among heterothallic yeasts is extremely variable in its strength. With some yeasts the mating reaction is so powerful that it is preceded by a visible agglutination when cells of compatible mating types are mixed.
This agglutination is immediately followed by zygote formation, although interestingly enough, some yeasts with a very strong agglutination reaction are relatively low in the percentage of asci. At the other extreme, there art species where the mating reaction is so weak that it is virtually impossible to detect by microscopic examination. In such yeasts, the preparation of nutritionally deficient mutants for each mating type has proved valuable in determining the sexual ability of the culture in question.
When weakly reacting mating types with individual nutritional deficiencies are mixed an occasional zygote will be formed which, when the mixed suspension is plated onto a basic minimal medium, is capable of growth whereas un-mated strains are not. By this technique, one zygote can be detected isolated, and studied from among the unmated cells of the originally mixed mating types.
It should be noted that while sexual differentiation in yeast strains exists, there is no implication that yeasts have reproductive structures to which the terms male and female can be applied. This term is normally reserved for highly differentiated male and female fusion cells, whereas the active structures in yeast are relatively unspecialized vegetative cells, ascospores, or sporidia.
However, it is common to designate these structures as gametes, particularly if they represent different mating types. Their designation as a and α A1, A2, A3, or A1B1, A1B2, etc., recognizes the inability to distinguish maleness or femaleness. In the case of homothallic vegetative cells which fuse prior to sporulation, such fusion may be considered to be a case of somatic conjugation.
Studies by various investigators have shown cases where yeasts and other fungi alternate between the haploid and diploid phases without the formation of sexual spores. This process, observed to occur in Aspergillus nidulans by Pontecorvo in 1954, is known as parasexuality. Subsequent to that time, similar observations have been made in other fungi; including some yeast species.
This alternation of haploid and diploid phases is of particular interest since it is functional not only in fungi without known sexual stages, but also in yeasts where a sexual stage has been well established. There could be a distinct advantage to yeast in which both haploid and diploid cells coexist. In the case of diploid cells where genes are paired, the presence of a recessive gene arising by mutation can spread, although its effect would be masked by the wild type, dominant gene.
In this manner, one would have a stable phenotype along with genetic variation. In the case of a haploid cell, however, with only a single set of chromosomes, the mutation would not be masked by a dominant gene and would express itself rapidly. Thus, with haploids and diploids coexisting, being genetically separated and being able to reproduce indefinitely in a vegetative phase, the yeast can have the advantages of haploidy or diploidy simultaneously.
Yeast Life Cycles:
In discussing the life cycles of yeasts, those forming asci will be discussed separately from those forming teliospores or basidia.
Ascosporogenous yeasts can be broadly subdivided into 2 classes, those in which the vegetative phase exists in the haploid condition and those in which the diplophase predominates. In the haploid class, such as the subgenus Zygosaccharomyces, the nucleus of the vegetative cell contains only a single set of chromosomes (1n).
Thus, there must be a fusion of 2 nuclei to establish the diploid (2n) condition before reduction division can begin and subsequent sporulation can take place. In those yeasts which exist vegetatively primarily as diploid cells, such as Saccharomyces cerevisiae, the ascus can develop directly from the vegetative cell by reduction division of the diploid nucleus followed by spore formation.
This general grouping of yeasts as haploid or diploid species is not a strict classification as there are certain yeasts in which haploid and diploid cells may occur side by side in the same culture. Thus, upon examination of a sporulating culture we may find asci which have arisen from a diploid cell and other asci which have obviously arisen from the conjugation of 2 haploid cells just prior to sporulation.
Haploid Vegetative Phase:
In ascosporogenous yeasts which characteristically exist as haploids in the vegetative phase, the diploid generation is usually limited to the zygote formed after fusion of 2 haploid cells and their nuclei. These yeasts normally have 1 of 3 types of life cycles.
In the first type of life cycle when conditions are favorable for sexual reproduction, the vegetative cells will form more or less distinct conjugation tubes. The tips of 2 conjugation tubes from 2 cells then grow together and plasmogamy (joining of the protoplasts) takes place.
The nuclei of the 2 cells then approach each other and karyogamy (nuclear fusion) occurs, thus forming a diploid nucleus. In most instances, meiosis or reduction division immediately follows karyogamy and the resultant 4 nuclei produce ascospores in a dumbbell-shaped ascus (the original zygote). Spore distribution between the 2 portions of the dumbbell is random so that asci containing 2 spores in each half, 3 to 1 or even 4 to 0, can be observed.
The second type of life cycle exhibited by some haploid yeasts varies from the first in that the haploid vegetative cell produces a Dud which, instead of separating in a normal fashion, stays attached to the mother cell, retaining a rather large connective opening. Nuclear division occurs (mitosis) and the 2 daughter nuclei move into the bud structure where karyogamy occurs.
Meiosis also occurs in the bud resulting in 2 or 4 haploid nuclei. Because meiosis does occur in this bud-like structure, it has been termed the “meiosis bud.” The haploid nuclei move back into the mother cell and ascospore formation takes place there. Haploid yeasts exhibiting this life cycle usually form only 1 or 2 spores per ascus and the extra nuclei presumably degenerate.
While no septum has been observed to be formed across the bud opening of cells in the genus Schwanniomyces, electron microscopy studies have revealed that in a species of Debaryomyces the bud is first separated from the mother cell by a septum which then dissolves, allowing the 2 cell nuclei to fuse and the life cycle to be completed. From morphological similarities among the asci of these genera, it may be assumed that one or the other of these methods is functional in other genera where species form asci from a mother cell-daughter cell pairing.
The third type of life cycle exhibited by haploid yeasts also involves the fusion of 2 “gametes,” but the fusion cell does not become the ascus. The 2 nuclei move to a specialized structure which becomes the ascus. In the young ascus the nuclei undergo fusion, reduction division, and subsequent ascospore formation. Examples of this are found in species of Eremascus and Nadsonia.
Diploid Vegetative Phase:
Ascosporogenous yeasts which spends their vegetative days in the diploid condition produce their spores within the vegetative cells, which become the asci. For these yeasts there are 4 means known by which the diploid condition in the vegetative cell can arise from the haploid ascospore.
(1) Two ascospores fused within the ascus before spores are dehisced. Fusion of the ascospores is followed by karyogamy of the 2 haploid, spore nuclei. Thus the first vegetative cell resulting from the conjugated spores is already a diploid cell. Such a life cycle occurs in Saccharomycodes ludwigii and also is found in certain strains of Sac- charomyces cerevisiae.
(2) The ascospores germinate by budding off relatively small sized, haploid, vegetative cells. This continues for a very limited number of generations with fusion then occurring between pairs of spore-bud- cells. This results in diploid, larger-sized cells which then continue to propagate as the vegetative phase.
(3) This life cycle is a variation of number (2) where only 1 of the spores germinates, producing a haploid cell which then fuses with a yet un-germinated spore from the same ascus. Again, this results in a diploid giving rise to the vegetative phase. This means of diploidization is also found in certain strains of Saccharomyces cerevisiae.
(4) In some yeasts known to exist vegetatively in a diploid phase, no conjugation of ascospores or ascospore bud-cells has been found. It has, however, been shown that the haploid nucleus of an individual ascospore may divide mitotically so that the germinating spore has 2 haploid daughter nuclei which then fuse prior to the formation of the first bud.
This diploid nucleus then divides mitotically so that the first bud coming from the single germinating ascospore already constitutes a diploid cell. This particular means of diploidization is obviously limited to homothallic yeasts. Species of Hanseniaspora reproduce in this fashion. There is also evidence that strains of Saccharomyces chevalieri also have this type of life cycle.
Life cycles of the basidiomycetous yeasts also show variations but, in general, after fusion of 2 haploid (1n) yeast cells, there is a stage not found in the ascomycetous yeasts. This is the development of a dikaryotic condition (1n plus 1n) where the 2 nuclei after plasmogamy do not fuse in the zygote but rather are incorporated in pairs into the cells of a mycelium by a clamp connection.
The clamp connection mechanism assures that 2 compatible nuclei are duplicated and propagated in subsequent cells of the mycelium. At the time of sporulation either thick-walled teliospores or thin-walled basidia are formed on the mycelium. After karyogamy and reduction division, sporidia are formed externally on the basidium or from a teliospore.
The teliospore germinates into a tube like promycelium upon which the sporidia are formed. The promycelium may be septate, with 4 cells being common, or aseptate. The sexual spores (sporidia) are not forceably discharged from the promycelium or basidia of these yeasts. They are formed by budding and further vegetative reproduction is also by this means.
In some teliospore-forming species, a self-sporulating, diploid phase is also known. It is believed that this phase is the result when a teliospore, after karyogamy has occurred, may fail to undergo reduction division so that the germinating buds are diploid. These diploid cells may reproduce asexually by budding or may develop into a uninucleate diploid mycelium without clamp connections. Teliospores formed on this type of mycelium may undergo reduction division so that the sporidia thus formed would partake in the usual-haploid type of life cycle.
It has also been reported that diploid cells can give rise to a dikaryotic mycelium when the diploid nucleus undergoes a “somatic reduction,” thus creating a dikaryon. Since many of the basidiomycetous yeasts have a tetrapolar compatibility system, fertile matings require the satisfaction of compatibility at 2 loci, whereas when this condition is met at only 1 of the 2 loci, the mycelium formed is unhealthy and does not form clamp connections or teliospores.
The determination of a yeast isolate’s ability to form asco- or basidio- spores is of prime importance in determining its identification. This determination can be complicated by the fact that some yeasts can grow in a vegetative state for an indefinite number of generations and that very specific conditions must be met before the sexual cycle can be induced.
Further, in heterothallic species, fertile matings and successful completion of the sexual cycle depend upon the mixing of compatible strains. Early workers were of the opinion that yeasts would sporulate only when conditions for growth became unfavorable. Today it is known that this is not necessarily true. Many types of yeast freshly isolated from nature often sporulate very heavily on the relatively rich media commonly used for isolation.
Further, yeasts cultured in a laboratory are propagated on much richer media than would normally be available to them in their natural habitat. However, many so-called domestic cultivated yeasts, for example- bakers’ and brewers’ yeasts, as well as many species commonly isolated from spoiled beverages and other food products, often sporulate poorly or not at all on media rich in nutrients. Some yeast species have a tendency to lose their ability to form sexual spores while others are not affected when they are held in laboratory culture for a period of years.
Innumerable media and various techniques have been used over the years for the purpose of inducing ascospore formation or to increase the percentage of sporulating cells. Yeast ascospores are somewhat more resistant to adverse conditions such as freezing, drying, and exposure to high temperatures and to harmful chemicals than are the vegetative cells.
The heat resistance of the ascospore is only a few degrees (6°-12°C) greater than the vegetative cells under the same conditions. Thus, by heating a cell suspension for a short time at mildly elevated temperatures, such as 55°- 60°C, vegetative cells can be killed but spores that might be present would still be viable.
Experience has shown that success in obtaining sporulation is best when the culture is well nourished and relatively young. In species of Saccharomyces it was found that young daughter cells which have not produced buds sporulate either very poorly or not at all. However, after producing one or more buds, sporulation occurs normally.
Fermentative (anaerobic) conditions do not promote sporulation. In Saccharomyces cereuisiae sporulation is enhanced when the vegetative cells are adapted for aerobic growth. However, it has been found that with some species of Hanseniaspora which produce 1 to 2 spheroidal spores, a reduced oxygen tension stimulates spore formation. When part of the vegetative cell inoculum is covered by a sterile cover slip, large numbers of asci may develop under the cover slip near the outer edge, but are infrequently found in the uncovered portion of the growth or-far under the cover slip.
In the laboratory most yeast will sporulate at room temperature (18°- 25°C) although reduced temperatures (12°-15°C) are beneficial to some species of Nadsonia and Metschnikowia.
As one might expect from the diversity of the many yeast species, there is no one medium capable of inducing sporulation for all of the yeasts. Most media are adjusted so that the pH is in the neutral to slightly acid range (pH 5.8-7.0). Certain media have been found to be better for some species than others. For example, acetate agar is commonly used for achieving sporulation with species of Saccharomyces, although potato extract glucose agai (PDA) works better for some haploid species of this genus.
Gorodkowa agar works well for most species of Debaryomyces; YM agar (yeast extract, malt extract maltose agar) induces sporulation of many species of Pichia and Hansenula and other yeasts. A medium based on vegetable juices (V-8), with or without added bakers’ yeast, is also an excellent sporulation medium for species of a number of genera. In certain special cases a medium composed of the substrate from which the yeast was isolated may be used.
Since heterothallic yeasts require compatible mating types, cultures in, which sporulation cannot be induced and which would be classified in the so-called imperfect genera should be mixed with similar cultures in various combinations, or, when the mating types for a species are known, mixed with the same mating types and re-observed for sporulation. Many yeast isolates formerly believed to be asporogenous are properly classified into their perfect genera by these techniques.
Yeasts being observed for ability to sporulate should be checked frequently over a period of several weeks because the time required to sporulate as well as the number of asci formed varies a great deal among the yeast species and even within strains of the same species.
Some can, produce spores in 1-2 days, whereas other yeasts may not form asci for a week or two or longer. Frequent examination is necessary because with many types of yeast the spores formed can also germinate on the same medium, and if observation were delayed or infrequent, one might not observe that sporulation had taken place.
Thus, although the ability or inability to produce sexual spores is not always easy to determine, the type of sexual spore formed is the primary criterion used by the taxonomist to place yeast into 1 of 3 subdivisions of fungi. The spores formed exhibit a wide diversity in number per ascus shape, size, surface markings, and color.
Generally, most of these features are quite constant for a given species. For some genera, all species have a similar morphology, although in other genera, spore morphology differs among the species, Sporulation among the yeasts also has made possible genetic studies.
However, from the standpoint of the yeast itself, it is the most important biological event in its life cycle and it governs to a large extent the evolutionary development of the various groups of the yeasts. Therefore, in spite of the time spent and difficulties encountered in determining yeast’s ability to sporulate, the importance is justified.
Man has used the ability of certain yeasts to carry out an alcoholic fermentation long before the responsible agent was recognized as a living thing. The everyday use of alcoholic beverages and panary products has also made large quantities of yeasts available, facilitating the work of the investigators elucidating the basic metabolic processes of living cells.
The “cell free extract” of the Buchners in 1897 provided the means for studying alcoholic fermentation in the absence of living (Sells and thus initiated the many studies which elucidated the intermediate steps that occur in the transformation of glucose to ethanol and carbon dioxide. It must be kept in mind that “fermentative” yeasts also possess the ability to oxidatively utilize glucose as well as many other compounds which cannot be fermented at all.
The ability to carry out an alcoholic fermentation varies in vigor from species to species and in fact from strain to strain. Actually, there are many types of yeast which are totally incapable of carrying out an alcoholic fermentation and thus are unable to derive energy or grow under anaerobic conditions.
In fact, fermentation being “la vie sans air” (life without air) is not quite accurate, Saccharomyces cerevisiae under strictly anaerobic conditions will cease its fermentation even though a fermentable substrate is still present, thus causing a “stuck” fermentation.
Apparently the presence of a small amount of oxygen is required for synthesis of certain vital compounds since supplementing a medium with ergosterol and oleic acid or with oleanoic acid will enable the fermentation to go to completion even if oxygen is absent.
Three basic rules regarding yeasts’ capabilities to ferment were formulated many years ago by Kluyver. The first rule is that if yeast is unable to ferment D-glucose, it cannot ferment any other sugar. The second rule states that if yeast can ferment D-glucose, then D-fructose and D-mannose will also be fermented. The third rule is that if yeast can ferment maltose, it cannot ferment lactose, and vice versa. Exceptions are known for the third rule in that there are a few yeasts capable of fermenting both maltose and lactose. Brettanomyces claussenii is an example of yeast able to ferment both disaccharides.
Aerobic metabolism is affected by the level of oxygen present and also concentration of the sugars. For example- both with Saccharomyces cerevisiae and with Candida utilis the Embden-Meyerhof pathway accounts for approximately 90% of the glycolytic metabolism.
Under aerobic conditions, however, the hexose monophosphate pathway is responsible for 6 to 30% glycolysis in Saccharomyces cerevisiae and for 3 to 50% in strains of Candida utilis. In contrast, species of Rhodotorula which are unable to ferment metabolize 60 to 80% of the glucose taken from the medium through the hexose monophosphate pathway.
The effect of substrate concentration can be illustrated for S. cerevisiae as follows. In media with glucose concentrations in excess of 5%, there is a total inhibition of synthesis of respiratory enzymes so that the yeast continues to ferment even though the medium is aerated. This phenomenon is known as the “glucose effect” or “Crabtree effect.”
Not all yeasts exhibit the Crabtree effect, e.g., species of Kluyveromyces and some haploid Saccharomyces. In contrast, a yeast (such as Saccharomyces cerevisiae) growing in low concentrations of glucose (0.1%) can shift from fermentation to respiration upon aeration of the medium.
The decrease in fermentative ability upon aeration was first demonstrated by Pasteur (Pasteur Effect) and industrially utilized by producers of bakers’ yeast and of feed yeasts. As the level of the sugar decreases in an aerated growth medium, there is a corresponding increase in the activity of the enzymes of the tricarboxylic acid cycle (TCA) and increased activity of enzymes involved in the glyoxylate cycle and in the electron-transport system. Since the enzymes for the TCA and glyoxylate cycles are located in the mitochondrial fraction of the yeast, the observation that the presence of sugar inhibits mitochondrial formation is not unexpected.
The TCA cycle starts with pyruvate’s being oxidized to acetyl coenzyme A instead of decarboxylation to acetaldehyde, which occurs during alcoholic fermentation. The β-oxidation of fatty acids also results in acetyl CoA. Acetyl CoA then condenses with oxaloacetate, giving rise to citrate. Subsequent reactions of the TCA cycle release 2 molecules of CO2 per cycle and generate ATP by oxidative phosphorylation. Several of the intermediate compounds also may be used in the synthesis of amino acids and other cellular constituents.
The glyoxylate cycle or bypass has been more recently elucidated and was found to function for the replenishment of TCA cycle intermediates—especially L-malate and succinate removed from the cycle due to the synthesis of cellular constituents. The glyaxylate cycle also serves as the mechanism by which yeasts can grow on 2-carbon molecules such as ethanol and acetate. A biotin-dependent reaction producing oxaloacetate from pyruvate and CO2 serves to supply additional oxaloacetate for incorporation into the TCA cycle.
The hexose monophosphate shunt pathway, also known as the pentose cycle, starts with glucose-6-phosphate which is oxidized to 6-phospho- gluconate. This reaction is dependent upon the reduction of NADP to NADPH2. Subsequently, 1 molecule of CO2 is released and an additional molecule of NADPH2 is formed. Aerobically, the cycle can result in the complete oxidation of glucose to 6CO2 and the formation of 12 molecules of NADPH2.
While the hexose monophosphate shunt does not generate high energy phosphate in the form of ATP, a portion of the NADPH2 formed is oxidized in other metabolic pathways and in this way does contribute to ATP formation. The NADPH2 is utilized in large part in synthetic reactions requiring reductions, such as the formation of lipids. The hexose monophosphate pathway also serves as a mechanism by which yeasts can respire pentoses and methyl pentose sugars if the yeasts have the proper pentose kinases and other enzymes that will convert the substrates into intermediates of the pentose cycle.
Growth Requirements of Yeast:
In order to grow, yeasts require oxygen, proper temperature and pH, utilizable organic carbon and nitrogen sources, and various minerals; and some require vitamins and other growth factors. All yeasts are able to utilize D-glucose, D-fructose, and D-mannose; although a particular yeast may utilize other carbon sources more efficiently.
Besides these 3 hexoses, another hexose, D-galactose, may be fermented, but this requires enzymatic adaptation by yeasts possessing the potential to ferment the sugar. Other monosaccharides and all L-sugars are un-fermentable although particular yeast may be able to utilize them by respiration.
In the fermentation of di-, tri-, or polysaccharides, the fermentation always goes through the hexose stage after enzymatic hydrolysis either at the cell surface or internally, depending upon the location of the specific enzymes. Consequently, if the particular hexose sugar is not fermented by the particular yeast, the di- or oligosaccharides are not fermented either.
Enzymes for the hydrolysis of sucrose, raffinose, melibiose, starch, and inulin apparently are situated outside of the cell membrane (plasma-lemma) although their precise location has still to be determined. Enzymes for the hydrolysis of other sugars such as maltose, lactose, cellobiose, and melezitose are located internally; the sugars are transported by specific permeases across the membrane and hydrolyzed within the cell.
Fermentation of polysaccharides such as starch and inulin (a fructose-containing polysaccharide) is possible by relatively few yeasts, and then generally at reduced rates of fermentation. The ability to ferment pentose sugars or methyl pentoses is not found in any yeast, although it is not unusual for these compounds to be utilized by respiration. Thus, among the yeasts which are able to ferment, glucose (fructose, mannose), galactose, sucrose, maltose, raffinose, melibiose, lactose, and trehalose are the most commonly utilized sugars.
In brief, the breakdown of a sugar and its transformation to ethanol and carbon dioxide proceed in the following manner- D-glucose, D-fructose, and D-mannose are all transported across the cell membrane by a common transport system termed “facilitated diffusion.”
In addition, these 3 hexoses are all phosphorylated by the same enzyme, yeast hexokinase, to the corresponding hexose-6-phosphates. Both the rate of transportation and the rate of phosphorylation of these sugars vary depending upon the sugar and also on the species, in fact, even on the strain of yeast.
Glucose-6- and mannose- 6-phosphate are subsequently transformed to fructose-6-phosphate. The other fermentable hexose, D-galactose, when utilized by a yeast, is transported by a separate, induced transport system, and once in the cell it is phosphorylated by a specific enzyme and transformed by 3 additional enzymes before it can enter the glycolytic cycle.
Hydrolysis to hexose components is obviously required before di-, tri-, and oligosaccharides may be fermented. After the hexoses are phosphorylated to the hexose-6-phosphate, a reaction requiring adenosine triphosphate (ATP) and other enzymes converts fructose-6-phosphate into fructose-1,6- diphosphate using another molecule of ATP.
The fructose-1,6-diphosphate is converted to pyruvic acid via the Embden-Meyerhof pathway of glycolysis. The net results are 2 molecules of pyruvate and the generation of 4 molecules of ATP, a net gain of 2 molecules of ATP. The 2 pyruvate molecules are subsequently decarboxylated yielding 2 molecules of CO2 and 2 molecules of acetaldehyde.
Finally, the 2 molecules of acetaldehyde are reduced to ethanol by alcohol dehydrogenase and the reduced form of the coenzyme nicotinic acid adenine dinucleotide (NADH), which had been formed during one of the intermediate steps of the Embden-Meyerhof pathway. The 2 molecules of ATP formed during the transformation of 1 molecule of hexose to 2 molecules of CO2 and 2 of ethanol are used as a supply of energy required for cell growth and for synthesis of storage reserve products (glycogen and trehalose).
It should be noted that under conditions of no growth, for example- in a medium containing glucose but no nitrogen source, the yeast cells will still convert about 70% of the glucose to ethanol and CO2 with the remainder going to reserve carbohydrates. When the glucose of the medium is depleted, it is felt that these reserve carbohydrates furnish the energy necessary for maintenance of the cell in replacement of the proteins and ribonucleic acids which are being constantly “recycled.”
The concentration of ethanol produced depends not only on the conditions of fermentation, but perhaps even moreso upon the species and particular strain of a species. Suitable strains of Saccharomyces cerevisiae, for example- strains of wine yeast or distillers’ yeast, can attain an ethanol concentration of 12 to 14% by volume with relative ease if a sufficient supply of fermentable sugar is supplied.
As the ethanol concentration increases above this level, the rate of fermentation is greatly reduced and will eventually cease. Ethanol levels of 18 to 20% by volume have been achieved with select strains and specific fermentation conditions. In contrast, species of Hanseniaspora (and the asporogenous forms in the genus Kloeckera), which frequently are predominant yeasts in the early stages of natural fruit fermentations, are relatively sensitive to the presence of alcohol (about 3 5 to 6.0% alcohol).
Of industrial interest is the ability of some yeast to ferment L-malic acid to ethanol and carbon dioxide. Some wine yeast (strains of Saccharomyces cerevisiae) has this ability, but species of Schizosaccharomyces (S. pombe and S. malidevorans) are more efficient. This property is especially of interest in the fermentation of grape and fruit musts of low sugar content and high levels of malic acid.
Yeasts can respire all sugars that they are capable of fermenting and in addition may respire a wide array of other organic compounds. In addition to the pentoses and methyl pentose sugars, compounds which yeasts can respire include organic acids, sugar alcohols, methanol, and ethanol,- as well as some aromatic compounds and hydrocarbons.
Only a limited number of yeasts can utilize the last 2 classes of compounds and then only a few of these. Hydrocarbons used are primarily limited to the n-alkanes, particularly those with 12 to 18 carbon skeletons. Candida lipolytica, C. tropicalis, and C. maltosa all grow well on these n-alkanes. A number of species of Schwanniomyces, Metschnikowia, De- baryomyces, and Pichia also can grow on these hydrocarbons.
Candida tropicalis can grow on a few aromatic compounds as well, and strains of Trichosporon cutaneum can metabolize many more. The use of methanol as a potential substrate for the production of single cell protein has received the attention of a number of investigators. Some species which grow well on methanol are Hansenula polymorphs, Pichia pastoris, Candida boidinii, and a recently described Torulopsis sonorensis, which was found in necrotic cacti.
The ability of yeasts to assimilate various compounds as a source of nitrogen also varies greatly among the yeasts. Among the inorganic sources of nitrogen, ammonium sulfate is utilizable by virtually all yeasts. In fact, most ammonium salts can supply nitrogen for the growth of yeasts, although some salts are more suitable than others.
The ability to use nitrate is much more restricted and is, in fact, a criterion used in yeast classification. Candida utilis is an example of industrially important yeast which utilizes nitrate. Bakers’ and brewers’ yeast (Saccharomyces cerevisiae strains) are unable to grow on nitrate. Nitrite is utilized by all of the yeasts which can use nitrate, although the toxicity of the nitrite ion varies among these yeasts, and caution must be used to assure that too high a concentration is not used.
All species of the genera Hansenula and Citeromyces can utilize nitrate and nitrite, as can some species in genera such as Rhodotorula, Torulopsis, and Candida. Yeasts are also known that have the ability to use nitrite but not nitrate. Some species of Debaryomyces have this ability and are often isolated from cured meats and similar products containing this compound.
Some investigators have published reports which claim that at least some Rhodotorula species can assimilate nitrogen from the air. However, this claim has been denied by others who have used an atmosphere containing radioactive nitrogen isotopes and who could not find any fixation of molecular nitrogen by the yeasts.
Amino acids also serve as a source of nitrogen for yeasts. While it was previously thought that yeasts could incorporate assimilated amino acids directly into proteins, it has more recently been shown that the utilization of a particular amino acid depends upon the ability of the particular yeast to deaminate the amino acid and to incorporate that nitrogen into other constituents of the cell. Glutamic and aspartic acids and their amines are easily deaminated or transaminated by most yeasts and serve as good sources of nitrogen.
In general, yeasts take up only the L-form of the amino acid, although certain D-isomers can be assimilated. In general, ammonium sulfate is a better source of nitrogen than any one single amino acid. In certain yeasts the assimilation of a particular amino acid may be either ‘ blocked or stimulated by the presence of ammonium sulfate in the medium.
Where mixtures of amino acids are present in addition to ammonium sulfate, the uptake of amino acids from the mixture is faster than the uptake of ammonium nitrogen. Studies have shown that di- and polypeptides can be assimilated by yeasts. In general, the growth of yeasts on peptides is inferior to that on amino acids or ammonium salts.
Other Organic Sources:
Of other compounds which can act as a source of nitrogen urea has been found to be utilized by virtually all yeasts and is as good a source as is ammonium sulfate. It was found, however, for a yeast to grow well the medium must also contain ample biotin. The ability to utilize purine and pyrimidine bases varies greatly among the yeasts, as does the growth response to the various bases. Bakers’ and brewers’ yeasts m general utilize very few whereas Candida utilis can assimilate a greater number of these nitrogen-containing compounds.
It has been shown for a number of yeasts that, during fermentation and growth/the yeasts actually excrete nitrogenous compounds into the medium. In many instances, these nitrogen-containing compounds are reabsorbed by the cell. Excreted compounds include amino acids, oligopeptides and nucleotides, components also to be found in the intracellular pool of nitrogenous compounds.
The requirement of yeasts for an exogenous source of vitamins varies widely, as some yeast can synthesize all of their required vitamins, whereas other yeasts have multiple requirements. In recognition of requirement variability only the ability or inability to grow in a medium lacking vitamins is presently used in classification. With the exception of meso-inositol, the vitamins serve catalytic functions, normally as part of a particular coenzyme. Meso-inositol serves a structural function in membrane synthesis where it is incorporated into the phospholipids.
The requirement for a particular vitamin in the medium is often qualified as to whether it is an absolute requirement, in which case the yeasts cannot grow if the vitamin is not regularly supplied, regardless of time or condition of growth, or whether it is a relative requirement, meaning that the yeasts can grow very slowly by synthesizing the vitamin at a very reduced rate, but that the yeasts will grow much more vigorously if the vitamin is supplied in the medium. Yeasts having absolute requirements for a particular growth factor find use as a biological assay tool.
Biotin is the most commonly required vitamin to be supplemented in the medium whereas riboflavin and folic acid are apparently synthesized in sufficient quantities by all yeasts. Vitamin Bl2 is not known to be required or even synthesized. In some yeasts there is an inter-conversion between pyrifloxine and thiamin so that a requirement for these cannot be determined in the presence of either vitamin in an assay medium.
In synthetic media the inclusion of 9 vitamins—biotin, pantothenic acid, folic acid, niacin, para-aminobenzoic acid, inositol, thiamin, riboflavin, and pyridoxine—is considered a complete supplementation. Adding high levels of vitamins, or in some cases their precursors, can “enrich” yeasts by taking advantage of their ability to concentrate vitamins, particularly the B complex, from a medium into the yeast cell.
Analysis of the minerals found in yeast cells show about 50 elements to be present, most in extremely minute amounts. Trace and small amounts of elements such as iron, copper, zinc, cobalt, calcium, and magnesium are added to the medium as they are required by yeasts as enzyme activators, structural stabilizers, and components of proteins, pigments, etc. Phosphorus is normally supplied as dihydrophosphate; potassium facilitates its uptake.
Monohydrophosphate is not taken up by the cell. Sulfur requirements are met by inorganic sulfates and to some degree by other sulfur- containing compounds. Some yeast can utilize the amino acid, methionine, or the tri-peptide, glutathione, as sources of sulfur but most yeast cannot use cystine or cysteine. Sulfite can be used, but a number of yeasts are sensitive to bisulfite and to sulfurous acid. This sensitivity is used industrially for inhibiting “wild yeasts” in certain fermentations, notably wine.
Besides adequately supplying yeast’s nutritional needs, attention to proper pH values and temperature of incubation are essential for desired activities and growth of the yeast. Actually most yeasts are relatively tolerant of a wide range of pH values; most will grow at pH 2,8-3.0 if hydrochloric acid is used to adjust the pH of the medium.
Lactic and acetic acids at low pH values are often inhibitory due to the high concentrations of the un-dissociated acid form which can pass through the cell membrane into the cytoplasm. Most yeast will also grow well at neutrality and in slightly alkaline conditions (pH 8-8.5). Optimum pH for growth varies from 4.5 to 6.5 for most species.
Temperature ranges for growth vary from relatively narrow, particularly for a few yeasts found associated with warm-blooded animals, to relatively broad ranges. Many types of yeast can grow at 0°C or slightly below but the rate of growth is extremely slow; some have a maximum of 18°-20°C, whereas others can grow at 46°-47°C. Most species encountered grow from about 5° to 30°-37°C with an optimum approximating 25°C.
While optimum conditions are known for many types of yeast, manipulation of – nutrient concentrations, pH, temperature, and oxygen tension are used in industrial fermentations to best meet the desired purposes as economically as possible. Industrial fermenters further improve their processes by careful selection of yeast strains. Strain improvement by genetic selection or mutation often results in strains performing many-fold better than the original wild type.
The following general references to the technology of yeasts provide additional information – White (1954); Ingram (1955); Roman (1957); Cook (1958); Prescott and Dunn (1959); Reiff et al. (1960, 1962); Rainbow and Rose (1963); Peppier (1967); Rose and Harrison (1969,1970,1971); Leche- valier and Pramer (1971); Reed and Peppier (1973); Miller and Litsky (1976); Phaff et al. (1978).