Yeast Bioprocessing: Recent Advances of Yeast Bioprocessing (With Diagram)

Yeasts are known to contribute significantly in the development of biotechnological processes.

Both mesophilic and thermophilic yeasts are making extraordinary advances in the knowledge of fundamental and applied bioprocessing systems at cellular, molecular and production levels.

Mesophilic yeasts like Saccharomyces cerevisiae containing covalently closed circular 2µ plasmid has been attempted to be used as a source of cloning vehicle for up gradation of brewer’s yeasts in terms of utilization of various carbohydrates like dextrin, maltose, lactose and flocculation characters.

For this recombination amplification of 2µ plasmid copy number increase has been attempted. This has added a new direction to yeasts technology. Therefore it has been necessary to examine the 2p plasmid characters of the yeast in understanding this new direction. Also, several thermophilic yeasts which are found in digestive tract of wild and domestic animals as well as in nature are contributing significantly in the development of yeast biotechnology.

Maximal growth temperatures of 40-50°C of thermophilic yeasts are not as high as those for thermophilic bacteria and fungi. Candida brassicae, C. acidothermophilum, Pichia etchelsii, Kluyveromyces hennenbergii etc. are few examples of thermophilic yeasts. In biotechnology these yeasts are playing significant role in overcoming many problems of product formation and giving newer in vivo proteolytic products using suitable bioprocessing strategy, and contributing towards downstream separation. Use of molecular biology techniques on these yeasts may add new dimensions towards new frontiers of industrial yeast biotechnology including the benefit in downstream separation.

More Recent Bioprocess Advances:

Yeasts are heterogeneous group of fungi. In common they possess predominantly unicellular morphology in at least one phase of their vegetative life cycles. Nuclear DNA base composition has proven to be a valuable exclusionary criterion for characterizing yeasts.

Likewise plasmid has a great role to play in recombination amplification under certain circumstances in several yeast strains of Saccharomyces species. Yeasts in nature and in bioprocessing circumstances may frequently encounter temperature shifts. Temperature shift causes several changes in cellular metabolism. One of the manifestations being transmit decrease in the production of ribosomal proteins. Upon temperature shift strains of yeasts carrying rna mutations are known.

It resulted different thermophilic strains of yeasts which are not true thermophilic but may belong to thermoduric. Both mesophilic and thermophilic yeasts show important characters of varying nature in terms of process biotechnology. Many yeasts of commercial importance have been shown to exhibit the flocculation property. This property has been used as an aid to downstream separation of bio-products.

Characteristic process biotechnology parameters of some thermophilic yeast has been used in newer bioprocessing for ethanol—a base chemical for product synthesis. In recent years using in vitro-in vivo events of cloning technology DNA recombination the flocculation characteristics of the yeast has been used to aid in quicker downstream separation of yeast cells.

Secretion of Foreign Proteins:

A great deal of advances in yeast genetics, molecular biology, physiological studies and cellular design engineering e.g. recombination DNA technology have taken place in recent years. It has increased the interest in this microorganism as an alternate host to E.coli for the production of foreign proteins of commercial importance.

The development of transformation methods and identification of many strong constitutive or inducible promoters in yeast has allowed the construction of a number of convenient expression vectors. For example the PGK promoter from Saccharomyces cerevisiae can direct the synthesis of phosphoglycerate kinase to levels equivalent to 15-30% of total cell protein.

Moreover, when the PGK promoter region is cloned into a high copy number, 2µ plasmid, the synthesis of PGK reaches 50-80% of total cell protein. Vectors based upon this system have been used to direct the expression of several heterologons proteins. Also, S. cerevisiae secretes several enzymes to the medium offering possibilities of harnessing this feature to secrete foreign proteins. Yeasts are, therefore, capable to render additional advantages like safety in food and health care products.

Yeast Signaling System in Bioprocessing:

The processing and secretion mechanism in yeast for the α mating factor involves synthesis of a large prepro 4α or prepro 3α subunit precursor. This is glycosylated in the endoplasmic reticulum (ER), trans-located to the golgi apparatus and processed by a protease encoded by the KEX2 gene.

Dipeptidy 1 aminopeptidase encoded by STE 13 cleaves the Glu-Ala peptide from N-terminus of the polypeptide before the mature a-factor is secreted to the medium. In yeast for secretion of proteins export mechanism may use different signal sequence. However, self secretion may also be possible. Export using the α-factor signal and export using the SUC 2 signal sequence could be observed in S. cerevisiae. Few important foreign proteins expressed and produced through redesigning of S. cerevisiae and using different signalling methods are given in Table 6.4.

It is evident that yeast has many features as a host for the synthesis of foreign proteins of health care and commercial value. The organism is easy to cultivate on a large scale bioprocessing and the use of, for example, the α-factor preprosequence, spliced to the product of interest, offers a potentially efficient secretion/export mechanism.

It has been indicated that it is possible to produce 10 mg per liter of a desired protein. By the advent of bio-molecular redesigning it appears likely that this can be raised significantly by further molecular design engineering principles to increase the initial level of expression and by the use of mutants with hyper-secretion activity.

Advances in Yeast Brewing by Cloning Technology:

In more recent years transformation of yeasts has been carried out. DNA is extracted and purified in an unmodified form from donor yeast cells. The native DNA has been modified with spheroplasts of the recipient yeast culture in presence of certain reagent and ion regenerating colonies. The regenerated colonies have been finally screened for obtaining successful transformants. Such a maltose/maltotriose system could be transformed.

There are strains of Saccharomyces that ferment only maltose and not maltotriose. Such strains lack the ability for uptake of the trisaccharide. However, by overcoming this permeability barrier to trisaccharide, the P-glucosidase system within such maltotriose negative strains can readily hydrolyze maltotriose to glucose units. A maltose positive/maltotriose negative haploid yeast strain could be developed by sequential treatments.

Maltotriose positive transformant could also be developed. However, this was similar to untransformed parent on worth-gelatin medium. These observations together with later findings indicated that successful transformations have been obtained for maltotriose uptake using native DNA as donor material into maltose positive/maltotriose negative yeast strains.

Flocculation Property

1. Aid to process biotechnology:

Cell recycle system has been reported to be a better processing strategy for alcohol production by yeast. In a cell recycle system the mash containing the cells are passed through a cell settler. In the settler cells are allowed to settle by flocculation through hindered settling phenomena and liquid is sent to a mash collector prior to distillation. For efficient operation of a settler the yeast cells must flocculate and settle with a high setting velocity.

It allows the dense viable cells to be recycled to the main fermenter to increase alcohol production. However, alcohol producing yeast strains are usually non-flocculating. Non flocculating strains take longer time to settle in the settler and required higher retention time of the cells in the reactor. For increasing settling velocity of yeast in the settler two approaches have been used.

The first approach attempts to increase settling velocity in a settler by inducing flocculation of the cells by suitable processing condition. In the second approach, however, genetic change in yeast strain is carried out by flocculating gene cloning technology.

2. Characteristic analysis:

In settler the flocculation of yeast is characterized through the estimations of floe diameter and floe density. The floe diameter (d_f, cm) may be estimated from the relation

in which d_p is the projected floe diameter on the screen perpendicular to the direction of settling (cm). A₂ is the rate of enlargement on the film through the close-up photography of the settler. A₂ is the enlargement through the projection on the screen.

The floc density can be calculated from its size and settling velocity by using a suitable settling velocity equation if the water temperature is known. The general equation for the terminal free settling velocity (W) of a discrete particle in water is given by

Where g is the gravitational acceleration 98 cm sec^-2, C_D is drag coefficient, p_s and p_w are density of the particle and water respectively, g. cm^-3, d is the particle diameter (cm). As C_D is a function of Reynolds number (Wd_s/v) and sphericity (ψ), therefore it was shown that when all the floes have Reynolds number (N_Re) less than 10⁶ in their settling the general form of C_D is given by

in which k is a constant depending on the sphericity. From available data on sphericity of ordinary floe its average value is known to be 0.8. Accordingly C_D for the floe particles has been given by (from plot of C_D vs N_Re).

Advances in Nutrition in Relation to Bioprocessing:

In ethanol production using Saccharomyces cerevisiae (ATCC 4126) it has been shown that an oxygen tension of 0.07 mm Hg was optimal. Below this oxygen tension the yeast becomes oxygen starved and ethanol productivity decreased.

At high oxygen tensions the yeast metabo­lism began to shift from anaerobic to acrobic and less ethanol was produced with a correspond­ing increased cell mass production. In continuous ethanol fermentations sometimes low fermen­tation rate is shown to be due to the lack of oxygen. It appears that even though ethanol fermen­tation is anaerobic process trace amount of oxygen is required for biosynthesis. It is reported that as a substitute of this trace oxygen unsaturated lipid, ergosterol can be used to the fermentation broth.

Likewise wort fermentation in beer production is largely anaerobic but this is not the case when the yeast is pitched into this wort and at that time some oxygen must be made available to the yeast. There is a need for oxygen because brewing yeasts in the absence of molecular oxygen are unable to synthesize sterols and unsaturated fatty acids.

Sterols and unsaturated fatty acids are essential in membrane for efficiency and beer flavour. Under aeration leads to suboptimal synthesis of essential membrane lipids. In turn it is reflected in limited yeast growth, a low fermentation rate and concomitant beer flavour problems. Over aeration results in over expending nutrients for the production of unnecessary yeast biomass, thus lowering fermentation efficiency because of the excess biomass and lower ethanol production.

Yeast Redesigning Advances:

1. Non-committed mutation:

Yeast mutational redesigning is a common occurrence throughout the growth and fermentation cycle. The mutation is usually recessive in nature. This is because of the loss of function of a gene. Since industrial strains are usually at least diploid, the dominant gene will function adequately in the strain and it will be physiologically normal. Only if the mutation takes place in both alleles will the character be expressed. If the mutation weakens the yeast, the mutated strain will not be able to compete and will soon be out grown by the other yeasts.

Normally three characteristic groups are routinely encountered resulting from spontaneous mutational redesigning of yeast which can be harmful to fermentation. These include (a) the tendency to the yeast strain to mutate from flocculescence to nonflocculescence, (b) the loss of ability to ferment maltotriose and components that are normally present in the worth in suboptimal quantities.

They are abundant in malt. As normal manufacturing procedures prevent their passing into the wort, oxygen must be supplied to allow their synthesis by yeast when ergosterol and an unsaturated fatty acid like oleic acid are added to wort, the requirement for oxygen disappears. When a brewer’s yeast is grown anaerobically it accumulates sterols and unsaturated lipids within the cells.

These lipids can be diluted to a degree by subsequent growth without negative effects. So cells prepared aerobically can grow to some extent anaerobically also. However, if yeast is harvested at the end of fermentation and used to inoculate a second batch of wort then oxygen is required.

This is because the new inoculum contains no reserves of the necessary lipids. Oxygen content of the wort at pitching is important to lipid metabolism in yeast fermentation. Another factor is the presence of respiratory deficient (R_D) mutants. The last group usually consists of cytoplasmic mutants.

The most commonly and frequently identified spontaneous mutant found in brewing yeast strains is the R_D or petite mutation. The R_D mutant arises spontaneously when a segment of the wild type mitochondrial genome, exercised by an illegitimate site, specific recombination is amplified.

It is due to formation of a defective mitochondrial genome. The mitochondria are then unable to synthesize certain proteins. This mutation shows non-Mendelian seggregation when crosses of the wild type with the mutant are carried out and may be termed non- committed mutation.

2. Frequency level and characters:

The R_d non-committed mutational redesigning occurs at frequencies between 0.5% and 5% of the yeast population. In some strains, however, figures as high as 50% have been reported. The mutant is characterised by deficiencies in mitochondria function resulting in a diminished ability to function aerobically. These redesigned yeast, are unable to metabolize non-fermentable carbon sources such as lactate, glycerol or ethanol. Many phenotypic changes occur because of the redesigning, including alterations in sugar uptake.