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310 Modern Industrial Microbiology and Biotechnology

The aeration through sparger holes is started as soon as mixing begins in the steam

sterilized fermentor. Water, mineral nutrients, yeasts and the blended molasses

containing 1% glucose are mixed. The amount of blended molasses added is calculated

so that the total sugar in the fermentor does not exceed 0.1%. Molasses is added

incrementally during the course of the fermentation as it is used up by the yeast, beyond

the 0.1% ceiling. The pH is maintained at pH 4-6 by the addition of alkali and the

temperature at 30°C by cooling. The amount of molasses to be added at predetermined

intervals is arrived at by experimentation. Automatic sensoring and self-adjusting

equipment for temperature pH, aeration, sugar, etc., are built into some modern

fermentors. Large amounts of heat are evolved and the cooling of the fermentor is very

important. 1-Ib dry wt. of yeast would require 4.3 Ib of molasses, 0.9 Ib of ammonia, 0.3 lb

of NH

, 1.1 lb of (NH

)

and 60 lb of air. Continuous fermentation has not been

widely used in baker’s yeast production. It is used (see below) for feed yeast production

from sulfite liquor.

16.1.3.3 Harvesting the yeast

The period of fermentation in the trade or production fermentor varies from 10 to 20 hours

depending on how much yeast is pitched into it; cells form from 3.5% to 5% dry wt. of the

broth. In some processes aeration is allowed to continue for 30-60 minutes at the end of

the feeding to allow unused nutrients to be used up, budding cells to divide so that most

cells are ‘resting’ at the beginning of the budding cycle. This ensures that the cells divide

somewhat ‘synchronously’ when growth resumes.

The fermentation broth is cooled and cells concentrated in centrifugal separators; they

are washed by resuspension in water and centrifugation until they are lighter in color.

The yeast cream resulting from this treatment contains 15-20% yeast cells. It is further

concentrated by passing over a rotary vacuum filter or through a filter press. Sometimes

the Mautner process is used to ensure a friable dry cream during vacuum filtration. This

latter process consists of adding before filtration 0.2-0.6% (w/v) sodium chloride, which

causes cell shrinking by osmosis. Excess salt is removed during filtration by spraying

water over the filtered yeast, so that the cells swell again. The resulting product has a dry

matter content of 28-30%.

The yeast may then be packaged as compressed yeast or active dry yeast. It may also be

converted into dried yeast for human or animal feeding as described further on in this

chapter.

16.1.3.4 Packaging

Baker’s yeasts may be packaged as moist (compressed) yeasts or as dried active yeast.

(i) Compressed yeast: The yeast product obtained after harvesting, is mixed with fine

particles of ice, starch, fungal inhibitors and processed vegetable oils (e.g. glyceryl

monostearate) which all help to stabilize it. It is then compressed into blocks of

small (1-5 Ib) blocks for household use or large (up to 50 Ib) for factory bakery

operations, stored at – 7 to 0°C and transported in refrigerated vans.

(ii) Active dry yeast: Dry yeast is more stable in that it can be used in areas or countries

where refrigeration is not available. In many developing countries baker’s yeast is

imported from abroad in the form of active dry yeast. For active dry yeast

Yeast Production 311

production special strains better suited for use and dry conditions may be used. It

has been found that when regular strains are used they perform better as dry yeasts

when they are subjected to a number of treatments. These treatments include

raising the temperature to 36°C (from about 30°C) towards the end of the

fermentation, addition of alcohol-containing spent broth (resulting from

centrifugation or finished yeast fermentation), synchronization of budding by

alternate feeding and starving. The reason for the benefit is not known.

Yeast cream of 30-38% content from filter pressing is extruded through a screen to form

continuous thread-like forms. These are then chopped fine and dried, using a variety of

driers: tray driers, rotary drum driers, or fluidized bed driers. The final product has a

moisture content of about 8% and may be packaged in nitrogen-filled tins. Sometimes

anti-oxidants may be added to the yeast emulsion to further ensure stability.

Fig. 16.1 Various Methods for Packaging Yeasts

16.2 FOOD YEASTS

Yeasts are used for food by man for the following reasons: to provide protein; to impart

flavor and to supply vitamins especially B-vitamins. Food yeasts are sometimes

prescribed medically when a deficiency of B-vitamins exists in a patient. Food yeasts

have several synonyms: dried yeast, inactive dried yeast, dry yeast, dry inactive yeast,

dried torula yeast, Saccaromyces siccum, sulfite yeast, wood sugar yeast, and xylose yeast.

The link between wood and the production of yeast for animal consumption is shown by

the last three names.

312 Modern Industrial Microbiology and Biotechnology

As food yeasts are consumed by man, stringent standards are imposed on the product

by governmental agencies, professional bodies and manufacturers. The standards of the

International Union of Pure and Applied Chemists (IUPAC) will be quoted here only as

an example, because they are the most comprehensive. The IUPAC requires that any

organism to be used as a food yeast belong to the family Cryptococcaceae, should be

unextracted, should have a fat content of not more than 20%, should contain no inert

fillers not indigenous to the yeasts, and should be free of Salmonella. The IUPAC has also

set upper limits for bacterial and fungal counts, lead, arsenic and lower limits for protein,

thiamin, riboflavin and niacin.

Too high a consumption of yeasts is detrimental to health because of the high RNA

content of yeasts, which the kidneys are unable to dispose of. This was discussed in the

previous chapter.

16.2.1 Production of Food Yeast

While baker’s yeasts are usually produced from molasses using special strains of Sacch.

cerevisiae food yeasts are produced from a wide variety of yeasts and substrates.

16.2.1.1 Yeasts used as food yeasts

Yeasts used as food yeasts are Saccharomyces cerevisiae, Saccharomyces carlbergiensis,

Saccaromyces fragilis, Candida utilis, and Candida tropicalis. Only Saccharomyces fragilis

(imperfect stage Candida pseudotropicalis) can utilize lactose hence it is used for the

fermentation of whey. Ethanol may be used as substrate for food yeast production; it is

however used only by Saccharomyces fragilis and Candida utilis. Candida utilis is the most

versatile of all the yeasts and will utilize a wider range of carbon and nitrogen sources

than any other, hence it is most widely used in food yeast preparations.

16.2.1.2 Substrates used for food yeast production

The most commonly used substrates are molasses, sulphite liquor, wood hydrolysate,

and whey. Since interest developed in single cell protein other unconventional sources

have been developed. These include hydrocarbons, alcohol and wastes of various types.

These have been discussed in the previous chapter. Only molasses, sulphite liquor, wood

hydrolysate and whey will be discussed.

(i) Molasses: Bakers yeast grown on molasses as described above may, after separation

from the spent liquor by centrifugation, be dried to yield food yeast. Drum-drying,

spray-drying or fluidized bed drying may be used to reduce the moisture content to

only about 5%. Sometimes food yeast is grown on molasses for that purpose per se.

Thus Candida utilis is grown fed-batch in Taiwan in Waldhof fermentors. The fed-

batch method using molasses is also used in South Africa.

Recently food yeasts using Candida utilis in continuous culture in molasses has

been grown in Cuba and Eastern Europe.

(ii) Sulfite liquor: The impetus to produce food and fodder yeast from sulfite liquor

(Chapter 4) derived from an attempt to reduce the pollution which would arise if

the wastes containing fermentable substrates were discharged directly into a

Yeast Production 313

stream. The use of continuous fermentation was attractive because the sulfite is

produced almost continuously in the operation of the pulp factory. In general a

Waldhof-type fermentor is used for the continuous production of yeasts from

sulfite waste. Liquors from various sources are usually blended. Thereafter, the

sulfite containing compounds are removed either by precipation with lime, by

aeration or by passing steam through it (steam stripping). The pH is adjusted from

about 2 to 5.5 using ammonia. The lowest pH consistent with high yield is usually

preferred in order to lessen the chances of contamination.

Ammonium, phosphate and potassium are monitored and supplied

continuously. The versatile and hardy yeast Candida utilis is usually used so that

biotin is not added. The yeast is harvested continuously and recovered by

removing liquor at the same rate as it is introduced. The effluent liquor containing

about 1% cell is concentrated to an 8% concentration by centrifuging. It is usually

washed with water by diluting and centrifuging to remove lignosulfnic acid. Yield

from sulfite liquor, whose assimilable matter content is usually low may be

increased by the addition of new carbon sources e.g. acetic acid and ethanol. The

liquor may be re-hydrolysed with H

thereby increasing the sugar content from

4% to about 24%. In some cases, addition of nutrients to the liquor e.g. yeast

hydrolysate or corn steep liquor leads to an increased yield of about 5%.

Simultaneously, more efficient organisms are usually also sought.

(iii) Production of food yeast from whey: The effluent which drains from the coagulum

from milk during cheese manufacture is known as whey. It contains

approximately 4% sugar (lactose), 1% mineral and some of the lactic acid which

enabled the coagulation of the milk protein. In countries where a lot of cheese is

produced, whey is a waste product but it is sometimes turned into good use in the

production of alcohol or yeasts. Very few yeasts metabolize lactose. Those which

do include Saccharomyces lactis, Kluyveromyces fragilis and its imperfect or

asporogeneous stage Candida pseudotropicalis. The whey is diluted, fortified with

ammonia, phosphate, minerals, yeast extract and then pasteurized at 80°C for

about 45 minutes. It is then inoculated with yeasts at pH 4.5 at an incubation

temperature of 30°. Any of the above yeasts could be used but in the United States

the preference is for K. fragilis. In many establishments the fermentation is

continuous and sugar, pH and minerals are monitored automatically. The yeast is

recovered by centrifugation and may be drum or spray dried.

16.3 FEED YEASTS

Feed yeasts are the same as food yeasts described above. The only difference is that less

rigid standards are imposed on the production of feed yeasts. Thus, feed yeasts intended

for animal feeding are usually obtained by drying out the whole fermentation broth, often

without washing.

Several thousands tons of yeasts are recovered from breweries around the world

annually. To be used as food yeasts, such yeast is ‘debittered’ of hop resins by repeated

washing with dilute alkali until the bitterness no longer exists. It is then slightly acidified

to about pH 5.5. Cells are recovered by centrifugation and spray – or drum-dried.

314 Modern Industrial Microbiology and Biotechnology

16.4 ALCOHOL YEASTS

Alcohol yeasts are those to be used in beer brewing wineries and distilleries for spirits of

industrial alcohol. In the production of alcohol yeasts, the aim is cell production. The

methods are generally similar to those already described for baker’s yeasts. Beginning

from a lypholized vial or tube, contamination is checked in a plate. A single colony (or

prefereably a single spore by micromanipulation) is picked and multiplied in

sequentially increasing amounts.

The yeasts used are specially selected strains of the following:

Brewing: Saccaromyces cerevisiae, Saccharomyces uvarum carlbergensis S. uvarum.

Wine: Saccharomyces cerevisiae, Sacch. bayanus, Sacch. beticus, Sacch. elipsoides.

Distillery Yeasts: Saccharomyces cerevisiae.

The medium used in the multiplication of the yeast is made of materials to be found in

the final fermentation. Thus for growing brewery yeasts wort is used, for distiller’s yeast

a rye-malt medium is used, and for wine grape juice is used.

Alcohol yeasts are usually recovered and reused for several rounds of fermentation

before being discarded.

16.5 YEAST PRODUCTS

Various products used in the food, pharmaceutical and related industries may be

produced from yeasts.

Yeast extracts are used in the preparation of soups, sausages, gravies, to which they

impart a meaty flavor. A well-known example is marketed in certain parts of the world as

‘marmite’. The extracts may be obtained by autolysing the yeasts and thereafter spray-

drying or drum-drying with or without extracting soluble materials from the autolysate.

The extract may also be obtained by hydrolyzing the yeast cells in acid solution. It is

neutralysed with sodium hydroxide, filtered, decolorized through charcoal and

concentrated to a syrup or spray-dried. Yeast products are usually fortified with the

flavoring compound, mono-sodium glutamate, extracts of animal or vegetable protein or

with yeast cells.

Yeast extracts are consumed for dietary purposes on pharmaceutical grounds as a

source of vitamins, mainly vitamin B

SUGGESTED READINGS

Boze, H., Moulin, G., Galzy, P. 1991. Production of Microbila Biomass. In: Biotechnology G. Reed,

T.W. Nagodawitana, (eds). VCH Weinheim Germany. pp. 167-220.

Burrows, S. 1979. In: Microbial Biomass. A.H. Rose, (ed.) Academic Press, New York, USA. pp. 32-

64.

Caron, C. 1991. Commercial Production of Bakers Yeast and Wine Yeast In: Biotechnology

G. Reed, T.W. Nagodawitana, (eds). VCH Weinheim Germany. pp. 321-350.

Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,

Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.

Scrimshaw, N.S., Murray, E.B. 1991. Nutritional value and Safety of “Single Cell Protein”. In:

Biotechnology G. Reed, T.W. Nagodawitana, (eds). VCH Weinheim Germany. pp. 221-240.

Insects are major pests of crops. Enormous losses occur when they attack various plant

parts, often transmitting disease in the process. Even after harvest insects attack stored

foods; this attack of stored foods is not limited to plant foods, but also includes animal

foods such as dried fish. Besides the loss they cause in agriculture and food, insects are

also vectors of various animal and human diseases.

In modern times insects have been controlled mainly with the use of chemicals. Over

the past decade or so there has been a move away from the sole use of chemical control,

and towards integrated control, which employs other methods as well as chemical

control. The reasons for this include non-specificity of chemical insecticides leading to

the destruction of pests as well as their natural predators, resistance to chemical

insecticides, concern for the environment and human health since the insecticides enter

drinking water from soil, and since some are toxic or carcinogenic. Finally due to

increased cost of petroleum on which many of these insecticides are based, their cost has

also increased.

17.1 ALTERNATIVES TO CHEMICAL INSECTICIDES

The alternatives to the use of chemicals include the following:

(a) Predators: Among vertebrates one of the best known is the use of fish especially

Gambusia affinis to eat mosquito larvae. Invertebrate predators include other larger

insects e.g. wasps, while plant predators include Utricularia (a bladder wort).

(b) Genetic manipulations: These include the production (by chemicals or by

irradiation) of large numbers of sterile males, whose mating does not result in

fertile eggs.

which act as sex attractants. The insects attracted are destroyed.

(d) The use of pathogens: Pathogens of insects are found among bacteria, fungi,

protozoa, viruses and nematodes. The idea of using pathogens to control insects

originated from studies of the diseases of the silkworm Bombyx mori. The pioneer

work of Bassi was followed by those of Le Conte, Pasteur, Hagen until Metchnikoff

actually tested the control of sugar beet pests with the fungus Metarrhizium

anisopliae in South Russia.

Production of

Microbial Insecticides

+0)26-4

!$ Modern Industrial Microbiology and Biotechnology

17.2 BIOLOGICAL CONTROL OF INSECTS

Biological control has been studied or practiced to a large extent in relation to agriculture,

food production and forestry. Its study and use in the control of insect vectors of disease

such as mosquitoes has been small in comparison. In 1976 the World Bank in

collaboration with the United Nations Development Programme (UNDP) and the World

Health Organization (WHO) put into operation a Special Programme for Research and

Training in Tropical Diseases. The diseases were malaria, trypanosomiasis, filariasis,

leishmaniasis, schistosomiasis and leprosy. Of these the first four are transmitted by

insect vectors. The WHO which administered the Programme also studied biological

control with respect to the insect-borne disease and ranked the organisms to be used in

order of effectiveness from time to time (Table 17.1). The organisms included bacterial,

fungi, nematodes, and fish, and targets are mainly mosquitoes-vectors of malaria and

yellow fever and black flies (Simulium spp), vectors of oncocerchiasis (river blindness).

For agricultural biological control however some viruses pathogenic to insects are also

used (Table 17.2). In this chapter only control using microorganisms will be discussed.

Table 17.1 Biological control agents as ranked in order of priority by the special program of the

World Health Organization 1980

Priority 1 Bacillus thuringiensis, serotype H-14 (bacterium)

Priority 2 Bacillus sphaericus, strain 1593 (bacterium)

Culicinomyces sp. (fungus)

Poecilia reticulata (fish)

Romanomermis culicivorax (nematode)

Toxorhynchites (predatory mosquitoes)

Zacco platypus (fish)

Priority 3 Aphanius dispar (fish)

Coelomomyces (fungi)

Lagenidium (fungus)

Leptolegnia (fungus)

Metarrhizium anisopliae (fungus)

Parasitoids in general (insects)

Romanomermis iyengari (nematode)

Stenopharyngodon idella (fish)

Priority 4 Group A

Aplocheilus (fish)

Baculoviruses (viruses)

Dimorphic Microspordia (protozoa)

Dugesia (Planaria)

Lutzia (predatory mosquito)

Octomyomermis muspratt (nematode)

Protozoa of snails (protozoan)

Tolypocladium

Group B

Entomophthorales (fungi)

Nosema algerae (protozoan)

Vavraia culicis (protozoa)

Production of Microbial Insecticides !%

17.2.1 Desirable Properties in Organisms to be Used for

Biological Control

The following are desirable in microorganisms to be used in the biological control of

insects:

(a) The agent should be highly virulent for the target insect, but should kill no other

insects.

(b) The killing should be done quickly so that in the case of crops, damage is kept as

low as possible, and in the case of vectors of disease before extensive transmission

of the disease occurs.

(d) The agent should not be harmful to man, animals or crops; in other words it should

be safe to use.

Table 17.2 Pathogenic viruses found in insects

Family Nucleic Particle Vertebrate and plant viruses resembling

Acid

Symmetry families of insect viruses

Vertebrate Plant

viruses viruses

1. Baculoviridae DNA Rod None None

(Baculovirus (occluded)

groups A,B,C)

2. Poxviridae

(Entomopox DNA ‘Brick’ Orthopoxvirus None

viruses) (occluded) Avipoxvirus Reoviruses

Capripoxvirus

Leporipoxvirus

Parapoxvirus

3. Reoviridae dsRNA Isometric Reovirus Plant

(Cytoplasmic (occluded) Orbivirus

polyhedrosis

viruses)

4. Irodioviridae DNA Isometric African Swine Fever Fungal

(Iridovirus) Frog Viruses 1-3 Algal

Lumphocystis virus

5. Parvoviridae ssDNA Isometric Parvovirus None

(Densovirus) Adeno-associated

Group

6. Picornaviridae RNA Isometric Enterovirus Small RNA

(Enterovirus; Viruses

unclassified (single

groups) polypeptide)

7. Rhabdoviridae RNA Bullet/ Vesiculovirus Plant rhab-

(Sigmavirus) bacciliform Lyssavirus dovirus

ds RNA, double stranded RNA, ss, single stranded DNA

!& Modern Industrial Microbiology and Biotechnology

(e) It should be technically amenable to cheap industrial production.

(f) When produced, it should be stable under the conditions of use such as under the

high temperature and ultra violet light of ordinary sunlight.

(g) It should be viable over reasonably long periods to permit storage and

transportation as necessary.

(h) It should ideally persist or recycle and/or be able to search for its host.

17.2.2 Candidates Which have been Considered as

Biological Control Agents

(i) Bacteria A large number of bacteria are pathogenic to insects including Bacillus spp.,

Pseudomonas sp. Klebsiella sp., Serratia marcescens. In practice, spore formers have been

developed commercially because they survive more easily in the environment then

vegetative cells, but especially because they are amenable mass production. The four

bacilli which have been produced for control purposes are:

(a) Bacillus thuringiensis: B. thuringiensis (commonly known as ‘Bt’) is an insecticidal

bacterium, marketed worldwide for control of many important plant pests–mainly

caterpillars of the Lepidoptera (butterflies and moths) but also mosquito larvae,

and simuliid blackflies that vector river blindness in Africa. Bt products represent

about 1% of the total ‘agrochemical’ market (fungicides, herbicides, and

insecticides) across the world. The commercial Bt products are powders

containing a mixture of dried spores and toxin crystals. They are applied to leaves

or other environments where the insect larvae feed. The toxin genes have also been

genetically engineered into several crop plants. The method of use, mode of action,

and host range of this biocontrol agent differ markedly from those of Bacillus

popilliae.

It is a complex of several organisms regarded by some as being variants of B.

cereus. There are 19 serotypes based on some flagellar or H-antigens. Serotype H3

and H3A are used in the United States on alfalfa, cotton, tobacco, spinach,

potatoes, tomatoes, oranges, and grapes. Serotype H14 attacks mosquitoes and

blackflies and will be discussed below. Bacilus thuringiensis produces at least three

toxins, a Phospholipase C, a water-soluble heat stable B-exotoxin potentially toxic

to mammals, and a crystalline, d-toxin or the parasporal body which is enclosed

within the sporangium (this will be discussed further below).

The crystalline d-toxin is the active principle against most insects. The spores

and crystals are released into the medium in most strains of B. thuringiensis

following the lysis of the sporangium.

(b) Bacillus moritai: This is used in Japan for the same purpose as B. thuringiensis

serotypes H3 and H3A.

against which it is used. Since it is an obligate parasite it is produced in the larvae

of the beetle.

(d) Bacillus thuringinensis var. israelensis (also known as serotype H14). This was

isolated in 1976 by Goldberg and Margalit from a mosquito breeding site in Israel.

It has proved very effective in killing mosquito larvae and the black fly (Simulium

Production of Microbial Insecticides !'

spp). So promising is it from results of various projects sponsored by the Special

Program of the WHO that it was expected that it would be produced on a large

scale in the US and Europe and probably on smaller scales in tropical countries. It

has a (nearly 100%) kill of mosquito larvae and shows no adverse effect on non-

target organisms. Unlike classical Bacillus thuringiensis it does not produce a beta-

toxin. Its killing effect is therefore based principally on its crystalline delta-toxin,

(d-toxin) which is resistant to both heat (surviving 80°C for 10 minutes and 60°C

for 20 minutes) and ultra violet light.

(e) Bacillus sphericus: Bacillus sphericus is an highly specific for mosquito larvae as

Bacillus thuringiensis, var israelensis (B.t.i.). However, whereas the lethality of B.t.i.

resides in toxic protein crystals formed during the spores of the organism, the toxin

of B. sphericus resides in the cell wall of the organism. The toxin of B. sphericus works

slowly (8-40 hours) compared with that of B.t.i. (2-10 hours). Bacillus sphericus

however, has the advantage of being able to lay dormant in muds or sewage ponds

and to recycle as susceptible mosquito larvae appear. Like B.t.i., it had reached

stage 4 of had WHO scheme for screening and evaluating biological agents for

control of disease vectors shown in Table 17.3.

(ii) Viruses: A large number of viruses has been isolated from insects. The advantages of

viruses as biological control agents is that they are specific. Seven groups of insect-

pathogentic viruses have been identified (Table 17.2). The most useful of them for

biological control purposes are the baculoviruses, which are easily recognizable because

the virus particles are included within a proteinaceous inclusion body large enough to be

seen under a light microscope. (These inclusion bodies, polyhedrons and granules, are

found in the nucleus of the host cell – hence they are nuclear polyhedrosis and

granuloses).

The baculoviruses are the best candidates for insect control because they are (a)

effective in controlling insect populations, (b) restricted to a host range of invertebrates,

because of the inclusion bodies.

Several experimental preparations are available and at least two (one each in the USA

and Japan) have been produced on a commercial scale. The preparations are ingested

when the insects consume leaves and other plant parts on which the virus particles have

been sprayed. After ingestion the polyhedral inclusion bodies dissolve within the mid-

gut; the released virions pass through the mid-gut epithelial cells into the haemocoel.

Death of the larvae occurs four to nine days after ingestion.

(iii) Fungi: All the four major groups of fungi, Phycomycetes, Ascomycetes, Fungi Imperfecti

and Basidiomycetes contain members pathogenic to insects. The great difficulty with

using fungi for biological control is that environmental conditions including

temperature and humidity must be adequate for spore germination and insect cuticle

penetration by the hyphae. Since these environmental conditions are not always assured

the result is that fungi are used for biological control only in a few countries especially the

USSR. Fungi which have been most widely used as Beauvaria bassiana and Metarrhizium

anisopliae. Others are Hirsutella thompsonii Verticillium and Aschersonia aleyrodis. H.

thompsonii is being developed commercially as acaricide, for killing mites which attack

plants, although a large number of other fungi attack mites. H. thomposonii has been