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! Modern Industrial Microbiology and Biotechnology

any hitherto unexplored waste source, what is required is to determine what

pretreatment, if any, is required and search for the appropriate organism which will grow

in the hydrolysate.

15.2 MICROORGANISMS USED IN SCP PRODUCTION

A list of selected organisms currently used in SCP production is given in Table 15.3.

Obviously that list is not exhaustive; new organisms may be discovered or new strains

may be developed from existing strains.

The properties required in industrial organisms in general have been described in

Chapter 1. In addition organisms to be used in SCP production should have the following

properties:

(a) Absence of pathogenicity and toxicity: It is obvious that the large-scale cultivation of

organisms which are pathogenic to animals or plants could pose a great threat to

health and therefore should be avoided. The organisms should also not contain or

produce toxic or carcinogenic materials.

(b) Protein quality and content: The amount of protein in the organisms should not only

be high but should contain as much as possible of the amino acids required by

man.

but it should possess acceptable taste and aroma.

(d) Growth rate: It must grow rapidly in a cheap, easily available medium.

(e) Adaptability to unusual environmental conditions: In order to eliminate contaminants

and hence reduce the cost of production, environmental conditions which are

antagonistic to possible contaminants are often advantageous. Thus, strains

which grow at low pH conditions or at high temperature are often chosen.

The heterotrophic microorganisms currently used are bacteria (and actinomycetes

and fungi (moulds and yeasts); protozoa have not been used in SCP production. Of the

substrates currently in use, the gaseous hydrocarbons (methane, propane, butane) are

almost exclusively attacked by bacteria. Liquid hydrocarbons (n-paraffins, gas oil, diesel

oil) and alcohols are utilized by both bacteria and yeasts. Of the carbohydrates sugar is

readily utilized by all the classes of microorganisms; a large number of them can also

utilize starch. Cellulose is not generally utilized directly by many microorganisms save

after treatment. Cellulose and other materials in peanut shells, carob beans, spoiled

fruits, corn and pea wastes, sugarcane bagasse, palm, cassava wastes have been used to

make SCP using the moulds Trichoderma sp., Glicladium sp., Geotrichum sp., Fusarium, and

Aspergilus. Paecilomyces variotii is used in the Pekilo sulfite liquor SCP method. Fungi

have the advantage that they are lower in RNA content and are easily harvested.

15.3 USE OF AUTOTROPHIC MICROORGANISMS IN

SCP PRODUCTION

Autotrophic organisms include the photosynthetic bacteria and algae. Most of the work

on SCP production by autotrophic microbes seems to be limited to the algae. It does not

Single Cell Protein !

Table 15.3 Organisms and substrates which have been used for single cell protein production

Gaseous hydrocarbons Bacteria Fungi

i) Methane Methanomones sp.

Methylococius capsulatus

Pseudononas sp.

Hyphomisobium sp. Mixed

Acinetobacter sp.

ii) Propane Flavobacterium sp.

Arthrobacter simplex

iii) Butane Nocardia paraffinica

Nocardia paraffinica

Liquid Hydrocarbons:

i) n-Alkanes (C

10 –

) Mycobacterium phlei Candida guillermondi

Nocardia sp. Candida lipolytica

carbon length

ii) n-Alkanes (unspecified) Candida Kofuensis

Candida tropicals

iii) Liquified petroleum gas Candida lipolylica

Candida rigida

iv) Gas oil Acinetobacter Candida tropicalis

Pseudomonas Candida lipolytica

v) Diesel oil Acromobacter delcavate

Alcohols Methylomonas Methanolica Torulopsis methansoba

Methanol Methyliphilus (Pseudomonas) Toralopsis methanolove

methylotrophus Candida boidini

Ethanol Methylomonas clara Hansemula polymorpha

Candid ethanomorphium

Candida tropicalis

Plant/Wood Wastes

Sulphite liquor Thermomonespore fusca Paecylomyces variotii

Cellulose pulping fines Brevibacterium sp. Candida utilis

Mesquite wood

Wheat bran Rhodopseudomonas glatinosa

Wastes from carb, Fusarium sp

Palms, papaya, etc. Aspergillus sp

Starch Wastes

Potato hydrolysate Rhodotromla rubra

Tapioca (Cassava) starch Candida tropicalis

Diary Wastes Endomycopsis

Libuligera

Whey Kluyveromyces fragilis

Trichosporon cutaneum

Sugar Wastes

Molasses Candida utilis

Chemical Industries Wastes:

Oxanone Waste Water Trichosporon cutaneum

Candida pseudotropicalis

Waste polyethylene

! Modern Industrial Microbiology and Biotechnology

appear that photosynthetic bacteria hold out much hope for use as SCP sources because

they require anaerobic conditions for photosynthesis. These conditions are difficult to

provide and maintain.

The annual production of the oceans and seas of the world which harbor the bulk of

the world’s algae is very high – some 550 x 109 tons – about 100 tons per annum for every

human being alive. Man consumes algae through fish for which the algae serve as food.

The algae themselves are rich in protein and could be harvested for this purpose.

However, the concentration of algae in marine water is only 3 mg/litre whereas for

economic viability the harvest should be at least 250 mg/litre. This is the first reason why

algae need to be cultivated and produced in high concentrations.

The second reason is that, when given adequate conditions, 20 tons (dry weight) of

algae having a protein concentration of 50% can be produced per acre of pond per

annum. In terms of the yield of digestible protein, this is 10-15 times greater than the soya

bean and 25-50 times more than the same area planted with corn. From the view point of

good energy the yield per acre of algae in terms of dietary energy is eight times as great as

sugar beet, and between 22 and 45 times as great as that of corn and potato respectively.

Third, in terms of water use for the same amount of protein, much less water is required

(Table 15.4).

Table 15.4 Comparison of water use for food production by some conventional crops and by

algae

Annual Protein Annual Water Ib Protein/

Yield (Ib/Acre) Consumption Acre/Feet/Acre Acre Food

Soya bean 576 2.0 288

Corn 240 2.0 120

Wheat 135 1.5 90

Algae 20,000 4.0 5,000

Capital investment involving various facets of the development of land, energy

consumption, and manpower utilization are about the same when conventional and

algal farming are compared.

Feeding tests in animals (using the green algae Scendemus and Cholorella) showed that

in general algae had beneficial effects if fed to animals in small amounts at a time. Feeding

in large amounts for long periods was more successful if they were supplemented by

proteins from other sources. Humans consuming foods containing algae, did so for

periods of up to 20 days with only minor abdominal upset due, probably, to the novelty of

the foods. However, due to the peculiar taste of the algae, such foods would probably be

more immediately acceptable in communities such as in Chad or in Mexico which have

developed a taste for the blue-green algae Spirulina through generations of consumption.

For the highest algal yields carbon dioxide is supplied to algae growing in day light;

where natural saline water rich in bicarbonates is available, such as is found in Chad

Republic or in Lake Texicoco in Mexico, supplementation with CO

is not necessary.

Effluents emanating from sewage treatment with their rich content of minerals would

be ideal for growing algae for animal feeding. The resulting algae should be heat-treated

to avoid any possibility of pathogen transmission.

Single Cell Protein !!

With the advantages of algae cultivation mentioned earlier, particularly the

comparatively low capital investment, the digestibility by ruminants and other animals

the possibility of integrating waste disposal with algae production and above all the

availability of sunlight and warm temperatures throughout the year, the tropics should

be the place for algae cultivation for animal feeding.

As can also be seen from Table 15.5 the production cost of protein per kilogram is very

favorable for SCP when compared with other protein sources.

Table 15.5 Production of various proteins (1980 figures, USA, $ dollars)

$ kg

-1

% Protein $ per kg protein

Beef 1.54 15 20.3

Pork 1.10 12 19.1

Poultry 0.66 20 6.6

Cheese 0.78 24 6.5

Soy flour 0.15 52 0.6

Peanut flour 0.15 59 0.5

Yeast from n-alkanes 0.42 53 1.4

Yeast from molasses 0.33 53 1.3

Fungi from celluloses 0.15 43 0.7

Algae 0.66 46 2.8

15.4 SAFETY OF SINGLE CELL PROTEIN

Probably on account of the novelty of SCP as food it has met with very strong opposition

especially in some countries, notably Japan and Italy. The public in the former country

had become aware of the health hazards of environmental pollution and in particular the

‘minimata disease’ which was due to the consumption of mercury from sea food

contaminated with it. The government was concerned with the possibility of the presence

of carcinogenic compounds in petroleum-grown SCP, with its limited and non-

renewable nature and because it was not conventional. The oil companies which had

been working on the SCP from petroleum-derived substrates switched over to working

with non-petroleum substrates. In Italy the concern was over the safe content of nucleic

acid in SCP, the polycyclic aromatic hydrocarbons, fatty acids containing odd-numbered

carbon skeletons and the presence of n-paraffins carried over from protein-grown yeasts

fed to farm animals. Evidence in support of the overall safety of SCP has been however,

presented and it is likely that SCP will eventually receive official approval.

The two examples given above are typical of the concern shown by the public and

organizations in many parts of the world, including some specialized agencies of the

United Nations, namely the World Health Organization (WHO), the Food and

Agriculture Organization (FAO) and the United Nations International Children’s

Emergency Fund (UNICEF). The concern for the nutritional completeness and

toxicological safety of novel protein foods designed for developing countries (solvent and

heat-extracted soy proteins, synthetic amino acids, flours from ground nut and cotton

seed, fish and leaf proteins, and microbial protein, etc.) led the WHO in 1955 to form the

Protein Advisory Group (PAG). WHO was joined by the two other above-mentioned

!" Modern Industrial Microbiology and Biotechnology

bodies in sponsoring the Protein Advisory Group (PAG) in 1960. The PAG appointed a

number of ad hoc working groups including an Ad hoc Working Group of SCP which

was formed in 1969. The PAG on SCP concluded that low levels of residual alkanes, and

the presence of odd-number fatty acids, or polycyclic hydrocarbons which are all derived

from petroleum do not constitute a danger in terms of carcinogenicity or toxicity. It has

also developed guidelines for the production, and nutritional and safety standards, of

SCP for human consumption. The International Union of Pure and Applied Chemists

(IUPAC) has a Fermentation Section which has prepared a set of standards and

specifications relating to the feeding of SCP to farm animals since these are ultimately

consumed by man. The two groups mentioned have similar protocols for determining

safety. These include microbiological examination for pathogens and toxin producers,

chemical analyses for heavy metals, nucleic acid content, presence of hydrocarbons,

safety tests on animals and protein quality studies.

15.4.1 Nucleic Acids and their Removal from SCP

Apart from the fears of carcinogenicity and toxicity from petroleum derivatives

mentioned above, both of which fears have been allayed in extensive studies, another

area of concern in SCP feeding is the consumption of high levels of nucleic acid. Man has

lost the enzyme uricase which oxidizes uric acid to the soluble and excretable allantoin.

When nucleic acid is eaten by man, it is broken up by nucleases present in the pancreatic

juice, and converted into nucleosides by intestinal juices before absorption. Guanine and

adenine are converted to uric acid, which as had been pointed out earlier cannot be

converted to the soluble and excretable allantoin. As a result when foods rich in nucleic

acid are consumed in large amounts, an unusually high level of uric acid occurs in the

blood plasma. Owing to the low solubility of uric acid, uricates may be deposited in

various tissues in the body including the kidneys and the joints when the diseases

known as kidney stones and gout may respectively result. In June, 1970 the PAG working

group of SCP established the upper limit of 2 gm nucleic acid per day in addition to the

quantity present in the usual diet for adults.

Some ordinary foods are high in nucleic acid (mostly RNA): liver, sardines, and fish

roe (caviar) contain 2.2. and 5.7 gm of nucleic acids per 100 gm of proteins respectively.

With SCP, comparable figures vary from 8 to 25. The proportion of nucleic acids in total

cell content of various micro-organisms is as follows: moulds, 2.5-6%; algae, 4-6%; yeasts,

6-11%; bacteria, up to 16%.

Various ways have been devised for the removal of nucleic acids from SCP.

(a) Growth and cell physiology method: The RNA content of cell is dependent on growth

rate: the higher the dilution rate (in continuous cultures) the higher the RNA/

protein ratio. In other words the higher the growth rate the higher the RNA content.

The growth rate is therefore reduced as a means of reducing nucleic acid. It must

however be borne in mind that high growth is one of the requirements of reducing

costs in SCP, hence the method may have only limited usefulness.

(b) Extraction with chemicals: Dilute bases such as NaOH or KOH will hydrolyze RNA

easily. Hot 10% sodium chloride may also be used to extract RNA. The cells

usually have to be disrupted before using these methods. In some cases the protein

may then be extracted, purified and concentrated.

Single Cell Protein !#

has been used to hydrolyze yeast RNA at 80°C at which temperature the cells are

more permeable.

(d) Activation of endogenous RNA: The RNAase of the organism itself may be activated

by heat-shock or by chemicals. The RNA content of yeasts have been reduced in

this way.

15.5 NUTRITIONAL VALUE OF SINGLE CELL PROTEIN

The nutritional value of SCP depends on the composition of the microbial cells used

especially their protein, amino acid, vitamin, and mineral contents. These to some extent

also depend on the conditions of growth of the organism. The FAO has set up reference

values for the amino acid content of proteins. On this basis, SCP derived from bacteria

and yeasts is deficient in methionine. Glycine and methionine are sometimes deficient in

molds. These can be improved by supplementation with small amounts of animal

proteins.

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.

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.

Litch Field, J. 1994. Foods, Nonconventional. Kirk-Othmer Encyclopedia of Chemical

Technology 10.

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

Rehm, (ed) Biotechnology. 2

Edit VCH Weinheim Germany. pp. 221-240.

306 Modern Industrial Microbiology and Biotechnology

Yeasts have interacted with man from time immemorial – from the time when he first

learnt that fruit juices developed into intoxicating drinks and that the dough produced

from his ground cereal can be leavened, although he did not understand these two

phenomena. Today yeasts which are produced and used in all the six continents of the

world form the single most produced micro-organisms in terms of weight. The estimated

world production (excluding the Eastern European countries) for 1977 is given in Table

16.1, which are clearly underestimates in such areas as Middle East/Asia and Africa. In

the United States.

It would be safe to double the figures in the table as today’s estimated production.

Baker’s yeast is manufactured by six major companies in the United States. These

companies are Universal Foods (Red Star Yeast), Fleischmanns, Gist-brocades, Lallemand

(American Yeast), Minn-dak, and Columbia. There are 13 manufacturing plants owned by

these companies. Table 16.2 lists the locations of these plants by manufacturer.

Table 16.1 Estimated yeast production (dry weight, tons) in 1977

Region Baker’s Yeast Food and Fodder Yeast

Europe (excluding Eastern) 74,000 160,000

North America 73,000 53,000

Middle east/Asia Countries 15,000 25,000

United Kingdom 15,000 -

South America 7,500 2,000

Africa 2,700 2,500

The purpose for which yeasts are used and the types of yeasts employed for each

purpose are given in Table 16.2. Of these, the production of baker’s yeasts has received

the greatest attention, followed by food and fodder yeasts. Due to recent interest in the

production of single cell protein, food and fodder yeasts may become as important as

baker’s yeasts in terms of total quantity produced. The chapter will discuss the large-

scale production of baker’s, food and fodder yeasts.

16.1 PRODUCTION OF BAKER’S YEAST

The use of yeasts in bread making is an ancient art, although man did not always

recognize the mechanism of the rise of dough. It is of interest to give a brief historical

Yeast Production

CHAPTER

Yeast Production 307

account of the development of the yeast industry. The dough of leavened bread whose

antiquity is testified by biblical records, was probably raised by a mixture of yeasts and

lactic acid bacteria. A small piece of successful dough was used as the inoculum for the

next batch, providing a type of early continuous culture. This system of course has been

largely abandoned except in a special type of sour bread produced in San Francisco,

California, United States.

From about the Middle Ages, bakery yeasts were obtained from winemaking and

brewing. But the quality of the yeast was variable and in the case of yeast obtained from

beer the product was bitter because of the hops in the beer. This period lasted until the

latter part of the 19th century when the work of Pasteur from 1855 to 1857 elucidated the

nature of yeasts.

The first major step in the development of baker’s yeast technology can be said to be the

so-called Vienna process introduced about 1860 in which grain mash meant for

anaerobic alcohol production was gently aerated so that a good quantity of yeast was

obtained. Thus two birds were killed with one stone – yeasts and alcohol being obtained

in one operation even if the quantities were less than would be optimal if either one or the

other alone were sought. The work of Pasteur later led to more vigorous aeration, thus

yielding more cells and less alcohol. As a result of grain shortages resulting from World

War I a shift was made from the use of grain to the use of beet molasses, supplemented by

ammonia and phosphate.

Table 16.2 Yeast manufacturing plants in the United States

Company Location of Plant

Lallemand (American yeast) Baltimore, Maryland

Columbia Headland, Alabama

Fleischmanns Gastonia, North Carolina

Memphis, Tennessee

Oakland, California

Sumner, Washington

Gist-brocades Bakersfield, California

East Brunswick, New Jersey

Minn-dak Wahpeton, North Dakota

Table 16.3 Major uses of yeasts

Use Yeasts involved

1. Bakery Saccheromyces cerevisiae

2. Beer brewing Sacch. uvarum (Sacch cerevisiae) Scch. cerevisiae

3. Food yeasts and feed yeasts Candida tropicalis Candida pseudotropicalis

Candida utilis Sacch. cerevisiae

Kluyveromyces fragilis (Sacch. fragilis

4. Feed yeasts Candida lipolytica

5. Wine making Saccharomyces cerevisiae var. elliposoides

6. Wine making (sparkling wines) Sacch. bayanus

7. Industrial alcohol/spirits Sacch. cerevisiae (Sacch. fragilis with whey)

8. Yeast-products (antolysates, biochemicals) Sacch. cerevisiae

308 Modern Industrial Microbiology and Biotechnology

The next major step in the development of baker’s yeast technology was the

introduction of fed-batch or incremental addition of nutrients rather than the introduction

of all nutrients at the beginning, as is the case in the classical batch method. The essence

of this system known as the Zulaut method is still used today in baker’s yeast

manufacture and ensures that an excess of molasses sugar which might lead to alcohol

production is avoided.

Another important development, the production of dried active yeast, was

necessitated by the need to provide troops fighting in far off lands a means of producing

bread, instead of the compressed yeast normally used in temperate countries.

Today’s production methods for baker’s yeast do not allow alcohol production

because of the vigorous aeration used. Furthermore the yield has increased from 3% in the

mid-19

century through 13% early in this century to the present-day yield of over 50%

dry weight of yeast.

16.1.1 Yeast Strain Used

Non-sporulating ‘torula’ yeasts have occasionally been used for baking; nowadays

however specially selected strains of Saccharomyces cerevisiae are used. For some time two

strains of baker’s yeasts were available: one was highly active but had poor stability

during storage; the other had poor activity but was highly stable in storage. Successful

breeding program were then undertaken to produce new strains from them.

New large-scale factory process for bread-making used in Western countries (the

Chorleywood Bread Process) involving sophisticated plants and computerization have

led to new demands on traditionally used yeasts. These new demands include the

fermentation of more complex sugars, high initial fermentation ability, faster adaptation

to maltose fermentation, and ability to reconstitute rapidly when prepared in the active

dry form. Yeast strains used for the modern fast-rising dough have been developed with

the following traditional and new physiological properties in mind.

(a) ability to grow rapidly at room temperature of about 20-25°C;

(b) easy dispensability in water;

in flour dough, rather than alcohol;

(d) good keeping quality, i.e., ability to resist autolysis when stored at 20°C;

(e) high potential glycolytic activity;

(f) ability to adapt rapidly to changing substrates;

(g) high invertase and other enzyme activity to hydrolyze the higher glucofructans

rapidly;

(h) ability to grow and synthesize enzymes and coenzymes under the anaerobic

conditions of the dough;

(i) ability to resist the osmotic effect of salts and sugars in the dough;

(j) high competitiveness i.e. high yielding in terms of dry weight per unit of substrate

used.

In many Eastern European countries no special yeasts are produced to cope with the

newer baking techniques mentioned above. Baking yeasts are not therefore produced

specially and a type of Vienna process is used, the yeast being obtained from grain mash

fermented for alcohol distillation.

Yeast Production 309

16.1.2 Culture Maintenance

The specially selected baking strains of Saccharomyces cerevisiae are apt to mutate and

therefore proper storage is most important. Of the various methods used, storage in liquid

nitrogen and the oil culture method in which sterile oil is placed over a slant of yeast and

refrigerated at 4°C are most widely used. Freeze drying is not highly used as it induces

loss of viability in many yeasts and a tendency towards mutation.

16.1.3 Factory Production

16.1.3.1 Substrate

The substrate usually used for baker’ yeast production is molasses. Where these are not

available or are too expensive any suitable sugar-containing substrate e.g. corn steep

liquor may of course be used. In the Soviet Union for example sulphite liquor is used for

both alcohol and baker’s yeast production. Ethanol has been used in laboratory studies,

but is yet to be used on a large scale.

Beet and cane molasses, when they are simultaneously available, are treated

separately: clarified, pH adjusted and sterilized. They are then mixed in equal amounts

so that the nutritional deficiency of one type is made up by the other (Chapter 4). Cane

sugar is particularly richer in biotin, panthothenic acid, thiamin and magnesium and

calcium; while beet molasses is much richer in nitrogen. Molasses composition is

however not constant and varies with the geographical area of growth, the factory

extraction of the sugar and other factors. When only one type of molasses is available,

deficiencies are made up by adding appropriate nutrients.

The molasses is clarified to remove inert colored material arising from colloidal

particles and which can impart undesirable color to the yeast. Clarification may be

achieved by precipitation with alum or calcium phosphate or by poly-electrolyte

flocculating agents such as alginates and polyacrylamides. Clarification also helps

reduce foaming. ‘Sterilization’ is achieved by heating at 100°–110°C for about an hour,

after the pH has been adjusted to pH 6-8 to prevent caramelization of the sugar.

Phosphorous, ammonium and smaller amounts of magnesium, potassium, zinc, and

thiamin are added for maximum productivity to the mixed molasses. Antifoam is

sometimes added.

16.1.3.2 Fermentor processes

The fermentor for baker’s yeast propagation is nowadays made of stainless steel. The

trade fermentor (i.e. the final fermentor) may be anything from 75 to 225 cubic meters. Of

this about 75% is occupied by the medium, the unused space being allowed for foaming.

The typical stirred tank fermentor with agitator baffles and sparging is not often used in

yeast growth because of the high initial and operating cost. Generally, baker’s yeast

fermentors are aerated only by spargers which are so arranged that large volumes of air

pass through per unit time: about one volume of air per volume of broth per minute.

Spargers of different types are available. It is most important that aeration be high and

constant. When the oxygen falls below 0.2 ppm anaerobic conditions set in and alcohol is

formed.