DDM 321 – Food and Industrial Microbiology

Fermentation – Upstream
Process

I.Manikkavasagan
Assistant Professor
Department of Food Safety and Quality Assurance
College of Food and Dairy Technology
Koduveli, Chennai – 600 052
 Upstream Process
Steps of upstream process
⮚ Industrially important microbes - Primary screening,
preparation of pure culture and selection of production strain at
lab scale level. Secondary screening and optimization of
production parameter of production strain at lab and pilot scale
⮚ Preservation of Microorganisms – to eliminate genetic change,
protect against contamination, and retain viability.
⮚ Strain improvement - Genetic modification of production strain
if required
⮚ Production media and inoculum development - optimization of
growth parameters at lab scale for product formation. The
production of sufficient pure and active microbial culture for
the inoculation in production vessel.
⮚ Sterilization - The sterilization of the inoculum and production
medium. Sterilization of fermenter and its auxiliary equipment.
Screening of industrially important microorganisms
• The tasks of both discovering the new microbial compounds
and improving the synthesis of known strains have become
more and more tedious.
• The microorganisms of industrial importance are generally,
bacteria, actinomycetes, fungi and algae.
• These organisms occur virtually everywhere, e.g., in air, water
and soil, on the surfaces of plants and animals, and in plant and
animals tissues. But most common sources of industrial
microorganisms are soils, lake and river mud.
• There are three important stages in the screening strategies
namely isolation, preservation and improvement of the strains
of commercial importance
Criteria used for choice of organisms
⮚ The nutritional characteristics of the organism: Organism
should be capable to utilize the ingredients present in the
medium to produce interested product.
⮚ The optimum temperature of the organisms: For instance, the
use of an organism having an optimistic temperature above
40oC considerably reduces the cooling costs of a large-scale
fermentation, and therefore, the use of such a temperature in
the isolation procedure may be beneficial
⮚ The reaction of the organism with the equipment to be
employed
⮚ The stability of the organism and its amenability to genetic
manipulation
⮚ The productivity of the organism, measured in its ability to
convert substrate into product and to give a high yield of
product per unit time.
⮚ The easy product recovery from the cultures.
⮚ It should be a high yielding strain
⮚ It should have stable biochemical characteristics
⮚ It should not produce undesirable substances
⮚ It should be easily cultivated on a large scale
⮚ The ideal isolation procedure commences with an
environmental source (frequently soil), which is highly
profitable to be rich in the desired types
⮚ Selective pressure may be used in the isolation of organism
that will grow on particular substrates in the presence of certain
compounds or under agricultural conditions adverse in their
types
⮚ If it is not possible to apply selective pressure for the
desired character it may be possible to design a procedure
to select for a microbial taxon which is known to show the
characteristics at a relatively high frequency. E.g. the
production of antibiotic by Streptomycin.
⮚ Alternately, the isolation procedure may be designed to
exclude certain microbial “weeds” and to encourage the
growth of more novel types
⮚ The advantages in the taxonomic description of taxa have
allowed the rational design of procedures for the isolation
of strains that may have a high probability of being
productive or are representatives of unusual groups.
⮚ The advances in pharmacology and molecular biology have
also enabled the design of more effective screening tests to
identify productive strains amongst the isolated organisms.
Primary screening
⮚ “Primary screening allows the detection & isolation of
microorganisms that possess potentially interesting
industrial application”
⮚ Primary screening separate out only a few microorganisms
having real commercial value.
⮚ Primary screening determines which microorganisms are
able to produce a compound without providing much idea
of the production or yield potential of the organisms
A. Primary screening of organic acid producing
microorganisms
⮚ Incorporation of a pH indicating dye such as neutral red or
bromothymol blue into a poorly buffered agar medium.
⮚ Greater buffer capacity of medium screen microbes having
capability to produce considerable quantities of the acid
⮚ Incorporation of calcium carbonate in the medium is also
used to screen organic acid producing microbes on the
basis cleared zone of dissolved calcium carbonate around
the colony
⮚ These screening approaches do not give idea that which
organic acid has been produced
⮚ Thus the colonies of microorganisms showing the potential
to produce any fermentation product should immediately
be purified and sub-cultured into appropriate medium to
be maintained as stock cultures for further testing.
B. Primary screening of antibiotic producing microorganisms
⮚ The simplest screening technique for antibiotic producers is
:Crowded Plate” technique
⮚ The technique is used to find out the microorganisms that
produce an antibiotic without giving much information of
sensitivity towards other microorganisms.
⮚ Procedure include dilution and spreading or pouring of soil
samples that give 300 or 400 or more colonies per plate
⮚ Colonies producing antibiotic activity are indicated by an
area of agar around the colony
⮚ Such a colony is sub-cultured to a similar medium and
purified by streaking, before making stock cultures. The
purified culture is then tested to find what types of
microorganisms are sensitive in the presence of these the
antibiotics i.e. “Microbial Inhibition Spectrum” (MIS).
⮚
⮚ The crowded plate procedure also does not necessarily select
an antibiotic producing microorganism, because the inhibition
area around the colony sometimes can be due to other
reason like….
⮚ (1) Marked change in the pH of the medium resulted due the
metabolism of the colony.
⮚ (2) Rapid utilization of critical nutrients in the vicinity of the
colony etc.
⮚ Thus further testing is required to confirm the inhibitory
activity associated with a microorganisms is whether
Secondary screening
Secondary screening allows further sorting out of microorganisms
obtained from PS having real value for industrial processes and
discarding of those lacking this potential
⮚ SS is conducted on agar plates, in flasks or small fermenter
containing liquid media
⮚ SS can be qualitative or quantitative in its approach
⮚ Secondary screening should give information about the
evaluation of the true potential of the microorganisms for
industrial usage
⮚ SS should determine whether microorganisms are actually
producing new chemical compounds not previously described
⮚ SS should reveal whether there is pH, aeration or other critical
requirements associated with particular microorganisms,
both for the growth of the organism and for the formation of
chemical products
⮚ SS should also detect gross genetic instability in microbial
cultures
⮚ SS should show whether certain medium constituents are
missing or possibly, are toxic to the growth of the organisms
or its ability to accumulate fermentation products
⮚ SS should determine whether the product has a simple,
complex, or even a macromolecular structure, if this
information is not already available
⮚ SS should show something of the chemical stability of the
product and of the product’s solubility picture on various
organic solvents
⮚ SS should show whether the product possesses physical
properties such as UV light absorption or fluorescence or
chemical properties that can be employed to detect the
compound during the use of paper chromatography or other
analytical methods and which also might be of value in
predicting the structure of the compound
⮚ In some case, for certain kinds of fermentation product
determinations should be made as to whether gross animal,
plant or human toxicity can be attributed to the fermentation
product, particularly if it is utilized (as are antibiotics) in
disease treatment
⮚ SS should reveal whether a product resulting from a microbial
fermentation occurs in the culture broth in more than one
chemical form and whether it is an optically or biologically
active material
⮚ SS should reveal whether the microorganisms are able to
chemically alter or even destroy their own fermentation
products
⮚ Secondary screening helps in predicting the approaches to be
utilized in conducting further research on the microorganisms
and its fermentation processes.
Strategies for isolation of industrially important microbes
⮚ The diversity of microorganisms may be exploited still by
searching for strains from the neutral environment able to
produce products of commercial value
⮚ The first stage in the screening of microorganisms of potential
industrial is their “isolation”
⮚ Isolation involves obtaining either pure or mixed cultures
followed by their assessment to determine which carry out the
desired reaction or produce the desired product
⮚ In some cases it is possible to design the isolation procedure in
such a way that the growth of producers is encouraged or that
they may be recognized at the isolation stage, whereas in other
cases organisms must be isolated and producers recognized at
a subsequent stage
⮚ It should be remembered that the isolate must carry out the
process economically and therefore the selection of the culture
Preservation techniques
⮚ The isolated cultures can be stored by various methods Storage at
reduced temperature
⮚ If the cultures are subcultured at 6-month interval, it can be stored
on agar slopes in refrigerator at 4°C or in freezer at -20°C. The time
of subculture may be extended to 1 year if the cultures are covered
with mineral oil For the long term preservation of cells that do not
survive freeze drying, liquid nitrogen (-156°C to-196°C) is the
suitable method for preservation of valuable stock cultures. This
technique involves growing a culture to the maximum stationary
phase and then resuspending the cells in a cryoprotective agent
such as 10% glycerol
⮚ Storage in a dehydrated form - Dried soil cultures are commonly
used especially for sporulating mycelia organisms
⮚ Lyophilization technique is used to store the culture in refrigerator
for 10 years. This is done by freezing the culture followed by its
drying under vaccum leading to the sublimation of water
⮚ The industrial cultures must be stored to eliminate genetic change,
protect against contamination and retain viability.
Strain Improvement
⮚ Wild type microbes usually produce a very low compound titre.
Therefore, In order to increase the productivity of the wild
strain, various improvement strategies are adopted.
⮚ Traditional methods for strain improvement are random
mutagenesis and selection. The selected strains are usually
subjected to successive cycles of mutagenesis and selection;
after several cycles, a large increase is yield is likely to be
obtained. Such a random approach is often a good start and
sufficient to generate a high producing strain.
⮚ Increased yields may be achieved by optimizing the culture
medium and growth conditions, but this approach will be
limited by the organism’s maximum ability to synthesize the
product. The potential productivity of the organisms is
controlled by its genome, therefore, the genome must be
modified to increase the potential yield.
⮚ The techniques and approaches used to genetically modify
strains, to increase the production of the desired product
are called strain improvement or strain development.
⮚ Genetic modification may be achieved by selecting
natural variants, by selecting induced mutants and by
selecting recombinants. There is a small probability of a
genetic change occurring each time when a cell divides,
hence a microbial culture will undergo a vast number of
such divisions, the culture will become more
heterogeneous.. However, variants have been isolated
which are superior producers and this has led to the
development of a natural product from a newly isolated
organism. Recombinant DNA technology has enabled the
production of heterologous products and has built on the
achievements of directed selection to increase yields of
conventional products still further.
Media for Industrial Fermentation
Characteristics of an Ideal production medium.
• Chemical composition: the production medium must have a
suitable chemical composition. Medium should contain a source
of carbon, a source of Nitrogen, growth factors and mineral salts.
• Precursors: In certain fermentations, the medium should supply
the required precursor for better yields of a desirable product.
• Buffering capacity: Maintenance of the pH in the optimum range
is necessary for making the process successful, since acidic and /or
basic compounds depending on the nature of the fermentation
process accumulate during the progress of the fermentation. To
control the pH of the medium, buffers should be added to the
medium (E.g. CaCO3). Media containing considerable quantities of
proteins, peptides, and amino acids possess good buffering
capacity in the pH range near neutrality. Additional buffering
capacity in this pH range also provided by phosphates (Mono and
dihydrogen potassium or Sodium phosphates).
• Avoidance of Foaming: Foaming is a serious problem in
fermentation industry, since it may help in contaminating the
fermentation medium and also causes other problems for the
fermentation. Hence defoamers (e.g. hard oil mixed with
octadecanol for penicillin fermentations) should be used for
controlling foam. These defoamers are added to the
production medium before sterilization or incorporated after
sterilization or added during the fermentation.
• Toxicity: the ideal production medium free from any toxic
effect on culture or product formation.
• Consistency: In aerobic fermentations, it is necessary to supply
sterile air into the medium. Under such circumstances, liquid
media allow the diffusion of air throughout the medium under
agitation. Fermentation media should not be viscous. Viscous
nature of the medium creates difficulty in the penetration of
the air interior of the medium. Air is not easily absorbed by the
liquid medium.
• Contamination: Certain conditions of the production
medium are helpful to check the contamination. For
example low pH values in citric acid production using
Aspergillus niger avoids contamination. Media having low
pH values may be sterilized at low temperatures.
• Recovery: Recovery of the desired product is an important
criteria. Components of the medium should be such that
separation and extraction of the product becomes easy and
cheaper.
• Availability of raw materials: Raw materials required for
designing of the production medium should be freely
available in large quantities at a reasonable price.
Raw materials as Media:
• Different types of raw material are used in different types of
industrial fermentation processes. Usually crude nutritive sources
are preferred, since they are economical. Mostly agricultural
products are utilized as a source of raw material in fermentation
industries.
• SACCHARINE MATERIALS: Sugar cane, sugar beets, molasses, and
fruit juices may be included in this category.
• Molasses: Molasses is a byproduct of the cane and beet sugar
industry. It is recovered at any one of several stages in the sugar
refining process. Chemical composition of sugarcane black strap is
variable. It depends on the quality and variety of the cane but also
on the process involved in the manufacture of sugar. About 95%of
the total sugar in cane molasses is fermentable. It is particularly rich
in biotin, pantothenic acid, thiamine, phosphorous and sulphur. The
organic nitrogen content is less than beet molasses, since it does
not contain betaine. Bu this substance is not assimilated by yeasts.
• Beet molasses are produced by the same processes employed
for cane molasses. Vitamins such as biotin, pyridoxine,
thiamine, pantothenic acid and inositol are also present. Beet
molasses have limited biotin. Therefore, in fermentations
involving yeast culture, a small amount of cane black strap
molasses or other biotin supplying material should be
incorporated in the production medium. Because yeast require
biotin for their growth. The largest utilization of cane black
strap molasses in India is in the alcohol industry, which utilizes
it for the manufacture of spirit, country liquors, rum, brandy,
gin and whisky.
• Fruit juices: Fruit juices contain soluble sugars. Grape juice
contain glucose 7 fructose. Therefore, fruit juices can be used
as a source of carbon in fermentation industries. Grapes are
used in the production of wine.
Cheese way: The straw coloured liquid produced as a
byproduct of cheese making is called cheese whey. It is a
major waste product for the cheese industry. It cannot be
disposed of without proper treatment. Therefore, it is
desirable to use it for useful products. It is also used as pig
feed. For lactic acid production and SCP production it is served
as raw material because it contains lactose, nitrogenous
substances including vitamins (e.g. vitamins) and inorganic
salts.
STARCHY MATERIAL: There are two main sources of
commercial starches
1. Cereals (Wheat, rice, maize)
2. Roots & tubers (potatoes, tapioca e.t.c)
• The moisture content of the grain is low where as that of
roots and tubers are very high. Starches require
pretreatment to bring about the conversion to fermentable
CELLULOSIC MATERIALS:
• Cellulosic materials are complex carbohydrate materials.
The cellulosic molecule is made up of the repeating units of
β-glucose. The formation of β -cellobiose requires two
molecules of β-glucose, which are linked through α 1, 4-
linkage. 1000 to 10,000 units of cellobiose are required to
form a simple linear polymer called cellulose. Units of
cellobiose are joined end to end through 1, 4-β - glucosidic
linkages. For this reason cellulosic materials require some
sort of pretreatment.
• Cellulosic materials are Sulfite waste liquor, wood molasses
and Rice straw.
Sulfite waste liquor:
⮚ In the manufacture of paper pulp, wood is subjected to hydrolysis
which is brought about with the help of Calcium bisulfite under
heat and pressure. This operation is called digestion process. At the
end of this process, the spent liquid is left and it is referred to as
sulfite waste liquor. It cannot be disposed of unless it is properly
treated.
⮚ Sulfite waste liquor contains 10 to 12 percent solids, of which sugars
make up about 20%. It contains sugars in the form of hexoses and
pentoses.
⮚ It is used in the industrial production of ethyl alcohol using
Saccharomyces cerevisiae and in the growth of Torula utilis cells for
animal feed. Saccharomyces cerevisiae requires hexoses whereas
Torula utilis requires both hexoses and pentoses.
⮚ Sulfite waste liquor cannot used directly as fermentation medium. It
contains free sulfur dioxide or sulfurous acid which is toxic to
microorganisms. These toxicants are removed by steam stripping or
precipitation with lime.
Wood molasses:
⮚ It is produced by acid hydrolysis of wood cellulose itself. This may
produce 65-85% fermentable sugars. Sulphuric acid of about 0.5%
concentration is used at a temperature range of 150 to 185oC.
Using a continuous process a syrup may be obtained from saw
dust. This syrup may contain 4 to 5% reducing sugars (a mixture of
glucose and pentoses) with an overall yield of 45 to 55%. It may
be subjected to concentration to give a kind of wood molasses.
Rice Straw:
⮚ Rice straw and related agricultural materials can serve as a good
source of cellulose. It is a poor quality animal feed in its natural
state because of its bulkiness, poor palatability, low protein
content and low digestibility. Numerous microorganisms are
capable of using cellulose for their growth. Rice straw has been
used as a fermentation medium in the production of silage and
single cell protein (SCP), mushroom cultivation e.t.c.
Hydrocarbons & Vegetable Oils:
⮚ Hydrocarbons used as fermentation substrates are usually
mixtures of various hydrocarbon components. These
fermentation raw materials are relatively cheap. However,
purified hydrocarbon fractions or hydrocarbon compounds
are more expensive.
⮚ Hydrocarbon substrates (e.g. gas oil and n-paraffins) are
used to produce single cell protein (SCP) products. In this
way biomass of yeasts (e.g. Candida lipolytica, Candida
kofuensis, Candida tropicalis) can be produced on a
significant scale under aerobic conditions.
Vegetable oils: Oils obtained by deoiling of vegetable seeds are
called vegetable oils. On the basis of their degree of
unsaturation, they may be grouped into following three major
classes:
⮚ a. Oleic (or ‗non-drying‘ type): these include olive and
groundnut oils.
⮚ b. Linoleic (or ‗semi drying‘ type): these have a higher content
of the double unsaturated fatty acid found in maize, sunflower
and cotton seed oils
⮚ c. Linolenic acid (or ‗drying type): These include linseed and
soya bean oils containing a fatty acid with three double bonds.
⮚ These oils may undergo drying if exposed to the atmosphere
due to the oxidation of the unsaturated components.
Commercial vegetable oils (e.g. maize oil) may be used in
conjunction with surface active agent as anti-foams or alone
as a nutrient source of carbon.
Nitrogenous Materials:
⮚ Corn steep liquor(CSL): The used steep water results from
the steeping of corn during the manufacture of starch,
gluten and other corn products this by product is subjected
to concentration to approximately 50% solids and this
concentrate is called corn steep liquor. Corn steep liquor
was originally found to be useful for penicillin production
specifically. But, it is now recognized as valuable in many
fungal antibiotic fermentation media. In addition to this, it
is also used in the manufacture of food stuffs.
⮚ Soya bean oil: the material left after deoiling of the soya
bean seeds is called soya bean meal. Soya bean meal
contains approximately 8% w/w nitrogen. This differs from
corn steep liquor, since soya bean meal is a much more
complex nitrogenous source than corn steep liquor, and
therefore not readily available to microbes. This is used as a
ingredient for fermentation media in the production of
streptomycin.
⮚ Pharmamedia: Pharmamedia is a clean, yellow, finely
ground powder prepared from the embryo of cotton seed.
It contains 56% w/w protein, 24% carbohydrate, 5% oil, and
5% ash. Ash, in turn, contains calcium, iron, chloride,
phosphorous and sulfate. It is used as an ingredient for
production media (e.g. Tetracycline production).
Distillers Solubles:
⮚ In the manufacture of alcohol using grain or maize, alcohol is
distilled from fermented grain or maize, leaving the residue
(containing 6 to 8% w/v total solids). The suspended solids
from the residue are eliminated by screening, leaving the
effluent. Thereafter effluent is subjected to concentration, until
the solid content reaches 35% w/v giving evaporator syrup‘.
This syrup is then drum dried to yield distillers solubles‘. This
may be used as a production medium component, since it
supplies nitrogen, together with many accessory food factors
(e.g. vitamin B complex).
⮚ Other natural organic nitrogenous materials are ground nut
meal, fish meal, bacto peptone, Difco yeast extract.
Precursors & Inducers:
⮚ Certain substances, which generally improves the yield or
quality of the product. These substances are known as the
precursors. These precursors are incorporated without any
major change in to the molecule of the fermentation product.
⮚ Ex: Phenyl acetic acid and Cobalt are being added for penicillin
G and Vitamin B12.
⮚ Corn steep liquor yields various pencillins but addition of
phenyl acetic acid determines the penicillin G production.
⮚ Proteases for various proteins, -amylases for starch, cellualse
for cellulose, pectinase for pectin and penicillin acylase for
phenyl acetic acid.
Repressors:
⮚ The substances which are being employed for the repression
of the industrial cultures are known as repressors.
⮚ Example 1. Media allowing restricted growth provides high
product yield as major portion of carbon and other
components of the medium are shunted to product
formation rather than to growth.
⮚ 2. Nitrogen sources such as soyabean meal and praline for
streptomycin; production is probably due to their slow
utilization, thus avoiding nitrogen metabolite repression.
⮚ 3. Aspergillus niger for gluconic acid production is first grown
on a medium that supports a rich growth as well as product
formation, then the mycelium is separated and placed in a
fresh medium high in carbon substrate (sugar) but lacking
combined nitrogen so that additional growth cannot occur.
Antifoams: Antifoams are surface active agents, reducing the
surface tension in the foams and destabilizing protein films by
hydrophobic bridges between two surface, displacement of
absorbed protein and rapid spreading on the surface of the film.
⮚ An ideal antifoam should have a fast action on the existing foam
but should not be metabolized by the microorganisms. It should
be cheap, heat sterilizable, non toxic, long acting and active at
low concentrations.
⮚ Examples: Stearyl alcohol and octyl decanol, esters, fatty acids,
cotton seed oil, linseed oil, castor oil, cod liver oil etc, silicones,
sulphonates.
⮚ If the oxygen transfer rate is severely affected by antifoam
addition, then mechanical foam breakers may have to be
considered as a possible alternative.
 Inoculum Development
The Development of Inocula for Bacterial Processes
• The main objective of inoculum development for traditional
bacterial fermentations is to decrease lag phase.
• A long lag phase is not only is wastage of time but also medium
consumed in maintaining a viable culture prior to growth.
• The size of the inoculum and its physiological condition affect
the length of the lag phase.
• Bacterial inocula should transfer when the cells are still
metabolically active.
• The age of the inoculum is particularly important in the growth
of sporulating bacteria, for sporulation induced at the end of
the logarithmic phase and the use of an inoculum containing a
high percentage of spores would result in a long lag phase in a
successive fermentation.
⮚ The commercial production of proteases uses five percent
inoculum of thermophilic Bacillus in logarithm phase.
⮚ A two-stage inoculum development programme of Bacillus
subtilis used for the production of proteases. Inoculum for a
seed fermenter was grown for 1 to 2 days on a solid or liquid
medium and then transferred to a seed vessel where the
organism was allowed to grow for a further ten generations
before transfer to the production stage.
⮚ The lag phase in plant fermenters eliminated by using
inoculum medium of the same composition as used in the
production fermenter and employing large inocula of actively
growing seed cultures in the production of bacterial enzymes.
⮚ Inoculum development programme at pilot- scale for the
production of vitamin B12 from Pseudomonas denitrificans
shown below (Spalla et al., 1989).
• The acetic-acid bacteria used in the vinegar process are
extremely sensitive to oxygen starvation therefore, it is
essential to use an inoculum in an active physiological state.
• The cells at the end of fermentation are use as inoculum for
the next batch by removing approximately 60% of the
culture and restoring the original level with fresh medium.
• In this process, there are enough chances of strain
degeneration and contaminant accumulation.
• However, strain stability is a major concern in inoculum
development for fermentations employing recombinant
bacteria.
• Plasmid stability and productivity in E. coli biotin
fermentation improved if stationary, rather than
exponential phase, cells used as inoculum due to loss of
plasmid in fermentation.
• In the lactic-acid fermentation, lactic acid inhibits the
production organism. Thus, production of lactic acid in the
seed fermentation may result in generation of poor quality
inoculum.
• High quality inoculum of Lactococcus lactis 10-1 on a
laboratory scale obtained using electrodialysis, which
reduced the lactate in the inoculum and reduced the length
of the lag phase in the production fermentation.
Development of Inocula for Anaerobic Bacterial Processes
• Clostridial Acetone-Butanol fermentation is anaerobic
process.
• However, the process was outcompeted by the
petrochemical industry but there is still considerable
interest in reestablishing the fermentation.
The inoculum development programme described by McNeil
and Kristiansen (1986) given as below
• Heat-shocked spore suspension inoculated into 150 cm3 of
potato glucose medium
• Stage 1 culture used as inoculum for 500 cm3molasses
medium
• Stage 2 culture used as inoculum for 9 dm3 molasses
medium
• Stage 3 culture used as inoculum for 90,000 dm3 molasses
medium
• The stock culture is heat shocked to stimulate spore
germination and to eliminate the weaker spores.
• The production stage inoculated with a very low volume.
• The use of such small inocula necessitates the achievement
of as near perfect conditions as possible to prevent
contamination and to avoid an abnormally long lag phase.
The Development of Inocula for Yeast Processes
• Industrial uses of yeasts are
• 1 The brewing of beer
• 2 The production of Baker’s Yeast (biomass) and
• 3 For the production of recombinant products from the
yeast
Brewing
• Yeast used to inoculate a fresh batch of wort from previous
fermentation or from propagator.
• It is common practice in the British brewing industry to use
the yeast from the previous fermentation.
• The brewing terms used to describe this process and, 'crop'
referring to the harvested yeast from the previous
fermentation and 'pitch' meaning to inoculate.
• One of the major factors contributing to the continuation of
this practice is the wort-based excise laws in the United
Kingdom where duty charged on the sugar consumed
rather than the alcohol produced.
• Thus, dedicated yeast propagation systems are expensive to
operate because duty charged on the sugar consumed by
the yeast during growth.
• The problems with this technique are chances of
contamination and degeneration of strains the most
common problem with the degenerated cell is the change
in the degree of flocculence and weakening of abilities of
the yeast.
• In breweries employing top fermentations in open fermenters,
the above dangers minimized by collecting yeast to be used for
future pitching from 'middle skimmings’'.
• As the head of yeast develops the surface layer, (the most
flocculent and highly contaminated yeasts) removed and
discarded and the underlying cells (the 'middle skimmings')
harvested and used for subsequent pitching.
• Therefore, the 'middle skimmings' contain cells which have the
desired flocculence and which have been protected from
contamination by the surface layer of the yeast head.
• The pitching yeast may be treated to reduce the level of
contaminating bacteria and remove protein and dead yeast
cells by such treatments as reducing the pH of the slurry to 2.5
to 3, washing with water, washing with ammonium persulphate
and treatment with antibiotics such as,polymixin, penicillin and
neomycin.
• However, traditional open vessels are becoming rare and the
bulk of beer brewed using cylindro-conical fermenters.
• In these systems, the yeast flocculates and collects in the cone
at the bottom of the fermenter where it is subject to the
stresses of nutrient starvation, high ethanol concentration, low
water activity, high carbon dioxide concentration and high
pressure, which decreases the viability and physiological state
of the yeast crop, would not be ideal for an inoculum.
• The situation is further complicate by the fact, that the
harvested yeast is stored rapidly to about one degree before it
is used as inoculum suspending in beer and storing in the
absence of oxygen.
• One of the key physiological features of yeast inoculum is the
level of sterol in the cells. Sterols are required for synthesis of
membrane but they are only produce in the presence of
oxygen.
• Thus, we have the irregularity of oxygen being required for
sterol synthesis yet anaerobic conditions are required for
ethanol production.
• This irregularity is resolved traditionally by aerating the wort
before inoculation.
• The difficulties outlined above and the likelihood of strain
degeneration and contamination mean that rarely used for more
than five to ten consecutive fermentations that necessitates the
periodical production of a pure inoculum.
• Pure inocula prepared by a yeast propagation scheme utilizing a
ten percent of inoculum volume at each stage in the programme
and employing conditions similar to those used during brewing.
• Continuous aeration used during the propagation stage, which
seems to have little effect on the beer produced in the
subsequent fermentation.
• Yeast inoculum produced in this way would also be sterol rich
obviating the need for aerated wort.
• The simplest type of propagator is a single stage system
resembling an unstirred aerated fermenter, which
inoculated with a shake-flask culture developed from a
single colony.
• Two-stage systems propagator operated semi-continuously.
It consisted of two linked vessels one point five and one
fifty cubic decimeters respectively.
• The smaller vessel filled with wort sterilized, cooled,
aerated, and inoculated with a flask-grown culture. After
growth for three to fourdays, the culture forced by air
pressure into the second vessel, which, filled with, sterilized
cooled wort and aerated.
• After mixing an aliquot of 1.5 dm3in second vessel, it is
force back into the first vessel. In a further 3 to 4 days, the
larger vessel contained sufficient biomass to pitch a
thousand cubic decimeters fermenter and the first vessel
contained sufficient inoculum for another second stage.
• However, although this procedure should produce a pure
inoculum there is a danger of strain degeneration occurring
in such a semi-continuous system.
• Baker's Yeast
• The commercial production of bakers' yeast involves the
development of an inoculum through a large number of
aerobic stages.
• Although the production stages, of the process, may not
operate under strictly aseptic conditions, a pure culture is
use for the initial inoculum thereby keeping contamination
to a minimum in the early stages of growth.
• The development of inoculum for the production of
bakers' yeast involve eight stages the first three
being aseptic while the remaining stages were
carried out in open vessels.
• The yeast pumped from one stage to the next or the
seed cultures may be centrifuge and washed before
transfer, which reduces the level of contamination.
• The yields obtained in the first five stages are
relatively low because they are not fed-batch
systems whereas the last three stages are fed-batch.
Thank You