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FERMENTATION TECHNOLOGY
FERMENTATION
Microbiologists consider fermentation as 'any
process for the production of a product by means of mass
culture of micro-organisms'.
Biochemists consider fermentation as 'an energygenerating process in which organic compounds act both
as electron donors and acceptors'; hence fermentation is
an anaerobic process where energy is produced without
the participation of oxygen or other inorganic electron
acceptors.
Moreover,
Fermentation is
usually
defined
as
a metabolic process
that
converts sugar to acids, gases and/or alcohol. It is also one method by which organisms derive
their energy when oxygen is lacking. It occurs in yeast and bacteria, but also in oxygen-starved
muscle cells, as in the case of lactic acid fermentation. The science of fermentation is known
as zymology.
Fermentation Process
Cellular Derivation of Energy
Once the glucose enters cytosol, Glycolysis begins. Glycolysis is a metabolic pathway
that breaks down glucose.
The first step, glycolysis, is common to all fermentation pathways:
C6H12O6 + 2 NAD+ + 2 ADP + 2 Pi 2 CH3COCOO + 2 NADH + 2 ATP + 2 H2O + 2H+
Pyruvate is CH3COCOO. Pi is phosphate. Two ADP molecules and two Pi are converted
to two ATP and two water molecules via substrate-level phosphorylation. Two molecules
of NAD+ are also reduced to NADH.
If oxygen is available Cellular Respiration (oxidative phosphorylation) will proceed
otherwise Fermentation allows glycolysis to continue.
In fermentation process, it turns the NADH and pyruvate produced in the glycolysis step
into NAD+ and various small molecules.
Fermentation is employed for preservation in a process that produces lactic acid as found in
such sour foods (food processing), as well as for producing alcoholic beverages such
as wine (fermentation in winemaking) and beer. Fermentation can even occur within the
stomachs of animals, such as humans.
Types of Fermentation
Fermentation reacts NADH with an endogenous, organic electron acceptor. Usually this is
pyruvate formed from the sugar during the glycolysis step. During fermentation, pyruvate is
metabolized to various compounds through several processes:
Ethanol
Fermentation,
aka
alcoholic
fermentation,
is
the
production
of ethanol and carbon dioxide. It occurs mostly in yeast and it is used to produce
alcoholic beverages such as wine.
Lactic Acid fermentation, produces lactic acid and it occurs in most organism including
humans. It is used to produce beverages such as buttermilk and food like cheese and
yogurt.
There are two means of producing lactic acid:
1. Homolactic Fermentation is the production of lactic acid exclusively
2. Heterolactic fermentation is the production of lactic acid as well as other acids and
alcohols.
Chemistry of Ethanol Fermentation
The chemical equation below shows the alcoholic fermentation of glucose,
whose chemical formula is C6H12O6. One glucose molecule is converted into
two ethanol molecules and two carbon dioxide molecules:
C6H12O6 2 C2H5OH + 2 CO2
C2H5OH is the chemical formula for ethanol.
Procedure
During glycolysis, the energy from the exothermic reaction is used to bind inorganic
phosphates to ADP and convert NAD+ to NADH. The two pyruvates are then broken down into
two acetaldehydes and give off carbon dioxide as a waste product. The two acetaldehydes are
then converted to two ethanol by using the H ions from NADH which is converted back to NAD+.
Chemistry of Lactic Acid Fermentation
Lactic Fermentation happens in our muscle cells this is what causes our muscles to burn
during hard exercise.
Procedure
From glycolysis, Pyruvate and NADH enter the fermentation process. Two NADH
molecules provide energy to convert pyruvate into lactic acid. As NADH is used, it is converted
back to NAD+ . Two molecules of NAD+ are recycled back to glycolysis. The recycling of NAD+
allows glycolysis to continue.
Homolactic Fermentation (producing only lactic acid) is the simplest type of

fermentation. The pyruvate from glycolysis undergoes a simple redox reaction,
forming lactic acid. It is unique because it is one of the only respiration processes to not
produce a gas as a byproduct. Overall, one molecule of glucose (or any six-carbon
sugar) is converted to two molecules of lactic acid: C 6H12O6 2 CH3CHOHCOOH
It occurs in the muscles of animals when they need energy faster than the blood can
supply oxygen. It also occurs in some kinds of bacteria (such as lactobacilli) and
some fungi. It is this type of bacteria that converts lactose into lactic acid in yogurt,
giving it its sour taste. These lactic acid bacteria can carry out either homolactic
fermentation, where the end-product is mostly lactic acid.
Heterolactic Fermentation, where some lactate is further metabolized and results in

ethanol and carbon dioxide, acetate, or other metabolic products, e.g.: C 6H12O6
CH3CHOHCOOH
+
C2H5OH
+
CO2
If lactose is fermented (as in yogurts and cheeses), it is first converted into glucose and
galactose (both six-carbon sugars with the same atomic formula): C 12H22O11 + H2O 2
C6H12O6
Heterolactic fermentation is in a sense intermediate between lactic acid fermentation,
and other types, e.g. alcoholic fermentation. The reasons to go further and convert lactic
acid into anything else are:
The acidity of lactic acid impedes biological processes; this can be beneficial to the
fermenting organism as it drives out competitors who are unadapted to the acidity;
as a result the food will have a longer shelf-life (part of the reason foods are
purposely fermented in the first place); however, beyond a certain point, the acidity
starts affecting the organism that produces it.
The high concentration of lactic acid (the final product of fermentation) drives the
equilibrium backwards (Le Chatelier's principle), decreasing the rate at which
fermentation can occur, and slowing down growth
Ethanol, that lactic acid can be easily converted to, is volatile and will readily
escape, allowing the reaction to proceed easily. CO2 is also produced, however it's
only weakly acidic, and even more volatile than ethanol.
Acetic acid is acidic, and not as volatile as ethanol; however, in the presence of
limited oxygen, its creation from lactic acid releases a lot of additional energy. It is a
lighter molecule than lactic acid, that forms fewer hydrogen bonds with its
surroundings (due to having fewer groups that can form such bonds), and thus more
volatile and will also allow the reaction to move forward more quickly.
If propionic acid, butyric acid and longer monocarboxylic acids are produced, the
amount of acidity produced per glucose consumed will decrease, as with ethanol,
allowing faster growth.
Aerobic Respiration
Fermentation does not necessarily have to be carried out in an anaerobic environment.
Even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to aerobic
respiration, as long as sugars are readily available for consumption (a phenomenon known
as the Crabtree effect).
In aerobic respiration, the pyruvate produced by glycolysis is oxidized completely,
generating additional ATP and NADH in the citric acid cycle and by oxidative
phosphorylation. However, this can occur only in the presence of oxygen. Oxygen is toxic to
organisms that are obligate anaerobes, and is not required by facultative anaerobic
organisms. In the absence of oxygen, one of the fermentation pathways occurs in order to
regenerate NAD+; lactic acid fermentation is one of these pathways.
Fermentation Technology
Fermentation technology is the oldest of all biotechnological processes. The term is

derived from the Latin verb fevere, which means 'to boil' . It is thought to have been first used in
the late fourteenth century in alchemy, but only in a broad sense. It was not used in the modern
scientific sense until around 1600.
Development of Fermentation Process

Fermentation has been used by humans for the production of food and beverages since
the Neolithic age.
6000BC- Bread making (involving yeast fermentation)
2500BC-Malting of barley, fermentation of beer in Egypt.
1787-Fabroni defined fermentation as a decomposition of one substance by another substance.
1814-Kirchhoff observed that a glutinous component of wheat is capable of converting starch to
sugar and dextriin.
1830-Robiquet and Boutron, also Chalard discovered the hydrolysis of amygdalin by bitter
almonds. Liebig and Whohler (1837) and Robiquet (1836) named the enzyme emulsin.
1833-Payen and Persoz separated active amylase from malt.
1837-Berzelius included fermentation under catalytic processes.
1838-Berzelius proposes the term catalysis, meaning a loosening down.
1858-Pasteur noted that green mould fermented only dextro tartaric acid and did not attack levo
tartaric acid.
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1862-Danielewski separated pancreatic amylase from trypsin by adsorption.
1870-Liebig developed a purely chemical theory of enzyme action.
1871-Pasteur showed that living yeast was necessary for fermentation. A difference was made
between organized ferments such as yeast and lactic acid-producing bacteria and unorganized
ferments such as pepsin and diastase.
1878-Kuhne designated the latter class of substances as enzymes, which means in yeast.
1883-Duclaux introduced the custom designating an enzyme by the substrate on which its
action was first observed and adding the suffix, -ase.
1898-Croft-Hill performed the first enzymatic synthesis, that of isomaltose.
1900-Catalysts of oxidation were considered as enzymes.
1909-Sorensen pointed out the dependence of enzyme activity on pH.
19001920 Ethanol, glycerol, acetone and butanol produced commercially by largescale fermentation.
1923-Citric acid fermentation plant using Aspergillus niger by Charles Pfizer.
1943-Submerged culture of Penicillium chrysogenum opens way for large -scale production
of penicillin.
1945-Production through fermentation process scaled up to make enough penicillin to treat
100,000 patients per year. Beginning of rapid development of antibiotic industry; during World
War II, research driven by 85% tax on excess profits, encouraged investment in research
and development for antibiotics.
1957-Commercial production of natural amino acids via fermentation facilitated the discovery of
Micrococcus glutamicus (later renamed Corynebacterium glutamicum) Glucose-isomerizing
capability of xylose isomerase reported
1960-Lysine produced on a technical scale
1961-First commercial production of MSG via fermentation
1965-Corn bran and hull replaces xylose as inducer of glucose (xylose) isomerase in
Streptomyces phaeochromogenus. Phenyl methyl ester of aspartic acid and phenylalanine
(aspartame) synthesized at G. D. Searle Co.
1967-Clinton Corn Processing ships fi rst enzymatically produced fructose syrup.
19791980-Energy-saving method for drying ethanol using corn (starch) and cellulose-based
adsorbents reported.
19771982 - Fermentation ethanol processes adapted by wet millers for fuel grade ethanol
Fermentation Process in Industries

Fermented products have applications as food as well as in general industry. Some
commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation.
Nearly all commercially produced enzymes, such as lipase, invertase and rennet, are made by
fermentation with genetically modified microbes. In some cases, production of biomass itself is
the objective, as in the case of baker's yeast and lactic acid bacteria starter cultures for cheese
making.
Classification of Fermentation Process used in Industries
Solid State Cultures

Microorganism grows on moist solid surface with little or
no free water like mushroom. Examples include fermented
bakery products such as bread or for the maturing of cheese.
Solid State Fermentation is also widely used to prepare raw
materials such as chocolate and coffee; typically cacao bean
fermentation and coffee bean skin removal are SSF processes
carried out under natural tropical conditions.
Submerged Cultures
Uses dissolved substrate e.g. sugar solution or a solid substrate

suspended in large amount of water to form slurry. Most
fermentation industries today use the submerged process for the
production of microbial products.
Types of Fermentation Process

The fermentation unit in industrial microbiology is analogous to a chemical plant in the
chemical industry. A fermentation process is a biological process and, therefore, has
requirements of sterility and use of cellular enzymic reactions instead of chemical reactions
aided by inanimate catalysts, sometimes operating at elevated temperature and pressure.
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Industrial fermentation processes may be divided into two main types, with various
combinations and
modifications. These are batch fermentations and continuous
fermentations.
Batch fermentations
A tank of fermenter is filled with the prepared mash of raw materials to be fermented.
The temperature and pH for microbial fermentation is properly adjusted, and
occassionally nutritive supplements are added to the prepared mash. The mash is
steam-sterilized in a pure culture process. The inoculum of a pure culture is added to the
fermenter, from a separate pure culture vessel. Fermentation proceeds, and after the
proper time the contents of the fermenter, are taken out for further processing. The
fermenter is cleaned and the process is repeated. Thus each fermentation is a
discontinuous process divided into batches.
Continuous fermentation
Growth of microorganisms during batch fermentation confirms to the characteristic
growth curve, with a lag phase followed by a logarithmic phase. This, in turn, is
terminated by progressive decrements in the rate of growth until the stationary phase is
reached. This is because of limitation of one or more of the essential nutrients. In
continuous fermentation, the substrate is added to the fermneter continously at a fixed
rate. This maintains the organisms in the logarithmic growth phase. The fermentation
products are taken out continuously. The design and arrangements for continuous
fermentation are somewhat complex.
Aerobic fermentations
A number of industrial processes, although

called 'fermentations',
are carried on by microorganisms under
aerobic conditions. In older aerobic processes
it was necessary to furnish a large surface
area by exposing fermentation media to air. In
modern fermentation processes aerobic
conditions are maintained in a closed
fermenter
with submerged
cultures. The
contents of the fermenter are agitated with au
impeller and aerated by forcing sterilized air
(Fig 1).
Anaerobic fermentations
Basically a fermenter designed to operate under micro-aerophilic or anaerobic conditions
will be the same as that designed to operate under aerobic conditions, except that
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arrangements for intense agitation and aeration are unnecessary. Many anaerobic
fermentations do, however, require mild aeration for the initial growth phase, and
sufficient N agitation for mixing and maintenance of temperature.
Component Parts of a Fermentation Process

1. Formulation of media (for both inoculum& production fermenter)
2. Sterilization
3. Inoculum development
4. Growth of the organism (optimal conditions)
5. Extraction / purification of product
6. Disposal of effluent
The microbes used for fermentation grow in (or on) specially designed growth
medium which supplies the nutrients required by the organisms. Varieties of media exist, but
invariably contain a carbon source, a nitrogen source, water, salts, and micronutrients.
Nutrient sources for industrial fermentation

Any Microbe requires Water, Oxygen, Energy source, Carbon source, Nitrogen source
and Micronutrients for the growth.
Carbon & Energy source + Nitrogen source + O2 + other requirements Biomass + Product +
byproducts + CO2 + H2O + heat
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Nutrient
Raw material
Carbon
Glucose
Corn sugar, Starch, Cellulose
Sucrose
Sugarcane, Sugar beet molasses
Lactose
Milk whey
Fats
Vegetable oils
Hydrocarbons
Petroleum fractions
Nitrogen
Protein
Soybean meal, Cornsteep liquor, Distillers' solubles
Ammonia
Pure
ammonia
or
ammonium
salts
Urea
Nitrate
Nitrate salts
Phosphorus source Phosphate salts
The Range of Fermentation Process

There are five major groups of commercially important fermentations:
Those that produce microbial cells (or biomass) as the product.
Those that produce microbial enzymes
Those that produce microbial metabolites.
Those that produce recombinant products.
Those that modify a compound which is added to the fermentation - the transformation
Some Important Fermentation Products
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Fermenters
Industrial fermentations are typically carried
out
in
large
tanks,
called fermenters or bioreactor. The heart of the
fermentation process is the fermenter. For aerobic
fermentations, air is typically used because it is
inexpensive to provide enough oxygen for cellular
respiration. Anaerobic fermentations, such as the
production of ethanol, typically do not require the
addition of any air, and only require agitation from
a mixer to keep the organisms suspended. Aerobic
fermentations may be conducted in a variety of fermenters, such as a bubble column or
apacked bed over which fermentation medium drips (as in the production of vinegar). Cooling is
typically required, since organisms produce waste heat as part of their metabolism.
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In general:
Stirred vessel, H/D 3
Volume 1-1000 m3 (80 % filled)
Biomass up to 100 kg dry weight/m3
Product 10 mg/l 200 g/l
Types of fermenter
Simple fermenters (batch and continuous)
Fed batch fermenter
Air-lift or bubble fermenter
Cyclone column fermenter
Tower fermenter
Other more advanced systems, etc
Fermenter in Antibiotic Production
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Fermenter in Antibiotic Production

Flow sheet of a multipurpose fermenter and its auxiliary equipment
14
Typical Fermenter
Microbes and nutrients are put into the fermenter and air is bubbled through so that the
microbes can respire aerobically. As carbon dioxide builds up the gas outlet releases it to avoid
build up of pressure. A water jacket surrounding the fermenter maintains an optimum
temperature so the proteins do not become denatured. Temperature, pH and oxygen probes are
linked to a computer which monitors the conditions inside the vessel. Paddle stirrers ensure that
the microbes, nutrients and oxygen are well mixed and distribute the heat evenly. The product is
run off from the bottom. It is separated from the microbes and purified so that it can be sold or
distributed.
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Microbial Growth Kinetics
Batch Culture
Batch culture is a closed system without any inlet or outlet streams as
nutrients are prepared in a fixed volume of liquid media. The inocula are
transferred and then the microorganisms gradually grow and replicate.
As the cell propagates, the nutrients are depleted and end products are
formed. The microbial growth is determined by cell optical density,
measured in a spectrophotometer, can be used as a measure of the
concentration of bacteria in a suspension. As visible light passes through
a cell suspension the light is scattered. Greater scatter indicates that
more bacteria or other material is present.
A growth curve can be divided into four phases: The lag phase shows almost no apparent cell
growth. This is the duration of time represented for adaptation of microorganism to the new
environment, without much cell replication and with no sign of growth. The length of the lag
phase depends on the size of the inocula. It is also results from the shock to the environment
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when there is no acclimation period (time required for bacteria to adapt to their new
environment). In exponential growth phase there is an appreciable amount of cells and they are
growing very rapidly, the cell number exponentially increases. The optical cell density of a
culture can then be easily detected. The rate of cell synthesis sharply increases. Finally, rapid
utilization of substrate and accumulation of products may lead to stationary phase where the cell
density remains constant. In this phase cell may start to die as the cell growth rate balances the
death rate. The dead cells and cell metabolites in the fermentation broth may create toxicity to
deactivating remaining cells. At this stage a death phase develops while the cell density
drastically drops it also shows an exponential decrease in the number of living cells in the media
while nutrients are depleted.
During the lag phase dX/dt and dS/dt are essentially zero. However as exponential growth
phase begins it is possible to measure dX/dt and dS/dt values which are very useful for defining
important microbial kinetic parameters. The exponential phase may be describe by the
equation:
where :
is the concentration of microbial biomass

is time
is the specific growth rate
Integrating the equation will give:
where :
is the concentration of microbial biomass

is the biomass concentration after the time interval
Taking natural logarithms the equation will becomes:
A plot of natural logarithm of biomass concentration against time should yield a straight line, the
slope of which would equal . During the exponential phase nutrients are in excess and the
organism is growing at its maximum specific growth rate
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some representative values of
for some range of microorganism
the nature of the limitation of growth may be explored by growing the organism in the presence
of a range of substrate concentrations and plotting the biomass concentration at stationary
phase against the initial substrate concentration.
Concentration
Where
is the concentration of biomass produced

is the yield factor
is the initial substrate concentration
is the residual substrate concentration
Specific Growth Rate

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Microbial growth kinetic relationships

Monod Kinetics
The most widely used expression for describing specific growth rate as a function of substrate
concentration is attributed to Monod (1942, 1949). This expression is:
Where:
is the residual substrate concentration

is the substrate utilization constant, numerically equal to substrate concentration
when
is half
some representative values of
for some range of microorganism
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Time-rate-of-change
Substrate utilization rate
First order kinetics

if S << KS
where substrate utilization is proportional to substrate concentration.
Zero order kinetics

Likewise if S >> KS
where substrate utilization rate is a constant
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Continuous Culture
In a continuous operation, one or more feed streams
containing
the
necessary
nutrients
are
fed
continuously, while the effluent stream containing the

cells,
products,
and
residuals
is
continuously
removed. A steady state is established by maintaining

an equal volumetric flow rate for the feed and effluent
streams. In so doing, the culture volume is kept
constant, and all nutrient concentrations remain at
constant steady state values. The condition in
continuous culture is different in batch culture due to
the continuous addition of fresh medium or nutrient to the vessel. Because of this, the
exponential growth is prolonged and it will continue until additional substrate is depleted. The
medium is continuously fed to the culture at a suitable rate in order to get continuous, steadystate cell production. The vessel that used as a growth container is continuous culture is called
bioreactor or chemostat.
The chemostat setup consists of a sterile fresh nutrient reservoir connected to a growth
chamber or reactor. Fresh medium containing nutrients essential for cell growth is pumped
continuously to the chamber from the medium reservoir. The medium contains a specific
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concentration of growth-limiting nutrient (Cs), which allows for a maximum concentration of cells
within the growth chamber. Varying the concentration of this growth-limiting nutrient will, in turn,
change the steady state concentration of cells (C c). Another means of controlling the steady
state cell concentration is manipulating the rate at which the medium flows into the growth
chamber. The medium drips into culture through the air break to prevent bacteria from traveling
upstream and contaminating the sterile medium reservoir.
The well-mixed contents of the vessel, consisting of unused nutrients, metabolic wastes, and
bacteria, are removed from the vessel and monitored by a level indicator, in order to maintain a
constant volume of fluid in the chemostat. This effluent flow can be controlled by either a pump
or a port in the side of the reactor that allows for removal of the excess reaction liquid. In either
case, the effluent stream needs to be capable of removing excess liquid faster than the feed
stream can supply new medium in order to prevent the reactor from overflowing.
Temperature and pressure must also be controlled within the chemostat in order to maintain
optimum conditions for cell growth. In order to prevent the reaction mixture from becoming too
acidic (cell respiration causes the medium to become acidic) or too basic, which could hinder
cell growth, a pH controller is needed in order to bring pH balance to the system.
The stirrer ensures that the contents of the vessel are well mixed. If the stirring speed is too
high, it could damage the cells in culture, but if it is too low, gradients could build up in the
system. Significant gradients of any kind (temperature, pH, concentration, etc.) can be a
detriment to cell production, and can prevent the reactor from reaching steady state operation.
The combination of growth and dilution within the chemostat will ultimately determine the growth
thus the change in biomass with time is
Where x is the cell mass,
is the specific growth rate and D is the dilution rate. A steady state
will be reached when

The mechanism underlying the controlling effect of the dilution rate is demonstrated by monod
equation:
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At steady state:
Where is the steady state concentration of substrate in the chemostat.

Concentration of cells in the chemostat at steady state
}]
Note:
If
> D, the utilization of substrate will exceed the supply of substrate, causing the growth rate
to slow until it is equal to the dilution rate

If
> d the amount of substrate added will exceed the amount utilized. Therefore the growth
rate will increase until it is equal to the dilution rate.

Steady state at
, such as a steady state can be achieve d and maintained as long as the
dilution rate does not exceed a critical rate

Critical dilution rate:
(
Biomass Productivity
The productivity of a culture system may be described as the output of biomass per unit time of
the fermentation.
Productivity for batch culture
Where:
is the output of the culture
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is the maximum cell concentration achieved at stationary phase
is the initial cell concentration at inoculation
is the time during which the organism grows at
is the time during which the organism is not growing at
and includes lag phase,
the deceleration phase and the periods of batching, sterilizing and harvesting.
Productivity for continuous culture
Where:
is the output of the culture

is the time period prior to the establishment of a steady state and includes vessel
preparation, sterilization and operation in batch culture prior to continuous operation
is the time period during which steady state conditions prevail
Fed Batch Culture

A fed-batch culture is a semi-batch operation in which the nutrients
necessary for cell growth and product formation are fed either
intermittently or continuously via one or more feed streams during the
course of an otherwise batch operation. The culture broth is harvested
usually only at the end of the operational period, either fully or partially
(the remainder serving as the inoculum for the next repeated run). This
process may be repeated (repeated fed-batch) a number of times if the
cells are fully viable and productive. Thus, there are one or more feed
streams but no effluent during the course of operation. The products
are harvested only at the end of the run. Therefore, the culture volume
increases during the course of operation until the volume is full. Thereafter, a batch mode of
operation is used to attain the final results. Thus, the fed-batch culture is a dynamic operation.
By manipulating the feed rates, the concentrations of limiting nutrients in the culture can be
manipulated either to remain at a constant level or to follow a predetermined optimal profile until
the culture volume reaches the maximum, and then a batch mode is used to provide a final
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touch. In so doing, the concentration of the desired product or the yield of product at the end of
the run is maximized.
Nomenclature
- concentration of microbial biomass
- time
- specific growth rate
- concentration of microbial biomass
- biomass concentration after the time interval
- yield factor
- initial substrate concentration
- residual substrate concentration
- substrate utilization constant
MEDIA FOR INDUSTRIAL FERMENTATION

Detailed investigation is needed to establish the most suitable medium for an individual
fermentation process, but certain basic requirements must be met by any such medium. All
micro-organisms require water, sources of energy, carbon, nitrogen, mineral elements and
possibly vitamins plus oxygen if aerobic. On a small scale it is relatively simple to devise a
medium containing pure compounds, but the resulting medium, although supporting satisfactory
growth may be unsuitable for use in a large scale process.
On a large scale one must normally use sources of nutrients to create a medium which
will meet as many as possible of the following criteria:
I. It will produce the maximum yield of product or biomass per gram of substrate used.
2. It will produce the maximum concentration of product or biomass.
3. It will permit the maximum rate of product formation.
4. There will be the minimum yield of undesired products.
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5. It will be of a consistent quality and be readily available throughout the year.
6. It will cause minimal problems during media making and sterilization.
7. It will cause minimal problems in other aspects of the production process particularly aeration
and agitation, extraction, purification and waste treatment.
The use of cane molasses, beet molasses, cereal grains, starch, glucose, sucrose and
lactose as carbon sources, and ammonium salts, urea, nitrates, corn steep liquor, soya bean
meal, slaughter-house waste and fermentation residues as nitrogen sources, have tended to
meet most of the above criteria for production media because they are cheap substrates.
However, other more expensive pure substrates may be chosen if the overall cost of the
complete process can he reduced because it is possible to use simpler procedures.
MEDIUM FORMULATION
Medium formulation is an essential stage in the design of successful laboratory
experiments, pilot-scale development and manufacturing processes. The constituents of a
medium must satisfy the elemental requirements for cell biomass and metabolite production and
there must be an adequate supply of energy for biosynthesis and cell maintenance. The first
step to consider is an equation based on the stoichiometry for growth and product formation.
Thus for an aerobic fermentation:
Carbon dioxide and energy source + nitrogen source + O2 + other requirement biomass +
products + CO2 + H2O + heat
This equation should be expressed in quantitative terms, which is important in the
economical design of media if component wastage is to be minimal. Thus, it should be possible
to calculate the minimal quantities of nutrients which will be needed to produce a specific
amount of biomass. Knowing that a certain amount of biomass is necessary to produce a
defined amount of product, it should be possible to calculate substrate concentrations
necessary to produce required product yields. There may be medium components which are
needed for product formation which are not required for biomass production. Unfortunately, it is
not always easy to quantify all the factors very precisely. A knowledge of the elemental
composition of a process micro-organism is required for the solution of the elemental balance
equation. This information may not be available so that data which is given in Table 4.2 will
serve as a guide to the absolute minimum quantities of N, S, P, Mg and K to include in an initial
medium recipe. Trace elements (Fe, Zn, Cu, Mn, Co, Mo, B) may also be needed in smaller
quantities. An analysis of relative concentrations of individual elements in bacterial cells and
commonly used cultivation media quoted by Cooney (1981) showed that some nutrients are
frequently added in substantial excess of that required, e.g. P, K; however, others are often near
limiting values, e.g. Zn, Cu. The concentration of P is deliberately raised in many media to
increase the buffering capacity. These points emphasize the need for considerable attention to
be given to medium design.
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Some examples of fermentation media:
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TYPES OF MEDIA
1. WATER
Water is the major component of all fermentation media, and is
needed in many of the ancillary services such as heating, cooling,
cleaning and rinsing. Clean water of consistent composition is
therefore required in large quantities from reliable permanent sources.
When assessing the suitability of a water supply it is important to
consider pH, dissolved salts and effluent contamination. The mineral
content of the water is very important in brewing, and most critical in
the mashing process, and historically influenced the siting of
breweries and the types of beer produced. Hard waters containing
high CaSO4 concentrations are better for the English Burton bitter beers and Pilsen type lagers,
while waters with a high carbonate content are better for the darker beers such as stouts.
Nowadays, the water may be treated by deionization or other techniques and salts added, or the
pH adjusted, to favour different beers so that breweries are not so dependent on the local water
source. Detailed information is given by Hough ci al. (1971) and Sentfen (1989).
2. ENERGY SOURCES
Energy for growth comes from either the oxidation of medium components or from light.
Most industrial micro-organisms are chemo-organotrophs, therefore the commonest source of
energy will be the carbon source such as carbohydrates, lipids and proteins. Some microorganisms can also use hydrocarbons or methanol as carbon and energy sources.
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A. CARBON SOURCES
Factors influencing the choice of carbon source
It is now recognized that the rate at which the carbon source is metabolized can often
influence the formation of biomass or production of primary or secondary metabolites. Fast
growth due to high concentrations of rapidly metabolized sugars is often associated with low
productivity of secondary metabolites. This has been demonstrated for a number of processes.
At one time the problem was overcome by using the less readily metabolized sugars such as
lactose (Johnson, 1952), but many processes now use semi-continuous or continuous feed of
glucose or Sucrose. Alternatively, carbon catabolite regulation might be overcome by genetic
modification of the producer organism.
The main product of a fermentation process will often determine the choice of carbon
source, particularly if the product results from the direct dissimilation of it. In fermentations such
as ethanol or single-cell protein production where raw materials are 60 to 77% of the production
cost, the selling price of the product will be determined largely by the cost of the carbon source
(Whitaker, 1973; Moo-Young, 1977). It is often part of a company development programme to
test a range of alternative carbon sources to determine the yield of product and its influence on
the process and the cost of producing biomass and/or metabolite. This enables a company to
use alternative substrates, depending on price and availability in different locations, and remain
competitive. Up to ten different carbon sources have been or are being used by Pfizer Ltd for an
30
antibiotic production process depending on the geo graphical location of the production site and
prevailing economics (Stowell, 1987).
Examples of commonly used carbon sources
CARBOHYDRATES
It is common practice to use carbohydrates as the carbon
source in microbial fermentation processes. The most widely
available carbohydrate is starch obtained from maize grain. It is
also obtained from other cereals, potatoes and cassava. Analysis
data for these substrates can be obtained from Atkinson and
Mavituna (1991a). Maize and other cereals may also be used
directly in a partially ground state, e.g. maize chips. Starch may
also be readily hydrolysed by dilute acids and enzymes to give a
variety of glucose preparations (solids and syrups). Hydrolysed
cassava starch is used as a major carbon source for glutamic acid production in Japan (Minoda,
1986). Syrups produced by acid hydrolysis may also contain toxic products which may make
them unsuitable for particular processes.
OILS AND FATS
Oils were first used as carriers for antifoams in antibiotic
processes (Solomons, 1969). Vegetable oils (olive, maize, cotton
seed, linseed, soya bean, etc.) may also be used as carbon
substrates, particularly for their content of the fatty acids, oleic,
linoleic and linolenic acid, because costs are competitive with
those of carbohydrates. In an analysis of commodity prices for
sugar, soya bean oil and tallow between 1978 and 1985, it would
have been cheaper on an available energy basis to use sugar
during 1978 to mid 1979 and late 1983 to 1985, whereas oil
would have been the chosen substrate in the intervening period
(Stowell, 1987).
Bader et al. (1984) discussed factors favouring the use of oils instead of carbohydrates.
A typical oil contains approximately 2.4 times the energy of glucose on a per weight basis. Oils
also have a volume advantage as it would take 1.24 dm3 of soya bean oil to add 10 kcal of
energy of a fermenter, whereas it would take 5 dm3 of glucose or sucrose assuming that they
are being added as 50% w/w solutions. Ideally, in any fermentation process, the maximum
working capacity of a vessel should be used. Oil based fed-hatch fermentations permit this
procedure to operate more successfully than those using carbohydrate feeds where a larger
spare capacity must be catered for to allow for responses to a sudden reduction in the residual
nutrient level (Stowell, 1987).
31
HYDROCARBONS AND THEIR DERIVATIVES
There has been considerable interest in hydrocarbons. Development work has been
done using n-alkanes for production of organic acids, amino acids, vitamins and co-factors,
nucleic acids, antibiotics, enzymes and proteins (Fukui and Tanaka, 1980). Methane, methanol
and n-alkanes have all been used as substrates for biomass production (Hamer, 1979; Levi et
al., 1979; Drozd, 1987; Sharp. 1989).
3. NITROGEN SOURCES
Factors influencing the choice of nitrogen source
Control mechanisms exist by which nitrate reductase, an enzyme involved in the
conversion of nitrate to ammonium ion, is repressed in the presence of ammonia (Brown et al.,
1974). For this reason ammonia or ammonium ion is the preferred nitrogen source. In fungi that
have been investigated, ammonium ion represses uptake of amino acids by general and
specific amino acid permeases (Whitaker, 197M. In Aspergillus nidulans, ammonia also
regulates the production of alkaline and neutral proteases (Cohen. 1973). Therefore, in mixtures
of nitrogen sources, individual nitrogen components may influence metabolic regulation so that
there is preferential assimilation of one component until its concentration has diminished.
The use of complex nitrogen sources for antibiotic production has been common
practice. They are thought to help create physiological conditions in the trophophase which
favour antibiotic production in the idiophase (Martin and McDaniel, 1977). For example, in the
production of polyene antibiotics, soybean meal has been considered a good nitrogen source
because of the balance of nutrients, the low phosphorus content and slow hydrolysis. It has
been suggested that this gradual breakdown prevents the accumulation of ammonium ions and
repressive amino acids. These are probably some of the reasons for the selection of ideal
nitrogen sources for some secondary metabolites.
32
Examples of commonly used nitrogen uses
Organic nitrogen may he supplied as amino acid, protein or urea. In many instances
growth will be faster with a supply of organic nitrogen, and a few microorganisms have an
absolute requirement for amino acids. It might be thought that the main industrial need for pure
amino acids would be in the deliberate addition to amino acid requiring mutants used in amino
acid production. However, amino acids arc more commonly added as complex organic nitrogen
sources which are non-homogeneous, cheaper and readily available. In lysine production,
methionine and threonine are obtained from soybean hydrolysate since it would be too
expensive to use the pure amino acids (Nakayarna, 1972a)
4. MINERALS
All micro-organisms require certain mineral elements for growth and metabolism
(Hughes and Poole, 1989, 1991). In many media, magnesium, phosphorus, potassium, sulphur,
calcium and chlorine arc essential components, and because of the concentrations required,
they must be added as distinct components. Others such as cobalt, copper, iron, manganese,
molybdenum and zinc are also essential hut arc usually present as impurities in other major
ingredients. There is obviously a need for batch analysis of media components to ensure that
this assumption can be justified, otherwise there may be deficiencies or excesses in different
batches of media. See Tables 4.7 and 4.8 for analysis of corn steep liquor and Pharmamedia,
and Miller and Churchill (1986) for analysis of other media ingredients of plant and animal origin.
When synthetic media are used the minor elements will have to be added deliberately. The form
in which the minerals are usually supplied and the concentration ranges arc given in Table 4.10.
As a consequence of product composition analysis, as outlined earlier in this chapter, it is
possible to estimate the amount of a specific mineral for medium design, e.g. sulphur in
penicillins and cephalosporins, chlorine in chlortetracycline.
Chelators
Many media cannot be prepared or autoclaved without the formation of a visible
precipitate of insoluble metal phosphates. Gaunt et al. (1984) demonstrated that when the
medium of Mandels and Weber (1984) was autoclaved, a white precipitate of metal ions formed,
containing all the iron and most of the calcium, manganese and zinc present in the medium.
33
5. GROWTH FACTORS
Some micro-organisms cannot synthesize a full complement of cell components and
therefore require preformed compounds called growth factors. The growth factors most
commonly required are vitamins, but there may also be a need for specific amino acids, fatty
acids or sterols. Many of the natural carbon and nitrogen sources used in media formulations
contain all or some of the required growth factors (Atkinson and Mavituna, 1991a). When there
is a vitamin deficiency it can often be eliminated by careful blending of materials (Rhodes and
Fletcher, 1966). It is important to remember that if only one vitamin is required it may he
occasionally more economical to add the pure vitamin, instead of using a larger bulk of a
cheaper multiple vitamin source. Calcium pantothenate has been used in one medium
formulation for vinegar production (Beaman, 1967). In processes used for the production of
glutamic acid, limited concentrations of biotin must be present in the medium. Some production
strains may also require thiamine (Kinoshita and Tanaka, 1972).
6. BUFFERS
The control of pH may be extremely important if optimal productivity is to be achieved. A
compound may be added to the medium to serve specifically as a buffer, or may also be used
as a nutrient source. Many media are buffered at about pH 7.0 by the incorporation of calcium
carbonate (as chalk). If the pH decreases the carbonate is decomposed. Obviously, phosphates
which are part of many media also play an important role in buttering. However, high phosphate
concentrations are critical in the production of many secondary metabolites.
7. OXYGEN REQUIREMENTS
It is sometimes forgotten that oxygen, although not added to an initial medium as such, is
nevertheless a very important component of the medium in many processes, and its availability
can be extremely important in controlling growth rate and metabolite production.
The medium may influence the oxygen availability in a number of ways including the following:
1. Fast metabolism
The culture may become oxygen limited because sufficient oxygen cannot be made
available in the fermenter if certain substrates, such as rapidly metabolized sugars which
lead to a high oxygen demand, are available in high concentrations.
34
2. Rheology
The individual components of the medium can influence the viscosity of the final medium
and its subsequent behaviour with respect to aeration and agitation.
3. Antifoams
Many of the antifoams in use will act as surface active agents and reduce the oxygen
transfer rate.
Hall et al. (1973) have recognized five patterns of foaming in fermentations:
1. Foaming remains at a constant level through-out the fermentation. Initially it is due to
the medium and later due to microbial activity.
2. A steady fall in foaming during the early part of the fermentation, after which it
remains constant. Initially it is due to the medium hut there are no later effects
caused by the micro-organism.
3. The foaming falls slightly in the early stages of the fermentation then rises. There are
very slight effects caused by the medium but the major effects arc due to microbial
activity.
4. The fermentation has a low initial foaming capacity which rises. These effects are
due solely to microbial activity.
5. A more complex foaming pattern during the fermentation which may be a
combination of two or more of the previously described patterns.
35
If excessive foaming is encountered there are three ways of approaching the problem:
1. To try and avoid foam formation by using a defined medium and a modification of
some of the physical parameters (pH, temperature. aeration and agitation). This
assumes that the foam is due to a component in the medium and not a metabolite.
2. The foam is unavoidable and antifoam should be used. This is the more standard
approach.
3. To use a mechanical foam breaker.
The Development of Inocula for Industrial Fermentations

Inocula are living organisms or an amount of material containing living organisms (such as
bacteria or other microorganisms) that is added to initiate or accelerate a biological process.
Criteria of Inocula:
Healthy, active state - minimize lag period

Available in sufficient quantities
Suitable morphological form
Free of contamination
Stable - retain its product forming properties
The process adopted to produce an inoculum meeting these criteria is called inoculum
development.
Difference between Inoculum and Production medium:
Inoculum grows in culture medium

Design of production medium is determined not only by nutritional requirements of
organism but also by requirements for maximum product formation
Both media can be of different composition
Examples of inoculum and production media:

Process
Griseofulvin
Inoculum Development Medium

Whey powder, Lactose} to give:
Lactose 3.5%
Nitrogen 0.05%
Corn-steep
Liquor solids 0.38%
(to give approx. 0.04% N)
36
Production Medium
Lactose
7%
Corn-steep
Liquor solids to give:
Nitrogen 0.2%
Limestone 0.8%
KH2PO4
0.4%
Clavunalic
Acid
Vitamin B12
Lysine
KH2PO4 0.4%
KCl
0.05%
Soybean flour 1.0%
Dextrin
2.0%
Plunoric L81
0.03%
(Antifoam)
KCl
0.1%
Soybean flour 1.5%

Oil
1.0%
KH2PO4
0.1%
(g dm-3)
Sugar beet molasses
70
Sucrose
Betaine
NH4H2PO4
0.8
(NH4)2SO4
2
MgSO4
0.2
ZnSO4
0.02
5-6 Dimethyl-benzimidazole 0.005
Cane molasses
5%
Corn-steep Liquor 1%
CaCO3 1%
Soybean meal hydrolysate -
(g dm-3)
105
15
3
2.5
0.2
0.08
0.025
20%
1.8%
Choice of Microorganism:
Nutritional characteristics - cheap medium

Optimum environmental conditions
Productivity - substrate conversion, product yield, rates.
Amenability to genetic manipulation
Ease of handling and safety (suitability)
Criteria for transfer of inoculum:
Physiological condition of inoculum major effect on performance of fermentation

Indicators of inoculum quality include dissolved oxygen, pH and CO 2
Yeast, Bacteria and Fungi common inocula
Safety Measures:
A. Use of laminar flow cabinets
Laminar Flows
Laminar air flows can maintain a working area devoid of contaminants. Many medical
and research laboratories require sterile working environments in order to carry out specialized
work. Laminar Flow Cabinets can provide the solution.
37
Laminar Flow Cabinets

Laminar Flow Cabinets create particle-free working environments by projecting air
through a filtration system and exhausting it across a work surface in a laminar or uni-directional
air stream. They provide an excellent clean air environment for a number of laboratory
requirements.
Types of Laminar Flow Cabinets:
a. Horizontal Laminar Flow Cabinets
Horizontal Laminar Flow Cabinets receive their name due to the direction
of air flow which comes from above but then changes direction and is
processed across the work in a horizontal direction. The constant flow of
filtered air provides material and product protection.
b. Vertical Laminar Flow Cabinets

Vertical Laminar Flow Cabinets function equally well as horizontal
Laminar Flow Cabinets with the laminar air directed vertically downwards
onto the working area. The air can leave the working area via holes in the
base. Vertical flow cabinets can provide greater operator protection.
Laminar Flow Cabinets are used:
to limit exposure of operators to aerosols and other possible infections

to protect the culture material from contamination
B. Asepsis must be maintained

Asepsis is the state of being free from disease-causing contaminants (such as bacteria,
viruses, fungi, and parasites) or, preventing contact with microorganisms.
C. Correct Standards must be applied

CLASS 1 - none or minimal hazard
CLASS 2 - ordinary potential hazard
CLASS 3 - Special hazard, require special containment
38
CLASS 4 - Extremely dangerous, may cause epidemic disease
CLASS 5 - Pathogens excluded by law
Methods of Storage and Preservation:
Storage at reduced temperatures:
1. Agar Slopes - refrigerator (4 oC), freezer (-20 oC), protect beads (-80 oC)
2. Liquid nitrogen (-150 to -196 oC)
Storage in dehydrated form;
1. Soil + culture dried. Used for fungi
2. Lyophilization \ freeze drying. Freezing of culture followed by drying under vacuum
which results in sublimation of cell water
Quality Control of Preserved Cultures
Each batch must be routinely tested.

Whatever method is used in preservation of stock cultures it is important to assess the
quality of the stocks
Each batch of cultures should be routinely checked to ensure the propagated strains
retain the correct growth characteristics, morphology (the study of the form or shape of
an organism) and product forming properties
Physiological Aspects
A. Lag phase - represents dead time with respect to process;
true lag = all of the population is retarded

apparent lag = part of population dead/ normal
39
Lag period may be due to:
1. Change in nutrients on transfer
2. Change in physical environment
3. Presence of inhibitor
4. Spore germination
5. Viability of culture on transfer
6. Size of inoculum
B. Number of generations during the growth cycle
For example, 6 - 7% biomass initially, then after 4 generations (doubling times), inoculum gives
100% final biomass.
Contamination and Instability

A. Consequences
Loss of productivity - media must support contaminant

Out compete and replace - e.g. in continuous systems
Contaminate product
Cause breakdown e.g. enzyme action
Complicate recovery e.g. polymers
Cause lysis (refers to the breaking down of a cell)
B. Avoidance
Pure inoculum
Aseptic conditions
Sterilize raw materials, additions + reactor, plant equipment etc.
C. Detection
Check using Microscope

Monitor pattern of pH, product, biomass formation
40
Inoculum Quality Control
A. Pure Culture - Tests
Cultural methods - slow
Loop dilution - involves the successive transfer (serial dilution) of bacteria from
the original culture to a series of tubes of liquefied agar
Streak plates - It is essentially a dilution technique that involves spreading a
loopful of culture over the surface of an agar plate.
Differential (designed to show the difference between different organisms grown
on it) /selective (designed to grow only specific bacteria but not others) plating
Direct methods - rapid (process requirement)
Yeast; Morphology, granulation, cell shape and size

Bacteria; Shape, Gram reaction
B. Test for Viability
Viable stain e.g. methylene blue, etc.
C. Test for Cell Concentration
Example from brewing - Sedimented volume
Instability
Organism has tendency to lose ability to produce product or some desirable
characteristic (e.g. yeast --> ability to flocculate - colloids come out of suspension in the
form of floc or flake)
Can occur at any stage during inoculum protocol (e.g. preservation, storage,
recovery from storage, in inoculum development unit or in production.
Can be major reason to reject a culture at industrial scale.
Any increase in scale (followed by an increased number of generations) will pose
greater problems if culture tends to degenerate.
Stability and performance of a culture during fermentation is influenced by:
Mode of substrate feeding

Nutrients
Temperature
41
Osmotic pressure
Oxygen
Intracellular product accumulation
Tolerance to product
Industrial Production
A. Development of Brewing Inoculum
It is common to use yeast from previous fermentation run to inoculate a fresh fermenter.
Problems:
1. Strain degeneration
Degree of flocculence
Degree of attenuation
2. Contamination
Wash with acid
3. Propagation
High level of asepsis
Environmental conditions may differ from brewing (e.g. media, sugars,
presence of air, pH, and temp.)
Reactor
B. Inocula for Fungal Process
Spore suspension - used at early stages

Inoculum affects morphology of fungus - can influence size of pellet or
floc.
Spore Suspension:
Solidified media e.g. agar media

Solid media e.g. cereal grains, bran, malt, flaked maize etc. (amount of water,
relative humidity of air, temp. are important)
Submerged culture - influenced by media
C. Aseptic Inoculation of Plant Fermenters

Transfer from seed tank to plant-scale reactor is carried out aseptically.
42
Critical Point in the Process and Involves:
Opening and closing a series of valves in a defined sequence

Sterilizing pipes\valves (usually with steam) in a defined sequence
D. Industrial Production of Lactic Starters

Unit Operations:
Biomass Production
- Raw Materials (nutrients)
- UHT Sterilization
- Fermentation
- Cooling: Cold storage
Finishing Operations
- Ultrafiltration
- Centrifugation
- Freeze/Spray dry
- Packaged at ambient
- Aseptic Filling
- Storage at -20oC
- Stored in liquid nitrogen
- Stored in dry ice
Reference:
Stanburry, P. F., Whitaker, A. and Hall, S. J., (2003), Principles of Fermentation and
Technology, 2nd edition
43

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FERMENTATION TECHNOLOGY

Homolactic Fermentation (producing only lactic acid) is the simplest type of

Heterolactic Fermentation, where some lactate is further metabolized and results in

Fermentation technology is the oldest of all biotechnological processes. The term is

Development of Fermentation Process

Fermentation Process in Industries

Classification of Fermentation Process used in Industries

Solid State Cultures

Uses dissolved substrate e.g. sugar solution or a solid substrate

Types of Fermentation Process

A number of industrial processes, although

Component Parts of a Fermentation Process

Nutrient sources for industrial fermentation

The Range of Fermentation Process

Those that produce microbial cells (or biomass) as the product.

Those that produce microbial enzymes

Those that produce microbial metabolites.

Those that produce recombinant products.

Some Important Fermentation Products

Stirred vessel, H/D 3

Volume 1-1000 m3 (80 % filled)

Biomass up to 100 kg dry weight/m3

Product 10 mg/l 200 g/l

Simple fermenters (batch and continuous)

Fed batch fermenter

Air-lift or bubble fermenter

Cyclone column fermenter

Other more advanced systems, etc

Fermenter in Antibiotic Production

Fermenter in Antibiotic Production

is the concentration of microbial biomass

Integrating the equation will give:

is the concentration of microbial biomass

Taking natural logarithms the equation will becomes:

for some range of microorganism

is the concentration of biomass produced

Specific Growth Rate

Microbial growth kinetic relationships

is the residual substrate concentration

some representative values of

for some range of microorganism

Substrate utilization rate

First order kinetics

where substrate utilization is proportional to substrate concentration.

Zero order kinetics

where substrate utilization rate is a constant

continuously, while the effluent stream containing the

removed. A steady state is established by maintaining

Where x is the cell mass,

will be reached when

Where is the steady state concentration of substrate in the chemostat.

to slow until it is equal to the dilution rate

rate will increase until it is equal to the dilution rate.

, such as a steady state can be achieve d and maintained as long as the

dilution rate does not exceed a critical rate

is the output of the culture

and includes lag phase,

is the output of the culture

Fed Batch Culture

MEDIA FOR INDUSTRIAL FERMENTATION

The Development of Inocula for Industrial Fermentations

Healthy, active state - minimize lag period

Difference between Inoculum and Production medium: