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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 energy-
generating 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:
C
6
H
12
O
6
+ 2 NAD
+
+ 2 ADP + 2 P
i
2 CH
3
COCOO
+ 2 NADH + 2 ATP + 2 H
2
O + 2H
+
Pyruvate is CH
3
COCOO
. P
i
is phosphate. Two ADP molecules and two P
i
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:
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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 C
6
H
12
O
6
. One glucose molecule is converted into
two ethanol molecules and two carbon dioxide molecules:
C
6
H
12
O
6
2 C
2
H
5
OH + 2 CO
2
C
2
H
5
OH 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
+.
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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
6
H
12
O
6
2 CH
3
CHOHCOOH
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
6
H
12
O
6
CH
3
CHOHCOOH + C
2
H
5
OH + CO
2
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
12
H
22
O
11
+ H
2
O 2
C
6
H
12
O
6
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. CO
2
is also produced, however it's
only weakly acidic, and even more volatile than ethanol.
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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.
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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 large-
scale 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.
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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 + O
2
+ other requirements Biomass + Product +
byproducts + CO
2
+ H
2
O + 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 m
3
(80 % filled)
Biomass up to 100 kg dry weight/m
3
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
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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 :
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
Where is the concentration of biomass produced
is the yield factor
Where: is the residual substrate concentration
.
some representative values of
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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Where:
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