Fermtech Exe

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FERMENTATION TECHNOLOGY

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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'.

Biochemistsconsider 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, Fermentationis usually defined as ametabolic process that

convertssugar to acids, gases and/oralcohol.It is also one method by which organisms derive

their energy when oxygen is lacking. It occurs inyeast andbacteria,but also in oxygen-starved

muscle cells, as in the case oflactic acid fermentation.The science of fermentation is known

aszymology.

Fermentation Process

Cellular Derivation o f 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 H 2O + 2H+

Pyruvateis CH3COCOO. Piisphosphate.TwoADP molecules and two Piare converted

to twoATP and two water molecules viasubstrate-level phosphorylation. Two molecules

ofNAD+are also reduced toNADH.

If oxygen is available Cellular Respiration (oxidative phosphorylation) will proceed

otherwise Fermentation allows glycolysis to continue.

In fermentation process, it turns theNADH and pyruvate produced in theglycolysis step

intoNAD+and various small molecules.

Fermentation is employed for preservation in a process that produceslactic acid as found in

such sourfoods (food processing), as well as for producing alcoholic beverages such

aswine (fermentation in winemaking) andbeer. Fermentation can even occur within the

stomachs of animals, such as humans.

Types of Fermentation

Fermentation reactsNADH with anendogenous,organicelectron acceptor. Usually this is

pyruvate formed from the sugar during the glycolysis step. During fermentation, pyruvate is

metabolized to various compounds through several processes:
http://en.wikipedia.org/wiki/Metabolismhttp://en.wikipedia.org/wiki/Sugarhttp://en.wikipedia.org/wiki/Alcoholhttp://en.wikipedia.org/wiki/Yeasthttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Lactic_acid_fermentationhttp://en.wikipedia.org/wiki/Zymologyhttp://en.wikipedia.org/wiki/Zymologyhttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/Phosphatehttp://en.wikipedia.org/wiki/Phosphatehttp://en.wikipedia.org/wiki/Phosphatehttp://en.wikipedia.org/wiki/Adenosine_diphosphatehttp://en.wikipedia.org/wiki/Adenosine_triphosphatehttp://en.wikipedia.org/wiki/Substrate-level_phosphorylationhttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Oxidative_phosphorylationhttp://en.wikipedia.org/wiki/NADHhttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/Glycolysishttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Lactic_acidhttp://en.wikipedia.org/wiki/Foodhttp://en.wikipedia.org/wiki/Fermentation_in_food_processinghttp://en.wikipedia.org/wiki/Winehttp://en.wikipedia.org/wiki/Fermentation_in_winemakinghttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/NADHhttp://en.wikipedia.org/wiki/Endogenyhttp://en.wikipedia.org/wiki/Organic_compoundhttp://en.wikipedia.org/wiki/Electron_acceptorhttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/File:Fermenting.jpghttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/Electron_acceptorhttp://en.wikipedia.org/wiki/Organic_compoundhttp://en.wikipedia.org/wiki/Endogenyhttp://en.wikipedia.org/wiki/NADHhttp://en.wikipedia.org/wiki/Beerhttp://en.wikipedia.org/wiki/Fermentation_in_winemakinghttp://en.wikipedia.org/wiki/Winehttp://en.wikipedia.org/wiki/Fermentation_in_food_processinghttp://en.wikipedia.org/wiki/Foodhttp://en.wikipedia.org/wiki/Lactic_acidhttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Glycolysishttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/NADHhttp://en.wikipedia.org/wiki/Oxidative_phosphorylationhttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotidehttp://en.wikipedia.org/wiki/Substrate-level_phosphorylationhttp://en.wikipedia.org/wiki/Adenosine_triphosphatehttp://en.wikipedia.org/wiki/Adenosine_diphosphatehttp://en.wikipedia.org/wiki/Phosphatehttp://en.wikipedia.org/wiki/Pyruvatehttp://en.wikipedia.org/wiki/Zymologyhttp://en.wikipedia.org/wiki/Lactic_acid_fermentationhttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Yeasthttp://en.wikipedia.org/wiki/Alcoholhttp://en.wikipedia.org/wiki/Sugarhttp://en.wikipedia.org/wiki/Metabolism


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Ethanol Fermentation, aka alcoholic fermentation, is the production

ofethanol andcarbon 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 producinglactic acid:

1. Homolactic Fermentationis the production of lactic acid exclusively

2. Heterolactic fermentationis the production of lactic acid as well as other acids and

alcohols.

Chemistry of Ethanol Fermentation

Thechemical equation below shows the alcoholic fermentation ofglucose,

whosechemical formula is C6H12O6. One glucose molecule is converted into

twoethanol molecules and twocarbon dioxide molecules:

C6H12O6 2 C2H5OH + 2 CO2

C2H5OH is thechemical formula forethanol.

Procedure

During glycolysis, the energy from the exothermic reaction is used to bind inorganic

phosphates to ADP and convertNAD+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 toNAD+.
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Chemistry of Lactic Ac id 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 NADHmolecules provide energy to convert pyruvate into lactic acid. As NADH is used, it is converted

back toNAD+. Two molecules ofNAD+are recycled back to glycolysis. The recycling ofNAD+

allows glycolysis to continue.

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

fermentation. The pyruvate from glycolysis undergoes a simpleredox reaction,

forminglactic 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 theblood cansupply oxygen. It also occurs in some kinds ofbacteria (such aslactobacilli) and

somefungi. It is this type of bacteria that convertslactose into lactic acid inyogurt,

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): C12H22O11+ 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.CO2is 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, theamount of acidity produced per glucose consumed will decrease, as with ethanol,

allowing faster growth.

Aerobic Respirat ion

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 modernscientific sense until around 1600.

Development of Fermentation Process

Fermentation has been used by humans for the production of food and beverages since

theNeolithic 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 madebetween 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.

19001920Ethanol, 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.

1977

1982- Fermentation ethanol processes adapted by wet millers for fuel grade ethanol

Fermentation Process in Industries

Fermented products have applications asfood as well as in generalindustry. Some

commodity chemicals, such asacetic acid,citric acid, andethanol are made by fermentation.

Nearly all commercially produced enzymes, such aslipase,invertase andrennet,are made by

fermentation withgenetically modified microbes.In some cases, production of biomass itself is

the objective, as in the case ofbaker's yeast and lactic acid bacteriastarter 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.

Continuou s 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 designedgrowth

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 fermentersor 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 froma 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/l200 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 :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 theorganism 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 concentrationwhen 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 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 thecells, 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, theexponential 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, steady-

state 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
https://controls.engin.umich.edu/wiki/index.php/File:Chemostat.jpg


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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 (Cc). 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 statewill 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 rateto slow until it is equal to the dilution rate

If > d the amount of substrate added will exceed the amount utilized. Therefore the growthrate 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 thedilution rate does not exceed a critical rate

Critical dilution rate:

( )

Biomass Product iv i ty

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 vesselpreparation, 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 individualfermentation process, but certain basic requirements must be met by any such medium. Allmicro-organisms require water, sources of energy, carbon, nitrogen, mineral elements andpossibly vitamins plus oxygen if aerobic. On a small scale it is relatively simple to devise amedium containing pure compounds, but the resulting medium, although supporting satisfactorygrowth 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 whichwill 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 aerationand 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 specificamount 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 inthe 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 micro-

organisms 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


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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).


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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 mixturesof 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.


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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.


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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 metabol ism

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.


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2. Rheology

The individual components of the medium can influence the viscosity of the final mediumand its subsequent behaviour with respect to aeration and agitation.

3. Anti foams

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 aredue 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.


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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 modif ication 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 Inoculum Development Medium Production Medium

Griseofulvin

Whey powder, Lactose} to give:

Lactose 3.5%

Nitrogen 0.05%

Corn-steep

Liquor solids 0.38%

(to give approx. 0.04% N)

Lactose 7%

Corn-steep

Liquor solids to give:

Nitrogen 0.2%

Limestone 0.8%

KH2PO4 0.4%


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KH2PO4 0.4%

KCl 0.05%

KCl 0.1%

Clavunalic

Acid

Soybean flour 1.0%

Dextrin 2.0%

Plunoric L81 0.03%

(Antifoam)

Soybean flour 1.5%

Oil 1.0%

KH2PO4 0.1%

Vitamin B12

(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

(g dm-3)

105

15

3

-

2.5

0.2

0.08

0.025

LysineCane molasses 5%Corn-steep Liquor 1%

CaCO31%

Soybean meal hydrolysate -

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 inoculummajor effect on performance of fermentation

Indicators of inoculum quality include dissolved oxygen, pH and CO2

Yeast, Bacteria and Fungicommon 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.


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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


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39

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


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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 acell)

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
http://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_(biology)http://en.wikipedia.org/wiki/Cell_(biology)


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Inoculum Quality Control

A. Pure Culture - Tests

Cultural methods - slow

Loop dilution- involves the successive transfer (serial dilution) of bacteria fromthe 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


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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. Ino cula for Fung al 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. Asept ic Inocu lat ion o f Plant Fermenters

Transfer from seed tank to plant-scale reactor is carried out aseptically.


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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. Ind ustr ial Product ion of L act ic 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

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