Biotechnology Unit 2:Fermentation Technology by Doctor-dr

Unit 2: Fermentation Technology

Fermentation

The modification or creation of goods with the aid of microbes is known as fermentation.
According to microbiologists, the word fermentation refers to any process that involves mass cultivation of a microbe to produce a product.
The term fermentation comes from the Greek word "fervere," which means "to boil," and refers to the action of yeast on yeast extracts or malted grain.
The appearance of boiling is due to the formation of CO2 bubbles during anaerobic sugar catabolism.
Since ancient times, it has been used to preserve and modify food and feed.

Fermentation Technology

Fermentation technology is the utilisation of organisms in large-scale commercial production of food, biomass, medicines, and alcoholic drinks.
Fermentation technology employs a variety of microorganisms from the following categories.

Category	Unicellular	Mutlicellular
Prokaryotes	Bacteria, cyanobacteria	Cyanobacteria
Eukaryotes	Yeast, algae	Fungi, algae

Microbes as a biocatalyst

An industrial microorganism must: generate a high yield of the desired product.
Grow quickly on low-cost culture medium in large quantities.
Be susceptible to genetic manipulation, such as mutation, and be genetically modified while being stable.
Make yourself non-pathogenic.

The range of fermentation process:

Fermentations that are commercially important fall into five categories:

Those who make microbial cells or biomass as a product, as well as those who make microbial enzymes
Those who are involved in the production of microbial metabolites
Those that create recombinant goods are known as recombinant producers.
Those who alter a chemical that is introduced to the fermentation process, also known as the transformation process.

1. Microbial Biomass Production

Fermentations that are commercially important fall into five categories:

The commercial production of microbial biomass can be divided into two major processes.
The production of Baker’s Yeast.
The production of microbial cells for human or animal food (single cell protein).
Microbial cells (biomass) are grown commercially as continuous culture on a large scale.

Single cell protein (SCP)

Algae, bacteria, yeasts, moulds, and mushrooms are dried and utilised as an excellent source of a complete protein known as "single cell protein (SCP)" that may be used as human food or animal feed.
The incubation of wheat flour for the making of ‘tandoori roti' is an excellent example of single cell protein synthesis.
The bacteria that make single cell protein eat a variety of things, from carbohydrates to hydrocarbons and petrochemicals. Other species use the gases as a substrate for fermentation, such as methane, ethane, propane, and n-butane.
Chlorella, Cellulomonas, Saccharomyces cerevisiae, Methylococcus capsulatus, and Aspergillus are some of the organisms utilised in single cell protein synthesis.

2. Microbial Enzymes

Fermentation technology may be used to generate microbial enzymes on a big scale.
Microbes produce enzymes into the media when grown, and these enzymes are recovered and utilised in a variety of sectors including detergent, food processing, brewing, and pharmaceuticals.
They're also employed in diagnostic, scientific, and analytical procedures.
Fermenting microorganisms create a variety of enzymes, including—
Glucoamylase, amylase, glucose isomerase, pectinase, cellulase, invertase, lactase, and lipase are all enzymes that break down glucose.
Glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, and restriction endonucleases, which are generated by Bacillus stearothermophillis, are examples of thermostable enzymes that may be utilised in industrial operations at high temperatures.

3. Microbial Metabolites

Many chemicals are generated during the metabolism of microbial cells, and many are released out of the cell, which may be readily collected and are highly helpful to humans and animals.
As a result, microbial cell fermentation is carried out on a large scale in order to get different metabolites.

The metabolites generated by microorganisms may be classified into two groups:

(a) Primary Metabolites

(b) Secondary Metabolites

(a) Primary Metabolites

Primary metabolites are metabolites that are created by a microbe's metabolism in order to maintain the microbe's minimal life process.
These metabolites are generated in large quantities at an early period of development, referred to as the trophophase.
Amino acids, nucleotides, proteins, nucleic acids, carbohydrates, lipids, and other primary metabolites are examples.
Many main metabolic products, including as ethanol, citric acid, lactic acid, acetic acid, acetone, formic acid, butanol, propionic acid, dihydroxy-acetone, and glycerol, are economically significant.

(b) Secondary Metabolites

Some microbial cultures generate chemicals that are not formed during trophophase and have no clear function in cell metabolism during stationary phase.
Secondary products of metabolism are known as idiophase chemicals, and the phase in which they are generated is known as idiophase.
The main metabolites are the source of the majority of secondary metabolites.
Hormones, enzyme inhibitors, antibiotics, alkaloids, poisonous pigments, vitamins, and other secondary metabolites are examples.
Primary metabolites are often discharged into the surrounding media, whereas secondary metabolites are trapped inside the cell and may only be retrieved when the cell walls have been lysed.

4. Recombinant Products

The use of recombinant DNA technology has increased the variety of fermented foods available.
Genes from higher species can be inserted into microbial cells, allowing recipient cells to produce foreign proteins.
E.coli, Saccharomyces cerevisiae, and filamentous fungus are some of the most commonly utilised bacteria as hosts for such systems.
Interferon, insulin, human serum albumin, epidermal growth factor, and other products generated by genetically engineered organisms are examples.
These processes are set up in such a manner that the products are released into the medium, the products are destroyed at a very low rate, and the foreign gene is expressed to its full potential.

5. Transformation or Modification of the substrate

Fermenting microorganisms are capable of converting more substrate into a more valued product.
Microbes operate as biocatalysts, which are more specific than chemical catalysts in that they allow the addition, removal, or alteration of functional groups at particular locations.
In comparison to chemical reagents, microbial reactions have the benefit of functioning at low temperatures and pressures without the need of potentially harmful heavy-metal catalysts.
Conversion of ethanol to acetic acid (vinegar), isopropanol to acetone, glucose to gluconic acid, sorbitol to sorbose (used in vitamin C production), and sterols to steroids are some examples.
The most frequently used fermentation biotechnology for the conversion of sterols into steroids, such as cortisone, hydroxycortisone, testosterone, and estradiol, is the manufacture of steroids.

Microbial Growth Kinetics

The kinetics of bacterial growth in a fermenter is described by microbial growth kinetics.

A microbe's development in a fermenter can be divided into the following stages.

1. Lag Phase

2. Exponential/Log Phase

3. Stationary Phase

4. Death/Decline Phase

1. The Lag Phase

In a fermentation process, it is the first significant phase of microbial growth.
It is the time when the cells adjust to their new surroundings.
At this point, cell density is at its lowest.

2. Exponential Phase

The logarithmic growth phase is also known as the Log phase.
The cells have adapted to their new surroundings.
The cells divide at a steady pace, resulting in an exponential growth in cell count.
At this point, the cell density is at its greatest.

3. The Stationary Phase

In a fermentation process, the third key phase of microbial development.
When the number of cells proliferating and dying is in balance, this occurs, and it can be caused by the following:
One or more critical growth components have been depleted.
Toxic by-products accumulate.
The induction of a recombinant gene is linked to stress.
The manufacturing of primary metabolites comes to a halt.
Production of secondary metabolites or non-growth-associated products may persist.

4. Death Phase

In a fermentation process, the fourth main phase of microbial development.
Also referred to as the decline stage.
The rate at which cells die is higher than the pace at which they divide.
The cells have generally eaten the majority of the nutrients in the medium and there isn't enough left for long-term survival.

Components of a Typical Fermentation process

1. Fermenter

2. Media

3. Inoculum

1. Fermenter

The fermentation process takes place in a container known as a fermenter or bioreactor.
These are employed in the industrial growth of microbial cells on a large scale under regulated circumstances.
The Fermenter's design and nature differ depending on the type of fermentation performed.
These range in size from a small laboratory fermenter to a large-scale industrial fermenter (several hundred litres capacity).

This closed metallic or glass vessel has the adequate arrangement for

aeration
mixing of media by agitation
temperature control by thermostat
pH control by pH meter
anti-foaming
control of overflow
sterilization of media and vessel
cooling
and sampling (removal of sample, while the Fermenter is on)

Type of fermenter:

Fermenters come in a variety of shapes and sizes.

External airlift for recycling Fermenter – a device that uses methanol as a substrate to produce bacterial biomass.
Internal airlift for recycling Fermenter – a device that uses oil as a substrate to produce yeast.
Fermenter with a tubular tower — Used to make beer, wine, and vinegar, among other things.
Nathan Fermenter is a kind of fermenter used in the brewing business.
Antibiotics are made in a stirred fermenter.

Types of Culture System:

Microbial system culture can be accomplished in a variety of methods.
The sort of culture method used is sometimes determined by the type of microbial system or the expected result.
Microbial cultures are categorised as follows, depending on the type of the desired product and the culture conditions:

1. Batch Culture

2. Fed-batch Culture

3. Continuous Culture

1. Batch Culture:

It is made up of a little amount of broth culture in a flask that has been infected with the bacterial or microbial inoculum and grows normally.
The medium includes a restricted quantity of nutrients that will be absorbed by the developing microorganisms for growth and multiplication, with the excretion of specific metabolites as products.
The nutrients in batch cultures are not replenished.
An early lag phase, a log phase or exponential growth phase, and a stationary phase make up the culture's growth phase.
During the log phase, nutrient consumption will be at its peak, resulting in the highest biomass output and product excretion.
The rate of growth slows and eventually stops at zero in the stationary phase.

2. Fed-batch Culture:

By providing fresh medium progressively at the conclusion of the log phase or the beginning of the stationary phase without removing cells, the batch culture can be converted to a semi-continuous or fed-batch culture.
As a result, the volume of culture will continue to grow as new media is introduced.
The fermenter is constructed in such a way that it can handle large quantities.
This technique can readily create a high cell density in the culture medium, something that a batch Fermenter may not be able to do.
Fed-batch culture is used in the industrial production of baker's yeast as biomass and penicillin as a secondary metabolite.

3. Continuous Culture:

Bacterial cultures may be kept in an exponential growth condition for lengthy periods of time utilising a continuous culture system.
The nutritional medium, including the raw material, is given at a rate equal to the volume of media with cells and product withdrawn from the culture in continuous culture.
The volume that has been withdrawn and the volume that has been added are the same.
In effect, neither the net volume nor the culture's chemical environment have changed.
Continuous cultivation is effective in industrial ethanol production.
Chemostats can be employed in continuous culture to keep a bacterial population at a constant density, which is more like bacterial growth in natural settings.

Chemostat:

The growing chamber in a chemostat is linked to a sterile media reservoir.
Fresh medium is continually fed from the reservoir after the growth has begun.
Some type of overflow drain keeps the volume of fluid in the growing chamber at a consistent level.
The bacterial cells multiply at the same pace that the overflow removes bacterial cells.

Procedure of fermentation

A specific bioreactor is chosen depending on the type of product required.
In liquid medium, a suitable substrate is introduced at a specified temperature, pH, and then diluted.
It contains the creature (microbe, animal/plant cell, subcellular organelle, or enzyme).
Then it's incubated for the prescribed amount of time at a certain temperature.
It's possible that the incubation will be aerobic or anaerobic.
By bubbling oxygen through the medium, aerobic conditions are generated.
Closed containers are used to generate anaerobic conditions, in which oxygen cannot penetrate into the medium and the oxygen existing above is replaced by carbon dioxide emitted.
Because some of the products are poisonous or at least inhibitive to the developing cell's development, they are eliminated after the prescribed time interval. The elimination of products is referred to as downstream processing.
The process's effluents must be disposed off.

Design of Industrial Fermentation Process

The fermentation process is divided in to two categories

1. Up stream Processing

2. Down stream Processing

1. Upstream Processing:

Pure culture of a specified organism in sufficient quantity and in the proper physiological condition is required for upstream processing.
A well crafted medium for organism development.
Sterilization of the medium, fermenters, and other apparatus.
A seed fermenter is a miniature fermenter used to create an inoculum that will start the fermentation process in the main fermenter.
The functional big fermenter is a production fermenter.

2. Downstream Processing:

Down stream processing includes:

Drawing the culture media in steady state
Cell separation
Collection of cell free supernatant
Product purification
Effluent treatment

Upstream Processing:

1. Formulation Mediator Microbial Culture

Microorganisms, like any other biological system, require energy, carbon, nitrogen, oxygen, iron and other minerals, micronutrients, and water to develop and multiply.
All of these nutrients are provided in the form of nutrient media, which are necessary for the growth and multiplication of microbial organisms.
Such nutritive media are known as synthetic media if the nutritive components are not of natural origin and may be synthesised in the laboratory using specific formulas.
There are a lot of commercially available nutrition media that have both salts and minerals in them. Semi-synthetic nutrition media are those that include such nutrients.
Semi- synthetic media include commercially available nutritional broth, trypticase soya broth (TSB), brain-heart infusion (BHI) broth, yeast extract, potato dextrose agar, and others.
These synthetic or semi-synthetic medium are recommended for laboratory-scale growth of bacteria and other microorganisms.
However, from a cost standpoint, these medium are not suggested for large-scale production.
The suggested medium for commercial use should be inexpensive and available all year.

Requirements of Microbial Medium

The following are the minimum components required in a microbial medium for cultivation of microbes in a laboratory:

Carbon source
Nitrogen sources
Microelements or trace elements
Growth factors
Anti-foams
Energy sources
Buffers
Water

1. Carbon source:

The carbon source's primary function is to supply energy and a carbon skeleton for the production of other biological molecules.
It consists of Sugars like glucose, lactose, and sucrose, complex polysaccharides like starch, glycogen cellulose, and a combination of other carbs, as well as other substances such cereal grain powders, cane molasses, and so on.

2. Sources of nitrogen:

Ammonium salts, urea, animal tissue extracts, amino acid combinations, and plant-tissue extracts are the most common nitrogen sources utilised in culture media.

3. Microelements (also known as trace elements):

Copper, cobalt, iron, zinc, manganese, magnesium, and other elements are required in tiny quantities.

4. Growth factors:

Growth factors are chemical substances that are required for cell growth and multiplication but cannot be produced by the cells themselves.
This category also includes certain amino acids and vitamins.

5. Anti-foams:

When the culture media is stirred for aeration, it might result in excessive foaming due to nutritive components such as starch, protein, and other organic material, as well as other products produced by the developing cells.
Anti-foaming agents are included in the media to prevent foam formation.
Anti-foam compounds include fatty acids like olive and sunflower oil, as well as silicones, which are widely employed in cell cultures.

6. Energy sources:

Carbon sources utilised in culture medium, including as carbohydrates, sugars, proteins, lipids, and others, can serve as energy sources for microbial cell growth and metabolism.

7. Buffers:

Calcium carbonate is used to buffer several media at a pH of around 7.0. (as chalk).
Phosphates, which are found in a variety of media, are also crucial in buffering.

8. Water:

Water is the foundation of all cultural medium, liquid or solid.
Single-distilled water or double-distilled water is commonly employed in scientific research.
However, when designing the medium needs and concentration for large-scale microbial growth for industrial applications, the pH and dissolved salts present should be taken into account.
Water is also necessary in the laboratory for a variety of additional functions such as cooling, heating, steaming, and so on.

2. Sterilization of Media and Fermenter

To avoid the growth of undesirable bacteria, the medium and fermenter must be sterilised.
The media, as well as the culture flasks, can be steam-sterilized with an autoclave or a pressure cooker if laboratory scale studies are carried out in 100 to 1,000 ml flasks, or in smaller quantities.
Steam sterilisation takes 15 to 20 minutes at 120°C under 15 psi pressure.
It is convenient to sterilise the fermenter as a whole, with or without medium, when microorganisms are cultured in a fermenter for large-scale operation.
By flowing steam via the sterilisation jacket or the coil around the fermenter, steam is utilised to sterilise the media and fermenter.
Steam may be fed into the fermentor through all holes while it is sterilised without media in it, enabling it to depart extremely slowly.
For 20 to 30 minutes, the steam pressure is kept at 15 psi.

3. Preparation of Inoculum for fermentation

It is essential that the culture used to inoculate a fermentation satisfy the following criteria:

It must be in a healthy, active state thus minimizing the length of the lag phase in the subsequent fermentation
It must be available in sufficiently large volumes to provide an inoculum of optimum size.
It must be in a suitable morphological form
It must be free of contamination
It must retain its product-forming capabilities
The process adopted to produce an inoculum meeting these criteria is called inoculum development
The quantity of inoculum normally used is between 3-10% of the medium volume

4. The Cultivation Process

First, the producing strain is grown in a tiny flask with nutrients and agar.
The flask is put in an incubator to ensure that the previously frozen/dried cells germinate at the appropriate temperature.
The cells are moved to a seed fermentor, which is a huge tank holding previously sterilised media, once the flask is ready.
Seed fermentation helps cells to proliferate while also allowing them to adapt to the environment and nutrients they will encounter later.
The cells are then moved to a bigger tank, the production fermentor, where conditions are closely monitored.
When the primary fermentation is finished, the broth (a combination of cells, nutrients, and enzymes) is available for downstream processing.

Downstream Processing

Downstream Processing:

It refers to the purification and recovery of fermentation products, as well as the recycling of recoverable components and efficient waste treatment and disposal.
This makes up the majority (about 85%) of the whole fermentation technology.
To begin, the broth is conditioned, which means that the cells have aggregated and formed big clumps, making separation simpler.
Heating, freezing, pH changes, antigen-antibody interactions, and other methods are used to condition the samples.
The conditioned broth is then utilised for constituent separation using methods such as sedimentation, flotation, filtration, ultra-filtration, centrifugation, and micro-filtration.

Cost Associated with Downstream Processing

Fermentation product extraction and purification may be complex and expensive.
Unfortunately, microbial product recovery costs might range from 15% to 70% of overall production expenses.
According to Atkinson and Mavituna (1991), industrial ethanol accounts for 15% of overall expenses, penicillin G accounts for 20%–30% of total costs, and enzymes account for up to 70% of total costs.
Extraction and purification expenses for products like recombinant proteins and monoclonal antibodies can account for 80–90% of overall processing costs.

Stages in a Downstream process

Downstream processing include:

1. Primary Unit Operations

2. Secondary Unit Operations

1. Primary Unit Operations of a Downstream Process

Removal of insolubles: it involves the removal of cells, cell debris or other particulate matter from the fermentation broth by techniques such as:

Filtration
Centrifugation
Sedimentation etc.

Product Isolation: it involves the removal of components whose properties vary markedly from that of desired product by techniques such as:

Solvent Extraction
Ultrafiltration
Precipitation

2. Secondary Unit Operations of a Downstream Process

Product Purification: it is done to separate contaminants closely resembling the product. It done by:

Affinity chromatography or ion exchange chromatography
Size exclusion and reverse phase chromatography
Crystallization
Fractional precipitation

Product polishing: it is the final processing step which ends with the packaging of the product in the form that is stable, easily transportable, and convenient. It is done by

Crystallization
Desiccation
Lyophilization and spray drying

Depending upon the product and its intended use, polishing may include steps for:

sterilization
dehydration
Blotting etc.

Obstacles in Good Recovery of Product

If a fermentation broth is analyzed at the time of harvesting, it will be discovered that the specific product may be present at a low concentration (typically 0.1–5 g dm−3) in an aqueous solution
The product may also be intracellular, heat labile, and easily broken down by contaminating microorganisms
All these factors tend to increase the difficulties of product recovery
To ensure good recovery or purification, the processing equipment must be of the correct type and also the correct size to ensure that the harvested broth can be processed within a satisfactory time limit
It should also be noted that each step or unit operation in downstream processing will involve the loss of some product
Hence, it is also important that the minimum number of operations possible are used to maximize product recovery.

Criteria for Correct Choice of Recovery Process

The choice of recovery process is based on the following criteria:

The intracellular or extracellular location of the product
The concentration of the product in the fermentation broth
The physical and chemical properties of the desired product
The intended use of the product
The minimal acceptable standard of purity
The magnitude of biohazard of the product or broth
The impurities in the fermenter broth
The marketable price for the product

Downstream processing depends upon product use

1. Enzyme preparation for animal feed supplement (e.g., phytase are not purified.

2. Enzyme for industrial use may be partially purified (e.g, amylase for starch industry)

3. Enzymes for analytical use (e.g. glucose oxidase) and pharmaceutical proteins (e.g., TPA) are very highly purified.

The recovery of an Extracellular Product

The removal of big solid particles and microbial organisms, generally by centrifugation or filtration, is the primary goal of the first step in the recovery of an extracellular product.
Using ultrafiltration, reverse osmosis, adsorption/ion-exchange/gel filtration, affinity chromatography, or precipitation, the broth is fractionated or extracted into main fractions in the following stage.
The product-containing fraction is then purified using fractional precipitation, chromatographic methods, and crystallisation to produce a finished product.
Modifications to this flow-stream are used to isolate other products.
Finally, the completed product might need to be dried.

The recovery of an intracellular product

Microorganisms have very strong cell walls that protect them. A variety of cell disintegration techniques have been developed in order to liberate their cellular contents.

The methods offered are divided into two categories:

Physicomechanical methods

Liquid shear
Solid shear
Agitation with abrasives
Freeze–thawing
Ultrasonication

Chemical and biological methods

Detergents
Osmotic shock
Alkali treatment
Enzyme treatment
Solvents

Drying of Products:

The drying of any product (including biological products) is often the last stage of a manufacturing process.

It involves the final removal of water or other solvents from a product, while ensuring that there is minimum loss in viability, activity, or nutritional value.

Drying is undertaken because:

The cost of transport can be reduced
The material is easier to handle and package
The material can be stored more conveniently in the dry state

To save money on heating during the drying process, it's critical to remove as much water as possible first using centrifuge or a filter press.
Driers are categorised according to the manner of heat transmission to the product and the degree of product agitation.
Drying the tray: Simple tray driers are used for various items, where the product is put on trays over which air is circulated in a heated oven.
Drying by contact: The product is touched with a heated surface in contact driers.
The drum drier is an example of this kind, which may be used to dry bioproducts that are more temperature stable.
A slurry is poured into a gently revolving steam-heated drum, where it evaporates and a scraper blade removes the dry result.

3. Spray drying: this method is most commonly used to dry biological materials that are in the form of a liquid or paste.

The material to be dried is atomized into minute droplets by a nozzle or by contact with a revolving disc rather than coming into contact with the heating surfaces.
The droplets are then sucked into a spiral stream of hot gas at a temperature of 150–250°C.
Because the droplets have a large surface area to volume ratio, they evaporate quickly and dry completely in a matter of seconds.

4. Freeze drying (sometimes referred to as lyophilization or cryodesiccation) is a crucial step in the manufacturing of many biologicals and medicines.

The substance is first frozen, then dried in a high vacuum by sublimation, followed by secondary drying to eliminate any remaining moisture.
The fact that this method does not damage heat-sensitive materials is a huge plus.

Effluent Treatment:

Raw ingredients are used in every fermentation factory, which are then transformed into a range of products.
Various volumes of a variety of waste products are created depending on the specific procedure.
Unused inorganic and organic media components, microbial cells and other suspended particles, waste wash water from cleaning operations, cooling water, water containing traces of solvents, acids, and alkalis, and so on are examples of typical wastes.

Treatment and disposal of effluents

The range of effluent-disposal methods, which can be considered are as follows:

The effluent is discharged to land, river, or sea in an untreated state
The effluent is removed and disposed of in a landfill site or is incinerated
The effluent is partially treated on site prior to the further treatment or disposal by one of the other routes
All of the effluent is sent to the sewage works for treatment
All the effluent is treated at the factory before discharge

It may be possible to recover waste organic material as a solid and sell it as a byproduct which may be an animal feed supplement or a nutrient to use in fermentation media

The marketable by-product helps to offset the cost of the treatment process

Historically, it was possible to dispose of wastes directly to a convenient area of land or into a nearby watercourse
This cheap and simple method of disposal is now very rarely possible, nor is it environmentally desirable
With increasing density of population and industrial expansion, together with greater awareness of the damage caused by pollution, the need for treatment and controlled disposal of waste has continued to grow