Bioproess/Fermentation Technology

The process of fermentation is defined as “a biological process that occurs in the absence of oxygen or under anaerobic conditions. The word “Fermentation” originates from verb of Latin origin “fervere” which means to boil. However in the modern times the Industrial fermentation is used for large- scale cultivation of microorganisms which are largely aerobic in nature. Instead of fermentation technology, the term Bioprocess technology is more in use as bioprocessing involves a variety of enzyme-catalysed reactions carried out by living cells (or cell free systems) in industrial setups. The most important equipment in use for bioprocessing is a Fermenter or a Bioreactor.

Bioreactor

A bioreactor is a device in which the microorganisms are cultivated and motivated to form the desired products by maintaining optimum conditions for growth and metabolic activity. A fermenter refers to the device used for the cultivation of prokaryotic cells e.g. bacteria, fungi etc. whereas a bioreactor is used for growing eukaryotic cells. A typical conventional bioreactor has cylindrical vessel with domed top and bottom generally made up of stainless steel. The reaction vessel which is surrounded by a jacket, is provided with a sparger at the bottom through which air (or other gases such as CO2 and NH3 (for pH maintenance) can be introduced. The reaction vessel also has side ports for pH, temperature and dissolved O2 sensors. The agitator shaft is connected to a motor at the bottom. Above the liquid level of the reaction vessel, connections for acid, alkali, antifoam chemicals and inoculum are located. The bioreactor is designed to work at very high temperatures (150-1800C), high pressure (377-412 kPa) and also to withstand vacuum which prevents its collapse while cooling.

 

Types of Bioreactors

Depending on the design of the reactor, the bioreactors are of following types:

a) Continuous stirred tank bioreactors - These bioreactors have a cylindrical vessel with motor driven central shaft which gives support to one or more agitators (impellers). The shaft is fitted at the bottom of the bioreactor. The diameter of the impeller is usually one third of the vessel diameter. The impellers are available in different designs like- Rustom disc, concave bladded, marine propeller etc. In stirred tank reactors, the air is added to the culture medium under pressure through a device called sparger. The sparger along with the impellers (agitators) enables better and efficient gas distribution through out in the vessel. The advantages of using stirred tank reactors are: the efficient transfer of gas to growing cells which keeps the growth of cells in healthy limits, stirring ensures good mixing of the contents, the operating conditions are flexible and the bioreactors are easily available which makes them commercially viable products.

b) Bubble column bioreactors - In these bioreactors, the gas or air is introduced at the base of the column through perforated pipes or plates, or metal microporous spargers. The vessel used for bubble column bioreactors is usually cylindrical with an aspect ratio (height o diameter ratio) of 4-6. The rate of flow of gas affects the O2 transfer and mixing.

c) Airlift bioreactors - Airlift bioreactors are commonly used for aerobic bioprocessing technology. In the airlift bioreactors, the medium of the vessel is divided into two interconnected zones by means of baffle or draft tube. The air/gas is pumped into one of the two zones referred to as ‘riser’ and the other zone that receives no gas is known as ‘downcomer’. The dispersion flows up the riser zone while the down flow occurs in the downcomer. Further there are two types of bioreactors: 1) Internal loop bioreactor - These bioreactors have a single container with a central draft tube that creates interior liquid circulation channels which keeps the volume and circulation at a fixed rate for fermentation. (2) External loop airlift bioreactor-These have an external loop to keep the liquid in circulation through separate independent channels. The modifications can be made in these bioreactors depending on the requirements of different fermentation processes.(3) Two stage airlift bioreactors -These bioreactors have two bioreactors which are basically used for the temperature dependent formation of products. The growing cells from one bioreactor (maintained at temperature 300C are pumped into another bioreactor (at temperature 420C). This is done because it is very difficult to increase the temperature quickly from 300C to 420C in the same vessel. The cells are grown in the first bioreactor and with the help of the fitted valves and a transfer tube and pump, they are transferred into the second bioreactor, where the actual bioprocessing takes place. (4) Tower bioreactors - In this type of bioreactor, a high hydrostatic pressure is generated at the bottom of the reactor which increases the solubility of O2 in the medium. Since the top is expanded, the pressure is reduced which helps in the expulsion of CO2. The cycle completes with the medium flowing back into the downcomer. The advantage with Tower bioreactor is that it has high aeration capacities without having moving parts.

d) Fluidized bed bioreactors - These bioreactors are mainly suitable to carry out reactions involving fluid suspended biocatalysts such as immobilized enzymes, immobilized cells, microbial flocs etc. The design of the bioreactors is such that the top is extended and the reaction column is narrow which retains the solids in the reactor and allows the liquids to flow out. To maintain an efficient operation of fluidized beds, gas is sparged to create a suitable gas-liquid-solid fluid bed. The recycling of the liquid ensures continuous contact between the reaction contents and biocatalysts which increases the efficiency of bioprocessing.

e) Packed bed bioreactors- A packed bed bioreactor consists of a bed of solid particles, with biocatalysts on or within the matrix of solids, packed in a column. The solids are generally porous or non-porous gels which maybe compressible or rigid in nature. The nutrient broth continuously flows over the immobilized biocatalyst and the products are released into the fluid from where they are removed. However, due to poor mixing, it is difficult to control the pH of packed bioreactors by the addition of acid or alkali.

f) Photobioreactors - These bioreactors are specialized for fermentation that can be carried out either by exposing to sunlight or artificial illumination. The photobioreactors are made up of glass or transparent plastic which are the solar receivers. The cell cultures are circulated through the solar receivers by using centrifugal pumps or airlift pumps. These bioreactors work in the temperature which ranges from 25-400C. In these bioreactors, the microorganisms e.g. microalgae, cyanobacteria etc. grow during the day time while the products (e.g. beta-carotene, asthaxanthin) are produced during the night.

Solid substrate Fermentation (SSF)

In some biotechnological processes, the growth of the microorganisms is carried out on solid substrates more or less in the absence of free water. Only approximately 15% of moisture is present which is essential for solid-substrate fermentation. Cereal grains, wheat bran, sawdust, wood shavings etc are some of the commonly used solid substrates for SSF. The technique of SSF is used for the production of edible mushrooms, cheese, soy sauce, and many enzymes and organic acids. It is carried out as non-aspetic process and therefore saves sterilization costs. The bioreactors used in this type of fermentation process have simple designs with simple aeration process and effluent treatment. The yield of the products is very high, at low energy expenditure. However, in this process only microorganisms, that can tolerate only low moisture content can be used. It also difficult to monitor O2 and CO2 levels in SSF. The slow growth of microorganisms, also become a limiting factor for product formation.

Working of a bioreactor

In the operation of a bioreactor, there are a few steps that are followed.

a) Sterilization - The most important requirement is to maintain aseptic or sterile conditions for aseptic fermentation. In order to achieve this, the air supplied during fermentation, he growth medium and the bioreactor it self and all it’s accessories are sterilized. There are two methods of sterilization that are followed: (1) In situ sterilization- In this, the bioreactor is filled with the required medium followed by injection of pressurized steam into the jacket or coil surrounding the reaction vessel. The whole system is heated to about 1200C and maintained at this temperature for about 20 minutes. However, this method is not energy efficient and prolonged heating destroys the vitamins and precipitates the components of the medium. (2) Continuous heat sterilization - In this, the empty bioreactor is first sterilized by injecting pressurized steam and the medium is rapidly heated to 1400C for a short period again by injecting the pressurized steam. This is an energy efficient method and also does not precipitate the medium components.

b) Aeration - Oxygen is stored in compressed tanks and is introduced at the bottom of the bioreactor through a ‘sparger’. Aeration of the fermentation medium supplies oxygen to the production microorganisms and remove carbon dioxide from the bioreactor. The gases released during the fermentation accumulate in the headspace and then pass out through an air outlet. The headspace is a vacant space on the upper part of the bioreactor and is generally about 20% of it’s volume. The air-lift system of aeration involves sparging of air done at the bottom of the fermenter with an upward flow of air bubbles. The aeration capacity of the system depends on the air-flow rate and the internal pressure. The stirred system of aeration involves increasing the aeration capacity by stirring using impellers driven by motor. The aeration capacity of the fermenter depends on the rate of stirring, rate of air flow and internal pressure.

c) Inoculation and sampling - The sterilized bioreactors with growth medium are inoculated with the production organisms. The size of the inoculum is generally 1-10% of the total volume of the medium. During the fermentation process, the samples are regularly withdrawn to check contamination and to measure the amount and quantity of product formed.

d) Control systems - Various factors like- pH, temperature, dissolved oxygen, adequate mixing, concentration of the nutrients, foam formation etc are continuously monitored to maintain optimal growth environment in the bioreactor. Very sensitive sensors are available which carry out automated monitoring of these variables. The ideal pH range for optimal growth of microorganisms is between 5.5- 8.5. The pH changes due to the release of metabolites into the medium by the growing microorganisms. The required pH level is maintained by adding acid or alkali followed by thorough mixing of the medium components. The optimal temperature is maintained by using the heating and cooling systems fitted in the bioreactor. Continuous monitoring of dissolved oxygen concentration is also a must for the optimal bioreactions. The oxygen is sparingly soluble in water (0.0084g/l at 250C) and is introduced into the bioreactor as the air bubbles.

The concentration of the nutrients is also important because the limiting concentrations of nutrients helps in the optimal product formation and the high nutrient concentrations have inhibitory effect on the microbial growth. Another important factor to control is the “foam formation”. The protein rich media is used in industrial fermentation which leads to ‘froth’ or ‘foam formation’ on agitation during aeration. Some antifoam chemicals lower the surface tension of the medium and causes the foam bubbles to collapse. Mineral oils with silicone or vegetable oils are also used as antifoam agents. The bioreactors can also be fitted with mechanical foam control devices which break the foam bubbles and throw back into the fermentation medium. The proper and continuous mixing of the microbial culture is very important to maintain optimal levels of oxygen in the nutrient medium and to prevent the accumulation of toxic metabolic products.

e) Cleaning - After the completion of the fermentation process, the products are ‘harvested’ (removal of contents for processing) and the bioreactor is prepared for the next round of fermentation after cleaning technically referred to as ‘turn around’. The cleaning of the bioreactors is carried out by using high-pressure water jets from the nozzles fitted into the reaction vessel. In order to maintain the cost effectiveness of the bioreactor, the time taken for turn around which is known as ‘down time’, is kept as short as possible.

 

Downstream processing (DSP)

The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing. The methods adopted for downstream processing depends on the nature of the end products, it’s concentration, stability, and the degree of purification required. Both intracellular metabolites as well as extracellular metabolites are isolated by DSP. The intracellular metabolites are products located within the cells e.g. vitamins, enzymes etc. The extracellular metabolites are the products present outside the cells e.g. most antibiotics, amino acids, alcohol, citric acid, enzymes like amylases, proteases etc. some products like vitamin B12, flavomycin etc are present both as intracellular as well as extracellular products.

Downstream processing involves a number of steps which are as follows:

a) Solid-liquid separation - The first step is to separate whole cells and other insoluble substances from the culture broth. This is done by using several methods:

1) Flotation- The process of aeration involves the bubbling of gas in to the liquid broth. The cells and other solid particles get adsorbed on gas bubbles which form a foamy layer which is collected and removed.
2) Flocculation - The cells or cellular debris form large aggregates and settle down which can be easily removed. Some flocculating agents like inorganic salt, organic poly-electrolyte, mineral hydrocolloid etc are often used to achieve appropriate flocculation.
3) Filtration - this is the most commonly used technique to separate the biomass and culture filtrate. The rate of filtration depends on many factors such as the size of the organism, presence of other organisms, viscosity of the medium, and temperature. Several filters like depth filters, absolute filters, rotary drum vacuum filters, membrane filters etc. are used. There are three major types of filtration processes used depending on the size of the particles- microfiltration, ultrafiltration, and reverse osmosis.
4) Centrifugation - The centrifugation is mostly used for separating solid particles from liquid phase. The technique of centrifugation is based on the principle of density differences between the particles to be separated and the medium. In the bioreactors, the continuous flow industrial centrifuges are used where there is a continuous feeding of the slurry and collection of clarified fluid. The solid deposits are intermittently removed. Various models of centrifuges used are: Tubular bowl centrifuge, Disc centrifuge, Multichamber centrifuge, Scroll centrifuge or decanter etc.

b) Release of intracellular products - The biotechnological products that are located with in the cells like vitamins, enzymes, etc are released in an active form for their further processing and isolation. The microorganisms or cells can de disintegrated or disrupted by physical, chemical, or enzymatic methods depending on the nature of the cells.

1) Physical methods of cell disruption are (i) Ultrasonication, (ii) Osmotic shock (used for releasing hydrolytic enzymes and binding proteins from gram-negative bacteria), (iii) Heat Shock treatment, (iv) High pressure homogenization, (v) Impingement which involves hitting a stationary surface or a second stream of suspended particles with a stream of suspended cells at high velocity and pressure. Microfluidizer is a device developed on the basis of the principle of impingement. (vi) Grinding with glass beads where the cells mixed with glass beads are subjected to a very high speed in a reaction vessel. The cells break as they are forced against the wall of the vessel by the beads
2) Chemical methods- Treatment with alkalies, organic solvents, and detergents lyse the cells to release the content. Alkali treatment is used for the extraction of some bacterial proteins. The organic solvents like methanol, ethanol, isopropanol, butanol etc also disrupt the cells. The organic solvent, toluene, which is commonly used, dissolves membrane phospholipids and creates the membrane pores to release intracellular contents. The ionic detergents denature the membrane proteins and lyse the cells e.g. cationic-cetyl trimethyl ammonium bromide or anionic-sodium lauryl sulfate. Non-ionic detergents are less reactive and also affect the purification steps.
3) Enzymatic methods - Lysozyme is the most commonly used enzyme which hydrolyses beta-1,4-glycosidic bonds of the mucopepide in bacterial cell walls e.g. gram positive bacteria. This enzyme is commercially available produced from hen egg white. For gram-negative bacteria, lysozymes are in use with EDTA to break the cells. When the cell wall gets digested by lysozyme, the periplasmic membrane breaks due to osmotic pressure, which releases the intracellular contents. Glucanase and mannanase along with proteases lyse the cell wall of yeast.

c) Concentration - The biological products are concentrated by getting rid of water which is present in the filtrate. Depending on the nature of the desired products and the cost effectiveness, the techniques used to concentrate the biotechnological products are evaporation, liquid-liquid extraction, membrane filtration, precipitation, and adsorption.

(i) Evaporation - The water from the broth is removed by the process of evaporation using evaporators. The evaporators use a heating device which supplies the steam. There is a unit for the separation of concentrated product and vapour and a condenser for condensing vapour. The commonly used evaporators are Plate evaporator, Falling film evaporator, Forced film evaporator, Centrifugal forced film evaporator.
(ii) Liquid-Liquid extraction - In liquid-liquid extraction, the biological products are concentrated by transferring the desired product from one liquid phase to another liquid phase. The process of liquid-liquid extraction is categorized as extraction of low molecular weight products and extraction of high molecular weight products.
(iii) Membrane filtration - This technique involves the use of a semipermeable membrane that selectively retains the particles/molecules that are bigger than the pore size while the smaller molecules pass through the membrane pores. The membranes are made up of polymeric materials such as polyethersulfone and polyvinyl difluoride. Now a days, microfilters and ultrafilters made up of ceramics and steel are being used which are easy to clean and sterilize. Pervaporation is a technique in which the volatile products are separated by a process of permeation through a membrane coupled with evaporation and is used to extract and concentrate volatile products. Perstraction- This technique is used to recover and concentrate hydrophobic compoundsvand is based on the principle of membrane filtration coupled with solvent extraction.
(iv) Precipitation - This is the most common method used to concentrate proteins and polysaccharides in industrial processes. The alteration in temperature and in pH, neutral salts, organic solvents, high molecular weight polymers (ionic and non ionic) etc are used in precipitation. Neutral salts like commonly used ammonium sulphate increases the hydrophobic interactions between protein molecules which leads to their precipitation. Ethanol, acetone and propanol are the commonly used organic solvents for protein precipitation which reduce the dielectric constant of the medium and increase the electrostatic interaction between the protein molecules. The precipitation process is carried out below 00C because the proteins get denatured by organic solvents. Polyethylene glycol (PEG) is a high weight non-ionic polymer that also precipitates the proteins by reducing the quantity of water available for protein salvation. In the category of ionic polymers e.g. polyacrylic acid, polyethyleneimine are used which form complexes with oppositely charged protein molecules and neutralize the charges. This leads to the precipitation of proteins. Besides this, the physical factors like increase in temperature, increase in pH, etc also leads to the precipitation of proteins. Besides these, the affinity precipitation (affinity interaction e.g. between antigen and antibody)and precipitation using ligands is also used.

d) Purification – The products of fermentation are purified by using the technique of chromatography. Chromatography consists of a stationary phase and a mobile phase. The porous solid matrix packed in a column constitutes the stationary phase and the mixture of the compound to be separated is loaded on this. The compounds are eluted by a mobile phase. A large number of matrices are commercially available for the purification of proteins e.g. agarose, cellulose, porous silica, cross linked dextran etc. Some of the commonly used chromatography techniques used are: Gel-filtration chromatography- In this technique, the separation of molecules is based on the size, shape and molecular weight using sponge-like gel beads with pores serving as molecular sieves for separation of smaller and bigger molecules. The Ion-exchange chromatography involves the separation of molecules based on their surface charges. It is useful for the purification of antibiotics, besides the purification of proteins. In ion exchange chromatography, the pH of the medium is very crucial because the net charge varies with pH. The ionic bound molecules are eluted from the matrix by changing the pH of the eluant or by increasing the salt concentration. The ion-exchangers are of two types- Cation exchangers- having negatively charged groups like carboxymethyl and sulfonate e.g. Dowex, HCR, Amberlite IR etc. Anion exchangers have positively charged groups like DEAE (diethylaminoethyl) e.g. Dowex SAR, Amberlite IRA etc. The Affinity chromatography is based on an interaction of a protein with an immobilized ligand. The ligand can be a specific antibody, substrate, substrate analogue or an inhibitor. The protein bound to the ligand is eluted out by changing the pH of the buffer, altering the ionic strength or by using another free ligand molecule.

e) Formulation- The maintenance of activity and stability of biotechnological products during storage and distribution is known as ‘formulation’. As proteins are highly sensitive and lose their biological activity, hence their formulations requires special care. In order to prolong their shelf life, certain stabilizing additives are added e.g. sugars (sucrose, lactose), salts (sodium chloride, ammonium sulphate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol). Proteins are formulated in the form of solutions, suspensions or dry powder. For some small molecules (antibiotics, citric acid), formulation is done by crystallization using salts. ‘Drying’ is an important component of product formulation which involves the transfer of heat to a wet product for removal of moisture. The commercially available dryers in use are- contact, convection and radiation dryers. Spray drying is used for drying large volumes of liquids where small droplets of liquid containing the product are passed through a nozzle directing over a stream of hot gas. After the evaporation of water, the solid particles are left behind.

Freeze drying or lyophilization is the most preferred method for drying and formulation of a wide range of products- pharmaceuticals, food stuff, bacteria, viruses etc. Freeze drying does not cause loss of biological activity of the desired product. Lyophilization is caused due to the sublimation of a liquid from a frozen state. The product with the liquid is frozen and then freeze dried under vacuum. After releasing the vacuum, the product containing vials are sealed e.g. penicillin is freeze dried directly in ampules.

It is ideal to integrate the fermentation and downstream processing to finally get the desired product. The efforts are on the integrate these steps and a limited success has also been achieved.

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