The process of fermentation is defined as “a biological process with the intention of occurs in the absence of oxygen or under anaerobic conditions. The word “Fermentation” originates from verb of Latin origin “fervere” which earnings to boil. However in the present era the Industrial fermentation is used pro large- extent encouragement of microorganisms which are largely aerobic in nature. Instead of fermentation equipment, the stretch Bioprocess equipment is more in aid as bioprocessing involves a variety of enzyme-catalysed reactions conceded made known by living cells (or cell emancipated systems) in manufacturing setups. The generally valuable equipment in aid pro 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 pro growth and metabolic endeavor. A fermenter refers to the device used pro the encouragement of prokaryotic cells e.G. Bacteria, fungi and that. While a bioreactor is used pro growing eukaryotic cells. A predictable square bioreactor has cylindrical vessel with domed top and underside commonly made up of stainless steel. The result vessel which is surrounded by a jacket, is provided with a sparger by the underside through which air (or other gases such as CO2 and NH3 (for pH maintenance) can be introduced. The result vessel furthermore has feature ports pro pH, warmth and dissolved O2 sensors. The agitator bar is connected to a motor by the underside. Above the liquid level of the result vessel, relations pro acid, alkali, antifoam chemicals and inoculum are located. The bioreactor is designed to bring about by very distinguished temperatures (150-1800C), distinguished pressure (377-412 kPa) and furthermore to survive 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 be inflicted with a cylindrical vessel with motor driven central bar which gives support to lone or more agitators (impellers). The bar is fitted by the underside of the bioreactor. The diameter of the impeller is ordinarily lone third of the vessel diameter. The impellers are unfilled in uncommon designs like- Rustom disc, bowl-shaped bladded, marine propeller and that. Inside stirred tank reactors, the air is added to the culture standard under pressure through a device called sparger. The sparger along with the impellers (agitators) enables better and efficient chatter distribution through made known in the vessel. The advantages of using stirred tank reactors are: The efficient conveying of chatter to growing cells which keeps the growth of cells in healthy limits, stirring ensures skilled mixing of the contents, the operating conditions are flexible and the bioreactors are straightforwardly unfilled which makes them commercially viable products.
B) Bubble discourse bioreactors - inside these bioreactors, the chatter or air is introduced by the corrupt of the discourse through perforated pipes or plates, or metal microporous spargers. The vessel used pro bubble discourse bioreactors is ordinarily cylindrical with an aspect ratio (height o diameter ratio) of 4-6. The rate of tide of chatter affects the O2 conveying and mixing.
C) Airlift bioreactors - Airlift bioreactors are commonly used pro aerobic bioprocessing equipment. Inside the airlift bioreactors, the standard of the vessel is on bad terms into two unified zones by earnings of baffle or recruit tube. The air/gas is pumped into lone of the two zones referred to as ‘riser’ and the other zone with the intention of receives thumbs down chatter is renowned as ‘downcomer’. The dispersion flows up the riser zone while the down tide occurs in the downcomer. Further here are two types of bioreactors: 1) Internal disk bioreactor - These bioreactors be inflicted with a single container with a central recruit tube with the intention of creates interior liquid passage channels which keeps the volume and passage by a fixed rate pro fermentation. (2) External disk airlift bioreactor-These be inflicted with an outdoor disk to keep the liquid in passage through separate self-determining channels. The modifications can be made in these bioreactors depending on the supplies of uncommon fermentation processes.(3) Two stage airlift bioreactors -These bioreactors be inflicted with two bioreactors which are basically used pro the warmth dependent formation of products. The growing cells from lone bioreactor (maintained by warmth 300C are pumped into a further bioreactor (at warmth 420C). This is made since it is very trying to boost the warmth quickly from 300C to 420C in the same vessel. The cells are developed in the initially bioreactor and with the help of the fitted valves and a conveying tube and pump, they are transferred into the following bioreactor, everywhere the real bioprocessing takes place. (4) Tower bioreactors - inside this type of bioreactor, a distinguished hydrostatic pressure is generated by the underside of the reactor which increases the solubility of O2 in the standard. Since the top is prolonged, the pressure is cut-rate which helps in the ejection of CO2. The cycle completes with the standard flowing back into the downcomer. The benefit with Tower bioreactor is with the intention of it has distinguished aeration capacities lacking having tender parts.
D) Fluidized bed bioreactors - These bioreactors are primarily apposite to involve made known reactions relating fluid floating biocatalysts such as immobilized enzymes, immobilized cells, microbial flocs and that. The design of the bioreactors is such with the intention of the top is extended and the result discourse is narrow which retains the solids in the reactor and allows the liquids to tide made known. To keep up an efficient surgical procedure of fluidized beds, chatter is sparged to create a apposite gas-liquid-solid fluid bed. The recycling of the liquid ensures unremitting friend linking the result 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 surrounded by the matrix of solids, packed in a discourse. The solids are commonly porous or non-porous gels which maybe compressible or rigid in nature. The nutrient gumbo continuously flows ended the immobilized biocatalyst and the products are released into the fluid from everywhere they are indifferent. However, due to poor mixing, it is trying to control the pH of packed bioreactors by the addition of acid or alkali.
F) Photobioreactors - These bioreactors are specialized pro fermentation with the intention of can be conceded made known either by exposing to sunlight or reproduction illumination. The photobioreactors are made up of schooner 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 bring about in the warmth which ranges from 25-400C. Inside these bioreactors, the microorganisms e.G. Microalgae, cyanobacteria and that. Grow all through the time calculate while the products (e.G. Beta-carotene, asthaxanthin) are produced all through the night.
Solid substrate Fermentation (SSF)
Inside approximately biotechnological processes, the growth of the microorganisms is conceded made known on solid substrates more or a reduced amount of in the absence of emancipated fill up. Only approximately 15% of dampness is bestow which is essential pro solid-substrate fermentation. Cereal grains, wheat cellulose, sawdust, wood shavings and that are approximately of the commonly used solid substrates pro SSF. The practice of SSF is used pro the production of edible mushrooms, cheese, soy sauce, and many enzymes and organic acids. It is conceded made known as non-aspetic process and therefore saves sterilization expenditure. The bioreactors used in this type of fermentation process be inflicted with unadorned designs with unadorned aeration process and sewage behavior. The yield of the products is very distinguished, by low energy expenditure. However, in this process single microorganisms, with the intention of can tolerate single low dampness content can be used. It furthermore trying to watch O2 and CO2 levels in SSF. The gradual growth of microorganisms, furthermore be converted into a limiting thing pro manufactured goods formation.
Working of a bioreactor
Inside the surgical procedure of a bioreactor, here are a hardly any steps with the intention of are followed.
A) Sterilization - The generally valuable requirement is to keep up aseptic or sterile conditions pro aseptic fermentation. Inside order to realize this, the air supplied all through fermentation, he growth standard and the bioreactor it self and all it’s accessories are sterilized. There are two methods of sterilization with the intention of are followed: (1) inside situ sterilization- inside this, the bioreactor is to the top with the vital standard followed by injection of hassled steam into the jacket or coil surrounding the result vessel. The total logic is heated to in this area 1200C and maintained by this warmth pro in this area 20 minutes. However, this method is not energy efficient and prolonged heating destroys the vitamins and precipitates the components of the standard. (2) Continuous excitement sterilization - inside this, the unfilled bioreactor is initially sterilized by injecting hassled steam and the standard is speedily heated to 1400C pro a fleeting cycle again by injecting the hassled steam. This is an energy efficient method and furthermore does not hurried the standard components.
B) Aeration - Oxygen is stored in compressed tanks and is introduced by the underside of the bioreactor through a ‘sparger’. Aeration of the fermentation standard equipment oxygen to the production microorganisms and remove carbon dioxide from the bioreactor. The gases released all through the fermentation accumulate in the headspace and at that time pass made known through an air outlet. The headspace is a vacant interval on the high part of the bioreactor and is commonly in this area 20% of it’s volume. The air-lift logic of aeration involves sparging of air made by the underside of the fermenter with an upward tide of air bubbles. The aeration room of the logic depends on the air-flow rate and the domestic pressure. The stirred logic of aeration involves increasing the aeration room by stirring using impellers driven by motor. The aeration room of the fermenter depends on the rate of stirring, rate of air tide and domestic pressure.
C) Inoculation and sampling - The sterilized bioreactors with growth standard are inoculated with the production organisms. The size of the inoculum is commonly 1-10% of the whole volume of the standard. During the fermentation process, the samples are evenly withdrawn to check contamination and to rate the amount and quantity of manufactured goods formed.
D) Control systems - Various factors like- pH, warmth, dissolved oxygen, passable mixing, concentration of the nutrients, foam formation and that are continuously monitored to keep up optimal growth background in the bioreactor. Very insightful sensors are unfilled which involve made known automated monitoring of these variables. The ideal pH range pro optimal growth of microorganisms is linking 5.5- 8.5. The pH changes due to the relief of metabolites into the standard by the growing microorganisms. The vital pH level is maintained by count acid or alkali followed by thorough mixing of the standard components. The optimal warmth is maintained by using the heating and cooling systems fitted in the bioreactor. Continuous monitoring of dissolved oxygen concentration is furthermore a should pro the optimal bioreactions. The oxygen is sparingly soluble in fill up (0.0084g/l by 250C) and is introduced into the bioreactor as the air bubbles.
The concentration of the nutrients is furthermore valuable since the limiting concentrations of nutrients helps in the optimal manufactured goods formation and the distinguished nutrient concentrations be inflicted with inhibitory effect on the microbial growth. Another valuable thing to control is the “foam formation”. The protein rich media is used in manufacturing fermentation which leads to ‘froth’ or ‘foam formation’ on agitation all through aeration. Some antifoam chemicals decrease the go up tension of the standard and causes the foam bubbles to collapse. Mineral oils with silicone or vegetable oils are furthermore used as antifoam agents. The bioreactors can furthermore be fitted with mechanical foam control diplomacy which break the foam bubbles and toss back into the fermentation standard. The proper and unremitting mixing of the microbial culture is very valuable to keep up optimal levels of oxygen in the nutrient standard 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 pro processing) and the bioreactor is prepared pro the then around of fermentation with cleaning technically referred to as ‘turn around’. The cleaning of the bioreactors is conceded made known by using high-pressure fill up jets from the nozzles fitted into the result vessel. Inside order to keep up the cost effectiveness of the bioreactor, the calculate taken pro curve around which is renowned as ‘down time’, is reserved as fleeting as doable.
Downstream dispensation (DSP)
The extraction and refining of a biotechnological manufactured goods from fermentation is referred to as downstream dispensation. The methods adopted pro downstream dispensation depends on the nature of the aim products, it’s concentration, stability, and the degree of refining vital. Both intracellular metabolites as well as extracellular metabolites are isolated by DSP. The intracellular metabolites are products located surrounded by the cells e.G. Vitamins, enzymes and that. The extracellular metabolites are the products bestow outside the cells e.G. Generally antibiotics, amino acids, alcohol, citric acid, enzymes like amylases, proteases and that. Approximately products like vitamin B12, flavomycin and that are bestow both as intracellular as well as extracellular products.
Downstream dispensation involves a digit of steps which are as follows:
A) Solid-liquid separation - The initially step is to separate total cells and other insoluble substances from the culture gumbo. This is made by using several methods:
1) Flotation- The process of aeration involves the fizzy of chatter in to the liquid gumbo. The cells and other solid particles make adsorbed on chatter bubbles which form a lathered up layer which is collected and indifferent.
2) Flocculation - The cells or cellular waste form generous aggregates and settle down which can be straightforwardly indifferent. Some flocculating agents like inorganic salt, organic poly-electrolyte, sandstone hydrocolloid and that are often used to realize appropriate flocculation.
3) Filtration - this is the generally commonly used practice to separate the biomass and culture dregs. The rate of filtration depends on many factors such as the size of the organism, presence of other organisms, viscosity of the standard, and warmth. Several filters like depth filters, absolute filters, gyratory drum vacuum filters, crust filters and that. 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 pro separating solid particles from liquid period. The practice of centrifugation is based on the opinion of density differences linking the particles to be separated and the standard. Inside the bioreactors, the unremitting tide manufacturing centrifuges are used everywhere here is a unremitting feeding of the slurry and collection of clarified fluid. The solid deposits are from time to time indifferent. Various models of centrifuges used are: Tubular bowl centrifuge, Disc centrifuge, Multichamber centrifuge, Scroll centrifuge or decanter and that.
B) Release of intracellular products - The biotechnological products with the intention of are located with in the cells like vitamins, enzymes, and that are released in an committed form pro their additional dispensation and isolation. The microorganisms or cells can de disintegrated or disrupted by corporal, compound, or enzymatic methods depending on the nature of the cells.
1) Physical methods of cell disruption are (i) Ultrasonication, (ii) Osmotic shock (used pro releasing hydrolytic enzymes and binding proteins from gram-negative bacteria), (iii) Heat Shock behavior, (iv) High pressure homogenization, (v) Impingement which involves hitting a stationary go up or a following spill of floating particles with a spill of floating cells by distinguished velocity and pressure. Microfluidizer is a device urban on the basis of the opinion of impingement. (vi) Grinding with schooner beads everywhere the cells diverse with schooner beads are subjected to a very distinguished alacrity in a result vessel. The cells break as they are mandatory hostile to the wall of the vessel by the beads
2) Chemical methods- Treatment with alkalies, organic solvents, and detergents lyse the cells to relief the content. Alkali behavior is used pro the extraction of approximately bacterial proteins. The organic solvents like methanol, ethanol, isopropanol, butanol and that furthermore disrupt the cells. The organic solvent, toluene, which is commonly used, dissolves crust phospholipids and creates the crust pores to relief intracellular contents. The ionic detergents denature the crust proteins and lyse the cells e.G. Cationic-cetyl trimethyl ammonium bromide or anionic-sodium lauryl sulfate. Non-ionic detergents are a reduced amount of reactive and furthermore affect the refining steps.
3) Enzymatic methods - Lysozyme is the generally commonly used enzyme which hydrolyses beta-1,4-glycosidic bonds of the mucopepide in bacterial cell walls e.G. Gram clear bacteria. This enzyme is commercially unfilled produced from hen egg white. For gram-negative bacteria, lysozymes are in aid with EDTA to break the cells. When the cell wall gets digested by lysozyme, the periplasmic crust breaks due to osmotic pressure, which releases the intracellular contents. Glucanase and mannanase along with proteases lyse the cell wall of mushroom.
C) Concentration - The biological products are concentrated by getting divest of fill up which is bestow in the dregs. 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, crust filtration, precipitation, and adsorption.
(i) Evaporation - The fill up from the gumbo is indifferent by the process of evaporation using evaporators. The evaporators aid a heating device which equipment the steam. There is a element pro the separation of concentrated manufactured goods and vapour and a condenser pro condensing vapour. The commonly used evaporators are Plate evaporator, Falling film evaporator, Forced film evaporator, Centrifugal mandatory film evaporator.
(ii) Liquid-Liquid extraction - inside liquid-liquid extraction, the biological products are concentrated by transferring the desired manufactured goods from lone liquid period to a further liquid period. The process of liquid-liquid extraction is categorized as extraction of low molecular consequence products and extraction of distinguished molecular consequence products.
(iii) Membrane filtration - This practice involves the aid of a semipermeable crust with the intention of selectively retains the particles/molecules with the intention of are larger than the stoma size while the less important molecules pass through the crust pores. The membranes are made up of polymeric equipment such as polyethersulfone and polyvinyl difluoride. Now a days, microfilters and ultrafilters made up of ceramics and steel are being used which are straightforward to clean and clean thoroughly. Pervaporation is a practice in which the precarious products are separated by a process of permeation through a crust coupled with evaporation and is used to extract and concentrate precarious products. Perstraction- This practice is used to recover and concentrate hydrophobic compoundsvand is based on the opinion of crust filtration coupled with solvent extraction.
(iv) Precipitation - This is the generally ordinary method used to concentrate proteins and polysaccharides in manufacturing processes. The alteration in warmth and in pH, neutral salts, organic solvents, distinguished molecular consequence polymers (ionic and non ionic) and that are used in precipitation. Neutral salts like commonly used ammonium sulphate increases the hydrophobic interactions linking protein molecules which leads to their precipitation. Ethanol, acetone and propanol are the commonly used organic solvents pro protein precipitation which reduce the dielectric constant of the standard and boost the electrostatic interaction linking the protein molecules. The precipitation process is conceded made known not more than 00C since the proteins make denatured by organic solvents. Polyethylene glycol (PEG) is a distinguished consequence non-ionic polymer with the intention of furthermore precipitates the proteins by sinking the quantity of fill up unfilled pro protein salvation. Inside the class 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 corporal factors like boost in warmth, boost in pH, and that furthermore leads to the precipitation of proteins. Besides these, the sympathy precipitation (affinity interaction e.G. Linking antigen and antibody)and precipitation using ligands is furthermore used.
D) Purification – The products of fermentation are purified by using the practice of chromatography. Chromatography consists of a stationary period and a mobile period. The porous solid matrix packed in a discourse constitutes the stationary period and the mixture of the compound to be separated is loaded on this. The compounds are eluted by a mobile period. A generous digit of matrices are commercially unfilled pro the refining of proteins e.G. Agarose, bran, porous silica, thwart linked dextran and that. Some of the commonly used chromatography techniques used are: Gel-filtration chromatography- inside this practice, the separation of molecules is based on the size, affect and molecular consequence using sponge-like gel beads with pores serving as molecular sieves pro separation of less important and larger molecules. The Ion-exchange chromatography involves the separation of molecules based on their go up charges. It is helpful pro the refining of antibiotics, above and beyond the refining of proteins. Inside ion chat chromatography, the pH of the standard is very crucial since the lattice 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 with a denial charged groups like carboxymethyl and sulfonate e.G. Dowex, HCR, Amberlite IR and that. Anion exchangers be inflicted with positively charged groups like DEAE (diethylaminoethyl) e.G. Dowex SAR, Amberlite IRA and that. The Affinity chromatography is based on an interaction of a protein with an immobilized ligand. The ligand can be a point antibody, substrate, substrate analogue or an inhibitor. The protein bound to the ligand is eluted made known by changing the pH of the memory, changing the ionic strength or by using a further emancipated ligand molecule.
E) Formulation- The maintenance of endeavor and stability of biotechnological products all through storage space and distribution is renowned as ‘formulation’. Equally proteins are highly insightful and lose their biological endeavor, therefore their formulations requires special trouble. Inside order to prolong their shelf life, particular 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 approximately small molecules (antibiotics, citric acid), formulation is made by crystallization using salts. ‘Drying’ is an valuable element of manufactured goods formulation which involves the conveying of excitement to a wet manufactured goods pro confiscation of dampness. The commercially unfilled dryers in aid are- friend, convection and radiation dryers. Spray drying is used pro drying generous volumes of liquids everywhere small droplets of liquid containing the manufactured goods are voted for through a needle directing ended a spill of sweltering chatter. After the evaporation of fill up, the solid particles are missing behind.
Freeze drying or lyophilization is the generally preferred method pro drying and formulation of a large range of products- pharmaceuticals, food stuff, bacteria, viruses and that. Freeze drying does not cause loss of biological endeavor of the desired manufactured goods. Lyophilization is caused due to the sublimation of a liquid from a frozen state. The manufactured goods with the liquid is frozen and at that time freeze dried under vacuum. After releasing the vacuum, the manufactured goods containing vials are sealed e.G. Penicillin is freeze dried frankly in ampules.
It is ideal to integrate the fermentation and downstream dispensation to irrevocably make the desired manufactured goods. The hard work are on the integrate these steps and a restricted accomplishment has furthermore been achieved.
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