of India) Project on - Creation of e-Contents on Fermentation Technology
Module-24: Introduction to Product Recovery & Purification
After the production of the desired fermentation product,the next most important step includes product recovery and purification. The recovery and purification of fermentation products refers Downstream Processing. Downstream processing includes many technologies for laboratory& industrial-scale separation of biological products. The Downstream Processing of fermentation products may be difficult and costly. Main purpose of this is to obtain a high-quality product as fast as possible at an effectiverecovery rate using least costs. The recovery costs of microbial products may vary from 15% to 70% of the total costs. It is an essential step in the manufacture of many products such as, 1. antibiotics, 2. hormones, 3. industrial enzymes, 4. natural fragrance and 5. flavor compounds. At the time of harvesting, the product concentration may be low in an aqueous solution, which may containmicro-organisms, cell debris, soluble and insoluble media components and other metabolic products. The product may also be intracellular, heat labile and easily damagedbycontaminating micro-organisms which increase the difficulties of product recovery. If product is heat labile,speed of operation may be the dominant factor in noble product recovery. To ensure the proper processing of harvested broth within a reasonable time limitprocessing equipment must be of the correct type andof correct size. The choice of recovery process is based on the following criteria: 1. The location of the product: Intracellular or Extracellular 2. The quantity of the product in the fermentation broth. 3. The proposed use of the product.
Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India
NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology
4. The marginal acceptable standard of purity.
5. The physical and chemical nature of the desired product. 6. The magnitude of bio-hazard 7. The impurities in the fermented broth. 8. The market potential of the product.
Stages of downstream processing
Stage – 1 Removal of Insoluble
The first stage for the recovery of an extracellular product aims to remove large solid particles and microbial cells. It is possible by various measures like by centrifugation, filtration, sedimentation, precipitation, flocculation, electro-precipitation, and gravity settling.
Stage – 2ProductIsolation
In this stage, the broth is extracted into major fractions using various techniques likeultrafiltration, reverse osmosis, adsorption/ion-exchange/ gel filtration or affinity chromatography, liquid- liquid extraction, two phase aqueous extraction, precipitation etc. It allows the removal of unnecessary components whose properties differdistinctly from that of the desired product.
Stage – 3 Product Purification
Once, the product-containing fraction is purified by fractional precipitation, further
more precise chromatographic techniques and crystallization is used to obtain a highly concentrated product which is essentially free from impurities. Steps in this stage are expensive because itrequires sensitive and sophisticated equipment which contributes a majorsection of the entire downstream processing expenditure.
Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India
NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology
Stage – 4 Product Polishing
It is the last processing steps which end with packaging of the product. It includes Crystallization, desiccation and spray drying. Sometime it alsoincludes operationsto sterilize the product by removing contaminants which otherwise affect product safety. In short the downstream processing involves the following major procedures:
i. Removal of microbial cells and other solid matter:Charged properties of
microbial cells to be exploited by electrophoresis and dielectrophoresis& flocculation characteristics and magnetic separations to beimproved byultrasonic treatment ii. Foam separation:It exploits differences in surface activity of materials. The material is selectively adsorbed to the surface of gas bubbles rising through a liquid.Then it separated and finally removed. iii. Precipitation:This is thechemical process in which solid gets formed in a solution or inside another solid.Various agentscan be used in precipitation like acids, bases, salts of sodium and ammonium, organic solvents, non-ionic polymers (polyethylene glycol), protein binding dyes etc. iv. Filtration: it is commonly used method which separate suspended particles from liquid or gas. It uses a porous medium that retain the suspended particles by allowing the liquid or gas to pass through.So it allows separation of solids by interfering a medium through which only the fluid can pass. v. Centrifugation:Centrifuge works using the sedimentation principle, where the centripetal speed causes separation of denser substances along the radial direction.Various types of centrifuges are used like the basket centrifuge, the tubular-bowl centrifuge, the solid-bowl scroll centrifuge etc. vi. Cell disruption:It is a method for releasing intracellular molecules. To release the cellular contents from their extremely tough cell wall a number of methods have been established. For examplephysico mechanical methods such as liquid shear, solid shear, freeze thawing, agitation with abrasives, and ultra-sonication. The chemical methods includeosmotic shock, usage of detergents, alkali treatment, and enzyme treatment.
Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India
NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology
vii. Liquid- liquid extraction:It is a method to separate compounds on the basis of
their relative solubilities in two different immiscible liquids.So, an extraction of a substance from one to another liquid phase in which the desired product is preferentially soluble is known as liquid-liquid extraction. It is also known as solvent extraction and partitioning. viii. Solvent recovery:Distillation is the best method for solvent recovery whichincludes three stages evaporation, vapour-liquid separation in a column and condensation. ix. Chromatography: In chromatography separation is based on differential partitioning between two phases that is mobile and stationary. Different types of chromatography techniques are used like adsorption chromatography, ion- exchange chromatography, gel permeation chromatography, affinity chromatography, reverse-phase chromatography and high-performance liquid chromatography. x. Drying:To allow minimum loss in viability, activity and nutritional value drying is necessary.It removes water from a heat-sensitive material assuringleast damage. xi. Crystallization:It is used for final purification of a diverse range of compounds including the recovery of organic acids and amino acids.
It must be remembered that the upstream and downstream processing are integral parts of an overall process. As they are interconnected, neither stage should be developed independently, as this might result in problems and unnecessary expenditure. So, by taking care of some steps in the upstream process productrecovery may be made easier. i.e. 1. Selection of test strain: By selecting test microorganisms that do not produce any pigment and/or undesirable metabolites. 2. Environmental setup: By adjusting the production environments to allow least production of undesirable secondarymetabolites. Beside this some process parameters should be checked and maintained like: 1. Time of harvesting 2. pHmaintenance during fermentation and harvesting
Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India
NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology
3. Temperature maintenance 4. Use of suitable chemicals for flocculation and separation The recovery and purification of many compounds may be achieved by a number of alternative ways. The decision to follow a particular way involves study of the following factors: 1. Capital expenses 2. Processing expenses 3. Probable yield 4. Product quality 5. Availability oftechnical skill 6. Requirements of waste disposal 7. Type of Processing: Continuous or batch 8. Automation 9. Health and safety There are so many problems associated with the product recovery program. For example, the recovery of extra cellular enzymes might be difficult as it needs immediate processing. One another problem is pigment production by test organisms because some time the pigment binds to the same resin as the enzyme. At the time of foam separation, selection of antifoam should be proper otherwise it affect ultrafiltration or ion exchange resins used in recovery stages. In short it should always be remembered that good recovery starts in the fermentation by the selection of proper upstream processing like the correct media and proper time of harvesting. The major problem currently faced in product recovery is transfer of small-scale preparative methods to the production scale without disturbingyield of the process, quality of the product and purity levelof the product.
Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India
NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology
References
Principles of Fermentation Technology: (2nd edition, by Peter F. Stanbury, Allan Whitaker and Stephen J. Hall, Butterworth-Heinemann, An imprint of Elsevier Science.) Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications) A Text Book of Industrial Microbiology: (2nd edition By WulfCrueger&AnnelieseCrueger) Biotechnology: Food Fermentation Microbiology, Biochemistry & Technology Vol. 1 & 2:(By V.K. Joshi & Ashok Pandey) Manual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L. Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard C. Willson and J. H. David Wu) Industrial Microbiology-An introduction: By Michael J. Waites, Neil L. Morgan, John S. Rockey and Gary Higton) Comprehensive Biotechnology-The Principles, Applications and Rugulations of Biotechnology in Industry, Agriculture and Medicine: (By Mrray Moo Young) Fermentation Technology : Up Stream Fermentation Technology- Vol-I: (By H. A. Modi-Pointer Publications) Fermentation Technology : Down Stream Fermentation Technology- Vol-II: (By H. A. Modi-Pointer Publications) Industrial Microbiology by Prescott and Dunn's: (4th edition, edited by Gerald Reed, CBR publications) Fermentation Technology: (By M.L. Srivastava, NAROSA publications) Industrial Microbiology: (By A.H. Patel) International student edition: Microbiology- A laboratory Manual: (4th edition. By James G. Chappuccino& Natalie Sherman) Bacteriological Techniques: (By F.J. Baker) Introduction to Microbial Techniques: (By Gunasekaran) Mannual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L. Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard C. Willson and J. H. David Wu)
