Downstream processing - industrial microbiology

Steps involved in fermentation products producing a viable product output.various steps and process were explained in them. A semester syllabus of undergraduate microbiology student in his/her semester -5 in paper -6 . I think this might be helpful to you and have a good response after reading this .thank you.

1. Downstream processing P. Kiran kumar 3 rd year BMC PAPER-6 Silver jubilee government college, kurnool [email protected]

2. Overview :- Definition Filtration Centrifugation Cell disruption Solvent extraction Output.

3. Downstream processing :- Definition :- The various stages of processing that occur after completion of fermentation or bioconversion stage of seperation, purification and packaging of products.

4. Stages in downstream processing :- Removal of insoluble Product isolation Product purification Product polishing. T he recovery and the purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or  fermentation  broth, including the recycling of salvageable components and the proper treatment and disposal of waste. 

5. Removal of insolubles:- Seperation of cells , cell debris or other particulate matter. Stages :- Filtration Centrifugation Sedimentation Floccuation Gravity setting.

6. Filtration :- This is a kind of mechanical operation that used for seperation of solids from fluids (liquids or gases )by interposing a medium to porous membrane through which the fluid can pass but solids in the fluuds are retained . The solids particles that deposited on the filter form of layer which is known as filter cake. All the solids particles are feeded and stopped by the cake and the cake grows at qhich particles are brought to its surface . All the fluids goes through the cake and fluid medium..

7. Continous Rotary Vaccum Filter :- It is one of the most commonly used type of filter fermentation.. The drum is pre coated prior to filtration. A small agent of coagulating is added to the broth before it is pumped before it is pumped into the filter. The drum rotates under vaccum and a thin layer of cells sticks to the drum. The thickness of the layer increases in the section designed for forming the cakes.

8. Points to be considered while selecting the filter medium :- Ability to build the soild. Minimum resistance to flow the filtrate. Resistance to chemical attack. Minimum cost. Long life.

9. Centrifugation :- Centrifugation is used to separate particles of 100 – 0.1 micrometer from liquid by gravitational forces. It depends on particles size , density difference between the cells and the broth and viscosity of broth. Use of the centrifugal force for the seperation of mixtures. More dense components migrate away from the axis of centrifuge . Less dense components migrate towards the axis . Types of centrifuges used are Tubular bowl centrifuge Multichamber centrifuge Disc bowl centrifuge etc………….

10. Sedimentation :- It is applicable only for large particles greater than 100 micrometer flocs. It is slow process and takes ~ 3hrs. It is used in process like activated sludge effluent treatment. It is free settling process depends only on gravity. Particles settling is a high particle density suspension (hindered settling ). Mostly applicable in every fermentors industries which are large scale or small scale……

11. Flocculation :- Process where a solute comes out of solution in the form of flocs or flakes. Particles finer than 0.1 micrometer in water remain continoulsy in motion due to electrostatic charge which cause them to repel each other. Once their electrostatic charge in neutralized ( use of coagulant ) the finer particles start to collide and combine together. These larger and heavier flocs of particels are called flocs.

12. Cell disruption :- Some product are intracellular including many enzymes and proteins required to disrupt the cell amd rrlease these products. E.g :- yeast Cell disruption can be achieved by both mechanical and non mechanical methods. Mechanical :- Sonication and liquid shear homogenization etc….. Non mechanical methods :- Autolysis and osmosis shock etc…….

13. Solvent extraction :- Reducing the volume of material to be handled and concentrating the product. Removal of those components whose properties vary markedly from that of desired product … Water Is the Che if impurity . Liquid liquid extraction Adsorption Precipitation Are some of the unit operations involved in them….

14. Liquid–liquid extraction  ( LLE ) :- Liquid–liquid extraction  ( LLE ), also known as  solvent extraction  and  partitioning , is a method to separate compounds or metal complexes, based on their relative  solubilities  in two different immiscible liquids, usually water (polar) and an organic solvent (non-polar).  There is a net transfer of one or more species from one liquid into another liquid phase, generally from aqueous to organic.  The transfer is driven by chemical potential, i.e. once the transfer is complete, the overall system of protons and electrons that make up the solutes and the solvents are in a more stable configuration (lower free energy). The solvent that is enriched in solute(s) is called extract. The feed solution that is depleted in solute(s) is called the  raffinate . 

15. Adsorption Adsorption  is the  adhesion  of  atoms ,  ions  or  molecules  from a gas, liquid or dissolved solid to a  surface . This process creates a film of the adsorbate on the surface of the adsorbent. This process differs from  absorption , in which a  fluid  (the absorbate) is  dissolved  by or  permeates  a liquid or solid (the absorbent), respectively.  Adsorption is a  surface phenomenon , while absorption involves the whole volume of the material. The term  sorption  encompasses both processes, while  desorption  is the reverse of it. Th e separation is based on the interaction of the adsorbate with the adsorbent. The adsorbent is the surface and adsorbate is the molecules of interest which are getting  adsorbed  on the adsorbent.

16. Ultrafiltration Ultrafiltration  (UF) is a pressure-driven barrier to suspended solids, bacteria, viruses, endotoxins and other pathogens to produce water with very high purity and low silt density.  Ultrafiltration  (UF) is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semi permeable membrane. The variety of  membrane filtration  in which forces like  pressure  or  concentration gradients  lead to a separation through a  semipermeable membrane .  Suspended solids  and  solutes  of high  molecular weight  are retained in them called retentate, while water and low molecular weight solutes pass through the membrane in the  permeate  (filtrate). This  separation process  is used in industry and research for purifying and concentrating macromolecular (10 3  - 10 6   Da ) solutions, especially  protein  solutions.

17. Precipitation :- Precipitation is the most commonly used technique in industry for the concentration of macromolecules such as proteins and polysaccharides. Further, precipitation technique can also be employed for the removal of certain unwanted byproducts e.g. nucleic acids, pigments. Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic), besides alteration in temperature and pH are used in precipitation. In addition to these non-specific protein precipitation reactions (i.e. the nature of the protein is unimportant), there are some protein specific precipitations e.g., affinity precipitation, ligand precipitation. The most commonly used salt is ammonium sulfate, since it is highly soluble, non­toxic to proteins and low-priced. Ammonium sulfate increases hydrophobic interactions between protein molecules that result in their precipitation. The precipitation of proteins is dependent on several factors such as protein concentration, pH and temperature.

18. Product purification :- Purification by Gel filtration Ion exchange Affinity Hydrophobic interactions.

19. Gel-filtration chromatography: This is also referred to as size-exclusion chromatography. In this technique, the separation of molecules is based on the size, shape and molecular weight. The sponge-like gel beads with pores serve as molecular sieves for separation of smaller and bigger molecules. A solution mixture containing molecules of different sizes (e.g. different proteins) is applied to the column and eluted. The smaller molecules enter the gel beads through their pores and get trapped. On the other hand, the larger molecules cannot pass through the pores and therefore come out first with the mobile liquid (Fig. 20.7). At the industrial scale, gel-filtration is particularly useful to remove salts and low molecular weight compounds from high molecular weight products.

20. Ion-exchange chromatography: It involves the separation of molecules based on their surface charges. Ion-exchangers are of two types (cation- exchangers which have negatively charged groups like carboxymethyl and sulfonate, and anion- exchangers with positively charged groups like diethylaminoethyl (DEAE). The most commonly used cation-exchangers are Dowex HCR and Amberlite IR, the anion-exchangers are Dowex SAR and Amberlite IRA. In ion-exchange chromatography, the pH of the medium is very crucial, since the net charge varies with pH. In other words, the pH determines the effective charge on both the target molecule and the ion-exchanger. The ionic bound molecules can be eluted from the matrix by changing the pH of the eluant or by increasing the concentration of salt solution. Ion-exchange chromatography is useful for the purification of antibiotics, besides the purification of proteins.

21. Affinity chromatography: This is an elegant method for the purification of proteins from a complex mixture. 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 immobilized ligand on a solid matrix can be effectively used to fish out complementary structures. The protein bound to the ligand can be eluted by reducing their interaction. This can be achieved by changing the pH of the buffer, altering the ionic strength or by using another free ligand molecule. The fresh ligand used has to be removed in the subsequent steps

22. Product formulations :- Formulation broadly refers to the maintenance of activity and stability of a biotechnological products during storage and distribution. The formulation of low molecular weight products (solvents, organic acids) can be achieved by concentrating them with removal of most of the water. For certain small molecules, (antibiotics, citric acid), formulation can be done by crystallization by adding salts. Proteins are highly susceptible for loss of biological activity; hence their formulation requires special care. Certain stabilizing additives are added to prolong the shelf life of protein. The stabilizers of protein formulation include sugars (sucrose, lactose), salts (sodium chloride, ammonium sulfate), polymers (polyethylene glycol) and polyhydric alcohols (glycerol). Proteins may be formulated in the form of solutions, suspensions or dry powders.

23. Product packaging :- Drying Freezing Space drying

24. Thanking you ……