AGM 312 Microbial Biotechnology (1+1) Page 48 E xp er im en t 11 Product recovery and cell separation Date: Down streaming refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals such as antibiotics, hormones, etc. Among the three steps of any fermentation process viz., upstreaming, fermentation, and down streaming, down streaming is the most critical step involving the recovery of the product and decides the economy of the final product. Down streaming involves the following four steps: 5. Removal of insoluble is the first step and involves the capture of the product as a solute in a particulate-free liquid. Typical operations are flocculation, floatation, filtration, centrifugation, sedimentation, and precipitation. 6. Product isolation is the removal of those components whose properties vary markedly from that of the desired product. Eg. Solvent extraction, adsorption, ultrafiltration, and precipitation. 7. Product purification is the removal of those contaminants that resemble the product very closely in physical and chemical properties. Eg. Chromatography, crystallization, or fractional precipitation. 8. Product polishing describes the final processing step and ends with the packaging of the product in a form that is stable, transportable in a convenient way. Several industrial microbial products are produced by culturing different microbial cultures in fermentation broth under favorable conditions. Prior to the production of target molecules like enzymes, antibiotics, organic acids, alcohols, etc., fermentation broth has to filter to be free from the cell mass. Different techniques are available to separate the biomass from fermentation broth. Techniques like centrifugation, filtration, sedimentation, flocculation, spray drying, and freeze-drying are available. Based on the scale, economics, product sensitivity, and purity of the products required, any one or combination of the above techniques is used. Centrifugation involves the application of centrifugal force to separate particles from a solution according to their size, shape, density, viscosity of the medium, and rotor speed. This involves separating molecules of different densities by spinning them in solution around a centrifugal axis (in a centrifuge rotor) at high speed. This process is used to separate two miscible substances. More-dense components of the mixture migrate away
AGM 312 Microbial Biotechnology (1+1) Page 49 from the axis of the centrifuge (move to the outside), while less-dense components of the mixture migrate towards the axis, i. e., move to the center. The first commercial scale centrifuge was designed in 1878 by the Swedish inventor De Laval to separate cream from milk. In 1923, Svedberg's invented the analytical ultracentrifuge, operating at 10 000 rpm. Centrifuges are used in different fields ranging from large-scale commercial applications to laboratory-scale scientific research. In microbiology, this process is used to separate microbial cells from the liquid media without cell disruption. This process also aids in separating microbial products containing liquids from cell debris and other solid materials. Filtration is a physical operation that separates solid particles from a suspended liquid by adding a physical filter medium through which only the liquid can pass. Solid particles that cannot pass through filter papers are retained and get separated on one side, while liquid particles pass through the filter medium is collected on another side. A liquid medium that passes through the filter medium is called the filtrate. Solid particles that cannot pass through the filter medium are described as oversize, and the fluid that passes through is called the filtrate. Materials required: 1. Each two hundred ml broth cultures of Lactobacillus and Yeast in the late log phase. 2. Laboratory centrifuge with 50 ml centrifuge tube loading capacity. 3. Filter paper with 0.45µ pore size and around 100 mm diameter. 4. Sterile glass funnels, Sterile 50 ml centrifuge tubes. Procedure: Cell separation using centrifuge technique: 1. Prepare two hundred ml nutrient broth for each culture of Lactobacillus and Yeast. 2. Inoculate the log phase cultures of Lactobacillus and Yeast in nutrient broth. 3. Incubate the cultures in a thermo-controlled orbital shaker at 160 rpm until the cultures reach the late log phase. 4. Take one set culture broth, fill in centrifuge tube to ¾th of the capacity.
AGM 312 Microbial Biotechnology (1+1) Page 50 5. Place the centrifuge tubes in a centrifuge rotor, ensure proper weight balance. 6. Run the centrifuge at 8000 rpm for 10 min. 7. Discard the supernatant. 8. Harvest the precipitated cell using a spatula, transfer to a Petri plate. 9. Dry the biomass by placing under hot air oven at 72°C, until the consistent weight is reached. 10. Calculate the yield of biomass per ml of the culture broth used. Cell separation using filtration technique: 1. Prepare two hundred ml nutrient broth for each culture of Lactobacillus and Yeast. 2. Inoculate the log phase cultures of Lactobacillus and Yeast in nutrient broth. 3. Incubate the cultures in a thermo-controlled orbital shaker at 160 rpm until the cultures reach the late log phase. 4. Filter the culture broth using a 0.45µ membrane filter using a glass funnel and conical flask. 5. Discard the culture filtrate. Dry the biomass present in the membrane filter by placing under hot air oven at 72 °C, until a constant weight is reached. 6. Calculate the yield of biomass per ml of the culture broth used. Review questions: What are the techniques available for cell separation? What is the principle behind the centrifugation technique?
AGM 312 Microbial Biotechnology (1+1) Page 51 What is Svedberg's unit? What is filtrate and oversize? Why are late log phase cultures preferred for cell harvesting? Work Done: References: Van Holde, K. E. (1998). Analytical ultracentrifugation from 1924 to the present: A remarkable history. Chemtracts – Biochemistry and Molecular Biology. 11:933-943. Ballou, David P.; Benore, Marilee; Ninfa, Alexander J. (2008). Fundamental laboratory approaches for biochemistry and biotechnology (2nd ed.). Hoboken, N.J.: Wiley. p. 235.ISBN 9780470087664.