Harvesting Technique In Downstream Processing

Bioreactor Bioprocessing

PRACTICAL 2 Harvesting Technique in Downstream Processing Objective To study the effectiveness of three different methods: sedimentation, centrifugation, and filtration; on solid-liquid separation of S. cerevisiae. Introduction The first step in the downstream processing of suspended cultures is a solid-liquid separation to remove the cells from the spent medium. Choice of solid-liquid separation method is influenced by the size and morphology of the organism (single-cell, mycelia, or aggregates), the specific gravity, viscosity, and rheology of spent fermentation medium. SEDIMENTATION: Use for primary yeast separation in the production of alcoholic beverages and in waste water treatment. Suitable only for large flocs. The rate of particle sedimentation is a function of both size and density; and the faster rate of sedimentation. CENTRIFUGATION: Normally used for solid-liquid separation to separate particles. It also used for liquid-liquid separation. Centrifugation depends on particles size and density difference between the cell and the medium. The choice of centrifuge depends on the size of the particles and density and the viscosity of the medium. Higher speed centrifuges are required for the separation of smaller microorganism, such as bacteria compared to yeast. FILTRATION: Conventional filtration of liquid containing suspended solids involves depth filters composed of porous media (cloth, glass wool, cellulose) that retain the solid and allow clarified liquid filtrate to pass through. Filtration technique is useful for fungus but less effective for bacteria. Methodology Microorganism The baker’s yeast was used in all experiment. This is hybrid yeast produced for commercial baker. Media Most practical industrial fermentation processes are based on complex media because of the cost and the choice of the nutrient and the ease of nutrient preparation. The well formulated defined medium was used in this experiment.

1

Bioreactor Bioprocessing

Compound Glucose Yeast extract KH2PO4 MgCl2 NH4Cl2H2O Technical Agar pH

Stock Culture Medium Concentration (g/L) 50 5 2 1 1 15 5.5

Centrifugation 15mL of samples were centrifuged at 4000rpm for 20min. The cell pellets were used in dry weight determination. Sedimentation 15mL samples were taken from shaking flasks. It was allowed to stand in normal condition. The cell pallets were used for dry cell weight determination. Filtration 15mL samples were filtered through 0.2µm nylon membrane filters (Whatman, 47mm) using vacuum pump. The pellets were used for dry cell weight determination. Results Centrifugation Sample Day 1 Day 2 Day 3 Day 4 Day 5

Dry weight (g) 0.0454 0.0740 0.1673 0.1930 0.5124

Sedimentation Sample Day 1 Day 2 Day 3 Day 4 Day 5

Dry weight (g) 0.0152 0.0175 0.0262 0.0374 0.0442

2

Bioreactor Bioprocessing

Filtration Sample Day 1 Day 2 Day 3 Day 4 Day 5

Dry weight (g) 0.0103 0.0176 0.0271 0.0417 0.0438

Discussion Centrifugation can be viewed as an extension of the conventional filtration and gravitational sedimentation. Centrifugal force replaces the pressure force in filtration and gravitational force in sedimentation, respectively. • Centrifugation and sedimentation utilize the difference in density of the particle and its surrounding fluid to separate particulates from the fermentation broth. Centrifugation efficiency is purely dependent on the particle size, shape, and density. • The biggest difference between sedimentation, centrifugation, and filtration is the yield, complexity, and time they take. Sedimentation is simplest and cheapest, but time consuming. Some of the yeast is removed with the liquid so total biomass may hard to get. Centrifugation requires more equipment: sterile bottles/flasks, liquid transfers, and a centrifuge. However, yield is higher than sedimentation. Filtration, if done right, can be quick. Liquid is passed through a sterile filter paper almost all of the yeast. There are some losses but not a complex procedure. • Sedimentation is often used in large-scale wastewater treatment processes as well as in traditional biosynthetic pathways involving fermentation. However, this process producing sludge that still has ample amount of liquid – which then will be dewatered by centrifugation. •

Conclusion Through this experiment, I become aware and understood about differences and effectiveness of three different methods: sedimentation, centrifugation, and filtration; on solid-liquid separation of S. cerevisiae References W.C. Yang, 2003. Handbook of Fluidization and Fluid-Particle Systems. Marcel Dekker, New York. 861p. Yang, S.T. and Basu, S. 2006. Bioseparation. In Lee, L. and Lee, S. (eds), Encyclopedia of Chemical Processing. pp 221-236. Taylor & Francis, New York.

3