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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.

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Bioreactor Bioprocessing

Stock Culture Medium


Compound Concentration (g/L)
Glucose 50
Yeast extract 5
KH2PO4 2
MgCl2 1
NH4Cl2H2O 1
Technical Agar 15
pH 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 Dry weight (g)
Day 1 0.0454
Day 2 0.0740
Day 3 0.1673
Day 4 0.1930
Day 5 0.5124

Sedimentation
Sample Dry weight (g)
Day 1 0.0152
Day 2 0.0175
Day 3 0.0262
Day 4 0.0374
Day 5 0.0442

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Bioreactor Bioprocessing

Filtration
Sample Dry weight (g)
Day 1 0.0103
Day 2 0.0176
Day 3 0.0271
Day 4 0.0417
Day 5 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.

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