About Downstream Processing, Homogenizer, Microfiltration Experiment and HPLC

1. DOWNSTREAM
PROCESSING
GROUP 4:
• NOPIA RAHMAT 1415546
• HANISA YASMIN 1427822
• WAN MAJIDAH 1423928
• SULIYANA 1415252
• AIN NUR SYAZWANI 1428692
• MUNA ALI AHMED 1036362
• INTAN AMIRAH 1426278

2. WHAT IS DOWNSTREAM PROCESSING?
• the recovery and 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
 Purity of product
 Processing time
 Yield losses
 cost

4. HOMOGENIZATION

5. What is homogenization?
• Process of converting 2 immiscible liquids into an emulsion
• Based on the use of pressure on liquids to subdivide particles
into the very smallest sizes and create a stable dispersion
ideal for further processing

6. PRINCIPLES OF HIGH
PRESSURE HOMOGENIZER
• By using pressure plunger, the liquid
product is pumped through a
specially designed adjustable valve
which creates high pressure
• This pressure generates high shear
force
• Cell disruption only accomplished
when
-sudden pressure drop upon
discharge
-impingement in the valve
-high liquid shear in the orifice

7. COMPONENTS
1.HIGH PRESSURE PLUNGER
2.ADJUSTABLE VALVE

8. ADVANTAGES & DISADVANTAGES OF HIGH PRESSURE
HOMOGENISER
ADVANTAGES
• PROCESS LARGE VOLUMES OF LIQUID SAMPLES
EFFECTIVELY
• DOES NOT DEPOSIT ANY MATERIALS INTO THE
SAMPLES
• FLEXIBLE TO MODULATE PROCESS STREAM
• PRODUCES VERY SMALL SUB-MICRON PARTICLE
SIZE
DISADVANTAGES
• HIGH COST
• NEED TO BE CLEANED FOR EVERY USAGE
• THE MACHINE IS LARGE & HEAVY
• NOT SUITABLE FOR SAMPLES WITH TOO MUCH
SOLID MATTER.

9. CROSSFLOW
MICROFILTRATION
EXPERIMENT

10. OBJECTIVES
• To study the principle of crossflow filtration and factors
affecting filtration processes
• To separates the yeast from the samples
• To observe the volume flow rate of the sample from the
filtration operation
• To study the relationship between filtration rate and
filtration time

11. INTRODUCTION
• Separative techniques are systematically required in
biotechnological processes in order to harvest microorganisms
and purify their metabolites products.
• Crossflow filtration such as microfiltration or ultrafiltration
is considered to be an efficient method to harvest microbial
cells from fermentation broth.
• The application of cross-flow membrane filtration appears to
offer some sources of specific industrial interest in term of
their efficiency and facility of exploitation
Application
Method
Type

12. This is the setup for the experiment of
micro crossflow filtration.
This is the permeate side where
the outlet water sample is being collected.
Sample tank
Measuring cylinder
Pump
GE healthcare Hollow Fiber Cartridge
(pore size 0.45μm and 420𝑐𝑚2
membrane area)

13. PART 1: RINSING
1. 500mL of warm distilled water are
poured into the sample tank
2. The motor speed was started
from 20rpm slowly increase to 250rpm
3. The permeate
(distilled water +
remained solute)
was transferred to the
measuring cylinder.

14. PART 2: MICROFILTRATION
1. 500mL yeast fermented sample
was poured into the tank.
2. The pump was switched on
to run the filtration.
3. The motor speed was started
from 20rpm and slowly
increased to 250rpm.
4. The pressure drop was
maintained at 2psi across inlet
and outlet.
5. All the permeate
(extracellular product + media)
was transferred to measuring
cylinder.
6. The time was taken and
recorded
for every 50mL of sample
entered the
measuring cylinder.
7. The filtration rate was
calculated.
8. All the retentate (yeast
cell) is
remained on the membrane
filtration
surface.

15. PART 3: BACK WASHED
1. The hollow fibre membrane
was reversed up-side-down.
2. 500 ml of warm distilled water
was poured into sample tank.
3. The pump is switched on
to run the filtration.
4. The motor speed was started
from
20 rpm and slowly increased to
250 rpm.
5. All the permeate
(cell + distilled water)
Was transferred to measuring
cylinder.
6. The back-wash procedure was
repeated by replacing the warm
distilled water with 500 mL of
0.1M NaOH solution.

16. • The pore size of the membrane (cellulose membrane) is 0.45
µm
• The substances that have size above 0.45 µm will be trapped in
the membrane tubes and leave as retentate
• Substances that have size below 0.45 µm, those that can pass
through is the permeate
DISCUSSION

17. Bioreactor 1 : Decreasing in flux over time
Bioreactor 2 : Increasing in flux over time
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12
filtrationrate(ml/s)
filtration time (min)
filtration rate vs filtration time
0.4
0.45
0.5
0.55
0.6
0.65
0 2 4 6 8 10 12 14
filtrationrate(ml/s)
filtration time(min)
filtration rate vs filtration time
Bioreactor 1 Bioreactor 2
Results

18.  Theoretically the flux rate should decrease due to fouling / cake build up
 The decreasing in flux which follows the dead-end filtration theory is due to cake build up (Sanjeev
Redkar & Davis, 1993)
 As the fluxes reaching steady or nearly steady
(i) cake growth stopped by the shear exerted at its surface
 As the shear rate increases, steady state flux increases
BIOREACTOR 1

19. Bioreactor 2
 The increasing pattern may due to some errors

20. Observation of the final results :
 The turbidity of the sample in bioreactor 2 is higher than in bioreactor 1
 Determine the concentration of permeate in bioreactor 2 is higher than in bioreactor 1
 Bioreactor 2 is more efficient

21. Errors & Precautions
• The bottle of the sample was not been shaken • Shake the bottle first, before the sample being
poured in the filtration tube
(i) Some substances might be deposited at the
bottom of the measuring cylinder
(ii) To allow all the substances being suspended
(iii) Increase the product
• Fouling • Perform backwash first before performing the
filtration process to reduce the huge amount of
fouling
(i) clean pore membrane
(ii) resulting in a better filtration
• Formation of humic acid on the surface of
membrane
• Prefiltration must be done to eliminate humic
acid and other useless substance to reduce the
rate of fouling (Wei & Zydney, 1999)

22. Conclusions
 All the objectives are managed to be achieved throughout the experiment .
 It is concluded that :
(i) The results in bioreactor 1 clearly showed that membrane fouling
(ii) Permeate flux decline due to accumulation of substances within membrane pores and/or onto
membrane surface
(iii) Decreasing in volume flow rate suggests the dead-end filtration theory is due to cake build up
(iv) In bioreactor 2, there are some errors observed during the experiment
(v) The cake build-up causes poor flow distribution
(vi) Decrease the efficiency and the performance of the hollow fiber membranes
(vii) Efficient removal of solids from the membrane surface, by the backwash, must be well
understood

23. Chromatography
to obtain 99% purity of the desired bioproduct in the mixture

24. Types of chromatography
 column chromatography
 ion-exchange chromatography
 gel permeation chromatography
 high-pressure liquid chromatography
 affinity chromatography.

25. High Pressure Liquid Chromatography
 Separation, and identification of amino acids, carbohydrates, lipids, nucleic acids,
proteins, steroids, and other biologically active molecules.
 Essential components of a HPLC device are solvent depot, high- pressure pump,
commercially prepared column, detector, and recorder.
 In this technique, use of small particles, and application of high pressure on the
rate of solvent flow increases separation power.

26. Types of HPLC
Normal phase
chromatography
 separates analytes based on
polarity (polar stationary
phase and a non-polar
mobile phase).
Reversed phase
chromatography
 a non-polar stationary phase
and an aqueous, moderately
polar mobile phase.