Downstream processing, homogenizing, microfiltration & 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
- 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
- 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.
- 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.