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KULIAH TEKNOLOGI FERMENTASI 29 November 2012

The choice of recovery process: The intracellular or extracellular location of the product The concentration of the product in the fermentation broth The physical and chemical properties of the desired product (as an aid to selecting separation procedures) The intended use of the product The minimal acceptable standard of purity The impurities in the bioreactor broth The marketable price for the product

Modification of the broth characteristics for faster handling and simpler equipment: Selection of microorganism which does not produce pigments or undesirable metabolites Modification of the fermentation condition to reduce the production of undesirable metabolites Precise timing of harvesting (optimum condition) pH control after harvesting Temperature treatment after harvesting Addition of flocculating agents Use of enzymes to attack cell walls

Tingkatan Proses Pemanenan Pengambilan bahan tidak terlarut Isolasi Fermentasi Filtrasi Ekstraksi

Produk Kons. (g/l) 0.1 5 1.0 5 5 50 Kwalitas (%) 0.1 1.0 0.2 2.0 1 10

Purifikasi
Produk akhir

Kromatografi
Kristalisasi

50 200
50 200

50 80
90 100

Pengambilan bahan tidak larut (Removal of Insolubles). Sedikit mengkonsentrasikan produk atau perbaikan produk. Filtrasi dan sentrifugasi. Isolasi Produk. Tidak spesifik, pengambilan bahan yang mempunyai sifat yang tersebar dibandingkan dengan produk yang diinginkan. Konsentrasi dan kwalitas produk mulai terjadi. Adsorpsi dan ekstraksi solven. Purifikasi. Teknik proses yang sangat selektif untuk menghasilkan produk dan mengambil bahan yang tidak diinginkan serupa dengan fungsi kimia dan sifat fisika. Khromatografi, elektrophoresis, dan presipitasi. Produk akhir. Kristalisasi

Pemisahan padatan dengan cairan dengan mendorong cairan melalui bahan padat atau medium filter

Rotary vacuum Filter

Bahan yang harus dilakukan perlakuan pendahuluan sebelum penyaringan karena bahan tersebut terlalu kental: Pemanasan Koagulasi dan Flokulasi Adsorpsi dengan bantuan filter, Diatomaceous Earth

1. Membrane (Module) 2. Pumps 3. Other


Piping Tanks Valves Flowmeter Manometer

4/14/2013

FEATURE Membrane Wall Thickness Film thickness Pore size

REVERSE OSMOSIS Asymmetrical 150mm 1mm <0.002um HMWC, LMWC,Sodiu m, Chloride, glucose, amino acids, proteins Tubular, spiralwound, plate & frame

NANO FILTRATION Asymmetrical 150mm 1mm <0.002um HMWC, mono,di-, and aligosaccharides, polyvalent anions Tubular, spiralwound, plate & frame

ULTRA FILTRATION Asymmetrical 150-250mm 1mm 0.02-0.2um

MICRO FILTRATION Symmetrical Asymmetrical 10-150mm various 0.2-5um

Rejects

Macromolecules, proteins, polysaccharid es, viruses Tubular, hollowfibre, spiralwound, plate & frame

Particulates, clay, bacteria

Membrane module

Tubular, hollowfibre, plate & frame CA, TFC, Ceramic, PVDF, Sintered <2 bar 50-1000 l/m2/hr

Material

CA, TFC

CA, TFC

CA, TFC, Ceramic

Pressure Flux

15-150 bar 10-50 (l/m2/hr)

5-35 bar 10-100 l/m2/hr

1-10 bar 10-200 l/m2/hr

Ultrafiltration

Microfiltration

RO membrane

MIKROFILTRASI
Tanpa pembentukan cake

Menggunakan membran: tipis dan microporous Lubang pori2nya kecil dan sangat monodisperse Mempunyai kemampuan menyaring partikel yang tidak diinginkan Membran mengikuti hukum Darcys untuk permeabilitas dan ketahanan yang tinggi thd aliran. Konvensional ketahanannya rendah. Perlu dilakukan pembersihan secara berkala Aliran yang melalui membran lebih rendah daripada aliran melalui conventional filter cake. Filter area per liter volume lebih besar daripada convensional. Type : Plate and frame, spiral wound and hollow fiber

Bacterial Cell Casein, whey

Lactose Minerals

Feed or Product
Initial material into system on feed side of membrane

Retentate or Concentrate
The fraction of the feed which is rejected by the membrane.

Permeate
The fraction of the feed which passes through the membrane

Spiral Wound Plate and Frame Tubular


Capilary Hollow fibre Pipe

Ceramic Zeolite Stainless Steel

Downstream protein purification by ultrafiltration concentration and diafiltration

Cold sterilization of pharmaceuticals Cell harvesting Sterile process filters for gas-phase Clarification of fruit juices, wine and beer Ultrapure water in semiconductor industry Metal recovery (colloidal (hydro)oxides) Waste water treatment Separation of oil-water emulsions Dehydration of lattices Pretreatment for RO
Eykamp, 1995; Mulder, 1998

Chemical Industry
Electro coat painting recovery Latex processing Textile size recovery Recovery of lubricant oils

Medical Applications
Kidney dialysis

Waste treatment Recovery of valuable products from effluents


Cheese whey
Cheryan, 1986

electro paint recovery, oil-water emulsions Beverages (juices) Dairy (milk, whey, cheese making) Food (gelatin, starch, sugar and proteins) Textile (sizing, dyes) Pharmaceutical (enzymes, antibiotics, pyrogens) Pulp and paper industry Leather industry Water purification
Eykamp, 1995; Mulder, 1998

choice of polymer, choice of solvent and nonsolvent, composition of casting solution, composition of coagulation bath, temperature of the casting solution and coagulation bath, evaporation time, location of the liquid-liquid demixing gap and crystallization behaviour of the polymer

Module Type
Characteristic Flat plate Spiral Wound Moderate (300-900) Shell and Tube Hollow Fibre

Packaging density (m2/m3)

Moderate (200-400)

Low High (9000-30000 (150-300)

Fluid management
Suspended solids capability Cleaning Replacement

Good

Good

High pumping costs


Good

Good

Moderate

Poor

Poor

Sometimes difficult Sheets or cartridge

Sometimes difficult Cartridge

Easy Tubes

Backflusing possible Cartridge

Memanfaatkan perbedaan densitas antara padatan dan cairan yang ada disekitarnya
Pemisahan suspensi antara padatan dan cairan secara perlahan dengan pengaruh gaya gravitasi disebut proses sedimentasi, sedangkan bilamana menggunakan kecepatan berdasarkan gaya sentrifugal disebut sentrifugasi. Sentrifugasi menghasilkan pasta sedangkan filtrasi menghasilkan dry cake

JENIS SENTRIFUGASI
Tubular Bowl, mempunyai tenaga yang kuat, dapat didinginkan, dan sangat cocok untuk kerja dengan protein Disc with a nozzle, Disc with intermittent Discharge, dan Basket, kombinasi sentrifuse dan filter
Transport of Sediment Solids content in feed, (vol %) 0-1 Maximum Throughput (m3/hr) 150

Bowl separator

Stays in Bowl

Solid injecting: nozzle separator


Solid injecting: slot separator Nozzle separator

Intermittent discharge through axial channels


Intermittent discharge through radial slot Continuous discharge

0.01 - 10
0.2 - 20 1 - 30

200
100 300

BIOMASS CENTRIFUGATIONS
Product Microorganisme Microorganism Bakers yeast Alcohol yeast Citric acid Enzyme Saccharomyces Saccharomyces Mold Bacillus Size (mm) 7 10 58 13 Relative throughput in Centrifuge 100 60 30 7 Type Separator

Nozzle Solids ejecting Solids ejecting Nozzle Solid ejecting

Digunakan untuk memecah sel. Method Chemical Technique Enzyme digestion Principle Cell wall digested, providing disruption Organic solvent dissolves in cell wall Cells ruptured by grinding with abrasives Cells broken with ultrasonic cavitation Stress or Product Gentle Cost Expensive Example Micrococcus lysodeikticus treated with egg lysosym Toluene disruption of yeast

Lipid dissolution

Moderate

Cheap

Mechanical Grinding

Moderate

Cheap

Ultrasonication

Harsh

Moderate

Cell suspension at least on a small scale

CHEMICAL METHOD Osmotic shock Solubilization by detergen; Sodium Dodecylsulfate (SDS) sebagai anionik Cetyltrimethylammonium Bromide (CTAB) sebagai kationik Sodium Taurocholate sebagai anioinik Sodium Sulfonat sebagai anionik Triton X-100 sebagai nonionik, polydisperse. Lipid dissolution, solvent; Toluene, Benzene, Chlorobenzene, Cumene, xylenes Alkene like decane Octanol

MECHANICAL METHOD Small scale Homogenization in Waring Blender, mycelial organisms and with other animal cells or tissue grinding, ultrasonication Large scale Homogenization and crushing

Batch Extraction

Differential Extraction

Variable Capacity

Extraction High Low

Adsorption

Selectivity

Moderate

High

Nature of equilibrium

Often linier; dilute solutes independent Steady state Emulsification; denaturation

Usually non linier; dilute solutes interact Unsteady; periodic Solids handling; compressible packing

Nature of operation Problems

Precipitation With non solvent: Antibiotik solvent : aceton, non solvent: water Pada suhu rendah akan didapatkan hasil yang tinggi dan mengurangi denaturasi Konsentrasi 0.05 0.2 M. Larutan dengan berat molekul yang besar memerlukan sedikit pelarut untuk inisiasi precipitasi With salts: salting out Anion : citrat>PO42->SO42->CH3COO->Cl->NO3Cation : NH4+>K+>Na+ Densitas presipitasi berbeda dengan densitas larutan With Temperature Ultra filtrasi Elution Chromatography; Adsorption, Ion exchange, Gel permeation, Affinity, Reverse phase, High performance liquid. Electrophoresis

Cells

Cell Suspension

Membrane

Cell Concentrate

Cell-free culture medium

Cell-free culture medium

Cross Flow Filtration

Dead End Filtration

After cells (or media) are harvested proteins may be purified/isolated Intracellular (inside cell) proteins are harder to purify
Require cell disruption, separation, removal of cell debris, DNA, RNA, lipid

Extracellular (outside cell) proteins are easier to purify


No cell disruption needed, just isolate

Vigorous Methods

Sonication French press Glass bead disruption


Gentle Methods

Enzymatic lysis Detergent lysis Freeze-thaw Osmotic lysis

Differential salt precipitation Differential solvent precipitation Differential temperature precipitation Differential pH precipitation Two-phase solvent extraction (PEG) Preparative electrophoresis Column chromatography

Most purifications require combinations of 2-3 steps

Precipitation:

Addition salts (ammonium and sodium sulphate), high molecular-weight polymers (dextrans and polyethylene glycol, and organic solvents (methanol, ethanol or aceton) Decrease the temperature (40C) can increase the enzyme precipitation The addition sometimes partial purify the product. The amount of addition must be earlier identified. Eg. for cellulase from Penicillium nalgiovense could be optimally precipitated at 80% of ammonium sulphate

Principle is to separate proteins (in tact) on the basis of their charge and their ability to migrate within a gel (jello-like) matrix A strong electric field is applied to the protein mixture for an extended period of time (hours) until the proteins move apart or migrate

The pH at which a protein has a net charge=0

Q = S Ni/(1

+ 10pH-pKi)

Transcendental equation

pKa Values for Ionizable Amno Acids Residue C D E pKa 10.28 3.65 4.25 Residue H K R pKa 6 10.53 12.43

Increasing pH + + + + + + + + + _ _ _ _ _ _ _ _ _ C A T H O D E

A N O D E

pI = 5.1

pI = 6.4

pI = 8.6

Separation of basis of pI, not Mw Requires very high voltages (5000V) Requires a long period of time (10h) Presence of a pH gradient is critical Degree of resolution determined by slope of pH gradient and electric field strength Keeps protein structure intact Can be scaled up to isolate mg to gms of protein in a single tube gel run

Most common (and best) approach to purifying larger amounts of proteins Able to achieve the highest level of purity and largest amount of protein with least amount of effort and the lowest likelihood of damage to the protein product Standard method for pharma industry

Can be done either at atmospheric pressure (gravity feed) or at high pressure (HPLC, 5002000 psi) Four types of chromatography:
Affinity chromatography Gel filtration (size exclusion) chromatography Ion exchange chromatography Hydrophobic (reverse phase) chromatography

Adsorptive separation in which the molecule to be purified specifically and reversibly binds (adsorbs) to a complementary binding substand (a ligand) immobilized on an insoluble support (a matrix or resin) Purification is 1000X or better from a single step (highest of all methods) Preferred method if possible

Step 1: Attach ligand to column matrix

Step 2: Load protein mixture onto column

Step 3: Proteins bind to ligand

Step 4: Wash column to remove unwanted material, elute later

Used in many applications Purification of substances from complex biological mixtures Separation of native from denatured forms of proteins Removal of small amounts of biomaterial from large amounts of contaminants

The ligand must be readily (and cheaply) available Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein Binding must not be too strong or weak Ideal KD should be between 10-4 & 10-8 M Elution involves passage of high salt or low pH buffer after binding

Ligand
AMP

Specificity
Enzymes with NAD cofactors an ATP dependent kinases

Arginine

Proteases such as prothrombin, kallikrein, clostripain Cibacron Blue Serum Albumin, Preablumin Dye Heparin Growth factors, cytokines, coagulation factors Protein A Fc region of immunoglobulins
Calmodulin EGTA-copper Calmodulin regulated kinases, cylcases and phosphatases Proteins with poly-Histidine tails

Molecules are separated according to differences in their size as they pass through a hydrophilic polymer Polymer beads composed of cross-linked dextran (dextrose) which is highly porous (like Swiss cheese) Large proteins come out first (cant fit in pores), small proteins come out last (get stuck in the pores)

Principle is to separate on basis of charge adsorption Positively charged proteins are reversibly adsorbed to immobilized negatively charged beads/polymers Negatively charged proteins are reversibly adsorbed to immobilized positively charged beads/polymers

Has highest resolving power Has highest loading capacity Widespread applicability (almost universal) Most frequent chromatographic technique for protein purification Used in ~75% of all purifications

Matrix is made of porous polymers derivatized with charged chemicals Diethylaminoethyl (DEAE) or Quaternary aminoethyl (QAE) resins are called anion exchangers because they attract negatively charged proteins Carboxymethyl (CM) or Sulphopropyl (SP) resins are called cation exchangers because they attract positively charged proteins

Strong ion exchangers (like SP and QAE) are ionized over a wide pH range Weak ion exhangers (like DEAE or CM) are useful over a limited pH range Choice of resin/matrix depends on:
Scale of separation Molecular size of components Isoelectric point of desired protein pH stability of the protein of interest

+
Net charge on protein
Attached to anion exchangers

9 pH

Attached to cation exchangers

_
Range of pH stability

DRYING Reason: The cost of transport can be reduced; the material is easier to be handle and package; can be more conveniently stored in the dry state; more stable than the liquid form. Instrument: spray dry, in this system the evaporative cooling protect the enzyme activity. CRYSTALLIZATION Is the best way to preserve the enzyme, but the method for most enzyme still to be developed. The enzyme should be pure.