KULIAH TEKNOLOGI FERMENTASI29 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 Modification of the broth characteristics for faster handling and simpler equipment:handling and simpler equipment:Selection of microorganism which does not produce Selection of microorganism which does not produce pigments or undesirable metabolitespigments or undesirable metabolitesModification of the fermentation condition to reduce Modification of the fermentation condition to reduce the production of undesirable metabolitesthe production of undesirable metabolites Precise timing of harvesting (optimum condition)Precise timing of harvesting (optimum condition) pH control after harvestingpH control after harvesting Temperature treatment after harvestingTemperature treatment after harvestingAddition of flocculating agentsAddition of flocculating agentsUse of enzymes to attack cell wallsUse of enzymes to attack cell walls

Tingkatan Produk

Proses Kons. (g/l) Kwalitas (%)

Pemanenan Fermentasi 0.1 – 5 0.1 – 1.0

Pengambilan bahan tidak terlarut

Filtrasi 1.0 – 5 0.2 – 2.0

Isolasi Ekstraksi 5 – 50 1 – 10

Purifikasi Kromatografi 50 – 200 50 – 80

Produk akhir Kristalisasi 50 – 200 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. Pumps3. Other

Piping Tanks Valves Flowmeter Manometer

04/08/23

FEATUREREVERSEOSMOSIS

NANOFILTRATION

ULTRAFILTRATION

MICROFILTRATION

Membrane Asymmetrical Asymmetrical AsymmetricalSymmetrical

Asymmetrical

Wall Thickness 150m 150m 150-250m 10-150m

Film thickness 1m 1m 1m various

Pore size <0.002um <0.002um 0.02-0.2um 0.2-5um

Rejects

HMWC, LMWC,Sodium, Chloride,

glucose, amino acids,

proteins

HMWC, mono-,di-, and

aligo-saccharides, polyvalent

anions

Macromolecules, proteins,

polysaccharides, viruses

Particulates, clay, bacteria

Membrane module

Tubular, spiral-wound, plate

& frame

Tubular, spiral-wound, plate

& frame

Tubular, hollow-fibre, spiral-wound, plate

& frame

Tubular, hollow-fibre, plate &

frame

Material CA, TFC CA, TFC CA, TFC, Ceramic

CA, TFC, Ceramic, PVDF,

Sintered

Pressure 15-150 bar 5-35 bar 1-10 bar <2 bar

Flux 10-50 (l/m2/hr) 10-100 l/m2/hr 10-200 l/m2/hr 50-1000 l/m2/hr

RO membrane

Ultrafiltration Microfiltration

MIKROFILTRASITanpa pembentukan cake

•Menggunakan membran: tipis dan microporous

•Lubang pori2nya kecil dan sangat monodisperse

•Mempunyai kemampuan menyaring partikel yang tidak diinginkan

•Membran mengikuti hukum Darcy’s 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 Minerals Lactose

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

Characteristic

Module Type

Flat plate Spiral Wound

Shell and

Tube

Hollow Fibre

Packaging density (m2/m3)

Moderate (200-400)

Moderate (300-900)

Low (150-300)

High (9000-30000

Fluid management

Good Good High pumping costs

Good

Suspended solids capability

Moderate Poor Good Poor

Cleaning Sometimes difficult

Sometimes difficult

Easy Backflusing possible

Replacement Sheets or cartridge

Cartridge Tubes 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 %)

Maximum Throughput

(m3/hr)

Bowl separator Stays in Bowl 0 - 1 150

Solid injecting: nozzle separator

Intermittent discharge through axial channels

0.01 - 10 200

Solid injecting: slot separator

Intermittent discharge through radial slot

0.2 - 20 100

Nozzle separator Continuous discharge 1 - 30 300

Product Microorganisme Relative throughput

in Centrifuge

Type Separator

Microorganism Size (m)

Baker’s yeast Saccharomyces 7 – 10 100 Nozzle

Alcohol yeast Saccharomyces 5 – 8 60 Solids ejecting

Citric acid Mold - 30 Solids ejecting

Enzyme Bacillus 1 – 3 7 Nozzle

Solid ejecting

BIOMASS CENTRIFUGATIONS

•Digunakan untuk memecah sel.

Method Technique Principle Stress or Product

Cost Example

Chemical Enzyme digestion

Cell wall digested, providing disruption

Gentle Expensive Micrococcus lysodeikticus treated with egg lysosym

Lipid dissolution Organic solvent dissolves in cell wall

Moderate Cheap Toluene disruption of yeast

Mechanical Grinding Cells ruptured by grinding with abrasives

Moderate Cheap

Ultrasonication Cells broken with ultrasonic cavitation

Harsh Moderate Cell suspension at least on a small scale

•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

CHEMICAL METHOD

•Small scale•Homogenization in Waring Blender, mycelial organisms and with other animal cells or tissue•grinding, •ultrasonication

•Large scale•Homogenization and crushing

MECHANICAL METHOD

Batch Extraction

Differential Extraction

Variable Extraction Adsorption

Capacity High Low

Selectivity Moderate High

Nature of equilibrium Often linier; dilute solutes independent

Usually non linier; dilute solutes interact

Nature of operation Steady state Unsteady; periodic

Problems Emulsification; denaturation

Solids handling; compressible packing

•PrecipitationPrecipitation•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>PO4

2->SO42->CH3COO->Cl->NO3

-

•Cation : NH4+>K+>Na+

•Densitas presipitasi berbeda dengan densitas larutan

•With Temperature•Ultra filtrasiUltra filtrasi•Elution ChromatographyElution Chromatography; Adsorption, Ion exchange, Gel permeation, Affinity, Reverse phase, High performance liquid.•Electrophoresis

Cell Suspension

Cell Concentrate

Cells

Cell-free culturemedium

Membrane

Cross Flow Filtration

Cell-free culturemedium

Dead EndFiltration

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

Sonication French press Glass bead

disruption

Enzymatic lysis Detergent lysis Freeze-thaw Osmotic lysis

Gentle Methods

Vigorous Methods

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

PrecipitationPrecipitation:• 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 = Ni/(1 + 10pH-pKi)

Residue pKa Residue pKa C 10.28 H 6 D 3.65 K 10.53 E 4.25 R 12.43

pKa Values for Ionizable Amno Acids

Transcendentalequation

ANODE

+++++++++

_________

CATHODE

Increasing pH

pI = 8.6pI = 6.4pI = 5.1

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, 500-2000 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 ligandto column matrix

Step 2: Load proteinmixture onto column

Step 3: Proteins bindto 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 Specificity

AMP Enzymes with NAD cofactors an ATP dependent kinases

Arginine Proteases such as prothrombin, kallikrein, clostripain

Cibacron Blue Dye

Serum Albumin, Preablumin

Heparin Growth factors, cytokines, coagulation factors

Protein A Fc region of immunoglobulins

Calmodulin Calmodulin regulated kinases, cylcases and phosphatases

EGTA-copper 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 (can’t 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

ch

arg

e o

n p

rote

in

4 5 6 7 8 9 pH

Attached tocation exchangers

Attached toanion exchangers

Range of pH stability

DRYINGDRYINGReason: 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.

CRYSTALLIZATIONCRYSTALLIZATIONIs the best way to preserve the enzyme, but

the method for most enzyme still to be developed. The enzyme should be pure.