CHAPTER 1

Cell Disruption

Learning Objectives
Recognize the classes and structures of cells for the recovery of bioproducts Chemical and mechanical cell disruption methods To choose appropriate chemical or mechanical methods for general classes of applications factors influencing efficiency of disruption process: Bead Mill and Homogenizer

Characteristic of fermentation broth

Contain complex aqueous mixtures of cells + soluble extracellular products + unconverted substrate/components Can be characterized by
Size Shape (morphology) Rheological (liq flow) behaviour of cells Concentration of cells Products By products Unconverted substrate/components

Size and morphology of cells

Bioproducts requires a large variety of cells as production host ranging from bacteria of 1 m to cellular agglomerates of > 4000 m Decreasing cell size
decreasing separation capacity Results in higher operational cost

Yeast, bacteria and animal cells are usually homogeneous suspended in liquid Mold
frequently form network of hyphi which increase viscosity Under certain conditions, molds form agglomerates called pellets Pellets are large (100-4000 m) easy to recover
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Size and morphology of cells

Bacteria may form slime layers depending on strain and fermentation conditions Slime forming bacteria are very difficult to separate due to
Slime tend to retain liquid Slime may block unit operation eg membrane Slime increase viscosity of broth decreases efficiency of unit op eg filtration and centrifugation

Cells
2 Types: Prokaryotic and Eukaryotic Prokaryotic = no membrane-enclosed nucleus
bacteria gram positive- stain with crystal violet gram negative weak stain with crystal violet

Eukaryotic = has nuclei and internal organelles

yeast, animal plant fungi (mold)
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Prokaryotic Cells

Eukaryotic and Prokaryotic Cells

Cells
Cell without cell wall
animal

Cells with cell wall

bacteria fungi (mold) yeast plant

Gram positive bacteria

Simple cell wall Gram positive bacteria stained with colours due to cell wall structure Surrounded by cytoplasmic membrane covered by a structural murein network composed of polysaccharides and amino acids Cytoplasmic membrane - phospholipids double layer (deformable) Murein layer is quite rigid and maintain characteristic shape of bacterium murein layer is much thicker than gram (-) gram (+) is more difficult to disrupt mechanically particularly susceptible to lysis by the antibacterial enzyme lysozyme Eg: Lactobacillus and Staphylococcus
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Gram negative bacteria

Complex cell wall multi layered envelops Murein layer (peptidoglycan) of cells wall is thinner and surrounded by outer membrane Eg Escherichia coli and Pseudomonas Outer membrane
Peptidoglycan Lipopolysaccharides + proteins

Periplasm
Liquid filled gap Important in bioprocessing recombinant proteins are secreted into it use osmotic shock to recover

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Gram Positive Gram Negative

Murein layer (10-80 nm) Cytoplasmic membrane (8 nm)

Periplasmic space

Outer membrane (8 nm)

Murein layer

Cytoplasmic membrane

(8 nm)
Periplasmic space

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Eukaryotic Cells
Yeast (unicellular), mold cells (multicellular, filamentous)
Thick cell walls (highly crosslinked structure)- Mainly composed of polysaccharides (glucans, mannans and chitins) Plasma membranes composed of phospholipids and lipoproteins

Eukaryotic = has nuclei and internal organelles

yeast fungi (mold) animal plant

Mammalian (Animal) cells

Animal cells do not have cell walls Animal cells are very fragile Cultured animal cells are several microns in size Spherical or ellipsoid

Plant cells
Very thick cell wall (cellulose and other polysaccharides)
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Eukaryotic Cells

Plant cells
can

be bigger Cell Wall - thick and robust

composed

of cellulose and other polysaccharides

difficult

to disrupt Cultured plant cells are less robust than real plant cells

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Eukaryotic Cells

Animal, plant and yeast cells

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PRETREATMENT OF SUSPENSION
After fermentation, suspension sometimes need to be pretreated before the product can be recovered. Some of the pretreatment are:
1. Cell disruption 2. Stabilization (eg cooling, adding protease inhibitor) 3. Removal of impurities 4. Sterilization (eg pasterurization) 5. Flocculation

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Cell Disruption
Requirement on Cell Disruption depends on Product Location
Intracellular require cell disruption to release these into the liquid medium Soluble and insoluble Eg: lipids, some antibiotics, baker yeast Extracellular Desired product in broth, just treat broth to isolate and purify product Do not require cell disruption Eg: some antibiotics, enzymes, polysaccharides, amino acids

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Recovery and Purification of Bio-Products

- Strategies to recovery and purify bio-products

Fermenter
Solid-liquid separation Cell products Cells

Supernatant
Recovery Purification

Cell disruption or rupture Cell debris

Crystallization and drying

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Cell Disruption
Many overproduced proteins are found clumped together in inclusion bodies (small nodules of insoluble protein segregated within cell) These non-secreted intracellular proteins must be separated from other cellular components before they can be purified
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List of intracelullar products

Traditional intracellular products rDNA intracellular products

Glucose isomerase
-galactosidase Phosphatase Ethanol dehydrogenase

Chymosin (yeast/E.coli)
Insulin (E.coli, mammalian) Immunoglobulin Interferons (mammalian)

Dnase, Rnase
NADH/NAD+ Alkaloids

Human growth hormone (E.coli)

Human serum albumin streptokinase

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Cell Disruption Methods

1. Disruption:

the cell envelope is physically broken, releasing all intracellular components into the surrounding medium
Physical/mechanical methods target on cell wall disruption 1. Bead mill/ball mill 2. Rotor-stator mill 3. French press 4. Ultrasonic vibration Chemical and physicochemical methods destabilizing the cell membrane 1. Detergents 2. Enzymes 3. Solvents 20 4. Osmotic shock

Cell Disruption Methods

The results of these methods are often evaluated in terms of
Activity level of a cellular enzyme released to the disrupted suspension Direct counting on suitably diluted samples by plating out technique or microscopic counting in a haemocytometer (stain cell)

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Chemical Methods

1. Detergent
Destabilizing cell membrane solubilizing phospholipids Creation of canals through cell membrane Rupture mammallian cells Bacterial cell need to use with lysozyme (break cell wall) Fungal (yeast and mould) need to weaken the cell wall first before detergent can act to cell membrane Detergent 3 categories
1. Cationic 2. Anionic 3. Non-ionic preferred since cause less damage to sensitive biological molecules (proteins, DNA)

Triton X Series, Tween Series Detergent need to be removed from product require additional purification, polishing step A lot of protein denature or precipitate with detergent try to avoid use detergent
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2. Solvent
Solvent type acetone, toluene, ether, phenylethyl alcohol, benzene, methanol, chloroform Others antibiotics, thionins, surfactants, chaotropic agent, chelates Act on cell membrane by solubilising phospholipids and denature protein Toluene can disrupt fungal cell wall Limitation (similar with detergents)
1. Need to remove from product 2. Denature proteins 3. Easier to remove than detergent
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3. Osmotic shock
Osmosis is the transport of water molecules from high- to a low- concentration region when these two phases are separated by a selective membrane. Water is easier to pass the membrane than other components. When cells are dumped into pure water, cells can swell and burst due to the osmotic flow of water into the cells. Procedures 1. Allow cell to equilibrate internal and external osmotic pressure in high sucrose medium (hypertonic) 2. The put in distilled water (hypotonic) rapid influx of water into the cell volume rapid expansion rupture cell membrane 3. Product released by osmotic is periplasmic substances/ located near surface of cell (proteins) without physical damage in recombinant and nonrecombinant gram negative bacteria

Mainly for mammalian cells red blood cells For bacterial and fungal cells, cell walls need to be weakened

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4. Enzymes
Limit to releasing periplasmic or surface product To disrupt cell wall Example 1: Lysozyme Enzyme (egg based enzyme)
Able to hydrolyse murein (wall) in gram (-) and gram (+) bacteria Cannot lyse cell membrane thus Combine with detergent to disrupt cell membrane Can also combine with osmotic or mechanical disruption methods Pretreatment with EDTA will enhance effectiveness of lysozyme

Example 2: Glucanase and Mannase

Combine with protease to degrade yeast cell wall

Example 3: Cellulase and Pectinase

To disrupt plant cells wall
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4. Enzymes
Enzymatic activity depends on T and pH. May require metal ions to enhance activity or specificity Cell wall degradation has several advantages
1. 2. 3. 4. Low energy consumption Specific reaction Small risk of product damage Harmless to environment

An overdose of enzyme is always requiredcost Limitations

1. High cost 2. Removal of lysozyme (enzyme) from the product 3. Presence of other enzymes (proteases) in lysozyme samples
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Example of Microbial cell wall degrading enzymes

Organisms Bacteria Fungi, yeast Enzymes peptidase chitinase Types of hydrolysed linkages Gly-gly, ala-gly, etc peptide bonds N-acetyl-b-Dglucosaminide (1,4)-Blinkages in chitin and chitodextrins (1,4)-linkages in cellulose

Algae

cellulases

Chemical Cell disruption methods

Method Chemical Technique 1. Osmotic Shock 2. Enzyme digestion Principle Osmotic rupture of membrane Cell wall digested causing rupture Stress
gentle

Cost
cheap

Examples Rupture of red blood cells M.Lysodeikticus treated with egg lysozyme

gentle

expensive

3. Solubilization
4. Lipid dissolution

Detergents solubilize membrane

Organic solvent dissolves in membrane , destabilizes Saponification of lipids dissolves membrane

gentle

moderate

Bile salts acting on E.coli

Toluene disruption of yeast

Moderate

cheap

5. Alkali Treatment

harsh

cheap

Nucleic acid extractions

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Mechanical Methods

Mechanical methods
Cell envelope is broken physically Equipment for mechanical cell disruption
1. Bead mill large scale; best for mycelial fungi and algae 2. Homogenizer large scale; suitable yeast and bacteria 3. Ultrasonic 4. Blender

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Mechanical methods
Technique 1. Waring blender Principle Cells chopped in blender or sheared Stress Moderate Cost Moderate Examples

2. Grinding with abrasives

Cells ruptured by grinding with abrasives

Moderate

Cheap

Most cell suspensions

3.Ultrasonication

Cells broken by sonic cavitation Cells broken by shear when forced through small hole Cells crushed between glass or steel balls or beads

Harsh

Expensive

Most cell suspensions Large scale treatment of cells suspensions Large scale treatment of cells suspensions and plant tissues

4. Homogenizer (orifice type)

Harsh

Moderate

5. Ball/Bead Mill

Harsh

Cheap

Larger scale Common in chemical and food industries

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Smaller scale

Animal tissues Mycelial organisms

Cell Breaking Equipments

Pestle homogenizer Vibrating bead mill ultrasonic

Blade blender Figure 3.4 Harrison

Pressure-shear disintegrator

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Electric Mortar Grinder

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1. Rotor-stator mill
Application: Tissue based material (plant and animal tissue) Operates in multi-pass mode; the disrupted material is sent back into the device for more complete disruption Typical rotation speed 10,000 to 50,000 rpm Mechanism disruption: High shear & turbulence

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1. Rotor-stator mill
Rotor

Consists of stationary block with a tapered cavity (stator) and a truncated cone shaped rotating object (rotor) Cells suspension is fed into tiny gap between rotating rotor and fixed stator Feed is drawn in due to rotation and expelled through outlet due to centrifugal force High shear rate and turbulence between rotor and stator disrupt cells

Disrupted cells

Cell suspension

Stator

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2. French press
Application: Small-scale recovery of intracellular proteins and DNA from bacterial and plant cells Typical volumes few milliliters to a few hundred milliliters Operating pressure : 10,000 50,000 psig

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2. French press

A cylinder fitted with a plunger is connected to a hydraulic press The cell suspension is placed within the cylinder and pressurized using the plunger The cylinder is provided with an orifice through which the suspension emerges at very high velocity in the form of a fine jet Cells disruption due to : high shear rates influence by the cells within the orifice An impact plate: the jet impinges further cell disruption

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3. Bead mill

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3. Bead mill
Cascading beads

Rolling beads

Cells being disrupted

Consist of tubular vessel (metal or glass) cell suspension is placed along with small metal or glass beads. The tubular vessel is then rotated about its axis and as a result of this the beads start rolling away from the direction of the vessel rotation. At higher speed some beads move up along with the curved wall of the vessel and then cascade back on the mass of beads and cells below. Cell disruption due to - grinding action of rolling beads - impact resulting from the cascading beads
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3. Bead mill

3. Bead mill

Bead milling can generate enormous amounts of heat Cryogenic bead milling : Liquid nitrogen or glycol cooled unit (+ for thermolabile material) Application: disrupting yeast cells, grinding animal tissue Small scale: Few kilograms of yeast cells per hour Large scale: Hundreds of kilograms per hour. Consists of horizontal or vertical grinding cylinder Central shaft is fitted with a number of impeller driven by electromotor via a belt Cell is agitated in suspension with small abrasive particles Impeller design based on efficient energy transfer to beads. A typical tip speed is 15 m/s
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3. Bead mill
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3. Bead mill

Cells break because of shear forces, grinding between beads and collisions with beads Beads disrupt cells to release biomolecules

Beads are moulded from wear resistance material Zirconium oxide Zirconium silicate Titanium carbide Glass Alumina ceramic

Kinetics of biomolecule release

First order Rate of product release

dCr K b (Crmax Cr ) dt
Kb = constant; depend on type of impeller, bead size, bead load, impeller speed, T, experimentally determined Crmax= max conc of product that can be released from biomass; determine experimentally Cr = conc of release product
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Kinetics of biomolecule release Kinetics of biomolecule release

Integration gives

Crm ax ln m ax K bt Cr Cr Cr Crm ax(1 exp(K bt ))

Batch mode with residence time t For continuous mode, mean residence time, residence time distribution, no. of CSTR in series should be taken into consideration

Multiple Pass Bead Mill

Cr Crmax(1 exp(Kbt ) N )
N = number of passes
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Factors influencing product release

Microorganism used cell wall thickness and composition and cell size 2. Location of product In cytoplasm In cell organelles cell little organ Periplasmic space (space within cell membrane and cell wall) 1.

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Factors influencing product release Factors influencing product release

3.

Type of bead mill

Bead loading Rate of release is enhanced by increase of bead load Packing density inside chamber : 80 90% depending on bead diameter Upper limit impose by high power consumption and difficulties in removing heat released from operation Bead diameter Smaller bead, faster disruption but not practical since smaller bead tend to float and difficult to retain in chamber Large scale : 0.4 mm lower limit of bead diameter 0.2 mm for laboratory mill Location of product inside cell also important to determine size of bead
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Factors influencing product release Factors influencing product release

4. Tip speed of impeller, U
Frequency of collision and intensity of shear produced by impeller disc are related to linear speed of its periphery. Within certain limit, specific rate of disruption is proportional to tip speed Kb = kU Limitation to high impeller tip speed (5-15 m/s) due to high energy consumption, high heat generation and erosion of beads inactivation of shear labile product
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Factors influencing product release Factors influencing product release

Type of impeller Figure 2

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Factors influencing product release Factors influencing product release

5. Cell concentration
Cell conc affect suspension rheology influence on product release Optimized cell conc by experiment decrease cell conc, decrease amt of generated heat but increase the power consumption per unit weight of treated cells

5. Temperature

Heat generated during milling if not removed will increase T Control T:coolant jacket whereby a cooling liq circulated
Increase Q, decrease t, residence time Cell disintegration first order, hence yield decrease as feed Q increase Increase Q, power consumption per unit mass decreases (So choose High Q) Recycling part of suspension can improve yield
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7. Residence time of cell suspension

4. Homogenizer
High pressure positive displacement pump Cell suspension is pumped through an adjustable orifice discharge valve Use High pressure 200-1000 bar by an instant expansion through a exiting nozzle
Pressure vary depending on type and conc of cells

During discharge, suspension passes between valve and seat

Back pressure is controlled by a hand wheel this provide pressure on seat via spring mechanism

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4. Homogenizer

Homogenizer

Bench scale = fitting pestle is reciprocated and / or rotated in a glass or steel cylinder industrial application= Waring Blender large scale= Bead mill and ball mill; shearing devices which pass particle suspensions through small orifices at high pressure Manual = French Press
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4. Homogenizer

Schematic of a Homogenizer

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High pressure cell homogenizer

Manton-Gaulin valve type homogenizer Most popular in biotech operations Sample feed enters valve chamber in pulsatile flow. Valve close and compress cell suspension against impact ring (inner wall of chamber)
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4. Homogenizer
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Homogenizer
Valve and seat subjected to abrasion. Abrasion material must be used (stellate and tungsten carbide) Different types of high efficiency discharge valves have been developed Conical Multi-pass splitstream

4. Homogenizer

Homogenizer

Cell Disruption is accomplished by 3 mechanisms

1. Impengement on valve 2. High liquid shear in orifice 3. And sudden pressure drop upon discharge causing an explosion of cells

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Scanning electron microscopy of disrupted cultures of Lactobacillus delbrueckii subsp. bulgaricus 11842. (a) Culture prior to disruption; (b)(d) culture after one, two or three passages through a Rannie highpressure homogeinizer operated at 135 MPa. Bar: 2 mm (Bury, 2000).

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1.

2.
3. 4. 5. 6.

Factors influencing efficiency of releasing product Microorganism used cell wall thickness and composition, cell size Location of product in cytoplasm, cell organelles, perisplasmic membrane Type of valve Pressure No of passages Temperature (rise)
Heat generated, hence increase T, product denature Control T:coolant jacket whereby a cooling liq circulated Higher T, reduce broth viscosity Constant P at increasing T has a positive effect on product release
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Cell Disruption Kinetics for Homogenizer

Rate of release first order. Similar to bead mill

Crm ax ln m ax K h f (p) N Cr Cr
N no of passages F-function of pressure difference

Cr Crm ax(1 exp( K h f (p) N )

Exposure time (t) in bead mills is replaced by no of passages through the homogenizer (N). f(p) is determined experimentally. Normally, f(p)= (p)

Thus

Cr Crm ax (1 exp( K h f (p ) N )
= constant depend on type of organism and physiological growth; 2.9 for yeast; 2.2 for E.coli
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5. Ultrasonic vibrators
Application: Bacterial and fungal cells Mechanism: Cavitation followed by shock waves High frequency formation of tiny bubbles bubbles collapse releasing mechanical energy (shockwave) ~ thousands atm pressure Frequency: 25 kHz Duration depends on cell type, sample size and cell concentration Bacterial cells (E. coli) 30-60 s Yeast cells 2-10 minutes Used in conjunction with chemical methods Cell barriers are weakened by small amounts of enzymes or detergents energy reduced
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Ultrasound generator

Ultrasound tip

Cell suspension

Laboratory Ultrasonicator
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Summary

Cells classification-prokaryotic (no nucleus bact) and eukaryotic (fungi, yeast, higher organism) Cell disruption methods Chemical Mechanical Cell membrane disruption normally use chemical methods

Cell wall disruption normally use physical methods

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