2 Cell Disruption 20022013

1

CHAPTER 1

Cell Disruption

2

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

3

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

4

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

5

• 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

Size and morphology of cells

6

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)

7

Prokaryotic Cells

Eukaryotic and Prokaryotic Cells

8

• Cell without cell wall– animal

• Cells with cell wall– bacteria

– fungi (mold)

– yeast

– plant

Cells

9

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

10

• 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

Gram negative bacteria

11

Gram Positive – Gram Negative

Periplasmic space

Murein layer (10-80 nm)

Cytoplasmic membrane (8 nm)

Periplasmic space

Murein layer

Cytoplasmic membrane (8 nm)

Outer membrane (8 nm)

12

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

• 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)

Eukaryotic = has nuclei and internal organelles•yeast•fungi (mold)•animal•plant

13

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

Eukaryotic Cells

14

Animal, plant and yeast cells

Eukaryotic Cells

15

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

16

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

17

Recovery and Purification of Bio-Products

- Strategies to recovery and purify bio-products

Fermenter

Solid-liquid separation

Recovery

Purification

SupernatantCellsCell products

Cell disruption or rupture

Cell debris

Crystallization and drying

18

• 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

Cell Disruption

19

List of intracelullar products

Traditional intracellular products rDNA intracellular products

Glucose isomerase Chymosin (yeast/E.coli)

-galactosidase Insulin (E.coli, mammalian)

Phosphatase Immunoglobulin

Ethanol dehydrogenase Interferons (mammalian)

Dnase, Rnase Human growth hormone (E.coli)

NADH/NAD+ Human serum albumin

Alkaloids streptokinase

20

Cell Disruption Methods1. Disruption:

the cell envelope is physically broken, releasing all intracellular components into the

surrounding medium

• Physical/mechanical methods – target on cell wall disruption1. Bead mill/ball mill2. Rotor-stator mill3. French press4. Ultrasonic vibration

• Chemical and physicochemical methods – destabilizing the cell membrane1. Detergents2. Enzymes3. Solvents4. Osmotic shock

21

• 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)

Cell Disruption Methods

22 Chemical Methods

23

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. Cationic2. Anionic3. 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

24

• 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 product2. Denature proteins3. Easier to remove than detergent

2. Solvent

25

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.

• Procedures1. 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 non-recombinant gram negative bacteria

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

weakened

26

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

27

• Enzymatic activity depends on T and pH. May require metal ions to enhance activity or specificity

• Cell wall degradation has several advantages1. Low energy consumption2. Specific reaction3. Small risk of product damage4. Harmless to environment

• An overdose of enzyme is always requiredcost• Limitations

1. High cost 2. Removal of lysozyme (enzyme) from the product3. Presence of other enzymes (proteases) in

lysozyme samples

4. Enzymes

28

Example of Microbial cell wall degrading enzymes

Organisms Enzymes Types of

hydrolysed linkages

Bacteria peptidase Gly-gly, ala-gly, etc peptide bonds

Fungi, yeast chitinase N-acetyl-b-D-glucosaminide (1,4)-B-linkages in chitin and chitodextrins

Algae cellulases (1,4)-linkages in cellulose

29

Chemical Cell disruption methodsMethod Technique Principle Stress Cost Examples

Chemical 1. Osmotic Shock

Osmotic rupture of membrane

gentle cheap Rupture of red blood cells

2. Enzyme digestion

Cell wall digested causing rupture

gentle expensive

M.Lysodeikticus treated with egg lysozyme

3. Solubilization

Detergents solubilize membrane

gentle moderate

Bile salts acting on E.coli

4. Lipid dissolution

Organic solvent dissolves in membrane , destabilizes

Moderate

cheap Toluene disruption of yeast

5. Alkali Treatment

Saponification of lipids dissolves membrane

harsh cheap Nucleic acid extractions

30 Mechanical Methods

31

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

32

Technique Principle Stress Cost Examples

1. Waring blender Cells chopped in blender or sheared

Moderate Moderate Animal tissuesMycelial organisms

2. Grinding with abrasives

Cells ruptured by grinding with abrasives

Moderate Cheap Most cell suspensions

3.Ultrasonication Cells broken by sonic cavitation

Harsh Expensive Most cell suspensions

4. Homogenizer (orifice type)

Cells broken by shear when forced through small hole

Harsh Moderate Large scale treatment of cells suspensions

5. Ball/Bead Mill Cells crushed between glass or steel balls or beads

Harsh Cheap Large scale treatment of cells suspensions and plant tissues

Mechanical methods

Larg

er

scale

Com

mon

in

ch

em

ical

an

d f

ood

in

du

str

ies

Sm

aller

scale

33Figure 3.4 Harrison

Cell Breaking EquipmentsPestle homogenizer

Blade blender

ultrasonic

Pressure-shear disintegrator

Vibrating bead mill

34

Electric Mortar Grinder

35

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

36

• 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

Cell suspension

Rotor

Stator

Disrupted cells

1. Rotor-stator mill

37

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

38

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

39

3. Bead mill

40

3. Bead millCascading beads

Cells being disrupted

Rolling beads

• 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

41

• 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

3. Bead mill3. Bead mill

42

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

3. Bead mill3. Bead mill

43

Kinetics of biomolecule release

• First order

• Rate of product release

• 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

)( maxrrb

r CCKdt

dC

44

• Integration gives

• 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

N = number of passes

))exp(1(

ln

max

max

max

tKCC

tKCC

C

brr

brr

r

Kinetics of biomolecule releaseKinetics of biomolecule release

))exp(1(max Nbrr tKCC

45

Factors influencing product release

1. 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)

46

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

Factors influencing product releaseFactors influencing product release

47

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

Factors influencing product releaseFactors influencing product release

48

– Type of impeller

– Figure 2

Factors influencing product releaseFactors influencing product release

49

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

7. Residence time of cell suspension– 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

Factors influencing product releaseFactors influencing product release

50

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

51

• 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

Homogenizer4. Homogenizer

52Schematic of a Homogenizer

4. Homogenizer

53

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)

54

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

Homogenizer4. Homogenizer

55

• Cell Disruption is accomplished by 3 mechanisms1. Impengement on valve

2. High liquid shear in orifice

3. And sudden pressure drop upon discharge causing an explosion of cells

Homogenizer4. Homogenizer

56

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 high-pressure homogeinizer operated at 135 MPa. Bar: 2 mm (Bury, 2000).

57

Factors influencing efficiency of releasing product

1. Microorganism used – cell wall thickness and composition, cell size

2. Location of product – in cytoplasm, cell organelles, perisplasmic membrane

3. Type of valve4. Pressure5. No of passages6. 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

58

Cell Disruption Kinetics for Homogenizer

• Rate of release – first order. Similar to bead mill

N –no of passagesF-function of pressure difference

• 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

= constant depend on type of organism and physiological growth; 2.9 for yeast; 2.2 for E.coli

))(exp(1(

)(ln

max

max

max

NpfKCC

NpfKCC

C

hrr

hrr

r

))(exp(1(max NpfKCC hrr

59

60

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

61

Laboratory Ultrasonicator

Cell suspension

Ultrasound tip

Ultrasound generator

62

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