Name:

ABDUL BASIT MUBEEN

Roll No:

SSP14-CE05

Assignment No. 1:

PHYSICAL & CHEMICAL CELL DISTRIBUTION TECHNIQUES

Date:

MAY 14, 2018

Submitted To:

DR. SAYED NADIR HUSSAIN

INTRODUCTION
Cell disruption is an essential part of biotechnology and the downstream
processes related to the manufacturing of biological products. The disruption of
cells is necessary for the extraction and retrieval of the desired products, as cell
disruption significantly enhances the recovery of biological products. Cell
disruption cannot be considered an isolated process, as it affects the physical
properties of the cell slurry, thus indirectly influencing further downstream
processes. Several types of cell disruption methods exist, as biological products
may be extracellular, intracellular or periplasmic. Cell disruption methods can be
categorised into mechanical methods and non-mechanical methods (Figure 1).
Mechanical methods are divided into solid shear methods and liquid shear
methods. Non-mechanical methods can be divided into physical methods,
chemical methods and enzymatic methods. This report will discuss some methods
from each category, as examples of the varying disruption methods available.

Figure 1 Methods of microbial cell disruption (Geciova J., 2002)

Different cells have different structures; hence they require different methods for
disruption. Cell walls act as additional disruption deterrents, with yeast cells being
particularly difficult to disrupt, as the cell wall limits the solvents access to the

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desired products. Other types of cells requiring disruption are bacterial cells,
moulds, plant cells, mammalian cells and ground tissue. Bacterial cells may have
different disruption methods, depending on whether they are gram positive or
gram negative, as the amount of peptidoglycan and the presence of an envelope
affect the overall process. Mammalian cells are the easiest to disrupt as they lack
a cell wall, unlike plant cells, which are more difficult to disrupt.

The drying of the cell mass enhances disruption methods and may help bring
down the costs. In some cases, more than one disruption method may be
necessary to achieve full product recovery. Factors that influence the selection of
disruption method include the susceptibility of the cells to disruption, product
stability, the ease of extraction from the cell debris, the speed of the method and
the cost of the method. Mechanical methods produce heat during the process, so
additional cooling systems are required when using mechanical cell disruption
methods.

Before cell disruption can take place, the cells must be separated from the culture
medium. Secreted extracellular components need to be decreased and unutilized
media components also need to be reduced. Ideally, the chosen cell disruption
method is appropriate for the cells being disrupted, has a well understood
mechanism, is sterilisable, containable and validated, with the possibility of
automation. Other beneficial properties include a continuous and compact
method, which is economical and efficient.

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PHYSICAL METHODS

The main principle of the mechanical disruption methods, is that the cells are
being subjected to high stress via pressure, abrasion with rapid agitation with
beads, or ultrasound. (Geciova, 2002) Some methods of disruption are cavitation,
shearing, impingement, or combination of those. Intensive cooling of the
suspension after the treatment is required in order to remove the heat that was
generated by the dissipation of the mechanical energy. Some high-pressure
methods can only be applied in laboratory scale, such as French press and Hughes
press. For industrial use, the bead mill and high-pressure homogenizer, are
suitable. (APV, 2009)

BEAD MILL

Bead mills have been originally used in the paint industry, and have been adapted
for cell disruption in both small scale and large scale production. (Geciova J.,
2002). It is an efficient way of disrupting different microbial cells as different
designs have been developed. The main principle requires a jacketed grinding
chamber with a rotating shaft, running in its center (figure 2). Agitators are fitted
with the shaft, and provide kinetic energy to the small beads that are present in
the chamber. That makes the beads collide with each other. The choice of bead
size and weight is greatly dependent on the type of cells. The diameter can affect
the efficiency of cell disruption in relation of the location of the desired enzyme in
the cell. The increased number of beads increases the degree of disruption, due
to the increased bead-to-bead interaction. The increased number of beads,
however, also affects the heating and power consumption. An optimal condition
for bead load is considered between 80 and 85% of the free volume. The discs run
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at a speed of 1500-2250 rpm. Glass beads with a diameter greater than 0.5 mm
are considered best for yeast cells, and diameter lesser than 0.5 mm is optimal for
bacterial cells. (APV, 2009) The process variables are: agitator speed, proportion
of the beads, beads size, cell suspension concentration, cell suspension flow rate,
and agitator disc design. (Chisti Y., 1986)

Main issues related to bead mills, are the high temperature rises with increase of
bead volume, poor scale-up, and most importantly, there is a high chance of
contamination. (Harrison S., 1991)

Figure 2 The basic principle of a bead mill

K is a function of the rate of agitation (1500-2250 rpm), cell concentration (30-

60% wet solids), beads diameter (0.2-1.0 mm), and temperature. Where R -
protein released (kg protein/kg biomass), Rm - max protein available, k -rate
constant and is a function of temperature. (Chisti Y., 1986)

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ULTRASOUND

Ultrasonic disruption is caused by ultrasonic vibrators that produce a high

frequency sound with a wave density of about 20 kHz/s (figure 3). A transducer
then converts the waves into mechanical oscillations through a titanium probe,
which is immersed into the cell suspension. Such a method is used for both
bacterial and fungal cell disruption. Bacterial cell can be disrupted in 30 to 60 sec,
and yeast between 2 and 10min. This method is usually used in combination with
a chemical method (mostly lysis). (Harrison S., 1991)

Figure 3 Schematic representation of ultrasonic disruption

Sonication can be very effective in small scale work; however, upscaling is very
poor. It has high energy requirements, as well as high health and safety issues,
due to noise. It is not continuous. (Chisti Y., 1986)

FRENCH PRESS AND HIGH PRESSURE HOMOGENISER

In a French press, or high pressure homogenization, the cell suspension is drawn

through a valve into a pump cylinder (figure 4). Then it is forced under pressure of
up to 1500 bar, through a narrow annular gap and discharge valve, where the
pressure drops to atmospheric. Cell disruption is achieved due to the sudden drop
in pressure upon the discharge, causing the cells to explode. This method is one of

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the most widely known and used methods. It is mostly used for yeast cells. It is a
vital unit in the dairy production industry, for milk homogenization. (Middleberg
A., 1995) By operating the press at higher pressures, the number of passes of the
slurry through it can be decreased in order to obtain the desired degree of
disruption. However, the operating pressure may be limited due to the
deactivation of certain heat-sensitive proteins, which may increase the number of
passages required. Hence, protein release is dependent on several factors:
temperature, intracellular location of the enzymes, number of passes, and
operating pressure. The process is dependent on biomass concentration. (APV,
2009)

The French press is a small scale method, whereas the homogenizer can be
applied to a large scale production. Homogenisers can vary in design and has a
high amount of solids, up to 50% of the feed. Heat generation is also high –
1.5ºC/1000 psi. (Geciova J., 2002)

Correlation between protein release and number of passes.

Protein release (R) is first order with respect to the number of passes (N). The
dependence of protein release on operating pressure (P 400-600 bar) can be
expressed as a function of the pressure raised to an exponent. (Middleberg A.,
1995)

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The results can be combined in the correlation, where R is protein released (kg
protein/kg biomass), Rm is max protein available, k is rate constant and is a
function of temperature, a is the pressure exponent. (Middleberg A., 1995)

Figure 4. Schematic representation of the basic principle of a French press.

THERMOLYSIS

Thermolysis has shown potential in becoming more common in large scale

production. Periplasmic proteins in G(-) bacteria are released when the cells are
heated up to 50ºC. Cytoplasmic proteins can be released from E.coli within 10min
at 90 ºC. Improved protein release has been obtained after short high
temperature shocks, than when at longer temperature exposures at lower values.
Unfortunately, the results are highly unreliable, as the protein solubility changes
with temperature fluctuations. (Middleberg A., 1995)

Freezing and thawing of a cell slurry can cause the cells to burst due to the
formation and melting of ice crystals. Gradual freezing, leading to the formation
of larger crystals, can cause an extensive damage to the cell. By combining this
method with cell grinding, this technique has shown great results. However, it is

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very costly, and restricted to small-scale laboratories. Some reports have also
shown loss of enzyme activity. (Harrison S., 1991)

DECOMPRESSION

During explosive decompression, the cell suspension is mixed with pressurized

subcritical gas for a specified time, depending on the cell type. The gas enters the
cell and expends on release, causing the cell to burst. Decompression has been
used in small scale laboratories for the disruption of E.coli. The technique has
shown promising results with yeasts, where it has the advantage that supercritical
CO2 is able to extract off-flavours that are caused by lipid components. This
technique is proving to be promising, being gentle on the cells, resulting in large
debris that are easier to remove in order to obtain the desired product.
Downsides, however, include its low efficiency and its high dependency on
pressure release and time of contact between the cell suspension and the gas.
Decompression chamber is shown in figure 5. (Harrison S., 1991)

OSMOTIC SHOCK

The proper functionality of cell’s processes usually requires strictly defined

chemical conditions. This means e.g. that cell’s internal pH or salt concentrations
should not deviate significantly from the optimal values. The optimal conditions
and ability to withstand suboptimal conditions are species specific. Cells have an
ability to actively control the internal conditions but sudden and major changes in
cell’s surrounding environment might lead to extreme shock which results in cell
death and disruption.

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Osmotic shock is a technology which can be utilized in biotechnical applications to
cause cell lysis. In this technology, cells are first exposed to either high or low salt
concentration. Then the conditions are quickly changed to opposite conditions
which leads to osmotic pressure and cell lysis (figure 6). The reason for that is that
water quickly flows from low salt concentration conditions towards conditions
with high salt concentration. Thus, if the cells are first exposed to high salt
concentration solution, water flows into cell after exposure to low salt
concentration. As a result, pressure in cell increases and cell explodes (Stanbury
et al. 2016). Conversely, if cell are exposed to high salt concentration (~1 molar
solution) after exposure to low concentration, water flows out of the cell which
leads to cell disruption.

Figure 6. Osmotic shock. Exposure of cells to either high or low salt concentration
causes cell disruption.

Osmotic shock is not commonly used method for cell disruption because of its low
efficiency. The efficient disruption would commonly require for example
enzymatic pre-treatment to weaken the cells. In addition, this technology requires

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addition of high amounts of salts and water usage is high. Also product may be
diluted which increases downstream processing costs.

CHEMICAL METHODS

In addition to physical and mechanical methods, several chemical methods for cell
disruption exist. These methods rely on utilization of chemical substances or
enzymes in disruption process. The mechanisms of actions are multiple, but the
most widely used methods act by destroying the cell wall by enzymes, osmotic
pressure, or by interfering or precipitating cell wall proteins. In addition, several
disruption methods can be combined to achieve desired efficiency. The
alternative strategies are reviewed in more detail below.

DETERGENTS

Detergents that are used for disrupting cells are divided into anionic, cationic and
non-ionic detergents. The common thing for all detergents is that they directly
damage the cell wall or membrane, and this will lead to release of intracellular
content (figure 7). One of the most commonly used anionic detergent is sodium
dodecyl sulfate (SDS) which reorganizes the cell membrane by disturbing protein-
protein interactions (Thermo Fisher Scientific | Detergents for Cell Lysis and
Protein Extraction). Another commonly used compound for cell lysis is Triton
X100, which is non-ionic detergent. Its mechanism of action is to solubilize
membrane proteins (Harrison 2011). In addition to these chemical compounds,
for example cationic detergent ethyl trimethyl ammonium bromide can also be
used for cell disruption. It is speculated that it acts on cell membrane
lipopolysaccharides and phospholipids (Stanbury et al. 2016).

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Figure 7. Cell disruption with detergents. Detergents interact with cell membrane
compounds which will lead to disassembly of cell membrane.

The disadvantage of using detergents for cell lysis is that many proteins will be
denatured in lysis process. Detergents may also disturb subsequent downstream
processing steps. Thus additional purification step may be required after cell lysis,
which limits their utilization in large scale

processes. However, detergents are commonly used for cell lysis in laboratory for
example once DNA, RNA or proteins are extracted from cells.

SOLVENTS

One additional method for chemical cell disruption is the utilization of chemical
solvents. Solvents which can be used for cell lysis include for example some
alcohols, dimethyl sulfoxide, methyl ethyl ketone or toluene (Stanbury et al.
2016). These solvents extract cell wall’s lipid components which leads to release

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of intracellular components. This method can be used with wide range of
production organisms but the problem can be that some proteins are denatured.
However, the advantage is that by the choice of solvent, it might be possible to
select the relished product. This method is not generally applied in large scale
processes.

In addition to solvents, cell lysis can be achieved by hydrolysing the cell wall by
alkali compound (pH 10.5-12-5). Disadvantage of this method is that chemical
costs for neutralization of alkali are high. In addition, the product may not be
stable in alkali conditions.

ENZYMES

Another strategy to achieve cell lysis is to use digestive enzymes which will
decompose the microbial cell wall (figure 8). Different cell types and strains have
different kind of cell walls and membranes, and thus the used enzyme depends
on microbe. For example, lysozyme is commonly used enzyme to digest cell wall
of gram positive bacteria. Lysozyme hydrolyzes β-1-4-glucosidic bonds in the
peptidoglycan (Crapisi et al. 1993). The cell wall of gram negative bacteria differs
from the cell wall of gram positive bacteria so lysozyme is not very efficient in the
case of gram negative cell wall.

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Figure 8. Enzymatic cell disruption concept. Enzymes degrade the cell wall
components which will lead to release of intracellular compounds.

The cell wall of yeast and fungi differs significantly from the cell wall bacteria. One
commonly used enzyme mixture for degradation of cell wall of yeast and fungi is
Zymolyase. It has for example β-1,3 glucanase and β-1,3-glucan laminaripentao-
hydrolase activities (Zymolyase | Yeast lytic enzyme). In addition, the enzymes
that are commonly used for degradation of cell wall of yeast and fungi include
different cellulases, pectinases, xylanases and chitinases.

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BIBLIOGRAPHY

1. APV, An SPX Brand (2009); Cell disruption by Homogenization;

http://www.apvhemisan.com/uploads/images/Cell_Disruption_by_Homog
enization_3006_01_ 06_2008_US.pdf
2. Chisti Y., Moo-Young M. (1986); Disruption of microbial cells for
intracellular products; Enzyme Microb. Technol., vol. 8, April; doi: 0141 --
0229/86/040194--11
3. Crapisi, A., Lante, A., Pasini, G., & Spettoli, P. (1993). Enhanced
microbial cell lysis by the use of lysozyme immobilised on
different carriers. Process Biochemistry, 28(1), 17–21.
4. Geciova J., Bury D., Jelen P. (2002); Methods for disruption of microbial
cells for potential use in the dairy industry- a review; International Dairy
Journal 12, 541–553; PII: S 0958-6946(02)00038-9
5. Harrison S. (1991); BACTERIAL CELL DISRUPTION: A KEY UNIT OPERATION
IN THE RECOVERY OF INTRACELLULAR PRODUCTS; Biotech. Adv. Vol. 9, pp.
217-240;
6. Harrison, S.T. L. (2011) Cell distruption. In: M. Moo-Young (Ed. in chief),
Comprehensive
7. Biotechnology, Volume 2, (2nd ed.) (pp. 619–639). Oxford: Elsevier.
8. Middelberg A., (1995) Process-scale disruption of microorganisms;
Biotechnology Advances, Vol. 13, No. 3, pp. 491-551; doi: 0734-
9750(95)02007-P
9. Banu N., Department of Biotechnology Vels University; Cell disruption
presentation; (2015); http://www.slideshare.net/eswar1810/cell-
distruption
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10.Stanbury, P., Whitaker, A. & Hall, S. (2016) Principles of Fermentation
Technology (Third Edition), Chapter 10.
11.Thermo Fisher Scientific | Detergents for Cell Lysis and Protein Extraction
[Online] Available: https://www.thermofisher.com/fi/en/home/life-
science/protein-biology/protein-biology-learning-center/protein-biology-
resource-library/pierce-protein-methods/detergents-cell-lysis-protein-
extraction.html
12.Zymolyase | Yeast lytic enzyme [Online]. Available:
13.https://www.zymoresearch.de/protein/enzymes/zymolyase-yeast-lytic-
enzyme

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