Biotech. Adv. Vol. 9, pp. 217-240,1991 0734 - 9750/91 $(3.(]0+ .

50
Printed in Great Britain. All Rights Reserved, © 1991 Pergamon Prt,.,s.splc

BACTERIAL CELL DISRUPTION: A KEY UNIT

OPERATION IN THE RECOVERY OF
INTRACELLULAR PRODUCTS

SUSAN T. L. HARRISON

Department of Chemical Engineering, University of Cambridge, Pembroke Street,

Cambridge, CB2 3RA, U.K.
Present Address: Department of Chemical Engineering, University of Cape Town,
Private Bag, Rondebosch 7700, South Africa

ABSTRACT

The need for microbial cell disruption has hindered the large scale production of commercial
biotechnological products of intraceUular derivation. The intracellular nature of many recombinant
products and the potential use of the bacterial storage product, PHB as a commodity thermoplastic have
renewed interest in the improvement of this unit operation. This paper provides a review of processes
of a mechanical, physical, chemical or biological nature used for cell disruption on both the laboratory
and large scale. Applicability of the techniques to large scale operation is discussed. Modification of
existing processes is suggested for the reduction of energy requirements and improved process
economics. The requirements for the liberation of granular intracellular products such as inclusion
bodies and virus-like yeast particles am distinguished from those for the liberation of soluble products,
mainly proteinaceous in nature.The integrated nature of the process with both upstream and
downstream processes is addressed. Finally, the recent approach of selective liberation of soluble
products of interest is reviewed.

KEYWORDS

cell disruption, intracellular product release, cell pcrmeabilisation, high pressure homogenisation, high
speed bead mills, chemical lysis, enzymic lysis, differential product release

217
218 S. T. L. HARRISON
INTRODUCTION

Increased interest is being shown in the efficient and cost-effective release of intracellular bacterial
products from their host micro-organisms. This is due to the predominantly intracellular location of
products of recombinant DNA technology, the overproduction of proteins as inclusion bodies and the
potential use of the intracellular storage product poly-13-hydroxybutyrate (PHB) as a biodegradable
thermoplastic. The nature of the cell disruption process may influence the extent of product recovery,
the ease of the subsequent purification steps, the nature of the suspensions to be processed and the
form and quality of the final product. These combined recovery processes are of prime importance in
process economics. It is estimated that in the production of recombinant proteins, 45% of the
equipment costs are associated with product recovery while only 14% can be attributed to the
fermentation process (27). The ratio of variable costs of recovery to those of fermentation vary from 1
to 3 for enzyme and antibiotic recovery up to 10 for the recovery of intracellular recombinant insulin
(19, 27).

Laboratory scale disruption processes have been well established for many years. However, processes
available for the large scale disruption of bacteria remain few. In addition, application of such
processes to the production of commodity products is frequently cost-prohibitive. In this review, a
brief description of the bacterial cell envelope is given to identify key components in its disruption. A
classification of cell disruption methods employed on both the laboratory and large scale follows in
which assessment of the applicability of the processes to both large scale operation and specific product
recovery is given. The integrated nature of the process with operations both upstream and downstream
is considered. This initiates discussion of the recent developments in which the preferential release of
the desired product is sought in place of general disruption and in which chemical or enzymic
treatments are coupled to mechanical cell disruption to enhance its efficiency.

THE B A C T E R I A L C E L L E N V E L O P E

The cell envelope of rnicro--organisms is a semi-rigid structure providing sufficient intrinsic strength to
protect the cell from osmotic lysis. Additionally, it forms a biologically active boundary between the
micro-organism and its external environment. In a typical Gram-negative bacterium, such as E.coli,
this consists of an elastic semi-permeable cytoplasmic membrane (innermost), an inter-membrane
periplasmie space, a thin rigid wall layer comprised of peptidoglycan, and a lipid-protein outer
membrane bilayer. Gram-positive bacteria such as Bacillus lack the outer membrane component, but in
turn possess a more dominant peptidoglycan structure. These envelope structures are readily
distinguished by their staining characteristics. While the composition of these envelopes have been well
reviewed (24, 30, 35, 63), a brief summary is useful to identify the nature of the structures to be
disrupted.

The cytoplasmic membrane provides the major interactive barrier between the internal cell environment
and the bulk media. Typically, the bilayer structure is approximately 4 nm wide (59) and is comprised
predominantly of phospholipid and protein. This biophysico-chemical system actively maintains
 BACTERIAL CELL DISRUPTION 219
concentration gradients, houses transport systems and is involved in ATP generation. It does not
provide any significant structural strength and is readily disrupted by osmotic shock in the absence of
the structural component layers.

The rigid peptidoglycan layer forms the basic structural framework of the cell envelope, providing its
mechanical strength. The basic peptidoglycan structure, similar in all bacteria, is comprised of linear
polysaccharide chains of aiternating N-acetyl-D-glucosamine (NAG) and N-acetyl-muramic acid
(NAM) residues joined by ~-(1-4) glycosidic bonds. The chains are cross-linked by a tetrapeptide of
the basic structure L-aianyl-D-glutamyl-L-R3-D-alanine attached to the C3 lactic acid side chain of the
NAM residue. The L-R3 amino acid residue may be one of several di-amino acids, frequently
diaminopimelic acid. The peptide branches of the parallel chains are further cross-linked. The resulting
rigid grid structure acts as a single macromolecular network to provide the shape, tensile strength and
osmotically protective nature of the cell envelope. Its strength is governed by the frequency of peptide
chains and their crosslinking (14, 24, 30, 35). The peptidoglycan layer in Gram-negative bacteria is
1.5 to 2.0 nm in thickness and typically accounts for 10 to 20% of the cell envelope in terms of dry
mass. It is spatially distinct from the cytoplasmic membrane and forms the major resistance to cell
breakage. The cell envelope of Gram-positive bacteria is composed of 50-80% peptidoglycan,
associated with teichoic acids, presenting a greater structural resistance to breakage.

The outer membrane specific to Gram-negative bacteria is comprised of protein, lipopolysaccharide and
phospholipid. It separates the peptidoglycan layer from the bulk medium environment, thereby
preventing their interaction. It is readily distinguished from the cytoplasmic membrane by the
anisotropic nature of its bilayer configuration and its narrow spectrum of protein molecules. Divalent
cations play an essential role in its stabilisation (24, 35, 63).

In conclusion, three layers of the cell envelope require consideration in the rupture of Gram-negative
bacterial ceils. The outer membrane protects the inner layers from direct chemical attack. The
peptidoglycan layer provides the mechanical strength of the cell. The cytoplasmic membrane may be
considered the biochemical boundary of the cell and the major player in permeability. In Gram-positive
cells, the peptidoglycan layer is not protected from the external environment by an outer membrane, but
provides greater structural strength.

While this review is chiefly confined to the disruption of bacteria, it is useful to consider the gross
distinctions between their cell envelopes and those of other micro-organisms of importance in
biotechnology, in particular the yeasts. The basic structural components of the yeast cell wall are
glucans, mannans and proteins which form a crosslinked polysaccharide-protein structure, the
molecular details of which remain under debate (24). The overall structure is somewhat thicker than
that of bacteria, typically 70 nm for Saccharomyces cerevisiae. As with bacteria, the role of the
membrane structure is predominantly biological. It is thus seen that while similarities may be drawn
between the mechanical disruption of these crosslinked polysaccharide-protein macromolecules on the
disruption of bacteria and yeasts, the applicability of chemical and biological disruption studies are
confined to a particular wall structure.
220 S. T. L. HARRISON
C L A S S I F I C A T I O N OF B A C T E R I A L DISRUPTION P R O C E S S E S

Broadly speaking, processes that have been applied to bacterial cell disruption can be classified as
mechanical, physical, chemical or biological. The classification of techniques reported in the literature
is summarised in Figure 1.

Cell Disruption

1
Mechanical methods Non-mechanical methods

1
I I
as solid physical chemical
I
biological
in suspension
I
I
pressure
I
mechanical -heat -alkali
I
-lysozyme
agitation -freezing -acid -Cytophaga
HPH -osmotic -detergents lysing enzymes
colloid mill shock -solvents -mutanolysin
impingement -dessieation -EDTA -autolysis
-gas de- -antibiotics -phage

1
grind
I
pressure
compression -chaotropic
-ultrasonic agents
-wall irdaibitors

I 4 cavitation
-bead mill Hughes press -hydrodynamic
-mortar + X-press cavitation
pestle

Figure 1 . Charac/erisation of Cell Disruntion Technioues.

C E L L DISRUPTION BY M E C H A N I C A L MEANS

Processes involving either solid shear (eg. bead milling, extrusion of frozen cells) or liquid shear (eg.
high pressure homogenisation) have been most frequently used in cell disruption. Some have found
application on a large scale. In general, the equipment used for large scale cell disruption has been
modified from that used for particle size reduction or the formation of emulsions in other industries.
Common disadvantages of mechanical disruption include high capital investment and energy costs.
 BACTERIALCELL DISRUPTION 221
protein inactivation by shear-associated denaturation at air-liquid interfaces (8) and excessive heating
due to energy dissipation.

Dispersion and Colloid Mills

Dispersion and colloid mills are used for the disruption of lightly bonded clusters and agglomerates as
well as for the formation of emulsions. They operate on the principle of high speed fluid shear and
cause very little grinding of individual particles. In general, the resultant particle size is 1 ~tm (74). As
there is a high dissipation of energy, considerable heating occurs. Colloid mills may have cone or disc
rotors that are smooth or grooved and rotate at some 3600 rpm. The rotor is separated from the fixed
stator by a minimum of 25 ~tm. The disruption of microbial cells has been demonstrated. High
throughput rates (10m3/h) and continuous operation are possible, but complications of heating and
rotor-stator wear have been reported (77).

High Pressure Homoeenisation

High pressure homogenisation (HPH), employing a valve and impactor arrangement, is one of the
most widely known methods for large scale cell disruption. A positive displacement piston pump is
used to draw the cell suspension through a check valve into the pump cylinder. On return of the piston,
the suspension is forced through the adjustable annular gap of a discharge valve and impinges on an
impact ring. The discharge pressure, regulated by a spring-loaded valve rod, controls the position of
the valve in relation to the valve seat.

The use of the HPH for microbial cell disruption has been mainly reported for yeasts (23, 28, 43).
From the study of Saccharomyces cerevisiae using a pressure range of 10-54 MPa, a biomass
concentration of 84-210 kg dry mass/m3 and a temperature range of 5-40°C (43), the following were
shown:

i. Protein release (a measure of cell disruption) is temperature dependent.

ii. The process is independent of biomass concentration in the range 84 to 170 kg dry mass/m3.
A dependence may be found at higher concentrations.
iii. In general loss of enzyme activity is not found on homogenisation (28).
iv. The rate of release of specific enzymes with respect to the extent of total protein release is dependent
on the intraceLlular location of these enzymes with periplasmic enzymes most readily released (28).
v. Protein release, R, is fin'st order with respect to the number of passes, N, through the homogeniser:

dR
-d--~= k'R. (1)

vi. The dependence of protein release on operating pressure, P, can be expressed as a function of the
pressure raised to an exponent.
222 S.T.L. HARRISON
A combination of (v) and (vi) results in the correlation (43):

where R is the protein released (kg protein/kg biomass), Rm is the maximum protein available for
release, k is the rate constant and a is the pressure exponent. The applicability of these findings to the
disruption of Gram-negative bacteria have been conftrmed in a study employing Alcaligenes eutrophus
in which cell concentrations of 95-260 kg dry mass/m3 were used (38). The dependence on pressure is
reduced with increasing operating pressure in excess of 50 MPa (23, 48). As disruption at these high
pressures is largely independent of biomass concentration, the cell concentration should be kept as high
as possible to achieve a high efficiency.

Table 1. The Effect of Micro-organism on Pressure reouired for Disruntion bv High

P r e s s u r e Homo~enisation.
Operating pressures quoted are those required to achieve 50% cell disruption on a single
pass using the Stansted cell disrupter (47).

Organism Type of Organism Shape Size Pressure applied

(~tm) (MPa)

E.coli Gram -ve bacterium rod 2-4 x 0.5 15

B.subtilis Gram +ve bacterium rod 1.5-3 x 0. 8 24

L.casei Gram +ve bacterium rod 4 x 0.4-0.7 31

Streptococcus Gram +ve bacterium ovoid 1.02 diam. 150

faecalis cocci
S.cerevisiae yeast oval 7-12 x 5-8 150

Aspergillus fungi filament. 68

fumigatis
Fusarium sp. fungi fdament 68

Chorella algae 48

Values of the constants, pressure exponent a and rate constant k of Equation 2 reported in the literature
suggest a dependence of the level of cell disruption achieved on the nature of the organism and its
culture conditions. Keleman and Sharpe (47) have used a homogeniser fitted with a ball and seat valve
(Stansted cell disrupter) to determine the relative forces required to disrupt a range of microbial cells.
Their results are tabulated in Table 1. All micro-organisms investigated showed a sigmoidal profile
 BACTERIAL CELL DISRUPTION 223
with respect to operating pressure with little disruption being observed below a threshold pressure.
However, distinctly different pressures were required to disrupt different micro-organisms. Gram-
negative bacteria are easier to disrupt than Gram-positive bacteria and filamentous fungi, which in turn
are easier to disrupt than unicellular yeasts. Ease of disruption appeared to be related to the cell wali
composition, size and shape and growth phase. The increased ease of disruption of the Gram-negative
bacterium Alcaligenes eutrophus with increased growth rate has been clearly shown (38). In addition,
culture conditions such as media composition also influence the efficiency of breakage (33).

Ultramicroscopic studies of the disruption of Gram-negative bacteria by HPH (40) have indicated two
stages in the disruption process. In the initial stage, cell rupture is observed as discrete fractures in the
electron dense peptidoglycan structure. The second phase involves the disintegration of the cell
structure and the total liberation of its intmcellular contents.

Conditions of high velocity shear, sudden decompression with resultant cavitational stresses,
turbulence and impingement are expected to exist within the process. Rapid pressure release (11, 12,
22, 29), impingement on a stationary plate (23) and hydrodynamic cavitation (36) are apparent as the
significant causative mechanisms of cell rupture. By modification of the impact ring in the high
pressure homogeniser, Keshavarz Moore et al (49) have illustrated a decrease in ceil disruption with
increasing distance before impact. Indeed, only 20% of the disruption obtained after a single pass
under normal conditions is achieved in the absence of the impact ring. This suggests that in the
conventional valve and impactor design, cell rupture may be chiefly attributed to impingement on a
stationary plate.

Commercial homogenisers may be operated at a maximum pressure of 55 MPa with a capacity of up to

53 m3/h. For operation at higher pressures (100 MPa), the capacity is severely reduced (0.11 m3/h);
hence multiple passes are generally required on a large scale. Multiple passes are accompanied by the
micronisation of cell debris which may hinder further downstream processing.

I m o i n e e m e n t Jets

In fluid energy or jet mills, there is a high energy release and a high order of turbulence which causes
particles to grind upon themselves and hence rupture (74). These jets may either impinge on a solid
surface or, in a counter-current design, impinge on each other. Engler and Robinson (23) report
efficient disruption of Candida utilis by the impingement of a high velocity jet of suspended cells
against a stationary surface and show it to be a first order process with respect to number of passes.
Indeed, it is postulated to provide the primary mechanism of cell rupture in the high pressure
homogeniser (49). Recently the effective use of counter current impingement jets in the disruption of
both yeast and bacteria has been favourably compared to HPH (50). Factors influencing the process
include nozzle geometry, inter-nozzle distance, jet velocity, number of passes and morphology of the
micro-organism. A 60% disruption of wild type E.coli and a 95% disruption of a recombinant E.coli
are reported on a single pass.
224 S. T. L. HARRISON
Hieh Soeed Bead Mills

The bead mills, originally developed for the pigment and dye-stuff industry, provide grinding and
dispersion by inter-particle collision and solid sheoa'. The bead mill consists of either a vertical or a
horizontal grinding chamber containing rotating discs or impellers mounted on a motor driven shaft.
These accelerate the glass or plastic beads to supply a grinding action. The grinding chamber has a
sieve plate or similar device to separate and retain the beads. An efficient cooling system, usually in the
form of a cooling jacket (and possibly a cooled impeller shaft), is required to dissipate the heat
generated. Horizontal units are preferred for cell disruption as the grinding action in the vertical units
may be reduced by the fluidising effect of the upward fluid flow on the beads (15, 48).

Studies of cell rupture using high speed bead mills have been carried out on yeasts (17, 41, 56, 58,
69), and both Gram-positive and Gram-negative bacteria (41, 69). This complex process is influenced
by a wide range of parameters relating to the number and energy of impacts taking place, the energy
transfer to the grinding elements, liquid shear, hydrodynamics and mixing. These parameters include
bead diameter, density and loading, cell concentration in feed, flow rate of feed, agitator speed and
configuration, geometry of the grinding chamber and temperature. In addition, the residence time,
nature and state of the cell envelope, and size of different organisms will affect the process.

In general, smaller bead sizes are more effective. Typically for yeast cells, beads of 0.2-2.8 mm
diameter are used with the range 0.25-0.5 mm being preferred. By using larger beads, enzymes located
in the periplasmic space can be preferentially released whereas smaller beads are required for the release
of cytoplasmic enzymes (69). Disintegration of bacteria in a bead mill is hampered by their size. Gram-
negative bacteria such as E.coff and A.eutrophus occupy approximately 1% of the volume of a yeast
cell. Reduction of bead size is required for efficient disruption. This is limited by the tendency of small
beads to float. The outcome is the necessity for repeated passes or increased residence time (Table 2)
and a decrease in processing rate by as much as a factor of 10 (69).

Cell breakage shows first order kinetics in which the rate of protein release is directly proportional to
the amount of unreleased protein. In a batch reactor, this is represented by:

where R is the protein released, Rm is the maximum protein available for release, t is the time of
treatment and k is a first order rate constant (17, 56). For continuous operation, this has been
expressed by viewing a mill as a series of CSTRs as seen in Equation 4 (56):

- j (4)
 BACTERIALCELL DISRUPTION 225
where x is mean residence time and j is the number of CSTRs in series. If the mill operates as a plug
flow reactor as illustrated by Mao and Moo-Young (58), factor f is unity. Should back mixing occur, f
is fractional. The rate constant is dependent on agitator speed, flow rate of suspension, yeast
concentration, bead size, bead volume and temperature. This subject has been extensively reviewed
(15, 24, 51, 69).

The advantage of high speed bead mills in the recovery of a granular product is the thorough
disintegration of the cells ensuring that the granules are released from the peptidoglycan structure. The
drawbacks for bacterial rupture include reduced suitability for small microbial cells and the
micronisation of cell debris, thereby complicating further separations.

Table 2. Effect of Tvoe and Size of M i c r o - o r g a n i s m on Cell Disruotion in a Hieh

Soeed Bead Mill (65).

Micro-organism Residence Time for Complete Disruption

(seconds)

Aerobacter aerogenes 96
Bacterium cyclo-oxydans 35
Escherichia coli 85
P eniciUium chryogenum <20
Saccharorayces cerevisiae < 17
Streptornyces noosus 85

Solid Pressure Sheaf

A complete and very efficient cell disintegration and general preservation of product properties is
achieved by pressing a frozen cell paste through a small slit or orifice under pressure. The X-press,
Hughes press and Chaikoff press all operate on this principle. Using a starting temperature of -25 to
-27oC and pressure of up to 550 MPa, a change of the ice crystal state from ice I to ice III with a 20%
decrease in volume will occur. This change in state, the abrasive action of the ice crystals and plastic
flow through the orifice are believed to be responsible for the cell rupture obtained (70). On a single
pass 90% disruption of S.cerevisiae is achieved.

These processes are efficient in rupturing a wide variety of biological material, even that of tough cell
wall structure. Lower contamination of cell fragments is achieved than in other devices (24, 46, 75).
226 S.T.L. HARRISON
Semi-continuous operation of the X-press at 10 kg/h has been reported (15). These low mass flowrates
indicate that this equipment remains limited to pilot plant applications thus far.

CELL DISRUPTION BY PHYSICAL MEANS

Cavitation

Vapour cavities can be formed in a liquid due to a local reduction in pressure. This may be caused by a
local increase in velocity (Bernoulli), rapid vibration of a boundary, ultrasonic vibrations, the
separation of a liquid column or the overall reduction of the static pressure. Subsequent collapse and
rebound of the cavities will occur until an increase in pressure causes their destruction. This process is
termed cavitation. Cavitation is associated with local pressure fluctuations of the order of 1000 MPa.
On the collapse of cavitation bubbles, a large amount of energy is released as mechanical energy in the
form of elastic waves which disintegrate into eddies. Cavitation is well-known for achieving a fine
dispersion of fragmented solid particles or gas bubbles in a liquid and for the damage it causes to
pumps and pipework. Two methods of generating cavitation have been considered with respect to cell
breakage: ultrasonic cavitation and hydrodynamic cavitation.

Doulah (20) proposed a disintegration mechanism for yeasts subjected to cavitation based on a drop
breakup mechanism in hydrodynamic fields in which breakage occurs when the dynamic pressure
difference across the drop exceeds its surface energy. Eddies of scale larger than the micro-organism
will move it from place to place, whereas eddies of a smaller scale will impart motions of various
intensities to the cell. Hence a pressure difference will be created across the cell. When this exceeds the
cell wall strength, the cell will disintegrate. As cell wall strength is generally unknown, a surface-
tension type force, s, was assigned to represent it:

0s
Esur - d (5)

where Esur is cell surface energy, 0 is a shape factor and d is the cell dimension (equivalent spherical
diameter). A largest stable cell size (dimension din) can then be described as a function of the energy
dissipation rate. While the resultant relationships illustrate the dependence on cell size shown
experimentally, the analogy of cell wall strength and surface tension is fundamentally unsound. The
cell wall is a structural component gaining its strength from its chemically bonded nature. Surface
tension, however, is attributed to the lower potential energy of a solvent molecule when in the bulk
solvent than when in a gaseous state or immiscible solvent owing to attractive forces and hydrogen
bonding between the molecules. Additionally, on the division of a droplet maintained by surface
tension, a number of like entities are formed. It has been clearly shown by transmission electron
microscopy of disrupted bacterial cells that this does not occur on the rupture of the peptidoglycan cell
wall (36).
 BACTERIAL CELL DISRUPTION 227
Ultrasonic Cavitation. Ultrasonic vibrations cover a frequency range extending upward from
20 kHz. Owing to the pressure fluctuations generated as the power input increases, intermolecular
cohesive forces cannot be maintained and cavitation results. This cavitation is both frequency and
intensity dependent and requires a pressure fluctuation of greater magnitude than the hydrostatic
pressure (34). It is an established laboratory technique for microbial cell disruption. Protein release
from brewer's yeast on exposure to ultrasound at 20 kHz and a power of 200 acoustic watts is
independent of biomass concentration up to 600 kg/m 3 wet weight. It is almost proportional to the
input acoustic power in the range 60-195 acoustic watts (77). The studies of Wase and Patel (78) using
S.cerevisiae, Bacillus cereus and E.coli conclusively showed that, irrespective of culture conditions,
the principal determining factor of susceptibility to ultrasonic disintegration is the mean cell volume.
Disruption is also dependent on the growth phase (16).

In principle, ultrasonic devices can be scaled up and operated continuously. They are in use in the
chemical industry; however, they are not used in the industrial scale disruption of micro-organisms.
This can be attributed to the excessive heating found. Most of the acoustic energy is absorbed by the
suspension and converted to heat, hence the efficient dissipation of heat is essential. In addition,
adiabatic compression of the medium by acoustic radiation and high deceleration result in high localised
temperatures (eg 240°C for bulk ambient temperature; 34). The inactivation of enzymes reported by
Lilly and DunniU (55) may result from ionisation and subsequent free radical formation. Cavities
generated by ultrasound are lens-shaped and do not possess spherical symmetry. Their collapse causes
a discharge owing to the asymmetrical distribution of electrons and, subsequently, ionisation (34).
Finally, ultrasonic cavitation produces very fine cell debris which may lead to subsequent processing
problems (15).

H v d r o d v n a m i e Cavitation. By pumping a cell suspension through a downstream constriction,

thereby causing a local increase in velocity, hydrodynamic cavitation will result. The study of the
passage of Alcaligenes eutrophus through a partially closed valve under conditions at which cavitation
is shown to exist indicated substantial cell rupture (36). Cell breakage is largely achieved on a single
pass and is enhanced by the augmentation of cavitation at elevated temperature and increased pump
discharge pressure. In addition, hydrodynamic cavitation appears favourable over ultrasonic cavitation
owing to the reduced energy input required for equivalent breakage and a decrease in the energy
dissipated to cause temperature elevation (36).

Other Phvsical Methods for Cell Disruption

Dessieation. This is one of the oldest techniques of cell rupture used in extraction processes. Freeze-
drying causes the least damage to bacterial cells. Slow drying in air, drum drying, drying with a
dessicant or treatment with a dehydrating solvent are more effective. Subsequent extraction of the
microbial powder in a suitable buffer results in the recovery of the product at low yields (24, 46, 73).
Despite the permeabilisafion of the cells, they generally remain morphologically intact (25).
228 S.T.L. HARRISON
Osmotic shock. The rapid dilution of a solution of high osmotic pressure or the rapid resuspension
of cells in a solution of high salt content shows only a small effect on microbial cells, owing to their
cell wall structure. This effect is further reduced in cells in the stationary phase (25). In Gram-negative
cells, periplasmic proteins may be released. To achieve the release of cytoplasmic components, the cell
walls must f'trst be weakened. While the technique is applicable to the disruption of fragile ceils such as
mammalian cells, the contamination of the product by high concentrations of salt (or other agent
causing high osmotic pressure) is undesirable (24, 46, 77).

T e m o e r a t u r e Extremes. Freezing and thawing of a cell paste causes cell disruption through the
formation and subsequent melting of ice crystals. Gradual freezing, resulting in larger crystals, causes
more extensive damage to the cells. Cell breakage is further enhanced by simultaneous grinding. To
date, this costly technique has been restricted to small scale applications and is slow with low yields
(73, 75). In addition, reports of a loss of enzyme activity has restricted further exploitation (52).

Investigations of the thermolysis of E.coli K12 over the temperature range 30 to 90°C showed a
significant amount of protein to be released during a 20 minute treatment at 90°C (79). Apparent
improved protein release was obtained on short high temperature shock compared to longer treatments
(~lh) at lower temperatures (30 to 70oc). However, interpretation of these results is complicated by
changes in protein solubility observed at elevated temperature (36). While electron microscopy
showing large fragments of cell debris supported cell disruption at 90°C, no quantitative measure of the
degree of disruption was obtained by this technique.

The pilot plant process currently used for the production of the biodegradable thermoplastic, poly-~-
hydroxybutyrate (PHB) employs a combination of elevated temperature and pressure for the disruption
of A.eutrophus (45). A similar process is reported for the production of yeast protein extracts for use in
the food industry (7). Operation may be batch or continuous. The application of heat in cell disruption
is, however, very limited owing to the intolerance of most biological products to elevated temperature.

C E L L DISRUPTION BY C H E M I C A L M E A N S

The use of 6N HC1 has been reported to hydrolyse dried Candida lipolytica cells. The process is slow,
requiring 6 to 12 hours, and results in the concomitant hydrolysis of proteins to amino acids (24).
Where the separation of soluble and insoluble cell constituents is desired, the use of acidic pH is
undesirable owing to its precipitation of many macromolecules.

Alkaline treatment at a pH of 11.5-12.5 for 20-30 minutes causes cell lysis (75). Wade (76) reported a
process improvement in which mechanical cell disruption in the extraction of L-asparaginase from
Erwinia was replaced by alkaline treatment at a pH in excess of pH 9.0 and preferably between 11.0
and 12.5. L-asparaginase was released in a soluble form. On disruption ofAlcaligenes eutrophus for
the recovery of PHB, treatment at pI-/10 and 45°C resulted in a 50% release of the available soluble
 BACTERIAL CELL DISRUPTION 229
protein (36). Alkaline conditions are reported in the preparation of protein concentrates for feeds from
microalgae, yeasts and bacteria. The pH range 11.0-11.5 is preferable (41). In this process, active
protein molecules are not required and the process is evaluated in terms of extractable nitrogen. Indeed,
most proteins are unable to tolerate such conditions.

The Use of C h a o t r o o i c Aeents

The chaotropic agents urea and guanidine hydrochloride are mentioned in the literature as mediators of
cell lysis (8, 44). Guanidine-HC1 has been shown to solubilise protein from E.coli membrane
fragments by altering the hydrophobicity of the aqueous medium. Such chaotropic agents may further
assist the ensuing extraction by aiding the dissociation of nucleic acids and nucleoproteins from
microbial products (18).

The Role of Deter~ents in Cell Envelot~e Solubilisation

Treatment of bacterial cells with sodium dodecyl sulphate (SDS) is a recognised means for the release
of cellular constituents in molecular biology (31). Consequently, detergents may be useful in the scale-
up of cell disruption. The role of detergents in membrane solubilisation by the solubilisation of proteins
and the perturbation of protein-lipid interactions is reviewed by Helenius and Simons (42). A brief
description follows of the effect of both anionic and non-ionic detergents on cell envelope constituents
and of their ability to destroy bacterial cell integrity.

Studies of E.coli show varying susceptibility of the outer membrane, cell wall and cytoplasmic
membrane to solubilisation by the anionic detergents sodium dodecyl sulphate (SDS) and N-lauroyl
sarcosinate (Sarkosyl) and the non-ionic detergent Triton X-100. In the absence of Mg 2+ ions both the
outer membrane and the inner membrane are readily solubilised by SDS and Triton X-100. The action
of Sarkosyl is specific to the inner membrane (26). The presence of Mg 2+ ions restricts the action of
Triton X-100 to the solubilisation of the inner membrane only (26, 68) and prevents the removal of the
inner membrane by Sarkosyl (26). This provides further confirmation of the important stabilising effect
of divalent cations on biological membrane structures. Membrane removal is independent of detergent
concentration in the range 0.5-2.0%. Some enhancement of the solubilisation of both the inner and
outer membranes is found with increasing temperature in the range 4-37oc. Little further improvement
results at higher temperatures (67, 71). Temperature independence is reported for Sarkosyl (26).

The ability of SDS to destroy cell integrity by the solubilisatlon of both the inner and outer membranes
is altered by the viability of the cells (71, 81). Woldringh and van Iterson (81) studied the time course
of events in E.coli following SDS treatment by both transmission electron microscopy and biochemical
analysis. They reported that inactivation of exponentially growing cells with potassium cyanide is
essential for SDS sensitivity. A defined sequence of ulu'astructural changes followed the addition of
0.05% (m/v) SDS at a cell concentration of 108 cells/cm 3. Dissolution of the cytoplasmic membrane
was rapidly effected (~30 s). After migration to the polar region in contact with the cytoplasmic
membrane, the nucleoplasm receded to a central position in the form of an axial filament. The
230 S.T.L. HARRISON
cytoplasm separated into two components, an amorphous electron-transparent protein region and an
electron-dense region comprised of RNA. Finally the nucleoplasm migrated to the boundary of the cell
and leakage occurred. This sequence required some 20 minutes and resulted in a residual cell shell
surrounded by an intact peptidoglycan layer enclosing RNA and a portion of the DNA fraction. The
amorphous protein fraction had been liberated. Conf'n-mafion is thus obtained of the inability of SDS to
disrupt the peptidoglycan layer. Treatment by boiling in 4% SDS is indeed a frequent step in the
preparation of bacterial peptidoglycan (66).

Hettwer and Wang (44) studied the combined effect of non-ionic Triton X-100 and the chaotropic agent
guanidine-HC1 which solubilises proteins from membrane fragments. Triton X-100 has a high binding
affinity for hydrophobic species and is thus effective in binding to and solubilising phospholipids from
both inner and outer membrane fragments. At Triton X-100 concentrations in the range 0.5-2.0% and
.-.0.1M guanJdine-HC1, a pronounced synergistic effect is observed. Protein release of less than 10% is
observed on exposure to either compound at these concentrations in the absence of the other. This
release is enhanced to 50% on their synergistic action. The synergistic action is attributed to the
preferential action of guanidine hydrochloride and Triton X-100 on the outer membrane and the
cytoplasmic membrane respectively under the conditions used (62). It enables satisfactory protein
release to be obtained for the extraction of several soluble intracellular enzymes without significant loss
of enzyme activity.

EDTA

Divalent cations, especially Mg 2+ ions are necessary for the stabilisation of the outer membrane of the
Gram-negative cell envelope. Their chelation by EDTA (ethylenediaminetetraacetic acid) causes
destabilisation or removal of the outer membrane. Solubilisation by detergents and bacteriolytic enzyme
action is thus enhanced by EDTA pretreatment. Coliform bacteria can lose 33 to 50% of their
lipopolysaccharide content and small amounts of protein and phospholipid during EDTA treatment.
These changes in the outer membrane may also influence the cytoplasmic membrane (25). Treatment
with EDTA alone causes extensive lysis of Pseudomonas aeruginosa (13, 21). This feature is expected
to be peculiar to the Pseudomonads. In E.coli, EDTA has been shown to assist in the induction of
autolysis. In other Gram-negative bacteria, increased cell permeability is observed.

Solvents

The use of solvents such as butanol and hot toluene in the disintegration of microbial cells has long
been recognised (73, 77, 80). On the addition of toluene to 10% of the biomass concentration, it is
absorbed into the cell wall lipids. Swelling and rupture result (9). In his review, Felix (25) reports a
25% loss of protein including cytoplasmic enzymes from E.coli on treatment with toluene in the
absence of Mg 2+ ions. The effect is dependent on both temperature and solvent concentration. TEM
studies show damage to the cytoplasmic membrane. The outer membrane remains intact. Treatment
with ether shows a less marked effect on E.coli; however, complete disorganisation of the cytoplasm
and disruption of the cell membrane of fungi is observed.
 BACTERIAL CELL DISRUPTION z31
Lopatina (57) has reported the effect of several organic solvents on the Gram-negative bacterium
Alcaligenes eutrophus Z-1. Complete disintegration of the cells is achieved on treatment in 50% ethyl
acetate at room temperature. The solvents butanol, 3-methyl-1-butanol (isoamyl alcohol), toluene and
chloroform cause 50 to 80% disintegration of the cell, but ceil wall fragments remain. While treatment
with isopropanol at room temperature shows no effect, boiling in alkaline isopropanol provides the
clearest separation of cell wails from ceil membranes.

It has been suggested that pretreatment of cells with acetone enhances mechanical rupture (3). Bhaduri
and Damchick (10) proposed a simple method for the disruption of bacteria by acetone. Treatment of
two volumes of a Gram-positive bacterial suspension with one volume of acetone at 4oc for 5 minutes
followed by treatment with 0.I volumes of I% SDS was shown in the laboratory to yield a similar
release of protein to sonication or agitation with glass beads. Disruption of the Gram-negative bacterial
cell wall has also been illustrated (36). Advantages of this potential technique include the lack of
specialised equipment required, the use of inexpensive reagents, the introduction of no extraneous
proteins and its wide applicability. Disadvantages include the need to recover the acetone used.

Belter et al. (9) suggest that solvents with similar solubility parameters will attack cells in a similar
manner. It is postulated that solvents can be chosen to maximise solubility of the cell envelope and
minimise product solubility. Unfortunately, such practical data are seldom available.

C E L L DISRUPTION BY B I O L O G I C A L M E A N S

• °

The use of enzymic cell lysis is advantageous owing to its biological specificity, mild operating
conditions, low energy requirements, low capital investment and the avoidance of harsh physical
conditions such as high shear stresses. Minimum damage to the product is ensured. The applicability of
different enzymes will be determined by the distinctly different structure of bacterial and yeast cell
wails. This discussion is limited to the disruption of bacteria.

Three types of bacteriolytic enzymes have been identified: glycosidases which split the polysaccharide
chains of the peptidoglycan backbone, acetylmuramyl-L-alanine amidases which cleave the
polysaccharide-polypepride junction, and endopepridases which split the polypeptide chains within
peptidoglycan (1). Examples include the well-known glycosidase, lysozyme, isolated from hen egg
white, the lyric enzyme system of Cyrophaga sp, a lyric protease of Micromonospora, a lyric protease
from Bacillus subrilis and an N-acetylmuramidase from Streptomyces globisporius known as
mutanolysin. Most bacteriolytic enzymes are not active on viable ceils, hence the biomass requires prior
sensitization by heat-inactivation, chemical pretreatment, freezing or lyophilisation (1, 32).

Lysozyme, currently the only bacteriolytic enzyme available commercially for large scale application,
attacks the 13-1,4 linkages of the polysaccharide chains of peptidoglycan. While Gram-positive bacteria
are directly susceptible to its action, prior removal or destabilisation of the outer membrane of the
232 5. T. L. HARRISON
Gram-negative cell envelope is required to expose peptidoglycan to attack. This may be achieved by the
removal of divalent cations, known to stabilise the outer membrane, by a chelating agent such as EDTA
or by a non-ionic detergent such as Triton X-100. Lysozyme treatment is generally conducted at
pH 6-7 and 35oc for 20-60 minutes (1, 77). The action of lysozyme is confirmed by observation of
morphological changes to the cell wall by transmission electron microscopy (TEM) (13, 31, 59). In a
hypotonic environment, disintegration of the cell will result.

The lytic enzyme system of Cytophaga sp is known to lyse both Gram-positive and Gram-negative
bacteria, as well as yeasts. The lysis of E.coli requires the removal of the outer membrane which may
be achieved by detergent treatment (1). However, the efficient lysis of Gram-negative Alcaligenes
eutrophus has been found in the absence of such pretreatment (39). The lysis of the Gram-positive
bacteria Bacillus and Corynebacterium by the Cytophaga lyric system is reported by LeCorre et al.
(53). High activity and the absence of significant product inhibition was found. Optimum operating
conditions reported are pH 9.2 and 45-50oc.

The lysis of the Gram-positive bacteria Streptococcus mutans and Micrococcus lysodecticus can be
achieved by mutanolysin. Subsequent to EDTA pretreatment, incubation at pH 6.8 and 37°C for
30 minutes in the presence of mutanolysin at a concentration of lllg/mg cells resulted in the complete
lysis of Streptococcus mutans (72).

The lyric protease ofMicromonospora sp has been shown to lyse both viable and non-viable bacteria of
Gram-positive or Gram-negative classification as well as certain yeasts and fungi (1, 32). Gram-
positive bacteria are particularly effectively disrupted. Owing to a protease present in the enzyme
preparation, amino acid release was also found.

At present the cost of these lyric enzymes and their limited availability preclude their use on a large
scale. Enzymic lysis of Gram-negative bacteria is further hindered by the general requirement of both
cell inactivation and pretreatment to disrupt the outer membrane. However, the preservation of products
of a labile nature, the low capital investment, the initiation of the production of Cytophaga lytic
enzymes on a pilot plant scale (4) and the potential use of immobilised enzyme systems suggest this as
a promising technique of the future.

Actively-growing micro-organisms produce enzymes to hydrolyse the polymeric structures of their

own cell wall material to allow normal wall growth. This is supported by the appearance of
discontinuities observed in the cell wall of rapidly growing cells by TEM when subjected to osmotic
shock (6). By triggering the overproduction of these enzymes, autolysis may result. This has been
exhibited in both Gram-positive (Bacillus) and Gram-negative (E.coli) bacteria (16, 54). Typically a
50% loss of culture turbidity or protein release (compared to that obtained on mechanical rupture) is
achieved in 1-2 hours. For E.coli, solubilisation of some 90% of the peptidoglycan is reported.
Autolysis may be triggered by an osmotic imbalance (eg. salt induced) or nutrient limitation in some
micro-organisms (46). Longer incubation times may be required (2-20h) and extensive protein
denaturation has been reported (24).
 BACTERIAL CELL DISRUPTION 233
Genetic engineering of the micro-organism may allow lysis to be induced under specific conditions.
This may be achieved by accentuating naturally occurring autolytic properties or by inducing a
stimulus-controlled lytic gene. For example, inserted genes may be placed under the control of a
temperature-induced region and only expressed on elevation of the temperature by some 10°C. This is
illustrated by the cloning of PHB genes into E.coli cells in which such a mechanism is operable.
However, in this instance the engineered cells currently remain unable to exploit the ability to
accumulate PHB to the degree of naturally occurring A.eutrophus H16 (64).

{~gll Disruotion bv Other Biological Agents

Cell wall synthesis may be blocked by inhibition of required enzymes or by the addition of antibiotics
such as penicillin (30). Such agents may be added in late growth phase; however, continued
biosynthesis and reproduction are essential for lysis to occur. Inhibition of cell wall synthesis is thus
only applicable to the recovery of growth associated products.

Certain antibiotics permeabilise the bacterial cell but do not cause its lysis. Polyene antibiotics
(eg. nystatin) permeabilise fungal ceils by fortrfng complexes with membrane steroids which distort
the cytoplasmic membrane (25). Polypeptide antibiotics such as polymyxin, gramicidin and tyrocidine
cause the disorganisation of bacterial cell membranes by binding specifically to the negatively charged
lipid layers. Leakage of cell constituents results (25, 60, 73). Such cell permeabilisation is useful in the
recovery of soluble intracellular products.

The hydrolysis of the host cell wall by an infecting phage is a well known phenomenon. Under heavy
infection, lysis may occur before phage nucleic acid enters the cell (24). However, the introduction of
phage on a large scale is totally unacceptable owing to the accompanying risk of uncontrolled phage
infection in the production facility (73).

T H E C O M B I N A T I O N OF M E C H A N I C A L OR P H Y S I C A L AND C H E M I C A L
PROCESSES.

It is apparent in this review that chemical treatments such as extreme alkaline pH or detergent treatment
increase the permeability of the cell, causing partial protein release but not cell breakage. While this
may suffice in the recovery of certain soluble products, more complete rupture may be required for the
release of other products and is essential for the recovery of granular products such as bacterial
inclusion bodies, yeast virus-like particles and PHB. Mechanical disruption, the obvious alternative,
poses high energy requirements and frequently causes the micronisation of cell debris, thereby
hindering further separations. On the combination of a chemical pretreatment such as alkaline pH,
increased monovalent cation concentration or detergent treatment with subsequent high pressure
homogenisation, it has been shown that the disruption of stationary phase A.eutrophus is greatly
enhanced. Maximum protein release is approached on a single pass, possibly at reduced operating
pressure (37). In a similar study, pretreatment of the yeast S.cerevisiae by the lytic enzyme Zymolase
234 S.T.L. HARRISON
(isolated from Oerskovia xanthineolytica) resulted in only some 5% lysis. However, subsequent
homogenisation using the Microfluidiser (Microfluidics Corp., Newton, MA, USA) enhanced cell
disruption from less than 40% in the absence of enzymic pretreatment to 70% in its presence under
defined conditions (5).

I N T E G R A T I O N OF C E L L DISRUPTION AND U P S T R E A M PROCESSES

In the review of bacterial disruption, several common features can be extracted. These pertain to the
influence of the micro-organism type and the culture conditions on disruption. In summary, cell
disruption is more readily accomplished following growth on a defined medium or on media depletion
than on a complex medium (33). Cell wall type markedly affects the ease of rupture (47), Resistance to
cell disruption is dependent on the growth phase and decreases with increasing growth rate (38). The
increased sensitivity of exponentially growing cells over stationary phase bacteria has been
demonstrated on high pressure homogenisation (38), disruption by the bead mill, ultrasound or
autolysis (16), on enzymic lysis (27, 32) and on osmotic shock (25). It is anticipated to result from the
spontaneous cleavage of the peptidoglycan structure required to lay down additional cell wall material
during active growth (6). Finally, the influence of cell size on rupture by ultrasound is well
documented (27, 78).

I N T E G R A T I O N OF CELL DISRUPTION AND D O W N S T R E A M PROCESSES

Obvious criteria for the selection of a cell disruption process include the extent of disruption achieved,
process economics and the tolerance of the product to the conditions employed. In addition, the process
chosen will largely influence the ensuing downstream process. This will be mediated through the
addition of exogenous biochemicals and salts, the solubility of both the desired product and debris, the
extent to which these fractions are released from the cell and the degree of micronisation of insoluble
cell debris. Finally, the rupture process may alter binding between or entanglement of the desired
product and other components, and the suspension rheology.

Recently, emphasis in cell disruption studies is shifting to re-define the aim of the operation not as the
complete destruction of the cellular structure while maintaining the product properties, but as the
selective release of the desired product from the biological factory with the minimum release of other
contaminating compounds. This selective approach has been reported for yeast by enzymic lysis (2)
and for both yeast and bacteria by chemical perrneabilisation of the cell (44, 61, 62).

S E L E C T I V E PRODUCT RELEASE

The approach of chemical permeabilisation to selective product release is based on the desire to limit the
biochemical components of the solution to proteins liberated from the cell and to retain the nucleic acids
and other cell debris in the form of intact cells readily separated from the suspending solution. The
synergistic effect of the chaotropic agent guanidine hydrochloride (0.1-2.0 M) and the non-ionic
detergent Triton X-100 (0.1-2.0%) on the yeast Pichia pastoris and the bacterium E.coli has been
 BACTERIAL CELL DISRUPTION 235
reported to result in a protein release similar to that obtained by mechanical or enzymic treatment (44,
61). Transmission electron microscopy studies clearly indicate the leakage of cytoplasmic material
without extensive disruption of the outer membrane or the peptidoglycan layer of E.coli (44).
Similarly, with Pichia pastoris no significant decrease in intact cell number was observed subsequent to
protein release (61). Protein release is thus attained without the requirement of elaborate equipment or
the complication of viscous nucleic acid solutions or micronised cell debris. Disadvantages include the
longer time scale (-24 hours) and the possible protein denaturation at high reagent concentrations.

The selectivity of chemical permeabilisation can be further enhanced to restrict protein release from
E.coli to the periplasmic proteins by treatment with guanidine alone. A 40 fold purification of
recombinant [~-lactamase from E.coli has been demonstrated by this technique (62). Owing to the
inability to effect secretion of recombinant products from E.coli, but recent advances in achieving the
accumulation of these foreign proteins in the periplasm, this selective release is of great significance in
the purification of recombinant proteins.

Differential product release by enzymic treatment has thusfar been confined to the Cytophaga lysing
system and a yeast substrate (2). By the use of lyric enzymes and a combination of osmotic support and
membrane stabilisers such as Zn 2+ ions, wall-associated proteins only are released into the supematant.
Following their removal, spheroplast lysis by osmotic imbalance will release the cytoplasmic products.
Finally, proteins located within the organelles may be released by detergent treatment. By comparison
to mechanical disruption or enzymic lysis, it is seen that increased release of specific proteins can be
obtained by this differential process while the overall protein release is reduced.

CONCLUSIONS

In the review of cell disruption techniques, it is seen that the method of choice for a particular process
will depend on the nature of the micro-organism, the nature and location of the product as well as its
tolerance to process conditions and the scale and value of its production. The cell disruption operation
cannot be considered in isolation. The present challenge to researchers in the field is its integration into
the overall production process. This is essential for full advantage to be taken of the influence of the
upstream fermentation and to use the unit operation to augment the ensuing recovery process.

ACKNOWLEDGEMENTS.

The author is appreciative of useful discussion with Dr. H.A. Chase and Dr. J.S. Dennis. The financial
support of an ERS awarded by Trinity College, Cambridge is gratefully acknowledged.

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