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New and revised methods of cell disruption that have been of recent interest include;

• The Microfluidiser • Critical Fluid Disruption • Rotor driven bead mills

(N.B. The first two methods stated above have also been studied using enzymic pre-treatment of the cell suspension)

The Microfluidiser

Small scale (lOml-6-12 1/hr) and production scale (20 - 11,000 L/h) models have been designed. The scale-up fi*om laboratory to pilot and production scale is linear. Advantages of using this new type of high pressure homogeniser are that it claims:

• no aerosol generation • aseptic operation facility • is steam sterilisable

• blocked disruption chamber can be cleaned by reverse flow

• small scale Microfluidisers can be immersed into an ice-bath to facilitate immediate cooling; a cooling coil can also be jacketed around the Microfluidiser.

As with the APV Manton-Gaulin type homogeniser the extent of disruption depends upon the operating pressure and number of passes used. Operating pressures can be up to 158 MPa.

In the Microfluidiser, two streams interact at high velocities and pressures in precisely defined microchannels. The mechanisms of cell disruption include shear, turbulence, and cavitation. 100% disruption ofE. coli has been claimed after one pass (White and Marcus, 1988). Sauer et al. (1989) reported that for the disruption of E. Coli strains using a Microfluidiser model Ml lOT, the disruption kinetics followed a first order rate equation based on that obtained by Hetherington et ai (1971) for the APV Manton-Gaulin type homogeniser. The exponent "b"

was found to vary between 0.28-0.94, depending upon the specific growth rate of the cells in continuous culture, type and concentration of cells.

log, R r R m a x " ^

(1.5)

For cells grown in continuous culture, recombinant strains were disrupted more readily than their native one. Cells grown in batch culture showed no significant difference between the disruption kinetics of the recombinant and native strains.

Baldwin and Robinson (1990) found that for baker’s yeast the Microfluidiser disrupted less than 40% of the cells after 5 passes. Their studies reported that enzymatic lysis combined with the Microfluidiser significantly enhanced the ability of the Microfluidiser model MllOT with approximately 100% disruption being obtained after four passes at a pressure of 95 MPa. The experimental results obtained by Baldwin and Robinson (1990) were described by modification of the Hetherington’s model to the form:

Ogc R m a x " R x R m a x - R

= k p . N \ P* (1.6)

Where R% = cells disrupted by enzymatic treatment alone; R = total cells disrupted after both enzymatic and homogenisation; kp is the rate constant; a and b are the exponents of pressure and number of passes, respectively, "a" was found to be 3.03, and "b" 1.3 for baker’s yeast.

This two stage disruption technique may be too costly on a large scale, unless the enzymic preparation can be recovered eflSciently.

Critical Fluid Disruption (CFD)

The method of critical fluid disruption (CFD) uses super critical fluids (SCF). Such fluids possess a combination of "gas-like" and "liquid-like" properties. The SCF penetrates like a gas and functions like a liquid. SCF have been used in the bioindustry particularly in the area of extraction and separation. The critical fluid disruption equipment consists of two major components; a piston injector and a pressure vessel. The process involves a sudden release of applied fluid pressure that allows penetration of the critical fluid into the cell body. The inflated microbial cells are than rapidly decompressed, where upon they rupture at their weakest points. Carbon dioxide and nitrogen are the typical super critical fluids used.

Lin et al (1991) extended the use of SCF, combined with pre-treatment of the suspension with a lytic enzyme, to the disruption of yeast. They used carbon dioxide as a primary SCF because it is nontoxic, nonflammable, inexpensive and physiologically safe. The critical temperature for carbon dioxide is just above ambient (i.e. critical temperature is 31.1 °C), which minimises the problems of thermal degradation (or dénaturation) of delicate biological materials. In addition its phase behaviour and other thermo-physical properties that are needed for process analysis are well studied. Volumes handled by these workers were approximately 50mL. Without enzyme pre-treatment, only 30% of the available protein was released. Pre-treatment with lytic enzymes enhanced significantly the disruption process.

The main advantages of this method are that there is insignificant cooling requirement, it is a gentle technique and it is scalable. However, there is little known in engineering terms about the use of this method in bioprocessing, and more research is required.

Rotor Driven Bead Mills

Mao and Moo-Young (1990) reported the use of a vertical cylindrical rotor instead of impellers to agitate beads in a bead mill. They found that flow patterns were found to be approximately those of plug-flow. Operation of these mills can either be continuous or batch mode. Ninety percent disruption has been observed for a rotational speed of 4000 rpm. At very high and low flow rates the cells and bead tended to separate.

Gaver and Huyghebaert (1990) used the CoBall-Mill, another new type of rotational bead mill. The disruption chamber of this mill is composed of a conical rotor fitting into a conical stator. The cell suspension is pumped continuously through the narrow gap, between the rotor and the stator, that is partially filled with disruption beads. The volume of the disruption chamber is only 25% of the disruption volume requirement of the conventional bead mill with the same throughput. The CoBall Mill also has a very eflScient cooling system.