Page 310 - Biosystems Engineering

Bioseparation Pr ocesses 287

the ice. The ice undergoes transformations in its crystal structure when
it passes through the hole because of the temperature and pressure
change. Heavy pressures (spring-loaded piston), which can apply pres-
2
sure above 2100 kg/cm , are needed to disrupt heavy cell pastes.
Liquid Homogenization
Liquid-based homogenization is the most widely used cell-disruption
technique for small volumes and cultured cells. Cells are lysed by
forcing the cell or tissue suspension through a narrow space, thereby
shearing the cell membranes. Three different types of homogenizers
are in common use:

1. The Dounce homogenizer consists of a round glass pestle that
is manually driven into a glass tube.
2. The Potter–Elvehjem homogenizer consists of a manually or
mechanically driven Teflon pestle shaped to fit a rounded or
conical vessel. The number of strokes and the speed at which
the strokes are administered influences the effectiveness of
Dounce and Potter–Elvehjem homogenization methods. Both
homogenizers can be obtained in a variety of sizes to accom-
modate a range of volumes.
3. A French press consists of a piston that applies high pressure
to a sample volume of 40 to 250 mL, forcing it through a tiny
hole in the press. Only two passes are required for efficient
lysis due to the high pressures used with this process. The
equipment is expensive, but the French press is often the
method of choice for breaking bacterial cells mechanically.

Ultrasonic Vibration
Ultrasonic waves of about 20 kg/s that are applied to bacterial sus-
pensions disrupt both the cell wall and the cell membrane; rod-shaped
are more readily broken than cocci, and gram-negative cells disinte-
grate more readily than gram-positive ones. This method is not as
successful for breaking fungal cells.
Ultrasonic waves in a liquid cause fluctuation in pressure, forming
bubbles, which grow to about 10 μm, begin to oscillate, and then
implode violently generating shock waves of several thousand atmo-
spheres and localized high temperatures. Free radicals are also gener-
ated in the solution. It is believed that rapid oscillations of the bubbles
are responsible for cell rupture, rather than damage from shock waves
or free radicals. Nonetheless, free radicals are important because they
damage enzymes released into solution. An electronic generator is
used to produce ultrasonic waves of 20 kc/s. A transducer converts
the waves to a titanium probe immersed in the cell suspension, cooled
in ice. This method seems to have possible industrial applications,
but, in practice, there are difficulties in dissipating the heat generated;