Page 311 - Biosystems Engineering

288 Cha pte r Ni ne

the free radicals tend to denature sensitive enzymes, and the cell
debris is extensively fragmented.

Other Methods
The bacterial cell membrane can be broken by physical stress such as
violent depression, osmotic shock, or rupture by freezing. Lytic agents
(cationic and anionic detergents, alkalis, bile salts, and solvents) also
have restricted use.
The freeze/thaw method is commonly used to lyse bacterial and
mammalian cells. This technique involves freezing a cell suspension
in a dry ice/ethanol bath or freezer and then thawing the material at
room temperature, or 37°C. This method of lysis causes cells to swell
and ultimately break because ice crystals form during the freezing
process and contract during thawing. Multiple cycles are necessary
for efficient lysis, and the process can be quite lengthy. However,
freeze/thaw has been shown to effectively release recombinant pro-
teins located in the cytoplasm of bacteria and is recommended for the
lysis of mammalian cells in some protocols. Bacterial cells can also be
destroyed by enzymes.

9.3.2 Centrifugal Separation
Solids that form a porous cake can be separated from liquids in a cen-
trifuge. A centrifuge uses centrifugal force to separate liquids from
solids. It is, essentially, a development of gravity filtering wherein the
force acting on the liquid is enormously increased by the use of cen-
trifugal force instead of being restricted to gravity. The slurry is fed to
a rotating basket having a slotted or perforated wall covered with a
filter medium such as canvas or metal cloth. Pressure, resulting from
the centrifugal action, forces the liquid through the filter medium
leaving the solids behind. If the feed to the basket is then shut off and
the cake of the solids are spun for a short time, much of the residual
liquid in the cake drains off the particles, leaving the solids much drier
then those from a filter press or vacuum filter.
The major types of filtering centrifugals are suspended batch
machines, which are discontinuous in their operation, automatic short-
cycle batch machines, and continuous conveyor centrifuges. In sus-
pended centrifugals, the filter media are canvas or other fabric or woven
metal cloth. The baskets range (see Fig. 9.3) from 30 to 48 in in diam-
eter and are 18 to 30 in deep, operating at speeds between 600 and
1800 rpm. It may be underdriven or top driven. The solids form a
cake 2 to 6 in thick inside the basket.
Immiscible liquids, like oil–water emulsions, can also be separated
by special types of centrifuge such as the Sharples Super Centrifuge
(Separation Equipment Sales, Inc., Albertson, New York), which oper-
ates at about 30,000 rpm.