Downstream processing - Pharmaceutical Biotechnology: Concepts and Aapplications - page 137

Table 6.1 Some chemical, physical and enzyme-based techniques that may
be employed to achieve microbial cell disruption
Treatment with chemicals:
detergents
antibiotics
solvents (e.g. toluene, acetone)
chaotropic agents (e.g. urea, guanidine)
Exposure to alkaline conditions
Sonication
Homogenization
Agitation in the presence of abrasives (usually glass beads)
Treatment with lysozyme
During the homogenization process a cell suspension is forced through an orifi ce of very narrow
internal diameter at extremely high pressures. This generates extremely high shear forces. As the
microbial suspension passes through the outlet point, it experiences an almost instantaneous drop
in pressure to normal atmospheric pressure. The high shear forces and subsequent rapid pressure
drop act as very effective cellular disruption forces, and result in the rupture of most microbial
cell types ( Figure 6.5). In most cases a single pass through the homogenizer results in adequate
cell breakage, but it is also possible to recirculate the material through the system for a second or
third pass.
An effi cient cooling system minimizes protein denaturation (denaturation would otherwise oc-
cur due to the considerable amount of heat generated during the homogenization process). Ho-
mogenizers capable of handling large quantities of cellular suspensions are now available, many
of which can effi ciently process several thousand litres per hour.
An additional method often employed to achieve microbial cell disruption, both at the labora-
tory level and on an industrial scale, involves cellular agitation in the presence of glass beads. In
such bead mills, the microorganisms are placed in a chamber together with a quantity of glass
beads of 0.2-0.3 mm in diameter. This mixture is then shaken/agitated vigorously, resulting
Figure 6.5 Diagrammatic representation of a cell homogenizer. This represents one of a number of
instruments routinely used to rupture microbial cells, and in some cases animal/plant tissue