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The high speed agitator bead mill was originally designed for the comminution of pigments in the paint and lacquer industry and is an established unit operation in the chemical engineering sector. The use of high speed bead mills for cell disruption was first reported by Zetalaki (1969). The design of the bead mill varies, though in general mills consist of either a vertical or a horizontal grinding chamber containing glass or steel beads, acting as the grinding element, and rotating disc impellers mounted, concentrically or off-centred, on a motor driven shaft. The units generate a significant amount of heat, and thus must be equipped with high capacity cooling systems for processing temperature sensitive materials. This requirement may restrict the size of the mill, but units from 0.1 to 20 litres are available.

In the bead mill suspensions of cells and glass beads are agitated by disc impellers rotating at high speeds. On agitation, the beads, which occupy 80-88% of the free working volume of the chamber cause disruption of the cells. It has been suggested by Rehacek and Schaefer (1977) that disruption may be caused by shear forces and also rolling of the grinding elements brought into motion by the agitator. Horizontal units are generally preferred for cell dismption as the grinding action in vertical mills is reduced due to fluidising effects of the upward fluid flow on the beads. Also, as vertical mills are fed from above there is a need for a

screen at the feed end to retain the beads inside the chamber, this is not the case in the horizontal mill. The bead mill can be operated in batch or continuous mode.

Kinetics of batch disruption

Kinetics of batch disruption of yeast cells have been reported to be first order with respect to the period of treatment, with the rate constant being a function of glass bead diameter, weight of beads used in the mill, agitator speed and design, cell concentration and temperature (Currie et al, 1972; Limon-Lason et al, 1979). The rate of disruption as measured by the rate of soluble protein release has been shown to be directly proportional to the amount of unreleased protein, see equation 1.1:

log. R r R m a x ” R

= k p . t (1.1)

where R = weight of protein released per unit weight of packed yeast; Rmax = maximum protein release; kp = first order rate constant; t = disruption time

Limon-Lason et al (1979) found that in larger mills and at higher impeller tip speeds (above 10 m/s) disruption did not follow first order kinetics as given by equation 1.1. However, diflBculties with temperature control in batch experiments led to rather inconclusive results and the limitations of the equation are not clear.

Limon-Lason et al (1979) also showed that for continuous disruption in a 5 L bead mill, the flow patterns were such that it could be considered as a series of continuous stirred tank reactors where each impeller was at the centre of a reactor. At a 0.6 L scale, however, there was considerable back-mixing between the reactors which had to be accounted for in a modified model.

Assuming a first order reaction and a CSTR in-series model, cell disruption was described by the following equation, for a 0.6 L bead mill (Limon-Lason et al, 1979):

R r

R r R

1 + ^ J

(1.2)

where x = mean residence time in the mill (=V/Q where V is the total volume of the mill and Q is the total throughput); j = the number of CSTRs in series (including fractions of CSTRs); kp, Rmax, and Rare as defined above in equation 1.1.

To incorporate back-flow between the CSTRs, x, mean residence time is written as: y

Q r =

1 + 2b

(1.3)

where, b/Q is the ratio of back-flow between the CSTRs to the overall flow rate (Q).

The effects of cell concentration reported in the literature are somewhat inconsistent though an optimal cell concentration for disruption of 40-50% cells (wet weight) has been reported (Kula, 1987). Further factors that affect milling are listed below:

Bead Size For the disruption of baker’s yeast small beads in the range of 0.5-0.28 mm have been reported to be most effective (Currie et al, 1972). Marfify and Kula (1974) also found that, for concentrations <60% w/v packed yeast, there was an increase in the disruption rate for decreasing bead diameter between the range 0.6-0.35 mm, but that the disruption rate fell as the bead diameter decreased to 0.2 mm. The optimum bead diameter for baker’s yeast disruption in a 20 L horizontal mill has been found to be 0.7 mm (Schutte et al, 1988). In practice the necessity for the continuous separation of the cell suspension from the grinding elements limits the size of the beads to 0.4 mm. Also, if the beads are too small they have a tendency to float. The location of the enzyme in the cell also influences the bead diameter used. Higher release of periplasmic enzymes has been obtained by using larger beads of 1.0 mm, whilst for cytoplasmic enzymes, smaller beads are more suitable (Schutte et al 1988).

Bead loading The volume fraction of the grinding chamber filled with glass beads afreets both the disruption achieved and the power requirements of the milling process. Increased bead loading increases cell disruption significantly, but loading of more than 88% is not suitable (Currie et al, 1972; Rehacek and Schaefer, 1977). Increased bead loading also necessitates more cooling.

Temperature The operating temperature should be maintained at 5 °C, to allow for minimum inactivation of the product. It has been reported that the rate constant is insensitive to the temperature of operation between the range 5 to 40 °C.

Agitator Speed Increasing the agitator speed generally increases the disruption rate constant (Currie et al, 1972, Marflfy and Kula, 1974). However, at higher tip speeds the rate of increase in disruption levels ofif. The main disadvantage of using very high tip speeds is the increased temperature rise and power consumption that accompanies such a change.

Agitator Design Limon-Lason et al (1979) found that in a 5L mill the dismption rate constant was always higher for polyurethane impellers, than for the stainless steel type, see figure 1.2. However, maximum protein release was always higher for the stainless steel impellers. This observation was explained by studying the mixing patterns caused by the impellers.

Figure 1.2; Details of (a) Polyurethane "open" i>-pe impeller (b) stainless steel "closed" type impeller (Limon Lason et a l 1979)

The polyurethane impeller because of its more "open" design creates a greater degree of mixing at any given distance from the impeller. The more "closed" stainless steel impeller design allows very high shear rates near the impeller, though its mixing properties are not as efficient.