# Centrifugation

Micro-organisms and other similar sized particles can be removed from a broth by using a centrifuge when filtration is not a satisfactory separation method. Although a centrifuge may be expensive when compared with a filter it may be essential when:

1. Filtration is slow and difficult.

2. The cells or other suspended matter must be obtained free of filter aids.

3. Continuous separation to a high standard of hygiene is required.

Non-continuous centrifuges are of extremely limited capacity and therefore not suitable for large-scale separation. The centrifuges used in harvesting fermentation broths are all operated on a continuous or semi-continuous basis. Some centrifuges can be used for separating two immiscible liquids yielding a heavy phase and light phase liquid, as well as a solids fraction. They may also be used for the breaking of emulsions.

According to Stoke's law, the rate of sedimentation of spherical particles suspended in a fluid of Newtonian viscosity characteristics is proportional to the square of the diameter of the particles, thus the rate of sedimentation of a particle under gravitational force is:

where Vg d g

= rate of sedimentation (m s"1) = particle diameter (m) = gravitational constant (m s~2) = particle density (kg m~3) = liquid density (kg m~3) = viscosity (kg m 1 s~J)

This equation can then be modified for sedimentation in a centrifuge:

where Vc = rate of sedimentation in the centrifuge (m s-1),

(o = angular velocity of the rotor (s -1), r = radial position of the particle (m). Dividing equation (10.6) by equation (10.5) yields

This is a measure of the separating power of a centrifuge compared with gravity settling. It is often referred to as the relative centrifugal force and given the symbol 'Z'.

It is evident from this formula that factors influencing the rate of sedimentation over which one has little or no control are the difference in density between the cells and the liquid (increased temperature would lower media density but is of little practical use with fermentation broths), the diameter of the cells (could be increased by coagulation/flocculation) and the viscosity of the liquid. Ideally, the cells should have a large diameter, there should be a large density difference between cell and liquid and the liquid should have a low viscosity. In practice, the cells are usually very small, of low density and are often suspended in viscous media. Thus it can be seen that the angular velocity and diameter of the centrifuge are the major factors to be considered when attempting to maximize the rate of sedimentation (and therefore throughput) of fermentation broths.

### Cell aggregation and flocculation

Following an industrial fermentation it is quite common to add flocculating agents to the broth to aid de-watering (Wang, 1987). The use of flocculating agents is widely practised in the effluent-treatment industries for the removal of microbial cells and suspended colloidal matter (Delaine, 1983).

It is well known that aggregates of microbial cells, although they have the same density as the individual cells, will sediment faster because of the increased diameter of the particles (Stokes law). This sedimentation process may be achieved naturally with selected strains of brewing yeasts, particularly if the wort is chilled at the end of fermentation, and leads to a natural clearing of the beer.

Micro-organisms in solution are usually held as discrete units in three ways. Firstly, their surfaces are negatively charged and therefore repulse each other. Secondly, because of their generally hydrophilic cell walls a shell of bound water is associated with the cell which acts as a thermodynamic barrier to aggregation. Finally, due to the irregular shapes of cell walls (at the macromolecular level) steric hindrance will also play a part.

During flocculation one or more mechanisms besides temperature can induce cell flocculation:

(a) Neutralization of anionic charges, primarily car-boxyl and phosphate groups, on the surfaces of the microbial cells, thus allowing the cells to aggregate. These include changes in the pH and the presence of a range of compounds which alter the ionic environment.

(b) Reduction in surface hydrophilicity.

(c) The use of high molecular weight polymer bridges. Anionic, non-ionic and cationic polymers can be used, though the former two also require the addition of a multivalent cation.

Flocculation usually involves the mixing of a process fluid with the flocculating agent under conditions of high shear in a stirred tank, although more compact and efficient devices have been proposed (Ashley, 1990). This stage is known as coagulation, and is usually followed by a period of gentle agitation when floes developed initially are allowed to grow in size. The underlying theoretical principles of cell flocculation have been discussed by Atkinson and Daoud (1976).

Nakamura (1961) described the use of various compounds for flocculating bacteria, yeasts and algae, including alum, calcium salts and ferric salts. Other agents which are now used include tannic acid, titanium tetrachloride and cationic agents such as quaternary ammonium compounds, alkyl amines and alkyl pyridinium salts. Gasner and Wang (1970) reported a many hundred-fold increase in the sedimentation rate of Candida intermedia when recoveries of over 99% were readily obtained. They found that flocculation was very dependent on the choice of additive, dosage and conditions of floe formation, with the most effective agents being mineral colloids and polyelectrolytes. Nucleic acids, polysaccharides and proteins released from partly lysed cells may also bring about agglomeration. In SCP processes, phosphoric acid has been used as a flocculating agent since it can be used as a nutrient in medium recycle with considerable savings in water usage (Hamer, 1979).

The majority of flocculating agents currently in use are polyelectrolytes, which act by charge neutralization and hydrophobic interactions to link cells to each other. In processes where the addition of some toxic chemicals is to be avoided, alternative techniques have been adopted. One method is to coagulate microbial protein which has been released from the cells by heating f0]-short periods. Kurane (1990) reports the use of bioflocculants obtained from Rhodococcus erythropolis. They are suggested as being safer alternatives to conventional flocculants. Warne and Bowden (1987) suggest the use of genetic manipulation to alter cell surface properties to aid aggregation. Flocculating agents such as cross-linked cationic polymers may also be used in the processing of cell lysates and extracts prior to further downstream processing (Fletcher et al., 1990) Bentham et al. (1990) utilized borax as a flocculating agent for yeast cell debris prior to decanter centrifuga-tion.

### The range of centrif uges

A number of centrifuges will be described which vary in their manner of liquid and solid discharge, their unloading speed and their relative maximum capacities. When choosing a centrifuge for a specific process it is important to ensure that the centrifuge will be able to perform the separation at the planned production rate, and operate reliably with minimum manpower. Large-scale tests may therefore be necessary with fermentation broths or other materials to check that the correct centrifuge is chosen.

Basket centrifuges are useful for separating mould mycelia or crystalline compounds. The centrifuge is most commonly used with a perforated bowl lined with a filter bag of nylon, cotton, etc. (Fig. 10.15). A continuous feed is used, and when the basket is filled with the filter cake it is possible to wash the cake before removing it. The bowl may suffer from blinding with soft biological materials so that high centrifugal forces cannot be used. These centrifuges are normally operated at speeds of up to 4000 rpm for feed rates of 50 to 300 dm3 m in " 1 and have a solids holding capacity of 30 to 500 dm3. The basket centrifuge may be considered to be a centrifugal filter.