POLYACRYLAMIDES.

Polyacrylamides and their derivatives are the most commonly used flocculants (Gregory, 1976). They are conveniently synthesised from the acrylamide monomer to produce a wide range of molecular weights and can be modified to be anionic, cationic or non-ionic in character. Charge density variations can be brought about by the number of functional groups per molecule. The main disadvantage of polyacrylamides is the toxicity of the monomer. Jackson eta l, (1975) details methods for the characterisation of polyacrylamides used for waste water treatment.

1.7.4 CHITOSAN.

Chitosan is a polyglucosamine usually of high molecular weight (10^) and a variable charge density, which is controlled by the number of amines per glucose unit. It is derived from the biopolymer chitin which is a poly-j8-(l-4)-N-acetyl-D-glucosamine; it can be partly or completely deacetylated. Like PEI, chitosan is a cationic polyelectrolyte; its chemical structure causes it to adopt a linear rigid structure in solution. Agerkvist et a l, (1988, 1990) showed that E.coli cell debris could be flocculated with chitosan, the process being sensitive to pH and ionic strength. The optimum flocculation doses were between 12 and 43 mg chitosan per gram dry cell weight in the pH range 4 to 6.9. The lower doses at lower pHs were attributed to the increase in cationic charge density (with lower pH), this also caused an increase in the hydrodynamic radius of the molecule. Increasing the ionic strength required greater quantities of polymer. These observations were in accordance with the charge-patch mechanism of flocculation. Degradation of the nucleic acids with DNAase and RNAase reduced the amount of polymer required to reach the optimum flocculation concentration, since the oligonucleotides did not precipitate with chitosan. Soluble protein loss from the supernatant was considerable (50 to 80%) of the enzyme of interest )3-galactosidase showing a 36 to 59% loss at the optimum dose. SDS-PAGE showed that high molecular weight protein was more likely to be removed. Possible mechanisms for protein removal include charge neutralisation and sweep flocculation, especially of the larger proteins. For smaller proteins the charge neutralisation is not complete, hence the protein remains soluble.

The addition of or caused a reduction in the optimum flocculation concentration, with the Al^^ having the greater affect (Agerkvist et al., 1990). The ions were shown to bind to the cell debris and hence reduce its surface charge, thus it required less electrostatic destabilisation; the degree of selective removal remained the same. Agerkvist et al, (in press) showed that the ionic strength could be used to modulate the selectivity of the flocculation process. At 0.5 M NaCl, cell debris and nucleic acids were extensively removed (95 and 88%), whilst most of the protein and 86% of the )3-galactosidase activity remained. Measurements of the sodium ion concentration showed that it did not change over the whole range, hence it did not interact with different species in the cell homogenate.

Agerkvist et al, (in press) reported that chitosans of different molecular weights produce little change in the optimum flocculation concentration, however lowering the charge density required considerably more flocculant. The lower molecular weight fraction gave broader range of good flocculation. The lowest charge density chitosan exhibited the weakest interaction with proteins however the removal of nucleic acids was similar to the high molecular weight samples, but increased amounts were required.

U rea was added in order to test for the hydrogen bonding capacity of chitosan in

E.coli homogenates. It reduced the effectiveness of chitosan as a flocculant with doses increased by 50% in order to achieve the same degree of flocculation. The results indicated that H-bonding was common in the flocculation process. The mechanism of cell debris flocculation was reported to be non-equilibrium bridging due to the relative insensitivity to molecular weight (on the cell debris flocculation), and the insensitivity of floes to shear. Protein unfolding reduced the effective charge density, improving its recovery, hence reducing the electrostatic interaction of protein and cationic polyelectrolyte. However unfolded protein would also uncover more hydrophobic groups, hence reducing the likelihood of charge neutralisation.

Low molecular weight chitosans are commercially available as nucleic acid and endotoxin removers (Karita Water Industries Ltd., Japan). For E.coli homogenates the use of chitosan reduced the D N A RNA and endotoxin levels to 0.9, 4 and 0.2% of the original. Removal of DNA from B.subtilis, S.cerevisiae and HeLa cell

homogenates gave similar reductions. A range of proteins tested (at pH 7.5) showed reductions of 10 to 30% in the presence of DNA (which reduced chitosan/protein interactions); the higher molecular weight and low isoelectric point proteins were more likely to be removed. Over 90% of the DNA was removed in all cases.

The advantage of chitosan as a flocculant is that its cationic character is strongly pH dependent, this allows a greater degree of modulation of the flocculation process compared to PEI. The major disadvantage of chitosan is its poor solubility; it requires acetic acid and salt solution in order to dissolve it. Also chitosan is insoluble above pH 8. Its poor solubility severely limits the biomass that can be flocculated and usually dilution is required. For example, Agerkvist et a l, (1990) grew E.co/f to 5 g DCW, this was then diluted 1:10 before flocculation was carried out. Normally

E xoli fermentations produce dry cell weights in the range 30 to 40 g L \