Colloids and Surfaces, 31 (1988) 259-264 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

259

Flocculation of Cellular Suspensions by Polyelectrolytes

A.A. BARAN

Institute of Colloid and Water Chemistry, Ukrainian Academy of Sciences, Vernadsky prospect 42, Kiev (U.S.S.R.)

(Received 7 April 1987; accepted in final form 14 July 1987)

In modern biotechnology many microorganisms are produced which are the raw material for ferments, antibiotics, proteins, etc. As a rule, stable disper- sions of microorganisms are obtained during cultivation; the aggregative and sedimentation stability of these systems is determined by different factors. There are the electric repulsion forces due to the ionic groups on the cell sur- face, the structural forces due to the polymolecular hydrate surface layers and the structural-mechanical forces arising in the protein-lipid layers on the cell-intermicellar liquid interface.

To obtain the end product of microbiological synthesis it is necessary to concentrate the cellular suspensions and to separate the biomasses from the solution. Due to the complex mechanism of stabilization of biological suspen- sions, simple electrolyte coagulation is inadequate in this case. New possibili- ties for concentrating cellular suspensions are offered by the use of high- molecular flocculants widely applied for water purification, thickening of tech- nological suspensions, dewatering of precipitates, etc.

We have tested a large number of different flocculants to concentrate the model bacterial suspensions Escherichia coli. Their characteristics are given in Table 1.

Purified industrial samples of polyethylene oxide (PEO ) , polyacrylamide (PAA), polyacrylic acid (PAC) , polyethyleneimine (PEI) and polyvinylben- zyltrimethylammonium chloride (PVBTAC ) were used. Polydiethylamino- ethylmetacrylate (polyDEAEMA) and its copolymers with a&amide (AA), acrylic acid (AC) and vinylpyrrolidone (VP ) were obtained by radical poly- merization as described in Refs [ 1,2]. The composition of the copolymers was determined by IR and NMR spectroscopy. Carboxymethylchitin and chitosan were obtained from chitin [ 31; alkylchitosans were prepared from chitosan according to the Eishweler-Clark reaction.

The molecular masses of the polymers were found from intrinsic viscosity, diffusion and light-scattering data.

As the measure of flocculation, the ratio of the cell numbers in the super-

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TABLE 1

Characteristics of polymers used

Polymer Monomeric unit Mol. mass

Polyethylene oxide (PEO) Polyacrylamide (PAA)

Polyacrylic acid (PAC)

Polyethyleneimine (PEI) Polyvinylbenzyltrimethylammonium

chloride (PVBTAC) Polydiethylaminoethylmetacrylate

(polyDEAEMA)

Copolymer DEAEMA with acrylic acid ( polyDEAEMA/AC )

Copolymer DEAEMA with acrylam- ide (polyDEAEMA/AA)

Copolymer DEAEMA with vinylpyr- rolidone (polyDEAEMA/VP )

Chitosan Alkylchitosans

-CH2-CH2-O- -CH, -CH-CONH2

I -CH2 -CH-COOH

I -CH,-CH,-NH- -CH2-CH-C6H,-CH2-N(CH3)3+C1-

-CH,- C -C-O(CH,),-N(C2H5),

&a d:

25,42,70 and 90 mol.% of DEAEMA in copolymer 4,11,39 and 77 mol.% DEAEMA in copolymer 9,16,34 and 65 mol.% DEAEMA in copolymer CzHs-02 (OH) ( NH2) CHzOH CzHr,-O2 (OH) (N) CH,OH

/\

Ri R2

l-3.106 l-lo6

0.5-l-lo6

0.5-1.105 5.105

4.105

up to 3.3.105

up to 8.7~10~

up to 6.5. lo5

0.5-1.5.105 up to 1.5.105

natant to those in the whole dispersion (determined 20 h after adding the flocculant ) was taken, as described in detail in Refs [ 431. As usual [ 4,5] the flocculant concentration in the system is expressed in micrograms of added polymer per billion cells.

The adsorption values were calculated from the material balance of polymer in the solution before and after its contact with cells. The polymer concentra- tion was determined interferometrically or by colloidal titration [ 431.

The electrophoretic mobility was measured by microelectrophoresis; the val- ues of the zeta potential were calculated with Smoluchowski equation, i.e. with- out taking into account the polarization of the double layer, which in our experiments was negligible.

It was shown that nonionic and anionic polymers - polyethylene oxide, polyvinyl alcohol, carboxylchitin, hydrolyzed polyacrylamide, polyacrylic acid, etc. - are weak flocculants for E. coli cells; the degree of extraction of cells from solution does not exceed 20% in this case.

Good flocculants for cellular suspensions are the flexible cationic polyelec- trolytes - polydiethylaminoethylmetacrylate and its copolymers with vinyl- pyrrolidone, a&amide and acrylic acid - which in 15-20 ,Wbillion concentration cause precipitation of 90% of cells from suspension (Fig. 1). The polymer concentration range at which the flocculation occurs is wide

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a

& -* , j.i g/billion. an3

Fig. 1. (left) Adsorption isotherms (a), zeta potentials of cells (b) and n/h of E. coli suspensions (c) in the presence of polyDEAEMA (1) , copolymer DEAEMA/AC with C = 1.8 (2), chitosan (3) and copolymer DEAEMA/AC with C =0.57 (4).

Fig. 2. (right) The dependence of n/h of E. coli suspensions on concentration of nonionic carbox- ~ethy~hitine (1) , hy~o~ethylchitine (2 f and cationic ~propylchi~~ (3 ) f chitosan (4 1, dimethylchitosan (5) and trimethylchitosan (6).

enough for practical purposes. From the data in Fig. 1 it can also be seen that increasing the charge density of polyelectrolytes (by increasing the DEAEMA content in the copolymer) enhances the flocculation.

The most effective flocculants for the cellular suspension are cationic poly- electrolytes of natural origin - chitosan and its derivatives. The effectiveness of the latter increases in series: isopropylchitosan < chitosan < dimethylchi- tosan < trimethylchitosan (Fig. 2). The addition of Z-10 fig/billion chitosans precipitates 98-99% of cells. This concentration is several times less than the flocculation concentration of flexible polyelectrolytes in spite of the fact that the molecular weight of the latter is 5-10 times higher. The high flocculation activity of chitosans is due to the rigidity of their macroions, which probably form thick adsorption layers on the cellular surfaces. So the effectiveness of

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polyelectrolytes is determined not only by their molecular weight but also by their molecular parameters in solution, depending on the charge density and rigidity of polymer chains as well.

To study the mechanism of flocculation, measurements of polymer adsorp- tion and its effect on the electrophoretic mobility of cells and degree of floc- culation were carried out. In these experiments the suspension of cells twice washed by physiological solution and resuspended in 0.1 M sodium chloride was used. As can be seen from Fig. 1, the amount adsorbed increases with increasing charge density of the polyelectrolytes, characterized by Donnan charge of elementary unit ( [ = e’/DkTl, where 1 is the distance between charged groups, and other symbols have their usual meanings). This confirms the dom- inant role of the electrostatic mechanism of adsorption of flexible polyelectro- lytes. There is good correlation between the adsorbed amount and the degree of zeta-potential decay during the adsorption of different polyelectrolytes. For polyDEAEMA and its copolymers the isoelectric point (IEP) is reached at the same flocculant concentrations (C,= 5-10 pg/billion) at which the minimum suspension stability is observed (Fig. 1). So the most probable mechanism of the flocculation of cellular dispersion is the neutralization of surface charge groups. At the same time the IEP of E. coli suspension in the presence of chi- tosans is reached at C,, which is much more than the flocculation concentra- tion, i.e. the flocculation in this case occurs long before the neutralization of surface groups. This is evidence of the contribution of nonelectric factors to flocculation; this factor is probably the “bridging” between cells via adsorbed polymers. This mechanism can operate in the case of rigid macroions of chi- tosans which undergo weak deformation during adsorption.

It was also found that the amount of flocculant necessary to obtain the same degree of precipitation is less for cells washed than for the initial culture sus- pension. This is due to the fact that in the latter case, interaction may occur between the reagent and substances in the intermicellar liquid as well as be- tween the reagent and the products of the metabolism and the components which form the cellular surface. This leads to a higher dosage of flocculant in comparison with the dispersion of “pure” cells.

Our experiments showed that increase of pH leads to an increase in the amount of flocculant. This may be caused by the cellular charge decrease with pH lowering (electrical repulsive forces between cells decrease, which favours “bridging” formation) and may be due to increasing ionization of amine groups of polyethyleneamides or chitosans. The latter promotes flocculation by the neutralization mechanism and by “bridging” (the linear dimensions of floc- culant increase with acidification).

The experimental results were obtained together with A.Ya. Teslenko, Yu.V. Medvedev and E.N. Lazarenko.

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REFEmNCES

1 Yu.P. Cherenkova, E.V. Silberman and G.N. Shvarceva, Zh. prikl. Khim. (USSR), 53 (1980) 378.

2 Y. Merle et al., in E.J. Goethall (Ed.), Polymeric Amines and Ammonium Salts, Pergamon Press, Oxford, 1980, p. 113.

3 R&L Muses, Chitin, Pergamon Prees, Oxford, 1977. 4 I.M. Solomentzeva, A.Ya. Teslenko, A.A. Baran, Yu.V. Medvedev and E.N. Lazarenko, Khim.

i Tekhnol. Vody (USSR), 5 (1983) 459. 5 Yu.V. Medvedev, E.N. Lazarenko, A.A. Baran and A.Ya. Teslenko, Khim. Tekhnol. Vody

(USSR), 7 (1985) 18.

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DISCUSSION

J.F. STAGEMAN (ICI Biological Products, Billingham, United Kingdom) (1) You attribute the relatively greater effectiveness of chitosan as a floc-

culating polymer for bacteria to its greater conformational rigidity, why should this be?

(2) If you reduce the background ionic strength for two more flexible, syn- thetic cationic polyelectrolytes you have tested, do you find that these mole- cules also become more effective flocculants due to the decreased screening and the resultant configurational restriction.

A.A. BARAN (Ukrainian Academy of Sciences, Kiev, U.S.S.R.) (1) The rigid macroions such as chitosan and its derivates undergo weak

deformation during adsorption; they form a thick adsorption layer on the cell surface. Therefore it favours more effective “bridging”.

(2) Yes, we have found such a behavior.