Characterization of zyxw €. zyxw co/i Cell Disintegrates from a Bead Mill and High Pressure Homogenizers Irene Agerkvist ' and Sven-Olof Enfors*-* 'Institute for Surface Chemistry, Box zyxwvut 560Z S-114 86 Stockholm, and 'Department of Biochemistry and Biotechnology, The Royal Institute of Technology, S-100 zyxwvuts 44 Stockholm, Sweden Received June 10, 1989iAccepted Feb 23, 1990 zyxwvuts The protein releases, the particle size distribution and the viscosity of disrupted E. zyxwvutsrqpo coli suspensions from Dyno Mill KDL, Manton Gaulin 15 M-8TA and Microfluidizer M-110 were determined. The effects of these parameters on sepa- ration of the cell debris from the protein solution by cen- trifugation and by filtration were also examined. All three disintegration methods investigated give approximately the same protein and enzyme releases but considerably differ- ent physical properties of the cell disintegrates which influ- ences centrifugation and filtration. The separation degree of biomass during centrifugation is only slightly affected by increasing degree of disruption (increasing protein releases) in the bead mill, while an increase in the degree of disrup- tion in the two high pressure homogenizers drastically reduces the centrifugal degree of separation. However, in- creasing degrees of disruption result in shorter filtration times during filtration for all three disintegration methods. The results show further that the cell concentration only has a minor influence on protein releases in the Microfluidizer high-pressure homogenizer, while an increase in the biomass content reduces the separability of the cell disinte- grate both in filtration and in centrifugation. INTRODUCTION In the downstream process of intracellular bacterial products, the first difficult step after cell disruption is to separate the cell debris from the protein solution. Typi- cally, processes for the removal of cell debris on a large scale involve centrifugation and/or filtration.' The per- formance of these processes depends on the physical properties of the cell debris suspension. Cell disruption techniques currently used on the in- dustrial scale are mechanical disintegrators, such as glass bead mills and high-pressure homogenizers, which produces cell disintegrates with physical properties un- favorable for separation by centrifugation and filtration. The problems arise from the small particle size of the cell debris, the wide particle size distribution, the high compressibility of the solids, the high viscosity and the small density difference between the solid and the liq- uid phase.' * zyxwvutsrqponm To whom all correspondence should be addressed. Biotechnology and Bioengineering, Vol. 36, Pp. 1083-1089 (1990) zyxwvuts 0 1990 John Wiley & Sons, Inc. To be able to predict the performance of cell debris separation it would be necessary to have information on some characteristic physical properties of the suspension after different disintegration methods. In the literature there exist little information on such properties. Quirk and Woodrow' have investigated parameters affecting the separation of bacterial enzymes from cell debris by tangential flow filtration. They examined the effects of organism/enzyme, method of cell breakage, and mem- brane characteristics. Mosqueira et al.4 studied the ef- fects of viscosity and density on the sedimentation performance of disrupted yeast suspensions. In this study, we have determined the protein re- leases, the particle size distribution, and the viscosity of disrupted zyxwv E. coli suspensions after application of dif- ferent disintegration methods. The effects of these parameters on the separability of the cell debris from the protein solution by centrifugation and by filtration were examined. The effect of cell concentration (dry weight of biomass) on separation performance were also examined. MATERIAL AND METHODS Materials The organism used was the p-galactosidase constitutive E. coli strain ATCC 15224. Commassie Brilliant Blue G (C.I. 42655) and o-nitrophenyl-P-D-galactopyranoside (ONPG) were purchased from Sigma Chemical Com- pany (St. Louis, MO). DNase (EC 3.1.21.1.) DN-25 and RNase Type I-AS both from bovine pancreas as well as bovine serume albumin A-4378 were likewise pur- chased from Sigma. The salts and other chemical used were all of the highest quality available. Cultivation and Cell Harvest The E. coli ATCC 15244 was cultivated in a loo0 L fer- menter (Electrolux Fermentation, Sweden) in a glycerol- mineral-salts medium at 37°C according to Veide et aL5 except for the concentrations of (NH4)'S04, which was CCC 0006-3592/90/01101083-07$0400