Bulmer, Mark Alastair; (1993) Selective flocculation for the improved recovery of intracellular proteins from bacterial homogenates. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mechanical disruption of bacterial suspensions to release soluble protein, also results in the formation of bio-colloidal material such as cell wall debris, nucleic acids etc. Its effective removal on an industrial scale (by centrifugation/ filtration), has proved to be difficult, due to the sub-micron size and colloidal nature of the debris. A number of downstream processing operations are required to remove these materials, due to their detrimental affect on high resolution operations. Destabilisation of homogenates using polymers has proved to be an effective method for selectively flocculating this material, thus facilitating its removal. Physico-chemical properties of the cationic polymer polyethyleneimine (PEI) were studied to determine how these influence flocculation effectiveness and mechanism. Potentiometric and colloid titrations provided charge density data, whilst dynamic light scattering and size exclusion chromatography yielded information concerning the hydrodynamic radii and molecular weight averages for various commercial samples. From this two grades of PEI (low and high molecular weight), were selected for further flocculation studies. Model systems consisting of bentonite, latex or washed cell debris were used to determine the effect of the flocculation process on the PEI molecular weight distribution. The charge difference between the polymer and material influenced which part of the molecular weight distribution was involved in the flocculation process. Other model systems studied included the interaction of PEI with phosphate, nucleic acids etc., to examine polymer interaction with homogenate components. Flocculation of cell debris under a variety of conditions showed that pH and ionic strength can be used to modulate the flocculation process especially with respect to soluble protein recovery. Phosphate addition to the polymer, before flocculation, reduced the charge density and resulted in improved protein recovery at low ionic strengths. Pre-flocculation of whole cells followed by disruption and re-flocculation, also resulted in greater protein recovery at lower doses. Determination of protein, nucleic acid, lipid and cell debris removal across a range of polymer doses enabled the optimum conditions for protein recovery to be determined. A radiolabelled assay for PEI was developed to quantify remaining PEI after flocculation; the minimum residual concentration occurred at the optimum flocculant dose.

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