Keshavarz, Elaheh; (1990) Studies of cell disruption in high pressure homogenisers. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this study several aspects of cell disruption in high pressure homogenisers were investigated. Firstly, the kinetics of disruption of Rhizopus nigricans, a filamentous fungus, was shown to exhibit major differences from those of unicellular microorganisms. The release of soluble protein was a weak function of pressure and number of passes. The maximum level of soluble protein release (R",) which appeared to vary with pressure was shown to be associated with micronisation of cell debris. A more specific assay for a soluble enzyme (ADH) showed no such apparent variation. The major effect of cell concentration was not on disruption kinetics but on structural characteristics of the suspension which resulted in homogeniser blockage at relatively low cell concentrations (20-22 g/L dry weight) and also influenced the harvesting and resuspension processes. Another aspect of cell disruption examined was the effect of valve assembly configuration on the disruption of both unicellular and filamentous microorganisms. Six valve units were characterised and several parameters were shown to be critical. Impact distance was an important factor in the disruption of filamentous R. nigricans cells, whilst valve geometry did not appear to have any notable effect. Valve unit configuration had a significant effect on the disruption of the unicellular microorganism, bakers'yeast. Maximum performance was observed with a 'knife-edge' valve seat configuration and valve geometries consisting of a flat valve seat and flat valve rod gave lowest yields. A reduction in the "land" width gave improved performance. Changing the valve rod to a cone shape also resulted in higher protein release. As with filamentous microorganisms, variation in the impact distance had a major effect on the performance of the homogeniser. The effect of homogenisation on the size distribution of yeast cell debris and microsomal fractions obtained from R. nigricans cells was also investigated. A reduction of at least 10X was observed in the size of the microsomes from one to 2 passes with no further decrease up to 4 passes. For all valve units tested, as the number of passes was increased, a reduction in the proportion of larger yeast debris particles was noted. In general, higher protein release was associated with a higher percentage of smaller particles except for a marginal trend observed for the 'flat valve' unit giving larger particles for equivalent protein release compared to other valves. Based on the findings for bakers' yeast an explanation of the mechanism of cell disruption was sought. A modified Bernoulli equation was used to define the flow velocities through the valve rod and valve seat for different valve unit configurations. The main disruption mechanism was shown to be impingement, the rate of cell breakage being related to the stagnation pressure or the maximum wall stress of the fluid jet. Decreased valve gap width and decreased impact distance both contributed to an increased cell disruption rate, the performance of the homogeniser being related to the product of these two dimensions for the range of valve seats and impact distances studied. Further experimental work to test the proposals has been defined. The design of an integrated homogenisation system consisting of a new commercial homogeniser and various ancillary items of equipment was undertaken to provide a suitable process unit for further research, taking into account the additional elements needed for containment.

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