Hornby, Stephen Patrick Powell; (1995) Downstream processing effects on microbial viability. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

An increasing number of biotechnology production processes require a whole cell recovery step during downstream processing. Viability loss at this stage is generally undesirable as it can lead to reduced yields and separation difficulties further downstream. Comparisons between the effects of different types of equipment on a single strain of cells is rare. Such information can be invaluable when designing new processes or equipment. This work was carried out in the following stages: • Production of identical batches of cells for testing; • Use cells to develop a reproducible assay test system; • Use the assay system to assess the damage of various concentration techniques on the viable cells. A reproducible batch fermentation for the production of Pseudomonas putida ML2 cells was developed. Both the inoculum and fermentation stages were carried out in fully defined media. The fermentation used a minimum salts media and fructose as a carbon source. The bioluminescent ATP assay was tested as a novel method for measuring cell damage. The action of separation was simulated at laboratory scale by the use of a high pressure homogeniser. The ATP assay was tested on growth and stationary phase cells and results were compared to standard assay methods such as plate counts and protein release. Cells in stationary phase appeared to undergo a metabolic activation and so ATP analysis proved to be unreliable. Protein assay and plate count techniques were developed to increase reproducibility and accuracy by the use of multiple repeats and statistical analysis. Batch centrifugation was examined, multichamber bowl centrifugation proved to be less damaging to cells than tubular bowl centrifugation. Continuous centrifugation was carried out using a disk stack centrifuge with intermittent solids discharge. Damage was observed between 0–20% loss of viability. Cells were sampled directly from the separation bowl as well as from the discharged solids. Little difference was observed, suggesting that damage occurs in either the separation or feed zones prior to discharge. Microfiltration was carried out using a specially devised laboratory scale rig, designed at UCL. Runs were carried out under a number of different operating conditions. Increased cell damage was observed at higher cell concentrations and transmembrane pressures. Unlike centrifugation, results the protein release levels are less than the measured loss of viability suggesting a different mode of inactivation. Hydraulic shear stresses were estimated across the range of operations tested, as well as in the fermenter. Examination of the microfiltration results suggested that the action of the system was similar to that of a high speed fermenter.

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