Deactivation and regeneration kinetics during bioconversions by immobilized Clostridium acetobutylicum

Abstract

The physiology and catabolic rates of calcium alginate-immobilized Clostridium acetobutylicum were studied under a variety of conditions, with an emphasis on the relationships between the activity and the environment of the immobilized cells during bioconversions of nitrogen-free glucose media and also during periods of activity regeneration under defined conditions. A number of experimental and analytical techniques were used to investigate these relationships, including measurements of product formation and substrate (glucose and nitrogen) consumption, quantification of immobilized cell concentrations by direct counts of several morphological types, and the in situ measurement of intracellular NAD(P)H-dependent fluorescence. In addition, a framework for the analysis of metabolic pathway rates from product and substrate concentration data was developed and applied to both suspended and immobilized cell cultivations of C. acetobutylicum. Two C. acetobutylicum strains were used in this work; one was the type strain, the sporogenous ATCC 824, and the other was ATCC 39236, which was found to be oligosporogenous. When the immobilized cell bioreactor feed was a medium deficient in nitrogen and required vitamin sources (to maintain the immobilized cells in a non-growing but viable state), the major causes of product-forming activity loss were initial cell lysis and sporulation; the latter effect was minor in the ATCC 39236 experiment. The addition of ammonia or ammonia plus vitamins to the feed during a regeneration period increased the total activity of the immobilized cell preparation, and this increased activity was not always due to cell growth. The patterns of product formation during repeated cycles of bioconversion (on nitrogen-free medium) and activity regeneration were discussed in terms of the changes in the adenylate energy charge that occurred in the course of alternating periods of nitrogen starvation and surplus. Product formation during bioconversion periods was affected by environmental conditions such as pH and the presence of some intermediate metabolites in the feed; these effects were related to distinct features of the C. acetobutylicum metabolic pathways. This work shows that knowledge of the metabolism and the underlying physiological responses of the cell to environmental changes can be used to understand and predict cell behavior.