Effects of pH and Added Metabolites on Bioconversions by Immobilized zyx No n -G row i n g zyxwv Clos tridium acetobutylicum Kenneth F. Reardon* and James E. Bailey Department of Chemical Engineering, California Institute of Technology, Pasadena, California 91 125 Accepted for publication December 28, 1988 zyxwvu The bioconversion activity of calcium alginate-immobi- lized Clostridium acetobutylicum ATCC 824 was investi- gated in a continuous reactor system utilizing a defined feed medium which did not support cell growth. The changes in biocatalytic activity with time were studied at different pH values as well as when different metabo- lites (butyric, acetic, and acetoacetic acids) were present in the feed stream. Although the nongrowing cells were metabolically active, the product distribution was shifted from solvent production to acidogenesis. Overall activity losses occurred due to cell lysis, sporulation, and the effects of nitrogen level on macromolecular turnover. These effects were minimized under some op- erating conditions (e.g., pH 61, resulting in significantly longer productivity lifetimes. INTRODUCTION Immobilized cell fermentations may utilize microorgan- isms that are nonviable, viable but nongrowing, or viable and growing. While the use of nonviable cells in immobi- lized biocatalysts may maximize the productivity and us- able lifetime for some processes, conversions requiring more than a few enzymes and/or cofactor regeneration dic- tate the use of viable cells. Growing, viable immobilized cells are in principle able to carry out any chemical con- version characteristic of the organism, but immobilized growing cell systems often experience problems such as a high suspended cell concentration in the reactor effluent, cell fouling of fluid passages in the bioreactor, and, if en- trapment techniques are utilized, physical damage to the immobilization matrix. At least in theory, viable zyxwvuts non- growing cells offer multistep, cofactor regenerating bio- conversions without the complications of cell growth (and suspended cell production). Also, the use of nongrowing immobilized cells should allow a higher fraction of sub- strate carbon to appear as product, rather than as biomass. zyxwvu In practice, systems in which immobilized, viable non- growing cells are used often display relatively rapid de- clines in the desired catalytic activity.'-5 Some reports on zyxwvu * Present address: Department of Agricultural and Chemical Engineer- ing, Colorado State University. Fort Collins, Colorado 80523. Biotechnology and Bioengineering, Vol. 34, Pp. 825-837 (1989) Q 1989 John Wiley & Sons, Inc. continuous immobilized fermentations involving significant cell growth have also indicated activity 10sses;2~~.~ this be- havior probably has different causes than that found in nongrowth systems. The underlying causes for loss of productivity in nongrowth immobilized cell systems un- doubtedly depend on the method used to obtain such a condition - nutrient limitation or starvation, or exposure to an inhibitory agent-and on the metabolic origin of the product, with differences expected between secondary metabolites and products typically associated with growth. The most common technique for growth limitation is nutrient starvation - usually the omission of a nitrogen source,238-'o vitamins or minerals,4s" or both nitrogen source and The use of hydroxyurea, a cell- cycle blocking agent, for the restriction of immobilized cell growth has also been reported.'* Possible causes for biocatalyst activity losses include the inactivation or protease-dependent degradation of enzymes, loss of cofac- tor regenerative capacity, degradation or diffusive release of cofactors, or loss of membrane integrity or function (i.e., the maintenance of various transmembrane gradi- ents), among others. In this study, factors affecting the decrease in biocata- lytic activity of calcium alginate-immobilized Clostridium acetobutylicum were investigated by varying pH and by the addition of metabolites (acetate, butyrate, and aceto- acetate) to the feed stream of a continuous-flow, packed- bed bioreactor. The feed medium contained no source of nitrogen nor the necessary growth factors p-aminobenzoic acid and D-biotin.I3 Product formation rates and immobi- lized cell concentrations were used to provide insights to the deactivation phenomena. MATERIALS AND METHODS Organism Clostridium acetobutylicum ATCC 824 was maintained in corn mash medium.I4 This medium was prepared by boiling an aqueous mixture of 5% (w/v) corn meal ("Argo" CCC 0006-3592/89/060825- 13$04.00