Biotechnology Letters Vol 8 No ii 817-822 (1986) Received September 30 IN SITU FLUORESCENCE MONITORING OF IMMOBILIZED CLOSTRIDIUM A CETOBUTYLICUM Kenneth F. Reardon, Thomas Schepert, and James E. Bailey* Department of Chemical Engineering California Institute of Technology Pasadena, California 91125 USA ABSTRACT The NAD(P)H-dependent culture fluorescence of immobilized Clostridium acetobutylicum was followed during a three-part fermentation involving product formation on minimal and nitrogen-free media. The fluorescence signal, together with a knowledge of the metabolic pathways of Cl. acetobutylicum, provides information on the state arid intracellular activ- ities of the immobilized bacteria not readily found by other methods. INTRODUCTION In situ fluorometry for the purpose of monitoring intracellular NAD(P)H levels was first reported by Duysens and Amesz (Duysens and Amesz, 1957), was developed signifi- cantly by Chance and coworkers (e.g., Chance and Legallais, 1959 and Chance et al, 1965), and has been applied to suspended cell fermentations by several research groups (Scheper and Schfigerl, 1986; Zabriskie and Humphrey, 1978). The ability to measure NAD(P)H levels in cells in intact rat heart tissue in vivo using fluorometry was demonstrated by Chance et al (Chance et al, 1965). This report describes a three-part experiment (growth, bioconversion, and regen- eration) utilizing calcium alginate-immobilized Clostridium acetobutylicum in which the immobilized cell NAD(P)H fluorescence was measured. Simultaneous measurements of substrate uptake and product concentrations, combined with culture fluorescence data, show interesting transient changes in the metabolic activities of the immobilized bacteria. MATERIALS AND METHODS Organism Clostridium acetobutylieum ATCC 824 was maintained in corn mash medium (5% corn meal and 0.05% cysteine, boiled for 1 hour). Corn mash cultures were used to inoculate (1%) working cultures grown in starch medium (soluble potato starch (Sigma), 40 g/L; yeast extract (Difco), 1 g/L; peptone (Difco), 1 g/L; NH4C1, 0.8 g/L; Na2HPO4-7H20, 0.9 g/Li KH2PO4, 0.4 g/L; MgSO4.TH20, 0.2 g/L; and 10 mL/L of a trace element solution consisting of: CaC12-2H20, 0.66 g/L; ZnSO4.7H20, 0.18 g/L, CuSO4-bH20, 0.16 g/L; MnSO4.4H20, 0.16 g/L; and COC12.6H20, 0.18 g/L) (F6rberg et al, 1983). Inocula were heat shocked for 60 seconds in a 90-95~ water bath before addition to the autoclaved starch medium. Three other media were used during the experiment. The growth medium was pre- pared from: glucose, 40 g/L; yeast extract, 5 g/L; peptone, 5 g/L; Na2HPO4.7H20, 0.9 g/L; KH2PO4, 0.4 g/L; CaCI2, 0.55 g/L; MgSO4.7H20, 0.2 g/L; NH4CI, 0.8 g/L; FeC13.6H20, 0.01 g/L; and 0.5 mL/L of the trace elements solution (F~rberg et al, 1983). t Current address: Institut ffir Technische Chemie, Universit~t Hannover, D-3000 Han- nover 1, Federal Republic of Germany 817