This article reviews most of the author’s studies on process development and reactor design for continuous microbial reactions. (1) Enzyme reactions of growing and non-growing microbial cells immobilized in agar gel beads were analyzed pertaining to the effects of external and internal diffusion of substrate on reaction kinetics. (2) Experimental correlations of production rates of β-fructosidase and acid phosphatase with dilution rate of continuous culture were simulated based on an operon model for enzyme regulation. (3) Population dynamics of an amylase-producing bacteria and their mutant were discussed in relation to enzyme productivity in a continuous culture of spore-forming bacteria. (4) Plasmid mobilization in a mixed population of donor, recipient, and helper cells was investigated in a continuous culture as a model study of accidental release of a genetically modified plasmid into a natural environment. (5) A production rate increase of up to 100-fold was achieved by cell-recycle culturing of continuous acetic acid fermentation using a filter module with a hollow fiber membrane. (6) The feasibility of a continuous surface culture for the biooxidation of organic substances was ascribed to an enhanced oxygen absorption rate in the presence of a microbial film on a liquid surface. (7) Simultaneous separation of inhibitory products using an electrodialysis module during some organic acid fermentations was effective for increasing production in a continuous culture.
The production of an antibiotic by free and immobilized cells of Streptomyces violatus through entrapment or adsorption on different materials was investigated. S. violatus entrapped in Ca-alginate beads gave low antibiotic activity compared to the free cell or adsorbed cell, while the adsorption of S. violatus on sponge cubes yielded the highest antibiotic concentration after 4 days of incubation in static cultures. A starch concentration of 10g/L was optimum for the production of the antibiotic by adsorbed cells. The weight and size of the sponge cubes used for immobilization influenced production of the antibiotic and the optimum weight and size of the sponge were 0.8g and 1.0cm3, respectively, yielding a maximum antibiotic production of 280μg/ml. Maximum antibiotic production was obtained at an initial pH value of 7.5 and in an inoculum size of 3ml (spore suspension) per 50ml. The production of the antibiotic in a fixed-bed bioreactor reached a maximum value after 2 days of incubation at a circulation rate of 30ml/h. The immobilized cells in the bioreactor were reused seven successive times over a period of 14 days.
The cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 is a modular enzyme comprised of a catalytic domain, a cellulose-binding domain and a cell wall-binding domain. Cellobiose-grown cells changed their adhesion ability to cellulose depending on the growth phase. On the other hand, carboxymethyl cellulose (CMC)-grown cells bound to cellulose regardless of their growth phase. The distribution of CBPA in the culture supernatant and cell fractions changed depending on the carbon source contained in the medium and growth phase. The cellobiose-grown cells harvested from the culture of the late stationary growth phase did not bind to cellulose, but their adhesion ability was recovered by treatment with recombinant CBPA. Moreover, cellobiose-grown cells harvested from the culture of an early exponential growth phase bound to cellulose, but their adhesion ability was inhibited by treatment with anti-CBPA antiserum. CBPA rapidly decreased the viscosity of CMC, indicating that CBPA was endoglucanase. The results obtained in this study indicate that CBPA plays an important role in the adhesion of E. cellulosolvens 5 cells to cellulose.