Computer-Mediated Addition of Fresh
Medium in Continuous Culture of Zymomonas
mobilis by Monitoring Weight Changes

H. D. Zakpaa1, Ayaaki Ishizaki1, and K. Shimizu2

department of Food Science and Technology, Faculty of Agriculture,
Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812, Japan
department of Biochemical Engineering and Science,

Faculty of Computer Science and Systems Engineering,

Kyushu Institute of Technology, Iizuka 820, Japan

A major problem encountered in pH-stat continuous culture of Zymomonas mobilis which employed growth-dependent pH changes to control the rate of addition of fresh medium into the culture was that of low dilution rate. Glucose concentration in the culture broth also became very low after the initiation of continuous fermentation.

A new control system for delivering fresh substrate into the fermentor is discussed that solves the problems of low dilution rate and low residual glucose. This new system involves linking the feeding of fresh substrate into the fermentor with the outflow of alkali (NaOH) from the alkali reservoir by monitoring weight changes of the two solutions. Dilution rates of about 0.33 h_1 and productivity values of about 11.00 g. Hlr1 could be achieved with this method, while maintaining the concentration of ethanol in the fermentor at about 33.00 gH when the ratio (a) of weight of glucose fed into the fermentor to the unit weight of alkali used in neutralizing acid produced in the broth was 25.00.

Zymomonas mobilis (/), a bacterium that occurs as motile short rods (2), has attracted considerable attention as a promising microorganism for large scale production of ethanol because of its unusual physiological and biochemical properties (/), and more recently because of its high efficiency in ethanol production.

Three main approaches namely; physiological (3, 4-9), genetic (10-16), and engineering (17-23) are being pursued with an aim towards improving the productivity of ethanol fermentation. With the engineering approach to fermentation process improvement, fermentors are operated in continuous mode instead of the more conventional batch mode, resulting in an increase in productivity. The genetic approach aims at increasing process productivity through the improvement of the metabolic characteristics of the organism employed, by attempting to correct a recognized weakness or deficiency, such as

© 1997 American Chemical Society

broadening the range of substrates which the organism can metabolize as carbon source. The physiological approach attempts to control parameters that affect process productivity, by varying environmental factors such as the chemical composition of the fermentation medium, concentration of essential nutrients or inhibitory substances, as well as pH and temperature (4-9, 24).

Computer technology has had significant impact on fermentation in recent times. The advent of inexpensive, computer-coupled fermentation systems has facilitated close monitoring and direct-digital control (DDC) of environmental variables such as temperature and dissolved oxygen concentration. These systems are now commonly used for on-line monitoring of several fermentation variables such as oxygen uptake rate, carbon dioxide evolution rate, and respiratory quotient. Such variables have been successfully used for identifying the physiological state of microorganisms in many fermentation processes (25, 26). The use of computer-coupled fermentation systems to control pH in DDC mode also allows for on-line measurements of the amount of acid produced and the acid — production rate during fermentation. These variables can prove significant in on­line characterization of an anaerobic fermentation process, like ethanol fermentation by Z mobilis.

Low dilution rate and low residual glucose concentration in the fermentor broth were encountered in the previous set-up of computer-mediated, pH — dependent addition of fresh substrate in continuous culture of Z. mobilis (27). In this paper an alternative but similar method of supplying fresh substrate to the culture is designed and tested. This approach involved linking the feeding of fresh substrate into the fermentor with the outflow of alkali (NaOH) from the alkali reservoir into the fermentor, achieved through the monitoring of weight changes. The rationale behind this method is that by monitoring the NaOH addition resulting from decrease in the pH of the culture broth, better control of glucose concentration in the fermentor can be accomplished This contrasts with previous methods (27), where fresh substrate was only fed when the pH in the fermenting culture rose above the set pH limit on the control unit as a result of excretion of NH4+ ions into the medium. In anaerobic culture employing Z. mobilis, this pH rise is delayed. By the time it occurred the cells had been starved of glucose and most had lost viability.