A segmented gas/liquid delivery system for continuous culture of microorganisms on insoluble substrates and its use for growth of Ruminococcus flavefaciens on cellulose

Abstract

An anaerobic continuous culture device was constructed that permits accurate delivery of media containing insoluble substrates, even at very low volumetric flow rates (<3 ml/h). The system consisted of: (1) a reservoir in which the medium slurry was mixed well by the combined use of stirring and diffusive gas sparging to suspend a cellulose substrate of small (< 45 μm) particle size; (2) a delivery system that segmented the slurry into small (~ 20μl) discrete liquid segments separated by intervening bubbles of CO2 gas; and (3) a stirred, temperature-controlled 2-l fermentation vessel. The device was used to examine substrate consumption, product formation, and cell yield by the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 under steady-state, cellulose-limited conditions at six different dilution rates (D) ranging from 0.017 to 0.101 h−1 (pH 6.4–6.6). Cellulose consumption decreased from 4.00 g/1 (at D=0.017 h−1) to 2.56 g/1 (at D=0.101 h−1). Increases in D resulted in a progressive shift toward production of more acetate and formate, and less succinate. Redox balance calculations revealed a deficiency in reduced products, probably due to the production of H2, which was not directly measured. Reducing sugar values remained low (0.05–0.10 g/1, as glucose) at all D values. The cellulose fermentation was characterized by a low maintenance coefficient (0.07 g cellulose/g cells per hour) and a high true growth yield (YG = 0.24 g cells/g cellulose, corrected for maintenance). Comparison of the data with literature values suggests that the fermentation of cellulose by this organism gives cell yields at least as great as the yields obtained from the fermentation of soluble sugars.