High Density Culture of Hyperthermophilic Archaeon Pyrococcus furiosus with a Perfusion Operation for Removal of Acetic Acid as Growth Inhibitor
Yutaka NAKASHIMADA, Kei-ichi NAKAE, and Naomichi NISHINO
Department of Molecular Biotechnology, Graduate School of Engineering Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739, Japan
In order to optimize the fermentation of P furiosus, the influence of the growth parameters has been studied such as medium composition, hydrodynamic stress and dissolved hydrogen concentration. Among such researches, the culture using a dialysis membrane reactor was the most effective for high density culture of P furiosus, suggesting that metabolic products removed by the dialysate may be responsible for the growth cessation during fermentation. Although this organism produces acetic acid and L-alanine besides hydrogen, candidate of the growth inhibitor seems to be the undissociated acetic acid because it is known that the undissociated acetic acid inhibits the growth of microorganisms in its metabolic products. However, our knowledge on inhibitory kinetics of acetic acid on the growth of hyperthermophile is limited. Therefore, at first, effect of acetate on the cell growth of P furiosus was studied in detail. And then, high density cultivations of P. furiosus with repeated fed- batch and a perfusion culture for controlling acetic acid concentration at low level were studied.
The inhibition of acetic acid on the cell growth could be expressed by a noncompetitive inhibition model, in which the inhibition constant, Kp and n of undissociated acetic acid on cell growth was estimated as 0.69 mM (= 25 mM of total acetic acid at pH 6.5, pKa=4.96, 98℃) and 1.0, respectively. This result indicates that the undissociated acetic acid is strong growth inhibitor. However, acetate formation can not be avoided because the formation is essential for ATP generation. Therefore, it is necessary to control acetate concentration in the culture as low as possible for keeping a higher growth rate and obtaining a higher cell density.
In order to remove the acetate inhibition, a repeated fed-batch culture using a filtration module was performed by feed forward control. Cells increased at 0.15 h-1 of specific growth rate while acetate concentration was kept below 5.0 mM as expected. At 12 h of culture, 0.5 g dry weight/1 of cell concentration was obtained which was 2.7-fold higher than that with the vial bottle although acetate concentration gradually increased in spite of the medium replacement. After that, however, it became impossible to continue the repeated-batch culture manually because the time intervals for the medium replacement became too short for the operation. Therefore, in order to keep acetate concentration at low level automatically, a perfusion culture was carried out where acetate removal, that is medium replacement, was coupled with pH stat due to a linear relationship between the decrease of medium pH and the production of acetate. In this culture, cells could grow at about 0.21 h-1 of specific growth rate and cell concentration reached 1.8 g dry cell/1 at 15 h culture, which was 10-fold higher than that of the batch culture with the vial bottle in this study, and acetate concentration could be kept below 8 mM.
