Accumulation of ectoine in the halotolerant Brevibacterium sp. JCM 6894

The optimum conditions for the intracellular synthesis of the cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyridine carboxylic acid) were determined using the halotolerant Brevibacterium sp. JCM 6894. The amount of ectoine synthesized in cells was quantitatively determined after extraction from cells grown under various conditions, such as different external osmolarities, incubation times, and types and concentrations of nutrients in the medium. The optimum condition for the synthesis of ectoine was confirmed to be consecutive cell transfers accompanied with the changes in the medium osmolarity as follows; cells grown in the presence of 0.3 M NaCl were transferred to medium containing 1.5-2 M NaCl. After incubation for 18-24 h a second transfer of the cells to fresh medium containing 2-3 M NaCl was carried out and the cells were incubated for a further 12-24 h. The addition of 50-100 mM glucose or a high concentration of yeast extract in the medium resulted in an increase in the intracellular concentration of ectoine as well as of the cell yield. The growth medium affording the highest ectoine productivity of strain JCM 6894 consisted not only of 2 M NaCl but also Polypepton (10.0 g/l), yeast extract (5.0 g/l), and glucose (9.0 g/l) as nutrients. The highest yield of ectoine, 800 mg/l culture, was obtained using the optimum medium and one osmotic upshock. This yield corresponded to about an 8-fold increase compared to that obtained using standard medium (2 M NaCl) without an osmotic shift. In addition, the ectoine which was synthesized in cells grown at high osmolarity was released well from the cells by osmotic downshock.