Analysis methods

Cell growth was monitored by sampling, centrifuging, and measuring the CDWs. For intracellular ectoine analysis, cells were collected by centrifuging at 6000×g for 10 min, washed twice with 1.5 M NaCl solution, resuspended into the distilled water, and passed through a French press (Aminco). After centrifugation at 8000×g for 10 min, two volumes ethanol were added to the supernant and the mixture were passed through a 0.25 μm filter for intracellular ectoine measurements. For extracellular ectoine measurement, the supernants were mixed with two volumes of ethanol and filtered as above described. The ectoine concentrations were quantified by a Shimadzu HPLC system (Kyoto, Japan) that equipped with a Venusil XBP NH2 column (4.6 × 250 mm) and a UV detector monitored at a wavelength of 204 nm. Analysis were carried out with acetonitrile/H₂O (60:40, V/V) as the mobile phase and a flow rate of 0.5 mL min⁻¹.

The mean consumption rate of ectoine in the secondary growth phase was estimated as

where Emax and Eend are the maximal and last concentrations of E, Tmax and Tlast are the correspondent time.