Shake flask cultivation
For culture maintenance, strains were grown in 500 mL round-bottom shake flasks containing 100 mL YP medium (10 g/l Bacto yeast extract, 20 g/l Bacto peptone) supplemented with 20 g/l glucose. Precultures were grown overnight in filter-sterilized synthetic medium (SM) at pH 6.0, prepared as described previously (Verduyn et al. , 1992a,b) and transferred to fresh medium for characterization in shakeflask.
For strain characterization in shake flasks, the (NH4)2SO4 in SM was substituted by 2.3 g/l urea and 6.6 g/l K2SO4, to provide an equimolar amount of nitrogen and prevent medium acidification due to ammonia assimilation (Luttik et al. , 2000). When required, formic acid was added to the medium to a final concentration of 1.2 g/l from a concentrated stock solution (99% w/w) prior to sterilization. Heat-sterilized glucose (110°C, 20 minutes) was aseptically added as carbon source after sterilization. For characterization, cultures were inoculated into 100 ml SM with 7.5 g/l (42 mM) glucose with and without 1.2 g/l (25 mM) formic acid in 500 ml round-bottom shake flasks. Shake flask cultures were incubated at 30°C in an Innova incubator shaker (New Brunswick Scientific, Edison, NJ, USA) set at 200 rpm and a throw of 2.5 cm.