For both species, increasing yields of sophorolipids were accompanied by decreasing concentrations of oleic acid, which was expected because of the incorporation of oleic acid into the sophorolipid molecule. The requirement for high aeration in production of sophorolipids was reported earlier (Guilmanov et al., 2002) and again shown in this study for both S. bombicola NRRL Y-17069 and Candida sp. NRRL Y-27208 (Table 2). The maximum yield of sophorolipids was obtained at a shaker speed of 350 r.p.m. Glucose concentration noticeably

affected sophorolipid production by both S. bombicola NRRL Y-17069 and Candida sp. NRRL Y-27208 (Table 2). For S. bombicola, 50 g L−1 glucose yielded Bleomycin 48.8 g L−1 sophorolipid, whereas 150 g L−1 glucose yielded 95.4 g L−1 sophorolipid. The increased sophorolipid

production was not fully reflected in the reduced concentration of residual oleic acid (Table 2), suggesting that a portion of the lipid moiety was synthesized by the yeast. During AZD6738 supplier production of sophorolipids, the pH of the culture medium declined from 4.5 to as low as 1.8. To sustain production, the pH was readjusted twice daily to 3.5 with 1 N NaOH. The precipitous decrease in pH during sophorolipid production and its impact on reducing yield was reported earlier by Gobbert et al. (1984). The solvent extracts obtained from all 26 strains examined were initially screened for the presence of sophorolipids by MALDI-TOF MS using techniques developed previously by Price et al. (2009). The spectra were characterized by molecular adduct ions for sophorolipids in the mass range

620–720 Da (Fig. 2). Major ions at m/z 711 and m/z 729 are respectively attributed to the [M+Na]+ molecular adduct ions for the lactone and free acid forms of the major diacetylated sophorolipid, 6′,6″-O-diacetyl-β-d-glucopyranosyl-21-O-β-d-glucopyranosyl-oxy-octadecenoic Vitamin B12 acid (Asmer et al., 1988). The observed 18 Da difference between these two ions corresponds to the mass difference between the free carboxylic acid form and the ester-linked 4′-O-lactone (Fig. 2). Less intense ions at m/z 669 and m/z 687 correspond to the monoacetylated forms of the major sophorolipids, and m/z 627 and m/z 645 correspond to the non-acetylated forms (Fig. 2). The 18 Da mass difference between these two sets of ions is again indicative of the free acid and lactone forms of the minor sophorolipids, and the 42 Da difference between di-, mono- and non-acetylated species is characteristic of O-linked acetyl groups (Price et al., 2009). Similar sophorolipid ions were also observed previously for C. bombicola by fast atom bombardment MS (Asmer et al., 1988; De Koster et al., 1995). The five species of the Starmerella clade tested that showed the most prominent production of sophorolipids: S. bombicola NRRL Y-17069, C. stellata NRRL Y-1446, the new species of Candida, NRRL Y-27208, C. riodocensis NRRL Y-27859 and C. apicola NRRL Y-2481, were further examined by MALDI-TOF MS.