Genomics of Rhodococcus for Biofuels Figure 4. Identification and purification of odd-carbon straight-chain fatty acids generated by Rhodococcus. a and b) GC-FID analysis of FAMEs synthesized during fermentation. Freeze-dried whole cells fermented on glucose of R. opacus PD630 (a) and R. jostii RHA1 (b). c and d) GC-FID analysis of FAMEs derived from TLC-purified TAGs of R. opacus PD630 (c) and R. jostii RHA1 (d) grown on glucose e) odd lipids C15:0, C17:0, and C17:1 increase during fermentation of both R. opacus PD630 and R. jostii RHA1. f) Rhodococcus grown on propionate generates mostly C15:0, C17:0, and C17:1 fatty acids that were used for purification and confirmation by mass spectrometry and structural 1H-NMR. These analyses demonstrated that the purified Rhodococcus odd-carbon fatty acids were straight-chain. doi:10.1371/journal.pgen.1002219.g004 carbons, that is in the range where most FAS type 1a products are released from the type 1a FAS (C16–18) [26]. Catabolic Phenotyping of Four Industrially Utilized Bacterial Species Rhodococcus fermentation of low-cost organic substrates into oil requires a more complete understanding of the catabolic capabilities of these species. We tested 190 organic compounds as the sole carbon source in time course growth assays with four soil-derived bacterial species including the Actinomycetales R. opacus PD630, R. jostii RHA1, Corynebacterium glutamicum 13032, and the Gram-negative b-proteobacterium Ralstonia eutropha H16. Four chemical categories of compounds including carboxylic acids, nitrogen containing, carbohydrates and alcohols, and oligosac- charides were tested in these bacterial time course growth assays. Compounds capable of supporting growth yielded growth values that were clustered hierarchically to show catabolic-relationships between growth substrates and the species being compared. Carbohydrates and alcohols are an important class of compounds to evaluate for converting natural organic streams such as cellulose and cellulose derived sugars, sugarcane, and beet sugars into biofuels. Seventeen oligosaccharides were screened for growth resulting in the identification of 13, 8, 3, and 0 growth substrates for R. opacus PD630, R. jostii RHA1, C. glutamicum 13032, and R. eutropha H16 respectively (Figure 5a). R. eutropha H16 showed no ability to degrade the oligosaccharides tested and relatively few monosaccharides were catabolized indicating that this species has many pathways to catabolize organic streams such as the rich pool of carboxylic acids defined below or fix CO2 by using the energy derived from splitting H2 [47,48,49]. Corynebacterium and Rhodococcus catabolized the disaccharides sucrose and maltose as well as the trisaccharide maltotriose. Rhodococcus species grew poorly on disaccaharide maltose but well on the trisaccharide maltotriose suggesting a possible membrane- transport preference. R. opacus PD630 gene OPAG_05551 is a glycogen hydrolase that contains the predicted activity to account PLoS Genetics | www.plosgenetics.org 8 September 2011 | Volume 7 | Issue 9 | e1002219