We present the bioconversion of second-generation cellulosic ethanol waste streams into biodiesel via oleaginous bacteria is a novel optimization strategy for biorefineries with substantial potential for rapid development. In this study, one- and two-stage alkali/alkali-peroxide pretreatment waste streams of corn stover were separately implemented as feedstocks in 96 h batch reactor fermentations with wild-type Rhodococcus opacus PD 630, R. opacus DSM 1069, and R. jostii DSM 44719<
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P-NMR, HPAECPAD, and SEC analyses, that the more rigorous and chemically-efficient two-stage chemical pretreatment effluent provided higher concentrations of solubilized glucose and lower molecular weight (70 300 g mol1 ) lignin degradation products thereby enabling improved cellular density, viability, and oleaginicity in each respective strain. The most significant yields were by R. opacus PD 630, which converted 6.2% of organic content with a maximal total lipid production of 1.3 g L1 and accumulated 42.1% in oils based on cell dry weight after 48 h.