BackgroundEconomical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts.ResultsIn this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to have superior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel biocompatible ionic liquid (IL) choline α-ketoglutarate, which produced 261 mg/L of bisabolene at bench scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high-gravity fed-batch bioreactor. Interestingly, R. toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars.ConclusionsThis study highlights the natural compatibility of R. toruloides with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0927-5) contains supplementary material, which is available to authorized users.

Differences in carbon assimilation pathways and reducing power requirements among organisms are likely to affect the role of the storage polymer poly-3-hydroxybutyrate (PHB). Previous researchers have demonstrated that PHB functions as a sole growth substrate in aerobic cultures enriched on acetate during periods of carbon deficiency, but it is uncertain how C 1 metabolism affects the role of PHB. In the present study, the type II methanotroph Methylocystis parvus OBBP did not replicate using stored PHB in the absence of methane, even when all other nutrients were provided in excess. When PHB-rich cultures of M. parvus OBBP were deprived of carbon and nitrogen for 48 h, they did not utilize significant amounts of stored PHB, and neither cell concentrations nor concentrations of total suspended solids changed significantly. When methane and nitrogen both were present, PHB and methane were consumed simultaneously. Cells with PHB had significantly higher specific growth rates than cells lacking PHB. The addition of formate (a source of reducing power) to PHB-rich cells delayed PHB consumption, but the addition of glyoxylate (a source of C 2 units) did not. This and results from other researchers suggest that methanotrophic PHB metabolism is linked to the supply of reducing power as opposed to the supply of C 2 units for synthesis.Poly-3-hydroxybutyrate (PHB) is a biologically produced, biodegradable polyester with properties similar to those of polypropylene and mechanical properties that can be tailored for different applications by changing the copolymer content of the polymer (1, 5, 27). Many bacteria accumulate PHB as a carbon storage polymer under conditions of unbalanced growth (i.e., nutrient deficiency and/or carbon excess) (1, 27). Although considerable efforts are currently being devoted to commercializing microbial PHB production, commercialization thus far has been limited because of the relatively high production cost of PHB compared to that of traditional petrochemical-based plastics, such as polyethylene and polypropylene (28). A major fraction of the production cost (30%) (7) is due to feedstocks, which are typically sugars such as glucose and sucrose (27).Methane is both an inexpensive feedstock for PHB production and a potent greenhouse gas; its use as a substrate for PHB production is therefore an effective means of carbon sequestration and an attractive alternative to sugar-based feedstocks. Methanotrophs have been shown to produce PHB (2, 18, 37, 41-44), and Wendlandt et al. (42) have reported PHB levels of up to 30 g liter Ϫ1 in a culture dominated by Methylocystis sp. GB 25 and maintained under nonaseptic conditions. While PHB production is well characterized in a variety of bacteria, comparatively few studies have investigated why bacteria accumulate PHB or the process of PHB consumption (16,20,33,36,41). In nonmethanotrophs, PHB consumption is linked to both short-term replication and long-term survival under carbon starvation. Handrick et al. (16) concluded that Ralstonia eutropha replicat...

Achieving low cost and high efficiency lignocellulose deconstruction is a critical step towards widespread adoption of lignocellulosic biofuels. Certain ionic liquid (IL)-based pretreatment processes effectively reduce recalcitrance of lignocellulose to enzymatic degradation but require either costly separations following pretreatment or novel IL compatible processes to mitigate downstream toxicity. Here we demonstrate at benchtop and pilot bioreactor scales a separation-free, intensified process for IL pretreatment, saccharification, and fermentation of sorghum biomass to produce the sesquiterpene bisabolene, a precursor to the renewable diesel and jet fuel bisabolane. The deconstruction process employs the IL cholinium lysinate ([Ch][Lys]), followed by enzymatic saccharification with the commercial enzyme cocktails Cellic CTec2 and HTec2. Glucose yields above 80% and xylose yields above 60% are observed at all scales tested. Unfiltered hydrolysate is fermented directly by Rhodosporidium toruloides -with glucose, xylose, acetate and lactate fully consumed during fermentation at all scales tested. Bisabolene titers improved with scale from 1.3 g L −1 in 30 mL shake flasks to 2.2 g L −1 in 20 L fermentation. The combined process enables conversion of saccharified IL-pretreated biomass directly to advanced biofuels with no separations or washing, minimal additions to facilitate fermentation, no loss of performance due to IL toxicity, and simplified fuel recovery via phase separation. This study is the first to demonstrate a separation-free IL based process for conversion of biomass to an advanced biofuel and is the first to demonstrate full consumption of glucose, xylose, acetate, and lactic acid in the presence of [Ch][Lys].