title: Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors.
abstract: BACKGROUND
  Lignocellulosic biomass has been investigated as a renewable non-food source for production of biofuels. A significant technical challenge to using lignocellulose is the presence of microbial growth inhibitors generated during pretreatment processes. Triacylglycerols (TAGs) are potential precursors for lipid-based biofuel production. Rhodococcus opacus MITXM-61 is an oleaginous bacterium capable of producing large amounts of TAGs on high concentrations of glucose and xylose present in lignocellulosic hydrolysates. However, this strain is sensitive to ligonocellulose-derived inhibitors. To understand the toxic effects of the inhibitors in lignocellulosic hydrolysates, strain MITXM-61 was examined for tolerance toward the potential inhibitors and was subjected to adaptive evolution for the resistance to the inhibitors.
  
  
  RESULTS
  We investigated growth-inhibitory effects by potential lignocellulose-derived inhibitors of phenols (lignin, vanillin, 4-hydroxybenzaldehyde (4-HB), syringaldehyde), furans (furfural and 5-hydroxymethyl-2-furaldehyde), and organic acids (levulinic acid, formic acid, and acetic acid) on the growth and TAG production of strain MITXM-61. Phenols and furans exhibited potent inhibitory effects at a concentration of 1 g L(-1), while organic acids had insignificant impacts at concentrations of up to 2 g L(-1). In an attempt to improve the inhibitor tolerance of strain MITXM-61, we evaluated the adaptation of this strain to the potential inhibitors. Adapted mutants were generated on defined agar media containing lignin, 4-HB, and syringaldehyde. Strain MITXM-61(SHL33) with improved multiple resistance of lignin, 4-HB, and syringaldehyde was constructed through adaptive evolution-based strategies. The evolved strain exhibited a two- to threefold increase in resistance to lignin, 4-HB, and syringaldehyde at 50% growth-inhibitory concentrations, compared to the parental strain. When grown in genuine lignocellulosic hydrolysates of corn stover, wheat straw, and hardwood containing growth inhibitors, strain MITXM-61(SHL33) exhibited a markedly shortened lag phase in comparison with that of strain MITXM-61.
  
  
  CONCLUSIONS
  This study provides important clues to overcome the negative effects of inhibitors in lignocellulosic hydrolysates on TAG production of R. opacus cells. The findings can contribute to significant progress in detoxified pretreatment of hydrolysates and development of more efficient strains for industrial TAG fermentations of R. opacus using lignocellulosic biomass.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kurosawa
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA ; Present address: Institute of Biotechnology, Department Applied and Molecular Microbiology Berlin University of Technology, Gustav-Meyer-Allee 25, Berlin, D-13355 Germany.
authors.forename: Josephine
authors.initials: J
authors.lastname: Laser
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA ; Engineering Systems Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Sinskey
keywords: Adaptation; Lignocellulose-derived inhibitor; Lignocellulosic fuel; Lipid-based biofuel; Rhodococcus opacus; Tolerance; Triacylglycerol