Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors.

Kazuhiko Kurosawa,Anthony J Sinskey,Josephine Laser

doi:10.1186/s13068-015-0258-3

Abstract

BackgroundLignocellulosic 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.ResultsWe 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-61SHL33 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-61SHL33 exhibited a markedly shortened lag phase in comparison with that of strain MITXM-61.ConclusionThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0258-3) contains supplementary material, which is available to authorized users.

Highlights

Lignocellulosic biomass has been investigated as a renewable non-food source for production of biofuels
Effects of individual lignocellulose-derived inhibitors on the growth and TAG production of R. opacus MITXM-61 In order to validate the elemental effects of lignocellulosederived inhibitors on the cell growth and TAG production of R. opacus, nine representative inhibitors, commonly found in lignocellulosic hydrolysates, were included in a defined medium at various concentrations
Strain MITXM61 was cultivated in the media containing 16 g L−1 glucose supplemented with lignin (0 to 2.0 g L−1), furfural (0 to 2.0 g L−1), HMF (0 to 2.0 g L−1), vanillin (0 to 1.0 g L−1), 4hydroxybenzaldehyde (4-HB) (0 to 0.5 g L−1), syringaldehyde (0 to 0.5 g L−1), levulinic acid (0 to 10 g L−1), formic acid (0 to 5.0 g L−1), or acetic acid (0 to 5.0 g L−1) as individual inhibitors

Summary

Introduction

Lignocellulosic biomass has been investigated as a renewable non-food source for production of biofuels. 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. This strain is sensitive to ligonocellulose-derived inhibitors. During the conventional pretreatment and hydrolysis of lignocellulose with dilute acid, the liberation of the monomeric sugars is accompanied by the generation of by-products [13,14] These by-products, and some materials present in the lignocellulose, are inhibitory to microbial metabolism, causing low yields and productivities in the fermentation processes [15,16,17]. Some studies suggest that the use of adaptive evolution to generate inhibitor-tolerant strains is a more effective method, as compared to the genetic engineering approach [50,51]

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