Lipid production from non-sugar compounds in pretreated lignocellulose hydrolysates by Rhodococcus jostii RHA1
Introduction
The current vision for biofuels mainly relies on microbial conversion of biomass-derived sugars to advanced biofuels and bioproducts. However, for any renewable product made using biomass, efficient utilization of most available carbons from biomass is required for an economical process [1,2]. Non-sugar compounds, including sugar degradation products such as furans (e.g. 5-hydroxymethylfurfural, furfural) and organic acids, and lignin degradation compounds such as phenolic compounds released into the hydrolysate during the pretreatment of lignocellulose can have a negative impact on biological processes [[3], [4], [5], [6]]. These compounds are usually considered as inhibitors to enzymes and microorganisms for cellulosic ethanol production and carbohydrate-based lipid production. Many previous studies focused on strains’ tolerance and detoxification ability of these inhibitory compounds [[7], [8], [9], [10], [11], [12], [13], [14]]. On the other hand, bioconversion of these non-sugar compounds to fungible fuel products is attractive but challenging. Unlike the classic glucose conversion to ethanol or lipid, relatively few studies have established a route to convert aromatic compounds or other biomass derived by-products to valuable products [15,16].
The oleaginous bacteria Rhodococcus sp. is one of the promising candidates for 2nd generation biofuel production because of its remarkable ability for lipid accumulation, which is up to 76% of cell dry weight (CDW) in Rhodococcus opacus PD630 on gluconate with high C/N ratio [17]. Accumulation of lipid, one of the in vivo storage compounds under nitrogen-limited conditions, is a common feature shared by many Rhodococcus strains [18]. Rhodococcus jostii RHA1 was reported to accumulate triacylglycerol (TAG) up to 55% CDW when fed on benzoate, an analog of lignin depolymerization products, during the transition to stationary phase [19]. Also, the first recombinant bacterial lignin-degrading enzyme DypB, which could depolymerize lignin macromolecules by cleaving Cα–Cβ bonds, was kinetically characterized in R. jostii RHA1 [20]. Indeed, lignin degradation by Rhodococcus was observed by Li et al. using pretreated poplar wood hydrolysate and alkali corn stover lignin as carbon sources for fermentation [1]. Their work suggested benzoate or its analogs as lignin-derived products in pretreated hydrolysis and fermentation broth supported by GC-MS and proteomics results. Furthermore, the variant Δatf8 mutant which was constructed in a previous study showed 20% greater TAG accumulation than the wild strain, giving the possibility of high lipid yield from lignin or lignin-derived aromatics [19].
In addition, R. jostii RHA1 is well known for its robust growth and adaption to various environmental conditions and remarkably versatile ability in catabolizing a wide range of compounds such as steroids [21], nitriles [22], aromatic acids [23], phenols [24], and biphenyl [25]. Its complete genome revealed broad catabolism and biosynthesis systems in R. jostii RHA1 [23,26]. Intensive studies have been conducted on the biofuel production from a number of individual biomass-derived compounds or whole biorefinery residues by Rhodococcus [16,27,28]. However, utilization of mixed non-sugar compounds by the strain is still not well-characterized, including fermentation kinetics and the competition or synergetic effects among multiple catabolism pathways, which are critical to design an integrated upgrading process of lignocellulosic biofuel production.
This study aimed at investigating the kinetics and lipid production capability of R. jostii RHA1 on sole or mixed carbon sources, including vanillin, vanillate, furfural, 5-HMF, and acetate, mixed with benzoate, which mimicked the complex components of the non-sugar compounds in the pretreated biomass hydrolysate.
Section snippets
Materials
LB broth was purchased from Sigma-Aldrich (St. Louis, MO). Sodium benzoate, furfural, vanillic acid, acetic acid, 5-formyl-2-furancarboxylic acid, 2,5-diformylfuran, 5-hydroxymethyl-2-furancarboxylic acid, furan 2,5-dicarboxylic acid, 2-furoic acid were purchased from Fisher Scientific (Pittsburgh, PA). 5-hydroxymethylfurfural (5-HMF) and vanillin were purchased from Acros Organics (Morris Plains, NJ).
Microorganism and fermentation medium
Rhodococcus jostii RHA1 was kindly provided by Dr. Lindsay Eltis (University of British
Growth of R. jostii RHA1 in presence of non-sugar compounds
Growth of R. jostii RHA1 on benzoate in the presence of five non-sugar compounds, including vanillin, vanillate, furfural, 5-HMF, and acetate, was examined. The growth test was first conducted on M9 agar plates containing benzoate in the presence of each tested compounds over a range of concentrations (Fig. 1). The plates were monitored for 6 days. On the control plates with addition of benzoate (up to 0.5 g L−1), colonies started to form 48 h post-inoculation, while the plates containing
Conclusion
The valorization of non-sugar compounds from biorefinery wastes to biofuels and bioproducts is an attractive but challenging strategy to improve the carbon efficiency of the entire biorefinery process for economic competitiveness. In this work, R. jostii RHA1 showed an excellent metabolic and catabolic capacity to degrade various non-sugar compounds, which are common in pretreated biomass hydrolysates, including vanillin, vanillate, furfural, 5-HMF and acetate. Different kinetics, specific
Funding
This work was supported by U.S. Department of Energy DOE EERE Awards (DE-EE0007104 and DE-EE0006112) with the Bioproducts, Science and Engineering Laboratory, Department of Biological Systems Engineering at Washington State University. A portion of the research was performed using EMSL (grid.436923.9), a DOE Office of Science user facility sponsored by the Biological and Environmental Research program. X.L. and Z.X. are grateful for support from the Pacific Northwest National Laboratory
Acknowledgement
The authors would like to thank Ms. Marie S. Swita who helped with collecting some GC-MS data for this project.
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