Elsevier

Bioresource Technology

Volume 100, Issue 20, October 2009, Pages 4843-4847
Bioresource Technology

Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides

https://doi.org/10.1016/j.biortech.2009.04.041Get rights and content

Abstract

Lignocellulosic biomass hydrolysis inevitably coproduces byproducts that may have various affects on downstream biotransformation. It is imperative to document the inhibitor tolerance ability of microbial strain in order to utilize biomass hydrolysate more effectively. To achieve better lipid production by Rhodosporidium toruloides Y4, we performed fermentation experiments in the presence of some representative inhibitors. We found that acetate, 5-hydroxymethylfurfural and syringaldehyde had slightly inhibitory effects; p-hydroxybenzaldehyde and vanillin were toxic at a concentration over 10 mM; and furfural and its derivatives furfuryl alcohol and furoic acid inhibited cell growth by 45% at around 1 mM. We further demonstrated that inhibition is generally additive, although strong synergistic inhibitions were also observed. Finally, lipid production afforded good results in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. Fatty acid compositional profile of lipid samples indicated that those inhibitors had little effects on lipid biosynthesis. Our work will be useful for optimization of biomass hydrolysis processes and lipid production using lignocellulosic materials.

Introduction

Lignocellulosic biomass, mainly composed of cellulose, hemicellulose and lignin, is the most abundant and renewable organic compound in the biosphere. To utilize biomass with a biochemical route more effectively, complete hydrolysis of lignocellulose is a prerequisite, because most microorganisms have much better bioconversion rate with monomeric carbohydrates. Unfortunately, the release of monosaccharides during hydrolysis is routinely accompanied by the generation of non-carbohydrate compounds, such as furfural and 5-hydroxymethylfurfural (HMF) from the dehydration of pentoses and hexoses, acetic acid from the acetyl group in hemicellulose, and phenolic compounds including syringaldehyde, p-hydroxybenzaldehyde, vanillin, etc. derived from lignin (Almeida et al., 2007, Palmqvist and Hahn-Hagerdal, 2000a, Palmqvist and Hahn-Hagerdal, 2000b). The distribution of these inhibitors in a given hydrolysate sample is depending on both the raw material and the operational conditions employed for hydrolysis (Marzialetti et al., 2008, Schirmer-Michel et al., 2008). These byproducts had various affects on microbial cell growth, metabolism, as well as on product titer, presenting a major challenge in biological conversion of biomass (Almeida et al., 2007). Previous studies investigated the toxic effects of aldehyde, alcohol and acid components from hemicellulose hydrolysate for ethanologenic Escherichia coli, and demonstrated that the toxicity of hydrolysate resulted from the aggregate effects of different compounds rather than a single agent (Zaldivar and Ingram, 1999, Zaldivar et al., 1999). Studies on yeasts with different inhibitors also reached similar conclusions (Palmqvist et al., 1999a, Palmqvist et al., 1999b). To deal with these problems, one possibility is to develop detoxification methods, such as further processing with active charcoal, overliming, ion-exchange resins, or inhibitor-degrading microorganisms (Nichols et al., 2005). On the other hand, engineering superior strains with global stress resistance using either traditional approach or rational design is also extensively pursued recently (Almeida et al., 2007, Larsson et al., 2001). Yet, it is equally important to identify strains or processes with excellent inhibitor tolerance.

Microorganisms that can accumulate lipids to more than 20% of their biomass are defined as oleaginous species (Ratledge and Wynn, 2002). Some yeast strains, such as Cryptococcus sp., Lipomyces sp., Rhodosporidium sp. and Rhodotorula sp. can accumulate intracellular lipids as high as 60% of its cell dry weight when using glucose as the carbon source (Li et al., 2007). Constitutive fatty acids of those lipids are mainly long chain ones that are quite similar to those of conventional vegetable oil. Therefore, oleaginous microorganisms have recently been suggested as alternative lipid producer to fuel a more sustainable biodiesel industry (Zhao et al., 2005). Making lipids through microorganisms is potentially a new technology of arable land-independent, continuous and controllable. However, the carbon sources for oleaginous microbes need extend to lignocellulosic biomass and related raw materials so that large volume of microbial lipids can be secured.

Our previous work demonstrated that oleaginous yeast Rhodosporidium toruloides Y4 was a powerful lipid producer capable of accumulating lipid over 70% and with a titer over 100 g/L (Li et al., 2007). In this study we took some representative byproducts found in biomass hydrolysate to check their effects on the lipid production. We demonstrated that R. toruloides Y4 was a robust strain for lipid production using biomass hydrolysates.

Section snippets

Yeast strain, media and chemicals

Oleaginous yeast R. toruloides Y4 was a domesticated strain of R. toruloides AS 2.1389 obtained from the China General Microbiological Culture Collection Center. Inoculum was grown in YPD liquid medium contained (g/L): glucose 20, yeast extract 10, and peptone 10, pH 6.0). YPD agar slants were made with YPD liquid medium supplemented with 20 g/L agar. The fermentation media contained (g/L): glucose 54, (NH4)2SO4 0.215, yeast extract 1.0, KH2PO4 0.4, and MgSO4·7H2O 1.5, pH 6.0. This medium had a

Results and discussion

To explore lipid production using inexpensive carbon sources, we conducted flask culture experiments by R. toruloides Y4 in the presence of some representative inhibitors found in biomass acid hydrolysate.

Conclusion

Release of monomeric carbohydrates from lignocellulose biomass routinely accompanied by the formation of various non-sugar compounds. Tolerance of these compounds is one of the most imperative characteristics for a successful biochemical process in order to utilize biomass. We demonstrated that R. toruloides Y4 had considerable ability to accumulate lipid in the presence of those representative inhibitors. We are now working on evolutionary engineering to further improve the stress resistance

Acknowledgements

Financial supports provided by the National High Technology Research and Development Program of China (2007AA05Z403) and the Knowledge Innovation Program of CAS (KGCX2-YW-336) are greatly acknowledged.

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