Abstract
BackgroundChemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.ResultsThermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.ConclusionsT. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0165-z) contains supplementary material, which is available to authorized users.
Highlights
Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, and restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF)
T. pseudethanolicus 39E furan aldehyde tolerance We initially investigated the growth tolerance of T. pseudethanolicus 39E to the furan aldehydes furfural and 5-hydroxymethylfurfural (5-HMF)
Higher growth rates and increased cell yield from the addition of subinhibitory concentrations of furfural and 5-HMF suggest that 39E metabolism is constrained by electron flow, which is relieved by the furan aldehydes serving as an alternative dissimilatory electron acceptor
Summary
Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, and restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may enable improvements in candidate CBP microorganisms Thermophilic bacteria, such as Clostridium thermocellum and Caldicellulosiruptor species have gained interest for their possible use as biocatalysts for converting lignocellulosic biomass into renewable fuels and chemicals [1,2,3,4]. Several bacterial phyla include thermophiles that are able to utilize plant cell walls directly through the action of complex (hemi)cellulase systems expressed either as free enzymes, cellulosomes, or multifunctional enzymes [5] Relying on these native enzymes in a bioprocessing scheme could substantially reduce or even eliminate the need for exogenous enzymes for cellulose solubilization with a resulting improvement in process economics [3,6,7]. Furfural is estimated to be responsible for 33% of the toxic effect of sugar cane hydrolysate on E. coli LYO1 [14]
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