Abstract
BackgroundPretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. The furan aldehydes hydroxymethylfurfural (HMF) and furfural have received increasing attention recently. In the present study, the effects of HMF and furfural on redox metabolism, energy metabolism and gene expression were investigated in anaerobic chemostats where the inhibitors were added to the feed-medium.ResultsBy cultivating the xylose-utilizing Saccharomyces cerevisiae strain VTT C-10883 in the presence of HMF and furfural, it was found that the intracellular concentrations of the redox co-factors and the catabolic and anabolic reduction charges were significantly lower in the presence of furan aldehydes than in cultivations without inhibitors. The catabolic reduction charge decreased from 0.13(±0.005) to 0.08(±0.002) and the anabolic reduction charge decreased from 0.46(±0.11) to 0.27(±0.02) when HMF and furfural were present. The intracellular ATP concentration was lower when inhibitors were added, but resulted only in a modest decrease in the energy charge from 0.87(±0.002) to 0.85(±0.004) compared to the control. Transcriptome profiling followed by MIPS functional enrichment analysis of up-regulated genes revealed that the functional group “Cell rescue, defense and virulence” was over-represented when inhibitors were present compared to control cultivations. Among these, the ATP-binding efflux pumps PDR5 and YOR1 were identified as important for inhibitor efflux and possibly a reason for the lower intracellular ATP concentration in stressed cells. It was also found that genes involved in pseudohyphal growth were among the most up-regulated when inhibitors were present in the feed-medium suggesting nitrogen starvation. Genes involved in amino acid metabolism, glyoxylate cycle, electron transport and amino acid transport were enriched in the down-regulated gene set in response to HMF and furfural. It was hypothesized that the HMF and furfural-induced NADPH drainage could influence ammonia assimilation and thereby give rise to the nitrogen starvation response in the form of pseudohyphal growth and down-regulation of amino acid synthesis.ConclusionsThe redox metabolism was severely affected by HMF and furfural while the effects on energy metabolism were less evident, suggesting that engineering of the redox system represents a possible strategy to develop more robust strains for bioethanol production.
Highlights
Pretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism
We focused on the quantification of the intracellular concentrations of NAD(P)+ and NAD(P)H, since in spite of the fact that the conversion of HMF and furfural to less inhibitory compounds is thought to proceed through NAD(P)H dependent reactions, there are surprisingly few reports on the actual impact of furan aldehyde stress on the intracellular levels of the redox factors
Since lignocellulosic hydrolysates contain a mixture of the furan aldehydes HMF and furfural, and the proposed mechanism of detoxification is similar for both compounds [2], both were added to the feed-medium of carbon-limited chemostats
Summary
Pretreatment of biomass for lignocellulosic ethanol production generates compounds that can inhibit microbial metabolism. Due to the recalcitrant nature of lignocellulosic materials, harsh conditions have to be used, which result in production of several by-products that can have inhibitory effects on microbial metabolism [1]. Two of these compounds, 5-hydroxymethyl furfural (HMF) and 2furaldehyde (furfural), have been subjects of extensive investigation due to their negative effects on microbial physiology, as reviewed in [2]. More robust ethanol producing microorganisms are needed to cope with such process constraints and there is a need to increase the understanding of the physiological responses to HMF and furfural in order to increase strain robustness
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