5-hydroxymethylfurfural and vanillin

For 2G ethanol production, pentose fermentation and yeast tolerance to lignocellulosic hydrolyzate components are essential to improve biorefinery yields. Generally, physicochemical pre-treatment methodologies are used to facilitate access to cellulose and hemicellulose in plant material, which consequently can generate microbial growth inhibitory compounds, such as furans, weak acids, and phenolic compounds. Because of the unsatisfactory yield of wild-type Saccharomyces cerevisiae during pentose fermentation, the search for xylose-fermenting yeasts tolerant to microbial growth inhibitors has gained attention. In this study, we investigated the ability of the yeasts Pichia guilliermondii G1.2 and Candida oleophila G10.1 to produce ethanol from xylose and tolerate the inhibitors furfural, 5-hydroxymethylfurfural (HMF), acetic acid, formic acid, ferulic acid, and vanillin. We demonstrated that both yeasts were able to grow and consume xylose in the presence of all single inhibitors, with greater growth limitation in media containing furfural, acetic acid, and vanillin. In saline medium containing a mixture of these inhibitors (2.5-3.5 mM furfural and HMF, 1 mM ferulic acid, 1-1.5 mM vanillin, 10-13 mM acetic acid, and 5-7 mM formic acid), both yeasts were able to produce ethanol from xylose, similar to that detected in the control medium (without inhibitors). In future studies, the proteins involved in the transport of pentose and tolerance to these inhibitors need to be investigated.

Topics: Biomass; Ethanol; Fermentation; Formates; Furaldehyde; Furans; Pentoses; Phenols; Pichia; Saccharomyces cerevisiae; Xylose

During the bioconversion of xylose to ethanol, Pichia stipitis cells are often inhibited by substances generated in the lignocellulosic hydrolysate. However, the response mechanism of P. stipitis to inhibitors has not been completely understood till date. With this aim, integrated transcriptomic and metabolomic analyses were performed on P. stipitis to investigate the interactive effects of three representative inhibitors [vanillin, 5-hydroxymethylfurfural (5-HMF), and acetic acid] present in lignocellulosic hydrolysates. The genes involved in carbohydrate metabolism were observed to significantly down-regulated in the presence of the three combined inhibitors in both lag and middle exponential phases. In addition, inhibitor addition induced amino acid metabolism (e.g., glutamine and asparagine syntheses), since the yeast cells required more amino acids in stressful conditions. The metabolomic analysis yielded similar results, particularly those related with the analysis of metabolic biomarkers including fatty acids, amino acids, and sugars. 70 intracellular metabolites were detected by gas chromatography coupled with mass spectrometry (GC-MS), and samples from different phases were clearly separated by principal component analysis (PCA). The large amount of specific responsive genes and metabolites highlighted the complex regulatory mechanisms involved in the fermentation process in the presence of the three combined inhibitors.

Topics: Acetic Acid; Amino Acids; Benzaldehydes; Ethanol; Fermentation; Furaldehyde; Lignin; Metabolome; Pichia; Principal Component Analysis; Transcriptome; Xylose

To obtain a global view of the dynamic phospholipids in Pichia stipitis during the ethanol fermentation in the presence of three representative inhibitors (vanillin, 5-hydroxymethylfurfural, and acetic acid). Considerable efforts have been expended to elucidate the biochemical mechanisms of inhibitors interaction with phospholipids. In this study, a comparative lipidomic analysis was performed using liquid chromatography-mass/mass spectrometry (LC-MS/MS) on P. stipitis. Partial least squares-discriminate analysis (PLS-DA) was used to deal with the large quantity of data generated using the systematic methods. PLS-DA revealed that phosphatidylinositol (PI) (PI34:1, PI34:2 and PI34:6), phosphatidylserine (PS) (PS34:1 and PS34:2), phosphatidylethanolamine (PE) (PE34:1 and PE34:2), and phosphatidylcholine (PC) (PC34:1, PC34:2, and PC34:3) were the predominant biomarkers. Further analysis of different classes of phospholipids indicated that: (a) the samples from three combined inhibitors condition during the lag phase possessed the lowest PI/PS value 1.4%, (b) alterations in PC/PE ratios with changes in inhibitors were coincident with the changes in xylose utilization rates, and (c) the levels of unsaturated and the relatively long chain phospholipids increased in the inhibitor-plus condition. These findings suggest that regulation of membrane properties with inhibitors might offer a way of self-protection of yeast to inhibitors stress.

Topics: Acetic Acid; Benzaldehydes; Chromatography, Liquid; Furaldehyde; Mass Spectrometry; Phospholipids; Pichia

Vanillin, furfural and 5-hydroxymethylfurfural (HMF) are representative fermentation inhibitors generated during the pretreatment process of lignocellulosic biomass in bioethanol production. These biomass conversion inhibitors, particularly vanillin, are known to repress translation activity in Saccharomyces cerevisiae. We have reported that the mRNAs of ADH7 and BDH2 were efficiently translated under severe vanillin stress despite marked repression of overall protein synthesis. In this study, we found that expression of VFH1 (YLL056C) was also significantly induced at the protein level by severe vanillin stress. Additionally, we demonstrated that the VFH1 promoter enabled the protein synthesis of other genes including GFP and ALD6 under severe vanillin stress. It is known that transcriptional activation of VFH1 is induced by furfural and HMF, and we verified that Vfh1 protein synthesis was also induced by furfural and HMF. The null mutant of VFH1 delayed growth in the presence of vanillin, furfural and HMF, indicating the importance of Vfh1 for sufficient tolerance against these inhibitors. The protein levels of Vfh1 induced by the inhibitors tested were markedly higher than those of Adh7 and Bdh2, suggesting the superior utility of the VFH1 promoter over the ADH7 or BDH2 promoter for breeding optimized yeast strains for bioethanol production from lignocellulosic biomass.

Topics: Benzaldehydes; Biomass; Furaldehyde; Gene Expression Regulation, Fungal; Gene Knockout Techniques; Lignin; Promoter Regions, Genetic; Protein Biosynthesis; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Stress, Physiological; Transcriptional Activation

To obtain a global insight into the dynamic protein expression pattern in Pichia stipitis during xylose fermentation in the presence of three representative inhibitors (acetic acid, vanillin and 5-hydroxymethylfurfural), proteins were extracted for quantitative proteomic analysis using 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) on a liquid chromatography-mass/mass spectrometry instrument. Interestingly, aconitase (Aco1p) and NAD-isocitrate dehydrogenase (Idh1p) were upregulated during the middle exponential phase in the presence of the three inhibitors during tricarboxylic acid cycle. We speculated that yeast cells adaptively increased the expression of the tricarboxylic acid cycle proteins to compensate for low NADH derived from glycolysis in the presence of the three inhibitors. Proteins related to amino acid metabolism, aminoacyl tRNA synthesis and stress response were also significantly affected in the presence of the three inhibitors. Taken together, quantitative proteomic analysis is capable of monitoring P. stipitis xylose fermentation under inhibitor conditions and identifying physiological changes, such as stress response.

Topics: Acetic Acid; Benzaldehydes; Chromatography, Liquid; Fermentation; Furaldehyde; Gene Expression Regulation, Fungal; Lignin; Pichia; Proteomics; Tandem Mass Spectrometry; Xylose

Lignocellulosic biomass conversion inhibitors such as vanillin, furfural, and 5-hydroxymethylfurfural (HMF) inhibit the growth of and fermentation by Saccharomyces cerevisiae. A high concentration of each fermentation inhibitor represses translation and increases non-translated mRNAs. We previously reported that the mRNAs of ADH7 and BDH2, which encode putative NADPH- and NADH-dependent alcohol dehydrogenases, respectively, were efficiently translated even with translation repression in response to severe vanillin stress. However, the combined effects of these fermentation inhibitors on the expression of ADH7 and BDH2 remain unclear. We herein demonstrated that exposure to a combined stress of vanillin, furfural, and HMF repressed translation. The protein synthesis of Adh7, but not Bdh2 was significantly induced under combined stress conditions, even though the mRNA levels of ADH7 and BDH2 were up-regulated. Additionally, adh7Δ cells were more sensitive to the combined stress than wild-type and bdh2Δ cells. These results suggest that induction of the ADH7 expression plays a role in the tolerance to the combined stress of vanillin, furfural, and HMF. Furthermore, we succeeded in improving yeast tolerance to the combined stress by controlling the expression of ALD6 with the ADH7 promoter. Our results demonstrate that the ADH7 promoter can overcome the pronounced translation repression caused by the combined stress of vanillin, furfural, and HMF, and also suggest a new gene engineering strategy to breed robust and optimized yeasts for bioethanol production from a lignocellulosic biomass.

Topics: Alcohol Dehydrogenase; Aldehyde Oxidoreductases; Benzaldehydes; Furaldehyde; Gene Expression Regulation, Fungal; Promoter Regions, Genetic; Protein Biosynthesis; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Conversion of lignocellulosic hydrolysate to biofuels is impeded by the toxic effects of inhibitors that are generated during pretreatment and hydrolysis processes. Here we describe a wild-type Clostridium sp. strain BOH3 with high tolerance to the lignocellulose-derived inhibitors and its capability to transform these inhibitors. Strain BOH3 is capable of tolerating over 60 mM furfural, 60 mM hydroxymethylfurfural, and 6.6 mM vanillin, respectively, and is able to convert 53.74 ± 0.37 mM furfural into furfuryl alcohol within 90 h. The high furfural tolerance and its biotransformation by strain BOH3, which is correlated to the high transcription levels of two short-chain dehydrogenase/reductases, enable strain BOH3 to produce 5.15 ± 0.52 g/L butanol from dilute sulfuric acid pretreated horticultural waste hydrolysate (HWH) that bypassed the detoxification step. The capability of strain BOH3 to produce butanol from un-detoxified HWH lays the foundation of cost-effective biofuel production from lignocellulosic materials.

Topics: Benzaldehydes; Biofuels; Biotransformation; Butanols; Clostridium; Fermentation; Furaldehyde; Furans; Hydrolysis; Lignin

An inhibitor-tolerance strain, Bacillus coagulans GKN316, was developed through atmospheric and room temperature plasma (ARTP) mutation and evolution experiment in condensed dilute-acid hydrolysate (CDH) of corn stover. The fermentabilities of other hydrolysates with B. coagulans GKN316 and the parental strain B. coagulans NL01 were assessed. When using condensed acid-catalyzed steam-exploded hydrolysate (CASEH), condensed acid-catalyzed liquid hot water hydrolysate (CALH) and condensed acid-catalyzed sulfite hydrolysate (CASH) as substrates, the concentration of lactic acid reached 45.39, 16.83, and 18.71 g/L by B. coagulans GKN316, respectively. But for B. coagulans NL01, only CASEH could be directly fermented to produce 15.47 g/L lactic acid. The individual inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), vanillin, syringaldehyde and p-hydroxybenzaldehyde (pHBal) on xylose utilization by B. coagulans GKN316 was also studied. The strain B. coagulans GKN316 could effectively convert these toxic inhibitors to the less toxic corresponding alcohols in situ. These results suggested that B. coagulans GKN316 was well suited to production of lactic acid from undetoxified lignocellulosic hydrolysates.

Topics: Bacillus; Benzaldehydes; Biomass; Bioreactors; Fermentation; Furaldehyde; Glucose; Hydrolysis; Industrial Microbiology; Lactic Acid; Lignin; Mutation; Temperature; Water; Xylose; Zea mays

Z. mobilis cell immobilization has been proposed as an effective means of improving ethanol production. In this work, polystyrene and corn silk were used as biofilm developmental matrices for Z. mobilis ethanol production with rice straw hydrolysate as a substrate. Rice straw was hydrolyzed by dilute sulfuric acid (H2SO4) and enzymatic hydrolysis. The final hydrolysate contained furfural (271.95 ± 76.30 ppm), 5-hydroxymethyl furfural (0.07 ± 0.00 ppm), vanillin (1.81 ± 0.00 ppm), syringaldehyde (5.07 ± 0.83 ppm), 4-hydroxybenzaldehyde (4-HB) (2.39 ± 1.20 ppm) and acetic acid (0.26 ± 0.08%). Bacterial attachment or biofilm formation of Z. mobilis strain TISTR 551 on polystyrene and delignified corn silk carrier provided significant ethanol yields. Results showed up to 0.40 ± 0.15 g ethanol produced/g glucose consumed when Z. mobilis was immobilized on a polystyrene carrier and 0.51 ± 0.13 g ethanol produced/g glucose consumed when immobilized on delignified corn silk carrier under batch fermentation by Z. mobilis TISTR 551 biofilm. The higher ethanol yield from immobilized, rather than free living, Z. mobilis could possibly be explained by a higher cell density, better control of anaerobic conditions and higher toxic tolerance of Z. mobilis biofilms over free cells.

Topics: Acetic Acid; Benzaldehydes; Biofilms; Biomass; Cells, Immobilized; Ethanol; Fermentation; Furaldehyde; Glucose; Hydrolysis; Oryza; Polystyrenes; Zea mays; Zymomonas

The inhibitory effects of furan derivatives [i.e. furfural and 5-hydroxymethylfurfural (5-HMF)] and phenolic compounds (i.e. vanillin and syringaldehyde) on dark hydrogen fermentation from glucose were comparatively evaluated. Phenolic compounds exhibited stronger inhibition on hydrogen production and glucose consumption than furan derivatives under the same 15mM concentration. Furan derivatives were completely degraded after 72h fermentation, while over 55% of phenolic compounds remained unconverted after 108h fermentation. The inhibition coefficients of vanillin (14.05) and syringaldehyde (11.21) were higher than those of 5-HMF (4.35) and furfural (0.64). Vanillin exhibited the maximum decrease of hydrogen yield (17%). The consumed reducing power by inhibitors reduction from R-CHO to RCH2OH was a possible reason contributed to the decreased hydrogen yield. Vanillin exhibited the maximum delay of peak times of hydrogen production rate and glucose consumption. Soluble metabolites and carbon conversion efficiency decreased with inhibitors addition, which were consistent with hydrogen production.

Topics: Benzaldehydes; Biofuels; Fermentation; Furaldehyde; Furans; Glucose; Hydrogen; Phenols

As one of the major microbes in the soy sauce fermentation, Candida versatilis enriches the flavor and improves the quality of soy sauce. In this study, a combination of five different GC-MS and LC-MS-based metabolome analytical approaches was used to analyze the intracellular, extracellular and whole metabolites of C. versatilis. Our results found out that a total of 132, 244 and 267 different metabolites were detectable from the intracellular, extracellular and whole part, respectively. When exposed to 0. 9 and 18 % salt, respectively, 114, 123 and 129 different intracellular metabolites, 184, 200 and 178 extracellular metabolites and 177, 188 and 186 whole metabolites were detected, respectively. Our data showed that salt enhances the metabolic capacity of C. versatilis, especially its amino acid and enhances the synthesis and secretion of some metabolites of C. versatilis, especially the aldehydes and phenols, such as vanillin, guaiacol and 5-hydroxymethylfurfural. Our data also showed that special attention has to be paid to the generation of biogenic amines when C. versatilis was treated with salt.

Topics: Benzaldehydes; Biogenic Amines; Candida; Furaldehyde; Guaiacol; Metabolome; Principal Component Analysis; Salt Tolerance; Sodium Chloride; Stress, Physiological

The influence of choice of flavour solvent, propylene glycol (PG) or triacetin (TA), was investigated during accelerated shelf life (ASL) testing of shortcake biscuits. Specifically, the differential effect on the stability of added vanillin, the natural baked marker compound 5-(hydroxymethyl)furfural (HMF), specific markers of oxidative rancidity (2,4-decadienal, 2,4-heptadienal), and the structural parameters of hardness and fracturability. Significantly more HMF was formed during baking of biscuits prepared with TA; these biscuits were also more stable to oxidative degradation and loss of vanillin during ageing than biscuits prepared with PG. Fresh TA biscuits were significantly more brittle than fresh PG biscuits. There was no impact of solvent choice on hardness. Sensory evaluation of hardness, vanilla flavour and oily off-note was tested during ASL testing. There was no significant impact of storage on sensory ratings for either the PG or TA biscuits.

Topics: Adult; Benzaldehydes; Cooking; Female; Flavoring Agents; Food Analysis; Food Storage; Furaldehyde; Humans; Male; Oxidation-Reduction; Propylene Glycol; Taste Perception; Triacetin; Young Adult

Zymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions.. We generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions.. We have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.

Topics: Acetates; Anti-Bacterial Agents; Bacterial Proteins; Benzaldehydes; Furaldehyde; Gene Deletion; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Lignin; Mutagenesis, Insertional; RNA-Binding Proteins; Saccharomyces cerevisiae; Stress, Physiological; Zymomonas

High cost of triacylglycerol lipid feedstock is the major barrier for commercial production of biodiesel. The fermentation of oleaginous yeasts for lipid production using lignocellulose biomass provides a practical option with high economic competitiveness. In this paper, the typical oleaginous yeast strains were screened under the pressure of lignocellulose degradation compounds for selection of the optimal strains tolerant to lignocellulose. The inhibitory effect of lignocellulose degradation products on the oleaginous yeast fermentation was carefully investigated. Preliminary screening was carried out in the minimum nutritious medium without adding any expensive complex ingredients then was carried out in the lignocellulosic hydrolysate pretreated by dilute sulfuric acid. Seven typical lignocellulose degradation products formed in various pretreatment and hydrolysis processing were selected as the model inhibitors, including three organic acids, two furan compounds, and two phenol derivatives. The inhibition of the degradation compounds on the cell growth and lipid productivity of the selected oleaginous yeasts were examined. Acetic acid, formic acid, furfural, and vanillin were found to be the strong inhibitors for the fermentation of oleaginous yeasts, while levulinic acid, 5-hydroxymethylfurfural, and hydroxybenzaldehyde were relatively weak inhibitors. Trichosporon cutaneum 2.1374 was found to be the most adopted strain to the lignocellulose degradation compounds.

Topics: Acetic Acid; Benzaldehydes; Biofuels; Fermentation; Formates; Furaldehyde; Levulinic Acids; Lignin; Rhodotorula; Trichosporon

To obtain in-depth information on the overall metabolic behavior of the new good xylitol producer Debaryomyces hansenii UFV-170, batch bioconversions were carried out using semisynthetic media with compositions simulating those of typical acidic hemicellulose hydrolysates of sugarcane bagasse. For this purpose, we used media containing glucose (4.3-6.5 g/L), xylose (60.1-92.1 g/L), or arabinose (5.9-9.2 g/L), or binary or ternary mixtures of them in either the presence or absence of typical inhibitors of acidic hydrolysates, such as furfural (1.0-5.0 g/L), hydroxymethylfurfural (0.01- 0.30 g/L), acetic acid (0.5-3.0 g/L), and vanillin (0.5-3.0 g/L). D. hansenii exhibited a good tolerance to high sugar concentrations as well as to the presence of inhibiting compounds in the fermentation media. It was able to produce xylitol only from xylose, arabitol from arabinose, and no glucitol from glucose. Arabinose metabolization was incomplete, while ethanol was mainly produced from glucose and, to a lesser less extent, from xylose and arabinose. The results suggest potential application of this strain in xyloseto- xylitol bioconversion from complex xylose media from lignocellulosic materials.

Topics: Acetic Acid; Arabinose; Ascomycota; Benzaldehydes; Fermentation; Furaldehyde; Glucose; Xylitol; Xylose

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.

Topics: Acetic Acid; Benzaldehydes; Biomass; Cellulose; Ethanol; Fermentation; Furaldehyde; Galactose; Hydrolysis; Lignin; Mannose; Mucor; Mycology; Time Factors; Xylitol; Xylose

Bioethanol production from lignocellulosic raw-materials requires the hydrolysis of carbohydrate polymers into a fermentable syrup. During the hydrolysis of hemicellulose with dilute acid, a variety of toxic compounds are produced such as soluble aromatic aldehydes from lignin and furfural from pentose destruction. In this study, we have investigated the toxicity of representative aldehydes (furfural, 5-hydroxymethlyfurfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) as inhibitors of growth and ethanol production by ethanologenic derivatives of Escherichia coli B (strains KO11 and LY01). Aromatic aldehydes were at least twice as toxic as furfural or 5-hydroxymethylfurfural on a weight basis. The toxicities of all aldehydes (and ethanol) except furfural were additive when tested in binary combinations. In all cases, combinations with furfural were unexpectedly toxic. Although the potency of these aldehydes was directly related to hydrophobicity indicating a hydrophobic site of action, none caused sufficient membrane damage to allow the leakage of intracellular magnesium even when present at sixfold the concentrations required for growth inhibition. Of the aldehydes tested, only furfural strongly inhibited ethanol production in vitro. A comparison with published results for other microorganisms indicates that LY01 is equivalent or more resistant than other biocatalysts to the aldehydes examined in this study.

Topics: Aldehydes; Benzaldehydes; Bioreactors; Cell Division; Colony Count, Microbial; Drug Synergism; Escherichia coli; Ethanol; Fermentation; Furaldehyde; Hydrogen-Ion Concentration; Temperature; Xylose