Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation

The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C. acetobutylicum in the presence of furfural and HMF were in the range of 15-85% and 23-78%, respectively, of the uninhibited Control, μ increased by 8-15% and 23-38% following exhaustion of furfural and HMF in the bioreactor. Using high performance liquid chromatography and spectrophotometric assays, batch fermentations revealed that furfural and HMF were converted to furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively, with specific conversion rates of 2.13g furfural and 0.50g HMF per g (biomass) per hour, by exponentially growing C. acetobutylicum. Biotransformation of these furans to lesser inhibitory compounds by C. acetobutylicum will probably enhance overall fermentation of lignocellulosic hydrolysates to butanol.     

Catalytic conversion of cellulose to chemicals in ionic liquid

A simple and effective route for the production of 5-hydroxymethyl furfural (HMF) and furfural from microcrystalline cellulose (MCC) has been developed. CoSO₄ in an ionic liquid, 1-(4-sulfonic acid) butyl-3-methylimidazolium hydrogen sulfate (IL-1), was found to be an efficient catalyst for the hydrolysis of cellulose at 150 °C, which led to 84% conversion of MCC after 300 min reaction time. In the presence of a catalytic amount of CoSO₄, the yields of HMF and furfural were up to 24% and 17%, respectively; a small amount of levulinic acid (LA) and reducing sugars (8% and 4%, respectively) were also generated. Dimers of furan compounds were detected as the main by-products through HPLC-MS, and with the help of mass spectrometric analysis, the components of gas products were methane, ethane, CO, CO_2, and H₂. A mechanism for the CoSO₄-IL-1 hydrolysis system was proposed and IL-1 was recycled for the first time, which exhibited favorable catalytic activity over five repeated runs. This catalytic system may be valuable to facilitate energy-efficient and cost-effective conversion of biomass into biofuels and platform chemicals.     

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C₆H₈O₃ and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.     

Expression of aldehyde dehydrogenase 6 reduces inhibitory effect of furan derivatives on cell growth and ethanol production in Saccharomyces cerevisiae

Yeast dehydrogenases and reductases were overexpressed in Saccharomyces cerevisiae D452-2 to detoxify 2-furaldehyde (furfural) and 5-hydroxymethyl furaldehyde (HMF), two potent toxic chemicals present in acid-hydrolyzed cellulosic biomass, and hence improve cell growth and ethanol production. Among those enzymes, aldehyde dehydrogenase 6 (ALD6) played the dual roles of direct oxidation of furan derivatives and supply of NADPH cofactor to their reduction reactions. Batch fermentation of S. cerevisiae D452-2/pH-ALD6 in the presence of 2 g/L furfural and 0.5 g/L HMF resulted in 20-30% increases in specific growth rate, ethanol concentration and ethanol productivity, compared with those of the wild type strain. It was proposed that overexpression of ALD6 could recover the yeast cell metabolism and hence increase ethanol production from lignocellulosic biomass containing furan-derived inhibitors.     

Production of 5-hydroxymethylfurfural in ionic liquids under high fructose concentration conditions

Acid-promoted, selective production of 5-hydroxymethylfurfural (HMF) under high fructose concentration conditions was achieved in ionic liquids (ILs) at 80 °C. A HMF yield up to 97% was obtained in 8 min using 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) catalyzed with 9 mol % hydrochloric acid. More significantly, an HMF yield of 51% was observed when fructose was loaded at a high concentration of 67 wt % in [C₄mim]Cl. Water content below 15.4% in the system had little effect on HMF yield, whereas a higher water content was detrimental to both reaction rate and HMF yield. In situ NMR analysis suggested that the transformation of fructose to HMF was a highly selective reaction that proceeded through the cyclic fructofuranosyl intermediate pathway. This work increased our capacity to produce HMF, and should be valuable to facilitate cost-efficient conversion of biomass into biofuels and bio-based products.     

Catalytic hydrolysis of lignocellulosic biomass into 5-hydroxymethylfurfural in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) from cellulose catalyzed by solid acids and metal chlorides was studied in the 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) under microwave irradiation. Among the applied catalysts, the use of CrCl₃/LiCl resulted in the highest yield of HMF. The effects of catalyst dosage (mole ratio of catalyst to glucose units in the feedstock) and reaction temperature on HMF yields were investigated to obtain optimal process conditions. With the 1:1 mol ratio of catalyst to glucose unit, the HMF yield reached 62.3% at 160 °C for 10 min. Untreated wheat straw was also investigated as feedstock to produce HMF for the practical use of raw biomass, in which the HMF yield was comparable to that from pure cellulose. After the extraction of HMF, [BMIM]Cl and CrCl₃/LiCl could be reused and exhibited no activity loss after three successive runs.     

Chromium halides mediated production of hydroxymethylfurfural from starch-rich acorn biomass in an acidic ionic liquid

Chromium halides were introduced for the sustainable production of hydroxymethylfurfural (HMF) from raw acorn biomass using an acidic ionic liquid. The free sugars (glucose and maltose) released by the acidic hydrolysis of the biomass were confirmed by the FT-IR absorption bands around 995–1014 cm⁻¹ and HPLC. FESEM analysis showed that the acorn biomass contains various sizes of starch granules and their structures were severely changed by the acidic hydrolysis. An optimal concentration of HCl for the HMF yields was 0.3 M. The highest HMF yield (58.7 + 1.3 dwt %) was achieved in the reaction mixture of 40% [OMIM]Cl + 10% ethyl acetate + 50% 0.3 M HCl extract containing a mix of CrBr₃/CrF₃. The combined addition of two halide catalysts was more effective in the synthesis of HMF (1.2-fold higher on average) than their single addition. The best productivity of HMF was found at 15% concentration of the biomass and at 50%, its relative productivity declined down to ca. 0.4-fold.     

Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae

 The physiological effects of 5-hydroxymethylfurfural (HMF) on Saccharomyces cerevisiae CBS 8066 in the presence and absence of furfural were studied. Experiments were carried out by pulse addition of HMF (2–4 g/l) as well as HMF (2 g/l) together with furfural (2 g/l) to batch cultivations of S. cerevisiae. Synthetic medium with glucose (50 g/l) as carbon and energy source was used. Addition of 4 g/l of HMF caused a decrease (approx. 32%) in the carbon dioxide evolution rate. Furthermore, the HMF was found to be taken up and converted by the yeast with a specific uptake rate of 0.14 (±0.03) g/g · h during both aerobic and anaerobic conditions, and the main conversion product was found to be 5-hydroxymethylfurfuryl alcohol. A previously unreported compound was found and characterized by mass spectrometry. It is suggested that the compound is formed from pyruvate and HMF in a reaction possibly catalysed by pyruvate decarboxylase. When HMF was added together with furfural, very little conversion of HMF took place until all of the furfural had been converted. Furthermore, the conversion rates of both furfural and HMF were lower than when added separately and growth was completely inhibited as long as both furfural and HMF were present in the medium. Received: 16 December 1998 / Received revision: 30 November 1999 / Accepted: 19 December 1999     

糠醛和5-羟甲基糠醛对大肠杆菌产丁二酸的影响

利用可再生生物质特别是木质纤维素水解液来生产平台化合物丁二酸,是目前研究的热点。虽然许多研究者相继报道了木质纤维素水解液对菌株生长和丁二酸生产存在一定抑制作用,但并没有水解液中各种抑制物对菌株影响的相关动力学研究及机理研究。我们选择了两种代表性木质纤维素水解液抑制物,即糠醛和5-羟甲基糠醛,系统研究了它们对大肠杆菌的生长和丁二酸生产的影响。结果表明：糠醛和5-羟甲基糠醛的初始抑制浓度均为0.8 g/L。当糠醛浓度大于6.4 g/L,5-羟甲基糠醛浓度大于12.8 g/L时,菌株生长完全受到抑制。在最高耐受浓度下,糠醛的存在使菌株生物量比对照菌株下降77.8%,丁二酸产量下降36.1%。5-羟甲基糠醛的存在使菌株生物量比对照菌株降低13.6%,丁二酸产量降低18.3%。糠醛和5-羟甲基糠醛具有明显的协同作用。体外酶活测定表明丁二酸生产途径中关键酶磷酸烯醇式丙酮酸羧化酶、苹果酸脱氢酶、富马酸还原酶均受糠醛和5-羟甲基糠醛抑制。研究结果对丁二酸生产用纤维素水解液的预处理和脱毒工艺开发具有指导作用,有利于实现丁二酸发酵生产的工业化。     

Biomass Conversion Inhibitors Furfural and 5-Hydroxymethylfurfural Induce Formation of Messenger RNP Granules and Attenuate Translation Activity in Saccharomyces cerevisiae

Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.     

Production of furans from rice straw by single-phase and biphasic systems

5-Hydroxymethylfurfural (HMF) and furfural, both of which can be derived from renewable sources, are key components for the production of different chemicals and fuels. In this study, rice straw, a cheap, abundant, and mainly unused agricultural waste, is converted to furans by a dilute acid hydrolysis process. The highest yield of HMF in a single-phase hydrolysis was 15.3 g/kg straw, attained at 180 °C during 3 h with 0.5% sulfuric acid, while the maximum yield of furfural, 59 g/kg straw, was obtained at 150 °C during 5 h. Different extracting solvents, including 2-PrOH, 1-BuOH, methyl isobutyl ketone (MIBK), and acetone at 180 °C for 3 h as well as tetrahydrofuran (THF) at 150 °C for 5 h were examined in biphasic systems. Use of the solvents generally improved the production of HMF compared to the single aqueous phase process. The best results of HMF production, more than 59 g/kg straw, were obtained in the systems containing either 2-PrOH or 1-BuOH. Using THF as an extracting solvent, a relatively high furfural yield, 118.2 g/kg straw, was obtained, and 96% of furfural produced in this system was extracted into THF during the process.     

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

Removal and recovery of furfural, 5-hydroxymethylfurfural, and acetic acid from aqueous solutions using a soluble polyelectrolyte

In the cellulosic ethanol process, furfural, 5-hydroxymethylfurfural (HMF), and acetic acid are formed during the high temperature acidic pretreatment step needed to convert biomass into fermentable sugars. These compounds can inhibit cellulase enzymes and fermentation organisms at relatively low concentrations (≥ 1 g/L). Effective removal of these inhibitory compounds would allow the use of more severe pretreatment conditions to improve sugar yields and lead to more efficient fermentations; if recovered and purified, they could also be sold as valuable by-products. This study investigated the separation of aldhehydes (furfural and HMF) and organic acid (acetic acid) inhibitory compounds from simple aqueous solutions by using polyethyleneimene (PEI), a soluble cationic polyelectrolyte. PEI added to simple solutions of each inhibitor at a ratio of 1 mol of functional group to 1 mol inhibitor removed up to 89.1, 58.6, and 81.5 wt% of acetic acid, HMF, and furfural, respectively. Furfural and HMF were recovered after removal by washing the polyelectrolyte/inhibitor complex with dilute sulfuric acid solution. Recoveries up to 81.0 and 97.0 wt% were achieved for furfural and HMF, respectively. The interaction between PEI and acetic acid was easily disrupted by the addition of chloride ions, sulfate ions, or hydroxide ions. The use of soluble polymers for the removal and recovery of inhibitory compounds from biomass slurries is a promising approach to enhance the efficiency and economics of an envisioned biorefinery.     

Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors

One major barrier to the economic conversion of biomass to ethanol is inhibitory compounds generated during biomass pretreatment using dilute acid hydrolysis. Major inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) inhibit yeast growth and subsequent fermentation. The ethanologenic yeast Saccharomyces cerevisiae demonstrated a dose-dependant inhibition by the inhibitors and has the potential to transform furfural and HMF into less toxic compounds of furfuryl alcohol and 2,5-bis-hydroxymethylfuran (also termed as furan-2,5-dimethanol (FDM)), respectively. For a sustainable and cost-competitive biomass-to-ethanol industry, it is important to develop more tolerant yeast strains that can, in situ, detoxify the inhibitors and produce ethanol. This study summarizes current knowledge and our understanding of the inhibitors furfural and HMF and discusses metabolic conversion pathways of the inhibitors and the yeast genomic expression response to inhibitor stress. Unlike laboratory strains, gene expression response of the ethanologenic yeast to furfural and HMF was not transient, but a continued dynamic process involving multiple genes at the genome level. This suggests that during the lag phase, ethanologenic yeasts undergo a genomic adaptation process in response to the inhibitors. The findings to date provide a strong foundation for future studies on genomic adaptation and manipulation of yeast to aid more robust strain design and development.The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Mixed inhibitions by methanol, furfural and acetic acid on xylitol production by Candida guilliermondii

Xylose production by Candida guilliermondii FTI 20037 was carried out in a synthetic medium in the presence of 0–100 g methanol l^–1, 0–0.7 g furfural l^–1 or 0–1.3 g acetic acid l^–1. Kinetic results show a mixed inhibition mechanism in all three cases. Maximum specific productivity and saturation constant for product formation were, in the absence of inhibition, 3.6 g_P g_X ^–1 h^–1 and 232 g_S l^–1, respectively, while the inhibition constants, K _i and K _i, were 17 and 50 g methanol l^–1, 0.62 and 7.0 g furfural l^–1, 0.69 and 3.5 g acetic acid l^–1, which suggests the following order of inhibition: furfural > acetic acid > methanol.     

An artificial neural network approach for studying phytoplankton succession

Artificial neural networks are used to model phytoplankton succession and gain insight into the relative strengths of bottom-up and top-down forces shaping seasonal patterns in phytoplankton biomass and community composition. Model comparisons indicate that patterns in chlorophyll aconcentrations response instantaneously to patterns in nutrient concentrations (phosphorous (P), nitrite and nitrate (NO₂/NO₃–N) and ammonium (NH₄–H) concentrations) and zooplankton biomass (daphnid cladocera and copepoda biomass); whereas lagged responses in an index of algal community composition are evident. A randomization approach to neural networks is employed to reveal individual and interacting contributions of nutrient concentrations and zooplankton biomass to predictions of phytoplankton biomass and community composition. The results show that patterns in chlorophyll aconcentrations are directly associated with P, NO₂/NO₃–N and daphnid cladocera biomass, as well as related to interactions between daphnid cladocera biomass, and NO₂/NO₃–N and P. Similarly, patterns in phytoplankton community composition are associated with NO₂/NO₃–N and daphnid cladocera biomass; however show contrasting patterns in nutrient– zooplankton and zooplankton–zooplankton interactions. Together, the results provide correlative evidence for the importance of nutrient limitation, zooplankton grazing and nutrient regeneration in shaping phytoplankton community dynamics. This study shows that artificial neural networks can provide a powerful tool for studying phytoplankton succession by aiding in the quantification and interpretation of the individual and interacting contributions of nutrient limitation and zooplankton herbivory on phytoplankton biomass and community composition under natural conditions.     

Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors generated from biomass pretreatment using dilute acid hydrolysis that interfere with yeast growth and subsequent fermentation. Few yeast strains tolerant to inhibitors are available. In this study, we report a tolerant strain, Saccharomyces cerevisiae NRRL Y-50049, which has enhanced biotransformation ability to convert furfural to furan methanol (FM), HMF to furan di-methanol (FDM), and produce a normal yield of ethanol. Our recent identification of HMF and development of protocol to synthesize the HMF metabolic conversion product FDM allowed studies on fermentation metabolic kinetics in the presence of HMF and furfural. Individual gene-encoding enzymes possessing aldehyde reduction activities demonstrated cofactor preference for NADH or NADPH. However, protein extract from whole yeast cells showed equally strong aldehyde reduction activities coupled with either cofactor. Deletion of a single candidate gene did not affect yeast growth in the presence of the inhibitors. Our results suggest that detoxification of furfural and HMF by the ethanologenic yeast S. cerevisiae strain Y-50049 likely involves multiple gene mediated NAD(P)H-dependent aldehyde reduction. Conversion pathways of furfural and HMF relevant to glycolysis and ethanol production were refined based on our findings in this study. The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W (GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.