Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli

Lignocellulose can be readily hydrolyzed into a mixture of sugars using dilute mineral acids. During hydrolysis, a variety of inhibitors are also produced which include aromatic alcohols from lignin and furfuryl alcohol from pentose destruction. Seven compounds were investigated individually and in binary combinations (catechol, coniferyl alcohol, furfuryl alcohol, guaiacol, hydroquinone, methylcatechol, and vanillyl alcohol). Aromatic alcohols and furfuryl alcohol inhibited ethanol production from xylose in batch fermentations primarily by inhibiting the growth of Escherichia coli LY01, the biocatalyst. The toxicities of these compounds were directly related to their hydrophobicity. Methylcatechol was the most toxic compound tested (MIC = 1.5 g/L). In binary combination, the extent of growth inhibition was roughly additive for most compounds tested. However, combinations with furfuryl alcohol and furfural (furaldehyde) appear synergistic in toxicity. When compared individually, alcohol components which are formed during hemicellulose hydrolysis are less toxic for growth than the aldehydes and organic acids either on a weight basis or a molar basis.     

Effect of binary combinations of selected toxic compounds on growth and fermentation of Kluyveromyces marxianus

The inhibitory effects of various lignocellulose degradation products on glucose fermentation by the thermotolerant yeast Kluyveromyces marxianus were studied in batch cultures. The toxicity of the aromatic alcohol catechol and two aromatic aldehydes (4-hydroxybenzaldehyde and vanillin) was investigated in binary combinations. The aldehyde furfural that usually is present in relatively high concentration in hydrolyzates from pentose degradation was also tested. Experiments were conducted by combining agents at concentrations that individually caused 25% inhibition of growth. Compared to the relative toxicity of the individual compounds, combinations of furfural with catechol and 4-hydroxybenzaldehyde were additive (50% inhibition of growth). The other binary combinations assayed (catechol with 4-hydroxybenzaldehyde, and vanillin with catechol, furfural, or 4-hydroxybenzaldehyde) showed synergistic effect on toxicity and caused a 60-90% decrease in cell mass production. The presence of aldehydes in the fermentation medium strongly inhibited cell growth and ethanol production. Kluyveromyces marxianus reduces aldehydes to their corresponding alcohols to mitigate the toxicity of these compounds. The total reduction of aldehydes was needed to start ethanol production. Vanillin, in binary combination, was dramatically toxic and was the only compound for which inhibition could not be overcome by yeast strain assimilation, causing a 90% reduction in both cell growth and fermentation.     

Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01

Hemicellulose residues can be hydrolyzed into a sugar syrup using dilute mineral acids. Although this syrup represents a potential feedstock for biofuel production, toxic compounds generated during hydrolysis limit microbial metabolism. Escherichia coli LY01, an ethanologenic biocatalyst engineered to ferment the mixed sugars in hemicellulose syrups, has been tested for resistance to selected organic acids that are present in hemicellulose hydrolysates. Compounds tested include aromatic acids derived from lignin (ferulic, gallic, 4-hydroxybenzoic, syringic, and vanillic acids), acetic acid from the hydrolysis of acetylxylan, and others derived from sugar destruction (furoic, formic, levulinic, and caproic acids). Toxicity was related to hydrophobicity. Combinations of acids were roughly additive as inhibitors of cell growth. When tested at concentrations that inhibited growth by 80%, none appeared to strongly inhibit glycolysis and energy generation, or to disrupt membrane integrity. Toxicity was not markedly affected by inoculum size or incubation temperature. The toxicity of all acids except gallic acid was reduced by an increase in initial pH (from pH 6.0 to pH 7.0 to pH 8.0). Together, these results are consistent with the hypothesis that both aliphatic and mononuclear organic acids inhibit growth and ethanol production in LY01 by collapsing ion gradients and increasing internal anion concentrations.     

Fungal metabolism of fermentation inhibitors present in corn stover dilute acid hydrolysate

Use of agricultural residues for ethanol production requires pretreatment of the material to facilitate release of sugars. Physical–chemical pretreatment of lignocellulosic biomass can, however, give rise to side-products that may be toxic to fermenting microorganisms and hinder utilization of sugars obtained from biomass. Potentially problematic compounds include furan aldehydes formed by degradation of sugars, organic acids released from hemicellulose side-groups, and aldehydes and phenolics released from lignin. A fungal isolate, Coniochaeta ligniaria NRRL30616, metabolizes furfural and 5-hydroxymethylfurfural (HMF) as well as aromatic and aliphatic acids and aldehydes. NRRL30616 grew in corn stover dilute-acid hydrolysate, and converted furfural to both furfuryl alcohol and furoic acid. Hydrolysate was inoculated with NRRL30616, and the fate of pretreatment side-products was followed in a time-course study. A number of aromatic and aliphatic acids, aldehydes, and phenolic compounds were quantitated by analytical extraction of corn stover hydrolysate, followed by HPLC–UV–MS/MS analysis. Compounds representing all of the classes of inhibitory side-products were removed during the course of fungal growth. Biological abatement of hydrolysates using C. ligniaria improved xylose utilization in subsequent ethanol fermentations.     

Effect of selected aldehydes found in the corncob hemicellulose hydrolysate on the growth and xylitol fermentation of Candida tropicalis

The effects of four aldehydes (furfural, 5‐hydroxymethylfurfural, vanillin and syringaldehyde), which were found in the corncob hemicellulose hydrolysate, on the growth and xylitol fermentation of Candida tropicalis were investigated. The results showed that vanillin was the most toxic aldehyde for the xylitol fermentation, followed by syringaldehyde, furfural and 5‐hydroxymethylfurfural. Moreover, the binary combination tests revealed that furfural amplified the toxicity of other aldehydes and the toxicities of other binary combinations without furfural were simply additive. Based on the fermentation experiments, it was demonstrated that the inhibition of aldehydes could be alleviated by prolonging the fermentation incubation, increasing the initial cell concentration, enhancing the initial pH value and minimizing the furfural levels in the hydrolysate evaporation process. The strategies that we proposed to suppress the inhibitory effects of the aldehydes successfully avoided the complicated and costly detoxifications. Our findings could be potentially adopted for the industrial xylitol fermentation from hydrolysates. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1181–1189, 2013     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

Tolerance of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> K35 to lignocellulose-derived inhibitory compounds

The hydrolysis which converts polysaccharides to the fermentable sugars for yeast’s lingocellulosic ethanol production also generates byproducts which inhibit the ethanol production. To investigate the extent to which inhibitory compounds affect yeast’s growth and ethanol production, fermentations by Saccharomyces cerevisiae K35 were investigated in various concentrations of acetic acid, furfural, 5-hydroxymethylfurfural (5-HMF), syringaldehyde, and coumaric acid. Fermentation in hydrolysates from yellow poplar and waste wood was also studied. After 24 h, S. cerevisiae K35 produced close to theoretically predicted ethanol yields in all the concentrations of acetic acid tested (1 ∼ 10 g/L). Both furans and phenolics inhibited cell growth and ethanol production. Ethanol yield, however, was unaffected, even at high concentrations, except in the cases of 5 g/L of syringaldehyde and coumaric acid. Although hydrolysates contain various toxic compounds, in their presence, S. Cerevisiae K35 consumed close to all the available glucose and yielded more ethanol than theoretically predicted. S. Cerevisiae K35 was demonstrated to have high tolerance to inhibitory compounds and not to need any detoxification for ethanol production from hydrolysates.     

Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant)

Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the production of ethanol. Gene arrays were used to identify expression changes that occurred in the mutant, LY01, during directed evolution to improve ethanol tolerance (defined as extent of growth in the presence of added ethanol). Expression levels for 205 (5%) of the ORFs were found to differ significantly (p < 0.10) between KO11 and LY01 under each of six different growth conditions (p < 0.000001). Statistical evaluation of differentially expressed genes according to various classification schemes identified physiological areas of importance. A large fraction of differentially expressed ORFs were globally regulated, leading to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+) in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the absence of ethanol. Other differences in gene expression provided additional clues that permitted experimentation. Tolerance appears to involve increased metabolism of glycine (higher expression of gcv genes) and increased production of betaine (higher expression of betIBA and betT encoding betaine synthesis from choline and choline uptake, respectively). Addition of glycine (10 mM) increased ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and KO11 but had no effect on growth in the absence of ethanol. Both glycine and betaine can serve as protective osmolytes, and this may be the basis of their beneficial action. In addition, the marAB genes encoding multiple antibiotic resistance proteins were expressed at higher levels in LY01 as compared to KO11. Interestingly, overexpression of marAB in KO11 made this strain more ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine deamination (sdaB) also appear beneficial for LY01. Addition of serine increased the growth of LY01 in the presence and absence of ethanol but had no effect on KO11. Changes in the expression of several genes concerned with the synthesis of the cell envelope components were also noted, which may contribute to increased ethanol tolerance.     

Polyamine Transporters and Polyamines Increase Furfural Tolerance during Xylose Fermentation with Ethanologenic Escherichia coli Strain LY180

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural.     

玉米秸秆酸解副产物对重组酿酒酵母6508-127发酵的影响

将木质纤维素类生物质如玉米秸秆等用稀酸水解预处理,在半纤维素水解为单糖的同时,水解液中还会产生一些可能对后续发酵有影响的副产物。本实验分别考查了在玉米秸秆稀酸水解液中检测出的乙酸、甲酸、香草醛、糠醛和羟甲基糠醛对重组木糖发酵菌株S. cerevisiae 6508-127生长和发酵的影响。结果表明,甲酸和乙酸对菌体生长的抑制强于乙醇生成,且甲酸的抑制程度远大于乙酸;2g/L香草醛可使菌体生长延滞期明显延长,而在较低浓度（≤1.2g/L）此现象不明显。糠醛在0.5-1.5g/L范围内对菌体生长有抑制作用,但使乙醇得率提高;羟甲基糠醛在0.2g/L浓度存在就使乙醇得率有明显降低,但使生物量得率提高;研究中还发现,糠醛、羟甲基糠醛和香草醛可被S. cerevisiae 6508-127代谢。     

Identification and characterization of fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw

A pilot plant for hydrothermal treatment of wheat straw was compared in reactor systems of two steps (first, 80°C; second, 190–205°C) and of three steps (first, 80°C; second, 170–180°C; third, 195°C). Fermentation (SSF) with Sacharomyces cerevisiae of the pretreated fibers and hydrolysate from the two-step system gave higher ethanol yield (64–75%) than that obtained from the three-step system (61–65%), due to higher enzymatic cellulose convertibility. At the optimal conditions (two steps, 195°C for 6 min), 69% of available C6-sugar could be fermented into ethanol with a high hemicellulose recovery (65%). The concentration of furfural obtained during the pretreatment process increased versus temperature from 50 mg/l at 190°C to 1,200 mg/l at 205°C as a result of xylose degradation. S. cerevisiae detoxified the hydrolysates by degradation of several toxic compounds such as 90–99% furfural and 80–100% phenolic aldehydes, which extended the lag phase to 5 h. Acetic acid concentration increased by 0.2–1 g/l during enzymatic hydrolysis and 0–3.4 g/l during fermentation due to hydrolysis of acetyl groups and minor xylose degradation. Formic acid concentration increased by 0.5–1.5 g/l probably due to degradation of furfural. Phenolic aldehydes were oxidized to the corresponding acids during fermentation reducing the inhibition level.     

Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR–XDH pathway

Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD⁺ to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120 h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50 g/L xylose in the presence of 40 mM furfural and most efficiently produced ethanol (15.70 g/L and 15.24 g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes.     

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.     

Liberation of fermentable sugars from soybean hull biomass using ionic liquid 1‐butyl‐3‐methylimidazolium acetate and their bioconversion to ethanol

Optimized hydrolysis of lignocellulosic waste biomass is essential to achieve the liberation of sugars to be used in fermentation process. Ionic liquids (ILs), a new class of solvents, have been tested in the pretreatment of cellulosic materials to improve the subsequent enzymatic hydrolysis of the biomass. Optimized application of ILs on biomass is important to advance the use of this technology. In this research, we investigated the effects of using 1‐butyl‐3‐methylimidazolium acetate ([bmim][Ac]) on the decomposition of soybean hull, an abundant cellulosic industrial waste. Reaction aspects of temperature, incubation time, IL concentration, and solid load were optimized before carrying out the enzymatic hydrolysis of this residue to liberate fermentable glucose. Optimal conditions were found to be 75°C, 165 min incubation time, 57% (mass fraction) of [bmim][Ac], and 12.5% solid loading. Pretreated soybean hull lost its crystallinity, which eased enzymatic hydrolysis, confirmed by Fourier Transform Infrared analysis. The enzymatic hydrolysis of the biomass using an enzyme complex from Penicillium echinulatum liberated 92% of glucose from the cellulose matrix. The hydrolysate was free of any toxic compounds, such as hydroxymethylfurfural and furfural. The obtained hydrolysate was tested for fermentation using Candida shehatae HM 52.2, which was able to convert glucose to ethanol at yields of 0.31. These results suggest the possible use of ILs for the pretreatment of some lignocellulosic waste materials, avoiding the formation of toxic compounds, to be used in second‐generation ethanol production and other fermentation processes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:312–320, 2016     

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 II: production of xylitol and ethanol in the presence of inhibitors

A systematic study was conducted characterizing the effect of furfural, 5-hydroxymethylfurfural (5-HMF), and acetic acid concentration on the production of xylitol and ethanol by a novel endophytic yeast, Rhodotorula mucilaginosa strain PTD3. The influence of different inhibitor concentrations on the growth and fermentation abilities of PTD3 cultivated in synthetic nutrient media containing 30?g/l xylose or glucose were measured during liquid batch cultures. Concentrations of up to 5?g/l of furfural stimulated production of xylitol to 77?% of theoretical yield (10?% higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in the presence of 5-HMF at concentrations of up to 3?g/l. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99?% of theoretical yield (15?% higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5?g/l of furfural, 5-HMF, and acetic acid, respectively. This yeast would be a potent candidate for the bioconversion of lignocellulosic sugars to biochemicals given that in the presence of low concentrations of inhibitors, its xylitol and ethanol yields are stimulated, and it is capable of metabolizing pretreatment degradation products.     

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor.     

Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF

Sugarcane bagasse was characterized as a feedstock for the production of ethanol using hydrothermal pretreatment. Reaction temperature and time were varied between 160 and 200°C and 5–20 min, respectively, using a response surface experimental design. The liquid fraction was analyzed for soluble carbohydrates and furan aldehydes. The solid fraction was analyzed for structural carbohydrates and Klason lignin. Pretreatment conditions were evaluated based on enzymatic extraction of glucose and xylose and conversion to ethanol using a simultaneous saccharification and fermentation scheme. SSF experiments were conducted with the washed pretreated biomass. The severity of the pretreatment should be sufficient to drive enzymatic digestion and ethanol yields, however, sugars losses and especially sugar conversion into furans needs to be minimized. As expected, furfural production increased with pretreatment severity and specifically xylose release. However, provided that the severity was kept below a general severity factor of 4.0, production of furfural was below an inhibitory concentration and carbohydrate contents were preserved in the pretreated whole hydrolysate. There were significant interactions between time and temperature for all the responses except cellulose digestion. The models were highly predictive for cellulose digestibility (R ² = 0.8861) and for ethanol production (R ² = 0.9581), but less so for xylose extraction. Both cellulose digestion and ethanol production increased with severity, however, high levels of furfural generated under more severe pretreatment conditions favor lower severity pretreatments. The optimal pretreatment condition that gave the highest conversion yield of ethanol, while minimizing furfural production, was judged to be 190°C and 17.2 min. The whole hydrolysate was also converted to ethanol using SSF. To reduce the concentration of inhibitors, the liquid fraction was conditioned prior to fermentation by removing inhibitory chemicals using the fungus Coniochaeta ligniaria.     

Morphogenetic effects of some volatile,organic compounds on Pestalotia rhododendri

Summary Some volatile alcohols, acids, esters, aldehydes and ketones influenced the morphogenesis of Pestalotia rhododendri when added through the gas phase. Linear growth, formation of aerial hyphae and production of conidial spores were determined. Mostly, increasing amounts of test substance inhibited linear growth and only one case of stimulation was noticed, viz. by nonenal.Many substances stimulated formation of aerial hyphae, particularly allyl alcohol, acetaldehyde and nonanal, but inhibitions were more common.Spore production was promoted by compounds from all the chemical classes investigated. The activity decreased as follows; alcohols > esters > acids, aldehydes > ketones. Inhibitions of spore production were also observed.The effects were reproducible on two different media. Pestalotia sydowiana exhibited the same main responses as the other Pestalotia species, but only two substances, furfural and n-propanol, were observed to promote spore formation of this species.