Conversion of furfural to furoic acid and furfuryl alcohol by Neurospora ascospores

Summary The metabolism of furfural was studied with regard to possible mechanisms by which the chemical induces germination in ascospores. Incubation of ascospores in furfural resulted in the uptake of a small percent of the furfural, and the conversion of the bulk of it to furoic acid which was in turn converted to furfuryl alcohol. Conversion also occurred in Neurospora mycelium and conidia with the order being furfural to furfuryl alcohol to furoic acid. Conversion appears to be a noninducible enzymatic process localized on the outer surface of the cell. Conversion was completely inhibited without preventing germination indicating that conversion is not involved in the breaking of dormancy in Neurospora ascospores.     

Cellular Localization of Protoporphyrinogen-Oxidizing Activities of Etiolated Barley (Hordeum vulgare L.) Leaves (Relationship to Mechanism of Action of Protoporphyrinogen Oxidase-Inhibiting Herbicides)

Barley (Hordeum vulgare L.) that had been malted for 5 d developed only a small amount of bound (inactive) limit dextrinase, and very little free (active) enzyme was detected. Continuation of malting for up to 10 d only slightly increased the amount of both bound and free forms. Grain grown under conditions of ample moisture (wet grown) for 5 d produced a much higher amount of bound enzyme but a similarly low amount of free enzyme compared to malting conditions. After 10 d of growth there was a decrease in the amount of bound enzyme and a large increase in the amount of free enzyme, such that almost all of the enzyme was present in the free form. A more detailed study of limit dextrinase development in wet-grown grains revealed that a bound form was rapidly produced soon after germination. Five to 6 d after germination the amount of bound enzyme decreased rapidly and a very low amount was found in grains 9 d after germination. Meanwhile, a free form appeared slightly later and its initial rate of development was slow. At about 5 d after germination, precisely when the bound enzyme began to decrease, the free form increased rapidly, so that by 9 d after germination nearly all the enzyme was in the free form. The release of bound limit dextrinase in vitro occurred by proteolytic modification through the action of cysteine proteinases that were kept active or activated by the presence of reduced thiols in the extraction medium. The presence of cysteine proteinases was confirmed by inhibition studies using the inhibitors iodoacetamide, N-ethylmaleimide, antipain, and leupeptin. In addition, most of the bound form of limit dextrinase was soluble in 0.2 M sodium acetate buffer (pH 5.0) following extraction at 30[deg]C for 16 h and centrifugation at 3000g.     

Induction of Dimorphism in the Basidiomycete Lenzites saepiaria

Sugars can serve as the germinant for basidiospores of the wood-rotting fungus Lenzites saepiaria. Hexoses sterilized by autoclaving were better germinants than hexoses that were sterilized by filtration. The degradation products in heated hexose which were responsible for the stimulation of germination were levulinic and formic acid. Another product of hexose degradation, hydroxymethyl furfural, had a marked effect on outgrowth of L. saepiaria basidiospores and on the development of mycelia. Basidiospores that germinated in the presence of hydroxymethyl furfural yielded large rounded bodies that, in some cases, developed as a chain of yeastlike cells. Addition of hydroxymethyl furfural to developing mycelia resulted in the production of chains of round yeastlike structures. Similar results were obtained by treating basidiospores or mycelia with phenethyl alcohol.     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

Characterization of the early synthesized DNA in germinating Triticum aestivum embryos

A radioactive DNA preparation was isolated from the post-mitochondrial supernatant fraction of thymidine-[¹⁴C] fed wheat embryos. The isolated sDNA preparation was similar to cytoplasmic non-mitochondrial DNA of other eukaryotic cells. The buoyant density and frequency of pyrimidine nucleotide clusters found for the sDNA were, essentially, the same as those found for the nuclear DNA. In contrast to DNA that can be leaked from nuclei or other DNA-containing organelles, the sDNA is firmly bound to a protein component. At an early germination stage (6–12 hr), the sDNA is the only newly-synthesized DNA fraction that can be isolated from the embryo homogenate. Considerable synthesis of nuclear and organellar DNA starts 18 hr after the beginning of germination, just prior to the first maximum of the cell divisions. It is concluded that wheat embryo cells contain cytoplasmic non-mitochondrial DNA and are able to resume its synthesis at an early germination stage, prior to the first post-dormant round of nuclear DNA replication.     

Effects of furfural on anaerobic continuous cultivation of Saccharomyces cerevisiae.

Furfural is an important inhibitor of yeast metabolism in lignocellulose-derived substrates. The effect of furfural on the physiology of Saccharomyces cerevisiae CBS 8066 was investigated using anaerobic continuous cultivations. Experiments were performed with furfural in the feed medium (up to 8.3 g/L) using three different dilution rates (0.095, 0.190, and 0.315 h(-1)). The measured concentration of furfural was low (< 0.1 g/L) at all steady states obtained. However, it was not possible to achieve a steady state at a specific conversion rate of furfural, q(f), higher than approximately 0.15 g/g.h. An increased furfural concentration in the feed caused a decrease in the steady-state glycerol yield. This agreed well with the decreased need for glycerol production as a way to regenerate NAD+, i.e., to function as a redox sink because furfural was reduced to furfuryl alcohol. Transient experiments were also performed by pulse addition of furfural directly into the fermentor. In contrast to the situation at steady-state conditions, both glycerol and furfuryl alcohol yields increased after pulse addition of furfural to the culture. Furthermore, the maximum specific conversion rate of furfural (0.6 g/g.h) in dynamic experiments was significantly higher than what was attainable in the chemostat experiments. The dynamic furfural conversion could be described by the use of a simple Michaelis-Menten-type kinetic model. Also furfural conversion under steady-state conditions could be explained by a Michaelis-Menten-type kinetic model, but with a higher affinity and a lower maximum conversion rate. This indicated the presence of an additional component with a higher affinity, but lower maximum capacity, either in the transport system or in the conversion system of furfural.     

Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.     

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。     

Cofactor Dependence in Furan Reduction by Saccharomyces cerevisiae in Fermentation of Acid-Hydrolyzed Lignocellulose

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.     

Effect of furfural on the growth of lactose-utilizing Candida Blankii 35

The effects of furfural on the growth of the lactose-utilizing yeast Candida blankii 35 were investigated using the method of continuous cultivation under conditions of carbon limitation and at dilution rates of 0.1 and 0.25 h⁻¹. The data obtained at dilution rate 0.1 h⁻¹ and 0.04% furfural showed a decrease in the yield of biomass by 6% and in the RNA content, but the synthesis of cell protein increased with 11.6% compared to the control. Treatment with 0.08% furfural induced significant changes in growth and biosynthesizing ability. A strong inhibitory effect of furfural was observed: the biomass yield decreased by half at 48 h and the culture the reached the control level of protein content. The effect of 0.02% furfural at 0.25 h⁻¹ dilution rate caused a significant reduction of biomass yield (34.4%) and the substrate utilization rate reached values higher by 52.4% at 48 h, but the protein-synthesizing ability of the cells slightly increased. The results showed that a treatment with 0.04 or 0.08% furfural caused significant disturbances of cell functions, the yields of biomass and protein drastically decreased and the culture was washed out. Data showed that the inhibitory effect of furfural on the growth and protein-synthesizing ability of Candida blankii 35 depends on the inhibitor concentration as well as the dilution rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.     

Effects of furfural on ethanol fermentation bySaccharomyces cerevisiae: Mathematical models

Different inocula with high yeast concentration were investigated as a means of overcoming the inhibitory effect of furfural in ethanol fermentation. In order to verify the toxicity of the furfural, a series of fermentation runs were made with 0.25, 5.50, and 9.00 g/L (dry weight) ofSaccharomyces cerevisiae inoculum and 1, 3, and 5 g/L of furfural. The extent of cell death occurring in the early phase of fermentation was dependent on the initial cell concentration. With high initial yeast concentration, the effect of furfural is canceled, because it is depleted at an early stage of fermentation. The ethanol weight yield averaged 0.45 on the basis of sugar consumed. The ethanol productivity and specific growth rate decreased with the increase of furfural concentration, and the inhibitory effect almost disappeared with high cell concentration (9 g/L). Mathematical models were developed that relate productivity and growth rate with furfural and cell concentration.     

A proteomic analysis of rice seed germination as affected by high temperature and ABA treatment

Seed germination is a critical phase in the plant life cycle, but the specific events associated with seed germination are still not fully understood. In this study, we used two‐dimensional gel electrophoresis followed by mass spectrometry to investigate the changes in the proteome during imbibition of Oryza sativa seeds at optimal temperature with or without abscisic acid (ABA) and high temperature (germination thermoinhibition) to further identify and quantify key proteins required for seed germination. A total of 121 protein spots showed a significant change in abundance (1.5‐fold increase/decrease) during germination under all conditions. Among these proteins, we found seven proteins specifically associated with seed germination including glycosyl hydrolases family 38 protein, granule‐bound starch synthase 1, Os03g0842900 (putative steroleosin‐B), N‐carbamoylputrescine amidase, spermidine synthase 1, tubulin α‐1 chain and glutelin type‐A; and a total of 20 imbibition response proteins involved in energy metabolism, cell growth, cell defense and storage proteins. High temperature inhibited seed germination by decreasing the abundance of proteins involved in methionine metabolism, amino acid biosynthesis, energy metabolism, reserve degradation, protein folding and stress responses. ABA treatment inhibited germination and decreased the abundance of proteins associated with methionine metabolism, energy production and cell division. Our results show that changes in many biological processes including energy metabolism, protein synthesis and cell defense and rescue occurred as a result of all treatments, while enzymes involved in methionine metabolism and weakening of cell wall specifically accumulated when the seeds germinated at the optimal temperature.     

Sugar cane arginase competes with the same fungal enzyme as a false quorum signal against smut teliospores

Sugar cane cultivars resistant to smut produce a pool of glycoproteins after experimental infection, one of them develops arginine activity. This arginase induces cytoagglutination of smut teliospores but impedes germination. Teliospores also secrete a fungal arginase that accelerates their own germination. This fungal arginase binds to teliospore cell walls. The affinity of this arginase for the ligand shows to be higher than that found for plant arginase. In fact, fungal arginase removes sugar cane arginase previously bound to their ligands in the cell wall whereas the inverse process is practically negligible. The enzymatic activity is required to the binding of the protein to teliospore cell wall but it is not related to the germination process. Thus, it can be concluded that the binding of fungal arginase to smut teliospores activates a signal transduction cascade that enhances germination.     

Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase.

The cell wall is a fundamental component in the response of plants to environmental changes. To directly assess the role of the cell wall we have increased the expression and activity of a cell wall associated peroxidase (TPX2), an enzyme involved in modifying cell wall architecture. Overexpression of TPX2 had no effect on wild-type development, but greatly increased the germination rate under high salt or osmotic stress. Differential scanning calorimetry showed that transgenic seeds were able to retain more water available for germination than wild-type seeds. Thermoporometry calculations indicated that this could be due to a lower mean pore size in the walls of transgenic seeds. Therefore, the higher capacity of transgenic seeds in retaining water could result in higher germination rates in conditions where the availability of water is restricted.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

Concentration-driven reverse membrane bioreactor for the fermentation of highly inhibitory lignocellulosic hydrolysate

Optimal production of lignocellulosic bioethanol is hindered due to commonly faced issues with the presence of inhibitory compounds and sequentially consumed sugars in the lignocellulosic hydrolysate. Therefore, in order to find a robust fermentation approach, this study aimed at enhancing simultaneous co-assimilation of sugars, and inhibitor tolerance and detoxification. Therefore, fermentation of toxic wheat straw hydrolysate containing up to 20 g/l furfural, using the concentration-driven diffusion-based technique of reverse membrane bioreactor (rMBR) was studied. The rMBR fermentation of the hydrolysate led to complete furfural detoxification and the conversion of 87 % of sugars into ethanol at a yield of 0.48 g/g. Moreover, when the toxicity level of the hydrolysate was increased to 9 g/l of initial furfural, the system responded exceptionally by reducing 89 % of the inhibitor while only experiencing about 25 % drop in the ethanol yield. In addition, using this diffusion-based set-up in extremely inhibitory conditions (16 g/l furfural), cells could detoxify 40 % of the furfural at a high initial furfural to cell ratio of 9.5:1. The rMBR set-up applied proved that by properly synchronizing the medium condition, membrane area, and inhibitor to cell ratio, some of the shortcomings with conventional lignocellulosic fermentation can be tackled, guaranteeing a robust fermentation.     

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.