Abstract: Alkaline detoxification strongly improves the fermentability of dilute-acid hydrolysates in the production of bioethanol from lignocellulose with Saccharomyces cerevisiae. New experiments were performed with NH4OH and NaOH to define optimal conditions for detoxification and make a comparison with Ca(OH)2 treatment feasible. As too harsh conditions lead to sugar degradation, the detoxification treatments were evaluated through the balanced ethanol yield, which takes both the ethanol production and the loss of fermentable sugars into account. The optimization treatments were performed as factorial experiments with 3-h duration and varying pH and temperature. Optimal conditions were found roughly in an area around pH 9.0/60 degrees C for NH4OH treatment and in a narrow area stretching from pH 9.0/80 degrees C to pH 12.0/30 degrees C for NaOH treatment. By optimizing treatment with NH4OH, NaOH, and Ca(OH)2, it was possible to find conditions that resulted in a fermentability that was equal or better than that of a reference fermentation of a synthetic sugar solution without inhibitors, regardless of the type of alkali used. The considerable difference in the amount of precipitate generated after treatment with different types of alkali appears critical for industrial implementation.
Abstract: When dilute-acid hydrolysates from spruce are fermented to produce ethanol, detoxification is required to make the hydrolysates fermentable at reasonable rates. Treatment with alkali, usually by overliming, is one of the most efficient approaches. Several nutrients, such as ammonium and phosphate, are added to the hydrolysates prior to fermentation. We investigated the use of NH4OH for simultaneous detoxification and addition of nitrogen source. Treatment with NH4OH compared favorably with Ca(OH)2, Mg(OH)2, Ba(OH)2, and NaOH to improve fermentability using Saccharomyces cerevisiae. Analysis of monosaccharides, furan aldehydes, phenols, and aliphatic acids was performed after the different treatments. The NH4OH treatments, performed at pH 10.0, resulted in a substantial decrease in the concentrations of furfural and hydroxymethylfurfural. Under the conditions studied, NH4OH treatments gave better results than Ca(OH)2 treatments. The addition of an extra nitrogen source in the form of NH4Cl at pH 5.5 did not result in any improvement in fermentability that was comparable to NH4OH treatments at alkaline conditions. The addition of CaCl2 or NH4Cl at pH 5.5 after treatment with NH4OH or Ca(OH)2 resulted in poorer fermentability, and the negative effects were attributed to salt stress. The results strongly suggest that the highly positive effects of NH4OH treatments are owing to chemical conversions rather than stimulation of the yeast cells by ammonium ions during the fermentation.
Abstract: Bioethanol can be produced from wood via acid hydrolysis, but detoxification is needed to achieve good fermentability. Overliming was investigated in a factorial designed experiment, in which pH and temperature were varied. Degradation of inhibitory furan aldehydes was more extensive compared to monosaccharides. Too harsh conditions led to massive degradation of sugars and formation of inhibiting acids and phenols. The ethanol productivity and yield after optimal overliming reached levels exceeding reference fermentations of pure glucose. A novel metric, the balanced ethanol yield, which takes both ethanol production and losses of fermentable sugars into account, was introduced and showed the optimal conditions within the investigated range. The findings allow process technical and economical considerations to govern the choice of conditions for overliming.
Abstract: Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step.
Abstract: Six anion-exchange resins with different properties were compared with respect to detoxification of a dilute-acid hydrolysate of spruce prior to ethanolic fermentation with Saccharomyces cerevisiae. The six resins encompassed strong and weak functional groups as well as styrene-, phenol-, and acrylic-based matrices. In an analytical experimental series, fractions from columns packed with the different resins were analyzed regarding pH, glucose, furfural, hydroxymethylfurfural, phenolic compounds, levulinic acid, acetic acid, formic acid, and sulfate. An initial adsorption of glucose occurred in the strong alkaline environment and led to glucose accumulation at a later stage. Acetic and levulinic acid passed through the column before formic acid, whereas sulfate had the strongest affinity. In a preparative experimental series, one fraction from each of six columns packed with the different resins was collected for assay of the fermentability and analysis of glucose, mannose, and fermentation inhibitors. The fractions collected from strong anion-exchange resins with styrene-based matrices displayed the best fermentability: a sevenfold enhancement of ethanol productivity compared with untreated hydrolysate. Fractions from a strong anion exchanger with acrylic-based matrix and a weak exchanger with phenol-based resin displayed an intermediate improvement in fermentability, a four- to fivefold increase in ethanol productivity. The fractions from two weak exchangers with styrene- and acrylic-based matrices displayed a twofold increase in ethanol productivity. Phenolic compounds were more efficiently removed by resins with styrene- and phenol-based matrices than by resins with acrylic-based matrices.
Abstract: A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.
Abstract: In addition to fermentable sugars, dilute-acid hydrolysates of lignocellulose contain compounds that inhibit fermenting microorganisms, such as Saccharomyces cerevisiae. Previous results show that phenolic compounds and furan aldehydes, and to some extent aliphatic acids, act as inhibitors during fermentation of dilute-acid hydrolysates of spruce. Treatment of lignocellulose hydrolysates with alkali, usually in the form of overliming to pH 10.0, has been frequently employed as a detoxification method to improve fermentability. A spruce dilute-acid hydrolysate was treated with NaOH in a factorial design experiment, in which the pH was varied between 9.0 and 12.0, the temperature between 5 and 80 degrees C, and the time between 1 and 7 h. Already at pH 9.0, >25% of the glucose was lost when the hydrolysate was treated at 80 degrees C for 1 h. Among the monosaccharides, xylose was degraded faster under alkaline conditions than the hexoses (glucose, mannose, and galactose), which, in turn, were degraded faster than arabinose. The results suggest that alkali treatment of hydrolysates can be performed at temperatures below 30 degrees C at any pH between 9.0 and 12.0 without problems with sugar degradation or formation of inhibiting aliphatic acids. Treatment with Ca(OH)2 instead of NaOH resulted in more substantial degradation of sugars. Under the harsher conditions of the factorial design experiment, the concentrations of furfural and 5-hydroxymethylfurfural decreased while the total phenolic content increased. The latter phenomenon was tentatively attributed to fragmentation of soluble aromatic oligomers in the hydrolysate. Separate phenolic compounds were affected in different ways by the alkaline conditions with some compounds showing an increase in concentration while others decreased. In conclusion, the conditions used for detoxification with alkali should be carefully controlled to optimize the positive effects and minimize the degradation of fermentable sugars.
Abstract: Sugarcane bagasse is a potential lignocellulosic feedstock for ethanol production, since it is cheap, readily available, and has a high carbohydrate content. In this work, bagasse was subjected to steam explosion pretreatment with different impregnation conditions. Three parallel pretreatments were carried out, one without any impregnation, a second with sulfur dioxide, and a third with sulfuric acid as the impregnating agent. The pretreatments were performed at 205 degrees C for 10 min. The pretreated material was then hydrolyzed using cellulolytic enzymes. The chemical composition of the hydrolyzates was analyzed. The highest yields of xylose (16.2 g/100 g dry bagasse), arabinose (1.5 g/100 g), and total sugar (52.9 g/100 g) were obtained in the hydrolysis of the SO2 -impregnated bagasse. The H2SO4 -impregnated bagasse gave the highest glucose yield (35.9 g/100 g) but the lowest total sugar yield (42.3 g/100 g) among the three methods. The low total sugar yield from the H2SO4-impregnated bagasse was largely due to by-product formation, as the dehydration of xylose to furfural. Sulfuric acid impregnation led to a three-fold increase in the concentration of the fermentation inhibitors furfural and 5-hydroxymethylfurfural (HMF) and a two-fold increase in the concentration of inhibitory aliphatic acids (formic, acetic, and levulinic acids) compared to the other two pretreatment methods. The total content of phenolic compounds was not strongly affected by the different pretreatment methods, but the quantities of separate phenolic compounds were widely different in the hydrolyzate from the H2SO4-impregnated bagasse compared with the other two hydrolyzates. No major differences in the content of inhibitors were observed in the hydrolyzates obtained from SO2-impregnated and non-impregnated bagasse. The fermentability of all three hydrolyzates was tested with a xylose-utilizing Saccharomyces cerevisiae strain with and without nutrient supplementation. The hydrolyzates of SO2-impregnated and nonimpregnated bagasse showed similar fermentability, whereas the hydrolyzate of H2SO4-impregnated bagasse fermented considerably poorer.
Abstract: Treatment with alkali, particularly overliming, has been widely used as a method for the detoxification of lignocellulose hydrolysates prior to ethanolic fermentation. However, the mechanisms behind the detoxification effect and the influence of the choice of cation have not been well understood. In this study, a dilute acid hydrolysate of spruce and an inhibitor cocktail consisting of six known inhibitors were used to investigate different alkali detoxification methods. The various treatments included the addition of calcium hydroxide, sodium hydroxide, potassium hydroxide, and ammonia to pH 10.0 and subsequent adjustment of the pH to 5.5 with either sulfuric or hydrochloric acid as well as treatment with the corresponding amounts of calcium, sodium, and potassium as sulfate or chloride salts at pH 5.5. An RP-HPLC method was developed for the separation of 18 different inhibitors in the hydrolysate, including furaldehydes and phenolics. Detection and quantification were carried out by means of UV, DAD, and ESI-MS in negative mode. Treatment of the spruce hydrolysate with alkali resulted in up to approximately 40% decrease in the concentration of furaldehydes. The effects on the aromatic compounds were complex. Furthermore, SFE was performed on the precipitate formed during alkali treatment to evaluate the inhibitor content of the precipitate, and the following RP-HPLC analysis implied that potential inhibitors were removed mainly through conversion rather than through filtration of precipitate. Parallel experiments in which sulfuric acid or hydrochloric acid was used for acidification to pH 5.5 after alkali treatment indicated that the choice of anion did not affect the removal of inhibitors. Detoxification with calcium hydroxide and ammonia resulted in better fermentability using Saccharomyces cerevisiae than detoxification with sodium hydroxide. The results from the experiments with the inhibitor cocktail indicated that the positive effects of alkali treatment are difficult to explain by removal of the inhibitors only and that possible stimulatory effects on the fermenting organism warrant further attention.
Abstract: This work describes a novel approach to detoxify lignocellulosic hydrolysates and facilitate the analysis of inhibitory compounds, namely supercritical fluid extraction (SFE). The efficiency of the fermentation of lignocellulosic dilute-acid hydrolysates depends upon the composition of the hydrolysate and the organism used. Furthermore, it has been shown that inhibitors in the hydrolysate reduce the fermentation yield. This knowledge has given rise to the need to identify and remove the inhibiting compounds. Sample clean-up or work-up steps, to provide a clean and concentrated sample for the analytical system, facilitate the characterization of inhibitors, or indeed any compound in the hydrolysates. Removal of inhibitors was performed with countercurrent flow supercritical fluid extraction of liquid hydrolysates. Three different groups of inhibitors (furan derivatives, phenolic compounds, and aliphatic acids) and sugars were subsequently analyzed in the hydrolysate, extracted hydrolysate, and extract. The effect of the SFE treatment was examined with respect to fermentability with Saccharomyces cerevisiae. Not only did the extraction provide a clean and concentrated sample (extract) for analysis, but also a hydrolysate with increased fermentability as well as lower concentrations of inhibitors such as phenolics and furan derivatives.
