Optimal conditions for alkaline detoxification of dilute-acid lignocellulose hydrolysates

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°C for NH₄OH treatment and in a narrow area stretching from pH 9.0/80°C to pH 12.0/30°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.     

The potential in bioethanol production from waste fiber sludges in pulp mill-based biorefineries

Industrial production of bioethanol from fibers that are unusable for pulp production in pulp mills offers an approach to product diversification and more efficient exploitation of the raw material. In an attempt to utilize fibers flowing to the biological waste treatment, selected fiber sludges from three different pulp mills were collected, chemically analyzed, enzymatically hydrolyzed, and fermented for bioethanol production. Another aim was to produce solid residues with higher heat values than those of the original fiber sludges to gain a better fuel for combustion. The glucan content ranged between 32 and 66% of the dry matter. The lignin content varied considerably (1-25%), as did the content of wood extractives (0.2-5.8%). Hydrolysates obtained using enzymatic hydrolysis were found to be readily fermentable using Saccharomyces cerevisiae. Hydrolysis resulted in improved heat values compared with corresponding untreated fiber sludges. Oligomeric xylan fragments in the solid residue obtained after enzymatic hydrolysis were identified using matrix-assisted laser desorption ionization-time of flight and their potential as a new product of a pulp mill-based biorefinery is discussed.     

Detoxification of lignocellulose hydrolysates with ion-exchange resins

Lignocellulose hydrolysates contain fermentation inhibitors causing decreased ethanol production. The inhibitors include phenolic compounds, furan aldehydes, and aliphatic acids. One of the most efficient methods for removing inhibiting compounds prior to fermentation is treatment of the hydrolysate with ion-exchange resins. The performance and detoxification mechanism of three different resins were examined: an anion exchanger, a cation exchanger, and a resin without charged groups (XAD8). A dilute acid hydrolysate of spruce was treated with the resins at pH 5.5 and 10.0 prior to ethanolic fermentation with Saccharomyces cerevisiae. In addition to the experiments with hydrolysate, the effect of the resins on selected model compounds, three phenolics (vanillin, guaiacol, and coniferyl aldehyde) and two furan aldehydes (furfural and hydroxymethyl furfural), was determined. The cation exchanger increased ethanol production, but to a lesser extent than XAD-8, which in turn was less effective than the an ion exchanger. Treatment at pH 10.0 was more effective than at pH 5.5. At pH 10.0, the anion exchanger efficiently removed both anionic and uncharged inhibitors, the latter by hydrophobic interactions. The importance of hydrophobic interactions was further indicated by a substantial decrease in the concentration of model compounds, such as guaiacol and furfural, after treatment with XAD-8.     

Ammonium hydroxide detoxification of spruce acid hydrolysates

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.     

Comparison of different methods for the detoxification of lignocellulose hydrolyzates of spruce

This study describes different detoxification methods to improve both cell growth and ethanol production by Baker's yeast, Saccharomyces cerevisiae. A dilute-acid hydrolyzate of spruce was used for the all detoxification methods tested. The changes in the concentrations of fermentable sugars and three groups of inhibitory compounds—aliphatic acids, furan derivatives, and phenolic compounds—were determined and the fermentability of the detoxified hydrolyzate was assayed. The applied detoxification methods included: treatment with alkali (sodium hydroxide or calcium hydroxide); treatment with sulfite (0.1% [w/v] or 1% [w/v] at pH 5.5 or 10); evaporation of 10% or 90% of the initial volume; anion exchange (at pH 5.5 or 10); enzymatic detoxification with the phenoloxidase laccase; and detoxification with the filamentous fungus Trichoderma reesei. An ion exchange at pH 5.5 or 10, treatment with laccase, treatment with calcium hydroxide, and treatment with T. reesei were the most efficient detoxification methods. Evaporation of 10% of the initial volume and treatment with 0.1% sulfite were the least efficient detoxification methods. Treatment with laccase was the only detoxification method that specifically removed only one group of the inhibitors, namely phenolic compounds. Anion exchange at pH 10 was the most efficient method for removing all three major groups of inhibitory compounds; however, it also resulted in loss of fermentable sugars.     

Influence of lignocellulose-derived aromatic compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces cerevisiae

Phenolic compounds released and generated during hydrolysis inhibit fermentation of lignocellulose hydrolysates to ethanol by Saccharomyces cerevisiae. A wide variety of aromatic compounds form from lignin, which is partially degraded during acid hydrolysis of the lignocellulosic raw material. Aromatic compounds may also form as a result of sugar degradation and dare present in wood as extractives. The influence of hydroxy-methoxy-benzaldehydes, diphenols/quinones, and phenylpropane derivatives on S. cerevisiae cell growth and ethanol formation was assayed using a defined medium and oxygen-limited conditions. The inhibition effected by the hydroxy-methoxy-benzaldehydes was highly dependent on the positions of the substituents. A major difference in inhibition by the oxidized and reduced form of a diphenol/quinone was observed, the oxidized form being the more inhibitory. The phenylpropane derivatives were examined with respect to difference in toxicity depending on the oxidation-reduction state of the γ-carbon, the presence and position of unsaturated bonds in the aliphatic side chain, and the number and identity of hydroxyl and methoxyl substituents. Transformations of aromatic compounds occuring during the fermentation included aldehyde reduction, quinone reduction, and double bond saturation. Aromatic alcohols were detected as products of reductions of the corresponding aldehydes, namely hydroxy-methoxy-benzaldehydes and coniferyl aldehyde. High molecular mass compounds and the corresponding diphenol were detected as products of quinone reduction. Together with coniferyl alcohol, dihydroconiferyl alcohol was identified as a major transformation products of conifery aldehyde.

     

Selection of anion exchangers for detoxification of dilute-acid hydrolysates from spruce

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.     

Comparison of the fermentability of enzymatic hydrolyzates of sugarcane bagasse pretreated by steam explosion using different impregnating agents

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°C for 10 min. The pretreated material was then hydrolyzed using celluloytic 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.     

Evaluation of Oxalate Decarboxylase and Oxalate Oxidase for Industrial Applications

Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching.     

Critical conditions for improved fermentability during overliming of acid hydrolysates from spruce

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.