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Anaerobic digestion of municipal waste is a promising technology for renewable energy production, notably for methane (CH4) production. Existing reactor designs have limitations that prevent efficient conversion and high throughput, especially of substrates high in suspended solids. Here, we introduce a novel compartmentalized reactor design encompassing controlled feed flow over fixed microbial biofilms for high rate CH4 production from enzymatically pre-hydrolyzed municipal solid waste. In a 240 liter working volume reactor, the biofilm generation was completed in less than 60 days and CH4 production was minimum ˜700 L day-1 in the ensuing months with a Hydraulic Retention Time (HRT) of 10 days or lower. When the organic load was gradually increased, a reactor productivity of 6 LCH4 Lreactor-1 day-1 was achieved. At this point, the reactor processed 20.8 gCOD Lreactor-1 day-1 at a HRT below 5 days, maintaining above 70% of the maximal Chemical Oxygen Demand (COD) conversion rate. Such fast conversion rates and high yields are far beyond what has been reported for other reactor designs, and are a crucial prerequisite for industrial realization of renewable biogas production from municipal solid waste and other organic waste streams.

Efficient methods for lignin characterisation are increasingly important as the field of lignin valorisation is growing with the increasing use of lignocellulosic feedstocks, such as wheat straw and corn stover, in biorefineries. In this study, we characterised a set of authentic lignin biorefinery samples in situ with no prior purification and minimal sample preparation. Lignin chemical formulas and lignin Fourier transform infrared (FTIR) spectra were extracted from mixed spectra by filtering out signals from residual carbohydrates and minerals. From estimations of C, H and O and adjustment for cellulose and hemicelluloses contents, the average chemical formula of lignin was found to be C9H10.2O3.4 with slight variations depending on the biomass feedstock and processing conditions (between C9H9.5O2.8 and C9H11.1O3.6). Extracted FTIR lignin spectra showed many of the same characteristic peaks as organosolv and kraft lignin used as benchmark samples. Some variations in the lignin spectra of biorefinery lignin residue samples were found depending on biomass feedstock (wheat straw, corn stover or poplar) and on pretreatment severity, especially in the absorbance of bands at 1267 and 1032 cm−1 relative to the strong band at ~1120 cm−1. The suggested method of FTIR spectral analysis with adjustment for cellulose and hemicellulose is proposed to provide a fast and efficient way of analysing lignin in genuine lignin samples resulting from biorefineries.

Effects of treatment time, citrate concentration, temperature, and pH on Si extraction from wheat straw prior to hydrothermal pretreatment were investigated for maximising Si removal and biomass recovery before biomass refining. With citrate, an almost linear negative correlation between Si content in the residual biomass and treatment temperature was observed up to 170 °C, yielding a Si removal of up to 97.7%. This high Si removal came at the expense of a low mass yield (down to 45%) in the insoluble lignocellulosic fraction. Optimum process conditions for high Si removal and high total mass yield were: 100 mM sodium citrate, 130 °C, 60 min, 2% w/v solids, and pH of ∼6.5 during extraction. Using the proposed process conditions, silica removal of up to 77% was achieved with a mass yield of 72.8%. This Si removal from the insoluble lignocellulosic fraction did not affect the enzymatic cellulose hydrolysis, neither negatively nor positively.

Elemental analysis by X-ray fluorescence spectrometry (XRF) of solid samples from a biorefinery process was performed to study the behaviour of mineral elements in a process involving hydrothermal pretreatment of biomass (wheat straw, corn stover, sugarcane bagasse, palm oil empty fruit bunches, poplar) followed by enzymatic hydrolysis and fermentation. For all the different biomasses, the biorefinery process concentrated silicon, aluminium, and calcium in the solid fraction, while potassium and magnesium were solubilised in the process and removed from the solid fraction. Sodium concentrations were in general low and they only increased in case of addition during the process. No general tendencies were observed for phosphorus, sulphur, and iron concentrations. A prerequisite for XRF elemental analysis was defining an average chemical formula for the organic matrix of process biomass samples. Based on ultimate elemental analysis of all biomasses, the formula for biomass was C6H8.4O3.5, which was used for all types of samples (raw biomass, pretreated biomass, and lignin residue) and can be used in future XRF analysis of samples of similar process and biomass feedstock as those used in this study.

Biorefineries aim to convert low value biomasses into high value products. The feedstock biomasses are often high-silica agricultural waste products such as rice straw, wheat straw, corn stover, sugarcane bagasse, or empty fruit bunches. This causes challenges, since silica is problematic in industrial processes, where it forms water-insoluble precipitates that are hard to remove, block filtration systems, and cause instrumental defects. In this paper we review various industries that experience issues with silica. These include paper pulping and waste-water treatment, where they try to solve their problems with silica in different ways. High pH and co-precipitation with mineral elements are some common ways of alleviating silica problems. Reviewing the literature for the fundamentals of silica revealed a complex chemistry that is not yet fully understood. Much is still to be learned about the interactions between silica and organic material as well as the mechanisms of silica precipitation and dissolution. Understanding the fundamental and complex chemistry of silica might help developing better solutions than those existing today, allowing efficient use of high silica biomasses in biorefineries. © 2014 Society of Chemical Industry and John Wiley & Sons, Ltd

The properties of slurries made of pyrolysis oil mixed with wood, char or ground char were investigated with respect to phase transitions, rheological properties, elemental compositions, and energy density. Also the pumping properties of the slurries were investigated at temperatures of 25, 40 and 60 °C and solid loadings from 0 to 20 wt%. The phase transitions of the wood slurry samples were observed at lower solid loadings compared to the char slurry samples. The apparent viscosity of the slurry samples was found to be considerably impacted by solid loading (0–20 wt%) and temperature (25–60 °C), especially in the phase transition region. The slurry viscosities with 20 wt% char loading, 20 wt% ground char loading and 15 wt% wood loading (at a shear rate of 100 s −1 ) are 0.7, 1.0 and 1.7 Pa.s, respectively at 60 °C and these values increases 1.2–1.4 times at 40 °C and 3–4 times at 25 °C. The wood, char and ground char slurry samples with 5–20 wt% solid loading obtain a volumetric energy density of 21–23 GJ/m 3 . The slurry sample with 20 wt% ground char having a d 80 of 118 μm was pumped successfully into a pressurized chamber (0–6 bar) while plugging appeared when the slurry samples with 15 wt% char having a d 80 of 276 μm was pumped into the pressurized chamber.

The degradation compounds formed during pretreatment when lignocellulosic biomass is processed to ethanol or other biorefinery products include furans, phenolics, organic acids, as well as mono- and oligomeric pentoses and hexoses. Depending on the reaction conditions glucose can be converted to 5-(hydroxymethyl)-2-furaldehyde (HMF) and/or levulinic acid, formic acid and different phenolics at elevated temperatures. Correspondingly, xylose can follow different reaction mechanisms resulting in the formation of furan-2-carbaldehyde (furfural) and/or various C-1 and C-4 compounds. At least four routes for the formation of HMF from glucose and three routes for furfural formation from xylose are possible. In addition, new findings show that biomass monosaccharides themselves can react further to form pseudo-lignin and humins as well as a wide array of other compounds when exposed to high temperatures. Hence, several aldehydes and ketones and many different organic acids and aromatic compounds may be generated during hydrothermal treatment of lignocellulosic biomass. The reaction mechanisms are of interest because the very same compounds that are possible inhibitors for biomass processing enzymes and microorganisms may be valuable biobased chemicals. Hence a new potential for industrial scale synthesis of chemicals has emerged. A better understanding of the reaction mechanisms and the impact of the reaction conditions on the product formation is thus a prerequisite for designing better biomass processing strategies and forms an important basis for the development of new biorefinery products from lignocellulosic biomass as well.

Fast pyrolysis of lignin from an ethanol plant was investigated on a lab scale pyrolysis centrifuge reactor (PCR) with respect to pyrolysis temperature, reactor gas residence time, and feed rate. A...

Fast pyrolysis may be used for sewage sludge treatment with the advantages of a significant reduction of solid waste volume and production of a bio-oil that can be used as fuel. A study of the infl...

A fast pyrolysis study on lignin and macroalgae (nonconventional biomass) and wood and straw (conventional biomass) were carried out in a pyrolysis centrifugal reactor at pyrolysis temperature of 550 °C. The product distributions and energy recoveries were measured and compared among these biomasses. The fast pyrolysis of macroalgae showed a promising result with a bio-oil yield of 65 wt % dry ash free basis (daf) and 76% energy recovery in the bio-oil while the lignin fast pyrolysis provides a bio-oil yield of 47 wt % daf and energy recovery in bio-oil of 45%. The physiochemical properties of the bio-oils were characterized with respect to higher heating value (HHV), molecular mass distribution, viscosity, pH, density, thermal behaviors, elemental concentrations, phase separation, and aging. The lignin and macroalgae oil properties were different compared to those of the wood and straw oils with respect to carbon and oxygen contents, HHV, thermal behaviors, and mean molecular weight. The HHV of wood, str...

In this study 26 new oligophenol cellulase inhibitors were discovered from wheat straw pretreatment liquors. By consideration of the reaction mechanisms for their formation it is proposed that these oligophenols are formed during hydrothermal biomass pretreatment by pentose self-condensation reactions involving aldol condensations, 1,4 additions to α,β unsaturated carbonyl compounds, 3-keto acid decarboxylations and oxidations. Furthermore, pentose reactions with phenolic lignin components are suggested. The identification of the central role of xylose in the reaction routes for oligophenolic inhibitor formation led to the solution to protect the reactive anomeric center in xylose. It is shown that protection of the anomeric center in in situ generated xylose with ethylene glycol monobutyl ether, during pretreatment of wheat straw, reduces the level of oligophenols by 73%. The results pave the way for implementation of new types of reactions that hinder inhibitor formation in lignocellulosic biomass processing.

Palm kernel press cake (PKC) is a residue of palm oil extraction, which was found to contain 48.5% of total carbohydrates of which 35.2% was mannan. The present study examines enzymatic hydrolysis of polysaccharides from the cell-wall material present in PKC to obtain monosaccharides that can be substrate in various fermentation processes such as ethanol production. The requirements for pretreatment were investigated and it was found that mannan in PKC was readily hydrolysed without any pretreatment. Several enzyme preparations were tested and Mannaway 25L was found as the best for releasing mannose, and Gammanase 1.0L worked well in degrading cellulose and mannose. Binary mixtures of enzymes were tested to increase the conversion, and 1:1 mixture of Mannaway 25L and Gammanase 1.0L showed good synergistic effect releasing 30% more mannose than the sum obtained using these enzymes individually. Using an enzyme loading of 2.3 mg protein/g PKC resulted in 63% of mannan in PKC being hydrolysed to mannose in 24 h, and in 96 h a total of 365 g mannose and glucose could be produced per kg PKC. Finally, PKC was hydrolysed and fermented using Saccharomyces cerevisiae with an ethanol yield of 125 g/kg PKC.

Efficient generation of a fermentable hydrolysate is a primary necessity in the utilization of fibrous plant biomass as a feedstock in bioethanol processes. Enzyme catalyzed hydrolysis of cellulose and heteroxylans in biomass feedstocks each require multiple enzyme activities to achieve degradation to fermentable monosaccharides. The minimal enzyme cocktail concept concerns identification of the minimal number, the minimal levels, and the optimal combination of the best performing key monoactive enzymatic activities to meet this requirement. Two major hypotheses lie behind this concept: 1. That the native multi-component profiles of crude “wild type” cellulolytic and/or xylan degrading enzyme preparations are not optimal for degradation of cellulose in pre-treated lignocellulosic biomass nor for degradation of heteroxylans in hemicellulose-rich product streams; 2. That it is possible to replace crude multienzyme preparations with designed combinations of the minimal number of required enzyme activities for biomass processing. This paper outlines the current strategies employed and the stage of development of minimal enzyme cocktails for enzymatic hydrolysis of cellulose and arabino-xylan in complex, genuine biomass substrates. The available data demonstrate the feasibility of the concept and illustrate the potential efficacy improvements obtainable by use of designed minimal enzyme cocktails for pre-treated lignocellulosic and hemicellulose-rich biomass substrates.

Hydrolysis of arabinoxylan is an important prerequisite for valorization of wheat endosperm materials, but the water insoluble arabinoxylan fraction of wheat endosperm cell walls has proven difficult to hydrolyze enzymatically. In this study, enzymatic hydrolysis of purified, water insoluble wheat arabinoxylan was examined and improved by combining detailed substrate analysis with analyses of product monosaccharides and oligosaccharides after treatments with rationally composed enzyme mixtures. Treatment of purified, water insoluble wheat arabinoxylan (substrate concentration 1.0%, w/w) for 48 h at pH 5, 50 °C with a combination of β-xylosidase from Trichoderma reesei and a hemicellulolytic fungal enzyme preparation from Humicola insolens, Ultraflo L, liberated 1.6 mg ferulic acid, 24 mg acetic acid, 51 mg arabinose, 167 mg xylose, and furthermore, solubilized 244 mg oligosaccharides per gram substrate dry matter. This yield was equivalent to solubilization of 45% by weight of the insoluble wheat arabinoxylan. Analysis of the solubilized oligomers by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis revealed presence of different feruloylated and acetylated pentose oligomers. The data thus signified the presence of acetyl groups in wheat arabinoxylan. Addition of acetyl xylan esterase from Aspergillus aculeatus on top of the Ultraflo L and β-xylosidase addition did however not facilitate additional degradation of the insoluble wheat arabinoxylan, whereas, addition of feruloyl esterase obtained from Aspergillus niger on top of the β-xylosidase and Ultraflo L blend solubilized an additional 1.9% (w/w) dry matter from the substrate and significantly changed the MALDI-TOF MS profile of the solubilized oligomers which became significantly less feruloylated after this enzyme treatment.

Generation of a fermentable hydrolysate from arabinoxylan is a first prerequisite in the utilization of wheat hemicellulose in the biofuel industry. This study examined the efficacy of four hemicellulolytic microbial enzyme preparations and one xylanase preparation in catalyzing degradation of purified water soluble and water insoluble wheat arabinoxylan—with particular emphasis on the catalytic degradation of water insoluble arabinoxylan. The effects of individual enzyme treatments were compared by assessing yields of arabinose, xylose, and xylobiose obtained under different reaction conditions of pH and temperature in response surface designs. In general, the monosaccharide yields obtained were lower on the water insoluble wheat arabinoxylan than on the water soluble. On both substrates, the Ultraflo L preparation from Humicola insolens was best in catalyzing arabinose and xylobiose release, while the Celluclast 1.5 L preparation from Trichoderma reesei was superior to all the other enzyme preparations in catalyzing xylose release. Treatments with 50:50 combinations of the enzyme preparations only resulted in a pronounced synergistic xylose release with a mixture of Ultraflo L:Celluclast 1.5 L. Examination of the progress of the substrate degradation indicated that the synergism on the insoluble arabinoxylan resembled what we have previously observed on soluble arabinoxylan, i.e. that the effect was a result of positive interaction in arabinose release and xylan depolymerization between the α- l -arabinofuranosidase (EC 3.2.1.55) and endo-1,4-β-xylanase (EC 3.2.1.8) activities present in Ultraflo L, and between the β-xylosidase (EC 3.2.1.37) activity present in the Celluclast 1.5 L. The results of treatments with combinations of Ultraflo L and purified T. reesei β-xylosidase – with both simultaneous and sequential addition, and with and without contemporary pH adjustments to optimize individual enzyme activities – strongly corroborated this conclusion.

A novel alpha-L-arabinofuranosidase (alpha-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. (1)H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1-->3)-alpha-L-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 degrees C. Two other novel alpha-L-arabinofuranosidases (alpha-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1-->2) and (1-->3)-alpha-L-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 alpha-AraF from H. insolens and the GH51 alpha-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 degrees C. This synergistic interaction between GH43 and GH51 alpha-AraFs was also evident when a GH43 alpha-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 alpha-AraFs being able to catalyse the removal of single-sitting (1-->2)-alpha-L- arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1-->3)-alpha-L-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.

The generation of a fermentable hydrolysate from arabinoxylan is an important prerequisite for utilization of wheat hemicellulose in production of ethanol or other value added products. This study examined the individual and combined efficiencies of four selected, commercial, multicomponent enzyme preparations Celluclast 1.5 L (from Trichoderma reesei ), Finizym (from Aspergillus niger ), Ultraflo L (from Humicola insolens ), and Viscozyme L (from Aspergillus aculeatus ) in catalyzing arabinose and xylose release from water-soluble wheat arabinoxylan in an industrial fermentation residue (still bottoms) in lab scale experiments. Different reaction conditions, i.e. enzyme dosage, reaction time, pH, and temperature, were evaluated in response surface and ternary mixture designs. Ultraflo L treatment gave optimal arabinose release: treatment (6 h, 60 °C, pH 6) with this enzyme preparation liberated up to 46% by weight (wt.%) of the theoretically maximal arabinose yield from the substrate. Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose and catalyzed release of up to 25 wt.% of the theoretical maximum xylose yield (6 h, 60 °C, pH 4). Prolonged treatment for 24 h with a 50:50 mixture of Celluclast 1.5 L and Ultraflo L at 50 °C, pH 5 exhibited a synergistic effect in xylose release and 62 wt.% of the theoretically maximal xylose yield was achieved. Addition of pure β-xylosidase from T. reesei to the Ultraflo L preparation released the same amounts of xylose from the substrate as the 50:50 mixture of Celluclast 1.5 L and Ultraflo L. The data thus signified that the synergistic effect in xylose release between Celluclast 1.5 L and Ultraflo L is the result of a three-step interaction mechanism involving α- l -arabinofuranosidase and different xylan degrading enzyme activities in the two enzyme preparations.

Hydrolysis of arabinoxylan is an important prerequisite for improved utilization of wheat hemicellulose in the ethanol fermentation industry. This study investigates the individual and combined efficiencies of three commercial, cellulytic and hemicellulytic enzyme preparations, Celluclast 1.5 L, Ultraflo L, and Viscozyme L, in catalyzing the liberation of arabinose and xylose from water-soluble wheat arabinoxylan. Ultraflo L was the best enzyme preparation for releasing arabinose, liberating 53 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 40 degrees C, pH 6). Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose, liberating 26 wt% of the theoretical maximum after 48 h of reaction (10 wt% enzyme/substrate ratio, 50 degrees C, pH 5). The 50:50 mixtures of the enzyme preparations showed no synergistic cooperation in arabinose release, but a synergistic interaction in xylose release was found between Ultraflo L and Celluclast 1.5 L. On the basis of high-performance anion exchange chromatography (HPAEC) analysis of the hydrolysates after enzymatic reaction, we propose that the observed synergism between Celluclast 1.5 L and Ultraflo L is the result of positive interaction between alpha-L-arabinofuranosidase and endo-1,4-beta-xylanase activities present in Ultraflo L that released arabinose, xylobiose and xylotriose, and beta-xylosidase activities in Celluclast 1.5 L, capable of catalyzing the hydrolysis of xylobiose and xylotriose to xylose.

Gasification of spent lignin pellets was used to obtain a gas suitable for energy production. Spent lignin was obtained from second-generation cellulosic ethanol demo plant using wheat straw as feedstock. Gasification of lignin did not give rise to any feeding problems, thus no significant changes were needed in the existing gasification installation. The rise of temperature and steam flow rate favoured the formation of H 2 , while hydrocarbons (C n H m ) and tar contents decreased. The increase of equivalent ratio (ER) also decreased hydrocarbons and tar contents, but syngas higher heating value (HHV) was reduced. The use of natural minerals improved lignin gasification. The presence of dolomite led to the highest H 2 and to the lowest C n H m and tar contents. Results obtained at bench-scale were confirmed at pilot-scale, as similar trends were obtained. However, as the residence time in pilot gasifier was higher, greater gas yields with higher H 2 and CH 4 concentrations were obtained, while tar contents decreased. After syngas hot cleaning and upgrading, the final syngas composition showed to be suitable for a wide range of applications (e.g. energy production and synthesis of chemicals), since it was substantially enriched in hydrogen, whereas tar and heavier gaseous hydrocarbons were completely destroyed.

Background: Mineral elements present in lignocellulosic biomass feedstocks may accumulate in biorefinery process streams and cause technological problems, or alternatively can be reaped for value addition. A better understanding of the distribution of minerals in biomass in response to pretreatment factors is therefore important in relation to development of new biorefinery processes. The objective of the present study was to examine the levels of mineral elements in pretreated wheat straw in response to systematic variations in the hydrothermal pretreatment parameters (pH, temperature, and treatment time), and to assess whether it is possible to model mineral levels in the pretreated fiber fraction. Results: Principal component analysis of the wheat straw biomass constituents, including mineral elements, showed that the recovered levels of wheat straw constituents after different hydrothermal pretreatments could be divided into two groups: 1) Phosphorus, magnesium, potassium, manganese, zinc, and calcium correlated with xylose and arabinose (that is, hemicellulose), and levels of these constituents present in the fiber fraction after pretreatment varied depending on the pretreatment-severity; and 2) Silicon, iron, copper, aluminum correlated with lignin and cellulose levels, but the levels of these constituents showed no severity-dependent trends. For the first group, an expanded pretreatment-severity equation, containing a specific factor for each constituent, accounting for variability due to pretreatment pH, was developed. Using this equation, the mineral levels could be predicted with R 2 > 0.75; for some with R 2 up to 0.96. Conclusion: Pretreatment conditions, especially pH, significantly influenced the levels of phosphorus, magnesium, potassium, manganese, zinc, and calcium in the resulting fiber fractions. A new expanded pretreatment-severity equation is proposed to model and predict mineral composition in pretreated wheat straw biomass

This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose-releasing and xylan-depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel alpha-l-arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water-soluble and water-insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 alpha-l-arabinofuranosidase from H. insolens (Abf II), the GH51 alpha-l-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-beta-xylanase from H. insolens (Xyl III), and a GH3 beta-xylosidase from Trichoderma reesei (beta-xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water-soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water-insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water-soluble wheat arabinoxylan, the water-insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme protein dosage levels were approximately 14, approximately 18, and approximately 27 times lower than the dosages used previously, when the same wheat arabinoxylan substrates were hydrolyzed with a combination of Ultraflo L and Celluclast 1.5 L, two commercially available enzyme preparations produced by H. insolens and T. reesei.

Enzymatic hydrolysis of arabinoxylan is an important prerequisite for the utilization of hemicellulose for ethanol fermentation or for making the low calorie sweetener xylitol by catalytic hydrogenation of the generated xylose. This study focus on cloning and characterization of two industrial relevant beta-xylosidases (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) from Talaromyces emersonii (betaXTE) and Trichoderma reesei (betaXTR) and a comparison of these in relation to hemicellulose hydrolysis using an industrial relevant substrate. Both beta-xylosidases were expressed in A. oryzae and subsequently purified. During the enzymatic hydrolysis of xylobiose, the reaction product of both enzymes was found to be beta-D-xylose proving that the hydrolysis is proceeding via a retaining reaction mechanism. Based on sequence similarities and glycosyl hydrolases family membership, the active site residues of betaXTE and betaXTR are predicted to be Asp 242 and Glu 441, and Asp 264 and Glu 464, respectively. The involvement in catalysis of these carboxyls was examined by modification using the carbodiimide-nucleophile procedure resulting in a complete inactivation of both enzymes. The degree of xylose release from vinasse, an ethanol fermentation by-product, by betaXTE and betaXTR was 12.1% and 7.7%, respectively. Using the beta-xylosidases in combination with the multicomponent enzyme product Ultraflo L, resulted in 41.9% and 40.8% release of xylose, respectively indicating a strong synergistic effect between the exo-acting beta-xylosidases and the endo-1,4-beta-xylanases and alpha-L-arabinofuranosidase in Ultraflo L. There seems to be no measurable differences between the two beta-xylosidases when used in this specific application despite the differences in specific activity and kinetic properties.

This study examined enzyme-catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, "vinasse", resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 degrees C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76-84%, 75-80%, and 43-47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme-catalyzed hydrolysis of arabinoxylan, beta-glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2-6.4 and 41-49 degrees C for arabinose release and from pH 4.9-5.3 and 42-46 degrees C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry matter. The study thus documents that enzymatic arabinoxylan hydrolysis of the vinasse significantly decreases the vinasse viscosity and that a compromise in the dry matter must be found if enzymatic efficiency must be balanced with monosaccharide yields.

We report here the preliminary characterization of oligosaccharides present in an enzyme-treated industrial fermentation residue using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ion trap mass spectrometry (ESI-ITMS), and gas chromatography mass spectrometry (GC-MS). After sample cleaning with carbon graphite columns, analysis of oligosaccharides present in the sample using MALDI-TOF-MS resulted in identification of molecular ions representing sodiated hexose and pentose oligo/polysaccharides. The GC-MS analyses revealed that the signals observed in the mass spectrum for hexose oligomers represent linear structures, whereas the pentose oligomers were identified as arabinoxylans with a (1--4) linked Xylp backbone where the Xylp residues were either not substituted or singly substituted with Araf branching residues at positions C-2 or C-3 of the Xylp ring. Analyses by ESI-ITMS of the signals corresponding to arabinoxylan oligosaccharides with four and five monosaccharide residues showed the presence of isomeric structures differing in degree of branching and localization of the branched residue along the Xylp backbone.

A fast pyrolysis study on lignin and macroalgae (non-conventional biomass) and wood and straw(conventional biomass) were carried out in a pyrolysis centrifugal reactor. The product distributions and energyrecoveries were measured and compared among these biomasses. The fast pyrolysis of macroalgae showed apromising result with on yield of 54 wt% dry ash free basis (daf) and 78% energy recovery in the bio-oil. Thephysiochemical properties of the bio-oils were characterized with respect to higher heating value, molecular massdistribution, viscosity, pH, density and elemental compositions. The lignin and macroalgae oil properties were quitedifferent to those of the conventional oils.