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    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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Biorefining of wheat straw using an acetic and formic acid based organosolv fractionation process
Snelders, J. ; Dornez, E. ; Benjelloun-Mlayah, B. ; Huijgen, W.J.J. ; Wild, P.J. de; Gosselink, R.J.A. ; Gerritsma, J. ; Courtin, C.M. - \ 2014
Bioresource Technology 156 (2014). - ISSN 0960-8524 - p. 275 - 282.
xylan-degrading enzymes - enzymatic-hydrolysis - lignocellulosic biomass - pretreatment - lignin - fermentation - residues - hemicellulose - bioethanol - cellulose
To assess the potential of acetic and formic acid organosolv fractionation of wheat straw as basis of an integral biorefinery concept, detailed knowledge on yield, composition and purity of the obtained streams is needed. Therefore, the process was performed, all fractions extensively characterized and the mass balance studied. Cellulose pulp yield was 48% of straw dry matter, while it was 21% and 27% for the lignin and hemicellulose-rich fractions. Composition analysis showed that 67% of wheat straw xylan and 96% of lignin were solubilized during the process, resulting in cellulose pulp of 63% purity, containing 93% of wheat straw cellulose. The isolated lignin fraction contained 84% of initial lignin and had a purity of 78%. A good part of wheat straw xylan (58%) ended up in the hemicellulose-rich fraction, half of it as monomeric xylose, together with proteins (44%), minerals (69%) and noticeable amounts of acids used during processing.
Characteristics of Wheat Straw Lignins from Ethanol-based Organosolv Treatment
Huijgen, W.J.J. ; Telysheva, G. ; Arshanitsa, A. ; Gosselink, R.J.A. ; Wild, P.J. de - \ 2014
Industrial Crops and Products 59 (2014). - ISSN 0926-6690 - p. 85 - 95.
industrial applications - enzymatic-hydrolysis - technical lignins - kraft lignin - fractionation - pretreatment - biomass - chemicals - bagasse
Non-purified lignins resulting from ethanol-based organosolv fractionation of wheat straw were characterized for the presence of impurities (carbohydrates and ash), functional groups (hydroxyl, carboxyl and methoxyl), phenyl-propanoid structural moieties, molar mass distribution and thermal behavior. In accordance with its herbaceous nature, the syringyl/guaiacyl-ratio of the wheat straw lignins was substantially lower than of Alcell lignin. In addition, the content of p-hydroxyphenyl and carboxyl groups is substantially higher for the wheat straw lignins. The non-purified organosolv lignins had a high purity with 0.4–5.2% carbohydrate impurities, both originating from lignin to carbohydrate complexes and residual organosolv liquor. The use of H2SO4 in the organosolv process improved the lignin yield, but at low acid doses increased the carbohydrate impurities. For applications where a low amount of carbohydrates is important, lignin from a high-temperature autocatalytic organosolv process was found to be preferred. The highest content of total hydroxyl groups was determined when lignins were produced using 30 mM H2SO4 as catalyst or 50% w/w aqueous ethanol as solvent for the organosolv process. Aliphatic hydroxyl groups, the most predominant type of hydroxyl groups present originating for a substantial part from residual carbohydrates, were found to decrease with reaction time and ethanol proportion of the organosolv solvent. The correlations between organosolv process conditions and lignin characteristics determined can facilitate the use of organosolv lignins in value-added applications such as in polymers and resins and as a feedstock for bio-based aromatics.
Characterization of an acetyl esterase from Myceliophthorathermophila C1 able to deacetylate xanthan
Kool, M.M. ; Schols, H.A. ; Wagenknecht, M. ; Hinz, S.W.A. ; Moerschbacher, B.M. ; Gruppen, H. - \ 2014
Carbohydrate Polymers 111 (2014). - ISSN 0144-8617 - p. 222 - 229.
bacterial polysaccharide xanthan - xylan esterases - carbohydrate esterases - xanthomonas-campestris - rheological properties - plant polysaccharides - enzymatic-hydrolysis - gum - transition - families
Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recogni-tion of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthorathermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activ-ity towards xanthan was only observed when xanthan molecules were in the disordered conformation.Optimal performance towards xanthan was observed at 53¿C in the complete absence of salt, a condi-tion favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulasesand analyzed for its repeating units using UPLC–HILIC–ELSD/ESI–MS. This showed that AXE3 specificallyremoves the acetyl groups positioned on the inner mannose and that acetyl groups positioned on theouter mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of allacetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed.
Characterization of Phosphorus in Animal Manures Collected from Three (Dairy, Swine, and Broiler) Farms in China
Li, G. ; Li, H. ; Leffelaar, P.A. ; Shen, J. ; Zhang, F. - \ 2014
PLoS ONE 9 (2014)7. - ISSN 1932-6203
magnetic-resonance-spectroscopy - enzymatic-hydrolysis - organic phosphorus - northwest germany - poultry litter - forms - soil - diets - speciation - fractions
In order to identify the phosphorus species and concentration in animal manure, we comparatively characterized phosphorus in dairy manure, swine manure, and broiler litter, using a sequential procedure, a simplified two-step procedure (NaHCO3/NaOH+EDTA), and a solution Phosphorus-31 Nuclear Magnetic Resonance (31P-NMR) spectroscopy procedure. In the sequential procedure, deionized water extracted 39, 22, and 32%; NaHCO3 extracted 48, 26, and 37%; NaOH extracted 8, 9, and 13.8%; and HCl extracted 3, 42.8, and 17% of the total phosphorus in dairy manure, swine manure and broiler litter, respectively. Total phosphorus extracted by the NaHCO3/NaOH+EDTA procedure was 7.5, 32.4, and 15.8 g P kg-1 for dairy manure, swine manure, and broiler litter, respectively. The solution 31P-NMR procedure detected that 9, 34, and 29% of total phosphorus was phytic acid in dairy manure, swine manure, and broiler litter, respectively. These results show that phosphorus forms, availability, and quantities differ between animal manures, which provides valuable information for P characterization of animal manures in China.
The influence of amylose-LPC complex formation on the susceptibility of wheat starch to amylase
Ahmadi-Abhari, S. ; Woortman, A.J.J. ; Oudhuis, A.A.C.M. ; Hamer, R.J. ; Loos, K. - \ 2013
Carbohydrate Polymers 97 (2013)2. - ISSN 0144-8617 - p. 436 - 440.
slow digestion property - native cereal starches - lipid complexes - enzymatic-hydrolysis - rice starch - in-vivo - digestibility - oligosaccharide - glucose - systems
This study was aimed to assess the role of lysophosphatidylcholine (LPC) in the development of slowly digestible starch (SDS). The influence of LPC, on the enzymatic degradation of diluted 9% wheat starch suspensions (w/w) was investigated, using an in vitro digestion method. Wheat starch suspensions containing 0.5–5% LPC (based on starch) were heated in a Rapid Visco Analyser (RVA) till 95 °C and subjected to enzyme hydrolysis by porcine pancreatic a-amylase at 37 °C for several digestion periods. In vitro digestion measurements demonstrated that complexing starch with 5% LPC leads to a 22% decrease in rate of reducing sugar compared to the reference while the samples containing 0.5% LPC showed an equal digestibility comparable to the control. A clear decrease in the formation of reducing sugars was observed in presence of 2–5% LPC, since the results after 15 min digestion imply the formation of SDS due to the formation of amylose-LPC inclusion complexes. The DSC measurements proved the presence of amylose-LPC inclusion complexes even after 240 min digestion demonstrating the low susceptibility of amylose-V complexes to amylase.
Performance of hemicellulolytic enzymes in culture supernatants from a wide range of fungi on insoluble wheat straw and corn fiber fractions
Gool, M.P. van; Toth, K. ; Schols, H.A. ; Szakacs, G. ; Gruppen, H. - \ 2012
Bioresource Technology 114 (2012). - ISSN 0960-8524 - p. 523 - 528.
xylan-degrading enzymes - enzymatic-hydrolysis - aspergillus-awamori - pretreatment - cellulose - arabinoxylans - fermentation - degradation - adsorption - substrate
Filamentous fungi are a good source of hemicellulolytic enzymes for biomass degradation. Enzyme preparations were obtained as culture supernatants from 78 fungal isolates grown on wheat straw as carbon source. These enzyme preparations were utilized in the hydrolysis of insoluble wheat straw and corn fiber xylan rich fractions. Up to 14% of the carbohydrates in wheat straw and 34% of those in corn fiber were hydrolyzed. The degree of hydrolysis by the enzymes depended on the origin of the fungal isolate and on the complexity of the substrate to be degraded. Penicillium, Trichoderma or Aspergillus species, and some non-identified fungi proved to be the best producers of hemicellulolytic enzymes for degradation of xylan rich materials. This study proves that the choice for an enzyme preparation to efficiently degrade a natural xylan rich substrate, is dependent on the xylan characteristics and could not be estimated by using model substrates.
In vitro pH-Stat protein hydrolysis of feed ingredients for Atlantic cod, Gadus morhua. 2. In vitro protein digestibility of common and alternative feed ingredients
Tibbetts, S. ; Verreth, J.A.J. ; Lall, S.P. - \ 2011
Aquaculture 319 (2011)3-4. - ISSN 0044-8486 - p. 407 - 416.
melanogrammus-aeglefinus l - salmon salmo-salar - apparent digestibility - rainbow-trout - juvenile haddock - nonstarch polysaccharide - gastrointestinal model - nutrient digestibility - enzymatic-hydrolysis - incorporation level
Using enzyme fractions isolated from the pyloric caeca of farmed Atlantic cod, the in vitro degree of protein hydrolysis (DH) of numerous conventional and novel feed ingredients were measured by a closed-system pH-Stat assay. Regression equations describing the relationship between in vivo apparent protein digestibility (ADC) and in vitro protein DH were used to predict in vitro protein ADC. The equations resulted in good correlation (
Hydrolysis of ß-casein by the cell-envelope-located PI-type protease of Lactococcus lactis: A modelling approach
Munoz-Tamayo, R. ; Groot, J. de; Bakx, E.J. ; Wierenga, P.A. ; Gruppen, H. ; Zwietering, M.H. ; Sijtsma, L. - \ 2011
International Dairy Journal 21 (2011)10. - ISSN 0958-6946 - p. 755 - 762.
acid bacteria - kappa-casein - streptococcus-cremoris - enzymatic-hydrolysis - milk - kinetics - proteolysis - strains - peptide - system
Lactic acid bacteria possess extracellular proteases that hydrolyze milk proteins. This work aimed to describe mathematically the hydrolysis of intact ß-casein by the PI-type protease of Lactococcus lactis, using a mutant strain that lacks the oligopeptide transport system. Experiments were performed under a broad range of initial protein concentrations (17–196 µm), at constant enzyme concentration or at constant initial enzyme/substrate ratio. Hydrolysis of the intact ß-casein was monitored and quantified. Four kinetic functions were evaluated to describe the hydrolysis: First-order, nth-order, Michaelis–Menten, and competitive inhibition kinetics. The hydrolysis rate was found to depend on the initial protein concentration, due to the micellisation behaviour of ß-casein. This effect was accounted for by modifying the kinetic functions. The modified competitive inhibition model provided the lowest mean square error. This model has only three parameters and described the hydrolysis of intact ß-casein effectively for a broad range of initial conditions.
Effect of neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by Caco-2 cells
Ou, K.Q. ; Liu, Y.Z. ; Zhang, L.B. ; Yang, X.G. ; Huang, Z.W. ; Nout, M.J.R. ; Liang, J. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)8. - ISSN 0021-8561 - p. 4894 - 4900.
in-vitro digestion - enzymatic-hydrolysis - culture model - phytic acid - milk - peptides - availability - bioavailability - dialyzability - solubility
Effects of enzymatic hydrolysates of whey protein concentrates (WPC) on iron absorption were studied using in vitro digestion combined with Caco-2 cell models for improved iron absorption. Neutrase- and papain-treated WPC could improve iron absorption; especially hydrolysates by Neutrase could significantly increase iron absorption to 12.8% compared to 3.8% in the control. Hydrolysates by alcalase had negative effects to the lowest at 0.57%. Two new bands at molecular weights (MW) around and below 10 kDa occurred at tricine-SDS-PAGE of hydrolysates by Neutrase, and one new band at MW below 10 kDa occurred in hydrolysates by papain. No new band was observed in hydrolysates by alcalase. Concentration of free amino acids indicated that, except for tyrosine and phenylalanine, amino acids in papain-treated hydrolysates were higher than that of alcalase, and no cysteine and proline were found in hydrolysates by alcalase. The results suggested that hydrolysate by Neutrase-treated WPC is a promising facilitator for iron absorption. Peptides of MW around and lower than 10 kDa and aspartic acid, serine, glutamic acid, glycin, cysteine, histidine, and proline may be contributors to enhancement
Optimization of the dilute maleic acid pretreatment of wheat straw
Kootstra, A.M.J. ; Beeftink, H.H. ; Scott, E.L. ; Sanders, J.P.M. - \ 2009
Biotechnology for Biofuels 2 (2009). - ISSN 1754-6834 - p. 31 - 31.
cellulose hydrolysis - enzymatic-hydrolysis - ethanologenic yeast - high-temperature - organic-acids - corn stover - pig diets - d-xylose - biomass - degradation
Background - In this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to €/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables. Results - When costs are disregarded, an almost complete glucan conversion to glucose can be reached (90% from solids, 7%-10% in liquid), after enzymatic hydrolysis. During the pretreatment, up to 90% of all xylan is converted to monomeric xylose. Taking cost factors into account, the optimal process conditions are: 50 min at 170°C, with 46 mM maleic acid, resulting in a yield of 65 €/Mg (megagram = metric ton) dry straw, consisting of 68 €/Mg glucose benefits (from solids: 85% of all glucan), 17 €/Mg xylose benefits (from liquid: 80% of all xylan), 17 €/Mg maleic acid costs, 2.0 €/Mg heating costs and 0.68 €/Mg NaOH costs. In all but the most severe of the studied conditions, furfural formation was so limited that associated costs are considered negligible. Conclusions - After the dilute maleic acid pretreatment and subsequent enzymatic hydrolysis, almost complete conversion of wheat straw glucan and xylan is possible. Taking maleic acid replenishment, heating, neutralization and furfural formation into account, the optimum in the dilute maleic acid pretreatment of wheat straw in this study is 65 €/Mg dry feedstock. This is reached when process conditions are: 50 min at 170°C, with a maleic acid concentration of 46 mM. Maleic acid replenishment is the most important of the studied cost factors
Effects of thermo-chemical pre-treatment on anaerobic biodegradability and hydrolysis of lignocellulosic biomass
Fernandes, T. ; Klaasse Bos, G.J. ; Zeeman, G. ; Sanders, J.P.M. ; Lier, J.B. van - \ 2009
Bioresource Technology 100 (2009)9. - ISSN 0960-8524 - p. 2575 - 2579.
corn stover - enzymatic-hydrolysis - wheat-straw - technologies - switchgrass - manure - acid
The effects of different thermo-chemical pre-treatment methods were determined on the biodegradability and hydrolysis rate of lignocellulosic biomass. Three plant species, hay, straw and bracken were thermo-chemically pre-treated with calcium hydroxide, ammonium carbonate and maleic acid. After pre-treatment, the plant material was anaerobically digested in batch bottles under mesophilic conditions for 40 days. From the pre-treatment and subsequent anaerobic digestion experiments, it was concluded that when the lignin content of the plant material is high, thermo-chemical pre-treatments have a positive effect on the biodegradability of the substrate. Calcium hydroxide pre-treatment improves the biodegradability of lignocellulosic biomass, especially for high lignin content substrates, like bracken. Maleic acid generates the highest percentage of dissolved COD during pre-treatment. Ammonium pre-treatment only showed a clear effect on biodegradability for straw.
Pressure-Aided Proteolysis of ß-Casein
Bruins, M.E. ; Creusot, N.P. ; Gruppen, H. ; Janssen, A.E.M. ; Boom, R.M. - \ 2009
Journal of Agricultural and Food Chemistry 57 (2009)12. - ISSN 0021-8561 - p. 5529 - 5534.
high hydrostatic-pressure - lactoglobulin-b - enzymatic-hydrolysis - milk-proteins - whey proteins - chymotrypsin - association - peptides - trypsin - immunoreactivity
ß-Casein, which is present in the form of micelles at atmospheric pressure, has been hydrolyzed during pressure treatment to improve the accessibility of the protein. Two proteolytic enzymes with different specificities were used. Trypsin was aimed at mainly hydrolyzing hydrophilic segments of ß-casein and chymotrypsin at hydrolyzing hydrophobic segments of ß-casein. Measurements on aggregation during hydrolysis at atmospheric pressure showed that probably not micelle disruption, but disruption of much larger aggregates, occurs in the process. Peptide profiles were measured via reversed-phase chromatography. Measurements on enzyme activity after pressure treatments showed that trypsin was inactivated by pressure, which could explain all differences in peptide profiles compared to atmospheric experiments. Pressure did not influence the reaction mechanism, probably because the hydrophilic part of ß-casein is sufficiently accessible. However, chymotryptic proteolysis under pressure yielded new peptides that could not be explained by a change in enzyme activity. Here, pressure altered the mechanism of hydrolysis, by changing either enzyme specificity or substrate accessibility, which led to different peptides that can have different properties
Modelling ethanol production from cellulose: separate hydrolysis and fermentation versus simultaneous saccharification and fermentation
Drissen, R.E.T. ; Maas, R.H.W. ; Tramper, J. ; Beeftink, H.H. - \ 2009
Biocatalysis and Biotransformation 27 (2009)1. - ISSN 1024-2422 - p. 27 - 35.
enzymatic-hydrolysis - lignocellulose - biomass - conversion - softwood - technology - inhibition - substrate - economics
In ethanol production from cellulose, enzymatic hydrolysis, and fermentative conversion may be performed sequentially (separate hydrolysis and fermentation, SHF) or in a single reaction vessel (simultaneous saccharification and fermentation, SSF). Opting for either is essentially a trade-off between optimal temperatures and inhibitory glucose concentrations on the one hand (SHF) vs. sub-optimal temperatures and ethanol-inhibited cellulolysis on the other (SSF). Although the impact of ethanol on cellobiose hydrolysis was found to be negligible, formation of glucose and cellobiose from cellulose were found to be significantly inhibited by ethanol. A previous model for the kinetics of enzymatic cellulose hydrolysis was, therefore, extended with enzyme inhibition by ethanol, thus allowing a rational evaluation of SSF and SHF. The model predicted SSF processing to be superior. The superiority of SSF over SHF (separate hydrolysis and fermentation) was confirmed experimentally, both with respect to ethanol yield on glucose (0.41 g g-1 for SSF vs. 0.35 g g-1 for SHF) and ethanol production rate, being 30% higher for an SSF type process. High conversion rates were found to be difficult to achieve since at a conversion rate of 52% in a SSF process the reaction rate dropped to 5% of its initial value. The model, extended with the impact of ethanol on the cellulase complex proved to predict reaction progress accurately.
Pretreatments to enhance the digestibility of lignocellulosic biomass
Hendriks, A.T.W.M. ; Zeeman, G. - \ 2009
Bioresource Technology 100 (2009)1. - ISSN 0960-8524 - p. 10 - 18.
steam-explosion pretreatment - wet oxidation pretreatment - ethanol-tolerant mutant - yellow-poplar sawdust - liquid hot-water - enzymatic-hydrolysis - clostridium-thermocellum - wheat-straw - corn stover - lime pretreatment
Lignocellulosic biomass represents a rather unused source for biogas and ethanol production. Many factors, like lignin content, crystallinity of cellulose, and particle size, limit the digestibility of the hemicellulose and cellulose present in the lignocellulosic biomass. Pretreatments have as a goal to improve the digestibility of the lignocellulosic biomass. Each pretreatment has its own effect(s) on the cellulose, hemicellulose and lignin; the three main components of lignocellulosic biomass. This paper reviews the different effect(s) of several pretreatments on the three main parts of the lignocellulosic biomass to improve its digestibility. Steam pretreatment, lime pretreatment, liquid hot water pretreatments and ammonia based pretreatments are concluded to be pretreatments with high potentials. The main effects are dissolving hemicellulose and alteration of lignin structure, providing an improved accessibility of the cellulose for hydrolytic enzymes.
Differential effects of mineral and organic acids on the kinetics of arabinose degradation under lignocellulose pretreatment conditions
Kootstra, A.M.J. ; Mosier, N.S. ; Scott, E.L. ; Beeftink, H.H. ; Sanders, J.P.M. - \ 2009
Biochemical Engineering Journal 43 (2009)1. - ISSN 1369-703X - p. 92 - 97.
saccharomyces-cerevisiae - high-temperature - fumaric-acid - cellulose hydrolysis - enzymatic-hydrolysis - rhizopus-oryzae - corn stover - pig diets - d-xylose - ethanol
Sugar degradation occurs during acid-catalyzed pretreatment of lignocellulosic biomass at elevated temperatures, resulting in degradation products that inhibit microbial fermentation in the ethanol production process. Arabinose, the second most abundant pentose in grasses like corn stover and wheat straw, degrades into furfural. This paper focuses on the first-order rate constants of arabinose (5 g/L) degradation to furfural at 150 and 170 °C in the presence of sulfuric, fumaric, and maleic acid and water alone. The calculated degradation rate constants (kd) showed a correlation with the acid dissociation constant (pKa), meaning that the stronger the acid, the higher the arabinose degradation rate. However, de-ionized water alone showed a catalytic power exceeding that of 50 mM fumaric acid and equaling that of 50 mM maleic acid. This cannot be explained by specific acid catalysis and the shift in pKw of water at elevated temperatures. These results suggest application of maleic and fumaric acid in the pretreatment of lignocellulosic plant biomass may be preferred over sulfuric acid. Lastly, the degradation rate constants found in this study suggest that arabinose is somewhat more stable than its stereoisomer xylose under the tested conditions
Hydrolysis of Brewers' Spent Grain by Carbohydrate Degrading Enzymes
Forssell, P. ; Kontkanen, H. ; Schols, H.A. ; Hinz, S.W.A. ; Eijsink, V.G.H. ; Treimo, J. ; Robertson, J.A. ; Waldron, K.W. ; Faulds, C.B. ; Buchert, J. - \ 2008
Journal of the Institute of Brewing 114 (2008)4. - ISSN 0046-9750 - p. 306 - 314.
enzymatic-hydrolysis - ferulic acid - baking properties - cereal products - cell-walls - barley - release - fiber - oligosaccharides - solubilization
In this work four commercial cellulase-hemicellulase mixtures with different activity profiles were used for solubilization of carbohydrates from brewers' spent grain (BSG). After the enzyme treatment, both the solubilised fraction and the unhydrolysed residue were characterized. Treatment with 5,000 nkat/g xylanase for 5 h at 50 degrees C resulted in the solubilisation of 13-14% of the BSG dry weight as monosaccharides. This corresponded to the solubilisation of 26-28% of the original carbohydrates and 30-34% of original arabinoxylans, depending on the enzyme cocktail used. The relatively low hydrolysis level indicates that the majority of the BSG biomass is rather recalcitrant towards the cellulose-hemicellulase enzyme mixtures applied in this study. The enzyme activity profile had a crucial impact on the chemistry of the oligosaccharides produced through the solubilisation of BSG. The presence of feruloyl esterase (FAE) activity in the enzyme cocktail resulted in the production of free ferulic acid, arabinoxylo-oligosaccharides and their corresponding monomers. However, when the enzyme mixture was devoid of FAE activity, ferulic acid was still bound to the oligosaccharides. The unhydrolysed fraction was still found to contain over 40% of carbohydrates after enzymatic treatment despite the extensive enzyme dosages used. The protein fraction remained largely unaffected (i.e. insoluble) by the carbohydrate-disrupting enzyme treatments. In addition to the recalcitrant carbohydrates, the residue was enriched with lignin and lipid type structures.
Pilot-scale conversion of lime-treated wheat straw into bioethanol: quality assessment of bioethanol and valorization of side streams by anaerobic digestion and combustion
Maas, R.H.W. ; Bakker, R.R.C. ; Boersma, A.R. ; Bisschops, I. ; Pels, J.R. ; Jong, E. de; Weusthuis, R.A. ; Reith, H. - \ 2008
Biotechnology for Biofuels 1 (2008). - ISSN 1754-6834
ethanol-production - lignocellulosic materials - saccharomyces-cerevisiae - enzymatic-hydrolysis - biomass - pretreatment - fermentation - acid - saccharification - softwood
The limited availability of fossil fuel sources, worldwide rising energy demands and anticipated climate changes attributed to an increase of greenhouse gasses are important driving forces for finding alternative energy sources. One approach to meeting the increasing energy demands and reduction of greenhouse gas emissions is by large-scale substitution of petrochemically derived transport fuels by the use of carbon dioxide-neutral biofuels, such as ethanol derived from lignocellulosic material. Results This paper describes an integrated pilot-scale process where lime-treated wheat straw with a high dry-matter content (around 35% by weight) is converted to ethanol via simultaneous saccharification and fermentation by commercial hydrolytic enzymes and bakers' yeast (Saccharomyces cerevisiae). After 53 hours of incubation, an ethanol concentration of 21.4 g/liter was detected, corresponding to a 48% glucan-to-ethanol conversion of the theoretical maximum. The xylan fraction remained mostly in the soluble oligomeric form (52%) in the fermentation broth, probably due to the inability of this yeast to convert pentoses. A preliminary assessment of the distilled ethanol quality showed that it meets transportation ethanol fuel specifications. The distillation residue, which contained non-hydrolysable and non-fermentable (in)organic compounds, was divided into a liquid and solid fraction. The liquid fraction served as substrate for the production of biogas (methane), whereas the solid fraction functioned as fuel for thermal conversion (combustion), yielding thermal energy, which can be used for heat and power generation. Conclusion Based on the achieved experimental values, 16.7 kg of pretreated wheat straw could be converted to 1.7 kg of ethanol, 1.1 kg of methane, 4.1 kg of carbon dioxide, around 3.4 kg of compost and 6.6 kg of lignin-rich residue. The higher heating value of the lignin-rich residue was 13.4 MJ thermal energy per kilogram (dry basis).
Improving the corn-ethanol industry: studying protein separation techniques to obtain higher value added product options for distillers grains
Brehmer, B. ; Bals, B. ; Sanders, J.P.M. ; Dale, B. - \ 2008
Biotechnology and Bioengineering 101 (2008)1. - ISSN 0006-3592 - p. 49 - 61.
enzymatic-hydrolysis - pretreatment - biomass - energy - stover
Currently in America the biofuel ethanol is primarily being produced by the dry grind technique to obtain the starch contained in the corn grains and subsequently subjected to fermentation. This so-called 1st generation technology has two setbacks; first the lingering debate whether its life cycle contributes to a reduction of fossil fuels and the animal feed sectors future supply/demand imbalance caused by the co-product dry distillers grains (DDGS). Additional utilization of the cellulosic components and separation of the proteins for use as chemical precursors have the potential to alleviate both setbacks. Several different corn feedstock layouts were treated with 2nd generation ammonia fiber expansion (AFEX) pre-treatment technology and tested for protein separation options (protease solubilization). The resulting system has the potential to greatly improve ethanol yields with lower bioprocessing energy costs and satisfy a significant portion of the organic chemical industry.
A stochastic model for predicting dextrose equivalent and saccharide composition during hydrolysis of starch by alpha-amylase
Besselink, T. ; Baks, T. ; Janssen, A.E.M. ; Boom, R.M. - \ 2008
Biotechnology and Bioengineering 100 (2008)4. - ISSN 0006-3592 - p. 684 - 697.
monte-carlo-simulation - bacillus-licheniformis - enzymatic-hydrolysis - soluble starch - kinetic-model - potato starch - amylopectin - amylolysis - enzymes - thermostability
A stochastic model was developed that was used to describe the formation and breakdown of all saccharides involved during -amylolytic starch hydrolysis in time. This model is based on the subsite maps found in literature for Bacillus amyloliquefaciens -amylase (BAA) and Bacillus licheniformis -amylase (BLA). Carbohydrate substrates were modeled in a relatively simple two-dimensional matrix. The predicted weight fractions of carbohydrates ranging from glucose to heptasaccharides and the predicted dextrose equivalent showed the same trend and order of magnitude as the corresponding experimental values. However, the absolute values were not the same. In case a well-defined substrate such as maltohexaose was used, comparable differences between the experimental and simulated data were observed indicating that the substrate model for starch does not cause these deviations. After changing the subsite map of BLA and the ratio between the time required for a productive and a non-productive attack for BAA, a better agreement between the model data and the experimental data was observed. Although the model input should be improved for more accurate predictions, the model can already be used to gain knowledge about the concentrations of all carbohydrates during hydrolysis with an -amylase. In addition, this model also seems to be applicable to other depolymerase-based systems
Effect of gelatinization and hydrolysis conditions on the selectivity of starch hydrolysis with alpha-amylase from Bacillus licheniformis
Baks, T. ; Bruins, M.E. ; Matser, A.M. ; Janssen, A.E.M. ; Boom, R.M. - \ 2008
Journal of Agricultural and Food Chemistry 56 (2008)2. - ISSN 0021-8561 - p. 488 - 495.
high hydrostatic-pressure - wheat-starch - enzymatic-hydrolysis - phase-transformations - temperature - mechanism - enzymes - stability - complexes - products
Enzymatic hydrolysis of starch can be used to obtain various valuable hydrolyzates with different compositions. The effects of starch pretreatment, enzyme addition point, and hydrolysis conditions on the hydrolyzate composition and reaction rate during wheat starch hydrolysis with ¿-amylase from Bacillus licheniformis were compared. Suspensions of native starch or starch gelatinized at different conditions either with or without enzyme were hydrolyzed. During hydrolysis, the oligosaccharide concentration, the dextrose equivalent, and the enzyme activity were determined. We found that the hydrolyzate composition was affected by the type of starch pretreatment and the enzyme addition point but that it was just minimally affected by the pressure applied during hydrolysis, as long as gelatinization was complete. The differences between hydrolysis of thermally gelatinized, high-pressure gelatinized, and native starch were explained by considering the granule structure and the specific surface area of the granules. These results show that the hydrolyzate composition can be influenced by choosing different process sequences and conditions.
Kinetics of Acid Hydrolysis of Water-Soluble Spruce O-Acetyl Galactoglucomannans
Xu, C. ; Pranovich, A. ; Vahasalo, L. ; Hemming, J. ; Holmbom, B. ; Schols, H.A. ; Willfor, S. - \ 2008
Journal of Agricultural and Food Chemistry 56 (2008)7. - ISSN 0021-8561 - p. 2429 - 2435.
molecular-weight changes - thermomechanical pulp - enzymatic-hydrolysis - degradation - chitosan - depolymerization - polysaccharides - products - polymers
Water-soluble O-acetyl galactoglucomannan (GGM) is a softwood-derived polysaccharide, which can be extracted on an industrial scale from wood or mechanical pulping waters and now is available in kilogram scale for research and development of value-added products. To develop applications of GGM, information is needed on its stability in acidic conditions. The kinetics of acid hydrolysis of GGM was studied at temperatures up to 90 °C in the pH range of 1¿3. Molar mass and molar mass distribution were determined using size exclusion chromatography with multiangle laser light scattering and refractive index detection. The molar mass of GGM decreased considerably with treatment time at temperatures above 70 °C and pH below 2. The molar mass distribution broadened with hydrolysis time. A first-order kinetic model was found to match the acid hydrolysis. The reaction rate constants at various pH values and temperatures were calculated on the basis of the first-order kinetic model. Furthermore, the activation energy, E, was obtained from the Arrhenius plot. The activation energy E was 150 kJ mol¿1 for acid hydrolysis of spruce GGM. The apparent rate constant during acid hydrolysis increased by a factor of 10 with a decrease in pH by 1 unit, regardless of temperature. In addition, gas chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were applied to study the released GGM monomers and oligomers. Keywords: Kinetics; acid hydrolysis; galactoglucomannans; oligosaccharides; stability.
Towards an optimal process for gelatinisation and hydrolysis of highly concentrated starch-water mixtures with alpha-amylase from B. licheniformis
Baks, T. ; Kappen, F.H.J. ; Janssen, A.E.M. ; Boom, R.M. - \ 2008
Journal of Cereal Science 47 (2008)2. - ISSN 0733-5210 - p. 214 - 225.
twin-screw extruder - wheat-starch - enzymatic-hydrolysis - extrusion-cooking - corn starch - sago starch - model - liquefaction - degradation - kinetics
The enzymatic hydrolysis of starch is usually carried out with 30¿35 w/w% starch in water. Higher substrate concentrations (50¿70 w/w%) were reached by using a twin-screw extruder for gelatinisation and for mixing enzyme with gelatinised starch prior to enzymatic hydrolysis in a batch reactor. The aim of this study was to determine which parameters are important for gelatinisation of wheat starch and to investigate the effects of different extrusion conditions on the enzymatic hydrolysis. After extrusion, the degree of gelatinisation was measured. During hydrolysis, the carbohydrate composition, the dextrose equivalent (DE) and the alpha-amylase activity were measured. Gelatinisation measurements showed that mechanical forces lowered the temperature required for complete gelatinisation. During hydrolysis experiments, high DEs were observed even if starch was not completely gelatinised during extrusion. Due to high substrate concentrations, the residual alpha-amylase activity remained high throughout enzymatic hydrolysis, although high temperatures were used. Increased substrate concentrations did not affect the carbohydrate composition of the product. Furthermore, the time required for the batch hydrolysis step could be varied by choosing a different enzyme-to-substrate ratio. This article provides a basis for detailed optimisation of this process to develop an industrial-scale process at high substrate concentrations.
A generic model for glucose production from various cellulose sources by a commercial cellulase complex
Drissen, R.E.T. ; Maas, R.H.W. ; Maarel, M.J. van der; Kabel, M.A. ; Schols, H.A. ; Tramper, J. ; Beeftink, H.H. - \ 2007
Biocatalysis and Biotransformation 25 (2007)6. - ISSN 1024-2422 - p. 419 - 429.
research-and-development - pretreated wheat-straw - enzymatic-hydrolysis - simultaneous saccharification - aspergillus-niger - saccharomyces-cerevisiae - fermentation process - ethanol - lignocellulose - technology
The kinetics of cellulose hydrolysis by commercially available Cellubrix were described mathematically, with Avicel and wheat straw as substrates. It was demonstrated that hydrolysis could be described by three reactions: direct glucose formation and indirect glucose formation via cellobiose. Hydrolysis did not involve any soluble oligomers apart from low amounts of cellobiose. Phenomena included in the mathematical model were substrate limitation, adsorption of enzyme onto substrate, glucose inhibition, temperature dependency of reaction rates, and thermal enzyme inactivation. In addition, substrate heterogeneity was described by a recalcitrance constant. Model parameters refer to both enzyme characteristics and substrate-specific characteristics. Quantitative model development was carried out on the basis of Avicel hydrolysis. In order to describe wheat straw hydrolysis, wheat straw specific parameter values were measured. Updating the pertinent parameters for wheat straw yielded a satisfactory description of wheat straw hydrolysis, thus underlining the generic potential of the model.
Comparison of methods to determine the degree of gelatinisation for both high and low starch concentrations
Baks, T. ; Ngene, I.S. ; Soest, J.J.G. van; Janssen, A.E.M. ; Boom, R.M. - \ 2007
Carbohydrate Polymers 67 (2007)4. - ISSN 0144-8617 - p. 481 - 490.
differential scanning calorimetry - high hydrostatic-pressure - partial molar volumes - potato starches - water-content - wheat-starch - birefringence measurements - crystalline polymorph - phase-transformations - enzymatic-hydrolysis
A general procedure was developed to measure the degree of gelatinisation in samples over a broad concentration range. Measurements based on birefringence, DSC (Differential scanning calorimetry), X-ray and amylose-iodine complex formation were used. If a 10 w/w % wheat starch-water mixture was used, each method resulted in approximately the same degree of gelatinisation vs. temperature curve. In case the gelatinisation of a 60 w/w % wheat starch-water mixture was followed as a function of the temperature, each method resulted in a different degree of gelatinisation vs. temperature curve. DSC and X-ray measurements are preferred, because they can be used to determine when the final stage of the gelatinisation process has been completed. Birefringence and amylose-iodine complex formation measurements are suitable alternatives if DSC and X-ray equipment is not available, but will lead to different results. The differences between the methods can be explained by considering the phenomena that take place during the gelatinisation at limiting water conditions. Based on the experimental data obtained with DSC and X-ray measurements, the gelatinisation of 10 w/w % and 60 w/w % wheat starch-water mixtures started at the same temperature (approximately 50 °C). However, complete gelatinisation was reached at different temperatures (approximately 75 °C and 115 °C for, respectively, 10 w/w % and 60 w/w % wheat starch-water mixtures) according to the experimental DSC and X-ray data. These results are in accordance with independent DSC measurements that were carried out. The Flory equation was adapted to provide a quantitative explanation for the curves describing the degree of starch gelatinisation as a function of the starch-water ratio and the temperature. The gelatinisation curves that were obtained with the model are in good agreement with the experimentally determined curves. The parameters Tm0, ¿Hu and ¿12 that resulted in the lowest sum of the squared residuals are 291 ± 63 °C, 29.2 ± 3.9 kJ/mol and 0.53 ± 0.05 (95% confidence interval). These values agree with other values reported in literature
Opposite Contributions of Glycinin- and ß-Conglycinin-Derived Peptides to the Aggregation Behavior of Soy Protein Isolate Hydrolysates
Kuipers, B.J.H. ; Koningsveld, G.A. van; Alting, A.C. ; Driehuis, F. ; Voragen, A.G.J. ; Gruppen, H. - \ 2006
Food Biophysics 1 (2006)4. - ISSN 1557-1858 - p. 178 - 188.
heat-induced gelation - soybean proteins - structural characteristics - emulsifying properties - enzymatic-hydrolysis - limited proteolysis - physical-properties - alpha-lactalbumin - gel properties - whey proteins
The aggregation behavior as a function of pH was studied for hydrolysates obtained by hydrolysis of soy protein isolate (SPI) and glycinin- and ß-conglycinin-rich protein fractions with subtilisin Carlsberg. The substrates were hydrolyzed up to degrees of hydrolysis (DH) of 2.2% and 6.5%. Compared with nonhydrolyzed SPI, a decrease in solubility was observed for the hydrolysates of SPI [0.8% (w/v) protein, I¿=¿0.03 M] around neutral pH. At pH 8.0, glycinin hydrolysates had a much lower solubility (~43% and 60%, respectively, for DH 2.2% and 6.5%) than SPI and ß-conglycinin-derived hydrolysates, which were almost completely soluble. Peptides that aggregated were all larger than 5 kDa, and as estimated by size-exclusion chromatography their composition was almost independent of the aggregation pH. The solubility of hydrolysates of SPIs with a varying glycinin and ß-conglycinin composition showed that glycinin-derived peptides are the driving force for the lower solubility of SPI hydrolysates. The solubility of SPI hydrolysates at pH 8.0 was shown not to be the sum of that of glycinin and ß-conglycinin hydrolysates. Assuming that the separate hydrolysis of glycinin and ß-conglycinin did not differ from that in the mixture (SPI), this indicates that ß-conglycinin-derived peptides have the ability to inhibit glycinin-derived peptide aggregation.
Starch hydrolysis under low water conditions: a conceptual process design
Veen, M.E. van der; Veelaert, S. ; Goot, A.J. van der; Boom, R.M. - \ 2006
Journal of Food Engineering 75 (2006)2. - ISSN 0260-8774 - p. 178 - 186.
thermostable alpha-amylase - niger glucoamylase-i - twin-screw extruder - enzymatic-hydrolysis - extrusion-cooking - corn starch - reactor - kinetics - glucose - liquefaction
A process concept is presented for the hydrolysis of starch to glucose in highly concentrated systems. Depending on the moisture content, the process consists of two or three stages. The two-stage process comprises combined thermal and enzymatic liquefaction, followed by enzymatic saccharification. The three-stage process starts with shear induced melting of starch, followed by enzymatic liquefaction and saccharification. At a low moisture content, the shear stress needed to completely melt corn starch is so high that significant enzyme inactivation cannot be avoided, which leads to a need for separating starch melting and liquefaction in two separate processing steps. Assuming the use of currently available enzymes, the final product composition was estimated to contain 69-93% glucose, starting with respectively 65% and 35% dry starch. These results showed that the formation of side-products, mainly isomaltose and isomaltotriose, increased with increasing dry matter content. Increasing the dry matter content from 35% to 65% resulted in increasing reactor productivity of 17%, while the amount of water that should be removed from the system was reduced by 87%. (c) 2005 Elsevier Ltd. All rights reserved.
Characterisation and foaming properties of hydrolysates derived from rapeseed isolate
Larré, C. ; Mulder, W.J. ; Sánchez-Vioque, R. ; Lazko, J. ; Bérot, S. ; Guéguen, J. ; Popineau, Y. - \ 2006
Colloids and Surfaces. B: Biointerfaces 49 (2006)1. - ISSN 0927-7765 - p. 40 - 48.
brassica-napus l. - emulsifying properties - functional-properties - enzymatic-hydrolysis - beta-lactoglobulin - plant-proteins - peptides - gluten - albumin - whey
Two hydrolysis methods used to obtain rapeseed isolate derivates were compared: chemical hydrolysis performed under alkaline conditions and pepsic proteolysis performed under acidic conditions. The mean molecular weights obtained for the hydrolysates varied from 26 to 2.5 kDa, depending on the level of hydrolysis. Further characterisation showed that, at the same level of hydrolysis, the chemical hydrolysates differed by their charges and hydrophobicity from those derived from enzymatic digestion. Analysis of the foaming properties showed, for both cases, that a limited degree of hydrolysis, around 3%, was sufficient to optimise the foaming properties of the isolate despite the different physicochemical properties of the peptides generated. The study of foaming properties at basic, neutral and acidic pHs showed that the hydrolysate solutions yielded dense foams which drained slowly and which maintained a very stable volume under the three pH conditions tested.
Production of glucose syrups in highly concentrated systems
Veen, M.E. van der; Goot, A.J. van der; Boom, R.M. - \ 2005
Biotechnology Progress 21 (2005)2. - ISSN 8756-7938 - p. 598 - 602.
enzymatic-hydrolysis - alpha-glucosidase - kinetic-model - wheat-starch - glucoamylase - optimization - mechanism - extruder - reactor
We have investigated the hydrolysis of maltodextrins in a high concentration (up to 70%), by means of enzymatic and acid catalysis. The study revealed that the equilibrium compositions of the catalyzed reactions were kinetically determined by the selectivity of the catalyst, the substrate concentration and the reaction time. A model comprising a set of two kinetic equations was used to describe the hydrolysis and condensation reactions of glucoamylase-catalyzed reactions, even to highly concentrated systems. Increased substrate concentration resulted in the formation of more condensation products. The enzyme inhibition was low and was found to be independent of the substrate concentration.
Shear induced inactivation of a-amylase in a plain shear field
Veen, M.E. van der; Iersel, D.G. van; Goot, A.J. van der; Boom, R.M. - \ 2004
Biotechnology Progress 20 (2004)4. - ISSN 8756-7938 - p. 1140 - 1145.
twin-screw extruder - enzymatic-hydrolysis - starch - deactivation - cellulase - reactor - transglutaminase - kinetics - enzymes - beta
A newly developed shearing device was used to study shear-induced inactivation of thermostable alpha-amylase in a plain shear field, under conditions comparable to extrusion. The results show that the inactivation can be described well with a first-order process, in which the inactivation energy largely depends on the shear stress, instead of specific mechanical energy or strain history. The resulting dependency of the rate of inactivation on the shear stress is very strong and nonlinear, which leads to the conclusion that in many cases the maximally applied shear stress determines the inactivation. Quantification of the inactivation rates gives design criteria for the application of enzymes in more viscous systems than conventionally used, provided that the reactor is designed such that no peak shear stresses occur.
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