Hydrolysis Of Pretreated Wheat Straw Research Articles

Partial characterization of β-glucosidase, β-xylosidase, and α-l-arabinofuranosidase from Jiangella alba DSM 45237 and their potential in lignocellulose-based biorefining

Jiangella alba DSM 45237 exhibited excellent extracellular β-glucosidase (1.03 ± 0.09 U/mL), β-xylosidase (16.29 ± 0.23 U/mL), and α-l-arabinofuranosidase (7.00 ± 0.09 U/mL) production in the growth media containing 15 g/L wheat straw pretreated with NaOH. The optimum temperature was 40 °C for β-glucosidase and β-xylosidase, whereas it was 50 °C for α-l-arabinofuranosidase. Among them, α-l-arabinofuranosidase was relatively stable at 60 and 70 °C. Enzymes showed maximum activity at pH 8.0. Enzymes, particularly β-glucosidase and α-l-arabinofuranosidase, were able to tolerate NaCl up to a final concentration of 12% (v/w). Among solvents, only ethanol and methanol increased the β-glucosidase activity. The majority of solvents did not significantly affect β-xylosidase activity but increased α-l-arabinofuranosidase activity. Except for phenol, other lignocellulose-derived compounds did not cause a significant activity loss in enzymes. Some of them, such as vanillic acid and acetic acid, have even increased the activity of enzymes. Hydrolysis of pretreated wheat straw using the crude enzyme from J. alba DSM 45237 released 160.9 mg/gds reducing sugars. Analysis of hydrolysis products with thin layer chromatography (TLC) showed that major products were 5C sugars. This is the first report related to the characterization of β-glucosidase, β-xylosidase, and α-l-arabinofuranosidase from J.alba DSM 45237 to date.

Evaluation of the action of Tween 20 non-ionic surfactant during enzymatic hydrolysis of lignocellulose: Pretreatment, hydrolysis conditions and lignin structure

The aim of this work was to study the effects of pretreatment process, hydrolysis condition and structural features of lignin on the improving action of surfactants (Tween 20) for enzymatic hydrolysis of pretreated wheat straw, and further to interpret the relation of these factors with the non-productive adsorption of cellulases on lignin. Tween 20 seemed to be more greatly improve cellulose conversion under harsher conditions. The surfactant showed more significant improvement for acid-pretreated substrates than oxidative-pretreated substrates. Highly-condensed lignin and phenolic hydroxyl groups showed much stronger adsorption ability to cellulases, while Tween 20 could well block the lignin–cellulase interactions recovering cellulose hydrolyzability. It was proposed that pretreatments altered lignin structures, resulting in the change of surface properties thus further impacting the lignin–cellulase interactions. Addition of Tween 20 could modify lignin surface properties to change its hydrophobicity, hydrogen bonding ability and surface charges, thus reducing the non-productive adsorption of proteins.

Properties important for solid-liquid separations change during the enzymatic hydrolysis of pretreated wheat straw.

The biochemical conversion of lignocellulosic biomass into renewable fuels and chemicals provides new challenges for industrial scale processes. One such process, which has received little attention, but is of great importance for efficient product recovery, is solid-liquid separations, which may occur both after pretreatment and after the enzymatic hydrolysis steps. Due to the changing nature of the solid biomass during processing, the solid-liquid separation properties of the biomass can also change. The objective of this study was to show the effect of enzymatic hydrolysis of cellulose upon the water retention properties of pretreated biomass over the course of the hydrolysis reaction. Water retention value measurements, coupled with 1H NMR T2 relaxometry data, showed an increase in water retention and constraint of water by the biomass with increasing levels of cellulose hydrolysis. This correlated with an increase in the fines fraction and a decrease in particle size, suggesting that structural decomposition rather than changes in chemical composition was the most dominant characteristic. With increased water retained by the insoluble fraction as cellulose hydrolysis proceeds, it may prove more difficult to efficiently separate hydrolysis residues from the liquid fraction with improved hydrolysis.

The effect of a lytic polysaccharide monooxygenase and a xylanase from Gloeophyllum trabeum on the enzymatic hydrolysis of lignocellulosic residues using a commercial cellulase.

Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). The enzymes were produced in the heterologous host Pichia pastoris. In the presence of an electron source, GtLPMO increased the activity of a commercial cellulase on filter paper, and the xylanase activity of GtXyn10A on beechwood xylan. Mixtures of GtLPMO, GtXyn10A and Celluclast 1.5L were used for hydrolysis of pretreated wheat straw. Results showed that a mixture of 60% Celluclast 1.5L, 20% GtXyn10A and 20% GtLPMO increased total reducing sugar production by 54%, while the conversions of glucan to glucose and xylan to xylose were increased by 40 and 57%, respectively. This suggests that GtLPMO can contribute to lignocellulose hydrolysis, not only by oxidative activity on glycosidic bonds, but also to hemicellulose through the oxidation of xylosyl bonds in xylan. The concerted action of these auxiliary enzymes may significantly improve large-scale recovery of sugars from lignocellulose.

The promotional effect of water-soluble extractives on the enzymatic cellulose hydrolysis of pretreated wheat straw

Enzymatic cellulose hydrolysis of pretreated wheat straw pulp to glucose is enhanced when the hydrolysis is performed in the presence of an aqueous extract of the wheat straw. A relative digestibility increase of about 10% has been observed for organosolv, alkaline and dilute acid pretreated wheat straw pulp (enzyme dose 2.5FPU/g pulp). At lower enzyme doses, the extract effect increases leading to an enzyme dose reduction of 40% to obtain a glucose yield of 75% within 48h using organosolv wheat straw pulp. Possibly, cellulase deactivation by irreversible binding to pulp lignin is reduced by competition with proteins in the extract. However, since the extract effect has also been demonstrated for lignin-lean substrates, other effects like improved accessibility of the pulp cellulose (amorphogenesis) cannot be excluded. Overall, this contribution demonstrates the positive effect of biomass extractives on enzymatic cellulose digestibility, thereby reducing costs for 2G biofuels and bio-based chemicals.

A reusable multipurpose magnetic nanobiocatalyst for industrial applications

A multipurpose magnetic nanobiocatalyst is developed by conjugating Pectinex 3XL (a commercial enzyme containing pectinase, xylanase and cellulase activities) on 3-aminopropyl triethoxysilane activated magnetic nanoparticles. The nanobiocatalyst retained 87% of pectinase, 69% of xylanase and 58% of cellulase activity after conjugation on modified nanoparticles as compared to their soluble counterparts. Thermal stability data at 70°C showed increase in enzyme stability after conjugation to nanoparticles and the kinetic parameters (Km and Vmax) remain unaltered after immobilization. The immobilized enzyme system can be successfully used upto 5th cycle after that slight decrease in enzyme activities was observed. The nanobiocatalyst retained high pectinase activities in organic solvents and chemical reagents as compared to free enzymes. DLS data shows that the nanoparticles size increases from 63nm to 86nm after immobilization. Atomic Force Microscopy data confirms the deposition of enzymes on the nanoparticles. The nanobiocatalyst was used for the clarification of pine apple and orange juice and was also used for the production of bioethanol. Hydrolysis of pretreated wheat straw produced 1.39g/l and 1.59g/l after treatment with free Pectinex 3xL and nanobiocatalyst respectively. The concentration of bioethanol also increases by 1.4 fold as compared to the free enzyme.

Development of Thermophilic Tailor-Made Enzyme Mixtures for the Bioconversion of Agricultural and Forest Residues.

Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5, and MtEG7), in the presence of other four “accessory” enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a) and β-glucosidase MtBGL3, were tested as a nine-component cocktail against one model substrate (phosphoric acid swollen cellulose) and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch, and spruce biomass, as forest residues). Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC), high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch). Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that tailor-made enzyme mixtures targeted toward a particular residual biomass can help maximize hydrolysis yields. The present work demonstrates the change from “one cocktail for all” to “tailor-made cocktails” that are needed for the efficient saccharification of targeted feed stocks prior to the production of biobased products through the biorefinery concept.

Dissecting the effect of chemical additives on the enzymatic hydrolysis of pretreated wheat straw

Chemical additives were examined for ability to increase the enzymatic hydrolysis of thermo-acidically pretreated wheat straw by Trichoderma reesei cellulase at 50°C. Semi-empirical descriptors derived from the hydrolysis time courses were applied to compare influence of these additives on lignocellulose bioconversion on a kinetic level, presenting a novel view on their mechanism of action. Focus was on rate retardation during hydrolysis, substrate conversion and enzyme adsorption. PEG 8000 enabled a reduction of enzyme loading by 50% while retaining the same conversion of 67% after 24h. For the first time, a beneficial effect of urea is reported, increasing the final substrate conversion after 48h by 16%. The cationic surfactant cetyl-trimethylammonium bromide (CTAB) enhanced the hydrolysis rate at extended reaction time (rlim) by 34% and reduced reaction time by 28%. A combination of PEG 8000 and urea increased sugar release more than additives used individually.

Co-cultivation of Trichoderma reesei RutC30 with three black Aspergillus strains facilitates efficient hydrolysis of pretreated wheat straw and shows promises for on-site enzyme production

Co-cultivation of fungi may be an excellent system for on-site production of cellulolytic enzymes in a single bioreactor. Enzyme supernatants from mixed cultures of Trichoderma reesei RutC30, with either the novel Aspergillus saccharolyticus AP, Aspergillus carbonarius ITEM 5010 or Aspergillus niger CBS 554.65 cultivated in solid-state fermentation were tested for avicelase, FPase, endoglucanase and beta-glucosidase activity as well as in hydrolysis of pretreated wheat straw. Around 30% more avicelase activity was produced in co-cultivation of T. reesei and A. saccharolyticus than in T. reesei monoculture, suggesting synergistic interaction between those fungi. Fermentation broths of mixed cultures of T. reesei with different Aspergillus strains resulted in approx. 80% efficiency of hydrolysis which was comparable to results obtained using blended supernatants from parallel monocultures. This indicates that co-cultivation of T. reesei with A. saccharolyticus or A. carbonarius could be a competitive alternative for monoculture enzyme production and a cheaper alternative to commercial enzymes.

Endo-exo Synergism in Cellulose Hydrolysis Revisited

Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and "steady state" conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the "steady state" rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on (14)C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios.

Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content

BackgroundThe recent discovery of accessory proteins that boost cellulose hydrolysis has increased the economical and technical efficiency of processing cellulose to bioethanol. Oxidative enzymes (e.g. GH61) present in new commercial enzyme preparations have shown to increase cellulose conversion yields. When using pure cellulose substrates it has been determined that both oxidized and unoxidized cellodextrin products are formed. We report the effect of oxidative activity in a commercial enzyme mix (Cellic CTec2) upon overall hydrolysis, formation of oxidized products and impact on β-glucosidase activity. The experiments were done at high solids loadings using a lignocellulosic substrate simulating commercially relevant conditions.ResultsThe Cellic CTec2 contained oxidative enzymes which produce gluconic acid from lignocellulose. Both gluconic and cellobionic acid were produced during hydrolysis of pretreated wheat straw at 30% WIS. Up to 4% of released glucose was oxidized into gluconic acid using Cellic CTec2, whereas no oxidized products were detected when using an earlier cellulase preparation Celluclast/Novozym188. However, the cellulose conversion yield was 25% lower using Celluclast/Novozym188 compared to Cellic CTec2. Despite the advantage of the oxidative enzymes, it was shown that aldonic acids could be problematic to the hydrolytic enzymes. Hydrolysis experiments revealed that cellobionic acid was hydrolyzed by β-glucosidase at a rate almost 10-fold lower than for cellobiose, and the formed gluconic acid was an inhibitor of the β-glucosidase.Interestingly, the level of gluconic acid varied significantly with temperature. At 50°C (SHF conditions) 35% less gluconic acid was produced compared to at 33°C (SSF conditions). We also found that in the presence of lignin, no reducing agent was needed for the function of the oxidative enzymes.ConclusionsThe presence of oxidative enzymes in Cellic CTec2 led to the formation of cellobionic and gluconic acid during hydrolysis of pretreated wheat straw and filter paper. Gluconic acid was a stronger inhibitor of β-glucosidase than glucose. The formation of oxidized products decreased as the hydrolysis temperature was increased from 33° to 50°C. Despite end-product inhibition, the oxidative cleavage of the cellulose chains has a synergistic effect upon the overall hydrolysis of cellulose as the sugar yield increased compared to using an enzyme preparation without oxidative activity.

Enzymatic hydrolysis optimization of microwave alkali pretreated wheat straw and ethanol production by yeast

Microwave alkali pretreated wheat straw was used for in-house enzyme production by Aspergillusflavus and Trichodermareesei. Produced enzymes were concentrated, pooled and assessed for the hydrolysis of pretreated wheat straw. Factors affecting hydrolysis were screened out by Placket–Burman design (PBD) and most significant factors were further optimized by Box–Behnken design (BBD). Under optimum conditions, 82% efficiency in hydrolysis yield was observed. After the optimization by response surface methodology (RSM), a model was proposed to predict the optimum value confirmed by the experimental results. The concentrated enzymatic hydrolyzate was fermented for ethanol production by Saccharomyces cerevisiae, Pichia stipitis and co-culture of both. The yield of ethanol was found to be 0.48gp/gs, 0.43gp/gs and 0.40gp/gs by S. cerevisiae, P. stipitis and by co-culture, respectively, using concentrated enzymatic hydrolyzate. During anaerobic fermentation 42.31μmol/mL, 36.69μmol/mL, 43.35μmol/mL CO2 was released by S. cerevisiae, P. stipitis and by co-culture, respectively.

Effect of cellobiase and surfactant supplementation on the enzymatic hydrolysis of pretreated wheat straw

Wheat straw is a suitable raw material for ethanol production since it has high cellulose content. The objective of this work was to evaluate the effect of cellobiase and surfactant on the enzymatic hydrolysis of lignocellulosic materials. For this purpose, wheat straw was first pretreated by organosolv digestion. The chemical s of raw and pretreated wheat straw were analyzed. Much of the hemicellulose and lignin were removed, and the relative cellulose content of pretreated wheat straw was 26.57% higher when compared to untreated wheat straw. Cellobiase was added into hydrolysate to improve the hydrolysis efficiency. Through experiments and analysis, the optimum cellobiase dosage was found to be 1/10 of the cellulase loading. Surfactant was also added into hydrolysate. Nonionic surfactant (Tween 80) exhibited better effect on improving enzymatic hydrolysis. When 0.06 g/g dry solids (DS) Tween 80 was also added into hydrolysate, the yield of glucose in hydrolyzate could reach 486 g/kg DS.

Application of a microassay method to study enzymatic hydrolysis of pretreated wheat straw

AbstractBACKGROUND: The conversion of lignocellulosic biomass to ethanol includes a disruptive pretreatment process followed by enzyme‐catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. As the cost and hydrolytic efficiency of enzymes are major factors that restrict the commercialization of biomass conversion processes, significant efforts are made nowadays to improve the enzymatic mixtures and make the process cost‐effective.RESULTS: In this work, enzymatic microassays have been developed and validated to test new different enzymatic formulations on real lignocellulosic substrates. Homogeneous handsheets from steam pretreated wheat straw were elaborated to be used as substrate. The microassay was adapted to test both water‐insoluble solids and the whole slurry as substrates. Results in hydrolysis microassays were comparable with those obtained in standard flask assays using pretreated wheat straw. Moreover, using the enzymatic microassays, two novel preparations have been evaluated, demonstrating the ability of microassays to discriminate between different enzymatic mixtures.CONCLUSIONS: This enzymatic microassay represents a rapid method to test the performance of new selected cellulase enzymes on real pretreated lignocellulosic substrates. This microassay will enable evaluation of enzyme components separately, or optimized mixtures. Copyright © 2010 Society of Chemical Industry

Analysis of methane potentials of steam-exploded wheat straw and estimation of energy yields of combined ethanol and methane production

Agrarian biomass as a renewable energy source can contribute to a considerable CO 2 reduction. The overriding goal of the European Union is to cut energy consumption related greenhouse gas emission in the EU by 20% until the year 2020. This publication aims at optimising the methane production from steam-exploded wheat straw and presents a theoretical estimation of the ethanol and methane potential of straw. For this purpose, wheat straw was pretreated by steam explosion using different time/temperature combinations. Specific methane yields were analyzed according to VDI 4630. Pretreatment of wheat straw by steam explosion significantly increased the methane yield from anaerobic digestion by up to 20% or a maximum of 331 l N kg −1 VS compared to untreated wheat straw. Furthermore, the residual anaerobic digestion potential of methane after ethanol fermentation was determined by enzymatic hydrolysis of pretreated wheat straw using cellulase. Based on the resulting glucose concentration the ethanol yield and the residual sugar available for methane production were calculated. The theoretical maximum ethanol yield of wheat straw was estimated to be 0.249 kg kg −1 dry matter. The achievable maximum ethanol yield per kg wheat straw dry matter pretreated by steam explosion and enzymatic hydrolysis was estimated to be 0.200 kg under pretreatment conditions of 200 °C and 10 min corresponding to 80% of the theoretical maximum. The residual methane yield from straw stillage was estimated to be 183 l N kg −1 wheat straw dry matter. Based on the presented experimental data, a concept is proposed that processes wheat straw for ethanol and methane production. The concept of an energy supply system that provides more than two forms of energy is met by (1) upgrading obtained ethanol to fuel-grade quality and providing methane to CHP plants for the production of (2) electric energy and (3) utility steam that in turn can be used to operate distillation columns in the ethanol production process.

Complete and efficient enzymic hydrolysis of pretreated wheat straw

A fractionation of wheat straw components in a two-step chemical pretreatment is proposed. Hemicelluloses were hydrolysed by dilute sulphuric acid, allowing a substantial recovery of crystalline xylose. Lignin was removed by means of a mild alkaline/oxidative solubilisation procedure, involving no sulphite or chlorine and its derivatives. The use of diluted reagents and relatively low temperatures, was both cheap and environmentally friendly. The pretreated material was nearly pure cellulose, whose enzymic hydrolysis proceeded fast and with high yields, leading to high glucose syrups of remarkable purity.

An approach in mathematical modeling of an upflow packed‐bed reactor for enzymatic hydrolysis of wheat straw

The behavior of a packed-bed reactor for enzymatic hydrolysis of pretreated wheat straw has been described by means of a mathematical model. The flow pattern has been evaluated by residence time distribution experiments. Small deviations from ideal plug flow behavior were found using the dispersion model. The kinetic model proposed for enzymatic hydrolysis of cellulosic fraction of pretreated wheat straw has been derived from batch experimental data. Variations of enzyme concentration throughout the straw bed have been approximately described using a ramp variation of adsorbed enzyme. The final explains qualitatively the experimental results.