The effects of nonionic surfactants on the pretreatment and enzymatic hydrolysis of recycled newspaper

The effects of surfactants on the pretreatment and enzymatic hydrolysis stages of recycled newspaper processing were examined. Newspaper substrate was pretreated with surfactants at 40°C and 400 rpm for 1 h, and the enzymatic digestibilities of the pretreated substrate were compared. NP-20 was 10–20% more effective as a surfactant than Tween-20 and Tween-80. To investigate the effects of the surfactants on the subsequent enzymatic hydrolysis stage, the newspaper was pretreated with NP-20 and then hydrolyzed in the presence of TW-20 or TW-80. TW-80 showed an approximate 7% higher digestibility than TW-20. The surfactant effect on the hydrolysis of the untreated newspaper was significant, whereas the surfactant effect on the hydrolysis of the surfactant-pretreated newspaper was marginal. When the digestibilities of the pure cellulose substrates (α-cellulose and filter paper) were examined, markedly different surfactant effects were observed. In contrast to the newspaper substrate, the surfactant-pretreated pure cellulose substrates had a significant effect on digestibility when they were hydrolyzed in the presence of a surfactant, indicating that the surfactant effect on digestibility is highly dependent on substrate type.     

Nutzung cellulosehaltiger Abprodukte bei der Biosynthese von Cellulasen

It was analysed the influence of different pretreatment methods of cellulosic materials to increase the productivity of the cellulase biosynthesis. The most effective pretreatment method for cotton cellulose is the thermomechanical destruction in a worm extruder (5–15 minutes) with hydrolysis catalysts. The enzymatic saccharification degree was increased about 4 fold. Utilizing a pretreated cellulose an increase of 70–120% in C₁-activity was found besides their level was stabilized in higher values. It was demonstrated that enzymatic hydrolysis parameters would be applied as model for biochemical utilization of cellulosic materials in the biosynthesis of cellulases.     

Enhancement of cellulose accessibility and enzymatic hydrolysis by simultaneous wet milling

It has been shown that the rate of enzymatic saccharification of cellulosic materials including “pure” cellulose (Whatman CF?11 cellulose), newsprint, lignocellulose (prehydrolyzed to remove hemicelluloses), and wood can be substantially increased by simultaneous wet milling. An enhanced hydrolysis rate was sustained above that observed for ball milling: providing a more extensive saccharification. The cellulosic substrates were wet milled with a variety of grinding elements, such as sand, glass beads, and stainless-steel beads, agitated in a shaker bath. Simultaneous hydrolysis was achieved with a 2% substrate slurry in a 0.1M acetate buffer at 45°C and pH 5. The effectiveness of this process was dependent upon the lignified matrix of the cellulose microfibrils, the grinding elements, and the oscillation frequency of the shaker bath. Wet milling “pure” cellulose for 48 hr, with 3.5 mm glass beads and 200 oscillations/min (opm), yielded 1031 mg reducing sugar/g substrates (93% saccharification) as compared to 483 mg (44%) for the ball-milled sample and 253 mg (23%) for the unmilled material. With the lignified substrates stainless-steel beads (3.5 mm) were more effective than glass. For lignocellulose 529 mg sugar/g substrate (93% saccharification) could be obtained by wet milling with cellulase for 24 hr. This was about three times greater than that of the ball milled (169 mg, 30%) and 10 times greater than that of the unmilled (52 mg, 9%) substrates. The method was also effective for wood particles (60 mesh) giving 143 mg sugar/g wood (approximately 38% saccharification) in 48 hr, whereas the ball-milled sample gave only 79 mg (21%) and the unmlilled substrate 38 mg (10%). These observations can be explained on the basis of the current crystalline theory for the morphology of the cellulosic microfibrils. The advantage of wet milling and simultaneous hydrolysis apparently depends on a continuous generation of accessible sites and sustained rapid hydrolysis rate as the saccharification proceeds, where in the pretreated substrates the hydrolysis rate slow down as the active sites are reduced.     

Hydrolysis of cellulose materials during successive treatment with cellulase from Penicillium funiculosum

Cellulase from Penicillium funiculosum exhibited different hydrolysis tendencies when acting on cellulose materials. Successive addition of fresh cellulase to enzymatic pre-treated substrates showed foolscap paper to be the most susceptible for enzymatic hydrolysis followed by filter paper, newsprint and microcrystalline cellulose.     

Study on crystal structures of enzyme-hydrolyzed cellulosic materials by X-ray diffraction

Different cellulosic materials were treated with different extraceller microbial enzymes. Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme and the type of cellulosic materials. Both endoglucanase and crude cellulase have pronounced effects on the structure of cellulose. The variation of crystal structure was found to attack preferentially the (0 0 2) crystal planes for cellulose I during enzymatic hydrolysis.     

Adsorption of cellulase on cellulose: Effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis

In the cellulase-cellulose reaction system, the adsorption of cellulase on the solid cellulose substrate was found to be one of the important parameters that govern the enzymatic hydrolysis rate of cellulose. The adsorption of cellulase usually parallels the rate of hydrolysis of cellulose. The affinity for cellulase varies depending on the structural properties of cellulose. Adsorption parameters such as the half-saturation constant, the maximum adsorption constant, and the distribution coefficient for both the cellulase and cellulsoe have been experimentally determined for several substrates. These adsorption parameters vary with the source of cellulose and the pretreatment methods and are correlated with the crystallinity and the specific surface area of cellulose substrates. The changing pattern of adsorption profile of cellulase during the hydrolysis reaction has also been elucidated. For practical utilization of cellulosic materials, the cellulose structural properties and their effects on cellulase adsorption, and the rate of hydrolysis must be taken into consideration.     

A functionally based model for hydrolysis of cellulose by fungal cellulase

A new functionally based kinetic model for enzymatic hydrolysis of pure cellulose by the Trichoderma cellulase system is presented. The model represents the actions of cellobiohydrolases I, cellobiohydrolase II, and endoglucanase I; and incorporates two measurable and physically interpretable substrate parameters: the degree of polymerization (DP) and the fraction of beta-glucosidic bonds accessible to cellulase, F(a) (Zhang and Lynd, 2004). Initial enzyme-limited reaction rates simulated by the model are consistent with several important behaviors reported in the literature, including the effects of substrate characteristics on exoglucanase and endoglucanase activities; the degree of endo/exoglucanase synergy; the endoglucanase partition coefficient on hydrolysis rates; and enzyme loading on relative reaction rates for different substrates. This is the first cellulase kinetic model involving a single set of kinetic parameters that is successfully applied to a variety of cellulosic substrates, and the first that describes more than one behavior associated with enzymatic hydrolysis. The model has potential utility for data accommodation and design of industrial processes, structuring, testing, and extending understanding of cellulase enzyme systems when experimental date are available, and providing guidance for functional design of cellulase systems at a molecular scale. Opportunities to further refine cellulase kinetic models are discussed, including parameters that would benefit from further study.     

The cellulosome and cellulose degradation by anaerobic bacteria

Despite its simple chemical composition, cellulose exists in a number of crystalline and amorphous topologies. Its insolubility and heterogeneity makes native cellulose a recalcitrant substrate for enzymatic hydrolysis. Microorganisms meet this challenge with the aid of a multi-enzyme system. Aerobic bacteria produce numerous individual, extra-cellular enzymes with binding modules for different cellulose conformations. Specific enzymes act in synergy to elicit effective hydrolysis. In contrast, anaerobic bacteria possess a unique extracellular multi-enzyme complex, called cellulosome. Up to 11 different enzymes are aligned on the non-catalytic scaffolding protein and thus ensure a high local concentration, together with the correct ratio and order of the components. These multi-enzyme complexes attach both to the cell envelope and to the substrate, mediating the proximity of the cells to the cellulose. Binding to the scaffolding stimulates the activity of each individual component towards the crystalline substrate. The most complex and best investigated cellulosome is that of the thermophilic bacterium Clostridium thermocellum, but a scheme for the cellulosomes of the mesophilic clostridia and the ruminococci emerges. Many crucial details of cellulose hydrolysis are still to be uncovered. Yet, a mechanistic model for the action of enzyme complexes on the surface of insoluble substrates becomes apparent and the application of enzymatic hydrolysis of cellulosic biomass can now be addressed.     

Enzymatische Hydrolyse und Ethanolgärung von Lignocellulosen

The kinetics of enzymatic hydrolysis of different lignocellulosic materials (wheat straw, newspaper and microcrystalline cellulose Avicel PH 101) was studied using the cellulase complexes from Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15 and MHC 22. The maximum yields of hydrolysis were obtained with wheat straw partially delignified with 1% NaOH as substrate, and using the enzyme from the mutants T. reesei M 6 and MHC 22. The possibility of simultaneous enzymatic hydrolysis and ethanol fermentation of wheat straw using the enzyme complex from M 6 and yeasts of the genus Candida and Torulopsis was also investigated. A good conversion of liberated glucose and cellobiose to ethanol was obtained, however, xylose was not fermented.     

The enzymatic hydrolysis rate of cellulose decreases with irreversible adsorption of cellobiohydrolase I

Protein adsorption onto solid substrates usually takes place in an irreversible fashion and this irreversible adsorption also occurs in some enzymatic reactions. In this work the adsorption behavior of intact β-1, 4-glucan-cellobiohydrolase (CBH I) from Trichoderma reesei onto microcrystalline cellulose was monitored by surface plasmon resonance and UV-spectral method. It was found that there existed reversible binding and irreversible binding of CBH I during its interaction with cellulose substrate. To evaluate the influence of adsorption on cellulose enzymatic hydrolysis, the reaction dynamics on pure cellulose were determined. A plot of the hydrolysis rate against the surface density of irreversibly adsorbed CBH I, revealed an inverse relationship in which an apparent decrease in the hydrolysis rate was observed with increasing surface density. Taken together, results presented here should be useful for modifying the binding characteristics of CBH I and making them more effective in cellulose hydrolysis.     

Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass

The switchgrass (SG) samples pretreated by cellulose solvent‐ and organic solvent‐based lignocellulose fractionation were characterized by enzymatic hydrolysis, substrate accessibility assay, scanning electron microscopy, X‐ray diffraction (XRD), cross polarization/magic angle spinning (CP/MAS) ¹³C nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). Glucan digestibility of the pretreated SG was 89% at hour 36 at one filter paper unit of cellulase per gram of glucan. Crystallinity index (CrI) of pure cellulosic materials and SG before and after cellulose solvent‐based pretreatment were determined by XRD and NMR. CrI values varied greatly depending on measurement techniques, calculation approaches, and sample drying conditions, suggesting that the effects of CrI data obtained from dried samples on enzymatic hydrolysis of hydrated cellulosic materials should be interpreted with caution. Fast hydrolysis rates and high glucan digestibilities for pretreated SG were mainly attributed to a 16.3‐fold increase in cellulose accessibility to cellulase from 0.49 to 8.0 m²/g biomass, because the highly ordered hydrogen‐bonding networks in cellulose fibers of biomass were broken through cellulose dissolution in a cellulose solvent, as evidenced by CP/MAS ¹³C‐NMR and FTIR. Biotechnol. Bioeng. 2011; 108:521–529. © 2010 Wiley Periodicals, Inc.     

Effect of adding surfactant for transforming lignocellulose into fermentable sugars during biocatalysing

Fuel ethanol is one of the most important alternative fuels used as a substitute for fossil fuel. Lignocellulose is the most abundant biomass resource for the production of fuel ethanol. However, the hydrolysis of lignocellulose requires high enzyme loading. In order to strengthen the process of enzyme hydrolysis of lignocellulose, surfactant-polyethylene glycol (PEG) was applied to the catalysis of lignocellulose into fermentable sugars. The effect of PEG on both the enzymatic hydrolysis and adsorption of cellulose were investigated. The addition of surfactant obviously facilitated enzymatic hydrolysis. In particular, upon addition of PEG4000, the enzyme catalytic efficiency increased by 51.06%. Meanwhile, the adsorption quantity of cellulase decreased by 11.25%. In addition, the mechanism of the effect of PEG on enzymatic hydrolysis and cellulase adsorption is discussed.     

Studies on the mechanism of enzymatic hydrolysis of cellulosic substances.

Most cellulosic substances contain appreciable amounts of cellulose and hemicellulose, which on enzymatic hydrolysis mainly yield a mixture of glucose, cellobiose, and xylose. In this paper, studies on the mechanisms of hydrolysis of bagasse (a complex native cellulosic waste left after extraction of juice from cane sugar) by the cellulase enzyme components are described in light of their adsorption characteristics. Simultaneous adsorption of exo- and endoglucanases on hydrolyzable cellulosics is the causative factor of the hydrolysis that follows immediately after. It supports the postulate of synergistic enzyme action proposed by Eriksson. Xylanase pretreatment enhanced the hydrolysis of bagasse owing to the creation of more accessible cellulosic regions that are readily acted upon by exo- and endoglucanases. The synergistic action of the purified exoglucanase, endoglucanase, and xylanse has been found to be most effective for hydrolysis of bagasse but not for pure cellulose. Significant quantities of glucose are produced in beta-glucosidase-free cellulase action on bagasse. Individual and combined action of the purified cellulase components on hydrolysis of native and delignified bagasse are discussed in respect to the release of sugars in the hydrolysate.     

Kinetic study on enzymatic hydrolysis of cellulose by cellulose from Trichoderma viride

Pure cellulose (Avicel) was hydrolyzed batchwise at 50 degrees C and pH 4.8 by cellulase from Trichoderma viride (Meicelase CEP). Then the effects of the crystallinity of cellulose as well as the thermal deactivation and product (cellubiose and glucose) inhibition to cellulose on the hydrolysis rate were quantitatively investigated. While these factor had evidently retarded the enzymatic hydrolysis of cellulose to a significant extent, the hydrolysis rates observed could not be explained. For practical purposes, an empirical, simple rate expression was developed which included only one parameter: a overall rate retardation constant. This empirical rate expression held for the hydrolysis of at least two kind of cellulosic materials: Avicel and tissue paper.     

Enzymatic hydrolysis of cellulose and various pretreated wood fractions

Three strains of Trichoderma-T. reesei C30, T. reesei QM9414, and Trichoderma species E-58-were used to study the enzymatic hydrolysis of pretreated wood substrates. ach of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which had been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis.     

Influence of steam pretreatment severity on post‐treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings

It is recognized that some form of post‐treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post‐treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post‐treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post‐treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post‐treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post‐treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post‐treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post‐treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used. Biotechnol. Bioeng. 2011;108: 2300–2311. © 2011 Wiley Periodicals, Inc.     

Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis

The susceptibility of cellulose to enzymatic hydrolysis is affected by the structural features of cellulosic materials. It has been suggested that the crystallinity and surface area of cellulose fibers are the most important structural features in this regard. This study investigated in depth the relative effects of these two structural features upon the enzymatic hydrolysis of cellulose and the change of the structural parameters of cellulose during the course of hydrolysis. It was found that the hydrolysis rate is mainly dependent upon the fine structural order of cellulose which can best be represented by the crystallinity rather than the simple surface area. Monitoring the changes in the structural parameters during the course of reaction showed that surface area is not a major limiting factor that slows hydrolysis in its late stages as has been suggested. This information concerning structural features is used to elucidate the mode of action of cellulase.     

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes.The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20-25%, liberating significantly more glucose than theoretically present in glucomannan.When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.     

A quantitative approach to the study of the adsorption/desorption of cellulase components in a crude cellulase mixture

Summary Chromatofocusing was used to separate and identify cellulase components for the study of their adsorption/desorption onto lignocellulosic substrates during cellulose hydrolysis. The separated cellulase components were characterized with respect to their M.W.s and enzymatic activities. Adsorption of the cellulase components onto five different cellulosic substrates was quantified. All the major cellulase components adsorbed to all the substrates studied with only minor differences observed in the amount of binding of each cellulase component.     

Effect of surfactants on separate hydrolysis fermentation and simultaneous saccharification fermentation of pretreated lodgepole pine

The effects of surfactants addition on enzymatic hydrolysis and subsequent fermentation of steam exploded lodgepole pine (SELP) and ethanol pretreated lodgepole pine (EPLP) were investigated in this study. Supplementing Tween 80 during cellulase hydrolysis of SELP resulted in a 32% increase in the cellulose‐to‐glucose yield. However, little improvement was obtained from hydrolyzing EPLP in the presence of the same amount of surfactant. The positive effect of surfactants on SELP hydrolysis led to an increase in final ethanol yield after the fermentation. It was found that the addition of surfactant led to a substantial increase in the amount of free enzymes in the 48 h hydrolysates derived from both substrates. The effect of surfactant addition on final ethanol yield of simultaneous saccharification and fermentation (SSF) was also investigated by using SELP in the presence of additional furfural and hydroxymethylfurfural (HMF). The results showed that the surfactants slightly increased the conversion rates of furfural and HMF during SSF process by Saccharomyces cerevisiae. The presence of furfural and HMF at the experimental concentrations did not affect the final ethanol concentration either. The strategy of applying surfactants in cellulase recycling to reduce enzyme cost is presented. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009