Effect ofMyrothecium sp. cellulase on different types of cellulose and cellulosic materials

Three types of cellulase preparations were applied to different types of cellulose and cellulosic materials. The action of these types of cellulase on cellulose powder was increased with the increase of enzyme concentration. Both carboxymethyl cellulose (CMC) and sodium carboxymethyl cellulose (Na-CMC) released high amounts of reducing sugar as affected by cellulase application. Different types of paper pulp were moderately hydrolyzed, while agricultural wastes were slightly hydrolyzed. Vegetable and fruits cellulose were equally hydrolyzed but at low rate. Pretreatment of cellulose or cellulosic materials by grinding or by swelling with phosphoric acid gave rise to increased hydrolysis by the enzyme. Cellobiose was detected chromatographically as an intermediate product of hydrolysis of both cellulose and carboxymethyl cellulose with glucose.     

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.     

An effective method for bioconversion of delignified waste-cellulose fibers from the paper industry with a cellulase complex

An effective method for production of glucose was developed using enzymatic hydrolysis of waste-cellulose fibers by the cellulase complex from Trichoderma reesei. However, these cellulosic materials are strongly resistant to direct enzymatic hydrolysis (the degree of degradation after 48 h of enzyme treatment did not exceed 14%) apparently due to the presence of various chemical substances used in the process of paper production. This adhesive “envelope” around the cellulose fibers was effectively pretreated with 0.25% H₃PO₄ and after that the free cellulose mass was extensively hydrolyzed by the cellulase complex (degree of degradation more than 80%). The HPLC analyses of the enzyme hydrolysates revealed glucose as the main component as well as some cellobiose and xylose.     

Effect of peracetic acid, sodium hydroxide and phosphoric acid on cellulosic materials as a pretreatment for enzymatic hydrolysis

Crystalline cellulose and cellulosic wastes have been treated with various concentrations of peracetic acid and other reagents at 100°C for various times, washed with water, ethanol and air dried. For each treated cellulose, the degree of enzymatic solubilization was measured with Trichoderma viride cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4]. Cellulosic wastes such as sunflower stalks, wheat straw and sugar-cane bagasse were solubilized effectively by the enzyme. Delignification of wheat straw with 1% sodium hydroxide and treatment of this straw with peracetic acid enhanced the degree of enzymatic solubilization. Infrared spectra of the untreated and treated cellulosic wastes were recorded.     

Effect of Maize Biomass Composition on the Optimization of Dilute-Acid Pretreatments and Enzymatic Saccharification

At the core of cellulosic ethanol research are innovations leading to reductions in the chemical and energetic stringency of thermochemical pretreatments and enzymatic saccharification. In this study, key compositional features of maize cell walls influencing the enzymatic conversion of biomass into fermentable sugars were identified. Stem samples from eight contrasting genotypes were subjected to a series of thermal dilute-acid pretreatments of increasing severity and evaluated for glucose release after enzymatic saccharification. The biochemically diverse set of genotypes displayed significant differences in glucose yields at all processing conditions evaluated. The results revealed that mechanisms controlling biomass conversion efficiency vary in relation to pretreatment severity. At highly severe pretreatments, cellulose conversion efficiency was primarily influenced by the inherent efficacy of the thermochemical process, and maximum glucose yields were obtained from cellulosic feedstocks harboring the highest cellulose contents per dry gram of biomass. When mild dilute-acid pretreatments were applied, however, maximum bioconversion efficiency and glucose yields were observed for genotypes combining high stem cellulose contents, reduced cell wall lignin and highly substituted hemicelluloses. For the best-performing genotype, glucose yields under sub-optimal processing regimes were only 10 % lower than the genotype-set mean at the most stringent processing conditions evaluated, while furfural production was reduced by approximately 95 %. Our results ultimately established that cellulosic feedstocks with tailored cell wall compositions can help reduce the chemical and energetic intensity of pretreatments used in the industry and improve the commercial and environmental performance of biomass-to-ethanol conversion technologies.     

Enzymatic hydrolysis of cellulosic materials by Sclerotium rolfsii culture filtrate for sugar production.

The hydrolysis of purified celluloses (cotton, Avicel, Cellulose-123, Solka Floc SW40) and cellulosic wastes (rice straw, sugarcane bagasse, wood powders, paper factory effluents) by Sclerotium rolfsii CPC 142 culture filtrate was studied. Factors which effect saccharification such as pH, temperature, enzyme concentration, substrate concentration, produce inhibition, adsorption, and inactivation of enzyme and particle size were studied. Virtually no inhibition (less than 3%) of cellulose hydrolysis by the culture filtrate was observed by cellobiose and glucose up to 100 mg/mL. Filter paper degrading enzyme(s) (but neither carboxymethylcellulase nor beta-glucosidase) was adsorbed on cellulose. The n value in the S. rolfsii system was calculated to be 0.32 for Avicel P.H. 101 and 0.53 for alkali-treated (AT) rice straw indicating penetration of cellulase into AT rice straw. In batch experiments at 10% substrate level, solutions containing 6 to 7%, 3.8 to 4.7%, 4.0 to 5.1%, and 4.2 to 4.9% reducing sugars were produced in 24 to 48 from AT rice straw. AT bagasse, alkali - peracetic acid treated mesta wood and paper factory sedimented sludge effluent, respectively. The main constituent in the hydrolysate from cellulose was glucose with little or no cellobiose, probably due to the high cellobiase content in the culture filtrate.     

Highly stable enzyme precipitate coatings and their electrochemical applications

This paper describes highly stable enzyme precipitate coatings (EPCs) on electrospun polymer nanofibers and carbon nanotubes (CNTs), and their potential applications in the development of highly sensitive biosensors and high-powered biofuel cells. EPCs of glucose oxidase (GOx) were prepared by precipitating GOx molecules in the presence of ammonium sulfate, then cross-linking the precipitated GOx aggregates on covalently attached enzyme molecules on the surface of nanomaterials. EPCs-GOx not only improved enzyme loading, but also retained high enzyme stability. For example, EPC-GOx on CNTs showed a 50 times higher activity per unit weight of CNTs than the conventional approach of covalent attachment, and its initial activity was maintained with negligible loss for 200 days. EPC-GOx on CNTs was entrapped by Nafion to prepare enzyme electrodes for glucose sensors and biofuel cells. The EPC-GOx electrode showed a higher sensitivity and a lower detection limit than an electrode prepared with covalently attached GOx (CA-GOx). The CA-GOx electrode showed an 80% drop in sensitivity after thermal treatment at 50°C for 4 h, while the EPC-GOx electrode maintained its high sensitivity with negligible decrease under the same conditions. The use of EPC-GOx as the anode of a biofuel cell improved the power density, which was also stable even after thermal treatment of the enzyme anode at 50°C. The excellent stability of the EPC-GOx electrode together with its high current output create new potential for the practical applications of enzyme-based glucose sensors and biofuel cells.     

Studies on the oxidative cross-linking of feruloylated arabinoxylans from wheat flour and wheat bran

Feruloylated arabinoxylans isolated from wheat flour and wheat bran were compared in their cross-linking behaviour with respect to viscosity properties and cross-linking products formed when various oxidative agents were applied to dilute solutions. Optimal conditions for each oxidative agent were investigated. In case of hydrogen peroxide and peroxidase, similar conditions were found for both types of arabinoxylans but wheat bran arabinoxylans gave a larger viscosity increase upon cross-linking than those of wheat flour.

When glucose, glucoseoxidase and peroxidase or ammonium persulphate were used as oxidative agents, differences in the concentration of reagent needed to induce cross-linking and in viscosity increase were observed. The distribution of coupling products for both types of arabinoxylans and the different oxidative treatments was approximately 5 : 3 : 1 : 1 for 8-5, 8-O-4, 8-8 and 5-5, respectively. The low ferulate recovery after oxidative treatment was assumed to be caused by formation of unknown compounds, such as higher oligomers and lignin-linked products.

A 1 : 1 mixture of flour arabinoxylan and feruloylated pectin showed a maximum synergistic effect on viscosity upon oxidative treatment using hydrogen peroxide and peroxidase. Both polysaccharides were shown to participate in cross-linking.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

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.     

Chemo-microbial conversion of cellulose into polyhydroxybutyrate through ruthenium-catalyzed hydrolysis of cellulose into glucose

Cellulose-derived glucose generated using the supported ruthenium catalyst was applied to poly(3-hydroxybutyrate) [P(3HB)] production in recombinant Escherichia coli. By the reaction with the catalyst at 220°C, 15-20 carbon mol% of cellulose was converted into glucose. The hydrolysate also contained byproducts such as fructose, mannose, levoglucosan, oligomeric cellulose, 5-hydroxymethylfurfural (5-HMF), and furfural together with unidentified compounds. Setting the reaction temperature lower (215°C) improved the ratio of glucose to 5-HMF, which was a main inhibiting factor for the cell growth. Indeed, the recombinant E. coli exhibited better performance on the hydrolysate generated at 215°C and accumulated P(3HB) up to 42 wt%, which was the same as the case of the same concentration of analytical grade glucose. The result indicated that the ruthenium-mediated cellulose hydrolysis has a potency as a useful biorefinery process for production of bio-based plastic from cellulosic biomass.     

Biotechnology report: single cell proteins from cellulosic wastes

Conventional sources of protein cannot meet the present or projected needs for human consumption. Single cell proteins from fermentation of petroleum and cellulosic wastes are likely sources of additional protein. The volume of cellulosic wastes is sufficient to supply all additional protein needs on a continuing basis for cellulose is a renewable resource. Both mesophilic and thermophilic microorganisms utilize cellulose at reasonable rates. Biodegradation of lignin and lignin–cellulose complexes constitutes a major obstacle to commercial utilization of cellulosic wastes. Thermophilic actinomyces appear to be the most effective organisms for single cell protein production from cellulosic wastes.     

Two-stage pretreatment of rice straw using aqueous ammonia and dilute acid

Liberation of fermentable sugars from recalcitrant lignocellulosic biomass is one of the key challenges in production of cellulosic ethanol. Here we developed a two-stage pretreatment process using aqueous ammonia and dilute sulfuric acid in a percolation mode to improve production of fermentable sugars from rice straw. Aqueous NH? was used in the first stage which removed lignin selectively but left most of cellulose (97%) and hemicellulose (77%). Dilute acid was applied in the second stage which removed most of hemicellulose, partially disrupted the crystalline structure of cellulose, and thus enhanced enzymatic digestibility of cellulose in the solids remaining. Under the optimal pretreatment conditions, the enzymatic hydrolysis yields of the two-stage treated samples were 96.9% and 90.8% with enzyme loadings of 60 and 15FPU/g of glucan, respectively. The overall sugar conversions of cellulose and hemicellulose into glucose and xylose by enzymatic and acid hydrolysis reached 89.0% and 71.7%, respectively.     

Economical factors in the assessment of various cellulosic substances as chemical and energy resources.

Economic factors in the assessment of various cellulosic substances as chemical and energy resources are many and complex. No substrate nor conversion process can be singled out as significantly advantageous. Agricultural wastes appear to have the best volume and availability characteristics. If glucose is to be the end product, then it will probably have to compete with corn syrup. If SCP is to be the end product, then productivities of 2-4 g/liter-hr must be achieved and the protein demand be such that the product can sell for at least $225/ton. If alcohol is to be the end product, then an intermediate product stream of glucose and other sugars must be obtained for 1-3cent/lb of fermentable sugars.     

Pretreatment of woody and herbaceous biomass for enzymatic saccharification using sulfuric acid-free ethanol cooking

A sulfuric acid-free ethanol cooking (SFEC) treatment was developed to achieve complete saccharification of the cellulosic component of eucalyptus and baggase flour, thereby avoiding the problems associated with the use of strong acid catalysts. Cutter-milled flours were exposed to an ethanol (EtOH)/water/acetic acid mixture in an autoclave. Enzymatic hydrolysis experiments of the pretreated samples demonstrated that almost complete conversion of the cellulosic components to glucose was achieved under optimal conditions. A large-scale trial revealed that there was little consumption of in-feed EtOH during SFEC; therefore, it is considered that most part EtOH used can be essentially recovered and reused. Field emission scanning electron microscopy showed that SFEC induced the formation of pores ranging in size from approximately 10 to several 100nm. It can be assumed that the porous surface was due to the partial removals of lignin and hemicellulose, which improved the accessibility of the enzyme onto the substrate.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Hydrolysis of rice hull by crosslinked Aspergillus niger cellulase

A. niger cellulase was crosslinked by glutaraldehyde to obtain a heat-stable enzyme preparation for rice hull cellulose hydrolysis. Under optimized crosslinking conditions of 0.12 M glutaraldehyde, pH 7.0, temperature 40 degrees C and at 45 min of crosslinking, a preparation having 15% more activity than free enzyme was obtained which also had considerable improvement in heat stability at 65 degrees C and 70 degrees C. Whereas the free enzyme lost 80% of its activity in 4 h at 65 degrees C, the crosslinked preparation lost only 30% activity. The crosslinked preparation hydrolyzed cellulosic biomass more effectively giving 2.2 mg/ml glucose and 52% corresponding saccharification in 4 h at 65 degrees C as compared to 14% saccharification by free enzyme under similar conditions.     

Hydrolysis of plant oils by means of lipase from Rhizopus nigricans

The crude enzyme powder from Rhizopus nigricans was immobilized by sorption and subsequent cross-linking with glutaraldehyde on collagen and polytetrafluoroethylene (PTFE) membranes. Lipolytic membranes were applied to plant oils hydrolysis. The comparison of the two types of membranes by calculating the time required to obtain 1 mole of free fatty acids (FFA) from 1 m² of membrane area, indicates that hydrophobic PTFE membrane is a better one in spite of the fact that the amount of protein sorbed on PTFE membrane is about three times smaller than that for collagen membrane. The hydrolysis of sunflower oil was the most efficient at the temperature of 37 °C and a pH of 7. At these conditions the specific activity after immobilization was about four times higher than that of the soluble enzyme.     

Effect of Fenton's pretreatment on cotton cellulosic substrates to enhance its enzymatic hydrolysis response

Fenton’s reagent that generates reactive hydroxyl radical species was evaluated for its effectiveness as a pretreatment agent on cotton cellulosic substrates to increase its susceptibility to cellulase enzyme. Response surface methodology was used to optimize four different process variables viz., time of reaction; substrate size and concentrations of Fe²⁺ and H₂O₂. Overall, the cellulose substrates treated at 0.5 mM concentration of Fe²⁺, 2% concentration of H₂O₂ for a reaction period of 48 h gave the highest enzyme activity as determined using the response surface methodology. Cellulose substrates with high aspect ratio recorded better enzyme response than that with low aspect ratio which is supported by copper number estimation. The cellulosic substrate prepared using a combination of optimized Fenton’s pretreatment conditions and/or enzyme hydrolysis were studied and characterized by atomic force microscopy and scanning electron microscopy. Additionally, degree of polymerization analysis gives further insight into the degradation during Fenton’s reaction.     

Integrated process of starch ethanol and cellulosic lactic acid for ethanol and lactic acid production

The sequential production of bioethanol and lactic acid from starch materials and lignocellulosic materials was investigated as ethanol fermentation broth (EFB) can provide nutrients for lactic acid bacteria. A complete process was developed, and all major operations are discussed, including ethanol fermentation, broth treatment, lactic acid fermentation, and product separation. The effect of process parameters, including ethanol fermentation conditions, treatment methods, and the amount of EFB used in simultaneous saccharification and fermentation (SSF), is investigated. Under the selected process conditions, the integrated process without additional chemical consumption provides a 1.08 acid/alcohol ratio (the broth containing 22.4 g/L ethanol and 47.6 g/L lactic acid), which corresponds to a polysaccharide utilization ratio of 86.9 %. Starch ethanol can thus promote cellulosic lactic acid by providing important nutrients for lactic acid bacteria, and in turn, cellulosic lactic acid could promote starch ethanol by improving the profit of the ethanol production process. Two process alternatives for the integration of starch ethanol and cellulosic lactic acid are compared, and some suggestions are given regarding the reuse of yeast following the cellulosic SSF step for lactic acid production.