Effect of pH on cellulase production of Trichoderma ressei RUT C30

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.     

The effect of adsorbed carboxymethyl cellulose on the cotton fibre adsorption capacity for surfactant

The research reported in this paper demonstrates that the capacity of cotton fibres to adsorb cationic surfactants as well as the rate of the adsorption process can be increased by adsorbing carboxymethyl cellulose (CMC) onto the fibre surfaces; in addition, the adsorption can be restricted to the fibre surface. CMC was deposited by means of adsorption from an aqueous solution. The adsorption of N-cetylpyridinium chloride (CPC) from an aqueous solution onto the CMC-modified fibres was measured using UV-spectrometric determination of the surfactant concentration in the solution. Adsorption onto the cotton fibres was studied in a weakly basic environment (pH 8.5) where cotton fibres are negatively charged and the CPC ion is positively charged. Modification of the fibres by adsorption of CMC introduces new carboxyl groups onto the fibre surfaces, thereby increasing the adsorption capacity of the fibres for CPC. The initial rate of adsorption of CPC increased proportionally with the amount of charge; however, this rate slowed down at high degrees of coverage on fibres with a high charge. The adsorption of cationic surfactant to the anionic surface groups was stoichiometric, with no indication of multilayer or admicelle formation. It was evident that the acidic group content of the fibres was the primary factor determining cationic surfactant adsorption to these fibres.     

Effects of nonionic surfactant and associative thickener on the rheology of polyacrylamide in aqueous glycerol solutions

The effect of a low molecular weight nonionic surfactant and an acrylic associative thickener on the rheology of polyacrylamide in aqueous glycerol solutions under steady shear was experimentally investigated. The nonionic surfactant (Tween20), associative thickener (Acrysol TT935) and polyacrylamide (Separan AP30) underwent complex molecular interactions in solution as reflected by rheological measurements. The surfactant also interacted with the glycerol solvent. The addition of surfactant in aqueous glycerol solutions reduced the surface tension, as well as the solution viscosity, at low surfactant concentration. The solution viscosity went through a minimum at certain surfactant concentration, depending on the composition of glycerol/water mixture, before increasing again. Similar behavior was found when the surfactant was added to the polyacrylamide solution, except there was an initial increase in the viscosity before the reduction. The associative thickener, Acrysol TT935 (an anionic acrylic emulsion copolymer) exhibited a strong affinity with polyacrylamide in solution, as indicated by a sharp increase in the solution viscosity. The dilute polyacrylamide solution became highly elastic in the presence of either the nonionic surfactant on the associative thickener. A threestage model was proposed to describe the surfactant/thickener/polymer interactions.     

Effect of ultraviolet light and heat on the properties of cotton cellulose

The changes in properties of cellulose brought about by ultraviolet (UV) irradiation, heat exposure and by a combination of both treatments were determined. The methods of characterizing the changes included breaking strength, color, yellowness index, thermal analysis, dye adsorption as a measure of changes in fine structure of the fibers, and Turnbull's Blue test as an empirical measure of carboxyl content. The exposures increased the color and yellowness of the samples as well as the carboxyl content and decreased dye adsorption and breaking strength. A shift of the decomposition endotherm of cellulose to lower temperatures was also noted. It appears that, under the exposure conditions used in this study, the changes in carboxyl content, color changes, yellowness index and breaking strength induced by heat are accelerated by an initial exposure to UV light but still simulate heat ageing alone. There also appears to be a correlation between breaking strength and dye uptake.     

Development and characterization of electrospun cellulose acetate nanofibers modified by cationic surfactant

Polymeric electrospun nanofibers have been gaining notoriety in the same way as their industrial applications, since the manufacturing of this type of material is simple and low-costed. In order to obtain fibrous polymeric material with small diameters and with reduced beads formation, a 2⁴ factorial experiment with triplicate at center point was performed. Cellulose acetate (CA) and cationic cetylpyridinium bromide (CPB) surfactant nanofibers were made using a homemade electrospinning apparatus. The assessed inputs were as follows: CA%, CPB%, flow rate, and applied voltage. From the analysis of the response surface methodology and scanning electron microscope (SEM), the optimal concentrations of CA and CPB for producing nanofibers were 21 w/v-% and 0.5 w/v-%, respectively, using a flow rate of 0.7 mL h⁻¹ and applied voltage of 18 kV. Fibers mats morphology shows average diameter of 0.2 μm and 7 nm pore size, as well as it was found that the single fiber unit presented nanoheterogeneity. Mechanical resistance of 2.70 MPa was obtained in the tensile strength test. The modification of CA by the addition of surfactant attributed better thermal and mechanical resistances to the nanofibers without, however, affecting their biodegradability and water resistance properties. The morphological characteristics of the newly obtained CA/CPB nanofibers combined with mechanical resistance provided subsidies to suggest that the as-obtained material presents potential to be applied as an air filter.     

Interaction between indigo and adsorbed protein as a major factor causing backstaining during cellulase treatment of cotton fabrics

A model microassay system was developed to measure indigo backstaining on cotton fabrics in the presence of enzymes on a small laboratory scale. Backstaining indexes for 11 cellulase samples were measured, and the enzymes were ranked from lower to higher backstaining. Two multienzyme cellulase preparations were separated into fractions using chromatofocusing on a Mono P column. Adsorption ability and backstaining properties of purified enzyme fractions were studied. Evidence was obtained that protein adsorption on cotton fabrics is a crucial parameter causing backstaining (both for crude cellulase samples and purified enzyme components).     

生物医用材料自增强工艺的研究现状及前景展望

介绍了生物医用材料自增强的概念,重点介绍了纺丝、熔体注塑和挤出、纤维集束模压成型、定向自由拉伸、固态挤出、成纤模压等自增强工艺,对这些自增强工艺的原理作了简要的介绍,并对生物医用材料的发展前景进行了展望。     

Effect of the addition of a nonionic surfactant on the complex poly(asparagine)--cationic surfactant

Poly(asparagine) (pAsn) at 0.1wt % in the presence of dodecyltrimethylammonium bromide (DTAB) and pentaoxyethylene octyl ether (C(8)E(5)) at 1:1 molar ratio leads to the formation of mixed DTAB/C(8)E(5) micelle-like aggregates onto the polypeptide as a total surfactant critical association concentration (cac) is reached, as revealed by surface tension measurements and NMR chemical shifts. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) capable of revealing spatial relationships among proximal protons has been performed on the pAsn-DTAB-C(8)E(5)-water system to study structural details of the surfactant-polypeptide aggregates. NOESY cross-peaks at sample temperature of 298.15 K indicate that the polypeptide interacts with the DTAB/C(8)E(5) micelle-like aggregates. The NOE intermolecular effects also show direct interactions between surfactant and polypeptide in the pAsn-DTAB-water system, whereas no interaction has been revealed in the pAsn-C(8)E(5)-water system. Furthermore, the experimental evidence suggest that the DTAB-polypeptide complex is mainly driven by the polar attraction between the two molecules.     

Effect of cellulase on the pore structure of bead cellulose

An enzymatic treatment with cellulases fromTrichoderma viride was investigated in its effect on the pore structure of different types of bead cellulose. One objective of this study was to establish a suitable procedure for combined enzymatic treatment and solvent exchange that would restore the original pore structure which the beads had before drying without causing major losses in mechanical stability. Another aim was to further increase the accessible pore space and internal surface area for separation of large molecular weight compounds with regard to Chromatographic applications. Finally, an attempt was made to extend the findings for unsubstituted beads to the derivatives carboxymethyl (CM) and diethylaminoethyl (DEAE) cellulose beads. The enzymatically treated samples were characterized by microscopic methods and porosity measurements such as mercury porosimetry, nitrogen sorption and size exclusion chromatography. It was found that under controlled conditions the low-porosity surface layer of dried beads could be removed making the internal pore space accessible without reducing the resistance to deformation of the beads. Additionally, a shift in pore size distribution towards larger pores was observed. Supplementary swelling treatments in solvents of high swelling power could substantially restore the former porosity of the dried beads but did not enhance the accessibility to the cellulases to a considerable extent. Internal pore volume and surface area of the derivatives were dramatically increased in the case of DEAE upon enzymatic hydrolysis, however, at the expense of mechanical stability, whereas CM was found to be less affected.     

Microemulsion polymerization of styrene using a polymerizable nonionic surfactant and a cationic surfactant

High polymer/surfactant weight ratios (up to about 15:1) of polystyrene microlatexes have been successfully produced by microemulsion polymerization using a small amount of polymerizable surfactant, ω-methoxypoly(ethylene oxide)₄₀ undecyl α-methacrylate macromonomer (PEO-R-MA-40), and cetyltrimethylammonium bromide (CTAB). After generating “seeding particles” in a ternary microemulsion containing only 0.2 wt% CTAB and 0.1 wt% styrene, the additional styrene containing less than 1 wt% PEO-R-MA-40 was added dropwise to the polymerized microemulsion for a period of about 4 h at room temperature. PEO-R-MA-40 copolymerized readily with styrene. The stable microlatexes were bluish-transparent at a lower polymer content and became bluish-opaque at a higher polymer content. Nearly monodisperse latex particles with diameters ranging from 50 to 80 nm and their molar masses ranging from 0.6 to 1.6 × 10⁶ g/mol could be obtained by varying the polymerization conditions. The dependence of the number of particles per milliliter of microlatex, the latex particle size and the copolymer molar mass on the polymerization time is discussed in conjunction with the effect of the macromonomer concentration. Received: 25 October/2000 Accepted: 2 February 2001     

Effect of copolymer structure on micellar behavior of acrylamide-octylphenylpoly (oxyethylene) acrylate surfactant

Acrylamide-octylphenylpoly (oxyethylene) acrylate copolymer (AM-C₈PhEO_nAc) surfactant is the copolymer of acrylamide (AM) and octylphenylpoly (oxyethylene) acrylate macromonomer (C₈PhEO_nAc). The effect of the copolymer structure on the micellar behavior in aqueous solution was studied using dynamic light scattering. It has been found that the length of ethylene oxide (EO) in the branch and the content of C₈PhEO_nAc in the copolymer surfactant have great effects on the size and distribution of the micelles. For AM-C₈PhEO₇Ac copolymer, at the concentration of 5 × 10⁻⁴ g/ml, the micellar size increases with the increase of C₈PhEO₇Ac content. However, for AM-C₈PhEO₁₀Ac copolymer, the result is the opposite; the micellar size decreases with the increase of C₈PhEO₁₀Ac content. Larger C₈PhEO_nAc content leads to narrower micellar distribution. For copolymer surfactants with equal C₈PhEO_nAc content, when the concentration of copolymer solution is the same, the copolymer with longer EO length forms smaller micelles. Received: 2 February 2000 Accepted: 6 October 2000     

Effect of cellulase adsorption on the surface and interfacial properties of cellulose

The surface properties of several purified cellulose (Sigmacell 101, Sigmacell 20, Avicel pH 101, and Whatman CF 11) were characterised, before and after cellulase adsorption. The following techniques were used: thin-layer wicking (except for the cellulose Whatman), thermogravimetry, and differential scanning calorimetry (for all of the above celluloses). The results obtained from the calorimetric assays were consistent with those obtained from thin-layer wicking – Sigmacell 101, a more amorphous cellulose, was the least hydrophobic of the analysed celluloses, and had the highest specific heat of dehydration. The other celluloses showed less affinity for water molecules, as assessed by the two independent techniques. The adsorption of protein did not affect the amount of water adsorbed by Sigmacell 101. However, this water was more strongly adsorbed, since it had a higher specific heat of dehydration. The more crystalline celluloses adsorbed a greater amount of water, which was also more strongly bound after the treatment with cellulases. This effect was more significant for Whatman CF-11. Also, the more crystalline celluloses became slightly hydrophilic, following protein adsorption, as assessed by thin-layer wicking. However, this technique is not reliable when used with cellulase treated celluloses.     

Effect of sodium hydroxide treatment of bacterial cellulose on cellulase activity

The synergistic action between Thermobifida fusca exocellulase Cel6B and endocellulase Cel5A on sodium hydroxide pretreated bacterial cellulose (BC) was determined. The activities of Cel6B and Cel5A were tested singly and both activities were dramatically increased on pretreated BC, especially in the early stage of hydrolysis. Cel5A, which attacks the cellulose chain randomly, showed a larger increase on NaOH treated BC than Cel6B. Mixtures of the two enzymes were also able to degrade NaOH treated BC faster than BC and the kinetics of the mixture differed from that of the individual enzymes. The degree of synergistic effect (DSE) on BC decreased dramatically with time of hydrolysis. However, the DSE on NaOH treated BC was almost constant throughout the incubation, with a smaller effect at higher NaOH concentrations. The change caused by NaOH did not increase the DSE, although each individual cellulase activity increased. This showed that synergistic activity was more effective on recalcitrant cellulose, which requires effective cooperation between the cellulase components for hydrolysis.     

Steady and dynamic rheological behaviors of sodium carboxymethyl cellulose entangled semi-dilute solution with opposite charged surfactant dodecyl-trimethylammonium bromide

The steady and dynamic rheological behaviors of sodium carboxymethyl cellulose (NaCMC) entangled semi-dilute solution filled with different concentrations of dodecyl-trimethylammonium bromide (C₁₂TAB) were investigated. The results reveal that the zero shear rate viscosity (η₀) and dynamic modules (G′and G″) increase with C₁₂TAB concentration (C_s), and there exist three scaling regions divided by two critical C₁₂TAB concentrations (C₁, C₂ and , respectively, from steady and dynamic tests). The increase of viscosity and modules with C_s is ascribed to formation of network due to C₁₂TAB micelles bridging NaCMC chains. The two critical C₁₂TAB concentrations implies that the structure evolution of NaCMC–C₁₂TAB complex is exposed to three states with increasing C_s, i.e., no network formation, network extent progressive formation and perfect network formation, respectively. Moreover, are a little lower than C₁, C₂, indicating that the dynamic test is more sensitive to detect the structure change of the complex as compared with steady test. Furthermore, it is found that as NaCMC concentration increases, , and increase.     

Influence of molecular characteristics of nonionic cellulose ethers on their interaction with ionic surfactant investigated by conductometry

The interaction between nonionic derivatives of cellulose, hydroxypropylmethyl cellulose (HPMC) and methyl cellulose (MC), and ionic surfactant, sodium dodecylsulfate (SDS) were investigated by conductometric titration method, at 30°C. Obtained titration curves show two break points: critical aggregation concentration (cac) defined as the concentration of SDS at which interaction starts, and polymer saturation concentration (psp) as the concentration at which interaction finishes. Changes of characteristic concentration breaks were determined in dependence on concentration and molecular characteristics of cellulose derivatives (degree of substitution (DS) and molecular mass, i.e. intrinsic viscosity). It was shown that the first break point, cac, is independent of polymer concentration; while the second break point, psp, increases as polymer concentration increases, as described by a linear correlation. The slopes of linear relationship justify the DS on the intensity of the cellulose derivatives–SDS interaction. Changes in the intrinsic viscosity of cellulose derivatives do not exhibit influence on the interaction with SDS.     

Synthesis and characterization of a novel double-tailed cationic surfactant

A novel double-tailed cationic surfactant, N,N′-didodecylacetamidinium bicarbonate, was prepared by reacting dimethylacetamide dimethyl acetal with dodecylamine, followed by reacting with dry ice. MSD, FTIR, ¹H NMR, ¹³C NMR, 2D-NMR were used to confirm its structure. The surfactant reduced the surface tension of pure water to 24.7 mN m⁻¹ and its critical micelle concentration was 8.75 × 10⁻⁵ M. Formation of vesicle was also observed.     

Real-time observation of the swelling and hydrolysis of a single crystalline cellulose fiber catalyzed by cellulase 7B from Trichoderma reesei

The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.