Corn Fiber: Structure, Composition, and Response to Enzymes for Fermentable Sugars and Coproducts

Corn (Zea mays L.) fiber, which is the seed coat and residual endosperm left after grain processing, is a low-value residue that contains carbohydrates and aromatic compounds that could provide value-added coproducts. Treatment of corn fiber with NaOH and assessment by gas chromatography indicated a prevalence of ferulic acid, with about 90% ester-linked in the cell walls. p-Coumaric acid was much lower at about 10% of the amount of ferulic acid. Histochemical reactions employing acid phloroglucinol and diazotized sulfanilic acid indicated the presence of phenolic acids in cell walls of the pericarp and aleurone layer. Various protocols were tested using milled corn fiber and pretreatment with commercial ferulic acid esterases before cellulase treatment, and dry weight loss and sugars and phenolic acids released into the filtrate were evaluated. Ferulic acid esterases effectively degraded corn fiber and released substantial amounts of ferulic acid and sugars (e.g., glucose and xylose) in the incubation medium. Light microscopy showed that ferulic acid esterase substantially disrupted the aleurone layer but caused little visible damage to the lignified pericarp cell walls. Amounts of compounds released varied with protocols, and one study with various milling methods showed that esterase pretreatment followed by cellulase released about 2.8 to 4.4 and 1.5 to 2.9 times more ferulic acid and glucose, respectively, than cellulase alone. The highest levels for one lot of corn fiber with esterase pretreatment followed by cellulase were 3.9 and 218 mg/g of ferulic acid and glucose, respectively.     

Grass lignocellulose

Grass lignocelluloses are limited in bioconversion by aromatic constituents, which include both lignins and phenolic acids esters. Histochemistry, ultraviolet absorption microspectrophotometry, and response to microorganisms and specific enzymes have been used to determine the significance of aromatics toward recalcitrance. Coniferyl lignin appears to be the most effective limitation to biodegradation, existing in xylem cells of vascular tissues; cell walls with syringyl lignin, for example, leaf sclerenchyma, are less recalcitrant. Esterified phenolic acids, i.e., ferulic and p-coumaric acids, often constitute a major chemical limitation in nonlignified cell walls to biodegradation in grasses, especially warm-season species. Methods to improve biodegradation in grasses, especially warm-season species. Methods to improve biodegradability through modification of aromatics include: plant breeding, use of lignin-degrading white-rot fungi, and addition of esterases. Plant breeding for new cultivars has been especially effective for nutritionally improved forages, for example, bermudagrasses. In laboratory studies, selective white-rot fungi that lack cellulases delignified the lignocellulosic materials and improved fermentation of residual carbohydrates. Phenolic acid esterases released p-coumaric and ferulic acids for potential coproducts, improved the available sugars for fermentation, and improved biodegradation. The separation and removal of the aromatic components for coproducts, while enhancing the availability of sugars for bioconversion, could improve the economics of bioconversion.     

Comparison of different methods for the detoxification of lignocellulose hydrolyzates of spruce

This study describes different detoxification methods to improve both cell growth and ethanol production by Baker's yeast, Saccharomyces cerevisiae. A dilute-acid hydrolyzate of spruce was used for the all detoxification methods tested. The changes in the concentrations of fermentable sugars and three groups of inhibitory compounds—aliphatic acids, furan derivatives, and phenolic compounds—were determined and the fermentability of the detoxified hydrolyzate was assayed. The applied detoxification methods included: treatment with alkali (sodium hydroxide or calcium hydroxide); treatment with sulfite (0.1% [w/v] or 1% [w/v] at pH 5.5 or 10); evaporation of 10% or 90% of the initial volume; anion exchange (at pH 5.5 or 10); enzymatic detoxification with the phenoloxidase laccase; and detoxification with the filamentous fungus Trichoderma reesei. An ion exchange at pH 5.5 or 10, treatment with laccase, treatment with calcium hydroxide, and treatment with T. reesei were the most efficient detoxification methods. Evaporation of 10% of the initial volume and treatment with 0.1% sulfite were the least efficient detoxification methods. Treatment with laccase was the only detoxification method that specifically removed only one group of the inhibitors, namely phenolic compounds. Anion exchange at pH 10 was the most efficient method for removing all three major groups of inhibitory compounds; however, it also resulted in loss of fermentable sugars.     

Evaluation of a Hypocrea jecorina Enzyme Preparation for Hydrolysis of Tifton 85 Bermudagrass

Tifton 85 bermudagrass, developed at the ARS-USDA in Tifton, GA, is grown on over ten million acres in the USA for hay and forage. Of the bermudagrass cultivars, Tifton 85 exhibits improved digestibility because the ratio of ether- to ester-linked phenolic acids has been lowered using traditional plant breeding techniques. A previously developed pressurized batch hot water (PBHW) method was used to treat Tifton 85 bermudagrass for enzymatic hydrolysis. Native grass (untreated) and PBHW-pretreated material were compared as substrates for fungal cultivation to produce enzymes. Cellulase activity, measured via the filter paper assay, was higher for fungi cultivated on PBHW-pretreated grass, whereas the other nine enzyme assays produced higher activities for the untreated grass. Ferulic acid and vanillin levels increased significantly for the enzyme preparations produced using PBHW-pretreated grass and the release of these phenolic compounds may have contributed to the observed reduction in enzyme activities. Culture supernatant from Tifton 85 bermudagrass-grown fungi were combined with two commercial enzyme preparations and the enzyme activity profiles are reported. The amount of reducing sugar liberated by the enzyme mixture from Hypocrea jecorina (after 192 h incubation with untreated bermudagrass) individually or in combination with feruloyl esterase was 72.1 and 84.8%, respectively, of the commercial cellulase preparation analyzed under the same conditions.     

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatmett or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60°C, 0.5∶1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.     

Wild olive fruits: Phenolics profiling,antioxidants, antimicrobial,thrombolytic and haemolytic activities

In this study, wild olive fruits were evaluated for the occurrence of phenolic antioxidant components and valuable nutrients which are distributed wildly in Soon valley of Pakistan. The shade-dried fruits of wild olive were extracted using different solvents to recover phenolic antioxidants. The highest concentration of extractable antioxidant components was recovered from tested fruits using aqueous ethanol compared to other solvents used. Crude concentrated extracts (CCEs) and phenolic rich fractions (PRFs) of tested fruits using hydroxyethanol were found to contain higher amount of total phenolic compounds and total flavonoid compounds along with superior biological attributes. According to ICP-OES analysis, potassium (17.96 g/kg) was the dominant macro element among other identified twenty-five minerals. The tested wild olive fruits juice was found to contain individual natural sugars including galactose (4.92 g/100 g dry weight), sucrose (2.75 g/100 g dry weight), glucose (0.73 g/100 g dry weight); and succinic acid (8.80 mg/100 g of dry matter) as major organic acid when analyzed on HPLC. Oleic acid (47.41 %) was the major monounsaturated fatty acid in the oil extracted from tested fruits. The concentration of phenolic antioxidants and biological activities vary significantly (p < 0.05) among extracting systems used. A strong correlation was also recorded among total phenolic (TP), total flavonoids (TF) and biological attributes of tested wild olive fruits. The results of this study explored wild olive fruits as a propitious source of natural phenolic components and valuable nutrients which reveal its potential use in the development of functional food and nutra-pharmaceuticals.     

Nutra-pharmaceutical potential and phytonutrients profiling of wild jujube fruits along with bioactivities studies

The present study reports the phenolic antioxidants and phytonutrients profiling in the wild jujube fruits which are naturally grown in Soon valley of Pakistan. Phenolic antioxidant components were recovered from shade-dried wild jujube fruits using various extracting solvents. Among all extracting solvents tested, aqueous ethanol recovered the maximum amount of extractable antioxidant compounds from the fruits of wild jujube. Crude concentrated extracts (CCEs) and phenolic rich fractions (PRFs) recovered from wild jujube fruits using aqueous ethanol contained higher concentration of total phenolics and flavonoids along with superior biological potential. ICP-OES analysis disclosed the occurrence of twenty-five minerals, where potassium (14.80 g/kg) and calcium (1.81 g/kg) were the dominant macro elements. The tested wild fruits juice was found to contain individual natural sugars including galactose (1.27 g/100 g dry weight), glucose (1.07 g/100 g dry weight), sucrose (0.70 g/100 g dry weight) and xylose (0.04 g/100 g dry weight); and gluconic acid (2.10 mg/100 g of dry matter) as dominant organic acid when analyzed on HPLC. The concentration of phenolic antioxidants and biological activities vary significantly (p < 0.05) among extracting systems used. A strong correlation was also recorded among total phenolic (TP), total flavonoids (TF) and biological attributes of tested wild fruits. The results of this study explored wild jujube fruits as a propitious source of natural phenolic components and valuable nutrients which advocate its potential use in the development of functional food and nutraceutical industry.     

Effects of External Enzymes on the Fermentation of Soybean Hulls to Generate Lipids by Mortierella isabellina

Hydrolytic enzymes were evaluated on the lipid accumulation via an oleaginous fungal species, Mortierella isabellina, cultivated on sugars released from soybean hulls. The weight loss of soybean hull, fungal growth, and lipid production were tested under different loads of hydrolytic enzymes. M. isabellina could not directly utilize cellulose and adding cellulase and ??-glucosidase significantly increased the cell growth and oil accumulation of M. isabellina on soybean hulls. The highest weight loss of soybean hulls was 47.80?% and the lipid production reached 0.14?g from 1?g of soybean hull when 12?U cellulase, 27.2?U ??-glucosidase, 2,278.56?U pectinase, and 15?U hemicellulase were added. Fatty acids (76.82?%) accumulated in M. isabellina were C16 and C18, which are suitable for biodiesel production. These results provide a new application for soybean hulls to be applied as the raw material for the production of biodiesel fuel, besides its traditional role as animal feed supplements.     

HPLC determination of organic acids, sugars, phenolic compositions and antioxidant capacity of orange juice and orange wine made from a Turkish cv. Kozan

Organic acids, sugars, phenolic compositions and antioxidant capacities of orange juice and orange wine obtained from the cv. Kozan of Turkey were determined. High-performance liquid chromatographic methods were used to identify and quantify of these compounds. Three organic acids (citric, malic and ascorbic acids) and three sugars (sucrose, glucose and fructose) were determined. The major organic acid was found as citric acid. With regard to sugars, sucrose was present in the largest amounts for orange juice and wine. A total of 13 phenolic compounds were identified and quantified in orange juice and wine, including hydroxybenzoic acids (2), hydroxycinnamic acids (5), and flavanones (6). Hesperidin, narirutin and ferulic acid were the most abundant phenolic compounds in orange juice and wine. Antioxidant activities of orange juice and wine were measured using the DPPH^• (2,2-diphenyl-1-picrylhydrazyl) assay, and the antioxidant capacity of orange juice was found to be higher than that of orange wine.     

Hydrolysis of Ammonia-pretreated Sugar Cane Bagasse with Cellulase, β-Glucosidase, and Hemicellulase Preparations

Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH₃: 1 biomass, 100 °C, 30 min) or with NH₄OH (0.5 g NH₄OH of a 28% [v/v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.     

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.     

Bioconversion of Kraft Paper Mill Sludges to Ethanol by SSF and SSCF

Paper mill sludge is a solid waste material composed of pulp residues and ash generated from pulping and paper making processes. The carbohydrate portion of the sludge has chemical and physical characteristics similar to pulp. Because of its high carbohydrate content and well-dispersed structure, the sludges can be biologically converted to value-added products without pretreatment. In this study, two different types of paper mill sludges, primary sludge and recycle sludge, were evaluated as a feedstock for bioconversion to ethanol. The sludges were first subjected to enzymatic conversion to sugars by commercial cellulase enzymes. The enzymatic conversion was inefficient because of interference by ash in the sludges with the enzymatic reaction. The main cause was that the pH level is dictated by CaCO₃ in ash, which is two units higher than the pH optimum of cellulase. To alleviate this problem, simultaneous saccharification and cofermentation (SSCF) using cellulase (Spezyme CP) and recombinant Escherichia coli (ATCC-55124), and simultaneous saccharification and fermentation (SSF) using cellulase and Saccharomyces cerevisiae (ATCC-200062) were applied to the sludges without any pretreatment. Ethanol yields of 75–81% of the theoretical maximum were obtained from the SSCF on the basis of total carbohydrates. The yield from the SSF was also found to be in the range of 74–80% on the basis of glucan. The SSCF and SSF proceeded under stable condition with the pH staying near 5.0, close to the optimum for cellulase. Decrease of pH occurred due to carbonic acid and other organic acids formed during fermentation. The ash was partially neutralized by the acids produced from the SSCF and SSF and acted as a buffer to stabilize the pH during fermentation. When the SSF and SSCF were operated in fed-batch mode, the ethanol concentration in the broth increased from 25.5 and 32.6 g/L (single feed) to 45 and 42 g/L, respectively. The ethanol concentration was limited by the tolerance of the microorganism in the case of SSCF. The ethanol yield in fed-batch operation decreased to 68% for SSCF and 70% for SSF. The high-solids condition in the bioreactor appears to create adverse effects on the cellulase reaction.     

Utilizing cellulase as a hydrogen peroxide stabilizer to combine the biopolishing and bleaching procedures of cotton cellulose in one bath

In this research, the stabilization effect of cellulase on the decomposition of hydrogen peroxide was investigated for the first time. It was concluded that, regardless of the decomposition mechanism, the cellulase protein could contribute significantly to peroxide stability. This effect stems from the formation of molecular hydrogen bonding between peroxide and cellulase protein or direct sequestering of free metal ions by amino acids in cellulase. Furthermore, based on this stability, a combined biopolishing and peroxide bleaching protocol was developed to improve cotton quality more efficiently. Afterwards, physicochemical properties such as the weight and strength loss, water absorbency, and carbonyl and carboxyl group content of treated cotton cellulose were measured to show the feasibility of the new method. Fourier-transform infrared (FT-IR) and X-ray diffraction (XRD) analyses indicated that the crystallinity index of cotton was increased due to the preferential hydrolysis of amorphous cellulose by cellulase.     

Phenolics content and antioxidant activity of tartary buckwheat from different locations

Two tartary buckwheat samples (Xingku No.2 and Diqing) grown at three locations were analyzed for free and bound phenolic content and antioxidant properties. Moreover, the relative contributions of variety and growing environment to phenolic content and antioxidant properties were determined, as well as correlations of these properties to growing conditions. The total phenolic contents varied from 5,150 to 9,660 μmol of gallic acid equivalents per 100 gram of dry weight (DW) of tartary buckwheat and the free phenolics accounted for 94% to 99%. Rutin content was in the range from 518.54 to 1,447.87 mg per 100 gram of DW of tartary buckwheat. p-Hydroxybenzoic, ferulic and protocatechuic acids were the prominent phenolic acids and other phenolics, including p-coumaric, gallic, caffeic, vanillic and syringic acids were also detected. Tartary buckwheat exhibited higher DPPH· and ABTS·+ scavenging activities and was more effective at preventing the bleaching of β-carotene in comparison with reference antioxidant and plant phenolics constituents. Additionally, growing conditions and the interaction between variety and environment may have more contribution than variety to individual phenolics and antioxidant properties of tartary buckwheat. Environmental parameters such as higher altitudes may also have an increasing effect on rutin and phenolic acids. This study suggests that tartary buckwheat has potential health benefits because of its high phenolic content and antioxidant properties. These components could also be enhanced by optimizing the growing conditions of a selected variety.     

Optimization of Brewery's spent grain dilute-acid hydrolysis for the production of pentose-rich culture media

Dilute-acid hydrolysis of brewery's spent grain to obtain a pentose-rich fermentable hydrolysate was investigated. The influence of operational conditions on polysaccharide hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.39–3.06. When the CS increased, the pentose sugars concentration increased to a maximum at a CS of 1.94, whereas the maximum glucose concentration was obtained for a CS of 2.65. The concentrations of furfural, hydroxymethylfurfural (HMF), as well as formic and levulinic acids and total phenolic compounds increased with severity. Optimum hydrolysis conditions were found at a CS of 1.94 with >95% of feedstock pentose sugars recovered in the monomeric form, together with a low content of furfural, HMF, acetic and formic acids, and total phenolic compounds. This hydrolysate containing glucose, xylose, and arabinose (ratio 10∶67∶32) was further supplemented with inorganic salts and vitamins and readily fermented by the yeast Debaryomyces hansenii CCMI 941 without any previous detoxification stage. The yeast was able to consume all sugars furfural, HMF, and acetic acid with high biomass yield, 0.68C-mol/C-mol, and productivity, 0.92 g/(L·h). Detoxification with activated charcoal resulted in a similar biomass yield and a slight increase in the volumetric productivity (11%).     

Phytochemical characterization and radical scavenging activity of Portulaca oleraceae L. leaves and stems

The present work aimed to proceed to the phytochemical characterization of purslane (Portulaca oleracea L.) and study of its antioxidant activity. Different samples were analyzed for their moisture and fat content, fatty acids, organic acids and phenolic compound composition and the scavenging effect of DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals was evaluated. Water was the major constituent of purslane stems (average 90.5%) and leaves (average 91.8%), while fat values varied from 0.11% to 0.57%. Twenty seven fatty acids were found in the leaves samples, being linolenic acid the most abundant, ranging from 27.7 to 39.1%, followed by palmitic (19.3–24.3%) and oleic acids (11.6–19.5%). Five different organic acids were detected namely fumaric, aconitic, citric, malic and oxalic acids. Oxalic and citric acids were the most abundant, while aconitic acid was present in the lowest content. Two phenolic acids were identified and quantified: 3-caffeoylquinic and 5-caffeoylquinic acids. Leaves presented higher amounts of phenolic compounds, being 3-caffeoylquinic acid more abundant in leaves and 5-caffeoylquinic acid in stems. The leaves revealed to be the part with highest antioxidant potential.     

The impact of enzyme characteristics on corn stover fiber degradation and acid production during ensiled storage

Ensilage can be used to store lignocellulosic biomass before industrial bioprocessing. This study investigated the impacts of seven commercial enzyme mixtures derived from Aspergillus niger, Trichoderma reesei, and T. longibrachiatum. Treatments included three size grades of corn stover, two enzyme levels (1.67 and 5 IU/g dry matter based on hemicellulase), and various ratios of cellulase to hemicellulase (C:H). The highest C:H ratio tested, 2.38, derived from T. reesei, resulted in the most effective fermentation, with lactic acid as the dominant product. Enzymatic activity during storage may complement industrial pretreatment; creating synergies that could reduce total bioconversion costs.     

Development of a Solid-Phase Extraction Procedure for the Simultaneous Determination of Polyphenols, Organic Acids and Sugars in Wine

A solid-phase extraction (SPE) procedure that fractionates wine samples into 2 sub-samples containing sugars and organic acids (sub-sample 1) and low molecular weight polyphenols (sub-sample 2), respectively, together with rugged LC procedures for their analyses are described. Wine is adjusted to pH 2.5 and loaded on a styrene-divinylbenzene (SDB) cartridge. The organic acids and sugars are eluted with 20 mM sulphuric acid and the monomeric polyphenols with ethyl acetate. Glucose and fructose are analysed by normal phase LC with evaporative light scattering detection and the organic acids by ion exclusion chromatography with UV detection at 210 nm. Analysis of the phenolic fraction is performed by reversed phase LC with diode array detection. Recoveries and repeatabilitys for 27 standard compounds (2 sugars, 7 organic acids and 18 polyphenols) are presented. The method represents an improvement in terms of productivity and robustness compared to currently used procedures.     

Identification and characterization of phenolic compounds in castor seed

Phenolic compounds, polyphenols, and flavonoids occur ubiquitously in plant kingdom, because they are important in plants for normal growth development and defense against infection and injury. Currently, there are no reports available on the phenolic compounds obtained from the residues of the oil-extracting process from defatted castor seed powder. Our studies, for the first time, in methanol-ether extract showed five low-molecular weight phenolic compounds namely p-coumaric acid, ferulic acid, o-coumaric acids, syringic, and cinnamic acids, which were in the soluble ether fractions. All these compounds showed strong absorbance at 240 nm.     

Possibilities for recycling cellulases after use in cotton processing: part II: Separation of cellulases from reaction products and released dyestuffs by ultrafiltration

The adsorption and activity of a total cellulase (Trichoderma reesei) was measured and compared on undyed and dyed cotton fabrics. Recovery of enzymes from the reaction mixture and by desorption from the cotton substrate was evaluated. About 80% of the initial protein could be recovered. The removal of released products (soluble reducing sugars and dyes) from the treatment liquor and subsequent concentration of cellulase proteins was performed using an ultrafiltration membrane. Strong protein-dye interactions made it impossible to separate efficiently the dyes from the enzyme-containing treatment liquors. The use of surfactants did not enhance cellulase desorption from cotton fabric. Although anionic surfactants have a deactivating effect on cellulases, this effect seems to be reversible, since after ultrafiltration the cellulase activity was similar to that of enzymes desorbed with buffer only. Humicola insolens cellulases were shown to be much more sensitive to anionic surfactant than T. reesei cellulases. The use of cellulases that bind reversibly to cellulose is suggested for achieving more efficient cellulase recycling and for reducing backstaining by dye-cellulase complexes.