Ethanol/water pulp enzymatic pretreatment: Chemical and FTIR-PCA analyses

Pulps obtained from ethanol/water cooking of sugarcane bagasse were treated at different times using xylanase enzyme obtained from Thermomyces lanuginosus IOC-4145 or commercially (Cartazyme HS, Sandoz Products Ltd.). The enzyme dosage was 18 IU per g of dry pulp and the time varied from 4 h to 12 h. When xylanase from T. lanuginosus was used, the kappa number and viscosity improved independently of the processing time used (4 h, 8 h, and 12 h). After chemical evaluation, the obtained pulps were classified using Fourier Transformed Infra-Red Spectroscopy and Principal Component Analysis. The results showed that the first three principal components explained more than 90 % of the total variance of the pulp spectra.     

Carboxymethylcellulose obtained by ethanol/water organosolv process under acid conditions

Sugar cane bagasse pulps were obtained by ethanol/water organosolv process under acid and alkaline conditions. The best condition of acid pulping for the sugarcane bagasse was 0.02 mol/L sulfuric acid at 160 degrees C, for 1 h, whereas the best condition for alkaline pulping was 5% sodium hydroxide (base pulp) at 160 degrees C, for 3 h. For the residual lignin removal, the acid and alkaline pulps were submitted to a chemical bleaching using sodium chlorite. Pulps under acid and alkaline conditions bleached with sodium chlorite presented viscosities of 3.6 and 7.8 mPa x s, respectively, and mu-kappa numbers of 1.1 and 2.4, respectively. The pulp under acid condition, bleached with sodium chlorite was used to obtain carboxymethylcellulose (CMC). CMC yield was 35% (pulp based), showing mass gain after the carboxymethylation reaction corresponding to 23.6% of substitution or 0.70 groups -CH(2) COONa per unit of glucose residue. The infrared spectra showed the CMC characteristic bands and by the infrared technique it was possible to obtain a substitution degree (0.63), similar to the substitution degree calculated by mass gain (0.70).     

Effect of dose of xylanase on bleachability of sugarcane bagasse ethanol/water pulps

Pulps obtained from the ethanol/water cooking of sugarcane bagasse were bleached with the xylanase enzyme obtained from the fungus Thermomyces lanuginosus IOC-4145 and with the commercial enzyme Cartazyme HS from Sandoz. By changing the enzyme dose from 4.3 to 36 IU/g of pulp, kappa number and viscosity were maintained when the xylanase from T. lanuginosus was used. On the other hand, by using Cartazyme HS, kappa number decreased by 17%, reaching 35.5. This pulp was further extracted with NaOH without a decrease in viscosity (10 cP), and pulp with a kappa number of 13 was obtained. Xylanases had no significant effect on the ethanol/water pulps.     

Ethanol production from cellulose by coupled saccharification/fermentation usingSaccharomyces cerevisiae and cellulase complex fromSclerotiun rolfsii UV-8 mutant

Using cellulase/hemicellulase complex of Sclerotium rolfsii UV-8 mutant and Saccharomyces cerevisiae for fermentation, the coupled saccharification/fermentation (CSF) of 15% AT-rice straw was carried out at 40 degrees C, pH 4.5 for the first 24 h and further incubation was performed at 30 degrees C for 72 h. Increasing the amount of cellulase activity from 3-12 IU FPA/g of substrate resulted in increased yields of ethanol from 1.5-3.6% in 96 h. It has been observed that the coupled system was advantageous over the two stage (separate hydrolysis/fermentation) system as it produced higher amounts of ethanol from cellulose (3.6% as compared to 2.3% ethanol from rice straw).     

High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol

Wheat straw was pretreated with dilute (0.5%) sulfuric acid at 140°C for 1 h. Pretreated straw solids were washed with deionized water to neutrality and then stored frozen at –20°C. The approximate composition of the pretreated straw solids was 64% cellulose, 33% lignin, and 2% xylan. The cellulose in the pretreated wheat straw solids was converted to ethanol in batch simultaneous saccharification and fermentation experiments at 37°C using cellulase enzyme fromTrichoderma reesei (Genencor 150 L) with or without supplementation with β–glucosidase fromAspergillus niger (Novozyme 188) to produce glucose sugar and the yeastSaccharomyces cerevisiae to ferment the glucose into ethanol. The initial cellulose concentrations were adjusted to 7.5, 10, 12.5, 15, 17.5, and 20% (w/w). Since wheat straw particles do not form slurries at these concentrations and cannot be mixed with conventional impeller mixers used in laboratory fermenters, a simple rotary fermenter was designed and fabricated for these experiments. The results of the simultaneous saccharification and fermentation (SSF) experiments indicate that the cellulose in pretreated wheat straw can be efficiently fermented into ethanol for up to a 15% cellulose concentration (24.4% straw concentration).     

Oxidation in Acidic Medium of Lignins from Agricultural Residues

Agricultural residues as sugarcane straw and bagasse are burned in boilers for generation of energy in sugar and alcohol industries. However, excess of those by-products could be used to obtain products with higher value. Pulping process generates cellulosic pulps and lignin. The lignin could be oxidized and applied in effluent treatments for heavy metal removal. Oxidized lignin presents very strong chelating properties. Lignins from sugarcane straw and bagasse were obtained by ethanol–water pulping. Oxidation of lignins was carried out using acetic acid and Co/Mn/Br catalytical system at 50, 80, and 115 °C for 5 h. Kinetics of the reaction was accomplished by measuring the UV-visible region. Activation energy was calculated for lignins from sugarcane straw and bagasse (34.2 and 23.4 kJ mol⁻¹, respectively). The first value indicates higher cross-linked formation. Fourier-transformed infrared spectroscopy data of samples collected during oxidation are very similar. Principal component analysis applied to spectra shows only slight structure modifications in lignins after oxidation reaction.     

Catalyzed sulphite and semichemical pulping

Very high yield sulphite pulps were produced by cooking black spruce wafers in pulping liquors at pH 7 or 10, containing 0.1% (on O.D. wood) of soluble anthraquinone (SAQ). These pulps had better strength properties relative to controls prepared without SAQ, breaking length and burst index being greater, on average, by 20%. Other improvements included: increased pulping rate, lower lignin contents at comparable pulp yields, and higher carbohydrate content at the same level of residual lignin in pulp (this resulted in an increase of total pulp yield by 2%). Results of cooks in liquors ranging in pH from 4 to 10, and under variable conditions of time (20–60 min) and temperature (120–160°C) suggested that: firstly, AQ does not act as a pulping catalyst at pH 4, and secondly, the sulphonate contents of AQ-catalyzed pulps are lower than those of the uncatalyzed controls. In the light of the lower sulphonate content, the higher strength is unexpected.     

Direct analysis of lignin and lignin-like components from softwood kraft pulp by Py-GC/MS techniques

Analytical pyrolysis combined with gas chromatography/mass spectrometry was used to analyse the structure and quantity of aromatic components, mainly guaiacyl and hydroxyphenyl derivatives, directly from chemical pulps. The quantity of aromatic degradation products was determined using a new external calibration method. The external standard was analyzed similarly to the pulp sample, and the combined area of the degradation products formed, normalized to the sample amount, was used for calibration. The method was sensitive enough to detect aromatics from fully bleached softwood pulps at a concentration level of 0.4 wt.%.The effect of bleaching on lignin structures in softwood pulps was studied by following the changes in guaiacyl-type degradation product distribution. The residual lignin structures that had been modified during cooking were removed during the course of bleaching. The residual lignin in fully bleached pulps therefore was found to bear features characteristic of native lignin in addition to increased oxidation. A striking enrichment of hydroxyphenyl-type aromatic pyrolysis products was observed during bleaching. It is suggested that they are derived not only from lignin but also from other pulp components.     

Biobleaching of nonwoody pulps using xylanase of Bacillus brevis BISR-062

The efficiency of xylanase of Bacillus brevis BISR-062 as a prebleaching agent was evaluated on three nonwoody pulps at two different pH values (7.0 and 8.5). Crude xylanase was found to have an optimum temperature and pH of 65-70 degrees C and 7.0, respectively. The stability of the enzyme was determined at two pH values (7.0 and 8.0), and it lost approx 50% of its activity at both values within 2 h at 50 degrees C. However, the enzyme was found to be effective as a prebleaching agent only with rice straw pulp. A maximum brightness gain of 6 points was obtained with this pulp at pH 7.0. The strength properties of the rice straw pulp at pH 7.0 also improved as the result of enzyme treatment.     

Alkaline peroxide mechanical pulping of wheat straw with enzyme treatment

Alkaline peroxide mechanical pulping (APMP) of wheat straw with enzyme treatment was studied. Instead of direct enzyme pretreatment on wheat straw, an alternative treatment method was used, in which coarse pulps from refiner defibrated wheat straw rather than wheat straw were pretreated with a crude enzyme containing mainly xylanase, then impregnated with alkaline H₂O₂ solution and further refined. The optimum conditions of enzyme treatment were xylanase dosage of 10–15 IU/g of oven-dried wheat straw, 90 min, 50–60°C, pulp consistency of 5–10%, and initial pH of 5.0, and those for chemical impregnation were 6% NaOH, 70–80°C, 60–90 min, and 4 to 5% H₂O₂. Enzyme treatment improved pulpability of wheat straw by the APMP process, and final pulp quality such as brightness, breaking length, and burst index of pulp. Pulp from the APMP process with enzyme treatment could be bleached to a brightness of 70.5% ISO by two-stage H₂O₂ bleaching sequence with only 4% H₂O₂, and breaking length of the bleach pulp reached 4470 m.     

Lime pretreatment of crop residues bagasse and wheat straw

Lime (calcium hydroxide) was used as a pretreatment agent to enhance the enzymatic digestibility of two common crop residues: bagasse and wheat straw. A systematic study of pretreatment conditions suggested that for short pretreatment times (1–3 h), high temperatures (85-135°C) were required to achieve high sugar yields, whereas for long pretreatment times (e.g., 24 h), low temperatures (50–65°C) were effective. The recommended lime loading is 0.1 g Ca(OH)₂/g dry biomass. Water loading had little effect on the digestibility. Under the recommended conditions, the 3-d reducing sugar yield of the pretreated bagasse increased from 153 to 659 mg Eq glucose/g dry biomass, and that of the pretreated wheat straw increased from 65 to 650 mg Eq glucose/g dry biomass. A material balance study on bagasse showed that the biomass yield after lime pretreatment is 93.6%. No glucan or xylan was removed from bagasse by the pretreatment, whereas 14% of lignin became solubilized. A lime recovery study showed that 86% of added calcium was removed from the pretreated bagasse by ten washings and could be recovered by carbonating the wash water with CO₂ at pH 9.5.     

Residual lignin inhibits thermal degradation of cellulosic fiber sheets

The market for cellulosic fiber based food packaging applications is growing together with the importance of improving the thermal durability of these fibers. To shed light on this, we investigated the role of residual lignin in pulp on the thermal stability of refined pulp sheets. The unbleached, oxygen delignified, and fully bleached pulp sheets were studied after four separate refining degrees. Comparison by Gurley air resistance, Bendtsen porosity, and the oxygen transmission rate tests showed that lignin containing sheets had better air and oxygen barrier properties than fully bleached sheets. Sheet density and light scattering coefficient measurements further confirmed that the lignin containing pulps underwent more intense fibrillation upon refining that changed the barrier properties of the sheets. Thermal treatments (at 225 °C, 20 and 60 min, in water vapor atmospheres of 1 and 75 v/v %) were applied to determine the thermal durability of the sheets. The results revealed that the residual lignin in pulps improved the thermal stability of the pulp sheets in the hot humid conditions. This effect was systematically studied by tensile strength, brightness, and light absorption coefficient measurements. The intrinsic viscosity results support the findings and suggest that lignin is able to hinder the thermal degradation of pulp polysaccharides. In spite of the fact that lignin is known to enhance the thermal yellowing of paper, no significant discoloration of the pulp sheets containing residual lignin was observed in the hot humid conditions (75 v/v %). Our results support the idea of lignin strengthening the thermal durability of paper.     

Potential Application of Alkaline Pectinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SS in Pulp and Paper Industry

Pectinase production from Bacillus subtilis SS was optimized under solid-state fermentation (5,943 U/g of dry bacterial bran). The pectinase produced was stable in neutral to alkaline pH range at 70 degrees C; therefore, the suitability of this pectinase in pulp and paper industry was investigated. The enzyme pretreatment process was optimized, and a pectinase dose of 5 IU/g of oven-dried pulp (10% consistency) at pH 9.5 temperature 70 degrees C after 150 min of treatment gave the best pretreatment to the pulp. An increase of 4.3% in brightness along with an increase of 14.8 and 65.3% in whiteness and fluorescence, respectively, whereas a 15% decrease in the yellowness of the pretreated pulp were observed. There was a 5.85% reduction in kappa number and 6.1% reduction in permanganate number along with a reduction in the chemical oxygen demand value. Significant characteristics showed by pectinase open new possibilities of application of this cellulase-free enzyme in the pulp and paper industry by reducing the negative environmental impact of chemicals apart from improving the properties of paper.     

Screening and Production Study of Microbial Xylanase Producers from Brazilian Cerrado

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by β-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 °C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0–5.5. They were stable in the pH range 5.0–10.0 and 5.5–8.5 for bacterial and fungal xylanase, respectively. The optimum temperatures were 55C and 60 °C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 °C.     

Fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol

Pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. Beet pulp was evaluated for conversion toethanol using recombinant bacteria as biocatalysts. Hydrolysis of pectin-rich residues followed by ethanolic fermentations by yeasts has not been productive because galacturonic acid and arabinose are not ferm entable toethanol by these organisms. The three recombinant bacteria evaluated in this study, Escherichia coli strain KO11, Klebsiella oxytoca strain P2, and Erwinia chrysanthemi EC 16 pLOI 555, ferment carbohydrates in beet pulp with varying efficiencies. E. coli KO11 is able to convert pure galactu ronic acid to ethanol with minimal acetate production. Using an enzyme loading of 10.5 filter paper un its of cellulase, 120.4 polygalactu ronase units of pectinase, and 6.4 g of cellobiase (per gram of dry wt sugar beet pulp), with substrate addition after 24 h of fermentation, 40 g of ethanol/L was produced. Other recombinants exhibited lower ethanol yields with increases in acetate and succinate production.     

Preliminary results on optimization of pilot scale pretreatment of wheat straw used in coproduction of bioethanol and electricity

The overall objective in this European Union-project is to develop cost and energy effective production systems for coproduction of bioethanol and electricity based on integrated biomass utilization. A pilot plan reactor for hydrothermal pretreatment (including weak acid hydrolysis, wet oxidation, and steam pretreatment) with a capacity of 100 kg/h was constructed and tested for pretreatment of wheat straw for ethanol production. Highest hemicellulose (C5 sugar) recovery and extraction of hemicellulose sugars was obtained at 190°C whereas highest C6 sugar yield was obtained at 200°C. Lowest toxicity of hydrolysates was observed at 190°C; however, addition of H₂O₂ improved the fermentability and sugar recoveries at the higher temperatures. The estimated total ethanol production was 223 kg/t straw assuming utilisation of both C6 and C5 during fermentation, and 0.5 g ethanol/g sugar.     

The effect of chemical and physical characteristics of spruce SEW pulps on enzymatic hydrolysis

Conifers, which are the most abundant biomass species in Nordic countries, USA, Canada and Russia, exhibit strong resistance towards depolymerization by cellulolytic enzymes. At present, it is still not possible to isolate a single structural feature which would govern the rate and degree of enzymatic hydrolysis. On the other hand, the forest residues alone represent an important potential for biochemical production of biofuels. In this study, the effect of substrate properties on the enzymatic hydrolysis of softwood was studied. Stem wood spruce chips were fractionated by SO₂–ethanol–water (SEW) treatment to produce pulps of varying composition by applying different operating conditions. The SEW technology efficiently fractionates different types of lignocellulosic biomass by rapidly dissolving hemicelluloses and lignin. Cellulose remains fully in the solid residue which is then treated by enzymes to release glucose. The differences in enzymatic digestibility of the spruce SEW pulp fibers were interpreted in terms of their chemical and physical characteristics. A strong correlation between the residual lignin content of SEW pulp and enzymatic digestibility was observed whereas cellulose degree of polymerization and hemicellulose content of pulp were not as important. For the pulps containing about 1.5 % (w/w) lignin, 90 % enzymatic digestibility was achieved at 10 FPU enzyme charge and 24 h of hydrolysis time.     

Oxidative lime pretreatment of high-lignin biomass

Lime (Ca[OH]₂) and oxygen (O₂) were used to enhance the enzymatic digestibility of two kinds of high-lignin biomass: poplar wood and newspaper. The recommended pretreatment conditions for poplar wood are 150°C, 6 h, 0.1 g of Ca(OH)₂/g of dry biomass, 9 mL of water/g of dry biomass, 14.0 bar absolute oxygen, and a particle size of −10 mesh. Under these conditions, the 3-d reducing sugar yield of poplar wood using a cellulase loading of 5 filter paper units (FPU)/g of raw dry biomass increased from 62 to 565 mg of eq. glucose/g of raw dry biomass, and the 3-d total sugar (glucose + xylose) conversion increased from 6 to 77% of raw total sugars. At high cellulase loadings (e.g., 75 FPU/g of raw dry biomass), the 3-d total sugar conversion reached 97%. In a trial run with newspaper, using conditions of 140°C, 3 h, 0.3 g of Ca(OH)₂/g of dry biomass, 16 mL of water/g of dry biomass, and 7.1 bar absolute oxygen, the 3-d reducing sugar yield using a cellulase loading of 5 FPU/g of raw dry biomass increased from 240 to 565 mg of eq. glucose/g of raw dry biomass. A material balance study on poplar wood shows that oxidative lime pretreatment solubilized 38% of total biomass, including 78% of lignin and 49% of xylan; no glucan was removed. Ash increased because calcium was incorporated into biomass during the pretreatment. After oxidative lime pretreatment, about 21% of added lime could be recovered by CO₂ carbonation.     

Benzylated pulps from sugar cane bagasse

An optimized ethanol/water process has been employed for the pulping of fibres from sugar cane bagasse. After pretreatment with aqueous NaOH, unbleached pulps were subjected to benzylation at 110 °C for different periods of time. The resulting purified products were characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), gel permeation chromatography (GPC) and infrared spectroscopy (IR). The results showed that benzylation proceeded to various extents depending on the reaction time, as assessed by weight gain. During the first 3 h, a loss of mass was observed due to the occurrence of benzylation of low molecular weight polyoses, which were eliminated in the purification step. After that period of time a drastic weight increase was observed probably because crystalline regions had developed. The samples with low degrees of benzylation were insoluble, whereas the more benzylated counterparts showed limited solubility in THF. Partially soluble samples and a completely soluble one showed very different GPC elution profiles. This may be attributed to the efficiency of the pre treatment which, in the latter case, employed more concentrated alkali. Thermogravimetric analyses showed that all samples were degraded above 310 °C. Glass transition temperatures ranged between 42 °C and 65 °C, increasing as the extent of benzylation increased This revised version was published online in August 2006 with corrections to the Cover Date.     

Production, bleaching, and characterization of pulp from Stipa tenacissima

Alfa grass pulping was successfully performed in hydro-organic acid medium under mild conditions (107°C, atmospheric pressure, cooking time: 3 h). Use of an acetic acid/formic acid/water mixture as pulping liquor was perfectly suitable for selective isolation of pulp, lignin, and hemicelluloses. The unbleached pulp obtained in good yield was first delignified by peroxyacids in organic acid medium and then bleached with hydrogen peroxide in a basic medium to give pulp offering good physico-chemical and mechanical characteristics.