Effect of xylanases on peroxide bleachability of eucalypt (E. globulus) kraft pulp

Industrial eucalypt (E. globulus L.) kraft pulp was treated with two commercial xylanase preparations Ecopulp^® TX-200A and Pulpzyme^® HC (endo-1,4-β-xylanase activity; EC 3.2.1.8) and bleached by totally chlorine-free (TCF) three-stage hydrogen peroxide bleaching sequence, without oxygen pre-delignification. The effect of enzymatic stage on pulp properties and bleachability has been studied and compared with reference (control) pulps, processed without enzyme addition. The similar mode of enzymatic action was noted for both xylanase preparations. Final brightness of 86% ISO was achieved after complete bleaching. Direct bleaching effect caused pulp brightening (by 1.2–1.5% ISO) and delignification (by 7–10%) immediately after the enzymatic stage. The maximal bleach boosting was shown after the first peroxide stage and then diminished, despite the progressive increase in delignification over the control. The loss in efficiency of xylanase treatment by the end of peroxide bleaching was associated with specific behavior of xylan-derived chromophores, i.e., hexenuronic acids.     

Preliminary studies on TCF bleaching of Pinus pinaster acetosolv pulps.

Oxygen pre-treatment of Pinus pinaster acetosolv pulps has been studied as a first step towards TCF bleaching. Using a 2(3) factorial design, the influence of temperature (80-120 degrees C), time (1-2 h) and NaOH concentration (1.5-3%) on pulp yield in the oxygen stage, chemical composition and physical properties of the pulps obtained was studied. Pulps pre-bleached with oxygen in the conditions selected as optimal (80 degrees C, 1 h with 2.25% NaOH) have been bleached with TCF sequences which included stages with hydrogen peroxide or hydrogen peroxide-oxygen under pressure. Even if high degrees of delignification were reached, with a reduction in Kappa number up to 95% and without important loss of viscosity, the carbohydrates degradation, especially hemicelluloses in the acetic acid delignification, reduces the strength potential of the pulps.     

Residual lignin studies of laccase-delignified kraft pulps

The delignification of chemical pulps with laccase and -hydroxybenzotriazole was explored employing a pre- and post-O₂ delignified softwood draft pulp. The delignification properties of laccase were shown to be improved with -hydroxybenzotriazole was used as a mediator instead of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). Analysis of the structure of residual lignin before and after laccase/ -hydroxybenzotriazole treatment indicated that the biobleaching system oxidizes the phenolic component of lignin and that the residual lignin is demethylated and significantly enriched in carboxylic acid groups.     

Bleaching Miscanthus x giganteusAcetosolv pulps with hydrogen peroxide/acetic acid. Part 1: Behaviour in aqueous alkaline media

Miscanthus x giganteus bark samples subjected to fractionation by the Acetosolv process under optimal conditions were bleached using hydrogen peroxide and acetic acid in aqueous media under alkaline conditions. The influence of the main operational variables in the bleaching of Acetosolv pulps of M. x giganteus (i.e. hydrogen peroxide concentration, 3–7%; temperature, 55–75 °C; pH 9–11), obtained after treatments, have been assessed on pulp yield, kappa number, viscosity and brightness of bleached pulps. For this purpose, a rotatable and orthogonal second-order factorial design of experiments was used, in order to identify the optimum operating conditions. The obtained empirical mathematical models demonstrate that, in general, the bleaching was efficient, achieving pulps with kappa numbers below 10. The chemical composition and physicochemical properties of the bleached pulps fulfilled the requirements for forthcoming bleaching stages. Moreover, an alkaline extraction stage to eliminate saponifiable groups of Acetosolv pulps was studied, as well as the necessity of use chelating agents in the stage with hydrogen peroxide.     

Effect of Residual Lignin Type and Amount on Bleaching of Kraft Pulp by Trametes versicolor

The white rot fungus Trametes (Coriolus) versicolor can delignify and brighten unbleached hardwood kraft pulp within a few days, but softwood kraft pulps require longer treatment. To determine the contributions of higher residual lignin contents (kappa numbers) and structural differences in lignins to the recalcitrance of softwood kraft pulps to biobleaching, we tested softwood and hardwood pulps cooked to the same kappa numbers, 26 and 12. A low-lignin-content (overcooked) softwood pulp resisted delignification by T. versicolor, but a high-lignin-content (lightly cooked) hardwood pulp was delignified at the same rate as a normal softwood pulp. Thus, the longer time taken by T. versicolor to brighten softwood kraft pulp than hardwood pulp results from the higher residual lignin content of the softwood pulp; possible differences in the structures of the residual lignins are important only when the lignin becomes highly condensed. Under the conditions used in this study, when an improved fungal inoculum was used, six different softwood pulps were all substantially brightened by T. versicolor. Softwood pulps whose lignin contents were decreased by extended modified continuous cooking or oxygen delignification to kappa numbers as low as 15 were delignified by T. versicolor at the same rate as normal softwood pulp. More intensive O₂ delignification, like overcooking, decreased the susceptibility of the residual lignin in the pulps to degradation by T. versicolor.     

Relationship between lignin structure and delignification degree in Pinus pinaster kraft pulps

This study examines the structure of residual and dissolved lignins from Pinus pinaster pulps obtained at different degrees of delignification by laboratory conventional kraft pulping. The cooking H factor was varied from 85 to 8049. The residual and dissolved lignin samples were characterised by elemental analysis, residual carbohydrate content, permanganate oxidation and 13C NMR spectroscopy. The reflectance factor of the pulps was also determined in order to tentatively correlate the delignification degree and residual lignin structure with the pulp colour. The obtained results confirmed that the delignification degree increases the condensation of the lignin structure, which might have an influence upon the observed increased pulp colour. The lack of selectivity of kraft pulping process in the case of more delignified pulps was also shown.     

A universal kinetic model for characterisation of the effect of chip thickness on kraft pulping

A kinetic model was developed to characterise the heterogeneous nature of kraft delignification kinetics, taking into account the effect of chip thickness. The final form of the model applied to kraft delignification can be represented by a first-order rate equation with a rate constant inversely proportional to a power function of chip thickness. Published laboratory results from kraft pulping of various lignocellulosic materials were used to validate the model. The outcomes confirm the significant effect of chip thickness on the delignification rate. The resultant model was used to predict the effect of chip thickness on the kappa distribution of kraft pulps.     

Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization.

Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).     

Delignification of sugarcane bagasse using glycerol-water mixtures to produce pulps for saccharification

This paper describes the organosolv delignification of depithed bagasse using glycerol–water mixtures without a catalyst. The experiments were performed using two separate experimental designs. In the first experiment, two temperatures (150 and 190 °C), two time periods (60 and 240 min) and two glycerol contents (20% and 80%, v/v) were used. In the second experiment, which was a central composite design, the glycerol content was maintained at 80%, and a range of temperatures (141.7–198.3 °C) and time (23–277 min) was used. The best result, obtained with a glycerol content of 80%, a reaction time of 150 min and a temperature of 198.3 °C, produced pulps with 54.4% pulp yield, 7.75% residual lignin, 81.4% delignification and 13.7% polyose content. The results showed that high contents of glycerol tend to produce pulps with higher delignification and higher polyoses content in relation to the pulps obtained from low glycerol content reactions. In addition, the proposed method shows potential as a pretreatment for cellulose saccharification.     

Isolation and characterisation of cell wall polymers from the heavily lignified tissues of olive (Olea europaea) seed hull

Cell wall material (CWM) was prepared from olive seed hull, which is heavily lignified and very tough. The material was cryomilled and delignified with chlorite/acetic acid for 9 h to give the holocellulose. Polymers were solubilised from the holocellulose by sequential extraction with cyclohexane-trans-1,2-diamine-NNN'N'-tetra-acetate (CDTA, Na salt), DMSO, 0.5, 1 and 4 KOH and 4 KOH + borate to leave the -cellulose residue. The suspension of -cellulose on neutralisation released a small amount of pectic material virtually free of xylan to give '-cellulose. The polymers from the various extracts were fractionated by graded precipitation with ethanol prior to anion-exchange chromatography, and selected fractions were subjected to methylation analysis. During delignification, glucuronoxylans with relatively low degrees of polymerisation (DP) and xylan-pectic polysaccharide complexes linked to degraded lignin were solubilised. A proportion of the xylan-pectic polysaccharide complexes were solubilised by 0.5 KOH. The major hemicellulosic polysaccharides of the olive seed hulls are glucuronoxylans, which occur as highly branched short chains, with DP of 30–60; or slightly branched chains with DP of 90–110. Partial acid hydrolysis of the major acidic xylan, gel-filtration chromatography and methylation analysis allowed us to propose a tentative structure for the major glucuronoxylan in which one residue of GlcpA occurs in each 14 continuously linked Xylp residues in a regular structure.     

On hexenuronic acid (HexA) removal and mediator coupling to pulp fiber in the laccase/mediator treatment

Flax soda/AQ pulps were treated with different fungal laccase-mediator combinations followed by physical and chemical characterization of the pulps to obtain a thorough understanding of the laccase/mediator effects on hexenuronic acid (HexA) removal and the coupling of mediator onto pulps for fiber functionalization. Large differences were found and the presence of lauryl gallate (LG) during Trametes villosa laccase (TvL) treatment (TvL + LG) resulted in a much larger reduction of pulp-linked HexA than the combination of p-coumaric acid (PCA) and Pycnoporus cinnabarinus laccase (PcL). A major portion of LG became attached to the pulp as revealed by an increase in the kappa number and further confirmed by thioacidolysis and ¹H NMR analysis of solubilized pulp fractions. Additional experiments with other chemical pulps and isolated pulp xylan and lignin revealed that HexA seems to be the sole pulp component attacked by TvL + LG. As a substrate for TvL, the reaction preference order is PCA > HexA > LG.     

The influence of pH on the viscous behavior of starch-poly(ethylene-co-acrylic acid) complexes

Starch-poly (ethylene-co-acrylic acid) (EAA) complexes were prepared by jet-cooking mixtures of either cornstarch, waxy cornstarch or high amylose cornstarch with aqueous ammonia dispersions of EAA (4% EAA based on the weight of starch). Viscosities (η) were determined at temperatures ranging from 80°C to 22°C, and plots of log η versus 1/T (K⁻¹) were prepared. When cooked with EAA, cornstarch and waxy cornstarch showed major changes in viscous behavior between 50°C and 60°C. Above 50–60°C, viscosity increased markedly with a reduction in temperature; however, viscosity increased slowly below 50–60°C with an apparent activation energy for the process approximating that of water itself. The temperature dependence of the measured viscosity from 80°C to 60°C could be attributed to the large increase in size and complexity of the flowing particles as individual amylopectin molecules were bound together by complexed EAA. Apparently, complexing is essentially complete at 50°C. When high amylose cornstarch was cooked in the absence of EAA, retrogradation produced a sharp increase in log η at temperatures below about 50°C. However, if EAA is present, association between amylose molecules apparently takes place via complex formation rather than retrogradation, since log η increases sharply at about 70–80°C. Also, in contrast to cornstarch and waxy cornstarch, log η versus 1/T plots for high amylose cornstarch did not level off at low temperatures. In general, viscosities increased with the pH of the system, particularly when η was measured at high temperatures. This could result from improved complexing ability of EAA under high pH conditions, possibly due to reduced micelle size and maximum extension of polymer chains from micelle surfaces.     

Studies on isolation and characterization of starch from oat (Avena nuda) grains

Starch from AC Hill oat grains (Avena nuda) was isolated and some of the characteristics determined. The yield of starch was 23·4% on a whole grain basis. The shape of the granule was polyhedral to irregular, with granules 6–10 μm in diameter. Lipids were extracted by acid hydrolysis and by selective solvent extraction with chloroform-methanol 2:1 v/v (CM) at ambient temperature, followed by n-propanol-water 3:1 v/v (PW) at 90–100°C. The acid hydrolyzed extracts which represented the total starch lipids (TSL) was 1·13%. The free lipids in the CM extract (1% TSL) was 6·2%, whereas the free and bound lipids in the PW extracts was 93.0%. Neutral lipids formed the major lipid class in the CM and PW extracts. The monoacyl lipid content in both CM and PW extracts was 61·0%. The total amylose content was 19·4%, of which 13·9% was complexed by native lipids. X-ray diffraction was of the ‘A’ type. Oat starch differed from wheat starch in showing a higher swelling factor, decreased amylose leaching, coleaching of a branched starch component and amylose during the pasting process, higher peak viscosity and set-back, low gel rigidity, greater susceptibility towards acid hydrolysis, greater resistance to -amylase action and a higher freeze-thaw stability. Furthermore, in comparison to wheat starch, the amylose chains of oat starch appear to be more loosely arranged in the amorphous regions, whereas in crystalline regions, oat starch chains are more compactly packed. Lipid removal from oat and wheat starches decreased their swelling factor, peak viscosity, set-back, gelatinization temperatures, freeze-thaw stability and paste clarity (at pH > 4·0), and increased their thermal stability, amylose leaching, enthalpy of gelatinization, susceptibility towards -amylase and paste clarity (at pH < 4·0). The results also showed that the properties of AC Hill oat starch is not representative of oat starch in general.     

Bioethanol production from tension and opposite wood of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> using organosolv pretreatment and simultaneous saccharification and fermentation

During tree growth, hardwoods can initiate the formation of tension wood, which is a strongly stressed wood on the upper side of the stem and branches. In Eucalyptus globulus, tension wood presents wider and thicker cell walls with low lignin, similar glucan and high xylan content, as compared to opposite wood. In this work, tension and opposite wood of E. globulus trees were separated and evaluated for the production of bioethanol using ethanol/water delignification as pretreatment followed by simultaneous saccharification and fermentation (SSF). Low residual lignin and high glucan retention was obtained in organosolv pulps of tension wood as compared to pulps from opposite wood at the same H-factor of reaction. The faster delignification was associated with the low lignin content in tension wood, which was 15% lower than in opposite wood. Organosolv pulps obtained at low and high H-factor (3,900 and 12,500, respectively) were saccharified by cellulases resulting in glucan-to-glucose yields up to 69 and 77%, respectively. SSF of the pulps resulted in bioethanol yields up to 35 g/l that corresponded to 85–95% of the maximum theoretical yield on wood basis, considering 51% the yield of glucose to ethanol conversion in fermentation, which could be considered a very satisfactory result compared to previous studies on the conversion of organosolv pulps from hardwoods to bioethanol. Both tension and opposite wood of E. globulus were suitable raw materials for organosolv pretreatment and bioethanol production with high conversion yields.     

Effect of organosolv and soda pulping processes on the metals content of non-woody pulps

In this work the effect of different pulping processes (ethyleneglycol, diethyleneglycol, ethanolamine and soda) of tow abounded raw materials (empty fruit bunches - EFB and rice straw) on the ash, silicates and metals (Fe, Zn, Cu, Pb, Mn, Ni and Cd) content of the obtained pulps have been studied. Results showed that pulps obtained by diethyleneglycol pulping process presented lower metals content (756 microg/g and 501 microg/g for EFB and rice straw pulp, respectively) than soda pulps (984 microg/g and 889 microg/g). Ethanolamine pulps presented values of holocellulose (74% and 77% for EFB and rice straw pulp, respectively), alpha-cellulose (74% and 69%), kappa number (18.7 and 18.5) and viscosity (612 and 90 6ml/g) similar to those of soda pulp, and lower lignin contents (11% and 12%).     

NH(4)OH-KOH pulping mechanisms and kinetics of rice straw

The mechanisms and kinetics of NH(4)OH-KOH mixture pulping rice straw were studied. When aqueous ammonia was mixed with a small amount of caustic potash (ratio of 1:5), three distinct delignification phases were observed in the pulping process: a bulk delignification phase from the beginning of the cooking period to 100 degrees C, a supplementary delignification phase from 100 degrees C to 155 degrees C lasting a further 45 min, and a residual delignification phase until the end of the cooking period. There were two silica removal phases; the first phase was from the beginning of the cooking period to 100 degrees C and the second phase was from 100 degrees C to the end of the cooking period. The rate of delignification reaction was first order with respect to residual lignin and 0.3 order with respect to [OH(-)]. The silica removal was pseudo-first-order with respect to residual silica and 0.6 order with respect to [OH(-)]. The activation energies of the delignification and removal of silica reactions were 35.6 and 30.9 kJ/mol, respectively.     

TCF bleaching of wheat straw pulp using ozone and xylanase. Part B: kinetic studies

The reaction kinetics of ozone bleaching of wheat straw pulp has been studied for the first time. The results were compared with eucalyptus pulp in order to know that both raw materials have a similar behaviour. Ozone treatments were carried out in a special reactor at low consistency (0.5% o.d.p.). The main variables were consumption of ozone by the pulp and application of a xylanase treatment (X) prior to the oxygen stage (O). The responses measured were kappa number, viscosity and brightness, to give the kinetic expressions for delignification, cellulose degradation and elimination of chromophore groups, along with calculation of selectivity. Cellulose degradation and elimination of lignin and chromophore groups show first-order kinetics in all cases. The kinetics of the enzyme pre-treatment effect shows similar behaviour in both raw materials, although the constants of delignification and elimination of chromophore are higher in straw pulp.     

Protective effect of CR 1409 (cholecystokinin antagonist) on experimental pancreatitis in rats and mice

CR 1409, a glutaramic acid derivative with competitive cholecystokinin-antagonistic activity, was administered IP and evaluated in comparison with proglumide (the model CCK-receptor antagonist), gabexate (protease inhibitor) and PGE₂ (cytoprotective) on two different models of experimental pancreatitis. Acute pancreatitis was induced in mice by six IP injections of 50 μg/kg caerulein at hourly intervals. The drugs were administered 30 minutes before each caerulein administration. Blood samples and pancreata were collected 3 hours after the last caerulein injection. In the second experiment, pancreatitis was induced in rats by injecting 0.3 ml 6% sodium taurocholate interstitially into the pancreas. The drugs were administered twice, 30 minutes before and 3 hours after taurocholate. The animals were killed 6 hours after laparotomy and blood samples and pancreata were collected. CR 1409 exhibited on both pancreatitis models a protective effect in a dose range of 0.3–10 mg/kg. Proglumide exhibited a protective activity at higher doses (200–400 mg/kg). Gabexate and PGE₂ were effective only in pancreatitis induced by taurocholate in a dose range of 30–60 mg/kg and 60–130 μg/kg respectively. These results, showing a high protective effect of CR 1409 on different models of acute pancreatitis, suggest an important role of CCK in the pathogenesis of pancreatitis.     

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp. Received: 21 March 1996 / Received revision: 11 July 1996 / Accepted: 19 July 1996