Two mannanases from Sclerotium rolfsii in total chlorine free bleaching of softwood kraft pulp

The plant pathogenic basidiomycete Sclerotium rolfsii produces a wide range of extracellular hemicellulolytic enzymes. To study the effect of β-mannanases in total chlorine free bleaching of softwood pulp, two purified β -mannanases from S. rolfsii, with molecular masses of 42 and 61 kDa, a xylanase preparation from S. rolfsii and combinations of these were tested in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution). A brightness increase of 1.6 and 1.9% ISO was obtained with the 42 and 61 kDa mannanase and a combination of each of these enzymes with xylanases gave a brightness increase of 2.5 and 2.8% ISO, respectively. The effect of mannanases and xylanases was nearly additive. Both mannanases alone caused a lower decrease of the kappa number as compared to xylanases. The mannanases differed in their ability to release oligosaccharides from different mannans. The 61 kDa mannanase liberated larger fragments and caused rapid depolymerisation of mannans, which seems to promote the bleaching of pulp.     

Family-10 and Family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp. Received: 6 January 1997 / Received revision: 10 April 1997 / Accepted: 19 April 1997     

Screening of cellulase-free fungal xylanases and evaluation of their performance on sulfite dissolving pulp

Cellulase-free xylanase preparations of different fungal origin lowered the xylan content of acid sulfite dissolving pulp from 5% (w/v) to less than 4%. This was not, though, related to the amount of reducing sugars released. The pulp bleachability improved in samples where xylan had been mostly degraded, however, the increment in brightness declined (from 2.0–2.9 to 1.0–1.2 brightness points) when the subsequent number of chemical bleaching steps increased from 1 to 5.     

Xylanase and Mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase and mannanase, singly or in combination, either sequentially or simultaneously. Enzymes were obtained from Streptomyces galbus NR that had been cultivated in a medium, containing either xylan of sugar cane bagasse or galactomannan of palm-seeds, when they were used as sole carbon sources from local wastes in fermentation media. No cellulase activity was detected. Incubation period, temperature, initial pH values and nature of nutritive constituents were investigated. Optimum production of both enzymes was achieved after 5 days incubation on a rotary shaker (200 rpm) at 35 degrees C and initial pH 7.0. Partial purification of xylanase and mannanase in the cultures supernatant were achieved by salting out at 40-60 and 60-80% ammonium sulphate saturation with a purification of 9.63- and 8.71-fold and 68.80 and 62.79% recovery, respectively. The xylanase and mannanase from S. galbus NR have optimal activity at 50 and 40 degrees C, respectively. Both enzymes were stable at a temperature up to 50 degrees C. Xylanase and mannanase showed highest activity at pH 6.5 and were stable from 5.0 to 8.0 and from 5.5 to 7.5, respectively. The partial purified enzymes preparations of xylanase and mannanase enzymes showed high bleaching activity, which is an important consideration for industry. Xylanase was found to be more effective for paper-bleaching than mannanase. When xylanase and mannanase were dosed together (simultaneously), both enzymes were able to enhance the liberation of reducing sugars and improve pulp bleachability, possibly as a result of nearly additive interactions. The simultaneous addition of both enzymes was more effective in pulp treatment than their sequential addition.     

Cellulose decomposing ability of Trichoderma in relation to their saprophytic survival

Abstract

Forty one strains of Trichoderma were isolated from four provinces in the Philippines and tested for their cellulose adequacy indices (CAI). T. harzianum strain no. 94-016 with the highest CAI was further tested for its saprophytic colonization in pots relative to the amount of inoculum. The higher the amount of inoculum of T. harzianum strain no. 94-016, the higher percentage colonization of rice straw in the soil. Further tests were done to compare the decomposition of rice straw on the surface and buried in the soil and the effect of moisture on soil decomposition. Buried rice straw inoculated with T. harzianum strain no. 94-016 watered on a daily basis provided better decomposition.     

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 °C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 °C and pH 6.5 for A. terricola, and 65 °C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 °C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t ₅₀ of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4–3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and β-xylosidase were detected which might act synergistically with xylanase.     

Two stages of treatments for upgrading bleached softwood paper grade pulp to dissolving pulp for viscose production

To convert bleached softwood paper grade pulp into dissolving pulp for viscose application, two stages of treatments consisting of enzymatic treatment and alkaline peroxide treatment were investigated. It was found that high reactivity (about 80%) of pulp could be achieved by endoglucanases (EG)-rich industrial cellulase treatment, and the α-cellulose content as well as the viscosity of enzymatically treated pulp can be further adjusted by the alkaline peroxide treatment with certain dosages of NaOH and H₂O₂ to finally meet the quality requirements of dissolving pulp. The resulting pulp with 68.7% of reactivity, 92.1% of α-cellulose content, and 506.9 mL/g of pulp viscosity could be obtained after the two stages of treatments. The appropriate dosage of EG-rich cellulase was 300 IU/g bone dry pulp in the stage of enzymatic treatment, while the suitable dosages of NaOH and H₂O₂ were 9 wt% and 1 wt%, respectively, in the stage of alkaline peroxide treatment.     

Dechlorination and decolorization of chloro-organics in pulp bleach plant E-1 effluents by advanced oxidation processes

Studies were conducted on the composition of chloro-organics in kraft-pulp bleach plant E-1 effluents and their response toward advanced oxidation processes, such as UV-, O(2)/UV-, O(3)/UV- and O(3)-H(2)O(2)/UV-photolysis processes with irradiation of 254 nm photons. The studies were extended to ozonation and O(3)-H(2)O(2) oxidation systems in alkaline aqueous solution. The effects of process variables included initial pH, addition of oxidant to the UV-photolysis system on the decolorization and dechlorination of the chloro-organics the E-1 bleaching effluents were also studied. The decolorization and dechlorination rate constants are increased in the presence of molecular oxygen in the UV-photolysis systems, but are decreased on addition of hydrogen peroxide. The dechlorination rate constants are increased appreciably on oxidation with ozone alone and a combination of ozone and hydrogen peroxide as compared to those of the corresponding UV-photolysis systems under aerial atmosphere.     

Dechlorination of chlorophenols found in pulp bleach plant E-1 effluents by advanced oxidation processes

Studies were conducted on the response of 2,4,6-trichlorophenol (1), 2,3,4,5-tetrachloro-phenol (2) and 4,5-dichloroguaiacol (3) toward advanced oxidation processes, such as UV-, O2/UV-, H2O2/UV-, O3/UV- and O3-H2O2/UV-photolyses with irradiation of 254 nm photons. The compounds 1-3 are among the chlorophenols found in the Kraft-pulp bleach plant E-1 effluents. The studies were extended to treatment of these compounds with ozonation and O3-H2O2 oxidation systems in alkaline aqueous solution. Except for the O2/UV-photolysis of 1 and H2O2/UV-photolysis of 2, the dechlorination of 1-3 by O2/UV- and H2O2/UV-potolyses were less effective than the corresponding N2UV-potolysis of 1-3. Guaiacol-type chlorophenols were more readily able to undergo dechlorination than non-guaiacol type chlorophenols by N2/UV-, O2/UV- and H2O2/UV-potolyses. In addition, the efficiency for the dechlorination of 1-3 by N2/UV-, O2/UV- and H2O2/UV-potolyses appeared to be dependent upon the inductive and resonance effects of substituents as well as number and position of chlorine substituent in the aromatic ring of the compounds. The dechlorination of 2 by treatment with O3 alone is slightly more effective than the corresponding the O3/UV-photlysis, whereas the dechlorination of 2 by treatment with the combination of O3 and H2O2 was slightly less effective than the corresponding O3-H2O2/UV-photolysis. In contrast, the dechlorination of 3 on treatment with O3 alone was slightly less effective than the corresponding the O3/UV-photolysis, whereas the dechlorination of 3 on treatment with the combination of O3 and H2O2 was slightly more effective than the corresponding the O3-H2O2/UV-photolysis. In the dechlorination of 2 and 3, chemical species derived from ozone and hydrogen peroxide in alkaline solution were dominant reactions in the O3/UV- and O3-H2O2/UV-photolysis systems as in the O3 and O3-H2O2 oxidation systems. Possible dechlorination mechanisms involved were discussed on the basis of kinetic data.     

Production,partial characterization and use of fungal cellulase-free xylanases in pulp bleaching

Seven fungal strains were screened for their ability to produce cellulase-free xylanases that could be used in pretreatment of sulphite pulp prior to bleaching. The potential xylanase producers were subjected to shake flask fermentations using four different carbon sources: wheat bran, corn cobs, oat spelts xylan and bleach plant effluent. When grown on corn cobs, Aspergillus foetidus (ATCC 14916) produced significant levels of xylanase (547.4 U/ml), accompanied however by 6.6 U/ml of cellulase activity. Two other strains, Aspergillus oryzae (NRRL 1808) and Gliocladium viride (CBS 658.70), produced high yields of cellulase-free xylanase on oat spelts xylan. The crude enzymes of these two isolates were characterized with respect to pH and temperature optima and stability in order to standardize the optimum conditions for their use on pulp. Although the two xylanases differed in their abilities to remove reducing sugars from pulp, their biobleaching abilities, when assessed in hydrogen peroxide delignification of pulp, were very similar: both of them increased brightness by 1.4 points and removed 7% of hemicellulose from pulp.     

Modification of high-lignin softwood kraft pulp with laccase and amino acids

This study demonstrates the potential of laccase-facilitated grafting of amino acids to high-lignin content pulps to improve their physical properties in paper products. Research studies have recently reported that increases in anionic fiber charge can improve strength properties of paper. In an effort to increase carboxylic acid groups, we developed a unique two-stage laccase grafting protocol in which fibers were initially treated with laccase followed by grafting reactions with amino acids. The bulk acid group content was measured, and a variety of amino acids including glycine (Gly), phenylalanine (Phe), serine (Ser), arginine (Arg), histidine (His), alanine (Ala), and aspartic acid (Asp) were examined. The effects of optimizing laccase dose, and amino acid structures, on fiber modification chemistry were studied. Histidine provided the best yield of acid groups on pulp fiber, and was used for the preparation of handsheets for physical strength testing. Laccase-histidine treated pulp showed an increase in strength properties of the resulting paper.     

Investigation of various adsorbents for their ability to bind aflatoxin B1

The contamination of animal feed with mycotoxins represents a worldwide problem for the animal industry. The most applied method for protecting animals against aflatoxicosis is the utilization of clay minerals. In the course of a research project adsorption experiments were performed in buffer solutions in order to evaluate the ability to bind Aflatoxin B₁ (AfB₁) at various pH-values. In order to investigate the strength of binding, the chemisorption index was calculated. Isothermal analysis was used to determine the values for the maximum adsorption capacity. Adsorption experiments in simulated gastrointestinal fluid and real gastric juice were carried out. Furthermore binding capability of the materials regarding selected vitamins was examined. Special attention was paid to the formation of AfB_2a during experimental conditions. Based on the obtainedin vitro results, highly promising sorbent materials were ranked for furtherin vivo studies. Some adsorbing bentonites were also analysed mineralogically, but the results did not indicate which smectite property influences the adsorption process for AfB₁ Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006 Financial support: Christian Doppler Society     

Modification of paper properties by the pretreatment of wastepaper pulp with Graphiumputredinis, Trichodermaharzianum and fusant xylanases

Graphiumputredinis, Trichodermaharzianum and fusant were used in the present study to produce extracellular xylanases, an important industrial enzyme used in pulp and paper industry produced in a minimal medium supplemented with oat spelt xylan (1%, w/v) pH 7.0 at 27+/-2 degrees C. The enzyme was purified to homogeneity by DEAE-Cellulose and Superdex 75 FPLC column, respectively. The enzyme was found to be a monomer as determined by SDS gel electrophoresis. The optimum pH and temperature for purified G. putredinis, T. harzianum and fusant xylanases were 5.0-6.0 and 50-70 degrees C, respectively. Pretreatment of paper pulp with G. putredinis, T. harzianum and fusant xylanases decreased pulp kappa number. Xylanases particularly that of fusant at 5 IU/g pulp concentration and 1.5% pulp consistency at 60 degrees C for 18 h followed by EDED process yielded good quality paper from waste paper pulp. A significant increase in pulp brightness and improvement in various pulp properties, viz. burst capacity, thickness and bulkness of the treated pulp were observed in comparison to the conventional chemical bleaching. Easy purification and high stability of these enzymes makes it amicable for industrial applications.     

Photosynthesis of mistletoes in relation to their hosts at various sites in tropical Brazil

 Chlorophyll a fluorescence parameters showing the instantaneous performance and carbon-isotope ratios reflecting long-term behaviour of leaves were determined for a large number of mistletoe/host-pairs in the cerrado belt of Brazil. Study sites were a very exposed rupestrian field, a semi-exposed savanna and a highly shaded gallery forest. The major question asked was if photosynthetic capacity of mistletoe leaves differed from that of the leaves of their respective hosts. It is shown that except for the very exposed rupestrian field site, photosynthetic capacity appeared to be similar in mistletoes and host leaves. The superior behaviour of host leaves in the rupestrian field was due to particularly expressed sun-plant characteristics of the host. However, mistletoes always had higher average stomatal conductances, lower leaf temperatures at similar or even higher irradiance and higher intercellular CO₂-partial pressures than hosts. Photosynthetic performance of mistletoe leaves was independent of whether a given mistletoe species parasitized aluminium-accumulating or non-accumulating host species in the cerrados with their aluminium-rich soils. Received: 7 April 1997 / Accepted: 20 August 1997     

Interconnection of physico-chemical characteristics of organic solvents and their denaturing ability in relation to proteins]

Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening of mannanases in actinomycetes and their potential application in the biobleaching of pine kraft pulps

Fifty actinomycete strains were screened for the production of mannanase activity during growth in both liquid and solid media. Streptomyces scabies CECT 3340 and Streptomyces ipomoea CECT 3341 were selected for their ability to produce high levels of mannanase (294.3 U/l and 242.9 U/l, respectively) during growth in liquid culture. β-Mannosidase (15.3 U/l) and α-galactosidase (7.7 U/l) activities were also detected in culture filtrate from S. scabies CECT 3340. Highest levels of mannanase activity for S. scabies CECT 3340 were achieved in media containing locust bean gum and asparagine (4.8 U mg⁻¹ protein) whilst in S. ipomoea CECT 3341 greatest activity was detected in media containing locust bean gum and yeast extract (13.2 U mg⁻¹ protein). No carboxymethylcellulase activity was detected. In biobleaching experiments, enzyme treatment, carried out with mannanase activity produced by S. ipomoea CECT 3341, followed by alkaline extraction of pine kraft pulp resulted in the release of colour (A ₄₆₅, 0.69) and chromophoric material from the pulp (A ₂₃₇, 12.9; A ₂₅₄, 6.9 and A ₂₈₀, 6.7). The ability of this enzyme complex to improve the bleaching of pine kraft pulps was also shown by a pulp brightness increase (2.4 units ISO) and a reduction in kappa number (from 21.4 units to 20.1 units) with the absence of variations on the viscosity values. Received: 23 February 1999 / Received revision: 1 April 1999 / Accepted: 6 April 1999     

In vitro genotoxic evaluation of conventional bleached and biobleached softwood pulp mill effluents

The effluents of pulp and paper mills contain about 300 different chemical compounds; many of them are mutagens and clastogens. Genotoxic studies have shown that chlorination stage liquors are significantly more genotoxic, in the Ames Salmonella assay, than the other process of lignin extraction, and that lyophilized effluents are genotoxic in cultured mammalian cells. Since these effluents from conventional bleaching stages are genotoxic, Chilean industries are interested in changing this process to a less toxic one, such as biobleaching using enzymes. In this study, we tested the in vitro genotoxicity of two types of effluents: an effluent obtained from a conventional radiata pine kraft-bleaching process (effluent D) and one derived from a biobleaching process with hemicellulase (effluent B). Both effluents were tested without any concentration or purification steps in the Ames Salmonella assay (TA100) and in the micronuclei (MN) and sister chromatid exchange (SCE) tests in CHO cells. The results showed that neither effluent induced base pair substitution mutations in the Ames Salmonella assay, and neither increased the micronucleus frequency in CHO cells. But, both increased the SCE frequencies in CHO cells, showing that this assay is more sensitive than the other ones, and that the two effluents contained chemical compounds in amounts enough to induce in vitro genotoxicity measured by the SCE induction.     

Mesenchymal progenitor cells in adult human dental pulp and their ability to form bone when transplanted into immunocompromised mice

The technique of tissue engineering is developing for the restoration of lost tissues. This new technique requires cells that fabricate tissue. Mesenchymal stem cells in bone marrow have been used as the cell source for this technique; however, dental pulp cells have recently been shown to possess stem-cell-like properties. We earlier demonstrated that dental pulp cells proliferate and produce an extracellular matrix that subsequently becomes mineralized in vitro. We now report that such dental pulp cells (first to eighth passage) produced bone instead of dentin when those cells were implanted into subcutaneous sites in immunocompromised mice with HA/TCP powder as their carrier. This evidence shows that dental pulp cells are the common progenitors of odontoblasts and osteoblasts, or dental pulp cells are mesenchymal stem cells themselves. It is expected that dental pulp cells can be a useful candidate cell source for tissue engineering, and contain the potential of new therapeutic approaches for the restoration of damaged or diseased tissue.     

Changes in developing pea (Pisum sativum) seeds in relation to their ability to withstand desiccation

In two separate experiments in 1970 and 1971 the germination ability of seeds that had been rapidly desiccated following harvesting from glasshouse-grown pea plants improved with time after fertilization. In 1970 the germination of seeds directly after harvesting also improved with time. In both years the readiness with which electrolytes were leached from dried seeds decreased with seed age, measured as time after fertilization. The germination ability of desiccated seeds, and of undried seeds in 1970, improved after the cessation of seed moisture increase on the parent plant. This improvement coincided, in 1970, with a fall in the readiness with which electrolytes could be leached from seeds and with a decline in the rate of oxygen uptake of seeds directly after harvest. Vital staining of dried seeds from harvest samples indicated the viability of the samples as determined by germination tests. In both years, when the cotyledons first showed complete staining, the staining was very intense; in 1970 this occurred in a harvest sample with a greater than 50% germination level, but in 1971 it occurred in a sample that showed incomplete staining in the embryo axis and less than 5 % germination. In 1971 the comparative rates of moisture loss from seeds, measured with a sensor element diffusion porometer, were very low at 25 days after fertilization, increased up to a peak at 35 days and fell to a rate at the final harvest (44 days) which was nearly three times faster than the initial rate. Thus, during the period of dehydration, after 37 days, the seeds were able to lose moisture. When seeds were allowed to lose moisture slowly after harvest, before being desiccated rapidly, their eventual condition as measured by leaching improved. Also, the faster the initial water loss preceding rapid desiccation the more readily were the dried seeds leached. It is suggested that seeds can only withstand rapid desiccation after the cessation of moisture increase and after some slow dehydration, which is accompanied by a slowing down in physiological activity.