Optimization of hydrogen peroxide in totally chlorine free bleaching of cellulose pulp from olive tree residues

The influence of the operating conditions used in the bleaching of olive wood trimmings pulp (viz. hydrogen peroxide concentration and time) on the yield, kappa index and viscosity of the resulting pulp and on strength-related properties of paper sheets was studied to determine the optimal bleaching conditions of this pulp. Hydrogen peroxide bleached pulps at different sequences (oxygen, ozone, chlorine dioxide and alkaline extractions) were compared. Hydrogen peroxide bleaching proved to be suitable for this pulp. Considerable improvements in viscosity were obtained with respect to other bleaching sequences such as oxygen, ozone and chlorine dioxide. Hydrogen peroxide bleaching decreased the kappa index 51.3% less than ozone bleaching, 25.0% less than chlorine dioxide (D) and 6.3% less combined chlorine dioxide-alkaline extraction (DE). To obtain kappa indices 50.9% and 37.9% lower than the index achieved by hydrogen peroxide, oxygen (LaO(p)) and ozone (LaO(LaZ)R) sequences respectively were needed. Lower-medium levels of hydrogen peroxide concentrations (1-3%) and high reaction times (210 min) proved to be suitable for bleaching of pulp olive trimming residues. This approach could be used on this residue to produce adequately bleached pulp.     

Biobleaching of Eucalyptus globulus kraft pulps: comparison between pulps obtained from exploded and non-exploded chips

The aim of this work was to evaluate the response to biobleaching of steam exploded kraft pulps and to compare the results with the controls. For this end, a laccase-mediator treatment using commercial laccase (Novozyme 51003) and a natural mediator (acetosyringone) were assayed, followed by alkaline extraction and hydrogen peroxide stages.Our approach resulted in exploded biobleached pulps with lower kappa number and improved optical properties compared to controls, even after subjecting pulps to accelerated ageing. Additionally, use of hydrogen peroxide was reduced. The LMS (laccase-mediator system) had a smaller impact on the properties of the bleached pulps and on hydrogen peroxide consumption than the steam explosion process did.     

Resilience and acclimation to bleaching stressors in the scleractinian coral Porites cylindrica

‘Resilience’, the capacity of the coral symbiosis with dinoflagellate algal symbionts (‘zooxanthellae’) to recover after bleaching, is a little-studied but crucial aspect of coral responses to bleaching stressors. This study investigated the response of the zooxanthella population in the coral Porites cylindrica after bleaching either naturally on a shallow subtidal reef or experimentally in response to elevated temperature and darkness. Coral resilience was influenced by the nature and duration of the stressor. Corals strongly bleached by natural stressors were less resilient than those that had been partially bleached; and a similar recovery profile was obtained for corals experimentally bleached by exposure to elevated temperature, in which recovery was slower for corals thermally-stressed 96 h than for 72 h. The opposite trend was evident for corals exposed to darkness, indicating that the bleaching trigger had a strong impact on coral resilience. When P. cylindrica recently recovered from bleaching was subjected to a repetition of bleaching stressors, it did not display acclimation, i.e. experience-mediated acquisition of resistance to bleaching stressors. The zooxanthella populations in all corals tested throughout the experiments were typed by PCR-RFLP as clade C, indicating that coral responses were not accompanied by any substantial change in zooxanthella composition at the cladal level.     

Bleach boosting and direct brightening by multiple xylanase treatments during peroxide bleaching of kraft pulps

The effects of multiple xylanase treatments were assessed during the peroxide bleaching of three pulps: Douglas-fir (kraft); Western hemlock (oxygen delignified kraft); and trembling Aspen (kraft). The addition of a xylanase treatment stage, either before or after the peroxide bleaching stage(s), resulted in the enhanced brightening of all pulps. A higher brightness was achieved using two enzyme treatments, one before and one after the peroxide stage(s). Both bleach boosting and direct brightening seemed to contribute to the enhancement of the peroxide bleaching. Compared to xylanase prebleaching, xylanase posttreatment of peroxide bleached pulps solubilized less lignin and chromophores and made smaller amounts of these materials alkaline soluble. Nevertheless, the final brightness achieved by xylanase posttreatment was similar or superior to that achieved with xylanase prebleaching of the corresponding unbleached pulps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 312-318, 1997.     

Arundo donax L. reed: new perspectives for pulping and bleaching. 5. Ozone-based TCF bleaching of organosolv pulps

Three selected alkali-based organosolv pulps (alkali-sulfite-anthraquinone-methanol (ASAM), alkali-anthraquinone-methanol (organocell) and ethanol-soda) from agrofibre crop giant reed (Arundo donax L.) were bleached by an ozone-based TCF (totally chlorine- free) bleaching sequence AZE(R)QP (where A is an acidic pulp pre-treatment, Z is an ozone stage, (E(R)) is an alkaline extraction in the presence of reducing agent, Q is a pulp chelating, P is a hydrogen peroxide stage) without oxygen pre-bleaching, and compared with a conventional kraft pulp used as a reference. The different response on bleaching conditions within each bleaching stage was noted for all tested pulps. The pulp bleachability, in terms of brightness improvement or lignin removal per unit of applied chemicals, was found higher for the organocell pulp. The ASAM and ethanol-soda pulps showed the highest bleaching selectivity, expressed by viscosity loss per unit of lignin removed or brightness improved. The overall bleaching results of organosolv pulps were superior to kraft.     

Extracting value from Eucalyptus wood before kraft pulping: effects of hemicelluloses solubilization on pulp properties

Eucalyptus globulus wood samples were subjected to autohydrolysis for extracting hemicelluloses, and the resulting solids were assayed as substrates for kraft pulping and further Totally Chlorine Free (TCF) bleaching. The susceptibility of treated solids to kraft processing was assessed under selected experiments covering the optimum experimental range. In order to establish a basis for comparison, samples of untreated wood were also subjected to kraft delignification. The best kraft pulps obtained from autohydrolyzed solids were subjected to an optimized TCF bleaching sequence involving double alkaline oxygen and pressurized H₂O₂ processing, and characterized using standard methods. The suitability of the final product obtained by autohydrolysis-kraft delignification-TCF bleaching for specific purposes is discussed on the basis of the experimental results.     

Identification of Paenibacillus sp. 2S-6 and application of its xylanase on biobleaching

A Paenibacillus sp. strain 2S-6 was isolated from the black liquor of the first brownstock washing stage of kraft pulping process and identified by its 16S rDNA sequence. This bacterial strain utilized a variety of saccharides and polysaccharides as carbon source, but neither lignin nor lipids. Crude xylanase from Paenibacillus sp. 2S-6 was produced in a 5 L laboratory fermenter at 37 °C, pH 7. After 24 h, up to 10.5 IU xylanase per mg of protein in the crude extract of fermentation broth was obtained. After two-stage ultrafiltration, the optimal activity of partially purified xylanase reached 60.51 IU/mg at 50 °C, pH 6. A major band indicating molecular weight of 33 kDa was shown on SDS-PAGE for the partially purified xylanase. After 4 h at 60 °C, 48.99% and 31.25% residual xylanase activities were demonstrated at pH 7 and 9, respectively. Efficacy of its xylanase on the bleaching agent saving was demonstrated by using 5 IU xylanase per gram oven-dried pulp prior to bleaching, referred as biobleaching. Identical levels of brightness and higher levels of viscosity were obtained for the xylanase pretreated eucalypt kraft pulps followed by a 20% reduction of the bleaching agent dosage in the first step of a commercial C₇₀/D₃₀-E_o-D bleaching sequence.     

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.     

Treatment of APMP pulping effluent based on aerobic fermentation with Aspergillus niger and post-coagulation/flocculation

A novel two-stage biological/flocculation process was developed for treating the pulping effluent from the alkaline peroxide mechanical pulping (APMP) process. In the first biological stage, the aerobic fermentation by using Aspergillus niger can decrease the chemical oxygen demand (COD) by about 60% while producing about 7 g/l of solid biomass. In the second stage (post-coagulation/flocculation), the residual COD, turbidity and color, can be further decreased by using alum and polyacrylamide (PAM). The overall removal efficiencies of COD, color and turbidity from the APMP pulping effluent by the above two-stage biological-coagulation/flocculation process were 93%, 92% and 99%, respectively, under the conditions studied.     

Chemotaxonomy of Hypericum genus from Portugal: Geographical distribution and essential oils composition of Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum

The geographical distribution and analysis of the essential oils of species from three sections of Hypericum L. (Guttiferae/Clusiaceae/Hypericaceae) from Portugal are presented. Hypericum perfoliatum (section Drosocarpium) grows wild in the centre and south of Portugal; Hypericum humifusum and Hypericum linarifolium are both from section Oligostema, the former occurring throughout the country, while the second is distributed mainly in the north and centre; Hypericum pulchrum (section Taeniocarpium) is confined to the littoral north of Portugal. The essential oils were obtained by distillation–extraction, hydrodistillation and distillation in a modified Marcusson apparatus from the dried aerial parts of the different populations and were analysed by GC and GC–MS. Monoterpene hydrocarbons constituted the main fraction in all oils (43–69%, 53–85%, 28–45% and 48–65% for H. perfoliatum, H. humifusum, H. linarifolium and H. pulchrum, respectively). Sesquiterpene hydrocarbons (2–13%, 6–18%, 21–27% and 16–18%, respectively) and a third fraction of non-terpenic compounds (20–29%, 3–16%, 2–14% and 5–11%, respectively) from the four species attained relatively high amounts in all oils. Within each species, no major differences were detected in the essential oil composition, despite the fact that different locations, phenological phases and extraction methodologies were used. Notwithstanding the dominance of α-pinene in all four species' oils, cluster and principal components analysis on the identified components showed that the range of α-pinene, β-pinene and n-nonane supported a separation of the four species. The essential oil composition of the four species showed some qualitative resemblances, which correlate well with the taxonomical classification based on morphological characters.     

High-light induced singlet oxygen formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (¹O₂*) in the intact and the Rieske-depleted cytochrome b₆f complexes (Cyt b₆f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b₆f can be bleached by light-induced ¹O₂*, and that the ¹O₂* production can be promoted by D₂O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, ¹O₂* was also detected in the Rieske-depleted Cyt b₆f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of ¹O₂* formation, a conclusion which draws further support from the generation of ¹O₂* with selective excitation of Chl a using monocolor red light.     

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.     

Isolation and characterization of Arthrobacter sp. HY2 capable of degrading a high concentration of p-nitrophenol

A soil bacterium strain, capable of using p-nitrophenol (PNP) as its sole source of carbon and energy, was isolated by enrichment on minimal salt medium (MSM). On the basis of a phylogenetic analysis of 16S rRNA gene sequences the bacterium is a species of Arthrobacter, closely related to Arthrobacter ureafaciens DSM 20126. This strain has an unusually high substrate tolerance for PNP degradation in MSM. Greatest degradation of PNP was observed at 30 °C and under slightly alkaline pH (pH 7–9) conditions. Effective degradation rates slowed as the concentration of PNP was increased. Addition of glucose from 0.1% to 0.5% generally enhanced the degradation of PNP at high concentration (400 mg/l) although acidification as a result of glucose metabolism had a negative effect on PNP depletion. Biodegradation of PNP at high concentration was greatly accelerated by β-cyclodextrin at a concentration of 0.5%, indicating that β-cyclodextrin could be a promising addictive for effective PNP bioremediation.     

Use of spent mushroom substrates from Agaricus subrufescens (syn. A. blazei, A. brasiliensis) and Lentinula edodes productions in the enrichment of a soil-based potting media for lettuce (Lactuca sativa) cultivation: Growth promotion and soil bioremediation

This study aimed to assess physicochemical and microbiological properties of fresh spent mushroom substrates (SMSs) – without post-crop heat treatment – from Agaricus subrufescens and Lentinula edodes production to optimize the use of these residues in the soil enrichment for lettuce growth promotion and soil remediation. Organic matter and C content of both SMSs were high. Fresh A. subrufescens SMS was a good source of N, P and K. On the other hand, L. edodes SMS presented a lower concentration of these nutrients and a high level of immaturity. Both SMSs presented high electric conductivity values (2.5–3.4 mS/cm). Microbiological analysis, based upon enumeration of culturable bacteria (thermophilic and mesophilic) and fungi, and also evolution of CO₂, showed that SMSs played higher microbial diversity than soil control. Laccase activity from A. subrufescens SMS tended to remain constant during a 2-month period, while L. edodes SMS presented low laccase activity throughout the same period. Agaricus subrufescens and L. edodes were able to grow on a PDA (Potato Dextrose Agar) media supplemented with different concentrations of atrazine (1–50 μg/ml), degraded the herbicide, attaining rates of 35% and 26%, respectively. On experiments of lettuce growth promotion using a soil-based potting media with different SMS rates, 5% and 10% (dw) rates of A. subrufescens SMS resulted in higher lettuce aerial dry weights than the rates of 25% and 40%, the chemical fertilization (NPK) and the control (soil). At 10% supplementation, lettuce aerial dry weight increased 2.2 and 1.3 times compared to the control and the NPK treatment, respectively. Protein content increased along with SMS rates. Fresh A. subrufescens SMS was an excellent supplement for lettuce growth promotion and showed potential for remediation of biocides possibly due to improved microbial diversity and enzymatic activity. Fresh L. edodes SMS was not a good fertilizer, at least under the conditions tested. However, microbiological analysis showed that promising results may be achieved when using fresh L. edodes SMS for soil remediation.     

Regeneration and molecular characterization of an intergeneric hybrid between Graphium putredinis and Trichoderma harzianum by protoplasmic fusion

The fungal strains Graphium putredinis and Trichoderma harzianum were selected as parents for fusant development. Protoplasts were isolated using the combination of lysing enzymes Novozym 234 and cellulase with 0.6 M KCl as osmotic stabilizer. The optimum conditions for release of viable protoplasts from the fungal mycelium viz. age of the mycelium, lytic enzymes, osmotic stabilizers, pH, incubation period and regeneration medium were determined. Intergeneric protoplast fusion was carried out using 50% polyethylene glycol with calcium chloride (CaCl₂) and glycine buffer and the conditions for effective protoplast fusion, viz. fusogen, osmotic stabilizer, pH, incubation period and regeneration medium were optimized. At optimum conditions, the regeneration frequency of the fused protoplasts on potato dextrose agar (PDA) medium and fusion frequency were calculated. The regeneration frequency on non-selective (PDA) and selective media (PDA amended with starch) was determined for the parental and fusant strains in which, fusant showed a higher rate of regeneration. Fusant formation was confirmed by morphological markers (colony morphology and spore size and shape) and genetical markers like, mycelial protein pattern, restriction digestion pattern and random amplified polymorphic DNA (RAPD) analysis. The efficiency of these parental strains and their intergeneric fusant in the production of hydrolytic enzymes – amylases (treatment plant for sago factory effluent), cellulases (bioethanol), xylanases (bleaching agents for waste paper pulp) and proteases (additives in commercial detergents) – have probable applications in various industrial processes.     

The morphological distinction between the postcranial skeleton of Cervalces/Alces and Megaloceros giganteus and comparison between the two Alceini genera from the Upper Pliocene-Holocene of Western Europe

Alces alces, Cervalces and Megaloceros giganteus are deer of approximately the same size whose limb bones could be exchanged if fragmentary so morphological features are needed, enabling identification of postcranials remains. In this work the limb bone features described by Pfeiffer (1999a) as distinguishing Cervalces latifrons from M. giganteus are re-analyzed and further developed, considering also the vertebral column, the articular girdles and the carpals and tarsal elements. This analysis is extended to the other Cervalces species (C. gallicus, C. carnutorum and C. scotti) and to the present-day moose Alces alces. It results in a substantial uniformity in the postcranial skeleton of the species of the genus Cervalces, confirming what has already been stated by Sher (1987), that the Alceini, in the Upper Pliocene, constituted an already well-defined morphological type that did not undergo further structural postcranial remodeling.     

Effects of NH4–N concentrations and gradient redox level on growth and allied biochemical parameters of Elodea nuttallii (Planch.)

Aquatic plants frequently encounter multiple stresses under natural conditions. Nuttall's water weed, Elodea nuttallii (Planch.) is a submerged aquatic macrophyte which has flexible ability to use different nutrient sources from various environments. However, recently the growth of E. nuttallii has been declining in waters of Japan and in the Chesapeake Bay, a large estuary in the United States. In the present experiment, we studied growth and survival capabilities of the plant under a gradient of redox conditions; from highly oxic (+400 to +440 mV) to extremely reduced (−180 to −120 mV) conditions. Reduced environment was prepared by adding glucose to growth medium and nitrogen gas bubbling, while the oxic environment was brought about by atmospheric air bubbling. In comparison to the oxic environment, growth rate and carbon–nitrogen content of the plants were significantly affected negatively at hypoxic and anoxic conditions. In hypoxic and anoxic environments, indole acetic acid (IAA), tissue nitrogen and chlorophyll levels were down-regulated, whereas hydrogen peroxide (H₂O₂), indole acetic acid oxidase (IAAO) and peroxidase (POD) levels were up-regulated. It was also found that high NH₄–N concentrations (10–40 ppm) affect the growth rate and biochemical parameters of the plant; however, in hypoxic and anoxic treatments the effects were more severe. We conclude that E. nuttallii is poorly tolerant to hypoxia/anoxia. Moreover, oxygen stress combined with high ammonium concentration act as important factors influencing distribution and abundance of this species.     

New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities

Bleaching of Oculina patagonica has been extensively studied in the Eastern Mediterranean Sea, although no studies have been carried out in the Western basin. In 1996 Vibrio mediterranei was reported as the causative agent of bleaching in O. patagonica but it has not been related to bleached or healthy corals since 2003, suggesting that it was no longer involved in bleaching of O. patagonica. In an attempt to clarify the relationship between Vibrio spp., seawater temperature and coral diseases, as well as to investigate the putative differences between Eastern and Western Mediterranean basins, we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. Two sampling points located in the Spanish Mediterranean coast were chosen for this study: Alicante Harbour and the Marine Reserve of Tabarca. A complex and dynamic assemblage of Vibrio spp. was present in O. patagonica along the whole year and under different environmental conditions and coral health status. While some Vibrio spp. were detected all year around in corals, the known pathogens V. mediteranei and V. coralliilyticus were only present in diseased specimens. The pathogenic potential of these bacteria was studied by experimental infection under laboratory conditions. Both vibrios caused diseased signs from 24 °C, being higher and faster at 28 °C. Unexpectedly, the co-inoculation of these two Vibrio species seemed to have a synergistic pathogenic effect over O. patagonica, as disease signs were readily observed at temperatures at which bleaching is not normally observed.     

Effectiveness and immunomodulation of chemotherapeutants against scuticociliate Philasterides dicentrarchi in olive flounder

Philasterides dicentrarchi is a histophagous scuticociliate causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis due to P. dicentrarchi infections was increased from May to July (40 ± 3.1% to 79.4 ± 1.7%) and it decreased from August to November (63 ± 2.3% to 30 ± 2.6%) in olive flounder farms at Jeju Island, South Korea during 2000-2006. The prevalence of mixed infection along with Vibrio spp. bacterial infection was 49 ± 7.2% than that of other mixed infection. At present no effective control measure for P. dicentrarchi infection has been described and large production losses continue. In the present study, formalin, hydrogen peroxide and Jenoclean chemotheraputants were used for bath treatment. Among Jenoclean at a low concentration of 50 ppm proved effective. The results were confirmed with in vitro motility assessments and morphological changes scoring system in P. dicentrarchi. On the other hand, similar trend was noted following hydrogen peroxide treatment at this concentration, but formalin was only moderately effective. Either hydrogen peroxide or Jenoclean are the promising compounds effective at low concentrations with short application time for P. dicentrarchi. Therefore, these substances were evaluated on day 10, 20 and 30 for their ability to enhance innate immune response and disease resistance against P. dicentrarchi in olive flounder after chemotheraputants bath treatment with 100 ppm for 30 min per day. All the tested immune parameters were enhanced by treatment with Jenoclean, but not formalin and hydrogen peroxide. These findings suggest that Jenoclean bath treatment can be used for ensuring the heath of cultured marine fish against internal parasites such as P. dicentrarchi.