Biochemical properties of a beta-mannanase and a beta-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan, Its activity was optimal at pH 4.5. The K(m) value with galactoglucomannan as substrate was 0.50 mg ml (1). One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 degrees C and pH 8.0. Its K(m) value with birchwood xylan as substrate was 1.65 mg ml (1). Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn(+2), Fe(+3), and Cu(+2) were strong inhibitors for the mannanase. (C) 2008 Elsevier Ltd. All rights reserved.

Technological advances and mechanistic basis for fungal biopulping

Biopulping fundamentals, technology and mechanisms are reviewed in this article. Mill evaluation of Eucalyptus grandis wood chips biotreated by Ceriporiopsis subvermispora on a 50-tonne pilot-plant demonstrated that equivalent energy savings can be obtained in lab- and mill-scale biopulping. Some drawbacks concerning limited improvements in pulp strength and contamination of the chip pile with opportunist fungi have been observed. The use of pre-cultured wood chips as inoculum seed for the biotreatment process minimized contamination problems related to the use of blended mycelium and corn-steep liquor in the inoculation step. Alkaline wash restored part of the brightness in biopulps and marketable brightness values were obtained by one-stage bleaching with 5% H2O2 when bio-TMP pulps were under evaluation. Considering the current scenario, the understanding of biopulping mechanisms has gained renewed attention because more resistant and competitive fungal species could be selected with basis on a function-directed screening project. A series of studies aimed to elucidate structural changes in lignin during wood biodegradation by C. subvermispora had indicated that lignin depolymerization occurs during initial stages of wood biotreatment. Aromatic hydroxyls did not increase with the split of aryl-ether linkages, suggesting that the ether-cleavage-products remain as quitione-type structures. On the other hand, cellulose is more resistant to the attack by C subvermispora. MnP-initiated lipid peroxidation reactions have been proposed to explain degradation of non-phenolic lignin substructures by C subvermispora, while the lack of cellobiohydrolases and the occurrence of systems able to suppress Fenton`s reaction in the cultures have explained non-efficient cellulose degradation by this biopulping fungus. (C) 2007 Elsevier Inc. All rights reserved.

Evaluation of Eucalyptus grandis Hill ex Maiden biopulping with Ceriporiopsis subvermispora under non-aseptic conditions

In biopulping, efficient wood colonization by a selected white-rot fungus depends on previous wood chip decontamination to avoid the growth of primary molds. Although simple to perform in the laboratory, in large-scale biopulping trials, complete wood decontamination is difficult to achieve. Furthermore, the use of fungal growth promoters such as corn steep liquor enhances the risk of culture contamination. This paper evaluates the ability of the biopulping fungus Ceriporiopsis subvermispora to compete with indigenous fungi in cultures of fresh or poorly decontaminated Eucalyptus grandis wood chips. While cultures containing autoclaved wood chips were completely free of contaminants, primary molds grew rapidly when non-autoclaved wood chips were used, resulting in heavily contaminated cultures, regardless of the C. subvermispora inoculum/wood ratio evaluated (5, 50 and 3000 mg mycelium kg(-1) wood). Studies on benomyl-amended medium suggested that the fungi involved competed by consumption of the easily available nutrient sources, with C. subvermispora less successful than the contaminant fungi. The use of acid-washed wood chips decreased the level of such contaminant fungi, but production of manganese peroxidase and xylanases was also decreased under these conditions. Nevertheless, chemithermomechanical pulping of acid-washed samples biotreated under non-aseptic conditions gave similar fibrillation improvements compared to samples subjected to the standard biodegradation process using autoclaved wood chips.

Laboratory and mill scale evaluation of biopulping of Eucalyptus grandis Hill ex Maiden with Phanerochaete chrysosporium RP-78 under non-aseptic conditions

Biopulping of Eucalyptus grandis wood chips with Phanerochaete chrysosporium RP-78 was evaluated under non-aseptic conditions in laboratory and mill wood-yard. The ability of P. chrysosporium to compete with indigenous fungi present in fresh wood chips was notorious under controlled laboratory experiments. A subsequent step involved an industrial test performed with 10-ton of fresh wood chips inoculated and maintained at 37 +/- 38 degrees C for 39 days in a biopulping pilot plant. Biotreated wood chips were pulped in a chemithermomechanical pulping mill. Net energy consumption during refining was 745 kWh ton(-1) and 610 kWh ton(-1) of processed pulp for control and biotreated wood chips, respectively. Accordingly, 18.5% net energy saving could be achieved. Biopulps contained lower shive content and had improved strength properties compared to control pulps. Tensile index improved from 25 +/- 1 N m g(-1) to 33.6 +/- 0.5 N m g(-1) and delamination strength from 217 +/- 19 kPa to 295 +/- 30 kPa.

Biopulping of Paddy Straw by Pleurotus eous

Biopulping being less energy intensive, inexpensive and causing lesser pollution, can be a viable alternative to chemical and mechanical pulping in paper and pulp industry. In view of shrinking forest reserves, agricultural residues are considered as an alternative raw material for making paper and board. By suitable treatment agriwaste can be converted into substrate for mushroom cultivation. Mushrooms of Pleurotus sp. can preferentially remove lignin from agriwaste with limited degradation to cellulose. The present study examines utilization of Pleurotus eous for biopulping of paddy straw by solid substrate fermentation. SMS, the mushroom growing medium that results from cultivation process, is a good source of fibre and can be pulped easily. Ligninases present in SMS were able to reduce lignin content to nearly half the initial amount by 21st day of cultivation. Highest cellulose content (% dry weight) was observed on 21st day, while cellulase production commenced from 28th day of cultivation. SEM images revealed that SMS fibres are still associated with non-cellulosic materials when compared to chemically (20% w/v NaOH) extracted fibres.

Relevance of extractives and wood transformation products on the biodegradation of Pinus taeda by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a promising white-rot fungus for biopulping. However, the underlying biochemistry involved in lignin removal and insignificant cellulose degradation by this species is not completely understood. This paper addresses this topic focusing on the involvement of ethanol-soluble extractives and wood transformation products in the biodegradation process. Cultures containing ethanol-extracted or in natura wood chips presented similar levels of extracellular enzymes and degradation of wood components. Fe3+-reducing compounds present in undecayed Pinus taeda were rapidly diminished by fungal degradation. Lignin-degradation products released during biodegradation restored part of the Fe3+-reducing activity. However, Fe3+ reduction was ineffective in presence of 0.5 mM oxalate at pH 4.5. Fungal consumption of Fe3+-reducing compounds and secretion of oxalic acid minimized the significance of Fenton`s reaction in the initial stages of wood biotreatment. This would explain limited polysaccharide degradation by the fungus that also lacks a complete set of hydrolytic enzymes. Scientific relevance of the paper: Ceriporiopsis subvermispora is a white-rot fungus suitable for biopulping processes because it degrades lignin selectively and causes significant structural changes on the wood components during the earlier decay stages. However, the intricate mechanism to explain lignin transformation and insignificant cellulose degradation by this species remains poorly understood. Some recent evidences pointed out for lipid peroxidation reactions as all initiating process explaining lignin degradation. On the other hand, alkylitaconic acids produced by the fungus via transformations of fatty acids occurring in wood showed to prevent polysaccharide degradation in Fenton reactions. In this context, one may conclude that the involvement of native wood substances or their transformation products in the overall wood biodegradation process induced by C subvermispora is still a matter of discussion. While free and esterified fatty acids present in wood extractives may be involved in the biosynthesis of alkylitaconic acids and in lipid peroxidation reactions, some extractives and lignin degradation products can reduce Fe3+, providing Fe2+ species needed to form OH radical via Fenton`s reaction. The present study focuses on this topic by evaluating the relevance of ethanol-soluble extractives and wood transformation products on the biodegradation of P. taeda by C subvermispora. For this, solid-state cultures containing ethanol-extracted and in natura wood chips were evaluated in details for up to 4 weeks. (C) 2007 Elsevier Ltd. All rights reserved.

Alkaline-sulfite chemithermomechanical pulping of Eucalyptus grandis biotreated by Ceriporiopsis subvermispora under varied culture conditions

The effect of different culture conditions have been evaluated concerning the extracellular enzyme activities of the white-rot fungus Ceriporiopsis subvermispora growing on Eucalyptus grandis wood. The consequence of the varied fungal pretreatment on a subsequent chemithermomechanical pulping (CTMP) was addressed. In all cultures, manganese peroxidase (MnP) and xylanase were the predominant extracellular enzymes. The biopulping efficiency was evaluated based on the amount of fiber bundles obtained after the first fiberizing step and the fibrillation levels of refined pulps. It was found that the MnP levels in the cultures correlated positively with the biopulping benefits. On the other hand, xylanase and total oxalate levels did not vary significantly. Accordingly, it was not possible to determine whether MnP accomplishes the effect alone or depends on synergic action of other extracellular agents. Pulp strength and fiber size distribution were also evaluated. The average fiber length of CTMP pulps prepared from untreated wood chips was 623 mu m. Analogous values were observed for most of the biopulps; however, significant amounts of shorter fibers were found in the biopulp prepared from wood chips biotreated in cultures supplemented with glucose plus corn-steep liquor. Despite evidence of reduced average fiber length, biopulps prepared from these wood chips presented the highest improvement in tensile indexes (+28% at 23 degrees Schopper-Riegler).

Linoleic acid peroxidation initiated by Fe(3+)-reducing compounds recovered from Eucalyptus grandis biotreated with Ceriporiopsis subvermispora

This work work evaluates linoleic acid peroxidation reactions initiated by Fe(3+)-reducing compounds recovered from Eucalyptus grandis, biotreated with the biopulping fungus Ceriporiopsis subvermispora. The aqueous extracts from biotreated wood had the ability to reduce Fe(3+) ions from freshly prepared solutions. The compounds responsible for the Fe(3+)-reducing activity corresponded to UV-absorbing substances with apparent molar masses from 3 kDa to 5 kDa. Linoleic acid peroxidation reactions conducted in the presence of Fe(3+) ions and the Fe(3+)-reducing compounds showed that the rate of O(2) consumption during peroxidation was proportional to the Fe(3+)-reducing activity present in each extract obtained from biotreated wood. This peroxidation reaction was coupled with in-vitro treatment of ball-milled E. grandis wood. Ultraviolet data showed that the reaction system released lignin fragments from the milled wood. Size exclusion chromatography data indicated that the solubilized material contained a minor fraction representing high-molar-mass molecules excluded by the column and a main low-molar-mass peak. Overall evaluation of the data suggested that the Fe(3+)-reducing compounds formed during wood biodegradation by C subvermispora can mediate lignin degradation through linoleic acid peroxidation. (C) 2010 Elsevier Ltd. All rights reserved.

Behavior of Ceriporiopsis subvermispora during Pinus taeda biotreatment in soybean-oil-amended cultures

Pinus taeda wood chips were treated with the biopulping fungus Ceriporiopsis subvermispora in soybean-oil-amended cultures The secretion of oxalic acid and the accumulation of thiobarbituric acid reactive substances were significantly increased in soybean-oil-amended cultures By contrast the secretion of hydrolytic and oxidative enzymes was not altered in the cultures Biotreated wood samples were characterized for weight and component losses as well as by in-situ thioacidolysis Residual lignins were also extracted from biotreated wood using a mild-non-razing extraction procedure The lignins were characterized by (31)P nuclear magnetic resonance ((31)P-NMR) spectroscopy Soybean oil amendment in the cultures was found to affect lignin degradation routes however it inhibited depolymerization reactions detectable in the residual lignin that was retained in the biotreated wood As a consequence chemithermomechanical pulping of the biotreated samples was not improved by soybean oil amendment in the cultures Crown Copyright (C) 2010 Published by Elsevier Ltd All rights reserved

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment. (C) 2009 Elsevier Inc. All rights reserved.

High-yield kraft pulping of Eucalyptus grandis Hill ex Maiden biotreated by Ceriporiopsis subvermispora under two different culture conditions

In the present study, it was evaluated how two different culture conditions for the biotreatment of Eucalyptus grandis by Ceriporiopsis subvermispora affect a subsequent high-yield kraft pulping process. Under the varied culture conditions investigated, different extracellular enzyme activities were observed. Manganese-peroxidase (MnP) secretion was 3.7 times higher in cultures supplemented with glucose plus corn-steep liquor (glucose/CSL) as compared to non-supplemented (NS) cultures. The biotreated samples underwent diverse levels of wood component degradation as losses of weight and lignin were increased in glucose/CSL cultures. Mass balances for lignin removal during kraft pulping showed that delignification was facilitated when both biotreated wood samples were cooked. Delignification efficiency did not correlate positively with MnP levels in the cultures. On the other hand, biopulps from NS and glucose/CSL cultures saved 27% and 38% beating time to achieve 288 Schopper-Riegler freeness during refining, respectively. Biopulps disposed of decreased tensile and tear resistances, thus easier refining of the biokraft pulps seems to be a consequence of less resistant fiber walls. Improved beatability of biopulps was tentatively related to short fibers and fines formation during refining. We suggest that to some extent polysaccharide depolymerization occurred during the biotreatment, which also resulted in diminished pulp yields in the case of glucose/CSL cultures.

Mecanismos envolvidos na biodegradação de materiais lignocelulósicos e aplicações tecnológicas correlatas

The biodegradation of lignocellulosic materials is an important natural process because it is responsible for the carbon recycling. When induced under controlled conditions, this process can be used for technological applications such as biopulping, biobleaching of cellulosic pulps, pre-treatment for subsequent saccharification and cellulosic-ethanol production, and increase of the digestibility in agroindustrial residues used for animal feed. In the present work, the enzymatic and non-enzymatic mechanisms involved in the biodegradation of lignocellulosic materials by fungi were reviewed. Furthermore, the technological applications of these extracellular metabolites are presented and discussed.

Uso de aditivos na biodegradação de madeira pelo fungo Ceriporiopsis subvermispora: efeito na peroxidação de lipídios dependente de manganês-peroxidase

Ceriporiopsis subvermispora is a selective fungus in the wood delignification and the most promising in biopulping. Through the lipid peroxidation initiated by manganese peroxidase (MnP), free radicals can be generated, which can act in the degradation of lignin nonphenolic structures. This work evaluated the prooxidant activity (based in lipid peroxidation) of enzymatic extracts from wood biodegradation by this fungus in cultures containing exogenous calcium, oxalic acid or soybean oil. It was observed that MnP significant activity is required to promote lipid peroxidation and wood delignification. Positive correlation between prooxidant activity x MnP was observed up to 300 IU kg-1 of wood.

Desenvolvimentos da biopolpação no Brasil

Biopulping is a technology which application can be advantageous to mechanical or chemical pulping. It presents benefits such as the creation of stronger pulp, as well as energy or chemicals savings. This paper gives an overview of the recent efforts to develop biopulping processes in Brazil as well as provides critical information on biopulping development worldwide. Eucalyptus grandis wood chips have been biotreated by Ceriporiopsis subvermispora in a 50-ton biopulping pilot-plant and used to produce TMP and CTMP pulps on a mill scale, Up to 18% and 27% energy savings have been observed for producing 450-470 CSFreeness TMP and CTMP pulps. Despite darker bio-TMP pulps are produced, one-stage bleaching with 5% H2O2 was sufficient to improve brightness values to 70% and 72% for bio-TMP and control pulps, respectively. Understanding biopulping mechanisms is also relevant because more resistant and competitive fungal species could be selected with basis on a function-directed screen-ing project. As far as the chemical changes induced by the fungus in wood are concerned, recent efforts have pointed out for two different types of wood transformations. One of them involves intense lignin depolymerization in short biotreatment periods, while the other indicates that esterification reactions of oxalate secreted by the Jungas on the polysaccharides chains increase the water saturation point of the fibers. Both transformations are expected to affect the fiber-fiber bonding and, consequently, the physical resistance of wood.

USE OF ADDITIVES IN THE WOOD BIODEGRADATION BY THE FUNGUS Ceriporiopsis subvermispora: EFFECT IN THE MANGANESE PEROXIDASE-DEPENDENT LIPID PEROXIDATION

USE OF ADDITIVES IN THE WOOD BIODEGRADATION BY THE FUNGUS Ceriporiopsis subvermispora: EFFECT IN THE MANGANESE PEROXIDASE-DEPENDENT LIPID PEROXIDATION. Ceriporiopsis subvermispora is a selective fungus in the wood delignification and the most promising in biopulping. Through the lipid peroxidation initiated by manganese peroxidase (MnP), free radicals can be generated, which can act in the degradation of lignin nonphenolic structures. This work evaluated the prooxidant activity (based in lipid peroxidation) of enzymatic extracts from wood biodegradation by this fungus in cultures containing exogenous calcium, oxalic acid or soybean oil. It was observed that MnP significant activity is required to promote lipid peroxidation and wood delignification. Positive correlation between prooxidant activity x MnP was observed up to 300 IU kg(-1) of wood.