Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe(3+)-reductants. Phenolates were the major compounds with Fe(3+)-reducing activity in both fungi and displayed Fe(3+)-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe(3+) and H(2)O(2) (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum-a model brown rot fungus-other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.

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.

Wet oxidation of recalcitrant lignin waters: Experimental and kinetic studies

The dissertation is based on four articles dealing with recalcitrant lignin water purification. Lignin, a complicated substance and recalcitrant to most treatment technologies, inhibits seriously pulp and paper industry waste management. Therefore, lignin is studied, using WO as a process method for its degradation. A special attention is paid to the improvement in biodegradability and the reduction of lignin content, since they have special importance for any following biological treatment. In most cases wet oxidation is not used as a complete ' mineralization method but as a pre treatment in order to eliminate toxic components and to reduce the high level of organics produced. The combination of wet oxidation with a biological treatment can be a good option due to its effectiveness and its relatively low technology cost. The literature part gives an overview of Advanced Oxidation Processes (AOPs). A hot oxidation process, wet oxidation (WO), is investigated in detail and is the AOP process used in the research. The background and main principles of wet oxidation, its industrial applications, the combination of wet oxidation with other water treatment technologies, principal reactions in WO, and key aspects of modelling and reaction kinetics are presented. There is also given a wood composition and lignin characterization (chemical composition, structure and origin), lignin containing waters, lignin degradation and reuse possibilities, and purification practices for lignin containing waters. The aim of the research was to investigate the effect of the operating conditions of WO, such as temperature, partial pressure of oxygen, pH and initial concentration of wastewater, on the efficiency, and to enhance the process and estimate optimal conditions for WO of recalcitrant lignin waters. Two different waters are studied (a lignin water model solution and debarking water from paper industry) to give as appropriate conditions as possible. Due to the great importance of re using and minimizing the residues of industries, further research is carried out using residual ash of an Estonian power plant as a catalyst in wet oxidation of lignin-containing water. Developing a kinetic model that includes in the prediction such parameters as TOC gives the opportunity to estimate the amount of emerging inorganic substances (degradation rate of waste) and not only the decrease of COD and BOD. The degradation target compound, lignin is included into the model through its COD value (CODligning). Such a kinetic model can be valuable in developing WO treatment processes for lignin containing waters, or other wastewaters containing one or more target compounds. In the first article, wet oxidation of "pure" lignin water was investigated as a model case with the aim of degrading lignin and enhancing water biodegradability. The experiments were performed at various temperatures (110 -190°C), partial oxygen pressures (0.5 -1.5 MPa) and pH (5, 9 and 12). The experiments showed that increasing the temperature notably improved the processes efficiency. 75% lignin reduction was detected at the lowest temperature tested and lignin removal improved to 100% at 190°C. The effect of temperature on the COD removal rate was lower, but clearly detectable. 53% of organics were oxidized at 190°C. The effect of pH occurred mostly on lignin removal. Increasing the pH enhanced the lignin removal efficiency from 60% to nearly 100%. A good biodegradability ratio (over 0.5) was generally achieved. The aim of the second article was to develop a mathematical model for "pure" lignin wet oxidation using lumped characteristics of water (COD, BOD, TOC) and lignin concentration. The model agreed well with the experimental data (R2 = 0.93 at pH 5 and 12) and concentration changes during wet oxidation followed adequately the experimental results. The model also showed correctly the trend of biodegradability (BOD/COD) changes. In the third article, the purpose of the research was to estimate optimal conditions for wet oxidation (WO) of debarking water from the paper industry. The WO experiments were' performed at various temperatures, partial oxygen pressures and pH. The experiments showed that lignin degradation and organics removal are affected remarkably by temperature and pH. 78-97% lignin reduction was detected at different WO conditions. Initial pH 12 caused faster removal of tannins/lignin content; but initial pH 5 was more effective for removal of total organics, represented by COD and TOC. Most of the decrease in organic substances concentrations occurred in the first 60 minutes. The aim of the fourth article was to compare the behaviour of two reaction kinetic models, based on experiments of wet oxidation of industrial debarking water under different conditions. The simpler model took into account only the changes in COD, BOD and TOC; the advanced model was similar to the model used in the second article. Comparing the results of the models, the second model was found to be more suitable for describing the kinetics of wet oxidation of debarking water. The significance of the reactions involved was compared on the basis of the model: for instance, lignin degraded first to other chemically oxidizable compounds rather than directly to biodegradable products. Catalytic wet oxidation of lignin containing waters is briefly presented at the end of the dissertation. Two completely different catalysts were used: a commercial Pt catalyst and waste power plant ash. CWO showed good performance using 1 g/L of residual ash gave lignin removal of 86% and COD removal of 39% at 150°C (a lower temperature and pressure than with WO). It was noted that the ash catalyst caused a remarkable removal rate for lignin degradation already during the pre heating for `zero' time, 58% of lignin was degraded. In general, wet oxidation is not recommended for use as a complete mineralization method, but as a pre treatment phase to eliminate toxic or difficultly biodegradable components and to reduce the high level of organics. Biological treatment is an appropriate post treatment method since easily biodegradable organic matter remains after the WO process. The combination of wet oxidation with subsequent biological treatment can be an effective option for the treatment of lignin containing waters.

Pulsed corona discharge as an advanced oxidation process for degradation of organic

Advanced oxidation processes (AOPs) have been studied and developed to suffice the effective removal of refractory and toxic compounds in polluted water. The quality and cost of wastewater treatment need improvements, and electric discharge technology has a potential to make a significant difference compared to other established AOPs based on energy efficiency. The generation of active oxidant species such as ozone and hydroxyl radicals by high voltage discharge is a relatively new technology for water treatment. Gas-phase pulsed corona discharge (PCD), where a treated aqueous solution is dispersed between corona-producing electrodes free of the dielectric barriers, was developed as an alternative approach to the problem. The short living radicals and ozone formed in the gas phase and at the gas-liquid interface react with dissolved impurities. PCD equipment has a relatively simple configuration, and with the reactor in an enclosed compartment, it is insensitive towards gas humidity and does not need the gas transport. In this thesis, PCD was used to study and evaluate the energy efficiency for degrading various organic compounds, as well as the chemistry of the oxidation products formed. The experiments investigate the aqueous oxidation of phenol, humic substances, pharmaceutical compounds (paracetamol, ibuprofen, indomethacin, salicylic acids, -estradiol), as well as lignin degradation and transformation to aldehydes. The study aims to establish the influence of initial concentration of the target pollutant, the pulsed discharge parameters, gas phase composition and the pH on the oxidation kinetics and the efficiency. Analytical methods to measure the concentrations of the target compounds and their by-products include HPLC, spectrophotometry, TOC and capillary electrophoresis. The results of the research included in this summary are presented in the attached publications and manuscripts accepted for publication. Pulsed corona discharge proved to be highly effective in oxidizing each of the target compounds, surpassing the closest competitor, conventional ozonation. The increase in oxidation efficiencies for some compounds in oxygen media and at lower pulse repetition frequencies shows a significant role of ozone. The role of the ·OH radicals was established in the surface reactions. The main oxidation products, formation of nitrates, and the lignin transformation were quantified. A compound specific approach is suggested for optimization of the PCD parameters that have the most significant impact on the oxidation energy efficiency because of the different characteristics and responses of the target compound to the oxidants, as well as different admixtures that are present in the wastewater. Further studies in the method’s safety (nitration and nitrosation of organic compounds, nitrite and nitrate formation enhancement) are needed for promoting the method.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

The continually growing worldwide hazardous waste problem is receiving much attention lately. The development of cost effective, yet efficient methods of decontamination are vital to our success in solving this problem.Bioremediation using white rot fungi, a group of basidiomycetes characterized by their ability to degrade lignin by producing extracellular LiP, MnP and laccase have come to be recognized globally which is described in detail in Chapter 1.These features provide them with tremendous advantages over other micro-organisms.Chapter 2 deals with the isolation and screening of lignin degrading enzyme producing micoro-organisms from mangrove area. Marine microbes of mangrove area has great capacity to tolerate wide fluctuations of salinitie.Primary and secondary screening for lignin degrading enzyme producing halophilic microbes from mangrove area resulted in the selection of two fungal strains from among 75 bacteria and 26 fungi. The two fungi, SIP 10 and SIP ll, were identified as penicillium sp and Aspergillus sp respectively belonging to the class Ascomycetes .Specific activity of the purified LiP was 7923 U/mg protein. The purification fold was 24.07 while the yield was 18.7%. SDS PAGE of LiP showed that it was a low molecular weight protein of 29 kDa.Zymogram analysis using crystal violet dye as substrate confirmed the peroxidase nature of the purified LiP.The studies on the ability of purified LiP to decolorize different synthetic dyes was done. Among the dyes studied, crystal violet, a triphenyl methane dye was decolorized to the greatest extent.

Optimization of Lignin Peroxidase, Manganese Peroxidase, and Lac Production from Ganoderma lucidum Under Solid State Fermentation of Pineapple Leaf

This study was undertaken to isolate ligninase-producing white-rot fungi for use in the extraction of fibre from pineapple leaf agriwaste. Fifteen fungal strains were isolated from dead tree trunks and leaf litter. Ligninolytic enzymes (lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac)), were produced by solid-state fermentation (SSF) using pineapple leaves as the substrate. Of the isolated strains, the one showing maximum production of ligninolytic enzymes was identified to be Ganoderma lucidum by 18S ribotyping. Single parameter optimization and response surface methodology of different process variables were carried out for enzyme production. Incubation period, agitation, and Tween-80 were identified to be the most significant variables through Plackett-Burman design. These variables were further optimized by Box-Behnken design. The overall maximum yield of ligninolytic enzymes was achieved by experimental analysis under these optimal conditions. Quantitative lignin analysis of pineapple leaves by Klason lignin method showed significant degradation of lignin by Ganoderma lucidum under SSF

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.

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.

Optimal recovery process conditions for manganese-peroxidase obtained by solid-state fermentation of eucalyptus residue using Lentinula edodes

Enzyme production is a growing field in biotechnology and increasing attention has been devoted to the solid-state fermentation (SSF) of lignocellulosic biomass for production of industrially relevant lignocellulose deconstruction enzymes, especially manganese-peroxidase (MnP), which plays a crucial role in lignin degradation. However, there is a scarcity of studies regarding extraction of the secreted metabolities that are commonly bound to the fermented solids, preventing their accurate detection and limiting recovery efficiency. In the present work, we assessed the effectiveness of extraction process variables (pH, stirring rate, temperature, and extraction time) on recovery efficiency of manganese-peroxidase (MnP) obtained by SSF of eucalyptus residues using Lentinula edodes using statistical design of experiments. The results from this study indicated that of the variables studied, pH was the most significant (p < 0.05%) parameter affecting MnP recovery yield, while temperature, extraction time, and stirring rate presented no statistically significant effects in the studied range. The optimum pH for extraction of MnP was at 4.0-5.0, which yielded 1500-1700 IU kg (1) of enzyme activity at extraction time 4-5 h, under static condition at room temperature. (C) 2011 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

Scale-up of diluted sulfuric acid hydrolysis for producing sugarcane bagasse hemicellulosic hydrolysate (SBHH)

Sugarcane bagasse was pretreated with diluted sulfuric acid to obtain sugarcane bagasse hemicellulosic hydrolysate (SBHH). Experiments were conducted in laboratory and semi-pilot reactors to optimize the xylose recovery and to reduce the generation of sugar degradation products, as furfural and 5-hydroxy-methylfurfural (HMF). The hydrolysis scale-up procedure was based on the H-Factor, that combines temperature and residence time and employs the Arrhenius equation to model the sulfuric acid concentration (100 mg(acid)/g(dm)) and activation energy (109 kJ/mol). This procedure allowed the mathematical estimation of the results through simulation of the conditions prevailing in the reactors with different designs. The SBHH obtained from different reactors but under the same H-Factor of 5.45 +/- 0.15 reached similar xylose yield (approximately 74%) and low concentration of sugar degradation products, as furfural (0.082 g/L) and HMF (0.0071 g/L). Also, the highest lignin degradation products (phenolic compounds) were rho-coumarilic acid (0.15 g/L) followed by ferulic acid (0.12 g/L) and gallic acid (0.035 g/L). The highest concentration of ions referred to S (3433.6 mg/L), Fe (554.4 mg/L), K (103.9 mg/L), The H-Factor could be used without dramatically altering the xylose and HMF/furfural levels. Therefore, we could assume that H-Factor was directly useful in the scale-up of the hemicellulosic hydrolysate production. (C) 2009 Published by Elsevier Ltd.

Análise de fluorescência de substâncias húmicas extraídas da água, solo e sedimento da Lagoa dos Patos - MS

The humic substances were extracted from sediments, water and soil close to the Lagoa dos Patos-MS. The characterization was performed through fluorescence emission. In the HSs it is possible to see strong indications of incorporation of compounds originating from lignin degradation, confirmed by fluorescence emission (EEM and synchronous spectra). These differences observed in HSs may be due to tropical regions displaying a great variety of vascular plants and aquatic macrophytes that contribute natural organic matter. The seasonality effect also causes the entrance of pedogenic material and the suspension of sediments by the action of winds.

Uma visão sobre a estrutura, composição e biodegradação da madeira

Wood is the main raw material used in the pulp and paper industry. It is a material that presents heterogeneous structure and complex composition, which results in a relatively resistant material to the biodegradation process. In the present review, we attempted to summarize the structural characteristics of wood and describe the chemical nature of its major components to, afterwards, comment about its biodegradation. The role of the enzyme manganese peroxidase in the lignin degradation by a selective white-rot fungus, Ceriporiopsis subvermispora, was highlighted.

Avaliação do uso de radiação micro-ondas e ultrassom combinados com agentes químicos como pré-tratamentos de bagaço de cana-de-açúcar para posterior sacarificação enzimática

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Seleção de espécies de basidiomicetos produtoras de ligninases para caracterização e aplicação das enzimas sobre corantes aromáticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)