Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi

Ethanol was produced by simultaneous saccharification and fermentation (SSF) from beech wood chips after bioorganosolve pretreatments by ethanolysis and white rot fungi, Ceriporiopsis subvermispora, Dichomitus squalens, Pleurotus ostreatus, and Coriolus versicolor. Beech wood chips were pretreated with the white rot fungi for 2-8 weeks without addition of any nutrients. The wood chips were then subjected to ethanolysis to separate them into pulp and soluble fractions (SFs). From the pulp fraction (PF), ethanol was produced by SSF using Saccharomyces cerevisiae AM12 and a commercial cellulase preparation, Meicelase, from Trichoderma viride. Among the four strains, C. subvermispora gave the highest yield on SSF. The yield of ethanol obtained after pretreatment with C. subvermispora for 8 weeks was 0.294 g g(-1) of ethanolysis pulp (74% of theoretical) and 0.176 g g(-1) of beech wood chips (62% of theoretical). The yield was 1.6 times higher than that obtained without the fungal treatments. The biological pretreatments saved 15% of the electricity needed for the ethanolysis.     

Anaerobic fermentation of woody biomass treated by various methods

Anaerobic fermentation was attempted to produce methane from the wood chip (Eucalyptus globulus). By the pretreatment of the wood chip using hot water with high temperature, NaOH, and steam explosion, the production of methane gas was enhanced. The pretreatment using steam explosion resulted in more amount of methane gas produced than the treatment using either hot water or 1% (w/w) NaOH with high temperature, and the steam explosion at a steam pressure of 25 atm and a steaming time of 3 min was the most effective for the methane production. The amount of methane gas produced depended on the ratio of weight of Klason lignin, a high molecular weight lignin, in the treated wood chip.     

Biochemical pulping of bagasse

This study deals with pretreatment of wheat straw with lignin-degrading fungi and its effect on chemical pulping. Ceriporiopsis subvermispora strains, which preferentially attack the lignin, were used for biochemical pulping of bagasse. Treatment of depithed bagasse with different strains of C. subvermispora reduced the kappa number by 10-15% and increased unbleached pulp brightness by 1.1-2.0 ISO points on chemical pulping at the same alkali charge. Bleaching of biopulps at the same chemical charge increased final brightness by 4.7-5.6 ISO points and whiteness by 10.2-11.4 ISO points. Fungal treatment did not result in any adverse effect on the strength properties of pulp.     

Ceriporic acid B, an extracellular metabolite of Ceriporiopsis subvermispora, suppresses the depolymerization of cellulose by the Fenton reaction

The white rot fungus, Ceriporiopsis subvermispora, is able to degrade lignin in wood without intensive damage to cellulose. Since lignin biodegradation by white rot fungi proceeds by radical reactions, accompanied by the production of a large amount of Fe3+-reductant phenols and reductive radical species in the presence of iron ions, molecular oxygen, and H2O2, C. subvermispora has been proposed to possess a biological system which suppresses the production of a cellulolytic active oxygen species, *OH, by the Fenton reaction. In the present paper, we demonstrate that 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B), an extracellular metabolite of C. subvermispora, strongly inhibited *OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus.     

Conversion of Japanese red cedar (Cryptomeria japonica) into a feed for ruminants by white-rot basidiomycetes

The potential of five white-rot fungi to convert Japanese red cedar (Cryptomeria japonica) into feed for ruminants was determined. Pleurotus ostreatus (ATCC 66376), Pholiota nameko (IFO 30373), Dichomitus squalens (CBS 432.34), Lentinula edodes (IFO 6654) and Ceriporiopsis subvermispora (ATCC 90467) were inoculated into chips of cedar sapwood. L. edodes was cultured at 25 °C, and the other fungi at 28 °C, for 4, 8, 12, or 20 weeks. The in vitro organic matter (OM) digestibility (IVOMD) in cedar wood cultured without fungus were between 0.047 and 0.068, while it was elevated to 0.446 by culturing with C. subvermispora and to 0.281 by culturing with L. edodes for 20 weeks. In contrast, the IVOMD were 0.200, or lower, in cedar wood cultured with P. ostreatus, P. nameko, or D. squalens. The in vitro gas production (IVGP) in cedar wood cultured with P. ostreatus, P. nameko, or D. squalens was 37 ml/g OM, or lower, while that in cedar not inoculated with fungus was between 4 and 17 ml/g OM. In contrast, the IVGP in cedar wood cultured with C. subvermispora for 20 weeks increased to 107 ml/g OM, and that in cedar wood cultured with L. edodes increased to 58 ml/g OM. Lignin degradability in cedar wood cultured with C. subvermispora and L. edodes for 20 weeks were 0.578 and 0.288, respectively. These changes in IVOMD and IVGP demonstrate that a selective white-rot fungus, C. subvermispora has the ability to convert cedar wood into feed for ruminants, although further increase is required before the cultured cedar wood would have widespread feed potential.     

Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.     

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a (sup14)C-labeled, polyethylene glycol-linked, high-molecular-weight (beta)-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C(inf(alpha)) and C(inf(beta)) to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C(inf(alpha))-C(inf(beta)) cleavage of (beta)-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), (sup14)C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 (mu)M to approximately 5 (mu)M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic (beta)-O-4 lignin model compounds between C(inf(alpha)) and C(inf(beta)) to release a benzylic fragment.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

Degradation of natural lignins and lignocellulosic substrates by soil-inhabiting fungi imperfecti

Abstract: The most powerful lignin-degraders among the 82 microbial strains isolated during a screening of ligninolytic microorganisms from forest soil were identified as Penicillium chrysogenum, Fusarium oxysporum and Fusarium solani . These fungi imperfecti mineralized 27.4%, 23.5% and 22.6% of ¹⁴C-labelled milled wood lignin (MWL) from wheat straw after 28 days of incubation in liquid media. Degradation of MWL from pine by P. chrysogenum was 8% and 19% when it was evaluated by spectrophotometry and Klason lignin, respectively, but this substrate was hardly mineralized. All fungi were able to attack the hemicellulosic, cellulosic and also lignin fractions of wheat straw during solid-state fermentation, F. solani being capable of degrading about 25% of both carbohydrates and lignin. When the selected fungi were tested for dye decolourization, they all readily attacked the polymeric dye Remazol brilliant blue R (RBBR) and also poly R-478 to a minor extent.     

Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius

Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [(sup14)C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn(sup2+) to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.     

Biopulping: An overview of developments in an environmentally safe paper-making technology

Abstract: Treatment of wood chips with lignin-degrading fungi prior to pulping has been shown to have great potential for mechanical as well as chemical pulping on a laboratory scale. Ceriporiopsis subvermispora , when grown on aspen or loblolly pine for 4 weeks, was found to be superior to other fungi. On aspen there was an energy savings of 47%, and an increase in burst and tear indices of 22% and 119%, respectively. With loblolly pine, energy savings amounted to 37%, while burst and tear indices increased by 41% and 54%, respectively. The weight loss was only 6%, but a decrease in optical properties had to be accepted. After sulfite cooking of wood chips pretreated for 2 weeks, the Kappa number decreased by 30% with hard- and softwood. Tensile and tear indices decreased by only 10%, while the brightness of unbleached pulp increased by 4% with birch. Information obtained by immunoelectron microscopy and differential staining led to the conclusion that the biopulping effect obtained after 2 weeks of incubation cannot be explained by the direct action of enzymes on lignin or polysaccharides. Instead, a low molecular mass agent is considered to be responsible for the biopulping effect. These results have changed the aims of biopulping from an emphasis on removing the bulk of lignin to an emphasis on a short-term process, lasting 2 weeks and yielding a low mass loss. Data on these kinetics of fungal development and the degree of asepsis will help to scale-up the process. An advanced chip pile is assumed to be the most feasible process design, rather than a controlled enclosed reactor.     

Alterations in structure, chemistry, and biodegradability of grass lignocellulose treated with the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus.

The white rot fungi Ceriporiopsis subvermispora FP-90031-sp and Cyathus stercoreus ATCC 36910 were evaluated for their ability to delignify Bermuda grass (Cynodon dactylon) stems and improve biodegradability. Compositional and structural alterations in plant cell walls effected by the fungi were determined by nuclear magnetic resonance spectroscopy, gas chromatography of alkali-treated residues, microspectrophotometry, and electron microscopy. Contaminating bacteria and fungi, which grew from unsterilized Bermuda grass stems, did not alter the improvement in grass biodegradability by either of the fungi from that of gas-sterilized stems. The biodegradation of stems by ruminal microorganisms, after treatment for 6 weeks with C. subvermispora or C. stercoreus, was improved by 29 to 32% and by 63 to 77%, respectively; dry weight losses caused by pretreatment with the fungi were about 20% over that in untreated, control stems. Both fungi preferentially removed aromatics to carbohydrates, and C. subvermispora removed proportionately more guaiacyl units than did C. stercoreus. Substantial amounts of ester-linked p-coumaric and ferulic acids were removed by both fungi, and about 23 and 41% of total aromatics (determined after 4 M NaOH direct treatment) were removed from the plant biomass after incubation with C. subvermispora and C. stercoreus, respectively. UV absorption microspectrophotometry indicated that ester-linked phenolic acids were totally removed from the parenchyma cell walls, and these cells were readily and completely degraded by both fungi. However, aromatic constituents were only partially removed from the more recalcitrant sclerenchyma cell walls, resulting in variation in electron density and random digestion pits after incubation with fiber-degrading bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran

Wheat bran, a by-product of the wheat milling industry, consists mainly of hemicellulose, starch and protein. In this study, the hydrolysate of wheat bran pretreated with dilute sulfuric acid was used as a substrate to produce ABE (acetone, butanol and ethanol) using Clostridium beijerinckii ATCC 55025. The wheat bran hydrolysate contained 53.1 g/l total reducing sugars, including 21.3 g/l of glucose, 17.4 g/l of xylose and 10.6 g/l of arabinose. C. beijerinckii ATCC 55025 can utilize hexose and pentose simultaneously in the hydrolysate to produce ABE. After 72 h of fermentation, the total ABE in the system was 11.8 g/l, of which acetone, butanol and ethanol were 2.2, 8.8 and 0.8 g/l, respectively. The fermentation resulted in an ABE yield of 0.32 and productivity of 0.16 g l⁻¹ h⁻¹. This study suggests that wheat bran can be a potential renewable resource for ABE fermentation.     

Time course of fungal removal of lipophilic extractives from Eucalyptus globulus wood

Free and esterified sitosterol, the main lipophilic constituents of eucalypt wood extractives, have been associated with the formation of pitch deposits during manufacturing of environmentally-sound paper pulp from Eucalyptus globulus wood. These, and other lipophilic compounds, were analyzed by gas chromatography-mass spectrometry in the course of wood treatments (up to 7 weeks) with four extractive-degrading fungi in order to optimize biotechnological control of pitch deposition in eucalypt pulp (with moderate loss of wood weight). In contrast to commercialized fungi used in pitch control, which are not able to degrade sitosterol, the fungi investigated in this paper produced a rapid decline of both free and esterified sterols in wood. The degradation rate of steroid hydrocarbons and squalene was moderate, and the amount of steroid ketones (probably formed during oxidative degradation of steroids) and triglycerides increased at different stages of wood treatment. Up to 95% removal of total steroids (including free and esterified sterols, steroid ketones and steroid hydrocarbons) by fungi was obtained at the end of wood treatment under the solid-state fermentation conditions used. The most promising results from the point of view of industrial applicability, however, were obtained after 1-2 weeks of treatment with either Phlebia radiata or Poria subvermispora, which enabled 70% steroid removal with a moderate wood weight loss of 1-4%.     

Effect of three strains of Pleurotus tuber-regium (Fr.) Sing. on chemical composition and rumen fermentation of wheat straw

This study was conducted to investigate changes in in vitro dry matter digestibility (IVDMD) and cell wall constituent degradation in wheat straw treated with 3 strains of the fungus Pleurotus tuber-regium (PT). The incubation of wheat straw for 30 days at 28 degrees C improved IVDMD from 30.3% (UWS-untreated wheat straw) to 47.1% for strain PT1, to 48.5% for PT4, and was unchanged IVDMD-29.9% -for PT5. The growth of fungi was accompanied by the dry matter loss of wheat straw: 31.5% for PT1, 20.9% for PT4, and 4.8% for PT5. Fungal treatment was characterized by increased crude protein and ash contents (%) in all fungi-treated straws and reduced hemicellulose and lignin content. It is evident that enzymes of all 3 PT strains preferentially degraded hemicellulose and lignin over cellulose. Wheat straw treated with PT1 (TWS-PT1), PT4 (TWS-PT4), and PT5 (TWS-PT5) and barley (80% : 20%) were used as the experimental diets at the fermentation in the artificial rumen. UWS with barley (80% : 20%) served as the control diet. The fermentation of experimental diets was accompanied with increased IVDMD and a very low degree of hemicellulose degradation. Total gas and methane productions were similar in all diets. Moreover, total volatile fatty acid (VFA) production (mmol day(-1)), mol % of acetate, propionate, butyrate, isobutyrate, and isovalerate were not influenced during the fermentation of experimental diets. From the stoichiometric relations, production, utilization, and recovery of metabolic hydrogen and organic matter fermented were unchanged. Only the recovery of metabolic hydrogen in TWS-PT5 was significantly increased in comparison to control diet. Total microbial production showed the tendency of lower values in experimental diets, and it was accompanied with a significant decrease of ammonia nitrogen (mg L(-1)). Finally the results showed that the strains of Pleurotus tuber-regium can improve the quality of wheat straw, but the loss of dry matter (DM) (mainly hemicellulose) limits the effective utilization of fungi-treated straw in ruminant digestion.     

Detection and characterization of a novel extracellular fungal enzyme that catalyzes the specific and hydrolytic cleavage of lignin guaiacylglycerol beta-aryl ether linkages.

Cleavage of the arylglycerol beta-aryl ether linkage is the most important process in the biological degradation of lignin. The bacterial beta-etherase was described previously and shown to be tightly associated with the cellular membrane. In this study, we aimed to detect and isolate a new extracellular function that catalyses the beta-aryl ether linkage cleavage of high-molecular lignin in the soil fungi. We screened and isolated 2BW-1 cells by using a highly sensitive fluorescence assay system. The beta-aryl ether cleavage enzyme was produced by a newly isolated fungus, 2BW-1, and is secreted into the extracellular fraction. The beta-aryl ether cleavage enzyme converts the guaiacylglycerol beta-O-guaiacyl ether (GOG) to guaiacylglycerol and guaiacol. It requires the C alpha alcohol structure and p-hydroxyl group and specifically attacks the beta-aryl ether linkage of high-molecular mass lignins with addition of two water molecules at the C alpha and C beta positions.     

麦草栽培姬菇配方的数学模型研究

借助二次通用旋转组合设计法研究了麦草栽培姬菇配方的数学模型,以探讨利用麦草替代棉籽壳的新配方.试验结果得出了配料的取值范围及最佳栽培配方,即麦草79％,麸皮15％,玉米粉4％,过磷酸钙1％,黄豆饼粉1％.在实际生产中很有应用价值.     

Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

Fungal degradation of lipophilic extractives in eucalyptus globulus wood

Solid-state fermentation of eucalypt wood with several fungal strains was investigated as a possible biological pretreatment for decreasing the content of compounds responsible for pitch deposition during Cl2-free manufacture of paper pulp. First, different pitch deposits were characterized by gas chromatography (GC) and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and different degradation patterns were observed. The results showed that some basidiomycetes that decreased the lipophilic fraction also released significant amounts of polar extractives, which were identified by thermochemolysis as originating from lignin depolymerization. Therefore, the abilities of fungi to control pitch should be evaluated after analysis of compounds involved in deposit formation and not simply by estimating the decrease in the total extractive content. In this way, Phlebia radiata, Funalia trogii, Bjerkandera adusta, and Poria subvermispora strains were identified as the most promising organisms for pitch biocontrol, since they degraded 75 to 100% of both free and esterified sterols, as well as other lipophilic components of the eucalypt wood extractives. Ophiostoma piliferum, a fungus used commercially for pitch control, hydrolyzed the sterol esters and triglycerides, but it did not appear to be suitable for eucalypt wood treatment because it increased the content of free sitosterol, a major compound in pitch deposits.