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.     

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.     

Main lipophilic extractives in different paper pulp types can be removed using the laccase–mediator system

Lipophilic extractives in wood and other lignocellulosic materials exert a negative impact in pulp and paper manufacturing causing the so-called pitch problems. In this work, the appropriateness of an enzymatic treatment using the laccase–mediator system for pitch biocontrol is evaluated. With this purpose, three pulp types representative for different raw materials and pulping processes—eucalypt kraft pulping, spruce thermomechanical pulping, and flax soda-anthraquinone pulping—were treated with a high-redox-potential laccase from the basidiomycete Pycnoporus cinnabarinus in the presence of 1-hydroxybenzotriazole as a redox mediator. The gas chromatography and gas chromatography/mass spectrometry analyses of the lipophilic extractives from the enzymatically treated pulps revealed that the laccase–mediator treatment completely or greatly removed most of the pitch-causing lipophilic compounds present in the different pulps including: (1) free and conjugated sitosterol in eucalypt paper pulp; (2) resin acids, sterol esters, and triglycerides in spruce pulp; and (3) sterols and fatty alcohols in the flax pulp. Different amounts of free and conjugated 7-oxosterols were found as intermediate products in the oxidation of pulp sterols. Therefore, the laccase–mediator treatment is reported as an efficient method for removing pitch-causing lipophilic compounds from paper pulps obtained from hardwood, softwood, and nonwoody plants.     

Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

Microbial and enzymatic control of pitch in the pulp and paper industry

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.     

Effect of oxygen, ozone and hydrogen peroxide bleaching stages on the contents and composition of extractives of Eucalyptus globulus kraft pulps

The effects of oxygen (O), ozone (Z) and hydrogen peroxide (P) bleaching stages on the composition and total amount of Eucalyptus globulus kraft pulp lipophilic extractives was studied. These bleaching stages led to the partial removal and to several oxidative transformations of fatty acids and sterols, the main lipophilic extractives found in the unbleached pulp. Unsaturated extractives were found to be partially degraded while saturated ones were, in general, stable. The oxygen and hydrogen peroxide bleaching stages were more effective than ozone in removing fatty acids from pulp, by dissolution in the liquid phase. On the other hand, the ozone stage was more effective in the oxidative degradation of sterols. Oxygen and hydrogen peroxide bleaching stages were also effective in sterols removal, but led to the formation of sterol oxidation derivatives, previously shown to be involved in the formation of pitch that accumulates in the bleaching filtrates.     

Influence of operation conditions on laccase-mediator removal of sterols from eucalypt pulp

The way how sterols, the main lipophilic compounds present in eucalypt kraft pulp, are eliminated by an enzymatic stage using the laccase-mediator system was evaluated. With this purpose laccase-mediator stage (L) was applied on an Eucalyptus globulus pulp under different operation conditions following a three-variable (laccase dose, mediator dose and reaction time) sequential statistical plan, to optimise the removal of sterols. The decrease in pulp sterol content during the enzymatic treatment was related to the decrease in kappa number and to brightness increase, as well as with the increase in some oxidation products of sitosterol (namely 7-oxositosterol and stigmasta-3,5-dien-7-one). The increase in reaction time from 1 to 5 h strongly reduced the sterol content, while no more sterols were eliminated during the 5–7 h period. Increasing the laccase dose from 1 to 20 U g⁻¹ of pulp produced a high reduction in pulp sterols, whereas the increase in mediator (1-hydroxybenzotriazole) dose (from 0.5 to 2.5% of pulp weight) had only a slight influence in removing sterols. Therefore, at 16 U g⁻¹ laccase dose, 0.5% mediator dose, 4 h of reaction, practically all the sterols were removed. Finally, it was demonstrated that sterols were more sensitive to a L stage (practically 100% of sterols were eliminated) than to a chlorine dioxide stage (54% of sterols eliminated).     

Degradation and detoxification of softwood extractives by sapstain fungi

Wood extractives (resin) cause pitch deposition problems and effluent toxicity in pulp and papermaking. The ability of six sapstaining fungi to degrade and detoxify extractive constituents in Scots pine sapwood was examined, and the results were compared with those obtained with the commercial depitching fungus Cartapip (Ophiostoma piliferum). Pestalotiopsis crassiuscula and O. piliferum were the best strains and they provided high reductions of total resin (50–60% in 6 weeks). Both strains were highly effective in the degradation of individual extractive components including triglycerides, diglycerides and free fatty acids. Although all strains displayed moderate to high pitch degradation, their detoxifying capacity was limited. Two important exceptions were Ceratocystis deltoideospora and O. piliferum that caused a 11–14-fold decrease in toxicity (Microtox bioassay). These results indicate the potential of wood pretreatment with the selected sapstain fungi for minimizing pitch problems and decreasing effluent toxicity in pulping.     

The biotechnological control of pitch in paper pulp manufacturing

At present, microbial and enzymatic preparations for the control of triglyceride-containing pitch deposits during the manufacture of mechanical and sulfite paper is commercially available. However, biotechnological products for pitch control in other pulping processes, such as alkaline pulping, are under development. These products include new fungi for the removal of steroids involved in pitch deposit formation in chlorine-free pulps, to be used as a biological pretreatment of wood before pulping. Simultaneously, tailor-made enzymes are being produced using protein-engineering techniques, enabling the specific removal of pitch contaminant compounds from paper pulp.     

Microscopy studies reveal delignification and sterol removal from eucalypt kraft pulps by laccase–HBT

Fungal laccases in the presence of mediators are powerful biocatalysts to degrade lignin. Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) have been successfully used to delignify eucalypt kraft pulp once integrated in a totally chlorine-free bleaching sequence. Real time delignification of kraft pulp by laccase–HBT was verified in situ by monitoring the loss of lignin autofluorescence during the enzymatic treatment using confocal laser scanning microscopy. The highest delignification of pulp fibers occurred over a very short time-span (5 min). Moreover, we demonstrate the removal of sterols, responsible for pitch deposits in hardwood kraft pulps, as an additional effect of laccase-HBT. Spherical structures between pulp fibers localized by low temperature scanning electron microscopy were removed by laccase–HBT. The use of filipin, a specific stain, revealed the sterol nature of many of these structures. At the end of the enzyme-aided bleaching sequence, the fluorescent sterols–filipin signals were almost completely absent.     

Effect of storage time on the composition and content of wood extractives in Eucalyptus cultivated in Brazil

Lipophilic wood extractives commonly referred to as pitch, cause significant problems for the pulp and paper industries. The reduction of these extractives is an important aspect that concerns industries around the world. In the present work the change in the amount and chemical composition of lipophilic extractives from Eucalyptus spp. stored for 20, 40, 60, 100, 140 and 180 days after harvesting was investigated. The results showed a decrease in extractives content with storage time, with the most significant decrease occurring 60 days after harvesting. In addition, fatty acids and sterols were the main classes of compounds responsible for the significant decrease in extractive content. Data were analyzed by principal component analysis. PC1 explains approximately 99% of the total variance, and beta-sitosterol was the major compound responsible for the differentiation. These studies demonstrate that in terms of economical aspects, quality of the pulp and paper and minimization of pitch formation, the best period of wood storage is 60 days.     

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.     

4-Desmethylsterol composition of citrus rootstocks of different salt exclusion capacity

Fibrous roots from seedlings of three citrus rootstocks (Rungpur lime, Kharna khatta and Etrog citron) grown hydroponically for 6 weeks in the presence or absence of 50 mM NaCl were analysed for their content of free, esterified, and glycosidic sterols. Leaf chloride analyses indicated that Rangpur lime was a good Cl- excluder and the other two rootstocks were Cl- accumulators.
On a dry weight basis and in the absence of NaCI only the free 4-desmethyIsterol levels showed significant rootstock differences. Kharna khatta had the highest, and Rangpur lime the lowest, free stcrol levels. Sitostcrol was the major component of all sterol fractions of Rangpur lime, the esterified fraction of the other rootstocks, and the glycosidic sterol fraction of Kharna khatta. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron in all cases and the ratio of apos;more-planar apos; to apos;less-planar apos; free sterols was highest in Etrog citron and lowest in Rangpur lime.
Treatment with 50 mM NaCI resulted in an increase in free sterol levels in Rangpur lime and a decrease in Kharna khatta. Steryl ester levels were unaffected in Rangpur lime but were significantly reduced in the other rootstocks. In all three sterol fractions the sitosterol/stigmasterol ratio was decreased. A decrease in the ratio of apos;more-planarapos; to apos;less-planarapos; free sterols was observed only in Kharna khatta and, more notably, Etrog citron. Salt-induced changes in the apos;more-planar apos; to apos;less-planar apos; free sterol ratio correlated well with salt exclusion capacity.     

Microscopy studies reveal delignification and sterol removal from eucalypt kraft pulps by laccase-HBT

Fungal laccases in the presence of mediators are powerful biocatalysts to degrade lignin. Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) have been successfully used to delignify eucalypt kraft pulp once integrated in a totally chlorine-free bleaching sequence. Real time delignification of kraft pulp by laccase-HBT was verified in situ by monitoring the loss of lignin autofluorescence during the enzymatic treatment using confocal laser scanning microscopy. The highest delignification of pulp fibers occurred over a very short time-span (5 min). Moreover, we demonstrate the removal of sterols, responsible for pitch deposits in hardwood kraft pulps, as an additional effect of laccase-HBT. Spherical structures between pulp fibers localized by low temperature scanning electron microscopy were removed by laccase-HBT. The use of filipin, a specific stain, revealed the sterol nature of many of these structures. At the end of the enzyme-aided bleaching sequence, the fluorescent sterols-filipin signals were almost completely absent.     

Elimination and detoxification of softwood extractives by white-rot fungi

The ability of several white-rot fungal strains to remove and detoxify acetone extractives (pitch or resin) in Scots pine sapwood was investigated in stationary laboratory batch assays. Fungal pretreatment provided up to 62% total pitch reduction and significant decreases in pitch toxicity. The best strains were Bjerkandera sp. strain BOS55, Stereum hirsutum and Trametes versicolor that eliminated over 93% of the problematic triglyceride fraction and 58–87% of other lipophilic extractive classes in only 2 weeks. Fungal removal of the wood extractives was accompanied by a 7.4–16.9-fold decrease in their inhibitory effect, as determined in the Microtox bioassay. Wood pretreatment by Bjerkandera sp. and T. versicolor caused limited losses of woody mass (less than 4% in 4 weeks); whereas S. hirsutum led to somewhat higher mass losses (7% in 4 weeks). These results indicate the potential of white rot fungi to control pitch deposition problems in pulping and to reduce the aquatic toxicity caused by naturally-occurring lipophilic extractives in forest industry effluents.     

Biological Processing of Pine Logs for Pulp and Paper Production with Phlebiopsis gigantea

Phlebiopsis gigantea (=Phanerochaete gigantea) is a white rot fungus that rapidly colonizes cut stumps, stems, and branches of pine. Two laboratory and several field studies showed that inoculation of red pine logs, Pinus resinosa, with P. gigantea reduced the pitch content of wood, facilitated bark removal, modified wood cells, and controlled detrimental sapstain. Isolations from inoculated logs revealed up to 100 and 80% colonization of the sapwood by P. gigantea after 8 weeks in the field and 32 days in the laboratory, respectively. Logs colonized by P. gigantea in both the laboratory and field showed a 9 to 71% reduction in pitch content, as well as a significant enhancement of bark removal. Examination with Simons' stain of refined wood fibers from inoculated logs revealed an increase in cell wall porosity. Blue stain fungi that cause dark discoloration of the sapwood were inhibited by inoculation with P. gigantea. These studies demonstrate that biological processing of logs with P. gigantea can result in substantial benefits to the pulp and papermaking process.     

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.     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

Sterol Changes during Germination of Nicotiana tabacum Seeds

The identity, composition, and concentration of the total, free, esterified, and glycosidic sterol fractions were determined during germination of tobacco seeds. The total, free, and esterified sterols increased, with stigmasterol and campesterol accounting for most of the increase. Steryl glycosides decreased during germination, and stigmasteryl and sitosteryl glycosides showed the largest decrease. During germination, sitosterol was the major sterol in all fractions but stigmasterol and campesterol showed the greatest changes. The fatty acid composition of the steryl esters and acylated steryl glycosides most closely resembled the di- and triglycerides.     

Lipids and lipolytic enzymes in the trunkwood ofRobinia pseudoacacia L. during heartwood formation

The radial distribution of membrane and storage lipids was determined in the trunkwood ofRobinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and fluctuations in the amount and composition of phospholipids, free sterols, steryl esters, diand triacylglycerols, and free fatty acids were investigated across the sapwood-heartwood boundary. The individual compounds were identified and quantified by thin layer chromatography, enzymatic and colorimetric assays, and by capillary gas chromatography. Phospholipids show a significant decrease towards the boundary area, and in the heartwood only trace amounts can be detected. The same pattern is observed for free sterols in the sapwood; in the heartwood, however, they reach maximum values with increasing depth of the trunk. Steryl esters exhibit a complementary behaviour by accumulating at the periphery of the heartwood. No concentration changes are found in the total amounts of diacylglycerols and free fatty acids. In contrast, the triacylglycerol concentration declines steadily across the trunk. With regard to qualitative composition, free fatty acids and the fatty acid moieties of the esterified constituents vary in their chain length from 14 to 24 carbon atoms and have up to three double bonds. A radial gradient in the ratio saturated/unsaturated fatty acids can be observed: except for the phospholipid fraction the relative amounts of unsaturated fatty acids increase in centripetal direction. Seven phospholipids were identified: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and phosphatidic acid, which constitutes the major proportion. In the sterol group, sitosterol is the most abundant component. The composition of the esterified sterols remains constant across the trunk cross-section, whereas the relative frequencies of individual free sterols change markedly.