Pre-treatment of Pinus radiata substrates by basidiomycetes fungi to enhance enzymatic hydrolysis

Pre-treatment is important step prior to enzymatic hydrolysis of ligno-cellulosic biomass in order to obtain renewable carbon source ca. glucose. Pinus radiata biomass including wood blocks, wood chips and steam exploded wood (SEW) were used to investigate the effect of fungal pre-treatment on glucose yield. Comparison was made using one white-rot fungus (Trametes versicolor) and three brown-rot fungi (Coniophora puteana, Antrodia xantha and Oligoporus placenta). This is the first study where SEW was treated with basidiomycetes and subsequent enzymatic hydrolysis gave 5 g glucose/l which is an order of magnitude greater compared to control biomass (0.5 g glucose/l). This enhanced glucose yield is due to the novel pre-treatment sequence used in this study.     

Biological degradation of resin acids in wood chips by wood-inhabiting fungi.

Resin acids in many pulp mill effluents are primary sources of toxicity to fish. Inconsistent biological detoxification of chlorinated and nonchlorinated resin acids in secondary treatment of pulp mill effluents is a continuing source of concern. An alternative approach to effluent detoxification is to remove or modify the toxic compounds present in wood chips prior to pulping. Results from experiments in which lodgepole pine sapwood chips were inoculated with several fungal candidates indicate that the total resin acid content can be reduced by up to 67% after fungal growth. Such a treatment could be an efficient and environmentally acceptable way for deresinating wood chips and so decreasing the toxicity of pulp mill effluents.     

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.     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

The effects of fungi pre-treatment of poplar chips on the kraft fiber properties

Poplar chips were pre-treated by Trametes versicolor for 1, 2 and 3 weeks. Pre-treated chips, after washing, have been air dried for kraft pulping to achieve pulp kappa number of about 20. Pulp samples have been analyzed by Bauer Mc Nett, Kajaani analyzer and SEM. The results indicated that fungi pre-treatment of chips can degrade lignin and carbohydrates and affect kraft pulping and fiber characteristics. Higher chemical charge in pulping, lower fine and higher long fiber fraction were observed in pre-treated pulp samples in comparison with others. Fiber length, cross sectional area, width, cell wall thickness and volume index were increased by increasing pre-treatment time while fine length, fiber coarseness and curl have been reduced. Based on the study findings, with respect to higher fiber length, lower fine, and lower fiber curl and coarseness, 2-weeks pre-treatment of chips was recommended to produce acceptable overall fiber properties in kraft pulping.     

Evaluating biopulping as an alternative application on oil palm trunk using the white-rot fungus Trametes versicolor

The objective of the research was to investigate the suitability of the white-rot fungus Trametes versicolor for biopulping using oil palm biomass as the substrate. Fungi are grown on solid-state cultures Kirk's enrichment media to determine the lignocellulolytic activities. Samples subjected to fungal pretreatment for periods of 1, 2, 3, and 4 weeks were investigated and compared to the untreated control. The crude enzyme extracts were assayed using specific substrates and enzyme activities were calculated. The highest level of laccase activity was 218.66 U/L; the peak activity of manganese peroxidase was 162.10 U/L, and lignin peroxidase is 42.56 U/L. The activity levels of cellulase and hemicellulase were insignificant in all extracts (53.30 and 1.50 U/L, respectively). When the chips were pulped mechanically the Kappa number, pulp yield, and screened pulp yield decreased significantly and paper strength increased marginally with the exposure time. Hand sheet properties were also improved significantly by fungal treatment. Weight loss, lignin loss, cellulose, and holocellulose loss were 8.45%, 9.35%, 4.58%, and 7.2%, respectively. Images from SEM seem to indicate a simultaneous type of decay pattern involving cell wall breakdown combined with lignin modification. Considering all its pulping and papermaking properties, the performance of T. versicolor is good and has potential for use in large-scale biotechnological processes.     

Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.     

Fourier transform infrared microscopy and imaging: detection of fungi in wood

FTIR microscopy was used to detect and discriminate the two wood decaying fungi Trametes versicolor and Schizophyllum commune in experimentally infected beech wood blocks. The distribution of fungal mycelium in wood was locally resolved and semiquantitatively recorded using FTIR microscopy combined with a focal plane array detector and image analysis. Cluster analysis revealed major differences between FTIR spectra recorded from wood fibers and empty vessel lumina and spectra from mycelium of both fungal species, irrespective of whether the fungi were grown on the surface of wood or inside vessel lumina. Species-specific clustering of spectra of fungal mycelium grown on the wood surface and inside vessel lumina demonstrated the potential of FTIR microscopy to discriminate among fungal species decaying wood.     

Bacterial biodegradation of extractives and patterns of bordered pit membrane attack in pine wood

Wood extractives, commonly referred to as pitch, cause major problems in the manufacturing of pulp and paper. Treatment of nonsterile southern yellow pine chips for 14 days with Pseudomonas fluorescens, Pseudomonas sp., Xanthomonas campestris, and Serratia marcescens reduced wood extractives by as much as 40%. Control treatments receiving only water lost 11% of extractives due to the growth of naturally occurring microorganisms. Control treatments were visually discolored after the 14-day incubation, whereas bacterium-treated wood chips were free of dark staining. Investigations using P. fluorescens NRRL B21432 showed that all individual resin and fatty acid components of the pine wood extractives were substantially reduced. Micromorphological observations showed that bacteria were able to colonize resin canals, ray parenchyma cells, and tracheids. Tracheid pit membranes within bordered pit chambers were degraded after treatment with P. fluorescens NRRL B21432. P. fluorescens and the other bacteria tested appear to have the potential for biological processing to substantially reduce wood extractives in pine wood chips prior to the paper making process so that problems associated with pitch in pulp mills can be controlled.     

Effect of Residual Lignin Type and Amount on Bleaching of Kraft Pulp by Trametes versicolor

The white rot fungus Trametes (Coriolus) versicolor can delignify and brighten unbleached hardwood kraft pulp within a few days, but softwood kraft pulps require longer treatment. To determine the contributions of higher residual lignin contents (kappa numbers) and structural differences in lignins to the recalcitrance of softwood kraft pulps to biobleaching, we tested softwood and hardwood pulps cooked to the same kappa numbers, 26 and 12. A low-lignin-content (overcooked) softwood pulp resisted delignification by T. versicolor, but a high-lignin-content (lightly cooked) hardwood pulp was delignified at the same rate as a normal softwood pulp. Thus, the longer time taken by T. versicolor to brighten softwood kraft pulp than hardwood pulp results from the higher residual lignin content of the softwood pulp; possible differences in the structures of the residual lignins are important only when the lignin becomes highly condensed. Under the conditions used in this study, when an improved fungal inoculum was used, six different softwood pulps were all substantially brightened by T. versicolor. Softwood pulps whose lignin contents were decreased by extended modified continuous cooking or oxygen delignification to kappa numbers as low as 15 were delignified by T. versicolor at the same rate as normal softwood pulp. More intensive O₂ delignification, like overcooking, decreased the susceptibility of the residual lignin in the pulps to degradation by T. versicolor.     

Wood decomposing abilities of diverse lignicolous fungi on nondecayed and decayed beech wood

We tested the decay abilities of 28 isolates from 28 lignicolous fungal species (Basidiomycota, Ascomycota and Zygomycota) with the pure culture test. We used beech wood powder in varying moisture conditions and decay stages (nondecayed, intermediately decayed and well decayed) as substrates. The weight loss in wood powder was -0.2-17.8%. Five isolates of Basidiomycota (Bjerkandera adusta, Mycena haematopus, Omphalotus guepiniformis, Trametes hirsuta, Trametes versicolor) caused high weight losses in nondecayed wood. We detected significant effects of decay stage on weight loss in wood in most isolates tested, whereas moisture content rarely had an effect on weight loss. Among Basidiomycota and Xylariaceae in Ascomycota weight loss was greater for nondecayed wood than for intermediately and well decayed wood. In contrast four isolates in Ascomycota (Scytalidium lignicola, Trichoderma hamatum, T. harzianum, T. koningii) caused substantial weight loss in intermediately and well decayed wood, although they rarely caused weight loss in nondecayed wood. Zygomycota caused low weight loss in wood. Wood decay stages also affected decomposition of wood chemical components. Acid-unhydrolyzable residue (AUR) decomposition was reduced, whereas holocellulose decomposition was stimulated by some strains of Basidiomycota and Ascomycota in well decayed wood. T. harzianum in particular caused significant weight loss of holocellulose in well decayed wood, although this fungus caused negligible weight loss of both AUR and holocellulose in nondecayed wood. We discuss these changes in the decay patterns of AUR and holocellulose with varying wood decay stages in relation to the role of fungal decomposition of woody debris in forests.     

Determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity

Beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (DMDHEU) to increasing weight percent gains (WPG). The resistance of the treated specimens against Trametes versicolor and Coniophora puteana, determined as mass loss, increased with increasing WPG of DMDHEU. Metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (TEA) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. Both methods revealed that the fungal activity was related with the WPG and the mass loss caused by the fungi. Still, fungal activity was detected even in wood blocks of the highest WPG and showed that the treatment was not toxic to the fungi. Energy production showed a higher consistency with the mass loss after decay than TEA; higher mass loss was more stringently reflected by higher heat production rate. Heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide.     

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.     

Extraction of xylans from hardwood chips prior to kraft cooking

The aim of this work is to define an extraction process of hemicelluloses from hardwood chips, prior to their transformation into paper pulp by the kraft process. Eucalyptus wood chips were submitted to autohydrolysis and acid hydrolysis treatments. Autohydrolysis means that no extra acid is added during the heating of the wood suspension, whereas sulphuric acid is added for the acid hydrolysis treatment. The liquor to wood ratio was 4, and temperature and time were varied so as to optimise the hemicelluloses extraction. Quantities of xylans varying between 12 and 38 g/l of hydrolysate (or between 9 and 15% on wood) could be extracted.Kraft cooking process was applied to the pre-hydrolysed wood chips. It was shown that they were easier to delignify than untreated wood chips, and the resulting pulp was also easier to bleach. The resulting cellulosic fibres were characterised for their papermaking properties: the higher the pentosan removal, the lower the strength properties of the pulps produced.     

Evaluation of mold,decay and termite resistance of pine wood treated with zinc- and copper-based nanocompounds

In this work, the resistance of black pine wood (Pinus nigra L.) vacuum-treated with zinc oxide, zinc borate and copper oxide nanoparticles against mold and decay fungi and the subterranean termites was evaluated. Some of the nanocompounds tested were forced with acrylic emulsions to avoid leaching. Results showed that mold fungi were slightly inhibited by nanozinc borate, while the other nanometal preparations did not inhibit mold fungi. Mass loss from fungal attack by Trametes versicolor was significantly inhibited by the zinc-based preparations, while the brown-rot fungus, Tyromyces palustris was not inhibited by the nanometal treatments. Notably, nanozinc borate plus acrylic emulsion imparted very high resistance in pine wood to the white-rot fungus, T. versicolor with a mass loss of 1.8%. Following leaching, all pine specimens treated with nanozinc borate, with or without acrylic emulsion, strongly inhibited termite feeding, i.e. mass losses varying at 5.2–5.4%. In contrast, the copper-based treatments were much less effective against the subterranean termites, Coptotermes formosanus. In general, nanozinc borate possessed favorable properties, that is, inhibition of termite feeding and decay by T. versicolor.     

Evaluation of accelerated decay of wood plastic composites by Xylophagus fungi

The resistance to fungal attack of wood plastic composites (WPCs) containing 40% polypropylene and 60% either pine, maple or oak, wt%/wt%, was examined. WPCs specimens were made using the hot press system. Resistance to decay was evaluated using soil block and agar tests. Test specimens were exposed to either white-rot fungi, Trametes versicolor or Phanerochaete chrysosporium, or the brown-rot fungi Gloeophyllum trabeum or Postia placenta for six or 12 weeks. Moisture content and weight loss were used to assess the extent of decay of WPCs. Rates of decay in WPCs exposed in soil-block tests were greater than those exposed in the agar. WPCs containing either maple or oak were more susceptible to fungal attack then those containing pine.     

Assessment of wood-inhabiting Basidiomycetes for biokraft pulping of softwood chips

Wood-inhabiting Basidiomycetes have been screened for various applications in the pulp and paper industry and it is evident that different fungi need to be used to suit the specific requirements of each application. This study assessed the suitability of 278 strains of South African wood-decay fungi for the pre-treatment of softwood chips for kraft pulping. The influence of these fungi on kappa number, yield and strength properties of pulp was evaluated. A number of these strains were more efficient in reducing kappa number than the frequently used strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispora. Six strains of Stereum hirsutum and a strain of an unidentified species were able to reduce the kappa number significantly without a significant influence on the pulp yield. Treatment of wood with two strains of S. hirsutum, one strain of Peniophora sp. and a strain of an unidentified species resulted in paper with improved strength properties.     

Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system.

Biobleaching of hardwood unbleached kraft pulp (UKP) by Phanerochaete chrysosporium and Trametes versicolor was studied in the solid-state fermentation system with different culture media. In this fermentation system with low-nitrogen and high-carbon culture medium, pulp brightness increased by 15 and 30 points after 5 days of treatment with T. versicolor and P. chrysosporium, respectively, and the pulp kappa number decreased with increasing brightness. A comparison of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase activities assayed by using fungus-treated pulp and the filtrate after homogenizing the fungus-treated pulp in buffer solution indicated that enzymes secreted from fungi were adsorbed onto the UKP and that assays of these enzyme activities should be carried out with the treated pulp. Time course studies of brightness increase and MnP activity during treatment with P. chrysosporium suggested that it was difficult to correlate them on the basis of data obtained on a certain day of incubation, because the MnP activity fluctuated dramatically during the treatment time. When brightness increase and cumulative MnP, LiP, and laccase activities were determined, a linear relationship between brightness increase and cumulative MnP activity was found in the solid-state fermentation system with both P. chrysosporium and T. versicolor. This result suggests that MnP is involved in brightening of UKP by white rot fungi.     

Bioremediation of paper and pulp mill effluents

Pulp and paper mill effluents pollute water, air and soil, causing a major threat to the environment. Several methods have been attempted by various researchers throughout the world for the removal of colour from pulp and paper mill effluents. The biological colour removal process uses several classes of microorganisms--bacteria, algae and fungi--to degrade the polymeric lignin derived chromophoric material. White rot fungi such as Phanerochaete chrysosporium, Corius versicolor, Trametes versicolor etc., are efficient in decolourizing paper and pulp mill effluents. Gliocladium virens, a saprophytic soil fungus decolourised paper and pulp mill effluents by 42% due to the production of hemicellulase, lignin peroxidase, manganese peroxidase and laccase.     

Fungal inoculum properties and its effect on growth and enzyme activity of Trametes versicolor in soil

The effect of fungal inoculum properties on colonization of nonsterile soil by three isolates of the white-rot fungus Trametes versicolor was investigated. Fungal inoculum properties were examined in separate experiments and were fungal inoculum composition, age of fungal inoculum, concentration of the inoculum and inoculation method. The fungal inoculum composition study compared pine versus poplar sawdust as the basic carrier with varying amounts of corn grit, corn meal and starch. The age of the fungal inoculum studied ranged from 3 to 21 days. The inoculum concentration gradually increased from 0 to 50% (v/v). The study assessing inoculation method compared mixing with layering techniques. The effect of moisture conditions of soil, sawdust and sand in combination with two inoculation methods (mixing versus point source inoculation) on colonization by T. versicolor was also determined. Colonization of soil was always assessed visually and enzymatically monitoring mycelial growth, biological potential (fluorescein diacetate assay) and laccase levels. Generally, the three different assessment methods correlated (P < 0.05) with each other. A fungal inoculum based on pine sawdust supported white-rot fungal growth in soil better than a poplar sawdust basis. Colonization of soil by T. versicolor was improved by increasing the corn content of the fungal inoculum. Younger (<7 days old) fungal inoculum resulted in better soil colonization than older (>10 days). A strong correlation (P < 0.001) was observed between the amount of fungal inoculum used in the soil augmentation and white-rot fungal colonization of soil. Inoculation of the fungal inoculum into soil by mixing was preferable over application in layers or point source inoculation. Moisture level did not influence biological potential measurements, but affected mycelial growth and laccase expression.