Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1–6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.     

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.     

Biopulping of lignocellulosic material using different fungal species: a review

Biopulping can be an alternative to the traditional methods of pulping. Biopulping use fungi that are known to be able to degrade wood as well as lignin constituent of wood. Amongst these white rot fungi are the most proficient biodegrader. The fungus is non sporulating and is a selective lignin degrader. It colonizes either on living or dead wood and decomposes all wood polymers including lignin and extractives making it to be extremely potential to be used in biopulping. The process of biopulping reduces the utilization of chemical in pulping industry and help in decreasing the environmental hazard caused by normal pulping. The present review deals with diverse aspects of biopulping and their ecological as well as economic significances.     

Lignin biodegradation and ligninolytic enzyme studies during biopulping of Acacia mangium wood chips by tropical white rot fungi

White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20–30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02–26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.     

Comparative studies of loblolly pine biodegradation and enzyme production by Argentinean white rot fungi focused on biopulping processes

The ability of eight white rot fungi: Coriolopsis rigida, Coriolus versicolor var. antarcticus, Peniophora sp., Phanerochaete sordida, Pycnoporus sanguineus, Steccherinum sp., Trametes elegans and Trametes villosa to selectively delignify loblolly pine (Pinus taeda) chips was studied. They were selected among 34 basidiomycetes from Argentina because of their capacity to decolorize Poly R-478 and Azure B. Fungal pretreatment caused changes in wood chemical composition as well as in physical structure. The present study allowed the identification of a new strain, potentially a candidate for use in softwoods biopulping processes. Results showed that P. sanguineus was able to reduce lignin content in 11% in 14 days of treatment, but also that P. taeda wood suffered notable structural changes of lignin and hemicelluloses during the treatment, as revealed from ¹³C CP-MAS NMR spectra. An increase of 15% in porosity of decayed wood confirmed physical changes due to fungal attack.     

Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. subvermispora with P. ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. ostreatus with C. subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. ostreatus with C. subvermispora or with P. rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications.     

Quantitative trait locus (QTL) analysis of wood quality traits in <Emphasis Type="Italic">Eucalyptus nitens</Emphasis>

To identify the chromosomal regions affecting wood quality traits, we conducted a genome-wide quantitative trait locus (QTL) analysis of wood quality traits in Eucalyptus nitens. This information is important to exploit the full potential of the impending Eucalyptus genome sequence. A three generational mapping population consisting of 296 progeny trees was used to identify QTL associated with several wood quality traits in E. nitens. Thirty-six QTL positions for cellulose content, pulp yield, lignin content, density, and microfibril angle (MFA) were identified across different linkage groups. On linkage groups (LG)2 and 8, cellulose QTL cluster with pulp yield and extractives QTL while on LG4 and 10 cellulose and pulp yield QTLs cluster together. Similarly, on LG4, 5, and 6 QTL for lignin traits were clustered together. At two positions, QTL for MFA, a physical trait related to wood stiffness, were clustered with QTL for lignin traits. Several cell wall candidate genes were co-located to QTL positions affecting different traits. Comparative QTL analysis with Eucalyptus globulus revealed two common QTL regions for cellulose and pulp yield. The QTL positions identified in this study provide a resource for identifying wood quality genes using the impending Eucalyptus genome sequence. Candidate genes identified in this study through co-location to QTL regions may be useful in association studies.     

Enhanced delignification of mixed wood pulp by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> laccase mediator system

An environmentally sound biobleaching to get high quality paper pulp from mixed wood pulp was attempted employing laccase from Aspergillus fumigatus VkJ2.4.5 for lignin removal. Laccase treatment was performed in the presence of a mediator N-hydroxybenzotriazole (HBT, 1.5% w/w), resulting into notably higher level of delignification of the pulp. Enzyme at 10 Ug⁻¹ of pulp at 50°C, pH 6.0, for 2 h with a pulp consistency of 10% was found suitable for enabling maximum decrease in the kappa number. The kappa number and yellowness decreased by 14 and 4% whereas ISO brightness improved by 7%. The presence of a characteristic peak at 280 nm indicated the presence of lignin in the effluent during biobleaching. Analysis of FTIR spectra of residual lignin revealed characteristic modifications following enzymatic bleaching by laccase mediator system (LMS). Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis.     

Screening of a fungus capable of powerful and selective delignification on wheat straw

Aims: To screen and characterize a novel fungus with powerful and selective delignification capability on wheat straw. Methods and Results: A fungus capable of efficient delignification under solid‐state fermentation (SSF) conditions on wheat straw was screened. After 5 days of incubation, 13·07% of the lignin was removed by fungal degradation, and 7·62% of the holocellulose was lost. Furthermore, 46·53% of the alkali lignin was removed after 2 days of liquid fermentation. The fungus was identified as Fusarium concolor based on its morphology and an analysis of its 18S rDNA gene sequence. The molecular weight distribution of lignin was evaluated by gel permeation chromatography. Enzyme assay indicated that the fungus produced laccase, cellobiose dehydrogenase, xylanase and cellulase during the incubation period. Intracellular lignin peroxidase, manganese peroxidase and laccase were produced during liquid fermentation. Conclusions: We have successfully screened a fungus, F. concolor, which can efficiently degrade the lignin of wheat straw, with slight damage to the cellulose, after 5 days of SSF. Significance and Impact of the Study: The newly isolated strain could be used in pretreatment of lignocellulose materials prior to biopulping, bioconversion into fuel and substrates for the chemical industry.     

Effect of physical and physicochemical pretreatments of wood for SCP production with Chaetomium cellulolyticum

Relatively poor SCP production (4.2 mg/L h) was obtained using C. cellulolyticum and ground aspen wood treated with steam at atmospheric pressure for 1 h. The percentage of protein in the final product increased to 21.4% at a specific growth rate of 0.15 h^?1 when the wood sample was treated with steam at a higher pressure (280 psig for 4 min) according to the "Stake" process. Alkali treatment (10% and 15% w/w at 121°C for 30 min), known to solubilize hemicelluloses and some of the lignin, gave intermediate results. More complete delignification of wood using NaClO₂ increased the protein composition in the final product to 37.9%, at a specific growth rate of 0.19 h^?1. Cellulose utilization was lowest (12.4%) in the case of the wood treated with steam at atmospheric pressure; it was higher at 75.3 and 78.5% for wood treated with NaOH at 10 and 15% w/w levels, respectively. The cellulose utilization was highest (90%) for wood treated with NaClO₂.     

Characterisation of the initial degradation stage of Scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) sapwood after attack by brown-rot fungus <Emphasis Type="Italic">Coniophora puteana</Emphasis>

In our study, early period degradation (10 days) of Scots pine (Pinus sylvestris L.) sapwood by the brown-rot fungus Coniophora puteana (Schum.: Fr.) Karst. (BAM Ebw.15) was followed at the wood chemical composition and ultrastructurelevel, and highlighted the generation of reactive oxygen species (ROS). An advanced decay period of 50 days was chosen for comparison of the degradation dynamics. Scanning UV microspectrophotometry (UMSP) analyses of lignin distribution in wood cells revealed that the linkages of lignin and polysaccharides were already disrupted in the early period of fungal attack. An increase in the lignin absorption A₂₈₀ value from 0.24 (control) to 0.44 in decayed wood was attributed to its oxidative modification which has been proposed to be generated by Fenton reaction derived ROS. The wood weight loss in the initial degradation period was 2%, whilst cellulose and lignin content decreased by 6.7% and 1%, respectively. Lignin methoxyl (–OCH₃) content decreased from 15.1% (control) to 14.2% in decayed wood. Diffuse reflectance Fourier-transform infrared (DRIFT) spectroscopy corroborated the moderate loss in the hemicellulose and lignin degradation accompanying degradation. Electron paramagnetic resonance spectra and spin trapping confirmed the generation of ROS, such as hydroxyl radicals (HO^∙), in the early wood degradation period. Our results showed that irreversible changes in wood structure started immediately after wood colonisation by fungal hyphae and the results generated here will assist in the understanding of the biochemical mechanisms of wood biodegradation by brown-rot fungi with the ultimate aim of developing novel wood protection methods.     

Evaluation of the Experimental Inoculation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis> and <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37°C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.     

Studies of growth conditions in wood for three white-rot fungi and their cellulaseless mutants

White-rot fungi, which have the ability to degrade all the wood components including lignin, are of great interest in biotechnological processes based on wood and other lignocellulosic materials. It was demonstrated earlier that enough lignin can be degraded to cause a decrease in the energy demand for production of thermomechanical pulp if wood chips are pretreated by cellulaseless mutants of white-rot fungi. This paper concerns the growth conditions in wood for three white-rot fungi and their cellulaseless mutants in order to determine optimal conditions for such pretreatment processes. The pH and temperature optima have been determined as well as the growth rate in wood. The results show that the growth rate in wood. at least for Cel 44 (a cellulaseless mutant of Sporotrichum pulverulentum), is not the rate-limiting step in delignification. From different mixtures of urea and NH₄H₂PO₄ the optimal nitrogen source was determined for the mutants. The optimal C/N ratio was found to vary between 160/1 and 400/1. It is suggested that the lower the C/N ratio, the faster the growth. It was also demonstrated that both water- and acetone-extractable substances in wood supported the growth of cellulaseless mutants. When some glucose was added to the wood, the weight loss caused by Cel 44 increased. All these observations support earlier findings that lignin in wood cannot be degraded by white-rot fungi unless a more easily metabolizable carbon source is used simultaneously.     

Laboratory studies on the life-histories of four enchytraeid worms (Oligochaeta) which inhabit sewage percolating filters

The life-histories of four enchytraeid worms, Lumbricillus rivalis, Enchy-traeus coronatus, E. buchholzi, and E. albidus which occur in sewage percolating filters, were studied under laboratory conditions at 8 , 15 and 20°C. The number of ova per cocoon varied from 0 to 50 (L. rivalis), 0 to 33 (E. coronatus), 1 to 9 (E. buchholzi) and 0 to 22 (E. albidus). The mean number of ova per cocoon was highest at 15°C for all species except E. coronatus which had a highest mean value at 8°C. The number of ova in cocoons was correlated with cocoon length (P < 0.001) for all species. Cocoon production usually increased with temperature ranging from 0.8 cocoons per adult per week at 8°C to 2.0 at 20°C for L. rivalis, and from 1–4 to about 2.6 for E. coronatus and E. buchholzi. The total number of ova produced by each E. coronatus (350 at 8°C to 550 at 20°C) was similar to that produced by each L. rivalis (600 at 8°C to 350 at 20°C) and was about five times greater than the total numbers produced by the other two species. Cocoon and ova production and the number of ova per cocoon varied with the age of the adult, usually reaching a peak soon after maturity. Hatching success was low and generally 40–50 % of ova failed to develop; subsequent mortality among immature worms was about 10–20%. Growth was more rapid at the higher temperatures; L. rivalis matured in about 26 days at 20°C, the clitellum forming when the worm was 13–14 mm long; data for the other species are 13 days and 5–6 mm (E. coronatus); 16 days and 3–4 mm (E. buchholzi); 28 days and 13–14 mm (E. albidus). The maturation period at 8°C was at least twice that at 20°C. The generation period (cocoon to cocoon) was about a month at 20°C for all species except E. albidus (2 months), but as some species had longer reproductive periods than others the actual number of generations per year was highest in E. buchholzi, 7.0 per year, and lowest in E. albidus, about 3.3 per year, At 8°C all four species had between 1.4 and 2.8 generations a year. A comparison of expected and observed population densities of L. rivalis and E. coronatus in a sewage percolating filter showed that neither achieved values approaching their potential summer densities although ample food was apparently available. Of the four species studied only E. buchholzi produced viable ova without pairing.     

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

An extracellular xylanase produced under optimal conditions by a thermophilic strain of Bacillus sp. XTR-10 was evaluated for its potential application in biobleaching of wood kraft pulp. Spectrophotometric analysis showed considerable release of lignin derived compounds and chromophoric material by the xylanase treated pulp samples. Xylanase was found to be effective in the liberation of reducing sugars in the pulp filtrates with increment in enzyme dose and reaction time. Eight hours pretreatment with 40 IU of xylanase/g of dry pulp resulted in 16.2% reduction of kappa number with 25.94% ISO increase in brightness as compared to the control. The same treatment slightly lowered the tensile strength and burst index, however. Enzyme pretreatment of the pulp saved 15% active chlorine charges in single step and 18.7% in multiple steps chemical bleaching with attainment of brightness at the level of the control. These results indicate the potential of enzymatic pretreatment of pulp for reduction in environmental discharge of hazardous waste from the pulp and paper industry.     

Biodegradation of eucalyptus urograndis wood by fungi

We focused in selecting four fungi, naturally living in Eucalyptus sp. fields, for application in accelerating stump decay. The wood-rot fungi Pycnoporus sanguineus (Ps), Lentinus bertieri (Lb) and Xylaria sp. (Xa) were isolated from Eucalyptus sp. field and the fungus Lentinula edodes (Led) was obtained from a commercial strain. All fungi were studied according to their capacity to degrade eucalyptus urograndis wood. In order to evaluate mass losses of seven years old eucalyptus urograndis' wood test blocks from heartwood were prepared added to glass flasks with red clay soil. The humidity of the soil was adjusted with 50 and 100% of its water retention capacity. Mass loss evaluations occurred at 30 until 120 days after eucalyptus wood degradation. Chemical analysis and soil pH were measured only in the last evaluation. Mycelial growth assays with potato-dextrose-agar, malt-agar and sawdust-dextrose-agar at three temperatures was carried out in order to get information about the best conditions of fungi growth. On the 120^th day, Ps and Lb showed good capacity of wood degradation by leading to a high mass loss in soil with highest humidity. These fungi were the best consumers of lignin, hemicellulose, cellulose and extractives, caused acidification in the soil. Ps and Lb had faster mycelial growth in sawdust-dextrose-agar, especially in high temperature, comparing to Xa and Led. Xa and Led are not good eucalyptus urograndis heartwood degraders, because they consume preferentially hemicellulose.     

Biodegradation of Chilean native wood species,Drimys winteri and Nothofagus dombeyi,by Ganoderma australe

Drimys winteri and Nothofagus dombeyi, two native Chilean wood species with high potential for pulp production, were biodegraded by Ganoderma australe. This fungus is known to provoke extensive and selective biodelignification of these wood species in the field. Under laboratory conditions, N. dombeyi underwent higher weight and component losses than D. winteri. In neither case was the lignin removal selective, because glucan loss was almost simultaneous with lignin degradation. The decayed wood chips became progressively discoloured throughout the biodegradation time. The brightness increase was only partly reversed in thermal reversion assays. Nothofagus dombey solubility in 1% NaOH increased by 13.7% after 9 weeks of biodegradation, while D. winteri solubility increased by 14.2% in a shorter period (6 weeks). In both cases, the solubility increase was proportional to the liquor absorbance increase at 272 nm, which indicates that the wood solubility in 1% NaOH was dependent of lignin solubilization.     

Two-stage fungal biopulping for improved enzymatic hydrolysis of wood

A novel two-stage, whole organism fungal biopulping method was examined for increasing the yield of enzymatic hydrolysis of wood into soluble glucose. Liriodendron tulipifera wood chips (1 g) were exposed to liquid culture suspensions of white rot (Ceriporiopsis subvermispora) or brown rot (Postia placenta) fungi and incubated at 28 °C, either alone in single-stage 30 day (one fungal species applied) or two-stage 60 day (both fungal species applied in alternative succession) treatments. Fungi grew in all treatments, but did not significantly decrease the percent carbohydrate content of the wood. Two-stage treatments differed significantly in mass loss depending on order of exposure, suggesting additive or inhibitory fungal interactions occurred. Treatments consisting of C. subvermispora followed by P. placenta exhibited 6 ± 0.5% mass loss and increased the yield of enzymatic hydrolysis by 67-119%. This significant hydrolysis improvement suggests that fungal biopulping technologies could support commercial lignocellulosic ethanol production efforts if further developed.