Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Evaluation of the selectivity of white rot isolates using near infrared spectroscopic techniques

Seventeen isolates from white rotted beech wood and six strains from a local culture collection were evaluated for their capability to delignify beech and spruce wood selectively. Six peroxidase-positive isolates were found using a colorimetric agar plate test (Poly R-478), and genetically identified by their internal transcribed spacer (ITS1) or 28S rDNA sequences. Colonised on beech and spruce wood veneers, some of the peroxidase-positive isolates caused selective white rot on both wood species. Weight loss and lignin content of the degraded veneers were estimated from FT-NIR spectra with established linear regression models and multivariate models based on partial least squares regression (PLSR). Weight loss of the samples was also determined gravimetrically. A measure for the relative selectivity of the strains for lignin degradation was formulated and the values were calculated. Two strains that were identified as Oxyporus latemarginatus and Trametes cervina exhibited high selectivity on spruce wood, but the lignin content of the decayed wood was higher than that degraded by the reference strain Ceriporiopsis subvermispora. One strain – identified as Phlebia tremellosa – led to a lower lignin content of beech wood but caused also comparably high weight loss and thus exhibited an overall lower selectivity. The NIR spectroscopic method proved to be convenient for the quick screening of selective white rot fungi. Furthermore, the results revealed that high selectivity for lignin degradation is much more pronounced in early degradation stages.     

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.     

Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.     

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.     

Extracellular Enzyme Activity Associated with Degradation of Beech Wood in a Central European Stream

The degradation of beech wood (Fagus sylvatica L.) was followed over 16 months in a central European upland stream, the Breitenbach. 1 cm³ cubes of beech wood were placed on the stream bed and sampled at monthly intervals. Besides mass loss, fungal biomass (ergosterol content) and lignin content, the activity of two extracellular enzymes was measured: β‐D‐glucosidase, an enzyme involved in the degradation of cellulose, and phenoloxidase, a ligninolytic enzyme. The suitability of the fluorigenic model substrate methylumbelliferyl‐β‐D‐glucoside for measuring β‐D‐glucosidase activity in wood from aquatic environments was tested. This technique is much more sensitive than the conventional photometric method. The beech wood was degraded at a constant rate of k = 0.00272 d^–1 across the entire 16‐month incubation period. There was a rapid onset of microbial colonisation, as witnessed by the initial detection of enzyme activity, after only 7 days of exposure. Lignin and ergosterol content as well as β‐glucosidase activity reached their highest values at the end of the 16‐month incubation period. Phenoloxidase activity increased rapidly to a maximum after 6 weeks, and then decreased to almost zero by the end of the experiment. The combination of biochemical techniques for measuring extracellular enzyme activities with measurements of mass loss, chemical composition and microbial colonisation provided valuable insights into the decomposition of wood in aquatic environments.     

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.     

Decomposition of Japanese beech wood by diverse fungi isolated from a cool temperate deciduous forest

We assessed 62 fungal strains in 31 species of wood decay fungi in the ability to decompose wood blocks of Japanese beech (Fagus crenata) under a pure culture condition. Fungi were collected in a cool temperate beech forest in Japan and isolated from the inside of beech logs and from sporocarps fruiting on logs and snags of beech that were different in diameter and decay class. Fungi in Holobasidiomycetidae showed marked decomposition of lignin and carbohydrate. These fungi were divided into three groups according to the pattern of lignin and carbohydrate utilization. Phanerochaete filamentosa decomposed lignin selectively. Lampteromyces japonicus, Steccherinum rhois, Trichaptum biforme, Stereum ostrea, Mycena haematopoda, Antrodiella albocinnamomea, Daedalea dickinsii, Daedaleopsis tricolor, Ganoderma tsunodae, and Trametes versicolor decomposed lignin and carbohydrates simultaneously. Psathyrella candolleana, Lenzites betulinus, and Trametes hirsuta decomposed carbohydrates selectively. Species in the Phragmobasidiomycetidae and in the Ascomycota caused low mass loss of wood.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Linoleic acid peroxidation initiated by Fe-reducing compounds recovered from Eucalyptus grandis biotreated with Ceriporiopsis subvermispora

This work evaluates linoleic acid peroxidation reactions initiated by Fe³⁺-reducing compounds recovered from Eucalyptus grandis, biotreated with the biopulping fungus Ceriporiopsis subvermispora. The aqueous extracts from biotreated wood had the ability to reduce Fe³⁺ ions from freshly prepared solutions. The compounds responsible for the Fe³⁺-reducing activity corresponded to UV-absorbing substances with apparent molar masses from 3 kDa to 5 kDa. Linoleic acid peroxidation reactions conducted in the presence of Fe³⁺ ions and the Fe³⁺-reducing compounds showed that the rate of O₂ consumption during peroxidation was proportional to the Fe³⁺-reducing activity present in each extract obtained from biotreated wood. This peroxidation reaction was coupled with in-vitro treatment of ball-milled E. grandis wood. Ultraviolet data showed that the reaction system released lignin fragments from the milled wood. Size exclusion chromatography data indicated that the solubilized material contained a minor fraction representing high-molar-mass molecules excluded by the column and a main low-molar-mass peak. Overall evaluation of the data suggested that the Fe³⁺-reducing compounds formed during wood biodegradation by C. subvermispora can mediate lignin degradation through linoleic acid peroxidation.     

Characterization of white zones produced on Pinus radiata wood chips by Ganoderma australe and Ceriporiopsis subvermispora

White zones produced on biodegraded Pinus radiata wood chips were characterized by micro-localized-FTIR (Fourier Transformed Infra Red) spectroscopy and scanning electron microscopy. Both techniques permitted assignment of the white zones to a selective lignin removal process. Although both fungi studied have degraded lignin selectively in these restricted superficial areas, chemical analysis of the wood chips indicated that Ganoderma australe removed 16% of the initial amount of glucan at the 20% weight loss level. Ceriporiopsis subvermispora did not remove glucan at weight loss values below 17%. Prolonged biodegradation resulted in reduction of white zones by G. australe, and increased white zones from C. subvermispora decayed samples. This revised version was published online in July 2006 with corrections to the Cover Date.     

Physiological Aspects of Biosynthesis of Lignin Peroxidases by Phanerochaete chrysosporium

Methods based on UV-visible diffuse reflectance spectroscopy were used to study the physiological aspects of lignin-peroxidase biosynthesis by Phanerochaete chrysosporium. Here we introduce the use of cytochrome aa₃ as an indicator of active fungal biomass and of its redox state to calculate the oxygen mass transport coefficient between the growth medium and the fungal cell interior. When lignin peroxidase biosynthesis was enhanced by the addition of Tween 80 or Tween 20 to the growth medium, a higher proportion of reduced cytochrome aa₃ and a higher oxygen diffusion barrier were observed compared with control cultures. In cultures supplemented with Tween 80 or Tween 20, a higher oxygen mass transport coefficient between the growth medium and the interior of the fungal cell was also found. The beginning of the lignin peroxidase activity in these cultures was found to coincide with a temporary cessation in the dry biomass increase and a reduction in the relative active-biomass concentration. During the lignin peroxidase activity, a decrease in the intracellular pH and an increase in the growth medium pH were determined in cultures supplemented with Tween 80.     

Extracellular Enzyme Production and Synthetic Lignin Mineralization by Ceriporiopsis subvermispora

The ability of the white rot fungus Ceriporiopsis subvermispora to mineralize ¹⁴C-synthetic lignin was studied under different culture conditions, and the levels of two extracellular enzymes were monitored. The highest mineralization rates (28% after 28 days) were obtained in cultures containing a growth-limiting amount of nitrogen source (1.0 mM ammonium tartrate); under this condition, the levels of manganese peroxidase (MnP) and laccase present in the culture supernatant solutions were very low compared with cultures containing 10 mM of the nitrogen source. In contrast, cultures containing a limiting concentration of the carbon source (0.1% glucose) showed low levels of both enzymes and also very low mineralization rates compared with cultures containing 1% glucose. Cultures containing 11 ppm of Mn(II) showed a higher rate of mineralization than those containing 0.3 or 40 ppm of this cation. Levels of MnP and laccase were higher when 40 ppm of Mn(II) was used. Mineralization rates were slightly higher in cultures flushed daily with oxygen, whereas laccase levels were lower and MnP levels were approximately the same as in cultures maintained under an air atmosphere. The presence of 0.4 mM veratryl alcohol reduced both mineralization rates and MnP levels, without affecting laccase levels. Lignin peroxidase activity was not detected under any condition. Addition of purified lignin peroxidase to the cultures in the presence or absence of veratryl alcohol did not enhance mineralization rates significantly.     

Selective Delignification of Aspen Wood Blocks In Vitro by Three White Rot Basidiomycetes

Aspen wood blocks were selectively delignified in the laboratory by Ischnoderma resinosum, Poria medulla-panis, and Xylobolus frustulatus. After 8 weeks only the outer surfaces of wood blocks were selectively delignified. The percentages of weight loss obtained after 4, 8, and 12 weeks showed that decay occurred at a relatively constant rate. Selectively delignified wood could be identified by using scanning electron microscopy only when lignin had been extensively removed from cell walls. X. frustulatus was able to form pockets of delignified wood throughout blocks after 12 weeks.     

Lignocellulose Degradation during Solid-State Fermentation: Pleurotus ostreatus versus Phanerochaete chrysosporium

Lignocellulose degradation and activities related to lignin degradation were studied in the solid-state fermentation of cotton stalks by comparing two white rot fungi, Pleurotus ostreatus and Phanerochaete chrysosporium. P. chrysosporium grew vigorously, resulting in rapid, nonselective degradation of 55% of the organic components of the cotton stalks within 15 days. In contrast, P. ostreatus grew more slowly with obvious selectivity for lignin degradation and resulting in the degradation of only 20% of the organic matter after 30 days of incubation. The kinetics of ¹⁴C-lignin mineralization exhibited similar differences. In cultures of P. chrysosporium, mineralization ceased after 18 days, resulting in the release of 12% of the total radioactivity as ¹⁴CO₂. In P. ostreatus, on the other hand, 17% of the total radioactivity was released in a steady rate throughout a period of 60 days of incubation. Laccase activity was only detected in water extracts of the P. ostreatus fermentation. No lignin peroxidase activity was detected in either the water extract or liquid cultures of this fungus. 2-Keto-4-thiomethyl butyric acid cleavage to ethylene correlated to lignin degradation in both fungi. A study of fungal activity under solid-state conditions, in contrast to those done under defined liquid culture, may help to better understand the mechanisms involved in lignocellulose degradation.     

Microbial Delignification with White Rot Fungi Improves Forage Digestibility

Three wild-type white rot fungi and two cellulase-less mutants developed from Phanerochaete chrysosporium K-3 (formerly Sporotrichum pulverulentum) were tested for their ability to delignify grass cell walls and improve biodegradation by rumen microorganisms. Fungal-treated and control stems of Bermuda grass were analyzed for their content of ester- and ether-linked aromatics by using alkali extraction and gas chromatography, for in vitro dry weight digestion and production of volatile fatty acids in in vitro fermentations with mixed ruminal microorganisms, for loss of lignin and other aromatics from specific cell wall types by using microspectrophotometry, and for structural changes before and after in vitro degradation by rumen microorganisms by using transmission electron microscopy. P. chrysosporium K-3 and Ceriporiopsis subvermispora FP 90031-sp produced the greatest losses in lignin and improved the biodegradation of Bermuda grass over that of untreated control substrate. However, C. subvermispora removed the most lignin and significantly improved biodegradation over all other treatments. Phellinus pini RAB-83-19 and cellulase-less mutants 3113 and 85118 developed from P. chrysosporium K-3 did not improve the biodegradation of Bermuda grass lignocellulose. Results indicated that C. subvermispora extensively removed ester-linked p-coumaric and ferulic acids and also removed the greatest amount of non-ester-linked aromatics from plant cell walls. Microscopic observations further indicated that C. subvermispora removed esters from parenchyma cell walls as well as esters and lignin from the more recalcitrant cell walls (i.e., sclerenchyma and vascular tissues). C. subvermispora improved in vitro digestion and volatile fatty acid production by ruminal microorganisms by about 80%, while dry matter loss due to fungi was about 20% greater than loss in untreated control stems. The chemical and structural studies used identified sites of specific fungal attack and suggested mechanisms whereby improvement occurred.     

Anthraquinone dyes decolorization capacity of anamorphic Bjerkandera adusta CCBAS 930 strain and its HRP-like negative mutants

Cultures of the anamorphic fungus Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930.     

Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete Ceriporiopsis subvermispora

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier Ceriporiopsis subvermispora when grown on a cellulose- and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one cdh gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of C. subvermispora CDH is cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.White rot fungi are the most efficient lignocellulose degraders in our ecosystem, and several species, e.g., Phanerochaete chrysosporium, Trametes versicolor, and Ceriporiopsis subvermispora, have been studied in great detail as model organisms for this complex process. The ability to degrade phenolic and nonphenolic lignin structures in wood has made these strains attractive for biotechnological applications mainly in the pulp and paper industry, where C. subvermispora exhibits a substantial advantage over P. chrysosporium and T. versicolor through its ability for selective removal of a large fraction of lignin without attacking the valuable cellulose (16, 38). The lignin-degrading system of these fungi is composed of extracellular enzymes together with low-molecular-mass cofactors (21, 46). Typically found ligninolytic enzymes are lignin peroxidase, manganese peroxidase (MnP), and laccase. The secretion pattern of these enzymes varies greatly in white rot fungi (22) and is influenced by culture conditions and medium composition. Whereas P. chrysoporium secretes high lignin and manganese peroxidase activities but no laccase activity (32, 33), C. subvermispora produces several MnP and laccase isoforms but no lignin peroxidase. T. versicolor is the only one of these model organisms known so far to express all three of these ligninolytic enzymes efficiently (5). Together with the cellulolytic enzyme system, these patterns of enzyme activities cause varied degrees of lignin and cellulose breakdown at different cultivation stages. The simultaneous attack of cellulose and lignin is the preferred strategy of T. versicolor, whereas C. subvermispora is a selective delignifier in the first stages of biotreatment, secreting only low activities of cellulolytic enzymes at a late culture stage (12, 23), and apparently lacks cellobiohydrolase activity (23).Cellobiose dehydrogenase (CDH; EC1.1.99.18; cellobiose (acceptor) 1-oxidoreductase) is an extracellular flavocytochrome secreted by some white rot and brown rot plant pathogenic and saprotrophic fungi from the dicaryotic phyla Basidiomycota and Ascomycota (50). It shows a strong preference for cellobiose and cello-oligosaccharides, which are oxidized to the corresponding lactones during the reductive half-reaction of the FAD cofactor, and further hydrolyze to aldonic acids in the bulk water. In the oxidative half-reaction FAD transfers two reduction equivalents to either one two-electron acceptor, e.g., various quinones, or to two one-electron acceptors, like complexed Fe(III) or Mn(II) ions. At low pH values (usually below 5.5), the heme cofactor can be involved in the electron transfer to one-electron acceptors. Even though CDH has been studied for a considerable time, the exact role and function of the two prosthetic groups are not fully understood. The pH optima with most electron acceptors are rather acidic, but oxygen, although a poor electron acceptor, is also reduced to H₂O₂ under neutral and alkaline conditions (30).In recent years CDH was shown to participate in the ligninolytic or cellulolytic metabolism of white rot fungi (3, 10, 24, 26, 50). The currently favored mechanism is the production of hydroxyl radicals through Fenton reaction chemistry by the ability of CDH to reduce Fe³⁺ to Fe²⁺ and to produce H₂O₂ (28, 31, 36, 37). CDH is believed to be involved in early stages of cellulose breakdown: knocking out the cdh gene in T. versicolor did not considerably affect its ability to grow on amorphous cellulosic substrates, while it could not grow on crystalline cellulose or recalcitrant substrates such as birch wood (13).Interestingly, no CDH activity has been reported so far from cultures of C. subvermispora, even though it is closely related to other white rotters producing this enzyme, e.g., Trametes spp. (35, 41) or Pycnoporus cinnabarinus (45). It has been speculated that the lack of CDH might contribute to the selectivity of C. subvermispora in degrading lignin while growing on wood. It was therefore the aim of our study to show unequivocally whether C. subvermispora carries a cdh gene and can produce the enzyme under certain growth conditions.     

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.     

Degradation of fluorene,anthracene, phenanthrene,fluoranthene, and pyrene lacks connection to the production of extracellular enzymes by Pleurotus ostreatus and Bjerkandera adusta

The degradation of polycyclic aromatic hydrocarbons (PAH) was studied in liquid cultures of Bjerkandera adusta and Pleurotus ostreatus during 7 weeks of cultivation. During only 3 days of incubation, B. adusta removed 56% and 38% of fluorene and anthracene, while P. ostreatus degraded 43% and 60% of these compounds; other PAH were degraded to a lower extent. Except for anthracene in cultures of P. ostreatus, all PAH were removed uniformly during the cultivation time but fluorene and anthracene were degraded faster than other PAH. Supplementation of liquid cultures with milled wood decreased the concentration of PAH in the solution and diminished the degradation of PAH. The fungi produced valuable activity of manganese-dependent peroxidase; laccase was secreted only by P. ostreatus and was strongly induced by the addition of milled wood. The production of the oxidative enzymes did not correlate directly to the metabolisation of PAH.