Fungal biodegradation of lignopolystyrene graft copolymers.

White rot basidiomycetes were able to biodegrade styrene (1-phenylethene) graft copolymers of lignin containing different proportions of lignin and polystyrene [poly(1-phenylethylene)]. The biodegradation tests were run on lignin-styrene copolymerization products which contained 10.3, 32.2, and 50.4% (wt/wt) lignin. The polymer samples were incubated with the white rot fungi Pleurotus ostreatus, Phanerochaete chrysosporium, and Trametes versicolor and the brown rot fungus Gloeophyllum trabeum. White rot fungi degraded the plastic samples at a rate which increased with increasing lignin content in the copolymer sample. Both polystyrene and lignin components of the copolymer were readily degraded. Polystyrene pellets were not degradable in these tests. Degradation was verified for both incubated and control samples by weight loss, quantitative UV spectrophotometric analysis of both lignin and styrene residues, scanning electron microscopy of the plastic surface, and the presence of enzymes active in degradation during incubation. Brown rot fungus did not affect any of the plastics. White rot fungi produced and secreted oxidative enzymes associated with lignin degradation in liquid media during incubation with lignin-polystyrene copolymer.     

Biological pretreatment of softwood Pinus densiflora by three white rot fungi

The effects of biological pretreatment on the Japanese red pine Pinus densiflora, was evaluated after exposure to three white rot fungi Ceriporia lacerata, Stereum hirsutum, and Polyporus brumalis. Change in chemical composition, structural modification, and their susceptibility to enzymatic saccharification in the degraded wood were analyzed. Of the three white rot fungi tested, S. hirsutum selectively degraded the lignin of this sortwood rather than the holocellulose component. After eight weeks of pretreatment with S. hirsutum, total weight loss was 10.7%, while lignin loss was the highest at 14.52% among the tested samples. However, holocellulose loss was lower at 7.81% compared to those of C. lacerata and P. brumalis. Extracelluar enzymes from S. hirsutum showed higher activity of ligninase and lower activity of cellulase than those from other white rot fungi. Thus, total weight loss and changes in chemical composition of the Japanese red pine was well correlated with the enzyme activities related with lignin- and cellulose degradation in these fungi. Based on the data obtained from analysis of physical characterization of degraded wood by X-ray Diffractometry (XRD) and pore size distribution, S. hirsutum was considered as an effective potential fungus for biological pretreatment. In particular, the increase of available pore size of over 120 nm in pretreated wood powder with S. hirsutum made enzymes accessible for further enzymatic saccharification. When Japanese red pine chips treated with S. hirsutum were enzymatically saccharified using commercial enzymes (Cellulclast 1.5 L and Novozyme 188), sugar yield was greatly increased (21.01%) compared to non-pretreated control samples, indicating that white rot fungus S. hirsutum provides an effective process in increasing sugar yield from woody biomass.     

Biodegradation of γ-hexachlorocyclohexane (Lindane) by a non-white rot fungus conidiobolus 03-1-56 isolated from litter

Biodegradation of chlorinated pesticide γ-hexachlorocyclohexane (lindane) by a nonwhite rot fungus Conidiobolus 03-1-56 is reported for the first time. Conidiobolus 03-1-56, a phycomyceteous fungus isolated from litter, completely degraded lindane on the 5th day of incubation in the culture medium, and GC-ECD studies confirmed that lindane removal did not occur via adsorption on the fungal biomass. Degradation studies using different medium compositions showed that nitrogen/carbon limiting conditions (stress conditions) and presence of veratryl alcohol, induced the secretion of extracellular oxidative enzymes, which enhanced the rate of lindance biodegradation. Under optimum nutrient-limiting conditions, GC-ECD and GC-MS analysis showed complete absence of any degradation metabolite, indicating that lindane was completely mineralized. Assays for tannic acid utilization and lignin peroxidase showed similar enzymatic profiles between Conidiobolus 03-1-56 and standard white rot fungi Pleurotus ostreatus 1200 and Trametes versicolor 1086. Although Conidiobolus 03-1-56 showed a reduced enzyme activity compared to white rot fungi, preliminary evidence indicates that enzymes responsible for lignin degradation by white rots play a key role in lindane degradation by Conidiobolus 03-1-56.     

Degradation of non-phenolic lignin by the white-rot fungus Pycnoporus cinnabarinus

High-molecular-weight lignin was methylated with diazomethane. The lignin (i.e., phenolic lignin) and methylated lignin (i.e., non-phenolic lignin) were mixed with fully bleached softwood pulp. Degradation of the lignin preparations by the white rot fungus Pycnoporus cinnabarinus was studied. After a 3-month incubation with the fungus, over 40% of the non-phenolic lignin and about 70% the phenolic lignin were degraded. The presence of phenolic hydroxyl groups in lignin greatly enhanced the degradation rate of lignin. This study reveals that P. cinnabarinus, an exclusively laccase-producing fungus, is capable of oxidatively degrading both phenolic and non-phenolic lignins. The ability of the fungus to degrade non-phenolic lignin suggests that a laccase/mediator system is involved in the complete degradation of lignin. After the fungal degradation of lignins, the content of carboxylic acids substantially increased for both phenolic and non-phenolic lignins.     

白腐菌的研究进展及其在重金属修复中的展望

白腐菌是一类特殊的丝状真菌,能降解多种污染物质,具有广谱、彻底、高效、无专一性的 特点,在生物修复中有广阔的应用前景。综述了白腐菌的分类、酶系、降解机理以及应用于有机 物污染的研究现状,特别介绍了白腐菌在重金属污染的生物修复的应用进展情况,包括白腐菌吸 附重金属的原理、在重金属污染的废水中的研究应用现状及在修复重金属污染土壤中需考虑的 因素。同时展望了白腐菌在重金属污染及复合污染的生物修复中的应用前景。     

Comparison of methods to evaluate the potential of fungal growth on decay of spruce wood after short-time treatment

Several analytical methods were compared to evaluate characteristic wood decaying fungi for their potential to depolymerise lignin on spruce wood particles. Wood samples were treated with the white rot fungi Phlebia brevispora, Ceriporiopsis subvermispora, Merulius tremellosus, Pycnoporus sanguineus, Trametes pubescens and with the brown rot fungus Gloeophyllum trabeum. The UV absorbancies of crude ethanol extracts, total extractives content from sequential extraction, ligninolytic enzyme activities, lignin solubilisation and decrease of lignin content were compared. It was shown, that, in early decay stages, UV absorbancies of crude ethanol extracts and total extractives content correlate well with lignin degradation, increase of acid soluble lignin and increased production of ligninolytic enzymes (total peroxidase). Lignin content was determined using FT-NIR spectroscopy as well as by wet-chemical analysis, indicating a very good correlation between the two methods. According to the different analytical methods, the tested fungi can be classified into three categories based on their characteristic behaviour: brown rot, “slow” and “fast” white rot.     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.     

Improvement of dry matter digestibility of water hyacinth by solid state fermentation using white rot fungi

Feeding value of water hyacinth biomass colonized by three species of white rot fungi during solid-state fermentation was investigated. All three organisms proved to be efficient degraders and enhanced dry matter digestibility. Loss of organic matter was maximum (23.6+/-0.1% dry wt) after 48 days by P. ostreatus. C. indica showed maximum cellulose degradation (18.5+/-0.1% dry wt) than other two fungi after 48 days of incubation. In all cases, an extensive removal of hemicellulose at the initial growth period and a delayed degradation of lignin were observed. Hemicellulolysis was maximum (46.3+/-0.1% dry wt) by C. indica, but delignification (14.2+/-0.2% dry wt) by P. sajor-caju after 48 days. The amount of reducing sugar in the degraded biomass decreased at early stages, but increased as degradation progressed in all three cases (maximum 1.1+/-0.05% dry wt after 48 days by C. indica). Soluble nitrogen content increased only during 16-32 days of incubation (highest 1.1+/-0.1% dry wt after 32 days by P. sajor-caju). Crude protein of the bioconverted biomass increased gradually up to 32 days but decreased thereafter (maximum 10.3+/-0.1% dry wt after 32 days by P. sajor - caju). Per cent change in in vitro dry matter digestibility of degraded substrates enhanced gradually after 8 days and reached maximum after 32 days but thereafter decreased (highest + 20.4+/-0.3% dry wt by P. sajor-caju). The results demonstrated the efficient degrading capacity of the test fungi and their potential use in conversion of water hyacinth biomass into mycoprotein-rich ruminant feed, more so by P. sajor-caju.     

Screening Wood Decayed by White Rot Fungi for Preferential Lignin Degradation

A screening procedure in which scanning electron microscopy was used indicated that 26 white rot fungi selectively removed lignin from various coniferous and hardwood tree species. Delignified wood from field collections had distinct micromorphological characteristics that were easily differentiated from other types of decay. The middle lamella was degraded, and the cells were separated from one another. Secondary cell wall layers that remained had a fibrillar appearance. Chemical analyses of delignified wood indicated that the cells were composed primarily of cellulose. Only small percentages of lignin and hemicellulose were evident. Delignified wood was not uniformly distributed throughout the decayed wood samples. White-pocket and white-mottled areas of the various decayed wood examined contained delignified cells, but adjacent wood had a nonselective removal of lignin where all cell wall components had been degraded simultaneously. This investigation demonstrates that selective delignification among white rot fungi is more prevalent than previously realized and identifies a large number of fungi for use in studies of preferential lignin degradation.     

New polymeric model substrates for the study of microbial ligninolysis.

Lignin model dimers are valuable tools for the elucidation of microbial ligninolytic mechanisms, but their low molecular weight (MW) makes them susceptible to nonligninolytic intracellular metabolism. To address this problem, we prepared lignin models in which unlabeled and alpha-14C-labeled beta-O-4-linked dimers were covalently attached to 8,000-MW polyethylene glycol (PEG) or to 45,000-MW polystyrene (PS). The water-soluble PEG-linked model was mineralized extensively in liquid medium and in solid wood cultures by the white rot fungus Phanerochaete chrysosporium, whereas the water-insoluble PS-linked model was not. Gel permeation chromatography showed that P. chrysosporium degraded the PEG-linked model by cleaving its lignin dimer substructure rather than its PEG moiety. C alpha-C beta cleavage was the major fate of the PEG-linked model after incubation with P. chrysosporium in vivo and also after oxidation with P. chrysosporium lignin peroxidase in vitro. The brown rot fungus Gloeophyllum trabeum, which unlike P. chrysosporium lacks a vigorous extracellular ligninolytic system, was unable to degrade the PEG-linked model efficiently. These results show that PEG-linked lignin models are a marked improvement over the low-MW models that have been used in the past.     

Comparative studies of lignin peroxidases and manganese-dependent peroxidases produced by selected white rot fungi in solid media

Abstract The effect of various incubation conditions and media composition on ligninolytic activity by selected strains of white-rot fungi was determined in solid media. When compared to conventional methods using liquid media or woody substrates, this method is fast, simple and also quantitative. Manganese-dependent peroxidase was easily detected in all strains studied. However, detection of lignin peroxidase required optimisation of both growth medium and enzyme assay conditions. Using this method, we showed that the role of nitrogen and oxygen in ligninolytic activity varies and that conditions must be optimised for each individual even within the same species. Furthermore, several white rot fungi produced manganesedependent peroxidase during the primary growth phase. Keywords: Manganese-dependent peroxidase; Lignin peroxidase; White rot fungus     

贝壳状革耳菌和黄孢平革菌固体培养酶系比较

白腐菌黄孢平革菌(Phanerochaete chrysosporium) 与贝壳状革耳菌(Panus conchatus)在类似自然状态的固体培养条件下酶的分泌情况有 较大差异。P.conchatus和P.chrysosporium的主要木素降解酶分别是漆酶和锰过氧化物酶 ;两种菌均产生较高水平的木聚糖酶;P.conchatus在整个培养过程中所产生的内切葡 聚糖酶、微晶纤维素酶和纤维二糖酶活力均比P.chrysosporium相应酶的活力低得多, 尤其是内切葡聚糖酶。研究结果初步揭示了P.conchaus降解木素的主要酶系及选择性降 解木素的原因。     

Effects of various media and supplements on laccase production by some white rot fungi

White rot fungi produce three main extracellular enzymes involved in ligninolysis; laccase, lignin peroxidase and manganese peroxidase. Though all white rot fungi do not produce all three enzymes, laccase occupies an important place in ligninolysis. The present paper reports its production by some white rot fungi; Daedalea flavida, Phlebia brevispora, Phlebia radiata and Polyporus sanguineus under different nutritional conditions. Of the various basal media tested, mineral salts malt extract broth proved to be the best medium for laccase production. Sugarcane bagasse proved to be the best laccase inducer among the various supplements added to different media.     

Glutathione depletion in isolated rat hepatocytes caused by styrene and the thermal degradation products of polystyrene

Depletion of reduced glutathione (GSH) was induced in isolated rat hepatocytes incubated with styrene or exposed for 120 min to products from oxidative thermal degradation of polystyrene. The depletion depended on the concentrations of styrene and on the degradation temperature. Styrene (1 mM) or products from degradation of polystyrene at 200°C (concentration of styrene in exposure atmosphere 0.7 ppm) had no detectable effect on glutathione levels in isolated hepatocytes. At higher degradation temperatures (250°C and 300°C, with styrene concentrations of 2.5 and 25 ppm, respectively) a rapid depletion was detected as well as with 3 mM styrene in incubation mixture. The latency of lactate dehydrogenase was affected neither by the polystyrene degradation products nor by the styrene added to the incubation mixture.     

Fermentation of woods by rumen anaerobic fungi

Abstract The potential of rumen anaerobic fungi for fermenting untreated woods has been assessed using two Neocallimastix species isolated from sheep. When a strain of N. frontalis was incubated for 11 days with wood from 12 hardwood (angiosperm) species, many woods were measurably fermented, with wood from Populus tremuloides (32%) and Fagus sylvatica (21%) being the most highly degraded. This N. frontalis solubilised celulose, hemicellulose and lignin in P. tremuloides wood. Lower degradation (17%) of P. tremuloides wood by a different species of Neocallimastix showed that the choice of fungus as well as the structure and chemistry of the wood influenced the amount of wood cell wall degraded by anaerobic fungi. The amount of degradation was not related to the length of fungal rhizoids.     

Bioremediation of Polycyclic Aromatic Hydrocarbons in Creosote-Contaminated Soil by Peniophora incarnata KUC8836

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are present in products made from creosote, coal tar, and asphalt. When wood pile treated with creosote is placed in soil, PAHs can contaminate it. Creosote has been used for wood preservation in the past and is composed of approximately 85% PAHs and 15% phenolic compounds. PAHs cause harmful effects to humans and the environment because of their carcinogenic and mutagenic properties. White rot fungi can degrade not only lignin, but also recalcitrant organic compounds such as PAHs. Among numerous white rot fungi used in previous studies, four species were selected to degrade PAHs in a liquid medium. From this evaluation of the degradation of PAHs by the four fungal isolates, two species were ultimately selected for the highest rates of removal. Following 2 weeks of incubation with Peniophora incarnata KUC8836, the degradation rates of phenanthrene, fluoranthene, and pyrene were 86.5%, 77.4%, and 82.6%, respectively. Mycoaciella bispora KUC8201 showed the highest degradation rate for anthracene (61.8%). Hence, bioremediation of creosote-contaminated soil with an initial concentration of 229.49 mg kg^?1 PAHs was carried out using the two selected fungi because they could simultaneously degrade 13 more PAHs than the comparison species. More importantly, isolates of P. incarnata KUC8836 were discovered as powerful degraders of PAHs by producing laccase and manganese-dependent peroxidase (MnP), with 1.7- and 1.1-fold higher than the comparison species, respectively. Therefore, the white rot fungus may be proposed for the removal of PAHs and xenobiotic compounds in contaminated environments.     

Degradation of alkali lignin by two ascomycetes and free radical scavenging activity of the products

Biodegradation and bioconversion of extracted alkali lignin was performed under varying concentrations of carbon and nitrogen sources, by two potential Ascomycetes ligninolytic fungus isolated from soil. Fungus, F10 was identified as Aspergillus flavus, while APF4 as Emericella nidulans based upon closed similarity with their morphology and high homology in 18S rRNA gene sequences. The alkali lignin degradation was checked in term of disappearance of lignin content and colority. Selected fungus, degraded 19–41.6% of alkali lignin (0.25%, w/v) within 21 days of incubation and reduced the colority up to 14.4–21%. The activity of ligninolytic enzymes was periodically checked. During alkali lignin degradation manganese peroxidase (13.31?U/ml), lignin peroxidase (13.73?U/ml) and laccase (0.05?U/ml) activities were observed (at highest level). The alkali lignin degradation products and functional group changes in degraded lignin were analysed through gas chromatography-mass spectroscopy (GC-MS) and solid state ¹³C-NMR spectroscopy, respectively. The functional group modifications in alkali lignin moiety, alter its biochemical property, thus fungal mediated modified alkali lignin was further tested for reactive free radical scavenging potential with respect to hydroxyl, nitric oxide and superoxide radicals. Results demonstrate that the alkali lignin undergo degradation in studied nutritional conditions (high-carbon low nitrogen) and consequently increase its free radical scavenging activity up to 1–18%.     

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.     

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.