Effect of substrate nitrogen on lignin degradation by Pleurotus ostreatus

In order to determine the effect of substrate nitrogen (N) on ligninolytic activity, Pleurotus ostreatus was grown in solid media containing either growth-limiting (1mM) or excess (10mM) NH₄Cl. After 25 days, ¹⁴C–CO₂ production from ¹⁴C-cornstover lignin in low-N medium was 3 times that in nitrogen (N)-rich medium. Supplementation of low-N medium with glucose (0.3%) further enhanced ligninolytic activity. Decolorization of an aromatic, polymeric dye, Poly R-481, in solid media was also greatest under N-limiting conditions.     

Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.     

Effect of Manganese on Lignin Degradation by Pleurotus ostreatus during Solid-State Fermentation

Lignin degradation by Pleurotus ostreatus was studied under solid-state fermentation (SSF) in chemically defined medium containing various levels of Mn. Degradation of [¹⁴C]lignin prepared from cotton branches to soluble products, as well as its mineralization to ¹⁴CO₂, was enhanced by the addition of Mn. The effect of malonate on lignin mineralization was most marked during the first 10 days of SSF, in a treatment amended with 73 μM Mn. A high concentration of Mn (4.5 mM) caused inhibition of both fungal growth and mineralization rates during the first 2 weeks of incubation. Addition of malonate reversed this effect because of chelation of Mn. Mn was found to precipitate in all treatments, with or without the addition of malonate. α-Keto-γ-methiolbutyric acid cleavage to ethylene, an indication of ^. OH production, was observed as early as 3 days of incubation in all treatments.     

Mn-peroxidase from Pleurotus ostreatus: The action on the lignin

Summary Three Mn-peroxidases from Pleurotus ostreatus have been isolated and purified. Mn-peroxidase III was homogeneous by isoelectrofocusing and electrophoresis. Isolated Mn-peroxidase isoenzymes were able to depolymerise lignosulfonates pretreated with ozone. Ozone caused significant alterations of lignosulfonate molecular size distribution resulting in appearance of two fractions of destruction products.     

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium

Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, 1,4-beta-glucosidase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase and 1,4-beta-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-beta-glucanase, 1,4-beta-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-beta-mannosidase in P. ostreatus.     

Wheat bran biodegradation by Pleurotus ostreatus: a solid-state carbon-13 NMR study

Solid-state (13)C nuclear magnetic resonance (NMR) and elemental analysis techniques were used to monitor the degradation of wheat bran by the white-rot fungus Pleurotus ostreatus during a 62-day cultivation period. The weight loss and in vitro organic matter digestibility of the substrate were also evaluated after fungal treatment. The (13)C NMR spectra of degraded wheat bran samples showed a lower content in carbohydrates and a higher content in aliphatic and carboxylic groups than the untreated control sample. In parallel, changes in the wheat bran elemental composition evidenced a decrease in carbon content and a concomitant increase in nitrogen and oxygen content during mycelium growth. These results clearly indicate the occurrence of progressive changes in the composition of wheat bran during fungal treatment and are interpreted in terms of preferential degradation of amorphous vs. crystalline polysaccharides by the fungal mycelium and accumulation of proteins in the substrate. At the end of the cultivation period, the treated samples experienced an average weight loss of 20% and an increase in organic matter digestibility of 17%.     

Influence of Pleurotus ostreatus inoculation on wood degradation and fungal colonization

The influence of Pleurotus ostreatus inoculation on wood degradation and on fungal community structure was studied. The experiments were performed on an organically poor fly ash deposit covered with a 10 cm layer of beech wood chips inoculated with P. ostreatus isolate ZIM76. Compared to non-inoculated wood chips, inoculation increased the temperatures and relative humidities and, in the first 6 months, accelerated Klason lignin degradation by 9% and also, after 17 months, increased iron translocation into wood chips by 30%. After 6 months, PCR-DGGE showed 22-28 and 13-21 fungal taxa in non-inoculated and P. ostreatus-inoculated beech chips, respectively. The differences in number of taxa and in the fungal community structure (based on Dice coefficient) between non-inoculated and inoculated wood chips diminished with time. The results indicate that the naturally occurring processes of wood degradation are as efficient as those occurring in sites inoculated with P. ostreatus.     

Production of manganese peroxidase by pellet culture of the lignin-degrading basidiomycete, Pleurotus ostreatus

Pleurotus ostreatus No. 42 produced the ligninolytic enzymes, manganese peroxidase (MnP) and laccase, in agitation culture in glucose/peptone/wheat-bran medium. Formation of mycelial pellets 1-2 mm in diameter was essential for the production of MnP; and the concentration of dissolved oxygen in the culture medium greatly influenced the production of MnP, a concentration over 5 ppm being necessary for MnP production. The maximal activity of MnP was obtained on days 7-9 of culture, after the consumption of nutrient glucose. Introduction of oxygen from the start of the cultivation caused large pellet formation, which resulted in a low MnP activity level. P. ostreatus No. 42 produced two MnP isozymes in agitation culture. The major isozyme, F-2, was 36.4 kDa and had a pI of 3.95. The MnP characteristics, Km values, dependence on Mn2+ and optimum pH showed the similarity between this isozyme and MnP 3, which was produced under different culture conditions. Analysis of the N-terminal amino acid sequence indicated the close similarity of F-2 to MnP 3.     

Effect of carbon source on the degradation of 2-naphthalenesulfonic acid polymers mixture by Pleurotus ostreatus in petrochemical wastewater

The ability of the white rot fungus Pleurotus ostreatus to biodegrade 2-naphthalenesulfonic acid polymers (2-NSAP) contained in a petrochemical wastewater was evaluated. The characterization of the wastewater and a LC–MS identification of polymers were performed. Biodegradation experiment was conducted in batches with and without the addition of a carbon source (glucose, corn starch, cellulose and lignin). It has been demonstrated that, in presence of a suitable carbon source, P. ostreatus is able to remove NSAP ranging from trimers to decamers. Interestingly, all the carbon sources utilized, despite the different velocities, allowed to degrade about 60–70% of the oligomers.Furthermore, respirometric tests showed that the fungal treatment was also able to significantly increase the bCOD/COD ratio, going from 9% (raw wastewater) up to 57%. This is clearly correlated with the enhancement in oligomers depolymerization confirming that the fungal action is not a mineralization of NSAP, but an increase of their biodegradability.     

Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus

Nineteen fungi were tested for their ability to degrade aflatoxin B1 (AFB1). An extracellular enzyme from the edible mushroom Pleurotus ostreatus showed afaltoxin-degradation activity detected by thin-layer chromatography (TLC). An enzyme with this activity was purified by two chromatographies on DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa by SDS-PAGE. Optimum activities were found in the pH range between 4.0 and 5.0 and at 25 degrees C. Also, degradation activity of several dyes in the presence of H2O2 was tested, resulting in the detection of bromophenol blue-decolorizing activity. Based on these data, we suggest this enzyme is a novel enzyme with aflatoxin-degradation activity. Fluorescence measurements suggest that the enzyme cleaves the lactone ring of aflatoxin.     

Growth of Azotobacter vinelandii in a solid-state fermentation of technical lignin

Azotobacter vinelandii was cultured on technical lignin, derived from Kraft pulping processes, for biofertilizer production in solid-state fermentation. The effects of the ratio of technical lignin to corn straw, initial water content, and material bed depth on the microorganisms were studied in detail. At 30 degrees C, technical lignin to corn straw at the ratio of 1:0.75, the bed depth of 5 cm, and 67% moisture content, A. vinelandii was grown and reached 4.2 x 10(10) cfu g(-1) dry rot after 36 h.     

Biodegradation of endocrine-disrupting phthalates by Pleurotus ostreatus

Biodegradation of endocrine-disrupting phthalates [diethyl phthalate (DEP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP)] was investigated with 10 white rot fungi isolated in Korea. When the fungal mycelia were added together with 100 mg/l of phthalate into yeast extract-malt extract-glucose (YMG) medium, Pleurotus ostreatus, Irpex lacteus, Polyporus brumalis, Merulius tremellosus, Trametes versicolor, and T. versicolor MrP1 and MrP13 (transformant of the Mn-repressed peroxidase gene of T. versicolor) could remove almost all of the 3 kinds of phthalates within 12 days of incubation. When the phthalates were added to 5-day pregrown fungal cultures, most fungi except I. lacteus showed the increased removal of the phthalates compared with those of the nonpregrown cultures. In both culture conditions, P. ostreatus showed the highest degradation rates for the 3 phthalates tested. BBP was degraded with the highest rates among the 3 phthalates by all fungal strains. Only 14.9% of 100 mg/l BBP was degraded by the supernatant of P. ostreatus culture in YMG medium in 4 days of incubation, but the washed or homogenized mycelium of P. ostreatus could remove 100% of BBP within 2 days even in distilled water, indicating that the initial BBP biodegradation by P. ostreatus may be attributed to mycelium-associated enzymes rather than extracellular enzymes. The biodegradation rate of BBP by the immobilized cells of P. ostreatus was almost the same as that in the suspended culture. The estrogenic activity of 100 mg/l DMP decreased during biodegradation by P. ostreatus.     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

不同木质纤维素诱导对糙皮侧耳液体发酵产漆酶活性的影响

以2株糙皮侧耳Pleurotus ostreatus栽培菌株为材料,研究在添加木屑、玉米芯和棉籽壳3种农林废弃物后,对其液体发酵产漆酶活性的影响。结果表明,不同糙皮侧耳菌株的漆酶活性具有极显著的差异(P0.001),仅第3天和第9天差异显著(P0.05);而不同培养基条件对漆酶活性也产生了极显著的影响(P0.001)。此外,不同菌株在产漆酶能力上存在一定的差异,在不同培养基条件下所表现出的差异也不一致。相比之下,在含棉籽壳的完全培养基中漆酶活性高于含木屑或玉米芯的完全培养基,说明棉籽壳对不同糙皮侧耳菌株产漆酶能力的诱导作用更强。     

Cultivation of Pleurotus ostreatus on weed plants

Oyster mushroom, Pleurotus ostreatus (Jacq.:Fr.) Kumm. ITCC 3308 (collected from Indian Type Culture Collection, IARI, New Delhi, India, 110012) was grown on dry weed plants, Leonotis sp, Sida acuta, Parthenium argentatum, Ageratum conyzoides, Cassia sophera, Tephrosia purpurea and Lantana camara. Leonotis sp. was the best substrate in fruit body production of P. ostreatus when it was mixed with rice straw (1:1, wet wt/wet wt) for mushroom cultivation. The fruiting time for P. ostreatus was also less on Leonotis sp. than on any other weed substrates tested in the present investigation. T. purpurea was the least suited weed for oyster mushroom cultivation. The main problem of oyster mushroom cultivation on weed substrates was found to be low yield in the second flush that could be overcome by blending weed plants with rice straw. The protein contents of the fruit bodies obtained from Cassia sophera, Parthenium argentatum and Leonotis sp. were not only better than rice straw but also from the rice straw supplemented weeds.     

Changes in the activities of enzymes involved in the degradation of butylbenzyl phthalate by Pleurotus ostreatus

Degradation of butylbenzyl phthalate (BBP) by the white rot fungus Pleurotus ostreatus and the activities of some degrading enzymes were examined in two different media containing 100 mg/l of the compound. P. ostreatus pregrown for 7 days in complex YMG medium was able to completely degrade BBP within an additional 24 h but degraded only 35 mg/l of BBP in 5 days of incubation in minimal medium. Fungal cell mass in the culture in YMG medium was higher in the presence than in the absence of BBP. The esterase activity of the fungal culture in YMG medium was higher than that in minimal medium and increased with the addition of BBP. On the contrary, laccase activity was higher in minimal medium and it did not increase upon the addition of BBP. General peroxidase activity increased for a few days after the addition of BBP to both media. The degradation of BBP and its metabolites by P. ostreatus thus may be attributed mostly to esterase rather than lignin-degrading laccase. In addition, the activities of the enzymes involved in BBP degradation and their changes varied significantly in the different media and culture conditions.     

Influence of Phanerochaete chrysosporium on microbial communities and lignocellulose degradation during solid-state fermentation of rice straw

To investigate the changes of microbial communities and influence of Phanerochaete chrysosporium during solid-state fermentation (SSF) of rice straw, phospholipid fatty acids (PLFA) and lignocellulose components were measured with periodical sampling. The results showed that the lignocellulose degrading ratios in SSF which was inoculated by P. chrysosporium and soil microorganisms were higher than those degraded by culturing a single species. The total amount of PLFAs, as an indicator of microbial biomass, reached the peak on day 6. Principal component analysis (PCA) of the PLFA compositions revealed that P. chrysosporium was well responsible for the succession of microbial community and showed that fungi were the predominant species at the end of the process. The correlation analysis between lignocellulose degrading ratio and PLFA profile in P. chrysosporium suggested that P. chrysosporium promoted lignin degrading as the main fungi with gram-positive bacteria.