Decolourization of Municipal Effluent and Sludge by Pleurotus sajor-caju and Pleurotus ostreatus

Summary The Americana Municipal Treatment Station, S?o Paulo, Brazil, manages 400 l of effluent s⁻¹, from domestic and textile origin, which produces an average of 20 t of sludge per day. The decolourization of the effluent and sludge by three strains of Pleurotus (Pleurotus sajor-caju F2, F6 and Pleurotus ostreatus) was evaluated. The strains of P. sajor-caju F2 and F6 were able to decolourize the sludge, while P. ostreatus was less efficient. Detoxification was appraised with three bioassays comprising the cnidarian Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. After exposure to fungi, effluent toxicity decreased but not that of its sludge. Strain P. sajor-caju F6 presented signs of toxicity shown by electron microscopy in the presence of the effluent. The three strains produced high amounts of manganese-peroxidase (Mn–P) and laccase in the presence of the sludge. Although P. ostreatus produced large amount of Mn–P and laccase enzymes, these enzymes did not result in decolourization of the sludge, suggesting that other factors are likely to be involved. Carbon content decreased only in the treatment with P. ostreatus.     

Infra-red spectroscopic analyses of banana waste degraded by oyster mushroom

Carbon, hydrogen and nitrogen analyses of banana leaf and pseudostem biomass revealed their potentiality as substrates for microorganisms. Infra-red (IR) spectra of both biomass show presence of cellulose, xylan and lignin. IR spectra of leaf and pseudostem biomass degraded in solid state fermentation (SSF) by two Pleurotus species (P. sajor-caju and P. ostreatus) for 40 days showed the utilization of cellulose, xylan and lignin by these microbes. Dynamics of various lignocellulolytic enzymes of Pleurotus species and analyses of carbon, hydrogen and nitrogen contents of degraded biomass supported the same. Both the Pleurotus species exhibited lignin consumption ability on both the substrates.     

Recycling of spent shiitake substrate for production of the oyster mushroom,Pleurotus sajor-caju

Pleurotus sajor-caju was produced on a basal medium containing spent shiitake substrate plus 10% wheat bran and 10% millet. An analysis of the fibrous composition of the spent shiitake substrate revealed that 85% of the original hemicellulose, 44% of the original cellulose and 77% of the original lignin was not consumed during production of a full crop (78% biological efficiency) of shiitake (63-day harvest period). To produce P. sajor-caju, the spent shiitake substrate was ground, air dried, supplemented, pasteurized with live steam and spawned. Highest yields (79% biological efficiency) of P. sajor-caju were obtained by supplementing the spent shiitake basal medium with 12% soybean and 1% CaCO₃. Increases in percentage biological efficiency and mushroom size were positively correlated with increasing levels of CaCO₃ added to the basal medium.     

Increasing <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> laccase production by culture medium optimization and copper/lignin synergistic induction

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2^6–2 where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.     

The effect of white-rot basidiomycetes on chemical composition andin Vitro digestibility of wheat straw

Five white-rot basidiomycetes were evaluated for their potential to improve ruminal degradation of wheat straw.Polyorus brumalis, Lyophyllum ulmarium III,Trametes gibbosa, Pleurotus ostreatus, and aPleurotus ostreatus mutant were incubated on wheat straw for 30 d at 28°C. Detergent fiber, crude protein andin vitro dry matter digestibility (IVDMD) were determined. The results showed increasing crude protein and ash contents in fungus-treated straw. IVDMD values were increased in straws treated withP. ostreatus, P. ostreatus mutant andT. gibbosa only. Relative to untreated wheat straw the detergent fiber content—neutral detergent fiber (NDF), and acid detergent fiber (ADF) was reduced in fungus-treated straw and out of three fractions—hemicellulose, cellulose and lignin, hemicellulose showed the largest proportionate loss whereas lignin the smallest one in all 5 samples of fungus treated straw.     

Biodegradation of Gossypol by Mixed Fungal Cultures in Minimal Medium

The fungal cultures, namely – Pleurotus sajor-caju MTCC 1806, Saccharomyces cerevisiae MTCC 6933 and Candida tropicalis MTCC 1406 and their combinations, C. tropicalis + S. cerevisiae, P. sajor-caju + S. cerevisiae and C. tropicalis + P. sajor-caju were grown in minimal medium containing 100 ppm of gossypol as the sole carbon and energy source. The culture supernatants of C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae had low residual gossypol levels of 29 and 25 ppm, respectively. In the present study, we attempted to isolate gossypol-degrading enzyme and biodegraded gossypol from the culture supernatants of C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae. The specific activity of laccase in the purified enzyme extracts of the C. tropicalis + S. cerevisiae and P. sajor-caju + S. cerevisiae treated samples was 425 and 224 U/mg, respectively. In SDS-PAGE, the gossypol-degrading enzyme was revealed as 3 bands of molecular weights ranging from 45 to 66 kDa. The characterization of biodegraded gossypol by FTIR analysis showed a reduction in aldehydes (C-H) stretches in samples treated with fungi. Mass spectrometry analysis revealed that the monoisotopic mass of the biodegraded gossypol was 474 g/mol.     

Growth of higher fungi on wheat straw and their impact on the digestibility of the substrate

Summary The influence of the growth of three higher fungi on the composition of wheat straw was investigated. Pleurotus pulmonarius, P. sajor-caju and Lentinus edodes grew very well on lignocellulosic substrates, breaking down a considerable amount of lignin. The initial lignin concentration of straw was halved after 12 weeks of fungal growth, doubling the enzymic digestibility. Together with lignin, the higher fungi consumed half of the amount of hemicellulose (i.e. 15%), leaving cellulose fairly intact, which should remain as an energy source for ruminants.     

Degradation of Chrysanthemum (Dendranthema grandiflora) wastes by Pleurotus ostreatus for the production of reducing sugars

In Colombia, a great amount of waste is generated during the cut-off and harvest stages in flowers culture. This study examines the possibility of degrading Chrysanthemum wastes by using Pleurotus ostreatus, Trametes versicolor, and Phanerochaete chrysosporium cultures; this has not been studied previously. The initial effect of fungi on the degradation of Chrysanthemum wastes were studied individually and in co-cultures. The highest degradation was by P. ostreatus. After that, the influence of pH and waste, copper, and manganese concentrations on reducing sugars concentration were determined in a submerged culture in 100 mL Erlenmeyer flasks. There was a significant effect of manganese and waste concentrations on sugar concentration, while the effect of copper concentration and pH were not significant. Following, the process was carried out in a 1.5 L reactor at the optimal values of the variables studied in Erlenmeyer flask but varying air injection from 0 to 2 vvm. The highest concentration of sugars was 21.2 g/L with 78% of glucose content at 6.3% w/v of waste, 7.5 mM of Mn and Cu and 2 vvm of air injection. Finally, laccase, Manganese peroxidase, endo-1,4-β-glucanase, exo-1,4-β-glucanase and 1,4-β-glucosidase were detected in the extract obtained under these conditions. The highest activities were obtained for laccase (4,694 U/L) and 1,4-β-glucosidase (9,513 U/L).     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

不同碳源和氮源对金针菇降解木质纤维素酶活性的影响

以3株栽培的金针菇Flammulina velutipes为材料,研究它们在玉米芯和棉子壳以及不同碳源、氮源培养条件下纤维素、半纤维素和木质素降解酶活性的规律。结果表明,不同金针菇菌株的羧甲基纤维素酶、木聚糖酶和漆酶活力显著不同（P<0.001）,同时,培养条件对羧甲基纤维素酶、木聚糖酶和漆酶的活力都有显著影响（P<0.001）。在简单碳源存在的条件下,金针菇的羧甲基纤维素酶和木聚糖酶活力远远低于复杂碳源培养基（P<0.05）。全营养培养基上生长的金针菇的羧甲基纤维素酶和木聚糖酶活力低于缺乏碳源和氮源的培养基（P<0.05）。漆酶活力在无简单氮源培养基上低于全培养基（P<0.05）和无葡萄糖培养基（P<0.05）,即复杂碳源和氮源培养基上的漆酶活力低于简单碳源和氮源培养基（P<0.05）。     

Decolorization of textile dyes by enzymatic extract and submerged cultures of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus sajor-caju PS2001 was screened in Petri dish plates to assess the dye-decolorizing ability of industrial textile dyes. P. sajor-caju PS2001 was also cultivated in solid-state fermentation containing sawdust of Pinus sp. and wheat bran to obtain the enzymatic extract, showing laccase and manganese-peroxidase activity, which was used to test the capacity to degrade the textile dyes. Additional tests of decolorization were performed in liquid cultures. Anthraquinone-type textile dyes proved to be substrates for the enzymatic system of P. sajor-caju PS2001. Cultures in Petri dish plates showed that the anthraquinone dye Reactive Blue 220 can act as a redox mediator for the enzymatic reactions involved in the decolorization process, and enables the azo dye degradation. Reactive Blue 220 and Acid Blue 280 were completely decolorized in 30 min and 60 min, respectively, during the tests with precipitated enzymatic extract, while the azo dyes showed resistance to degradation. Additionally, in submerged cultures with dyes, veratryl alcohol oxidases and lignin peroxidase activities were observed. These results suggest that the strain P. sajor-caju PS2001 has great potential for use in the bioremediation technology of recalcitrant pollutant such as textile effluents.     

Protein enrichment and digestibility of soft rush (<Emphasis Type="Italic">Juncus effusus</Emphasis>) and rice residues using edible mushrooms <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> and <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus species are found to be among the most efficient lignocellulolytic types of white-rot fungi. Rice is the main grain cultivated in the extreme south of Brazil. Defatted rice bran and straw are by-products of low aggregate value. Soft rush (Juncus effusus) is a common native plant also very abundant in the region. In the present work, we evaluated changes in substrate composition after growth of two white-rot fungal species: Pleurotus ostreatus and Pleurotus sajor-caju, aiming to increase protein content and digestibility from substrates through solid fermentations and obtain edible mushrooms of high aggregate value. For that, defatted rice bran, defatted rice straw and soft rush were utilized as substrate. The influence of the variables thermal treatment temperature of substrate, substrate moisture and concentration were evaluated on the protein content, digestibility and biological efficiency. The highest protein enrichment of rice bran in P. sajor-caju-fermented medium was due the fact that there was no fructification in these media, while for the P. ostreatus-fermented medium, part of the synthesized protein was converted into mushrooms. The highest protein enrichments were verified in medium with 80% moisture and 25% soft rush (47.1% using P. ostreatus and 49.0% using P. sajor-caju). A higher digestible protein increase was obtained for both species in media with 70% moisture and 25% soft rush.     

Changes in phenol oxidases and superoxide dismutase during fruit-body formation of <Emphasis Type="Italic">Pleurotus </Emphasis>on sawdust culture

 Peroxidase and laccase activities increased rapidly up to the formation of primordia and then declined throughout the entire stage of fruiting. In the case of Pleurotus ostreatus, the level of Mn-dependent peroxidase was very low in primordia and fruiting stages but gradually increased with the growth of the fruit-body, whereas no activity was detected in Pleurotus sajor-caju during all growth stages. Superoxide dismutase activity was observed mainly at the fruiting stages. These results show that changes in concentration of lignin-related enzymes are associated with the fruiting process. Received: December 11, 2000 / Accepted: March 28, 2002     

Cultivation of oyster mushrooms (<Emphasis Type="Italic">Pleurotus</Emphasis> spp.) on various lignocellulosic wastes

Cultivation of speciality mushrooms on lignocellulosic wastes represents one of the most economically and cost-effective organic recycling processes. Three species of Pleurotus, namely P. columbinus, P. sajor-caju and P. ostreatus were experimentally evaluated on untreated organic wastes including chopped office papers, cardboard, sawdust and plant fibres. Production studies were carried out in polyethylene bags of about 1 kg wet weight with 5% spawning rates of substrate fresh weight in a custom-made growth room especially designed for spawn run and cropping. The conversion percentage from dry substrate weight to fresh mushroom weight (biological efficiency) was determined. The highest biological efficiency was noted with P. columbinuson cardboard (134.5%) and paper (100.8%), whereas P. ostreatus produced maximum yield on cardboard (117.5%) followed by paper (112.4%). The overall yield of P. sajor-cajuwas comparatively low (range 47–78.4%). The average number of sporophore flushings ranged between 5 and 6 times. The findings that P. columbinus and P. ostreatus are superior to P. sajor-caju are consistent with previous reports elsewhere. Further evaluation of P. columbinus alone on different bagging systems containing partially pasteurized office papers as a growing substrate revealed that polyethylene bags resulted in 109.4% biological efficiency in contrast to pottery (86%), plastic trays (72%) or polyester net (56%). The above findings reveal an opportunity for commercial implication of oyster mushroom especially P. columbinus for utilization of different feasible and cheap recyclable residues.     

Microbial protein from leguminous and non-leguminous substrates

The biodegradation of leguminous and non-leguminous organic materials by Pleurotus sajor-caju and P. ostreatus was studied. Comparisons were made between mushroom yield on both types of substrates. The conversion percentage from dry substrate to fresh mushroom (biological efficiency) was determined. Mushrooms were analyzed for their protein content, carbohydrates, percentages of ash, fat content and crude fibre. The energy value of fruit bodies was computed. The yield obtained from leguminous substrates was significantly high as compared to that yield which was obtained from non-leguminous substrates. The lowest yield of P. sajor caju (10 g ± 0.3 g) and P. ostreatus (6.5 g ± 1.2 g) was obtained using the Bougainuillea substrate and the highest one (519 g ± 16 g, P. sajor-caju and 488 g ± 18 g, P. ostreatus) using nitrogen-fixing leguminous Leucaena leucocepholea. The nitrogen content in fruit bodies was found to be higher with leguminous substrate than with non-leguminous ones.     

Evaluation of holocellulase production by plant-degrading fungi grown on agro-industrial residues

Agaricus brasiliensis CS1, Pleurotus ostreatus H1 and Aspergillus flavus produced holocellulases when grown in solid and submerged liquid cultures containing agro-industrial residues, including sugar cane bagasse and dirty cotton residue, as substrates. These isolates proved to be efficient producers of holocellulases under the conditions used in this screening. Bromatological analysis of agro-industrial residues showed differences in protein, fiber, hemicellulose, cellulose and lignin content. Maximal holocellulase activity (hemicellulase, cellulase and pectinase) was obtained using solid-state cultivation with 10% substrate concentration. In this case, remarkably high levels of xylanase and polygalacturonase activity (4,008 and 4,548 IU/l, respectively) were produced by A. flavus when grown in media containing corn residue, followed by P. ostreatus H1 with IU/l values of 1,900 and 3,965 when cultivated on 5% and 10% sugar cane bagasse, respectively. A. brasiliensis CS1 showed the highest reducing sugar yield (11.640 mg/ml) when grown on medium containing sugar cane bagasse. A. brasiliensis was also the most efficient producer of protein, except when cultivated on dirty cotton residue, which induced maximal production in A. flavus. Comparison of enzymatic hydrolysis of sugar cane bagasse and dirty cotton residue by crude extracts of A. brasiliensis CS1, P. ostreatus H1 and A. flavus showed that the best reducing sugar yield was achieved using sugar cane bagasse as a substrate.     

Production,partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10⁻⁶ IU/ml) and manganese peroxidase (24.11 × 10⁻⁶ IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × ⁻⁶ IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.     

Isolation and characterization of a thermostable cellulase-producing <Emphasis Type="Italic">Fusarium chlamydosporum</Emphasis>

A thermostable cellulase-producing fungus, HML 0278, was identified as Fusarium chlamydosporum by morphological characteristics and ITS rDNA sequence analysis. HML 0278 produced extracellular cellulases in solid-state fermentation using sugar cane bassage as the carbon source. Native-PAGE analysis demonstrated that this fungus strain was capable of producing the three major components of cellulases and xylanase, with a yield of 281.8 IU/g for CMCase, 182.4 IU/g for cellobiohydrolase, 135.2 IU/g for β-glucosidase, 95.2 IU/g for filter paper activity, and 4,720 IU/g for xylanase. More importantly, the CMCase and β-glucosidase produced by HML 0278 showed stable enzymatic activities within pH 4–9 and pH 4–10, and at temperatures below 70 and 60°C, respectively.     

Delignification of Wheat Straw by Pleurotus spp. under Mushroom-Growing Conditions

Pleurotus sajor-caju, P. sapidus, P. cornucopiae, and P. ostreatus mushrooms were produced on unsupplemented wheat straw. The yield of mushrooms averaged 3.6% (dry-weight basis), with an average 18% straw weight loss. Lignin losses (average, 11%) were lower than cellulose (20%) and hemicellulose (50%) losses. The cellulase digestibility of the residual straw after mushroom harvest was generally lower than that of the original straw. It does not appear feasible to simultaneously produce Pleurotus mushrooms and a highly delignified residue from wheat straw.     

Industrial Dye Decolorization by Laccases from Ligninolytic Fungi

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized. Received: 26 May 1998 / Accepted: 7 August 1998