Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity

Summary The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l⁻¹ and from 55 to 390 U l⁻¹, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5-fold, respectively.     

Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita,Volvariella volvacea and Pleurotus spp.

Ten selected wild and commercial strains of Pleurotus ostreatus,Pleurotus eryngii,Pleurotus pulmonarius, Agrocybe aegerita andVolvariella volvacea were cultivated on three agricultural wastes, i.e. wheat straw (WS), cotton waste (CW) and peanut shells (PS). All species demonstrated significantly higher colonization rates on WS and CW than on PS. WS supported faster growth of A. aegerita and Pleurotus spp., whereas V. volvacea performed better on CW. Comparison of growth rates on composted and non-composted WS and CW substrates revealed that in the latter case faster colonization was achieved, particularly for Pleurotus spp. However, one commercial strain of V. volvacea presented higher growth rates when the composted CW medium was used. Furthermore, earliness in the fructification of P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW substrates, while WS favoured earliness of P. eryngii and A. aegerita. Similarly, high sporophore yields were obtained by P. ostreatus and P. pulmonarius on both wastes, whereas WS enhanced yield and basidioma size of P. eryngii and A. aegerita strains and CW production of V. volvacea. The substrates cellulose:lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of P. ostreatus and P. pulmonarius; in addition, positive correlation was also detected for carbon:nitrogen ratio and mushroom yield in P. eryngii and A. aegerita and between cellulose content and mushroom yield for V. volvacea strains.     

Use of various coffee industry residues for the cultivation of Pleurotus ostreatus in solid state fermentation

Studies were carried out to evaluate the feasibility of using coffee industry residues, viz. coffee husk, coffee leaves and spent coffee ground as substrates in solid state fermentation (SSF) to cultivate edible mushrooms Pleurotus. Eight strains of Pleurotus ostreatus and two strains of Pleurotus sajor‐caju were screened on a medium prepared from aqueous extract of coffee husk and agar. Based on best mycelial growth (9.68 mm/day) and biomass production (43.4 mg/plate in 9 days at 24°C), the strain P. ostreatus LPB 09 was selected for detailed studies. SSF was carried out using these substrates under different moisture conditions (45–75%) and spawn rates (2.5–25%). In general, although a 25% spawn rate appeared superior, the 10% spawn rate was recommended for all the three substrates in view of the process economics, as there was not any significant difference in the increase with 10 to 15%. The ideal moisture content for mycelial growth was 60–65% for coffee husk and spent coffee ground, and 60–70% for coffee leaves. The biological efficiency (BE), which is defined as the ratio of the weight of fresh fruiting bodies to the weight of dry substrate, multiplied by 100, and which indicates the fructification ability of the fungus for utilizing the substrate, was best with coffee husk. With coffee husk as the substrate, the first fructification occurred after 20 days of inoculation, and the biological efficiency reached about 97% after 60 days. When coffee leaves were used as the substrate, no fructification was observed even upon prolonged cultivation. With spent ground as the substrate, the first fructification occurred 23 days after inoculation and the biological efficiency reached about 90% in 50 days. There was a significant decrease in the caffeine and tannin contents (61 and 79%, respectively) of coffee husk after 60 days. It was remarkable to observe that caffeine was adsorbed onto the fruiting body (0.157%), indicating that it was not completely degraded by the fungal culture. However, no tannins were found in the fruiting body, indicating that the fungal strain was capable of degrading them. The results showed the feasibility of using coffee husk and spent coffee ground as substrates without any pre‐treatment for the cultivation of edible fungi in SSF, and provided one of the first steps towards an economical utilization of these otherwise unutilized or poorly utilized residues.     

Cultivation of Pleurotus ostreatus and other edible mushrooms

Pleurotus ostreatus is the second most cultivated edible mushroom worldwide after Agaricus bisporus. It has economic and ecological values and medicinal properties. Mushroom culture has moved toward diversification with the production of other mushrooms. Edible mushrooms are able to colonize and degrade a large variety of lignocellulosic substrates and other wastes which are produced primarily through the activities of the agricultural, forest, and food-processing industries. Particularly, P. ostreatus requires a shorter growth time in comparison to other edible mushrooms. The substrate used for their cultivation does not require sterilization, only pasteurization, which is less expensive. Growing oyster mushrooms convert a high percentage of the substrate to fruiting bodies, increasing profitability. P. ostreatus demands few environmental controls, and their fruiting bodies are not often attacked by diseases and pests, and they can be cultivated in a simple and cheap way. All this makes P. ostreatus cultivation an excellent alternative for production of mushrooms when compared to other mushrooms.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Cultivation of the oyster mushroom (Pleurotus ostreatus) on wood substrates in Hawaii

Summary Five non-native, aggressively growing trees, Falcataria moluccana (Miquel) Barneby & Grimes, Casuarina equisetifolia L. ex J. R. & G. Forst, Eucalyptus grandis Hill ex Maid, Psidium cattleianum Sabine, and Trema orientalis (L.) Blume, were evaluated for suitability as substrate for outdoor cultivation of the oyster mushroom, Pleurotus ostreatus (Jacq.: Fr.) Kumm., in Hawaii. An existing shade house was modified for mushroom production and proved to be an adequate fruiting site. Nitrogen-fixing trees (C. equisetifolia, T. orientalis, and F. moluccana) supported greater yield (275.5, 272.4 and 268.8 g/bag, respectively), biological efficiency (70.1, 78.5, and 74.0%, respectively), and flush number (3.0, 3.2, and 3.5) than non-fixers. P. cattleianum supported significantly lower yield (190.5 g/bag) and biological efficiency (44.2%). Mean crop period was 51 days and was not affected by the wood substrate. Similarly, substrate did not have a significant impact on the concentration of nutrients or moisture in fruit bodies. Taste preferences were noted in mushrooms grown on different substrates; those grown on C. equisetifolia were most flavourful and preferred in one taste test.     

Isolation of a gram-positive bacterium effective in suppression of brown blotch disease of cultivated mushrooms,Pleurotus ostreatus andAgaricus bisporus, caused byPseudomonas tolaasii

A Gram-positive bacterium was isolated from a rottingPleurotus ostreatus fruiting body that markedly reduced the level of extracellular toxins (i.e., tolaasins) produced byPseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The isolated bacterium is saprophytic but not parasitic nor pathogenic toP. ostreatus. A low ratio, ca. 10⁻³ cells of the isolated bacterium for oneP. tolaasii cells, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease inP. ostreatus andAgaricus bisporus. The suppression of the disease development, however requires the initial cell density equivalent to ca. 10⁻¹ cells of the isolated bacterium for one cells of the pathogen. The effects is ascribed to the inactivation of tolaasin by the live, suppressive bacterial cells, and not to metabolites secreted from the organism into culture media. Examination by conventional bacteriological tests and with testing kits, i.e., MicroStation^TMSystem Release 3.5 (Biolog Inc., Hayward, CA), ATB Expression (bioMerieux Inc. Japan) and VITEK (bioMerieux Inc. Japan), failed to assign the organism to any defined bacterial genus. The suppressive bacterium may be useful in future for the development of biocontrol system and/or the construction of genetically modified edible fungi resistant to the disease caused byP. tolaasii.     

Degradation of endosulfan during substrate preparation and cultivation of Pleurotus pulmonarius

Summary Endosulfan is an insecticide used on many vegetable crops. In mushroom cultivation, vegetable materials used as a growth substrate may contain residues of endosulfan that may accumulate in the final mushroom biomass. After preparing the substrate, it is subjected to pasteurization and/or composting and then inoculated with the desired fungus. The purpose of this research was to determine the rate and extent of endosulfan reduction from a grass substrate that was either composted or sterilized by autoclaving. In addition, the rate and extent of removal of endosulfan from substrate colonized with Pleurotus pulmonarius was determined. The degradation of 65 mg/kg endosulfan was analyzed on both, the substrate preparation and the culture of P. pulmonarius on the grass Digitaria decumbens. During composting in presence of Ca(OH)₂ for 120 h, the concentrations of α and β endosulfan were reduced by 61.4 and 49.5% respectively, significantly higher compared with the control (without Ca(OH)_2,) in which the reduction was 38.5%. After sterilization the concentration of α and β endosulfan was reduced by 84.8 and 87.5% respectively. After the colonization of substrate by P. pulmonarius (15 days after spawning) α and β endosulfan were reduced by 96% and at the end of cultivation (35 days after spawning) were reduced by 99%. When carpophores were analyzed, residues of α and β endosulfan were observed between 0.019–0.084 mg/kg. The results showed that α and β endosulfan were partially removed during the preparation of substrate and entirely eliminated during fungal colonization on the substrate.     

Comparative culturing of Pleurotus spp. on coffee pulp and wheat straw: biomass production and substrate biodegradation

The results of the cultivation of six strains of Pleurotus (P. djamor (2), P. ostreatus (2) and P. pulmonarius (2)) on coffee pulp and wheat straw are presented. Metabolic activity associated with biomass of each strain was determined, as well as changes in lignin and polysaccharides (cellulose and hemicellulose), phenolic and caffeine contents in substrate samples colonized for a period of up to 36 days. Analysis were made of changes during the mycelium incubation period (16 days) and throughout different stages of fructification. Greater metabolic activity was observed in the wheat straw samples, with a significant increase between 4 and 12 days of incubation. The degradation of polysaccharide compounds was associated with the fruiting stage, while the reduction in phenolic contents was detected in both substrates samples during the first eight days of incubation. A decrease was observed in caffeine content of the coffee pulp samples during fruiting stage, which could mean that some caffeine accumulates in the fruiting bodies.     

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.     

Lignocellulolytic enzymes profile during growth and fruiting of Pleurotus ostreatus on wheat straw and tree leaves

Cultivation of two commercial Pleurotus ostreatus (oyster mushroom) strains was performed in plastic bags. Tree leaves appeared to be an excellent growth substrate for the conversion into fruiting bodies with biological efficiency of 108-118%. The level of enzyme activity was strongly regulated during the life cycle of mushrooms. However, despite the quantitative variations, each strain had a similar pattern of enzyme accumulation in fermentation of both substrates. Laccase and MnP activities were high during substrate colonization and declined rapidly during fruiting body development. On the contrary, in substrate colonization P. ostreatus expressed comparatively low activity of hydrolases. When primordia appeared, the activity of these enzymes sharply increased. Both cellulase and xylanase activity peaked at the mature fruiting body stage. When mushrooms shifted to the vegetative growth, the activity of ligninolytic enzymes again gradually increased, whereas the activity of hydrolases decreased.     

Mitochondrial DNA and its Inheritance in Pleurotus ostreatus and P. pulmonarius

Six stocks each of the basidiomycetes Pleurotus ostreatus and P. pulmonarius were checked for intraspecific polymorphism and inheritance of mitochondrial DNA (mtDNA). Polymorphisms were extensive in both species after digestion with the endonucleases Hae III and Hind III, and they were stock-specific with one exception. The range of mtDNA sizes was similar for both taxa, lying between approximately 56 kb and 70 kb for P. ostreatus and 60 kb and 78 kb for P. pulmonarius. The inheritance of mtDNA was uniparental throughout. In pairings of monokaryons exhibiting specific mtDNA types, only one parental mtDNA type was recovered in the resulting dikaryons; while the mitochondria of the donor mycelium spread out, the mitochondria of the recipient mycelium apparently are lost. Certain mtDNA types were more often inherited than others. By examining this dominance of mtDNA types one could establish parentage for both species.     

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes

Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.     

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.     

Enzymes produced by <Emphasis Type="Italic">Ganoderma australe</Emphasis> growing on wood and in submerged cultures

Enzymes produced by Ganoderma australe in solid-state fermentation and submerged cultures were evaluated. Strain A464 produced laccase activity in liquid medium and in solid-state cultures containing Drimys winteri or Eucalyptus globulus wood chips, while MnP and LiP activities were not detected. On the other hand, strain A272 cultured for 75 days on E. globulus presented MnP activity of 719 IU/kg of wood. The suitability of D. winteri wood as a substrate enabling MnP production was checked with a well-documented MnP-producing basidiomycete, Ceriporiopsis subvermispora, which produced MnP activity of 327 IU/kg of wood in 9-day-old cultures. Data from two different G. australe strains (A272 and A464) indicated that MnP secretion depended on strain origin as well as on culture conditions.     

Purification and characterization of extracellular laccase fromPleurotus ostreatus

Laccase (EC 1.10.3.2) from the culture filtrate of a strain of white rot basidiomycetePleurotus ostreatus was purified using DEAE-Toyopearl 650M and butyl-Toyopearl 650M column chromatographies and Superdex 75 HR 10/30 fast protein liquid chromatography. Molecular weight of the purified laccase was about 55,000, and the isoelectric point was 3.0. The optimum pH for enzyme activity was 6.5, and the optimum temperature was 50°C. This enzyme contained 7.4% sugar and two copper atoms per molecule. The substrate specificity was similar to those of other fungal laccases. Comparison of the N-terminal amino acid sequence of theP. ostreatus laccase with those fromPleurotus ostreatus Florida,Coriolus hirsutus, Phlebia radiata, basidiomycete PM1 (CECT 2971),Trametes villosa, Pycnoporus cinnabarinus, Ceriporiopsis subvermispora, andAgaricus bisporus showed 95, 65, 60, 55, 55, 55, 50, and 35% similarity, respectively, in the first 20 residues. No similarity in this region was detected with laccases fromNeurospora crassa, Aspergillus nidulans, andCryptococcus neoformans.     

Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species

Pleurotus eryngii, P. ostreatus and P. pulmonarius produced laccase (Lac) both under conditions of submerged fermentation (SF) and solid-state fermentation (SSF) with all of the investigated carbon and nitrogen sources. The highest levels of Lac activity were found in P. eryngii, under SF conditions of dry ground mandarine peels and in P. ostreatus, strain No. 493, under SSF conditions of grapevine sawdust.High levels of peroxidases activities were occurred in P. ostreatus, strain No. 494, and P. pulmonarius, under SSF conditions of grapevine sawdust, whereas in SF, these activities were either very low or absent.After purification of extracellular crude enzyme mixture of investigated species and strain which were grown in the medium with the best carbon sources, the Lac activity measurements revealed two peaks in P. eryngii, three peaks in both P. ostreatus strains and three in P. pulmonarius. Results obtained after purification also showed that the levels of phenol red oxidation in absence of external Mn²⁺ were higher than phenol red oxidation levels in presence of external Mn²⁺.In the medium with the best carbon sources (mandarine peels and grapevine sawdust, respectively), both P. eryngii and P. ostreatus, strain No. 493, showed the highest Lac activity with (NH₄)₂SO₄, as a nitrogen source, with a nitrogen concentration of 20 and 30 mM, respectively.In P. ostreatus, strain No. 494, and P. pulmonarius, the best nitrogen sources for peroxidases production were peptone in a concentration of 0.5% and NH₄NO₃ with a nitrogen concentration of 30 mM, respectively.     

Mycelial yield of<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> using thinned apple,pear, and peach on submerged culture

The effect of thinned fruits, apple, pear and peach, on the mycelial growth of mushrooms was investigated. The growth of mycelia with the addition of thinned fruit was clearly better than that in the control for all the tested mushrooms. The growth rate ofPleurotus ostreatus was faster than any other mushroom. The optimal concentrations of thinned apple, pear, and peach in a solid culture were 1.0%, 1.0%, and 3%, respectively, while in a liquid culture the optimal concentrations were 5,0%, 3.0%, and 5.0%, respectively. WhenPleurotus ostreatus was incubated in a 20-L pilot scale fermenter with 10 L of a liquid medium containing 3% thinned fruit at 25°C and 6 vvm for 10 days, the mass-production of mycelia was 74.2 g/10 L (apple), 96.2 g/10 L (pear), and 86.3 g/10 L (peach). The mycelial yield ofPleurotus ostreatus in a medium containing thinned fruit was 2≈3 times higher than that in the control.     

Ligninolytic enzymes activities of Oyster mushrooms cultivated on OMW (olive mill waste) supplemented media, spawn and substrates

Ligninolytic enzymes activities (laccases, peroxidases (total, MnP and MiP) and aryl-alcohol oxidase (AAO)) were measured during the cultivation of six commercial Pleurotus sp. strains on MMP media, on cereal grains (spawn) and on straw substrates (the three commonly utilized cultivation steps to obtain fruiting bodies) supplemented with several concentrations of autoclaved (OMW) or gamma-irradiated (iOMW) olive mill waste. Results indicated that all the strains were able to grow on MMP media and spawn containing up to 30% OMW and iOMW and on straw substrates mixed with 50% OMW. None of the strains showed AAO activity and there was not a single strain which showed the highest laccases and peroxidases activities, independently of the utilized substrate. Pleurotus mycelia adjusted their enzymatic mechanisms depending on their variety, type of substrate, concentration of OMW or iOMW added. OMW was a better supplement to use than iOMW because OMW induced higher exo-enzymes activities.     

rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.