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.     

Cellulase production by the edible mushroom Volvariella diplasia

Volvariella diplasia produced cellulolytic enzymes (550 U CM-cellulase and 69 U filter-paper cellulase/l) when grown in shake culture at pH 5.4 and 28°C with 0.5% cellulose powder as carbon source. Alkali-treated as well as untreated cellulosic substrates were hydrolysed by both enzymes (sp. act. 2.75 U/mg protein), with cellobiose and glucose as the end products.The author is with NCIM, Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, Maharashtra, IndiaNCL Communication No. 6168.     

Isolation of a novel N-acetylglucosamine-specific lectin from fresh sclerotia of the edible mushroom Pleurotus tuber-regium

An N-acetylglucosamine-binding lectin with a molecular mass of 32kDa was isolated from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Its N-terminal sequence exhibited some similarity to that of Agaricus bisporus lectin. The isolation procedure was simple, involving (NH(4))(2)SO(4) precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on N-acetyl-D-glucosamine-agarose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin exhibited hemagglutinating activity toward trypsinized rabbit erythrocytes but not toward untrypsinized rabbit erythrocytes.     

Bioconversion of sugar cane crop residues with white-rot fungiPleurotus sp.

Four mushroom strains ofPleurotus spp. were cultivated on sugar cane crop residues for 30 days at 26°C. Biochemical changes affected the substrate as a result of fungal growth, in terms of nitrogen, lignin, cellulose and hemicellulose contents. All strains showed a strong ligninolytic activity together with variable cellulolytic and xylanolytic action.Pleurotus sajor-caju attacked lignin and cellulose at the same rate, showing a degradation of 47% and 55%, respectively. A better balance was shown by theP. ostreatus-P. pulmonarius hybrid, which exhibited the poorest cellulolytic action (39%) and the highest ligninolytic activity (67%). The average composition of mushroom fruit bodies, in terms of nitrogen, carbohydrates, fats and amino acid profiles, was determined. Crude protein and total carbohydrate varied from 23% to 33% and 36% to 68% of dry matter, respectively. Fat ranged from 3.3% to 4.7% and amino acid content from 12.2% to 22.2%. Slight evidence for a nitrogen fixing capability was encountered in the substrate to fruit body balance.     

Differential Patterns of Constitutive Intracellular Laccases of the Vegetative Phase of Pleurotus Species

The constitutive intracellular laccase activity of ten strains of Pleurotus spp. was determined in vitro and by zymograms, using different substrates. Differences in the in vitro activities were observed between all the strains; however, zymogram patterns were only similar for strains within same species, independently of any of the three substrate (2,6-dimethoxyphenol, p-anisidine or o-tolidine) used. The differences observed in the number and positions of the isoforms in the gel suggest that laccase zymograms can be used to differentiate species of this organism.     

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.     

Lignocellulolytic enzyme profiles of edible mushroom fungi

One of the most economically-viable processes for the bioconversion of many types of lignocellulosic wastes is represented by edible mushroom cultivation. Lentinula edodes, Volvariella volvacea and Pleurotus sajor-caju are three important commercially cultivated mushrooms which exhibit varying abilities to utilise different lignocellulosics as growth substrate. Examination of the lignocellulolytic enzyme profiles of the three species show this diversity to be reflected in qualitative variations in the major enzymic determinants (i.e. cellulases, ligninases) required for substrate bioconversion. For example, L. edodes, which is cultivated on highly lignified substrates such as wood or sawdust, produces two extracellular enzymes which have been associated with lignin depolymerisation in other fungi, (manganese peroxidase and laccase). Conversely, V. volvacea, which prefers high cellulose-, low lignin-containing substrates produces a family of cellulolytic enzymes including at least five endoglucanases, five cellobiohydrolases and two -glucosidases, but none of the recognised lignin-degrading enzymes.J.A. Buswell, Y.J. Cai, S.T. Chang, J.F. Peberdy and S.Y. Fu are with the Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong. J.A. Buswell and S.T. Chang are also with the Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, Hong Kong. J.F. Peberdy is also with the Department of Life Science, University of Nottingham, United Kingdom. S.Y. Fu is also with, and H.-s. Yu is with the Institute of Chemistry, Academia Sinica, Guangzhou, China.     

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.     

Biodegradation of lignocellulosic waste by Aspergillus terreus

Biodegradation of lignocellulosic waste by Aspergillus terreus is reported for the first time. This isolate produced 250 CMCase (carboxymethyl cellulase or endoglucanase) U.ml^-1 and biodegraded hay and straw during 3 days and the biomass production on straw was 5g.L^-1dry weight from 0.25 cm² inoculated mycellium. This strain secreted endocellulases and exocellulases in the culture medium, but some of the enzymes produced, remained cell membrane bound. Cell bound enzymes were released by various treatments. The highest amount of endoglucanase and exoglucanase was released when the cells were treated with sonication. Aspergillus terreus was added to two tanks containing sugar wastewater and pulp manufacturing waste, as a seed for COD removal. This fungus reduced the COD by 40–80 percent, also, ammonia was reduced from 14.5 mM to 5.6 mM in sugar beet wastewater. The effects of crude enzyme of this fungus for COD removal was studied.     

A heteropolysaccharide from aqueous extract of an edible mushroom, Pleurotus ostreatus cultivar: structural and biological studies

A water soluble polysaccharide isolated from the hot aqueous extract of Pleurotusostreatus cultivar was found to contain d-glucose and d-galactose in a molar ratio of nearly 7:1. Structural investigation was carried out using acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies (¹H, ¹³C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above mentioned experiments the structure of the repeating unit of the polysaccharide was established as:This heteroglycan stimulates macrophages, splenocytes, and thymocytes.     

Rapid strain classification and taxa delimitation within the edible mushroom genus Pleurotus through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy

Fourier transform infrared (FT-IR) spectroscopy has been successfully applied for the identification of bacteria and yeasts, but only to a limited extent for discriminating specific groups of filamentous fungi. In the frame of this study, 73 strains - from different associated hosts/substrates and geographic regions - representing 16 taxa of the edible mushroom genus Pleurotus (Basidiomycota, Agaricales) were examined through the use of diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. A binary matrix, elaborated on the basis of presence/absence of specific absorbance peaks combined with cluster analysis, demonstrated that the spectral region 1800-600 cm(-1) permitted clear delimitation of individual strains into Pleurotus species. In addition, closely related species (e.g., Pleurotus ostreatus and Pleurotus pulmonarius) or taxa of the subgenus Coremiopleurotus demonstrated high similarity in their absorbance patterns, whereas genetically distinct entities such as Pleurotus dryinus, Pleurotus djamor, and Pleurotus eryngii provided spectra with noteworthy differences. When specific regions (1800-1700, 1360-1285, 1125-1068, and 950-650 cm(-1)) were evaluated in respect to the absorbance values demonstrated by individual strains, it was evidenced that this methodology could be eventually exploited for the identification of unknown Pleurotus specimens with a stepwise process and with the aid of a dichotomous key developed for this purpose. Moreover, it was shown that the nature of original fungal material examined (mycelium, basidiomata, and basidiospores) had an effect on the outcome of such analyses, and so did the use of different mycelium growth substrates. In conclusion, application of FT-IR spectroscopy provided a fast, reliable, and cost-efficient solution for the classification of pure cultures from closely related mushroom species.     

New uses of food waste: application to laccase production by Trametes hirsuta

Diverse food wastes, apple, orange and potato, were screened for laccase production, under solid-state fermentation conditions, by the white-rot fungus Trametes hirsuta. Potato peelings gave the highest activity, reaching about 5000 U l^–1 within 8 days. These values are higher than those reported to date.     

Bioconversion of lignocellulosic waste from selected dumping sites in Dar es Salaam,Tanzania

The poor management of solid wastes in Tanzania urban centers is a chronic problem that has increasingly become a source of environmental pollution. Bioconversion offers a cheap and safe method of not only disposing these wastes, but also it has the potential to convert lignocellulosic wastes into usable forms such as reducing sugars that could be used as food. This paper reports a preliminary study on the physical characteristics, acid pretreatment, saccharification by cellulase from Trichoderma reesei and fermentation by Saccharomyces cerevisiae of the lignocellulosic component of the solid wastes collected from various dumping sites located in Kinondoni Municipality, Dar es Salaam city. The results showed that overall, the lignocellulosic component constitute about 50% of solid wastes dumped in the study areas. Maximum production of reducing sugars was obtained after 6 h of saccharification while highest concentrations of bioethanol were achieved after 48 h of fermentation. Microbial bioconversion of lignocellulose component yielded up to 21% bioethanol.     

The structure of a polysaccharide from Fraction-II of an edible mushroom, Pleurotus florida

A water-soluble polysaccharide was isolated from Fraction-II of the aqueous extract of the fruit bodies of the mushroom, Pleurotus florida. Compositional analysis, methylation analysis, periodate oxidation study, Smith degradation, and NMR studies (1H, 13C, DQF-COSY, TOCSY, NOESY, HSQC, and HMBC) revealed the presence of the following repeating unit in the polysaccharide: [structure: see text].     

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.     

The identification of a novel <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> dsRNA virus and determination of the distribution of viruses in mushroom spores

Double-stranded RNAs and virus particles were identified in Pleurotus ostreatus strain Shin-Nong in Korea. Isometric virus particles with a diameter of 33 nm were purified, which are similar to other Pleurotus viruses reported previously. This strain contains 5 dsRNAs, 8.0, 2.5, 2.4, 2.0, and 1.8 kb in size. The virus particles contain 2 dsRNAs, designated RNA-1 (2.5 kb), and RNA-2 (2.4 kb) which is a typical pattern of Partitiviridae. A non-encapsidated dsRNA of about 8.0 kb also was identified. Partial cDNA from RNA-1 was cloned, and sequence analysis revealed that this gene codes for RdRp. The comparison of the sequence from partial cDNA clone showed 35% amino acid homology with the C-terminal end of the RdRp gene of Helicobasidum mompa virus and Rosalinia necatrix virus. Specific primers designed from the partial sequences successfully amplified RT-PCR product from the infected mycelium and a single spore culture. We used these primers to determine the pattern of distribution of viruses in spores. Of the 96 different single spore cultures generated from Shin-Nong strain, a specific RT-PCR product was identified in 25 cultures, indicating that about 26% of basidiospores contain viruses.     

Evaluation of cellulase recycling strategies for the hydrolysis of lignocellulosic substrates

Recycling of cellulases should lower the overall cost of lignocellulosiic bioconversion processes. In this study, three recycling strategies were evaluated to determine their efficiencies over five successive rounds of hydrolysis. The effect of lignin on recycling was examined by comparing water-washed, steam-exploded birch (WB; 32% lignin) and WB which had been further extracted with alkali and peroxide (PB; 4% lignin). When the cellulases were recovered from the residual substrates after partial hydrolysis of both substrates, the recovered cellulase activity toward the mixture of fresh and residual substrates decreased after each recycling step. When the cellulases in the supernatants were also recycled, up to 20% more activity could be recovered. In both of these cases, the recovered activities did not correspond to the activities expected from the amount of cellulase protein recovered during recycling. The best recovery was obtained when the cellulases were recovered from both the residue and the supernatant after complete hydrolysis of the PB substrate. In this case, all of the originally added cellulase activity could be recovered for four consecutive hydrolysis rounds. However, when the same recycling strategy was carried out using the WB substrate, the recovered cellulase activity declined quickly with each recycling round. In all three of the recycling strategies, lower cellulase activities were recovered from the substrates with higher lignin contents. (c) 1995 John Wiley & Sons, Inc.     

Utilization of mucin by oral Streptococcus species

The ability of oral Streptococcus strains to utilize oligosaccharide chains in mucin as a source of carbohydrate was studied in batch cultures. Pig gastric mucin, as a substitute of human salivary mucin, was added to chemically defined medium containing no other carbohydrates. Strains of S. mitior attained the highest cell density, while mutans streptococci: S. mutans, S. sobrinus, S. rattus, grew very little in the medium with mucin. S. mitis, S. sanguis, and S. milleri in decreasing order, showed intermediate growth. Mucin break-down as measured by sugar analyses indicated that oligosaccharide chains were only partially degraded. Every strain produced one or more exoglycosidases potentially involved in hydrolysis of oligosaccharide. The enzyme activities occurred mainly associated with the cells, and very little activity was found in the culture fluids. The relationships between glycosidase activities and growth, or mucin degradation were not always clear.     

Production and characterization of somatic hybrids raised through protoplast fusion between edible mushroom strains Volvariella volvacea and Pleurotus florida

Twelve somatic hybrid lines were raised through polyethylene glycol-mediated intergeneric protoplast fusion between Volvariella volvacea and Pleurotus florida using a double selection method. Four hybrid lines belonging to two distinct colony morphology types could be maintained in culture. Basidiocarps could be generated from two hybrid lines, one of which showed resemblance to the P. florida parent while the other showed intermediate parental morphology. Hybridity of the fusant lines was established on the basis of colony morphology, mycelial growth rate, hyphal traits, fruit body morphology, isozyme and RAPD analysis. Four isozyme activities were analyzed to provide biochemical criteria for detection of polymorphism among the hybrids and their parents. Using 20 random decamers a total of 203 RAPD bands ranging from 0.09 to 1.6 kb were observed. The UPGMA method of clustering exhibited two major phylogenetic clusters, the first of which comprised of the parents, two fruit body generating hybrid lines and their subsequent fruit body derived lines, while the two non-fruit body generating hybrid lines formed the out group of the first major cluster. Screening of a Pleurotus type hybrid line having high biological efficiency and generation of a temperature tolerant Pleurotus type line through backcross mating between a non-fruit body generating somatic hybrid and V. volvacea parent are the key achievements of this fusion programme.