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.     

Seven <Emphasis Type="Italic">Armillaria</Emphasis> species identified from Hokkaido Island,northern Japan

Sixty-two isolates from basidiocarps of Armillaria spp. were obtained from Hokkaido Island, northern Japan. Six species (Armillaria cepistipes, A. gallica, A. nabsnona, A. ostoyae, A. sinapina, and an undescribed species, “Nag. E”) were identified by pairing tests with known tester strains and one subspecies (Armillaria mellea subsp. nipponica, a non-heterothallic form of A. mellea) was identified by its macro- and micro-morphological characters of the basidiocarps. This is the first case of “Nag. E” being reported from Hokkaido Island.     

Mycelium growth kinetics and optimal temperature conditions for the cultivation of edible mushroom species on lignocellulosic substrates

The influence of environmental parameters on mycelial linear growth ofPleurotus ostreatus, P. eryngii, P. pulmonarius, Agrocybe aegerita, Lentinula edodes, Volvariella volvacea andAuricularia auricula-judae was determined in two different nutrient media in a wide range of temperature, forming the basis for the assessment of their temperature optimaV. volvacea grew faster at 35°C,P. eryngii at 25°C,P. ostreatus andP. pulmonarius at 30°C,A. aegerita at 25 or 30°C andA. auricula-judae at 20 or 25°C depending on the nutrient medium used andL. edodes at 20 or 30°C depending on the strain examined. The mycelium extension rates were evaluated on seven mushroom cultivation substrates: wheat straw, cotton gin-trash, peanut shells, poplar sawdust, oak sawdust, corn cobs and olive press-cake. The mycelium extension rates (linear growth and colonization rates) were determined by the ‘race-tube’ technique, and were found to be the highest on cotton gin-trash, peanut shells and poplar sawdust forPleurotus spp. andA. aegerita. Wheat straw, peanut shells and particularly cotton gin-trash supported fast growth ofV. volvacea, whereas wheat straw was the most suitable substrate forL. edodes andA. auricula-judae. Supplemented oak sawdust and olive press-cake were poor substrates for most species examined, white almost all strains performed adequately on corn cobs.     

The relationship betweenPleurotus ostreatus andAspergillus flavus and the production of aflatoxin

The relationship betweenPleurotus ostreatus andAspergillus flavus in common mixed culture on various substrates was investigated. It was found thatP. ostreatus, similarly to some other higher fungi, can liquidate coloniesof A. flavus. This fungus does not produce aflatoxin and chromatographically similar compounds. On straw, corn cobs, millet and wheatA. flavus produced aflatoxin after a 3-week cultivation. A subsequent cultivation of P.ostreatus led to detoxication of straw and corn cobs but millet and wheat were not detoxicated. Cultivation of P.ostreatus in the presence of 40–100 μg of aflatoxin B₁ per g substrate did not result in detoxication of the material even after 34 d but the results showed that the aflatoxin concentration decreased to about one-fourth of the added amount.     

Kinetics of wheat straw solid-state fermentation with Trametes versicolor and Pleurotus ostreatus — lignin and polysaccharide alteration and production of related enzymatic activities

Summary The kinetics of straw solid-state fermentation (SSF) with Trametes versicolor and Pleurotus ostreatus was investigated to characterize the delignification processes by these white-rot fungi. Two successive phases could be defined during straw transformation, characterized by changes in respiratory activity, changes in lignin and polysaccharide content and composition, increase in in-vitro digestibility, and enzymatic activities produced by the fungi. Lignin composition was analysed after CuO alkaline degradation, and decreases in syringyl/guaiacyl and syringyl/p-hydroxyphenyl ratios and cinnamic acid content were observed during the fungal treatment. An increase in the phenolic acid yield, revealing fungal degradation of side-chains in lignin, was produced by P. ostreatus. The highest xylanase level was produced by P. ostreatus, and exocellulase activity was nearly absent from straw treated with this fungus. Lactase activity was found in straw treated with both fungi, but lignin peroxidase was only detected during the initial phase of straw transformation with T. versicolor. High levels of H₂O₂-producing aryl-alcohol oxidase occurred throughout the straw SSF with P. ostreatus. Offprint requests to: A. T. Martínez     

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10–20% SW, 70–80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6–21.8% lignin, 33.1–55.2% cellulose and 14–53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.     

Comparative secretome of white-rot fungi reveals co-regulated carbohydrate-active enzymes associated with selective ligninolysis of ramie stalks

In the present research, Phanerochaete chrysosporium and Irpex Lacteus simultaneously degraded lignin and cellulose in ramie stalks, whereas Pleurotus ostreatus and Pleurotus eryngii could depolymerize lignin but little cellulose. Comparative proteomic analysis of these four white-rot fungi was used to investigate the molecular mechanism of this selective ligninolysis. 292 proteins, including CAZymes, sugar transporters, cytochrome P450, proteases, phosphatases and proteins with other function, were successfully identified. A total of 58 CAZyme proteins were differentially expressed, and at the same time, oxidoreductases participated in lignin degradation were expressed at higher levels in P. eryngii and P. ostreatus. Enzyme activity results indicated that cellulase activities were higher in P. chrysosporium and I. lacteus, while the activities of lignin-degrading enzymes were higher in P. eryngii and P. ostreatus. In addition to the lignocellulosic degrading enzymes, several proteins including sugar transporters, cytochrome P450 monooxygenases, peptidases, proteinases, phosphatases and kinases were also found to be differentially expressed among these four species of white-rot fungi. In summary, the protein expression patterns of P. eryngii and P. ostreatus exhibit co-upregulated oxidoreductase potential and co-downregulated cellulolytic capability relative to those of P. chrysosporium and I. lacteus, providing a mechanism consistent with selective ligninolysis by P. eryngii and P. ostreatus.     

Limited degradation of industrial,synthetic and natural lignins by Serratia marcescens

Summary Serratia marcescens was found to degrade kraft lignin by only 15%. When ¹⁴C-radiolabelled lignocelluloses and DHP lignins were used as substrates the bacterium mineralized to ¹⁴CO₂ only 1.1–1.9% and 0.4–0.8% of the lignins respectively. However, some 44.4% of the ¹⁴C--DHP lignin was recovered as soluble radiolabelled products.     

Involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation by the wood-rotting fungusPleurotus ostreatus

The mycelium of the wood-rotting fungus,P. ostreatus, contains NAD-dependent glutamate synthase inhibited by azaserine.l-Glutamine andl-glutamate are the most important free amino acids in the mycelium. Feeding of the mycelium with nitrogenous substrates showed thatl-glutamate,l-aspartate andl-alanine are interconnected by way of transaminases. After the inhibition of glutamine synthetase by methionine-S-sulfoximine the synthesis ofl-glutamate was inhibited and the level of all free amino acids decreased. The¹⁵N-NMR spectra of mycelia after the addition of¹⁵NH₄Cl confirmed that the GS/GOGAT is the only pathway of ammonia assimilation inP. ostreatus and NAD-glutamate dehydrogenase should be the deaminating enzyme.     

Improved conditions for protoplast formation and transformation of Pleurotus ostreatus

Conditions suitable for the production and regeneration of Pleurotus ostreatus protoplasts from dikaryotic mycelia were examined. Three commercially available muralytic enzymes, including Sigma lysing enzyme, Novozym 234 and Novozym 234 LP, were used for production of protoplasts. Over 2 × 10⁷ protoplasts per gram fresh weight mycelia were obtained within 1.5 h by using each of these three enzymes. The colony regeneration rate was up to 13% on potato-dextrose-agar medium containing 0.8 m mannitol. Genetic transformation was based on positive selection for resistance to hygromycin B (HmB) using the plasmid vector pAN7-1 and accomplished by either electroporation or a polyethylene glycol (PEG)-divalent cation method. P. ostreatus strains used in this study have innate sensitivity to HmB at a critical inhibitory concentration of between 40–50 g/ml. Selection for HmB resistance of this fungus, indicative of transformation, resulted in 3–48 HmB-resistant colonies per microgram of pAN7-1 per 10⁷ viable protoplasts. No significant differences were apparent when either transformation protocol or either P. ostreatus strain was used. The best electrical condition found for the electrotransformation of P. ostreatus is at a field strength of 2.6–2.8 kV/cm with a capacitance of 25F and a parallel resistance of 800 ohms, corresponding to a time constant range of 10–14 ms. Correspondence to: P. A. Lemke     

Decay rate and substrate quality of fine roots and foliage of two tropical tree species in the Luquillo Experimental Forest,Puerto Rico

Decomposition rates, initial chemical composition, and the relationship between initial chemistry and mass loss of fine roots and foliage were determined for two woody tropical species, Prestoea montana and Dacryodes excelsa, over a gradient of sites in two watersheds in the Luquillo Experimental Forest, Puerto Rico. At all locations, fine roots decayed significantly more slowly than foliage during the initial 6 months.Substrate quality of the initial tissue showed marked differences between roots and foliage when using cell wall chemistry, secondary chemistry and total elemental analysis as indices. Quantity of acid detergent fiber (ADF) (non-digestible cell wall fiber) and lignin content were higher for roots than leaves: D. excelsa roots had 55.3% ADF and 28.7% lignin while leaves had 36.2% ADF and 11.8% lignin; P. montana roots had 68.0% ADF and 26.8% lignin while leaves had 48.5% ADF and 16.1% lignin. Aluminum concentrations were higher in fine roots (843 mg kg^–1 in D. excelsa, 1500 mg kg^–1 in P. montana) than leaves (244 mg kg^–1 in D. excelsa, 422 mg kg^–1 in P. montana), while calcium concentrations were higher in foliage (5.5 mg g^–1 in D. excelsa, 7.8 mg g^–1 in P. montana) than roots (3.4 mg g^–1 in D. excelsa, 3.1 mg g^–1 in P. montana). Nitrogen did not show any trend with tissue or species type. A linear model between mass remaining after 6 months and initial tissue chemistry could be developed only for calcium (r²=0.64).     

Decomposition patterns of unprocessed and processed lignocellulosics in a freshwater fish pond

In an attempt to recycle the lignocellulosic wastes like Eichhornia crassipes, Salvinia cucullata and rice (Oryza sativa) straw as manurial inputs in freshwater fish pond ecosystem, a decomposition experiment was carried out in litter bags in an oligotrophic freshwater fish pond environment, with the above mentioned three substrates in unprocessed and microbially processed forms. The loss rates, associated microbial groups, oxygen consumption patterns and other related parameters like carbon, nitrogen, phosphorus, cellulose, hemicellulose and lignin were analysed. The mean daily dry matter loss rates (unprocessed: 10.44>6.97>1.97 and processed: 11.03>8.21>3.67) and oxygen uptake rate (unprocessed: 0.675>0.571>0.568 mg O₂ g^–1 h^–1 and processed: 0.592>0.424>0.407 mg O₂ g^–1 h^–1) in raw and processed substrates were in the sequence Eichhornia > rice straw > Salvinia. The oxygen consumption pattern almost covariated with variations in temperature of pond water, daily dry matter loss rates and fungal counts on substrates. During the decay, the percentage of N and P increased whereas that of C decreased, resulting in lowering of C/N and C/P ratios of the substrates. The structural polymeric fractions like cellulose and hemicellulose decreased along with dry matter whereas the lignin content increased after an initial decrease due to loss of other structural carbohydrates resulting in apparent per cent gain of lignin. A higher number of different heterotrophic bacterial groups was observed in the processed substrates as compared to their raw counterparts. However, cellulolytic bacterial numbers were found to fluctuate through the study period. The fungal load was found to be decreasing gradually as the decay progressed. In this study, bacteria were found to be the prominent microbial group responsible for the decay. The nitrogen-fixing, phosphatase-producing and phosphorus-solubilising bacterial groups were observed to play an important role in lowering the C/N and C/P ratios of the decomposing substrates during decay.     

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.     

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.     

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.     

Yield,size, nutritional value,and antioxidant activity of oyster mushrooms grown on perilla stalks

Perilla is an edible medical plant with rapidly increasing acreage in China. In this study, we investigated the potential of perilla stalks (PSs) as an alternative substrate for the cultivation of oyster mushrooms (Pleurotus ostreatus). P. ostreatus was cultivated on cottonseed hulls (CSH) alone or mixed with PSs in different ratios. The production parameters, physical characteristics, nutritional values, and antioxidant activity of mushrooms cultivated on different substrate mixtures were determined. The addition of PSs to CSH significantly improved the growth rate, yield, biological efficiency, and proximate composition and shortened the cultivation cycle. Cultivation on PSs alone increased the amino acid content in P. ostreatus fruiting bodies and the antioxidant activity of mushroom extracts. The PS75 (25% CSH + 75% PS) substrate was deduced to be the most effective substrate on the basis of yield and biological efficiency obtained in a large area where perilla had been planted. The results demonstrate that mixtures of PS with CSHs could be used as novel, practical, and easily accessible alternative substrates for P. ostreatus cultivation.     

Degradation of 14C-labelled poplar wood lignin by selected white-rot fungi

Summary Of eight white-rot fungi examined, seven fungi grew on nitrogen-limited poplar wood meal medium and degraded ¹⁴C-lignin in wood meal to ¹⁴CO₂. Increased oxygen enhanced both the rate and extent of degradation. However, whereas Pleurotus ostreatus, Pycnoporus cinnabarinus 115 and Pycnoporus cinnabarinus A-360 degraded 12–17% of ¹⁴C-(U)-lignin of poplar wood to ¹⁴CO₂ also in an air atmosphere, Sporotrichum pulverulentum, Phlebia radiata 79 and Phanerochaete sordida 37 degraded only 1–5% under these conditions. Addition of cellulose and glucose to the poplar wood medium stimulated degradation of ¹⁴C-(RING)-lignin of poplar wood by Phlebia radiata 79 but repressed degradation by Polyporus versicolor and Pleurotus ostreatus. Cellulose added to the wood meal medium had no effect on the degradation of lignin by Phanerochaete sordida 37 and Sporotrichum pulverulentum but glucose slightly repressed lignin degradation by these fungi. Those white-rot fungi which were considered as preferentially lignin attacking fungi could degrade ¹⁴C-(RING)-lignin of poplar wood efficiently under 100% oxygen. They did not require an extra energy source in addition to wood meal polysaccharides for rapid ring cleavage and they degraded up to 50–60% of the ¹⁴C-lignin to ¹⁴CO₂ in 6–7 weeks at a maximum rate of 3–4% per day.These results were reported in part at the Journées Internationales d'Etudes du Groupe Polyphenols, 29. 9.–1. 10. 1982, Université Paul Sabatier, Toulouse, France     

Production of extracellular enzymes by two Pleurotus species using banana pseudostem biomass

The degradation of lignocellulosic biomass of banana pseudoste was investigated during solid state fermentation (SSF) by P. ostreatus and P. sajor-caju. Both organisms proved to be efficient degraders of banana pseudostem biomass. P.ostreatus degraded hemicellulose (40% of dry weight, d.w.) better than cellulose (17.5% of d.w.) and lignin (10% of d.w.). P. sajor-caju also degraded hemicellulose (31% of d.w.) better than cellulose (12.4% of d.w.) and lignin (6% of d.w.). In both cases, a preferential removal of hemicellulose during the initial growth period and a delayed degradation of lignin were observed. The kinetics of cellulolytic, hemicellulolytic and lignolytic enzyme production in liquid culture were also examined. The activities of CMCase and β-glucosidase were highest at 16 days of growth and avicelase activity was at its maximum after 24 days (CMCase - 1.1 IU/ml, β-glucosidase - 0.09 IU/ml in the case of P. ostreatus; CMCase - 1.0 IU/ml, β-glucosidase - 0.087 - IU/ml in the case of P. sajor-caju.). Xylanase and laccase activity reached their maximum after day 16 and day 24 of incubation, respectively. (Xylanase - 1.1 IU/ml and laccase 3.0 IU/ml in the case of P. ostreatus; xylanase - 1.0 IU/ml and laccase - 3.6 IU/ml in the case of P. sajor-caju.). The efficient degrading capacity of test fungi demonstrated their potential use in the conversion of banana pseudostem biomass into mycelial protein-rich fermented animal feed.     

Foliar and Litter Nutrients, Nutrient Resorption, and Decomposition in Hawaiian Me t rosideros polymorpha

The native tree Metrosideros polymorpha dominates Hawaiian forests across a very wide range of soil fertility, including both sites where forest production is limited by nitrogen (N) and others where it is limited by phosphorus (P). Five long-term fertilization experiments have further broadened the range of nutrient availabilities experienced by Metrosideros. Adding P to P-limited sites increased foliar P concentrations threefold and litter P concentrations up to 10-fold; lignin concentrations decreased, and the decomposability of leaf litter increased from 32%–35% to 36%–46% mass loss in the first year. Adding N to N-limited sites increased leaf and litter N concentrations by only 15%–20%, with little or no effect on the decomposability of tissue. Received 22 January 1998; accepted 4 May 1998.