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.     

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.     

PRODUCTION OF LIGNINOLYTIC ENZYMES BY SOLID-STATE FERMENTATION USING Pleurotus eryngii

Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ability to produce various ligninolytic enzymes such as laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) by solid-state fermentation (SSF), which was carried out using a support substrate from the fruit juice industry. The chemical content of grape waste from this industry was studied. Also, the production patterns of these extracellular enzymes were researched during the growth of the organism for a period of 20 days and the protein, reducing sugar, and nitrogen levels were monitored during the stationary cultivation. The highest Lac activity was obtained as 2247.62 ± 75 U/L on day 10 in the presence of 750 µM Mn²⁺, while the highest MnP activity was attained as 2198.44 ± 65 U/L on day 15 in the presence of 500 µM Mn²⁺. Decolorization of methyl orange and reactive red 2 azo dyes was also achieved with ligninolytic enzymes, produced in SSF of P. eryngii.     

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.     

Effect of growth substrate,method of fermentation,and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml⁻¹) and xylanase (135 U ml⁻¹) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l⁻¹). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.     

Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.     

Waste-reducing cultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffee pulp: changes in the production of some lignocellulolytic enzymes

Fruiting body production for one strain of Pleurotus ostreatus and three strains of P. pulmonarius was evaluated on coffee pulp pasteurized at 80 °C for 1 h. Based upon three harvests per strain, the single P. ostreatus line was found to display a 40-day culture cycle, whereas the three P. pulmonarius strains completed their cycles after more than 50 days of incubation. These time periods were notably shorter than those observed in previous studies using other growth substrates. Nevertheless, yields expressed as biological efficiencies were not significantly different among strains, fluctuating between 125 and 138%. Extracellular enzymatic activity was also monitored for P. ostreatus and P. pulmonarius (one strain only). To do this, samples of mycelium-bearing substrate were taken every 4 days throughout the incubation period. Care was taken to represent all developmental stages, including primordial and fruiting bodies. Samples were either lyophilized and then analysed or, in some cases, analysed immediately without lyophilization. Hydrolase activity (i.e. endoglucanase (CMC) and cellobiohydrolase (CBH)) was found to depend on developmental stage, showing peak production during fruiting body formation. On the other hand, oxidase activity-(i.e. laccase (LAC) and Mn-peroxidase (MnP)) was associated with phenol degradation. Nevertheless, in the case of oxidases developmental timing differences were also observed. Specifically, LAC activity was detected as early as 8 days after inoculation in non-lyophilized samples, whereas MnP appeared near the end of the incubation period. No LAC activity was observed in lyophilized samples. This study concludes that coffee pulp might be successfully employed in the cultivation of mushrooms, not only because important extracellular enzymes are produced by mushrooms when grown upon this substrate, but also because the abbreviated cultivation cycle associated with this medium favours commercial processes. Commercialization might be further improved if strains specifically adapted to this novel substrate are selected.     

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.     

Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

Evaluation of Burma Reed as Substrate for Production of Pleurotus eryngii

Burma reed (Neyraudia reynaudiana), a giant C₄ grass, was included in substrate at the rates of 0, 20, 40 and 66 % to partially or wholly substitute sawdust and cottonseed hulls to evaluate its suitability for Pleurotus eryngii cultivation. Inclusion of 20 and 40 % Burma reed did not significantly affect linear mycelial growth, dry matter loss, spawn run period and fructification, and achieved high fruiting body yields and biological efficiency of 336.67 g/bag, 67.33 % and 342.15 g/bag, 68.43 %, respectively, which were not significantly different from 350.08 g/bag to 70.02 % obtained from the control substrate. Enzyme assay revealed that on the mixed substrates laccase and manganese peroxidase activity were significantly enhanced, but cellulase was significantly reduced in the middle stage of incubation as compared with the control substrate. Even on Burma reed substrate without sawdust and cottonseed hulls, fruiting body yield (313.56 g/bag) and biological efficiency (62.71 %) were satisfactory, although significantly lower than that on the control substrate. Therefore, Burma reed was a promising potential substrate for P. eryngii production to largely substitute sawdust and cottonseed hulls.     

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.     

Interaction of Trace Elements and Ligninolytic Enzymes in <Emphasis Type="Italic">Pleurotus eryngii</Emphasis>

Considering the fact that some trace elements have influence on the growth and physiological processes in the fungi, the aim of this study was to resolve the question of whether Fe, Zn, and Se, added to the medium in the different forms, affect activity of selected Pleurotus eryngii ligninolytic enzymes participating in mandarine peels submerged fermentation. Compared with the control, Fe and Zn showed stimulatory effect on laccase activity. Reduction in the activity was emphasized with cultivation time, but it was slower in the presence of Zn than in the presence of Fe. Among studied Se forms, SeO₂ was the most unfavorable Se source for laccase activity. All tested trace elements, independent on form, showed stimulatory effect on the activity of Mn-oxidizing peroxidases on the seventh day of cultivation. Based on the results, it could be concluded that the presence of the tested trace elements can advance various biotechnological processes among which is biodegradation of agricultural wastes, potential environmental pollutants.     

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     

Genetic diversity,core collection and breeding history of Pleurotus ostreatus in China

Pleurotus ostreatus is one of the most widespread and favourably cultivated mushrooms in China. The cultivated strains of this species have been frequently exchanged domestically and internationally, but no detailed breeding history has been documented. The frequent domestic and international exchange of strains combined with a non-detailed historical documentation of breeding might have led to confusion about strain names and genetic background. In this study, 91 strains of P. ostreatus, including 57 cultivated and 34 wild strains, were analysed using 21 simple sequence repeat markers developed from the genomic sequence of “P. cf. floridanus”. Among the cultivated strains, 46 were found to possess different allelic patterns. The remaining 11 strains were clustered into five groups, each with their own private alleles, suggesting that 10.5% (6/57) of the cultivated strains were previously labelled with improper names. Our analyses indicate that wild strains harbored greater genetic diversity than the cultivated strains. With regard to the cultivation history of P. ostreatus, the cultivated strains in China have three sources: direct introduction from Europe, domestication from wild strains from China, or hybridisation of the European and Chinese strains. Furthermore, we propose a core collection of P. ostreatus with 34 strains, including 13 wild and 21 cultivated strains. The allele retention proportion of the core collection for the entire collection was 100%.     

Effects of concentrated carbon dioxide on the fruiting of several cultivated basidiomycetes (II)

Edible basidiomycetesFlammulia velutipes andPleurotus ostreatus were cultivated in the usual manner on media based on sawdust and rice bran, and the cultures were exposed to slowly flowing CO₂-enriched air (550 (control), 3,000, 6,000, and 9,000µl/l) for seven days at different stages of cultivation. When the cultures were exposed at the primordium stage (less than 10 mm in length), length and yield of fruit-bodies increased and pileus expansion was slightly inhibited inF. velutipes, while inP. ostreatus length increased, yield decreased, and pileus expansion was greatly inhibited. When the cultures with fruit-bodies larger than 10 mm were exposed, length and yield were insensitive and pileus expansion was greatly inhibited inF. velutipes, while inP. ostreatus length was insensitive, but pileus expansion was heavily damaged by trumpet-like deformation and yield decreased. The different action of CO₂ on the two species appeared to be due to the different anatomical structures of their fruit-bodies.     

Genetic Variability and Population Structure of the Mushroom Pleurotus eryngii var. tuoliensis

The genetic diversity of 123 wild strains of Pleurotus eryngii var. tuoliensis, which were collected from nine geographical locations in Yumin, Tuoli, and Qinghe counties in the Xinjiang Autonomous Region of China, was analysed using two molecular marker systems (inter-simple sequence repeat and start codon targeted). At the variety level, the percentage of polymorphic loci and Nei’s gene diversity index for P. eryngii var. tuoliensis was 96.32% and 0.238, respectively. At the population level, Nei’s gene diversity index ranged from 0.149 to 0.218 with an average of 0.186, and Shannon''s information index ranged from 0.213 to 0.339 with an average of 0.284. These results revealed the abundant genetic variability in the wild resources of P. eryngii var. tuoliensis. Nei’s gene diversity analysis indicated that the genetic variance was mainly found within individual geographical populations, and the analysis of molecular variance revealed low but significant genetic differentiation among local and regional populations. The limited gene flow (Nm = 1.794) was inferred as a major reason for the extent of genetic differentiation of P. eryngii var. tuoliensis. The results of Mantel tests showed that the genetic distance among geographical populations of P. eryngii var. tuoliensis was positively correlated with the geographical distance and the longitudinal distances (r_Go = 0.789 and r_Ln = 0.873, respectively), which indicates that geographical isolation is an important factor for the observed genetic differentiation. Nine geographical populations of P. eryngii var. tuoliensis were divided into three groups according to their geographical origins, which revealed that the genetic diversity was closely related to the geographical distribution of this wild fungus.     

Solid state production of polygalacturonase and xylanase by Trichoderma species using cantaloupe and watermelon rinds

Different solid state fermentation (SSF) sources were tested such as cantaloupe and watermelon rinds, orange and banana peels, for the production of polygalacturonase (PG) and xylanase (Xyl) by Trichoderma harzianum and Trichoderma virens. The maximum production of both PG and Xyl were obtained by T. harzianum and T. virnes grown on cantaloupe and watermelon rinds, respectively. Time course, moisture content, temperature, pH, supplementation with carbon and nitrogen sources were optimized to achieve the maximum production of both PG and Xyl of T. harzianum and T. virens using cantaloupe and watermelon rinds, respectively. The maximum production of PG and Xyl of T. harzianum and T. virens was recorded at 4–5 days of incubation, 50–66% moisture, temperature 28–35°C and pH 6–7. The influence of supplementary carbon and nitrogen sources was studied. For T. harzianum, lactose enhanced PG activity from 87 to 120 units/g solid, where starch and maltose enhanced Xyl activity from 40 to 55–60 units/g solid for T. virnes. Among the nitrogen sources, ammonium sulphate, ammonium nitrate, yeast extract and urea increased PG activity from 90 to 110–113 units/g solid for T. harzianum. Similarly, ammonium chloride, ammonium sulphate and yeast extract increased Xyl activity from 45 to 55–70 units/g solid for T. virens.     

Edible mushrooms from olive oil mill wastes

The biological remediation of olive oil mill wastes has been attempted several times in the past through the use of different types of microbes. Among them, a relatively large array of fungi were studied for neutralizing the heavy pollutant effects and/or for converting these wastes into new value-added products. The present investigation was aiming at examining whether olive oil mill wastes could be exploited for the cultivation of mushrooms of the genus Pleurotus. At a preliminary stage, two Pleurotus species, i.e. P. eryngii and P. pulmonarius, were tested for their ability to colonize an olive press-cake (OPC) substrate supplemented with various dilutions of raw olive mill wastewater (OWW). Some important cultural characters related to mushroom production (earliness, yield, biological efficiencies and quality of basidiomata) were estimated. The outcome revealed different cultural responses for each Pleurotus species examined; the P. pulmonarius strain showed better earliness values and P. eryngii, although it was a slow growing fungus, produced basidiomata in high yields and of a very good quality. On the other hand, the OPC substrate supplemented with low concentrations of OWW (12.5% v/w) behaved satisfactorily as regards the fungal colonization rates and mushroom yield, but when the addition of higher rates of raw, untreated OWW (75–100% v/w) was attempted then the Pleurotus strains were completely unable to grow. The optimal concentration of OWW for Pleurotus mycelial growth was assessed through measurements of the biomass produced in liquid nutrient media and was found to lie within the 25–50% range, depending on the Pleurotus species and on the properties of the substrates examined. Furthermore, the phytotoxic effects that the spent liquid medium possessed were examined in comparison with the phytotoxicity of the raw liquid waste. The prospects of exploiting olive oil mills wastes for mushroom cultivation is discussed.