Successful large-scale production of fruiting bodies of Laetiporus sulphureus (Bull.: Fr.) Murrill on an artificial substrate

Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5–6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40–45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200–300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.     

Mass separation and in vitro immunological activity of membrane-fractionated polysaccharides from fruiting body and mycelium of Agaricus subrufescens

Membrane technology has been applied to separate polysaccharides from Agaricus subrufescens (ASPs). The membrane-retained fractions and unfractionated preparations have been tested for in vitro immunological activity. Both the microfiltration (MF) and ultrafiltration (UF1) membranes were able to separate high-molecular weight polysaccharides from fruiting body (ASP-FB) and submerge-fermented mycelium (ASP-SmF) extracts. All fractions showed immunostimulatory effects on RAW 264.7 macrophages, measured by TNF-??, iNOs gene expression, and NO production. In contrast, antibody and proliferation levels in B lymphoblastoid SKW 6.4 cells were significantly increased after treatment with ASP-FB, but did not with ASP-SmF preparations. The ASPs- and LPS-induced stimulation could be differentiated by the finding that polymyxin B, a specific inhibitor of LPS, did not significantly affect the immunoactivating response and proliferation activity of ASPs on macrophages and B cells, respectively. Furthermore, the ASP-FB treatment was unable to induce IL-6 production by B cells unlike LPS activation, sustaining distinct signaling pathways for ASP-FB and LPS. The overall results provided additional information about the action of ASPs on the immune system and support the membrane method to separate and concentrate high-molecular weight ASPs for immunopharmacological and biotechnological applications.     

Comparative Study of Metals Accumulation in Cultured In Vitro Mycelium and Naturally Grown Fruiting Bodies of Boletus badius and Cantharellus cibarius

Cantharellus cibarius Fr. (chanterelle) and Boletus badius Pers. (bay bolete) harvested from natural sites in Poland were used to derive in vitro cultures. The optimal medium composition for cultures was developed. Concentrations of the chosen elements (Zn, Cu, Fe, Mg, Ni, and Cd) in mycelium samples were measured by means of atomic absorption spectrometry. Fe concentration in the analyzed mushroom materials was in the range 215.4–680.3 μg/g dry weight. Mean values of Mg were respectively (in micrograms per gram dry weight) 541.8 for mycelium of C. cibarius cultured in vitro and 1,004.1 for C. cibarius fruiting bodies and 928.9 for the mycelium of B. badius cultured in vitro and 906.4 for B. badius fruiting bodies. The mean concentrations of Zn were 442.7 μg/g dry weight in mycelium from in vitro cultures of B. badius and 172.1 in B. badius fruiting bodies and 131.9 in the case of C. cibarius in mycelium from in vitro cultures and 95.5 for the C. cibarius fruiting bodies. Cu exhibited a reversal tendency, i.e., the element concentrations in naturally grown mushrooms were significantly higher (43.57 μg/g dry weight for C. cibarius and 43.54 μg/g for B. badius) than in cultured in vitro mycelium (12.47 μg/g for C. cibarius and 4.17 μg/g for B. badius). Ni was found in lowest concentrations ranging from 0.33 to 1.88 μg/g dry weight. Toxic metal Cd was found in relatively high concentrations in naturally grown species (0.79 μg/g dry weight—1.02). The lowest was the concentration of Cd in C. cibarius mycelium from in vitro culture—0.06 μg/g dry weight—a bit higher than it was in the B. badius mycelium (0.21 μg/g).     

Polluting macrophytes Colombian lake Fúquene used as substrate by edible fungus Pleurotus ostreatus

Invasive aquatic plants from Lake Fúquene (Cundinamarca, Colombia), water hyacinth (Eichhornia crassipes C. Mart.) and Brazilian elodea (Egeria densa Planch.) have been removed mechanically from the lake and can be used for edible mushrooms production. The growth of the oyster mushroom (Pleurotus ostreatus) on these aquatic macrophytes was investigated in order to evaluate the possible use of fruiting bodies and spent biomass in food production for human and animal nutrition, respectively. Treatments included: water hyacinth, Brazilian elodea, sawdust, rice hulls and their combinations, inoculated with P. ostreatus at 3 %. Water hyacinth mixed with sawdust stimulated significantly fruiting bodies production (P = 3.3 × 10^?7) with 71 % biological efficacy, followed by water hyacinth with rice husk (55 %) and elodea with rice husk (48 %), all of these have protein contents between 26 and 47 %. Loss of lignin (0.9–21.6 %), cellulose (3.7–58.3 %) and hemicellulose (1.9–53.8 %) and increment in vitro digestibility (16.7–139.3 %) and reducing sugars (73.4–838.4 %) were observed in most treatments. Treatments spent biomass presented Relative Forage Values (RFV) from 46.1 to 232.4 %. The results demonstrated the fungus degrading ability and its potential use in aquatic macrophytes conversion biomass into digestible ruminant feed as added value to the fruiting bodies production for human nutrition.     

Development of antler-type fruiting bodies of Ganoderma lucidum and determination of its biochemical properties

Following the importance of antler-type fruiting bodies of Ganoderma lucidum, in this study, the impact of main growth parameters such as ventilation and light on the development of antler-type fruiting bodies has been investigated together with the determination of physico-chemical properties of antler fruiting bodies. For this, the primordia bags of G. lucidum were kept under controlled ventilation to adjust the CO₂ produced by the mushrooms owing to its respiration under light and dark conditions. The bioactive compounds such as phenolics, flavonoids, water-soluble polysaccharides and ganoderic acid showed a two-fold increase in the antler-type fruiting bodies as compared to normal kidney-shaped fruiting bodies. It is assumed from this study that the antler type fruiting bodies are developed due to restricted ventilation which causes an increase in the level of CO₂ gas in the air as a result of respiration of mushroom. The shape and colour of antler fruiting bodies again dependent on the light provided in the growth chamber. This study also proves that with the manipulation of light and ventilation antler-type fruiting bodies of G. lucidum could be developed with higher quantity of bioactive compounds and with higher antioxidant potential.     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

The first set of expressed sequence tags (EST) from the medicinal mushroom Agaricus subrufescens delivers resource for gene discovery and marker development

Agaricus subrufescens is one of the most important culinary-medicinal cultivable mushrooms with potentially high-added-value products and extended agronomical valorization. The development of A. subrufescens-related technologies is hampered by, among others, the lack of suitable molecular tools. Thus, this mushroom is considered as a genomic orphan species with a very limited number of available molecular markers or sequences. To fill this gap, this study reports the generation and analysis of the first set of expressed sequence tags (EST) for A. subrufescens. cDNA fragments obtained from young sporophores (SP) and vegetative mycelium in liquid culture (CL) were sequenced using 454 pyrosequencing technology. After assembly process, 4,989 and 5,125 sequences were obtained in SP and CL libraries, respectively. About 87 % of the EST had significant similarity with Agaricus bisporus-predicted proteins, and 79 % correspond to known proteins. Functional categorization according to Gene Ontology could be assigned to 49 % of the sequences. Some gene families potentially involved in bioactive compound biosynthesis could be identified. A total of 232 simple sequence repeats (SSRs) were identified, and a set of 40 EST-SSR polymorphic markers were successfully developed. This EST dataset provides a new resource for gene discovery and molecular marker development. It constitutes a solid basis for further genetic and genomic studies in A. subrufescens.     

pH-induced flocculation,indirect electrocoagulation,and hollow fiber filtration techniques for harvesting the saltwater microalga Dunaliella

This research assessed the efficacy of three harvesting methods on a strain of Dunaliella viridis. While there is strong potential to use lipids from microalgae as a feedstock for biofuels to replace petroleum-based fuel, at present microalgal harvesting for biofuel production is not yet economically feasible or energy efficient. pH-induced flocculation (by adjusting the pH of exponentially growing cells), indirect electrocoagulation (applying aluminum hydroxide coagulant to culture), and hollow fiber filtration (separating biomass from medium using tangential flow) were compared as potential harvesting mechanisms for small-scale (3–10 L) and large-scale (30–150 L) volumes of D. viridis. Both pH-induced flocculation and electrocoagulation yielded significant biomass recovery (>95 %), but both methods required removal of added chemicals and/or coagulant before the medium could be reused. In contrast, hollow fiber filtration did not require added chemicals or coagulant, and as another advantage, the filtrate was successfully reused as culture medium without apparent detrimental effects on cell size, cell shape, or cell production. When high salinity stress was imposed on the concentrate produced from the filtration method, total fatty acids (FAs) did not increase. However, total FAs did significantly increase after hollow fiber filtration (49 %) in comparison to FA content before filtration (36 %). This research indicates that hollow fiber filtration as a commercial harvesting mechanism offers attractive advantages as a harvesting mechanism for microalgae such as Dunaliella, relative to pH-induced flocculation and indirect electrocoagulation.     

Stimulation of the formation of fruiting bodies of the fungusLentinus tigrinus (Bull.) Fr. by growth regulators

Growth regulators, indole-3-acetic acid (IAA), gibberellic acid (GA₃), and kinetin (KIN), were used in different concentrations to stimulate the initiation and further development of the fruiting bodies of the fungusLentinus tigrinus. Vegetative mycelium of the fungus was cultivated on cellulose cylinders soaked with a synthetic nutrient solution or with a 3% malt extract. When the mycelium covered the surface of the cylinders, further cultivation was carried out in graduated concentrations of the growth regulators mentioned above. The number of developed fruiting bodies showed that the optimum IAA and GA₃ concentrations were in both media 300 p.p.m. The optimum concentration of kinetin was 400 p.p.m. The individual growth regulators influenced characteristically also the shape of the fruiting bodies and changed in them the natural level of endogenous growth regulators. The addition of IAA into the medium raised the level of endogenous auxins in the cap. The presence of gibberellic acid and of kinetic in the medium resulted in an increase in the level of these regulators in the fruiting bodies.     

Chemical Composition of the Edible Mushroom Pleurotus ostreatus Produced by Fermentation

Pleurotus ostreatus `Florida' was grown in submerged liquid culture. The biomass yield of the fungus, grown for 3 days in 2-liter fermentors, where the mycelial pellets measuring 5 mm in diameter were formed, was 11.7 g (dry weight)/liter. Comparing the chemical constituents of fruiting bodies produced on cotton straw and mycelial pellets revealed several similarities in total nitrogen, protein, glycogen, fatty acids, RNA, and ash content. Differences were observed in the contents of six amino acids. Although the total fatty acid content was similar, there were more saturated fatty acids in the mycelium. Cell wall composition, typical for basidiomycetes, was observed in both mycelium and fruiting bodies, with laminarin as the main polymer.     

Lactarius deliciosus and Pinus radiata in New Zealand: towards the development of innovative gourmet mushroom orchards

The cultivation of Lactarius deliciosus (saffron milk cap) in New Zealand began in 2002 when fruiting bodies were produced in an Otago plantation of Pinus radiata seedlings artificially mycorrhized by L. deliciosus. In 2007, 42 P. radiata seedlings mycorrhized by L. deliciosus under controlled conditions were planted in a grass field at Plant and Food Research (Lincoln, Canterbury). The effects of pine bark mulch application and initial degree of mycorrhization of seedlings were examined to determine their influence on tree growth, development of mycorrhizae (i.e. their multiplication on the root system and their degree of branching) and fruiting body production. Mulch application increased tree growth significantly over 4 years. High initial mycorrhization slightly stimulated tree growth over 2 years. The initial degree of mycorrhization was positively, but not strongly, related to the persistence and development of L. deliciosus mycorrhizae and rhizomorphs based on root sample analyses 2 years after planting. However, mulching strongly reduced the proportion of highly branched L. deliciosus mycorrhizae compared with poorly ramified ones. A positive correlation was observed between the fruiting of L. deliciosus and the development of mycorrhizae. Mulching delayed the onset of fruiting body production. In 2010, fruiting bodies were produced only from non-mulched trees with eight of these (38 %) producing a total of 12 fruiting bodies. In 2011, 19 non-mulched trees (90 %) and 9 mulched trees (45 %) produced 143 and 47 fruiting bodies, respectively, totalling 190 fruiting bodies. By 2012, 19 non-mulched trees (90 %) and 13 mulched trees (65 %) produced 333 and 236 fruiting bodies, respectively, totalling 569 fruiting bodies (c. 30 kg). This study presents new information on factors influencing the onset of fruiting and the development of yields in a plantation of P. radiata mycorrhized by L. deliciosus. Projected yields as high as c. 300 kg/ha from the third year of production reiterate the feasibility of farming saffron milk cap in P. radiata plantations in New Zealand. Continued monitoring of this site and development of similar trials will provide important knowledge for the optimisation of yields in commercial saffron milk cap orchards.     

Analysis of indole compounds from the fruiting bodies and the culture mycelia of Sarcodon imbricatus

Amounts of non-hallucinogenic indole compounds were determined in methanolic extracts from the fruiting bodies and biomass of Sarcodon imbricatus cultured in vitro. In both extracts there were non-hallucinogenic indole compounds present, l-tryptophan, tryptamine and serotonin. Additionally, melatonin was found also in the fruiting bodies. The total amount of indole compounds was 89.38 mg/100 g d.w. in the fruiting bodies and 8.45 mg/100 g d.w. in the cultured mycelia. The leading compound in the fruiting bodies and the mycelium was serotonin (52.02 mg/100 g d.w. and 3.03 mg/100 g d.w., respectively). This main indole compound was isolated and identified using spectral methods.     

Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?

Storage conditions of the spawn of edible fungi are of major importance to facilitate the production of mushrooms. Here, standard storage conditions at 10 °C or 15 °C were used and the potential of colonization of standard European compost by the tropical species Agaricus subrufescens was assessed during the spawn running phase. Two lignocellulolytic activities, laccase and CMC-cellulase, were enhanced after storage compared to control as well as substrate transformation, as described by the aromaticity ratio and a humification ratio calculated from NMR data. This result indicates that mycelium growth probably occurred during storage at 10 or 15 °C, leading to a larger amount of biomass in the inoculum. Moreover, the microbial functional diversity of the substrate was favored, showing that the electivity of the substrate was maintained. Thus, these findings indicate that recommendations for the mushroom producers can be established for A. subrufescens cultivation under European standard conditions.     

Utilization of fat and degradation of cholesterol by pleurotus spp.

Plant oil was found to stimulate the formation of biomass inPleurotus ostreatus, P. ostreatus formFlorida andP. cornucopiae. The highest increase of the delipidized dry substance was found inP. ostreatus. The supernatant after submerged fermentation contained active emulsifiers. Fruiting bodies and mycelium ofP. ostreatus did not contain cholesterol. Cholesterol added to homogenates of the fruiting bodies was degraded in a temperature-dependent manner. Degradation of cholesterol in the fermentation medium was slower.     

Preservation of Agaricus subrufescens strains at low temperature by using cultures on sorghum grains

BackgroundOne of the main problems for the preservation of genetics resources of Agaricus subrufescens is to maintain the viability of the strains because the mycelium is very sensitive to cooling and therefore it ages rapidly.AimsEvaluate the viability of A. subrufescens strains stored as cultures on sorghum grain (spawn) at different temperatures.MethodsEighteen strains of A. subrufescens and three strains of Agaricus bisporus were studied. Spawn's viability was evaluated under the following conditions: (1) control at 25 °C (C), (2) cooling to 4 °C (R) and (3) freezing in liquid nitrogen at ?196 °C (LN). Samples were recovered from week 4 every 2 weeks until week 12 and week 24 in C and R, whereas in LN samples were recovered at 4, 12 and 24 weeks. Viability was evaluated in 50 seeds, by strain and condition, recovering the mycelium in Petri dishes with potato dextrose agar medium (PDA). Mycelium growth was also evaluated on PDA after 14 days of recovery.ResultsMost strains showed 100% viability and they were recovered usually in 1 day. In LN the viability ranged between 84 and 100% depending on the strain, but in some cases recovery took more than 10 days. Mycelial growth decreased gradually over time and although the results show significant differences between treatments C and R, the decline is associated with ageing of the mycelium rather than the treatment itself.ConclusionsCulture on sorghum grain and storage at low temperature is an interesting way to preserve genetic resources of A. subrufescens.     

Pressure drop increase by biofilm accumulation in spiral wound RO and NF membrane systems: role of substrate concentration,flow velocity,substrate load and flow direction

In an earlier study, it was shown that biofouling predominantly is a feed spacer channel problem. In this article, pressure drop development and biofilm accumulation in membrane fouling simulators have been studied without permeate production as a function of the process parameters substrate concentration, linear flow velocity, substrate load and flow direction. At the applied substrate concentration range, 100–400 μg l^?1 as acetate carbon, a higher concentration caused a faster and greater pressure drop increase and a greater accumulation of biomass. Within the range of linear flow velocities as applied in practice, a higher linear flow velocity resulted in a higher initial pressure drop in addition to a more rapid and greater pressure drop increase and biomass accumulation. Reduction of the linear flow velocity resulted in an instantaneous reduction of the pressure drop caused by the accumulated biomass, without changing the biofilm concentration. A higher substrate load (product of substrate concentration and flow velocity) was related to biomass accumulation. The effect of the same amount of accumulated biomass on the pressure drop increase was related to the linear flow velocity. A decrease of substrate load caused a gradual decline in time of both biomass concentration and pressure drop increase. It was concluded that the pressure drop increase over spiral wound reverse osmosis (RO) and nanofiltration (NF) membrane systems can be reduced by lowering both substrate load and linear flow velocity. There is a need for RO and NF systems with a low pressure drop increase irrespective of the biomass formation. Current efforts to control biofouling of spiral wound membranes focus in addition to pretreatment on membrane improvement. According to these authors, adaptation of the hydrodynamics, spacers and pressure vessel configuration offer promising alternatives. Additional approaches may be replacing heavily biofouled elements and flow direction reversal.     

The Effect of Gas Sparging in Cross‐Flow Microfiltration of 2,3‐Butanediol Fermentation Broth

Recovery of 2,3‐butanediol from a fermentation broth entails the separation of cells and other suspended solids as the initial step for subsequent separation stages. The aim of this work was to study the cross‐flow filtration of broth in the fermentation of 2,3‐butanediol from blackstrap molasses by Klebsiella oxytoca (NRRL B‐199). A plate type laboratory scale cross‐flow microfiltration unit with a 0.2‐μm cellulose acetate membrane was employed for this purpose. Preliminary results showed that the permeate flux would decline rapidly due to fouling caused by the natural impurities of blackstrap molasses, and modifications of the conventional cross‐flow filtration would be essential to achieve a filtration rate appropriate for practical purposes. In this work, the permeate flux was enhanced by air sparging, which scoured the membrane surface of colloidal deposits and allowed a practical filtration rate to be maintained. The average permeate flux increased by 39 % and 54 % for an air sparging rate of 0.5 L/min and 1.0 L/min respectively, in the case of an initial biomass concentration of 4.66 g/L. For an initial biomass concentration of 14.2 g/L, the flux increased by 105 % and 146 % for the gas rate of 0.5 and 1.0 L/min, respectively. It may be concluded that gas sparging is beneficial in cross‐flow filtration of thick suspensions like a fermentation broth.     

Cultivation and utility of <Emphasis Type="Italic">Piptoporus betulinus</Emphasis> fruiting bodies as a source of anticancer agents

Piptoporus betulinus is a wood-rotting basidiomycete used in medicine and biotechnology. However, to date, no indoor method for cultivation of this mushroom fruiting bodies has been developed. Here we present the first report of successful production of P. betulinus mature fruiting bodies in artificial conditions. Four P. betulinus strains were isolated from natural habitats and their mycelia were inoculated into birch sawdust substrate supplemented with organic additives. All the strains effectively colonized the medium but only one of them produced fruiting bodies. Moisture and organic supplementation of the substrate significantly determined the fruiting process. The biological efficiency of the P. betulinus PB01 strain cultivated on optimal substrate (moisture and organic substance content of 55 and 65 and 25 or 35 %, respectively) ranged from 12 to 16 %. The mature fruiting bodies reached weight in the range from 50 to 120 g. Anticancer properties of water and ethanol extracts isolated from both cultured and nature-derived fruiting bodies of P. betulinus were examined in human colon adenocarcinoma, human lung carcinoma and human breast cancer cell lines. The studies revealed antiproliferative and antimigrative properties of all the investigated extracts. Nevertheless the most pronounced effects demonstrated the ethanol extracts, obtained from fruiting bodies of cultured P. betulinus. Summarizing, our studies proved that P. betulinus can be induced to fruit in indoor artificial culture and the cultured fruiting bodies can be used as a source of potential anticancer agents. In this respect, they are at least as valuable as those sourced from nature.     

Quantification of extraradical mycelium of Tuber melanosporum in soils from truffle orchards in northern Spain

Quantification of extraradical mycelium of black truffle (Tuber melanosporum) has been carried out in a natural truffle ground and in seven truffle orchards (around 20 years old) established in Tierra Estella and Valdorba sites, within the natural distribution area of the black truffles in Navarre (northern Spain). Specific primers and a Taqman® probe were designed to perform real-time PCR with DNA extracted from soil samples. Amplification of T. melanosporum DNA was obtained from 131 out of the 160 soil samples. The detection limit of the technique was 1.48 μg mycelium/g of soil. The extraradical mycelium biomass detected in the soil from the natural truffle ground was significantly greater (up to ten times higher) than the mycelium biomass detected in any of the orchards. Soil from productive, nonirrigated orchards in the Tierra Estella site contained significantly more extraradical mycelium than the rest of orchards irrigated, productive of T. brumale, or nonproductive. The comparison of soil mycelium biomass in nonirrigated evergreen oak orchards in both sites showed significantly more mycelium biomass in the Tierra Estella site. This study is the first attempt to quantify extraradical mycelium of T. melanosporum in the soil using Taqman® probes. The obtained quantitative results are of special interest to evaluate the fungal response to cultural treatments and to monitor the dynamics of the extraradical mycelium of T. melanosporum in the soil.