Valorization of low-cost, carbon-rich substrates by edible ascomycetes and basidiomycetes grown on liquid cultures.

Three ascomycetes (Morchella vulgaris AMRL 36, M. elata AMRL 63, Tuber aestivum AMRL 364) and four basidiomycetes strains (Lentinula edodes AMRL 124 and 126, Agaricus bisporus AMRL 208 and 209) were screened for their ability to grow on liquid static flask cultures of glucose, glycerol, molasses and waste flour-rich hydrolysates with C/N ratio of 20 and produce biomass, exopolysaccharides and lipids. The profile of lipid fatty acids was also assessed. Selected strains were furthermore cultivated in C/N = 50. Results showed that substrate consumption, biomass formation and secondary metabolites production were strain, substrate and C/N ratio dependent. The maximum biomass (X), lipid (L) and exopolysaccharides (EPS) values noted were Xmax = 25.2 g/L (C/N = 20; molasses) and Lmax = 6.51 g/L (C/N = 50; rice cereal hydrolysates) by T. aestivum strain AMRL 364 and EPSmax = 2.41 g/L by M. elata strain AMRL 63 (C/N = 50; molasses), respectively. When C/N ratio of 50 was applied, biomass, lipid production and substrate consumption seem to be negatively affected in most of the trials. The adaptation and capability of the mushroom strains to be cultivated on substrates based on agro-industrial waste streams and infant food of expired shelf date offers the opportunity to set a circular oriented bioprocess.

Spent mushroom substrate for a second cultivation cycle of Pleurotus mushrooms and dephenolization of agro-industrial wastewaters.

Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour and used as substrate for a new cultivation cycle of the oyster mushrooms Pleurotus ostreatus and Pleurotus pulmonarius. The bioconversion efficiency of mushrooms produced over substrate (BE%) used and the chemical composition of sporophores were evaluated. The concentration of mycelial mass, crude exopolysaccharide content and laccase enzyme activity were also determined at the supplemented SMS before inoculation, at 50% and 100% of colonization stages in the new cultivation and in the final re-utilized SMS. The laccase enzyme was extracted to examine SMS potential for the dephenolization of olive mill and winery wastewaters. Results showed that both Pleurotus species exhibited BE over 185%, demonstrating this bioprocess could represent a promising strategy to convert SMS into nutritional food. Data also indicate the strong positive impact that SMS could have in the solid wastes' management and agribusiness enhancement.

Upgrading Grape Pomace through Pleurotus spp. Cultivation for the Production of Enzymes and Fruiting Bodies.

Grape pomace, a by-product derived from winery industries, was used as fermentation media for the production of added-value products through the cultivation of two Pleurotus species. Solid-state (SSF), semiliquid (SLF), and submerged (SmF) fermentations were carried out using grape pomace as substrate. The effect of the different fermentations on the consumption of phenolic compounds, the production of mycelial mass and enzymes was evaluated using P. ostreatus and P. pulmonarius. The production of fungal biomass and enzymes was influenced by the fermentation mode. The maximum biomass values of ~0.5 g/g were obtained for both P. pulmonarius and P. ostreatus in SmF. Laccase production was induced in SSF and a maximum activity of 26.247 U/g was determined for P. ostreatus, whereas the highest endoglucanase activity (0.93 U/g) was obtained in the SmF of the same fungi. Analysis of phenolic compounds showed that both strains were able to degrade up to 79% of total phenolic content, regardless the culture conditions. Grape pomace was also evaluated as substrate for mushroom production. P. pulmonarius recorded the highest yield and biological efficiency of 14.4% and 31.4%, respectively. This study showed that mushroom cultivation could upgrade winery by-products towards the production of valuable food products.

Evaluation of Biomass and Chitin Production of Morchella Mushrooms Grown on Starch-Based Substrates.

Morchella sp. is one of the most expensive mushrooms with a high nutritional profile. In this study, the polysaccharide content of Morchella species was investigated. Specifically, mycelium growth rate, biomass production, sclerotia formation, and glucosamine and total polysaccharides content of six Morchella species grown on a starch-based media were evaluated. Submerged fermentations in potato dextrose broth resulted in a glucosamine content of around 3.0%. In solid-state fermentations (SSF), using potato dextrose agar, a high linear growth rate (20.6 mm/day) was determined. Increased glucosamine and total polysaccharides content were observed after the formation of sclerotia. Biomass and glucosamine content were correlated, and the equations were used for the indirect estimation of biomass in SSF with agro-industrial starch-based materials. Wheat grains (WG), potato peels (PP), and a mixture of 1:1 of them (WG-PP) were evaluated as substrates. Results showed that the highest growth rate of 9.05 mm/day was determined on WG and the maximum biomass yield (407 mg/g) on WG-PP. The total polysaccharide content reached up to 18.4% of dried biomass in WG-PP. The results of the present study proved encouraging for the efficient bioconversion of potato and other starch-based agro-industrial waste streams to morel biomass and sclerotia eliciting nutritional and bioactive value.

Valorization of spent oyster mushroom substrate and laccase recovery through successive solid state cultivation of Pleurotus, Ganoderma, and Lentinula strains.

Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour in various proportions to obtain C/N ratios of 10, 20, and 30, and their effect was evaluated in successive cultivation of Pleurotus ostreatus, Pleurotus pulmonarius, Ganoderma adspersum, Ganoderma resinaceum, and Lentinula edodes strains with respect to mycelium growth rate, biomass concentration, recovery of the enzyme laccase and crude exopolysaccharides, and also with additional fruiting body production. All fungi showed the highest growth rate on unamended SMS (C/N 30), with G. resinaceum being the fastest colonizer (Kr = 9.84 mm day-1), while biomass concentration maximized at C/N 10. Moreover, supplementation affected positively laccase activity, with P. pulmonarius furnishing the highest value (44,363.22 U g-1) at C/N 20. On the contrary, L. edodes growth, fruiting, and laccase secretion were not favored by SMS supplementation. Fruiting body formation was promoted at C/N 30 for Ganoderma and at C/N 20 for Pleurotus species. Exopolysaccharide production of further studied Pleurotus strains was favored at a C/N 20 ratio, at the initial stage of SMS colonization. The obtained results support the potential effective utilization of supplemented SMS for laccase production from Ganoderma spp. and for new fruiting body production of Pleurotus spp.

Adaptation of Volvariella volvacea metabolism in high carbon to nitrogen ratio media.

Increasing glucose concentration (up to 46%) in the culture medium of Volvariella volvacea resulted in a noticeable biomass increase (63.82%) and intracellular polysaccharide (IPS) production (32.84% in absolute and 51.34% in relative values). In high glucose concentrations, decreased biomass yields on glucose consumed were recorded. A non-negligible lipid was synthesized (up to 32.22%, w/w) in the first growth steps. Lipids and IPS were decreased as fermentation proceeded. Fatty acid composition of lipids was not remarkably affected by the C/N ratio imposed, whereas the concentration of neutral lipids increased in the high C/N ratio media. Phospholipid was the richest in saturated fatty acids lipid fraction. The major phospholipid classes, i.e. phosphatidyl-choline and phosphatidyl-inositol, were differentially affected by the high glucose concentrations, as the former increased its proportion and the latter decreased. Linoleic was the predominate fatty acid. C/N ratio and fermentation time affected IPS composition, as glucose increased in a high C/N ratio, while fructose and mannitol decreased.

Patterns of major metabolites biosynthesis by different mushroom fungi grown on glucose-based submerged cultures.

The biosynthetic potential of four basidiomycetes (Agrocybe aegerita, Flammulina velutipes, Ganoderma applanatum and Pleurotus pulmonarius) and one ascomycete (Morchella esculenta) was examined in regard to biomass, intracellular (endopolysaccharides and lipids) and extracellular (exopolysaccharides) compounds' production in liquid media with glucose as substrate, in static and agitated cultures. Exopolysaccharides' production presented significant negative correlation with biomass, endopolysaccharides and lipids, while biomass was positively related to the production of endopolysaccharides and lipids. Maximum values of biomass, endo- and exo-polysaccharides obtained were quite impressive: P. pulmonarius produced 22.5 g/L of biomass, A. aegerita 60.4 % (w/w) of endopolysaccharides and F. velutipes 1.2 g/L of exopolysaccharides. Polysaccharides and lipids synthesized at the early growth stages were subjected to degradation as the fermentation proceeded. Mycelial lipids of all strains were highly unsaturated, dominated by linoleic acid, whereas glucose was the main building block of endopolysaccharides. The ability of the examined mushroom fungi to synthesize in high quantities biomass and polysaccharides, products with biotechnological and medicinal interest, renders these fungi as potential candidates in sugar-based bio-refineries.

Mushroom polysaccharides and lipids synthesized in liquid agitated and static cultures. Part II: study of Volvariella volvacea.

Volvariella volvacea strains were studied in relation with their ability to produce biomass, lipids and polysaccharides. Firstly, screening of four strains (AMLR 188, 190, 191 and 192) was performed in agar cultures, where the mycelial growth rate of the strains was measured, and in static liquid cultures, where the production of biomass, the biosynthesis of total cellular lipids and the consumption of glucose were monitored. For all strains, biomass production was significant (13-15 g l(-1)) and total lipid in dry weight (%, w/w) ranged from 3 to 12 %. Afterwards, a detailed kinetic analysis of mycelial biomass, extra- and intra- cellular polysaccharides (EPS, IPS, respectively) as well as lipid production by a V. volvacea selected strain was conducted in submerged static and agitated cultures. Maximum values of 15 g l(-1) biomass, ~1.0 g l(-1) EPS and 5.5 g l(-1) IPS were recorded. Agitation did not have severe impact on biomass, EPS and IPS production, but it increased total lipid in dry weight quantities. EPS, IPS and lipid in dry weight values decreased with time. Glucose was the major cellular carbohydrate detected. Total fatty acid analysis of cellular lipids was performed for all V. volvacea strains and linoleic acid (Δ9,12)C18:2 was predominant. Neutral lipids constituted the major fraction of cellular lipids, but their quantity decreased as fermentation proceeded. Phospholipids were the most saturated lipid fraction.

Mushroom polysaccharides and lipids synthesized in liquid agitated and static cultures. Part I: screening various mushroom species.

The effect of four synthetic media containing glucose (initial concentration 30 g l(-1)) on mycelial growth, exopolysaccharides (EPS) and cellular lipids production was examined in 11 mushroom species after 12 and 16 days of culture in static- and shake-flasks. Fatty acid analysis of cellular lipids produced was also performed. Agitation had a positive effect on biomass production, glucose consumption and lipid biosynthesis. Media that favoured the production of biomass were not suitable for EPS biosynthesis and vice versa. Biomass values varied from ~1.0 g l(-1) (Lentinula edodes) to ~19 g l(-1) (Pleurotus ostreatus), while the highest EPS quantity achieved ranged between 1.6 and 1.8 g l(-1) (for Ganoderma lucidum and L. edodes, respectively). Quantities of total cellular lipids varied between 2.5 and 18.5 % w/w, in dry mycelial mass for the fungi tested. Lipid in dry weight values were influenced by the medium composition. Cellular lipids presented noticeable quantities of poly-unsaturated fatty acids like linoleic acid. Compared to most of the mushrooms tested, lipids of Volvariella volvacea were more saturated. The ability of several mushroom species of our study to produce in notable quantities the above-mentioned added-value compounds renders these fungi worthy for further investigations.

Residue evaluation of famoxadone and trifloxystrobin in cultivated mushrooms.

Dissipation of the fungicides famoxadone and trifloxystrobin in basidiocarps of Agaricus bisporus was studied in mushroom growing rooms. The mushroom samples taken at all three consecutive production flushes following single or split applications of the fungicides were extracted with solvents and the residues were determined by using a gas chromatograph equipped with an electron capture detector (GC-ECD). Recoveries from the fortified control samples ranged from 87 to 105%. Following drench applications at 0.1-1 g/m2 of culture bed area, the highest famoxadone residue determined in basidiocarps was 0.1447 mg/kg. Analysis of trifloxystrobin revealed a quantitative relationship between the application rate (0.8-1.8 g/m2) and the residue levels of both the parent compound and its acid metabolite. The maximal combined residues of trifloxystrobin and its metabolite were 0.1313 mg/kg. Short- and long-term dietary risk assessment for both fungicides was carried out using consumption data from World Health Organization and the UK Pesticide Safety Directorate's Ten Consumer Model. The potential acute and chronic residue intakes via mushroom consumption were below toxicologically significant indicators.