Mycelial Yield Research Articles

Extracellular lipase production by a sapwood-staining fungus, Ophiostoma piceae

The extracellular lipase production of a sapwood-staining fungus, Ophiostoma piceae, grown in liquid media, was optimally active at pH 5.5 and 37°C. Although glucose, fructose, sucrose, starch and dextrin, as carbon sources for growth gave similar mycelial yields, which were higher than those obtained with arabinose, galactose or raffinose, the cells growing on those carbohydrates produced little extracellular lipase. However, both high biomass and lipase activity were obtained when plant oils (olive, soybean, corn, sunflower seed, sesame, cotton seed or peanut) were used as carbon sources. Among the nitrogen sources examined, Casamino acids gave the best growth, whereas (NH4)2SO4 gave the best lipase production. The highest lipase productivity seen was obtained in a medium with olive oil as carbon source and a combination of (NH4)2SO4and peptone as nitrogen source.

Insect cuticle and yeast extract effects on germination, growth, and production of hydrolytic enzymes byNomuraea rileyi

Larval cuticle ofHelicoverpa (Heliothis)zea and yeast extract added to a minimal medium (MM) induced germination of conidia ofNomuraea rileyi whereas sterile distilled water or MM alone did not. Yeast extract increased mycelial yield, but when cuticle was added, mycelial yield significantly decreased. Proteases and chitinases ofN. rileyi were only expressed when cuticle was added to the MM.

Convenient and effective methods for in vitro cultivation of mycelium and fruiting bodies of Lentinus edodes

Three strains were selected from 18 commercial strains of the shiitake mushroom, Lentinus edodes , on the basis of rate and density of mycelial growth. Comparison of biomass production between stationary and shake flask cultures was strain-dependent, strain Le-465 producing higher yields from shaken than stationary cultures while no differences were recorded in Le-11 and Le-103. Among 5 media tested for mycelial growth yield, a molasses medium gave the highest biomass after 14 d, but by day 28 yield from that medium was the second lowest. By day 28, a chemically defined medium was the best for biomass yield. Homogenization of mycelia provided an excellent uniform source of inoculum for replicate trials. Homogenization of the inoculum increased the yield of mycelium by day 28, when fruiting started. Chemically-defined media were best for fruiting of L. edodes . There was not much difference in fruiting performance between cultures incubated at a uniform 25 °C and those incubated in a 14/22° cycle. Supplementation of flask cultures with vermiculite promoted growth and fruiting. In flask cultures kept in a normal incubator a cotton stopper, as opposed to one made of plastic foam, was most suitable for growth and fruiting. Inclusion of a carbon dioxide trap was not necessary. The best fruiting in vitro was obtained by inoculation on to solid supports, like a wire mesh or agar, in crystallizing dishes which were kept within a plastic chamber whose air-flow and humidity could be regulated with air-vents and an air-pump. Under such conditions, vermiculite addition to the media did not enhance fruiting.

Effects of culture conditions on amylase production by some soil fungi

Twenty-one species of soil fungi have been screened for amylolytic activity. Six species designated as active enzyme producers were used in the experiments: Aspergillus flavus, Cladosporium herbarum, Cunninghamella echinulata, Fusarium culmorum, Paecilomyces divaricata, and Penicillium notatum. The results obtained revealed that, in most cases, enzyme production and dry weight yield of mycelium were not positively correlated. An incubation period of 8 days at 27°C and an initial pH range of 4–6 were most favourable for enzyme production by all fungi studied. A 1.5% concentration of soluble starch was the most suitable substrate for all the studied fungi although rice and corn flours appeared to be preferred by A. flavus, F. culmorum, P. divaricata and P. notatum. Ammonium nitrate at 0.5% concentration provided a suitable nitrogen source for all fungi although productivity by A. flavus and P. divaricata was enhanced by the presence of casein and urea. A C: N ratio of 3:1 was the most favourable for all fungi investigated. A concentration of 10 μg/L SrCl2 favoured productivity by all fungi. Preferential utilization of nutrients caused changes in pH towards alkalinity which repressed productivity. 21 Bodenpilz-Arten wurden auf ihre amylolytische Aktivität untersucht. 6 Arten waren aktive Enzymproduzenten und wurden weiter optimiert: Aspergillus flavus, Cladosporium herbarum, Cunninghamella echinulata, Fusarium culmorum, Paecilomyces divaricata und Penicillium notatum. Die Ergebnisse offenbarten, daß in der Mehrzahl der Fälle die Enzymproduktion und der Trockengewichtsertrag des Myzels nicht positiv korrelierten. Bei allen untersuchten Pilzen war für die Enzymproduktion eine Inkubationszeit von 8 Tagen bei 27°C und ein Start-pH-Wert von 4–6 am günstigsten. Eine Konzentration von 1,5% löslicher Stärke war am geeignetsten, Reis und Korngetreidemehl wurden von A. flavus, F. culmorum, P. divaricata und P. notatum vorgezogen. Ammoniumnitrat (0,5%) ist die günstigste N-Quelle, allerdings wurde bei A. flavus und P. divaricata durch Anwesenheit von Casein und Harnstoff die Produktivität erhöht. Ein C : N-Verhältnis von 3:1 war für alle Pilze am günstigsten. Bevorzugte Verwertung von Nährsubstrat verursacht eine pH-Veränderung zur Alkalität, welche die Produktivität erniedrigt.

Physiological Studies of Two Fungi Isolated from Nymphaea Odorata

Physiological studies of Dichotomophthoropsis nymphaearum and Sclerotium hydrophilum were undertaken to determine the growth parameters for these two fungi. Optimum or near-optimum growth of D. nymphaearum occurred at temperatures of 25 and 30 C. Optimum temperature for growth of S. hydrophilum was 30 C, although maximum sclerotial production occurred at 25 C. Both fungi were able to utilize all of the carbon sources tested. Dichotomophthoropsis nymphaearum gave lower mycelial yields on xylose, while S. hydrophilum grew poorly on maltose. Both fungi utilized inorganic and organic nitrogen sources, but mycelial yields were lower when ammonium sulphate served as the nitrogen source. Glycine was also a poor nitrogen source for S. hydrophilum. Thiamine was required by both isolates of D. nymphaearum studied and by one of the isolates of S. hydrophilum. Light was not found to be essential for conidium production by D. nymphaearum or for the initiation of sclerotia by S. hydrophilum, although it did affect maturation of these structures. Pairing of six isolates of each fungus in all possible combinations and on several media failed to give evidence of a sexual response or production of a sexual state.

Physiological Studies of Two Fungi Isolated from Nymphaea odorata

Physiological studies of Dichotomophthoropsis nymphaearum and Sclerotium hydrophilum were undertaken to determine the growth parameters for these two fungi. Optimum or near-optimum growth of D. nymphaearum occurred at temperatures of 25 and 30 C. Optimum temperature for growth of S. hydrophilum was 30 C, although maximum sclerotial production occurred at 25 C. Both fungi were able to utilize all of the carbon sources tested. Dichotomophthoropsis nymphaearum gave lower mycelial yields on xylose, while S. hydrophilum grew poorly on maltose. Both fungi utilized inorganic and organic nitrogen sources, but mycelial yields were lower when ammonium sulphate served as the nitrogen source. Glycine was also a poor nitrogen source for S. hydrophilum. Thiamine was required by both isolates of D. nymphaearum studied and by one of the isolates of S. hydrophilum. Light was not found to be essential for conidium production by D. nymphaearum or for the initiation of sclerotia by S. hydrophilum, although it did affect maturation of these structures. Pairing of six isolates of each fungus in all possible combinations and on several media failed to give evidence of a sexual response or production of a sexual state.

The effect of carbon sources on the single cell proteins and extracellular enzymes production byChrysonilia sitophila (TFB 27441 strain)

The single cell protein and extracellular enzyme production from a lignocellulolytic fungus,Chrysonilia sitophila, using different carbon sources were evaluated. The mycelial dry mass composition showed a high protein (39.2%) and low nucleic acid content (3.3%), as well as carbohydrate, fatty acid, fiber, and ash levels comparable with single cell proteins currently studied. Mycelial protein showed amino acid content similar to or higher than FAO standard requirements. The amino acid, fatty acid, and carotenoid composition, as well as mycelial mass yield and enzyme production, were dependent on the carbon source used. Glucose, saccharose, cellobiose, cellulose, microcrystal-line cellulose, lactose, and rice hull as carbon sources were studied.

Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer.

Production of eicosapentaenoic acid byMortierella fungi

AbstractMycelia of arachidonic acid‐producing fungi belonging to the genusMortierella were found to be rich sources of 5,8,11,14,17‐cis‐eicosapentaenoic acid (EPA). Production of EPA by these fungi was observed only when they were grown at low temperature (6–16 C). EPA comprised 5–20% of the total extractable mycelial fatty acids in most strains tested. No significant accumulation of EPA was observed on incubation at high temperature (20–28 C), at which the other major mycelial C‐20 fatty acid, arachidonic acid, was still efficiently produced. In a study on the optimization of the culture conditions for EPA production by a selected fungiM. alpina 20–17, a medium containing glucose and yeast extract as major carbon and nitrogen sources, respectively, was found to be suitable. Periodic feeding of glucose during growth of the fungus and cultivation at high temperature (20 C) during the early growth phase followed by temperature shift to 12 C were found to be effective at increasing mycelial yield and reducing cultural period, respectively. Under the optimal culture conditions, the EPA production reached 0.49 mg/ml of culture broth (29 mg/g dry mycelia). This value accounted for 13.5% of the total fatty acids in the extracted lipids. Other major fatty acids in the lipids were palmitic acid (6.0%, by weight), stearic acid (5.3), oleic acid (6.2), linoleic acid (3.0), γ‐linolenic acid (3.5) and arachidonic acid (60.0).

Effect of heavy metals on growth and survival of Macrophomina phaseolina (Tassi) Goid.

The effects of Cd, Co, Ni and Zn on growth and survival of Macrophomina phaseolina were studied in vitro. Cd, Ni and Co at 500 μg ml−1 inhibited growth by 78.8%, 73.6% and 11.8%, respectively, after 4 days at 25 ± 1°C. The mycelial dry weight yield was enhanced by 2.1% at 100 μg ml−1 Zn. The population of the pathogen declined in soil amended separately with Cd, Co, Ni and Zn. Cd (4000 mg kg−1) proved to be the most toxic, by completely inhibiting the survival of the pathogen in soil after 20 days.

Botrytis cinerea: a study of the immunological properties during growth. Incidence of antibodies against B. cinerea in a group of patients with aspergillosis.

When growing Botrytis cinerea in Sabouraud medium, different phases of growth could be recognized using pH, mycelium yield, and culture filtrate antigens as parameters of growth. Immunological characteristics of the different phases (early, intermediate, and late) of growth were measured using IgE and IgG binding by enzyme allergosorbent test and enzyme-linked immunosorbent assay, respectively, and quantities of precipitating components (double immunodiffusion and rocket immunoelectrophoresis). The number of precipitating components was maximal in culture filtrate extracts during late phase (III) of growth. In contrast, both IgE and IgG binding were already maximal in the early phase of growth (9 days of cultivation). In a group of patients with elevated IgE antibodies against Aspergillus fumigatus, about half of them also showed elevated IgE antibodies against B. cinerea. This indicates that multiple exposition is a cause of multiple sensitization; however, cross-reactivity in some of these sera cannot be excluded.

Folylpolyglutamate Deficiencies in Two Methionine-requiring Mutants of Neurospora crassa

Summary The growth of two Neurospora crassa methionine auxotrophs ( met -6, 35809, FGSC 1330 and mac , 65109, FGSC 3609), that have lesions affecting folylpolyglutamate synthetase, was examined by culture in defined, liquid media. Both mutants responded to L-methionine supplements but additions of homocysteine, cysteine, serine, formate, thymine and adenine, supplied singly or in various combinations were not effective in stimulating growth. Mycelial yields after 22 hr growth in 0.5 mM L-methionine-supplemented media were comparable to those of a wild type strain (Lindegren A, FGSC 853) cultured in defined minimal medium. Pulse feeding radio-labelled cysteine, glycine, serine and formate showed that both mutants had less ability to generate methionine than the wild type. Standard microbiological assays, conjugase treatments and DEAE-cellulose chromatography showed that folylpolyglutamates were lacking in met -6 and mac but accounted for about 70% of the wild type folate pool. The polyglutamyl chain lengths of these folates were determined following tritium labelling, acid cleavage and Chromatographic separation as diazo dye derivatives. The wild type contained mainly tri- and hexaglutamyl folates. In met -6, mono- and diglutamates predominated but in mac only monoglutamates were found. Extracts of met -6: mac heterokaryons contained folates that were hexaglutamate derivatives. These findings provide additional evidence that folylpolyglutamates have an essential role in the methionine synthesis of N. crassa .

Submerged Production of Edible Mushroom Mycelium

The edible mushroom Morchella esculenta, previously adapted to a peat extract culture medium, has been grown using acid Sphagnum peat extracts as the only substrate source. Extracts obtained by autoclaving peat mixed with 1.5% (v/v) H2S04 for 2h at 121°C were utilized in shaker flask fermentations in order to evaluate the incidence of different factors on the growth. Sets of different conditions for inoculum ratio, temperature, initial pH, fermentation time and agitation speed were tested. The results are expressed as mycelial yield (g of dry mycelium produced per g of carbohydrate consumed). The best combination of operating variables in the range of values investigated, were: 7% (v/v) inoculum ratio, 26°C, pH 6.0, 6 days and 150RPM.

Improved media for growth and sporulation of Sporidesmium sclerotivorum

Additional nutritional and environmental factors affecting growth and sporulation of the beneficial mycoparasite, Sporidesmium sclerotivorum, were determined. A liquid medium composed of mineral salts, glucose, glutamine, thiamine, biotin, and succinic acid supported good growth and sporulation of isolate CS-5 within 5 weeks at 25 °C. Growth was substantially greater in a medium with KH2PO4, CaCl2, minor elements, and ferric–potassium salt of EDTA (FeKEDTA) than in media with single omissions of these ingredients. Iron supplied as FeCl3 or FeKEDTA at 10−5 M or greater markedly stimulated production of conidia. Supplementation of the medium with Casamino acids or with Soytone and yeast extract decreased the lag phase slightly but did not affect the maximum growth rate nor final mycelial and spore yield. Succinic acid (0.2%) buffered the medium against a rapid drop in pH and thereby promoted growth. The growth rate was independent of glucose concentrations of 0.5 to 2%, but the total mycelial yield was dependent upon the total amount of glucose furnished. Conidia developed primarily on mycelial mat surfaces, and maximum spore yields were favored by cultivation in shallow depths of medium.

Ca2+-dependence and metabolic status of an obligate thermophile,Thermoactinomyces vulgaris, under shake culture conditions

Ca2+ stimulates germination ofT. vulgaris spores. There is a higher mycelial yield as well as higher protein, DNA, RNA and free Pi content in cultures grown in the presence of Ca2+ as compared to those grown in the absence of this divalent cation.

Growth and Synthesis of Aflatoxin by Aspergillus parasiticus in the Presence of Sorbic Acid

Two media [basal (M1) and enriched (M2)] containing potassium sorbate (0–300 ppm as sorbic acid) were inoculated with spores (104 – 106/flask) of Aspergillus parasiticus and incubated for 5 days at 28 C. The greater the amount of sorbate added, the higher was the pH of the media after incubation and the smaller was the yield of mold mycelium. Intermediate amounts of sorbate sometimes resulted in greater accumulation of aflatoxin than when media were free of sorbate. Sorbate more effectively inhibited mold growth and aflatoxin production in medium M2 than M1 and when the small rather than the large inoculum was used. A second trial was done with 106 or 105 spores/flask of M2 (ca. 27 ml) and 105 spores/flask of M2 (ca. 27 ml) containing sorbate (200 ppm of sorbic acid). Cumulative data for mold growth. pH and content of aflatoxin in the medium showed that relative effects of different treatments changed during the incubation period. An index to measure the capacity of molds to synthesize aflatoxins was developed. Application of the index indicates that sorbate delayed mold growth but did not inhibit biosynthesis of aflatoxin. The ability to synthesize aflatoxin was greatest in the early stages of mold growth and then decreased linearly as mold growth progressed.

Effect of oxygen transfer rate on the composition of the pectolytic enzyme complex of Aspergillus niger

AbstractOptimal agitation and aeration conditions [assuring O2 transfer rates (OTR) from 12 to 179 mmol L−1 h−1] were determined for pectin lyase (PL) synthesis of an Aspergillus niger strain. Components of the pectolytic enzyme complex were also investigated in order to determine whether their O2 demand is identical with or different from that of pectin lyase. Should the latter be the case, a possibility would be given to produce enzyme complexes of different agitation and aeration conditions. According to our results, mycelium yield of Aspergillus niger attained a maximum at an OTR of 100 mmol L−1 h−1. The yields of the various pectolytic enzymes reached maxima at different OTRs. Pectin lyase production was the highest (0.555 µmol min−1 mL−1) at an OTR of 60 mmol L−1 h−1. Endopolygalacturonase (PG) production showed a maximum at the OTR of 49 mmol L−1 h−1 with a second peak at 100−135 mmol O2 L−1 h−1. Pectin esterase (PE) synthesis showed a maximum at on OTR of 12−14 mmol L−1 h−1, while both apple juice clarifying and macerating activities gave two maxima at 14 and 60 mmol L−1 h−1 due to the optima of PE and endo‐PG. Macerating activity showed a high value at OTR optimal for PL production as well.

Propagation of Lentinus edodes on modified malt sprouts medium for amylase production

The moisture, ash, total nitrogen and total organic carbon contents of malt sprouts extract and a synthetic medium used for fungal amylase production were determined and compared. The results revealed that these constituents varied greatly in the two media. Accordingly, various sources of carbon (glucose, maltose, dextrin and soluble starch), of nitrogen (ammonium nitrate, ammonium sulphate, peptone and yeast extract) and of mineral salts (calcium carbonate, potassium dihydrogen phosphate, magnesium sulphate, sodium chloride and zinc sulphate) were introduced into the media in order to modify the medium composition for Lentinus edodes propagation to attain maximum production of amylase. High amylolytic activity and mycelium yield were attained in the presence of dextrin (2.0%) and yeast extract (0.5%), potassium dihydrogen phosphate and calcium carbonate (0.05%).

Título en español.

El mosto de destilerías de ron resultó útil para el cultivo de hongos cuyo micelio puede utilizarse como alimento suplementario para animales. Se obtuvieron reducciones altas en el BOD y azúcares totales del mosto. El contenido en nitrógeno bajó a medida que aumentó el rendimiento de micelio, indicando que el hongo lo utiliza para su crecimiento. Se observó un aumento en el pH del mosto cuando los hongos crecieron. De 12 cepas de hongos dos compararon favorablemente con la cepa control. Se observaron variaciones en el contenido proteico del micelio entre las cepas investigadas. Las reducciones en el BOD del mosto fueron proporcionales al rendimiento de micelio. Con las cepas que dieron mejores resultados se obtuvieron reducciones de 30-53% BOD al cabo de 7 días de crecimiento.

Edible mushrooms as producers of amylases

Five varieties of mushroom— Absidia blakesleeana, Agaricus bisporus, Lentinus edodes, Peziza auburouniv and Polyporus sulphureus—were qualitatively examined for their ability to hydrolyse starch. Quantitative estimation was made to compare the amylolytic activity of the five types of mushroom propagated on a synthetic medium with that of Aspergillus niger NRRL 337. Lentinus edodes proved to be the most promising strain for amylase production. The amylolytic activity of Lentinus edodes reached its maximum (621 U/ml) after seven days of incubation, the dry mycelium yield being 7·5 g/l with a protein content of 34%. Lentinus edodes was then propagated on various extracts of food wastes—malt sprouts, navel orange peel, potato peel, rice bran, corn steeping water and rice steeping water. Malt sprouts extract proved to be a promising medium for Lentinus edodes propagation as well as for amylase production.