Optimization of nutrient medium for submerged cultivation of Ganoderma lucidum (Curt.: Fr.) P. Karst

The dependence of the amount of the grown vegetative mycelium of Ganoderma lucidum on the composition of the nutrient medium has been studied under conditions of submerged cultivation. The medium was optimized using full factorial and steepest ascent experimental designs. The addition of two carbon sources to the medium considerably improved the submerged growth of the fungus. An optimized medium provided for a high yield (20-20.95 g/l) of the morphologically homogeneous mycelium and shortened the cultivation period to 3-4 days.     

Glucose oxidase ofAspergillus niger

The authors studied the effect of the various components of synthetic nutrient medium on glucose oxidase production in submerged cultivation ofAspergillus niger. It was found that the optimal glucose concentration was 3.5–6%. The only suitable source of nitrogen was nitrate nitrogen. If the medium contained ammonia nitrogen, glucose oxidase was not formed. The addition of citric acid to the medium very effectively stimulated theQ _{O 2} of the mycelium. Calcium added in the form of calcium nitrate had the same effect. A decrease in the Mg²⁺ ion concentration raised the activity of the enzyme, while inhibiting growth of the mycelium. If the initial pH was less than 4, glucose oxidase production was inhibited and did not start until the pH rose in the course of fermentation. Differences in the initial pH affected not only production of the enzyme, but also the formation of acids and the morphological appearance of the submerged mycelium. On the basis of the findings the synthetic medium for submerged cultivation ofAspergillus niger was modified, resulting in a 50–100% increase in glucose oxidase production as compared with the original medium.     

Dedikaryotization of higher fungi in submerged culture

Shaker experiments were done with submerged propagation of the oyster pleurotus (Pleurotus ostreatus/Jacq. ex. Fr./Kummer). The starting material was dikaryotic or monokaryotic mycelium obtained under stationary conditions. During submerged cultivation in a wort-containing medium on a cyclic shaker at 240 r.p.m. in flasks with articulated surface, dedikaryotization took place and the culture was predominantly monokaryotic after 10–14 days. Agitation of the medium favours the formation of monokaryotic forms. The typical mushroom flavour is associated with the dikaryotic form of mycelium so that submerged cultivation does not produce higher fungal mycelium in its aromatic form.     

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids.

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.     

Chitin-glucan complex production by Schizophyllum commune submerged cultivation

Chitin-glucan complex is a fungal origin copolymer that finds application in medicine and cosmetics. Traditionally, the mycelium of Micromycetes is considered as an industrial chitin-glucan complex source. Basidiomycete Schizophyllum commune submerged cultivation for chitin-glucan complex production was studied. In different S. commune strains chitin-glucan complex composed 15.2 +/- 0.4 to 30.2 +/- 0.2% of mycelium dry weight. Optimized conditions for chitin-glucan complex production (nutrient medium composition in g/l: sucrose - 35, yeast extract - 4, Na2HPO4*12H2O - 2.5, MgSO4*7 H2O - 0.5; medium initial pH 6.5; aeration intensity 21 of air per 11 of medium; 144 hours of cultivation) resulted in 3.5 +/- 0.3 g/l complex yield. Redirection of fungal metabolism from exopolysaccharide synthesis to chitin-glucan complex accumulation was achieved most efficiently by aeration intensity increase. Chitin-glucan complex from S. commune had the structure of microfibers with diameter 1-2 microm, had water-swelling capacity of 18 g/g, and was composed of 16.63% chitin and 83.37% glucan with a degree of chitin deacetylation of 26.9%. S. commune submerged cultivation is a potent alternative to Micromycetes for industrial-scale chitin-glucan complex production.     

Features of lipogenesis of basidiomycetes Pleurotus ostreatus and Flammulina velutipes in cultures on various media

The compositions of free fatty acids (FA) in the mycelia of oyster cap (Pleurotus ostreatus (Jacq. ex Fr.) Kumm.) and honey mushroom (Flammulina velutipes (Curt. ex Fr.) Sing.) and the effect of mycelium cultivation conditions on the composition and proportions of individual FA were investigated. Palmitic and linoleic acids were found to be major acids produced by P. ostreatus growing on solid agar medium and in a submerged culture with a synthetic medium. The composition of minor FA in P. ostreatus was dependent on cultivation conditions. Surface cultivation of its mycelium yielded pentadecanoic, octadecenoic, and stearic acids. Submerged cultivation additionally yielded undecanoic, myristic, hexadecenoic, and lignoceric acids. The composition of free FA in F. velutipes showed no significant differences from that of P. ostreatus. Variation in the C/N ratio in the cultivation medium affected both the FA composition in P. ostreatus and F. velutipes and the relationship between saturated and unsaturated FA.     

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.     

Lipogenesis in the Basidiomycetes Pleurotus ostreatusand Flammulina velutipes Cultivated on Different Media

The compositions of free fatty acids (FA) in the mycelia of oyster cap (Pleurotus ostreatus (Jacq. ex Fr.) Kumm.) and honey mushroom (Flammulina velutipes (Curt. ex Fr.) Sing.) and the effect of mycelium cultivation conditions on the composition and proportions of individual FA were investigated. Palmitic and linoleic acids were found to be major acids produced byP. ostreatus growing on solid agar medium and in a submerged culture with a synthetic medium. The composition of minor FA in P. ostreatuswas dependent on cultivation conditions. Surface cultivation of its mycelium yielded pentadecanoic, octadecenoic, and stearic acids. Submerged cultivation additionally yielded undecanoic, myristic, hexadecenoic, and lignoceric acids. The composition of free FA in F. velutipesshowed no significant differences from that of P. ostreatus. Variation in the C/N ratio in the cultivation medium affected both the FA composition in P. ostreatus and F. velutipes and the relationship between saturated and unsaturated FA.     

Developmental cycle and the cytomorphological characteristics of the oleandomycin producer Streptomyces antibioticus

The developmental cycle and cytomorphological features of the industrial strain OL-1 and its variant 0968 of the oleandomycin-producing organism were studied. Variant 0968 was obtained as a result of exposure of the spores of strain OL-1 to UV light. When grown under submerged conditions in flasks with the rich medium, the strains were characterized by a complete developmental cycle consisting of three generations of the hyphae. Every generation had a tendency for formation of submerged spores. The UV-induced variant differed from the industrial strain by higher levels of the antibiotic accumulation which correlated with higher rates of the spore germination. The strains were characterized by polymorphism of the mycelium and formation of submerged spores during their cultivation which is likely to prolong the antibiotic synthesis from 120 to 216 hours from the inoculation moment. The long-term selection of the oleandomycin-producing organism on the rich medium markedly changed the culture genotype and resulted in significant changes in the developmental cycle under submerged cultivation conditions. The data may be used for the microscopic control of the process of oleandomycin production.     

Production of tannase (tannin acyl hydrolase E.C. 3.1.1.20) by a strain of Aspergillus niger

Summary Culture of a strain of Aspergillus niger on medium containing 3 % tannic acid yielded tannase (tanin acylhydrolase), evidenced both in the culture medium and the mycelium. Fermentation in submerged culture at constant air flow gave mycelium with high tannase activity.     

培养条件对里氏木霉306菌体形态和t-PA生物合成的影响

里氏木霉（Trichoderrna reesei）306是能够生物合成组织型纤溶酶原激活剂（t-PA）的基因工程菌株。在对其液态深层培养时,发现随培养夸件和培养时间的变化,其菌体能以松散和菌丝球的两种形态存在。菌体形态和t-PA的产生密切相关。培养基中无机盐和表面活性剂的种类和添加量以及接种量和pH等培养条件是影响里氏木霉306菌体形态和t-PA合成的主要因素。在液态深层培养过程中,菌体以松散的菌丝体形态生长,形成纸浆状发酵液,利于t-PA的合成。     

Morphogenetic action of trichothecin on Trichothecium roseum]

Trichothecin and some conditions of cultivation, especially high concentrations of carbon favour differentiation of the submerged mycelium of Trichothecium roseum, i.e. formation of submerged conidia, bud forming cells, chlamidospores, chains of barrel-shaped cells capable of germination and development of new generations of the submerged mycelium. Biosynthesis of trichothecin is connected with growth of these generations of the mycelium which are characterized by a high dehydrogenase activity. Synthesis of fibrinolytic enzymes is also possible in the period of growth of a weakly differentiated mycelium.     

Growth and Pigmentation of Epicoccum nigrum in Submerged Culture

Growth and pigmentation of Epicoccum nigrum was studied in submerged culture in various media. Red pigmentation of the mycelium and of fermentation broth was obtained only in a medium containing glucose and yeast autolysate. This pigmentation occurred at the time of maximal production of carotenoids. Observations on the submerged culture of this organism in flasks and in fermentors are described, and the details of a standardized procedure for the production of carotenoid pigments are given.     

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.     

Novel laccase—producing ascomycetes

Screening of ascomycetes producing laccases during growth on agar medium or submerged cultivation in the presence of various natural sources of carbon and energy (grain crops and potato) was carried out. The conditions of submerged cultivation of the most active strains (Myrothecium roridum VKM F-3565, Stachybotrys cylindrospora VKM F-3049, and Ulocladium atrum VKM F-4302) were optimized for the purpose of increasing laccase activity. The pH-optima and substrate selectivity of laccases in the culture liquid of the strains in relation to ABTS and phenolic compounds (2,6-dimethoxyphenol, syringaldazine, ferulic acid, p-coumaryl alcohol, and coniferyl alcohol) were investigated. High laccase activity at neutral pH was shown for the culture liquids of M. roridum VKM F-3565 and S. cylindrospora VKM F-3049 strains that provides prospects for using laccases of these strains in various cell biotechnologies.     

美味牛肝菌液体深层培养工艺条件的探索

对美味牛肝菌液体深层培养的工艺条件进行探索,得出其最佳摇瓶条件为:温度24～28℃,种龄3天,接种量10%,振荡转速为150r/min,培养时间为5天,在最佳工艺条件下菌丝体干重可达24.8g/L。     

A mitochondrial respiratory mutant of Podospora anserina obtained by short-term submerged cultivation of senescent mycelium

A spontaneous long-lived isolate of Podospora anserina obtained by relatively short-term submerged cultivation of the wild-type senescent culture and conventionally termed ??immortal?? was shown to be a cox1 mutant. As a respiratory mutant, the isolate in question is characterized by dysfunction of the cytochrome respiratory chain, activation of alternative respiration leading to a low level of reactive oxygen species production, and the lack of accumulation of ??-senDNA, the specific factor of P. anserina senescence. Absence of visible vegetative incompatibility was shown in the fungal mutants carrying respiratory defects. It was discovered that the P. anserina female sex organs could be fertilized not only by microconidia but also by the fragments of vegetative mycelium. Partial nonobservance of monoparental female inheritance of mitochondria associated with fertilization by vegetative mycelium was also revealed.     

Enhanced Production of Ganoderic Acids and Cytotoxicity of Ganoderma lucidum Using Solid-Medium Culture

Submerged cultures of Ganoderma lucidum are used to produce fungal mycelium, which is used as a functional food and in the production of various triterpenoids, including ganoderic acids (GAs). Specific culture approaches that produce fungal mycelium with high levels of GAs and good biological activity are critical in the functional food industry. In this study, a solid-medium culture approach to producing mycelium was compared to the submerged culture system. Production of GAs, biomass, intracellular polysaccharides, and cytotoxicity of the cultured mycelium were compared as between solid and submerged culture. Growing G. lucidum strains on solid potato dextrose agar medium increased biomass, the production of ganoderic acid 24 (lanosta-7,9(11), 24-trien-3α-o1-26-oic acid), GAs, and total intracellular polysaccharides as compared to fungi grown in submerged culture. Triterpenoid-enriched methanol extracts of mycelium from solid-medium culture showed higher cytotoxicity than those from submerged culture. The IC(50) values of methanol extracts from solid-medium culture were 11.5, 8.6, and 9.9 times less than submerged culture on human lung cancer cells CH27, melanoma cells M21, and oral cancer cells HSC-3 respectively. The squalene synthase and lanosterol synthase coding genes had higher expression on the culture of solid potato dextrose medium. This is the first report that solid-medium culture is able to increase GA production significantly as compared to submerged culture and, in the process, produces much higher biological activity. This indicates that it may be possible to enhance the production of GAs by implementing mycelium culture on solid medium.     

Russian Article pp. 207–211

During the submerged cultivation of Trichoderma sp. 414 on a medium with 2% cellulose containing plant substrats an enzyme system – exocellobiohydrolase (C₁-enzyme), endogluconase (C_x-enzyme), β-glucosidase and xylase – catalizing the cellulose hydrolysis was synthesized. The process of enzyme biosynthesis by the microbial strain under the conditions of two-step cultivation in flasks was optimized. The influence of different sources of carbohydrate – avicel, micricel, maize stalk and straw – on the activity of the synthesized enzymes was studied. This activity depends on induction properties and the concentration of the used substrats. During the cultivation of Trichoderma sp. 414 on a medium containing avicel and wheat bran the activity of cellobiohydrolase reaches 40 U/ml and this of endogluconase – 520 U/ml. When the cultivation was performed on a medium containing wheat bran and straw the activity of xylanase reaches 240 U/ml.