Study of the chemical makeup of the mycelium from an active strain of Act. rimosus and from an inactive mutant in relaiton to oxytetracycline biosynthesis]

Chemical composition of the mycelium of the active and inactive mutants of Act. rimosus grown under conditions favourable for oxytetracycline biosynthesis on the starch or maltose medium and under favourable conditions on the glucose medium was studied. It was shown that according to its chemical composition the above strains did not practically differ. When grown on the starch medium the mycelium of both strains contained great amounts of carbohydrates and comparatively small amounts of nucleic acids and nitrogen. Replacement of starch in the medium by glucose or maltose induced significant changes in the mycelium composition: the synthesis of intracellular polysaccharides was markedly suppressed and the synthesis of nucleic acids and nitrogen containing compounds increased. RNA was the main nucleic acid in both strains on starch and glucose media. The content of DNA was low and did not practically change. The mycelium of both strains contained small amounts of lipids which did not significantly change during the process of cultivation and did not correlate with the antibiotic activity.     

Distribution of Cellular Carbohydrates during Development of the Mycelium and Fruitbodies of Flammulina velutipes

Flammulina velutipes (Curt. ex Fr.) Sing. was grown on potato-glucose solution freed of most starch. Glucose uptake and dry weight changes in the colony indicated that the large fruitbodies derived their substrates partly from glucose remaining in the medium and partly from cellular constituents stored in the mycelium and small fruitbodies. Changes in the amounts of low molecular weight carbohydrates, glycogen, and four cell wall polysaccharide fractions were followed in the mycelium and fruitbodies. Trehalose, arabitol, and smaller amounts of mannitol were the main stored low molecular weight carbohydrates. A large net loss of these compounds occurred in the mycelium and small fruitbodies after their growth ceased. The carbohydrates accumulated in the large fruitbodies, but were also partly metabolized in the colony. Reducing sugars were minor components, and included about 30 to 50% glucose and a small undetermined quantity of fructose. Glycogen was the main storage carbohydrate in the mycelium, and was also stored in the small fruitbodies. It was broken down in both structures during growth of the large fruitbodies which accumulated only small amounts. During the same period, almost 45% of the maximum amount of cell wall polysaccharides were degraded in the small fruitbodies, but not in the mycelium.     

Study of certain carbohydrate metabolism enzymes in an active oxytetracycline producer strain and in an inactive mutant in relation to antibiotic biosynthesis]

The activity of the glycolysis enzymes, i.e. aldolase and pyruvate decarboxylase and the enzymes of the pentose cycle, i.e. transketolase were investigated in the process of cultivation of an active strain and inactive mutant of Act. rimosus under conditions favourable for oxytetracycline biosynthesis on starch medium and under unfavourable conditions on glucose medium. It was shown that the aldolase and transketolase activity in the inactive mutant was higher on the starch medium as compared to the active strain, while the activity of pyruvate dekarboxylase was lower. The above difference between the both strains was preserved on the glucose medium and the activity of aldolase and transketolase in both strains increased, while the activity of pyruvate dekarboxylase remained at the same level.     

Carbohydrate utilization by Trichomonas gallinae: effects on growth and metabolic activity under nongrowth conditions

Trichomonas gallinae used 13 of 29 carbohydrates for growth. Quantitative relationships between final populations, acid production, and cellular glycogen contents varied depending on the substrate. The effect of growth on different carbohydrates on the subsequent utilization of carbohydrates by cells under nongrowth conditions was studied by measuring carbohydrate uptake, changes in cellular glycogen content, and gas production. Two major utilization patterns were found. Cells grown on maltose or starch used these substrates well, but cells grown on other sugars did not. All cells used glucose, fructose, galactose, and mannose, but cells grown on maltose or starch did not use them as well as cells grown on other sugars. All cells used ribose slightly but not xylose or arabinose. Turanose, a disaccharide yielding high populations in growth medium, was not used under nongrowth conditions.     

Conditions of cultivation, composition, and biological activity of mycelium of Flammulina velutipes (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karat. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5-10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6-78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages--central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Conditions of cultivation,composition, and biological activity of mycelium of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karst. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5–10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6–78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages—central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.     

Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: I. Autotrophic conditions

The effect of the microalgae-growth promoting bacterium Azospirillum brasilense on accumulation of total carbohydrates and starch in two species of Chlorella (Chlorella vulgaris and Chlorella sorokiniana), when the bacterium and each microalga were jointly immobilized in alginate beads was studied under autotrophic conditions for 144h in synthetic medium. The interaction of the bacterium with the microalgae enhanced accumulation of total carbohydrate and starch. Cells of Chlorella accumulated the highest amounts of carbohydrate after incubation for 24h. Yet, this did not coincide with the highest affinity and volumetric productivity measured in these cultures. However, after incubation for 72h, mainly in jointly immobilized treatments of both microalgae species, the cultures reached their highest total carbohydrate content (mainly as starch) and also the highest affinity and volumetric productivity. These results demonstrate the potential of A. brasilense to affect carbohydrates and starch accumulation in Chlorella spp. when both microorganisms are co-cultured, which can be an important tool for applications of microalgae.     

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.     

Beta-glucanase and chitinase biosynthesis in a culture of a mycophilic strain of Trichoderma viride

The production of extracellular 1,3-, 1,6-beta-glucanases and chitinase was studied during submerged cultivation of a Trichoderma viride strain 3/78 on various carbon sources: glycerol, glucose, lactose, sucrose, laminaran, starch, pustulan, chitin, and Agaricus bisporus fruit bodies. The synthesis of these enzymes and cellulase was studied also under the conditions of depression at low concentrations (10(-2) and 10(-3)M) of the first five aforementioned carbon sources as well as cellobiose, gentiobiose, N-acetyl-beta-D-glucosamine and 0.1% chitooligosaccharides and A. bisporus cell walls. The experiments were conducted with the washed mycelium of this strain grown for 2 days in a medium with glycerol as a carbon source. The results indicated that 1,3- and 1,6-beta-glucanases of the strain were of the constitutive nature and were repressed by such carbon sources as glycerol and glucose. Chitinase and cellulase were shown to be inducible enzymes. Chitinase was induced by N-acetyl-beta-D-glucosamine, chitooligosaccharides and A. bisporus cell walls as well as by lactose when the fungus was grown on this carbon source. Cellulase biosynthesis was induced by lactose, cellobiose and gentiobiose.     

Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity.

Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N-nitrosoguanidine together with selective enrichment on the glucose analog 2-deoxyglucose. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively. C. acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain. The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C. acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone. Localization studies demonstrated that the amylolytic activities of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested.     

Effect of carbohydrates in the culture medium on amino acid composition of a cephalosporin C producer

The intracellular and extracellular amino acid composition of an auxotrophic methionine-deficient strain of Acremonium chrysogenum was studied in respect of the content of various carbohydrates in the fermentation medium. In the presence of glucose, an intensive involvement of exogenous DL-methionine into the cell metabolism was observed at earlier stages than in the presence of dextran or succrose. The total content of intracellular amino acids was lower in the cells grown on the medium with glucose. The production of cephalosporin C depended on the intracellular content of methionine, glutamic acid and lysine.     

Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity.

Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N-nitrosoguanidine together with selective enrichment on the glucose analog 2-deoxyglucose. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively. C. acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain. The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C. acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone. Localization studies demonstrated that the amylolytic activities of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested.     

Effect of Carbohydrate Demand on the Remobilization of Starch in Stolons and Roots of White Clover (Trifolium repens L.) after Defoliation

White clover plants were grown from stolon tips in growth cabinetsand then defoliated. Thereafter, changes in the contents ofnon-structural carbohydrates such as starch, sucrose, glucose,fructose, maltose, and pinitol in stolons and roots were monitored.Initial contents of carbohydrate reserves, photosynthetic supplyof new carbohydrates and carbohydrate demand after defoliationwere varied by growing the plants at various CO₂ partial pressures,by varying the extent of defoliation and by removing eitherroots or stolon tips at the time of defoliation. Remobilization of carbohydrate reserves in stolons increasedproportionally to their initial contents and was greater whenplants had been severely defoliated, suggesting that carbohydrateswere remobilized according to availability and demand. Starchwas the predominant reserve carbohydrate. Starch degradationwas associated with decreased contents of sucrose, glucose andfructose in young stolon parts and roots but not in old stolonparts suggesting that starch degradation was not strictly controlledby the contents of these sugars. A decrease in the demand forcarbohydrates by removal of roots did not decrease starch degradationbut increased the contents of sucrose, glucose, and fructose.Removal of stolon tips decreased starch degradation and contentsof sucrose, glucose, and fructose. The results suggest thatstarch degradation was controlled by a factor other than sucrose,glucose, and fructose which was exported from stolon tips, e.g.gibberellin. Key words: White clover, storage carbohydrates, remobilization, regrowth     

Differences in the carbohydrate composition between the yeastlike and mycelial cells of Mucor hiemalis

The use of sporangiospores from a 20-day culture for inoculation enabled us to obtain yeastlike cells of strain Mucor hiemalis F-1431 (for which capacity for dimorphic growth was not previously studied) by cultivation in liquid medium under aerobic conditions. The carbohydrate composition of the fat-free biomass of the mycelial and yeastlike forms grown in the same culture under aerobic conditions was studied. In the fat-free biomass consisting of yeastlike cells, as compared to the mycelium, the contents of chitin and glucose decreased from 25 and 30.9% of dry biomass to 14 and 17.5% of dry biomass, respectively. We failed to detect any changes in the contents of fucose, galactose, mannose, and uronic acids among the heteropolysaccharide monomers of fungal cells of different morphotypes, which is probably due to the aerobic cultivation conditions. It was suggested that the composition of heteropolysaccharide monomers does not play such a significant role in the formation of yeastlike cells in the culture grown under aerobic conditions as the content of chitin, the main supporting biopolymer.     

Calcium ions and the differentiation of Streptomyces hygroscopicus 155, a producer of an antibiotic complex]

The effect of Ca2+ on differentiation of Streptomyces hygroscopicus 155 and its inactive variant 155-0 was studied. Addition of Ca2+ to the medium induced formation of the aerial mycelium in the inactive variant and accelerated formation of the aerial mycelium in the parent strain. The inhibitory effect of EGTA, verapamil, nifedipin, chlorpromazine and dilthiazeme on the aerial mycelium formation demonstrated the physiological role of Ca2+ in the process. Addition of pandavir (nigericin) and azalomycin B, the antibiotics produced by the streptomycete, induced formation of the aerial mycelium in the inactive variant. The effect was higher in the presence of Ca2+. Streptomyces hygroscopicus 155 and its inactive variant synthesized a proteolytic complex containing metalloproteases and trypsin-like proteases. The total proteolytic activity of the inactive variant was lower than that of the parent strain. Addition of Ca2+ to the medium stimulated their proteolytic activity. The inducing action of the antibiotics produced by the parent strain on differentiation of S.hygroscopicus 155-0 and the increase of their action in the presence of Ca2+ suggested that they controlled the differentiation and that such a function of the antibiotics expressed itself through the Ca2+ signal system.     

Changes in the fungus-specific,soluble-carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius

A simple and convenient culture system has been developed for the analysis of ectomycorrhiza formation under controlled conditions. Rapid and synchronous mycorrhiza synthesis was observed when thin and even layers of Pisolithus tinctorius (Pers.) hyphae were brought at once into contact with the entire root system of 3-month-old Picea abies (L. Karst) plants. Suitable fungal layers were grown on cardboard with limiting glucose supply in the medium to maximize radial growth. The glucose was almost consumed by the time the fungus had spread over the whole cardboard and was ready for inoculation of the roots. At this stage, the fungus contained trehalose and arabitol as the main soluble carbohydrates. A few hours after the assembly of the culture system, contacts between roots and aerial hyphae were observed and a sheath was formed 3 days later, suggesting very rapid ectomycorrhiza formation under these conditions. The pool of soluble carbohydrates of the inoculum, i.e. the extramatrical mycelium, declined after inoculation of the roots and was almost zero after 2 weeks. The supply of carbon by the plant was then sufficient for the fungus to expand the soluble pool efficiently in both the mycorrhizas and the extramatrical mycelium. The kinetics of the carbohydrate pool and the observed differentiation of the short roots to mycorrhizas imply that in our culture system fully functional symbiosis was established no later than 14 days after the plants were inoculated with the fungus.     

Glycoside hydrolase enzymes present in the zoospore and vegetative growth stages of the rumen fungi Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate, grown on a range of carbohydrates

The rumen fungi Neocallimastix patriciarum, Piromonas communis, and a morphologically distinct but unidentified isolate were cultivated on the polysaccharides starch, cellulose, xylan, and their principal component monosaccharides and disaccharides, and the range and specific activities of the glycoside hydrolases formed were monitored using gluco-oligosaccharide and p-nitrophenyl glycoside substrates. A wide range of enzyme activities was detected in preparations from vegetative growth and zoospores of all three isolates. Enzyme activity was also present in the culture medium. The specific activities were affected by the carbohydrate source available in the growth medium, although the more active hydrolases involved in the degradation of plant structural and storage polysaccharides were formed on all seven carbohydrate sources evaluated. Enzyme activities were increased in the zoospore, vegetative, and extracellular preparations after growth on the appropriate structurally related disaccharide or polysaccharide. The hemicellulolytic glycosidases (alpha-L-arabinofuranosidase, beta-D-xylosidase) were most active after growth on xylan, whereas alpha-/beta-glucosidase activity was increased with the corresponding glucan as growth substrate. However, whereas wide-ranging beta-glucosidase activity was detected following growth on maltose or starch, the alpha-glucosidase activities of P. communis were lower or undetectable in vegetative preparations grown on glucose or the beta-glucans cellobiose and cellulose.     

Comparison of Growth and Primary Shunt Product Formation by Claviceps purpurea Cultured on Succinic Acid and Glucose as Carbon Sources

Growth on a medium containing succinic acid as the sole carbon source produced 1 g (dry weight) of mycelium per liter of medium by 50 days of incubation, whereas 25 g of mycelium was produced in 10 days when glucose was also present in the medium. Primary shunt metabolism took place during growth on succinic acid in spite of the extremely slow growth. Mycelia grown on succinic acid contained a higher percentage of residual mycelium and phosphate, but a lower percentage of mannitol, carbohydrate, lipid, and water-soluble nitrogen, than mycelia grown on a mixture of glucose and succinic acid. Thus, although primary shunt metabolism is favored by rapid growth on a rich, balanced sugar medium, it can also take place during extremely restricted growth in a medium containing succinic acid as the sole carbon source.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates.

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.