Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture

AIMS: The nutritional requirements for mycelial growth of Cordyceps sinensis in semi-synthetic liquid media were investigated. The results provide a basis for further physiological study and industrial fermentation of the fungus. METHODS AND RESULTS: Nutritional requirements, including 17 carbohydrates, 16 nitrogen compounds, nine vitamins, four macro-elements, four trace-elements and eight ratios of carbon to nitrogen, were studied for their effects on the mycelial growth in submerged cultures of C. sinensis by using one-factor-at-a-time and orthogonal matrix methods. Among these variables, sucrose, peptone, folic acid, calcium, zinc and a carbon to nitrogen ratio 12 : 1 were identified as the requirements for the optimum mycelial growth. The concentrations of sucrose, peptone and yeast extract were optimized and the effects of medium composition on mycelial growth were found to be in the order sucrose > yeast extract > peptone. The optimal concentration for mycelial growth was determined as 50 g l(-1) sucrose, 10 g l(-1) peptone and 3 g l(-1) yeast extract. CONCLUSIONS: Under optimal culture conditions, over 22 g l(-1) of mycelial biomass could be obtained after 40 days in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Cordyceps sinensis, one of the most valued medicinal fungi, is shown to grow in axenic culture. This is the first report on nutritional requirements and design of a simplified semi-synthetic medium for mycelial growth of this psychrophilic species, which grows slowly below 20 degrees C. The results of this study will facilitate research on mass production of the fungus under defined culture conditions.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

Different proportions of laccase isoenzymes produced by submerged cultures of Trametes versicolor grown on lignocellulosic wastes

The white-rot fungus Trametes versicolor grown in submerged culture produced two laccase isoenzymes, LacI and LacII. Addition of insoluble lignocellulosic materials into the culture medium increased the total laccase activity. The proportion of laccase isoenzymes also changed depending on the lignocellulosic material employed, with ratios of activity LacII/LacI from 0.9 (barley straw) to 4.4 (grape stalks). Besides, this proportion played an important role in the dye decolourisation.     

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.     

The production and activity of extracellular amylase by Rhizoctonia bataticola

Rhizoctonia bataticola produced the highest amounts of amylase in medium containing starch than that lacking starch within the 10 days of culture. Doubling the concentration of starch in the growth medium resulted in a near doubling of the amylase activity. Amylase production by the fungus is related to the type of carbon source in the medium with maximum amylase produced in medium containing starch. The maximum activity of the enzyme was detected in extracellular filtrates obtained from 4 days cultures. After this period, amylase activity decreased at first, and then increased through the 10 days incubation period. The fungus produced maximum levels of amylase prior to attainment of maximum mycelial biomass. Peak activity of the extracellular amylase was recorded at a temperature and pH range of 20–25°C and 4–5 respectively. The role of the exoenzyme in the deterioration of stored food products and its possible use in industrial fermentation processes are discussed.     

Production of Dihomo-gamma-Linolenic Acid by a Delta5-Desaturase-Defective Mutant of Mortierella alpina 1S-4

A mutant, which has low Delta5-desaturase activity, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was shown to be a novel potent producer of dihomo-gamma-linolenic acid (DHGA). On submerged culture under optimal conditions for 6 days at 28 degrees C in a 10-liter fermentor, the mutant produced 3.2 g of DHGA per liter of culture broth (123 mg/g of dry mycelia), which accounted for 23.4% of the total mycelial fatty acids. Mycelial arachidonic acid amounted to only 19 mg/g of dry mycelia (0.5 g/liter of culture broth), which accounted for 3.7% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (11.0%), stearic acid (12.8%), oleic acid (22.7%), linoleic acid (8.9%), gamma-linolenic acid (6.5%), and lignoceric acid (7.8%). More than 97 mol% of the DHGA produced was found in the triglyceride fraction irrespective of the growth temperature employed (12 to 28 degrees C).     

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.     

The production of extracellular mucilaginous material (ECMM) in two wood-rotting basidiomycetes is affected by growth conditions

The ability of two wood-decay basidiomycetes to produce extracellular mucilaginous material (ECMM) and its relationship with total biomass production has been investigated. Growth and ECMM production by the white-rot fungus Coriolus versicolor and the brown-rot fungus Gloeophyllum trabeum were assessed in liquid culture under different nutritional and environmental conditions. Nutritional, pH and temperature factors all influenced significantly the proportion of ECMM in the total biomass produced. When total biomass production was reduced due to unfavorable growth conditions (stress), the proportion of ECMM in the biomass was elevated. The results are discussed with regard to the possible role(s) of ECMM in the responses of these fungi to stress.     

Determination of the parameters for producing a biobinder from wood: a mathematical modeling of the transformation of lignocellulose substrate by the fungus Panus tigrinus

A biochemical scheme for the transformation of wood lignocellulose during enzymatic hydrolysis of polysaccharides and lignin destruction in reactions involving free radicals was developed, and a corresponding mathematical model was constructed. Processing (fermentation) of wood particles by the fungus Panus tigrinus in a submerged culture for producing a biobinder of wood composites--woodchip boards and fiber-boards--is considered. The mathematical model was used to study the technological parameters that influence the production of enzymes and fungal biomass and the level of free radical accumulation in the substrate, i.e., the factors determining the production of the biobinder. The optimal values of these parameters were determined, namely: the specific surface of wood particles, amounting to 2000 cm2/g; processing time of 56 h; and an initial concentration of 3.0 g/l of fungal biomass in the submerged culture.     

Production of Dihomo-γ-Linolenic Acid by a Δ5-Desaturase-Defective Mutant of Mortierella alpina 1S-4

A mutant, which has low Δ5-desaturase activity, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was shown to be a novel potent producer of dihomo-γ-linolenic acid (DHGA). On submerged culture under optimal conditions for 6 days at 28°C in a 10-liter fermentor, the mutant produced 3.2 g of DHGA per liter of culture broth (123 mg/g of dry mycelia), which accounted for 23.4% of the total mycelial fatty acids. Mycelial arachidonic acid amounted to only 19 mg/g of dry mycelia (0.5 g/liter of culture broth), which accounted for 3.7% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (11.0%), stearic acid (12.8%), oleic acid (22.7%), linoleic acid (8.9%), γ-linolenic acid (6.5%), and lignoceric acid (7.8%). More than 97 mol% of the DHGA produced was found in the triglyceride fraction irrespective of the growth temperature employed (12 to 28°C).     

Prospects of using marine actinobacteria as probiotics in aquaculture

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l⁻¹) and EPS production (0.6 g l⁻¹) in submerged flask culture were achieved in a medium containing 30 g l⁻¹ maltose, 2 g l⁻¹ soy peptone, and 2 mM MnSO₄·5H₂O at an initial pH 2.0 and temperature 25°C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l⁻¹, respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.     

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.     

Production of polygalacturonase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> grown on tomato pomace and its chromatographic behaviour on immobilized metal chelates

Tomato pomace and pectin were used as the sole carbon sources for the production of polygalacturonase from a strain of Coriolus versicolor in submerged culture. The culture of C. versicolor grown on tomato pomace exhibited a peak of polygalacturonase activity (1,427 U/l) on the third day of culture with a specific activity of 14.5 U/mg protein. The production of polygalacturonase by C. versicolor grown on pectin as a sole carbon source increased with the time of cultivation, reaching a maximum activity of 3,207 U/l of fermentation broth with a specific activity of 248 U/mg protein. The levels of different isoenzymes of polygalacturonase produced during the culture growth were analysed by native PAGE. Differential chromatographic behaviour of lignocellulosic enzymes produced by C. versicolor (i.e. polygalacturonase, xylanase and laccase) was studied on immobilized metal chelates. The effect of ligand concentration, pH, the length of spacer arm and the nature of metal ion were studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). The adsorption of these lignocellulosic enzymes onto immobilized metal chelates was pH-dependent since an increase in protein adsorption was observed as the pH was increased from 6.0 to 8.0. The adsorption of polygalacturonase as well as other enzymes to immobilized metal chelates was due to coordination of histidine residues which are available at the protein surface since the presence of imidazole in the equilibration buffer abolished the adsorption of the enzyme to immobilized metal chelates. A one-step purification of polygalacturonase from C. versicolor was devised by using a column of Sepharose 6B-EPI 30-IDA-Cu(II) and purified enzyme exhibited a specific activity of about 150 U/mg protein, final recovery of enzyme activity of 100% and a purification factor of about 10. The use of short spacer arm and the presence of imidazole in equilibration buffer exhibited a higher selectivity for purification of polygalacturonase on this column with a high purification factor. The purified enzyme preparation was analysed by SDS-PAGE as well as by "in situ" detection of enzyme activity.     

Production and molecular characteristics of four groups of exopolysaccharides from submerged culture of Phellinus gilvus

AIMS: The objective of the present study was to determine the optimal culture conditions for the production of four groups of exopolysaccharides (EPSs) in Phellinus gilvus by submerged culture and to investigate their molecular properties by multi-angle laser-light scattering (MALLS) analysis. METHODS AND RESULTS: The optimal temperature and initial pH for the production of both mycelial biomass and EPSs in P. gilvus by submerged flask cultures were found to be 30 degrees C and pH 9.0, respectively. Glucose and corn steep powder were the most suitable carbon and nitrogen source for both mycelial biomass and EPS production. Optimal medium composition was determined to be glucose 30 g l-1, corn steep powder 5 g l-1, MgSO4 1.23 g l-1, KH2PO4 0.68 g l-1, and K2HPO4 0.87 g l-1. Four groups of EPSs (Fr-I, II, III, and IV) were obtained from the culture filtrates by gel filtration chromatography on Sepharose CL-4B and characterized by size exclusion chromatography (SEC) coupled with MALLS. The weight average molar mass (Mw) of Fr-I, Fr-II, Fr-III and Fr-IV were determined to be 8.628 x 106 (+/-129 420), 1.045 x 106 (+/-19 855), 61.09 x 104 (+/-1244), and 33.55 x 104 (+/-134) g mol-1, respectively. CONCLUSIONS: Under optimal culture conditions, the maximum EPS production in a 5-l stirred fermenter indicated 5.3 g l-1 after 11 days of fermentation. The SEC/MALLS analysis revealed that Fr-I, which has extremely high molecular weight, was presumably an aggregate of complex polysaccharides forming a compact globular shape; whereas Fr-II was nearly spherical, Fr-III and Fr-IV were rod-like chains in an aqueous solution. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of high amounts of EPSs from liquid-culture of the basidiomycete, P. gilvus. The SEC/MALLS approach used in this study could be useful in providing greater insight into the characterization of the mushroom polysaccharides without carrying out elaborate fractionation procedures prior to analysis.     

Growth morphology of Streptomyces akiyoshiensis in submerged culture: influence of pH, inoculum, and nutrients.

Most media in which the growth of shaken submerged cultures of Streptomyces akiyoshiensis was examined did not support the formation of well-dispersed mycelial suspensions. Investigation of the culture conditions promoting dispersed growth showed the pH of the culture medium to be of critical importance; an initial value of 5.5 minimized aggregation of the mycelium while supporting adequate biomass production. In cultures started at this pH, spore inocula gave better mycelial dispersal than did vegetative inocula; with spore inocula, growth morphology was also less affected by inoculum size. The composition of the nutrient solution influenced the extent of mycelial dispersal; slow growth was often associated with clumping but no clear correlation was observed between pellet formation and the ability of carbon or nitrogen sources to support rapid growth. Increasing the phosphate concentration from 0.5 to 15 mM caused a modest decrease in mycelial aggregation. Conditions promoting a well-dispersed mycelium suitable for studying the physiological control of secondary metabolism also supported the formation of 5-hydroxy-4-oxonorvaline by S. akiyoshiensis.     

Effect of culture conditions on antifouling compound production of a sponge-associated fungus

Microorganisms associated with invertebrate hosts have long been suggested to be a source for bioactive metabolites. In this study, we reported that a sponge-associated fungus, Letendraea helminthicola, produced two antifouling compounds: 3-methyl-N-(2-phenylethyl) butanamide and cyclo(D-Pro-D-Phe). To optimize the production of these antifouling compounds, we then examined the production of compounds under different culture conditions (temperature, salinity, pH, and carbon and nitrogen sources). This fungus grew well and produced more compounds at temperatures between 18 and 30°C; the fungus grew well at 75 parts per thousand (ppt) salinity but produced the highest amount of antifouling compounds at 30 and 45 ppt. The optimal initial pH value for mycelial growth was 5.5 to 6.5, whereas the production of the antifouling compounds was maximized at pH 3.5 and 4.5. Glucose and xylose (as carbon sources) increased the production of antifouling compounds. Yeast extract and peptone (as nitrogen sources) maximized the production of mycelial biomass and antifouling compounds. Our results indicate that culture conditions greatly affect the production of bioactive compounds from mycelial fungal cultures as exemplified by strain L. helminthicola and that the conditions favorable for fungal growth may not be the best conditions for bioactive compound production.     

Influence of humidity on production of pelleted fungal inoculum

One of the practical problems in scaling-up the production of fungal inocula for environmental applications is how to provide essential humidity for fungal growth. Pelleted solid substrate was used as a fungal biomass carrier. It was coated with alginate or agar hydrogels that contained mycelial fragments of the white-rot fungi Trametes versicolor or Irpex lacteus. To follow fungal growth and formation of mycelial coat over pelleted substrate, the fluorescein-diacetate hydrolysing activity (FDA) assay and visual inspection were used. Both fungi were able to overgrow the pelleted substrate in 5–6 days, at a relative humidity (RH) of 86.3% or higher. The enrichment of alginate hydrogel with nutrients or coating of pelleted substrate with more hydrophilic agar hydrogel enabled I. lacteus to overgrow the pellets at a lower RH of 83.6%. Fungal inocula produced at lower RH possessed lower final moisture contents and had greater mechanical strength. Conditioning of T. versicolor mycelial fragments, by a 3-h incubation in fresh growth medium, enhanced fungal growth over the pelleted substrate. A mathematical model was used to simulate and to explain moisture distribution in a hydrogel-coated pellet and the formation of mycelial coat, for various conditions of fungal inocula production.     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

Determination of the parameters for producing a biobinder from wood: A mathematical modeling of the transformation of lignocellulose substrate by the fungus Panus tigrinus

A biochemical scheme for the transformation of wood lignocellulose during enzymatic hydrolysis of polysaccharides and lignin destruction in reactions involving free radicals was developed, and a corresponding mathematical model was constructed. Processing (fermentation) of wood particles by the fungus Panus tigrinus in a submerged culture for producing a biobinder of wood composites—woodchip boards and fiberboards—is considered. The mathematical model was used to study the technological parameters that influence the production of enzymes and fungal biomass and the level of free radical accumulation in the substrate, i.e., the factors determining the production of the biobinder. The optimal values of these parameters were determined, namely: the specific surface of wood particles, amounting to 2000 cm²/g; processing time of 56 h; and an initial concentration of 3.0 g/l of fungal biomass in the submerged culture.