A Unique Pattern of Mycelial Elongation of Blakeslea trispora and Its Effect on Morphological Characteristics and β-Carotene Synthesis

The mycelial morphology of Blakeslea trispora was of crucial importance in the production of beta-carotene in submerged cultures of B. trispora. After the spores were inoculated, the time-course variation of mycelial morphology was closely examined under the microscope. With the addition of the non-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern of mycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance of mycelia with very short length, which allowed a well-dispersed growth of B. trispora without significant pellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungal culture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But the short mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition, because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation. The 20-fold increase in specific yield of beta-carotene (mg beta-carotene produced per g mycelium) was achieved with this drastic change in the pattern of mycelial elongation. The reason for this result might be more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in the well-dispersed mycelial cultures of B. trispora.     

Production of the fungal biocontrol agent Ulocladium atrum by submerged fermentation: accumulation of endogenous reserves and shelf-life studies

A method was developed for the induction of submerged conidiation of Ulocladium atrum Preuss (isolate 385) for the first time, using an oatmeal extract broth. Two inoculum types were produced by this process: spores and mycelial fragments. Spore production was stimulated by reducing the broth water potential (psi) to -2.1 MPa and adding 20 mM calcium chloride. In contrast, mycelial fragments were dominant at -7.0 MPa psi. Maximum total inoculum (mycelial fragments and conidia) yields were approximately 2 x 10(7) ml(-1) after 9 days incubation at 25 degrees C at 100 rpm. Biomass from liquid cultures responded to water-stress by accumulating increased concentrations of endogenous sugar alcohols (polyols), particularly glycerol. Long-term shelf-life studies showed that submerged inoculum from cultures subjected to an intermediate water-stress (-2.1 MPa psi) and containing enhanced levels of glycerol (> 300 mg g(-1) freeze-dried material) retained viability significantly better (P < 0.05) than that from unstressed cultures, when assessed on agar with fully available water. This level of viability was comparable to that of aerial U. atrum spores from a 4-week solid-substrate fermentation on oat grains. However, in contrast to aerial spores, the ability of submerged biomass to germinate in drier conditions declined significantly after 6 months.     

A Unique Pattern of Mycelial Elongation of Blakeslea trispora and Its Effect on Morphological Characteristics and β-Carotene Synthesis

The mycelial morphology of Blakeslea trispora was of crucial importance in the production of β-carotene in submerged cultures of B. trispora. After the spores were inoculated, the time-course variation of mycelial morphology was closely examined under the microscope. With the addition of the non-ionic surfactant (Span 20: Sorbitan monolaurate, E493) to the culture medium, a unique pattern of mycelial elongation was observed: 1) slow formation of germ tubes from spores and 2) appearance of mycelia with very short length, which allowed a well-dispersed growth of B. trispora without significant pellet aggregation. Span 20 appears to act like a paramorphogen. Without Span 20, however, the fungal culture finally formed a big clump of mycelium owing to heavy cross-linking of long mycelia. But the short mycelium maintained in the course of cultivation seemed to be irrelevant to growth inhibition, because the final concentration of dry mycelium was much higher with Span 20 after 3-day cultivation. The 20-fold increase in specific yield of β-carotene (mg β-carotene produced per g mycelium) was achieved with this drastic change in the pattern of mycelial elongation. The reason for this result might be more effective mass transfer and/or enhanced sensitivity to environmental oxidative stress in the well-dispersed mycelial cultures of B. trispora.     

巴西蘑菇原种培养基的筛选

以麦草、稻草、棉籽壳、玉米秸、木屑、麦粒、玉米粒和麦麸为原料 ,从 2 0种培养基中筛选出 2种适合巴西蘑菇菌丝生长的原种培养基。用这 2种培养基生产的菌种 ,菌丝粗壮、洁白、浓密 ,萌发快。     

Biomass production from Trichoderma viride in nonconventional oat medium

Oatmeal, an alternative, renewable, and low‐cost substrate, was used for the production of Trichoderma viride spores by submerged fermentation. The nonconventional oat medium was only supplemented with potato peptone, which is a green source of nitrogen for the microorganism. Because particles are suspended in the nonconventional oat medium, the characterization was based on viscosity, average particle diameter, size distribution, and porosity of the particles. Because of the complexity of the fungal biomass extraction, the dry weight and protein content were used as methods for quantifying the growth of T. viride. The inversion between the proportion of mycelia and spores was captured in the microscopic image analysis during the fermentation process. After 60 h, spores began to appear, accounting for most of the form present at 120 h of fermentation. The decrease in pH and the increase in glucose concentration during fermentation indicate that glucan hydrolysis occurs and that glucose is released into the medium. The potential for industrial applications of submerged fermentation with oats for biomass production of T. viride is noted in the results. This simple and easily controllable process has several advantages, including the use of low‐cost substrates for the propagation of a microorganism that is widely used in scientific and commercial settings. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Formulation of filamentous fungi for bioremediation

Mycelia of Marasmiellus troyanus embedded in calcium alginate granules with corn cob grits as a nutritive amendment were viable after one year with refrigeration but inviable when stored at room temperature. With refrigeration, Phanerochaete chrysosporium mycelia and spores embedded in alginate were both viable after one year. At room temperature, spores encapsulated in alginate granules gave good viability while mycelial formulations did not. In all trials, corn cob grits was superior to saw dust for extending shelf life. Corn cob grits-amended granules of both species were able to germinate and grow in both uncontaminated soil and chemical waste-contaminated soil. © Rapid Science Ltd. 1998     

Production of conidia of Alternaria cassiae with alginate pellets

Formulations of alginate-encapsulated mycelia are used to generate spores for mycoherbicidal application to weed-infested fields and for bulk production of spore-based products. Spore yield of such formulations is a primary determinant of product efficacy. A number of parameters of the alginate process were studied to develop an optimal alginate formulation for field application of Alternaria cassiae, a mycoherbicide for sicklepod (Cassia obtusifolia). The composition of the fermentation medium and of the filler used in formulation and the fermentation time were important variables. The addition of nutrients to the mycelial homogenate after fermentation increased sporulation but the amount and ratio of nutrients in the fermentation medium had a greater influence on spore yield from pellets. Optimal sporulation resulted from mycelia produced during a 60- to 70-h fermentation in 2.4% dehydrated potato dextrose broth and 14% V-8 vegetable juice and entrapped in pellets containing corn cob grits as the filler.     

灰树花孔菌固体发酵基质抗氧化活性成分研究

选用燕麦、大豆、玉米、麸皮、大米、荞麦6种培养基质对灰树花孔菌进行固体发酵及抗氧化活性研究。结果表明：灰树花孔菌最佳抗氧化发酵基质为燕麦大豆培养基（燕麦16.25%,大豆83.75%）,最佳发酵时间为8d。灰树花孔菌燕麦大豆发酵基质抗氧化活性成分种类丰富,含量高。其中总黄酮物质40.86μg/g,总三萜5.94mg/g,多酚8.51mg/g,还原糖13.10mg/g,花色苷70.23μg/g,维生素C 24.57mg/g,维生素E 0.33mg/g,谷光甘肽（GSH）0.84g/g,SOD酶活性299.74U/g。     

Micromorphological features of Pleurotus pulmonarius (Fr.) Quel. and P. ostreatus (Jacq.) P. Kumm. strains in pure and binary culture with yeasts

Micromorphology of oyster mushrooms Pleurotus ostreatus (Jacq.) Quel. and P. pulmonarius (Fr.) Quel. was studied in pure and binary culture with yeasts (Cryptococcus laurentii 1629, Rhodotorula minuta 2790, Sporidiobolus salmonicolor (see text for symbol) 31A-11, Candida krusie 3452, Pichia holstii 3438). The cultures were cultivated on malt-agar and water agar. Various mycelial structures were described: strands, rings, thin searching mycelium, clamps, crystals, head-like offshoots, mycelial fragments, chlamydospores, and coralloid hyphae. Vegetative mycelia interact in different ways (forming anastomoses, strands, system of thin anucleate hyphae) within the same culture. Head-like offshoots of mycelial cells, previously regarded as spores of asexual reproduction, appeared to lack nuclei and to be filled with polyphosphates. Coralloid hyphae, which induce yeast cell lysis after direct contact, were detected only in binary culture with yeasts under condition of nitrogen deficit. The same way of feeding is typical for carnivorous mushrooms.     

Confining mycelial growth to porous microbeads: a novel technique to alter the morphology of non-newtonian mycelial cultures

In an effort to alter the filamentous morphology of Penicillium chrysogenum cells, a technique was developed to confine the growth of the mycelia to porous celite beads. The pore matrix of these beads was found to be very effective for entrapping mycelial cells and spores. The entrapped spores were used to initiate the fermentations in shake flask cultures. Significant increases in final cell densities were obtained in the confined cell cultures reaching up to 60 g/L cells. This is nearly double the cell concentration attainable in free cell cultures grown in the absence of beads. Cell loadings up to 0.55 g cells per bead were obtained in the confined cell cultures. In the later stages of the fermentations, the specific oxygen uptake rates in the confined cell cultures were found to decrease with respect to free cell cultures.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

A COMPARATIVE STUDY OF THE GROWTH OF WILD OATS (AVENA FATUA L. AND A. LUDOVICIANA DUR.) AND OF CULTIVATED CEREALS WITH VARIED NITROGEN SUPPLY

Growth analysis of wild oats ( Avena fatua and A. ludoviciana ) grown in pots with different levels of nitrogen supply showed many similarities to spring barley, winter oats and winter wheat.
Small differences that could affect competition between wild oats and cereals occurred mainly in the seedlings. Wild oat seedlings were smaller than the corresponding cultivated cereals in total dry weight, total nitrogen content, leaf area and number of shoots. However, very young wild oat plants had higher net assimilation rates than the cultivated cereals and soon caught up and passed them. The difference in net assimilation rate did not persist, and in the later stages of growth differences in dry-matter production depended mainly on differences in leaf area. Another important difference between wild oats and cultivated cereals was that 98–100% of the wild oat seeds and none of the crop seeds were dormant 2 months after harvest.
Ear emergence in wild oats spread over a longer period, the range of ear heights was greater and the tallest ears were taller than in the corresponding cultivated cereals. Assimilation in the ear appeared to account for less of the total dry matter of the plants of wild and cultivated oats than of wheat. The wild oats produced more seeds per plant than the cultivated cereals, but the 1000-grain weight, and hence the total dry weight of seeds, was lower in the weeds than in the crop.
Addition of nitrogen to the soil affected the growth of the wild oats in the same ways as the cultivated cereals; they took up the same amount of nitrogen per plant as winter oats and winter wheat but more than spring barley.
It is concluded that wild oats are most susceptible in the seedling stage to competition from the crop and that nitrogenous fertilizer applied to an infested field is unlikely to alter the balance between the yields of crop and of wild oats.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

Free amino acid pools of Trichoderma aureoviride during conditions of glucose-limited growth and glucose starvation

Glucose-limited and glucose-starved cultures of Trichoderma aureoviride were analyzed for the size and composition of the mycelial free amino acid pool. In glucoselimited mycelia the pool size increased as a function of the specific growth rate above a value of ca. 0.08 h^-1 and this was due principally to increasing concentrations of alanine and glutamic acid. During glucose starvation, the net pool size decreased only by ca 20% although a transient elevation of free amino acids was observed, the latter being attributed to the turnover of mycelial proteins. The amino acid pool compositions were categorized according to their ionic nature and, although no particular group varied significantly in its percentage contribution to the total pool size of growing mycelia, the observed variations during starvation were mostly attributable to the basic and acidic amino acids. Comparisons are made of the results with those obtained for other species of filamentous fungi and some possible explanations for the observed variations are discussed.     

Isolation and identification of a new hypocrellin A-producing strain Shiraia sp. SUPER-H168

A new hypocrellin A-producing strain, Shiraia sp. SUPER-H168, was isolated from tissues of bamboo, Brachystachyum densiflorum. The morphology of this strain was characterized with a light microscope and a scanning electronic microscope. The mycelia, conidia, pycnidia of fungus were observed. Small pycnidia (10-20 microm in length) full of small conidia appeared on the mycelia, which were first reported in this study. The 18S rDNA region of this strain was amplified and sequenced. Then a neighbor-joining tree of 18S rDNA was constructed. According to the result of analysis, the strain SUPER-H168 was proved to belong to the genus Shiraia. Hypocrellin A was produced by solid-state fermentation, extracted by acetone, isolated by preparative RP-HPLC, and identified by RP-HPLC, ESI-MS and ultraviolet-visible absorbing scanning with diode array detection. The HA production could reach 2.02 mg/g dry solid substrate.     

Factors Affecting the Antigenicity of Trichophyton rubrum

Nitrogen determinations, performed upon the mycelia of Trichophyton rubrum, indicated that both the total nitrogen to mycelial weight ratio and the protein nitrogen to mycelial weight ratio decreased as the age of the mycelia increased. An increase in nitrogen concentration in the medium produced an increase in the total nitrogen to mycelia weight ratio, but did not necessarily increase the protein nitrogen to mycelial weight ratio. The optimal nitrogen source concentration which produced the highest protein nitrogen to mycelium ratio was found to be considerably less than that recommended in most standard Sabouraud medium formulations. Antisera to antigen preparations, grown on low concentrations of Multipeptone, produced more lines in the gel diffusion reaction than did antisera to antigens grown on standard concentrations of Multipeptone. Antisera to antigenic preparations from 2-week-old mycelia exhibited better and sometimes more lines than those of antigens prepared from 1- or 3-week-old mycelia, regardless of the nitrogen concentration in the medium. Dialysis and storage of the antigen produced no change in the quality of the precipitin lines, even though both processes involved considerable loss of Lowry protein. Immunofluorescence studies showed that young mycelia were more antigenic than the old mycelia, since a substantial degree of cell wall fluorescence was exhibited by the young mycelia, especially at the hyphal tips. Older mycelia lacked this fluorescence. An extracellular antigen was also found to be associated with the young mycelia, but cytoplasmic fluorescence was not observed.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Effect of L-amino acids on Mucor rouxii dimorphism.

Mucor rouxii organisms growing aerobically and exponentially on a well-defined minimal medium are able to differentiate as yeasts or as mycelia, depending on the amino acid as the nitrogen source. When certain amino acids were used as the nitrogen source, spores differentiated only as hyphae, whereas other amino acids gave rise to other morphological forms having different ratios of yeasts to hyphae. In both hyphal and yeast cultures, an aerobic metabolism was predominant, as shown by determining several metabolic parameters such as oxygen tension, glucose consumption, ethanol production, and CO2 release. A complete conversion of yeasts to hyphae was obtained by the appropriate change in the amino acid used as nitrogen source. By preparing spheroplasts from mycelial cultures and transferring them to media with amino acids that induce yeast formation, a 50% yield in the reverse transformation was achieved. A correlation between the change in pH of the medium and cell morphology was observed in different growth conditions. Decrease in the pH of the medium preceded the appearance of hyphae. Also, when the initial pH of the medium was increased, aspartate-containing cultures developed mainly as mycelia, instead of yeasts, with a corresponding decrease in the final pH.     

A SURVEY OF WILD OATS (AVENA FATUA AND A. LUDOVICIANA) IN ENGLAND AND WALES IN 1951

In 621 samples of wild oats, collected in England and Wales by N.A.A.S. and N.I.A.B. Officers, only two species ( Avena fatua and A. ludoviciana ) were found but both were very variable. A. fatua occurred in all wheat- and barley-growing areas, in both winter and spring corn and on all soil types. A. ludoviciana occurred (with two exceptions) only within approximately 80 miles radius of Oxford, mainly on heavy soila and chiefly in winter corn.
The amount of wild oats present appeared to depend on the frequency in the rotation of corn or other crops in which wild oats would shed seed. Deep ploughing often increased the infestation. The data did not show whether a field could be freed of wild oats by grassing down for a number of years, nor what is the maximum period of survival of buried wild oat seeds.