Effect of Culture Conditions on Ergosterol as an Indicator of Biomass in the Aquatic Hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 μg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 μg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r² = 0.83 to 0.98) and highly significant (P 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

Fungal biomass associated with decaying leaf litter in a stream

Summary Fungal biomass, measured as ergosterol content, was determined on alder leaf litter incubated during autumn in a softwater Pyrenean stream. The ergosterol content of the leaf litter increased rapidly to a maximum of 462 μg/g detrital dry mass. Ergosterol contents of aquatic Hyphomycetes grown in shake culture were typically ≤5 mg/g mycelial dry mass. Using the corresponding ergosterol-to-biomass conversion factor of 200, peak fungal mass accounted for 9.2% of total system mass, or 10.2% of leaf dry mass. For the period of highest activity (incubation days 7–28), net fungal production on leaf litter was estimated as 2.3 mg d⁻¹ g⁻¹ leaf mass. A conservative estimate of the growth efficiency for the same period was 105 mg mycelial mass per gram leaf mass degraded, assuming that non-leaf organic matter did not constitute an important carbon source supporting fungal production.     

Pellet size affects mycelial ergosterol content in aquatic hyphomycetes

Ergosterol was measured in mycelia of seven species of aquatic hyphomycetes grown in malt-extract broth. The harvested 21 d old pellets were grouped into 5-6 classes based on size, which were analyzed separately. In all but one species, there was a significant, positive correlation between the amount of ergosterol per unit mass and pellet diameter. Ignoring this correlation could result in the misleading conclusion that there is no relationship between mycelial mass and its absolute ergosterol content. The highest ergosterol concentrations were close to the average generally used to convert the amount of ergosterol in environmental samples to fungal biomass; the average was about half that value.     

Ergosterol-to-Biomass Conversion Factors for Aquatic Hyphomycetes

Fourteen strains of aquatic hyphomycete species that are common on decaying leaves in running waters were grown in liquid culture and analyzed for total ergosterol contents. Media included an aqueous extract from senescent alder leaves, a malt extract broth, and a glucose-mineral salt solution. Concentrations of ergosterol in fungal mycelium ranged from 2.3 to 11.5 mg/g of dry mass. The overall average was 5.5 mg/g. Differences among both species and growth media were highly significant but followed no systematic pattern. Stationary-phase mycelium had ergosterol contents 10 to 12% lower or higher than mycelium harvested during the growth phase, but these differences were only significant for one of four species examined. Availability of plant sterols in the growth medium had no clear effect on ergosterol concentrations in two species tested. To convert ergosterol contents determined in field samples to biomass values of aquatic hyphomycetes, a general multiplicative factor of 182 is proposed. More accurate estimates would be obtained with species-specific factors. Using these in combination with estimates of the proportion of the dominant species in a naturally established community on leaves resulted in biomass estimates that were typically 20% lower than those obtained with the general conversion factor. Improvements of estimates with species-specific factors may be limited, however, by intraspecific variability in fungal ergosterol content.     

Ergosterol as an indicator of mould growth on wood in relation to culture age, humidity stress and nutrient level

Changes in ergosterol content in cultures of Penicillium brevicompactum and Aspergillus versicolor on wood with time, changes in humidity or addition of glucose solutions to wood were studied with HPLC. Lowering of the humidity level caused a very large decline in ergosterol content of cultures of P. brevicompactum on wood over a 10 day period, although small amounts remained after this time. After an initial increase, up to an inoculation time of 45 days, reductions were also observed in control samples maintained at 100% RH, but these were smaller. The amount of ergosterol decreased to very low levels in wood impregnated with low levels of glucose during a 93 day incubation period. Ergosterol concentration in hyphae produced in surface liquid cultures was shown to be higher in mycelia growing on media enriched with nitrogen or with more available nutrients. The concentration of ergosterol in the mycelia of P. brevicompactum in surface liquid cultures varied by a factor of 5 from 2 to 10 mg g⁻. The results clearly show that ergosterol present in solid materials in mainly related to active biomass. With certain prerequisites, ergosterol determinations could also be used for total fungal biomass estimations on wood.     

Lead and cadmium uptake in the marine fungi Corollospora lacera and Monodictys pelagica

This study provides observations on the effects of lead and cadmium ions on the growth of two species of marine fungi, Corollospora lacera and Monodictys pelagica. On solid media lead appeared to have no effect on the radial rate of growth of fungi. Exposure to increasing cadmium concentrations on solid media resulted in significant reduction (p < 0.05) in the radial mycelial growth rates of both fungi, especially in M. pelagica. These results reveal significant difference in species sensitivity toward cadmium and, essentially, insensitivity toward lead exposure. In liquid cultures, the metal content of mycelia (metal mass found in mycelium, in mg), and the concentration of metal in dry mycelium (metal mass in 1g of mycelium, in mg g(-1)) were both found to increase (p < 0.05) with the increase in the metal cation concentration, while mycelium dry mass decreased. As it was observed on solid media, cadmium cation affected more severely (p < 0.05) the growth of M. pelagica in liquid cultures. Ergosterol content of mycelia of C. lacera exposed to increasing cadmium cation concentration decreased, similarly to the trend observed for dry mycelial mass. It was found that ca. 93% of all lead sequestered by C. lacera is located extracellularly. M. pelagica was found to bioaccumulate over 60 mg of cadmium and over 6 mg of lead per 1 g of mycelium, while C. lacera bioaccumulated over 7 mg of cadmium and up to 250 mg of lead per 1 g of mycelium. Overall, the results indicate that both metal ions affect the growth of marine fungi with lead being accumulated extracellularly in the mycelia. Both metals accumulated by fungi may then enter the marine ecosystem food web, of which marine fungi are integral members.     

Mathematical models of mycelium growth and ergosterol synthesis in stationary mould culture

Aims:  To develop mathematical models for mycelium growth and ergosterol formation in conditions of periodic stationary culture; to verify possibilities of applying a model describing the relationship between ergosterol content and mycelium quantity.
Methods and Results:  The mould growth and ergosterol formation models covering all phases of mould growth are described using a modified logistic equation with the addition of an exponential function. The correlation between ergosterol and mycelium biomass depended on the growth phase of mould. Meaningful relation was obtained for initial two phases, when both parameters were growing equally. The quadratic function for estimation of the biomass based on ergosterol content was formulated. The error resulting from the application of this function in initial phases of moulds growth was small at 5–7%, in the following phases it was at 11–31%.
Conclusions:  Mycelium biomass can be precisely determined basing on the ergosterol content, when we know the moulds growth phase. In natural environments, without the information about growth phases, it will be possible, but with the higher error.
Significance and Impact of the Study:  Presented model based on the ergosterol content making possible to estimate the quantity of mycelium in moulds cultures and natural environments.     

The extraction and quantification of ergosterol from ectomycorrhizal fungi and roots

Recently, ergosterol analysis has been used to quantify viable fungal biomass in resynthesized ectomycorrhizae. An objective of our study was to quantify ergosterol in a range of ectomycorrhizal isolates under differing growth conditions. In addition, we tested the applicability of the method on field-collected roots of ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal plants. Quantification of sitosterol as a biomass indicator of plant roots was also undertaken. Ergosterol was not detected in roots of uninoculated Betula populifolia seedlings, and sitosterol was not detected in an ectomycorrhizal fungal isolate but was present in birch roots. Ergosterol was produced in all isolates examined, which represented the major orders of ectomycorrhizal fungi. The range of values obtained, from 3 to nearly 18 g ergosterol mg^-1 dry mass, agrees well with reported values for other mycorrhizal and decomposer fungi. Hyphal ergosterol was the same during growth on phytic acid and KH₂PO₄. Reduction of growth temperature from 25° C to 15° C had little effect on ergosterol content of cultures harvested at similar growth stages. Ergosterol and sitosterol were detected in field-collected ectomycorrhizae of B. populifolia and Pinus sylvestris and VA mycorrhizae of Acer rubrum and Plantago major. Both ergosterol content and ergosterol to sitosterol ratios were significantly lower in VA mycorrhizae than ectomycorrhizae. Calculations of viable fungal biomass associated with field-collected roots were in agreement with those reported by others using the method on resynthesized ectomycorrhizae. Estimates of total mass could be obtained for field-collected B. populifolia roots by a simultaneously using ergosterol to estimate fungal biomass and sitosterol to estimate root mass. Some potential applications and limitations of sterol quantification in studies of mycorrhizal physiology and ecology are discussed.     

Ergosterol content of Rhizopus oligosporus NRRL 5905 grown in liquid and solid substrates

Summary The ergosterol content of Rhizopus oligosporus NRRL 5905 varied between 2–24 g/ mg biomass dry matter when grown in laboratory media and was found to be influenced by the substrate composition.When grown on a natural substrate (soya beans) the ergosterol content was considerably higher (estimated at approx. 60–90 g/mg biomass dry matter).In laboratory media, the ergosterol content was also influenced by the extent of aeration and the growth phase of the mycelium; within the range of 25° C–35° C, the incubation temperature did not influence the ergosterol content significantly.In view of these variations, ergosterol should not be used as a chemical index for the quantification of biomass grown in static solid-substrate fermentations with limited mass transfer, e.g. tempe or oncom.     

Influence of age on ergosterol content in mycelium of Aspergillus ochraceus

Ergosterol has been suggested as a paramoter for studying the content and evolution of fungal infestation in different products. Important differences in this parameter have been described for different genera and species. A study of the relationship between ergosterol content and dry mass in a strain of Aspergillus ochraceus , which is able to produce an antifungal agent, has therefore been carried out in order to use this parameter to accurately evaluate the fungal mass of this strain. We report herein our research work to determine the ergosterol content of the mycelial mass of our mould and how it changes with the fungal age.     

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelial biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture practices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during July and August but low as ectomycorrhizal development continued into the growing season. Percentages of mycelial dry weight over lateral roots decreased from 9% in July to 2.5% in November because seedling lateral root dry weight accumulated faster than mycelial dry weight. Total ergosterol per seedling increased from July through February. As lateral root dry weight ceased to increase during winter months, ectomycorrhizal mycelia became the major carbohydrate sink of pine seedlings. No distinctive seasonal pattern of soil ergosterol content was observed. The impact of ectomycorrhizal fungi on plant carbohydrate source-sink dynamics can be quantitatively estimated with ergosterol analysis but not with conventional visual determination.     

Estimation of fungal biomass in a solid substrate by three independent methods

Summary Dry matter increase of Agaricus bisporus mycelium in liquid culture, was found to be directly proportional to quantities of fungal derived chitin and extracellular laccase for up to 28 days following inoculation. Fungal ergosterol showed a similar relationship to mycelial growth which was sustained for 56 days of culture. In cultures grown on autoclaved rye grain a correlation was found between the three biochemical indices and linear extension of the mycelium. Each method gave a similar biomass estimate for cultures grown on cereal grains.     

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.     

Effect of pollutants on the ergosterol content as indicator of fungal biomass

Ergosterol content was determined in 20 white-rot fungi isolates and the values ranged from 2380 to 13 060 μg g⁻¹ fungal biomass. Significant changes of ergosterol content according the physiological stage for Bjerkandera adusta 4312 and Coriolopsis gallica 8260 were found, showing the highest values during the stationary phase. However, in the case of Phanerochaete chrysosporium 3642, no changes were detected during growth. The effect of pollutants, such as heavy metals and fungicides, on the ergosterol content of C. gallica was determined. Heavy metals (Cu 80 ppm, Zn 50 ppm or Cd 10 ppm) and fungicides (thiram 3 ppm or pentachlorophenol 1.5 ppm) at concentrations that reduce the metabolic activity between 18% and 53% (pollutant-stressed cultures) did not affect the ergosterol content. Only the fungicide zineb (25 ppm) reduced significantly the ergosterol content in biomass basis. In soil experiments with Cu (80 ppm) or thiram (10 ppm) after 15 and 30 days of incubation, the ergosterol content in soil was linearly correlated to the fungal biomass C in both polluted and control soil cultures. The ergosterol content was independent of the presence or the absence of pollutants. Thus, these results indicate that ergosterol can be a useful indicator for fungal biomass in polluted soils, and can be applied for monitoring bioremediation processes.     

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.     

Estimation of mycelial biomass of arbuscular mycorrhizal fungi associated with the annual legume Kummerowia striata by ergosterol analysis

The biomass of internal and external mycelia of an arbuscular mycorrhizal (AM) fungus, Gigaspora margarita Becker & Hall, symbiotic with the annual legume, Kummerowia striata (Thunb.) Schindler, was estimated in a sterile culture experiment. When ergosterol, which is a component of fungal cell membranes, was measured in the mycorrhizal roots and soil at 20, 40, 60 and 80 days after inoculation with the AM fungus, the content of ergosterol in the roots increased from 0.036 g per plant (at 20 days) to 1.85 g per plant (at 80 days). Ergosterol content in the soil also increased with time, but the ratio of external to internal mycelial biomass decreased from 24.7 at 40 days to 5.6 at 80 days after sowing. The average ergosterol concentration in the external mycelia of G. margarita was 0.63 mg g^–1. It was estimated that at 80 days after inoculation, the biomass of internal and external mycelia of the AM fungus accounted for approximately 16 and 92% of root biomass, respectively. For comparison, ergosterol content in the roots of K. striata growing in the field was also measured. The results suggest that AM fungi can be a large sink of the carbon that is assimilated by the host plants.     

Ergosterol contents of some wood-rotting basidiomycete fungi grown in liquid and solid culture conditions

Ergosterol contents of six wood-rotting basidiomycetes were analyzed under different cultivation conditions. Four white-rot and two brown-rot fungi were cultivated in liquid synthetic medium with low nutrient nitrogen (2 mM) and 0.1% glucose, and ergosterol in mycelial biomasses were measured weekly for 35 days. The highest ergosterol content per fungal dry mass in the white-rot fungi was found in Phanerochaete chrysosporium being 2100 μg g⁻¹, while in Ceriporiopsis subvermispora it was 1700 μg g⁻¹, Phlebia radiata 700 μg g⁻¹, and Physisporinus rivulosus 560 μg g⁻¹. In brown-rot fungi the ergosterol content was in Poria placenta 2868 μg g⁻¹ and in Gloeophyllum trabeum 3915 μg g⁻¹. On agar media, P. chrysosporium and P. radiata reached the highest ergosterol value in 7 days, while in wood block cultures the ergosterol contents were quite stable. The conversion factors for ergosterol-to-fungal biomass varied from 48 and 243, which were lower than values for ascomycetous soil fungi reported in the literature.     

Ecomethodology for organoosmotrophs: Prokaryotic unicellular versus eukaryotic mycelial

Although they are very unlikely to play large direct roles in water-column microbial loops, eukaryotic mycelial decomposers (the mycelial true fungi, eumycotes, and zoosporic fungi, oomycotes) have the potential to be important secondary producers in decaying plant material in shallow aquatic systems. Their secondary productivity may lead to important exchanges of material with microbial loops: output of ascospores, conidia, zoosporic flagellates, leaked lysates, and particles of decayed plants containing mycelium; input of dissolved organics and inorganic nutrients. Development of methods for ecological study of the aquatic mycelial eukaryotic decomposers has not advanced as rapidly as that for the prokaryotes of microbial loops, probably because (1) there are fewer aquatic microbial ecologists with mycological training and inclination than with prokaryotic leanings; and (2) the mycelial decomposers are difficult to work with, because they produce their mycelial mass virtually entirely within opaque solid substrates. Direct microscopic methods have emerged as prime tools for the measurement of prokaryotic mass, whereas an index-chemical assay (ergosterol) is currently the most efficient way to measure the mass of eumycotes. For measuring productivity of prokaryotes of microbial loops, microbial ecologists may choose from several (>10) published and field-tested methods, involving direct microscopy or monitoring of radiotracers. Extensive reviews of distribution and dynamics of aquatic bacterial mass and productivity have appeared. For measuring productivity of eukaryotic mycelial decomposers, one has only two published methods from which to choose, a direct-microscopic and a radiotracer method, neither of which has had adequate field testing. We are, furthermore, much less well equipped to obtain mass and productivity information for the poorly known mycelial oomycotes than we are for the eumycotes. Application of productivity techniques and nucleic-acid technology, may within the next decade allow knowledge of ecology of aquatic eukaryotic mycelial decomposers to advance to levels approaching that for the prokaryotes of microbial loops.     

Comparison of ATP and Ergosterol as Indicators of Fungal Biomass Associated with Decomposing Leaves in Streams

ATP and ergosterol were compared as indicators of fungal biomass associated with leaves decomposing in laboratory microcosms and streams. In all studies, the sporulation rates of the fungi colonizing leaves were also determined to compare patterns of fungal reproductive activity with patterns of mycelial growth. During leaf degradation, ATP concentrations exhibited significant, positive correlations with ergosterol concentrations in the laboratory and when leaves had been air dried prior to being submerged in a stream. However, when freshly shed leaves were submerged in a stream, concentrations of ATP and ergosterol were negatively correlated during degradation. This appeared to be due to the persistence of leaf-derived ATP in freshly shed leaves during the first 1 to 2 weeks in the stream. Estimates of fungal biomass from ergosterol concentrations of leaf litter were one to three times those calculated from ATP concentrations. ATP, ergosterol, and sporulation data generally provided similar information about the fungi associated with decomposing leaves in streams during periods when fungi were growing. Ergosterol concentrations provide a more accurate indication of fungal biomass in situations in which other organisms make significant contributions to ATP pools.     

Ergosterol Content in Various Fungal Species and Biocontaminated Building Materials

This paper reports the ergosterol content for microbial cultures of six filamentous fungi, three yeast species, and one actinomycete and the ergosterol levels in 40 samples of building materials (wood chip, gypsum board, and glass wool) contaminated by microorganisms. The samples were hydrolyzed in alkaline methanol, and sterols were silylated and analyzed by gas chromatography-mass spectrometry. The average ergosterol content varied widely among the fungal species over the range of 2.6 to 42 μg/ml of dry mass or 0.00011 to 17 pg/spore or cell. Ergosterol could not be detected in the actinomycete culture. The results for both the fungal cultures and building material samples supported the idea that the ergosterol content reflects the concentration of filamentous fungi but it underestimates the occurrence of yeast cells. The ergosterol content in building material samples ranged from 0.017 to 68 μg/g of dry mass of material. A good agreement between the ergosterol concentration and viable fungal concentrations was detected in the wood chip (r > 0.66, P ≤ 0.009) and gypsum board samples (r > 0.48, P ≤ 0.059), whereas no relationship between these factors was observed in the glass wool samples. For the pooled data of the building materials, the ergosterol content correlated significantly with the viable fungal levels (r > 0.63, P < 0.0001). In conclusion, the ergosterol concentration could be a suitable marker for estimation of fungal concentrations in contaminated building materials with certain reservations, including the underestimation of yeast concentrations.