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Department of Biological Sciences, Mangalore University, Mangalagangotri, D.K. 574 199, Mangalore, Karnataka, India
Liliya G. Nikolcheva
Felix Bärlocher 1
Department of Biology, Mount Allison University, Sackville, New Brunswick E4L 1G7, Canada
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ABSTRACT |
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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.
Key words: ergosterol-biomass ratio, fungal biomass
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INTRODUCTION |
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, 2001; Gessner et al 2003). It is relatively easy to extract and quantify and essentially is restricted to the membranes of living cells of true fungi. Incorporation of a radiolabeled precursor, 14C-acetate, allows in situ estimates of fungal growth and production (Newell and Fallon 1991, Newell 2001, Gessner et al 2003). Accurate biomass estimates depend on a reasonably constant ratio between the quantity of an indicator molecule and fungal biomass. In a group of five dominant aquatic hyphomycetes, the ergosterol content of mycelia was 3.5–10.1 µg g–1 (Gessner and Schwoerbel 1991). In a group of 14 strains, belonging to 12 species, it varied between 2.3 and 11.5 µg g–1 (Gessner and Chauvet 1993). These results lead to a recommended ergosterol-to-biomass conversion factor of 182–200 (assuming an average ergosterol content of 5.5 or 5.0 µg g–1; Gessner and Chauvet 1993, Gessner and Newell 2002). It has been shown, however, that culture conditions can profoundly affect this ratio. For example, under varying nutrients and oxygen conditions the ergosterol-to-biomass ratio varied by a factor of up to 14 (range: 0.8–11.0 µg g–1; Charcosset and Chauvet 2001).
Variable results were described by Bermingham et al (1995a). In nine species, mycelial mats were subdivided and the wet weights and absolute ergosterol contents of these subunits were measured. A significant positive relationship was found in only three of the nine species. These results have been criticized (Fell and Newell 1998) on the grounds that, when subdividing a single, homogeneous mycelial mat, all parts should contain identical ergosterol concentrations. This should result in a positive correlation between ergosterol quantity and fungal biomass. At least in part, the discrepancy has been attributed to large errors when converting wet weight to dry weight. This calibration was done for two species (Bermingham et al 1995a). The coefficient of determination, R2, which indicates how much of the total variability can be attributed to the linear correlation, was 0.84 for Flagellospora curvula (P < 0.05) and 0.36 for Alatospora acuminata (P < 0.05), respectively. It has been suggested (Gessner 1997, Fell and Newell 1998, Newell 2001) that the apparently nonsignificant correlation between ergosterol and mycelial dry mass was due largely to this lack of accurate biomass determination.
There is, however, another possibility that might have contributed to the apparently illogical results of Bermingham et al (1995a). The assumption is that all parts of a mycelial mat contain identical ergosterol concentrations. By extension, we might conclude that all mycelium within a single flask should be homogeneous. In shaken cultures, however, it is not uncommon to observe pellets of variable sizes. They all are nominally of the same age (period since inoculation and incubation), but the relative contributions of peripheral (younger) and internal (older) hyphae clearly will differ, as they might in subsections of a large mycelial mat.
The objective of the current study was to determine if the amount of ergosterol, in fact, does vary with pellet size. If this is the case, could random mixing of size classes result in a nonsignificant correlation between ergosterol amounts and mycelial biomass, as observed by Bermingham et al (1995a)? For our study, we used two species whose ergosterol had been measured under a variety of conditions (Gessner and Schwoerbel 1991, Gessner and Chauvet 1993, Bermingham et al 1995a, Charcosset and Chauvet 2001), as well as five previously unstudied species.
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MATERIALS AND METHODS |
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Mycelium production. – Cultures were maintained on solid media (1% malt extract, 0.25% yeast extract, 1.5% agar). Agar plugs (8 mm diam) were cut from the edge of an actively growing, 2–4 wk old colony and used to inoculate 125 mL of 1% malt extract and 0.25% yeast-extract broth (one plug per 250 mL Erlenmeyer flask). The flasks were incubated on a rotary shaker (150 rpm) at 20 C. After 21 d, pellets were harvested, freeze-dried in aluminum trays (5 µm Hg, 16 h) and sorted into 5–6 size classes based on diameter, using a ruler and a low-power stereo-microscope. As far as possible, near-spherical pellets were used. Measuring pellets was much easier with freeze-dried pellets, which tended to maintain their shapes indefinitely when handled carefully. The original agar plug used for inoculation was excluded from these analyses.
Ergosterol measurements. –
The method for microwave-assisted ergosterol extraction was modified from Young (1995). Freeze-dried fungal pellets (25–50 mg) were ground in liquid nitrogen and placed in glass tubes (15 mL, 2 cm diam, with Teflon-lined screw caps). Methanol (2 mL) and NaOH (0.5 mL, 2 M) were added, and the tubes were capped. Six extraction tubes were placed in one 250 mL Teflon-coated plastic bottle, which was capped. The plastic bottle containing extraction tubes was microwaved at 50% power for 95 s (Kenmore Microwave, Model No. 85055). After cooling to room temperature (10–15 min), the glass tubes were removed from the plastic bottle. The solution in each tube was neutralized with 1 mL of 1 M HCl, and ergosterol was extracted with three consecutive hexane washes. The combined hexane fractions were evaporated and the residue was dissolved in 1 mL of methanol. The solution was injected into a high-performance liquid chromatography C18 column (Varian, Palo Alto, California) and eluted with methanol at 1.5 mL min–1 (5.3 min elution time). Ergosterol content was estimated by comparison of peak areas with those of external standards. Losses of external standards due to the extraction process were 
1%. Three replicate measurements were taken for each pellet size.
Statistical analyses. – Linear regressions were done with GLMStat for Macintosh, version X 5.7.3 (http://members.ozemail.com.au/~kjbeath/glmstat.html). Simulated random sampling was performed with Resampling Stats, version 4.1 for Macintosh (www.statistics.com).
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RESULTS |
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In all but one species (A. rubescens), ergosterol concentration increased significantly with pellet size (FIG. 1, TABLE I). The increase was most pronounced in C. aquatica, T. setigerum and T. aquatica, where ergosterol concentrations more than doubled between smallest and largest pellet size.
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. The 15 generated samples randomly were subdivided into five groups. The five groups were interpreted to represent steps in a calibration series with equidistant increases (e.g., Group 1 representing three samples of 10 mg, Group 2 three samples of 12.5, Group 3 three samples of 15.0, Group 4 three samples of 17.5 mg, Group 5 three samples of 20 mg). Absolute ergosterol amounts were generated by multiplying the ergosterol concentration (obtained by random sampling of the data from T. aquatica) with the biomass of the group. Absolute ergosterol amounts then were regressed against the biomass of the groups. This was repeated 10 000 times. When the range of mycelial biomass from the smallest to the largest sample was only twofold (e.g. ranging from 10 to 20 mg), 13% of all regressions were not significant at 
= 0.05, and 37% were not significant at = 0.01 (TABLE II). When the range of biomass was fivefold (e.g., from 10 to 50 mg), <0.01% of regressions were not significant at = 0.05 (2% at = 0.01).
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DISCUSSION |
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showed that ergosterol concentrations can increase considerably within the first few days of growth.
We tested if the increase in ergosterol concentrations with increasing pellet size could interfere with the expected positive correlation between absolute ergosterol amounts and mycelial biomass. In the resampling model, when the difference in mycelial biomass between the smallest and the largest sample is twofold, 37% of the regressions of absolute ergosterol amounts with mycelial biomass are not significant at = 0.01. When the range in mycelial biomass between the smallest and the largest sample increases by a factor of 10, only 0.01% of the regressions are not significant at = 0.01. Intuitively, this makes sense: In an extreme case, all high ergosterol concentrations might by chance be assigned to the group with the smallest biomass and all low concentrations to the group with the largest biomass. It is possible that the ergosterol concentrations double from smallest to largest pellet size (e.g., in C. aquatica, T. aquatica). If the range of analyzed biomasses doubles as well and if the lowest ergosterol concentration is assigned to the group with the highest biomass, and vice versa, we would expect a flat line (i.e., no correlation). When we extend the range of biomasses analyzed, it quickly will overcome the effect of doubling ergosterol concentrations from small to large pellets. In the current study we showed that ergosterol concentration can more than double with increasing pellet size; in four of the nonsignificant regressions in Bermingham et al (1995a), the ratio of high to low biomass was approximately 2. If ergosterol concentrations vary to a similar extent within a mycelial mat, random sampling in fact might have contributed to the observation of nonsignificant correlations between total ergosterol amount in subsamples of mycelial mat and their mass.
Of the seven species in this study, two previously have been examined for ergosterol content. For Clavariopsis aquatica grown in mineral salts plus glucose solution or in malt-extract broth, Gessner and Chauvet (1993) found values of 4.6 and 4.3 µg g–1, respectively, while mycelium grown in leaf-extract broth had a concentration of 8.0 µg g–1 (current study: 4.6 µg g–1 in malt-extract broth). In static cultures of C. aquatica, ergosterol concentration of the same species was 2.0 (Bermingham et al 1995a) and 1.4 µg g–1 (Charcosset and Chauvet 2001). Our values for Heliscus lugdunensis vary between 4.1 and 4.8 µg g–1; by contrast, Bermingham et al (1995a) found only 0.6 µg g–1. This low value again might be attributed to the fact that they used static cultures (Charcosset and Chauvet 2001).
To our knowledge, ergosterol concentrations of the remaining five species never have been determined before. Their values range between 1.6 (A. crassa, 2 mm pellets) and 4.8 µg g–1 (T. setigerum, 12 mm pellets). The grand average of our measurements (all pellet sizes, all species) was 3.0 µg g–1, corresponding to 55–60% of the conventional ergosterol-to-biomass conversion factor. Gessner and Chauvet (1993) suggested that more accurate estimates of fungal biomass in environmental samples might be obtained by using species-specific factors, combined with estimates of how naturally established communities are subdivided among the various species. This has been done traditionally by counting spores (or estimating total spore volumes) produced by individual species and assuming that this accurately reflects mycelial biomasses on the substrate (Bärlocher and Schweizer 1983, Gessner and Chauvet 1993). Quantitative ELISA, based on specific monoclonal antibodies, suggests that this might not be the case (Bermingham et al 1997).
Another potential source of error is the fact that environmental conditions, or the status of the mycelium, can greatly expand the range of ergosterol concentrations within species. In aquatic hyphomycetes, oxygen scarcity in static cultures has been associated with very low ergosterol amounts (Charcosset and Chauvet 2001). On the other hand, glucose deficiency may stimulate ergosterol accumulation. Other factors (not restricted to aquatic hyphomycetes) include age of the colony, its physiological status, substrates and temperature (Newell and Statzell-Tallman 1982, Newell 1994, Newell et al 1987, Gessner et al 2003). To account for these factors, it obviously is desirable to determine the amount of ergosterol of mycelia under conditions closely resembling those on leaves in a stream. It is not clear, however, to what extent this is possible. For example, what is the oxygen available to hyphae growing in a single leaf exposed to the current? In the center of a densely packed accumulation of leaves? Can localized scarcity be compensated for by translocation within the mycelial network? What are the concentrations of simple sugars within the leaf, where breakdown of structural polymers and uptake of monosaccharides presumably overlap? The most accurate calibration intuitively is likely to be based on comparing in situ mycelial biomass estimated by direct observation of hyphae and ergosterol measurements. Microscopic estimates unfortunately are time consuming and may be subject to severe errors (Newell 2000).
Charcosset and Chauvet (2001) concluded that ergosterol measurements in environmental samples provide reasonably robust estimates of total aquatic hyphomycete biomass across different species and external conditions, provided oxygen and nutrient concentrations used for calibrations accurately reflect the conditions prevailing in streams. The current study lends some support to this statement, provided over or underestimates by a factor of up to 2–3 are acceptable. The use of quantitative ELISA, based on moncoclonal antibodies, may allow narrowing this margin of error (Bermingham et al 1995b, 1996, 1997) by using species-specific conversion factors. Some initial results suggest that production of some antigens may be less influenced by certain factors (temperature, age of colony, dissolved metals) than ergosterol concentrations.
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FOOTNOTES |
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1 Corresponding author. E-mail: fbaerlocher{at}mta.ca
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LITERATURE CITED |
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Bermingham S.L, Dewey FM, Maltby L. 1995a. A critical assessment of the validity of ergosterol as an indicator of fungal biomass. Mycol Res 99:479–484.
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