Applied Soil Ecology 17 (2001) 285–289

Short communication
A rapid method to extract ergosterol from
soil by physical disruption
Ping Gong∗ , Xin Guan, Ernst Witter
Department of Soil Sciences, Swedish University of Agricultural Sciences, P.O. Box 7014, S-75007 Uppsala, Sweden
Received 1 June 1999; accepted 15 February 2001

Abstract
A physical disruption method was developed to extract ergosterol from soil samples. The optimised procedure was to extract
4 g of moist soil with 6 ml of methanol by shaking for 1 h at 320 rpm in the presence of 4 g of glass beads. Approximately
102% of the spiked pure ergosterol (1, 2 and 30 ␮g g−1 soil) was recovered from nine agricultural soils using this method.
As a biomarker of fungal biomass, the ratio of ergosterol content to microbial biomass in soil increased with the decreasing
of soil pH, implying that acidic soil environment favoured fungi rather than bacteria. Results from this study indicate that the
physical disruption method is a potentially promising method for ergosterol extraction in soil. © 2001 Elsevier Science B.V.
All rights reserved.
Keywords: Ergosterol; Soil; Physical disruption

1. Introduction 1993)), the Grant and West (G&W) method is still

Ergosterol has been used as a measure of fungal
biomass because it is the endogenous and predom-
inant sterol found only in fungal cell membranes
(Weete, 1989), certain microalgae, and protozoa
(Newell, 1992). The measurement of ergosterol was
first applied by Grant and West (1986) to soil as a
biomarker of fungi and to monitor the changes of
live fungal biomass in soil (West et al., 1987). Al-
though some new techniques have been developed
(e.g. microwave-assisted extraction (Young, 1995)
and supercritical fluid extraction (Young and Games,
∗ Corresponding author. Present address: Biotechnology Research
Institute, National Research Council of Canada, 6100 Royalmount
Avenue, Montreal, Que., H4P 2R2 Canada. Tel.: +1-514-496-8087;
fax: +1-514-496-6265.
E-mail address: ping.gong@nrc.ca (P. Gong).
the most commonly used one for soil ergosterol de-
termination. Nevertheless, the G&W method, which
was adopted from a method originally developed to
detect fungal contamination in higher plants (Seitz
et al., 1977, 1979), has been subsequently evaluated
(Davis and Lamar, 1992; Stahl and Parkin, 1996) and
modified by a number of workers on the purpose of
improving the extraction efficiency and simplifying
the extraction procedure (Eash et al., 1996; Zelles
et al., 1987). For example, some workers reduced
the soil sample size, increased the soil-to-solvent
ratio and added KOH at the onset of the extraction
process (Eash et al., 1996; Zelles et al., 1987). Re-
cently, Djajakirana et al. (1996) further concluded
that saponification could be dispensed.
Previous studies have shown that physical disrup-
tion is an efficient means of lysing microbial cells

0929-1393/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
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286 P. Gong et al. / Applied Soil Ecology 17 (2001) 285–289

in soil (Marstorp and Witter, 1999; Marstorp et al.,
2000). In the present study, we thus hypothesised that
fungal cells in soil could be ruptured by violent phys-
ical actions (shaking or beating) and hence ergosterol
could be released into the extractant (methanol). Dif-
ferent physical disrupting conditions were compared
and the optimised procedure was used for extracting
ergosterol from spiked and non-spiked soils. The ob-
jective of this study was to further simplify the ex-
traction procedure by replacing the time-consuming
reflux step with physical disruption. The further sim-
plified method aimed to be especially suitable for the
handling of a large number of samples.
2. Materials and methods
2.1. Soils
Soils of two texture types were used, one sandy
loam (clay 10%, silt 23% and sand 67%) and the other
clay loam (clay 37%, silt 41% and sand 23%). The
latter soil received eight treatments of different N fer-
tilisers and organic amendments as part of the Ultuna
long-term soil organic matter experiment started in
1956 (Witter, 1996). Some selected characteristics of
these soils are shown in Table 1.
2.2. Ergosterol quantification
A Waters 2690 HPLC equipped with an UV detector
(Photodiode Array Detector 996), a pump (600 Con-
troller), an autosampler (717 Delivery System), and
a C18 reverse-phase column was used for analysing
ergosterol. The UV detection was set at 282 nm, and
methanol was used as the mobile phase at a flow rate
of 1 ml min−1 . Prior to analysis, methanol was de-
gassed for 15 min in an ultrasonic water bath (Ban-
delin Sonorex Super RK510H) at full power. The col-
umn pressure was maintained at 660–700 psi. Under
such conditions, the retention time of ergosterol was
15.5–16.5 min. Ergosterol (purity ≥ 90%) was pur-
chased from Sigma and was used for constructing a
standard curve and for spiking as well.
2.3. Optimisation of the disruption conditions
for extraction
In order to increase the disruption intensity, the
addition of glass beads in combination with shaking
or beating was tested with two soils (soil A and E
in Table 1). Four grams of moist soil was combined
with or without 4 g acid-washed glass beads (2 g
of 212–300 ␮m diameter and 2 g of 710–1180 ␮m
diameter) in a 20 ml scintillation vial (Packard Instru-
ment, Meriden, CT, USA). After the addition of 6 ml
methanol, the vial was vortexed for 10 s, followed by
shaking for 1 h at 320 rpm on an orbital HT-TR225
shaker (INFORS AG, Bottmingen, Switzerland) or
beating for 15 min at 2000 oscillations min−1 . After
shaking or beating, the soil–methanol mixture was
allowed to precipitate for 15 min, and an aliquot of
1.5 ml supernatant was transferred into a 3 ml Ep-
pendorf microtube. The microtube was centrifugated
for 10 min at 11,000 oscillations min−1 and 5◦ C in a

Table 1
Selected characteristics of the soils used in this study
Soil Texture Treatment Organic C (%) pHa Double-stranded Microbial biomass
DNA (␮g g−1 )a (␮g C g−1 )a
A Sandy loam No treatment 1.6 6.1 NDb NDb
B Clay loam Fallow 1.0 6.1 23.7 111
C Clay loam Cropped, non-fertilised 1.2 6.4 36.2 192
D Clay loam Ca(NO3 )2 1.5 7.3 51.6 259
E Clay loam (NH4 )2 SO4 1.4 3.8 23.3 101
F Clay loam Straw 1.7 6.6 67.5 293
G Clay loam Green manure 1.8 6.3 67.3 314
H Clay loam Farmyard manure 2.2 7.0 95.0 465
I Clay loam Sewage sludge 2.7 5.0 50.8 164
a Double-stranded DNA was determined by the bead beating method while microbial biomass C by the fumigation-extraction method

(Guan, 1998).
b ND: not determined.
 P. Gong et al. / Applied Soil Ecology 17 (2001) 285–289 287

Hettich Universal 30RF centrifugator (Firma An-
deas Hettich, Tuttlingen, Germany). The supernatant
(0.9 ml) was then filtered (0.2 ␮m), and 0.5 ml of
the filtrate was filled in a 1 ml sample tube that was
loaded to an auto-sampler for HPLC analysis.
2.4. Recovery of spiked ergosterol from soil
Ergosterol dissolved in 1 ml methanol was added to
4 g moist soil at levels of 1, 2 and 30 ␮g g−1 soil and
was allowed to equilibrate in soil for 30 min before
extraction. The optimised procedure was followed to
extract both spiked and non-spiked soil samples. Re-
covery was estimated as the difference in ergosterol
extracted from the spiked and the non-spiked soil, di-
vided by the amount of ergosterol spiked.
3. Results and discussion
Different disruption conditions were compared with
respect to extraction efficiency. As shown in Fig. 1,
the addition of beads significantly improved the ex-
traction efficacy of ergosterol from soil E, but not from
soil A. Compared to the no-treatment control (i.e. no
shaking and beating), shaking or beating significantly
increased the amount of ergosterol extracted from both
soils. Previous studies have shown that the beating
with beads treatment is highly efficient in lysing soil
microorganisms (Marstorp et al., 2000). However, in
the present study the highest efficacy was observed
with the treatment of shaking with beads, which was
taken as the optimum procedure and was used in fur-
ther experiments.
No matter which soil was spiked, the recovery of
2 ␮g ergosterol g−1 was all above 90% with average
values ranging from 93 to 109% (Table 2). At spik-
ing levels of 1 and 30 ␮g g−1 of ergosterol, the re-
coveries were also within this range (data not shown).
These recoveries are significantly higher than those
reported by others (less than 85%) (Eash et al., 1996;
Stahl and Parkin, 1996). The coefficient of variance
was lower than 7.4%, implying that the simplified pro-
cedure was highly reproducible (Table 2). Statistical

Fig. 1. Extraction efficiency of different physical disruption treatments as evaluated by the yield of indigenous fungal ergosterol from two
soils. Mean (column) ± S.D. (error bar) (n = 4). Significant difference for each soil is indicated by different letters (P < 0.05, ANOVA).
288 P. Gong et al. / Applied Soil Ecology 17 (2001) 285–289

Table 2
Indigenous ergosterol content and the recovery of 2 ␮g ergosterol g−1 spiked in soils
Soila Indigenous ergosterol (␮g g−1 )b Recovery of 2 ␮g ergosterol g−1

Average (%)c CV (%)d

A 1.15 ± 0.04 109 0.5

B 0.34 ± 0.02 101 3.5
C 0.80 ± 0.07 105 5.6
D 1.25 ± 0.15 105 5.0
E 1.22 ± 0.13 98 3.9
F 1.97 ± 0.12 106 7.4
G 2.00 ± 0.19 100 5.5
H 1.15 ± 0.34 93 13
I 2.05 ± 0.21 98 4.9
Pooled samples (n = 36) 102 7.1
a Each soil was sampled from four replicated plots. See Table 1 for treatment each soil received.
b Mean ± S.D. (n = 4).
c Mean of four replicated plots.
d Coefficient of variance.

analysis of the data (ANOVA) indicates that the soil
factor does not affect the extraction efficacy, i.e. the
recovery. Therefore, the recovery of ergosterol is in-
dependent of soil.
Although different fungi have different ergosterol
contents, Montgomery et al. (2000) have shown that a
general conversion factor of 3.65 ␮g ergosterol mg−1
fungal biomass can be applied to soil and plant fungi.
If taking the fumigation labile C as a measure of
total microbial biomass (Bc ), the ratio of the ergos-
terol content to Bc can be considered as a measure
that indicates the relative contribution of fungi to
the whole microbial biomass. As shown in Fig. 2,
interestingly, this ratio decreases as the soil pH in-
creases, which is in agreement with those findings
from earlier studies (Djajakirana et al., 1996). This

Fig. 2. Relationship between the ergosterol content-to-biomass C (or DNA) ratio and soil pH in eight soils (soil B to I in Table 1).
 P. Gong et al. / Applied Soil Ecology 17 (2001) 285–289
implies that long-term acidification may lead to an
increase of fungal biomass (Ruess et al., 1996), and
that in acidic soil fungi could contribute relatively
more biomass to the whole soil microbial communi-
ties, probably due to that acidic environment is more
favourable for fungi than for bacteria (Pennanen et al.,
1998).
Further experiments should be carried out to com-
pare the simplified method developed in this study
with the conventional G&W method. It is also impor-
tant to validate this method with soils of known fungal
biomass, which may provide direct evidence on the
extraction efficacy of ergosterol from soil fungi. So
far, however, no studies have been documented with
regard to this aspect, probably owing to the fact that
there is no method that can directly and precisely mea-
sure soil fungal biomass.
In summary, the optimised procedure of physical
disruption is efficient in terms of extracting ergosterol
from spiked soil and may replace the time-consuming
and resource-intensive refluxing procedure. This
physical diruption method for ergosterol extraction
and determination is also highly reproducible and
cost-effective. The recovery of ergosterol from spiked
soil was averaged at 102% and was not affected by
soil texture. Using this method, it makes possible to
analyse a large amount of samples in a short period
of time.
Acknowledgements
The Swedish Institute is acknowledged for provid-
ing P. Gong with financial support.
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