FEMS Microbiology Ecology 53 (2005) 445–453

www.fems-microbiology.org

Lead and cadmium uptake in the marine fungi Corollospora

lacera and Monodictys pelagica

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Michael A.S. Taboski a, Thomas G. Rand a, Adam Piórko b,*

a
Department of Biology, Saint MaryÕs University, Halifax, Nova Scotia, Canada B3H 3C3
b
Department of Chemistry, Saint MaryÕs University, Halifax, Nova Scotia, Canada B3H 3C3

Received 24 June 2004; received in revised form 7 November 2004; accepted 3 February 2005

First published online 10 March 2005

Abstract

This study provides observations on the effects of lead and cadmium ions on the growth of two species of marine fungi, Corol-
lospora lacera and Monodictys pelagica. On solid media lead appeared to have no effect on the radial rate of growth of fungi. Expo-
sure 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 1 g 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.

2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Marine fungi growth; Lead; Cadmium; Metal uptake; Sequential elution

1. Introduction
The toxicity of heavy metals to living systems is well
recognized and a significant research effort has been di-
rected toward the study of their cycling, levels in differ-
ent habitats, mechanisms of uptake, toxicity to living
systems, and remediation of polluted environments.
Their toxicity toward terrestrial fungi has been studied
*
Corresponding author. Fax: +1 902 496 8104.
E-mail addresses: michael.taboski@utoronto.ca (M.A.S. Taboski),
thomas.rand@smu.ca (T.G. Rand), adam.piorko@smu.ca (A. Piórko).
for over 30 years and the results have been exhaustively
reviewed by Gadd [1], Wainwright and Gadd [2], Ram-
say et al. [3], Baldrian [4] for white-rot fungi and by Ley-
val et al. [5] for mycorrhizal fungi. The role of cations in
biodegradation of wood by brown rot fungi, including
metal cation toxicity toward these fungi, has also been
reviewed by Jellison et al. [6]. Bioaccumulation of
various metals by living and dead fungi in polluted
environments has also been studied to establish a
biotechnological potential of fungi, along with other
microorganisms, to control pollution by heavy metals
at the source [7–10]. Overall, these areas of study on
terrestrial fungi are advanced, although a number of

0168-6496/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2005.02.009
446 M.A.S. Taboski et al. / FEMS Microbiology Ecology 53 (2005) 445–453
questions related to bioavailability of heavy metals
including their speciation in fungal habitats and mecha-
nisms of solubilization and immobilization still remain
and require further evaluations [11–13].
However, the same cannot be said for marine fungi,
for which only a few papers have been published, as
far as we are aware. Babich and Stotzky revealed that
the growth of some marine fungi exposed to nickel
was less depressed in the presence of magnesium com-
pared to their growth in the presence of nickel alone
[14]. Hicks and Newell [15] found that exposing Phaeos-
pharia typharum, a salt marsh fungus, to mercury at me-
tal concentration of 0.74 mg l 1 resulted in no
significant change in glucosamine content or growth dif-
ferences compared to the cultures grown in the absence
of mercury. In another study, Vala et al. searched for the
chelators located on the external surface of mycelia,
which facilitate sequestration of iron from seawater.
Some 13 strains of facultative marine fungi were exam-
ined for the presence of siderophores, 10 of which pro-
duced carboxylate and/or hydroxamate siderophores in
the iron-deficient media [16].
Urban waste can have a significant impact on the dis-
tribution of chemical contaminants in marine sediments
and, consequently, by biogeochemical equilibria, on
marine ecosystems. In our area, in Halifax Harbor, the
highest concentrations of the metals such as copper
(up to 250 lg g 1), zinc (up to 500 lg g 1), mercury (up
to 2.5 lg g 1), lead (up to 500 lg g 1), and cadmium (up
to 2.5 lg g 1) in surficial sediments have been found in
areas of organic rich sediments, near sewage outfalls
[17]. Cadmium and lead are both toxic metals, which
can be released into the environment as a result of indus-
trial activities and burning of fuels. Cadmium is more
toxic to living systems than lead, although their chemical
properties are similar [1,18]. While there is a significant
body of literature on the toxicity and bioaccumulation
of these metals in terrestrial fungi [1–6,19–24] and some
marine organisms, including crustaceans [25,26] and
protozoans [27], their effect on marine fungi is unknown.
As marine fungi are important members of the inshore
micro-biota [13,15,28–30] they are exposed to metal ions
and complexes in large harbors and effects of such
exposure on fungi and co-habitants are unknown. The
objectives of our study were to evaluate the effects of
lead and cadmium on the growth of Corollospora lacera
(Ascomycotina: Halosphaeriaceae) and Monodictys
pelagica (Deuteromycotina: Dematiaceae) [31] as model
marine fungi. These species are commonly found
in coastal Atlantic Canada waters and exhibit good
growth in artificial media. Study objectives were
achieved by measuring their radial growth rate, myce-
lium dry weight and ergosterol content as features of
fungal biomass. Additionally we also evaluated metal
concentration and accumulation in the mycelia of both
species.
2. Materials and methods
2.1. Fungi, solvents, glassware, and reagents
Cultures of C. lacera (ATCC 34603) isolated from
sand grains from Bermuda and M. pelagica (ATCC
22771) from submerged balsa wood in Maryland were
used in the study.
All solvents used in the study were of HPLC grade
while acids used for glassware cleaning were of reagent
grade. Acids used for digestions and extractions were
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of trace metal grade. All glassware in this study was acid
washed with either concentrated sulfuric acid in the case
of all culture and extraction glassware or concentrated
nitric acid in the case of the metal analysis. The glass-
ware was then further cleaned with five washes with ul-
tra-pure water, followed by a methanol wash (Fisher,
HPLC grade) and a final wash with dichloromethane
(Caledon, HPLC grade). Artificial seawater was pre-
pared according to literature [32].
Cd standards for metal analysis were prepared using
SCP Science AA 1000 lg l 1 Cd standard solution. Pb
standards were prepared from ACP 1000 lg g 1 Pb
standard solution. HPLC grade ergosterol (>98%, Flu-
ka) was used as a standard in ergosterol analyses.
2.2. Instruments
Ergosterol analyses were performed using a Varian
HPLC system equipped with a UV–VIS Detector and
a l-Bondapak C18 RP column (0.42 lm, 300 ·
3.9 mm). For metal analysis, a Perkin–Elmer Atomic
Absorption Spectrometer 3300 was employed. Each
sample series run was preceded by the measurement of
standards. All samples were analyzed in triplicate and
the readings were averaged to obtain a final value.
2.3. Radial growth rate measurements
The fungi were sub-cultured directly from the ATCC
slant cultures onto Petri dishes containing a medium
comprising 50 g cornmeal (Difco), and 15 g agar (Difco)
per liter of artificial seawater and incubated at 25C for
14 days. After this, circular fungal plugs (4 mm in diam-
eter) were removed from the cultures with a sterilized
metal cork borer and transferred, fungal side up, to
the center of new Petri dishes containing 35 g Czapek-
Dox broth (Difco), and 15 g agar per liter of artificial
seawater. This medium (pH 7.5) was amended with
either Pb chloride (Fisher) or Cd chloride (Fisher) to fi-
nal cation concentrations of 10 mg l 1, 50 mg l 1,
100 mg l 1, 250 mg l 1, 500 mg l 1, respectively. We
also prepared the medium with 500 mg l 1 Pb nitrate
(Fisher). The control medium contained neither Pb
nor Cd. The plates, with five replicates per cation
treatment were incubated at 22 C for 10–15 days and
 M.A.S. Taboski et al. / FEMS Microbiology Ecology 53 (2005) 445–453
the diameters of mycelial growth, in two directions at
90 angles were measured. The radial growth rates
(mm per day) were determined for each species and at
each cation concentration.
2.4. Liquid medium cultures
Inocula from the original ATCC cultures were sub-
cultured on agar slants consisting of 2% salt water malt
extract agar (SWMEA composition: 20 g malt extract,
and 15 g agar per liter of artificial seawater) and homog-
enized in 50 ml of sterile, distilled water. Ten milliliter
aliquots of the homogenized mycelia were used to inoc-
ulate 250 ml Erlenmeyer flasks each containing 125 ml
of a culture medium containing 3 g ammonium chloride,
0.2 g ferrous sulfate heptahydrate, 2 g potassium dihy-
drogen phosphate, 2 g peptone, 2 g yeast extract, 2 g
malt extract and 20 g of glucose per liter of ultra-pure
water, prepared according to Strongman et al. [32]. Ali-
quots of 250 ml of culture medium were prepared for
each species, and the four flasks were placed on a G10
gyrotary shaker at 146 rpm for 7 days.
Cation-containing media cultures were prepared, typ-
ically, by adding 20 ml inoculum to a 500 ml Erlenmeyer
flask or a 1 L Roux bottle containing 200 ml (for shaken
cultures) or 400 ml, respectively (for stationary cul-
tures), of 35 g of Czapek-Dox medium (Difco) per liter
of artificial seawater. The medium was amended with
the addition of either Pb chloride (Fisher) or Cd chloride
(Fisher) to provide final cation concentrations. For each
species there was also a control with no cation added,
and all the experiments were completed in triplicate. Fi-
nal pH values for solutions were as follows: for controls
– pH 7.31; for Pb amended media – pH 7.31–7.27; for
Cd amended media – 7.29–6.82 (lower values for Cd
at 250 and 500 mg l 1); for samples of Halifax Harbor
water pH varied from 7.83 to 8.01. Compositions of
our solutions were not modified in order to compensate
for changes in ionic strength, osmotic pressure, or pH
resulting from introduction of metal-containing solutes.
Both species grew consistently well under such
conditions.
Corollospora lacera cultures were inoculated in Erlen-
meyer flasks and grown in a shaker for 15 or 30 days at
146 rpm at room temperature. M. pelagica cultures were
inoculated in Roux bottles placed on a stationary slant
of approximately 45 to ensure maximum surface area
and grown for 30 days at room temperature.
For the M. pelagica cultures exposed to Pb, the myce-
lia and medium were separated under vacuum filtration
(Whatman No. 2 paper), while Cd exposed M. pelagica
cultures were separated using gravity filtration (sterile
cheesecloth). After gentle washing with de-ionized water
the mycelia were placed into clean glass Petri dishes and
autoclaved at 395 K and 1.03 · 105–1.38 · 105 Pa for
15 min to prevent contamination in the storage before
447
analysis. The mycelia then were dried to a constant mass
and the masses were recorded. The cultures of C. lacera
exposed to Pb and Cd were treated as M. pelagica
above, although Pb nitrate was used instead of Pb chlo-
ride and the cultures were incubated on a shaker for 15
days. A white precipitate was observed in all cultures for
both fungal species and both cations at concentrations
50 mg l 1 or higher, although its composition was not
investigated.
2.5. Sequential elution
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A sequential elution procedure [33–35] was employed
to determine the Pb location within mycelium of C. lac-
era. It recovers metal ions from three locations – inter-
cellular (metal ions between the cells remaining even
after washing, reflecting the composition of the growth
medium), extracellular (metal ions bound to the polar
groups on the outer cell wall surface), and intracellular
(metal ions transported into the cell). At the end of each
growth period, the mycelia were separated from the li-
quid media, placed in 250 ml Erlenmeyer flasks and
rinsed briefly in ultra-pure water to remove residual
medium solution. To remove intercellular elements, the
mycelial samples were eluted with two 20 ml washings
of ultra-pure water for 40 and 30 min. The combined
washings from each sample were filtered into 50 ml poly-
propylene centrifuge tubes and acidified to pH 1.0–2.0
(0.2 M HNO3) for intercellular Pb analysis. To remove
extracellular elements, the mycelia were washed with
two 20 ml portions of 100 mM EDTA (Fisher, pH 4.5)
for 40 minutes and 30 minutes. The washings were col-
lected by filtration through cheesecloth into 50 ml poly-
propylene centrifuge tubes and acidified to pH 1.0–2.0
(0.2 M HNO3) for extracellular Pb analysis. The remain-
ing mycelial samples were dried over Drierite and the
dry weight mass was determined. A small sample of each
dry mycelium was treated with concentrated HNO3 to
dissolve intracellular elements and then diluted with
0.2 M HNO3 for intracellular Pb analysis. The samples
from all three locations were prepared in this way and
their Pb content was subsequently analyzed using the
flame atomic absorption spectrometer.
2.6. Flame atomic absorption spectrometer analysis
Metal cation content of the mycelia was analyzed
using atomic absorption spectrometry using 0.2 M
HNO3 (Fisher, Trace Metal Grade) and following the
standard EPA methods (EPA-600/4-82-055, method
213.1 for Cd and method 239.1 for Pb). Cd samples were
analyzed at wavelengths 228.8 or 326.1 nm, while Pb-
containing samples were analyzed at 261.4 and
283.3 nm wavelengths depending on whether or not
the sample was within the linear range of the selected
wavelength. The results are presented as total metal
448 M.A.S. Taboski et al. / FEMS Microbiology Ecology 53 (2005) 445–453
which is the metal content of mycelia (mass of the metal
found in dry mycelium, in mg), and the concentration of
metal in dry mycelium (mass of the metal in 1 g of dry
mycelium, in mg g 1).
2.7. Ergosterol extraction
Extraction of the dried mycelia was accomplished
using a simplified method for the analysis of fatty acids
and ergosterol adapted from Paterson and Amado [36]
with consideration given to reports by other authors
[37–39]. The dried mycelium of one replicate from each
species and treatment was ground to a fine powder
using a mortar and pestle. The powder was added to
a 25 ml screw-capped test tube with 4.5 ml of 1% pyro-
gallol in methanol (Fisher, HPLC grade) and 0.5 ml of
50% potassium hydroxide (BDH) and the content
hydrolyzed in a shaking water bath for 1 h at
120 rpm at 60 C. After cooling to room temperature,
2.5 ml of de-ionized water was added, followed by
5 ml of hexane (Fisher, HPLC grade), and the samples
were vortexed at the highest setting for 2 min. Vortexed
material was then centrifuged at 2200g for 3 min; the
upper hexane layer was transferred to a clean 15 ml
screw capped test tube while hexane extraction of water
layers was repeated. The hexane extracts for each sam-
ple were combined and evaporated to dryness. The res-
idue was re-dissolved in 5 ml of hexane. The solutions
were filtered through a 0.22 lm polyvinylidene fluoride
(PVDF) filter in polypropylene housing into clean vials.
Using a method of Newell [36], the ergosterol extracts
were analyzed using HPLC with methanol as the sol-
vent at a flow rate of 2 ml min 1 and the UV–VIS
detection at 282 nm.
2.8. Statistical analysis
All statistical analyses were done using Minitab
13.30. The Anderson–Darling test was used to deter-
mine if data were parametric or non-parametric. The
significance (p < 0.05) of differences among means for
all experimental results on solid media, the 30-day Pb
uptake experiments, and all Cd uptake experiments in li-
quid media was evaluated using one-way ANOVA for
parametric data. The Kruskall–Wallis test for non-para-
metric data was used to compare the means for 15-day
C. lacera Pb uptake and for the sequential elution exper-
iments. Correlation analysis was completed using the
Pearson correlation method.
3. Results
Exposure of C. lacera and M. pelagica to Pb and Cd
resulted in a variety of responses. These were manifest as
species-specific differences in growth rates, as significant
changes in mycelial mass and as sequestration of metal
ions by mycelium.
3.1. Lead – radial growth on solid media
Exposure of C. lacera and M. pelagica to increasing
Pb concentrations in solid media did not significantly al-
ter growth rates compared to controls. Comparison of
C. lacera growth on media containing either Pb chloride
or Pb nitrate revealed that there was no significant dif-
ference between growth rates at concentrations
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500 mg l 1. However, there was a significant difference
between the control and the two treatments (p < 0.05)
with fungal growth rates slightly higher on both Pb salts.
The highest growth rate for the nitrate (observed
2.5 ± 0.1 against 1.4 ± 0.6 mm day 1 for control) was
expected, as this anion is a nitrogen source for fungal
growth.
3.2. Lead – liquid media
Exposure of C. lacera to increasing Pb concentrations
in liquid culture resulted in marked colony morphology
and color changes. Colonies formed in the presence of
Pb were larger, irregularly shaped, and grayish com-
pared to control colonies, which were spheroidal and
greenish. These morphological colony features were
most apparent at the highest Pb concentration
(500 mg l 1). Compared to controls, C. lacera mycelial
mass increased (p < 0.05) at Pb concentrations 6250
mg l 1 after 30 days of exposure and decreased at
P10 mg l 1 at 15 days post-inoculum (PI) (Table 1). To-
tal Pb and mycelial Pb concentrations also increased
(p < 0.05) in colonies exposed to increasing Pb concen-
trations at both 15 and 30 days PI. Both total Pb and
mycelial Pb concentration were significantly higher
(p < 0.05) in C. lacera colonies harvested at 15 days
compared to those harvested at 30 days PI. The correla-
tion coefficients for mycelial dry mass and mycelial Pb
content were 0.97 for C. lacera grown for 15 days
and 0.61 for C. lacera grown for 30 days. Pb content
of intercellular, extracellular, and intracellular mycelial
fractions and total dry mycelia of C. lacera (shaken,
15 days) increased with increasing Pb concentration in
the medium (Table 2). Compared with control values,
these differences were all significant (p < 0.05). While
Pb was detected in both extracellular and intracellular
mycelial compartments (intercellular Pb refers to metal
ions loosely associated with mycelium although not re-
moved by washing prior to metal analysis), distribution
was uneven. The majority of the Pb was present in the
extracellular fraction, which contained 93.6–95.6% of
the total Pb in the mycelia while only 1.7–5.5% of the to-
tal Pb was found in the intracellular fraction.
Unlike the marked colony changes observed in
C. lacera exposed to Pb in liquid culture, M. pelagica
 M.A.S. Taboski et al. / FEMS Microbiology Ecology 53 (2005) 445–453 449

Table 1 M. pelagica with increasing Pb concentration, values

Effect of Pb(II) on the growth and Pb uptake by Corollospora lacera measured in this species were markedly lower than those
and Monodictys pelagica in liquid media
measured in C. lacera especially at the highest Pb con-
Initial Pb(II) Mycelium Total metala Metal concentration centration in the cultures (29.6 mg Pb/52.4 mg Pb g 1
concentration dry mass (mg) (mg Pb g 1
(mg l 1) (g) mycelium)b
for C. lacera vs. 9.28 mg Pb/6.72 mg Pb g 1 for M.
pelagica).
Corollospora lacera (15 days, shaken)c
0 0.39 ± 0.02 0 0
10 0.45 ± 0.05 1.6 ± 0.1 (S) 3.5 ± 0.5 (S) 3.3. Cadmium – radial growth on solid media
50 0.42 ± 0.02 6.6 ± 0.6 (S) 15.7 ± 1.3 (S)
100 0.35 ± 0.02 12.9 ± 2.2 (S) 36.2 ± 4.1 (S) Exposure of C. lacera and M. pelagica to increasing
250 0.23 ± 0.04 (S) 26.3 ± 3.9 (S) 116.2 ± 11.2 (S) Cd concentrations resulted in depressed or no radial
500 0.17 ± 0.01 (S) 44.9 ± 3.8 (S) 259.0 ± 36.0 (S)

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mycelial growth on solid media (Fig. 1). C. lacera
Corollospora lacera (30 days, shaken)d growth was significantly (p < 0.001) depressed compared
0 0.61 ± 0.56 0 0
to controls at P50 mg l 1 Cd for up to two days PI, fol-
10 0.71 ± 0.13 (S) 0.16 0.22
50 0.73 ± 0.06 (S) 1.85 2.53 lowed by modest growth at all Cd concentrations
100 0.77 ± 0.11 (S) 2.63 3.39 (0.5 ± 0.1 vs. 2.5 ± 0.1 mm day 1for control or ca.
250 0.77 ± 0.04 (S) 5.50 7.13 20% of the control growth rate for the highest Cd con-
500 0.56 ± 0.06 (S) 29.6 52.4 centration). C. lacera responses to Cd were significantly
Monodictys pelagica (30 days, stationary)d different (p < 0.001), indicating sensitivity of the species
0 1.42 ± 0.37 0 0 to this metal at higher concentrations. Exposure to
10 1.70 ± 0.11 0.43 0.25 increasing Cd concentrations depressed growth of M.
50 1.56 ± 0.09 1.17 0.75
100 1.37 ± 0.28 6.50 4.74
pelagica for up to 6 days PI at 10 mg l 1, and suppressed
250 1.09 ± 0.53 3.89 3.57 growth at P50 mg l 1. Growth rates of both species ex-
500 1.38 ± 0.08 9.28 6.72 posed to Cd were significantly depressed (p < 0.001)
Data are expressed as either the mean or means ± SD; S – significant compared to those of Pb exposed cultures and the
(p < 0.05). Dry mass of mycelia was measured in three replicates. controls.
Metal content in mycelia was measured in three replicates (15 days
experiment) and two replicates (30 days experiments). 3.4. Cadmium – liquid media
a
Mass of the metal found in mycelium, in mg.
b
Mass of the metal in 1 g of mycelium, in mg g 1.
c
Significance of differences among means was evaluated using the In liquid cultures, C. lacera (shaken, 15 days) and M.
Kruskall–Wallis test. pelagica (stationary, 30 days) mycelial dry masses de-
d
Significance of differences among means was evaluated using one- creased significantly (p < 0.05) as Cd concentration in-
way ANOVA. The other two parameters (n = 2) could not be creased compared to the controls (Table 3). At
analyzed.
500 mg l 1, C. lacera dry mass decreased to ca. 80% of
control dry mass while M. pelagica dry mass decreased
Table 2
Cellular location of Pb(II) in Corollospora lacera (15 days, shaken, in
liquid media)
3
Initial Pb(II) Metal concentrationa (mg of Pb g 1
of M. Pelagica C. lacera
Radial Growth Rate (mm day -1)

concentration (mg l 1) mycelia) 2.5

Intercellular Extracellular Intracellular
2
0 0 0 0
**
10 0.04 ± 0.02 3.31 ± 0.52 0.18 ± 0.01
50 0.15 ± 0.01 14.71 ± 0.52 0.86 ± 0.02 1.5
**
100 0.45 ± 0.12 33.92 ± 7.97 1.85 ± 0.46
250 3.09 ± 0.03 110.99 ± 0.05 1.99 ± 0.05 1 ** **
500 10.30 ± 0.02 243.20 ± 1.30 5.99 ± 0.09 **
0.5
Data are expressed as means ± SD of three replicate experiments for
each concentration; data at all five Pb concentrations are significant
(p < 0.05, Kruskall–Wallis test). 0
a
Mass of the metal in 1 g of mycelium, in mg g 1. 0 10 50 100 250 500
Cadmium Concentration (mg l -1)

Fig. 1. Radial growth rates of Corollospora lacera and Monodictys

showed only modest change. Compared to controls,
mycelial mass change for this species was non-signifi-
cant, even at the highest concentration (Table 1). Addi-
tionally, even if both total and mycelial Pb increased in
pelagica on solid media amended with Cd(II) ranging in concentration
from 0 to 500 mg l 1. The bars reflect mean values of five replicates.
Error bars show 99% confidence intervals. Double asterisks indicate
highly significant (p < 0.001, one-way ANOVA) differences.
450 M.A.S. Taboski et al. / FEMS Microbiology Ecology 53 (2005) 445–453

Table 3 Mycelial ergosterol content of C. lacera in liquid cul-

Effect of Cd(II) on the growth and Cd uptake by Corollospora lacera ture decreased significantly (p < 0.05) from 15.5 ± 0.1 lg
(15 days, shaken) and Monodictys pelagica (30 days, stationary) in
liquid media
in the control cultures to 9.3 ± 0.1 lg in cultures con-
taining 500 mg l 1 Cd (Table 4). The strong correlation
Initial Cd(II) Mycelium Total metala Metal concentration
concentration dry mass (mg) (mg Cd g 1
between ergosterol content and the mass of dry mycelia
(mg l 1) (g) mycelium)b (r = 0.94) in our experiment indicates that this com-
Corollospora lacera (15 days, shaken)
pound remains a reliable indicator of fungal biomass
0 0.51 ± 0.09 0 0 even in environments significantly contaminated with
10 0.52 ± 0.07 0.08 0.15 metals.
50 0.50 ± 0.05 (S) 0.58 1.15
100 0.47 ± 0.01 (S) 0.89 1.90
250 0.39 ± 0.01 (S) 1.33 3.37

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500 0.42 ± 0.02 (S) 3.11 7.42
4. Discussion
Monodictys pelagica (30 days, stationary)
In this study two marine fungi, C. lacera and M. pel-
0 1.42 ± 0.37 0 0
10 0.36 ± 0.32 (S) 0.41 1.12 agica, were exposed to two toxic metal cations com-
50 0.33 ± 0.03 (S) 2.44 7.42 monly found in the environment, Pb(II) and Cd(II).
100 0.26 ± 0.03 (S) 5.28 20.7 We attempted to assess the toxicity of Pb and Cd to
250 0.33 ± 0.05 (S) 17.9 54.3 these species by studying their radial growth rate on so-
500 0.24 ± 0.08 (S) 16.3 66.7
lid media and growth of biomass in liquid media, and to
Data are expressed as either the mean or means ± SD; S – significant evaluate biosorption of metals by the species in liquid
(p < 0.05); dry mass of mycelia was measured in three replicates. Metal
cultures. Our study revealed that C. lacera and M. pelag-
content in mycelia was measured in two replicates. Significance of
differences among means was evaluated using one-way ANOVA. The ica are sensitive to Pb and Cd cations in vitro. This re-
other two parameters (n = 2) could not be analyzed. sult suggests that the local levels of these metals in
a
Mass of the metal found in mycelium, in mg. Halifax Harbor sediments and waters (due to biogeo-
b
Mass of the metal in 1 g of mycelium, in mg g 1. chemical equilibria) could restrict distribution and/or
growth of these fungi in this area. Marine fungi are
to ca. 20% of that of the control. However, for both spe- important members of marine ecosystems serving
cies, total and mycelial Cd concentration increased with mostly as decomposers [28], and as food for meiofauna,
increasing Cd concentration of media. The correlation microarthropods and benthic organisms [9,12,13,28],
coefficients for mycelial dry mass and Cd content were among others. Restricted distribution and/or growth of
negative: for C. lacera grown for 15 days r = 0.78, fungi due to toxic concentrations of these metals could
for M. pelagica grown for 30 days r = 0.46. The myce- impact on biodegradation processes and food web com-
lial mass in C. lacera was lower than that of M. pelagica position and interactions within Pb and Cd contami-
in the absence of metals. However, in the presence of nated sediments in Halifax Harbor. Given the
metal, total metal content and mycelial Cd concentra- importance of saprophytic marine fungi in marine food
tions in M. pelagica were comparatively higher webs, the possibility that marine fungi can accumulate
(3.11 mg Cd/7.42 mg Cd g 1 for C. lacera vs. 16.3 mg toxic metal ions in nature should be explored.
Cd/66.7 mg Cd g 1 for M. pelagica at highest Cd Corollospora lacera and Monodictys pelagica exhib-
concentration). ited different growth responses towards Pb and Cd expo-
sure. Exposure of both species to Pb had essentially no
effect on the growth rates on solid media and in liquid
Table 4
cultures, with one exception. At the highest Pb concen-
Effect of Cd(II) on the ergosterol level in Corollospora lacera (15 days,
shaken culture, liquid media) trations in liquid cultures of C. lacera grown for 15 days
we observed significant (p < 0.05) decrease in dry mass
Initial Cd(II) Mycelium Total ergosterola Ergosterol
concentration dry mass (g) (lg) (lg erg. g 1 of mycelium. In contrast, exposure to Cd on solid media
(mg l 1) (±0.001 g) myc.)b depressed the growth rate of C. lacera and suppressed
0 0.467 15.5 ± 0.1 33.2 ± 0.1 the growth of M. pelagica at concentrations above
10 0.490 15.8 ± 0.1 32.2 ± 0.2 10 mg l 1. In liquid cultures Cd significantly reduced
50 0.482 13.1 ± 0.2 27.1 ± 0.4 the M. pelagica biomass, while C. lacera biomass was af-
100c 0.462 0.000 0.000 fected to a lesser degree. That difference in growth re-
250 0.391 6.1 ± 0.1 15.6 ± 0.2
sponses of the two species toward these two metals
500 0.410 9.3 ± 0.1 22.7 ± 0.2
were observed is not surprising since Cd is known to
Data are expressed as means ± SD from three replicate measurements
be more toxic than Pb. However, in cultures exposed
for the ergosterol content in mycelia.
a
Mass of the ergosterol found in mycelium, in lg. to Cd, C. lacera and M. pelagica exhibited species-spe-
b
Mass of the ergosterol in 1 g of mycelium, in lg g 1 mycelium. cific sensitivity manifest as differences in the growth
c
Sample contaminated. rates on solid media. Differences in species sensitivity
 M.A.S. Taboski et al. / FEMS Microbiology Ecology 53 (2005) 445–453
have been reported previously in selected terrestrial
basidiomycetes grown in the presence of metals [21]
but apparently not in marine fungi.
The species responses to metal exposure revealed that
the type of culture medium influences the fungal growth.
Cultivation of the fungi on solid media with Cd resulted
in a significant and abrupt inhibition of growth rates for
both species. In contrast, in liquid media containing this
metal both species exhibited far more gradual inhibition
of growth. For Pb amended cultures this medium influ-
ence was clearly observed only in the case of C. lacera
grown on solid media and for 15 days in liquid media.
It is unclear what mechanism accounts for this difference
in growth performance on solid vs. liquid media. Possi-
bly, the solid medium amended with metal salts appears
to represent an environment with dispersed metal cat-
ions that are immobilized by interaction with other mol-
ecules and ions present in solid medium. Thus, the
fungus growing on the solid medium would encounter
the metal cations in fixed positions, on the periphery
of mycelial growth. Growth of terrestrial fungi in such
heterogeneous media has been studied recently
[3,40,41] and a negative fungal chemotropism was re-
ported [42]. The authors of these studies proposed that
‘‘foraging’’ mode of fungal growth prevails in such med-
ia types, particularly under limited carbon conditions.
Under these conditions, fungal growth was toward car-
bon sources and away from immobilized toxic metal
ions. Ramsay et al. [3] reported that both copper and
Cd were capable to disrupt this exploratory growth
phase by different mechanisms. In the case of Cd, most
of the fungal biomass was located in the interior of the
colony at the expense of reduction in radial growth
and in number of hyphal branches. The Cd concentra-
tion used in their work (0.1 mM or 11.2 mg l 1) parallels
the concentration range at which M. pelagica was still
growing in our experiments (10.0 mg l 1) while its
growth was totally depressed at higher (50.0 mg l 1)
concentration. That C. lacera grew under all Cd concen-
trations we used (up to 500 mg Cd l 1) suggests that this
species is resistant to Cd toxicity, although defensive
mechanisms are unknown.
Conceivably, this resistance may be due to incorpora-
tion and sequestration of Cd cations in mycelial walls,
just as it seems to be in the case of Pb. For Pb, we found
that color of C. lacera mycelial pellets in Pb enriched li-
quid media was different from that in control pellets.
Colony pigmentation change was observed in Zalerion
maritimum exposed to pesticides [29] and in many other
studies of fungal growth on solid media and in liquid
cultures [1,3,4,20,21,43]. In our experiments the majority
of accumulated Pb was found in the extracellular com-
partment, i.e. cell wall, and this result may indicate the
involvement of functional groups of cell wall [1,44,45]
and pigments such as melanins [1,46], in sequestering
the Pb ions. Ashkenazy et al. [44] found that biosorption
451
of Pb in Saccharomyces uvarum occurs predominantly
via complexation by exposed carboxyl and other anionic
groups, with significant contribution by uncharged,
nitrogen-containing groups of the cell walls. Brady and
Duncan [45] also found that biosorption can be achieved
through complexation by cell wall and membrane
hydroxylated components. Cellular location of some
metal cations such as copper [47], zinc [48] and Cd
[19,49–55] was investigated in terrestrial fungi. These
metals were found distributed extracellularly, and intra-
cellularly in soluble fractions and vacuoles, although
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percentage metal distribution at these sites varied. White
and Gadd [49] studied uptake and cellular distribution
of copper, cobalt and Cd in strains of Saccharomyces
cerevisiae cultured on elevated concentrations of metals.
For Cd they found increased uptake of metal in the sol-
uble fractions of the cell and decrease in the bound frac-
tion, which in one strain contained ca. 25% of the total
intracellular Cd. Kunst and Roomans [50] found that
polyphosphate granules localized in or close to the cell
vacuoles, which are the major storage vesicles for heavy
metal ions in Saccharomyces cerevisiae pre-treated with
phosphate. Orlovich and Ashford [51] followed up on
aforementioned studies and determined the exact vacuo-
lar location for polyphosphate granules incorporation.
Favero et al. [52] found that 80% of Cd was bound to
the hyphal cell walls in their study of basidiomycete
Pleurotus ostreatus. In the exposure of fungus Paxillus
involutus to Cd for up to 12 h Blaudez et al. [19] found
that Cd was nearly equally distributed among cell wall,
cytoplasm and vacuole. In shorter exposures (0.5 h) the
distribution was 48%, 32%, and 20%, respectively. Joho
et al. [53–55] reported that Cd-sensitive and Cd-resistant
strains of Saccharomyces cerevisiae differed in the distri-
bution of Cd after exposure. The Cd-resistant strain was
found to contain metal mostly in the intracellular solu-
ble fraction (up to 90% of metal) while in the Cd-sensi-
tive strain metal was associated predominantly with the
insoluble intra- and extracellular fractions (up to 40% in
intracellular soluble fraction).
In liquid cultures based on artificial salt water, com-
plex equilibria involving metal cations, their complexes,
other ions present in solution, acidic ions released by
fungus, and ions either deposited or/and absorbed by
fungus are established. Metal cations may exist in solu-
tion in the form of free cations, Me2+ (or hydrated
forms) which are toxic, and complexes, most probably
of the type MeCl1+, MeCl02 and MeCl13 [18,25,26,56],
which decrease the concentration of the toxic form. As
a result of foraging fungal actions, extracellular enzymes
as well as citrate and oxalate ions, are released by fungi
[1,11]. All these molecules can form complexes with met-
als, and oxalate complexes of both Pb and Cd are insol-
uble in water [11]. Metal ions may also be complexed by
the functional groups present on the mycelial surface
[1,44–46]. These actions likely contribute to the fungal
452 M.A.S. Taboski et al. / FEMS Microbiology Ecology 53 (2005) 445–453
resistance against the action of toxic metals. Finally, the
metal cations may be moved across the external mem-
brane, predominantly by the action of energy-dependent
transport systems [1]. Such a transport may proceed at a
reduced rate in the presence of external factors, for
example in the presence of magnesium in seawater as re-
ported by Babich and Stotzky [14], thus increasing fun-
gal resistance to toxic metals. Intracellular metal ions
may be eventually stored in cellular compartments,
although they remain subjected to equilibria by influx–
efflux processes [1,19,22]. The interplay of all these equi-
libria may alter the effective concentration of cations in
solution and lower their relative toxicity. In fact, the
concentration of metals in all solutions at the time of
mycelia separation was lower than initial cation concen-
trations (data not shown) in our experiments. Addition-
ally, the fungal access to a soluble carbon source
(saccharose) is less restricted in solution than in solid
medium due to solute mobility, and the liquid medium,
based on an artificial salt water, contains some compo-
nents reported to alleviate toxic action of cations in
studies on terrestrial [1,22,52,57,58] and marine [14]
fungi.
Bioaccumulation of metal cations by fungi was also
evident in our study. We found that M. pelagica is capa-
ble to bioaccumulate over 60 mg of Cd and over 6 mg of
Pb per 1 g of mycelium, while C. lacera is capable of
accumulating over 7 mg of Cd and up to 250 mg of Pb
per 1 g of mycelium. Terrestrial fungi were shown to
bioaccumulate levels up to 190 mg g 1 for Cd and up
to 350 mg g 1 for Pb [1,2,4,7–10,22,52,59,60].
The results of our in vitro studies demonstrated spe-
cies-specific responses of the marine fungi C. lacera and
M. pelagica to the presence of either Pb or Cd ions in so-
lid and liquid media. The mechanisms by which these
fungi apparently minimize the toxic effects of metals re-
main unknown. Further studies of toxic effects of Cd
and Pb upon C. lacera and M. pelagica will likely pro-
vide insight into defensive mechanisms of marine fungi,
mechanisms of metal uptake, and impact of absorbed
metals on other components of marine food web.
Acknowledgments
The authors thank the anonymous reviewers and the
Editor (Dr. M. Häggblom) for helpful comments.
Financial support provided by Saint MaryÕs University
through both Senate and General Research Grants to
A.P. is gratefully acknowledged.
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