Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Effects of temperature on metal tolerance and the

accumulation of Zn and Pb by metal-tolerant fungi isolated
from urban runoff treatment wetlands
D. Purchase, L.N.L. Scholes, D.M. Revitt and R.B.E. Shutes
Urban Pollution Research Centre, Department of Natural Sciences, Middlesex University, The Burroughs, Hendon, London, UK

Keywords
Beauveria bassiana, metal accumulation,
Rhodotorula mucilaginosa, runoff,
temperature.
Correspondence
Diane Purchase, Department of Natural
Sciences, School of Health and Social
Sciences, Middlesex University, The
Burroughs, London NW4 4BT, UK.
E-mail: d.purchase@mdx.ac.uk
2008 ⁄ 0372: received 3 March 2008,
revised 11 September 2008 and accepted 16
September 2008
doi:10.1111/j.1365-2672.2008.04082.x
Abstract
Aims: To investigate the ability of two fungi to accumulate Zn and Pb, the
effect of temperature on their metal tolerance and possible mechanisms
involved in metal accumulation.
Methods and results: Beauveria bassiana and Rhodotorula mucilaginosa isolated
from constructed wetlands receiving urban runoff were grown in modified
glycerol asparagine medium containing elevated levels of Zn and Pb at 30C.
Beauveria bassiana accumulated up to 0Æ64% of available Zn and 8Æ44% of Pb.
The corresponding values for R. mucilaginosa were up to 2Æ05% for Zn and
16Æ55% for Pb. Radial growth of colonies grown at 4 and 30C on agar con-
taining Zn or Pb indicated that metal tolerance was not seriously affected by a
decrease in temperature. Transmission electron microscopy and emission dis-
persion x-ray spectrophotometry suggested that the mechanism of resistance in
B. bassiana may be associated with the precipitation of Pb (possibly in the
form of oxalates).
Conclusion: The processes of biosorption could potentially occur throughout
the year with both living and dead cells able to accumulate metals.
Significance and Impact of the Study: Identified precipitation processes could
be an important mechanism in metal removal in wetland substrates serving as
long-term storage sinks.

outlet zone, where it is collected before being discharged

Introduction
Heavy metals are common pollutants present in urban
runoff and, as they cannot be chemically or biologically
degraded, their removal is generally based on immobiliza-
tion processes (Ruttens et al. 2006; Sneddon et al. 2006)
and ⁄ or extraction from the contaminated compartment
(Sen Gupta and Bhattacharyya 2006). Constructed
wetlands provide a possible solution for the treatment
of metal contaminated water and both horizontal
sub-surface flow and surface flow wetlands have been
used for this purpose in the United Kingdom (Ellis et al.
2003). These systems typically consist of a rectangular bed
planted with aquatic plants (e.g. Phragmites australis or
Typha latifolia) and are normally lined with an imperme-
able membrane. The influent passes slowly through the
filtration medium or substrate in a horizontal path to the
into a receiving water body. During its passage through
the wetland vegetation and substrate, the wastewater
makes contact with a network of aerobic, anoxic and
anaerobic zones where various physical (e.g. sedimenta-
tion, filtration, adsorption), chemical (e.g. complexation,
precipitation and cation exchange) and biological (e.g.
plant uptake and microbial-mediated reactions) pollutant
removal processes take place. Constructed wetlands have
been used to remove metals from domestic wastewater
(Bragato et al. 2006), from acid mine drainage (White-
head et al. 2005; Sheoran and Sheoran 2006), metal-
contaminated urban stormwater runoff (Birch et al. 2004;
Lee et al. 2006; Weiss et al. 2006) and from highway run-
off (Pontier et al. 2004; Revitt et al. 2004). The removal
efficiencies vary for different metals with 48% being
observed for Ni (Lesage et al. 2007), 83% for Pb (Nelson

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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1163–1174 1163
Wetland fungal metal accumulation
et al. 2006) to over 90% for Fe (Whitehead et al. 2005)
and Al (Lesage et al. 2007).
Laboratory studies have shown that the rate of nutrient
removal in constructed wetlands is optimal at 30C (Wood
et al. 1999), becomes inhibited at 10C and decreases rap-
idly at 6C (Werker et al. 2002). Seasonal temperature vari-
ations also affected the nutrient removal performance of
macrophytes grown in microcosms containing 56 mg l)1 N
and 31 mg l)1 P with removal rates being higher in sum-
mer than in winter (Picard et al. 2005). Little research has
been carried out to study the impact of seasonal variation
or temperature changes on heavy metal removal in the dif-
ferent components of a wetland system such as substrate,
biomass and macrophytes.
Wetlands are known to provide favourable environ-
ments for a wide range of micro-organisms with a ready
supply of carbon and other nutrients for growth. In addi-
tion, wetland plants and sediments provide ideal sub-
strates for the attachment of micro-organisms. Studies
of the microbiology of both constructed and natural wet-
land substrates have found that microbial diversity
and numbers were comparable (Duncan and Groffman
1994), with bacteria being the most abundant group
(4Æ6 · 106 CFU g)1), followed by actinomycetes
(1Æ3 · 105 CFU g)1) and then fungi (4Æ0 · 104 CFU g)1)
(Hatano et al. 1994). These values are lower than typical
soil counts by c. 2–3 orders of magnitude. Nevertheless,
constructed wetlands are often viewed as ‘natural’ micro-
bial degradation systems, where the aim is to maximize
contact time between micro-organisms and the water
requiring treatment. Micro-organisms are involved in
many important pollutant removal processes such as the
biodegradation of organic matter, nitrification and deni-
trification, binding of heavy metals through biosorption,
and precipitation of metals as insoluble salts (Kosolapov
et al. 2004). Both aerobic and anaerobic micro-organisms
are involved in these processes of pollutant removal, deg-
radation and immobilization.
A number of species of metal-resistant fungi have been
isolated from heavy metal-polluted sediments and soil
(e.g. Kameo et al. 2000; Villegas et al. 2005; Zafar et al.
2007). Fungi play an important role in the removal of
metals through their ability to bind metals to the cell wall
forming a chitin-metal complex (Franco et al. 2004; Zhou
et al. 2005), through production of metallothioneins
(Kameo et al. 2000; Jaeckel et al. 2005) and oxalates
(Fomina et al. 2005), and through extracellular accumula-
tion in the mycelia (Taboski et al. 2005). Christie et al.
(2004) have reviewed the evidence that arbuscular mycor-
rhizal (AM) fungi can exert protective effects on host
plants in metal-contaminated soil by possibly binding or
immobilizing metals. However, in greenhouse experi-
ments on AM assemblages collected from Florida wetland
1164 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1163–1174
D. Purchase et al.
plants, flooding to a depth of 2Æ5 cm nearly eliminated
AM fungal colonization (Ipsilantis and Sylvia 2007). The
results of this study suggest that AM fungi will be present
in horizontal sub-surface flow constructed wetlands but
that they may have limited populations in surface flow
systems.
Diverse microbial communities significantly contribute
to biogeochemical element cycles (Jin and Kelley 2007)
and the removal of toxic heavy metals in wetland systems
(Groudeva et al. 2001). Relatively little information is
available on the importance of the different microbial
groups responsible for organic matter degradation (Tietz
et al. 2008), and even less well understood is the heavy
metal bioremediation role of fungi in constructed wet-
lands. Two Zn- and Pb-tolerant fungi, Beauveria bassiana
and Rhodotorula mucilaginosa, were isolated from two
constructed wetlands in Dagenham and Brentwood in
outer London, England. The minimum inhibitory concen-
trations (MIC) of B. bassiana and R. mucilaginosa for Zn
were 2000 and 200 lg g)1 respectively; and for Pb, 1000
and 100 lg g)1 respectively (Scholes et al. 1999). This
paper examines the effect of Zn and Pb on cell growth
and metal accumulation over 21 days. The effects of tem-
perature on colony growth and metal tolerance as well as
the possible mechanisms involved in metal accumulation
resistance are also investigated.
Materials and methods
Culture condition
A modified glycerol asparagine (GA) medium based on
the work by Dietz and Thayer (1980) was used to culture
B. bassiana and R. mucilaginosa. This is a defined
medium with minimal interactions between metals and
the ingredients and at a pH range where metal salts are
soluble. The modified GA medium contained asparagine
(1Æ14 g), glycerol (20 ml), 3 [N-morpholino] propanesulf-
onic acid (MOPS; 1Æ37 g) and agar (20 g) in 1 l distilled
water at pH 7Æ0–7Æ4. The isolates were incubated at 30C
in a shaking incubator for the duration of the metal accu-
mulation experiments.
For the variable temperature experiments, the two iso-
lates were grown on glycerol yeast extract agar (GYEA)
containing glycerol (20 ml), yeast extract (4 g), NaNO3
(2 g), KH2PO4 (1 g), MgSO4Æ7H2O (0Æ5 g), FeSO4Æ7H2O
(0Æ01 g), agar (15 g) in 1 l distilled water at 4 and 30C.
Assessment of metal accumulation
Metals associated with the cells, cell mass and the viable
counts were determined at the beginning of the experiment
and at intervals of 1, 7, 14 and 21 days. The isolates were
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D. Purchase et al. Wetland fungal metal accumulation

grown in 50 ml of GA broth containing Zn (100 or
200 lg g)1, equivalent to 1Æ5 or 3 lmol l)1) or Pb (40 or
100 lg g)1, equivalent to 0Æ2 or 0Æ5 lmol l)1) at 30C in a
shaking incubator. Analytical grade metal nitrate salts
(Fisher, UK) were used to provide the required metal con-
centrations. Metal- and inoculum-free controls were estab-
lished and all experiments were carried out in duplicate.
Fungal biomass was harvested by vacuum-filtration
through autoclaved, preweighed Q-MA filters (particle
retention 0Æ6 lm) and the dry cell mass was determined.
The cells and associated filter were then digested in 10 ml
of concentrated nitric acid. The samples were evaporated
to dryness on a sand bath at 100C. The residues were
dissolved in 1% nitric acid, filtered through a Whatman
No. 42 filter paper and made up to a final volume of
100 ml. Heavy metals present in the samples were analy-
sed by inductively coupled plasma atomic emission spec-
troscopy (ICP-AES). Additional filters were also digested
as blanks. These procedures were repeated at intervals of
1, 7, 14 and 21 days.
The percentages of metals accumulated by B. bassiana
and R. mucilaginosa over 21 days were calculated by com-
paring the metal determined in the cell biomass with the
amount of metal initially available in a 50 ml medium
sample. The calculation was based on the following for-
mula, assuming 1 ml of the medium weighs 1 g.
mB
% accumulation ¼  100;
V M
where m is the accumulated metal concentration in bio-
mass (mg g)1), B is the cell biomass (g), V is the volume
of the medium (ml) and M is the metal concentration in
the medium (mg ml)1).
Effect of metals on colony growth at different
temperatures
An inoculum was obtained from fungal colonies growing
on GYEA medium for 7 days at 30C. A series of small
squares (c. 200 mm2 in area) were excised and placed
colony side down on replicate GA plates containing either
Pb (at 40, 200 or 400 lg g)1) or Zn (at 30, 400 or
800 lg g)1). The control contained no added metal.
Radial growth of the colonies was measured at regular
intervals over 35 days.
Transmission electron microscopy (TEM) and x-ray
analysis
Both isolates were grown on GA plates as well as plates
containing Pb (at 40, 200 or 400 lg g)1) or Zn (at 30,
400 or 800 lg g)1) for 7 days at 30C. The specimens
were fixed with osmium tetroxide, stained with uranyl
acetate and lead citrate and embedded in Spurr’s resin.
A cross-section of the samples was cut using a microtome
to a thickness of 60–70 nm. The samples were examined
with a Philips EM301G TEM operating at an accelerating
voltage of 80 kV. Metal locations were determined by
X-ray analysis using a TEM (Zeiss EM10C) operating at
an accelerating voltage of 80 kV.
Statistical analysis
All statistical analyses were carried out using a Minitab
statistical package. t-test and Mann–Whitney tests were
used to analyse normally and non-normally distributed
data, respectively.
Results
Effects of Zn and Pb on cell growth
The final viable counts of B. bassiana and R. mucilaginosa
grown in control conditions and media containing added
Zn (100 and 200 lg g)1) and Pb (40 and 100 lg g)1) are
shown in Table 1. Viable counts of B. bassiana in the
control flasks showed a marked increase of 241% after
day 1 but cell viability decreased by 45% and 60% in the

Table 1 Mean viable counts (CFU ml)1) for fungi grown in modified GA medium (control) compared with the same condition containing
200 lg g)1 Zn or 100 lg g)1 Pb (Beauveria bassiana) and 100 lg g)1 Zn or 40 lg g)1 Pb (Rhodotorula mucilaginosa). The cultures were
incubated at 30C for 21 days

B. bassiana R. mucilaginosa

Culture containing Culture containing Culture containing Culture containing

Days Control 200 lg g)1 Zn 100 lg g)1 Pb Control 100 lg g)1 Zn 40 lg g)1 Pb

0 7Æ9 ± 0Æ3 · 104 3Æ8 ± 2Æ3 · 104 4Æ3 ± 1Æ7 · 104 2Æ0 ± 0Æ3 · 107 1Æ5 ± 0Æ1 · 107 1Æ5 ± 0Æ7 · 107
1 2Æ7 ± 0Æ5 · 105 2Æ1 ± 0Æ7 · 104 1Æ7 ± 1Æ4 · 104 4Æ7 ± 0Æ7 · 107 1Æ8 ± 0Æ6 · 106 5Æ6 ± 4Æ0 · 105
7 9Æ5 ± 7Æ8 · 103 1Æ4 ± 1Æ2 · 104 2Æ4 ± 1Æ4 · 103 5Æ8 ± 2Æ9 · 107 3Æ9 ± 1Æ5 · 103 4Æ9 ± 5Æ2 · 103
14 1Æ6 ± 1Æ4 · 104 1Æ7 ± 0Æ8 · 103 18 ± 9 4Æ8 ± 0Æ1 · 107 3Æ2 ± 0Æ0 · 102 5Æ2 ± 2Æ5 · 102
21 5Æ1 ± 4Æ0 · 104 6Æ8 ± 7Æ4 · 103 0 2Æ8 ± 0Æ5 · 107 56 ± 31 ND

ND, no data.

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Wetland fungal metal accumulation D. Purchase et al.

Table 2 Cell biomass (mg) ± SD for fungi grown in modified GA medium (control) compared with the same condition containing 200 lg g)1 Zn
or 100 lg g)1 Pb (Beauveria bassiana) and 100 lg g)1 Zn or 40 lg g)1 Pb (Rhodotorula mucilaginosa). The cultures were incubated at 30C for
21 days

B. bassiana R. mucilaginosa

Culture containing Culture containing Culture containing Culture containing

Days Control 200 lg g)1 Zn 100 lg g)1 Pb Control 100 lg g)1 Zn 40 lg g)1 Pb

0 4Æ52 ± 0Æ10 3Æ48 ± 0Æ12Æ76 3Æ65 ± 2Æ33 19Æ45 ± 12Æ7 18Æ44 ± 8Æ00 18Æ44 ± 15Æ75
1 6Æ25 ± 3Æ52 2Æ20 ± 0Æ26 2Æ66 ± 0Æ83 23Æ39 ± 4Æ57 9Æ12 ± 1Æ34 8Æ23 ± 1Æ03
7 13Æ91 ± 5Æ68 10Æ07 ± 10Æ55 3Æ35 ± 1Æ53 22Æ04 ± 2Æ94 7Æ72 ± 1Æ39 7Æ13 ± 0Æ04
14 18Æ52 ± 0Æ61 10Æ51 ± 2Æ93 3Æ11 ± 0Æ78 21Æ11 ± 0Æ21 6Æ72 ± 0Æ15 6Æ79 ± 0Æ64
21 7Æ67 ± 1Æ98 11Æ94 ± 11Æ35 1Æ57 ± 1Æ08 26Æ15 ± 0Æ0 8Æ82 ± 0Æ42 8Æ09 ± 0Æ35

cultures containing Zn or Pb, respectively. Cell viability
of R. mucilaginosa increased by 222% after day 7 but
decreased by 88% and 96% in the cultures containing Zn
and Pb, respectively. Cell biomass in the media contain-
ing Zn and Pb also showed a substantial reduction com-
pared with the control over the 21-day period, with the
exception of B. bassiana, which demonstrated a limited
increase in biomass after day 14 in the medium contain-
ing 200 lg g)1 of Zn (Table 2).
Metal accumulation by Beauveria bassiana and
Rhodotorula mucilaginosa
Zn and Pb accumulation via adsorption and ⁄ or absorp-
tion and the percentage of metal accumulated based on
cell biomass by the two isolates over a 21-day period are
shown in Tables 3a and b. Rhodotorula mucilaginosa
appeared to accumulate higher levels of Zn over the first
24 h than B. bassiana, but the levels of accumulation of
both fungi stabilized at c. 6 mg g)1 by day 7 and showed
a similar decline afterwards. By the end of the experimen-
tal time period, the levels of metal determined in associa-
tion with B. bassiana and R. mucilaginosa were 2Æ62 and
3Æ02 mg g)1, respectively. The maximum Pb accumulation
for B. bassiana was recorded by day 14 (mean of
135Æ83 mg g)1). The concentrations of Pb found in
R. mucilaginosa were consistently lower with the maxi-
mum accumulation (mean of 46Æ49 mg g)1) recorded by
day 14.
The percentage ranges of Zn accumulated by B. bassi-
ana and R. mucilaginosa were 0Æ10–0Æ64% and 0Æ53–
2Æ05%; and for Pb, 1Æ89–8Æ44% and 6Æ68–16Æ55%, respec-
tively. The percentage of Zn accumulated by B. bassiana
was 0Æ10% during the first day of metal exposure but
increased to 0Æ64% by day 7 followed by a subsequent
reduction to 0Æ29% by day 21. In contrast, the percentage
of Pb rapidly achieved 6Æ57% and remained constant at
this level on day 7. The maximum level was recorded on
day 14 at 8Æ44%, but had reduced to 1Æ89% by day 21
(Table 3b). For R. mucilaginosa, the initial percentage of
Zn accumulated was 2Æ05% followed by a gradual reduc-
tion over the 21 days of the experiment to 0Æ53%. The
percentage of Pb accumulated by R. mucilaginosa was ini-
tially similar to that of B. bassiana but increased substan-
tially by day 7 to 16Æ55%. Subsequent decreases resulted
in a final uptake percentage, after 21 days, of 11Æ71%.
Effects of Zn and Pb on cell growth at different
temperatures
The average growths of B. bassiana colonies at 30C,
expressed as colony area are presented in Fig. 1a and b.

Table 3a Accumulation of Zn per cell dry weight (mg g)1) ± SD by Beauveria bassiana and Rhodotorula mucilaginosa. The cultures were
incubated at 30C for 21 days, the control contained no added metal, B. bassiana and R. mucilaginosa was grown in media containing 200 and
100 lg g)1 (3 and 0Æ5 lmol l)1) Zn, respectively. The percentage accumulation of Zn is based on a comparison between metal available in a
50-ml culture (mg) and metal concentration associated with the cell biomass (mg)

B. bassiana R. mucilaginosa

Metal accumulated per Metal accumulated per

Days Control cell dry weight (mg g)1) % accumulation Control cell dry weight (mg g)1) % accumulation

1 0Æ23 ± 0Æ01 4Æ76 ± 1Æ79 0Æ1 0Æ06 ± 0Æ03 11Æ30 ± 0Æ96 2Æ05
7 0Æ06 ± 0Æ04 6Æ39 ± 3Æ86 0Æ64 0Æ04 ± 0Æ01 6Æ44 ± 1Æ09 0Æ99
14 0Æ04 ± 0Æ02 5Æ23 ± 1Æ57 0Æ54 0Æ02 ± 0Æ00 4Æ98 ± 1Æ79 0Æ67
21 0Æ19 ± 0Æ06 2Æ62 ± 0Æ26 0Æ29 0Æ03 ± 0Æ00 3Æ02 ± 0Æ39 0Æ53

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D. Purchase et al. Wetland fungal metal accumulation

The different growth rates of B. bassiana on a metal-

free medium and plates containing 40, 200 and
400 lg g)1 Pb at 30C are illustrated in Fig. 1b. After
7 days, similar growths were recorded on the control
plate and the plate containing 40 lg g)1 Pb but the latter
demonstrated a preferential growth rate after 21 days and
by the end of the experiment (35 days), the colony size
was 23% larger than the control. In contrast, the colony
sizes on the plates containing 200 and 400 lg g)1 Pb were
similar but only slightly smaller than the control (4%
reduction). The overall colony sizes on the plates contain-
ing Pb are larger compared with those containing Zn.
This is illustrated for the average colony sizes after
35 days on the plates containing the same concentration
(400 lg g)1) of Pb and Zn, which were measured as 1248
and 991 mm2, respectively.
The results obtained for the growth of B. bassiana on
metal-free controls and plates containing Zn and Pb at
4C are presented in Fig. 2a and b. The growth of the iso-
lates on all the plates showed a clear lag phase between
10 and 21 days. The growth patterns of the colonies on
the control and plates containing 30 lg g)1 Zn were simi-
lar, as were the trends observed on the plates containing
400 and 800 lg g)1 Zn. However, the latter demonstrated
Figure 1 (a) Average growth of Beauveria bassiana on the control and clearly reduced growth rates between 21 and 35 days
plates containing Zn at 30C. ( , Control; , 30 lg g)1 Zn; , (Fig. 2a). The temperature effect is illustrated by the
)1 )1
400 lg g Zn; , 800 lg g Zn). (b) Average growth of B. bassiana
reduced Zn growth at 4C, which is between 11% and
on control and plates containing Pb at 30C. ( , Control; ,
44% of that observed at 30C (Fig. 1a). However, the
40 lg g)1 Pb; , 200 lg g)1 Pb; , 400 lg g)1 Pb).
growth of B. bassiana remained substantial at 4C achiev-
ing 74% (control), 62% (30 lg g)1 Zn), 70% (400 lg g)1
Beauveria bassiana showed optimal growth on metal-free Zn) and 88% (800 lg g)1 Zn) of the growth recorded for
control plates, with decreasing colony growth on plates the same Zn concentrations at 30C.
containing Zn concentrations in the order 400 lg g)1 > Colony growth of B. bassiana on plates containing
30 lg g)1 > 800 lg g)1 (Fig. 1a). Similar patterns were 40 lg g)1 Pb showed limited stimulation at 4C (Fig. 2b)
observed in both duplicates although they are not shown with the colony sizes matching those on the control after
in the diagram. After 35 days, the colony surface area on 35 days. The increasing inhibitory effect of Pb was
the Zn plates represented 66%, 76% and 58% of that of observed on plates containing 200 and 400 lg g)1 Pb
the control, for Zn concentrations of 30, 400 and such that by day 35, the growth areas on these plates were
800 lg ⁄ g respectively. between 22% and 38% lower than those at 30C.

Table 3b Accumulation of Pb per cell dry weight (mg g)1) ± SD by Beauveria bassiana and Rhodotorula mucilaginosa. The cultures were
incubated at 30C for 21 days, the control contained no added metal, B. bassiana and R. mucilaginosa were grown in media containing 100
and 40 lg g)1 (0Æ5 and 0Æ2 lmol l)1) Pb, respectively. The percentage accumulation of Pb are based on comparison between metal available in a
50-ml culture (mg) and metal concentration associated with the cell biomass (mg)

B. bassiana R. mucilaginosa

Metal accumulated by Metal accumulated by

Days Control cell dry weight (mg g)1) % accumulation Control cell dry weight (mg g)1) % accumulation

1 0Æ20 ± 0Æ16 123Æ74 ± 6Æ92 6Æ57 0Æ04 ± 0Æ02 16Æ28 ± 2Æ72 6Æ68
7 0Æ14 ± 0Æ08 98Æ39 ± 4Æ54 6Æ58 0Æ08 ± 0Æ00 46Æ49 ± 2Æ45 16Æ55
14 0Æ11 ± 0Æ22 135Æ83 ± 64Æ52 8Æ44 0Æ15 ± 0Æ14 31Æ92 ± 0Æ67 10Æ79
21 0Æ07 ± 0Æ02 60Æ42 ± 20Æ94 1Æ89 0Æ29 ± 0Æ02 29Æ26 ± 0Æ04 11Æ71

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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1163–1174 1167
Wetland fungal metal accumulation
Figure 2 (a) Average growth of Beauveria bassiana on control and
plates containing Zn at 4C. ( , Control; , 30 lg g)1 Zn;
)1 )1
400 lg g Zn; , 800 lg g Zn). (b) Average growth of B. bassi-
ana on control and plates containing Pb at 4C. ( , Control;
40 lg g)1 Pb; , 200 lg g)1 Pb; , 400 lg g)1 Pb).
The growth of R. mucilaginosa was inhibited at
400 lg g)1 Zn and 200 lg g)1 Pb (results not shown). At
30C, a Pb concentration of 40 lg g)1 appeared to stimu-
late growth (Fig. 3a) such that the colony size had only
doubled by day 35. However, the differing behaviour of
B. bassiana was illustrated by the demonstrated 11-fold
increase in colony area under corresponding conditions
(Fig. 1b). Minimum growth occurred for R. mucilaginosa
at 4C on all the plates (Fig. 3b), with the highest
recorded growth being 14% over the 35 days in the pres-
ence of 30 lg g)1 Zn.
Examination of Beauveria bassiana using TEM and
energy-dispersive x-ray (EDX) spectrometry
The results obtained in the viable count experiments
(Table 1) suggest that B. bassiana has a greater tolerance
of Pb and Zn than R. mucilaginosa. This was investigated
further by using TEM and EDX to examine B. bassiana
both before and after exposure to metals. A TEM of the
cross-sections of hyphae of B. bassiana grown on a metal-
free control medium at ·10 000 magnification is provided
in Fig. 4a. The average hypha diameter is estimated to be
1168 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1163–1174
D. Purchase et al.
,
Figure 3 (a) Average growth of Rhodotorula mucilaginosa on control
and plates containing selected Zn and Pb concentrations at 30C. ( ,
,
Control; , 30 lg g)1 Zn; , 40 lg g)1 Zn). (b) Growth of R. muci-
laginosa on control and plates containing selected Zn or Pb concentra-
tions at 4C. ( , Control; , 30 lg g)1 Zn; , 40 lg g)1 Pb).
2Æ84 ± 0Æ56 lm. Fungal cell walls are clearly apparent, as
are various internal structures such as nuclei, mitochon-
dria, vacuoles and lipid droplets. The pale or light cell in
the centre of the photograph is likely to be an older, dead
cell. The cross-sections of hyphae grown on a medium
containing 800 lg g)1 Pb (at ·10 000 magnification) are
shown in Fig. 4b. The average diameter of the hyphae is
1Æ44 ± 0Æ47 lm. The cell walls and some internal struc-
tures are apparent but additional electron dense areas
associated with the cell wall, with the inside of the cell
itself, and with the medium surrounding the cells can be
observed. The peripheral cells are very pale, which sug-
gests that they are dead. In contrast, the hyphae grown
on a medium containing 800 lg g)1 Zn (Fig. 4c) appear
to be much healthier, with darker cell contents and inter-
nal structures that are clearly visible. Electron dense areas
are not visible around the cell wall or the cross-sections
of hyphae grown on plates containing Zn, confirming
that B. bassiana did not accumulate Zn as efficiently as
Pb (Table 3a).
EDX indicated that the electron dense areas identified in
Fig. 4b corresponded to the detection of a high level of Pb.
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D. Purchase et al.
Figure 4 (a) Cross-section of hyphae of Beauveria bassiana grown on
a metal-free control medium (·10 000). (b) Cross-section of hyphae
of B. bassiana grown on a medium containing 800 lg of Pb g)1
(·10 000). The electron dense areas can be observed in the medium
(e.g. location 1), on the cell wall (e.g. location 2) and inside the cell
(e.g. location 3). (c) Cross-section of hyphae of B. bassiana grown on
a medium containing 800 lg of Zn g)1 (·10 000).
Figure 5a shows the locations in the region of a cross-
section of hyphae of B. bassiana grown on a medium
containing 80 lg g)1 Pb that have been subjected to EDX
spot analysis. The results of such analyses for electron
dense areas on and inside a hypha are shown in Fig. 5b
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1163–1174
Wetland fungal metal accumulation
Figure 5 (a) Locations of the spot analysis on the cross-section of
hyphae of Beauveria bassiana grown on a medium containing
800 lg g)1 Pb. (b) Energy-dispersive spectrometric (EDX) analysis of
location 1. (c) EDX analysis of location 2.
and c, respectively. The elevated Pb peaks suggest the
ability of B. bassiana to accumulate and precipitate Pb
intracellularly and to adsorb Pb to the cell wall. A
detailed elemental map of the hyphae cross-section envi-
ronment indicated that the majority of Pb was located in
the medium rather than associated with the cell walls
(result not shown).
1169
Wetland fungal metal accumulation
Discussion
Effects of Zn and Pb on cell growth
Initial viable count results show that the respective series
of flasks inoculated with either B. bassiana or R. mucilag-
inosa were inoculated with a similar number of cells to
within an order of magnitude (Table 1). The reduction in
B. bassiana viable count in the control on day 7 (Table 1)
probably resulted from nutrient exhaustion. The subse-
quent increase of viability may be attributed to cells that
have undergone autolysis releasing nutrient back into the
medium. In contrast, the viable counts in the Zn- and
Pb-spiked flasks of both species show a gradual decrease
over the experimental time period, indicating that the
viability of both species decreases with the length of expo-
sure time. The viable counts for B. bassiana in the
Pb-spiked flasks decreased more rapidly in comparison
with the viable counts reported in the Zn-spiked flasks,
supporting earlier work by Scholes et al. (1999), which
reported that both species could tolerate higher levels of
Zn in comparison with Pb.
The relationship between cell mass (see Table 2) and
viable count is not clear, although an overall decrease in
the viable count of spiked flasks with time is associated
with an overall decrease in the cell mass, with the excep-
tion of B. bassiana in flasks spiked with Zn, where the
viable count decreases as the cell mass increases. A factor
in this trend could be that all flasks were inoculated from
a plate rather than a broth as B. bassiana tends to grow
in a pellet form making liquid inoculation with a consis-
tent number of cells difficult. However, using inoculum
from a plate means a mixture of different growth stages
may be selected with the possibility that some growth
stages may be more able to tolerate metals than others.
Alternatively, the possibility of sporulation as a contribu-
ting factor cannot be discounted.
Accumulation of Zn and Pb by Beauveria bassiana and
Rhodotorula mucilaginosa
The amounts of Pb and Zn detected in association with
cells grown in the metal-free control were significantly
lower than those grown in a culture containing metal
(P < 0Æ05; Tables 3a and b). After standardizing the
results by taking into account metal availability and fun-
gal biomass, B. bassiana was found to accumulate higher
levels of Pb than Zn. Several other studies have also
reported a greater binding capacity for Pb in comparison
with other metals for a range of biomass types, which
may be related to its large atomic weight and ionic radius
(Holan and Volesky 1995; Gopal et al. 2002; Taboski
et al. 2005).
1170 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1163–1174
D. Purchase et al.
Accumulation of metals by the isolates, in addition to
substantial decreases in cell viability, may be interpreted
in different ways. It could indicate that biosorption
(metabolic-independent accumulation) is the primary
mechanism of metal accumulation, whereas, e.g. binding
of Pb to cell walls could cause them to rupture, resulting
in both cell death and high levels of metal accumulation
following the exposure of new, internal binding sites
(Gadd 1990). However, these results could also indicate
that bioaccumulation (metabolic-dependent accumula-
tion) was the major mechanism for metal accumulation,
with cells bioaccumulating metals intracellularly to such a
degree that a rapid reduction in cell viability resulted.
However, the results of this study do not provide enough
evidence to conclude whether biosorption or bioaccumu-
lation is the more important mechanism in B. bassiana.
It has been reported that as cell mass increases, the
amount of metal removed from the solution also
increases, but the actual amount of metal accumulated
per gram of cell decreases (Horikoshi et al. 1981; Jun-
ghans and Straube 1991; Gadd 1993). It is thought that as
cell density increases, the adhesion between cells reduces
the relative amount of surface area available for binding.
In this study, although higher cell mass corresponded to
a higher amount of Pb removal from solution, it also cor-
responded to a higher percentage of metal accumulated.
By day 7, the amount of Pb increased by 247% whereas
the amount of Zn associated with R. mucilaginosa fell by
over 50%. These data suggest that the accumulation of Pb
in R. mucilaginosa is nonmetabolic-dependent but that
Zn was accumulated primarily within living cells. Hence,
as the viability of the isolate decreases, the amount of Zn
accumulated also decreases. The Mg transport systems of
both the yeast Saccharomyces cerevisiae and the bacterium
Escherichia coli have been reported to take-up Zn, among
other ions (Portier and Palmer 1989). A similar mecha-
nism may be responsible for Zn accumulation by R. mu-
cilaginosa. In contrast, the amount of Pb accumulated by
R. mucilaginosa showed a significant increase from 1 to
7 days. It is possible that the initial accumulation of Pb
acted as a nucleation site around which further metals
were then deposited. A similar pattern was observed in
R. aurantiaca where the biosorption of Pb took place
rapidly within 5 min following the Langmuir sorption
model (Cho et al. 2004).
After day 14, the mean cell mass of R. mucilaginosa in
the control medium remained unchanged, whereas those
in media containing Zn or Pb fell slightly. The amount of
Zn removed from the medium also decreased, supporting
the theory that Zn accumulation may be primarily a met-
abolic-dependent process. Similarly, the amount of Pb
removed from the medium also showed a decrease. How-
ever, this could be because of the lysis of cells following
ª 2009 The Authors
D. Purchase et al.
death, which resulted in the release of metals back into
solution. Alternatively, precipitated metals may be no
longer associated with cell matter and therefore may not
be collected by the filter.
The percentage of Zn accumulated was the lowest at
day 21, which correlates with the lowest viable count
recorded and also supports the supposition that part of
the removal mechanism is metabolic-dependent. In con-
trast, the percentage of accumulated Pb increased at day
21, probably because of the increase in cell mass and the
subsequent increase in biosorption.
There is a clear difference in the percentage of Pb accu-
mulated between the two strains, with the percentage
accumulated by R. mucilaginosa being substantially higher
than that accumulated by B. bassiana (Table 2). Differ-
ences between percentages of metals accumulated by each
strain are thought to be associated with a wide range of
factors such as differences in morphology, cell wall com-
position and structure, type, concentration, ionic radius
and atomic weight of the selected metal, production of
extracellular products and mechanisms of cell transport
and compartmentalization (Gadd 1993). Although the
ability of both strains to accumulate Zn and Pb (and in
particular the accumulation of Pb by R. mucilaginosa) has
been clearly demonstrated, it is difficult to speculate at
this stage which of these factors are primarily responsible
for the differences in the amount accumulated by each
isolate and whether adsorption or absorption plays a
more significant role in the tolerance of metals.
Effects of temperature on colony growth at different
temperatures
A comparison of colony growth of B. bassiana and
R. mucilaginosa under the same nutrient conditions, at
the same temperature and in the presence of the same
concentrations of Zn (30 lg g)1) or Pb (40 lg g)1)
clearly demonstrates the greater tolerance and robustness
of B. bassiana to these metals (e.g. Figs 1a, 1b and 3a). It
is unclear why 30 lg g)1 of Zn appeared to be more toxic
to B. bassiana than 400 lg g)1; however, the difference is
not significant at the 95% confidence level.
The growth on plates containing 40 lg g)1 Pb was
greater than the control when grown at 30C, which sug-
gests that at low concentrations Pb stimulates the growth
of B. bassiana and R. mucilaginosa. Stimulation of micro-
bial growth by low concentration of Pb has been previ-
ously reported (Liao et al. 2007). In comparison with the
growths on plates incubated at 30C, fungal growths at
4C were lower and a concentration of 40 lg g)1 of Pb
did not appear to stimulate growth (Figs 2b and 3b).
However, the differences in growth at the two tempera-
tures were not significant at the 95% confidence level
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 1163–1174
Wetland fungal metal accumulation
(P = 0Æ1732 and 0Æ436 for B. bassiana and R. mucilagin-
osa, respectively). This has implications for the year-
round treatment ability of constructed wetland treatment
systems receiving urban runoff. Minimum water tempera-
tures recorded at the sampling locations were 5Æ3 and
5Æ6C at the Dagenham and Brentwood wetland sites,
respectively (results not shown). At the Dagenham site,
where flow is generally low, the temperature of the sedi-
ment surface would be approximately the same as that in
the overlying water, with sediment temperature increasing
relative to water temperature with increasing sediment
depth. Therefore, it could be assumed that sediment tem-
perature (at least at the time when samples were col-
lected) was at a minimum of 5Æ3C. The results of this
study suggest that B. bassiana is able to both grow and
tolerate metals at 4C and that there is a year-round bio-
logical activity in the wetlands.
Distribution of Zn and Pb in Beauveria bassiana
Examination of B. bassiana using TEM indicated the
absence of electron dense areas on any of the plates con-
taining Zn, which is consistent with the results of the
metal accumulation experiments, which showed that
B. bassiana did not have a great ability to bind Zn
(Table 3a). The healthy appearance of cells grown on
plates containing Zn compared with those containing the
same concentration of Pb again suggests that B. bassiana
is more tolerant to Zn than Pb. Examination of the iso-
late grown on media containing Pb indicated the presence
of electron dense areas associated with cell walls, within
the cells and in the medium surrounding the cells
(Fig. 4b). These electron dense areas were not present on
metal-free controls or samples grown in medium contai-
ning Zn. EDX analysis and elemental mapping were
carried out, and confirmed that these electron dense areas
were because of the presence of Pb (Fig. 5a–c).
Chitin and chitosan are important components of fun-
gal cell walls and both have been shown to sequester
metal ions (Franco et al. 2004; Zhou et al. 2005). Pb
uptake by Rhizopus nigricans was reported to be primarily
because of the binding of Pb to the amine-N of chitin,
which then acted as a nucleation site for the further
deposition of Pb (Zhang et al. 1998). Almost all filamen-
tous fungi contain chitin in their cell walls, and such a
mechanism may therefore have also been involved in the
accumulation of Pb by B. bassiana. The presence of pre-
cipitated Pb in the medium surrounding the hyphae may
indicate that the release of metabolites by the isolate also
results in the precipitation of Pb. It has been shown that
Zn and Pb are bound predominantly to phosphoryl
(c. 95%) and minor carboxyl groups (about 5%) in
Penicillium chrysogenum (Sarret et al. 1998). It is possible
1171
Wetland fungal metal accumulation
that B. bassiana exhibits similar binding affinity. How-
ever, owing to the complex nature of the cell wall compo-
sition and structure, it is unlikely that all the insoluble Pb
is present as insoluble phosphate.
Beauveria bassiana is an insect pathogen and is known
to produce citric and oxalic acids, which are thought to
reduce protein bonding in the insect cuticle thereby
enabling the penetration of hyphae (Charnley 2003).
Fomina et al. (2005) indicated that Beauveria caledonica
solubilized Cd, Cu, Pb and Zn minerals that were then
precipitated as oxalates. The release of oxalic acid by
B. bassiana may be responsible for the precipitation of Pb
in the medium surrounding the hyphae. The precipitation
of metals as oxalate crystals is highly desirable as insolu-
ble metal oxalates are resistant to further solubilization,
with only a few bacteria and fungi being known that are
able to readily degrade them (Sayer and Gadd 1997).
Potential applications
This study has shown that selected metal-tolerant micro-
organisms are capable of rapid adaptation to metal
removal at sites receiving contaminated wastewaters. Both
living and dead cells are able to accumulate significant
amounts of Zn and Pb, through bioaccumulation and
biosorption processes, enabling micro-organisms to play
an important role in the treatment of urban runoff, espe-
cially in winter conditions when the macrophytes die
back. The precipitation of metals by fungi, such as B. bas-
siana, represents a removal mechanism for urban runoff
treatment wetlands in which the substrates act as long-
term storage sinks. The results have implications for
urban runoff treatment wetland design, through the selec-
tion of wetland plants and substrates to support specific
microbial populations in terms of their year-round pollu-
tant removal potentials.
Acknowledgements
The authors acknowledge the support of the Engineering
and Physical Sciences Research Council and the Environ-
ment Agency England and Wales for the wetland research
and monitoring programme, and the excellent technical
assistance of Manika Choudhury and Alan LaGrue.
Thanks are also given to Drs Tony Brian (King’s College
London) and Alice Wavely (St Thomas’ Hospital, Lon-
don) for their assistance with the EDX analysis.
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