Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 115-118, 2005

Accepted
Accumulation of Cu and Its Oxidation State in Tremolecia atrata (Rusty-Rock Lichen)
Mycobiont
H. Fujii,a K. Hara,a K. Komine,a T. Ozaki,b T. Ohnuki,b and Y. Yamamoto*, aaFaculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-nakano, Akita 010-1195, Japan
Advanced Science Research Center, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-1195, Japanb

Received: November 15, 2004; In Final Form: April 21, 2005
We studied the association of a Cu tolerant mycobiont, Tremolecia atrata (rusty-rock lichen) with Cu. T. atrata
mycobiont cell aggregates, which grew into a sperical shape, rapidly absorbed Cu into their inner and outer parts.
The EDS/SEM study showed that Cu was more highly accumulated in the inner part than the outer part of theaggregates. The XANES study revealed that the Cu absorbed by the T. atrata mycobiont was both monovalentand divalent. These results suggested that the T. atrata mycobiont’s high tolerance to Cu is attributable to its
ability to store Cu(I) inside the cytoplasm and to adsorb Cu(II) on the cell wall.

1. Introduction
Lichens, symbiotic organisms combining a fungus (mycobiont) and an alga (photobiont), can be found ubiquitously evenin environments extremely unfavorable to other living things.
They grow in the polar region, burning deserts, salty seashores,
extremely high mountains, and heavy metal mines. Their highadaptability is achieved through their intricate cooperative
lifestyle. Algae provide photosynthetic products for fungi,
which produce metabolic substances to overcome the stressfound in those unfavorable environments. Many researchers1−3
have reported the accumulation of heavy metals, such as Cu,
Cd, and U, in lichens growing in mines and nearby smelters.
Brown et al.4 proposed three principle mechanisms for the
metal accumulation by lichens: extracellular uptake through an
ion exchange process, intracellular accumulation, and the trapping of particles with metals in their body frame. However,
the tolerance mechanisms of lichens at the molecular level
have not been clear until now. Their tolerant properties could
be used for bioremediation of toxic metals and biomonitoring
toxic metal pollutions, once the associations at the molecularlevel, such as the adsorption sites on cell walls and the transport mechanisms to cytoplasms, are clearly understood.
In order to elucidate the tolerance mechanisms of lichens at
the molecular level, the roles of mycobionts and photobionts
must be examined separately. Yamamoto et al.5 succeeded inisolating about 250 mycobionts from their symbionts andfound that some mycobionts are highly tolerant to Cu. In thisstudy, the distribution of Cu and its chemical state in the
mycobiont-cell aggregates of spherical-shaped Tremolecia
atrata (rusty-rock lichen) having Cu tolerance were examinedusing scanning electron microscopy equipped with energydispersive spectrometry (EDS/SEM) and X-ray absorption
near edge structure (XANES). The effect of Cu on the growthof the T. atrata mycobiont was also examined by a batch
method.
2. Experimental
Sample preparation. A T. atrata mycobiont separated by
Yamamoto et al.5 from natural lichens was used. The cells
were cultured in a malt-yeast medium (MY)6 composed of 20*Corresponding author. E-mail: yyamamoto@akita-pu.ac.jp.
FAX: +81-18-872-1678

g/L malt extract and 2 g/L yeast extract. Before inoculation,
the medium had a pH of 5.8.
Effect of Cu on the growth of T. atrata mycobiont-cell
aggregates. About 100 mg of fresh T. atrata mycobiont-cell
aggregates were inoculated into 100 mL Erlenmeyer flasks
containing 25 mL MY medium with 1.2 mM Cu(II)SO4. The
pH of the medium was about 4.7. The cell aggregates wereincubated on a rotary shaker at 120 rpm in the dark at 20 ˚C. At
1, 7, and 14 days after inoculation, the culture was filtered witha 125 µm nylon mesh filter, and the mycobiont cell-aggregateson the filter were collected and weighed. The amount of Cu
accumulated in the T. atrata mycobiont was determined bymeasuring the Cu concentration in the medium solution filteredthrough a 0.22 µm membrane filter (ADVANTEC MFS, Inc.,DISMIC-25) using induced coupled plasma atomic emission
spectroscopy (ICP-AES: Shimadzu, ICP-7000).
SEM/EDS. After 24 hours of incubation with Cu as
mentioned above, the cell aggregates were collected, washed 1time with purified water (pH ~ 6.8), and freeze-dried. The
resulting spherical-shaped cell aggregates, with diameters of
about 1 mm, were sliced into 1 µm sections. Morphologicalobservations and P, S, K, and Cu contents were determinedusing scanning electron microscopy (SEM) equipped withenergy dispersive spectrometry (EDS) (JEOL JMS-6330 F).
The EDS spectra were collected between 0 and 20 keV.
XANES. The oxidation state of Cu in the cell aggregates of
the T. atrata mycobiont and Saccharomyces cerevisiae were
examined by X-ray absorption near edge structure (XANES)
spectroscopy in the fluorescence mode at the BL27B line at theHigh Energy Accelerator Research Organization, Tsukuba,
Japan. The XANES spectra for the Cu Kα1 were collected.
About 1 g of fresh T. atrata mycobiont-cell aggregates were
inoculated into 100 mL Erlenmeyer flasks containing 25 mL of
the MY medium with 1.2 mM Cu(II)SO4. The medium showeda pH of 4.7 before inoculation. The cell aggregates were incubated on a rotary shaker at 120 rpm for 24 hours in the dark at
20 ˚C. The oxidation state of Cu in the yeast S. cerevisiae wasalso analyzed. About 1 g of fresh S. cerevisiae cells were incubated in the MY medium with 1.2 mM Cu(II)SO4 at 120 rpm
for 24 hours at 20 ˚C. The medium showed a pH of 4.7 before
inoculation. Cells of these two species were collected bycentrifugation and the cells were washed with purified waterand then with 0.001 M HCl. After being freeze-dried, the cells
were sealed threefold in polypropylene bags after exchanging
the air in the bags with argon gas. Antioxidants made of zeolite© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

Fujii

116 J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005
were put in the second bag to prevent the oxidation of Cu(I) in
the cells. As standards for mono- and di-valent copper,
Cu(I)Cl and Cu(II)SO4, respectively, mixed with BN (about 10
w/w%) in each sample were used. The oxidation of Cu(I)Cl
was also prevented by the antioxidants.

outer areas.
Figure 4 shows the sliced cell aggregates grown in the MY
medium with 1.2 mM Cu for 14 days. The spherical cellaggregates were broken into small pieces in the process of
slicing. Figures 5A and B illustrate the EDS spectra for the

3. Results
Counts

A
S
Cu

P

K
Cu

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Energy/ keV

BSCounts

Effect of Cu on the growth of T. atrata mycobiont cellaggregates. Figure 1 shows the time courses of growth of T.
atrata mycobiont cell aggregates in the MY media with 1.2
mM Cu and the amounts of Cu accumulated in the cell aggregates. The growth was expressed as the relative growth ratio
calculated by dividing the dry weight at 1, 7, and 14 days after
contact by the initial dry weight. The cell aggregates grew
rapidly in the first 1 day after inoculation. The relative growthratio at 1 day was approximately 2.3, and then reached about
3.0 and 3.4 at 7 and 14 days, respectively.
The amounts of Cu accumulated in the cell aggregatesincreased up to 7 days after inoculation; the amounts wereabout 1.0 and 2.4 mg/g dry weight at 1 and 7 days, respectively.
However, the amounts of Cu at 14 days was 2.3, indicating that
the Cu accumulation did not increase between 7 and 14 days.
SEM/EDS. Figure 2A shows a photo image of T. atrata
mycobiont cell aggregates grown in the MY medium with Cu
for 1 day and Figure 2B, an SEM image of the rectangular areain Figure 2A. As shown in Figure 2A, the centers of the spherical-shaped cell aggregates were hollow. A morphologicaldifference was observed between the outer and inner parts of
the hollow area. The outer part seemed to be dense with cells,
while the matrix seemed harsh in the inner part. Figures 3A
and B show EDS spectra for the inner and outer areas, respectively, indicated by the squares in Figure 2B. In both areas,
peaks corresponding to Kα emissions of P, S, and K weredetected at 2.01, 2.31, and 3.31 keV, respectively. The Kαemission of Cu was detected at 8.04 keV in both the inner and

Cu

P
K
Cu

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Energy/ keV
Figure 3. EDS spectra for the inner (A) and outer areas (B), respectively, indicated by the squares in Figure 2B.

b
14.0

Relative growth ratio

3.5

12.0

3.0

10.0

2.5
2.0

8.0

1.5

6.0

1.0

4.0

0.5

2.0

0.0

Cu accumulation/ mg g-1dw

4.0

aFigure 4. Sliced cell aggregates grown in MY medium with 1.2 mM
Cu for 14 days. The bar in the figure corresponds to 100 µm in length.

0.0
0

7

14

P

B
B

i
inner

A
K
Cu

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Energy/ keV
P

ou
outer

S

Cu

CountsA

CountsContact time/ day

Figure 1. Growth of T. atrata mycobiont cell aggregates in the MY
media with 1.2 mM Cu (●) and the Cu accumulated in the cell aggregates from the medium (◇). The growth was expressed as a relative
growth ratio calculated by dividing the fresh weight at 1, 7, and 14
days after contact by the initial fresh weight. The Cu concentration
accumulated in the cell aggregates was calculated by dividing theweight of Cu (mg) by the dry weight of the cell aggregates.

100 µm

S

B

Cu

K
Cu

10 µm

0.0

Figure 2. A photo image of T. atrata mycobiont cell aggregatesgrown in MY medium with Cu for 1 day (A) and an SEM image ofthe area indicated by the rectangle in Figure 2A (B). The bar in
Figure 2B corresponds to 10 µm in length.

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

Energy/ keV
Figure 5. EDS spectra for the areas a (A) and b (B) indicated by thesquares in Figure 4.

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005 117

Accumulation of Cu and Its Oxidation State

A

B
(a)

(a)

(b)

Counts (arbitrary)

Counts (arbitrary)

(b)

(c)

(c)

(d)
(d)

-10

0

10

20 30
Energy/ eV

40

50

-10

0

10

20 30
Energy/ eV

40

50

Figure 6. Kα1 edge Cu XANES spectra (A) and their first derivates(B) for Cu in (a) the T. atrata mycobiont, (b) S. cerevisiae, (c) Cu(I)Cl,and (d) Cu(II)SO4. The energy calibration was performed to fix thepeak maximum of the first derivative of the spectrum for the metal Cu
to be 0 eV.

square areas, a and b, in Figure 4. We previously showed that
S was distributed in the T. atrata mycobiont cell aggregatesmost homogeneously among these 4 elements. 7 The peakheight of S at 2.31 keV in these two spectra was fixed to be thesame so that a comparison of the peak strength for P, K, andCu in these spectra could be easily performed. The peakheight of P was almost the same in areas a and b, while that of
K in area b was slightly larger than that in area a. The peakheight of Cu in area b was larger than that in area a.
XANES. Figures 6A and B show the Kα1 edge Cu XANES
spectra and their first derivatives for Cu in (a) T. atrata mycobiont, (b) S. cerevisiae, (c) Cu(I)Cl, and (d) Cu(II)SO4. Theenergy was calibrated by fixing the peak maximum of the first
derivative of the spectrum for the metal Cu to be 0 eV (datanot shown). As shown in Figure 6A, the spectrum shape of Cu
was quite different in each sample. The Cu in the T. atrata
mycobiont (Figure 6A(a)) and Cu(II)SO 4 (Figure 6A(d))
showed a peak at around 20 eV, while Cu(I)Cl showed a peakat around 5 eV (Figure 6A(c)). Two peaks at around 5 and 15
eV were observed in S. cerevisiae (Figure 6A(b)). On theother hand, the first derivative spectra (Figure 6B) showed a
couple of outstanding peaks. Three peaks at around 2.5, 7.5,
and 12.5 eV were observed in the T. atrata mycobiont (Figure
6B(a)), while the spectrum of Cu in S. cerevisiae had only one
marked peak at around 2.5 eV (Figure 6B(b)). The spectrumof Cu(I)Cl had a characteristic peak of monovalent Cu ataround 2.5 eV (Figure 6B(c)). The spectrum of Cu(II)SO 4
showed two large peaks of divalent Cu at around 7.5 and 12.5
eV (Figure 6B(d)).
4. Discussion
Rapid growth was observed in T. atrata mycobiont cell aggregates, even though Cu was accumulated in the aggregates,
implying that Cu did not seriously discourage growth. The
XANES study showed that the peak positions of Cu in the T.
atrata mycobiont were at the energies for monovalent (2.5 eV
for Cu(I)Cl) and divalent (7.5 and 12.5 eV for Cu(II)SO4) ions,indicating that Cu in the T. atrata mycobiont was a mixture ofmonovalent and divalent Cu. The peak position of Cu in S.
cerevisiae was at ~ 2.5 eV, indicating that the predominant Cu

species in S. cerevisiae was monovalent. The requirement of Cu
by microorganisms is usually very low. In contrast, the presenceof hydrated Cu(II) ions at higher levels is toxic to microbial
cells.8, 9 The toxicity of Cu to microorganisms, such as lichens10
and yeasts11 has been widely reported. These microorganisms
have detoxification mechanisms for Cu. Copper(II) with a hightoxicity is reduced to Cu(I) by glutathione (GSH).12 Copper
induces metallothionein (MT) in S. cerevisiae and Cu(I)-MT isformed 13 as well as Cu(I)-GSH. 14 Copper complexed withorganic substances shows less toxicity. As the predominant Cu
in S. cerevisiae was monovalent, Cu(II) incorporated in S. cerevisiae cells was rapidly reduced to Cu(I) by biosubstances suchas GSH. Monovalent Cu was observed also in the T. atrata
mycobiont. The photobionts of lichens produce phytochelatin in
response to exposure to heavy metals.15 Mycobionts play a rolein producing GSH,15 which then is converted to MT. MT, therefore, is ubiquitously found in fungi.16 These facts suggest that
the Cu accumulated in the T. atrata mycobiont as Cu(I) is also
bound to GSH and MT.
In contrast to Cu in S. cerevisiae, Cu in the T. atrata mycobiont
was both monovalent and divalent. Cervantes and GutierrezCorona17 reported that the ability of fungi to accumulate highlevels of heavy metals is due to the high capacity of their cellwall components, such as chitin and melanin, to adsorb themetals as well as to the effective storing inside cells, where themetals bind with GSH and MT. Fungal melanin has carboxyl
and hydroxyl functional groups that have a high affinity withheavy metals.18 Purvis et al.18 showed that the toxic heavy metal
U is strongly adsorbed on melanin in the cell walls of lichens.19
It is known that fungal melanin shows affinities in the order ofCu(II) > Ca(II) > Mg(II) > Zn(II). Moreover, the production ofmelanin in soil fungus is reported to be accelerated in responseto the exposure to Cu(II).20 The adsorption of Cu on the melaninin cell walls would effectively prevent Cu absorption into thecytoplasm. These facts can explain that the amount of Cu(I) in
the T. atrata mycobiont was small compared to that in S. cerevisiae, because the T. atrata mycobiont was in an aggregatedform and the cell wall components were well developed. Somefungal species produce a blue-colored mycelium in which Cu is
accumulated.21, 22 However, no blue-colored mycelium wasobserved in the T. atrata mycobiont. These findings suggestthat the Cu existing as Cu(II) in the T. atrata mycobiont adsorbs
to cell wall components such as melanin and chitin, providing
the T. atrata mycobiont with high tolerance to Cu.
The cell aggregates used had been pre-cultured in a medium
without Cu before use. Therefore, the SEM/EDS study signified that Cu reached the inner area of the cell aggregates within
24 hours. The concentration of Cu was higher in the inner part
than the outer part. This morphological observation showedthat the senescence of cell aggregates proceeded mostly in thecenter, resulting in production of a hollow there. In the part
where the senescence proceeds, cells lyse and only cell wallcomponents remain. These facts indicate that the Cu accumulated in the cell aggregates by adsorption on cell wall components such as melanin and chitin is divalent.
The high tolerance of the T. atrata mycobiont to Cu is
attributable to two mechanisms. The first is achieved throughthe reduction of Cu(II) to Cu(I) in the cytoplasm to store Cu(I)as low-toxicity Cu complexes, such as Cu(I)-MT and Cu(I)GSH. The second is the adsorption of Cu(II) on cell wallcomponents such as melanin. Rizzo et al.23 reported that the
toxic metals accumulated on melanin in fungi protect the fungi
from antagonistic microorganisms. This study showed thepossibility that one of the strategies of the T. atrata symbiont
to overcome the difficulty of living in a hostile environment isto protect the symbiont through the accumulation of toxic
metals on cell wall components of the T. atrata mycobiont,
which thereby limits the access of enemies to the symbiont.

118 J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005
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