Chemosphere 62 (2006) 14901499

www.elsevier.com/locate/chemosphere

Heavy metal ion inuence on the photosynthetic growth

of Rhodobacter sphaeroides
Livia Giotta a,*, Angela Agostiano b,c, Francesca Italiano b,
Francesco Milano c, Massimo Trotta c
a

Dipartimento di Scienza dei Materiali, Universita` degli Studi di Lecce, strada per Arnesano, 73100 Lecce, Italy
b
Dipartimento di Chimica, Universita` degli Studi di Bari, via Orabona, 4, 70126 Bari, Italy
c
CNR, Istituto per i Processi Chimico-Fisici, Sezione di Bari, via Orabona, 4, 70126 Bari, Italy
Received 13 January 2005; received in revised form 31 May 2005; accepted 12 June 2005
Available online 3 August 2005

Abstract
The potential of purple non-sulphur bacteria for bioremediation was assessed by investigating the ability of Rhodobacter sphaeroides strain R26.1 to grow photosynthetically in heavy metal contaminated environments. Bacterial cultures
were carried out in articially polluted media, enriched with the transition metal ions Hg2+, Cu2+, Fe2+, Ni2+, Co2+,
2

MoO2

4 , and CrO4 in millimolar concentration range. For each investigated ion the eect on growth parameters was
evaluated. The analysis of concentrationeect curves revealed a dierentiated response, indicating that diverse mechanisms of tolerance and/or resistance are involved. Adaptation or selection procedures were not applied, leading to
assess intrinsic abilities of coping with these contaminants. The microorganism proved to be highly tolerant to heavy
2+
metal exposure, especially towards Co2+, Fe2+ and MoO2

and Co2+ were found to decrease the cel4 . In addition Ni
lular content of light harvesting complexes. A characteristic behavior was observed with mercuric ions, which produced
a signicant increase of the lag-phase.

2005 Elsevier Ltd. All rights reserved.
Keywords: Purple bacteria; Heavy metal toxicity; Phototrophic growth; Concentrationeect curves; EC50; Heavy metal ion eects on
cellular pigments; Bioremediation

1. Introduction
The role of microorganisms in the mobilization and
immobilization of heavy metals in the environment
spans from the biogeochemical cycles to their biotechnological applications in bioremediation (White et al.,
1997; Gadd, 2000). Due to the increased interest in this
topic, the interaction between metal ions and microor-

Corresponding author. Fax: +39 0832 297282.

E-mail address: livia.giotta@unile.it (L. Giotta).

ganisms was widely investigated in the last 10 years.

No general rules can be derived from these investigations: the cellular response diers depending upon the
organism, the metal ion, its concentration and its oxidation state. A number of dierent uptake and resistance
mechanisms have been identied and recently reviewed
(Silver, 1996; Nies, 1999; Bruins et al., 2000; Valls and
de Lorenzo, 2002).
Because of the unique advantages of solar radiation
used as energy source, photosynthetic organisms are
particularly recommended to be employed in bioremediation processes devoted to the degradation or recovery

0045-6535/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2005.06.014

L. Giotta et al. / Chemosphere 62 (2006) 14901499

of pollutants from contaminated environments. The

potentialities of these organisms in terms of heavy metal
tolerance and biosorption abilities have been extensively
explored. Several investigations concerning the interactions between heavy metal ions and photosynthetic
microorganisms such as green algae (Chlamydomonas
reinhardtii (Danilov and Ekelund, 2001), Chlorella vulgaris (Rai et al., 1994a,b)), cyanobacteria (Spirulina platensis (Singh and Kumar, 1994), Microchaete tenera
(Zaccaro et al., 2001), Microcystis sp. (Pradhan and
Rai, 2001), Gloeothece magna (Mohamed, 2001), Nostoc
linckia (El-Enany and Issa, 2000)) and proteobacteria
(R. sphaeroides (Moore and Kaplan, 1992), Rhodospirillum rubrum (Watt and Ludden, 1999)) have been
reported. Inuence on photosynthetic activity and
pigment content was in some cases observed (Rai
et al., 1994b; Horne et al., 1998; Danilov and Ekelund,
2001; Zaccaro et al., 2001) even though the heterogeneity of the metal stress response suggests that the interaction cannot be conned to the sole photosynthetic
apparatus.
This paper represents a preliminary study on the
inuence played by metal ions on the photosynthetic
growth of the purple non-sulphur bacterium R. sphaeroides strain R26.1 (Feher and Okamura, 1978). This carotenoidless mutant, known for its widely investigated
reaction center, can grow aerobically in the dark while
its phototrophic growth requires anaerobic conditions
to avoid serious photo-oxidative damages triggered by
oxygen and light. In order to investigate the abilities of
this microorganism to grow photosynthetically in highly
contaminated environments, anaerobic cultures were
carried out in liquid media enriched with transition
metal cations such as Hg2+, Cu2+, Fe2+, Ni2+, Co2+
and oxyanions such as MoO2

and CrO2

4
4 . Growth
parameters were evaluated by tting the modied Gompertz equation to the growth curves. The analysis of
these parameters revealed a variable response and a generally high level tolerance to heavy metal contamination.
In addition, eects on the content of photosynthetic
pigmentprotein complexes were reported.

2. Materials and methods

2.1. Growth media
The Feher/Okamura group (San Diego) kindly provided cells of R. sphaeroides strain R26.1. Bacterial cells
were grown in the medium 27 of the German Collection
of Microorganisms and Cell Cultures. This medium,
containing heavy metal ions as trace elements, namely
Zn2+, Fe2+, Mn2+, Co2+, Cu2+, Ni2+ and MoO2

4 , was
termed plain. Before starting any experiment, cells
were plated out on plain medium in order to obtain a
fresh stock of bacteria ready to inoculate. Metal ion-

1491

contaminated media were obtained by dissolving the

appropriate amount of salts (NiCl2, CuCl2, HgCl2,
CoCl2, FeSO4, K2CrO4, Na2MoO4) into the plain broth.
Complete solubilization of Ni2+, Co2+ and Cu2+ was
achieved by using citric acid as a chelant in 1:1 molar
ratio with respect to the ion. The Fe2+ solution added
to the media was prepared using FeSO4, Na2SO4 and
citric acid with a 1:1:2 molar ratio, ushing the solution
with N2 to avoid rapid iron oxidation. Plain medium
supplemented with citric acid was used to check for
any chelant-related eect on the bacterial growth. Even
at the highest concentration achieved (50 mM) this organic acid did not inuence the bacterial growth ruling
out any chelant related eect.
2.2. Photosynthetic growth
The photosynthetic growth was monitored following
the changes in the absorption at 535 nm using a single
wavelength digital photometer (Mod. LP1W, Dr. Lange,
Germany). For these measurements R. sphaeroides cells
were grown into 8 ml glass vials (1 cm diameter) that tted in the cuvette-holder. The inoculated vials were
sealed and kept in the dark for 46 h for allowing the
consumption of residual oxygen and then exposed to
the light. The illumination of the cultures was achieved
by 100 W tungsten lament light bulbs placed at 25 cm
from the vessels (120 lE m
2 s
1). The pigment specic
absorption is minimal at 535 nm, so that only cellular
increment accounts for absorbance changes at this wavelength value. The linearity between sample absorbance
and bacteria concentration was checked counting the
viable cells on Petri dishes for dierent values of the culture turbidity, and was found to hold up to 0.6 absorbance units. Above this value a correction factor was
required. The relationship linking the absorbance of
the culture at 535 nm to the cellular concentration was
obtained tting a fourth order polynomial function to
the calibration curve with a Marquardt least-squares
algorithm (Bevington and Robinson, 1992).
The anaerobic growth curves were obtained by plotting the logarithm of the relative population size, ln(N/
N0), against the time elapsed from the light exposure.
The growth parameters, namely maximum growth rate
(lmax) and lag-phase duration (k), were determined tting the modied Gompertz equation (Zwietering
et al., 1990, 1992) to growth curves. In the modied
Gompertz equation:
ln

n
hl  e
io
N
A exp
exp max k
t 1 ;
N0
A

N is the cell concentration at the time t, N0 is the cell

concentration at the time 0 and A, equal to ln(Nst/N0),
is the asymptote of the curve, i.e., the logarithm of
the maximum relative population size reached at the

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L. Giotta et al. / Chemosphere 62 (2006) 14901499

stationary phase. The cell cultures behavior at death

phase was not investigated.

et al., 1985) and the traces normalized to the same protein concentration.

2.3. Toxicity evaluation

2.5. Pigment assay

For each heavy metal, cultures were carried out

simultaneously in media supplemented with dierent
ion concentrations and in the plain broth, used as control. The growth parameters observed into the plain
medium were used to estimate the maximum growth rate
ratio (lmax,metal/lmax,plain) and the population size ratio
at the stationary phase (Ametal/Aplain). These dimensionless parameters eliminate any eect on bacterial growth
(i.e., light intensity, temperature, inoculum condition,
etc.) other than heavy metal contamination, allowing a
direct comparison of dierent growth experiment data.
Moreover these normalized quantities, expected to range
from zero to one, are suitable as toxicity indicators and
plotted against the metal ion concentration in the
growth medium, provide the concentrationeect
curves. These plots were tted by the two-parameter logistic equation:

The total amount of chlorines, i.e., bacteriochlorophyll a and bacteriopheophytin a, was estimated extracting the photosynthetic pigments from the cells with
acetone. 1 N HCl was added to the extraction mixture
in a 1:1 volume ratio in order to convert any chlorophyll
molecules into pheophytin. The cell debris was sedimented by centrifugation and discharged while the
supernatant was evaporated under nitrogen stream.
The resulting aqueous suspension containing the pigments was re-extracted with diethylether. A visible spectrum was acquired on the ether fraction and the total
pigment content was estimated using 67.6 mM
1 cm
1
as extinction coecient for bacteriopheophytin at
749 nm (Smith and Benitez, 1955).

1

b ;
x
1
x0

x0 provides directly the EC50 value, i.e., the eective ion

concentration able to reduce the quantity under investigation by the 50% of the observed plain medium value. b
is correlated to the steepness of the curve and indicates
how eective is the pollutant concentration in inducing
its noxious eect once begun.
2.4. Spectrophotometric measurements
In order to check any eect on the photosynthetic
apparatus, optical spectra of R. sphaeroides R26.1 suspensions at the early stationary phase were recorded in
the wavelength range 6001000 nm using a double-beam
spectrophotometer Jasco 7800-S. For obtaining comparable data, the original spectra were subtracted of the
scattering and normalized to the same cellular concentration. The intensity of the protein-bound bacteriochlorophyll band at 865 nm was used to estimate the
amount of light harvesting (LH) complexes into the cells
(Horne et al., 1998).
Before recording the soluble cell extracts visible spectra, harvested cells were broken by two passages
through a French pressure cell at 8200 kPa. Heavy particles, included the photosynthetic membranes, were
sedimented at 150 000g for 90 min and the resulting
supernatant was analyzed spectrophotometrically. In
order to compare the spectra of soluble fractions isolated from dierent cultures, their total protein content
was determined by bicinchoninic acid assay (Smith

3. Results
3.1. Inuence on lag-phase, maximum growth rate
and population size
Addition of millimolar amounts of divalent cations
as cobalt, copper, nickel and iron produced a decrease
of both the maximum growth rate and the population
size at the stationary phase, while the lag-phase duration
appeared substantially unchanged. Growth curves obtained in presence of increasing concentrations of cobalt,
shown in Fig. 1a, depict this common feature. A completely dierent behavior was observed with mercuric
ions (Fig. 1b), which produced a signicant increase of
the lag-phase duration at concentration as low as
1 lM whereas no substantial eect on the growth rate
and on the cellular yield was observed up to
[Hg2+] = 0.03 mM. At [Hg2+] = 0.05 mM no increment
of the population size was observed even after 10 d of
light exposure. The complete set of growth parameters
data of all investigated ions is presented in Table 1.
2

The oxyanions CrO2

4 and MoO4 were found to cause
a slight increase of the lag-phase duration, even though
this delay was induced at rather high (above 0.5 and
50 mM, respectively) concentrations. Both the anions
proved to inuence the lmax, while the eect on the cellular concentration at the stationary phase was conned
to the chromium case. Interestingly molybdate enrichment of growth media at concentration as high as
60 mM did not produce a substantial decrease of the
population size at the stationary phase. It should tough
pointed out that in the case of slowly growing cultures,
the Gompertz equation gave high standard deviations,
particularly in estimating A, as in 50 and 60 mM molybdate cases.

L. Giotta et al. / Chemosphere 62 (2006) 14901499

Fig. 1. Growth curves of R. sphaeroides R26.1 in culture media

supplemented with Co2+ (a) and Hg2+ (b). Experimental points
were tted using the modied Gompertz equation (see text for
details).

3.2. Concentrationeect curves

Mo, Co, Ni, Cu and Fe are involved as essential elements in proteobacteria biochemical processes (Kassner
and Kamen, 1968). Concentrationeect curves for these
metals would show a detrimental eect also for concentration values signicantly lower than the optimal ones.
Heavy metal starvation eects, however, are not included in the purpose of this work and low concentrations were consequently not investigated.
The concentrationeect curves, showing the drop in
the maximum growth rate upon addition of the metal
ions under investigation, are shown in Fig. 2. The
EC50 values, summarized in Fig. 2h, ranged from
0.03 mM for mercury to 21 mM for molybdenum.
On the basis of the heavy metal ion inuence on
the growth rate the following toxicity scale was identi2
ed: Hg2 > Cu2 > CrO2

> Co2 > Fe2 >
4  Ni
2

MoO4 . The lmax and A EC50 values are summarized
in Table 2. With the exclusion of Hg, all investigated
chemicals show an A EC50 value signicantly higher
than the respective lmax EC50.
3.3. Eect on the photosynthetic apparatus
The optical spectra of R. sphaeroides R26.1 cellular
suspensions in the region 6001000 nm show the typical

1493

broad and intense absorption band at 865 nm due to

the overlapping of both light harvesting complex I
(LHI) and light harvesting complex II (LHII) bacteriochlorophyll molecules (B-875 and B-850 respectively).
The LHII complexes of this mutant lack of the putative
ligand of B-800 bacteriochlorophylls (Davidson and
Cogdell, 1981), therefore only a weak absorption peak
due to reaction center bacteriochlorophylls is detectable
at 800 nm. Fig. 3a shows the spectra, subtracted of the
scattering and normalized to the same cellular concentration (108 cells ml
1), of cultures grown into Ni2+
and Co2+ supplemented media. These heavy metal ions
produced a drop of the amount of pigmentprotein
complexes resulting in a lowering of the major peak
compared to unpoisoned cells. Extraction of total pigments from Ni2+ and Co2+ exposed cells allowed to better quantify the inhibitory eect of these ions. Fig. 3b
shows the spectra of bacteriopheophytin isolated from
Co2+ supplemented cultures following acidication of
the total pigment extract. The number of pigment molecules per cell was found to decrease from 77 104 to
69 104 units upon addition of 1 mM CoCl2 to the
growth medium, while a more dramatic drop down to
44 104 molecules per cell was observed exposing the
bacteria to a vefold higher concentration. The EC50
value obtained from pigment reduction was found
6 mM in the case of cobalt. Interestingly the porphyrin
Soret band of the soluble fraction of bacterial cells
exposed to Co2+ was found to increase simultaneously
with the decrease of chlorines in the photosynthetic
membrane. Fig. 3c shows the spectra of soluble fractions
isolated from plain and Co2+ supplemented cultures. No
substantial variation of pigment content was observed
for the remaining heavy metal ions.

4. Discussion
4.1. Heavy metal stress response
The method here described for the bacterial growth
parameters analysis gave a rich variety of data, which
represent a good starting point for further investigations. The use of the parameter EC50 instead of MIC
(minimal inhibitory concentration) allowed to assess
the heavy metal ion toxicity with the following advantages: (1) the uncertainly on each value was easily quantiable by regression procedures, (2) a toxicity parameter
was determined also for metals (iron, molybdenum)
which did not completely inhibit the bacterial growth,
even at the highest tested concentration, (3) the eect
on various aspects of the bacterial growth was evaluated
separately (growth rate, population size, pigment
content).
When grown photosynthetically, R. sphaeroides
R26.1 showed a signicant tolerance to relatively high

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L. Giotta et al. / Chemosphere 62 (2006) 14901499

Table 1
Complete set of data determined through analysis of growth curves of R. sphaeroides R26.1 in presence of heavy metal ions
Concentration (mM)

Lag-phase (h)

lmax (h
1)

lmax Ratio

A Ratio

Hg

0.001
0.005
0.01
0.03
0.05

71
56 1
58 1
53 1
132 17

0.067 0.003
0.062 0.003
0.061 0.002
0.065 0.003
0.06 0.03

3.68 0.03
3.53 0.02
3.56 0.02
3.52 0.02
ND
NG

1
0.92 0.08
0.90 0.08
0.96 0.10
0.88 0.10

1
0.85 0.05
0.89 0.05
0.85 0.05
ND

Cu2+

6 10
5
0.01
0.05
0.1
1

33
23
33
29

0.050 0.007
0.045 0.006
0.040 0.005
0.016 0.004

2.38 0.07
2.26 0.08
2.11 0.07
1.26 0.13
NG

1
0.9 0.2
0.8 0.2
0.32 0.13

1
0.88 0.13
0.76 0.11
0.33 0.07

CrO2

4

0.1
0.2
0.5
1
10

13 1
10 1
11 2
29 4
23 3

0.144 0.007
0.117 0.007
0.114 0.012
0.045 0.005
0.018 0.003

4.02 0.03
3.79 0.03
3.73 0.06
3.46 0.12
0.41 0.01
NG

1
0.81 0.08
0.79 0.12
0.31 0.05
0.12 0.03

1
0.79 0.05
0.74 0.07
0.57 0.08
0.027 0.001

Ni2+

8 10
5
0.1
0.2
0.3
0.4
1

31
41
31
31
21

0.113 0.005
0.127 0.006
0.117 0.006
0.077 0.004
0.055 0.004

3.33 0.03
2.80 0.03
2.74 0.03
2.44 0.03
2.01 0.04
NG

1
1.13 0.11
1.04 0.10
0.69 0.06
0.48 0.06

1
0.59 0.03
0.55 0.03
0.41 0.02
0.27 0.02

Co2+

8 10
4
0.1
0.2
0.5
1
2
5
10
20

31
61
71
51
42
15
23
05

0.122 0.008
0.084 0.004
0.082 0.006
0.079 0.007
0.060 0.005
0.046 0.008
0.041 0.004
0.025 0.004

3.44 0.03
3.34 0.03
3.13 0.05
3.14 0.05
3.21 0.08
3.22 0.16
2.47 0.07
1.86 0.10
NG

1
0.69 0.08
0.67 0.09
0.65 0.10
0.49 0.08
0.38 0.09
0.34 0.06
0.21 0.05

1
0.91 0.06
0.73 0.06
0.75 0.06
0.80 0.08
0.81 0.16
0.38 0.04
0.21 0.03

Fe2+

0.013
0.5
1
6
12
40

93
10 3
43
55
0 16
07

0.046 0.003
0.047 0.003
0.051 0.004
0.032 0.003
0.021 0.005
ND

4.28 0.09
4.29 0.09
4.62 0.10
4.46 0.13
3.4 0.3
2.17 0.10

1
1.04 0.15
1.13 0.17
0.70 0.11
0.46 0.15
ND

1
1.01 0.18
1.4 0.3
1.2 0.3
0.41 0.16
0.12 0.02

MoO2

4

1 10
4
1
5
10
15
20
30
50
60

91
61
81
41
03
03
10 3
74 32
54 61

0.118 0.008
0.093 0.003
0.096 0.004
0.094 0.005
0.063 0.006
0.056 0.006
0.063 0.006
0.026 0.010
0.020 0.010

3.84 0.04
3.85 0.03
3.67 0.03
3.97 0.04
4.00 0.08
3.86 0.10
3.84 0.09
4.3 0.6
5.6 1.4

1
0.79 0.08
0.81 0.09
0.80 0.10
0.54 0.08
0.48 0.08
0.54 0.09
0.22 0.10
0.17 0.10

1
1.00 0.07
0.84 0.06
1.14 0.09
1.17 0.15
1.01 0.14
0.99 0.13
1.12 0.17
1.5 0.4

Heavy metal ion

2+

Italic values in the rst row of each series of data represent the quantities relevant to the plain medium used as control. Only chromium
and mercury were not present as trace elements in the plain medium. ND indicates the numerical value that could not be determined.
NG indicates the absence of cellular growth.

concentrations of toxic ions. EC50 values for this purple

bacterium are generally higher than MICs found for

Escherichia coli (Nies, 1999). MIC values are by denition higher than the corresponding EC50 for the same

L. Giotta et al. / Chemosphere 62 (2006) 14901499

1495

Fig. 2. Concentrationeect curves relevant to the decrease of the maximum growth rate upon addition of Hg2+ (a), Cu2+ (b), CrO2

4
(c), Ni2+ (d), Co2+ (e), Fe2+ (f) and MoO2

4 (g) to the culture media. Plots were tted by using the two-parameter logistic equation (see
text for details). The histograms (h) show the growth rate EC50 for all investigated heavy metal ions.

Table 2
Comparison between the eective-concentration values (EC50)
able to reduce the growth rate (lmax) and the relative population size at the stationary phase (A) by 50% of the value
observed into the plain medium
Metal ion
2+

Hg
Cu2+
CrO2

4
Ni2+
2+
Co
Fe2+
MoO2

4

EC50 lmax (mM)

EC50 A (mM)

0.03 0.02
0.08 0.01
0.34 0.05
0.38 0.03
0.8 0.4
10 2
21 4

0.02 0.01
0.10 0.01
0.69 0.05
0.43 0.06
12 2
39 8
>60

organism; therefore our data clearly indicate that the

purple bacterium under investigation is more tolerant

to heavy metals than E. coli. It should be stressed that

this ability to cope with heavy metals is not the result
of a selection process, in contrast with highly tolerant
bacteria isolated from metal contaminated environments
(Rubikas et al., 1997; Hassen et al., 1998) or from naturally metal-rich habitats (Mengoni et al., 2001) where
steady stress conditions exercise a constant pressure towards specic mutations. In fact metal ion enriched
media were inoculated with R. sphaeroides R26.1 cells
growing exponentially into the plain medium, avoiding
any adaptation or selection procedures. Moreover the
response of R. sphaeroides R26.1 to heavy metal ion
exposure appeared signicantly variable depending on
the nature of the ion. The wide range of EC50 observed
indicates that dierent toxicity, tolerance and/or resistance mechanisms are involved. For divalent cations,
however, the toxicity appeared to increase upon decrease

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L. Giotta et al. / Chemosphere 62 (2006) 14901499

Fig. 3. (a) Qy transition absorption band of protein-bound bacteriochlorophylls of R. sphaeroides cells grown in plain medium (solid
line) and in media supplemented with Co2+ 5 mM (dashed line) and Ni2+ 0.5 mM (dash-dotted line). The scattering was subtracted and
the band intensity normalized to the same cell concentration (108 CFU/ml). (b) Visible spectra of total bacteriopheophytin extracted
from R. sphaeroides cells grown into plain medium (solid line) and in media supplemented with 1 mM (dash-dotted line) and 5 mM
(dashed line) CoCl2. The complete conversion of bacteriochlorophyll into bacteriopheophytin was performed by acidication. Each
trace, suitably normalized, represents the spectrum of the pigment isolated from 108 cells and solubilized in 1 ml of diethylether. (c)
Visible spectra of soluble fractions isolated from R. sphaeroides cells grown into plain medium (solid line) and in media supplemented
with 1 mM (dash-dotted line) and 5 mM (dashed line) CoCl2. The traces were normalized to the same protein concentration (1 mg/ml).
The cuvette path length was 1 cm in all spectra.

of the respective sulde solubility product Ksp, as expected by the increased ability to bind to the SH groups
of sensitive enzymes (Fig. 4).

Hg2+ poisoning experiments revealed a singular

behavior in terms of inuence on lag-phase and growth
rate. Lag-phase duration appeared strongly increased by

L. Giotta et al. / Chemosphere 62 (2006) 14901499

Fig. 4. Linear relation between the metal cation toxicity

indicator (lmax EC50) and the solubility product (Ksp) of the
relative sulde. The correlation factor was 0.91.

Hg2+ addition in spite of no substantial eect on the

growth rate. These ndings suggest that a resistance
mechanism, activated by Hg2+ ions and employing the
lag-phase interval to be expressed, might be involved.
Moreover the lag-phase duration increment upon increase of mercuric ion concentration may indicate the
course of a detoxication process, which becomes longer
the higher is the amount of ions to inactivate. Both
Gram-positive and Gram-negative bacteria demonstrated resistance to mercury and the biochemical reactions involved in the detoxication mechanism have
been extensively studied (Komura and Izaki, 1971; Summers, 1986; Brown et al., 2002). On the basis of the present data a similar process involving reduction of Hg2+ to
the less toxic and volatile Hg0 cannot be excluded for R.
sphaeroides R26.1.
Particularly interesting is the extremely low toxicity
observed for molybdate. The nding that no substantial
decrease of cellular mass at the stationary phase was detected even at concentration as high as 60 mM suggests
that an ecient resistance and/or tolerance mechanism
is involved for this anion. In spite of the high concentration in the growth medium, the molybdenum accumulation observed into the cells was poor (data not shown).
This is in agreement with a tight regulation of molybdate
transport and accumulation mechanisms reported for
other bacteria such as E. coli and Klebsiella pneumoniae
(Pienkos and Brill, 1981; Corcuera et al., 1993). For both
these organisms it was indeed observed that the higher is
the molybdate level in the medium, the lower are the rate
of transport and the accumulation extent. Since the three
genes modA, modB and modC, encoding for the proteins
involved in molybdenum regulation, were identied also
in R. capsulatus (Grunden and Shanmugam, 1997), the
hypothesis that a tight molybdenum uptake regulation
also exists in R. sphaeroides appears further justied.
The tolerance towards Co2+ also appeared particularly high compared to that of other microorganisms

1497

such as S. platensis (Singh and Kumar, 1994) or Pseudomonas aerouginosa (Hassen et al., 1998). Only Ni2+ and
Co2+ were found to be detrimental for the LH complexes in the microorganism under investigation. This effect appeared strictly associated to the decrease of the
population size reached at the stationary phase, indicating that the negative inuence on the photosynthetic
machinery may account signicantly for Ni2+ and
Co2+ toxicity. Pigment content evaluation revealed that
LH reduction is a consequence of bacteriochlorophyll
biosynthesis drop. Co2+-induced inhibition of pigment
biosynthesis (pyoverdin and pyocyanin) was also observed in P. aerouginosa (Hassen et al., 1998), suggesting
that this may be an eect shared by dierent proteobacteria. Although a survey of the mechanisms of heavy metal toxicity is beyond the scope of this discussion it is
likely that interference with magnesium transport and
regulation might explain Ni and Co eects on maximum
population size and pigment content. The CorA transport system is the primary Mg2+ inux pathway in Bacteria and Archea (Smith and Maguire, 1998; Moncrief
and Maguire, 1999) and the antagonistic eect of cobalt
on magnesium uptake through this transport system is
well documented in literature (Nelson and Kennedy,
1971; Park et al., 1976; Jasper and Silver, 1978; Gibson
et al., 1991). In addition the simultaneous increase of
intracellular porphyrins and Fe-porphyrins observed in
Co-supplemented cultures might be strictly related to
the reduction of bacteriochlorophyll synthesis. The
amount of protoporphyrin available at the branch point
for Fe-chelatase might be dependent on Mg-chelatase
activity (Walker and Willows, 1997; Schubert et al.,
1999), which in turn may be aected either by lack of
magnesium or cobalt abundance.
4.2. Perspectives for bioremediation
The response towards CrO2

4 is particularly interesting due to the extreme toxicity of this widespread contaminant (Wong and Trevors, 1988; Yassi and
Nieboer, 1988). The involvement of an ecient resistance mechanism for chromate in R. sphaeroides is suggested by both the high EC50 values and the lag-phase
lengthening induced by this anion at concentration
above 0.5 mM. The ability of this purple bacterium to
anaerobically reduce chromate to the less toxic Cr3+
was recently reported (Nepple et al., 2000) together with
the isolation of a cytoplasmic chromate reducing enzyme. This reducing ability linked to the high here reported tolerance makes R. sphaeroides an excellent
candidate for chromate bioremediation.
Zn2+ and Cd2+ biosorption properties of steam sterilized R. sphaeroides biomass have recently been investigated and the nature of active metal binding sites on the
cell envelop was identied (Seki et al., 1998). On the
other hand active intracellular sequestration to prevent

1498

L. Giotta et al. / Chemosphere 62 (2006) 14901499

exposure to essential cellular components is one of possible mechanisms for heavy metal resistance (Mallick,
2004) and many reports have been published showing
metal inclusions into the cell body (Ariskina et al.,
2004). In presence of such accumulation mechanisms,
living cells might show enhanced uptake activity compared to non-living biomass, rendering them more
ecient in bioremediation processes. The reports concerning heavy metal accumulation abilities of living cells
of R. sphaeroides are to date restricted to the case of
tellurite, selenite and rare earth metal oxides (Moore
and Kaplan, 1992; Bebien et al., 2001). The information
acquired by the present screening allows expecting that
metabolically active processes account also for the high
tolerance observed towards the ions investigated in the
present work. With regard to Co2+ and Ni2+ the bacteriochlorophyll biosynthesis inhibition represents an indirect proof that both ions do enter the cytosol. In
agreement with this preliminary nding, measurements
performed by inductively coupled plasma-atomic emission spectrometry indicated that signicant amounts of
nickel and cobalt are retrieved in R. sphaeroides biomass
harvested from supplemented media. The cell biomass
elemental analysis is currently under investigation in
our laboratory and will be presented in a further paper.
These data are therefore expected to elucidate the real
potential of this purple bacterium in biotechnological
processes devoted to the recovery of toxic metal ions.
On the other hand the tolerance observed towards heavy
metals allows to propose the employment of this photoetherotrophic bacterium in the degradation of organic
matter and/or organic pollutants in metal contaminated
environments.

Acknowledgements
The authors wish to thank Dr. Marina Mattoni, Dr.
Panajota Stavrou, Dr. Grazia Mancini and Dr. Elaisa
Sardella for their help. Thanks also to Prof. Gianluigi
Ercolani and Dr. Fabio Mavelli for helpful discussion.
This work was made possible thanks to the nancial support of the grants: Meccanismi Molecolari della Fotosintesi (FIRB-MIUR), Con-MIUR 2002 and Progetto
Giovani Ricercatori of the University of Bari (2001).

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