Hydrometallurgy 104 (2010) 518523

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Hydrometallurgy
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / h yd r o m e t

In situ bioremediation of contaminated soils in uranium deposits

S. Groudev , I. Spasova, M. Nicolova, P. Georgiev
University of Mining and Geology Saint Ivan Rilski, Soa 1700, Bulgaria

a r t i c l e

i n f o

Available online 16 June 2010

Keywords:
Uranium
Non-ferrous metals
Soil bioremediation
Uranium complexes
Sulphate-reducing bacteria
Iron-reducing bacteria

a b s t r a c t
Experimental plots consisting of acidic and alkaline soils heavily contaminated with radionuclides (mainly U
and Ra) and non-ferrous metals (mainly Cu, Zn, Cd and Pb) were treated in situ under real eld conditions
using the activity of the indigenous soil microora. This activity was enhanced by suitable changes of some
essential environmental factors such as pH and water, oxygen and nutrient contents of the soil. The
treatment was connected with solubilization and removal of contaminants from the top soil layers
(horizon A) due to the joint action of the soil microorganisms and leach solutions used to irrigate the soils
(mainly acidophilic chemolithotrophic bacteria and diluted sulphuric acid in the acidic soil, and various
heterotrophs and bicarbonate and soluble organics in the alkaline soil). The dissolved contaminants were
removed from the soil prole through the drainage soil efuents or were transferred to the deeply located
soil subhorizon B2 where they were precipitated as the relevant insoluble forms (uranium as uraninite, and
the non-ferrous metals as the relevant sulphides) as a result of the activity of the sulphate-reducing bacteria
inhabiting this soil subhorizon.
2010 Elsevier B.V. All rights reserved.

1. Introduction
The contamination of soils, sediments, surface and groundwaters
with uranium, its decay products and different heavy metals is a great
environmental problem in countries in which mining and processing
of uranium ores are applied. Such contaminants can result in
radioactive and chemical exposures that directly and indirectly lead
to disease.
The remediation of contaminated waters and sediments in the
uranium deposits is a common practice. Various methods based on both
active and passive treatment systems have been applied, including such
using different organisms, mainly bacteria and plants. In some cases, the
treatment is connected not only with the remediation of the
contaminated waters and sediments but also with the recovery of
some valuable components from them, e.g. of uranium and some
non-ferrous metals.
However, the remediation of contaminated soils located within or
near the uranium deposits is a more difcult problem. Traditional efforts
to manage such soils often focus on their removal by extraction followed
by off-site treatment/disposal as well as on the in situ monitored natural
attenuation or passive capping using the installation of clean, inert
material over the contaminated soil.
In situ management of contaminated sites is potentially less
expensive and less risky than ex-situ management (Knox et al.,
2008). In situ chemical leaching using various lixiviants (bicarbonate,

Corresponding author. Tel.: +359 28687396.

E-mail address: groudev@mgu.bg (S. Groudev).
0304-386X/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.hydromet.2010.02.027

mineral acids and some organic complexing agents) is applied to

solubilize uranium and other contaminants and in this way to remove
them from the soil (Osiensky and Williams, 1990; Phillips et al., 1995).
Such treatment is possible only in sites with suitable geologic and
hydrogeologic conditions and requires the construction of collector
systems that prevent the undesirable seepage of contaminated
pregnant solutions into the environment. In some cases such pregnant
solutions can be used as sources for recovery of uranium and other
metals. A system of this type was suggested by Phillips et al. (1995) to
concentrate uranium from contaminated soils by a two-stage process:
rstly, extraction of uranium from the soil with bicarbonate and then
precipitation of uranium from the soil efuents with U6+-reducing
microorganisms. In any case, the contaminated soil efuents must be
subjected to cleanup before recycling or other possible use.
Another type of in situ methods is connected with the immobilization of uranium and heavy metals inside the soil by converting them into
their least soluble or toxic forms or by encapsulation in solid products of
high structural integrity (Mulligan et al., 2001).
Some in situ remediation methods of the above-mentioned
two-types are based on the activity of some soil microorganisms
able to solubilize or precipitate uranium and other heavy metals
(Suzuki and Suko, 2006). This activity is enhanced by suitable changes
in the levels of some essential environmental factors such as pH, Eh,
and oxygen, water and nutrient contents in the soil. These changes are
achieved mainly by irrigating the soil with solutions able to solubilize
and maintain the contaminants in soluble forms or to facilitate their
precipitation in the soil (Groudev et al., 2005). In practice, the
remediation is achieved as a result of the joint actions of the soil
microorganisms and the relevant irrigating solutions.

S. Groudev et al. / Hydrometallurgy 104 (2010) 518523

Another group of in situ bioremediation methods is based on the

ability of some plants to accumulate uranium and heavy metals from
contaminated soils via their root systems (Hinchee et al., 1995).
Considerable portions of the bioaccumulated contaminants are then
transferred to the plant biomass located above ground. This biomass is
periodically removed and burned to ashes. These ashes are suitable for
disposal or can be used for recovering some valuable components.
Different variants of the above methods have been applied in several
countries, mainly in Northern America and Europe. Some data about the
application of such methods for remediation of contaminated soils in
two uranium deposits in Bulgaria are shown in this paper.
The uranium deposit Curilo, located in Western Bulgaria, for a long
period of time was a site of intensive mining activities including both
open-pit and underground techniques as well as in situ leaching of
uranium. The mining operations were ended in 1990 but until now both
the surface and ground waters and soils within and near the deposit are
heavily contaminated with radionuclides (mainly uranium and radium)
and toxic heavy metals (mainly copper, zinc, and cadmium). Some soil
plots located in this area were treated in situ by means of methods based
on the activity of the indigenous soil microora. This activity was
enhanced by suitable changes in the levels of some essential
environmental factors such as pH and water, oxygen and nutrient
contents in the soil.
The polymetallic Rossen deposit is located in the Vromos Bay area,
near the Black Sea coast, Southeastern Bulgaria. Some agricultural lands
located in this area have been contaminated with radionuclides (mainly
uranium and radium) and heavy metals (mainly copper, cadmium and
lead) as a result of mining and processing of polymetallic ores. Several in
situ methods were applied to treat contaminated soils in this area
(chemical leaching with different reagents such as bicarbonate, mineral
and organic acids, phytoremediation by herbaceous plants able to
accumulate some of the above-mentioned contaminants). Enhanced
natural attenuation consisting to periodic ploughing and addition of
organic substrates and sources of N and P to stimulate the growth and
activity of some indigenous microorganisms (mainly sulphate and
iron-reducing bacteria) able to reduce and precipitate uranium as
uraninite, and to precipitate the non-ferrous metals as the relevant
insoluble sulphides. Some of these methods (mainly the bicarbonate
leaching) were partially efcient and decreased the concentrations of
some contaminants and the toxicity of the soil but caused a negative
effect on its structure and composition, as well as on its microora. The
best results were achieved by an in situ bioremediation method based on
the enhanced activity of the indigenous soil microora.
The two eld operations described in this paper (in the Curilo
deposit and in the Vromos Bay area) are typical examples for in situ
bioremediation of acidic and alkaline soils, respectively.
2. Materials and methods
2.1. Soil plots in the Curilo deposit
Two soil plots located in the Curilo deposit consisted of a leached
cinnamonic forest soil and were 180 m2 in size each. The soil prole was
100 cm deep (horizon A, 30 cm; horizon B, 50 cm; horizon C, 20 cm) and
was underlined by intrusive rocks with a very low permeability. Data
about the chemical composition and some essential geotechnical
parameters of the soil are shown in Table 1. The contaminants were
located mainly in the upper soil layers (in the horizon A).
The soil treatment in both plots was connected with the initial
solubilization of contaminants. Water acidied with sulphuric acid to
pH in the range of 2.83.5 was used as leach solution. Periodically, this
solution was supplemented with ammonium and phosphate ions in
concentrations sufcient to maintain their concentrations in the soil
pore solution in the range of about 20 and 10 mg/l, respectively. The
irrigation rate and acidity of the leach solutions were adjusted in
connection with the levels of the local natural rainfall and temperature

519

Table 1
Characteristics of the soils in the plots in the Curilo deposit before and after the
treatment.
Parameters
Chemical composition, %
SiO2
Al2O3
Fe2O3
P2O5
K2O
N total
S total
S sulphidic
Carbonates
Humus
pH (H2O)
Net neutralization potential, kg CaCO3/t
Bulk density, g/cm3
Specic density, g/cm3
Porosity, %
Permeability, cm/h
Particle size, mm (%)
1.000.25
0.250.01
b0.01

Before treatment

After treatment

77.4
12.5
2.35
0.14
2.12
0.10
1.72
1.54
0.14
2.10
4.40
44.8
1.32
2.68
51
10.4

78.1
12.0
1.58
0.10
1.70
0.08
0.79
0.71
0.01
1.41
3.21
21.4
1.27
2.62
46
8.2

18.9
49.5
31.6

18.1
50.9
31.0

to maintain the water-lled porosity in the soil upper layers (mainly in

the horizon A) at about 60%, and the pH of the soil pore solution within
the range of about 3.03.5. This level of the soil moisture was the
optimum for the activity of the aerobic microorganisms inhabiting these
soil layers. The pH of the soil pore solution was higher than the optimal
values for the acidophilic chemolithotrophic bacteria but was still
suitable for their growth and activity. Lower pH values had a negative
effect on the soil structure and composition. The upper soil layers were
ploughed up periodically to enhance the natural aeration. The
contaminants dissolved in the rst plot were removed from the soil
prole through the soil efuents. Periodically, usually once per
13 weeks, higher irrigation rates were applied for ushing these
contaminants from the soil prole. The contaminants dissolved in the
second plot were transferred to the deeply located soil subhorizon B2
where they were immobilized as a result of the activity of the indigenous
sulphate-reducing bacteria. Water solutions of dissolved organic
compounds (lactate and acetate) and ammonium and phosphate ions
were injected through vertical boreholes to this soil subhorizon to
enhance the bacterial activity.
The owsheet included also a system to collect the soil efuents and
to avoid the migration of contaminants into the environment. The
system consisted of several ditches, boreholes and wells located in
suitable sites in the plots. The soil efuents collected by this system were
then treated by constructed wetlands located near the plots to remove
the dissolved contaminants. The wetlands were characterized by a
mixed (surface/subsurface) water owpath and by abundant water and
emergent vegetation and a diverse microora. Typha latifolia and
Phragmites australis were the prevalent plant species in the wetlands but
representatives of the genera Scirpus, Juncus, Eleocharis, Carex and Poa
were also present, as well as several algae.
2.2. Soil plot in the Vromos Bay area
A soil plot located in the Vromos Bay area consisted of a typical
cinnamonic soil and was 400 m2 in size. The soil prole was 95 cm deep
(horizon A, 30 cm; horizon B, 50 cm; horizon C, 15 cm). Data about
some essential parameters of the soil are shown in Table 2.
The contaminants were located mainly in horizon A. Solubilization of
these contaminants was achieved by means of a system in which a
mixture of solid biodegradable organic substrates (cow manure, plant
compost and hay) were added to the horizon A at the start of treatment
and after 6 and 8 months in amounts of 50 kg mixture/t dry soil. The

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S. Groudev et al. / Hydrometallurgy 104 (2010) 518523

Table 2
Characteristics of the soil in the plot in the Vromos Bay area before and after the
treatment.
Parameters
Chemical composition, %
SiO2
Al2O3
Fe2O3
P2O5
K2O
N total
S total
S sulphidic
Carbonates
Humus
pH (H2O)
Net neutralization potential, kg CaCO3/t
Bulk density, g/cm3
Specic density, g/cm3
Porosity, %
Permeability, cm/h
Particle size, mm (%)
N1.00
1.000.25
0.250.01
b0.01

Before treatment

After treatment

73.8
14.0
3.29
0.10
3.25
0.19
1.40
0.82
4.51
3.50
7.52
49.8
1.50
2.80
48
8.2

74.3
13.7
3.02
0.08
2.97
0.15
0.99
0.51
2.21
2.82
7.81
21.3
1.43
2.73
44
7.3

6.0
11.1
34.3
48.6

5.5
10.4
36.1
48.0

plant biomass consisted of about 44% cellulose, 28% hemicellulose, 21%

lignin and 7% ash. The soil was irrigated by water solutions containing
relatively low concentrations of sodium bicarbonate (0.0100.025 M),
as well as some nutrients (mainly ammonium and phosphate ions).
Periodic moulching of the soil was also applied which, together with the
higher irrigation rates and water-lled porosity (6570%), reduced
oxygen availability and maintained a slightly positive redox potential in
this soil horizon (in the range of about 150250 mV). System for
enhancing the in situ activity of the indigenous sulphate-reducing
bacteria in the subhorizon B2, as well as a system for collecting the soil
drainage efuents was also constructed. The soil efuents were treated
by means of a constructed wetland located near the soil plot.
2.3. Monitoring of the soil
A detailed sampling procedure was carried out to characterize the
soil and the subsurface geologic and hydrogeologic conditions. Surface
and bulk soil samples up to a depth of 2 m were collected by an
excavator. Drill hole samples were collected up to a depth of 8 m.
Elemental analysis of the samples was performed by digestion and
measurement of the ion concentration in solution by atomic absorption
spectrometry and induced coupled plasma spectrometry. Mineralogical
analysis was carried out by X-ray diffraction techniques.
The main geotechnical characteristics of the site, such as permeability
and wet bulk density, were measured in situ using the sand-core method
(U.S. Environmental Protection Agency, 1991). True density measurements were carried out in the laboratory using undisturbed core samples.
Such samples were also used for determination of their acid generation
and net neutralization potentials using static acidbase accounting tests.
The bioavailable fractions of the pollutants were determined by leaching
the samples with DTPA and EDTA (Sobek et al., 1978). The mobility of the
pollutants was determined by the sequential extraction procedure
(Tessier et al., 1979). The toxicity of soil samples was determined by the
EPA Toxicity Characteristics Leaching Procedure (U.S. Environmental
Protection Agency, 1990).
The isolation, identication and enumeration of microorganisms were
carried out by methods described elsewhere (Karavaiko et al., 1988;
Widddel and Hansen, 1991; Widdel and Bak, 1991; Groudeva and
Tzeneva, 2001; Hallberg and Johnson, 2001). The bacterial activity in situ
in the plots located in the Curilo deposit was determined by the technique

described by Karavaiko and Moshniakova (1971) with some modications (Groudev and Groudeva, 1993).
The ecotoxicity of the soils before, during and after the treatment
towards different test-organisms was determined by the methods
described earlier (Groudev et al., 2007).
3. Results and discussion
3.1. Soil treatment in the Curilo deposit
The leaching of contaminants in the horizon A in the plots located
in the Curilo deposit was efcient and within a period of 20 months
(including a 2-month pause in irrigation during the cold winter
months) their concentrations were decreased below the relevant
permissible levels (Table 3). The analysis of microora in this soil
horizon revealed that it was characterized by a rich diversity of
microorganisms (Fig. 1). The mesophilic acidophilic chemolithotrophic bacteria related to the species Acidithiobacillus ferrooxidans, At.
thiooxidans and Leptospirillum ferrooxidans were the prevalent microorganisms in the horizon A. These bacteria were able to oxidize the
sulphide minerals present in the soil and to solubilize their metal
components. The non-ferrous metals (Cu, Zn and Cd) were solubilized
mainly in this way and as the relevant free cations were removed from
the soil prole of the rst plot by drainage efuents. The hexavalent
uranium was readily solubilized by the sulphuric acid present in the
irrigating solutions or generated in the soil as a result of the oxidation
of sulphides, mainly of pyrite.
The tetravalent uranium was solubilized as a result of its prior
bacterial oxidation to the hexavalent state. The bacterial oxidation of
sulphides and tetravalent uranium was carried out by the well known
direct and indirect (via the oxidation of Fe2+ to Fe3+) mechanisms. The
oxidative activity of these chemolithotrophic bacteria and their ability to
x CO2 as a carbon source depended on some essential environmental
factors such as pH and temperature (Table 4), and the availability of
substrates, oxygen and nutrients (mainly sources of N and P). High
numbers of bacteria and efcient oxidation rates were achieved during
the rst 56 months of treatment when most of the sulphide sulphur
and soluble iron were still present in the soil (in the horizon A) and
considerable portions of sulphides were well exposed. The maintenance
of concentrations of dissolved oxygen higher than 56 mg/l in the pore
solution (by means of periodic ploughing and control of irrigation)
resulted in relatively fast growth and oxidation, while at concentrations
lower than 2 mg/l these processes were much slower. Concentrations of
ammonium and phosphate ions in the range of about 1525 mg/l for
Table 3
Contents of contaminants in the horizon A of the soil from the plots in the Curilo
deposit.
Parameters
Contents of contaminants [ppm]
Before treatment
After treatment
Permissible levels for soils with pH 4.15.0
Permissible levels for soils with pH b 4.1
Bioavailable fractions [ppm]
(a) By DTPA leaching
Before treatment
After treatment
(b) By EDTA leaching
Before treatment
After treatment
Easily leachable fractions, ppm
Before treatment
After treatment
Inert fractions, ppm
Before treatment
After treatment

Ra

Cu

Zn

Cd

68
8.0
10
10

510
65
65
65

190
35
40
20

215
48
60
30

4.6
0.4
1.5
0.5

12
1.4

105
10

5.3
0.7

41
5.0

19
1.2

120
10

5.1
4.6

50
45

35
4.1

28
3.5

0.6
0.04

25
1.9

15
1.7

0.35
0.01

68
3.7

62
4.8

1.0
0.02

21
18

28
25

0.35
0.30

Notes: The contents of radium are shown in Bq/kg dry soil or in Bq/l.

S. Groudev et al. / Hydrometallurgy 104 (2010) 518523

521

carried out also by sulphate-reducing bacteria as well as by some Fe3+reducing bacteria. Portions of iron and manganese were precipitated as
Fe(OH)3 and MnO2 as a result of the prior bacterial oxidation of Fe2+ and
Mn2+ to Fe3+ and Mn4+, respectively. Radium and portions of the heavy
metals and uranium were removed by sorption on the living and dead
plant biomass and on the clay minerals present in the wetland.
The contaminants dissolved in the horizon A of the second soil plot
were also transferred by the drainage waters to the deeply located soil
layers (in the subhorizon B2) but here they were precipitated as the
relevant insoluble forms (sulphides and UO2) as a result of the
enhanced activity of the indigenous sulphate-reducing bacteria. The
concentrations of contaminants in the efuents from this plot were
lower than the relevant permissible levels for water intended for use
in the agriculture and/or industry.
The monitoring of the soil toxicity revealed that it was connected
with the concentrations of contaminants in the soil pore solutions and
of their bioavailable fractions in the soil. The toxicity during the
treatment was initially increased in comparison with that before the
treatment. However, after reaching the maximum during the period
from the 3rd to the 8th month since the start of the treatment the
toxicity then steadily decreased and at the end of the treatment was
considerably lower than that before the treatment (Table 5). The data
from Table 3 revealed that the toxicity of this soil was due not only to
the radionuclides and heavy metals but also to the high acidity, which
was further increased during the treatment.
Fig. 1. Microorganisms in the Curilo soil before and during the treatment. Horizon A
pH before treatment 4.40; during treatment 4.403.21; subhorizon B2 pH before
treatment 5.32; during treatment 5.324.82; 1 aerobic heterotrophic bacteria; 2
acidophilic chemolithotrophs able to oxidize Fe2+ and/or S; 3 chemolithotrophs able to
oxidize S at pH 4 9; 4 nitrifying bacteria; 5 fungi; 6 anaerobic heterotrophic
bacteria; 7 sulphate-reducing bacteria; 8 Fe3+-reducing bacteria; 9 denitrifying
bacteria.

each of these ions were needed for the optimum growth and activity of
these bacteria. A portion of the complex uranyl sulphate anions formed
in the acidic leach solutions was absorbed on the positively charged
surface of the soil minerals, mainly iron oxides and alumosilicates.
Regardless of this, a considerable portion of uranium was also removed
from the soil prole through the drainage efuents. This was connected
with the saturation and passivation of the active sites on the mineral
surfaces.
The pregnant soil efuents from the rst plot were treated efciently
in the constructed wetland. The dissolved non-ferrous metals and iron
were precipitated mainly as the relevant insoluble sulphides by the
sulphate-reducing bacteria inhabiting the wetland. Uranium was zprecipitated mainly as uraninite (UO2) as a result of the reduction of U6+ to U4+
Table 4
Microbial activity in situ at different environmental conditions in the plots in the Curilo
deposit.
Sample and conditions of testing

Fe2+
oxidized for
5 days, g/l

14
CO2 xed for
5 days, counts/min.
ml (g)

Soil efuents with pH of 3.8 + Fe2+

(10 g/l) at 911 C
Soil efuents with pH of 3.2 + Fe2+
(10 g/l) at 911 C
Soil efuents with pH of 3.2 + Fe2+
(10 g/l) at 1618 C
Soil efuents with pH of 3.2 + Fe2+
(10 g/l) at 2123 C
Soil suspensions in K nutrient medium
(with 10 g/l Fe2+ and pH 3.8) at 911 C
Soil suspensions in K nutrient medium
(with 10 g/l Fe2+ and pH 3.2) at 911 C
Soil suspensions in K nutrient medium
(with 10 g/l Fe2+ and pH 3.2) at 1618 C
Soil suspensions in K nutrient medium
(with 10 g/l Fe2+ and pH 3.2) at 2123 C

0.511.40

15004100

0.912.84

26007700

1.544.21

440011200

1.906.44

500016200

0.601.61

15004400

0.993.05

28007900

1.454.86

440012500

2.057.11

550018100

3.2. Soil treatment in the Vromos Bay area

The system established in the horizon A of the soil plot in the
Vromos Bay area was favorable for the growth and activity of several
aerobic and facultatively aerobic microorganisms, including cellulosedegrading bacteria and fungi (Fig. 2). This resulted in an efcient
removal of radionuclides and heavy metals from this soil horizon
within 20 months of treatment (Table 6).
The biodegradation of biopolymers resulted in increasing the
concentrations of monomers (mainly monosaccharides and organic
acids) in the pore solution of the soil and maintained a relatively
stable pH in the system (in the range of about 7.58.0), regardless of
the irrigation with hydrocarbonates and the solubilization of
carbonates from the soil. In this system U4+ was oxidized to U6+ by
some heterotrophic bacteria producing peroxides. The hexavalent
uranium was solubilized as different complexes: mainly with
carbonate [UO2(CO3)2
and UO2(CO3)4
2
3 ], but also with carbonate
and calcium [CaUO2(CO3)2
and Ca2UO2(CO3)3] (Bernhard et al.,
3
2001) and with organic monomers such as carbonic and humic acids
(Finch and Murakami, 1999).
Radium also was solubilized as such complexes. The non-ferrous
metals (Cu, Zn, Cd and Pb) were solubilized mainly as complexes with
organic acids. Small portions of these metals, mainly of the lead, were
solubilized as complexes with chloride ions. It must be noted that
small portions of the non-ferrous metals in the soil were present as
Table 5
Toxicity of the soils from the plots in the Curilo deposit before and after the treatment.
Test-organisms

Bacillus cereus
Pseudomonas putida
Lactuca sativa
Trifolium repens
Avena sativa
Lumbricus terrestris

Toxicity
Before treatment

After treatment

After treatment

(pH 4.4)

(pH 3.2)

(pH 4.4 by lime)

40
30
40
40
30
20

40
40
40
50
40
10

80
100
NOEC at 100
NOEC at 100
90
60

Notes: The toxicity was expressed as the lowest observed effect concentration (LOEC)
at different contents (in wt.%) of contaminated soil in a mixture with a clean soil of the
relevant type; NOEC = no observed effect concentration.

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S. Groudev et al. / Hydrometallurgy 104 (2010) 518523

Table 7
Sulphate-reducing bacteria in the subhorizon B2 in the soil plot in the Vromos Bay area.
Sulphate-reducing bacteria

Cells/g dry soil

Before treatment

Desulfovibrio (mainly D. desulfuricans)

Desulfobulbus (mainly D. elongatus)
Desulfococcus (mainly D. postgatei)
Desufobacter (D. multivorans)
Desulfotomaculum (non-identied species)
Desulfosarcina (D. variabilis)
Desulfomonas (non-identied species)

10 10
0103
0102
0102
ND
101102
0101

During treatment
103106
103105
102104
103105
0101
102104
0102

Note: ND = not detected.

Fig. 2. Microorganisms in the Vromos Bay soil before and during the treatment.
Horizon A pH before treatment 7.52; during treatment 7.527.81; subhorizon B2
pH before treatment 8.02; during treatment 8.028.24; 1 aerobic heterotrophic
bacteria; 2 cellulose-degrading aerobic bacteria; 3 nitrifying bacteria; 4
chemolithotrophs able to oxidize S at pH 49; 5 fungi; 6 anaerobic heterotrophic
bacteria; 7 cellulose-degrading anaerobic bacteria; 8 sulphate-reducing bacteria;
9 Fe3+-reducing bacteria; 10 denitrifying bacteria.

the relevant sulphides. The decrease in the content of sulphidic

sulphur and the increase in the number of basophilic chemolithotrophs in the soil were indications for the role played by these bacteria
in the solubilization of metals. Most of them were related to the genus
Thiobacillus, mainly to the species T. thioparus and T. denitricans, as
well as to the species Halothiobacillus neapolitanus. These bacteria
oxidized the elemental sulphur which was generated as a result of the
chemical and electrochemical oxidation of sulphides and was
precipitated on the surface of these minerals as passivation lms
(Groudev et al., 2008). The removal of these passivation lms

Table 6
Contents of contaminants in the horizon A of the soil from the plots in the Vromos Bay
area.
Parameters
Contents of contaminants, ppm
Before treatment
After treatment
Permissible levels for soils with pH N7.0
Bioavailable fractions, ppm
(a) By DTPA leaching
Before treatment
After treatment
(b) By EDTA leaching
Before treatment
After treatment
Easily leachable fractions, ppm
Before treatment
After treatment
Inert fractions, ppm
Before treatment
After treatment

Ra

41
280
7.1
55
10
65

18
1.4
7.1
0.9
18
1.2
4.6
4.1

Cu

Zn

Cd

Pb

611
242
280

251
99
370

7.3
1.7
3

268
109
80

60
5

64
12

32
0.8
5.5 0.1

28
10

25
3.0

41
3.5

17
0.44
2.8 0.02

46
10

73
109
8.2
9.9

62
1.5
7.7 0.02

53
6.8

30
23

62
59

52
48

104
99

Notes: The contents of radium are shown in Bq/kg dry soil or in Bq/l.

1.4
1.2

enhanced the sulphide oxidation. The metal ions liberated during

this oxidation were maintained in solution as the above-mentioned
complexes.
The dissolved radionuclides and heavy metals were transported by the
drainage solutions into the more deeply located soil layers (in the
horizon B). In the top layer of this horizon, under anaerobic conditions and
a negative redox potential at levels relatively closed to zero, rich and
diverse populations of denitrifying and iron-reducing bacteria were
present. The ability of iron-reducing bacteria to reduce the hexavalent
uranium to the tetravalent state is well known (Karavaiko et al., 1988;
Lovley et al., 1991; Anderson et al., 2003). On the other side, the
denitrication is a process which maintains uranium in solution. This is
due to the fact that the nitrogen intermediates formed during this process
4+
(NO
to U6+ both directly and indirectly
2 , NO and N2O) oxidize U
(Osiensky and Williams, 1990; Senko et al., 2002). The indirect oxidation
is connected with the oxidation of Fe2+ to Fe3+ by these intermediates. As
a result of this oxidation, poorly crystalline Fe3+-oxide minerals are
formed and they are able to oxidize U4+ to U6+ more efciently than the
nitrogen intermediates. The rate of U4+ oxidation coupled to microbial
denitrication is increased when aqueous Fe2+ is added to a solution
containing U4+ and nitrite. The Fe3+-oxide minerals produced during
the chemical oxidation of Fe2+ by nitrite are poorly crystalline and
oxidized U4+ efciently.
Fe2+ ions were solubilized in the upper layers of the horizon B as a
result of the reduction of Fe3+-oxide minerals by the iron-reducing
bacteria. The injection of nitrite ions to these soil layers stimulated the
microbial denitrication and in this way decreased the uranium
precipitation (Elias et al., 2003; Finneran et al., 2002). However, more
intensive precipitation of uranium occurred in the deeply located soil
layers inhabited by iron and mainly by sulphate-reducing bacteria,
regardless of the fact that the CaU6+CO3 and some U6+organics
complexes are quite refractory to both chemical and biological
reduction (Brooks et al., 2003). In some cases sulphate-reducing
bacteria were able to reduce the U6+ present in some complexes but
these complexes were not degraded and U4+ remained in the solution.
In most cases the microbial sulphate reduction was enhanced by
injecting dissolved organic compounds such as lactate and acetate to the
deeply located soil layers. However, it must be noted that the high
contents of dissolved organic carbon (higher than 2530 mg/l) inhibited
the precipitation of uranium. It is well known that long-term
amendments of uranium contaminated soils with organic electron
donors result in reoxidation of the initially bioreduced uranium (U4+)
under reducing conditions (Mulligan et al., 2001). In such cases CaU
CO3 complexes are formed due to the increased concentrations of some
microbial metabolites, including hydrocarbonate ions. Furthermore, the
reduction of U6+ decreases when sulphate-reducing bacteria not able to
carry out an enzymatic reduction of this ion are dominant over the
iron-reducing bacteria (Vrionis et al., 2005; Ortiz-Bernad et al., 2004).
Considerable portions of the non-ferrous metals were precipitated in
the deeply located soil layers (mainly in the subhorizon B2) as the
relevant insoluble sulphides as a result of the activity of sulphatereducing bacteria (Table 7). Most of the uranium was precipitated as
uraninite also by these bacteria. It must be noted that relatively small

S. Groudev et al. / Hydrometallurgy 104 (2010) 518523

Table 8
Toxicity of the soils from the plot in the Vromos Bay before and after the treatment.
Test-organisms

Bacillus cereus
Pseudomonas putida
Lactuca sativa
Trifolium repens
Avena sativa
Lumbricus terrestris

Toxicity
Before treatment (pH 7.52)

After treatment (pH 7.81)

40
40
50
50
30
30

NOEC at 100
100
NOEC at 100
NOEC at 100
90
90

Notes: The toxicity was expressed as the lowest observed effect concentration (LOEC)
at different contents (in wt.%) of contaminated soil in a mixture with a clean soil of the
same type; NOEC = no observed effect concentration.

portions of uranium and non-ferrous metals were removed from the

percolating drainage waters by means of sorption on the soil particles
and by the hydroxides and oxides of iron and manganese present in the
soil. However, the sorption was an essential mechanism for the removal
of radium and lead from these waters. Small amounts of the insoluble
(Pb, Ra)SO4 were also detected in the soil. There was a clear tendency for
the initially adsorbed uranium and non-ferrous metals to be subjected
to reduction in the course of time.
In any case, the efuents from the soil prole still contained
dissolved radionuclides and heavy metals but in concentrations lower
than the relevant permissible levels.
The soil toxicity initially increased during the treatment but then
steadily decreased and at the end of the operation was much lower than
that of the contaminated soil before treatment (Table 8).
4. Conclusions
The monitored natural attenuation and liming of the acidic soils are
still the most largely applied in situ methods for treatment of soils
contaminated with radionuclides and heavy metals. However, the
application of in situ bioremediation based on the activity of the
indigenous soil microorganisms is steadily increasing under the form of
several variants. The treatment connected with solubilization of
contaminants and ushing the soil prole usually increases the toxicity
of the soil during the operation. It must be noted, however, that the
duration of such operations is much shorter than the long periods of
spontaneous natural leaching of the soils. Furthermore, regardless of the
fact that the natural leaching generates pregnant solutions with lower
concentrations, it is difcult to control the distribution of these solutions
and to avoid the contamination of other ecosystems. In any case, after
the end of the operation for removal or detoxication of the
contaminants, the treated soils are subjected to some conventional
melioration procedures such as liming (if necessary), grassing, moulching, addition of fertilizers and animal manure as well as periodic
ploughing and irrigation.
Acknowledgements
Parts of this work were nancially supported by the Bulgarian National
Fund Scientic Research under the project Center of Biotechnologies for
the Environment and Related Health Problems (CENBIOHEALTH).
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