Environ. Sci. Technol. 2000, 34, 1081-1087
An Integrated Method Incorporating
Sulfur-Oxidizing Bacteria and
Electrokinetics To Enhance Removal
of Copper from Contaminated Soil
GIACOMO MAINI,§ AJAY K. SHARMAN,§
GARRY SUNDERLAND,‡
CHRISTOPHER. J. KNOWLES,† AND
S I M O N A . J A C K M A N * ,†
IBS-Viridian Ltd., 114-116 John Wilson Business Park,
Thanet Way, Whitstable, Kent CT5 3QT, U.K., EA Technology
Ltd., Capenhurst, Chester CH1 6ES, U.K., Oxford Centre for
Environmental Biotechnology, Department of Engineering
Science, University of Oxford, Parks Road, Oxford OX1 3PJ,
and NERC Institute of Virology & Environmental
Microbiology, Mansfield Road, Oxford OX1 3SR, U.K.
The combination of bioleaching and electrokinetics for
the remediation of metal contaminated land has been
investigated. In bioleaching, bacteria convert reduced sulfur
compounds to sulfuric acid, acidifying soil and mobilizing
metal ions. In electrokinetics, DC current acidifies soil, and
mobilized metals are transported to the cathode by
electromigration. When bioleaching was applied to silt
soil artificially contaminated with seven metals and amended
with sulfur, bacterial activity was partially inhibited and
limited acidification occurred. Electrokinetic treatment of
silt soil contaminated solely with 1000 mg/kg copper nitrate
showed 89% removal of copper from the soil within 15
days. To combine bioleaching and electrokinetics sequentially,
preliminary partial acidification was performed by
amending copper-contaminated soil with sulfur (to 5%
w/w) and incubating at constant moisture (30% w/w) and
temperature (20 °C) for 90 days. Indigenous sulfuroxidizing bacteria partially acidified the soil from pH 8.1 to
5.4. This soil was then treated by electrokinetics yielding
86% copper removal in 16 days. In the combined process,
electrokinetics stimulated sulfur oxidation, by removing
inhibitory factors, yielding a 5.1-fold increase in soil sulfate
concentration. Preacidification by sulfur-oxidizing bacteria
increased the cost-effectiveness of the electrokinetic
treatment by reducing the power requirement by 66%.
Introduction
In the U.K. alone, 200 hundred years of industrialization has
resulted in an estimated 200 000 contaminated sites (1). A
significant number of these contain cocktails of toxic
chemicals that provide a hazard to both human health and
the environment. The Confederation of British Industry has
recently estimated the cost of remediating this contaminated
land at £ 20bn (1). The implementation of the U.K. 1995
* Corresponding author phone: +44 1865 281630; fax: +44 1865
281696; e-mail: [email protected].
§ IBS-Viridian Ltd.
‡ EA Technology Ltd.
† University of Oxford and NERC Institute of Virology & Environmental Microbiology.
10.1021/es990551t CCC: $19.00
Published on Web 02/17/2000
 2000 American Chemical Society
Environment Act, increases in landfill tax. and a commitment
by the U.K. government to build 60% of new houses on
previously developed sites (2) have increased the pressure
to develop effective remediation technologies. Over 60% of
sites are co-contaminated with metals and organics. The
metals are frequently found as complex mixtures. and many
former industrial sites contain highly toxic metals such as
cadmium, nickel. and arsenic. Therefore, in the U.K., as in
other countries, the potential for developing these sites
together with the possibility that metals may leach into
groundwater or enter the food chain through plant material
means that remediation of metal-contaminated land is a
priority. There are currently few comprehensive remediation
technologies available for such sites.
Within the mining industries, technologies have been
developed for the recovery of valuable metals from ores and
rock/soil materials. Indeed the use of bacteria in bioleaching
has become a prominent method of recovery (3). Naturally
occurring iron- and sulfur-oxidizing bacteria from mining
wastes have been isolated and strains selected for their ability
to solubilize metals from different substrates (4). In this
respect, the bacteria involved in bioleaching processes are
able to convert metal sulfides to their respective sulfates,
thereby transforming them from insoluble to soluble salts.
The metals can be recovered by washing and further
processing. This established technology is now being extended to investigate the leaching of contaminating metals
from soils as a bioremediation technique (5). The application
of mixed cultures of bacteria has proved successful in
mobilizing metals within different soils. Difficulties, however,
may be presented by inhibition of microbial activity due to
the mobilized metals, with the potential for the entire
bioleaching process to come to a halt (6). The presence of
anionic species is also known to inhibit sulfur oxidation as
pH values are reduced (7). The potential for metals to leach
from soils means that to avoid groundwater contamination
the process has either to be conducted ex situ, or, if in situ,
with a metal removal system.
Electrokinetics is an emerging engineering technique for
the remediation of contaminated land (8-10). The application of a direct current to soil, by insertion of electrodes,
leads to the generation of hydrogen ions at the anode and
hydroxyl ions at the cathode. These migrate into the soil
and the hydrogen ions can displace adsorbed metal ions
into the pore fluid of the soil. By manipulation of the
conditions surrounding the cathode, an acidic pH can be
maintained throughout the soil. Metal ions, once solubilized,
can be transported by electromigration through the soil and
recovered at the cathode. The process has been effective in
both model and real systems and has been applied to sites
in the United States (11) and in Europe (12).
The combination of bioleaching and electrokinetics has
the potential to overcome several of the limitations of the
individual techniques with the possibility that some of the
combined attributes may prove to be synergistic. In particular,
electrokinetics mobilizes metal ions provided their speciation
is appropriate. Metals as hydroxides or oxides may be
solubilized by the electrokinetic acidification, but those
present as insoluble sulfides, a common speciation in former
gasworks sites and mining wastes, will not be extracted by
this method. However, the bacteria involved in bioleaching
processes can convert metal sulfides to sulfates, thereby
enabling their solubilization and subsequent transport by
electromigration. In addition, the directional transport of
metal ions by electrokinetics is a useful complement to
bioleaching as solubilized metals can be removed at the
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TABLE 1. Characteristics of Standardized Silt Soil
parameter
pH
soil density (kg/dm3)
extractable phosphorus (mg/L)
particle size, diameter (mm)
cationic exchange capacity (me/100 g)
soil buffering capacity (g/kg) (calcd as the
amount of [H+] from sulfuric acid
required to acidify the soil to pH 2)
permeability, k (m/s)
copper concn (mg/kg)
calcium carbonate (%)
total sulfur (%)
total nitrogen (%)
organic matter (%)
organic carbon (%)
C:N ratio
value
8.1
1.350
24
<2
21.7
0.52
1.81 × 10-11
35.5
2
0.08
0.25
3.40
1.97
7.9:1
cathode for straightforward downstream processing. A further
advantage is the potential for preliminary bioleaching prior
to electrokinetics to reduce the overall time scale and cost
of electrokinetic remediation. By amendment of the soil with
inexpensive sulfur (below $ 250/tonne) and simply allowing
the soil bacteria to metabolize, predictable sulfate generation
and acidification can be achieved. Little intervention, in terms
of man hours, is required in comparison to acid washing of
soil with relatively more expensive and hazardous chemicals
(e.g. sulfuric acid). This preliminary bioleaching stage forms
an important part of the current study.
Electrokinetics together with bioremediation has been
used to effect the movement and degradation of phenol by
an industrial/government consortium in the United States
(13). The same consortium has developed this technology
up to a full-scale field test to move trichloroethylene (TCE)
into treatment zones in which it is degraded by reaction with
zerovalent iron (14). The introduction of nutrients into soil
by electrokinetics (15, 16) and movement of other organics
have also been investigated (17), but there has been little use
of combined techniques for remediation of metal contamination. The behavior of sulfur-oxidizing bacteria at acidic
pH values has been well studied (18), and the acidophilic
bacterium Thiobacillus ferrooxidans has been used for
combined electro/bioleaching processes for metal recovery
(19, 20). The paucity of data concerning microbial activities
in soil in electric fields led us, therefore, to investigate the
behavior of sulfur-oxidizing bacteria and heterotrophs in the
presence of a DC current. We established that, in the presence
of soil, both Thiobacillus thiooxidans-like organisms and
heterotrophs were protected from the otherwise deleterious
effects of the electric current and indeed stimulation of
metabolic activity was observed (21).
The purpose of the current study is to combine soil
acidification by indigenous sulfur-oxidizing bacteria with
electrokinetic remediation for removal of copper ions from
contaminated soil.
Experimental Section
All studies were conducted using a standardized silt soil
obtained from Wye College, University of London (see Table
1 for characteristics). The soil was contaminated by spraying
a solution of metals onto the soil and mixing to effect a
homogeneous contamination. For preliminary studies, a
cocktail of six metals was used, added as nitrates, at
concentrations of 10 mg/kg Cd, 500 mg/kg Cr, 500 mg/kg
Cu, 200 mg/kg Ni, 2000 mg/kg Pb, and 2000 mg/kg Zn, plus
200 mg/kg molybdate. These concentrations were chosen
such that the soil was characterized as contaminated
according to U.K. government guidelines (22). Following these
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preliminary bioleaching experiments with static soil and soil
slurries, it was decided to focus upon a single contaminating
metal for electrokinetic studies. This would enable a clearer
elucidation of the complementary effects of bioleaching and
electrokinetic metal removal. Copper was chosen as the
contaminant and applied as 1000 mg/kg copper nitrate.
Static Soil Experiment. Contaminated and uncontaminated soils were amended with sulfur (mesh size 50) at two
different concentrations (0.5 and 5% w/w) by slow addition
of sulfur with constant mixing. Water was added to 30% (w/
w). Soils were set aside in trays with a soil depth of 10 cm
at constant temperature (20 °C) and moisture (maintained
by regular dry weight measurement and addition of water)
for up to 42 days. After 21 and 42 days, samples were taken
and analyzed for pH (by suspending 1 g of soil in 10 mL of
water) and the concentration of sulfate. Sulfate analysis was
by addition of 10 mL of 0.1 M HCl to 1 g of soil and mixing.
Following centrifugation, barium chloride was added to
samples, precipitating barium sulfate, which was analyzed
by spectrophotometry.
Soil Slurry Experiment. Soil slurries were prepared from
contaminated and uncontaminated silt soil amended with
sulfur and added to deionized water at 30% (w/v) to give a
total volume of 300 mL in 500 mL conical flasks. Slurries
were incubated with shaking (160 rpm) at 30 °C, and samples
were taken for analysis of pH and sulfate concentration.
Electrokinetic Studies. The apparatus for performing
electrokinetics is outlined in Figure 1. A soil cell system was
constructed with six individual compartments each being
6.5 cm long between anode and cathode, 3 cm wide and 4
cm deep, and containing approximately 150 g of moist soil.
This arrangement allowed samples to be taken at a range of
time points. Within each compartment, soil could be sliced
into five centimeter-wide sections to enable measurements
of pH, moisture content, and metal ions. pH was determined
by suspending 1 g of soil in 10 mL of water and using a
standard pH meter. Moisture content was determined by
drying soil at 110 °C for 24 h and comparing wet and dry
weights. Metal ions in soil were analyzed by Electron
Diffraction X-ray Fluorescence (ED-XRF; CPL Laboratories,
Derbyshire, U.K.). Anodes were of carbon felt material (8.5
cm wide, 7 cm deep, and 1.5 cm thick), and stainless steel
mesh (14 cm long and 5 cm wide folded in the center) was
used for cathodes. Each cathode was located in a compartment (8.5 cm wide, 7 cm deep containing approximately 80
mL of liquid) bordered by a semipermeable membrane to
allow ions and water to pass from soil to cathode. This enabled
a catholyte solution to be contained within the cathode
compartment and recirculated through an ion exchange
column for recovery of metals. The total volume of the
catholyte system was approximately 500 mL with a recirculation rate of approximately 5 mL/min. A pH control unit
was incorporated to maintain an acidic pH (pH 4.5) in this
compartment through addition of acetic acid. An aqueous
feed was attached to the anode, and electrical connections
were attached to a direct current power pack.
A first series of experiments used silt soil contaminated
with 1000 mg/kg Cu(NO3)2 and adjusted to 30% (w/w)
moisture. The experiment shown here is a representative of
a series of three studies which were conducted under identical
conditions and yielded the same results. A current density
of 3.72 A/m2 electrode surface area was applied at a constant
current with 8 M acetic acid added to the catholyte to
maintain an acidic pH, and 0.05 M H2SO4 was added to the
anode at 60 mL/day.
In a second set of experiments, the same soil was amended
with 5% (w/w) sulfur and 30% (w/w) moisture and allowed
to incubate for 90 days at 20 °C prior to electrokinetics. Two
electrokinetic experiments were conducted using this soil:
one with 3.72 A/m2 current density and the other with the
FIGURE 1. Electrokinetic apparatus. The power pack was able to deliver up to 2 A and 60 V. Acid reservoir contained 8 M acetic acid.
The pH control unit was set to add acetic acid when the pH in the catholyte reservoir, as measured by a pH probe, increased above pH
4.5. Recirculating pumps were used to maintain a flow of catholyte through the ion exchange column and the mixing reservoir at approximately
5 mL/min. Anolyte feed contained either 0.05 M sulfuric acid (first run) or water (second run) and was supplied at approximately 60 mL/day.
The box containing the anode and cathode was also filled with soil, and the movements of water and metal ions are shown.
TABLE 2. pH and Sulfate Contents of Metal-Contaminated and
Uncontaminated Soils Amended with 0.5 and 5% (w/w) Sulfur
(S) and Incubated at 20 ˚C at 30 % (w/w) Moisturea
21 days
42 days
sample
pH
sulfate
mg/kg soil
pH
sulfate
mg/kg soil
0.5% S, uncontaminated
0.5% S, contaminated
5% S, uncontaminated
5% S, contaminated
control
4.87
6.46
5.01
6.28
7.78
4910
820
5120
1640
40
4.31
6.39
3.38
7.05
7.74
8590
1200
13770
1320
0
a The control soil was not amended with sulfur but was maintained
at constant (30% w/w) moisture for the duration of the experiment. The
pH of the soil at the start of the experiment was 8.1.
current density increased to 7.44 A/m2 after 7 days. In both
cases, 8 M acetic acid was added to the catholyte, and
deionized water was added to the anode at 60 mL/day.
Metal was recovered using a column packed with ionexchange resin (Amberlite IRC-718). The column was replaced when copper adsorption became visible. Copper was
eluted from the ion-exchange resin using a 15% (v/v) HCl
solution and analyzed by Atomic Absorption Spectroscopy
(AAS). The total run times were 15 and 16 days for the first
and second experiments, respectively. Soil metal concentrations were determined by ED-XRF.
Results
Static Soil Experiment. Contaminated and uncontaminated
silt soils were amended with sulfur at 30% (w/w) moisture
and incubated (Table 2).
In the case of soil that had not been contaminated by
addition of a cocktail of metals, sulfate was produced, and
the pH was reduced, whereas with metal-contaminated soil,
sulfate production was lower and the pH of the soil was not
as much reduced. With 5% sulfur amendment, there was a
10-fold difference in the level of sulfate production between
contaminated and uncontaminated samples, which was
accompanied by a pH difference of approximately 3.7 units.
Soil Slurry Experiment. Silt soil was amended with sulfur
to 5% (w/w) and made into a slurry at 30% (w/v). The results
in terms of the change in pH and sulfate production for metalcontaminated and uncontaminated soils are shown in Figure
2.
The pH profile shows that in uncontaminated soil
amended with 5% (w/w) sulfur acidification to approximately
pH 1 could be achieved in 55 days. However, when this soil
was contaminated with metals, the pH was only reduced to
pH 2.5 over this time scale. Amendment of uncontaminated
and contaminated soils with 0.5% (w/w) sulfur (results not
shown) led to acidification to pH 4 in both cases. Sulfate
generation above 2000 mg/kg was only seen with uncontaminated soil amended with 5% (w/w) sulfur. In all other
flasks in which sulfur was added, sulfate generation reached
1000-1500 mg/kg. This lower level of sulfate was sufficient
to reduce the pH of the soil to between 2.5 and 4.5, but it was
not sufficient for further acidification. Separate analyses of
microbial populations by plating techniques indicated that
during the transition from pH 8 to pH 4, the predominant
species of sulfur-oxidizing bacteria present were neutrophilic
in terms of their growth pH (S. A. Jackman, unpublished
results), whereas below pH 4, acidophilic bacteria, similar to
Thiobacillus thiooxidans, predominated (21). It has been
demonstrated that at lower pH values metal ions such as
copper and zinc become soluble as ionic species in soil (23).
At this point, therefore, their toxicity to soil bacteria may
increase. Nitrates and other anions are also more toxic to
sulfur oxidizing bacteria at low pH values, due to their
protonation and potential movement into cells, destroying
the membrane potential, ∆ψ (7). These toxic effects could
cause the reduction in sulfur metabolism by indigenous
sulfur-oxidizing bacteria under these conditions.
As a link to the electrokinetic experiments, which were
performed upon soil containing a single metal contaminant,
this copper-contaminated soil (1000 mg/kg copper nitrate)
was tested in a soil slurry (5% w/v) containing 10% (w/w)
sulfur. Similar profiles for pH and sulfate generation were
found as with the soil contaminated with a cocktail of seven
metals.
Electrokinetic Experiments. Standard silt soil contaminated to 1000 mg/kg with Cu(NO3)2 was subjected to
electrokinetics. No sulfur was added to the soil, and therefore
no bacterial sulfur oxidation would be expected. The data in
terms of metal recovery at the cathode and overall pH of the
soil are displayed in Figure 3.
Over 90% of copper ions were removed at the cathode
within 15 days of initiation of the electrokinetic treatment.
Similarly, there was removal of 70-90% of the calcium and
manganese. Analyses of magnesium and iron were also
performed, but their movement was negligible under the
electric field. The soil temperature was between 25 and 35
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FIGURE 2. Acidification and sulfate production in silt soil slurries amended with sulfur. Uncontaminated silt soil was compared to soil
contaminated with a cocktail of seven metals. Data points for uncontaminated soil are shown by solid symbols and those for contaminated
soil by open symbols. Each flask (500 mL) contained 300 mL of 30% (w/v) soil slurry not amended with sulfur (diamonds) or amended with
5% w/w sulfur (squares). Solid lines are for pH values and dotted lines for sulfate concentration.
FIGURE 3. Metal recovery from the catholyte and the change in pH of the soil during electrokinetics. Recoveries are expressed as a
percentage of the starting metal concentrations in the soil. Copper ions are shown as triangles, calcium as squares, and manganese as
circles. pH is shown in diamonds with dotted lines.
°C. The operating voltage remained at an average of 12.2 V
for the first 9 days of the experiment, rising to an average of
38.0 V for the remaining 6 days. The operating current was
100 mA, and the average voltage over the whole experiment
was 23.0 V. Soil moisture content was maintained at between
20 and 25% (w/w) over the course of the experiment. Water
balance determinations showed that 91% of water added to
the apparatus, in terms of soil moisture and liquid added to
the electrodes, was recovered at the end of the experiment.
The pH and copper profiles across the soil, which was
divided into five centimeter-wide sections from anode to
cathode, were determined and are shown in Figure 4.
The mass balance for copper was 100.1% as recovered
within soil samples (assayed by ED-XRF) plus from the
catholyte by ion exchange (assayed by AAS). Addition of
sulfuric acid (approximately 86 mg/kg soil) at the anode was
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significantly below the buffering capacity for the soil,
indicating that the acidic pH was as a result of the electrokinetics. Measurement of chloride and acetate concentrations
demonstrated migration of these ions toward the anode.
Chloride concentration was reduced from 818 mg/kg to less
than 30 mg/kg across the soil within 24 h. Acetate concentration in the soil increased as the ion migrated into it from
the catholyte. After 7 days, concentrations of approximately
10 000 mg/kg were determined across the whole area between
the anode and cathode.
A second experiment was conducted, also with silt soil
contaminated with copper nitrate to 1000 mg/kg and with
the moisture adjusted to 30% (w/w), but, in this case, sulfur
was added to 5% (w/w). In this experiment, the soil was
preincubated for 90 days at 20 °C prior to the application of
DC current, during which time the pH of the soil reduced
FIGURE 4. Copper concentration and pH profile across soil during electrokinetics of silt soil contaminated with 1000 mg/kg copper nitrate.
Individual time points are shown for time 0 (squares), 2 days (diamonds), and 15 days (circles). Solid lines are for copper concentration
and dotted lines for pH.
FIGURE 5. Sulfate concentration across soil during electrokinetics.
The soil was sectioned into five equal portions, the data for which
are shown at 1-cm distances from the anode. Results (( SEM) are
displayed for time 0 (squares), 3 days (diamonds), 10 days (circles),
and 16 days (triangles).
from 8.1 to 5.4 due to the activity of the sulfur-oxidizing
bacteria. Average sulfate production was 3101 mg/kg soil.
After the incubation period, the soil was packed into an
electrokinetic reactor, and a DC current was applied under
the same conditions as for the previous experiment with the
exception that water was added to the anode as opposed to
sulfuric acid. Copper removed from the soil was collected by
ion exchange, and soil samples were taken during the course
of the experiment. Analyses of the soil pH and copper
concentration revealed very similar profiles to those displayed
in Figure 4, with pH reduced to below 3.0 in 9 days. The
average copper concentration across the soil was 114 mg/kg
after 16 days. Soil moisture content was maintained at
between 24 and 31% (w/w), and the soil temperature was
between 20 and 27 °C over the course of the experiment. The
applied voltage remained at an average of 6.3 V for the course
of the experiment, apart from a peak of 28.2 V at day 8. Overall,
the average voltage was 7.7 V, with an operating current of
100 mA. Figure 5 shows the sulfate profile across the soil
during electrokinetics.
The removal of copper from the soil at the cathode was
80% after 10 days and 86% after 16 days treatment. The final
mass balance for copper was 108%. Calcium and manganese
were also readily mobilized and removed from the soil,
whereas iron and magnesium showed negligible movement.
Sulfate appeared to concentrate within the center of the
electrokinetic apparatus, but, as the electrokinetics progressed, it moved toward and into the anode compartment.
Initially, the soil contained 3101 mg/kg sulfate. After 10 days,
the level increased to an average of 15 939 mg/kg, an increase
of 5.1-fold. Within this same 10-day period, the average
copper concentration in the soil was reduced from 1000 mg/
kg to 186 mg/kg. After this time, the sulfate content of the
soil reduced to an average of 6417 mg/kg after 16 days as the
sulfate migrated into the anode compartment. Movements
and concentrations of chloride and acetate were similar to
those in the previous experiment.
An identical experiment in which the relative current
density was increased to 7.44 A/m2 after 7 days led to greater
movement of sulfate ions toward the anode such that the
soil was cleared to less than 1000 mg/kg sulfate between the
electrodes.
The power consumption for each of the electrokinetic
runs could be calculated from the applied voltage and current
and the run time. For the first experiment the total electrode
surface area was 0.0162 m2. With an operating current of 0.1
A, this yields a current density of 6.2 A/m2. The average applied
voltage was 23 V, over a distance of 6.5 cm between the
electrodes. Assuming a linear increase in voltage at increasing
distance between the electrodes, this gives a voltage of 354
V/m. Power consumption was therefore 786 kWh/m3. At a
power cost of $0.05/kWh and total run time of 15 days,
assuming 1 m3 of soil is approximately 1.5 tonne, the total
cost of metal removal was therefore $26.2 per tonne. When
the soil was preincubated with sulfur-oxidizing bacteria,
power consumption was 281 kWh/m3, yielding a cost of $9.3
per tonne, a cost reduction of 66%. The cost of sulfur, at
below $250/tonne, and a relatively low input in terms of
man hours for monitoring and sampling do not greatly affect
this cost reduction.
Discussion
The potential of combining bioleaching by sulfur-oxidizing
bacteria with electrokinetics for metal mobilization and
recovery has been investigated. Initial experiments with sulfur
amendment of contaminated soil demonstrated that acidification was partially inhibited in comparison with uncontaminated soil in both static soil experiments and in soil
VOL. 34, NO. 6, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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slurries. Static experiments showed continuing production
of sulfate and reduction in pH in uncontaminated soil up to
42 days. In comparison, after 21 days, there was no further
increase in sulfur oxidation in contaminated soil amended
with 5% sulfur. In soil slurries, enhanced sulfur oxidation
and acidification might be expected due to increased aeration
and dilution of the soil with water. While a reduction in pH
to below pH 3 was observed, in comparison to no significant
pH change in static experiments, sulfate production remained
below 2000 mg/kg indicating that inhibition of bacterial
activity was still occurring. Inhibition of bacterial metabolism
by metal ions has been studied for a wide range of bacteria
(23), and with acidophilic sulfur-oxidizing bacteria, copper
and other ions have been shown to be inhibitory (24),
although the concentration of copper in this experiment may
not have been sufficient to produce the level of inhibition
observed. Anions, especially nitrate, have also been shown
to be inhibitory at acidic pH values (7). Nitrate concentrations
were significant in these experiments, and therefore this anion
could be a significant contributor to the inhibition of bacterial
metabolism. The pH change from neutral to pH 4-5 was
accompanied by an alteration in the populations of indigenous sulfur-oxidizing bacteria in the soil. Neutrophiles were
replaced by acidophiles. This pH change also affected the
solubility of contaminating metal ions, with those such as
copper and zinc becoming more soluble. The emergence of
the acidophilic bacteria was therefore combined with the
mobilization of inhibitory metal ions and the increased
toxicity of anions. The acidification process therefore slowed
markedly.
Electrokinetic treatment of the soil generated protons at
the anode and hydroxyl ions at the cathode. While protons
entered the soil and migrated toward the cathode by
electromigration, hydroxyl ions were titrated at the cathode
with acetic acid to maintain the pH at 4.5 in this compartment.
This process led to the soil being rapidly acidified to
approximately pH 2 within a period of 9 days. At the same
time, mobilized copper ions migrated toward the cathode
by electromigration and entered the cathode chamber where
they were pumped through an ion exchange column.
Removal of copper was 85% complete within 15 days when
soil had not been preacidified due to the activity of sulfuroxidizing bacteria. For preacidification experiments, a time
scale of 90 days was chosen for incubation with sulfur, to
maximize sulfate production and acidification based upon
the results of earlier experiments. The observation that, in
static experiments, bacterial activity had ceased after 21 days
in the presence of seven contaminating metals, whereas in
uncontaminated soil, activity continued up to 42 days (and
possibly beyond) meant that in studies with a single
contaminating metal, a time scale of 90 days might be
expected to ensure maximal sulfate generation and acidification. When the soil was allowed to preacidify, there were
two marked differences in the overall run parameters. First,
copper removal was achieved with much lower power
consumption. The pH of the soil had already been reduced
by 2 units due to sulfur oxidation, and therefore the amount
of hydrogen ions required to acidify the soil to pH 2 and the
power required to generate these ions were reduced. In
commercial application in a field situation, the cost of power
is a major component of the overall cost of the process. In
addition, any reduction in the time scale for electrokinetic
remediation will also affect the cost in terms of manpower
and requirements for equipment. By amending soil at an
earlier stage with little further intervention, the soil can be
prepared for electrokinetic treatment.
A second benefit of the preacidification experiment is
that sulfate production by sulfur-oxidizing bacteria was
significantly stimulated when the electric current was applied.
One of the effects of the electrokinetics was to enable
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acidification of the soil below pH 4.5. This appeared to
overcome the resistance which the sulfur-oxidizing bacteria
encounter in attempting to reduce the pH of the soil to below
pH 3. The rise in sulfate production accompanied removal
of copper ions from the soil by electrokinetics. Experimental
studies have also demonstrated that nitrate is rapidly removed
(by electromigration toward the anode) from silt soil by
electrokinetics (S. A. Jackman, unpublished results), although
it is also possible to speculate that microaerophilic sulfur
oxidizers were able to reduce nitrate in the soil, thereby
relieving its inhibitory effects. It is possible that this removal
of inhibitory metals and anions was sufficient for the activity
of the sulfur-oxidizers to be stimulated. However, the rate of
sulfate production in the soil reached an average of 894 mg/
kg/day between days 3 and 7 and 1311 mg/kg/day between
days 7 and 10. In comparison, sulfate production in uncontaminated soil in the static soil experiment was only 328
mg/kg/day. Clearly, the rate of sulfate production was
significantly increased in the presence of the electrokinetic
treatment. It has been demonstrated previously that the
activity of sulfur-oxidizing bacteria is stimulated in soil slurries
in the presence of an electric current (21). The soil used in
these experiments was identical, and the microbial populations would be expected to be similar. The additional effects,
which may be seen in soil treated with electrokinetics, include
an increase in microbial activity due to a temperature rise
of between 3 and 13 °C. There is also the generation of oxygen
at the anode and the subsequent movement of oxygenated
water into the soil. Since sulfur oxidation is very oxygendependent, any increase in oxygen in the soil would be
expected to positively affect this process. The stimulation of
sulfur-oxidizing activity also accompanied an increase in
acetic acid (measured as acetate) movement from the
electrode and hence its concentration in the soil. A concentration of 10 000 mg/kg corresponded to approximately
670 mM acetate in the pore fluid of the soil. Experiments by
Alexander and co-workers (25) have demonstrated that, at
a concentration of 10 mM (pH 3), acetic acid accumulates
in the cytoplasm of T. ferrooxidans and partially inhibits the
oxidation of ferrous iron. The findings of the present study
in which the soil contained much higher concentrations of
acetic acid clearly demonstrate that these were not inhibitory
to sulfur oxidation by sulfur-oxidizing bacteria. An additional
experiment, in which a higher current density was used
resulted in removal of sulfate from the soil to below 1000
mg/kg, is of significance. One of the major drawbacks of
bioleaching technology is the high production of sulfate from
sulfur/sulfide leading to soil contaminated with sulfate. The
selective and efficient removal of sulfate from soil under
electrokinetic processing following metal removal may
therefore lead to a cleaner and more effective technology.
In summary, the combination of preacidification by sulfuroxidizing bacteria followed by electrokinetics is cost-effective
in reducing the power input for electrokinetics. The electrokinetic treatment also appears to stimulate the activity of
sulfur-oxidizing bacteria by removal of inhibitory ions and
other positive effects of the electric current upon soil
microbial activity. These synergistic effects are promising
for future experiments in which complex mixtures of metal
ions may be present, and bacteria may be required to mobilize
metals from sulfides. The methodology has potential for a
range of contaminated sites including former gasworks and
wastes from mining.
Acknowledgments
This work was supported by the U.K. Department of Trade
and Industry and the Engineering and Physical Sciences
Research Council under the LINK Biological Treatment of
Soils and Water Initiative.
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Received for review May 13, 1999. Revised manuscript received December 21, 1999. Accepted December 21, 1999.
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