The effects of direct electric current

on the viability and metabolism of

acidophilic bacteria
Simon A. Jackman,* Giacomo Maini, Ajay K. Sharman, and
Christopher J. Knowles*

*Oxford Centre for Environmental Biotechnology, University of Oxford, Department of

Engineering Science, Oxford, United Kingdom and NERC Institute of Virology and Environmental
Microbiology, Oxford, United Kingdom, IBS-Viridian Ltd., Whitstable, Kent, United Kingdom

The application of direct electric current to soil to remove metal ions is an emerging remediation technology. The
combination of this approach with bioremediation requires that soil bacteria be viable and metabolically active
under the applied currents (20 mA cm22) and their imposed acidic conditions. In this study, sulfur-oxidizing
bacteria (SOBs; a mixed culture and pure culture of Thiobacillus ferrooxidans) growing on elemental sulfur, and
the acidophilic heterotroph Acidiphilium SJH, growing on glucose, have been chosen as representative
organisms. In liquid culture, low cell densities of T. ferrooxidans and Acidiphilium SJH were inactivated by the
current; however, a high cell density of SOBs was able to recover activity when the current was switched off, and
a high density of Acidiphilium SJH was able to grow despite the presence of current. In soil slurries (5, 10, and
30% w/v silt soil), indigenous SOBs were metabolically active in the presence of current; sulfate production was
enhanced. There was also enhanced glucose consumption of Acidiphilium SJH in 10 and 30% slurries; however,
no protective effect or increased metabolism occurred when T. ferrooxidans was introduced into soil slurries.
1999 Elsevier Science Inc.

Keywords: Sulfur-oxidizing bacteria; Thiobacillus ferrooxidans; Acidiphilium; direct electric currents

Introduction surface charge, on a cell. This measurement has important

The behavior of bacteria in electric fields has been studied
for several decades to test their viability, metabolism, and
movement. Specifically, viability studies have focused on
the use of pulsed very high voltages for sterilization
purposes.1,2 Metabolic studies have utilized electrodialysis
for the removal of end product inhibition during fermenta-
tion3 and have also been applied to the electrochemical
regeneration of ferrous iron substrate for iron-oxidizing
bacteria in bioleaching processes.4 Bacteria have also been
moved in electric fields by dielectrophoresis (the applica-
tion of an alternating current5) which has proven remark-
ably effective in separating different bacteria. Under direct
current, the electrophoretic mobility of bacteria can been
determined and related directly to the zeta potential, or
Address reprint requests to Dr. S. A. Jackman, University of Oxford,
Oxford Ctr. for Environ. Biotechnol., Department of Engineering Science,
Parks Road, Oxford OD1 3PJ, United Kingdom
Received 30 June 1998; revised 6 September 1998; accepted 28 September
1998
implications for the binding of ionic species (e.g., metal
ions) and associated metabolic effects.
The approach we have adopted is to investigate DC
current effects on bacteria as a prelude to combining
bioremediation and electrokinesis in a soil environment.
Electrokinesis is the application of a direct electric current
to soil. Specific directional movement of metal ions and
organic species can be achieved under acidic conditions; the
technique is therefore emerging in its application to the
remediation of contaminated land. The combination of
electrochemical treatment with bioremediation may enable
the removal of metal ions which are often inhibitory to
bacterial activity, thereby enabling complete remediation of
the soil.6 For this hybrid approach, it is vital to know how
the bacteria will behave under acidic soil conditions in the
presence of an electric current. While we have cited several
studies of electrical effects on bacteria, none of these have
focused on soil especially under acidic conditions. It is
bacterial behavior under these conditions which is the object
of this study.
For the purposes of this study, we have chosen sulfur-

Enzyme and Microbial Technology 24:316 324, 1999

1999 Elsevier Science Inc. All rights reserved. 0141-0229/99/$see front matter
655 Avenue of the Americas, New York, NY 10010 PII S0141-0229(98)00128-8

oxidizing bacteria (SOBs; inorganic sulfur oxidizers7) and
the Acidiphilium (organic heterotrophs8) which form a
significant part of the overall microbiology of the soil
environment under acidic conditions. SOBs oxidize reduced
sources of sulfur including metal sulfides to produce acidic
sulfates.9 The most commonly found SOBs within acid
environments are Thiobacilli,10 certain of which, including
Thiobacillus ferrooxidans, also oxidize Fe21 to Fe31. As an
iron oxidizer, T. ferrooxidans has been investigated within
bioleaching processes. Several groups have looked at cou-
pling microbial oxidation of Fe21 to electrochemical regen-
eration for enhancing growth of the organisms11 and the
overall leaching process.12,13 As such, SOBs are the most
characterized acidophiles in terms of their behavior in the
presence of metal ions10 and in electric fields. Acidophilic
heterotrophs from the genus Acidiphilium are commonly
found in conjunction with chemolithoautotrophs within
bioleaching environments. We have therefore selected Aci-
diphilium SJH for our study, monitoring growth and metab-
olism on glucose at pH 2.5 as a model system. Our approach
has been to examine the behavior of SOBs in pure (T.
ferrooxidans) and mixed culture (indigenous organisms
from silt soils), and acidophilic heterotrophs in an electric
field in liquid suspension and in soil slurries with the
intention that, provided these early studies demonstrate that
the microbiology is feasible, future work will be able to
concentrate on testing soils undergoing bioremediation and
electrokinesis.
Materials and methods
Materials
The strain of T. ferrooxidans used in all pure culture experiments
was ATCC 23270. A mixed culture of sulfur-oxidizing bacteria
was isolated from silt soil by enrichment techniques at pH 2.5.
Acidiphilium SJH was a kind gift from Dr. Barrie Johnson,
University of Wales, Bangor, U.K. Sulfur, mesh size 100, was
from Aldrich Chemical Co. Ltd. (Poole, U.K.). All other chemicals
were of analytical grade or purer and were purchased from
BDH-Merck Ltd. (Poole, U.K.) or Fisher Chemical Co. (Lough-
borough, U.K.).
The soil used in these experiments was silt obtained from Wye
College, University of London, U.K. This soil has been fully
characterized in terms of particle size, pH (8.1), cation-exchange
capacity (21.7 meq/100 g), calcium carbonate (2% w/w), extract-
able phosphorus (24 mg l21), total sulfur (0.08% w/w), total
nitrogen (0.25% w/w), organic matter (3.4% w/w), organic carbon
(1.97% w/w), and C:N ratio (7.9:1).
Methods
T. ferrooxidans was grown at 30C in a shaking incubator at 160
rpm in a basic salts solution with the following composition (g
l21): (NH4)2SO4, 0.15; KCl, 0.05; MgSO4 z 7H2O, 0.5; KH2PO4,
0.05; Ca(NO3)2, 0.01; FeSO4, 100 mm; trace elements solution, 0.5
ml; adjusted to pH 2.5 with H2SO4 (conductivity, 0.8 mS). The
trace elements solution comprised (g l21): ZnSO4 z 7H2O, 10;
CuSO4 z 5H2O, 1; MnSO4 z 4H2O, 1; CoSO4 z 7H2O, 1; Cr2(SO4)3 z
15H2O, 0.5; Na2B4O7 z 10H2O, 0.5; NaMoO4 z 2H2O, 0.5; NaVO3,
0.1; in 0.01 m H2SO4. Sulfur was used as the growth substrate at
0.5% (w/v).
The mixed culture of SOBs was maintained in semicontinuous
culture at a temperature of 20 22C in the following medium (g
Enzyme Microb. Technol., 1999, vol. 24, April/May 1
Effects of direct electric current: S. Jackman et al.
l21): KH2PO4, 6.8; NH4Cl, 5.35; MgSO4 z 7H2O, 0.98; CaCl2 z
2H2O, 1.32; FeSO4 z 7H2O, 0.027; adjusted to pH 3.5 with 5 m HCl
(conductivity, 2.35 mS). To this was added 2.5 ml l21 trace
elements solution with the following composition (g l21): MnCl2 z
4H2O, 1.98; CuSO4 z 5H2O, 0.25; ZnSO4 z 7H2O, 0.92; CoCl2 z
6H2O, 0.12; H3BO3, 0.31; Na2MoO4 z 2H2O, 0.12; KI, 0.08;
Na2SeO4, 0.001; NiCl2 z 6H2O, 0.0018. As sulfur was consumed
by the SOB activity, it was replenished every 57 days to ensure
that the concentration of sulfur in the culture was maintained at
approximately 0.5% (w/v). As sulfate was produced by SOB
activity, the pH was readjusted to a value of 3.5 with 5 m sodium
hydroxide solution. Every 4 6 weeks, two thirds of the culture
was removed and fresh medium was added.
Sulfur-free inocula of all SOBs were produced by settling the
sulfur in a vertical glass cylinder for 10 min. The majority of the
bacteria remained in suspension and were removed, centrifuged at
12,000 g for 10 min, and resuspended in fresh medium. Chloride
was omitted from the medium in electric field studies to avoid
generation of chlorine gas at the anode.
Acidiphilium SJH was cultured in a medium comprising 2.17 g
l21 ammonium sulfate, 0.87 g l21 magnesium sulfate heptahy-
drate, and 0.43 g l21 tryptone soy broth, pH 2.5 containing 10 mm
glucose (conductivity, 4.45 mS).
All experiments with DC current utilized platinized titanium
paddle electrodes to ensure that the anode and cathode were inert
and unreactive. The electrodes were 1 cm2 in the paddle area each
attached to a rod with a spacing between them of approximately 2
cm. The current used was 20 mA (3 V), equivalent to 200 A m22
current density, similar to those used in electrokinetic soil reme-
diation. Experiments were performed using culture volumes of 100
ml in 250-ml plastic conical flasks in an orbital incubator shaker at
30C and 160 rpm. Electrodes were inserted through a rubber bung
and were attached to a DC power supply (Iso-Tech, RS Compo-
nents, Corby, U.K.) set to deliver a constant current.
Sulfate determination was by ion chromatography with sepa-
ration on a Metrosep anion dual 2 column fitted with a PRP1 guard
column followed by chemical suppression and conductivity detec-
tion. The mobile phase for analyses was 6 mm Na2CO3 (elution
time 6 min) for suspension cultures and 2.4 mm NaHCO3/2 mm
Na2CO3 (elution time 15 min) for soil slurries. With soil slurries,
some sulfate may be adsorbed to soil particles and therefore total
exchangeable sulfate was determined by transferring 1 ml of the
soil slurry into a microfuge tube and adding 50 ml 2 m HCl. Tubes
were shaken for 5 min and centrifuged at 12,000 g for 5 min. The
supernatant was used for analysis. Glucose concentrations in
experiments with Acidiphilium SJH were measured using the
GOD/Perid kit supplied by Boehringer Mannheim Diagnostics
(Lewes, U.K.). Protein was determined by the method of Brad-
ford14 using bovine serum albumin as a standard. To determine the
protein content of samples containing SOBs, it was important to
avoid complications due to the presence of sulfur particles to
which some of the cells would be attached; therefore all samples
were sonicated (20 3 1 s; 40 W) and sodium hydroxide added to
0.5 m. Following incubation at room temperature for 10 min,
treated samples were centrifuged (12,000 g for 3 min) and the
supernatant was used for the protein assay.
An overlay plating technique was used to determine the ability
of SOBs to grow following exposure to DC current. The overlay
technique has been described in detail by Johnson.15 To comple-
ment the investigation of growth potential, the viability of the
SOBs was assessed by staining techniques and confocal micros-
copy. The BacLight system contains propidium iodide, which is
accumulated by dead cells and stains nucleic acids fluorescent red,
and SYTO 9, which is actively transported into live cells and stains
their nucleic acids fluorescent green. It has previously been applied
to a variety of bacterial and yeast cells including Pseudomonas
317
Papers
fluorescens in which total and viable cells were determined by
fluorescence microscopy.16
Results and discussion
Growth and metabolism of T. ferrooxidans ATCC
23270 in liquid culture
T. ferrooxidans ATCC 23270 was grown on sulfur. Cells
were harvested in the exponential phase (protein concentra-
tion, 60 mg ml21) and a sulfur-free inoculum was prepared
at the same cell concentration. From this inoculum, 20 ml
was added to 80 ml medium in each of four plastic conical
flasks. Sulfur was added to 0.5% (w/v) and flasks were
incubated. Two flasks were exposed to a DC current; two
were controls. After 28 h, the current was switched off in
one of the two DC current flasks. Changes in pH and sulfate
concentration over time are shown in Figure 1A and protein
levels are displayed in Figure 1B. Figure 1 shows that in the
absence of current, T. ferrooxidans was able to metabolize,
producing sulfate and reducing the pH of the medium.
Growth was also evident from an increase in protein
concentration. When exposed to 200 A m22 DC current,
both cell metabolism and growth ceased and did not revive
within 50 h after the current was switched off.
318 Enzyme Microb. Technol., 1999, vol. 24, April/May 1
Figure 1 Exposure of cultures of T. ferrooxidans ATCC
23270 to 200 A m22 DC in shake flasks. Cells were
incubated in chloride-free medium with 0.5% (w/v) sulfur.
Solid symbols indicate the control flask which received no
current. Open symbols are in the presence of current with
squares denoting the flasks in which the current was
maintained throughout the experiment and circles for the
flask in which the current was switched off after 28 h. pH
profile (dashed lines) and sulfate levels (solid lines) (A)
and protein levels (B)
Growth and metabolism of a mixed population of
sulfur-oxidizing bacteria in liquid culture
Cells of a mixed culture of SOBs were harvested at a high
concentration (60 mg ml21) from semicontinuous culture,
washed with fresh medium pH 3.5, and resuspended to 60
mg ml21 in medium containing 0.5% (w/v) sulfur as growth
substrate. To determine whether cellular inactivation by DC
current is related to the conductivity of the growth medium,
flasks were set up incorporating 0; 0.05; and 0.1 m starting
sulfate concentrations, corresponding to 2.35; 26.7; and
46.2 mS conductivity, respectively. Current was applied and
switched off part way through to investigate whether the
bacteria recover from the DC effects.
With all three starting sulfate concentrations, the behav-
ior of the bacteria was identical in terms of acidification,
sulfate production, and protein levels. The combined data
for the cells in the absence of added sulfate are displayed in
Figure 2. An identical experiment was also performed with
a higher current of 500 A m22 to see if this had any effect,
but the results in terms of pH and sulfate levels were very
similar. At both current densities, control flasks showed
acidification of the medium and production of sulfate with
little growth of the bacteria (they are already at a high cell
density). With the electric current, metabolism is halted and
protein levels decrease slightly. When the current is

switched off, there is some recovery of metabolic activity
with no increase in protein levels. In all experiments, once
the electric current was switched off, sulfate production
commenced and thereafter followed a linear profile. Inacti-
vation of cellular metabolism by DC current was verified by
determining the growth potential of the cells by overlay
plating. SOBs not exposed to current grew well in 10 14
days and distinct white colonies were observed on the
plates, but no growth was seen with the cultures exposed to
current. The viability of the SOBs was also assessed by
staining techniques and confocal microscopy. As applied to
SOBs, viable cells in the control flasks were visible as they
fluoresced green and were motile or attached to sulfur
particles. In contrast, the cells exposed to DC current were
not motile; there was clear evidence of red fluorescence
which is indicative of lysed cells. Although some recovery
of metabolism following DC treatment was possible, the
majority of SOB cells were not viable and were incapable of
growth.
Soil slurries amended with sulfur; behavior of
indigenous SOBs
Soils contain indigenous populations of SOBs. Sulfur
amendment to such soils will therefore probably stimulate
the metabolism and growth of these bacteria. Five, ten, and
thirty percent (w/v) slurries of silt soil were prepared in
chloride-free T. ferrooxidans medium containing 0.5%
(w/v) elemental sulfur and were incubated in the presence
and absence of DC.
The results of this experiment in terms of pH changes
and sulfate production are shown in Figure 3. From each of
the sulfate profiles, the maximum rate of sulfate production
and the lag phase prior to sulfate production were deter-
mined and are shown in Table 1. Figure 3A indicates that at
all of the slurry concentrations, in the presence and absence
of current, the soil was acidified during the time frame of
the experiment. Control flasks (not shown) containing
sterilized soil exhibited no change in pH. With each slurry
concentration, acidification proceeded more rapidly in the
presence of the DC current than in control flasks. The
sulfate analyses (Figure 3B) show the corresponding in-
creases in sulfate concentration with time. The most impor-
tant conclusion from this experiment is that the sulfur-
oxidizing bacteria are metabolically active throughout the
Enzyme Microb. Technol., 1999, vol. 24, April/May 1
Effects of direct electric current: S. Jackman et al.
Figure 2 Exposure of a mixed culture of sulfur-
oxidizing bacteria to 200 A m22 DC in flasks incu-
bated in chloride-free medium with 0.5% (w/v)
sulfur at 30C and 160 rpm. Solid symbols indicate
the control flasks which received no current. Open
symbols are in the presence of current. Solid lines
are for sulfate concentration; dashed lines for pH;
and dotted lines for protein level
time course of the experiment. This contrasts with the liquid
culture experiment with a mixed culture of SOBs, derived
from a silt soil, in which the cells were inactivated by the
DC current. The presence of the soil therefore exerts a
protective and possibly activating effect on the viability and
metabolism of the SOBs.
The acidification profiles show two very distinct phases
with a rapid decrease from pH 7 to pH 4 within the first
120 h in all flasks followed by a decrease from pH 4 to pH
1.5 which takes place after different lengths of time at pH 4.
The soil has a buffering capacity and may therefore adsorb
significant sulfate at pH 4 before any further decrease in pH.
Alternatively, there may be a switching between neutro-
philic SOBs, operating from pH 7 4, and acidophilic SOBs
which operate below pH 4. The sulfate profiles (Figure 3B)
show a low level of production associated with the pH
change from 7 to 4 followed by much greater sulfate
production to lower the pH from 4. Prior to sulfate analysis,
all soil slurries were pretreated by addition of hydrochloric
acid to release exchangeable sulfate. This should avoid any
complications arising from soil buffering capacity and
adsorption phenomena. The dominant determinant of sul-
fate and pH levels is therefore most likely to be related to
the activity of the different microbial populations.
The differences between the acidification and sulfate
profiles in the presence and absence of the DC current are
very marked, with the application of the DC current leading
to more rapid acidification of the medium. The maximum
rate of sulfate production (Table 1) is not very different
between the 5, 10, and 30% slurries. There is little distinc-
tion between the flasks with and without DC current. In
contrast, the lag phase data is very different. With control
flasks, the lag phase for the 5% slurry is half the time of
those for the 10 and 30% slurries, which are approximately
equal. In the presence of the DC current, all the lag phases
are reduced, but the extent differs, with a 33% reduction for
the 5% slurry, and 67% and 9% reductions for the 10 and
30% slurries, respectively. This indicates that there is a time
lag before the second phase of acidification, probably
related to the activation of the acidophilic SOBs. This time
lag is dependent on both the concentration of the soil slurry
and the presence or absence of the DC current.
In addition to the chemical analyses, overlay plates were
used to analyze the populations of bacteria present toward
319
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Figure 3 Exposure of silt soil slurries, amended with

0.5% (w/v) sulfur, to 200 A m22 DC current. Slurries
were prepared in chloride-free medium and incubated
at 30C and 160 rpm. Solid symbols indicate the control
flasks which received no current and open symbols are
in the presence of current. Squares denote 5% (w/v)
slurries and circles and triangles 10% and 30% slurries,
respectively. The pH profile in each flask (A) and the
sulfate levels in the 5, 10, and 30% slurries, respectively
(B)

the end of the experiment. Samples were taken from the processes, an enhancement in the availability of the sulfur
flasks and plated out on ferrous sulfate and ferrous sulfate/ substrate and/or accessibility of the bacteria to the substrate
tetrathionate plates. Growth of colonies was only observed may be increased or more specialized microbial events may
on the latter plates. This indicated that the bacteria present be occurring such as the formation of biofilms.
were only able to oxidize sulfur compounds and were not
iron oxidizers. This defines the population as sulfur oxidiz- Metabolism of T. ferrooxidans in sterilized
ers such as T. thiooxidans and not iron/sulfur oxidizers such soil slurries
as T. ferrooxidans.
The protective effects of soil slurries on sulfur-oxidizing To investigate the behavior of a pure culture of T. ferrooxi-
bacteria in the presence of DC current is encouraging for dans in the presence of soil and DC current, the silt soil was
future electrokinetic studies. The increased rate of acidifi- sterilized by autoclaving (121C for 15 min, repeated three
cation with the DC current in soil slurries is both intriguing times with 24-h intervals), and 5, 10, and 30% (w/v) slurries
and difficult to interpret. There may be an increase in the were prepared in chloride-free T. ferrooxidans growth
activity of the bacteria due to the stimulation of metabolic medium. The pH of each slurry was adjusted to 2.5 with
sulfuric acid and 5 ml inoculum of T. ferrooxidans was
added (to 95 ml slurry). Cells had been grown to the
Table 1 Sulfate production in silt soil slurries amended with
0.5% (w/v) sulfur during incubation exponential phase and harvested (sulfur-free) as described
in Materials and methods. Flasks were incubated in the
Lag phase Sulfate production
presence and absence of DC current and the results in terms
(h) (ppm h21) of pH profile and sulfate production are displayed in
Figures 4A and 4B, respectively.
5% Slurry control 180 89.3 The initial pH values for 10 and 30% slurries did not
5% Slurry 1 DC current 120 64.9 remain at 2.5 but rose to 2.8 and 3.5, respectively. This was
10% Slurry control 350 47.9 due to the difficulty in accurately adjusting the pH values of
10% Slurry 1 DC current 120 58.1 concentrated soil slurries at the start of the experiment. The
30% Slurry control 330 55.6
30% Slurry 1 DC current 300 43.7 close correlation between the conditions for each set of
slurries (control and DC current) and the high activity of T.
Lag phase is that prior to the start of sulfate production. The ferrooxidans below pH 3.5 indicate that these pH discrep-
maximum rate of sulfate production is calculated from Figure 3 ancies should not mask the findings concerning microbial

320 Enzyme Microb. Technol., 1999, vol. 24, April/May 1


activity. Indeed, the results show very marked differences
between the absence and presence of the DC current. With
the 5 and 10% control slurries, the pH values rapidly
decreased from 2.5 to below 1.5 in 120 h. The pH of the
30% control slurry remained at 3.5 for the first 120 h before
decreasing to below 1.5 in 264 h. In marked contrast in the
presence of DC current, the pH values within the flasks did
not change significantly following the initial 24-h period
and remained at pH values of 2.5, 2.8, and 3.5 for the 5, 10,
and 30% slurries, respectively. These results are corrobo-
rated by the sulfate analyses which demonstrate that
whereas sulfate production occurs throughout the course of
the experiment with the control flasks, there is no produc-
tion when in the presence of the DC current. The results are
very different to those with indigenous SOBs in which
protection and stimulation of the bacterial activity was
observed. Clearly, the same protection is not effective with
the introduced organisms.
Growth and metabolism of a heterotroph,
Acidiphilium SJH, in liquid culture
Acidiphilium SJH was grown in 100 ml medium containing
10 mm glucose at pH 2.5 and was exposed to DC current.
The results of the analyses are shown in Figure 5 for
glucose utilization and growth as protein concentration.
Growth in terms of increased protein concentration corre-
Enzyme Microb. Technol., 1999, vol. 24, April/May 1
Effects of direct electric current: S. Jackman et al.
Figure 4 Exposure of T. ferrooxidans to 200 A m22 DC
current within a pre-sterilized soil slurry. Incubation was
in chloride-free medium in the presence of 0.5% (w/v)
sulfur at 30C and 160 rpm. Data is presented for 5%
(squares), 10% (circles), and 30% (triangles) slurries in
the absence (solid symbols) and presence (open sym-
bols) of current. pH profile (A) and sulfate production (B)
lated well with glucose utilization, with the control flask (no
current) consuming 10 mm glucose within 55 h and the
maximum protein concentration being reached at this time.
In contrast, no growth or glucose utilization was observed
when the DC current was present over a time period of 98 h.
In a second experiment, a glucose concentration of 100
mm was selected to ensure maximal growth of the bacteria
and to provide sufficient substrate for the bacteria to
continue metabolizing once cells reached confluence. With
cells grown to late exponential phase and inoculated into
100 ml medium to a concentration of 3 mg ml21, the pattern
of growth in the presence and absence of current was
identical to that described above; however, when a higher
initial cell concentration (12 mg ml21) was present, growth
occurred in the presence of current. Three flasks were set up
under each condition and the results of one of each are
shown in Figure 6. In the control flask, glucose was utilized
to approximately 60 mm in 192 h with the protein level
increasing rapidly in the first 24 h and then slowly to 300 mg
ml21 after 192 h. In contrast, when DC current was present,
glucose utilization was significantly faster, to 20 mm re-
maining after 192 h. This was accompanied by a greater
increase in protein concentration to approximately 400 mg
ml21.
The results which we have obtained for low concentra-
tions of Acidiphilium SJH are similar to those for T.
321
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Figure 5 Growth and utilization of glucose by Acidiphilium SJH
at pH 2.5 with a substrate concentration of 10 mM glucose.
Closed symbols denote the control in which no current has been
applied and open symbols are for 200 A m22 DC. Glucose
concentration (solid lines) and protein concentration (dashed
lines) are displayed
ferrooxidans in liquid culture in that the bacteria are
inactivated in the presence of the DC current whereas
growth and glucose utilization is rapid in control flasks. In
both of these situations, the cell concentrations are relatively
low and therefore an effect such as an inactivating or
destructive interaction with the electrode surface would lead
to a more rapid reduction in numbers than can be countered
by the growth of the bacteria. With higher initial cell
concentrations, Acidiphilium SJH was able to overcome the
inactivating DC effects. The higher apparent utilization of
glucose by Acidiphilium SJH at high cell concentrations in
the DC field may be due to continuous destruction of a
proportion of the bacterial population with growth of more
cells, and concomitant increased glucose utilization, to
maintain a high cell density.
Metabolism of Acidiphilium SJH in sterilized silt
soil slurries
Soil slurries containing Acidiphilium SJH utilized the same
medium as for liquid cultures. Soil was sterilized as de-
scribed for the T. ferrooxidans experiments and the bacteria
introduced as an inoculum from a culture grown to late
exponential phase. A 5-ml inoculum of bacteria was used
and the slurry pH was adjusted to pH 2.7 (control) and 2.8
(1 current) for the 5% slurries; pH 3.3 (control) and 3.2 (1
current) for 10% slurries; and 3.6 (control) and 3.4 (1
current) for 30% slurries. These pH values remained con-
stant throughout the time frame of the experiment. The
changes in glucose concentrations over time are displayed
in Figure 7. Glucose was utilized in all slurries in the
presence and absence of DC current. The order of utilization
of glucose in control flasks was 5% . 10% . 30% (w/v)
slurries. In contrast, in the presence of a DC current, this
order was reversed and the Acidiphilium in the 5% (w/v)
slurry took 192 h to degrade the glucose.
These findings agree with the hypothesis that the soil can
protect certain bacteria from the effects of the DC current
whether this be direct interaction with the electrodes or
322 Enzyme Microb. Technol., 1999, vol. 24, April/May 1
Figure 6 Growth of Acidiphilium SJH on 100 mM glucose at pH
2.5. Incubation was at 30C and 160 rpm. Open symbols indicate
flasks with 200 A m22 DC and solid symbols are for control
flasks. Solid lines follow glucose concentration and broken lines
are for protein concentration
exposure to the current. The 5% (w/v) slurry has a low soil
concentration and therefore a large proportion of the bacte-
ria may still be exposed and inactivated when the current is
on. The 10 and 30% slurries are progressively more protec-
tive. What remains unexplained is why the cultures in the
presence of the electric current are more active than con-
trols. Approximate protein analyses, which are interfered
with by soil humic components, thereby preventing absolute
quantification, indicate that increased metabolism of glu-
cose correlated with an increase in the number of cells
present. There was no abiotic glucose breakdown by the DC
current. The increase in metabolism of glucose in the
presence of the current is the same phenomenon as with
indigenous SOBs and the same possible reasons for in-
creased bacterial activity or bioavailability can be
postulated.
Conclusions
The overall results from this work demonstrate that in liquid
culture, low densities of both sulfur-oxidizing bacteria and
heterotrophs are inactivated by DC current at 200 A m22 (3
V). This inactivation appears to occur irrespective of the
conductivity of the medium. Cellular metabolism could not
be reactivated within the time frame of our experiments by
switching off the current. With high cell densities of SOBs,
some revival of the cells occurred and with a high density of
Acidiphilium SJH, it appeared that growth of the bacterium
was more rapid than the rate of destruction by the electric
field. Sale and Hamilton2 showed that the killing of cells
required high pulsed voltages (25 kV cm21 and 40 100 ms
pulse duration) and that this related to neither interaction
with the products of electrolysis nor with temperature
changes but rather a direct effect of the current on the cells.
Other recent studies have supported this in demonstrating
that electric currents affect the orientation of membrane
lipids and consequently, cell viability.17 In a bioleaching
environment, positive voltages as low as 1 V have been
shown to be deleterious to T. ferrooxidans activity;12

Furthermore, when the voltage drop across the cell wall
exceeds 0.4 2 V, the wall breaks down.1 There is therefore
evidence from both high and low voltage studies that cell
walls and membranes can be disrupted. The results which
we have obtained with different cell densities suggest that
inactivation is dependent on the concentration of cells
present. It is therefore most likely that inactivation occurs
by interaction of the bacteria with the surfaces of the
electrodes, resulting in cell wall or membrane degradation
by oxidation (cathode) or reduction (anode) during the
application of the current.
The presence of a soil slurry has two major effects on
cellular metabolism and growth which were observed with
indigenous SOBs and with introduced Acidiphilium SJH in
sterilized soil slurries. Firstly, the bacteria are protected
from inactivation by the DC current and secondly, the DC
current stimulates the metabolism of sulfur and glucose. The
protective effect might be because the bacteria may be able
to avoid direct contact with the electrodes by being located
within the soil matrix. It is also possible that penetration of
the bacteria into particles and the formation of structures
such as biofilms may protect the cells from the effects of the
current passing through their walls and membranes. It is
more difficult to explain the stimulatory effects of the
current. There is the possibility that the current stimulates
the metabolism of individual bacteria, but it is more likely
that the effects relate to the accessibility of the substrate to
the organism. It is to be expected that DC current will tend
to break ionic interactions between components of the
slurries. If glucose or sulfur become sorbed to soil surfaces
in slurries and these interactions are broken by the current,
they may become more available to the bacteria as they are
maintained in solution (i.e., an increase in bioavailability).
The same could also be said of the interactions between the
bacteria and the soil. In electrokinesis, the electrical field
generates hydrogen ions which can replace metal ions
adsorbed to soil surfaces, thereby releasing them into the
pore fluid in which they can be transported by electromi-
gration and electroosmosis through the soil.18 Electro-
phoretic movement of particles including clay minerals and
also bacteria19 has been demonstrated within the soil matrix.
Electrokinesis is therefore fundamentally characterized by
changes in the interactions between species and the move-
ment of species. It is therefore not unreasonable to postulate
that changes in interactions and movements occur within the
Enzyme Microb. Technol., 1999, vol. 24, April/May 1
Effects of direct electric current: S. Jackman et al.
Figure 7 Glucose utilization by Acidiphilium SJH in soil
slurries. Data is presented for 5% (squares), 10% (circles),
and 30% (triangles) slurries in the absence (solid sym-
bols) and presence (open symbols) of 200 A m22 DC
current
more simplified soil slurries. Another possible explanation
for increased bacterial activity is the formation of biofilms
on the soil surfaces which have enhanced metabolic capa-
bilities. T. ferrooxidans exhibits tolerance to a wider range
of temperature, pH, and product concentration when oxidiz-
ing ferrous iron within a biofilm.20 These changes may be
attributed to alterations in the physiology of the microor-
ganisms when attached to a surface. If DC currents were to
cause stress to the organisms, which is a common cause of
biofilm formation, then it is possible that this might in turn
lead to increased bacterial activity.
The inability of the soil slurries to have any protective
effects on T. ferrooxidans ATCC 23270 contrasts with the
results from indigenous SOBs and suggests that there may
be differences between introduced and indigenous organ-
isms which could relate to their abilities to form stabilizing
and protective interactions with soil particles. Because the
soil slurries were presterilized, there should not be any
detrimental effect of the soil microbiota on the survival of
the introduced species; however, the DC effects were
marked even at 30% (w/v) slurry concentrations. Given that
interaction with electrode surfaces is unlikely for bacteria
within the soil matrix undergoing electrokinesis, this effect
may not be relevant to the introduction of organisms for
combined bioremediation and electrokinetic treatments, but
it is an interesting phenomenon which merits further
investigation.
In summary, both heterotrophic and autotrophic bacteria
have been shown to survive in soil slurries in the presence
of DC current and therefore we can have reasonable
confidence that their viability, metabolism, and potential for
remediation are maintained within the soil matrix under an
electric field. It remains to extend these studies into soil
matrices undergoing electrokinesis. The findings of in-
creased bacterial metabolism, for whatever reason, are
particularly encouraging. If these can be transferred into the
compact soil, then electrokinesis may be significant in
enhancing bioremediation. While some attempts have been
made at combination approaches for bioremediation and
electrokinesis, these have usually been limited to the elec-
trokinetic transport of organic species from a contaminated
zone into a treatment area. There is therefore a separation
between the techniques.21
The supply of nutrients and maintenance of moisture
levels by electrokinesis within soils have been investigated
323
Papers
to enhance bioremediation,22,23 but the removal of contam-
inating metal ions by electromigration, oxygenation of
water entering the system from the anode, the warming of
the soil by the DC current, and the findings of this study
should provide further stimulation for microbiological
processes.
Acknowledgments
This work was supported by the Engineering and Physical
Sciences Research Council as part of the DTI LINK
Biological Treatment of Soils and Water Programme. We
are grateful to our collaborators Garry Sunderland and
Lawrence Parr at EA Technology Ltd. for their help and
advice in performing these studies.
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