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Environmental Pollution xx (2006) 1e8

www.elsevier.com/locate/envpol

Impact of electrokinetic remediation on microbial communities within

PCP contaminated soil
G. Lear a,b,1, M.J. Harbottle a,b, G. Sills b, C.J. Knowles a,c, K.T. Semple d, I.P. Thompson a,*
a
NERC-CEH-Oxford, Virology and Environmental Microbiology, Mansfield Road, Oxford, OX1 3SR, UK
b
Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK
c
Department of Earth Sciences, University of Oxford, Banbury Road, Oxford, OX2 6PN, UK
d
Department of Environmental Science, Faculty of Science and Technology, Lancaster University, Lancaster, LA1 4YQ, UK
Received 21 August 2005; received in revised form 4 June 2006; accepted 14 June 2006

Electrokinetics negatively impacted soil.

Abstract

Electrokinetic techniques have been used to stimulate the removal of organic pollutants within soil, by directing contaminant migration to
where remediation may be more easily achieved. The effect of this and other physical remediation techniques on the health of soil microbial
communities has been poorly studied and indeed, largely ignored. This study reports the impact on soil microbial communities during the
application of an electric field within ex situ laboratory soil microcosms contaminated with pentachlorophenol (PCP; 100 mg kg1 oven dry
soil). Electrokinetics reduced counts of culturable bacteria and fungi, soil microbial respiration and carbon substrate utilisation, especially close
to the acidic anode where PCP accumulated (36 d), perhaps exacerbated by the greater toxicity of PCP at lower soil pH. There is little doubt that
a better awareness of the interactions between soil electrokinetic processes and microbial communities is key to improving the efficacy and
sustainability of this remediation strategy.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Electrokinetic; Soil remediation; Soil health; Carbon substrate utilisation; Pentachlorophenol

1. Introduction
Electrokinetics provides a physical method for the extrac-
tion of both metals and organic chemicals from contami-
nated sites, stimulating a directed movement of pollutants
in response to the presence of an electric current (Maini
et al., 2000). The applied current produces hydrogen ions
(Hþ) at the anode and hydroxyl ions (OH) at the cathode,
with a resulting pH gradient (Acar and Alshawabkeh, 1993).
* Corresponding author. Tel.: þ44 1865 281630; fax: þ44 1865 281696.
E-mail address: ipt@ceh.ac.uk (I.P. Thompson).
1
Present address: Williamson Centre for Molecular Environmental Science,
School of Earth, Atmospheric and Environmental Sciences, University of
Manchester, Manchester, M13 9PL, UK.
During this process, ions, ion complexes and particles are
caused to migrate (via electromigration) to electrodes of
opposite charge as a consequence of their orientation within
the electric field, such that positively charged particles are
stimulated to migrate to the negatively charged cathode,
and vice versa. Dipolar interactions between the negatively
charged surfaces of clay particles also encourage the
transport of water and the solutes within it, towards the
cathode, via electroosmosis (Acar et al., 1995). Electro-
mobile contaminants are therefore stimulated to migrate to
positions where they are amenable to removal, for example,
via the collection and treatment of contaminated electrolyte
solutions or precipitates formed close to the electrodes.
Such processes now support an active remediation industry
(e.g. Geokinetics BV, Rotterdam, ND), incorporating a range

0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2006.06.037

Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
Pollution (2006), doi:10.1016/j.envpol.2006.06.037
 ARTICLE IN PRESS
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2 G. Lear et al. / Environmental Pollution xx (2006) 1e8
of novel applications for the removal of both organic and
heavy metal contaminants in soil (e.g. LasagneÔ, Elektro-
KleanÔ, Electro-SorbÔ; Virkutyte et al., 2002).
An important requirement before new methods for the
remediation of contaminated land are accepted is to demon-
strate that they are effective, and that they have few negative
impacts on soil microbial communities and therefore soil
health. Although little is currently known about the effect of
electrokinetics on exposed soil microbial communities, recent
studies have suggested that no serious impact on microbial
health occurs when it is applied to pristine, non-contaminated
soils (Lear et al., 2004). However, the influence of electroki-
netics on soil health in the presence of a pollutant is not
thought to have been previously investigated. It therefore re-
mains possible that using electrokinetics to remove pollutants
from contaminated land may affect soil microbial communi-
ties, possibly aggravating the toxic influence of contaminants
and impairing the ability of the microbial community to intrin-
sically remediate any further incidences of pollution.
Pentachlorophenol (PCP) has been widely used as a wood
preservative and biocide since commercial production began
in the 1930s, with worldwide annual production reaching
90 000 tonnes in 1983 (IRPTC, 1983). Pentachlorophenol is
regarded toxicologically as a priority pollutant (Wild et al.,
1993), being a suspected human carcinogen, hepatoxic agent
and teratogen (McAllister et al., 1996). In mammals, PCP un-
dergoes oxidative dechlorination in the liver to form tetra-
chlorohydroquinone (TCHQ) which may damage the liver,
kidneys and immune system (EPA, 1986). Concerns about
the safety of PCP have meant its use is now banned in many
countries within the Northern Economic Divide. However, of
all of the simple chlorinated phenols, PCP is thought to be
the most resistant to microbial degradation (McAllister
et al., 1996) and therefore its historic presence in the environ-
ment is a matter of ongoing concern (Muir and Eduljee, 1999),
emphasising the need for cost-effective remediation strategies
to treat PCP contaminated land.
The electrokinetically induced movement of pentachloro-
phenol has previously been demonstrated to occur within an
unsaturated soil (Harbottle et al., 2001). PCP has an acid disso-
ciation constant (pKa) of 4.7 in water, whilst the natural pH of the
soil in this study was pH 7.8. Therefore, the initially ionic PCP
moves via electromigration towards the anode, but is later
affected by the reverse flow of electroosmosis when entering
the zone of low pH (i.e. <4.7) that develops close to the anode.
The biocidal properties of PCP have been shown to impact soil
microbial communities (Scheunert et al., 1995; Martins et al.,
1997), especially under acidic conditions as the more toxic
non-ionic pentachlorophenate is formed, so impacting
negatively on degradation rates (Salminen and Haimi, 1998).
Thus, the aim of this study was to develop a better understanding
of the interactions between the soil, PCP contaminant, and
indigenous microbial communities, during the application of
electrokinetic technology. The experimental regime used in
the present study was the progression of an earlier investigation
(Lear et al., 2004), which examined the effect on a pristine soil,
with no history of site contamination.
Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
Pollution (2006), doi:10.1016/j.envpol.2006.06.037
2. Methods
To determine the effect of an electric field on microbial counts and activity
within soil contaminated with PCP (100 mg kg1 oven dry soil) and inoculated
with the PCP mineralising bacterium Sphingobium sp. UG30 (Leung et al.,
1997), two treatments were constructed, in triplicate. Three electrokinetic car-
tridges were exposed to an applied current (3.14 A m2), using Isotech
IPS601A benchtop power supplies (Southport, UK), whilst three more were
established as negative controls, with no current applied. Both treatments
were inoculated with 1  108 cells g1 oven dry soil of Sphingobium sp.
UG30. This experimental regime was essentially as reported earlier (Lear
et al., 2004) in a parallel study, which provided complete details of the elec-
trokinetic technique and sampling procedure, also outlining electrokinetically
induced changes in temperature, pH and conductivity within the soil car-
tridges. However, in the previous study, no PCP or degradative strain was
added.
2.1. Soil electrokinetic cartridges
Electrokinetic experiments were undertaken within a separable acrylic car-
tridge system (Fig. 1; inner cartridge size 13.0  5.4  5.9 cm), as described
by Harbottle et al. (2001), but the present study used smaller cartridge dimen-
sions. Cartridges were designed so that soil could be treated and stored within
the detached central chamber for any length of time, and easily slotted into the
remainder of the electrokinetic system, as required. Porous Daramic (Daramic
Inc., Sélestat, De) membranes at either end separated the soil within the central
cartridge from the ionic solutions which contained the anode and cathode. The
membranes prevented the passage of soil into the neighbouring chambers,
whilst allowing a flow of water between them. Clamps and rubber seals
bonded the three compartments to a base, reducing leakage, whilst ensuring
chambers remained amenable to dismantle and reassemble. Graphite carbon
electrodes (5.0  5.0  0.8 cm) were used to provide an electrical current,
housed within the exterior electrode chambers. Holes (10 mm diameter)
were bored into the graphite electrodes to enable a flow of water within
each compartment. Chambers were placed within sealed plastic containers
(opened only for sampling) in order to collect PCP-derived 14CO2 associated
activity, facilitating its subsequent analysis. Electroosmotic flow was deter-
mined by measuring liquid loss from a Mariotte bottle, used to keep a constant
head of fluid within the anode chamber.
The soil used within this study was taken from the Oxford University Field
Station, Oxford, and is an Evesham series heavy clay, having 53% clay, 25%
sand and 22% silt (Lilley et al., 1996). Prior to use, all soil was air-dried and
sieved (<2 mm), re-moistened to a water content of 18e21% (v:w), and stored
at 4  C. Soil (0.5 kg) was added to each of six cartridges (statically compacted
at a pressure of 50 kPa in 100 g layers, with the final height matching the level
of the outflow ports in the electrode chambers). PCP (200 mg ml1) was added
to each cartridge in 500 ml of de-gassed, deionised, water, applied to the top of
the chamber. The employment of radiolabelled contaminants provides a supe-
rior method of tracking the fate of pollutants within soil. Therefore the PCP
amendment comprised a component of [UL-14C]PCP (Sigma, UK), providing
a final activity of 125 kBq g1 PCP within the soil. The leachate was collected
and recycled three times and the cartridges allowed to equilibrate for 24 h to
attain a saturation ratio of approximately 70%. This procedure has been
perfected to achieve a concentration of w100 mg PCP kg1 oven dry soil,
and a water content of 40e44% (v:w). Cartridges were stored in sealed con-
tainers for 5 d in the dark at 4  C, to facilitate a period of soil recovery.
In order to determine the effect of electrokinetics on the ability of micro-
organisms to degrade PCP, a PCP mineralising bacterial strain, Sphingobium
sp. UG30 was utilised (Leung et al., 1997). Sphingobium sp. UG30 was
cultured in a minimal salts medium, containing L-glutamic acid (K2HPO4,
0.65 g l1; KH2PO4, 0.19 g l1; MgSO4$7H2O, 0.1 g l1; NaNO3, 0.5 g l1;
1 1 
L-glutamic acid, 4.0 g l ; FeSO4, 0.003 g l ) and incubated (28 C,
140 rpm) in 250 ml Erlenmeyer flasks. Culture density was routinely moni-
tored at OD600 nm, and where required, converted to bacterial numbers, as
described by Wall and Stratton (1994). Soil chambers were inoculated using
a point augmentation procedure. Briefly, to each chamber, aliquots (100 ml)
of a concentrated bacterial suspension (containing 1.7  1010 cells ml1),
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G. Lear et al. / Environmental Pollution xx (2006) 1e8 3

13.0cm
Porous
Daramic
Membrane Anode
Chamber

Acrylic
5.9cm Chamber

Cathode Soil
Chamber

Sampled on day 0
Sampled on day 7
Sampled on day 14
Sampled on day 26
Sampled on day 36
5.4cm

Fig. 1. Schematic of the sampling regime within the soil electrokinetic chamber. Nine soil cores were taken across the length of each chamber in increments of
1.44 cm (first core 0.72 cm from anode chamber), 0, 7, 14, 26 and 36 d after the current was applied. Inner chamber dimensions 13.0  5.4  5.9 cm. Graphite
electrode dimensions: 5.0  5.0  0.8 cm.

harvested and washed in phosphate buffer (K2HPO4, 0.65 g l1; KH2PO4,
0.19 g l1) during the mid-exponential growth phase, were evenly distributed
at depths of 5 mm, 30 mm and 55 mm (utilising 36, 24 and 36 evenly spaced
injection points, respectively). This procedure allowed an average cell density
of w1  108 cells g1 oven dry soil of Sphingobium sp. UG30 to be achieved
for the entire chamber. Many soils, including the one used in this study, lack
a significant biomass of PCP degrading microorganisms (Mueller et al., 1991;
Seech et al., 1991). Consequently, the specific interaction of the contaminant
with an individual, inoculated degradative strain may be studied in detail.
Soil samples were removed from the chambers immediately prior to the
application of electrokinetics (0 d) and then after a further 7, 14, 26 and
36 d. This was achieved by dividing the chambers into five lanes, such
that a fifth of the soil cartridge was sampled during each sampling time,
as indicated in Fig. 1. Metal tubing (14 mm inner diameter, Oxford Instru-
ments Superconductivity, Oxford, UK) was employed to extract soil cores
(w5 g) from nine locations, evenly spaced along the transect between the
anode and cathode chambers. Soil pH, temperature, conductivity and volt-
age were determined as previously described (Harbottle et al., 2001; Lear
et al., 2004), with similar results. During the entire study, the pH of the
catholyte was maintained at pH 7 with the addition of sulphuric acid which
had the effect of maintaining electroosmotic flow throughout the
experiment.
2.2. Determination of the loss and extractability of
[UL-14C]PCP-associated activity in soil
[14C]PCP-associated activity was assessed by sequential extraction, first
agitating 0.5 g soil with 10 ml water and then 10 ml acetonitrile, on a rotary
shaker (6 rpm, 24 h), centrifuging the sample (5000 rpm, 15 min) and remov-
ing the supernatant between stages. Supernatant (6 ml) was combined with
12 ml UltimaGold XR scintillation fluid (PerkinElmer, USA) and activity
counted using a Wallac Liquid Scintillation Counter (1217 Rackbeta;
Groningen, NL) for 5 min at a wavelength of 104e165 nm. Water was chosen
as an initial extractant to provide a measure of the amount of PCP which is
easily desorbed, or present within the pore water, whilst acetonitrile extracted
the majority of the remaining, more recalcitrant PCP. Non-extractable
[14C]PCP-associated activity was determined using a combustion method as
similarly undertaken by Reid et al. (2000). Soil samples (1 g) were packed
into paper combustion cones, prior to analysis using a sample oxidiser (Model
307). Carbosorb-E and Permofluor-E were used to trap CO2, and as a scintil-
lant, respectively. Combustaid (100 ml) was added to each sample prior to the
3 min combustion process. The Packard sample oxidiser and respective liquid
agents were from Canberra Packard, UK. This combination of procedures pro-
vided a mass balance of the total [14C]PCP-associated activity within the soil.
The respirometric method of Reid et al. (2001) was used to assess the ex-
tent of PCP degradation within the soil. The breakdown of [14C]PCP releases
14
CO2, which may be trapped within a NaOH solution, where measurements of
14
CO2 evolution can be directly correlated to the loss of PCP. Each sealed ex-
perimental treatment housed a glass scintillation vial containing 3 ml of 1 M
NaOH solution to collect evolved CO2. Traps were regularly removed and so-
lutions replaced. Liquid scintillation fluid (16 ml, Ultima Gold, Perkinelmer,
USA) was added to each vial and the activity of the trapped 14CO2 was deter-
mined by liquid scintillation counting following a 24 h rest period.
2.3. Analysis of soil microbial numbers and activity
Culturable counts of soil bacteria and fungi, soil microbial respiration and
carbon substrate utilisation patterns were examined using methods previously
outlined (Lear et al., 2004). Counts of culturable soil bacteria and fungi were
examined across the cartridge transect by incubating soil dilutions on Plate
Count Agar (Oxoid Ltd., Hampshire, UK; containing 100 mg l1 cyclohexa-
mide, as an antifungal agent) and potato dextrose agar (Oxoid Ltd.; containing
320 mg l1 AureomycinÒ as an antibacterial agent), respectively. Soil micro-
bial respiration was monitored using a modified method of Öhlinger (1996)

Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
Pollution (2006), doi:10.1016/j.envpol.2006.06.037
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4 G. Lear et al. / Environmental Pollution xx (2006) 1e8

which quantifies CO2 production by recording the pH change of a NaOH so- 120
lution, determined by colorimetric analyses. The ability of the soil microbial (i)
100

Percentage14C-PCP-Associated Activity Recovered

community to utilise a range of different carbon sources was assessed by
the colour development of a tetrazolium dye within BIOLOG EcoplatesÔ 80
(Hayward, CA, USA), allowing both spatial and temporal differences in the
soil microbial communities to be determined. 60

40
2.4. General statistical analysis
20

(Relative to Day 0)
Bacterial and fungal counts were calculated per gram of oven dry soil and 0
log-transformed [log10(x þ 2)] to improve the homogeneity of the variance of 0 6 12 18 24 30 36
the data. Data were analysed using the statistical package GENSTAT (GEN-
STAT V release 6.1). Unless otherwise stated, analyses of variance (ANOVAs) 120
(ii)
were constructed using a general two-way design where position between elec- 100
trodes was a fixed effect and sampling time was a repeated measures factor.
Standard methods in GENSTAT were used to test the homogeneity of variance 80
and normality of the data to ensure it remained within the required parameters
for the statistical test. t-tests were paired and two-tailed. BIOLOGÔ data from 60
each plate were transformed to facilitate the confounding effects on rates of 40
colour development caused by differing inoculation densities, using the fol-
lowing formula, (S  C )/B  109, where S is the sample well OD620 nm, C 20
is the control well OD620 nm and B is bacterial CFU g1 oven dry soil.
0
0 6 12 18 24 30 36

3. Results Time (Days)

Fig. 3. Percentage [14C]PCP-associated activity recovered over 36 d (relative

By the end of the study (36 d), a pH profile developed to activity measured on 0 d) from soil inoculated with w1  108 cells
across the length of the electrokinetic chambers from pH 3.8 Sphingobium sp. UG30 g1 dry soil (i) without current applied (ii) with current
applied (3.14 mA m2); data are [14C]PCP-associated activity recovered via
close to the anode (2.2 cm) to pH 7.9 close to the cathode
[14C]PCP trapping ( ); acetonitrile extraction ( ); water extraction ( ). Er-
(2.2 cm), as similarly detailed within Lear et al. (2004). The ror bars are 1  SE (three replicates).
distribution of PCP within the soil of the electrokinetic
chambers concentrated towards the anode after 36 d of electro-
kinetic exposure; the result of electromigration. [14C]PCP-
-associated activity extracted from experimental or control
associated activity increased by 13% close to the anode
treatments (t ¼ 0.68, df ¼ 14, p ¼ 0.504). No 14C activity
(1.3 cm), with no associated increase within the control
was detected from the soil following sequential extraction us-
cartridges (Fig. 2). Over the duration of study, a decrease
ing the combustion method, suggesting a complete removal of
was observed of water-extractable PCP (Fig. 3), more so
PCP.
within the electrokinetic treatment than the control (17  5%
Culturable numbers of both bacteria and fungi remained
(SE) and 46  4% (SE) of original soil 14C activity after
constant within the control treatments over the experimental
36 d, respectively). However, on the final sampling day, no
period (i.e. 6.74  0.04SE, 3.34  0.04SE Log10 (xþ2) CFU
significant difference was detected between total [14C]PCP
g1 oven dry soil of bacteria and fungi, respectively; Fig. 4).
However, bacterial counts decreased (4.3%, Log10(xþ2) units)
Recovered from Treatments as

140 within the electrokinetic cell as compared to the control treat-

a Percentage of Original PCP-

ment (t ¼ 2.53, df ¼ 58, p ¼ 0.014; all samples combined).

120
The application of electrokinetics reduced culturable counts
Associated Activity

100 of both bacteria and fungi close to the anode (0.7 cm) by
80
17%, and 30%, respectively (0 d as compared to all other
time-points combined). Bacterial counts increased within the
60 inoculated soils, reflecting the addition of Sphingobium cells
40 (as compared to 6.50.03 Log10 (xþ2) CFU g1 oven dry
soil (Lear et al., 2004), all samples combined (t ¼ 3.81,
20
df ¼ 48, p ¼ 4.00  104)). The application of electric current
0 significantly (p ¼ 0.037) reduced soil microbial respiration
14C

0 2 4 6 8 10
Distance from Anode (cm)
Fig. 2. Total PCP associated activity recovered from soil after 36 d of applied
current in each treatment (3.14 mA m2) from soil cartridges inoculated with
1  108 cells Sphingobium UG30 sp. g1 oven dry soil; data are 14C-activity
recovered as a percentage of original PCP-associated activity inoculated (:)
without current applied; (6) with current applied; Error bars are 1  SE;
three replicates.
Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
Pollution (2006), doi:10.1016/j.envpol.2006.06.037
12
below that of the control by 21 d (i.e. 3.320.33SE and
3.93  0.42SE mg CO2 g1 oven dry soil, respectively;
Fig. 5). Furthermore, respiration was lowest close to the
anode end (0.72 cm) of the electrokinetic chambers; only
2.55  0.57SE mg CO2 g1 oven dry soil.
Colour development profiles of the BIOLOG EcoPlatesÔ
showed the preferential order of carbon substrate utilisation
 ARTICLE IN PRESS
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G. Lear et al. / Environmental Pollution xx (2006) 1e8 5

Fig. 4. Colony forming units of soil fungi (left) and bacteria (right) (i) without electrokinetics (ii) with electrokinetics, sampled along the cartridge transect from
anode to cathode for each sampling time-point during electrokinetic treatment initially contaminated with 100 mg PCP kg1 oven dry soil. Data are log-trans-
formed log10(x þ 2) CFU g1 oven dry soil. Sampling timepoints: () 0 d; (:) 7 d; (A) 14 d, (-) 26 d; (d) 36 d; pooled standard error ¼ 0.06 and 0.26
for each treatment respectively; an ANOVA for the combination is shown on the graph (L, sampling position; S, sampling timepoint; P, probability.) Error
bars are 1  SE; three replicates.

for the control treatment to be amino acids > amines z
carboxylic acids z carbohydrates z phenols > polymers (as
determined by the greatest absorbance (OD620 nm) after 10 d
incubation (data not shown)). Within soil exposed to the
electric field, the order of carbon substrate utilisation was
amines > polymers > amino acids > carboxylic acids > phe-
nols > carbohydrates (Fig. 6). The extent of carbon substrate
utilisation was significantly less with an electric field applied
(x¼ 7.4 and 40.9, with and without an applied current, respec-
tively; t ¼ 11.7, df ¼ 1112, p ¼ 6.54  1030). Both the rate
6 p
0.632
mg CO2 g-1 Oven Dry Soil
and extent of carbon substrate utilisation was inhibited
adjacent to the anode (0.72 cm), with no visible utilisation
of carbon source occurring by 36 d.
4. Discussion
The toxic effect of PCP is thought to be due to its ability to
uncouple oxidative phosphorylation (Steiert et al., 1988) and
alter the composition of bacterial membranes (Dercová
et al., 2004). The impact of even low concentrations (50e100
mg kg1 dry soil) of PCP have been described as severe
(Chaudri et al., 2000), reducing soil microbial counts (Chaudri

5
0.037 et al., 1996; Martins et al., 1997; Salminen and Sulkava,
1997), biomass, microbial respiration rates (Zelles et al.,
4
1986) and substrate induced respiration (Scheunert et al.,
3 1995). In the present study, PCP reduced fungal counts as
compared to a non-contaminated soil (4.9  0.1 log10(x þ 2)
2 fungal CFU g1 dry soil; Lear et al., 2004). Bacterial counts
were higher, reflecting the addition of a Sphingobium
1 inoculum, but declined towards the anode of the electrokinetic
treatment over the duration of the study. However, within the
0
2 5 8 11 control treatment (where no electrokinetics was applied), the
Distance from Anode (cm) application of PCP stimulated the range of carbon sources
utilised by the soil microbial count as compared to a PCP
Fig. 5. Soil respiration rate in each treatment (red, control (no applied current);
blue, electrokinetic), sampled along the cartridge transect from anode to
free, non-electrokinetic treatment (Lear et al., 2004). This is
cathode on 21 d. Data are mg CO2 g1 oven dry soil; Pooled standard thought to be a stress-induced response as, following the
error ¼ 0.99 and 0.45 for each treatment respectively; an ANOVA for the loss of PCP susceptible soil fauna, the availability of resources
sampling position is shown on the graph. Bars are 1  SE; three replicates. may have provided energy and nutrients available to the
Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
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6 G. Lear et al. / Environmental Pollution xx (2006) 1e8
Fig. 6. Colour development profiles (OD620) of BIOLOG EcoplatesÔ incubated
over 10 d for soil taken (i) 12.7, (ii) 6.5, and (iii) 0.7 cm from the anode, respec-
tively, at the final timepoint (36 d). Treatment was 100 mg PCP kg1 oven dry
soil, exposed to electrokinetics (3.13 mA cm1) Data were transformed OD620
values grouped as an average for each of the six carbon substrate types in the BI-
OLOGÔ EcoPlate: >, polymers; B, amino acids; d, carbohydrates; -, car-
boxylic acids; 6, phenols; , amines. Error bars are 1  SE; three replicates.
saprophytic microbial community. As a consequence of this
selection, a shift in the composition of the microbial commu-
nity may have resulted, to one able to utilise a greater amount
of carbon per cell than the initial community. Hence, carbon
substrate utilisation increased, but without any associated
detectable increase in soil respiration, or culturability.
Previous studies revealed that the addition of a Sphingobium
inoculum did not significantly affect patterns of carbon
substrate utilisation (data not shown), and this was perhaps
a consequence of its poor survival in PCP contaminated soil. In-
deed, loss of cell viability of PCP degraders has been observed
in both liquid culture media (Gonzalez and Hu, 1991) and soil
(Karlson et al., 1995). This may account for the observation
that the application of electrokinetics did not accelerate the
loss of PCP in contaminated soil, the very purpose for which
such an approach may be applied in soil remedial applications.
Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
Pollution (2006), doi:10.1016/j.envpol.2006.06.037
Importantly, electrokinetics appeared to increase the proportion
of PCP extractable only with acetonitrile, as compared to water.
You and Liu (1996) revealed the increased desorption of PCP
from soil at higher pH. It is therefore suggested that the acid-
front caused by the application of current may have increased
the extent of PCP sorption to the soil interface within the elec-
trokinetic treatments, which could inhibit the efficacy of the
physical remediation strategy.
Bioaugmentation failed to reduce PCP concentrations suffi-
ciently to remove its toxic effect, as observed by a decrease in
carbon substrate utilisation and soil microbial respiration. The
addition of PCP affected the order of utilisation of carbon sour-
ces within the BIOLOGÔ plates. However, it remains difficult
to hypothesise why these changes may have occurred as few
publications exist regarding the significance of the order of sub-
strate utilisation, in environmental terms. Nevertheless, the
greater use of L-glutamic acid observed within this study is
a likely consequence of the greater number of PCP degrading
organisms within the soil; glutamic acid is the precursor of
glutathione, which PCP degrading bacteria have to synthesise
in order for degradation to occur (Leung et al., 1997).
Both the application of PCP and electrokinetics were
previously shown to induce changes in the soil microbial
community (Martins et al., 1997; Lear et al., 2004, respec-
tively), the latter reducing bacterial numbers close to the
anode. In addition to any direct impact of electrokinetics on
the microbial community, the movement of PCP towards the
anode end of the electrokinetic cell, as detected in this study
(similarly observed in Harbottle et al., 2001) may have caused
further disruption to the microbial community. It has been
suggested that PCP may be more toxic under acidic condi-
tions, as PCP is in its phenolic, lipophilic form. Moreover,
the most significant influence of electrokinetics on the soil is
thought to be due to changes in soil pH, as oxidation reactions
at the anode generate an acid front, whilst reduction at the
cathode produces a base front (Acar and Alshawabkeh,
1993). Although the pH was controlled in the cathode
chamber, acidic conditions developed in the soil close to the
anode (pH w 2, as similarly observed in Lear et al., 2004),
which may have caused stress to exposed soil microorganisms,
reducing their tolerance to the pollutant. Hence, the decrease
in culturable counts of both bacteria and fungi may have
been enhanced by the combination of low pH and soil PCP
toxicity that interacted synergistically to make conditions
even more harmful. It is known that PCP may retain its
biocidal activity, even in its bound form by releasing small
amounts of soluble residues (Scheunert et al., 1995). We there-
fore suggest the prevailing acidic conditions acted to increase
the toxicity of PCP within the soil. That counts of soil bacteria
and fungi were not more greatly impacted (at least close to the
anode) could be the result of the soil pH profile and PCP
distribution within the microclimate of the soil, which
combined to produce a protective effect. Microorganisms lo-
cated in the inner compartment of soil microcosms may
have been relatively unaffected, as they were not exposed to
the same pH profile or PCP toxicity as those at the exterior
(Martins et al., 1997), or within the free pore water.
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G. Lear et al. / Environmental Pollution xx (2006) 1e8
Our previous studies demonstrated that microbial respiration
may increase close to the anode even when culturable counts of
soil microorganisms decrease (Lear et al., 2004). It is possible
that this was a stress-induced response. However, when apply-
ing electrokinetics to a PCP contaminated soil, microbial respi-
ration was greatly reduced, especially close to the acidic anode
(23%), a likely consequence of the significant decline in micro-
bial counts and biomass. PCP contamination reduced the extent
to which the polymers were utilised, whilst increasing the uti-
lisation of the simpler amino acids and amines.
In conclusion, the results of this study (as Lear et al., 2004)
demonstrate that the application of electrokinetics to soil alters
both the physico-chemical characteristics of the soil and the ex-
posed microbial community. The direct effect of the applied
current on soil bacteria could not be ascertained, this influence
being inseparable from other electrochemically induced soil
changes such as soil acidification close to the anode. However,
the combined effects of PCP and electrokinetic exposure
reduced microbial counts, respiration and carbon substrate
utilisation potential greatly, probably in part at least, as a conse-
quence of the increased toxicity of PCP at lower soil pH. Further
studies are now required to examine the impact of electrokinet-
ics and other physical remediation strategies on soil microbial
communities, also assessing the long-term sustainability of
such approaches. Whilst the small-scale laboratory-based mi-
crocosms used within this study allowed differing treatment re-
gimes to be easily assessed and regulated, we must remain
cautious of extrapolating such findings to the field scale. Here
non-regular influences including soil heterogeneity and chang-
ing weather patterns will further influence results and process
design may vary, conforming to the constraints of time, eco-
nomics and land ownership. In addition to the influence of elec-
trokinetics, mass transfer processes may also have a greater
effect on soil pH and PCP distribution. It is hoped that future
work will begin to examine the effects of electrokinetics on
soil microbial communities at the field-scale, an issue that has
so far been widely overlooked. This will help to ensure that
the ability of soils to microbially remediate further incidences
of pollution are not unnecessarily compromised by otherwise
well-intentioned remedial intervention.
Acknowledgements
This work was funded via an Engineering and Physical
Sciences Research Council (EPSRC) CASE studentship with
CEH-Oxford. The majority of the experimental apparatus
used was expertly constructed by Mr C. Waddup within the
Department of Engineering Science, University of Oxford.
We thank Paula Clasper and Tatiana Bouchard at the Univer-
sity of Lancaster, UK, for their kind assistance in using the
soil oxidation equipment.
References
Acar, Y.B., Alshawabkeh, A., 1993. Principles of electrokinetic remediation.
Environmental Science and Technology 27, 2638e2647.
Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
Pollution (2006), doi:10.1016/j.envpol.2006.06.037
7
Acar, Y.B., Gale, R.J., Alshawabkeh, A.N., Marks, R.E., Puppala, S.,
Bricka, M., Parker, R., 1995. Electrokinetic remediation e basics and
technology status. Journal of Hazardous Materials 40, 117e137.
Chaudri, A.M., McGrath, S.P., Knight, B.P., Johnson, D.L., Jones, K.C., 1996. Tox-
icity of organic compounds to the indigenous population of Rhizobium legumi-
nosarum biovar Trifolii in soil. Soil Biology and Biochemistry 28, 1483e1487.
Chaudri, A.M., Lawlor, K., McGrath, S.P., 2000. Pentachlorophenol utilisation by
indigenous soil microorganisms. Soil Biology and Biochemistry 32, 429e432.
Dercová, K., Čertı́k, M., Mal’ová, A., Sejáková, A., 2004. Effect of chlorophe-
nols on the membrane lipids of bacterial cells. International Biodeteriora-
tion and Biodegradation 54, 251e254.
EPA, 1986. Chemical Fact Sheet: Pentachlorophenol, Environmental Protection
Agency (US EPA) gopher://ecosys.drdr.Virginia.EDU/00/library/gen/
toxics/pentachlorophenol. 2002
Gonzalez, J.F., Hu, W.S., 1991. Effect of glutamate on the degradation of
pentachlorophenol by Flavobacterium sp. Applied Microbiology and
Biotechnology 35, 100e104.
Harbottle, M.J., Sills, G.C., Thompson, I.P., Jackman, S.A., 2001. Movement
of pentachlorophenol in unsaturated soil by electrokinetics. Proceedings of
the Third Symposium and Status Report on Electrokinetic Remediation,
Karlsruhe, Germany, April 17, pp. 1e13.
IRPTC, 1983. Data Profile on Pentachlorophenol. International Register of
Potentially Toxic Chemicals. United Nations Environment Program,
Geneva.
Karlson, U., Miethling, R., Schu, K., Scholtz Hansen, S., Uotila, J., 1995. Bio-
degradation of PCP in soil. In: Hinchee, R.E., Wilson, J.T. (Eds.), Biore-
mediation of Recalcitrant Organics 3(7). Batelle Press, 380 pp.
Lear, G., Harbottle, M.J., van der Gast, C.J., Jackman, S.A., Knowles, C.J.,
Sills, G.C., Thompson, I.P., 2004. The effect of electrokinetics on soil
microbial communities. Soil Biology and Biochemistry 36, 1751e1760.
Leung, K.T., Cassidy, M.B., Shaw, K.W., Lee, H., Trevors, J.T., Lohmeir-
Vogel, E.M., Vogel, H.J., 1997. Pentachlorophenol biodegradation by
Pseudomonas spp. UG25 and UG30. World Journal of Microbiology and
Biotechnology 13, 305e313.
Lilley, A.K., Fry, J.C., Bailey, M.J., Day, J., 1996. Comparison of aerobic
heterotrophic taxa-isolated from four root domains of mature sugar beet
(Beta vulgaris). FEMS Microbiology Ecology 21, 231e242.
Maini, G., Sharman, A.K., Knowles, C.J., Sunderland, G., Jackman, S.A.,
2000. Electrokinetic remediation of metals and organics from historically
contaminated soil. Journal of Chemical Technology and Biotechnology 75,
657e664.
Martins, J.M., Monrozier, L.J., Chalamet, A., Bardin, R., 1997. Microbial response
to repeated applications of low concentrations of pentachlorophenol in Alfisol
under pasture. Chemosphere 35, 1637e1650.
McAllister, K., Lee, H., Trevors, J.T., 1996. Microbial degradation of
pentachlorophenol. Biodegradation 7, 1e40.
Mueller, J., Middaugh, D.P., Lantz, S.E., Chapman, P.J., 1991. Biodegradation
of creosote and pentachlorophenol in contaminated groundwater: chemical
and biological assessment. Applied and Environmental Microbiology 57,
1277e1285.
Muir, J., Eduljee, G., 1999. PCP in the freshwater and marine environment of
the European Union. The Science of the Total Environment 236,
41e56.
Öhlinger, R., 1996. Soil respiration by titration. In: Schinner, F., Öhlinger, R.,
Kandeler, E., Margesin, R. (Eds.), Methods in Soil Biology. Springer-
Verlag, London, pp. 95e98.
Reid, B.J., Stokes, J.D., Jones, K.C., Semple, K.T., 2000. Nonexhaustive
cyclodextrin-based extraction technique for the evaluation of PAH bio-
availability. Environmental Science and Technology 34, 3174e3179.
Salminen, J.E., Sulkava, P.O., 1997. Decomposer communities in contami-
nated soil: Is altered community regulation a proper tool in ecological
risk assessment of toxicants? Environmental Pollution 97, 45e53.
Salminen, J.E., Haimi, J., 1998. Responses of the soil decomposer community
and decomposition processes to the combined stresses of pentachlorophe-
nol and acid precipitation. Applied Soil Ecology 9, 475e481.
Scheunert, I., Attar, A., Zelles, L., 1995. Ecotoxicological effects of soil-bound
pentachlorophenol residues on the microflora of soils. Chemosphere 30,
1995e2009.
 ARTICLE IN PRESS
+ MODEL

8 G. Lear et al. / Environmental Pollution xx (2006) 1e8

Seech, A.G., Trevors, J.T., Bulman, T.L., 1991. Biodegradation of pen-
tachlorophenol in soil: the response to physical, chemical, and
biological treatments. Canadian Journal of Microbiology 37,
440e444.
Steiert, J.G., Thoma, W.J., Ugurbil, K., Crawford, R.L., 1988. P-31 Nuclear
magnetic-resonance studies of effects of some chlorophenols on Escheri-
chia-coli and a pentachlorophenol-degrading bacterium. Journal of
Bacteriology 170, 4954e4957.
Virkutyte, J., Sillanpaa, M., Latostenmaa, P., 2002. Electrokinetic soil remedi-
ation. The Science of the Total Environment 289, 97e121.
Wall, A., Stratton, G., 1994. Effects of a chromated-copper-arsenate wood pre-
servative on the growth of a pentachlorophenol degrading bacterium.
Water, Air and Soil Pollution 82, 723e737.
Wild, S., Harrad, S.J., Jones, K.C., 1993. Chlorophenols in digested UK
sewage sludges. Water Research 27, 1527e1534.
You, C.N., Liu, J.C., 1996. Desorptive behaviour of chlorophenols in
contaminated soil. Water Science and Technology 33, 263e270.
Zelles, L., Scheumert, I., Korte, F., 1986. Comparison of methods to test
chemicals for side-effects on soil-microorganisms. Ecotoxicology and
Environmental Safety 12, 53e69.

Please cite this article in press as: G. Lear et al., Impact of electrokinetic remediation on microbial communities within PCP contaminated soil, Environmental
Pollution (2006), doi:10.1016/j.envpol.2006.06.037