Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Enhanced biotransformation of TCE using plant terpenoids

in contaminated groundwater
J.R.-M. Brown1,2, I.P. Thompson3, G.I. Paton2 and A.C. Singer4
1 Centre for Ecology and Hydrology, Oxford, UK
2 School of Biological Sciences, University of Aberdeen, Aberdeen, UK
3 Department of Engineering Science, University of Oxford, Kidlington, Oxfordshire, UK
4 Centre for Ecology and Hydrology, Wallingford, UK

Keywords
biostimulation, carvone, cumene,
groundwater, plant terpenes, secondary plant
metabolites, TCE.
Correspondence
Andrew C. Singer, Centre for Ecology and
Hydrology, Maclean Building, Benson Lane,
Crowmarsh Gifford, Wallingford OX10 8BB,
UK. E-mail: acsi@ceh.ac.uk
2009 ⁄ 1000: received 5 June 2009, revised
and accepted 9 September 2009
doi:10.1111/j.1472-765X.2009.02738.x
Abstract
Aims: To examine plant terpenoids as inducers of TCE (trichloroethylene) bio-
transformation by an indigenous microbial community originating from a
plume of TCE-contaminated groundwater.
Methods and Results: One-litre microcosms of groundwater were spiked with
100 lmol 1)1 of TCE and amended weekly for 16 weeks with 20 ll 1)1 of the
following plant monoterpenes: linalool, pulegone, R-(+) carvone, S-()) carv-
one, farnesol, cumene. Yeast extract-amended and unamended control treat-
ments were also prepared. The addition of R-carvone and S-carvone, linalool
and cumene resulted in the biotransformation of upwards of 88% of the TCE,
significantly more than the unamendment control (61%). The aforementioned
group of terpenes also significantly (P < 0Æ05) allowed more TCE to be
degraded than the remaining two terpenes (farnesol and pulegone), and the
yeast extract treatment which biotransformed 74–75% of the TCE. The micro-
bial community profile was monitored by denaturing gradient gel electrophore-
sis and demonstrated much greater similarities between the microbial
communities in terpene-amended treatments than in the yeast extract or una-
mended controls.
Conclusions: TCE biotransformation can be significantly enhanced through the
addition of selected plant terpenoids.
Significance and Impact of the Study: Plant terpenoid and nutrient supplemen-
tation to groundwater might provide an environmentally benign means of
enhancing the rate of in situ TCE bioremediation.

have been pursued as a means for remediating TCE, as

Introduction
Accidental and historical release of chlorinated compounds
such as TCE has resulted in extensive contamination of the
subsurface environment (Major et al. 1991; Shapiro et al.
2004). A combination of TCE’s physical and chemical
properties (high solubility and miscibility) (Moran 2007),
lends it to the formation of a dense, nonaqueous phase in
the subsurface (Lee 2007), resisting biotransformation
(Vogel et al. 1987) and dispersing over wide areas.
The pump and treat approach is one of the primary
mechanisms of TCE-contaminated groundwater remedia-
tion. In recent years, bioremediation and biostimulation
pump and treat is less cost-effective when applied to low-
level TCE-contaminated sites (Aulenta et al. 2005; Da
Silva et al. 2006).
TCE biostimulation has been demonstrated using
growth substrates and cometabolites such as toluene
(Hopkins and McCarty 1995), phenol (Hopkins et al.
1993) and methanol (DiSpirito et al. 1991; Kang et al.
2001). However, there are concerns about the suitability
of using highly toxic chemicals to induce TCE degrada-
tion within the environment (http://www.epa.gov/
OGWDW/contaminants/basicinformation/toluene.html, US
EPA 2000; Brautbar and Williams Ii 2002). Consequently,

ª 2009 The Authors

Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 769–774 769
Terpene-enhanced TCE biotransformation
it would be beneficial to seek alternative substrates that
would stimulate TCE-degradation without causing addi-
tional environmental concern.
A novel avenue for selecting suitable cometabolites is the
use of plant secondary metabolites. Plant secondary metab-
olites are low molecular weight plant products (Hadacek
2002) that are traditionally considered to be nonessential
for the basic metabolic processes of the plant (Dixon 2001).
Terpenes are the main components of essential oils of
plants, such as limonene from lemon oil, carvone from
spearmint oil and pinene from pine oil. A wide range of
terpenes have already been reported to stimulate microbial
degradation of xenobiotic compounds, such as polychlori-
nated biphenyls (PCB) (Gilbert and Crowley 1997; Singer
et al. 2000, 2003; Crowley et al. 2001; Oh et al. 2003). The
monoterpene cumene has previously been demonstrated to
stimulate TCE degradation in pure culture (Dabrock et al.
1992; Suttinun et al. 2004) and in soil (Suttinun et al.
2004). Hence, we hypothesized that an even wider range of
plant secondary metabolites should be applicable to
enhancing TCE biodegradation owing to many structural
similarities within the terpenoids.
In this study, we constructed one-litre microcosms to
compare TCE biotransformation by the indigenous
microbial community from a TCE-contaminated ground-
water plume. Groundwater amended with TCE
(100 lmol l)1) was spiked with one of six plant second-
ary metabolites: 1, 6-octadien-3-ol (linalool), R-(+)-
p-menth-4 (8)-en-3-one (pulegone), 2-cyclohexen-1-one
[R-(+) carvone], 2-cyclohexen-1-one [S-()) carvone],
2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl (farnesol) and
isopropylbenzene (cumene), with one treatment receiving
only yeast extract and the control remaining unamended.
TCE biotransformation was confirmed by headspace anal-
ysis-gas chromatography. The microbial communities
were monitored by denaturing gradient gel electrophoresis
(DGGE; community structure).
Materials and methods
Materials
TCE (99% purity) and S-()) carvone (98% purity), cum-
ene (99% purity), linalool (98% purity) were obtained
from Sigma Aldrich (York, UK), R-(+) carvone (98%
purity) from Lancaster (Morecambe, UK) and both pule-
gone (92% purity) and farnesol (96% purity) from Arcos
Organics (Leicestershire, UK).
Field site
Samples were collected in 1000-ml PTFE-lined screw-
capped glass bottles with no headspace from boreholes of
770 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 769–774
J.R.-M. Brown et al.
a TCE-contaminated site located in southern England.
The site covers an area of c. 3 km2 and is characterized
by a shallow chalk aquifer, protected by London clay
overlain by Silchester Gravels of Eocene age. The ground-
water contains a plume of TCE at a concentration
between 5 and 620 lmol l)1. A total of 27 boreholes rep-
resentative of the plume were sampled against a hydraulic
gradient within an area of 25 000 m2. The boreholes used
in this study contained TCE at a moderate to high level
(150–400 lmol l)1). Groundwater samples located outside
of the plume served as a control.
Microcosm preparation
Microcosms were prepared in the 1000-ml sampling bot-
tle by sparging with nitrogen to remove the ‘native’ TCE
and any additional co-contaminating volatile organics. All
microcosms were spiked with an initial concentration of
100 lmol l)1 of TCE. Each bottle was amended with
20 ll l)1 of substrate: linalool, pulegone, cumene, farne-
sol, R-(+) carvone, and S-()) carvone. The concentration
of 20 ll l)1 was chosen as it was below the concentration
of the least soluble of the terpenoids; hence, it was
assumed that all microcosms had equivalent exposure to
each of the treatment compounds. Control microcosms
were set up with no substrate and a sterilize blank which
was autoclaved at 121C, 15 psi, for 15 min. A micro-
cosm was also set up with 20 mg l)1 of yeast extract as a
terpene-free carbon source control. The nine microcosm
treatments were set up in triplicate. Microcosms were
kept at 22C in the dark with weekly addition of the
respective substrate (20 ll l)1). A small headspace volume
(c. 10 ml) was maintained in the bottles to minimize par-
titioning of TCE between the liquid and gas phase.
Hence, microaerophilic and anaerobic conditions likely
persisted in the microcosms during the majority of the
study except directly following the respiking of the meso-
cosms each week, at which point each microcosm was
vigorously shaken providing limited aeration. Samples
were withdrawn using a glass pipette directly after the
bottles had been shaken and placed directly into a 20-ml
borosilicate gas chromatograph-headspace vial and fitted
with a PTFE-lined septum crimp cap.
Analytical methods
Samples (5 ml) were amended with 50 ll of 5 mol l)1
NaOH to terminate biological activity and 2-ll dibromo-
benzene as an internal standard. The vial was placed in a
headspace sampler (Agilent 7964; Agilent Technologies,
Berkshire, UK) in shake mode, with an oven temperature
of 70C, loop temperature of 110C and a transfer line
temperature of 90C to the gas chromatograph inlet. A
ª 2009 The Authors
J.R.-M. Brown et al.
75-m · 0Æ53-mm DB-624 capillary column with a 3-lm
film (Agilent Technologies) was used to separate TCE.
The temperature programme of the Agilent 6890 gas
chromatograph (Agilent Technologies) used was as fol-
lows: 50C hold for 4 min, 40–280C for 5 min, hold for
6 min at 300C. The electron capture detector (ECD)
operated at a temperature of 300C; helium was the car-
rier gas with a flow rate of 1Æ0 ml min)1. The makeup gas
was nitrogen at 60 ml min)1. The data were recorded
using the ChemStation Software Rev A.10.02 (Agilent
Technologies). Calibration curves were prepared from
serial dilution of TCE in methanol.
Samples (1 ml) were removed from each microcosm
weekly for 16 weeks and analysed for chloride (Chen
et al. 2005) using ion chromatography on a Dionex AS50
(Dionex Corporation, Leeds, UK), employing an isocratic
pump, an Ionpac ASH-HC column autosampler and elec-
trochemical detector. Results were corrected for back-
ground chloride.
Molecular biological investigations
Total nucleic acids from the indigenous microbial com-
munity were extracted from 50 ml of groundwater sam-
ples by a combination of bead beating, enzymatic lysis
and solvent extraction for DGGE analysis, as previously
described (Griffiths et al. 2000). A 200-bp product span-
ning the V3 region of the 16S rDNA was amplified by
PCR as previously described (Muyzer et al. 1993), in a
total volume of 50 ll containing universal 16S primers
530r (5¢-GTA TTA CCG CGG CTG CTG-3¢) and the GC
clamped 338F (5¢-CGC CCG CCG CGC CCC CGC CCC
GGC CCG CCG CCC CCG CCC ACT CCT ACG GGA
GGC AGC-3¢). Approximately 1 ll of template was
amplified with 1Æ5 ll of Taq polymerase (Sigma Chemi-
cals, Dorset, UK), 0Æ5 ll of each primer, 0Æ5 ll of
100 mmol l)1 deoxynucleoside triphosphate on an MJRe-
search PTC-225 (Peltier Thermal Cycler; MJ Research
Instruments, Watertown, MA, USA). The temperature
TCE concentration mmol l–1
120
100
80
60
40
20
0 0
0 2
Figure 1 TCE concentration (lmol l)1) in microcosms during the experiment with different terpenoid (left panel) and control (right panel) treat-
ments: (d) linalool; (s) farnesol; (.) pulegone; (4) R-carvone; ( ) S-carvone; (h) cumene; (r) sterilized control; ()) TCE control and ( ) yeast
extract.
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 769–774
Terpene-enhanced TCE biotransformation
programme was as follows: 95C denaturation for 2 min
followed by 35 cycles at 95C for 60 s, annealing at 60C
for 60 s, 72C extension for 90 s and a single step of
72C for 30 min. DGGE was performed with the Bio-Rad
D Code system (Bio-Rad, Hercules, CA, USA). The PCR
product was loaded onto a 10% (w ⁄ v) acrylamide gel
containing a linear denaturant gradient of 30–60% [100%
denaturant consisted of 7 mol l)1 urea and 40% (v ⁄ v)
formamide parallel to the direction of electrophoresis].
Gels were run in a 0Æ5· TAE buffer at 60C and 100 V
for 16 h. Gels were stained with 0Æ5· TAE buffer contain-
ing SyberGold (Molecular Probes, Paisley, UK) and pho-
tographed using the VersaDOC Imaging System and the
Quantity One software (Bio-Rad). Band and profile anal-
ysis were determined using Phoretix 1D analysis soft-
ware (Phoretix International, Newcastle upon Tyne, UK).
Statistical analysis
One-way anova with Tukey HSD (honestly significant
difference) was performed by using spss 16.0 (SPSS Inc.,
Chicago, IL). The correlation coefficient r is only stated if
their significance level was at least 95% (P < 0Æ05). For
the analysis of the relationship between DGGE profiles
agglomerative hierarchical clustering was performed by
the unweighted-pair group method using arithmetic aver-
ages (UPGMA) with squared Euclidean distances which
was displayed as a dendrogram.
Results
Biodegradation experiments were carried out with
groundwater samples from a TCE-contaminated aquifer
with the substrates (i.e., terpenes and yeast extract).
Figure 1 illustrates the biotransformation of TCE in the
presence of individual terpenes, yeast extract and the una-
mended control. Biotransformation was calculated as the
per cent loss of TCE when compared to the autoclaved
control treatment, while the rate of TCE biotransformation
TCE concentration mmol l–1
120
100
80
60
40
20
4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time (weeks) Time (weeks)
771
Terpene-enhanced TCE biotransformation J.R.-M. Brown et al.

was the difference in TCE removed over the 16-week per- TCE
iod (112 days). R-(+) carvone-amended, S-()) carvone-
Yeast extract
amended, cumene-amended, and linalool-amended
groundwater resulted in significantly greater TCE Farnesol
biotransformation after 16 weeks, 82–88% (4–7 nmol (R )-Carvone
TCE ⁄ day; P < 0Æ05), than the treatments with pulegone,
Pulegone
farnesol and yeast extract (74–75%; 9–10 nmol TCE ⁄ day)
as well as the unamended control (61%; 15 nmol (S )-Carvone
TCE ⁄ day; P < 0Æ001). Cumene
Chloride originating from dechlorination of TCE
Linalool
increased sharply at week 5 in the unamended control
and in the yeast extract-amended treatment, whereas all
the other treatments showed a sharp increase in chloride
at week 7 (Fig. 2). R-(+) carvone accumulated the highest 0 2 4 6 8 10 12 14 16
amount of chloride by week 9 (181 lmol l)1) followed by Similarity index
S-()) carvone and cumene (133 and 122 lmol l)1),
Figure 3 Unweighted-pair group method using arithmetic averages
respectively. The sterilized control flask showed no degra-
dendrogram showing the similarity of denaturing gradient gel electro-
dation of TCE and no chloride accumulation. phoresis patterns of 16S rDNA extracted from the different micro-
The DGGE profile of the groundwater community cosms treatments.
between treatments was expectedly similar at the start of
the experiment, but a decline in the number of opera-
tional taxonomic units (OTUs) was observed from week
Discussion
1 through 13 (data not shown). The DGGE demonstrated
two major clusters of microbial community fingerprints: Terpene addition significantly increase the TCE biotrans-
(i) the unamended TCE treatment and the yeast extract- formation compared to the unamended control. The
amended treatment, and (ii) all the terpene-amended treatments containing R-(+) carvone, S-(+) carvone,
treatments (Fig. 3). Within the terpene cluster, there were cumene and linalool significantly stimulated the native
three subclusters (% TCE biotransfomation): (i) farnesol community to biotransform 82–88% of the TCE present
(74%) and R-(+) carvone (88%); (ii) pulegone (75%), in the respective microcosm treatment compared to the
S-()) carvone (86%) and cumene (87%); and (iii) lin- unamended treatment. Dabrock et al. (1992) observed
alool (82%). Notably, the two isomers S-carvone and 100% and 71% degradation of TCE with Pseudomonas sp.
R-carvone did not cluster together. JR1 and Rhodococcus erythropolis BD1 (isolated from
groundwater and TCE ⁄ toluene contaminated soil), using
cumene as a growth substrate (Dabrock et al. 1992), a
result which is consistent with the findings in this study
200 where 87% of the TCE was biotransformed in the pres-
ence of cumene. Other terpenes such as p-cymene, gera-
Chloride concentration (mmol l–1)

niol, citronellol, anise oil, cuminic acid and cinnamyl

150 alcohol were also used by Dabrock et al. (1992), but
<10% of TCE degradation was observed. Suttinun et al.
(2004) investigated the effect of terpenes such as cumene,
100 limonene, S-()) carvone and pinene at different con-
centrations on the rates of TCE (10 ppm) degradation
in vitro by pure Rhodococcus gordoniae cells. The authors
50 found that all of the terpenes induced greater biotransfor-
mation of TCE than the unamended control, with the
highest percent biotransformation stemming from the
0
0 2 4 6 8 10 12 14 16 18 cumene treatment, which achieved 72% TCE removal
Time (weeks) (Suttinun et al. 2004). The authors also reported signifi-
Figure 2 Chloride concentration (lmol l)1) in microcosms during the
cant changes in the amount of TCE removed depending
experiment with different treatments: (d) linalool; (s) farnesol; (.) on the concentration of terpene employed; pinene was
pulegone; (4) R-carvone; ( ) S-carvone; (h) cumene; (r) sterilized only effective in the range of 5–10 mg l)1, carvone at
control; ()) TCE control and ( ) yeast extract. 5–25 mg l)1 and cumene at 10–50 mg l)1, while limonene

ª 2009 The Authors

772 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 769–774
J.R.-M. Brown et al.
yielded significantly greater removal of TCE at all the
tested concentrations 5–50 mg l)1. The results from this
study are consistent with that of Suttinun et al. (2004)
and further extend the spectrum of terpenoids capable of
stimulating TCE biotransformation.
Chloride accumulation was used as an indication of
TCE dechlorination and biotransformation. In compari-
son to the untreated and yeast extract-amended micro-
cosm, there was a delay in chloride production by
2 weeks in the terpene-amended treatments (Fig. 2).
Although the data was not statistically significant, cell
counts by flow cytometry suggest that cell growth was
greater in terpene-amended microcosms when compared
to the control (data not shown). Hence, the delay in TCE
dechlorination in terpene-amended treatments might be
indicative of competitive exclusion of the TCE for the
more energy and carbon-rich terpene substrate.
Evident from the DGGE community fingerprints, terp-
enes enriched for a microbial community distinguishable
from the terpene-free treatments. Despite slight shifts in
the microbial community within the terpene-amended
community fingerprints, this did not appear to corre-
spond to any functional differences with regard to TCE
biotransformation. The readily distinguishable microbial
communities generated from R-(+) carvone and S-())
carvone addition yielded similarly functioning communi-
ties with regard to TCE biotransformation. These results
are consistent with a previous report that demonstrated
the capacity for river water to evolve into both function-
ally similar and distinguishable microbial communities
after exposure to the terpene isomers R-carvone and
S-carvone (Lehmann et al. 2008).
In summary, the results from this study provide evi-
dence that secondary plant metabolites such as R-(+)
carvone, S-()) carvone, linalool, pulegone and farnesol
are readily utilized by groundwater bacteria and can be
used as inducers to stimulate TCE biotransformation at a
rate in excess of unamended controls. Terpenes are non-
toxic, natural alternative substrates and as such can be
regarded as benign if released into the environment and
are often very effective at low concentrations (Singer et al.
2003). To our knowledge, this is the first study to use
pulegone, linalool and farnesol to stimulate degradation
of TCE.
Acknowledgements
The authors would like to thank the EPSRC and the
AWE plc for their financial support, Sean Amos of AWE
plc for providing groundwater samples, Ken Killham and
Graeme I. Paton for their support, Andrew Whiteley for
his help with flow cytometry and Hong Li for his help
with chloride analysis.
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 769–774
Terpene-enhanced TCE biotransformation
References
Aulenta, F., Fina, A., Potalivo, M., Papini, M.P., Rossetti, S.
and Majone, M. (2005) Anaerobic transformation of tetra-
chloroethane, perchloroethylene, and their mixtures by
mixed-cultures enriched from contaminated soils and sedi-
ments. Water Sci Technol 52, 297.
Brautbar, N. and Williams Ii, J. (2002) Industrial solvents and
liver toxicity: risk assessment, risk factors and mechanisms.
Int J Hyg Environ Health 205, 479–491.
Chen, S.P., Huang, T. and Sun, S.G. (2005) A new method of
ion chromatography technology for speedy determination
and analysis in organic electrosynthesis of glyoxylic acid. J
Chromatogr A 1089, 142–147.
Crowley, D.E., Luepromchai, E. and Singer, A.C. (2001)
Metabolism of xenobiotics in the rhizosphere. In Pesticide
Biotransformation in Plants and Microorganisms: Similarities
and Divergences ed. Hall, J.C., Hoagland, R.E. and Zabloto-
wicz, R.M. pp. 333–352. Washington, DC: American
Chemical Society.
Da Silva, M.L.B., Daprato, R.C., Gomez, D.E., Hughes, J.B.,
Ward, C.H. and Alvarez, P.J.J. (2006) Comparison of bio-
augmentation and biostimulation for the enhancement of
dense nonaqueous phase liquid source zone bioremedia-
tion. Water Environ Res 78, 2456–2465.
Dabrock, B., Riedel, J., Bertram, J. and Gottschalk, G. (1992)
Isopropylbenzene (cumene) – a new substrate for the iso-
lation of trichloroethene-degrading bacteria. Arch Microbiol
158, 9–13.
DiSpirito, A.A., Gulledge, J., Shiemke, A.K., Murrell, J.C.,
Lidstrom, M.E. and Krema, C.L. (1991) Trichloroethylene
oxidation by the membrane-associated methane monooxy-
genase in type I, type II and type X methanotrophs.
Biodegradation 2, 151–164.
Dixon, R.A. (2001) Natural products and plant disease resis-
tance. Nature 411, 843–847.
Gilbert, E.S. and Crowley, D.E. (1997) Plant compounds that
induce polychlorinated biphenyl biodegradation by
Arthrobacter sp. strain B1B. Appl Environ Microbiol 63,
1933–1938.
Griffiths, R.I., Whiteley, A.S., O’Donnell, A.G. and Bailey, M.J.
(2000) Rapid method for coextraction of DNA and RNA
from natural environments for analysis of ribosomal DNA-
and rRNA-based microbial community composition. Appl
Environ Microbiol 66, 5488–5491.
Hadacek, F. (2002) Secondary metabolites as plant traits: cur-
rent assessment and future perspectives. Crit Rev Plant Sci
21, 273–322.
Hopkins, G.D. and McCarty, P.L. (1995) Field-evaluation of
in-situ aerobic cometabolism of trichloroethylene and
3 dichloroethylene isomers using phenol and toluene
as the primary substrates. Environ Sci Technol 29, 1628–
1637.
Hopkins, G.D., Semprini, L. and McCarty, P.L. (1993)
Microcosm and in situ field studies of enhanced
773
Terpene-enhanced TCE biotransformation
biotransformation of trichloroethylene by phenol-utilizing
microorganisms. Appl Environ Microbiol 59, 2277–2285.
Kang, J.M., Lee, E.Y. and Park, S. (2001) Co-metabolic biodeg-
radation of trichloroethylene by Methylosinus trichosporium
is stimulated by low concentrations methane or methanol.
Biotechnol Lett 23, 1877–1882.
Lee, S. (2007) Enhanced dissolution of TCE in NAPL by TCE-
degrading bacteria in wetland soils. J Hazard Mater 145,
17–22.
Lehmann, K., Crombie, A. and Singer, A.C. (2008)
Reproducibility of a microbial river water community to
self-organize upon perturbation with the natural chemical
enantiomers, R- and S-carvone. FEMS Microbiol Ecol 66,
208–220.
Major, D., Hodgins, E.W. and Butler, B.J. (1991) Field and
laboratory evidence of in situ biotransformation of tetra-
chloroethene to ethene and ethane at a chemical transfer
facility in north Toronto ed. Hinchee, R.E. and Olfenbut-
tel, R.F. pp. 147–171. Boston, MA: Butterworth-Heine-
mann.
Moran, M.J. (2007) Chlorinated solvents in groundwater of
the United States. Environ Sci Technol 41, 74–81.
Muyzer, G., Dewaal, E.C. and Uitterlinden, A.G. (1993) Profil-
ing of complex microbial-populations by denaturing gradi-
ent gel-electrophoresis analysis of polymerase chain
reaction-amplified genes-coding for 16s ribosomal-RNA.
Appl Environ Microbiol 59, 695–700.
774 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 769–774
J.R.-M. Brown et al.
Oh, E.T., Koh, S.C., Kim, E., Ahn, Y.H. and So, J.S. (2003)
Plant terpenes enhance survivability of polychlorinated
biphenyl (PCB) degrading Pseudomonas pseudoalcaligenes
KF707 labeled with gfp in microcosms contaminated with
PCB. J Microbiol Biotechnol 13, 463–468.
Shapiro, S.D., Busenberg, E., Focazio, M.J. and Plummer, L.N.
(2004) Historical trends in occurrence and atmospheric
inputs of halogenated volatile organic compounds in
untreated groundwater used as a source of drinking water.
Sci Total Environ 321, 201–217.
Singer, A.C., Gilbert, E.S., Luepromchai, E. and Crowley, D.E.
(2000) Bioremediation of polychlorinated biphenyl-
contaminated soil using carvone and surfactant-grown
bacteria. Appl Microbiol Biotechnol 54, 838–843.
Singer, A.C., Crowley, D.E. and Thompson, I.P. (2003)
Secondary plant metabolites in phytoremediation and
biotransformation. Trends Biotechnol 21, 123–130.
Suttinun, O., Lederman, P.B. and Luepromchai, E. (2004)
Application of terpene-induced cell for enhancing biodeg-
radation of TCE contaminated soil. Songklanakarin J Sci
Technol 26, 131–142.
US EPA (2000). Health Effects Notebook for Hazardous Air
Pollutant, Technology Transfer Network Air Toxics Website,
Toluene. http://www.epa.gov/ttn/atw/hlthef/toluene.html.
Vogel, T.M., Criddle, C.S. and McCarty, P.L. (1987) ES&T
critical reviews: transformations of halogenated aliphatic
compounds. Environ Sci Technol 21, 722–736.
ª 2009 The Authors