Advances in Microbiology, 2012, 2, 284-294
http://dx.doi.org/10.4236/aim.2012.23034 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Identification and Characterisation of a Bacterial Isolate
Capable of Growth on Trichloroethylene as the Sole
Carbon Source
Piyali Mukherjee, Pranab Roy*
Department of Biotechnology, Burdwan University, Burdwan, India
Received April 23, 2012; revised May 28, 2012; accepted June 13, 2012
ABSTRACT
The aim of this research work was to isolate microbes from soil, to investigate their potential use as effective bioreme-
diation tools for trichloroethylene—a potent environmental pollutant. The isolate showing good growth in presence of
TCE was named PM102. Microbiological characterisation of the PM102 isolate showed that it was a gram negative rod.
Detailed structure was revealed by scanning electron microscopy. pH and temperature optima, salt tolerance and opti-
mum TCE concentration for growth of PM102 were determined. The ability of this bacterium to degrade TCE was
studied in acidic and neutral pH by biochemical test and chloride release. Five TCE inducible bands were detected in
the protein profile of the isolate as studied by SDS PAGE. A major TCE inducible band of 51.61 kDa was excised from
the gel and injected into rabbit to raise specific antibody. The bacterium was identified as Stenotrophomonas malto-
philia PM102 by 16S rDNA amplification and sequencing. The 16S rRNA gene sequence has been deposited in the
NCBI GenBank with accession number JQ797560. This genus has not been described previously as being capable of
TCE degradation. We report for the first time a Stenotrophomonas sp. that grows on TCE as the sole carbon source.
Keywords: Trichloroethylene; Sole Carbon Source; Bioremediation
1. Introduction
Trichloroethylene (molecular formula: C2HCl3)—a vola-
tile, colourless, nonflammable liquid, was discovered by
Emil Fischer in 1864 while working on the preparation of
tetrachlorethane [1]. Germany began the first commercial
production of trichloroethylene in 1910 and the US fol-
lowed in 1925. Through the years, the amount of TCE
production in the world has increased dramatically. Ac-
cording to the World Health Organisation (WHO), TCE
is also present in the air we breathe. In 2000, concentra-
tions of TCE measured in urban areas worldwide showed
background levels between <17 and 109 ng/m3 (actual
guidelines recommend 5 ng/m3) [2]. In rural areas, levels
between 0.10 and 0.68 μg/m3 have been reported [3]
TCE is commonly used for cleaning of metals by vapour
degreasing and as a solvent in adhesives, lubricants, pes-
ticides, electronics, printing, paper, pulp and textile in-
dustries [4]. Pioneered by Imperial chemical industries in
Britain, its development was hailed as an anaesthetic
revolution. TCE is the most frequently reported soil and
groundwater contaminant on the National priority list of
the US Environmental Protection Agency [5]. Despite its
low water solubility, TCE leaches into the residual gro-
undwater. It is highly persistent with a half life of days to
several weeks and difficult to degrade. Exposure to TCE
is associated with a number of adverse health effects: it is
primarily a central nervous system depressant and was
found to be carcinogenic in experimental animals [6]. On
September 28, 2011, EPA released the final health as-
sessment for trichloroethylene (TCE) to the Integrated
Risk Information System (IRIS) database, where it has
been characterized as carcinogenic to humans and a hu-
man noncancer health hazard affecting the kidneys, liver,
male reproductive organs and developing foetus. In re-
cent years, much focus has been laid on the use of mi-
croorganisms for the elimination of TCE as it is com-
paratively low-cost and easier than other conventional
processes of decontamination.
A variety of aerobic bacteria have been reported to
degrade the pollutant trichloroethylene (TCE) come-
tabolically when they are grown on aliphatic hydrocar-
bons [7,8], aromatic hydrocarbons [9-14], ammonium
salt [15,16], or propane [17]. There are very few reports
of bacterial growth on TCE as the sole carbon and energy
source. Kitayama (1997) had reported Pseudomonas
aeruginosaJI104 as a potential microbe that can use TCE
*Corresponding author.
C
opyright © 2012 SciRes. AiM

P. MUKHERJEE, P. ROY 285
as a sole carbon source [18]. A Bacillus sp. was also re-
ported to be able to grow on medium with TCE as the
sole carbon source [19]. Stenotrophomonas sp. has been
found to play important role in biodegradation of keratin
[20], geosmin [21], atrazine [22], p-nitrophenol and 4-
chlorophenol [23] and monocyclic hydrocarbons [24].
Xanthobacter sp. grown on propene has also been found
to degrade TCE [25]. The PM102 isolate reported in this
study belongs to the class γ proteobacteria (Xanthomo-
nadaceae). This is the first instance where trichloro-
ethylene degradation activity has been found in the genus
related to Stenotrophomonas, which grows on TCE as the
sole carbon source.
2. Materials and Methods
2.1. Collection of Soil Samples
Soil samples were collected in sterile plastic bags, from
the waste disposal site of the industrial belt in Asansol
and Dhanbad. Asansol is a major coal mining and indus-
trial city with a number of iron and steel industries.
Dhanbad district across the Barakar river in Jharkhand,
India, is also a major mining area. All soil samples were
stored at –20˚C.
2.2. Isolation of Microorganisms from Soil
Samples
The microorganisms were isolated by serial dilution and
plating technique. 10 g soil sample each was suspended
in 100 ml sterile distilled water. The soil particles were
allowed to settle down and 1ml of the supernatant was
transferred to 9 ml sterile distilled water containing 0.9%
NaCl. Serial dilutions up to 10–6 were performed. 0.1ml
from all tubes were spread on plates containing minimal
medium with TCE (2 µl/ml). The minimal medium was
composed of (g/l): KH2PO4: 3; Na2HPO4: 6; NaCl: 0.5;
NH4Cl: 1; MgSO4.7H2O: 0.5; CaCl2: 0.05; agar: 2%; pH
7.4. The plates were incubated at 37˚C for 48 hrs.
2.3. Screening of Microorganisms on Medium
with TCE as the Sole Carbon Source
Trichloroethylene (GC 99.5%) was obtained from Merck
Ltd. India. 10 colonies were randomly selected from the
serial dilution plates and tested for their ability to grow
on TCE as the sole carbon source. The colonies were
transferred to tubes containing 5 ml of minimal broth
with 0.2% TCE. The minimal medium composition was
same as above (agar omitted). All 10 tubes were incu-
bated at 37˚C for 72 hrs. Growth was absent in 4 tubes.
Fujiwara test was done to check for TCE degrading ac-
tivity in the other 6 isolates. Three isolates designated:
PM101, PM102 and PM103 showed positive result in
Fujiwara reaction. The PM102 isolate showing maximum
TCE degradation activity was chosen for further investi-
gation. The isolate was grown and subcultured in mini-
mal medium slants with 0.2% TCE.
2.4. Morphological and Biochemical
Characterisation
The pure colony thus isolated was characterised by vari-
ous biochemical tests using the Bergey’s manual of De-
terminative Bacteriology, as summarised in Table 1.
The bacterial culture was streaked on respective plates
containing tryptone agar, EMB (eosin methylene blue)
agar and blood agar. The plates were incubated at 37˚C
for 24 hrs. The different colony characters were re-
corded.
Scanning Electron Microscopy
The isolate was grown in 10 ml LB and pelleted by cen-
trifugation. The cell pellet were washed with phosphate
buffer and fixed with gluteraldehyde (4% v/v) for 4 hrs.
Table 1. Growth in presence of different carbon source was
observed. No growth of the PM102 cells was seen in pres-
ence of sucrose even after 72 hrs. Even 3% sucrose did not
give any growth. The PM102 cells were able to grow in me-
dium containing 0.5% and 1% of glucose, fructose and
maltose.
Shape Short rods
Lactose fermentation Non fermentative
Gram character -
Oxidase -
Indole -
H2S +
Catalase +
Methyl red -
Vogues proskauer +
Amylase +
Gelatin +
Endospore formation -
Growth Aerobic
pH range 5 - 8
Optimum pH 5.5
Temperature range 30˚C - 42˚C
Optimum temperature 33˚C
NaCl range 0.01 M - 1 M
Optimum salt concentration 0.1 M
Growth in presence of TCE 0.1% - 0.4%
Optimum TCE concentration 0.3%

P. MUKHERJEE, P. ROY 286
After centrifugation, the cells were dehydrated by gradu-
ally lowering the concentration of alcohol: 95%, 70%,
50%. Cells were incubated 30 minutes in each of the al-
cohol concentrations followed by washing. Finally, the
cells were diluted with 50% alcohol. One drop of this cell
suspension was placed on a cover slip and studied with
Hitachi, S530 SEM at University Science Instrument
Centre, Burdwan University (Figures 1(a) and (b)).
2.5. Physiological Characterisation
2.5.1. pH
PM102 cells were grown in minimal medium with only
TCE as the carbon source (0.2%) at 33˚C. pH was varied
from 3 to 13. Optimum pH for growth was determined.
pH was rechecked after the experiment.
2.5.2. Temperature
PM102 cells were grown in minimal medium with 0.2%
peptone and 0.2% TCE and incubated at temperatures
ranging from 20˚C to 45°C. Growth was determined by
measuring O.D. at 620 nm after 24 hours.
(a)
(b)
Figure 1. (a) Scanning electron micrograph of a single bac-
terium PM102; (b) Scanning electron micrograph of a col-
ony of PM102.
2.5.3. N aCl Concentration
Cells were grown in minimal medium at 33˚C, with salt
concentration from 0.01 M to 1 M. Optimum salt con-
centration for growth was determined.
2.5.4. TCE Concentration
The cells were grown at pH 5 in minimal medium with
TCE as the sole carbon source at 33˚C. The amount of
TCE added was varied from 0.1% to 0.5%. We know that
excess TCE can inhibit growth as it becomes toxic for
the bacteria. Thus, how much TCE can be endured by the
PM102 isolate was observed.
2.5.5. Growth in Presence of Different Carbon Source
The PM102 cells were grown in minimal medium with
0.2%, 0.5% and 1% of each of the carbon source respec-
tively: glucose, fructose, maltose and sucrose. The ex-
periment was carried out in sidearm flasks and O.D. was
taken at 620 nm at 24 hrs, 48 hrs and 72 hrs respectively.
2.6. Identification of the PM102 Isolate by 16 S
rRNA Gene Amplification and Sequencing
2.6.1. D NA Extracti on
Cells obtained from 30 ml culture were resuspended in
lysis buffer [4.5 ml TE buffer, 100 µl 10 mg/ml ly-
sozyme, 450 µl of 10% SDS, 10 µl of 20 mg/ml pro-
teinase K] and incubated for 1 hour at 37˚C. DNA was
purified by phenol chloroform extraction and ethanol
precipitation (Sambrook et al. 1989).
2.6.2. 16S rRNA Gene Sequence and Phylogenetic
Analysis
PCR was carried out by using the universal primers: 27F:
5’ AGA GTT TGA TCC TGG CTC AG 3’ and 1492R:
5’ TAC GGY TAC CTT GTT ACG ACT T 3’. The am-
plification conditions were: 94˚ for 3 min for the first step,
30 cycles comprising of 94˚ for 1 min, 52˚ for 45 sec, 72˚
for 1 min and the final extention step of 72˚ for 3 min.
The PCR product obtained was purified and sequenced
by an automated DNA sequencer. The forward and re-
verse sequences were assembled through DNABaser
V35.0 software and submitted to GenBank through
NCBI online sequence submission tool: Bankit. MEGA5
software was used to construct phylogenetic tree by
aligning PM102 sequence with reference sequences ob-
tained from NCBI GenBank.
2.7. Determination of TCE Degradation Activity
by Fujiwara Test
Fujiwara test was performed to calculate the amount of
trichloroethylene remaining in the medium inoculated
with the PM102 isolate. In this reaction, polychlorinated
hydrocarbons, in presence of alkali and pyridine gives a

P. MUKHERJEE, P. ROY 287
red coloured compound [26].
PM102 cells were grown in 50 ml King’s B me-
dium (Na2HPO4—1 g/l, K2HPO4—3 g/l, NH4Cl—1 g/l,
MgSO4·7H2O—0.4 g/l) with 0.2% TCE, at pH 5 and pH
7 respectively. Cells were pelleted by centrifugation and
suspended in 10ml phosphate buffer with 0.3% TCE in
acetone. As TCE is insoluble in water, it was dissolved in
acetone. pH of the buffer was set at pH 5 and pH 7. 2 ml
aliquot was taken at the beginning (just after inoculation)
and after every 30 minutes interval up to 120 minutes
and treated with 2 ml 5N NaOH and 2 ml pyridine fol-
lowed by heating at 80˚C for two minutes. Absorbance of
the upper red phase was recorded at 470nm by spectro-
photometer. A control was set up with E. coli. The result
of this test was repeated thrice and mean value of ab-
sorbance are given. A standard curve was plotted by
varying TCE concentration from 0.01% to 0.48%, from
which the amount of TCE remaining at each time point
was calculated.
2.8. Monitoring TCE Degradation by Chloride
Release
As chloride present in minimal medium interferes with
this experiment, a chloride free minimal medium—
MCl, was formulated (K2HPO4—3 g/l, Na2HPO4—1 g/l,
(NH4)2SO4—1 g/l, MgSO4·7H2O—0.4 g/l). 10 drops of
0.3 M K2CrO4 was added to the chloride containing me-
dium and titrated against 10 mM AgNO3 taken in a bu-
rette. Initially, a white precipitate of AgCl is formed but
when free chloride is no longer left in the medium, the
solution turns reddish brown due to the formation of
Ag2CrO4. The reaction mechanism is:
Ag+ + Cl– = AgCl
2Ag+ + = Ag2CrO4
4
CrO
PM102 cells were grown in 50 ml of MCl and at pH 5
and pH 7 and harvested by centrifugation. Amount of
TCE added in this experiment was kept within the solu-
bility range of TCE in water i.e. 1.28 g/l (0.128%). The
cells thus obtained were suspended in 10 ml MCl broth
with 0.12% TCE at pH 5 and pH 7 respectively and ti-
trated against 10 mM AgNO3 after every 3 hrs interval,
from 0 hrs (just after suspension) to 30 hrs. A standard
curve was plotted by varying the concentration of NaCl
from 2 mM to 9 mM, from which the concentration of
free chloride released after the respective time intervals
were calculated. The experiment was done in triplicate
and mean values of the readings are given.
2.9. Protein Profile of the Isolate
The cells were grown in 50 ml minimal medium pH 7,
with 0.2% peptone, 0.2% TCE + 0.2% peptone and 0.2%
TCE respectively at 33˚C for 48 hrs. The cell pellet ob-
tained was suspended in 1 ml lysis buffer (10 mM PBS,
1% SDS, 0.1 mM PMSF) for 1 hr at 37˚C. The suspen-
sion was centrifuged at 10,000 rpm for 10 minutes at 4˚C
and supernatant was collected. Protein content was
measured in each sample by Bradford assay. 40 µg of
protein was loaded in each well of a 12% gel and SDS
PAGE was carried out. The gel was stained with Cooma-
ssie Blue R 250 and destained with 30% destaining solu-
tion. The gel was observed in the gel documentation sys-
tem (Vilbur Lourmat, France) and analysed with Quan-
tum Capt software.
2.10. Immunisation of Rabbit to Raise Specific
Antibody
A single TCE inducible band of 51.6 kDa was cut from
the gel and homogenised by crushing with a sterile glass
rod in Eppendrof tube containing 500 µl autoclaved wa-
ter. A rabbit was injected subcutaneously at 4 to 5 dif-
ferent sites with this protein homogenate mixed 1:1 with
Freund’s complete adjuvant twice (once per month) fol-
lowed by two more injections of the same protein ho-
mogenate mixed 1:1 with Freund’s incomplete adjuvant.
To check serum antibody titre, dot blot assay was done. 3
µl of the different dilutions (1:10, 1:100 and 1:1000) of
antigen i.e., total bacterial protein obtained from TCE +
peptone grown cells was spotted onto nylon membrane. 1
mg/ml BSA in different volumes (1 µl, 2 µl, 3 µl) was
also spotted on the membrane as control.
2.11. Western Blot
The SDS PAGE gel, after electrophoresis (unstained)
was electroblotted onto nitrocellulose membrane (Sigma
Aldrich USA) at 45 volts for 3 hours. The nitrocellulose
membrane was blocked with 3% milk powder for 1 hour
at room temperature and washed thrice with buffer A(10
mM tris HCl pH8, 1 mM EDTA pH8, 0.05% tween 20
and 0.9% NaCl), followed by incubation in 1:100 dilu-
tion of antiserum in the same buffer, at 4˚C overnight.
Then the membrane was washed with buffer A thrice,
five minutes each and incubated in 1:15,000 dilution of
Goat antirabbit IgG coupled to alkaline phosphatase
(Sigma Aldrich USA) in buffer A for 2 hours at room
temperature. The membrane was again washed in buffer
A, 5 minutes each, for three times and equilibriated for
20 minutes in alkaline phosphatase buffer (100 mM tris
HCl pH 9.5, 100 mM NaCl and 5 mM MgCl2). The mem-
brane was stained with BCIP/NBT (5-Bromo,4-Chloro,
3-Indolyl phosphate/Nitrobluetetrazolium) in alkaline
phosphatase buffer and kept in the dark.
2.12. Preadsorption of the Serum Antibody
10 µl of the peptone grown cellular proteins were spotted
on small strips of nitrocellulose membrane and air dried.

P. MUKHERJEE, P. ROY 288
These strips were immersed in 1:100 dilution of the an-
tiserum in buffer A and incubated by gentle shaking for 1
hour. The strips were removed and the antiserum thus
obtained contained antibodies specific only for TCE in-
duced proteins. This process was repeated until all the
antibodies against the common antigens were removed.
3. Results
3.1. Morphological, Biochemical and
Physiological Characterisation
Colony formed on tryptone agar was smooth, glistening,
opaque with entire margins and pale yellow in colour.
Growth on blood agar plates were green in colour with a
pungent ammonia odour while growth on eosin methyl-
ene blue (EMB) agar plate was of a pale tan colour indi-
cating PM102 to be a lactose nonfermenter. Gram nature
of the isolate was confirmed by treating a smear of the
culture on a slide with 3% NaOH. Gram negative PM102
instantly lysed and became viscous whereas a control
gram positive bacillus was not affected by the alkali
treatment [27].
The results of morphological, biochemical and physio-
logical characterisation of the PM102 isolate are summa-
rised in Table 1.
SEM micrograph reveals that the cells are rod shaped
of length 2.8 µm and diameter 0.5 µm. The cells appear
to glue to each other forming clusters that may be due to
the presence of lipopolysaccharides on the cell surface.
(Figures 1(a) and (b)).
3.2. Identification by 16S rRNA Gene
Amplification and Sequencing
The PCR product obtained was checked in 1.2% agarose
gel with 100 b.p. ladder as marker. Bands were obtained
at 1.4 kb (Figure 2). Nucleotide BLAST analysis of the
16S rRNA gene sequence of PM102 with 16S rRNA
gene sequences retrieved from NCBI GenBank, showed
99% similarity with Stenotrophomonas maltophilia strain
ATCC 19861 which belongs to the class Proteobacteriae
and phylum Xanthomonadaceae.
GenBank accession no.: The 16S rRNA gene sequence
of PM102 was deposited to NCBI GenBank under the
accession no. JQ 797560.
3.3. Phylogenetic Analysis
Phylogenetic tree constructed through MEGA5 software
using the neighbour joining method revealed that the
PM102 clustered homolog were Pseudomonas hibisicola
(Acc No. NR024709) and Pseudomonas geniculata NR-
9024708). PM102 showed the highest level of sequence
similarity with Stenotrophomonas maltophilia. (Acc. No.
NR040804) (Figure 3).
Figure 2. 1.2% agarose gel of the 16S rDNA PCR product
of PM102. Lanes 2, 3 and 5 contain PCR product of 1.4 Kb.
Lane 4 had water as control. Lane 1 contains the 100 b.p.
ladder.
3.4. Determination of TCE Degrading Activity
by Fujiwara Test
Out of the three isolates: PM101, PM102 and PM103, the
PM102 isolate showed maximum TCE degradation ac-
tivity (Figure 4(a)). The Fujiwara test for TCE degrada-
tion by PM102, showed a decrease in absorbance corre-
sponding to decrease in colour intensity of the upper
phase with time, thus confirming TCE degradation. The
control setup with E. coli gave a parallel graph indicating
no change in colour. A standard curve was set up by per-
forming the Fujiwara reaction with varying concentra-
tions of TCE. The percentage of TCE remaining in dif-
ferent set of experiments for PM102 was calculated from
the standard curve. The percentage of TCE remaining
after 120 minutes at pH 7 was 0.035% while at pH 5 was
0.069%, of the 0.3% added initially (Figures 4(b) and
(c)). From this TCE disappearance assay, it was calcu-
lated that 90% TCE was degraded at pH 7 while 77%
TCE was degraded at pH 5.
3.5. Monitoring TCE Degradation by Chloride
Release
To further confirm that PM102 was capable of TCE
mineralisation, a simple titration experiment was per-
formed to measure the release of chloride ions from TCE
by the PM102 isolate. The amount of chloride released
by the degradation of 0.12% TCE by PM102 was found
to be 1.5 mM at pH 7 and 1.45 mM at pH 5 after 30
hours respectively. Interestingly, it was noted that chloride

P. MUKHERJEE, P. ROY 289
PM102 ( JQ797560)
S tenotrophomonas maltophilia A TCC 19861 ( NR040804)
Stenotr ophomonas maltophilia IAM 12423 (NR041577)
Stenotrophomonas rhizophila e-p 10 (NR028930)
Xanthomonas axonopodis LMG 538 (NR026317 )
Xylella fastidiosa PL 788 (NR041783)
Lysobacter brunescens KCTC 12130 (NR041004)
Silani mona s l enta 25-4 (NR 02 5815)
Fulvimonas soli LMG 19981 (NR025465)
Dyella qinsenqisoli G Soil 3046 (NR041370)
Frateuria aurantiaDSM 6220 (NR040947)
Stenotropho monas hu mi R- 32729 (NR042568)
Stenotrophomonas acidaminiphila AMX 19 (NR025104)
Stenotrophomonas kor eensis TR 6 - 01 (NR041019)
Pseudomonas hibiscicola A TCC 19867 (NR024709)
Pseudomonas geniculata A TCC 1 9374 ( NR024708)
0.005
Figure 3. Neighbour joining tree based on PM102 16S rRNA gene sequence comparison with 16S rRNA gene sequences re-
trieved from the NCBI GenBank, constr ucted using MEGA 5 software. The numbers in pare ntheses are GenBank accession
numbers.
(a) (b)
(c)
Figure 4. (a) Comparison of TCE degradation activity of the three isolates: PM101, PM102, PM103 by Fujiwara test; (b) Line
graph showing the decrease in absorbance corresponding to the catabolism of TCE by PM102, measured at pH 5 and pH 7
respectively. Secondary vertical axis shows the % of TCE remaining at different time intervals as calculated from the stan-
dard curve. Fujiwara reaction was carried out with 0.3% TCE, after every 30 minutes interval, till 120 minutes; (c) Standard
curve of % of TCE plotted against absorbance. As % of TCE added increases, intensity of colour formed after the Fujiwara
eaction also increases, causing a rise in O.D. recorded at 470 nm. r

P. MUKHERJEE, P. ROY 290
released at pH 5 commenced after a lag period of 9 hours.
(Figures 5(a) and (b)). The theoretical amount of chlo-
ride released from 0.12% TCE is 13.3 mM, considering
that all the TCE added remains in solution.
3.6. Protein Profile of the Isolate
From the total protein profile (Figure 6(a)) obtained by
culturing PM102 in the presence and absence of TCE, 5
prominent TCE inducible bands were observed in lane 5,
11 and 12 (TCE). These bands were absent in the ab-
sence of TCE as seen in lane 6 and 8 (peptone). These
bands are also present in lanes 2, 3, 4, 7, 9 and 10 (TCE
+ peptone). The molecular weights of these 5 TCE in-
duced proteins are 90.25, 51.61, 38.83, 35.14 and 20.47
kDa respectively. The 5 proteins that are produced in
response to TCE may be involved in the degradation of
TCE. The TCE inducible proteins can be clearly identi-
fied in the densitogram (Figure 6(b)).
3.7. Immunoblotting
As seen in the dot blot assay, the antiserum reacted with
all the dilutions of TCE + peptone grown proteins but no
reaction was obtained with BSA (Figure 7). In he west-
ern blot analysis with the total antiserum, bands were
observed in response to TCE + peptone grown proteins
as well as peptone grown proteins (Figure 8(b)). After
the antiserum was preadsorbed on peptone grown pro-
teins, response was obtained only for TCE + peptone
grown proteins. The preadsorbed antiserum did not react
with peptone grown proteins (Fig ure 8(c )).
4. Discussion
Trichloroethylene, one of the major groundwater pollut-
ants throughout the world, is not only an occupational
health issue but also a public health concern [Hazardous
substance data bank, 2009]. Two important challenges
are encountered when one is dealing with bioremediation
of TCE. First of all, TCE is almost immiscible in water
with a solubility of 1.28 g/l. Thus, when one is studying
TCE degradation, one has to either keep the concentra-
tion of TCE within this solubility range or dissolve it in
some other organic solvent like ethanol, ether or chloro-
form—the latter option gets discouraged when one wants
to study TCE as the sole carbon source. Studies are un-
derway to demonstrate TCE degradation by an enzymatic
assay based on product formation rather than TCE dis-
appearance assay. This would allow the monitoring of
TCE degradation rates more accurately. Many microbes
have been isolated and characterised that can degrade
TCE under laboratory conditions. The second major
challenge is to see how effectively these microbes can
perform in the given set of environmental conditions. Out
of 10 colonies only 3 gave positive reaction in Fujiwara
test while maximum degradation was achieved with the
PM102 isolate. The PM102 isolate was shown to be able
to grow and degrade TCE under neutral as well as acidic
pH. This could extend its application to acidic soils as
well. There are many reports on cometabolic degradation
of TCE and the use of microbial consortia to enhance
overall TCE catabolism but research on pure cultures that
can grow on TCE as the sole carbon source, as in this
(a) (b)
Figure 5. (a) Plot showing the volume of silver nitrate corresponding to the amount of chloride released. 0.128% TCE was
added initially in a 10 ml reaction volume with PM102 cells. 2 ml of this suspension was taken for titration against silver ni-
trate, at every 3 hrs interval, up to 30 hrs. The secondary vertical axis shows concentration of chloride released by TCE min-
eralisation by PM102, at different time intervals, as calculated from the standard curve; (b) Standard curve of chloride esti-
mation plotted by varying concentration of NaCl.

P. MUKHERJEE, P. ROY 291
(a)
Lane 6 peptone Lane 7 TCE + peptone Lane 5 TCE
(b)
Figure 6. (a) 12% SDS PAGE of PM102 grown in peptone, TCE + peptone and only TCE. Lanes 6 and 8 show proteins ex-
tracted from PM102 grown in 0.2% peptone. Lanes 2, 3, 4, 7, 9 and 10 contains proteins obtained from PM102 grown in
0.2% TCE with 0.2% peptone. Lanes 5, 11 and 12 contains proteins extracted from the same isolate grown in 0.2% TCE.
Lane 13 contains BSA while lane 1 has mole cular weight marker of medium and low range. All molecular weight markers are
in kDa. (b) Densitometric analysis of the 12% gel by Quantum Capt software showing the differential expression of proteins
under varying culture c onditions –0.2% peptone, 0.2% TCE with 0.2% peptone and 0.2% TCE.
paper, will provide in depth knowledge at the metabolic
and genetic level.
4.1. Protein Profile
One of the best techniques for detecting changes in me-
tabolism is proteomics i.e. determining the complete
change in protein production in the cell when it is ex-
posed to different culture conditions. When PM102 was
exposed to TCE, peptone and TCE + peptone, 5 promi-
nent bands were detected in presence of TCE which were
absent in presence of peptone, as illustrated clearly in the
densitogram. These 5 proteins may be involved in the
catabolic pathway that leads to TCE degradation.
4.2. Monitoring TCE Mineralisation by Chloride
Release
In the titration experiment, it was interestingly noted that
chloride release at pH 5 commenced after 9 hours
whereas at neutral pH, chloride was release could be de-
tected before 3 hours. The lag period noticed at pH 5

P. MUKHERJEE, P. ROY 292
coincided with the growth pattern of PM102. The growth
of PM102 at pH 5 started after 72 hrs whereas at pH 6
and 7 started after 48hrs. After 72 hrs, growth at pH 5 was
more than that of pH 6 and 7. Thus, at pH 5, there was a
longer lag period which may be due to the fact that acidic
pH poses a stress condition to the growth and survival of
the isolate. Many bacteria have been known to produce
exopolysaccharides in response to environmental stress
like low pH and starvation [28,29]. PM102 produced a
creamy white pellicle at pH 5 that turned the medium
yellowish. Nature of this suspension was determined to
be a polysaccharide by ethanol precipitation. Presence of
carbohydrate was confirmed by acid hydrolysis and
Molisch test.
4.3. Immunoblotting
Western blot analysis carried out with rabbit antisera
should have given bands in response to the 51.16 kDa
protein (TCE induced) that was injected for immunisa-
tion of the rabbit. Somehow, the rabbit antiserum was
found to cross react with the 46.84 kDa protein in addi-
tion to the 51.16 kDa band. This 46.84 kDa protein is
common to both TCE and peptone. Thus, in Western blot
with the total antisera, the nitrocellulose membrane re-
vealed bands in lane containing proteins from TCE +
peptone grown cells as well as only peptone grown cells.
When the antiserum was preadsorbed with peptone
grown cellular proteins, all the common antibodies were
removed. The preadsorbed antiserum contained antibod-
ies specific to the 51.16 kDa TCE induced protein and
thus did not give any signal in response to the peptone
grown cellular antigen. This gives a definite proof that
the PM102 isolate does actually express proteins that are
specific for TCE degradation.
Figure 7. Dot blot assay. Spots 1, 2, 3 contained 3 µl of the
different dilutions of Ag (TCE + peptone grown proteins).
Spots A, B, C had 1 µl, 2 µl and 3 µl of 1mg/ml BSA.
(a) (b) (c)
Figure 8. (a) 12% SDS PAGE. Lane 1: Molecular weight marker, Lanes 2, 3, 4: TCE + peptone, Lane 5: TCE and Lane 6:
peptone. Molecular weights of protein markers are in kDa; (b) Western blot with total antiserum. Bands seen for TCE +
peptone as well as peptone grown proteins; (c) Western blot with antiserum Preadsorbed on peptone grown proteins. Band
een against TCE + peptone grown proteins. No reaction against peptone grown proteins. s

P. MUKHERJEE, P. ROY 293
5. Conclusions
The isolate PM102 was capable of degrading 90% TCE
at pH 7 after 48 hours of growth and 77% TCE at pH 5
after 72 hours of growth. Chloride release due to the
mineralisation of TCE by PM102 was also monitored
over a 30 hours time period. Through polyphasic identi-
fication strategy [30], this TCE degrader was found to be
a gram negative, non lactose fermenting Xanthomonad,
highly similar (99%) to Stenotrophomonas sp. By study-
ing changes in total protein expression of this isolate un-
der different culture conditions, 5 distinguishable TCE
induced bands were observed that were not present in
presence of some other carbon source such as peptone.
These differentially expressed proteins pinpoint towards
a complex catabolic pathway involving different multi-
component enzyme systems that may be involved in TCE
degradation. There is a proposed pathway of TCE deg-
radation but the exact mechanism is still unknown [31].
Further attempts will be made to isolate the genes re-
sponsible for TCE degradation and to unravel the degra-
dation mechanism in detail.
Thus, this novel isolate proves to be a promising bio-
remediation tool for eradicating a potential carcinogen
like trichloroethylene from our environment.
6. Acknowledgements
This research work was supported by The Department of
Science and Technology (DST), New Delhi, India. The
authors sincerely thank DST for the grant of INSPIRE
fellowship (Innovation in Science Pursuit for Inspired
Research). We also acknowledge Mrigendranath Mondal
endowment to our Department.
REFERENCES
[1] J. Parkhouse, “Trichloroethylene,” British Journal of An-
aesthesia, Vol. 37, No. 9, 1965, pp. 681-687.
doi:10.1093/bja/37.9.681
[2] Fact Sheet, “Trichloroethene in Indoor and Outdoor Air,”
New York State Department of Health, 2005.
http://www.victorny.org/Data/FileManager/PageDownloa
ds/Town/Modock/TCE_Facts.pdf
[3] Chapter 5.15, “Trichloroethylene, Air Quality Guide-
Lines,” World Health Organization, 2000.
http://www.euro.who.int/document/aiq/5_15trichloroethyl
ene.pdf
[4] “Documentation of Threshold Limit Values and Biologi-
cal Exposure Indices,” 6th Edition, American Conference
of Governmental Industrial Hygienists, Cincinnati, 1991.
[5] “Substances Found at Proposed and Final NPL Sites
through Update Number Three,” US Environmental Pro-
tection Agency, Washington DC, 1985, Document NPL-
U3-6-3.
[6] G. J. Hathaway, N. H. Proctor, J. P. Huges and M. L.
Fischman, “Chemical Hazards of the Workplace,” 3rd
Edition, Van Nostrand Reinhold, New York, 1991.
[7] R. Oldenhuis, R. L. J. M. Vink, D. B. Janssen and B.
Witholt, “Degradation of Chlorinated Aliphatic Hydro-
carbons by Methylosinus trichosporium OB3b Expressing
Soluble Methane Monooxygenase,” Applied and Envi-
ronmental Microbiology, Vol. 55, No. 11, 1981, pp. 2819-
2826.
[8] Alvarez-Cohen and P. L. McCarty, “Product Toxicity and
Cometabolic Competitive Inhibition Modeling of Chlo-
roform and Trichloroethylene Transformation by Metha-
notrophic Resting Cells,” Applied and Environmental Mi-
crobiology, Vol. 57, No. 4, 1991, pp. 1031-1037.
[9] K. J. Malachowsky, T. J. Phelps, A. B. Teboli, D. E. Min-
nikin and D. C. White, “Aerobic Mineralization of Tri-
chloroethylene, Vinyl Chloride, and Aromatic Compo-
unds by Rhodococcus Species,” Applied and Environ-
mental Microbiology, Vol. 60, No. 2, 1994, pp. 542-548.
[10] M. J. K. Nelson, S. O. Montgomery and P. H. Pritchard,
“Trichloroethylene Metabolism by Microorganisms That
Degrade Aromatic Compounds,” Applied and Environ-
mental Microbiology, Vol. 54, No. 6, 1988, pp. 604-606.
[11] Y. Kamagata, T. Kanagawa, R. Kurane, H. Zhang, S.
Hanada, T. Shigematsu and K. Shibuya, “Burkholderia
kururiensis sp. nov., a Trichloroethylene (TCE)-Degrad-
ing Bacterium Isolated from an Aquifer Polluted with
TCE,” International Journal of Systematic and Evolu-
tionary Microbiology, Vol. 50, No. 2, 2000, pp.743-749.
doi:10.1099/00207713-50-2-743
[12] B. R. Folsom, P. J. Chapman and P. H. Pritchard, “Phenol
and Trichloroethylene Degradation by Pseudomonas ce-
pacia G4: Kinetics and Interactions between Substrates,”
Applied and Environmental Microbiology, Vol. 56, No. 5,
1990, pp. 1279-1285.
[13] L. P. Wackett and D. T. Gibson, “Degradation of Tri-
chloroethylene by Toluene dioxygenase in Whole-Cell
Studies with Pseudomonas putida Fl,” Applied and Envi-
ronmental Microbiology, Vol. 54, No. 7, 1988, pp. 1703-
1708.
[14] M. S. Shields and M. J. Reagin, “Selection of a Pseudo-
monas cepacia Strain Constitutive for the Degradation of
Trichloroethylene,” Applied and Environmental Microbi-
ology, Vol. 58, No. 12, 1992, pp. 3977-3983.
[15] M. E. Rasche, M. R. Hyman and D. J. Arp, “Factors Lim-
iting Aliphatic Chlorocarbon Degradation by Nitrosomo-
nas europaea: Cometabolic Inactivation of Ammonia
Monooxygenase and Substrate Specificity,” Applied and
Environmental Microbiology, Vol. 57, No. 10, 1991, pp.
2986-2994.
[16] D. Arciero, T. Vannelli, M. Logan and A. B. Hooper,
“Degradation of Trichloroethylene by the Ammonia-
Oxidizing Bacterium, Nitrosonionas europaea,” Bioche-
mical and Biophysical Research Communications, 1989,
Vol. 159, No. 2, pp. 640-643.
doi:10.1016/0006-291X(89)90042-9
[17] L. P. Wackett, G. A. Brusseau, S. R. Householder and R.
S. Hanson, “Survey of Microbial Oxygenases: Trichloro-
ethylene Degradation by Propane-Oxidizing Bacteria,”

P. MUKHERJEE, P. ROY 294
Applied and Environmental Microbiology, Vol. 55, No.
11, 1989, pp. 2960-2964.
[18] P. Pant and S. Pant, “A Review: Advances in Microbial
Remediation of Trichloroethylene (TCE),” Journal of En-
vironmental Sciences, Vol. 22, No. 1, 2010, pp. 16-126.
doi:10.1016/S1001-0742(09)60082-6
[19] K. Dey and P. Roy, “Degradation of Trichloroethylene by
Bacillus sp.: Isolation Strategy, Strain Characteristics, and
Cell Immobilization,” Current Microbiology, Vol. 59, No.
3, 2009, pp. 256-260. doi:10.1007/s00284-009-9427-6
[20] S. Yamamura, Y. Morita, Q. Hasan, K. Yokoyama and E.
Tamiya, “Keratin Degradation: A Cooperative Action of
Two Enzymes from Stenotrophomonas sp.,” Biochemical
and Biophysical Research Communications, Vol. 294, No.
5, 2002, pp. 1138-1143.
doi:10.1016/S0006-291X(02)00580-6
[21] B.-H. Zhou, R.-F. Yuan, C.-H. Shi, L.-Y. Yu, J.-N. Gu
and C.-L. Zhang, “Biodegradation of Geosmin in Drink-
ing Water by Novel Bacteria Isolated from Biologically
Active Carbon,” Journal of Environmental Sciences, Vol.
23, No. 5, 2011, pp. 816-823.
doi:10.1016/S1001-0742(10)60458-5
[22] S. Rousseaux, A. Hartmann and G. Soulas, “Isolation and
Characterization of New Gram-Negative and Gram-Posi-
tive Atrazine Degrading Bacteria from Different French
Soils: Sandrine,” FEMS Microbiology Ecology, Vol. 36,
No. 2-3, 2001, pp. 211-222.
doi:10.1111/j.1574-6941.2001.tb00842.x
[23] Z. Liu, C. Yang and C. Qiao, “Biodegradation of p-Ni-
trophenol and 4-Chlorophenol by Stenotrophomonas sp.,”
FEMS Microbiology Ecology, Vol. 277, No. 2, 2007, pp.
150-156. doi:10.1111/j.1574-6968.2007.00940.x
[24] G. Urszula, G. Izabela, W. Danuta and L. Sylwia, “Isola-
tion and Characterization of a Novel Strain of Stenotro-
phomonas maltophilia Possessing Various Dioxygenases
for Monocyclic Hydrocarbon Degradation,” Brazilian
Journal of Microbiology, Vol. 40, No. 2, 2009, pp. 285-
291.
[25] M. W. Reij, J. Kieboom, J. A. M. De Bont and S. Hart-
mans, “Continuous Degradation of Trichloroethylene by
Xanthobacter sp. Strain Py2 during Growth on Propene,”
Applied and Environmental Microbiology, Vol. 61, No. 8,
1995, pp. 2936-2942.
[26] M. S. Moss and H. J. Rylance, “The Fujiwara Reaction:
Some Observation on the Mechanism,” Nature, 1966, Vol.
210, pp. 945-946. doi:10.1038/210945a0
[27] T. Gregersen, “Rapid Method for Distinction of Gram
Negative from Gram Positive Bacteria,” Applied Micro-
biology and Biotechnology, Vol. 5, No. 2, 1978, pp. 123-
127.
[28] C. C. Ling, “Bacterial Polysaccharides: Current Innova-
tions and Future Trends,” Caister Academic Press, Nor-
folk, 1999.
[29] M. Dilworth, F. G. Rynne, J. M. Castelli, A. I. Vivas-
Marfisi and A. R. Glennt, “Survival and Exopolysaccha-
ride Production in Sinorhizobium rneliloti WSM419 Are
Affected by Calcium and Low pH,” Microbi ology , Vol.
145, 1999, pp. 1585-1593.
[30] P. Van Damme, B. Pot, M. Gillis, P. Devos, K. Kersters
and J. Swings, “Polyphasic Taxonomy, a Consensus Ap-
proach to Bacterial Systematic,” Microbiological Reviews,
Vol. 60, No. 2, pp. 407-438.
[31] M. Whittaker, D. Monroe, D. Junoh and S. Anderson,
“TCE Degradation Pathway,” 2006.
http://unbbd.msi.umm.edu