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Tributyltin (TBT) Biodegradation and Bioremediation by Alkaliphilic, Hassan, 2017
Tributyltin (TBT) Biodegradation and Bioremediation by Alkaliphilic, Hassan, 2017
Tributyltin (TBT) Biodegradation and Bioremediation by Alkaliphilic, Hassan, 2017
PII: S0141-8130(17)33210-5
DOI: https://doi.org/10.1016/j.ijbiomac.2017.11.025
Reference: BIOMAC 8505
Please cite this article as: Hamdy A.Hassan, SugE belongs to the Small Multidrug
Resistance (SMR) Protein Family involved in Tributyltin (TBT) Biodegradation and
Bioremediation by Alkaliphilic Stenotrophomonas chelatiphaga HS2, International
Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.11.025
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SugE belongs to the Small Multidrug Resistance (SMR) Protein Family involved in
Hamdy A. Hassan
Current address: Department of Biological Science, Faculty of Science and Humanity Studies at Al-
E-mail: hamdy.hassan@gebri.usc.edu.eg
1
ABSTRACT
Tributyltin (TBT) used in a variety of industrial processes, subsequent discharge into the
environment, its fate, toxicity and human exposure are topics of current concern. TBT degradation
by alkaliphilic bacteria may be a key factor in the remediation of TBT in high pH contaminated
sites. In this study, Stenotrophomonas chelatiphaga HS2 were isolated and identified from TBT
contaminated site in Mediterranean Sea. S. chelatiphaga HS2 has vigor capability to transform TBT
into dibutyltin and monobutyltin (DBT and MBT) at pH 9 and 7% NaCl (w/v). A gene was
amplified and characterized from strain HS2 as SugE protein belongs to SMR protein family, a
reverse transcription polymerase chain reaction analysis confirmed that SugE protein involved in
the TBT degradation by HS2 strain. TBT bioremediation was investigated in stimulated TBT
contaminated sediment samples (pH 9) using S chelatiphaga HS2 in association with E. coli BL21
(DE3)-pET28a(+)-sugE instead of S chelatiphaga HS2 alone reduced significantly the TBT half-life
from 12d to 5d, although no TBT degradation appeared using E. coli BL21 (DE3)-pET28a(+)-sugE
alone. This finding indicated that SugE gene increased the rate and degraded amount of TBT and is
Introduction
Tributyltin (TBT) has different commercial uses, include wood treatment and preservation,
antifouling of boats (in marine paints), antifungal action in textiles and industrial water systems,
such as cooling tower and refrigeration systems, wood pulp and paper mill systems, and breweries
[1-2].
TBT exposure are generally proposed either directly by ingestion of contaminated seafood
or indirectly by exposure from household items containing butyltin compounds [3]. TBT leads to
the contamination of foodstuff and beverages like drinking water and wine [4]. Several studies
2
either in vivo or in vitro have suggested that potential adverse effects of TBT in humans include
cardiovascular, respiratory and reproductive deficiencies [4] and also suggest that TBT has a role
in the human immune response by affecting on the lymphocytes B and reducing natural killer
Generally, TBT is more toxic than dibutyltin (DBT) and monobutyltin (MBT) [5]. Some
countries continued using TBT, although International Maritime Organization has been banned it
[6].
TBT is toxic to most of bacterial strains collected from sediments, where Gram- positive bacteria
were more sensitive to TBT [7-8]. The resistance of bacteria to TBT can be happened by three
methods, firstly: removing of butyl groups from the tin atom, this lead to DBT and MBT formation,
secondly: through a multidrug efflux pump by pumping out TBT [9] thirdly: some bacterial cells
have the capability of TBT bioaccumulation [10]. TBT can be degraded by bacteria either Gram-
negative or Gram-positive bacteria [8,11,12], but still agape which gene/s responsible for TBT
Alkaliphiles represent 1-10% from the aerobic organisms and most of alkaliphiles are also
halophiles, because alkaliphiles require some sodium ions to grow [16-17]. Hydrocarbons wastes in
high pH industrial wastewaters can be treated by alkaliphiles [18]. Moderate alkaliphiles and
halophiles can be used in different promising application compared with other extremophiles
[16,19,20].
Small multidrug resistance (SMR) proteins are found in most of the bacterial plasma
membranes [21] that allow resistance to lipophilic cations and antibiotics especially quaternary
ammonium compounds (QAC) [22]. SMR proteins are multidrug transporters family in bacteria
with short length (105-150 amino acids), they have a wide diversity in structure and function [22-
24]. Suppressor of groEL mutations (SUG) is a major protein subclass from the SMR protein family
allowing bacteria to resist quaternary cation compound (QCC) [22, 24]. SugE was discovered in
3
Aeromonas molluscorum Av27, as sugE involved in TBT resistance without indication for its role
effects, which usually has a high public acceptance and can often be carried out on site without
changing the environment. [25-26], where bioaugmentation is considered one of the most efficient
unique metabolic profiles are used to treat the contaminated sites [28]. Most experiments dealing
Sphingobium and Achromobacter [29-31] and also with Gram-positive bacteria as Rhodococcus,
Mycobacterium and Bacillus [32-34]. Thus, biological methods using microorganisms or microbial
consortia capable of pollutant degradation have a great appeal in their potential application for
environmental remediation. GMOs and their genetic transfer of catabolic genes using molecular
biology is a possible method for engineering or enhancing remediation genes to the pollutants in
To our knowledge, there is no halo or alkaliphilic bacteria from Mediterranean Sea have been
isolated and reported to exhibit resistance or possess a degradation and remediation capability to
TBT. The objective in this study, is to isolate and characterize alkaliphilic bacterium from the
Mediterranean Sea, Abu Qir- coastline, Egypt, that have the capability to degrade TBT into DBT
and MBT and try to find gene/s responsible for the biodegradation and bioremediation of TBT by
4
TBT contaminated samples were collected from water and sediments in the Mediterranean Sea,
Abu Qir-coastline, Egypt. Samples were placed in sterile bottles. Enrichment cultures were
prepared by adding 1 ml of water sample to 9 ml of mineral medium (MM) [36] with TBT (125
μM), 3% NaCl (w/v) and pH 9, the cultures were incubated at 30°C, and 10% of the culture was
transferred after one month to fresh MM containing pH 7 – 11. MM agar plates supplemented with
TBT were spread with dilutions of the culture and incubated for 7 days. The purified isolate was
selected and identified by 16S rRNA; three primers were used for the amplification of 16S rRNA.
3’) [37], full-length sequence (1492bp) of the 16S rRNA was obtained, and aligned using Clustal W
implemented in MEGA software version 3, 1 [38]. The phylogenetic tree was constructed using
Phylogeny.Fr [39].
HS2 sample was spread on MM containing 125μM TBT, The isolate was successively
transferred to MM plates containing TBT concentrations (0- 3) mM. The pure isolate was tested to
the antibiotic sensitivity according to the Clinical Laboratory Standard Institute [40].
Bacterial pure culture from HS2 was grown in minimal medium (MM) containing 2 mM of TBT
until growth reached late exponential phase. Cells harvested, and resuspended to an OD600 nm of 5.
The effect of salt and pH on TBT biodegradation was determined at (0–12%) NaCl (w/v) and pH
5
To quantify the growth rate of S. chelatiphaga HS2 and TBT disappearance, HS2 was grown as
described above and the cultures were harvested during the late of exponential growth phase by
centrifugation at 7000 rpm for 10 min. Cells were washed twice with 50mM phosphate buffer (pH
9) and resuspended in liquid MM to give an OD600nm of 0.1. Degradation of TBT was tested in
sterilized glass tubes containing 2ml cell suspension (OD600nm = 0.1) and 2mM of TBT as sole
carbon source. The test tubes were incubated at 150 rpm and 30°C. For the estimation of the colony
forming units (CFU) aliquots were serially diluted, 100μl aliquots were plated on solid LB medium
and the CFUs counted after 2 days incubation at 30°C. Uninoculated tubes and tubes without
substrate served as controls. For TBT analysis the sample vial was purged with nitrogen gas to
achieve an inert atmosphere chamber to exclude oxygen and water. A 90 μl solution of 0.8M n-
hexylmagnesium bromide was added and derivatized for 30min. The reaction was stopped by
adding 1mL of 2M HCl and the solution was set aside for 30 min. The organic phase was separated
by n-hexane and moisture was removed with anhydrous ammonium sulfate, 1 μl was injected to gas
chromatography (Agilent 7890A GC system) with flame ionization detector (FID). The GC
analyses were performed under flow of nitrogen (2 ml/min) on HP-5 column (30 m length, 0.25 mm
I.D., 0.25 μM film thickness). The oven temperature was increased from 80˚C to 320 ˚C by a rate of
3˚C/min. TBT and Dibutyltin dichloride (DBT), purchased from Sigma Aldrish, Germany) were
injected to GC-FID to act as standards according to their retention time and peak area
Genomic DNA was extracted from the strain HS2 grown on MM + Na2CO3 (pH 9) and salinity
(NaCl 7%) in the presence of TBT as a sole carbon source. The isolated DNA was screened for the
nucleotide sequences determined in this study were compared with existing sequences in GenBank
6
2.6 Extraction of mRNA, cDNA synthesis, and RT-PCR
To assess constitutive expression, S chelatiphaga HS2 was grown on TBT (2 mM) and was
grown in parallel on Glucose (2 mM) as the sole carbon source, the cultures were harvested during
exponential growth by centrifugation. Total RNA was isolated from 3 ml of TBT- or Glucose-
grown cells. Harvested cells were resuspended in 100 µl water and immediately processed as
previously described [43]. The RNA was subsequently purified using the RNeasy kit (Qiagen), and
2 µl of the eluted RNA (60µl) was separated in 1% agarose gels and stained with ethidium bromide.
cDNA was synthesized from 1 µl of total RNA using a RevertAid First Strand cDNA Synthesis Kit
(Thermo Scientific) from RNA templates. The kit uses RevertAid Reverse Transcriptase (RT), a
supplied with the kit, effectively protects RNA templates from degradation. The reverse
transcription reaction mixtures were serially diluted (3.2-fold) with nuclease-free water (Qiagen),
and 1 µl of each dilution was subjected to amplification by PCR using the primer set TBTRNAF
3’) to SugE gene fragment were amplified, purified and sequenced to verify their identity.
The suitable expression in E.coli, sugE was cloned into the expression vector pET28a (+). The sugE
gene was amplified by PCR with the primers pairs which was added a Nde I and Sac I site
(DE3) for expression. Strain BL21 (DE3)-pET28a(+)-sugE was grown in LB with antibiotic to an
OD600 of 0.6, at which 0.5 mM IPTG was added to induce the protein expression, after one hour it
7
2.8 Bioremediation of TBT in the sediments
Sediment from Mediterranean Sea, Abu Qir-coastline, Egypt was homogenized and spiked with
TBTCl at 150µg kg-1 to obtain a control sample, 2 kg of spiked sediment was transferred into a 2L
sterilized glass flask. Three enhanced samples each has 2kg of spiked sediment pH 9 in 2L
sterilized glass flask, one was inoculated by 300 mL of a cell culture from E. coli BL21 (DE3)-
pET28a(+)-sugE, prepared previously to obtain approximately 108 cells g-1 of sediment, the second
was inoculated by 300 mL of a cell culture from S. chelatiphaga HS2 to get 108 cells g-1 of
sediment and the third was inoculated with 150ml from E. coli BL21 (DE3)-pET28a(+)-sugE and
150ml from S. chelatiphaga HS2 (1:1) tell 108 cells g-1 of sediment. The samples were aerated and
incubated at 28⁰C to start the degradation. Subsequently, the samples were homogenized and water
was replenished before sediment was collected periodically up to 20 d. Analysis of TBT compounds
were carried out to determine degradation performance as described previously with [44-45]
The 16S rRNA sequence for the reported strain in this study S. chelatiphaga HS2, has been
deposited in the GenBank database under accession numbers (KR780649) and SMR gene (sugE
3. Results
In this study alkaliphilic bacterial culture capable of growing on TBT as a sole source of carbon and
energy was isolated from alkaline and saline water samples from Port of Abu Qir, Egypt. The
isolate could grow at pH 7.5-10, with optimum growth at pH 9 and could also grow at 1.5-10%
NaCl, with optimum growth at 7% NaCl. The isolate couldn’t grow at lower pH7 or lower
salinity1.5%. 16S rRNA sequence result suggested that the isolates were phylogenetically most
8
closely related to Stenotrophomonas chelatiphaga strain LPM-5 with 98% similarity and was
TBT and its degradation products (DBT and MBT) were measured during incubation for 40 hours
in MM containing TBT concentrations (0-3mM) and NaCl (7%) at pH 9, and there is no growth or
TBT degradation on the control (media without inoculation). The results showed that HS2 strain
can grow at high TBT concentrations (up to 3mM) and its best growth was at 2mM TBT
concentration. Moreover, HS2 showed a decrease in TBT percentage and generation of DBT and
MBT as degradation products. This indicated that this bacterial strain has the capability process to
reduce the TBT concentration. There is no degradation appeared by increasing salinity above 10 %
Strain HS2 degraded more than 90 % of the TBT, converting 50% into DBT and 35% into
MBT in the presence of its best conditions for growth ( pH 9, 7% salinity and 30˚C). The
degradation proceeded according to the postulated scheme shown in (Fig. 1). Phylogenetic analysis
of 16S rRNA gene for Stenotrophomonas chelatiphaga HS2 showed 98% similarity with
KB2. (Fig. 2)
TBT degradation with S. chelatiphaga HS2 in the presence of the best conditions (pH 9, 7%
NaCl and 30˚C) showed a decrease in TBT concentration profile between 10 hours and 30 hours
consistent with the rise of bacterial growth (Fig. 3), whereas the degradation rate declined along
with the available nutrients between 30 hours and 50 hours, achieving more than 95 % of
degradation within 50 hours. The degradation of TBT contrasted with generation of DBT and MBT
as intermediate products. DBT concentration increases during incubation, reaching around 50% of
the original TBT concentration (2mM), MBT was detected with 35% during this period of
9
incubation, the cultured media showed the brown, yellow and white color coincided with the
postulated products that could be inorganic tin in the form of insoluble compounds. S. chelatiphaga
HS2 strain has the capability to use TBT as carbon source in a mineral salt medium with 7% salinity
and pH 9.
S. chelatiphaga HS2 degraded TBT into DBT and MBT as less toxic compounds. TBT
degradation has been determined by using GC-FID [46] and standard TBT and DBT with retention
time (21.020 and 14.226min), the derivatives solution for HS2 bacterial sample showed peaks with
the same retention time for TBT and DBT (Fig. 4), while MBT was predicted from the sample
because it is less polar than DBT and TBT, this results indicated that HS2 has effective debutylation
The results showed HS2 was sensitive to all antibiotics tested except Ampicillin, amoxicillin-
clavulanic acid30 mcg/disc as ß-lactams antibiotics, and this confirm that there is no potential risk
A gene was amplified using specific primers [24]. The obtained fragment was 315bp, whose
deduced 104 amino acids sequence have high similarity (97%) with Av27-sugE gene from
Aeromonas molluscorum Av27, that is involved in TBT degradation by this strain [24], (91%)
highly homologous with the small multidrug resistance (SMR) family SMR from Aeromonas rivuli
and (83%) to the Aeromonas hydrophila-sugE belonging to the small multidrug resistance (SMR)
family, which includes genes involved in the transport of lipophilic drug, where HS2-sugE in this
study showed ~70% highly similarity with the other reprted sugE protein from the strains
Rhizobium sp. NFR03, Rhizobium sp. 9140, and Methylobacterium sp. 174MFSha1.1 (Fig. 5).
10
3.5 Expression of HS2-sugE gene in S. chelatiphaga HS2
To verify whether HS2-sugE gene expressed in response to TBT, RT-PCR experiments were
performed with total RNA extracted from S chelatiphaga HS2 growing on TBT and Glucose (Fig.
6A) [43, 49]. RT-PCR amplification products of the expected 300-bp size were observed with 3pg
of RNA extracted from the culture grown on TBT (Fig 6B), whereas no product was detected with
RNA extracted from the culture grown on fructose (Fig. 6C), indicating that gene transcripts are
induced at least 107 fold and also specifically induced in the presence of TBT, in addition, no
amplification products were observed in controls devoid of reverse transcriptase. Sequencing of the
approximately 300-bp product confirmed that it was identical to the corresponding sugE gene
The degradation of TBT in the sediments by S chelatiphaga HS2 was confirmed by adjusting
with E. coli BL21 (DE3)-pET28a(+)-sugE in the autoclaved sediments spiked with TBT 150µg/ k
g-1 was superior in the enhancement of TBT degradation (Fig 7B) than the inoculation by S
chelatiphaga HS2 only (Fig 7A) and deceases half-life of TBT degradation from 12d to 4d in both
inoculations DBT and MBT were appeared as result of TBT transformation, in other side there is no
any degradation for TBT either in presence of E. coli BL21 (DE3)-pET28a(+)-sugE alone or in the
control.
4. Discussion
Tributyltin (TBT) is considered as recalcitrant compound and has a toxically effect to a large
number of aquatic organisms [50-52]. Some microorganisms were as TBT resistance [53-57],
although the toxicity effect of TBT on the organisms either eukaryotic or prokaryotic [4]. S.
11
chelatiphaga HS2 showed the optimum degradation capability at pH 9 and 7% NaCl, where TBT
degradation by Aeromonas molluscorum Av27 and Aeromonas veronni Av27, which used TBT as
carbon source in a mineral salt medium [8] and showed an optimum growth with salinity 4% and
pH 9 [58-59], by increasing the salinity over 7% or alkalinity over pH 9 effect negatively on the
growth and the TBT degradation capability of HS2 as observed with most extremely halotolerant
Understanding alkaliphiles diversity and identifying its microorganisms that play a key role in
TBT degradation are important for defining new strategies for TBT bioremediation. Most of the
studies have been carried out bacterial degradation of TBT at not extreme conditions [9, 24], but
very few reports are known about biodegradation of TBT at extreme environments such as hyper
saline and alkaline conditions using pure bacterial isolates [58-59]. The polluted wastes either from
ships or factories drains in the seas or oceans often have high pH and their bioremediation using
nonalkaliphilic microorganisms is difficult because high pH and salt inhibit their growth and the
HS2 was most closely related to S. chelatiphaga LPM-5 which is aerobic EDTA degrading
bacterium [64] and S. maltophilia KB2 which is triphenyltin degrading bacterium [65]. HS2 was
sensitive for most of antibiotics indicating that it is non-pathogenic bacterium and could be suitable
for TBT bioremediation [59]. The bacterial resistances to the antibiotics have effect on human
health and may be toxic [66] as a result of horizontal gene transfer through plasmids, transposons
and integrons lead to rapid appearance of antibiotic resistance between environmental bacteria [67-
69].
supposed to be different either among bacterial genera or even the same species or strains [9, 24],
for this reasons molecular studies on TBT degraders received great attention specially genes and its
rolls in the mechanisms of TBT degradation, moreover it is not known whether alkaliphilic bacteria
12
have novel genes and pathways to degrade TBT in comparison with the identified genes by
nonalkaliphiles.
The obtained gene from strain HS2 was very similar to sugE from Aeromonas molluscorum
Av27, which has the capability to degrade TBT into DBT and MBT, and then complete
debutylation happened and free Tin obtained. The results of RT-PCR experiments showed that
SugE gene expressed only in the presence of TBT as direct evidence that S chelatiphaga HS2 is
TBT degrader and considered as a novel haloalkaliphilic TBT degrader and could be effective
candidate bacterium for TBT bioremediation in the halophilic and alkaliphilic TBT contaminated
sites.
Most xenobiotics bioremediation studies by bioaugmentation with single strains inoculation have
been carried out using Gram-negative bacteria [28], where S chelatiphaga HS2 is Gram-negative.
Inoculation natural attenuation sediments spiked by TBT with Enterobacter cloacae resulted in
half-life reduction to 10d at pH 7.5 [45]. S. chelatiphaga HS2 has the capability to degrade TBT in
half-life 12d at pH 9 in autoclaved sediments spiked by TBT without any natural attenuation, the
halfe-life of TBT reduced to only 4d using S chelatiphaga HS2 in association with E. coli BL21
(DE3)-pET28a(+)-sugE, this results confirmed that sugE overexpressed and increased the TBT
degradation resulting in reducing TBT half-life. Many studies dealt with using genetically modified
bacterial strain for hydrocarbon degradation in soil to enhance the ability of new genetically
modified bacterial strains to degrade a wide range of hydrocarbon pollutants and increase the
Laboratory-constructed strain E. coli D11-inoculated in soil contaminated with 2,4-D has the
capability to degrade 2,4-D more rapidly than the inoculated 2,4-D degrader R. eutropha JMP134
[70]. These results indicated that laboratory-constructed strains could be considered for xenobiotics
using E. coli BL21 (DE3)-pET28a(+)-sugE alone, this indicated that sugE could not start the
13
debutylation of TBT and could be another gene in S chelatiphaga HS2 initiate TBT degradation and
5. Conclusions
The finding from this study is to isolate and characterize alkaliphilic bacterium that has the
capability to degrade TBT into DBT and MBT. S. chelatiphaga HS2 was obtained, and could use
TBT as a carbon source in minimal media with 7% salinity and pH 9. S. chelatiphaga HS2 was the
most effective TBT degrader, non-pathogenic and transform most of TBT into DBT and MBT.
Molecular studies have been done to clarify that system in the TBT resistant of S. chelatiphaga
HS2, new gene was amplified and identified as sugE gene, a reverse transcription polymerase chain
reaction analysis in HS2 strain confirmed that sugE protein was contributed in the TBT degredation.
The study suggests that, to get more effectiveness bioremediation in sediments spiked with TBT, it
could be by inoculation the sediments by S. chelatiphaga HS2 together with E. coli BL21 (DE3)-
pET28a(+)-sugE. This study also mentioned for the first time that, it could be another gene initiate
the TBT degradation and then sugE gene make the TBT debutylation. S. chelatiphaga is novel
alkaliphilic strain and its TBT degradation capability has never been reported previously. Moreover,
HS2 strain is an effective candidate bacterium for TBT bioremediation in the halophilic and
Acknowledgements
14
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22
Figures Captions
Fig. 2 Phylogenetic tree based on 16S rRNA gene sequences showing the relationship
Stenotrophomonas strains. The strain followed by the accession numbers and degraded substrate“.
“TPT” Triphenyltin, “EDTA” Ethylenediaminetetra acetic acid, and “TBT” Tributyltin degrader.
Fig. 3. Growth of S chelatiphaga HS2 on 2mM TBT as a carbon source. Growth was monitored by
following colony-forming units (CFU), TBT depletion and formation of DBT and MBT were
assessed by GC.
Fig. 4 Identification by GC-FID of DBT, and MBT as metabolites produced from TBT by S.
chelatiphaga HS2 panel (C). panel (A) and (B) are authentic standard for TBT and DBT with
Fig. 5 Phylogenetic tree shows the relatedness HS2-SugE protein. The dendrogram was calculated
using Phylogeny.fr based on protein sequence alignments. HS2-SugE from S. chelatiphaga HS2 is
shown by arrow.
Fig. 6 RNA extraction from S. chelatiphaga HS2 on TBT (panel A lanes 1, 2 respectively) and
Fructose (panel A lanes 3, 4). RT-PCR amplification of SugE gene from S. chelatiphaga grown on
TBT (Panel B) or Fructose (Panel C) M, molecular weight marker Hyperladder 1 (Bioline). cDNA
generated from template RNA was serially diluted (3.2-fold) with nuclease-free water and 1 µl of
23
each dilution was subjected to amplification by PCR Panel B (Lanes 1 - 8), Panel C (lanes 1- 6).
Negative controls panel B (lanes 9-11), included RT and PCR reactions devoid of reverse
Fig. 7 Degradation and transformation of TBT into DBT and MBT in autoclaved sediment spiked
chelatiphaga HS2 and (B) inoculation with S. chelatiphaga HS2 and E. coli BL21 (DE3)-
pET28a(+)-sugE .
24
Fig. 1 Proposed TBT debutylation pathway [46- 56]
S. maltophilia TPH-6 KM386988 (Petroleum H)
Fig. 2 Phylogenetic tree based on 16S rRNA gene sequences showing the relationship
Stenotrophomonas strains. The strain followed by the accession numbers and degraded
xylenes, “TPT” Triphenyltin, “EDTA” Ethylenediaminetetra acetic acid, and “TBT” Tributyltin
degrader.
TBT
DBT
S. chelatiphaga HS2 (cfu) MBT
10 2,5
8
log cfu/ml
1,5
1
6
0,5
5
4 0
0 10 20 30 40 50
Incubation time (h)
Fig. 3. Growth of S chelatiphaga HS2 on 2mM TBT as a carbon source. Growth was monitored
by following colony-forming units (CFU), TBT depletion and formation of DBT and MBT were
assessed by GC.
A B C
Fig. 4 Identification by GC-FID of DBT, and MBT as metabolites produced from TBT by S.
chelatiphaga HS2 panel (C). panel (A) and (B) are authentic standard for TBT and DBT with
retention time (21.02min) and (14.25min) respectively.
Fig. 5 Phylogenetic tree shows the relatedness HS2-SugE protein. The dendrogram was
Fructose (panel A lanes 3, 4). RT-PCR amplification of SugE gene from S. chelatiphaga grown
cDNA generated from template RNA was serially diluted (3.2-fold) with nuclease-free water
and 1 µl of each dilution was subjected to amplification by PCR Panel B (Lanes 1 - 8), Panel C
(lanes 1- 6). Negative controls panel B (lanes 9-11), included RT and PCR reactions devoid of
Fig. 7 Degradation and transformation of TBT into DBT and MBT in autoclaved sediment
with S. chelatiphaga HS2 and (B) inoculation with S. chelatiphaga HS2 and E. coli BL21
(DE3)-pET28a(+)-sugE .