Complete degradation of dimethyl phthalate by biochemical cooperation of the Bacillus thuringiensis strain isolated from cotton field soil

Article abstract of DOI:10.1039/c4ra09465d

Dimethyl phthalate (DMP), a phthalate ester, is widely used in cosmetics, perfumes, and plasticizers. It has been classified as a suspected endocrine disruptor by many countries. The present study describes the biodegradation of DMP by a new aerobic bacterium, isolated from soil samples of a cotton field by an enrichment culture technique utilizing DMP as the sole source of carbon and energy. The isolate was identified as Bacillus thuringiensis based on the morphological and biochemical characteristics as well as gene sequence analysis. Bacillus thuringiensis grows best in a mineral salt medium of pH 7.0 at 30°C incubation for 48 hours. The effects of temperature, inoculum size, substrate concentration and incubation time on DMP degradation were also studied. Bacillus thuringiensis is able to biodegrade 400 mg L^-1 of DMP under aerobic conditions with 99% degradation potential. A combination of GC and GC-MS analysis revealed a complete DMP biodegradation pathway. The results indicate that Bacillus thuringiensis may prove a promising source for DMP bioremediation at a commercial scale.

Full text of DOI:10.1039/c4ra09465d

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Complete degradation of dimethyl phthalate
by biochemical cooperation of the Bacillus
Cite this: RSC Adv., 2014, 4, 55960
thuringiensis strain isolated from cotton field soil†
a
a
Muhammad Ali Surhio, Farah N. Talpur, Shafi M. Nizamani,a Farah Amin,a
*
Chui Wei Bong,b Choon Weng Lee,b M. A. Ashrafc and Muhammad Raza Shahd
Dimethyl phthalate (DMP), a phthalate ester, is widely used in cosmetics, perfumes, and plasticizers. It has
been classified as a suspected endocrine disruptor by many countries. The present study describes the
biodegradation of DMP by a new aerobic bacterium, isolated from soil samples of a cotton field by an
enrichment culture technique utilizing DMP as the sole source of carbon and energy. The isolate was
identified as Bacillus thuringiensis based on the morphological and biochemical characteristics as well
as gene sequence analysis. Bacillus thuringiensis grows best in a mineral salt medium of pH 7.0 at
30 ꢀC incubation for 48 hours. The effects of temperature, inoculum size, substrate concentration
and incubation time on DMP degradation were also studied. Bacillus thuringiensis is able to
Received 4th September 2014
Accepted 17th October 2014
biodegrade 400 mg Lꢁ1 of DMP under aerobic conditions with 99% degradation potential.
A
combination of GC and GC-MS analysis revealed a complete DMP biodegradation pathway. The results
indicate that Bacillus thuringiensis may prove a promising source for DMP bioremediation at a
commercial scale.
DOI: 10.1039/c4ra09465d
www.rsc.org/advances
Natural processes such as hydrolysis and photo decompo-
sition have been previously reported for degradation of phtha-
lates from environment.6–8 Unfortunately degradation of DMP
under abiotic conditions has been reported to be slow and
insignicant process.3
Introduction
Dimethyl phthalate (DMP) is widely used as a plasticizer in the
plastic industry during the manufacturing of compounds such
as latex, cellulose acetate lms and plastic. DMP leaches into
the environment from tubing, dishes, paper, and containers via
the general use of plastic.1 DMP molecules are bound physically
to the plastic structure; they are easily released from plastic
products and leached into the environment.2 Moreover it is a
relatively stable compound in the natural environment with a
half-life of z20 years.3 Therefore, DMP has been frequently
identied in diverse environmental samples including
groundwater, river water, drinking water, open ocean water, soil
humates, lake and marine sediments.4 DMP promotes chro-
mosome injuries in human leucocytes and known to be an
endocrine disrupting chemical (EDC) that interferes with the
reproductive system and normal development of animals and
humans.5
Breakdown by microorganisms plays a major role in DMP
degradation and bacterial activities bring about actual assimi-
lation of various compounds under eco-friendly conditions.
Several investigators have demonstrated successful degradation
of DMP by microbes under aerobic conditions in soil, natural
water and wastewater.9 Different bacterial strains have been
isolated with the ability to degrade DMP and their isomers from
activated sludge, mangrove sediment, wastewater, etc.9–11 The
microbial strains such as Sphingomonas paucimobilis, Pseudo-
monas uorescens, Pseudomonas aureofaciens, Xanthomonas
maltophilia,12 Rhodococcus rubber,13 Pseudomonas uorenscens
FS1,14 Flavobacterium sp.,15Candida rugosa16 and Arthrobacter
sp.17 have reported as to degrade DMP. Studies have shown that
bioremediation can offer a potential solution for the conversion
of phthalic acid esters into harmless end products such as CO2
and H2O.18 However; many authors have not described the
degradation pathways, although few have reported the conver-
sion of DMP into monomethyl phthalate (MMP) and phthalic
acid (PA).
aNational Centre of Excellence in Analytical Chemistry, University of Sindh,
Jamshoro-76080, Pakistan. E-mail: farahtalpur@hotmail.com; Tel: +92-222-772065
bInstitute of Biological Sciences, Faculty of Science Building, University of Malaya,
50603, Kuala Lumpur, Malaysia
cDepartment of Geology, Faculty of Science, University of Malaya, 50603, Kuala
Lumpur, Malaysia
Hence in the present study, an aerobic bacterial strain
Bacillus thuringiensis was enriched from cotton eld soil.
Bacillus thuringiensis is a soil dwelling bacterium and is deadly
to insects but it is non-pathogenic to human beings. The
dH.E.J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi 75270, Pakistan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra09465d
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biodegradation potential of this strain is investigated rst time puried by streaking on LB Nutrient agar plates and then
in literature for DMP degradation and its end product in re-streaked onto agar plates with and without DMP to
present study. The inuences of environmental factors on conrm their degradation abilities. Isolates showing growth
degradation ability were also examined. Gene sequence and GC- in the presence of DMP were selected for further degradation
MS were employed to analyze the microbial strain structure and study.
DMP degradation intermediate respectively.
Identication and characterization of bacteria
Experimental
Gram reaction and cell morphology were determined by
observing stained cells under a light microscope. Biochemical
Chemicals
tests for the identication of bacteria were performed using
standard methods.22 The bacterial isolates of pure culture were
further characterized using 16S rRNA gene sequencing
methods. To extract the genomic DNA of bacterial strains for
16S rRNA gene amplication, we used the phenol :
chloroform : isoamyl alcohol (25 : 24 : 1) extraction and ice-
cold ethanol precipitation method. DNA was resuspended in
Dimethyl phthalate (DMP) 99% purity was purchased from
Scharlau (Barcelona, Spain). Luria–Bertani (LB) medium was
procured from Caisson laboratories inc (USA). n-Hexane and
chloroform was procured from Sigma-Aldrich (Steinheim,
Germany). All other chemical reagents used were of analytical
grade.
ꢀ
Tris–EDTA (TE) buffer (pH 8.0) and stored at ꢁ20 C. For iso-
Source of microorganisms
lated strain, one pair of universal primers was used for the
amplication of the 16S rDNA gene: the forward primer, 27F
(50-AGAGTTTGATCMTGGCTCAG-30), and the reverse primer,
1525R (50-AAGGAGGTGWTCCARCC-30) were used to produce
an amplicon of approximately 1498 bp. Polymerase chain
reaction (PCR) was performed in a 15 mL reaction mixture
containing 10 ꢂ PCR buffer, 1.5 mM of MgCl2, 0.4 mM of each
primer, 3 mL of DNA template (approximately 200 ng mLꢁ1),
The bacteria used in the degradation experiments were isolated
from cotton eld soil collected from village of district
Mirpurkhas, Sindh, Pakistan. The soil samples were stored in
plastic bags and kept at 4 ꢀC in refrigerator until start of
experiments. The soil was air dried at room temperature (28 ꢀC).
Next the soil ground gently in mortar & pestle and passed
through a 2 mm-mesh sieve to remove stones and plant debris
before the physico-chemical testing. The physical and chemical
properties of the soil are given as ESI.†
0.2 mM of dNTPs, and 0.5
U
of Taq polymerase
(FinnzymesDyNAzyme™ II, Finland). PCR amplication was
carried out according to the following conditions: initial
Enrichment and isolation of bacteria
ꢀ
denaturation at 94 C for 2 min; 35 cycles of denaturation for
ꢀ
The mineral salt medium (MSM) used in all experiments 30 s at 94 C, annealing for 40 s at 55 ꢀC, extension for 90 s at
including enrichment experiments contained (Lꢁ1): (NH4)2SO4 72 ꢀC; and a nal extension at 72 ꢀC for 7 min. PCR products were
1 g, KH2PO4 0.8 g, K2HPO4 0.2 g, MgSO4$7H2O 0.5 g, FeSO4 then puried using the QIAquick® PCR Purication Kit
0.01 g, CaCl2 0.05 g, NiSO4 0.032 g, Na2B4O7$10H2O 0.021 g, (QIAGEN, Germany) as reported earlier by Lee et al.23 The 16S
(NH4)6Mo7O24$H2O 0.0144 g, ZnCl2 0.023 g, CoCl2$H2O 0.021 g, rDNA sequences of each isolates were compared to GenBank
CuCl2$2H2O 0.01 g and MnCl2$4H2O 0.03 g.19 The pH of MSM entries using Basic Local Alignment Tool (BLAST) in order to
was adjusted to 7.0 with 0.1 M HCl or NaOH and then sterilized obtain a preliminary affiliation.
ꢀ
by autoclaving for 20 min at 121 C.
The agar plates were prepared by adding 20 g of Luria–
Bertani (LB) medium, 15 g Agar and the minimal salt medium
Sample preparation for AFM imaging
consisting of (Lꢁ1): K2HPO4 4.5648 g, KH2PO4 3.4023 g, The bacterial culture was grown in 100 mL MSM at 30 ꢀC.
Na2HPO4$12H2O 5.3721 g, NH4Cl 0.8024 g, CaCl2 0.0368 g, Bacteria were harvested at mid-exponential growth phase and
MgSO4$7H2O 0.1232 g and FeCl3$6H2O 0.0014 g, at pH 7.0.20 washed three times in phosphate-buffered saline (PBS) (con-
The enrichment procedure was similar to that described by tained 5 mM KH2PO4, 25 mM Na2HPO4, and 120 mM NaCl, pH
Chao et al.21 with some modications. Initially, 2.0 g of soil adjusted to 7.4 by using NaOH) by low-speed centrifugation, so
was added to a 250 mL Erlenmeyer ask containing 100 mL of as to avoid shear, and resuspended in PBS. The obtained solu-
MSM solution amended with DMP (1 mg Lꢁ1). The suspen- tion was vortexed for 1 min and sonicated (KQ 500-DE) for 30
sion was incubated for 7 days in the dark at 30 ꢀC in an orbital min. About 30 mL solution was taken out and deposited on a
incubator shaker operated at 150 rpm. Subsequently, 2 mL of freshly cleaved mica surface, and then the sample was analyzed
the enrichment culture was serially transferred four times to through atomic force microscopy (AFM). The images for MSM
fresh medium, each time containing a higher concentration culture was also taken directly without washing with PBS buffer
of DMP (3–10 mg Lꢁ1) and incubated under the same condi- aer appropriate dilution.
tions. Then the nal enrichment was streaked onto the
Topographic images at room temperature were recorded
agar plates and incubated one week at 30 ꢀC. Presumptive with AFM 5500 (Agilent, USA). Silicon nitride probes with a
colonies were picked on the basis of differences in colony, triangular so cantilever (Veeco, model MLCT-AUHW) with a
morphology and coloration and re-streaked onto agar plates nominal value of the spring constant of 0.01 N mꢁ1 and 0.1 N
amended with the DMP. The bacterial isolates were further mꢁ1 being used in the non-contact topography measurements.
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DMP degradation experiment
Statistical analysis
Bacterial cultures were grown in the MSM on 30 ꢀC at Excel XL State (Microso Corp., Redmond, WA) was used to
150 rpm, growing cells were then harvested aer 48 hours. For perform tests for statistical signicance. Student t-test was
degradation experiment 100 mL MSM was prepared in 250 mL performed to assess the signicance of the differences between
Erlenmeyer ask sterilized and supplemented with DMP percent degradation and experimental variables at probability
at various concentrations (100–1000 mg Lꢁ1) and then inoc- level of 0.05.
ulated with freshly harvested culture and incubated at
30 ꢀC. Aer incubation period, 30 mL of media containing
degraded DMP and n-hexane was added directly to separating
funnel and shaken vigorously for few minutes. The
Results and discussion
Identication and characterization
The strain was Gram-positive, rod-shaped and was a facultative
anaerobe. In addition, this strain exhibited positive reaction for
spore formation and negative reaction for mannitol test. The
16S rDNA partial gene sequence of this bacterium were aligned
and compared with the 16S rDNA bacterial gene sequences in
GenBank and the results indicated that it bore the closest
relationship to Bacillus thuringiensis with 97% of sequence
similarity (GenBank accession no. KF218168). The strain was
also identied as Bacillus thuringiensis through its morphology,
physiochemical characteristics and 16S rDNA sequence.
However, there have been no comprehensive studies of DMP
biodegradation by this bacterium. Only one paper is available
on concurrent degradation of dimethyl phthalate (DMP) during
production of Bacillus thuringiensis based biopesticides.24
resulting heavy emulsion was centrifuged (5000 rpm, 5 min)
and the aqueous phase was extracted. The hexane phase
was evaporated to dryness and reconstitute for further anal-
ysis. All the degradation experiments were performed in
triplicate.
GC-FID conditions for substrate analysis
The substrate analysis was done by using RT-2560 (0.25 mm ID)
100 m long polar capillary column, with the gas chromatograph
(GC-8700 PERKIN ELMER). Nitrogen was used as a carrier gas,
ꢀ
oven temperature was increased from 150 to 200 C at a ramp
rate of 11 ꢀC minꢁ1 and then nal temperature 220 ꢀC at a ramp
rate of 2 ꢀC minꢁ1 held for 20 min, injection volume was 2 mL,
injector and detector temperature were kept 240 ꢀC and 260 ꢀC
respectively.
Bacterial cell morphology by AFM analysis
The observation of the morphology of bacterial cell and their
ultrastructures is fundamental for understanding the structure
and behaviour of bacteria, since morphology is one way for
bacteria to cope with their environment and gain a competitive
advantage.25 The surface morphology of isolated strain in
various environmental conditions (buffer and media) was
observed. Freshly cleaved mica is an appropriate substrate for
visualization of bacteria. The images of isolated bacterial strain
were taken from bacterial culture harvested from growing
medium and imaged in PBS and MSM (Fig. 1a and b). Although
bacteria imaged in PBS gave universally good results in the
qualitative tests (Fig. 1a), bacterial images comparable to those
derived from mineral media were obtained by AFM. In all cases,
the bacteria maintained a hydrated appearance with no
evidence of collapse of the cell wall. Isolated bacterial strain
have different sizes, cells are longer and thicker as seen in
Fig. 1b. The obtained parameters characterizing the bacteria
dimensions for isolated strain are summarized in as: length
2.75 mm, width 0.75–1.75 mm, height 0.55 mm. The preparation
technique used gives a monolayer and bacterium is clearly seen
in AFM images (Fig. 1a). Most of bacteria are packed close to
one another, forming compact coverings due to surrounding
medium (Fig. 1b). Packing of bacteria leads to a reduced contact
area and may contribute to greater collective stability. In all
cases examined attachment was robust enough to allow indi-
vidual bacterium to be repeatedly imaged.
Spectrophotometric determinations
The microbial biomass in the culture asks was determined
spectrophotometrically by measuring optical density at 600 nm
(OD600) using UV-visible spectrophotometer (Biochrom Libra
S22).
Analysis of DMP-derived degradation products by GC-MS
To detect end products aer the course of incubation, the
samples extracted above were concentrated to 1 mL and then
subjected to GC-MS analysis using a Agilent 6890 N gas chro-
matograph, Injector auto sampler 7683-B, equipped with a
MS-5975 inert XL mass selective mass spectrometer (GC-MS,
Agilent Technologies, Santa Clara, USA). Gas chromatography
was performed on a Capillary column HP-5MS (30 m ꢂ 0.25 mm
ꢂ 0.25 mm); temperature maintained at 190 ꢀC for 1 min,
ꢁ1
ꢀ
ꢀ
increased from 15 C min to 300 C for 15 min, then cooled
rapidly to 60 ꢀC, using ultra high purity helium as carrier (ow
rate, 0.8 mL minꢁ1), injector and detector temperature were
kept 310 ꢀC and 350 ꢀC respectively. For MS spectra collected in
the positive electron ionization (EI) mode, the source temper-
ature was 250 ꢀC with an electron ionization energy of 70 eV, the
mass range was 50–550 m/z, and 1 mL of the extracted sample
was injected in split mode (10 : 1). The NIST Mass Spectral
Search Program (National Institutes of Standard and Tech-
nology, Gaithersburg, MD) was used to identify signals by
Biodegradation of DMP
comparison to the retention time and mass spectra of authentic A microbial strain capable of mineralizing DMP under aerobic
compounds.
conditions was enriched and isolated from cotton eld soil.
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Similarly Jianlong et al.4 has also reported complete degra-
dation of DMP in soil bioaugmented with acclimated activated
sludge at much slower rate i.e. within 15 days. Authors sug-
gested that the mixed microbial consortium can enhance the
degradation rates of DMP in the presence of Pseudomonas
uorescens, Pseudomonas aureofaciens and Sphingomonas
paucimobilis, possibly due to the coordinated metabolic
interaction of different bacterial strains.19 However in present
study single strain B. thuringiensis was capable of degrading
DMP in 72 h incubation time. Table 1 shows the comparison of
degradation potential of DMP by B. thuringiensis with other
species which are reported in literature.
Inuence of pH and time on growth of DMP degrading
bacteria
The culture medium hydrogen ion concentration greatly inu-
enced DMP degrading bacterial growth as pH limited enzyme
activities is shown in Fig. 3a. The growth of DMP degrading
bacteria increased rapidly when pH was increased from 5.0 to
7.0. The highest growth was achieved at pH 7.0. When the pH
exceeded 9.0, the growth decreased dramatically. Therefore,
from this study it was found that pH 7.0 was optimal for the
growth of B. thuringiensis sp.
Fig. 3b displays the effects of the time on the growth of
B. thuringiensis. The OD600 increased signicantly (P < 0.05) with
the increase of time from 2 to 48 hours, with a slight increase in
the growth rate when the time was increased to 53 hours.
However, when the time exceeded 53 hours, the microbial
biomass decreased notably. Hence 48 hours were optimal for
B. thuringiensis growth.
Fig. 1 AFM images of Bacillus thuringiensis in (a) PBS (b) MSM media.
Effects of temperature on DMP biodegradation
Bacterial growth is temperature sensitive. As shown in Fig. 4a, the
DMP degradation rate increased rapidly (P < 0.05) as temperature
increased from 25 ꢀC to 30 ꢀC. The highest DMP degradation rate
(about 99%) for B. thuringiensis sp. was achieved at 30 ꢀC.
ꢀ
Degradation decreased at temperatures above 30 C, indicating
that the optimum temperature was 30 ꢀC. The optimum
temperatures for microbes in degrading various organic pollut-
ants have been reported in the range of 30 ꢀC to 38 ꢀC.2,29,30
Fig. 2 GC-FID chromatogram of (a) control and (b) reaction with B.
thuringiensis containing DMP at 400 mg Lꢁ1
Effects of inoculum size on DMP degradation
.
Since biodegradation is highly dependent on inoculum
concentration,31 the effect of inoculum concentrations on the
Bacterial strain could utilize DMP as the sole carbon and energy
source. Results have demonstrated that DMP was completely
degraded by B. thuringiensis as evident from GC-FID chro-
matogram for control of DMP (Fig. 2a) as compared to reaction
with B. thuringiensis (Fig. 2b). It can be clearly seen that the DMP
was degraded very quickly, with more than 99% removal in less
than 72 h. Control experiment was also carried out by auto-
claving and not inoculating the ask, the results showed that no
obvious degradation was observed. This suggested that abiotic
loss of phthalates can be neglected during this study.
Table 1 % Degradation potential of DMP by Bacillus thuringiensis used
in present study in comparison with other bacterial species
Degradation potential
S. no. Micro-organism employed (%)
Reference
1.
2.
3.
4.
5.
Arthrobacter sp.
Thauera sp.
Rhodococcus sp.
Sphingomona spaucimobilis >90
Bacillus thuringiensis 99
75
22
93
17
26
27
28
This study
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Fig. 3 (a) Effect of pH and (b) time on growth of Bacillus thuringiensis.
degradation of DMP was studied (Fig. 4b). Under aerobic
conditions, degradation was only 85% when 2 mL inoculums
made up to 100 mL medium, degradation increased to 99%
when 5 mL inoculums made up to 100 mL medium. However,
further increase in the inoculums concentration did not
increase the degradation efficiency (P > 0.05). On the contrary
inoculums concentrations greater than 5 mL to 100 mL medium
resulted in a decrease in degradation efficiency to 87%. Thus,
considering the degradation efficiency and treatment costs, an
inoculums concentration of 5 mL in 100 mL medium was the
most appropriate for aerobic degradation of DMP.
The decrease in degradation efficiency with inoculums
concentrations was observed previously under both anaerobic
and aerobic conditions.32 Increasing inoculums concentration
did not enhance the DMP degradation, because increasing
population of bacteria also increases the bacterial competition
for the resources (e.g., food), and therefore limits bacterial
growth when the food in the limited environment becomes
depleted.33
Fig. 4 Effect of (a) temperature (b) inoculum size and (c) concentra-
tion on DMP biodegradation.
concentration of DMP increased to 600 mg Lꢁ1, the degradation
rate may reach to 96% within 72 h.
Luo et al.34 reported that Trichosporon sp. could degrade DMP
at much lower concentration i.e. 21.5 mg Lꢁ1 and its degrada-
tion rate just reached to about 70% aer 24 days incubation.
Jianlong et al.4 also reported that the degradation rate of DMP
(100 mg Lꢁ1) in the soil augmented with acclimated sludge
reached to 90% aer 15 days incubation time. Therefore,
B. thuringiensis is much efficient in degrading DMP compared
with previous reports. Furthermore, the results of our study are
evident that B. thuringiensis can tolerate high concentration i.e.,
1000 mg Lꢁ1. Similarly Wang et al.,35 has reported that the free
cells of several bacillus sp. can tolerate 400 mg Lꢁ1. In addition
Hai and Yun-xia36 has reported that the green alga Dunaliella
tertiolecta can hydrolyze DMP to the product PA with the
Effects of DMP concentration on degradation efficiency
The effect of various initial concentrations of DMP on biodeg-
radation efficiency was subsequently determined under optimal
conditions. When DMP was initially added in a range of
100–400 mg Lꢁ1, degradation proles were approximately
similar (Fig. 4c). On the other hand, when the substrate
concentration was increased to the range of 400–1000 mg Lꢁ1
the degradation signicantly decreased (P < 0.05).
,
The results (Fig. 4c) also showed that B. thuringiensis can
completely degrade DMP within 72 h when the initial concen-
tration is not higher than 400 mg Lꢁ1. When the initial
maximum tolerance level reaching up to 300 mg Lꢁ1
.
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Kinetics of DMP biodegradation
Biochemical degradation pathway
DMP biodegradability by B. thuringiensis sp. was investigated at To explore the pathways of DMP degradation by B. thuringiensis
initial DMP concentration 400 mg Lꢁ1, pH 7.0 and 30 ꢀC. First- sp., metabolites of DMP were identied by GC-MS. Two major
order kinetics has been frequently used to describe the metabolites besides the parent compound DMP, MMP and PA
biodegradation kinetics of organic pollutants1.4,28,37 Here, DMP were detected and identied during the course of degradation
biodegradation by B. thuringiensis sp. was assumed to t to the by comparing the mass spectrum at a particular retention time
rst-order kinetic equation, with the form:
with the published mass spectra from National Institute of
Standards and Technology (NIST) Database (Fig. 6a–c). Ester
hydrolysis of DMP resulting in MMP and PA was carried out in
two steps before aromatic ring cleavage and full mineralization.
In the rst step, MMP was formed and followed by hydrolysis of
MMP to PA presumably by the same hydrolytic enzyme. The
above results are similar to those previously reported.9,40 Based
on the above results, a tentative metabolic pathway for the
degradation of DMP by B. thuringiensis sp. is proposed in
Fig. 6d.
ln C ¼ ꢁKt + A
where C is the DMP concentration, K the rst-order kinetic
constant, t time, and A is a constant. The biodegradation half-
life of the DMP rst-order reaction can be expressed as:
t1/2 ¼ ln 2/K
Similarly, the proposed degradation biochemical pathway of
PAEs involves sequential cleavage of the ester bond to yield the
phthalate monoester and then PA which can be further
metabolized to produce carbon dioxide and water.36,41–44
The tting of DMP biodegradation to experimental data
using the rst-order kinetic model and the kinetic equation is
shown in Fig. 5.
Here, the calculated results indicated that the rst-order
model was a good t. When the initial concentration of DMP
was 400 mg Lꢁ1, the rate constant (K) was independent of
initial concentration and the rst-order equation could be
expressed as ln C ¼ ꢁ0.0616t + A with 0.9783 R2 value, the
biodegradation half-life was 11.25 h. Jianlong et al.4 reported
that in soil augmented with acclimated sludge the biodegra-
dation half-life for DMP (100 mg Lꢁ1) was 2.29 days On other
hand Wang et al.38 reported that biodegradation half-life for
DMP alone (10 mg Lꢁ1) by using digested sludge was 1 day.
Donglei, et al.39 reported that half-life for DMP (100 mg Lꢁ1
)
biodegradation by using Arthrobacter sp. half-life was
2.25 days. Hence in present study DMP was degraded at much
higher concentration and rate.
Fig. 6 Mass spectrum of (a) dimethyl phthalate (b) monomethyl
Fig. 5 Kinetics of DMP biodegradation.
phthalate (c) phthalic acid and (d) proposed degradation pathway.
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DMP degrading bacterium was isolated from cotton eld soil.
Based on 16S rDNA partial gene sequencing analysis, the
dominating bacterial strain B. thuringiensis sp. was identied.
This study investigated the optimal pH and time for growth of
DMP degrading microbes in MSM and the effects of inoculums
size, substrate concentration and time on DMP degradation.
MMP and PA could be monitored in metabolic DMP interme-
diates. The B. thuringiensis sp. metabolized DMP completely at
higher concentrations, suggesting that B. thuringiensis sp.
represents a novel tool for removing DMP from contaminated
waste.
´
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