Degradation of dimethyl carboxylic phthalate esterby Burkholderia cepacia DA2 isolated from marinesediment of South China Sea

Yali Wang AElig Bo Yin AElig Yiguo Hong AElig Yan Yan AEligJi-Dong Gu

Accepted 9 July 2008 Published online 24 July 2008

Springer Science+Business Media LLC 2008

Abstract Burkholderia cepacia DA2 isolated from

marine sediment of the South China Sea is capable of

utilizing dimethyl phthalate (DMP) as the sole source of

carbon and energy During the transformation of DMP in

batch culture its corresponding degradation intermediates

were identified as monomethyl phthalate (MMP) and

phthalate acid (PA) sequentially over the time of incuba-

tion The biodegradation biochemical pathway of DMP

was DMP to MMP and then to PA before mineralization

Degradation of DMP by B cepacia DA2 was also depen-

dent upon DMP-induction and the initial concentrations of

DMP affected the degradation rate Degradation kinetics fit

well with the modified Gompertz model The optimum pH

and salinity was 60 and 5 respectively for DMP

degradation by B cepacia DA2 This study showed that the

indigenous microorganisms of the deep-ocean sediments

are capable of DMP degradation completely

Keywords Dimethyl phthalate Degradation pathway Endocrine-disrupting Burkholderia Deep-ocean sediments

Introduction

Dimethyl phthalate (DMP) is an important synthetic

organic compound widely used as plasticizers and addi-

tives in plastics manufacturing and moulding to improve

mechanical properties of the plastic resin particularly

flexibility and softness (Giam et al 1978) It is typically

used in cellulose-ester based plastics such as cellulose

acetate and butyrate DMP and its intermediates are sus-

pected to be responsible for functional disturbances in the

nervous systems and liver of animals Widely known as an

endocrine-disrupting chemical it may also promote chro-

mosome injuries in human leucocytes and also interfere

with the reproductive systems and normal development of

animals and humans (Staples et al 1997 Jobling et al

1995 Lottrup et al 2006) Therefore the US Environ-

mental Protection Agency (US EPA) has listed this

chemical together with others as a priority pollutant (US

EPA 1992)

DMP as a short-chained carboxylic diester easily

migrates into environment including surface marine waters

freshwaters and sediments (Zhao et al 2004) Effective and

economical removal of these phthalates from the environ-

ment is critically important and scientifically challenging

Currently available removal methods for phthalates include

biodegradation advanced oxidation coagulation and

adsorption (Iturbe et al 1991) Although coagulation by

flocculation is useful for the removal of organic micropol-

lutants and its removal mechanism has been reported

(Thebault et al 1981) coagulation by ferric chloride was

not very effective and efficient Adsorptive removal by

activated carbon b-cyclodextrin macroreticular resin and

diatomite was effective (Adhoum and Monser 2004 Murai

et al 1998 Zhang et al 2007) but these methods could not

eliminate the chemicals from the environment completely

Y Wang B Yin Y Hong Y Yan

Key Laboratory of Tropical Marine Environment Dynamics

(LED) South China Sea Institute of Oceanography

Chinese Academy of Sciences 164 Xingang Road West

Guangzhou 510301 Peoplersquos Republic of China

J-D Gu (amp)

School of Biological Sciences The University of Hong Kong

Pokfulam Road Hong Kong SAR Peoplersquos Republic of China

e-mail jdguhkucchkuhk

123

Ecotoxicology (2008) 17845ndash852

DOI 101007s10646-008-0247-4

Advanced oxidation including photo-active TiO2 process

has been proved to be effective but the method may have

potential problem because of the release of catalyst into the

environment (Xu et al 2006 2007b) Among the various

treatments microorganisms are believed to be capable of the

complete destruction of phthalate ester in the environments

(Staples et al 1997 Cheung et al 2007) but accumulation

of degradation intermediates has been documented in

selective environment (Wang et al 2003 Fan et al 2004)

which prevent further transformation of the degradation

intermediates The bioconversion of phthalates under both

aerobic and anaerobic conditions has been investigated and

their biodegradation by activated sludge (Iturbe et al 1991

Wang et al 2003 2004 1996 1997 Fan et al 2004) and

mangrove sediment (Gu et al 2005 Li and Gu 2006 2007

Cheung et al 2007) has also been demonstrated Several

studies were also conducted on biodegradation of this class

of compounds in terms of the microorganisms involved or

the biochemical pathways of degradation but the bacterial

strains are mostly derived from waste treatment system or

coastal environment impacted by pollution and eutrophi-

cation (Wang et al 2003 2004 1996 1997 Fan et al 2004

Gu et al 2005 Li and Gu 2006 2007 Cheung et al 2007

Xu et al 2007a)

To our knowledge little information is available on the

degradation of DMP by marine microorganisms though

isomers of carboxylic diester have been studied by this

research group (Wang and Gu 2006a 2006b) Therefore

the objectives of this study were to enrich and isolate

bacteria from deep-ocean sediments able to transform

dimethyl phthalate (DMP) and to propose a biochemical

pathway of degradation

Materials and methods

Chemicals

DMP MMP and PA were purchased from Aldrich-Sigma

Chemicals (St Louis Missouri USA) with [99 purity

All other chemical reagents used were also of analytical

grade and solvents of HPLC grade

Sediment sampling

The marine sediments sample were collected from a depth

of 1340 m on ocean floor of South China Sea which was

located at 109250790 9170380 (longitude latitude) by a

scientific survey ship of the South China Sea Institute of

Oceanography Chinese Academy of Sciences in Guang-

zhou PR China The samples were taken on 14 May 2002

and preserved immediately at -20C after being collected

before use

Enrichment culture and isolation of microorganisms

The minimum salt medium (MSM) contained the following

(mg l-1) (NH4)2SO4 1000 KH2PO4 200 K2HPO4 800

MgSO4 7H2O 500 FeSO4 10 CaCl2 50 NaCl 500 and

the medium was adjusted to pH 70 plusmn 01 with dilute HCl

or NaOH A stock solution was prepared by dissolving

DMP 400 mg l-1 directly in MSM to form a saturated

solution After passing through 02 lm membrane filter

(Gelman Ann Arbor Michigan USA) on a pre-sterilized

vacuum filtration apparatus the medium solution was

transferred to individual Erlenmeyer flasks that were pre-

viously sterilized

About 05 g marine sediments were introduced to each

sterile Erlenmeyer flask containing 150 ml culture medium

as described above The cultures were incubated in a

shaker at 150 rpm and 30C After 1 week of incubation

([80 depletion of DMP) about 1 ml aliquot culture was

aseptically taken and transferred into a new Erlenmeyer

flask containing sterile culture medium identical to the

initial one This enrichment culture process was carried

out successively for three times of transferring (Gu 2008)

bacteria in the final culture were plated on Noble agar

(Difco Lab Detroit Michigan USA) plates containing

MSM and DMP for isolation and further purification of the

bacterial isolates was conducted on Noble agar plates

(Difco Lab Detroit Michigan USA) containing MSM and

DMP by streaking technique Single colonies on the agar

plates after purification were examined for their morpho-

logies and further analysis based on 16S rRNA gene

Effect of pH and salinity

Similar to degradation experiments described above pH

was further adjusted to the projected value of 40 50 60

70 80 and 90 by either dilute HCl or dilute NaOH during

the medium preparation before making up to the volume

Salinity of 5 10 15 and 20 was made by including the

necessary amount of NaCl in this study The medium was

then inoculated and incubated in the same way as in the

degradation experiments

Degradation experiments

DMP (400 mg l-1) was dissolved in the MSM as the cul-

ture medium The culture medium was sterilized by passing

through 02 lm membrane filter (Pall Gelman Laboratory

Ann Arbor Michigan USA) The Bacterial isolate was first

cultured in LB medium overnight and 1 ml of such culture

was centrifuged at 40009 g for 5 min in 15 ml Eppendorf

centrifuge tube and the cell pellet was washed with the

culture medium three times and transferred into 150 ml

of the sterilized culture medium Periodically samples of

846 Y Wang et al

123

culture (2 ml) were taken and preserved at -20C for

further chemical analysis All experiments were performed

in triplicates with the control without bacteria and sterilized

cells

Substrate induction

To prepare the DMP-induced cells the bacterial isolate was

cultured in MSM containing DMP (400 mg l-1) to mid-log

phase and in contrast the DMP non-induced cells were

cultured in LB medium to mid-log phase One millilitre of

either of the cultures was centrifuged washed re-sus-

pended and adjusted to an OD600 value of approximately

01 with MSM The cell suspension (10 ml) was inocu-

lated into a fresh MSM containing DMP (400 mg l-1) to

determine degradation capability Periodically samples of

culture (2 ml) were taken and 1 ml was preserved at -

20C for further chemical analysis and the other 1 ml was

used for OD measurement All experiments were per-

formed in triplicates with the control without bacteria and

the autoclave killed cells

Analysis of substrate and metabolites

The frozen aliquot samples were thawed centrifuged and

filtered through 02 lm syringe filter (Iwaki Glass Japan) and

analyzed for the concentration of substrates and intermediates

by HPLC (Agilent 1100 Series USA) equipped with a diode

array detector and a Hypersil ODSC8 (125 9 40 mm)

chromatography column The mobile phase consisted of

methanolmdash001 moll phosphate (pH 30) (4060 vv) at

30C and a rate of 10 ml min-1 The UV absorption spectra

of DMP and its intermediates were measured at 240 nm

Quantifiation of the chemical concentrations was achieved

using external standards and calibration

The microbial biomass in culture flasks was determined

by optical density measurements at 600 nm spectrophoto-

metrically (OD600) using a UV 2100 spectrophotometer

(UNICO Instrument Co Shanghai PR China)

Results and discussion

Isolation and characterization of bacteria

Initial enrichment cultures showed that DMP was degraded

by microorganisms enriched from the marine sediments

taken from South China Sea Over serial enrichment

transfers those bacteria capable of utilization of DMP were

further selected in the enrichment cultures (Gu 2008) After

three times of enrichment transfers three bacterial isolates

capable of growth on DMP as the sole source of carbon and

energy were isolated in pure culture Among them isolate

DA2 a purple gram-negative shot rod-shaped bacterial

strain was the most effective in degrading DMP in sub-

sequent assays This strain was further characterized using

16S rRNA gene sequencing methods (Wang et al 1996)

The 16S rRNA partial gene sequences of the bacteria were

aligned and compared with the 16S rRNA bacterial gene

sequences in the GenBank and indicated that it belongs to

the genus Burkholderia cepacia with similarity of 99

(Fig 1) The partial nucleotide sequence of Burkholderia

cepacia DA2 was deposited in the GenBank database under

accession number EU600235

Burkholderia sp K301

Burkholderia sp Ff54

Burkholderia sp J62

Burkholderia cepacia AW201

Burkholderia cepacia RREM25

Burkholderia cepacia a8

Burkholderia cepacia ATCC 49709

Burkholderia vietnamiensis Ja2

Burkholderia sp BCB-16

Burkholderia cepacia BC2311-6

Burkholderia cepacia Yabuuch

Burkholderia cepacia ATCC 17759

DA2

Burkholderia cepacia ESR63

Burkholderia cepacia RS2

Escherichia coli rrnH

Escherichia coli K12 D15061

98

100

100

83

67

33

31

22

18

5

15

22

19

100

Fig 1 A phylogenetic 16S

rDNA-based tree showing

relationships between the strain

DA2 and selected members of

the family Burkholderia

Degradation of dimethyl carboxylic phthalate ester by Burkholderia cepacia DA2 847

123

Many species from the genus Burkholderia have been

described for different contaminated environments Some

Burkholderia strains such as JS150 are known to have

multiple oxygenase pathways for the utilization of aromatic

compounds (Haigler et al 1992 Johnson and Olsen 1997)

Chang and Zylstra (1999) have cloned the genes from

Burkholderia cepacia ATCC17616 which encode specific

enzymes for the degradation of phthalate and a key inter-

mediate protocatechuate However no study of DMP

biodegradation by bacteria under aerobic conditions using

marine sediment as a source of microorganisms has been

reported

Biochemical degradation pathway

The initial concentration of DMP was 400 mg l-1 by

directly dissolving the substrate in culture medium in DMP

degradation experiments DMP in culture flasks declined

slowly from 400 mg l-1 after an initial lag phase of 6 days

and then decreased more rapidly after 6 days (Fig 2) By

day 9 DMP concentration was undetectable in the culture

At the same time an increase of OD600 values in the cul-

ture medium was observed corresponding to the DMP

decrease Final biomass reached 0274 after 9 days of

incubation No depletion of DMP concentration or increase

in the values of OD600 was noticed in the abiotic controls

and no growth was detected in the controls without DMP

over the whole duration of the incubation This indicates

that DMP was utilized as the sole source of carbon and

energy since no other carbon was present in culture med-

ium except for the mineral salts

The metabolites from degradation of DMP were detec-

ted and then identified using information of both the

retention time on HPLC and UV-visible spectrum of a

metabolite matched with the standards Information of

National Institute of Standards and Technology (NIST) was

also used to reach a confirmation of the identification Two

degradation intermediates were identified as monomethyl

phthalate (MMP rt 19636 min) and phthalate acid (PA

rt 16636 min) during DMP degradation by B cepacia

DA2 under aerobic condition (Fig 3a) During degradation

of DMP (400 mg l-1) MMP appeared and accumulated by

day 8 as an intermediate and the concentration increased to

389 mg l-1 (Fig 2) A very slow concentration decrease

was observed after 8 days of incubation and then

decreased to zero The amount of PA was also produced

during the first 12 days of degradation but PA was com-

pletely degraded after 15 days In sterile mineral medium

without inoculation of the bacterial strain DMP did not

change and neither MMP nor PA was detected throughout

the experiment period

Together with the previous results the biochemical

pathway for DMP degradation under aerobic conditions was

proposed (Fig 3b) Degradation of DMP followed two

steps of ester bond hydrolysis resulting in MMP and PA

before the cleavage of aromatic ring The aerobic biodeg-

radation of DMP involves several steps of biochemical

transformation before the substrate becomes fully miner-

alized (Fig 3b) In some cases the initial step in the

degradation of phthalate esters was a de-esterification

reaction (Kurane et al 1984 Niazi et al 2001) The

microbial metabolism of DMP by the B cepacia DA2 in

our investigation was initiated by an initial ester hydrolysis

to form MMP and methanol and followed by a further

hydrolysis of MMP to PA presumably by the same hydro-

lytic enzyme The results were similar to those previously

reported on degradation of this class of esters by microor-

ganisms isolated from activated sludge mangrove sediment

and deep-ocean sediment (Shen et al 2004 Li and Gu

2006 Wang and Gu 2006a 2006b) It should be pointed out

that the initial hydrolysis of the identical ester bonds could

be carried out by two different bacterial species indicating

the highly specificity of the two hydrolytical enzymes to the

substrates (Li et al 2005a 2005b Li and Gu 2006 2007)

MMP and PA are the acidic organic intermediates so

the two steps are acid-producing processes and the pH

value of the culture medium is expected to show a

decrease Degradation of PA is an acid-consuming process

in which organic acid PA is mineralized to CO2 and water

(Niazi et al 2001) However there was no apparent

decrease of pH value during the degradation of DMP by the

B cepacia DA2 in this study In the current study the pH

of the culture media showed a trend of decrease from ini-

tially 70 to 65ndash60 (data not shown) This result was also

different from two reconstituted consortia capable of

degrading DMP with pH increase from 70 to 76ndash79

(Wang et al 2003) In this study once the enzyme system

0

50

100

150

200

250

300

350

400

450

0 2 4 6 8 10 12 14

time(days)

Con

cent

ratio

n (m

g l-1

)

0

005

01

015

02

025

03

OD

600

DMP

MMP

PA

control

OD600

Fig 2 Degradation of DMP by Burkholderia cepacia DA2 isolated

from deep-ocean sediment as a sole carbon and energy source and two

degradation intermediates monomethyl phthalate and phthalate

848 Y Wang et al

123

of the bacteria is activated they are capable to remove the

intermediates quickly from the medium so that the pH

value would not drop to their sensitive level during the

degradation of DMP

DMP-induction on degradation rate and assessment

Bacterial growth often displays a sigmoidal curve with

three distinctive phases namely maximum specific growth

rate lag time and stationary phase Many models have

been developed to describe the bacterial growth curve

(Schepers et al 2000 Richards 1959) Among them the

Gompertz model (Eq 1) was found to be the most suitable

model to fit the growth data based on Lactobacillus plan-

tarum (Zwietering et al 1990)

X frac14 Aexp expume

Aethk tTHORN thorn 1

h in oeth1THORN

In the above equation X A um and k are defined biomass

concentration asymptotic phase and maximum growth

rate and lag phase time respectively Based on Eq 1 the

corresponding equation for substrate transformation was

deduced by Fan et al (2004) as following

S frac14 S0 1 exp expRme

S0

k teth THORN thorn 1

eth2THORN

where S is the substrate concentration (mg l-1) S0 is

the initial substrate concentration (mg l-1) Rm is the

maximum substrate transformation rate (mg l-1 day-1) kis the lag phase time (d) and t is the incubation time (d)

For the bacterial growth curve the modified Gompertz

model in Eq 2 fit the experiment data very well which is

simple and easy to use for the number of parameters in

equations is only 2 (Rm and k) Li et al (2005a 2005b) also

used this formula to analyze DMP DMI and DMT trans-

formation kinetics In our study both biomass growth and

substrate depletion curves were describe well by the

modified Gompertz model with high correlation coefficient

(R2 [ 099) (data not shown) There was an apparent lag

min0 25 5 75 10 125 15 175 20 225

mAU

0

50

100

150

200

250

300

350

68

40

82

56

16

636

18

836

20

361 2

101

8

COOCH3

COOCH3

Dimethyl Phthalate

COOH

COOCH3

Monomethyl Phthalate

COOH

COOH

Phthalic Acid

CO2 + H2O

CH3OH CH3OH

a

b

Fig 3 (a) A representative HPLC chromatograph showing the

metabolic intermediates when dimethyl phthalate (DMP) was metab-

olized as the sole source of carbon and energy (DMP rt 21018 min

MMP rt 19636 min and PA rt 16636 min) and (b) a proposed

biochemical pathway for degradation of dimethyl phthalate by

Burkholderia cepacia DA2

0

100

200

300

400

500

600

0 1 2 3 4 5 6 7 8 9 10 11 12 14

time (days)

DM

P (

mg

l-1)

0

005

01

015

02

025

03

035

OD

600

Fig 4 Relationship between DMP-induction of the bacterium Burk-holderia cepacia DA2 and the subsequent DMP degradation (j)

DMP (mg l-1) with induction (m) DMP (mg l-1) without induction

(h) OD600 with induction and (D) OD600 without induction

Degradation of dimethyl carboxylic phthalate ester by Burkholderia cepacia DA2 849

123

phase (k 6750) observed when degradation was not

induced at an initial concentration of 500 mg l-1 (Fig 4)

and OD600 of the non-induced bacterial biomass increased

to the maximum after 10 days However with DMP-

induction DMP was rapidly degraded to below detection in

the culture medium in 5 day (Fig 4) and the lag phase (k)

was only 2618 days and OD600 of the induced bacteria

was 0321 in 5 days Both the maximum substrate trans-

formation rate of DMP non-induced and induced (Rm)

were 2111203 mg l-1 day-1 and 3241772 mg l-1 day-1

respectively The results also showed that DMP-induction

enhanced the rate of the degradation and shortened the lag

phase

It is known that the initial step in both the aerobic and

anaerobic mineralization of phthalic acid esters is hydro-

lysis of the ester side chains resulting in formation of

monoalkyl phthalate and phthalate (Gu et al 2005 Xu

et al 2005 Cheung et al 2007 Xu et al 2007a) There-

fore the primary step in the catabolic pathway for

degradation is de-esterification reaction by esterase DMP-

induction improves the esterase activity to activate the

utilization of DMP through hydrolysis of the diester into

the corresponding monoester Once the first step has been

performed further degradation may be carried out imme-

diately Similar results have also been observed on di-n-

butyl phthalate (DBP) (Zhou et al 2005)

Effect of DMP concentrations

In order to determine the effect of initial DMP concentra-

tions on degrading efficiency data analysis of the

biodegradation of DMP by B cepacia DA2 at initial con-

centrations of 200ndash800 mg l-1 with the Gompertz model is

shown in Table 1 Lag phase increased with the increase of

DMP concentrations from as little as 0664 d to as long as

11554 d DMP was rapidly degraded after the lag phrase

the maximum transformation rate Rm was observed with

the highest DMP concentration indicating that the initial

concentration of DMP may play an important role affecting

the degradability of DMP Once a bacterial population is

established to breakdown a formerly recalcitrant com-

pound degradation rate can be accelerated

Although degradation of DMP by microorganisms from

different environments has been reported there were only a

few reports in the literature on DMP degradation by pure

cultures of Bacillus (Sivamurthy and Pujar 1989) Chlo-

rella (Yan et al 1995) and Sclerotium (Sivamurthy et al

1991) To our knowledge no study of DMP biodegradation

at such a high concentration by a pure bacterial strain under

aerobic condition using marine sediment as a source of

bacteria has been reported in the literature Furthermore

B cepacia DA2 also completely degraded DMT DMI

under aerobic conditions (data not shown)

Effects of pH and salinity

Effect of the pH on the DMP degradation by B cepacia

DA2 analyzed using the modified Gompertz model is

showed in Table 2 At 400 mg l-1 DMP the maximum

transformation rate (Rm) and shortest lag time were

achieved at pH 60 B cepacia DA2 could degrade DMP

between pH 50 and 90 and was totally inhibited at pH

40 When pH value was increased from 6 to 9 lag time

was extended from1 day to 74 days and the degradation

rate was lowered from 512 to 120 (Table 2) Degradation

of DMP was particularly sensitive to low pH (Fan et al

2004 Li and Gu 2007 Xu et al 2005 2007a Wang et al

2003) since acids including phthalic acid were generated

from the de-esterification of phthalate esters

Salinity is a significant parameter in the ocean ecosys-

tem B cepacia DA2 degraded DMP in the salinity range

from 0 to 10 and exhibited the highest Rm (3611851)

and the shortest lag phase (k 1383) at 0 salinity

(Table 3) The Rm declined with the increase of salinity at

the same time higher salinity reduced the bacterial growth

resulting in a longer k in this study The bacteria failed to

grow at 20 salinity

Table 1 Comparison of calculated maximum transformation rate lag

phase and the correlation coefficient using the modified Gompertz

model at different initial DMP concentrations (pH 70 and 5salinity)

DMP concentration (mg l-1) Rm (mg l-1 day-1) k (days) R2

200 1414517 0664 09988

400 3194393 2041 09992

500 3241772 2618 09929

600 7814164 6614 09944

800 8083521 11554 09931

Table 2 Comparison of calculated maximum transformation rate lag

phase and the correlation coefficient using the modified Gompertz

model at different initial medium pH values (400 mg l-1 DMP and

5 salinity)

pH Rm (mg l-1 day-1) k (days) R2

40 ND [20

50 4790332 0839 09991

60 5119834 0656 09986

70 3194393 2041 09992

80 1793590 4517 09924

90 1199446 7357 09987

ND no data available (below detection limit)

No detectable bacterial growth observed during the whole period of

experiment

850 Y Wang et al

123

Conclusions

Burkholderia cepacia DA2 from the marine sediments of

South China Sea can utilize DMP as the sole source of

carbon and energy DMP could be mineralized completely

ie converted into carbon dioxide and water by B cepacia

DA2 through degradation intermediates MMP and PA The

optimum range of pH and salinity for DMP degradation

under aerobic conditions were 60 and 5 respectively

Induction of bacteria by DMP can shorten the lag phase

prior to initiation of the degradation

Acknowledgments This research was supported by a Nature Sci-

ence Doctoral Grant from Guangdong Province (06301430) and a

Young Innovation Grant of South China Sea Institute of Oceanog-

raphy Chinese Academy of Sciences (07SC011009)

References

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activated carbon application to the treatment of industrial

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jseppur200311011

Chang H Zylstra GJ (1999) Characterization of the phthalate

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Cheung JKH Lam RKW Shi MY Gu J-D (2007) Environmental fate

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under anoxic sulfate-reducing conditions Sci Total Environ

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Fan Y Wang Y Qian P Gu J-D (2004) Optimization of phthalic acid

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modeling degradation Int Biodeter Biodegr 5357ndash63 doi

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Giam CS Chah HS Nef GS (1978) Phthalate ester plasticizers a new

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Gu J-D (2008) Microbial transformation of organic chemicals in

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QY (ed) Mineral-organic matter-microorganism interactions

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Gu J-D Li J Wang Y (2005) Biochemical pathway and degradation

of phthalate ester isomers by bacteria Water Sci Technol

52(8)241ndash248

Haigler BE Pettigrew CA Spain JC (1992) Biodegradation of

mixtures of substituted benzenes by Pseudomonas sp strain

JS150 Appl Environ Microbiol 582237ndash2244

Iturbe R Moreno G Elefsiniotis P (1991) Efficiency of a phthalate

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Jobling S Reynolds T White R Parker MG Sumpter JP (1995) A

variety of environmentally persistent chemicals including some

phthalate plasticizers are weakly estrogenic Environ Health

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Johnson GR Olsen RH (1997) Multiple pathways for toluene

degradation in Burkholderia sp strain JS150 Appl Environ

Microbiol 634047ndash4052

Kurane R Suziki T Fukuoka S (1984) Purification and some

properties of phthalate ester hydrolyzing enzyme from Nocardiaerythropolis Appl Microbiol Biotechnol 29378ndash383

Li J Gu J-D (2006) Biodegradation of dimethyl terephthalate by

Pasteurella multocida Sa follows an alternative biochemical

pathway Ecotoxicology 15391ndash397 doi101007s10646-006-

0070-8

Li J Gu J-D (2007) Complete degradation of dimethyl isophthalate

requires the biochemical cooperation between Klebsiella oxytocaSc and Methylobacterium mesophilicum Sr isolated from wet-

land sediment Sci Total Environ 380181ndash187 doi101016

jscitotenv200612033

Li JX Gu J-D Pan L (2005a) Transformation of dimethyl phthalate

dimethyl isophthalate and dimethyl terephthalate by Rhodococ-cus rubber Sa and modeling the processes using the modified

Gompertz model Int Biodeter Biodegr 55223ndash232 doi

101016jibiod200412003

Li JX Gu J-D Yao J-H (2005b) Degradation of dimethyl terephthal-

ate by Pasteurella multocida Sa and Sphingomonas paucimobilisSy isolated from mangrove sediment Int Biodeter Biodegr

56158ndash165 doi101016jibiod200507001

Lottrup G Andersson AM Leffers H Mortensen GK Toppari J

Skakkebaeek NE et al (2006) Possible impact of phthalates on

infant reproductive health Int J Androl 29(1)172ndash180 doi

101111j1365-2605200500642x

Murai S Imajo S Takasu Y Takahashi K Hattori K (1998) Removal

of phthalic acid esters from aqueous solution by inclusion and

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doi101021es970463d

Niazi JH Prasad DT Karegoudar TB (2001) Initial degradation of

dimethylphthalate by esterases from Bacillus species FEMS

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tb10565x

Richards FJ (1959) A dexible growth function for empirical use J

Exp Bot 10290ndash300 doi101093jxb102290

Schepers AW Thibault J Lacroix C (2000) Comparison of simple

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modeling of Lactobacillus helveticus growth in pH-controlled

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Shen P Wang YY Gu J-D (2004) Degradation of phthalate acid and

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10643ndash664

Sivamurthy K Pujar BG (1989) Bacterial degradation of

dimethylterephthalate J Ferment Bioeng 68375ndash377 doi

1010160922-338X(89)90015-9

Sivamurthy K Swamy BM Pujar B (1991) Transformation of

dimethylterephthalate by the fungus Sclerotium rolfsii FEMS

Microbiol Lett 7937ndash40 doi101111j1574-69681991

tb04500x

Staples CA Peterson DR Parkerton TF Adams WJ (1997) The

environmental fate of phthalate esters a literature review

Chemosphere 35667ndash749 doi101016S0045-6535(97)00195-1

Table 3 Comparison of calculated maximum transformation rate lag

phase and the correlation coefficient using the modified Gompertz

model at different medium salinity (400 mg l-1 DMP and pH 70)

Salinity () Rm (mg l-1 day-1) k (days) R2

0 3611851 1383 09992

5 3194393 2041 09992

10 2515628 2812 09929

15 1794935 9335 09923

20 ND [20 ND

ND no data available (below detection limit)

No significant bacterial growth observed during the whole period of

experiment

Degradation of dimethyl carboxylic phthalate ester by Burkholderia cepacia DA2 851

123

Thebault P Cases JM Fiessinger F (1981) Mechanism underlying the

removal of organic micropollutants during coagulation by an

aluminium or iron salt Water Res 15183ndash189 doi101016

0043-1354(81)90110-X

US EPA (1992) and update Code of federal regulations 40 CFR Part

136 US EPA

Wang YY Fan YZ Gu J-D (2003) Aerobic degradation of phthalic

acid by Comamonas acidovorans Fy-1 and dimethyl phthalate

ester by two reconstituted consortia from sewage sludge at high

concentrations World J Microbiol Biotechnol 19811ndash815 doi

101023A1026021424385

Wang Y Fan Y Gu J-D (2004) Dimethyl phthalate ester degradation

by two planktonic and immobilized bacterial consortia Int

Biodeter Biodegr 5393ndash101 doi101016jibiod200310005

Wang YP Gu J-D (2006a) Degradability of dimethyl terephthalate by

Variovorax paradoxus T4 and Sphingomonas yanoikuyaeDOS01 isolated from deep-ocean sediment Exotoxiology

15549ndash557 doi101007s10646-006-0093-1

Wang Y Gu J-D (2006b) Degradation of dimethyl isophthalate by

Viarovorax paradoxus strain T4 isolated from deep-ocean

sediment of the South China Sea J Hum Ecol Risk Assess

12236ndash247 doi10108010807030500531521

Wang J Liu P Qian Y (1996) Biodegradation of phthalic acid esters

by an acclimated activated sludge Environ Int 22737ndash774 doi

101016S0160-4120(96)00065-7

Wang J Liu P Qian Y (1997) Biodegradation of phthalic acid esters

by immobilized microbial cells Environ Int 23775ndash778 doi

101016S0160-4120(97)00089-5

Xu XR Li HB Gu J-D (2005) Biodegradation of an endocrine-

disrupting chemical di-n-butyl phthalate ester by Pseudomonasfluorescens B-1 Int Biodeter Biodegr 559ndash15 doi101016

jibiod200405005

Xu XR Li HB Gu J-D (2006) Simultaneous decontamination of

hexavalent chromium and methyl tert-butyl ether by UVTiO2

process Chemosphere 63254ndash260 doi101016jchemosphere

200507062

Xu XR Li HB Gu J-D (2007a) Photocatalytic reduction of

hexavalent chromium and degradation of di-n-butyl phthalate

in aqueous TiO2 suspensions under ultraviolet light irradia-

tion Environ Technol 281055ndash1061 doi101080

09593332808618866

Xu XR Li HB Gu J-D Li X-Y (2007b) Kinetics of n-butyl benzyl

phthalate degradation by a pure bacterial culture from the

mangrove sediment J Hazard Mater 140194ndash199 doi101016

jjhazmat200606054

Yan H Ye C Yin C (1995) Kinetics of phthalate ester biodegradation

by Chlorella pyrenoidosa Environ Toxicol Chem 14931ndash938

doi1018971552-8618(1995)14[931KOPEBB]20CO2

Zhang W Xu Z Pan B (2007) Assessment on the removal of

dimethyl phthalate from aqueous phase using a hydrophilic

hyper-cross-linked polymer resin NDA-702 J Colloid Interface

Sci 311382ndash390 doi101016jjcis200703005

Zhao X-K Yang G-P Wang Y-J (2004) Adsorption of dimethyl

phthalate on marine sediments Water Air Soil Pollut 157179ndash

192 doi101023BWATE000003888057430c3

Zhou QH Wu ZB Cheng SP He F Fu GP (2005) Enzymatic

activities in constructed wetlands and di-n-butyl phthalate (DBP)

biodegradation Soil Biol Biochem 371454ndash1459 doi

101016jsoilbio200501003

Zwietering MH Jongenburger I Rombouts FM vanrsquot Riet K (1990)

Modeling of the bacterial growth curve Appl Environ Microbiol

561875ndash1881

852 Y Wang et al

123