Degradation of Di-N-Butyl Phthalate by Microbacterium Aoyamense

Atpm-11 Isolated from Waste Water Treatment Plant

Ke Zhang

, Wei Chen and Jia Chen

College of Civil Engineering, Sichuan Agricultural University, Dujiangyan,Sichuan, 611830, China

Email: zhangke@sicau.edu.cn

Keywords: Di-n-butyl phthalate (DBP), microbacterium aoyamense ATPM-11, biodegradation, characteristics

Abstract: An efficient di-n-butyl phthalate bacterial strain ATPM-11 was isolated from activated sludge of waste water

treatment plant (WWTP). Based on its morphological, physiobiochemical characteristics and 16S rRNA

gene sequence, strain ATPM-11 was identified as Microbacterium aoyamense sp. The degradation

characteristics were investigated under different environmental conditions. The results showed that the

optimal temperature and pH for DBP degradation by ATPM-11 was 25 ℃ and 8.0, respectively. Under

these conditions, ATPM-11 could effectively degraded more than 83% of DBP at 400 mg/L. The diversity

of degradable substrates showed that strain ATPM-11 could degrade phthalate (DMP), DEP and DOP

efficiently. Therefore, this bacterial strain has potential to be used in DBP bioremediation.

1 INTRODUCTION

Phthalic acid esters (PAEs), one of the synthetic

organic compounds, are the wildly used and a higher

productivity as plasticizers, adhesive, additives,

paint solvent and Printing inks in the world (Li et al.,

2005). However, with the broad use of plastic

products, the phthalic acid esters abound in the

environment and they can migrate into the soil and

rainwater, thus enter the water system, which may

harm aquatic organisms and human health (Bai et al.,

2012). Di-methyl phthalate (DMP), di-n-butyl

phthalate (DBP) and di-n-octyl phthalate (DOP)

have been listed as priority pollutants by China

National Environmental Monitoring Center and the

US Environmental Protection Agency (Wang et al.,

2008). PAEs can be degraded by chemical and

physical methods, but microbial technology was

regarded as the most efficient way duo to it high

efficiency and low toxicity (Wan, 2012). The

hydrolysis and photolysis of DBP in the natural

environment are very slow and are difficult to

degrade. The physical method mainly consists of

humic acid or activated carbon adsorption, relying

on the strong pore structure and adsorption capacity

of adsorbent to remove DBP in water (Li et al.,

2013). The chemical method is mainly

photocatalytic degradation, which is the removal of

DBP in water by ultraviolet light. Although physical

and chemical methods have a good effect on the

removal of DBP in water body, there are obvious

defects, such as the final destination of DBP

attached to the adsorbent. In comparison, the

biological method is low cost and high efficient.

(Guo et al., 2007; Ding, 2012; Zheng et al., 2007)

Presently, several PAEs-degrading bacterial

strains belonging to the Gordonia sp.( Sarkar et al.,

2013), Enterobacter sp.( Fang et al., 2010) and

Arthrobacter sp.( Wen et al., 2014). They can be

isolated from different environments, while their

degrading efficiencies in other PAEs were low and

far from meeting the actual pollution control

requirements.Therefore, in order to improve the

biodegradation rate of phthalate esters, it’s necessary

to isolate highly effective degradation bacteria(Li et

al., 2014).

In the study, a DBP-degrading bacterium was

isolated from active sludge and identified by 16S

rDNA sequence. The biodegradation kinetics and

different environmental factors affecting this process

were investigated.And this study is expected to

improve current understanding of the bioremediation

of DBP and find higher effective DBP-dergading

strains.

Zhang, K., Chen, W. and Chen, J.

Degradation of Di-N-Butyl Phthalate by Microbacterium Aoyamense Atpm-11 Isolated from Waste Water Treatment Plant.

In Proceedings of the International Workshop on Environment and Geoscience (IWEG 2018), pages 11-15

ISBN: 978-989-758-342-1

2 MATERIALS AND METHODS

2.1 Reagents and Chemicals

DBP (99.5% purity) for the experiment was

purchased from Chengdu Kelong Chemical Reagent

Co., Ltd., All the chemical reagents were of

analytical grade and all solvents(Ethyl acetate and

methanol) were of HPLC grade purchased from

Tianjing kemiou Reagent Co., Ltd China. The

minimal medium (MM) contained (1L): MgSO

7H2O 0.5 g, K

HPO

1.70 g, FeSO

4•

O 0.05 g,

and NaNO

0.5 g, (NH

)

1.0 g,Na

MoO

0.0024

g, CaCl

2•

O 0.04 g,FeCl

0.0018 g.The nutrient

broth (NB) for bacteria enrichment consisted of beef

extract 3g, peptone 5 g, NaCl 5 g, pH 7.2.Nutrient

agar plates were made using Nutrient Broth (NB)

supplemented with 2% agar.

2.2 Enrichment and Isolation of Dbp

Strains

The enrichment procedure was according to Wu

(Wu et al., 2010) with some modifications. Initially,

5.0 g of sludge was added to a 500-ml Erlenmeyer

flask containing 200 ml of MM solution amended

with concentration of 100 mg/l DBP. The

suspension was incubated for 6 days in the dark at

25 ℃according to pre-experiment on a rotary shaker

operated at 140 rpm. Subsequently, 2ml of the

enrichment culture was serially transferred five

times to fresh medium incubated under the same

conditions. At the same time, in the process of

transfer, containing a higher concentration of DBP

200–500 mg/L each time. Then the final enrichment

was streaked onto MM agar plates supplemented

with a mixture of DBP (500 mg/L) and incubated 1

week at 25℃. Presumptive colonies were picked on

the basis of differences in colony morphology and

coloration and re-streaked onto MM agar plates

amended with DBP. The bacterial isolates were

further purified by streaking on Nutrient Agar plates

and then re-streaked onto MM agar plates with and

without DBP to confirm their degradation abilities.

Isolates can grow in the presence of DBP but not in

their absence were selected for further study.

2.3 Amplification of 16S rDNA

Extraction kit (Sangon Corporation, Shanghai,

China) was used for the extraction of bacterial

genomic DNA according to the manufacturer’s

instructions. Further identification was performed by

16S rDNA gene sequencing. and then about 1500 bp

length of 16S rRNA was amplified through PCR by

using the bacterial universal primer 27F (50-

AGAGTTTGATCCTGGCTCAG-30) and 1492R

(50-GGCTACCTTGTTACGACTT-30). PCR was

performed ( Bio-Rad USA) under the following

conditions: preheated at 95 ℃ for 2 min; then

denatured at 94 ℃ for 1 min, annealing at 56 ℃ for

1 min, extended at 72 ℃ for 3min for 30 cycles, last

extended at 72 ℃ for 8 min.

2.4 Sequence Analysis of Strain

Purified PCR product was directly sequenced. The

sequence data of the closest relatives were retrieved

from NCBI database and aligned with CLUSTALW

with all parameters set at their default values. A

phylogenetic tree was then constructed using the

neighbor-joining method with MEGA 6.0 software.

The trees were validated using bootstrap analysis

performed with 1000 replicates.

2.5 Degradation Experiments of

Microbacterium Aoyamense

ATPM-11

The following environmental factors were assayed

to investigate their effects on DBP degradation

within 60 h of cultivation at a 140 rpm shaking rate.

Temperature (10, 15, 20, 25 and 30 ℃); Initial pH

value (4.0, 5.0, 6.0, 7.0, 8.0, 9.0); Initial DBP

concentration (100 mg/L, 200 mg/L,300 mg/L,400

mg/L,500 mg/L). Other PAEs (DOP, DEP, DMP,

DEHP and DPP).

IWEG 2018 - International Workshop on Environment and Geoscience

Figure 1: Phylogenetic tree derived from 16S rRNA gene sequence of ATPM-11 and sequences of related species.

Distances were calculated using neighbor-joining method.

2.6 Analysis Method

Concentration of DBP in the supernatant solution

was performed using high performance liquid

chromatography (HPLC) (Aglient 1200 series). The

column temperature was 40℃. The volume of the

injected samples was 40μl; Chromatography column

was Inertsil ODS-2151-K. 6× 150 mm. Ethyl acetate

was added to each sample, which was vigorously

shaken for 5 min, the aqueous and organic phases

were separated by centrifugation at 8000 rpm for 5

min. The aqueous phase was extracted twice with

equal volume of ethyl acetate. Ethyl acetate phase

was dried over anhydrous sodium sulfate and

evaporated, then dissolved in 10 ml of methanol.

RESULTS AND DISCUSSION

3.1 Isolation and Identification of the

DBP-degrading Bacterium

After 35 days enrichment, one strain showed high

biomass and high degradation efficiency was

selected for further investigation. Phylogenetic tree

of the 16Sr RNA gene revealed strain

Microbacterium aoyamense ATPM-11 clustered

with members of the genus Microbacterium, and

had a 100% sequence similarity with

Microbacterium aoyamense JCM 14900 (AB234028)

(Figure 1).

3.2 Effects of Temperature on DBP

Biodegradation

The strain was cultivated under condition of 25℃

and pH 8. The DBP (500 mg/L) degradation

efficiencies under 10, 15, 20,25and 30 ℃ were

tested. The effects of temperature on the degradation

of DBP in the culture medium were tested after

incubation 60 h. The results indicate that the

degradation is best at the temperature of 25 ℃, and

The degradation rate was 83% (Figure 2). The

degradation rate was only 30%. There is no

significant difference between 20 ℃ and 25℃

(P<0.05).

10 15 20 25 30

degradation(%)

Tem perature(



Figure 2: Effects of temperature on DBP biodegradation.

3.3 Effects of Initial pH on DBP

Biodegradation

Figure 3 shows the results of pH (4.0–9.0) on DBP

biodegradation at an initial concentration of DBP at

400 mg/L. Strain could effectively degraded DBP

when pH ranged from 7.0 to 9.0. The optimal

Degradation of Di-N-Butyl Phthalate by Microbacterium Aoyamense Atpm-11 Isolated from Waste Water Treatment Plant

degradation pH for DBP degradation by this

bacterial strain were 8.0. The degradation rate could

reach up to 93%. The degradation under acid

conditions is poor. The optimal pH values in

degrading of DBP are consistent with the other

study, where the optimal pH for Di-n-butyl phthalate

(DBP) Degradation by strain H-2 ranged from 7.0 to

9.0. (Lei et al., 2014).

456789

100

Degradation rate (%)

Figure 3: Effects of initial pH on DBP biodegradation.

3.4 Effects of Initial Concentration on

DBP Biodegradation

The experiment was conducted under different DBP

concentrations (100,200,300,400,500,600 mg/L) to

investigate the influence of concentration on DBP

degradation rate. As shown in Figure 4, when the

initial concentration was 200 mg/L, Microbacterium

aoyamense ATPM-11 had the highest degradation

rate of DBP. The degradation rate was slightly lower

than 200 mg/L when the concentration was 100mg/L.

when the concentration gradually increased from

200mg/L to 600mg/L, the degradation rate reached

the lowest value of 69%.

100 200 300 400 500 600

100

Degradation rate (%)

Concentration(mg/L)

Figure 4: Effects of initial concentration on DBP

biodegradation.

3.5 Degradation of Other PAEs by

Bacteria

In order to investigate the degradation ability of the

consortium to other commonly used PAEs in

environment, the consortium was cultured in MSM

supplemented with DBP, DOP, DEP, DMP, DEHP

and DPP at 30 °C. Figure 5 shows that

Microbacterium aoyamense ATPM-11 can also

degrade the other APEs. The strain could degraded

DEP and DMP with high efficiency up to 94 %.

DEP DMP DOP DEHP DPP

100

Degradation rate (%)

PAEs

Figure 5: Degradation of other commonly used PAEs by

bacteria.

4 CONCLUSIONS

A broad-spectrum and efficient di-n-butyl phthalate

(DBP)-degrading bacterial strain Microbacterium

aoyamense ATPM-11 was isolated from activated

sludge of waste water treatment plant (WWTP). The

strain Microbacterium aoyamense ATPM-11 could

completely degrade DBP and the degradation rate

was up to 93%. The temperature 25 ℃ and pH 8.0

are the optimal conditions for DBP degradation by

strain ATPM-11. Microbacterium aoyamense

ATPM-11 degrades DBP faster in alkalinity than in

acidity. The strain Microbacterium aoyamense

ATPM-11could also degrade other commonly used

phthalates like (DMP), DEP and DOP.

IWEG 2018 - International Workshop on Environment and Geoscience

REFERENCES

Bai YH, Xu K and Zhao DB 2012 Exposure assessment of

Di (2-ethylhexyl) phthalate of plastic food packaging

materials. Modern Food Science and Technology

28(10) 1423-1428

Ding K 2012 Experimental research on carbon for the

adsorption of DBP from water and dielectric barrier

discharge [D]. Nanchang: Nanchang University

Fang CR, Yao J, Zheng YG, Hu LF and Jiang CJ 2010

Dibutyl phthalate degrationby Enterobacter sp. T5

isolated from municipal solid waste inland fill

bioreactor. International Biodeterioration and

Biodegradation 64(6) 442-446

Guo D, Li Y, Zhao Y and Zhou J 2007 Activated carbon

adsorption of di-n-butyl phthalate, nonyl phenol and

bisphenol A in Yellow River water. Water Wastewater

Eng 33(1) 30-33

Li JL, Shao XL , Liu SL, Yao K, Zhao Q, Hu XJ and

Deng WQ 2014 Screening and identification of

dibutyl phthalate degradation strains. Modern Food

Science and Technology 30(10) 10-113

Li JX, Gu JD and Li P 2005 Transformation of dimethyl

phthalate, dimethyl isophthalate and dimethyl tereph

Li thalate by Rhodococcus rubber Sa and modeling

the process using themodified gompertz model.

International Biodeterioration and Biodegradation

55(3) 223-232

Li YH,Yi YC,Wu J, Wu J and Na W 2013 Isolation of a

humus-reducing bacterium strain and its

biodegradation of pollutants [J]. ChinJ Appl Environ

Biol 19(3) 506-510

Lei J, Chen Y, Long J, Guo YM, Yan ZY,Zhu Y and Gu

PQ 2014 Isolation and Identification of a Di-n-butyl

phthalate (DBP)-Degrading Strain H-2 and Its

Degradation Characteristics. Journal of Food Science

35(15) 203-206

Sarkar J, Chowdhury P P and Dutta T K 2013 Complete

degradation ofdi-n-octyl phthalate by Gordonia sp.

strain Dop5 [J].Chemosphere 90 2571-2577

Wan Y 2012 Study on Adsorption-desorption

characteristics of dibutyl-phthalate on humic acid [D].

Chongqing: Southwestern University

Wang P, Wang S and Fan CQ 2008 Atmospheric

distribution of particulate- and gas-phase phthalic

esters (PAEs) in a metropolitan city, Nanjing, East

China, Chemosphere 12 (72) 1567–1572

Wen ZD, Gao DW and Wu WM 2014 Biodegradation and

kineticanalysis of phthalates by an Arthrobacter strain

isolated from constructed wetland soil. Applied

Microbiology and Biotechnology 1-8

Wu XL, Liang RX, Dai QY, Jin DC, Wang YY and Chao

WL 2010 Complete degradation of di-n-octyl

phthalate by biochemical cooperation between

Gordonia sp. strain JDC-2 and Arthrobacter sp. strain

JDC-32 isolated from activated sludge. Journal of

Hazardous Materials 176 262–268

Zheng HH, Zhao LW and Tao J 2007 Photo degradation

of dibutyl phthalate in flowing water. Chin J Public

Health 23(2) 210-211

Degradation of Di-N-Butyl Phthalate by Microbacterium Aoyamense Atpm-11 Isolated from Waste Water Treatment Plant