Paper in press
Zhu et al.
Isolation and Characterization of Terephthalic Acid
Aerobic Degrading Bacteria
1Wang
1 College
J., 1Li X. D., 1Qiu J. P. and 2Zhu H. X.
of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
of Environmental Sciences, Insitute of Light Industry and Food Engineering, Guangxi
University, Nanning, 530004, China.
2 Department
Abstract
From an expanded granular sludge bed reactor (EGSB) treating of pure terephthalic acid (PTA) wastewater, an
aerobic bacteria was isolated, and identified. It could effectively degrade terephthalic acid (TA). Aerobic bacteria was
Pseudomonas aeruginosa. It was able to degrade 1000 mg/L TA within 24 h with degradation rate of 99%. It is
extracellular enzyme.
Keywords: Terephthalic acid (TA), aerobic, degradation, isolation, identification.

Corresponding author: Department of Environmental Sciences, Insitute of Light Industry and Food
Engineering, Guangxi University, Nanning, 530004, China.
1
PAPER IN PRESS
The aerobic shaking culture incubator DKY-2
was made from Shanghai Duke Automation
Equipment Company Limited.
Introduction
Terephthalic acid (TA) is a main material used to
produce
polyesters,
of
which
polyethylene
terephthaiate is the most commonly used for bottles,
films and textile fibers. During the production of TA
and about 70% of polyester textiles undergoing this
treatment called "caustic treatment", wastewater with
a high concentration of TA is generated. Terephthalic
acid (TA) can be a variety of microbial decomposition,
but also can be used as the sole carbon source and
some microbial degradation. The TA degradation
microbes are gram negative Pseudomonas sp.,
Alcaligenes sp., Corynebacterium parvum sp. and
Arthrobacter sp., Bacillus sp., gram negative, gram
positive bacillus, Methanosarcina and methanothrix
soehngenii. Degradation of TA by microorganisms
mostly
are
gram
negative
microorganisms
Pseudomonas sp., most of them belong to the aerobic
bacteria. Aerobic degradation rate is much quickly
than the anaerobic degradation. Feng YY et al.,
isolated an aerobic Pseudomonas sp. strain PA-18
taking TA as sole carbon source from the sludge pool
of Yangzi Petrochemical Limited Liability Company
wastewater treatment plant, in the optimum
condition was 37 ℃, pH 7, aerobic treatment PTA
concentration of 3000 mg/L, the degradation rates on
PTA was more than 95%, and the Chemical oxygen
demand (CODcr) removal rate could reach as high as
85%. Microbial is catalyst for biological wastewater
treatment.
Therefore,
the
TA
degrading
microorganisms were screened, isolated, and
researched on the degradation characteristics of TA,
for the efficient biological treatment, has important
theory significance and the practical significance,
especially for anaerobic treatment of PTA wastewater.
Isolation Method of TA Degrading Bacteria
Taking 1 g sludge from an expanded granular
sludge bed (EGSB) reactor which was operated for 1
year, into the pre-sterilized, 250 ml Erlenmeyer flask
containing 150 ml TA inorganic medium with 1000
mg/L of TA. Then placed them into the aerobic
shaking culture incubator to enrichment cultivate, at
35°C, until the clear medium became cloudy,
transferred 10% (10 ml) to the TA inorganic salt
medium for the next batch static culture. Gradually
increased TA concentration to 3000 mg/L. Drawing
0.1 ml this liquid medium, transferred to TA
inorganic salt agar solid medium with TA
concentration of 3000 mg/L, repeatedly separated and
purified of bacteria.
3 bacteria strains were isolated, which were
aerobic bacteria, and according to their growth rate in
TA inorganic salt culture medium, further screened
one aerobic superior strain as the experimental
strains, named JD-1.
Identification Methods on Bacteria
According to the Bacteria Identification Manual
to carry out the following identification test: Gram
stain, Oxidase test positive, Acetyl methyl methanol
(V.P.) test, Methyl red (M.R.) experiment, 42°C
growth test and Gelatin liquefaction test.
Pyocyanin test: cultivated the bacteria in LB
culture medium until the logarithmic growth phase,
added chloroform 3~5ml in vitro, shaking, settled for
a moment, transferred the chloroform to another test
tube, adding 1mol/L hydrochloric acid solution about
1ml, shaking and standing for a moment, if there
appears pink in hydrochloric acid solution layer,
namely pyocyanin test positive, at the same time do
the negative control.
Materials and Methods
Medium and Reagent
16S rDNA PCR Amplification and Sequence
Analysis
Inorganic salt culture Murashige and Skoog
medium (MS, pH 7) including: 0.1 g MgSO 4·7H2O,
0.02 g CaC12·2H2O, 0.68 g KH2PO4, 1.73 g K2HPO4,
0.03g MnSO4·H2O, 1.0g NH4NO3, 0.03g FeSO4·7H2O,
add distilled water to 1000 mL. TA inorganic salt
culture medium: according to the need of experiment
in MS culture medium supplemented with
appropriate concentration of TA (dissolved TA in MS
dissolved NaOH solution). Lysogeny broth (LB)
medium (pH 7): 10 g peptone, 5 g beef extract, 5 g
NaCl, distilled water to 1000mL. Its solid medium (i.e.
nutrient agar) was added 1.8% agar into LB medium.
Acetyl methyl methanol (V.P.) and methyl red (M.R.)
experimental culture medium: 5 g peptone, 5 g
glucose, 5 g NaCl, distilled water to 1000mL.
Extracting DNA of the strain per CTAB method,
which used as 16S rDNA template in PCR
(Polymerase
chain
reaction)
amplification.
Amplification of 16S rDNA adopted bacterial
universal primer. The sequence of the forward primer
BSF8/20 was: 5'-AGAGTTTGATCCTGGCTCAG-3';
the
reversed
primer
BSR1541/20
was:
5'AAGGAGGTGATCCAGCCGCA-3'.
Using the Blastn program made the homologous
comparison between the obtained nucleic acid 16S
rDNA
sequence
and
GenBank
data.
(http://www.ncbi.nlm.nih.gov/blast).
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Zhu et al.
Results and Discussion
(PHB). The colony of JD-1 was circular smooth, edge
regular, surface smooth and moist, shiny, appeared
green in LB medium, in the pure TA medium also
have a small amount of blue and green pigment
diffusion (as shown in Fig. 2). Gram negative bacilli,
oxidase test was positive, pyocyanin test was
negative. The nitrate reducing produce gas test, 42 ℃
growth test, hydrolysis and liquefaction test of gelatin
were all positive, the M.R test (methyl red test), V.P
test (acetyl methyl carbinol test) were all negative,
identification of the strains was Pseudomonas
aeruginosa sp.
Physiological and Biochemical Identification of
the Bacteria
The research showed that, the isolated strains
JD-1 was gram negative. JD-1 was aerobic bacteria
(facultative anaerobic), could produce ink green
pigment when grew on LB medium. The results of
electron microscope showed that the JD-1 was rodshaped or elliptic with terminal flagellum (Fig. 1).
Their size was (0.5-0.8) μm × (0.9-1.6) μm. No spores
and no accumulation of poly-beta-hydroxybutyrate
Fig. 1: The scanning electron microscope photograph of bacterium JD-1.
Fig. 2: The bacterial colony photograph of bacterium JD-1. (Left: LB substrate; Right: TA substrate).
In order to double verify the identification, the
shown in Table 1. Also the strain was identified as
automatic identification card made of bioMerieux
Pseudomonas aeruginosa.
VITEK-2 (France) was used to identify the strain, as
Table 1: Results of VITEK Test.
RHANAG+
RIBINOACE+
LAT+
ALA+
MAN+
ARAPROP+
CAP+
VALT+
2KG+
30BU+
pOBE+
SER-
SACGLU+
CIT+
PRO+
3
MALSAL+
HIS+
MNT+
ITA+
MEL+
5KGSOR-
SUBFUCGLYGmOBE-
PAPER IN PRESS
biochemical and molecular biological method for
species identification results: aerobic TA degradation
bacteria was Pseudomonas aeruginosa.
Molecular Biology Identification Results of the
Bacteria
Total DNA was extracted from the strain,
amplification of full-length 16S rDNA sequence using
bacterial universal primers, was about 1.5 Kb DNA
fragment. Part of the 16S rDNA was sequenced, the
sequence was submitted to GenBank nucleotide
database registration, was given access number, the
strain JD-1 was identified as No. EF489294 (502bp).
The 16S rDNA sequences of the strain was input
into the GenBank, to make the homology comparison
by Blastn program. The result showed that, 16S
rDNA sequence of JD-1 reached 98% of homology with
its of Pseudomonas aeruginosa. Combined with
TA Degradation Characteristics
Put 150 ml TA medium (1000 mg/L) into 250ml
triangle bottle, seeded with 0.5 ml fresh seed solution
(JD-1 strain), aerobic static cultivated in the shaker
incubator with 150 r/min, at 35 ℃. After a certain
period, measured the pH value of medium and the
degradation rate of TA. Determination method on TA
was
the
First
Order
Derivative
UV
Spectrophotometric.The results were in Table 2.
Table 2: Degradation efficiency of TA by JD-1 strain (Unit: %).
Duration, h
Strain JD-1
0
0
12
67
24
99
Max (%)
99
From table 2, it is quickly to degrade TA by JD-1
bacteria under aerobic conditions. The initial pH was
7, pH value of the aerobic solution increased
gradually.
were carried out on the NOVA-4 computer data
processing system.
Analysis of TA Degradation Products
For aerobic degradation, took a sample to add
some acid every 4 h. Before the first 8h, all samples
had a large number of white precipitate in solution
after dosed acid, which suggesting that most of TA
samples were not transformed by bacteria, when at
12h, took samples to dose acid solution, only a small
amount of white precipitate (about 1/5 of the former),
which suggested most of the TA in the samples had
been transformed into other substances by bacteria;
at 24 h, after dosed acid to samples, there was no
white precipitate, namely all TA in the culture
solution was converted into other substances. From
the GC/MS chromatogram Fig.3 of aerobic
degradation products of TA, degradation and
metabolites products were multiplicate, the product
was 52. The retrieval by computer, the main peaks
were benzene, alkanes and small molecule alcohol,
acid, ester etc. Given that many TA degradation
pathways have been studied clearly, this paper did
not focus on them.
Analysis Method of Degradation Products
Each 0.5 ml fresh seeds was inoculated in 8 sets
of Erlenmeyer flasks of 150 ml containing sterilization
of PTA medium, in aerobic flasks in the enrichment
culture, under the condition of 35 ℃, aerobic shaking
conditions a 24 h, taking one every 8 h for anaerobic
flasks, taking one every 4 h for aerobic flasks,
processing according to the following method:
centrifugation at 3000 r/min for 20 min, then the
supernatant was concentrated to 25 ml at 50 ℃, and
dosing H2SO4 to adjust to pH 2, double filter paper
filter. Aerobic filter liquid was collected separately,
taking three times of extraction with 200 ml ether.
The merger of three extract, passed through a glass
funnel filtration with 30-40g anhydrous Na2SO4, then
separately poured into a cylinder of 500ml, purge
concentrated to about 3 ml by nitrogen. The treated
samples were by analyzed by GC/MS-QP2010NC gas
chromatography/mass spectrometry made of USA
Angilent Instrument Company, qualitative analyses
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Zhu et al.
Fig. 5: GC-MS chromatogram of aerobic degradation production of TA by bacterium JD-1.
Characteristics of TA Degrading Enzyme
Acknowledgements
For the aerobic bacteria (JD-1 bacteria):
inoculated a loop culture from slope culture of TA
bacteria cultivated for 24 h at 35 ℃, into a 150 ml
Erlenmeyer flask containing 100 ml LB culture
medium, shaking cultivated for 12 h, then at the
logarithmic growth phase. Taking 10 ml culture,
using microporous membrane filter sterilized liquid
culture, 7 ml was added to 100 ml sterile TA (1000
mg/L) medium, 35 ℃ shaking culture, TA
concentration was measured by regular sampling.
Another 3 ml bacterium medium was added to
another containing 100 ml sterile TA culture medium,
was used as control under the same cultivate
conditions. Although the results showed that the
degradation ability of sterilizing liquid culture was
not poor than liquid culture on TA difference, but also
can quickly eventually degrade TA. The enzyme of
JD-1 bacteria degrading TA also existed at the
external of cell, it is the extracellular enzyme. The
difference between endoenzyme and ectoenzyme was
an important cause led to different TA degradation
rate. Extracellular enzyme decomposition rate was
faster.
This work was financially supported by the
Natural Science Foundation of China (Project no
51108261), and the Start Program for Young Talent of
Shanghai Jiao Tong University (Project no
12X100040059). The authors acknowledge Wei LI
from Instrumental Analysis Center, Shanghai Jiao
Tong University for their assistance in SEM analyses.
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Conclusions
The main conclusions of the research are as
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An aerobic TA degradation bacterium was
isolated from the EGSB reactor, which named JD-1.
The result of species identification by biochemical and
molecular biological method: aerobic degradation of
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degradation rate of JD-1 aerobic was 99% on 1000
mg/L TA in 24 h. Its enzyme was the extracellular
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