International Biodeterioration & Biodegradation 93 (2014) 70e77

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Biodegradation of N-Methylpyrrolidone by Paracoccus sp. NMD-4 and

its degradation pathway
Shu Cai a, Tianming Cai b, Shiyang Liu b, Qian Yang b, Jian He a, *, Liwei Chen b, *, Jiang Hu b
a
The College of Life Science, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China
b
The College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: N-Methylpyrrolidone (NMP), a kind of nitrogen-containing heterocyclic pollutant, is widely used in
Received 26 December 2013 chemical industry. Microbial degradation is an important environmental fate process in soil and water,
Received in revised form however, the microbial metabolic mechanism is still unknown. Strain NMD-4, capable of utilizing NMP as
28 April 2014
the sole source of carbon and nitrogen, was isolated from the activated sludge of a pesticide plant in
Accepted 28 April 2014
Available online
Jiangsu, China, and identified as Paracoccus sp. based on its physiologicalebiochemical properties, as well
as 16S rRNA gene sequence analysis. The degradation characteristic of NMP by strain NMD-4 was studied
in a liquid culture, and the metabolic pathway of NMP by the strain was investigated. Two metabolites, 1-
Keywords:
N-Methylpyrrolidone
methyl-2,5-pyrrolidinedione and succinic acid, were detected and identified by liquid chromatography-
Paracoccus sp. mass spectrometry analysis, and a plausible microbial degradation pathway of NMP was proposed by the
Biodegradation first time.
Metabolic pathway Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction degradation mechanism in the environment, and a lot of studies

N-Methylpyrrolidone (NMP) is a nitrogen-containing heterocy-
clic compound with a five-membered lactam structure. Its advan-
tages include a strong polarity, good inertia, low viscosity, non-
corrosiveness and low volatility (Lane DJ, 1991). The high produc-
tion of NMP wide usage as a solvent and intermediate in chemical
industry may result in its release to the environment through
various waste streams (Rhee et al., 1997). NMP is difficult to remove
because of its high solubility in water. According to the Toxics
Release Inventory Data by American Conference of Governmental
Industrial Hygienists (2008), NMP is released into the environ-
ment by chemical industries at a volume of 2450 tons. NMP shows
certain toxic to human’s health. E.g., it causes headache and irri-
tation to the respiratory system and eyes (Beaulieu et al., 1993);
skin contact with NMP may also cause abortion (Solomon et al.,
1996; Bower et al., 1997). The Environmental Protection Agency
of the USA and the European Union have recognized NMP as
causing developmental toxicity. The Food and Drug Administration
of the USA placed NMP into class 2 of residual solvents that should
be limited in pharmaceutical products due to their inherent toxic-
ities. Therefore, it is necessary to investigate its behavior and
* Corresponding authors. Tel./fax: þ86 25 84395002.
E-mail addresses: hejian@njau.edu.cn (J. He), clw@njau.edu.cn (L. Chen).
concerned the absorption, migration, volatilization and conversion
of NMP in soil and water have been reported. The Koc of 1-methyl-
2-pyrrolidone is estimated as 5(SRC), using a log Kow of
0.38
(Sasaki H et al., 1988) and a regression-derived equation (US EPA;
Estimation Program Interface (EPI) Suite. Ver. 4.1. Jan, 2010). Ac-
cording to a classification scheme (Swann RL et al., 1983), this
estimated Koc value suggests that 1-methyl-2-pyrrolidone is ex-
pected to have very high mobility in soil. 1-Methyl-2-pyrrolidinone
had Rf values of 0.74, 0.65, 0.67, and 1.0 in silt, loam, clay and sand,
respectively, in laboratory soil thin layer chromatography (TLC)
experiments (Shaver TN, 1984) which is consistent with significant
mobility in soil (SRC). The Henry’s Law constant for 1-methyl-2-
pyrrolidone is 3.20  10
9 atm-cu m/mole (SRC) using a fragment
constant estimation method (Kim et al., 2000). This Henry’s Law
constant indicates that 1-methyl-2-pyrrolidone is expected to be
essentially nonvolatile from water surfaces (Lyman et al., 1990). 1-
Methyl-2-pyrrolidone’s Henry’s Law constant indicates that vola-
tilization from moist soil surfaces may not occur (SRC).
In soil, NMP is expected to have a very high mobility with an
estimated Koc of 4.6, and a low volatilization from soil surfaces
based upon its vapor pressure. According to Japanese MITI (Min-
istry of International Trade and Industry) data, 73 percent of the
Theoretical BOD was reached in 4 weeks in a static die-away system
using an activated sludge with the initial NMP present at 100 mg/L,
suggesting that microbial degradation is an important

http://dx.doi.org/10.1016/j.ibiod.2014.04.022
0964-8305/Ó 2014 Elsevier Ltd. All rights reserved.
 S. Cai et al. / International Biodeterioration & Biodegradation 93 (2014) 70e77
environmental fate process in soil and water (Chem Inspect Test
Inst., 1922). However, to the best of our knowledge, there is no
report about the degradation of NMP by a pure culture and its
microbial metabolic mechanism.
In this study, an NMP-degrading strain, NMD-4, was isolated
from the activated sludge of a pesticide plant in Jiangsu, China.
Strain NMD-4 was identified as Paracoccus sp. This strain could use
NMP as the sole carbon and nitrogen sources for growth and
completely degrade 500 mg L
1 NMP within 24 h. The degradation
characteristic of NMP by strain NMD-4 was studied in a liquid
culture, and the degradation pathway of the NMP by the strain was
also investigated.
2. Materials and methods
2.1. Materials and media
The activated sludge used for enrichment was collected from a
pesticide factory in Jiangsu, China. NMP and chromatographic-
grade dichloromethane was purchased from Sinopharm Chemical
Reagent Co. Ltd. Molecular biology reagents were purchased from
TaKaRa Biotechnology Co. Ltd (Dalian, China). All other reagents
used in this study were of analytical grade. A mineral salts medium
(MSM) (1.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO47H2O, 1.0 g NaCl
per liter water, pH 7.0) was used in this study for enrichment, and a
LuriaeBertani (LB) medium (10.0 g NaCl, 10.0 g peptone and 5.0 g
yeast extract per liter water, pH 7.0) was used for strain culture. For
solid media, 15 g per liter of agar powder was added. When
necessary, NMP was added to the media at an appropriate con-
centration. All media were sterilized by autoclaving at 121  C for
20 min.
2.2. Enrichment and strain isolation
An activated sludge sample (5 g) was added to 100 ml of MSM
containing 500 mg L
1 NMP as the sole carbon and nitrogen source.
The sample was incubated on a rotary shaker at 160 rpm at 30  C.
After 3 days, the culture became turbid, and 5 ml of the culture was
inoculated in fresh MSM containing 500 mg L
1 NMP and incubated
for another 3 days. The decrease of NMP was monitored by HPLC as
described below. Enrichment cultures capable of degrading NMP
were diluted and spread onto MSM agar containing 500 mg L
1
NMP. Colonies grown on these plates were picked up, purified by
repeated streaking, and tested for the ability to degrade NMP. In
this study, four strains capable of degrading NMP were isolated.
Among them, strain NMD-4, which processed the highest NMP-
degrading ability, was selected for further investigation.
2.3. Identification of the strain
The NMP-degrading bacterium was identified according to
Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994).
Genomic DNA was extracted by high-salt precipitation (Sambrook
and Russel, 2001), and the 16S rDNA sequence was amplified by
PCR using standard procedures with a bacterial universal primer set
27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -TACGGC-
TACCTTGTTACGACTT-30 ) (Lane, 1991; Marchesi et al., 1998). A
fragment corresponding to E. coli 16S rDNA positions 8e1513 was
amplified. The PCR product was purified using an Axy-Prep PCR
Purification Kit (AxyGen) and determined by the Nanjing Jinsirui
Biological Technique Company (Applied Biosystems, model 3730).
Closely related 16S rDNA sequences from GenBank were aligned
using Clustal_X, and phylogenetic analysis was performed with the
software MEGA version 3.0 (Kumar et al., 2004). Phylogenetic trees
were generated using the neighbor-joining method according to
71
the Kimura 2-parameter model and evaluated by bootstrap ana-
lyses based on 1200 resamplings (Li et al., 2009a,b). DNAeDNA
hybridizations were performed according to the method of Ezaki
et al.
2.4. Inoculum preparation for degradation studies
Strain NMD-4 was precultured in 5 ml of LB medium at 30  C
with shaking at 160 rpm for 12 h. Cells were harvested by centri-
fugation at 6000 rpm for 3 min, washed twice, and resuspended in
fresh MSM (OD600nm ¼ 1.0).
2.5. Degradation of NMP by strain NMD-4 in relation to the
bacterial growth
An inoculum (1 percent, v/v) was inoculated into a 500 ml flask
containing 200 ml MSM supplemented with 500 mg L
1 of NMP.
Each treatment was performed in triplicate, and control experi-
ments without inoculation or without substrate were carried out
under the same condition. The cultures were incubated on a rotary
shaker at 160 rpm and 30  C. At regular intervals, 5 ml samples
were collected from each flask. Bacterial growth was monitored by
measuring the optical density of culture samples at 600 nm
(OD600). The disappearance of NMP was monitored by high-
performance liquid chromatography (HPLC), and the metabolites
were identified by HPLCeMass spectrometry (MS) as described
below.
2.6. Biodegradation kinetics
The NMP-degrading strain incubated in LB medium for 48 h was
centrifuged at 6000 g for 10 min in 50 mL Eppendorf centrifuge
tube. The cell pellet was washed twice with phosphate buffer saline
(PBS, pH 7.4) and then suspended in NaCl solution (0.85 percent).
Then the bacteria suspension was prepared for the next biodegra-
dation experiments. 4.0 ml of bacteria suspension was added into
250 ml flasks containing 100 ml MSM with 500 mg L
1 NMP to
make the final cell density 1.0  108e1.0  109 cfu mL
1 .The flasks
was placed in rotary shaker (160 r min
1) at 30  C. At regular in-
tervals, 5 ml samples were collected from each flask and the sam-
ples were used to determine the concentration of NMP. Cell
counting was performed with LB plates by the plate dilution
technique, and colonies were counted after 72 h of incubation at
28  C.
2.7. Effects of the temperature, pH, metal ions, initial substrate
concentration and dissolved oxygen concentration on NMP
degradation
Two milliliter inoculums were incubated in 200 ml of MSM
containing 500 mg L
1 NMP for 24 h at different temperatures (20,
25, 30, 35, 40 and 45  C), under different pH conditions (5.0e10.0,
in increments of 1.0 pH units) and with different concentrations
(0.1 mM and 1 mM) of metal ions (Cd2þ, Cu2þ, Co2þ, Mn2þ, Zn2þ and
Ni2þ). The effects of initial NMP concentrations of 500 mg L
1,
750 mg L
1, 1000 mg L
1, 1500 mg L
1, and 2000 mg L
1 were
investigated. The effects of oxygen level on NMP degradation was
investigated by inoculating the strain into 50 mL, 75 mL and 125 mL
MSM containing 500 mg L
1 NMP, respectively. The cultures were
incubated on a rotary shaker at 160 rpm and 30  C. At regular in-
tervals, 2 ml samples were collected from each flask. The biomass
was monitored by OD600, and the concentration of the NMP was
determined by HPLC. Experiments were conducted in three parallel
flasks and the concentration of NMP in each flask was indepen-
dently determined.
72 S. Cai et al. / International Biodeterioration & Biodegradation 93 (2014) 70e77

2.8. Analytical method

The concentration of NMP was determined by HPLC (FL2200,

Fuli analytical instrument Co. Ltd, Zhejiang, China). The separation
column used for HPLC (internal diameter of 4.6 mm and length of
25 cm) was filled with C18. The mobile phase was methanol:water
(20:80, V:V), and the flow rate was 1.0 ml min
1. The detection
wavelength was 214 nm, and the injection volume was 20 mL. The
metabolites produced during NMP degradation were identified by
HPLC-MS. The conditions for the HPLC/UV in HPLC-MS were as
followed: The type of the columns were Agilent XDB-C18
5 cm  0.46 cm 1.8 mm columns, and methanol and ultrapure
water (20:80, V:V) was used as mobile phase, the flow rate was
1 ml min
1. MS analyses were performed in ESI mode with an
Agilent G6410B Triple Quad Mass Spectrometer.

2.9. Nucleotide sequence accession numbers

Fig. 1. Time course of NMP biodegradation and growth of Paracoccus sp. strain NMD-4
on mineral salt medium containing 500 mg L
1 NMP.
The GenBank Accession number for the 16S rRNA gene sequence
of strain NMD-4 is KF715850.
amount of the NMP added in the medium suggested a possible
3. Results and discussion negative correlation between the growth of the strain and the NMP
concentration: the high the NMP concentration was, the slower the
3.1. Isolation and identification of the NMP-degrading strain strain grew. The decrease of the bacteria growth at higher NMP
concentration is most likely due to substrate inhibition.
After two rounds of enrichment, the enrichment culture could
degrade about 75 percent of 500 mg L
1 NMP within 3 days. A 3.3. Biodegradation kinetics
number of bacterial strains capable of degrading NMP were isolated
from the enrichment culture. One isolate, designated NMD-4, was The biodegradation experiments were carried out at 30  C, pH
selected for further study due to its strongest NMP-degrading 7.0, with the initial NMP concentration of 500 mg L
1. As shown in
ability. This strain was able to degrade over 90 percent of Fig. 3, the cell number of strain NMD-4 (colony forming units) kept
500 mg L
1 NMP in MSM within 24 h incubation and utilized NMP relatively stable (about 8.5 108 cfu/ml) during the whole process,
as carbon and nitrogen sources for growth. whereas the biodegradation efficiency of NMP were 28.25 percent,
When grown on LB agar, colonies of the strain are pale white, 60.20 percent, 79.89 percent and 90.64 percent, when incubation
circular and convex. Cells are non-spore-forming, gram-negative, for 4 h, 8 h, 12 h and 16 h, respectively. Based on these results, the
non-motile, strictly aerobic and globular or globular-rod-shaped biodegradation of NMP was fitted with the first-order kinetics
(Fig. S2). The optimal growth of strain NMD-4 was observed at shown as follows:
pH 7.0e8.0 and a temperature of 30  C in LB medium. Positive for
nitrate reduction, but negative for hydrolysis of starch and Tweens dC=dt ¼
kC
20, urease, arginine dihydrolase, gelatinase, glucose fermentation
The integrated form of the above equation is
and indole production. Utilizes L-arabinose, D-glucose, D-mannitol,
arabinose, succinic acid, valeric acid, D-sorbitol, D-mannose, pro-
C ¼ C0 $e
kt
pionic acid, acetic acid, malate, L-histidine, sodium lactic acid, D-
fructose, methylamine and propionic acid. Genomic DNA was iso- where C is the NMP concentration at time t, C0 is the NMP con-
lated from the bacterium and the 16S rRNA gene sequence was centration at time zero k is the first-order rate constant. According
amplified by PCR. Phylogenetic analysis of the 16S rRNA gene
sequence (Fig. S3) revealed that strain NMD-4 belonged to Para-
coccus sp. and formed a subclade with Paracoccus bengalensis JJJT
(99.92 percent similarity) and Paracoccus versutus ATCC 25364T
(99.93 percent similarity). On the basis of 16S DNA gene analysis,
the strain was identified as Paracoccus sp.

3.2. Growth and NMP biodegradation

3.2.1. Utilization of NMP as the sole source of carbon and nitrogen

for growth of strain NMD-4
As shown in Fig. 1, the growth of the strain entered a log phase
after a lag phase of approximately 4 h, the OD600 increased from
0.012 to 0.789 within 24 h; correspondingly, The concentration of
NMP slowly decreased in the first 4 h and then sharply declined,
and finally the added NMP was completely degraded within 24 h.

3.2.2. Effect of the initial concentration of NMP on its degradation

Fig. 2 shows the effect of the initial concentration of NMP on the Fig. 2. The effect of different NMP initial concentration on growth of NMD-4 strain in
growth of strain NMD-4. Comparison of the biomass with the mineral salt medium.
 S. Cai et al. / International Biodeterioration & Biodegradation 93 (2014) 70e77
Fig. 3. (a) Time course of biodegradation of NMP and growth of strain NMD-4; (b) The
first order kinetic of the biodegradation of NMP by strain NMD-4.
to the fitted result, the first-order rate constant was 0.137, and the
biodegradation half-life of NMP was 5.05 h.
3.4. NMP degradation characteristics
3.4.1. Effect of temperature on the degradation of NMP
Fig. 4(a) shows that after 24 h of cultivation, when the tem-
peratures were 20  C, 25  C, 30  C, 35  C and 40  C, 23 percent, 82
percent, 91 percent, 85 percent and 42 percent of the added NMP
were degraded, respectively. These results indicated that NMP was
degraded quickly at temperatures ranging from 25  C to 35  C and
the most optimal temperate was 30  C.
3.4.2. Effect of pH on the degradation of NMP
As shown in Fig. 4(b), when the pH was between 6 and 8, more
than 80 percent of the added 500 mg L
1 NMP was degraded by the
strain within 24 h, the highest NMP removal rate was achieved at
pH 7.0. When the pH was below 6 or above 9, the NMP biodegra-
dation process was severely inhibited.
3.4.3. Effects of heavy metal ions on NMP biodegradation
The results of Fig. 4(c) revealed that both 1.0 mM and 0.1 mM of
Ni2þ, Cd2þ and Mn2þ severely inhibited the degradation of NMP.
Addition of 1.0 mM Cu2þ or Co2þ strongly inhibited the degradation
activity, whereas addition of 0.1 mM of Cu2þ or Co2þ showed a
slight inhibitory effect. However, addition of 1.0 mM or 0.1 mM
Zn2þ showed little inhibition effect on the degradation of NMP.
3.4.4. Effects of dissolved oxygen concentration on NMP
biodegradation
As we know, at the culture shaking at the same speed, the more
bulk the culture volume is, the less the oxygen dissolved in the
culture. The data in Table 1 revealed a negative correlation between
the degradation rate of NMP and the liquid volume, suggesting that
73
Fig. 4. Effect of temperature (a), pH (b) and metal ions (c) on NMP degradation
efficiency.
oxygen level is an important factor in the degradation of NMP, thus
high dissolved oxygen level can promote the NMP degradation
efficiency.
3.5. The metabolic pathway of NMP biodegradation by strain
NMD-4
The metabolites produced during NMP degradation were iden-
tified by HPLC-MS. The mass spectrum of NMP and its metabolites
after 12 h of cultivation and 24 h of cultivation are shown in Figs. 5e
6.
As shown in Fig. 5(a) and (b), the most prominent protonated
molecular ions were found at m/z ¼ 100.3 [M þ H]þ, m/z ¼ 122.5
[M þ Na]þ, m/z ¼ 136.4 [M þ Na]þ, and m/z ¼ 117.4 [M
H]
, and the
compounds corresponding to these protonated molecular ions
were designated as products A, B, C, and D respectively.
Fig. 6(a) shows that the positive-ion chemical ionization of
product A was 100.1 [M þ H]þ, enabling the assignment of the
molecular ion (Mþ) at m/z ¼ 99. Product A had characteristic
second-order MS fragment ion peaks at m/z ¼ 58.10 and 69.10. We
proposed that peak m/z 58.10 was due to the loss of CH3NH2 and
peak m/z 69.10 was due to the loss of CH2eC]O. Product A was
identified as N-methyl-2-pyrrolidone. Product B showed a base
peak at m/z ¼ 122.0, also enabling the assignment of the molecular
ion at m/z ¼ 99.0. We proposed peak m/z 122 was due to the
74 S. Cai et al. / International Biodeterioration & Biodegradation 93 (2014) 70e77

Table 1 at m/z ¼ 99.0, 75.8, 85.3 and 54.8. As it can be seen m/z 99 fragment
Influence of oxygen factor to the biodegradation of NMP by strain NMD4. loosing H2O molecule, m/z 75.8 fragment loosing H2O and CH2, m/z
Liquid volume (mL) Degradation ratea (%) 85.3 fragment loosing CH2eC]O and m/z 54.8 corresponding to
50 95  5
H2C]CHeCH]O. Thus product D was identified as succinic acid.
75 93  4
125 84  2
4. Discussion
a
Results are average value of triplicate measurements  deviation.

Nitrogen-containing heterocyclic compounds, such as pyridine,

addition of Naþ. Thus, product B was identified as N-methyl-2-
pyrrolidone. The positive-ion chemical ionization of product C
was 136.4 [M þ Na]þ, enabling the assignment of the molecular ion
(Mþ) at m/z ¼ 113. As shown in Fig. 6(b), product C had charac-
teristic second-order MS fragment ion peaks at m/z ¼ 67.8, 92.3,
78.4, and 96.9. The initial protonation of the carbonyl group, as
occurs in C, makes the further elimination of CH3eC]O, CH3eNe
C]O and CH2eC(NHeCH3) ¼ O. Product C was identified as 1-
methyl-2,5-pyrrolidinedione. The negative-ion chemical ioniza-
tion of product D was 117.4 [M
H]
, enabling the assignment of the
molecular ion (M
) at m/z ¼ 118. Fig. 6(c) demonstrates that
product D had characteristic second-order MS fragment ion peaks
quinolone and NMP, are widely used in chemical industry, and also
are important environmental contaminants. Therefore, their
degradation mechanisms are of great interest. Microbial degrada-
tion is considered to be one of the most important factors for
removing nitrogen-containing heterocyclic pollutants from the
environment. To date, several bacterial strains capable of degrading
some other nitrogen-containing heterocyclic pollutants, such as
pyridine and quinolone, have been reported (Kaiser et al., 1996).
Strain NMD-4 was an efficient NMP-degrading strain, which could
mineralize 500 mg L
1 NMP within 24 h in a very small inoculum.
The strain was identified as Paracoccus sp. To our knowledge, strain
NMD-4 is the first NMP-degrading strain reported for the genus

O
Na+

C
a

OH

O
O

OH
D b

Fig. 5. HPLC-MS analysis of metabolites formed during NMP degradation by strain NMD-4 (a) 12 h extract, (b) 24 h extract.
 S. Cai et al. / International Biodeterioration & Biodegradation 93 (2014) 70e77 75

Fig. 6. Tandem mass spectrometry spectrum of NMP and its metabolites: (a) NMP þ Hþ (b) 1-methyl-2,5-pyrrolidinedione (c) succinic acid.
76 S. Cai et al. / International Biodeterioration & Biodegradation 93 (2014) 70e77
CH CH
O
N N O O
O O
CH3NH2
Fig. 7. Proposed metabolic pathway of NMP by strain NMD-4.
Paracoccus. Representatives of the genus Paracoccus are saprophytic
soil and water microorganisms (Siller et al., 1996; Tsubokura et al.,
1999), bioreactors (Lipski et al., 1998; La et al., 2005), activated
sludge (Katayama et al., 1995; Liu et al., 2006), root nodules
(Deng et al., 2011), cornetfish (Kim et al., 2010) and the marine
environment (Pukall et al., 2003; Berry et al., 2003; Lee et al., 2004;
Kim et al., 2006). They are metabolically versatile and can degrade a
wide range of toxic organic pollutants, such as diflubenzuron,
propanil, chlorpropham dimethoate (Sun et al., 2013), acetobathlor
(Ni et al., 2011), methylamine, N,N-dimethylformamide (Urakami
et al., 1990), and acetone (Siller et al., 1996).
The pathway of NMP metabolism in strain NMD-4 was firstly
studied by metabolite identification, and the proposed pathway is
shown in Fig. 7. In strain NMD-4, NMP is oxidized to form 1-methyl-
2,5-pyrrolidinedione. The next step is the cleavage of the two CeN
bonds of 1-methyl-2,5-pyrrolidinedione to form succinaldehyde,
and then succinaldehyde was oxidized to succinic acid, which then
entered the Krebs cycle which is the central carbon metabolic
pathway, and was completely mineralized. This pathway in the
strain was different from those in mammals, mice and human body,
degradation of NMP was initialed by 5-hydrolation or 2-reduction
to form 5-hydroxy-N-methyl-2-pyrrolidone or 2-hydroxy-N-
methylsuccinimide respectively; and the further metabolic
pathway of 5-hydroxy-N-methyl-2-pyrrolidone and 2-hydroxy-N-
methylsuccinimide in mice and human are still unknown.
The metabolic mechanism of NMP in strain NMD-4 is also
different from other previously reported nitrogen-containing het-
erocyclic pollutants, pyridine and quinoline. In strain NMD-4, the
degradation of NMP was initialed by oxidation to introduce a
carbonyl group at the 5-position carbon, whereas, there were two
general proposed microbial degradation pathways of pyridine:
under anaerobic condition, degradation of pyridine was initiated by
either ring reduction or ring hydroxylation, and under aerobic
condition, pyridine was degraded through aerobic reduction
(Holcenberg et al., 1969; Watson et al., 1975); microbial degradation
of quinoline was transformed by hydrolation, which then followed
by cleavage of either pyridine ring or aromatic ring (Grant et al.,
1976; Bennett et al., 1985; Fetzner, 1998).
Thus, in light of its relatively high degradation activities and
ability to mineralize NMP, strain NMD-4 is a good candidate for the
study of the metabolic mechanism of NMP, and has the potential
application in removing NMP from industrial wastewater.
Acknowledgments
This work was supported by Taihu lake recovery scientific
project of Jiangsu Province, China (grant no. TH2012203 and
TH2013210). The authors would like to thank Experiment Platform
owned by the College of Resources and Environmental Sciences,
Nanjing Agricultural University, for providing experimental
equipment.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ibiod.2014.04.022.
OH
O O CO2+H2O
OH
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