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Isolation and characterization of microorganisms degrading nylon 4

in the composted soil


Koichiro Tachibana, Kazuhiko Hashimoto
*
, Masato Yoshikawa, Haruki Okawa
Department of Applied Chemistry, Faculty of Engineering, Kogakuin University, Nakano-cho, Hachioji, Tokyo 192-0015, Japan
a r t i c l e i n f o
Article history:
Received 5 December 2009
Received in revised form
16 March 2010
Accepted 20 March 2010
Available online 27 March 2010
Keywords:
Nylon 4
Biodegradation
Bacteria
Fungi
Soil
Compost
a b s t r a c t
Two kinds of microorganisms, a bacterium (KT-1 strain) and a fungus (KT-2 strain), degrading nylon 4
(polyamide 4), which was easily prepared by the anionic ring-opening polymerization of 2-pyrrolidone,
were isolated from the composted soil with the utilization of enrichment cultures and the culture
using nylon 4 as a carbon source. KT-1 and KT-2 strains were identied as neighboring species to
Stenotrophomonas sp. and Fusarium sp., respectively, by their morphological properties and the
nucleotide sequences. These strains were conrmed to grow in the culture medium containing nylon 4
powders as a carbon source. In addition, nylon 4 lm was decomposed in both mineral media con-
taining KT-1 and KT-2 strains, respectively, and disappeared within two months. The MALDI TOF-MS
analysis of nylon 4 recovered during the biodegradation test suggest that the isolated KT-2 strain
recognize the acyllactam or carboxy chain end and degrade them or their neighboring amide bond.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Synthetic plastics have been widely used as fundamental
materials for human life because of excellent properties and low
prices. Among them, polyesters have been recently developed also
as biodegradable plastics, and nowadays have been a great majority
of biodegradable plastics [1e6]. The biodegradability of synthetic
polyesters may be mainly caused by the low substrate specicity of
hydrolytic enzymes such as esterase, lipase, and so on.
However, the degradation of synthetic polyamides (nylons) is
known to be generally slower than that of the corresponding
synthetic polyesters through both biological and hydrolytic
processes under natural conditions, althoughthe polyamides contain
amide bonds similar to peptide bonds in proteins and synthetic
polypeptides [7e9]. Their high resistance to degradation is caused
mainly by the high symmetry of their molecular structures and
strong intermolecular hydrogen bonds, which result in highly crys-
talline morphology.
Therefore only a few investigations on the biodegradation of
nylons having relatively simple repeating units had been reported
[10e12]. Deguchi et al. reported that the white lot fungi strain
IZU-154, a kind of lignin-degrading microorganisms, degraded
nylon 66 lms through oxidative processes [13e16].
We found that a lm of nylon 4 (polyamide 4), which was
obtained easily by the ring-opening polymerization of 2-pyrroli-
done, was decomposed in the composted soil gathered from
Nagoya University Farm and disappeared within four months
[17e19]. Three species of microorganisms which belong to asco-
mycetous fungi were isolated as candidates for microorganisms
participating in the degradation of nylon 4. However, unfortunately
the degradation activity of the isolated strains was lost during the
repetitive subcultures.
Nylon 4 was found to degrade in the activated sludge, too, by the
researchers in National Institute of Advanced Industrial Science and
Technology in Japan [20e22]. They isolated the Pseudomonas strain
ND-11 degrading nylon 4 from the activated sludge. These results
suggest that nylon 4 specically degrades in the natural environ-
mental conditions in contrast to the other synthetic polyamides.
Since g-aminobutyric acid and 2-pyrrolidone, a repeating unit
for nylon 4, are known to be prepared from plants such as starch
and so on [23], nylon 4 will be one of promising bio-based poly-
mers. Therefore the further clarication of the degradation mech-
anism of nylon 4 is necessary for development of novel bioplastics.
In the present work, two kinds of microorganisms degrading
nylon 4, a bacterium and a fungus, were isolated from the com-
posted soil, and the degradation behaviors of nylon 4 in the pres-
ence of the microorganisms were examined [24]. The degradation
* Corresponding author. Tel.: 81 42 628 4616; fax: 81 42 628 5647.
E-mail address: hashimot@cc.kogakuin.ac.jp (K. Hashimoto).
Contents lists available at ScienceDirect
Polymer Degradation and Stability
j ournal homepage: www. el sevi er. com/ l ocat e/ pol ydegst ab
0141-3910/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymdegradstab.2010.03.031
Polymer Degradation and Stability 95 (2010) 912e917
ability of the strains was preserved even after freezing more than
one year.
2. Experimental
2.1. Materials
Nylon 4 (M
n
, 1.3 10
3
and M
w
, 3.0 10
4
) having benzamide
and acyllactam at each chain end was prepared by the anionic
ring-opening polymerization of 2-pyrrolidone using N-acyllactam
and potassium t-butoxide as an initiator and a catalyst in
a similar method to that described in our previous articles
[17e19,25e27]. The average molecular weight was controlled by
the mole ratio of the catalyst to the monomer to be from
1.3 10
3
to 3.0 10
4
. The number average molecular weight of
the low molecular weight nylon 4 (M
n
, 1.3 10
3
) was estimated
from the
1
H NMR and TOF-MS analyses and the weight average
molecular weight of the relatively high molecular weight one
(M
w
, 3.0 10
4
) was determined by viscometry [28]. Nylon 6 (M
w
,
3.7 10
4
) provided by Teijin Co. (Tokyo, Japan) was puried by
reprecipitation using 2,2,2-triuoroethanol (TFE) and acetone as
a solvent and a precipitant, respectively. Nylon 46 (M
w
, 2.0 10
4
)
was prepared by the polycondensation of nylon 46 salt.
2.2. Preparation of the nylon 4 lm and powder
Nylon 4 (M
w
3.0 10
4
, 0.7 g) solution in 2,2,2-triuoroethanol
(10 mL) was poured into at glass plate and dried at room temper-
ature for one week to give the corresponding lm. The lm was
immersed and rinsed in deionized water and dried in vacuo. The
powder of nylon 4 (80 mesh) was prepared bygrinding in a ball mill.
2.3. Preparation of the solid agar medium suspending
the nylon 4 powder
Agar (15 g), KH
2
PO
4
(1.0 g), NaCl (0.03 g), MgSO
4
$7H
2
O (0.14 g),
FeSO
4
$7H
2
O (0.03 g), CaCO
3
(0.01 g) and 1 mL of mixed trace
elements (MnSO
4
$7H
2
O 145.1 mg, H
3
BO
3
188.6 mg, CuSO
4
$5H
2
O
86.4 mg, ZnSO
4
$5H
2
O 103.4 mg, Co(NO
3
)
2
$6H
2
O 108.8 mg, and H
2
O
300 mL) were dissolved in distilled water and diluted to 1 L (pHwas
xed at 7.2). After addition of nylon 4 powder (0.1 g) as a carbon
source, the medium was sterilized in an autoclave at 121

C for
15 min and then poured into Petri dishes.
2.4. Isolation of microorganisms in the composted soil
The soil was gathered from Nagoya University Farm (Japan),
which had been composted mainly with cow manure and grass for
more than ten years (pH, 7.5e7.6). Several pieces of nylon 4 lms
(1 5 cm) were buried in the composted soil packed in the
container made of polypropylene, and kept in an atmosphere at
80% relative humidity at 25

C. A month later, the lm was taken
out from the composted soil and immersed in the normal saline
solution. The inoculums were spread on the nylon 4 agar and
cultured stationarily at 25

C for a few days. Some of colonies that
grewon the nylon 4 agar were streaked onto fresh media. After this
procedure was repeated several times, two kinds of colonies were
isolated. These isolated microorganisms were characterized by
their morphological observation and further identication entrus-
ted to Techno Suruga Co. in Shizuoka (Japan).
2.5. BOD (biochemical oxygen demand) test
In order to estimate the biodegradability of polyamides by the
isolated microorganisms, a value of BOD was determined with
a 100F-type BOD tester (TAITEC Co., Japan) according to the
procedure described in JIS K6950 (ISO 14851) [29]. Nylon 4 (M
n
1.3 10
3
or M
w
3.0 10
4
, 100 mg) was used as a carbon source in
the testing mixture and measurement was progressed at 25

C for 7
days. The molecular weight of nylon 4 recovered on the BOD test
using the relatively high molecular weight one was measured by
viscometry.
2.6. Degradation of nylon 4 lm with the isolated microorganisms
At rst a liquid mediumwas prepared fromKH
2
PO
4
(1.0 g), NaCl
(0.03 g), MgSO
4
$7H
2
O (0.14 g), FeSO
4
$7H
2
O (0.03 g), CaCO
3
(0.01 g)
and a mixture of trace elements (1 mL) per liter of distilled water. A
sheet of nylon 4 lm (20e30 mg) was immersed in the liquid
medium at pH 7.2 in each test tube, which was sterilized in an
autoclave at 121

C for 15 min before use. The mixture of the nylon
4 lmwith the liquid mediumin each test tube was inoculated with
1 mL of the culture solution which was obtained by incubation of
the isolated microorganism at 25

C on a shaker until the growth
was attained to the stationary phase in the nutrient broth or potato
dextrose broth. These testing mixtures were incubated with
constant shaking at 25

C for several weeks. Nylon 4 lmwas taken
out every week and its weight was measured, and then immersed
in the fresh medium. Such procedure was repeated for several
times.
2.7. Characterization of nylons
1
H NMR and
13
C NMR analyses were performed with a JEOL
JNM-ECX-400 Fourier transform high-resolution spectrometer. A
mixture of 2,2,2-triuoroethanol with chloroform-d
1
(volume ratio,
1:4) was used as a solvent, and tetramethylsilane was as an internal
standard. The relative viscosity of nylon 4 in m-cresol solution was
measured using an Ubbelhode viscometer at 25

C [28].
Matrix assisted laser desorption/ionization time of ight
(MALDI TOF) mass spectra were measured on a Perceptive
Biosystems model Voyager Elite ST (DE) mass spectrometer. Ions
were generated using nitrogen laser operating at wavelength
337 nm. Linear-mode acquisition methods optimized for positively
charged ion in the m/z 500e2000 mass range were adopted. 2-(4-
Hydroxyphenylazo)benzoic acid (HABA) and sodium iodide (NaI)
were used as a matrix and an ionizing agent, respectively.
Scanning electron microscopic image (SEM) of the nylon 4 lms
recovered after the degradation test with the isolated microor-
ganisms was taken with a Hitachi S-2600H scanning electron
microscope (Hitachi, Japan) at 25.0 kV of accelerating voltage.
3. Results and discussion
3.1. Isolation of microorganisms in the soil
As described in the experimental section, the agar medium
suspending the nylon 4 powder was used for the cultivation and
isolation of microorganisms degrading nylon 4. Two colonies
propagating particularly fast were puried by the repeated
streaking on the fresh nylon 4 medium. The one was identied as
a bacterium(KT-1 strain) and the other was as a fungus (KT-2 strain)
from the morphological observation as shown in Figs. 1 and 2,
respectively.
KT-1 strain was aerobic, nonsporeforming, gram negative,
motile and rod-shaped bacterium (cell size, 0.8 1.5e2.5 mm).
From the results of homology search using BLAST [30] for data base
of bacterial type strain, the 16S rDNA base sequence of KT-1 strain
shared high homology with those derived from Stenotrophomonas
and revealed 99.6% identity with those of Stenotrophomonas
K. Tachibana et al. / Polymer Degradation and Stability 95 (2010) 912e917 913
maltophilia [31] LMG958 strain. Therefore KT-1 strain was identi-
ed to be Stenotrophomonas sp. which was closely related to
S. maltophilia.
KT-2 strainyielded as a colony of the white woolliness on potato
dextrose agar (PDA). The 28S rDNA-D1/D2 base sequence of KT-2
strain showed homology with the base sequence of genus Fusarium.
From these results, KT-2 strain was identied as Fusarium sp.
3.2. Biodegradation of nylon 4 powder estimated from the BOD test
Biochemical oxygen demand (BOD) values of KT-1 and KT-2
strains were determined using nylon 4 powders, mixture of
glucose and glutamic acid (for positive control) and nylon 6
powders (for negative control) as a carbon source. The values of
both strains began to increase after about one day in accompany
with the metabolism of nylon 4, while they didnt increase even
after one week on the test using nylon 6 as a carbon source. In
addition, both strains did not multiply on the medium containing
other nylons (ex. nylon 46) as a carbon source, too, although the
corresponding data was not shown in Fig. 3. These results indicate
that both strains degraded nylon 4 exclusively but not nylon 6 and
nylon 46.
Although the lag phase for about one day was observed on the
metabolism of nylon 4 by both strains as shown in Fig. 3, the
BOD values of both strains on the medium containing glucose and
glutamic acid as carbon sources for positive control began to
increase after shorter lag (8e12 h). From these results, the isolated
microorganisms are inferred to contact with the water-soluble
carbohydrate and amino acid more frequently than the high
molecular weight nylon 4 powders.
The degrees of biodegradation of nylon 4 by KT-1 and KT-2
strains were tried to be evaluated from the BOD values of both
strains (290 ppm and 230 ppm, respectively) after the test using
nylon 4 powder as a carbon source for a week in Fig. 3. The
percentage of biodegradation (D
t
) can be estimated from the ratio
of the BOD value to the theoretical oxygen demand (ThOD) as
shown in the equation (1).
D
t
BOD=ThOD 100 (1)
The ThOD of nylon 4 could be calculated from the oxidation by
microorganisms expressed by the equation (2), if ammonia were
not oxidized.
C
4
H
7
NO 4.5O
2
/4CO
2
2H
2
O NH
3
(2)
Since the molecular weight of the repeating unit of nylon 4
(C
4
H
7
NO) is 85.1, the value of ThOD (mg per mg of nylon 4) is
calculated as follows:
ThOD 4:5 32:0=85:1 1:69mg=mg
It is consistent with the value calculated from Japanese Indus-
trial Standards (JIS) [29].
Since the concentration of nylon 4 in the mixture was 100 mg in
200 mL of solution, the value of ThOD (ppm) is transformed to be
845 ppm.
ThOD f1:69 100=200g 10
3
845ppm
Therefore the percentage of biodegradation of nylon 4 by KT-1
and KT-2 strains was estimated to be 34% and 27%, respectively. The
maximum value of D
t
by KT-2 was attained to be about 60% for
three weeks, and it did not increase afterwards. The proliferative
process of this strain is inferred to be changed to the stable phase
from the log phase. When the culture solution was inoculated into
the fresh medium containing nylon 4 as a carbon source, the strain
degraded nylon 4 and grew again. Even after the D
t
value was
Fig. 1. Microphotograph of KT-1 strain (Stenotrophomonas sp.).
Fig. 2. Microphotograph of KT-2 strain (Fusarium sp.).
Fig. 3. BOD curves of KT-1 and KT-2 strains determined using nylon4 and nylon6
powder as a carbon source. C; KT-1 strain, nylon 4 powder: -; KT-2 strain, nylon4
powder: B; KT-1 strain, nylon 6 powder: ,; KT-2 strain, nylon 6 powder.
K. Tachibana et al. / Polymer Degradation and Stability 95 (2010) 912e917 914
attained to be constant, all the microorganisms should not be dead
but take a rest. Therefore the growth environment of the micro-
organisms may be changed by the accumulation of any products
and the dead cell, lack of the nutrition, the increase of the cell
population and so on.
Even after the biodegradation test by KT-2 strain for three weeks
(D
t
60%), nylon 4 remained in the culture, of which the average
molecular weight was almost the same as the original one. This
suggests that the biodegradation by the strain proceeded on the
surface of the polyamide powder.
3.3. Biodegradation of nylon 4 lm
Nylon 4 lms were immersed in the broths containing minerals
and the isolated microorganisms (KT-1 and KT-2 strains) for two
months. As shown in Fig. 4, the weight of the recovered lms was
found to decrease with the incubation time and to disappear within
two months, whereas it did not change in the absence of the strains.
These results indicate that nylon 4 was degraded really by the
isolated strains.
Even after the cultivation in the broths containing both strains
for ve weeks, the lms keeping their original shapes were
recovered, although their weight decreased drastically. Taking
account of our previous work that the average molecular weight of
nylon 4 recovered after the biodegradation in the composted soil
was almost the same as that of the original one [17], such biodeg-
radation is supposed to proceed just on the surface of the nylon 4
lm, which can contact with the strains.
Fig. 4. Change in weight of nylon 4 lm during the immersion in the broth containing
minerals and microorganisms. C, KT-1 strain; -, KT-2 strain; , blank.
Fig. 5. SEM images of the surface of nylon 4 lms during the immersion in the broth containing minerals and KT-1, or KT-2 strains. (A), before immersion; (B), after immersion for
one week in the presence of KT-1 strain; (C), after immersion (ve weeks, KT-1 strain); (D), after immersion (one week, KT-2 strain); (E), after immersion (ve weeks, KT-2 strain).
K. Tachibana et al. / Polymer Degradation and Stability 95 (2010) 912e917 915
The surface of nylon 4 lms recovered after the submerged
cultivation was observed by scanning electron microscopy (SEM)
and shown in Fig. 5.
The SEM images in Fig. 5A, B show that the surface of the lm
began to be damaged after the submerged cultivation with KT-1
strain for one week, although it was at and smooth before the
cultivation. After the cultivation for ve weeks, the widespread and
deep damage due to the encroachment probably by KT-1 strain was
observed on the lm surface as shown in Fig. 5C. Fig. 5D, E also
indicate that the partial stripping and deep hollows were found on
the surface after the immersion in the broth containing KT-2 strain.
On the other hand, the nylon 4 lm surface did not change in the
absence of the strain even after ve weeks. Therefore, such
behavior should correspond to the biological degradation process
fromthe surface of the nylon lmwith the isolated microorganisms
(KT-1 and KT-2 strains).
In addition, the average size of the hollows on the surface of
nylon 4 lms in Fig. 5B, C was smaller than that in Fig. 5D, E,
which may correspond to the size of the strains. The lm was
damaged uniformly, because it was inferred to be evenly coated
with the multiplied KT-1 strain uniformly. In the case of KT-2
strain in Fig. 5D, E, the nylon 4 lm was heterogeneously
damaged since the hypha was guessed to take in a peripheral
substrate, crawl on the surface of the substrate and/or dive into
the lm.
3.4. Structural analysis of nylon 4
In Fig. 6 are shown the MALDI TOF mass spectra of low
molecular weight nylon 4 (M
n
, 1.3 10
3
) prepared from 2-pyrro-
lidone (A), and recovered during the biodegradation test (B). The
sample used for Fig. 6B was obtained by the cultivation with KT-2
Fig. 6. MALDI TOF mass spectra of (A) original nylon 4, and (B) recovered nylon 4 after the biodegradation test with KT-2 strain for one week. B, nylon 4 having benzamide and
acyllactam at each chain end; O, nylon 4 having benzamide and carboxy chain ends; P, nylon 4 having benzamide and sodium carboxylate chain ends.
K. Tachibana et al. / Polymer Degradation and Stability 95 (2010) 912e917 916
strain under the same condition as in the experimental Section 2.5
for about two days.
It was hard to estimate the decrease of the molecular weight of
the nylon 4 during the test directly from the TOF mass analysis,
because the quantitative recovery of the nylon 4 after the test was
difcult and the signals of which the mass numbers were less than
600 in the TOF mass spectra were also difcult. Therefore we
discussed mainly the conversion of the acyllactam-type chain end
during the test.
The cationized molecule with sodium iodide, [M Na]

was
clearly detected for all samples in Fig. 6. The mass numbers
denoted by open circle in Fig. 6 correspond to those of the nylon 4
molecules having benzamide and acyllactam groups at each chain
end. They are the main signals in both spectra (Fig. 6A, B). The
latter spectrum (Fig. 6B) has two kinds of another signals marked
by open triangle and open inverted triangle which are assignable
to the nylon 4 molecules having benzamide and carboxy groups at
each chain end, and their sodium salts, respectively. During the
biodegradation, the acyllactam-type chain end may be converted
to the carboxy-type chain end. At any rate, all the nylon 4 mole-
cules recovered after the biodegradation were found to have the
benzamide end groups. These results support that nylon 4 chains
are degraded sequentially from the acyllactam- or carboxy-type
chain end but not through the random hydrolytic scission of
amide bond. Therefore, the isolated KT-2 strain may recognize the
structure of acyllactam- or carboxy-type chain end accurately. The
isolation and analysis of the enzyme generated by the microor-
ganism are inferred to be necessary for the investigation of the
sequence recognized by the microorganism and of the degradation
mechanism of nylon 4.
4. Conclusion
In summary, two strains isolated from the composted soil, were
found to degrade a typical synthetic polyamide, nylon 4, and they
were identied as Stenotrophomonas sp. and Fusarium sp. from
their DNA analysis. Nylon 4 was decomposed probably by their
exo-type extracellular enzyme. At least, KT-2 strain is inferred to
recognize the acyllactam- or carboxy-type chain end structure and
degrade them or their neighboring amide bond in nylon 4.
Acknowledgement
The authors would like to thank Dr. Yoshimi Ben-no and
Dr. Motofumi Suzuki in RIKEN Research (Japan) for their experi-
mental guidance and helpful discussion.
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