APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1997, p. 329331 0099-2240/97/$04.

000 Copyright 1997, American Society for Microbiology

Vol. 63, No. 1

Nylon Biodegradation by Lignin-Degrading Fungi

TETSUYA DEGUCHI,1* MASAAKI KAKEZAWA,1
AND

TOMOAKI NISHIDA2

Chemical, Polymer, Biotechnology Laboratory, Bio-Technology Research Section, Kobe Steel, Ltd., Kobe 651-22,1 and Department of Forest Resources Science, Faculty of Agriculture, Shizuoka University, Shizuoka 422,2 Japan
Received 29 April 1996/Accepted 30 October 1996

The biodegradation of nylon by lignin-degrading fungi was investigated. The fungus IZU-154 signicantly degraded nylon-66 membrane under ligninolytic conditions. Nuclear magnetic resonance analysis showed that four end groups, CHO, NHCHO, CH3, and CONH2, were formed in the biodegraded nylon-66 membranes, suggesting that nylon-66 was degraded oxidatively. Synthetic polymers, including nylon, are a growing environmental concern because they are generally nondegradable, and recent research has been focused on the biological treatment of plastic wastes and the development of biodegradable plastics. The amide bond (CONH) which occurs widely in natural polymers, like polyamino acids (e.g., proteins), is easily hydrolyzed by proteolytic enzymes. However, very little is actually known about the biodegradation of amide bonds present in the main polymer chain of nylon. On the other hand, although lignin is a natural polymer and a major component of woody plants, it is extremely resistant to attack by most microorganisms because of its random and complex structure. At present, white rot fungi are the best known and most effective lignindegrading microorganisms. In this study, we report the biodegradation of nylon-66 by the white rot fungus IZU-154. Nylon-66 membranes (Sartrius) were placed on various agar media and incubated at 30C with white rot fungus IZU-154, which had been isolated in our laboratory (1215). Basal medium was prepared by dissolving 10 g of glucose, 24 mmol of ammonium tartrate (AT), 1 g of KH2PO4, 0.2 g of NaH2PO4, 0.5 g of MgSO4 7H2O, 15 g of agar, and trace amounts of thiamine-HCl, CaCl2, FeSO4, ZnSO4, and CuSO4 in 1 liter of water. After 20 days of incubation, the nylon-66 membranes were harvested, washed with water, dried under vacuum, and dissolved in hexauoroisopropanol (HFIP) containing 10 mM triuoroacetate. The dissolved samples were subjected to gel permeation chromatography to determine changes in molecular weight distribution. Two identical HFIP-80m columns (Showa Denko), HFIP containing 10 mM triuoroacetate as a mobile phase at a ow rate of 1 ml/min, and a refractive index detector were used. Weight-average molecular weights (Mws; dened as Ni Mi2/ Ni Mi, where Ni is the number of molecules and Mi is the molecular weight) and number-average molecular weights (Mns; dened as Ni Mi/ Ni) were calculated based on polymethylmethacrylate standards. Table 1 shows the Mws and Mns of nylon-66 membranes incubated with IZU-154 for 20 days under various nutritional conditions. Signicant reductions in Mw and Mn were observed when either AT or glucose was limited, but no reduction was observed with the basal medium. These results suggest that nylon degradation can be triggered in the same manner as lignin degradation by starvation for either carbon or nitrogen (7, 8). The reduction in molecular weight was accompanied by morphological disintegration of the nylon-66 membrane (Fig. 1). In addition, the nylon-degrading activities of well-known white rot fungi, Phanerochaete chrysosporium (ATCC 34541) and Trametes versicolor (IFO 7043), on the AT-free medium were investigated for 20 days. The Mws of nylon membrane incubated with these fungi were 11,482 and 27,108, respectively. This result demonstrates that both fungi are able to degrade the nylon-66 membrane as well as IZU-154. It has been reported that Flavobacterium sp. strain K172, which is isolated from nylon waste, is able to degrade nylon oligomers and that two hydrolases are involved in the degradation. The two hydrolases were active only towards the cyclic dimer and linear oligomer, respectively, and inactive toward the natural amide bonds tested (9, 10). The authors, therefore, concluded that the degrading activity was newly acquired in the nylon waste (16, 17). In this study, we demonstrated that all white rot fungi used were able to degrade nylon-66, even though these fungi have never been adapted to growth on nylon. Therefore, it is apparent that these fungi possess the latent ability to degrade nylon. The nylon-66 membranes incubated with IZU-154 on the AT-free and basal media for 20 days were subjected to nuclear magnetic resonance (NMR) analysis as degraded and undegraded nylon samples. The harvested membranes were washed with water, dried under vacuum, and dissolved in HFIP. 1H NMR, 13C NMR, and 15N NMR spectra were measured with a Bruker AC300P in HFIP plus CDCl3 (3:1) at 300.13, 75.5, and 30.4 MHz, respectively. Chemical shifts were given on the (parts per million) scale with tetramethylsilane as an internal standard. As shown in Fig. 2, several new peaks were observed in the spectrum of degraded nylon. The carbon resonated at

TABLE 1. Nylon degradation under various nutritional conditions

Nutritional conditions AT concn (mM) Glucose concn (g/liter) Nylon-66 membrane Mw Mn

* Corresponding author. Mailing address: Biotechnology Research Section, Chemical, Polymer, Bio-Technology Laboratory, Kobe Steel, Ltd., Takatsukadai 1-chome, Nishi-ku, Kobe 651-22, Japan. Phone: 81-78-992-5732. Fax: 81-78-993-2035. 329

Controla 24.0 8.0 1.2 0.0 24.0 24.0 24.0

10.0 10.0 10.0 10.0 0.5 0.25 0.0

84,832 84,751 84,784 71,168 5,523 81,245 11,025 5,983

49,709 49,565 49,650 28,010 2,296 36,159 4,325 2,511

a The control was incubated for 20 days on the AT-free basal medium but not inoculated.

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DEGUCHI ET AL.

APPL. ENVIRON. MICROBIOL.

FIG. 1. Morphological disintegration of a nylon-66 membrane.

14 was assigned to CH3 carbon by distortionless enhancement by polarization transfer experiments. The carbons resonating at 166 and 210 were assigned to CHO carbons by twodimensional (C-H) analysis in which these carbons were found to be coupled with the hydrogens resonating at 8.97 and 9.62,

respectively. In addition, the carbon resonating at 166 was considered to be adjacent to the nitrogen atom based on its chemical shift. The nitrogen resonating at 105 was assigned to CONH2 nitrogen by distortionless enhancement by polarization transfer experiments and its chemical shift. NMR data indicates that four end groups, CHO, NHCHO, CH3, and CONH2, were formed in the biodegraded nylon-66 membrane. This suggests that the hydrolysis of amide bonds did not occur because no formation of the primary amine at an end group was observed and that at least two different oxidative cleavages occurred. The formation of these four end groups can be explained by a thermal oxidative degradation mechanism in which methylene groups adjacent to a nitrogen atom were attacked by oxygen; subsequently, nylon was degraded further by chain reactions (3, 11, 18). The formations of CONH2 and CHO may be caused by the cleavage of a C-N bond in NHCH2. The formations of NHCHO and CH3 may be caused by the cleavage of a C-C bond in CH2-CH2 adjacent to a nitrogen atom. The effect of manganese on nylon-66 degradation was also investigated. Manganese is known to play an important role in biological oxidation. In lignin biodegradation by white rot fungi it is a very important cofactor, especially as a regulator of enzyme production and as an active mediator in the manganese peroxidase catalytic cycle (1, 2, 47). AT-free medium

FIG. 2. NMR spectra of nylon-66 membranes treated with IZU-154. (a)

13

C NMR spectra; (b)

15

N NMR spectra.

VOL. 63, 1997 TABLE 2. Effect of Mn(II) on nylon degradation

MnSO4 concn (mM) Nylon-66 membrane Mw Mn

NYLON DEGRADATION BY LIGNIN-DEGRADING FUNGI

331

0 0.01 0.1 1 10

74,152 34,009 23,629 14,147 23,046

31,909 11,961 8,601 5,229 9,637

containing manganese sulfate was inoculated with IZU-154 and incubated for 7 days, and then the nylon-66 membrane was placed on the grown mycelium. Table 2 shows the Mws and Mns of nylon-66 incubated with IZU-154 for 24 h. Nylon degradation was evidently accelerated by addition of manganese, and the maximal effect was obtained with 1 mM MnSO4. The effects of other metals, 1 mM FeSO4, Fe2(SO4)3, ZnSO4, and CuSO4, were negligible (data not shown). In this study, we present the rst report of nylon degradation by white rot fungi. Our results concerning nutritional regulation, the microorganisms having this activity, and the acceleration by manganese suggest that nylon-degrading activity is closely related to the ligninolytic activity of these microorganisms. The purication and characterization of the enzyme(s) catalyzing nylon degradation are currently under study.
We thank Y. Uesono for his helpful discussions and comments in the various stages of this work and H. Yasuda for the NMR analysis.
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