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Keywords:

  • Aliphatic polycarbonate;
  • Biodegradation;
  • Lipolytic enzyme;
  • Pseudomonas sp.;
  • Variovorax sp.

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Bacteria that degrade an aliphatic polycarbonate, poly(hexamethylene carbonate), were isolated from river water in Ibaraki Prefecture, Japan, after enrichment in liquid medium containing poly(hexamethylene carbonate) suspensions as carbon source, and dilution to single cells. Four of the strains, 35L, WFF52, 61A and 61B2, degraded poly(hexamethylene carbonate) on agar plate containing suspended poly(hexamethylene carbonate). Degradation of poly(hexamethylene carbonate) was confirmed by gel permeation chromatography. Besides poly(hexamethylene carbonate), the strains were found to degrade poly(tetramethylene carbonate). The strains were characterized morphologically, physiologically, and by 16S rDNA sequence analysis. Strains 35L and WFF52 were tentatively identified as Pseudomonas sp. and Variovorax sp., respectively, while strains 61A and 61B2 constitute an unidentified branch within the β subclass of the Proteobacteria.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Development of biodegradable plastics is being extensively promoted as one of the solutions to plastic waste disposal. Aliphatic polyesters are currently considered the most promising materials for the production of biodegradable plastics. Strains of microbial degraders for biodegradable plastics were reported for: naturally occurring biodegradable polymer, poly-β-hydroxybutyrate (PHB) [1, 2]; and artificially synthesized polymers, poly(ethylene adipate) [3], poly(ε-caprolactone) [1], poly(tetramethylene succinate) [4] and poly(lactic acid) [5].

Aliphatic polycarbonates are also biodegradable [6–9], and with their chemical property of resistance to water are thought to provide a new possibility for designing biodegradable plastics with unique properties, as raw materials of polyurethanes, copolymers, and as matrix of polymer blends. We have tested the populations of polycarbonate-degrading microorganisms using poly(ethylene carbonate) as a substrate [8]. Of the colonies plated out, 0.2–5.7% showed activities of decreasing the turbidity of the suspended poly(ethylene carbonate), confirming the distribution of polycarbonate-degrading microorganisms in the natural environment. However, a detailed characterization of the degrading microorganisms has not yet been carried out. We previously examined the susceptibility of polycarbonates to commercial lipolytic enzymes [9]. Enzymatic degradation of poly(tetramethylene carbonate) into low-molecular-mass components was demonstrated by lipoprotein lipase (from Pseudomonas sp., Toyobo, Japan). However, besides 1,4-butanediol and carbonic acid, diester was detected as a dead-end product of enzymatic degradation. For practical use of biodegradable plastics in place of non-degradable ones, it is necessary to prove their degradation by activities present in the environment.

To investigate the degradation of aliphatic polycarbonates in the natural environment, we tried in the present study to isolate environmental microorganisms that degrade aliphatic polycarbonates, and also tried to characterize the isolates morphologically, physiologically, and phylogenetically.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Isolation of polycarbonate-degrading microorganisms

The sample water was obtained from the Hanamuro river in Ibaraki Prefecture, Japan. The samples were incubated in the liquid medium containing suspended poly(hexamethylene carbonate) (PHC, Fig. 1a) described below, at 30°C with vigorous shaking. The achievement of degradation was monitored by the decrease of turbidity in the medium due to degradation of PHC suspension. Pure strains of PHC degrading microorganisms were obtained by repeated application of subcultures with dilution to single cells. The degradation activity of the isolates was checked by the formation of clear zones around the colonies on agar plates prepared with the same medium.

image

Figure 1. Chemical structures of (a) PHC and (b) di(6-hydroxyhexyl) carbonate.

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2.2Culture media containing suspended polymers

The medium containing PHC (number-average molecular mass: Mn= 2000; Toagosei Co., Japan) was prepared by the protocol reported earlier [10] with slight modifications. PHC was suspended into the medium as a substrate polymer at a final concentration of 0.1% (w/v). The medium also contained (per liter), 1 g of (NH4)2SO4, 0.1 g of yeast extract (Difco, USA), 20 mg of CaCl2·2H2O, 10 mg of NaCl, 10 mg of FeSO4·7H2O, 0.5 mg of Na2MoO4·2H2O, 0.5 mg of Na2WO4·2H2O, 0.5 mg of MnSO4, 60 mg of surfactant (Plysurf A210G: Daiichi Kogyo Seiyaku, Japan), 0.2 g of KH2PO4 and 1.6 g of K2HPO4 (pH 7.0). The liquid medium was dispensed into test tubes in 10-ml aliquots and then autoclaved. The agar plates were prepared by addition of 1.5% agar and poured into petri dishes after autoclaving.

The medium containing poly(tetramethylene carbonate) (PTC [9]; Mn= 2000; Asahi Chemical Industry Co., Japan) was prepared in the same way.

2.3Analytical methods in the degradation of polymers

Analysis of the bacterial degradation products was performed as reported previously [9]. Strains were precultured in the PTC/PHC medium at 30°C with vigorous shaking and transferred into a fresh PTC/PHC medium (100 μl of cell suspension containing 2×103 cfu was inoculated into 10 ml medium). After incubation the culture medium was dried with a rotary evaporator at 40°C and the degradation products from PTC/PHC were extracted with chloroform (10 ml).

Chromatograms of gel permeation chromatography (GPC) were taken by an HLC-8020 GPC system (Tosoh Co., Japan) with two wide-spectrum TSK gel columns (GMHHR-M and GMHHR-L). Chloroform was used as an eluent with a flow rate of 1.0 ml min−1 and the measurements were performed at 40°C. NMR spectra and gas chromatograms were taken by a model JNM-EX 270 FT-NMR spectrometer (JEOL, Japan) and a model GC-14B gas chromatograph (Shimadzu, Japan), respectively, in the conditions reported previously [9].

Authentic 1,6-hexanediol and 1,4-butanediol were purchased from Nacalai Tesque Co., Japan. Authentic diesters were prepared from enzymatic digestion of PTC and PHC by the method previously described [9]. The production and the purity of diester products were confirmed as follows.

Di(6-hydroxyhexyl) carbonate (formula given in Fig. 1b): 1H-NMR (δ ppm: number of protons, coupling, assignment), 4.13 (4H, t, 1 and 1′-H), 3.63 (4H, t, 6 and 6′-H), 1.99 (2H, s, hydroxyl-H), 1.69 (4H, t-t, 2 and 2′-H), 1.58 (4H, t-t, 5 and 5′-H) and 1.40 (8H; m; 3, 3′, 4 and 4′-H); 13C-NMR (δc ppm), 155.5 (carbonyl-C), 67.9 (1 and 1′-C), 62.7 (6 and 6′-C), 32.5 (5 and 5′-C), 28.7 (2 and 2′-C), 25.5 (4 and 4′-C) and 25.4 (3 and 3′-C).

Di(4-hydroxybutyl) carbonate [9]: 1H-NMR, 4.18 (4H, t, 1 and 1′-H), 3.68 (4H, t, 4 and 4′-H), 1.78 (4H, t-t, 2 and 2′-H), 1.66 (4H, t-t, 3 and 3′-H) and 1.63 (2H, s, hydroxyl-H); 13C-NMR, 155.3 (carbonyl-C), 67.7 (1 and 1′-C), 62.3 (4 and 4′-C), 28.9 (3 and 3′-C) and 25.2 (2 and 2′-C).

2.4Characterization of isolates

The accumulation of PHB was tested by Nile blue A staining using the method of Ostle et al. [11]. Nile blue A staining, Gram staining, morphology and motility were observed with an Aristoplan phase-contrast microscope equipped with an Ortomat E camera system (Ernst Leitz, Wetzlar, Germany). Some standard physiological tests, e.g., oxidase and catalase production, nitrate reduction, and denitrification reaction, were carried out by conventional methods [12]. Most biochemical properties, including enzymatic activities and acid production from carbohydrates, were determined using substrate disks of a commercial identification kit (Minitek; BBL Microbiology Systems, USA).

2.5Phylogenetic study

Almost full-length 16S rRNA genes were amplified by polymerase chain reaction (PCR) from chromosomal DNA extracted and purified by the method of Marmur [13] using an AmpliTaq polymerase kit (Perkin Elmer, USA) and a pair of primers targeted for conserved regions of eubacteria homologous or complementary to positions 8–27 and 1492–1511 (Escherichia coli numbers [14, 15]). The PCR products were cloned using a pT7Blue T-Vector Kit (Novagen, USA) with E. coli JM109 (Takara, Japan). All the procedures were performed according to the standard protocols of the kits. The DNA inserts were sequenced with an ABI 373A DNA sequencing system (Applied Biosystems, USA). Sequence uniformity of 20–30 clones was determined.

The 16S rRNA gene sequences were compared with previously published sequences in GenBank-EMBL DNA sequence data libraries. Phylogenetic analysis was performed with GENETYX 9.0 (Software Development Co., Japan) and CLUSTAL W1.6 [16] programs. The nucleotide sequences determined in the present study have been deposited in the DDBJ/EMBL/GenBank data libraries under accession numbers: AB003623, strain 61A clone group 1; AB003624, strain 61A clone group 2; AB003625, strain 61B2 clone group 1; AB003626, strain 61B2 clone group 2; AB003627, strain WFF52; and AB003628, strain 35L.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1The new strains

A total of 10 PHC-degrading strains with distinct colony types were isolated from the water samples. All of the strains had activity to reduce the turbidity of opaque PHC suspension in liquid culture. Four of the isolates had comparatively strong activity to make clear zones on the PHC suspension plates. The clear zones began to appear 2 days after inoculation. The four strains were also culturable on agar plates of 0.08% nutrient broth (Difco) while maintaining PHC-degrading activity, whereas the other strains lost degrading activities during cultivation in the medium lacking PHC. The four isolates were designated strains 35L, WFF52, 61A and 61B2.

The colonies of the four strains (Fig. 2) were surrounded by clear zones on the opaque plate of suspended PHC. Strain 35L formed a disk-shaped white colony that was thin in the peripheral region. Strain WFF52 spread a flat yellowish colony from a central dot. Degradation of suspended PHC on the agar plates was most visible in the colonies of strain WFF52, by their rapid growth with high motility and by the permeability of their degrading enzyme into the medium. The other two strains, 61A and 61B2, initially formed white colonies which exhibited pink in the course of the growth. The colonies of strain 61A were smooth and soft. The transparency of clear zones produced by strain 61A was the highest among those by the new isolates. It is likely that the enzyme activity of PHC degradation produced by strain 61A was the strongest. Strain 61B2 resembled strain 61A but the colonies grew more slowly and larger. The colonies of strain 61B2 were hard and less moist. Flocculation in liquid culture was often formed by this strain. These facts suggest that the cells of strain 61B2 exhibit behavior causing coagulation.

image

Figure 2. Colonies of PHC-degrading strains: (a) 35L; (b) WFF52; (c) 61A; (d) 61B2. The colonies were grown for 5 days on agar plates with suspended PHC.

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3.2Confirmation of degradation of polymers by strain 61A

The degradation process of PHC in the culture medium incubated with the strains was confirmed in detail using GPC. Fig. 3 shows the changes in GPC chromatograms of PHC incubated with strain 61A. The main peak (retention time at around 14 min) was identified as the substrate polymer (PHC, Mn= 2000), whose area was decreased by incubation with strain 61A. Little degradation occurred during 12 h of incubation; the chromatogram of the sample incubated for 12 h (Fig. 3a) was almost identical to that of the sample before inoculation. The other minor peaks extracted from the culture medium were unidentified. (The peaks were observed at the same retention times in any extract from different kinds of substrate polymer medium. It is possible that the components of the minor peaks were complex products resultant from autoclaving of other substances in the culture medium.) After 24 h of incubation, a large peak at retention time 20 min appeared which was identified as a peak of diester, i.e., di(6-hydroxyhexyl) carbonate (Fig. 3b, see Section 2). The peak disappeared after an additional 24 h of incubation (Fig. 3c).

image

Figure 3. Changes in GPC chromatograms in the time course of PHC (Mn= 2000) degradation after the inoculation of strain 61A (2×103 cfu): (a) 12 h; (b) 24 h; (c) 48 h. The arrow shows the peak of di(6-hydroxyhexyl) carbonate.

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The four strains also made clear zones on the PTC suspension plate. The degradation of PTC was also determined in the same way. Observed changes in time course of PTC degradation were almost identical with that of PHC degradation (Fig. 4 a–c). Similar to the processes of PHC degradation, temporal accumulation and disappearance of diester, di(4-hydroxybutyl) carbonate (Fig. 4b, a peak at retention time 20.2 min, see Section 2) was observed.

image

Figure 4. Changes in GPC chromatograms in the time course of PTC (Mn= 2000) degradation after the inoculation of strain 61A (2×103 cfu): (a) 12 h; (b) 24 h; (c) 48 h. The arrow shows the peak of di(4-hydroxybutyl) carbonate.

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The diesters (arrows in Fig. 3 b and Fig. 4b) were dead-end products of enzymatic degradation by lipoprotein lipase [9]. The temporal accumulation of the diester products strongly suggests that the lipolytic enzymes, similar to what we discovered previously, are the key enzymes of PHC/PTC degradation also in microbial processes.

In the case of microbial degradation, the diesters were short-lived (Fig. 3 c and Fig. 4c). This is due to the secondary degradation pathways (esterases degrading the diesters) existing in the strains. Unlike the enzymatic degradation [9], no monomeric diols (1,6-hexanediol or 1,4-butanediol) were observed by GPC or gas chromatography in the entire course of degradation. On the other hand, evolution of organic acids that was not observed in the enzymatic degradation was observed. Adipic acid (PHC) or succinic acid (PTC) and some other unidentified acids were detected from the microbial degraded samples.

3.3Characterization and phylogenetic analysis of new strains

The isolates were characterized by the standard tests listed in Table 1. As the strains did not grow on bouillon media, they were cultured on nutrient broth agar plates for the tests.

Table 1.  Biochemical characteristics of new isolatesa
Characteristics35LWFF5261A61B2
  1. a+, positive reaction; −, negative reaction. All four strains were negative for the following characteristics: Gram stain; degradation of extracellular PHB; denitrification; hydrolysis of esculin and salicin; ornithine decarboxylase; urease; indole production; acid production from raffinose, maltose, trehalose, cellobiose, lactose, sorbitol, mannitol, rhamnose and glycerol; and positive for the following characteristics: PHB accumulation, oxidase and utilization of citrate.

MorphologyStraight rodStraight rodRodCurved rod
 0.5×2–4 μm0.5×3–5 μm0.5×2 μm0.5×3–5 μm
Motility+++
Catalase++
Nitrate reduced to nitrite+
Starch hydrolysis++
β-Galactosidase (ONPG)++
Arginine dihydrolase+
Lysine decarboxylase++
Acid production from    
 Glucose++
 Mannose+
 Arabinose+++
 Xylose++

The phylogenetic tree implied from 16S rRNA gene sequences (Fig. 5) indicates that the strains belong to the β or γ subclass of Proteobacteria. Strain 35L was classified in the γ subclass as a close relative of Pseudomonas aureofaciens and P. chlororaphis with similarities of 97.9% and 97.7%, respectively. Other strains were classified in the β subclass. Strain WFF52 was classified as a close relative of Variovorax paradoxus with a similarity of 99.0%. The sequences of strains 61A and 61B2 were almost identical, except for a single base, and made a distinct cluster in the β subclass. They made no obvious cluster with other specified genera (Fig. 5). A pair of slightly different sequences were detected in both clones from the two strains (shown as clone group 1 and clone group 2). The detection was reproducible in at least four distinct amplification experiments and the strains are thought to have multicopies of the 16S rRNA gene with slight mutation in their cells.

image

Figure 5. Phylogenetic positions of new isolates (nucleotide sequence accession numbers AB003623–AB003628) among Proteobacteria derived from analysis of 16S rRNA genes by the method of neighbor-joining [17]. Positions with alignment gaps and unidentified bases were not taken into consideration. The percentages of 1000 bootstrap trials that support each topological element are indicated beside the nodes.

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The new isolates were the first strains found and isolated as degraders of aliphatic polycarbonates. The specificity of the polycarbonate-degrading activity was not so strict, since all of the PHC-degrading isolates also had PTC-degrading activity. Although they require yeast extract or nutrient broth as cosubstrates for optimum growth, they have obvious activity to convert PHC/PTC into biomass and minerals under the conditions, and have a function to restore the synthetic polymers into natural carbon cycle. The phylogenetic study (Fig. 5) showed that the four strains were classified into Proteobacteria but all were not closely related to each other. This result indicates that the polycarbonate-degrading activity is not a property limited to a specified organism of a restricted group.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

We wish to thank Akira Iwamoto (JSP Co.) and Satoshi Hanada (RITE: Research Institute of Innovative Technology for the Earth) for technical advice.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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