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  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 ; 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 . 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 .
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 . 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 : 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. . 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 . 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).
Almost full-length 16S rRNA genes were amplified by polymerase chain reaction (PCR) from chromosomal DNA extracted and purified by the method of Marmur  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  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.