Culture
Ideonella sakaiensis was cultured as described previously4,13 with several modifications. Briefly, I. sakaiensis was pre-cultured in NITE Biological Resource Center (NBRC) 802 medium (1.0 w/v% polypeptone, 0.2 w/v% yeast extract, and 0.1 w/v% MgSO4) at 30 °C and harvested by centrifugation (5000×g, 5 min, 4 °C). The cell pellet was resuspended in YSV medium (0.01 w/v% yeast extract, 0.02 w/v% sodium hydrogen carbonate, 0.1 w/v% ammonium sulfate, 0.01 w/v% calcium carbonate, 0.1 v/v% vitamin mixture [0.25 w/v% thiamine-HCl, 0.005 w/v% biotin, and 0.05 w/v% vitamin B12], and 1 v/v% trace elements [0.1 w/v% FeSO4·7H2O, 0.1 w/v% MgSO4·7H2O, 0.01 w/v% CuSO4·5H2O, 0.01 w/v% MnSO4·5H2O, and 0.01 w/v% ZnSO4·7H2O] in 10 mM phosphate buffer; pH 7.4). The suspension was inoculated into a Petri dish (90 mm in diameter) containing 30 mL YSV medium supplemented with ≈ 300 mg of oyster shells as a pH adjusting agent (YSVO medium) in the presence or absence of 10 g (≈ 560 pieces) of PET granules shaped like an elliptic cylinder [2 (minor axis) × 3 (major axis) × 3 (height) mm]; 5.8% crystallinity determined by differential scanning calorimetry (Bell Polyester Products, Inc., Yamaguchi, Japan), 0.5 w/v% of disodium terephthalate (TPA-2Na), EG, or a mixture of 0.5 w/v% TPA-2Na and 0.5 w/v% EG as the carbon source to adjust the absorbance measured at 660 nm to 0.002. The culture dish was placed in a reciprocal shaker set at 50 strokes/min at 30 °C. After filtering the culture fluid through a 5 µm pore size poly-vinylidene difluoride filter (Merck Millipore, Billerica, MA) to remove the small broken pieces of oyster shells, cells were harvested, lyophilized using an FZ-2.5 freeze-dry system (Labconco, Kansas City, MO), and weighed. As a portion of the cells was trapped by the filter, the DCW was corrected using the amounts of protein before and after filtration. Briefly, cells were mixed with an equal volume of 10 w/v% sodium dodecyl sulfate (SDS) and heated at 90 °C for 10 min to lyse the cells and solubilize the cellular protein, and the protein concentration was determined with a DC protein assay kit (BioRad, Hercules, CA) with comparison to a calibration curve using bovine serum albumin. The PET granules were washed with 1 w/v% SDS, distilled water, and then ethanol. After drying in air and a desiccator, the granules were weighed to determine the weight reduction.
Fluorescence microscopy
PHA that accumulated in the cells was stained with Nile red (Fujifilm Wako Pure Chemical, Osaka, Japan)14. A glass slide was coated with 0.01 w/v% poly-l-lysine (Sigma-Aldrich, St. Lois, MO) for cell attachment. The culture suspension (100 µL) was placed on the poly-l-lysine-coated glass slide for 10 min and then aspirated. The cells attached to the glass slide were incubated with a drop of 10 µg/mL Nile red in D-PBS(−) (Nacalai Tesque, Kyoto, Japan) for 30 min, washed with D-PBS(−), mounted with Prolong Diamond antifade medium (Thermo Fisher Scientific, Waltham, MA) for immobilization, and covered with a coverslip. Cells were observed using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan). The frame of each individual cell was manually identified, and the cell area was calculated using the BZ-X800 Analyzer software (Keyence). PHA formation was visualized using a tetramethylrhodamine isothiocyanate filter set (λem ≈ 554 nm, λex ≈ 570 nm). The fluorescence intensity of the Nile red-stained dot assembly was calculated on average using the BZ-X800 Analyzer software.
TEM
The I. sakaiensis cells grown on PET granules for 6 days were harvested by centrifugation, fixed with phosphate-buffered 2% glutaraldehyde, and post-fixed with 2% osmium tetraoxide for 3 h on ice. Next, the samples were dehydrated using an ethanol gradient and embedded in epoxy resin. Ultrathin sections of the specimen were stained with uranyl acetate and lead, and subjected to TEM using the H-7600 transmission electron microscope (Hitachi, Tokyo, Japan).
Polymer extraction and analyses
Samples were prepared as described previously15 with several modifications. The lyophilized cells of I. sakaiensis were suspended in chloroform with vigorous stirring for 1 day, and the extract was filtered through a polytetrafluoroethylene filter (pore size, 0.2 µm). The filtrate was mixed with twofold volume of 35% methanol:35% ethanol:30% water (v/v/v), and the precipitate was dried in vacuo and then dissolved in chloroform. To determine the molecular weight of the polymer, the chloroform-soluble fraction was analyzed using a size exclusion chromatography system equipped with a RI-2031Plus refractive detector (Jasco, Tokyo, Japan) and two in-line TSKgel GMHHR-M columns with a TSK-guard column HHR-H (Tosoh, Tokyo, Japan) at 40 °C. Chloroform was used as the mobile phase and passed through the column at a flow rate of 1 mL/min. The molecular weight was estimated by comparison with poly(methylmethacrylate) (PMMA) standards (PMMA calibration kit M-M-10, Agilent Technologies, Santa Clara, CA). To analyze the polymer chemical structure, the chloroform-soluble fraction was further purified by precipitation in cold methanol. The precipitate was dissolved in deuterated chloroform (CDCl3) and analyzed via proton nuclear magnetic resonance (1H NMR) (JNM-ECX400P, Jeol, Tokyo, Japan).
PHA quantification and composition analysis
The cellular PHA content and composition were determined by GC after direct methanolysis of the dried cells in the presence of 15% sulfuric acid at 100 °C for 140 min, as described previously16. The GC analysis was performed using GC-2014 (Shimadzu, Kyoto, Japan) equipped with an InertCap 1 capillary column (30 m × 0.23 mm; GL Sciences, Tokyo, Japan) and a flame ionization detector. PHA composition was further determined by mass spectrometry.
