Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymatically degrade PET has been thought to be limited to a few fungal species, biodegradation is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, we isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymatically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.

A Gram-stain-negative, aerobic, non-spore-forming, rod-shaped bacterium, designed strain 201-F6 T , was isolated from a microbial consortium that degrades poly(ethylene terephthalate) (PET) collected in Sakai city, Japan, and was characterized on the basis of a polyphasic taxonomic study. The cells were motile with a polar flagellum. The strain contained cytochrome oxidase and catalase. It grew within the pH range 5.5-9.0 (optimally at pH 7-7.5) and at 15-42 ºC (optimally at 30-37 ºC). The major isoprenoid quinone was ubiquinone with eight isoprene units (Q-8). C 16 : 0 , C 17 : 0 cyclo, C 18 :1 !7c and C 12 : 0 2-OH were the predominant cellular fatty acids. The major polar lipids were phosphatidylethanolamine, lyso-phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The G+C content of genomic DNA was 70.4 mol%. Phylogenetic analysis using the 16S rRNA gene sequences showed that strain 201-F6 T was affiliated to the genus Ideonella, and was closely related to Ideonella dechloratans LMG 28178T (97.7 %) and Ideonella azotifigens JCM 15503 T (96.6 %). Strain 201-F6 T could be clearly distinguished from the related species of the genus Ideonella by its physiological and biochemical characteristics as well as by its phylogenetic position and DNA-DNA relatedness. Therefore, the strain represents a novel species of the genus Ideonella, for which the name Ideonella sakaiensis sp.

Yang et al. suggest that the use of low-crystallinity poly(ethylene terephthalate) (PET) exaggerates our results. However, the primary focus of our study was identifying an organism capable of the biological degradation and assimilation of PET, regardless of its crystallinity. We provide additional PET depolymerization data that further support several other lines of data showing PET assimilation by growing cells of Ideonella sakaiensis. W e appreciate the Comment by Yang et al.(1) and are grateful for this opportunity to explain the context of how our work builds on previous studies. The intent of our study was to isolate and describe a microorganism that can degrade and assimilate poly(ethylene terephthalate) (PET) (2). Therefore, we only cited the pioneering works that report specific microorganisms able to grow on PET (3, 4). In a previous study, Sharon and Sharon (5) confirmed microbial PET degradation by semiisolated microorganisms, where the involvement of Nocardia species was implied by microscopic observation. However, the contribution of other microorganisms was not ruled out, nor was the possibility that the PET was degraded by the resting cells harboring PET-hydrolyzable enzymes. We identified the PET hydrolase (PETase) gene based on amino acid sequence identity with a known hydrolase from Thermobifida fusca (TfH) that exhibited PET-hydrolyzing activity (6), similar to other PET-hydrolyzing enzymes (7-10). We further referred cutinase from Humicola insolens (HiC) (11) and seven other enzymes in the supplementary materials [table S2 in (2)]. The functions of these enzymes, particularly TfH, LCC (9), and FsC (10), greatly contributed to the understanding of PETase enzymology.With the intention of isolating a specific microorganism that can use PET for growth, even if the PET is mostly in the amorphous form, the extent of crystallinity is of secondary importance. We described the motivation for using low-crystallinity (1.9%) PET and the observation that the structure of crystallized PET hampered the enzymatic hydrolysis of its ester linkages (8, 12) in our Report.Based on the proton nuclear magnetic resonance and gel permeation chromatography profiles of the degraded PET film, we concluded that the degradation by the consortium termed "no. 46" proceeded from the PET surface (2). To support this inference, we analyzed the surface of the PET film using x-ray photoelectron spectroscopy (XPS). The XPS spectrum for degraded PET film by no. 46 showed that a new peak appeared at 0.6 eV higher in binding energy than that observed for the O 1s peak of C-O-C linkage (532.5 eV), indicating the formation of the C-O-H linkage by hydrolysis of the ester linkage, which should either be of hydroxyl or carboxyl groups. To accurately determine the surface functional groups, each group was labeled with heptafluorobutyryl chloride (for -OH) and 1,1-carbonyldiimidazole (for -COOH) (13) and analyzed by XPS. Consequently, the peaks of F 1S (687.5 eV) and N 1S (398.5 eV) after degradation dramatically increased (Fig. 1A). The ...

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