Draft Genome Sequences of Nine Environmental Bacterial Isolates Colonizing Plastic

Plastic is omnipresent in our environment, and there is no sustainable recycling solution. Many pollutants can be degraded by microorganisms, and degradation is often induced by colonization of the material by the organism. Aiming to contribute a biological solution to the plastic problem, we isolated nine bacterial strains colonizing plastic. Environmental samples (wastewater [WW], seawater [SW], and soil [S]) were obtained from a wastewater treatment plant (Lyngby-Taarbæk Forsyning A/S, Kongens Lyngby, Denmark [55°48′07.4″N, 12°32′20.8″E]) on 14 September 2018, from a harbor (Hellerup, Denmark [55°43′55.5″N, 12°34′51.0″E]) on 17 September 2018, and from a site near the university campus (55°47′07.8″N, 12°30′49.7″E) on 11 October 2018. On the marine and terrestrial sides, plastic pieces (seawater plastic [SWP] and soil plastic [SP]) were also collected. Microbial communities were removed from plastic by sonication for 5 min, and all environmental samples were inoculated at a starting concentration of 10⁶ cells/ml (as determined by SYBR gold staining, filtration, and microscopy) in OECD301 medium (WW, S, and SP samples) or OECD306 medium (SW and SWP samples) (1, 2) with a piece of plastic (18 by 18 mm by 0.05 mm), i.e., poly-l-lactic acid (ME331050; Goodfellow), polyethylene terephthalate (ES301250; Goodfellow), or low-density polyethylene (ET311150; Goodfellow), or a cover glass (18 by 18 mm) (631-1567; VWR) as a control. OECD301 and OECD306 media contain 8.5 mg/liter KH₂PO₄, 217.5 mg/liter K₂HPO₄, 334 mg/liter Na₂HPO₄·2H₂O, 5 mg/liter NH₄Cl, 36.4 mg/liter CaCl₂·2H₂O, 22.5 mg/liter MgSO₄·7H₂O, and 0.25 mg/liter FeCl₃·6H₂O in either distilled water (OECD301) or seawater (OECD306) prepared with 2.5% sea salts (Sigma). Cultures were incubated in the dark at 16°C without shaking. After 14 days, the plastic pieces were transferred to fresh OECD301 or OECD306 medium and sonicated for 5 min. The samples were diluted 10-fold and plated onto OECD301 or OECD306 medium with 1.5% agar, 1% polypeptone, and 0.2% yeast extract. After incubation at 16°C for 7 days, colonies that were unique to each plastic type and different from the control samples were isolated by restreaking onto OECD301 or OECD306 1.5% agar plates with 1% polypeptone and 0.2% yeast and incubation for 4 days at 16°C.

Genomic DNA was extracted using the NucleoSpin tissue kit (740952; Macherey-Nagel). Five hundred nanograms of DNA per strain was submitted for sequencing at the Novo Nordisk Center for Biosustainability (Technical University of Denmark, Lyngby, Denmark) using the NextSeq 500/550 midoutput kit v2 (Illumina) for 150-bp paired-end sequencing on a MiSeq Illumina platform. The sequence data were analyzed with KBase v1.8.9 (3). The read quality was assessed using FastQC v0.11.5 (4) and Trimmomatic v0.36 (5) (sliding window size:4; sliding window minimum quality:15; post tail crop length:140; head crop length:10; leading minimum quality: 3; trailing minimum quality:3; minimum read length:36). The genomes were assembled using SPAdes v3.13.0 (6), and quality and metrics were analyzed using QUAST v4.4 (7). The level of contamination was assessed using CheckM v1.0.8 (8). The assemblies were automatically annotated with NCBI PGAP v5.0 (9). Species phylogeny was analyzed using autoMLST (10) (Table 1). Six strains had <95% estimated average nucleotide identity (ANI) with respect to genomes of type strains (11) and thus could represent novel bacterial species.

This work was financially supported by a Danish National Research Foundation grant to the Center for Microbial Secondary Metabolites (CeMiSt) (grant DNRF137).