Alcanivorax bacteria as important polypropylene degraders in mesopelagic environments

ABSTRACT

Plastics are causing serious problems in the sea and settling even to abyssal depths. Polypropylene (PP) is the second most common plastic product and thus would constitute a large fraction of plastics in the sea. The biodegradation of PP has not been clearly shown. In this study, Alcanivorax bacteria (mainly Alcanivorax borkumensis) were indicated to be enriched most abundantly on liquid PP and on its structurally similar branched alkane, pristane, in mesopelagic water. An Alcanivorax isolate probably of A. borkumensis showed the highest liquid PP-degrading activity among the isolates. These results indicate that Alcanivorax bacteria could be major degraders of PP in mesopelagic environments. Alcanivorax bacteria did not use liquid PP as the sole carbon and energy source. Short PP was preferentially degraded, and PP of all lengths appeared to be degraded. Liquid PP was degraded more efficiently at 10°C than at 20°C, and correspondingly, higher concentrations of another carbon source were required at 10°C. Regarding the degradation of solid PP, an initial degradation sign, oxidation, was detected, but a weight loss of at least 1% was not detected. By using this study as a model, various PP-degrading microbes would start to be clarified.

IMPORTANCE

PP biodegradation has not been clearly shown (it has been uncertain whether the PP structure is actually biodegraded or not). This is the first report on the obvious biodegradation of PP. At the same time, this study shows that Alcanivorax bacteria could be major degraders of PP in mesopelagic environments. Moreover, PP biodegradation has been investigated by using solid PP as the sole carbon source. However, this study shows that PP would not be used as a sole carbon and energy source. Our data thus provide very important and key knowledge for PP bioremediation.

INTRODUCTION

Conventional plastics are durable materials used for disposable products, resulting in unwanted release into the sea. Such uncontrolled plastics could be physically and chemically harmful to wildlife and humans (1, 2). There are numerous reports of plastic debris settling to the sea floor even at abyssal depths (1). Polyethylene ([PE] having an n-alkane structure) and polypropylene ([PP] having a branched alkane structure) dominate plastic production. Therefore, large amounts of these alkanes could accumulate on the sea floor and enrich the degrading bacteria there. There are many reports on the biodegradation of PE structures (n-alkanes) (3). However, no reports exist on the biodegradation of PP structures.

Alcanivorax bacteria have often dominated in crude oil-contaminated temperate seawater and have been identified as key microorganisms in alkane degradation in marine environments (4–12). Alcanivorax bacteria are heterotrophs using limited carbon sources, including alkanes (11). Accordingly, an abundance of Alcanivorax bacteria can be an indicator of alkane contamination in marine environments. The ability of Alcanivorax bacteria to use n-alkanes and isoprenoid-derived branched alkanes (pristane and phytane) is shown to be high using crude oil as an alkane source (8, 10). Consistently, Alcanivorax bacteria have dominated in seawater enriched on pristane (4, 13). However, other alkane-degrading bacteria have dominated in seawater enriched on n-alkanes (4, 12, 13). This may suggest that Alcanivorax bacteria can grow better on n-alkanes in the presence of other crude oil components than other n-alkane-degrading bacteria (3, 14). n-Alkanes appear more degradable than branched alkanes (8, 10, 15–18). Some n-alkane degraders clearly degrade isoprenoid-derived branched alkanes, but others do not (8, 10, 15, 16). PP is more branched than isoprenoid-derived branched alkanes and could be more recalcitrant to biodegradation.

The biodegradation mechanism of n-alkanes is rather well understood (19), while that of isoprenoid-derived branched alkanes is poorly understood. Biodegradation of isoprenoid-derived branched alkanes is indicated to involve oxidation of the isopropyl terminus, which is followed by β-oxidation (16, 20, 21). It is conceivable that PP is also degraded through the same pathway. Although genes for isoprenoid-derived branched alkane degradation have not been identified, pristane has induced transcription of alkB, p450, and almA genes (22). This could suggest that the genes responsible for the hydroxylation of n-alkanes are involved in the hydroxylation of the isopropyl terminus.

The biodegradability of PE and PP would be greatly promoted by abiotic factors, such as UV radiation, that cause chain cleavage (reduction of molecular mass) and oxidation (23–25). Bacteria and fungi associated with PE biodegradation have been reported (26). PE has been biodegraded generally at up to 10% (wt/wt) weight loss in more than 2 months (26). An Alcanivorax borkumensis bacterium has been shown to degrade PE by weight loss (2%–3%) and oxidation in 80 days (27). Compared to PE, PP biodegradation has scarcely been reported. Bacillus (28, 29), Pseudomonas (28), and Rhodococcus (29) bacteria have been suggested to degrade PP as evaluated by weight loss, morphological deterioration, and oxidation modification. Aspergillus fungi have also been indicated to degrade PP by forming colonies using PP as the sole carbon source (23, 25). In this study, obvious PP biodegradation and important PP-degrading bacteria were investigated from mesopelagic environments.

RESULTS AND DISCUSSION

Enrichment of A. borkumensis bacteria on liquid PP in mesopelagic water

Mesopelagic water (depth of 374 m) was incubated with liquid PP at 10 (original temperature of the mesopelagic water), 20, and 50°C to enrich PP-degrading bacteria. Liquid PP oil appeared to disappear at 10°C but not at 20 and 50°C. The inhibitory effect of pressures equivalent to a depth of 1500 m on the biodegradation of hydrocarbons is small (18). These data suggest that PP could be biodegraded preferably in mesopelagic environments than in surface marine environments. As PP could be considered to be biodegraded by pristane-degrading bacteria, the mesopelagic water was also incubated with pristane at 10, 20, and 50°C. Pristane appeared to disappear at all the temperatures tested, implying PP biodegradation potential at 20 and 50°C. The mesopelagic water was also similarly incubated with n-hexadecane (C₁₆). C₁₆ seemed to disappear at 20 and 50°C but not at 10°C (insoluble at 10°C), suggesting that PE could be biodegraded preferentially in surface marine environments.

Bacteria enriched on liquid PP and pristane at 10°C were examined by sequencing their 16S rRNA gene fragments (Fig. 1). In liquid PP-enriched seawater, except for Colwellia and Phaeobacter bacteria, Alcanivorax bacteria were the most abundant (Fig. 1). In pristane-enriched seawater, Alcanivorax bacteria dominated (Fig. 1). In the original mesopelagic water at Muroto, Alcanivorax and Colwellia bacteria are undetectable and potential Phaeobacter bacteria (with at least 95% 16S rRNA gene sequence identity to the type strain of the type species) are suggested to be present at less than 0.13% (30). Almost all Phaeobacter bacteria in liquid PP-enriched seawater (Fig. 1) were from Phaeobacter porticola: sequences with 99%–100% identities to the sequence of P. porticola P97^T constituted 21.5% of 400 randomly selected sequences from the enriched seawater. The P. porticola bacteria are undetectable in the original mesopelagic water at Muroto (all sequences from the mesopelagic water show less than 97.5% identity to strain P97^T sequence) (30). Colwellia bacteria have been indicated to degrade very short-chain alkanes, ethane, and propane, by stable isotope probing (31). However, there is no direct evidence that Colwellia bacteria degrade long-chain alkanes. In addition, almost all sequences in Colwellia from liquid PP-enriched seawater (Fig. 1) were closely related to Colwellia psychrerythraea 34H (36% of 400 randomly selected sequences from liquid PP-enriched seawater showed 98%–100% identities to strain 34H sequence), where alkane hydroxylase genes are not found (32). Regarding Phaeobacter, there has been no report on alkane degradation. These data suggest that Colwellia and Phaeobacter bacteria would not degrade alkanes (except for the very short-chain alkanes) and that liquid PP might not be highly degraded in mesopelagic water (Fig. 1). The major sequences in Alcanivorax in both enriched seawaters (Fig. 1) were from A. borkumensis: A. borkumensis SK2^T sequences were deduced to constitute 5.0% and 41.3% of the total bacterial sequences from liquid PP- and pristane-enriched seawaters, respectively (with 99%–100% identities; deduced from 400 randomly selected sequences from each enriched seawater). These data indicate that A. borkumensis bacteria could be major PP degraders in mesopelagic environments. A. borkumensis bacteria have been indicated to be major n-alkane degraders in the deep sea (at 1,170–1,210 m depth) (33), supporting that A. borkumensis bacteria are active down to the deep sea (10).

Fig 1.

Bacterial community composition at the genus level in liquid PP- or pristane-enriched mesopelagic water. Indications are given for the composition with a frequency of 0.5% or more. Sequences identified as Erythrobacter were indicated to be of Qipengyuania by BLAST (due to reclassification), and thus Qipengyuania is indicated in parentheses.

Isolation of a bacterium showing the highest liquid PP-degrading activity

Bacteria enriched on liquid PP and pristane were isolated on agar plates. Colonies of different morphologies were selected, and the isolates were subjected to repetitive extragenic palindromic sequence PCR (rep-PCR) analysis for strain typing. The isolates from the liquid PP- or pristane-enriched seawater were grown in SW medium (a seawater-based medium) with liquid PP or pristane, respectively, at 10°C for 4 weeks and analyzed by GC-MS. An isolate exhibiting the highest liquid PP-degrading activity was selected and designated strain sw2. An isolate showing the highest pristane-degrading activity (degraded 98%–99% of pristane added) was also selected and designated strain MB7. Strain MB7 showed the same rep-PCR pattern as strain sw2, indicating that these two strains are identical or close relatives. These results support the hypothesis that PP and isoprenoid-derived branched alkanes are biodegraded through the same mechanism.

Identification of the isolate probably as A. borkumensis

The 16S rRNA gene fragment sequences of strains sw2 (1403 bp; accession no. LC739329) and MB7 (673 bp; accession no. LC738853) were 100% identical to that of A. borkumensis SK2^T, suggesting that these strains belong to A. borkumensis. Alcanivorax strain A29 (100% identical in the 16S rRNA gene sequence to A. borkumensis SK2^T) has been suggested to show the highest n- and branched alkane-degrading activities among at least Alcanivorax bacteria in temperate surface seawater (10). Growth temperatures of strains sw2 and A29 were compared on dR2A-SW plates. Strain sw2 grew at 4–42°C, optimally at 20°C, while strain A29 grew at 4–37°C, optimally at 28°C. The relatively low optimum growth temperature and tolerance to the high temperature of strain sw2 may imply that this strain was adapted to cold temperatures and can grow at rarely hot temperatures in mesopelagic environments.

Liquid PP-degrading activity of Alcanivorax bacteria

The growth of strains sw2 and A29 on C₁₆, pristane, and liquid PP was tested on ONR7a medium (an inorganic medium) plates at 10°C. Both strains grew on C₁₆ and pristane but not on PP, showing that PP is not used as a sole carbon and energy source. The growth on C₁₆, pristane, and liquid PP was also tested on 1/5 MB plates at 10°C. Both strains grew fastest on C₁₆, slower on pristane, and slowest on liquid PP. This suggests that PP was degraded when a carbon and energy source was supplied. On the 1/5 MB plates at 10°C, strain sw2 (from mesopelagic water) grew faster on liquid PP and pristane than strain A29 (from surface seawater), while strain A29 grew faster on C₁₆ than strain sw2. These results show that Alcanivorax bacteria from mesopelagic environments could have been adapted to branched alkane degradation.

Colwellia strain T11-10 and Phaeobacter strain d2A-3a, showing 99.8% and 99.5% 16S rRNA gene sequence identities to the major sequences in Colwellia (potentially Colwellia psychrerythraea) and Phaeobacter (probably Phaeobacter porticola), respectively, from liquid PP-enriched seawater (Fig. 1), had been obtained from mesopelagic water (unpublished). Their growth on liquid PP and pristane was tested on ONR7a and SW medium plates at 10°C. Colwellia strain T11-10 did not grow on pristane on both medium plates. Its growth on liquid PP was not clear. The growth of Phaeobacter strain d2A-3a on liquid PP and pristane was also not clear. On the other hand, strain sw2 grew on liquid PP and pristane on SW medium plates (the growth on ONR7a medium plates was as described above). These results suggest that Colwellia and Phaeobacter bacteria did not degrade liquid PP and pristane in mesopelagic water.

Degradation of liquid PP at 28°C was examined in SW medium supplemented with 0.025% (vol/vol) C₁₆ or 0.025% (wt/vol) pyruvate using strain A29 for 4 weeks. Pyruvate was used because it is a central metabolite probably used by most bacteria and would not be an inducer of alkane hydroxylase. Liquid PP was degraded at 28°C similarly with both C₁₆ and pyruvate (Fig. 2A). Together with the observation that Alcanivorax strains grew on liquid PP on 1/5 MB plates, the results indicate that any carbon and energy source would support the PP-degrading activity of Alcanivorax bacteria.

Fig 2.

Degradation of liquid PP in a seawater-based medium by strain A29 or sw2 with or without C₁₆ or pyruvate at 28 (A), 20 (B), or 10℃ (C) for 4 weeks. Total ion chromatograms are shown. Concentrations of C₁₆ (vol/vol) or pyruvate (wt/vol) are indicated. Pentamers to hexadecamers detected are indicated as numbers above the peaks. Liquid PP in noninoculated control samples is shown in gray and at a slightly left position. These are normalized to equivalent n-dodecane added just prior to extraction. *, contaminated peaks. (B) is from Fig. S1, while (C) is from Fig. S2 and S3 in online supplementary file 1.

An optimal concentration of the supplemented carbon source for liquid PP-degrading activity was investigated in SW medium supplemented with 0%–3% (wt/vol) pyruvate using strain A29 at 20°C for 4 weeks (Fig. S1). The activity gradually increased up to 0.5% pyruvate and then decreased above 0.5% pyruvate (Fig. 2B; Fig. S1). The activity of strain sw2 was also investigated in the same way with 0.1 and 0.2% (wt/vol) pyruvate. Strain sw2 showed somewhat lower liquid PP-degrading activity compared to strain A29 at 20°C (data not shown). At 10°C using strain A29 or sw2, the highest degradation activity was observed with 1%–2% pyruvate (Fig. 2C; Fig. S2 and S3). Strains A29 and sw2 preferentially degraded short PP and appeared to degrade all lengths of PP (Fig. 2; S1 to S3). Higher activity was observed at 10°C than at 20°C, and correspondingly, higher concentrations of pyruvate were required at 10°C (Fig. 2B and C; Fig. S1 to S3).

Liquid PP-degrading activity of Colwellia strain T11-10 and Phaeobacter strain d2A-3a was investigated in SW medium supplemented with 0.2% (wt/vol) pyruvate at 10°C for 4 weeks. As this liquid PP used was contaminated with C₁₆ that had naturally been derived from the liquid PP, C₁₆-degrading activity was also examined. As suggested by the plate assay, strains T11-10 and d2A-3a did not show liquid PP- and C₁₆-degrading activities (data not shown). Colwellia and Phaeobacter bacteria would have grown faster than the other bacteria by using organic carbon in mesopelagic water in the presence of liquid PP at 10°C. Consistently, Colwellia psychrerythraea is psychrophilic (https://lpsn.dsmz.de). Phaeobacter porticola is a mesophile able to grow at 4°C and potentially produces antibiotics (34). Overall, the results indicate that A. borkumensis bacteria could be major and important PP degraders in mesopelagic environments.

In natural mesopelagic water, liquid PP appeared to disappear at 10°C but not at 20°C, although liquid PP-degrading activity of strain sw2 appeared higher at 20°C than at 10°C in SW medium without addition of another carbon source for 4 weeks (data not shown; the activity at 20°C was comparable to that of strain A29 at 20°C without addition of another carbon source) (Fig. 2B). This may be due to other bacteria that would help in degrading PP in mesopelagic environments.

Oxidation of PP film by the Alcanivorax isolate

Degradation of the PP film was examined in SW medium supplemented with 2% (wt/vol) pyruvate using strain sw2 at 10°C for 38 days. Weight loss of 1% or more was not detected with strain sw2. However, we analyzed the surface of the PP film using X-ray photoelectron spectroscopy (XPS). The C1s XPS spectra showed a new peak and two new shoulders at approximately 288–286 eV for PP films incubated with strain sw2, indicating the formation of O=C–O, C=O, and C–O linkages (each peak at approximately 288, 287, and 286 eV, respectively) (Fig. S4). The O=C–O, C=O, and C–O linkages were implied to be increased by 1.6–1.8, 0.5–1.4, and 2.2–5.2 atomic% of the total C compared to the controls, respectively (the resultant increased total oxygen-linked carbon, 5.4–7.4 atomic%) (Fig. S5). The O1s XPS spectra also suggested the formation of C–O, –OH, and C=O linkages by strain sw2 (Fig. S4). The O/C atomic ratio of the film was increased by strain sw2 (Table S1). These results suggest that the PP film was oxidated enzymatically with strain sw2 but that fluidity and fragmentation of PP would be the key to its efficient degradation.

PP biodegradation has been investigated by using solid PP as the sole carbon source and the degradation rates were compared by the weight loss (23, 25, 28, 29), although this weight loss could be due to biodeterioration. In contrast, the approach using liquid PP and GC in this study clearly showed the PP biodegradation and that PP would not be used as a sole carbon and energy source. Using this present study as a model, various PP-degrading microbes would start to be unveiled.

MATERIALS AND METHODS

Seawater

Mesopelagic water used for formulating media and isolating bacteria was collected from the coastal area of Muroto, Kochi, Japan (33°15′02″ N, 134°11′06″ E, depth of 374 m) by the mesopelagic water pumping facility of Muroto City (Aqua Farm). The original temperature of the mesopelagic water was approximately 10°C. Surface seawater used for formulating media was also collected from the coastal area of Muroto (33°18′ N, 134°11′ E, depth of 0.5 m).

Liquid PP- and pristane-enriched seawaters

Twenty milliliters of nonsterilized fresh mesopelagic water collected in June 2019 was incubated in a glass beaker with 0.5-µl liquid PP (PP oligomers; described below) and with gentle shaking at 10°C. When the liquid PP oil seemed to disappear on the surface of the seawater (on the 13th and 36th days after starting the cultivation), 0.5-µl liquid PP was added to the seawater (1.5-µl liquid PP was added in total). When liquid PP oil seemed to slightly disappear again (on the 51st day), this liquid PP-enriched seawater was stocked at −80°C after mixing with glycerol at a final concentration of 20% (vol/vol) until the isolation of bacteria. The liquid PP was prepared by the San-ei Kogyo Corporation by heating solid PP (containing additives at less than 3%, wt/wt) at 370°C (35). The resultant liquid PP consisted of terminal monoolefins, PP, and terminal diolefins as dominant, second dominant, and minor components, respectively, which was confirmed by the San-ei Kogyo Corporation with GC-MS and partly NMR.

Pristane-degrading bacteria were also enriched in the same way using pristane instead of liquid PP unless stated otherwise. Twenty milliliters of fresh mesopelagic water was incubated with 0.5-µl pristane (Synthetic, Wako). When pristane oil disappeared (on the 12th, 20th, and 28th days), 0.5-µl pristane was added to the seawater (2-µl pristane was added in total). When pristane oil seemed to disappear again (on the 38th day), the pristane-enriched seawater was stocked at −80°C after mixing with glycerol (20%, vol/vol). Mesopelagic water was similarly incubated with liquid PP or pristane at 20 and 50°C or with C₁₆ at 10, 20, and 50°C.

Bacterial community composition

The liquid PP- or pristane-enriched seawaters were filtered through 0.22-µm pore size membranes (MF-Millipore membrane, Millipore) to collect the bacteria. These membranes were completely dried at 50–55°C and stocked at – 80°C before being used for DNA extraction for bacterial community analyses. DNA was extracted from the microorganisms on the membrane by ISOIL for Beads Beating (Nippon Gene, Tokyo) using a bead beater (FastPrep-24 5G, MP Biomedicals, CA, USA). DNA was purified with a DNeasy PowerClean Pro Cleanup Kit (Qiagen).

The 16S rRNA gene fragments were amplified by hemi-nested PCR. The first primers comprised the 16S rRNA gene sequence of Pro341F (36) and Q-1046R (37). The second primers (38) had the priming regions of Pro341F and Pro805R (36). Preparation of reaction mixtures (36) and amplification (39) were performed as described previously.

Sequencing was conducted on MiSeq (Illumina, CA, USA) using MiSeq Reagent Kit v3 (600 cycles). Primer sequences were removed with Cutadapt ver 1.18 with default parameters. Fastq-join (40) was used for merging overlapping pair-end reads. Sequences that had quality value scores of 20 or more for more than 99% of the sequence were selected through FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Chimeric sequences were removed using UCHIME (41, 42). The remaining 52,078 and 31,752 sequences for the liquid PP- and pristane-enriched seawaters, respectively, were deposited in GenBank/EMBL/DDBJ under accession numbers TAAF01000001-TAAF01052078 and TAAG01000001-TAAG01031752, respectively. Nonbacterial sequences were further removed using the RDP Classifier (43). A total of 52,062 and 31,723 bacterial sequences were analyzed for the community analysis of the liquid PP- and pristane-enriched seawaters, respectively. The sequence was assigned to the genus by the RDP Classifier with a confidence threshold of 80% and to species by BLAST (44).

Isolation of bacteria

Bacteria in the liquid PP- or pristane-enriched seawater at 10°C were isolated at 10°C on four types of medium plates (1.5%, wt/vol, agar; 9 cm in diameter): (i) an SW medium plate covered with 0.8-µl liquid PP or pristane; (ii) a marine broth 2216 (MB; BD) plate; (iii) a dMB plate, which contained 0.1-L distilled water, 0.9-L surface seawater, and 3.74-g MB; and (iv) dR2A-SW, which contained 1-L mesopelagic water and 1.82-g R2A agar (BD). SW medium contained 1-L mesopelagic water, 1-g NH4NO3, 0.2-g K2HPO4, and 25-mg iron(III) citrate n-hydrate. The plates were incubated at 10°C for up to 33 days. The bacterial colonies were purified on the original isolated medium plate at 10°C. Isolates sw2 and MB7 were deposited in NBRC (NITE Biological Resource Center, Japan) under numbers NBRC 114995 (as strain PP10sw2) and NBRC 115939 (as strain Pr10MB7), respectively. These strains were indicated to be obtained on all four types of plates, as isolates with the same rep-PCR pattern were obtained on all of them.

Reference bacteria

Alcanivorax sp. A29 (NBRC 105772) (10) was obtained from NBRC. Phaeobacter sp. d2A-3a had been obtained from mesopelagic water, and Colwellia sp. T11-10 had been obtained from the surface of a fish living in mesopelagic water (our unpublished results). These two strains, d2A-3a and T11-10, were deposited in NBRC under numbers NBRC 115947 and NBRC 115940, and their accession numbers of the 16S rRNA gene fragment sequence were LC738854 and LC738855, respectively.

Degradation of alkanes and GC-MS analysis

For selecting the PP- or pristane-degrading isolates, each isolate freshly grown on the originally isolated medium plate was inoculated with a toothpick into 2-ml SW medium with 0.8-µl liquid PP or pristane. This culture was incubated for 4 weeks with shaking at 10°C. For comparison of the liquid PP-degrading activity, bacteria freshly grown on a dR2A-SW plate were inoculated into 2-ml SW medium with 0.8-µl liquid PP and with the indicated amount of sodium pyruvate (wt/vol) or C₁₆ (vol/vol). This culture was then incubated for 4 weeks with shaking at 10, 20, or 28°C. Noninoculated sterile samples were similarly incubated without the addition of pyruvate or C₁₆ and served as controls.

Hydrocarbons were extracted twice from the cultures by shaking vigorously with 2-ml n-heptane for 1 minute. Just before extraction, 0.027-µl n-dodecane was added as an internal standard to evaluate degradation. Sodium sulfate was added to the n-heptane extracts to dehydrate them, and the supernatants were concentrated to approximately 150 µl by N₂ purging. The concentrated extracts were analyzed by GC-MS using a 6890N gas chromatograph as described previously (8) unless stated otherwise. To investigate the degradation of liquid PP, C₁₆ (naturally derived from the liquid PP), or pristane, their peak areas obtained with GC-MS total ion monitoring were normalized by the peak area for n-dodecane and compared to those from the controls.

Growth temperature

Growth temperatures of strains sw2 and A29 were tested at 4, 10, 20, 28, 37, 42, and 50°C on dR2A-SW plates for 44 days. The growth was checked by visual inspection.

Utilization of alkanes as carbon sources

All strains were pregrown on dR2A-SW plates. Strains sw2 and A29 were plated on ONR7a medium (an inorganic medium) (45) plates (1.5%, wt/vol, agar; purified for microbial cultures, Nacalai, Japan) covered with 0.8-µl liquid PP, pristane, or C₁₆. The strains were also plated on 1/5 MB plates, which contained 0.2-L distilled water, 0.8-L surface seawater, 7.48-g MB, and 15-g agar, covered with 0.8-µl liquid PP, pristane, or C₁₆. The strains were also plated on ONR7a medium plates and 1/5 MB plates that were not covered with liquid PP, pristane, or C₁₆ as controls. Plates were incubated at 10°C for 3 weeks on ONR7a medium plates and for 7 days on 1/5 MB plates. Similarly, strains d2A-3a and T11-10 were plated on ONR7a medium plates covered with 0.8-µl liquid PP or pristane and on SW medium plates covered with 0.8-µl liquid PP or pristane. Strain sw2 was also plated on SW medium plates covered with 0.8-µl liquid PP or pristane. Strains d2A-3a, T11-10, and sw2 were also plated on the same medium plates that were not covered with liquid PP or pristane as controls. These plates were incubated at 10°C for 36 days. The growth was checked by visual inspection.

Degradation of PP film

PP film of Torayfan no. 3501 (Toray Advanced Film Co., Ltd) containing additives at less than 0.1% (wt/wt) was used as solid PP. PP films were 254 nm UV-irradiated for 74 or 75 hours with UV Crosslinker CL-1000 (Funakoshi, Japan) until they could crack easily when pinched. While UV-irradiated, they were floated on Milli-Q water (Merck) 11.5 cm from the inside bottom.

Strain sw2 freshly grown on dR2A-SW plates was inoculated into 4.8-ml SW medium supplemented with 2% (wt/vol) sodium pyruvate and 18- to 22-mg UV-irradiated PP films. This culture was incubated for 38 days with shaking at 10°C. Noninoculated sterile samples were similarly incubated and served as controls. Then, the films were incubated in 2% (w/v) sodium dodecyl sulfate with gentle shaking at 50°C for 4 hours to lyse biofilm on the surface and were washed with water at 50°C. The film surface was stroked with a cotton swab with water when possible to remove biofilm. The dry weight was measured using an ionizer (STABLO-AP, Shimadzu).

XPS

The hydroxyl, carbonyl, and carboxyl groups on the film surface were quantified by XPS on a Quantera SXM (Ulvac-PHI, Kanagawa, Japan) with monochromatic AlKα X-ray radiation (ϕ 100 µm; 280 or 55 eV for survey or narrow scan, respectively) with a takeoff angle of 45°. The surface charge was neutralized simultaneously by irradiation with 10 V Ar⁺ and 20 μA e^– beams.

Other methods

Rep-PCR for genomic fingerprints of bacteria, performed with primers REP1R-I and REP2-I (46), and 16S rRNA gene sequencing were conducted as described previously (8).

AFTER EPUB

[This article was published on 20 November 2023 with incomplete data accession information. The Data Availability statement and the Materials and Methods were modified to include the missing information and two superfluous related supplemental material files were deleted in the version posted on 5 December 2023. In addition, superfluous text was deleted from the legend of Fig. 2 in the current version, posted on 8 December.]

DATA AVAILABILITY

The 52,078 sequences from the liquid PP-enriched seawater and the 31,752 sequences from pristane-enriched seawater were deposited in GenBank/EMBL/DDBJ under accession numbers TAAF01000001 to TAAF01052078 and TAAG01000001 to TAAG01031752, respectively. FASTA files for the two sets of sequences are available at https://www.ncbi.nlm.nih.gov/Traces/wgs/TAAF01?display=download and https://www.ncbi.nlm.nih.gov/Traces/wgs/TAAG01?display=download, respectively.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.01365-23.

SUPPLEMENTAL FILE 1. aem.01365-23-s0001.pdf.

Fig. S1–S5 and Table S1

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.