Introduction

Plastic wastes have led to numerous environmental problems since they are not degradable. Recently, efflux of microplastics has been identified as one of the causes of environmental destruction [1]. Biodegradable plastics have attracted attention as the potential solution to the aforementioned problems. Poly(ethylene succinate) (PESu), an aliphatic polyester and a chemosynthetically biodegradable plastic, has a high melting temperature (104 °C) and good mechanical properties comparable to those of polypropylene [2]. Poly(3-hydroxybutyrate) (P(3HB)), which is a fully biobased microbially biodegradable aliphatic polyester, also exhibits good mechanical and thermal properties comparable to those of commodity plastics [3].

PESu can be hydrolyzed in vitro by P(3HB) depolymerases produced by P(3HB)-degrading microbes because these depolymerases can recognize the dimer unit (−O-CH2-CH2-O-CO-CH2-CH2-CO-O-)2 of PESu as an analog of the tetramer unit (−O-CH(CH3)-CH2-CO-)4 of P(3HB) [4,5,6]. Although both PESu and P(3HB) can be degraded by P(3HB) depolymerases, they differ in their environmental degradability [2, 7, 8]. P(3HB) shows stable biodegradability under various conditions, while PESu hardly degrades in seawater. This indicates that the environmental degradability of PESu cannot be accounted for by the enzymatic degradation caused by P(3HB) depolymerases.

Our previous study showed all fungi opted by PESu-degrading ability degraded not only PESu but also P(3HB), suggesting that P(3HB) degraders were closely related to PESu degraders in fungi [5]. On the other hand, most PESu-degrading bacteria, which are mainly composed of Gram positive species, did not degrade P(3HB) [9, 10]. Therefore, the difference in the environmental degradability between PESu and P(3HB) could be related to the difference in the type of bacterial degraders.

Thus, in this study, we focus on bacterial strains to understand the aforesaid difference in environmental degradability between P(3HB) and PESu. We isolated P(3HB)- and PESu-degrading bacteria from 68 environmental samples and characterized them. Further, the relation between the abundances of P(3HB)- and PESu-degrading microbes in each environment and their environmental degradabilities was investigated.

Experimental

Chemicals

Poly(ethylene succinate) (PESu) (Mn = 4.8 × 104, Mw/Mn = 1.9) was donated by Nippon Shokubai Co., Ltd., Tokyo. Poly(3-hydroxybutric acid)(P(3HB)) (Mn = 6.8 × 104, Mn/Mw = 2.6) was donated by Mitsubishi gas chemical Co., Ltd., Tokyo. To purify the polymers, they were dissolved in chloroform, then reprecipitated by the addition of methanol. The films of PESu and P(3HB) were prepared by solvent-cast techniques from chloroform solutions of polyesters using glass Petri dishes as casting surfaces. Other chemicals were purchased from Tokyo chemical industry Co., Ltd., Wako pure chemical industry Co., Ltd., Kanto chemical Co., Inc., or Sigma-Aldrich Japan Co. and used without further purification.

Environmental samples

Forty-seven soil samples were collected from the following environments: forest (5 locations: location no. A, S, T, U, AO (Table S1)), schoolyard (1: AX), campus (7: B, AV, BE, BF, BG, BH, BI), city area (8: E, F, G, H, I, J, K, V), riverside (2: N, Y), sludge (8: M, Q, AE, AI, AN, BB, BC, BD), park (5: K, O, P, AP, AQ), field (9: Z, AF, AM, AR, AS, AT, AU, AY, AZ), and roadside (2: AC, BA). Eleven freshwater samples were collected from the following environments: snow (1: C), river (5: L, R, X, AA, AD), lake (2: D, AB), pond (2: AG, AH) and fountain (1: W). Ten seawater samples were collected from coastal waters (10: AJ, AK, AK’, AK” AL, AL’, BJ, BK, BL, BM). Further information on sampling locations and sample aspects are listed in Table S1.

Media and culture conditions

PESu was dissolved in 30 mL dichloromethane. The solution was emulsified with TOMY ultrasonic disruptor UD-200 in 1 L of basal medium containing the following components: 4.6 g/L of KH2PO4, 11.6 of Na2HPO4·12H2O, 0.5 of MgSO4·7H2O, 0.1 of FeCl3·6H2O, 1.0 of NH4Cl, 0.1 of Yeast extract, 0.1 of Plysurf, pH 7.0. Dichloromethane was removed by stirring at 40 °C. P(3HB) powder was directly mixed into the basal medium. The composition of Luria-Bertani(LB) broth contain the following components: 10 g/L of tryptone peptone, 5.0 of yeast extract, 5.0 of NaCl. Agar (15 g/L) was added to the media to solidify the medium. Medium was autoclaved for 15 min at 121 °C before use [9].

Bacterial strains

Bacterial reference strains used in this study were listed in Table 1.

Table 1 Bacterial strains used in this study
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Plate count method

The total numbers of viable microbes and polymer-degrading microbes in the samples were counted by the plate count and clear zone methods, respectively [11]. After 1 g of environmental sample was dispersed in 10 mL of sterile physiological saline, it was left for 10 min. A series of dilutions was prepared from 10−1 to 10−5 by adding 50 μL of the sample suspensions to 450 μL sterile water. Fifty microliter of the supernatants were plated onto both LB and polymer containing solid plates as described in Media and culture conditions section. The plates were sealed with plastic tapes and incubated at 30 °C for 1 week. Colonies formed on LB plates and clear zones formed on polymer containing plates were counted and the numbers were expressed as colony- (c.f.u.) and clear zones-forming units (cz.f.u.) per g of samples, respectively.

Isolation of polymer-degrading bacteria

PESu- and P(3HB)-degrading bacteria were isolated from environmental samples by clear zone methods with agar plate that contained each polymer. The bacteria screened for P(3HB)-degrading activity on P(3HB)-containing plates were defined as P(3HB)-degrading bacteria, whereas the bacteria screened for PESu-degrading activity on PESu-containing plates were done as PESu-degrading bacteria. Further information on sampling locations and sample aspects are listed in Table S1. Colony formed clear zone on the polymer-containing plate was further purified by streak culture on LB plate. The purity of isolate was confirmed with the combination of polymer-containing plate and LB plate.

Phylogenetic analysis

16S rRNA genes were amplified by PCR method. The forward amplification primers were 16Sf, 5′-GTTTGATCATGGCTCAG-3′ (corresponding to positions 36–53 in the 16S rDNA nucleotide sequence of E. coli) and 27f, 5′ -AGAGTTTGA TCCTGGCTCAG-3′ (positions 8–27). The reverse amplification primers were 16Sr, 5′ -TACCTTGTTACGACTTCA-3′ (positions 1517–1533) and 1525r, 5′-AAAGGAGGTGATCCAGCC-3′ (positions 1543–1525). Reactions were carried out in 20 μL volumes containing 1.25 U ExTaq DNA polymerase (TaKaRa, Japan), 100 nM each primer (16Sf, 16Sr, 27f and 1525r), 25 mM each dNTP (dATP, dTTP, dCTP, and dGTP), 2 μL of 10 × ExTaq buffer (TaKaRa, Japan), and colony. The PCR cycle parameters were as follows: preheating for 2 min at 94 °C, denaturation at 94 °C for 20 s, annealing at 55 °C for 30 s, extension at 72 °C for 5 min and for 10 min. After 25 cycles, the PCR products were analyzed by means of an electrophoresis with 1.5% (wt./vol.) agarose gel, and then ligated to pMD20 (TaKaRa, Japan). The 16S rDNA sequence was determined by dideoxy method and then was compared with Genbank data using the program blast via NCBI site (http://www.ncbi.nlm.nih.gov/BLAST/). The 16S rDNA sequences were aligned by the program ClustalW W via DDBJ site (http://clustalw.ddbj.nig.ac.jp). A phylogenetic tree was constructed according to the neighbor-joining method [12] with the program MEGA 5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods [13].

Substrate degradation test

PESu and P(3HB) hydrolytic abilities of isolates were examined by clear zone method. Solid polyester-containing plate inoculated with bacteria incubated for 1 week at 30 °C. The hydrolytic activities of them were evaluated by the distance between the center of colony and the edge of clear zone.

Biochemical oxygen demand (BOD)-biodegradability of polyester sample with seawater as an inoculum

Three seawaters (Oarai (location no.: BJ, (Table S1)), Niigata (BK) and Atami (AL)) were used as inocula for BOD-biodegradability test. All tests were performed under aerobic conditions in a temperature-controlled BOD reactor (Taitec Japan) at 25 °C with stirring. A sample film (area: 1 × 1 cm, weight: about 5 mg) was placed in a 300-ml BOD reactor, and 197.4 m1 of seawater were added into the reactor as an inoculum. Three seawaters were used as inocula for the biodegradation test. In addition, 2.6 ml of mineral solution was added to the seawater. The mineral solution contained the following: 0.085 g/L of KH2PO4, 0.2175 of K2HPO4, 0.446 of Na2HPO42H2O, 0.017 of NH4Cl, 0.225 of MgSO4.7H2O, 0.275 of CaCl2 and 0.0025 of FeC13.6H2O. The biodegradation test was carried out at 25 °C for 28 d, and the BOD data was measured once a day. The BOD-biodegradability of polymer sample was calculated by subtracting the BOD of the control blank (BODb) from that of the test solution (BODt) and by dividing the value (BODt-BODb) by the theoretical oxygen demand (ThOD) of the test sample [8].

Nucleotide sequence accession numbers

Nucleotide sequences of the isolated strains 16S rRNA genes were deposited in the DDBJ nucleotide sequence database. Figure 3 shows each strain accession numbers.

Results

Characteristics of environmental samples and abundances of P(3HB)- and PESu-degrading microbes in respective viable microbe counts

Viable P(3HB)- and PESu-degrading microorganisms in 68 environmental samples were counted. The abundances of the P(3HB)- and PESu-degrading microorganism counts in the viable microorganism counts in each sample were calculated (Table S1). Figure 1 shows the relationship between the viable microorganisms counts and the P(3HB)- and PESu-degrading microorganism counts in each environmental sample. The number of viable microorganisms in soil, freshwater, and seawater samples were 1.3 × 105 to 1.6 × 108 (median:5.3 × 106, average:1.1 × 107), 2.3 × 102 to 3.2 × 105 (9.6 × 103, 6.9 × 104), and 1.6 × 102 to 3.1 × 105 c.f.u./g (7.9 × 102, 5.4 × 103), respectively. The abundance of the P(3HB)-degrading microorganism counts in viable microorganism counts in environmental samples was 0.0030–43.8% (median:3.9, average:10.0) [soil: 0.0030–33% (4.4, 9.3), freshwater: 0.59–25% (15, 13), seawater: 0.24–43.8% (2.5, 9.7), indicating that these microorganisms existed in all the environmental samples used in this study. On the other hand, the abundance of the PESu-degrading microorganism counts in the viable microorganism counts in environmental samples was 0.0–16.7% (median: 0.4, average: 2.6). In contrast, PESu-degrading microorganisms were not detected in 11 environmental samples. The abundances of the PESu-degrading microorganism counts in the viable microorganism counts in soil and freshwater samples were 0.0–16.7% (median: 0.7, average: 2.6) and 0.03–14.8% (3.6, 4.7), respectively. In particular, PESu degraders were undetected in all the 10 seawater samples. In addition, the abundances of the PESu-degrading microorganism counts in the viable microorganisms counts at intermediate temperatures tended to be lower than those of P(3HB)-degrading microorganisms in each environmental sample.

Fig. 1
figure 1

Relationship between viable microorganism counts and P(3HB)- and PESu-degrading microorganism counts at a mild condition. Open squares, open triangles, and open circles indicate the ratios of P(3HB)-degrading microorganisms to viable microorganism counts in soil, freshwater, and seawater samples, respectively. Closed squares, closed triangles, and closed circles indicate the ratios of PESu-degrading microorganisms to viable microorganism counts in soil, freshwater, and seawater samples, respectively

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BOD-biodegradability of P(3HB) and PESu in seawater

Figure 2 shows BOD-biodegradation curves of P(3HB) and PESu films in 3 seawater samples at 25 °C. The BOD-biodegradabilities of P(3HB) and PESu within 28 days in seawater were 26–50% (average: 39, standard deviation: 11.9) and 0.0–4.4% (1.7, 2.4), respectively. This implies that P(3HB) was mineralized in all 3 seawater samples, whereas PESu was hardly biodegraded.

Fig. 2
figure 2

BOD biodegradation curves of P(3HB) and PESu films in seawater. Open and closed symbols indicate the biodegradability on P(3HB) and PESu, respectively. Niigata(square symbols, location no.:BK), Oarai(triangle symbols, BJ), and Atami(circle symbols, AL) seawaters were respectively used as inocula for BOD degradability testing

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Phylogenetic analysis of P(3HB)- and PESu-degrading bacterial isolates

Ten P(3HB)-degrading bacteria were isolated from 3 kinds of environments: soil, freshwater, and seawater. On the other hand, 32 PESu-degrading bacteria were isolated from soil and freshwater. Figure 3 shows the phylogenetic tree based on 16S rDNA sequences of 10 strains P(3HB)- and 16 strains PESu-degrading bacterial isolates in this study, together with 15 reference strains, source of isolates (location), and P(3HB) and PESu degradation abilities of these bacteria at 30 °C [9, 14, 15]. P(3HB)-degrading bacteria, which were isolated from each environmental sample, belonged to the phyla Proteobacteria (3 strains), Actinobacteria (3), and Firmicutes (4), whereas 29 of the 32 PESu-degrading bacterial isolates did to the genus Bacillus. Only one of the 42 isolates ((BH-2B) besides the 11 reference strains considered herein could degrade both P(3HB) and PESu.

Fig. 3
figure 3

Phylogenetic tree of P(3HB)- or PESu- degrading bacteria based on 16S rDNA sequence date and the ability of clear zone formation on the plates containing P(3HB) and PESu. +++; A radius of clear zone was larger than 5 mm. ++; A radius was larger than 3 mm. +; A radius was larger than 1 mm. -; No clear zone was formed around the isolate. ND; Experiments have not been done

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Discussion

P(3HB) is biodegraded in various environments: soil, estuarine sediment, freshwater, seawater, deep sea, anaerobic sewage, aerobic sewage, and compost [7, 8, 16, 17]. Nishida et al. showed that the abundances of the P(3HB)-degrading microorganism counts in the viable microorganism counts in 10 soil samples were 0.2–11.4% [11]. This study revealed P(3HB)-degrading microorganisms existed in all 68 environmental samples, namely, 47 soils, 11 freshwaters, and 10 seawaters (abundance: 0.0030–43.8%). These collectively suggest that P(3HB)-degrading microorganisms would exist almost everywhere. Previous studies have also shown that P(3HB)-degraders are more distributed in various environments as compared with microorganisms that degraded other polyesters [11, 18]. On the other hand, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), which is a copolymer of 3HB and 3-hydroxyvalerate (3 HV), differed in its biodegradation rates depending on the exposure environment. Complete degradation of PHBV occurred in 350 weeks in seawater but in 40 weeks in estuarine sediments [7]. In this study, BOD-biodegradabilities of P(3HB) in seawater over 28 days reached 39 ± 11%, while a previous study reported that the biodegradability in freshwater was 68 ± 23% for 28 days [8]. These data suggest that the rate of P(3HB) biodegradation in seawater would be much lower than that in other environments. The abundances of the P(3HB)-degrading microorganism counts in the viable microorganisms did not depend on the environment (Fig. 1). On the other hand, the viable microbe counts decreased in the following order: soil > freshwater > seawater. Therefore, the number of P(3HB)-degrading microorganisms in each environment increased in the order seawater < freshwater < soil, suggesting that the biodegradation rate of P(3HB) in each environment is related to the respective P(3HB)-degrader counts. Taken together, we conclude that the low biodegradation rate in seawater is caused by a small number of P(3HB)-degrading microorganisms.

Our previous studies have shown that PESu was degraded to a very small extent in seawater environments, although it was degraded well by the P(3HB) depolymerases produced by P(3HB) degraders [6, 8]. Here, PESu-degrading microorganisms were not detected in 16% of the samples and were particularly undetected in the seawater samples, because of the extremely low BOD biodegradability of PESu in seawater (Fig. 2). This suggests that PESu-degraders would be heterogeneously distributed in the environments and are not always in agreement with P(3HB)-degraders. Thus, we conclude that the low biodegradability of PESu in seawater could be due to the extremely low PESu-degrading bacteria counts. At high temperatures (50 °C), the abundances of the PESu-degraders were much higher (20–80%) [10] than those (0–17%) under mild conditions (30 °C). The relatively small number of viable microbes at high temperatures would be responsible for such a high abundance.

We previously demonstrated that PESu-degrading fungi are a subgroup of P(3HB)-degrading fungi since all of PESu-degrading fungal isolates also degraded P(3HB) [5]. Meanwhile, it has been reported that the abundance of P(3HB)-degrading fungi among marine isolated fungi (6.72%) is much lower than that of terrestrial isolates (~68%) [19, 20]. In seawater, the abundance of eukaryotes such as fungi was much smaller than that of prokaryotes such as bacteria [21]. These suggest that distribution of bacterial strains, rather than fungal strains, in seawater would have a greater influence on the degradation of PESu. Therefore, we focused on the domain bacteria that degrade P(3HB) and PESu.

P(3HB)-degrading bacteria were detected in all the environmental samples used in this study. In addition, the identified genera, which belonged to 3 phyla, indicated the involvement of a broad spectrum of bacteria in P(3HB) biodegradation in various environments. Thus far, a variety of bacterial strains belonging to 5 phyla, which include 36 genera [16, 17, 22,23,24,25,26,27,28,29,30,31,32,33,34], have been isolated as P(3HB)-degraders from environments. Considering that P(3HB) is degraded by a broader spectrum of bacteria as compared with other biodegradable polyesters, it would show good environmental degradability.

Meanwhile, PESu-degrading mesophiles could be classified into a narrow spectrum of bacteria, which mostly belong to the genus Bacillus (29/32 isolates). In addition, 3 PESu-degrading bacterial isolates were contained in the phylum Proteobacteria, genera Acidovorax, Pseudomonas, and Acinetobacter. Some other genera have also been isolated as mesophilic PESu-degrading bacteria, such as Streptomyces, Pseudomonas, Brevundimonas, and Leptothrix [35,36,37,38]. Taken together, these suggest that PESu would be degraded under mild conditions by bacteria belonging to the phyla Firmicutes, Proteobacteria, and Actinobacteria. Thus, we conclude that such Firmicutes are major PESu-degraders in mesophilic environments, except for marine environments.

Our previous study revealed that several P(3HB) depolymerases showed hydrolysis activity for not only P(3HB) but also PESu [6]. This may be owing to the substrate specificity of the P(3HB) depolymerase that recognizes two units of PESu as an analog of a 3HB tetramer. This hypothesis was supported by the crystalline structure of the P(3HB) depolymerase from Penicillium funiculosum [4]. Although P(3HB) depolymerases showed PESu-degrading activity, most P(3HB)-degrading bacteria including strains Delftia acidovorans JCM 10172 and Ralstonia pickettii T1 could not hydrolyze PESu in this study (Fig. 3). These results indicate that most of the bacterial P(3HB) depolymerases are inducible enzymes produced by P(3HB) and its hydrolysates, whereas they are never induced by PESu. Thus, P(3HB)-degrading bacteria would typically not be involved in the biodegradation of PESu in most environments.

This study reveals that Bacillus bacteria are the major PESu-degraders in mesophilic environments, but the PESu-degrading Bacillus isolated in this study could not degrade P(3HB) (Fig. 3). The 17 mesophilic PESu-degrading Gram-positive bacteria isolated in a previous study could not degrade P(3HB) [9]. These suggest that most of the PESu-degrading bacteria do not hydrolyze PESu by a P(3HB) depolymerase but by other esterases in natural environments.

Fig. 4 shows the relationship between the factors contributing to the biodegradation of PESu and P(3HB) and each environment. The fungal group that degrades PESu is a subgroup of the P(3HB)-degrading fungal group, whereas PESu-degrading bacteria, a few of which exist in marine environments, do not always correspond to P(3HB)-degrading bacteria. Thus, the difference among the degrading bacterial strains for each polyester could be responsible for the difference in environmental degradability between P(3HB) and PESu.

Fig. 4
figure 4

Relationship between the environmental degradability of P(3HB) and PESu, and the distribution of P(3HB)- and PESu-degrading microorganisms in environments

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