Isolation of poly[d-lactate (LA)-co-3-hydroxybutyrate)]-degrading bacteria from soil and characterization of d-LA homo-oligomer degradation by the isolated strains
Introduction
Polyhydroxyalkanoates (PHAs) are bacterial polyesters produced from renewable resources. Currently, a PHA copolymer, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx) or PHBH], is commercially manufactured as a commodity plastic by Kaneka Corp. (Japan). PHAs have attracted increasing attention because they exhibit superior biodegradability in various natural environments and controlled aerobic and anaerobic conditions [1,2]. In contrast, poly(l-lactic acid), the most widespread chemically synthesized bio-based plastic, is a compostable plastic that is degraded efficiently only under managed conditions [3]. There is limited information on the biodegradability of poly(d-lactic acid) (PDLA) (see Discussion).
A broad range of bacteria and fungi have been isolated from various environments as degraders of naturally occurring PHAs such as P(3HB) and PHBH by using clear zone formation on emulsified PHA-containing agar plates [1]. Techniques such as denaturing gradient gel electrophoresis and metagenomics have revealed that the microbial community varies depending on environmental factors such as soil, freshwater, and sea in different areas [[4], [5], [6], [7], [8], [9]]. For example, PHA-degrading soil bacteria have been identified to belong to the genera Acidovorax, Acinetobacter, Arthrobacter, Bacillus, Burkholderia, Cytophaga, Cupriavidus, Mycobacterium, Nocardiopsis, Pseudomonas, Rhizobium, Variovorax, Stenotrophomonas, Xanthomonas, and Zoogloea and bacterium Ellin [1,[6], [7], [8]].
In 2008, artificial PHAs containing unusual d-lactate (LA) units were synthesized using the engineered PHA synthase PhaC1PsSTQK, lactate-polymerizing enzyme (LPE) [10]. The artificial polymer, P(d-LA-co-3HB), possesses favorable physical properties such as semitransparency and flexibility [11,12]. P(d-LA-co-3HB) shares part of its structure with PDLA, and, therefore, the biodegradability of the copolymer has been of interest.
The screening of soil bacteria that can degrade P(67 mol% d-LA-co-3HB) resulted in the isolation of a Betaproteobacteria (Burkholderiales) bacterium, Variovorax sp. C34 [13]. PHA depolymerases (PhaZs) play a central role in PHA biodegradation [14]. PhaZ derived from Variovorax sp. C34 (PhaZVs, Accession#, BAR87946.1) is capable of hydrolyzing all four types of ester bonds in P(67 mol% d-LA-co-3HB), i.e., the linkages of 3HB–3HB, 3HB-LA, LA-3HB, and LA-LA. However, the enzyme did not hydrolyze high-molecular-weight PDLA. The assay using d-LA homo-oligomers indicated that hydrolysis of the LA-LA linkage in the d-LA homopolymer by PhaZs was limited by the degree of polymerization (DP) of the substrate, and an inverse relationship was observed between the DP and hydrolysis efficiency [15]. The results prompted us to investigate the bacterial group that can degrade P(d-LA-co-3HB) and whether they exhibit the DP-dependent degradation of the d-LA homo-polymer.
The aim of this study was, therefore, to isolate the P(d-LA-co-3HB)-degrading bacterial group in the soil and evaluate their capacity of consuming d-LA homo-oligomers with different DPs, which were prepared via the finely controlled chemical polymerization. Using the candidate isolates, we discuss the biodegradability of the target polymers in terms of the hydrolysable DP of d-LA homo-oligomer together with a molecular dynamic study.
Section snippets
P(d-LA-co-3HB) production
P(d-LA-co-3HB) was biosynthesized using batch jar fermentation. The polymers with 64 and 67 mol% LA were obtained in two batches. Seed culture of the recombinant E. coli strain JWMB1 (a dual-gene-knockout mutant ΔpflAΔdld of parent strain BW25113) harboring pTV118NpctphaC1(ST/FS/QK)AB [16] was prepared in 100 mL of Luria broth (LB) containing 100 mg/L ampicillin and 100 mg/L kanamycin in a flask for 7 h at 30 °C with reciprocal shaking at 120 rpm. The seed culture was used to inoculate 4 L of
Bacterial screening by using the P(d-LA-co-3HB) plate
Of a total of 216 soil samples, 12 bacteria were isolated as single colonies forming clear zones on the plate containing emulsified P(64 mol% d-LA-co-3HB) (Fig. S1). The isolated bacteria were identified on the basis of the homology search for the 16S rRNA region (Table 1). Seven Variovorax sp. and three Acidovorax sp., classified as Betaproteobacteria (Burkholderiales), were identified. In addition, another Betaproteobacteria (Burkholderiales), Burkholderia sp., and a Brevibacillus sp.
Conclusion
We successfully isolated 11 P(d-LA-co-3HB)-degrading bacteria. Most of the isolated bacteria were Variovorax and Acidovorax related to previously identified natural PHA-degrading soil bacteria. The genera Variovorax and Acidovorax presumably possess two types of PhaZs in their genomes, and PhaZ with CD2 could be involved in P(d-LA-co-3HB) degradation. The isolates partially consumed DLA20 (DP ranging from 10 to 30) and hardly consumed DLA40 (DP ranging from 20 to 60), suggesting that the d
Funding
This work was supported by JSPS KAKENHI, Japan, Grant Numbers 17K00619 to T.O., S.T., K.M., and C.H. and 17K05025 to S.S.
CRediT authorship contribution statement
Chiaki Hori: Writing - original draft, Investigation, Formal analysis. Tomohiro Sugiyama: Investigation. Kodai Watanabe: Investigation. Jian Sun: Investigation. Yuu Kamada: Investigation. Toshihiko Ooi: Methodology, Investigation. Takuya Isono: Investigation. Toshifumi Satoh: Methodology. Shin-ichiro Sato: Methodology, Investigation. Seiichi Taguchi: Writing - review & editing, Conceptualization. Ken'ichiro Matsumoto: Conceptualization, Supervision, Writing - original draft, Investigation.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Ms. Ayane Yamamoto for technical assistance.
References (33)
- et al.
Effects of polyhydroxyalkanoate degradation on soil microbial community
Polym. Degrad. Stabil.
(2016) - et al.
Degradation of polyhydroxyalkanoates in eutrophic reservoir
Polym. Degrad. Stabil.
(2007) - et al.
Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli
J. Biotechnol.
(2011) - et al.
Effect of monomeric composition on the thermal, mechanical and crystalline properties of poly[(R)-lactate-co-(R)-3-hydroxybutyrate]
Polymer
(2017) - et al.
Enzymatic characterization of a depolymerase from the isolated bacterium Variovorax sp. C34 that degrades poly(enriched lactate-co-3-hydroxybutyrate)
Polym. Degrad. Stabil.
(2014) - et al.
High-cell density culture of poly(lactate-co-3-hydroxybutyrate)-producing Escherichia coli by using glucose/xylose-switching fed-batch jar fermentation
J. Biosci. Bioeng.
(2019) - et al.
Purification and characterization of new bio-plastic degrading enzyme from Burkholderia cepacia DP1
Protein Expr. Purif
(2019) - et al.
Degradation of poly(d-lactic acid) by a thermophile
Polym. Degrad. Stabil.
(2003) - et al.
Degradation and applications of polyhydroxyalkanoates
Can. J. Microbiol.
(1995) - et al.
Biodegradability of modified plastic films in controlled biological environments
Environ. Sci. Technol.
(1992)
Biodegradability and biodegradation of poly(lactide)
Appl. Microbiol. Biotechnol.
Molecular characterization of the bacterial community in biofilms for degradation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films in seawater
Microb. Environ.
Biofilm formation and degradation of commercially available biodegradable plastic films by bacterial consortiums in freshwater environments
Microb. Environ.
Biodegradation of polyhydroxyalkanoates by soil microbial communities of different structures and detection of PHA degrading microorganisms
Appl. Biochem. Microbiol.
Biodegradation of polyhydroxyalkanoates in natural soils
J. Sib. Fed. Univ. Biol.
A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme
Proc. Natl. Acad. Sci. U.S.A.
Cited by (10)
Modification of poly(lactate) via polymer blending with microbially produced poly[(R)-lactate-co-(R)-3-hydroxybutyrate] copolymers
2024, International Journal of Biological MacromoleculesApplication of MicroResp™ for quick and easy detection of plastic degradation by marine bacterial isolates
2024, Marine Environmental ResearchToward the production of block copolymers in microbial cells: achievements and perspectives
2024, Applied Microbiology and BiotechnologyEnzymatic degradation
2022, Poly(lactic acid): Synthesis, Structures, Properties, Processing, Applications, and End of Life
- 1
Present address: Department of Chemistry for Life Sciences and Agriculture, Faculty of Life Sciences, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156–8502, Japan.