Anaerobic Biodegradation of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) under Flooded Paddy Soil Conditions

Abstract

Plastic pollution in aquatic environments has become a global concern, and agricultural plastics are one potential source. Biodegradable plastics have been developed as an alternative; however, their degradation behavior under flooded soil conditions remains unclear. Therefore, we herein investigated the degradation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), a biodegradable polymer, using an in vitro system simulating paddy soil environments. Microcosm experiments were conducted under different temperatures (14, 25, and 35°C) and soil-water ratios (1:1, 1:10, 1:100, and 1:1,000) to evaluate PHBH degradation and associated gas production. The results obtained showed that temperature and the soil-water ratio both significantly affected PHBH degradation and gas production. PHBH was almost completely degraded under the flooded soil condition (soil–water ratio 1:1) at 35°C, and was accompanied by the production of large amounts of methane and carbon dioxide, suggesting the involvement of anaerobic microbial processes. Degradation and gas production were markedly reduced under lower soil-content conditions. An amplicon sequencing anal­ysis of samples showing the highest degradation revealed the higher relative abundance of several anaerobic bacteria, including Ilyobacter delafieldii, Clostridium homopropionicum, and Syntrophomonas zehnderi, than in a control sample. These results suggest the involvement of an anaerobic metabolic network in the turnover of PHBH-derived intermediate metabolites. The present results will contribute to a more detailed understanding of the environmental fate of biodegradable plastics in paddy field environments.

The leakage of plastic waste into aquatic environments has recently become a serious global environmental issue (Jambeck et al., 2015). Agricultural activities also contribute to this problem because plastics are widely used for greenhouses, low tunnels, and mulch films. In Japan, agricultural plastics account for 1.4% of total plastic consumption, approximately 120,000 tons annually, with a recycling rate of about 70% in 2022 (MAFF, 2020). Nevertheless, some agricultural plastics, such as the polyolefin- or polyurethane-based polymer shells of controlled-release fertilizers, may persist in soil for extended periods. After the release of nutrients, residual microcapsules may be brought to the soil surface by tillage and eventually transported into aquatic environments (Sudo and Iwama, 2025). Katsumi et al. (2021) reported that about 70% of the microcapsules discharged from a single field remained in the soil for at least two years. In addition, approximately 0.067–0.076% of the total number of microcapsules that accumulated in paddy soils was discharged during the irrigation season. Once released into aquatic environments, these plastic particles may cause harm to organisms and act as carriers of pollutants or microorganisms (Zettler et al., 2013; Fu et al., 2021).

To mitigate these issues, biodegradable plastics have been developed. For example, polyethylene (PE) undergoes photo-oxidation under ultraviolet light, which reduces the molecular weight of the polymer and enables subsequent microbial degradation (Restrepo-Flórez et al., 2014). Polylactic acid (PLA) is another biodegradable polymer that hydrolyzes slowly at ambient temperatures and efficient microbial degradation generally requires elevated temperatures (≥50°C), such as those found in composting facilities (Zaaba and Jaafar, 2020). These materials often degrade slowly or incompletely in natural environments, where conditions markedly fluctuate (Sintim and Flury, 2017; Haider et al., 2019). Efficient degradation often requires the stable conditions found in industrial composting systems. Therefore, biodegradable plastics may persist in agricultural environments for extended periods, similar to non-biodegradable plastics. This limitation is relevant in paddy fields, where coated fertilizers are often applied under flooded conditions. However, the degradation behavior of biodegradable plastics under flooded soil conditions has not been investigated in sufficient detail.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), a copolymer of hydroxybutyrate and hydroxyhexanoate, is a microbial polyester synthesized from plant oils by bacteria (e.g., Aeromonas caviae). The incorporation of 3-hydroxyhexanoate (3HH) units reduces the crystallinity of the polymer, resulting in superior flexibility (Cai and Qiu, 2009) and biodegradability to PHB (Wang et al., 2004). PHBH is biodegradable under aerobic and anaerobic conditions (Fukuda, 2017). In aquatic environments, microorganisms colonize the polymer surface and form biofilms that facilitate polymer degradation. Previous studies suggested the contribution of Alteromonadaceae and Burkholderia in marine and freshwater environments, respectively, to the degradation of PHBH (Morohoshi et al., 2018a; 2018b). Although the anaerobic degradation of PHB by microorganisms, such as Ilyobacter delafieldii, has been reported (Janssen and Harfoot, 1990), the degradation of PHBH and the associated microbial community structure in anaerobic paddy soils remain unclear, particularly with respect to how environmental conditions and microbial community dynamics affect its degradation. Flooded soils restrict oxygen diffusion, and microbial activity quickly creates reducing conditions (Boie et al., 2025). In the present study, we constructed an in vitro system simulating paddy field conditions, varied the soil-water ratio, and analyzed microbial communities to evaluate PHBH degradation and characterize microbial community dynamics and also to examine the potential involvement of specific taxa in its anaerobic degradation.

Materials and Methods

Surface soil was collected from paddy fields operated by Nakajo Agricultural Products (Tainai city, Niigata Prefecture) in 2020. The soil was stored outdoors under polyethylene sheeting for approximately one year to allow for natural decomposition. Before the experiment, visible impurities, including rice straw and plant roots, were manually removed. The pH and electrical conductivity (EC) of the soil were 6.1 and 1.20 mS cm^–1, respectively. Commercially available PHBH grade PHBH 131A (Kaneka Corporation), supplied as cylindrical pellets (3.1‍ ‍mm×4.5 mm), was used as the test material. The exact 3HH molar fraction was 6 mol%.

Decomposition experiments were conducted for 137 days in 100-mL glass vials. Paddy soil, PHBH, and deionized water were mixed thoroughly and sealed with a rubber stopper and aluminum cap. Each vial contained 1.02±0.02 g of PHBH. To investigate the effects of soil volume on degradation, the soil-to-water ratio was adjusted to four weight ratios: 1:1, 1:10, 1:100, and 1:1,000. After the soil was thoroughly homogenized, deionized water was added according to the predetermined weight ratio, and the mixture was dispensed into each vial to 80 g. Soil weights corresponding to each ratio were 40, 8, 0.8, and 0.08 g, respectively. Incubations were conducted at 14°C, 25°C, and 35°C. Control vials without PHBH were prepared for comparison. Twenty-four experimental conditions were established, and each was prepared in triplicate.

Gas production was measured every few days for all treatments by collecting headspace gas with a gas-tight glass syringe using the internal pressure of the vial. All measurements were performed at 25°C to ensure consistent measurement conditions. Gas composition was analyzed using a GC-2014 gas chromatograph (Shimadzu Corporation) equipped with a TCD detector. Argon G3 was used as the carrier gas, with a Shincarbon-ST 50–80 column (2 m; Shinwa Chemical). The carrier gas flow rate, column oven temperature, and anal­ysis time were 50.0 mL‍ ‍min^–1, 150°C, and 3 min, respectively. A 1-mL headspace gas sample was analyzed. Methane and carbon dioxide concentrations were calculated from retention times and peak areas and converted to molar amounts. Total gas production and its time-dependent changes were analyzed using a two-way ANOVA with Tukey’s HSD test and a linear mixed-effects model (LMM), respectively.

At the end of the experiment (day 137), PHBH was removed from the samples, washed with deionized water, thoroughly dried, and weighed. Residual PHBH was recovered by sieving and visually identified. The practical detection limit was selected by the minimum particle size recoverable during the sieving process (45 μm). The degradation rate (%) was calculated as (W₀–W_t)/W₀×100, where W₀ and W_t represent PHBH weights before and after the experiment, respectively. PHBH surface morphology was exami­ned using a super depth-of-field microscope (VHX-6000/VHX-D510; KEYENCE).

Microbial community profiling was performed using samples in which PHBH degradation was pronounced (35°C, soil-water ratio 1:1). Soil samples were collected on days 69 and 137 of the experiment. One soil sample from the control treatment on day 137 (35°C, 1:1) was used as a reference. DNA extraction and an amplicon sequencing anal­ysis were performed by Biological Technology Research using the ISOFECAL for Beads Beating kit (NIPPON Genetics) and LabAid 824s DNA Extraction Kit (ZEESAN Biotech).

The V4 region of the bacterial 16S rRNA gene was amplified using the primers 515F/806R in a two-step PCR approach and sequenced on an Illumina MiSeq platform in the 300-bp paired-end mode. Sequence data were deposited in the DDBJ Sequence Read Archive under BioProject accession number PRJDB40671. Sequence data were processed in QIIME2 (version 2022.8). Noise and chimeric sequences were removed with DADA2, and amplicon sequence variants (ASVs) were inferred. Rarefaction was performed to normalize the sequencing depth to a minimum of 33,214 reads per sample for comparisons of microbial communities. A taxonomic classification was conducted using the Greengenes database (version 13_8), which was part of the service provider’s standard pipeline. Species-level identifications were additionally exami­ned using BLAST searches against the NCBI database for validation (Sayers et al., 2025), while primary ecological interpretations were based on broader taxonomic patterns at the genus level. Differentially abundant ASVs were identified with DESeq2, applying a false discovery rate (FDR) threshold of 0.05. Alpha diversity was calculated using the Shannon index in QIIME2 (version 2023.5.1) with the core-metrics-phylogenetic pipeline. Beta diversity was calculated using Bray–Curtis dissimilarity and weighted UniFrac distances, and visualized using a principal coordinate anal­ysis (PCoA).

Statistical anal­yses were performed in R (version 4.5.2). Differences between two groups were evaluated using the Wilcoxon rank-sum test, and multiple comparisons were analyzed by a one-way ANOVA followed by Tukey’s post hoc test. The significance of differences was defined as P<0.05.

Results

Total gas production on day 137 is shown in Fig. 1. In controls, gas production was negligible at all temperatures and soil-water ratios. In contrast, in the PHBH-amended treatments, temperature and the soil-water ratio both significantly affected gas production, and their interaction was also significant (two-way ANOVA, P<0.05). At 25°C and 35°C, gas production significantly decreased with reductions in the soil percentage (Tukey’s HSD, P<0.05). At 14°C, minimal gas production was observed in the soil-water ratio 1:1 group, but did not significantly differ from that in the other ratio groups. In the 1:1 and 1:10 groups, gas production was significantly higher at 25°C and 35°C than at 14°C (P<0.05), whereas no significant difference was observed between temperatures in the 1:100 and 1:1,000 groups.

Fig. 1. Cumulative gas production after 137 days in control and PHBH treatments. Error bars represent the standard deviation (n=3). Within each temperature, different letters indicate significant differences among ratios in PHBH treatments (Tukey’s HSD test, P<0.05).

Temporal changes in methane and carbon dioxide production in the PHBH-amended treatments are shown in Fig. 2 and 3. At 35°C, the production of both gases was significantly higher in the 1:1 group than in the 1:10 and 1:100 groups (P<0.05). At 25°C, the 1:1 group also showed significantly higher values than the 1:10 group (P<0.05). Almost no production of either gas was observed at 14°C. The LMM anal­ysis revealed that the soil-water ratio significantly affected methane and carbon dioxide production (P<0.001). Furthermore, the production of both gases increased over time (CH₄: P<0.05, CO₂: P<0.01), and the pattern of the increase differed depending on the soil-water ratio (P<0.05). Moreover, a significant interaction between temperature and the soil-water ratio was observed (P<0.001), indicating that the effect of temperature on gas production was dependent on the soil-water ratio.

Fig. 2. Time course of cumulative methane production in PHBH treatments at 35°C (A), 25°C (B), and 14°C (C). Error bars represent the standard deviation (n=3).

Fig. 3. Time course of cumulative carbon dioxide production in PHBH treatments at 35°C (A), 25°C (B), and 14°C (C). Error bars represent the standard deviation (n=3).

At the end of the experiment, PHBH was removed and the degradation rate was calculated (Table 1). PHBH degradation reached 100% in the 1:1 group at 35°C, with no visible residues, whereas the degradation rate markedly decreased with reductions in the soil percentage. At 25°C, degradation was generally limited, although 36.3% degradation was observed in the 1:1 group. At 14°C, 19.3% degradation was observed in the 1:1 group despite negligible gas production.

Table 1.Biodegradation rate of PHBH (%) after 137 days (mean±SD), with an initial PHBH mass of 1.02±0.02 g. “n.d.” (not detected) indicates values below the practical detection limit of the method.

Temperature (°C)	Soil-to-water ratio
	1:1	1:10	1:100	1:1,000
35	100.0±0.0	59.7±12.7	41.0±20.8	4.2±3.0
25	36.3±7.2	17.8±8.0	1.1±1.5	0.5±0.2
14	19.3±2.0	4.8±4.3	n.d.	n.d.

A two-way ANOVA indicated that temperature and the soil-water ratio both significantly affected the PHBH degradation rate (P<0.001), and a significant interaction between these two factors was also observed (P=0.003). Tukey’s multiple comparison test revealed that at 35°C, degradation rates in the 1:1 and 1:10 groups were significantly higher than in the 1:1,000 group (P<0.001), and the degradation rate in the 1:1 group was also significantly higher than that in the 1:100 group (P=0.0006). At 25°C, the degradation rate in the 1:1 group was significantly higher than those in the 1:100 and 1:1,000 groups (P<0.05). At 14°C, no significant differences were observed among the soil-water ratio groups. Furthermore, degradation rates in the 1:1 and 1:10 groups were significantly lower at 14°C than at 25°C and 35°C (P<0.001). Similarly, significant differences were observed in the 1:100 group (P<0.05), whereas no significant differences were found among temperatures in the 1:1,000 group. Before the experiment, the PHBH surface was smooth and flat, whereas a rough and irregular surface structure was observed after the incubation (Fig. S1). Surface roughness generally increased at higher degradation rates.

Amplicon sequencing was performed on soil samples (n=3) collected from the treatment showing the highest PHBH degradation rate (35°C, 1:1, PHBH-amended) on days 69 and 137. One soil sample from the control treatment on day 137 (35°C, 1:1) was used as a reference. A total of 264,762 reads were obtained after quality filtering, from which 3,809 ASVs were identified. The DESeq2 anal­ysis detected 47 ASVs showing significant differences (FDR<0.05). The relative abundance of 25 of these ASVs was high in samples collected on day 69. These ASVs belonged to the classes Deltaproteobacteria and Clostridia.

The relative abundance anal­ysis showed changes in community composition from days 69 to 137. At the phylum level (Fig. 4A), the PHBH-amended treatments were dominated by Euryarchaeota, Firmicutes, and Proteobacteria. The relative abundance of Euryarchaeota increased from days 69 to 137, whereas Firmicutes and Proteobacteria were relatively abundant on day 69. In the control, the percentage of archaea was negligible (0.91%), whereas the‍ ‍bacterial phyla Proteobacteria, Acidobacteria, and Actinobacteria were dominant.

Fig. 4. Soil microbiome changes in the 35°C, 1:1 PHBH-amended soil group, which showed the highest PHBH degradation rate, on days 69 and 137. A control soil sample collected on day 137 under the same conditions (35°C, 1:1) was used for comparison. (A) Phylum-level composition, (B) archaeal genera, and (C) bacterial genera.

A total of 617 ASVs (16.2%) were classified at the genus level (Fig. 4B and C). In the PHBH-amended treatments, hydrogenotrophic methanogens (Methanobacterium and Methanocella) and acetoclastic methanogens, such as Methanosarcina, were detected and remained dominant throughout the incubation period. Among bacteria, Rhodoplanes was prominent in the control, but occurred at lower levels in the PHBH-amended treatments. On the other hand, Clostridium (an obligate anaerobic Gram-positive rod-‍shaped bacterium), the fatty acid β-oxidizing bacterium Syntrophomonas, and the filamentous bacterium Anaerolinea were generally more abundant (Fig. 4C).

Overall, 430 ASVs (37 archaea and 393 bacteria) were identified at the species level with ≥97% similarity, representing 11.3% of the total. Fig. 5 shows a heatmap of sequences with ≥0.01% relative abundance in all PHBH-amended samples. In this figure, taxonomic groups are divided into several categories (No. 1, No. 2–7, No. 8–9, No. 10–18, and No. 19–47). The functional assignments of taxa were based on information available in public databases (e.g., NCBI) and representative studies describing the metabolic characteristics of key taxa (McInerney et al., 1981; Dörner and Schink, 1990; Janssen and Harfoot, 1990; Sousa et al., 2007). For example, No. 1 included I. delafieldii, a PHB-degrading bacterium (Janssen and Harfoot, 1990), while No. 10–18 included methanogenic archaea. Among these, the species consistently detected in all PHBH-amended samples, but not in the single control sample were Clostridium homopropionicum, Syntrophomonas zehnderi, I. delafieldii, Syntrophomonas cellicola, Magnetospirillum gryphiswaldense, Flavisolibacter ginsengisoli, Azonexus caeni, and Clostridium magnum, as well as seven methanogenic archaeal species. Furthermore, Desulfitobacterium metallireducens, Geobacter metallireducens, and Geomonas terrae were more abundant in the PHBH-amended treatments than in the single control sample.

Fig. 5. Heatmap of sequences with ≥0.01% relative abundance in all PHBH-amended soil samples. A control soil sample collected on day 137 under the same conditions (35°C, 1:1) was used for comparison. Relative abundance is indicated by a color gradient from green (low) to red (high).

Alpha diversity was consistently lower on day 137 than on day 69 for all samples (the Wilcoxon rank-sum test, P=0.1). Beta diversity showed relatively large effect sizes (R²=0.38–0.56) for both Bray-Curtis and weighted UniFrac distances, despite the lack of significance (P=0.1), which may have been due to the limited sample size (n=3 per group). One sample showed clear separation in the PCoA plot (Fig. S2), which may have contributed to the observed effect sizes.

Discussion

The present results showed that the degradation of PHBH and associated gas production were strongly affected by temperature and the soil-water ratio. In the 1:1 treatment simulating flooded paddy fields, gas production indicated that the majority of PHBH was degraded within approximately 100 days at 35°C. In contrast, at 25°C, only 36.3% of PHBH had degraded after 137 days, indicating strong temperature dependence. This result is consistent with previous findings showing that the degradation of PHB-based polymers was strongly affected by temperature (Wang et al., 2004; Cai and Qiu, 2009). Lotto et al. (2004) demonstrated that PHB and PHBV completely degraded within 104 days at 46°C in compost, whereas only about 50% degradation occurred after 321 days at 24°C. Laszakovits et al. (2026) found that the biodegradation rate of PHBH in three types of agricultural soils increased with temperature over a range of 5–35°C. Similarly, Gil-Castell et al. (2020) reported that only 0.5% of PHBH (0.5 wt%) degraded in 1,000 g of compost after 90 days at 25°C. These studies, all conducted under aerobic conditions, suggest that the degradation of PHB-based polymers is strongly dependent on temperature. However, in flooded environments, such as paddy soils, oxygen is rapidly depleted, leading to anaerobic conditions (Liesack et al., 2000). Under these conditions, the decomposition of organic matter proceeds via anaerobic microbial processes, producing methane and carbon dioxide. The present results indicate that the addition of PHBH was associated with increased gas production under anaerobic conditions; however, the carbon source of the gases was not identified. The experiment was terminated at 137 days, and degradation and gas production may have continued beyond this period.

In the low soil-content treatments, the degradation rate significantly decreased. PHBH remained exposed above the soil surface in these treatments, limiting contact with microorganisms and their secreted degradative enzymes. In addition, microbial abundance and nutrient availability may be lower at reduced soil volumes. The anaerobic decomposition of organic matter generally proceeds through a metabolic cascade involving hydrolysis, fermentation, syntrophic fatty acid oxidation, and methanogenesis (Schink, 1997; Stams and Plugge, 2009). Hydrolytic bacteria, fatty acid-oxidizing bacteria, and methanogenic bacteria were detected in the PHBH-amended treatment (Fig. 5). These results are consistent with the potential involvement of a metabolic network. Therefore, sufficient soil contact plays a critical role in promoting PHBH degradation in paddy field environments. Furthermore, the soil-water ratio showed a significant interaction with temperature: even at 35°C, degradation may be delayed if PHBH is exposed in water or on the water surface, which has implications for long-term environmental persistence.

Gas production and residual PHBH levels were measured to evaluate degradation and mineralization. In the 1:1 soil-water treatment at 35°C, methane and carbon dioxide production levels were higher than in the other treatments, and PHBH had nearly disappeared. This result indicates that gas production was closely associated with PHBH degradation, with degradation to low-molecular-weight compounds and subsequent mineralization likely proceeding in tandem. The plateau observed at 35°C may also have been affected by limited nutrient availability in the system. Conversely, although a certain degree of degradation (19.3%) was observed at 14°C, gas production was negligible for most of the incubation period and only slightly increased toward the end in the 1:1 treatment. Therefore, degradation and gas production may not have been directly coupled under low-‍temperature conditions. Methanogenic activity is generally highly dependent on temperature and decreases at low temperatures (Conrad, 2020). In addition, the limited activity of syntrophic microorganisms may further constrain downstream processes, leading to the accumulation of intermediate metabolites, such as volatile fatty acids, under low-temperature conditions. The ester bonds in biodegradable polyesters, such as PHBH, are known to be susceptible to hydrolytic cleavage, even in the absence of enzymatic activity; however, these reactions proceed slowly under low temperatures and neutral conditions. The pH of the soil used in the present study was 6.1, which is nearly neutral. Therefore, PHBH degradation may still occur even at low temperatures, albeit at a slower rate. Collectively, the present results suggest that temperature-limited microbial processes and abiotic hydrolysis both contribute to PHBH degradation under low-temperature conditions. However, their relative contributions were not assessed in the present study because abiotic controls were not included. Further investigations are needed, for example, by quantifying intermediate metabolites and using stable isotope-labeled PHBH to trace carbon flow during degradation. Notably, gas production in the control treatments was similarly negligible across all conditions. This result may reflect the removal of visible plant-derived organic materials, such as roots, husks, and straw, prior to the experiment, which reduced the availability of readily degradable organic substrates.

Amplicon sequencing revealed that the genera Clostridium, Syntrophomonas, and Anaerolinea were more abundant in the PHBH treatment than in the reference control sample. At the species level, I. delafieldii, C. homopropionicum, and S. zehnderi were relatively abundant in the PHBH treatment. Given the limited sample size (n=3), the DESeq2-based results on differential abundance need to be interpreted with caution due to limited statistical power and a potential risk of false positives. Although several taxa identified in the present study harbor genes encoding proteins annotated as PHB depolymerase-related esterases in the NCBI protein database, only a limited number of these taxa have been experimentally confirmed to‍ ‍degrade PHB. Among them, I. delafieldii is an obligate‍ ‍anaerobe that has been reported to secrete PHB-degrading enzymes extracellularly and to utilize PHB as a fermentation substrate (Janssen and Harfoot, 1990). C. homopropionicum ferments 3-hydroxybutyrate (3HB) and other compounds to produce acetate and butyrate (Dörner and Schink, 1990). S. zehnderi is a metabolically specialized symbiont capable of degrading long-chain fatty acids and is involved in syntrophic metabolism in association with methanogenic archaea (McInerney et al., 1981; Sousa et al., 2007). Although correlation-based anal­yses were not feasible due to the limited sample size and the focus on a single treatment condition (35°C, 1:1), the observed co-occurrence of functionally relevant taxa under conditions showing active PHBH degradation is consistent with a proposed anaerobic degradation pathway. In this conceptual pathway, PHBH is initially depolymerized into monomers or oligomers, followed by fermentation to short-chain fatty acids, syntrophic oxidation, and ultimately methanogenesis. Bacteria possessing PHB depolymerases, such as I. delafieldii, may contribute to the initial depolymerization of PHBH, releasing oligomers or monomers, such as 3HB and 3HH. Members of the genera Clostridium and Anaerolinea may subsequently ferment these intermediates to short-chain fatty acids, including acetate and butyrate. Subsequently, the genus Syntrophomonas, including S. zehnderi, may participate in the syntrophic oxidation of butyrate and related fatty acids in cooperation with methanogenic archaea, thereby producing acetate and hydrogen, which may support acetoclastic and hydrogenotrophic methanogenesis, respectively (Thauer et al., 2008). This interpretation is consistent with the co-occurrence of Methanosarcina, which includes acetoclastic methanogens, and hydrogenotrophic methanogens, such as Methanobacterium and Methanocella, in Fig. 4B. However, these interpretations are based on indirect evidence, and direct causal relationships have yet to be clarified.

Differences in the community composition between days 69 and 137 suggest temporal shifts in microbial assemblages during PHBH degradation. Although a clear stage-wise transition cannot be conclusively confirmed from the present data, the observed changes are broadly consistent with changes in gas production and the metabolic potential of the detected taxa, suggesting a link to degradation processes. Further studies on functional genes and enzyme activities are needed to clarify whether the taxa detected directly contribute to PHBH degradation or respond to intermediate degradation products. Future research that incorporates more comprehensive sampling across multiple time points and environmental conditions will enable a more robust evaluation of the relationships between microbial community composition, gas production, and PHBH degradation dynamics.

In conclusion, the present study identified soil-plastic contact and temperature as critical factors governing the degradation of biodegradable plastics under flooded soil conditions. The degradation process was accompanied by methane production, which may be associated with shifts in the soil microbial community and the possible formation of an anaerobic metabolic network involving PHBH-derived intermediates. These results indicate the potential of PHBH as an organic carbon source by microbial communities under flooded soil conditions, contributing to anaerobic carbon cycling. However, the observed degradation cannot be attributed solely to microbial activity because abiotic hydrolysis may also contribute, particularly under lower-temperature conditions. Similar pathways may also occur in other biodegradable plastics, highlighting the need to evaluate appropriate application levels. Since this study was based on laboratory experiments, long-term field studies are required to assess the environmental impact in paddy field systems. In actual paddy field environments, redox conditions fluctuate due to water management practices, such as drainage and intermittent drying (e.g., mid-season drainage). These shifts between oxic and anoxic conditions may affect microbial degradation pathways, potentially promoting aerobic degradation processes during oxic phases while suppressing methanogenesis and favoring anaerobic mineralization under flooded conditions.

Acknowledgements

This work was supported by JSPS KAKENHI grant number JP25K15470. The authors thank Kaneka Corporation for kindly providing the PHBH used in this study. The authors used AI-assisted tools for English language editing and improved readability. All scientific content and interpretations were reviewed and verified by the authors.

Citation

Satone, H., Watanabe, T., Abe, K., and Nagashima, Y. (2026) Anaerobic Biodegradation of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) under Flooded Paddy Soil Conditions. Microbes Environ 41: ME26036.

https://doi.org/10.1264/jsme2.ME26036

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