Enhanced biodegradable polyester film degradation in soil by sequential cooperation of yeast-derived esterase and microbial community.

The degradation of biodegradable plastics poses a significant environmental challenge and requires effective solutions. In this study, an esterase derived from a phyllosphere yeast Pseudozyma antarctica (PaE) enhanced the degradation and mineralization of poly(butylene succinate-co-adipate) (PBSA) film in soil. PaE was found to substitute for esterases from initial degraders and activate sequential esterase production from soil microbes. The PBSA film pretreated with PaE (PBSA-E) rapidly diminished and was mineralized in soil until day 55 with high CO2 production. Soil with PBSA-E maintained higher esterase activities with enhancement of microbial abundance, whereas soil with inactivated PaE-treated PBSA film (PBSA-inact E) showed gradual degradation and time-lagged esterase activity increases. The fungal genera Arthrobotrys and Tetracladium, as possible contributors to PBSA-film degradation, increased in abundance in soil with PBSA-inact E but were less abundant in soil with PBSA-E. The dominance of the fungal genus Fusarium and the bacterial genera Arthrobacter and Azotobacter in soil with PBSA-E further supported PBSA degradation. Our study highlights the potential of PaE in addressing concerns associated with biodegradable plastic persistence in agricultural and environmental contexts.

Complete Genome Sequence of Caballeronia sp. Strain NK8 (MAFF311271), a Chlorobenzoate-Degrading Bacterium.

Caballeronia sp. strain NK8 grows on 3-chlorobenzoate and shows chemotaxis toward 3-chlorobenzoate and its degradation products, such as chlorocatechols. Complete genome sequencing revealed a 9.2-Mb genome consisting of three chromosomes and four plasmids. The genes for degradation of 3-chlorobenzoate and chlorocatechols were located on plasmids pNK81 and pNK84, respectively.

Fungal community dynamics during degradation of poly(butylene succinate-co-adipate) film in two cultivated soils in Japan.

Fungi play an important role in the degradation of biodegradable plastics (BPs) in soil. However, little is known about their dynamics in the soil during the degradation of BPs. We studied the community dynamics of BP-degrading fungi during poly(butylene succinate-co-adipate) (PBSA) film degradation in two different types of soils using culture-dependent and culture-independent methods. The Fluvisol and the Andosol soils degrade embedded PBSA films at high and low speeds, respectively. The number of PBSA emulsion-degrading fungi that increased in the Fluvisol soil was higher than that in the Andosol soil after embedding with PBSA films. We succeeded in detecting internal transcribed spacer 1 (ITS1) regions those matched that of the fungi by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in both soils. Our results suggest that fungal community analyses using PCR-DGGE in combination with BP degraders isolation techniques enables the monitoring of BP films-degrading fungi.

High-throughput method for the evaluation of esterase activity in soils.

We describe a method to quickly evaluate soil esterase activity using p­nitrophenyl valerate as the substrate. Unwanted coloration of the control samples was suppressed by cooling. Esterase activity can be evaluated using arbitrary amounts of soil. Sample dispensation was simplified and the number of examinations per soil sample reduced.

Enzymatic degradation of poly-butylene succinate-co-adipate film in rice husks by yeast Pseudozyma antarctica in indoor conditions.

Agricultural mulch films made from biodegradable polymers (BP) have been used to decrease the burden of plastic waste recovery and recycling. However, their degradations depend largely on environmental conditions and sometimes do not proceed as desired. Yeast strains of Pseudozyma antarctica often isolated from rice husks were found to secrete an esterase to degrade BP films. Poly-butylene succinate-co-adipate (PBSA) films buried in unsterilized rice husks with 60% (w/w) moisture degraded rapidly compared to that buried in field soil. The type strain of P. antarctica JCM 10317 added as cell suspension onto sterilized rice husks with PBSA film grew rapidly forming filamentous growth on the surface of rice husks and films. BP-degrading enzyme secreted by the growing cells was adsorbed on the surface of film and decomposed the film. Addition of rice husk-derived P. antarctica strains also showed BP film degradation activity in sterilized rice husks. In the light of these findings, we suggest that techniques for disposal of used BPs which combine plastics with unutilized residual plant materials piled at the side of agricultural fields be developed.

Enhancement of Biodegradable Plastic-degrading Enzyme Production from Paraphoma-like Fungus, Strain B47-9.

To improve the productivity of Paraphoma-like fungal strain B47-9 for biodegradable plastic (BP)-degrading enzyme (PCLE), the optimal concentration of emulsified poly(butylene succinate-co-adipate) (PBSA) in the medium was determined. Emulsified PBSA was consumed as a sole carbon source and an inducer of PCLE production by strain B47-9. Among the various concentrations of emulsified PBSA [0.09-0.9% (w/v)] used in flask cultivation, 0.27% yielded the maximum enzyme activity within a short cultivation period. To evaluate the residual concentration of emulsified PBSA in culture, emulsified PBSA in aliquots of culture supernatant was digested in vitro, and the concentration of released monomerised succinic acid was determined. Regardless of the initial concentration of emulsified PBSA in medium, PCLE activity was detected after residual succinic acid decreased below 0.04 mg/mL in culture broth. Jarfermentation was performed at a 0.27% PBSA concentration. Among the various airflow rates tested, 1 LPM resulted in a PCLE production rate of 1.0 U/mL/day. The enzyme activity in the resulting culture filtrate (4.2 U/2 mL) was shown to degrade commercial BP films (1 × 1 cm, 20 µm thickness) within 8 hours.

Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils.

The relationship between degradation speed of soil-buried biodegradable polyester film in a farmland and the characteristics of the predominant polyester-degrading soil microorganisms and enzymes were investigated to determine the BP-degrading ability of cultivated soils through characterization of the basal microbial activities and their transition in soils during BP film degradation. Degradation of poly(butylene succinate-co-adipate) (PBSA) film was evaluated in soil samples from different cultivated fields in Japan for 4 weeks. Both the degradation speed of the PBSA film and the esterase activity were found to be correlated with the ratio of colonies that produced clear zone on fungal minimum medium-agarose plate with emulsified PBSA to the total number colonies counted. Time-dependent change in viable counts of the PBSA-degrading fungi and esterase activities were monitored in soils where buried films showed the most and the least degree of degradation. During the degradation of PBSA film, the viable counts of the PBSA-degrading fungi and the esterase activities in soils, which adhered to the PBSA film, increased with time. The soil, where the film was degraded the fastest, recorded large PBSA-degrading fungal population and showed high esterase activity compared with the other soil samples throughout the incubation period. Meanwhile, esterase activity and viable counts of PBSA-degrading fungi were found to be stable in soils without PBSA film. These results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is. This could be due to the rapid accumulation of secreted esterases in these soils.

A putative porin gene of Burkholderia sp. NK8 involved in chemotaxis toward ß-ketoadipate.

Burkholderia sp. NK8 can utilize 3-chlorobenzoate (3CB) as a sole source of carbon because it has a megaplasmid (pNK8) that carries the gene cluster (tfdT-CDEF) encoding chlorocatechol-degrading enzymes. The expression of tfdT-CDEF is induced by 3CB. In this study, we found that NK8 cells were attracted to 3CB and its degradation products, 3- and 4-chlorocatechol, and ß-ketoadipate. Capillary assays revealed that a pNK8-eliminated strain (NK82) was defective in chemotaxis toward ß-ketoadipate. The introduction of a plasmid carrying a putative outer membrane porin gene, which we name ompNK8, into strain NK82 restored chemotaxis toward ß-ketoadipate. RT-PCR analyses demonstrated that the transcription of the ompNK8 gene was enhanced in the presence of 3CB.