In the present study, we determined the potential of using BP degrading- microorganisms for accelerating the degradation of used BP mulch films in agricultural fields. Several studies concerning fungi that possess the ability to degrade plastics have already been reported. A Fusarium solani
f. sp. pisi
, a plant pathogenic fungus degraded BP of poly (ϵ-caprolactone) (PCL) emulsion (Murphy et al. [1996
]). Heterologously expressed cutinases of plant pathogenic fungi (F. solani
f. sp. pisi, Alternaria brassicicolaAspergillus fumigates
and Humicola insolens
) degraded PCL film (Baker et al. [2012
]). Aspergillus oryzae
, a fungus widely used in traditional Japanese fermentation industries, degraded PBS and PBSA mulch film (Maeda et al. [2005
]). To address safety concerns regarding the use of microorganisms in the agricultural fields, in this study, we performed our microbial isolation from gramineous crop plants. Our results showed that 4.5% of the isolated fungal strains degraded PBSA emulsion. We have previously reported in a related study that 2% to 100% of yeast populations isolated from seed rice husks degraded PBSA emulsion (Kitamoto et al. [2011
]). These findings thus demonstrate that phylloplane is an important source of BP-degrading eukaryotic microorganisms. Further analysis and identification of the evaluated fungal strains could be expected to give us detailed information about the diversity and activities of BP-degrading phylloplane-derived fungi.
Among the isolated strains, B47-9 was found to have the strongest ability to degrade PBSA and PBS films on agarose medium, and was the only strain that resulted to a detectable PBSA emulsion-degrading activity in liquid culture. Strain B47-9 is identified as an imperfect fungi belonging to Ascomycota, and is closely related to Phoma. To the best of our knowledge, this is the first report on a Phoma-related fungus which possesses a BP-degrading ability.
Strain B47-9 degraded almost all the commercially available polymer-blended BP mulch film mounted on indoor-sterilized soil environment (Figure
b’). Our analysis of the commercially available mulch film used in this study revealed that it is composed of PBS, PBSA, and PBAT at a weight-based ratio of 47:37:17. This observation showed that strain B47-9 can degrade various BP components. On the unsterilized soil, the lower degradability of the polymer-blended commercially available mulch film (Figure
c’) compared with PBSA (Figure
d) and PBS (Figure
e) films indicated that PBAT is degraded more slowly than PBSA and PBS in natural soil environment.
We observed how the strain grew on the film surface and degraded the film on sterilized soil. Vigorous growth of gray-colored mycelia of B47-9 was observed on the surface of polymer blended film (Figure
b). Under the SEM, we observed the strain grew on the surface of film, producing breaks and holes on the film along the direction of its hyphal growth (Figure
b, b’). These observations support our assumption that B47-9 may have secreted an enzyme capable of directly degrading the film. As indicated by the arrows in figure
b’, it was clear that the degradation event proceeded along one direction of the film. The same degradation characteristics was also observed under SEM in poly [(R
)-3-hydroxyputyrate] (p(3HB)) film after partial degradation by PHB depolymerase (Iwata [2005
]). It is well known that the amorphous region is etched faster than the crystal one (Vert [2005
]). BP-degrading enzyme of strain B47-9 may have also degraded the polymer blended mulch film in a similar way.
On the surface of the film mounted on unsterilized soil, networks of mycelia and conidia of soil-derived Penicillium
sp. were observed (Figure
c). A magnified part of the same film is shown in Figure
c’, where the arrow (x
) indicates the film-degrading hyphae, which we presumed to be strain B47-9, along with other hyphae that did not degrade the film, designated by arrow (y
), which were believed to be soil-derived microorganisms. These observations support our presumption regarding the existence of microbial competition between strain B47-9 and native soil fungi.
To further confirm the contribution of inoculated strain B47-9 to the film degradation on unsterilized soil, the transition in the fungal diversity of unsterilized soil as well as the growth of B47-9 were analyzed by PCR-DGGE. The populations of these filamentous fungal species (Aspergillus
sp.) and Acanthamoeba
sp., which commonly inhabit the soil (Rosenberg et al. [2009
]), were found to increase in the presence of PBSA film.
In contrast, in the PBSA film-covered soil sample treated with B47-9 culture filtrate, the following bands were detected after 1
week incubation: d
(soil-delived fungi), f
(B47-9) and j
). These results corresponded to our SEM observations of the degraded film surface as described earlier. The bands for soil-derived fungi disappeared after 2
weeks incubation, and this was construed as due to the degradative activity of native soil fungi. Those of B47-9 (f
) and Acanthamoeba
), however, were still detected, which in fact, appeared as dominant bands. These results indicated that strain B47-9 could remain dominant in the soil only when PBSA film was mounted on it. Furthermore, increments of soil-derived Acanthamoeba
sp. strains were observed only when the soil was covered with PBSA film. Acanthamoeba
is found in a variety of soil, and has been reported to assist in rapidly changing the composition of bacterial community in the soil (Rosenberg et al. [2009
]) by increasing the digestible nutrients which support the growth of soil microorganisms, and transporting and dispersing microorganisms into new environmental niches (Schuster [2002
]). Furthermore, Acanthamoeba
spp. strains possess degradative activities for soil microorganisms, such as yeasts and fungi (Steenbergen et al. [2001
]). There is also a report regarding a strain of Acanthamoeba castellani
which is known to have depolymerizing activity for polyhydrozybutyarate (PHB) (Anderson et al. [2005
]), a bacterial storage polyester. It is, therefore, possible that Acanthamoeba
spp. strains observed in this study prefer to grow around PBSA film, and any of them may have contributed to the changes in microbial community composition.
In this study, we isolated and confirmed that a fungal strain B47-9 has directly contributed to accelerating the degradation of soil-mounted film. Since this strain B47-9 was isolated from healthy leaf of barley, it may be assumed that it can be safely utilized for acceleration of degradation of BP mulch film after use. The establishment of an economical and environment-friendly technique for BP degradation using fungi, such as strain B47-9, could be expected to provide a viable solution to the plastic disposal problem in agriculture in the future.