Biodegradation of PBSA Films by Elite Aspergillus Isolates and Farmland Soil

Abstract

Plastic films are widely used in current agricultural practices; however, most mulch films used are discarded and buried in the land after harvest, having adverse environmental impacts. To solve this environmental problem, the demand for biodegradable mulch has been increasing in recent years. Polybutylene succinate-co-adipate (PBSA) is a biodegradable polymer with good ductility and can be used for packaging and mulching. In this study, we isolated two elite fungal strains for PBSA degradation from farmlands, i.e., Aspergillus fumigatus L30 and Aspergillus terreus HC, and the latter showed better degradation ability than the former. It is noteworthy that biodegradation of PBSA by A. terreus is reported for the first time, which revealed unique characteristics. In the soil burial test, even the soil with relatively poor degradation ability could be improved by the addition of elite fungal mycelia. In substrate specificity analyses of soil samples, PBSA could induce the synthesis of lipolytic enzymes of indigenous microbes to degrade substrates with medium and long carbon chains in soil. Furthermore, PBSA residues or fungal mycelia supplementation in soils had no adverse effect on the seed germination rate, seedling growth, or mature plant weight of the test green leafy vegetable. Taken together, the results of this study not only advance our understanding of the biodegradation of PBSA films by filamentous fungi but also provide insight into improving the efficiency of biodegradation in soil environments.

Keywords: Aspergillus; biodegradation; ecotoxicity; lipolytic enzyme; phytotoxicity; polybutylene succinate-co-adipate (PBSA).

Figure 1

Phylogenetic tree of ITS and clear zone formation on PBSA agar plates. (A) A phylogenetic tree of two selected fungal strains (Aspergillus terreus HC and Aspergillus fumigatus L30) was constructed from a comparison of sequences of approximately 400 bp of the internal transcribed spacer (ITS) region using neighbor-joining analysis of a distance matrix with Kimura’s two-parameter model. Bootstrap values (expressed as percentages of 1000 replications) greater than 75% are shown at branch points. A reference fungal strain, Aspergillus oryzae RIB40 (ATCC42149), was used as a control in this study, and Fusarium nisikadoi was used as an outgroup. The scale bar represents 0.05 substitutions per nucleotide position. (B) Morphology of the elite PBSA-degrading fungal strain.

Figure 2

Colonization of fungal strains on PBSA plastic films and their degradation abilities. (A) Biodegradation PBSA films after 30 days of incubation. Twelve pieces of PBSA films (size: 2.5 × 5.0 cm²; thickness: 50 μm) were cocultured with A. terreus HC, A. fumigatus L30, and A. oryzae RIB40 (ATCC42149) at 30 °C in a carbon-free basal medium. The upper panel shows the attached hyphae of individual strains on the surface of PBSA plastic films after 30 days of incubation. The bottom panel shows the surface of plastic films after hyphae removal by rinsing with DDW. (B) Weight loss of PBSA plastic film against time. The sampling times were 0, 14, 30 days after incubation. The values are expressed as the mean ± standard deviation of three biological replicates (p < 0.05; Tukey’s post hoc ANOVA test).

Figure 3

Determination of the effect of replacement or addition of fresh medium on PBSA plastic film degradation after 60 days of incubation. (A) Weight loss of PBSA film against time with A. fumigatus strain L30. (B) Weight loss of PBSA film against time with A. terreus HC. w/o medium change: maintaining the original medium during the whole incubation period (60 days); replacement of all culture medium (100 mL) or addition of 30 mL of fresh medium were carried out on the 30th day of incubation. The value of individual treatment is expressed as the row mean ± standard deviation. Statistical analyses were based on the degradation rates (g/day) on the 30th (in uppercase letters A and B) and 60th (in lowercase letters a and b) days of incubation.

Figure 4

Scanning electron microscopic images of plastic films degraded by two elite fungal strains. PBSA films degraded by A. terreus fumigatus L30 (A) and HC (B) after 30 days of incubation. (Aa) The attachment and network of A. terreus L30 hyphae on the surface of PBSA film, (Ab) cracks and holes on the plastic surface, (Ac) irregular cavity in internal of plastic film. (Ba) A. fumigatus HC hyphae intertwined inside the plastic film and formed holes on the plastic surface, (Bb) cracks and holes on the plastic surface, and (Bc) internal erosion of the plastic film.

Figure 5

NMR spectra of PBSA films after degradation by A. terreus HC. (A) PBSA film without degradation. (B) PBSA film degraded by A. terreus HC for 30 days. Compared with the original PBSA film, the PBSA film, after 30 days of biodegradation, showed a lower number average molecular weight (Mn) but a similar monomer composition ratio. Peaks 1–5 correspond to the main signals of protons shown in (A,B). Peak 1 represents succinic acid (SA), peaks 2 and 3 represent adipic acid (AA), and peaks 4 and 5 represent 1,4-butanediol (BDO).

Figure 6

Soil burial test for biodegradation of PBSA films. (A) Schematic diagram of the device for the soil burial test. (B) A piece of degraded PBSA film before analysis. (C) Biodegradation of PBSA films buried in summer soil. (D) Biodegradation of PBSA films buried in winter soil. PBSA plastic films were buried in NTU farmland soil that was collected from late summer (September) or winter (December) in 2020. Low (10 mL of mycelial suspension) or high (50 mL of mycelial suspension) doses of A. terreus HC fungal mycelia were inoculated into the soil. The values for the respective weights of PBSA film are the mean ± standard deviation of triplicate samples (p < 0.05; Tukey’s post hoc ANOVA test).

Figure 7

Lipolytic enzyme activities in the A. terreus HC culture broth and soil. (A) The lipolytic enzyme activities of A. terreus HC in the culture broth were determined by chromogenic nitrophenyl esters with different chain lengths as substrates (i.e., p-nitrophenyl esters). The supernatant of the A. terreus HC culture was collected from the culture fluid incubated with PBSA film and A. terreus HC for 60 days. (B) The lipolytic enzyme activities of soil. (C) The lipolytic enzyme activities of soil supply with A. terreus HC. (D) The lipolytic enzyme activities of soil buried with PBSA. (E) The lipolytic enzyme activities of PBSA buried soil supply with A. terreus HC. The assay was conducted after 30 days of PBSA degradation in soil. The substrates used were as follows: C2, 4-nitrophenyl acetate; C4, 4-nitrophenyl butyrate; C8, 4-nitrophenyl caprylate; C10, 4-nitrophenyl decanoate; C12, 4-nitrophenyl dodecanoate; and C16, 4-nitrophenyl palmitate. The results are presented as the mean ± standard deviation (p < 0.05; Tukey’s post hoc ANOVA test).

Figure 8

Phytotoxicity assessment of PBSA plastic film residues or A. terreus HC on plant growth. Seeds of Chinese cabbage were cultivated in soil containing 30-day degraded PBSA film residues to determine the seedling vigor for assessing phytotoxicity. (A) The biomass of Chinese cabbage seedlings was assessed after 8 days of germination. (B) The phenotype of Chinese cabbage at 21 days of cultivation. (C,D) Fresh and dry weights of shoots after 21 days of cultivation in soil containing PBSA residues. Ns: no significant difference among the treatments. The results are presented as the mean ± standard deviation (p < 0.05; Tukey’s post hoc ANOVA test).