Dual Application of p-Nitrophenol Alkanoate-Based Assay for Soil Selection and Screening of Microbial Strains for Bioplastic Degradation

Abstract

With an increase in the commercialization of bioplastics, the importance of screening for plastic-degrading strains and microbes has emerged. Conventional methods for screening such strains are time-consuming and labor-intensive. Therefore, we suggest a method for quickly and effectively screening plastic-degrading microbial strains through dual esterase assays for soil and isolated strains, using p-nitrophenyl alkanoates as substrates. To select microbe-abundant soil, the total amount of phospholipid fatty acids (PLFAs) included in each soil sample was analyzed, and esterase assays were performed for each soil sample to compare the esterase activity of each soil. In addition, by analyzing the correlation coefficients and sensitivity between the amount of PLFAs and the degree of esterase activity according to the substrate, it was confirmed that substrate pNP-C2 is the most useful index for soil containing several microbes having esterase activity. In addition, esterase assays of the isolated strains allowed us to select the most active strain as the degrading strain, and 16S rRNA results confirmed that it was Bacillus sp. N04 showed the highest degradation activity for polybutylene succinate (PBS) as measured in liquid culture for 7 days, with a degradation yield of 99%. Furthermore, Bacillus sp. N04 showed degradation activity against various bioplastics. We propose the dual application of p-nitrophenyl alkanoates as an efficient method to first select the appropriate soil and then to screen for plastic-degrading strains in it, and conclude that pNP-C2 in particular, is a useful indicator.

Keywords: Soil; esterase assay; phospholipid fatty acid; polybutylene succinate; screening method.

Fig. 1. Schemes for soil esterase activity dependent screening process.

(A) Existing methods for screening plasticdegrading strains. (B) New method of screening plastic-degrading strains proposed in this study.

Fig. 2. Comparison of PBS degradation yields with each soil sample.

Degradation yield after PBS degradation in each soil for 14 days at 42°C was confirmed.

Fig. 3. Relative abundances of bacteria, gram-positive bacteria, gram-negative bacteria, fungi, and actinomycete in six soils as determined using phospholipid fatty acid (PLFA) analysis.

The six soil samples subjected to PLFA analysis were (A) Soil A, (B) Soil B, (C) Soil C, (D) Soil D, (E) Soil E, and (F) Soil F.

Fig. 4. Esterase activity assay for six soil samples.

(A) Comparing esterase activity of six soils using each supernatant. (B) Comparison of color changes to yellow after 30 min of reaction.

Fig. 5. Correlation analysis for soil esterase activity and PLFAs.

Regression analysis of the total amount of PLFA in each soil and the absorbance value at 405 nm for a p-nitrophenyl ester substrate containing (A) pNP-C2, (B) pNP-C4, (C) pNPC6, (D) pNP-C8, (E) pNP-C10, and (F) pNP-C12.

Fig. 6. Comparison of esterase activity for strains isolated from Soil A.

Esterase activity analysis of strains isolated from Soil A was conducted using p-nitrophenyl ester substrate containing (A) pNP-C2, (B) pNP-C4, (C) pNP-C6, (D) pNP-C8, (E) pNP-C10, and (F) pNP-C12. (G) Phylogenetic tree of Bacillus sp. N04 according to 16S rRNA sequencing.

Fig. 7. Confirmation of PBS degradation by Bacillus sp. N04 over the incubation period.

(A) Clear zone test for 14 days on solid medium containing PBS plastic emulsion in TSB. (B) GC-MS results for degradation yield of PBS according to degradation time (days 0, 3, 5, and 7). (C) The visual appearance of PBS films undergoes alterations as a result of degradation. Changes in the degradation of PBS films are observed at different incubation periods (0, 3, 5, and 7 days). Representative images of surface changes observed by (D) scanning electron microscopy (SEM).

Fig. 8. Ability of Bacillus sp. N04 to degrade various plastics.

The degradation potential of Bacillus sp. N04 for several plastics containing (A) PCL, (B) PBAT, (C) PLA, and (D) PHB was confirmed for 7 days at 42°C in TSB liquid medium.