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. 2021 Dec 23:12:778828.
doi: 10.3389/fmicb.2021.778828. eCollection 2021.

Evaluation of PET Degradation Using Artificial Microbial Consortia

Xinhua Qi  1   2 , Yuan Ma  1   2 , Hanchen Chang  1   2 , Bingzhi Li  1   2 , Mingzhu Ding  1   2 , Yingjin Yuan  1   2
Affiliations

Evaluation of PET Degradation Using Artificial Microbial Consortia

Xinhua Qi et al. Front Microbiol. .

Abstract

Polyethylene terephthalate (PET) biodegradation is regarded as an environmentally friendly degradation method. In this study, an artificial microbial consortium composed of Rhodococcus jostii, Pseudomonas putida and two metabolically engineered Bacillus subtilis was constructed to degrade PET. First, a two-species microbial consortium was constructed with two engineered B. subtilis that could secrete PET hydrolase (PETase) and monohydroxyethyl terephthalate hydrolase (MHETase), respectively; it could degrade 13.6% (weight loss) of the PET film within 7 days. A three-species microbial consortium was further obtained by adding R. jostii to reduce the inhibition caused by terephthalic acid (TPA), a breakdown product of PET. The weight of PET film was reduced by 31.2% within 3 days, achieving about 17.6% improvement compared with the two-species microbial consortium. Finally, P. putida was introduced to reduce the inhibition caused by ethylene glycol (EG), another breakdown product of PET, obtaining a four-species microbial consortium. With the four-species consortium, the weight loss of PET film reached 23.2% under ambient temperature. This study constructed and evaluated the artificial microbial consortia in PET degradation, which demonstrated the great potential of artificial microbial consortia in the utilization of complex substrates, providing new insights for biodegradation of complex polymers.

Keywords: Polyethylene terephthalate; artificial microbial consortia; biodegradation; ethylene glycol; terephthalic acid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Construction of a two-species microbial consortium for BHET degradation. (A) Fusion of the PETase gene to different signal peptides resulted in different levels of PETase secretion, and therefore different levels of the product pNP. The species in the control group were wild-type B. subtilis. (B) Comparison of the effects of PETase and MHETase on BHET. All experiments were performed at least in triplicate. Error bars indicate standard deviation. *p < 0.05, **p < 0.01 (one-sided t-test).
FIGURE 2
FIGURE 2
Schematic diagram of PET degradation by synthetic microbial consortia. (A) Bs_PETase (B1) and Bs_MHETase (B2) were engineered to secrete PETase and MHETase to degrade PET and a two-species microbial consortium was constructed. (B) Due to PETase inhibition by TPA, R. jostii (Rj) was added, resulting in a three-species microbial consortium. (C) To further utilize EG, P. putida (Pp) was introduced and a four-species microbial consortium was constructed.
FIGURE 3
FIGURE 3
Optimization of the two-species microbial consortium. (A) Comparison of BHET degradation by single bacterial species and the two-species microbial consortium. B1, Bs_PETase; B2, Bs_MHETase. The control group the BHET degradation by wild-type B. subtilis. (B) Time required for the two-species microbial consortium to completely degrade 2 g/L BHET with different ratios of bacteria added at inoculation. (C) Concentration of TPA over time during degradation of PET. (D) Effects of adding TPA, EG and TPA + EG on PET weight loss. The addition of exogenous degradation products significantly inhibited the degradation of PET compared with B1B2 (only using Bs_PETase and Bs_MHETase). (E) Effects of adding TPA, EG, or TPA + EG on BHET degradation. (F) Effects of adding TPA and EG on Bs_PETase (B1) and Bs_MHETase (B2). All experiments were performed at least in triplicate. Error bars indicate standard deviation. *p < 0.05, **p < 0.01 (one-sided t-test).
FIGURE 4
FIGURE 4
Construction of the three-species microbial consortium. (A) Concentration of TPA during incubation with R. jostii (dark blue circles) and OD600 of R. jostii grown in LB medium with (orange triangles) or without (light blue squares) 2 g/L TPA. (B) PET degradation by the three-species microbial consortium with different starting ratios of Bs_PETase: Bs_MHETase: R. jostii. (C) Concentrations of BHET (orange triangles and light blue squares) and TPA (yellow rhombuses and dark blue circles) over time when incubated with different microbial consortia. (D) PET degradation by Bs_PETase, Bs_MHETase, the two-species microbial consortium, and the three-species microbial consortium. All experiments were performed at least in triplicate. Error bars indicate standard deviation. **p < 0.01 (one-sided t-test).
FIGURE 5
FIGURE 5
PET degradation by four-species microbial consortia. (A) Weight loss (degradation) of PET film incubated with different consortia in Wn medium. (B) Concentration of TPA incubated with different consortia in Wn medium. (C) Optimization of the initial ratio of Bs_PETase/Bs_MHETase/R. jostii to P. putida in the four-species microbial consortium in Wn medium. (D) Optimization of the initial ratio of Bs_PETase/Bs_MHETase/P. putida to R. jostii in the four-species microbial consortium in Wn medium. All experiments were performed at least in triplicate. Error bars indicate standard deviation. *p < 0.05, **p < 0.01 (one-sided t-test).
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