Interactions between biodegradable microplastics and Bacillus subtilis - Pseudomonas putida dual-species biofilm model

Abstract

Biodegradable plastics have been developed as an alternative to conventional fossil-based, non-degradable plastics to solve the environmental issues caused by their widespread use and inappropriate disposal. Among all biodegradable plastics, polyhydroxyalkanoates (PHA) have gained significant attention due to their biological origin, rapid biodegradability, and excellent biocompatibility. Microplastics, defined as plastic fragments smaller than 5 mm, are generated during the use and subsequent disposal of all kinds of plastics, including PHA. Microplastics are reported to pose potential risks to environmental and human health, considering they accumulate and transfer across multiple ecosystems, which raise significant scientific and societal concern.

Throughout the plastics biodegradation process in the natural environment (e.g., freshwater, soil, and sediments), biofilms form on the surface of microplastics, creating a protective microenvironment that supports microbial survival and accelerates biodegradation. Natural biofilms are typically more heterogeneous and complex than their laboratory counterparts, consisting of multiple microbial species, which exhibits diverse metabolic and biochemical interactions. However, limited research has focused on elucidating the interactions between PHA microplastics and mixed biofilms that develop during their degradation.

The objectives of this study were: (1) To establish a dual-species biofilm model, which would serve as a simplified model of natural multi-species biofilms, enabling a controlled and systematic investigation of the interactions between PHA microplastics and biofilms; (2) To examine how PHA microplastics influence biofilm properties, including biomass, morphology, and species composition within biofilm; (3) To investigate the biodegradation of PHA microplastics mediated by biofilm activity, including morphology and physicochemical composition changes of PHA material.

The dual-species biofilm model developed in this study consisted of Pseudomonas putida and Bacillus subtilis, two bacterial species that are widespread in natural environments. PHA micro beads with a diameter of 50 μm were used to simulate PHA microplastics, while glass beads of the same size served as inert controls. Biofilm biomass was assessed by using biochemical assays, including crystal violet staining, protein quantification and polysaccharides quantification. The morphology and species composition of the biofilms were characterized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). PHA micro beads particle size and morphology alteration during biodegradation process were measured through dynamic image particle size analyzer and SEM imaging. Additionally, alterations in the physicochemical composition of PHA micro beads were characterized with thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS).

The results demonstrated that PHA microbeads promoted the formation of dual-species biofilms compared to the glass bead control under the tested condition. Within the dual-species biofilm, an antagonistic interaction between P. putida and B. subtilis was observed. PHA micro beads underwent rapid biodegradation in 24 and 72 hours induced by biofilm, as evidenced by reduced particle size, irregular morphology, and structural changes. The physicochemical properties of PHA micro beads also changed during the biodegradation process, as indicated by TGA and FT-IR results.

This study provides new insights into the interactions between PHA microplastics and an artificially established dual-species biofilms, offering a simple framework for understanding PHA biodegradation in natural environments and contributes to a better understanding of microplastic pollution at the ecosystem scale.

Date of Award	15 Jul 2026
Original language	English
Awarding Institution	University of Nottingham
Supervisor	Enrico Marsili (Supervisor), Jodi Woan-Fei Law (Supervisor) & Honglei Zhang (Supervisor)