Plastics are widely produced and utilized in the textile and packaging industries due to their lightweight, ductility, high transparency, and rigidity. In 1950, global plastic production was approximately 2 million tons annually. By 2015, around 6.3 billion tons of plastic waste had been generated, with only 9% recycled, 12% incinerated, and the remaining 79% accumulating in landfills or natural environments. Excessive plastic consumption and inadequate recycling management have led to severe plastic/microplastic pollution, imposing significant burdens on ecosystems and posing serious threats to human health. As one of the most widely used plastics globally, polyethylene terephthalate (PET) stands out for its excellent chemical stability and mechanical properties. However, the inherent chemical inertness of PET’s ester bonds and aromatic nuclei presents major degradation challenges. In China alone, approximately 24 million tons of discarded polyester (containing over 85% PET) require effective annual treatment to mitigate environmental pollution.

Current PET Recycling Methods

he current PET processing methods, including landfilling, incineration, mechanical and chemical treatment, all cause serious environmental pollution and require significant energy consumption. Biocatalysts have the advantage of high selectivity under mild process conditions. By using biocatalysts, PET can be recovered into its monomers and then re polymerized, which is widely regarded as an ideal and environmentally friendly solution for PET recycling and treatment. However, there are still technical and cost barriers for the industrialization of PET recycling through biotechnology.
As shown in Figure 1, traditional biotechnology mainly consists of three parts: 1. pretreatment (preparation of culture medium and sterilization); 2. Fermentation cultivation; 3. Purification of the product.
In Part 1, traditional microbial cultivation is susceptible to contamination by miscellaneous bacteria, thus requiring complex high-pressure sterilization facilities and huge energy consumption. In addition, a large amount of freshwater resources need to be consumed to allocate culture media to provide nutrients for engineering bacteria.
In Part 2, antibiotics are usually added to further avoid contamination (which may accelerate the emergence of drug-resistant strains). In addition, the expression level of PET hydrolase produced by natural PET degrading microorganisms (such as i. sakaiensis) is often insufficient to meet industrial demand, therefore it is necessary to develop effective recombinant expression systems to reduce production costs.
In Part 3, as PET hydrolases typically exhibit optimal PET degradation activity under alkaline conditions, PET is degraded into soluble terephthalate salts (such as Na2TPA), and then terephthalic acid (TPA) is obtained through acidification precipitation. Specifically, 1kg of waste PET can be degraded and acidified with HCl to obtain 0.865kg of TPA, while the resulting wastewater will contain at least 0.609kg of NaCl. Directly discharging a large amount of saline wastewater generated by acidification may cause secondary pollution of wastewater and waste of by-product resources.




 
Here, in order to solve the problems of microbial contamination, high energy consumption, high freshwater consumption, and low yield in traditional biotechnology, as well as the problem of saline wastewater generated by TPA purification during PET enzymatic hydrolysis, based on the team's existing scientific research achievements (industrialized high-performance enzyme assemblies and full process technology for waste PET enzymatic regeneration), this study aims to develop an economical, efficient, and environmentally friendly PET biodegradation process. Based on host screening and byproduct recycling (Figure 1), the new process provides an effective approach to address the challenges associated with traditional PET bioremediation.



 

Efficient protein expression ability of V. natriegens in new processes

 
Due to its rapid growth rate and abundant ribosome count, the novel host V. natriegens exhibits excellent biosynthesis rate and protein expression ability. Two different highly active PET hydrolases (IsPETasePA mutant and LCCICCG mutant) were successfully used in V Soluble expression in natriegens (Figure 2). Compared with Escherichia coli, V. natriegens showed higher protein expression levels, which may be attributed to V. natriegens under the same induction conditions The growth rate of natriegens is faster. The protein expression levels of IsPETasePA mutant and LCCICCG mutant increased by 87.3% and 65.8%, respectively. For PET biodegradation, IsPETasePA and LCCICCG from two different hosts have similar degradation effects on PET over time, indicating the use of V The expression of proteins in natriegens does not affect the activity and stability of enzymes, further indicating that V The potential of natriegen as an efficient host for expressing PET hydrolase.




Fig.2 Comparative analysis of protein expression levels and enzyme activity of PET hydrolase expressed in V. natriegens and E. coli. (a) SDS-PAGE of IsPETasePA and LCCICCGexpressed by V. natriegens and E. coli; (b) protein expression quantity of IsPETasePA and LCCICCG expressed by V. natriegens and E. coli; (c-d) the growth of V.natriegens during heterologous expression of PET hydrolase: (c)before the addition of IPTG; (d) after the induction of 24 h. (e-f) the effect of PET enzymatic hydrolysis by IsPETasePA(e) or LCCICCG (f) expressed in V. natriegens and E. coli.

The ability of new technology to resist pollution and save freshwater resources

 
Fig.3 The industrial potential of V. natriegens as a heterologous expression host for PET hydrolase. (a,c) Protein quantity and enzyme activity of IsPETasePA (a) or LCCICCG (c) expressed by V. natriegens under non-sterile conditions; (b,d) SDS-PAGE analysis of IsPETasePA(b) or LCCICCG  (d) expressed by V. natriegens under non-sterile conditions; (e-f) Protein expression quantity and enzyme activity of IsPETasePA(e) or LCCICCG  (f) expressed by V. natriegens cultivating in seawater-based medium under non-sterile conditions

Waste recycling and utilization of the new PET enzymatic hydrolysis process

PET hydrolase usually exhibits the best PET degradation activity under alkaline conditions. PET is degraded into soluble terephthalate, which requires acidification to obtain TPA and generates a large amount of saline wastewater. The IsPETasePA mutant can degrade PET under mild reaction conditions and exhibits high levels of protein expression. It has been selected as a biocatalyst to develop an economically efficient and environmentally friendly PET biodegradation process. Based on rapidly growing halophilic host V natriegens， The new process cleverly reuses the saline wastewater generated during the Na2TPA acidification process as a culture medium. V. Natriegens can grow normally in saline wastewater culture medium and maintain the expression of IsPETasePA at normal levels (Figure 4). Therefore, a novel PET biodegradation process for wastewater and by-product recycling has been developed.

 

In addition, in the new process, the halophilic bacterium V As an anti pollution host (Figure 1), natriegens effectively avoided the huge energy consumption caused by sterilization (energy reduction of 2.48 times) (Table 1). Meanwhile, through efficient protein expression, the rapidly growing V Natriegens significantly reduced the cost of chemicals (fermentation enzyme production cost decreased by 47.9%, Table 2). Therefore, the new PET bioremediation process greatly reduces the energy consumption and cost input of PET biodegradation, highlighting its industrial application potential.




Based on the novel chassis cell V The efficient expression system of natriegens and the new process of by-product recycling have the advantages of high efficiency and low cost, which can help promote the industrialization of PET biological recycling and achieve clean recycling of "white pollution".

 

 
This article is a joint research and development achievement between Yuantian Biotechnology (Tianjin) Co., Ltd. and the Enzyme Engineering Research Group of Tianjin University. Dr. Zhou Yu, Master Shen Bowen, and Associate Researcher You Shengping from the Enzyme Engineering and Technology Research Group of Tianjin University are the co first authors of this article, and Professor Qi Wei is the corresponding author.
Special thanks to Professor Ni Jun from Shanghai Jiao Tong University for providing halophilic V. for the engineering bacteria in this article Natriegens chassis cells.

Original link：https://www.sciencedirect.com/science/article/abs/pii/S096085242301341X