Towards biorecycling of plastics: Isolation and characterization of Pseudomonas capeferrum TDA1, a bacterium capable to degrade polyurethane mono- and oligomers

During the last 50 years, plastic industry has grown exponentially with an estimated 8300 million metric tonnes of plastic produced to date. Regardless of the large variety of polymers available, 99% are entirely fossil-fuel based which compromises its degradability after use. Major synthetic polymers in use today are polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyurethane (PU) and polyethylene terephthalate (PET). The current methods for disposing of plastic waste mainly include landfilling, incineration, mechanical and chemical recycling. Despite of the significant improvement of these technologies, it is still necessary to overcome several limitations and deficiencies. Polyurethane (PU) is a synthetic polymer used as raw material in several industries. In 2015, PU global production reached 27 million metric tons, making the sixth most-used plastic worldwide. The main constituents of polyurethane are isocyanates, polyols and chain extenders. Unfortunately, the mismanaged plastic has spread out in different habitats across our planet including cold marine areas and uninhabited places, threatening wildlife and ecosystems. In order to avoid further contamination, it is necessary to transform plastic waste by restoring functional properties, providing added value and exploring new application areas that could provide economic benefits in a long- term perspective. In the last 10 years, a transition from a linear economy to a sustainable, bio-based circular economy has become fundamental to cope the fossil fuel-driven climate change and global plastic pollution. This transformation involves industrial and basic research strongly focused on biotechnology and bioprocesses. Within this transition, microorganisms are key players due to the wide diversity of enzymes and metabolic pathways that could be used for the development of sustainable processes and biomaterials. Recently, microorganisms with plastic-degrading potential have been regularly identified in different environments such as waste disposal, landfills, plastic refineries, open dumps, etc. Selective pressure and evolution of genetically flexible mechanisms have contribute to metabolize anthropogenic compounds, which it has been noted in several enzymatic reactions designed for the efficient degradation of a wide variety of recalcitrant substrates, leading to novel metabolic pathways. Even though several bacterial genera have been reported in the degradation of environmental pollutants, Pseudomonas species are amongst the most cited degraders of aromatic hydrocarbons and plastic polymers. The genus Pseudomonas incorporates one of the most complex, diverse, and ecologically significant group of bacteria on the planet. Members of this genus are found in large numbers in all the major natural environments (terrestrial, freshwater, and marine) and form intimate associations with plants and animals. This universal distribution suggests a remarkable degree of physiological and genetic adaptability. In fact, Pseudomonas have been most frequently linked with PU degradation. Chemically, polyester-based PUs are semi-crystalline structures containing hydrolysable ester and urethane bonds that are fragmented by extracellular enzymes (hydrolases), releasing oligomeric and monomeric building blocks. For instance, amines, alcohols, acids, aromatics, and other residues, such as EG (ethylene glycol), 1,4-butanediol (BDO), adipic acid (AA) ,4′-methylenedianiline (MDA) and 2,4- toluene diamine (2,4-TDA) are constantly present during PU degradation. However, MDA and 2,4-TDA are considered environmental pollutants, which represent a major risk for species in the aquatic and terrestrial areas. This fragmentation of the polymer is known as depolymerization and it is essential for strengthening recycling processes that use plastic waste as feedstock. The broad spectrum of building blocks might be used as carbon and energy source for microorganisms that degrade these compounds and/or use them for the production of higher-value elements. This latter is considered a promising upcycling strategy to reduce fossil-fuel plastic waste and promote new waste management strategies. Previous studies have revealed that extracellular enzymes are essential for biofilm formation on the polymer surface, reducing the resistance and durability of plastic materials. This first step promotes microbial attachment and further degradation. Enzymes with hydrolytic and proteolytic activity have been detected in spherical structures called outer membrane vesicles (OMVs) in several Pseudomonas species. Generally, OMVs play a key role in establishing inter- and intra-species communication, acquisition of nutrients, stress response, delivery of toxins, adhesion and virulence factors, biofilm formation, etc. Even though numerous bacterial strains and enzymes are involved in degradation processes, the complete catabolic mechanism is not totally understood yet. This thesis also centers on the characterization of outer membrane vesicles for extracellular degradation of a polyurethane oligomer and elucidation of the degradation pathway for the polyurethane monomer 2,4-diaminotoluene (2,4-TDA) by Pseudomonas capeferrum TDA1. In the first chapter, bacterial isolation from soil samples and the subsequent protocols to quantify biodegradation of polyurethane building blocks were fully described. The isolated strain was able to use a PU oligomer and 2,4-TDA as sole source of carbon. The latter compound also served as nitrogen source. These results provided a key insight into the catabolic mechanism of the soil bacterium as a potential PU monomer and oligomer-degrader. The second chapter described the identification of the isolated strain as Pseudomonas sp. by partial 16S rRNA gene sequencing, membrane fatty acid profile and structural gene for the cis/trans isomerase (cti). In addition, genomic DNA was isolated from bacterial cells grown on succinate and utilized for whole genome sequencing in order to detect catabolic genes related to aromatic compounds degradation. Preliminary, enzymes involved in the metabolic pathway were identified, which eventually led to a suggested degradation pathway for Pseudomonas sp. grown on 2,4-TDA. The strain was identified as Pseudomonas capeferrum (type strain WCS358) using the full 16S rRNA gene sequence. The third chapter reported a new method of RNA extraction from Pseudomonas capeferrum TDA1 growing on 2,4-TDA. Phenols and catechols are central intermediates of the aromatics biodegradation that can be easily oxidized to yield the corresponding quinones, which interfere with nucleic acids and tend to co- precipitate or degrade RNA. The chemical process is regulated by the activity of polyphenol oxidases enzymes, which have been identified in several Pseudomonas species previously. This optimized protocol incorporated several modifications including the use of a carrier, pooled samples and a final cleaning up step that could improve it significantly, yielded a high-quality RNA measured by A260/A280, A260/230 ratios (2.02 ± 0.16, 1.95 ± 0.01, respectively) from cells grown on 2,4-TDA compared to standard assays. Moreover, RIN (RNA integrity number) values were analyzed and samples with a RIN higher than 7.0 were selected for downstream applications, confirming the RNA quality. Finally, the fourth chapter evaluated the transcriptional changes in Pseudomonas capeferrum TDA1 grown on 2,4-TDA using RNA-seq. From all the expressed genes, one third were overexpressed in comparison to the control (succinate). These alterations in the gene expression demonstrates that aromatic compounds trigger adaptive responses that modify the transcriptional regulation mechanism including important changes not only in the catabolic system, but also in other patterns related to bacterial cell physiology and biofilm formation. In order to evaluate extracellular degradation, OMVs isolated from P. capeferrum TDA1 grown on a PU oligomer were tested for hydrolytic activity. Purified OMVs showed higher esterase activity compared to cell pellets. Relative OMV yields in TDA1 raised significantly in PU oligomer (0.28 ± 0.05%) compared to succinate (0.09 ± 0.01%). This three-fold increased activity could demonstrate that the release of OMV is part of the adaptive mechanisms of bacteria to stressful environmental conditions. The macromolecular degradation may occur through the action of both periplasmic and membrane-bound hydrolases harbored inside of OMVs and can be considered as a supporting mechanism for biodegradation. The results of this thesis present a further understanding of the transcriptome response in P. capeferrum TDA1 exposed to a PU monomer, suggest a model for extracellular degradation involving OMVs and propose a complete catabolic mechanism for the biodegradation of polyester-based PU containing intra and extracellular enzymes. Moreover, further studies on biological degradation of PU will contribute to redesign plastic polymers considering biodegradable building blocks and improving biocatalytic degradation, which could provide a sustainable use of PU plastic waste in the future