OUTER PLANET 49 PETplanet Insider Vol. 24 No. 09/23 www.petpla.net OUTER PLANET SIU doctoral student explores how to produce high-value chemicals from breakdown products PET meets microbes Based on an article by Tim Crosby Southern Illinois University (SIU) Carbondale graduate student researcher Lakshika Dissanayake, who is earning her doctorate in microbiology, is working on an economical and environmentally friendly process that uses microbial factories to turn plastic waste into a value-added material. Much of the work centres on creating a novel bacterium that can break down PET by expressing certain enzymes. The process, which utilises a genetically engineered microbe known as Erwinia aphidicola LJJL01, could be the key to creating a biologically based plastics upcycling system. “Biological plastic upcycling,” refers to producing high-value products from plastic waste using biological reactions and microbes instead of chemicals. Scientists have identified a few microbes that degrade PET, as well as the corresponding PET hydrolase enzymes. Previous studies have successfully characterised and engineered these enzymes to selectively depolymerise PET into original monomers, such as terephthalic acid and ethylene glycol. Dissanayake, an Environmental Research & Education Foundation Scholar, hopes to take that work a step further. “Researchers have developed potential hybrid biological and chemical plastic upcycling strategies,” Dissanayake said. “But a whole microbial system for plastic upcycling is yet to be developed.” A key to developing such a system is finding the right kind of “bug” to mix in with the waste streams and then improving on it. Erwinia aphidicola LJJL01 seemed promising, because of its presence in waste charcoal used in plastic recycling. Fashioning the perfect creature With a promising microbe on deck, Dissanayake and her doctoral adviser, Lahiru Jayakody, assistant professor in the School of Biological Sciences, set to work studying it. “First, we conducted a comprehensive characterisation of it, to identify its growth conditions and metabolic potential of the strain,” Dissanayake said. The researchers next used advanced synthetic biological tools, including a method known as plasmidbased transformation. That method involves transferring outside DNA into a host cell, which will then carry both a bacterial origin of replication and an antibiotic resistance gene that researchers can use as a selectable marker in bacteria. They also used CRISPR-Cas9 technology, a genetic engineering technique, to engineer the bacterium’s genome for expression and secretions of the enzymes they wanted. CRISPR-Cas9 allows researchers to edit genes by precisely cutting DNA and then letting natural DNA repair processes take over. The enzymes produced use hydrolysis to break down PET by splitting large molecules into smaller ones. These souped-up, engineered versions of the bacterium showed great promise in tests at SIU. “Right now, we are engineering the metabolic pathways of the bacterium to produce high-value chemicals from PET breakdown products,” Dissanayake said. “We have successfully demonstrated we can cause the expression and secretion of these enzymes, and we confirmed that the strains we engineered can completely deconstruct PET, and that they can even outperform commonly used platform strains in other plastic upcycling studies.” www.siu.edu SIU doctoral student Lakshika Dissanayake tests whether an engineered microbe can upcycle plastic waste. (Photo: Russell Bailey)
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