MARINE MEDAKA (ORYZIAS MELASTIGMA) AS A BEHAVIORAL MODEL ORGANISM FOR INVESTIGATING ENVIRONMENTAL STRESS: IMPLICATIONS FOR CHEMICAL COMMUNICATION

ABSTRACT

THE DEMAND FOR PLASTIC HAS LED TO ENORMOUS PLASTIC WASTE IN THE ENVIRONMENT, WHICH PERSIST AND NEGATIVELY IMPACT THE ECOSYSTEMS. POLYETHYLENE TEREPHTHALATE (PET) IS ONE OF THE MOST COMMON THERMOPLASTIC POLYMERS AVAILABLE ON THE MARKET. THE CONCERNS ABOUT PLASTIC WASTE GENERATED AN INTEREST IN STRATEGIES TO ENHANCE ITS BIODEGRADATION AND FINDING ALTERNATIVE POLYMERS. IN THIS WORK WAS INVESTIGATED THE POSSIBILITY OF USING BACTERIA TO DEGRADE PET AND TO PRODUCE BIOPLASTICS (POLYHYDROXYALKANOATES, PHAS). FINALLY, THE INTEGRATION OF THE TWO PROCESSES WAS TESTED. OVERALL, THE WORK AIMED TO INVESTIGATE THE POTENTIAL TO RECYCLE PET INTO BIOPLASTIC USING BACTERIA. THE POTENTIAL OF BACTERIAL CONSORTIA FROM VARIOUS ENVIRONMENTAL SAMPLES TO DEGRADE PET GRANULES IN LIQUID MATRIX WAS INVESTIGATED. . THE RESULTS REVEALED MAXIMUM PET GRANULES DEGRADATION OF 1.1 % BY ONE OF THE TESTED CONSORTIA. PET DEGRADATION INTERMEDIATE TEREPHTHALIC ACID (TPA) WAS NOT DETECTED AT THE END OF 55 DAYS. FOURIER-TRANSFORM INFRARED SPECTROSCOPY (FTIR) RESULTS SHOWED MAJOR SPECTRAL PEAK SHIFTS AND BENDS ON PET CHEMICAL STRUCTURE COMPARED TO NON-INOCULATED CONTROL. THE BIODEGRADATION OF PET FILMS BURIED IN THE SOIL (A), WITH MANGROVE PLANTS (B), AND BIOAUGMENTED WITH A BACTERIAL CONSORTIUM (C) WAS ALSO INVESTIGATED. THE EXPERIMENTS WERE CONDUCTED FOR 270 DAYS AT AMBIENT CONDITIONS. THE RESULTS REVEALED NO DIFFERENCE BETWEEN TREATMENTS IN THE DEGRADATION, WITH A MAXIMUM WEIGHT LOSS OF 0.118 % IN THE BIOAUGMENTED TREATMENT. NEVERTHELESS, SCANNING ELECTRON MICROSCOPE (SEM) AND FTIR RESULTS INDICATED SIGNIFICANT SURFACE CHANGES, SPECTRAL PEAK SHIFTS, AND STRETCHES IN PET CHEMICAL STRUCTURES. BACTERIAL CONSORTIA ISOLATED FROM THE SOIL OF THE EXPERIMENTAL TREATMENTS WERE ASSESSED FOR DEGRADATION OF PET MONOMERS, TPA AND MONOETHYLENE GLYCOL (MEG), AND INTERMEDIATE BIS(2-HYDROXYETHYL) TEREPHTHALATE (BHET). THE CONSORTIA WERE INOCULATED IN FLASKS CONTAINING MINIMAL MEDIA WITH 1000 MG/L TPA OR BHET OR1113 MG/L MEG AS THE SOLE CARBON SOURCE. RESULTS SHOWED COMPLETE DEGRADATION OF TPA AND SIGNIFICANT DEGRADATION OF BHET (96.09%), AND MEG (83.65%) BY THE CONSORTIA. IN THE SECOND PART OF THE STUDY, BACTERIA WERE ISOLATED FROM VARIOUS ENVIRONMENTAL SAMPLES AND SCREENED FOR PHA PRODUCTION USING SUDAN BLACK B STAINING ON COLONIES AND SMEARED GLASS SLIDES. TRANSMISSION ELECTRON MICROSCOPE IMAGES WERE CAPTURED TO CONFIRM THE INTRACELLULAR PHA INCLUSIONS. A TOTAL OF 35 ISOLATES WERE SCREENED FOR PHA, AND 22 SHOWED POSITIVE STAINING. THE ISOLATE SHOWING HIGHER LEVELS OF PHA SYNTHESIS (EC2-30-3) WAS IDENTIFIED BASED ON 16S RRNA GENE SEQUENCE AS BACILLUS SP. AND SELECTED FOR PET MONOMERS DEGRADATION AND FERMENTATION CULTURES FOR PHA PRODUCTION. IT WAS CULTURED IN MINIMAL (MOREIRA ET AL., 2013) MEDIA WITH 1000 MG/L TPA AND 1113 MG/L MEG AS THE CARBON SOURCE FOR EIGHT DAYS. THE ISOLATE GREW BETTER IN MEDIA CONTAINING MEG, WHICH WAS SELECTED AS A SUBSTRATE MODEL FOR PHA FERMENTATION. TO INTEGRATE PET MONOMERS BIODEGRADATION AND PRODUCTION OF PHA, THE ISOLATE WAS CULTURED IN 0.2 % MEG. A CONTROL WITH 0.2 % OF GLUCOSE WAS PREPARED, AND THE CULTURES WERE INCUBATED FOR 96 HOURS. BACILLUS SP. EC2-30-3 SHOWED HIGHER PHA ACCUMULATION IN MEDIA SUPPLIED WITH MEG (40.31%) THAN GLUCOSE (25.53%). THIS IS THE FIRST REPORT SHOWING THAT BACILLUS SP. USES PET MONOMER AS CARBON SOURCE TO PRODUCE A BIOPOLYMER. FTIR RESULTS OF THE EXTRACTED PHA IDENTIFIED ITS FUNCTIONAL UNITS AS C–H, CH3, C=O, AND C–O GROUPS. THE ABSORPTION BANDS OBTAINED ARE CLOSELY RELATED TO THE STRUCTURE OF PHB. THE STUDY THUS CONFIRMED THE ABILITY OF THE ISOLATED BACTERIA TO DEGRADE PET MONOMERS AND PRODUCE BIOPOLYMERS. THE RESULTS OF THIS WORK OPEN THE POSSIBILITY FOR UPSCALING THE USE OF BACTERIA TO MITIGATE THE IMPACT OF PET ON THE ENVIRONMENT WHILE PRODUCING ENVIRONMENTALLY FRIENDLY BIOPLASTICS