Uklanjanje fenola i boja iz otpadne vode prirodnim i rekombinantnim oksidativnim enzimima

Abstract

Boje i halogenovani fenoli koji imaju dezaktivirano aromatiĉno jezgro ĉine znaĉajnu kategoriju veoma toksiĉnih i teško razgradljivih zagaĊivaĉa u raznim industrijskim granama. Glavni cilj ove disertacije je bio dobivanje jeftinih rastvornih i imobilizovanih enzima za uklanjanje fenola i boja iz otpadnih voda. Korišćeno je ĉetiri prirodna (nativna) enzima (polifenoloksidaza iz krompira, tri lakaze na sporama izolovanih sojeva Bacillus amyloliquefaciens, lakaza iz gljive Trametes versicolor, kisele i bazne izoforme peroksidaze iz rena) i dva rekombinantna enzima (hloroperoksidaza iz Caldariomyces fumago proizvedena u Aspergillus niger i lakaza iz soja B. amyloliquefaciens 12B1 proizvedena u Escherichia coli). Djelimiĉno preĉišćena polifenoloksidaza (PPO) iz krompira je imobilizovana na razliĉitim nosaĉima. Od dobijenih biokatalizatora, tri sa najvećim aktivnostima PPO, Eupergit C250L-PPO, Celit-PPO i CelulozaM-PPO, su testirani u reaktoru za uklanjanje fenola, p-hlorfenola i p-bromfenola. U sluĉaju 2,5 mM supstrata sa Eupergit C250LPPO, postignuto je oko 45% razgradnje p-bromfenola, dok su p-hlorfenol i fenol razgraĊeni 35% odnosno 20%. Testirana je i sposobnost višestruke upotrebe Eupergit C250L-PPO imobilizata za uklanjanje p-hlorfenola. Iz eksperimenata sinteze novog nosaĉa sa pipcima i njegove primjene za imobilizaciju PPO može se zakljuĉiti da je imobilizovana PPO bila znatno otpornija na denaturaciju u odnosu na solubilni enzim. Biokatalizator je testiran u šaržnom reaktoru za uklanjanje p-hlorfenola i p-bromfenola iz vodenih rastvora. Postignuto je uklanjanje pomenutih fenola preko 90% pri koncentraciji fenola 100 mg/L. Za oba halogenfenola TC-PPO je pokazao stepen uklanjanja od preko 90% u prva tri ciklusa, nakon ĉega efikasnost opada do 60% nakon šest ciklusa od po 8 ĉasova. Rastvornom PPO pod optimizovanim uslovima moguće je ukloniti 93-99.9% boje nakon tretmana u trajanju od 1 ĉas sa 424-1700 U/mL PPO, zavisno od boje. Pokazano je da je optimalno pH za proces obezbojavanja bilo 3,0. Obezbojavanje je postignuto uz formiranje nerastvornih polimera koji su uklonjeni filtrovanjem ili centrifugiranjem. Formiranje polimera potvrĊeno je infracrvenomspektroskopijom...

Dyes and phenols containing halogens which tend to deactivate the aromatic nuclei constitute a significant category of highly toxic and difficult-to-degrade pollutants from a wide variety of industries. Main goal of this dissertation was the production of inexpensive soluble and immobilized enzymes for removal of phenols and dyes from wastewater. Four native enzymes (potato polyphenoloxidase, three laccases on spores of isolated strains of Bacillus amyloliquefaciens, laccase from Trametes versicolor, acidic and basic isoforms of horseradish peroxidase) and two recombinant enzymes (chloroperoxidase from Caldariomyces fumago produced in Aspergillus niger and laccase from B. amyloliquefaciens 12B1 strain produced in Escherichia coli). Partially purified potato polyphenol oxidase (PPO) was immobilized onto different commercial and laboratory produced carriers. The three of the obtained biocatalysts, with the highest PPO activities, namely Eupergit C250L-PPO, Celite-PPO and CelluloseM-PPO, were tested in the batch reactor for phenol, p-chlorophenol and pbromophenol removal. In the case of 2.5 mM substrates with Eupergit C250L-PPO, around 45% removal of p-bromophenol was achieved, while p-chlorophenol and phenol were removed 35% and 20%, respectively. The reusability of Eupergit C250L-PPO for the removal of p-chlorophenol has been tested. From experiments of synthesis of new tentacle carrier and its application for PPO immobilization we can conclude that immobilized PPO was more resistant to denaturation when compared with its soluble counterpart. Biocatalyst was tested in the batch reactor for p-chlorophenol and pbromophenol removal from aqueous solution. More than 90% removal was achieved for both halogenophenols at concentration of 100 mg/l from aqueous solution. For both halogenophenols TC-PPO works with over 90% removal during first three cycles which decrease to 60% removal efficiency after six cycles each of 8 hours duration. With soluble PPO, under optimized conditions 93-99.9% removal of dyes was achieved after 1h using 424 – 1700 U/mL of PPO, depending on dye. Optimum pH for decolorization process was found to be 3.0. Decolorization was accomplished via insoluble polymers formations that were separated by filtration or centrifugation. Polymer formation was confirmed with infrared spectroscopy...