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

Abstract

Boje i halogenovani fenoli koji imaju dezaktivirano aromatiĉno jezgro ĉineznaĉajnu kategoriju veoma toksiĉnih i teško razgradljivih zagaĊivaĉa u raznimindustrijskim granama. Glavni cilj ove disertacije je bio dobivanje jeftinih rastvornih iimobilizovanih enzima za uklanjanje fenola i boja iz otpadnih voda. Korišćeno je ĉetiriprirodna (nativna) enzima (polifenoloksidaza iz krompira, tri lakaze na sporamaizolovanih 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 izsoja B. amyloliquefaciens 12B1 proizvedena u Escherichia coli).Djelimiĉno preĉišćena polifenoloksidaza (PPO) iz krompira je imobilizovana narazliĉitim nosaĉima. Od dobijenih biokatalizatora, tri sa najvećim aktivnostima PPO,Eupergit C250L-PPO, Celit-PPO i CelulozaM-PPO, su testirani u reaktoru za uklanjanjefenola, 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 fenolrazgraĊeni 35% odnosno 20%. Testirana je i sposobnost višestruke upotrebe EupergitC250L-PPO imobilizata za uklanjanje p-hlorfenola. Iz eksperimenata sinteze novognosaĉa sa pipcima i njegove primjene za imobilizaciju PPO može se zakljuĉiti da jeimobilizovana PPO bila znatno otpornija na denaturaciju u odnosu na solubilni enzim.Biokatalizator je testiran u šaržnom reaktoru za uklanjanje p-hlorfenola i p-bromfenolaiz vodenih rastvora. Postignuto je uklanjanje pomenutih fenola preko 90% prikoncentraciji fenola 100 mg/L. Za oba halogenfenola TC-PPO je pokazao stepenuklanjanja od preko 90% u prva tri ciklusa, nakon ĉega efikasnost opada do 60% nakonšest ciklusa od po 8 ĉasova. Rastvornom PPO pod optimizovanim uslovima moguće jeukloniti 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 uklonjenifiltrovanjem ili centrifugiranjem. Formiranje polimera potvrĊeno je infracrvenomspektroskopijom...

Dyes and phenols containing halogens which tend to deactivate the aromaticnuclei constitute a significant category of highly toxic and difficult-to-degradepollutants from a wide variety of industries. Main goal of this dissertation was theproduction of inexpensive soluble and immobilized enzymes for removal of phenols anddyes from wastewater. Four native enzymes (potato polyphenoloxidase, three laccaseson spores of isolated strains of Bacillus amyloliquefaciens, laccase from Trametesversicolor, acidic and basic isoforms of horseradish peroxidase) and two recombinantenzymes (chloroperoxidase from Caldariomyces fumago produced in Aspergillus nigerand laccase from B. amyloliquefaciens 12B1 strain produced in Escherichia coli).Partially purified potato polyphenol oxidase (PPO) was immobilized ontodifferent commercial and laboratory produced carriers. The three of the obtainedbiocatalysts, with the highest PPO activities, namely Eupergit C250L-PPO, Celite-PPOand CelluloseM-PPO, were tested in the batch reactor for phenol, p-chlorophenol and pbromophenolremoval. In the case of 2.5 mM substrates with Eupergit C250L-PPO,around 45% removal of p-bromophenol was achieved, while p-chlorophenol and phenolwere removed 35% and 20%, respectively. The reusability of Eupergit C250L-PPO forthe removal of p-chlorophenol has been tested. From experiments of synthesis of newtentacle carrier and its application for PPO immobilization we can conclude thatimmobilized PPO was more resistant to denaturation when compared with its solublecounterpart. Biocatalyst was tested in the batch reactor for p-chlorophenol and pbromophenolremoval from aqueous solution. More than 90% removal was achieved forboth halogenophenols at concentration of 100 mg/l from aqueous solution. For bothhalogenophenols TC-PPO works with over 90% removal during first three cycles whichdecrease to 60% removal efficiency after six cycles each of 8 hours duration. Withsoluble PPO, under optimized conditions 93-99.9% removal of dyes was achieved after1h using 424 – 1700 U/mL of PPO, depending on dye. Optimum pH for decolorizationprocess was found to be 3.0. Decolorization was accomplished via insoluble polymersformations that were separated by filtration or centrifugation. Polymer formation wasconfirmed with infrared spectroscopy...