Biochemical characterization of bacteria isolated from oil-contaminated sites and their application for bioremediation and production of exopolysaccharides

Nafta i rafinisani proizvodi prerade nafte prilikom eksploatacije, transporta, skladištenja i akcidentalnih izlivanja zagađuju životnu sredinu i ispoljavaju toksične efekte po živi svet u njoj uključujući i čoveka. S druge strane, naftom zagađena životna sredina predstavlja lokalitet u kome žive brojni mikroorganizmi koji imaju sposobnost degradacije različitih zagađujućih supstanci. Cilj ove disertacije je bio ispitivanje primena različitih mikroorganizama izolovanih iz lokaliteta zagađenih naftom, za dva procesa, bioremedijaciju i proizvodnju egzopolisaharida. Istraživanja vezana za bioremedijaciju su podeljena u dva dela. Prvi deo studije ispituje sposobnost izolovanog konzorcijuma mikroorganizama, degradera ugljovodonika, u procesima bioremedijacije izuzetno kompleksnih lokaliteta, kao što su isplačne jame. Isplačne jame predstavljaju zemljane rezervoare u koje se odlaže otpad, nastao tokom ekspoloatacije nafte i zemnog gasa. Na početku procesa napravljen je bioremedijacioni supstrat dobijen mešanjem zemljišta sa ruba isplačne jame i rastresitog sedimenta bogatog naftnim ugljovodonicima. U bioremedijacioni supstrat je dodat izolovan konzorcijum mikroorganizama. Pokazano je da nakon 180 dana konzorcijum sa visokom efikasnošću razgrađuje naftne ugljovodonike i smanjuje njihovu koncentraciju za 95,3%. Ovako visok procenat razgradnje ukazuje da je u konzorcijumu koji je korišćen u postupku bioremedijacije prisutna velika količina raznovrsnih mikroorganizama što je metagenomskom analizom i potvrđeno. Naime, analiza je pokazala da su dominantno prisutni rodovi Gordonia, Bacteroides, Candidatus Macondimonas, Brucella, Parvibaculum, Mycobacterium. Navedeni rodovi su u literaturi ranije istaknuti kao degraderi naftnih ugljovodonika. Prednost metagenomske analize se ogleda u tome, što pored mikroorganizama koji se mogu gajiti u laboratoriji, uspešno identifikuje i nekulturabilne autohtone mikroorganizme prisutne u uzorku. U drugom delu istraživanja proučavana je efikasnost ex situ postupka bioremedijacije kontaminiranog zemljišta, pored praćenja stepena mikrobiološke razgradnje naftnog polutanta, određen je i njegov grupni sastav, kao i promene u količini frakcija tokom studije. Dodatni kvalitet ovog istraživanja je i ispitivanje nastanka huminskih kiselina tokom procesa bioremedijacije. Tokom postupka, količina naftnih ugljovodonika je smanjena u visokom procentu, za 86,6% u odnosu na početnu vrednost. Analizom uzoraka tokom bioremedijacije je utvrđeno da se sa procesom biodegradacije paralelno odvija i humifikacija. Pokazano je da je nakon 150 dana postupka bioremedijacije količina huminskih kiselina porasla za 26,5%, pri čemu su nastale huminske kiseline bile bogatije aromatičnim strukturama i kiseoničnim funkcionalnim grupama. Ovako obogaćeno zemljište, ranije zagađen ekosistem koji je tretiran bioremedijacijom, ponovo omogućava život mikroorganizmima i biljkama, jer huminske kiseline u njemu pozitivno utiču na mikrobni rast i njihov metabolizam, indukuju sintezu enzima i predstavljaju redoks aktivna jedinjenja. U disertaciji je izolovan i ispitivan mikroorganizam prisutan u sredini zagađenoj naftnim ugljovodonicima, koji ima sposobnost produkcije egzopolisaharida. Mikroorganizam CH-KOV3, izolovan iz kanala otpadnih voda Pančevo, u preliminarnim laboratorijskim testovima je pokazao potencijal za sintezu egzopolisaharida. Izolovana bakterija je u okviru teze molekularno identifikovana kao vrsta roda Brachybacterium, a sekvenca 16s rRNK zavedena u NCBI bazu podataka pod pristupnim brojem KC881303.1. Daljom analizom izolata je utvrđeno da pripada grupi nepokretnih bakterija koje ne proizvode spore, a rastu u prisustvu kiseonika. Ovoj Gram-pozitivnoj bakteriji je pomoću 2D GCxGC-MS tehnike određen masnokiselinski profil ukupnih lipida, od kojih su dominantno prisutni bili ai-C15, i-C15, ai-C17, i-C17, i-C16, n-C16, n-C18 i C18:2 metil estri masnih kiselina. Izolat je okarakterisan i pomoću MALDI-TOF MS tehnike, a rezultat je značajan jer dobijen proteinski profil predstavlja jedinstven ˮotisak prstaˮ i do danas je jedini publikovan spektar roda Brachybacterium. Tokom studija produkcije polisaharida upotrebom izolovane bakterije Brachybacterium sp. CH- KOV3 je utvrđeno da ona proizvodi najveću količinu egzopolisaharida pri temperaturi od 28 °C i pH vrednosti 7, na podlozi sa 500 g/L saharoze. Pri ovim uslovima je dobijeno 45,2 g/L egzopolisaharida, što je čak 5 puta veća količina u poređenju sa ranije publikovanim rezultatima za rod Brachybacterium. Detaljna strukturna analiza polisaharida pokazala je da je proizvedeni egzopolisaharid fruktanskog tipa, relativno visoke molekulske mase sa β(2,6) glikozidnom vezom u osnovnom nizu i grananjem u položaju β(2,1), što odgovara egzopolisaharidu levanu.During their exploitation, transport, storage and accidental spills, oil and refined oil products pollute the environment and have toxic effects on living organisms including humans. On the other hand, such oil-polluted environments are localities inhabited by numerous microorganisms that have the ability to degrade some of the pollutants. The aim of this dissertation was to investigate the application of various microorganisms isolated from oil-contaminated sites for two processes, bioremediation and exopolysaccharide production. Research related to bioremediation included two studies. The first study examined the involvement of an isolated consortium of microorganisms, hydrocarbon degraders, in the bioremediation processes in extremely complex localities, such as mud pits. Mud pits are earthen reservoirs in which waste generated during the exploitation of oil and natural gas is disposed of. In the study, at the beginning of the process, a bioremediation substrate was obtained by mixing the soil from the edge of a mud pit with loose sediment rich in petroleum hydrocarbons. A consortium of microorganisms was isolated, cultivated and then added to the bioremediation substrate. After 180 days of bioremediation, the consortium decomposed petroleum hydrocarbons with high efficiency and reduced their concentration by 95.3%. Such a high percentage of hydrocarbon degradation indicates that large numbers of various microorganisms are present in the consortium used in the bioremediation process, which was confirmed by metagenomic analysis. Namely, the analysis showed that the genera Gordonia, Bacteroides, Candidatus Macondimonas, Brucella, Parvibaculum and Mycobacterium predominate in the consortium. These genera have previously been highlighted in the literature as petroleum hydrocarbon degraders. The advantage of metagenomic analysis is reflected in the fact that in addition to microorganisms that can be grown in the laboratory, it successfully identifies uncultured indigenous microorganisms present in the matrix. In the second study, on the efficiency of ex situ bioremediation of contaminated soil, in addition to monitoring the degree of microbiological degradation of petroleum pollutants, the group composition of the petroleum pollutants was determined, as well as changes in the amounts of fractions during the study. An additional quality of this research is the investigation of the formation of humic acids during the bioremediation process. During the bioremediation procedure, the amount of petroleum hydrocarbons in the soil was reduced by a high percentage, i.e. by 86.6% compared to the initial value. Analysis of soil samples taken during bioremediation showed that humification takes place in parallel with the biodegradation process. In fact, after 150 days of the bioremediation process, the amount of humic acids increased by 26.5%, with the resulting humic acids being richer in aromatic structures and oxygen functional groups than at the start. This resulting enriched soil, obtained from a previously polluted ecosystem that was treated by bioremediation, allows microorganisms and plants to thrive, because it contains humic acids that have a positive effect on microbial growth and metabolism, induce enzyme synthesis and act as redox active compounds. A microorganism from a petroleum hydrocarbon-contaminated environment which could produce exopolysaccharides was isolated and examined. The microorganism CH-KOV3, isolated from the Pančevo wastewater canal, showed potential to synthesize exopolysaccharides in preliminary laboratory tests. The isolated bacterium was molecularly identified as a species belonging to genus Brachybacterium, and the 16s rRNA sequence was entered into the NCBI database under accession number KC881303.1. Further analysis of the isolate determined that it belongs to the group of immobile bacteria that do not produce spores, but grow in the presence of oxygen. This Gram-positive bacterium was studied by the 2D GCxGC-MS technique to determine the fatty acid profile of its total lipids; predominant lipids are ai-C15, i-C15, ai-C17, i-C17, i-C16, n-C16, n-C18 and C18:2 fatty acid methyl esters. The isolate was also characterized by the MALDI-TOF MS technique, and the result is significant because the protein profile obtained represents a unique fingerprint and, to date, is the only published spectrum for the genus Brachybacterium. Studies into polysaccharide production using the isolated Brachybacterium sp. CH-KOV3 showed it produces the largest amount of exopolysaccharides at 28 °C and pH 7, on a medium with 500 g/L sucrose. Under these conditions, 45.2 g/L of exopolysaccharide were obtained, which is five times higher than the previously published results for genus Brachybacterium. Detailed structural analysis showed the exopolysaccharide produced by Brachybacterium CH-KOV3 was a fructan-type polysaccharide of relatively high molecular weight, with β(2,6) glycosidic bonds in the main chain and branching in the β(2,1) position, which corresponds to the exopolysaccharide levan