Biohemijska karakterizacija bakterija izolovanih iz naftom zagađenih lokaliteta i njihova primena za bioremedijaciju i proizvodnju egzopolisaharida
Biochemical characterization of bacteria isolated from oil-contaminated sites and their application in bioremediation and exopolysaccharide production
Докторанд
Žerađanin, AleksandraМентор
Beškoski, VladimirЧланови комисије
Avdalović, JelenaGojgić-Cvijović, Gordana
Vujisić, Ljubodrag
Jovančićević, Branimir
Метаподаци
Приказ свих података о дисертацијиСажетак
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 supstr...at 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 mixi...ng 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