Banca de QUALIFICAÇÃO: ALINE CARDOSO CARLOS

Uma banca de QUALIFICAÇÃO de DOUTORADO foi cadastrada pelo programa.
DISCENTE : ALINE CARDOSO CARLOS
DATA : 17/12/2018
HORA: 14:00
LOCAL: Sala Carl Peter von Dietrich - Departamento de Bioquímica
TÍTULO:

PROSPECTION OF GENES INVOLVED IN THE BIODEGRADATION OF PETROLEUM AND DERIVATIVES

PALAVRAS-CHAVES:

metagenomic libraries, bioremediation, degradation

PÁGINAS: 94
GRANDE ÁREA: Ciências Biológicas
ÁREA: Bioquímica
RESUMO:

Residues generated by oil exploration form a large environmental liability, which need adequate treatment. In this way, the development of environmental bioremediation strategies has been increasing in recent years. Areas contaminated by petroleum are relevant sources of microorganisms and research of genetic resources for this purpose, due to the occurrence of microorganisms with degradation capacity and the use of hydrocarbons as carbon source. In this context, the metagenomic approach was used in the present work to identify genes and microorganisms associated with hydrocarbon biodegradation and biosurfactant synthesis, from the functional screening of the metagenomic library and the isolation of bacteria from the wells petroleum. A library of 1000 clones was obtained in plasmid and expressed in E. coli, whose environmental DNA was extracted from residue samples from petroleum drilling fluid. The clones were functionally screened, using qualitative and quantitative tests, such as the colorimetric test based on the reduction of DCPIP (2,6-dichlorophenol indophenol), which allows the detection of hydrocarbon degradation. The DCPIP test revealed 60 clones positive for degradation activity, of which 25 were sequenced and their sequences compared in GeneBank (https://www.ncbi.nlm.nih.gov/genbank/) using the BLAST packet. The prediction of ORFs was made by the ORF finder program (https://www.ncbi.nlm.nih.gov/orffinder/), which allowed the identification of ORFs involved in aliphatic and aromatic hydrocarbon degradation pathways. Growth curves, using petroleum as carbon source, corroborated with the results obtained in the DCPIP test, revealing degradation ability by the clones. Six clones showed a degradation rate between 50 and 80%, which is quite significant when compared to E.coli DH10BØ (host strain), which showed a rate of 17%, thus confirming that the degradation verified is caused by the eDNA inserts present in the clones. These clones were selected for assembly of two consortia (clones and clones + bacterial isolate) which was used for bioremediation of the residue from which they were obtained. In this assay, high metabolic activity was observed through the dehydrogenase activity during the monitored period (28 days). The degradation rates were 14 and 42% of n-alkanes for the clones and clones + isolated consortia, respectively, suggesting potential of application of the consortia in bioremediation processes of environments contaminated with petroleum. The sequencing of consortium forming clones through the Ion Torrent Platform showed the presence of genes involved in different pathways as well as key genes involved in the degradation of aliphatic and aromatic hydrocarbons. The isolated bacteria were identified by sequencing the 16S rRNA by the Sanger method. The 16S rRNA sequences obtained from isolated strains were submitted to phylogenetic analyzes that suggest the occurrence of new strains belonging to the clades Brevibacillus and Bacillus. Tests carried out suggest hydrocarbon degradation capacity and the production of biosurfactants. The functional approach of metagenomic libraries used in the present work has proved to be a good strategy for the prospection of enzymes active in the degradation of petroleum hydrocarbons. In addition the identification of enzymes responsible for different cellular functions acting in the degradation, expands our knowledge about the process of hydrocarbon degradation.

MEMBROS DA BANCA:
Interno - 1453487 - KATIA CASTANHO SCORTECCI
Interno - 1544647 - MATHEUS DE FREITAS FERNANDES PEDROSA
Presidente - 1674709 - VIVIANE SOUZA DO AMARAL