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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2022.tde-11102022-172512
Document
Author
Full name
Ana Carolina de Araujo Butarelli
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2022
Supervisor
Committee
Pellizari, Vivian Helena (President)
Araujo, Welington Luiz de
Saia, Flavia Talarico
Silva, Aline Maria da
 
Title in Portuguese
Perfil taxonômico e funcional de microrganismos metanotróficos em regiões de exsudação de metano do Atlântico Sudoeste
Keywords in Portuguese
Methylomicrobium sp.
Bacia de Santos
Gás do efeito estufa
Metanotrofia
Recuperação de MAGs
Abstract in Portuguese
O metano é um dos principais gases produzidos em sedimentos marinhos, podendo ter origem biótica ou abiótica. Esse importante gás do efeito estufa, quando liberado na coluna dágua, é mitigado graças à atividade de microrganismos metanotróficos, ou seja, microrganismos capazes de usar metano como única fonte de carbono e energia. Este trabalho foi realizado na região Bacia de Santos, que é uma bacia sedimentar localizada na região do talude do Atlântico Sudeste, representando a maior região produtora de petróleo e gás natural do país. O objetivo foi explorar a diversidade microbiana metanotrófica em amostras de sedimentos oriundos de regiões com potencial para serem áreas de exsudação ativa de metano, uma vez que apresentam as feições geológicas de montes carbonáticos e pockmarcks, através de técnicas dependentes e independentes de cultivo. As amostras foram coletadas em 10 pontos, e inoculadas em meio de cultura NMS + 3,5% NaCl e metano:ar (1:1) nas culturas aeróbias e metano:argônio (1:1) nas culturas anaeróbias, à 20 ºC, durante 8 meses. O DNA das culturas foi extraído e o gene 16S rRNA sequenciado pela plataforma Illumina Miseq. A cromatografia gasosa foi realizada para identificar se houve oxidação de metano. As análises de diversidade alfa e beta revelaram que não há diferença na riqueza das espécies, mas sim na estrutura das comunidades, de acordo com os tratamentos utilizados. O Filo Proteobacteria foi dominante na análise taxonômica dos consórcios, com destaque para a classe Gammaproteobacteria. Foram identificados 3 genêros de bactérias metanotróficas em quatro consórcios microbianos que tiveram consumo de metano confirmado por cromatografia gasosa, sendo eles Methylomicrobium, Methylomonas e Methylovulum. Essas amostras foram então enviadas para sequenciamento metagenômico para a recuperação de MAGs associadas à microrgansimos metanotróficos. Das 90 MAGs recuperadas, 2 foram anotadas taxonomicamente como pertencentes ao gênero Methylomicrobium sp. e que possivelmente são espécies novas, denominadas LECOM 001 e 002. Ambas as MAGs possuem genes associados ao ciclo do nitrogênio, ciclo do enxofre e somente em LECOM 002 o operon pmoABC foi recuperado, indicando que ela pode sim oxidar metano. Esses MAGs ainda revelaram uma possível associação desses organismos à fixação de nitrogênio em regiões montes carbonáticos de mar profundo e são os primeiros registros de genomas deste gênero recuperados na Bacia de Santos. Estes resultados representam também novas evidências da presença de exsudação fria na região dos montes carbonáticos Alpha Crucis, motivando a continuidade da pesquisa de metanotróficos na região.
 
Title in English
Taxonomic and functional profile of methanotrophic microorganisms in methane exudation regions of the Southwest Atlantic
Keywords in English
Methylomicrobium sp.
Greenhouse gas
Metanotrophy
Recovery of MAGs
Santos Basin
Abstract in English
Methane is one of the main gases produced in marine sediments, and may have a biotic or abiotic origin. This important greenhouse gas, when released into the water column, is mitigated thanks to the activity of methanotrophic microorganisms, that is, microorganisms capable of using methane as the only source of carbon and energy. This work was carried out in the Santos Basin region, which is a sedimentary basin located in the Southeast Atlantic slope region, representing the largest oil and natural gas producing region in the country. The objective was to explore the methanotrophic microbial diversity in sediment samples from regions with potential to be areas of active methane exudation, since they present the geological features of carbonate mounds and pockmarcks, through dependent and independent cultivation techniques. The samples were collected at 10 points, and inoculated in culture medium NMS + 3.5% NaCl and methane:air (1:1) in the aerobic cultures and methane:argon (1:1) in the anaerobic cultures, at 20 ºC, for 8 months. DNA from cultures was extracted and the 16S rRNA gene sequenced using the Illumina Miseq platform. Gas chromatography was performed to identify whether there was methane oxidation. The analysis of alpha and beta diversity revealed that there is no difference in species richness, but in the structure of communities, according to the treatments used. The Phylum Proteobacteria was dominant in the taxonomic analysis of the consortia, with emphasis on the class Gammaproteobacteria. Three genera of methanotrophic bacteria were identified in four microbial consortia that had methane consumption confirmed by gas chromatography, namely Methylomicrobium, Methylomonas and Methylovulum. These samples were then sent for metagenomic sequencing for the recovery of MAGs associated with methanotrophic microorganisms. Of the 90 MAGs recovered, 2 were taxonomically annotated as belonging to the genus Methylomicrobium sp. and that are possibly new species, named LECOM 001 and 002. Both MAGs have genes associated with the nitrogen cycle, sulfur cycle and only in LECOM 002 the pmoABC operon was recovered, indicating that it can indeed oxidize methane. These MAGs also revealed a possible association of these organisms to nitrogen fixation in deep sea carbonate mound regions and are the first records of genomes of this genus recovered in the Santos Basin. These results also represent new evidence of the presence of cold seepage in the region of the Alpha Crucis carbonate mounds, motivating the continuity of methanotrophic research in the region.
 
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Release Date
2024-10-10
Publishing Date
2022-12-02
 
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