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SIMONE TESSARO

ESTUDO DA COMUNIDADE DE BACTÉRIAS METANOTRÓFICAS

EM UMA CRONOSSEQUÊNCIA DE SOLOS DA AMAZÔNIA

Dissertação apresentada ao Programa de Pós-

Graduação em Microbiologia do Instituto de

Ciências Biomédicas da Universidade de São Paulo,

para obtenção do título de Mestre em Ciências.

Área de concentração: Microbiologia

Orientadora: Dra. Vivian Helena Pellizari

Versão corrigida. A versão original eletrônica

encontra se disponível tanto na Biblioteca do ICB

quanto na Biblioteca Digital de Teses e

Dissertações da USP (BDTD)

São Paulo

2012

RESUMO

TESSARO, S. Estudo da comunidade de bactérias metanotróficas em uma

cronossequência de solos da Amazônia. 2012. 94 f. Dissertação (Mestrado em

Microbiologia) - Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo,

2012.

Bactérias metanotróficas são conhecidas por utilizarem o metano como única fonte de

carbono e energia, e possuírem importante papel na regulação do fluxo líquido de metano

entre o solo e a atmosfera. O gás metano é um dos principais gases que contribuem para o

efeito estufa no planeta, tendo uma ação 25 vezes maior do que a do CO2, em relação à

retenção do calor. A Amazônia possui importante papel no ciclo global do carbono, possuindo

um significativo reservatório desse composto. O desmatamento da floresta original para

conversão a pastagem é dos principais problemas enfrentados pela Amazônia. As mudanças

no uso do solo levam a alterações na comunidade microbiana, podendo alterar as suas

funções. O objetivo deste trabalho foi pesquisar a presença e a diversidade metanotrófica a

partir de amostras de uma cronossequência de solos da Amazônia, onde ocorreu conversão de

floresta à pastagem, e posteriormente a vegetação secundária, e verificar se tal manejo do solo

influencia na estrutura da comunidade metanotrófica. Para isso técnicas de cultivo e

enriquecimento, construção de bibliotecas do gene pmoA e a aplicação da técnica de

DNA‐SIP foram utilizadas. A partir de amostras de solo de floresta primária, secundária e de

pastagens, foram realizados enriquecimentos com a finalidade de avaliar o consumo de

metano, e realizar extração do DNA total para construir bibliotecas do gene funcional pmoA

para o estudo da comunidade metanotrófica. Uma amostra de pastagem foi também utilizada

para a aplicação da técnica de DNA‐SIP 13CH4, resultando na análise de sequências de três

bibliotecas do gene rRNA 16S. As bibliotecas do gene pmoA indicaram a presença de

bactérias metanotróficas das Famílias Methylocystaceae e Methylococcaceae, sendo que a

maioria das sequências obtidas foram agrupadas com sequências relacionadas à família

Methylocystaceae e ao gênero Methylocaldum. Os resultados da avaliação de consumo de

metano demonstram que a comunidade consumidora de metano está ativa para todas as

amostras avaliadas. No experimento de DNA‐SIP 13CH4, a recuperação do DNA marcado

ocorreu após o consumo de 0,6 mmol de metano no microcosmo do tempo de incubação t2.

As bibliotecas do gene rRNA 16S do DNA total do solo revelaram a presença bactérias

distribuídas por diversos Filos relacionados a solo. Nas bibliotecas construídas a partir do

DNA em que foi incorporado o 13CH4, foram identificados micro-organismos relacionados ao

ciclo do metano, como as metanotróficas pertecentes as famílias Methylocystaceae e

Methylococcaceae, além de outros grupos de micro-organismos cujo papel no ciclo do metano

não é totalmente conhecido, como os pertecentes a família Methylophilaceae e ao Filo

Armatimonadetes (OP10). Apesar de incerto o processo de como ocorre à utilização dos

metabólitos da oxidação do metano por estes micro-organismos, a presença destes no

ambiente pode ser de grande importância.

Palavraschave: Bactérias metanotróficas. Oxidação do metano. Uso do solo. Amazônia.

Pastagem. Gene pmoA.

ABSTRACT

TESSARO, S. Study of the methanotrophic community present in chronosequence soils

from Amazon. 2012. 94 p. Masters thesis (Microbiology) - Instituto de Ciências Biomédicas,

Universidade de São Paulo, São Paulo, 2012.

Methanotrophic bacteria are known to utilize methane as a single source of carbon and

energy, and have an important role in regulating methane flux between soil and atmosphere.

Methane is a major gas contributing to global warming on Earth, taking an action 25 times

greater than CO2, relative to heat retention. The Amazon forest plays an important role in the

global carbon cycle, having a significant carbon reservoir. Deforestation of the original forest

for the conversion to pasture is a major problem faced by the Amazon forest. Changes in land

use lead to changes in soil microbial community, altering the functions of this community.

The objective of this study was to investigate the presence and the diversity of

methanotrophic bacteria in samples of Amazon forest chronosequence soils, where there have

been a conversion of forest to pasture, and then to secondary forest, and verify if such soil

management influences the structure of the methanotrophic community. Enrichment and

cultivation techniques, gene libraries of pmoA gene and application of the DNA-SIP technique

were used to achieve this goal. From primary, secondary forest and pasture soil samples,

enrichments were performed in order to evaluate the consumption of methane. Functional

gene pmoA libraries werw constructed from total DNA extracted from soils in order to study

the methanotrophic community. A pasture sample was used in the DNA-SIP 13CH4 technique,

resulting in three sequence analysis of 16S rRNA gene libraries. The pmoA gene libraries

indicated the presence of methanotrophic bacteria from Families Methylocystaceae and

Methylococcaceae, and most of the sequences were grouped with sequences related to

Methylocystaceae Family and Methylocaldum. The evaluation of methane consumption

showed that the methane consuming community is active for all samples. In the DNA-SIP

13CH4 experiment, the recovery of labeled DNA occurred after 0.6 mmol of methane were

consumed in the microcosm, - incubation time t2. Gene libraries of rRNA 16S from total

DNA of soil, revealed the presence of bacteria over several Phyla related to soil. In gene

libraries constructed from labeled DNA, methane cycle microorganisms were identified such

as the Methylocystaceae, and Methylococcaceae methanotrophic Families. Other groups of

microorganisms like Methylophilaceae Family and Armatimonadetes Phylum (OP10) bacteria

were also found, but their role in the methane cycle is not fully understood. The knowledge

about the process of metabolites from the methane oxidation by these microorganisms may

be uncertain, although the presence of this microorganisms in the environment may be

important.

Keywords: Methanotrophic bacteria. Methane oxidation. Amazon. Soil use. Pastures. pmoA

gene.

16

1 INTRODUÇÃO

O metano é um dos principais gases que contribuem para o aquecimento do planeta, tendo

uma ação 25 vezes maior do que a do CO2 em relação à retenção do calor (DALTON, 2005;

INTERGOVERNAMENTAL PANEL ON CLIMATE CHANGE, 2007). O protocolo de

Kyoto considera de suma importância o controle das emissões deste gás para a atmosfera,

intensificando-se assim as pesquisas que visam esclarecer o papel dos micro-organismos

envolvidos com o ciclo do metano em diversos ambientes.

As bactérias metanotróficas formam um grupo fisiologicamente distinto pela sua

habilidade em usar o metano como única fonte de carbono e energia, desenvolvendo assim um

papel importante no ciclo do metano (HANSON; HANSON, 1996). Estas bactérias possuem

grande potencial na redução de emissões de CH4 para a atmosfera, pois podem formar uma

espécie de barreira biológica no solo (VISVANATHAN et al., 1999), regulando o fluxo

líquido de CH4 entre o solo e a atmosfera (VERCHOT et al., 2000).

Os níveis de emissões de CH4 na Amazônia estão aumentando com o passar dos anos

(FEIGL et al., 2002; STEUDLER et al., 1996). Desde os anos 70, a Amazônia passa por um

processo contínuo de desmatamento, sendo que, muitas destas áreas desmatadas são utilizadas

na agricultura e na formação de pastagens para a pecuária (NEPSTAD et al., 2008). Muitas

destas pastagens são utilizadas na criação de gado por um determinado período, sendo

posteriormente abandonadas, surgindo então uma floresta secundária (FEIGL et al., 2002).

As investigações sobre a estrutura e composição das comunidades microbianas do solo da

região Amazônica tem-se intensificado, assim como estudos que tentam compreender qual o

impacto gerado pelas mudanças no manejo do solo para estas comunidades (JESUS et al.,

2009). O entendimento de como estas comunidades microbianas respondem às perturbações

ambientais é crítico para a manutenção e restauração de funções importantes do ecossistema

(WU et al., 2006).

Dessa forma, buscando caracterizar a microbiota e compreender as respostas das

comunidades microbianas do solo da Amazônia, frente a diferentes formas de uso, o projeto

denominado - Monitoramento da Microbiota da Floresta Amazônica - (“Amazon Rainforest

Microbial Observatory” - ARMO), ao qual este trabalho está integrado, reuniu pesquisadores

da Universidade de São Paulo, Michigan State University, University of Massachusetts at

Amherst, University of Oregon e University of Texas at Arlington.

As amostras do projeto ARMO são advindas da Fazenda Nova Vida, em Rondônia, estado

este que possui uma das maiores áreas de floresta já comprometida pelo desmatamento. A

17

Fazenda Nova Vida também integra um dos locais de pesquisa do LBA-ECO (Large Scale

Biosphere-Atmosphere Experiment in Amazonia), sendo este um dos maiores programas de

investigação internacional para estudar as consequências do desmatamento nos níveis local,

regional e global.

Dentro deste contexto, o presente trabalho tem como objetivo pesquisar a diversidade

taxonômica e funcional de bactérias metanotróficas a partir de amostras de uma

cronossequência de solos da Amazônia, onde ocorreu a conversão de floresta à pastagem, e

posteriormente à vegetação secundária, e compreender se tal manejo do solo influencia na

estrutura da comunidade metanotrófica.

Para atingir os objetivos foram empregadas as técnicas clássicas de cultivo e avaliação de

consumo de CH4 in vitro, além de técnicas de biologia molecular como a construção de

bibliotecas do gene pmoA e a Marcação de Ácidos Nucleicos com Isótopos Estáveis” (“DNA ‐

Stable Isotope Probing” - DNA‐SIP). A técnica de DNA-SIP tem se mostrado de grande

importância para a microbiologia ambiental, pois possibilita estabelecer uma ligação entre

filogenia e funcionalidade (CÉBRON et al., 2007 a,b).

77

6 CONCLUSÕES

 As amostras de pastagens (P11, P72 e P04) apresentaram um consumo de metano mais

rápido, em relação às amostras de floresta (F) e floresta secundária (S);

 as sequências obtidas a partir das bibliotecas do gene pmoA com amostras de solo de

floresta primária, floresta secundária e pastagens, foram relacionadas em sua maioria

com sequências pertencentes à família Methylocystaceae;

a técnica de DNA‐SIP possibilitou identificar a comunidade metanotróficas ativa,

além de outros grupos de micro‐organismos, como os pertencentes à família

Methylophilaceae e ao gênero Methylocaldum, além do Filo OP10, cujo papel no ciclo

do metano ainda é incerto;

 o protocolo utilizado para a técnica de DNA-SIP com metano marcado, mostrou-se

eficiente para o estudo de bactérias metanotróficas. No entanto para a amostra

estudada foi verificada a importância de diminuir o tempo de incubação;

78

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