Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-05-18T17:44:09Z No. of bitstreams: 1 2014 - Dayana da Silva Correia.pdf: 2038600 bytes, checksum: 031920d278558b902310d93f752920d7 (MD5) / Made available in DSpace on 2018-05-18T17:44:09Z (GMT). No. of bitstreams: 1 2014 - Dayana da Silva Correia.pdf: 2038600 bytes, checksum: 031920d278558b902310d93f752920d7 (MD5) Previous issue date: 2014-03-26 / CAPES / CNPq / The symbiosis between soil invertebrates and micro"organisms is a major ally in promoting the decomposition of plant residues in the soil. The micro"organisms in turn, have an immense genetic diversity and play crucial roles in maintaining ecosystems. One of these functions is the production of extracellular enzymes that assist the mineralization of organic matter. The possibility of developing new biotechnological processes based on the exploration of microbial diversity is immense, due to the great variability that exists between biological systems and it can be optimized to improve the agricultural production systems in a sustainable manner. The objective of this work was to study the profile of the bacterial community and cellulolytic potential of bacteria isolated from three different species of invertebrates saprophages. The experiments were performed at the Experimental Station of Embrapa Agrobiology, Serop?dica, Rio de Janeiro State. Millipede, of the Trigoniulus corallinus species, were collected in piles of plant compounds from local experimental field sites, which were subsequently incubated for 60 days under six different diets. Bacterial diversity in the intestinal tract of invertebrates was analyzed by PCR"DGGE of 16S rDNA gene amplification by PCR electrophoresis in denaturing gradient gel (DGGE); two domains were used, Bacteria and Actinobacteria. Some bands of the DGGE gel were extracted and sequenced. To assess the potential for production of cellulases in response to the presence of carboxy methyl cellulose (CMC) of isolates, the technique Congo red stain was used and the values were expressed as means (Ie) enzymatic index. From the highest values of (Ie) twenty" three bacteria were selected for the analysis of 16S rDNA. After the phylogenetic identification, the cellulolytic potential was rated, through cellulolytic activity of endoglucanase (CMCase), and endo" and exoglucanases (FPase) tests. To determine the molecular weight and activity of the enzymes polyacrylamide gels (SDS"PAGE) and zymography were performed. The results obtained in the technique of DGGE, the profiles of DGGE bands, showed that the intestinal microbiota of the invertebrates has distinct bacterial groups. It is possible to infer that, despite the communities having similar abundance, such as in the Trigoniulus corallinus and Cubaris murine species, the groups that make up this abundance were different among the invertebrate?s species. From the clones of the incised bands, three phyla members, Proteobacteria, Firmicutes and Bacteroidetes, were sequenced. Through phylogenetic analysis, it was possible to identify the 23 species of bacteria. Presenting two distinct Actinomycetes and Firmicutes phylum, the largest genus identified was Streptomyces, followed by an isolated Bacillus, Paenibacillus, and Staphylococcus. The intestinal tract of the three species of saprophages invertebrates showed to be an adequate environment for prospection of bacteria with cellulolytic efficiency, with high potential for future biotechnological studies. / A simbiose entre invertebrados do solo e microrganismos ? um grande aliado no auxilio da decomposi??o de res?duos vegetais presentes no solo. Os microrganismos por sua vez, apresentam uma imensa diversidade gen?tica e desempenham fun??es cruciais na manuten??o dos ecossistemas, uma dessas fun??es ? a produ??o de enzimas extracelulares que auxiliam na mineraliza??o da mat?ria org?nica. A possibilidade de desenvolver novos processos biotecnol?gicos com base na prospec??o da diversidade microbiana ? imensa, em decorr?ncia da grande variabilidade que existe entre os sistemas biol?gicos e que podem ser aperfei?oados para melhorar os sistemas de produ??o agr?colas de forma sustent?vel. O objetivo deste trabalho foi estudar o perfil da comunidade bacteriana e potencial celulol?tico de bact?rias isoladas de tr?s diferentes esp?cies de invertebrados sapr?fagos. Os experimentos foram montados no Campo Experimental da Embrapa Agrobiologia, Serop?dica, Rio de Janeiro. Gong?los, da esp?cie Trigoniulus corallinus, foram coletados em pilhas de compostos vegetais presentes em torno do campo experiemental, que posteriormente foram incubados durante 60 dias, sob seis diferentes dietas. A diversidade bacteriana do trato intestinal dos invertebrados foi analisada atrav?s da t?cnica de PCR"DGGE por amplifica??o do gene 16S rDNA PCR por eletroforese em gel com gradiente desnaturante (DGGE); foram utilizados dois dom?nios Bacteria e Actinobact?ria. Algumas bandas do gel de DGGE foram extra?das e sequenciadas. Para avaliar o potencial quanto ? produ??o de celulases em resposta ? presen?a de carboxi"metil"celulose (CMC) das bact?rias isoladas, foi utilizada a t?cnica de colora??o vermelho Congo, e os valores foram expressos atrav?s de (Ie) ?ndice enzim?tico. A partir dos maiores valores de (Ie), foram selecionadas vinte e tr?s bact?rias para a an?lise de sequenciamento do gene 16S rDNA. Ap?s a identifica??o filogen?tica, foi avaliado o potencial celulol?tico, atrav?s de testes de atividade celulol?tica de endoglucanases (CMCase) e endo e exoglucanases (FPase). Para determinar a massa molecular e atividade das enzimas foram realizados g?is de poliacrilamida (SDS"PAGE) e zimograma. Os resultados obtidos na t?cnica de DGGE, os perfis de bandas de DGGE mostrou que a microbiota intestinal dos invertebrados, det?m grupos bacterianos distintos. Pode"se inferir neste ponto, que apesar das comunidades possu?rem abund?ncia similar, como as esp?cies de Trigoniulus corallinus e Cubaris murina, os grupos que comp?em esta abund?ncia foram diferentes entre as esp?cies de invertebrados. A partir dos clones de bandas incisadas, foram sequ?nciados membros de tr?s filos, Proteobacteria, Firmicutes e Bacteroidetes. Atrav?s da an?lise filogen?tica, foi poss?vel identificar as 23 esp?cies de bact?rias. Apresentando dois filos distintos Actinomicetos e Firmicutes, o maior g?nero identificado foi Streptomyces, seguido de um isolado para Bacillus, Paenibacillus e Staphylococcus. O trato intestinal das tr?s esp?cies de invertebrados sapr?fagos revelou ser um ambiente h?bil ? prospec??o de bact?rias com efici?ncia celulol?tica, com alto potencial para futuros estudos biotecnol?gico.