Prospecção de microrganismos isolados de ninho de espuma de Leptodactylus vastus com potencial biotecnológico

Abstract

Anuran foam nests are stable biostructures used for the deposition and maintenance of eggs and tadpoles out of the water. These biopumps have several functions for offspring development, such as oxygen and nutrient supply, protection against ultraviolet rays and against predators, and increased growth rate. There are few studies aimed at the exploration of the microbiota associated with the anuran foam nests, making them valuable sources of prospecting of new biomolecules with biotechnological potential. The aim of the present work was to isolate bacteria from Leptodactylus vastus foam nest, its molecular identification through the sequencing of the 16S RNAr gene, and to investigate morphotypes with amylase, protease, lipase, antimicrobial and emulsifying activities. The L. vastus foam nests were collected in the private reserve of natural heritage of Monte Alegre, in the Pacatuba-CE mountains in the rainy season. Serial dilutions were made with nest and puddle samples for plating viable counts using the scattering technique. The dilutions 10-1, 10-2, 10-3, and 10-4 were plated in ATGE medium (15 g / L agar, tryptone 5 g / L, glucose 1 g / L, yeast extract 2.5 g / L), and incubated at room temperature for 16 h. The 35 morphotypes were selected and stored at -80 ° C in TGE broth (tryptone 5 g / L, glucose 1 g / L, yeast extract 2.5 g / L) with glycerol 50% (v / v). For the enzyme activities test, each isolate was reactivated and cultured in ATGE supplemented with starch (0.1% w / v) for amylases, skim milk (1% w / v) and gelatin (3%) for proteases, and tributyrin (1% v / v) for lipases. For the emulsifying, antibacterial and antifungal activities supernatants grown in TGE with optical density at 600 nm equal to 1.0 were used. For the extraction of DNA the thermolysis protocol was used and the amplification of the 16S RNAr gene was performed through PCR using primers 27F and 1525R. Of the 14 morphotypes isolated from foam nest samples, 4 (11.4%) presented amylase activity, 2 (5.7%) presented lipase activity, 5 (14.3%) presented casease proteinase activity and 3 (8,7%) for gelatin. The supernatants of Lvs 02, 15 and 16 presented emulsifying activity. Antifungal activity against fungus Fusarium sp. was observed in the Lv 05 cell suspension and in the cell suspension and supernatant of the Lv 15 morphotype. These results point out the potential of the microbiota associated with the L. vastus nest as a source of biomolecules with biotechnological applications.