Prospecção de microrganismos isolados de ninho de espuma de Leptodactylus vastus e Physalaemus cuvieri com potencial biotecnológico.

Abstract

Foam nests are one of the many reproductive mode found in amphibians, constituting a strategy to increase egg fertilization. They are known to protect eggs and tadpoles against UV radiation, predators and to provide oxygen supply. For these functions, its biochemical composition and structure are essential. Frog foam nests have as major components surfactant proteins and carbohydrates, besides a microbial community. So far, there are no studies concerning the associated microbiota in foam nests and their role in tadpole development nor they biotechnological potential. Hence, this study aimed to identify the cultivable foam nests bacterial community of Leptodactylus vastus and Physalaemus cuvieri and to prospect enzyme-producing microorganisms of biotechnological interest. Frog foams nests were collected at the na Reserva Particular do Patrimônio Natural de Monte Alegre, in Pacatuba/CE. Samples were serially diluted and plated using the spread plate technique. For comparative purposes, water and soil samples where the nests of were placed were also plated. After total viable counting, the morphotypes found in the foam nests plates were isolated and stored in glycerol 20% (v/v) stock in freezers at -80° C. The isolates had their DNA extracted by thermolysis and CTAB 2X, followed by the 16S rRNA gene amplification and sequencing by the Sanger method. For enzymatic tests, isolates were grown in ATGE plates supplemented with tributyrin (0.1% v/v), starch (0.1% w/v), gelatin (3% w/v) and skim milk (1 % w/v), to verify lipase, amylase and protease activities, respectively. The nests had a viable count average of 2.82 x 107 CFU/mL for P. cuvieri and 2.74 x 107 CFU/mL for L. vastus, while environmental samples had 3.33 x 104 CFU/mL in water where the foam nest of P. cuvieri was disposed and 1.41 x 106 CFU/mL for the soil where the foam nest of L. vastus was disposed. Compared with the water and soil samples, the foam nests presented approximately 1000 times greater than those of the water and 10 times larger than the soil. A total of 153 morphotypes were isolated and molecular identification showed the presence of 32 genera of bacteria, in which 11 were common for both species. In the enzymatic activities, 23 isolates of P. cuvieri showed positive results for amylase, 66 for proteases and 56 for lipases. On the other hand, L. vastus showed 14 amylase, 45 proteases, and 41 lipases. Our results show that foam nests provide a selection environment for microorganisms with biotechnological potential for the discovery of novel biomolecules.