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Bioprospecting is underway to replace nitrogen fertilizers with bacteria that fix atmospheric nitrogen (N2) and convert it into a form that the plant is capable of assimilating, such as Gluconacetobacter diazotrophicus, an obligate endophytic bacterium and the only species of its kind capable of fixing nitrogen. This bacterium is also endowed with other characteristics, such as the biosynthesis of levan, an important biomolecule for the scientific community, as it can be used in the pharmaceutical industry as a hypocholesterolemic, anticarcinogenic, prebiotic and modified drug release agent. The aim of this research was to isolate, morphologically characterize and select levain-producing strains from endophytic bacteria with characteristics similar to G. diazotrophicus associated with two varieties of sugarcane grown in the São José do Rio Preto region (SP).