2.4. Functional characterization of isolates

The isolated and purified bacterial strains were analyzed for enzymatic production, biological fixation of atmospheric nitrogen and the ability to degrade phosphate minerals, according to the methodology of analysis of functional groups of microorganisms, described by Albino and Andrade (2006). Cross-sectional measurements of halos and colonies were taken to calculate the enzyme index.

The amilolytic activity of the isolates was assessed via the same method, using the AMILO culture medium (Pontecorvo et al., 1953; Oliveira et al., 2010) and development was carried out by applying 10% lugol solution to the plates. The lugol reacts with the starch, producing a purple color and allowing for the visualization of the degradation halos. The production of cellulases followed the same methodology, applying the isolates in the CELULO culture medium (Wood, 1980). Development is achieved by applying 0.5% Congo red solution to the plates, removed with saline solution (0.85% NaCl) after 30 min. Cellulose becomes red and the degradation halos appear yellow.

Phosphate solubilizers were analyzed by inoculating the isolates onto the SF culture medium (Sylvesterbradley et al., 1982), which does not require any further development, but requires an eight-day incubation period. Therefore, Petri dishes should receive a greater volume of culture medium. The medium, which is opaque at the moment in which it receives the bacteria, becomes clear around colonies capable of solubilizing phosphate crystals. The phosphate solubilization index (ISF) was calculated through ISF = A/B, where A was the diameter of the halo (phosphate solubilization) including the colony and B consisted only of the diameter of the colony (Montañez et al., 2012).

The biological fixation of N was analyzed by inoculating the isolates in the BNF culture medium (Döbereiner and Day, 1976). Since the N-fixing microorganisms are microaerophilic, the plates must be wrapped in plastic film and incubated at 28 ± 1 °C for five days. N fixation is revealed by the appearance of a blue halo around the colonies.