Bacillus spp. Produtoras de biofloculantes e hidrolases extracelulares, entre estirpes de solo/lodo que sintetizam polihidroxialcanoatos e/ou surfactantes

Abstract

Enzymes are extremely attractive biological catalysts in industrial conversion processes due to their ability to promote reactions with high enantioselectivity and in milder conditions than chemicals, as well as biological flocculants are advantageous for easy biodegradation and safety to living beings. Both are easily obtained from microorganisms that naturally inhabit environments where these molecules are necessary to favor their metabolism. In the present work, four bacterial isolates were used, two from the Atlantic Forest but cultivated for 40 years with sugarcane (LBPMA strains: APF.SG3-Isox and ACO.PR1-Isox), previously selected by high tolerance to agrochemicals, and two from agro-industrial sludge (LBPMA: BDLJ2 and BDL07) (Coruripe-AL, Brazil), sorted for producing polyhydroxyalkanoates and biosurfactants. Each lineage was submitted to biochemical and molecular tests for identification purposes and then submitted to qualitative / semi-quantitative tests (cultures in solid medium containing specific substrate) and quantitative (submerged cultures containing specific substrates) of cellulolytic, proteolytic, lipolytic activity and bioflocculant producer. Based on the morphological properties and analyzes of the 16S rDNA sequences, the isolates LBPMA-APF.SG3-Isox, LBPMA-ACO.PR1-Isox, LBPMA-BDLJ2 and LBPMA-BDL07 were identified respectively as B. megaterium, B. toyonensis, B. thuringiensis and B. pumilus, and all were able to present the studied activities. The maximum indices for protease and lipase activity in solid culture were obtained by B. toyonensis (IE = 4.55 and 2.41, respectively), while the maximum cellulolytic activity in solid medium (IE = 1.40) was obtained by B. pumilus. In liquid medium, the strains of B. pumilus and B. thuringiensis showed the highest cellulolytic activity in the first 48 h. The maximum lipolytic activity of the three isolates analyzed in submerged culture containing olive oil as a specific substrate (0.274 U. mL-1 for Bacillus thuringiensis LBPMA-BDLJ2, 0.450 U. mL-1 for Bacillus pumilus LBPMA-BDL07 and 0.552 U. mL-1 for Bacillus megaterium LBPMA- APF.SG3), also corresponded to the maximum cell growth at 72 h of incubation. Concerning proteolytic activity in submerged culture, B. thuringiensis showed the same maximum activity as B. pumilus, but in a shorter incubation period. All isolates secreted the tested enzymes and bioflocculat at the constant temperature of 37 ± 1 ° C, without changes in the pH over time. The flocculant activity increased with the incubation time, initially reaching the maximum value of 57% at 72 h for B. pumilus culture, while for B. thuringiensis, B. toyonensis and B. megaterium this was 33%, 21% and 34%, respectively, after 24 h of incubation. These initial results stimulate the need for optimization of the pH (3, 5, 7 and 9) and nitrogen [Urea, Peptone and (NH4)2SO4] and carbon sources (Maltose, Sucrose and Glucose) variables of the medium for flocculation using such strains of the Bacillus genus. Sucrose and Maltose were the best sources of carbon, whilst Urea was the prefered source of nitrogen for two of the tested isolates (B. pumilus and B. toyonensis), followed by (NH4)2SO4 (B . megaterium) and Peptone (B. thurigiensis). The best pH values for the production of bioflocculant were 5.0 (B. toyonensis e B. thuringiensis) and 3.0 (B. pumilus e B. megaterium). The FTIR-ATR spectra of the extracted flocculants revealed the presence of the carboxyl, hydroxyl and methoxy functional groups as responsible for the flocculant activity of the studied strains, thus being characterized as polysaccharides.