SELECTION OF FACTORS AFFECTING THE GROWTH AND INDOLEACETIC ACID PRODUCTION OF BACILLUS ALTITUDINIS 19RS3

ONETTO, ANDREA LILIANA; MASONEVES, CAMILA IVON; CORTESE, ILIANA JULIETA; CASTRILLO, MARIA LORENA; BICH, GUSTAVO ANGEL; ZAPATA, PEDRO DARIO; LACZESKI, MARGARITA ESTHER

Rondônia

Simposio; SIMICRON-I Simposio de Microbiologia de Rondônia: Saúde, Ambiente e Innovacao; 2021

Introduction: Biofertilizers are products composed of livingmicroorganisms that exhibit properties associated with plant growth(PGP), of which the most frequently reported are nitrogen fixation,phosphate solubilization, siderophore production and auxin synthesis.Microorganisms can produce and release auxins as secondarymetabolites, with indoleacetic acid (IAA) being the most frequentlyreported. The IAA is synthesized from L-tryptophan, but tryptophanindependentsynthetic pathways are also described. One feature limitingthe application of biofertilizers is obtaining an enough amount (growth) ofthe endophytic bacteria or the product of interest in a short period of time.Objective: This study aimed to assess the influence of several factors onthe growth and production of IAA of Bacillus altitudinis 19RS3, a PGPbacteria isolated from yerba mate (Ilex paraguariensis St Hil.) roots.Methods: A half-fraction experimental design with six factors and twolevels was used. The variables growth medium (nutrient broth ortrypticase soy broth), initial pH (7.0 or 7.5), temperature (31 or 34 °C),agitation (static or 120 rpm), incubation time (24 or 48 h), and addition ofL‑tryptophan (0 or 100 ppm) were evaluated. Azospirillum brasiliense sp.245 was used as positive control. Flasks containing 15 mL of thecorresponding medium, pH value, and IAA concentration were prepared.Twenty-four hours old stock cultures were adjusted to 0.5 of McFarland?sscale and 150 μL were added to each flask. Flasks were incubated at thecorresponding temperature, time, and agitation speed according to eachexperimental run. The growth was determined by measuring the opticaldensity (OD) at 600 nm in a spectrophotometer. Production of indoles wasdetermined by a colorimetric method at 535 nm using Salkowski?sreagent. The concentration of indolic compounds in μg mL wascalculated using a concentration curve for indoleacetic acid. Theexperimental matrix for the screening (consisting of a set of 32 runs) wasdesigned and analysed using the software Statgraphics Centurion XV.Factors with p-values of less than 0.05 were considered to have significanteffects on the response (growth or indole synthesis). Results: For B.altitudinis 19RS3, agitation increased growth, while IAA production wasincreased by the addition of L-tryptophan and incubation time, anddecreased by temperature. The highest growth (OD of 2.49) wasregistered using trypticase soy broth, initial pH of 7.5, 34 °C, agitation,without addition of L-tryptophan at 24 h, while the lowest growth (OD of0.39) was registered using nutrient broth, pH of 7.5, 34 °C, agitation,without L-tryptophan at 48 h. The highest IAA production (32.39 μg mL )was obtained using nutrient broth, pH of 7.5, 31 °C, agitation, addition of100 ppm of L-tryptophan for 48 h. The lowest IAA value (0.22 μg mL )was obtained using nutrient broth, pH of 7.0, 34 °C, agitation, addition of100 ppm of L-tryptophan for 48 h. Conclusion: Identification of thesefactors will allow us to proceed with the optimisation of the growthconditions for B. altitudinis 19RS3, which is key for reducing productioncosts in the development of a bioproduct for stimulating the growth ofyerba mate plants.