“DESENVOLVIMENTO DE FORMULAÇÕES PARA CONSÓRCIO BACTERIANO ENTRE Azospirillum brasilense E Pseudomonas fluorescens COM POTENCIAL USO COMO INOCULANTE BIOLÓGICO”

Abstract

Biological inoculants represent a promising aspect in contemporary agriculture, playing a fundamental role in promoting agricultural sustainability. The growing demand for multifunctional biotechnological products, characterized by a longer shelf life and a favorable cost-benefit ratio for producers, which at the same time prioritize the preservation of the ecosystem and human health, has remained constant in the global. Biological inoculants emerge as highly effective catalysts for increasing agricultural productivity, while significantly mitigating the negative impact associated with the excessive use of chemical fertilizers, thus contributing to the promotion of sustainable agriculture. The objective of this work was to develop liquid formulations for bacterial consortium between Azospirillum brasilense Ab-V5 and Ab-V6 and Pseudomonas fluorescens ATCC 13525 that guarantee cell survival and viability for a commercially viable period. For this, five formulations were developed (F1, F2, F3, F4 and F5) and tested in comparison to two control formulations (RC, Nfb). The best formulation (F4) was selected for adding five protectors in different concentrations: PVP (0.10%, 0.5% and 1%), PVA (0.15%, 0.25% and 0.5%), Trehalose (5mM, 10mM and 20mM), malic acid (0.15%, 0.25% and 0.5%), and citric acid (5mM, 10mM and 15mM), aiming to evaluate cell stability. To evaluate the Colony Forming Units (CFU), 1 mL of inoculum (1x108 CFU/mL) was used in 100 mL of culture medium, incubation occurred at 180 rpm at 30 °C for 48 hours. The performance of the formulations and protectors was evaluated according to the methodology established by MAPA (Normative Instruction, N°30 of 2010). In associated cultivation of the Ab-V5 and Ab-V6 strains of A. brasilense, the formulations F2, F3 and F4 stood out among those tested by showing significant growth in relation to the control. For this reason, P. fluorescens isolates were selected for cultivation. To evaluate growth in a bacterial consortium, the A. brasilense and P. fluorescens strains were grown in two ways: 1) Simultaneous cultivation of the three strains. 2) Cultivation with an interval in growth time with the association of P. fluorescens, after 24 hours of previous cultivation of A. brasilense. F4 was selected for semi-pilot scale testing, carried out in flasks with a volume of 10 L, in which 1.87% of each strain were inoculated in 8 L of culture medium (F4), with the addition of protectors at different concentrations. and incubated in an air-conditioned room with a temperature of 30 °C, under sterile aeration. The inoculation of the flasks with the strains occurred respecting the time interval of 24 hours of growth between A. brasilense and P. fluorescens. None of the protectors showed a significant difference greater than F4 without the addition of protectors at the end of 90 days of evaluation in relation to cell stability and viability. Viable cells were counted using the conventional method and qPCR, and it was found that the viable cell counting method using qPCR was efficient and could optimize the quality control process of liquid inoculants. The results demonstrated that the F4 formulation, with or without protector, presented satisfactory viability and microbial stability, in accordance with current legislation. Furthermore, F4 presents itself as a viable candidate for future industrial scaling processes.