Polymers selection for a liquid inoculant of Azospirillum brasilense based on the Arrhenius thermodynamic model

Abstract

Plant growth promoting bacteria (PGPB) enhances the growth of their hosts and can protect them from biotic and abiotic stresses. Bacterial inoculants contain one or more of these beneficial strains in a carrier material, which must be able to maintain the viability of the cells during the time of storage, and also guarantee the biological activity of the strains once applied in the soil. These inoculants can be solid, liquid, gel or oil-based, depending on the characteristics of the strains and the shelf life expected by the producers. In this study, we used a method of accelerated degradation to select a polymer and a concentration to maintain cell stability of a liquid inoculant based on the strain C16 Azospirillum brasilense. A screening at 45°C was made to compare the protectant effect of five polymers on the viability of the strain (p/v): carrageenan (1.5%), sodium alginate (1%), trehalose (10 mM), polyvinylpyrrolidone (2%), glycerol (10 mM) and phosphate saline buffer as control. Carrageenan and sodium alginate showed significant differences in cell viability over the use of other polymers (P < 0.05). We evaluated cell viability with these two polymers at three concentrations and three different temperatures (4, 28 and 45°C) for 60 days and determined the bacterial degradation rates. Based on the Arrhenius thermodynamic model, we calculated the time required to reduce cell concentration in three log units, and observed that the protectant activity of each polymer and each concentration depends on the temperature of storage. Cell viability was best preserved in all treatments at 4°C. In general, alginate prolonged cell viability at 28°C, and carrageenan at 45°C. Alginate at 1% and carrageenan at 0.75% showed a stable behavior (superior to the control) in the three evaluated temperatures, so we conclude that they can be used for a formulation of a liquid inoculant based on the strain C16 of A. brasilense.

Development of liquid rhizobial inoculants and pre-inoculation of alfalfa seeds

“…temperature for a long time without compromising the viability of bacteria and their effectiveness. The physical and chemical properties of applied polymers should protect cells against desiccation, sedimentation and cell death [34]. In addition, the use of sodiumalginate (1 g L -1 ) in the inoculant formulation provided successful survival during 60 days of storage at 28°C [34].…”

“…The physical and chemical properties of applied polymers should protect cells against desiccation, sedimentation and cell death [34]. In addition, the use of sodiumalginate (1 g L -1 ) in the inoculant formulation provided successful survival during 60 days of storage at 28°C [34]. Because of this, room temperature was selected and a satisfactory survival of rhizobia was obtained.…”

Enhancement of drought tolerance on guinea grass by dry alginate macrobeads as inoculant of Bacillus strains

“…Thus, we selected the ratio 80:20 to perform the immobilization process of Bacillus megaterium XT14 strain. The biodegradation rate of the polymer matrix was directly related to its composition, preparation method and biological activity of the microorganisms (Bashan et al 2016; Cortes-Patino and Bonilla 2015). With respect to the biological activity, the results previously described and those reported by Vogelsang and Østgaard (1996), the alginate exposed to non-sterile systems, such as wastewater and soil, can be degraded by microorganisms.…”

Biotechnological production of plant inoculants based on nitrogen-fixing bacteria

“…Cultivation experiments were carried out at 28±2 °C with an agitation rate of 120 rpm for two days, while the optimized composition of the ABRA medium was as follows (g/l): glutamate 28.33, yeast extract 2.92, K 2 HPO 4 •3H 2 O 1.34, MgSO 4 •7H 2 O 0.5 and FeCl 3 0.02. The same medium and the same strain were used in the study performed by Cortés-Patiño and Bonilla (2015) under slightly different cultivation conditions: bioprocess duration was set to 27 h, and 1 % of inoculum was applied for inoculation of the ABRA medium. Azospirillum brasilense Sp245 was grown in the 5 lbenchtop bioreactor (working volume 3 l) for 37 h at 30 °C, pH 6.8, with dissolved oxygen concentration (DO) 3 %, and agitation rate 50-500 rpm (adjusted automatically to maintain the set DO value), while inoculum (100 ml) was prepared for 14 h at 30 °C and 180 rpm (Ona et al, 2005).…”

“…After polymerization and filtration to separate liquid phase, beads were inoculated in BTB-1 medium and cultivated for 18 h at 30 °C and 120 rpm to maintain the same concentration of bacteria in the microbeads that are reduced by the polymerization process. Beads were consequently dried at 37 °C for 24 h. Cortés- Patiño and Bonilla (2015) have investigated several polymers and protectants and their protective activity in terms of Azospirillum brasilense C16 survival at 45 °C: carrageenan (1.5 %), sodium alginate (1 %), trehalose (10 mM), polyvinylpyrrolidone (2 %) and glycerol (10 mM). Each polymer or protectant was added to cultivation broth in the amount of 10 % (v/v) prior to incubation at 45 °C for 15 days.…”