JIA-2019-11

2546 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 microspheres. This can be explained by the increase in microsphere crosslink density (Aslani and Kennedy 1996) and homogeneity (Al-Musa et al . 1999). The rate of alginate gelation is an important factor in controlling microcapsule homogeneity and strength. Slower gelation produces more uniform structures with better mechanical properties than faster gelation systems. At higher calcium ion concentrations the microspheres formed faster, becoming less homogeneous than those formed at lower calcium ion concentrations. Homogeneous microspheres allow a higher rate of diffusion due to uniform pore size, whereas inhomogeneous delays diffusion throughout network structure. The amount of released calcium ions from ALG/Ca microsphere increased with increasing initial calcium ions concentration, whereas the amount of released calcium cations in the presence of T . viride spores decreased. This may be ascribed to the change in microsphere structure due to the electrostatic repulsions between alginate chains and T . viride spores and by mechanical interactions, as well as by stronger calcium binding to the T . viride within the microsphere. 4. Conclusion New agroformulations containing alginate microspheres loaded with a biocontrol agent ( Trichoderma viride ) and macronutrient (calcium ions) for plant protection and nutrition were prepared. Results pointed out remarkable differences in physicochemical properties and the release behavior of bioactive agents from alginate microspheres depending on initial calcium concentration and presence of T . viride spores. Investigation of intermolecular interactions using FTIR spectroscopy revealed complex interactions between all components in a microsphere involving mainly interactions with hydroxyl and carboxylate functional groups. Negatively charged T . viride spores bind calcium ions primarily by electrostatic interactions. An increase of calcium chloride concentration increased the number of spores in suspensions and in microspheres in a concentration-dependent manner. Visible mycelium observed around microspheres were generated by germination inside matrix and germ tubes protruding out of microspheres. Calcium alginate microspheres provide a supportive environment of T . viride germination. In the range of investigated calcium ion concentration, the best biomass yield in microspheres prepared at 1 mol dm –3 of initial calcium chloride concentration indicated the benefits of microspheres prepared at this concentration. Release profiles of T . viride showed good fit into power law equation. The initial release of T . viride from microspheres prepared at higher calcium chloride concentration was quicker than those prepared at low concentration of calcium chloride due to the smaller microsphere size. Fitting to simple Korsmeyer-Peppas empirical model revealed the underlying release mechanism of T . viride is the anomalous transport kinetics determined by a combination of the two diffusion mechanisms and Type II transport (polymer swelling and relaxation of the polymeric matrix). The increase in n value with calcium ion concentration indicates the prevailing influence of the polymer swelling and relaxation on the rate of T . viride release indicating that the transition of glassy structure to rubbery state is slower on less swelled microspheres. The fraction of released T . viride biomass above 1 indicated germination. The release of calcium ions is controlled by Fickian diffusion through microspheres. A decrease in the release rate is in accordance with the effect of increasing calcium concentration on the strength of the alginate network structure. The presence of T . viride spores affects the release rate and amount of calcium released; both are smaller in comparison with ALG/Ca microspheres. This may be ascribed to the germ tubes formation inside the microsphere matrix and their penetration through the surface in the surrounding medium. The results of this study showed that physicochemical properties of calcium alginate microspheres loaded with T . viride spores could be controlled by adjusting microsphere structure with a concentration of calcium ions. Depending on the microsphere structure, various bioactive agents release rates and patterns can be accomplished. By adopting a systematic experimental approach, optimum conditions for microsphere preparation can be reached to achieve protection and nutrition effects on the plants over an extended period of time. An understanding of intermolecular interactions between bioactive agents and the delivery system as well as mechanisms controlling the release of active agents enhance the ability to control active agents release behavior and may aid in developing new microspheres with specifically tailored properties. Table 4 Variation of the release constant ( k ), exponent ( n ), and correlation coefficient ( R 2 ) of calcium released from ALG/Ca and ALG/(Ca+ Tv ) microspheres prepared at various initial calcium chloride concentrations, c i (CaCl 2 ) c i (CaCl 2 ) (mol dm –3 ) k (ALG/Ca, h –1 ) n R 2 k (ALG/(Ca+ Tv ), h –1 ) n R 2 0.5 9.5×10 –4 0.03 0.98 6.5×10 –4 0.07 0.99 1.0 8.5×10 –4 0.04 0.99 6.1×10 –4 0.04 0.98 1.5 8.4×10 –4 0.10 0.98 5.9×10 –4 0.03 0.99 2.0 5.0×10 –4 0.20 0.99 5.2×10 –4 0.02 0.99