JIA-2019-11

2537 Slaven Jurić et al. Journal of Integrative Agriculture 2019, 18(11): 2534–2548 performed at room temperature. Microspheres were stained with fluorescent dye (eosin, 0.01%). Loading efficiency, loading capacity and swelling degree of microspheres Detailed procedures for the determination of loading efficiency (LE), loading capacity (LC) and swelling degree (S w ) were previously described (Vinceković et al . 2016, 2017). The concentration of calcium cations was determined at λ=650 nm and concentration of T . viride spores (expressed as the number of spores per 1 g of dry microsphere, NS g –1 ) at λ=550 nm by a spectrophotometer (Shimadzu, UV-1700). LE (%) was expressed as the percentage of total available calcium c tot (mol dm –3 ) and calculated by the equation: LE =( c load / c tot )×100 (1) where c load = c tot – c f , and c f is a concentration of calcium ions (mol dm –3 ) in filtrate. The loading efficiency of T . viride spores in microspheres was determined by the same procedure. No T . viride spores presence in filtrate after microspheres preparation indicated almost 100% of loading. Calcium loading capacity (mmol g –1 ) expressed as a calcium ions mmol per 1 g of dry microspheres was calculated by the equation: LC Ca =( C Ca × V / w c ) (2) where C Ca (mmol dm –3 ) is a concentration of calcium ions in the sample, V (dm 3 ) is a volume of the sample and w c (g) is a weight of microspheres. T . viride spore loading capacity expressed as the number of spores per 1 g of dry microspheres (NS g –1 ) was calculated by the equation: LC Tv =( C NS × V / w c ) (3) where C NS (NS dm –3 ) is a concentration of spores in the sample, V (dm 3 ) is a volume of the sample, and w c (g) is a weight of microspheres. The swelling degree (%) was calculated using the equation: S w = w t – w 0 w 0 (4) where w t (g) is the weight of the swollen microspheres, and w 0 (g) is their initial weight. All measurements were replicated three times and results are presented as the mean values. In vitro T. viride and calcium ions release from microspheres The release experiments frommicrosphere are designed to achieve conditions as close to the intended application in the soil and in hydroponic conditions. Samples for measurements were prepared by dispersing 4 g of microspheres in 100 mL of deionized water and allowed to stand without stirring during experiments. At appropriate time intervals, dispersion was stirred for 60 s, aliquots were withdrawn and the spore count was determined spectrophotometrically. The release experiments from microspheres were carried out at room temperature (~293 K). Results are presented as the fraction of released bioactive agents using the equation: f = R t R tot (5) where f represents the fraction of released T . viride , f Tv , or calcium ions, f Ca , R t is the amount of T . viride (NS mL –1 ) or calcium ions (mol dm –3 ) released at time, t and R tot is the total amount of T . viride spores or calcium ions loaded in microspheres. All measurements were replicated three times and results are presented with the standard deviation of the means. Statistical analysis The results were statistically analyzed with Microsoft Excel 2016 and XLSTAT Statistical Software add-in. The data are shown as mean value±standard deviation. 3. Results and discussion The results are presented and discussed in two sections. In the first section, the interaction between T . viride spores and calcium ions are analysed. In the second section, essential physicochemical properties of microspheres prepared at various calcium chloride concentrations without and with T . viride spores are described. 3.1. The interaction between T. viride spores and calcium ions In order to test T . viride spores bioactivity in the presence of various calcium chloride concentrations, growth and sporulation were examined in suspensions, inoculated on PDA substrate or loaded in microspheres. Morphology, size and charge of T. viride spores Fig. 1 presents a SEM image of dry T . viride spores and FM images of spores suspended in calcium chloride solutions. Dried T . viride spores appear similar in size (around 3.6 µm) and shape to Penicillium (Ye et al . 2002) and Aspergillus (Kwon-Chung and Sugui 2013) spores (Fig. 1-A). Oval shaped spores showed deep surface holes, which may have been related to the loss of parts of the spore wall or even material inside. T . viride spores were relatively easily suspended in water (Fig. 1-B) indicating prevailing surface hydrophilicity. Spores suspended in water at a concentration of 1.8×10 –6 mL –1 showed negative zeta potential (around –9 mV) which arises from various functional groups such as carboxyl, hydroxyl, and amine. This is in accordance with the analysis of the relationship between relative surface hydrophobicity and surface electrostatic charge. Singh et al . (2004) found an inverse relationship between cell surface electrostatic charge (zeta potential ranged from –11 to –42 mV) and cell