Optimization of the Phosphate-Solubilizing Fungus, Aspergillus japonicus SA22P3406, in Solid-State Cultivation by Response Surface Methodology

Abstract

Phosphorus (P) is one of the essential macronutrients for plant growth and reproduction. Plants acquire P from the soil solution as phosphate anions. However, phosphate anions are extremely reactive and may be immobilized through precipitation with cations such as Ca²⁺, Mg²⁺, Fe³⁺ and Al³⁺, depending on the particular properties of the soil and as a result, the phosphate is highly insoluble and unavailable to plants. Application of phosphate-solubilizing fungi (PSF) has been added as fertilizer to increase P uptake and plant growth. The aim of this study was to use response surface methodology to determine the optimal factors of the phosphate-solubilizing fungus Aspergillus japonicus SA22P3406 for use in solid state cultivation, specifically the pH, temperature and ratio of water to dry rice bran (RWB). A. japonicus SA22P3406 was screened from 24 soil samples. Primary screening was carried out by a 10-fold serial dilution of the soil samples at 10^-4 to 10^-6 and then spread on Pikovskaya agar supplemented
with 1.0% w/v of tricalcium phosphate at 37°C for 3-5 d. One hundred and fifty five fungal isolates produced high ratios of clear zone to colony on Pikovskaya agar supplemented with 0.003% w/v of rose bengal at 37°C for 7 d. The ratios were calculated by dividing the area of the clear zone by the area of the colony. Secondary screening was performed by measuring the available phosphate in liquid medium from the 62 fungal strains by CRD and DMRT at 5% probability. Two fungal strains, A. niger SA07P3332 and A. japonicus SA22P3406, were selected as most suitable for solubilizing tricalcium phosphate and producing the highest available phosphate levels of 3.01 and 2.99 mg P₂O₅ ml^-1, respectively. A. japonicus SA22P3406 was cultivated in 5.0 g rice bran to study the optimal conditions using response surface methodology with a central composite design. The optimum conditions of pH, temperature and RWB were 7.37, 36.0°C and 82.10% v/w of dry rice bran, respectively. These conditions were used in the
solid-state cultivation and a sporulation curve was determined. The fungal sporulation had a lower than average decadic logarithm of practical quantitative sporulation of 8.46 compared with the estimated quantitative sporulation of 8.53 at the 5th day of incubation.