Optimization of Ice Nucleation Activity of a Newly Isolated Pseudomonas sp. IRL.INP۱ Using Rice Bran Based on Response Surface Methodology

Introduction: Several isolated species from symptomatic frozen leaves and soil produce ice nucleation proteins and have been exploited for their Ice Nucleation Activity (INA). Ice nucleation proteins can be employed as promising substances for biotechnological applications such as artificial snow-making, cryopreservation of tissues, and the condensation of ice nuclei in clouds. Considering INA has a direct relationship with bacterial growth, optimization of the culture medium seems indispensable. In this study, the INA of a newly isolated Pseudomonas sp. IRL.INP۱ was evaluated.Materials and Methods: Plackett–Burman was applied for screening several cost-effective carbon and nitrogen sources affecting bacterial growth and INA. The response surface method was employed for medium optimization. Moreover, biomass, whole-cell protein, specific growth rate, and INA were estimated.Results: Rice bran, ammonium sulfate, temperature, and olive oil had significant effects on the INA of Pseudomonas sp. IRL.INP۱. Results demonstrated that ۵% rice bran, ۳۱ °C, ۱.۰% olive oil, and ۶ g/L ammonium sulfate led to the best INA. The final optical density at ۶۰۰ nm was ۲.۳. Also, ۱.۹۴ g/L biomass, ۱.۷۵ µg/µl whole-cell protein, and ۰.۲۶ specific growth rate (day-۱) were obtained, and INA was observed after ۵ sec at -۵ °C.Conclusions: The present research is the first study using agricultural waste for INA optimization. Since rice bran is a cost-effective agro-waste and a qualified replacement for glucose, it can be utilized as a promising substrate for bacterial growth and INA.

Keywords:

Pseudomonas , rice bran , Olive oil , Placket-Burman Statistical Design , Response Surface Methodology