Background and Objectives: Psychrotrophic bacteria can produce enzymes at low temperatures; this provides a wide biotechnological potential, and offers numerous economical advantages over the use of mesophilic bacteria. In this study, extracellular protease production by psychrotrophic Rheinheimera sp. (KM459533) was optimized by the response surface methodology. Materials and Methods: The culture medium was tryptic soy broth containing 1% (w v-1) skim milk. First, the effects of variables were independently evaluated on the microbial growth and protease production by one-factor-at-a-time method within the following ranges: incubation time 24-120 h, temperature 15-37°C, pH 6- 11, skim milk concentration 0-2% (w v-1), and inoculum size 0.5-3% (v v-1). The combinational effects of the four major variable including temperature, pH, skim milk concentration, and inoculum size were then evaluated within 96 h using response surface methodology through 27 experiments. Results and Conclusion: In one-factor-at-a-time method, high cell density was detected at 72h, 20°C, pH 7, skim milk 2% (w v-1), and inoculum size 3% (v v-1), and maximum enzyme production (533.74 Uml-1) was achieved at 96h, 20°C, pH 9, skim milk 1% (w v-1), and inoculum size 3% (v v-1). The response surface methodology study showed that pH is the most effective factor in enzyme production, and among the other variables, only temperature had significant interaction with pH and inoculum size. The determination coefficient (R2=0.9544) and non-significant lack of fit demonstrated correlation between the experimental and predicted values. The optimal conditions predicted by the response surface methodology for protease production were defined as: 22C, pH 8.5, skim milk 1.1% (w v-1), and inoculum size 4% (v v-1). Protease production under these conditions reached to 567.19 Uml-1. The use of response surface methodology in this study increased protease production by eight times as compared to the observed before optimization.

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