The culture conditions for the production of carrageenase were optimized using one-factor-at-atime method combined with orthogonal array design. With the one-factor-at-a-time method revealed optimal conditions for carrageenase production were 24 h of fermentation period, 28 °C incubation temperature at pH 8.0 with NaNO 3 as nitrogen source and carrageenan as carbon source in MMS media. Further optimization of carrgeenase production by using orthogonal experimental design L 9 (3 4 ) with four factors, temperature, pH, NH 4 NO 3 and carrageenan with their relevant levels revealed optimised conditions for carrageenase production were temperature of 28 °C, pH 8.0, 2 g L -1 NaNO 3 and 2 g L -1 carrageenan. The order of the factors affecting the fermentation process was found to be temperature > pH > NaNO 3 > carrageenan. The temperature played a significant role on the carrageenase production. Higher carrageenase yield with activity of 0.542 ±0.045 U ml -1 was obtained in the optimised medium when compared to those of basal medium. Carrageenase hydrolysed products of carrageenan were identified by LC-ESI-MS as neocarrabiose, neocarrabiose-4 sulfate, neocarratetraose, neocarratetraose-4 sulfate, anhydrogalactose, galactose, galactose-4 sulphate and sulphate.

The culture conditions for the production of extracellular agarase by Pseudomonas aeruginosa ZSL-2 were optimized using One-Factor-At-A-Time combined with orthogonal array design. One-Factor-At-A-Time method investigates the effect of time, temperature, NaCl, carbon sources, nitrogen sources and pH on agarase production. The optimized culture conditions obtained from the statistical analysis were temperature of 30 °C, pH 8.5, NH4NO3 2 g L-1 and agar 3 g L-1. The L9 orthogonal array design was used to select the fermentation parameters influencing the yield of agarase. The order of the factors affecting the fermentation process was found to be NH4NO3 > pH > agar > temperature, with temperature playing a significant role on the agarase production (p < 0.10). The higher yields than those in basal media culture were obtained in the final optimized medium with activity of 0.439 ± 0.013 U ml-1. Extracellular agarase hydrolysed agar into a range of oligosaccharides which were analysed by LC-ESI-MS spectrometry as anhydrogalactose, galactose, agarobiose, agarotetrose and agarohexaose.

The optimization of solid-substrate medium and fermentation conditions for the production of enzyme carrageenase by a Pseudomonas aeruginosa ZSL-2 was achieved for the first time by employing an orthogonal array testing strategy (OATS). Of the four different substrates tested, wheat bran showed maximum production of carrageenase enzyme. The factors that influence the solid-substrate fermentation (SSF) conditions such as, moisture content, moistening agent, temperature, pH, inoculum size, additional carbon source and the fermentation period for the production of enzyme were studied by one-factor at a time and also by L9(3 4 ) an orthogonal array method. The maximal production of k-carragenase (7.44 U/g) dry bacterial bran was achieved at moisture level 1:2.5 (w/v; wheat bran to moisture level), moistening agent IV, inoculum size (10%), temperature 37°C and 48 h of fermentation. The effects of various parameters like medium, inoculum size, temperature, moistening agent, moisture level, incubation period, and supplementation of other carbon sources on the production of carrageenase by SSF using OATS is investigated and the results are presented and discussed.

The culture conditions for the production of extracellular agarase by Pseudomonas aeruginosa ZSL-2 were optimized using One-Factor-At-A-Time combined with orthogonal array design. OneFactor-At-A-Time method investigates the effect of time, temperature, NaCl, carbon sources, nitrogen sources and pH on agarase production. The optimized culture conditions obtained from the statistical analysis were temperature of 30 °C, pH 8.5, NH 4 NO 3 2 g L -1 and agar 3 g L -1 . The L 9 orthogonal array design was used to select the fermentation parameters influencing the yield of agarase. The order of the factors affecting the fermentation process was found to be NH 4 NO 3 > pH > agar > temperature, with temperature playing a significant role on the agarase production (p < 0.10). The higher yields than those in basal media culture were obtained in the final optimized medium with activity of 0.439 ± 0.013 U ml -1 . Extracellular agarase hydrolysed agar into a range of oligosaccharides which were analysed by LC-ESI-MS spectrometry as anhydrogalactose, galactose, agarobiose, agarotetrose and agarohexaose.

The culture conditions for the production of carrageenase were optimized using one-factor-at-a-time method combined with orthogonal array design. With the one-factor-at-a-time method revealed optimal conditions for carrageenase production were 24 h of fermentation period, 28 °C incubation temperature at pH 8.0 with NaNO3 as nitrogen source and carrageenan as carbon source in MMS media. Further optimization of carrgeenase production by using orthogonal experimental design L9 (34) with four factors, temperature, pH, NH4NO3 and carrageenan with their relevant levels revealed optimised conditions for carrageenase production were temperature of 28 °C, pH 8.0, 2 g L-1 NaNO3 and 2 g L-1 carrageenan. The order of the factors affecting the fermentation process was found to be temperature > pH > NaNO3 > carrageenan. The temperature played a significant role on the carrageenase production. Higher carrageenase yield with activity of 0.542 ±0.045 U ml-1 was obtained in the optimised medium when compared to those of basal medium. Carrageenase hydrolysed products of carrageenan were identified by LC-ESI-MS as neocarrabiose, neocarrabiose-4 sulfate, neocarratetraose, neocarratetraose-4 sulfate, anhydrogalactose, galactose, galactose-4 sulphate and sulphate