Submerged Alginate Biopolymer production by Azotobacter chroococcum Isolated from soil

The purpose of this study was submerged production of alginate biopolymer by Azotobacter sp. which was isolated from soil and elevation of production by experiment design for optimization of culture conditions. Soil samples were cultured in Azotobacter broth medium enriched by 0.1% phenol as sole carbon source. Initial identification of Azotobacter sp. was done by morphological and biochemical properties and the isolated bacterium was exactly identified by PCR amplification of nifH-g1 region in Azotobacter genome. Production of alginate biopolymer was estimated by gravimetric method in culture supernatant after separation of Azotobacter cell biomass in Clementi medium. Also, the produced biopolymer was confirmed as alginate by biological method via immobilization of Vibrio fischeri ATCC 7744 cells in it and detection of bioluminescence production by immobilized cells. Then the factors affecting alginate biopolymer production by the isolated Azotobacter sp. including glucose, lactose and molasses as carbon sources (20 g L-1); yeast extract, urea and ammonium sulfate as nitrogen sources (6 g L-1); temperature (24, 25 and 26°C) and pH values (7.2, 7.4 and 7.6) were optimized in Clementi medium using Taguchi statistical method (Qualitek 4 software). The isolated bacterium was characterized as Azotobacter chroococcum after alignment of amplified genome region in Gene bank. The highest amount of alginate biopolymer obtained in optimal conditions using lactose and yeast extract as nutritional sources in 25oC and pH=7.6. The amount of polymer production was equal to 3.58 g L-1 in mentioned conditions. Also, the main effect was belonged to carbon source and the interaction between carbon and nitrogen sources was reported as the most effective interactions between analyzed factors.