Production of extracellular polymer in Azotobacter and biosorption of metal by exopolymer

Two Azotobacter strains were isolated from alkaline and acid soils. The production of alginate and exopolymer from these two strains showed that, strain AC2 produced high polymer in 2% beet molasses or 1% sucrose broth and addition of nitrogen sources (yeast extract) reduced production of this polymer. The optimum condition for production of maximum polymer production (7.5 mg/ml) was at 200 rpm shaking, pH 7 without addition of nitrogen sources. The production of polymer was reduced at pH 4. The polymer adsorbed Cu, Zn, and Fe, at 15.5, 20 and 25 mg, respectively.


Exo-polysaccharide extraction
Several different media were used to produce exopolysaccharides.The media for maximum polysaccharide production in Azotobacter contains the following 20 g/L sucrose, 3.2 g/L K2HPO4, 0.8 g/L KH2PO4, 0.4 g/L MgSO4.7H2O,0.2 g/L NaCl, 0.020 g/L FeSO4.6H2O,0.03 g/L Na2MoO4, 0.05 g/L CaCO3 at pH 7.2.The bacteria was grown on the optimal media and incubated at 20°C at 200 rpm.The cells were centrifuged at 9000 rpm in 1 mM EDTA.The supernatant was removed and equal volume of cold acetone was added.The precipitated was collected by centrifugation at 20,000 rpm for 30 min.

Biomass as metal biosorption
The harvested biomass was washed with deionized water and then dried at 60°C for 24h in an oven.The growth rate was obtained by optical density of 600 nm.

Bioadsorption experiments
Bioadsorption experiments were conducted using separate solutions containing 10PPM Cu, Fe and Zn 2+ in distilled water.Known amount of polysaccharide or bacteria cells mixed with each metal solution.The reaction mixture was agitated at 125 rpm on rotary shaker.After 1 h of contact time, the pellet was obtained by centrifugation of mixture at 10000 rpm.Metals concentration was measured using a varian AA-10 atomic absorption spectrophotometer.Bioadsorption experiments were carried out in duplicate and average values were used in the analysis.Bioadsorption capacity, i.e. amount of metal ion (mg) bioadsorbed per g (dry mass or polysaccharide) was calculated using the following equation (3).

RESULTS AND DISCUSSION
Two Azotobacter strains, AC1 and AC2, were isolated from dry and wet soils.These isolates were grown in different media.In all media AC2 grew better than AC1 (Figure 1).Both strains had good growth in sucrose (1%).However AC2 produced maximum (7.5 mg/ml) exopolymer in media with sucrose as the only carbon source (Figure 2).AC2 had maximum growth at 4% sucrose (Figure 3).However this strain had maximum exopolymer in 1% sucrose (Figure 2).Different concentration of beet molasses were used for production of polymer.The maximum growth and exopolymer production of AC2 was on 2% beet molasses (Figures 4  and 5).Addition of vitamin, different nitrogen sources (Ammonium salts, yeast extract and peptone) did not effect exopolymer production in Azotobacter.However, shaking had significant effect on exopolymer production by Azotobacter AC2 (Table 1).Production of maximum polymer was at 30ºC, 200 rpm shaking during 4 days in 1% sucrose.The production of this polymor at pH 4 and pH 8 was reduced significantly.This polymer biosorbe metals more than cells.The biosorption of Cu, Zn, Fe Afr.J. Biotechnol.Table 1.The effect of O2 and nitrogen sources on exopolymer production in Azotobacter AC2 grown on Sucrose (1%).
The removal of toxic metals from waste waters has directed attention to biosorption based on the metal binding capacities of algae, bacteria, fungi and yeast as potential metal sorbents (Veglio and Boelchini, 1998;Say et al., 2001).Also various biological materials like live and dead cells of mucor (Yan. and Viraraghavan, 2000), DNA (Sponer et al., 1998;Jaroslav et al., 1997) outer membrane of Escherichia coli (Hoyle and Beveridge, 1983;Ferris and Beveridge, 1986) and microbial envelope can be used for metal removal (Weppen and Homburg, 1995).In this work it was shown that exopolysaccharide from Azotobacter had high capacity to bisorbe metals and sucrose is the best substrate to produce this polymer.
at (100 rpm) without yeast extract as nitrogen sources 5 Shaking at (100 rpm) with vitamin 4