1. Sterile mineral medium composition:

KNO3 : 25g, MgSO4 • 7H2O: 10 g, KH2PO4: 4 g, K2HPO4: 1 g, FeSO4 • 7H2O: 1 g, H3BO3: 2.86 |g, MnCl2 • 4H2O: 1.81 |g, ZnSO4 • 7H2O: 0.11 |g, CuSO4 • 5H2O: 0.09 |g, Na2MoO4: 0.021 |g/L. Axenic Chlorella cultures were incubated for 5-6 d under continuous agitation (150 rpm), light intensity of 31.8 W/m2 , and at 27-30°C.

2. Azospirillum brasilense strain Cd DMS 1843 was grown in liquid nutrient broth (Sigma) or M9 nitrogen-free medium (composition g/L: Na2HPO4: 6 g, KH2PO4: 3 g, NaCl: 1.5 g, MgSO4 • 7H2O: 1 M; CaCl2: 0.01 M, malic acid: 20%) at 30 ± 2°C and agitation (120 rpm) for 17 h.

3. As immobilization normally reduces the number of bacteria in the beads, incubate the beads overnight in diluted nutrient broth (1:10). After washing thrice in sterile solution the beads are ready for application.

4. To date, operations of algal biosensors are based on two major principles. In the first type, the operating principle is based on the inhibition of the activity of AP. AP activity is determined with methylumbelliferoyl phosphate (MUP) as sub strate, which is converted into methylumbelliferone (MUF), a fluorescent compound by the action of alkaline phosphatase.

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