Procedure

1. Empty Nestle 1.5 litre water bottle.
2. Pour Dasani 1.5 litre water into the empty Nestle bottle.
3. Remove 24 ml water
4. Add 7.5ml of each of the Complete Salts solution, phosphate solution, and Acetate solution into the water. *Use 7.5 ml of Sulphur-free salts solution for the sulphur deficient media.
5. Add 1.5 ml of the complete trace elements into the water. * Use 1.5ml of sulphur-free trace elements for the sulphur deficient media.
6. Remove 10 ml of the media.
7. Add 10 ml of Chlamydomonas Reinhardtii algae to the media.
8. Shake the bottle and cover with lid, leaving the spout open.
9. Grow for 3 days. *Note the sulphur deficient must be closed immediately.
10. Repeat for 5 regular (for 4 different concentrations of copper and 1 control) bottles and 1 sulphur deficient bottle.

1. Add 0.786 g of Copper (II) Sulphate Pentahydrate to 1 litre of water. *This creates a solution where each 1.5 ml will increase the copper concentration of a 1.5 litres of water by 0.2 ppm. (for calculations refer to log book)
2. For 0.8ppm Cu: Add 3.0 ml (will give 0.4ppm + 0.2ppmx2 = 0.8ppm) of the copper sulphate solution to the culture media.
3. For 1.0ppm Cu: Add 4.5 ml (all calculations are similar as above, refer to log book) of the copper sulphate to the culture media.
4. For 1.6ppm Cu: Add 9 ml of the copper sulphate solution to the culture media.
5. For 2.0ppm Cu: Add 12 ml of the copper sulphate solution to the culture media.
6. Close the spouts on the bottles, including the control culture which has a normal concentration of 0.4 ppm Cu.

1. Put sealant tape on tubing A so that it forms a tight seal when put inside tubing B.
2. Slide tubing A into B.
3. Put sealant tape on the point where they join, make sure there is no leak.
4. Attach the plastic connecter tightly into the open end of tubing A.
5. Put balloon on the plastic connecter, make sure it is tight.
6. Repeat for all 6 bottles.
7. Attach the 6 gathering apparatus’s to the 6 different bottles. **

1. Grow algae on 12 hour light/dark cycle approximately 15 cm from two florescent light bulbs, maintain 25°C.
2. Let the control and copper media grow for 3 days before attaching the gathering apparatus. Attach the gathering apparatus of the sulphur deficient immediately. **
3. After 3 days attach the gathering apparatus to the copper and control cultures. This will now close the system which will then become anaerobic eventually.
4.  Over the next 7 days (3+ 7 =10 days for the total experiment) monitor the bottles and if needed close the spout to mix the algae if it is settled mostly at the bottom
5. On the last day tap the bottles and try to squeeze out any remaining bubbles.

(fig 6-3) Algae bottle, trail 2, final day of experiment gas bubbles can be seen

(fig 6-4) Algae, 400x microscope view