1. Applications of Biotechnological Processes Enhancing Nitrogen Fixing

2. Nitrogen fixation Revision Nitrogen is an element required for the amino groups of proteins. Nitrogen is taken up by plants in the form of ammonium or nitrate. These supplies of nitrogen are usually limiting. Nitrogen fixing bacteria convert atmospheric nitrogen into ammonia. Nitrogen fixing bacteria can be free living e.g. Azotobacter or be in symbiosis with plants (root nodules) e.g. Rhizobium.

3. Enzyme nitrogenase is responsible for the conversion of nitrogen to ammonia. Nitrogenase operates in anaerobic conditions but nitrogen fixation is an ATP demanding reaction (aerobic respiration is therefore necessary). Leghaemoglobin in root nodules binds to oxygen to prevent it disabling nitrogenase activity. Leghaemoglobin releases oxygen slowly for aerobic respiration to take place.

4. Rhizobia Contain a set of nif genes that are responsible for the coding of nitrogenase. However the expression of nif genes alone is not sufficient for nitrogen fixation to take place. Scientists have attempted to transfer nif genes to other bacteria e.g. E.coli and directly into plant cells. However nitrogenise expression has not been successful

5. Enhancing nitrogen fixing Farmers can add Rhizobia inoculum cultures to their legume fields (this reduces the need for artificial fertiliser and increases crop yield). Mutant Rhizobia have been engineered so that the nif genes are never “switched off”. The effector complex which binds to the nif genes to prevent expression in high nitrogen environments, is prevented from binding

6. There are many species of Rhizobia. A single species will form a symbiosis with only certain legumes (there is a degree of specificity between plant and bacteria). This specificity is due to the type of plasmid carried by the Rhizobia. The plasmid produces enzymes that are responsible for producing signal molecules that are detected by the plant. It is possible to transfer the plasmid of one Rhizobia species to another Rhizobia.

7. The plasmid from (a) is transferred to Rhizobium (b), to create (c) that can infect pea and carry out good fixation. (b)(c)(a) Infects pea plants Moderate growth Moderate fixation Infects clover plants Good growth Good fixation Infects pea plants Good growth Good fixation

8. An ideal inoculum will:  Have a large number of bacteria  Have been mutated to ensure that nif genes are permanently switched on  Have the correct plasmid for the target plant species Past Paper Questions 2004 Q1(ii)