Question

You think you have nitrogen fixers in an anaerobic part of your Winogradsky column. You cap the column with 15N2 gas (a non-radioactive N isotope) and want to identify the active N2 fixers in this region of the column both metabolically and molecularly. Describe in detail the approach you would take to identify the active nitrogen fixers

Answer 1

For the given question first, we have to isolate the bacteria present in the anaerobic part of the Winogradsky column as pure culture and we need to grow them as a pure culture in the anaerobic condition. After that, for each culture, we need to perform an acetylene reduction test. This test is special for the nitrogen-fixing bacteria because they will have an enzyme called nitrogenase. For this test, the bacteria will be kept in a gas-tight vessel and the vessel will be filled with 5-10% of acetylene. In the presence of this acetylene, the enzyme will lose its affinity towards dinitrogen and start reducing acetylene to ethylene. As ethylene is not necessary for the bacteria to grow, they will release it in the air and we need to collect the air from the vessel. Then we have to perform gas chromatography to check the percentage of ethylene in the air. the change in ethylene percentage will show which bacterial culture can fix nitrogen.

For confirming we can choose specific bacteria and do specific tests for nitrogen fixtion invitro.