I will assay for the best nitrogenase activity of my nitrogen-fixing bacterial isolates. Can you suggest an easy protocol to do this experiment? I will be making an ethylene standard, however, I don't have any idea on how to do it. We will be analyzing the activity by GC. Thank you very much.
Dear Sir. Concerning your issue about how to make ethylene standard for Acetylene reduction Assay. Nitrogenase activity was determined with an acetylene reduction assay (ARA). Each bacterial isolate was grown in a 20 ml test tube containing 10 ml of N-free semi-solid medium for 72 h at 30°C. Each test tube was sealed with a rubber stopper, and 1 ml of acetylene gas was added to the air in the headspace (10 ml). The test tubes were incubated at 30°C for 24 h. One ml of each gas sample from the headspace was assayed for ethylene (C2H4) production by gas chromatography (GOW MAC series 750, New Jersey, USA) equipped with a hydrogen flame ionisation detector (FID) and a Porapack N column. Nitrogenase activity was calculated as nmol of ethylene per tube per h. To examine the effect of yeast extract on nitrogenase activity, ARA was performed as mentioned above, but N-free semi-solid medium was added with yeast extract at 0%, 0.005%, 0.01% and 0.05%. For more details, I think the following below links may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437324/pdf/tlsr-26-1-41.pdf
http://shodhganga.inflibnet.ac.in/bitstream/10603/84883/20/19_publication.pdf
Thanks
Hi Sir Isam, Thank you very much for your answer. This helps. Before adding acetylene gas to the tube, do I need to remove equal volume of air from the tube?
Kindly explain me how to remove the air from the tube for acetylene reductase assay?
Hi Mr. Tiwari and Mr. Xu,
I used a sterile syringe (the smallest size). You have to cover your tube with rubber septum so you could immediately remove air and insert acetylene gas inside the tube.
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