Hello, I spent lots of time to figure out ARA method to determine nitrogen fixation by free living in soil. However, I am not getting significance difference between ethylene peaks of control (no soil) and soil sample. Could improve my experiment?
I used following parameter:
1. Prepared 1140mL jar with rubber septum.
2. Put 100g moisture soil into the jar and seal with rubber septum.
3. A control (no soil) will set up simultaneously.
4. Replace 10% of 1040mL air volume in to the jar (104mL) from by acetylene using gas-tight syringe (10%/100×1040=104mL) into the jar. One set up prepared for control (no soil) with same acetylene ratio.
5. Jar set up left for 24h incubation at 22C temp. After 24h incubation, inject 20ml of N2 to make pressure mix well and withdraw 20mL gas samples and inject into 12mL already evacuated tube.
9. A 0.5mL amount sample analysed with GC (FID). As a standard 22ppm ethylene was used. As result, for control the peak height is 2396 and for soil sample (100g) it is 2513. It is calculated with 22ppm, get 4.38ppm ethylene in control and 4.86ppm ethylene in soil sample.
However, i think it is not good formation of ethylene in to 1125mL headspace.
In the nitrogen fixation process atmospheric nitrogen is reduced to ammonia in the presence of nitrogenase. ATP is the source of energy necessary for the cleavage and reduction of nitrogen into ammonia. The synthesis of ATP require more phosphorus, as well as nitrogenase is an oxygen sensitive enzyme and Cu, Mo, Co, B are necessary for N fixation.
A problem that is inherent in Acetylene reduction assay (ARA) is the need to calibrate the rates of ethylene production with the actual rate of N fixation. The commonly used ratio of 3:1 for acetylene reduced per N2 fixed is not always valid. I think the problem you are facing may be due to deficient P in your soil or you have less oxygen or your soil may be deficient in available Cu, Mo, Co or B which discourage the nitrogenase activity. You can go for Harber process to estimate your soil ability for N fixation.
Mr. Kumar,
Your methodology for quantifying ethylene is appropriate for assessing ethylene production. Apparently your soil has a low population of free-living N2 fixing bacteria or the free-living bacteria have low nitrogenase activity due to some nutrient limitation. This could be Mo, Fe , Ca, P and others. Mo is in the active site of nitrogenase and therefore crucial for enzymatic function.. Mo availability in soil increases as soil pH increases and is very low in acidic soils (pH < 5.0). What is the pH of the soil that you used in the test? If P was deficient, energy production to support N2 fixation would be impaired.
I hope this information proves helpful for you .
Dan Israel
Thank you for answering. I tried different ARA protocol with different type of soil. However, i am not getting good peaks of ethylene formation comparative to control (no soil).
Mr. Kumar,
Have you determined the nutrient status of these soils as mentioned in previous response?
Dan Israel
Hi Dan,
I determined pH for those soil, which are in between 6 to 7.5. I have not more idea about to determine nutrients. So, i did not do still. Could you please share protocol/method for that.
However, I used different location's soil and stored in freeze until use after those were left for 5 days at room temp to thaw and equilibrate. And just one soil sample is from native prairie that sampled in September. Still is there any chance to not have nutrients? Thanks...
Mano,
Yes, I would do soil tests on your samples to determine if any essential plant nutrients are limiting for plant growth . Mo,Fe and P are closely related to nitrogenase function. If these nutrients are limiting to plant growth then it is likely microbial function would be limited as well.
Dan
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