That nitrogenase produces hydrogen gas had been known for a while; this study wanted to see how different nutrient limitations affected this phenomenon.
They grew Azotobacter chroococcum on mannitol B medium in continuous culture, with limitations in carbon, sulfate, oxygen, or dinitrogen. Then they measured acetylene reduction, hydrogen production, and oxygen consumption in vivo and the former two on purified nitrogenase. Also update hydrogenase activity directly.
What They Saw
There was very little hydrogen evolved by oxygen-limited cultures in air, unless the uptake hydrogenase was inhibited by acetylene first. Replacing air with an argon/oxygen/CO2 mix (without nitrogen) also increased hydrogen evolution to a similar level. With both treatments, the hydrogen seen was much higher.
When limited in sulfate, the hydrogen produced under air (with hydrogenase active) or under argon mix seemed much higher (at least proportional to the amount of protein in cells).
Under argon mixes with different proportions of oxygen, hydrogen evolution seemed highest around 10% oxygen. With nitrogen instead of argon, the peak was similar. Too much or too little oxygen was not good. And despite the high amount of hydrogen, around 10% (actually between 6-12%) was when the most nitrogen was being fixed too, such that the ratio of hydrogen produced to nitrogen fixed was as low as 1 (or 0.5, when sulfate-limited).
In carbon-limited cultures, though, the optimum oxygen value was 3%, though oxygen consumption increased as oxygen increased, at least up to 6%.
In vitro, the hydrogen-nitrogen ratio increased as the ratio of dinitrogenase to dinitrogenase reductase increased, though higher levels of ATP decreased this effect. Sulfate limitation didn't really affect this finding.
What This Means
This makes sense; some oxygen is required to generate ATP to power the nitrogenase, but too much oxygen requires extra carbon to detoxify it, so there's less energy for nitrogenase.
It seems like a lack of dinitrogenase reductase or ATP reduces the ability to fix nitrogen instead of just producing hydrogen. I wonder if dinitrogenase has any effect on its own in the absence of ATP or its other component.
Chemically it's unclear how or why nitrogenase produces hydrogen, but it seems to be an essential part of the nitrogen fixation process.
When limited in sulfate, the hydrogen produced under air (with hydrogenase active) or under argon mix seemed much higher (at least proportional to the amount of protein in cells).
Under argon mixes with different proportions of oxygen, hydrogen evolution seemed highest around 10% oxygen. With nitrogen instead of argon, the peak was similar. Too much or too little oxygen was not good. And despite the high amount of hydrogen, around 10% (actually between 6-12%) was when the most nitrogen was being fixed too, such that the ratio of hydrogen produced to nitrogen fixed was as low as 1 (or 0.5, when sulfate-limited).
In carbon-limited cultures, though, the optimum oxygen value was 3%, though oxygen consumption increased as oxygen increased, at least up to 6%.
In vitro, the hydrogen-nitrogen ratio increased as the ratio of dinitrogenase to dinitrogenase reductase increased, though higher levels of ATP decreased this effect. Sulfate limitation didn't really affect this finding.
What This Means
This makes sense; some oxygen is required to generate ATP to power the nitrogenase, but too much oxygen requires extra carbon to detoxify it, so there's less energy for nitrogenase.
It seems like a lack of dinitrogenase reductase or ATP reduces the ability to fix nitrogen instead of just producing hydrogen. I wonder if dinitrogenase has any effect on its own in the absence of ATP or its other component.
Chemically it's unclear how or why nitrogenase produces hydrogen, but it seems to be an essential part of the nitrogen fixation process.
Reference:
Walker, C. C., Partridge, C. D. P. & Yates, M. G. The Effect of Nutrient Limitation on Hydrogen Production by Nitrogenase in Continuous Cultures of Azotobacter chroococcum. J. Gen. Microbiol. 124, 317–327 (1981).
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