Friday, February 20, 2015

022 - The Influence of Fixed Nitrogen on Azotobacter

Azotobacter fixes nitrogen very well, but if fixed nitrogen is already available, it'll use that first. Makes sense; why waste the energy?

Others had already found that Azotobacter could use nitrogen in various forms—nitrates, ammonium, peptides. But their methods were somewhat coarse and clumsy.

What They Wanted to Know
Burk and Lineweaver wanted to use better techniques (for the time) to investigate Azotobacter's nitrogen physiology more thoroughly.

What They Did
They measured oxygen consumption by organisms grown in something pretty close to what is called Burk medium today. They also measured cell growth by direct cell counting. This experiment was done with both A. chroococcum and A. vinelandii.

They claim that changes in growth rate correlate well with changes in oxygen consumption. This means oxygen consumption can be a proxy for growth/nitrogen fixation, though the correlation is not the same in all conditions for all strains. Also, growth efficiency increases as growth rate increases, so less oxygen is consumed. So it's hard to extrapolate much.

Then, with some cultures, they added humic acid to influence the growth rate (it seemed to increase it). And measured growth rate and respiration rate in various atmospheres, each with 20% oxygen, varying concentrations of nitrogen, and the remainder hydrogen.

They also tried growing cultures with different forms of fixed nitrogen to see which inhibited nitrogen fixation.

What They Observed
The Relation of Growth and Respiration
With or without humic acid, the respiration rate increased as the proportion of nitrogen gas in the headspace increased, though the final value was a lot higher when humic acid was present. In terms of the ratio of the increase of growth over the increase of respiration rate, this also increased as nitrogen proportion increased, and when humic acid was present it increased more. So as nitrogen increased, the proportionality of growth and respiration changed.

It also seemed like the more humic acid, the better the growth and respiration, up to about 0.5 mg/mL (above which the effect leveled off). And adding fixed nitrogen in the form of ammonia definitely helped, the more the better up to 0.05 mg/mL; and the ratio of growth increase to respiration increase was much higher than with humic acid or nitrogen gas. So the type of nitrogen provided affects this ratio.

Growth Efficiency Depends on Oxygen Concentration
They also noticed that this ratio increases greatly as the oxygen concentration goes down. This makes sense, since less oxygen is being consumed, but it means the efficiency of growth goes up, as we've seen in other studies (090, 105). Presence or lack of ammonia doesn't seem to affect this.

Inhibition of Nitrogen Fixation
In 21% oxygen, with the remainder either nitrogen or hydrogen (man, 21:79% oxygen:hydrogen seems like a recipe for explosions), the type of gas didn't affect the respiration of cells when added fixed nitrogen was above 5 mg/L; below this amount, the cells started fixing nitrogen, and then growth and respiration increased more with nitrogen gas than with hydrogen.

When they added nitrate instead of ammonia, they observed the same thing: 5mg/L was sufficient to inhibit nitrogen fixation, at least as measured by amount of growth.

And in either case, the amount of nitrogen in the cells remains pretty constant regardless of whether cells are fixing it or just taking it up already fixed.

What This Means
Azotobacter's ratio of growth to respiration probably varies so much because its respiration rate can vary so much, up to 3 times its own dry weight in glucose per hour, which is 25 times more than yeast. It's just interesting that growth and respiration are so loosely connected; in some conditions, growth can change 89-fold while respiration only changes 5-fold.

I'm not really clear on why the humic acid has the effect it does.

It seems that the type of fixed nitrogen doesn't matter when it comes to inhibiting nitrogen fixation, at least between ammonia and nitrate. Other forms might be different; the authors cite some other work that showed that peptone might have less inhibitory effect (or perhaps just as much).

Overall, some interesting basic observations of Azotobacter physiology, made only a few decades after A. vinelandii was discovered.

Burk, D. & Lineweaver, H. The Influence of Fixed Nitrogen on Azotobacter. J. Bacteriol. 19, 389–414 (1930).

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