106 - Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

What They Wanted to Know

As mentioned last time (105), as the carbon-to-nitrogen ratio of culture medium increases (and the carbon becomes a lot more available than fixed nitrogen), Azotobacter vinelandii biomass stays fairly level for a time, and then starts increasing; it's like two different phases. This depends on the oxygen exposure somewhat (at low oxygen, biomass increases more linearly; at high oxygen, it doesn't increase much at all, at least at the C/N ratios tested), but is a definite phenomenon at some levels.

The hypothesis is that, when there's not much more carbon than fixed nitrogen, there's not enough nitrogen to produce much more biomass (nitrogen is limiting), and there's not enough carbon to make the cells start fixing nitrogen (because that takes a lot of energy; so carbon is also limiting). But as carbon increases, the cells start up their nitrogenase, and nitrogen stops being limiting, so biomass increases.

In this paper, Bühler, Oelze, and colleagues wanted to see if this was actually what was happening in the cells, by testing nitrogenase activity directly.

What They Did

As before, they grew A. vinelandii CA in a chemostat, but this time they measured nitrogenase activity by acetylene reduction (nitrogenase can reduce acetylene/ethyne to ethylene/ethene, which is easy to measure). They also measured total nitrogen and protein contents of the culture, corrected for added ammonium. And to make triple-sure, they did Western blots on samples of culture, using antibodies targeting nitrogen-fixing proteins.

What They Observed

As in 105, protein/nitrogen content remained fairly constant at a mid-range oxygen level up to a point as sucrose increased, and beyond that point, it increased proportionally along with dry weight.

For nitrogenase activity, they saw that the higher the oxygen, the higher the C/N ratio had to be before the cells had detectable nitrogenase activity (and the lower the peak activity at the highest carbon level). After nitrogenase started, it increased up to a certain C/N level, then leveled off. 

They give a formula for how to calculate the C/N ratio when nitrogenase starts working. And based on that, they figured out that cells started fixing nitrogen when the ammonium they were given was not enough for production of biomass from the sucrose they were given. Which makes sense. That happens at about 14 mmol ammonium per gram of protein.

Finally, they wanted to figure out whether nitrogenase proteins needed to be synthesized from scratch in ammonium-grown cells, or whether they were already present to some extent, just not active. So they used Western blots to look at nitrogenase proteins from cells at various C/N ratios. The lowest ratio showed no nitrogenase activity and no visible nitrogenase protein on the blot; mid-range showed slight activity and the faintest of bands; and the highest showed good activity and solid, visible bands. Flavodoxin proteins, related to electron transport to nitrogenase, showed up at all ratios, interestingly.

What This Means

It appears from this that cells have to produce nitrogenase proteins from scratch as C/N ratios increase, but I'm not sure it's clear that inactive versions of the proteins would show up on the blot. Maybe the antibodies they used to detect the active versions don't work well on inactive versions. It's possible.

The other possibility is that A. vinelandii does keep inactive nitrogenase around for short periods, but eventually breaks it down, and the cells in this study were kept too long in nitrogen-sufficient conditions, so they had to re-synthesize nitrogenase. This would make sense too.

In order to explain the nitrogenase regulation, the authors say it's tempting to say the need for respiratory protection is why cells don't fix nitrogen until C/N ratios are high enough, but clarify that another explanation could be that the cells just have enough fixed nitrogen until a certain point. A tricky conclusion to a tricky series of studies. Perhaps I will revisit later.

Citation: Bühler, T. et al. Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii. Archives of Microbiology 148, 247–251 (1987).

105 - Control of respiration and growth yield in ammonium-assimilating cultures of Azobacter vinelandii

What They Wanted to Know

As discussed in the previous posts, Bühler, Oelze, and others knew that Azotobacter vinelandii could fix nitrogen at high oxygen levels, but weren't sure how: the respiratory protection hypothesis, that the cells increased their respiration to consume all the oxygen, only seemed to make sense at relatively low levels of oxygen (below 30% saturation); respiratory activity doesn't increase very much beyond a certain point.

Also, since nitrogenase requires a large amount of energy, it's possible the respiration might just be increasing to provide for it. In 089, this lab showed that increasing the fixed nitrogen provided to A. vinelandii led to lower respiratory activity. So, similar to 090 which looked at substrate use efficiency in nitrogen-fixing conditions, here they wanted to see how the efficiency changed when fixed nitrogen was provided.

What They Did

As in previous, they grew A. vinelandii OP (aka CA) in chemostats. They added various levels of sucrose as a substrate and ammonium chloride as fixed nitrogen. They also added sodium citrate, 0.05g/L, to keep the iron they provided from precipitating.

They measured respiratory activities in different states based on oxygen consumption, and also cell dry weights and protein contents, as well as residual sucrose and ammonium.

What They Observed

At the lowest oxygen level (5%), biomass increased almost linearly with increasing sucrose. But as oxygen increased, biomass stayed constant at lower sucrose levels (up to 13-20 mM), and then rose linearly but not as steeply as at low oxygen. At high oxygen (60%), biomass barely rose at all. This was all with the same amount of ammonium added. So, at a given sucrose level, more oxygen meant less biomass. This is consistent with previous studies (090).

They also tried keeping the sucrose constant and varying the amount of ammonium, which affected the carbon-to-nitrogen ratio. They saw similar patterns of biomass vs. C/N ratio, with shifts in the biomass increase at higher ratios, as they had seen when varying the sucrose.

Residual sucrose and ammonium were always very low, so it was all being consumed, and thus limiting. Ratios of dry weight to protein contents were always constant, so there didn't seem to be any nutrient storage going on, even at high sucrose levels.

In nitrogen-limited states at low C/N ratios, yields of biomass were higher, though they decreased as sucrose increased. This is sorta the opposite of what was seen in purely nitrogen-fixing cultures (090), where yield increased as dilution rate (and thus, amount of sucrose) increased. They leveled off when cultures started fixing nitrogen. Even with ammonium though, higher oxygen meant lower yields.

Similarly, respiratory activity increased as C/N ratio increased, up until nitrogen-fixing started; the higher the oxygen, the higher the respiration. Though at a given C/N, higher oxygen always meant higher respiration, unlike in previous studies where it leveled off, though maybe the ranges of sucrose concentrations were different. Also, they measured both respiratory capacity and actual respiration, and cells always seemed to be using only about 50% of their capacity.

What This Means

The way to understand this is that at low sucrose levels, there's only enough ammonium to support a certain amount of biomass production, and not enough sucrose to make it worth turning on nitrogenase, but as the sucrose increases (or ammonium decreases), it becomes more worthwhile.

So C/N ratios seem to control respiratory capacity and activity. That kinda explains why respiration might level off at higher C/N ratios, when nitrogen-fixing activity has started: cells fix as much nitrogen as the carbon level permits, keeping the C/N ratio constant, so the respiration level is constant also. I guess. Look for more discussion about that in the next post.

One last cool thing about this paper: they give the ratios of the main components of A. vinelandii cells, based on the thesis of one H.W. van Verseveld in 1979. The composition is C₆H_10.8N_1.5O_2.9. Useful for calculating molar yields.

Given this, it appeared that the cells converted between 20-30% of the sucrose they consumed into biomass, getting rid of the rest of it, at the lowest oxygen level (5%). At 60% oxygen saturation, they only assimilated 5-10%. Overall, the results aren't really consistent with respiratory protection of nitrogenase, since these were cells grown with ammonium. Interesting.

Citation: Bühler, T. et al. Control of respiration and growth yield in ammonium-assimilating cultures of Azotobacter vinelandii. Arch. Microbiol. 148, 242–246 (1987).