Tuesday, September 30, 2014

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 C6H10.8N1.5O2.9. 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).

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