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).

090 - Dependency of growth yield, maintenance and Ks-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

What They Wanted to Know

Considering Azotobacter vinelandii's high respiration rates at higher oxygen concentrations, it seemed like its energy metabolism was uncoupled from respiration, meaning that it was respiring without getting as much energy from it, proportionally. This meant that its maintenance energy, the amount of food it needs just to remain the same, should be pretty high in some conditions. So Kuhla and Oelze wanted to examine maintenance requirements and growth yields in more depth, at different levels of oxygen.

What They Did

Similar to previous posts, they grew A. vinelandii OP (aka CA) in a chemostat, limiting its carbon diet and controlling its oxygen exposure, under nitrogen-fixing conditions. They adjusted dilution rates (by adjusting the rate that fresh medium flowed into the reactor) and oxygen saturations.

They also tested different carbon substrates: grams per liter of sucrose, glucose, or acetate. They measured cell dry weights in different conditions, and protein content, nitrogen content, and residual substrates.

What They Observed

First they ruled out the possibility of nutrient storage as a confounding factor, by increasing the amount of food the bacteria received. This didn't really affect the nitrogen content of the cells much, so they didn't seem to be storing it for later. So protein content is a good proxy for total biomass.

Protein content increased as dilution rate increased, not surprising because it meant they were getting more food. At the lowest oxygen level, though, this increase leveled off before too long (around 0.1 h^-1), but even after leveling off the values were still much higher than at any other oxygen level, so they might've just run out of oxygen. Speaking of oxygen, as the saturation increased, the protein content decreased at a given dilution rate.

Growth yields followed almost exactly the same pattern as protein content. So the higher the oxygen, the less efficiently cells used the substrate.

They used a couple methods to calculate maintenance requirements in different conditions (including consumption vs. growth rate from 199 and 1/Y vs. 1/D from 065), but they gave similar results, at least for sucrose and glucose. As expected, maintenance increased as oxygen increased, at least for sucrose, but the maintenance on acetate at mid-range oxygen seemed relatively low and the yield relatively high, so it seems like acetate is used more efficiently.

What This Means

Oxygen above a certain level seems to place extra demands on a cell, preventing it from converting as much of the substrate it consumes into biomass. Exactly what these demands are is unclear though, but growing on acetate seems to reduce the demands somewhat, at least proportional to the amount of carbon consumed.

The authors calculated that, theoretically, A. vinelandii could convert up to 26.2% of the carbon it consumed into biomass, when fixing nitrogen. Presumably this would be significantly higher if not for the requirement to fix nitrogen.

Speaking of respiratory protection again, the expectation would be that maintenance requirements and oxygen levels would be linearly related, but this was only true at lower oxygen levels, suggesting that at higher oxygen, respiration isn't as good at removing oxygen.

Citation: Kuhla, J. & Oelze, J. Dependency of growth yield, maintenance and K_s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii. Arch. Microbiol. 149, 509–514 (1988).

089 - Whole Cell Respiration and Nitrogenase Activities in Azotobacter vinelandii Growing in Oxygen Controlled Continuous Culture

This paper was basically a sequel to 098, by most of the same authors.

What They Wanted to Know

The question that Post, Kleiner, and Oelze wanted to answer in this paper was in regard to Azotobacter vinelandii's ability to protect its nitrogenases from the damaging effects of oxygen. Azotobacter is an obligate aerobe, so this is always an issue, but how it protects its sensitive enzymes was not clear.

The theory was that A. vinelandii employs respiratory protection, in which it consumes oxygen at so high a rate that oxygen cannot build up to toxic levels in the cell. If oxygen does build up too high, or there isn't enough substrate available to consume it all, A. vinelandii can reversibly change the conformation of its nitrogenase so it is protected, though it cannot fix nitrogen in this state, so the cell effectively goes dormant.

The idea of respiratory protection comes from the observation that A. vinelandii can only fix nitrogen aerobically when there is adequate substrate available to maintain high enough rates of respiration. If there's no substrate from which to get electrons to dump onto oxygen, the system doesn't work. It takes time to increase respiration rates, so this process doesn't cope well with sudden increases in oxygen. This is when conformational change helps. In theory.

What They Did

They grew A. vinelandii OP (aka CA) in a chemostat, limiting its carbon (at two different levels) and controlling oxygen exposure, either with nitrogen gas or ammonium as a source of N. Similar to 098. Oxygen was always kept higher than limiting, so they could measure exactly how much there was; I wonder if that was the best range to observe though, and it means that 0% wasn't really anaerobic.

They measured cell protein contents and nitrogenase and respiratory activities, as well as residual sucrose.

What They Observed

Cell protein levels were always higher in ammonium-grown cells, not surprisingly, and in both N conditions they rose a bit as oxygen rose to about 3% saturation, and then dropped, leveling off at around 30%. At higher carbon, N-fixing cells took a bit longer to level off, at about 50% oxygen.

Protein yield followed a similar pattern, dropping as oxygen increased up to 30%. Carbon level didn't affect N-fixing cells' yield, but ammonium-grown cells had higher yields with lower carbon levels.

The pattern of respiratory activity was similar to the above, but inverted: it rose between 1 and 30% oxygen saturation, then remained pretty constant. Nitrogen status didn't affect it much at the higher carbon level, and was always higher than the lower carbon values, but at the lower carbon it was about double when fixing nitrogen compared to when grown with ammonium.

Nitrogenase activity decreased quickly up to about 3% saturation, then gradually up to 100%. Carbon level didn't matter.

Then they tried holding the oxygen constant at 45% and increasing the dilution rate (how fast new medium flowed into the reactor, diluting out the contents). Respiration increased linearly with dilution rate, as did protein content and nitrogenase activities at first, but at a point (around D = 0.25 h^-1), the protein content dropped off and nitrogenase activity increased greatly.

Finally, instead of gradual increases in oxygen saturation, they adapted cells to one level and then suddenly changed it to a higher level for 7 minutes, then dropped it back. Regardless of the starting saturation or new peak of oxygen, the cells always switched off their nitrogenase activity when exposed to a larger amount of oxygen. They started it up again when the oxygen dropped back down, but not at the same level as before.

What This Means

Cell activity seemed to level off around 30% oxygen saturation, so either that's more than they can use, or their ability to deal with it has peaked and doesn't need to increase any more. However, a sudden large increase does cause them to suddenly shut down, even if they wouldn't have shut down with a gradual increase to the same level, so there's something else going on.

It's interesting to note the lower yields as oxygen increased, indicating that A. vinelandii was sorta wasting the carbon to deal with the oxygen. It wasn't just when fixing nitrogen though, so it might not be specifically to protect the nitrogenase. Hard to say from just this.

Inconsistent with the respiratory protection hypothesis is the large increase in nitrogenase activity at higher dilution rates without a simultaneous increase in respiration, while oxygen remained the same. Also the fairly constant rates of respiration and nitrogenase activity as oxygen increased above 30% to 100%; we would expect respiration to rise and nitrogenase to drop more severely.

So respiratory protection might be important at some levels of oxygen, but possibly not all.

Citation: Post, E., Kleiner, D. & Oelze, J. Whole Cell Respiration and Nitrogenase Activities in Azotobacter vinelandii Growing in Oxygen Controlled Continuous Culture. Arch. Microbiol. 134, 68–72 (1983).

144 - Experiments on the Transformation and Fixation of Nitrogen by Bacteria

This old and somewhat hard-to-find report (Update: apparently now it's on Google Books; see link below) by J.G. Lipman details the discovery of Azotobacter vinelandii, discovered in Vineland, New Jersey in the early 1900s.

Lipman's goal was to study the nitrogen-related behaviors of soil bacteria in general, and specifically to isolate nitrogen-fixing bacteria.

Behaviors of soil microbes in nitrogen-rich or nitrogen-poor media
When soil is added to nitrogen-rich medium, such as meat extract, many species grow. Some soluble nitrogen becomes insoluble (incorporated into bacterial cells/biomass), but more is broken into simpler forms, including nitrogen gas, so the total amount of nitrogen in the culture decreases.

But when there is a minimal medium of certain salts and organic compounds but not much fixed nitrogen, the amount of nitrogen in the medium increases when soil is added, because bacteria fix nitrogen gas.

So two results from the same soil in different media. This makes sense. Different bacteria grow to different levels in each.

Some of the flasks of these two kinds of media were inoculated with soil from Freehold, either pasteurized or not, or similar soils from Vineland. Others were kept sterile or inoculated with a single species (one B. pyocyaneus). These each differed in appearance after incubating for a number of days.

In high-nitrogen medium, the non-pasteurized soils reduced the total nitrogen the most, as should be expected.
In low-nitrogen medium, pasteurized Vineland soil actually remained sterile, but the others grew, and those increased the amount of nitrogen. Apparently the Freehold soil had more spore-forming organisms, which are more likely to survive pasteurization.

In the Freehold soil, nitrogen-fixers seemed to consist mostly of spore-forming organisms, while in the Vineland soil the main organism appeared as large, aerobic diplococci (paired roundish cells), which Lipman labeled Azotobacter (i.e. nitrogen bacteria).

Also noteworthy: non-pasteurized Vineland soil fixed more nitrogen than any of the others, by far.

Isolation of Azotobacter vinelandii
Trying to isolate Azotobacter from soil samples was somewhat difficult for Lipman, because small bacilli kept overgrowing his cultures, but eventually he found a good medium to select for the desired bacteria. Through certain tests (growth preferences, motility, shape of cells), Lipman distinguished his culture from Beijerinck's Azotobacter chroococcum isolate, naming it instead Azotobacter vinelandii (because it came from Vineland).

A. vinelandii Characteristics
Lipman noted A. vinelandii's growth characteristics in various media, and observed in some that the cells accumulated deposits of what he called fat, which looked like small globules, giving the body a granular appearance.

He also noted that the cells produce a bright yellow pigment that diffuses out from the cells, especially when well-aerated. He tried to figure out what the pigment is, but apparently a fire at the research station interfered with that goal.

A. vinelandii Fixing Nitrogen
Lipman did an experiment with his bacteria, inoculating several flasks of broth with them and testing how much nitrogen they fixed. He found that, for a given volume of culture, being in a bigger flask meant fixing more nitrogen; i.e. more surface area was helpful. Apparently oxygen is important.

Then he tried different forms of nitrogen (or none at all): potassium nitrate, peptone, or ammonium chloride, in two different quantities. After 18 days, all cultures had the same appearance, with growth and yellow pigment. The amount of fixed nitrogen in them was pretty similar for all. So it seemed like adding fixed nitrogen, at least in these low amounts, didn't affect nitrogen fixation much.

For some reason, Lipman also observed that adding filter paper to the culture more than doubled the amount of nitrogen fixed. Maybe extra carbon? Seems improbable. When they weighed the paper after removing it from the culture, it didn't seem to have decreased at all, so apparently it wasn't consumed.

A. vinelandii Carbon Substrates
Lipman tried growing the bacteria on a variety of carbon sources, to see which it could use: ethanol, glycerine, mannite (mannitol) + soil, propionate, succinate, and citrate. It seemed to grow on all of them to some extent; perhaps best on mannitol, then glycerine, then ethanol and propionate, then a bit on succinate, and very slightly on citrate. This is paralleled in the amounts of nitrogen fixed in each one.
They tested some other substrates, but didn't complete the experiments before the fire mentioned above. After 48 hours, A. vinelandii seemed to be growing on glucose, sucrose, and dextrin, but not lactose.

A. vinelandii and Bacillus 30
Apparently there was some mysterious bacterium, discovered around the same time as A. vinelandii, which, when grown together with A. vinelandii, enhanced the latter's nitrogen fixation for some reason, though by itself B. 30 couldn't fix very much, if any.
When grown together, A. vinelandii seemed to fix from 2-4 times more nitrogen. Considering that B. 30 seems to produce a large amount of hydrogen, this could be one way it helps nitrogen fixation, but I'm not sure exactly how that works.
None of the other soil microbes discovered around the same time seemed to help A. vinelandii fix nitrogen very much.

Adding pasteurized soil from different places also seemed to help A. vinelandii fix nitrogen, especially Vineland soil, but it wasn't clear why for that either.

Other Factors Influencing Nitrogen Fixation
Adding iron to the medium seemed to help increase nitrogen fixation a great deal.

There were some other experiments, but I didn't think them very important. Check it out if you're curious.

Citation: Lipman, J. G. Experiments on the Transformation and Fixation of Nitrogen by Bacteria. New Jersey State Agric. Exp. Sta. Ann. Rep. 24, 217-285 (1903).