453 - Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum

This study looked at hydrogen and its relationship to different enzymatic processes in Azotobacter chroococcum.

What They Saw
The organism was grown in continuous culture with limited oxygen, 5% glucose. Samples were removed and sparged with different mixtures of argon, oxygen, and deuterium. Gases are measured by mass spectrometer.

Their figure is pretty confusing, poorly designed, and poorly described, but as far as I can tell, when they added either 80% argon with 20% oxygen or 70% argon, 20% oxygen, and 10% deuterium, the oxygen goes down to near zero within about 5 minutes either way, at which point hydrogen production starts increasing and deuterium uptake slows down or stops. So it seems like oxygen is required for hydrogenase to work.

They tried again with the addition of some carbon monoxide and acetylene to inhibit hydrogenase. This didn't really change the oxygen consumption, but deuterium consumption was a lot lower. Hydrogen evolution was the same.

What This Means
The deuterium consumption is by hydrogenase, of course, and it seems like oxygen is a necessary electron acceptor for it to function (in the absence of something else). But it seems like the increase in hydrogen evolution is not because hydrogenase stopped working, but rather because nitrogenase started, as oxygen stopped interfering. This is something we've seen before.

Oxygen is important for many things: it provides energy by accepting electrons, powering the nitrogenase and allowing more hydrogen production. It accepts electrons from hydrogenase, enabling hydrogen oxidation. It inhibits nitrogenase, reducing hydrogen production. Pretty confusing.

Reference:

Lespinat, P.-A., Gerster, R. & Berlier, Y. Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum. Biochimie 60, 339–341 (1978).

307 - The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity

This study looked at the influence of different sources of nitrogen on the activity of nitrogenase and hydrogenase in Azotobacter chroococcum.

What They Saw
They grew cells in batch or continuous culture with sodium nitrate, ammonium acetate or chloride, or dinitrogen gas. Cultures were either carbon- or sulfate-limited. Dissolved oxygen was kept above zero. Nitrogenase activity was measured by acetylene reduction and hydrogenase by methylene blue (or by adding H-T with radioactive tritium and measuring radioactivity of resulting water when oxygen was the electron acceptor).

There was about twice as much hydrogenase activity when cells were fixing nitrogen in batch than when they had either ammonium or nitrate. They cite other results in A. chroococcum and A. vinelandii that showed higher activity with nitrate than ammonium though, but still less than when fixing nitrogen. This might be because cells have to adapt to use nitrate, and they'll be fixing nitrogen before that happens. But care is necessary because activity changes over the course of a batch culture, increasing throughout exponential phase even when standardized by protein concentration.

In continuous cultures, they started growing with ammonium, then switched to nitrogen-free, watched what happened with hydrogenase and nitrogenase, and then pulsed a limited amount of ammonium. As expected, nitrogenase activity started up and rose to a plateau, then immediately stopped when ammonium was added, and restarted when it was removed. Hydrogenase activity showed a similar pattern, though more delayed, and it never went all the way to zero.

In sulfate-limited culture, hydrogenase activity was lower even when fixing nitrogen, about the same as with ammonium in carbon limitation, and when going from fixing to non-fixing (with ammonium), the activity declined a bit but then went back up to about the same level. Results were similar going from non-fixing to fixing. So with sulfate limitation, nitrogen source doesn't matter much.

Also with nitrogen-fixing cells in sulfate limitation, when they increased the dilution rate, hydrogenase activity decreased but nitrogenase increased. The decrease wasn't linear, though; it leveled off.

They also tested whether adding hydrogen to the atmosphere of an ammonium-grown culture would influence hydrogenase activity, and it did! Hydrogenase activity doubled. This was not the case with sulfate limitation though, only carbon limitation. They didn't test nitrogen-fixing cells.

What This Means
The continuous culture experiments helped overcome the constantly changing activity in batch cultures.

That hydrogenase activity lags behind nitrogenase activity increase when ammonium runs out could be explained by the last observation: maybe nitrogenase starts producing hydrogen (as it does) and this stimulates hydrogenase activity.

They reasoned from the data that excess carbon might inhibit hydrogenase activity somehow (like catabolite repression). I'm not sure that makes sense, but it seems possible, and does fit with the data from batch cultures, sulfate limitations, and increasing dilution rates. Interesting.

Reference:

Partridge, C. D. P., Walker, C. C., Yates, M. G. & Postgate, J. R. The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity. J Gen Microbiol 119, 313–319 (1980).

225 - O2-repression of nitrogenase synthesis in Azotobacter chroococcum

Klebsiella pneumoniae only fixes nitrogen when oxygen levels are very low (or absent), so oxygen represses nitrogenase synthesis. This study looks at whether the same is true in Azotobacter chroococcum; it seems unlikely, since this organism is known for its very aerobic nitrogen fixation; however, it is possible to stress this organism with oxygen such that it may shut down nitrogenase, at least temporarily.

What Robson Saw
He grew A. chroococcum in chemostats with Burk medium with mannitol, with or without ammonium for nitrogen, and stressed them with oxygen either by moving ammonium-grown cells to ammonium-free medium or by suddenly increasing aeration in nitrogen-fixing cells. He looked at levels of nitrogenase proteins (by labeling with radioactive sulfur isotopes) and nitrogenase activity.

At initial low oxygen levels, activity increased and radioisotype-labeled protein levels were relatively high (indicating high levels of nitrogenase protein synthesis). Upon oxygen shock, activity went to zero and protein synthesis levels dropped a lot. When the stress was relieved, both measures increased again.

With ammonium removed and then re-added, things were similar: nitrogenase activity went up and synthesis gradually increased to a plateau, but decreased when more ammonium was added. It did take a relatively longer time for nitrogenase activity to pick up after ammonium was removed, about 80 minutes.

Along with nitrogenase, flavodoxin and the small protein that protects nitrogenase from oxygen by temporarily inactivating it both showed up in radiolabeling. The former matched nitrogenase synthesis patterns, but the latter was fairly constant.

What This Means
It seems that oxygen stress can repress synthesis of nitrogenase even in Azotobacter, though radiolabeling might not be the best method for studying this question. This adds another layer to Azotobacter's protection of its enzymes from oxygen.

Reference:

Robson, R. L. O₂-repression of nitrogenase synthesis in Azotobacter chroococcum. FEMS Microbiology Letters 5, 259–262 (1979).

216 - Effect of Dissolved Oxygen on Growth Yield and Aldolase Activity in Chemostat Culture of Azotobacter vinelandii

This study looked at carbon- or oxygen-limited cultures of Azotobacter vinelandii and the effects on enzymes of the TCA cycle: aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, and isocitrate lyase.

What They Saw
They grew A. vinelandii in chemostats with Burk medium with glucose at different agitation speeds and different dilution rates.

At the second-lowest agitation, dissolved oxygen was low and a bit of glucose was residual in the outflow (about 0.44 g/L); this increased as D increased. Growth yield and carbon dioxide production didn't change much as D changed. Things were similar in the lowest agitation, except there was less biomass and presumably more residual sugar.

At the second-highest agitation, residual glucose was very low up to D = 0.2, then went up, so the culture seemed to switch from glucose-limited to oxygen-limited at that point. Biomass increased up to that point too, and then decreased. Growth yield and CO₂ production changed inversely, with yield increasing up to a certain D before 0.2 and then leveling off. Once leveled off, values were similar to those from lower aerations.

So overall for all aerations, as dissolved oxygen increased, growth yield from glucose decreased; growth became less efficient. Carbon dioxide production increased though, indicating that the carbon was being consumed but going toward that gas, complete oxidation.

In terms of enzyme activities, of the four tested enzymes, only aldolase increased as oxygen increased. Below is their model of what effect this has:

Fig 4, Nagai et al. 1971

The rise in aldolase activity meant that carbon was cycling through the pentose cycle more instead of moving on to the TCA cycle (which could lead to greater ATP generation and anabolism), so that explains the increase in CO₂ production and decreased growth yield.

Reference:

Nagai, S., Nishizawa, Y., Onodera, M. & Aiba, S. Effect of Dissolved Oxygen on Growth Yield and Aldolase Activity in Chemostat Culture of Azotobacter vinelandii. J Gen Microbiol 66, 197–203 (1971).

197 - Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Considering how oxygen-sensitive nitrogenase is, it might be expected that enzymes specifically involved in oxygen detoxification (such as catalase and superoxide dismutase (SOD)) might be involved in protecting such oxygen-sensitive enzymes, especially since ramping up respiration in response to increased oxygen might also ramp up the production of reactive oxygen species. This study investigates the activity of SOD and catalase in Azotobacter vinelandii at different oxygen levels.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats with different levels of oxygen, with 3 or 15 g/L sucrose. They extracted enzymes from samples and assayed them for SOD or catalase activity, and also by electrophoresis.

As oxygen saturation increased from 1% to 90%, SOD activity increased linearly (when standardized to total protein); the increase was slightly faster at lower oxygen when standardized by number of cells (probably because cell size increases as oxygen increases, 098). The sucrose concentration didn't affect things. When cells were given ammonia, SOD activity was about 2x lower.

Based on electrophoresis, they concluded that the SOD is iron-containing, rather than manganese. They tried adding manganese but still didn't see any Mn-SOD.

It didn't seem like catalase activity increased with increasing oxygen, standardized by protein. The increase standardized by cells was much more apparent.

What This Means
It seems like SOD at least might contribute to A. vinelandii's protection of its nitrogenase enzyme from oxygen.

Reference:

Dingler, C. & Oelze, J. Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations. Arch. Microbiol. 147, 291–294 (1987).

195 - Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations

Obviously oxygen levels have a big effect on nitrogen-fixing Azotobacter. This study looked into specific effects on levels of different proteins related to nitrogen fixation, at different oxygen levels.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats with 3 g/L sucrose at different oxygen levels (or dilution rates). They measured nitrogenase activity and purified nitrogenase components as well as flavodoxin and FeSII protein.

Unlike in previous studies (111, 165, 183), increasing the oxygen levels didn't seem to reduce nitrogenase activity (in steady state), at least not in the range they tried (except maybe a little at low levels). At all different levels, nitrogenase activity (i.e. acetylene reduction) correlated only with dilution rate.

With Western blots, they found that levels of different nitrogenase proteins (and others) didn't really vary much across different oxygen levels; they were always about 10% of total protein. And if the activity doesn't vary, this means the proportion of active enzyme is constant too. The enzyme activity does match previous numbers though (106).

Trying to grow cells in ammonia, the two components of nitrogenase disappeared, flavodoxin decreased, but FeSII remained constant.

They tried measuring nitrogenase levels at different dilution rates and oxygen levels. At the lowest D, nitrogenase increased as oxygen increased, but it remained pretty constant at higher rates (as shown before). In contrast to the earlier data though, levels didn't seem to increase consistently with increasing D; it could be that the same quantity of enzyme is less active at lower D, probably related to the flow of electrons to the enzymes.

Then they tried inhibiting protein synthesis with chloramphenicol, at either low or high oxygen. The culture started to wash out, of course. Levels of the four proteins didn't change much with oxygen or with time passed after addition of the antibiotic, but nitrogenase activity decreased greatly over time (while respiratory activity didn't change much). This wasn't due to damage to the nitrogenase components; nitrogenase extracts had just as much activity as cells grown without chloramphenicol. Somehow the activity is inhibited.

What This Means
As suggested before, it seems like the absolute presence/concentration of oxygen doesn't determine its toxicity so much as the ratio of oxygen to availability of energy and reducing equivalents. So if there's enough energy and electrons available to nitrogenase, it can keep going up to high levels of oxygen.

Reference:

Dingler, C., Kuhla, J., Wassink, H. & Oelze, J. Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations. J Bacteriol 170, 2148–2152 (1988).

183 - Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii

Later investigators criticized the previous study (182) as too simplistic, not explaining the total potential of the nitrogenase system. Azotobacter seems to make three flavodoxins, not just azotoflavin; flavodoxin II seems like the important one but it wasn't known how it got reduced; and ferredoxin might not be involved at all.

So this study grew A. vinelandii with and without ammonium, then observed the differences in its redox systems.

What They Saw
They grew cells in a chemostat with ammonium, and then removed samples and washed with nitrogen-free medium to remove the fixed nitrogen. They measured nitrogenase activity of these samples and labeled newly formed proteins with radioactive sulfur compounds.

They observed that nitrogenase activity correlated well with rate of respiration in different conditions, so they wondered if the two might be linked.

After they removed the fixed nitrogen from cells that had been growing with it, they observed nitrogenase activity within 20 minutes. Then the activity increased linearly over time for at least 40 minutes in this condition. On protein gels, they observed the nitrogenase proteins produced quickly, within 5 minutes, and flavodoxin II showed up some time later. There are some others of uncertain identity, and some interesting ones showing up only in the membrane protein fraction.

What This Means
The linear relationship between respiration and nitrogenase activity has a number of possible explanations. Extra respiration could mean higher membrane potential or ATP levels, so more energy for nitrogenase, or there could be more enzymes (or enzymes that are more active) to transport electrons to nitrogenase. The former seems unlikely, since Azotobacter uncouples respiration from energy generation at higher oxygen levels, so extra respiration doesn't necessarily mean more energy. And extra enzymes seems unlikely too, based on the protein results.

So it seems like increased nitrogenase activity might be due to increased transport of electrons to the enzyme, though it's not clear how that happens.

Reference:

Klugkist, J., Haaker, H. & Veeger, C. Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii. European Journal of Biochemistry 155, 41–46 (1986).

178 - Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii

This study looks at what it takes for oxygen stress to induce Azotobacter vinelandii to shut off its nitrogenase.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats, fixing nitrogen, with limited carbon (3 g/L of sucrose, acetate, or citrate). They stressed the culture with oxygen by increasing the aeration for 6-minute periods. They measured acetylene reduction, and nitrogen fixation directly (by fixed nitrogen increase). They also measured oxygen levels going in and coming out, to determine consumption.

As usual, with sucrose, they observed that respiration rates increased as oxygen levels rose. At a given oxygen level, respiration also increased as the dilution rate D increased. The amount of respiration increase wasn't the same at different dilution rates though, even with the same change in oxygen. The oxygen maintenance requirement increases as oxygen increases, but not linearly (the rate of increase goes down).

With acetate or citrate, the oxygen maintenance coefficient (and respiration at a given oxygen level) was much lower than with sucrose. Also as D increased, respiration with citrate increased linearly, but with acetate it leveled off at some point.

The rate of nitrogen fixation depended only on D, and increased linearly with D. The carbon source didn't affect it.

When they did the oxygen challenges, they found that up to D = 0.15 h^-1, increasing the oxygen shut off nitrogenase completely. Above that D, the shut-off was less severe. With a less severe challenge, there was less shut-off at the same D too, as expected. Substrate didn't seems to matter.

They couldn't measure a change in respiration from oxygen stress directly, because it was too short, but they knew that cultures grown in acetate or citrate couldn't increase their respiration because they had already consumed all the substrate. There was still residual sucrose though, but the amount didn't seem to change with oxygen challenge, so they concluded that respiration didn't suddenly increase.

Finally they tried controlling nitrogenase activity by giving cells small amounts of ammonium, not enough to repress nitrogen fixation, just reduce it. So giving cells 1 mM ammonium when D = 0.16 resulted in the same nitrogenase activity as when D = 0.06 with no ammonium. And they found that an equivalent oxygen challenge led to the same amount of nitrogenase shut-off.

What This Means
So the rate of oxygen consumption doesn't affect how severe the nitrogenase shut-off is, only the rates of substrate feeding and nitrogenase activity, and more so the latter.

This is kinda weird, because if respiration is how the cells protect nitrogenase from oxygen by removing it (respiratory protection), then higher respiration should correlate with higher nitrogenase activity, but it doesn't seem to here. Also, oxygen level and oxygen consumption should correlate linearly, but they don't, especially considering different carbon substrates.

The authors propose that, instead of respiratory protection, the cells' redox state is what matters: nitrogenase requires a reduced state to function, and more oxygen leads to a more oxidized state. Reduction is made possible by the carbon substrate, which provides energy and electrons; at higher dilution rates, more reduction is possible, so the nitrogenase activity can be higher. That also explains why at higher D, the same oxygen challenge leads to less nitrogenase shut-off, because the change in the redox state is less severe. That way, the cells don't need to create an anaerobic environment in their cytoplasm, just maintain a low redox potential and good flow of electrons.

Respiratory protection as a concept is still useful, since it is still true that the cells' respiration increases as oxygen increases when fixing nitrogen, to allow nitrogenase to function; it's just the details that have been challenged here.

Reference:

Kuhla, J. & Oelze, J. Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii. J. Bacteriol. 170, 5325–5329 (1988).

175 - Regulation of the Tricarboxylic Acid Cycle and Poly-β-hydroxybutyrate Metabolism in Azotobacter beijerinckii Grown under Nitrogen or Oxygen Limitation

When Azotobacter grows in oxygen-limited conditions, one expects that reducing equivalents such as NADH and NADPH would accumulate (since the electrons have no good acceptor). This could inhibit the TCA cycle. This study looked at different catabolic enzymes and their behavior under different nutrient limitations.

What They Saw
They grew A. beijerinckii in a chemostat with oxygen or (alleged) nitrogen limitations. As seen before, low oxygen induced PHB formation to store the carbon and as an electron sink. Adding extra oxygen caused a drop in PHB and dry weight, and then removing it reversed the effect. Enzymes for PHB synthesis showed a similar pattern.

NADH oxidase and enzymes involved in the TCA cycle of carbon catabolism (2-oxoglutarate dehydrogenase and isocitrate dehydrogenase) tended to increase activity as oxygen increased, and vice versa. NADH oxidase (part of the electron transport chain), though, fell after the initial increase and then rose again at the second increase. This enzyme seems like an important part of the genus's respiratory protection.

Some other TCA enzymes, citrate synthase and pyruvate dehydrogenase, didn't change much with oxygen changes.

They saw again that the lower the oxygen, the higher the growth yield (amount of biomass produced per unit sucrose), though probably some of this was due to PHB formation increasing the dry weight. The amount of sucrose consumed increased as oxygen increased, indicating less efficient growth.

With NADH/NAD+, when oxygen limitation was first imposed, the ratio rose greatly but then went down again quickly, and remained mostly steady with some fluctuations.

What This Means
The recovery of NADH/NAD+ ratio is likely due to the formation of PHB acting as an electron sink.

Azotobacter is kind of an interesting mix of aerobic and facultative organisms; they need oxygen, but not too much, and excess is harmful, so their respiratory systems are tightly regulated to deal with multiple different levels. The ability to fix nitrogen puts them in kind of a different category.

Reference:

Jackson, F. A. & Dawes, E. A. Regulation of the Tricarboxylic Acid Cycle and Poly-β-hydroxybutyrate Metabolism in Azotobacter beijerinckii Grown under Nitrogen or Oxygen Limitation. J. Gen. Microbiol. 97, 303–312 (1976).

165 - The Behaviour of Azotobacter chroococcum in Oxygen- and Phosphate-limited Chemostat Culture

This study looked at Azotobacter chroococcum behavior when limited in oxygen or phosphate.

What They Saw
They grew A. chroococcum in chemostats, similar to previous studies (163). In their allegedly oxygen-limited culture, biomass levels fell as dilution rate increased, while carbon dioxide production fell and then rose again for some reason; they claim it's just random, and that CO₂ was actually fairly constant (or blame it on consistency of electricity in Britain). They also observed that as dilution rate decreased, the amount of PHB in the cells rose. They could tell the cells were oxygen-limited because when more oxygen was added, it disappeared pretty quickly.

When cells were phosphate-limited, extra oxygen was pretty toxic; the cells couldn't handle it. Plating cells out after this challenge revealed that there was very little viability, if any. In contrast, cells that had been oxygen-limited didn't seem to mind extra oxygen much, especially when they were grown on medium with fixed nitrogen.

What This Means
It fits pretty well with previous results. PHB seems to be an electron sink, useful when cells don't have enough oxygen to receive all their electrons.

The fact that oxygen didn't reduce cells' viability when plated on medium with fixed nitrogen suggests that the cells' problem with extra oxygen is lack of ability to fix nitrogen immediately, rather than loss of viability. Then the cells ramp up respiration to consume the extra oxygen—respiratory protection.

Reference:

Lees, H. & Postgate, J. R. The Behaviour of Azotobacter chroococcum in Oxygen- and Phosphate-limited Chemostat Culture. J Gen Microbiol 75, 161–166 (1973).

164 - Reassessment of Maintenance and Energy Uncoupling in the Growth of Azotobacter vinelandii

Maintenance energy reflects a cell's inefficiency, the energy it needs but that does not go toward growth; it may go toward maintaining cellular components somehow, or just reflect waste. These possibilities aren't easily distinguishable.

Previous results looked weird for A. vinelandii when its glucose feed was suddenly changed, so Nagai and Aiba wanted to clarify their understanding of this bacterium's maintenance and yield values.

They grew A. vinelandii ATCC9046 in chemostats with 5 or 8 g/L glucose, 0.05 g/L sodium citrate, and other things in Burk medium, limited either in glucose or oxygen.

The important equations are:

1: v = m + μ_x/Y_G

and

2: Q_O2 = m_o + μ_x/Y_GO

which, being interpreted, mean that 1) the specific rate of glucose use (mmol glucose/mg bacteria/h) = maintenance (same units) + biomass growth rate/true yield (g bacteria/mol glucose). Which makes sense: maintenance takes glucose, and growth takes more, so adding them together with the growth rate you get the total glucose use;

and 2) the specific rate of respiration (mmol oxygen/mg bacteria/h) = respiration maintenance (same units) + biomass growth rate/true oxygen yield (mg bacteria/mmol oxygen). Which is parallel to the first, except with oxygen instead of glucose.

So with these equations in mind, they measured specific glucose use and specific respiration rate over a series of dilution rates (equivalent to growth rates):

Positive slopes indicate oxygen-limited points, negative slopes glucose-limited points, at different agitation speeds.
Nagai and Aiba, 1972

So from these graphs and the equations, v and Q_O2 can be the y in the equation of a line (y = mx + b), while D is the x, and so 1/Y_GO or 1/Y_G become the slopes, and the y-intercept is m, maintenance.

What's weird is with the glucose-limited points, the slope of the line (and thus the growth yield) is negative (so the amount of biomass should decrease as the glucose or oxygen increases); are glucose and oxygen toxic in this case? The other thing is m, which is within a reasonable range when oxygen-limited (around 0.8 mmol glucose/g bacteria/h, comparable to other organisms), gets super-high when carbon-limited: between about 19.5 and 26 mmol glucose/g bacteria/h, depending on the agitation speed and glucose concentration. The explanation for this is likely energy-uncoupled growth: when cells increase their use of substrates without increasing their growth rate (like with respiratory protection, and oxygen-wasting system).

The maintenance requirement of oxygen, or oxygen wasting, increases almost linearly as the amount of oxygen present increases. So the reason that the yields were negative when carbon-limited was that the cells receive more carbon per hour as D increases, so they produce more cells, but the rate of consumption remains the same. Therefore a lower proportion is being wasted per cell, but the same amount overall. So increasing the amount of substrate doesn't decrease the amount of biomass, but rather the 1/Y_GO term encompasses both actual yield and substrate-wasting values, and the latter is negative because it goes down proportional to the number of cells as D increases, and the negative outweighs the positive in this case. So the true growth yield is still positive.


Reference:

Nagai, S. & Aiba, S. Reassessment of Maintenance and Energy Uncoupling in the Growth of Azotobacter vinelandii. J Gen Microbiol 73, 531–538 (1972).

153 - The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

This is a study on what factors initiate production of large amounts of PHB in Azotobacter beijerinckii.

What They Saw
In batch with 5 g/L glucose, bacteria started producing PHB near the end of the exponential phase. They stopped when they had consumed all the glucose, and then consumed the PHB, but this didn't help them increase in density, possibly because the PHB itself had been a proportion of the bacterial density; it seemed to permit the increase in actual biomass.

When glucose was 20 g/L, the cells continued producing PHB and the bacterial dry weight kept increasing long after exponential phase ended; the polymer got up to 74% of the dry weight.

Using an oxygen electrode, they observed that PHB production didn't start until dissolved oxygen reached 0%, at which point exponential phase was over. So they thought it might be oxygen limitation that induced the production, unlike in other organisms where nitrogen limitation is the inducer. But to be sure that it was really oxygen and not nitrogen, they turned to chemostats.

They found that nitrogen limitation didn't induce PHB formation at any dilution rate, though growth yield increased as D increased. In contrast, in oxygen-limited conditions, the PHB content (as a proportion of dry weight) and yield seemed to decreased as D increased, starting around 45% and going down to 20%, while growth yield seemed to peak at mid-range and then fall. Glucose limitation didn't induce PHB either, though some was produced at lower dilution rates.

The sudden imposition of oxygen limitation on nitrogen-limited cultures immediately induced PHB formation, and content increased over at least 10 hours. Dry weight and OD initially went up but then back down below what it had been, possibly reflecting more efficient growth until oxygen was completely depleted. They don't show it, but claim that PHB went back down from 45% to 20% after 32 hours.

What This Means
The authors speculate that the limitation of low oxygen comes in at the level of the TCA cycle; acetyl-CoA stops being oxidized as much as before, so it starts going toward PHB synthesis.

So limited oxygen might be the best condition for these microbes: they have some but not too much, their growth is more efficient, and they produce a nice storage polymer to save food for harder times.

Reference:

Senior, P. J., Beech, G. A., Ritchie, G. A. F. & Dawes, E. A. The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii. Biochem. J. 128, 1193–1201 (1972).

141 - Effect of Oxygen and Nitrogen Limitation on Poly-β-Hydroxybutyrate Biosynthesis in Ammonium-Grown Azotobacter beijerinckii

Azotobacter produces PHB polymer under nutrient limitations (other than carbon); the authors wanted to see whether nitrogen-fixing conditions were required for this process. This may affect the influence of oxygen on the organism, since it doesn't require respiratory protection.

What They Saw
The capsuleless strain of A. beijerinckii they used in this study accumulated up to 70% of its dry weight as PHB in batch culture, like its parent, whether or not it was fixing nitrogen. It started accumulating right when the culture became oxygen-limited.

So they grew A. beijerinckii in low-oxygen continuous culture with ammonium. Oxygen was about 1.75%, flowing at 0.4 liters per minute. As the dilution rate increased, dry weight and PHB proportions decreased, and the cells consumed less of the available carbon and nitrogen. PHB only got up to 50% though.

When they reduced the ammonium and kept oxygen constant at about 5% saturation, they saw constant PHB at 1% of dry weight and all the ammonium was consumed (so, ammonium-limiting growth), but total dry weight and carbon consumption showed the same pattern as before.

They tried even lower levels of oxygen to see if they could get PHB up to 70% of dry weight in continuous like in batch cultures, and succeeded, when oxygen was only 0.275% of the inflow, and dilution rates were fairly high (0.18 h^-1). Actually at very low oxygen, higher dilution rate meant higher PHB content, then the trend reversed at a bit higher oxygen, and then returned at an even higher level. The reversal took place when the total biomass had peaked in the highest dilution rate:

Ward et al. 1977

What This Means
As in a previous study (114), the question came up of what oxygen limitation actually means: is it the point at which oxygen is the only nutrient holding cells back from growth, or is it the point at which they change their metabolism to start producing PHB? It is poorly defined.

The biggest difference seen between nitrogen-fixing and nitrogen-assimilating conditions is the high-low-high pattern seen with ammonium, compared to a steady decrease seen when fixing nitrogen. So it seems like the main difference may only be that nitrogen fixation requires so much more energy.

Reference:

Ward, A. C., Rowley, B. I. & Dawes, E. A. Effect of Oxygen and Nitrogen Limitation on Poly-β-Hydroxybutyrate Biosynthesis in Ammonium-Grown Azotobacter beijerinckii. J Gen Microbiol 102, 61–68 (1977).

113 - The hydrogen cycle in nitrogen-fixing Azotobacter chroococcum

Azotobacter chroococcum makes hydrogen when fixing nitrogen, but its uptake hydrogenase reoxidizes hydrogen. It wasn't clear what the purpose of this reoxidation is, or in what conditions it's useful, or how much hydrogen is produced in different conditions, so this study aimed to find out.

What They Saw
When they had bacteria in carbon-free broth, with hydrogen present in the atmosphere, the bacteria were able to reduce acetylene with nitrogenase using only energy from the hydrogen. No such activity was observed without hydrogen present. Even when mannitol was added up to 2 g/L, adding hydrogen still increased the acetylene reduction activity, though the proportion of activity attributable to hydrogen decreased as mannitol increased, though surprisingly it leveled off above zero even when mannitol wasn't the limiting nutrient anymore; it's possible that electron transfer from hydrogen works differently.

They also tried increasing oxygen levels with a little mannitol; when hydrogen was absent, oxygen became inhibitory about twice as fast as when hydrogen was present, so hydrogenase seems to help protect the nitrogenase. The effect went down to around zero as mannitol increased though.

They looked at hydrogen production when fixing nitrogen with various limitations (carbon, nitrogen, oxygen) in continuous culture; unlike in batch culture, cells seemed to evolve significant hydrogen. They compared hydrogen produced in air to that produced when replacing air with argon to get the proportion of nitrogenase activity going to hydrogen (presumably in air, the remainder goes to actually fixing nitrogen), and found that under oxygen or nitrogen limitation (whatever that means here), the proportion was 40-50% going to hydrogen. In carbon limitation, it was lower, around 13%, but they said that hydrogenase activity was higher in this case (for some reason) so it doesn't represent the true proportion (since not all hydrogen is observed).

What This Means
Since hydrogen could protect nitrogenase from oxygen, it seems like its electrons go to oxygen through the respiratory chain rather than to power nitrogenase activity.

It is somewhat puzzling that the hydrogenase would work so well when acetylene is present, since acetylene has been shown to inhibit the hydrogenase (112). They observed that in this study too. So it's possible that the hydrogenase might be even more useful when acetylene is not present. But 40% acetylene is required to completely inactivate hydrogenase, whereas they only used 8% in the activity assays.

40-50% electron flux going to hydrogen is higher than estimated by others, at least for the molybdenum nitrogenase, but it's unclear the effect of the limitations imposed.

Here's the model they propose:

Walker and Yates, 1978

Reference:

Walker, C. C. & Yates, M. G. The hydrogen cycle in nitrogen-fixing Azotobacter chroococcum. Biochimie 60, 225–231 (1978).

108 - The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-Fixing, Carbon-Limiting Conditions in Continuous and Batch Cultures

Since hydrogen is an energy-rich gas, and nitrogenase produces hydrogen, one would expect that diazotrophs that can re-oxidize the hydrogen they produce (using uptake hydrogenases) would have a competitive advantage over those that lack an uptake hydrogenase. However, results of previous studies of this question, in this and other organisms, have been mixed (019,065).

This study is another comparison of Azotobacter chroococcum strains, one with and three without an uptake hydrogenase, in a variety of conditions.

What They Saw
As the dilution rate increased in carbon-limited nitrogen-fixing conditions, the wild-type strain's growth yield remained relatively constant, while the three mutants' yields were noticeably lower at most rates. There was no noticeable difference when fixed nitrogen was provided. When oxygen or sulfate were limiting, there didn't seem to be much difference between strains.

When the strains were mixed together in equal densities in continuous culture, the mutants seem to overtake the wild-type a couple times at lower dilution rates, but the wild-type always took over at higher rates. With ammonium added, there was no consistent pattern.

What This Means
The mutants in this study were obtained by random mutagenesis, so the lack of hydrogenase wasn't necessarily the only difference from wild-type. Still, the three strains seemed to give some fairly consistent results.

The advantage seen in the wild-type occurred in low-density populations or high dilution rates, so it seems like the hydrogenase helps recover energy (or protect nitrogenase from oxygen) rather than preventing inhibition by hydrogen itself.


Reference:
Aguilar, O. M., Yates, M. G. & Postgate, J. R. The Beneficial Effect of Hydrogenase in Azotobacter chroococcum Under Nitrogen-Fixing, Carbon-Limiting Conditions in Continuous and Batch Cultures. J. Gen. Microbiol. 131, 3141–3145 (1985).

136 - Effect of Oxygen Concentration and Growth Rate on Glucose Metabolism, Poly-β-Hydroxybutyrate Biosynthesis and Respiration of Azotobacter beijerinckii

This is a study on Azotobacter beijerinckii and the effects of oxygen levels on its respiration, PHB production, and other parameters.

They varied the oxygen in the inflowing gas between 0 and atmospheric (20%), and also varied dilution rates (when not holding D at 0.1 h^-1).

What They Saw
As oxygen decreased, the PHB content of cells increased up to about 50% of the dry weight near 0% oxygen. Activities of PHB-producing enzymes increased also, while glucose-6-phosphate dehydrogenase activity remained constant. Below about 5% oxygen, the culture seemed oxygen-limited (no dissolved oxygen was detectable). They also saw increased respiratory activity and carbon dioxide production as oxygen increased, and indication of respiratory protection of nitrogenase.

When they increased the dilution rates (from 0.1 up to 0.2), they saw an increase in the activity of each enzyme tested. PHB content decreased as D increased, which is inconsistent with the increase in PHB enzyme activities, but it's likely that enzymes breaking down PHB were more active too.

The Entner-Doudoroff pathways seems to be the main glucose metabolism pathway in A. beijerinckii.

Reference:
Carter, I. S. & Dawes, E. A. Effect of Oxygen Concentration and Growth Rate on Glucose Metabolism, Poly-β-Hydroxybutyrate Biosynthesis and Respiration of Azotobacter beijerinckii. J. Gen. Microbiol. 110, 393–400 (1979).

163 - Effect of Oxygen on Growth of Azotobacter chroococcum in Batch and Continuous Cultures

This is another chemostat study of Azotobacter chroococcum and the effects of oxygen on various physiological parameters.

They grew the bacteria on mannitol B medium (apparently Burk medium but with mannitol in place of sucrose) or B₆ (B, modified for chemostats by adding some trace elements and nitrilotriacetic acid (to keep everything in solution). They measured oxygen with an electrode and regulated the gases flowing through.

A nutrient was considered limiting if decreasing its feed decreased bacterial growth proportionally, except for nitrogen gas, whose limitation was diagnosed by exclusion (i.e. no other nutrient addition could increase growth).

They measured dry weights and mannitol consumed.

What They Saw
In initial batch cultures, inoculated A. chroococcum grew only those with low aeration (or low oxygen exposure) when fixing nitrogen, but given ammonium it could grow in up to 40% oxygen with high aeration.

Then they tried continuous culture with different oxygen levels, between about 1% and 60% of the gas flow (with the rest nitrogen), at D = 0.2 h^-1. They saw the highest yield of biomass remain fairly constant between 10 to almost 60% oxygen. Nitrogenase efficiency (fixed N per substrate consumption) was highest at lowest oxygen, dropped some up to 20%, and then dropped very low at 30% or higher.

Dalton and Postgate 1968, fig 1

Respiratory activity plateaued between 20 and 50%.

Then they tried limiting the carbon at 20% oxygen. This led to inhibitory oxygen levels quickly when oxygen was increased (by raising agitation from 680 to 1100rpm or raising oxygen from 20 to 50%). So carbon limitation led to oxygen hypersensitivity, when fixing nitrogen. The same was true of phosphate limitation.

What This Means
It supports the idea of respiratory protection (respiring carbon quickly and inefficiently to reduce oxygen to protect oxygen-sensitive nitrogenase); when carbon is unavailable, the cells are more sensitive to oxygen when fixing nitrogen. Also the efficiency increases as oxygen levels decrease.

Reference:
Dalton, H. & Postgate, J. R. Effect of Oxygen on Growth of Azotobacter chroococcum in Batch and Continuous Cultures. J. Gen. Microbiol. 54, 463–473 (1968).

114 - The growth of nitrogen-fixing Azotobacter chroococcum in continuous culture under intense aeration

This study attempted to precisely define the transition from oxygen limitation to oxygen sufficiency in Azotobacter chroococcum.

They grew the bacteria in a chemostat with agitation up to 1750 rpm and different dilution rates of 0.1-0.3 h^-1 in a liquid volume of 200 mL, measuring dissolved oxygen with a probe, in mannitol-containing B₆ medium.

At such high feeding rates, the culture could be adapted to any level of oxygen (up to the 1750-rpm agitation limit with 20% oxygen in the flow), so that the probe was reading 0% (since it was all consumed). They tried increasing the proportion of oxygen in the flow up to 50%, but this was too much; the cells couldn't tolerate it.

What They Saw
As oxygen increased above atmospheric levels (20%) the carbon dioxide produced increased also, at all dilution rates, up to the 50% oxygen mark, when some of the cultures couldn't take it anymore. This was still true when proportional to biomass.

Biomass didn't change much with higher oxygen at lower dilution rates, though at higher rates the higher oxygen levels resulted in greater biomass. The composition of biomass didn't change much with dilution rate or oxygen level; at a low dilution rate and 20% oxygen, PHB production was about 19% of the biomass, but no more than 6% in any other condition. RNA also decreased from around 20% to closer to 10%. "Polysaccharide" was 3-4% in lower D values but 7-12% in higher, and protein increased from around 60-70 to 70-80%.

Carbon inputs and outputs were pretty balanced over different D and oxygen values; when outputs were noticeably less than inputs, there was a noticeable drop in pH (seeming to indicate incomplete oxidation of substrate).

For some reason, cells didn't do as well at lower D values; there was a loss of viability.

What This Means
The authors thought that measuring dissolved oxygen or oxygen transfer directly was not adequate to determine how much oxygen was actually getting to cells, because neither measure matched well with the amount of carbon dioxide being produced (which should correlate well with oxygen consumption). Only CO₂ production, O₂ consumption, or growth are reliable to measure oxygen transfer.

Still, it's hard to know exactly what "oxygen limitation" means; does it start when increasing the oxygen leads to an increase in biomass? Or when cells start producing PHB (which apparently is a much lower level)? The authors suggest the former as the better definition; this seems odd though, because they observed that biomass increased as oxygen increased up to the point when oxygen became intolerable and the cells washed out.

And yet, the cultures did seem to be oxygen-limited at 20% oxygen. The maintenance coefficient calculated from CO₂ production at 20% oxygen corresponded to that which others calculated in A. vinelandii in oxygen-limited cultures (0.0055 mmol O₂/mg dry weight per hour), and the respiratory index (mmol CO₂ produced per mg cell growth) approached the value indicative of oxygen limitation. Above this value, excess carbon dioxide is produced, indicating respiratory protection in the cells (oxidizing the substrate to use up all the oxygen).

Reference:

Hine, P. W. & Lees, H. The growth of nitrogen-fixing Azotobacter chroococcum in continuous culture under intense aeration. Can. J. Microbiol. 22, 611–618 (1976).

112 - The Effect of Nutrient Limitation on Hydrogen Production by Nitrogenase in Continuous Cultures of Azotobacter chroococcum

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.

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).

111 - Localization and activities of nitrogenase, glutamine synthetase and glutamate synthase in Azotobacter vinelandii grown in oxygen-controlled continuous culture

Previous research showed that A. vinelandii forms intracellular membrane structures in certain oxygen conditions (098). This could function to protect the sensitive nitrogenase from oxygen, except that one study seemed to disprove any such enzyme localization; this study was inadequate though.

So Röckel, Oelze and colleagues decided to test this hypothesis better, using oxygen-controlled continuous culture. They also tested localization of glutamine and glutamate synthases (which incorporate nitrogenase-fixed nitrogen into biomass).

What They Saw

First, the more oxygen saturation, the less nitrogenase activity (and protein content in general) they observed. In cell-free extracts, nitrogenase was only found in the soluble fraction (and thus wasn't membrane-bound). Glutamine synthase also seems all soluble.

With glutamate synthase, on the other hand, as oxygen increased, membrane-bound activity increased while soluble activity decreased.

What This Means

It's not impossible that the soluble-seeming enzymes may have a weak attachment to membranes that couldn't be resolved in this study, but it didn't seem like the membranes affected them much. The increase in membrane-bound glutamate synthase might be a form of stabilization with increasing oxygen.

Reference:

Röckel, D., Hernando, J. J., Vakalopoulou, E., Post, E. & Oelze, J. Localization and activities of nitrogenase, glutamine synthetase and glutamate synthase in Azotobacter vinelandii grown in oxygen-controlled continuous culture. Archives of Microbiology 136, 74–78 (1983).