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).

215 - Respiratory protection of nitrogenase in Azotobacter vinelandii

This study is pretty similar to 214, on how oxygen levels influence the respiratory chain of Azotobacter vinelandii.

What They Saw
They grew cells in batch at low aeration, and then increased the aeration to expose cells to excess oxygen.

The results were similar to 214: when aeration increased, cells stopped growing until respiration had ramped up and leveled off. Despite the large increase in respiration, cellular ATP levels dropped 40%. P/O ratios decreased as respiration increased to accommodate the increased oxygen, at least for NADH dehydrogenase.

They tried adding chloramphenicol again, since last time it didn't affect the growth lag, but this time observed that it lessened the increase in respiration about half, and prevented cytochrome a₂ and other dehydrogenase increases.

What This Means
The M/N ratio, meaning maintenance M (moles ATP consumed per weight of cells over time) over phosphorylating efficiency N (P/O ratio x 2), explains doubling of respiratory activity upon increase in oxygen only if M increases or N decreases. It doesn't seem like M does increase, so N seems to be decreasing, because respiration uncouples from phosphorylation.

A. vinelandii seems to have different branches in its respiratory chain, which allows it to tolerate different and suddenly changing levels of oxygen.

Reference:

Jones, C. W., Brice, J. M., Wright, V. & Ackrell, B. A. C. Respiratory protection of nitrogenase in Azotobacter vinelandii. FEBS Letters 29, 77–81 (1973).

214 - The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects

This study looks at how oxygen levels influence the respiratory chain in Azotobacter vinelandii.

What They Saw
They grew cells with high or low aeration (based on volume of culture in the same size flask), then isolated respiratory membranes and measured P/O ratios.

The cells grew and quickly used up all the dissolved oxygen. With high aeration, they grew much faster and leveled off once the oxygen was gone, and with low aeration the growth was slower (not even really logarithmic) but continued long after the oxygen was gone. Respiratory activities were 2-5x higher when cells were growing logarithmically with excess oxygen than when oxygen was limited.

If they suddenly increased the aeration when oxygen had run out, respiratory activity increased back up to high levels (1000 μl/h/mg dry weight). Cells didn't start growing again until it had leveled off. This lag was the same when chloramphenicol (which inhibits protein synthesis) was present, suggesting that it wasn't due to the synthesis of new enzymes for respiration.

When oxygen was being consumed, cytochrome and oxidase levels were pretty constant (except cyt o oxidase, which increased), but when oxygen ran out, levels of c₄, c₅, and b₁ increased quickly and a₂ more slowly. Cytochrome o oxidase and a₂ oxidase also increased a lot, a₁ less so. The increase of o was fastest and greatest.

P/O ratios were similar to those seen before at maximum, but they didn't reach this maximum until oxygen was mostly used up late in the logarithmic growth phase, at least for NADH dehydrogenase.

With low aeration, cytochrome levels were pretty constant (since oxygen ran out almost immediately), increasing just a little, except for cyt a₂ oxidase which went up pretty constantly. P/O ratios were pretty level too, and fairly high, similar to when oxygen ran out in high aeration.

What This Means
This kind of pattern fits in well with those seen in other obligate aerobes. Cytochromes probably increase during oxygen limitation to try to compensate for the limitation. But the low P/O ratios at high oxygen makes sense in light of respiratory protection; respiration is uncoupled from energy generation.

Reference:

Ackrell, B. A. C. & Jones, C. W. The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects. Eur. J. Biochem. 20, 29–35 (1971).

352 - The Respiratory System of Azotobacter vinelandii: 1. Properties of Phosphorylating Respiratory Membranes

Similar to 161, this study looks at elements of Azotobacter vinelandii's respiratory chain and P/O ratios thereof.

What They Saw
They isolated respiratory membranes (basically pieces of cell with the respiratory chain intact but not much else, I think) and looked at oxygen reduction with different electron donors.

They saw P/O levels up to 1.1 with NADH and about 0.7 with malate, higher than in 161.

Reference:

Ackrell, B. A. C. & Jones, C. W. The Respiratory System of Azotobacter vinelandii: 1. Properties of Phosphorylating Respiratory Membranes. Eur. J. Biochem. 20, 22–28 (1971).

213 - Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum

When respiratory protection fails in Azotobacter, it can temporarily inactivate its nitrogenase to protect it, by association with another protein, called FeSII or Shethna. This study purifies this whole complex (nitrogenase and FeSII) and investigates its characteristics in A. chroococcum.

What They Saw
The more pure the nitrogenase, the less protection from oxygen inactivation they observed. But while crude extract had the most protection, more pure forms were pretty similar until the protective FeSII protein was absent, in which case the nitrogenase was rapidly inactivated. Magnesium ions (or possibly other divalent ions) were also necessary for this stabilization.

This protective protein was 14 kDa, orange in color, and had 2 Fe and 2 S atoms, so a 2Fe-2S center (thus the name). This version seems smaller than the A. vinelandii version though, which is 23 kDa. In stable complexes, the three components (dinitrogenase, dinitrogenase reductase, and FeSII) were present in about 1:1:1 ratios.

What This Means
This FeSII (with Mg ions) appears to be sufficient to protect the nitrogenase complex from oxygen, stabilizing it even outside of the cellular environment. This stability is perhaps not complete though, since crude extracts did show more activity after exposure to oxygen.

Reference:

Robson, R. L. Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum. Biochem J 181, 569–575 (1979).

198 - Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments

This study looks at oxygen interactions with dinitrogenase reductase (DNR) in Azotobacter chroococcum (and Klebsiella pneumoniae) to see if it's possible that the enzyme can be oxidized without being totally inactivated.

When the DNR was present in high enough levels relative to the amount of oxygen (4-fold molar excess), it was protected from inactivation by oxygen: it seemed to reduce superoxides to peroxide (and then water if catalase doesn't get to it first) before the reactive oxygen species could harm it. Superoxide seems to be the harmful form of oxygen for DNR. They suggest this helps with high respiration rates (i.e. consumption of oxygen). They calculated that if 10% of the cell's protein were DNR, it could account for pretty much all of the oxygen consumption. This is unlikely, but it could still be a significant part of respiratory protection. They call this "autoprotection."

Reference:

Thorneley, R. N. & Ashby, G. A. Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments. Biochem J 261, 181–187 (1989).

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).

099 - Oxygen and Hydrogen in Biological Nitrogen Fixation

Oxygen is pretty toxic to nitrogen fixation enzymes, so organisms or the people studying them need to take steps to protect them. They lose more than half their activity within minutes exposed to air. The dinitrogenase reductase is more sensitive than the dinitrogenase itself, at least the Mo version. Some can retain some activity even up to an hour in air. But the FeMo-cofactor, when extracted, is even more sensitive than the dinitrogenase reductase. Overall, it seems that the metal-sulfur centers are the most sensitive parts.

And yet, there are nitrogen-fixing species that are obligate aerobes, or even oxygenic. How do they do it?

Azotobacter has been shown to increase its respiration while its growth efficiency decreases as oxygen increases, seeming to waste the oxygen: this has been called "respiratory protection." The mechanism for this is not simple though; it involves carefully regulated shifts in respiratory components throughout the whole catabolic system.

Azotobacter also has the ability to reversibly inactivate its nitrogenase if respiratory protection is not possible (such as in carbon-limited conditions, or upon a sudden increase in oxygen). This seems to depend on FeSII protein (aka Shethna), though it is suggested that there may be other mechanisms.

When oxygen is too high and cells' supply of fixed nitrogen runs out, production of nitrogenase may be regulated (no sense making an enzyme when it can't function). This regulation may be done by the products of nifAL genes.

Azotobacter also produces gummy alginate which might have a role in protection from oxygen, but non-gummy strains (such as CA) have been isolated that don't seem especially oxygen-sensitive. I wonder if they have higher rates of respiration though, or if they might be more sensitive in carbon-limited conditions.

Nitrogenase also produces hydrogen gas, whether or not it's reducing anything else. This reaction seems separate from the nitrogen fixation reaction, since some things can inhibit the latter without inhibiting the former. Acetylene seems to inhibit hydrogen production though. Nitrogen can't compete with hydrogen for electrons completely, even with pure pressurized nitrogen; the enzyme always produces at least 1 mol hydrogen for each mol nitrogen gas fixed.

Of course, this hydrogen usually doesn't just escape; Azotobacter and other diazotrophs recapture it with their uptake hydrogenase. The exact purpose this serves is not clear though.

Reference:

Robson, R. L. & Postgate, J. R. Oxygen and Hydrogen in Biological Nitrogen Fixation. Ann Rev Microbiol 34, 183–207 (1980).

374 - The Azotobacteriaceae

This was a very interesting review of the Azotobacter family from more than fifty years ago. It was interesting to see observations in this paper that I had made myself in my own research.

It discusses the taxonomy of Azotobacter somewhat: the genus includes A. chroococcum, A. beijerinckii, and A. vinelandii of course, and A. agile (which I'm not sure is considered a real separate species now); other alleged species (A. indicum for example) seemed like they should be separated into another genus, Beijerinckia. A. chroococcum was the first, discovered by Martius Beijerinck in 1901.

Characteristic features of Azotobacter are their large size, short thick rod-shaped cells (often found in pairs), nitrogen fixation (despite being obligate aerobes), and poor growth on digestions of meat extracts and such, like LB (something I've noticed myself). The cells change shape depending on their conditions though, which can be confusing. Worse, they can be difficult to isolate from contaminating strains. They also form resistant, dormant cysts in some conditions, though I don't think I've observed this personally. They also can form storage granules of different kinds which are observable under a microscope.

They're pretty versatile in their ability to use different carbon compounds. They can use alcohols (ethanol, propanol, butanol, etc), organic acids (acetate, citrate, butyrate, etc), and saccharides (glucose, fructose, galactose, sucrose, etc.). This depends on the species and strain somewhat; some seem to be able to use lactose, others not. Some can use starch and some other polysaccharides. Some can even use cyclic compounds (benzoic acid, phenol, salicylic acid) which are generally toxic. There are some things they can't use, such as xylose, methanol, and formic acid. Their respiration rate can be very high, the highest observed in nature (at that time, at least).

Their versatility regarding nitrogen compounds seems to be lower though. They can fix nitrogen, of course, and use basic inorganic forms (ammonia, nitrate) and some common organic forms (urea, glutamate, asparagine), but otherwise are limited. So they don't grow well on complex forms such as protein digestions (peptone, tryptone).

Otherwise, as represented in Burk medium, they need phosphorus, sulfur, potassium, calcium, magnesium, iron (amount depending on whether they were fixing nitrogen), and of course molybdenum or vanadium helped when fixing nitrogen too. Other trace elements or vitamins seem unnecessary, at least in many conditions.

The review suggests that azotobacters can produce compounds that stimulate or inhibit plant roots; I wonder if that is true.

The organisms are obligate aerobes, of course, capable of tolerating very high levels of oxygen, especially when not fixing nitrogen. They're mesophiles, preferring around 30ºC. Preferred pH depends on the strain, but around 6-8 is typical.

Some have actually reported that the weather can affect their growth, especially high-pressure areas, but this hasn't been confirmed.

Many have noticed that azotobacters seem to mutate fairly frequently; this is probably due to transposons and natural competence.

How much nitrogen do azotobacters actually fix in soils? It's hard to tell, of course, because it depends on many things and it's hard to measure the contribution of a single genus in such a complex environment, so it couldn't be said.

Jensen, 1954

Reference:

Jensen, H. L. The Azotobacteriaceae. Bacteriol. Rev. 18, 195–214 (1954).

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).

182 - The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity

Nitrogenase requires a steady flow of reductant to reduce nitrogen gas to ammonia. This study looks at how electrons move from catabolism to the nitrogenase in Azotobacter vinelandii. Previous studies seemed to isolate two components of the transport chain to nitrogenase: azotoflavin and azotobacter ferredoxin. But it was still unknown how these compounds themselves were reduced, since A. vinelandii can't reduce them directly from pyruvate as others can (such as Clostridium pasteurianum).

What They Saw
They extracted cell components and fractionated them, separating the carriers mentioned above from nitrogenase. Then they tested the potential of NADH and NADPH to reduce these carriers, hypothesizing that these reductants are used for many other redox reactions.

This reaction seems energetically unfavorable because the redox potential of NAD(P)H is higher than that of ferredoxins, but it has been shown to happen before, especially when the NAD(P)H/NAD(P)⁺ ratio is high. So it's plausible.

They found that NADPH seemed to be able to reduce azotoflavin, and could support nitrogenase activity, at least in vitro. NADH did not seem to have the same activity. Adding extra azotoflavin or ferredoxin increased activity too.

There was one more factor that they didn't identify or include in their assays, one that they replaced with spinach ferredoxin-NADP⁺ reductase. Presumably it is some reductase in A. vinelandii that they didn't purify right. They did figure out which fraction contained it though, and it restored most activity. It could be denatured by mild heating, apparently.

Of other substrates that might support nitrogenase activity, only those linked to NADP⁺ seemed to be useful in vitro: malate, glucose-6-phosphate, alpha-ketoglutarate, and isocitrate. And apparently NADP⁺-linked isocitrate dehydrogenase is pretty common in A. vinelandii cells.

So it seems like NADPH is the donor that starts the electron transport branch to nitrogenase.

Reference:

Benemann, J. R., Yoch, D. C., Valentine, R. C. & Arnon, D. I. The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity. Biochim. Biophys. Acta 226, 205–212 (1971).

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).

156 - Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

This study was intended to study carbon metabolism in Azotobacter and the formation of PHB.

What They Saw
The enzymes they studied (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 6-phosphogluconate dehydratase, 3-deoxy-2-oxo-6-phosphogluconate aldolase, citrate synthase, and isocitrate dehydrogenase) all seemed to be inhibited by NADH and/or NADPH.

What This Means
PHB synthesis consumes reduced nucleotides such as NADH, which could reduce their inhibitory effects when oxygen is limited.

Reference:

Senior, P. J. & Dawes, E. A. Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii. Biochem. J. 125, 55–66 (1971).

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).

097 - Aeration in fermentations

Providing oxygen in culture conditions can be tricky. It should be done homogeneously across time and space in the reactor, so all cells experience the same levels all the time. But homogenizing aeration adequately and measuring real-time dissolved oxygen levels are difficult tasks, because oxygen is not very soluble in aqueous solutions.

Azotobacter vinelandii, for example, is an obligate aerobe, so it needs some level of oxygen, but too much can be harmful when the cells are fixing nitrogen (or, at least, reduces the efficiency of the cells' product formation). So optimizing A. vinelandii culture requires determining the optimal oxygen level and how to reach that level.

What They Did
Phillips and Johnson had a reactor with pH and dissolved oxygen electrodes, aerated by agitation and sparging, and sensors of oxygen and CO₂ in the exhaust gas. So a pretty nice setup.

They grew various organisms, including A. vinelandii strain O, in media with 5% glucose and measured their oxygen demands. Sometimes they added ammonia as fixed nitrogen, sometimes they didn't.

It seemed like A. vinelandii's oxygen utilization rate was the lowest, compared to E. coli, Aspergillus, and Penicillium; all of these remained constant as oxygen tension increased. It seems odd though.

In the reactor, when fixing nitrogen, A. vinelandii seemed to use excess sugar just to get rid of oxygen; its oxygen demand was much higher than expected for the growth, yield, and number of cells observed. When they added ammonia so the cells didn't fix nitrogen, they claim the same effect was observed, but I don't really understand how they reached that conclusion. Their graphs seem kind of messed up, not fitting their descriptions very well.

What This Means
Overall, the conclusion was that cells don't need more oxygen than just enough to keep the level above a critical threshold. Measuring dissolved oxygen is good for keeping above this threshold, but it doesn't help determine how much extra oxygen is needed if levels drop below measurable amounts. But when oxygen demand and uptake rates are measured, the oxygen deficit can be measured, except maybe with A. vinelandii which is more complicated.

Reference:

Phillips, D. H. & Johnson, M. J. Aeration in fermentations. Journal of Biochemical and Microbiological Technology and Engineering 3, 277–309 (1961).

161 - On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids

The authors wanted to study oxidative phosphorylation (generating ATP through respiration) in Azotobacter vinelandii and another related to oxygen, nitrogen fixation, and hydrogen oxidation by hydrogenase.

What They Saw
They grew A. vinelandii strain OP (aka CA) in chemostats, at D = 0.1 h^-1, limited in oxygen or nitrogen, then broke up the cells and isolated membrane vesicles anaerobically.

The P/O ratio is how much ATP is produced by moving 2 electrons through the electron transport chain to reduce one oxygen atom. The pattern of P/O over a range of oxygen levels is similar for different electron donors (NADH, malate, hydrogen, and NADH + hydrogen): it goes up to a peak, then falls as dissolved oxygen levels rise above the limit of detection. ATP production mostly levels off near that peak too (except with hydrogen, where it declines). The peak occurs at higher oxygen levels with NADH and malate than with hydrogen though. The height of the peak is 0.7 (ATPs per O reduced) for NADH and hydrogen, but only 0.5 with malate.

They tried adding acetylene up to 20%, but didn't see any indication that hydrogenase was inhibited. Previous studies showed that 40% acetylene was required to show inhibition, so it's not surprising. They also found that the hydrogen branch seemed to be very efficiently coupled to phosphorylation, and doesn't seem to involve flavoproteins.

What This Means
The lower values from hydrogen at high oxygen is probably due to inactivation of the hydrogenase, which is sensitive to oxygen.

The fall in P/O ratio is likely due to excess oxygen going through a different cytochrome branch, with cytochrome b to cytochrome d, which isn't involved in ATP production, so respiration and ATP are decoupled.

It also appears that the electrons from hydrogen oxidation don't travel through the same branch of the electron transport chain as electrons from carbon sources; hydrogenase has its own branch.

Reference:

Laane, C., Haaker, H. & Veeger, C. On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids. European Journal of Biochemistry 97, 369–377 (1979).