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).