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).

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).