Tuesday, April 21, 2015

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).

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).

No comments:

Post a Comment