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|
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.
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).