144 - Experiments on the Transformation and Fixation of Nitrogen by Bacteria

This old and somewhat hard-to-find report (Update: apparently now it's on Google Books; see link below) by J.G. Lipman details the discovery of Azotobacter vinelandii, discovered in Vineland, New Jersey in the early 1900s.

Lipman's goal was to study the nitrogen-related behaviors of soil bacteria in general, and specifically to isolate nitrogen-fixing bacteria.

Behaviors of soil microbes in nitrogen-rich or nitrogen-poor media
When soil is added to nitrogen-rich medium, such as meat extract, many species grow. Some soluble nitrogen becomes insoluble (incorporated into bacterial cells/biomass), but more is broken into simpler forms, including nitrogen gas, so the total amount of nitrogen in the culture decreases.

But when there is a minimal medium of certain salts and organic compounds but not much fixed nitrogen, the amount of nitrogen in the medium increases when soil is added, because bacteria fix nitrogen gas.

So two results from the same soil in different media. This makes sense. Different bacteria grow to different levels in each.

Some of the flasks of these two kinds of media were inoculated with soil from Freehold, either pasteurized or not, or similar soils from Vineland. Others were kept sterile or inoculated with a single species (one B. pyocyaneus). These each differed in appearance after incubating for a number of days.

In high-nitrogen medium, the non-pasteurized soils reduced the total nitrogen the most, as should be expected.
In low-nitrogen medium, pasteurized Vineland soil actually remained sterile, but the others grew, and those increased the amount of nitrogen. Apparently the Freehold soil had more spore-forming organisms, which are more likely to survive pasteurization.

In the Freehold soil, nitrogen-fixers seemed to consist mostly of spore-forming organisms, while in the Vineland soil the main organism appeared as large, aerobic diplococci (paired roundish cells), which Lipman labeled Azotobacter (i.e. nitrogen bacteria).

Also noteworthy: non-pasteurized Vineland soil fixed more nitrogen than any of the others, by far.

Isolation of Azotobacter vinelandii
Trying to isolate Azotobacter from soil samples was somewhat difficult for Lipman, because small bacilli kept overgrowing his cultures, but eventually he found a good medium to select for the desired bacteria. Through certain tests (growth preferences, motility, shape of cells), Lipman distinguished his culture from Beijerinck's Azotobacter chroococcum isolate, naming it instead Azotobacter vinelandii (because it came from Vineland).

A. vinelandii Characteristics
Lipman noted A. vinelandii's growth characteristics in various media, and observed in some that the cells accumulated deposits of what he called fat, which looked like small globules, giving the body a granular appearance.

He also noted that the cells produce a bright yellow pigment that diffuses out from the cells, especially when well-aerated. He tried to figure out what the pigment is, but apparently a fire at the research station interfered with that goal.

A. vinelandii Fixing Nitrogen
Lipman did an experiment with his bacteria, inoculating several flasks of broth with them and testing how much nitrogen they fixed. He found that, for a given volume of culture, being in a bigger flask meant fixing more nitrogen; i.e. more surface area was helpful. Apparently oxygen is important.

Then he tried different forms of nitrogen (or none at all): potassium nitrate, peptone, or ammonium chloride, in two different quantities. After 18 days, all cultures had the same appearance, with growth and yellow pigment. The amount of fixed nitrogen in them was pretty similar for all. So it seemed like adding fixed nitrogen, at least in these low amounts, didn't affect nitrogen fixation much.

For some reason, Lipman also observed that adding filter paper to the culture more than doubled the amount of nitrogen fixed. Maybe extra carbon? Seems improbable. When they weighed the paper after removing it from the culture, it didn't seem to have decreased at all, so apparently it wasn't consumed.

A. vinelandii Carbon Substrates
Lipman tried growing the bacteria on a variety of carbon sources, to see which it could use: ethanol, glycerine, mannite (mannitol) + soil, propionate, succinate, and citrate. It seemed to grow on all of them to some extent; perhaps best on mannitol, then glycerine, then ethanol and propionate, then a bit on succinate, and very slightly on citrate. This is paralleled in the amounts of nitrogen fixed in each one.
They tested some other substrates, but didn't complete the experiments before the fire mentioned above. After 48 hours, A. vinelandii seemed to be growing on glucose, sucrose, and dextrin, but not lactose.

A. vinelandii and Bacillus 30
Apparently there was some mysterious bacterium, discovered around the same time as A. vinelandii, which, when grown together with A. vinelandii, enhanced the latter's nitrogen fixation for some reason, though by itself B. 30 couldn't fix very much, if any.
When grown together, A. vinelandii seemed to fix from 2-4 times more nitrogen. Considering that B. 30 seems to produce a large amount of hydrogen, this could be one way it helps nitrogen fixation, but I'm not sure exactly how that works.
None of the other soil microbes discovered around the same time seemed to help A. vinelandii fix nitrogen very much.

Adding pasteurized soil from different places also seemed to help A. vinelandii fix nitrogen, especially Vineland soil, but it wasn't clear why for that either.

Other Factors Influencing Nitrogen Fixation
Adding iron to the medium seemed to help increase nitrogen fixation a great deal.

There were some other experiments, but I didn't think them very important. Check it out if you're curious.

Citation: Lipman, J. G. Experiments on the Transformation and Fixation of Nitrogen by Bacteria. New Jersey State Agric. Exp. Sta. Ann. Rep. 24, 217-285 (1903).