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Nitrogen fixation is the biological reaction where N₂ gas is converted into NH₃. Ammonia is a form of nitrogen that can be readily utilized for biosynthetic pathways, whereas N₂ is essentially unusable. Nitrogen fixation, therefore, is a process critical in the completion of the nitrogen cycle. Bacteria and Archaea are the only living organisms known to be able to fix nitrogen and make it available for use in the biosynthesis of proteins and nucleic acids. Higher organisms are unable to perform this process.

Nitrogen fixation is carried out by the extremely oxygen labile enzyme complex nitrogenase, that is activated under fixed nitrogen limiting conditions. This enzyme reduces nitrogen gas to ammonia using the following reaction:

16-24 ATP + 8H⁺ + 8e^- + N₂ -> 16-24 ADP + P_i + 2NH₃ + H₂

As can be seen, this reaction is extraordinarily energy-expensive. Because of this, nitrogen fixation is the method of last resort and expression of the nitrogenase enzyme complex is tightly regulated by the cell. Some organisms even turn off the already synthesized enzyme during unfavorable conditions (post-translational regulation).

Since nitrogenase is inactivated in the presence of oxygen this presents an interesting problem for Azotobacter, a strict aerobe. Azotobacter species solve this problem by protecting their oxygen labile enzymes in two ways. One, special auxiliary proteins are produced which cause nitrogenase and other O₂ sensitive enzymes involved in nitrogen fixation to aggregate when exposed to oxygen. Two, the organism has one of the highest respiration rates ever recorded (about five times that of E. coli), which creates a nearly anoxygenic environment in the cytoplasm of the cells. Azotobacter and several other bacteria carry out nitrogen fixation as free living soil organisms (Azotobacter and Klebsiella pneumoniae, for example) and others carry it out in symbiotic association with plants (Rhizobia and Frankia).

In symbiotic nitrogen fixation, the plant helps the bacteria protect its nitrogenase from oxygen exposure.The plant also provides nutrients and housing for the bacteria, in return the bacteria generate a useable form of nitrogen for the plant.

In this experiment we will isolate Azotobacter from soil or water

Isolation of Azotobacter

Organisms in the genus Azotobacter are chemoorganotrophs, capable of using sugars, alcohols and salts of organic acids for growth. During growth, many species will produce water soluble and water insoluble pigments, causing cultures and colonies of the organism to appear in shades of yellow, green, red and brown. While growing on sugars, some Azotobacter will produce copious amounts of an extracellular polysaccharide (EPS). Often in this laboratory the microorganisms isolated will produce so much EPS that the culture will have the appearance of cream pudding.

Under nutrient limiting conditions, the organism forms resting structures called cysts. Cysts can be described as vegetative cells encapsulated in a desiccation resistant coat. They are very resistant to drying and the encysted bacteria can survive for many years in this state.

The enrichment for the Azotobacter is based on the ability of the organism to fix nitrogen aerobically. Also, the large size of the bacteria (greater than 2 mm in diameter and 3-7 mm in length) helps to screen for the desired organism. The bacteria are found in many soils and in water. In this experiment we will be isolating Azotobacter species from plain old, garden variety soil and water.

Period 1

Materials

Soil samples from one or more sites will be available for those who didn't bring in their own.

Flask of Nitrogen-Free Broth

Empty 250-300 ml stoppered flask

Procedure

1. Record the details about the sample you are using (source, date of collection). Place about half a teaspoonful of soil in the empty flask. Add the Nitrogen-Free Broth up to about the 50 ml mark.
2. Incubate the flask at 30°C for 2-5 days.

Period 2

Materials

2 plates of Nitrogen-Free Agar

N-free broth enrichment after incubation

1. Examine the flask for a film of surface growth. The common (and easily-recognized microscopically) nitrogen-fixer Azotobacter is a strict aerobe, and its growth may be profuse.
2. Prepare a wet mount - preferably of the surface film if it is present - and observe with the phase microscope. Note the variety of different cell types which may be motile or non-motile. Some things to keep in mind:
• Azotobacter - a common, strictly aerobic nitrogen-fixer - is generally seen as relatively large, oval-shaped cells, usually in pairs. Other nitrogen-fixers may not be as distinctive, such as Klebsiella - a short, facultatively anaerobic, non-motile rod which is also common in many soils.
• Often the enrichments are deep enough such that strictly anaerobic nitrogen-fixers can grow. Many strains of Clostridium fix nitrogen, and they are usually distinguishable by the presence of endospores which swell the cell. These organisms are responsible for any frothy bubbles and rancid smell (both due to certain end-products of fermentation).
• Non-nitrogen-fixing organisms may arise as the amount of fixed nitrogen compounds provided by the nitrogen-fixers) increases; these organisms will not be distinguishable except for occasional protozoa, some of which may be seen ingesting bacteria whole. (Note that your flask may have such a rudimentary food chain!)
3. Streak a plate of Nitrogen-Free Agar using the enrichment (preferably from any surface growth growth) for isolated colonies and incubate at 30°C for 1-2 days. (If incubated too long, rapid and profuse growth of the colonies of nitrogen-fixers may cause overgrowth such that finding isolated colonies will be impossible; re-streaking may become necessary.)

Period 3

Materials

2 or more slants each of Nitrogen-Free Agar and an all-purpose medium (such as Trypticase Soy Agar)

Procedure

1. Examine the plates, noting the variety of colonies present. Would there be a possibility of non-nitrogen-fixers growing among the nitrogen-fixers? Make wet mounts and gram stains of two or more of the well-isolated colonies. Are Azotobacter-like cells apparent for each colony chosen? (Unless the growth is unusually mucoid, Azotobacter will stain gram-negative.) If not, at least note the cell morphology and Gram reaction. Further tests would be necessary to make an identification of organisms other than Azotobacter. (What would it take to identify Klebsiella?)
2. From each of the colonies examined, inoculate a slant of Nitrogen-Free Agar and a slant of an all-purpose medium. Considering what we are testing for here, why is it ultra-critical that we can only be inoculating pure cultures? Incubate both slants at 30°C for 2-5 days.

Growth of Azotobacter on Nitrogen-free medium slant and on all purpose medium

Period 4

1. Examine the slants. On which medium would growth indicate nitrogen-fixation? Note why it is essential that the slants be inoculated with pure cultures - which should be the rule anyway!

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