11-4 Nitrogen fixing bacteria
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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 + Pi + 2NH3 + H2
Figure 11-10 Azotobacter at 1000 x
The appearance of Azotobacter vinelandii in the phase microscope at 400X. Note the large, ovoid shape of the cells.
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 go so far as to be able to turn 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.
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
2 plates of Nitrogen-Free Agar
Figure 11-11 Nitrogen-free broth enrichment
The appearance of the broth after incubation (A). Note the ring of growth at the surface of the medium. A phase contrast micrograph of the enrichment at 400X (B).
2 or more slants each of Nitrogen-Free Agar and an all-purpose medium (such as Trypticase Soy Agar)
Figure 11-12 Azotobacter colonies on N-free medium
Azotobacter colonies (left) are often slimy, due to synthesis of exopolysaccharide, and pigmented. A wet mount of an isolated colony off the plate at 1000X magnification. Note the large size of the cells. Both cysts (phase bright ovals) and vegetative cells (phase dark bacilli) are visible.
Figure 11-13 Growth of isolates on all-purpose and nitrogen free medium
Growth of nitrogen-fixing isolates on all-purpose medium (APM) and nitrogen-free medium (NF). Notice how the microbes grow to a much lower density of NF, this is due to the energy requirements of nitrogen fixation.