Need a Fix? Nitrogen Fixation and Reduction in Our Unknown Soil Microbe

      In order to further characterize our unknown soil microbe, this week's lab focused on the process of nitrate reduction. This process is an integral part of the larger nitrogen cycle that alters atmospheric nitrogen into a usable form by plants and back into its atmospheric form. This process can be seen as a balancing act between the nitrogen that is "fixed" by other species of bacteria the nitrate that is removed from the soil by "denitrifying" bacteria and accounts for the majority of the nitrogen that enters the atmosphere. They are essential for the prevention of excessive levels of soil nitrate, which is important for public health and prevent eutrification (an overabundance of nutrients) of water sources.

      Only certain species of bacterial microbes have evolved the ability to reduce nitrate. This process typically occurs in locations that do not provide enough oxygen for aerobic respiration. Though nitrate reduction provides a microbe with a source of energy, it does not produce nearly as much energy as oxygen does in aerobic respiration. Thus, it would make sense that only bacterial species that are commonly found in oxygen-defficient locations would evolve the ability to reduce nitrate.

     We were able to use a simple test to determine whether our unknown microbe was capable of reducing nitrate. Three samples were inoculated with E. coli, P. aergoginosa, and our unknown sample, respectively, along with a control that did not contain a microbe. Samples were incubated for 48 hours. A smaller tube, known as a Durham tube, was located within the inoculated sample tube. If a bubble formed in the top of the Durham tube, it would indicate that our microbe was able to reduce nitrate (NO3) to its gaseous atmospheric state (N2). As seen in the photo below, this was not the case for our microbe. If the microbe were able to reduce nitrate to another form, such as nitrite (NO2), we would see the solution turn red after adding a solution of sulfanilic acid and alpha-naphthylamine to the inoculated sample. After adding the solution, our inoculated sample turned red (bottom photo). Thus, we concluded that our microbe was able to reduce nitrate to nitrite.

Results after 48 hours. Bubble formed in Durham tube in P. aeroginosa only.

Result following addition of sulfanilic acid and alpha-naphthylamine.

Up to this point, the dichotomous key has indicated that our unknown bacterium is Clostridium. According to the internet sources listed below, this identification still holds true with the results of this week's experiment. Come back next week for more details!!

http://en.wikipedia.org/wiki/Nitrogen_fixation
http://en.wikipedia.org/wiki/Denitrifying_bacteria

- Austin