Blog Post #8

Last week in lab, Hannah and I did an experiment to determine if our soil microbe reduced nitrate or not.  Nitrogen is a basic element of life and is a component of both proteins and nucleic acids.  Nitrogen is very abundant in the atmosphere; however, it is not in the accessible for that living organisms need it to be in.

In order to test if our soil microbe was a nitrate reducer or not, we used a positive control (P. Auroginosa), a negative control (E. Coli), and our soil microbe and inoculating 3 cultures (1 of each).  Each of the samples were incubated for 24-48 hours.  Our soil microbe is a fast grower, so we returned 24 hours later.

A snapshot before we returned to lab 24 hours later

The second day, we returned to observe the Durham tube in each sample.  If there were bubbles in the Durham tube, then the organism was not a fermenter.  However, our sample did not have any bubbles present so we added 8 drops of reagent A and 8 drops of reagent B and mixed well.  Once the drops were added, the sample turned red.  This means that the microbe reduced nitrate to nitrite!!  Below are snapshots of our Durham tubes during the experiment.

After reagent A & B were added, our sample turned red

Reagent A & B that were added to our soil sample

In order to further understand nitrate reduction, let me explain some information from a broader, biological context.  Most bacteria use the cellular structures called flagella for motility.  They attach to the cell surface and provide a 'swimming' movement.  There are also flagellar arrangements for bacteria that can be determined by staining and microscopic observation.  

Nitrate reduction indicates that the organism can use NO3- as an electron acceptor.  Also, nitrite may be reduced to either NO, N20, N2, and NH3 depending on the enzyme system of the organism and the atmosphere it is growing in.  Nitrate reduction often involves a shift to anaerobic metabolism.  Nitrate reduction plays a key role in the nitrogen cycle.  I think that some microbes evolved to be nitrate reducers, because nitrate reduction is one of the most fundamental biological processes that accounts for tons of inorganic nitrogen.  Simply stated, since nitrogen is a basic element for life, nitrogen reductase evolved in order to maintain the nitrogen cycle, which allows biological processes to be carried out successfully. 

I think that some microbes did not evolve to be nitrate reducers, because nitrogen was in a form that was accessible and usable to that particular organism.  Organisms that did not evolve to be nitrate reducers were able to use nitrogen in the form that it was already in, rather then converting it to a different form. 

Along our journey to identify our soil microbe, we have been keeping track by using a dichotomous key.  Below is the identification of our mysterious soil microbe thus far:

1. Gram positive

2. Morphology, Rod Shaped (Bacilli)

3. Non-acid fast organism

4. Catalase activity

5. Endospore positive

6. Motile

7. Nitrate reduction

Citations:

http://www.nature.com/scitable/knowledge/library/the-nitrogen-cycle-processes-players-and-human-15644632

http://www.microbelibrary.org/library/laboratory-test/3660-nitrate-and-nitrite-reduction-test-protocols