This past week in lab Dylan and I performed a Nitrate Reduction Test on a positive control, a negative control, and our unknown bacteria. This test has three different purposes that can be sought out through experimentation:

• The utilization of nitrate as a nitrogen source for growth, which is nitrate assimilation.
• The generation of metabolic energy by using nitrate as a terminal electron acceptor, which is nitrate respiration.
• The dissipation of excess reducing power for redox balancing, which is nitrate dissimilation.

Nitrate has proven to be significant in the fields of biochemistry, medicine, and environmental microbiology. Nitrate reduction is also very helpful when it comes to identifying different strains of bacteria; some of the more popular strains are Enterobacteriaceae, Neisseria, and Corynebacterium. We plan to use nitrate reduction to continue on in the never-ending search to  identify our UNKNOWN!

Why get irate about fixin' nitrate?

• Nitrogen is a vital element for amino acids and nucleic acids, which are vital biological processes in all organisms

• N2 gas is the major available source of nitrogen, but is highly inert due to the strength of the N2 triple bond
• Some bacteria have evolved to convert this N2 into usable forms such as NO3-Example- Rhizobium grow mutualistic with plants.Plants provide rhizobium with carbohydrates, and rhizobium give plants usable NO3- 
• These plants are then eaten by omnivores, which assimilate the Nitrogen of the eaten plant 
• These animals eventually die, and their nitrogen is utilized by other organisms and fungi

• Thus the circle of Nitrogen (and life) continues

• Information gathered via: http://www.visionlearning.com/en/library/Earth-Science/6/The-Nitrogen-Cycle/98

Nitrogen fixers vs. Non-Nitrogen

• We've previously discussed Nitrogen fixation and the benefits seen in the Nitrogen cycle, so why would it be favorable for any microbe to preform the opposite reaction and reduce nitrates?
• Microbes that reduce nitrate use it as a means of an electron acceptor in anaerobic conditions.
• This requires a low O2 concentration, allowing these microbes to be better competitors in habitats with low O2.
• These microbes are often found deeper in soil than are nitrogen fixers
• The reduction of Nitrates reaching these deep soil nitrogen reducers prevents the leeching of nitrogen into deeper soil
• The Nitrate reduction process is also used in sewage sanitation as well as industrial waste sanitation in order to remove ammonia and nitrates from waste water
• Information gathered via: https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/microbial-metabolism-5/anaerobic-respiration-49/nitrate-reduction-and-denitrification-314-7650/

In order to determine if our unknown bacteria reduced nitrate (NO3-) to nitrite (NO2-) the following procedure was followed:

 

Day One

• Three cultures ( a positive control, a negative control, and our unknown) were inoculated and then incubated for 24-48 hours.
  • But keep in mind that slow growing bacteria may need a longer incubation period to get the desired results!

Day Two

• Dylan and I returned back to the lab to check our cultures after 48 hours and saw that our cultures had not really reached a point for good observation, so another 24 hour incubation period was added to the 48 hours of incubation that had already occurred.

Day Three

• The Durham tubes were observed to see if any bubbles had formed inside, meaning that the controls and the unknown would produce nitrogen gas (N2).
• If there are bubbles present (which there weren’t) and the organism is not a known fermenter, then the most possible scenario is that the microbes reduce nitrate to N2.
  • If these bubbles are present then no further test need to be run, and the microbe is classified as a nitrate reducer!
• BUT…if no bubbles are present upon observation after incubation then a set of further steps must be taken in order to identify the microbes.
• First 8 drops of reagent A and 8 drops of reagent B are added to the cultures and mixed thoroughly.
  • If the sample then turns red, it is determined that the microbe does in fact reduce nitrate to nitrite.
  • BUT…if the sample does not turn red, then a small amount of zinc must be added to the culture.
    • If when the zinc is added the sample turns red, then it is determined that the microbe is not capable of nitrate reduction and the test is complete.
    • BUT…if the sample does not turn red after the addition of zinc, then it is to be decided that the microbe does reduce nitrate, but it is into something other than nitrate.

Results

• Upon completion of our Nitrate reduction test, we found:
• No bubbles-  Our microbe does not reduce NO3- to N2
• No color change after application of reagent A and B- Our microbe does not reduce NO3- to NO2-
• Color change after application of Zinc- Our microbe is unable to preform Nitrate Reduction

What the heck is this microbe?

• At this point Devin and I have had a mixed bag in the way of results for our experiments.
• Pleomorphic (between cocci and bacillus)
• Gram stain results were inconclusive (a perfect color between our + and - controls)
• Not acid fast
• No catalase activity
• Ferments carbohydrates via aerobic and anaerobic metabolism
• Endospore former
• No Motility
• No nitrate reduction abilities
• As proposed earlier our microbe looks as if it could be of the genus Streptomyces
• Unfortunately, according to http://terpconnect.umd.edu/~asmith/ALSACE/Discussion.html similar experiments showed results in Acid fast, and fermentation experiments that yielded different results.
• Maybe this week we can re-do these experiments and try to eliminate all doubt for our microbe being Streptomyces.
• Terpconnect also reports that Streptomyces are antibiotics producers, so this could be an additional experiemejnt

So, until further experiments are conducted, it seems as though we are still at a stand still with identifying our microbe. With that said we could still be on to something with Streptomyces, and will continue to look into further testing for this genus.


Tune in next week and checkout the results of our hemolysis experiment!


Is our organism fastidious? Find out next week!