Nitrogen Cycle

What is nitrification?

conversion of NH4+ to NO2- to NO3-

Describe peptone broth, including

• testing for

• complex or chemically defined

• main component of broth

• main differential component

• enzyme or pathway product

• positive indicator and org

• negative indicator and org

• ammonification, creation of NH4+

• complex

• peptides

• OPA reagent, TCA in OPA added to create NH4+ if capable

• ammonification pathway product

• pos: Citrobacter freundii, blue-black

• neg: yellow-green color, blank control

Describe nitrate broth, including

• testing for

• complex or chemically defined

• main differential component

• enzyme or pathway product

• positive indicator and org

• negative indicator and org

• nitrate reduction

• complex

• sulfamic acid and zinc dust

• nitrate reductase enzyme

• pos: nitrogen gas before zinc dust added (P. aerogenes), fizzing after sulfamic acid (E. coli) or no gas after zinc dust added

• neg: nitrogen gas after zinc dust added

What are genuses of common denitrifiers in soil?

Pseudomonas, Bacillus, Clostridium

What do rhizobia look like and what is unique about them?

• nitrogen fixers with nitrogenase

• gram-negative irregular bacilli

Describe how nitrate broth works to pinpoint what organisms reduce NH3 to

• 1. bubble present in durham tube - nitrate reduction to n2 gas

• 2. no gas in durham tube, add sulfamic acid. fizzing occurs - nitrate reduction to nitrite

• 3. no fizzing with sulfamic acid, add zinc dust - negative for nitrate reduction

Gas is present in the durham tube of nitrate broth. What does this mean?

positive for nitrate reduction to gas

Prolonged fizzing occurs after the addition of sulfamic acid to nitrate broth. What does this mean and what occured?

nitrate reduction to nitrite. sulfamic acid reduces nitrite to n2 gas

Prolonged fizzing occurs after the addition of zinc dust to nitrate broth. What does this mean and what occured?

negative for nitrate reduction. reduces nitrate to n2 gas, so nitrate not reduced

No prolonged fizzing occurs after the addition of zinc dust to nitrate broth. What does this mean and what occured?

Nitrate reduced to something other than nitrite or n2, no nitrate detected in broth

What are the roles of TCA and OPA in peptone broth when testing for ammonification?

• TCA - creates acidic condition, increases NH4+ in broth

• OPA - reacts with NH4, indicating Nh3/NH4 production

What are the positive and negative controls for nitrate reduction and what do they look like?

• pos: nitrogen gas before zinc dust added (P. aerogenes), fizzing after sulfamic acid (E. coli) or no gas after zinc dust added

• neg: nitrogen gas after zinc dust added

What are the positive and negative controls for ammonification and what do they look like?

• pos: Citrobacter freundii, blue-black

• neg: yellow-green color, blank control