Lecture 24: Nitrogen Cycle

• Nitrogen is essential to all life • Large impacts on the environment • Large nitrogen reserves are inert (‘hidden’), small ones are actively recycled (air; 80% N2)

• Based on oxidation states 2. Forms are transformed by microbes

• di-nitrogen (N2) in the atmosphere • N-fixation via microbes to organic N in soil • This can the be mineralised by microbes to ammonia (NH3) • Ammonia can undergo nitrification from NH3 to NO2- (nitrite) to NO3- (nitrate) • Nitrate can be reduced via denitrification to NO2-, to NO, to N2O, back to N2 in the atmosphere • “Anammox” (anaerobic ammonia oxidation) also occurs, where NO2- and NH3 is converted directly back to N2

• Both ammonia (NH3) and nitrate (NO3-) can be up-taken by plants • Big challenge in agriculture • For good crop yields, fertilizers provide additional NH3 and NO3- • NO3- (nitrate) can leak into oceans/rivers via “leaching”, which is bad for aquatic ecosystems • N2O (nitrous oxide) is a powerful green house gas (300x more than CO2, so if isn’t restored to N2 can be harmful a. can be prod from denitrification and nitrification

• Nitrate (NO3-) is very mobile in soil so can leak into nearby rivers/oceans via a process called “leaching” • Bad for aquatic ecosystems

• Done in two ways: a. Biological b. Anthropogenic (industrial) • Only process where atmospheric N2 is incorporated into biological matter • Two different types of organisms able to carry out this process: a. Free living b. Symbiotic

• Rhizobium - root nodules of plants 2. Cyanobacteria - lichen

• Cyanobacteria 2. Azotobacter

• Nitrogenase (nif): catalyses conversion of N2 into NH3 a. N2 + 3H2 -> 2NH3 -> AAs -> proteins • V. energy-costly: triple bond of N2 hard to break • Tightly regulated • Oxygen sensitive

• “Haber-Bosch process” a. how N fertilisers are made • Req. temps of 300-500C & pressures of 150-250 bars • ~50% of worlds human population would not exist without this process

"Mineralisation" a. Organic N to NH3 (ammonia) b. Makes it available for plants and microbes c. a lot less specialized; done by many plants and microbes

Ammonia to NO2- (nitrite) to NO3- (nitrate) 1. Important for fertiliser loss because NO3- isn't stable in soil so can be lost 2. Two steps: a. NH3 -> NO2- i. carried out by AMO (ammonia oxidisers) ii. e.g., Nitrosomonas b. NO2- -> NO3- i. carried out by nitrite oxidisers ii. e.g., Nitrobacter 3. Can lead to leaching; causing eutrophication/environmental pollution 4. Produces NO and N2O which are climate active gases 5. Enzymes involved require: a. O2 as an e- acceptor b. CO2 as carbon source c. NH3 & NO2- as e- donors

• First discovered by Grace Frankland and isolated by Sergei Winogradsky • Low cell densities and growth rates - difficult to study • Typical soil AOB (nitrosospira and nitrosomonas) belong to beta- proteobacteria, typical marine AOB (nitrosococcus) to gamma-proteobacteria

• AOA = Ammonia oxidising archaea • Soil fosmid 54d9 contained 16S and 23S ribosomal subunits, enabling identification of the fosmid as an archaeon • amoA and amoB encode for subunits of “ammonia monooxygenase” - a key enzyme in the global nitrogen cycle • Some ammonia oxidiser can also use urea and cyanate as substrates • Significance? A WHOLE DIFFEERNT DOMAIN OF LIFE! • AOA can nitrify in conditions where AOB struggle (acidic soils, open ocean) - THEIR NICHE a. N. devanaterra

• Stands for “complete ammonia oxidation into nitrate” • Single organism that can do both ammonia and nitrite oxidation • Nitrospira sp. - nitrite oxidiser which acquired ammonia oxidation genes via horizontal gene transfer

1. stands for "anaerobic ammonium oxidation" 2. especially significant to N turnover in ocean and is large single source of dinitrogen gas on earth 3. Chemolithoautotrophic bacteria use NH4+ as e- donor and NO2- as acceptor 4. Uses "ladderane" membrane lipids that form acellular "anammoxosome" organelle a. site of membrane-bound enzyme combining nitrite and ammonia to N2. Hydrazine is an intermediate 5. New low-cost method for N-removal in wastewater treatment 6. at least 3 genera of bacteria: a. Brocadia b. Kuenenia c. Scalindua 7. Process patented: NH4+ + NO2- -> N2 + 2H2O

• Diverse; carried out by all three domains of life • sequential formation of N2 • anaerobic • Nitrogen is the electron acceptors - essentially like our oxygen in respiration a. lots of organisms can switch between the two for energy • Performed primarily by heterotrophic bacteria. E.g., a. Paracoccus denitrificans b. various pseudomonads • Some autrophic found: a. Thiobacillus denitrificans • nirS and nirK encode nitrite reductase (key marker gene for population studies) a. never present together

• agriculture: remove nitrogen from field (BAD THING) 2. Wastewater treatment: removes nitrogen from system. Prevents eutrophication of receiving waters

• N2O and NO crucial to troposphere a. N2O powerful greenhouse gas in lower atmosphere b. NO reacts in lower atmosphere and produces NO2 (nitrogen dioxide) • Prod. is result of different processes: a. Denitrification b. Nitrification

1. Sustainable intensification of food production 2. Improving soil N use efficiency a. slow release fertilisers b. Commercial nitrification inhibitors (e.g., DCD, nitrapyrin) c. Demand driven fertilisation