Nitrogenous Compounds in Water. Evolution of the Nitrogen Cycle Unlike carbon or oxygen, nitrogen is...

Nitrogenous Nitrogenous Compounds in WaterCompounds in Water

Evolution of the Nitrogen Evolution of the Nitrogen CycleCycle

Unlike carbon or oxygen, nitrogen is not very available to life.

It’s conversion requires biological activity

nitrogen cycle is required by life, but also driven by it.

Cycle is rather complex and has evolved as the atmosphere became oxygenated.

As we know, Earth’s original atm was oxygen-poor.


Earliest forms of nitrogen-reducing bacteria had to have been anaerobic.

other option: NH4+ already existed in

some form.Today, these ancient N-fixers either

only exist in anaerobic environments or the N-fixing apparati are carefully guarded from intracellular oxygen.


As Earth’s atmosphere became more O2-rich, more NO3 became available.

This created niches occupied by organisms that could reduce NO3 to NH3 (many higher plants can do this).

Converting NO3 back to N2 (denitrification) is an arduous process and has evolved more recently.

Composition of AtmosphereComposition of Atmosphere The atmosphere is The atmosphere is

primarily composed of primarily composed of nitrogen (Nnitrogen (N22, 78%), , 78%), oxygen (Ooxygen (O22, 21%), and , 21%), and argon (Ar, 1%). argon (Ar, 1%).

A number of other very A number of other very influential components influential components are also present: the are also present: the water (Hwater (H22O, 0 to 7%), O, 0 to 7%), "greenhouse" gases or "greenhouse" gases or ozone (O, 0 to 0.01%), ozone (O, 0 to 0.01%), carbon dioxide (COcarbon dioxide (CO22, , 0.01to 0.1%).0.01to 0.1%).

Gaseous NitrogenGaseous Nitrogen Nitrogen is the major gas in the

atmosphere. After oxygen, second limiting factor. Constitutes 78.1% of total gases in air. Solubility in water is largely dependent

upon two physio-chemical factors: temperature and salinity.

At saturation/equilibrium it has higher values than oxygen or CO2.

Nitrogen Saturation Nitrogen Saturation ValuesValues

Generalized Nitrogen Generalized Nitrogen CycleCycle

Nitrogen dynamics in the environment involves some fairly complex cycling.

N is relatively unreactive as an element.

cyclic conversions from one form to another are mainly mediated by bacteria.

Cycle occurs in both aerobic and anaerobic environments.

nitrogen cycle

Cycling of NitrogenCycling of Nitrogen

Four processes Four processes participate in the participate in the cycling of nitrogen cycling of nitrogen through the through the biosphere:biosphere:

1) Nitrogen fixation1) Nitrogen fixation2) Decay2) Decay3) Nitrification3) Nitrification4) Denitrification4) Denitrification

Microorganisms play Microorganisms play major roles in these major roles in these processes processes

Simplified diagram of the Simplified diagram of the nitrogen cycle that is nitrogen cycle that is

established in a saltwater established in a saltwater aquariaaquaria

Process 1: Nitrogen FixationProcess 1: Nitrogen Fixation

Three “Fixation” Three “Fixation” processes are processes are responsible for responsible for most of the most of the nitrogen fixation nitrogen fixation in the biosphere in the biosphere are …are …

Atmospheric Atmospheric fixation by fixation by lightning. lightning.

Biological fixation Biological fixation by certain microbes by certain microbes - alone or in a - alone or in a symbiotic symbiotic relationship with relationship with plants. plants.

Industrial fixation.Industrial fixation.

Process 1: Process 1: FixationFixation Nitrogen fixation refers

to the conversion of N2 to either NO3 or NH4 by bacteria.

Terrestrial systems: soil bacteria in root nodules of legumes.

Aquatic systems: blue green algae.

Biological, meteorological, industrial transformations also occur.

Process 2:Process 2:DecayDecay

The proteins made by plants enter and pass The proteins made by plants enter and pass through food webs just as carbohydrates do. At through food webs just as carbohydrates do. At each trophic level, their metabolism produces each trophic level, their metabolism produces organic nitrogen compounds that return to the organic nitrogen compounds that return to the environment, chiefly in excretions.environment, chiefly in excretions.

The final beneficiaries of these materials are The final beneficiaries of these materials are microorganisms of decay.microorganisms of decay.

They breakdown the molecules in excretions They breakdown the molecules in excretions and dead organisms into ammonia (NHand dead organisms into ammonia (NH33).).

Nitrogen FixationNitrogen FixationType of Fixation N2 fixed (1012 g per year)

Non-biological

industrial About 50

combustion About 20

lightning About 10

Total About 80

Biological

Agricultural land About 90

Forest + nonag land About 50

Sea About 35

Total About 175

Process 3: Process 3: NitrificationNitrification

The term nitrification refers to the conversion of ammonium to nitrate (pathway 3-4 opposite).

Responsible: nitrifying bacteria known as chemoautotrophs.

These bacteria gain their energy by oxidizing NH3,

while using CO2 as a source of carbon to synthesize organic compounds.

The nitrogen cycle, once more!

Process 4: Process 4: DenitrificationDenitrification

By this process, NO3 in soil or water is converted into atm N2, nitric oxide or nitrous oxide.

This must occur under anaerobic conditions (anaerobic respiration).

Presence of O2 can reverse the reaction.

Again, mediated by bacteria (Pseudomonas sp., Alkaligenes sp. and Bacillus sp.).

Denitrification = step 5, above

Aquatic Nitrogen Aquatic Nitrogen CyclingCycling

For aquaculturists, cycling transforms usually begin with the decomposition of organic matter from either plant or animal sources.

major source in aquaculture: feeds

Ultimately excreted as amine groups on amino acids or excreted in soluble form primarily as NH3/NH4

+, other compounds.

amino acid

Release of NHRelease of NH33

NH3 separated from organic protein via microbial activity.

Process referred to as deaminification or ammonification.

NH3 is released to water column (mineralization) and assimilated into primary productivity (NH3 + H+ --> NH4

+). ammonification is

heterotrophic, under aerobic or anaerobic conditions.

ammonification


NH3 and NH4+ are both either assimilated

by aquatic plants for growth or nitrified (oxidized) to NO3

- (nitrate).Nitrate can also be used as a growth

substrate (Guillard’s F medium).Two step process:

NH4+ + 1.5O2 NO2

- + 2H+ + H2O

NO2- + 0.5O2 NO3

-

Note: these are oxygen-driven reactions.


Conversion of ammonia (NH3) to nitrate (NO3

-) is via chemoautotrophic bacteria. First step by Nitrosomonas sp. second step by Nitrobacter sp. Both steps/reactions use NH4

+ and NO2- as

an energy source, CO2 as a carbon source. This is a non-photosynthetic type of growth.


Reaction runs best at pH 7-8 and 25-30oC. however; under low DO, it runs in reverse. NO3

- is converted to NO2= and other forms.

Can go all the way backwards to NH3.

Occurs in the hypolimnion under eutrophic (stagnant) conditions.

REM: nitrogen also fixed by leguminous plants, free living bacteria, blue-green algae.

Magnitude of this transform not well studied.

Nitrogen: Nitrogen: aqueous aqueous formsforms

Gaseous form of nitrogen (N2) is most prevalent. Followed by: nitrite, nitrate, ammonia or

ammonium. Nitrite is seldom a problem unless DO levels are

low (to be discussed later). Ratio of NH3:NH4

+ rises with pH. Unfertilized ponds: TAN (NH3 +NH4

+) = 0.05-0.075 mg/L.

Fertilized ponds: TAN = 0.5 mg/L, 0.075 mg NO3

-

Nitrogen Nitrogen AmendmentsAmendments

Nitrogen added as fertilizer to ponds: urea

Immediately upon addition, it starts to decline.

Only small portion detectable from metabolic processes.

Plants typically take it up, die, mud deposit.

Inorganic nitrogen typically denitrified in the hypolimnion.

High afternoon pH = increased volatization.

urea

Nitrogen Equilibria: Nitrogen Equilibria: NHNH33/NH/NH44

++

Ammonia (NH3) is toxic to fish/inverts.

pH affects proportion of NH3/NH4

+. As pH increases, NH3

increases. Calculation example

TAN = 1.5 mg/L, 26oC, pH = 8.6

Answer: 0.35 mg NH3/L

Affect of pH/temp on NH3/NH4+

equilibria

More on AmmoniaMore on Ammonia As mentioned, initial source: feed, direct

source: excretion. Can calculate daily dosage/loading if you know:

NPUNPU and % protein in feed. NPU is 0.4 (approx.) for most aquaculture feeds Equ.: (1.0 - NPU)(pro/6.25)(1000) = g NH3/kg

feed. For 1.0 ha pond receiving 50 kg of 28% protein

feed/day, loading is 1,345 g NH3. Dilution in 10 x 106 L is 0.135 mg NH3/L. If NPU stays constant, NH3 production increases

with increased feeding.

Ammonia ToxicityAmmonia Toxicity Both NH3 and NH4

+ are toxic to fish/inverts: As medium NH3 increases, ability to excrete

internal NH3 decreases (fighting gradient). Blood/tissue NH3 increases causes increase in

blood pH. Result: imbalance in enzyme activity, reduced

membrane stability. Increased O2 consumption by tissues, gill

damage, reduced O2 transport (Root/Bohr, but other direction).

Reduced growth, histological changes in gills/other organs.

Ammonia ToxicityAmmonia Toxicity

Short term exposure toxic at 0.7-2.4 mg/L.

96 hr LC50 varies from 0.5-3.8 mg/L for most fish.

Toxicity tolerance varies due to biological variability of different strains of species.

Eggs are most tolerant (fish). Larvae least tolerant, older = more

tolerant. Same probably holds true for inverts.

Ammonia ToxicityAmmonia Toxicity

Species 96- hour LC50 (mg/L NH3) Pink salmon 0.08- 0.1 Brown trout 0.50- 0.70 Rainbow trout 0.16- 1.10 Largemouth bass 0.9- 1.4 Common carp 2.2 Channel catfish 0.50- 3.8 Shrimp 5.71

Ammonia Toxicity in Ammonia Toxicity in PondsPonds

NH3 is more toxic when DO levels are low.

However, toxic effect is probably nullified by resultant increase in CO2.

Thus, increased CO2 = decreased NH3.

Increased CO2 = decreased pH. In some cases, fish have been shown

to acclimate to increases in NH3.

Nitrite (NONitrite (NO22--) Toxicity) Toxicity

Nitrite reacts with hemoglobin to form methemoglobin.

In process, iron converted from ferrous (Fe2+) to ferric (Fe3+) form.

Ferric form of iron cannot bind with oxygen Blood changes from red to brown, appears

anemic. Those fish having methemoglobin reductase

enzyme can convert iron moeity back to ferrous

Maybe same for crustaceans?


Recovery from nitrite toxicity usually occurs when fish are transferred to better water.

Complete recovery can occur in 24 h. How does it get into system in first place? Nitrite is quickly transported across gill

membrane by lamellar chloride cells. Cells can’t distinguish between NO2

- and Cl- Thus: nitrite absorption regulated by

nitrite:chloride ratio in medium.


Nitrite is about 55 times more toxic in freshwater vs. 16 ppt seawater.

Question: Can you add NaCl to water to reverse nitrite toxicity?

24 hr LC50 values vary tremendously in fish.

Safe bet: authors say 4.5 mg/L


Species 48- or 96- hr LC50 (mg/L NO2- N)

Rainbow trout 0.19- 0.39

Chinook salmon 0.88

Common carp 2.6

Channel catfish 7.1- 13

Largemouth bass 140

Guadeloupe bass 160

Shrimp, freshwater 8.5- 15.4

Shrimp, saltwater 45- 204 mg/L

Nitrate (NONitrate (NO33--) )

ToxicityToxicityNitrate builds up in ponds, like

nitrite, when ponds are cooler.Nitrobacter does not function well

under cool or cold water conditions.however, nitrates are least toxic

form of soluble nitrogen.Effects are similar to nitrite toxicity,

but values of levels are much higher.

Nitrate ToxicityNitrate ToxicitySpecies 96- hr LC50 (mg/L NO3- N)

Guppy 180- 200

Guadeloupe bass 1,260

Chinook salmon 1,310

Rainbow trout 1,360

Channel catfish 1,400

Bluegill 420- 2,000

Shrimp Who knows???

Today, we discussed Today, we discussed Nitrogenous CompoundsNitrogenous Compounds

Nitrogen cycleNitrogen cycleNitrogen FixationNitrogen FixationNitrificationNitrificationDenitrificationDenitrificationNitrogen cyclingNitrogen cyclingNitrogen equilibriaNitrogen equilibriaToxicityToxicity

Nitrogenous Compounds in Water. Evolution of the Nitrogen Cycle Unlike carbon or oxygen, nitrogen is...

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