1

Biogeochemistry of WetlandsS i d A li tiS i d A li ti

Institute of Food and Agricultural Sciences (IFAS)

Science and ApplicationsScience and Applications

Wetland Biogeochemistry LaboratorySoil and Water Science Department

NITROGEN

6/22/2008 P.W. Inglett 16/22/2008 WBL 16/22/2008 16/22/2008 WBL 1

Instructor :Patrick Inglettpinglett@ufl.edu

Soil and Water Science DepartmentUniversity of Florida

Nitrogen

IntroductionN Forms, Distribution, Importance

Basic processes of N CyclesExamples of current researchExamples of applications

6/22/2008 P.W. Inglett 2

Key points learned

2

Learning ObjectivesIdentify the forms of N in wetlands

Nitrogen

yUnderstand the importance of N in

wetlands/global processesDefine the major N processes/transformationsUnderstand the importance of microbial activity

in N transformations Understand the potential regulators of N

6/22/2008 P.W. Inglett 3

processesSee the application of N cycle principles to

understanding natural and man-made ecosystems

Plant biomass NPlant biomass N

Nitrogen CyclingNitrogen Cycling

AEROBICAEROBIC

Organic N

Plantuptake

NO3-

Litterfall

Peataccretion

Nitrification Mineralization.

[NH4+]s

NH4+

[NH4+]s

NH4+NO3

-

N2 N2O (g)NH3

Volatilization

N2

Nitrogen Fixation

Water Water ColumnColumn

6/22/2008 P.W. Inglett 4

ANAEROBICuptake

Adsorbed NH4+

Denitrification

accretion

Organic N

[ 4 ]s[ 4 ]s

NN2, N2O (g)

MicrobialBiomass N

3

Forms of NitrogenForms of Nitrogen

Organic Nitrogen• Proteins• Amino Sugars• Nucleic Acids

U

Inorganic Nitrogen• Ammonium (NH4

+)• Nitrate N (NO3

-)• Nitrite N (NO2

-) Nit id (N O)

6/22/2008 P.W. Inglett 5

• Urea • Nitrous oxide (N2O)• Dinitrogen (N2)

Solid Phase:

Gaseous Phase:

N TransformationsN Transformations

Aqueous

Particulate N

Bound: NH4+

NO3- NO2

-

N2N2O

6/22/2008 P.W. Inglett 6

NH4+

NO3-

NO2-

Aqueous Phase:

DON

Particulate NDIN

4

Reservoirs of NitrogenReservoirs of Nitrogen

Lithosphere 163,600 x 1018 gAtmosphere 3,860 x 1018 gHydrosphere 23 x 1018 gBiosphere 0.28 x 1018 g

6/22/2008 P.W. Inglett 7

p g

Total Soil NForms of NitrogenForms of Nitrogen

Organic Inorganic

6/22/2008 P.W. Inglett 8

NH4+

Clay-fixed NH4+

ProteinAmino sugarsHeterocyclic N

5

1. Ammonification/Mineralization

Nitrogen TransformationsNitrogen Transformations

2. Immobilization3. Nitrification4. Denitrification5. Dissimilatory nitrate reduction to

ammonia (DNRA)

6/22/2008 P.W. Inglett 9

ammonia (DNRA)6. Nitrogen fixation7. Ammonia volatilization

NH4+

Organic N

8e-

1

5

2

Transformation of N SpeciesTransformation of N Species

NO3-

NO2-

N2O

NH3

2 e-

2e-

3e-

2e-

3

6

4NH2OH 4e-

2e-

7

4

33

6/22/2008 P.W. Inglett 10

+5+3+10

-3

N2

Oxidation Number

1e-

3e

+2 +4-1-2

4

6

Nitrogen CycleNitrogen Cycle

Plant/Algae

Nit ifi D it ifi

Org-N NH4+ NO3

-

N Fi

6/22/2008 P.W. Inglett 11

Nitrifiers Denitrifiers

N2/N2ON2

N-FixersMicrobial Biomass

Productivity Productivity

6/22/2008 P.W. Inglett 12

7

Greenhouse Gas Emissions Greenhouse Gas Emissions

6/22/2008 P.W. Inglett 13

Biological N2 fixationDry and wet depositionNon-point sourcesWastewaters

Nitrogen BudgetNitrogen Budget

Plant biomassMicrobial biomassSoil organic NPorewater (DIN, DON)Exchangeable N

6/22/2008 P.W. Inglett 14

VolatilizationOutflowGaseous lossesPlant harvest

Clay fixed NH4-N

8

Ammonium Ion Wet Deposition1985-2003

1985

2003

6/22/2008 P.W. Inglett 15

http://nadp.sws.uiuc.edu/isopleths/

Nitrate Ion Wet Deposition1985-20031985

2003

6/22/2008 P.W. Inglett 16

9

Inorganic N Wet Deposition1985-20031985

2003

6/22/2008 P.W. Inglett 17

Plant biomass NPlant biomass N

Biological Nitrogen FixationBiological Nitrogen Fixation

AEROBICAEROBIC

Organic N

Plantuptake

NO3-

Litterfall

Peataccretion

Nitrification Mineralization.

[NH4+]s

NH4+

[NH4+]s

NH4+NO3

-

N2 N2O (g)NH3

Volatilization

N2

Nitrogen Fixation

Water Water ColumnColumn

6/22/2008 P.W. Inglett 18

ANAEROBICuptake

Adsorbed NH4+

Denitrification

accretion

Organic N

[ 4 ]s[ 4 ]s

NN2, N2O (g)

MicrobialBiomass N

10

Type of fixation N2 fixed (1012 g yr-1)Non-biological

Nitrogen FixationNitrogen Fixation

Industrial about 50Combustion about 20Lightning about 10Total about 80BiologicalAgricultural land about 90F t d i lt l l d b t 50

6/22/2008 P.W. Inglett 19

Forest and non-agricultural land about 50Sea about 35Total about 175

Biological NBiological N22 FixationFixationN N + 8H+ + 8e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi

6/22/2008 P.W. Inglett 20

Nitrogenase

11

Biological NBiological N22 FixationFixation

Prokaryotes

AzotobacterBeijerinckiaKlebsiella

C anobacteria

Aerobic

ClostridiumDesulfovibrioCyanobacteria

Anaerobic

RhizobiumFrankia

Azospirillum

Associated w/ Plants

y

6/22/2008 P.W. Inglett 21

Cyanobacteria

Biological NBiological N22 FixationFixation

HeterocystsHeterocysts (cyanobacteria)Leghaemoglobin (rhizobium)ExopolysaccharidesTemporal isolation

6/22/2008 P.W. Inglett 22

Temporal isolation

http://www.bact.wisc.edu/Microtextbook/ images/book_4/chapter_2/2-53.jpg

12

y

Temporal IsolationTemporal Isolation

Nitr

ogen

ase

activ

ity

Non-heterocystous

Heterocystous

6/22/2008 P.W. Inglett 23

12:00Time (h)24:00 12:00

Biological NBiological N22 FixationFixationRegulators?Regulators?

LightLightConcentration of Oxygen Growth limitation

Macro nutrients (C, P) Co-factors (Fe, Mo)

6/22/2008 P.W. Inglett 24

Inc. Growth

Inc. N Demand Species

shiftInc. N2Fixation

13

Plant biomass NPlant biomass N

Nitrogen MineralizationNitrogen Mineralization

AEROBICAEROBIC

Organic N

Plantuptake

NO3-

Litterfall

Peataccretion

Nitrification Mineralization.

[NH4+]s

NH4+

[NH4+]s

NH4+NO3

-

N2 N2O (g)NH3

Volatilization

N2

Nitrogen Fixation

Water Water ColumnColumn

6/22/2008 P.W. Inglett 25

ANAEROBICuptake

Adsorbed NH4+

Denitrification

accretion

Organic N

[ 4 ]s[ 4 ]s

NN2, N2O (g)

MicrobialBiomass N

Organic NitrogenOrganic Nitrogen

ProteinsAmino acids (30-50% of total organic N)

Nucleic Acids 2-5 % of total organic N

Amino Sugars3-13% of total organic N

6/22/2008 P.W. Inglett 26

3-13% of total organic N

Urea

14

Proteins

A i idPeptides

Protein DecompositionProtein Decomposition

Amino acidsPeptides

Proteases PeptidasesAla

Phe

Asp

L

Pro

Arg

Pro

Ala

Phe

Asp

Lys

Pro

Arg

6/22/2008 P.W. Inglett 27

Pro

Lys

N - C - C - OHOR1

HH

HN - C - C - OH

OR2

HH

HN - C - C - OH

OR3

HH

H

N - C - C - NOR1

- C - C - N

OR2

- C - C - OHOR3H

- 2H2O

6/22/2008 P.W. Inglett 28

HH HH HH

Peptide Bonds

15

N - C - C - OHOR1

HH

HN - C - C - OH

OR2

HH

HN - C - C - OH

OR3

HH

H

HH HH HH

N - C - C - NOR1

- C - C - N

OR2

- C - C - OHOR3H

+ 2H2O

6/22/2008 P.W. Inglett 29

HH HH HH

Peptide Bonds

EnzymeHydrolysis

Detrital MatterComplex PolymersCellulose, Hemicellulose,

Proteins, Lipids, Waxes, Lignin

MonomersSugars, Amino Acids

Fatty Acids

Leaching

Amino acidsElectron Acceptors:O2, NO3

-, Mn4+, Fe3+, SO4

2-, CO2

Uptake

Bacterial Cell

6/22/2008 P.W. Inglett 30

Products:CO2, H2O,

NH4+, Mn2+,

Fe2+,S2-,CH4

ATPEnergy

NH4+

NH4+

16

Urea DecompositionUrea Decomposition

C = O + H2O CO2 + 2NH3

NH2

NH2

Urease

6/22/2008 P.W. Inglett 31

Urea DecompositionUrea Decomposition

Urease activity (14C Tracer)

6/22/2008 P.W. Inglett 32

[Thorén (2007)]

17

AmmonificationAmmonification[Mineralization]

Organic N Ammonium N[B kd f O i M tt ][Breakdown of Organic Matter]

ImmobilizationImmobilization

Inorganic N Organic N

6/22/2008 P.W. Inglett 33

Inorganic N(NH4-N and NO3 -N)

Organic N

[Buildup of Organic Matter]

Ammonification/NAmmonification/N--MineralizationMineralizationOrganic N

AmmoniumRR

NN--ImmobilizationImmobilizationInorganic N Organic N

NH4+

NH2C

H

-HOOC NH2C

H

-HOOC

RR

6/22/2008 P.W. Inglett 34

NH4+

NO3-

NH2C

R

H

-HOOC NH2C

R

H

-HOOC

18

y = 10.7 x + 2.2R2 = 0.78 25

Microbial Biomass C & NMicrobial Biomass C & N[Everglades[Everglades-- WCAWCA--2A]2A]

0

5

10

15

20

crob

ial b

iom

ass

C(g

C k

g-1 )

6/22/2008 P.W. Inglett 35

0

0 0.5 1.0 1.5 2.0

Microbial biomass N (g N kg-1)

Mic

White and Reddy, 2000

Decomposition Decomposition Microbial Biomass

C/N ratio = 10Effi i f b i il tiEfficiency of carbon assimilation

Aerobic = 20-60% Anaerobic = 10%

Plant Detritus [100 units]40% carbon (dry weight basis) = 40 units CAmount of C assimilated during decomposition

6/22/2008 P.W. Inglett 36

Amount of C assimilated during decompositionAerobic = 16 units [40% efficiency]Anaerobic = 4 units [10% efficiency]

19

Carbon/Nitrogen RatioCarbon/Nitrogen Ratio

Nitrogen Demand?Mi bi l Bi C/N ti 10Microbial Biomass C/N ratio = 10

Aerobic = 1.6 unitsAnaerobic = 0.4 units

Critical Plant Detritus Nitrogen?Aerobic = 1.6 %Anaerobic = 0.4%

6/22/2008 P.W. Inglett 37

Critical Carbon/Nitrogen Ratio?Aerobic = [40% C]/[1.6% N] = 25Anaerobic = [40% C]/[0.4% N] = 100

Carbon/Nitrogen RatioCarbon/Nitrogen Ratio

Mineralization [M] vs. Immobilization [I]

Aerobic I > M = C/N ratio > 25M > I = C/N ratio < 25

AnaerobicI M C/N ti 100

6/22/2008 P.W. Inglett 38

I > M = C/N ratio > 100M > I = C/N ratio < 100

20

C/N = 15ease

N Mineralization/ImmobilizationN Mineralization/Immobilization[Aerobic][Aerobic]

C/N = 35

C/N = 100norg

anic

N R

ele

6/22/2008 P.W. Inglett 39

Decomposition (days)

In

Immobilization

n R

atio 40

N Mineralization/ImmobilizationN Mineralization/Immobilization[Aerobic][Aerobic]

Mineralization

arbo

n/N

itrog

en

10

20

30

6/22/2008 P.W. Inglett 40

Ca

0

Decomposition Period [days]0 150

C/N ratio of soil humus/microbial biomass

21

Regulators of AmmonificationRegulators of Ammonification

Substrate qualityC/N ratio of the substrateC/N ratio of the substrateSecondary compoundsN forms (soluble vs. structural compounds)

Microbial ActivityExtracellular enzyme activity

6/22/2008 P.W. Inglett 41

Extracellular enzyme activitySupply of electron acceptors (Redox)TemperaturepH

400

Mineralizable NitrogenMineralizable Nitrogen[Everglades[Everglades--WCAWCA--2A]2A]

y = 0.317 x + 52.4R2 = 0.76

100

200

300

400

Min

eral

izat

ion

Rat

e(m

g N

kg-

1d-

1 )

6/22/2008 P.W. Inglett 42

00 100 200 300 400

Extractable NH4+ (mg N kg-1)

M

22

Plant biomass NPlant biomass N

Fate of AmmoniumFate of Ammonium

AEROBICAEROBIC

Organic N

Plantuptake

NO3-

Litterfall

Peataccretion

Nitrification Mineralization.

[NH4+]s

NH4+

[NH4+]s

NH4+NO3

-

N2 N2O (g)NH3

Volatilization

N2

Nitrogen Fixation

Water Water ColumnColumn

6/22/2008 P.W. Inglett 43

ANAEROBICuptake

Adsorbed NH4+

Denitrification

accretion

Organic N

[ 4 ]s[ 4 ]s

NN2, N2O (g)

MicrobialBiomass N

Nitrogen Uptake by Rice (Nitrogen Uptake by Rice (1515N)N)

T t l N t kTotal N uptake

Added 15N uptake

N-uptake fromNitr

ogen

upt

ake

6/22/2008 P.W. Inglett 44

upta e oSoil organic N

Growing season

23

NH +

Ammonium AdsorptionAmmonium Adsorption-- AerobicAerobic

---

- Ca2+

K+NH4

+

Mg2+

Ca2+

NH4

K+

Mg2+

Ca2+

Ca2+

K+

-

-

-

--

--

-

--

------

-

-

- K+

K+

Ca2+ NH4+

NH4+ Mg2+

6/22/2008 P.W. Inglett 45

Adsorbed [Solids]

Dissolved[Pore water]

-- - Ca2+Mg2+

K+K+

Ammonium AdsorptionAmmonium Adsorption-- AnaerobicAnaerobic

NH4+

C 2+----

- -K+

Ca2+

Mg2+

Fe2+

Mn2+Fe2+

4

K+

Mg2+

Ca2+

Ca2+

K+

-

-

--

--

-

--

- ------

-

-K+

NH4+

NH4+ Mg2+

Mn2+

Fe2+

Fe2+

Mn2+

Mn2+

6/22/2008 P.W. Inglett 46

Adsorbed [Solids]

Dissolved[Pore water]

K- - Ca2+Mg2+

K+Fe2+

24

6/22/2008 P.W. Inglett 47

Ammonium Fixation Ammonium Fixation Illite Illite -- Clay MineralClay Mineral

NH4+K+NH4

+Mg2+ ---

--

---

- --NH4

+K+

Mg2+

Ca2+ K+ Al-O-OH layerSi-O layer

Si O layer

Ca2+

NH4+K+NH4

+Mg2+ -- --- - --NH4

+NH4+

NH4+

Fixed Ammonium

NH4+K+NH4

+Mg2+ ---

--

--- - -

- --

-NH4

+

NH4+

NH4+

NH4+

- - -Interlayer Space

Si-O layer

NH +

Interlayer Space

6/22/2008 P.W. Inglett 48

Mg2+

Mg2+

NH4+

Ca2+K+

K+

--- - - -NH4

+

25

Adsorption/Desorption EquilibriumAdsorption/Desorption Equilibrium

NH +NH4

+

SoilNH4

+

NH4+ 4

NH4+NH4

+

NH +

NH4+

NH4+

6/22/2008 P.W. Inglett 49

NH4+NH4

+ NH4

Fixed Exchangeable Solution

Adsorption/Desorption EquilibriumAdsorption/Desorption Equilibrium

NH4+

NH4+

NH4+

NH4+

NH4+

NH4+

6/22/2008 P.W. Inglett 50

NH4+

NH4+

NH4+

26

(+)

NH4+

ads = K × [NH4+] + NH4

+fixed

S = K × C + S0

K, Partition Coefficient

E NH4-C0

mm

oniu

m A

dsor

bed

(mg

N k

g-1 )

( )

6/22/2008 P.W. Inglett 51

Porewater ammonium (mg N l-1)

Am

(-)S0

Ammonium Adsorption Ammonium Adsorption -- SalinitySalinity

6/22/2008 P.W. Inglett 52

27

Increasing salinityIncreasing salinity

6/22/2008 P.W. Inglett 53

NH3(g)

Ammonia FluxAmmonia Flux

(Volatilization)(Volatilization)

Water

2H+ + CO32- CO2 + H2O

Photosynthesis

Respiration

O2 + CH2O CO2 + H2O NH3(g)

NH4+NH3(aq) + H+

6/22/2008 P.W. Inglett 54

Soil

Water

NH4+

(adsorbed)

28

Diel pH changes in the Diel pH changes in the Water ColumnWater Column

7

8

9

10

pH

System with high photosynthesis

System with low photosynthesis

6/22/2008 P.W. Inglett 55

12:00

6

7

Time (h)

24:00 12:00

Ammonia VolatilizationAmmonia Volatilization(assuming high pH)(assuming high pH)

SAV/Periphyton DensityFloodwater depth Plant density

6/22/2008 P.W. Inglett 56

Temperature Wind speed Soil CEC

29

Ammonia VolatilizationAmmonia Volatilization

NH + = NH + H+ TemperatureNH4 = NH3 + HHigh floodwater pH High soil pHHigh algal activityPlant density

TemperatureCation exchange capacity of soilWind speedFloodwater depth

6/22/2008 P.W. Inglett 57

Plant biomass NPlant biomass N

NitrificationNitrification

AEROBICAEROBIC

Organic N

Plantuptake

NO3-

Litterfall

Peataccretion

Nitrification Mineralization.

[NH4+]s

NH4+

[NH4+]s

NH4+NO3

-

N2 N2O (g)NH3

Volatilization

N2

Nitrogen Fixation

Water Water ColumnColumn

6/22/2008 P.W. Inglett 58

ANAEROBICuptake

Adsorbed NH4+

Denitrification

accretion

Organic N

[ 4 ]s[ 4 ]s

NN2, N2O (g)

MicrobialBiomass N

30

NH + → NO → NO

NitrificationNitrification

NH4+ → NO2

- → NO3-

Obligate aerobes (O2 e- acceptor)Common nitrifiers (autotrophs)MethanotrophsH t t h

6/22/2008 P.W. Inglett 59

Heterotrophs

Sites of NitrificationSites of NitrificationAerobic

Water column

O2 bic

Soil

Aerobic Soil/FloodwaterInterface

6/22/2008 P.W. Inglett 60

AerobicRoot Zone

O2

Aer

ob

Ana

erob

ic S

oil

O2

31

NitrificationNitrification

NH4+ → NO2

- Nitrosomonas (‘Nitroso’ spp.)

NO2- → NO3

- Nitrobacter (‘Nitro’ spp.)

Obligate aerobes (O2 e- acceptor)Energy/e- source: NH4

+

Carbon source: CO

6/22/2008 P.W. Inglett 61

Carbon source: CO2

NitrificationNitrificationStep I: Nitrosomonas

NH4+ + 2H2O → NO2

- + 6e- + 8H+

ΔGo = -65 kcal/mole

1½ O2 + 6e- + 6H+ → 3H2O

NH4+ + 11/2O2 → NO2

- + 3H2O + H+

Step II: Nitrobacter ΔGo = -18 kcal/mole

2NO 2H O 2NO 4 4H+

6/22/2008 P.W. Inglett 62

2NO2- + 2H2O → 2NO3

- + 4e- + 4H+

O2 + 4e- + 4H+ → 2H2O

2NO2- + O2 → 2NO3

-

32

B t i d f i

Heterotrophic NitrificationHeterotrophic Nitrification

Bacteria and fungiPoorly studiedOnly dominant under high C/low N conditionsMany facultative (reduce oxidized N to N2)

6/22/2008 P.W. Inglett 63

2

“Nitrifier denitrification”

NitrificationNitrification

AmmoniaAmmoniaNH3 toxic to nitrifying bacteriapH7 - 8.5 optimumSulfide

6/22/2008 P.W. Inglett 64

S2- toxic to nitrifying bacteria

33

NitrificationNitrification

Ideal Conditions Inhibited

C

NH4+ NH4

+

NO2-

NO2-NO3

-

NO3-

6/22/2008 P.W. Inglett 65

C

Time Time

Oxygenases Hydroxylamineid d t

NitrificationNitrification

NH4+ NH2OH [NOH]

NO2-N2O NO3

-

yg oxidoreductase

Chemical

2 e- 2 e-

2 e-

2 e-

Oxygenstress

6/22/2008 P.W. Inglett 66

NO2N2O NO3Nitrite

reductaseNitrite

reductase

34

Inhibited NitrificationInhibited Nitrification

NO3-NH4

+ N2

N2O

NO

Nitrifiers Denitrifiers

NO

N2O

‘Leaky Pipe’ Model:

6/22/2008 P.W. Inglett 67

22

Regulators: Ammonia concentration

NitrificationNitrification

Ammonia concentrationOxygen availabilityAlkalinity and CO2

TemperatureNitrifying population

6/22/2008 P.W. Inglett 68

pHCEC

35

Organic NOrganic N

AmmonificationVolatilization

(to atmosphere)

NHNH44++ NHNH44

++Soluble Adsorbed

[NH[NH33 ]](g)(g)

[NO[NO33-- ]]

-

-

--

-

-

-

Soi

l Par

ticle

-

ant/M

icro

bial

U

ptak

e

6/22/2008 P.W. Inglett 69

Live Organic N

Live Organic N

Pl

Anaerobic Ammonium Oxidation: Anammox

Anaerobic Ammonium Oxidation: Anammox

6/22/2008 P.W. Inglett 70Kuypers et al. 2003. Nature 422:608–611.

36

Anaerobic Ammonium OxidationAnaerobic Ammonium Oxidation(ANAMMOX)(ANAMMOX)

5NH4+ + 3NO3

- → 4N2 + 9H2O + 2H+

NH + + NO - → N + 2H O

6/22/2008 P.W. Inglett 71

NH4 + NO2 → N2 + 2H2O

ANAMMOXANAMMOX

6/22/2008 P.W. Inglett 72

37

Plant biomass NPlant biomass N

DenitrificationDenitrification

AEROBICAEROBIC

Organic N

Plantuptake

NO3-

Litterfall

Peataccretion

Nitrification Mineralization.

[NH4+]s

NH4+

[NH4+]s

NH4+NO3

-

N2 N2O (g)NH3

Volatilization

N2

Nitrogen Fixation

Water Water ColumnColumn

6/22/2008 P.W. Inglett 73

ANAEROBICuptake

Adsorbed NH4+

Denitrification

accretion

Organic N

[ 4 ]s[ 4 ]s

NN2, N2O (g)

MicrobialBiomass N

Pasteur, L. 1859. Sur la fermentation nitreuse. Bull. Sot. Chim. Fr. 1:21.

“Nitrates, which the juice of beetroot naturally includes, decompose, and great quantities of nitrous vapor form at the surface of the vats”

6/22/2008 P.W. Inglett 74

38

Dehérain andDehérain and Maquenne, 1882Maquenne, 1882. Sur la reduction des nitrates dans la terre arable. C. R. Acad. Sci. 95: 691-693

“From experience”:Nitrate is reduced under certain conditions

in arable land releasing nitrogen gasNitrate reduction occurs in arable soil

which contains a high organic matter

6/22/2008 P.W. Inglett 75

g gWe have observed that this reduction

occurs when the soil atmosphere is completely stripped of oxygen

Denitrification NO → N

Nitrate ReductionNitrate Reduction

5 e-NO3 → N2

Dissimilatory nitrate reduction to ammonia (DNRA)NO3

- → NH4+

Assimilatory nitrate reduction to i

8 e-

6/22/2008 P.W. Inglett 76

ammoniaNO3

- → NH4+ → Proteins

39

Nitrate ReductionNitrate Reduction

AssimilatoryNH + uptake for

Dissimilatory Functions as an electronNH4

+ uptake for growth - biosynthesisNH4

+ concentration-main regulatorGrowth linkedOccurs mainly in

Functions as an electron acceptorOxygen concentration main regulatorLinked to catabolic reactions

6/22/2008 P.W. Inglett 77

aerobic soilsInsensitive to O2

Occurs in anaerobic soilsSensitive to O2

Nitrate ReductionNitrate ReductionDenitrification

NO - /NO -→ NDNRA

NO -→ NH +NO3 /NO2 → N25 e- transferred Low electron pressureEh = 200 – 300 mVO2 transient

diti

NO3 → NH48 e- transferredHigh electron pressureEh = < 0 mVLake sediments or

t tl d

6/22/2008 P.W. Inglett 78

conditionsFacultative anaerobes

permanent wetlandsObligate anaerobes

40

Low O content Low Eh

Regulators of DenitrificationRegulators of Denitrification

Low O2 content - Low Eh Presence of Electron Acceptor- N OxidesFacultative Anaerobes - EnzymesElectron Donor - Carbon Source

Organic carbon compoundsReduced sulfur compounds

Temperature

6/22/2008 P.W. Inglett 79

Nitrate diffusion/flux to anaerobic zonesPlant root density

Sites of Nitrification/DenitrificationSites of Nitrification/DenitrificationAerobic

Water column

O2 bic

Soil

Aerobic Soil/FloodwaterInterface

6/22/2008 P.W. Inglett 80

AerobicRoot Zone

O2

Aer

ob

Ana

erob

ic S

oil

O2

41

NO3- NO2

- [HNO] [H2N2O2]

Dissimilatory Nitrate ReductionDissimilatory Nitrate Reduction

NO3 NO2 [HNO] [ 2 2 2]

NH2OH N2OBiological

[HNO] = Nitroxyl[H2N2O2] = Hyponitrite

6/22/2008 P.W. Inglett 81

NH4+ N2

[ 2 2 2] ypNH2OH = Hydroxylamine

DNRA Denitrification

Abiotic Nitrate ReductionAbiotic Nitrate Reduction(‘Chemo(‘Chemo--denitrification’)denitrification’)

Abiotic Nitrate ReductionAbiotic Nitrate Reduction(‘Chemo(‘Chemo--denitrification’)denitrification’)

6/22/2008 P.W. Inglett 82

42

Abiotic Nitrate ReductionAbiotic Nitrate ReductionAbiotic Nitrate ReductionAbiotic Nitrate Reduction

6/22/2008 P.W. Inglett 83

Lithotrophic Nitrate ReductionLithotrophic Nitrate ReductionLithotrophic Nitrate ReductionLithotrophic Nitrate Reduction

OthOther potential e- donors:S2-, H2

6/22/2008 P.W. Inglett 84

43

w/ added NO3-

N.R. Coupled to Fe(II) Oxidation:Paddy Soils

N.R. Coupled to Fe(II) Oxidation:Paddy Soils

w/o added NO3

-

w/ added NO3

-

w/o added

6/22/2008 P.W. Inglett 85

Ratering and Schnell. 2001. Env. Microbiol. 3(2), 100-109.

Fe(II) Fe(III)

NO3-

NO3--control

N.R. Coupled to Fe(II) Oxidation N.R. Coupled to Fe(II) Oxidation

NO3- + Fe(II) → NO2

- → Fe(III) + N2

Fe(III)-culture

NO3--culture

6/22/2008 P.W. Inglett 86

Straub, et al. 2007. Appl. Env. Microbiol. 62: 1458-1460.

Fe(III)-control

44

N2N2

NO3-NO3-

NH4+NH4+ NO2

-NO2-

O2/Fe3+

6/22/2008 P.W. Inglett 87

CH4?

6/22/2008 P.W. Inglett 88

45

Low O2 content - Low Eh Presence of Nitrogenous Oxides

Regulators of DenitrificationRegulators of Denitrification

Presence of Nitrogenous OxidesHeterotrophs: Carbon Source

Organic carbon compounds, DOCLithotrophs: Electron Source

Reduced Fe, Mn, S compounds, H2

TemperatureNitrate diffusion/flux to anaerobic zones

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Nitrate diffusion/flux to anaerobic zonesPlant root density

Nitrate uptake, O2 loss

DenitrificationDenitrificationDenitrificationDenitrificationWetlandWetland Denitrification RateDenitrification Rate

Houghton Lake WetlandInflow ZoneInterior Zone

(mg N kg-1 day-1)7411

Orange County ESAWT 91

Inflow ZoneInterior Zone

6/22/2008 P.W. Inglett 90

Everglades-WCA-2a61

1Interior Zone

Inflow ZoneInterior Zone

46

Coupled NitrificationCoupled Nitrification--DenitrificationDenitrification

Nitrification rate depends on:Ammonification rateAmmonium flux into aerobic zone

Denitrification rate depends on:Nitrification rateNitrate flux into anaerobic zones

Nitrification-denitrification occurs in:

6/22/2008 P.W. Inglett 91

Flooded soil-water column with aerobic-anaerobic interfacesAerobic-anaerobic interfaces in the root zoneWater-table fluctuations/ alternate flooding and draining

Coupled NitrificationCoupled Nitrification--DenitrificationDenitrificationCoupled NitrificationCoupled Nitrification--DenitrificationDenitrification

6/22/2008 P.W. Inglett 92From: Reddy (1989)

47

10

Everglades –WCA-2AEverglades –WCA-2ANitrate source/ High C/Low Eh Nitrate depleted/

High C/Low Eh

Nitrate source/ Low C,P/Low Eh

trific

atio

n R

ate

g N

kg

-1hr

-1)

4

6

8

10

With GlucoseWithout Glucose

6/22/2008 P.W. Inglett 93

Den

i(m

g

Distance (km)

0

2

0 2 4 6 8 10

Denitrification Walls:(Schipper et al. 2003)

48

Supply of NO2-

ANAMMOX vs. DenitrificationANAMMOX vs. DenitrificationANAMMOX vs. DenitrificationANAMMOX vs. Denitrification

pp y 2Low NO2

- favors Denitrification, High NO2- (>10mM)

inhibits ANNAMOX

Organic matter availabilityHigher DOC favors Denitrification

Temperature

6/22/2008 P.W. Inglett 95

Temperature ANNAMOX dominant @ low Temp.

6/22/2008 P.W. Inglett 96

49

Supply of NO3-

Denitrification vs. DNRADenitrification vs. DNRADenitrification vs. DNRADenitrification vs. DNRA

Supply of NO3Low NO3

- favors DNRA

Organic matter availabilityHigh DOC favors DNRA

Hydropattern

6/22/2008 P.W. Inglett 97

Denitrifiers are largely facultative

ON

(Of added 15NO3-)

6/22/2008 P.W. Inglett 98

50

6/22/2008 P.W. Inglett 99

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51

Potential FatePotential FatePotential FatePotential FateLow Organic

Matter (Low C:EA)

NO

NO3-

NO2-

N2

N2ONH4

+

N2

N2O

N

N2

Dissim

ila

H+

NO

6/22/2008 P.W. Inglett 101

High Organic Matter

(High C:EA)AerobicAnaerobic

NH4+

N2

atory

Permanently Anaerobic

Potential FatePotential FatePotential FatePotential Fate

6/22/2008 P.W. Inglett 102

Burgin and Hamilton. 2007. Front Ecol Environ 5(2): 89–96.

52

Plant biomass NPlant biomass N

Nitrogen CyclingNitrogen Cycling

AEROBICAEROBIC

Organic N

Plantuptake

NO3-

Litterfall

Peataccretion

Nitrification Mineralization.

[NH4+]s

NH4+

[NH4+]s

NH4+NO3

-

N2 N2O (g)NH3

Volatilization

N2

Nitrogen Fixation

Water Water ColumnColumn

6/22/2008 P.W. Inglett 103

ANAEROBICuptake

Adsorbed NH4+

Denitrification

accretion

Organic N

[ 4 ]s[ 4 ]s

NN2, N2O (g)

MicrobialBiomass N