BIOGEOCHEMISTRY OF NITROGEN I.Introduction II.N-cycle, and the Biochemistry of N III.Global N...

BIOGEOCHEMISTRY OF NITROGENBIOGEOCHEMISTRY OF NITROGEN

I. Introduction

II. N-cycle, and the Biochemistry of N

III. Global N Patterns/Budget (Galloway et al. 1995)

IV. Patterns of N at the HBEF

V. Inputs, Effects and Management of Anthropogenic N in the Northeast

I. INTRODUCTION

Nitrogen is a difficult element to study. Nitrogen has many different species, phases and oxidation states.

N is an important element because:

1. It is a macronutrient (protein);

2. At elevated concentrations, it may cause adverse environmental effects (NH3, NH4

+, NO2-, NO3

-).

Reduced Oxidized

-III 0 +I +II +III +IV +V

NH4+ N2 N2O NO NO2

- NO2 NO3-

NH3 (molecular N)(nitrous oxide)

(nitric oxide)

(nitrite)(nitrogen dioxide)

(nitrate)

Org N

(g, aq, s) (g) (g) (g) (aq) (g) (aq)

Nitrogen is an interesting element because some pools (N2, Org N) are large and generally unavailable.

II. N-CYCLE, AND THE BIOCHEMISTRY OF NN Utilization

1. Assimilatory - used for biosynthetic reactions (amino acid production), not directly used in energy metabolism - All living organisms require N.

2. Dissimilatory processes - Nitrogen is taken up in a particular form (oxidized or reduced), for specialized reactions involving ATP production and excretion of a N product. Dissimilatory N is not incorporated into the physical or biochemical structure of an organism - Only a few specialized organisms can utilize dissimilatory processes.

N Assimilation

Nitrogen in biomass largely occurs as the reduced oxidation state (-III), so this is the energetically favored form of N. However, NO3

- is generally preferred by plants. This may be due to greater mobility of NO3

-. Energy must be expended by plants or microbes to extract NH4

+ from soil sediments. Also competition of NH4+ with other

cations on enzymes.

Plants/microorganisms can commonly assimilate NH4+, NO3

- in water or soil. Organic N is rarely used as an N source. Some coniferous trees have been shown to assimilate dissolved organic N.

N-cycle, and the Biochemistry of N (cont.)

If N is taken in as NH4+, it is directly used by organisms in biosynthesis.

If N is assimilated as NO3-, it must be reduced within the cell.

Two enzymes are involved:

1. Nitrate reductase - contains molybdenum

NO3- + NADH + H+ = NO2

- + H2O + NAD+

2. Nitrite reductase

NO2- + 3NADH + 5H+ = NH4

+ + 2H2O + 3NAD+

Some organisms have the unique characteristic to assimilate molecular N - nitrogen fixation.

This process requires the enzyme, nitrogenase, which is a complex protein containing iron, molybdenum and inorganic S as part of its structure.

The process is extremely energy-intensive, as you might expect, to break a triple bond.

N N


Only a few species of microorganisms can fix nitrogen. These include free-living organisms (asymbotic, e.g. Clostribium, Azobacter, Azospirillum, and Anabena) and organisms in symbiotic relationships with roots (e.g. Rhizobium, Frankia).

N2 + 10H+ + 8e- + nATP + nH2O = 2NH4+ + H2 + nADP + nH2PO4

-

where: n = 12 - 20 (exact number uncertain)

Ammonium assimilation occurs by two enzymatic routes:

1. Glutamine synthetase

COO- CONH2

CH2 CH2

CH2 + NH4++ATP = CH2 + ADP + H2O

CHNH3+ CHNH3

+

COO- COO-

glutamate glutamine


2. Glutamate dehydrogenase

COO- COO-

CH2 CH2

CH2 + NADH + H+ + NH4+ = CH2 + NAD + H2O

C = O CHNH3+

COO- COO-

-ketoglutarate glutamate


In addition, Glutamate synthase is used in plants and microorganisms to convert amido-nitrogen of glutamine back to glutamate for amino acid systems.

Glutamate synthase

COO- CONH2 COO-

CH2 CH2 CH2

CH2 + NADH + H+ + CH2 = 2 CH2 + NAD+

C = O CHNH3+ CHNH3

+

COO- COO- COO-

-ketoglutarate glutamine glutamate


Mineralization

Mineralization is the decomposition of organic matter to inorganic matter. This is accomplished by heterotrophic microbes. The release of N is generally thought to be a by-product of the use of soil organic C as an energy source.

R - NH2 = NH3 + H2O = NH4+ + OH-

Mineralization of organic matter is critical to the supply of nutrients to vegetation in terrestrial environments (see Table).

Mineralization is directly related to the nitrogen content of soil and the availability of organic carbon. Vegetation with high C/N in litter generally shows low rates of mineralization in soil.

Urea

NH2

ureaseC = O + 2H2O + 2H+ = 2NH4

+ + H2CO3

NH2

Percentage of the annual requirement of nutrients for growth in the Northern Hardwoods Forest at Hubbard Brook, New Hampshire, that could be supplied by various sources of available nutrients*

Process N P K Ca Mg

Growth requirement (kg ha-1 yr-1) 115.4 12.3 66.9 62.2 9.5

Percentage of the requirement that could by supplied by:

Intersystem inputs

Atmospheric

Rock weathering

Intrasystem transfers

Reabsorption

Detritus turnover (includes return in throughfall

and stemflow)

18

0

31

69

0

1

28

67

1

11

4

87

4

34

0

85

6

37

2

87

*Calculated using Eqs. 6.2 and 6.3. Reabsorption data are from Ryan and Bormann (1982). Data for N, K, Ca, and Mg are from Likens and Bormann (1995) and for P from Yanai (1992).


Nitrogen Dissimilation

Nitrification - the oxidation of NH4+

NH4+ + 2O2 = NO3

- + H2O + 2H+

Two different species of lithotrophic organisms are responsible for this reaction.

Nitrosomonas

ammonia oxidaseNH4

+ + 3/2 O2 = NO2- + 2H+ + H2O

This oxidation/reduction sequence is not direct but includes an electron transport chain in which 1 mol of ATP is produced per mol of NH4

+ oxidized.

This sequence is continued by the organism.

Nitrobacter

nitrite oxidaseNO2

- + ½O2 = NO3-

The electron produced from the oxidation of NO2- is also coupled with an electron

transport cycle producing 1 mol of ATP.


Nitrification can also be accomplished by heterotrophic bacteria.

Nitrification is an important process because many factors influence it and because it converts nitrogen from an immobile form (NH4

+) to a mobile form (NO3-).

Because the organisms which mediate nitrification reactions are specific populations, they are easily disrupted.

1. Lithotrophic organisms use inorganic C (CO2) to produce organic C through the Calvin cycle. This process is very energy intensive so these organisms have slow growth rates.

2. Nitrifiers, require well-oxygenated conditions.

3. Very sensitive to toxicants, trace metals.

4. Sensitive to pH (< 6?).

N2O and NO are released via nitrification.


Denitrification

Denitrification is the process by which N is used as the terminal electron acceptor in a reduction reaction.

This may be conducted by species: Pseudomonas, Bacillus, Vibrio and Thiobacillus.

Because organisms favor O2 reduction due to energetics, denitrification only proceeds under anaerobic conditions.

Organisms produce 2 mol ATP per mol NO3- reduced.

The process proceeds through an electron transport chain. The reductant is generally organic matter, generally sugars or simple compounds (methanol used in waste water treatment). Reduced sulfur compounds can also be used (sulfur, sulfide). These electrons are transferred to the electron transport chain where the reduction occurs.

In this process, NO3- is first reduced to NO2

-.

NO3- + NADH + H+ = NO2

- + NAD+ + H2O

Through this process, ATP is produced.


Subsequent reactions may occur:

NO2- + 2H+ + e- = NO + H2O

NO2- + 3H+ + 2e- = ½N2O + 3/2 H2O

NO2- + 4H+ + 3e- = ½N2 + 2H2O

NO + 2H+ + 2e- = ½N2 + H2O

½N2O + H+ + e- = ½N2 + ½H2O

The overall reaction to N2 is

C6H12O6 + 24/5 NO3- + 24/5 H+ = 6CO2 + 12/5 N2 + 42/5 H2O

The "leaky pipe" hypothesis suggests that trace gases, N2O and NO, are by-products of nitrification and denitrification.

Mechanisms of N Immobilization

1. Plant assimilation

2. Microbial (thought to predominate)

bacteria C5H7O2Nfungal higher C:N

Critical C:N 20-25

Above, microbial growth is N limitedLittle N leaching

Below, microbial growth is C limitedN leaching occurs

3. Nitrification, distribution of NH4+, NO3

-

Abiotic immobilization

Cation exchange X- - Na+ + NH4+ = X- - NH4

+ + Na+

No significant mechanism for abiotic immobilization of NO3- (anion exchange

weak).


N-Volatilization

NH4+ participates in an acid-base reaction.

NH4+ = NH3 + H+ ; pKa = 9.1

NH3 also has the ability to volatilize.

NH3 aq = NH3 g

As a result, NH3 can volatilize, but the reaction is only quantitatively important under high pH conditions.

Forest soils are generally acidic, so NH3 volatilization is an insignificant process.

In agricultural lands, application of fertilizer (manure) can result in high pH conditions and significant NH3volatilization.


Stable Isotopes of N

Stable isotopes of N can provide insight into biogeochemistry.

1. Addition tracer experiments

2. Natural abundance observations

There are two stable isotopes of N:

air composition

15N = 0.0037

14N = 0.9963

15N/14N = 1/272

Nitrogen isotopes are reported in values of per mil relative to atmospheric air.

Delta notation


15

15

N =

N

N sample

N

N std

N

N std

x 1000

15

14 14

15

14

Let's consider an example:

sample 15N = 0.00371

std 15N = 0.00370

Note that this example suggests that the sample is slightly enriched in 15N relative to the standard (+ sign).

A negative value would indicate that the sample is depleted relative to the standard.

In most terrestrial ecosystems, 15N values range from -10 to +15 %o. In absolute abundance, this represents a range of 0.3626 to 0.3718 atom % 15N.

Rule of thumb Organisms prefer the light isotope (14N) over the heavy isotope (15N) in transformations (see figures).


15 0 99629

0

N sample =

0.00371 0.00370

0.996300.00370

0.99630

x 1000

= 2.7 %

.

δ 15N

SOM

NO3

SOM

t1

t2

15N

N mineralization

SOM

NH4

+

NH4 + nitrification

NO 3

-

Isotope Enrichment Effect on Ammonium


Several factors influence the degree of fractionation:

1. Specific process.

2. Size of the pool. Large pools exhibit large fractionation, small pools exhibit little fractionation.

3. Temperature.

The 15N of a cumulative product is always lighter than the residual reactant. Consider denitrification. Say that this process fractionates by 5, 10, 20 %o from an initial NO3

- of 0 %o. The first bit of product (N2) is lighter than the reactant by the fractionation factor. As the reaction proceeds to completion, the product becomes progressively heavier until, at the end, it reaches its initial composition. The reactant also becomes progressively heavier until it is used up.


N Fractionation

Process Qualitative Charge Literature

N fixation small -3 to +1 %o

Assimilation

microbial small -1.6 to +1 (-0.52)%o

plant small -2.2 to +0.5 (-0.25)%o

Mineralization small -1 to +1 %o

Nitrification large -12 to -29 %o

Volatilization large > 20 %o

Sorption/desorption small 1 to 8 %o

Denitrification large -40 to 5 %o


Observations in the Literature

Terrestrial Ecosystem Compartments

1. Plants are slightly depleted.2. Organic soils are enriched.3. Mineral soils are more enriched.

The 15N of plants is similar to what they assimilate (little fractionation). Variations in plant 15N are due to:

1. rooting depth; deeper roots more enriched

2. NO3- vs. NH4

+ preference; NH4+ more enriched

Rates of N Cycling

In general, 15N increases in ecosystems with increased rates of N cycling due to fractionation associated with nitrification and NO3

- loss.

This is sometimes quantified as an enrichment factor (15N leaf - 15N soil).

See observations from Walker Branch, TN and Hubbard Brook.

Pardo et al., 2002 (Can. J For Res)


Food Web Studies

Food web studies show an enrichment in 15N.

N isotope scientists like to say you are what you eat, plus 3 %o.

See figure.

Use of 18O and 15N as a Tracer of Ecosystem N Retention

There are some drawbacks to using 15N as an ecosystem tracer due to its relatively narrow range. 18O associated with NO3

- offers additional information as a tracer.

Durke et al. (1994) used 15N and 18O together to evaluate the retention of atmospheric NO3

- to forests in Germany.

See tables.

WALLEYESHADYELLOW PERCHZEBRA MUSSEL FLESHDAPHNIASESTONSEDIMENT

C

-35 -30 -25

N

6

8

10

12

14

16

Adult Walleye

Young-of-the-Year Fish(e.g., Yellow Perch and Gizzard Shad)

Benthic Macroinvertebrates

Zebra Mussels

Benthic AlgaePhytoplankton

Zooplankton(e.g., Daphnia)

Pseudofeces

Adult Yellow Perch

Where does this nitrate come from: atmosphere (rain/snow) or soil?We need to know sources and sinks if we want to control N pollution

Stable isotopes can help answer this question

– Number of neutrons can vary:

• Nitrogen: 7 (99.6%) 14N & 8 (0.4%) 15N• Oxygen: 8 (99.8%)16O & 10 (0.2%) 18O

– The relative isotopic abundances of an element depend on sources of the element and various (physical, chemical and biological) isotopic fractionation processes.

o/ o

o

o/oo

Atmospheric

Derived from Soil Nitrification

N and O Isotopes of Nitrate

-10 0 10 20 30

0

20

40

60

80

o/ o

o

o/oo

Atmospheric

Derived from Soil Nitrification

N and O Isotopes of Nitrate

-10 0 10 20 30

0

20

40

60

80

Atmospheric/SnowSurface Waters

Archer Creek Catchment in Arbutus Watershed

Piatek et al. (2005)

III. GLOBAL N PATTERNS/BUDGET

Across the Earth, N largely occurs as N2 in the atmosphere (78%) and in the ocean and in soil.

Nitrogen is divided into two broad classes:

1. Reactive- NOy = NOx (NO + NO2) + any oxidized N with a single atom of N

- NHx = NH4+ + NH3

- organic N

2. Unreactive - N2

- N2O

- organic N (soil)

See tables.

Global N Patterns/budget (cont.)

Table 1. Estimates of the active pools in the global nitrogen cycle.

million tonnes N

AirN2 3 900 000 000N2O 1 400

LandPlants 15 000Animals 200 of which people 10Soil organic matter 150 000 of which microbial biomass 6 000

SeaPlants 300Animals 200In solution or suspension 1 200 000 of which NO3

--N 570 000 of which NH4

+-N 7 000Dissolved N2 22 000 000


Table 2. Production of combined nitrogen gases by land, sea and air.

Gas

Atmospheric-stock,million tonnes N

Residence timein atmosphere

Annual production, million tonnes N per year

NH3 <1 6 days 54 ± 8

N2O 1400 170 years 14 ± 7

NOx <1 5 days 48 ± 15


Table 3. Distribution of nitrogen (g m-2) between plant biomass and above-ground litter and plant uptake in difference bioclimate zones. Calculated from Baztlevich and Soderlund and Svenson.

Biomass(g N m-2)

Litter(g N m-2)

Turnover timein litter (yr)

Plant uptake(g m-2 yr-1)

Polar areas 12 106 66 1.6

Boreal areas 15 76 12 6.4

Sub-boreal areas 57 10 1.0 10.0

humid 137 22 14.5

semi-arid 22 5 11.0

arid 13 3 4.3

Subtropical areas 73 9 0.4 21.2

humid 161 18 37.8

semi-arid 68 10 20.7

arid 22 3 11.0

Tropical areas 165 6 0.2 29.3

humid 287 9 46.4

semi-arid 88 6 21.6

arid 8 1 3.4

Total terrestrial 94 23 1.3 17.2


Preindustrial N budget

The transfer of reactive to unreactive N was balanced.

N2, N2O produced by denitrification in oceans and soil.

NH3 is released by volatilization.

NH4+ = NH3(aq) + H+ ; pKa = 9.3

NH3(aq) = NH3(g)

This process occurs only under high pH conditions.

NH3 is released by burning of plants.

NH3 is very reactive and has a short residence time in the atmosphere.

NH3 + H2O = NH4+ + OH-


NO can be formed by

1. Oxidation of N2 by lightning;

2. Soil microbes;

3. Burning of biomass.

NO, NOx are very reactive and have a short residence time in the atmosphere.

In the preindustrial world, N inputs were largely retained where they were deposited.Nitrogen is a tightly conserved element in terrestrial environments because it is the growth limiting nutrient.


NH4+ - relatively immobile form of N

a. Strongly assimilated by biota due to energetics;b. Abiotically retained on soil cation exchange sites.

NO3- - relatively mobile from of N

a. No significant mechanism of abiotic retention.

Nitrification is a key process regulating the mobility of N.

Riverine fluxes of N are thought to be 75 - 120 kg N/km2-yr and this is thought to largely occur as particulate organic N.

Galloway et al.: N Fixation: Anthropogenic Influence

(Tg N/yr)

(Tg N/yr)



Anthropogenic Sources

Human activity has had a profound effect on the N cycle.

Three processes largely contribute to this disturbance, through anthropogenic nitrogen fixation.

Both thermal and fuel NOx can be significant, but fuel NOx is often the dominant source.

1. Energy production - under high temperature combustion processes, unreactive nitrogen is converted to reactive nitrogen by two processes.

a. Thermal NOx

1000oK

N2 + O2 2NO

b. Fuel NOx - the oxidation of organic N in fuels

Natural gas - very low 0%Coal - up to 3%


2. Fertilizer-Most anthropogenic fertilizers are either NH3 or urea produced from NH3.

This material is produced by the Haber process.

4N2 + 12H2 = 8NH3

This is a very energy intensive process if natural gas is the energy source for H2, as it usually is.

3CH4 + 6H2O + 4N2 = 3CO2 + 8NH3

3. Production of legumes and other crops allows for the conversion of N2 to reactive N by increasing biological nitrogen fixation.

Legumes include:SoybeansGround nuts (peanuts)Pulses (lentils)Forage (alfalfa, clover)

(Tg N/yr)

(Tg N/yr)


0

2

4

6

1850 1870 1890 1910 1930 1950 1970 1990 20100

50

100

150

200

Population Haber Bosch C-BNF Fossil Fuel Total Nr

Global Population and Reactive Nitrogen TrendsGlobal Population and Reactive Nitrogen Trends

From Galloway et al. 2003.

Hum

an P

opul

atio

n (b

illio

ns)

Tg N

yr-1

Natural N Fixation


All three categories of anthropogenic nitrogen fixation have increased, but most significant is fertilizer consumption. The rate of fertilizer consumption is increasing particularly in Asia.

There are two critical questions in response to this change.

1. What is the fate of this fixed N; and

2. What are the effects of this increase in fixed N?

IV. PATTERNS OF N AT THE HBEF (HBEF W6, CPW, BNA)

1965 1970 1975 1980 1985 1990 1995 2000

SO

2 E

mis

sio

ns

Mill

ion

sh

ort

to

ns

2

4

6

8

10

12

14

16

with Canadian Emissionsw/out Canadian Emissions

Year

1965 1970 1975 1980 1985 1990 1995 2000

NO

x E

mis

sio

ns

Mill

ion

sh

ort

to

ns

0

2

4

6

8

Source Area Includes: VT, MA, NY, NH, CT, RI, ME, OH, PA, DC, MD, NJ, DE, MI, VA, WV, QUE, ONT

SO2 Emissions - 24 hr source area (Million metric tons)

0 2 4 6 8 10 12 14 16

Hub

bard

Bro

ok

Pre

cipi

tatio

n S

O4

(eq

/L)

0

20

40

60

80

Y= 3.82X + 5.73r ²=0.764

NOx Emissions - 24 hr source area (Million metric tons)

0 2 4 6 8

Hub

bard

Bro

okP

reci

pita

tion

NO

3

(eq

/L)

0

5

10

15

20

25

30

35

Y= 7.31X - 24.99r ²=0.393

NO3- High hardwood elevation

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Month

um

ol L

-1

Oa

Bh

Bs

Stream

Seasonal Patterns NO3-

Growing Season

DON SFB elevation

0

5

10

15

20

25

30

35

40

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

month

um

ol L

-1 Oa

Bh

Bs

Stream

Dissolved organic nitrogen (DON)

• Stream water [DON] > Mineral Soil [DON]Stream water [DON] > Mineral Soil [DON]

• Seasonal increase in N cycling activities at the HBEF.Seasonal increase in N cycling activities at the HBEF.

Increased N Cycling

Horizonal Patterns 1992-2003

High hardwood elevation Nitrogen Flux

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

Stream

Bs

Bh

Oa

TF

ppt

mmol m-2 yr-1

NH4

NO3

DON

Spruce Fir Birch Elevation Nitrogen Flux

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

Stream

Bs

Bh

Oa

TF

ppt

mmol m-2 yr-1

NH4NO3DON

1 High Hardwood Elevation Nitrogen Flux

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

Stream

Bs

Bh

Oa

TF

ppt

mmol m-2 yr-1

NH4NO3DON

2 Low Hardwood Elevation Nitrogen Flux

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

Stream

Bs

Bh

Oa

TF

ppt

mmol m-2 yr-1

NH4NO3DON

3

(a)

(b)

(c)

Bormann et al.

(1977) 1965-76 1990-95

Inputs

Bulk Precipitation 464 524 549

Dry Deposition 52 54

N-fixation 36 36

Total Inputs 464 560 639

Outputs

Streamwater

DIN 286 310 64

DON 38 38

Denitrification 0 0

Total Outputs 286 348 102

Changes in Pools

Biomass 643 1328 167

Forest Floor 550 0 0

Mineral Soil 314 314

Net Retention 1014 1430 56

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BIOGEOCHEMISTRY OF NITROGEN I.Introduction II.N-cycle, and the Biochemistry of N III.Global N...

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