The Origin of Nitrogen Isotopic
Signatures in Algae
Peter K. Swart
RSMAS/MGG
Stable Isotope Facility
University of Miami
Stable Isotope Facility
• Founded in 1986
• Four Operational Stable Isotope Instruments
– Two which are dedicated for carbonate material
– One which does C, N isotopes on solids as well as
O2, N2O and DIC in waters (>400,000 analyses since
1996)
– One which does H and O isotopes in water as well as
C isotopes in atmospheric gases
– IC, ICP-OES, XRD
Acknowledgments
• Graduate Students
– Kathryn Lamb
– Courtney Drayer
• Colleagues
– Bill Anderson
– Mark Altabet
• Funding
– BNP, EPA, NOAA
1. WHAT ARE Nitrogen isotopes?
2. Why Nitrogen Isotopes?
3. What are the ranges of N isotopes in the
environment?
4. What do different values mean?
5. EPA Funded project
6. Current Research
1. Kathyrn Lamb (Ph.D. Defense Dec. 8th)
2. Courtney Drayer
What are Nitrogen Isotopes?
ƒAll elements have more than
one isotope.
ƒSome of these are stable and
some unstable (radioactive)
ƒNitrogen has two stable
isotopes (14N and15N)
ƒChanges in the abundance of
the less abundant isotope 15N
relative to 14N reflect biological
and physical processes and
can be used as tracers of
such processes
ƒAll organisms contain N and
therefore some 15N
How are Changes Reported?
• Changes in the concentration of nitrogen
isotopes are reported relative to
atmospheric nitrogen in parts per thousand
or per mille (‰).
• If a substance has a value of 0 ‰ then it
has the same composition as atmosphere
nitrogen.
• If is has a higher values it contains more
15N, if it has lower values then more 14N
15
δ N ( / 00 ) =
15
0
14
N / N sample
15
14
N / N s tan dard
− 1*1000
1. WHAT ARE Nitrogen isotopes?
2. Why Nitrogen Isotopes?
3. What are the ranges of N isotopes in the
environment?
4. What do different values mean?
5. EPA Funded project
6. Current Research
1. Kathyrn Lamb (Ph.D. Defense Dec. 8th)
2. Courtney Drayer
Why
• Nitrogen Isotopes give information on
sources and processes of nitrogen
– Sources: Nitrogen isotopes have been
quoted as being the DNA of sewage, with
heavy or more positive values being derived
from sewage.
– Processes: Nitrogen give information on the
nature of transformation processes
1. WHAT ARE Nitrogen isotopes?
2. Why Nitrogen Isotopes?
3. What are the ranges of N isotopes in the
environment?
4. What do different values mean?
5. EPA Funded project
6. Current Research
1. Kathyrn Lamb (Ph.D. Defense Dec. 8th)
2. Courtney Drayer
1. WHAT ARE Nitrogen isotopes?
2. Why Nitrogen Isotopes?
3. What are the ranges of N isotopes in the
environment?
4. What do different values mean?
5. EPA Funded project
6. Current Research
1. Kathyrn Lamb (Ph.D. Defense Dec. 8th)
2. Courtney Drayer
Modified from Nadelhoffer and Fry (1994)
Atm
N2
α = 1.00
Denitrification
α = 1.028
N2
+
+4
H
C
+5
NH
2N 3
2
+4
N
O3
O2
O
H2
7
+
Nitrogen Fixation
N2O
N2 + 8H+ + 8e- + 16ATP = 2NH3 + H2 + 16ADP + 16 Pi
α = ~1.000
Æ
NH3
NO
αH = 1.020
O
2
5C
NO2
NO3-
Organic Matter
Assimilation
NO2α = ~1.002-1.004
NH4+
Volatilization
-20‰
0
2N
PON
NH4+ Æ NH3
O
2 -+
α = ~1.000
α = 1.020
DON
O
2
Æ
2N
O
3 -
Mineralization
NO2Nitrification
2N
Assimilation and
α = ~1.002-1.004
DON Æ NH4
H4
α = ~1.000
++
3O
2
α = 1.015 - 1.035
Æ
2N
O
2
-
+2
NH4+
H
2O
4H +
Lamb 2006
+ve
“You are what you eat… plus a
few per mille”
Nitrogen Isotopes
12
8
“
4
0
1
2
4
Trophic Level or Food Chain
8
Nitrogen as Indicator of Sewage
– (Heikoop et al. 2000a,b;
Sammarco et al., 1999;
Risk and Erdmann, 2000;
Costanzo et al., 2001,
2004).
12
11
N-15
• Numerous papers have
been published which
report the use of stable
isotopes in benthic
organisms in order to
distinguish sewage
13
10
9
8
7
0
20
10
40
30
60
50
80
70
Distance from Source
Risk and Erdman 2000
(km)
100
90
Fig. 3.6. δ15N values of algae in Moreton Bay, Australia where the city of Brisbane occupies the western shore. High δ15N
values along the western shore indicate N pollution inputs from watershed rivers and local sewage treatment facilities.
The coastal pollution plumes are hard to identify by conventional measurements of ammonium and nitrate nutrients,
because tides rapidly disperse nutrients and algae use up the nutrients during growth in algal blooms of the region. But the
isotope values persist as nutrients are incorporated into the algae, tracing the nitrogen linkage to coastal inputs. Results are
contoured for macroalgae that were incubated 4 days in situ at approximately 100 sites in September 1997, then analyzed
for δ15N (Costanzo et al. 2001). This δ15N work continues now as a monitoring technique termed “sewage plume
mapping” (Costanzo et al. 2005). Reprinted from Marine Pollution Bulletin 42:149-156, S.D. Costanzo, M.J. O’Donohue,
W.C. Dennison, N.R. Loneragan, and M. Thomas, A new approach for detecting and mapping sewage impacts. Copyright
2001, with permission from Elsevier.
Nitrogen Isotopes of Brevard
County
14
12
10
Nitrogen
• In South Florida
values have been
suggested to
represent input of
anthropogenic
nitrogen linked to
precipitation (Data
measured on Codium
isthmocladum).
8
6
4
2
0
May
July
June
Lapointe 1997
September
August
Nitrogen
• Other studies which have shown that sewage
derived nitrogen has more negative values
compared to normal planktonic nitrogen (Wada
and Hattori, 1975). In that particular study the
δ15N of the effluent only had a value of +2.5 ‰
while the planktonic δ15N was greater than 9‰.
• Similar positive planktonic δ15N values have also
been reported by other workers
• (Wada and Hattori, 1978; Sweeney and Kaplan, 1980;
Montoya; Peterson & Howarth, 1987; Goering et al. 1990;
Harrigan et al., 1990).
Nitrogen
• One study (Rogers, 2003)
sampled a sewage outfall
which had been
depositing sewage for
over 10 years at a
location.
• The value was +2 to +3
per mille.
• After three weeks the
value had increased and
within 9 months the value
was the same as algae
further away (+7 per
mille).
Other Possible Factors
• Different organisms have different values
• Different parts of plants and animals have
different values
• Different age plants have different values
• Different organic molecules have different
values
• Different times of the year have different
values as physiological rates change
1. WHAT ARE Nitrogen isotopes?
2. Why Nitrogen Isotopes?
3. What are the ranges of N isotopes in the
environment?
4. What do different values mean?
5. EPA Funded project
6. Current Research
1. Kathyrn Lamb (Ph.D. Defense Dec. 8th)
2. Courtney Drayer
Atm
N2
Denitrification
α = 1.028
+
5C
H
O
2
+4
H
+4
N
O3
Æ
2N 2
C
+5
O2
O
H2
7
+
Nitrogen Fixation
N2O
N2 + 8H+ + 8e- + 16ATP = 2NH3 + H2 + 16ADP + 16 Pi
α = ~1.000
NH3
NO
NO2
Organic Matter
Assimilation
α = ~1.002-1.004
NO3-
PON
Volatilization
2N
NH4+ Æ NH3
O
2 -+
α = ~1.000
α = 1.020
DON
O
2
Æ
2N
O
3 -
Mineralization
NO2Nitrification
2N
Assimilation and
α = ~1.002-1.004
DON Æ NH4
H4
α = ~1.000
++
3O
2
α = 1.015 - 1.035
Æ
2N
O
2
-
+2
NH4+
H
2O
4H +
Lamb 2006
NH4+
NO3-
α = 1.020
70
50
40
30
20
10
0
-10
-20
5
4
3
2
Concentration of NH4+
Nitrification
1
0
Dissolved NH4+ or NO3-
60
NH4
NO3
NH4+
NO3-
α = 1.020
70
40
NH4
60
35
NO3
50
30
40
25
30
20
20
15
10
10
0
5
-10
0
-20
5
4.5
4
3.5
3
2.5
Concentration of NH4+
2
1.5
1
Dissolved NH4+ or NO3-
P lant or Algae
45
Assimilation
Mineralization
α=1.000
NH4+
Assimilation (α=1.004)
NO3-
Nitrification
α = 1.020
NH4+
Flux from Sediment
NO3Upwelling
Denitrification
α = 1.020
Nitrogen Isotopes
10
9
Bulk
8
Nitrate
7
Ammonia
6
5
4
3
2
1
0
0.2
0.4
0.6
Nitrate
0.8
1
Initial 15/14
Alpha nitrification
NH4 Fluxsediments
Nitrification rate
Initial NH4
0.007
1.02
0.2
0.06
10
Alpha Assimilation NH4
1.002
Alpha Assimilation NO3
1.002
NH4 Assimilation Rate
0.02
NO3 Assimilation Rate
0.054
Denitrification Rate
0.01
Alpha Denitrification
1.02
10
9
Nitrogen Isotopes
8
7
6
5
4
Bulk
3
Nitrate
2
Ammonia
1
0
0.1
0.3
0.5
Nitrate
0.7
0.9
Initial 15/14
Alpha nitrification
NH4 Fluxsediments
Nitrification rate
Initial NH4
0.007
1.02
0.2
0.06
10
Alpha Assimilation NH4
1
Alpha Assimilation NO3
1.003
NH4 Assimilation Rate
0.02
NO3 Assimilation Rate
0.053
Denitrification Rate
Alpha Denitrification
0.01
EPA Experiment
• Grow strains of algae (Gracillaria sp, Ulva
sp. and Agardhiella sp.) under different
ratios of ammonium and nitrate.
– High ammonium, low nitrate (nitrate will have
an isotopic label + 40‰)
– Low ammonium, high nitrate (ammonium will
have isotopic label +40‰)
– Zero nitrate
– Zero ammonium
1. WHAT ARE Nitrogen isotopes?
2. Why Nitrogen Isotopes?
3. What are the ranges of N isotopes in the
environment?
4. What do different values mean?
5. EPA Funded project
6. Current Research
1. Kathyrn Lamb (Ph.D. Defense Dec. 8th)
2. Courtney Drayer (Biscayne Bay and point north)
Martinique November 2003 (Giliam)
Martinique 2006 (Judy Lang)
Biotic (fish) wastes
1%
Florida Bay
0%
Anthropogenic
wastes
0%
Gulf of Mexico
1%
Atmospheric
deposition
20%
Atmospheric
Deposition
NH4 efflux
10%
N fixation
68%
Upwelling
0%
Anthropogenic
Wastes
Florida Bay
Upwelling
Groundwater ?
NH4 Efflux
Sediments
N fixation
(via seds and flora)
Biotic
Wastes
GOM Gyre
Waters
Lamb 2006
Nutrient Impacted Reefs
-8
-9
Affected
-10
Carbon
-11
-12
-13
-14
-15
Reference
-16
-17
0
2
4
6
Nitrogen
Heikoop et al. (2000)
8
10
12
Comparison of Florida Corals
-8
-9
Affected
-10
Carbon
-11
-12
Key Largo
-13
-14
-15
Reference
-16
-17
0
2
4
6
Nitrogen
Heikoop et al. (2000) Swart et al (2005)
8
10
12
δ15N (per mil)
14
-15
-17
12
-19
10
-21
8
-23
-25
6
-27
4
-29
2
-31
0
-33
Jun-00 Aug-00 Oct-00 Dec-00 Feb-01 Jun-01 Sep-01 Nov-01 Feb-02 Apr-02 Jun-02 Aug-02
Date
δ13C (per mil)
Nitrogen
Carbon
(‰)
(‰)
Lamb and Swart (unpublished)
-15
Dec-05
Nov-05
Oct-05
Sep-05
Aug-05
Jul-05
Jun-05
May-05
Apr-05
Mar-05
Feb-05
Jan-05
Dec-04
Nov-04
Oct-04
Sep-04
Aug-04
Jul-04
Jun-04
May-04
Apr-04
Mar-04
Feb-04
Jan-04
Temporal trends in δ15N and δ18O (none)
35
30
25
20
15
10
5
0
-5
-10
Data from Lamb, Swart and Altabet (unpublished)
Spatial trends in δ15N and δ18O
δ15N NO3-
δ15N (‰)
15
10
5
0
-5
C
PK
M
WB
SI
ROD
O
M
TR
PI
O
Lamb, Swart, Altabet (unpublished)
70
60
δ18O (‰)
50
NO3- in
precipitation
40
30
20
Synthetic
NO3- fertilizers
10
NH4+
Fertilizers
0
Sewage wastes
and manure
Soil N
-10
-10
-5
0
5
10
15
20
25
30
35
40
δ15N (‰)
Lamb, Swart, Altabet (unpublished)
-7
-9
Seagrass
δ13C ‰
-11
S. chry
-13
S. vir
S. atom
S. cro
S. auro
-15
-17
S. dor
A. bah
Dictyota
K. sec
Turf
Halimeda
-19
-21
0
1
2
3
4
5
6
7
δ15N ‰
B
Lamb and Swart (unpublished)
8
9
Are N Isotopes the DNA of
Sewage?
• Are N Isotopes the DNA of Sewage?
– No
• Can they provide valuable information
about the system and process?
– Yes, but a system wide approach needs to be
utilized and not only N isotopes
– A range of physical factors need to be
considered in addition to other chemical
measurements (tracers and other stable
isotope systems)
Should we Use N Isotopes?
• N isotopes should not be used in isolation
• When studying a system all possible
components of the system should be
studied so that an idea is obtained of
natural variability which exists in the
system and the fractionation which occurs
during normal transformations of nitrogen
compounds understood.