Training managment training 14-18 feb,2021

1. USE OF15N AND 13C ISOTOPES
TECHNIQUES IN AGRICULTURE
RESEARCH
Dr. Md. Azizul Haque
Chief Scientific Officer (CC)
Soil Science Division
BINA, BAU campus, Mymensingh
Email: azizul_bina@yahoo.com
Paper presented in the training course “Reseaech Managenent” to be held at BINA,
Mymensingh, 14-18 Feb. 2018.

2. Some Basic Concepts on
Stable Isotopes

3. Construction of Matter
It is a general name of nucleus or atoms that
have specific numbers of protons and neutrons.
For a given nuclide, Z represents its proton
number, N its neutron number and A its mass
number which equals Z+N.
A = Z + N
For 13C: 13 = 6 + 7

4. Isotopes
Nuclides having the same number of protons but
different numbers of neutrons are called isotopes.
For examples:
12C, 13C, 14C
14N, 15N

5. Stable isotopes:
For example, 12C and 13C, 14N and 15N, 16O, 17Oand 18O.
Parent, Daughter
Stability of Nuclides:
Stability of Nuclides and Radioactive Decay
The unstable ones spontaneously transfer to other types of
nuclides through one of the decay mechanisms
14C, 137Cs
In isotope geochemistry, the radioactive isotope is referred to as
the parent, and the nuclide resulting from the decay of the
parent is called the daughter.
Unstable isotopes (radioactive isotope):

6. Fig.1 A Part of the Chart of the Nuclides
At low mass, stable nuclides have ~ equal number of
protons and neutrons.
http://atom.kaeri.re.kr/

7. Isotopic Abundance and excess
For a given isotope, its abundance is the percentage of
total atom number of an element.
15N abundance= The total atom% 15N in a sample (a)
Natural abundance(a0): atom% 15N natural
abundance in nature is generally about (0.3663
atom%15N±0.0004)
Atom %15N excess in a compound= Total atom% 15N-
natural abundance = (a-a0)

8. n
‘
light’
isotope ‘
heavy’
isotope
14N
7
14N
7
99.63 % 0.37 %
15N
7
15N
7
Bio-element Stable isotopes
NITROGEN:

9. Atomic
number
Element Mass
number
Varied range of
abundance（%）
The average
abundance
（%）
1 H 1 99.9918-99.9770 99.985
2 0.0230-0.0082 0.015
6 C 12 98.99-98.86 98.9
13 1.14-1.01 1.1
7 N 14 99.639-99.625 99.63
15 0.375-0.361 0.37
8 O 16 99.7771-99.7539 99.762
17 0.0407-0.035 0.038
18 0.2084-0.1879 0.2
14 Si 28 92.41-92.14 92.3
29 4.73-4.57 4.67
30 3.14-3.01 3.1
16 S 32 95.253-94.638 95.02
33 0.780-0.731 0.75
34 4.562-4.001 4.21
36 0.0199-0.0153 0.02
Table 1. Abundances of some isotopes

10. Isotopically Substituted Molecule
Definition:
Because an element includes a few isotopes ， the same
compound consists of different species, and these species are
called isotopically substituted molecules of the compound.
For example, H2O has 9 isotopically substituted molecules.
Like 1H2O16,
1H2O18, 2H2O18 etc,

11. Isotope Effect
Isotope effect is the slight differences of chemical
and physical properties existing among different
isotopes of the same element or different
isotopically substituted molecules of the same
compound.

12. Properties State Temperature H2
16O D2
16O
Molecular weight 18.01629 20.02948
Density Liquid 25(°C) 0.996781 1.104211
Melting point(°C) 105 Pa 0 3.813
Boiling point (°C) 105 Pa 100.00 101.43
Relative vapour
pressure (105 Pa)
Liquid 20(°C) 1.171 1.000
Heat capacity (J
mol-1 0C -1)
Liquid 25(°C) 76.074 84.7827
Ice Melting point 38.4767 45.1755
Melting heat (J mol-
1)
Liquid Melting point 6012 6343
Vaporization heat
(J mol-1)
Liquid Boiling point 40691 41562
Dielectric constant Liquid 25(°C) 78.25 78.54
Table 2. Slight differences of chemical and physical
properties between H2
16O and D2
16O

13. Causes of Isotope Effect
Different move velocity
Difference in Mass
Different Zero
Point Energy
Different chemical
reaction rate
Different spectral
characters
For example, the mass of D2
16O is 20, H2
16O is 18.
The former is slower in process of diffusion than the latter;
D2
16O has a higher zero point energy than H2
16O, resulting in
the former has a lower reaction rate in chemical reactions than the
latter.
Different zero point energy also results in different spectral
characters between D2
16O and H2
16O.
Same in case of C and N isotopes.

14. Expression of Isotope Data

15. The relative isotope abundance of 15N in a particular
compound (N2, Urea) is defined by the ratio between the
amount of the isotope 15N (mol) and the amount of the total
chemical nitrogen containing the isotope 14N and 15N (mol) .
The unit of the relative isotope abundance of 15N is
consequently 1. (=mol/mol). This can be explained by the
following equation:
a/100= n(15)/ {n(14)+n(15)}= n(15)/nN
Relative isotope abundance can be either expressed in
1) 15N abundance (atom%15N)
a=n(15)*100/{n(14)+n(15)}=n(15)/nN*100 (atom%)
Or
δ-notation:
δ values indicate small deviation per mill (%0) in isotope ratio of a sample
related to that of a standard (usually atmospheric N)

16. where X* and X0 are atom numbers of heavy isotope and light
isotope of element X, respectively.
R = X*/X0
RH2O = (18O/16O)H2O
（2）
R value is the ratio of atom number of heavy isotope to that of
light isotope
D/H, 13C/12C, 15N/14N, 18O/16O, 34S/32S
RH2O = (D/H)H2O
Isotopic Ratio (R)

17. δ-notation:
δ(‰) = 〔(Rsam - Rst) /Rst〕×103
1000
N
N
N
N
N
N
N
st
14
15
st
14
15
sam
14
15
15










































(3)
where Rsamand Rst indicate the R of the standard matter and sample,
respectivly
Note that the unit of -notation is per mil.

18. Again simplify the equation 3
δ(‰) = (Rsam /Rst-1)×103
This isotopic ratio is mostly used for precision
measurements in natural abundance range. Common
standard is atmospheric nitrogen with an accepted value
of 0.3663±0.0004 at.%N

19. 15N Tracer Techniquies in Agricultural
Research
15N has become an isotope of broad application
for the understanding of biological and/or
chemical process affecting the -
Nitrogen cycle and
the movement of N compounds in agricultural
systems.

20. 1) N turnover in soil.
2) Distribution of applied N in soil organic matter.
3) Genotypic difference in N uptake and use
4) Fertilizer N utilization:
• Fertilizer management practices (Timing,
placement, sources, etc.
•Interactions with another agronomic factors
(irrigation, plant species, cultivars, land
preparations etc.)
• Fertilizer N balance
Extensively use of 15N as tracer in soil –
plant systems

21. 5) Recovery of N from crop residues
6) Nitrogen movement in soil
7) Nitrogen gaseous losses (Volatilization,
denitrification etc.)
8) Nitrogen leaching losses
9) Environmental of nitrogen use
10) Degradation of organic chemicals added to soils
11) Biological nitrogen fixation both symbiotic and
non symbiotic, identification of N-fixing systems,
Measurement of biological N fixation in field.
12) Distribution of N among plant parts
13) N metabolic studies in plants and animals.

24. :

25. Ndff=
N derived from soil (Ndfs)=N yield – Ndff (kg/ha)
%N derived from soil (%Ndfs)= 100-%Ndff
This is also called fertilizer use efficiency or fertilizer recovery

26. For example:

27. Dinitrogen fixation
Nitrogen fixation is a process by which nitrogen (N2) in the
atmosphere is converted into ammonia (NH3).
Nitrogen fixation, natural and synthetic, is essential for all forms of
life because nitrogen is required to biosynthesize basic building
blocks of plants, animals and other life forms, e.g., nucleotides for
DNA and RNA and amino acids for proteins.

28. Biological N fixation
Biological nitrogen fixation (BNF) occurs when
atmospheric nitrogen is converted to ammonia
by an enzyme called nitrogenase.
The reaction for BNF is:
N2 + 8 H+ + 8 e− → 2 NH3 + H2

29. Traditional method:
Nitrogen difference method
Biological N fixation by plants:
Quantification

32. 15N isotopic method
• 15N2 reduction method
• 15N isotpic dilution method
• 15N natural abundance method

34. 15N2 reduction method

37. Sampling and measurement
1. Sampling: different parts of plant
2. Total Nitrogen: Distillationed method (semi
Micro Kjeldhal Method)
3. 15N measurement: Mass spectrometer

38. N2 fixation:
Ndfa%= Atom% Excess of plant ÷
Atom% Excess of 15N2 in sample
Ndfa=Ndfa%*Total N amount in plant /100
Fixation rate=Ndfa/Wgt. of plant/time peroid

44. 15N dilution method
Since there are only two sources of N for Non fixing crop (NF) or reference crop
(1)

45. Fig.1.

46. (2)
Fig. 1
2
(3)
Since according to equation 3

47. (4)
Derivation of equation 4:
Equation 4 is derived from eq.1, 2 and 3 as follows;
From eq. 3:
2
(5)
1

48. From equation 1 and 5:
Or
(6)
From equation 3 and 6
Or
(7)
Or

49. The equation 7 can also be written as
(8)
(9)
(10)
The equation from (7) to (10) should always keep in mind
for calculation of different N fraction in biological N2
fixation in Isotopic dilution (ID) method
Ndfa: Nitrogen derived from atmosphere, Ndff: N derived from fertilizer,
Ndfs: N derived from soil
F: fixing crop; NF: Non fixing crop

50. 2

53. Advantages & Disadvantages of Natural
Abundance Method
Advantages
 No tracer or isotope require.
 Very useful for natural ecosystems e.g. trees, for
which it is very difficult to label.
Disadvantages
Small differences in 15N abundance.
Require highly precise isotope ratio mass
spectrometer (IRMS) to trace.
 Often small differences between 15Nabundance in
soils and air.

54. Use of 15N isotopes in organic residue studies
Use of 15N in organic fertilizer has been significantly
advanced of our understanding of N release from
organic materials:
Two main approches
1)Direct techniques- where plant residues or OM are
labelled directly and fate of 15N is traced
2) Indirect- Soil is labelled and the dilution of 15N in the
crop receiving the residue is measured.

55. the equation 11.
(11)
Ndfr: N derived from residue
15N DIRECT LABELLING TECNIQUE OF RESIDUE

56. %N recoveryfrom residue canbe calculated as:
%N recovery from residue=

57. 15N INDIRECT LABELLING TECNIQUE OF RESIDUE
as
In

58. Exercise 1:
In a field experiment 80 kgN/ha in the form of 15N labelled urea
was applied to a maize crop. The maize was harvested at
tasseling time with a dry matter yield of 4 tons/ha and a plant
sample had 0.67%15N abundance and 3% total N content. The
applied fertilizer had 1.37% 15N abundance.
Please find out:
i) What was the total amount of N yield/uptake of the crop?
ii) What was % N derived from fertilizer (%Ndff) in crop?
iii) What was %N derived from soil (Ndfs) in crop ?
iv) How much of N derived from fertilizer in crop ?
V) What was the %fertilizer N utilization or recovery by the
crop?

59. Hints for solution: Ex.1
1.
2. %Ndff in plant
%15N a.e. in plant= 0.67-0.37= 0.30
%15N a.e. in fertilizer= 1.37-0.37=1.00
3. %Ndfs in plant=100 - %Ndff
4.
5.

60. Some basic concept about carbon
isotopes in Environment

61. 7Isotope Fractionation
Isotope fractionation is a phenomenon that
isotope abundance always changes in chemical
and physical processes. It is caused by isotope
effect.
For example.
Decomposition of Soil organic matter (SOM)

62. From this plot, we can see that soil
organic carbon content (C%)decreased
with soil depth, suggesting that the
extent of decomposition of soil organic
matter increased with depth. However
13C of soil organic matter (SOM)
increased with depth. It indicates that
the residual SOM enriched more and
more 13C with decomposition because
12C enters the respired CO2 more
easily relative to 13C. The change of
isotope abundances in the process of
SOM decomposition is Isotope
Fractionation.
1
Fig. 1. Carbon contents (a) and 13C
values (b) of soil organic
matter and above –ground
plants

63. 1) Equilibrium Fractionation:
Types of Isotope Fractionation
When a system is in isotopic equilibrium, we define the
fractionation occurring at the time as equilibrium
fractionation.
Character/Result:
Isotopic abundances in both substrate and product do not
change with time
H2
18O + C16O2  H2
16O + C18O2
Example:
This is an exchange reaction of O isotope between H2O
and CO2.when the reaction is in equilibrium, O isotopic
abundances in both H2O and CO2 keep constant

64. 2) Kinetic Fractionation:
Kinetic Fractionation is the isotope fractionation that
isotopic abundances in both substrate and product
Change with time and evolution of chemical
reaction

65. Fig.2. Kinetic Isotope Fractionation in
molecular diffusion

66. PDB vs. V-PDB
The PDB standard matter has been used up, now a days, a
marble (its serial number is NBS-19) has been used for the
new standard matter for carbon isotope. And the new standard
is called V-PDB. The 13C of NBS-19 is 1.95‰ relative to the
PDB.
Earlier, 13C data were reported relative to PDB, since then,
13C data have been reported relative to V-PDB.
V-PDB is different from PDB, so the 13C data relative to V-
PDB differs from the data relative to PDB.
About 13C standard materials

67. Some Isotopes Standard Matter
International Atomic Energy Agency, AEA;
National Institute of Standard and Technology, NIST,USA
Isotopes Standard Matters R values
C PDB (Peedee Belemnite) 13C/12C=(11237.2±
90)×10-6
N Air 15N/14N=
(3676.5±8.1)×10-6
H SMOW, Standard Mean Ocean
Water
D/H=(155.76±0.10)
×10-6
O SMOW, Standard Mean Ocean
Water
18O/16O=
(2005.20±0.43)×10-
6
S CDT, Canyon Diablo (Troilite) 34S/32S=0.0450045
±93

68. The C3 & C4 photosynthetic pathways
• C3 Calvin cycle
• 95% of plant species
• E.g. wheat, potato, beet,
many fruits & vegetables
• d values -22 to -30‰
• Relatively depleted in
carbon-13
• C4 Calvin cycle
• 1% of plant species
• E.g. maize,cane,sorghum
• d values -9 to -14‰
• Relatively enriched in
carbon-13
C4 C4

69. 13
C isotope abundance (%)
carbonates
natural gas marine organisms marine HCO3
-
coal, oil atmosph. CO2
C3 plants C4 plants
CAM plants
terrestrial animals
| |
13
C‰ versus PDB
1.07
1.06 1.08 1.09 1.10
-50 -40 -30 -20 0
-10 +10
1.11 1.12
maize
cane
95% of all
plants
wheat
pasture
Increasing carbon-13 content

70. Role of Organic Matter in Agriculture
The study of soil organic matter (SOM) is becoming increasingly important
as world agriculture attempts to increase sustainability of soils.
The use of green manures, the return of residues to the soil, the use of
pasture leys and additions of organic amendments are often used in
attempts to increase SOM which can have large benefits on both the
chemical and physical fertility of the soil.
Carbon is the energy source, which drives many of the nutrient cycles that
occur in the soil.
A ready supply of accessible carbon is necessary for a continuous supply of
soil nutrients and to maintain soil structure
OM is very low in Bangladesh soils. It is less than 1%. A good soil should
have 2-5% OM.
Proper management of SOM is very urgent issue for increased crop
production as well as sustainable agriculture of the country.
13C isotopic technique is an unique tool for soil organic matter studies in
various soil pools.

71. Role of 13C in Organic Matter studies
•It can often be difficult to study the SOM pools and soil chemical and
physical fertility because of the large amount of background carbon
present in the soils.
•The use of carbon isotopes can provide an easy method of tracing the
additions of different plant materials to soil carbon fractions and its
influence on soil properties such as soil nutrients and soil structure.
•The 13C of SOM is comparable to that of the source plant material
(Schwartz et al., 1986).
•Thus every change in vegetation between C3 and C4 plants results in a
corresponding change in the 13C value of the SOM (Lefroy et al., 1995).
•This means that when C3 plants are grown in soils, which had previously
been under C4 vegetation (or vice versa) there is virtually an in situ
labeling of the organic matter incorporated into the soil.
•SOM, C mineralization, soil respiration etc. also can be studied with 13C
enriched organic materials applied into the soil .

72. Carbon isotopes varied in nature
• Ratio of 13C to 12C in the atmosphere can vary with altitude, latitude
and temperature as well as by some biological processes (Lefroy et al.,
1995).
• When plants fix carbon during photosynthesis there is a degree of
discrimination between the amount of 13C and 12C.
• Discrimination occurs during the carboxylation step in photosynthesis,
with greater discrimination against 13C in C3 (Calvin cycle) plants than
in C4 (Hatch-Slack cycle) plants, due to the greater discrimination in
the primary carboxylation step of C3 plants.
• This primary carboxylation step is catalysed by the enzyme ribulase
biphosphate carboxylase (RuBP) resulting in a lower 13C:12C ratio in
C3 plants than in C4.
• CAM plants (crassulacian acid metabolism) show variable
discrimination, but it is more often similar to C4 plants.
• The 13C:12C ratio is generally measured as 13C .
• A C4 species such as maize will have a 13C value of approximately –
12‰ whereas in a C3 species such as wheat or rice it will be
approximately –26‰.

73. MEASUREMENT OF 13C
13C is most often determined in CO2 produced from a solid sample
combusted in a stream of oxygen.
The two pieces of equipment most commonly used are the Leco and
Carlo-Erba furnaces linked to a mass spectrometer set to measure the
mass 45/44 ratio.
The results are expressed as 13C (‰), which is not the absolute isotope
ratio but that relative to a standard.
The original standard used as a limestone fossil of Belemnitella
americana (PDB) from the Cretanaceous Pee Dee formation in South
Carolina, USA.
Since this material is no longer available other standards which have
been cross calibrated are used.

74. Calculation of proportion of added residues
remaining in the soil
• The proportion of soil C derived from the C3 (or C4) plant can be
calculated from (Equation-1)
1
If the total carbon content C of the soil is known then the absolute
quantity X of carbon from the C3 (or C4) plants can be determined from
(Equation-2)
2
Eq.3
3

75. Example for calculation
= 0.27 x 100= 27% C derived from maize residue
C derived from maize leaf:

77. Production of 13C enriched plant materials
13C labelling techniques of plant.
• Plants can be grown in the greenhouse in pot containing 3 kg of quartz sand.
• The seedlings are watered with a nutrient solution.
• At sufficient growth stage, the plants are pulse labelled with 13C.
• This can be achieved by placing the plants in a gas tight Perspex air-conditioned chamber
(Figure 3), which contained a vial of concentrated lactic acid.
• Ten mL of 0.5 M 99 atom % 13C sodium bicarbonate solution can be added to the lactic
acid to release labelled CO2 ..
• Two and four hours after the initial injection 10 ml of unlabelled sodium bicarbonate
solution can be added to the acid, this ensure most of the labelled CO2 is taken up by the
plants.
• CO2 concentration is monitored throughout the labelling period using a conventional infra
red gas analyser.
• The labelling procedure is repeated twice weekly for two weeks and the plants harvested 3
days after the last injection.
• Plant material can separated in to leaves and shoots, dried at 70 °C and ground for 13C
mass spectrometry analysis (Optima, Micromass, UK).
• This 13C enriched material can be used for C mineralization or OM studies in soil

78. Dual labelling of cowpea plants with 13C and 15N
15N labeled from urea sol. in pot
13C labeling through generation of 13CO2 from NaH13CO3
injected into Lactic acid solution
Tent for 13CO2 feeding
Fig. 3. 13C labelling techniques
Injection port of chamber

79. 100
soil
to
added
residue
of
C
CO
respired
of
C
%Cdfr 2

 13
13


Cdfr= Carbon derived from residue
Carbon respiration study from soil or labeled residue
Figure 4 . Sampling jar with gas sampling ports

80. 2. TCEA/EA-CF IR MS:
 Thermo-Scientific
GV (before Micro Mass, UK)
In this method heavier and lighter isotopes will be separated
in a elctro-magnetic field, after it has been ionized.

89. THANKS