Karen Casciotti

Woods Hole Oceanographic Institution

Stable Isotopes and Microbial

Biogeochemistry

Part 1:
Introduction to isotopes and
applications using isotope tracers.
Sea Surface Chlorophyll

SeaWiFS annual average chlorophyll

Global sea surface nitrate distribution

Data from World Ocean Atlas 2005 plotted in ODV

 Marine Nitrogen Cycle
Atmospheric deposition
Atmospheric N2 CO2 N2O and continental inputs

N2O
Dissolved N2 Particulate NH4+
Nitrogen Fixation N DON
Assimilation NO3-
Surface ocean
Deep ocean Vertical mixing/
upwelling
Nitrification
Remineralization NH 4+ NO3- Sedimentary
N2O Denitrification
and Anammox

Water column
Denitrification
and Anammox

Sediment Burial
 Marine Nitrogen Cycle
Atmospheric deposition
and continental inputs

N2 Nitrogen Particulate
Fixation N
Surface ocean
Deep ocean
NH 4+ NO3-
Sedimentary
Denitrification
Water column and Anammox
Denitrification
and Anammox

Input/Output
Sediment Burial
Processes
 Marine Nitrogen Cycle
Atmospheric deposition
and continental inputs

Nitrogen N2O
N2 Fixation Particulate NH4 +

N
DON
Assimilation NO3-
Surface ocean
Deep ocean Nitrification Vertical mixing/
NO3- upwelling
Sinking/ NH 4+
Sedimentary
Remineralization Denitrification
and Anammox
Water column
Denitrification
and Anammox

Sediment Burial Internal cycling

 Some underlying questions…

• What forms of nitrogen support phytoplankton growth in

the oligotrophic ocean?

• How do phytoplankton communities adapt to different

nutrient supplies?

• How is the inventory of oceanic nitrogen regulated?

• What are the feedbacks between chemistry, climate, and

microbial activities?

• Who is there and what are they doing?

 Overview of Lecture 1

• Introduction to stable isotopes

– What are isotopes
– Why are they useful/interesting

• Applications of tracer-level isotopes to microbial

biogeochemistry:
– Bulk rate measurements
– Metabolic probing with stable isotopes
– Taxon-specific rate measurements
 Overview of Lecture 2

• Applications of natural abundance isotopes

to microbial biogeochemistry:
– Isotopic fractionation
– Natural variations in isotope ratios--record a
history of microbial, chemical, and physical
processes
– Applications and interpretations
• Example 1: Global N budget
• Example 2: Sources of N to the euphotic zone
What are isotopes?
What are isotopes?

14
N
7P
7 e-
7N

15
N
7P
7 e-
8N
 What are isotopes?

• Stable vs. radioactive 12

C: 98.90 %
13
C: 1.10 %
14
C: 10-10 %

14
N: 99.63 %
15
N: 0.37 %

16
O: 99.76 %
17
O: 0.038 %
18
O: 0.20 %
 Notation

• 15
N: The isotope, its molar amount or concentration

• 15
F: Fractional abundance = 15N/(14N+15N)
• 15Atom % = 15F x 100%

• 15
R: Isotope ratio = 15N/14N

• 15N: Delta value (‰) = (15R/15Rstd -1) x 1000

 Isotopes of nitrate

16
O - 16
O - 16
O -

14
N 15
N 14
N

16
O 16
O 16
O 16
O 18
O 16
O

14
NO3- 15
NO3- N16O218O-
 Isotope Tracers

N2 N2 N2

NH4+ NH4+ NH4+

NO3- NO3- NO3-
15
NO3-
15
NO3- 15
NO3-
 Stable isotope measurements

Isotope ratios are measured by mass

spectrometry:

• Gas sample ionized in the ‘ion source’

• Ions accelerated into the ‘flight tube’

• Separation by mass/charge ratio

• Detection
Stable isotope measurements
 Online sample purification for N2O

He, N2O,
H2O, CO2 Open split
Volatile organics He, N2O
He

GC
Mg(ClO4)2
CarbaSorb
Sample -60º C
-60º C LN2 LN2
Nafion

vial

EA combustion to N2: 1-5x10-9 moles of N Mass Spec

N2O GC-IRMS: 2-10 x 10-9 moles of N
 Overview of Lecture 1

• Introduction to stable isotopes

• Applications of tracer-level isotopes to
microbial biogeochemistry:
– Bulk rate measurements
– Metabolic probing with stable isotopes
– Taxon-specific rate measurements
 ‘New Production’
Atmospheric deposition
N2 CO2 and continental inputs

N2 Biomass NH4+
DON
NO3-
Surface ocean

NO3-
Export of C, N

Deep ocean
 Bulk rate measurements

Concentration
NO3-
NO3-
ChlA
Nitrate Assimilation

Time
 Bulk rate measurements

N2

Concentration
NH4+
NO3-
ChlA
Nitrate Assimilation
Nitrogen Fixation
Ammonium Assimilation
NO3-
Ammonification
Nitrification
Time
 Bulk rate measurements

15
At%
N2 15
Atom % PN

Concentration
NH4+
15
NO3-
ChlA
Nitrate Assimilation
Nitrogen Fixation
Ammonium Assimilation NO3-
Ammonification
Nitrification
Time
 Bulk rate measurements

15
At%
N2 15
Atom % NO3-

Concentration
NH4+
15
NO3-
15
Atom % PN

Nitrate Assimilation Rate PN

from 15N incorporation:
• Directly proportional to NO3-
15
At% NO3-
• Inversely proportional to
[PN] Time
 Bulk rate measurements

15
At%
N2
15
Atom % NO3-

Concentration
NH4+
15
NO3-
15
Atom % PN

Nitrate Assimilation Rate PN

from 15N incorporation:
• Directly proportional to NO3-
15
At% NO3-
• Inversely proportional to
[PN] Time
 Bulk rate measurements

15
At%
N2 15
Atom % NO3-

Concentration
NH4+
15
NO3-
15
Atom % PN

Nitrate Assimilation PN
Nitrogen Fixation
Ammonium Assimilation NO3-
Ammonification
Nitrification
Time
 Bulk rate measurements

15
At%
N2 15
Atom % NO3-

Concentration
NH4+
15
NO3-
15
Atom % PN

Nitrate Assimilation PN
Nitrogen Fixation
Ammonium Assimilation NO3-
Ammonification
Nitrification
Time
 New Production
Atmospheric deposition
N2 CO2 and continental inputs

N2 Biomass NH4+
DON
NO3-
Surface ocean

NO3-
Export of C, N

Deep ocean
 Bulk rate measurements

15
At%
N2
15
Atom % NO3-

Concentration
NH4+ 15
Atom % PN
NO3-
15
Atom % PN

Which organisms were PN

responsible for the uptake?
NO3-
15
Atom % PN
What forms of N are different
members of the community
using? Time
 Overview of Lecture 1

• Introduction to stable isotopes

• Applications of tracer-level isotopes to
microbial biogeochemistry:
– Bulk rate measurements
– Metabolic probing with stable isotopes
– Taxon-specific rate measurements
 Approaches

• Sort the whole cells or sort

the biomarkers…
DNA • Uptake of isotope label
Lipids RNA from substrates into
Pigments biomarkers:
Proteins – Lipids
– DNA
– RNA
Substrates* Products – Pigments
– Proteins
• Separation and analysis of
isotopically labeled
biomolecules
 Stable Isotope Probing (SIP)
• Sample is incubated with heavy
DNA* isotope labeled substrate
Lipids RNA*
Pigments • DNA or RNA is extracted after
Proteins incubation

• ‘Heavy’ nucleic acids are

Substrates* Products separated from ‘light’

• ‘Heavy’ nucleic acids are

interrogated by:
– PCR
– Fingerprinting (T-RFLP,
DGGE)
– Cloning
– Sequencing
 SIP Example 1:
Probing with 13CH3OH
Separate
Extract by
DNA density

13
CH3OH

Remove
fractions
Interrogate
DNA fractions

Neufeld et al., 2008 Environmental Microbiology

 SIP Example 1:
Probing with 13CH3OH
Separate
Extract by
DNA density

12
CH3OH

Remove
fractions
Interrogate
DNA fractions

Neufeld et al., 2008 Environmental Microbiology

 SIP Example 1:
Probing with 13CH3OH
“Interrogate DNA fractions”

• 16S rRNA DGGE of all

fractions from 13CH3OH
(experimental) incubations
and 12CH3OH (control)
incubations

• Excise, reamplify, and

sequence unique bands

Neufeld et al., 2008 Environmental Microbiology

 SIP Example 1:
Probing with 13CH3OH
“Interrogate DNA fractions”
• Whole-genome amplification of high-density DNA
• Fosmid library construction with amplified DNA (17-40 kb
fragments)
• Screened for methanol dehydrogenase genes.
• Shotgun sequenced fosmid containing genes of interest.
• Identified Methylophaga

Neufeld et al., 2008 Environmental Microbiology

 SIP Example 2:
15
N-DNA SIP
• DNA has less N than C
– Lower resolving power
Low density – 15N gradients: 0.016 g ml-1
– 13C gradients: 0.036 g ml-1
– G+C gradients: 0.050 g ml-1
Increasing Increasing
G+C 15
N (or 13C)
content content

High density

Buckley et al., 2007a Applied and Environmental Microbiology

 SIP Example 2:
15
N-DNA SIP
• DNA has less N
– Lower resolving power
Low density – 15N gradients: 0.016 g ml-1
– 13C gradients: 0.036 g ml-1
– G+C gradients: 0.050 g ml-1
Increasing Increasing
G+C 15
N (or 13C)
content content

High density

Buckley et al., 2007a Applied and Environmental Microbiology

 SIP Example 2:
15
N-DNA SIP

Increasing Increasing
G+C 15
N (or 13C)
content content Increasing Increasing
G+C 15
N (or 13C)
content content

Secondary density gradient

With bis-benzimide (AT intercalating agent)
Separates according to G+C content

Buckley et al., 2007a Applied and Environmental Microbiology

 SIP Example 2:
Probing nitrogen fixing communities
Separate
Extract by
DNA density Secondary
Density
15
N2 separation

Phylogenetic
information =
Discovery of
novel diazotrophs

Buckley et al., 2007b Applied and Environmental Microbiology

 SIP Strengths and Limitations

• Probing the unculturable (and unpredictable) for metabolic

function

• Assimilatory processes

• Large isotopic enrichments needed

– Lots of substrate added
– Long incubations
– Cross-feeding

• G+C content
 Overview of Lecture 1

• Introduction to stable isotopes

• Applications of tracer-level isotopes to
microbial biogeochemistry:
– Bulk rate measurements
– Metabolic probing with stable isotopes
– Taxon-specific rate measurements
 Taxon-specific activities
• Sample is incubated with
isotopically labeled substrate

DNA* • Cells sorted physically or

Lipids* RNA* specific RNA molecules
Pigments* captured
Proteins*
• Isotopic content of isolated
cells or molecules are
Substrates* Products determined by:
– Mass spectrometry (stable
isotopes)
– Scintillation counting
(radioisotopes)
– SIMS?
Taxon-Specific Activity Example 1:
N uptake by Prochlorococcus
FLOW-SIP:
Community incubation
with 15N-substrates
Flow-cytometric cell
sorting
Isotopic analysis of
sorted cells by
EA/IRMS
Taxon-specific N uptake
rates.
Casey et al., 2007 Geophysical Research Letters
 Taxon-Specific Activity Example 2:
N limitation of Prochlorococcus
Initial
32
P 32
P
Extract 32
P
RNA 33
P 32
P
33
PO43- 33
P
33 P
33
P

Final
32
P
33
P
P 33 P
32

P
33
P

32
Van Mooy and Devol, 2008 Limnology and Oceanography
 Taxon-Specific Activity Example 2:
N limitation of Prochlorococcus
Results:
• NH4+ enhanced
– total 33P uptake
– RNA synthesis
rates
– Pro. -specific
RNA synthesis
rates
• PO43- and NO3-
did not

Van Mooy and Devol, 2008 Limnology and Oceanography

 Taxon-Specific Activity Example 2:
N limitation of Prochlorococcus
Initial
32
P 32
P
Extract 32
P
RNA 33
P 32
P
33
PO43- 33
P
33 P
33
P

Final
32
P
33
P
P 33 P
32

P
33
P

32
Van Mooy and Devol, 2008 Limnology and Oceanography
 Secondary Ion Mass Spectrometry
(SIMS)

Secondary Ions 12
C14N-, 12C15N-
Primary Ion Beam

~ 50,000 “ion counts”

At about 2% ionization
efficiency and 80% ion
transmission->
~ 5x10-18 moles of N

Courtesy of Francois Herreard, CAMECA

Current Work: The Stable Isotope Array

• Massively parallel
15
N2 RNA capture
15
NO3- technique
15
NH4+
15
N 15
Norg
• High resolution, high
Extract RNA sensitivity 15N/14N or
13
C/12C analyses by
Hybridize to microarray (Nano)SIMS

Measure
15
N/14N
 New Production
Atmospheric deposition
N2 CO2 and continental inputs

N2 Biomass NH4+
DON
NO3-
Surface ocean

NO3-
Export of C, N

Deep ocean
 References and Further Reading

• Neufeld et al., 2008 Environmental Microbiology 10: 1526-1535

• Whitby et al., 2001 Letters in Applied Microbiology 32: 398-401

• Buckley et al., 2007a Applied and Environmental Microbiology 73:

3189-3195

• Buckley et al., 2007b Applied and Environmental Microbiology 73:

3196-3204

• Casey et al., 2007 Geophysical Research Letters 34: L10604

• Van Mooy and Devol, 2008 Limnology and Oceanography