Molecular Tools and Technologies

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Molecular Tools and Technologies
Nathaniel E. Ostrom Dept. of Zoology and Center
for Global Change and Earth Observations,
Michigan State University Tim Filley Dept. of
Earth and Atmospheric Sciences, Purdue
University Stefan Scherer Dept. of Atmospheric,
Oceanic and Space Sciences, University of
Michigan
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Recommendations of NEON Stable Isotope Network
Meeting Sept. 16-17, Park City
Utah Development of a Stable Isotope
Network - what parameters should be
measured? - what parameters should the network
have the capacity to measure? - how often? -
laboratory vs in situ measurements - emerging
technologies and applications - quality control
(QA/QC) - instruction and interpretation -
focus on broad spatial and temporal scales
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Need for Isotope monitoring on across multiple
scales
Candell et al., 2000 Ecosystems 3 115-130
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Isotope monitoring on temporal scales Mauna Loa
CO2 record
NOAA Climate Monotoring and Diagnostics
Laboratory http//www.cmdl.noaa.gov/ccgg/iadv/
What measurements should we collect today that
will enable us to monitor ecosystem change over
the next 50 years?
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Isotope monitoring on temporal scales Net
Biological Productivity
- Seasonal changes in the concentration of
atmospheric CO2 shown as a function of 10o
latitudinal bands (Conway et al., 1988, Tellus
40B, 81-115.
- Seasonal changes in the O2/N2 ratio of air
reflects rates of Net Biological Production of
the southern hemisphere oceans. - Long-term
decliine in O2/N2 reflects consumption from
burning of fossil fuels.
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Use of stable isotopes in monitoring ecosystem
water and CO2 exchange
Advantage of Isotopes Unique labeling of flux
components - Photosynthesis tends to enrich the
atm. - Respiration tends to deplete atm. in 13C
and 18O - Leaf transpiration and soil
evaporation are isotopically distinct - Root and
Soil respiration can have distinct 13C values
Yakir and Sternberg, 2000 Oecologia 123 297-311.
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Extending the scale of stable isotope data from
patches to Landscapes
Aircraft
Keeling Plot Model - Reveals Isotopic
composition of CO2 source mixing with atm. -
application at multiple scales allows
apportionment to the landscape level
Flux Tower
Flux Chamber
Real-time and continuous monitoring
Flanagan and Ehleringer, 1998 TREE 13 10-14.
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Requirements and Structure of the NEON Stable
Isotope Network
A need for real-time and continuous isotope
measurements
N2O Flux from the KBS LTER, Alfalfa (unpublished
data S. Bohm and G.P. Robertson)
Trace gas fluxes tend to be episodic - in situ
instrumentation may be the only effective means
to characterize flux Some trace gases can not
be stored for later analysis - e.g. NO
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Isotope monitoring on spatial scales North
Pacific Transition Zone
GIS surface profile of nitrogen isotope data for
NRWD
P.H. Ostrom, unpublished data
Chlorophyll concentrations within the study
siteImplied relationship between Isotope values
and nutrient concentrations
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Examples of core measurements proposed as part of
the NEON Stable Isotope Network
Atmosphere Application CO2 d13C, d18O
anthropogenic input, water use efficiency N2O
d15N, d18O, S.P. microbial sources H2O d18O,
d2H regional climate change, hydrolgic
cycle dO2/Ar net primary production Hydrospher
e H2O d18O, d2H regional climate change,
watershed change NO3- d15N, d18O sources,
denitrification DOM d13C, d15N, d34S sources,
biogeochemical cycling POC d13C, d15N, d34S
sources, biogeochemical cycling O2 d18O
GPP, ratio of R to P Biosphere Tree rings
d13C and d18O Carbon sources, water use
efficiency Mice d13C, d15N C3 vs. C4, climatic
induced diet changes
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Requirements and Structure of the NEON Stable
Isotope Network
18 parameters x 15 sites x 40 subsites x 26
meas/yr 280,800 samples/yr At 5,000 samples/yr
the network needs approx. 60 mass spectrometers 4
mass spectrometers/site Cost approx. 15-30
million Does not include novel samples or
additional parameters likely to be
assumed Solution Each of the 15 NEON sites
needs a central mass spectrometry facility -
houses 4 to 6 mass spectrometers - centralized
training facility
Environmental Isotope Geochemistry Laboratory at
MSU
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Requirements and Structure of the NEON Stable
Isotope Network
National NEON Mass Spectrometry Facility -
houses large, expensive or novel
instrumentation - e.g. accelerator mass
spectrometry, SIMS - e.g. compound specific 14C
analysis - challenging or novel analyses -
e.g. organic compound specific d13C, d15N, d2H -
center for Quality Control and standardization -
national training facility - center for novel
instrument design
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Examples of NEON Instrumentation Secondary Ion
Mass Spectrometry
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Anaerobic oxidation of methane in ocean cold
seep environmentsOrphan et al., 2002

• Background
• Large reservoirs of methane exist dissolved,
  cyrstalized or as free gas beneath the seafloor.
  Little escapes to the oxic water column and is
  instead oxidized to CO2 by microbes in anearobic
  sediments. The amount of Anaerobic Methane
  Oxidation (AOM) is equivalent to 5 to 20 of the
  total methane flux to the atmosphere.
• No microbe capable of anaerobic growth solely on
  methane has been cultured.
• The specific pathways and microbes involved in
  AOM are not well understood.
• Recent isotope work on lipd biomarkers and 16S
  rRNA gene surveys suggest involvement of two
  archeal phylogenic groups ANME-1 and ANME-2
• ANME-2 found to be physically associated with
  with a sulfate-reducing partner (Fig. 2).
• Approach
• Sediments were collected from beneath
  chemosynthetic clam beds (Fig. 1) and bacterial
  mats from Eel River Basin cold methane seep.
• A combined approach using fluorescent in situ
  hybridization (FISH) and secondary ion mass
  spectrometry was used to identify microbial cells
  and obtain isotope data on individual cells

Fig. 1. Deep ocean cold seep environment showing
bed of Calyptogena sp. Clams.
Archaea
A
B
Sulfate Reducer
Fig. 2.   Individual cells and cell aggregates of
ANME-1 and ANME-2 archaea from ERB sediments,
visualized with fluorescent-labeled
oligonucleotide probes. (A) Color overlay of
archaeal ANME-1 rods visualized with the
ANME1-862 probe labeled with fluorescein (in
green), and Desulfosarcina spp. stained with the
DSS_658 probe labeled with Cy-3 (in red). (B)
Color overlay of a layered ANME-2/DSS aggregate
showing a core of ANME-2 Archaea (hybridized with
EelMSMX932 probe), surrounded by sulfate-reducing
Desulfosarcina (hybridized with DSS658 probe)
imaged by laser scanning confocal microscopy.
(Orphan et al., PNAS 99 7663-7668)
5 mm
5 mm
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Anaerobic oxidation of methane in ocean cold seep
environmentsOrphan et al., 2002

• Results
• Marked depletions in 13C were found in ANME-1 and
  ANME-2 cells providing strong evidence of
  utilization of 13C depleted methane by AOM (Fig.
  3)
• ANME-2 cells showed stonger depletions in 13C
  toward the centers of clusters with
  Desulfosarcina sp.
• Many unidentified bacteria and a diatom cell
  exhibite normal 13C enriched values indicating
  plankton (photosynthesis) derived carbon
• Other bacteria exhibited isotope values
  intermediate between methane and plankton values
• Conclusion
• ANME-1 and ANME-2 definitively carry out AOM in
  cold seep environments. This process, however,
  is not restricted to clusters with sulfate
  reducing bacteria. The observation of 13C values
  between methane and plankton suggests that other
  microbes within the seep community beneifit
  indirectly from the AOM process.

Fig. 3.   Ion microprobe measurements of 13C
profiles for individual cells and cell aggregates
recovered from methane-seep samples underlying
clams or bacterial mats (PC-21 and PC-45). The x
axis represents the time course Cs ion beam
exposure for each individual cell profile.
Individual cell profiles are indicated by a line
connecting the 13C values measured over time
during Cs ion-beam exposure. Dashed lines show
13C values for DIC and methane in sample PC-21 as
indicated. Mono-species ANME-2 aggregate (no.
1). ANME-2/DSS aggregates (nos. 2 and 3).
Individual ANME-1 rods (nos. 4-7 and 9-13).
ANME-1 rod aggregates (nos. 8 and 14). Bacterial
filaments hybridized with general bacterial
oligonucleotide probe Eub338 (nos. 15-23).
Unidentified microbial aggregate stained with
DAPI (nos. 24 and 25). Diatom frustule (no. 26).
The analytical precisions shown (1 ) are
appropriate within each depth profile, but do not
account for the uncertainty in the calibration of
the y axis (5 in this case).
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Examples of NEON Instrumentation In situ Mass
Spectrometry
Mass SURFER test deployment
Mass SURFER a robust instrument that
can function for long periods at water depths as
deep as 4000m. The RFMS has the distinction of
ranging in mass detection from 1 to gt100,000 amu
(atomic mass units), maintaining high mass
resolution (mass/charge gt1000), a high
sensitivity of lt1 ppb while consuming lt10
watts power.
Above is a Lysozyme run, mixed in ethanol, 10-6
molar. The lower image shows the linearity of
RFMS over wide range of concentrations for
lysozyme. Dilutions were prepared in 5050 DD
water-ethanol.
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Examples of NEON Instrumentation Magnetic Sector
with Microfarraday Array
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Examples of NEON Instrumentation Magnetic Sector
with Microfarraday Array
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Examples of NEON Instrumentation Spectroscopic
Approaches to Stable Isotopes
CO2
13C-CO2
18O-CO2

• Tunable diode laser absorption spectroscopy
• 13C-CO2, 18O-CO2, 18O-H2O
• Laser Isotope Spectroscopy for 13CO2
• Fourier-transform infrared spectroscopy
• 15N, 18O-N2O