Molecular Tools and Technologies

1. 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

2. 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

3. Need for Isotope monitoring on across multiple scales Candell et al., 2000 Ecosystems 3: 115-130

4. 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?

5. 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.

6. 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.

7. 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.

8. 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Stable Isotope Network A need for real-time and continuous isotope measurements: N2O Flux from the KBS LTER, Alfalfa (unpublished data S. Bohm and G.P. Robertson) Trace gas fluxes tend to be episodic - in situ instrumentation may be the only effective means to characterize flux Some trace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

9. 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 site:Implied relationship between Isotope values and nutrient concentrations

10. Examples of core measurements proposed as part of the NEON Stable Isotope Network Atmosphere Application CO2d13C, d18O anthropogenic input, water use efficiency N2Od15N, d18O, S.P. microbial sources H2Od18O, d2H regional climate change, hydrolgic cycle dO2/Ar net primary production Hydrosphere H2Od18O, d2H regional climate change, watershed change NO3-d15N, d18O sources, denitrification DOMd13C, d15N, d34S sources, biogeochemical cycling POCd13C, d15N, d34S sources, biogeochemical cycling O2d18O GPP, ratio of R to P Biosphere Tree ringsd13C and d18O Carbon sources, water use efficiency Miced13C, d15N C3 vs. C4, climatic induced diet changes

11. 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

12. 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

13. Examples of NEON Instrumentation: Secondary Ion Mass Spectrometry

14. 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

15. 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).

16. 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 >100,000 amu (atomic mass units), maintaining high mass resolution (mass/charge >1000), a high sensitivity of <1 ppb while consuming <10 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 50:50 DD water-ethanol.

17. Examples of NEON Instrumentation: Magnetic Sector with Microfarraday Array

18. Examples of NEON Instrumentation: Magnetic Sector with Microfarraday Array

19. 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