1. Deciphering the structure and function of complex microbial communities is a central theme in microbial ecology.

2. Stable Isotope Probing or MAR-FISH The addition of substrates labeled with stable or radioactive isotopes to microbial communities has been applied to the identification of microorganisms which consume specific compounds in the environment. Identification can be performed by using molecular tools after the separation of labeled DNA or RNA from unlabeled nucleic acids. The recently developed combination of FISH and microautoradiography (MAR) allows one to record the substrate uptake profiles of probe-identified prokaryotes at the single-cell level under different environmental conditions.

3. Microbial Respirometry Radiotracers, e.g. 3 H, 14 C and 35 S, quantify rates, but are also incorporated into biomass. Because they are incorporated into biomass they can be indirectly used to relate phylogeny to physiology. Radioactive tracers such as 3 H and 14 C also occur naturally.

4. Detection Limits 14 C total = 14 C respiration product + 14 C tracer contaminant + 14 C environmental background + counter background 100% PMC = 13.56 cpm/gC = 80  mol MC required for AMS 80  mol MC = 10 -4 cpm

5. 14 C environmental background in deep subsurface Steady-state 1% PMC CO 2 %PMC = P'e - dt CH 4 CH 3 COOH biomass CO 2

6. 35 S autoradiography – Krumholz et al. 1998  L's of 35 S SO4 dripped on to freshly fracture core Place Ag foil between core faces Incubate anaerobically at in situ temperature 35 S SO4 microbially reduced to 35 S AgS Image foil with phosphor image screen 35 S SO4 contaminant limits detection to 40 cpm vs. 25 cpm counter background by cold distillation process (Kallmeyer et al. 2004).

7. A. B. C. D.

8. DNA Microarray Technology The goal is to develop a method which allows simultaneous monitoring of the diversity and substrate incorporation by complex microbial communities with DNA microarray technology. Apply this method to subsurface environments where incorporation rates are slow.

9. Example: Ammonia Oxidizing Bacteria (AOM's) – Adamczyk et al. 2003 9 nCi of 14 C per  g of rRNA Extracted rRNA was fluorescently labeled 0.5  g of rRNA was hybridized to the DNA microarray with 125-  m diameter spots. Scanned with  -Imager for the individual spots but 14 C not detectable.

10. 14 C + DNA microarray CONT – no 14 C grown N. Eutropha Eubacterial probe Noneubacterial probe NONSENSE – nonsense probe Nso190+1225 - AMO probes NSV443 – Nitrospira probe NEU – Nitrosomonad probe <1% incorporation of 14 C into the rRNA Detection limit was 19,000 CPM per  g of rRNA 125  m 500  m 1000  m

12. rRNA Isotope Microarrays and Subsurface Environments Response time shorter for RNA than DNA RNA changes do not require growth Transfer of 14 C from one organism to another can be monitored over time

13. CCD w/ White LED's & UV laser fluorescence Avalanche Photodiode & pulsed white light Cintered Filter Array Borehole & Fracture Imaging Fluid & Particle Analyses Sample Retrieval & Storage Mini-syringe pumps for lysing solution and bioluminescent sensors : NAD/NADH 2 ATP CoM Borehole Instrumentation for Detection of Biosustainable Energy and Compounds 52 mm 360 o rotation Surface Spectroscopy Retractable Sampling Syringe Circulating Pump Fluid reaction chamber Diode detector array On board Computer & transformers pH, Eh, T, P, O 2 Sensor array (not shown) Fiber optic cable Microcanti lever array