SCIENTIFIC APPROACHES

While drilling cores for microbiology, contamination tests will be done routinely. Perfluorocarbon tracer (PFT) will be added to the drilling fluid, and the penetration of this tracer solute from the periphery towards the center of cores will be checked. Previous contamination tests combining PFT and bacterial-sized plastic beads have shown this contamination test to be efficient and to provide a useful indicator of potential bacterial contamination from the drilling fluid (Smith et al., 2000).

The study of deep subsurface microorganisms and their activity is a methodological and experimental challenge at the frontiers of modern marine microbiology and biogeochemistry. Many of the studies planned for this cruise are "first time" for ODP and overall for the deep biosphere, and methods and concepts will need to be further developed, refined, or even completely changed. The following selection of scientific approaches was developed on the basis of extensive discussions and experiences of many colleagues and should enable a major step towards our understanding of the deep biosphere. The approaches are, however, still very much in the development phase and will need refinement before they may be recommended for future routine application.

Enumeration, Identification, and Isolation of Microorganisms
Direct cell counts after fluorescent deoxyribonucleic acid (DNA) staining is well established as a method to obtain total numbers of intact cells (e.g., Parkes et al., 2000). The method allows also a description of cell sizes and morphologies and of the spatial structure of the community. Alternative staining techniques (e.g., to discriminate live and dead cells) will be considered.

DNA- and ribonucleic acid (RNA)-based techniques will be applied for the (postcruise) analysis of microbial diversity and activity. Samples will be taken and frozen (liquid N₂ and/or -86°C) for later DNA/RNA extraction and polymerase chain reaction (PCR) amplification and cloning (e.g., Rochelle et al., 1992). Sequence libraries will later be established based on 16S ribosomal DNA (rDNA) to provide a phylogenetic characterization of the microbial populations. This will be supplemented with a denaturing gradient gel electrophoresis (DGGE) separation and sequencing of dominant RNA molecules. Through the amplification and sequencing of selected messenger RNA molecules, the expression of certain key functional genes may be analyzed. Also real-time PCR may be used to quantify selected types of rRNA and messenger RNA (mRNA).

The microbial populations will also be fingerprinted (postcruise) by other molecular techniques, e.g., terminal-restriction fragment length polymorphism (T-RFLP) or membrane lipid biomarkers, including bacterial phospholipid fatty acids (PLFAs) and diverse archaeal lipids. The carbon isotopic composition of these biomarkers will be analyzed to identify the organic substrates of the corresponding microorganisms.

To obtain pure cultures of important microbial representatives, different methods of viable counts, enrichment, and isolation will be applied (and initiated on board) (e.g., Parkes et al., 1995). Through decimal dilutions in different growth media, MPN viable counts of the corresponding microorganisms will be made. Although such counts may greatly underestimate the true population sizes, the highest positive dilutions provide optimal material for a subsequent isolation of dominant members of the microbial communities. Because the subsurface microorganisms expectedly grow extremely slowly, the isolation of truly indigenous species may take several years. Alternative techniques, such as single cell isolation by laser tweezers, will also be considered.

The physiological types of microorganisms targeted for isolation will include sulfate reducers, methanogens, methane oxidizing consortia, fermenters, manganese reducers, and iron reducers.

Activity of Subsurface Microorganisms
Pathways and rates of microbial processes will be analyzed by different approaches. Among these, direct experimental measurements of specific processes will be done based on incubation of samples with radioactively labeled substrates. The application of radiotracers on the JOIDES Resolution is unique for Leg 201, and the prerequisite laboratory facilities, experimental procedures, and handling protocols are therefore described in some detail in a following section.

Experimental Estimation of In Situ Activities
Because of very low process rates and correspondingly slow turnover of electron acceptors or donors, it is not possible to experimentally determine the metabolic activity in subsurface sediments simply from changes in chemical concentrations over time. Instead, radioactively labeled substrates may be added in trace amounts to sediment samples, upon which the bacteria metabolize the radiotracer with the same relative turnover as the indigenous substrate. The sensitivity of process rate measurements may thereby be increased many thousand fold (Jørgensen, 1978; Whelan et al., 1986; Tarafa et al., 1987; Cragg et al., 1992).

Microbial processes that will be analyzed experimentally include sulfate reduction, methanogenesis, methane oxidation, and the turnover of selected small organic molecules such as acetate. Because of its universal and regulatory importance for anaerobic sediment metabolism, the turnover of tritiated H2 may also be studied. The radiotracer incubation experiments will be initiated as soon as possible after retrieval of core samples and will run during the duration of the cruise. Further experiments on the effect of temperature, pressure, or certain substrates will be continued postcruise. The processing of samples and determination of metabolic rates will be done postcruise because of the requirement for special radioisotope laboratory facilities.

Additional indicators of microbial activity will include the measurement of adenosine triphosphate (ATP). The concentration of ATP in individual cells is related to their metabolic rate.

Modeling of In Situ Activities
Major pore water constituents involved in biological processes include bicarbonate, ammonium, sulfate, methane, hydrogen sulfide, and manganese ions. Through the downcore analysis of these chemical species and diffusion diagenesis modeling of their production or consumption, the depth distribution of several primary microbial processes can be quantified. This approach, however, is dependent on the correct determination of transport coefficients of pore fluid and solutes. Some of the sites to be drilled on the Peruvian shelf are known to include a subsurface brine intrusion advecting offshore through a porous sediment interval. Also, the basement rock underlying the equatorial Pacific sites may be porous enough to allow significant fluid transport. The physical properties of cored sediments will therefore be closely related to modeled solute transports. A comparison of modeled and experimentally determined processes will improve the confidence in the resulting rates.

Geochemistry of the Deep Biosphere
Many dissolved constituents in the pore water are substrates or products of microbial metabolism and will be analyzed to determine these metabolic processes and their downcore changes. The analytical program will include a broad spectrum of inorganic anions and cations, dissolved gases such as CH₄, H₂S, and H₂, small organic molecules such as volatile fatty acids, as well as the total dissolved organic carbon (DOC).

The interactions between microbial processes in different sediment layers and the ambient sediment composition and geochemistry will be studied in particular for the biologically relevant elements of carbon, nitrogen, phosphorous, sulfur, iron, and manganese. The solid phase analyses will include the quantity and quality of organic material, organic carbon, and nitrogen, organic and inorganic phosphate, carbonates, sulfur-iron minerals, and metal oxides. Stable isotope analyses of carbon, nitrogen, and sulfur species will provide additional information on their original sources and their transformations at depth in the sediment. This information will be used to differentiate ongoing processes and to determine the role of depositional environment in the geological past for the present-day subsurface microbial activity.