General Sampling Scheme: Subcores will be taken from boxcores and multicores and immediately transferred to a refrigerator or ice tray. The upper 5 cm of sediment will be sampled at 0.5 cm intervals. Between 5 and 10 cm, samples will be collected at 1.0 cm intervals. Finally, a deep horizon, 14--15 cm, will be sampled. Measurements will be normalized to the dry weight of the sediment sample.
I will use phospholipid, ester-linked fatty acids to assess community composition, as they have proven to be reproducible, capable indicators of a wide suite of microbes encountered in the environment. The fatty acids esterified to the phospholipid will be methylated by mild alkaline transesterification of the polar lipid fraction (Guckert et al., 1985). The resulting fatty acid methyl esters will be separated and quantified (Guckert et al., 1985). Structural verification will be done using a HP5995A capillary gas chromatograph/mass spectrometer. Fatty acid profiles will be expressed in absolute values (pmoles/gram dry weight of sediment) and as percentages of the total molar recovery.
I will assess microbial utilization of carbon at three stations: 0, 5, and 9N. These stations encompass the range of organic-carbon degradation rates reported by Martin et al. (1990). Sediment will be homogenized, then diluted with filtered (0.2 µm) seawater from the box core or multicore. Aliquots of the slurry will be withdrawn into 3-ml syringes containing a C-glutamic acid/seawater solution, the needles stuck into rubber stoppers, and the syringes placed in cylinders. The cylinders will be pressurized commensurate with the depth at which the sediment was collected. Time-course measurements will be made; samples will be decompressed at 24, 48, 96, and 144 hours and their contents processed as follows. The syringes' contents will be injected into serum bottles containing acid and CO will be collected. Macromolecules will be precipitated with cold acid and collected on membrane filters, which will be dried, then oxidized prior to radioassay (Novitsky and Karl, 1986). Results of the CO assay will represent respiration, those of the macromolecule assay, assimilation. A battery of control samples will be tested for effects of depressurization, abiotic uptake of radioactivity, stimulation of activity by the glutamic acid, and effects of slurrying on microbial activity.
I will use radiolabeled nucleic-acid precursors to estimate growth and production of the microbial community. Growth will be evaluated from the turnover of the adenine nucleotide pool. Production will be estimated based on the incorporation of radiolabel from H-adenine into microbial RNA and DNA. These measurements are interrelated when H-adenine is the precursor; measurement of biomass and production can be made contemporaneously. A bonus to using H-adenine to label DNA is that RNA also is labeled; the ratio of labeled DNA to labeled RNA will be used to monitor the growth stage of the microbial populations.
Sediments will be processed as described above for measurements of carbon-utilization rates, except that the added radioactive material will be 2-H-adenine. Once the samples are pressurized, time-course measurements will be made; samples will be decompressed at 24, 48, 96, and 144 hours and H-RNA, H-DNA, H-ATP and ATP will be extracted and radioassayed. Macromolecules will be collected by filtering the contents of a syringe, then washing the filter with cold acid. Radioactive RNA and DNA will be separated and measured using the technique of Craven and Karl (1984) for marine sediments. Both labeled and unlabeled ATP will be extracted as described above in the ``Biomass'' section. Thus, the specific activity of the ATP pool at each time point will be known and the turnover of the adenine nucleotide pool can be determined (Karl and Bossard, 1985; Karl et al., 1987). Rates of RNA and DNA synthesis will be calculated from the average rate of isotope incorporation divided by the integral of the ATP pool specific activity (Winn and Karl, 1984). Controls analogous to those described for glutamic acid will be run and HO produced through catabolism of radiolabeled adenine will be measured.
There is reason to consider that the disturbance caused by slurrying sediment may yield artifactual data. To evaluate this possibility, I will make a series of comparisons, comparable to those described in Dobbs et al. (1989), between results obtained from slurries and from cores.