Sample collection, preparation, and storage:
FBOM was collected from stream beds with a hand vacuum pump into a 2 L collecting jar. The intake line was fitted with a 1 mm stainless steel screen, allowing benthic material to be wet-sieved during sampling. Samples were transferred to polystyrene jars (500 ml) and stored during field sampling in an insulated chest with stream water and ice. In the laboratory, a slurry was prepared by decanting excess stream water from the jars and mixing, keeping FBOM suspended while subsampling. Subsamples were dispensed using 1, 3, or 5 ml plastic syringes with enlarged openings. All laboratory analyses involving slurry began immediately upon return from FBOM collection which took 8-12 hours.
Because of significant perturbation (e.g., suction, sieving, mixing, and preparing slurries) of FBOM during sampling and subsampling, we performed laboratory analyses described in the following sections to measure potential activity rates. These rates do not reflect in-stream FBOM activity rates, but rather give us a relative sense of the variability of rates among treatments and over time.
Denitrification potential rates
Denitrification potential was determined as N2O emission by anaerobically incubating FBOM supplemented with glucose and NaNO3 (Martin et al., 1988). Duplicate 5-ml slurry samples in 25-ml Erlenmeyer flasks were capped with rubber stoppers and purged for 3 min with argon at a rate of at least 120 cc /min. In the middle of the purge, the flasks were shaken gently to ensure removal of air bubbles. The flasks were allowed to incubate for 1 h at 24 degrees C, and then 2 ml of sterile solution of 1 mM glucose and 1 mM NaNO3 were injected through the stopper, and 2 ml of headspace gas were withdrawn with the syringe. Flasks were allowed to incubate for another hour at 24 degrees C. At zero and 2 h time, 0.5 ml of headspace gas was removed from the flasks and assayed for N2O in a gas chromatograph equipped with an electron capture detector. All gas chromatographs had stainless steel columns packed with Poropak-Q, either 50/80 or 80/100 mesh (Water Associates, Inc., Medford, MA).
Respiration rates
To determine respiration rates, duplicate 5-ml subsamples were placed in 25-ml Erlenmeyer flasks, and then capped with rubber stoppers. After incubating for 1 h at 24 degrees C, 0.5 ml of headspace gas was analyzed for CO2 in a gas chromatograph fitted with a thermal conductivity conductor. After incubating for an additional 2 h under the same conditions, a second headspace gas sample (0.5 ml) was collected and analyzed.
Acetylene reduction rates
Putative acetylene reduction rates were determined by the acetylene reduction method (Weaver and Danso, 1994). Samples were prepared in the same way as for denitrification potential, except that the headspace gas contained 1.5% O2, 12.5% acetylene, and 86% argon. After 24 h of incubation, 0.5 ml of headspace gas was removed and analyzed for ethylene in a gas chromatograph fitted with a flame ionization detector. A control was analyzed for ethylene production in the absence of acetylene.
Mineralizable nitrogen and extractable ammonium concentrations
For mineralizable nitrogen measurements, the microbial conversion of organic N to inorganic N in the form of NH4-N was measured by the "anaerobic" incubation method (Koeney, 1982). Duplicate 10-ml sediment subsamples were added to 50-ml screw-topped test tubes, which then were completely filled with deionized water (53 ml), capped, and incubated at 40 degrees C for 7 d. After incubation, subsamples were transferred to 25-ml Erlenmeyer flasks, and 53 ml of 4 M KCl and 0.4 ml of 10 M NaOH were added to each. The flasks were shaken for 1 h, and then analyzed with a selective ion electrode (Corning ammonium electrode, Medford, MA). Extractable ammonium concentrations were measured by adding 50 ml of 2 M KCl to duplicate 10-ml subsamples in 250-ml Erlenmeyer flasks. The flasks were capped, shaken while incubating for 1 h in the presence of 0.4 ml 10 M NaOH, and analyzed with a selective ion electrode to determine KCl-extractable ammonium concentration. Net mineralization was calculated as mineralizable N ( extractable ammonium to account for ammonium present prior to incubation.
Enzyme activities
Phosphatase activity was determined according to the spectrophotometric assay of Tabatabai and Bremner (1969) as modified by Zou et al. (1992) One ml of 50 mM p-nitrophenyl phosphate substrate was added to duplicate 1-ml subsamples containing suspended FBOM. The tubes were shaken and then placed, along with duplicate controls with no phosphatase substrate addition, in a 30 degrees C water bath for 1 h. After incubation, 1 ml of 50 mM Sigma 104 phosphatase substrate was added to the controls; reactions were stopped immediately with the addition of 2 ml 0.5 M NaOH and 0.5 ml 0.5 M CaCl2. The assay mixtures were centrifuged for 5 min at 500 x g. A 0.2-ml subsample of the supernatant was diluted with 1.8 ml deionized water, and the optical density was measured at 410 nm. A standard curve was prepared from 0.02 to 1.00 ?mol ml-1 p-nitrophenol. Enzyme activity was expressed as ?mol p-nitrophenol released gdw-1 h-1. The B-glucosidase activity assay required the same general procedure as was used for phosphatase activity, except that the substrate was 1 ml 10 mM p-nitrophenyl ?-D glucopyranoside, the incubation period was 2 h, and 2 ml 0.1 M tris[hydroxymethyl]aminomethane at pH 12.0, instead of 0.5 M NaOH, were added to terminate the reaction.
Total carbon and nitrogen
Total C and N were determined by dry combustion with a Carlo-Erba NA 1500 Series II high-temperature combustion furnace on oven-dried subsamples ground to pass through a 250-mm sieve.
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