Denitrification potential was measured as N2O production in FBOM slurries incubated in an AR atmosphere and amended with glucose and NaNO3 (Martin et al. 1988). Duplicate 5 mL FBOM slurry samples in 25 mL Erlenmeyer flasks were capped with rubber stoppers and purged for 3 minutes with argon at 120 cc/min. The flasks were gently shaken to remove air bubble and incubated at 24°C for 1h. After this initial incubation, 2 mL of a sterile 1 mM glucose and 1 mM NaNO3 solution was injected through the stopper and 2 mL of headspace removed. Incubation was continued at 24°C for an additional 3 h. After 1 and 3 hours, a gas chromatograph (GC) equipped with an electron capture detector was used to measure N2O concentrations.
Putative nitrogen fixation rates were measured by acetylene reduction (Weaver and Danso 1994). Samples were prepared as for denitrification except the headspace was replaced with 1.5% O2, 12.5% acetylene and 86% argon. After the samples had incubated for 24 h, ethylene concentrations were measured on a GC equipped with a flame ionization detector. A control without acetylene was analyzed for endogenous ethylene production.
Respiration was measured on duplicate 5 mL FBOM slurry samples in 25 mL Erlenmeyer flasks capped with rubber stoppers. Slurries were incubated at 24°C for 3 h. At 1 and 3 h., the headspace was analyzed for CO2 on a GC fitted with a thermal conductivity conductor.
s-glucosidase activities were measured using the spectrophotometric assay of Tabatabai and Bremner (1969), as modified by Zou et al. (1992). One mL of 10 mM p-nitrophenyl s-D glucopyranoside substrate was added to duplicate 1 mL subsamples containing suspended FBOM. The tubes were shaken and then placed in a 30°C water bath for 2 hours, along with duplicate controls with no s-glucosidase substrate addition. After incubating, 1 mL of 10 mM p-nitrophenyl s-D glucopyranoside was added to the controls and all reactions were immediately stopped with the addition of 0.5 mL of 0.5 M CaCl2 and 2 mL of 0.1 M tris[hydroxymethyl]aminomethane at pH 12.0. The mixtures were centrifuged for 5 minutes at 500 x g. From the supernatant, 0.2 mL of solution was diluted with 2.0 mL deionized water and the optical density measured at 410 nm. A standard curve was prepared from 0.02-1.0 Fmol mL-1 p-nitrophenol. Phosphatase followed the same general procedure as for s-glucosidase, except the substrate used was 1 mL of 50 mM p-nitrophenyl phosphate, incubation period was 1 hour, and 2 mL of 0.5 M NaOH instead of 0.1 M tris[hydroxymethyl]aminomethane, were added to terminate the reaction.
Mineralizable nitrogen measurements were determined using the 7 d anaerobic incubation method (Keeney 1982). Duplicate 10 mL FBOM samples in 50 mL screw-topped test tubes were filled to the top edge with deionized water, capped and incubated at 40°C for 7 d. After incubation an equal amount of 4 M KCl was added, shaken for 1 hour in the presence of 0.4 mL 10M NaOH and analyzed for NH4-N using a selective ion electrode (Corning ammonium electrode, Medford, MA). The value for extractable ammonium was subtracted from the total to account for background levels of ammonium prior to incubation.
Extractable ammonium was extracted by adding 50 mL of 2 M KCl to duplicate 10 mL samples in 250 mL Erlenmeyer flasks. Flasks were capped, shaken while incubating for 1 hour in the presence of 0.4 mL 10 M NaOH and analyzed using a selective ion electrode to determine KCl-extractable ammonium concentration.
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Bonin, L. H., R. P. Griffiths, and B. A. Caldwell. 2000. Nutrient and microbial characteristics of fine benthic organic matter in mountain streams. J. North Amer. Benth. Soc. 19:235-249.
Bonin, L. H., R. P. Griffiths, and B. A. Caldwell. Spatiotemporal nutrient distribution of fine particulate organic matter in low-order Oregon Cascade streams. Soil Biol. Biochem. (in review 5/01).
