All experiments were performed by incubating soils in gas-tight containers with and without acetylene gas (10% by volume) and measuring the increase in nitrous oxide (N2O) in the headspace over time. Rates of N2O production without acetylene reflected N2O production, while rates with acetylene, which inhibits the reduction of N2O to N2, reflect rates of denitrification (defined here as N2O + N2 production). Each treatment was run in triplicate (first and second experiments) or with four replicates (third experiment). Rates of N2O production were determined by the increase in N2O over time, and the linear rate of increase is reported as umol gws-1 d-1. The ration of N2O production to denitrification (N2O:DNF ratio) was determined on a per mole basis.
Experiment 1: Surficial (0-2 cm) agricultural soils were collected from organically farmed and conventionally farmed agricultural fields in Lancaster, PA and exposed to gradients in five physicochemical parameters. Soils were returned to the laboratory and homogenized. For each treatment, about 20 g of fresh soil was placed into each of six 410 mL gas-tight incubation jars, amended as appropriate, flushed with nitrogen gas to remove oxygen, and acetylene was added to 3 of the 6 jars (to 10% volume). Jars were then incubated for between 12 and 24 hours, with 10 mL headspace samples removed periodically to determine the change in nitrous oxide concentration. N2O was determined within one day by gas chromatography (Agilent Technologies 6850 Series II with electron capture detector). Salinity was amended by adding 10 mL of water of varying salinities to each incubation jar made by diluting 30 g kg-1 seawater (made from by adding 350 mM NaCl, 45.5 mM MgCl2, 24.2 mM Na2SO4, 8.9 mM CaCl2, 2 mM NaHCO3, and 0.5 mM KCl from salts) with deionized water for salinities of 0, 1, 3, 5, 10, and 30 g kg-1. Zinc was amended by adding 10 mL of 0, 0.05, 0.1, 0.25, 0.5 or 1.0 g Zn L-1 (made by diluting a zinc chloride solution with deionized water). Temperature treatments (20, 30, 37, 43, and 52°C) were achieved by adding 10 mL of deionized water to each jar and incubating them at various temperatures. The pH was amended by adding 10 mL of water to each jar with pH values that raised or lowered the ambient soil pH by +3, +2, +1, 0, -1, -2, and -3. Moisture treatments were achieved by air-drying the soil for several days and adding deionized water to 20 g of soil in jars to achieve 0.0, 0.05, 0.09, 0.17, 0.33, and 0.50 g water g-1 soil (weight:weight). Each jar also received 1 mM nitrate and 2 mM glucose. A total of 360 incubations were performed in the first experiment.
Experiment 2: Estuarine soils were collected from three sites along the salinity gradient in the Scheldt River estuary Belgium/Netherlands. Soil cores were sectioned and the surface section (0-2 cm) from several cores taken at each site was homogenized and 2 g of fresh sediment was placed into six 38 mL headspace vials. 10 mL of varying salinity water (achieved by mixing 0.7 um filtered site water collected from the tidal freshwater site and from the ocean near the laboratory) was added to each vial to achieve salinities of 0 (ambient), 1, 3, 5, 10, 15, and 30 g kg-1 for the tidal freshwater sediment, 0, 1, 3, 5 (ambient), 10, 15, and 30 g kg-1 for the oligohaline sediment, 0, 3, 5, 10, 24 (ambient), and 30 g kg-1 for the mesohaline sediment. 2 mM nitrate and 4 mM glucose was added to each jar, the headspace was purged with helium to remove oxygen, and acetylene (10% final volume) was added to the headspace of 3 jars from each treatment. Jars were incubated for about 1 day with headspace samples removed into separate, evacuated headspace vials for determination of N2O concentration. N2O was analyzed on a Shimadzu GC8 gas chomatograph with electron capture detection.
Experiment 3: Surficial (0-2 cm) tidal freshwater soils from the Rancocas River, NJ were collected, homogenized, and 0.75 L was placed into four large 4 L flasks. Each flask received 0.75 L of either deionized water (S=0 press treatment) or water of a salinity of 20 g kg-1 (S=20 press treatment) made from salts as described above), flushed with nitrogen to remove oxygen, and incubated for six months under gentle mixing. Each flask was amended with 0.4 mM nitrate and 0.8 mM glucose weekly to avoid substrate limitation. A 200 mL subsample of the soil slurry was removed from each flask on days 0, 7, 14, 21, 35, 49, 70, 110, and 181 and split (10 mL each) into 20 gas tight headspace jars (410 mL). The headspace jars were amended with 10 mL of water of various salinities to achieve 0.0, 4.3, 7.6, 16.9, and 25.6 g kg-1 salinity pulse treatments for the S=0 press treatment (four jars of each) and 3.5, 7.4, 11.6, 20.0, and 28.3 g kg-1 for the S = 20 treatment (four jars of each). The differences in final salinity reflect the difference in press treatment starting salinity. The vials were amended with 0.4 mM nitrate and 0.8 mM glucose, capped, and purged with nitrogen gas. Acetylene was added to two of the jars to achieve 10% by volume, and the jars were incubated at room temperature for < 1 day with subsamples of the headspace removed (10 mL in gas tight syringe) several times during the timecourse. N2O was analyzed by gas chromatography within 1 day of collection on an ilent Technologies 6850 Series II with electron capture detector. On days 7, 35, and 110, the soils that had been incubated without acetylene were frozen at -80 degrees for determination of functional gene expression. DNA was extracted using the MoBio PowerSoil DNA isolation kit and RNA was extracted following Q Sepharose chromatography optimized for soils with high humic acid content. The RNA was reverse transcribed to cDNA with Invitrogen SuperScript III. DNA and cDNA was quantified using Quant-iT PicoGreen and RiboGreen, respectively. Nucleic acids were normalized to 3 ng μL-1 and functional genes were quantified via quantitative PCR (qPCR) on a Stratagene MX-3005p thermocycler using nirS primers from Braker et al. (1998) and nosZ primers from Henry et al. (2006). Gene expression was defined as the cDNA:DNA ratio for each functional gene.
Braker, G., Fesefeldt, A., and Witzel, K. P.: Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples, Applied and Environmental Microbiology, 64, 3769-3775, 1998.
Henry, S., Bru, D., Stres, B., Hallet, S., and Philippot, L.: Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils, Applied and Environmental Microbiology, 72, 5181-5189, 10.1128/aem.00231-06, 2006.
