High rates of biological nitrogen fixation (BNF) are
commonly reported for tropical forests, but most studies have been conducted in
regions that receive substantial inputs of molybdenum (Mo) from atmospheric
dust and sea-salt aerosols. Even in these regions, the low availability of Mo
can constrain free-living BNF catalyzed by heterotropic bacteria and archaea.
We hypothesized that in regions where atmospheric inputs of Mo are low and
soils are highly weathered, such as the southeastern Amazon, Mo would constrain
BNF. We also hypothesized that the high soil acidity, characteristic of the
Amazon Basin, would further constrain Mo availability and therefore soil BNF.
We conducted two field experiments across the wet and dry seasons, adding Mo,
phosphorus (P), and lime alone and in combination to the forest floor in the
southeastern Amazon. We sampled soils and litter immediately, and then weeks
and months after the applications, and measured Mo and P availability through
resin extractions and measured BNF by the acetylene reduction assay. The
experimental additions of Mo and P increased their availability and the lime
increased soil pH. While the combination of Mo and P increased BNF at some time
points, BNF rates did not increase strongly or consistently across the study as
a whole, suggesting that Mo, P, and soil pH are not the dominant controls over
BNF. In a separate short-term laboratory experiment, BNF did not respond
strongly to Mo and P even when labile carbon was added. We postulate that high
nitrogen (N) availability in this area of the Amazon, as indicated by the
stoichiometry of soils and vegetation and the high nitrate soil stocks, likely
suppresses BNF at this site. These patterns may also extend across highly
weathered soils with high N availability in other topographically stable
regions of the tropics.
Geographic descriptionTanguro Ranch, an 800 km2 working farm in eastern Mato Grosso State, Brazil
Geographic coordinates(13°04’ S, 52°23’ W)
Time periodBegin date: October 2016
End date: February 2019
MethodologyFertilization experiment and sample collection
To evaluate the effect of the fertilization on free-living BNF, we sampled soil and litter from experimental plots before the experiment and three and four times after fertilization in the dry and wet seasons, respectively. In the October dry season experiment, we collected separate soil and litter samples two days prior and one day, 11 days, and three months after fertilization. In the February wet season experiment, we collected samples one week prior and one day, one week, one month, and 11 months after fertilization. For each sampling event, we weighed soils on the day of collection, incubating them overnight using the acetylene reduction assay (ARA) (described below), and initiated incubations of litter samples the following morning.
Free-living BNF by the acetylene reduction assay (ARA)
We measured free-living BNF in soil and litter using the ARA method (Hardy et al. 1968, Barron et al. 2009). For each incubation, we added about 15 grams of litter or 25 grams of fresh surface soil to 125 mL gas-tight jars with lids fitted with septa (Fisher Scientific, 501215190), sealed them, and added a 10% atmosphere of acetylene gas generated from calcium carbide (Santa Cruz Biotechnology, 278804). We incubated litter samples for 8-10 hours and soil samples for 14-18 hours at ambient temperature in an open-air greenhouse environment.
After incubations, we collected 30 mL headspace into pre-evacuated gas-tight vials (Teledyne Tekmar 22mL) fitted with thick butyl septa (Geo-Microbial Technologies, Inc.). To measure moisture and to express ARA rates per gram dry weight after the ARA was terminated, we weighed litter and soil before and after drying for two days at 65°C or 105°C, respectively. For the field experiment, we measured the ethylene concentrations in the gas samples at Cornell University using a Shimadzu 8-A gas chromatograph equipped with a flame ionization detector (330°C) and a Porapak-N 80/100 column at 70°C with two standard curves daily and check standards every ten samples.
We measured the soil pH in a 1:2 soil (field moisture) to distilled water ratio by weight, stirred and equilibrated for 30 minutes with a pH meter standardized at pH 7 and 4 daily using a single junction, epoxy body, sealed electrode (Cole-Parmer).
Resin-extractable and total soil Mo and P
We extracted available Mo and P from soils using anion-exchange beads following the procedures of Wurzburger et al. (2012) for mineral soils. We conducted extractions with a bead:soil:water ratio of 1:5:50 where the anion-exchange resin beans (Dowex 1x4-200) were loaded into acid-washed tubing capped at both ends with Nitex bolting cloth (243 µm) held by plastic clamps to expose the beads to the solution in 50 mL vials. The soil solution was shaken for 16 hrs at 80 rpm, and we rinsed the resins with DI to remove excess soil particles, eluted the resins with 10% trace-metal grade HNO3 on the shaker for another hour in a separate 50 mL vial, and then filtered the final solution. Procedural blanks and spiked check standards in DI were also run with each set.
To measure total soil Mo and P, samples were ground in an alumina ceramic ring and puck shatterbox, oven-dried at 105°C, ashed at 550°C for four hours, and digested in concentrated trace-metal grade nitric acid (HNO3) using the 3052 EPA microwave-assisted acid digestion of siliceous and organically based matrices. The digests were centrifuged, filtered, and diluted. Resin Mo and P, total Mo, and Mo and P contamination in the fertilizer and lime were measured on an Elan DRC-e quadrople Inductively Coupled Plasma Mass Spectrometer (ICP-MS) at the State University of New York College of Environmental Science and Forestry (SUNY ESF). Total P concentrations were measured on a Perkin Elmer Optima 3300DV Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES).