Denitrification rates under salt exposure for soils from four stormwater basins in Ithaca, New York
Abstract
Description: This data represents denitrification rates as measured while conducting a denitrification enzyme assay (DEA) for soils from stormwater basins, with additions of salts of varying concentration to the basin soils. Sites: Stormwater basins on Cornell University campus in Ithaca, New York, USA.Two wet basins, two dry basins. Within site measurements:At each basin site, soils were collected from 5 locations. There are two reference locations just outside of each stormwater basin, in soil that is not receiving stormwater inputs. There are three 'Basin' locations located within each stormwater basin, and thus are influenced by inflowing stormwater, inflowing salts and other pollutants, and other conditions in each basin. Methods:The general DEA method is described in McPhillips and Walter (2015), referenced below. For full details on methodology, including chloride additions to incubation media, see the additional protocol document included with this repository. Column definitions in .csv filesSite- One of the four different stormwater basin sitesTreatment- the concentration of chloride added as NaCl to the denitrification incubation mediaMeasure Location- the location of soil sampling relative to the basin, either Reference or BasinDenit_mgN_kg_hr- Denitrification rate, in mg N/kg/hr, as determined by methodology described in the protocol Reference for general DEA method (without chloride additions to soils): McPhillips, L.E. and M.T. Walter. 2015. Hydrologic conditions drive denitrification and greenhouse gas emissions in stormwater detention basins, Ecological Engineering, 85: 67-75, doi:10.1016/j.ecoleng.2015.10.018.
Full text
Protocol: Denitrification Enzyme Assay (DEA) w/ Chloride Manipulations Author: Lauren McPhillips Date: September 2014 Overview • Lab assay to measure ‘denitrification potential’ of soils • Soils are incubated with ample nutrients (carbon and nitrate) + a range of chloride concentrations to see what the potential of the soil’s microbial community is to denitrify under various salinity regimes • Acetylene is used to inhibit full denitrification (reduction to N2) so that denitrification rate can be measured as production of N2O, where N2O is easily measured via gas chromatograph Equipment • Gas chromatograph with Electron Capture Detector (ECD) for N2O analysis and (optional) Flame Ionization Detector (FID) for CH4 analysis • Assembled gas vials that are compatible with GC autosampler • Ultra high purity nitrogen (UHP NI 300 from Airgas) • Tedlar bag (e.g. 1092312 from Fisher Sci) fitted with short length of Masterflex tygon tubing and luer-lock valve (for nitrogen) • Acetylene gas • 21 L Tedlar bags with septa, for acetylene and nitrogen • 23 g1 needles • 10 mL syringe • Evacuation manifold • DEA media o 200 mg/L NO3--N, 1000 mg/L glucose + variable concentrations of chloride o Recipe: In a 500 mL volumetric flask add: 0.72 g KNO3, 0.5g glucose ▪ Chloride treatments: ▪ 0 g Cl/L= add no Cl (just use regular media of nitrate + glucose) ▪ 0.5 g (500mg) Cl/L (added as NaCl) ▪ 5 g Cl/l ▪ 10g Cl/L ▪ 25g Cl/L • 125 mL glass bottles with butyl rubber septa and crimps (same as used for GC vials) or 4 oz mason jars with rubber septum fitted in lid • Data sheet (columns: date, site, rep, wet weight (g), start time for t=0, t=20, t=40, and t=60 minutes) • Timer • Shaker table capable of 125 rpm • Bucket to dump soil solution after incubation
Methods Initial prep • Make DEA media, store in fridge • Wash bottles • Print data sheets • Fill one gas bag with acetylene and another with N2 • Evacuate and label gas vials (4 per soil sample + standards) • Take soil samples (Homogenize 2-3 5cm cores per site) o Sieve soil and remove gravel >2mm • Remove subset of soil and dry at 105C o Calculate % SoilH2O as (mass of wet soilmass of dry soil)/(mass of wet soil) Performing assay • Weigh 2 reps of 5 g (± 0.5g) of sieved, homogenized soil into bottles • Add 10mL of DEA media to each jar and screw cap on tightly • Create anoxic conditions in bottles: evacuate and flush o Evacuate jars for 1 min each o Insert needle from large N2 bag into septa and gently compress bag for 30 sec o Evacuate and flush with N2 again Incubation • Add 10mL acetylene from small gas bag to each flask. Pump up and down 3 times and start timer. Take out 15mL gas sample and inject into evacuated vial. This is T=0min sample. • Record start times. Note the order that you take the samples, and maintain consistency for each timepoint so that time intervals are correct. • Place flasks on shaker table at 125 rpm (or speed 2 on our shaker) for ~20min • At T=20min, take out 12mL gas sample from each flask and inject into evacuated vial. Add 12mL N2 from small gas bag. • Put flasks back on shaker table for ~15min, until T = 40min • At T=40min, take out 12mL gas sample from each flask and inject into evacuated vial. Add 12mL N2 • Put flasks back on shaker table for ~15min, until T = 60min • At T=60min, take out 12mL gas sample from each flask and inject into evacuated vial. This is the end of the incubation. Note: If preparing for more assays, you can use down time to weigh more samples, wash and dry DEA bottles, prepare gas standards, evacuate vials, label vials, etc. References Overall methodology:
Groffman, P. M., E. A. Holland, D. D. Myrold, G. P. Robertson, and X. Zou. 1999. Denitrification. in Robertson, G. P., D. C. Coleman, C. S. Bledsoe, and P. Sollins. Standard Soil Methods for Long-Term Ecological Research. Oxford University Press. pp 272-288. Additional References: S. Wang, B. McGill. 2012. DEA Protocol and Calculations. http://www.biology.duke.edu/wrightlab/ Hofmockel K. DEA Protocol. http://kirstenhofmockel.org/sites/kirstenhofmockel.org/files/DEA_Hofmockel_1.pdf Murray, R.E. and R. Knowles (1999) Chloramphenicol inhibition of denitrifying enzyme activity in two agricultural soils, Applied and Environmental Microbiology, 65(8): 3487-3492. Pell et al. 1996. Potential denitrification activity assay in soilwith or without chloramphenicol? Soil Biology & Biochemistry 28:393-398. Calculations The output from the gas chromatograph is in area, which is then converted to ppm by N2O gas volume. In other words, it is given as one volume unit of N2O for every one million volume units of other gases in the sample. The calculations that then convert this number to ppb (parts per billion) of N by soil dry weight are as follows. 1. Create linear regression for gas standards to transform peak area into concentration in ppm per volume. 2. Divide concentration by 24 (the gas constant, assuming T~298K and P~1 atm) to get the number of moles per unit volume. ppmv(ppm per volume) = uL/L = nL/mL (nL/mL)*(nM N2O/24 nL)=> nM/mL 3. Multiply the result of step 2 by 44 (molecular weight) to get the nanograms of N2O/mL. (nM/mL)*(44 ng N2O/nM)=> ng N2O/mL 4. Multiply the result of step 3 by 0.636 to get the nanograms of N/mL. (ng N2O/mL)*(0.636 ng N/ng N2O)=> ng N/mL 5. Multiply the result of step 4 by the headspace in the bottle/jar to get the nanograms of N evolved.
Headspace volume, in mL=> volume of the jar (118 mL for 4oz mason jar, 160 mL for ‘125 mL’ serum bottle) minus the volume of the soil (mass of wet soil in mL is approximately = to volume in mL) minus the media (10 mL) in the flask (ng N/mL)*(mL of headspace)=> ng N 6. Divide the liquid volume (volume of media + volume of water in the soil) by the headspace. Multiply this by 0.615 (Bunsen coefficient) and by the result of step 5 to get the dissolved N gas present in the liquid in the flask. This should be very small. (ng N)* [0.615*(media volume, mL + (wet weight of soil*soil H2O %))] / headspace in mL Note: Calculate % SoilH2O as (mass of wet soilmass of dry soil)/(mass of wet soil) from samples dried separately 6. Add the results of step 5 and step 6 to get the total N (ng) gas produced. Divide this by the dry weight of the soil (wet weight of soil – wet weight of soil*soil H2O %) put in the flask to get the N gas produced per gram of dry soil, parts per billion. (ng N2O in headspace+ ng dissolved N2O)/dry weight of soil, in grams => ng N/g 7. Get the slope from Timepoint 1 to Timepoint 4 to get a rate in parts per billion per hour (ng/g/h). Hint: in Excel, rather than having to make a graph and do a regression for each sample, you can use the LINEST function. E.g. =LINEST(y-values, x-values, TRUE, 1)