Gill Ecosystem and Global Change Ecology Lab
Protocol: Inorganic Nitrogen, Net N mineralization, and Net Nitrification
Created by: Lafe Conner (with input from Debbie Rigby’s protocol)
Edited by:
Date Last Modified: 6 February 2016
Soil Sampling Protocol for Inorganic N and net N Mineralization
Items needed: Soil gouge auger (small diameter silver colored)
Soil auger with compact slide hammer (red and black 5 cm diameter)
Clear plastic tube liners that fit in the slide hammer auger
Large zip-lock storage bags
Sharpie permanent marker
Cooler with ice packs
1. Mark gouge auger with a piece of tape at 15cm (same depth as the plastic tubes)
2. Place a clear plastic tube liner in the 5cm auger with the slide hammer
3. Pound this into the ground up to the depth of the tube
4. Remove the clear tube from the auger
5. Place the clear tube with soil in it back in the hole
6. Take 3 soil cores 15 cm deep with the gouge auger and place these into the zip-lock bag
7. Label the bag with the site information, the date, and mark it as “initial”
8. Place the soil sample in the cooler as soon as possible and keep it cool
9. Leave the clear tube in the soil at the site for 28 days then retrieve it
10. Bring the samples back to the lab and process them within 24 hours
11. Keep samples in the refrigerator before extraction
12. After extraction place the liquid extract into the freezer and leave the bags of soil open so
they can dry (if you plan to use these sample for soil texture analysis or to measure
matrix potential)
Soil inorganic N:1
Sample preparation for NO3 and NH4
Use also for Net Nitrogen Mineralization and Net Nitrification
2
Items needed: 125 ml Erlenmeyer flasks
50 ml centrifuge tubes
50 ml syringe
Parafilm
0.5 M K2SO4 -Dissolve 87.13 g in 1 liter double DI water
1.
2.
3.
4.
5.
6.
Tare balance with a 125 mL Erlenmeyer flask
Label the flask with the sample ID, use permanent marker
Weigh 15 g (+/-0.5 g) of soil into the flask
Record the exact mass of the soil on the data sheet and the electronic spreadsheet
Weigh an empty soil tin
Record the number (label) and mass of the tin on the data sheet and the electronic
spreadsheet
7. Weigh 4 g (+/-0.5 g) of soil into a soil tin
8. Record the mass of the soil in the tin on the datasheet and the electronic spreadsheet
9. Weigh approximately 12-18 samples
10. Dispense 30 mL of 0.5 M K2SO4 into tube and cap the tube (1:2 weight to volume ratio
of 0.5 M K2SO4, see reagent recipe above for 0.5 M K2SO4)
11. Shake for 60 minutes on shaker at low speed (100 rpm)
12. While the samples are shaking weigh the remaining samples and prepare the funnels and
filters to filter the samples
13. Use the funnels and Whatman filter paper (44, 11 or 12.5 cm diameter)
14. Place the filters in the funnels above the labeled centrifuge tubes (label tubes with the
experiment name, “N” for nitrogen, the date, and the Sample ID). Use permanent marker
to label the tubes.
15. Pour the supernatant into the filter and let stand for approximately 1 hour until the liquid
has filtered through to the centrifuge tube
16. Cap the centrifuge tubes and place them into the freezer
17. Place the soil tins into the drying oven for 48 hours on low
18. Reweigh the tins and record their dry mass on the data sheet and the electronic
spreadsheet
19. store at -20C until the samples are analyzed
20. Wash the dishes using soap, rinse three times with regular water and three times with DI
water
21. Use acetone to remove the marker (use ventilation hoods while working with acetone)
Ammonium, NH4+
1
2
The original protocol for the lab work was written by Debbie Rigby and modified by Lafe Conner.
If using this procedure for net N mineralization you will take two soil cores in the field. Bring one back to the lab
and analyze it (initial), but leave the other in the field for 28 days (incubated). For net N mineralization sum the
NH4+ and NO3- values from the initial core and subtract this value from the sum of NH 4+ and NO3- from the
incubated core. You can also calculate net nitrification by subtracting the initial NO3- value from the incubated NO3value.
Preparing reagents3
Equipment and Chemical list:
100 ml volumetric
100 ml container with cap
Weigh paper
Parafilm
Gloves
Nanopure water
Sodium nitroprusside (sodium nitroferricyanide) (Na2[Fe(CN)5NO]·2H2O)
Sodium salicylate (C7H5NaO3 )
Sodium citrate (C6H5Na3O7 )
Sodium tartrate (C4H4Na2O6)
Sodium hydroxide (NaOH)
Bleach
Reagent A:
1. Fill a 100 ml volumetric 1/2 full with nanopure water
2. Pre-weigh and add the following:
 0.05 g sodium nitroprusside (sodium nitroferricyanide)
 13 g sodium salicylate
 10 g sodium citrate
 10 g sodium tartrate
3. Cap the top with parafilm and shake until all the chemicals are dissolved
4. Fill the volumetric to the meniscus with a water bottle labeled “nanopure water”
5. Pour the liquid into an new container and label it “Reagent A”
Warnings:
33
These notes are from Megan Taylor who ran this procedure for the 2012 samples.
“For this procedure I made reagents A and B. I stored reagent A in a container covered in foil to
block out any light. Both reagents were stored in a refrigerator for up to 3 weeks. I put the
sample liquid into microcentrifuge tubes and would use a microcentrifuge to make the white
precipitate go to the bottom of the tube before extracting the sample. I created a 6 ppm standard.
I would put 430ul K2SO4 into 6 microcentrifuge tubes. Then I would mix in the 6 ppm. Then
take the mixture from that tube and put it into the next tube and so on. This created standards
with values of 3, 1.5, .75, .375, .1875, and 0 ppm. When putting the samples into the plates, I
would put 200ul of the samples first. Then add 50ul of reagent A and 50ul of reagent B. Then I
would wait 1 hour and use the spectrophotometer to read the samples. I created a graph from the
standard ppm and abs values. I then used the regression equation to convert the absorbance
values of the samples to ppm.
Samples a blue color when read”
For samples with NH4+ concentrations higher than 3 ppm, discovered after the first run, we ran them a second time
after diluting the sample 1:1 with 0.5 M K2SO4 (diluted by ½). If the diluted sample was still higher than 3 ppm we
diluted it 1:2 with 0.5 M K2SO4 (diluted by 1/3).



Nanopure water is the clean laboratory water that is used in molecular and
analytical chemistry. If you think it is contaminated or the amount in the nalgene
is low please refill it with more nanopure from the Genetics Molecular Lab
When weighing chemicals make sure you tare the balance and wear gloves
Reagent A is light sensitive, so wrap aluminum foil around the bottle for
storage
Reagent B:
1. Weigh 6 g sodium hydroxide (NaOH)
2. Using a 100 ml container with cap, dissolved the 6 g NaOH in 100 ml water
3. Add 2 ml bleach with a pipette or graduated cylinder
Reagent A and B can be stored for several weeks in the refrigerator
Creating an NH4+ standard and standard curve
Making a 100 ppm stock solution
Equipment and chemical list:
500 ml volumetric
Ammonium sulfate ((NH4)2SO4)
Parafilm
1.
2.
3.
4.
Fill a 500 ml volumetric 1/2 full with nanopure water
Add 0.236 g of (NH4)2SO4
Cap the top with parafilm and shake until all the chemicals are dissolved
Label and store in a 500 ml glass bottle
This is for a 100 ppm stock solution that is used to generate you standard curve
Warning:
 When creating the standards and analyzing samples wear gloves
Making a standard curve for soils with NH4+ concentrations up to 3 ppm.
This is the standard curve that we use for most wildland soils since inorganic N does not usually
hang around in this form. The standard curve includes 6 standards: 3, 1.5, 0.75, 0.375, 0.1875,
and 0. To calculate the standards we use the following equation:
C1 x V1= C2 x V2
Where C1 = 100 ppm, C2 = standard ppm, and V2 = 1000 uL, V1 = the amount of 100 ppm stock
that you will add. The remaining 1000 uL is added to the standards as 0.5 M K2SO4.
Table. Recipe for NH4+ standards
Standard (ppm)
0.5 M K2SO4 (uL)
100 ppm (uL)
0
0.1875
0.375
0.75
1.5
3
1000
998.125
996.25
992.5
985
970
0
1.875
3.75
7.5
15
30
1.
2.
3.
4.
In a microcentrifuge tube add 1.5 mL of the 100 ppm stock
Label microcentrifuges with the names for the 6 standards
Add the calculated amount of 0.5 M K2SO4 in uL to each standard using the same pipette
Add the calculated amount of 100 ppm stock in uL using a new pipette tip for each
standard
5. Cap the tubes and invert them 5-times to mix the solution
Analyzing soils for NH4+
Equipment and Chemical list:
96 well plate
Reagent A
Reagent B
Pippette
Pippette tips
1. Find a clean acid-washed 96 well plate. The maximum volume that one of these wells
hold is 350 uL
2. Pipette 200 uL sample into each well. Make sure to fill out the data sheet to label each
well
3. Load two wells with each sample and average the values also make sure there are not
unexpectedly large differences between any two measures
4. Pipette in each well 50 uL of A, and 50 uL of B to each well. Make sure to save pipette
tips by using one tip for A and B for an entire plate (2 tips total)
5. Cover the plate with the top
6. Record the time
7. After approximately an hour measure/record the absorbance at 650 nm. Make sure there
are no bubbles in the sample. You must be trained and approved by Dr. St. Clair to use
the spectrophotometer
Warning:
 We are measuring the following ammonium concentrations:
For soils with low ammonium use the following-low range, 0 to 1 ppm N, use 800 uL
sample, 200 uL A, and 200 uL B. Lower limit is approximately 0.02 ppm
Extra notes for soils with higher or lower NH4+ concentrations
You will need to adjust these ratios so they fit in the 96-well plate
For up to 10 ppm N, use 20 uL sample, 110 uL A, and 110 uL B. Lower limit is approximately
0.2 ppm N. If your soil values do not fall within this range please see below. Remember to also
change your standard curve.
For higher concentrations, dilute reagent A 1:1 with nanopure water and use the following
sample to reagent ratios:
For up to 20 ppm N, use 40 uL sample, 500 uL A, and 500 uL B. Lower limit is
approximately 0.4 ppm N.
For up to 60 ppm, use macrocuvets, 45 uL sample, 1500 uL A, and 1500 uL B. Lower
limit is about 1 ppm N.
Nitrate, NO3Preparation of NO3- Reagent4
Equipment and Chemical list:
0.5 M HCl- add 8.33 ml HCl to 200 ml DI water
Vanadium (III) Chloride (remake often)
Sulfanilamide
N-(1-naphthyl)ethylenediamine dihydrochloride
Weigh paper
200 ml container or Erlenmeyer flask
1.
2.
3.
4.
5.
6.
Pour approximately 200 ml 0.5 M HCl into a bottle
Weigh and add approximately 0.5 g vanadium(III) chloride
Dissolve with gentle swirling (side to side shaking)
Weigh and add about 0.2 g sulfanilamide
Weigh and add about 0.01 g N-(1-naphthyl)ethylenediamine dihydrochloride
Dissolve all chemicals by gently swirling
*The reagent is stable for about a week in the refrigerator or indefinitely if frozen. If
there are any problems with the standard, try to fix it by remaking the vanadium chloride.
4
These notes are from Megan Taylor, who ran this procedure for the 2012 samples.
“For this procedure I made the NO3 reagent and stored half of it in the freezer and half of it in
the fridge. It lasts about 1week in the fridge. Then I would unfreeze the other half and use it for
about a 1 week if necessary. I put the sample liquid into microcentrifuge tubes and would use a
microcentrifuge to make the white precipitate go to the bottom of the tube before extracting the
sample. I created a 5 ppm standard. I would put 430ul K2SO4 into 4 of the 5 microcentrifuge
tubes. Then I would mix in the 5 ppm into the tube without K2SO4. I would do this again with
the next tube (meant to be the 2.5ppm standard) Then take the mixture from that tube and put it
into the next tube and so on. This created standards with values of 5, 2.5, 1.25, .625, .3125, and 0
ppm. When putting the samples into the plates, I would put 150ul of the samples first. Then add
150ul of the NO3 reagent. Then I would wait 6-8 hours and use the spectrophotometer to read
the samples. I created a graph from the standard ppm and abs values. I then used the regression
equation to convert the absorbance values of the samples to ppm.
Samples a pink color when read”
For samples with NO3- concentrations higher than 5 ppm, discovered after the first run, we ran them a second time
after diluting the sample 1:1 with 0.5 M K2SO4 (diluted by ½). If the diluted sample was still higher than 3 ppm we
diluted it 1:2 with 0.5 M K2SO4 (diluted by 1/3).
Creating a NO3- standard and standard curve
Making a 100 ppm stock solution
Equipment and chemical list:
500 ml volumetric
Potassium Nitrate (KNO3)
Parafilm
1.
2.
3.
4.
Fill a 500 ml volumetric 1/2 full with nanopure water
Add 0.361 g of (KNO3)
Cap the top with parafilm and shake until all the chemicals are dissolved
Label and store in a 500 ml glass bottle
This is for a 100 ppm stock solution that is used to generate you standard curve
Warning:
 When creating the standards and analyzing samples wear gloves
Making a standard curve for soils with NO3- concentrations up to 5 ppm.
This is the standard curve that we use for most wildland soils since inorganic N does not usually
hang around in this form. The standard curve includes 6 standards: 5, 2.5, 1.25, .625, .3125, and
0 ppm. To calculate the standards we use the following equation:
C1 x V1= C2 x V2
Where C1 = 100 ppm, C2 = standard ppm, and V2 = 1000 uL, V1 = the amount of 100 ppm stock
that you will add. The remaining 1000 uL is added to the standards as nanopure.
Table. Recipe for NO3- standards
Standard (ppm)
0.5 M K2SO4 (uL)
100 ppm (uL)
0
0.3125
0.625
1.25
2.5
5
1000
996.875
993.75
987.5
975
950
0
3.125
6.25
12.5
25
50
1.
2.
3.
4.
In a microcentrifuge tube add 1.5 mL of the 100 ppm stock
Label microcentrifuges with the names for the 6 standards
Add the calculated amount of 0.5 M K2SO4 in uL to each standard using the same pipette
Add the calculated amount of 100 ppm stock in uL using a new pipette tip for each
standard
5. Cap the tubes and invert them 5-times to mix the solutions
Analyzing soils for NO3Equipment and Chemical list:
96 well plate
Nitrate Reagent
Pippette
Pippette tips
1. Find a clean acid-washed 96 well plate. The maximum volume that one of these wells
hold is 350 uL
2. Pipette 150 uL sample into each well. Make sure to fill out the Nitrate 96-well run data
sheet for each name.
3. Load two wells with each sample and average the values also make sure there are not
unexpectedly large differences between any two measures
4. Pipette in each well 150 uL the Nitrate reagent to each well. Make sure to save pipette
tips by using one tip all the wells.
5. Cover the plate with the top
6. Record the time and wait at least 6-8 hours
7. At room temperature, color is maximum after about 6-8 hours, and stable for at least 2
days. Read absorbance at 540 nm against a reagent blank (0 ppm standard). Be sure there
are no bubbles in the wells.
8. You must be trained and approved by Dr. St. Clair to use the spectrophotometer
Measuring the concentration:
Using the spectrophotometer
-Read absorbance at 650 nm for NH4 against a reagent blank (0 ppm standard) after about an
hour.
-Read absorbance at 540 nm for NO3 against a reagent blank (0 ppm standard) after 6-8 or before
2 days.
1.
2.
3.
4.
5.
6.
Plug in the instrument
Turn on the computer
Turn on the spectrophotometer machine
Open Softmax pro version 5
Open Excel
The tray will open automatically. Put in the plate. Push the “Drawer” button to close the
tray holder.
7. Click settings and set wavelength to 650 nm (NH4) or 540 nm (NO3)
8. Push read to read samples
9. Copy the results into an Excel sheet
10. If reading more samples repeat steps 6-9. If not, remove sample. Push “Drawer” to close
tray.
11. Turn off the computer
12. Turn off the machine
13. Unplug the machine
14. Cover the machine
Calculating ppm N in the soil from the absorbance readings
1. Calculate the mass of soil that was extracted by finding the percent of soil in the 4 g of
soil dried in the soil tin and multiplying that by the mass of the soil + water extracted
a. To find the percent of soil in the tin, divide the dry mass of soil by the wet mass
of soil (remember to subtract the mass of the tin to get wet and dry mass of soil)
(Table 1).
GramsOfSoilExtracted
TinNumber
WeightOfTin
WetSoilPlusTin
DrySoilPlusTin
MassDrySoil
MassWetSoil
ProportionDrySoil
Mass of Soil
15.04
16
1.03
5.05
4.13
3.1
4.02
0.771144
11.59801
15.07
3
1.03
5.03
4.47
3.44
4
0.86
12.9602
15.07
2
0.99
5.02
4.25
3.26
4.03
0.808933
12.19062
15
45
1.03
5.03
4.02
2.99
4
0.7475
11.2125
15.01
11
1.02
5.02
4.54
3.52
4
0.88
13.2088
Table 1. Sample calculation of soil mass
2. Use the absorbance values for the standards to create a standard calibration line (The Rsquared of this line should be 0.99 or higher) (Fig. 1).
Figure 1. Example of standard line used to calculate ppm NH4
3. Use the equation of the line to calculate the concentration of NH4 and NO3 in the samples
(Each set of standards and samples will produce its own standard line)
4. Multiple the ppm N in the sample by the dilution factors to calculate N in the Soil
a. Calculate dilution factors using this equation:
Dilution Factor = Total Volume / Aliquot Volume (measured sub-volume of the
original sample)
b. The first dilution factor is approximately 3 = (Mass of soil + 30 mL K2SO4) /
Mass of soil
c. The second dilution factor for NH4+ is 1.5 = (200 uL Sample +50 uL Reagent A
+50 uL Reagent B) / 200 uL Sample
d. The second dilution factor for NO3- is 2 = (150 uL Sample + 150 uL Reagent) /
150 uL Sample
e. If the sample had more than 3 ppm it was diluted again, giving a third dilution
factor of 2 = (100 uL Sample + 100 uL K2SO4) / 100 uL Sample
f. If the diluted sample still had more than 3 ppm it was diluted again, giving a third
dilution factor of 3 = (100 uL Sample + 200 uL K2SO4) / 100 uL Sample
g. For the third dilution factor we can use a 1 if there was no third dilution, a 2 if the
sample was diluted by half, and a 3 if the sample was diluted by one third.
5. When you multiple the ppm N in the sample by the dilution factors you should end up
with ppm N in the soil, this is the value you analyze and report
For Calculating Net N Mineralization rates
1. Sum the concentration of NH4+ and NO3- in the initial soil samples
2. Sum the concentration of NH4+ and NO3- in the incubated soil samples
3. Subtract the initial N values from the incubated values
4. Divide this amount by the number of days of the incubation (usually 28)
5. This gives you the net mineralization rate in ppm per day (usually reported as ug N/g soil
/day)
For Calculating Net Nitrification rates
1. Subtract the initial NO3 concentration from the incubated NO3 concentration
2. Divide this amount by the number of days of the incubation (usually 28)
3. This gives you the net nitrification rate in ppm per day (usually reported as ug NO3/g soil
/day)