Nitrification Potential in Soils Workshop

In this workshop high school students taking advancement placement biology or chemistry learn the processes in the nitrogen cycle with emphasis on nitrification. The workshop is best for students who have taken an advanced placement course in biology or chemistry at their high school. The workshop can be used for groups of up to 20 students.

Project overview

The students work in groups to formulate nitrification-related hypotheses. After reviewing their hypotheses, the students design experiments to test them. The students collect soil samples on a field trip and bring them to the laboratory to measure their potential nitrifying activity. Students create posters to present their findings and open them up for discussion among their peers. The presentation may be open to the public if desired.

Number of sessions

The workshop is covered over approximately 8 sessions : one lecture and hypotheses formation; two laboratory sessions to familiarize students with the methodology; one field trip for students to collect samples; two laboratory sessions to analyze their own samples; one session for poster design; and one session to present posters and discuss findings.

Inquiry-based learning objectives

  • Formation of appropriate (testable) scientific questions,
  • Use of calibration (standard) curves,
  • Drawing conclusions with emphasis on comparison with other classmates' conclusions as well as with the literature.

    Information resources

    URL links are provided for further information such as detailed methodology or background including global importance of the nitrogen cycle and consequences of its imbalance. The URL links lead to the most up-to-date research.


    Students learn the importance of nitrification in the nitrogen cycle and its potential applications to remediate the environment. Students learn how to formulate hypotheses, and to propose and implement experiments. Students get acquainted with laboratory procedures, techniques, and recording and interpretation of data. They practice communication skills conveying their findings to an audience. Students receive constructive feedback from the instructor and peers regarding their insight, hypotheses formation, laboratory technique, and communication skills.

    Suggested schedule for the workshop

    1. Lecture

      Nitrogen cycle overview. The major processes in the nitrogen cycle are presented and discussed. The techniques to measure nitrification are introduced.

      Lecture components

        1. Nitrogen cycle: The three major processes in the nitrogen cycle are nitrogen fixation, nitrification and denitrification.
        2. Nitrification: It is composed of two steps, ammonia oxidation and nitrite oxidation.
        3. Methodology: A method to measure nitrification activity is presented. The concept of potential nitrifying activity is introduced. In this workshop potential nitrifying activity is expressed in µg of nitrite/g dry soil/day. Emphasis is made on determining the potential nitrification activity of the soil samples under the same conditions to make valid comparisons. The steps in the protocol are: i) sample collection; ii) slurry preparation; iii) ammonia addition to provide the substrate; iv) chlorate addition to block further nitrite oxidation; v) nitrite content measurements in a sample to test content before incubation and in a sample after incubation.
        4. Discussion: The lecture ends with a discussion of the factors influencing nitrification in the soil. Examples of what one can expect under the different scenarios are given.
        5. Examples of hypotheses: The composition of the microbial population affects the rates of nitrification. The pH of the soil affects the availability of ammonia to nitrifiers. The effectiveness of nitrogen-based fertilizers is influenced by nitrification. Soil composition affects nitrification.
        6. Suggestions for discussion: Where and when is it desirable to inhibit nitrification? Where and when is it desirable to enhance it? How can nitrification be of use in cleaning the environment? What other aspects of nitrification are worth testing?
    2. Laboratory Session #1.
      Students learn and practice the methodologies they will use when conducting their own experiments to study nitrification. Students are given soil samples to measure potential nitrifying activity. Sample incubations are started. Instructions to prepare standard curves are given and discussed. Example calculations are given. Students show their calculations and make comparisons to other measurements found in the literature.

    3. Laboratory Session #2.
      Samples from Laboratory Session #1 are analyzed and conclusions are drawn.
    4. Field trip.
      Each group of students has agreed upon a question (an hypothesis) to test in the field. Students could collect soil samples at different depths or across a transect (up to 6 samples). Some students may want to test the reproducibility or heterogeneity of a soil by taking several samples form a single site. The amount of soil collected to constitute a sample should be at least 200 g (more if several different treatments are intended).
    5. Laboratory Session #3.
      Students start the incubations as described in Laboratory Session #1 using the collected samples.
      If time and resources allow, sample the soil during the incubation period more than two times to determine whether nitrite production (nitrifying activity) is linear. Additional soil samples may be treated with lime, a fertilizer mix, or sodium chloride (table salt), or they may be incubated at a higher temperature, exposed to light from a slide projector, etc. The effects of these on potential nitrifying activity can then be compared.
    6. Laboratory Session #4.
      Students carry out the nitrite measurements as in Laboratory Session #2. Students compare the potential nitrifying activity in the sites sampled. Students compare the potential nitrifying activity in the alternative incubations, if any. Students determine the reproducibility of the sampling technique.
    7. Poster design and construction session.
      Students organize their data and draw conclusions to be documented in a poster.
    8. Exposition Session.
      Students present their posters and discuss them with their peers. The session may be open to the public to give students a wider audience. Allow time for the students to share their experiences during the workshop and receive feedback about their participation.

    Protocol to estimate nitrifying potential in soils

    The protocol is adapted from the method described in Schmidt and Belser, 1995; Methods of Soil Analysis. SSSA. Pages 171 to 177. Students should have access to laboratory coats, gloves and safety glasses.

    To measure potential nitrifying activity in the soil samples the following materials (easily obtained from a scientific products supplier) and solutions are necessary.

        1. Two 125 ml Erlenmeyer flasks and at least twenty ~10 ml test tubes per soil sample.
        2. Aluminum foil to hold 20 g soil samples.
        3. Funnels and filter paper.
        4. Phosphate buffer: ~50 g of potassium phosphate dibasic and ~5 g of potassium phosphate monobasic in 1 liter of distilled water made to pH=8 using a pH meter.
        5. Ammonium sulfate solution: 25 g ammonium sulfate in 1 liter of distilled water.
        6. Potassium chlorate solution: 6 g potassium chlorate in 100 ml distilled water.
    1. Soil slurries preparation and incubation
        1. Weigh two 20 g soil samples to analyze for potential nitrifying activity. One of the 20 g samples will be used to determine nitrification activity. The other 20 g sample will be used to determine the dry weight of the soil and should be placed on a piece of aluminum foil of known weight.
        2. Place the 20 g of soil in a 125 ml flask, add 90 ml of phosphate buffer and 0.2 ml of ammonium sulfate solution (total volume should be about 100 ml).
        3. Add 2 ml of chlorate solution to stop the second step in nitrification (oxidation of nitrite to nitrate). This permits the accumulation of nitrite.
        4. Place the flask on a rotary shaker for 5 min.
        5. Remove the flask and allow to stand for approximately 5 minutes.
        6. Filter the upper part of the soil slurry to obtain 15 ml. Divide the filtrate into three clean tubes (about 5 ml per tube). All tubes should be properly labeled for identification.
        7. Place the filtrate samples in a refrigerator to slow down nitrification. Alternatively, an inhibitor of nitrification can be added such as merthilolate (ethylmercurithiosalicylic acid, sodium salt) 1% (wt/vol). These samples will be used as reference to compare the soil after 48-hour incubation (next session).
        8. The remnant soil in the flask is incubated ~48 hours at room temperature. At the end of the incubation, repeat the filtering procedure collecting three 5 ml samples in clean tubes. Alternative treatments should be sampled in the same fashion.
    2. Dry weight determination of the soil
        1. The second 20 g soil sample is placed in an oven at 45°C for at least 24 hours.
        2. Determine the weight after the drying time. The aluminum foil weight is subtracted. The dry weight of the soil samples is used in the calculations.
    3. Standard curve preparation to measure nitrite concentration

      Prepare the following solutions:
        1. Nitrite stock solution: 10 mg of sodium nitrite per liter.
        2. Sulfanilamide solution: 10 g of sulfanilamide in 1 liter of 1.5 M HCl
        3. NNEQ solution: 200 mg of n-(1-napthyl) ethylene diamine-2HCl in 1 liter of distilled water.

      Prepare eight 12 ml test tubes with the following solutions mixing thoroughly:
        1. Tube 1: 0.5 ml stock solution and 4.5 ml water
        2. Tube 2: 2.5 ml of tube 1 and 2.5 ml distilled water
        3. Tube 3: 2.5 ml of tube 2 and 2.5 ml distilled water
        4. Tube 4: 2.5 ml of tube 3 and 2.5 ml distilled water
        5. Tube 5: 2.5 ml of tube 4 and 2.5 ml distilled water
        6. Tube 6: 2.5 ml of tube 5 and 2.5 ml distilled water
        7. Tube 7: 2.5 ml of tube 6 and 2.5 ml distilled water, mix and dispose 2.5 ml
        8. Tube 8: 2.5 ml of distilled water.

      Add 0.2 ml of sulfanilamide solution to each tube followed by 0.2 ml of NNEQ. Mix thoroughly and allow to stand for 30 min. The color of the solution should change to a vivid violet color. Measure the absorbance of the samples at 540 nm using a spectrophotometer. The instrument should be calibrated to read zero absorbance using the solution in Tube 8.

    4. Measurement of nitrite concentration in the soil samples
      Prepare tubes containing aliquots of the filtrates (i.e. from the start of the incubation and at the end of the incubation). For example, prepare 4 tubes containing 0.25, 0.5, 1.0, 1.5 and 1.75 ml of the filtrate made to a final volume of 2.5 ml with water. Measure the nitrite concentration in the same way it was measured in the preparation of the standard curve. If the filtrates have a brown color make a duplicate for each tube without adding sulfanilamide and NNEQ and use to blank the instrument for each dilution. For colored samples, one will need 10 ml of filtrate (i.e. in step 1f). Alternatively to clear a high background sample one can add to the tubes a few drops of a flocculant solution prepared dissolving 7.35 g CaCl2 and 10.15 g MgCl2 in 100 ml H2O
    5. Calculations
      Calculate the µg of nitrite per gram of dry soil per day. Determine the variation among replicate samples.

    This workshop has contributions from K. Halsey, D. Arp, and L. Sayavedra-Soto from Oregon State University, USA and from C. Beedlow from Corvallis High School District 509J