Nitrification and Wastewater Reclamation Workshop

In this workshop high school students learn about the link between nitrification and wastewater reclamation. The effects of metals on the process of nitrification are introduced. The workshop is best-suited for students who have some biology or chemistry background. This workshop is well suited for up to 20 students.

Project overview

Untreated wastewater contains high levels of nutrients that include ammonia. The introduction of untreated wastewater into a body of water often causes large algae blooms. The algae bloom persists until all nutrients in the body of water are consumed, at which point the algae die and begin to decay. While this is occurring, most of the dissolved oxygen is depleted killing other aquatic organisms including fish, invertebrates and plants. This phenomenon is called eutrophication.

Wastewater treatment facilities prevent eutrophication of bodies of water by removing the nutrients such as ammonia. For this, the treatment facilities rely on a biological process in which nitrifying bacteria play a key role. Nitrifying bacteria consume the ammonia in the wastewater transforming it to nitrate, which in turn serves as substrate to denitrifying bacteria (Arp et al. 2002).

Nitrosomonas europaea
, is a lithoautotrophic nitrifying bacterium that carries out the first step in the oxidation of ammonia (NH3) to nitrite (NO2-) (Chain et al. 2003). Nitrifying bacteria are sensitive to metals sometimes found in wastewater (e.g. copper, zinc, or chromium).
Students participating in this workshop examine the effect of different levels of metals on the growth of N. europaea. The students correlate the growth of N. europaea to the nitrifying activity (ability to oxidize NH3 to NO2-).

Number of sessions

The workshop is covered over approximately 4 to 5 sessions: one lecture; one laboratory session to familiarize students with the methodology and prepare samples; follow-up laboratory sessions to analyze samples in the spectrophotometer; one session for poster design; and one session to present posters and discuss findings.

Learning objectives

  • To raise awareness of the consequences of pollution in the nitrogen cycle,
  • To compare the results of the experiments with current environmental issues (e.g. link),
  • To practice presenting scientific research to peers and the public.

    Information resources

    URL links are provided for further information such as detailed methodology or background including the importance of nitrification and consequences of its imbalance. The URLs links lead to the most up-to-date research in nitrification (e.g. Nitrate in Lake Superior: On the Rise).


    Students learn the importance of nitrification in the reclamation of wastewater. Students get acquainted with laboratory procedures, and gather and interpret data. Students learn the effect of different metals to the efficacy of nitrification in the reclamation of wastewater The students convey 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

      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. Eutrophication is introduced. Effects of pollution in nitrification are discussed.
          3. Methodology: A method to measure nitrifying activity is presented. The concept of nitrifying activity in water reclamation is introduced. The steps in the workshop protocol are: i) sample preparation and inoculation; ii) addition of the metals to the sample cultures; and iii) measuring spectrophotometerically nitrifying activity (nitrite accumulation).
          4. Discussion: The lecture ends after discussing the factors that influence nitrification with emphasis on water reclamation. Examples of what one can expect under different scenarios are given. Sample questions: 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 are other pollutants that may affect nitrification? What other aspects of nitrification are worth testing?
    2. Laboratory Session #1.
      Students learn and practice the methodologies and prepare cultures and stock solutions. Students are given N. europaea to inoculate their samples. The treatments are prepared. The incubations are started. The treatments with a given metal may be divided among the students. Variations of the incubation may be tried for large groups.
    3. Subsequent periodic sampling.
      Samples from Laboratory Session #1 are taken and analyzed with the spectrophotometer, ideally every 12 hours. After three to four days of incubation the experiments are stopped and one last sampling is taken. Data is processed and conclusions are drawn.

    4. Poster design and construction session.
      Students organize their data in single groups or with other groups and construct a poster.

    5. Exposition Session.

      Students present their posters and discuss the outcomes 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.

      Suggested questions for discussion in the presentation session: How do metal concentrations affect the amount of NO2- produced (nitrification)? How do different metals affect nitrification? 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?


    Suggested materials

        1. A viable culture of N. europaea. The culture is started using the protocol described in link. The bacterium may be purchased from ATCC or requested from a laboratory researching nitrification.
        2. Erlenmeyer flasks (125 ml capacity).
        3. Stock 0.1 mM solutions: Cupric sulfate (250 mg/ liter), zinc sulfate (287 mg/liter), chromium chloride (266 mg/liter) and ferrous sulfate (278 mg/liter). These reagents may be purchased from a scientific products supplier.
        4. Sterile growth medium (see recipe).
        5. Spectrophotometer.
    1. Cultures preparation and incubation.
        1. Using an aseptic technique, pipette 3 ml of N. europaea culture into flasks containing 47 ml of fresh growth medium in sterile flasks.
        2. Add from the stock solutions the appropriate metal as follows
          Control culture = no metal addition
          Test culture 1 = 10 µl of the stock solution (~0.2 µM)
          Test culture 2 = 50 µl of the stock solution (~1 µM)
          Test culture 3 = 100 µl of the stock solution (~2 µM)
          Test culture 4 = 500 µl of the stock solution (~10 µM)
          Test culture 5 = 1000 µl of the stock solution (~20 µM)
        3. Incubate the cultures for two to four days with gentle agitation, preferably at 30°C. The cultures ideally are sampled every 12 hours (e.g. collecting 2 ml each sampling time using an aseptic technique). If a sample cannot be analyzed immediately it can be frozen or an inhibitor of nitrification added (e.g. merthilolate 1% wt/vol (ethylmercurithiosalicylic acid, sodium salt)). The stored samples can then be analyzed at a later date.
        4. Place the sample of the cell culture in a cuvette and measure its absorbance in a spectrophotometer at wavelengths 600 nm (OD600 representing growth), and at 352 nm and 400 nm (OD352 and OD400 to estimate nitrite accumulation). Using litmus paper or a pH meter measure the pH of the sample. Record the findings in a chart .
        5. Calculate the amount of NO2- (nitrite) present in mM (millimoles per liter) using the following formula:
          [NO2-] = (A352 - A400)/0.0225
          After all of the data has been gathered, make charts for NO2- production vs. time, growth (OD600) vs. time and pH vs. time In each chart place time on the X-axis and the other variable on the Y- axis. Include them in a poster as appropriate.

    This workshop has contributions from T. Radniecki, L. Semprini, D. Arp, K. Halsey and L. Sayavedra-Soto from Oregon State University, USA