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GROWING Nitrosomonas europaea:

Factors to Consider and the Recipe for Making Medium


From the
Laboratory of Daniel J. Arp
Oregon State University, Corvallis OR 97331, USA

Nitrosomonas europaea is an autotrophic nitrifying bacterium that gains all of its energy for growth from the oxidation of ammonia to nitrite as follows:

Equation: 2H+ + 2e- + NH3 + O2 --> NH2OH + H2O --> NO2- + 5H+ + 4e-

Most nitrifying bacteria have a relatively long generation time (8-72 hours) and their isolation from the environment is a long-drawn out process that may take up to 6 months.
However, from an ATCC culture, or once they are growing, they are relatively easy to grow and keep viable. The ATCC 19718 N. europaea stock culture has a generation time of approximately 8 hours.
There are several factors worth considering in successfully growing a nitrifying bacterium such as N. europaea. Lack of growth is usually due to an error in one or a combination of the following factors:

  • pH of the growth medium. Ammonia (NH3), not ammonium (NH4+; pKa= 9.25) is the natural growth substrate of N. europaea. The availability of ammonia, which is supplied as ammonium salts, is dependent on the pH of the medium. During growth, the medium becomes acidified (see equation), which results in a progressive decrease in the amount of free NH3 in solution. In the laboratory, N. europaea is grown at a pH between 7.8 and 8.0 using a relatively strong buffer to minimize the drop in pH. If N. europaea is not growing reasonably well, a possible cause might be low pH.

  • Water quality. N. europaea is relatively sensitive to metals. While one can grow them well on tap deionized water, more consistent results are obtained when using double distilled water. For example, N. europaea is sensitive to copper and zinc, so if it is not growing well, metal contamination might be the cause of poor growth (e.g. from distillation equipment, containers, faucets, etc.).

  • Rubber. Because N. europaea is strictly lithotrophic; any compound that inhibits ammonia oxidation will hamper growth. Sulfur-containing compounds are very effective growth inhibitors and are found in unexpected places such as red-rubber stoppers. Many volatile sulfur compounds are used in the vulcanization of rubber and these compounds tend to leach out. Sulfur compounds can inhibit ammonia oxidation at concentrations as low as a few ppm.

  • Heterotrophic microorganisms. Nitrifying bacteria may be preyed on by heterotrophic microorganisms as they grow or may excrete compounds that heterotrophic microorganisms can use for growth. Contamination with heterotrophic microorganisms is the bane of nitrifying cultures; once they contaminate a culture of nitrifying bacteria, they are hard to eliminate. You may want to maintain several storage cultures that can be kept viable in a refrigerator for up to 2 months. It is also possible to store the cultures in DMSO (7%) or in Glycerol (35%) at -80°C for long-term storage.

  • Viability. Propagate the cultures every three to four days with actively growing cells. Because 4-day old cells are close to peak activity, they will grow quickly in the fresh culture flasks, minimizing the likelihood of heterotrophic contamination. A culture started with cells from long-term storage (i.e. at -80°C in 7% DMSO or in 35% Glycerol) can take up to a month to show first appreciable growth.

  • Light. Ammonia oxidation is inhibited by intense light. Consider wrapping the flasks in foil or incubating them in a darkened room if you are still having problems growing N. europaea.


    THE MEDIUM RECIPE


    Step 1

    Put 900 ml of deionized water in a 2-liter Erlenmeyer flask.
    Add in sequence:
    3.3 g (NH4)2SO4 (50mM);
    0.41 g KH2PO4
    0.75 ml 1 M MgSO4 stock solution
    0.2 ml 1 M CaCl2 stock solution
    0.33 ml 30 mM FeSO4 /50 mM EDTA stock solution
    0.01 ml 50mM CuSO4 stock solution

    Sterilize it using an autoclave

    Step 2

    Put 400 ml of deionized water in a beaker. Add:
    27.22 g KH2PO4
    2.4 g NaH2PO4
    Adjust pH to 8.0 with 10 N NaOH, and bring it to a final volume of 500 ml with deionized water.
    Sterilize in an autoclave in 100 ml fractions in 250-500 ml Erlenmeyer flasks.

    Step 3
    Prepare 500 ml of 5% (w/v) Na2CO3 (anhydrous)
    Sterilize it in an autoclave

    HOW TO GROW N. europaea

    1) Add 1 x 100 ml aliquot of solution prepared in Step 2 to the flask prepared in Step 1.
    2) Now add 8 ml of the solution prepared in Step 3 to the flask prepared in Step 1.
    3) Finally add 10 ml of 3-day old culture to the flask prepared in Step 1, and incubate it on a rotary shaker (100-150 RPM) at 30°C.
    The bacterium should reach approximately OD600nm~0.1 in three to four days.

    HOW TO MONITOR THE GROWTH OF N. europaea

    The simplest way to monitor the growth of N. europaea is to follow the release of nitrite into the growth medium. The medium contains NH4+ that is stoichiometrically converted to NO2-. We follow nitrite production using a spectrophotometer by monitoring the optical drensity (OD) difference between 352 nm and 400 nm. The nitrite concentration is determined using a milimolar extinction coefficient of 0.0225 mM-1. The nice thing about this assay is that it can be performed directly by sampling the medium with the cells.

    NO2- concentration (mM) = (OD352 - OD400)/0.0225

    N. europaea gives low-growth yields, so do not be surprised if nitrite levels are 20 mM and the medium shows little growth, i.e. looks vaguely turbid. Typical yields from this system are between 0.1-0.2 g wet/weight cells per liter of medium.