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Nitrifying Bacteria, N-BART™ Quality Control

Nitrification serves as the major route by which ammonium is aerobically oxidized to nitrate. There are two steps to nitrification process:

		(1)			(2)
	NH4+ ----------» NO2- ----------» NO3-
	     Ammonium	Nitrite	      Nitrate
               

Nitrifying bacteria are divided according to which of the above reactions they are able to perform:

Group 1 - step (1) only - Nitrosofiers -Nitrosomonas
Group 2 - step (2) only - Nitrifiers -Nitrobacter

The polarized relationship between the nitrifying and the denitrifying bacteria is a problem in the testing of natural samples since the two groups are either producing or utilizing nitrate respectively. In developing a biodetection system for the nitrifying bacteria in natural samples, the terminal product (nitrate) may not be recoverable because of the intrinsic activities of the denitrifying bacteria which are also likely to be present and active in the sample. It is because of this difficulty that the N-BART restricts itself to detecting the nitrosofiers that generate nitrite. This nitrite will be oxidized to nitrate by the nitrifiers only to reappear when reduced back to nitrite by any intrinsic denitrification occurring in the sample.

The nitrifying bacteria are an important indicator group for the recycling of organic nitrogenous materials from ammonium (the end point for the decomposition of proteins) to the production of nitrates. In waters, the presence of an aggressive population of nitrifiers is taken to indicate that there is a potential for significant amounts of nitrate to be generated in waters which are aerobic (rich in oxygen). Nitrates in water are a cause of concern because of the potential health risk particularly to infants who have not yet developed a tolerance to nitrates. In soils, nitrification is considered to be a very significant and useful function in the recycling of nitrogen through the soil. Nitrate is a highly mobile ion in the soil and will move (diffuse) relatively quickly while ammonium remains relatively "locked" in the soil. In some agronomic practices, nitrification inhibitors have been used to reduce the "losses" of ammonium to nitrate.

A common use for the presence of aggressive nitrifying bacteria in waters is that these bacteria signal the latter stages in the aerobic degradation of nitrogen-rich organic materials. Aggressive presence of nitrifying bacteria in water can be used to indicate that there is a potential for the water to have been polluted by nitrogen-rich organics from such sources as compromised septic tanks, sewage systems, industrial and hazardous waste sites and is undergoing an aerobic form of degradation. Nitrification and denitrification are essentially parallel processes that function in reverse sequence of each other. It is recommended that, where a high aggressivity is determined, waters should be subjected to further evaluation as a hygiene risk through a subsequent determination for the presence of nitrates. In soils, the presence of an aggressive denitrifying bacterial population may be taken to indicate that the nitrification part of the soil nitrogen cycle is functional. Nitrification is fundamentally an aerobic process in which the ammonium is oxidatively converted to nitrate via nitrite. Nitrite produced by the denitrification of nitrate may also be oxidized back to nitrate.

Reaction Patterns
This test is an unusual test in that the presence of nitrifying bacteria is detected by the presence of nitrite in the test vial after a standard incubation period of five days. Nitrification involves the oxidation of ammonium to nitrate via nitrite. Unfortunately, in natural samples, there are commonly denitrifying bacteria present in the water and these reduce the nitrate back to nitrite. If denitrification is completed, this nitrite may be reduced further to dinitrogen gas (under anaerobic conditions). That is why this test is laid upon its side with three balls to provide a moistened highly aerobic upper surface where nitrification is most likely to occur. The reagent administered in the reaction cap detects nitrite specifically by a red color reaction. This test is interpreted by the amount of pink-red coloration generated, and the location of this color.

PP - Pink-red color on roughly half the ball (Reaction 1)
RP - All balls are reddened, solution may be pale pink (Reaction 2)
DR - Balls and the solution are reddened (Reaction 3)

This test is different from the other BART tests in that a chemical reagent is added to detect the product (nitrite) after a standard incubation period. The typical reactions are described below:

PP - Partial Pink on the Balls
Clear solution but a pink reaction may be generated on the FID hemispheres indicating that nitrification has just begun and the nitrite detected is in the biofilm on the balls.

RP - Red Deposits and Pink Solution
Reaction causes a light pink solution with red deposits all over the three balls. Nitrite is now present in solution as well as in the biofilms on the balls.

DR - Dark Red Deposits and Solution
Reaction causes dark red solution with heavy red deposits on ball. High concentrations of nitrite have been detected indicating an aggressive level of nitrification has occurred in the test period.

RPS (Reaction Pattern Signatures )
The reaction represents the population size and does not reflect the variety of microorganisms present in the water sample:

  • PB Small population of nitrifiers (<102 nitrifiers/ml) associated with aerobic slime-forming bacteria in a consortium
  • RB Moderate population of nitrifiers (>102 and < 105 nitrifiers/ml) forming a major component in the bacterial flora
  • RT Dominant population of nitrifiers (>105 nitrifiers/ml)

    Hygiene Risk
    The presence of an aggressive population of nitrifying bacteria in water is taken to indicate that there is a potential for significant amounts of nitrate to be generated in waters, which are aerobic. This may indicate a potential health risk particularly to infants who have not yet developed a tolerance to nitrates. It is recommended that, where a high population is determined, waters should be subjected to further evaluation as a hygiene risk, through subsequent determination for the presence of nitrates.

    N-BART™
    This test detects the nitrifying bacteria that are able to oxidize ammonium (NH4) to nitrite (NO2) and on to nitrate (NO3). This test uses a selective medium for the bacteria able to oxidize ammonium to nitrite by examining chemically for the nitrite product. The additional two balls used in this test provide a larger solid: medium: air area on the upper hemispheres of the three balls. This encourages nitrification in the liquid film over the balls. In the early stages, the first (product) nitrite is detected at these sites. A reactant cap is used to detect the presence of nitrite that is generated during the early stages of nitrification. If the sample being tested also contains denitrifying bacteria, nitrite may again be created by the reduction of nitrate (denitrification). This test method has been developed in consideration of the greater likelihood of nitrite being detectable rather than the (product) nitrate. Note that this test cannot function in water samples with a natural nitrite level of greater than 3.0 ppm. Water samples with greater than 28 ppm of nitrite will automatically turn the liquid medium to a yellow color when the reaction cap test is applied.

    N-BART medium
    Circular white crystalline opaque deposit remains clustered around the central peg in the basal cone. This extension may be 5 to 8 mm in radius with a defined largely smooth edge. In the normal event of the confirmation of sterility, there is a change in the characteristics of the liquid medium forming in the test vial. These are detailed in Table Forty below.

    N-BART Reaction Cap
    This cap is a small screw type white plastic cap which can be screwed down onto the inner test vial. This cap contains rough porous paper disc fitted within the inner flange. When viewed from the under side, this disc is colored. It varies from a solid pink to a yellowish pink center with a darker pink perimeter. This color is generated by the reactants used to detect nitrite in the test medium. While there is a variation in the color, it has been found that this does not affect the accuracy of the test method.

    Table Forty Medium Diffusion in a Sterile N-BART Inner Test Vial to Confirm a Negative Reaction
    Time (days) Color Basal Lower column Upper column 0.25 Grey Clear Clear 0.5 Clear Clear Clear 10.0 Clear Clear Clear

    *Note that the liquid medium is crystal clear and has been generated using sterile distilled or deionized water. Natural water samples can cause minor chemical reactions which may be seen through an intensification of the color in the diffusion front and occasionally crystalline deposits may form along the floor of the test vial.

    Table Forty-One Characterization of Medium Diffusion in a Sterile N-BART inner test vial
    Time (days) Color Contamination Basal Mid-column 1.0 Clear Clear Cloudy 10.0 Clear Clear Turbid, possible gassing

    The N-BART is a relatively specialized test in which the medium will not support a wide range of contaminants. Where there is contamination, the initial expression of growth is a light clouding (day 1) which gradually intensifies causing the medium to go turbid. If there are any complete denitrifiers among the contaminants, gassing may occur.

    Confirmation of the Selective Media Composition in the N-BART
    In order to confirm the suitability of the selective medium for the biodetection of the various bacteria recognized by this test method, it is recommended that the following A.T.C.C. (American Type Culture Collection) strains be applied to the N-BART biodetectors to determine the standard reaction patterns. Each culture should be prepared as a 7 day culture incubated at 25oC to reach the stationary growth phase. The cultural techniques to be used are referenced in Verhagen et al. (1993) "Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Soil Columns" in Journal of Appl. and Environmental Microbiology. Inoculation of the inner test vial should be with a 0.1ml suspension of the broth culture in 15 ml of the sterile Ringer's solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated. 7.75 ml of this inoculated solution should be applied directly to the test vial. Do not shake the vial. Incubate at 22 to 24oC for five days and observe for activities and reactions after applying the reactant cap following the standard procedure. Typical results are listed below for the recommended A.T.C.C. strains in Table Forty-Two.

    Table Forty Two Cultural Characterization of the N-BART
    A.T.C.C. Genus/species Characterization/solution 25391 Nitrosomonas winogradski clouding, red reaction, nitrite + 27853 Pseudomonas aeruginosa -, no reaction, nitrite negative 19718 Nitrosomonas europae clouding, red reaction, nitrite +

    QC Procedure for the N Reactant Cap
    Prepare or obtain analytical grade solutions of sodium nitrite stock solution and dilute standard sodium nitrite solution (5mg and 50mg N-NO2/L). These analytical solutions should be labeled nitrite 5, and nitrite 50 and should be freshly prepared using sterile distilled water and aseptic techniques. The protocol for testing the reactant cap would be similar for all solutions. This protocol would involve the following steps:

    A. Sodium nitrite stock solution (solution A)
    Dissolve 100mg NaNO2 in 50ml distilled water. Transfer it to a 200-ml volumetric flask. Add distilled water to mark line. Final concentration is 500mg/L.

    B. Dilute standard NaNO2 solution:

    Solution B1: Pipet 0.5ml solution A to a 50-ml volumetric flask. Add distilled water to mark line. Final concentration is 5mg/L.

    Solution B2: Pipet 5.0ml solution A to a 50-ml volumetric flask. Add distilled water to mark line. Final concentration is 50mg/L.

    Prepare the solutions before use.

    C. Prepare three tubes. Put one white ball in each tube. Pipet 15mls of solution B1 into each tube. Prepare another three tubes. Put one white ball in each tube. Pipet 15mls of solution B2 into each tube. Screw on D-RX cap. Turn over onto caps for 30 minutes. Turn back and observe the color change after 3 hours. For the tube with solution B1, the solution color will be pink. For the tube with solution B2, the solution color will be yellow.

    Refer to Table Forty-Three for the interpretation of the observations.

    Table Forty-Three Reaction Confirmation for the QC on the N-BART Reactant Cap
    Solution Color Absorption Nitrite, 5 pink 0.36 @500nm Nitrite, 50 yellow 0.30 @580nm

    Note: In weak reactions for the nitrite solutions, a weakened reaction giving lower color generation and absorption values suggests a degenerating reactant cap.

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