4. DETERMINATION OF AMMONIA AND NITRATE UPTAKE IN LAKE WATER WITH ENZYME KINETICS

Plants, both microphytes and macrophytes utilize the various forms of nitrogen in different ways. Nitrogen is most actively utilized in forms of ammonia and nitrate but numerous nitrogen-containing organic compounds are also suitable as nitrogen sources for plants. The determination of ammoniauptake and nitrate-uptake is important with regard to quantitative description of nitrogen cycle in water bodies.

The rate of nitrogen assimilation may be calculated from the increase in phytoplankton biomass on addition of nutrients (Thomas, 1953). The best method for determining the ammonia and nitrate uptake is that of measuring the uptake of ¹⁵N-labelled substrate. However, it requires equipment such as ¹⁵N-analyser and mass spectrometer. The method of measuring the reduction in concentrations of ammonia and nitrate in water at different substrate levels, with simultaneous inhibition of substrate producting/consuming processes is a suitable method for uptake studies.

4.1 PRINCIPLE

The decrease in the concentrations of ammonia and nitrate is measured at natural, twofold, threefold, fourfold and fivefold substrate concentrations, during a short incubation time (with indophenol blue and cadmium reduction method), with the simultaneous inhibition of nitrification with N-serve. The kinetic parameters, ^Vmax ^Km and T are calculated with the Michaelis-Menten equation.

4.2 MATERIAL REQUIRED

• reaction flask

• nurser bottle with rubber teat (300 cm³)

• beakers (600 cm³)

• graduated pipette (5 cm³)

• Perlon string

• lead sinker

• tools necessary for ammonia and nitrate determination

4.3 CHEMICALS

• Ammonium stock solution (i): (250 μmol NH₄⁺ - N cm^-3) 4.13 g (NH₄)₂SO₄ is dissolved and volume made up to 250 cm³ with ammonia-free distilled water.

• Diluted ammonium stock solution (ii): (25 μmol NH₄⁺ - N cm^-3). 10 cm³ is taken from stock solution (i) and diluted to 100 cm³ with ammonia-free distilled water (always fresh solution should be prepared).

• Diluted ammonium stock solution (iii): (2.5 μmol NH₄⁺ - N cm^-3). 10 cm³ is taken from stock solution (ii) and diluted to 100 cm³ with ammonia-free distilled water (always fresh solution should be prepared).

• Nitrate stock solution (i): (250 μmol NO₃-N cm³).6.32 g KNO₃ is dissolved and volume made up to 250 cm³ with ammonia-free distilled water.

• Diluted nitrate stock solution (ii): (25 μmol NO₃-N cm³).10 cm³ of stock solution (i) is diluted to 100 cm³ with ammonia-free distilled water (always fresh solution should be made).

• Diluted nitrate stock solution (iii): (2.5 μmol NO₃-N cm^-3).10 cm³ of stock solution (ii) is diluted to 100 cm³ with ammonia-free distilled water.

• N-serve solution: 1 g solid N-serve dissolved in 100 cm³ alcohol (96%).

• Chemicals necessary for ammonia - and nitrate-determination.

4.4 PROCEDURE

The ammonia and nitrate concentrations of the water sample are determined and natural substrate concentrations c (μmol N . cm^-3) are calculated. The nutrient-enriching stock-solution is chosen based on the ranges, as given below:

Further diluted stock solutions may be prepared as necessary, and the standard quantity of the suitable substrate increase stock solution (V_s) can be calculated.

Approximately 500 cm³ water sample is measured into each of the 12 graduated beakers and 0.25 cm³ N-serve solution added and thoroughly mixed.

To adjust the different substrate concentrations, multiple amounts of the standard quantity of ammonia and nitrate (see above) are pipetted using 5 cm³ pipette with an accuracy of ± 0.01 cm³.

Each reaction flask is filled up completely after mixing the solution thoroughly and the remaining solution is saved. The flasks are closed with rubber teats and suspended with Perlon string for in situ incubation of four hours at a water depth from where the sample was taken. In the meantime, ammonia and nitrate concentrations are determined in the remaining part of the sample (^Cb, i NH₄-N and ^Cb, i NO₃-N). Reaction flasks are opened after incubation and ammonia and nitrate concentrations are determined (^Ca, i NH₄-N and ^Ca, i NO₃-N).

4.5 CALCULATION

Uptake rates at each substrate concentration are calculated as follows:

and

If the rate of decrease in substrate concentration is linear with the time of incubation, the uptake rate values are equal to the so-called initial reaction rate. Michaelis-Menten (1913) suggested the following equation to describe the correlation between the initial rate of enzyme reactions and the substrate concentrations.

The so-called Michaelis-Menten equation:

M_max and K_M value can be obtained from the curve (V_max) is the intersect of the curve, where the increase in uptake rate is not corresponding with the increase of substrate concentration, i.e., a substrate concentration corresponding to the half of v_max (Fig. 3).

More reliable v_max and K_M values can be obtained if one of the linear forms of Michaelis-Menten equation is plotted. A possible linear form corresponding to y = mx + b: with this option v_max and K_M can be measured more accurately from the section of axes or can be determined from the rise of the line (Fig. 4).

Lines are plotted from measurements as can be the characteristics of the enzyme reaction obtained from the two variance linear regression analysis. If the regression analysis is orthogonal, kinetic parameters can be clearly made out regardless of the linearization.

The complete set of kinetic parameters (maximum rate of uptake, v_max; maximum natural substrate concentration K_M + S_n; time of exchange T) can be obtained with a slight modification of the Michaelis-Menten equation:

Separating the pre-incubation concentration to a natural and an additional substrate concentration (C_b, i = C_n + C_a,i) the following equation can be described applying the linearizing method of Hanes.

Plotting (C_n + C_a,i) ^vi values as the function of ^Ca,i, the time of exchange (T) can be obtained (Fig. 5).

Figure 3

Figure 4

Figure 5

With the knowledge of maximum natural substrate concentration (K_M + C_n), the maximum rate of uptake (v_max) and the natural substrate concentration (C_n), the actual rate of uptake (v) and the Michaelis constant (K_M) can be calculated. Thus the rate of ammonia and nitrate uptake (v) as related to a unit volume of lake water will be:

The rate of ammonia and nitrate uptake (w) in the whole water column of the lake as related to a unit volume, can be obtained by summing the rate values (v_i) measured in the partial columns.

where: = the depth of water

h_i = the length of partial columns

Surface rate (φ) to characterize the ammonia and nitrate uptake as related to a unit region of a lake is the following:

Ammonia and nitrate uptake in an extended region of the lake during a longer period of time can be expressed by summing the uptake values (I_ik) of different regions in different periods of time.