Biochemical Pathways

Denitrification is a two-step process in which the first step is a conversion of nitrate into nitrite. The second step carries nitrite through two intermediates to nitrogen gas. This two-step process is normally termed "dissimilation". Each step in the denitrification process is catalysed by a separate enzyme system.

Denitrifiers are also capable of an assimilation process whereby nitrate (through nitrite) is converted into ammonia. Ammonia is then used for the nitrogen requirements of the bacteria cells. The step or steps, from nitrite to hydroxylamine are not fully known.

Table 4.2 Methods for the identification of denitrifying bacteria.

Method Reference

Chromatographic techniques Payne (1973)

Tood and Nuner (1973)

MPN-technique Tood and Nuner (1973)

Measurements of the enzymatic activity plates Lenhard (1969)

If ammonia is already present, for example in a nitrification plant, assimilation of nitrate need not occur to satisfy cell requirements.

The transfer of electrons from the carbon source (the electron donor) to nitrate or nitrite (the electron acceptor) to promote the conversion into nitrogen gas, will be discussed in detail in Section 4.4. It involves the "electron transport system" of the denitrifiers and consists of the release of energy from the carbon source for the use in the growth of the organism. This electron transport system is identical to that used for respiration by organisms oxidizing organic matter aerobically, except for one enzyme. Because of this very close relationship, many facultative bacteria can shift between using nitrate (nitrite) or oxygen rapidly and without difficulty.

Most investigators consider oxygen an inhibitor in the denitrification process. But some species have been reported to denitrify in systems with oxygen tension still as high as 0.2 atm. Table 4.3 show the metabolic processes in biological denitrification.

There is also evidence that nitrification and denitrification may occur simultaneously in soil or when applying special porous media, as for example clinoptilolite. Though not fully explained, these phenomena may occur in anaerobic micro-zones in otherwise aerobic systems (Masuda etal. 1987,1990; Watanabe 1990; Halling-Sorensen and Hjuler 1992; 1993).

Many nitrate-reducing bacteria exhibit both dissimilatory and assimilatory behaviour. From an engineering point of view the ratio between dissimilated and assimilated nitrogen is of interest, as it is more desirable to produce nitrogen gas than to produce organic nitrogen bound in bacteria. Christensen and Harremoes (1977) and Painter (1970) indicate the yield coefficient for denitrifying bacteria Ycjenu to be approximately 0,4 mg VSS per mg N03" - N. If the nitrogen content in the organic matter is 10%, then 0,04 mg N is assimilated for every 1 mg N03" - N converted into nitrogen gas.

An electron transport system for nitrate reduction is shown in Table 4.3, example 3. The steps from the electron donor to the cytochrome are always identical, while the final steps depend upon the final electron acceptor (nitrate, nitrite etc.).

Different species of bacteria may have slightly different electron transport systems, in particular in respect to quinone and cytochrome (Painter 1970).

For each of the steps in the dissimilatory nitrate reduction sequence a reductase enzyme has been isolated (Mudrack 1971).

Table 4.3 Metabolic processes in biological denitrification.

1 : Dissimilatory nitrate reduction (denitrification). N03" -> N02" -ยป NO -> N20 N2

2: Assimilatory nitrate reduction (synthesis). N03" -> N02" -> X -> NH2OH Org. N

3: Possible electron transport system of the first step of denitrification.

e" donor -> NAD FAD Quinone Cytochrome -> Nitratereductase N03

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