The use of oxygen as the final electron acceptor is more energtically favored than the use of nitrate. By oxygen respiration the energy yield per mole of glucose is 686 kcal/mole and by nitrate dissimilation the energy yield per mole glucose is only 570 kcal/mole.
The greater free energy released for oxygen favors its use whenever it is available. Therefore, denitrification must be conducted in an anoxic environment to ensure that nitate, rather than oxygen, serves as the final electron acceptor. Methanol, ethanol, acetic acid, have been most frequently used as the electron donor in experiments, rather than glucose.
Using methanol as an electron donor and neglecting synthesis, denitrification can be represented as a two-step process as shown in equations (4.1) and (4.2). First step:
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