Dehydrogenase Activity of Soils 8.1. OVERVIEW Objective: To demonstrate the dehydrogenase activity associated with soil microorganisms. + Set up two soil incubations: i) unamended control soil; and ii) soil + ‘glucose. Also set up a blank—no soil + Incubate all vials with triphenyltetrazotium chloride (TTC) under saturated soil conditions for 1 week + Extract both soil treatments and the blank for reddish colored triphenyl formazan (TPF) through the use of methanol + Analyze TPF concentrations spectroscopically to give an estimate of dehydrogenase activity 8.2. THEORY Dehydrogenase are enzymes that are found in all living organisms (Maier cet al., 2000). These enzymes take part in many reactions invoiving the trans- for of pairs of electrons. An example of a dehydrogenase mediated electron transfer is given in Figare 8-1. In catabolic reactions, ie., reactions involving the modification of complex or high-energy compounds to simpler or low- ‘energy compounds, dehydrogenases catalyze the transfer of electron pairs from some substrate to NAD* forming NADH. NADH then transfers the electrons to another compound, thereby serving as an electron transfer inter- ‘mediary. In anabolic reactions (the opposite of catabolic reactions), NADP” is involved instead. Two hydrogen atoms “tag” along to keop the charge balanced; it is the electrons that are being transferred. As dehydrogenases also take part in the electron transfer system of aerobic organisms, the activ- ity of these enzymes isa measure of respization along with geacral metabolic activity. [By far the most commonly used version of the dehydrogenase assay is that ‘of Casida ot al. (1977). This assay usually iavolves incubating soil mixed with a solution of the competitive NAD" inhibitor, 2,3.5-triphenyltetrazolium chloride (TTC) with soil in airtight containers to exclude oxygea. Here the TTC serves as the ultimate electron acceptor during respiration. The soil should be freshly collected as research has shown that dehydrogenase assay results are adversely affected by storage, even at 4°C (Ross, 1970), Exclusion of oxygen favors the transfer of the eiectrons to TTC, reducing the pale yellow, slightly water-soluble compound to the insoluble rod dye triphenyl formazan (IPF) (see Figure 8-1). In fertile soils high in soil organic matter (Figure 8-2), 20 nuteient amend- ‘meat is needed, and in fact, one of the advantages of the dehydrogenase assay over other eazyme assays is that it does not require any amendments, EXPERIMENTFgWe £1 Normal Reaction NAD* is reduced to NADH through a transfer of two electrons land two hydrogen ions in the presence of dehydrosenase from numerous compounds (only one Of the two hydrogen ions actually participate in the reaction and, therefore.only one is depicted here), [Natural Coenzyme Here, the chemical transformations occurring to NAD* during its reduction to NADH are depicted, The incoming H’ (in extra bold face type) is reduced in the presence of the dehydrogenase enzyme. The NADH is used to reduce ather compounds ‘Synthetic Coenzyme Analog TTC 2.35-Triphenyltetrazolium chloride (TTC) accepts one H* and two electrons ia # manner similar éo NAD" Hence, TTC is being reduced to teipheny! ‘mazan in the presence of dehydrogenase, acts as a competitive NAD" inhibitor. However, alike NADI, the triphenyl formazan is a metabolic dead end. This poisons the system docs make it possible to measure metabolic activity due to the red color. volved but and does not preferentially stimulate any group of microorganisms. However, addition of an organic amendment is necessary in soils of low fer- tility status, such as desert soils (Klein et al., 1971). After incubation, the soils are extracted with a solvent to remove the TPF, ‘Casida et al. (1977) used ethanol, which has become the standard. Following extraction, the concentration of TPF in the svil is determined by absorption spectrophotometry. The dissolved analyte, TPF, absorbs light in the visible region of the spectrum, with A= 485 nm best for analytical purposes. Over a certain range of concentration, the degree to which the TPF absorbs 485nm. light is a linear function of concentration of TPF in the methanol solution.