Dehydrogenase activity measurement

The microbial oxidation of organic substances under aerobic conditions is linked to a membrane-bound electron transfer chain with O₂ as a final electron acceptor. The electron transport system is coupled with the synthesis of ATP, the so-called "oxidative phosphorylation.

It became clear in the 1930s that the measurement of one or more enzyme activities in the respiratory chain could be used as an index for the total oxidative activities of the cell therefore, dehydrogenase activity in soil has been used as a measure for overall microbial activity (Alef, 1995).

One of the most frequently used methods to estimate dehydrogenase activity in soil is based on the use of triphenyltetrazolium chloride (TTC) as an artificial electron acceptor (Alef, 1995). The TTC is water soluble, has a redox potential of about -0.08 V and functions as an electron acceptor for several dehydrogenases (Alef, 1995). Dehydrogenases, similarly to other enzymes reduce TTC to triphenyl formazan (TPF). Nearly all microorganisms reduce TTC to TPF, which can be colorimetrically estimated. Therefore the method is based on the reduction rate of TTC to TPF in soil after incubation for 24 hours at 30°C.

Chemicals and solutions:

1. Tris-HCl buffer: Dissolve 12.1 g of Tris (hydroxy methyl)-aminomethane in 700 mL distilled water, adjust with HCI to pH 7.6. Bring up with distilled water to 1000 ml.

            2. TTC solution: 1.5 g TTC is dissolved in 80 ml Tris-HCl buffer, then made up with the same buffer to 100 ml.

           3. TPF standard solution: 50 mg TPF is dissolved in 80 ml acetone and brought up with acetone to 100 ml. Pipette 0, 0.5, 1.0, 2.0, 3.0, 4.0 ml and 5 mL of TPF standard solution in a 50 mL volumetric flasks, add 8.3 ml Tris buffer and make up with acetone to 50 mL. The optical density of the calibration series is measured at 546 nm and the calibration curve is prepared.

Procedure:

20-20 g of field-moist soil and 10-10 mL TTC solution is weighed into 100 mL tubes sealable with rubber stoppers then shaken thoroughly for 20 hours at 25 °C. Instead of the TTC solution only 10-10 mL of Tris-HCl buffer is added to the blank. After incubation 40 mL acetone is added to each sample and then shaken for 2 more hours. The acetone extracts the produced TPF. After 10 minute settling the soil suspension is filtered and the optical density of the clear supernatant is measured against the blank at 546 nm (red colour) by a SANYO SP55 UV/VIS spectrophotometer.

Evaluation and interpretation of the results:

The TPF concentrations are read from the calibration curve, after having the absorbance readings, then the dehydrogenase enzyme activity/g soil is calculated based on the below formula:

Dehydrogenase enzyme activity = (μg TPF/ml*VA+TTC)/(szt*nt)

where

szt: dry weight of moist soil (g), 

VA+TTC:  volume of added acetone and TTC solution (ml)

nt: the weight of the moist soil (g).