**3.5. Temperature**

Many researchers have studied effect posed by temperature incubation on soil DHA and/or on soil microorganisms abundance (Subhani et al., 2001; Ghaly & Mahmoud, 2006; Trasar-Cepeda et al., 2007). Taking into account the important fact that DHA is found inside the viable soil microbial cells only, its activity must be the highest at a temperature close to optimum temperature for microorganisms growth and their development (Wolińska & Stępniewska, 2011).

It is known that, the rate of enzyme catalysis generally increases with increase in temperature until the unfavorable temperature, at which enzyme becomes denaturized and hence its activity reduces (Wolińska & Stępniewska, 2011).

Our investigations were concentrated on investigations of DHA changeability at temperature range 5-30ºC, what reflect natural changes of soil temperature during seasons. Surface layer (0-20 cm) of *Mollic Gleysol* was used for experiment. Soil samples were incubated at the following temperatures: 5, 10, 20 and 30ºC. DHA was measured after 30 h incubations at proper temperature and after ethanol extractions. Absorbance was tested at λ=485 nm (UV-1800 Shimadzu). Received results are presented in Fig. 7.

We found growing, linear trend for DHA with increase of temperature at the range from 5 to 30ºC, what we described by R2=0.97. The differences between DHA level estimated at 5 and 30ºC were significant (*P*<0.01), analogically like between 5 and 20ºC (*P*<0.05). The lowest values of DHA at 5ºC were found, where DHA equaled 1.259 (µg TPF g-1 min-1\*10-6), whereas the same soil sample incubated at 30ºC reached DHA level of 3.149 (µg TPF g-1 min-1\*10-6), what was by c.a. 60% higher in relation to DHA level from 5ºC. Quite high DHA level (2.741 µg TPF g-1 min-1\*10-6) was also estimated at 20ºC, where mentioned value was only by 13% lower than maximum DHA, from 30ºC.

**Figure 7.** The dependence between DHA and temperature incubation in the *Mollic Gleysol*, according to Wolińska & Stępniewska (unpublished data). Averaged values of three replicates with standard deviations are presented

Casida et al. (1964) indicated that incubation of soil samples at 37ºC increased of soil DHA above the value normally observed at lower temperatures.

Trevors (1984) described positive significant correlation among DHA and temperature in the range from 5 to 70ºC and determined r coefficient on the level of 0.99\*. Moreover, study by Trasar-Cepeda et al. (2007) reported that increased temperatures up to 57-70ºC enhanced the product formation in the reaction catalyzed by soil dehydrogenases increased with, explained by the fact that specific substrate (TTC), used for DHA determination, is chemically reduced at high temperatures.

Analogically, Subhani et al. (2001) noted positive correlation in soil samples incubated at 10, 25 and 40ºC (under constant moisture – flooded conditions), what confirmed by r=0.82\*.

As suggested by Cirilli et al. (2012) optimum temperature for soil DHA is 30ºC, what is in agreement with our findings. Similarly, Brzezińska et al. (1998) indicated that under laboratory conditions DHA demonstrated the highest activity at 28-30ºC.
