**3.6. Season of the year**

192 Dehydrogenases

**Figure 6.** Relationship between DHA and pH values in the *Mollic Gleysol* (n=18, r=-0.70\*\*), according to

pH

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 &

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

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

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%

Wolińska & Stępniewska (unpublished data)

DHA (µg TPF g-1 min-1)

lower than maximum DHA, from 30ºC.

hence its activity reduces (Wolińska & Stępniewska, 2011).

λ=485 nm (UV-1800 Shimadzu). Received results are presented in Fig. 7.

**3.5. Temperature** 

Stępniewska, 2011).

Seasonal variations in both microbial biomass and soil enzymatic activities reflect the combine effects of temperature, moisture, substrate availability and other environmental factors. Dehydrogenases belong to the enzymes displaying strong fluctuations in their activities caused by season of the year, as they are in close relationships with dynamic of microbial activity.

Yuan & Yue (2012) stated the highest DHA level in autumn season and the lowest value of DHA in winter time. The study performed by Piotrowska & Długosz (2012) indicated that DHA level in *Luvisols* revealed significantly higher values in April (by 96%) than in August, probably due to intensive winter wheat growth with an increased secretion of substrates such as polysaccharides, organic acids, which may have affected the growth and activity of microorganisms.

Dehydrogenase Activity in the Soil Environment 195

and is the reason of slight DHA increase during this time. Moreover, taking into account that DHA is present inside viable microbial cells, its activity must be the highest at temperature 20-30ºC (temperature characteristic for summer and early autumn), close to the optimum temperature for microbial growth, activity and development (Wolińska &

Some of environmental factors have ability to affect negatively on DHA, by reducing its activity. In the role of enzyme inhibitors usually different molecules are involved, which by binding to enzymes activation sites are the reason of prohibition the enzymes from catalyzing its reaction, and finally decrease their activity. The most important soil factors

Depth of the soil profile is one of the most known and popular environmental factor reducing soil DHA level. It is well known that the highest microorganisms abundance is in the surface layer of the soil profile (till to the depth of 30 cm), at the deepest part of the soil the number of microbial cells is limited, and consequently also DHA level display

**Figure 9.** DHA (µgTPFg-1min-1) at different depth of the *Mollic Gleysol* profile (n=18, *P*<0.001), according

The confirmation of the above statement might be the Fig. 9, where effect of depth on DHA in *Mollic Gleysol* is presented. The highest level of DHA we noted in surface layer (0-20 cm),

**4. Soil factors inhibiting dehydrogenase activity** 

inhibiting soil DHA are described below.

**4.1. Depth of the soil profile** 

to Wolińska founding (unpublished data)

diminishing trend.

Stępniewska, 2011).

Similarly, our investigations demonstrated the highest level of DHA in *Eutric Fluvisol* sample taken in May (0.0087 µg TPF g-1 min-1), than in the same soil type taken in October, where DHA was reduced by 42.5 % (Fig. 8). Quite high level of DHA (lower by 14.9% from its maximum reaching in May) we also noted in July. Moreover, we did not found significant differences (*P*>0.05) in DHA values during autumn season, where DHA remained on similar level equaled 0.000598 (µg TPF g-1 min-1) and 0.0005 (µg TPF g-1 min-1), for September and October, respectively.

**Figure 8.** Effect of the season of the year on soil DHA in the *Eutric Fluvisol*, according to Wolińska & Stępniewska (unpublished data). Averaged values of three replicates with standard deviations are presented

Analogical trend like our observations, related to high Oxidoreductases activity at the time form spring to autumn was noted by Januszek (1993). A study by Włodarczyk (2000) performed on *Orthic Luvisol* sample, showed that DHA demonstrated seasonal pattern and reached the highest values in September, whereas the lowest in winter time. Similarly effect noted Tripathi et al. (2007), who indicated maximum DHA in September and its reduction in January.

Spring season is strongly connected with increase in microbial activity, intensification of oxido-reduction reactions and temperature change, what is indirectly impacted with DHA, and is the reason of slight DHA increase during this time. Moreover, taking into account that DHA is present inside viable microbial cells, its activity must be the highest at temperature 20-30ºC (temperature characteristic for summer and early autumn), close to the optimum temperature for microbial growth, activity and development (Wolińska & Stępniewska, 2011).
