**3.2. Soil aeration state (redox potential and oxygen diffusion rate)**

Oxygen diffusion rate (ODR) is usually considered to be the most critical proximal regulator of microbial activities (Hutchinson, 1995). Moreover, it is often assumed that a decrease of soil water content (higher value of pF), cause a significant (*P*<0.001) increase of ODR and redox potential (Stępniewski et al., 2000; Wolińska & Bennicelli, 2010). The available literature shows that low ODR level, ranged below its critical values (35 µg O2 m-2s-1), is favorable and optimal for DHA (Stępniewski et al., 2000; Brzezińska et al., 2001; Wolińska & Bennicelli, 2010).

We confirmed that dehydrogenases are sensitive enzymes, indirectly depended on the soil aeration status (Wolińska & Bennicelli, 2010). Based on performed measurements we found, that pF constitutes a significant factor, determining ODR in the soil environment, as well as its DHA level (*P*<0.01). The reoxidation processes, occurring in the direction from pF 0 to pF 3.2, were the reason of DHA inhibition and stimulation of ODR level in the *Rendzina Leptosols* and *Eutric Fluvisol* soil samples (Fig. 2). We also stated that soil DHA at pF 3.2 was lower by about 60.86%, in comparison to the activity estimated at pF 0.

186 Dehydrogenases

Stępniewska, 2011).

and Weaver et al (2012).

**DHA response** 

*Rendzina Leptosols* 0-20

*Eutric Fluvisol* 0-20

(R) (95% LSD method, n=15), according to Wolińska & Bennicelli, 2010

than aerobically incubated soils (Trevors, 1984; Subhani et al., 2001).

**3.2. Soil aeration state (redox potential and oxygen diffusion rate)** 

correlations are undoubtedly connected with the fact that the metabolism and the survival of soil microorganisms are also strongly impacted by the availability of water (Uhlirova et al., 2005), what is essential for microbial survival and activity. Consequently, low water availability can inhibit microbial activity by lowering intracellular water potential, and thus by reducing of hydration and enzymes activity (Wall & Heiskanen, 2003). Periods of moisture limitation may affect microbial communities through starvation. Thus, the most common environmental stress for soil microorganisms is perhaps drought (Wolińska &

It was shown in many studies that DHA is significantly influenced by water content and dropped with the decrease of soil humidity. For example, Gu et al. (2009) observed higher DHA level (even by 90%) in flooded soil, rather than in non-flooded conditions. The higher DHA values in flooded conditions agreed also with results presented by Zhao et al. (2010)

**Depth**

50-60

50-60

**Table 1.** Statistical significance of differences between DHA and pF described by correlation coefficient

The decline of DHA with an increase of pF value, could be also explained by the fact, that flooding of soil with water significantly increased the electron transport system (Wolińska & Stępniewska, 2011). Dehydrogenases however, are responsible for electron transport in the soil environment. It was also reported that DHA is higher in flooded, anaerobically soils,

Oxygen diffusion rate (ODR) is usually considered to be the most critical proximal regulator of microbial activities (Hutchinson, 1995). Moreover, it is often assumed that a decrease of soil water content (higher value of pF), cause a significant (*P*<0.001) increase of ODR and redox potential (Stępniewski et al., 2000; Wolińska & Bennicelli, 2010). The available literature shows that low ODR level, ranged below its critical values (35 µg O2 m-2s-1), is favorable and optimal

We confirmed that dehydrogenases are sensitive enzymes, indirectly depended on the soil aeration status (Wolińska & Bennicelli, 2010). Based on performed measurements we found, that pF constitutes a significant factor, determining ODR in the soil environment, as well as its DHA level (*P*<0.01). The reoxidation processes, occurring in the direction from pF 0 to pF

for DHA (Stępniewski et al., 2000; Brzezińska et al., 2001; Wolińska & Bennicelli, 2010).

\*, \*\*, \*\*\* - indicate significance at the 5, 1 and 0.1% level, respectively, n.s. – not significant differences

**(cm) pF** 



**Figure 2.** The response of soil DHA to varied aeration factors (pF and ODR), at surface layers of *Rendzina Leptosols* and *Eutric Fluvisol* , during reoxidation process (according to Wolińska & Bennicelli, 2010). Averaged values of three replicates with standard deviations are presented

The Figure 3. demonstrates that low oxygen diffusion rate (2.8-25 µg O2 m-2 s-1) was optimal for DHA, what was also confirmed by correlation coefficient (Wolińska & Bennicelli, 2010; Wolińska, 2010). Our results and founding's are compatible with work of Stępniewski et al. (2000), Brzezińska et al. (2001), and Yang et al. (2005).

Statistical relationships between DHA and ODR, determined for two soil types (*Rendzina Leptosols* and *Eutric Fluvisol*) by Wolińska & Bennicelli (2010) are presented in Table 2. At every case negative correlations DHA-ODR were determined.

**Figure 3.** Relationship between DHA (µgTPFg-1min-1 \* 10-6) and ODR (µg m-2 s-1), in surface layer of different mineral Polish soil types (n=315, *P*<0.05), according to Wolińska (2010)


Dehydrogenase Activity in the Soil Environment 189

/NO2-

, Fe3+/Fe2+,

Negative correlations DHA-Eh were also described by Brzezińska et al. (1998), who determined r=-0.75\*\*\*, r=-0.83\*\*\* and r=-0.87\*\*\* for temperature 10, 20 and 30ºC, respectively,

Mentioned relationships DHA-Eh have significant negative character, what means that increase of soil DHA level is indirectly connected with decrease of Eh values, as most of microorganisms, which are responsible for DHA prefer rather anaerobic conditions, and belong to obligate anaerobes. What is more, anaerobic conditions are consequence of flooding and decrease of oxygen availability in soil environment. Competition for oxygen limits aerobic processes and the subsequent oxygen deficiency creates local anaerobic microsites, which stimulates growth of anaerobic bacteria (Wolińska & Stępniewska, 2011), and finally DHA. Also, in the absence of oxygen in the soil a decline of Eh and the reduction of oxidized forms (nitrate, Mn4+, Fe2+ and SO42-) takes place. Bohrerova et al. (2004) reported

and Mn4+/Mn2+. In the literature data, it was also assumed that DHA is strongly affected by

Soil organic matter (OM) has important effects not only on soil enzymes activities but first of all on microorganisms activities. Soil OM has been considered as an indicator of soil quality (similarly like dehydrogenases,) because of its character of nutrient sink and source that can enhance soil physical and chemical properties, and also promote biological activity (Salazar et al., 2011). Interestingly, not only amount of OM in the soil is important but most of all its quality, as OM affects the supply of energy for microbial growth and enzyme production

It is evident that soil enzymatic activity is strongly connected with soil OM content. The higher OM level can provide enough substrate to support higher microbial biomass, hence higher enzyme production (Yuan & Yue, 2012). Several authors reported positive correlation between DHA and OM content (Chodak & Niklińska, 2010; Moeskops et al., 2010; Romero

Zhang et al. (2010) indicated also that as well DHA and CaCO3 correlated with OM content, and what is more DHA, OM and CaCO3 were correlated with each other in their spatial distribution, suggesting that abundant OM content contributed to the formation of

Salazar et al. (2011) hypothesized that activities of dehydrogenases in different forest ecosystems are involved in the carbon cycling, and they also reported their positive relationships. Dehydrogenases, are highly associated with microbial biomass (MB), which in

We also investigated effect posed by total organic carbon (TOC) and response of DHA in the agricultural used *Mollic Gleysol*, taken from Kosiorów village (SE part of Poland). We

turn affects on decomposition of OM and the release of CaCO3 (Zhang et al., 2010).

that the most common ions forming the redox couples of soil include NO3-

both Fe as Mn presence in the soil (Brzezińska et al., 1998; Włodarczyk et al., 2002).

and by Stępniewski et al. (2000), and Nyak et al. (2007).

**3.3. Organic matter content** 

(Fontaine et al., 2003).

pedogenic calcium carbonate.

et al., 2010; Zhao et al., 2010; Yuan & Yue, 2012).

\*, \*\*, \*\*\* - indicate significance at the 5, 1 and 0.1% level, respectively, n.s. – not significant differences

**Table 2.** Statistical significance of differences between DHA and ODR described by correlation coefficient (R) (95% LSD method, n=15)

Redox potential (Eh) is the next, important, environmental factor, which expresses the tendency of an environment to receive or to supply electrons in solution (Stępniewski et al., 2005). The well-oxygenated soils are characterized by high values of Eh (600-800 mV), in quite well-oxygenated soils Eh ~ 500-600 mV, whereas in anaerobic conditions drop of Eh below 300 mV or even lower values were observed (Pett-Ridge & Firestone, 2005; Stępniewski et al., 2000).

It is well known, that Eh play a crucial role in regulating microbial activity as well as community structure (Pett-Ridge & Firestone, 2005; Song et al., 2008), and affecting on soil enzymatic activity, especially DHA. Brzezińska et al. (1998) indicated that among all aeration parameters, Eh plays the most important role in determining soil DHA level. Similar conclusions were also reported by Włodarczyk et al. (2001) and Menon et al. (2005).

We founded significant negative relationships between DHA and Eh (Fig. 4) at surface layers of *Mollic Gleysols*, *Eutric Fluvisols*, *Rendzina Leptosols* and *Haplic Phaeozems*, where determined correlation coefficients equaled as follows: r=-0.91\*, r=-0.43\*, r=-0.47\*\* and r=- 0.48\*\* (Wolińska, 2010).

**Figure 4.** Relationship between soil DHA level and Eh at *Mollic Gleysol* (n=9, r=-0.91\*), according to Wolińska (2010)

Negative correlations DHA-Eh were also described by Brzezińska et al. (1998), who determined r=-0.75\*\*\*, r=-0.83\*\*\* and r=-0.87\*\*\* for temperature 10, 20 and 30ºC, respectively, and by Stępniewski et al. (2000), and Nyak et al. (2007).

Mentioned relationships DHA-Eh have significant negative character, what means that increase of soil DHA level is indirectly connected with decrease of Eh values, as most of microorganisms, which are responsible for DHA prefer rather anaerobic conditions, and belong to obligate anaerobes. What is more, anaerobic conditions are consequence of flooding and decrease of oxygen availability in soil environment. Competition for oxygen limits aerobic processes and the subsequent oxygen deficiency creates local anaerobic microsites, which stimulates growth of anaerobic bacteria (Wolińska & Stępniewska, 2011), and finally DHA. Also, in the absence of oxygen in the soil a decline of Eh and the reduction of oxidized forms (nitrate, Mn4+, Fe2+ and SO42-) takes place. Bohrerova et al. (2004) reported that the most common ions forming the redox couples of soil include NO3- /NO2- , Fe3+/Fe2+, and Mn4+/Mn2+. In the literature data, it was also assumed that DHA is strongly affected by both Fe as Mn presence in the soil (Brzezińska et al., 1998; Włodarczyk et al., 2002).
