## 4 Discussion
### Microbial Stress Indicators  
Various approaches for studying stress affecting soil microorganisms are available. To characterize the initial state of microbial communities in this study, it was decided to use the combination of selected enzymatic activities and PLFA profiling as used in previous studies [11; 36; 45]. Based on this data, it could be gathered that the OIL variant was the most beneficial one for microorganisms because it exhibited the highest amount of living microbial biomass (PLFA\({}_{\text{tot}}\)), soil respiration and dehydrogenase activity, referring to overall soil microbial activity. Most likely, microorganisms capable of utilizing petroleum contamination as a source of carbon and energy were present since they are generally ubiquitous in soils [62].  
For stress effect determination during the experiment we focused explicitly on \(cy/pre\) PLFA stress indicator, which has proven to be suitable for microbial development monitoring in previous environmental studies, for example, on phytoremediation [11], the effect of tourist traffic in caves [45] and effect of oxidation agents during bioremediation [36]. The increase of cyclopropyl fatty acids in microbial membranes in stress conditions is explained as protective mechanisms occurring during the physiological change from exponential growth to stationary phase (cyclopropyl fatty acids are less rapidly biodegraded), and they are considered indicators of growth inhibition [47; 63]. This is most likely caused by insufficient nutrients or their suboptimal ratio, but it can have other reasons such as pollution or soil compaction [45; 47]. Calculation of this indicator is conditioned by the presence of G-bacteria, yet in disturbed and contaminated soils, the abundance of G-bacteria is usually high [47]. That was also our case; the G-PLFA dominated in all samples.  
Initial \(cy/pre\) PLFA results in our soils were contrary to total microbial biomass and activity, i.e. the highest \(cy/pre\) values were found in OIL soil. The lowest \(cy/pre\) stress indicator was determined in AGR soil, the median value was in MIL. These initial values correlated with the stress of plants. Nevertheless, the disadvantage of this indicator is that it cannot identify the specific cause of stress. Dominantly, the \(cy/pre\) values are connected with nutrition deficiencies (in our case, the ratios of C:N and C:P are very likely). However, the contamination concentration and synergy of contaminants or high pH and Ca content could also increase stress. During two vegetation seasons in AGR and MIL \(cy/pre\) PLFA continuously decreased below significant stress value 0.4 (MIL) or stayed at a non-stressed level (AGR). The same development was reported in our previous pot study with miscanthus in soil with elevated concentrations of As, Zn, Cu, Sr and Zr [11]. On the contrary, in the OIL variant with soil from petroleum sludge, it increased continuously.  
That can lead to the hypothesis that well-developed plat roots in AGR and MIL soil could help with microbial community stabilization and stress reduction, while a weak small plant with stunted roots in OIL soil did not support soil microorganisms. That is in agreement with some previous conclusions. [PERSON] et al. [58] reported increased soil biological activities (basal soil respiration and selected enzymatic activities) and increased microbial biomass carbon in a field planted with miscanthus compared to an unplanted area. The short-term effect of miscanthus cultivation on soil microorganisms compared to plot with conventional wheat cropping system with annual crops in soil contaminated by long-term wastewater irrigation was studied by [PERSON] et al. [64]. According to their results, miscanthus stimulated several genera of bacteria that are involved in plant-bacteria symbioses such as _Rhizobiales_ and _Nitrospira_.