**3. Results**

#### *3.1. Hydrocarbon Biodegradation*

The analysis of the biodegradation kinetics shows no significant influence of low and moderate levels of biostimulation (NP1 and NP2) on the distribution of most hydrocarbon fractions: total petroleum hydrocarbons (TPH), alkanes, aromatic, and polyaromatic hydrocarbon compounds (Figure 1). The exception is the fraction of polycyclic aromatic hydrocarbons (PAH), where a statistically significant improvement in the effectiveness of biological decomposition is found in the variants with low and moderate levels of nitrogen supplementation (by 10.8% in the NP1 variant, and 15.2% in the NP2 variant, respectively) (Figure 1D). Among all the analyzed groups of hydrocarbon compounds, the inhibitory effect of excessive supplementation with nitrogen compounds is noted. The strongest biodegradation inhibition is noted in the case of PAH, with only 13.9% of this fraction of hydrocarbon compounds degraded after 7 days.

**Figure 1.** Biodegradation kinetics of selected hydrocarbon fractions: TPH (**A**), alkanes (**B**), aromatic compounds (**C**), and PAH (**D**) in the experimental variants.

#### *3.2. Microbial Activity*

Cytometric analysis shows no significant effect of low and moderate doses of nitrogen on the metabolic activity of microbial cells after 24 h of the biodegradation process. In the variant NP3 (excessive biostimulation), a 5.6% decrease in metabolic activity is noted (Figure 2).

The second assessment of metabolic activity, carried out after 168 h of the experiment, shows significant, but slight, changes in the variants NP2 and NP3 (Figure 3). The moderate level of biostimulation (NP2) has a positive effect on the activity of microorganisms (3.2% increase, compared to the control sample). Moreover, excessive biostimulation (NP3) causes a significant decrease in microbial activity (by 17.6%, compared to the control sample) (Figure 3).

**Figure 2.** Cytometric analysis of microbial metabolic activity in experimental variants after 24 h of biodegradation. The metabolically active population (Q2) is marked in green, the metabolically inactive population (Q1) is marked in purple.

**Figure 3.** Cytometric analysis of microbial metabolic activity in experimental variants after 168 h of biodegradation. The metabolically active population (Q2) is marked in green, the metabolically inactive population (Q1) is marked in purple.

## *3.3. Biodiversity Analysis*

The results of the selected biodiversity coefficients analysis are presented in Table 2. Excessive levels of biostimulation (NP3) contribute to a significant decrease in biodiversity (OTU number, Simpson's index, and phylogenetic diversity). In other experimental variants, no significant differences are noted.

**Table 2.** Microbial biodiversity coefficients in the analyzed variants after 168 h of the biodegradation process.


The analysis of the taxonomic structure of bacterial populations shows significant differences between the variants characterized by a low level of nitrogen compounds (NP0 and NP1), and the variants in which the amount of these compounds is at the optimal, and over-optimal, level (NP2 and NP3). In the samples with the levels of nitrogen compounds below the optimal concentration, the domination of the Sphingobacteria (26% for NP0 and 25% for NP1) and Alphaproteobacteria (24% for NP0 and 27% for NP1) is noted. The variants with a lower ratio of carbon to nitrogen show a much higher abundance of the Gammaproteobacteria (39% for NP2 and 41% for NP3) and Betaproteobacteria (24% for NP2 and 28% for NP3). A detailed taxonomic analysis is presented in Figure 4. It should be mentioned that the share of the orders Sphingobacteriales and Xanthomonadales decreases significantly in variants with optimal and excessive levels of nitrogen compounds. The Burkholderiales order shows the opposite trend. Moreover, excessive biostimulation contributes to a large decrease in the share of the Pseudomonadales and Rhizobiales orders, compared to the trials with optimal, or suboptimal, supplementation.

**Figure 4.** The relative abundance of the bacterial orders in the experimental variants after 168 h of the biodegradation process. Taxa with relative abundance below 1% are excluded from the analysis.

The linear discriminant analysis effect size (LEfSe) allows for the selection of the most differentially abundant taxa between a deficient nitrogen level and a sufficient nitrogen level in experimental variants. (Figure 5). The taxa particularly responsive to changes in the supply of nitrogen compounds include Gammaproteobacteria and Betaproteobacteria, as well as the orders Xanthomonadales, Burkholderiales, Sphingomonadales, Flavobacteriales, and Sphingobacteriales.
