*3.4. Prediction of Metabolic Properties*

The analysis of the predicted metabolic potential for the degradation of PAH compounds in individual bacterial taxa is presented in Figure 6. In all experimental variants, the orders of Sphingobacteriales and Rhizobiales are characterized by a high-predicted representation of genes encoding enzymes involved in PAH biodegradation.

**Figure 6.** The relative predicted abundance of genes participating in PAH degradation in microbial orders in analyzed experimental variants.

#### **4. Discussion**

Biostimulation is considered a promising method of supporting hydrocarbon biodegradation processes, by eliminating element deficiencies, mainly nitrogen and phosphorus [12,14–16]. Biostimulation can be combined with bioaugmentation techniques, which use microorganisms characterized by a high biodegradation potential, in both single strain and consortia. These microorganisms are often isolated from permanently contaminated

environments, and they are well described in the literature [12,17,18]. Methods using the enzymatic potential of microbes are considered effective for the degradation of most hydrocarbon fractions. However, there are some limitations in the effective decomposition of aromatic compounds, which belong to the fraction that is extremely resistant to biological degradation [40–42]. Similar conclusions are noted in our research, where the aromatic and polyaromatic fractions are the group of compounds most difficult to decompose. The conducted experiment confirms the increase in the efficiency of biological decomposition of the most problematic fraction, PAH, under the influence of low and optimal supplementation with nitrogen compounds (NP1 and NP2 variants). Interestingly, excessive biostimulation contributes to the inhibition of biological decomposition of all analyzed fractions of hydrocarbon compounds. Similar trends are mentioned in other studies [43,44]. Uncontrolled biostimulation can be counterproductive in the context of hydrocarbon remediation.

The decrease in biodegradation efficiency due to the excessive supply of nitrogen compounds is associated with a decrease in metabolic activity, which is noted both after 24 h (by 3.2%) and after 168 h (by 17.6%) of the experiment. The toxic effect of excessive concentrations of nitrogen compounds on the population of microorganisms could be related to the progressive accumulation of their metabolism products, such as ammonia and ammonium ions, or its oxidation products—nitrates. The phenomenon of the toxicity of ammonia and ammonium ions is very well characterized in the context of the impact on higher organisms, while, in the field of microbiology, this topic still requires detailed research [45]. Moreover, the research by Leejeerajumnean et al. (2000) shows that microorganisms can differ significantly in the level of tolerance to the action of ammonia compounds [46]. This fact may be related to the decrease in alpha-biodiversity observed in the NP3 variant, which suggests the presence of an important selection factor. It is believed that microbial populations have a relatively high metabolic flexibility and functional redundancy; however, they react relatively quickly, with a change in taxonomic structure, to the action of variable, selective environmental factors [47]. This phenomenon is confirmed in the conducted genetic analyzes. Changes in the dominant bacterial classes in variants with a moderate and high supply of nitrogen compounds (NP2 and NP3) are observed. There is a dominance of the Gammaproteobacteria and Betaproteobacteria classes, as well as the Xanthomonadales and Burkholderiales orders, which suggests that these classes and orders are particularly important during the population response to changes in the supply of nitrogen compounds. Similar trends are observed in an earlier study [48]. The researchers find an increase in the share of Gammaproteobacteria and Xanthomonadales in soils subjected to a long-term fertilization process with nitrogen compounds. Therefore, it can be assumed that these changes are not accidental.

It can be also postulated that those dominant taxa, which are also the most differentially abundant between deficient and sufficient nitrogen variants, such as Xanthomonadales and Burkholderiales, are more competitive than the other bacteria groups in the use of nitrogen compounds. In an earlier study, Sun et al. (2021) emphasize that long-term exposure to nitrogen compounds results in the dominance of microbial groups associated with ureolysis, and the nitrification and denitrification processes. It is found that the microbial groups that harbor the same or similar functional genes involved in nitrogen use can coexist together. This functional redundancy is well described in the literature, and it is believed that it may increase the stability of microbial metapopulations in the long term [49].

The LEfSe analysis identifies the most significant taxonomic differences between two classes of nitrogen supply (deficient and sufficient). The Xanthomonadales, Burkholderiales, and Sphingomonadales orders may be of key importance in the biological degradation of hydrocarbon fractions in environments with a periodically variable supply of nitrogen compounds, as well as in intensive biostimulation technologies. However, due to their high sensitivity to the variability of environmental parameters, this groups of microorganisms should not play a bioindicative role.

PICRUSt analysis reveals that the Sphingobacteriales and Rhizobiales orders have a high genetic potential for biodegradation of the PAH fraction. The nitrogen-fixing Rhizobiales order seems to be particularly important. The decrease in its share in the NP3 variant, found after 168 h of biodegradation, can be related to the reduced degradation efficiency of PAH. The biodegradation potential of PAH by microorganisms belonging to the Rhizobiales order is described in the literature. It is believed that Rhizobiales effectively support the process of PAH bioremediation in environments with a deficiency of nitrogen compounds, due to the activity of genes responsible for nitrogen binding [50]. It can be suggested that the quantitative analysis of the Rhizobiales order is helpful in estimating the effectiveness of PAH natural attenuation in areas with a deficiency of nitrogen compounds, and the validity of bioremediation techniques supported by biostimulation. However, it should be mentioned that the process of biological decomposition of polycyclic aromatic hydrocarbons is a complex process. Many studies indicate the importance of cometabolism and metapopulation interactions in the degradation of this group of xenobiotics [51–53]. Therefore, it is not possible to indicate only one group of microorganisms that clearly determines the effectiveness of biological decomposition of PAH.

Based on the current knowledge, it is difficult to make any hypotheses regarding the mechanisms that drive the observed population changes of microorganisms and modifications of the genetic pool in the context of the biological decomposition of hydrocarbon compounds, including PAH. However, it should be emphasized that properly optimized supplementation with nitrogen compounds has a positive effect on the processes of hydrocarbon remediation, if the dose of the biostimulator is optimally selected. Careless application of nitrogenous substances above optimal levels may contribute to the inhibition of biodegradation processes.
