**1. Introduction**

Intensive exploitation of liquid fossil fuels, and the migration of hydrocarbon compounds to the natural environment, have a highly negative influence on terrestrial and aquatic ecosystems. Due to the high resistance to biodegradation, and the potential for biomagnification, this pollution is considered to be one of the most serious environmental threats [1–3]. Due to the complexity of the chemical structure, and the diversity of physical and chemical properties, individual groups of hydrocarbons differ in bioavailability, toxicity, and the potential for biological decomposition [3,4].

Many biotic and abiotic factors affect the biological decomposition rate of hydrocarbon compounds [3–8]. The abiotic factor, i.e., the supply of microelements such as nitrogen, phosphorus, sulfur, calcium, magnesium, and potassium, is crucial in the functioning of the cellular metabolism of microorganisms that perform biodegradation processes [9]. In

**Citation:** Staninska-Pi ˛eta, J.; Czarny, J.; Juzwa, W.; Wolko, Ł.; Cyplik, P.; Piotrowska-Cyplik, A. Dose–Response Effect of Nitrogen on Microbial Community during Hydrocarbon Biodegradation in Simplified Model System. *Appl. Sci.* **2022**, *12*, 6012. https://doi.org/ 10.3390/app12126012

Academic Editors: Amanda Laca Pérez and Yolanda Patiño

Received: 18 May 2022 Accepted: 10 June 2022 Published: 13 June 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

contaminated areas, deficiencies in biogenic elements and inhibition of bioremediation processes may occur very quickly, as a result of an excessive supply of carbon compounds, and the activity of microorganisms [10]. Research suggests that the share of nutrients, including the C:N ratio, should be at a level similar to that in live cells. The optimal values for the C:N ratio are in the range from 100:5 to 100:15 [9,11]. The wide range of the optimal ratio of these elements may result from the different rates of decomposition of individual hydrocarbon groups, and the limited bioavailability of biogenic elements, depending on abiotic and biotic environmental factors [9].

One of the most popular bioremediation technologies, based on the knowledge of the influence of nutrient supply, is the biostimulation technique. Targeted compensation of deficiencies in the environmental niche results in metabolic activation of indigenous microflora, and the improvement of the biodegradation efficiency of xenobiotics. This compensation may consist of the application of deficient nutrients and electron acceptors, in the form of both organic and inorganic compounds [12,13]. Many studies prove that biostimulation is beneficial for effective hydrocarbon degradation [14–16]. A popular strategy is also to combine bioaugmentation and biostimulation techniques, in order to maximize remediation effects [12,17,18].

Despite the many advantages of bioremediation technologies, and their widespread social acceptability, they have a number of limitations. These include the differentiation of the metabolic potential of microorganisms living in different environments, problems related to the optimization of the scale of necessary implementations, and the difficulties in extrapolating the results of tests carried out on a laboratory scale [19,20]. It is emphasized that, in the case of biostimulation with nitrogen compounds, the excessive supply of nutrient may negatively affect the efficiency of biological decomposition [21–23].

An innovative context in the improvement and understanding of the mechanisms of bioremediation processes is the approach to the issues within the field of synthetic microbiology, assuming a multitude of biotic and abiotic interactions in the microbial community. According to the assumptions of the synthetic approach, simple interactions between genotypically different microorganisms may contribute to the creation of properties that are difficult to predict at the consortium level [20,24,25]. Therefore, there is a need to acquire and analyze data on the molecular mechanisms of the microbial transformation of hydrocarbon compounds, and the influence of environmental factors and degradation processes. The aim of this study was to comprehensively assess the effect of nitrogen compounds on an environmental consortium with high hydrocarbon degradation potential. Both the enzymatic efficiency and metabolic activity, as well as the overall genetic potential of individual members of the metapopulation, were taken into account. This type of analysis allows for the improvement of planning an effective and predictable ecosystem remediation process.

#### **2. Materials and Methods**

#### *2.1. Microbial Consortium Isolation*

The environmental consortium with a high potential for biodegradation of hydrocarbons was isolated from soil matrix. The soil sample was taken from the area of railway sleepers impregnation plant in Solec Kujawski (Poland): 53◦04 40.7 N 18◦14 23.6 E. A detailed description of the soil sampling site is presented in previous publications [8,26].

The isolation protocol was based on our prior experience [8]. A 10 g sample of the soil was added into 90 mL of sterile saline, and shaken for 4 h (150 rpm). After the soil particles sedimented, the obtained supernatant was added to a non-selective nutrient broth, containing enriched broth (BTL, Łód ´z, Poland) and a 2% of glucose (Sigma-Aldrich, Darmstadt, Germany). Bacteria were incubated for 7 days at 25 ◦C in aerobic conditions provided by continuous shaking (150 rpm). Finally, the biomass was centrifuged (10 min, 4000 rpm), washed twice, and suspended in saline solution. The microorganism suspension was normalized to OD600 = 0.7 (Helios Delta Vis, ThermoFisher Scientific, Waltham, MA, USA), and used as inoculum for the biodegradation experiments.

#### *2.2. Experimental Design*

The biodegradation experiment was performed in flasks equipped with septum and vent caps with a semi-permeable DURAN® membrane, in order to ensure the best oxygenation of the culture medium. Each experimental flask included 50 mL of mineral medium described in our previous studies [8], 2 g of diesel oil (PKN Orlen, Płock, Poland), and 250 μL of optimized microbiological inoculum. Casein peptone (Merck, Darmstadt, Germany) was used as the nitrogen source (Table 1). According to the manufacturer's declarations, the total amount of nitrogen was 14%, of which 4% was the amine nitrogen fraction. Based on the literature data, the nitrogen content in diesel fuel was assumed to be 0.13% [27], and the carbon to nitrogen ratio was assumed to be as 10:1 [11]. Biodegradation was carried out for 168 h at 25 ◦C. The aerobic conditions were provided by continuous shaking (150 rpm). The experiment was performed in three replicates.

**Table 1.** Characteristics of the experimental variants.

