*Article* **Dose–Response Effect of Nitrogen on Microbial Community during Hydrocarbon Biodegradation in Simplified Model System**

**Justyna Staninska-Pi ˛eta 1,\*, Jakub Czarny 2, Wojciech Juzwa 3, Łukasz Wolko 4, Paweł Cyplik 3,\* and Agnieszka Piotrowska-Cyplik 1,\***


**Abstract:** Knowledge about the influence of C:N ratio on the biodegradation process of hydrocarbon compounds is of significant importance in the development of biostimulation techniques. The purpose of this study was to assess the impact of nitrogen compounds on the environmental consortium during the process of biological decomposition of hydrocarbons. The experimental variants represented low, moderate, and excessive biostimulation with nitrogen compounds. The metabolic activity of the consortium was tested using the flow cytometry technique. The efficiency of the biodegradation of hydrocarbons of the consortium, based on the gas chromatography method, and metapopulation changes, based on the analysis of V4 16srRNA sequencing data, were assessed. The results of the research confirm the positive effect of properly optimized biostimulation with nitrogen compounds on the biological decomposition of polycyclic aromatic hydrocarbons. The negative impact of excessive biostimulation on the biodegradation efficiency and metabolic activity of microorganisms is also proven. Low resistance to changes in the supply of nitrogen compounds is demonstrated among the orders Xanthomonadales, Burkholderiales, Sphingomonadales, Flavobacteriales, and Sphingobacteriales. It is proven that quantitative analysis of the order of Rhizobiales, characterized by a high-predicted potential for the decomposition of polycyclic aromatic hydrocarbons, may be helpful during biostimulation optimization processes in areas with a high nitrogen deficiency.

**Keywords:** microbial community; hydrocarbon biodegradation; biostimulation; next generation sequencing
