Microorganisms can serve as Single Cell Proteins (SCP) used as an alternative and non-conventional protein source to meet the protein shortage and increasing demand. The objective of this research was to characterize growth and biomass composition of the bacterium Cupriavidus necator, cultivated on various nitrogen (ammonium sulfate and urea) and carbon (glucose, formic acid and oleic acid) sources. Under well-controlled conditions, a mono-factorial approach was employed to study the impact of the nature of the substrates on biomass growth and composition in bioreactors. Cultures were performed in batch culture mode, except for formic acid for which a fed-batch mode was selected due to its toxicity on cell growth. Results were compared in terms of growth, cell morphology, cell membrane permeability, biomass composition in proteins, nucleic acids, amino acids and polyhydroxyalkanoates. The results showed high protein amounts and an impact of substrate on biomass composition. Independently, ammonium sulfate, as nitrogen source, and formic acid, as carbon source provided better results in term of growth and biomass composition. The highest total proteins (83 %DW), the lowest PHA amount (1.7 %DW) and the lowest changes in cell size and granularity profiles were obtained using the organic acid, showing the more attractive SCP composition. Despite interesting amino acid contents, the results showed that their distribution is affected by the nature of the substrates.