Aim: The aim of the present study was to investigate the phenotypic and genotypic variability of two strains of Acidithiobacillus ferrooxidans genus during growth in sewage sludge. Methods and Results: Compared with A. ferroaxidans cells grown in mineral medium, those grown in sewage sludge demonstrated remarkable changes in ultrastructure (transmission electron microscopy) and significantly elongated lag phases. These latter cells also lacked carboxysomes and rusticyanin, showed lower level of cytochromes and exhibited modifications to their outer membrane proteins (SDS-PAGE). Restriction fragment length polymorphism analysis using pulsed-field gel electrophoresis showed that most restriction fragments were highly conserved and shared by strains grown under different conditions. However, in relation to cells grown in mineral medium, sludge-grown A. ferrooxidans lacked a number of restriction fragments, clearly indicating structural changes to the chromosomal DNA of the organism. Conclusions: In combination, the results of this study provide evidence of adaptive responses by chemolithoautotrophic aciclophilic A. ferroaxidans to facilitate growth in sewage sludge. Significance and Impact of the Study: The obtained results are important from scientific as well as industrial application point of view, because they confirmed that A. ferrooxidans highly sensitive to organic compounds bacteria is useful in biotechnologies of heavy metal removal from shale ore, polluted soils and sewage sludge containing organic hazardous compounds.