The low rank coal lignite stands out to be a profuse source of energy in Turkey and accounts for nearly 43% of its total fossil fuel production. However, the high sulfur content associated with lignite averts its application in several sectors due to various health and environmental issues. In the present work, biodesulphurization of Turkish lignite was studied in 1 and 20 L aerated bioreactors using an iron oxidizing acidophile, Leptospirillum ferriphilum (L. ferriphilum). Additionally, the effect of Span 80 (S80) on biodesulphurization of the sample was studied, under the optimum concentration derived from our previous shake flask study, in order to notice its effect under scale-up aerated bioreactor conditions. Under lab scale (using 1 L bioreactors), in the absence of S80, L. ferriphilum could desulphurize a maximum of 65.6% sulfur from the lignite sample, while 56% desulphurization was achieved in the presence of 0.05% (v/v) S80. Further scale-up studies under semipilot conditions (in 20 L reactors) indicated a maximum desulphurization of 63% from the sample in absence of S80. The pH, oxidation–reduction potential (ORP), and Fe2+ iron and total iron concentrations were periodically monitored in all the reactors during the course of the experiments. XRD and FTIR characterization of the lignite samples pre and post biodesulphurization provided valuable information on the structural and phase changes due to the microbial action.