## 4 Conclusions  
Organosulfur compounds (OSCs) in petroleum pose significant challenges to refining processes and environmental compliance due to catalyst poisoning and SO2 emissions. Biodosulfurization (BDS) via the selective 4S pathway offers a green alternative to hydrodesulfurization (HDS) but is limited by low biocatalyst concentrations and cells' desulfurization activity (CDA). This study addressed these challenges through a comparative evaluation of batch, repeated batch and fed-batch cultivation strategies for _Rhodococcus qingshengii_ IGT8. Among the three cultivation modes tested, the fed-batch process proved to be the most effective in achieving high biomass concentrations and maintaining elevated levels of CDA. In contrast, batch and repeated batch cultivations exhibited lower productivity and a marked decline in CDA over time, with no performance gains observed beyond the first replacement in the repeated batch regime.  
The application of the biomass obtained from fed-batch cultivation directly in a two-phase bubble column reactor demonstrated the functional robustness of the process. Complete desulfurization of model sulfur compounds (DBT and 4-MDBT) was achieved without the need for further cell processing, while partial but concurrent desulfurization in mixed-substrate systems confirmed the catalyst's applicability to more complex matrices. Overall, this work establishes fed-batch cultivation as an efficient and scalable method for producing active biocatalysts suitable for industrial biodegradation applications.  
Future research should focus on exploring the genetic or metabolic engineering of _R. qingshengii_ IGT8 to improve enzyme activity, broaden substrate specificity and increase resistance to inhibitory compounds such as aromatic hydrocarbons or desulfurization by-products like 2-HBP. In parallel, the identification and characterization of other microorganisms with high biodegradation potential could expand the range of available biocatalysts. Additionally, future studies could investigate bioreactor design improvements, such as optimizing bubble column or airlift configurations as well as packed-bed reactors with immobilized cells to enhance mass transfer and overall process efficiency. Scaling up the process to pilot and industrial levels will be essential to assess its technical feasibility, economic viability and environmental impact.  
**Author Contributions:** Conceptualization, D.M., D.K. and D.G.H.; methodology, D.M. and D.G.H.; software, P.D.G.; validation, K.D., S.K. and G.P.; formal analysis, O.M., K.D. and S.K.; investigation, K.D., S.K. and G.P.; resources, D.M. and D.G.H.; data curation, O.M., S.K. and P.D.G.; writing--original draft preparation, D.M.; writing--review and editing, D.M. and D.G.H.; supervision, D.M. and D.G.H. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This work was co-financed by the European Regional Development Fund of the European Union, and Greek national funds through the Operational Program \"Competitiveness, Entrepreneurship, and Innovation\", under the call RESEARCH-CREATE-INNOVATE (grant number: T1 EDK-02074, MIS 5030227).  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The data are contained within the article.  
**Conflicts of Interest:** The authors declare no conflicts of interest.