## 1 Introduction  
Carbon and hydrogen are the most abundant elements in crude oil followed by sulfur. Depending on the origin of the crude oil, sulfur compounds account for 0.1% to 8% (_w/w_) [1]. Various forms of sulfur compounds are present in crude oil, e.g., hydrogen sulfide, mercaptans, thiophenes, benzothiophenes and dibenzothiophenes [2, 3]. The organosulfur compounds (OSCs) are unwanted in the refining process since they can deactivate the catalyst during the processing of crude oil and also can corrode pumping equipment and pipelines [2; 4]. Furthermore, upon combustion they emit large amounts of sulfur dioxides which are harmful for the environment and human health [5]. As a result, current environmental regulations have become increasingly stringent, demanding very low sulfur contents in diesel and gasoline. The recommended limit of sulfur content set by the Environmental Protection Agency (EPA) of the USA is 15 ppm for gasoline and 30 ppm for diesel [6]. On the other hand, EU regulations are more strict concerning the sulfur content in gas-oil (commercial fuel used as an alternative to road diesel, also known as red diesel), which was set at 10 ppm [7]. Hydrodesulfurization (HDS) is the current industry standard for the removal of sulfur from the OSCs present in crude oils; it is conducted under a high temperature and pressure in the presence of an expensive chemical catalyst [8; 9]. HDS is not very effective in removing recalcitrant molecules, mainly from the dibenzothiophene (DBT) chemical group, which adds up to 60% of the total sulfur content in crude oils [10]. According to [PERSON] & Ball, [11] the susceptibility of OSCs in HDS follows the subsequent order: thiophene (TH) > alkylated TH > benzothiophene (BT) > alkylated BT > dibenzothiophene (DBT) and alkylated DBT without substituents at the fourth and sixth positions > alkylated DBT with alkyl substituents at the fourth and sixth positions. Deep (ultra-deep) HDS (sulfur levels below 15 ppm) on the other hand requires higher temperatures (\(\geq\)425 \({}^{\circ}\)C), a higher pressure (\(\geq\)250 psi) and more expensive catalysts in order to remove recalcitrant OSCs [9]. That leads to an increase in the operational and capital costs as well as an increase in the greenhouse gas (GHG) and H\({}_{2}\)S emissions [5; 12]. Biodesulfurization (BDS) is a green approach for the removal of sulfur from the recalcitrant OSCs; it has been proposed as a complementary technology to HDS treatment for petroleum products and can contribute to the reduction in GHG and H\({}_{2}\)S emissions. BDS involves the use of microorganisms capable of removing the sulfur from the recalcitrant OSCs and produces ultra-low sulfur fuels [2; 11]. In general, microorganisms have two pathways for OCS metabolism/biodesulfurization, i.e., the oxidative C-C cleavage pathway (Kodama pathway) and the oxidative C-S cleavage pathway (4S pathway) [13; 14]. The 4S pathway is a four-step enzymatic process that converts DBT to 2-hydroxybiphenyl (HBP) and sulfate. More specifically, DBT is converted initially to DBT-sulfoxide (DBTO), and then to DBT- sulfone (DBTO\({}_{2}\)). Both reactions are catalyzed by the action of a DBT-monooxygenase (DszC) and an NADH-flavin mononucleotide oxidoreductase (DszD) supplying the FMNH\({}_{2}\) needed. DBT-sulfone is then converted to 2-hydroxybiphenyl-2-sulfinic acid (HBPS) by the action of a DBT-sulfone monooxygenase (DszA) and DszD. Finally, 2-hydroxybiphenyl sulfate desulfinase (DszB) converts HBP-sulfinate to 2-HBP and sulfate [13]. The 4S pathway is of great biotechnological importance since it is a non-destructive process leaving the carbon skeleton of OSCs intact, thus maintaining the calorific value of the fuel [1].