BDS biocatalyst

Biodesulfurization (BDS) is the excision (liberation or removal) of sulfur from organosul-fur compounds, including sulfur-bearing heterocycles, as a result of the selective cleavage of carbon-sulfur bonds in those compounds by the action of a biocatalyst. Biocatalysts capable of selective sulfur removal, without significant conversion of other components in the fuel are desirable. BDS can either be an oxidative or a reductive process, resulting in conversion of sulfur to sulfate in an oxidative process and conversion to hydrogen sulfide in a reductive process. However, the reductive processes have been rare and mostly remained elusive to development due to lack of reproducibility of the results. Moderate reaction conditions are employed, in both processes, such as ambient temperature (about 30°C) and pressure. 

Other Rhodococcus strains similar to those described above in terms of the desulfurization ability have also been isolated [83], The purpose of identifying such Rhodococcus strains, in several cases, appears to be the development of in-house biocatalysts for BDS application. The specificity of the desulfurizing strains of organosulfur compounds in addition to DBT has also been studied (Table 3). 

A BDS patent [106] was awarded for the use of biocatalysts belonging to the group of Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus, or Corynebac-terium. One of the strains P. putida was further developed by mutation of the parent strain to obtain organic solvent-resistant mutants [107], The mutated strains were screened by selective cultivation in the presence of 0.1% to 10% by volume (v/v) of concentrations of a toxic organic solvent. The specific mutated strains obtained were P. putida No. 69-1 (PERM BP-4519), P. putida No. 69-2 (PERM BP-4520), and P. putida No. 69-3 (PERM BP-4521). 

In this section, we will consider the methodologies used for genetic engineering of biocatalysts for desulfurization and the biocatalysts developed so far via various technologies. The application of genomic techniques as reported in patent literature associated with BDS is described first. 

Further research in improving the BDS activity of the biocatalysts was targeted towards the search of co-catalysts and co-factors to enhance overall desulfurization rates as well as promoters to enhance enzyme expression. This research resulted in identification of NADH and FMNH2 as co-factors essential for electron transfer and related oxidoreductase enzymes as co-catalysts as described in detail below. Additionally, other bacterial strains were also investigated as hosts and are reported below. 

In addition to desulfurization activity, several other parameters are important in selecting the right biocatalyst for a commercial BDS application. These include solvent tolerance, substrate specificity, complete conversion to a desulfurized product (as opposed to initial consumption/removal of a sulfur substrate), catalyst stability, biosurfactant production, cell growth rate (for biocatalyst production), impact of final desulfurized oil product on separation, biocatalyst separation from oil phase (for recycle), and finally, ability to regenerate the biocatalyst. Very few studies have addressed these issues and their impact on a process in detail [155,160], even though these seem to be very important from a commercialization point of view. While parameters such as activity in solvent or oil phase and substrate specificity have been studied for biocatalysts, these have not been used as screening criteria for identifying better biocatalysts. 

Due to the water requirement of biocatalytic systems, BDS is typically carried out as a two-phase aqueous-oil process. However, increased sulfur removal rates could be accomplished by using an aqueous-alkane solvent catalytic system [46,203,220,255], The BDS catalytic activity depends on both, the biocatalysts and the nature of the feedstock. It can vary from low activity for crude oil to as high as 60% removal for light gas-oil type feedstocks [27,203,256], or 70% for middle distillates, 90% for diesel, 70% for hydrotreated diesel, and 90% for cracked feedstocks [203,256], The viscosity of the crude oil poses mixing issues in the two-phase oil-water systems however, such issues are minimal for distillate feedstocks, such as diesel or gasoline [257]. 

Significant effort has been devoted to the development of separation methods for the post-BDS stage. Simple approach employing two phase separation methods like filtration or use of centrifugal forces have been studied as well as newer approaches including modification of biocatalyst to facilitate separation have been studied. Information has been obtained from patents as well as publications in open literature. 

A microbial biocatalyst in context of BDS is defined as a microorganism expressing enzymes capable of removing sulfur selectively from organosulfur compounds. The definition of a biocatalyst, in general, also includes use of one or more enzymes, by themselves or in a cellular extract (used either in suspended form or carrier-supported form) for removal of sulfur. Additionally, biocatalyst can be a microbial consortium as well. Aerobic as well as anaerobic pathways for sulfur removal have been reported. The anaerobic routes, however, have been plagued with lack of reproducibility preventing further development. 

Improving functionality may involve a complex biocatalytic system including more than one biocatalyst, as it is the case in BDM reactions. Additionally to the BDS for instance, another biocatalyst active for BDM [395,406], which consists of a heme oxygenase or a Cytochrome reductase could be used to widen up the functionality. 

Most of the known microorganisms to be active for BDS (by the date that patent [408] was introduced), were considered in this invention. Furthermore, their mutational or engineered derivatives, enzymes, cell-free extracts, recombinant enzymes, recombinant DNA, plasmids, vectors, and fragments were also incorporated in the intellectual property document. The mentioned operating conditions regard ambient temperature, mechanical agitation and a 1 9 biocatalyst/petroleum ratio. 

It should be noted that Diversa was involved in a BDS study from 2003 to 2006 in collaboration with PetroStar, Inc. They acquired many of the strains developed by EBC and investigated the potential of desulfurizing diesel using these strains and conducted further genetic engineering and host strain manipulation to develop better biocatalysts. 

Deep desulfurization method of fossil fuels, comprising a first step of HDS and a BDS step for the removal of HDS sulfur refractory compounds, using an effective amount of a biocatalyst. The fuel is incubated in the presence of one or more BDS-active microorganisms, which converts the organic sulfur compounds into water-soluble inorganic sulfur. Then, in a separation stage, the products of the incubation are separated into a deeply desulfurized liquid fossil fuel, and the water-soluble inorganic sulfur. 

A BDS method using a transformed microorganism containing a recombinant DNA molecule of Rhodococcus origin. This transformed microorganism expresses a BDS-active biocatalyst. (Use in BDS of the biocatalyst protected in Refs. [37-39])... 

US5733773 [46] desulfurization of fossil fuel with flavoprotein. comprising DNA which encodes a flavoprotein and DNA of Rhodococcal origin which encodes a protein biocatalyst active for BDS. The DNA biomolecule is not a Rhodococcus genome. 

An isolated DNA molecule comprising DNA which encodes a group III alcohol dehydrogenase and DNA which encodes a BDS-active biocatalyst via nicotinamide adenosine dinucleotide-dependent manner. 

A BDS method consisting in the incubation of a mixture formed by a fossil fuel and an aqueous phase containing a biocatalyst and a rate-enhancing amount of a protein having NADH FMN oxidoreductase activity or enzymatically active mutant thereof. The oxidoreductase has the amino acid sequence set forth in SEQ ID No. 2, described in the original document (see Ref. [57]). A separation stage is also claimed. 

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