Biodesulfuración de fracciones petrolíferas con "pseudomonas putida" CECT5279optimización del proceso

Abstract

Sulfur dioxide emissions from fuel combustion are the mayor contributor to the acid rain and air pollution. For that reason governments are increasing their attention on the reduction of the limit of the sulphur content in oil fractions after refining. Actually, hydrodesulphurization is the most profitable process employ toremove sulphur from crude oil. This process is capable of reducing sulphur limits since 300-500 ppm of inicial concentration until next to 50 ppm. Nevertheless, regulations of UE and USA are demanding sulphur limits lowest than 10 ppm in diesel oil and gasoline on 2010. Conventional HDS process would need a high temperature and pressure conditions to remove recalcitrant molecules such as dibenzothiphene (DBT) and its derivates (Cx-DBT).and it would be much more expensive. Biodesulphurization is defined as a biological process in which biocatalyst is employed to remove sulphur compounds from oil fractions. These compounds can be converted to a final sulphur free compound, without change fuel properties under mild pressure and temperature conditions, which permits the reduction of the energetic consumption and operative costs. Currently, it seems that the most profitable way to desulphurize oils is by combined processes, hydrotreating the oil fractions first, following by the biological process. By this option, the recalcitrant compounds could be selectively removed, and 10 ppm of final sulphur content could be achieved, according to law restricitions. In this work, the biodesulfurization process using the GMO Pseudomonas putida CECT5279 has been studied. This bacterium carries the genes dszABC from Rhodococcus erythropolis IGTS8, and the gene hpaC. from Escherichia Coli. Therefore, it is capable to remove DBT and Cx-DBT following a biochemical route similar to the 4S pathway, which was elucidated for the wild strain R. erythropolis. This work approaches some problems relevant for the industrial application of the biodesulphurization process. The production of the biocatalyst using different bioreactor configurations, as well as, the scale up of the growing process has been carried out. In the same way, kinetics study of resting cell reactions has been conducted for the degradation of DBT and Cx-DBT, using orbital shaker and stirred tank reactor as working scales, both in aqueous and biphasic conditions. On the other hand, different studies to optimize the biodesulphurization process have been done. These include the improvement of Cx-DBT transfer between aqueous and oil phases, reuse of biocatalyst and characterization and enhancement of the aeration conditions. All of these operational conditions and modification of the reaction media composition for biphasic reactions have been used for the experiments conducted at different working scales with the model oils assayed, as well as or the treatment of a real hydrotreated diesel sample. On the chapter of the biocatalyst production and scale up of the growing process, a comparative study of the kinetics of the growth has been carried out for two different bioreactor configurations (air-lift and stirred tank reactors), based on the protocol proposed by Martin y col., 2004. For the biomass production using air-lift reactor, it has been studied the aeration conditions to maximize the growth and the evolution of the desulfurization capability with the growing time. Simultaneously, it has been characterized the oxygen mass transfer conditions, and the consumption of oxygen for the biocatalyst along the production process. On the other hand, due to the high amount of biocatalyst required for biodesulphurization experiments performed in stirred tank reactor, it has been done a scale up of biomass production process until 15L stirred tank bioreactor. This scale up process has been based on the maintenance of the aeration conditions and it has permitted to get enough biocatalyst without losing desulphurization capability. On the next chapter, it has been studied the kinetics of degradation of DBT and Cx-DBT: 4-metyl-DBT, 4,6-dimetyl-DBT and 4,6dietyl-DBT, individually and mixed. These model compounds are the most abundant in the last oil fraction before hydrodesulphurization process. It has been obtained the kinetic parameters that describe the degradation of the Cx-DBT compounds, in aqueous and biphasic reaction media. The effect on the biodesulphurization yield of the presence of several mixtures of sulphur compounds and the cellular age employed for the treatment of them was evaluated as well. This work includes the characterization and biodesulphurization of a real hydrotreated diesel sample from REPSOL YPF. Using HPLC-DAD, it was identified the more abundant aromatic sulphur compounds. The biodesulphurization o this sample was conducted into a 2L stirred tank reactor using the operational conditions established for the model oil. In the last chapter it has been presented different issues to improve the resting cell biodesulphurization process. It is recognized that the problem of DBT transfer between liquids layer could be the limiting step of the biodesulphurization process. For that reason, it has been studied the influence of the addition of several surfactants (Tween 85, Tween 20, SDS and Triton X-100) and ethanol as co-solvent. With the aim to enhance the sulphur compound bioavailability, stabilizing the emulsion and reducing the mass transfer problems. It has been established the best surfactant, and the dosage to be used to increase the biodesulphurization yield. In relation to these assays for the enhancement o biodesulphurization process, it was study the possibility of the reducing equivalent regeneration by the addition of several carbon sources into the resting cell reaction media. The evaluation of the use of pyruvate, succinate and citrate for the degradation of DBT and two of their metabolites according to the 4S pathway was assayed in aqueous and biphasic conditions and orbital shaker. Another factor that it has been studied was the reuse of the biocatalist. It was investigated the possibility of three cycles of biodesulfurization using DBT and two intermediates of the 4S route as substrates, in aqueous phase. As well as, it was evaluated the effect of the addition of pyruvate in the reuse of the biocatalyst. Once it was established the optimal operational conditions for the biodesulfurization process in orbital shaker, these were used in stirred tank bioreactor to determinate the oxygen consumption both for the cellular maintenance and for the biodesulfurization process of DBT in biphasic reaction media. In the same way, it was evaluated the effect of the air supply on the DBT biodesulphurization yield.