ES2232290B1 - PROCEDURE FOR CULTIVATION OF THE RECOMBINING PSEUDOMONAS (CECT 5279) FOR USE IN THE DESULFURATION OF DIBENZOTIOPHENE. - Google Patents

Abstract

Culture procedure of the recombinant Pseudomonas putida (CECT 5279) for use in the desulfurization of dibenzothiophene. The invention consists of a process for producing in a fermenter the recombinant strain of Pseudomonas putida CECT5279 which is used as a biocatalyst to desulfurize dibenzothiophene. The pre-circle is prepared in LB medium (Luria - Bertani) and the production in aerated commercial bioreactor using basal saline medium (BSM) with 20g / L of glutamic acid at a temperature of 30 ° C. The expression of the 4S desulfurization pathway genes is induced within 4 hours after the production of the microorganism has begun, using IPTG. This invention therefore indicates the culture medium and the appropriate operating conditions to improve and increase the production scale of this biocatalyst with a view to its use at the industrial level.

Description

Culture procedure of the recombinant Pseudomonas putida (CECT 5279) for use in the desulfurization of dibenzothiophene.

Technical sector

The invention claims a method of production of high quantities of a biocatalyst with desulfurizing capacity that uses glutamic acid as a source of carbon in a minimum growth medium (BSM, Basal Salino Means, medium). It describes the experimental method to lead to carry out the aforementioned production using operating conditions optimal. This process has considerable advantages over other processes of obtaining cells with the aforementioned ability to selective removal of sulfur from organic molecules previously described.

State of the art

The desulfurization of oil through the use of microorganisms has been studied during the last part of the last century (McFarland et al. (1998) Crit. Rev. Microbiol. 4, 99; McFarland (1999) Curr. Opin. Microbiol 2, 257). The investigations that have been carried out have served to develop biotechnological methods to remove sulfur from oil before combustion. The strict regulations that have governments imposed on the emission of sulfur into the atmosphere have been the true drivers of this development (McFarland et al. (1998) Crit. Rev. Microbiol. 4, 99).

The breakthrough in the field of oil biodesulfurization occurred with the isolation and characterization of the Rhodoccocus erythropolis IGTS8 (ATCC53968) bacteria, a Gram-positive bacterium obtained by the Institute of Gas Technology in Chicago that has been the source of inspiration for the development of many biocatalysts and processes (US Patents 5,356,801, 5,358,870, 5,358,813, 5,198,341, 5,132,219, 5,344,778, 5,104,801, 5,002,888, 5,804,433, 5,846,813, 5,879 .914, 5,733,773). This microorganism has a metabolic pathway called the "4S" pathway capable of transforming the dibenzothiophene (DBT) molecule into 2-hydroxybiphenyl (2HBP) and sulfite (McFarland et al. (1998) Crit. Rev. Microbiol. 4, 99; McFarland ( 1999) Curr. Opin. Microbiol. 2, 257), thus producing the removal of the sulfur atom from the DBT without loss of carbon atoms in its structure, which is of great interest to the oil and petrochemical industry. It should be noted that DBT is the molecule that most profusely has been used as a model compound to determine the desulfurizing capacity of a microorganism and, therefore, has been chosen for the studies described in this patent. The "4S" desulfurization pathway of R. erythropolis IGTS8 consists of four enzymes called DszA, DszB, DszC and DszD (Gray et al. (1996) Nature Biotechnol. 14, 1705). About the function of these enzymes it is known that the DszA and DszC proteins are FMNH2-dependent monooxygenases that oxidize DBT, the DszB enzyme is a desulfinase that releases the sulfur atom from the structure of the oxidized DBT, and the enzyme DszD is a flavin: NADPH reductase that provides the FMNH2 that DszA and DszC enzymes need. It is also known that the dszABC genes, which encode the enzymes DszA, DszB and DszC, respectively, are forming an operon located in a plasmid, while the dszD gene, which encodes the DszD reductase, is located on the chromosome of the bacteria (Denome et al. (1994) J. Bacteriol. 176, 6707).

Although almost all the biodesulphurization processes described to date have been carried out with wild microorganisms, a few cases have recently been described in which recombinant bacteria of R. erythropolis IGTS8 (US Patents 5,733,773, have been used for biodesulfurization of DBT) 5,789,914, 5,804,433). In these cases, attempts have been made to increase the desulfurizing capacity of the wild bacteria of R. erythropolis IGTS8 by adding genes by genetic engineering techniques. That is, it has tried to improve a microorganism that already had the ability to desulfurize. However, it is interesting that not many studies have been carried out to try to develop recombinant microorganisms capable of desulfurizing DBT from bacteria that did not initially possess such capacity. The first description of one of these cases was made when the dszABC genes of R. erythropolis IGTS8 were donated in a strain of Escherichia coli (Denome et al. (1994) J. Bacteriol. 176, 6707), although only the desulfurizing capacity due to the fact that the strains were capable of growing in a minimum culture medium containing DBT as the sole source of sulfur. Apparently, the few data provided in this work suggest that the desulfurizing capacity of DBT of these recombinant E. coli strains was low. More recently, the construction of several recombinant strains of the genus Pseudomonas that carried the dszABC genes of R. erythropolis IGTS8 and that were capable of growing in a minimal culture medium containing as the sole source of sulfur DBT (Gallardo et al. (1997) J. Bacteriol. 179, 7156). In this case, it was found that as a consequence of the desulfurization of the DBT, 2HBP was released to the culture medium and that the recombinant Pseudomonas grew faster in this minimal medium than the strain R. erythropolis IGTS8. In view of these results, it could be stated that the recombinant Pseudomonas strains were more effective than R. erythropolis IGTS8 in the desulfurization of DBT under the culture conditions tested. However, it was not found when these recombinant strains of Pseudomonas were capable of desulfurizing DBT in a reactor capable of resting cells (resting cells), the process biodesulfuración oil most used at industrial scale (McFarland et al. (1998) Crit. Rev. Microbiol. 4, 99). On the other hand, it should also be noted that in these recombinant Pseudomonas strains only the desulfurization, dszA, dszB , and dszC genes had been introduced but not the dszD gene whose product also participates in the process. It was assumed that the recombinant Pseudomonas could provide an endogenous flavin: NADPH reductase that would provide FMNH2 to the enzymes DszA and DszC, however the tests carried out with these strains indicated that desulfurization was less efficient than expected. For this reason, a procedure was recently described by which organisms capable of efficiently desulfurizing DBT can be obtained after donating the dszABC genes involved in the desulfurization of this compound, from the R. erythropolis IGTS8 bacteria, together with the hpaC gene that encodes flavin: NADH HpaC reductase from E. coli W (Galán et al. (2000) J. Bacteriol. 182, 627), in a bacterium of the genus Pseudomonas (Spanish Patent Application 200000661).

In the present patent a method has been developed to obtain Pseudomonas putida KTH2 cells (pESOX3) (hereinafter referred to as Pseudomonas putida CECT5279) with maximum desulfurizing capacity. For this, the optimal operating conditions (temperature, pH, dissolved oxygen, initial concentration of nitrogen source) have been determined when a minimum growth medium is used in which the carbon source is glutamic acid.

Description of the invention Cultivation procedure of recombinant Pseudomonas putida (CECT 5279) for use in desulfurization of dibenzothiophene

The present invention describes a process by which the cultivation and growth of the genetically modified bacterium Pseudomonas putida CECT5279 is carried out under the ideal operating conditions (inoculum preparation, culture medium, pH, temperature, dissolved oxygen) to obtain the greater desulfurizing capacity of the model compound (dibenzothiophene), using a short period of time in relation to those used with cells of other microorganisms with the aforementioned natural capacity.

The production process of a dibenzothiophene biocatilizer is based on the improvement of the sulfur elimination capacity of organosulfurized molecules using the dibenzothiophene model that has the recombinant bacterium Pseudomonas putida KTH2 (pESOX3) obtained through Genetic Engineering and deposited in a model the Spanish Type Culture Collection (CECT) such as Pseudomonas putida CECT5279.

At the origin of this invention it was thought that the operating conditions employed during the growth of said bacterium should have a high influence on the desulfurization capacity thereof. To demonstrate that the aforementioned operating conditions have a high influence on obtaining 2HBP from dibenzothiophene when P. putida CECT5279 grown on glutamic acid is used, we first proceeded to determine the influence of the presence of ammonium in the medium of culture. Secondly, the influence of temperature was determined. Thirdly, the influence of oxygen transfer was determined. The results obtained allowed us to establish the optimal operating conditions in a flask fermentation system to improve both the production efficiency of the catalyst and its catalytic capacity, and, therefore, represents a significant advance in the development of the desulfurization process.

Next, we examine the possibility of applying this knowledge to the development of the production of the P. putida CECT5279 biocatalyst by fermentation in a bioreactor. Therefore, the last stage of this invention consisted of optimizing the operating conditions in a bioreactor to obtain the best yields of the biocatalyst both in terms of its biomass and in terms of its desulfurization power. For this, it was necessary not only to use an optimized culture medium as described above, but also to find the right balance between the production of biomass, the induction of the desulfurization system and the amount of oxygen supplied to the bioreactor. The results obtained show that under the conditions developed in the bioreactor it is possible to obtain a considerable improvement in production with respect to the procedure initially developed for flask production.

Description of the figures

To facilitate the understanding of the invention and being part of this descriptive report are accompanied by a series of figures.

Figure 1 shows the experimental results obtained in the experiments performed at 30 ° C in a flask using an orbital incubator (250 rpm) using BSM medium (see Example 1) to perform the experiments with 20 g / L of acid glutamic as a carbon source by adding or not ammonium to the medium of cultivation The initial starting concentration is 0.1 g / l of biomass obtained as specified in Example 1. The monitoring of biomass, pH, ammonium and desulfurizing capacity in Samples have been carried out as specified in Example 1. The experimental results regarding capacity are observed desulfurization in percentage numerical values for each of The experiments represented.

The results are shown in Figure 2 experimental results obtained in the experiments performed different temperatures (28, 30 and 32 ° C) in a flask using a orbital incubator (250 rpm) using BSM medium (see Example 1) to perform the experiments with 20 g / L of glutamic acid as source of carbon and ammonium as a source of nitrogen to the medium of culture. The initial starting concentration is 0.1 g / l of biomass obtained as specified in Example 1. The monitoring of biomass, pH and desulfurizing capacity in the samples has been carried out as specified in Example 1. It is observe the experimental results regarding the ability to desulfurization in percentage numerical values for each of The experiments represented.

Figure 3 shows the experimental results obtained in the experiments performed at 30 ° C in a flask using an orbital incubator (250 r.p.m.) and in a fermenter 2L commercial workload - with IPTG from the start of the experiment and IPTG added at 4 hours -, using medium BSM (see Example 1) to perform the experiments with 20 g / L of glutamic acid as a source of carbon and ammonium as a source of nitrogen. The initial starting concentration is 0.1 g / l of biomass obtained as specified in Example 1. The monitoring of biomass, pH and desulfurizing capacity in the samples has been carried out as specified in Example 1. It is observe the experimental results regarding the ability to desulfurization in percentage numerical values for each of The experiments represented.

Embodiment of the invention

To the present invention related to a Production procedure of a biocatalyst, the following examples.

Example 1 Analysis of the desulfurizing capacity of DBT of Pseudomonas putida CECT5279 grown in flask and orbital incubator in the presence and absence of ammonium in the culture medium performed in the following stages Preparation of culture medium

For the studies on the growth of the microorganism Pseudomonas putida CECT5279 described in this patent, the minimum medium called BSM (Basal Saline Medium) formed by the following compounds in the following concentrations has been used as basic culture: monosodium phosphate monohydrate 4 g / L ; Dipotassium phosphate trihydrate 4 g / L; magnesium chloride hexahydrate 0.0245 g / L; calcium chloride dihydrate 0.001 g / L; iron (III) chloride hexahydrate 0.001 g / L; tetracycline 25 µg / mL; 2 mM magnesium sulfate; 2% glycerol; glutamic acid 20 g / L. Adjust to pH 7 with NaOH. In addition, 2 g / L ammonium chloride is added to this medium as a source of nitrogen, with the exception of the test in which the absence of this compound is studied (example 1). The desulfurizing capacity inducer: isopropyl-? -D-thiogalactopyranoside (IPTG) 2 µL / mL is always added but the IPTG test can be carried out from the beginning (examples 1, 2 and 3) or added after 4 hours after starting growth (example 3).

Preinocle Preparation

In all cases, a previously standardized method of inoculum preparation has been used, which provides total reproducibility of the experimental results. For the preparation of the pre-circle, P. putida CECT5279 bacteria have been used, which has been preserved in Eppendorf tubes of 1.5 mL capacity with a solution of LB medium (Yeast extract 5 g / L; tryptone 10 g / L; 10 g / L sodium chloride) and 50% glycerin at a temperature of -18 ° C for a maximum period of three weeks. The pre-circle is prepared in LB medium by inoculating Erlenmeyer flasks with 50 mL of LB medium, with 50 µL of the contents of the Eppendorf and incubating them in an orbital incubator at 30 ° C and 250 rpm for 12 hours. This preinocle is used as described in each example.

Analytical techniques used Biomass analysis

For monitoring the growth of microorganism employed a method of analysis of the biomass concentration (g / L) based on turbidity measurement of the broth, that is to say an optical method that relates the absorbance that present the bacteria, in wavelengths of the spectrum visible, with the dry weight of the microorganism. It consists, so therefore, in measuring the absorbance of the sample to a certain length Wave, in front of a target. The wavelength used for this Analysis was 600 nm and the target used, distilled water. He Biomass analysis during experiments is performed according to the following experimental protocol: A sample of the orbital bioreactor or incubator and diluted with distilled water, of so that the measured absorbance enters within the linear region of The calibration curve. The absorbance value obtained at 600 nm is turned into concentration through the equation:

C_ {biomass} \ (g / L) = 0.621 \ Abs \ 600 nm}

Nitrogen substrate concentration measurement

The nitrogen concentration (as salt of ammonium) has been measured using a membrane electrode semipermeable house ORION, model 720A. The procedure followed for the analysis of ammonium has been the following: It is taken a sample and diluted if necessary to a volume of 10 mL and a solution consisting of: 5 M NaOH, 0.5 M EDTA, is added, 10% methanol and thymol blue, until pH values are reached greater than 10 (when the blue thymus indicator) turns colorless to bluish). To ensure that the reaction takes done correctly, 10 drops of the solution are always added, Shaking the sample constantly. The calibration is done introducing two patterns of known concentration (10 and 100 p.p.m.). The calibration concentration range is 0 to 100 p.p.m. The patterns are analyzed as indicated 5 and the pending is calculated and stored by the team so that, a Once the calibration is done, the concentration is read directly.

Analysis of the 4S desulfurization path compounds

It is performed by high-performance liquid chromatography. resolution. The chromatograph used for this purpose is of the brand HEWLETT PACKARD, 1100 series, with "diode array" detector. The column used is of type C-8 of 3 µm (150 x 4.6 mm); As eluent an acetonitrile / water mixture has been used Mili Q (50:50), with a flow rate of 1 L / min and at a temperature of 25ºC, measuring in the detector at a wavelength of 240 nm. In these Conditions are identified: DBT, at an elution time of 14.9 minutes, 2HBP, 5.9 minutes, dibenzothiophene sulfone (DBTO_ {2}), which appears at 4 minutes and dibenzothiophene sulfoxide (DBTO) at 2.7 minutes. The calibration equations obtained are the following:

C_ {DBT} (µM) = 0.00478 \ Area_ {14.9 min} - 0.283

C_ {HBP} (µM) = 0.03536 \ Area_ {5.9 min} - 0.621

C_ {DBTO_ {2}} (µM) = 0.0095 \ cdot Area_ {4 min} - 0.41

C_ {DBTO} (\ mu M) = 0.03 • A 2.7 min - 0.06

The other compound of the 4S route, the hydroxybiphenylsulfinite (HBPSi) is analyzed at pH between 1 and 2 to be detected with the UV-VIS detector in form closed - called sultine -. It is analyzed using a C18 column 3 µm (150 x 4.6 mm), a mixture used as eluent acetonitrilolagua Mili Q (50:50), with a flow rate of 1 mL / min at temperature of 25 ° C. The compound is detected at 1.99 minutes in a 210 nm wavelength, being the calibration equation of the concentration depending on the area obtained from the peak:

C_ {HBPSi} (µ M) = 0.021 \ Area_ {1.99 min} - 0.6

In summary, the protocol for the sample preparation for monitoring the desulfurization is as follows: 0.5 mL of sample taken each time to test cells at rest (resting cells). 0.5 mL of acetonitrile is added and then centrifuged at 9000 rpm and 4 ° C for 10 minutes. An aliquot of the supernatant is taken to be analyzed by HPLC and detect DBT, 2HBP, DBTO_ {2} and DBTO. The area of each peak corresponding to each compound allows its concentration to be calculated using the corresponding calibration equation. Subsequently, the contents of the vials are acidified with HCI (10 µL) to reach a pH between 1 and 2 and analyzed with column C18 to detect HBPSi.

Desulfurization capacity analysis

The analysis is performed by desulfurization system resting cells (resting cells). The embodiment protocol is as follows: it is based on a sample whose cells have been taken during growth in the BSM medium and stored in 50% glycerol at -18 ° C. The samples are thawed and their biomass concentration is measured, to know the inoculum volume necessary to perform the desulfurization test with a concentration of 0.75 g / L of said biomass. In parallel, DBT is added to the HEPES buffer at pH 8 (40 mL in Erlenmeyer 250 mL), to have an initial DBT concentration of 25 µM. The buffer with the DBT is left in an incubator for 15 minutes at a temperature of 30 ° C and 250 rpm. Then the previously set amount of biomass is inoculated in the Erlenmeyer. Desulfurization is monitored by taking samples every 30 minutes for 3 hours. These samples will be subsequently analyzed by HPLC, as indicated. The parameter indicating the desulfurizing capacity is what has been called the percentage of desulfurization, whose value is obtained according to the following expression:

% BDS = \ frac {C_ {HBP}} {C_ {DBT}} x 100

The objective of these experiments was to know the influence that the introduction of ammonium in the culture medium has on the capacity of desulfurization P. putida CECT5279 during the different stages of growth when an amino acid such as glutamic acid is used as a carbon source. To observe this influence, two experiments were performed. In the first one, 34 mM ammonium chloride plus 136 mM glutamic acid was used as the nitrogen source. In the second of them, 136 mM glutamic acid was used as the sole source of nitrogen.

The experiments were carried out as follows. 50 mL of the BSM medium (salts and glutamic acid) are distributed in 250 mL Erlenmeyer flasks. Subsequently, the IPTG (100 µL) is introduced, to have a concentration of 0.2 mM, and the tetracycline (50 µL), so that the concentration is 50 µL. Ammonium chloride is also added in half of the flasks. The flasks are inoculated with the pre-circle prepared as previously explained, so that the starting concentration in them is 0.1 g / L of biomass. The inoculated Erlenmeyer flasks are introduced into the orbital incubator with a stirring of 250 rpm and a temperature of 30 ° C. Samples (one Erlenmeyer flask per point) are taken and processed, as indicated below, in order to monitor the process and for subsequent desulfurization test ( resting cells ).

During the development of the cultures, the analysis and monitoring of the biomass, ammonium, glutamic acid, pH and desulfurization capacity were carried out. The volume of the Erlenmeyer (50 mL) is centrifuged at 9000 rpm for 10 min at 4 ° C, in order to separate the broth from the cells, and thus stop the growth and evolution of the samples. The broth is preserved in sterile tubes and placed in a freezer for preservation until the analysis of the different components of the broth. The sediment with the cells is washed with 50 ml of saline and centrifuged again under the conditions mentioned above. The supernatant will be removed and the sediment will be resuspended in 10 mL of 50% glycerol for preservation until the desulfurization test (resting cells ) is performed.

The results are shown in Figure 1 obtained from the aforementioned experiments, noting that there are differences in the evolution of biomass and pH throughout the experiment (20 hours) between the crops that they contain ammonium chloride and those that do not contain it. In the cultures that do not contain ammonium chloride produce a progressive accumulation of ammonium because the microorganism must  use the hydrocarbon part of the molecule to a greater extent glutamic acid and leftover ammonia, which leaves in the broth. Without However, when ammonium ion is introduced into the broth, a decrease in concentration during the first 10 hours of fermentation, then producing an increase in ammonium at same speed as in the previous case.

The desulfurizing capacity of the microorganism obtained under these conditions is performed and calculated as indicated above. While the cells grown in The presence of ammonium has a maximum desulfurization of around 70%, those obtained without ammonium do not exceed in any case 25% desulfurization. This result indicates the importance of ammonium to obtain cells with desulfurizing capacity.

Example 2 Analysis of DBT desulfurizing capacity of Pseudomonas putida CECT5279 grown in flask and orbital incubator at different temperatures

It is developed as in Example 1, where to check the influence of the operating temperature on the desulfurization capacity developed by P. putida CECT5279 cells, experiments were carried out at different temperatures in a culture medium containing ammonium chloride. . The experiments were carried out as follows. For each experiment performed at a given temperature, Erlenmeyer flasks were prepared with culture medium containing ammonium chloride as described in example 1. The inoculated Erlenmeyer flasks were introduced into the orbital incubator with a stirring of 250 rpm at the temperatures under study. , ie 28 ° C, 30 ° C, and 32 ° C. Samples (one Erlenmeyer flask per point) were taken and processed and analyzed as indicated in example 1.

The results are shown in Figure 2 comparisons of the three commented experiments, observing that the maximum level of desulfurization is obtained when the cells are grown at a temperature of 30 ° C.

Example 3 Analysis of the desulfurizing capacity of DBT of Pseudomonas putida CECT5279 in a reactor of 2 L of work volume

As in Example 1, to determine the influence of oxygen transport (or the amount of dissolved oxygen that the microorganism can access) in the preparation of cells with DBT desulfurization capacity, an experiment was conducted using a commercial reactor type Stirred tank of the Braun Biotech brand (currently Sartorius ), model Biostat B, using a tank with a working volume of 2 L, with pH, dissolved oxygen and temperature measurement sensors.

In the reactor you can control the amount of air (flow) and stirring speed on a scheduled basis to avoid the existence of cellular damage caused by stress hydrodynamic In this way, it is possible to maintain oxygen dissolved in the system in non-limiting values for growth of an aerobic microorganism as is the employee. Another objective of This experiment is the determination of the impact of stress oxidative in the production of the biocatalyst.

The experimental procedure used in Experiments performed on commercial fermenter described to continuation. First the composition of the culture medium is similar to that used in Example 1, using a volume of 2 L, the salts of the BSM medium being sterilized separately (whose pH has been adjusted to 7) and the carbon source (acid glutamic). The carbon source is introduced sterile (using a peristaltic pump of the equipment for this) once the temperature of the vessel has dropped to values close to 50 ° C. A once the salts and the carbon source are mixed, and with the reactor preheated to a temperature at 30 ° C the antibiotic is introduced tetracycline In parallel the inoculum is prepared as it has been previously discussed in Example 1, and known the concentration of biomass present in it, the inoculation is carried out in the fermenter with the necessary volume (not exceeding 200 mL) so that the initial biomass concentration is 0.1 g / L. In a first experiment, keeping the preheated reactor at a temperature at 30 ° C (example 2) the IPTG is then introduced (0.2 mM) that acts as inducer of the desulfurization system. In a second experiment the IPTG was introduced into the reactor after of the 4 hours of fermentation.

An air flow is introduced into the reactor (L air / L medium / min) to the broth, increasing during I experience the agitation from an initial value of 100 r.p.m. until a maximum of 350 r.p.m. to avoid the existence of cellular damage caused by hydrodynamic stress. This increase is made to compensate for dissolved oxygen consumption and try to maintain the value of its amount in solution in values slightly greater than 0% saturation.

To track the evolution of this system during the process, the taking of the samples at different times, as well as their analysis. Biomass, pH and capacity analysis were performed. desulfurization A volume of 50 is extracted in each sample mL of the reactor. Each sample is processed and analyzed as described. in example 1.

The results are shown in Figure 3 obtained under conditions of limitation of dissolved oxygen (flask in orbital incubator) and in the case of maintaining the aforementioned variable in values far from a possible limitation (tank type reactor agitated and airy). In this second case the results are shown obtained when the IPTG inducer is added at the beginning of the fermentation and after 4 hours of fermentation.

First, Figure 3 shows that when the IPTG inductor is added from the beginning of the fermentation it is observed that both the growth of the microorganism as the ability to remove sulfur from the DBT molecule is less when there is no oxygen limitation. This is due to oxidative stress that this microorganism suffers from hyperproduction of the enzyme HpaC oxidoreductase. As the inductor IPTG has been introduced since the beginning of the growth of the microorganism a large amount of the enzyme HpaC is produced, the which generates from the beginning too much FMNH_ {2} who in turn in the presence of oxygen it generates a lot of hydrogen peroxide that at not being able to be eliminated by the bacteria causes cell damage severe that decreases its desulfurization capacity.

In view of the result of the experiment Previously, it was thought that the method to be used to produce cells with high desulfurization capacity in a reactor would consist of perform an initial growth of the bacteria in the absence of IPTG inductor, so that after 4 hours of starting it, when the amount of biomass is higher, you can enter the IPTG in the reactor. Performed this experiment, the results are shown in Figure 3. In this figure you can see how the introducing the IPTG at 4 hours influences highlighted in the desulfurization capacity that those cells develop, achieving desulfurization values greater than those obtained to date with any other procedure, since either in reactor or in orbital incubator.

Therefore, after examples 1, 2 and 3 that have been exposed, the steps to follow to obtain a culture of Pseudomonas putida CECT5279 with high desulfurizing capacity are the following:

to)

Starting from a frozen stock of microorganism a first pre-circle is performed in culture medium LB contained in 250 mL Erlenmeyer flasks. This crop is incubate at 30 ° C for 12 hours in Orbital Incubator. TO then, with the cells grown at this stage, a second culture in LB medium under the same conditions as before but setting the initial biomass concentration at 0.1 g / L.

b)

A Once the preinocle is obtained, it is transferred to the 2 L bioreactor which Contains a BSM synthetic culture medium whose composition is: monosodium phosphate monohydrate 4 g / L; dipotassium phosphate trihydrate 4 g / L; 2 g / L ammonium chloride; magnesium chloride hexahydrate 0.0245 g / L; calcium chloride dihydrate 0.001 g / L; chloride iron (III) hexahydrate 0.001 g / L; tetracycline 25 µg / mL; 2 mM magnesium sulfate; 2% glycerol; glutamic acid 20 g / L; isopropyl-? -D-thiogalactopyranoside (IPTG) 2 µL. pH 7 with NaOH.). The initial concentration of Biomass in the reactor is set at 0.1 g / L. The conditions of operation in the aerated bioreactor are 28-32ºC of temperature and agitation, which will increase from 100 rpm to 350 rpm The inductor of the desulfurizing capacity IPTG must be add within 4 hours of having performed the inoculum in the reactor.

C)

Through this procedure you they will obtain cells with high desulfurization capacity from 10 hours of growth in the environment and conditions described.

Claims (4)

1. Procedure of culture of the recombinant Pseudomonas putida (CECT 5279) for its use in the desulfurization of dibenzothiophene, characterized in that the culture of said bacterium includes the following operations:

to)

Use of a preinoculum obtained at from frozen stock cells and using culture medium LB (Yeast extract 5 g / L; tryptone 10 g / L; sodium chloride 10 g / L) to perform preinoculars.

b)

Use of a culture medium synthetic and minimal BSM: monosodium phosphate monohydrate 4 g / L; Dipotassium phosphate trihydrate 4 g / L; 2 g / L ammonium chloride; magnesium chloride hexahydrate 0.0245 g / L; calcium chloride 0.001 g / L dihydrate; iron (III) chloride hexahydrate 0.001 g / L; tetracycline 25 µg / mL; 2 mM magnesium sulfate; 2% glycerol; acid glutamic 20 g / L; isopropyl-? -D-thiogalactopyranoside (IPTG) 2 µm / mL. pH 7 with NaOH).

2. Method of cultivation of the recombinant Pseudomonas putida (CECT 5279) for use in the desulfurization of dibenzothiophene, according to claim 1, characterized in that the operating temperature used is between 26 and 34 ° C, presenting a greater desulfurization capacity at 30 ° C. 3. Method of culturing a biocatalyst for the desulfurization of dibenzothiophene using a recombinant bacterium of Pseudomonas putida , according to claim 1, characterized in that the IPTG inducer is introduced after 4 hours of carrying out the inoculum in the growth medium. 4. Production process of a biocatalyst for desulfurization of dibenzothiophene using a recombinant bacterium of Pseudomonas putida , according to previous claims, characterized in that it is carried out in a bioreactor or in an orbital incubator.