4S Pathway Research Articles

Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds

The 4S pathway is the most studied bioprocess for the removal of the recalcitrant sulfur of aromatic heterocycles present in fuels. It consists of three sequential functional units, encoded by the dszABCD genes, through which the model compound dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) molecule. In this work, a set of synthetic dsz cassettes were implanted in Pseudomonas putida KT2440, a model bacterial “chassis” for metabolic engineering studies. The complete dszB1A1C1-D1 cassette behaved as an attractive alternative — to the previously constructed recombinant dsz cassettes — for the conversion of DBT into 2HBP. Refactoring the 4S pathway by the use of synthetic dsz modules encoding individual 4S pathway reactions revealed unanticipated traits, e.g., the 4S intermediate 2HBP-sulfinate (HBPS) behaves as an inhibitor of the Dsz monooxygenases, and once secreted from the cells it cannot be further taken up. That issue should be addressed for the rational design of more efficient biocatalysts for DBT bioconversions. In this sense, the construction of synthetic bacterial consortia to compartmentalize the 4S pathway into different cell factories for individual optimization was shown to enhance the conversion of DBT into 2HBP, overcome the inhibition of the Dsz enzymes by the 4S intermediates, and enable efficient production of unattainable high added value intermediates, e.g., HBPS, that are difficult to obtain using the current monocultures.

Influence of oxygen transfer on Pseudomonas putida effects on growth rate and biodesulfurization capacity.

The growth rate and desulfurization capacity accumulated by the cells during the growth of Pseudomonas putida KTH2 under different oxygen transfer conditions in a stirred and sparged tank bioreactor have been studied. Hydrodynamic conditions were changed using different agitation conditions. During the culture, several magnitudes associated to growth, such as the specific growth rate, the dissolved oxygen concentration and the carbon source consumption have been measured. Experimental results indicate that cultures are influenced by the fluid dynamic conditions into the bioreactor. An increase in the stirrer speed from 400 to 700 rpm has a positive influence on the cell growth rate. Nevertheless, the increase of agitation from 700 to 2000 rpm hardly has any influence on the growth rate. The effect of fluid dynamics on the cells development of the biodesulfurization (BDS) capacity of the cells during growth is different. The activities of the intracellular enzymes involved in the 4S pathway change with dissolved oxygen concentration. The enzyme activities have been evaluated in cells at several growth time and different hydrodynamic conditions. An increase of the agitation from 100 to 300 rpm has a positive influence on the development of the overall BDS capacity of the cells during growth. This capacity shows a decrease for higher stirrer speeds and the activity of the enzymes monooxygenases DszC and DszA decreases dramatically. The highest value of the activity of DszB enzyme was obtained with cells cultured at 100 rpm, while this activity decreases when the stirrer speed was increased higher than this value.

Evaluation of the Conformational Stability of Recombinant Desulfurizing Enzymes from a Newly Isolated Rhodococcus sp.

Metabolic pathways of aerobic bacteria able to assimilate sulfur can provide biocatalysts for biodesulfurization of petroleum and of other sulfur-containing pollutants. Of major interest is the so-called "4S pathway," in that C-S bonds are specifically cleaved leaving the carbon skeleton of substrates intact. This pathway is carried out by four enzymes, named Dsz A, B, C, and D. In view of a possible application of recombinant Dsz enzymes in biodesulfurization treatments, we have investigated the structural features of enzymes cloned from a Rhodococcus strain isolated from polluted environmental samples and their resistance to temperature (20-95°C) and to organic solvents (5, 10, and 20% v/v methanol, acetonitrile, hexane, and toluene). Changes in protein structures were assessed by circular dichroism and intrinsic fluorescence spectroscopy. We found that all Dsz proteins are unfolded by temperatures in the range 45-60°C and by all solvents tested, with the most dramatic effect being produced by toluene. These results suggest that stabilization of the biocatalysts by protein engineering will be necessary for developing biodesulfurization technologies based on Dsz enzymes.

Isolation and characterization of an interactive culture of two Paenibacillus species with moderately thermophilic desulfurization ability

To isolate and characterize novel thermophilic bacteria capable of biodesulfurization of petroleum. A culture containing two Paenibacillus spp. (denoted "32O-W" and "32O-Y") was isolated by repeated passage of a soil sample at up to 55°C in medium containing dibenzothiophene (DBT) as sulfur source. Only 32O-Y metabolized DBT, apparently via the 4S pathway; maximum activity occurred from 40 to 45°C, with some activity up to at least 50°C. 32O-W enhanced DBT metabolism by 32O-Y (by 22-74% at 40-50°C). With sulfate as sulfur source, 32O-Y and 32O-W grew well up to 58 and 63°C, respectively. Selection of a mixed culture of 32O-Y and 32O-W at 54°C increased DBT metabolism 36-42% from 40 to 45°C. Genome sequencing identified desulfurization gene homologs in the strains consistent with their desulfurization properties. The 32O-Y/32O-W culture may be a useful starting point for development of an improved thermophilic petroleum biodesulfurization process.

Biocatalytic desulfurization of thiophenic compounds and crude oil by newly isolated bacteria.

Microorganisms possess enormous highly specific metabolic activities, which enable them to utilize and transform nearly every known chemical class present in crude oil. In this context, one of the most studied biocatalytic processes is the biodesulfurization (BDS) of thiophenic sulfur-containing compounds such as benzothiophene (BT) and dibenzothiophene (DBT) in crude oils and refinery streams. Three newly isolated bacterial strains, which were affiliated as Rhodococcus sp. strain SA11, Stenotrophomonas sp. strain SA21, and Rhodococcus sp. strain SA31, were enriched from oil contaminated soil in the presence of DBT as the sole S source. GC-FID analysis of DBT-grown cultures showed consumption of DBT, transient formation of DBT sulfone (DBTO2) and accumulation of 2-hydroxybiphenyl (2-HBP). Molecular detection of the plasmid-borne dsz operon, which codes for the DBT desulfurization activity, revealed the presence of dszA, dszB, and dszC genes. These results point to the operation of the known 4S pathway in the BDS of DBT. The maximum consumption rate of DBT was 11 μmol/g dry cell weight (DCW)/h and the maximum formation rate of 2-HBP formation was 4 μmol/g DCW/h. Inhibition of both cell growth and DBT consumption by 2-HBP was observed for all isolates but SA11 isolate was the least affected. The isolated biocatalysts desulfurized other model DBT alkylated homologs. SA11 isolate was capable of desulfurizing BT as well. Resting cells of SA11 exhibited 10% reduction in total sulfur present in heavy crude oil and 18% reduction in total sulfur present in the hexane-soluble fraction of the heavy crude oil. The capabilities of the isolated bacteria to survive and desulfurize a wide range of S compounds present in crude oil are desirable traits for the development of a robust BDS biocatalyst to upgrade crude oils and refinery streams.

Sulfur Removal from Dibenzothiopheneby Newly Isolated Paenibacillusvalidus Strain PD2 and Process Optimizationin Aqueous and Biphasic (Model-Oil) Systems

Dibenzothiophene (DBT) is an organic sulfur compound which remains in oil after hydrodesulfurization (HDS) process and can be removed by biodesulfurization (BDS). A new strain of Paenibacillus validus (strain PD2) was isolated from oil contaminated soils that is able to desulfurize DBT. HPLC analysis and Gibbs assay showed that this strain was capable to convert DBT to 2-Hydroxybiphenyl (2-HBP) as final product. The presence of dszC gene confirmed that DBT desulfurization occurred through the 4S pathway. Maximum growth and the highest induction in dsz operon obtained in the presence of dimethyl sulfoxide (DMSO) as sole sulfur source. DBT concentration, temperature and pH were optimized statistically for growing and resting cells by using Response Surface Methodology (RSM). All parameters in growing cells had a significant effect on 2-HBP production during BDS of DBT by P. validus PD2, although in resting cells temperature in range of 20-40 degrees C was not a significant factor. Maximum BDS for growing cells was obtained at 0.41 mM DBT concentration, pH 6.92 and temperature 31.23 degrees C. For resting cells, optimum pH, temperature and DBT concentration were 6.62, 27.73 degrees C and 7.86 mM respectively. The results of this study showed that high concentrations of DBT could be desulfurized by P. validus strain PD2 in model-oil. Thus, the isolated strain could be introduced as a proper candidate for biodesulfurization of organic sulfur in the oil industry.

Isolation and Identification of Dibenzothiophene Biodesulfurizing Bacteria

This study aimed to obtain efficient bacteria capable of desulfurizing dibenzothiophene (DBT). For this purpose forty oil contaminated soil samples were collected from different sites in Iraq. It was found that three isolates (M9, M19 and S25) had the ability to desulfurize DBT (cleave C-S bond) and converted it to 2-hydroxybiphenel (2-HBP) or other phenolic end products. This suggests the involvement of the 4S pathway in the desulfurizing of DBT via a specific cleavage of only the C-S bond by these isolates. These isolates were identified as Pseudomonas aeruginosa. The result also showed that P. aeruginosa S25 was the most efficient one for removing sulfur from DBT. The GC/MS analysis for DBT after growth of P. aeruginosa S25, indicated that 12.89 % of DBT was consumed (consumption of sulfur), and the product (2-HBP) was further converted to 2-MBP (addition of a methyl group) and this could be a novel pathway for consuming DBT.

A novel Bacillus pumilus-related strain from tropical landfarm soil is capable of rapid dibenzothiophene degradation and biodesulfurization.

BackgroundThe presence of organic sulfur-containing compounds in the environment is harmful to animals and human health. The combustion of these compounds in fossil fuels tends to release sulfur dioxide in the atmosphere, which leads to acid rain, corrosion, damage to crops, and an array of other problems. The process of biodesulfurization rationally exploits the ability of certain microorganisms in the removal of sulfur prior to fuel burning, without loss of calorific value. In this sense, we hypothesized that bacterial isolates from tropical landfarm soils can demonstrate the ability to degrade dibenzothiophene (DBT), the major sulfur-containing compound present in fuels.ResultsNine bacterial isolates previously obtained from a tropical landfarm soil were tested for their ability to degrade dibenzothiophene (DBT). An isolate labeled as RR-3 has shown the best performance and was further characterized in the present study. Based on physiological aspects and 16 s rDNA sequencing, this isolate was found to be very closely related to the Bacillus pumillus species. During its growth, high levels of DBT were removed in the first 24 hours, and a rapid DBT degradation within the first hour of incubation was observed when resting cells were used. Detection of 2-hydroxybiphenyl (HBP), a marker for the 4S pathway, suggests this strain has metabolical capability for DBT desulfurization. The presence of MgSO4 in growth medium as an additional sulfur source has interfered with DBT degradation.ConclusionsTo our knowledge, this is the first study showing that a Bacillus strain can metabolize DBT via the 4S pathway. However, further evidences suggest RR-3 can also use DBT (and/or its derivative metabolites) as carbon/sulfur source through another type of metabolism. Compared to other reported DBT-degrading strains, the RR-3 isolate showed the highest capacity for DBT degradation ever described in quantitative terms.The potential application of this isolate for the biodesulfurization of this sulfur-containing compound in fuels prior to combustion was discussed.

Improvement of Biodesulfurization Rate of Alginate Immobilized Rhodococcus erythropolis R1.

Background:Sulfur oxides released from the burning of oil causes severe environmental pollution. The sulfur can be removed via the 4S pathway in biodesulfurization (BDS). Immobilization approaches have been developed to prevent cell contamination of oil during the BDS process.Objectives:The encapsulation of Rhodococcus erythropolis R1 in calcium alginate beads was studied in order to enhance conversion of dibenzothiophene (DBT) to 2-hydroxy biphenyl (2-HBP) as the final product. Also the effect of different factors on the BDS process was investigated.Materials and Methods:Calcium alginate capsules were prepared using peristaltic pumps with different needle sizes to control the beads sizes. Scanning electron microscopy and flow cytometry methods were used to study the distribution and viability of encapsulated cells, respectively. Two non-ionic surfactants and also nano Ƴ-Al2O3were used with the ratio of 0.5% (v/v) and 1:5 (v/v) respectively to investigate their BDS efficiency. In addition, the effect of different bead sizes and different concentrations of sodium alginate in BDS activity was studied.Results:The 2% (w/v) sodium alginate beads with 1.5mm size were found to be the optimum for beads stability and efficient 2-HBP production. The viability of encapsulated cells decreased by 12% after 20 h of desulfurization, compared to free cells. Adding the non-ionic surfactants markedly enhanced the rate of BDS, because of increasing mass transfer of DBT to the gel matrix. In addition, Span 80 was more effective than Tween 80. The nanoƳ-Al2O3 particles could increase BDS rate by up to two-folds greater than that of the control beads.Conclusions:The nano Ƴ-Al2O3 can improve the immobilized biocatalyst for excellent efficiency of DBT desulfurization. Also the BDS activity can be enhanced by setting the other explained factors at optimum levels.

Genetic Analysis of Benzothiophene Biodesulfurization Pathway of Gordonia terrae Strain C-6

Sulfur can be removed from benzothiophene (BT) by some bacteria without breaking carbon-carbon bonds. However, a clear mechanism for BT desulfurization and its genetic components have not been reported in literatures so far. In this study, we used comparative transcriptomics to study differential expression of genes in Gordonia terrae C-6 cultured with BT or sodium sulfate as the sole source of sulfur. We found that 135 genes were up-regulated with BT relative to sodium sulfate as the sole sulfur source. Many of these genes encode flavin-dependent monooxygenases, alkane sulfonate monooxygenases and desulfinase, which perform similar functions to those involved in the 4S pathway of dibenzothiophene (DBT) biodesulfurization. Three of the genes were found to be located in the same operon, designated bdsABC. Cell extracts of pET28a-bdsABC transfected E. coli Rosetta (DE3) converted BT to a phenolic compound, identified as o-hydroxystyrene. These results advance our understanding of enzymes involved in the BT biodesulfurization pathway.

Deep-desulfurization of dibenzothiophene and its derivatives present in diesel oil by a newly isolated bacterium Achromobacter sp. to reduce the environmental pollution from fossil fuel combustion

Thiophenic compounds viz. dibenzothiophene (DBT) and its derivatives present in fossil fuels contribute significantly to air pollution. Here we report for the first time the efficacy of a strain of Achromobacter sp., isolated from a contaminated petroleum–oil soil sample, at efficiently cleaving carbon–sulfur (C–S) bonds in DBT under mesophilic growth conditions. This bacterium utilized DBT and its derivative viz. 4-methyl DBT as the sole source of sulfur and degraded them to 2-hydroxybiphenyl (2-HBP). The 2-HBP was finally converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP suggesting that this bacterium followed sulfur-specific pathway (4S pathway) for deep-desulfurization. Further, 2-MBP is much less pollutant than DBT and therefore, it reduces the environmental pollution from fossil fuel combustion. Gas chromatography and X-ray fluorescence analyses revealed that the resting cells of Achromobacter sp. reduced 7.1% of the total sulfur content of diesel oil, obtained typically by treating 5mL of diesel oil with 14g of dry cell, without compromising the quality of diesel oil. For the industrial application, DBT desulfurization was optimized by using response surface methodology. Our study demonstrates that Achromobacter sp. is a potential candidate for biodesulfurization of diesel oil with anticipated application as an eco-friendly biodesulfurizing agent.

Exploring the mechanism of biocatalyst inhibition in microbial desulfurization.

Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 μM with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC50s) for DszA, DszB, and DszC were measured to be 60 ± 5 μM, 110 ± 10 μM, and 50 ± 5 μM, respectively. The fact that the IC50s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation.

Fructophilic behaviour of Gordonia alkanivorans strain 1B during dibenzothiophene desulfurization process

Biodesulfurization (BDS) aims at the removal of recalcitrant sulfur from fossil fuels at mild operating conditions with the aid of microorganisms. These microorganisms can remove sulfur from dibenzothiphene (DBT), a model compound, or other polycyclic aromatic used as sulfur source, making BDS an easy and environmental friendly process. Gordonia alkanivorans strain 1B has been described as a desulfurizing bacterium, able to desulfurize DBT to 2-hydroxybiphenyl (2-HBP), the final product of the 4S pathway, using d-glucose as carbon source. However, both cell growth and desulfurization can be largely affected by the nutrient composition of the growth medium, due to cofactor requirements of many enzymes involved in the BDS biochemical pathway. In this study, the main goal was to investigate the influence of several sugars, as carbon source, on the growth and DBT desulfurization ability of G. alkanivorans strain 1B. The results of desulfurization tests showed that the lowest values for the growth rate (0.025 hour(-1)) and for the overall 2-HBP production rate (1.80 μm/hour) by the strain 1B were obtained in glucose grown cultures. When using sucrose, the growth rate increase exhibited by strain 1B led to a higher biomass productivity, which induced a slightly increase in the 2-HBP production rate (1.91 μm/hour), conversely in terms of 2-HBP specific production rate (q2-HBP) the value obtained was markedly lower (0.718 μmol/g/hour in sucrose versus 1.22 μmol/g/hour in glucose). When a mixture of glucose and fructose was used as carbon source, strain 1B reached a value of q2-HBP=1.90 μmol/g/hour, close to that in fructose (q2-HBP=2.12 μmol/g/hour). The highest values for both cell growth (μ=0.091 hour(-1)) and 2-HPB production (9.29μm/hour) were obtained when strain 1B was desulfurizing DBT in the presence of fructose as the only carbon source, indicating a fructophilic behaviour by this bacterium. This fact is in agreement with the highest value of biomass productivity by strain 1B be in fructose, which resulted in a higher amount cells fulfilling the DBT-desulfurization. The greater number of functional cells conducted to a more effectiveness BDS process by strain 1B, as they attained a q2-HBP about 74% higher than in glucose grown cultures. Moreover, this significant BDS enhancement can better be observed in terms of the overall 2-HBP production rate, which increased over 5-fold, from 1.80 μm/hour (in glucose) to 9.29 μm/hour (in fructose).

Crystallization and preliminary structural analysis of dibenzothiophene monooxygenase (DszC) fromRhodococcus erythropolis

Dibenzothiophene (DBT) and its derivatives are typical sulfur compounds found in fossil fuels. These compounds show resistance to the hydrodesulfurization treatment that is commonly used in industry. Dibenzothiophene monooxygenase (DszC) is responsible for the oxidation of DBT, which is the first and the rate-limiting step in the DBT enzymatic desulfurization 4S pathway. In this study, the crystal structure of DszC from Rhodococcus erythropolis DS-3 is reported. The crystal of native DszC belonged to space group P1, with unit-cell parameters a = 96.16, b = 96.27, c = 98.56 Å, α = 81.03, β = 67.57, γ = 85.84°. To determine the phase, SAD X-ray diffraction data were collected from a SeMet-derivative DszC crystal, which also belonged to space group P1, with unit-cell parameters a = 95.379, b = 95.167, c = 94.891 Å, α = 87.046, β = 70.536, γ = 79.738°. Further structural analysis of DszC is in progress.

Thermophilic desulfurization of dibenzothiophene and different petroleum oils by Klebsiella sp. 13T

Biodesulfurization (BDS) has the potential to desulfurize dibenzothiophene (DBT) and its alkylated derivatives, the compounds that are otherwise refractory to hydrodesulfurization (HDS). Thermophilic microorganisms are more appropriate to be used for BDS applications following HDS. The aim of the present study was to isolate a thermophilic microorganism and to explore its commercial relevance for BDS process. The desulfurizing thermophilic strain was isolated and enriched from various soil and water samples using sulfur free medium (SFM) supplemented with DBT. Microbiological and genomic approach was used to characterize the strain. Desulfurization reactions were carried out using DBT and petroleum oils at 45°C followed by different analytical procedures. We report the isolation of a thermophilic bacterium Klebsiella sp. 13T from contaminated soils collected from petroleum refinery. HPLC analysis revealed that Klebsiella sp. 13T could desulfurize DBT to 2-hydroxybiphenyl (2-HBP) at 45°C through 4S pathway. In addition, adapted cells of Klebsiella sp. 13T were found to remove 22-53% of sulfur from different petroleum oils with highest sulfur removal from light crude oil. Klebsiella sp. 13T is a potential candidate for BDS because of its thermophilic nature and capability to desulfurize petroleum oils.

Comparative modeling of DszC, an enzyme in biodesulfurization, and performing in silico point mutation for increasing tendency to oil.

Desulfurization protein named DszC from Rhodococcus erythropolis is the key enzyme for biodesulforization of dibenzothiophene (DBT) in 4S pathway, which is a pathway with four enzymes. DszC enzyme biodesulfurizes DBT and its derivatives in oil components and biphasic systems. It functions well at the oil- water interface. In this study point mutation performed in DszC enzyme regarding to increase protein hydrophobicity and stability for application in immobilized form. 3D model of DszC predicted using Phyre2, SAM-T08 and M4t servers. I-Mutant 2 server used to determine potential spots for point mutation, and Molegro Virtual Docker (MVD) used for performing point mutation on 3D model. Hydrophobicity plots generated by Bioedit version 7.0.8.0 in Kyte-Doolittle scale indicated that protein hydrophobicity is increased after mutation. Also protein stability increased 26.11 units in scale of DDC2.

Roles of sulfite oxidoreductase and sulfite reductase in improving desulfurization by Rhodococcus erythropolis

Rhodococcus erythropolis has been widely studied for desulfurization. However, activity levels required for commercial application have not been achieved. A major limitation of the current work in biodesulfurization is inadequate information regarding sulfur metabolism generally, and in particular the metabolism of the sulfur obtained from dibenzothiophene (DBT) metabolism via the 4S pathway. In this work, we have investigated the possible routes taken by the sulfur from DBT to convert into biomass or other metabolites. We propose two alternate hypotheses. In the first, we hypothesize that the cell can convert via sulfite reductase (SR) the sulfite from the metabolism of DBT into sulfide that can be assimilated into biomass. However, in the process, it may convert any excess sulfite into extracellular sulfate via sulfite oxidoreductase (SOR) to avoid the toxic effects of sulfite. In the second, we speculate that the cell cannot assimilate the sulfite directly into biomass via SR. It must first use SOR to produce extracellular sulfate, and then recapture that sulfate into biomass via SR. Thus, either way, we propose that SOR and SR activities, in addition to dsz genes and cofactors, may be critical in increasing desulfurization levels significantly. In particular, we suggest that the simultaneous increase in SOR activity and decrease in SR activity can enable increased desulfurization activity.

Metabolic engineering of hydrophobic Rhodococcus opacus for biodesulfurization in oil–water biphasic reaction mixtures

An organic solvent-tolerant bacterium, Rhodococcus opacus B-4, was metabolically engineered to remove sulfur from dibenzothiophene (DBT), a component of crude oil. The resulting recombinant strain ROD2-8 constitutively expressed the Rhodococcus erythropolis IGTS8 genes dszA, dszB, and dszC, encoding dibenzothiophene sulfone monooxygenase, 2-(2'-hydroxyphenyl) benzenesulfinate desulfinase, and dibenzothiophene monooxygenase, respectively, of the 4S pathway to avoid transcriptional inhibition by the sulfate end-product. Unlike the wild-type strain, ROD2-8 grew in mineral salts medium containing DBT as the sole sulfur source. Under aqueous conditions, ROD2-8 resting cells converted greater than 85% of DBT to 2-hydroxybiphenyl (2-HBP), although the consumption rate by ROD2-8 cells precultured on DBT as the sole sulfur source was 3.3-fold higher than that of cells cultured in complex medium. Notably, DBT consumption rates increased by 80% in oil-water biphasic reaction mixtures with n-hexadecane as the organic solvent, and resting cells were predominantly localized in the emulsion layer. Desulfurization activity in biphasic reaction mixtures increased with increasing concentrations of DBT and was not markedly inhibited by 2-HBP accumulation. Intracellular concentrations of DBT and 2-HBP were significantly lower under biphasic conditions than aqueous conditions. Our findings suggest that the enhanced desulfurization activity under biphasic conditions results from the combined effects of attenuated feedback inhibition and reduced mass transfer limitations due to 2-HBP diffusion from cells and accumulation of both substrate and biocatalyst in the emulsion layer, respectively. Therefore, the solvent-tolerant and hydrophobic bacterium R. opacus B-4 appears suitable for biodesulfurization reactions in solvents containing a minimum ratio of water.

The Effect of Elimination of the dszC Gene in Energy Consuming of Biodesulfurization in Broken 4S Pathway

In this study, the dszC gene was eliminated in the 4S pathway to evaluate the energy consuming in the biodesulfurization process. Therefore, a new recombinant plasmid was constructed harboring the dszAB gene of Rhodococcus erythropolis IGTS8 was transferred into E. coli DH5α. In addition, another construct harboring the complete dsz operon was constructed and to compare the energy consumption of the biodesulfurization process. The biodesulfurization activity in the complete 4s pathway was higher than that of the broken 4s pathway, approximately up to 1.4-fold. Therefore, the energy consuming in the complete 4s pathway was less than that of the broken 4s pathway. In this report, we showed that the existence of the third gene (dszC) is necessary for reduction of energy consuming of the desulfurization process, and a certain concentration of substrate (DBT sulfone, a product of dszC gene) causes increasing of the biodesulfurization activity and more than this has no affection.

Mixtures of Pseudomonas putida CECT 5279 cells of different ages: Optimization as biodesulfurization catalyst

Sulfur content in fossil fuels is known to be the most important anthropogenic cause of sulfur oxide emissions to the atmosphere. In order to avoid health, environmental and technical problems caused by this compound, legislation imposes restrictive limitations to fuel sulfur content. Biodesulfurization (BDS) can become a complementary technology to hydrodesulfurization (HDS) to face this situation. Pseudomonas putida CECT 5279 is a genetically modified microorganism which can act as a desulfurizing biocatalyst. This microorganism has the ability of performing the metabolic 4S pathway from Rhodococcus erythropolis IGTS8, in order to desulfurize DBT, as a model compound. Maximum in vivo activities of monooxygenase enzymes (DszA and DszC) are shown when late exponential growth phase is reached (23h), while desulfinase enzyme DszB presents a maximum activity during the early exponential growth phase (5h), as previously reported [1]. Also, it has been proved that the combined utilization of these two cell ages yields excellent results when used as biocatalyst for desulfurization [2]. The aim of this work is to optimize the ratio and total biomass concentration of both 5h and 23h growth time cells in a complex biocatalyst for desulfurization by performing resting cells biodesulfurization assays using dibenzothiophene (DBT) as sulfur model compound. The best combination of cells was determined aiming for the highest desulfurization in the shortest time of operation while investing the minimum concentration of biomass. A particular cell mixture, containing 66.7% of 23h growth time cells, was found to work as the most effective desulfurization biocatalyst.