Rhodococcus Erythropolis IGTS8 Research Articles

Bacterial Biological Factories Intended for the Desulfurization of Petroleum Products in Refineries

The removal of sulfur by deep hydrodesulfurization is expensive and environmentally unfriendly. Additionally, sulfur is not separated completely from heterocyclic poly-aromatic compounds. In nature, several microorganisms (Rhodococcus erythropolis IGTS8, Gordonia sp., Bacillus sp., Mycobacterium sp., Paenibacillus sp. A11-2 etc.) have been reported to remove sulfur from petroleum fractions. All these microbes remove sulfur from recalcitrant organosulfur compounds via the 4S pathway, showing potential for some organosulfur compounds only. Activity up to 100 µM/g dry cell weights is needed to meet the current demand for desulfurization. The present review describes the desulfurization capability of various microorganisms acting on several kinds of sulfur sources. Genetic engineering approaches on Gordonia sp. and other species have revealed a variety of good substrate ranges of desulfurization, both for aliphatic and aromatic organosulfur compounds. Whole genome sequence analysis and 4S pathway inhibition by a pTeR group inhibitor have also been discussed. Now, emphasis is being placed on how to commercialize the microbes for industrial-level applications by incorporating biodesulfurization into hydrodesulfurization systems. Thus, this review summarizes the potentialities of microbes for desulfurization of petroleum. The information included in this review could be useful for researchers as well as the economical commercialization of bacteria in petroleum industries.

Biodesulfurization of Dibenzothiophene by Decorating Rhodococcus erythropolis IGTS8 Using Montmorillonite/Graphitic Carbon Nitride

Fossil fuels are the main sources of human energy, but their combustion releases toxic compounds of sulfur oxide. In the oil industry, using the optimal methods to eliminate sulfur compounds from fossil fuels is a very important issue. In this study, the performance of montmorillonite/graphitic carbon nitride (a new hybrid nanostructure) in increasing the biodesulfurization activity of Rhodococcus erythropolis IGTS8 was investigated. X-ray diffraction, Fourier-transform infrared spectroscopy, field emission scanning electron microscopy and transmission electron microscopy were used for the characterization of the nanoparticles. The effective factors in this process were determined. Optimum conditions for microorganisms were designed using the Design Expert software. Experiments were performed in a flask. The results indicated that the biodesulfurization activity of a microorganism in the presence of the nanostructure increases by 52%. In addition, in the presence of the nanostructure, the effective factors are: 1. concentration of the nanostructure; 2. concentration of sulfur; 3. cell concentration. In the absence of the nanostructure, the only effective factor is the concentration of sulfur. Through analysis of variance, the proposed models were presented to determine the concentration of the 2-hydroxy biphenyl produced by the microorganisms (biodesulfurization activity) in the presence and absence of the nanostructure. The proposed models were highly acceptable and consistent with experimental data. The results of a Gibbs assay showed that the biodesulfurization efficiency of in the presence of the nanostructure was increased by about 52%, which is a very satisfactory result. The biodesulfurization activity of decorated cells in a bioreactor showed a significant increase compared with nondecorated cells. Almost a two-fold improvement in biodesulfurization activity was obtained for decorated cells compared with free cells.

Biodesulfurization of Dibenzothiophene and Its Alkylated Derivatives in a Two-Phase Bubble Column Bioreactor by Resting Cells of Rhodococcus erythropolis IGTS8

Biodesulfurization (BDS) is considered a complementary technology to the traditional hydrodesulfurization treatment for the removal of recalcitrant sulfur compounds from petroleum products. BDS was investigated in a bubble column bioreactor using two-phase media. The effects of various process parameters, such as biocatalyst age and concentration, organic fraction percentage (OFP), and type of sulfur compound—namely, dibenzothiophene (DBT), 4-methyldibenzothiophene (4-MDBT), 4,6-dimethyldibenzothiophene (4,6-DMDBT), and 4,6-diethyldibenzothiophene (4,6-DEDBT)—were evaluated, using resting cells of Rhodococcus erythropolis IGTS8. Cells derived from the beginning of the exponential growth phase of the bacterium exhibited the highest biodesulfurization efficiency and rate. The biocatalyst performed better in an OFP of 50% v/v. The extent of DBT desulfurization was dependent on cell concentration, with the desulfurization rate reaching its maximum at intermediate cell concentrations. A new semi-empirical model for the biphasic BDS was developed, based on the overall Michaelis-Menten kinetics and taking into consideration the deactivation of the biocatalyst over time, as well as the underlying mass transfer phenomena. The model fitted experimental data on DBT consumption and 2-hydroxibyphenyl (2-HBP) accumulation in the organic phase for various initial DBT concentrations and different organosulfur compounds. For constant OFP and biocatalyst concentration, the most important parameter that affects BDS efficiency seems to be biocatalyst deactivation, while the phenomenon is controlled by the affinities of biodesulfurizing enzymes for the different organosulfur compounds. Thus, desulfurization efficiency decreased with increasing initial DBT concentration, and in inverse proportion to increases in the carbon number of alkyl substituent groups.

Investigating the effect of starch/Fe3O4 nanoparticles on biodesulfurization using molecular dynamic simulation.

The application of dibenzothiophene (DBT) as a source of energy leads to air pollution. The key solution to overcome this drawback is desulfurization. Magnetic nanoparticles have shown an excellent performance in the desulfurization of dibenzothiophene. In this study, molecular dynamic (MD) simulation was considered for the first time to gain insight about the molecule interactions in the biodesulfurization (BDS) process of DBT using Rhodococcus erythropolis IGTS8, in the presence and absence of starch/magnetic nanoparticles. According to the MD simulation results, the density of the system in the presence of starch/Fe3O4 was ascending while in the absence of these nanoparticles, the density was descending. Starch/magnetic nanoparticles caused more rapid equilibrium state in the biodesulfurization process. The energy diagram showed that magnetic nanoparticles decrease the energy fluctuation and increase the difference of non-bounding energy and potential energy (8 times) compared to (BDS) without nanoparticle, which reflects higher bounded energy in the system using starch/magnetic nanoparticles. The height of RDF peak in the presence of starch/Fe3O4 was 4 times more than the RDF peak in the absence of nanoparticle. In addition, the nanoparticles decreased the fluctuations around optimal temperature in BDS up to 5% compared to other state.

Inhibition Mechanisms of Rhodococcus Erythropolis 2'-Hydroxybiphenyl-2-sulfinate Desulfinase (DszB).

Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2'-Hydroxybiphenyl-2-sulfinate desulfinase (DszB), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced and purified via Escherichia coli BL21 to test these hypotheses. Activity and the resulting conformational change of DszB in the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and was inhibited via competitive binding of the product, HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of DszB bound to common, yet structurally diverse, crude oil contaminants 2',2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root-mean-square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that the combination of a molecule's hydrophobicity and nonspecific interactions with nearby functional groups contributes to a competitive inhibition mechanism that locks DszB in a closed conformation and precludes substrate access to the active site.

Simulation-based protein engineering of R. erythropolis FMN oxidoreductase (DszD)

The sulfur contents of fossil fuels have negative impacts on the environment and human health. The bio-catalytic desulfurization strategies and the biological refinement of fossil fuels are a cost-effective process compared to classical chemistry desulfurization. Rhodococcus erythropolis IGTS8 is able to metabolize the organic sulfur compound by the unique genes cluster (i.e. DszA, B, C and D genes) in the 4S metabolic pathway. The dszD gene codes a key enzyme for sulfur reduction in the gene cluster. In this study, the structure of the DszD enzyme was predicted and then the key residues toward FMN binding were identified which were Thr62, Ser63, Asn77, and Ala79. To investigate the effect of manipulation in key residues on the enzymatic activity of the DszD, different mutations were performed on key residues. The molecular docking simulation showed that A79I and A79N mutants have the lowest binding free energies compared to the wild-type enzyme in binding with FMN substrate. A 50 ns molecular dynamics (MD) simulation performed using GROMACS software. The RMSD and RMSF analysis showed that two mutants are more stable than the wild-type enzyme during MD simulation. The binding free energies between FMN substrate and complexes were calculated and analyzed by the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) method. The experimental results showed that the enzyme activity for the oxidoreductase process toward biodesulfurization increased 1.9 and 2.3 fold for A79I and A79N mutants, respectively.

Investigation of Desulfurization Activity, Reusability, and Viability of Magnetite Coated Bacterial Cells.

BackgroundMagnetic separation using magnetic nanoparticles can be used as a simple method to isolate desulfurizing bacteria from a biphasic oil/water system.ObjectivesMagnetite nanoparticles were applied to coat the surface of Rhodococcus erythropolis IGTS8 and Rhodococcus erythropolis FMF desulfurizing bacterial cells, and the viability and reusability of magnetite-coated bacteria evaluated by using various methods.Material and MethodsMagnetite nanoparticles were synthesized through a reverse co-precipitation method. Glycine was added during and after the synthesis of magnetite nanoparticles to modify their surface and to stabilize the dispersion of the nanoparticles. The glycine-modified magnetite nanoparticles were immobilized on the surface of both oil-desulfurizing bacterial strains. Reusability of magnetite-coated bacterial cells was evaluated via assessing the desulfurization activity of bacteria via spectrophotometry using Gibb’s assay, after the separation of bacterial cells from 96h-cultures with the application of external magnetic field. In addition, CFU and fluorescence imaging were used to investigate the viability of magnetite-coated and free bacterial cells.ResultsTEM micrographs showed that magnetite nanoparticles have the size approximately 5.35±1.13 nm. Reusability results showed that both magnetite-coated bacterial strains maintain their activity even after 5 × 96h-cycles. The viability results revealed glycine-modified magnetite nanoparticles did not negatively affect the viability of two bacterial strains R. erythropolis IGTS8 and R. erythropolis FMF.ConclusionsIn conclusion, the glycine-modified magnetite nanoparticles have great capacity for immobilization and separation of desulfurizing bacteria from suspension.

Isolation and identification of a TetR family protein that regulates the biodesulfurization operon

Biodesulfurization helps in removal of sulfur from organosulfur present in petroleum fractions. All microorganisms isolated to date harbor a desulfurization operon consisting of three genes dszA, -B and -C which encode for monooxygenases (DszA & C) and desulfinase (DszB). Most of the studies have been carried out using dibenzothiophene as the model organosulfur compound, which is converted into 2 hydroxybiphenyl by a 4S pathway which maintains the calorific value of fuel. There are few studies reported on the regulation of this operon. However, there are no reports on the proteins which can enhance the activity of the operon. In the present study, we used in vitro and in vivo methods to identify a novel TetR family transcriptional regulator from Gordonia sp. IITR100 which functions as an activator of the dsz operon. Activation by TetR family regulator resulted in enhanced levels of desulfurization enzymes in Gordonia sp. IITR100. Activation was observed only when the 385 bp full length promoter was used. Upstream sequences between − 385 and − 315 were found to be responsible for activation. We provide evidence that the TetR family transcription regulator serves as an activator in other biodesulfurizing microorganisms such as Rhodococcus erythropolis IGTS8 and heterologous host Escherichia coli. This is the first report on the isolation of a possible transcriptional regulator that activates the desulfurization operon resulting in improved biodesulfurization.

Evaluation of desulfurization activity of SPION nanoparticle-coated bacteria in the presence of magnetic field

Sulfur content in diesel fuel is an environmental concern because sulfur is converted to sulfur oxides (Sox) during combustion, which not only contributes to acid rain, but poisons the catalytic converter for exhaust emission treatment. Hydrodesulfurization (HDS) method, a conventional procedure used for desulfurization of petroleum, is not efficient for desulfurization of aromatic components such as dibenzothiophene (DBT). However, biological processes require relatively mild conditions (low pressures and low temperatures) and also in BDS bacteria are able to uptake the sulfur without breaking the structure of the compounds which results in maintaining the value of fuel. In this study, functionalized magnetic iron oxide nanoparticles were synthesized and coated on Rhodococcus erythropolis IGTS8 cells to evaluate the biodesulfurization activity. Fe3O4 nanoparticles and silica-coated magnetite nanoparticles were synthesized. The morphology and properties of the synthesized nanoparticles were examined by SEM, FT-IR, XRD, DLS and zeta potential measurements. In the next stage, the bacterial cells were coated with nanoparticles and the desulfurizing activity was tested with Gibbs’s assay, HPLC and external magnet field. The results from Gibb’s assay showed that bacteria coated with functionalized nanoparticles have more desulfurization ability compared to that of uncoated bacterial cells. An external magnetic field showed rapid separation of coated bacterial cells from the reaction mixture. Thus, the necessity for a more efficient process is required to enhance the use of bionanotechnology and design a recombinant strain with a stable activity to improve the efficiency of biodesulfurization.

Comparing E. coli Expression and Enzyme Kinetics of Wildtype and Codon Optimized 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) from Nocardia asteroides A3H1 and Rhodococcus erythropolis IGTS8

Dibenzothiophene (DBT) and its derivatives comprise up to 60% of the organosulfur contamination of crude oil. The enzyme 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) catalyzes the carbon‐sulfur bond cleavage in the final, and rate‐limiting step in the biodesulfurization of DBT. The wildtype dszb genes from Nocardia asteroides A3H1 (A3H1) and Rhodococcus erythropolis IGTS8 (IGTS8) were expressed with and without chaperone proteins under different induction conditions. Yield of active enzyme was optimal when co‐expressed in E. coli in the presence of GroEL and GroES. Codon optimized dszb genes were synthesized, optimizing E. coli codon usage and minimizing GC content. The codon optimized dszb genes from A3H1 and IGTS8 were expressed with and without chaperone proteins under different induction conditions. GroEL and GroES were both still required for optimal enzyme activity. Protein aggregation was observed using whole cell FT‐IR spectroscopy in all cases, but was significantly larger in induced cells in the absence of chaperones.Support or Funding Information NSF CHE 1461175 James Madison University Department of Chemistry and Biochemistry This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhancement of Microbial Biodesulfurization via Genetic Engineering and Adaptive Evolution.

In previous work from our laboratories a synthetic gene encoding a peptide (“Sulpeptide 1” or “S1”) with a high proportion of methionine and cysteine residues had been designed to act as a sulfur sink and was inserted into the dsz (desulfurization) operon of Rhodococcus erythropolis IGTS8. In the work described here this construct (dszAS1BC) and the intact dsz operon (dszABC) cloned into vector pRESX under control of the (Rhodococcus) kstD promoter were transformed into the desulfurization-negative strain CW25 of Rhodococcus qingshengii. The resulting strains (CW25[pRESX-dszABC] and CW25[pRESX-dszAS1BC]) were subjected to adaptive selection by repeated passages at log phase (up to 100 times) in minimal medium with dibenzothiophene (DBT) as sole sulfur source. For both strains DBT metabolism peaked early in the selection process and then decreased, eventually averaging four times that of the initial transformed cells; the maximum specific activity achieved by CW25[pRESX-dszAS1BC] exceeded that of CW25[pRESX-dszABC]. Growth rates increased by 7-fold (CW25[pRESX-dszABC]) and 13-fold (CW25[pRESX-dszAS1BC]) and these increases were stable. The adaptations of CW25[pRESX-dszAS1BC] were correlated with a 3-5X increase in plasmid copy numbers from those of the initial transformed cells; whole genome sequencing indicated that during its selection processes no mutations occurred to any of the dsz, S1, or other genes and promoters involved in sulfur metabolism, stress response, or DNA methylation, and that the effect of the sulfur sink produced by S1 is likely very small compared to the cells’ overall cysteine and methionine requirements. Nevertheless, a combination of genetic engineering using sulfur sinks and increasing Dsz capability with adaptive selection may be a viable strategy to increase biodesulfurization ability.

Biodesulfurization of Dibenzothiophene by Two Bacterial Strains in Cooperation with Fe3O4, ZnO and CuO Nanoparticles

Fossil fuels contain a considerable amount of sulphur which after burning will have negative effects such as generating acid rain in the environment. Biodesulfurization is assumed to be a promising process for desulfurization due to no need for extreme conditions and furthermore breaking the C-S bond by microorganisms without destruction of the heterocyclic structure, through a pathway called 4S. In this study dibenzothiophene (DBT), as a model target compound was applied for sulfur removal by a cooperative system of two bacterial strains; Rhodococcus erythropolis IGTS8 and Pseudomonas aeruginosa PTSOX4, and Fe3O4, ZnO and CuO nanoparticles. Results of spectrophotometry and further HPLC analysis demonstrated that addition of the ZnO nanoparticles into the microbial culture, significantly resulted in the increment of desulfurization rate and conversion of DBT to 2-hydroxybiphenyl. The maximum value of almost 1.4 fold improvement in biodesulfurization activity was obtained for P. aeroginusa PTSOX4 in the presence of ZnO nanoparticles.

DBT desulfurization by decorating Rhodococcus erythropolis IGTS8 using magnetic Fe3O4 nanoparticles in a bioreactor.

Today, crude oil is an important source of energy and environmental contamination due to the continued use of petroleum products is a matter or urgent concern. In this work, two technological platforms, namely, the use of a robust desulfurizing bacteria and the use of nanotechnology to decorate the surface of the bacteria with nanoparticles (NP), were combined to enhance biodesulfurization (BDS). BDS is an ecologically friendly method for desulfurizing petroleum products while avoiding damage to the hydrocarbons due to the high temperaturesnormally associated with physical desulfurization methods.First, a bacterium known to be a good organism for desulfurization (Rhodococcus erythropolis IGTS8) was employed in cell culture to remove a recalcitrant sulfur molecule from a common sulfur-containing compound found in crude petroleum products (dibenzothiophene). 2-Hydroxybiphenyl (2-HBP) produced as a consequence of the BDS of dibenzothiophene was determined using Gibbs' assay. The synthesized NP were characterized by field emission scanning electron microscope, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, and vibrating sample magnetometer. The field emission scanning electron microscope and transmission electron microscopy images showed the size of the NP is 7-8 nm. The decorated cells had a long lag phase, but the growth continued until 148 h (at OD600 = 3.408) while the noncoated bacteria grow until 96 h before entering the stationary phase at OD600 = 2.547. Gibbs' assay results showed that production of 2-HBP by decorated cells was 0.210 mM at t = 148 h, while 2-HBP production by nondecorated cells was 0.182 mM at t = 96 h. Finally, the experiments were repeated in a fermenter.

Improving the Biodesulfurization of Crude Oil and Derivatives: A QM/MM Investigation of the Catalytic Mechanism of NADH-FMN Oxidoreductase (DszD).

The development of biocatalytic desulfurization strategies of petroleum and its derivatives could result in more economic alternatives than the widely used chemical desulfurization. The organism Rhodococcus erythropolis IGTS8 has been shown to metabolize organic sulfur compounds through a mechanism known as 4S pathway, which involves four enzymes (DszA, DszB, DszC, and DszD) and has been explored in biodesulfurization. Here we have applied QM/MM methods to study the catalytic mechanism of the enzyme DszD, a NADH-FMN oxidoreductase that occupies a central place on the 4S pathway by catalyzing the formation of the FMNH2 that is used by the two monooxynases in the cycle: DszA and DszC. In addition, to clarify the catalytic mechanism of this enzyme, this study analyzed in detail the role played by the active site Thr residue and of Asn and Ala enzyme mutants. The results help to explain previous experimental evidence and suggest new strategies for improving biodesulfurization through an increase in the activity of DszD.

Fatty acid methyl ester production from industrial waste by Rhodococcus erythropolis IGTS8 and Rhodococcus gordoniae R3

Fatty acid methyl ester production from industrial waste by Rhodococcus erythropolis IGTS8 and Rhodococcus gordoniae R3

Designing a New Recombinant Indigenous Klebsiella oxytoca ISA4 by Cloning of dsz Genes

A bacterium designated as a Klebsiella oxytoca ISA4 was isolated from oil-contaminated soil; it not only is unable to catabolize dibenzothiophene as a sole source of carbon and energy but also produces biosurfactant. In order to enhance the efficiency of biodesulfurization, the dszABC genes from Rhodococcus erythropolis IGTS8 were amplified by specific primers. PCR ampilicon of 3.8 kb was obtained and cloned using pVLT31 vector in native K. oxytoca ISA4. Further analysis by Gibbs assay and gas chromatograph revealed that recombinant K. oxytoca ISA4 has the highest desulfurization ability (48%) in comparison with R. erythropolis IGTS8 (42%) and Pseudomonas aeruginosa pTSOX4 (46%).

Effect of fluiddynamic conditions on growth rate and biodesulfurization capacity of Rhodococcus erythropolis IGTS8

The growth rate and desulfurization capacity accumulated by the cells during the growth of Rhodococcus erythropolis IGTS8 under different fluid dynamic conditions in a stirred and sparged tank bioreactor have been studied. Hydrodynamic conditions were changed using different stirrer speeds and gas flow rates. It was observed that the growth was strongly dependent on the stirrer speed employed. Oxygen transfer limitation was observed at low stirrer speeds (from 100 to 250rpm). In contrast, at higher stirrer speeds, cell damage was caused by hydrodynamic stress in the turbulent bulk of the broth, yielding again a decrease in growth for stirrer speeds higher than 450rpm. Moreover, increasing the agitation from 100 to 450rpm has a positive influence on the development of the desulfurization capacity of the cells during growth, yet this capacity shows a dramatic decrease for higher stirrer speeds. Nevertheless, the change of the air flow rate hardly has any influence on the growth rate and no hydrodynamic stress effect has been detected between 1 and 10Lmin−1. A regime analysis of the characteristic times for oxygen mass transfer, oxygen uptake and mixing under different agitation conditions has been made. It was found that the minimum stirrer speed necessary for a satisfactory performance of the bioreactor from the point of view of the cells oxygen demand was 350rpm, while at stirrer speeds over 450rpm, growth rate and desulfurization capacity are both negatively affected.

Specific oxygen uptake rate as indicator of cell response of Rhodococcus erythropolis cultures to shear effects

Specific oxygen uptake rate, qO2, on Rhodococcus erythropolis IGTS8 cultures has been measured under different fluid dynamic conditions in a STBR, changing stirrer speed, between 100 and 700rpm, and air flow rate, between 1 and 10Lmin−1. Experimental results indicate that qO2 values for R. erythropolis cultures can be influenced by the fluid dynamics inside the bioreactor. Under non oxygen-limited conditions, the oxygen consumption rate by the cells does not depend on the oxygen transfer rate; hence, the specific oxygen uptake rate is constant. However, when the stirrer speed is increased over a limit value, around 450rpm in the culture studied, a change is clearly detected and, as higher stirrer speeds are used, specific oxygen uptake rate suffers a significant decrease. Nevertheless, when the hydrodynamics are varied changing the air flow rate, between 1 and 10Lmin−1, this variable does not affect the oxygen uptake rate. According to these results, specific oxygen uptake rate measurement provides a good indication of whole stress generated by the hydrodynamic conditions into the bioreactor. A simple method to determine changes into the cell response due to the fluid dynamic environment in the bioreactor is herein proposed. According to this method, shear effects on the culture can be detected by the change on specific oxygen uptake rate measured at laboratory scale, employing the usual gas in-gas out dynamic method. This assay combines oxygen transfer, oxygen uptake rate and possible cell damage in a one simple method that can considerably simplify and accelerate fundamental research on the development and scale-up of bioprocesses.

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.

Enhancement of desulfurization activity by enzymes of the Rhodococcus dsz operon through coexpression of a high sulfur peptide and directed evolution

Removal of sulfur from petroleum improves the performance of refineries and reduces air pollution from fuel combustion. Biodesulfurization can selectively remove sulfur from petroleum, but improved biocatalysts are needed. This study illustrates a new approach to obtain improved desulfurization biocatalysts. A synthetic gene (Sulpeptide, S1) encoding high proportions of the sulfur containing amino acids methionine and cysteine was designed, constructed and cloned, as part of the Rhodococcus dszABC (desulfurization) operon, in vector pRESX, under control of the Rhodococcus kstD promoter. pRESXdszABC and pRESXdszAS1BC were electroporated into desulfurization-negative Rhodococcus opacus and the strains were transferred through 40 passages in medium with dibenzothiophene (DBT) as the sole sulfur source. Modest increases in desulfurization activity were achieved after 10 passages selecting for rapid growth with DBT. After selection for 40 passages, both the dszABC and dszAS1BC expressing R. opacus strains showed a greater than 20-fold increase in specific desulfurization ability, and exceeded the specific activity of the control, desulfurization positive strain Rhodococcus erythropolis IGTS8. This study serves as a proof of concept that engineering with Sulpeptide genes, combined with selective pressure for rapid growth with DBT, can drive evolution of improved desulfurization biocatalysts.