DBT degradation enhancement by decorating Rhodococcus erythropolis IGST8 with magnetic Fe3O4 nanoparticles

Biodesulfurization (BDS) of dibenzothiophene (DBT) was carried out by Rhodococcus erythropolis IGST8 decorated with magnetic Fe₃O₄ nanoparticles, synthesized in‐house by a chemical method, with an average size of 45–50 nm, in order to facilitate the post‐reaction separation of the bacteria from the reaction mixture. Scanning electron microscopy (SEM) showed that the magnetic nanoparticles substantially coated the surfaces of the bacteria. It was found that the decorated cells had a 56% higher DBT desulfurization activity in basic salt medium (BSM) compared to the nondecorated cells. We propose that this is due to permeabilization of the bacterial membrane, facilitating the entry and exit of reactant and product, respectively. Model experiments with black lipid membranes (BLM) demonstrated that the nanoparticles indeed enhance membrane permeability. Biotechnol. Bioeng. 2009;102: 1505–1512. © 2008 Wiley Periodicals, Inc.     

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.     

Cultivation of a desulfurizing bacterium, Mycobacterium strain G3

Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO₄, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO₄ or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO₄. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.     

Biodesulfurization of dibenzothiophene by <Emphasis Type="Italic">Gordonia</Emphasis> sp. AHV-01 and optimization by using of response surface design procedure

A novel desulfurizing bacterium has been isolated from oil-contaminated soils in Khuzestan. The ability for dibenzothiophene desulfurization and its biochemical pathway were investigated. The bacterium was identified as Gordonia sp. AHV-01 (Genbank Accession no. HQ607780) by 16S rRNA gene sequencing. HPLC results and Gibb’s assay were shown that dibenzothiophene desulfurized via 4S-pathway. Maximum growth (0.426 g dry cells/L) and produced 2-hydroxybiphenyl (63.1 μM) were observed at 120 h of cultivation. By using of response surface design procedure the optimization of pH, temperature and rotary shaker round on the desulfurization reaction of isolate AHV-01 were performed. The optimum conditions were determined at pH of 7.0, temperature of 30°C and rotary shaker round of 180 rpm. At these conditions, the dibenzothiophene desulfurization activity was increased and maximum 2-hydroxybiphenyl production was detected 70.29 μM at 96 h. According to these results, isolate AHV-01 was capable to desulfurize dibenzothiophene via 4S-pathway and likely it can be useful to reduce organic sulfur contents of crude oil.     

Biodesulfurization of dibenzothiophene by a newly isolated Rhodococcus erythropolis strain

A new dibenzothiophene (DBT) desulfurizing bacterium was isolated from oil-contaminated soils in Iran. HPLC analysis and PCR-based detection of the presence of the DBT desulfurization genes (dszA, dszB and dszC) indicate that this strain converts DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The strain, identified as Rhodococcus erythropolis SHT87, can utilize DBT, dibenzothiophene sulfone, thiophene, 2-methylthiophene and dimethylsulfoxide as a sole sulfur source for growth at 30 °C.The maximum specific desulfurization activity of strain SHT87 resting cells in aqueous and biphasic organic–aqueous systems at 30 °C was determined to be 0.36 and 0.47 μmol 2-HBP min⁻¹ (g dry cell)⁻¹, respectively. Three mM DBT was completely metabolized by SHT87 resting cells in the aqueous and biphasic systems within 10 h. The rate and the extent of the desulfurization reaction by strain SHT87 suggest that this strain can be used for the biodesulfurization of diesel oils.     

Efficient production of desulfurizing cells with the aid of expert system

An expert system was used to achieve the high production of desulfurizing cells of Rhodococcus erythropolis KA 2-5-1. By adding a proper amount of sulfur containing component with the aid of the expert system, we could avoid excess feeding which resulted in the lowering of desulfurizing activity and starvation which caused serious damage to cell growth. In order to determine the addition amount by the expert system, the data of the amount of chemical elements contained in the cells were used as a reference for comparison with the medium components present. Culture experiments were carried out using a 5l jar fermentor with several kinds of media whose components were determined based on the inferred results with the aid of the expert system. We could restrict the amount of the sulfur component addition so that sulfur was a growth-limiting factor; in contrast, the amounts of other elements were sufficient for growth.As a result, the maximum specific production rate of 2-hydroxy biphenyl (2HBP) and the maximum cell concentration were 20mmolkg-drycells(-1)h(-1) and of 45g-drycellsl(-1), respectively. At 100h of cultivation, 1g/l of dibenzothiophene (DBT) was converted to 2HBP within 20h, i.e., 10mmolkg-drycells(-1)h(-1) of specific desulfurization activity, and the specific activity remained stable for a long period in the culture experiment.     

Ralstonia eutropha as a biocatalyst for desulfurization of dibenzothiophene

The potential of Ralstonia eutropha as a biocatalyst for desulfurization of dibenzothiophene (DBT) was studied in growing and resting cell conditions. The results of both conditions showed that sulfur was removed from DBT which accompanied by the formation of 2-hydroxybiphenyl (2-HBP). In growing cell experiments, glucose was used as an energy supplying substrate in initial concentrations of 55 mM (energy-limited) and 111 mM (energy-sufficient). The growing cell behaviors were quantitatively described using the logistic equation and maintenance concept. The results indicated that 2-HBP production was higher for the energy-sufficient cultures, while the values of the specific growth rate and the maintenance coefficient for these media were lower than those of the energy-limited cultures. Additionally, the kinetic studies showed that the half-saturation constant for the energy-limited cultures was 2 times higher than the energy-sufficient ones where the inhibition constant (0.08 mM) and the maximum specific DBT desulfurization rate (0.002 mmol g_cell ⁻¹ h⁻¹) were almost constant. By defining desulfurizing capacity (D _DBT) including both the biomass concentration and time to reach a particular percentage of DBT conversion, the best condition for desulfurizing cell was determined at 23% g_cell L⁻¹ h⁻¹ which corresponded with the resting cells that were harvested at the mid-exponential growth phase.

     

De-repression and comparison of oil–water separation activity of the dibenzothiophene desulfurizing bacterium, <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. G3

Expression of the desulfurization genes (dsz) in Mycobacterium sp. G3 is repressed by sulfate, which is the product of biodesulfurization. An expression clone, pSMTABC, was constructed by placing the dsz genes downstream of the hsp60 promoter and the constructed plasmid was electroporated into G3. The recombinant strain G3-1 desulfurized dibenzothiophene in the presence of 0.5 mM sulfate while the Dsz phenotype was completely repressed in the wild-type strain. However, there was no significant increase in the amount of desulfurization enzymes in G3-1. In addition, G3 had superior separation of diesel oil–water separation activity compared to E. coli, which is superior to desulfurizing rhodococci.     

Continuous desulfurization of dibenzothiophene with Rhodococcusrhodochrous IGTS8 (ATCC 53968)

Desulfurization of a model fuel system consisting of hexadecane and dibenzothiophene (DBT) by Rhodococcus rhodochrous IGTS8 was demonstrated in a 2-L continuous stirred tank reactor (CSTR). The reactor was operated in a semicontinuous and continuous mode with and without recycling of the model fuel. A constant volumetric desulfurization activity A(t), (in mg HBP L(-1) h(-1)) was maintained in the reactor with a feeding strategy of fresh cell suspension based on a first-order decay of the biocatalyst. Maximum desulfurization rates, as measured by specific desulfurization activity, of 1.9 mg HBP/g DCW h were attained. Rates of biocatalyst decay were on the order of 0.072 h(-1). Theoretical predictions of a respiratory quotient (RQ) associated with this biotransformation reaction agree well with experimental data from off-gas analysis. In addition, the ratio of the specific desulfurization activity a(t), (in mg HBP/g DCW h) of recycled and fresh biocatalyst was determined and evaluated.     

Demonstration of the carbon-sulfur bond targeted desulfurization of benzothiophene by thermophilic Paenibacillus sp. strain A11-2 capable of desulfurizing dibenzothiophene

Paenibacillus sp. strain A11-2, which had been primarily isolated as a bacterial strain capable of desulfurizing dibenzothiophene to produce 2-hydroxybiphenyl at high temperatures, was found to desulfurize benzothiophene more efficiently than dibenzothiophene. The desulfurized product was identified as o-hydroxystyrene by GC-MS and 1H-NMR analysis. Benzothiophene was assumed to be degraded in a way analogous to the 4S pathway, which has been well-known as a mode of dibenzothiophene degradation. These results suggest that benzothiophene desulfurization may share at least partially the reaction mechanism with dibenzothiophene desulfurization.     

Biodesulfurization of dibenzothiophene by Gordonia sp. AHV-01 and optimization by using of response surface design procedure

A novel desulfurizing bacterium has been isolated from oil-contaminated soils in Khuzestan. The ability for dibenzothiophene desulfurization and its biochemical pathway were investigated. The bacterium was identified as Gordonia sp. AHV-01 (Genbank Accession No HQ607780) by 16S rRNA gene sequencing. HPLC results and Gibb's assay were shown that dibenzothiophene desulfurized via 4S-pathway Maximum growth (0.426 g dry cells/L) and produced 2-hydroxybiphenyl (63.1 microM) were observed at 120 h of cultivation. By using of response surface design procedure the optimization of pH, temperature and rotary shaker round on the desulfurization reaction of isolate AHV-01 were performed. The optimum conditions were determined at pH of 7.0, temperature of 30 degrees C and rotary shaker round of 180 rpm. At these conditions, the dibenzothiophene desulfurization activity was increased and maximum 2-hydroxybiphenyl production was detected 70.29 microM at 96 h. According to these results, Isolate AHV-01 was capable to desulfurize dibenzothiophene via 4S-pathway and likely it can be useful to reduce organic sulfur contents of crude oil.     

Analysis of biodesulfurization of model oil system by the bacterium,strain RIPI-22

The execution of 4S process (sulfur-specific pathway) by growing cells of the newly isolated strain RIPI-22 and its resting cells in both aqueous reaction system and in two-phase system were investigated. The time for maximum desulfurization activity of the strain in resting state and in model oil system (hexadecane containing dibenzothiophene) was 7 h. Using taguchi design procedure the effects of cell density, pH and phase ratio on the desulfurization reaction were studied. The extent of desulfurization was hardly dependent on the pH where as the volume ratio of hydrocarbon-aqueous phase significantly affected the desulfurization activity.On the basis of the determined optimum desulfurizing conditions, the biodesulfurization pattern of DBT was studied and kinetic parameters were calculated.     

Microbial Conversion of Petro-sulfur Compounds

For the purpose of proving possibility of desulfurization from petroleum oil by microbial procedures, first approach was made to obtaining microorganisms capable of converting petro-sulfur compounds. Dibenzothiophene was used in this study as a model of the sulfur compounds in heavy oil.

Six strains of microorganisms were isolated with dibenzothiophene from various soils. These strains produced organic acid compounds containing sulfur from dibenzothiophene. As a result of the taxonomic studies, three of the strains were found to be new species belonging to the Genus Pseudomonas, so the authors proposed to assign the names, Pseudomonas abikonensis nov. sp. to one strain, and Pseudomonas jianii nov. sp. to the other two strains.     

Site-directed mutagenesis enhances the activity of NADH-FMN oxidoreductase (DszD) activity of Rhodococcus erythropolis

Microbial desulfurization is potentially an alternative process to chemical desulfurization of fossil fuels and their refined products. The dibenzothiophene desulfurizing system of Rhodococcus erythropolis includes DszD which is an NADH-dependent FMN oxidoreductase with 192 residues that is responsible for supplying reducing equivalents in the form of FMNH₂ to monooxygenases, DszA and DszC. We performed amino acid sequence comparisons and structural predictions based on the crystal structure of available pdb files for three flavin reductases PheA2, HpaC_Tt and HpaC_St with the closest structural homology to IGTS8 DszD. The Thr62 residue in DszD was substituted with Asn and Ala by site-directed single amino acid mutagenesis. Variants T62N and T62A showed 5 and 7 fold increase in activities based on the recombinant wild type DszD, respectively. This study revealed the critical role of position 62 in enzyme activity. These results represent the first experimental report on flavin reductase mutation in R. erythropolis and will pave the way for further optimization of the biodesulfurization process.     

Developments in destructive and non-destructive pathways for selective desulfurizations in oil-biorefining processes

Biocatalytic desulfurization is still not a commercial technology, but conceptual engineering and sensitivity analyses have shown that the approach is very promising. The purpose of this paper is to investigate further some aspects of the biodesulphurization pathways, discussing the non-destructive pathway with the well-known Rhodococcus rhodochrous IGTS8. Findings revealed byproducts, such as 2′-hydroxybiphenyl (HBP), sulfite and sulfate, obtained by the desulfurization of dibenzothiophene (DBT), to exert an inhibiting effect. The results suggest that IGTS8 may follow two different metabolic pathways in stationary-growth-phase cells or under growing conditions. The first pathway is characterized by oxidative steps, which convert DBT to DBT sulfoxide and to DBT sulfone. The sulfone is transformed to 2-(2′-hydroxyphenyl)benzene sulfinate and then to HBP and sulfite by a sulfinic acid hydrolase. In the second pathway the sulfone is further oxidized to 2-(2′-hydroxyphenyl)benzene sulfonate and then to HBP and sulfate by a sulfonic acid hydrolase. Experiments using benzene sulfonic acid suggest that the sulfonic acid hydrolase is an induced enzyme. Received: 8 June 1998 / Received revision: 1 October 1998 / Accepted: 2 October 1998     

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus–E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source. Received: 4 December 2001 / Accepted: 7 January 2002     

Biodesulfurisation of DBT with Pseudomonas putida CECT5279 by resting cells: Influence of cell growth time on reducing equivalent concentration and HpaC activity

Dibenzothiophene (DBT) biodesulfurisation (BDS) route using a genetically modified organism, Pseudomonas putida CECT 5279, is studied. Tests of BDS with whole cells and with homogenized cells are carried out by taking samples of the cells during growth. The influence of the growth phases in the evolution of the intermediates of the 4S DBT desulfurising route is shown.Conversions of the five key compounds of the 4S route (DBT, DBTO, DBTO₂, HBPS and HBP) are measured. DBT conversion values are maximal with cells obtained after 30 h of growth time. HBP conversion values do not coincide with DBT conversion values, the maximum HBP production is obtained with cells grown for 10 h. A greater intermediate DBTO and DBTO₂ accumulation in broth is produced with cells obtained at 5 and 10 h of growth time. Nevertheless, the accumulation in broth of HBPS, another intermediate, is considerably lower than that observed with cells obtained at 23, 30 and 45 h of growth time.Also, the concentration of the reducing equivalents (NADH and FMNH₂) and flavin-oxido-reductase activity inside the cells is measured. This showed that the concentration of the reducing equivalents and the activity of the HpaC enzyme in the P. putida cytoplasm do not limit BDS rate.The influence of 4S compound transport across cellular membrane is studied by comparison of results obtained by resting cell assays (whole cells) and with homogenized cells assays (disrupted cells). The results show that there is no accumulation of any compound inside the cells, and that the transport rate across the cellular membrane does not limit the overall biodesulfurisation rate.     

Identification and characterization of a novel heparin‐binding peptide for promoting osteoblast adhesion and proliferation by screening an Escherichia coli cell surface display peptide library

Heparin/heparan sulfate (HS) plays a key role in cellular adhesion. In this study, we utilized a 12‐mer random Escherichia coli cell surface display library to identify the sequence, which binds to heparin. Isolated insert analysis revealed a novel heparin‐binding peptide sequence, VRRSKHGARKDR, designated as HBP12. Our analysis of the sequence alignment of heparin‐binding motifs known as the Cardin–Weintraub consensus (BBXB, where B is a basic residue) indicates that the HBP12 peptide sequence contains two consecutive heparin‐binding motifs (i.e. RRSK and RKDR). SPR‐based BIAcore technology demonstrated that the HBP12 peptide binds to heparin with high affinity (K_D = 191 nM ). The HBP12 peptide is found to bind the cell surface HS expressed by osteoblastic MC3T3 cells and promote HS‐dependent cell adhesion. Moreover, the surface‐immobilized HBP12 peptide on titanium substrates shows significant increases in the osteoblastic MC3T3‐E1 cell adhesion and proliferation. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Multi-criteria comparison of resting cell activities of bacterial strains selected for biodesulfurization of petroleum compounds

Five isolates able to utilize dibenzothiophene (DBT) as a sole sulfur source and to convert it to 2-hydroxybiphenyl (HBP) with high rates were selected to investigate their potentialities as biocatalysts of a diesel oil biodesulfurization process. Conventional and chemotaxonomic analyses and 16S ribosomal DNA (rDNA) sequencing showed that these strains belonged to the Rhodococcus/Gordonia cluster. The desulfurizing activities of resting cells were compared under various conditions to evaluate their stability in both aqueous and organic media, their sensitivity to the presence of hexadecane and their sulfur substrate selectivity. In spite of their taxonomic similarity, the five strains exhibited different properties. This diversity was not confirmed by the analysis of the desulfurizing genes by amplification and sequencing of large fragments of dszA, dszB, dszC, and dszD genes which revealed that four of the five selected strains had a dsz genotype identical to those of the reference strain, Rhodococcus erythropolis IGTS8.     

Enhanced biodesulfurization by expression of dibenzothiophene uptake genes in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The transfer of dibenzothiophene (DBT) and its derivatives into cells is a critical step for biodesulfurization. The desulfurization reactions of resting cells and cell lysate were studied, which showed that the desulfurization rate of DBT, especially 4, 6-dimethyldibenzothiophene (4, 6-DMDBT) in Rhodococcus erythropolis LSSE8-1 was seriously affected by the transfer into cells. The inhibited effect of NaN₃ on desulfurization reactions was studied, which confirmed that the transfer of DBT into cells was an active transport in R. erythropolis LSSE8-1. The uptake-genes of DBT and its derivatives (HcuABC) of Pseudomonas delafieldii R-8 were introduced into the specific desulfurization bacterium, R. erythropolis LSSE8-1. Compared with the wild type, the strains bearing HcuABC genes showed a higher desulfurization activity. The desulfurization ratio of DBT showed a 19% increase, and 13% increase of 4, 6-DMDBT.