Deep desulfurization of diesel oil and crude oils by a newly isolated Rhodococcus erythropolis strain

The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as alpha-hydroxy-beta-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30 degrees C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30 degrees C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.     

Deep Desulfurization of Diesel Oil and Crude Oils by a Newly Isolated Rhodococcus erythropolis Strain

The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as α-hydroxy-β-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30°C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30°C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.     

Biodesulfurization in biphasic systems containing organic solvents

Biphasic systems can overcome the problem of low productivity in conventional media and have been exploited for biocatalysis. Solvent-tolerant microorganisms are useful in biotransformation with whole cells in biphasic reactions. A solvent-tolerant desulfurizing bacterium, Pseudomonas putida A4, was constructed by introducing the biodesulfurizing gene cluster dszABCD, which was from Rhodococcus erythropolis XP, into the solvent-tolerant strain P. putida Idaho. Biphasic reactions were performed to investigate the desulfurization of various sulfur-containing heterocyclic compounds in the presence of various organic solvents. P. putida A4 had the same substrate range as R. erythropolis XP and could degrade dibenzothiophene at a specific rate of 1.29 mM g (dry weight) of cells(-1) h(-1) for the first 2 h in the presence of 10% (vol/vol) p-xylene. P. putida A4 was also able to degrade dibenzothiophene in the presence of many other organic solvents at a concentration of 10% (vol/vol). This study is a significant step in the exploration of the biotechnological potential of novel biocatalysts for developing an efficient biodesulfurization process in biphasic reaction mixtures containing toxic organic solvents.     

Chemostat approach for the directed evolution of biodesulfurization gain-of-function mutants

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.     

Microbial desulfurization of alkylated dibenzothiophenes from a hydrodesulfurized middle distillate by Rhodococcus erythropolis I-19.

Rhodococcus erythropolis I-19, containing multiple copies of key dsz genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 micromol g dry cell weight(-1) min(-1), more than 25-fold higher than that for wild-type bacteria. According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes. Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S. XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones. I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs. Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane. Apparent saturation rate constant (K(0)) and half-saturation rate constant (K(1)) values were calculated to be 2.8 micromol g dry cell weight(-1) min(-1) and 130 ppm, respectively. However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior. A competitive-substrate model suggested that the apparent K(1) values for each group of Cx-DBTs increased with increasing alkylation. Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure.     

Genome sequence of Rhodococcus erythropolis XP, a biodesulfurizing bacterium with industrial potential

Rhodococcus erythropolis strains have shown excellent characteristics in petroleum oil biodesulfurization. Here we present the first announcement of the draft genome sequence of an efficient biodesulfurizing bacterium named R. erythropolis XP (7,229,582 bp). The biodesulfurizing genes dszABC are located on a plasmid, while the flavin reductase gene dszD is located on the chromosome.     

Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5-99.8%.     

Sulfur-selective desulfurization of dibenzothiophene and diesel oil by newly isolated Rhodococcus sp. strains

New desulfurizing bacteria able to convert dibenzothiophene into 2-hydroxybiphenyl and sulfate were isolated from contaminated soils collected in Mexican refineries. Random amplified polymorphic DNA analysis showed they were different from previously reported Rhodococcus erythropolis desulfurizing strains. According to 16S rRNA gene sequencing and fatty acid analyses, these new isolates belonged to the genus Rhodococcus. These strains could desulfurize 4,6-dimethyldibenzothiophene which is one of the most difficult dibenzothiophene derivatives to remove by hydrodesulfurization. A deeply hydrodesulfurized diesel oil containing significant amounts of 4,6-dimethyldibenzothiophene was treated with Rhodococcus sp. IMP-S02 cells. Up to 60% of the total sulfur was removed and all the 4,6-dimethyldibenzothiophene disappeared as a result of this treatment.     

Sulfonate desulfurization in Rhodococcus from wheat rhizosphere communities

Organically bound sulfur makes up about 90% of the total sulfur in soils, with sulfonates often the dominant fraction. Actinobacteria affiliated to the genus Rhodococcus were able to desulfonate arylsulfonates in wheat rhizospheres from the Broadbalk long-term field wheat experiment, which includes plots treated with inorganic fertilizer with and without sulfate, with farmyard manure, and unfertilized plots. Direct isolation of desulfonating rhizobacteria yielded Rhodococcus strains which grew well with a range of sulfonates, and contained the asfAB genes, known to be involved in sulfonate desulfurization by bacteria. Expression of asfA in vitro increased >100-fold during growth of the Rhodococcus isolates with toluenesulfonate as sulfur source, compared with growth with sulfate. By contrast, the closely related Rhodococcus erythropolis and Rhodococcus opacus type strains had no desulfonating activity and did not contain asfA homologues. The overall actinobacterial community structure in wheat rhizospheres was influenced by the sulfur fertilization regime, as shown by specific denaturing gradient gel electrophoresis of PCR amplified 16S rRNA gene fragments, and asfAB clone library analysis identified nine different asfAB genotypes closely affiliated to the Rhodococcus isolates. However, asfAB -based multiplex restriction fragment length polymorphism (RFLP)/terminal-RFLP analysis of wheat rhizosphere communities revealed only slight differences between the fertilization regimes, suggesting that the desulfonating Rhodococcus community does not specifically respond to changes in sulfate supply.     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

Microtitre plate assay for biofilm formation, production and utilization of hydroxybiphenyl by Rhodococcus sp. isolated from gasoline-contaminated soil

Gasoline-contaminated soil from Isfahan, Iran was selected to isolate a bacterium capable of desulfurizing dibenzothiophene (DBT). The isolated strain was named R1 and identified as Rhodococcus erythropolis through biochemical tests as well as sequencing of 16S rRNA gene. This strain could efficiently produce 2-hydroxybiphenyl (HBP) from DBT via the 4S metabolic pathway. The highest HBP amount was produced at 2 mM DBT with addition of glucose (10 g l(-1)), ethanol (3 g l(-1)), glycerol (2 g l(-1)) or succinate (10 g l(-1)) as carbon sources at pH 7. Highest respiration and growth rates were observed by microplate titration on 0.1 mM HBP, and addition of 0.2 mM HBP to glucose (1 g l(-1)) and DBT (0.3 mM) could inhibite the respiration of the isolate. The isolated strain could grow up to 0.4 mM of HBP when it is used with mineral sulfur as sole sulfur source. To the best of our knowledge this is the first report on a microtiter assay for the production and utilization of HBP by Rhodococcus.     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC)catalysis,the first and also the key step in the microbial DBT desulfurization,is the conversion of DBT to DBT sulfone (DBTO2).In this study,dszC of a DBT-desulfiaizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank.The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E.coli BL21 strain.The expression amount of DszC was about 20% of total supernatant at low temperature.The soluble DszC in the supematant was purified by Ni2+ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity.Only one band was detected by Western-blotting,which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3,paC5) and Rhodococcus sp.DS-3.The activity of purified DszC was 0.36 U.DszC can utilize the organic compound such as DBT and methyl-DBT,hut not DBT derivates such as DBF,which has no sulfur or inorganic sulfur.     

Construction of random transposition mutagenesis system in Rhodococcuserythropolis using IS1415

Recent studies on the metabolic activities of genus Rhodococcus have shown rhodococci to be of important use in industrial, pharmaceutical and environmental biotechnology. The increasing economic significance of Rhodococcus encourages renewed efforts to characterize their genetic systems, as Rhodococcus genetics are still poorly understood. The goal of this study is to adapt a transposon system for use in creating random mutagenesis in Rhodococcus erythropolis. A plasmid carrying IS1415, a member of IS21 family identified from Rerythropolis, has been constructed and designated as pTNR. pTNR is a non-replicating transposon tool introduced into target cells by electroporation. During its transposition, the transposable-marker gene is separated from the open reading frames (istAB) of IS1415, which should avoid secondary transposition. Transposition of pTNR into wild-type R. erythropolis created mutagenesis with a high efficiency of 1.23x10(6)mutants per microgram plasmid DNA. However, it could also be transposed into other Rhodococcus spp. at lower frequencies in comparison with that of R. erythropolis. It has been indicated by Southern hybridization that the generated kanamycin-resistant mutants were resulted from single transposition event of pTNR. The results also revealed that the transposable-marker gene of pTNR was randomly inserted into the chromosomal DNA of R. erythropolis. The affected DNA regions carrying the transposed DNA element could be conveniently recovered for further characterization using a plasmid rescue procedure. Sequence data of the insertion sites of 40 random mutants analyzed indicated that transposition of pTNR generated 6-bp direct target duplications in 36 cases, while in the remaining four mutants; it generated 5- or 7-bp target duplications (two cases each). This study concluded that pTNR could be served as an efficient genetic tool for construction of random mutagenesis system in Rhodococcus species.     

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.     

The surfactant tween 80 enhances biodesulfurization

In biocatalytic conversions, substrates and products may display inhibitory or toxic effects on the biocatalyst. Rhodococcus erythropolis 1awq could further remove sulfur from hydrodesulfurized diesel oil, and the biodesulfurization was enhanced by the surfactant Tween 80. Tween 80 was shown to decrease the product concentration associated with the cells, reducing product inhibition.     

Degradation of hydrocarbons and alcohols at different temperatures and salinities by Rhodococcus erythropolis DCL14

Rhodococcus erythropolis DCL14 cells were able to metabolise C5-C16 hydrocarbons and C1-C12 alcohols as sole carbon and energy sources, both at 15 and 28 degrees C. Metabolic activity was also observed at 1.00%, 1.95% and 2.50% sodium chloride. Almost complete degradation of n-, iso- and cyclo-alkanes and aromatic compounds present in fuel oil was achieved after 9 months, 60% being consumed in the first three months. The results from the conditions tested here suggest that this type of bacterium could be involved in bioremediation processes in marine environments such as the Atlantic, Pacific and Indian Ocean.     

Riding the sulfur cycle--metabolism of sulfonates and sulfate esters in gram-negative bacteria

Sulfonates and sulfate esters are widespread in nature, and make up over 95% of the sulfur content of most aerobic soils. Many microorganisms can use sulfonates and sulfate esters as a source of sulfur for growth, even when they are unable to metabolize the carbon skeleton of the compounds. In these organisms, expression of sulfatases and sulfonatases is repressed in the presence of sulfate, in a process mediated by the LysR-type regulator protein CysB, and the corresponding genes therefore constitute an extension of the cys regulon. Additional regulator proteins required for sulfonate desulfonation have been identified in Escherichia coli (the Cbl protein) and Pseudomonas putida (the AsfR protein). Desulfonation of aromatic and aliphatic sulfonates as sulfur sources by aerobic bacteria is oxygen-dependent, carried out by the alpha-ketoglutarate-dependent taurine dioxygenase, or by one of several FMNH(2)-dependent monooxygenases. Desulfurization of condensed thiophenes is also FMNH(2)-dependent, both in the rhodococci and in two Gram-negative species. Bacterial utilization of aromatic sulfate esters is catalyzed by arylsulfatases, most of which are related to human lysosomal sulfatases and contain an active-site formylglycine group that is generated post-translationally. Sulfate-regulated alkylsulfatases, by contrast, are less well characterized. Our increasing knowledge of the sulfur-regulated metabolism of organosulfur compounds suggests applications in practical fields such as biodesulfurization, bioremediation, and optimization of crop sulfur nutrition.     

Glycogenformation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630

Members of the genus Rhodococcus were investigated for their ability to produce glycogen during cultivation on gluconate or glucose. Strains belonging to Rhodococcus ruber, Rhodococcus opacus, Rhodococcus fascians, Rhodococcus erythropolis and Rhodococcus equi were able to produce glycogen up to 0.2–5.6% of cellular dry weight (CDW). The glycogen content varied from 0.8% to 3.2% of CDW in cells of R. opacus PD630, which is a well-known oleaginous bacterium, during the exponential growth phase, when cultivated on diverse carbon sources. Maltose and pyruvate promoted glycogen accumulation by cells of strain PD630 to a greater extent than glucose, gluconate, lactose, sucrose or acetate. This strain was able to produce triacylglycerols, polyhydroxyalkanoates and glycogen as storage compounds during growth on gluconate, although triacylglycerols were always the main product under the conditions of this study. Cerulenin, an inhibitor of de novo fatty acid synthesis, inhibited the accumulation of triacylglycerols from gluconate and increased the content of polyhydroxyalkanoates (from 2.0% to 4.2%, CDW) and glycogen (from 0.1% to 3.0%, CDW). An increase of the polyhydroxyalkanoates and glycogen content was also observed in two mutants of R. opacus PD630, which produced reduced amounts of triacylglycerols during cultivation of cells on gluconate.     

An oil-degrading bacterium: Rhodococcus erythropolis strain 3C-9 and its biosurfactants

AIMS: To isolate a biosurfactant-producing bacterium and find new products within its culture. METHODS AND RESULTS: A biosurfactant-producing bacterium identified as Rhodococcus erythropolis (3C-9 strain) was isolated from seaside soil. When n-hexadecane was supplied as the sole carbon source, two types of biosurfactants (free fatty acids and glycolipids) were detected in the supernatant of the bacterial culture by use of thin layer chromatography (TLC). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the former consisted of at least 12 free fatty acids of chain lengths from C(9) to C(22); and the latter contained 2 kinds of glycolipids (a glucolipid and a trehalose lipid), which were detected by use of TLC, as well as GC-MS. The hydrophobic moieties of both glycolipids consisted of seven types of straight-chain fatty acids of varying compositions, with chain lengths ranging from C(10) to C(18). It was also noted that biosurfactants of strain 3C-9 were produced in a manner that was growth-related and cannot be synthesized from water-soluble substrates. The effects to enhance the solubility of polycyclic aromatic hydrocarbons and the degradation rate of hexadecane were also tested. CONCLUSIONS: The biosurfactants produced by strain 3C-9 of R. erythropolis included two kinds of glycolipids, as well as free fatty acids. These biosurfactants were notably different from those of previously reported Rhodococcus species, both in terms of their structure and chemical composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain 3C-9 of R. erythropolis is a competitive candidate for use in oil spill cleanup operations, or in new biosurfactant exploration. The findings in this report show that Rhodococcus is a natural reservoir of new biosurfactants.     

Distribution and application of mycobactins for the characterization of species within the genus Rhodococcus

Representatives of 11 species of Rhodococcus were examined for their ability to synthesize mycobactin, a lipid-soluble siderophore, following iron-limited growth on solidified glycerol/asparagine medium. Rhodococcus bronchialis, R. terrae and R. rubropertinctus formed mycobactins, whereas the remaining species (R. coprophilus, R. equi, R. erythropolis, R. rhodnii, R. rhodochrous, R. ruber, R. maris and R. luteus) failed to synthesize these compounds even under conditions of strictly iron-limited growth. The mycobactins from R. terrae and R. rubropertinctus showed close similarity by thin-layer chromatography and high-performance liquid chromatography and could be easily distinguished from that of R. bronchialis.