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Applied and Environmental Microbiology, August 2003, p. 4689-4696, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4689-4696.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Growth of Rhodosporidium toruloides Strain DBVPG 6662 on Dibenzothiophene Crystals and Orimulsion

Franco Baldi,1,2* Milva Pepi,2 and Fabio Fava3

Department of Environmental Sciences, Cà Foscari University, S. Marta, Dorsoduro 2137I-30121 Venice,1 Interuniversity Consortium for Environmental Chemistry, Marghera, I-30175 Venice,2 DICASM, Faculty of Engineering, University of Bologna, I-40136 Bologna, Italy3

Received 29 January 2003/ Accepted 2 June 2003


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ABSTRACT
 
Strains DBVPG 6662 and DBVPG 6739 of Rhodosporidium toruloides, a basidiomycete yeast, grew on thiosulfate as a sulfur source and glucose (2 g liter-1 or 10.75 mM) as a carbon source. DBVPG 6662 has a defective sulfate transport system, whereas DBVPG 6739 barely grew on sulfate. They were compared for the ability to use dibenzothiophene (DBT) and related organic sulfur compounds as sulfur sources. In the presence of glucose as a carbon source and DBT as a sulfur source, strain DBVPG 6662 grew better than DBVPG 6739. In the presence of thiosulfate as a sulfur source, the two yeast strains did not use DBT, DBT-sulfone, benzenesulfonic acid, biphenyl, and fluorene. When the two strains were grown in the presence of glucose, strain DBVPG 6662 transformed 27% of the DBT present (10 µM) at a rate of 0.023 µmol liter-1 h-1 in 36 h. Traces of 2,2'-dihydroxylated biphenyl were transiently accumulated under these conditions. When the same strain was grown on glucose in the presence of a higher concentration of DBT (0.5 g liter-1), mainly in an insoluble form, the whole surface of the DBT crystals was colonized by a thick mycelium. This adherent structure was imaged by confocal microscopy with fluorescent concanavalin A, a lectin that specifically binds glucose and mannose residues. When DBVPG 6662 was grown on glucose in the presence of a commercial emulsion of bitumen, i.e., orimulsion, 68% of the benzo- and dibenzothiophenes and DBTs was removed after 15 days of incubation. The fungus adhered by hyphae to orimulsion droplets. When cultivated in the presence of commercial emulsifier-free fuel oil containing alkylated benzothiophenes and DBTs and having a composition similar to that of orimulsion, strain DBVPG 6662 removed only 11% of the total organic sulfur that occurs in the medium and did not adhere to the oil droplets. These results indicate that strain DBVPG 6662 is able to utilize the organic sulfur of DBT and a large variety of thiophenic compounds that occur extensively in commercial fuel oils by physically adhering to the organic sulfur source.


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INTRODUCTION
 
Biological removal of sulfur from fossil fuels to decrease the quantity of the sulfur oxides produced during their combustion is a challenge for microbiologists and other scientists. With prokaryotes, biocleaning of fossil fuels has only been partially successful because chemolithotrophic bacteria and archaea (8, 29, 30) only remove inorganic sulfur. Various scale-up processes are available for removal of inorganic sulfur, but no industrialized process for organic sulfur degradation has been developed. The removal of organic sulfur, i.e., the sulfur covalently bound in complex organic molecules, such as mercaptans, thiophenols, thioethers, dithioethers, and dibenzothiophene (DBT), is being investigated by many laboratories, and differently adapted microbial cultures are being tested. Many strains are not useful because they mineralize the carbon skeleton (9, 12, 13, 21, 24, 25). Bacteria that desulfurize organic sulfur compounds without metabolizing the carbon skeleton are less common and generally use a sulfur-selective oxidative pathway (12, 15, 18, 30, 35, 36). Isolation of new microorganisms capable of carbon-sulfur bond-targeted desulfurization is still of primary interest. Bacteria belonging to the gram-positive domain (7, 17, 19, 34), mainly from the genus Rhodococcus (22, 27), are responsible for DBT desulfurization. Few gram-negative bacteria are capable of organic sulfur degradation; some recently reported ones are Sphingomonas (26) and Paenibacillus (20) spp. Among eukaryotic organisms, Cunninghamella elegans has been reported to desulfurize DBT (9). This fungus grows on DBT by forming DBT-5-oxide and DBT-5-dioxide but not biphenyl. The fungus Paecylomyces sp. carries out sulfur-specific oxidation of DBT by producing 2,2'-dihydroxybiphenyl (11). No yeasts have been reported to grow desulfurizing DBT and/or related thiophenic compounds that extensively occur in industrial fuel oils.

Rhodosporidium toruloides strain DBVPG 6662 was isolated from industrial waste several years ago (2, 31, 32) and grows on thiosulfate as a sulfur source in the presence of glucose as a carbon source. The aim of the present study was to investigate and characterize the ability of this strain to utilize DBT and related organic sulfur compounds that occur in fossil and oil fuels as sulfur sources.


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MATERIALS AND METHODS
 
Yeast cultures in organic and inorganic sulfur sources.
Two strains from the industrial yeast collection of the Dipartimento di Biologia Vegetale Università di Perugia (DBVPG), belonging to the European Culture Collection Organization, were compared for organic sulfur utilization: DBVPG 6739, which grows to a very limited extent on sulfate as a sulfur source, and DBVPG 6662, which does not. The two strains were routinely grown in Bacto Sabouraud Dextrose Broth (Difco, Detroit, Mich.) and incubated overnight at 28°C in a rotary drum. Cells were harvested by centrifugation at 6,000 x g for 20 min and washed twice with 10 mM PIPES [(piperazine-N,N'-bis(2-ethanesulfonic-acid)] buffer (Sigma, Milan, Italy) at pH 7.4. They were then inoculated into 250-ml conical flasks containing 50 ml of a minimal medium (MM) consisting of 2 g of yeast nitrogen base liter-1 without amino acids (Difco) and 0.011 mM ammonium chloride (Aldrich, Milan, Italy), which replaced ammonium sulfate, to lower the sulfate content of the MM to 3.1 mM. The carbon source was 2 g of D-glucose (Aldrich) liter-1 (10.75 mM); sulfate (Aldrich), thiosulfate (Aldrich), or sulfite (Aldrich) was used, at concentrations ranging from 0.080 to 6.2 mM, as a sulfur source.

To demonstrate that DBT is a sulfur source for these yeast strains, inorganic sulfur was replaced with an organic sulfur source such as 500 mg of DBT (Merck, Milan, Italy) liter-1 (2.71 mM) with or without 2 g of D-glucose liter-1 in MM. For a control, fluorene (Aldrich), a sulfur moiety-free DBT analog, was also tested at 500 mg liter-1 (3.0 mM) as a potential carbon source for both yeast strains in MM with thiosulfate (5 mM) as a sulfur source. A further control experiment was also performed with MM plus glucose (2 g liter-1) but no added sulfur source.

Yeast biomass.
Yeast biomass was determined by (i) counting cells stained with acridine orange by epifluorescence microscopy (Axiophot; Zeiss, Oberkochen, Germany), (ii) analyzing total protein by the Bradford method (3), (iii) measuring A600 with a UV-visible light spectrophotometer (Shimadzu UV-160), and (iv) weighing the dried biomass on a filter. Glucose concentration was determined as described by Miller (23). Growth yields were determined at several different initial glucose concentrations in the presence of DBT. The yield determination was based on glucose consumption, which was measured by treating 1.5-ml samples of the yeast culture with 1.5 ml of a reagent containing 1% (wt/vol) dinitrosalicylic acid, 0.2% (wt/vol) phenol, 0.05% (wt/vol) sodium sulfite, and 1% (wt/vol) sodium hydroxide (all chemicals were from Aldrich). The glucose concentration was then determined spectrophotometrically at 575 nm (A575).

Studies of oxygen consumption.
The yeast strains were pregrown on MM containing 2 g of D-glucose liter-1 (10.75 mM) and 50 µM DBT. DBT-induced cells were harvested in the exponential growth phase (by centrifugation at 6,000 x g for 20 min), washed twice with phosphate buffer (pH 7.4), and resuspended in 5 ml of fresh MM. A 100-µl aliquot of the resulting cell suspension was added to 3 ml of fresh phosphate buffer and introduced into the measuring cell of an oxygen monitor (model 5300; Yellow Springs Instrument Co., Yellow Springs, Ohio) equipped with a Clark-type oxygen electrode. DBT, benzothiophene (Aldrich), DBT-sulfone (Aldrich), benzenesulfonic acid (Aldrich), biphenyl (Aldrich), and fluorene were dissolved in N,N'-dimethylformamide (Carlo Erba, Milan, Italy) and added to cell suspensions to a final concentration of 50 µM. Depending on the experiment, 10.75 mM glucose or 5 mM thiosulfate was also added to the assay vessel as a carbon or sulfur source, respectively. Oxygen consumption was measured at 28°C. The instrument was calibrated constantly with air-saturated fresh MM. The measurements were corrected for endogenous respiration and chemical substrate oxidation. Yeast concentration was expressed in terms of total cell protein determined by the Bradford method (3).

DBT biodegradation in liquid cultures.
DBT biodegradation by the two R. toruloides strains was studied in cells growing in batch cultures. The strains were inoculated into sterile conical flasks (1 liter) containing 200 ml of sterile MM amended with 10 µM DBT (by spiking each culture with 0.2 ml of a 10 µM DBT solution in dimethylformamide) as a sulfur source and 10.75 mM glucose as a carbon source. An identical culture was developed with no inoculum as an abiotic control. All flasks were closed with sterile aluminum foil and incubated at 28°C in static mode to reduce abiotic DBT loss due to volatilization. DBT and its potential biodegradation metabolites were extracted from the cultures. Duplicate 10-ml samples were removed from each culture at pre-established times. Each sample was placed in a 20-ml vial equipped with a Teflon-coated butyl stopper and an aluminum crimp sealer, and the samples were subjected to batch solvent extraction with 1 ml of hexane in an ultrasonic bath. The organic phase was then analyzed through a Beckman high-performance liquid chromatography (HPLC) system equipped with a Beckman Ultrasphere octyldecyl silane column (4.6 by 25 mm; particle diameter, 5 µm) and a Beckman Coulter 168 System Gold diode array detector operating at 240 and 250 nm. The column temperature was 35°C; the injection volume was 20 µl. The solvents used were water acidified with 1% (vol/vol) acetic acid (A) and methanol acidified with 1% (vol/vol) acetic acid (B). The solvent gradient method used was as follows: initial solvent composition, 60% A and 40% B; isocratic elution for 15 min; solvent composition changed to 20% A and 80% B in 20 min; isocratic elution for 8 min; and back to the initial solvent composition for 12 min. Five standard solutions ranging from 5 to 100 µM DBT were used for calibration. Extraction efficiency was determined by the standard addition method; it was typically found to be 93% ± 3%. Five replicates of the same sample gave a coefficient of variation of 7.3%. The DBT detection limit of the method was 0.54 µM.

The water phase resulting from DBT extraction was then acidified to pH 2.5 with trichloroacetic acid (2 M; Aldrich) and subjected to two successive batch extractions with 5 ml of ethyl acetate to recover polar metabolites of DBT degradation. The organic phase obtained was evaporated to dryness under an N2 stream. The residue collected was redissolved in methanol to a final volume of 0.5 ml. Qualitative and quantitative analyses of the main DBT desulfurization organic metabolites, such as DBT-sulfone, benzothiophene, benzenesulfonic acid, biphenyl, and 2,2'-dihydroxybiphenyl, that occur in the hexane fraction and in the 20-fold-concentrated ethyl acetate fraction were done by the same HPLC procedure. One-milliliter samples were also periodically taken from each of the yeast cultures for analysis of the main inorganic metabolites of DBT desulfurization. Each sample was harvested and centrifuged at 11,000 x g for 3 min (Eppendorf 5415), and 0.2 ml of the resulting supernatant was analyzed for sulfite concentration by the iodometric method (14) and for sulfate by ion chromatography. In the latter case, the anion concentration was measured with a Dionex DX-120 IC system equipped with an IonPac AS14 column (4 by 250 mm) and a conductivity detector combined with an ASRS-Ultra conductivity suppressor system (Dionex Corporation, Sunnyvale, Calif.). The eluent was a solution of 3.5 mM Na2CO3 and 1.0 mM NaHCO3 prepared in ultra-resi-analyzed water; the flow rate was 1.2 ml min-1, and the injection volume was 20 µl. A linear four-point calibration curve (range, 0.2 to 20.0 mg/liter, i.e., 0.0021 to 0.21 mM) for SO42- was developed by using a pure standard of this compound. The limit of SO42- detection was 0.052 µM.

Morphology changes and cell adhesion determined by CSLM.
Morphology changes in strain DBVPG 6662 from vegetative cell to hypha formation were monitored in MM with 2 g of glucose liter-1 (10.75 mM) in the presence of DBT crystals at a concentration (500 mg liter-1) much higher than the water solubility of DBT. Strain 6662 was also grown in MM with 2 g of glucose liter-1 amended with 1 g of orimulsion 4097 or with fuel oil (MTZ 4098) containing organic sulfur (determined as described below). The culture was sampled at pre-established times throughout the experiment, and the collected sample was analyzed by confocal scanning laser microscopy (CSLM). A confocal microscope (MRC-500; Bio-Rad Microscience Division) mounted on a Nikon Microphot microscope was used to observe strain DBVPG 6662 growing on MM with DBT crystals (500 mg liter-1), orimulsion, or fuel oil. Yeast colonization of crystals was checked after different periods of inoculation. To determine polysaccharide production during growth on the different thiophenic compounds, a 1-ml aliquot of suspension was incubated for 1 h with 5 µl of concanavalin A (ConA; 1 mg ml-1; Sigma) conjugated with fluorescein isothiocyanate (FITC; C7642; Sigma) (5). Individual CSLM images were horizontal high-resolution optical thin sections (10 nm) (6). A krypton-argon laser with maximum emission lines at 488 nm was used to measure the excitation source of the fluorescent lectin. Black and white photographs were taken with T-max 100 ASA film (Kodak, Rochester, N.Y.). Image analysis of recorded sections of samples was carried out with Bio-Rad Comos software.

Determination of organic sulfur in orimulsion and fuel oil.
Strain DBVPG 6662 was also tested for the ability to take up sulfur from organic sulfur in commercial fuels. The yeast was transferred five times at 28°C in MM containing 2 g of glucose liter-1 (10.75 mM) and 1 mM thiosulfate as a sulfur source to avoid DBT contamination. The strain was then inoculated (0.021 ± 0.005 mg of protein ml-1) into duplicate flasks (250 ml) containing 50 ml of MM with glucose (10.75 mM) and 1 g (20 g liter-1) of orimulsion 4097 or of MTZ 4098 fuel oil (with a medium sulfur content of 1.5%, wt/wt). These materials were kindly supplied by the ENEL Waste Treatment Research Center (Brindisi, Italy). Orimulsion is a fuel consisting of natural bitumen dispersed in fresh water (26 to 30%, wt/wt), stabilized by addition of a patented surfactant (33). The qualitative and quantitative analyses of the organic sulfur compounds that occur in the cultures amended with the two commercial products were carried out at the beginning and end (after 15 days) of the culture incubation period. The batches of whole cultures were extracted (in three successive steps) with dichloromethane in accordance with Environmental Protection Agency SW-846 method 3510B (10). Extracts were dried on a column of anhydrous sodium sulfate (Aldrich) and carefully concentrated to a final volume of 10 ml. Aliquots (1 ml) of the resulting solution were diluted to 30 ml with dichloromethane. A 2-cm column with activated silica gel, pretreated at 150°C for 24 h, was used to remove particles and very high-molecular-weight components as previously described (16) for oil spill characterization. Aliquots of 1 µl were injected in the splitless mode in a gas chromatograph-mass spectrometer (Finnigan Magnum) equipped with an SPB-5 fused silica column (0.32-µm film thickness; 30 by 0.32 m [inside diameter]; Supelco). The carrier gas was helium at 1,110 kPa. The injector temperature was 200°C. The analytical conditions were injection and holding for 1 min at 35°C and then a temperature increase of 10°C min-1 to 300°C. Compounds were detected by ion trap mass spectrometry. The ion trap operated at a nominal voltage of 70 eV, scanning 80 to 300 atomic mass units s-1. DBT and benzothiophene were identified by analysis of standard solutions and mass spectra. Methylated residues of these sulfur compounds were also identified by ion profile (m/z 148, 162, and 176 for methylbenzothiophene, dimethylbenzothiophene, and trimethylbenzothiophene, respectively; m/z 198, 212, and 226 for methyl-DBT, dimethyl-DBT, and trimethyl-DBT, respectively). Methylated residues were quantified with respect to the sum of the peaks of each class of compounds and normalized to DBT.


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RESULTS
 
Yeast growth on different sulfur species.
R. toruloides strain DBVPG 6662 cultivated in the presence of different inorganic sulfur sources grew on thiosulfate (>1 mM) but not at all on sulfate or sulfite (up to 80 µM). Strain DBVPG 6739 grew on a lower concentration of thiosulfate (<0.080 mM) and slightly on sulfate but not on sulfite (Fig. 1). Strain DBVPG 6662 also grew on high concentrations of DBT at a rate of 5.67 mg ml-1 day-1 but only in the presence of glucose as a carbon and energy source. It did not grow on DBT as a combined source of energy, carbon, and sulfur. Strain DBVPG 6739 grew at a slower rate (0.715 mg ml-1 day-1) under the same laboratory conditions. Neither strain grew on fluorene, as a DBT analog lacking a sulfur moiety, in the presence of thiosulfate (Fig. 2).



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  		FIG. 1. Growth of R. toruloides strains DBVPG 6662 ({triangleup}, {circ}, {square}) and DBVPG 6739 ({blacktriangleup}, •, {blacksquare}) in the presence of the inorganic sulfur sources thiosulfate ({triangleup}, {blacktriangleup}) sulfite ({circ}, •), and sulfate ({square}, {blacksquare}). The mineral medium contained a background sulfate level of about 300 mg liter-1 (3.1 mM).



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  		FIG. 2. Growth curves of R. toruloides strains DBVPG 6662 ({triangleup}, {circ}, {square}) and DBVPG 6739 ({blacktriangleup}, •, {blacksquare}) in the presence of DBT with glucose 10.75 mM ({triangleup}), DBT without glucose ({circ}), and fluorene with 5 mM thiosulfate ({square}).

The metabolism of DBT, DBT-sulfone, and benzenesulfonic acid, along with that of biphenyl, fluorene, and the solvent N,N'-dimethylformamide, by the two yeast strains was also studied through O2 consumption measurements. None of the organic compounds tested was oxidized by R. toruloides DBVPG 6662 and DBVPG 6739 in the absence of glucose (Table 1). However, in the presence of glucose, DBT, DBT-sulfone, and benzenesulfonic acid significantly sustained the growth of DBVPG 6662 and poorly sustained that of strain DBVPG 6739. In the presence of D-glucose with no organic sulfur source, the yeast strains did not grow. Fluorene, biphenyl, and N,N'-dimethylformamide were not oxidized by DBT-induced cells of both strains in the presence of thiosulfate (Table 1).


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  		TABLE 1. Substrate-dependent oxygen uptake by cells of R. toruloides DBVPG 6662 and DBVPG 6739a

To demonstrate direct DBT transformation, two batch experiments were performed with both strains in glucose-amended MM with dissolved DBT (10 µM). DBT was found to be metabolized partially by both cultures. Strains DBVPG 6662 and DBVPG 6739 exhibited average DBT degradation rates (calculated by subtracting the average depletion rate measured in the abiotic culture from those recorded in the parallel inoculated cultures) of 0.023 and 0.0088 µmol liter-1 h-1, respectively, with average percentages of removal of the initially occurring DBT of 24 and 17%, respectively, after 36 h of incubation (Fig. 3). The DBT depletion observed in the strain 6739 cultures was only slightly faster and higher than that observed in the corresponding abiotic controls. In fact, a detectable amount of the spiked DBT was also found to disappear over time in the abiotic controls (Fig. 3); analyses carried out on the headspace gas of identical cultures described in parallel in hermetically closed microcosms (data not shown) indicate that the detected DBT abiotic losses were mostly due to volatilization phenomena. Traces of 2,2'-dihydroxylated biphenyl were transiently accumulated in the DBVPG 6662 cultures, whereas none of the metabolites sought, such as DBT-sulfone, sulfate, and sulfite, was produced in either the DBVPG 6662 or the DBVPG 6739 cultures. The detected compound in DBVPG 6662 cultures was characterized as 2,2'-dihydroxylated biphenyl, as it coeluted with pure 2,2'-dihydroxybiphenyl under a variety of different HPLC analytical conditions and displayed a diode array UV-visible light absorption spectrum identical to that of the pure compound. Biphenyl was detected (at 0.0654 ± 0.0032 µM) in both cultures and in the control from the beginning of the experiment. This compound was neither produced nor metabolized throughout the whole experiment; it was found to be an impurity in the DBT product used in the study.



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  		FIG. 3. Consumption of DBT (10 µM) in liquid cultures of R. toruloides strains DBVPG 6662 ({blacksquare}) and DBVPG 6739 (•) and in an uninoculated culture (control) ({circ}) throughout a 36-h experiment. Results are means of single determinations on duplicate cultures; error bars not visible are smaller than the symbols and show ranges of duplicate samples.

Glucose metabolism and biomass growth in the DBT-amended cultures were studied through a parallel experiment carried out under the same conditions. DBVPG 6662 metabolized glucose rapidly and extensively (Fig. 4), exhibiting a doubling time of 5.9 h, producing 0.139 mg of protein liter-1, consuming 1.5 mg of D-glucose, and giving a yield of 0.085. By contrast, strain DBVPG 6739 metabolized glucose weakly (Fig. 4) and exhibited a doubling time of 20.0 h, a cell production of 0.016 mg of protein liter-1, a glucose consumption of 0.13 mg, and a cell yield of 0.01.



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  		FIG. 4. Consumption of D-glucose (2 mg liter-1) as a carbon and energy source by cultures of R. toruloides strains DBVPG 6662 ({triangleup}) and DBVPG 6739 ({circ}) and in an uninoculated culture (+) during 120 h of incubation at 28°C in the presence of dissolved DBT (10 µM) as a sulfur source. Simultaneous determination of the biomass (micrograms of protein per milliliter) that occurred in the cultures of strains DBVPG 6662 and DBVPG 6739 is also shown ({blacktriangleup} and •, respectively). Results are means of single determinations on duplicate cultures; error bars not visible are smaller than the symbols and show ranges of duplicate samples.

DBVPG 6662 growth in the presence of DBT crystals and commercial fossil fuels.
Given its ability to use DBT and other organic sulfur compounds as a source of sulfur, strain DBVPG 6662 was subjected to further physiological and metabolic investigations. In particular, when DBVPG 6662 interacted with DBT crystals, it underwent a morphologic transformation from yeast cells to hyphae. This event was first investigated by confocal microscopy with the lectin ConA-FITC. This lectin specifically binds the carbohydrate residues of D(+)-glucose and D(+)-mannose (1). The cell wall of R. toruloides is known to contain glucose, mannose, galactose, and glucosamine, but only mannose seems to be involved in the adhesion process (9). DBVPG 6662 cells growing in MM with glucose (10.75 mM) and DBT (500 mg liter-1) were found to adhere to and colonize only DBT crystals (Fig. 5A) and not the walls of the culture vessel. At the start, the contact with DBT caused a significant reduction in cell volume. The smallest cells (size, 1.4 ± 0.13 µm) showed a huge concentration of ConA fluorescence at the cell envelope (Fig. 5B). Yeast cells (size, 5 ± 0.34 µm) were not, or only faintly, stained by ConA. After 6 days of incubation, hypha formation was induced and the cells formed a thick mycelium (Fig. 5C) on the DBT crystal surface. Confocal microscopy image analysis of a series of 20 scanned sequential optical planes (x and y), 0.6 µm in thickness, and therefore equivalent to 12 µm in the z plane, showed mycelium rich in exopolysaccharides (Fig. 5D), which were concentrated mainly at the poles of elongated hyphal cells (Fig. 5E). The mycelium developed and branched, forming a large epiphytic biomass solidly attached to the hydrophobic DBT crystals.



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  		FIG. 5. (A) Cells of R. toruloides strain DBVPG 6662, observed in transmission mode, at the beginning of their colonization of DBT crystals (500 mg ml-1). Bar, 25 µm. (B) The same specimen observed in fluorescence mode after staining with ConA-FITC; smaller yeast cells became highly fluorescent because of exopolysaccharide production. Bar, 25 µm. (C) After 6 days of incubation, DBT crystals, observed in transmission mode, were intensively colonized by a mycelium produced by the basidiomycete R. toruloides. Bar, 25 µm. (D) The same specimen observed in fluorescence mode after staining with ConA-FITC. Hyphae are producing a large amount of exopolysaccharide. Bar, 25 µm. (E) Detail showing that the largest amounts of exopolysaccharide were produced at the polar regions of elongated cells. Bar, 5 µm.

Strain DBVPG 6662 was also investigated for the ability to use organic sulfur in commercial fossil fuels. The strain was found to grow significantly on glucose in the presence of orimulsion, i.e., a bitumen amended with an emulsifying agent and water. However, the same strain grew only slightly in the presence of the MTZ 4098 fuel oil (data not shown). Both commercial products contain a number of sulfur compounds heavier and more hydrophobic than DBT, such as methylated, dimethylated, and trimethylated benzo- and dibenzothiophenes, which constituted about 96% (wt/wt) of the orimulsion and 80% (wt/wt) of the fuel oil (Table 2). The growth of the strain throughout the 15 days of incubation resulted in the removal of about 68% of the organic sulfur initially detected in the orimulsion-amended culture and of 11% in that in the fuel oil culture. DBVPG 6662 cells were found to adhere to and aggregate orimulsion droplets (Fig. 6A) by producing the ConA-positive exopolysaccharides. This was followed by a significant reduction in cell size and polar adhesion of cells to produce hyphae (Fig. 6B), extending almost to the same growth pattern as on DBT crystals. At the end of the incubation period (day 15), the hyphae were longer than those on DBT crystals (Fig. 6C) and they trapped orimulsion droplets. Staining of specimens with ConA-FITC showed clearly that hyphae were rich in exuded exopolysaccharides. The orimulsion droplet surface was strongly fluorescent, being coated by this polysaccharide biofilm (compare Fig. 6D and B).


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  		TABLE 2. Concentrations of the main organic sulfur compounds of orimulsion and fuel oil detected in sterile controls and the biologically active DBVPG 6662 cultures after 15 days of incubation



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  		FIG. 6. (A) Cells of R. toruloides strain DBVPG 6662, observed in transmission mode, starting to colonize the hydrophobic surface of orimulsion droplets. (B) The same specimen observed in fluorescence mode after staining with ConA-FITC. Yeast cells adhering to orimulsion became fluorescent because of exopolysaccharide production. Cells reduced their size and formed elongated aggregates (arrows). (C) After 15 days of incubation, development of long hyphae by R. toruloides was observed in transmission mode. Mycelium entrapped orimulsion droplets. (D) The same specimen observed in fluorescence mode after staining with ConA-FITC. Orimulsion droplet became coated by exopolysaccharides, suggesting that strain 6662 has an intrinsic emulsifying activity different from that of the emulsifying agent already present in orimulsion.


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DISCUSSION
 
The present study originated from previous research on Cr(VI) resistance in yeasts (2, 31) in which it was found that R. toruloides strain DBVPG 6662 has a variety of transport systems for reduced forms of sulfur and an inefficient sulfate transport system. This was naturally shut down to avoid uptake of chromate, a natural competitor for sulfate uptake (2, 28). The basidiomycete yeast was isolated from chromate-rich industrial wastes and was the only microorganism capable of growing at Cr(VI) concentrations of up to 500 µg ml-1 (31).

Several experimental findings indicate that R. toruloides DBVPG 6662 requires reduced forms of sulfur for growth. Indeed, the strain grew on reduced forms of inorganic sulfur sources, such as thiosulfate, and of organic sulfur sources, such as cysteine, methionine, and thioglycolic acid (32), along with DBT, DBT-sulfone, and benzenesulfonic acid (Fig. 1 and 2 and Table 1). The utilization of the organic sulfur compounds is strictly dependent on the presence of glucose in the culture medium. A lack of growth (Fig. 1 and 2) or oxygen consumption (Table 1) in media with such organic sulfur sources in the absence of glucose and a lack of growth and an absence of oxygen consumption in the presence of fluorene (a DBT analogue with no sulfur moiety) in the presence of thiosulfate indicate that the reduced S-aromatic compounds were used exclusively as a sulfur source by the strains. The absence of detectable sulfate or sulfite production in these cultures further supports this hypothesis. Unfortunately, little is known about the nature and fate of the organic metabolites resulting from sulfur removal from the metabolized organic sulfur compounds; indeed, only traces of 2,2'-dihydroxybiphenyl were detected in the DBT- and glucose-amended culture of strain DBVPG 6662, which was the strain that exhibited the greatest activity on the range of organic sulfur sources utilized by the two yeasts used in the study (Fig. 1, 2, and 3). Our inability to detect other metabolites of DBT desulfurization probably resulted from the small content of the organic sulfur compounds consumed by the two strains in these tests (Fig. 3). Therefore, the metabolites were produced in quantities too small for detection by the analytic techniques used in this study.

When grown in the presence of DBT at a concentration in excess of its water solubility, strain DBVPG 6662 extensively colonized the surface of DBT crystals in the culture medium (Fig. 5). This is an expected feature of this basidiomycetous strain, which is a common phylloplane epiphyte that typically adheres to plant surfaces (5). However, the fact that growth did not occur on the inner surface of the culture vessel suggests that intimate contact between yeast cells and the solid sulfur source is essential for efficient uptake of the sulfur nutrient. To better investigate this phenomenon, the lectin ConA, a good specific marker for the trimannosyl core of N-linked glycans (1) to which the lectin strongly binds, was used to monitor glycosylation during yeast cell adhesion to DBT crystals. The lectin used in this study turned out to be useful for identification of morphological and biochemical changes in the glycosylation of outer cell layers in response to adaptation to DBT. On the basis of the confocal microscopy of DBVPG 6662 cells growing on DBT crystals (Fig. 5), a significant decrease in cell size and a high level of mannoprotein production seemed to be the prevalent adaptive mechanisms through which the strain performed DBT metabolism and sulfur assimilation. The development of a thick wall of glycoproteins probably protected the cells and/or contributed to DBT dissolution, thus permitting gradual and controlled sulfur uptake. This hypothesis is supported by findings according to which a wild-type R. toruloides strain adheres to surfaces by means of mannoproteins (i.e., glycoproteins containing mannose), which accounted for 56% of the surface glycoproteins (4, 5), mostly concentrated in the pole regions of cells (5).

Strain DBVPG 6662 was also found to massively adhere to and colonize orimulsion droplets by extensively coating their surface with glycoproteins (Fig. 6). However, no cell adhesion or colonization was observed on droplets of fuel oil exposed to the strain under identical culture conditions. The two commercial products used were similar in organic sulfur compound composition and content (Table 2). Thus, the extensive colonization that was observed only on orimulsion droplets may be ascribed to the fact that orimulsion incorporates an emulsifier, which, according to the literature (36), may have significantly enhanced the apparent water solubility and/or the dissolution rate in water of the highly hydrophobic organic sulfur compounds, making them more available to yeast cells. The finding that a higher and broader removal of the organic sulfur compounds was observed in the culture with orimulsion (68%) than in the culture with fuel oil (11%) (Table 2) seems to support the hypothesis according to which intimate contact between cells or mycelium and the organic sulfur source is required for effective sulfur source transformation and assimilation in strain DBVPG 6662.

In conclusion, R. toruloides strain DBVPG 6662 exhibited an ability to utilize a large variety of organic sulfur compounds, including those that occur in commercial sulfur-rich products such as orimulsion and fuel oils, as sulfur sources. Intimate contact between yeast cells and the sulfur source, provided by yeast colonization of organic sulfur crystals or oil emulsion droplets, appears to be essential for more efficient organic sulfur source metabolism and sulfur uptake. The yeast seems to selectively remove sulfur from the organic molecules without using their carbon skeleton as a carbon and energy source. The transient accumulation of 2,2'-dihydroxybiphenyl, along with our inability to detect other DBT desulfurization organic metabolites, does not, however, exclude the possibility that organic intermediates of the process were degraded by the yeast through cometabolism. Anyhow, the yeast selectively utilizes DBT and thiophenic compounds but not many of their structurally correlated sulfur-free hydrocarbons, and this suggests that it may be used successfully for fuel and fossil oil industrial desulfurization.

The results of this study are also of interest because they document for the first time that (i) the yeast strain R. toruloides DBVPG 6662 is able to utilize a large variety of organic sulfur compounds as sulfur sources, (ii) complex mixtures of alkylated benzo- and dibenzothiophenes can be significantly metabolized and used as sulfur sources by a naturally occurring eukaryotic microorganism, and (iii) complex thiophenic fractions that occur in the commercial product orimulsion can be efficiently removed by R. toruloides strain DBVPG 6662, which can combine effective biotransformation of such sulfur fractions with the ability to produce an abundant mycelium. This biomass can be easily removed from the oil bulk.


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ACKNOWLEDGMENTS
 
This research was financed by ENEL.

We thank G. Maspero (ENEL-Milan) for analysis of the organic sulfur in orimulsion and fossil fuel and G. Varallo (ENEL, Waste Treatment Research Center, Brindisi, Italy) for supplying commercial oil samples.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Environmental Sciences, Cà Foscari University, Calle Larga, S. Marta, Dorsoduro 2137, 30121 Venice, Italy. Phone: 39-041-2578432. Fax: 39-041-2348901. E-mail: baldi{at}unive.it. Back


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Applied and Environmental Microbiology, August 2003, p. 4689-4696, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4689-4696.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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