biodesulfurization of refractory organic sulfur compounds in fossil fuels

$: Biodesulfurization of Refractory Organic Sulfur Compounds in Fossil Fuels$
Biotechnology Advances 25 (2007) 570–596www.elsevier.com/locate/biotechadv

Research review paper

Biodesulfurization of refractory organic sulfurcompounds in fossil fuels

Mehran Soleimani, Amarjeet Bassi ⁎, Argyrios Margaritis

Department of Chemical and Biochemical Engineering The University of Western Ontario London, Ontario, Canada N6A 5B9

Received 22 May 2007; accepted 25 July 2007Available online 31 July 2007

Abstract

The stringent new regulations to lower sulfur content in fossil fuels require new economic and efficient methods fordesulfurization of recalcitrant organic sulfur. Hydrodesulfurization of such compounds is very costly and requires high operatingtemperature and pressure. Biodesulfurization is a non-invasive approach that can specifically remove sulfur from refractoryhydrocarbons under mild conditions and it can be potentially used in industrial desulfurization. Intensive research has beenconducted in microbiology and molecular biology of the competent strains to increase their desulfurization activity; however, eventhe highest activity obtained is still insufficient to fulfill the industrial requirements. To improve the biodesulfurization efficiency,more work is needed in areas such as increasing specific desulfurization activity, hydrocarbon phase tolerance, sulfur removal athigher temperature, and isolating new strains for desulfurizing a broader range of sulfur compounds. This article comprehensivelyreviews and discusses key issues, advances and challenges for a competitive biodesulfurization process.© 2007 Elsevier Inc. All rights reserved.

Keywords: Biodesulfurization; 4S pathway; dsz gene cluster; Rhodococcus; IGTS8; Recalcitrant sulfur compounds; Dibenzothiophene; Oxidativedesulfurization

Abbreviations: ATCC, American Type Culture Collection; BMIM, 1-Butyl-3-methylimidazolium; BPSi, sultine; BPSo, sultone; BT,benzothiophene; DBT, dibenzothiophene; DBT-MO, dibensothiophene-monooxygenase; DBTO, dibenzothiophene-5-oxide, DBT sulfoxide;DBTO2, dibenzothiophene-5,5-dioxide, DBT sulfone; DBTO2-MO, dibenzothiophene-5,5-dioxide-monooxygenase; DEDBT, diethyl dibenzothio-phene; DHBP, dihydroxy biphenyl; DMDBT, dimethyl dibenzothiophene; DMSO, dimethyl sulfoxide; dsz, desulfurization; EMIM, 1-Ethyl-3-methylimidazolium; FB, fed-batch; FBR, fluidized bed reactor; FCC, fluid catalytic cracking; HBFT, 3-hydroxy-2-formyl-benzothiophene; HBP,Hydroxybiphenyl; HBPS, 2′-hydroxybiphenyl-2-sulfinate; HBPSi, 2′-hydroxybiphenyl-2-sulfinate, sulfinate; HBPSo, 2′-hydroxybiphenyl-2-sulfonate, sulfonate; HDEBP, 2-hydroxy-3,3′-diethylbiphenyl; HDS, hydrodesulfurization; IL, ionic liquids; MBT, methyl benzothiophene; MDBT,methyldibenzothiophene; MeSO4, methyl sulfate; NCBI, National Center for Biotechnology Information; NTG, 1-methyl-2-nitro-1-nitrosoguani-dine; OcSO4, octylsulfate; ORF, open reading frame; PA, pyruvic acid; PCMB, p-chloromercuribenzoic acid; PF6, hexafluorophosphate; sox, sulfuroxidation; TH, thiophene; TMDBT, trimethyl dibenzothiophene.⁎ Corresponding author. Tel.: +1 519 661 2111x88324; fax: +1 519 661 3498.E-mail address: [email protected] (A. Bassi).

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571M. Soleimani et al. / Biotechnology Advances 25 (2007) 570–596

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5712. Sulfur in crude oil and coal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5723. Desulfurization methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572

3.1. Hydrodesulfurization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5733.2. Desulfurization by ionic liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5733.3. Biological sulfur removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574

3.3.1. Destructive biodesulfurization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5743.3.2. Anaerobic biodesulfurization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5743.3.3. Specific oxidative desulfurization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

4. Feasibility and economy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5915. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

1. Introduction

Combustion of sulfur-containing compounds in fossilfuels emits sulfur oxides, which can cause adverseeffects on health, environment and economy. Amongsulfur oxides, SO2 is abundant and is produced in thelower atmosphere. Furthermore, SO2 can be the cause ofsulfate aerosol formation. The aerosol particles have anaverage diameter of 2.5 μm that can be transported intothe lungs and cause respiratory illnesses (World Bank,1999; Atlas et al., 2001). SO2 can react with moisture inthe air and cause acid rain or low pH fogs. The acidformed in this way can accelerate the erosion of historicalbuildings. It can be transferred to soil, damage thefoliage, depress the pH of the lakes with low buffercapacity and endanger the marine life (EPA, 2006).

As SO2 is transported by air streams, it can be producedin one area and show its adverse impact in another remoteplace thousands of kilometers away. Therefore, to controlSO2 emissions, international cooperation is required. Since1979, Canada, the United States and the European nationsin particular have signed several agreements to reduce andmonitor SO2 emissions.Most of these agreements targetedtransport fuel because it was one of the important sourcesof SO2 emission. In 1993, the CleanAir Act proscribed thesales and supply of diesel oil with sulfur concentrationmore than 500mg/kg. In 1998, the EuropeanUnion set thetarget for sulfur content in diesel fuel at the levels of 350and 50 mg/kg for the years 2000 and 2005, respectively(Knudsen et al., 1999). Similar initiatives were taken inCanada and the United States to reduce the sulfur in dieselfuel and gasoline. New sulfur regulations took effect inCanada and theU.S. from June 1, 2006 to reduce the sulfurcontent in on-road diesel fuel and gasoline from500mg/kgand 350 mg/kg to 15 mg/kg and 30 mg/kg, respectively

(Avidan et al., 2001; EC, 2006). The maximum allowablesulfur content in diesel is targeted at 10 mg/kg by 2010 inthe US (Kilbane, 2006).

Sulfur removal is also important for the newgeneration of engines, which are equipped with nitrogenoxides (NOx) storage catalyst. However, sulfate pro-duced by sulfur in the fuel has poisoning effects on thecatalyst. Sulfate is highly thermostable and can saturatethe reduction sites on the catalyst. Therefore, the spaceavailability for the reduction of NOx decreases and thecatalyst loses its efficiency. To overcome this problem,treatment procedures must be devised or improved toreach a sulfur level less than 10 mg/kg (König et al.,2001; Song et al., 2000).

In general, two different methods can be used to lowerthe impact of sulfur oxide emissions: pre-combustionand post-combustion treatment methods. Pre-combus-tion treatment method has more advantages compared tothe post-combustion method. For instance, in the case offlue gas treatment, post-combustion method can beapplicable. However, this method deals with hotcorrosive effluents and it is expensive. Quality monitor-ing of the treated flue gas at every treatment location ispractically not possible. Furthermore, this method islimited and cannot be extended to all applications. On theother hand, pre-combustion treatment of fossil fuelreduces the sulfur oxide emissions regardless of the typeof the combustion process. Since desulfurization sites arenot as numerous, quality monitoring is much easier(Kilbane, 1989; Reichmuth, 2002).

Most sulfur in fossil fuel can be removed easily.However, there is one part, known as refractory organicsulfur, which is very difficult to remove. The currentmethods, which can remove the refractory part, operateunder extremely invasive conditions. They are very

Fig. 1. Chemical structure of typical organic sulfur compounds infossil fuel. The 4, 6-dimethyl dibenzothiophene (4, 6-DMDBT) isconsidered as a refractory alkylated DBT. The alkylated DBTs aredifferent in type of substituents, number of substituents and their bondposition on benzene ring (Kropp and Fedorak, 1998).

572 M. Soleimani et al. / Biotechnology Advances 25 (2007) 570–596

costly and produce considerable amount of carbondioxide. There are other methods, such as microbialdesulfurization, which have shown good potential forremoving refractory sulfur under mild conditions.However, all these methods have their own advantagesand disadvantages.

Microbial desulfurization is an environmentallyfriendly method that can remove sulfur from refractoryorganic compounds under ambient temperature andpressure without lowering the calorific value of the fuel.Such a feature has been the reason to conduct extensivestudies to develop methods by which desulfurizationunder mild condition can be viable. In this paper wepresent a comprehensive review on different types ofbiodesulfurization and focus on the advances taken andthe challenges involved on the most promising biode-sulfurization type. A concise description about thecurrent industrial desulfurization method and desulfur-ization by ionic liquids have also been provided.

2. Sulfur in crude oil and coal

Sulfur-containing compounds in crude oil and coalare generally divided into two major groups: inorganicsulfur and organic sulfur. The average amount of sulfurcontent in the U.S. coals has been reported to beapproximately 1.04–5.25%. In most samples, thepredominant portion of sulfur in coal is inorganic(pyritic), however, organic sulfur sometimes accountsfor more than 50% (up to 70%) of the total sulfur contentin coal. Pyritic sulfur makes up discrete physical entitieswithin coal and they might be from very big nodules tosub-micron crystal inclusions. Pyritic sulfur is effec-tively removed by various physical and chemicaltechniques such as heavy media separation, magneticseparation, leaching, selective agglomeration and floa-tation (Kawatra and Eisele, 2001). Organic sulfur in coalbelongs to thiols, sulfides, disulfides and thiophenes(Fig. 1). Application of physical methods to removeorganic sulfur from coal is impossible, because organicsulfur is finely distributed throughout the coal. (Kawatraand Eisele, 2001).

After carbon and hydrogen, sulfur is considered as themost abundant element in petroleum. Crude oils withhigher density contain more sulfur compounds. Distilla-tion fractions with higher boiling point contain higherconcentrations of sulfur compounds (Kropp and Fedorak,1998; Schulz et al., 1999). The average amount of totalsulfur in crude oil may vary from 0.03 wt.% to 7.89 wt.%.As in coal, organic and inorganic sulfur-containingcompounds naturally occur in crude oil. Inorganicsulfur-containing compounds include elemental sulfur,

H2S and pyrite. These compounds may be in dissolved orsuspended form. Organic sulfur compounds in crude oilare generally aromatic or saturated forms of thiols,sulfides and heterocycles. Among these, aromatic com-pounds such as dibenzothiophene (DBT) or its derivativesare of significant importance because they have higherboiling points (more than 200 °C) and it is difficult toremove them from atmospheric tower outlet streams (e.g.middle distillates) (Kawatra et al., 2001; Shennan, 1996).Benzothiophene (BT), non-β, single β and di-β-substi-tuted benzothiophenes (B.P.N219 °C) are the typicalthiophenic compounds that are found in diesel.

Among organic sulfur compounds some are consid-ered recalcitrant. These compounds are chiefly stablearomatic sulfur-containing compounds, which needmore invasive desulfurization procedure to removetheir sulfur atom. The refractory portion of distillate/diesel fuels is attributed to thiophenic compounds suchas DBT derivatives with 4 and/or 6 alkyl substitutinggroups (McHale, 1981).

3. Desulfurization methods

There are various desulfurization methods to removesulfur from fossil fuels. Among these, hydrodesulfur-ization (HDS) is currently considered as the mostimportant one. Desulfurization by ionic liquids (ILs) andbiological methods have also shown good potential to


be a plausible substitution for HDS or to be used in linewith HDS in future refining systems.

3.1. Hydrodesulfurization

Generally, removal of organic sulfur from fossil fuel isdifficult, because sulfur can only be detached from theorganic molecule when certain chemical bonds arecleaved. Breakage of suchbonds requires high temperatureand pressure. One of the current technologies to reducesulfur in middle distillate/diesel fuels is known ashydrodesulfurization (HDS). In HDS, the sulfur atom insulfur compounds is reduced to H2S on CoMo/Al2O3 orNiMo/Al2O3 catalyst in the presence of H2 gas. H2S is thencatalytically air oxidized to elemental sulfur. Depending onthe hydrocarbon type and degree of desulfurization, HDSmay occur at 200–425 °C and 150–250 psi H2. To reachlower concentration of sulfur (b15 mg/kg) highertemperature and pressure are required.

Although the concentrations of BT and DBT areconsiderably decreased by HDS (Monticello, 1998) and ithas been commercially used for a long time, it has severaldisadvantages: (i) For refractory sulfur compounds, HDSrequires higher temperature, pressure and longer resi-dence time. This makes the process costly due to therequirement of stronger reaction vessels and facilities(McHale, 1981); (ii) for older units, which are not compe-tent tomeet the new sulfur removal levels, erection of newHDS facilities and heavy load of capital cost is inevitable;(iii) HDS removes paraffinic sulfur compound such asthiols, sulfides and disulfides effectively. However, somearomatic sulfur-containing compounds such as 4- and 4,6-substituted DBT, and polyaromatic sulfur heterocycles(PASHs) are resistant to HDS and form themost abundantorganosulfur compounds after HDS (Monticello, 1998;Ma et al., 1994); (iv) The hydrogen atmosphere in HDSresults in the hydrogenation of olefinic compounds andreduces the calorific value of fuel. To increase the calorificvalue, the HDS-treated stream is sent to the fluid catalyticcracking (FCC) unit, which adds to the cost (Eβer et al.,2004; Hernández-Maldonado and Yang, 2004); (v)although HDS is considered a cost-effective method forfossil fuel desulfurization, the cost of sulfur removalfrom refractory compounds by HDS is prohibitive. Atlaset al. (2001) estimated the cost of lowering the sulfurcontent from 500 to 200 mg/kg to be approximately onecent per gallon. To reduce the sulfur content from 200 to50 mg/kg, the desulfurization cost would be four timeshigher.

The average sulfur content of the remaining crude oilis increasing (Kilbane, 2006). Therefore, in order toeconomize the desulfurization process and avoid CO2

production, resulting from energy intensive processessuch as HDS, milder desulfurization methods are to bedeveloped.

3.2. Desulfurization by ionic liquids

Ionic liquids (ILs) are organic salts that are in liquidstate at temperatures below 100 °C. Ionic liquids arepredicted to take the place of organic solvents, becausethey have no measurable vapor pressure below theirdecomposition temperature and can be designed to havedifferent properties depending on their structure.Desulfurization by ILs is based on extraction theoriesand it is a mild process. Organic ions in ILs can bedesigned in numerous varieties and combine together tomake practically unlimited number of ionic liquids(Freemantle, 2004). Among these, imidazolium basedionic liquids, such as [BMIM][PF6], [EMIM][BF4],[BMIM][MeSO4], [BMIM][AlCl4], [BMIM][OcSO4],have demonstrated a high selective partitioning forheterocyclic sulfur-containing molecules such as DBT,single β and di-β methylated DBTs.

Selection of ions for ionic liquids used in organicsulfur removal from fuel oils is very important. Some ofthe chlorometallate ILs such as the ones with [BMIM][AlCl4] show good selectivity for sulfur removal;however, they are very sensitive to air and moistureand may cause alkene polymerization in fuel (Haunget al., 2004). The size of anions in ILs was also found tobe rather important in extraction of DBT from oil phase.Bigger anions such as [OcSO4]

− could extract DBTsmore effectively than smaller anions (e.g. [PF6]

− or[CF3SO3]

−) (Bösmann et al., 2001). Eβer et al. (2004)have reported that imidazolium ions with larger alkylsubstitution groups are better solvents for DBT removal.However, they found that alkyl groups beyond certainsize lowered the selectivity. For instance, although[BMIM][OcSO4] demonstrated a high partitioning forDBTs, it could also considerably dissolve non-sulfurorganic molecules especially cycloalkanes and aro-matics. Therefore, it was not considered a selective ionicliquid for DBT compounds.

There is an increasing trend on desulfurization offossil fuels by ILs. The purpose of research on ILs infuture refineries is to economize desulfurization energyrequirements, and to decrease CO2 production that isassociated with other desulfurization processes such asHDS. The recovery and recycling of ILs duringdesulfurization process is difficult (Earle et al., 2006).Organic solvent extraction techniques can be used torecycle or recover ILs; however, loss of solvents duringthe extraction process is inevitable and undesirable.


3.3. Biological sulfur removal

One of the alternative options to remove sulfur fromfossil fuel is by biological methods. Sulfur atom forms0.5–1% of bacterial cell dry weight. Microorganismsrequire sulfur for their growth and biological activities.Sulfur generally occurs in the structure of some enzymecofactors (such as Coenzyme A, thiamine and biotin),amino acids and proteins (cysteine, methionine, anddisulfur bonds) (Kertesz, 1999; Stoner et al., 1990).Microorganisms, depending on their enzymes and meta-bolic pathways, may have the ability to provide theirrequired sulfur from different sources. Some microorgan-isms can consume the sulfur in thiophenic compoundssuch as DBT and reduce the sulfur content in fuel.Desulfurization by microorganisms is potentially advan-tageous. Firstly, it is carried out in mild temperature andpressure conditions; therefore, it is considered as anenergy-saving process (an advantage over HDS). Sec-ondly, in biological activities, biocatalysts (enzymes) areinvolved; therefore, the desulfurization would be highlyselective (an advantage over ILs). In this paper threedifferent types of biodesulfurization processes aredescribed: destructive, anaerobic and specific. Amongthese processes, specific biodesulfurization has gainedmore attention; therefore, a detailed review on thisdesulfurization type will be provided.

3.3.1. Destructive biodesulfurizationAs the first step in the development of biodesulfuriza-

tion process, many bacterial species that had the ability toconsume DBTs as their energy source were isolated fromtheir natural habitats. However, the first attempts forbiodesulfurization seemed to fail because the isolatedmicrobial species could not specifically remove sulfurfrom DBTs. Some of the isolated microorganisms coulduse thiophenic compounds as carbon and sulfur sources(Laborde and Gibson, 1997; Malik, 1980; Kirshenbaum,1961). Some other metabolized DBTs as carbon sourceand, in a series of oxidizing steps, converted them intoseveral water-soluble compounds (Kodama et al., 1970,1973; Yamada et al., 1968; Monticello et al., 1985). Theaccumulation of these water-soluble end products signif-icantly inhibited microbial growth and DBT oxidation.The target of the attack in this oxidation is one of thephenyl rings, which results in the breakage of one bond ora fragment from the phenyl ring. This microbial treatmentis not considered a sulfur removal approach becausesulfur is not specifically removed from the molecule.Aromatic compounds other than those having sulfur inthem can also be the target for the cleavage of aromaticbonds. Due to the undesired breakage of carbon–carbon

bonds in benzene rings, this type of desulfurization isconsidered to be a destructive process.

The oxidative and carbon-destructive series ofenzymatic actions that attack carbon atoms in DBTphenyl ring is known as Kodama pathway (McFarlandet al., 1998) and it consists of three main steps includinghydroxylation, ring cleavage, and hydrolysis (Gupta andRoychoudhury, 2005) as shown in Fig. 2.

Several different genera have been reported to carryout desulfurization through DBT carbon-destructivepathway. However, the majority of investigations havefocused on Pseudomonas cultures (Hartdegen et al.,1984). In 1985, Monticello et al. showed that Kodamapathway for DBT degradation was plasmid associated inat least two Pseudomonas species, P. alcaligenes andPseudomonas putida. These two species harbored asingle plasmid with an approximate molecular weight of55 MDa. The same plasmid was also reported to mediatethe biodegradation of other aromatic compounds such asnaphthalene and salicylate. In cases where MDBT wassubjected to Kodama desulfurization, it was found thatthe carbon–carbon cleavage occurred on the benzenering that had no substituting group to form correspondingmethyl 3-hydroxy-2-formyl-benzothiophene (methylHFBT). No significant carbon–carbon breakage wasobserved with 4,6-DMDBT (Saftić et al., 1993; Kroppet al., 1997). A 9.8 kbDNA fragmentwas found to encodethe dibenzothiophene degrading enzymes in Kodamapathway. The fragment was made up of ten open readingframes (ORF's) larger than 100 amino acids in length. TheORF C lacked a ribosomal binding site and it wasseemingly not a functional gene. Therefore, the functionalORF's were designated as doxABDEFGHIJ (dox forDBT oxidation). The same gene fragment was found tocontrol degradation of naphthalene and phenanthrene(Denome et al., 1993b).

3.3.2. Anaerobic biodesulfurizationSeveral anaerobic strains have demonstrated the ability

to remove organic sulfur, e.g. DBT. Kim et al. (1990)investigated the specific desulfurization by Desulfovibriodesulfuricans M6. This anaerobic strain could degrade96% and 42% of BT and DBT, respectively. Metaboliteanalyses proved that the strain could convert DBT tobiphenyl and H2S. Some anaerobic microorganisms, suchas Desulfomicrobium scambium and Desulfovibrio long-reachii, have been reported to have the ability to de-sulfurize only about 10% of DBT dissolved in kerosene.The GC analyses of samples showed unknown metabo-lites, indicating that the bacteria had possibly followed apathway different from common anaerobic pathways(Yamada et al., 2001).

Fig. 2. Kodama enzymatic pathway on dibenzothiophene. The oxidation in most cases reaches the step where 3-hydroxy-2-formyl-bnzothiophene(HBFT) and pyruvic acid (PA) are produced. The steps after production of HFBTare not fully known. HFBT is chemically unstable and it is probablymineralized in nature (Bressler and Fedorak, 2001).


Under anaerobic conditions, oxidation of hydrocar-bons to undesired compounds such as colored and gum-forming products is minimal (McFarland, 1999). Thisadvantage can be counted as an incentive to continueresearch on reductive biodesulfurization. However,maintaining an anaerobic process is extremely difficultand the specific activity of most of the isolated strainshave been reported to be insignificant for DBTs(Armstrong et al., 1995).

3.3.3. Specific oxidative desulfurizationThe process of sulfur removal by the Kodama pathway

was shown to be destructive as it was not specific in

removing the sulfur fromDBT thiophene ring. Therefore,attempts were made to isolate a variety of aerobic andanaerobic strains that could possibly remove sulfurnondestructively. All these attempts; however, were futileand gave rise to the isolation of more strains with carbon-destructive pathway (Stevens and Burgess, 1987).

Kilbane (1989) proposed a hypothetical oxidativedesulfurization pathway that, if ever existed in nature,could specifically remove sulfur from DBT. Thepathway was named as 4S and implied consecutiveoxidation of DBT sulfur to sulfoxide (DBTO), sulfone(DBTO2), sulfinate (HPBS) and hydroxybiphenyl(HBP) as shown in Fig. 3.

Fig. 3. Proposed sulfoxide–sulfone–sulfinate–sulfate (4S) pathway.All the metabolites are practically immiscible in water. DBT has thelowest (1.0 mg/L) and DBTO has the highest water solubility (320 mg/L) at 25 °C (Seymour et al., 1997). The released sulfate is consumed bythe cell through sulfur assimilation pathways (BBD, 2007).


Several researchers tried to isolate microorganismsthat could remove sulfur nondestructively. Afferden etal. (1990) isolated Brevibacterium sp. DO, which duringthe growth associated condition, could consume DBT asthe sole sulfur and carbon sources. The pathway of DBTdegradation by this strain was different from Kodama,and partly similar to 4S pathways. This bacterial straincould transform DBT to DBTO and then to DBTO2.Sulfur in DBTO2 was removed to form SO3

2− and SO42−

consecutively. With the removal of sulfur, DBTO2 wasconverted to benzoate and then was mineralized to waterand carbon dioxide (Afferden et al., 1990). Although thedesulfurization pathway was reported to be partlysimilar to the 4S pathway, it was still carbon destructivebecause the aromatic compounds were degraded duringthe desulfurization process.

An Arthrobacter sp. K3b (also known as DBTS2)was also reported to act similar to Brevibacterium sp.DO. This strain (Arthrobacter sp. K3b) could transform

DBTO2 (but not DBT) to sulfate and benzoate. Theproduced benzoate could then be used in the TCA cycle(Nojiri et al., 2001; Dahlberg et al., 1993). As in the caseof Brevibacterium sp. DO, the desulfurization processby Arthrobacter sp. K3b was also carbon destructivebecause of similar reasons mentioned above.

A group of fungi e.g. Cunnighamella elegans wasshown to have the ability to oxidize sulfur in DBT toDBTO and then to DBTO2. However, with these fungalstrains no further degradation was noted and sulfurcould not be detached (Holland et al., 1986; Crawfordand Gupta, 1990). DBT non-specific desulfurization ofcoal by a mixed culture of yeasts (Hansenula spp.) wasreported by Stevens and Burgess (1989). The mixedculture could increase the desulfurization activity by thesecretion of a surfactant-like matrix.

The performed research proved that there was nonatural specificity for the oxidation of C–S bonds.Therefore, in 1990, The Institute of Gas Technology(IGT), Chicago, IL attempted to develop new strains viaunnatural selective mutation. In this attempt, IGT used amixed culture soil sample, which was contaminated withsulfur-containing hydrocarbons. The sample was addedto a continuous coal bioreactor where it was exposed to amutagen (NTG; 1-methyl-3-nitro-1-nitrosoguanidine).After several months it was observed that a certain groupof microbial species could survive in the bioreactor.These species showed specific C–S incision activity andthey could remove 90% of organic sulfur from coal. Thismicrobial group was named as IGTS7 (Kilbane, 1990;Kilbane, 1992b; ATCC, 2007). IGTS7 consisted of atleast 7 different colony forms with different populations.Among these seven colony types, two strains of Rho-dococcus rhodochrous and Bacillus sphaericus, whichhad the lowest abundance, proved to have desulfuriza-tion capabilities. These two mutant strains wereconsequently named as R. rhodochrous IGTS8 (ATCC53968) and B. sphaericus IGTS9 (ATCC 53969)(Monticello and Kilbane, 1994; Kilbane, 1996). Differ-ent similar species from American Type CultureCollection (ATCC) were examined and compared withthese two strains. The comparison showed that these twostrains had a unique capability of specific sulfur removalthat could not be observed in their similar species. IGTS8and IGTS9 had a slower growth rate in comparison withthe other members of IGTS7. The co-cultivation ofIGTS8 with Enterobacter agglomerans (a biodesulfur-ization-negative member of IGTS7) with only DBT asthe sole sulfur source showed growth fluctuation. TheIGTS8 population was higher at the beginning of growthand it initially had a faster growth rate. However, after alag, the Enterobacter species could grow faster than

1 Greek; erythro-: red, -polis: city.


IGTS8 and formed the predominant species of the mixedculture. The result explained that the sulfate ion formedduring 4S pathway was released into the growth mediumand could be assimilated by other microorganisms(Kilbane and Bielaga, 1990). Kayser et al. (1993) alsoreported similar growth with different population ratio ofIGTS8 to Enterobacter cloacae (a desulfurizationnegative species). The lag time of E. cloacae was longerwhen the initial (time zero) ratio of E. cloacae to IGTS8increased. Final population ratio of E. cloacae to IGTS8in mixed culture also depended on their initial ratio. Theco-cultivation of IGTS8 with E. cloacae could notincrease desulfurization activity and the highest sulfurremoval activity was observed with pure culture IGTS8.Kilbane and Jackowski (1992) cultivated IGTS8 strainon water-soluble coal derived compounds. The treatmentshowed a reduction of 30–40% of organic sulfur in 24 h.Carbon to sulfur and Btu to sulfur ratios in thisinvestigation proved that sulfur could be removed fromDBTwhile the calorific value remained constant. It wasreported that desulfurization of solubilized coal wouldresult a higher sulfur removal. A higher desulfurizationyield was observed with biological solubilization thanthe one with chemical solubilization (Baek et al., 2002).To date, several genera have been reported to havespecific desulfurization activity. However, most of thembelong to Rhodococcus genus.

3.3.3.1. Microbiology of Rhodococcus genus. Thegenus Rhodococcus belongs to the phylum and class ofActinobacteria, order of Actinomycetales, and family ofNocardiaceae (NCBI, 2007). Rhodococci possess avariety of plasmids from small circular to large andlinear ones (Dosomer et al., 1988; Kayser, 2002), and incomparison to most bacteria, they have a thick cellenvelope, which may give higher resistance for theassimilation of required compounds (Carrano et al.,2001). Rhodococci cells are hydrophobic because ofpeptidoglycan layer of long aliphatic mycolic acid chainsin their cell wall. Taking advantage of the hydrophobicity,Rhodococci can attach to oil/water interface whilegrowing in aqueous–hydrocarbon system (Neu, 1996;Borole et al., 2002). All Rhodococcus species form cocci,rods and extensively branched mycelia with commonlyno aerial hyphae. With the exception of some strains,which require thiamine, Rhodococci grow on standardcultivation media at 30 °C, and they require 1–3 days toform visible colonies on solid media. Rhodococci areGram-positive, obligate aerobes, catalase-positive, par-tially lysozyme sensitive, non-motile, non-endospore ornon-conidia forming bacteria. They are chemoorgano-trophic and have oxidative type of metabolism.

3.3.3.2. Rhodococcus erythropolis IGTS8. R.erythropolis1 was first isolated from soil (Goodfellow,1992; Finnerty, 1992; Larkin et al., 1998) and it isclassified as a saprophyte Rhodococcus. It has no aerialhyphae and similar to all other species of Rhodococci itis an aerobe. Many well-known desulfurization compe-tent microorganisms, e.g. IGTS8, N1-43, D-1, KA2-5-1and I-19, are strains of R. erythropolis. Among theseIGTS8 has been studied most extensively. R. erythro-polis IGTS8 was isolated by Kilbane and Bielaga (1990)and was used by Energy Biosystems Corp. (EBC) forthe development of their commercial microbial desul-furization plan. The strain IGTS8 is a Gram-positive rodshaped bacterium with approximate length of 0.5 μm.On nutrient agar medium IGTS8 forms cream colorcolonies. After some time, however, they turn intopeach-colored colonies.

The strain R. erythropolis IGTS8, for a while afterbeing patented, was not fully identified; therefore, it wasreferred to as Rhodococcus sp. IGTS8. To determine thespecies of IGTS8, its membrane lipids were extracted bysolvent and analyzed by gas chromatography. Thecomparison of the results with available chromatogramsfrom a variety of species proved the strain to belong torhodochrous species (Kilbane, 1992a,b). The strain wasregistered by the name R. rhodochrous IGTS8 and wasdeposited with the ATTC. In 1997, nucleotide sequenceanalysis of 16S rRNA operon from this strain proved itto be a R. erythropolis IGTS8 (GenBank accession no.AF001265). However, as this strain is a patentedculture, in some publications it is still referred to as R.rhodochrous IGTS8 (Bressler et al., 1998; ATTC, 2007;NCBI, 2007).

The desulfurization genes of IGTS8 are located on aplasmid. The IGTS8 desulfurization trait, and thereforeits desulfurization plasmid, is stable. The desulfurizationtrait of the strain is fully maintained by subculturing.Furthermore, heat treatment of the strain has shown noimpact on loss of plasmid. However, 5 to 20 s of UVemission (254 nm at 3.5 cm distance) to IGST8 mayhave an adverse effect on its sulfur removal capability.Rhodococcus sp. UV1 is a UV desulfurization negative(dsz-) mutagenized strain of IGTS8, which has lost itsability for specific desulfurization. UV1 strain wasprepared for genetic and cloning studies on IGTS8(Denome et al., 1993a). IGTS8 is the first stable carbon–sulfur cleaving bacteria. Available records show thatprior to IGTS8, Isbister and Doyle (1985) had isolatedthe strain Pseudomonas sp. CB1 (ATCC 39381), whichcould degrade DBT by incision of carbon–sulfur bond.


Pseudomonas sp. CB1, however, lost its sulfur specificdesulfurization trait and it could no longer besubstituted.

3.3.3.3. Molecular biology of specific desulfurization.Since the isolation and identification of R. erythropolisIGTS8, considerable research has been conducted onmolecular biology of this and other similar strains, inorder to obtain a better control over the machinery ofspecific sulfur removal. The first questions that shapedin mind regarding the desulfurization genes were asfollows. (i) Was the sulfur removal controlled bygenomic DNA or a plasmid was involved in desulfur-ization? (ii) What was the size of desulfurization DNA?(iii) Could the genes be transferred to other strains? (iv)How many genes were involved in specific desulfur-ization? (v) If desulfurization was controlled by morethan one gene, what was the duty of each one? (vi)Under what conditions could the genes be induced orrepressed?

In 1993, Denome et al. (1993a) isolated a highmolecular weight plasmid pSOX from IGTS8 cells. Theplasmid was partially digested by the restrictionendonuclease into ∼20 kb fragments. The fragmentswere then ligated into the cosmid pLAFR5. Theconstructed cosmid was later transduced into E. coliS17-1. The cloned cosmids were isolated and electro-porated into Rhodococcus sp. UV1 (an IGTS8 mutantwithout desulfurization ability). After electroporation,few UV1 colonies gained desulfurization morphologyand could transform DBT to DBTO2 and 2-HBP. Resultsfrom the pulsed-field gel electrophoresis showed that thedesulfurization genes were located on a plasmid.However, the cosmid transferred into UV1 (recipientstrain) could not be recovered because it had integratedinto the chromosomal DNAof UV1. To narrow down theDNA region in which the desulfurization gene waslocated, the chromosome-integrated cosmid wasdigested by various restriction enzymes and the obtainedfragments were individually transformed into UV1. Thecolonies that could demonstrate desulfurization traitcarried the desulfurizing DNA fragment. In this way, thetarget DNA was cleaved shorter by various restrictionenzymes until a 4.0 kb BsiWI–BstBI fragment (theshortest desulfurization positive fragment) was reachedwhich could give desulfurization morphology to UV1.

The transduction of a plasmid that carried 4.0 kbBsiWI–BstBI fragment to E. coli S17-1 gave nodesulfurization morphology to the strain (Denome et al.,1993a). Later on, it was found that a lac promotercontrolled the desulfurization gene andE. coli could showdesulfurization phenotype with the same gene in opposite

direction (i.e. BstBI–BsiWI). Therefore, the orientation ofligated desulfurization DNA to lac promoter was crucialfor E. coli to demonstrate desulfurization morphology(Denome et al., 1994). The first attempts to expressdesulfurization genes by transforming desulfurizationpositive plasmid into R. fasciance were unsuccessful.Rhodococcus species appeared to have some endonucle-ase restriction systems, which could cleave the SalIrecognition sites in the transformed plasmid. Transfer ofmethylated plasmid into R. fasciance could blind therecognition sites and gave desulfurization trait to thespecies (Denome et al., 1993a).

The DNA sequence of BstBI–BsiWI fragment,which was responsible for the desulfurization or sulfuroxidation trait in IGTS8, encoded three open readingframes (ORF's). This proved that sulfur oxidation ofDBT was controlled by three sulfur oxidation (sox)genes, which in order of their location on the fragmentwere called as soxA, soxB, and soxC. To specify theresponsibility of each gene in DBT desulfurization,soxC, soxA, and soxAB were cloned in E. coli. Thespecies was consequently grown on DBTand DBTO2 asthe sole sulfur substrates. The strain containing soxCcould convert 98% of DBT to DBTO2. However, nofurther sulfur oxidation to produce 2-HBP was ob-served. This expression confirmed that the gene soxCwas involved in the first steps of specific desulfurization(DBT–DBTO2 conversion). Although the E. coli,which had soxA, could consume DBTO2, no 2-HBPwas detected. The species retaining both soxA and soxBcould not oxidize DBT but could oxidize DBTO2 to 2-HBP. The results obtained by these experiments provedthat both soxA and soxB were responsible for theoxidation of DBTO2 to 2-HBP. The gene soxAconverted DBTO2 to an intermediate metabolite,which was the substrate for soxB enzyme. The genesoxB could finally oxidize the intermediate metaboliteto 2-HBP. The desulfurization genes and their encodedenzymes are commonly indicated as sox. The abbrevi-ation sox was later replaced by dsz (desulfurization)(Denome et al., 1994). The dsz genes and their proteinswere compared in databases such as GenBank 83,EMBL 39 and Swiss-Prot 28. As the search showed nosignificant homology, it was deduced that the desulfur-izing genes dszA, dszB and dszC encoded undiscoveredenzymes (Piddington et al., 1995). Denis-Larose et al.(1997) reported that the three dsz genes in IGTS8 werelocated on a 150 kb mega-plasmid. This plasmid wasearlier reported to be 120 kb by Denome et al. (1994).

To develop the biodesulfurization process, it wasimportant to know under what conditions the desulfur-ization genes were expressed or repressed. Li et al.


(1996) investigated the effect of various sulfur sourcessuch as dimethyl sulfoxide (DMSO), cysteine, methio-nine, and sulfate on dsz gene expression. In a set ofbatches, IGTS8 was grown in media whose sulfur sourcewas 15 mM of DMSO plus increasing amounts ofcysteine, methionine or sulfate. The desulfurizationexpression was measured after 48 h. The results showedthat desulfurization activity decreased when cysteine,methionine or sulfate concentration in the media in-creased. In comparison, methionine had a strongerrepression effect. When concentration of these inhibitorsreached above 375 μM, desulfurization activity wasstrongly repressed. The batches with desulfurizationphenotype, dszA, dszB and dszC proteins could clearlybe observed on Coomassie blue SDS-polyacrylamideelectrophoresis gel. However, in batches with nodesulfurization activity (the ones with Cys, Met, orsulfate repression) dsz protein bands were non-existent.The differences on gels could be a sign of dsz generepression caused by other sulfur sources such as sulfate.This repression was found to be due to binding arepressor protein next to the dsz promoter, which waslocated within 385 bp upstream of dszA. Deletion anal-ysis indicated that the promoter was localized to theregion from −121 to −44. Apart from promoter, the385 bp fragment had at least three dsz affecting elements(some overlapping the promoter region). The regionfrom −263 to −244 proved to reduce dsz repression.However, deletion of the region did not affect repressionof expression. The region from −144 to −121 could binda protein such as an activator. The region between −98and −57 could be a repressor binding site (Li et al.,1996).

3.3.3.4. Enzymes involved in specific desulfurization.The three genes in IGTS8 encoded three desulfurizationenzymes, known as dszABC. The response of theseenzymes, or the cell free extract, to some sulfur-contain-ing substrates in some cases proved to be different fromthe cell theywere originated from. It was observed that thepresence of sulfate in DBT desulfurization by cell freeextract had no inhibition effect on the desulfurizationenzymes. However, sulfate ion could suppress desulfur-ization when the whole cell was used for sulfur removal.For instance, resting cells of Rhodococcus sp. SY1 werereported to have no DBT desulfurization ability in amedium containing sulfate (Omori et al., 1995). Ohshiroet al. (1996a,b,c) studied the effect of sulfate on DBTdesulfurization activity withwhole cells ofR. erythropolisD-1. A concentration of 0.3 mM of sulfur sources such asDBT, DBTO2, methanesulfonic acid, sodium sulfate, anddimethyl sulfone could all support growth (approx. an

average of 3.4 at OD660). However, enzymatic desulfur-ization activity was only detected in cultures with DBTorDBTO2 as their sole sulfur source (approx. 1.5–1.9 nmol/min/mg by Bradford assay). In media containing sodiumsulfate, methanesulfonic acid or dimethyl sulfone nodesulfurization activity was detected. Addition of 0.1 mMof another sulfur source (sodium sulfate or methanesul-fonic acid) to DBT in the media had no negative effect ongrowth. However, the levels of detected desulfurizingenzymes were considerably lower (approx. 0.32 nmol/min/mg by Bradford assay). Sodium sulfate concentra-tions of 0.5 mM and methanesulfonic acid concentrationsof 1 mM completely stopped specific activity. Sulfaterepression effect could be observed when sulfateconcentration was higher than 0.02 mM (Ohshiro et al.,1996a,b,c). Accumulation of 2-HBP was extremely toxicfor both growth and desulfurization activities. Increase of2-HBP from 0.0 to 1.0 mM decreased the relativedesulfurization activity of cell free extract from 100% to20%. Growth ofR. erythropolisD-1 was inhibited with 2-HBP concentrations higher than 0.2 mM. The growth wasfully suppressed with concentrations as much as 0.6 mMof 2-HBP (Ohshiro et al., 1996a).

Ohshiro et al. (1994, 1995b) had noticed that whendesulfurization enzymes were used in vitro, theydemonstrated a very low activity. They showed that indialyzed cell free extract NADH, FAD or FMN wasrequired for DBT desulfurization. At 35 °C, the additionof FAD or FMN to desulfurization enzymes, up to10 μM, had an increasing effect on enzymatic activity.The peak of activity was observed at 10 μM. HigherFMN or FAD concentrations decreased the desulfuriza-tion activity. FAD or FMN concentrations up to 1 mMcompletely inhibited the activity of the enzymes. Duringpurification process of desulfurization enzymes, bothdszC and dszA enzymes lost their activities. However,the enzymes activation could be regained after theaddition of the Rhodococcus extract with no dszC anddszA. Therefore, dszC and dszA were assumed torequire some other factor for their activity. Dependenceof dszC and dszA activity on an “FMN reduction andNADH oxidation” system indicated that an NADH:FMN oxidoreductase (also called as flavin reductase)had to be involved in desulfurization pathway to providedszC and dszA enzymes with the reduced flavin. TheNADH:FMN oxidoreductase, which is called frdA(purified from IGTS8), was reported to have a molecularweight of 25 kDa on SDS-PAGE. Comparison of aminoacid sequence of this enzyme with available proteindatabase showed no considerable homology. The novelpurified enzyme was colorless indicating that it did nothave any chromophore such as FMN (Gray et al., 1996;


Brenda, 2007). Deleting frd gene gave rise to loss ofdesulfurization trait in IGTS8. However, the strain couldgrow in sulfate containing media. Matsubara et al.(2001) purified and characterized NADH:FMN reduc-tase of R. erythropolis D-1. This enzyme, as in IGTS8,contained no chromophore. The molecular weight ofthis enzyme, obtained by gel filtration, was determinedto be 86 kDa. SDS-PAGE run showed that this enzymewas a homotetramer, consisting of four identical 22 kDasubunits. The enzyme's optimum pH and temperaturewere reported to be 6.0 and 30 °C and it was stronglyinhibited in the presence of heavy metals such as Ag+,Cu2+ and Hg+. The enzyme was stable below 50 °C andcould keep its activity after 30 min of heat treatment at80 °C. The specific activity and N terminal amino acidsequence of flavin reductase originated from both D-1and IGTS8 were significantly similar to an extent thatthey could be considered identical enzymes (Matsubaraet al., 2001).

Gray et al. (1996) studied the desulfurizationkinetics with crude desulfurization enzymes of IGTS8and DBT. Kinetic studies on the conversion of DBT toDBTO and then DBTO2 with the addition of NADH,FMN and flavin reductase revealed that the conver-sions were catalyzed by dszC, in two consecutive steps(DBT→DBTO→DBTO2). The conversion of DBT toDBTO had a constant rate of 0.06/min while constantrate of DBTO to DBTO2 was 0.5/min that was almostten times faster. It was reported that during the con-version of DBTO2 to 2-HBP an intermediate com-pound (HPBS) was accumulated. The production rateof HPBS from DBTO2, which was catalyzed by dszA,was approximately five times faster than its consump-tion rate to 2-HBP (catalyzed by dszB). As describedearlier (Lei and Tu, 1996), presence of DBTO wasdifficult to detect because it was readily consumed.Therefore, conversion of HPBS to 2-HPB was regardedas the slowest step in 4S pathway (Gray et al., 1996).The purified desulfurization enzymes had no absorp-tion in the visible region. Therefore, it was deducedthat they had no chromophore (Tanner et al., 1996; Leeet al., 1995; Ohshiro et al., 1997, 1999).

In many enzymatic redox reactions, transformationof one target molecule to another may depend on tworedox functions in series. One single enzyme or twoloosely associated ones may be in charge of the targetreaction (Vetrova et al., 2007). Oxidative conversions ofDBT→DBTO and DBTO→DBTO2 occur through twoloosely associated oxygenases; one NAD(P)H:FMNoxidoreductase and one final monooxygenase (dszC ordszA). In DBT/DBTO enzymatic oxidation by dszC/dszA and O2, one oxygen atom is transferred to the

substrate and the other is reduced to H2O. There isevidence that both NADH and NADPH can be used inDBT desulfurization by R. erythropolis D-1 and IGTS8.Desulfurization with presence of NADH has shown tobe 10 times more effective in R. erythropolis D-1 (Xiet al., 1997).

The purified dszC enzyme showed to have a peak ofabsorption at 281 nm. No absorption peak was observedin the region 300–700 nm. Since flavin and heme bothhave absorption peaks in 300–700 nm, it was presumedthat no such prosthetic group was bound to dszC enzyme.However, dependence of dszC enzyme on FMN (flavinmononucleotide) was evident. It was observed that up to79% of DBT could be converted to DBTO2 when DBTwas in contact with dszC enzyme, FMN, NADH andflavin reductase P (or NADH:FMN oxidoreductase) in airsaturated 5 mM phosphate buffer (pH 7.0). Incubation ofDBTO and dszC enzyme with the same conditions led tothe full conversion of DBTO to DBTO2. This might beone of the reasons why no intermediate could be found inDBT desulfurization pathway. When flavin reductase P(FRP) was in solution, it could provide FMNH2 toenhance dszC enzyme activity. The obtained results fromDBT and DBTO conversion to DBTO2 proved that dszCenzyme was a catalyst for DBT and DBTO conversion toDBTO2 and supported the idea that DBTO was anintermediate of DBT to DBTO2 oxidation (Lei and Tu,1996; Tanner et al., 1996). The optimum activity of dszCoriginated from D-1 was reported to have stable activityup to 40 °C (Ohshiro et al., 1997, 1999). Denome et al.(1994) reported that dszC obtained from IGTS8 was a45.0 kDa enzyme with 417 amino acids. Lei and Tu(1996) overexpressed dszC gene in E. coli, lysed the cellsand purified the dszC enzyme by batch adsorption, phenyland DEAE sepharose. The gel filtration chromatographyof dszC enzyme showed a molecular weight of 86 kDa,which was approximately twice as much as the onereported earlier (Piddington et al., 1995; Denis-Laroseet al., 1997). The HPLC size exclusion chromatographyshowed that, dszC (DBT monooxidase) was a tetramerand had a molecular weight of 180 kDa (Gray et al.,1996).

In 1994, Denome et al. (1994) determined that dszAof IGTS8 was a 49.6 kDa protein with 453 amino acids.The dszA (DBTO2 monooxidase) was in fact a dimerwith a molecular weight of 100 KDa (Gray et al., 1996).Ohshiro et al. (1999) determined the dszA molecularweight in the strain D-1 to be 97 kDa with two 50 kDasubunits. As in dszC, the enzyme dszA requires NADH,FMN and flavin reductase to catalyze DBTO2 intoHPBS. The turnover number and Km for dszA are 1 s−1

and 1 μM, respectively. The reaction rate of dszA was


reported to be 5 to 6 times higher than dszC (Gray et al.,1996). The optimum dszA activity, originated from thestrain D-1, was found to be at 35 °C and pH 7.5. Theoptimum temperature of the flavin reductase requiredfor the dszA activity was the same as the one for dszA.However, flavin reductase showed its highest activity atpH 6. The activity of flavin reductase at dszA optimumcondition (T=35 °C, pH 7.5) was 80% of its activity atpH 6 (Ohshiro et al., 1999).

Addition of metal ions, Fe3+, Fe2+, Cu+, to the pureenzymes dszA and dszC did not increase the desulfur-ization rate. The desulfurization rate was not eitherrepressed or inhibited by the addition of EDTA. Theresults showed that these enzymes had no metal cofactor(Li et al., 1996; Gray et al., 1996). Similar experimentson dszA, purified from the strain D-1, proved that dszAactivity was inhibited by 8-quinolinol, p-chloromercur-ibenzoic acid (PCMB), EDTA and 2,2′-bipyridine.Presence of PCMB inhibited the activity of flavinreductase; however, no inhibition was observed byEDTA (Ohshiro et al., 1997).

The dszB (HPBS desulfinase) is regarded as the lastenzyme in 4S pathway. The dszB in the strain IGTS8was determined to be 39.0 kDa with 365 amino acids(Denome et al., 1994). HPLC size exclusion chroma-tography also confirmed that dszB (HPBS desulfinase)was a monomer (Gray et al., 1996). The enzyme dszBwas determined to have a turnover number of 2/min,indicating that the dszB was the rate-determiningenzyme. Protein bands on SDS-10% polyacrylamidegel showed that in IGTS8, dszB is expressed less thandszAC. The dszB originated from R. erythropolisKA2-5-1 was also purified. The enzyme subunit onSDS-PAGE was determined to be 40 kDa. Gel filtrationshowed the molecular weight of the enzyme to be43 kDa, which proved that this enzyme, like dszB ofIGTS8, was a monomer. Similarity of molecular weight,identical N terminal sequence (verified up to the first 23amino acids) could suggest that the dszB enzymes inKA2-5-1 and IGTS8 were identical. The optimumactivity of this enzyme was determined to be at 35 °Cand pH 7.5. The enzyme demonstrated heat stability upto 28 °C. However, temperatures above 30 °C drasti-cally decreased the enzyme's relative activity (Naka-yama et al., 2002). The dszB required no metal ions forits activity. Cysteine proved to be associated withcatalytic function of dszB, as the enzyme was stronglyinhibited by the presence of 1 mM of some reagentssuch as Ag+, Cu2+, Hg2+, or N-ethylmaleimide. Theoptimum temperature, pH, Km, and kcat for dszB (HPBSdesulfinase) of IGTS8 were reported to be 35 °C, 6.0–7.5, 0.90 μM, and 0.022 s−1, respectively (Watkins

et al., 2003). The Km was found to be somewhatdifferent from what was reported by Gray et al. (1996).No metal was detected to enhance HPBS desulfinaseactivity; however, Cu2+ and Zn2+ proved to besignificant inhibitors (Watkins et al., 2003). Inhibitionof 2-HBP is due to ortho-hydroxyl group. Therefore,biphenyl or 4,4′-DHBP has no or negligible inhibitionto dszB. 2-HBP(N0.5 mM) and its derivatives such as2,2′-DHBP and 2,3-DHBP proved to be competitiveinhibitors for the substrate HBPSi and had KI values of0.25, 2, 0.4 mM, respectively (Nakayama et al., 2002).

A recent report on dszB crystal structure revealedthat the overall fold of dszB enzyme (EC 3.13.1.3)resembles the ones of periplasmic substrate bindingproteins. Cysteine modifying reagents and hydroxylgroup on biphenyl inhibit dszB activity. The dszB isapproximately 60×50×40 Å, monomeric and ovalshaped and belongs to (or evolved from) the phosphatebinding proteins. The active site cavity of dszB includesCys and a glycerol molecule. The glycerol molecule isestimated to be responsible for protein stability. ThedszB overall structure and the proposed catalystmechanism have been described by Lee et al. (2005b).

3.3.3.5. Development of specific desulfurization pathway.With increasing the knowledge about the desulfurizationenzymes, reactants and intermediate substrates, themachinery of the pathway could be better understood.In 1993, Olson et al. (1993) characterized the DBTdesulfurization intermediates, which were produced byIGTS8. The analysis of the GC/FID and GC/FTIR/MSchromatograms revealed the abundance of DBT and 2-hydroxybiphenyl. However, smaller and less noticeablepeakswere also observedwhich could show the formationof some other metabolic intermediates. The mainintermediates in this characterization were detected asDBTO, DBTO2, 2′-hydroxybiphenyl-2-sulfonate(HBPSo), and 2′-hydroxybiphenyl-2-sulfinate (HBPSi)(Fig. 4).

The transformation sequence was proposed to occurthrough R1, R2 and R3 in stationary phase. It wassuggested that IGTS8 cells could not grow on sultine asthe sole sulfur, and growing cells could transform DBTvia the reactions R1, R4, R5 and R6. In this study therewas not enough evidence to support R7 occurrence.

Gallagher et al. (1993) carried out a set of IGTS8cultivations with different sulfur sources but identicalbasal salt mixture, carbon source (glycerol), and othergrowth conditions. The sulfur sources considered for thegrowth included sulfate ion, DBT, DBTO, DBTO2,sultine or sultone and only a single sulfur compound waspresent in each sample run. The samples were taken at

Fig. 4. Modified 4S desulfurization pathway (Gallagher et al., 1993; Olson et al; 1993). Samples were detected at growth stationary phase. With DBTas the sole sulfur source, small amounts of DBTO and sulfonate (HBPSo) were detected. However, the main product was determined to be 2-HBP.When DBTO was used as the sole sulfur source, considerable amount of DBTwas detected in samples. The result supported the idea that R1 was anequilibrium reaction. Small amount of sulfonate (HBPSo) but very large amount of 2-HPB was also detected. In samples with DBTO2 as the solesulfur source, small amounts of DBTO, HBPSi, HBPSo but very large amount of 2-HBP were detected. Therefore, R4 was deduced to be anequilibrium reaction. With sultine, large amounts of HBPSi, HBPSo and 2-HBP were observed in samples. Presence of sultone only gave rise toconsiderable production of 2-HBPSO. It has been observed that in acidic conditions, HBPSi and HBPSo shift, under equilibrium, towards theproduction of sultine and sultone, respectively. 2,2′-HBP was only detected in exponential phase sample with sultone as the sole sulfur source.


stationary phase. The growth trend with sulfate ion,DBT, DBTO2 and sultone were similar and exponential(0.06–0.04 h−1 approx.; 12–17 h). However, IGTS8demonstrated faster growth in presence of richer media.Cultivation of IGTS8 with sultine demonstrated nogrowth. A weak growth (more linear than exponential)was observed for the IGTS8 cultivation with DBTO.Linear growth was attributed to the limitation caused bysulfur source or enzymatic kinetic rate. Based on theanalysis of the data obtained a modified 4S pathway wasproposed (Gallagher et al., 1993).

Although in most cases selectivity of enzymaticreactions in an advantage; however, in biodesulfurization,specific sulfur removal from broader range of sulfurcompounds is desired. Kayser et al. (1993) showed thatDBT, DBTO and DBTO2 could be metabolized to 2-HBP.

Thianthrene could also be desulfurized and form 2-hydroxy diphenyl sulfone and phenol. Diphenyl sulfoxideand 2-aminophenyl disulfide, showing some inhibitoryeffects, could be used as sulfur sources. Sulfur removal inall these compounds occurred through carbon–sulfurcleavage. Some sulfur compounds such as 1,3-dithiane,1,3-propanethiol, thianaphthene, tolyl disulfide, phenylsulfoxide, and sulfanilamide proved to be specificinhibitors for IGTS8 growth even in the presence ofDBT. IGTS8 could grow in the presence of the specificinhibitors if sulfate ionwas added to themedium; however,specific desulfurization could not be induced. Addingmethionine, thiamine, cystine, and L-cysteine as the solesulfur source could not support growth. DBT desulfuriza-tion by IGTS8 was reported to occur in the presence ofsulfate ion. However, sulfate concentrations above 20 mM

Fig. 5. The 4S pathway dependingmay end upwith the production of 2-HPBor 2,2′-DHBP. ThemechanismofHBPSi toHBPSo is not completely known(Oldfield et al., 1997). It is also believed that 2,2′-DHBP is produced during growing of the cells and not in stationary phase (Gallagher et al., 1993).


caused inhibition on DBT desulfurization. The minimumamount of sulfur required for bacterial growth ranges from0.25 to 1 mM, which causes no inhibition for DBTdesulfurization (Kilbane, 1992b).

It was known that bacterial strains such as IGTS8required oxygen for their metabolic activities. In order tofind the origin of oxygen atoms in conversion of DBT toDBTO and DBTO2 with dszC, a 50:50% supply of

16O2:18O2 was fed to the system. The formation of

DBT16O2, DBT18O2 and DBT16O18O supported theconcept that oxygen atoms from two different oxygenmolecules were involved in DBT oxidation (Gray et al.,1996). The study of dszA catalysis with 18O2 andDBTO2 proved that oxygen gas is involved inconversion of DBTO2 to HBPS. The mass spectroscopydata revealed that the phenolic oxygen on HBPS was


18O, indicating that the reaction catalyzed by dszA wasan oxygen dependent step. Therefore, no sulfoneoxygen could be rearranged and transferred to benzenering carbon after C–S bond cleavage. Incubation of R.erythropolis IGTS8 with 35S labeled DBT released35SO3 in medium which proved that the 35SO3 ion wasoriginated from the labeled sulfur in [35S]DBT (Oldfieldet al., 1997). Stoichiometrically, the overall reaction of4S pathway, which ends up with the production 2-HBP,maybe summarized by the following reaction (Fig. 5):

DBT þ 4NADH þ 2Hþ þ 3O2→HBPþ 4NADþ þ SO−2

3 þ 3H2O: ð1ÞThe first step of the reaction (at neutral pH), which iscatalyzed by dszC, may be written as:

DBT þ 2NADH þ 2Hþ þ 2O2→DBTO2

þ 2NADþþ2H2O: ðdszCÞð2Þ

The function of dszC in specific desulfurizationpathway is clear and all studies conducted so far agreethat the conversion of DBT to DBTO2 is NADH-dependent and FMNH2 is also involved in reactions as aco-substrate. The produced DBTO2 may end up withDBTO2→HBPSi−→HBP or DBTO2→HBPSi−→ -HBPSo→BPSo→DHBP. When DBTO2 reaction isdriven towards the production of HBP the followingstoichiometry is expected (to show the charge balanceclearly, a negative charge is shownwithHBPSi orHBPSo).

DBTO2 þ 2NADH þ 2Hþ þ O2→HBPSi−

þ 2NADþH2O ðdszAÞð3Þ

HBPSi− þ H2O→HBP þ 2Hþ

þ SO−23 : ðdszBÞ

ð4ÞDBTO2 conversion in favor of DHBP is expected to

occur as follows.

HBPSi−→HBPSo−ðMechanism not fully knownÞ ð5Þ

HBPSo− þ Hþ→BPSo þ H2O ð6Þ

BPSo þ 2NADH þ O2→DHBP þ 2NADþ

þ SO−23 : ðdszAÞ

ð7ÞSulfite ion produced in 4S pathway cannot be

metabolized by IGTS8. Setti et al. (1994) showed that

IGTS8 could not grow in a medium with sulfite ion asthe sole sulfur source. However, sulfate ion couldstrongly support growth. Some researchers believe thatDBTO2 is converted to sulfonate by direct oxygenationand then it can convert to HBP or DHBP under acidic orbasic condition.

In cultivation of IGTS8 with DBT, DBTO andDBTO2 very small amount of sultine (BPSi) is detected.However, due to instability of sultine in neutral pH, it ishydrolyzed to HBPSi− in equilibrium reaction. The genedszA is responsible for the conversion of DBTO2→ -HBPSi−. Presence of sultone (BPSo), as an intermedi-ate, was also detected in DBT desulfurization by IGTS8.The whole cell IGTS8 can specifically remove sulfurfrom sultone and convert it to DHBP. Therefore, thegene dszA is responsible for two reactions: DBTO2 toHBPSi− and BPSo to DHBP (Olson et al., 1993;Oldfield et al., 1998).

The equilibrium reaction of HBPSo to BPSo can beshifted in favor of the production of BPSo at pH 8.0 and30 °C. Activity of dszA in the reaction BPSo→DBHPcan also shift the equilibrium HBPSo↔BPSo towardsthe production of BPSo. The mechanism of HBPSi− toHBPSo conversion required more investigation (Mon-ticello et al., 1997; Ohshiro et al., 1999).

In the pathway that leads to the production of HBPSiand HBP, dszA was proposed to have binding sites forNADH, FMNH2 and DBTO2. An oxygen moleculereacts with FMNH2 to make FMNH 4a-hydroperoxide(FMN-OOH). An NADH molecule releases a hydride(H:−) ion, which can target FMNH-OOH and decom-pose it to FMNH 4a-hydroxide (FMN-OH) andhydroxide ion. The hydroxide ion can attack C–Sbond in DBTO2 and cleave it to HBPSi

−. The FMN-OHloses an H2O and converts to FMN, which can in turnreact with oxygen molecule again (Oldfield et al., 1997).When O2 concentration in reaction mixture is high,FMNH2 reoxidation may occur non-enzymatically. Inorder to enhance enzymatic FMNH2 reoxidation and tosuppress oxygen non-enzymatic competition, the dis-solved oxygen must be kept at very low concentration(McFarland et al., 1998). The dszB was proposed to be anucleophilic enzyme, which can target water molecule.The hydroxide ion released by this reaction can attachsulfur atom in HBPSi− to form a sulfonate branch.Sulfonate is a weak base and acts as a leaving group(HSO3

−). Therefore, it is detached from the rest of themolecule and leaves a carbanion intermediate. Thecarbanion can in turn obtain an H+ from the protonateddszB (Fig. 6) (Oldfield et al., 1997).

The dszB was proposed to be a nucleophilic enzyme,which can target water molecule. The hydroxide ion

Fig. 6. The proposed mechanism for the conversion of DBTO2 to 2-HBP. The conversion of DBTO2 to HPBSi and HBPSi to 2-HBP is catalyzed bydszA and dszB, respectively (Bressler et al., 1998; Oldfield et al., 1997).


released by this reaction can attach sulfur atom inHBPSi− to form a sulfonate branch. The sulfonate,which is a weak leaving group base and acts as a goodleaving group (HSO3

−), is detached from the benzenering in HBPSi− and leaves a carbanion intermediate.The sulfonate can in turn obtain an H+ from theprotonated dszB (Oldfield et al., 1997).

3.3.3.6. Process kinetics and dynamics. The researchon process kinetics of specific biodesulfurization wasfirst focused on solid–aqueous phase systems such ascoal–water or DBT–water. In most experiments, DBTin the form of fine particles, was used as the modelorganic sulfur and was contacted with aqueous phasecontaining desulfurizing bacteria or enzyme (Sagardia etal., 1975; Finnerty, 1993). The research on biodesulfur-ization gradually shifted towards sulfur removal frombenzothiophenic compounds in liquid hydrophobicmedia such as diesel. In this section both solid–aqueousand oil–aqueous phase systems are discussed.

In DBT–aqueous phase system, the growth rate ofIGTS8 proved to be dependent on the type of carbonsource and on the concentration of carbon and sulfursources. It was observed that succinate was a bettercarbon source for the growth of IGTS8 and the straincould start growth after a shorter lag time and reach thestationary phase faster. Although IGTS8 growth seemedto increase when further DBTwas added to the aqueous

medium, the effect of DBT concentration on growth wasless than the effect of carbon source concentration. Theincreased growth due to adding more DBT to themedium was attributed to increase of DBT solid surfacearea and higher solid–liquid mass transfer rate. A fastergrowth was observed when DBTO2, which is moresoluble in water, was used as the sole sulfur sourceinstead of DBT. The production of HBP was observed tobe growth associated and followed the same trend asgrowth did (Setti et al., 1994).

Wang and Krawiec (1996) studied the desulfurizationprocess of fine DBT particles by three different R.erythropolis strains (N1-36, N1-34, andQ1a-22) in batchand fed-batch cultures. The organic sulfur in the form ofDBT or DBTO2 (0.1 mM) was added as fine crystals inenmeshed nylon filters. The temperature, agitation, andaeration rates were set at 30 °C, 300 rpm and 1.5 vvm,respectively. Process pHwasmonitored and controlled at6.0. The initial glucose concentration in batch culturesand working glucose concentration in fed-batch culturewas 3.0 g/L. With the strain N1-36, within 10–35 h offermentation, small amounts of DBTO but no amount ofDBTO2 could be detected. For all the strains used, 2-HBPwas found to be the predominant converted productand it could be detected at early exponential phase. Thestrains N1-36 and Q1a-22 had the highest growth ratewithin pH 6.0 to 8.0. For N1-36 the highest cellpopulation, the highest desulfurization, and the highest


specific desulfurization occurred at pH 6.5, 6.0 and 5.5,respectively. Although at pH 5.5 the process had a peakof specific desulfurization, the cell growth was extreme-ly low. The specific growth rates of N1-36 with DBTandDBTO2 as sulfur source were determined to be 0.153 h−1

and 0.180 h−1, respectively. In these experiments, asreported before by Setti et al. (1994), a better growth anddesulfurization rate was observed with DBTO2. DBT orDBTO2 desulfurization was observed to repress when200 μM MgSO4 was present in the media (Wang andKrawiec, 1996).

Wang et al. (1996) investigated the kinetics ofDBTO2 desulfurization with R. erythropolis N1-36 in a355 mL continuous culture. In this process DBTO2 wasconsidered as the limiting substrate and the desulfuriza-tion at ten different dilution rates was examined.Throughout the steady state run the temperature wasmaintained at 30 °C, which was the best growth temper-ature. Maximum specific growth rate and saturationconstant of the strain were determined to be 0.2 h−1 and0.39 μM DBTO2, respectively μ. Activation energy ofthe process was also obtained by running the steady stateculture at different temperatures using Arrhenius equa-tion. Dilution rate for different temperatures was set at0.12 h−1, which was in the safe margin of steady stateregion (below μmax).

Ohshiro et al. (1995a,b) carried out a set ofexperiments with R. erythropolis H-2 to investigatedesulfurization of DBT dissolved in various hydropho-bic solvents. It was noted that R. erythropolis H-2 couldconvert DBT to 2-HBP even when DBT was dissolvedin a hydrophobic phase. Relatively long chain alkanes(e.g. hexadecane) supported the growth of the strainwhile other hydrocarbons such as shorter alkanes, p-xylene, styrene or toluene were toxic to the strain re-gardless of adding glucose to the media. Presence ofsome other hydrocarbons in desulfurization media couldimprove DBT sulfur removal. For instance, DBT desul-furization was the highest with 40% w/v of kerosene inthe medium. The best operating temperature for R.erythropolis H-2 was found to be 37 °C. Temperaturesbeyond 40 °C had a strong negative effect on DBTdesulfurization (Ohshiro et al., 1996a,b,c).

The desulfurization activity of R. erythropolisIGTS8, as a biocatalyst in the DBT–hexadecane system,was found to follow the first order decay with decayconstant of 0.072 h− 1. The decay constant wasdetermined for the hydrocarbon to aqueous phase ratioof 1:1 v/v at 2 vvm, 30 °C and pH 7. Respiratoryquotient by off-gas analysis in continuous desulfuriza-tion (constant desulfurization activity) was determinedto be 0.52, which was close to the theoretical value of

0.57 when glucose was used as substrate (Schillinget al., 2002).

As explained before in this paper, biodesulfurizationmay occur with cell free extract or pure enzymes;however, use of the whole cells as biocatalyst is a morereasonable approach for DBT specific desulfurization.Purifying enzymes and providing NADH and FAMH2

for the reaction is costly. The desulfurization activity ofthe whole cell biocatalyst (0.4 gDBT removed/gdwbiomass h) has been shown to be higher than that of thecell free extract (0.01 gDBT removed/g protein h) (Settiet al., 1997).

To further enhance the biodesulfurization activity bywhole cell, a high cell density (with resting cells of freshor freeze-dried cells) may be contacted with organicsulfur. Sandip et al. (1997) studied biodesulfurization inan aqueous–hydrocarbon phase by lyophilized IGTS8.In this system, freeze-dried IGTS8 cells, which wererevived in the minimum amount of water was theaqueous phase, while the DBT dissolved in hexadecanewas the hydrocarbon phase. It was noted that IGTS8 lost20% of its desulfurization activity after freeze-drying;however, after 10 weeks of storage at −80 °C, theactivity decreased only by 2.8%. Use of surfactants inaqueous–hydrocarbon system increased the desulfuriza-tion yield. However, it was found that the desulfurizationefficiency depended on the addition order of hydrocar-bon, water (including basic salt medium), surfactant andbacteria. The highest desulfurization yield was obtainedwhen freeze-dried bacteria were added to the emulsifiedmixture of hydrocarbon, water and surfactant. Amountof water used to revive the lyophilized IGTS8 also turnedout to be of high importance. When the strain was notfully hydrated (less than 1.25 mL/gdw of cells),desulfurization activity of IGTS8 considerably declineddue to insufficient water concentration. However, incases where the strain was completely revived (1.25 mL/gdw of cell), IGTS8 maintained its desulfurizationactivity for very high hydrocarbon/water w/w% ratio(even up to 90%).

The strain, Nocardia globerula R-9, has also showndesulfurization phenotype in water–hydrocarbon phase.Mingfang et al. (2003) used the freeze-dried strain toinvestigate the kinetics of DBT sulfur removal. The Km

and Vmax values for the desulfurization of DBTwith thisstrain were determined to be 0.7 mmol dm−3 and11.0 mmol sulfur kg−1 h−1, respectively. The Km andVmax were obtained under one to one ratio of dodecane:water biphasic condition with 100 mg freeze-dried cellin 5 cm3 phosphate buffer at 30 °C and pH 7.0.

As biodesulfurization by the high density of restingcells was much more efficient, several researchers began


to work on various methods by which they could obtainthe highest cell density. The cells produced could thenbe harvested and used in biodesulfurization as restingcells. Honda et al. (1998) investigated the effect of ahigh cell density IGTS8 culture on DBT desulfurization.The strain IGTS8 was first grown in a 1 L bioreactorwith fed-batch (FB) medium (containing sulfate ion assulfur source) at pH and temperature of 7.0 and 30 °C.Different carbon sources were tried with FB feed. Thereactor was operating under cascade control mode tomaintain dissolved oxygen level at 2–5 mg/L. Someorganic acids such as acetic acid could effectivelysupport IGTS8 growth. A buffer mixture (acetic acid/ammonium acetate) was fed to the system both as acarbon source and as a pH-adjusting agent. No growthwas observed when pyruvate or formate was present inthe medium as the carbon source. Ammonium andacetate could support growth; however, growth wasdrastically inhibited when ammonium or acetate con-centration reached 3 g/L. The yield of YX/acetate andYX/NH3

were calculated to be 0.6 gdw cells/g acetate and0.67 gdw cells/g NH3. The highest biomass productionwas observed when the feed had a ratio of 1.72 g aceticacid per g acetate. In this condition, a biomass density of33 g dry weight/L was reported which could conse-quently be used for DBT desulfurization in a sulfate freemedium. Although fresh medium was fed to the system,growth stopped after 28 h due to some unknown reason.It was reported that the DBT desulfurization trait couldbe induced in the rinsed and centrifuged biomass uponincubation for 3–4 h in a medium with DBT as the solesulfur source. The IGTS8 cultivation, under the samegrowth condition but with DBTas the sole sulfur source,could produce a much lower biomass density of1.1 gdw/L. The reason for such a considerabledifference in biomass density due to the change insulfur source was attributed to the production of HBPin the medium. Growth inhibition was observed at0.2 mM HBP concentration (Honda et al., 1998).

In another attempt to reach a high cell density ofdesulfurizing bacteria, Chang et al. (2001) obtained acell concentration of 92.6 g/L with a different strain,Gordonia nitida CYKS1. The induction time when theywere transferred to a DBT containing medium was 4 h(close to what was reported by Honda et al. (1998) withIGTS8). In another set of experiments, R. erythropolisKA-2-5-1 was used as a desulfurization strain. It wasfound that the cultivation with ethanol as the carbonsource could result in a cell density of 37 g/L and aspecific desulfurization activity of 135.5 mmol HBP kgdry cell weight−1 h−1. Comparison of glucose, glyceroland ethanol as carbon source showed that 0.1% w/v

ethanol is metabolized by R. erythropolis KA-2-5-1more easily. It was assumed that consumption of ethanolas carbon source might supply more NADH to providethe required FMNH2 for desulfurization (ethanol+NAD+→ acetaldehyde +NADH+H+) (Yan et al.,2000). Ethanol concentration above 1.0% was reportedto decrease both specific growth rate and enzymespecific activities and proved to be toxic to cells (Wangand Krawiec, 1996).

Konishi et al. (2005) studied the effect of exponentialfeed on a fed-batch culture of R. erythropolis KA2-5-1to obtain a high cell density. The initial volume was 2 Lout of 5 L of bioreactor volume. In this cultivation,ethanol concentration (the carbon source) was main-tained at 1 g/L and DBT was linearly fed (0.5 g/L) for24 h. After this period, DBT was fed exponentially(Fn+ 1=Fn exp(μsΔt)) with μs of 0.03–0.21 h−1. Atsmaller exponential coefficient, μs, higher specificdesulfurization rate was observed although the growthrate was slow because of sulfur limitation. With μs of0.03–0.06, the specific desulfurization was detected tobe 120–130 mmol 2-HBP (kg cell)−1 h−1. Feeding withhigher μs (0.12 h−1) gave rise to a significant decline inspecific desulfurization activity. The reason for thisrepression was attributed to the presence of excess sulfurin the growth medium.

Resting cells have also been used for biodesulfuriza-tion of crude oil or FCC fraction. In such hydrocarbonphases, DBT may be present in several alkyl substitutedforms, which are generally shown as CX-DBT. Forinstance, C1-DBT refers to a methylated DBT wheremethyl group is on 1, 2, 3 or 4 carbon position of thebenzene ring; C2-DBT refers to an ethylated DBT or abi-methylated DBT where the substituted group islocated on 1, 2, 3, or 4 carbon position of the benzenering. In hydrotreated FCC branch, DBT is mostly inalkylated form. Folsom et al. (1999) used a partlyhydrotreated FCC sample to study the biodesulfuriza-tion by R. erythropolis I-19 (a genetically modifiedstrain of IGTS8). The strain I-19 demonstrated a higherspecific desulfurization rate because of overexpressionof dsz enzymes in its cells. For desulfurization thebiocatalyst phase (12.5 g dry weight cell of I-19/L) wascontacted with FCC samples in a 3:1 water to oil ratio.The sample contained mercaptans, sulfidic compounds,DBT and alkylated CX-DBTs. Among dibenzothio-phenes, DBT concentration had the lowest and C2-DBT isomers had the highest concentrations (C2-DBTNC3-DBTNC1-DBTNC4-DBTNC5-DBTNDBT).It appeared that desulfurization of different DBTs didnot occur simultaneously. The desulfurization startedwith DBT and other middle boiling point sulfur


compounds and then it shifted toward C1-DBTs whenDBT concentration was decreased. After 3 h when themajority of DBT and C1-DBT were desulfurized, sulfurremoval in heavier alkylated DBTs was detected. Thedesulfurization rates were also different (initial desul-furization rate: C2-DBTNC1-DBTNC3-DBTNC4-DBTNC5-DBT). It was proposed that the desulfuriza-tion rate was a function of concentration and degree ofalkylation (Folsom et al., 1999).

The resting cells of R. erythropolis KA2-5-1, isolatedby Petroleum Energy Center, Tokyo, Japan (PEC), wasalso reported to decrease total sulfur of a hydrotreatedgas oil from 900 mg/kg to 310 mg/kg (Ohshiro andIzumi, 1999). This strain, KA2-5-1, contained identicaldsz genes with the ones found in IGTS8 and it couldspecifically remove sulfur from DBT and its alkylatedhomologs dissolved in n-tetradecane. The desulfuriza-tion activity was reported to depend on size, locationand number of alkylation. The highest desulfurizationactivity of 74.2 mmol kg dry cell−1 h−1 was observedfor DBT while the lowest desulfurization activity of8.68 mmol kg dry cell−1 h−1 was observed for 3,4,6,7-tetramethyl DBT (Kobayashi et al., 2000). Largersubstituents proved to have a stronger negative effecton desulfurization. For instance, the relative desulfur-ization activity of KA2-5-1 resting cells with 4-MDBT,4,6-DMDBT, 4-Propyle-DBT, 3,4,6-TMDBT, 4,6-DEDBT, 3-propyle-4,8-DMDBT was approximately78%, 55%, 41%, 37%, 27%, and 14%, respectively.However, no desulfurization was detected with 4,6-dipropyl DBT or 2-n-hexyl DBT. Almost similardesulfurization properties have been observed in otherstrains such as Rhodococcus sp. KT462 (Tanaka et al.,2002), and Mycobacterium phlei GTIS10 (Kayser et al.,2002).

Desulfurization of DBTalkylated homologs by KA2-5-1 cell free extract was reported to occur approximatelyat the same time and desulfurization was found to followthe Michaelis–Menten kinetics. The biodesulfurizationkinetic constants of Km and Vmax were obtained whenKA2-5-1 resting cells (8.75 g dry cell/L) were contactedwith equal volume of n-tetradecane in which a mixtureof DBT, 4-MDBT, 4,6-DMDBT, 3,4,6-TMDBT wasequally dissolved (each 0.78 mM). The Vmax linearlydecreased from 54.5 to 10.8 mmol kg dry cells−1 h−1

from DBT to 3,4,6-TMDBT. The value of Km increasedwith alkylation number of DBTs in the mixture, from0.55 to 0.65 mM (Kobayashi et al., 2000).

Desulfurization of crude oils with frozen paste of R.erythroplis IGTS8 was reported in a crude oil:water(1:3) system. The crude oils selected for the experimentswere originally different but they were set to have

identical concentrations of DBT derivatives. With thesame operating conditions, the rate of sulfur removal ineach crude oil sample was different and this differencewas due to the presence of specific inhibitors in crudeoils. Furthermore, the desulfurization yield per gram drycell was approximately one tenth of the value for thecondition in which a DBT/hydrocarbon phase was usedin lieu of crude oil. To avoid the formation of extremelyviscose paste in crude oil, which could be caused bystabilization of asphaltene groups with buffer salts,surfactant (0.2 mL Petrolite MU2000/L) was added tothe reactor (Kaufman et al., 1999).

R. erythropolis XP (Yu et al., 2006), a strain isolatedfrom soil, was reported to have the ability to removerefractory sulfur compounds in hydrotreated diesel oilwithout the adverse impact of toxic component reportedby Kaufman et al. (1999). The specific desulfurizationof this strain was determined to be 4 μmol (gdw cells)−1

h−1 and the final concentration of recalcitrant sulfur waslowered from 259 mg/kg to 14 mg/kg (94.5% reduction)after 24 h. This concentration could potentially meet thedefined requirements for ultra low sulfur fuels.

In a bigger scale, a decent bioprocess design must beconsidered for biodesulfurization. One of the earliest pilotplants for DBT desulfurization was reported by EnergyBiosystems Corp. (EBC). In this process R. erythropolisIGTS8 was used as the microbial strain. The processbegan with STRs batch experiments using different waterto oil ratios from 0.1:1.0 to 5:1 with a capacity of 0.5–5barrels of oil per day. Then the batch mode operationswere further developed to CSTR operations. Temperatureand pH of the process were controlled at 30 °C and 6–8,respectively. Water to oil ratio was 3:1 and the reactiontime was 1 h. The specific productivity of biocatalyst wasobtained to be 8–30 mg HBP/h/g. However, McFarlandet al. (1998) reported that a productivity of 600 mg HBP/h/g is required to compete with other desulfurizationmethods. After the STR reaction time, the biphasic har-vest was transferred to a centrifuge to separate the oil andaqueous phase. The oil phase left the unit as the product.The aqueous phase, containing biocatalyst, was sent to asulfate precipitation unit where the produced sulfate ioncould be removed from the aqueous phase in the form ofgypsum. The aqueous phase containing negligibleamount of sulfate was then recycled back to the reactor.

To increase the mass transfer efficiency between thetwo phases and to promote the desulfurization activityfor a given strain, a fine emulsion might be produced. Insmall scale, there are many efficient methods to emulsifytwo immiscible phases. In larger scale, preparing anemulsion system that can be quickly separated is not aneasy feat. In most cases, to achieve such an emulsion,


considerable amount of power in the form of mixing orpumping is dissipated. Many studies have been carriedout to obtain finer emulsion systems with less powerconsumption. Electrically driven emulsion phase con-tactors (EPC) are special contactors that focus electricalforce on interface of immiscible phases to disperse thenonconductive phase into fine droplets (Scott andWham, 1989). Kaufman et al. (1997) took advantageof this property to emulsify aqueous and DBTcontaininghexadecane phase using a pulsed DC high voltage (up to40 kV). In this reactor the hexadecane/DBT was thecontinuous phase on which the aqueous/IGTS8 phasewas sprayed. Droplets produced by EPC had averagediameters of 1–10 μm and 30–100 μm. The biggerdiameters were believed to form during sampling. Theamount of aqueous phase required in this method is lessthan 5%. Therefore, for a given volume of EPC,considerably higher volumes of hydrocarbon phasecould be treated. The fine droplets reduce the desulfur-ization mass transfer limitations. The power consump-tion in the EPC was about 3 W/L, which is considered tobe very low. This bioreactor was introduced by OakRidge National Laboratory (ORNL).

Fine emulsion increases biodesulfurization yield(Borole et al., 2002); however, the separation ofhydrocarbon and water phase takes a long time and itis also difficult to recover hydrophobic biocatalyst fromhydrocarbon phase without loss (Yu et al., 1998). Oneof the possible ways to maintain the cell level in thereactor may be by immobilization. The immobilizedcells can then be used in different operation modese.g. fluidization. Argonne National Laboratory (ANL)introduced a fluidized bed reactor (FBR) in whichgasoline was aerated in another unit outside FBR. Thebiocatalyst was immobilized on hydrophilic beads andwas fluidized by oxygenated gasoline. The streamleaving the FBR was driven to a solid–liquid cyclone.The treated gasoline could totally leave the cyclone as thefinal product or could be partly recycled back to theFBR. This system was also equipped with a biocatalystregeneration system (McFarland et al., 1998).

Naito et al. (2001) reported that they could entrap R.erythropolis KA2-5-1 in a photo cross-linkable resinprepolymer known as ENT-4000. By this method theynot only maintained biomass concentration in thereactor, but they also managed to increase longevity ofthe biocatalyst to 900 h. The longest longevity reportedby that time was 150 and 192 h. Desulfurization by un-immobilized resting cells had a higher average desul-furization rate. The slower rate observed by immobili-zation was attributed to diffusion limitations. However,as no water was involved, the separation was much

easier and no cell leakage was reported out of the ENT-4000 entrapment.

Lee et al. (2005a) investigated diesel oil desulfuriza-tion in a combination of air-lift/stirred tank reactor,which consisted of two coaxial cylinders. Air wassparged in the inner cylinder, which acted as a riser. Ashaft with two impellers was installed in the innercylinder: one axial type impeller in the lower part of theriser to help better flow and one disk type impeller ontop of the riser to mix the phases. The downcomer, thespace between inner and outer cylinder, was filled witheight strings of nylon fibers in which living cells of G.nitida CYKS1 were immobilized. The process wascontrolled at 30 °C and pH 7; however, no furtherinformation was reported regarding the mixing speed,amount of air supplied, aqueous to hydrocarbon ratio orcycle time. This process proved to reduce the sulfurcontent in diesel oil from 202–250 mg/kg to 76–90 mg/kg in 72 h. Since living cells were immobilized in nylonfibers, population of suspended cells increased by time.

A two-step biodesulfurization process has beenreported for Rhodococcus globerulus DAQ3 that wasfound to have a high stability in aqueous–hydrocarbonsystems. The desulfurization in the first step took 24 h.The hydrocarbon phase and aqueous phase (containingDAQ3) were then separated. The aqueous phase wasdriven to a fresh medium to be regenerated. Therecovered hydrocarbon phase was sent to the secondbiodesulfurization unit to be contacted with fresh restingcells. This procedure reduced the activity inhibitionduring DBT sulfur removal. The cell density, pH,temperature, hydrocarbon to aqueous phase ratio andreaction time in each step were 20 g/L, 7.0, 30 °C, 1:10,and 24 h, respectively (Yang and Marison, 2005).

3.3.3.7. Research on improving specific desulfurization.During recent years, the research on improving theefficiency of biodesulfurization has increased. Thisresearch mostly focuses on three general areas: (i)designing or isolating new strains with higher specificactivity for broader range of refractory compounds, (ii)designing or isolating new strains with higher hydrocarbontolerance, and (iii) designing or isolating new strainswhich are able to desulfurize refractory compounds underthermophilic conditions (e.g. 50 °C).

DBT by itself is not counted as a recalcitrant sulfur-containing compound, because it can be easily removedby HDS. However, some of its derivates, such as 4,6-alkylated DBTs, are extremely difficult to remove. Theresearch on the potential approaches to desulfurize thesecompounds will have an important effect in futurerefractory compounds desulfurization (Monticello,


1998). In 1995, Lee et al. (1995) reported the isolationof an Arthrobacter strain that could specifically cleavesulfur from sterically hindered DBTs. In this research4,6-diethyldibenzothiophene (4,6-DEDBT) was used asthe sulfur source and it was converted to 2-hydroxy-3,3′-diethylbiphenyl (HDEBP). Apart from 4,6-DEDBT, other organic sulfur compounds such asDBT, DBTO2, 4,6-DMDBT, dimethyl sulfoxide andphenothiazine could be metabolized by this species. Nogrowth was observed when sulfur sources such asthiophene, 2-phenylthiophene and 2-(1-naphthyl) thio-phene were added as the sole sulfur source. The lack ofgrowth proved that the added organic sulfurs could notbe metabolized by the Arthrobacter species. When DBTor 4,6-DEDBT was separately added to the growthmedium, they were consumed by the same rate. In caseswhere both DBT and 4,6-DEDBT were present in themedium, DBT was first metabolized by Arthrobacterstrain. Consumption of 4,6-DEDBT started when themajor part of DBT was oxidized. This preference couldbe due to the fact that sulfur atom in DBT is easilyaccessible, while in 4,6-DEDBT sulfur is hindered withalkyl groups.

The strain R. erythropolis H-2 proved to have arelatively broad range of biodesulfurization. The straincould grow in a medium with DBT derivatives such as3,4-benzoDBT, 2,8-DMDBT, 2,8-DMDBT, and 4,6-DMDBT as the sole sulfur source and specificallydesulfurize them (Ohshiro et al., 1996a,b,c). The strainR. erythropolis T09 was claimed to have the capabilityto desulfurize benzothiophenic compounds even in thepresence of DBTs. This strain could grow well in sulfursources including BT, 3-MBT, 5-MBT, and thiophene 3-carboxilic acid. However, compounds such as 7-ethylBT, 2,7-diethyl BT, and 5,7-diethyl BT, DBT or DBTO2

weakly supported the growth (Matsui et al., 2000).Grossman et al. cultivated Rhodococcus sp. strain

ECRD-1 in a medium with hydrocarbon:water ratio of1:4. The hydrocarbon phase was a hydrotreated middledistillate oil with the total sulfur content of 669 mg/kg.The oil had only 5% of DBT and the majority of theremained 95% was alkylated DBTs. The final sulfurconcentration was detected to be 56 mg/kg after 7 daysof cultivation in rotary shaker at 25 °C, pH 7(controlled), and 200 rpm (Grossman et al., 2001).

A bacterial strain Microbacterium sp. ZD-M2 wasisolated from sludge, which could desulfurize DBT, 4,6-DMDBT, BT, thiophene and diphenylsulfide (each0.2 mM). Thiophene, BT, DBT, and 4,6-DMDBT weretotally desulfurized in 46, 72, 58, and 72 h, respectively.The strain could desulfurize 70% of diphenylsulfidewithin 72 h. The difference in desulfurization activities

supports the probability that dissimilar enzymes mightbe involved in substrate transformation (Li et al., 2005).

Until 2006, no microbial strain was observed to havethe ability to desulfurize benzothiophenic compoundswith large alkyl substituents, such as 2-n-hexyl DBT.Recently, a Gram-negative strain, Sphingomonas sub-arctica T7b, was isolated in Japan and was found tohave the ability to desulfurize alkylated BT's with longalkyl chains, such as 4,6-dipropyl DBT, 4,6-dipentylDBT, 4-hexyl DBT, and 5-dodecyl BT (Gunam et al.,2006).

Biodesulfurization with high hydrocarbon phasetolerance is considered an advantage because lessamount of water is required for biodesulfurization. R.globerulus DAQ3, which was recently isolated from oil-contaminated soil, demonstrated the highest specificDBT desulfurization activity and stability in oil:watersystems. A model diesel with 1452 μM DBT/hexade-cane was contacted with the same volume of 10 gdwcells/L of aqueous phase. It was reported that restingcells of DAQ3 could transform 92% and 100% of DBTin 9 h and 24 h, respectively. Desulfurization withIGTS8 at identical condition resulted in 29% and 32%desulfurization. The DBT desulfurization by DAQ3proved to be significantly higher than the one reportedby IGTS8 (Yang and Marison, 2005).

In desulfurization of petroleum, there are manydifferent compounds (solvents) that have inhibitory effecton desulfurization competent strains. Pseudomonas spp.was found to be an ideal candidate for biodesulfurizationin petroleum, because they are organic solvent tolerantand have a high growth rate. With the properties noted,dszABC genes from R. erythropolis XP was cloned intoP. putida Idaho to construct a solvent-tolerant, desulfuriz-ing P. putida A4. This strain, when contacted with sulfurrefractory compounds dissolved in hydrocarbon solvent,maintained the same substrate desulfurization traits asobserved in R. erythropolis XP. Resting cells of P. putidaA4 could desulfurize 86% of DBT in 10% (v/v) p-xylenein 6 h. In the first 2 h, the desulfurization occurred with arate of 1.29 mM DBT (gdw cell)−1 h−1. No DBTreduction was noticed when the experiment was repeatedwith R. erythropolis or P. putida Idaho at identicalconditions (Tao et al., 2006).

Biodesulfurization is preferred to occur underthermophilic conditions, because it enhances the biode-sulfurization rate and the operating temperature wouldbe closer to FCC or HDS outlet streams. Highertemperature decreases oil viscosity and makes moleculardisplacement easier. The first thermophilic strain withdesulfurization trait, Paenibacillus sp. A11-2, wasisolated by Ishii et al. (2000). The strain proved to


have a different desulfurization gene cluster (tdsABC),which was 73%, 61% and 52% homologous withdszABC genes.

Bacillus subtilis WU-S2B and M. phlei WU-FI arealso thermophilic strains that can specifically cleavecarbon–sulfur bond of DBT and its alkylated homologsup to 52 °C. The DBT desulfurization gene cluster inWU-S2B was identified and named as bdsABC. TheDNA and amino acid sequencing of bds genes with theones of IGTS8 showed 61.0% homology (Kirimura etal., 2004).

To increase the biodesulfurization activity, Reich-muth et al. (1999) studied the desulfurization ability of acloned E. coli DH10B strain that contained the plasmidspDSR2 and pDSR3. The plasmid pDSR2 contained aVibrio harvey NADH:FMN oxidoreductase gene whilepDSR3 encoded enzymes, which converted DBT toHBP. In plasmid pDSR3 the native desulfurizationcontrol element had been removed. Therefore, E. coliDH10B/pDSR3 could show its desulfurization trait evenin the presence of sulfate ion or undefined rich mediasuch as LB. When E. coli DH10B/pDSR3 was placed ina medium with DBT as the sole sulfur source,desulfurization activity stopped at 0.2 mM HBP. Therepression was attributed to insufficient oxidoreductasesupplied by E. coli DH10B/pDSR3. As in IGTS8, E.coli, has an intrinsic NADH:FMN oxidoreductase.However, the oxidoreductase level proved to beinsufficient for overexpressed dszABC. Desulfurizationactivity was tested with a cloned E. coli DH10B/pDSR2/pDSR3; however, no significant desulfurizationactivity change was observed under different amounts ofinducers.

In search for the development of a method to providerequired supply of reduced flavin to DBT oxygenationsystem, Galán et al. (2000) used hpaC flavin reductaseoriginated from E. coli Wand contact it, in vitro, with asystem of dszABC purified enzymes and an NADHsource. They also used catalase in the desulfurizationmedium to minimize the probability of H2O2 formation,which might be produced by non-enzymatic reoxidationof FMNH2 under high oxygen concentration. Additionof hpaC flavin reductase increased DBT desulfurization7–10 times in 30 min. The enzyme hpaC flavin re-ductase and the oxidoreductase originated from IGTS8were from the same subfamily of flavin:NAD(P)Hreductase. To verify if hpaC flavin reductase couldincrease desulfurization in resting cells, a recombinantGram-negative strain, P. putida KTH2/pESOX3, wasdesigned to contain both dszABC and hpaC genes. Toeliminate the effect of sulfate inhibition resulted by DBTdesulfurization, the dszABC gene cluster in pESOX3

was designed to express under the control of the Ptacpromoter. Results obtained from desulfurization byKTH2/pESOX3 strain showed that the bacteria couldkeep its desulfurization phenotype even in sulfatecontaining media. Designing a plasmid, which encodesright amount of FMN:NADH reductase to dszABCenzymes is crucial to reach an optimum desulfurizationpotential in this bacterium. Insufficient FMN:NADHreductase would make NADH supply as the limitingstep in DBT oxidation. On the other hand, highconcentration of FMNH2 will give rise to H2O2

formation, which would be lethal to cells (Gaudu etal., 1994; Galán et al., 2000).

4. Feasibility and economy

Inorganic sulfur removal can be carried out bydifferent methods. Although several biological desul-furization methods e.g. by Gram-negative Thiobacillusferrooxidans, have been known (Detz and Barvinchak,1978; Kopacz, 1986) for inorganic sulfur removal, otherphysical methods are efficient and economical (Richard-son, 1981; Kargi and Robinson, 1982).

The organic sulfur compounds are divided in twogroups: A portion that can be removed by cost-effectiveprocedures such as HDS, and the second part, which arerecalcitrant. In order to meet the ultra low sulfur contentregulations, the refractory part should also be desulfur-ized. Hydrodesulfurization is able to remove therefractory sulfur; however, for these compounds, theprocess is very expensive and it is performed under veryhigh temperature and pressure conditions. Consideringthe growing trend of fuel consumption and exploitingpetroleum resources, the remaining part of petroleum ismore viscose and has high sulfur content whosedesulfurization will make final desulfurized fuel producteven more expensive. Taking advantage of an efficientmild desulfurization process will include severalbenefits. It lowers the final fuel price, economizes thedesulfurization energy consumption and produces loweramount of pollutants. Removing sulfur by microbialapproaches is one of the methods that can potentially beused after HDS unit in near future and replace HDS infar future.

The first bacterial strain (R. erythropolis IGTS8)with which sulfur could be removed specificallywithout breaking C–C bond was introduced by Kilbane(1992a,b) (Institute of Gas Technology). Later on, otherpotentially competent strains were isolated or designed.In biodesulfurization, two immiscible phases are con-tacted with each other. Although the cell membrane of thestrains (e.g. in Rhodococci) involved in desulfurization


typically are hydrophobic in nature and can fairly toleratehydrocarbon phase, there are many other toxic com-pounds in oil that, above certain concentration, candrastically inhibit biocatalysts. It has been observed thatbearing dsz genes is not sufficient to ensure equaldesulfurization phenotype among various desulfurizingstrains. Different strains with similar dsz genes maydemonstrate different activity, stability and selectivity(Abbad-Andaloussi et al., 2003). Considering the varietyof components in crude oil or oil fractions, a differentprocess operation or strain may be used to reach ultra lowsulfur content.

Biological and biotechnology processes are usuallyregarded to be expensive and tailored for the productionof low volume high value products. The output ofbiological desulfurization process is a high volume lowvalue product and the process is different from mostcommon biological procedures. The biological sulfurremoval is a slow process and it requires water. In anindustrial scale, the size of the two-phase reactor thatmight meet the required desulfurization is unimaginable(Vazquez-Duhalt et al., 2002). Therefore, a bioprocessthat consumes lower aqueous:hydrocarbon phase ratio isdefinitely preferred. Enzymes, in comparison to micro-organisms, require less water to function. However,since several enzymes, NADH, and FADH2 are involvedin biodesulfurization and their activity is not high, use ofthe whole cell as biocatalyst is more practical. Anotherpoint to consider is the hydrocarbon variety. Organicsulfur is found in many different refinery fractions.However, a fraction which has a higher refractorycompounds and a lower volume may be a decenthydrocarbon phase for desulfurization. Elimination orreduction of water required for biodesulfurization is alsoanother challenge (Borgne and Quintero, 2003).

Microbial desulfurization, similar to other microbialprocesses, is slow and requires much attention to havereproducible results. In this method, either resting cellsmight be used for desulfurization, or microorganismsmight be grown in an aqueous/oil or coal two-phasesystem. Cultivating living microorganism in two-phase(aqueous/oil or coal) systems would be impractical;because, the process would be too slow with very lowdesulfurization yield (Shennan, 1996).

Production of resting cells by high cell densityprocedure may be cross contaminated with othermicroorganisms that can attack C–C bonds or canconsume nutrients and grow at higher rate. Furthermore,resting cells production requires huge facilities toprepare cells and to maintain biocatalyst activity whileit is shipped to the desulfurization site (Yamada et al.,2001).

Biodesulfurization from process point of view at leastconsists of feedstock preparation, microorganism/bio-catalyst preparation, desulfurization in bioreactor, andseparation and recovery. Among all the stated items,preparation of biocatalyst with long half-life, high andreproducible specific activity is important.

To date, the most important challenge to approachindustry level biodesulfurization is the search to isolate astrain with higher biodesulfurization activity or todesign a recombinant biocatalyst with a stable activityto work in tandem with refining pace (Hirasawa et al.,2001). Apart from this, more work is required to obtainhigher desulfurization specific activity by increasing thedriving force from one phase to another and preventingthe accumulation of inhibitors. One of the points thatmight accelerate biodesulfurization is to eliminate thecooling time required after HDS (Konishi et al., 1997).

Biodesulfurization is estimated to have 70–80%lower CO2 emissions. In case of reaching adequatebiodesulfurization efficiency level, the capital costrequired for an industrial biodesulfurization process ispredicted to be two third of the one for an HDS process.Biodesulfurization operating cost is also expected to be15% lower (Vazquez-Duhalt et al., 2002). The operatingcost of a biodesulfurization unit is estimated to be 10–15% lower than a HDS unit (Kaufman et al., 1997;McFarland et al., 1998).

5. Conclusions

This paper presented the key issues, advances andchallenges on the path to reach a competitive biodesulfur-ization process. Microbial desulfurization in nature isdifferent from other more common biotechnologyprocesses. The delicate enzymes of desulfurizationcompetent cells have to obtain part of their substratesfrom a different phase in which their survival is notpossible. Since the first time that a specific sulfur removalwas introduced, research on this field has been steadilycontinued. Many other strains have been isolated orcloned and different methods have been tried. The mostserious problem in the implementation of biodesulfuriza-tion as an alternative industrial approach to produce ultralow sulfur content lies in the isolation or design of amicrobial strain with higher efficiency. Any small successthat provides the possibility to remove sulfur at highertemperature, with higher rate, or longer stability ofdesulfurization activity is considered a significant steptoward industry level biodesulfurization. Moreover,process development and unexpected problems thatmight occur in big scale operations should be considered.Upon success, the process may operate in a line after


hydrodesulfurization unit. Total desulfurization of fossilfuel by microbial approach is not expected to occur inearly future and more research is needed to design arecombinant strain with a broader range of target sulfurcompounds or to use successive desulfurizing microbialsystems with high potency. Most researches on desulfur-ization of refractory compounds have been performedwith simple model fuel to understand the nature ofdesulfurization. Dealingwith genuine fossil fuel will openup new challenges to solve.

Acknowledgements

The authors thank the Natural Sciences and Engi-neering Research Council of Canada (NSERC) forsupporting this research via allocating the DiscoveryGrant to A. Bassi and A. Margaritis.

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