Fuel 92 (2012) 245–249

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Fuel

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Reduced coke formation and aromaticity due to chloroperoxidase-catalyzedtransformation of asphaltenes from Maya crude oil

Marcela Ayala ⇑, Edna L. Hernandez-Lopez, Lucia Perezgasga, Rafael Vazquez-DuhaltInstituto de Biotecnologia, UNAM Av., Universidad 2001 Col., Chamilpa, Cuernavaca, Morelos 62210, Mexico

a r t i c l e i n f o

Article history:Received 31 March 2011Received in revised form 29 June 2011Accepted 30 June 2011Available online 18 July 2011

Keywords:AsphalteneChloroperoxidaseReactivityCokeHydrogen peroxide

0016-2361/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.fuel.2011.06.067

⇑ Corresponding author. Tel.: +52 777 3291619; faxE-mail address: maa@ibt.unam.mx (M. Ayala).

a b s t r a c t

The transformation of the porphyrin-free asphaltene fraction from a heavy Maya crude oil was catalyzedby chloroperoxidase from Caldariomyces fumago (CPO) in a ternary system with low water content of6.3%. The total turnover number (TTN) in this system was determined for lyophilized CPO in the presenceof sucrose as cryoprotector and for a covalent bioconjugate with chitosan. The covalent conjugation ofCPO with chitosan increased the TTN by 10-fold. Fluorescence due to aromatic groups decreased by24% in treated asphaltenes, indicating significant changes after the biotransformation. On the other hand,the solubility profile indicated that transformed asphaltenes are less soluble in toluene and more prone toprecipitate in the presence of hexane, compared to untreated asphaltenes, which could be related to theintroduction of polar atoms. Energy-dispersive X-ray spectroscopy (EDS) showed that the content of chlo-rine increased six-fold on an atomic basis. Finally, enzymatically transformed asphaltenes are more reac-tive and thus generate less coke, as evidenced by thermal degradation under nitrogen atmosphere.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Asphaltenes are the most recalcitrant fraction in crude oil,defined as nC7–C5 insoluble fraction of crude oil. Thus, asphaltenesare a heterogeneous, complex mixture of hundreds of moleculessharing solubility properties. This fraction concentrates most ofthe heteroatoms (sulfur, oxygen, nitrogen, V and Ni) present incrude oil. It is also characterized by a high degree of aromaticity.Asphaltenes from heavy crude oils may contain up to 50% carbonin aromatic structures [1]. This high degree of aromaticity com-bined with the presence of short alkyl chains may explain its recal-citrancy; although there are reports on microorganisms (bacteriaand fungi) growing in the presence of asphaltenes, other carbonand energy sources are also available for growth. Most frequentlythese alternative sources come from the inoculum, the matrix in so-lid fermentations or the presence of other components of crude oil[2–4]. The growth of microorganisms on asphaltenes as sole sourceor carbon and energy is controversial [5]. Enzymatic transformationof the petroporphyrin-rich fraction of asphaltenes was firstreported using soluble chloroperoxidase from Caldariomycesfumago (CPO) [6], and also with a chemically-modified cytochromec [7]. The CPO catalyzed transformation lead to metal release frompetroporphyrins and also from model porphyrins [6]. However, thereaction efficiency in terms of spent biocatalyst was low due toenzyme instability. CPO is a fungal enzyme able to catalyze a

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number of oxidative reactions using hydrogen peroxide as electronacceptor, either in the presence or absence of halogen ions.CPO-catalyzed reactions that could involve functional groups orcertain moieties present in asphaltenes include: phenolic oxidation,halogenation of aromatic moieties, sulfoxidation of heteroaromat-ics and sulfides, oxygen insertion in nitrogen-containing heterocy-cles such as imidazole and alkene epoxidation, among others [8]. Inthis work, we report for the first time the biotransformation of theporphyrin-free asphaltenes from Maya crude oil, a heavy Mexicanblend, using CPO-based biocatalysts. Total turnover number (TTN)for asphaltenes transformation was determined for CPO lyophilizedin the presence of sucrose as cryoprotectant and CPO covalentlyattached to chitosan. The effect of the biotransformation on severalphysicochemical properties of asphaltenes was also studied anddiscussed.

2. Materials and methods

2.1. Chloroperoxidase purification

CPO from C. fumago was kindly donated by Prof. Michael A.Pickard. The enzyme was purified as described elsewhere to anRz (A400nm/A280nm) of at least 1.2 [9].

2.2. Asphaltenes purification and petroporphyrin removal

Maya crude oil was kindly donated by Dr. Jorge A. Aburto fromthe Mexican Petroleum Institute. The asphaltene fraction was

246 M. Ayala et al. / Fuel 92 (2012) 245–249

recovered from crude oil and purified based on previously de-scribed procedures [7]. Briefly, 400 mL of pentane were added to10 g of crude oil, followed by stirring for 30 min and sonicationfor another 30 min. The sample was left still overnight, protectedfrom light. The precipitate was recovered after centrifugation at10,000 rpm for 15 min and dissolved in 10 mL of toluene. The pro-cedure was repeated with this sample. The petroporphyrin fractionwas removed by acetone extraction as described elsewhere [10].Briefly, 6 g of asphaltenes were dissolved in 300 mL of tolueneand stirred for 2 h. Then, 3 L of acetone were added and the mix-ture was stirred for 3 h, protecting the flask from light. The precip-itate was recovered after centrifugation at 12,000 rpm for 20 minand extensively washed with acetone.

2.3. Lyophilized biocatalyst preparation

A 1000-fold molar excess of sucrose (0.22 g) was added to aconcentrated CPO solution (500 lL, 150 lM) in 60 mM phosphatepH 6. The solution was lyophilized and maintained under desicca-tion with P2O5 before use.

2.4. Bioconjugate preparation

For CPO covalent conjugation to chitosan, 45 nmol of CPOwould be reacted with a 1000-fold molar excess of chitosan(0.8 g of a 1% chitosan solution) in the presence of a 500-fold molarexcess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in50 mM phosphate buffer pH 5. The final volume was 1 mL; thereaction was gently stirred for 4 h at room temperature and exten-sively washed by ultrafiltration with a 50,000 Da cutoff membrane.

2.5. CPO-chitosan bioconjugate characterization

For stability in the ternary system, around 400 pmol of eitherfree CPO or CPO-chitosan were added to 1 mL of the ternary sys-tem, adjusting the final water concentration to 6.3% (v/v). Thermalstability was determined by incubating 0.5 to 0.7 nM of free CPO orCPO-chitosan in a 60 mM phosphate solution pH 5 at 60 �C. Ali-quots (2–50 lL) were withdrawn at different times and the resid-ual activity was determined. Residual activity was measuredwith the halogenation assay described elsewhere [11]. Data wasobtained as the mean of triplicates and adjusted to a first order de-cay model. Activity with petroporphyrins was measured as de-scribed before [6].

2.6. CPO-catalyzed asphaltenes transformation

Free-petroporphyrin asphaltenes were dissolved in dichloro-methane to a concentration of 2.5 mg/mL. Six lL of this solutionwere added to a final concentration of 15 lg/mL in 1 mL of the reac-tion mixture containing hexane–isopropanol-buffer (46:47.7:6.3, v/v). The buffer phase was 100 mM citrate–phosphate buffer pH 2.8containing 6.67 mM KCl. The reaction was started by addition of0.5 mM H2O2 and a variable amount of the enzyme. Reaction pro-gress was monitored by Excitation–emission fluorescence matrix(EEFM) as described below. Further additions of enzyme and perox-ide were performed to obtain maximum conversion. Controls withonly enzyme or only peroxide were also carried out. All experi-ments were performed at least in triplicate.

2.7. Excitation–emission fluorescence matrix (EEFM)

Changes in asphaltenes fluorescence were monitored by obtain-ing emission profiles (350–600 nm) at several excitation wave-lengths, from 320 to 450 nm. The reaction mixture was analyzeddirectly, without further treatment. Proper controls with the

ternary system hexane–isopropanol-buffer without asphaltenesand containing only enzyme, only peroxide or both showed nofluorescence. Measurements were performed at least in triplicate.Experimental error was less than 10%. The matrix was visualizedwith MatLab and the fluorescence was expressed as the volumeunder the surface of this matrix.

2.8. Solubility profiles

Two volumes of water were added to 10 mL of the reaction mix-ture with asphaltenes and CPO. The organic phase was recoveredand passed through an anhydrous sodium sulfate bed. The solventwas evaporated under vacuum. The residue was dissolved indichloromethane and the absorbance at 350 nm was adjusted toa value of 1. One mL of this solution was placed in clean vialsand dichloromethane was evaporated. Hexane and toluene wereadded at different proportions to the dry solid and the mixturewas incubated for 1 h at 35 �C. The mixture was centrifuged andthe supernatant absorbance at 350 nm was measured. An identicalprocedure was followed for control asphaltenes. Profiles were per-formed in triplicate.

2.9. Energy-dispersive X-ray spectroscopy (EDS)

A thin film of gold–platinum was deposited over the samples bysputtering. EDS coupled to a scanning electron microscope (SEM)was performed in a LEO 1450 VP SEM, at 5–10 keV. Elemental com-position for selected atoms is reported as the mean of three mea-surements in different regions of the sample.

2.10. Thermogravimetric analysis (TGA)

Dynamic TGA were performed in a TA Instruments SDT Q60.Samples were heated from 25 to 900 �C at 10 �C/min under a nitro-gen atmosphere (100 mL/min).

3. Results and discussion

Asphaltenes from Maya crude oil are characterized by a highcontent of sulfur, more than 7%, as well as an aromaticity degreeof 0.49–0.52 [12,13]. This asphaltene fraction was enzymaticallytreated in a ternary system containing a low amount of water.The ternary system generates a microemulsion that was previouslyshown to sustain chloroperoxidase activity during the oxidation ofstyrene [14]. The system containing hexane–isopropanol-buffer in46–47.7–6.3% v/v was the most favorable for halogenation activity.However, the enzyme is very unstable in this system. In order toimprove CPO performance in the ternary system, the enzymewas lyophilized in the presence of sucrose. The goal was to gener-ate a biocatalyst able to disperse in the organic medium. Further-more, it is well established that the presence of excipientsprotects enzymes when exposed to organic solvents [15]. Fluores-cence due to aromatic structures, solubility properties and thermo-gravimetric analysis were measured in order to elucidate the effectof enzymatic transformation of asphaltenes. The fluorescence ma-trix, which can be associated to the aromatics content, showed a24% maximum decrease after several additions of hydrogen perox-ide and/or enzyme, as shown in Fig. 1.

On the other hand, control experiments with only hydrogenperoxide or enzyme showed less than 5% fluorescence decrease.According to our results, fluorescence loss could be related to theextent of conversion of asphaltenes and it could represent an alter-native, fast and sensitive method to estimate reaction conversion(Ayala M., unpublished work). By quantifying the minimumamount of enzyme required to achieve the 24% fluorescence

Fig. 1. EEFM of (A) control asphaltenes and (B) CPO-treated asphaltenes in the reaction mixture.

M. Ayala et al. / Fuel 92 (2012) 245–249 247

decrease, we estimated a total turnover number (TTN) of 0.8 lg ofasphaltenes transformed per 1 lg of enzyme. In order to increasethe catalytic efficiency of CPO, we covalently attached the enzymeto chitosan, a hydrophilic polymer of D-glucosamine that is bothbiodegradable and commercially available and presents a numberof advantages for enzyme immobilization, such as displaying highaffinity for proteins and a high concentration of reactive functionalmoieties [16]. The catalytic properties of the chitosan-CPO conju-gate are presented in Table 1.

Thermal stability as well as activity of the bioconjugate in aque-ous media is similar to the soluble enzyme. A 50% decrease of sta-bility during incubation in the ternary system was observed.Nevertheless, the bioconjugate performance is enhanced in thepresence of the substrate. For instance, using this conjugate as bio-catalyst, TTN increased 10-fold, from 0.8 for sucrose–lyophilizedCPO to 8.1 lg asphaltenes/lg CPO for the chitosan bioconjugate.To further prove this observation we compared the soluble CPOand the bioconjugate in a previously reported reaction of petro-porphyrin transformation, using as reaction medium a ternary sys-tem based on toluene–isopropanol-buffer [6]. As shown in Table 1,the bioconjugate displayed a two-fold higher specific activity inthis system. It has been reported for peroxidases that the presenceof the substrate increases the stability of the enzymes [15,17,18]. Itmay be possible that the more hydrophilic microenvironment cre-ated by chitosan enhances the reaction rate by increasing the localconcentration of polar reactants (e.g. H2O2 and Cl�); this effectcould also explain the lower stability in the absence of substrate.It is known that peroxidases, once activated with hydrogen perox-ide and in the absence of an exogenous substrate, are able to ex-tract electrons from the heme group or nearby aminoacidresidues, leading to inactivation [19]. This result highlights theneed to engineer the biocatalyst in order to improve its operationalperformance. Further biocatalyst engineering could thus increasethe TTN of CPO in the type of reactions described here.

Table 1Characterization of CPO-chitosan bioconjugate in aqueous and low water content systems

Biocatalyst property

Enzyme load (U/g chitosan)Halogenation activity (s�1)a

Thermal stability at 60 �C (t½, min)a

Stability in the ternary system (t½, min)b

Total turnover number for petroporphyrin-free asphaltenes (TTN, lg asphaltenes/lgSpecific activity with petroporphyrins (D(A410nm/A390nm) s�1 lmol�1)c

a In aqueous media.b In ternary system hexane–isopropanol-buffer.c In ternary system toluene–isopropanol-buffer.

Regarding Fig. 1, the fluorescence decreased symmetrically inthe studied emission–excitation range. It is well known that thesize and composition of the aromatic ring cluster influences thefluorescence emission wavelength [20]; for asphaltenes, it couldbe expected that the larger the number of fused aromatic rings,the larger the emission wavelength [21,22]. Thus, the symmetricfluorescence decrease after CPO transformation suggests that thebiotransformation is not selective and the enzyme may be catalyz-ing the oxidation of a broad range of aromatic species. The lowspecificity displayed by CPO has been already described using assubstrates polyaromatic hydrocarbons (PAH) with a variable num-ber of aromatic rings, as shown in Table 2.

We have previously found that PAH are halogenated in the pres-ence of CPO, hydrogen peroxide and chloride. No quinones orhydroxylated compounds were detected under these conditions[24]. In the case of asphaltenes, an EDS analysis revealed an in-crease in chlorine content after the enzymatic transformation, asshown in Table 3. The increase in chlorine content may representan important drawback, leading to refinery corrosion. Whereasinorganic chlorides are efficiently removed in the desalter units,organic chlorides remain in the crude oil due to insolubility inthe aqueous phase. They may decompose at high temperaturesgenerating HCl, which has to be neutralized by the addition ofamines. However, given that asphaltenes concentrate in the resi-due, it is possible that corrosion management arising from CPO-treated material could be confined to a few units. In the case ofoxygen, no changes in the atomic content could be detected. Sur-prisingly, the sulfur content dropped by 40%. In asphaltenes, sulfuris present mostly as thiophenes and sulfides [25,26]. CPO catalyzesthe conversion of organosulfur compounds to sulfoxides and sulf-ones [27–31]. It is thus very likely that as sulfur compounds be-come more polar upon CPO-treatment, they may partition to theaqueous-alcohol phase during the extraction and thus artificiallyreduce the sulfur concentration in the organic phase.

.

Chloroperoxidase form

Soluble Bioconjugate CPO-chitosan

NA 770 (±15)568 (±91) 452 (±113)23 1919 20

enzyme) 0.8 8.116 30

Table 3Atomic content of selected elements in control and CPO-treated asphaltenes.

Element Control CPO-treated

C 1 1O 0.1537 0.1325S 0.0153 0.0091Cl 0.0004 0.0025

Table 4Wavelength number regions of transformed functional groups.

Functional groups Wavelength number interval (cm�1)

Increase–OH 3600–3200Ketones, aldehydes 1725–1680Sulfites, sulfates 1420–1350 and 1200–1145–OH alkyl or C–Cl aromatic 1150–1040 or 1100–1130Nitro aromatic 865–1180C–Cl alkyl 830–560

DecreaseAromatic 1600, 900–800

Table 5Mass variation with temperature of control and CPO-treated asphaltenes. Initialweight of 2.688 mg and 0.739 mg, respectively.

Control asphaltenes CPO-treated asphaltenes

Temperature range (�C) Weight (%) Temperature range (�C) Weight (%)

25–310 10.14 20–88 2.99310–540 41.85 88–290 15.71540–900 8.911 290–570 45.43Residue 39.15 570–680 5.55

680–900 8.66Residue 21.72

Table 2PAH substrates for CPO bearing different number of aromatic rings in their structure[23,24].

Number of aromaticrings in PAH

CPO PAH substrates

1 Azulene2 Naphthalene, acenaphthene, biphenylene,

fluorene3 Anthracene, 2-methylanthracene, 9-

methylanthracene, phenanthrene4 Triphenylene, fluoranthene, pyrene, 7,12-

dimethylbenzanthracene5 Benzo[a]pyrene, 7-methyl benzo[a]pyrene6 Benzo[ghi]perylene

248 M. Ayala et al. / Fuel 92 (2012) 245–249

The solubility profiles of treated asphaltenes were performed toinvestigate the tendency to precipitate of biotransformed asphalt-enes. It is known that aromatic compounds, such as resins, interactwith asphaltenes molecules favoring its solubility in an otherwisealiphatic mixture [22,32]. Thus, the reasoning behind the solubilityprofiles is that a higher solubility in hexane–toluene mixtureswould imply a higher stability in crude oil. The solubility profilesin Fig. 2 show that CPO-treated asphaltenes are slightly less proneto solubilize in the presence of higher proportions of toluene. Thisobservation suggests that interactions among CPO-treated asphalt-enes molecules are larger and/or stronger than among untreatedasphaltenes molecules. This could be expected if treated asphalt-enes show higher polarity, which could be the case due to oxida-tion and chlorination as discussed above (Table 3).

FTIR measurements also support the introduction of polar moi-eties, as sulfoxide, nitro and carbonyl signals can be detected inbiotransformed asphaltenes, as listed in Table 4. The appearanceof signals corresponding to hydroxyl, carboxyl, aldehyde, sulfoxide,sulfone and sulfonate groups has been previously described for thecytochrome c-treated petroporphyrin-rich fraction of asphaltenes[7]. Interestingly, the aromatic signals in the 1600 and 900–800 cm�1 region decrease in treated asphaltenes, in accordanceto the results obtained by EEFM.

Fig. 2. Solubility profile of control (�) and CPO-treated asphaltenes (d).

In order to assess the effect of the biotransformation on asphalt-enes reactivity, thermogravimetric analyses were performed underan inert nitrogen atmosphere. Thermogravimetry (TG) under nitro-gen has been previously used to study the thermal stability ofasphaltenes [33–38]. The most complex aromatic systems remainas non-volatile residue (usually 45%) normally called coke [39].As shown in Table 5, the level of coke in the enzyme-transformedasphaltenes is significantly lower (21.72%) than the appropriatecontrols (39.15%). Thermal degradation under an inert atmospherehas been related to reactivity. Thus, we may conclude that bio-transformed asphaltenes are more reactive and produce less cokethan untreated asphaltenes. Temperature ranges obtained for con-trol and treated asphaltenes also confirm alterations in reactivity.The temperature ranges in Table 5 are defined by the fluctuatingslope of the thermogravimetric curve. It can be observed in Table5 that a more complex degradation profile arises for CPO-treatedasphaltenes. Comparing similar temperature ranges, the materialdecomposing below 290–310 �C increases by 85% after enzymatictreatment, whereas the material decomposing above 540–570 �Cincreases by 45%.

Asphaltenes raise trouble in petroleum processing. During thehydroprocessing of heavy feedstock, asphaltenes act as coke pre-cursors leading to catalyst deactivation and thus limiting the effi-ciency of conversion and refining of heavy oils [40]. Underhydroprocessing conditions, asphaltenes undergo a multitude ofreactions changing their structure [41–46]. Nevertheless, it hasbeen observed that asphaltene content of the feed does not corre-late with coke deposition, but it is the asphaltenes quality thatplays a more important role in catalyst deactivation [47,48].

On the other hand, asphaltenes conversion technology has beenalmost limited to thermal processes, such as visbreaking, delaycoking, fluid coking and flexicoking. Asphaltenes conversion usingconventional coking processes is considered non-environmentallyappropriate, because of the large amount of coke produced. Othertechnologies such as asphaltene pyrolysis, gasification or partialoxidation have also been proposed to transform asphaltenes. Nev-ertheless, asphaltenes gasification seems to be the most promisingtechnology providing that investment costs could be reduced andthe synthesis gas could be integrated into a petrochemical complex

M. Ayala et al. / Fuel 92 (2012) 245–249 249

[49]. In addition, the thermal conversion kinetics of petroleummacromolecules is not only important for thermal conversion pro-cesses, such as visbreaking and coking, but is also the primarycracking mode for hydroconversion and represents the undesiredside reaction for fluid catalytic cracking [46]. Asphaltenes hydro-conversion is an environmentally acceptable solution to transformasphaltenes with high sulfur, nitrogen and metals content intoclean fuels, leaving no byproducts to be disposed of. However,the existing technology is capital intensive and this has limited fur-ther applications.

4. Conclusions

For all technologies currently available to convert asphaltenesand treat heavy feedstocks, it is important to start with highlyreactive asphaltenes, in order to reduce coke formation. Thus, thefinding that an enzymatic transformation leads to a more reactiveasphaltenes that generates 45% less coke during thermal decompo-sition, shows the potential application of biotechnology in the oilindustry. The unique catalytic properties of chloroperoxidase couldserve as a template for the design of useful catalysts (from proteinengineering through biomimetics) to tackle environmental, pro-cessing and refining issues in the crude oil industry.

Acknowledgments

Authors acknowledge funding from BP Products North AmericaInc. We are also grateful to Rosa Roman for technical assistance.

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