Bioresource Technology 100 (2009) 4050–4053

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Short Communication

Sitosterol bioconversion with resting cells in liquid polymer based systems

Filipe Carvalho, Marco P.C. Marques, Carla C.C.R. de Carvalho, Joaquim M.S. Cabral, Pedro Fernandes *

IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 January 2009Received in revised form 12 March 2009Accepted 13 March 2009Available online 11 April 2009

Keywords:Silicone oil4-Androstene-3-17-dioneSitosterolBioconversionResting cells

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.03.044

* Corresponding author. Tel.: +351 218419065; faxE-mail addresses: pedro.fernandes@ist.utl.pt, pe

(P. Fernandes).

The use of a biocompatible water-immiscible organic phase as a substrate and product pool has beenacknowledged as an effective tool to overcome the low volumetric productivity of aqueous bioconversionsystems involving hydrophobic compounds. The growing environmental and public health awareness isnevertheless leading to restrictions in the use of organic solvents in industrial processes, in order to ren-der these more environmentally friendly. Different approaches are hence being assessed for the design ofalternative bioconversion media, involving the use of supercritical fluids, ionic liquids and natural oilsand liquid polymers, among others.

In this work, the use of liquid polymers as key components in the bioconversion media for a multi-stepmicrobial bioconversion was assessed. The model system used was the selective cleavage of the side-chain of b-sitosterol by free resting cells of Mycobacterium sp. NRRL B-3805, a well established industrialmulti-enzymatic process involving the use of nine catabolic enzymes in a fourteen-step metabolic path-way. High product yields were obtained when silicone B oil was used as substrate carrier/product pool,both in single oil and in oil:buffer two liquid phase system.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The use of organic solvents as substrate and/or product reser-voir, in order to improve the efficiency of bioconversion systems,is possibly the most widely chosen approach to overcome the tox-icity and/or low solubility of useful compounds (Heipieper et al.,2007; Kim et al., 2007). Most of the organic solvents commonlyused as carriers present nevertheless some drawbacks, namelythe damaging effects on microbial cells, along with their explosiveand environmentally hazardous nature (Schmid et al., 1998; Dézielet al., 1999; Kim et al., 2007). Alternative approaches that retainthe advantages of organic solvents, namely solubilization of hydro-phobic compounds to high concentration, while avoiding thedrawbacks have thus been sought after and presented (Kimet al., 2007; Wang, 2007). One of these is based in the use of liquidpolymers as components of the bioconversion media, either inpolymer:polymer systems or in polymer:salt solution systems(Zijlstra et al., 1998). This strategy, which provides a mild environ-ment to the biocatalysts and is also environmentally friendly (Zijl-stra et al., 1998; Raghavarao et al., 2003), has been scarcely appliedto sterol/steroid bioconversions, namely to D1-dehydrogenation ofcortisol (Kaul and Mattiasson, 1986; Santos et al., 1991) and side-chain cleavage of cholesterol (Flygare and Larsson, 1989) and ofphytosterols (Kutney et al., 2000; Stefanov et al., 2006). Flygare

ll rights reserved.

: +351 218419062.dro.fernandes@mail.ist.utl.pt

and Larsson (1989) performed cholesterol bioconversions usingimmobilized resting cells in a polymer:polymer:detergent mediaand an initial substrate concentration of only 1.0 g l�1, whereasKutney et al. (2000) and Stefanov et al. (2006) performed phytos-terol bioconversions using growing cells in a polymer:aqueousmedia system. In the present work the feasibility of using free rest-ing Mycobacterium sp. cells for sitosterol side-chain cleavage to 4-androstene-3,17-dione (AD), in a polymer based medium is ad-dressed. The phytosterol used was recovered from tall-oil (Diaset al., 2002) and cells were occasionally grown using waste glyc-erol recycled from a lab-scale biodiesel production systems as partof the carbon source. When a given bioconversion can be per-formed by either growing or resting cells, and provided cell viabil-ity is not compromised, in case this feature affects catalyticactivity, the use of resting cells can be advantageous over growingcells. The former approach allows for a more focused evaluation ofthe bioconversion itself, given that the complex metabolic path-ways involved in microbial cell growth are not present and thusdo not have to be taken into consideration, and there is no needfor sterile environment, the whole leading to a simpler system(Mutafov et al., 1997; Wang et al., 2005; Cánovas et al., 2007).The use of a single liquid phase allows on the other hand highervolumetric productivities, and in the overall simplifies the designof reaction media, selection of operational conditions and down-stream processing. High product yields were observed for initialsitosterol concentrations of 5.0 g l�1 (12 mM) both in silicone oilmedia and in silicone oil:buffer two-phase systems. Thepoly(methylphenylsiloxane) oil, henceforth termed silicone B oil,

F. Carvalho et al. / Bioresource Technology 100 (2009) 4050–4053 4051

proved thus to provide a suitable media for sitosterol side-chaincleavage with microbial cells.

Table 1Solubility of sitosterol in the liquid polymers tested.

Polymer Sitosterolsolubility(mM)

Temperature(�C)

Comments

PPG 425 12(±2.5) 3584(±5.9) 100 Crystallization of

sitosterol after additionto the aqueous phase (2)

PPG 2000 12(±1.8) 35 (1)72(±2.9) 100 (2)

Silicone A 10(±1.0) 35 (1)20(±2.5) 100 (2)

Silicone B 10(±1.7) 35 (1)44(±3.5) 100 (2)

2. Methods

2.1. Reagents

Yeast extract and potato dextrose agar were obtained from Dif-co (Detroit, MI, USA). Ammonium chloride and dioctyl phthalate(DOP) was supplied by Merck-Schuchardt (Hohenbrunn, Ger-many), glycerol was from Riedel-de-Häen (Seelze, Germany). Sitos-terol (for preparation of analytical standards) was from Acros(Geel, Belgium), otherwise it was obtained from tall-oil as de-scribed elsewhere (Dias et al., 2002). Tween 20, 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione (ADD) and progesteronewere obtained from Sigma (St Louis, MO, USA). Silicone oils (hightemperatures), namely poly(dimethylsiloxane), henceforth termedsilicone A, and silicone B, were obtained from Acros Organic (NewJersey, USA). Polypropylene glycol (PPG) 425 and 2000 were alsoobtained from Acros Organic (New Jersey, USA). All other chemi-cals were of analytical or high-performance liquid chromatography(HPLC) grade and purchased from various suppliers.

2.2. Microorganisms, growth conditions and bioconversion

Mycobacterium sp. NRRL B-3805 cells were maintained in potatodextrose agar slants (42 g l�1) and grown in 1 l Erlenmeyer flaskscontaining 200 ml of a defined medium composed of (%, w/v) glyc-erol (2), NH4Cl (0.4), Tween� 20 (0.08), MgSO4 � 7H2O (0.02), b-sitosterol (0.1), prepared in pH 7 di-sodium/potassium phosphatebuffer (100 mM). Incubation was performed for 48 h at 30 �C and200 rpm in orbital shakers Aralab Agitorb 2001C (Portugal) with25 mm shaking diameter. Active microbial cells were harvestedby filtration in qualitative filter paper, thoroughly washed withpH 7 di-sodium/potassium phosphate buffer (100 mM), and storedat �20 �C as a wet cell paste (roughly 200 mg dry cell weight pergram wet paste) until use (Staebler et al., 2004).

Bioconversion runs were performed at 35 �C in 15 ml magneti-cally stirred (800 rpm) reactors, closed with sealing tape, contain-ing 1 ml of bioconversion media, to which 25 mg of wet cell pastewere added. The bioconversion media was composed either by1 ml of a 12 mM solution of sitosterol in a given liquid polymeror in DOP, or by a two-phase system composed of similar volumesof the sterol rich solution, in either polymer or in DOP, and a Tris-HCl, pH 7.0, 100 mM, buffered phase. A sacrificial vessel approach,based on the methodology described by Ferreira-Torres et al.(2005), was used. Periodically, the whole volume of two replicatereactors was collected and centrifuged (14.000 rpm � 5 min). A gi-ven volume of the DOP or polymer phase was collected and dilutedin a solution of progesterone (0.2 g l�1, internal standard) in n-hep-tane. For the two-phase systems, a given volume of the aqueousphase was extracted twice with a three-fold volume of the internalstandard solution. Triplicate samples were prepared for each reac-tor. Steroid quantification was performed by HPLC.

2.3. Partition and solubility data

In order to assess the ability of the selected polymers to solubi-lize the substrate, given amounts (100 mg) of sitosterol were addedto 5 ml of each liquid polymer until saturation occurred, as checkedvisually by formation of insoluble phase. The process was per-formed in magnetically stirred vessels, under controlled tempera-tures of either 35 �C or 100 �C. For each polymer, eight replicateruns were made. The supernatant was collected and diluted in asolution containing the internal standard for assessment of sitos-

terol concentration. Sterol solubility was determined based on anaverage value of the eight runs for each polymer. Occasionally, sat-urated solutions incubated at 100 �C were allowed to cool to 35 �C,to check for substrate crystallization or similar phenomena.

Partition of substrate and product between oil and aqueousphases were assessed as follows: a volume of 5 ml of polymer con-taining 8 mM of AD and/or sitosterol was added to 5 ml of aqueousmedia. After overnight magnetic stirring (at 30 �C), phase separa-tion was achieved by centrifugation (4500 rpm, 30 min, 30 �C).The aqueous phase was collected and the amount steroid/sterolwas determined by HPLC analysis. Triplicate runs were carried out.

2.4. Sterol/steroid analysis

HPLC analysis (Lichrospher Si-60 column, 5 m particle size,Merck, Germany) with 1 ml min�1 isocratic elution was performedto determine products concentration, with UV detection at 215 nm(sitosterol) and 254 nm (AD and ADD). The mobile phase was com-posed of n-heptane and ethanol (90:10, v/v). Samples were ex-tracted with n-heptane containing n-heptane, containing 0.2 g l�1

progesterone as internal standard, prior to injection into the HPLCcolumn.

3. Results

Given the promising results obtained with silicone oil and PPGas substrate carriers in AD production from phytosterols withgrowing Mycobacterium sp. cells (Kutney et al., 2000; Stefanovet al., 2006), the feasibility of using specific types of such liquidpolymers as substrate carriers was tentatively addressed. Polye-thers displayed a far higher sterol solubilization capability, partic-ularly at higher temperatures, although this feature is notparticularly noticeable at 35 �C (Table 1).

Free resting Mycobacterium sp. cells were introduced into thedifferent liquid polymers tested as reaction media (Fig. 1). Sincethe threshold for sitosterol solubilization at 35 �C was about 10–12 mM (Table 1), 12 mM sitosterol solutions used in bioconversiontrials were prepared by incubating at higher temperatures, trans-ferred to the individual reactors and allowed to cool to 35 �C priorto addition of the biocatalyst. Crystallization was not observed inthe process. In the bioconversion runs performed, Silicone B clearlyemerged as the most suitable substrate carrier among the liquidpolymers tested. Given the promising results, silicone B was alsotested in a two-liquid phase system and compared to the perfor-mance of the well established DOP-buffer bioconversion system(Staebler et al., 2004) (Fig. 2). Both substrate and AD were shownin preliminary trials to partition preferably to the silicone phase,since partition coefficient between oil and aqueous phase of 44(±6) and 1.2 � 102 (±2 � 101) were observed for sitosterol andAD, respectively. Similar partition coefficients were tentatively

0

2

4

6

8

10

12

0 20 40 60 80 100 120 140

AD

pro

duct

ion

(mM

)

Bioconversion time (h)

Fig. 1. Time course of AD production from sitosterol upon incubation in silicone A(squares), silicone B (triangles), PPG 425 (closed dots), PPG 2000 (open dots).Incubation was performed at 35 �C.

0

2

4

6

8

10

12

0 20 40 60 80 100 120

AD

pro

duct

ion

(mM

)

Bioconversion time (h)

Fig. 2. Time course of AD production from sitosterol upon incubation in buffer:silicone B (triangles) and in buffer:DOP (closed dots). Incubation was performed at35 �C.

4052 F. Carvalho et al. / Bioresource Technology 100 (2009) 4050–4053

evaluated for DOP:aqueous systems, but only vestigial concentra-tion of either AD or sitosterol were observed in the aqueous phase,which prevented the determination of a statistically significant va-lue, yet data suggest partition coefficient values clearly in excess ofone order of magnitude in both cases. Under similar operationalconditions the silicone oil:buffer system proved to out-performthe phthalate based system

4. Discussion

The higher activity observed during incubation in silicone Bwhen compared to the other liquid polymers tested could eventu-ally be ascribed to the bulkier nature of this polymer, due to thepresence of a benzyl residue in the silicone chain, when com-pared to silicone A, which may lead to diverse interaction pat-terns with cell membrane. A similar trend was previouslyreported when assessing the diversity in catalytic activity andcell-solvent interaction when mycobacterial cells were incubatedin the presence of phthalates with different branched structures(Cruz et al., 2004a). On the other hand, low biocatalytic activitywas observed when the use of polypropylene glycol with differentmolecular weights was evaluated as bioconversion media, unlikethe reports by Kutney et al. (2000) and by Stefanov et al. (2006).However, it should be pointed out that, along with performingsuch bioconversion with growing cells, both works were carried

out in two-phase water:oil systems, where the aqueous phasewas predominant. Stefanov et al. (2006), working with CaradolED 56–10�, a commercial PPG 2000 from Shell, reported howeverthat increasing the oil:water volumetric phase ratio up to 1:1 ledto a marked decrease in both cell growth and bioconversion rate.Microbial cell growth has been reported to be considerably unaf-fected when performed in the presence of PEG (polyethylene gly-col) 4000 or higher (Zijlstra et al., 1998), hence there may besome deleterious effect, particularly when the lower molecularweight PPG was used.

In the present work, AD yields close to 10 mM were obtained in4–5 days of incubation, for an initial substrate concentration of12 mM (referred to the polymer/organic solvent phase), with a bio-catalyst concentration of 5 mg dry cell weight ml�1. Flygare andLarsson (1989) reported bioconversion yields of cholesterol to amixture of AD and ADD close to 80%, in roughly 3–4 days, usingan initial cell concentration of 10 g l�1, in an aqueous two-phasesystem composed of polyvinylpyrrolidone 40000, dextran T-500and Brij 35. The initial substrate concentration was only of1.0 g l�1 (2.6 mM). Staebler et al. (2004) optimized a DOP-bufferbioconversion system for sitosterol bioconversion and reported a10 mM AD yield in 24 h, using roughly 9 mg dry cell weightml�1, in a mechanical stirred vessel. When working under similarconditions of biocatalyst concentration and stirring, as in the pres-ent work, the DOP-buffer bioconversion was slightly less efficientthan the silicone B-buffer system. Cruz et al. (2004b) reported anAD yield close to 10 mM after roughly 30 h in a monophasic DOPsystem, operating in a magnetically stirred reactor with forced aer-ation, yet a biocatalyst concentration of 16 mg dry cell weight ml�1

was used. Kutney et al. (2000) reported conversion yields of 90%for initial substrate concentration of 12 mM after 7 days of incuba-tion in a PPG/complex fermentation media, which is a bit less effec-tive than the present process. Again using growing cells, Stefanovet al. (2006) reported full conversion of a 24 mM initial concentra-tion of sitosterol in a 5 days period, in a silicon oil/complex mediavolumetric phase ratio of 3:7. Increase of the oil phase clearly de-creased the efficiency of the process, which is probably unfeasiblein a single oil system, as described in the present work. Wang et al.(2006) reported an ADD yield of 40 mM, from an initial sitosterolconcentration of 60 mM, upon a 6-day incubation period of restingmycobacterial cells in an optimized cloud point system. A biocata-lyst concentration of 200 g cell paste l�1 was nevertheless required.These authors compared resting and growing cells for performingthis particular bioconversion and concluded for the advantages ofthe former system.

5. Conclusions

Silicone B, a non-volatile and non-toxic oil, was shown to pro-vide a suitable environment for the microbial side-chain cleavageof sitosterol, both in single liquid phase systems and in oil:aqueoustwo liquid phase systems, using resting bacterial cells and rela-tively high substrate concentration. The encouraging results ob-tained in the present work are prone to pave the way for furtherwork envisaging optimization on the bioconversion system anddeeper insight on polymer:cell interaction.

Acknowledgements

M.P.C. Marques, P. Fernandes and C.C.C.R. de Carvalho acknowl-edge Fundação para a Ciência e Tecnologia (Portugal) for financialsupport in the form of PhD Grant SFRH/BD/24433/2005 and pro-gramme Ciência 2007. This work was partially funded by researchproject POCI/SAU-MMO/59370/2004 from Fundação para a Ciênciae a Tecnologia (Portugal).

F. Carvalho et al. / Bioresource Technology 100 (2009) 4050–4053 4053

References

Cánovas, M., García, V., Bernal, V., Torroglosa, T., Iborra, J.L., 2007. Analysis ofEscherichia coli cell state by flow cytometry during whole cell catalyzedbiotransformation for L-carnitine production. Process Biochemistry 42, 25–33.

Cruz, A., Fernandes, P., Cabral, J.M.S., Pinheiro, H.M., 2004a. Solvent partitioning andwhole-cell sitosterol bioconversion activity in aqueous-organic two-phasesystems. Enzyme and Microbial Technology 34, 342–353.

Cruz, A., Angelova, B., Fernandes, P., Cabral, J.M.S., Pinheiro, H.M., 2004b. Study ofkey operational parameters for the side-chain cleavage of sitosterol by freemycobacterial cells in bis-(2-ethylhexyl) phthalate. Biocatalysis andBiotransformation 22, 189–194.

Déziel, E., Comeau, Y., Villemur, R., 1999. Two-liquid-phase bioreactors forenhanced degradation of hydrophobic/toxic compounds. Biodegradation 10,219–233.

Dias, A.C.P., Fernandes, P., Cabral, J.M.S., Pinheiro, H.M., 2002. Isolation of abiodegradable sterol-rich fraction from industrial wastes. BioresourceTechnology 82, 253–260.

Ferreira-Torres, C., Micheletti, M., Lye, G.J., 2005. Microscale process evaluation ofrecombinant biocatalyst libraries: application to Baeyer–Villigermonooxygenase catalysed lactone synthesis. Bioprocess and BiosystemsEngineering 28, 83–93.

Flygare, S., Larsson, P.O., 1989. Steroid transformation in aqueous two-phasesystems: side-chain degradation of cholesterol by Mycobacterium sp. Enzymeand Microbial Technology 11, 752–759.

Heipieper, H.J., Neumann, G., Cornelissen, S., Meinhardt, F., 2007. Solvent-tolerantbacteria for biotransformations in two-phase fermentation systems. AppliedMicrobiology and Biotechnology 74, 961–973.

Kaul, R., Mattiasson, B., 1986. Extractive bioconversion in aqueous two-phasesystems. Applied Microbiology and Biotechnology 24, 259–265.

Kim, P.-Y., Pollard, D.J., Woodley, J.M., 2007. Substrate supply for effectivebiocatalysis. Biotechnology Progress 23, 74–82.

Kutney, J.P., Milanova, R.K., Vasilev, C.D., Stefanov, S.S., Nedelcheva, N.V., 2000.Process for the microbial conversion of phytosterols to androstenedione andandrostadienedione. United States Patent 6071714.

Mutafov, S., Angelova, B., Avramova, T., Boyadjieva, L., Dimova, I., 1997. Theinducibility of 9a-steroid hydrolxylating activity in resting Rhodococcus sp. CellsProcess Biochemistry 7, 585–589.

Raghavarao, K.S.M.S., Ranganathan, T.V., Srinivas, N.D., Barhate, R.S., 2003. Aqueoustwo phase extraction – an environmentally benign technique. CleanTechnologies and Environmental Policy 5, 136–141.

Santos, R.A., Caldeira, J.C.O., Pinheiro, H.M., Cabral, J.M.S., 1991. Steroidbioconversion in a novel aqueous two-phase system. Biotechnology Letters13, 349–354.

Schmid, A., Kollmer, A., Mathys, R.G., Witholt, B., 1998. Developments toward large-scale bacterial bioprocesses in the presence of bulk amounts of organic solvents.Extremophiles 2, 249–256.

Staebler, A., Cruz, A., van der Goot, W., Pinheiro, H.M., Cabral, J.M.S., Fernandes, P.,2004. Optimization of androstenedione production in an organic – aqueoustwo-liquid phase system. Journal of Molecular Catalysis B: Enzymatic 29, 19–23.

Stefanov, S., Yankov, D., Beschkov, V., 2006. Biotransformation of phytosterols toandrostenedione in two phase water–oil systems. Chemical and BiochemicalEngineering Quarterly 20, 421–427.

Wang, Z., 2007. The potential of cloud point system as a novel two-phasepartitioning system for biotransformation. Applied Microbiology andBiotechnology 75, 1–10.

Wang, Z., Zhao, F., Chen, D., Li, D., 2005. Cloud point system as a tool to improve theefficiency of biotransformation. Enzyme and Microbial Technology 36, 589–594.

Wang, Z., Zhao, F., Chen, D., Li, D., 2006. Biotransformation of phytosterol to produceandrosta-diene-dione by resting cells of Mycobacterium in cloud point system.Process Biochemistry 41, 557–561.

Zijlstra, G.M., de Gooijert, C.D., Tramper, J., 1998. Extractive bioconversions inaqueous two-phase systems. Current Opinion in Biotechnology 9, 171–176.