Whole-Cell Biocatalysis for 1-Naphthol Production in ... · fold increases in 2-naphthol and phenol concentrations using dioctyl phthalate as the organic solvent. McIver et al. (23)

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2009, p. 6545–6552 Vol. 75, No. 200099-2240/09/$08.00�0 doi:10.1128/AEM.00434-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Whole-Cell Biocatalysis for 1-Naphthol Production in Liquid-LiquidBiphasic Systems�

S. V. B. Janardhan Garikipati, Angela M. McIver, and Tonya L. Peeples*Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242

Received 20 February 2009/Accepted 11 August 2009

Whole-cell biocatalysis to oxidize naphthalene to 1-naphthol in liquid-liquid biphasic systems was per-formed. Escherichia coli expressing TOM-Green, a variant of toluene ortho-monooxygenase (TOM), was usedfor this oxidation. Three different solvents, dodecane, dioctyl phthalate, and lauryl acetate, were screened forbiotransformations in biphasic media. Of the solvents tested, lauryl acetate gave the best results, producing0.72 � 0.03 g/liter 1-naphthol with a productivity of 0.46 � 0.02 g/g (dry weight) cells after 48 h. The effects ofthe organic phase ratio and the naphthalene concentration in the organic phase were investigated. The highest1-naphthol concentration (1.43 g/liter) and the highest 1-naphthol productivity (0.55 g/g [dry weight] cells)were achieved by optimization of the organic phase. The ability to recycle both free cells and cells immobilizedin calcium alginate was tested. Both free and immobilized cells lost more than �60% of their activity after thefirst run, which could be attributed to product toxicity. On a constant-volume basis, an eightfold improvementin 1-naphthol production was achieved using biphasic media compared to biotransformation in aqueousmedia.

Biocatalysis has emerged as an important technology in in-dustrial organic synthesis for the production of chemical syn-thons and high-value products (29, 34, 37). Biocatalysis offersthe advantage of performing reactions under mild conditionsand provides an environmentally benign approach for chemicalreactions (1, 38). Oxygenases are a class of enzymes that havegreat potential and versatility for catalyzing reactions that aregenerally not accessible by chemical routes with high regio-,stereo-, and enantioselectivities (6, 27, 42, 43). Oxygenasesintroduce either one or two atoms of molecular oxygen intoorganic molecules using NADH or NADPH as a cofactor. Toeliminate the addition of a costly cofactor, whole cells express-ing oxygenases are generally used (34, 43).

One of the potential applications of biocatalysis utilizingoxygenases is the oxidation of naphthalene to 1-naphthol.1-Naphthol has wide applications in the manufacture of dyes,drugs, insecticides, perfumes, and surfactants (2, 7, 17). Tao etal. (39) have compared the reaction rates and regioselectivitiesof various wild-type and modified monooxygenases for theoxidation of naphthalene to 1-naphthol. Of the monooxygen-ases tested, the best enzyme for the oxidation of naphthaleneto 1-naphthol was a toluene ortho-monooxygenase (TOM)variant, TomA3(V106A), also known as TOM-Green. TOMwas isolated from Burkholderia cepacia G4 and consists of an�2�2�2 hydroxylase (encoded by tomA1, tomA3, and tomA4)with two catalytic oxygen-bridged binuclear iron centers, anNADH-oxidoreductase (encoded by tomA5), a protein (en-coded by tomA2) involved in electron transfer between oxi-doreductase and hydroxylase, and a relatively unknown subunit(encoded by tomA0) (26, 36). TOM-Green was produced by

directed evolution of TOM with one amino acid change in thealpha-subunit of the hydroxylase (7, 33). TOM-Green retainedhigh regioselectivity (98%) and was sevenfold faster than wild-type TOM.

There has been considerable effort to identify and charac-terize oxidative biocatalysts for 1-naphthol production (7, 11,26, 33, 36, 40, 41). However, this process is not economicallyfeasible owing to the very low optimum concentration of naph-thalene (0.1 mM [7], which is less than the solubility of naph-thalene, 0.23 mM [28]) and the toxicity of both naphthaleneand 1-naphthol (38, 44). Substrate loading has to be increased,and the toxicities of both naphthalene and 1-naphthol have tobe minimized to make the process feasible. As a consequence,biotransformations in water-organic solvent biphasic mediahave been developed (8, 9, 12, 21, 45, 46). The use of a secondphase consisting of an organic solvent not only increases sub-strate loading but also maintains low concentrations of toxiccompounds in the aqueous phase (4). The organic solventchosen is critical for achieving the benefits of biphasic media.Two main criteria for solvent selection are a high distributioncoefficient for the product and biocompatibility with microor-ganisms (3, 4). Biocompatibility is generally correlated with thelogP of the solvent, which is the logarithm of the partitioncoefficient in an octanol-water system, and organic solventswith logP values greater than 4 are generally biocompatiblewith microorganisms (19). However, the correlation of activ-ity with logP is specific to the microorganism, and the criticallogP above which solvents are biocompatible has to be iden-tified for each microorganism (8, 16).

Biphasic systems have been widely used for reactions involv-ing a toxic substrate and/or product to enhance productivity orto improve recovery of the product (22–25, 31, 38). Oxidationof naphthalene has also been improved using biphasic reac-tions (13, 23, 35, 38). Tao et al. (38) used a biphasic system for2-naphthol and phenol production using toluene 4-moooxyge-nase and its variant TmoA(I100A). They obtained 10- to 21-

* Corresponding author. Mailing address: Department of Chemicaland Biochemical Engineering, University of Iowa, Iowa City, IA 52242.Phone: (319) 335-2251. Fax: (319) 353-1415. E-mail: [email protected].

� Published ahead of print on 21 August 2009.

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fold increases in 2-naphthol and phenol concentrations usingdioctyl phthalate as the organic solvent. McIver et al. (23) usednaphthalene dioxygenase to oxidize naphthalene to cis-(1R,2S)-1,2-naphthalene dihydrodiol using dodecane as the or-ganic solvent and obtained a productivity of 1.7 g/g (dryweight) cells/h in the first 6 h. In spite of the significant im-provements achieved by using a biphasic system for variousreactions, application of this strategy to 1-naphthol productionhas not been explored yet. Considering the high toxicities ofnaphthalene and 1-naphthol (38), biphasic reactions can en-hance the productivities. In this work, a biphasic system wasused to increase 1-naphthol productivities with whole cells ofEscherichia coli expressing the TOM-Green enzyme. Organicsolvents were screened, and solvents suitable for high 1-naph-thol productivity were identified. The organic phase was opti-mized by studying the effects of the naphthalene concentrationand the organic phase ratio. The stability of the biocatalyst forrecycling was also tested.

MATERIALS AND METHODS

Chemicals and bacterial strain. Dodecane, lauryl acetate, naphthalene,1-naphthol, and sodium alginate were purchased from Sigma (St. Louis, MO).Dioctyl phthalate and CaCl2 were purchased from Fischer Scientific (HanoverPark, IL). Luria-Bertani (LB) broth was purchased from Difco (Lawrence, KS).E. coli TG1/pBS(Kan)TOM-Green expressing the TOM-Green enzyme waskindly donated by Thomas K. Wood (Texas A&M University). PlasmidpBS(Kan)TOM-Green expresses TOM-Green (tomA012345) from a lac pro-moter. The lac promoter yields constitutive expression of TOM-Green genes dueto the high copy number of the plasmid and the lack of a lacI represser (7, 40).

Toxicity experiments. The cells were grown until early log phase in 250-mlshake flasks, and then growing cells (5 ml) were added to 20-ml sterile screw-capvials. Naphthalene or 1-naphthol dissolved in 50 �l dimethyl formamide (DMF)was added to the growing cells to obtain final concentrations of 0.05 g/liter, 0.1g/liter, 0.5 g/liter, and 1 g/liter. The growth was monitored by determining theoptical density at 660 nm (OD660). Due to the low solubilities of naphthalene and1-naphthol in water, the cosolvent DMF was used to suspend the compounds inthe aqueous phase. A positive control experiment in which 50 �l DMF was addedwithout naphthalene or 1-naphthol was also performed.

Cell viability determination. A Becton Dickinson LSR II flow cytometer at theUniversity of Iowa Flow Cytometry Facility was used to measure cell viability.For this analysis an L-701 Invitrogen Molecular Probes (Carlsbad CA) LIVE/DEAD BacLight bacterial viability kit was employed (20). Aqueous samples

were collected after 3 h of biotransformation for the analysis. Flow cytometry wasperformed as suggested by the supplier.

HPLC analysis. High-performance liquid chromatography (HPLC) was usedto quantify 1-naphthol and naphthalene, using a method similar to the methodused previously (23). A series 1100 Agilent HPLC with a photodiode arraydetector and a Supelcosil LC-PAH 5 �m column (25 cm by 4.6 mm) at roomtemperature was used for this analysis. Aqueous samples were diluted 1/2 inacetonitrile. Organic samples were injected directly without dilution. Both or-ganic and aqueous samples were centrifuged to separate cell debris and werefiltered with 0.2-�m polytetrafluoroethylene filters.

The mobile phase consisted of deionized water with 0.2% (vol/vol) glacialacetic acid and acetonitrile (Optima grade; Fisher Scientific) with gradient elu-tion. The water-acetonitrile elution profile included a linear gradient from 65:35(vol/vol) at zero time to 100:0 at 7 min, followed by a linear gradient to 65:35 at8 min and equilibration until 10 min. The mobile phase flow rate was 1.5 ml/min,and the injection volume was 10 �l. Naphthalene and 1-naphthol were analyzedat 272 nm and were detected at retention times of 5.7 and 3.9 min, respectively.The integrated areas of the elution peaks were used to calculate the concentra-tions of naphthalene and 1-naphthol in each phase.

Distribution coefficient measurement. Different organic solvents (500 �l) con-taining 0.75 g/liter 1-naphthol were added to 500 �l of phosphate buffer in 2-mlmicrocentrifuge tubes. The two phases were mixed by vortexing them for 30 s fivetimes with 1-min intervals between treatments. The two phases were analyzedusing HPLC. A distribution coefficient was calculated by determining the ratio ofthe 1-naphthol concentration in the organic phase to the 1-naphthol concentra-tion in the aqueous phase.

Biotransformation. All biotransformations were conducted in 250-ml Erlen-meyer flasks with a 50-ml working volume at 30°C and 200 rpm. Fresh LBmedium was inoculated with an overnight culture of E. coli TG1/pBS(Kan)TOM-Green cells. Cells were grown to late log phase (OD660, �1.6), when the LBmedium appeared to be green due to the production of indigo and isatin (7, 10).Cells were harvested by centrifugation (�10,000 � g), washed with phosphate

FIG. 1. Toxicity of naphthalene and 1-naphthol for E. coli cells expressing TOM-Green. Cells were grown in LB medium at 37°C, and thecontrol cells were grown in the absence of naphthalene or 1-naphthol. OD, optical density.

FIG. 2. Oxidation of naphthalene to 1-naphthol using whole cellsexpressing TOM-Green.

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buffer (pH 7.2), and resuspended in 0.5 volume of phosphate buffer (pH 7.2) toincrease the cell density. The medium was supplemented with 20 mM glucoseand 100 mg/liter kanamycin. Resuspended cells (30 ml) were used to measure thedry weight of cells. For aqueous biotransformation, naphthalene at a final con-centration of 0.5 g/liter was added to 50 ml of resuspended cells using DMF asa cosolvent due to the low water solubility of naphthalene. For biphasic biotrans-formations, the desired volume of an organic solvent with a known concentrationof dissolved naphthalene was added to the aqueous phase of resuspended cells toobtain a final volume of 50 ml. Aqueous and organic samples were taken at 3, 6,24, and 48 h and analyzed using HPLC. The results shown below are averages ofthree identical experiments.

Organic phase optimization. Four different organic phase ratios (20, 40, 60,and 80% in phosphate buffer) were evaluated in the bioconversion experiment,and for each organic phase ratio four different concentrations of naphthalene(20, 40, 60, and 70 g/liter) were used. The formation of 1-naphthol in the laurylacetate phase was monitored using HPLC.

Immobilization. Immobilization of cells was performed using a method similarto the method described previously (23). E. coli TG1/pBS(Kan)TOM-Green cells(360 ml) were grown to late log phase (OD660, �1.6) and harvested by centrif-ugation at �10,000 � g for 10 min. The cells were washed with Tris buffer (pH7.2). The cells for each experiment were immobilized together and later dividedand placed into six separate flasks. A 3% sodium alginate solution was preparedusing 120 ml of deionized water. A 1% CaCl2 solution was prepared as thegelation agent using deionized water. Both the sodium alginate and CaCl2 solu-tions were autoclaved at 121°C for 15 min. The sodium alginate solution wasallowed to cool to room temperature, and the CaCl2 solution was cooled to 4°C.The pelleted cells were resuspended in 25 ml of sterilized deionized water. Thecells and sodium alginate solution were mixed by stirring them for 5 min on a stirplate. The mixture was added dropwise to the stirred gelation agent using a 60-mlsyringe with an 18-gauge needle. The mixture was stirred for 1 h to harden it. Theresulting calcium alginate beads were 1 to 2 mm in diameter. After hardening,the beads were removed from the solution and washed twice with sterilizeddeionized water. The immobilized cells were divided equally among six sterileflasks containing 19.5 g of beads each.

Immobilized cell biotransformation. The immobilized biocatalyst was sus-pended in 250-ml Erlenmeyer flasks with a working volume of 50 ml. The beadswere suspended in 30 ml of Tris-HCl buffer (pH 7.2) supplemented with 20 mMglucose and 100 mg/liter kanamycin. The solvent phase (20 ml) was added tobegin the reaction, and the flasks were shaken at 200 rpm and 37°C. An HPLCanalysis was done using samples of the solvent phase.

RESULTS

Substrate and product toxicities. Whole cells of E. coli TG1expressing TOM-Green were used for oxidation of naphtha-lene to 1-naphthol. The toxicities of both naphthalene and1-naphthol for the E. coli TG1 strain expressing TOM-Greenare shown in Fig. 1. Naphthalene inhibited cell growth even ata low concentration, 0.05 g/liter. The inhibition of growth in-

creased as the concentration of naphthalene increased to 0.5g/liter, and no growth was observed with 1 g/liter naphthalene.The inhibitory effect of 1-naphthol was greater than that ofnaphthalene, and no growth was observed even with 0.5 g/liter1-naphthol. These results are comparable to the results ofsimilar work done previously (38). Therefore, maintaining lowconcentrations of the substrate and the product is critical formaintaining the cell viability and the activity for the reaction.

1-Naphthol production. The oxidation of naphthalene to1-naphthol using whole-cell TOM-Green is shown in the Fig. 2.The TOM-Green enzyme uses molecular oxygen and NADHas a cofactor. Oxidation of naphthalene to 1-naphthol wasperformed in an aqueous medium, and 0.04 g/liter of 1-naph-thol was obtained after 24 h using E. coli TG1/pBS(Kan)TOM-Green. To improve 1-naphthol production, biphasic biotrans-formations were performed with a 40% organic phase and 40g/liter naphthalene dissolved in a solvent. The organic solventchosen is critical for achieving the maximum benefits frombiphasic reactions. Three solvents, dodecane, dioctyl phthalate,and lauryl acetate, were chosen for screening. Table 1 showsthe distribution coefficients and structures of the three solventsused for 1-naphthol production. Dodecane (23, 35) and dioctylphthalate (35, 38) were used previously to improve 1,2-naph-thalene dihydrodiol and 2-naphthol productivities, respec-tively. Lauryl acetate was chosen because it has a high distri-bution coefficient for 1-naphthol. Although dodecane has a lowdistribution coefficient for 1-naphthol, it was used to study theeffect of 1-naphthol partitioning. All three solvents have logPvalues greater than 4. The biocompatibility of the solvents wasconfirmed by assaying cell viability using flow cytometry. Theviability of E. coli/pBS(Kan)TOM-Green cells was approxi-mately 99% after 3 h of exposure to a 40% organic phase withany of the three solvents (dodecane, lauryl acetate, or dioctylphthalate).

Biphasic biotransformations were performed using the threesolvents. Figure 3 shows the 1-naphthol concentration in theorganic phase and the 1-naphthol productivity expressed ingrams of 1-naphthol formed per gram (dry weight) of cellswhen 40 g/liter naphthalene was in the organic solvent. Whendodecane was used, 1-naphthol was formed within 3 h in abiotransformation, and after this the cells lost their activity.

TABLE 1. Distribution coefficients of the three solvents for 1-naphthol

Organic solvent Structure LogPa % Partitioned intoorganic phase

Distributioncoefficient

Dodecane 6.1 77.5 3.4

Dioctyl phthalate 8.7 98.5 66

Lauryl acetate 7.0 98.4 61

a LogP values were obtained from reference 19.

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However, when either lauryl acetate or dioctyl phthalate wasused, 1-naphthol was formed at a steady rate, approximately0.06 g/liter/h, for 6 h. Production of 1-naphthol continued at alower rate up to 48 h. The formation of 1-naphthol was signif-

icantly greater when either lauryl acetate or dioctyl phthalatewas used than when dodecane was used. After 48 h, the con-centrations of 1-naphthol obtained with lauryl acetate and withdioctyl phthalate were higher (approximately 10-fold higher[0.724 � 0.03 g/liter] and 7-fold higher [0.52 � 0.022 g/liter],respectively) than the 1-naphthol concentration obtained withdodecane (0.075 g/liter). A comparison of lauryl acetate anddioctyl phthalate showed that lauryl acetate gave slightlyhigher concentrations of 1-naphthol. Although both of thesesolvents have high distribution coefficients for 1-naphthol,dioctyl phthalate has a high viscosity and requires a longermixing time to reach equilibrium. Therefore, lauryl acetategave the best results, with 0.46 � 0.02 g 1-naphthol/g (dryweight) cells produced after 48 h.

Optimization of organic phase. After identification of laurylacetate as the best of the solvents tested, the organic phaseratio and the naphthalene concentration had to be optimizedto achieve the best results. Different organic phase ratios andnaphthalene concentrations were tested using lauryl acetatefor 1-naphthol production, and their effects are shown in theFig. 4. 1-Naphthol productivity was increased when either thenaphthalene concentration in the organic phase was increasedfrom 20 to 70 g/liter or the organic phase ratio was increasedfrom 20 to 60%. Higher naphthalene concentrations in the

FIG. 3. 1-Naphthol concentration in the organic phase with E. colicells expressing TOM-Green in biphasic media. The reaction condi-tions were as follows: 40% organic phase with 40 g/liter naphthalene,30°C, and 200 rpm. The error bars indicate standard deviations basedthree identical experiments. CDW, dry weight of cells.

FIG. 4. Effects of the naphthalene concentration and the organic phase ratio on the 1-naphthol concentration in the organic phase when lauryl acetateis used as the organic solvent. The reaction conditions were as follows: total working volume, 50 ml; 30°C; and 200 rpm. CDW, dry weight of cells.


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organic phase allow more naphthalene to partition into theaqueous phase, thereby increasing the naphthalene bioavail-ability. A higher organic phase ratio also promotes the reactionby allowing better partitioning of 1-naphthol, thereby minimiz-ing its toxicity and improving productivity. Improvement in1-naphthol productivity with an increase in the organic phaseratio was also observed previously (38). However, the 1-naph-thol productivity decreased when the organic phase ratio wasincreased from 60 to 80%. For the different reaction conditionstested, the highest 1-naphthol concentration (1.43 g/liter) andthe highest 1-naphthol productivity (0.55 g/g [dry weight] cells)were observed after a reaction time of 48 h.

Although 1-naphthol productivity increases with an increasein the organic phase ratio, the 1-naphthol concentration in theorganic phase decreases. Therefore, the reaction conditionscan be optimized either for high 1-naphthol productivity, whichimpacts upstream processing costs, or for a high 1-naphtholconcentration in the organic phase, which impacts downstreamprocessing costs. In order to find the optimum reaction condi-tions, the effects of the naphthalene concentration and theorganic phase ratio were determined after 24 h, and the resultsare shown in Fig. 5. Although a high organic phase ratio and ahigh naphthalene concentration improve productivity, the datain Fig. 5 demonstrate that the optimum conditions for thereaction can be achieved at organic phase ratios and naphtha-lene concentrations lower than the maximum evaluated levelswithout a significant decrease in productivity. The optimumconditions for high 1-naphthol productivity were an organicphase ratio of 40% and a naphthalene concentration of 60g/liter. The optimum conditions for a high 1-naphthol concen-tration were an organic phase ratio of 20% and a naphthaleneconcentration of 60 g/liter. Table 2 shows the amount of1-naphthol formed for each condition and the percentage ofnaphthalene converted to 1-naphthol. The maximum conver-sion value, 3.5%, was obtained with 20 g/liter naphthalene andan organic phase ratio of 20%. Under the optimum conditionsfor high 1-naphthol productivity and a high 1-naphthol con-

centration in the organic phase, naphthalene conversion valuesof 1.3% and 2.3%, respectively, were obtained.

Recycling of biocatalyst. Recycling of the biocatalyst im-proves the process economics. Recycling experiments wereperformed to test the stability of the cells. The stability of bothfree cells and calcium alginate-immobilized cells was tested.Immobilized cells produced approximately 40% of the 1-naph-thol produced by free cells (0.196 � 0.014 g 1-naphthol/g [dryweight] cells for immobilized cells, compared to 0.49 � 0.01 g1-naphthol/g [dry weight] cells for free cells after 6 h of bio-transformation with an organic phase ratio of 40% and 60g/liter naphthalene). The decrease in immobilized cell activitycould have been due to mass transfer limitations due to the

FIG. 5. Optimization of the organic phase for 1-naphthol formation using E. coli cells expressing TOM-Green with lauryl acetate as the organicsolvent: 1-naphtol concentration in the organic phase expressed as a function of the naphthalene concentration in the organic phase and the organicphase ratio. The reaction conditions were as follows: reaction time, 24 h; 30°C; and 200 rpm. CDW, dry weight of cells.

TABLE 2. Naphthalene conversion values with different naphthaleneconcentrations in the organic phase and different organic phase

ratios after 48-h reactions (total reaction volume, 50 ml)

Naphthaleneconcn (g/liter)

Organic phaseratio (%)

Amt of1-naphthol (mg)

%Conversion

20 20 7.0 3.540 9.5 2.460 6.8 1.180 2.4 0.3

40 20 11.2 2.840 13.0 1.660 9.3 0.880 2.8 0.2

60 20 13.6 2.340 15.6 1.360 10.7 0.680 3.3 0.1

70 20 14.3 2.040 16.4 1.260 11.8 0.680 3.5 0.1


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calcium alginate beads. A similar decrease in the activity ofimmobilized cells compared to free cells was observed previ-ously (23). The reaction time for recycling experiments is crit-ical in determining the stability of the biocatalyst, consideringits exposure to toxic products, toxic substrates, and organicsolvents. Optimum conditions for 1-naphthol productivity (or-ganic phase ratio of 40% and 60 g/liter naphthalene) were usedfor the recycling experiment. As shown in Fig. 5 for 1-naphtholproduction under optimum reaction conditions for high pro-ductivity, 1-naphthol was produced at essentially a linear rateup to 6 h and at a reduced rate thereafter. Therefore, tworeaction times, 6 h and 12 h, were chosen to test the stability ofcells at the two reaction rates. The percentages of activityretained by free and immobilized cells for four cycles areshown in Fig. 6. In the 6-h recycling experiment, free cellsshowed greater retention of activity. Approximately 40% of theactivity of free cells was retained for the second run, comparedto 20% for immobilized cells. The activities decreased furtherfor the third and fourth runs. In the 12-h recycling experiment,both free and immobilized cells lost most of their activity, andonly 20% of the activity was retained for the second run.

DISCUSSION

Because of the wide applications of 1-naphthol and the ver-satility of toluene monooxygenases, production of 1-naphtholusing whole cells expressing TOM-Green in a biphasic systemwas evaluated. Efficient in situ removal of the toxic compound1-naphthol from the aqueous phase was critical for improving1-naphthol productivity. The high distribution coefficients oflauryl acetate and dioctyl phthalate for 1-naphthol enabled 10-and 7-fold improvements in 1-naphthol productivity, respec-tively, compared to the productivity with dodecane. The higher1-naphthol productivities obtained with lauryl acetate and withdioctyl phthalate than with dodecane suggest that the dynamicsof 1-naphthol partitioning into the organic phase plays a majorrole in maintaining cellular activity and improving 1-naphtholproductivity. Moreover, inefficient partitioning of 1-naphtholresults in accumulation of the toxic product in the aqueousphase, thereby lowering the cellular activity and 1-naphthol

productivity. Similar improvements in productivity were ob-tained by using solvents with high distribution coefficients forthe product 2-naphthol (38). Compared to biotransformationsin the aqueous systems, on a constant-volume basis, an eight-fold improvement in 1-naphthol production was obtained usinglauryl acetate as the second phase in a biphasic system (16.4 mg1-naphthol [with 70 g/liter naphthalene and an organic phaseratio of 60%] in the biphasic system, compared to 2 mg 1-naph-thol [0.04 g/liter 1-naphthol] with the aqueous medium for50-ml reactions).

The stability of E. coli/pBS(Kan)TOM-Green for 1-naphtholproduction in a biphasic system was also tested, and more than60% of the activity was lost for the second run after recycling.Similar recycling for biphasic biotransformation has been per-formed previously (23) to produce nontoxic cis-1,2-naphtha-lene dihydrodiol, and the activity was retained for up to fourruns for 6 h of recycling. However, in the reaction examinedhere, the product, 1-naphthol, is very toxic and could be themain reason for the loss of activity during recycling (38). More-over, diffusional limitations for immobilized cells may result in1-naphthol accumulation in the beads that would adverselyaffect immobilized cell activity (18).

The 1-naphthol concentrations in the organic phase and thenaphthalene conversion values obtained in this work are com-parable to results obtained previously for a similar compound,2-naphthol (38). The low level of naphthalene conversion isdue to high substrate loading and the toxicity of the product,1-naphthol. The naphthalene added to the biphasic system canbe recycled along with the organic solvent to improve theeconomics. Although higher product concentrations and con-version values were obtained for oxidation reactions in a bi-phasic system (5, 31), the high toxicity of 1-naphthol is themain factor limiting the production of higher 1-naphthol con-centrations. Additional organic solvents can be tested based onbiocompatibility and high selectivity for 1-naphthol. However,the toxicities of solvents limit the use of biphasic systems. Useof solid-liquid biphasic systems eliminates the effects of solventtoxicity, and recently, the use of thermostable polymers hasbeen demonstrated to improve production of toxic chemicals,such as 3-methyl catechol (30). Alternatively, solvent-tolerant

FIG. 6. Recycling of the biocatalyst after 6 and 12 h using both free and immobilized cells. The reaction conditions were as follows: 30°C and200 rpm. The bars indicate the averages of three identical experiments, and the error bars indicate the standard deviations.


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strains can be used to express the enzyme to improve biocat-alyst stability in the presence of solvents that are generally toxicto microorganisms (14). Solvent-tolerant strains have beenused previously for production of toxic products, such as3-methyl catechol, in the presence of toxic organic solvents (14,15, 32). More work is being done to improve 1-naphthol pro-duction using solvent-tolerant strains and biphasic systems.

Conclusions. Whole cells of E. coli expressing TOM-Greenwere used for oxidation of naphthalene to 1-naphthol in bi-phasic media. Biphasic reactions, in which there is decreasedproduct toxicity due to in situ movement of the product intothe organic phase and increased substrate loading, increase1-naphthol productivity. Of the solvents tested, lauryl acetategave the best results, production of �0.72 g/liter 1-naphthol inthe organic phase with a productivity of 0.46 g/g (dry weight)cells after 48 h with an organic phase ratio of 40% and with 40g/liter naphthalene. The effects of the organic phase ratio andnaphthalene concentration on 1-naphthol production were in-vestigated. The highest 1-naphthol concentration, 1.43 g/literin the organic phase, and the highest productivity, 0.55 g/g (dryweight) cells, were obtained by varying the organic phase ratioand naphthalene concentration. The recycling ability of thebiocatalyst was tested using both free cells and immobilizedcells. There was significant loss of activity for both free andimmobilized cells that could be attributed to product toxicity.The production of 1-naphthol was enhanced by use of bio-catalysis and liquid-liquid biphasic reactions. On a constant-volume basis, an eightfold improvement in 1-naphthol produc-tion was obtained using biphasic systems with lauryl acetate asthe solvent compared to biotransformation in aqueous me-dium. Both the solvent and the naphthalene substrate could berecycled. However, the product concentrations have to be in-creased to at least 50 to 100 g/liter to make the process indus-trially feasible (29). More work will be done to increase theproduct concentrations by using solvent-tolerant strains thatare more stable in toxic environments.

ACKNOWLEDGMENTS

This work was done with support from NSF grant EEC-0310689 andthe Center for Environmentally Beneficial Catalysis. The Flow Cytom-etry Facility is funded through user fees and the generous financialsupport of the Carver College of Medicine, Holden ComprehensiveCancer Center, and Iowa City Veteran’s Administration MedicalCenter.

We thank Thomas Wood for kindly donating E. coli TG1 expressingTOM-Green. The cell viability data were obtained at the Flow Cytom-etry Facility, which is a Carver College of Medicine Core ResearchFacilities/Holden Comprehensive Cancer Center Core Laboratory atthe University of Iowa.

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