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Whole-cell biocatalysis to oxidize naphthalene to 1-naphthol in liquid-liquid biphasic systems was performed. Escherichia coli expressing TOM-Green, a variant of toluene ortho-monooxygenase (TOM), was used for this oxidation. Three different solvents, dodecane, dioctyl phthalate, and lauryl acetate, were screened for biotransformations in biphasic media. Of the solvents tested, lauryl acetate gave the best results, producing 0.72 ± 0.03 g/liter 1-naphthol with a productivity of 0.46 ± 0.02 g/g (dry weight) cells after 48 h. The effects of the organic phase ratio and the naphthalene concentration in the organic phase were investigated. The highest 1-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 immobilized in calcium alginate was tested. Both free and immobilized cells lost more than ~60% of their activity after the first run, which could be attributed to product toxicity. On a constant-volume basis, an eightfold improvement in 1-naphthol production was achieved using biphasic media compared to biotransformation in aqueous media.
Biocatalysis has emerged as an important technology in industrial organic synthesis for the production of chemical synthons and high-value products (29, 34, 37). Biocatalysis offers the advantage of performing reactions under mild conditions and provides an environmentally benign approach for chemical reactions (1, 38). Oxygenases are a class of enzymes that have great potential and versatility for catalyzing reactions that are generally not accessible by chemical routes with high regio-, stereo-, and enantioselectivities (6, 27, 42, 43). Oxygenases introduce either one or two atoms of molecular oxygen into organic molecules using NADH or NADPH as a cofactor. To eliminate the addition of a costly cofactor, whole cells expressing oxygenases are generally used (34, 43).
One of the potential applications of biocatalysis utilizing oxygenases 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 et al. (39) have compared the reaction rates and regioselectivities of various wild-type and modified monooxygenases for the oxidation of naphthalene to 1-naphthol. Of the monooxygenases tested, the best enzyme for the oxidation of naphthalene to 1-naphthol was a toluene ortho-monooxygenase (TOM) variant, TomA3(V106A), also known as TOM-Green. TOM was isolated from Burkholderia cepacia G4 and consists of an α2β2γ2 hydroxylase (encoded by tomA1, tomA3, and tomA4) with two catalytic oxygen-bridged binuclear iron centers, an NADH-oxidoreductase (encoded by tomA5), a protein (encoded by tomA2) involved in electron transfer between oxidoreductase 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 the alpha-subunit of the hydroxylase (7, 33). TOM-Green retained high regioselectivity (98%) and was sevenfold faster than wild- type TOM.
There has been considerable effort to identify and characterize oxidative biocatalysts for 1-naphthol production (7, 11, 26, 33, 36, 40, 41). However, this process is not economically feasible owing to the very low optimum concentration of naphthalene (0.1 mM , which is less than the solubility of naphthalene, 0.23 mM ) and the toxicity of both naphthalene and 1-naphthol (38, 44). Substrate loading has to be increased, and the toxicities of both naphthalene and 1-naphthol have to be minimized to make the process feasible. As a consequence, biotransformations in water-organic solvent biphasic media have been developed (8, 9, 12, 21, 45, 46). The use of a second phase consisting of an organic solvent not only increases substrate loading but also maintains low concentrations of toxic compounds in the aqueous phase (4). The organic solvent chosen is critical for achieving the benefits of biphasic media. Two main criteria for solvent selection are a high distribution coefficient for the product and biocompatibility with microorganisms (3, 4). Biocompatibility is generally correlated with the logP of the solvent, which is the logarithm of the partition coefficient in an octanol-water system, and organic solvents with logP values greater than 4 are generally biocompatible with microorganisms (19). However, the correlation of activity with logP is specific to the microorganism, and the critical logP above which solvents are biocompatible has to be identified for each microorganism (8, 16).
Biphasic systems have been widely used for reactions involving a toxic substrate and/or product to enhance productivity or to improve recovery of the product (22-25, 31, 38). Oxidation of naphthalene has also been improved using biphasic reactions (13, 23, 35, 38). Tao et al. (38) used a biphasic system for 2-naphthol and phenol production using toluene 4-moooxygenase and its variant TmoA(I100A). They obtained 10- to 21-fold increases in 2-naphthol and phenol concentrations using dioctyl phthalate as the organic solvent. McIver et al. (23) used naphthalene dioxygenase to oxidize naphthalene to cis-(1R,2S)-1,2-naphthalene dihydrodiol using dodecane as the organic solvent and obtained a productivity of 1.7 g/g (dry weight) cells/h in the first 6 h. In spite of the significant improvements achieved by using a biphasic system for various reactions, application of this strategy to 1-naphthol production has not been explored yet. Considering the high toxicities of naphthalene and 1-naphthol (38), biphasic reactions can enhance the productivities. In this work, a biphasic system was used to increase 1-naphthol productivities with whole cells of Escherichia coli expressing the TOM-Green enzyme. Organic solvents were screened, and solvents suitable for high 1-naphthol productivity were identified. The organic phase was optimized by studying the effects of the naphthalene concentration and the organic phase ratio. The stability of the biocatalyst for recycling was also tested.
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 (Hanover Park, IL). Luria-Bertani (LB) broth was purchased from Difco (Lawrence, KS). E. coli TG1/pBS(Kan)TOM-Green expressing the TOM-Green enzyme was kindly donated by Thomas K. Wood (Texas A&M University). Plasmid pBS(Kan)TOM-Green expresses TOM-Green (tomA012345) from a lac promoter. The lac promoter yields constitutive expression of TOM-Green genes due to the high copy number of the plasmid and the lack of a lacI represser (7, 40).
The cells were grown until early log phase in 250-ml shake flasks, and then growing cells (5 ml) were added to 20-ml sterile screw-cap vials. Naphthalene or 1-naphthol dissolved in 50 μl dimethyl formamide (DMF) was added to the growing cells to obtain final concentrations of 0.05 g/liter, 0.1 g/liter, 0.5 g/liter, and 1 g/liter. The growth was monitored by determining the optical density at 660 nm (OD660). Due to the low solubilities of naphthalene and 1-naphthol in water, the cosolvent DMF was used to suspend the compounds in the aqueous phase. A positive control experiment in which 50 μl DMF was added without naphthalene or 1-naphthol was also performed.
A Becton Dickinson LSR II flow cytometer at the University 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 was performed as suggested by the supplier.
High-performance liquid chromatography (HPLC) was used to quantify 1-naphthol and naphthalene, using a method similar to the method used previously (23). A series 1100 Agilent HPLC with a photodiode array detector and a Supelcosil LC-PAH 5 μm column (25 cm by 4.6 mm) at room temperature was used for this analysis. Aqueous samples were diluted 1/2 in acetonitrile. Organic samples were injected directly without dilution. Both organic and aqueous samples were centrifuged to separate cell debris and were filtered with 0.2-μm polytetrafluoroethylene filters.
The mobile phase consisted of deionized water with 0.2% (vol/vol) glacial acetic acid and acetonitrile (Optima grade; Fisher Scientific) with gradient elution. 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 at 8 min and equilibration until 10 min. The mobile phase flow rate was 1.5 ml/min, and the injection volume was 10 μl. Naphthalene and 1-naphthol were analyzed at 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 concentrations of naphthalene and 1-naphthol in each phase.
Different organic solvents (500 μl) containing 0.75 g/liter 1-naphthol were added to 500 μl of phosphate buffer in 2-ml microcentrifuge tubes. The two phases were mixed by vortexing them for 30 s five times with 1-min intervals between treatments. The two phases were analyzed using HPLC. A distribution coefficient was calculated by determining the ratio of the 1-naphthol concentration in the organic phase to the 1-naphthol concentration in the aqueous phase.
All biotransformations were conducted in 250-ml Erlenmeyer flasks with a 50-ml working volume at 30°C and 200 rpm. Fresh LB medium 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 LB medium 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 buffer (pH 7.2), and resuspended in 0.5 volume of phosphate buffer (pH 7.2) to increase the cell density. The medium was supplemented with 20 mM glucose and 100 mg/liter kanamycin. Resuspended cells (30 ml) were used to measure the dry weight of cells. For aqueous biotransformation, naphthalene at a final concentration of 0.5 g/liter was added to 50 ml of resuspended cells using DMF as a cosolvent due to the low water solubility of naphthalene. For biphasic biotransformations, the desired volume of an organic solvent with a known concentration of dissolved naphthalene was added to the aqueous phase of resuspended cells to obtain 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 of three identical experiments.
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 lauryl acetate phase was monitored using HPLC.
Immobilization of cells was performed using a method similar to 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 centrifugation at ~10,000 × g for 10 min. The cells were washed with Tris buffer (pH 7.2). The cells for each experiment were immobilized together and later divided and placed into six separate flasks. A 3% sodium alginate solution was prepared using 120 ml of deionized water. A 1% CaCl2 solution was prepared as the gelation agent using deionized water. Both the sodium alginate and CaCl2 solutions were autoclaved at 121°C for 15 min. The sodium alginate solution was allowed 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. The cells and sodium alginate solution were mixed by stirring them for 5 min on a stir plate. The mixture was added dropwise to the stirred gelation agent using a 60-ml syringe with an 18-gauge needle. The mixture was stirred for 1 h to harden it. The resulting calcium alginate beads were 1 to 2 mm in diameter. After hardening, the beads were removed from the solution and washed twice with sterilized deionized water. The immobilized cells were divided equally among six sterile flasks containing 19.5 g of beads each.
The immobilized biocatalyst was suspended in 250-ml Erlenmeyer flasks with a working volume of 50 ml. The beads were suspended in 30 ml of Tris-HCl buffer (pH 7.2) supplemented with 20 mM glucose and 100 mg/liter kanamycin. The solvent phase (20 ml) was added to begin the reaction, and the flasks were shaken at 200 rpm and 37°C. An HPLC analysis was done using samples of the solvent phase.
Whole cells of E. coli TG1 expressing TOM-Green were used for oxidation of naphthalene to 1-naphthol. The toxicities of both naphthalene and 1-naphthol for the E. coli TG1 strain expressing TOM-Green are shown in Fig. Fig.1.1. Naphthalene inhibited cell growth even at a low concentration, 0.05 g/liter. The inhibition of growth increased as the concentration of naphthalene increased to 0.5 g/liter, and no growth was observed with 1 g/liter naphthalene. The inhibitory effect of 1-naphthol was greater than that of naphthalene, and no growth was observed even with 0.5 g/liter 1-naphthol. These results are comparable to the results of similar work done previously (38). Therefore, maintaining low concentrations of the substrate and the product is critical for maintaining the cell viability and the activity for the reaction.
The oxidation of naphthalene to 1-naphthol using whole-cell TOM-Green is shown in the Fig. Fig.2.2. The TOM-Green enzyme uses molecular oxygen and NADH as a cofactor. Oxidation of naphthalene to 1-naphthol was performed in an aqueous medium, and 0.04 g/liter of 1-naphthol was obtained after 24 h using E. coli TG1/pBS(Kan)TOM-Green. To improve 1-naphthol production, biphasic biotransformations were performed with a 40% organic phase and 40 g/liter naphthalene dissolved in a solvent. The organic solvent chosen is critical for achieving the maximum benefits from biphasic reactions. Three solvents, dodecane, dioctyl phthalate, and lauryl acetate, were chosen for screening. Table Table11 shows the distribution coefficients and structures of the three solvents used for 1-naphthol production. Dodecane (23, 35) and dioctyl phthalate (35, 38) were used previously to improve 1,2-naphthalene dihydrodiol and 2-naphthol productivities, respectively. Lauryl acetate was chosen because it has a high distribution coefficient for 1-naphthol. Although dodecane has a low distribution coefficient for 1-naphthol, it was used to study the effect of 1-naphthol partitioning. All three solvents have logP values greater than 4. The biocompatibility of the solvents was confirmed by assaying cell viability using flow cytometry. The viability of E. coli/pBS(Kan)TOM-Green cells was approximately 99% after 3 h of exposure to a 40% organic phase with any of the three solvents (dodecane, lauryl acetate, or dioctyl phthalate).
Biphasic biotransformations were performed using the three solvents. Figure Figure33 shows the 1-naphthol concentration in the organic phase and the 1-naphthol productivity expressed in grams of 1-naphthol formed per gram (dry weight) of cells when 40 g/liter naphthalene was in the organic solvent. When dodecane was used, 1-naphthol was formed within 3 h in a biotransformation, and after this the cells lost their activity. However, when either lauryl acetate or dioctyl phthalate was used, 1-naphthol was formed at a steady rate, approximately 0.06 g/liter/h, for 6 h. Production of 1-naphthol continued at a lower rate up to 48 h. The formation of 1-naphthol was significantly greater when either lauryl acetate or dioctyl phthalate was used than when dodecane was used. After 48 h, the concentrations of 1-naphthol obtained with lauryl acetate and with dioctyl 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 with dodecane (0.075 g/liter). A comparison of lauryl acetate and dioctyl phthalate showed that lauryl acetate gave slightly higher concentrations of 1-naphthol. Although both of these solvents have high distribution coefficients for 1-naphthol, dioctyl phthalate has a high viscosity and requires a longer mixing time to reach equilibrium. Therefore, lauryl acetate gave the best results, with 0.46 ± 0.02 g 1-naphthol/g (dry weight) cells produced after 48 h.
After identification of lauryl acetate as the best of the solvents tested, the organic phase ratio and the naphthalene concentration had to be optimized to achieve the best results. Different organic phase ratios and naphthalene concentrations were tested using lauryl acetate for 1-naphthol production, and their effects are shown in the Fig. Fig.4.4. 1-Naphthol productivity was increased when either the naphthalene concentration in the organic phase was increased from 20 to 70 g/liter or the organic phase ratio was increased from 20 to 60%. Higher naphthalene concentrations in the organic phase allow more naphthalene to partition into the aqueous phase, thereby increasing the naphthalene bioavailability. A higher organic phase ratio also promotes the reaction by allowing better partitioning of 1-naphthol, thereby minimizing its toxicity and improving productivity. Improvement in 1-naphthol productivity with an increase in the organic phase ratio was also observed previously (38). However, the 1-naphthol productivity decreased when the organic phase ratio was increased from 60 to 80%. For the different reaction conditions tested, the highest 1-naphthol concentration (1.43 g/liter) and the 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 increase in the organic phase ratio, the 1-naphthol concentration in the organic phase decreases. Therefore, the reaction conditions can be optimized either for high 1-naphthol productivity, which impacts upstream processing costs, or for a high 1-naphthol concentration in the organic phase, which impacts downstream processing costs. In order to find the optimum reaction conditions, the effects of the naphthalene concentration and the organic phase ratio were determined after 24 h, and the results are shown in Fig. Fig.5.5. Although a high organic phase ratio and a high naphthalene concentration improve productivity, the data in Fig. Fig.55 demonstrate that the optimum conditions for the reaction can be achieved at organic phase ratios and naphthalene concentrations lower than the maximum evaluated levels without a significant decrease in productivity. The optimum conditions for high 1-naphthol productivity were an organic phase ratio of 40% and a naphthalene concentration of 60 g/liter. The optimum conditions for a high 1-naphthol concentration were an organic phase ratio of 20% and a naphthalene concentration of 60 g/liter. Table Table22 shows the amount of 1-naphthol formed for each condition and the percentage of naphthalene converted to 1-naphthol. The maximum conversion value, 3.5%, was obtained with 20 g/liter naphthalene and an organic phase ratio of 20%. Under the optimum conditions for high 1-naphthol productivity and a high 1-naphthol concentration in the organic phase, naphthalene conversion values of 1.3% and 2.3%, respectively, were obtained.
Recycling of the biocatalyst improves the process economics. Recycling experiments were performed to test the stability of the cells. The stability of both free cells and calcium alginate-immobilized cells was tested. Immobilized cells produced approximately 40% of the 1-naphthol produced by free cells (0.196 ± 0.014 g 1-naphthol/g [dry weight] cells for immobilized cells, compared to 0.49 ± 0.01 g 1-naphthol/g [dry weight] cells for free cells after 6 h of biotransformation with an organic phase ratio of 40% and 60 g/liter naphthalene). The decrease in immobilized cell activity could have been due to mass transfer limitations due to the calcium alginate beads. A similar decrease in the activity of immobilized cells compared to free cells was observed previously (23). The reaction time for recycling experiments is critical in determining the stability of the biocatalyst, considering its exposure to toxic products, toxic substrates, and organic solvents. Optimum conditions for 1-naphthol productivity (organic phase ratio of 40% and 60 g/liter naphthalene) were used for the recycling experiment. As shown in Fig. Fig.55 for 1-naphthol production under optimum reaction conditions for high productivity, 1-naphthol was produced at essentially a linear rate up to 6 h and at a reduced rate thereafter. Therefore, two reaction times, 6 h and 12 h, were chosen to test the stability of cells at the two reaction rates. The percentages of activity retained by free and immobilized cells for four cycles are shown in Fig. Fig.6.6. In the 6-h recycling experiment, free cells showed greater retention of activity. Approximately 40% of the activity of free cells was retained for the second run, compared to 20% for immobilized cells. The activities decreased further for the third and fourth runs. In the 12-h recycling experiment, both free and immobilized cells lost most of their activity, and only 20% of the activity was retained for the second run.
Because of the wide applications of 1-naphthol and the versatility of toluene monooxygenases, production of 1-naphthol using whole cells expressing TOM-Green in a biphasic system was evaluated. Efficient in situ removal of the toxic compound 1-naphthol from the aqueous phase was critical for improving 1-naphthol productivity. The high distribution coefficients of lauryl acetate and dioctyl phthalate for 1-naphthol enabled 10- and 7-fold improvements in 1-naphthol productivity, respectively, compared to the productivity with dodecane. The higher 1-naphthol productivities obtained with lauryl acetate and with dioctyl phthalate than with dodecane suggest that the dynamics of 1-naphthol partitioning into the organic phase plays a major role in maintaining cellular activity and improving 1-naphthol productivity. Moreover, inefficient partitioning of 1-naphthol results in accumulation of the toxic product in the aqueous phase, thereby lowering the cellular activity and 1-naphthol productivity. Similar improvements in productivity were obtained by using solvents with high distribution coefficients for the product 2-naphthol (38). Compared to biotransformations in the aqueous systems, on a constant-volume basis, an eightfold improvement in 1-naphthol production was obtained using lauryl acetate as the second phase in a biphasic system (16.4 mg 1-naphthol [with 70 g/liter naphthalene and an organic phase ratio of 60%] in the biphasic system, compared to 2 mg 1-naphthol [0.04 g/liter 1-naphthol] with the aqueous medium for 50-ml reactions).
The stability of E. coli/pBS(Kan)TOM-Green for 1-naphthol production in a biphasic system was also tested, and more than 60% of the activity was lost for the second run after recycling. Similar recycling for biphasic biotransformation has been performed previously (23) to produce nontoxic cis-1,2-naphthalene dihydrodiol, and the activity was retained for up to four runs for 6 h of recycling. However, in the reaction examined here, the product, 1-naphthol, is very toxic and could be the main reason for the loss of activity during recycling (38). Moreover, diffusional limitations for immobilized cells may result in 1-naphthol accumulation in the beads that would adversely affect immobilized cell activity (18).
The 1-naphthol concentrations in the organic phase and the naphthalene conversion values obtained in this work are comparable to results obtained previously for a similar compound, 2-naphthol (38). The low level of naphthalene conversion is due to high substrate loading and the toxicity of the product, 1-naphthol. The naphthalene added to the biphasic system can be recycled along with the organic solvent to improve the economics. Although higher product concentrations and conversion values were obtained for oxidation reactions in a biphasic system (5, 31), the high toxicity of 1-naphthol is the main factor limiting the production of higher 1-naphthol concentrations. Additional organic solvents can be tested based on biocompatibility and high selectivity for 1-naphthol. However, the toxicities of solvents limit the use of biphasic systems. Use of solid-liquid biphasic systems eliminates the effects of solvent toxicity, and recently, the use of thermostable polymers has been demonstrated to improve production of toxic chemicals, such as 3-methyl catechol (30). Alternatively, solvent-tolerant strains can be used to express the enzyme to improve biocatalyst stability in the presence of solvents that are generally toxic to microorganisms (14). Solvent-tolerant strains have been used previously for production of toxic products, such as 3-methyl catechol, in the presence of toxic organic solvents (14, 15, 32). More work is being done to improve 1-naphthol production using solvent-tolerant strains and biphasic systems.
Whole cells of E. coli expressing TOM-Green were used for oxidation of naphthalene to 1-naphthol in biphasic media. Biphasic reactions, in which there is decreased product toxicity due to in situ movement of the product into the organic phase and increased substrate loading, increase 1-naphthol productivity. Of the solvents tested, lauryl acetate gave the best results, production of ~0.72 g/liter 1-naphthol in the 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 40 g/liter naphthalene. The effects of the organic phase ratio and naphthalene concentration on 1-naphthol production were investigated. The highest 1-naphthol concentration, 1.43 g/liter in the organic phase, and the highest productivity, 0.55 g/g (dry weight) cells, were obtained by varying the organic phase ratio and naphthalene concentration. The recycling ability of the biocatalyst was tested using both free cells and immobilized cells. There was significant loss of activity for both free and immobilized cells that could be attributed to product toxicity. The production of 1-naphthol was enhanced by use of biocatalysis and liquid-liquid biphasic reactions. On a constant-volume basis, an eightfold improvement in 1-naphthol production was obtained using biphasic systems with lauryl acetate as the solvent compared to biotransformation in aqueous medium. Both the solvent and the naphthalene substrate could be recycled. However, the product concentrations have to be increased to at least 50 to 100 g/liter to make the process industrially feasible (29). More work will be done to increase the product concentrations by using solvent-tolerant strains that are more stable in toxic environments.
This work was done with support from NSF grant EEC-0310689 and the Center for Environmentally Beneficial Catalysis. The Flow Cytometry Facility is funded through user fees and the generous financial support of the Carver College of Medicine, Holden Comprehensive Cancer Center, and Iowa City Veteran's Administration Medical Center.
We thank Thomas Wood for kindly donating E. coli TG1 expressing TOM-Green. The cell viability data were obtained at the Flow Cytometry Facility, which is a Carver College of Medicine Core Research Facilities/Holden Comprehensive Cancer Center Core Laboratory at the University of Iowa.
Published ahead of print on 21 August 2009.