39[Noda Institute for Scientific Research GRANT]2006 Young Investigator Research Grant
Outline of Research Result

Development of a Water/Solvent-multiphase Bioprocess with
Solvent Tolerant Bacteria
Kohsuke HONDA
Graduate School of Engineer, Osaka University

Research aims
The use of organic solvents as reaction media has the potential to greatly expand the repertoire of biocatalytic transformations. Although it has been noticed that many enzymes can retain their activities in organic solvents containing little or no water, most of the work so far has involved
relatively simple, hydrolytic enzymes1. Therefore, attentionhas been paid to the development of a whole-cell catalyst
that functions in nonaqueous environments.
Rhodococcus opacus B4, which has recently been isolated from a gasoline-contaminated soil, is highly tolerant
to a variety of organic solvents2. During the course of thisstudy, it was found that the bacterial cells avidly adhere to
the organic phase when they were cultured in organic-aqueous biphasic media. Interestingly, wet cells of the bacteria
were also shown to be dispersible in anhydrous organic solvents. Here, it is reported that R. opacus B4 is able to retaintheir metabolic activity even in anhydrous organic solventsand to mediate bioconversion as a whole-cell catalyst.
Methods
Viability and bioconversion assays. R. opacus B4 wasaerobically cultivated on Tryptic Soy Broth (TSB). Cells
collected from 5 ml of the culture were suspended in 2 mlof an organic solvent in a screw-capped tube. The cell suspension was incubated with shaking at 30°C. After the incubation for 1 to 5 days, cells were collected by centrifugation, resuspended in an adequate volume of 0.85% NaCl,and then plated onto TSB agar plates. The plates were incubated at 30°C for 3 days for colony formation.
For bioconversion assays, the genes encoding four components of benzene dioxygenase, namely, bnzA1, bnzA2,bnzA3, and bnzA42, were constitutively expressed under thetac promoter in a recombinant plasmid pROPtacBnzA. R.
opacus B4 bearing the expression plasmid was grown in
5 ml of TSB, collected by centrifugation, and suspended in
2 ml of an organic solvent containing 5 mg ml1 indole.
Bioconversion of indole to indigo was performed at 30°Cfor 12 h with shaking.
Metabolizing activity in BEHP. Wet cells were suspended in bis(2-ethylhexyl) phthalate (BEHP) to give a cellconcentration of 2.5% (w/v), and then oleic acid was added
to the suspension at a final concentration of 10 mg ml1.
Ten milliliters of the mixture was transferred into a 50-ml

baffled flask, and incubated at 30°C with orbital shaking.
An aliquot of the mixture was sampled at time intervals of
3 h. After removing the cells by centrifugation, remaining
oleic acid in the supernatant was fluorescence-labeled with
9-anthryldiazomethane and determined by HPLC.
Indigo production. Wet cells of R. opacus B4 (pROPtacBnzA) were suspended in BEHP at a concentration of
2.5% (w/v). Indole and oleic acid were added to the cellsuspension at concentrations of 9 and 10 mg ml1, respectively. Ten milliliters of the mixture was transferred into a50-ml baffled flask and incubated at 30°C with orbital shaking. An aliquot of the mixture was sampled at time intervalsof 3 h. After removing the cells by centrifugation, indigo
production was assessed by measuring the absorbance at600 nm of the supernatant.
Results
Viability and biocatalytic ability in organic solvents
Wet cells of R. opacus B4 were well dispersible in anhydrous organic solvents. The hydrophobicity of an organic
solvent is given by the logarithm of the partition coefficientin a mixture of n-octanol and water (log Pow). A solventwith a lower log Pow shows a higher toxicity to microorganisms3. Fig. 1 shows the cell viabilities of R. opacus B4 in
organic solvents having different values of log Pow. R. opacus B4 cells were able to survive in middle- and long-chain
n-alkanes, BEHP, and oleyl alcohol for at least 5 days. It
has been reported that R. opacus B4 was able to grow on amedium containing 10% (v/v) monoaromatics, including
benzene, toluene, xylene, and ethylbenzene2. However, thebacterium was unable to survive in essentially water-freemonoaromatics. The biocatalytic ability was also investigated using the conversion of indole to indigo as a modelreaction (Fig. 1). No significant relationship was observed
between cell viability and indigo production. Among thetested solvents, BEHP was most suitable for indigo production.
Metabolizing activity in BEHP
When R. opacus B4 cells were incubated in BEHP containing 1% (w/v) oleic acid, oleic acid concentration significantly decreased with time as shown in Fig. 2. The consumption rate was nearly constant during 12 h of incubation. Approximately 64% of the initial amount of oleic acidwas consumed by 12 h. No significant decrease in the40Fig. 1. Cell viability of and indigo production by R. opacus B4 in organic solvent. The initial concentration of the bacterial suspension was 2.35109cells ml1. The value of log Pow is shown in parentheses.

Fig. 2. Oleic acid consumption by intact (squares) or heat-killed (circles) cells of R. opacus B4 in BEHP.

amount of oleic acid was observed in blind tests with heatkilled cells.
Indigo production
Because bioconversions catalyzed by benzene dioxygenase require NADH as a cofactor, NADH regeneration isessential for reactions. Bioconversion of indole to indigo byR. opacus B4 transformants was carried out in anhydrous
BEHP by employing oleic acid as an oil-soluble energysource (Fig. 3). The rates of indigo production were not different for the initial 6 h, regardless of whether oleic acidwas present. However, the difference in the rate of indigo
production became significant after 6 h of incubation. In thepresence of oleic acid, the indigo concentration reached71.6 m g ml1 by 24 h, which was approximately 1.5-foldhigher than that observed in the absence of oleic acid.

Fig. 3. Indigo production from indole by R. opacus B4 in BEHP. Experiments were performed with (squares) or without (circles) 10 mg ml1 ofoleic acid.

immiscible hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including ntetradecane, oleyl alcohol, and BEHP. R. oapcus B4 transformants carrying pROPtacBnzA catalyzed the oxidation ofindole to indigo in anhydrous BEHP. The transformants
were able to catabolize oleic acid in BEHP and thus regenerate NADH. This approach is potentially applicable toother cofactor-dependent enzymatic conversions of waterimmiscible compounds.
References1)

Conclusion
In the present study, the author developed a single-phasesolvent culture technique using a solvent-tolerant bacterium
R. opacus B4 as a whole-cell biocatalyst. R. opacus B4 hasa high hydrophobicity and exhibits a high affinity for water-2)

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