Organic co-solvent effects on the bioconversion of (R)-(+)-limonene to (R)-(+)-α-terpineol

Vol. 33, No. 7, pp. 755-761, 1908 ~'; 1908 Elsevier Science Ltd. All rights rcsct~'cd Printed in Great Britaia 11()32-05~12/0,~ $ - - see front manor

Process' B i o c h e m i s t r y

ELSEVIER PII:

S0032-9592{98)00046-6

Organic co-solvent effects on the bioconversion of (R)-( + )-limonene to (R)-( + )- -terpineol Qiang Tan and Donal F. Day Audubon Sugar Inst., Louisiana Agricultural Experimental Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA (Received 8 December 1997; revised version received 17 March 1998~accepted 22 March 1998)

Abstract

The effects of 22 organic solvents were tested on the bioconversion of (R)-(+)-Iimonene to (R)-( +)-~-terpineol by free and immobilized P d~gitatum mycelia. Free cells showed the highest bioconversion activity in water-organic co-solvent single-phase systems. The relative activity increased 2.4 fold with dioctylphthalate (1'5%, v/v) and 2.2 fold with ethyl decanoate (1.5%, v/v). Immobilized cells showed the greatest bioconversion in detergent reversed micelle systems. When Tween 80 (0.1%) or Triton 100-X ((/.1%) was added into an immobilized P. digitatum bioconversion system, the relative bioconversion activity increased 2 fold. Immobilization protected the fungal cells from the cytotoxic effects of organic solvents. Dioctylphthalate 1-5% to 2% (v/v) was the optimum concentration range for free cell bioconversion, and Tween 80 0'5% to 1% (v/v) was the optimum concentration range for immobilized cell bioconversion. © 1998 Elsevier Science Ltd. All rights reserved

Kevwords: organic co-solvent, bioconversion, limonene, ~-terpineol, t: digitatum.

non-toxic usually have restricted solvating power and consequently are of limited use. This problem is compounded bv the fact that different cell types, lines, or individual strains vary considerably in their response to a given solvent, even under the same physiological conditions [6]. Thus, selection the solvent becomes the key determining factor for biotransformations in organic solvent systems. ~-Terpineol (CjoH~sO) is the most important of the monocyclic monoterpenc alcohols. Its annual consumption has been estimated at over 13 000 kg, which places it among the top 30 commonly used flavour compounds [7]. The commercial production process requires hydration by aqueous mineral acid of pinenc or turpentine oil to the cis-terpin hydrate, followed by partial dehydration to 2-terpineol [8]. D-( +)-Limonene (C~oH~,), the primary terpene from citrus juice, can bc converted to (R)-(+)-~-terpineol by P. digitatum ( N R R L 12(12) [9]. However, limonene like many other flavour substances is poorly water-soluble and is toxic to cells at high concentrations. This study reports on

Introduction

Most volatile aroma chemicals are produced synthetically [1]. Drawbacks to chemical synthesis of these compounds are lack of specificity in the syntheses and a consumer perception that 'natural' food additives are better. Bioconversion processes potentially provide methods for the selective production of 'natural' flavours and fragrances. Most flavour substrates are insoluble in aqueous media and/or are highly cytotoxic [2-6], requiring either two-phase or organic solvent systems for bioconversion. Use of the 'right' organic solvent (1) increases the concentration of poorly water-soluble substrates: (2) reduces product and/or substrate inhibition; (3) reduces mass-transfer limitations; and (4) alters the partitioning of the substrate/product [6]. The greatest problem in using organic solvents with viable cells lies not with the system or reactor employed but rather in the choice of solvent, Most organic solvents are highly cytotoxic and/or inhibitory. Those that are 755

756

Q Tan, D. E Day

the selection of appropriate organic systems for the bioconversion of limonene to 7-terpineol. Materials and methods

Chemicals (R)-(+)-, (S)-(-)-limonene and authentic ~-terpineol were purchased from the Aldrich Chemical Company (Milwaukee, WI, USA). The purities of 2-terpineol, (R)-(+)- and (S)-(-)-limonene were 98%, 97%, and 95%, respectively. Prior to use, these compounds were purified by silica gel chromatography. The final purities of these terpenes, determined by gas chromatography, were (R)-, (S)-limonene and ~-terpineol, 98.8%, 96.1%, and 99-3% respectively. Sodium alginate, low viscosity, used for fungal immobilization, was purchased from the Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals or solvents were of the best available commercial grade.

Organism and growth conditions Stock cultures of Penicillium digitatum (NRRL 1202) were maintained on potato dextrose agar (PDA, Difco) slants. After sporulation, they were stored at 4°C. Spores were collected from two-week-old PDA plates, inoculated from stock cultures, and maintained at 25°C. The growth medium contained 10 g glucose/litre, 10 g peptone/litre, 20 g malt extract/litre, and 3 g yeast extract/litre. The pH of the medium was adjusted to pH 7.0 with NaOH (40%, w/v) prior to sterilization. 5 ml aliquots of spore suspensions (1-3 × 107 spores/ ml) were transferred aseptically into 250ml Edenmeyer flasks containing 50 ml of growth medium and incubated for 12h. This culture was transferred to fresh medium, in 1:10 (v/v) ratio, 12 h prior to use in bioconversion studies. The cultures were grown on a rotary shaker (NBS Model G25-KC rotary shaker, NBS Co., Edison, NJ) at 28°C and 100 rpm.

Immobilization of P. digitatum on calcium alginate 30 ml suspensions of germinated P. digitatum spores (early log phase, 6 h growth) containing 24.5 mg dry weight/ml of fungi were transferred into 200 ml of fresh medium and then allowed to grow for 6 h at 100 rpm and 28°C. This culture broth was suspended into 230 ml of 10% (w/v) sodium alginate, dissolved in the same medium, to produce 5% (w/v) of sodium alginate in the final mixture. The mixture was pumped, dropwise, through a tube of diameter 0.01 inch into 400 ml of cold 0.2 M CaCl2 solution. Beads were aged for 1 h in the CaCI2 solution, then removed by filtration and washed twice with a 0.9% sterile NaC1 solution, once with sterile water, and twice with sterile 0.01 M pH 7 citrate-phosphate buffer. They were stored wet at 4°C until use.

Bioconversion activity Bioconversion activity of P. digitatum mycelia was determined by aseptically withdrawing for analysis 5 ml samples from the 120h old growth culture. For immobilized cells, 3 g of wet, immobilized beads in 5 ml of pH 7 citrate phosphate buffer (0"05 M) were taken. To each sample, 50 ~l of limonene and 1.5-2% of a test organic solvent was added, and the mixture was vortexed for 30 s. The solvents were: o-xylene, cyclohexane, p-cymen, ethyldecanoate, butylbenzoate, tetradecane, dimethylphthalate, methanol, ethanol, n-amyl alcohol, hexane, isooctane, dimethyl sulphoxide, Tween 80, polyethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, glycerol, Triton 100-X, and dioctylphthalate. This reaction mix was incubated, with shaking at 100 rpm, for 12 h at 28°C and then extracted with 5 ml of re-distilled diethyl ether. An internal standards solution (50 Ill) containing two standards (tetradecane and l-decanol) was added to each reaction mixture prior to extraction. Tetradecane and 1-decanol are standards for limonene and ~-terpineol (~T), respectively. Both chemicals were prepared in methanol as 5000 ppm stock solutions. The samples were vortexed for 30 s, and then anhydrous sodium sulphate (4 g) was added to break the emulsion that formed. The ether fraction was separated, dried over anhydrous Na2SO4, and concentrated to 0-5 ml under a stream of nitrogen prior to gas chromatography. The relative activity was obtained by comparison with the control without solvent addition. The analytical methods were the same as previously reported [9].

Optimization of dioctylphthalate and methanol concentration for free cell bioconversion Dioctylphalate at 1.5% was found to improve significantly the yield of ~-terpineol produced by free cells. The concentration of this organic co-solvent was optimized to improve of the bioconversion process. Seven different concentrations (0; 5000; 10000; 15000; 20000; 30000; 40000 ppm; v/v) of dioctylphthlate were tested. Bioconversion activity from each test was determined as described above. Methanol is reported to improve the rate of bioconversion of steroids [10]. In this study, methanol (1.5%, v/v) was found to improve ~-terpineol production by free cells. The optimum concentration of this solvent was determined. Seven different concentrations (0; 1000; 5000; 10000; 20000; 30000; 40000; 80000 ppm; v/v) of methanol in the reaction mixture were investigated. Zero methanol addition was used as a control. ~-Terpineol production from the methanoltreated samples was compared with the controls. This test was repeated three times, and the mean results reported.

Bioconversion of'limonene to 7-terpineol Optim&ation of Tween 80 concentration for immobilized cell bioconversion

757

Results

Bioconversion of limonene in organic medium T w e e n 80 was f o u n d to i m p r o v e the b i o c o n v c r s i o n yield o f l i m o n e n e to ~-terpineol with i m m o b i l i z e d cells. T h e o p t i m u m T w e e n 80 c o n c e n t r a t i o n was d e t e r m i n e d b a s e d on triplicate tests o f different c o n c e n t r a t i o n s (0: 10; 100; 200; 250; 500, 1000; 5000; 10000; 20000; 40000 and 8 0 0 0 0 p p m ) in the r e a c t i o n mixture. Bioconversion activities of i m m o b i l i z e d cells with T w e e n 80 were d e t e r m i n e d as above.

22 different organic solvents were tested for their ability to e n h a n c e this bioconversion. T h e results are shown in T a b l e s 1 and 2. F o u r co-solvents e n h a n c e d the b i o c o n v e r s i o n by free cells. D i o c t y l p h t h a l a t e (1.5%, v/v) and ethyl d e c a n o a t c (1.5q4, v/v) i n c r e a s e d the bioconversion yields m o r e than 2 fold (Table 1). M e t h anol, which is often used to i m p r o v e the reaction rate in steroid bioconversions [12], increased the yield by

Table 1. Organic solvents and surfactants as co-solvents for enhancement and none inhibition on free and immobilized cells for the bioconversion of limonene to ~-terpineol Solvent and concentration (%,, v/v)

control (no solvent) Tween 80 (0'1%) Triton 100-X (0' 1% ) polyethylene glycol (2%) propylene glycol (2%) dipropylene glycol (2%) dioctylphthalatc (1.5%.) ethyl decanoate (1.5 ,,~) methanol (1-5%) glycerol ( 2 ~ ) dimethyl sulphoxide (2c~)

Log P "

NA ---- 1.4 t> - 1.4 h 9.6 4-9 I).76 - 3"0 " -1.3

Immobilized cells

Free cells

7-Terpineol (mg/g dry cells in beads)

Relative activity ((;)

>Tcrpincol (mg/g dry cells)

Relative activity (C/c,)

85.2±5.1 186.2+__ 16"8 170.5 ± 27.3 132.5 + 6.7 112-1 ± 6-8 112.5 f. 7-9 78-5 ± 13.3 76.3-+ 14-4 81.7 ±5.7 107.2 _± 16.4 91.5±5.4

ll)0 218 +0 200 + 16 155 + 5 131 L 6 132 ~7 NS NS NS NS NS

197.8± 16.0 185.9 ~=. - _~7.q 1543 ±33-~ 242.4 :~ 17.1 203.7 ± 2().4 210.4± 19-0 544.2 ± 76.2 436.1 +51).2 228.3 ± 18-2 215"6 -~34"6 219.7 ~- 19.7

100 NS NS 123 ± 7 NS NS 243 ± 14 220± I'~ 115 +8 NS NS

" Values following Laane et al. [11]. h Values were calculated based on Laane et al. [ll].NS means no significant diffcrencc of relative activity compared with the control.

Table 2. Organic solvents as co-solvents for inhibition of free anti immobilized cells for the hioconversion of limonene to :~-terpineol Solvent anti concentration (%, Wv)

control (no solvent) tripropylene glycol (2%) dimethylphthalate (1'5%) diethylphthalate (1-5%) o-xylene (1"5%) cyclohexane (1.5%) p-cymen (1.5%) butylbenzoate (1.5%) tetradecacane (1.5%) ethanol (2%) n-amyl alcohol (2%) hexane (2%) isooctane (2%:)

Log P ~'

NA - 1.5 h 2.3 3.3 3.1 32 4.1 3.7 7-6 -0.24 1'3 3.5 4"5 "

Immobilized cells

Free cells

:~-Terpineol (mg/g dry cells in beads)

Relative activity (54)

>Terpincol (mg/g OU, cells)

Relative activity ((7()

85.2 + 5" 1 0 101.5 ± 15.2 30'4 + 3.6 13.2±0.5 38.9_+ 27 50.4_+6"1 40.6_+ 6"5 49.3 ± 6-4 70-6 ± 5.7 1.5+0.0 38.5 + 3.5 55"1 + 11-6

1{10 IN NS IN IN IN IN IN IN NS IN IN IN

197.8 + 16.0 0 87.0 +_ 15.6 34.6 + 3.7 (1 0 1&7+0.7 22.3 + 1.5 66.9 -+ 5-2 t) I-1 + 10.0 0 53-5 ± 2-6 1/18.7+ 15.2

100 IN IN IN IN IN IN IN IN IN 1N IN IN

" Values following Laane et al. [11]." Values were calculated based on Laanc ct al. [11].NS means no significant difference of relative activity compared with the control.IN means inhibition effect of relative activity c~mpared with the control.

Q. Tan, D. E Day

758

15% at a concentration of 1.5% (v/v). Polyethylene glycol (2%, v/v) produced a small improvement in yield, 23_+7%. The free cell bioconversion was inhibited by tripropylene glycol, dimethylphthalate, diethylphthalate, o-xylene, cyclohexane, p-cymen, butylbenzoate, tetradecacane, ethtanol, n-amyl alcohol, hexane, and isooctane at concentrations between 1.5% and 2% (v/v) (Table 2). Organic co-solvents had different effects on immobilized cells compared with free cells. The addition of non-ionic surfactants (Tween 80 and Triton 100-X) increased the bioconversion yield about 2 fold (Table 1). Primary and secondary propylene glycol had little effect on bioconversion activity. Dioctylphthalate (1"5%, v/v) and ethyl decanoate (1.5%, v/v) had no significant effect on bioconversion by immobilized cells. Glycerol and dimethyl sulphoxide, as co-solvents at 2% (v/v) concentration, had no effect (enhancement or inhibition) on this bioconversion. Organic solvents were less cytotoxic to immobilized cells than to free cells. The protective effect of immobilization in this system has been reported previously [10].

400

110 100

350

90

A B

300

i

250

8o 70

O

60

200

~

150

50

~ C .2 P

40

C ou

.2 30 ~

G

.~_ -~ 100 Q a:

20 50

•

Relative activity to control (%) Bioconversion extent (%)

0 3.6

I 3.8

I 4

I 4.2

10

I 4.4

I 4.6

0 4.8

Dioctylphthalate concentration (Log ppm)

Fig. 2. Effect of dioctylphthalate concentration on free cell bioconversion extent and activity relative to the control (10ll%) without dioctylphthalate. Dioctylphthalate concentration determined from 5 ml of 12 h mycelia in broth. Values are an average of three trials. Error bars show SD.

Threshold effects of dioctylphthalate and methanol on free cell bioconversion Dioctylphthalate and methanol concentrations were optimized for free cells (Figs 1 and 2). The highest product yield was obtained at a methanol concentration of 5000 ppm (v/v). At this concentration, yield increased by 45%. The extent of bioconversion, the ratio of c~-terpineol produced to the amount of substrate, limonene, added, increased from 27.3% to 39.5%. Cytotoxic effects due to the solvent were 160

100

140

90

A

Bioconversion with reversed micelle .systems

80

120 --

70

~ 100 O

60

'~

~,

80

50

.2

60

.0

E

30

.9

'~ "6

40

®

m

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2O 2O o 2.5

f

] - -Ae - Bi~.v~r~ion e~.t ~) [ 3

I 3.5

I 4

lO

\\

I q

~L

•

observed when the methanol concentration was greater than 20000 ppm. A concentration of 20000 ppm (2%, v/v) dioctylphthalate in the mixture produced the highest ~-terpineol yields. At this concentration, the product yield increased about 2.5 fold. The extent of bioconversion increased from 35.6% (control) to 92.4%. When dioctylphthalate concentration was above or below this value, the product yield decreased. Cytotoxicity by this solvent was not obvious until the concentration was greater than 4% (v/v) (Fig. 2).

o

4.5

Methanol concentration (Log ppm)

Fig. 1. Effect of methanol concentration on free cells bioconversion extent and activity relative to the control (100%) without methanol. Free cells were 12h old P. digitatum mycelia. Values are average of three trials. Error bars show the SD.

Reversed micelles were produced by adding non-ionic surfactants (Tween 80 and Triton 100-X) to the bioconversion system. These surfactants, at 0.1% concentration, increased the bioconversion yield of immobilized cells by greater than 2 fold. The surfacrants did not alter the bioconversion by free cells (Table 1). Tween 80 over a range of 100-40000ppm increased the bioconversion yield by immobilized cells. The yield increased more than three fold when Tween 80 concentrations were between 5000 and 40000 ppm (Fig. 3). Because of potential difficulties with product recovery at high concentrations of Tween 80, the practical concentration of Tween 80 for this bioconversion is between 0.5% and 1% (v/v). The extent of bioconversion was 46% when 2% (v/v) substrate was used.

Bioconversion time-course with Tween 80 addition The effect of addition of Tween 80 (1%, v/v) on the bioconversion rate is shown in Fig. 4. Tween 80

Bioconversion of fimonene to ~-terpineol 500

759

Discussion

6o

450

-

50

400

o 350

.o. • -,

40

a00

,~

250

30 ._o .~

200

20

> 150

o

100 Relative activity to control (%) I 50

~

0 0.5

I 1

I 1.5

I 2

I 2.5

10

I

Bioconver on extent (%)

I 3

I 3.5

I 4

I 4.5

I 5

0 5.5

Tween 80 concentration (Log ppm)

Fig. 3. Effect of Tween 80 concentration on bioconvcrsion extent and relative activity to the control (100%, no Tween 80 addition) with immobilized P. digitatum mycelia. Each assay contained 3g of immobilized cells in 5 ml of pH 7 citrate phosphate buffer (0.05 M). Values are an average of three trials. Error bars show the SD. inhibited the bioconversion activity by free cells. However, Tween 8(1 enhanced the bioconversion yield for immobilized cells, increasing the rate of bioconversion (Fig. 4). With Tween 80, the bioconversion for immobilized cells reached the maximum yield by 72 h. Without Tween 80, the reaction took more than 120 h to reach the maximum yield. 1.4

3.5

~_

,,

_

E

g

l0

0.8

2

=- 0.6

1.s .~

~0.4

, ~

"~. ,.e 0

0 0

24

48

72

96

120

144

168

192

216

Time (h)

Fig. 4. Time course of ~-terpineol production by free and immobilized P. digitatum myeelia with and without 1% (v/v) Tween 80 addition. For free cells, each assay contained 5 ml

of 12h old cells in broth. For immobilized cells, 3g of immobilized cells in 5 m l p H 7 citrate phosphate buffer (0.05 M) were used for each assay.

Organic co-solvents can be applied to bioconversion systems in three different ways, in a single-phase, two-phase, or reversed micelles system. True singlephase systems are produced when water-miscible co-solvents are added to the medium to improve the solubility of normally insoluble compounds [6]. This may reduce mass-transfer limitations, increasing reaction rates. Two-phase systems have a continuous and a discontinuous phase, formed by two (or more) immiscible liquids. The aqueous phase normally contains the biocatalyst, either dissolved, colloidal, or in insoluble form (quite possibly immobilized) and is sometimes known as the 'biophase'. Organic solvents make up the non-aqueous phase and include the substrate/product, typically in low concentrations. Reversed micellcs, also called microemulsions, are thermodynamically stable, 'single-phase" systems, where the addition of an appropriate amphiphile or detergent (surfactant) permits the single-phase coexistence of otherwise mutually insoluble aqueous and organic media. Based on many studies with single solvents, a widespread consensus has emerged regarding the parameters that define the appropriate organic solvent. The generalized case is that biocatalyst stability decreases as log P increases, where l o g P is the logarithm of the octanol:water partition coefficient of the solvent. Stability reaches a minimum ti)r log P values between 11 to 2 for enzymes, and bctween 2 and 4 for microorganisms. Above these ranges incrcasing log P of the solvent (or fl)r that matter substrate) results in increased biocatalyst stability [2,11,13-15]; i.e. biocatalysts arc more stable in less polar solvents. The transition point from cytotoxicity to non-toxicity for solvents typically occurs between a log P of 3-5 [16]. Limonene is a lipophilic compound with a low solubility in aqueous media. The following strategies were applied to increase bioconversion rates: (1) addition of single-phase water-organic co-solvent systems (methanol, dimethyl sulphoxide, propylene glycol, glycerol, and several esters); (2) use of rcvcrsed micelle systems using non-ionic surfactants (Twcen 80 and Triton 100-X); and (3) the use of organic solvent two-phase systems. Generally, all of the water-organic, co-solvent, single-phase systems tested showed some enhancement of bioconversion. Methanol at 0.5% (v/v) increased the yields 50% for bioconversion with free cells. At methanol concentrations above 2.5% (v/v), yields rapidly declined. This critical point effect has been reported for many organic co-solvents [6]. Methanol has been used to enhance steroid bioconversions [17, 18]. The greatest yield improvement was seen using the water-organic, co-solvents esters, dioctylphthalate and ethyl decanoate. They increased product yields by 2.5 and 2.2 fold, respectively. Lilly ct al. [19] has reported that these two solvents and dodecane (all of them logP > 4) improve the rate of A~-dehydrogen -

760

Q. Tan, D. E Day

ation of hydrocortisone by Arthrobacter simplex. The optimum concentration of dioctyl phthalate was determined to be about 2% (v/v) for the terpineol bioconversion, whereas Lilly et al. [19]used equal amounts of organic and aqueous phases. In the hydrocortisone biotransformation, the conversion rates were tested after 2 h of reaction. Bioconversion of limonene by P digitatum typically takes more than 12 h. Exposure time is an important factor that influences the cytotoxicity of organic solvents [6]. The non-ionic surfactants Tween 80 and Triton X-100 produced reversed micelles which significantly improved the bioconversion yields of immobilized P. digitatum mycelia, but had no effect on free cell yields. The mycelial support, Ca-alginate, is hydrophilic, while the substrate is lipophilic, undoubtedly reducing substrate contact for immobilized cells. Comparative studies have been performed on bioconversion activity in hydrophilic (H-gel) and lipophilic (l-gel) gels [20-22]. For transformations of highly hydrophobic substrates, such as steroids, terpenoids, and various organic compounds, lipophilic gel-entrapped biocatalysts are more effective. The solubility of the substrates as well as their products, is enhanced and the hydrophobicity of the gels leads to rapid diffusion through gel matrices. Owing to product recovery and downstream process considerations, a 0-5% to 1% (v/v) concentration of Tween 80 for immobilized mycelia is recommended for this bioconversion, even though the highest activity was obtained with a 4% Tween 80 concentration. There were no significant changes in product yields between 0-5% and 4% (v/v) Tween 80. Bioconversion decreased when the Tween 80 concentrations were above 4%. This may not be true inhibition but rather a loss in product recovery. Tween 80 had no effect on bioconversion by free cells. The walls of vegetative mycelia are naturally lipophilic such that even without detergent, the surrounding mycelia may be saturated with substrate. A non-ionic surfactant, Prawozell Won-100, has been reported to enhance hexadecane hydroxylation, which is catalysed by the NADPH-cytochrome P-450 reductase from the yeast Lodderomyces elongisporus [23] and Tween 80 has been used to accelerate cholesterol degradation rates by Mycobacterium strain DP [24]. Isooctane enhances the microbial conversion of /3-ionone with Aspergillus niger [12] but this solvent inhibited limonene bioconversion by P digitatum. Methanol is toxic to /Monone conversion [12] but enhanced limonene bioconversion. The effects of hexane on both conversions were similar. The log P values of both isooctane and tetradecane are above 4, but the relative bioconversion activities were only 55% and 34%, respectively, of the control. Hailing [5] pointed out that the log P value is an appropriate parameter to measure the tendency of solvents or other organic molecules to partition between phases of differing polarity, but the reaction involves the direct

effects of the bulk organic phase, or the solvation of other species within in it, then logP of the solvent is not an appropriate parameter. The best organic co-solvent system for limonene bioconversion was dependent upon the usage mode of the biocatalyst. For free cells a water-organic co-solvent system using either dioctylphthalate or ethyl decanoate produced the highest product yields. For immobilized mycelia, a reversed micelle system using Tween 80 or Triton X-100 was best. The 'so-called' log P rule was useful only for general selection of the best solvents. It did not hold all test cases.

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Bioconversion of lirnonene to ~-terpineol

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