Organic media selection for the extraction of β-hydroxyisobutyric acid produced by microbial biotransformation

Organic media selection for the extraction ofb-hydroxyisobutyric acid produced by microbialbiotransformationRosa Leon,1 Francesco Molinari,2 Duarte MF Prazeres1 and Joaquim MS Cabral1*1Centro de Engenharia Biologica e Quımica, Instituto Superior Tecnico, Av Rovisco Pais, 1096 Lisbon, Portugal2Dipartimento Scienze Tecnologie Alimentari Microbiologiche, Universita degli Studi di Milano, Milan, Italy

Abstract: The aim of this work is to evaluate both the toxic effect of different organic media on the

stereospeci®c oxidation of 2-methyl-1,3-propanediol to R-(ÿ)-b-hydroxyisobutyric (HIBA) in two-

phase systems and the extraction ability and selectivity of these non-water miscible phases. Apart from

traditional solvents, speci®c organic acid-complexing carriers like TOPO, TOA and Aliquat 336

dissolved in different diluents have been studied. Special interest has been focused on the effect of the

concentration of the organic phase extractants and the pH of the aqueous phase on the extraction

system. TOPO dissolved in isooctane enabled higher Kp values at lower concentrations to be attained

and resulted in lower toxicity, but its extractive capacity is strongly dependent on the pH. Our results

suggest that using a compromise pH value between optimum for bioconversion and extraction, TOPO

dissolved in isooctane can be successfully used as an extractive phase for HIBA production in a two-

phase system.

# 2000 Society of Chemical Industry

Keywords: two-phase bioconversions; TOPO; hydroxyisobutyric acid

NOTATIONCHAo

Total acid concentration in the organic

phase (moldmÿ3)

CHAwTotal acid concentration in the aqueous

phase (moldmÿ3)

HIBA Hydroxyisobutyric acid

KHA Ionization constant of the acid in the

aqueous phase (moldmÿ3)

Kp Partition coef®cient, =Co/Cw

(dimensionless)

Kp* Partition coef®cient at very low pH values

KS Solvation constant (molÿ1dm3)

p Solvation number

TOA Trioctylamine

TOPO Trioctylphosphine oxide

w/v Weight/volume

a Separation factor=Kp(HIBA)/Kp(diol)

(dimensionless)

s Standard deviation

Subscriptso Organic phase

w Aqueous phase

1 INTRODUCTIONMany organic acids can be obtained by fermentation

or by incomplete oxidation of the corresponding

primary alcohols. The latter method is an old and

established way to produce different acids, such as

acetic or gluconic acids, at industrial scale and it may

also be used to produce aliphatic acids employed as

natural ¯avours or chiral intermediates.1 Regio- and

enantioselective oxidation of diols can afford hydroxy-

carboxylic acids, such as b-hydroxyisobutyric acid

(HIBA), to be used as chiral building blocks.2

Although the production of carboxylic acids by

microbial means is very attractive, its industrial

application is still severely hampered by the low

productivity compared with conventional chemical

methods and by the dif®culties concerned with

product isolation.3 Improved productivity and con-

centration of the product can be achieved by in situ

product recovery.4±6 The integration of the biocataly-

tic and the extractive processes can circumvent

inhibition caused not only by the product, but also

by the substrate, since this can be maintained at the

desired concentrations by a controlled addition at the

same rate as its consumption.7 Moreover, extractive

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:617±624 (2000)

* Correspondence to: Joaquim MS Cabral, Centro de Engenharia Biologica e Quımica, Instituto Superior Tecnico, Av Rovisco Pais, 1096Lisbon, Portugal(Received 21 September 1998; revised version received 1 March 1999; accepted 28 March 1999)

2000 Society of Chemical Industry. J Chem Technol Biotechnol 0268±2575/2000/$17.50 617

bioconversions can reduce the formation of by-

products and the costs of the downstream processing.8

Liquid/liquid extractive systems offer a simple way

to remove products from an aqueous phase, where

most of the bioconversions take place, into a second

immiscible liquid phase. It is therefore crucial to ®nd

an extractant able to remove quantitatively and

selectively the desired product. These two require-

ments are related to the partition coef®cient (Kp) and

the separation factor (a), respectively. The extraction

of organic acids with pure organic solvents is often

complicated by their polarity.3 Other extractive agents

such as phosphorus-bonded oxygen donor extractants

(alkyl phosphates or alkyl phosphine oxides), which

establish quite speci®c and strong interactions with the

protoned form of the acid through donor bonds,

aliphatic amines, which react with organic acids

forming ammonium salts or ion pairs which are

soluble in the organic phase and quaternary ammo-

nium salts have been proposed for the extraction of

organic acids.3,9±11 A critical problem is often the

toxicity of the extractant to the biocatalyst.12

In the present study the extraction ef®ciency of

different organic media for the recovery of hydroxy-

isobutyric acid (HIBA) and their biocompatibility with

a newly isolated bacterium able to perform the

selective and quantitative oxidation of 2-methyl-1,3-

propanediol to HIBA is evaluated.

2 MATERIALS AND METHODS2.1 Chemicals2-Methyl-1,3-propanediol (99%), trioctyl phosphine

oxide (90%), trioctylamine (98%) and Aliquat1 336

were supplied by Aldrich (Sigma-Aldrich Co Ltd,

UK). Oleyl alcohol of technical grade was purchased

from BDH Chemicals Ltd, Poole, UK. Hexane,

isooctane, dodecane, octanol and ethyl acetate, all of

reagent grade, were obtained from Riedel-de HaeÈn,

Seelze, Germany. The agar and the yeast extract were

manufactured by DIFCO, Detroit, USA. Other

chemicals and media components, all of analytical

grade, were supplied by Merck, Darmstadt, Germany.

2.2 Microorganism and culture conditionsA bacterium (called ALEI) isolated from ripened fruits

and capable of growth on 2-methyl-1,3-propanediol as

sole carbon source was maintained on 5% (w/v)

glucose, 1% (w/v) yeast extract, 3% (w/v) CaCO3,

1.5% (w/v) agar, pH 6.5. The bacterium was Gram-

negative, rod-shaped, pale and cells occurred in pairs

or chains. No further characterization was performed.

Cells grown on slopes for 48h were used to inoculate

25cm3 of growth media consisting of mannitol 2.5%

(w/v) and yeast extract 1% (w/v), pH 5. Fivecm3 of

this preculture, grown for 24h, were used to inoculate

200cm3 of the same growth medium. Cells were

grown in 2dm3 Erlenmeyer ¯asks at 28°C and

200rpm orbital shaking for 24h, harvested by cen-

trifugation, washed and used for biotransformations

and biocatalytic stability experiments.

2.3 BiotransformationsBiotransformations were carried out in 100cm3

Erlenmeyer ¯asks containing 10cm3 of citrate buffer

(0.1moldmÿ3 pH 5.0) with 5mgcmÿ3 of substrate at

28°C under orbital shaking (200rpm). Samples were

periodically withdrawn, centrifuged and the super-

natant analysed by HPLC. Under these conditions a

complete molar conversion of the diol into HIBA was

observed within 5h. Biocatalytic activity was calcu-

lated from the slope of the product concentration in

the aqueous medium (gdmÿ3) versus time (h) plots.

2.4 Partition coefficientsPartition coef®cients of substrate and product were

determined by dissolving a known amount of these

compounds in 5cm3 of the aqueous phase at the

chosen pH, adding the same volume of different

organic phases and stirring vigorously for 30s on a

mixer. After 1h at room temperature the concentra-

tion in the aqueous phase was calculated by HPLC.

The concentration in the organic phase was calculated

by mass balance and partition coef®cients (Kp) were

calculated as the ratio between equilibrium concentra-

tions of the target compounds in the organic and

aqueous phases. All Kp values shown are averages for

at least three different measurements (standard devia-

tion, s<5%).

2.5 Solvent toxicity assaysTo study the in¯uence of the solvent on the

biocatalytic capacity of ALEI bacterium the cells

harvested at the end of the exponential phase of

growth were resuspended in the bioconversion

medium with saturation concentration of the indicated

solvents or organic mixtures and bioconversions were

carried out in the conditions previously described (pH

5.0, 28°C, 200rpm). Biocatalytic activity, expressed

as gdmÿ3hÿ1 of product, was determined after 1h of

bioconversion in the presence of the indicated solvent.

2.6 Catalytic stability assaysCells harvested at the end of the exponential phase of

growth were resuspended in the bioconversion buffer

(0.1moldmÿ3 citrate buffer pH 5). Organic solvent

was then added, until saturation concentration or a

second visible phase appeared (phase volume ratio

1:1) and the cells were incubated at the temperature

and conditions indicated. Aliquots (10cm3) were

periodically withdrawn, centrifuged, washed and

resuspended in the bioconversion medium with

5mgcmÿ3 of substrate and used to test the biocatalytic

activity as indicated above.

2.7 Analytical determinations2-Methyl-1,3-propanediol and (b)-hydroxyisobutyric

acid were determined by HPLC on a Polyspher OA

HY column, the mobile phase was 0.01moldmÿ3

618 J Chem Technol Biotechnol 75:617±624 (2000)

R LeoÂn et al

H2SO4. A ¯ow rate of 0.3cm3 minÿ1 and a refractive

index detector were used. The results are the average

of two replicates (standard deviation, s<3%).

3 RESULTS AND DISCUSSION3.1 Extraction of hydroxyisobutyric acid (HIBA)and 2-methyl-1,3-propanediol by different organicsolventsTable 1 shows the values of partition coef®cients (Kp)

for the diol and HIBA between water (pH 2.0) and

different extractant solutions. Inert hydrocarbons such

as hexane, isooctane and dodecane showed no extrac-

tion capacity at all. Esters, like ethyl acetate, and

aliphatic alcohols have a higher capacity to interact

with HIBA but the values of Kp are still quite low.

Substances capable of more speci®c interactions are

necessary for the extraction of organic acids like HIBA

due to its low hydrophobicity (log Poct=ÿ0.5). The

use of solutions containing trioctylphosphine oxide

(TOPO) and Aliquat 336, a phosphine oxide and a

quaternary ammonium salt respectively allowed for

high extraction of HIBA. The structures and complex-

ing reactions for the various carriers are shown in Fig

1. Trioctyl amine (TOA) was a poor extractant for

HIBA, although ternary amines are often described as

adequate extractants for organic acids.10 The percen-

tage of the extractive agent is limited by its solubility in

the organic solvent chosen. TOA solutions higher than

20% (w/v) were only possible in oleyl alcohol. Aliquat

336 was easier to dissolve in all the solvents tested,

although in some cases three phases appear after

adding the aqueous phase for extraction experiments.

TOPO solubility was also limited to 20% (w/v) in all

cases except in oleyl alcohol where 50% (w/v) solu-

tions were obtained.

Partition coef®cients with TOPO and TOA de-

pended not only on the percentage of the extractant

Table 1. Partition coefficients for HIBA (acid) and 2-methyl-1,3-propanediol(diol) between water (pH 2) and different organic extracting solutions

Solvent Extracting agent a Kp (acid) Kp (diol) a

Alkanes ± 0.00 0.00 0

Octanol ± 0.31 0.18 1.72

Ethyl acetate ± 0.18 0.10 1.86

Oleyl alcohol ± 0.08 0.04 1.81

Oleyl alcohol Aliquat 336 (20%) 0.23 0.09 2.56



Hexane Aliquat 336 (50%) 0.56 0.20 2.80

Hexane Aliquat 336 (80%) 0.92 0.32 2.88

Isooctane Aliquat 336 (20%) 0.23 0.08 2.88



Dodecane Aliquat 336 (20%) 0.25 0.09 2.78



± Aliquat 336 (100%) 1.38 0.49 2.82

Oleyl alcohol TOA (20%) 0.14 0.05 2.80



Hexane TOA (20%) 0 0 0

Isooctane TOA (20%) 0 0 0

Dodecane TOA (20%) 0 0 0

Oleyl alcohol TOPO (5%) 0.11 0.03 3.67



Hexane TOPO (20%) 0.53 0.15 3.53

Isooctane TOPO (20%) 0.53 0.15 3.53

Dodecane TOPO (20%) 0.54 0.16 3.38

a The ratio of the extracting agent to the solvent is expressed as w/v

percentage.

Figure 1. Organic acid extractiveagents structures and chemicalinteractions.

J Chem Technol Biotechnol 75:617±624 (2000) 619

Extraction of b-hydroxyisobutyric acid

agent, but also on the nature of the organic solvent

used. The partition coef®cient for TOPO dissolved in

oleyl alcohol is much lower than when it is dissolved in

aliphatic hydrocarbons. The only advantage of oleyl

alcohol is the higher solubility of TOPO in it. However

a higher proportion of TOPO leads to lower biocom-

patibility, as will be shown later.

Concerning selectivity, it can also be observed in

Table 1 that the diol substrate is also extracted by all

the complexing carriers tested, as well as by ethyl

acetate and octanol and that it is not extracted at all by

aliphatic hydrocarbons. Nevertheless the separation

factor (a) is higher when speci®c extractive agents are

used, with values between 2.53 and 3.75. The com-

parison of the Kp values obtained for the extraction of

HIBA with the values reported in the literature for the

extraction of other organic acids (Table 2) shows that

HIBA is as effectively extracted as other hydroxyacids

like lactic acid13 and more ef®ciently extracted than

polycarboxylic acids like citric acid. But Kp values for

other organic acids without hydroxylic or second

carboxylic groups (eg acetic acid) are higher, as would

be expected. The inductive effect of these groups over

the carboxylic group causes a decrease in the

extractability of the organic acid.3

In all cases it is observed that the Kp of HIBA

increased in a linear fashion for increasing amounts of

the extractive agent in the organic mixture. Figure

2(A) shows this tendency in the case of TOPO. In Fig

2(B) the logarithm of the molar concentration of

TOPO is plotted against log Kp. If we consider the

reactions taking place during the extraction process, ie

the ionization of the acid in the aqueous phase and the

formation of the acid±extractant agent abduct, the

following expression can be deduced:

Kp � CHAo

CHAw

� �HA � Sp�o�HA�w � �Aÿ�w

� KS�HA�w�S�po�HA�w �

KHA�HA�w�H��

� KS�S�po1�KHA�HA�w

�H��

�1�

Since the Kp values were determined at low pH,

[H�]�KHA, thus:

Kp � KS � �S�polog Kp � log KS � p � log�S�o

�2�

where S is the concentration of the extractive agent

and p is the solvation number, or number of molecules

of extractive agent which are solvating the acid. The

value of p can be easily estimated from the slope of this

linear representation and usually corresponds to the

number of carboxylic groups of the acid molecule. In

the case of HIBA a p value of 1.03 was obtained (Fig

2), thus indicating a stoichiometric reaction.

3.2 Toxicity of extractant phaseToxicity studies were performed to determine the

maximum extractive agent concentration tolerated by

the cells of the ALEI bacterium. When contacting an

organic solvent with an aqueous solution a certain

number of solvent molecules are dissolved in the

aqueous phase which can interact with cell membranes

and cause disruption of their essential functions.14±16

This is termed molecular toxicity. Osborne and

coworkers17 proposed that a critical solvent concen-

tration in the cell membrane exists at which complete

loss of biocatalytic activity occurs, and only solvents

able to reach this critical membrane concentration are

toxic to the cells. The more polar the solvent is the

higher will be its aqueous solubility and the easier it

will be for it to reach the critical toxic concentration.

Table 2. Partition coefficients of some typical organic acids between waterand three different organic solvents

Kp

Organic acid Hexane

Ethyl

acetate

TOPO/

hydrocarbon Reference

Citric acid 0 0.009 ± 3

Lactic acid 0 0.1 0.6±0.8 3, 11

HIBA 0 0.097 0.53 ±

Propionic acid 0.005 1.75 ± 3

Acetic acid 0.02 ± 4.7 9

Figure 2. (A) Kp values for HIBApartition between water (pH 2) anddifferent molar concentrations of TOPOdissolved in isooctane. TOPOconcentration expressed in percentage(w/v) is also shown in parenthesis.(B) Logarithm of the concentration ofTOPO (mmoldmÿ3) against thelogarithm of Kp.


R LeoÂn et al

Between the different parameters used to correlate

solvent polarity, log Poct is the most generally used.12

We tested the molecular toxicity of different organic

solvents on the biocatalytic activity of the ALEI

bacterium (Table 3). The more polar solvents, such

as ethyl acetate or octanol, were very toxic, while the

hydrophobic ones were compatible. Pure Aliquat 336

and TOA were shown to be very toxic. The bioconver-

sion rates were reduced to 14.7 and 11.5% of the

control value respectively. It was not possible to test

the toxicity of pure TOPO since this product is solid at

room temperature. Solutions of these extractive agents

in oleyl alcohol were also evaluated. Increasing the

concentration of TOPO until 50% had no effect on the

bioconversion rate, while concentrations of Aliquat

and TOA above 20% were severely inhibitory. Oleyl

alcohol was used to dissolve the acid-complexing

carriers because it enabled higher solubility of TOPO.

It is noteworthy that with isooctane, dodecane and

oleyl alcohol the bioconversion rates were even higher

than in the aqueous system. An increase of the

catalytic activity of whole cells in the presence of

organic systems was previously described for yeast

fermentation in the presence of small quantities of

n-dodecane and per¯uorocarbon18 and for other

fermentative processes with emulsi®ed organic sol-

vents.19,20 Organic solvents can act like oxygen

vectors, increasing the volumetric oxygen transfer

coef®cient and the fermentation rates. Small quantities

of solvent (1±4%) are enough to observe this effect.18

Organic solvents may also permeabilize cell mem-

branes, enhancing substrate and product transport

into/from the cellular cytoplasm where the majority of

the reactions take place.21 Either of these facts could

explain the bioconversion rate increase observed in the

case of isooctane and dodecane. Although speci®c

organic acid extractive agents are described by the

majority of authors as very toxic substances,11 several

examples of extractive fermentations using organic

mixtures containing these substances have been

described10,11,22 and some processes based on them

have been patented.23

3.3 Influence of TOPO on catalytic stabilityThe catalytic stability of the ALEI bacterium was

evaluated by incubating the cells at 28°C in biotrans-

formation buffer with and without orbital stirring.

Catalytic stability was also veri®ed in the presence of

the biotransformation buffer saturated with the

organic solvent (20% TOPO dissolved in isooctane)

without agitation, where only molecular toxicity is

signi®cant and in the presence of a second distinct

phase (ratio aqueous/organic 1:1) with agitation. In

these conditions an emulsion was formed and phase

toxicity also played an important role (Fig 3). In both

Table 3. Toxic effect of different solventson bioconversion activity of ALEI cells.Bioconversion rate, expressed asgdmÿ3hÿ1 of product, was determinedafter 1h of bioconversion in the presenceof aqueous saturation concentration ofthe indicated solvent. Oleyl alcohol wasused to dissolve all the acid-complexingcarriers studied

Kind of solvent Solvent Bioconversion rate (%) Log Poct

Control 100

Alkanes Hexane 76 3.5

Isooctane 108 4.5

Dodecane 120 6.7

Alcohols and esters Ethyl acetate 1.8 0.6

Octanol 0.5 2.9

Oleyl alcohol 108 7.0

P-bonded oxygen extractants TOPO (5% w/v) 107

TOPO (20% w/v) 107

TOPO (50% w/v) 100

Ternary amines TOA (20% w/v) 96.9

TOA (50% w/v) 49.7

TOA (100% w/v) 11.5

Quaternary ammonium Aliquat 336 (20% w/v) 102

Aliquat 336 (50% w/v) 46.9

Aliquat 336 (100% w/v) 14.7

Figure 3. Biocatalytic stability of the ALEI bacteria incubated underdifferent conditions. Cells were incubated at 28°C in biotransformationbuffer with (*) and without (*) stirring, biotransformationbuffer�substrate 5mgcmÿ3 (~) and biotransformation buffer in thepresence of saturation concentration of the organic solvent without stirring(&) or in the presence of a distinct organic phase with stirring (&). Theorganic solvent was 20% (w/v) TOPO dissolved in isooctane. A controlincubation at 4°C (^) is also shown. The arrow (;) represents the additionof substrate (5mgcmÿ3) to the incubation buffer.



cases the organic solvent was 20% (w/v) TOPO

dissolved in isooctane.

In the absence of TOPO, the oxidative activity is

quite stable during the ®rst 12h, while, for longer

times biocatalytic activity tended to decrease, with

only 43% of residual activity after 24h of incubation

for shaken cells and 69% for those not shaken.

Incubating in the presence of the substrate of the

bioconversion, 2-methyl-1,3-propanediol, led to an

important stabilization of the activity, which remained

constant at 100% for the ®rst 24h. The addition of the

substrate to the incubation medium after 24h allowed

partially inhibited cells to recover original activity.

This stabilizing effect of the substrate is very important

in the perspective of developing a continuous bio-

transformation process. Control incubation at 4°Cwas also carried out. No reduction of activity was

observed for more than 3 days at 4°C. Cell incubation

in the presence of 20% (w/v) TOPO dissolved in

isooctane negatively affected the catalytic stability,

causing a faster loss of biocatalytic activity. Shaking

enhances this decrease.

3.4 Influence of the pH on the partition coefficientAll previous experiments were carried out at pH 2,

since low pH values at which the acid is protoned are

the most favourable for its extraction. However such

an acidic pH is not the optimal for the studied

bioconversion, which showed a maximum rate at pH

6. To perform the bioconversion and the extraction

integrated in one unique step, a compromise between

optimum pH values for bioconversion and extraction

should be used.

While the extractive capacity of the organic phase

containing TOPO is strongly dependent on the pH, a

system containing Aliquat 336 as extractive agent

showed similar Kp values for all the studied range

(2±8.5) (Fig 4). TOPO can only interact with the

protoned form of the acid and consequently signi®cant

extraction will only take place for pH values lower than

the pKa of the acid. Aliquat 336 is really acting as an

ionic exchanger that exchanges Clÿwith the carboxylic

anion (Fig 1), therefore removing HIBA from the

aqueous phase also at high pH. The majority of the

organic acid-producing fermentations have optimum

pH values between 5 and 7, which is far from the pKa

of most organic acids. For this reason many authors

have studied the use of quaternary amines integrated

extraction/fermentation processes. This is the case of

lactic acid (pKa 3.86) which is optimally extracted by

Aliquat 336.24 Strong toxicity problems however make

their use dif®cult, it being necessary to minimize the

contact between the cell and the organic phase by

immobilizing the cells8 or by using supported liquid

membranes, where the extractant is contained in the

membrane that separates two liquid phases.25 In the

case of HIBA, a relatively high pKa value, 4.7, makes it

possible to have a high percentage of the protoned

form of the acid at relatively high pH values, allowing

the use of TOPO as extractant at pH values near the

optimum for the bioconversion.

All previous partition coef®cient calculations were

performed with HIBA dissolved in distilled water as an

aqueous phase to avoid interference of the buffers. In a

real biphasic bioconversion/extraction process, a buf-

fer solution and cells will be present in the extraction

system, therefore the effect of these factors on Kp was

also studied. In Fig 5 the values of Kp are plotted for

different pH values and in different extraction systems.

In all cases 20% (w/v) TOPO was used as organic

extractive agent, being the aqueous phase where

hydroxyisobutyric acid was dissolved: distilled water

in cases A and B, 0.1moldmÿ3 citrate buffer in case C

and 0.1moldmÿ3 citrate buffer with cells of bacterium

ALEI (100mg FWcmÿ3) in case D; and the organic

solvent for the solution of TOPO oleyl alcohol in case

A and isooctane in cases B, C and D. Kp variations due

to the organic solvent used to dissolve TOPO have

been discussed previously. In this ®gure the differences

Figure 4. Influence of pH on Kp values for HIBA partition between waterand solutions 20% (w/v) of TOPO (*) and Aliquat 336 (&) dissolved inoleyl alcohol.

Figure 5. Influence of pH on Kp for HIBA partitioning in different aqueous/organic systems. In all cases 20% (w/v) TOPO was used as organicextractive agent, the aqueous phase being: distilled water (^, &),0.1moldmÿ3 citrate buffer (*) or 0.1moldmÿ3 citrate buffer with cells(100mg FWcmÿ3) (*) and the organic solvent for the dissolution of TOPOoleyl alcohol (^) or isooctane (&, *, *).


R LeoÂn et al

found for the Kp values calculated in systems where the

aqueous phase was or was not buffered can be

observed. This enhancement of the extraction capacity

in the presence of citrate buffer could well be due to a

`salting out' effect. This effect has been described by

several authors who reported the `salting out' of the

organic products to the extraction solvents when salts

were added to the fermentation broth, increasing

Kp.11,26 Also the presence of cells caused an increase in

Kp. The exact reasons for this increase were not

determined.

The sharp decrease in Kp between pH 4 and 5 that

can be observed in Fig 5 occurs because of the pKa of

the HIBA, which is equal to 4.7. At a pH of 4, 84% of

the acid is in the protoned form while only 34% is

protoned at pH 5. At pH 6 the proportion of the acidic

form is only 1.3% so little extraction takes place. It is

interesting to note that the Kp for the alcohol is not

signi®cantly affected by the pH so the selectivity for

the extraction of the acid at low pH values is higher, as

shown in Table 4.

The importance of the pH on the partition

coef®cient for the extraction of organic acids has been

reported.10,11,27 Hatzinikolaou and Wang28 studied

the in¯uence of the pH on the Kp values for the

extraction of butyric acid from a fermentation broth

with oleyl alcohol and dodecanol and developed

equations to predict the evolution of the distribution

coef®cient with the pH as shown below:

Kp �K �p

1�KHA

H�

� � � K �p1� 10pHÿpKa

�3�

Kp* represents the partition coef®cient for very low pH

values. When this theoretical relationship is applied to

the partition coef®cient of HIBA in a system with 20%

(w/v) TOPO in isooctane as organic phase, very good

accuracy was found between the experimental and

predicted data (data not shown).

3.5 Effect of other factorsThe in¯uence of the temperature on Kp was tested at

22, 30, 38 and 50°C without ®nding signi®cant

variation between the values obtained. Also no

in¯uence of the presence of different concentrations

of 2-methyl-1,3-propanediol on the Kp for the acid was

found. The Kp value for the acid in the presence of diol

concentrations as high as 40mgcmÿ3 was exactly the

same as that Kp value obtained in the absence of

alcohol in the same conditions. Although some

authors have found that Kp could depend on the

solute concentration for speci®cally interacting chemi-

cals solvents like TOPO,6 the in¯uence of the HIBA

concentration on the extraction by 20% (w/v) TOPO

in isooctane, over a range from 3 to 12mgcmÿ3, was

not observed.

4 CONCLUSIONSHydroxyisobutyric acid (HIBA) can be produced by

microbial oxidation of 2-methyl-1,3-propanediol with

a newly isolated bacterium. To improve the produc-

tivity of the bioconversion a selective and in-situ

extraction of the product must be found. The

extraction of the diol and the acid with different

organic phases, pure or containing extractive agents,

was measured to determine a system for selective

extraction of the biotransformation product. Hydro-

phobic organic solvents, such as isooctane and oleyl

alcohol, containing TOPO or Aliquat 336 gave better

separation factors (a) between substrate and product.

TOPO enabled higher Kp values at lower concentra-

tions and resulted in lower toxicity, but its extraction

ability strongly decreases at pH values around 5±6

which are suitable for the biotransformation. A

compromise can be reached by using a medium for

biotransformation with a pH very close to the pKa of

the acid (4.7). An extractive system based on the use of

TOPO could be used in extractive bioconversions

assisted by membranes, where the organic phase and

the cells are maintained apart, improving cell stability

and minimizing toxic effects.

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Organic media selection for the extraction of β-hydroxyisobutyric acid produced by microbial biotransformation