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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
Oleyl alcohol Aliquat 336 (50%) 0.52 0.19 2.74
Oleyl alcohol Aliquat 336 (80%) 0.91 0.36 2.53
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
Isooctane Aliquat 336 (50%) 0.57 0.18 3.17
Isooctane Aliquat 336 (80%) 0.95 0.31 3.06
Dodecane Aliquat 336 (20%) 0.25 0.09 2.78
Dodecane Aliquat 336 (50%) 0.57 0.19 3.00
Dodecane Aliquat 336 (80%) 0.93 0.34 2.74
± Aliquat 336 (100%) 1.38 0.49 2.82
Oleyl alcohol TOA (20%) 0.14 0.05 2.80
Oleyl alcohol TOA (50%) 0.18 0.06 3.00
Oleyl alcohol TOA (80%) 0.20 0.07 2.86
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
Oleyl alcohol TOPO (20%) 0.20 0.06 3.33
Oleyl alcohol TOPO (50%) 0.45 0.12 3.75
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.
620 J Chem Technol Biotechnol 75:617±624 (2000)
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.
J Chem Technol Biotechnol 75:617±624 (2000) 621
Extraction of b-hydroxyisobutyric acid
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 (&, *, *).
622 J Chem Technol Biotechnol 75:617±624 (2000)
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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