J. Chem. T echnol. Biotechnol.

1998, 71, 315È325

Alkanol Removal from the Apolar Phase of a

Two-Liquid Phase Bioconversion System.
Part 1: Comparison of a Less Volatile and a
More Volatile in-situ Extraction Solvent for
the Separation of 1-Octanol by Distillation
Renata G. Mathys,* Oemer M. Kut” & Bernard Witholt

Institute of Biotechnology, Swiss Federal Institute of Technology, ETH HoŽnggerberg, HPT, CH-8093
ZuŽrich, Switzerland

(Received 27 August 1996 ; revised version received 30 May 1997 ; accepted 15 June 1997)

Abstract : Biocatalytic systems can be used for the regio- and stereospeciÐc syn-
thesis of oxidized alkanes and aromatic compounds, such as aliphatic and aro-
matic alcohols, aldehydes and epoxides. These reactions are typically carried out
in two-liquid phase media. The biocatalyst is usually a natural microorganism,
often a Pseudomonas, or a genetically altered host, a Pseudomonas or E. coli
recombinant typically, which grows in the aqueous phase, while the substrate
and product are present in an organic bulk phase. Oxidation products formed in
these systems must be puriÐed after separation of the two liquid phases. We have
evaluated the performance of distillation for the separation of the product 1-
octanol by examining a more volatile (octane) and a less volatile (hexadecene)
in-situ extraction system. The separation performance of the two systems has
been compared based on recovery efficiency, energy cost and number of required
process units. Results showed that a less volatile extractant compared favorably
in terms of number of product separation unit steps, decreased operating and
energy cost to the use of a more volatile extraction solvent. In addition, a major
disadvantage of the more volatile in-situ extraction process was the coloring of
the bottom product of the Ðrst distillation step, in which the product is contained
in this case. Such modiÐcations can be implemented into an upstream and down-
stream process of bioconversions to improve the overall system and to reduce
downstream processing cost. ( 1998 SCI

J. Chem. T echnol. Biotechnol. 71, 315È325 (1998)

Key words : two-liquid fermentation system ; downstream processing ; in-situ

extraction ; distillation ; 1-hexadecene ; n-octane ; 1-octanol

* To whom correspondence should be addressed.

” Present address : Chemical Engineering Department, Swiss Federal Institute of Technology, ETH Zentrum, CAB, CH-8092
ZuŽrich, Switzerland.
315
( 1998 SCI. J. Chem. T echnol. Biotechnol. 0268-2575/97/$17.50. Printed in Great Britain
316
INTRODUCTION
Downstream processing often dominates the overall
economic performance of many interesting new bio-
conversion processes.1h4 The simplicity or complexity
of such separation unit operations depends on the
physical and chemical properties of system components,
the nature of the organism used and the mechanism
involved in the release of the product of interest ; and,
consequently, can be governed by the recovery cost.
When such new laboratory bioproducts are created, it is
important to investigate the technical and economic fea-
sibility of promising new bioconversions during an early
stage of product development.
One of the potential applications of bioconversions is
in the production of Ðne and medium priced chemi-
cals.5h7 One area of interest is that of regio- and stereo-
speciÐc oxidations of aliphatic and aromatic
hydrocarbons by bacterial oxygenases, because the
directed insertion of oxygen into unactivated molecules
is generally not possible via classical organic synthesis.
Such bioconversions can be carried out by organisms
able to grow on water-immiscible organic compounds
in two-liquid phase systems, in which the cells occupy
the water phase and the substrate forms or is dissolved
in the apolar phase. During growth, the cells oxidize
substrates or co-substrates to products. Thus, Pseudo-
monas oleovorans has been used to oxidize n-alkanes
and n-alkenes to the corresponding n-alkanols, alkanoic
acids and epoxides ;8 to oxidize substrates such as alka-
loids and steroids9,10 and to produce a variety of Ðne
chemicals11 from aldehydes and related apolar com-
pounds.
Normally, P. oleovorans not only oxidizes substrates,
but it also utilizes the products for growth. Since this
leads to massive product losses it is necessary to
uncouple cell growth from the desired biotransfor-
mation. As an example, the oxidation of n-alkanes to
1-alkanols has been achieved by introducing the
alkane hydroxylation system of P. oleovorans into a
mutant which cannot grow on alkanols, Pseudomonas
putida PpS81 (alcA81). The resulting recombinant
P. putida (GPp11) could be grown on a water-soluble
carbon source, such as glucose or pyruvate, while
at the same time the cells oxidized alkane to
1-alkanol without further oxidation or utilization of
1-alkanol.12
In these two-liquid phase fermentation processes
there is in-situ extraction of 1-alkanol into the apolar
phase (alkane). To obtain the product it is necessary to
separate the apolar phase from the polar phase, after
which it can be recovered from the organic phase.
Several methods can in principle be used for this. Based
on a literature analysis, we have concluded that of these,
distillation and pervaporation are today the best
options for the separation of volatile products and sub-
R. G. Mathys, O. M. Kut, B. W itholt
strates.
In order to investigate the technical and economical
feasibility of recovering organic products from a two-
liquid fermentation system, we have taken the oxidation
of n-octane to 1-octanol by P. putida (Gpp11) as a suit-
able model. It is not clear whether the large-scale pro-
duction of 1-octanol from n-octane is of economic
interest. However, 1-octanol is used to a certain extent
for the manufacturing of detergents, perfumes and insec-
ticides, as well as esters.13 Product prices vary from
4É50 $ kg~1 (98% pure) for bulk quality14 to 23É00 $
kg~1 for 99É5% pure 1-octanol,15 while substrate prices
vary from 0É45 $ kg~1 (94% pure) for bulk quality16
to 50É40 $ kg~1 for 99% pure n-octane.15 The per-
formance characteristics of this bioconversion are well
documented,12 and the product separation process
evaluated here and in the accompanying paper can
easily be adapted to many related two-liquid phase oxi-
dations. As a result, we consider the n-octane to 1-
octanol bioconversion as a useful paradigm for other
similar processes.
The Ðrst step in processing a two-liquid phase fer-
mentation system is to separate the apolar, product-
containing, phase from the aqueous, cell-containing,
phase. In this paper we compare the subsequent separa-
tion by distillation of 1-octanol from a more volatile
(octane) and a less volatile (hexadecene) bulk apolar
phase. To determine how the original two-liquid phase
system might a†ect subsequent puriÐcation of 1-octanol
comparisons are made between a model system consist-
ing of pure chemicals versus a system consisting of an
apolar phase separated from a simulated fermentation
system. Under practical conditions, varying amounts of
aqueous phase and cells may contaminate the apolar
phase. This will a†ect the efficiency of the distillation
step, and hence the obtained product purity and atten-
dant costs. These e†ects are examined in detail in the
accompanying paper.17 The results obtained permit the
calculation of the energy requirements and recovery effi-
ciency under various conditions.
Finally, prior information on the upstream bio-
conversion and the accompanying paper17 on the
downstream processing of 1-octanol have been used to
model integrated bioconversion and processing systems
for the production of 10 000 tons per year of 1-octanol.
Comparisons are also made between batch and contin-
uous systems. These systems and their economic per-
formance are described in a third paper (to be
published). Based on this a cost/beneÐt analysis can be
made for the puriÐcation of 1-octanol to di†erent
extents and under di†erent conditions. This in turn will
allow the design of the most suitable distillation column
and a solvent recovery step for a given desired product
puriÐcation step. This approach can be extended to
other carrier solvents and substrateÈproduct com-
binations, once the thermodynamic properties of such
combined mixtures are known.
Separation of 1-octanol by distillation
2 MATERIALS AND METHODS
2.1 Process media
2.1.1 Separation of apolar and polar phases from
fermentation medium
P. oleovorans GPo1 was grown in E2 medium and on
n-octane (20% (v/v)), in a 3 dm3 bioreactor, as described
by Preusting et al.18 At the end of the fermentation, the
two phases were separated by centrifugation as
described below. The polar phase (80% (v/v)) contained
12 g dm~3 P. oleovorans GPo118 in E2 medium. The
apolar phase (AP) consisted of n-octane, which con-
tained emulsifying components : about 2 g (dry weight)
dm~3 of bacterial surfactant (90% lipopolysaccharides
and 10% other membrane components),19 about 10%
water, and contaminating proteins and bacterial cells.
2.1.2 Model apolar phases
The two model apolar phases consisted of a bulk
solvent, either the organic substrate or an organic bulk
solvent with the organic substrate dissolved in it, and
the product. The more volatile octane-based apolar
phase (OAP) was prepared by adding 1-octanol to the
fermentation medium (0É9% (v/v) relative to total
volume) to a Ðnal volume of 5% (v/v) relative to the
octane apolar phase at the end of a fermentation. Fol-
lowing phase separation, the OAP consisted of 95%
(v/v) octane and 5% (v/v) 1-octanol. The less volatile
hexadecene-based apolar phase (HAP) was prepared by
separating the octane apolar phase from the fermenta-
tion medium and replacing it with a mixture of hexa-
decene (17% (v/v) relative to total volume), the octane
apolar phase (2É1% (v/v) relative to total volume), and
1-octanol (0É9% (v/v) relative to total volume) to make
up an equivalent apolar phase, which consisted of 85%
(v/v) hexadecene, 11% (v/v) octane and 4% (v/v) 1-
octanol. To simulate conditions in the bioreactor
(metabolic activities), the model fermentation media
were mixed at 2000 rpm for another 10 h, before the
organic phase was separated from the medium.
The apolar phase was separated from the aqueous
fermentation broth prior to distillation using a labscale
tubular-bowl centrifuge from CEPA, Germany. The
centrifuge was operated at 26 000 rpm, corresponding
to 16 000g. Small amounts (1È3% (v/v) with respect to
the apolar phase) of emulsiÐed component impurities
separated out with the apolar phase, which however
settled instantaneously in the holding vessel. Before dis-
tillation the emulsiÐed components were separated from
the apolar phase by decanting. The amount of emulsi-
Ðed substance remaining in the organic phase was
quantiÐed by centrifugation (18 000g, 30 min, 0¡C) and
the gel was weighed after removal of the solvent
mixture.
317
After phase separation the OAP consisted of 94É3%
(w/v) octane, 5É7% (w/v) 1-octanol, 0É9& (w/v) polar
phase and about 0É3È0É4% (w/v) emulsiÐed substance.
The HAP consisted of 84É1% (w/v) hexadecene, 11É7%
(w/v) octane, 4É2% (w/v) 1-octanol, 1É2& (w/v) water
and about 0É2È0É3% (w/v) emulsiÐed substance.
2.1.3 Simulated organic phases
DeÐned mixtures, made up from pure chemicals and
containing only the volatile compounds, were used to
optimize the column performance for atmospheric dis-
tillation and to compare the separation efficiency
obtained with these mixtures to those of the model
apolar phases separated from the two-liquid fermenta-
tion system. The simulated octane apolar phase (SOAP)
consisted of 94É9% (w/v) octane and 5É1% (w/v) 1-
octanol ; and the simulated hexadecene apolar phase
(SHAP) was made up of 83É6% (w/v) hexadecene, 12É4%
(w/v) octane and 4% (w/v) 1-octanol.
All of the chemicals and hydrocarbons used were of
the best purity available (Fluka, Buchs, Switzerland). n-
Octane (98É5% purity) and 1-hexadecene (94% purity)
were purchased from Acros (Geel, Belgium).
2.2 Analytical procedures
Measurements of n-octane, 1-hexadecene and 1-octanol
concentrations were done using a computer-controlled
capillary gas chromatograph (Fison Instruments,
HRGC MEGA 2 series). Samples were prepared as
follows : 1% (v/v) organic phase (directly or after freez-
ing and thawing) was added to pure n-hexane with 1%
(v/v) 2-octanol as internal standard. These prepared
samples were analyzed by split injection into a CP-SIL-
5CB (25 m long with a 0É45 mm internal diameter) cap-
illary column (supplied by Chrompack, The
Netherlands) with hydrogen as carrier gas, and peaks
were detected with a Ñame ionization detector (FID).
The samples were eluted at an initial temperature of
200¡C for 2 min, followed by a linear increase of
10¡C min~1 to reach the Ðnal temperature of 280¡C, at
an injector temperature of 200¡C and a detector tem-
perature of 280¡C. The compounds were quantiÐed
from the integrated GC signal by the internal standard
method, suing reagent grade standards.
Cis ] trans Decalin used in vacuum distillation
column performance measurements, as standard indica-
tor, were determined by gas chromatography (Pye
Unicam, Series No. 304) with an FID. Samples were
analyzed by direct injection into a silicon oil DC550 on
chromosorb AW packed glass column (2É1 m long with
a 4 mm internal diameter) using hydrogen as carrier gas
318
at an injector temperature of 260¡C, a detector tem-
perature of 280¡C and a column temperature of 115¡C.
Cell densities, expressed as gram cell dry weight
dm~3 water phase, were measured optically at 450 nm
and determined as previously described.20
The presence of polar phase (water-content) in the
apolar phase was analyzed by the KarlÈFischer-
Colorimetric titration diaphragm method21 using a
Metrohm 652KF coulometer.
For the determination of the vaporÈliquid equi-
librium (VLE) of the systems n-octane/1-octanol, 1-
octanol/1-hexadecene and n-octane/1-hexadecene
respectively, a LABODEST VLE apparatus from
Fischer (Germany) was used. Experiments were done at
constant pressure, using pure chemicals, in accordance
with the guidelines of the manual and as described by
Grassman and Widmer.22
2.3 Distillation details
2.3.1 Determination of V L E data for n-octane,
1-octanol and 1-hexadecene mixtures
To design an optimized distillation separation system
VLE data of the components are necessary. Since VLE
data for the two apolar phase systems could not be
located in the literature, they had to be determined
experimentally and predicted by correlation. For a
highly non-ideal system the non-random two liquid
(NRTL) activity coefficient model was used, which con-
tains a non-randomness parameter (c ) and is therefore
ij
applicable for a large variety of mixtures.23 Since ideal
behavior of the gas phase can only be considered for
Fig. 1. Schematic diagram of the experimental batch distillation apparatus used for atmospheric and vacuum distillation. Tn,
Thermocouple Pt-100 n \ 1, 2, 3 ; T1, bottom temperature regulation ; T2, column temperature regulation ; T3, head temperature
regulation ; P1, vacuum meter.
R. G. Mathys, O. M. Kut, B. W itholt
rough estimations, the RedlichÈKwongÈAspen equation
of state24 was used for the vapor phase.
2.3.2 Equipment details
Figure 1 shows a schematic diagram of the resulting
experimental batch distillation apparatus (QVF,
Germany) used in this study. A glass distillation column
had to be built, taking into consideration that distilla-
tion was carried out under atmospheric and vacuum
conditions and giving special attention to the fact that
either the distillate or bottom product fraction of the
mixture was extremely small. The column used had a
diameter of 30 mm, a total height of 0É635 m and a
plate stack height of 0É34 m. To minimize liquid hold-
up, to avoid a possible build-up of foam and to provide
good contacting efficiency and high pressure drop effi-
ciency the column was packed with Sulzer material,
type CY made from stainless steel woven gauze
(material 1É4404). One pack was 0É16 m high and con-
sisted of two actual stages (measured) for the octane/1-
octanol separation and 1É9 actual stages (measured) for
the hexadecene/octane and 1-octanol separation. The
column and the reÑux divider were isolated with a silver
plated high vacuum mantle. The reÑux ratio was con-
trolled by a magnetic valve, where special design atten-
tion had to be given to ensure that no dead volume
would be trapped, because of the small fraction of the
lighter component during vacuum distillation of SHAP
and HAP. The reboiler at the bottom of the column had
a total volume of 2 dm3 and was operated at a working
volume of 1É5 dm3. The distillate was cooled in the
overhead condenser at the top of the column and col-
lected in the fractional distillate receiver. The total
height of the distillation apparatus was 2 m. For the
vacuum distillations a membrane pump from Vacu-
Separation of 1-octanol by distillation
ubrand MZ 2C (9É8È0É02 MPa) was used. To ensure
smooth and steady boiling and to remove the air before
start-up, nitrogen was bubbled through the distillation
column throughout the experiment. The reboiler
(bottom) and reÑux divider (head) temperature, the
minimum level of the bottom liquid in the still, the
reÑux ratio and the pressure were electronically con-
trolled. The distillation column was mounted in a plex-
iglass housing so that the exhaust gases could be
channelled for venting and generally, for safety pur-
poses.
The distillation column efficiency was determined by
measuring the actual stages at inÐnite reÑux ratio.25
The atmospheric pressure column performance was
characterized with the SOAP mixture. The tray effi-
ciency for the vacuum distillation was determined in
accordance with the guidelines of Onken and Arlt26
using cis ] trans Decalin, which is especially suited for
studying the performance of vacuum distillation
columns.
2.3.3 Optimization of distillation
Organic phase samples of the feed mixture were taken
from the still shortly before and after the distillation
experiment. For optimization of the column per-
formance, three fractions of the distillate were collected
in a fractional separation device during distillation, after
which a sample of the distillate was collected at the end
of each experimental run from the distillate receiver.
The distillation column was optimized both for atmo-
spheric and vacuum distillation using the two simulated
organic phases. Optimized runs of both OAP and HAP
(model apolar phases) and SOAP and SHAP (simulated
organic phases) were each repeated three times. The
average deviation from the mean between three suc-
cessful runs ranged from 1É8 to 2É5% for all optimized
runs.
Values for both mass- and energy balances were
determined experimentally. Energy consumption during
the whole distillation period was measured with an
energy and power measuring device (EMU 1É24 and
EMU 1É28). Active and reactive power consumptions of
induction devices were taken into consideration.
2.3.4 Evaluation, design and optimization of modeled
data
For the correlation of our measurements and sub-
sequent modeling we used a commercial software
package (ASPEN, Version 9.1, 1994) which has ther-
modynamic models, regression algorithms, mass and
energy balance calculations and which permits cost cal-
culations for the selected process equipment design.
The design and initial optimization of the distillation
column was modeled using RADFRAC, a rigorous
model for simulating all types of continuous single and
multistage vaporÈliquid fractionation operations, based
on the method of Boston and Sullivan.27
319
3 RESULTS
3.1 Two-liquid phase bioconversion and downstream
processing system
3.1.1 Suitability of the extraction solvent in the
bioconversion system
In the simplest bioconversion system analyzed here the
substrate to be oxidized is present as a bulk phase (20%
(v/v) with respect to the total fermentation medium) and
therefore serves as the extractant. In this system the
bulk organic component (n-octane, b.p. at 1 atm,
T \ 125É7¡C) is more volatile than the product (1-
octanol, b.p. at 1 atm, T \ 195É2¡C) and the amount of
1-octanol formed during the fermentation process gen-
erally amounts to less than 5% (v/v) of the octane bulk
apolar phase (OAP). This poses problems : the product
remains in the still where all fermentation impurities
remain, and long operation times are necessary to distill
the more volatile bulk component, which requires more
energy than needed when the product is distilled o†.28
Also, octane being Ñammable, there is an explosion risk.
A bulk apolar phase during fermentation, at ratios of
about 10È20% (v/v) relative to the total fermentation
medium, is required, as it permits generally good cell
growth,29 for optimal alkane oxidation and can also
help to reduce product toxicity.9 A more suitable apolar
phase for distillation separation should consist therefore
of a less volatile bulk apolar phase component, which
retains fermentation impurities, and which reduces the
Ñowrate and energy necessary to distill the product,
provided the substrate (octane in these experiments) can
be supplied in much smaller quantities than when it is
both the substrate and the apolar bulk phase, as in the
OAP system. Moreover, a suitable solvent for extractive
bioconversion has to fulÐl several more requirements,
such as : a high partitioning coefficient for the product ;
limited formation of stable emulsions with the fermenta-
tion broth ; it must be non-toxic to and non-
metabolizable by microorganisms ; it must be
immiscible with the aqueous phase, easily separable
from the product, non-Ñammable, cheap and available
in large quantities. Further, the added complexity and
cost of a ternary system has to be minimized by choos-
ing a third solvent which exhibits good separation effi-
ciency (no azeotrope formation) between all compounds
and which has to be recycled to minimize added pro-
duction cost.
Generally, alkanes and alkenes are good candidates
as inert bulk phases. In particular, those with a boiling
point higher than the substrate and product are of
interest, because it is easier to remove the product from
the bulk phase by distillation and the explosion risk
decreases with increasing carbon number. Hexadecane
(b.p. 286É6¡C) and hexadecene (b.p. 274¡C), both having
a high and similar volatility di†erence, were found to
meet the above criteria. They are not metabolized by P.
320 R. G. Mathys, O. M. Kut, B. W itholt

oleovorans GPo1 and are not toxic as experimentally
observed with batch fermentation tests in 250 cm3
Erlenmeyer Ñasks, with bacterial concentrations of 1 g
dm~3, which showed that hexadecane or hexadecene
did not inhibit the growth of P. oleovorans GPo1. Both
the substrate (n-octane) and the product (1-octanol) par-
titioned into these bulk solvents, leaving no octane and
only 0É05È0É08& 1-octanol in the polar phase after
phase separation. Since hexadecene, a fossil fuel deriv-
ative gained through the petroleum production, can be
purchased in large quantities at reasonable cost, we
used it as a bulk solvent in the experiments described
below. Thus, the new apolar phase mixture consisted of
85% hexadecene (v/v), c. 10È20% octane (v/v) and
4È5% 1-octanol (v/v).
3.1.2 Downstream processing
A proposed product separation and puriÐcation process
as part of a continuous bioconversion recovery oper-
ation is shown in Fig. 2.
Figure 2(A) illustrates the process for the more vola-
tile bulk OAP system, which consists of a two- or three-
step distillation, depending on whether solvent recovery
is done to recycle the organic feed back to the fermenter

Fig. 2. Flow-diagram of product separation process after fermentation/phase separation of the OAP system (A) and the HAP
system (B). A ; 1, Octane separation step (atmospheric distillation) ; 2, 1-octanol separation/puriÐcation step (vacuum distillation) ; 3,
solvent recovery tower. B ; 1, Hexadecene separation/puriÐcation step (vacuum distillation) ; 2, octane/1-octanol separation step
(atmospheric distillation).
Separation of 1-octanol by distillation 321

or whether the heavy solvent is wasted. Octane is

separated from 1-octanol by an atmospheric distillation
process (step 1). In this process 95% (v/v) of the feed
Ñowrate is octane which is now recycled with high
purity to the fermenter. Since the bottom product (1-
octanol) retains a signiÐcant amount of concentrated
impurities from the fermentation medium, a second
puriÐcation step is necessary. For this second step, the
addition of a higher boiling point solvent, such as hexa-
decene, will require vacuum distillation. The remaining
bottom product now consists of the heavier solvent and
concentrated fermentation medium purities. Hexa-
decene, or any other suitable heavy solvent, will subse-
quently have to be recovered in a third distillation step
so that impurities are not recycled back to the column ;
or the bottom product will have to be disposed of as
waste material when colored or otherwise decomposed.
Figure 2(B) shows the process for the less volatile
bulk HAP system, which consists of a two-step distilla- Fig. 3. Isobaric vaporÈliquid equilibrium curves for di†erent
tion. Here, the product and the remaining feed solvent mixtures of alkanes, alkenes and 1-alkanols. 1, octane/
are separated from the inert bulk apolar phase in a Ðrst hexadecene (p \ 0É5 MPa) ; 2, 1-octanol/hexadecene
step, which is accomplished by a separation/puriÐcation (p \ 0É1 MPa) ; 3, octane/1-octanol (p \ 1É0 MPa) ; 4, octane/
1-octanol (p \ 9É8 MPa). (Points measured-curves NRTL).
process requiring vacuum distillation. Based on the
work of Wubbolts et al.9 the ratio of apolar phase
mixture of the HAP system used throughout this study
for the vacuum distillation. Figure 3 shows the experi-
was 85 : 11 : 4% v/v (bulk solvent : substrate : product).
mental binary VLE data for the di†erent mixtures. This
Thus, the bottom product hexadecene has a Ñowrate of
phase diagram indicates that each of these mixtures
85% (v/v) with respect to the feed Ñowrate and includes
could be separated quite well. The ternary mixture, at
only minor impurities from the fermentation medium.
the earlier speciÐed compound ratio, was distilled and
The bottom product can be recycled directly to the bio-
showed good separation results. Therefore, we did not
reactor : because the impurities are considerably less
collect VLE data for this ternary system.
concentrated with the high HAP Ñowrate ; since no
Based on the VLE data, the resulting binary NRTL
coloring occurs ; and because the concentration of
activity coefficient parameters were calculated using a
impurities into the distillation column remains constant
process simulator (ASPEN), which provided such phase
at a constant efficiency of the phase separation process
equilibria calculations with di†erent activity coefficient
between the fermentation and distillation step. As a
correlations. These are listed in Table 1 and are needed
result no solvent recovery step of the bottom product is
to model and optimize separation performance for the
required for the HAP system in step 1. The distillate
design of a suitable distillation column. A constant
consists of puriÐed octane [11% (v/v)] and 1-octanol
value had to be assigned to the nonrandomness inter-
[4% (v/v)] and is separated in a second step atmo-
action parameter (c ). Renon and Prausnitz23 found
spheric distillation process, where the ratio of octane to ij
that the optimum value for c was between 0É40 and
1-octanol is approximately 73 to 27% (v/v). The top ij
0É55. The best Ðt was found at 0É43 (Table 1), with
product of the second (octane) step can be recycled to
which a good correlation of the experimental VLE data
the fermenter and the product (1-octanol) is recovered
could be obtained (Fig. 3).
from the bottoms.

3.3 Optimization of the OAP and the HAP systems

3.2 Optimization of solvent system : VLE data for
various binary mixtures of n-octane, 1-octanol and
1-hexadecene
Isobaric experiments were conducted at atmospheric
pressure and at reduced pressures of 0É1È1É0 MPa. The
latter were the lowest vapor pressure, for which the
individual mixtures did not require additional cooling,
under ambient temperature. This also allowed the opti-
mization of the pressure setting over a range of values
Today, most distillation operations are estimated using
commercial process simulators. We also used such mod-
eling for preliminary estimation of the distillation
apparatus design parameters and to optimize the
column performance. However, the e†ect of fermenta-
tion impurities on distillation efficiency cannot be pre-
dicted by computer simulations. Therefore, predicted
equipment design parameters were used to construct the
distillation column as described in Section 2 with which
322 R. G. Mathys, O. M. Kut, B. W itholt

TABLE 1
NRTL Parameters of Experimental VLE Data

Mixture Pressure k k k k k Sum of p

aij aji bij bji cij
(MPa) squares

n-Octane/1-octanol 1É0 and 9É8 [3É939 43 1É074 509 2158É689 [430É7878 0É43 26É628 1É1261
n-Octane/1-hexadecene 0É5 0É994 912 8 [10É010 91 [758É9131 4354É353 0É43 69É002 2É6268
1-Octanol/1-hexadecene 0É1 1É576 371 [11É345 44 [337É88 4435É136 0É43 9É261 0É917 55

k \ interaction parameter, p \ standard deviation, a \ refers to van der Waals internal pressure coefficient, b \ refers to van der
Waals volume coefficient, c \ nonrandomness constant for binary ij interactions, ij \ indices for components.

modeled optimization values were experimentally veri-
Ðed and Ðne-tuned for the simulated organic phases.
Optimized data for the OAP and HAP system are
shown in Table 2 where di†erent experimental runs are
compared in terms of mass and energy balances. The
control parameters of the experimental distillation pro-
cesses which have been optimized are the number of
actual plates, the reÑux ratio and the pressure for
vacuum distillation. All distillation runs were main-
tained at constant reÑux ratio (R), which is by deÐnition

TABLE 2
Mass and Energy Balance

Component OAP systemÈstep 1 HAP systemÈstep 1 HAP systemÈstep 2

(Fig. 2(A)) (Fig. 2(B)) (Fig. 2(B))
Distillate from
SOAP OAP SHAP HAP apolar phase

Feed :
Hexadecene (g) È È 910É0 962É5 È
Octane (g) 994É0 979É3 134É8 134É2 792É5 (73É5%)
1-Octanol (g) 53É1 59É5 43É9 48É4 286É3 (26É5%)
Distillate :
Hexadecene (g) È È 0 0 È
Octane (g) 992É2 977É3 134É8 134É2 787É9
1-Octanol (g) 5É0 4É9 41É2 46É0 4É8
Bottom product :
Hexadecene (g) È È 910É0 962É5 È
Octane (g) 1É8 2É0 È È 4É6
1-Octanol (g) 48É1 54É6 2É7 2É4 281É5
Feed :
Fraction of 1-octanol/octane (%) 5É3 6É1 32É6 36É1 36É1
Distillate :
Purity of light compound (octane) (%) 99É5 99É5 È È 99É4
Purity of binary mixture (octane/1-octanol) (%) È È 100 100 È
Percent of product (1-octanol) recycled 9É4 8É2 È È 1É7
(% 1-octanol in distillate/1-octanol in feed)
Bottom product :
Purity of product (1-octanol) (%) 96É5 96É4 È È 98É4
Purity of heavy compound (hexadecene) (%) È È 99É7 99É75 È
Percent of product (1-octanol) recycled È È 6É2 5É0 È
(% 1-octanol in bottom/1-octanol in feed)
Number of actual trays 4 4 3É8 3É8 4
ReÑux ratio 1 1 1É5 1É5 1
Pressure (MPa) 9É6 9É6 0É5 0É5 9É6
Final temperature in still (¡C) 235 231 175É0 174É0 192
Duration of distillation run during steady state (min) 40 44 23É0 25É0 31
Reactive energy consumption (W h) È È 68É0 91É0 È
Energy requirement during steady state (W h) 361É0 398É0 201É0 215É0 282É0
Energy requirement of vacuum pump (W h) È È 73É5 78É0 È
Energy/mass of product recovered (kW h kg~1) 7É48 7É3 6É66 6É37 1É0
Energy cost/mass of product recovered (US$ kg~1) 0É67 0É66 0É60 0É57 0É09
Separation of 1-octanol by distillation
the ratio of the liquid returning to the column (dm3) to
the distillate (D). The pressure was optimized between
0É5 and 1É0 MPa, since the di†erence between the distil-
lation temperature and cooling water temperature
becomes so small below 0É5 MPa that additional
cooling would be required, consuming more energy ;
above 1É0 MPa energy requirements became too high.
The batch system was operated at total reÑux “to equili-
brateÏ before starting product removal. For extrapo-
lation of the results to a continuous system, energy
requirement data were compared during steady state
operation, i.e. when product removal began. Neverthe-
less, it has to be kept in mind that batch distillation, in
contrast to continuous distillation, is always a transient
process, since the composition of the mixture and con-
sequently, the temperature and the volume in the still
are changing over time. Also, liquid hold-up of the dis-
tillate from condenser, column and column packings,
which will return to the still every time when a charge is
Ðnished, contribute to an increased impurity of the
bottom product.
The measured energy requirements of the distillation
process, given in Table 2, include electrical energy
required for reboiler duty, reÑux, pumps and process
control and are reported as a function of the recovered
product. No heat integration has been considered in
this study. Clearly however, the use of an integrated
heat loop which recovers the exothermic heat of the
bioconversion reaction and recovery of overhead con-
denser heat, supplied back to the column reboiler,
would lower the overall energy requirements of this
system. Reactive power consumption of the induction
motor is reported in Table 2 for the HAP system, but
was not added to the total energy cost, since reactive
energy consumption is usually compensated by install-
ing consumption controlled capacitors in plants. Energy
requirements were calculated at an average cost of
9 US{ kW h~1.
3.4 Comparison of the OAP and the HAP systems
The comparison of Table 2 shows that energy costs of
the OAP system are about 10% higher than for the
HAP system (step 1). The purity of the HAP (step 1)
distillate can be considered satisfactory at the reported
column setting, and bottom product efficiency will be
improved in a continuous distillation, when the e†ect of
column hold-up is eliminated. Based on similar energy
expenditures of the Ðrst distillation step, the purity of
the OAP effluents, especially that of the bottoms which
contain the product, is considerably lower than that of
the distillate of the HAP system, which contains the
same product.
The second step distillation of the MAP was only
done experimentally for the HAP system (Fig. 2(B)),
because the results of the OAP distillation showed that
323
the performance of this system was not such to be
pursued further. The results for the second step HAP
distillation are reported in Table 2. The energy cost per
kg of product recovered is small compared with the Ðrst
step of the process, because of the large amount of
product recovered relative to the total amount of feed
into the distillation column.
Total energy cost for product recovery of the HAP
system, including steps 1 and 2 (Fig. 2(B)), amount to
approximately 0É67È0É78 US$ kg~1 product recovered.
3.5 E†ect of fermentation impurities on the efficiency
and energy requirements of the OAP and HAP systems
The data in Table 2 also give a comparison of the two
model systems (OAP and HAP) to the performance of
the corresponding simulated organic phases (SOAP and
SHAP), which contained no impurities from the fermen-
tation step. The results show negligible di†erences
between both the SOAP and the OAP, and the SHAP
and HAP, based on mass and energy balance compari-
sons. Small di†erences were mainly accounted for by
the di†erent ratios of product to bulk solvent (OAP), or
of product to light component mixture (HAP). A major
di†erence however, between the SOAP and the OAP
system was the strongly colored bottom product of the
OAP, resulting in inferior product quality. Coloring was
observed to start at 130È150¡C. During atmospheric
distillation the OAP system reached a temperature of
140È150¡C at steady state under total reÑux, rising up
to 235¡C when batch operated.
4 DISCUSSION
This paper illustrates some of the possibilities on how
di†erent parameters, such as extraction solvent, bottom
and distillate Ñowrate, and pressure can be varied in
order to improve the puriÐcation of a biotech-
nologically produced medium cost chemical, with the
aim to minimize the downstream processing cost for a
given desired product purity.
4.1 Optimization of solvent system
1-Hexadecene is a good apolar solvent for use in two-
liquid phase whole cell bioconversions, because it is
essentially inert with respect to the microorganisms, and
because it is a good extractant for apolar products
formed in the bioconversion.
In this paper we have shown that hexadecene is also
very useful in subsequent product separation. Fermen-
tation tests in a 3 dm3 continuous stirred reactor with a
20% (v/v) hexadecene apolar phase (HAP) and a micro-
organism concentration of 7È11 g dm~3 in the aqueous
phase resulted in a less emulsiÐed apolar phase com-
pared with the octane apolar phase (OAP) system and
324
microbial growth was not inhibited. Phase separation of
the HAP system using centrifugation also showed
improved separation capabilities relative to the OAP
system. Further studies related to the characteristics of
apolar phases in the two-liquid fermentation systems,
especially for phase separation purposes, have also been
undertaken in our laboratory. In general, alkanes and
alkenes are well suited as in-situ extraction solvents and
for further product separation using distillation.
However, when di†erent compounds are produced in
the fermentation process, VLE data and other ther-
modynamic properties of the apolar phase system have
to be obtained for optimizing distillation performance.
4.2 Comparison of the OAP and the HAP systems
The results of this study show that obtaining the
product as a distillate rather than bottom product after
the Ðrst distillation step is favorable. Total distillation
costs (production and capital investment) are higher in
the latter case, because either a third solvent recovery
step will be required and because the fermentation
residue in the still decomposes, which contributes to
increased disposal and, consequently, fresh solvent cost.
Thus, it is useful to modify the upstream process by
altering the extraction solvent, to reduce subsequent
process steps and to avoid the decomposition of the
bottom product, resulting in lower energy cost of the
overall distillation, especially in the Ðrst step.
The Ðrst distillation step of the HAP system (Table 2)
gave slightly better results than that of the OAP system
in terms of energy requirements and much better results
in terms of product recovery. The purity of the bottom
product from the OAP system (step 1) was improved to
98É8%, and to 99É9% in the distillate, when the reÑux
ratio was increased to 3. However, this parameter
change resulted in energy costs of 1É10 US$ kg~1
product recovered for the OAP system leading to a cost
di†erence between the OAP (step 1) and the HAP (step
1) system of about 45%. The energy cost of the modeled
second step OAP distillation process was found to be
similar to that of the second step HAP distillation. As a
result, when the additional energy cost of a third step
OAP solvent recovery was added, the total energy cost
of the OAP system surpassed that of the HAP system.
Thus, the proposed HAP separation was found to be
superior, both in terms of product purity and energy
cost than the process based on the OAP system. The
actual energy cost as a function of the recovered
product is very sensitive to, and will strongly depend
on, the degree of substrate conversion during the fer-
mentation process. This e†ect, which is dependent on
biocatalytic activity, is likely to be similar in both the
OAP and HAP fermentation. The impact of di†erences
in the feed fraction “1-octanol/octaneÏ on energy cost/
mass of product recovered between the SOAP/OAP
R. G. Mathys, O. M. Kut, B. W itholt
and SHAP/HAP, respectively, can be seen in Table 2.
This e†ect increases with increasing product to solvent
ratio. This shows how signiÐcantly production costs are
inÑuenced by the energy input into a distillation
column, as a function of the distillate Ñowrate.28 In
addition, for both systems OAP and HAP liquid
hold-up due to batch operation decreases the purity of
the bottom product.
We observed coloring of the bottom product of the
OAP system, as a result of the polymerization of sugars
from the trace amounts of fermentation medium impu-
rities remaining in the apolar phase after phase separa-
tion (OAP and HAP), and found this to be a function of
temperature and time. Even very small fermentation
impurities in the apolar phase resulted in a colored
bottom product when the bottom temperature exceeded
130¡C and the residence time of the mixture in the
bottoms was long. In contrast, no coloring of the
bottom product was observed during vacuum distilla-
tion of the HAP, where the temperature was 100È110¡C
at steady state under total reÑux and reached 175¡C
only at the end of a charge. Hence, di†erences between
the OAP and HAP systems are due to a large extent to
the depleting volume in the pot during batch operation.
When operating continuously, the OAP heavy com-
ponent product in the bottoms has a very small Ñowrate
(5% (v/v) of total Ñowrate) and consequently a long
residence time, compared with the HAP system where
the Ñowrate is 85% (v/v) with respect to the total Ñow-
rate, resulting in a rapid turnover of the bottom
product.
Besides the smaller energy cost per kg of product
removed in the second step HAP distillation due to the
ratio of product to total feed, the Ñowrate into the con-
tinuous distillation column of step 2 (see Fig. 2(B)) will
only be 15% with regard to the feed Ñowrate of step 1.
Distillate and bottom product purity can be improved,
if required, either by small reÑux ratio increments or by
the addition of one or two more actual trays, without
signiÐcant additional energy requirements as was
demonstrated experimentally. After the reÑux ratio was
changed to 3, the energy cost of step 2 increased by only
0É02 US$ kg~1 of product recovered.
In the accompanying paper,17 the e†ect of contami-
nation of the apolar phase by aqueous fermentation
medium components on subsequent distillation per-
formance are considered. With this information, it is
possible to determine the dependency of product distil-
lation on prior phase separation efficiency under
various conditions, based on which both the phase
separation and the distillation can be optimized.
Overall fermentation/recovery economics are dependent
on raw material prices of the solvents. Bulk quantity
solvent prices were obtained from Shell16 at 0É45 US$
kg~1 for both n-octane and 1-hexadecene and the
solvent loss can be estimated to be similar for both
systems at 5È10% (v/v), for steps 1 and 2 (Fig. 2(B)) or
Separation of 1-octanol by distillation
steps 1 and 3 (Fig. 2(A)). However, if the solvent is not
recovered in the OAP system (step 3), solvent losses will
be higher for this process. Other costs include
equipment costs, which will be higher for the OAP
system when comparing a three-step distillation process
to the HAP two-step distillation process. The overall
economics of the fermentation and distillation systems
will be described elsewhere.
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