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Extractive fermentation with non-ionic surfactants to

enhance butanol production

Pradip B. Dhamole a,c, Zhilong Wang a,d, Yuanqin Liu a, Bin Wang a, Hao Feng a,b,*
Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Department of Biotechnology, Sinhgad College of Engineering, Pune 411041, India
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China

article info abstract

Article history: One of the major limitations in butanol fermentation is the end product toxicity which
Received 29 October 2011 limits the butanol yield and increases the downstream processing costs. In this work,
Received in revised form a range of non-ionic surfactants (Triton X 114, L64, L62LF, L61, and L62) was tested to
6 February 2012 enhance the acetoneebutanol (AB) production, and to extract and separate butanol from
Accepted 9 February 2012 the fermentation broth. In biocompatibility tests, a volume fraction of 3% L62, L62LF, and
Available online 25 February 2012 L61 did not show inhibition to AB production in 72-h fermentation using Clostridium pas-
teurianum. Three-percent L64 reduced the AB yield whereas Triton X 114 (3%) inhibited the
Keywords: AB production. Further optimization with L62 at 6% resulted in a butanol yield of 225%
Butanol higher than the control. The partition coefficient of butanol in L62-water two phase
Biofuel systems in cloud point extraction ranged from 3 to 4. A considerable enrichment of butanol
Non-ionic surfactant (6 times) was achieved in the surfactant-rich phase over the control. In addition, the
Extractive fermentation downstream process volume was reduced by 4e6 times. Butanol was separated from the
Biocompatibility surfactant-rich phase (obtained from model system) by evaporation between 120 and
130  C. The butanol was enriched in the condensate reaching a concentration of 106.8 g l1,
under which butanol automatically separated into two phases. The L62 was recovered by
evaporation and reused for 3 times without affecting the partition coefficient, volume
reduction, and butanol recovery in the surfactant-rich phase. The results demonstrated
that the L62 not only significantly enhanced the butanol production but also functioned as
a good extractant for separating butanol from the fermentation broth.
2012 Elsevier Ltd. All rights reserved.

1. Introduction 30% higher energy content (29.2 MJ L1) over ethanol

(19.6 MJ L1), lower vapor pressure, less volatile, less flam-
In recent years, a renewed and growing interest in the mable, and mixable with gasoline [1e4]. However, since the
production of acetone, butanol and ethanol (ABE) has been discovery of ABE fermentation a century ago, biobutanol
stimulated by the increasing demand for advanced biofuels. production has faced numerous difficulties, preventing it from
Butanol is the main product of ABE fermentation, and has becoming a commercially viable process. There are two inter-
some attractive properties. The advantages of butanol include related outstanding challenges facing the commercialization

* Corresponding author. Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 382F-AESB, 1304
West Pennsylvania Avenue, Urbana, IL 61801, USA. Tel.: 1 217 244 2571; fax: 1 217 333 9329.
E-mail address: haofeng@illinois.edu (H. Feng).
0961-9534/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 1 2 e1 1 9 113

of biobutanol today. The first is the end-product toxicity to the micelle concentration and/or cloud point, surfactant mole-
fermenting microorganisms. Butanol at low product concen- cules usually assemble themselves into many kinds of struc-
trations (normally < 20 g L1) causes cell growth inhibition and tures i.e. micelles, lamellar, hexagonal, etc. These nano-sized
premature termination of ABE fermentation [5]. The toxicity of micellar assemblies formed facilitate removal of targeted
butanol has been ascribed to passive proton flux by butanol compound (depending on its properties).
causing membrane leaking [6], disruption of the lipid struc- Hence, in this work, we have explored a non-ionic surfac-
ture in cell membranes that alters membrane-bound enzyme tant aqueous solution system for overcoming the end product
activity [7], and membrane fluidity in the presence of butanol (butanol) toxicity and separation of butanol from the non-
[8]. Newly developed strains, such as Clostridium beijerinckii ioninc surfactant micelle aqueous solution by cloud point
P260, have shown improved butanol tolerance, but the extraction. The same hypothesis is used to relieve butanol
maximum yield of butanol is still ca. 22.27 g L1 after hydro- toxicity during fermentation in non-ionic surfactant micelle
lysate detoxification [9]. The other challenge is the high energy aqueous solution, resulting in a significant increase in butanol
cost of recovering butanol from the fermentation broth. Due yield. Experiments were carried out with non-ionic surfac-
to the low concentration (w20 g L1) in the broth and the high tants to find its butanol capturing capacity and those with
boiling point of butanol (117  C), removing water to obtain high butanol capturing capacity were tested further for its
purified butanol is an expensive process [10e12]. To attack the biocompatibility. The surfactant resulting in high butanol
low concentration butanol recovery problem, scientists have production than control (i.e., without surfactant) was used for
explored various techniques for butanol separation, including concentrating and separating butanol. The separation was
adsorption [13,14], pervaporation [15,16], perstraction [17,18], carried out after fermentation by incubating the broth at
liquideliquid extraction [19,20], and gas stripping [21,22]. a fixed temperature. A model system was used to study
Extractive fermentation is a potential method to eliminate further downstream processing of butanol and recovery of
the product inhibition and thus increase the final product surfactant. Recovered surfactant was reused to capture
concentration. The concentrated product in the extraction butanol.
phase during fermentation could save the cost in down-
stream process. Extractive fermentation of butanol has been
conducted in organic solvent-aqueous solution two-phase 2. Materials and methods
systems [23e26] and in aqueous solution-polymer two-phase
systems formed by polymer polypropylene glycol (PPG) [27]. 2.1. Chemicals
Kumn [28] carried out an extractive fermentation of ethanol in
polyethylene glycol (PEG)-dextrin aqueous two-phase system. Glucose, yeast extract, K2HPO4, KH2PO4, ammonium acetate,
The key challenge of extractive fermentation in aqueous para-amino benzoic acid, thiamine, biotin, MgSO4.7H2O,
solution-organic solvent two-phase systems is the biocom- MnSO4.H2O, FeSO4.7H2O, and NaCl of analytical grade were
patibility of the organic solvent to the bacteria [29] whereas purchased. Non-ionic Pluronic surfactants L61, L62, L62LF, and
that of the PEG-dextrin aqueous two-phase system it is the L64 were provided by BASF, USA as a gift sample. The letter L
high price of the polymer dextrin. A number of non-ionic in the nomenclature denotes that the surfactant is liquid.
surfactant aqueous solution forming cloud point systems at The first number in the surfactant name indicates the
above a certain temperature have been developed as a novel molecular weight range of the hydrophobe (i.e., PPO) whereas
medium for extractive microbial fermentation [30e33]. The the second number signifies the weight percentage of hydro-
main advantages of extractive fermentation in a cloud point phile (i.e., PEO). Thus, L61 has a weight to volume fraction of
system include that the biocompatibility to the bacteria is 10% hydrophile whereas L62 and L64 have 20% and 40%
improved in comparison to that of organic solvent-aqueous hydrophile, respectively.
solution two-phase systems, and the cost of PEOePPOePEO
block copolymers is lower than that of dextrin in an aqueous 2.2. Culture and cell propagation
two-phase system. Aqueous solution of a non-ionic surfactant
at a temperature above the cloud point (the temperature at Acetoneebutanol producing strain Clostridium pasteurianum
which the copolymer solution starts to separate) forms (NRRL B-598) obtained from the ARS culture collection centre
a surfactant rich phase (coacervate) and surfactant diluted (NRRL, Peoria, IL, USA) was used as a model organism because
phase. An organic compound presenting in the non-ionic a high yield strain was not available. The strain was activated
surfactant aqueous solution should unevenly partitioned into following the procedure provided by the supplier. Stock
those two phases. Such a scheme is called cloud point solutions were prepared before starting the fermentation. The
extraction (CPE). CPE has many advantages which include buffer stock solution consisted of K2HPO4 (50 g L1), KH2PO4
mild environment, simplicity, effectiveness of operation and (50 g L1), and ammonium acetate (220 g L1). The vitamin
easy scale up. Surfactants are amphiphilic in nature i.e. stock solution contained 0.19 g L1 para-amino benzoic acid,
having a polar part and an apolar part which interact with 0.19 g L1 thiamine, and 0.19 g L1 biotin whereas the
interfaces. Amphiphilic characteristics are critically depen- composition of minerals stock solution was 20 g L1
dent on the molecular properties, such as total molecular MgSO4.7H2O, 1 g L1 MnSO4.H2O, 1 g L1 FeSO4.7H2O, and
weight, relative block size and block sequence as well as 1 g L1 NaCl. Before fermentation the strain was reactivated by
thermodynamic parameters, such as temperature and pres- incubating 1 ml of refrigerated strain into 25 mL of Difco
sure. Water solubility of the surfactant is due to hydrophilic Infusion broth (35 g L1). 0.25 mL of the buffer stock solution
group which is an ionic or highly polar group. Above critical was added to the infusion broth before inoculation. The flasks
114 b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 1 2 e1 1 9

were incubated at 32  C for 36 h and the cells thus obtained micelle diameters were measured by dynamic light scattering
were used for fermentation. at a fixed scattering angle of 90 at 23  C with a NICOMP 380
ZLS Particle Sizer.
2.3. Butanol capturing capacity (BCC) of surfactants
2.7. Extraction of butanol from fermentation broth
A dialysis cell (Scienceware, Bel-Art Products, NJ) and UF
membrane was used in determining the relative butanol After the fermentation, cells were separated from the
capturing capacity of a variety of surfactants. Total volume of fermentation broth by centrifugation at 13,130  g for 5 min to
the cell was 18 mL which was divided into two compartments obtain a clear fermentation broth. It was then incubated in
by a membrane, with each compartment of 9 mL. One side of a water bath (Poly Science Digital Temperature Controller,
the cell was filled with distilled water whereas the other side Niles, IL) at different temperatures between 35 and 70  C to
was filled with a solution containing surfactant (30 g L1), obtain a phase separation. The clear phase separation was
butanol (50 g L1), and glucose (60 g L1). The dialysis cells observed at 70  C on incubation for 30 min when a volume
were kept at 30  C throughout the study. Samples were fraction of 6% L62 was present. This resulted in a two phase
collected from the water side on a periodic basis till the steady formation with lower phase rich in surfactant and the upper
concentration of butanol was observed (data not shown). The phase being the aqueous phase. Samples were collected from
butanol capturing capacity was defined as amount of butanol the separated phases and analyzed for butanol concentration.
captured per unit amount of surfactant.
2.8. Separation of butanol from surfactant rich phase
2.4. Fermentation
C. pasteurianum produces very low amount of butanol
A fermentation medium consisting of glucose (60 g L1), yeast (6e8 g L1) [34]. Due to unavailability of a high yielding strain to
extract (1 g L1), buffer stock solution (1 ml L1), mineral stock the authors, fermentation was carried out with C. pasteurianum
solution (1 ml L1), and vitamin stock solution (1 ml L1) was and separation studies were conducted with model systems.
used. Nitrogen was purged after the inoculation and every A model system consisting 50 g L1 butanol and 6% L62
time the sample was withdrawn to maintain the anaerobic (optimum for fermentation) was chosen to resemble the
conditions. Flasks were incubated at 32  C for butanol process with high butanol producing strains. The model
fermentation. Fermentation was carried out with and without solution (containing 50 g L1 butanol and a volume fraction of
surfactant (control). Surfactant addition was carried out after 6% L62) was incubated at 38  C to obtain the surfactant rich
autoclaving the fermentation medium and before inoculation. phase [35]. The surfactant rich phase thus obtained was used
for further downstream processing. Butanol and water from
2.5. Analysis the surfactant rich phase was separated by evaporation
between 120 and 130  C and the vapor phase was condensed in
Acetone and butanol in fermentation was determined by a condenser using tap water. The feed, condensate and
a gas chromatograph unit (GC Hewlett Packard 5890 Ser- concentrate were analyzed for butanol concentration, and
ies II, Avondale, PA) equipped with an auto sample their volumes were recorded for material balance calculations.
injector (HP 7673A Automatic Injector) and a flame
ionization detector (FID). The column used was DB-WAX 2.9. Reuse of L62
30 m  0.250 mm  0.25 mm fused silica capillary column
(J & W scientific, Agilent Technologies, Germany). The oven The L62 used in extraction of butanol (50 g L1) was separated
temperature was programmed from 40  C to 190  C at following the above mentioned process. The concentrate
20  C min1. The injector and detector temperature was set to containing L62 was reused for butanol extraction. The L62
220  C and 250  C, respectively. The carrier gas was He at concentrate was added into a solution having a total butanol
0.72 mL min1 flow rate and acetonitrile was used as an concentration of 50 g L1 and the solution was incubated at
internal standard. Peaks, areas and percentages were calcu- 38  C to obtain a surfactant-rich phase and an aqueous phase.
lated using Agilent Technologies GC Chemstation software Samples were collected from both phases for butanol analysis.
(Agilent Technologies, Germany). Glucose was estimated The surfactant-rich phase was again processed to recover L62
using HPLC system (Waters Corporation, Milford, MA) e2695 following the above procedure and was reused for butanol
separation module and a Waters 2414 refractive index extraction for the third time.
detector monitored by an Empower pro software version 6.2.
Aminex column (HPX-87P 300 mm  7.8 mm) equipped with
Microguard Carbo-P cartridge (30 mm  4.6 mm) from Biorad 3. Results and discussion
(Hercules, CA) was used.
3.1. Screening of surfactant
2.6. Characterization of surfactant in the model system
The relative butanol capturing capacity of the selected
H NMR spectra of the L62 solutions were recorded with surfactants (non-ionic and triblock co-polymer) (Triton X 114,
a Varian Unity 400 MHz NMR spectrometer using D2O as the L61, L62, L64, L62LF) was first examined with the dialysis cell
solvent in 5 mm NMR tubes. The residual signal of the solvent method at 30  C. As shown in Table 1, 1 kg of Triton X 114 and
was used as a reference in all the spectra (D2O, d 4.8). The L62 co-polymer L62LF can capture 0.6 and 0.52 kg of butanol,
b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 1 2 e1 1 9 115

Table 1 e Butanol capturing capacity of some selected A 7

surfactants at 30  C. Control
Surfactant Butanol capturing Cloud point 6 L62LF
Triton X114
capacity, (kg kg1) temperature ( C)
5 L64
Triton X 114 0.6 25
L62LF 0.52 28

Butanol (g L )

L62 0.32 32 4
L64 0.06 58
L61 Insoluble in water 24

respectively; much higher than that of the other 3 polymers. 2

Butanol captured and the cloud point of the surfactant is also
included in Table 1. It can be seen that non-ionic surfactants 1
having a low cloud point (than 30  C) captured high amount of
butanol at 30  C. It is assumed that micelle formation
(for Triton X 114 and L62LF) is better above cloud point. Hence, 0 10 20 30 40 50 60 70 80
Triton X 114 entrapped the highest amount of butanol whereas
Time (hour)
L64 just captured very little butanol since the operating
temperature (30  C) is lower than the cloud point (58  C) of L64. B 2.0
In control fermentation (i.e. without surfactant) maximum Control
butanol produced was 4.75 g L1 after 120 h. Fermentation was
Triton X114
continued till 168 h, however butanol concentration remained L43
1.5 L64
unchanged. Corresponding unutilized glucose concentration
was observed to be 42.7 g L1 (Initial glucose 60 g L1) which
Acetone (g L )


clearly indicated that butanol fermentation was inhibited at

4.75 g L1 of butanol. Similar studies with C. pasteurianum
(NRRL B-598) are reported in the literature however in
those studies higher inhibition levels (6e7.5 g L1) were
observed which could be attributed to different medium
composition [34]. 0.5
The biocompatibility of the surfactants was evaluated with
an AB fermentation test with C. pasteurianum where 3% L62
was added in a fermentor and the butanol yield over a 72-h
period was monitored (Fig. 1). Among the 5 surfactants 0.0
examined, Triton X 114 totally inhibited AB production while 0 10 20 30 40 50 60 70 80
a significant reduction in butanol yield when compared to the Time (hour)
control (without surfactant) was observed for L64. L61 and
L62LF produced nearly the same amount of butanol as the Fig. 1 e Biocompatiblity of different surfactants for AB
control. In the case of L64, the acetone and butanol produced fermentation. (A) Butanol production and (B) Acetone
were about 50% of the control, respectively. Notably, the production. Concentration of surfactant [ 3%, volume
fermentation with L62 enhanced the AB fermentation. Addi- fraction. Temperature [ 32  C.
tion of 3% L62 increased the acetone as well as butanol
production by little over 70% (Fig. 1A and B).
The toxicity of surfactants follows the order: ionic capacity. Therefore, for Triton X 114 having a high butanol
surfactant > non-ionic surfactant > polymeric non-ionic capturing ability, because of its high HLB values, is toxic to the
surfactant [30]. All the pluronic surfactants studied in this microbes. Only the L62, which had a moderate butanol
work are polyoxypropylene (PPO) epolyoxyethylene (PEO) capturing capacity but a low HLB, enhanced the butanol
block copolymers. The toxicity of a surfactant can be corre- production (0.32 kg kg1 of surfactant). Therefore, L62 was
lated with its hydrophileelipophile balance (HLB) value. used in all the subsequent AB fermentation tests.
Generally, an increase in HLB is accompanied by an increase
in the toxicity of the surfactant to microbes. For Triton X 114 3.2. Effect of surfactant concentration
and L64 the HLB values are 12.3 and 12 to 18, respectively and
hence were toxic to the microbes. Other three surfactants Screening of surfactants showed that a volume fraction of 3%
(L61, L62 and L62LF) have HLB values in the range of 1e7 and of L62 enhanced the acetone and butanol production (Fig. 1).
hence are non-toxic to the microorganisms. L61 is insoluble in It was desirable to find out the optimum amount of L62 which
water and does not extract butanol from the fermentation; will further increase the AB production. Since the AB yield
hence no relief to the microbes from butanol. Further, the remained increasing at the end of 72-h fermentation, showing
butanol yield during fermentation with a surfactant is related the potential to further increase the AB production, if the
to both the surfactant toxicity and its butanol capturing fermentation is continued over an extended period. It was
116 b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 1 2 e1 1 9

thus decided to study the effect of surfactant concentration on 12

fermentation over a period of 5 days. As can be seen from
Fig. 2, an increase in surfactant concentration from 3% to 6% Butanol (control)

Acetone/Butanol Produced (g L )
10 Butanol (6% L62)

improved the acetone and butanol production by 25%. Acetone (control)
However, a further increase in surfactant concentration (9%) Acetone (6% L62)
did not enhance AB production. The results suggested that 6% 8
L62 was the optimum for fermentation. Fig. 3 shows the time
course of AB fermentation in presence of 6% L62. It can be seen
that the AB production was higher from day 3 onwards. At the 6
end of 120 h, the AB produced in the presence of L62 was 225%
higher than that of the control. The fermentation was
continued till 8 days but no further increase in AB was
observed (data not shown).
It was assumed that L62 might have formed micelles and 2
entrapped butanol resulting in high butanol production
during fermentation. To confirm this, studies were carried out
to check the micelle formation by the surfactant in a model
0 20 40 60 80 100 120
system at fermentation temperature (32  C) with NMR and
dynamic light scattering. The 1H NMR spectrum (Fig. 4) clearly Time (hour)
shows the methyl proton signal of both L62 (d 1.1) and butanol
Fig. 3 e Time course of AB fermentation in the presence of
(d 0.8). If L62 formed micelles and entrapped butanol, the NMR
a volume fraction of 6% L62.
signal of butanol should decrease or disappear. However, from
the integral of the peaks the ratio of L62/butanol was similar to
the theoretical ratio (1: 0.5). The NMR data thus indicated that
butanol was free and in an un-trapped form in the model
fermentation process was used to separate and concentrate
system. The particle size measurement results (Table 2) also
butanol from the fermentation broth. An increase in temper-
demonstrated that there were no micelles detected in the
ature above the cloud point resulted in two phases with upper
model system (L62 (6.0%) butanol (3 or 30 g L1)) because the
phase being aqueous and the lower one rich in L62. The
measured particle sizes (10e11 nm) were similar to that of
butanol was concentrated in surfactant rich phase. To sepa-
single L62 molecule (L62 (0.001%), 8 nm) and much smaller
rate the surfactant rich phase and to enrich butanol in the
than the size of L62 micelles (L62 (6%), 130 nm). It can be
surfactant rich phase, the entire broth was first incubated at
concluded that no micelles formed in the model system and
different temperatures to find out the phase separation
the increase in AB production might be attributed to the
conditions. It was noted that the presence of cells increased
attachment of butanol to the monomers of L62.
the turbidity and no clear phase separation was observed.
Therefore, cells were separated from the fermentation broth
3.3. Concentration of butanol from fermentation broth by centrifugation to obtain a clear fermentation broth. The
by phase separation clear supernatant was then incubated at different tempera-
tures to separate the surfactant-rich phase from the aqueous
Butanol produced during the fermentation needs to be sepa- phase. Two phase formation was observed at 70  C after
rated from the fermentation broth. The L62 added during the incubating the supernatant for 30 min Table 3 shows the
partition of butanol in aqueous phase and surfactant rich
phase. The butanol was enriched in the surfactant-rich phase,
as shown by a partitioning coefficient of 3.5. Furthermore, the
10 volume was reduced by a factor of 6 (i.e. the initial volume/
Butanol Produced (g L )

surfactant rich phase volume 6), resulted in a significant


0 2 4 6 8 10

L62 Added in the Medium (%)

Fig. 2 e Effect of amount of L62 (in volume fraction) on AB Fig. 4 e 1H NMR spectrum of a model system (L62 (6.0%,
fermentation (Data after 120 h of fermentation). volume fraction) D butanol (3 g LL1)).
b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 1 2 e1 1 9 117

Table 2 e Particle size of co-polymer L62 in the solution.

% (v/v) Diameter at 32  C (nm)

L62 (0.001%) 8  4
L62 (6%) 130  38
L62 (6%) butanol (0.3 g L1) 10  4
L62 (6%) butanol (3 g L1) 11  3

reduction in downstream process volume and hence energy

usage in butanol recovery. It needs to be emphasized here that
in the control system (without L62) only 5 g L1 of butanol was
produced by C. pasteurianum whereas with the surfactant
system the surfactant rich phase contained 30 g L1 butanol Fig. 5 e Separation of butanol from the surfactant rich
representing a 6 times enrichment. Therefore, the addition of phase by evaporation (temperature [ 120e130  C).
L62 during the fermentation not only increased the fermen-
tation yield, it also reduced the process volume and enriched
the surfactant rich phase significantly.
achieved; this will further increase the production and reduce
Since C. pasteurianum used in this study produces very low
the downstream separation costs.
amount of butanol, to represent the butanol fermentation
with high yield strains (such as C. beijerinckii BA101), down-
stream processing was carried with a model system contain- 3.4. Separation of butanol from surfactant rich phase
ing higher butanol concentration (50 g L1) and 6% L62 and reuse of surfactant
(optimum for fermentation). In the presence of 6% L62 C.
pasteurianum produced 10.71 g L1 of butanol which is 225% The surfactant rich phase obtained after incubating the
higher than control. It is anticipated that high butanol aqueous solution containing butanol (50 g L1) and L62 (6%)
producing strains will produce butanol in presence of was used for further studies. Fig. 5 shows the material balance
biocompatible L62 (6%) in a similar manner. Thus a strain over the evaporation unit and composition of the feed,
producing 20e22 g L1 butanol may produce 45e50 g L1 condensate and concentrate. Water and butanol were
butanol. obtained in the condensate and the high butanol concentra-
The phase diagram of L62 with 50 g L1 butanol was tion (106.8 g L1) resulted in two phase formation with the
reported in our earlier work [35]. It was observed that the upper phase being butanol. Over 95% butanol was recovered
butanol concentration had a significant effect on phase from the surfactant rich phase. Very little butanol remained in
separation. Butanol lowers the cloud point. In the presence of the surfactant (2.1 g L1). Since the entire separation system is
50 g L1 butanol and 6% L62, the phase separation took place at closed, the high boiling point L62 is retained in the concen-
38  C (model system). When the fermentation broth contained trate. Further, the minimum amount of butanol that remained
10 g L1 butanol, a clear phase separation was observed at in the L62 implies that surfactant can be reused for butanol
70  C whereas with 50 g L1 butanol the phase separation extraction. Hence, tests were conducted to examine the
occurred at 38  C (model system) [35]. Consequently, for a high reuse of L62 for butanol extraction. The results are included in
butanol producing strain, the temperature required for Table 4. The basic parameters of extractive fermentation,
phase separation will be close to fermentation temperature. i.e., the volume ratio of non-ionic surfactant-rich phase
If a phase separation takes place during fermentation, to aqueous phase, partitioning coefficient of 1-butanol and
a continuous extraction of the produced butanol can be 1-butanol concentration, were unchanged in the second and

Table 3 e Separation of L62 from the aqueous phase.

Fermentation broth Model system containing
(after fermentation) 50 g L1 butanol and 6% L62

Initial concentration of butanol, g L1 11.8 50

Concentration of butanol in surfactant rich phase, g L1 30.1 95
Concentration of butanol in aqueous phase, g L1 8.71 35.18
Concentration factor 2.55 1.9
Initial volume, mL 12 12
Volume of surfactant rich phase, mL 2 3
Phase volume ratio (Aqueous phase : 6: 1 4: 1
Surfactant rich phase)
Partition coefficient (Butanol in surfactant 3.5 2.7
rich phase/Butanol in aqueous phase)
118 b i o m a s s a n d b i o e n e r g y 4 0 ( 2 0 1 2 ) 1 1 2 e1 1 9

Table 4 e Repeated utilization of recovered polymer non-ionic surfactant in the L62-rich phase (Butanol [ 50 g LL1,
L62 [ 6%, Incubation temperature [ 38  C).
Run Volume ratio Partitioning coefficient Butanol in surfactant Butanol in aqueous
phase, g L1 phase, g L1

First 4 2.6 94.82 36.47

Second 4 2.7 93.3 34.55
Third 4 2.87 95.8 33.38

third runs in comparison to that of first run. Therefore, the energy production and Conservation: proceedings,
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