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Pradip B. Dhamole a,c, Zhilong Wang a,d, Yuanqin Liu a, Bin Wang a, Hao Feng a,b,*
a
Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
b
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
c
Department of Biotechnology, Sinhgad College of Engineering, Pune 411041, India
d
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, PR China
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.
* 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.
doi:10.1016/j.biombioe.2012.02.007
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
1
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
Butanol (g L )
L62
-1
L62 0.32 32 4
L64 0.06 58
L61 Insoluble in water 24
3
L62
-1
Acetone/Butanol Produced (g L )
10 Butanol (6% L62)
-1
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
4
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
0
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
12
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 )
-1
0
0 2 4 6 8 10
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
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
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
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