enhancement of n-butanol production by in situ butanol removal using permeating–heating–gas...
TRANSCRIPT
Accepted Manuscript
Enhancement of n-butanol production by in situ butanol removal using perme-ating-heating-gas stripping in acetone-butanol-ethanol fermentation
Yong Chen, Hengfei Ren, Dong Liu, Ting Zhao, Xinchi Shi, Hao Cheng, NanZhao, Zhenjian Li, Bingbing Li, Huanqing Niu, Wei Zhuang, Jingjing Xie,Xiaochun Chen, Jinglan Wu, Hanjie Ying
PII: S0960-8524(14)00654-3DOI: http://dx.doi.org/10.1016/j.biortech.2014.04.107Reference: BITE 13403
To appear in: Bioresource Technology
Received Date: 1 March 2014Revised Date: 28 April 2014Accepted Date: 29 April 2014
Please cite this article as: Chen, Y., Ren, H., Liu, D., Zhao, T., Shi, X., Cheng, H., Zhao, N., Li, Z., Li, B., Niu, H.,Zhuang, W., Xie, J., Chen, X., Wu, J., Ying, H., Enhancement of n-butanol production by in situ butanol removalusing permeating-heating-gas stripping in acetone-butanol-ethanol fermentation, Bioresource Technology (2014),doi: http://dx.doi.org/10.1016/j.biortech.2014.04.107
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
1
Enhancement of n-butanol production by in situ butanol
removal using permeating-heating-gas stripping in
acetone-butanol-ethanol fermentation
Yong Chen1, Hengfei Ren
1, Dong Liu, Ting Zhao, Xinchi Shi, Hao Cheng, Nan
Zhao,Zhenjian Li, Bingbing Li, Huanqing Niu, Wei Zhuang, Jingjing Xie, Xiaochun
Chen, Jinglan Wu, Hanjie Ying*
State Key Laboratory of Materials-Oriented Chemical Engineering, College of
Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology,
Nanjing, China
1These authors are equally contributed to this work.
* Corresponding author. Tel./fax: +86 25 86990001.E-mail address: [email protected] (H.
Ying).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
2
ABSTRACT:
Butanol recovery from acetone-butanol-ethanol (ABE) fed-batch fermentation using
permeating-heating-gas stripping was determined in this study. Fermentation was
performed with Clostridium acetobutylicum B3 in a fibrous bed bioreactor and
permeating-heating-gas stripping was used to eliminate substrate and product
inhibition, which normally restrict ABE production and sugar utilization to below 20
g/L and 60 g/L, respectively.In batch fermentation (without permeating-heating-gas
stripping), C. acetobutylicum B3 utilized 60 g/L glucose and produced 19.9 g/L ABE
and 12 g/L butanol, while in the integrated process 290 g/L glucose was utilized and
106.27 g/L ABE and 66.09 g/L butanol were produced. The intermittent gas stripping
process generated a highly concentrated condensate containing approximately 15%
(w/v) butanol, 4% (w/v) acetone, a small amount of ethanol (<1%), and almost no
acids, resulting in a highly concentrated butanol solution [~70% (w/v)] after phase
separation. Butanol removal by permeating-heating-gas stripping has potential for
commercial ABE production.
Keywords:
ABE fermentation, Permeating-heating-gas stripping, Fed-batch fermentation, Cell immobilization
1. Introduction
The bioproduction of acetone, butanol, and ethanol (ABE) by solventogenic clostridia
such as Clostridium acetobutylicum was previously the second largest
biotechnological industry (Jones and Woods, 1986), and has attracted renewed
interest in recent years. Butanol can be used as a fuel extender and has excellent fuel
characteristic such as higher energy content, high miscibility with gasoline and diesel
fuel, and low vapor pressure (Qureshi and Blaschek, 1999a). In addition,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
3
fermentation-derived butanol is preferred in the food industry (for food flavor
extraction) as petroleum-derived butanol has the potential for carcinogen carryover
(Formanek et al., 1997). For these important reasons, bio-based butanol is favored.
Additionally, production of butanol by fermentation could help relieve the
dependence on foreign oil in China.
Solvent toxicity, due mainly to n-butanol, is a major factor that negatively affects
the economic feasibility of acetone-butanol-ethanol (ABE) fermentation. Butanol
toxicity leads to microbial growth inhibition, which results in low product titer, yield,
and productivity (Mariano and Filho, 2012; Setlhaku et al., 2013). Over the past three
decades, extensive research and development efforts have focused on solutions to
overcome the issue of butanol toxicity. One approach is to improve butanol tolerance
of strains by using genetic techniques, including mutagenesis and metabolic
engineering (Papoutsakis, 2008; Zheng et al., 2009; López-Contreras et al., 2010), and
this has resulted in the development of mutants that can produce up to 20 g/L of
butanol (Chen and Blaschek, 1999; Ezeji et al., 2010). However, compared to ~10%
(w/v) ethanol obtained in yeast fermentation, a butanol yield of 2% (w/v) is too low
and uneconomical due to high energy consumption in the recovery by distillation.
Another approach is the coupling of butanol removal with fermentation in situ,
thus reducing inhibition and improving fermentation productivity (Ezeji et al., 2007b;
Groot et al., 1992; Qureshi et al., 1992; Roffler et al., 1988). Several online butanol
recovery methods have been reported, including adsorption (Nielsen and Prather,
2009; Qureshi et al., 2005), liquid–liquid extraction (Barton and Daugulis, 1992;
Roffler et al., 1987a), pervaporation (Matsumura et al., 1988; Qureshi and Blaschek,
1999), and gas stripping (Groot et al., 1989; Qureshi and Blaschek, 2001). Gas
stripping is the most studied of these methods because it is a relatively simple process,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
4
which does not affect the fermentation culture and which can be applied continuously
(Qureshi and Blaschek, 2001). However, gas stripping typically removes a large
amount of water with butanol and requires a higher energy input due to its lower
butanol selectivity compared to that of other separation techniques (Qureshi and
Blaschek, 2001; Qureshi et al., 2005; Vane, 2008). To improve the integrated
fermentation-gas stripping process for butanol production, optimization of gas
stripping conditions, fermentation broth temperature, and a better understanding of its
effects on ABE fermentation are necessary.
All previous studies on integrated ABE fermentation with gas stripping were
conducted at relatively low butanol concentrations of 5 g/L or less to minimize
butanol toxicity, which could not only inhibit the fermentation, but also induce
sporulation and cause culture degeneration (Kashket and Cao, 1993). However, gas
stripping would be more efficient with a higher butanol concentration (8 g/L or
higher), at which the condensed vapor from gas stripping would have a butanol
concentration higher than its solubility (~7.8 g/100 mL water) and would thus result
in a highly concentrated organic phase with ~80% (v/v) butanol, as seen in this
study.
In this study, the removal of acetone, butanol, and ethanol in water solutions by
permeating-heating-gas stripping was characterized with the goal of investigating
and demonstrating its feasibility in an integrated fermentation for higher butanol
production with C. acetobutylicum B3. The fermentation kinetics in a fibrous bed
bioreactor (FBB) (Huang et al., 2004) and the effect of permeating-heating-gas
stripping on butanol production in fed-batch fermentation with highly concentrated
substrate were also studied. Fed-batch fermentation with intermittent
permeating-heating-gas stripping was capable of producing a highly concentrated
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
5
ABE product and of significantly reducing energy and water usage in n-butanol
production, and these results are compared with those from previous studies in this
manuscript.
2. Materials and methods
2.1. Bacterial strains and culture medium
C. acetobutylicum B3 is an adaptive mutant strain derived from CGMCC 5234 (China
General Microbiological Culture Collection Center, Beijing, China), and was used in
all fermentations. This strain was previously isolated from soil, and the adaptive
mutant strain selected for after UV mutagenesis. C. acetobutylicum B3 was cultured
in solid reinforced clostridia medium (RCM) for routine growth and modified P2
medium (P2 medium containing 10 g/L glucose as the sole carbohydrate) for seed
culture, at 37°C in an anaerobic chamber (Bug Box, Ruskinn Technology, Leeds, UK)
(Liu et al., 2013). The growth medium used for main fermentation contained the
following components: carbon source (60 g/L glucose), phosphate buffer (0.5 g/L
KH2PO4 and K2HPO4), ammonium acetate (2.2 g/L), vitamins (1 mg/L
para-amino-benzoic acid, 1 mg/L thiamine, and 0.01 mg/L biotin), and mineral salts
(0.01 g/L MnSO4·H2O, 0.01 g/L NaCl, 0.2 g/L MgSO4·7H2O, and 0.01 g/L
FeSO4·7H2O). Antifoam TBP was added to prevent excessive foaming.Glucose,
phosphate buffer, and ammonium acetate were autoclaved together at 121°C for 15
min, and vitamins and mineral salts were sterile-filtered. The final medium had a pH
of 4.5–5.0.
2.2. Fermentation system with permeating-heating-gas stripping
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
6
The integrated fermentation system with permeating-heating-gas stripping consisted
of F for immobilized cell fermentation, G for isolating bacteria and fermentation
broth,G (4-L working volume; New Brunswick Scientific Co., New Brunswick, NJ)
with controlled temperature and pH, E and J (300 mm × 600 mm2; E for cooling the
fermentation broth and J for vapor condensation), and D、H and I for recirculation of
the fermentation broth and gas (Fig. 1). The fiber bed bioreactor (FBB) was
constructed using a stainless steel column (400 mm × 635 mm2, 1.5-L working
volume) packed with 90 g of cotton towel and stainless steel trestle (Silva and Yang,
1995). The spinner flask, two custom-built coil condensers, and FBB were autoclaved
separately for 30 min and aseptically connected after sterilization. The system was
flushed with nitrogen to ensure an oxygen-free environment. For batch fermentation
without gas stripping, the system was operated without connecting to the condensers
and the hollow fiber module.
2. Experimental set-up for gas stripping tests
Solutions of acetone, butanol, and ethanol, alone or in combinations, were prepared in
demineralized water at the indicated concentrations. The solutions (3 L) were placed
in a 5-L spinner flask with impellers (Rushton type, stirrer speed; 100 rpm) at the
bottom and below the solution surface. Gas was sparged through the solutions through
a sparger attached to a metal mold (plurality; 0.5–1 µm holes) at the bottom of the
spinner flask below the lower impellor. The gas outflow first passed through a
condenser on top of the spinner flask (temperature set at 70 °C) before being led
through J (95%ethanol was used as coolant,10 L/min) with the temperature set at
-5 °C to -10 °C. The gas was then led through a water flask containing 5 L
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
7
demineralized water placed in an ice water bath before being released to the
atmosphere.
ABE model solutions were preheated to 70 °C while stirring (100 rpm). Gas
stripping [1 L N2/(1 L liquid vol·min)] was initiated when solvent solutions reached
the final temperature. At the same time, the gas outlet was connected to J and wash
water flasks. Samples (3 mL) were periodically taken out from the bioreactor and
stored at -20 °C until used. At the end of the experiment, the volumes of the solution
in the spinner flask, the condensate (estimated), and the wash water were determined.
Water loss by vaporization from the spinner flask was between 0.6 and 1.0 mL/h. The
condensate was quantitatively collected by washing the flask with water several times.
Samples of the diluted condensate and the wash water were stored at -20 °C until
used.
2.4. Cell immobilization and reactor start-up
The FBB and the spinner flask containing 3.6 L of P2 medium were flushed with
nitrogen for 30 min until oxygen-free. The spinner flask was then inoculated with 400
mL of actively growing cells (12 h) and then maintained at 37 °C and pH 4.5 (with
the addition of 2N NaOH and 1N HCl), and agitated at 100 rpm for 18–24 h until cell
growth reached an optical density (OD600) of over 8.0. Cells in the fermentation broth
were then recirculated through the FBB for 15–18 h for immobilization onto the
fibrous matrix until cell density in the broth ceased to decrease. The broth was
replaced with fresh P2 glucose medium to allow growth of the cells in the FBB. The
medium was changed again and the process repeated several times until a stable and
high cell density in the FBB was reached. Throughout the process, temperature was
maintained at 37 °C and pH controlled at 4.5.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
8
2.5. Batch and fed-batch ABE fermentations
Batch fermentation kinetics was studied in the FBB system without gas stripping at
37 °C and pH 4.5 with a glucose concentration of 60 g/L.Before starting a new
batch,the medium in the reactor system was drained, and 4 L of fresh medium
pumped into the spinner flask and recirculated through the FBB. Broth samples were
taken from the spinner flask periodically until all glucose was consumed. For
fed-batch fermentation, additional glucose was used to initiate the fermentation. After
26 h, the butanol production reached approximately 10 g/L and the bacteria and
fermentation broth were separated. The temperature was increased to 70 °C and the
fermentation broth was gas stripped with fermentation gas (H2 and CO2) through I
(gas circulation pump)at 1.5 L/min in a closed circuit to prevent any loss. The gas
stream containing volatile compounds (mainly acetone, butanol, and ethanol) was
then cooled in the J (95%ethanol was used as coolant,10 L/min) at -5 °C to -10 °C.
The condensate was collected at the bottom of the condenser in a round-bottom flask.
Broth samples were taken from the spinner flask for analysis of sugar and product
concentrations throughout the fermentation. Highly concentrated glucose was added
to the spinner flask when the sugar content in the fermentation broth was low; it also
replaced the water lost due to gas stripping. For extended fed-batch fermentation,
additional yeast extract (0.5 g/L, equivalent to 1.5 g in a 3.0-L fermentation system)
was also added with the glucose to avoid exhaustion of nutrients. The condensate
collected in the solvent collector was measured and analyzed for ABE content and
volume after three batch fermentation cycles. The fed-batch fermentation with gas
stripping was maintained for 175 h (15 cycles) to evaluate long-term performance and
stability.
2.6. Analytical methods
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
9
Cell density was measured using a BioMate™ 3 spectrophotometer (Thermo
Scientific, Waltham, MA, USA) at 600 nm. Glucose, acetic acid, and butyric acid
concentrations were measured by HPLC (Agilent 1100 series; Hewlett–Packard, Palo
Alto, CA, USA) with a refractive index detector, using an Aminex HPX-87H ion
exclusion column (300 × 7.8 mm; Bio-Rad Laboratories, Hercules, CA, USA) with
5.0 mM H2SO4 used as the mobile phase (0.6 mL/min) at 50 °C. Acetone, ethanol,
butanol, and acetoin were analyzed by gas chromatography (GC) using an Agilent
HP-INNOWAX column (60 m × 250 µm × 0.5 µm) with a flame ionization detector.
The injector and detector temperatures were set at 180°C and 220°C, respectively.
Samples were first filtered through a 10-µL syringe filter with an injection volume of
1 µL. The column temperature was initially held at 70 °C for 0.5 min and then raised
by 20 °C/min until reaching 190 °C, where temperature was held for 4 min.
3. Results and discussion
3.1. Gas stripping model of ABE
Removal of acetone, butanol, and ethanol from model solutions was studied at 37 °C
(cultivation temperature of C. beijerinckii B3). The concentrations and ratios of ABE
in the model solutions detected were based on those typically obtained after batch
fermentations by C. beijerinckii B3. The solvent removed by stripping gas from the
model solutions or fermentation broth was condensed in J (-5 °C to -10 °C). ABE
mixtures resulted in a two-phase system in solvent collector; a saturated
water/butanol/acetone/ethanol lower phase with an organic (butanol/acetone/ethanol)
upper phase. Gas stripping of butanol and other organic solvents from an aqueous
solution can be modeled as a first-order process according to the following equation
(Truong and Blackburn, 1984; Ezeji et al., 2005):
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
10
Rs = -dCs/dt = Ksa·Cs
[Rs: removal rate, g/(L·h); Cs: concentration of solvent, g/L; Ksa: removal rate
constant, h-1
]
Ksa can be determined from the exponential best-fit curve following the equation:
Cs = Cs0-K
sa.t
(Cs0: concentration of solvent at 0 h, g/L; t: time, h)
Based on the above equation, the gas-stripping rate will increase proportionally with
changes in concentration and Ksa. The Ksa values for acetone, butanol and ethanol
were determined using model solutions of butanol and ABE (Table 1). At 70 °C, Ksa
for butanol was approximately 2-fold higher than the Ksa at 37 °C, thus the butanol
removal rate from a solution at 70 °C was much faster (Fig.2A). At concentrations
higher than 10 g/L, the removal of butanol at 70 °C was constant and amounted to 1.6
g/(L·h) under the applied conditions (Fig. 2A). The removal rate of butanol at 37 °C
from model solutions with a mixture of ABE (6, 12, and 2 g/L acetone, butanol, and
ethanol, respectively) was not affected by the presence of other solvents (Fig. 2A), has
similar Ksa value for a model solution of butanol only(Table 1). However, at 70 °C
the Ksa for butanol in a model ABE mixture solution was nearly half that of a model
solution containing only butanol (Table 1). Accordingly, the removal rate of butanol
at 70 °C was increased (Fig. 2A), indicating that the higher temperatures facilitate
butanol removal and that other polar metabolites such as acetone and ethanol should
be minimized.
The increase was much less than seen for butanol (approximately 2.5-fold) and
therefore a large difference in the removal rate of these two solvents is present at this
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
11
temperature (Fig. 2B). According to Raoult’s law, the vapor pressure of a volatile
compound in a dilute solution increases linearly with its molar concentration in the
solution. Therefore, the removal rates for ABE by gas stripping would increase with
increasing concentration, as shown in Fig. 2. Butanol has both the highest stripping
rate and the highest concentration in the condensate collected, suggesting that the gas
stripping process is more selective in separating butanol from water, though butanol
has a higher boiling temperature and lower vapor pressure than acetone, ethanol, and
water at 37 °C. At 37 °C, Ksa for butanol was approximately 2-fold higher than for
ethanol and acetone (Table 1). The Ksa increased more than 1.5-fold at 70 °C,
indicating that the temperature of the model solution affects recovery.
The effect of medium components (including antifoam) and C. beijerinckii B3
cells on the removal of ABE was determined using fermentation broth from a C.
beijerinckii B3 batch culture. ABE was added at concentrations of 6, 12, and 2 g/L,
respectively. It should be noted that the presence of cells would have a negative effect
on gas stripping, resulting in significantly lower stripping rates (Fig. 3). The gas
stripping data indicated that gas stripping would be a highly efficient method for
butanol isolation and purification if the concentration in the fermentation broth was
higher than 8 g/L (Fig. 2 and 3).
Therefore, for the removal of butanol from fermentation broth by gas stripping,
the butanol concentration should be maintained above 8 g/L and the free cell
concentration should be as low as possible. On the other hand, butanol is highly toxic
to clostridia (Maddox, 1988; Soni et al., 1987; Xue et al., 2012), and its concentration
should be kept as low as possible during ABE fermentation. To compromise between
these two opposing requirements, ABE fermentation with online gas stripping in this
study was designed to operate at 8 g/L butanol and 70 °C.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
12
3.2 Batch fermentation
Batch fermentation with P2 medium initially containing 60 g/L glucose produced 12
g/L butanol, 6.1 g/L acetone, and 1.8 g/L ethanol when the fermentation terminated at
48 h with approximately 8 g/L glucose remaining in the medium (Fig. 4). Halting of
the fermentation and lack of full glucose utilization was due to butanol toxicity, which
strongly inhibited the cells at a concentration of 10 g/L butanol, as indicated by the
declining cell density and reduced butanol production starting at 35 h. Acetic and
butyric acids were produced during the first 10 h and then were re-assimilated by cells
for solvent production, exhibiting typical two-phase ABE fermentation with
acidogenesis followed by solventogenesis (Jones and Woods, 1986).The yields of
batch ABE fermentation were as follows: butanol, 0.2 g/g; acetone, 0.1 g/g; ethanol,
0.03 g/g; and total ABE, 0.33 g/g. Solvent and butanol productivities were 0.42 and
0.26 g/(L·h), respectively.
C. acetobutylicum B3 and CGMCC 5234 typically can only produce up to 8–10
g/L butanol due to sporulation onset and degeneration caused by the accumulated
butanol (Ezeji et al., 2003; Maddox, 1988; Xue, 2012). C. acetobutylicum B3 in the
FBB system was able to produce up to 12–14 g/L butanol and was able to tolerate and
produce butanol at a much higher concentration (14 g/L). The hyper-butanol
production and tolerance of C. acetobutylicum B3 would permit usage in long-term
fed-batch fermentation with permeating-heating-gas stripping to control the butanol
concentration at a moderate level of 7–9 g/L, as demonstrated in the study discussed
below.
3.3. Fed-batch ABE fermentation coupled with permeating-heating-gas stripping
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
13
In this study, fed-batch ABE fermentation was conducted, with the pH controlled at
approximately 4.5. The kinetics of fed-batch fermentation with intermittent gas
stripping for butanol recovery from the fermentation broth for a total of fifteen batch
cycles in 175 h are shown in Fig. 5. Over this period, a total of 290 g/L glucose was
consumed, and 66.09 g/L of butanol, 30.55 g/L of acetone, and 10.03 g/L of ethanol
(total ABE: 106.27 g/L) were produced (Fig. 5A). In the fed-batch fermentation,
permeating-heating-gas stripping was started at 38 h to intermittently remove solvents
from the fermentation when the butanol concentration was higher than 8 g/L, and
concentrated glucose was pulse-fed into the reactor when the glucose level in the
fermentation broth was lower than 14 g/L. The condensate containing the product
solvents was collected and analyzed for each batch cycle, and the total ABE
production was estimated based on the ABE in the fermentation broth and the
condensate. The fed-batch fermentation with intermittent gas stripping maintained
relatively stable ABE production, even with the butanol concentration in the
fermentation broth fluctuating between 7 and 10 g/L (Fig. 5B), indicating a dynamic
equilibrium between solvent production and removal. Comparing the cumulative ABE
production (Fig. 4A) with the ABE concentration in the reactor (Fig. 5B), it is evident
that gas stripping was effective in removing butanol from the fermentation broth,
maintaining levels at 8 g/L or below throughout the fermentation, and thus alleviating
butanol toxicity and allowing the fermentation to continue for an extended period.
A highly concentrated butanol solution of 14–17% (w/v) was obtained in the
condensate collected at the end of every three feeding cycles. However, acetone was
less than 4% (w/v) and only a small amount of ethanol (<1%) and very little acids
were obtained in the condensate.
These results indicate that permeating-heating-gas stripping was more selective in
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
14
removing butanol than acetone and ethanol, and did not remove much butyric and
acetic acids, which are not volatile at the fermentation pH of 4.5.
The overall product yields from glucose were (g/g): butanol, 0.23; acetone, 0.10;
ethanol, 0.03; and total ABE, 0.36. Compared to the first batch, the second feeding
cycle had higher solvent production and glucose consumption rates, likely due to
increased viable cell density in the FBB. However, both solvent productivity and
glucose utilization rate decreased after the seventh feeding cycle and dropped
significantly from the eighth cycle to the fifteenth cycle (Fig. 5A), indicating
decreased cell viability and productivity. Due to the extended fermentation time, dead
cells and non-active cells were present with active solvent-producing cells in the
fermenter (Qureshi et al., 1988; Mollah and Stuckey, 1993). A previous report
demonstrated ABE fermentation failure due to exhaustion of nutrients, and the
addition of nutrients led to an increase in glucose consumption and cell concentration
(Ezeji et al., 2003, 2004b, 2005). Nutrients supplementation with yeast extract in
subsequent feeding cycles allowed for maintenance of stable fermentation for an
extended period, though it did not restore the higher reactor productivity.
The fermentation performance during every three feeding cycles in the fed-batch
fermentation is shown in Fig. 6. Consistent results were obtained in all 15 batch
cycles, indicating that the fermentation process was stable and sustainable for
long-term operation without significant culture degeneration, as observed in
conventional ABE fermentation (Gapes et al., 1996; Xue et al., 2012). As expected,
the condensate contained a high butanol concentration (150.6 g/L) that separated into
two phases, with the upper organic phase containing (703.4 g/L) butanol,58 g/L
acetone,14 g/L ethanol, and no acids. The lower aqueous phase contained 78.8 g/L
butanol, 37 g/L acetone, 9.6 g/L ethanol, and 0.4 g/L acids.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
15
Compared to batch fermentations without permeating-heating-gas stripping, more
butanol was produced while acetone and ethanol production was negligibly affected
(Table 2). The overall butanol and ABE productivity were significantly higher than
those in batch fermentation without integrated permeating-heating-gas stripping.
Clearly, removing butanol by permeating-heating-gas stripping not only relieved the
butanol toxicity and thus increased the fermentation rate, but also increased the
butanol yield. The effects of permeating-heating-gas stripping on ABE fermentation
will be discussed further in the next section.
3.4. Effects of permeating-heating-gas stripping on ABE fermentation
Gas stripping removes only volatile compounds, which are mainly ABE and a very
small amount of acids. The continuous removal of butanol from the fermentation
broth allows the fermentation to consume more substrate at a higher rate with higher
ABE productivity. In this study, butanol and ABE productivities increased 35% and
40.1%, respectively, compared to fermentation without permeating-heating-gas
stripping. Permeating-heating-gas stripping preferentially removed butanol over
acetone from the fermentation broth, not only alleviating butanol toxicity but also
altering the fermentation kinetics (Fig. 2B). Typical ABE fermentations produce
butanol and acetone at a 2:1 ratio, as seen in the batch fermentation (Table 2). In
contrast, the overall butanol/acetone ratio was significantly higher than 2(2.2–2.4) in
this study, though butanol yield increased approximately 10% (from 0.20 to 0.23 g/g),
which could be attributable to enhance acid re-assimilation. As both acetic and butyric
acids produced in acidogenesis remained in the fermentation broth, they would be
re-assimilated via solventogenesis versus accumulated. This was also evidenced by
the stable concentration levels of both acetic and butyric acids in the fermentation
broth throughout the fed-batch fermentation.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
16
In permeating-heating-gas stripping, the solvent removal rate and selectivity (over
water) generally increase with increasing solvent concentration. It is clear that
permeating-heating-gas stripping is more effective in separating and concentrating
butanol than acetone from the aqueous solution, especially at higher concentrations.
n-Butanol has a low solubility (7.7% w/w, at 20°C) in water and undergoes phase
separation when the concentration is higher than 8%. When the butanol concentration
in the solution was approximately 8 g/L, butanol in the condensate increased to 150
g/L. After phase separation, the upper organic phase contained approximately 700 g/L
or 91% (v/v) of butanol, and the aqueous phase contained approximately 8% (w/v) of
butanol. This phase separation would simplify the butanol purification process
significantly and reduce the energy input necessary for butanol removal from ABE
fermentation broth (Vane, 2008; Ezeji et al., 2005). However, maintaining the butanol
level above 8 g/L in the fermentation is required for gas stripping to attain the high
butanol concentration in the condensate necessary for organic/aqueous phase
separation.
4. Conclusions
This study demonstrates the feasibility of producing n-butanol in ABE fermentation
with intermittent permeating-heating-gas stripping. Permeating-heating-gas
stripping is an efficient method to relieve butanol toxicity and increase reactor
productivity and substrate utilization in ABE fermentation for the recovery of butanol.
With periodic nutrient supplementation, the integrated fermentation process
maintained a stable productivity and high butanol yield for an extended period,
making the process attractive for the industrial production of biobutanol.
Acknowledgements
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
17
This work was supported by grants from the National Outstanding Youth Foundation
of China (Grant No.: 21025625), the National High-Tech Research and Development
Program of China (863) (Grant No.: 2012AA021200), the National Basic Research
Program of China (973) (Grant No.: 2011CBA00806), the National Key Technology
R&D Program (2012BAI44G01),the National Natural Science Foundation of China
(Grant No.:201390204), the Program for Changjiang Scholars and Innovative
Research Team in University (Grant No.: IRT1066), Jiangsu Provincial Natural
Science Foundation of China (Grant No.: SBK 201150207), and the Priority
Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
References
1. Barton WE, Daugulis AJ. 1992. Evaluation of solvents for extractive butanol fermentation with Clostridium
acetobutylicum and the use of poly(propylene glycol) 1200. Appl Microbiol Biotechnol 36,632–663.
2. Chen CK, Blaschek HP. 1999. Acetate enhances solvent production and prevents degeneration in Clostridium
beijerinckii BA101. Appl Microbiol Biotechnol 52,170–173.
3. Ezeji, T. C., Karcher, P. M., Qureshi, N., & Blaschek, H. P. 2005. Improving performance of a gas
stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation. Bioprocess and
biosystems engineering, 27(3), 207–214.
4. Ezeji TC, Qureshi N, Blaschek HP. 2003. Production of acetone, butanol and ethanol by Clostridium
beijerinckii BA101 and in situ recovery by gas stripping. World J Microbiol Biotechnol 19,595–603.
5. Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2004b. Acetone–butanol–ethanol (ABE) production from concentrated
substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping. Appl.
Microbiol.Biotechnol. 63, 653–658.
6. Ezeji TC, Qureshi N, Blaschek HP. 2007b. Production of acetone butanol(AB) from liquefied corn starch, a
commercial substrate, using Clostridium beijerinckii coupled with product recovery by gas stripping.J Ind
Microbiol Biotechnol 34,771–777.
7. Formanek, J., Mackie, R. & Blaschek, H.P. 1997. Enhanced butanol production by Clostridium beijerinckii
BA101 grown in semidefined P2 medium containing 6 percent maltodextrin or glucose.Applied and
Environmental Microbiology 63, 2306–2310.
8. Gapes JR, Nimcevic D, Friedl A. 1996. Long-term continuous cultivation of Clostridium beijerinckii in a
two-stage chemostat with on-line solvent removal. Appl Environ Microbiol 62:3210–3219.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
18
9. Groot, W.J., van der Lans, R.G.J.M. & Luyben, K.Ch.A.M. 1989. Batch and continuous butanol fermentation
with free cells:integration with product recovery by gas stripping. Applied Microbiology and Biotechnology
32,305–308.
10. Groot WJ, Lans RGJM, Luyben KChAM. 1992. Technologies for butanol recovery integrated with
fermentations. Process Biochem 27,61–75.
11. Huang WC, Ramey DE, Yang ST. 2004. Continuous production of butanol by Clostridium acetobutylicum
immobilized in a fibrous bed bioreactor.Appl Biochem Biotechnol 113–116,887–898.
12. Liu, D., Chen, Y., Li, A., Ding, F., Zhou, T., He, Y., ... & Ying, H. 2013. Enhanced butanol production by
modulation of electron flow in Clostridium acetobutylicum B3 immobilized by surface adsorption. Bioresource
technology, 129, 321–328.
13. López-Contreras, A.M., Kuit, W., Siemerink, M.A.J., Kengen, S.W.M., Springer, J.,Claassen, P.A.M., 2010.
Production of longer-chain alcohols from lignocellulosic biomass: butanol, isopropanol and 2,3-butanediol. In:
Waldron, K. (Ed.),Bioalcohol Production. Woodhead Publishing, Cambridge (UK), pp. 415–460.
14. Maddox IS. 1988. The acetone–butanol–ethanol fermentation: Recentprogress in technology. Biotechnol Genet
Eng Rev 7:189–220.
15. Mariano, A.P., Filho, R.M., 2012. Improvements in biobutanol fermentation and their impacts on distillation
energy consumption and wastewater generation. Bioenerg. Res. 5, 504–514.
16. Matsumura M, Kataoka H, Sueki M, Araki K. 1988. Energy saving effect of pervaporation using oleyl alcohol
liquid membrane in butanol purification.Bioprocess Eng 3,93–100.
17. Mollah, A.H. & Stuckey, D.C. 1993. Feasibility of in-situ gas stripping for continuous acetone-butanol
fermentation by Clostridium acetobutylicum.Enzyme and Microbial Technology 15, 2002–07.
18. Nielsen DR, Prather KJ. 2009. In situ product recovery of n-butanol using polymeric resins. Biotechnol Bioeng
102,811–821.
19. Papoutsakis ET. 2008. Engineering solventogenic clostridia. Curr Opin Biotechnol 19,420–429.
20. Qureshi N, Blaschek HP. 1999. Butanol recovery from model solutions/fermentation broth by pervaporation:
Evaluation of membrane performance.Biomass Bioenerg 17,175–184.
21. Qureshi, N. Blaschek, H.P. 1999a Production of acetone butanol ethanol (ABE) by a hyper-producing mutant
strain of Clostridium beijerinckii BA101 and recovery by pervaporation. Biotechnology Progress 15, 594–602.
22. Qureshi, N., Blaschek, H.P., 2001. Recovery of butanol from fermentation broth by gas stripping. Renew.
Energy 22 (4), 557–564.
23. Qureshi N, Hughes S, Maddox IS, Cotta MA. 2005. Energy-efficient recovery of butanol from model solutions
and fermentation broth by adsorption. Bioprocess Biosyst Eng 27,215–222.
24. Qureshi N, Maddox IS, Friedl A. 1992. Application of continuous substrate feeding to the ABE fermentation:
Relief of product inhibition using extraction, perstraction, stripping, and pervaporation. Biotechnol
Prog8,382–390.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
19
25. Qureshi, N., Paterson, A.H.J., Maddox, I.S., 1988. Model for continuous production of solvents from whey
permeate in a packed bed reactor using cells of Clostridium acetobutylicum immobilized by adsorption onto
bonechar. Appl. Microbiol.Biotechnol. 29, 323–328.
26. Roffler SR, Blanch HW, Wilke CR. 1987a. In-situ recovery of butanol during fermentation. Part 1: Batch
extractive fermentation. Bioproc Eng 2,1–12.
27. Roffler SR, Blanch HW, Wilke CR. 1988. In situ extractive fermentation of acetone and butanol. Biotechnol
Bioeng 31,135–143.
28. Setlhaku, M., Heitmann, S., Górak, A., & Wichmann, R. 2013. Investigation of gas stripping and pervaporation
for improved feasibility of two-stage butanol production process. Bioresource technology, 136, 102–108.
29. Silva, E.M., Yang, S.T., 1995. Kinetics and stability of a fibrous-bed bioreactor for continuous production of
lactic from unsupplemented acid whey. J. Biotechnol.41, 59–70.
30. Truong, K.N., Blackburn, J.W., 1984. The stripping of organic chemicals in biological treatment processes.
Environ. Prog. 3 (3), 143–152.
31. Vane LM. 2008. Separation technologies for the recovery and dehydration of alcohols from fermentation
broths. Biofuels Bioprod Biorefin 2,553–588.
32. Xue, C., Zhao, J., Lu, C., Yang, S. T., Bai, F., & Tang, I. 2012. High-titer n-butanol production by Clostridium
acetobutylicum JB200 in fed‐batch fermentation with intermittent gas stripping. Biotechnology and
bioengineering, 109(11), 27462–756.
33. Zheng YN, Li LZ, Xian M, Ma YJ, Yang JM, Xu X, He DZ. 2009. Problems with the microbial production of
butanol. J Ind Microbiol Biotechnol 36,1127–1138.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
20
Tables
Table 1. Removal rate constant (Ksa) for gas stripping of solvents from model solutions.
Solvent Cs0 (g/L) Ksa(h-1)
37°C 70°C
Acetone (ABE) 6 0.025 ± 0.02 0.088 ± 0.003
Butanol (ABE) 12 0.055 ± 0.003 0.122 ± 0.002
Ethanol (ABE) 2 0.024 ± 0.02 0.079 ± 0.003
Butanol 6 0.056 ± 0.003
Butanol 10 0.161 ± 0.015
Model solutions included butanol (6 and 10 g/L) or a mixture of acetone, butanol, and ethanol (6,
12, and 2 g/L, respectively). The flow rate of the stripping gas was set at 1 vvm, the bioreactor
temperature kept at 37 °C or 70 °C, and the stirring speed was 100 rpm.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
21
Table 2. Kinetics of batch fermentation and fed-batch fermentation with gas
stripping.
Batch fermentation Fed-batch fermentation with
permeating-heating-gas stripping
Substrate Glucose (60 g/L) Glucose (600 g/L)
Fermentation time (h) 47 175
Glucose consumed (g/L) 60 290
Glucose consumption rate
[g/(L·h)]
1.28 1.66
Acetone production (g/L) 6.1 30.15
Butanol production (g/L) 12 66.09
Ethanol production (g/L) 1.8 10.03
Total ABE production (g/L) 19.9 106.27
Butanol/acetone ratio (g/g) 1.9 2.2
Acetone yield (g/g) 0.10 0.10
Butanol yield (g/g) 0.2 0.23
Ethanol yield (g/g) 0.03 0.03
ABE yield1 (g/g) 0.33 0.36
Acetone productivity[g/(L·h)] 0.13 0.17
Butanol productivity [g/(L·h)] 0.26 0.38
Ethanol productivity [g/(L·h)] 0.04 0.06
ABE productivity2 [g/(L·h)] 0.42 0.61
Product yields and productivities are based on three replicates in each batch fermentation, and on
the fifteen batch data shown in Fig. 4 for the fed-batch fermentation with permeating-heating-gas
stripping.
ABE yield1(g/g) was calculated as the ratio of ABE produced (g) to glucose consumed (g).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
22
ABE productivity2 [g/(L·h)] was calculated as the ratio of ABE concentration (g/L) to the
fermentation time (h).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
23
Figure captions
Figure 1. Integrated fermentation system with permeating-heating-gas stripping for in
situ butanol recovery
Figure 2. Removal rates of butanol from model solutions of butanol alone and from a
20 g/L ABE mixture (A) and removal rates of acetone, butanol, and ethanol from the
ABE mixture (B) at 37 °C and 70°C by gas stripping.The ratio of butanol, acetone,
and ethanol in the mixture was 6:3:1, by weight. Symbols: (A and B) Open symbols,
37 °C, solid symbols, 70 °C. (A) Stars, butanol only; diamonds, butanol in ABE
mixture. (B) Squares, butanol; diamonds, acetone; triangles, ethanol all in ABE
mixture.
Figure 3. Effects of ABE concentration and cells in the broth on gas stripping.
Figure 4. Kinetics of batch ABE fermentation by C. acetobutylicum B3 at 37 °C, pH
4.5.
Figure 5. Kinetics of fed-batch ABE fermentation by C. beijerinckii B3 with
intermittent permeating-heating-gas stripping for butanol recovery. (A): Glucose
concentration profiles and cumulative ABE production. (B) Concentrations of
products in the fermentation broth.
Figure 6. Comparison of the performance of every three feeding cycles in fed-batch
fermentations with permeating-heating-gas stripping. (A) ABE titers in condensate;
(B) ABE yield; (C) ABE productivity.
G
5 h
F
L
E
D
B
A
C
H
I
J
N
K M
A: Substrate storage tank B: Feed pump C: Spinner flask D: Liquid circulation pump E: Custom-built condenser
pipe F: Fiber bed bioreactor(FBB) G: Hollow fiber module H: Liquid circulation pump I: Gas circulation pump
J: Custom-built condenser pipe K: Cold water bath L: Pump M: Solvent collector N: pH controller
Figure 1
0 2 4 6 8 1 0 1 2 1 40 . 0
0 . 4
0 . 8
1 . 2
1 . 6BA
R em o
v a l r a
t e [ g /
(L⋅h)
]
B u t a n o l ( g / L )0 2 4 6 8 1 0 1 2 1 4
0 . 0
0 . 4
0 . 8
1 . 2
1 . 6
S o l v e n t s ( g / L )
Figure 2
0 2 4 6 8 1 0 1 2 1 40 . 0
0 . 1
0 . 2
0 . 3
0 . 4
0 . 5
0 . 6
0 . 7
0 . 8 B u t a n o l B u t a n o l w i t h c e l l sA c e t o n eA c e t o n e w i t h c e l l sE t h a n o l E t h a n o l w i t h c e l l s
R em o
v al r a
t e [ g /
(L×h)]
C o n c e n t r a t i o n i n b r o t h ( g / L )
Figure 3
0 1 0 2 0 3 0 4 0 5 00
1 0
2 0
3 0
4 0
5 0
6 0
7 0
Prod
ucts (g
/L)
G l u c o s e A B EB u t a n o lA c e t o n eE t h a n o lA c e t i c a c i d B u t y r i c a c i d
T i m e ( h )
G lu c o
s e ( g /
L )
024681 01 21 41 61 82 02 2
Figure 4
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 002468
1 01 21 41 61 82 02 22 4
T i m e ( h )
A
B A B E B u t a n o l A c e t o n e E t h a n o l A c e t i c a c i d B u t y r i c a c i d
C on c
e nt a t
i o n ( g
/ L )
T i m e ( h )
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 00
1 02 03 04 05 06 07 0
G l u c o s e G i
u co s e
( g/ L )
A B E B u t a n o l A c e t o n e E t h a n o l
Cumu
lative
prod
uctio
n (g/L
)
01 02 03 04 05 06 07 08 09 01 0 01 1 01 2 0Figure 5
0 3 6 9 1 2 1 50 . 00 . 10 . 20 . 30 . 40 . 50 . 60 . 7
0 3 6 9 1 2 1 50 . 0 00 . 0 50 . 1 00 . 1 50 . 2 00 . 2 50 . 3 00 . 3 50 . 4 00 . 4 5
0 3 6 9 1 2 1 50
5 0
1 0 0
1 5 0
2 0 0
2 5 0
C
B
A
]
B a t c h #
A B E B u t a n o l A c e t o n e E t h a n o l
A B E B u t a n o l A c e t o n e E t h a n o l
A B E B u t a n o l A c e t o n e E t h a n o lFigure 6
Highlights
Gas stripping model of ABE from model solutions was characterized.
Integrated fermentation system with gas stripping for in situ butanol recovery.
Fed-batch fermentation with solvent removal at 70°C has been demonstrated.
Permeating-heating-gas stripping greatly enhances butanol productivity and yield.