Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Department of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA
h i g h l i g h t s
" High-titer n-butanol was produced by C. acetobutylicum in a brous bed bioreactor.
" In situ gas stripping for butanol separation alleviated butanol toxicity in the fermentation.
" Two-stage gas stripping produced butanol at a high titer of >400 g/L (500 g/L ABE).
" This process can produce n-butanol with greatly reduced energy consumption.
a r t i c l e
i n f o
Article history:
Available online 25 July 2012
Keywords:
ABE fermentation
Butanol
Clostridium acetobutylicum
Fibrous bed bioreactor
Two-stage gas stripping
a b s t r a c t
Two-stage gas stripping for butanol recovery from acetone-butanol-ethanol (ABE) fermentation with
Clostridium acetobutylicum JB200 in a brous bed bioreactor was studied. Compared to fermentation
without in situ gas stripping, more ABE (10.0 g/L acetone, 19.2 g/L butanol, 1.7 g/L ethanol vs. 7.9 g/L acetone, 16.2 g/L butanol, 1.4 g/L ethanol) were produced, with a higher butanol yield (0.25 g/g vs. 0.20 g/g)
and productivity (0.40 g/Lh vs. 0.30 g/Lh) due to reduced butanol inhibition. The rst-stage gas stripping
produced a condensate containing 175.6 g/L butanol (227.0 g/L ABE), which after phase separation
formed an organic phase containing 612.3 g/L butanol (660.7 g/L ABE) and an aqueous phase containing
101.3 g/L butanol (153.2 g/L ABE). After second-stage gas stripping, a highly concentrated product containing 420.3 g/L butanol (532.3 g/L ABE) was obtained. The process is thus effective in producing
high-titer butanol that can be puried with much less energy.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Butanol is an important chemical and advanced biofuel, which
can be produced through acetonebutanolethanol (ABE) fermentation by various Clostridium spp. (Qureshi et al., 2008). As a substitute for petroleum fuel, butanol is superior to ethanol due to its
higher energy density, less corrosiveness to existing infrastructure
and better compatibility with gasoline (Gheshlaghi et al., 2009;
Drre, 2007). However, one of the biggest challenges for biobutanol production is intensive energy consumption in product recovery from the dilute fermentation broth and low butanol yield and
productivity due to the co-production of acetone and ethanol as
well as severe butanol toxicity to cells (Ezeji et al., 2010; Nicolaou
et al., 2010; Qureshi and Blaschek, 2001b).
Metabolic engineering and mutagenesis of Clostridia have been
explored to provide solutions for enhancing butanol tolerance and
productivity (Huang et al., 2010; Papoutsakis, 2008; Zheng et al.,
Corresponding author. Tel.: +1 614 292 6611; fax: +1 614 292 3769.
E-mail address: yang.15@osu.edu (S.T. Yang).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.07.062
2009). However, compared to 1215 ethanol achieved in fermentation with yeasts (Xue et al., 2010), the 1.22.0 butanol produced by
solventogenic Clostridia in ABE fermentation is much lower and
not economically competitive for butanol recovery by conventional
distillation (Chen and Blaschek, 1999). Therefore, in situ butanol removal from the fermentation system by adsorption (Nielsen and
Prather, 2009; Qureshi et al., 2005), liquidliquid extraction (Rofer et al., 1987; Rofer et al., 1988), pervaporation (Matsumura
et al., 1988; Qureshi and Blaschek, 1999) and gas stripping (Qureshi and Blaschek, 2001a; Qureshi et al., 2008) has been used to mitigate butanol inhibition and improve productivity. However, a
common problem of these techniques is their low efciency in
recovering butanol with high concentration even though the productivity of the fermentation system could be improved
effectively.
Among butanol recovery methods, gas stripping has the advantages of simple operation, no harm to culture, and low energy input
and capital investment for facilities (Evans and Wang, 1988; Ezeji
et al., 2007a; Garcia et al., 1986; Qureshi and Blaschek, 2001a;
Zheng et al., 2009). However, the solvent concentration obtained
397
Strain
Condensate
Butanol g/L
ABE g/L
Batch
C. acetobutylicum P262
<5/8.1
27.2/5.2
1.98.8/11.9
26/7.4
1.33.2/11.8
3.514.6/16.2
1743
35.9
175.6b
420.3c
34.8
150.5
101152
75.9
20120
42.782.5
2766
45.9
227.0b
532.3c
53.7
195.9
151203
26.7
38.3996
C. beijerinckii BA101
C. beijerinckii P260
C. acetobutylicum JB200
Continuous
Fed-batch
C. acetobutylicum P262
C. acetobutylicum JB200
C. acetobutylicum P262
C. beijerinckii BA101
3.4/3.3
810/19.1
615/20.4
24.5/5.3
29.6/11.9
25.5/13.7
Reference
, Unavailable.
a
Vacuum was applied intermittently for 1.5 h at 4 h interval to remove ABE vapor.
b
Butanol and ABE in the condensate collected from the rst-stage gas stripping.
c
Butanol and ABE in the nal product collected from the two-stage gas stripping process.
2. Methods
2.1. Culture and media
Clostridium acetobutylicum JB200 derived from ATCC 55025 was
used in this study. The seed culture was prepared in the Clostridial
growth medium (CGM) containing 30 g/L glucose, 2 g/L yeast extract, 1 g/L Tryptone, minerals, and vitamins in a phosphate buffer
as described in Lu et al. (2012), and incubated at 37 C for 16 h
until active growth was observed. ABE fermentation was studied
using the P2 medium containing: glucose (80 g/L), yeast extract
(1 g/L), KH2PO4 (0.5 g/L), K2HPO4 (0.5 g/L), ammonium acetate
(2.2 g/L), vitamins (1 mg/L para-amino-benzoic acid, 1 mg/L thiamin and 0.01 mg/L biotin), and mineral salts (0.2 g/L MgSO47H2O,
0.01 g/L MnSO4H2O, 0.01 g/L FeSO47H2O, 0.01 g/L NaCl), prepared
according to the procedures described previously (Qureshi and Blaschek, 1999). The media were sterilized by autoclaving at 121 C
and 15 psig for 30 min. All solutions were purged with nitrogen
for 1 h through a sterile 0.2 lm lter, either before or after
autoclaving.
398
Fig. 1. Experimental setup for ABE fermentation with two-stage gas stripping. 1, stirred tank fermentor; 2, brous bed bioreactor; 3, pH probe; 4, temperature probe; 5,
thermostat water inlet; 6, thermostat water outlet; 7, condenser; 8, rst-stage condensate storage tank; 9, cold-water bath; 10, second-stage condensate storage tank; 11,
cooling water inlet; 12, cooling water outlet; 13, peristaltic pump.
100
35
Glucose
Acetone
Butanol
Ethanol
ABE
Acetic
Butyric
OD
30
80
25
Glucose, g/L
15
40
10
5
0
10
20
30
40
50
60
Time, h
35
B 100
Glucose
ABE
Acetone
Acetic
Butanol
Butyric
Ethanol
OD
30
80
25
Gas stripping
Glucose, g/L
20
20
60
60
20
15
40
10
20
the organic phase from the rst-stage stripping for further product
purication. Liquid samples were taken from the spinner ask, and
the rst- and second-stage condensate storage tanks for analysis.
5
0
10
20
30
40
50
Time, h
Fig. 2. Time course proles of ABE fermentation by C. acetobutylicum JB200
immobilized in a brous bed bioreactor. (A) Batch fermentation without in situ gas
stripping; (B) Batch fermentation with in situ gas stripping starting at 28 h. Dashed
lines indicate the total production including products collected in the gas stripping
condensate and those remained in the fermentation broth.
399
Batch fermentation
without gas stripping
Batch fermentation
with gas stripping
80.0 1.1
0.3 0.1
1.30
53
7.9 0.1
16.2 0.9
1.4 0.2
25.5 1.2
0.20 0.01
0.32 0.02
0.30 0.02
0.48 0.02
1.2 0.1
0.9 0.1
2.1 0.2
80.0 1.1
0
1.64
48
10.3 0.2
19.8 0.8
1.7 0.1
31.8 1.1
0.25 0.01
0.40 0.01
0.41 0.02
0.66 0.02
0.4 0.1
1.7 0.1
2.1 0.2
butanol from the aqueous phase formed with the condensate collected from the rst-stage gas stripping. The compositions of the
condensates collected from the rst and second-stage gas stripping
are summarized in Table 3.
The solubility of butanol in water at 20 C is 7.7% (w/w) and
phase separation occurs spontaneously when butanol concentration is higher than 8%. The condensate of the rst-stage gas stripping contained 42.7 g/L acetone, 175.6 g/L butanol, 8.6 g/L
ethanol, 0.4 g/L acetic acid, and 0.8 g/L butyric acid. The butanol
concentration in the condensate was high enough for effective
phase separation, generating an organic phase containing 39.3 g/L
acetone, 612.3 g/L butanol and 9.1 g/L ethanol, and an aqueous
phase containing 43.4 g/L acetone, 101.3 g/L butanol, 8.5 g/L ethanol, 0.5 g/L acetic acid, and 0.7 g/L butyric acid. It is noted that no
acid was present in the organic phase and the butanol concentration in the aqueous phase was higher than the butanol solubility
in water because of the presence of acetone. The second gas stripping of the aqueous phase produced a highly concentrated condensate with 118.7 g/L acetone, 336.6 g/L butanol, and 22.1 g/L
ethanol, which after combining with the organic phase from the
rst-stage gas stripping gave a nal product mixture of 94.0 g/L
acetone, 420.3 g/L butanol, 18.0 g/L ethanol, and no acid. When
butanol is concentrated to such a high level, energy consumption
in nal product recovery as fuel or solvent by existing technologies
such as distillation can be reduced signicantly. Gas stripping is
not an energy-intensive process, as little energy is required in
pumping the stripping gas through the fermentor and the cooling
water through the condenser for recovering the solvent vapors.
Compared to steam stripping and distillation used in the conventional ABE fermentation recovery process, the two-stage gas stripping process would consume much less energy because only a
small amount of water needs to be removed in the nal dewatering
and product purication step.
Table 3
Product compositions and volumes obtained from rst and second stage gas stripping.
First-stage gas stripping
Acetone, g/L
Butanol, g/L
Ethanol, g/L
ABE, g/L
Acetic acid, g/L
Butyric acid, g/L
Total acids, g/L
Volume, mL
a
b
+
Condensate
Organic phase
Aqueous phase
42.7 3.2
175.6 12.9
8.6 0.9
226.9 17.0
0.4 0.02
0.8 0.03
1.2 0.05
350 1
39.3 4.8
612.3 40.8
9.1 1.2
660.7 46.8
None
None
None
50 1
43.4 2.1
101.3 5.4+
8.5 0.4
153.2 7.9
0.5 0.03
0.7 0.03
1.2 0.06
300 1
118.7 4.3
336.6 17.4
22.1 2.3
477.4 24.0
None
None
None
75 1
94.0 5.6
420.3 23.5
18.0 2.1
532.3 31.2
None
None
None
125 1
Using the simulated feed solution formulated based on the composition of the aqueous phase obtained in the rst-stage gas stripping.
The mixture of the organic phase from the rst-stage gas stripping and the condensate from the second-stage gas stripping.
Butanol concentration was higher than its solubility (77 g/L) in water due to the presence of acetone.
A 120
500
80
400
Butanol in solution
Acetone in solution
Ehanol in solution
Butanol in condensate
Acetone in condensate
60
40
300
200
Ethanol in condensate
20
100
600
12
15
18
Ethanol
12
Time, h
20
40
60
80
100
B 600
30
500
25
400
Selectivity
Acetone
16
100
20
Butanol
400
300
Butanol
200
Acetone
Ethanol
100
20
15
10
Butanol
20
40
60
80
100
Acetone
Ethanol
20
40
60
80
100
In addition, the selectivity for butanol, which is dened as the concentration ratio between the condensate and feed solution, is much
higher than those for acetone and ethanol, and is generally higher
than 10. Clearly, gas stripping is efcient for butanol recovery
when the butanol concentration is higher than 510 g/L, allowing
phase separation in the condensate to result in a highly concentrated butanol solution.
However, the butanol concentration in the condensate should
be as high as possible for the energy efciency of further butanol
purication and dewatering by distillation or other technologies.
Therefore, in order to capture the full energy-saving potential of
gas stripping, a second-stage gas stripping of the aqueous phase
from the rst-stage gas stripping is necessary. As demonstrated
in the present study, second-stage gas stripping can recover more
than 80% of the butanol (100 g/L) present in the rst-stage
aqueous phase and produce a condensate with more than 330 g/L
of butanol. The more than threefold increase in the butanol concentration would reduce at least 67% of the energy required in
the nal dewatering of biobutanol for fuel application. Overall,
gas stripping can increase butanol concentration from less than
2% (w/v) to over 40% (w/v) and the energy saving thus can be more
than 90% compared to conventional ABE fermentation without gas
stripping. It is thus desirable to use two-stage gas stripping for
butanol recovery to further reduce energy consumed in biobutanol
production.
401
References
Chen, C.K., Blaschek, H.P., 1999. Acetate enhances solvent production and prevents
degeneration in Clostridium beijerinckii BA101. Appl. Microbiol. Biotechnol. 52,
170173.
Drre, P., 2007. Biobutanol: an attractive biofuel. Biotechnol. J. 2, 15251534.
Ennis, B., Marshall, C.T., Maddox, I.S., Paterson, A.H.J., 1986. Continuous product
recovery by in-situ gas stripping/condensation during solvent production from
whey permeate using Clostridium acetobutylicum. Biotechnol. Lett. 8, 725730.
Evans, P.J., Wang, H.Y., 1988. Enhancement of butanol formation by Clostridium
acetobutylicum in the presence of decanol-oleyl alcohol mixed extractants. Appl.
Environ. Microbiol. 54, 16621667.
Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2003. Production of acetone, butanol and
ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping.
World J. Microbiol. Biotechnol. 19, 595603.
Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2004. AcetoneButanolEthanol (ABE)
production from concentrated substrate: Reduction in substrate inhibition by
fed-batch technique and product inhibition by gas stripping. Appl. Microbiol.
Biotechnol. 63, 653658.
Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007a. Bioproduction of butanol from
biomass: from genes to bioreactors. Curr. Opin. Biotechnol. 18, 220227.
Ezeji, T.C., Qureshi, N., Blaschek, H.P., 2007b. Production of acetone butanol (AB)
from liqueed corn starch, a commercial substrate, using Clostridium beijerinckii
coupled with product recovery by gas stripping. J. Ind. Microbiol. Biotechnol. 34,
771777.
Ezeji, T.C., Milne, C., Price, N.D., Blaschek, H.P., 2010. Achievements and perspectives
to overcome the poor solvent resistance in acetone and butanol-producing
microorganisms. Appl. Microbiol. Biotechnol. 85, 16971712.
Garcia, A., Lannotti, E.L., Fischer, J.L., 1986. Butanol fermentation liquor production
and separation by reverse osmosis. Biotechnol. Bioeng. 28, 785791.
Gheshlaghi, R., Scharer, J.M., Moo-Young, M., Chou, C.P., 2009. Metabolic pathways
of clostridia for producing butanol. Biotechnol. Adv. 27, 764781.
Huang, H., Liu, H., Gan, Y.R., 2010. Genetic modication of critical enzymes and
involved genes in butanol biosynthesis from biomass. Biotechnol. Adv. 28, 651
657.
Lu, C.C., Zhao, J.B., Yang, S.T., Wei, D., 2012. Fed-batch fermentation for butanol
production from cassava bagasse hydrolysate in a brous bed bioreactor with
continuous gas stripping. Bioresour. Technol. 104, 380387.
Maddox, I.S., Qureshi, N., Roberts-Thomson, K., 1995. Production of acetonebutanol-ethanol from concentrated substrates using Clostridium acetobutylicum
in an integrated fermentation-product removal process. Process Biochem. 30,
209215.
Matsumura, M., Kataoka, H., Sueki, M., Araki, K., 1988. Energy saving effect of
pervaporation using oleyl alcohol liquid membrane in butanol purication.
Bioprocess Eng. 3, 93100.
Mariano, A.P., Qureshi, N., Filho, R.M., Ezeji, T.C., 2011. Bioproduction of butanol in
bioreactors: new insights from simultaneous in situ butanol recovery to
eliminate product toxicity. Biotechnol. Bioeng. 108, 17571765.
Nielsen, D.R., Prather, K.J., 2009. In situ product recovery of n-butanol using
polymeric resins. Biotechnol. Bioeng. 102, 811821.
Nicolaou, S.A., Gaida, S.M., Papoutsakis, E.T., 2010. A comparative view of metabolite
and substrate stress and tolerance in microbial bioprocessing: from biofuels and
chemicals, to biocatalysis and bioremediation. Metab. Eng. 12, 307331.
Oudshoorn, A., Van der Wielen, L.A.M., Straathof, A.J.J., 2009. Assessment of options
for selective 1-butanol recovery from aqueous solutions. Ind. Eng. Chem. Res.
48, 73257336.
Papoutsakis, E.T., 2008. Engineering solventogenic clostridia. Curr. Opin. Biotechnol.
19, 420429.
Qureshi, N., Maddox, I.S., 1991. Integration of continuous production and recovery
of solvents from whey permeate: use of immobilized cells of Clostridium
acetobutylicum in a uidized bed reactor coupled with gas stripping. Bioproc.
Eng. 6, 6369.
Qureshi, N., Maddox, I.S., Friedl, A., 1992. Application of continuous substrate
feeding to the ABE fermentation: relief of product inhibition using extraction,
perstraction, stripping, and pervaporation. Biotechnol. Prog. 8, 382390.
Qureshi, N., Blaschek, H.P., 1999. Butanol recovery from model solutions/
fermentation broth by pervaporation: evaluation of membrane performance.
Biomass Bioenerg. 17, 175184.
Qureshi, N., Blaschek, H.P., 2001a. Recovery of butanol from fermentation broth by
gas stripping. Renew. Energy 22, 557564.
Qureshi, N., Blaschek, H.P., 2001b. Evaluation of recent advances in butanol
fermentation, upstream, and downstream processing. Bioproc. Biosyst. Eng.
24, 219226.
Qureshi, N., Hughes, S., Maddox, I.S., Cotta, M.A., 2005. Energy-efcient recovery of
butanol from model solutions and fermentation broth by adsorption. Bioprocess
Biosyst. Eng. 27, 215222.
Qureshi, N., Saha, B.C., Hector, R.E., Hughes, S.R., Cotta, M.A., 2008. Butanol
production from wheat straw by simultaneous saccharication and
fermentation using Clostridium beijerinckii: Part I Batch fermentation.
Biomass Bioenerg. 32, 168175.
Rofer, S.R., Blanch, H.W., Wilke, C.R., 1987. Extractive fermentation of acetone
and butanol: process design and economic evaluation. Biotechnol. Prog. 3,
131140.
Rofer, S.R., Blanch, H.W., Wilke, C.R., 1988. In situ extractive fermentation of
acetone and butanol. Biotechnol. Bioeng. 31, 135143.
4. Conclusions
A two-stage gas stripping process was developed for in situ
butanol recovery from ABE fermentation. The rst-stage gas stripping was initiated at butanol concentration above 8 g/L, which not
only alleviated butanol inhibition, but also made the condensate
containing enough butanol for efcient phase separation. After
the organic phase was collected, the aqueous phase was subjected
to second-stage gas stripping, resulting in a nal product mixture
containing more than 400 g/L butanol (500 g/L ABE), which was
20-fold higher than that achieved in conventional ABE fermentation. Therefore, this process can signicantly reduce energy consumption in nal product recovery.
Acknowledgements
This work was supported in part by the Ohio Department of
Development-Third Frontier Advanced Energy Program (Tech 08036), the National Science Foundation STTR program (IIP0810568, IIP-1026648), and Advanced Research Projects AgencyEnergy (DE-AR0000095). Financial support from the Fundamental
Research Funds for the Central Universities (DUT11RC(3)77), China
Postdoctoral Science Foundation (20110491527) and China Scholarship Council (2009100607) is also acknowledged.
402
Vane, L.M., 2008. Separation technologies for the recovery and dehydration of
alcohols from fermentation broths. Biofuels Bioprod. Bioren. 2, 553588.
Xue, C., Zhao, X.Q., Bai, F.W., 2010. Effect of the size of yeast ocs and zinc
supplementation on continuous ethanol fermentation performance and
metabolic ux distribution under very high concentration conditions.
Biotechnol. Bioeng 105, 935944.
Xue, C., Zhao, J.B., Lu, C.C., Yang, S.T., Bai, F.W., Tang, I.C., 2012. High-titer n-butanol
production by Clostridium acetobutylicum JB200 in fed-batch fermentation with