FEMS Microbiology Letters, 363, 2016, fnw023
doi: 10.1093/femsle/fnw023
Advance Access Publication Date: 5 February 2016
Research Letter
R E S E A R C H L E T T E R – Biotechnology & Synthetic Biology
Synergistic effect of calcium and zinc on glucose/
xylose utilization and butanol tolerance of Clostridium
acetobutylicum
Youduo Wu1 , Chuang Xue1 , Lijie Chen1,∗ , Wenjie Yuan1 and Fengwu Bai1,2
1
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China and 2 School
of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
∗
Corresponding author: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning, China. Tel: +86-0411-8470-6308;
Fax: 0411-8470-6308; E-mail: [email protected]
One sentence summary: Calcium and zinc play synergistic roles in glucose/xylose utilization and butanol tolerance of Clostridium acetobutylicum.
Editor: Michael Sauer
ABSTRACT
Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield
and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was
investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and
glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose
utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol
and ABE production of 16.1 and 25.9 g L−1 were achieved from 69.3 g L−1 glucose with butanol/ABE productivities of 0.40 and
0.65 g L−1 h−1 compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L−1 h−1 obtained in the control respectively
without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved
xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L−1 butanol). Under the same conditions,
calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested
that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum.
Keywords: Clostridium acetobutylicum; calcium; zinc; synergistic effect; xylose; butanol tolerance
INTRODUCTION
Currently, the dramatic fluctuations of oil prices and significantly increasing demands for fuels have aroused resurgent interest and motivation to search for the renewable feedstocksbased energy that alleviates the prevailing overdependence on
fossil fuels and thus is preferred as alternative biofuels in
terms of the low-cost and widely abundant feedstocks (Jang
et al. 2012a; Guo, Song and Buhain 2015). Biobutanol typically
produced via acetone-butanol-ethanol (ABE) fermentation by
Clostridium species has been widely acknowledged as one of the
most promising alternatives superior to bioethanol (Jones and
Woods 1986; Xue et al. 2013). However, this fermentative process was less economically feasible and competitive than the
petrochemical-based butanol (Ezeji et al. 2010; Gu et al. 2011).
Of these strict anaerobic bacteria, C. acetobutylicum is an important solvent-producing microbe due to its natural capability of
fermenting a wide range of raw fermentable sugars, including
polysaccharides, disaccharides and monosaccharides.
In nature, lignocellulosic biomass such as agricultural
residues including rice straw, wheat straw and corn stover are
regarded as the most promising candidate feedstocks (Qureshi
et al. 2010a,b, 2013; Jang et al. 2012b; Ranjan, Khanna and
Received: 15 November 2015; Accepted: 29 January 2016
C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2016, Vol. 363, No. 5
Moholkar 2013), which consist mainly of cellulose and hemicellulose that can be hydrolyzed into sugar mixture containing
pentoses (mostly xylose) and hexoses (mostly glucose). However, butanol fermentation from the xylose-based media is
usually unfavorable and undesired due to low butanol production, productivity and yield, which could be ascribed to insufficient xylose utilization of C. acetobutylicum, glucose-mediated
catabolic repression and drastic butanol inhibition on xylose
transport/metabolism (Grimmler et al. 2010; Ren et al. 2010;
Bruder et al. 2015). Nevertheless, the availability of a costeffective and high-producing medium for efficient conversion
of xylose remains a major issue and significant hurdle for economic ABE fermentation based on these renewable and sustainable feedstocks.
As a matter of fact, the efficiency of ABE fermentation inevitably depends on not only the regulatory response to each
sugar but also the essential availability of nutrients in media. With the development of omics and genetic tools, the detailed understandings of regulatory and metabolic networks of
C. acetobutylicum have been expanded (Servinsky et al. 2010;
Ren et al. 2012; Tracy et al. 2012; Lütke-Eversloh 2014), and
thus considerable efforts on the genetics and physiology of
C. acetobutylicum have been expended to develop biochemical
engineering strategies for improving ABE fermentation (Xiao
et al. 2011; Jin et al. 2014; Lütke-Eversloh 2014; Wu et al.
2015a). Nevertheless, more information with a desirable trait
that derive from considerable impacts of examining conditions or available nutrients on more productive fermentation
should be well taken into consideration to maximize the performance of ABE fermentation and thus propose a substantial scope for better understanding of the functional mechanisms of sugar utilization, central carbon metabolism and stress
response.
Specially, CaCO3 and ZnSO4 ·7H2 O are two important chemical additives successfully applied in ABE fermentation as previously described (El Kanouni et al. 1998; Richmond, Han and Ezeji
2011; Wu et al. 2013). CaCO3 enhances ABE fermentation due to
not only its pH-buffering effects but also broader effects of calcium at the cellular and protein levels on sugar utilization, acids
re-assimilation, butanol production and butanol tolerance (Han
et al. 2013). Zinc plays significant roles stimulating metabolic activities with respects to sugar utilization, cell growth and acids
re-assimilation as well as initiation of solventogenesis (Wu et al.
2013), implying calcium and zinc might be functionally synergistic for ABE fermentation. The objective of present study
was to investigate the synergistic effect of calcium and zinc cosupplementation on ABE fermentation and butanol tolerance by
C. acetobutylicum. This study provides a novel and fundamental
bioprocess engineering strategy for significantly improved ABE
fermentation by Clostridium species, which is essential for commercial exploitation of lignocellulosic feedstocks-based biofuels
production.
KH2 PO4 , 0.5; MgSO4 . 7H2 O, 0.2; MnSO4 . H2 O, 0.01; FeSO4 . 7H2 O,
0.01; CH3 COONH4 , 3.22; para-amino-benzoic acid, 0.01; biotin,
0.01. Glucose, xylose and glucose/xylose mixtures with three ratios of 2:1, 1:1 and 1:2 were selected as the carbon source(s) with
initial sugar(s) concentration of ∼70 g L−1 . Specially, as high as
3.22 g/L CH3 COONH4 was used for maintaining the C/N ratio in
the medium. CaCO3 and ZnSO4 ·7H2 O with their optimum concentrations of 4 g L−1 and 0.001 g L−1 was supplemented into
fermentation medium (Richmond, Han and Ezeji 2011; Wu et al.
2013), as the exogenous source of calcium and zinc to investigate their synergistic effect on batch ABE fermentation compared to the control experiments under the other three conditions with neither, calcium or zinc supplementation. It should
be noted that as low as ∼1810 ± 41.4 μg/L calcium and ∼35
± 2.1 μg/L zinc were detected in the control medium without CaCO3 or ZnSO4 ·7H2 O supplementation, which might derive
from other metal salts or yeast extract. All media were sterilized at 121◦ C for 15 min. After inoculation, the initial medium
pH was adjusted to 5.5 using 3 M H2 SO4 or 3 M KOH. All the
chemicals and reagents used were of analytical grade or equivalent and purchased from Sangon (Shanghai Sangon Company,
China).
Batch ABE fermentation
Batch ABE fermentation was conducted using a stirred tank
(1.5BG−4−3000, Shanghai Baoxing Engineering, China) with a
working volume of 1.1 L under anaerobic condition as previously
described (Wu et al. 2013). Samples of 4 mL were collected for
analysis of biomass density, residual sugar(s), acids and ABE. All
fermentation experiments were triplicated or more at 37.5◦ C and
150 rpm, and the mean values were presented with standard error bars.
Butanol-shock experiments
Butanol-shock experiments were further performed in 250 mL
screw-capped bottles containing 100 mL fermentation medium
using xylose as sole carbon source with/without supplement(s)
to investigate the synergistic effect of calcium and zinc on xylose utilization and butanol biosynthesis by C. acetobutylicum.
All experiments were anaerobically generated by purging with
filtered nitrogen for 10 min to remove dissolved oxygen and
initiated with 10% (v/v) inoculation of active cell suspension followed by butanol addition into the medium to a final butanol concentration of 2, 4, 6, 8 and 10 g L−1 , respectively. Each experiment was conducted in triplicate or more
at 37.5◦ C and 150 rpm of stirring for 120 h. Samples of 1 mL
were taken at 12 h intervals for residual xylose and ABE
determination.
Analytical methods
MATERIALS AND METHODS
Strain, pre-culture and media
The strain C. acetobutylicum L7 used in this study was derived
from C. acetobutylicum ATCC 824 after long-term adaptation as
previously documented (Wu et al. 2013). The pre-culture medium
and anaerobically cultured condition were as previously described (Wu et al. 2013). The standard fermentation medium
composed of (g L−1 ): sugar(s), 70; yeast extract, 2; K2 HPO4 , 0.5;
The spectrophotometer (Thermo Spectronic, USA) used for the
optical density (OD620 ) measurement, which was converted to
cell dry weight by using a predetermined correlation (Wu et al.
2013). Sugars and acids were analyzed with a high performance
liquid chromatography (Waters 1525 HPLC) and ABE were determined by a gas chromatography (Alilgent 6890A GC) as previously described (Wu et al. 2013). The standard compounds of
acids and ABE were of quality HPLC gradient grade and purchased from Sigma-Aldrich.
1.7 ± 0.1
1.3 ± 0.1
3.8 ± 0.2
2.2 ± 0.1
2.4 ± 0.2
2.8 ± 0.2
3.0 ± 0.2
3.7 ± 0.3
38.1 ± 1.0
45.2 ± 1.3
46.9 ± 1.5
49.7 ± 1.9
Neitherc
Zincc
Calcium
Both
7% xylose as sole carbon source
3
a
The fermentation media were supplemented with 4 g L−1 CaCO3 and 0.001 g L−1 ZnSO4 ·7H2 O as the exogenous source of calcium and zinc. All four conditions were expressed as follows. Neither, without CaCO3 and ZnSO4 ·7H2 O
supplementation; zinc, with only ZnSO4 ·7H2 O supplementation; calcium, with only CaCO3 supplementation; both, with CaCO3 and ZnSO4 ·7H2 O supplementation.
b
Data adapted from Wu et al. (2013).
c
Data adapted from Wu et al. (2015b).
2.39
4.29
1.85
3.72
0.5 ± 0.0
0.5 ± 0.0
0.8 ± 0.1
1.0 ± 0.1
6.3 ± 0.3
8.3 ± 0.4
8.8 ± 0.4
10.5 ± 0.6
4.4 ± 0.2
5.2 ± 0.3
4.8 ± 0.3
6.0 ± 0.4
3.0 ± 0.2
2.0 ± 0.1
4.0 ± 0.3
2.5 ± 0.2
6.4 ± 0.2
6.7 ± 0.4
8.1 ± 0.5
8.6 ± 0.5
0.8 ± 0.1
0.7 ± 0.1
1.2 ± 0.1
1.0 ± 0.1
0.9 ± 0.1
0.5 ± 0.1
2.2 ± 0.2
1.7 ± 0.2
4.2 ± 0.1
5.2 ± 0.2
5.8 ± 0.2
6.5 ± 0.3
55.4 ± 1.8
55.0 ± 0.8
67.7 ± 1.3
69.3 ± 1.1
Neitherb
Zincb
Calcium
Both
7% glucose as sole carbon source
Supplement
0.08/0.13
0.10/0.17
0.10/0.17
0.15/0.24
0.21/0.35
0.23/0.38
0.21/0.34
0.23/0.37
6.88
11.45
4.24
5.96
1.3 ± 0.1
1.7 ± 0.1
0.8 ± 0.1
1.2 ± 0.1
11.7 ± 0.4
12.6 ± 0.3
14.4 ± 0.8
16.1 ± 0.9
Ethanol
Butanol
Acetone
Butyrate
Acetate
Max OD620
)
(g L
−1
a
Sugar utilized
0.17/0.29
0.18/0.31
0.19/0.31
0.21/0.35
0.18/0.30
0.32/0.53
0.36/0.58
0.40/0.65
(Butanol/ABE)
(g L−1 h−1 )
(Butanol/ABE)
(Ratio)
Productivity
Products titer (g L−1 )
Table 1. Comparative results of ABE fermentation using glucose and xylose as sole carbon source with/without supplement(s).
The comparative results of ABE fermentation using glucose and
xylose as sole carbon source under all four conditions were
shown in Table 1. After calcium and zinc was co-supplemented
into the medium, as high as 69.3 g L−1 glucose was utilized following 40 h of fermentation. The cell growth was significantly
stimulated with a peak OD620 of 6.5 at 24 h compared to those of
4.2 at 28 h, 5.8 at 28 h and 5.2 at 20 h observed under the other
three conditions with neither, calcium or zinc supplementation.
The maximum butanol and ABE production of 16.1 and 25.9 g L−1
were achieved with butanol/ABE productivities of 0.40 and 0.65 g
L−1 h−1 , which were almost 2-fold higher than those of only 0.18
and 0.30 g L−1 h−1 obtained in the control with neither supplements. Similarly, when xylose was used as sole carbon source,
calcium and zinc co-supplementation resulted in the highest xylose utilization of 49.7 g L−1 and butanol production of 10.5 g L−1 .
Also, drastic increase on cell growth was observed with a peak
OD620 of 3.69 at 32 h compared to those of 2.42 at 32 h, 2.53 at
48 h and 2.45 at 40 h under the other three conditions.
As shown in Table 2, when glucose/xylose (2:1) mixture was
used as carbon sources, 10.6 g L−1 butanol and 17.7 g L−1 ABE
were produced from 52.7 g L−1 total sugars (47.5 g L−1 glucose
and 5.2 g L−1 xylose) with butanol/ABE productivities of 0.22 and
0.37 g L−1 h−1 in the control with neither supplements. Strikingly,
after calcium and zinc were co-supplemented into the medium,
as high as 13.9 g L−1 butanol and 23.0 g L−1 ABE were rapidly
achieved from 64.7 g L−1 total sugars (48.5 g L−1 glucose and
16.2 g L−1 xylose) with butanol/ABE productivities improved to
0.35 and 0.58 g L−1 h−1 . Cell growth was greatly enhanced with
a peak OD620 of 6.6. Notably, xylose utilization was restored to
different extent after glucose was completely depleted based
on the observations that 16.2 g L−1 xylose was further utilized
whereas only 5.2, 10.8 and 6.6 g L−1 xylose under the other
three conditions. Similar improvements were observed during
fermentations using the other two glucose/xylose mixtures (1:1
and 1:2) as carbon sources respectively. For example, in the glucose/xylose (1:1) medium supplemented with calcium and zinc,
butanol and ABE production were increased to 11.8 and 19.8 g L−1
with improved sugar utilization of 54.7 g L−1 (36.0 g L−1 glucose
and 18.7 g L−1 xylose). Furthermore, the comparative results under all four conditions were as summarized in Fig 1, the specific
glucose/xylose consumption rate, specific growth rate and specific butanol production rate were significantly facilitated, indicating the effect of calcium and zinc was conceivably synergistic
with respects to glucose/xylose utilization, cell growth, acids reassimilation and butanol biosynthesis.
Considering that the glucose/xylose utilization and cell
growth of C. acetobutylicum are highly dependent on the environmental pH as previously documented (Roos, Mclaughlin and
Papoutsakis 1985; Reardon and Bailey 1989), it was obvious that
CaCO3 supplementation led to enhanced glucose/xylose utilization, cell growth as well as excess acids production due to higher
pH maintained above 5.0 resulting from its pH-buffering effect.
Even so, the fermented xylose was limited to 47 g L−1 due to
slower growth rate and longer metabolic transition (Fond et al.
1986). Although the effects of pH on batch ABE fermentation by
C. acetobutylicum XY16 have been investigated using glucose, a
maximum ABE production and productivity of 20.3 g L−1 and
0.63 g L−1 h−1 were achieved via a two-stage controlled-pH strategy (Guo et al. 2012). Jiang et al. (2014) reported that more xylose
ABE/Acids
Synergistic effect of calcium and zinc on cell growth,
glucose/xylose utilization and butanol biosynthesis
Yield
RESULTS AND DISCUSSION
(g g−1 )
Wu et al.
(g L−1 )
Acetate
1.8 ± 0.2
1.0 ± 0.1
2.5 ± 0.2
2.1 ± 0.1
2.9 ± 0.2
2.3 ± 0.2
4.2 ± 0.3
2.7 ± 0.2
3.2 ± 0.2
2.7 ± 0.2
3.9 ± 0.3
3.0 ± 0.2
Max OD620
47.5 ± 1.2/5.2 ± 0.3
46.6 ± 1.3/6.6 ± 0.4
48.3 ± 1.4/10.8 ± 0.7
48.5 ± 1.8/16.2 ± 1.0
4.2 ± 0.2
5.0 ± 0.2
5.8 ± 0.4
6.6 ± 0.4
36.0 ± 1.1/8.8 ± 0.7
35.9 ± 1.7/12.5 ± 0.9
35.6 ± 1.3/15.9 ± 1.1
36.0 ± 1.6/18.7 ± 1.4
3.9 ± 0.2
4.5 ± 0.3
5.0 ± 0.4
5.7 ± 0.3
Neither
Zinc
Calcium
Both
24.0 ± 0.8/17.3 ± 1.1
23.3 ± 0.8/21.6 ± 1.6
23.8 ± 0.6/26.6 ± 0.9
24.1 ± 0.9/30.0 ± 1.4
3.6 ± 0.2
4.1 ± 0.2
4.4 ± 0.2
4.9 ± 0.3
7% glucose/xylose (1:2) mixture as carbon sources
Neither
Zinc
Calcium
Both
7% glucose/xylose (1:1) mixture as carbon sources
Neither
Zinc
Calcium
Both
7% glucose/xylose (2:1) mixture as carbon sources
Supplement
Glucose/Xylose utilized
2.4 ± 0.2
1.8 ± 0.1
3.3 ± 0.2
2.2 ± 0.2
2.1 ± 0.2
1.5 ± 0.1
2.9 ± 0.3
1.9 ± 0.2
2.7 ± 0.21
2.3 ± 0.1
2.3 ± 0.2
1.2 ± 0.1
Butyrate
4.2 ± 0.3
4.7 ± 0.3
5.0 ± 0.2
5.6 ± 0.4
4.7 ± 0.4
5.0 ± 0.3
5.4 ± 0.3
6.5 ± 0.5
6.1 ± 0.4
6.6 ± 0.5
7.2 ± 0.6
7.6 ± 0.6
Acetone
Products titer (g L−1 )
7.0 ± 0.4
8.5 ± 0.5
9.4 ± 0.6
10.6 ± 0.6
8.5 ± 0.5
9.6 ± 0.4
10.3 ± 0.5
11.5 ± 0.6
10.6 ± 0.6
11.4 ± 0.5
12.5 ± 0.8
13.9 ± 0.9
Butanol
0.7 ± 0.0
0.8 ± 0.1
0.8 ± 0.1
1.0 ± 0.1
0.9 ± 0.1
1.0 ± 0.1
1.0 ± 0.1
1.3 ± 0.1
1.0 ± 0.1
1.0 ± 0.1
1.2 ± 0.1
1.5 ± 0.1
Ethanol
Table 2. Comparative performance of ABE fermentation using glucose/xylose mixtures as carbon sources with/without supplement(s).
2.13
3.11
2.25
3.31
2.82
4.11
2.35
4.20
3.93
5.76
4.35
6.97
(Ratio)
ABE/Acids
0.17/0.29
0.19/0.31
0.19/0.30
0.20/0.32
0.19/0.31
0.20/0.32
0.20/0.32
0.21/0.35
0.20/0.34
0.21/0.36
0.21/0.35
0.21/0.35
(Butanol/ABE)
Yield (g g−1 )
0.09/0.15
0.11/0.18
0.14/0.22
0.17/0.27
0.15/0.25
0.18/0.30
0.23/0.38
0.26/0.44
0.22/0.37
0.26/0.43
0.31/0.52
0.35/0.58
(Butanol/ABE)
(g L−1 h−1 )
Productivity
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FEMS Microbiology Letters, 2016, Vol. 363, No. 5
Figure 1. Comparative analysis of batch ABE fermentation using glucose (A), xylose (B) and glucose/xylose (2:1) mixture (C) as carbon source(s) with/without
supplement(s).
was consumed with the pH maintained at 4.8 when xylose was
used as sole carbon source. Moreover, with the addition of ammonium acetate in cassava medium, the ABE productivity by C.
acetobutylicum was increased to 0.40 g L−1 h−1 (Gu et al. 2009).
Similarly, the addition of 70 mM ammonium acetate led to not
only more glucose utilization but also increased ABE production and productivity of 17.8 g L−1 and 0.37 g L−1 h−1 due to its
buffering capacity for the pH maintained above 5.0 during fermentation (Boonsombuti et al. 2014). On the other hand, it has
been demonstrated that calcium showed more favorable effects
on ABE fermentation with respects to sugar utilization, acids reassimilation, ABE production and butanol tolerance of C. beijerinckii at the cellular and protein levels. El Kanouni et al. (1998)
Wu et al.
5
Figure 2. Impact of calcium and zinc co-supplementation on xylose utilization and butanol biosynthesis using xylose medium without butanol addition (A) and under
various butanol-shock conditions by adding butanol to the final concentrations of 2 g L-1 (B), 4 g L-1 (C), 6 g L-1 (D), 8 g L-1 (E) and 10 g L-1 (F).
reported that addition of 3 g L−1 CaCl2 resulted in reduced residual xylose from 30 to 2.3 g L−1 only when the pH was adjusted
using 2 M NaOH. Han et al. (2013) found that 0.5 g L−1 CaCl2 addition into the medium resulted in increased total ABE production
of 17 g L−1 by C. beijerinckii NCIMB 8052 compared to that of
15 g L−1 in the fermentation treated with 0.5 g L−1 CaCO3 . Notably, CaCl2 concentrations above 0.5 g L−1 were found to be toxic
to C. beijerinckii due to its bacteriostatic and bactericidal effects
on the microorganism.
In our study, xylose utilization was still strongly repressed
by glucose under all four conditions. This repression was ascribed to not only glucose competition with xylose in the transport system but also glucose-mediated catabolite repression on
key enzymes located in the xylose metabolism pathway (Vanzyl
et al. 1993; Tangney et al. 2003). According to previous studies,
xylose-pregrown cells of C. acetobutylicum could subsequently
convert xylose when glucose was exhausted in batch fermentation (Fond et al. 1986). The addition of 10 g L−1 CaCO3 could
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FEMS Microbiology Letters, 2016, Vol. 363, No. 5
contribute to the improved xylose utilization by xylosepregrown C. acetobutylicum in spite of the persistence of glucosemediated catabolic repression, resulting in as high as 11.5 g L−1
butanol and 15.8 g L−1 ABE produced from a mixture of 30 g L−1
glucose and 30 g L−1 xylose (El Kanouni et al. 1998). In a recent
study, 10.0 g L−1 butanol was produced from 50.1 g L−1 xylose using immobilized C. acetobutylicum CGMCC 5234 cells compared to
8.5 g L−1 butanol from 39.8 g L−1 xylose using suspended cells.
When mixture of 30 g L−1 glucose and 30 g L−1 xylose was used
as carbon sources, 11.1 g L−1 butanol was produced from
56.5 g L−1 total sugars (30 g L−1 glucose and 26.5 g L−1 xylose)
with immobilized cells, compared to 8.7 g L−1 butanol from 46.9
g L−1 total sugars (30 g L−1 glucose and 16.9 g L−1 xylose) with
suspended cells (Chen et al. 2013). In addition, C. acetobutylicum
ATCC 824 was metabolically engineered by overexpression of
a transaldolase (talA) from E. coli for improved xylose utilization and ABE production with apparent CCR (Cu et al. 2009). Significant co-utilization of glucose/xylose without apparent CCR
was achieved by inactivation of the ccpA gene and adding
10 g L−1 CaCO3 as pH-controlled strategy, leading to restored cell
growth and improved butyrate re-assimilation with maximum
butanol/ABE production of 12.1 g L−1 and 18.0 g L−1 . However,
an unexpected drawback remained in that large amounts of acetate and butyrate were produced by this C. acetobutylicum ATCC
824ccpA strain without CaCO3 supplementation (Ren et al. 2010).
Synergistic roles of calcium and zinc for improving
butanol tolerance
Under various butanol-shock conditions, the comparative profiles of xylose utilization and butanol production were shown
in Fig. 2. The xylose utilization drastically decreased as the initial concentration of additional butanol increased. Surprisingly,
the inhibitory effect of butanol on xylose utilization was greatly
alleviated by co-supplementing both calcium and zinc. For example, when 4 g L−1 butanol was added into the medium, xylose utilization of 43.8 g L−1 and butanol production of 10.0 g L−1
were achieved by C. acetobutylicum, which was close to the results observed without butanol stress and almost 2-fold higher
compared to those of 24.4 and 4.7 g L−1 obtained in the control with neither supplements. When the initial concentration
of additional butanol was increased to 8 g L−1 , as high as
34.7 g L−1 xylose was utilized for relatively higher butanol production of 6.4 g L−1 . Under this condition, it should be noted
that calcium led to 28.8 g L−1 xylose utilized, which was consistent with the results obtained by El Kanouni et al. (1998). It was
of great importance to find that sole zinc supplementation also
contributed to a xylose utilization of 22.6 g L−1 , implying zinc
could facilitate butanol tolerance of C. acetobutylicum to some
extent.
In nature, butanol has been reported to be a potent inhibitor
to xylose metabolism and could lead to more severely inhibitory
effect on ABE fermentation from xylose than that from glucose
(Bowles and Ellefson 1985; Ezeji et al. 2010), which resulted in 50%
inhibition on the initial sugar transport rate and incorporation
into cell in the presence of 4.5 g L−1 butanol and 7.0 g L−1 butanol
for xylose respectively, while 7 g L−1 butanol and 10.5 g L−1 butanol for glucose. More importantly, 8 g L−1 butanol could completely inhibit the cell growth on xylose while 14 g L−1 butanol
for glucose. The xylose and glucose permease were strongly inhibited by 8 and 12 g L−1 butanol respectively (Ounine et al. 1985).
Previous study reported that when 8 g L−1 butanol was added
to the medium at the exponential growth phase, xylose uptake
could be altered with no more than 30 g L−1 xylose utilized
under pH-controlled condition while increased xylose utilization of 43 g L−1 was achieved in the presence of 10 g L−1 CaCO3
(El Kanouni et al. 1998). Similar results were observed under iron
limitation conditions, the addition of 10 g L−1 CaCO3 could enhance xylose utilization up to 41 g L−1 compared to that of only
16.5 g L−1 without CaCO3 supplementation (Junelles et al. 1988).
Until recently, proteomic analysis has been performed to elucidate the role of calcium in ABE fermentation by C. beijerinckii
NCIMB 8052. The results showed that calcium has regulatory effects at the cellular and protein levels associated with butanol
tolerance by inducing the heat shock response (Han et al. 2013).
More importantly, more xylose was utilized by supplementing
zinc under various butanol-shock conditions while no studies
have been reported on the improved butanol tolerance by zinc,
implying zinc might be a critical factor involved in butanol stress
response of C. acetobutylicum. In fact, it has been demonstrated
that both the ethanol tolerance and thermal tolerance could be
significantly improved by zinc supplementation in the continuous ethanol fermentation using self-flocculating yeast (Zhao
et al. 2009).
Despite substantial progress in the development of biochemical engineering strategies for the ABE fermentation from various low-cost and renewable feedstocks, there is still a need for
further improvements to meet the industrial requirements. This
study highlighted the synergistic roles of calcium and zinc in improving ABE fermentation by C. acetobutylicum associated with
glucose/xylose utilization, cell growth, acids re-assimilation, butanol biosynthesis and butanol tolerance, implying a potential
reprogramming of cellular metabolic network and redistribution
of carbon flux by calcium and zinc. Therefore, manipulation of
calcium and zinc supplementation is thus an effective engineering strategy for the development of industrial ABE production.
Further elucidation of the underlying molecular mechanisms is
needed for better understanding the regulatory mechanism on
the physiology of C. acetobutylicum.
FUNDING
This work was supported by the National Natural Science
Foundation of China (21376044 and 21576045) and the National High-Tech Research and Development Program of China
(2011AA02A208 and 2012AA021205).
Conflict of interest. None declared.
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