2.bio Butanol Production
2.bio Butanol Production
Regular Article
a r t i c l e i n f o a b s t r a c t
Article history: Our recent work showed that Chlorella vulgaris JSC-6 can accumulate over 50% carbohydrates per dry
Received 25 May 2014 weight, which mainly contain glucose and xylose as the monosaccharides. In this study, synthetic mixed
Received in revised form 12 July 2014 sugars simulating the hydrolyzed biomass of C. vulgaris JSC-6 (primarily containing glucose and xylose
Accepted 4 August 2014
at a ratio of 5:16.5:1) was used as the carbon source for bio-butanol production with Clostridium aceto-
Available online 13 August 2014
butylicum ATCC824. The growth and product formation kinetics based on glucose, xylose, and mixtures
of the two sugars were determined using Monod-type and MichaelisMenten models, respectively. C.
Keywords:
acetobutylicum ATCC824 can grow faster and produce butanol more efciently on glucose, while the per-
Butanol
Fermentation
formance was markedly poorer when using xylose. The butanol production kinetics were quite similar
Carbohydrates when using glucose alone or using mixed sugars (glucose to xylose ratio = 5:1 or 6.5:1), resulting in a
Growth kinetics maximum butanol production rate of 0.890.93 g/h/L. This work demonstrated the feasibility of using
Production kinetics the microalgae-based carbohydrates as the feedstock for biobutanol production with C. acetobutylicum
Microalgae ATCC824.
2014 Elsevier B.V. All rights reserved.
1. Introduction cost [3]. A cheaper way to produce butanol is thus required before
its widespread commercial utilization.
Today about 80% of global energy demand is produced from The use of Clostridial species (such as Clostridium acetobutylicum,
fossil fuels [1]. However, rising concerns over energy security Clostridium beijerinckii and Clostridium saccharoperbutylaceton-
and climate change make biofuels (such as biodiesel, ethanol, icum) via ABE fermentation is the most commonly used process
and butanol) attract increasing amounts of attention as potential for butanol production [4]. Clostridium sp. are known to have the
sources of clean, renewable energy. Among these, biobutanol is capability of utilizing simple and complex sugars, including pen-
especially promising, since it is less corrosive, less hydroscopic, tose and hexose, as well as CO2 , H2 and CO [58]. Butanol has
and can potentially replace ethanol as a gasoline additive due to traditionally been produced by ABE fermentation using carbon sub-
several advantages, such as low vapor pressure (reduced emis- strates obtained from corn, sugar beets and sugar cane, potatoes,
sions), high energy density (greater miles per gallon), and blending tapioca and millet [5,9,10]. However, in order to obtain enough
options (increased concentration) [2]. The current production scale fermentable carbon substrates without getting into the competi-
of biobutanol is only second to that of bioethanol, although the tion with food supplies, other efcient feedstocks are now being
butanol that is now produced is mainly used as an industrial sol- considered, such as cellulosic materials or microalgal biomass [11].
vent, rather than an alternative energy, due to the high production Moreover, butanol production using food wastes or plants is ben-
ecial only when the biomass is produced in a sustainable way,
without having adverse impacts on the environment. The concept
of photonol has recently has been proposed to address this issue,
Corresponding author at: Department of Chemical Engineering, National Cheng
based on the direct conversion of solar energy in the production
Kung University, Tainan, Taiwan. Tel.: +886 6 2757575x62651; fax: +886 6 2357146.
Corresponding author. of fermentation end products, such as biofuels, by photosynthetic
E-mail addresses: [email protected] (J.-S. Chang), [email protected] microalgae [5]. In this approach, H2 O, CO2 and solar energy are used
(N.-Q. Ren). as inputs for the production of fermentation products [5]. This is
http://dx.doi.org/10.1016/j.bej.2014.08.007
1369-703X/ 2014 Elsevier B.V. All rights reserved.
Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 221
referred to as photofermentation, and is a process that is likely 2.3. ABE fermentation using glucose and xylose as sole or dual
to make biofuel production more economically attractive when carbon sources
its photosynthetic efciency is further improved [12,13]. The use
of microalgal biomass as a feedstock for biobutanol production is The butanol fermentation was conducted at a controlled tem-
thus one of the most promising approaches to the production of perature of 37 C. The initial pH of the culture was 6.0, and this
biobutanol, although few studies have examined this issue. More was then maintained at 4.5 with NaOH (1 N). The batch cultiva-
information is thus needed on the characterization and kinetics tion of C. acetobutylicum ATCC824 was carried out in a 250 mL glass
of microalgae-based biobutanol production, in order to aid in the vessel and the working volume was 100 mL. Glucose alone, xylose
design of bioreactors, scale up the process, and eventually achieve alone, and the mixture of the two with a glucose to xylose ratio of
sustainable commercialization of this renewable fuel. 5:1 or 6.5:1 (simulating the sugar composition of the hydrolyzed
Our previous works showed that isolated Chlorella vulgaris C. vulgaris JCS-6), were used as the carbon source for biobutanol
strains can accumulate over 50% carbohydrates per dry weight fermentation. The concentrations of 20, 40, 60, and 80 g/L were
under autotrophic condition, and have been successfully used as a examined for the batch butanol fermentation.
feedstock to produce bioethanol and biohydrogen [1418]. The car-
bohydrates produced from the microalga grown under autotrophic
2.4. Analytical methods
conditions consist mainly of glucose and xylose, which account
for 44.9% and 6.14% of dry cell weight, respectively. Mixotorophic
The carbohydrate content and prole in the microalgal biomass
cultivation of microalage can greatly shorten cultivation time for
were determined using a modied quantitative saccharication
the biomass growth and carbohydrate accumulation. The current
(QS) method (Huang) originally reported by the National Renew-
work was thus undertaken to evaluate the feasibility of using
able Energy Laboratory (NREL), USA [19]. In general, after grown
this carbohydrate-rich microalga cultivated under mixotrophic
under mixotrophic conditions for nearly 11 days, the broth of
condition as feedstock for biobutanol production. Before using
C. vulgaris JSC-6 culture was centrifuged at 6000 rpm for 3 min,
hydrolysate of real microalgal biomass for biobutanol fermenta-
washed three times, and then lyophilized. The lyophilized microal-
tion with a C. acetobutylicum strain, it is useful to obtain clearer cell
gal biomass was dissolved into 3 mL 72% (w/w) sulfuric acid in a
growth and butanol production properties with pure and mixed
water bath at 30 C for about 60 min to completely dissolve the
mono sugars (i.e., glucose and xylose) as the carbon source. There-
biomass. The liquid samples were then mixed with 87 mL water to
fore, in this study, a simulated hydrolysate of the microalgae-based
reach an acid concentration of 4% (w/w), and the acidmicroalga
carbohydrates (i.e., an appropriate ratio of glucose and xylose) was
mixture was autoclaved at 121 C for 20 min for hydrolysis of car-
used as the carbon source to characterize the kinetic properties of
bohydrates to obtain sugars. The supernatant of the hydrolyzed
bio-butanol fermentation.
microalgal biomass was neutralized with solid CaCO3 and analyzed
using a high-performance liquid chromatograph (HPLC) equipped
2. Materials and methods with a refraction index detector (RID-10A, Waters, USA) for the
assay of monosaccharides (glucose, xylose). The column used was
2.1. Microalgal strain and its cultivation conditions ICSep 156ICE-COREGEL 87H3 (Transgenomic, USA) [20]. The solu-
ble metabolites produced during the ABE fermentation were also
The microalga used in this study was C. vulgaris JSC-6, which is detected with the same HPLC device used for sugar analysis men-
a UV-mutated strain of C. vulgaris ESP6 isolated from fresh water tioned above following the procedures described in our earlier
in Southern Taiwan [1416]. The basal medium used to grow the work [21]. The mobile phase for sugar and other soluble metabo-
microalga consisted of (g/L): KNO3 , 1.25; KH2 PO4 , 1.25; MgSO4 , 1.0; lites production was 0.008 N H2 SO4 with a ow rate of 0.4 mL/min,
CaCl2 , 0.111; FeSO4 , 0.0498; EDTA 2 Na, 0.5. Mixotrophic cultiva- the injection sample volume was 2.0 mL and the column tempera-
tion was used, in which carbon dioxide (2.5%) was continuously ture was controlled at 70 C. The retention time for glucose, xylose,
supplied to the microalgal culture at a ow rate of 0.2 vvm, and acetate, butyrate, ethanol and butanol were 13.8, 14.8, 22.6, 32.1,
0.3 g/L acetate and 0.8 g/L butyrate was added to the basal medium, 32.6, 55.2 min, respectively.
as mentioned above. The organic acids were used as the carbon The gas products (mainly H2 and CO2 ) were measured by a
source for the heterotrophic growth of C. vulgaris, since they are the gas meter and analyzed by gas chromatography (Model 9800,
predominant soluble metabolites in the dark fermentation culture China Chromatography, Taipei, Taiwan) with a thermal conductiv-
associated with Clostridium sp. [14]. The microalga was grown in a ity detector [22].
1-L glass photobioreactor illuminated by an external light source Dry cell concentrations were obtained based on the optical den-
(TL5 uorescent lamp) mounted on both sides of the photobioreac- sity (OD) values measured at 685 nm with a spectrophotometer
tor at a light intensity of 150 mol/m2 /s. The initial optical density (model U-2001, Hitachi, Tokyo, Japan) via appropriate calibration.
(measured at 685 nm) and pH used for the microalga cultivation
were 2.0 and 7.0, respectively. The liquid sample was collected from
3. Results and discussions
the sealed glass vessel to determine the microalgae cell concentra-
tion, carbohydrate content in the microalga, pH, and residual nitrate
3.1. Carbohydrate composition of the biomass of C. vulgaris JSC-6
concentration over time.
to xylose represents different cultivation period of C. vulgaris JSC- poor when the xylose concentration was lower than 20 g/L. How-
6 as carbohydrate content will accumulate after nitrogen source ever, when the xylose concentration was increased to 4080 g/L,
exhausted [17]. the performance was better, giving a similar butanol yield and
productivity of 0.320.38 mol/mol xylose and 0.120.14 g/L/h,
3.2. Butanol fermentation performance at different sugar respectively (Table 2). During ABE fermentation with C. aceto-
concentrations butylicum ATCC824, a maximum H2 production of 73087352 mL/L
was obtained at xylose concentrations of 6080 g/L (Table 2). More-
Carbohydrates are the main carbon source used in the culture over, as the xylose concentration increased from 20 to 80 g/L, the
medium for ABE fermentation using Clostridial species [23]. Butanol xylose utilization efciency decreased from 99 to 70% (Table 2). As
production with a theoretical yield of about 90% was obtained when shown in Tables 1 and 2, the butanol production performance when
glucose was used as the sole carbon source. Since the objective using glucose as the carbon source is signicantly better than when
of this work is to use microalgae-based carbohydrates (containing using xylose.
mainly glucose and xylose after hydrolysis) as the carbon source for Since the results of the composition analysis show that the ratio
butanol production, glucose and xylose were employed as sole and of glucose and xylose content in the microalgal biomass ranged
dual carbon sources, based on the results of the composition anal- from 5:1 to 6.5:1 (data not shown), a mixed sugar with a glucose-
ysis of C. vulgaris biomass, to investigate the kinetics of butanol to-xylose ratio of 5:1 was then used as the carbon source for
production when using the major monosaccharides originating butanol fermentation. As shown in Fig. 3, the concentration of
from microalgal carbohydrates as the carbon source. Moreover, the butanol increased from 1.36 to 11.5 g/L as the total sugar con-
concentration of the carbon source usually plays a crucial role in the centration increased from 20 to 80 g/L. Although a higher total
efciency of anaerobic butanol fermentation [24]. Therefore, differ- sugar concentration resulted in a higher nal butanol concentra-
ent concentrations of carbon sources (glucose, xylose and mixed tion, as well as a slightly higher butanol productivity, a similar
sugars at a total concentration of 20 g/L, 40 g/L, 60 g/L and 80 g/L) maximum butanol yield of 0.420.47 mol/mol sugar was observed
were used to examine the effects of this concentration on the per- when the total sugar concentration was 4080 g/L (Table 3). On the
formance of ABE fermentation. other hand, H2 production increased as the sugar concentration
The process of butanol production in ABE fermentation can increased. However, the maximum H2 accumulation (7756 mL/L)
be divided into the acid production phase (acidogenesis) and the and yield (2.13 mol/mol sugar) were obtained at a total sugar con-
solvent production phase (solventogenesis) [4]. In the acid produc- centration of 60 g/L and 40 g/L, respectively (Table 3). Moreover,
tion phase, the growth of C. acetobutylicum is accompanied by the when the mixed sugar was used as the carbon source, the C. aceto-
production of organic acids (mainly acetate and butyrate) and H2 butylicum ATCC824 strain preferred to utilize glucose over xylose.
(Fig. 1). In the solvent production phase, the organic acids gener- The glucose utilization efciency was 99100% for the xylose con-
ated in the earlier phase are converted to produce acetone, ethanol centration of 2060 g/L, while a lower glucose utilization of 96.6%
and butanol (Fig. 1). Since glucose accounts for over 80% of total was observed when the xylose concentration was increased to
microalgal sugars, according to the composition analysis (Section 80 g/L. In contrast, the xylose utilization was only 2044% (Table 3).
3.1), the butanol fermentation was rst carried out at different To determine the effect of the glucose to xylose ratio in the
concentrations of glucose (Fig. 1). mixed sugars on the ABE fermentation performance, another
At a glucose concentration of 20 g/L, a higher nal butyric glucose-to-xylose ratio (i.e., 6.5:1) was also used to carry out ABE
acid concentration and lower solvent concentration were detected, fermentation (Fig. 4). This increased glucose-to-xylose ratio did not
suggesting that butanol fermentation should be carried out at affect the trends of solvent generation and H2 accumulation, as
a relatively higher carbon source concentration to achieve bet- indicated in Table 4. These results also show that the butanol pro-
ter butanol producing performance. The concentration of butanol duction performance using mixed sugar with a glucose-to-xylose
increased from 1.85 to 13.03 g/L as the glucose concentration was ratio of 5:1 and 6.5:1 is quite similar to that obtained from using
increased from 20 to 80 g/L. The butanol yield increased as the glucose alone as the carbon source. Based on the butanol produc-
glucose concentration increased from 20 to 60 g/L, reaching a max- tion results and taking the sugar utilization efciency into account,
imum butanol yield and productivity of 0.45 mol/mol glucose and the preferable total sugar concentration for butanol fermentation
0.33 g/L/h, respectively, with nearly complete glucose utilization is thus 60 g/L.
(Table 1). A further increase in glucose concentration to 80 g/L
resulted in an increase in maximum butanol production and a sim-
ilar butanol yield, but the glucose utilization efciency became 3.3. Kinetics of cell growth and product (H2 , solvent, and butanol)
much lower (87.6%) (Table 1). formation using different compositions of carbon sources
Based on the metabolism of Clostridial species, the fermenta-
tion process always produces both liquid (ABE) and gaseous (H2 ) To date, little research has been done to describe the kinetics of
biofuels [24,26]. As indicated in Table 1, a maximum H2 accumu- cell growth and product generation in relation to the current study,
lation (10,021 mL/L) and yield (1.76 mol/mol glucose) were also and thus two kinetic models were used to describe the ABE fer-
obtained at a glucose concentration of 60 g/L. A further increase mentation system in this work. The dependence of the cell growth
in glucose concentration from 60 to 80 g/L led a decrease in both rate of C. acetobutylicum ATCC824 on glucose, xylose and mix sugar
H2 production and yield. These results suggest that the best glucose concentrations is described by the following Monod-type kinetic
concentration for butanol fermentation would be 60 g/L. model (Eq. (1)) [27]:
Butanol fermentation was also carried out using different con-
centrations of xylose as the carbon source, with the results shown
max S
in Fig. 2. It can be see that butanol was successfully produced using = (1)
xylose, but a long lag phase (1626 h) occurred when the xylose KS + S
concentration was increased from 20 to 80 g/L. This lag time is much
longer than that observed when using glucose as a carbon source where denotes the specic growth rate (h1 ), S denotes the
(Figs. 1 and 2). The concentration of butanol increased from 1.34 carbon substrate concentration (g/L), max denotes the maximum
to 8.89 g/L when the xylose concentration was increased from 20 specic growth rate (h1 ), and KS denotes the half-saturation con-
to 80 g/L (Table 2). The butanol production performance was quite stant (g/L).
Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 223
12000
(a) 20 g/L 70 6 12000
(b) 40 g/L 70 6
H2 accumulation (ml/L)
10000 60 5 10000 60 5
50 50
Biomass (g/L)
Biomass (g/L)
8000 4 8000 4
40 40
6000 3 6000 3
30 30
4000 2 4000 2
20 20
2000 10 1 2000 10 1
0 0 0 0 0 0
80 14 80 14
20 4 20 4
2 2
0 0 0 0
5 5
pH
5.0 5.0
2 2
4.5 1 4.5 1
4.0 0 4.0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Time (h) Time (h)
12000
(c) 60 g/L 60 6 (d) 80 g/L
12000 60 6
H2 content in biogas (%)
H2 accumulation (ml/L)
10000 50 5
10000 50 5
Biomass (g/L)
8000 40 4
Biomass (g/L)
8000 40 4
6000 30 3
6000 30 3
4000 20 2
4000 20 2
2000 10 1
2000 10 1
0 0 0
80 14 0 0 0
80
Solvent production (g/L)
14
12
Glucose conc. (g/L)
12
60
10 60
10
8
40 8
6 40
6
20 4
20 4
2
2
0 0
0 0
5
Organic acid conc. (g/L)
6.0 5
Organic acid conc. (g/L)
6.0
4
4
5.5
3 5.5
pH
3
pH
5.0
2 5.0
2
4.5 1 4.5 1
4.0 0
4.0 0
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
Time (h)
Time (h)
H2 accumulation (ml/L) Glucose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)
Fig. 1. BioH2 and ABE fermentation performance at different glucose concentrations (2080 g/L). (Operating condition: temperature: 37 C; initial pH: 6.0; controlled nal
pH: 4.5; agitation rate: 200 rpm.)
224 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230
(a) 20 g/L 70 7
(b) 40 g/L 70 7
8000 8000
H2 accumulation (ml/L)
60 6 60 6
6000 50 5 6000 50 5
Biomass (g/L)
Biomass (g/L)
40 4 40 4
4000 4000
30 3 30 3
20 2 20 2
2000 2000
10 1 10 1
0 0 0 0 0 0
80 14 80 14
20 4 20 4
2 2
0 0 0 0
5 5
Organic acid conc. (g/L)
pH
5.0 5.0
2 2
4.5 1 4.5 1
4.0 0 4.0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
(c) 60 g/L (d) 80 g/L
70 7 70
8000 8000
H2 content in biogas (%)
H2 accumulation (ml/L)
60 6 60 6
6000 50 5 50
Biomass (g/L)
6000
Biomass (g/L)
40 4 40 4
4000 4000
30 3 30
20 2 20 2
2000 2000
10 1 10
0 0 0 0 0 0
80 14 80 14
Solvent production (g/L)
60 60
10 10
8 8
40 40
6 6
20 4 4
20
2 2
0 0 0 0
5 5
Organic acid conc. (g/L)
6.0 6.0
4 4
5.5 5.5
3 3
pH
pH
5.0 5.0
2 2
4.5 1 4.5 1
4.0 0 4.0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
H2 accumulation (ml/L) Xylose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)
Fig. 2. BioH2 and ABE production performance at different xylose concentrations (2080 g/L). (Operating conditions: temperature: 37 C; initial pH: 6.0; controlled nal pH:
4.5; agitation rate: 200 rpm.)
Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 225
(a) 20 g/L 70 6
(b) 40 g/L 70 6
H2 accumulation (ml/L)
60 5 60 5
50 50
Biomass (g/L)
Biomass (g/L)
6000 4 6000 4
40 40
3 3
4000 30 4000 30
2 2
20 20
2000 2000
10 1 10 1
0 0 0 0 0 0
14 14
pH
5.0 5.0
2 2
4.5 1 4.5 1
4.0 0 4.0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
(c) 60 g/L (d) 80 g/L
70 6 70 6
8000
H2 content in biogas (%)
8000
H2 accumulation (ml/L)
60 5 60 5
50 50
Biomass (g/L)
Biomass (g/L)
6000 4 6000 4
40 40
3 3
4000 30 4000 30
2 2
20 20
2000 2000
10 1 10 1
0 0 0 0 0 0
14 14
Solvent production (g/L)
10 10
40 8 40 8
6 6
20 4 20 4
2 2
0 0 0 0
5 5
Organic acid conc. (g/L)
6.0 6.0
4 4
5.5 5.5
3 3
pH
pH
5.0 5.0
2 2
4.5 1 4.5 1
4.0 0 4.0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Time (h) Time (h)
H2 accumulation (ml/L) Glucose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)
Xylose (g/L)
Fig. 3. BioH2 and ABE fermentation performance at different mixed sugar concentrations (2080 g/L). The ratio of the glucose and xylose concentration is 5:1. (Operating
conditions: temperature: 37 C; initial pH: 6.0; controlled nal pH: 4.5; agitation rate: 200 rpm.)
226 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230
12000
(a) 20 g/L 70 6
(b) 40 g/L
12000 70 6
60
H2 accumulation (ml/L)
10000 5 10000 60 5
50
Biomass (g/L)
50
Biomass (g/L)
8000 4 8000 4
40 40
6000 3 6000 3
30 30
4000 2 4000 2
20 20
2000 10 1 2000 1
10
0 0 0 0 0 0
70 14 70 14
pH
5.0 5.0
2 2
4.5 1 4.5 1
4.0 0 4.0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Time (h) Time (h)
(c) 60 g/L (d) 80 g/L
12000 70 6 12000 70 6
H2 content in biogas (%)
60
H2 accumulation (ml/L)
10000 5 10000 60 5
50
Biomass (g/L)
50
Biomass (g/L)
8000 4 8000 4
40 40
6000 3 6000 3
30 30
4000 2 4000 2
20 20
2000 10 1 2000 1
10
0 0 0 0 0 0
70 14 70 14
Solvent production (g/L)
50 10 50 10
40 8 40 8
30 6
30 6
20 4
20 4
10 2
10 2
0 0
0 0
5
Organic acid conc. (g/L)
5
Organic acid conc. (g/L)
6.0
6.0
4
4
5.5
5.5
3
pH
3
pH
5.0
2 5.0
2
4.5 1 4.5 1
4.0 0
4.0 0
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
Time (h)
Time (h)
H2 accumulation (ml/L) Glucose (g/L) pH
Acetone (g/L) Acetate (g/L)
H2 content in biogas (%) Butyrate (g/L)
Ethanol (g/L)
Biomass (g/L) Butanol (g/L)
Xylose (g/L)
Fig. 4. BioH2 and ABE fermentation performance at different mixed sugar concentrations (2080 g/L). The ratio of the glucose and xylose concentration was 6.5:1. (Operating
conditions: temperature: 37 C; initial pH: 6.0; controlled nal pH: 4.5; agitation rate: 200 rpm.)
Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 227
Table 1
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different glucose concentrations.
H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(mL/L) glucose) (acetone:butanol:ethanol) production (g/L) glucose) productivity (g/L/h)
Table 2
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different xylose concentrations.
H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(mL/L) xylose) (acetone:butanol:ethanol) production (g/L) xylose) productivity (g/L/h)
Table 3
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different total mixed sugar concentrations with a glucose-to-xylose ratio of 5:1.
Glucose Xylose H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(%) (%) (mL/L) sugar) (acetone:butanol:ethanol) production (g/L) glucose) productivity (g/L/h)
In addition, the dependences of gas product (bio-H2 ) and solvent The effects of various xylose concentrations on cell growth
product (bio-butanol and total solvent production) were evaluated and product formation rates were also investigated, with the
by the MichaelisMenten (MM) model (Eq. (2)) [24]: model simulation results shown in Figs. 5 and 6, respectively.
The Monod-type model tted the cell growth data with an R2
vmax S value of 0.96, while the MM model also ts the data well
v= (2)
Km + S (R2 = 0.920.97). The parameters (max and KS ) were 0.18 h1 and
4.21 g/L, respectively (Table 5). The vmax and Km values for H2
where v represents the specic product production rate (L/h/L for production were 0.45 L/h/L and 6.29 g/L, for solvent production
H2 , and g/h/L for liquid production), vmax represents the maximum 0.77 g/h/L and 45.43 g/L, and for butanol production 0.63 g/h/L and
specic product production rate (L/h/L, g/h/L for butanol or g/h/L 57.73 g/L, respectively (Table 5).
for solvent), and Km represents the Michaelis constant (g/L). The growth kinetics of C. acetobutylicum ATCC824 on mixed sug-
The dependence of cell growth and product production rates ars at a glucose-to-xylose ratios of 5:1 and 6.5:1 are shown in
on glucose concentration is shown in Figs. 5 and 6, respectively. Fig. 5, while simulations with the Monod-type model gave max
The estimated parameters for the Monod-type model (max and values of 0.31 h1 and 0.34 h1 , respectively, which are quite simi-
KS ) are 0.35 h1 and 7.43 g/L, respectively (Table 5). Furthermore, lar (Table 5). Kinetic modeling with the MM model also shows that
the MM type model ts the experimental results for the kinetics the bio-H2 and ABE production performances with the two glucose
of H2 , solvent, and butanol with an R2 value of 0.95 (Fig. 6a), 0.97 to xylose ratios were also very similar in terms of the maximum
(Fig. 6b) and 0.99 (Fig. 6c). The vmax and Km values for H2 production production rate (i.e., vmax ) and the dynamic trends (as represented
are 1.25 L/h/L and 23.12 g/L, for solvent production 1.22 g/h/L and by the Km value) (Table 5). Nevertheless, the vmax is slightly higher
58.91 g/L, and for butanol production 0.93 g/h/g VSS and 65.28 g/L, when a higher glucose to xylose ratio is used. These results indicate
respectively.
Table 4
BioH2 and ABE production performance of Clostridium acetobutylicum ATCC824 at different total xylose concentrations with a glucose-to-xylose ratio of 6.5:1.
Glucose Xylose H2 accumulation Yield (mol/mol Solvent production (g/L) Maximum butanol Yield (mol/mol Overall
(%) (%) (mL/L) sugar) (acetone:butanol:ethanol) production (g/L) glucose) productivity (g/L/h)
Table 5
Kinetics of cell growth, bioH2 production, and butanol production with Clostridium acetobutylicum ATCC824 using different carbon sources.
Carbon source Monod type model MichaelisMenten model for MichaelisMenten model for MichaelisMenten model for
H2 production butanol production solvent production
max (h1 ) KS (g/L) R2 max (L/h/L) Km (g/L) R2 max (g/h/L) Km (g/L) R2 max (g/h/L) Km (g/L) R2
Glucose 0.35 7.43 0.96 1.25 23.12 0.95 0.93 65.28 0.99 1.22 58.91 0.97
Xylose 0.18 4.21 0.96 0.45 6.29 0.97 0.63 57.73 0.94 0.77 45.43 0.96
Mixed (G/X = 5.0) 0.31 4.54 0.99 0.83 13.38 0.98 0.89 59.25 0.99 0.97 66.76 0.96
Mixed (G/X = 6.5) 0.34 4.67 0.99 0.94 16.11 0.96 0.90 62.16 0.99 1.00 66.33 0.97
Table 6
Electron mass balances (as mg COD) according to the results using glucose, xylose and mixed sugars.
Carbon source Substrate CODin CODres CODSMP Cell yield (g cell/g CODBio CODH2 CODsum Missing electron
conc. (g/L) (mg COD)a (mg COD)b (mg COD)c substrate) (mg COD)d (mg COD)e (mg COD)f equivalents (%)g
Mixed sugar (G:X = 5.0) 20 2133 303 1114 0.15 489 158 2065 3.2
40 4267 403 2801 0.09 628 391 4223 1.0
60 6400 723 3797 0.07 724 507 5751 10.1
80 8533 1242 4682 0.07 875 556 7355 13.8
Mixed sugar (G:X = 6.5) 20 2133 255 1075 0.15 489 207 2027 5.0
40 4267 282 2753 0.10 681 333 4049 5.1
60 6400 592 3707 0.08 828 495 5622 12.2
80 8533 1079 4525 0.07 857 504 6966 18.4
a
CODin : mg COD of initial sugar, calculated with sugar concentration (mg COD/L) 0.1 L.
b
CODres : mg COD of residual sugar, calculated with CODin,glucose (1 glucose conversion) + CODin,xylose (1 xylose conversion).
c
CODSMP : mg COD of soluble microbial products (SMP).
d
CODBio : mg COD of biomass in the reactor, calculated with mg cell 1.63 mg COD/mg cell; assuming that cell formula is C5 H7 O2 N.
e
CODH2 : mg COD of H2 evolved, calculated with mol H2 16 g COD/mol H2 1000 mg/g.
f
CODsum : mg COD of residual sugar, SMP, biomass, and H2 .
g
Calculated with (1 CODsum /CODin ) 100%.
Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230 229
Average H2 production rate (L/h/L) different cultivation stage of C. vulgaris JSC-6 (i.e., different status
1.4 Glucose
a
Xylose of nitrogen deciency) is all suitable for bio-butanol fermentation
1.2
Mixed sugar (Glucose:Xylose = 5:1) with C. acetobutylicum. The kinetic parameters determined in this
Mixed sugar (Glucose:Xylose = 6.5:1) study thus represent new information that is of great importance in
1.0 understanding the kinetic properties of producing butanol and H2
from microalgae-based carbohydrates. Nevertheless, future stud-
0.8 ies should also use the real hydrolysate of the microalgal biomass
as a carbon source for ABE fermentation, in order to conrm the
0.6 observations obtained in the current work by using a simulated
synthetic mixed sugar carbon source mimicking the microalgal car-
0.4
bohydrates.
0.2
3.5. Electron mass balance of the substrate and products
0.0
0 20 40 60 80 100 The electron mass balances (based on the level of chemical oxy-
Sugar concentration (g/L) gen demand (COD)) of the substrates and products were obtained
for all the experiments to verify whether the quantitative analyses
of the gaseous and liquid metabolites from ABE fermentation were
reasonable. The missing electron equivalent (MEE) is an indicator
Average solvent production rate (g/h/L)
0.4
This work demonstrated the feasibility of using the simulated
hydrolysate of C. vulgaris JSC-6 (primarily containing glucose and
0.2 xylose at a ratio of 5:16.5:1) as the carbon source to produce biobu-
tanol with C. acetobutylicum ATCC824. Kinetic studies show that the
maximum butanol production rate (vmax ) on glucose and xylose
0.0
alone was 0.93 and 0.63 g/h/L, respectively, indicating that glucose
0 20 40 60 80 100
is a more effective carbon source than xylose for ABE fermentation.
Sugar concentration (g/L) In the mixed sugar systems, the bio-butanol production was pre-
dominantly affected by glucose, while the utilization efciency of
xylose by C. acetobutylicum ATCC824 was quite poor (ranging from
20 to 50%).
Average butanol production rate (g/h/L)
Glucose
c
Xylose Acknowledgements
0.6 Mixed sugar (Glucose:Xylose = 5:1)
Mixed sugar (Glucose:Xylose = 6.5:1)
This work was supported by Open Project of State Key Labora-
tory of Urban Water Resource and Environment, Harbin Institute of
Technology (HCK201205). This work is also supported by National
0.4 Natural Science Foundation of China (grant nos. 51008105 and
51121062) and Academician Workstation Construction in Guang-
dong Province (2012B090500018). The authors also gratefully
acknowledged the support by Taiwans Ministry of Science and
0.2
Education (MOST) under grant numbers of 103-2221-E-006-190-
MY3 and 103-3113-E-006-006.
0.0 References
0 20 40 60 80 100
[1] C.Y. Chen, K.L. Yeh, R. Aisyah, D.J. Lee, J.S. Chang, Cultivation, photobioreactor
Sugar concentration (g/L) design and harvesting of microalgae for biodiesel production: a critical review,
Bioresour. Technol. 102 (2011) 7181.
Fig. 6. Kinetics of (a) H2 production, (b) ABE production, and (c) butanol production [2] N. Qureshi, A. Lolas, H.P. Blaschek, Soy molasses as fermentation substrate for
of Clostridium acetobutylicum ATCC824 using glucose alone (), xylose alone (), production of butanol using Clostridium beijerinckii BA101, J. Ind. Microbiol.
mixed sugar (glucose-to-xylose ratio 5:1) (), and mixed sugar (glucose-to-xylose Biotechnol. 26 (2001) 290295.
ratio = 6.5:1) () as the carbon source. (Symbols: experimental data; curves: model [3] C.L. Cheng, P.Y. Che, B.Y. Chen, W. Lee, L. Chien, J.S. Chang, High yield bio-butanol
prediction.) production by solvent-producing bacterial microora, Bioresour. Technol. 113
(2012) 5864.
[4] S.Y. Lee, J.H. Park, S.H. Jang, L.K. Nielsen, J. Kim, K.S. Jung, Fermentative butanol
production by Clostridia, Biotechnol. Bioeng. 101 (2008) 209228.
[5] Y. Jang, J. Lee, A. Malaviya, D.Y. Seung, J.H. Cho, S.Y. Lee, Butanol production
from renewable biomass: rediscovery of metabolic pathways and metabolic
engineering, Biotechnol. J. 7 (2012) 186198.
230 Y. Wang et al. / Biochemical Engineering Journal 91 (2014) 220230
[6] K. Michael, C. Held, S. Hujer, H. Liesegang, A. Wiezer, A. Wollherr, A. Ehrenreich, [18] S.H. Ho, S.W. Huang, C.Y. Chen, T. Hasunuma, A. Kondo, J.S. Chang, Bioethanol
W. Liebl, G. Gottschalk, P. Durre, Clostridium ljungdahlii represents a micro- production using carbohydrate-rich microalgae biomass as feedstock, Biore-
bial production platform based on syngas, Proc. Natl. Acad. Sci. 107 (2010) sour. Technol. 135 (2013) 191198.
1308713092. [19] G. Moxley, Y.H.P. Zhang, More accurate determination of acid-labile carbohy-
[7] P.C. Munasinghe, S.K. Khanal, Biomass-derived syngas fermentation into drates in lignocellulose by modied quantitative saccharication, Energy Fuels
biofuels: opportunities and challenges, Bioresour. Technol. 101 (2010) 21 (2007) 36843688.
50135022. [20] C.L. Cheng, J.S. Chang, Hydrolysis of lignocellulosic feedstock by novel cellulases
[8] B.P. Tracy, S.W. Jones, A.G. Fast, D.C. Indurthi, E.T. Papoutsakis, Clostridia: originating from Pseudomonas sp. CL3 for fermentative hydrogen production,
the importance of their exceptional substrate and metabolite diversity Bioresour. Technol. 102 (2011) 86288634.
for biofuel and biorenery applications, Curr. Opin. Biotechnol. 23 (2012) [21] Y.C. Lo, C. Chen, C. Lee, J.S. Chang, Photo fermentative hydrogen production
364381. using dominant components (acetate, lactate, and butyrate) in dark fermenta-
[9] D.T. Jones, D.R. Woods, Acetone-butanol fermentation revisited, Microbiol. Rev. tion efuents, Int. J. Hydrogen Energy 36 (2011) 1405914068.
50 (1986) 484. [22] C.Y. Chen, M.-H. Yang, K.-L. Yeh, J.S. Chang, Biohydrogen production using
[10] N. Qureshi, T.C. Ezeji, J. Ebener, B.S. Dien, M.A. Cotta, H.P. Blaschek, Butanol sequential dark and photo fermentation processes, Int. J. Hydrogen Energy 33
production by Clostridium beijerinckii. Part I: Use of acid and enzyme hydrolyzed (2008) 47554762.
corn ber, Bioresour. Technol. 99 (2008) 59155922. [23] G.P. de Silva, M. Mack, J. Contiero, Glycerol: a promising and abundant carbon
[11] C.L. Cheng, Y.C. Lo, K.S. Lee, D.J. Lee, C.Y. Lin, J.S. Chang, Biohydrogen production source for industrial microbiology, Biotechnol. Adv. 27 (2009) 3039.
from lignocellulosic feedstock, Bioresour. Technol. 102 (2011) 85148523. [24] C.L. Cheng, P.Y. Che, B.Y. Chen, W.J. Lee, L.J. Chien, J.S. Chang, High yield
[12] S.A. Angermayr, K.J. Hellingwerf, P. Lindblad, M.J.T. de Mattos, Energy biotech- bio-butanol production by solvent-producing bacterial microora, Bioresour.
nology with cyanobacteria, Curr. Opin. Biotechnol. 20 (2009) 257263. Technol. 113 (2012) 5864.
[13] K.J. Hellingwerf, M.J.T. De Mattos, Alternative routes to biofuels: light-driven [26] V.V. Zverlov, O. Berezina, G.A. Velikodvorskaya, W.H. Schwarz, Bacterial acetone
biofuel formation from CO2 and water based on the photanol approach, J. and butanol production by industrial fermentation in the Soviet Union: use of
Biotechnol. 142 (2009) 8790. hydrolyzed agricultural waste for biorenery, Appl. Microbiol. Biotechnol. 71
[14] C.H. Liu, C.Y. Chang, Q. Liao, X. Zhu, J.S. Chang, Biohydrogen production by a (2006) 587597.
novel integration of dark fermentation and mixotrophic microalgae cultivation, [27] Y.C. Lo, W.M. Chen, C.H. Hung, S.D. Chen, J.S. Chang, Dark H2 fermentation
Int. J. Hydrogen Energy 38 (2013) 1580715814. from sucrose and xylose using H2 producing indigenous bacteria: feasibility
[15] C.H. Liu, C.Y. Chang, Q. Liao, X. Zhu, J.S. Chang, Photoheterotrophic growth and kinetic studies, Water Res. 42 (2008) 827842.
of Chlorella vulgaris ESP6 on organic acids from dark hydrogen fermentation [28] A. Procentese, F. Raganati, G. Olivieri, M.E. Russo, P. Salatino, A. Marzocchella,
efuents, Bioresour. Technol. 145 (2013) 331336. Continuous xylose fermentation by Clostridium acetobutylicumkinetics and
[16] C.H. Liu, C.Y. Chang, C.L. Cheng, D.J. Lee, J.S. Chang, Fermentative hydrogen energetics issues under acidogenesis conditions, Bioresour. Technol. 164 (2014)
production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal 155161.
biomass as feedstock, Int. J. Hydrogen Energy 37 (2012) 1545815464. [29] E.N. Efremenko, A.B. Nikolskaya, I.V. Lyagin, O.V. Senko, T.A. Makhlis, N.A.
[17] S.H. Ho, S.W. Huang, C.Y. Chen, T. Hasunuma, A. Kondo, J.S. Chang, Charac- Stepanov, O.V. Maslova, F. Mamedova, S.D. Varfolomeev, Production of biofuels
terization and optimization of carbohydrate production from an indigenous from pretreated microalgae biomass by anaerobic fermentation with immobi-
microalga Chlorella vulgaris FSP-E, Bioresour. Technol. 135 (2013) 157165. lized Clostridium acetobutylicum cells, Bioresour. Technol. 114 (2012) 342348.