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Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum
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Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum Md. Saiful Islam a,d, Chen Zhang a, Kun-Yan Sui b, Chen Guo a,c, Chun-Zhao Liu a,b,* a State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China b School of Material Science and Engineering, Qingdao University, Qingdao 266071, PR China c Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China d University of Chinese Academy of Sciences, Beijing 100049, PR China
article info
abstract
Article history:
This study was focused on investigating the potential of hydrogen and volatile fatty acid
Received 6 July 2016
(VFA) coproduction. Sweet sorghum stalks (SS) were used as substrate along with Clostridium
Received in revised form
thermocellum and Clostridium thermosaccharolyticum as production microbes. Inoculation ratio
2 September 2016
of C. thermosaccharolyticum to C. thermocellum (0:1e1.5:1 and 1:0 v/v), substrate concentra-
Accepted 15 September 2016
tions (2.5e15.0 g/L) and inoculation time intervals of C. thermosaccharolyticum followed by
Available online xxx
C. thermocellum (0e48 h) were investigated. Experimental data showed that higher yields of hydrogen and VFA were obtained in the co-culture than their individual cultures. The
Keywords:
optimum conditions for the highest yield of products found as 1:1 inoculation ratio of both
Hydrogen
strains, 24 h of time gap between C. thermosaccharolyticum followed by C. thermocellum after
Volatile fatty acid
the first inoculation and 5 g/L of substrate concentration. The maximum yield of products
Sweet sorghum stalks
was observed as hydrogen (5.1 mmol/g-substrate), acetic acid (1.27 g/L) and butyric acid
Co-culture
(1.05 g/L) at optimum conditions. The results suggest that SS can be used for simultaneous
Fermentation
production of hydrogen and VFA employing co-culture of C. thermocellum and C. thermosaccharolyticum strains. This approach can contribute to the sustainability of biorefinery. © 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Conversion of renewable biomass for production of biofuels and high-value chemicals provides lots of advantages to the biobased economy. Biomass is the only renewable resources
which can be a raw material for the production of fuels, chemicals and materials [1]. In recent years, researchers have studied the production of hydrogen, VFA, ethanol, methane from biomass [2e6]. In the global energy infrastructure, biomass plays a vital role in the generation of power and heat along with fuels and chemicals [7]. Fermentative hydrogen
* Corresponding author. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. Fax: +86 10 82622280. E-mail address: [email protected] (C.-Z. Liu). http://dx.doi.org/10.1016/j.ijhydene.2016.09.117 0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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production from lignocelluloses is more attractive compared to other processes due to low cost involvement and simplicity to control the system [8e10]. During the fermentative production of hydrogen, VFAs are also produced along with hydrogen [11]. Hydrogen is high energy content alternative energy carrier and VFAs are the key precursors of fuels and other value added products [12,13]. Hence, production of hydrogen and VFAs from renewable biomasses is crucial. Sugars are the suitable energy sources for microbial fermentation [14] and also components of biomass substrates. Because of this, different types of renewable biomass, agricultural wastes and energy crops have been studied as raw materials for hydrogen and VFA production [15e17]. Sweet sorghum has high sugar and less lignin content compared to other lignocelluloses, which is favorable for microbial fermentation. There are only a few reports on hydrogen and VFA production from sweet sorghum bagasse and sweet sorghum juice [6,18,19], some of the materials were pretreated and some others were not. Pretreatment methods are cost intensive and time consuming. To scale-up the fermentation process from direct conversion of lignocellulosic materials without pretreatment would be attractive and cost effective. Some authors have reported fermentative hydrogen and VFAs production from lignocelluloses such as: corn stalks [20e22], agricultural wastes [23], wheat straw [24], barley straw [25], oat straw [26]. However, to the best of our knowledge, there has been no report on coproduction of hydrogen and VFA by co-culture of strains from promising sweet sorghum stalks without pretreatment. It has been demonstrated that cellulolytic bacteria, Clostridium thermocellum can degrade cellulose and hemicelluloses from lignocellulosic materials without pretreatments and produce soluble sugars along with hydrogen and acetic acid. On the otherhand, Clostridium thermosaccharolyticum convert soluble sugars into hydrogen, acetic acid and butyric acid [27,28]. So co-culture of both strains can improve the fermentation products yield by increasing the production of hydrogen and VFAs specially butyric acid, which is commercially more valuable than other VFAs [20,29,30]. The objective of this study was to explore the optimum conditions of the important parameters that affect hydrogen and VFA production via thermophilic fermentation of sweet sorghum stalk from co-culture of C. thermocellum and C. thermosaccharolyticum.
Mikroorganismen and Zellkulturen (DSMZ). The cultivation medium for both strains was CM4 with slight modification. The medium (pH 7) contained (g/L) 1.5-KH2PO4, 3.8K2HPO4$3H2O, 4.7-(NH4)2SO4, 1.6-MgCl2$6H2O, 0.013-CaCl2, 5.0-yeast extract, 1.25 103-FeSO4$7H2O, 1.0 103-resazurin and 0.5-L-cysteine HCl. The C. thermocellum was cultured for 3 days and C. thermosaccharolyticum was cultured for 24 h before used as inoculums. Both strains were cultured into a fresh CM4 medium. 5 g/L of microcrystalline cellulose and 10 g/L of sucrose were used as carbon sources for preparation of C. thermocellum and C. thermosaccharolyticum inoculums respectively.
Materials and methods
Analytical methods
Materials and chemicals
Optical density of inoculum was measured by UV-1800 spectrophotometer (AOE Instruments) at 600 nm following the procedure reported [27]. Hydrogen gas was determined by gas chromatography (Agilent GC 7890) equipped with thermal conductivity detector (TCD) and two columns (Plot Q polymer column and a molecular sieve column) separated by a switch valve. The carrier gas used was N2 at a flow rate of 23 mL/min and air was used as purging gas at 40 mL/min. The temperature of the injector, column and detector was 150, 45 and 250 C respectively. For VFA sample preparation, the liquid broth after fermentation was centrifuged for 5 min at 12,000 rpm and 0.6 mL of supernatant were collected. Then 0.2 mL of 6% (w/w) formic acid and 0.4 mL deuterium-depleted (DD) water was
Sweet sorghum stalks were collected from the Sonid Youqi Farm of Inner Mongolia, China. The sample was dried at 60 C and grinded to 1 mm powder. The main components of SS were: cellulose 22.11, hemicellulose 19.15, lignin 8.11 and total soluble sugar 32.50 (% w/w, on dry weight basis). All chemicals were analytical grade and purchased from Beijing Chemical Reagents Company (Beijing, China).
Microorganisms and culture condition C. thermocellum DSM7072 and C. thermosaccharolyticum DSM572 were obtained from the Deutsche Sammlung von
Experimental design Batch experiments for hydrogen and VFA production using sweet sorghum stalk were performed in 135 mL anaerobic glass bottle with media working volume of 50 mL. SS powder samples were added before degassing. Each bottle was sealed with a rubber stopper and screw cap. By using water circulation vacuum pump degassed the bottle and gassed by nitrogen purging. Degassed and gassed similarly three times to ensure the anaerobic condition and finally remove the gas from the anaerobic bottle by a syringe. The fermentation bottles were autoclaved at 115 C for 20 min. Then inoculums were added into the anaerobic bottles under sterile condition and kept at 55 C for fermentation. For each experiment, total inoculums were 10% by volume for both co-culture and mono-culture by C. thermocellum and C. thermosaccharolyticum with optical density at 600 nm (OD600 1.10e1.19). To optimize the inoculation ratio of bacterial strains, C. thermosaccharolyticum/C. thermocellum (v/v), 0.05:1, 0.25:1, 0.5:1, 1:1, 1.5:1, 0:1 (monoculture of C. thermocellum) and 1:0 (monoculture of C. thermosaccharolyticum) inoculums were used into CM4 medium with 10 g/L of sweet sorghum stalks. Substrate concentrations were optimized by using different concentrations of SS (2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 g/L) and to optimize C. thermocellum inoculation time, it was inoculated into the liquid culture medium after 0 (simultaneous), 12, 24, 36 and 48 h of C. thermosaccharolyticum inoculation. Each batch of experiment was conducted in triplicate and control experiments were carried out at the same time containing only medium without substrate.
Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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added to the collected sample. Formic acid was added to acidify the sample and to release carboxylic acids for GC analysis. The mixture was centrifuged at 15,000 rpm for 30 s. 1 mL of obtained liquid was filtered using 0.22 mm filter and collected into a glass GC vial. VFAs were measured by gas chromatography (Agilent GC 7890) equipped with flame ionization detector (FID) and a fused-silica capillary column (ZB-WAX) of 30 m 0.25 mm 0.25 mm. The column temperature program was 75 C for 0.25 min, increased to 130 C at the rate of 10 C/ min and further increased to 220 C at the rate of 80 C/min, 220 C for 5.7 min. The carrier gas used was N2 at a flow rate of 25 mL/min and H2 was used as fuel gas at 40 mL/min. Air was used as a purge gas at 300 mL/min. The temperature of injector and detector was 200 and 300 C respectively. The injection volume was 1 mL with a split ratio of 100:1. Cellulose, hemicellulose and lignin of sweet sorghum stalk and residual solids after fermentation were measured using the methods of Goering and Van Soest [31]. These parameters were calculated on the basis of residual total solid (TS) of fermented liquid broth and TS was determined by the methods of American Public Health Association [32]. Total sugar was determined by using the method [33] and pH of the fermented liquid was measured by pH meter. All data were presented as mean ± standard deviation.
Results and discussion Effect of the inoculation ratio of C. thermosaccharolyticum to C. thermocellum on hydrogen and VFA production In this study, hydrogen and VFA, mainly acetic acid and butyric acid were produced from raw sweet sorghum stalks. Results of the monoculture experiment of C. thermocellum and C. thermosaccharolyticum strains for hydrogen and VFA production showed that, C. thermosaccharolyticum produced (3.27 mmol/g-substrate) 4 times higher hydrogen than C. thermocellum (0.83 mmol/g-substrate) (Fig. 1A). It has been found that in the fermented liquid, C. thermosaccharolyticum produced acetic acid (1.18 g/L) and butyric acid (0.97 g/L) where as C. thermocellum only produced acetic acid (0.78 g/L) as main products VFA (Fig. 1B and C). Similar results have been reported by Li & Liu and Liu et al. [20,34] using corn stalks and microcrystalline cellulose. Studies have shown that C. thermocellum is a cellulolytic bacteria responsible for breaking down the sugar polymers (cellulose and hemicelluloses) to its monomeric unit, which is carbon source for hydrogen producing bacteria [34]. Hence, in this study both strains C. thermosaccharolyticum and C. thermocellum have
C. thermosaccharolyticum to C. thermocellum inoculation ratio
C. thermosaccharolyticum to C. thermocellum inoculation ratio
0.05:1 0.25:1 0.5:1 1:1 1.5:1 0:1 1:0
3.0
A
1.5
0.6 0.3 0.0
0
20
40
60
80
100
0
Fermentation time (h)
1.2
0.8
B
0.4
0.0
0
20
40
60
80
Fermentation time (h)
100
Soluble sugar (g/L) Acetic acid to butyric acid ratio
0.05:1 0.25:1 0.5:1 1:1 1.5:1 0:1 1:0
20
40
60
80
100
Fermentation time (h)
C. thermosaccharolyticum to C. thermocellum inoculation ratio
Acetic acid (g/L)
C
3
D Soluble sugar Acetic acid/Butyric acid Hemicellulose degradation Cellulose degradation
40
2 32 1 24
Hemicellulose degradation (%) Cellulose degradatio (%)
0.0
0.05:1 0.25:1 0.5:1 1:1 1.5:1 0:1 1:0
0.9 Butyric acid (g/L)
H2 (mmol/g-substrate)
4.5
0 0.05:1 0.25:1 0.5:1 1:1 1.5:1 Inoculation ratio of C. thermosaccharolyticum to C. thermocellum (h)
Fig. 1 e Effect of the inoculation ratio of C. thermosaccharolyticum to C. thermocellum on A. hydrogen (A), acetic acid (B), butyric acid (C) production and substrate degradation (D) with simultaneous inoculation in the co-culture process. Values are means of triplicate ± standard deviation. Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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been used to increase the yields of hydrogen and VFA. Recent studies also have shown that co-culture of cellulolytic bacteria with hydrogen-producing bacteria increased hydrogen yield from single culture [30]. Fig. 1(AeC) has shown the results of the co-culture experiments. Hydrogen, acetic acid and butyric acid yields were higher than any of the mono-culture. In all the cases, hydrogen and VFA production had reached the peak after 72 h of inoculation. With the increase of C. thermosaccharolyticum ratio from 0.05:1 to 1:1 in co-culture process both hydrogen and VFA production were increased, but acetic acid/butyric acid ratio decreased (Fig. 1D). More butyric acid production suggests the increased contribution of C. thermosaccharolyticum in the fermentation process because C. thermocellum only produced acetic acid. Geng et al. [29] also reported similar results in cellulose fermentation by co-culture of C. thermocellum and Clostridium thermopalmarium. Maximum hydrogen was produced at the inoculation ratio of 1:1, which was 4.27 mmol/g-substrate. Acetic acid and butyric acid concentrations were 1.23 and 1.02 g/L respectively in the fermented broth. Li & Liu [20] reported that 0.25:1 ratio of C. thermosaccharolyticum to C. thermocellum produced the maximum yields of hydrogen and VFA from corn stalks. Since the soluble sugar content of sweet sorghum stalk was high (32.5%) compared to other lignocellulosic materials, higher ratio of C. thermosaccharolyticum (1:1) was required to consume higher soluble sugar present. This could be the reason of increasing the yields of hydrogen and VFAs.
Butyric Acid (g/L)
3 2 20
40
60
80
100
1.0
0.0
Substrate concentration 2.5 g/L 5 g/L 7.5 g/L 10 g/L 12.5 g/L 15 g/L
B
0.5
0
20
40
60
Time (h)
0
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40
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Time (h)
8 Soluble sugar (g/L)/pH
Acetic Acid (g/L)
1.0
C
0.5
Time (h) 1.5
Substrate concentration 2.5 g/L 5 g/L 7.5 g/L 10 g/L 12.5 g/L 15 g/L
80
100
Soluble sugar D pH Hemicellulose degradation Cellulose degradation
6
48 40 32 24
4
16
2
8 0
Hemicellulose degradation (%) Cellulose degradatio (%)
H2 (mmol/g-substrate)
4
0
Optimal substrate concentration for hydrogen and VFA production was determined by using various initial substrate concentrations ranging from 2.5 g/L to 15 g/L. Recent studies show that substrate concentration has a significant effect on the conversion of lignocellulosic materials into hydrogen [30,35]. As shown in Fig. 2(AeC), maximum production of hydrogen and VFA were obtained when 5 g/L of sweet sorghum stalk was used for fermentation. At this substrate concentration, hydrogen yield was 4.91 mmol/g-substrate and the main liquid products in fermentation broth were acetic
A
7.5 g/L 15 g/L
5
1
Effect of substrate concentration to optimize the maximum conversion for co-culture of C. thermosaccharolyticum to C. thermocellum on hydrogen and VFA
1.5
Substrate concentration 2.5 g/L 5 g/L 10 g/L 12.5 g/L
6
Cellulose and hemicelluloses contents in residues after fermentation decreased while lignin content increased (lignin data not shown) in all the co-culture processes. Because cellulolytic bacteria, C. thermocellum hydrolyzed cellulose and hemicelluloses to provide soluble sugars for C. thermosaccharolyticum, this strain consumed produced sugars and increased the products yield of hydrogen and VFA. Fig. 1D showed that cellulose and hemicelluloses degradation from the fermented liquid residue also supported the results of hydrogen and VFA. There was almost no soluble sugar was detected in the end liquid. Hydrogen and VFA production increased with the increased degradation of cellulose and hemicelluloses.
3 6 9 12 15 Initial substrate concentration (g/L)
Fig. 2 e Effect of substrate concentration for co-culture of C. thermosaccharolyticum and C. thermocellum on hydrogen (A), acetic acid (B) and butyric acid (C) production, and substrate degradation (D) with simultaneous inoculation. Values are means of triplicate ± standard deviation. Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
B
0.5
0.0
Soluble sugar (g/L) Acetic acid to butyric acid ratio
3
2
1
0 12 24 36 48 Co-culture time of C. thermosaccharolyticum followed by C. thermocellum (h) Soluble sugar C Acetic acid/Butyric acid Hemicellulose degradation Cellulose degradation
48
40
32
24 0
0 12 24 36 48 Co-culture time of C. thermosaccharolyticum followed by C. thermocellum (h)
Fig. 3 e Effect of inoculation time of C. thermosaccharolyticum followed by C. thermocellum on hydrogen (A), VFA production (B) and end liquid (C) in the co-culture process. Values are means of triplicate ± standard deviation.
acid and butyric acid and the maximum yield recorded was 0.91 g/L and 0.70 g/L respectively. Fig. 2(AeC) has also shown that, the products yield of hydrogen and VFA were decreased when the initial substrate concentration increased from 5 g/L to 15 g/L and decreased at the initial substrate concentration of less than 5 g/L. Previous studies have reported that higher substrate concentration
Table 1 e Comparison of hydrogen and VFA production performance using various lignocellulosic substrate.
1.0
[30] [41] [42] [43] [34] This study 1.5 ~0.07 1.8e2 0.64 ~0.16 1.05 2.03 ~0.17 1.8e2 1.51 ~0.17 1.27 10.4 3.23e3.48 8.1 2.97 1.8 5.10
Acetic Acid Butyric Acid
Hemicellulose degradation (%) Cellulose degradatio (%)
VFA Concentration (g/L)
1.5
37 60 38 37 60 55
0 12 48 24 36 Co-culture time of C. thermosaccharolyticum followed by C. thermocellum (h)
12 5 10 5 5 5
0
Microcrystalline cellulose Corn stalk/wheat straw, corn cob Microcrystalline cellulose Avicel Microcrystalline cellulose Sweet sorghum stalk
2
5
C. acetobuyilicum X9 and C. thermocellum B9 T. thermosaccharolyticum M18 with M2, M13 and W16 C. acetobutylicum X9 and Ethanoigenens harbinense B49 Enterococcus gallinarum G1 and Ethanoigenens harbinense B49 C. thermocellum JN4 and T. thermosaccharolyticum GD17 C. thermosaccharolyticum DSM572 and C. thermocellum DSM7072
4
Substrate
A
Microorganism
H2 Yield (mmol/g-substrate)
6
Substrate Temperature H2 yield Acetic acid Butyric acid Reference concentration (g/L) ( C) (mmol/g) (g/L) (g/L)
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Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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could not produce higher hydrogen probably as a result of higher hydrogen partial pressure [36]. Researchers also have shown that high initial substrate concentration led to the accumulation of by-products and might have led to unfavorable thermodynamic state of the culture, which inhibited further substrate degradation [37]. With increase in initial substrate concentrations, VFA concentration increased in fermentation broth along with produced hydrogen partial pressure. This increased acidic conditions decreased the pH of fermented liquid (Fig. 2D) and hence it may also inhibit further growth of the strains and as a result slowed down the degradation of substrate [38,39], because both strains are neutrophilic in nature (they are working at the pH range of 5e8). Likewise, relatively low hydrogen and VFA yields were detected for the initial substrate concentration of less than 5 g/L. This might be because of cellular respiration and growth of microbes [36]. Fig. 2D has shown that, almost complete degradation of soluble sugars occurred at the initial substrate concentration of 2.5 g/L to 10 g/L after that at the end liquid there was nondegraded soluble sugar. It has also shown that cellulose and hemicellulose degradation decreased when the initial substrate concentration increased, which has also supported the experimental results.
Optimization of inoculation time gap between C. thermosaccharolyticum followed by C. thermocellum To establish the effect of inoculation time on hydrogen and VFA production, C. thermosaccharolyticum was inoculated into the liquid culture medium followed by the inoculation of C. thermocellum at different time intervals of 0 (simultaneous), 12, 24, 36 and 48 h. C. thermosaccharolyticum was inoculated first and then C. thermocellum. Inoculation time gap between C. thermocellum after C. thermosaccharolyticum of 0e24 h, yields of hydrogen and VFA were increased with inoculation time interval and at more than this time gap yields were decreased (Fig. 3A and B). Fig. 3A has also shown that the maximum hydrogen yield was 5.1 mmol/g-substrate in the co-culture process of C. thermocellum inoculation after 24 h of C. thermosaccharolyticum inoculation. At this time, acetic acid and butyric acid concentration in the liquid broth were 1.27 and 1.05 g/L respectively (Fig. 3B). Though hydrogen production was not significantly increased, the increment of acid production was significant, and one of our goals was to increase the production of commercially more valuable butyric acid. With time, more butyric acid was produced than hydrogen and acetic acid possibly because of hydrogen consuming pathway to produce butyric acid suggested by Prins [40]. This results also supported by other report [36], due to higher hydrogen partial pressure; butyric acid was produced by consuming hydrogen. Because of same reason, with inoculation time after 24 h, butyric acid has decreased less than the other yields. In Fig. 3C, it has shown that acetic acid/butyric acid ratio decreased with time, it is also supported the above mentioned results. Because sweet sorghum stalks have high soluble sugar (32.5%), after consumption of most of this sugar by C. thermosaccharolyticum by 24 h, C. thermocellum has been added and it started to cell growth by using a little amount of sugars then
degraded cellulose and hemicelluloses to produce more soluble sugars along with hydrogen and VFA. Li & Liu [20] also reported the similar results. Fig. 3C has also shown that, almost all soluble sugars were degraded and maximum cellulose and hemicelluloses degradation occurred when inoculation time gap was 24 h between two strains, resulted in higher hydrogen and VFA. The hydrogen and VFA results by co-culture experiment were comparable to that reported in other studies (Table 1). The results have little differences in yields. This could be due to differences in bacterial strain, medium and fermentation condition.
Conclusions This study demonstrates the optimization of the key factors that affect hydrogen and VFA (acetic acid and butyric acid) production from sweet sorghum stalks by the co-culture of C. thermosaccharolyticum DSM572 and C. thermocellum DSM7072 strains. To facilitate a better understanding and scale-up the technology, optimization of the process parameters is necessary. The highest hydrogen yield was 5.1 mmol/g-substrate and the end products in the fermentation liquid were acetic acid and butyric acid, which were 1.27 g/L and 1.05 g/L respectively. The optimum conditions were 1:1 inoculation ratio of both strains, inoculation of C. thermosaccharolyticum followed by C. thermocellum after 24 h of first inoculation and concentration of substrate 5 g/L SS. The results suggest that sweet sorghum stalks have potential to co-production of hydrogen and VFA from co-culture of C. thermosaccharolyticum and C. thermocellum and also shows the applicability of sweet sorghum stalk as a feedstock of lignocellulosic biorefinery.
Acknowledgments This work was financially supported by the National Basic Research Program (973 Program) of China (No. 2013CB733600), the National Natural Science Foundation of China (No. 21476242), the National Key Technology Research and Development Program of China (No. 2015BAK45B01), the Quality Inspection public Welfare Program of China (No. 201410020), and CAS-TWAS President’s Fellowship for International PhD Students.
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Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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[23] Guo XM, Trably E, Latrille E, Carrere H, Steyer J-P. Hydrogen production from agricultural waste by dark fermentation: a review. Int J Hydrogen Energy 2010;35:10660e73. [24] Kongjan P, O-Thong S, Kotay M, Min B, Angelidaki I. Biohydrogen production from wheat straw hydrolysate by dark fermentation using extreme thermophilic mixed culture. Biotechnol Bioeng 2010;105(5):899e908. [25] Panagiotopoulos I, Bakker R, de Vrije T, Claassen P, Koukios E. Dilute-acid pretreatment of barley straw for biological hydrogen production using Caldicellulosiruptor saccharolyticus. Int J Hydrogen Energy 2012;37:11727e34. [26] Arriaga S, Rosas I, Alatriste-Mondragon F, Razo-Flores E. Continuous production of hydrogen from oat straw hydrolysate in a biotrickling filter. Int J Hydrogen Energy 2011;36:3442e9. [27] Cheng XY, Liu CZ. Hydrogen production via thermophilic fermentation of cornstalk by Clostridium thermocellum. Energy Fuels 2011;25:1714e20. [28] Levin DB, Carere CR, Cicek N, Sparling R. Challenges for biohydrogen production via direct lignocellulose fermentation. Int J Hydrogen Energy 2009;34:7390e403. [29] Geng A, He Y, Qian C, Yan X, Zhou Z. Effect of key factors on hydrogen production from cellulose in a co-culture of Clostridium thermocellum and Clostridium thermopalmarium. Bioresour Technol 2010;101:4029e33. [30] Bao H, Chen C, Jiang L, Liu Y, Shen M, Liu W, et al. Optimization of key factors affecting biohydrogen production from microcrystalline cellulose by the co-culture of Clostridium acetobutylicum X 9 þ Ethanoigenens harbinense B2. RSC Adv 2016;6:3421e7. [31] Goering HK, Van-Soest PJ. Forage fiber analyses, apparatus, reagents, procedures and some applications. Agricultural handbook No. 379. Washington DC, USA: USDA; 1970. [32] APHA. Standard methods for the examination of water and wastewater. 20th ed. Washington DC, USA: American Public Health Association, American Water Works Association and Water Environment Federation; 1998. [33] Gerhardt P, Murray RGE, Wood WA, Krieg NR. Methods for general and molecular bacteriology. 1994. p. 518. ASM, Washington DC, ISBN 1-55581-048-9. [34] Liu Y, Yu P, Song X, Qu Y. Hydrogen production from cellulose by co-culture of Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17. Int J Hydrogen Energy 2008;33:2927e33. [35] Alibardi L, Cossu R. Composition variability of the organic fraction of municipal solid waste and effects on hydrogen and methane production potentials. Waste Manag 2015;36:147e55. [36] Qiu C, Zheng Y, Zheng J, Liu Y, Xie C, Sun L. Mesophilic and thermophilic biohydrogen production from Xylose at various initial pH and substrate concentrations with microflora community analysis. Energy Fuels 2016;30:1013e9. [37] Rodriguez J, Kleerebezem R, Lema JM, Van Loosdrecht MCM. Modeling product formation in anaerobic mixed culture fermentations. Biotechnol Bioeng 2006;93:592e606. [38] Lin CY, Hung CH, Chen CH, Chung WT, Cheng LH. Effects of initial cultivation pH on fermentative hydrogen production from xylose using natural mixed cultures. Process Biochem 2006;41:1383e90. [39] Chen W, Chen S, Kumarkhanal S, Sung S. Kinetic study of biological hydrogen production by anaerobic fermentation. Int J Hydrogen Energy 2006;31:2170e8. [40] Prins RA. Biochemical activities of gut microorganisms. In: Clarke RTJ, Bauchop T, editors. Microbial ecology of the gut. New York: Academic Press; 1977. p. 73e184. [41] Cao GL, Zhao L, Wang AJ, Wang ZY, Ren NQ. Single-step bioconversion of lignocellulose to hydrogen using novel
Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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moderately thermophilic bacteria. Biotechnol Biofuels 2014;7:82. [42] WANG A. Bioaugmented hydrogen production from microcrystalline cellulose using co-culturedClostridium acetobutylicum X9 and Ethanoigenens harbinense B49. Int J Hydrogen Energy 2008;33:912e7.
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Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum Md. Saiful Islam a,d, Chen Zhang a, Kun-Yan Sui b, Chen Guo a,c, Chun-Zhao Liu a,b,* a State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China b School of Material Science and Engineering, Qingdao University, Qingdao 266071, PR China c Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China d University of Chinese Academy of Sciences, Beijing 100049, PR China
article info
abstract
Article history:
This study was focused on investigating the potential of hydrogen and volatile fatty acid
Received 6 July 2016
(VFA) coproduction. Sweet sorghum stalks (SS) were used as substrate along with Clostridium
Received in revised form
thermocellum and Clostridium thermosaccharolyticum as production microbes. Inoculation ratio
2 September 2016
of C. thermosaccharolyticum to C. thermocellum (0:1e1.5:1 and 1:0 v/v), substrate concentra-
Accepted 15 September 2016
tions (2.5e15.0 g/L) and inoculation time intervals of C. thermosaccharolyticum followed by
Available online xxx
C. thermocellum (0e48 h) were investigated. Experimental data showed that higher yields of hydrogen and VFA were obtained in the co-culture than their individual cultures. The
Keywords:
optimum conditions for the highest yield of products found as 1:1 inoculation ratio of both
Hydrogen
strains, 24 h of time gap between C. thermosaccharolyticum followed by C. thermocellum after
Volatile fatty acid
the first inoculation and 5 g/L of substrate concentration. The maximum yield of products
Sweet sorghum stalks
was observed as hydrogen (5.1 mmol/g-substrate), acetic acid (1.27 g/L) and butyric acid
Co-culture
(1.05 g/L) at optimum conditions. The results suggest that SS can be used for simultaneous
Fermentation
production of hydrogen and VFA employing co-culture of C. thermocellum and C. thermosaccharolyticum strains. This approach can contribute to the sustainability of biorefinery. © 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Conversion of renewable biomass for production of biofuels and high-value chemicals provides lots of advantages to the biobased economy. Biomass is the only renewable resources
which can be a raw material for the production of fuels, chemicals and materials [1]. In recent years, researchers have studied the production of hydrogen, VFA, ethanol, methane from biomass [2e6]. In the global energy infrastructure, biomass plays a vital role in the generation of power and heat along with fuels and chemicals [7]. Fermentative hydrogen
* Corresponding author. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China. Fax: +86 10 82622280. E-mail address: [email protected] (C.-Z. Liu). http://dx.doi.org/10.1016/j.ijhydene.2016.09.117 0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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production from lignocelluloses is more attractive compared to other processes due to low cost involvement and simplicity to control the system [8e10]. During the fermentative production of hydrogen, VFAs are also produced along with hydrogen [11]. Hydrogen is high energy content alternative energy carrier and VFAs are the key precursors of fuels and other value added products [12,13]. Hence, production of hydrogen and VFAs from renewable biomasses is crucial. Sugars are the suitable energy sources for microbial fermentation [14] and also components of biomass substrates. Because of this, different types of renewable biomass, agricultural wastes and energy crops have been studied as raw materials for hydrogen and VFA production [15e17]. Sweet sorghum has high sugar and less lignin content compared to other lignocelluloses, which is favorable for microbial fermentation. There are only a few reports on hydrogen and VFA production from sweet sorghum bagasse and sweet sorghum juice [6,18,19], some of the materials were pretreated and some others were not. Pretreatment methods are cost intensive and time consuming. To scale-up the fermentation process from direct conversion of lignocellulosic materials without pretreatment would be attractive and cost effective. Some authors have reported fermentative hydrogen and VFAs production from lignocelluloses such as: corn stalks [20e22], agricultural wastes [23], wheat straw [24], barley straw [25], oat straw [26]. However, to the best of our knowledge, there has been no report on coproduction of hydrogen and VFA by co-culture of strains from promising sweet sorghum stalks without pretreatment. It has been demonstrated that cellulolytic bacteria, Clostridium thermocellum can degrade cellulose and hemicelluloses from lignocellulosic materials without pretreatments and produce soluble sugars along with hydrogen and acetic acid. On the otherhand, Clostridium thermosaccharolyticum convert soluble sugars into hydrogen, acetic acid and butyric acid [27,28]. So co-culture of both strains can improve the fermentation products yield by increasing the production of hydrogen and VFAs specially butyric acid, which is commercially more valuable than other VFAs [20,29,30]. The objective of this study was to explore the optimum conditions of the important parameters that affect hydrogen and VFA production via thermophilic fermentation of sweet sorghum stalk from co-culture of C. thermocellum and C. thermosaccharolyticum.
Mikroorganismen and Zellkulturen (DSMZ). The cultivation medium for both strains was CM4 with slight modification. The medium (pH 7) contained (g/L) 1.5-KH2PO4, 3.8K2HPO4$3H2O, 4.7-(NH4)2SO4, 1.6-MgCl2$6H2O, 0.013-CaCl2, 5.0-yeast extract, 1.25 103-FeSO4$7H2O, 1.0 103-resazurin and 0.5-L-cysteine HCl. The C. thermocellum was cultured for 3 days and C. thermosaccharolyticum was cultured for 24 h before used as inoculums. Both strains were cultured into a fresh CM4 medium. 5 g/L of microcrystalline cellulose and 10 g/L of sucrose were used as carbon sources for preparation of C. thermocellum and C. thermosaccharolyticum inoculums respectively.
Materials and methods
Analytical methods
Materials and chemicals
Optical density of inoculum was measured by UV-1800 spectrophotometer (AOE Instruments) at 600 nm following the procedure reported [27]. Hydrogen gas was determined by gas chromatography (Agilent GC 7890) equipped with thermal conductivity detector (TCD) and two columns (Plot Q polymer column and a molecular sieve column) separated by a switch valve. The carrier gas used was N2 at a flow rate of 23 mL/min and air was used as purging gas at 40 mL/min. The temperature of the injector, column and detector was 150, 45 and 250 C respectively. For VFA sample preparation, the liquid broth after fermentation was centrifuged for 5 min at 12,000 rpm and 0.6 mL of supernatant were collected. Then 0.2 mL of 6% (w/w) formic acid and 0.4 mL deuterium-depleted (DD) water was
Sweet sorghum stalks were collected from the Sonid Youqi Farm of Inner Mongolia, China. The sample was dried at 60 C and grinded to 1 mm powder. The main components of SS were: cellulose 22.11, hemicellulose 19.15, lignin 8.11 and total soluble sugar 32.50 (% w/w, on dry weight basis). All chemicals were analytical grade and purchased from Beijing Chemical Reagents Company (Beijing, China).
Microorganisms and culture condition C. thermocellum DSM7072 and C. thermosaccharolyticum DSM572 were obtained from the Deutsche Sammlung von
Experimental design Batch experiments for hydrogen and VFA production using sweet sorghum stalk were performed in 135 mL anaerobic glass bottle with media working volume of 50 mL. SS powder samples were added before degassing. Each bottle was sealed with a rubber stopper and screw cap. By using water circulation vacuum pump degassed the bottle and gassed by nitrogen purging. Degassed and gassed similarly three times to ensure the anaerobic condition and finally remove the gas from the anaerobic bottle by a syringe. The fermentation bottles were autoclaved at 115 C for 20 min. Then inoculums were added into the anaerobic bottles under sterile condition and kept at 55 C for fermentation. For each experiment, total inoculums were 10% by volume for both co-culture and mono-culture by C. thermocellum and C. thermosaccharolyticum with optical density at 600 nm (OD600 1.10e1.19). To optimize the inoculation ratio of bacterial strains, C. thermosaccharolyticum/C. thermocellum (v/v), 0.05:1, 0.25:1, 0.5:1, 1:1, 1.5:1, 0:1 (monoculture of C. thermocellum) and 1:0 (monoculture of C. thermosaccharolyticum) inoculums were used into CM4 medium with 10 g/L of sweet sorghum stalks. Substrate concentrations were optimized by using different concentrations of SS (2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 g/L) and to optimize C. thermocellum inoculation time, it was inoculated into the liquid culture medium after 0 (simultaneous), 12, 24, 36 and 48 h of C. thermosaccharolyticum inoculation. Each batch of experiment was conducted in triplicate and control experiments were carried out at the same time containing only medium without substrate.
Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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added to the collected sample. Formic acid was added to acidify the sample and to release carboxylic acids for GC analysis. The mixture was centrifuged at 15,000 rpm for 30 s. 1 mL of obtained liquid was filtered using 0.22 mm filter and collected into a glass GC vial. VFAs were measured by gas chromatography (Agilent GC 7890) equipped with flame ionization detector (FID) and a fused-silica capillary column (ZB-WAX) of 30 m 0.25 mm 0.25 mm. The column temperature program was 75 C for 0.25 min, increased to 130 C at the rate of 10 C/ min and further increased to 220 C at the rate of 80 C/min, 220 C for 5.7 min. The carrier gas used was N2 at a flow rate of 25 mL/min and H2 was used as fuel gas at 40 mL/min. Air was used as a purge gas at 300 mL/min. The temperature of injector and detector was 200 and 300 C respectively. The injection volume was 1 mL with a split ratio of 100:1. Cellulose, hemicellulose and lignin of sweet sorghum stalk and residual solids after fermentation were measured using the methods of Goering and Van Soest [31]. These parameters were calculated on the basis of residual total solid (TS) of fermented liquid broth and TS was determined by the methods of American Public Health Association [32]. Total sugar was determined by using the method [33] and pH of the fermented liquid was measured by pH meter. All data were presented as mean ± standard deviation.
Results and discussion Effect of the inoculation ratio of C. thermosaccharolyticum to C. thermocellum on hydrogen and VFA production In this study, hydrogen and VFA, mainly acetic acid and butyric acid were produced from raw sweet sorghum stalks. Results of the monoculture experiment of C. thermocellum and C. thermosaccharolyticum strains for hydrogen and VFA production showed that, C. thermosaccharolyticum produced (3.27 mmol/g-substrate) 4 times higher hydrogen than C. thermocellum (0.83 mmol/g-substrate) (Fig. 1A). It has been found that in the fermented liquid, C. thermosaccharolyticum produced acetic acid (1.18 g/L) and butyric acid (0.97 g/L) where as C. thermocellum only produced acetic acid (0.78 g/L) as main products VFA (Fig. 1B and C). Similar results have been reported by Li & Liu and Liu et al. [20,34] using corn stalks and microcrystalline cellulose. Studies have shown that C. thermocellum is a cellulolytic bacteria responsible for breaking down the sugar polymers (cellulose and hemicelluloses) to its monomeric unit, which is carbon source for hydrogen producing bacteria [34]. Hence, in this study both strains C. thermosaccharolyticum and C. thermocellum have
C. thermosaccharolyticum to C. thermocellum inoculation ratio
C. thermosaccharolyticum to C. thermocellum inoculation ratio
0.05:1 0.25:1 0.5:1 1:1 1.5:1 0:1 1:0
3.0
A
1.5
0.6 0.3 0.0
0
20
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100
0
Fermentation time (h)
1.2
0.8
B
0.4
0.0
0
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Fermentation time (h)
100
Soluble sugar (g/L) Acetic acid to butyric acid ratio
0.05:1 0.25:1 0.5:1 1:1 1.5:1 0:1 1:0
20
40
60
80
100
Fermentation time (h)
C. thermosaccharolyticum to C. thermocellum inoculation ratio
Acetic acid (g/L)
C
3
D Soluble sugar Acetic acid/Butyric acid Hemicellulose degradation Cellulose degradation
40
2 32 1 24
Hemicellulose degradation (%) Cellulose degradatio (%)
0.0
0.05:1 0.25:1 0.5:1 1:1 1.5:1 0:1 1:0
0.9 Butyric acid (g/L)
H2 (mmol/g-substrate)
4.5
0 0.05:1 0.25:1 0.5:1 1:1 1.5:1 Inoculation ratio of C. thermosaccharolyticum to C. thermocellum (h)
Fig. 1 e Effect of the inoculation ratio of C. thermosaccharolyticum to C. thermocellum on A. hydrogen (A), acetic acid (B), butyric acid (C) production and substrate degradation (D) with simultaneous inoculation in the co-culture process. Values are means of triplicate ± standard deviation. Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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been used to increase the yields of hydrogen and VFA. Recent studies also have shown that co-culture of cellulolytic bacteria with hydrogen-producing bacteria increased hydrogen yield from single culture [30]. Fig. 1(AeC) has shown the results of the co-culture experiments. Hydrogen, acetic acid and butyric acid yields were higher than any of the mono-culture. In all the cases, hydrogen and VFA production had reached the peak after 72 h of inoculation. With the increase of C. thermosaccharolyticum ratio from 0.05:1 to 1:1 in co-culture process both hydrogen and VFA production were increased, but acetic acid/butyric acid ratio decreased (Fig. 1D). More butyric acid production suggests the increased contribution of C. thermosaccharolyticum in the fermentation process because C. thermocellum only produced acetic acid. Geng et al. [29] also reported similar results in cellulose fermentation by co-culture of C. thermocellum and Clostridium thermopalmarium. Maximum hydrogen was produced at the inoculation ratio of 1:1, which was 4.27 mmol/g-substrate. Acetic acid and butyric acid concentrations were 1.23 and 1.02 g/L respectively in the fermented broth. Li & Liu [20] reported that 0.25:1 ratio of C. thermosaccharolyticum to C. thermocellum produced the maximum yields of hydrogen and VFA from corn stalks. Since the soluble sugar content of sweet sorghum stalk was high (32.5%) compared to other lignocellulosic materials, higher ratio of C. thermosaccharolyticum (1:1) was required to consume higher soluble sugar present. This could be the reason of increasing the yields of hydrogen and VFAs.
Butyric Acid (g/L)
3 2 20
40
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1.0
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Substrate concentration 2.5 g/L 5 g/L 7.5 g/L 10 g/L 12.5 g/L 15 g/L
B
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Time (h)
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Time (h)
8 Soluble sugar (g/L)/pH
Acetic Acid (g/L)
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C
0.5
Time (h) 1.5
Substrate concentration 2.5 g/L 5 g/L 7.5 g/L 10 g/L 12.5 g/L 15 g/L
80
100
Soluble sugar D pH Hemicellulose degradation Cellulose degradation
6
48 40 32 24
4
16
2
8 0
Hemicellulose degradation (%) Cellulose degradatio (%)
H2 (mmol/g-substrate)
4
0
Optimal substrate concentration for hydrogen and VFA production was determined by using various initial substrate concentrations ranging from 2.5 g/L to 15 g/L. Recent studies show that substrate concentration has a significant effect on the conversion of lignocellulosic materials into hydrogen [30,35]. As shown in Fig. 2(AeC), maximum production of hydrogen and VFA were obtained when 5 g/L of sweet sorghum stalk was used for fermentation. At this substrate concentration, hydrogen yield was 4.91 mmol/g-substrate and the main liquid products in fermentation broth were acetic
A
7.5 g/L 15 g/L
5
1
Effect of substrate concentration to optimize the maximum conversion for co-culture of C. thermosaccharolyticum to C. thermocellum on hydrogen and VFA
1.5
Substrate concentration 2.5 g/L 5 g/L 10 g/L 12.5 g/L
6
Cellulose and hemicelluloses contents in residues after fermentation decreased while lignin content increased (lignin data not shown) in all the co-culture processes. Because cellulolytic bacteria, C. thermocellum hydrolyzed cellulose and hemicelluloses to provide soluble sugars for C. thermosaccharolyticum, this strain consumed produced sugars and increased the products yield of hydrogen and VFA. Fig. 1D showed that cellulose and hemicelluloses degradation from the fermented liquid residue also supported the results of hydrogen and VFA. There was almost no soluble sugar was detected in the end liquid. Hydrogen and VFA production increased with the increased degradation of cellulose and hemicelluloses.
3 6 9 12 15 Initial substrate concentration (g/L)
Fig. 2 e Effect of substrate concentration for co-culture of C. thermosaccharolyticum and C. thermocellum on hydrogen (A), acetic acid (B) and butyric acid (C) production, and substrate degradation (D) with simultaneous inoculation. Values are means of triplicate ± standard deviation. Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
B
0.5
0.0
Soluble sugar (g/L) Acetic acid to butyric acid ratio
3
2
1
0 12 24 36 48 Co-culture time of C. thermosaccharolyticum followed by C. thermocellum (h) Soluble sugar C Acetic acid/Butyric acid Hemicellulose degradation Cellulose degradation
48
40
32
24 0
0 12 24 36 48 Co-culture time of C. thermosaccharolyticum followed by C. thermocellum (h)
Fig. 3 e Effect of inoculation time of C. thermosaccharolyticum followed by C. thermocellum on hydrogen (A), VFA production (B) and end liquid (C) in the co-culture process. Values are means of triplicate ± standard deviation.
acid and butyric acid and the maximum yield recorded was 0.91 g/L and 0.70 g/L respectively. Fig. 2(AeC) has also shown that, the products yield of hydrogen and VFA were decreased when the initial substrate concentration increased from 5 g/L to 15 g/L and decreased at the initial substrate concentration of less than 5 g/L. Previous studies have reported that higher substrate concentration
Table 1 e Comparison of hydrogen and VFA production performance using various lignocellulosic substrate.
1.0
[30] [41] [42] [43] [34] This study 1.5 ~0.07 1.8e2 0.64 ~0.16 1.05 2.03 ~0.17 1.8e2 1.51 ~0.17 1.27 10.4 3.23e3.48 8.1 2.97 1.8 5.10
Acetic Acid Butyric Acid
Hemicellulose degradation (%) Cellulose degradatio (%)
VFA Concentration (g/L)
1.5
37 60 38 37 60 55
0 12 48 24 36 Co-culture time of C. thermosaccharolyticum followed by C. thermocellum (h)
12 5 10 5 5 5
0
Microcrystalline cellulose Corn stalk/wheat straw, corn cob Microcrystalline cellulose Avicel Microcrystalline cellulose Sweet sorghum stalk
2
5
C. acetobuyilicum X9 and C. thermocellum B9 T. thermosaccharolyticum M18 with M2, M13 and W16 C. acetobutylicum X9 and Ethanoigenens harbinense B49 Enterococcus gallinarum G1 and Ethanoigenens harbinense B49 C. thermocellum JN4 and T. thermosaccharolyticum GD17 C. thermosaccharolyticum DSM572 and C. thermocellum DSM7072
4
Substrate
A
Microorganism
H2 Yield (mmol/g-substrate)
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Substrate Temperature H2 yield Acetic acid Butyric acid Reference concentration (g/L) ( C) (mmol/g) (g/L) (g/L)
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Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117
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could not produce higher hydrogen probably as a result of higher hydrogen partial pressure [36]. Researchers also have shown that high initial substrate concentration led to the accumulation of by-products and might have led to unfavorable thermodynamic state of the culture, which inhibited further substrate degradation [37]. With increase in initial substrate concentrations, VFA concentration increased in fermentation broth along with produced hydrogen partial pressure. This increased acidic conditions decreased the pH of fermented liquid (Fig. 2D) and hence it may also inhibit further growth of the strains and as a result slowed down the degradation of substrate [38,39], because both strains are neutrophilic in nature (they are working at the pH range of 5e8). Likewise, relatively low hydrogen and VFA yields were detected for the initial substrate concentration of less than 5 g/L. This might be because of cellular respiration and growth of microbes [36]. Fig. 2D has shown that, almost complete degradation of soluble sugars occurred at the initial substrate concentration of 2.5 g/L to 10 g/L after that at the end liquid there was nondegraded soluble sugar. It has also shown that cellulose and hemicellulose degradation decreased when the initial substrate concentration increased, which has also supported the experimental results.
Optimization of inoculation time gap between C. thermosaccharolyticum followed by C. thermocellum To establish the effect of inoculation time on hydrogen and VFA production, C. thermosaccharolyticum was inoculated into the liquid culture medium followed by the inoculation of C. thermocellum at different time intervals of 0 (simultaneous), 12, 24, 36 and 48 h. C. thermosaccharolyticum was inoculated first and then C. thermocellum. Inoculation time gap between C. thermocellum after C. thermosaccharolyticum of 0e24 h, yields of hydrogen and VFA were increased with inoculation time interval and at more than this time gap yields were decreased (Fig. 3A and B). Fig. 3A has also shown that the maximum hydrogen yield was 5.1 mmol/g-substrate in the co-culture process of C. thermocellum inoculation after 24 h of C. thermosaccharolyticum inoculation. At this time, acetic acid and butyric acid concentration in the liquid broth were 1.27 and 1.05 g/L respectively (Fig. 3B). Though hydrogen production was not significantly increased, the increment of acid production was significant, and one of our goals was to increase the production of commercially more valuable butyric acid. With time, more butyric acid was produced than hydrogen and acetic acid possibly because of hydrogen consuming pathway to produce butyric acid suggested by Prins [40]. This results also supported by other report [36], due to higher hydrogen partial pressure; butyric acid was produced by consuming hydrogen. Because of same reason, with inoculation time after 24 h, butyric acid has decreased less than the other yields. In Fig. 3C, it has shown that acetic acid/butyric acid ratio decreased with time, it is also supported the above mentioned results. Because sweet sorghum stalks have high soluble sugar (32.5%), after consumption of most of this sugar by C. thermosaccharolyticum by 24 h, C. thermocellum has been added and it started to cell growth by using a little amount of sugars then
degraded cellulose and hemicelluloses to produce more soluble sugars along with hydrogen and VFA. Li & Liu [20] also reported the similar results. Fig. 3C has also shown that, almost all soluble sugars were degraded and maximum cellulose and hemicelluloses degradation occurred when inoculation time gap was 24 h between two strains, resulted in higher hydrogen and VFA. The hydrogen and VFA results by co-culture experiment were comparable to that reported in other studies (Table 1). The results have little differences in yields. This could be due to differences in bacterial strain, medium and fermentation condition.
Conclusions This study demonstrates the optimization of the key factors that affect hydrogen and VFA (acetic acid and butyric acid) production from sweet sorghum stalks by the co-culture of C. thermosaccharolyticum DSM572 and C. thermocellum DSM7072 strains. To facilitate a better understanding and scale-up the technology, optimization of the process parameters is necessary. The highest hydrogen yield was 5.1 mmol/g-substrate and the end products in the fermentation liquid were acetic acid and butyric acid, which were 1.27 g/L and 1.05 g/L respectively. The optimum conditions were 1:1 inoculation ratio of both strains, inoculation of C. thermosaccharolyticum followed by C. thermocellum after 24 h of first inoculation and concentration of substrate 5 g/L SS. The results suggest that sweet sorghum stalks have potential to co-production of hydrogen and VFA from co-culture of C. thermosaccharolyticum and C. thermocellum and also shows the applicability of sweet sorghum stalk as a feedstock of lignocellulosic biorefinery.
Acknowledgments This work was financially supported by the National Basic Research Program (973 Program) of China (No. 2013CB733600), the National Natural Science Foundation of China (No. 21476242), the National Key Technology Research and Development Program of China (No. 2015BAK45B01), the Quality Inspection public Welfare Program of China (No. 201410020), and CAS-TWAS President’s Fellowship for International PhD Students.
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Please cite this article in press as: Islam MdS, et al., Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.09.117