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An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 4 ) 1 e6
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An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate Lei Zhao a, Guang-Li Cao a,b,**, Ai-Jie Wang a, Hong-Yu Ren a, Nan-Qi Ren a,* a
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China b School of Life Science and Technology, Harbin Institute of Technology, Harbin 150090, China
abstract Keywords:
Anaerobic sequencing batch reactor (ASBR) process offers great potential for H2 production
Hydrogen production
from wastewaters. In this study, an ASBR was used at first time for enhanced continuous
Thermoanaerobacterium thermo-
H2 production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium
saccharolyticum W16
thermosaccharolyticum W16. The reactor was operated at different hydraulic retention times
Cornstalk hydrolysate
(HRTs) of 6, 12, 18, and 24 h by keeping the influent hydrolysate constant at
Anaerobic sequential batch reactor
65 mmol sugars L1. Results showed that increasing the HRT from 6 to 12 h led to the H2
Hydraulic retention time
production rate increased from 6.7 to the maximum of 9.6 mmol H2 L1 h1 and the substrate conversion reached 90.3%, although the H2 yield remained at the same level of 1.7 mol H2 mol1 substrate. Taking into account both H2 production and substrate utilization efficiencies, the optimum HRT for continuous H2 production via an ASBR was determined at 12 h. Compared with other continuous H2 production processes, ASBR yield higher H2 production at relatively lower HRT. ASBR is shown to be another promising process for continuous fermentative H2 production from lignocellulosic biomass. Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Concerns about the excessive development of fossil fuels, as well as the related environmental problems caused by their usage, have promoted research for the production of alternative fuels [1]. Hydrogen gas (H2) is regarded as a promising alternative because it is an inexhaustible, clean, and renewable energy carrier and owns the potential to eliminate all of the problems that the fossil fuels create [2]. Of all the H2
production ways, biological H2 production is one of the alternative methods which is gaining more and more attention in recent years. Bio-H2 production processes can be classified as photosynthetic bio-H2 production and anaerobic bio-H2 production [3,4]. Compared these two methods, anaerobic bio-H2 process is more favourable owning to its simpler to operate and easy to control system, higher H2 production rates, and broader substrates utilization spectra, such as renewable organic waste [5,6].
* Corresponding author. Fax: þ86 451 86282008. ** Corresponding author. State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China. Fax: þ86 451 86402695. E-mail addresses: [email protected] (G.-L. Cao), [email protected] (N.-Q. Ren). http://dx.doi.org/10.1016/j.ijhydene.2014.05.167 0360-3199/Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 4 ) 1 e6
Lignocellulosic biomass, the underutilized low value renewable resource with the largest abundance on earth, is regarded as one of the most promising substrate for anaerobic bio-H2 production [7,8]. In China, lignocellulosic biomass mainly contains residues after agricultural crop harvest, forestry and related industries, energy crops, as well as municipal waste [9]. Among them, agricultural residues, which is produced at a rate of more than 700 million tons per year accounted for the largest proportion of lignocellulose and applied the greatest potential for bio-H2 production. However, the crystallinity of cellulose and the chemical resistance of lignin gives lignocellulose its recalcitrant nature [1], restricting the efficiency of direct utilization of parent biomass. Thus, to realize the biological conversion of lignocellulosic biomass to H2, lignocellulosic biomass should be hydrolyzed to fermentable sugars (mainly pentose and hexose) by pretreatment and enzymatic hydrolysis processes at first, and then fermented by organisms which could utilize both pentose and hexose [10]. Although several species of anaerobic bacteria or microflora have been reported owns potential to produce H2 from lignocelllulosic hydrolysate, the reported rates and yields of fermentative H2 production are not high enough [10e12]. Currently, most researches focused on developing different operating modes of bioreactors, such as upflow anaerobic sludge blanket bioreactor (UASB), semicontinuous reactor, and continuous stirred tank reactor (CSTR) to cultivate anaerobic bacteria for improving H2 production [13e16]. However, the performance of bioreactors was limited by low substrate utilization, especially pentose utilization, and biomass retention capacity. As a result, for enhanced H2 production from lignocellulosic biomass hydrolysate, development of a bioreactor which could improve H2 production efficiency and make fully use of pentose and hexose contained in the hydrolysate is crucial. Anaerobic sequencing batch reactor (ASBR) holds some advantages over other systems, including relative ease of operation, flexibility, high biomass retaining, low operation costs and solid/liquid separation in a single vessel [17,18]. Consequently, ASBR has been widely used in the H2 production processes from wastewater [19,20]. However, no studies have been demonstrated that ASBR could apply into continuous H2 production process from lignocellulose hydrolysate to enhance H2 production. In addition, in the previous study, an efficient and environmentally friendly biological pretreatment and hydrolysis procedure has been established [21]. Therefore, ASBR process in this research was used to improve H2 production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium thermosaccharolyticum W16. Moreover, different operating strategies were studied in this research to explore optimal operational parameters.
cornstalk hydrolysate [21]. The microorganism was cultivated in 100 ml anaerobic bottles containing 60 ml culture medium at 60 C for 18 h with a rotation speed of 130 rpm, and then the fermentation broth was stored as inoculum.
Reactor setup and operation The schematic of the investigated ASBR unit in this research is shown in Fig. 1. The laboratory-scale ASBR was a sealed glass cylinder with a total volume of 800 ml. The reactor contained 490 ml culture medium was purged with N2 for 30 min to create anaerobic condition, after that, the reactor was airtight and autoclaved at 121 C for 15 min. By inoculating 10 ml of seed inoculum into the ASBR, the reactor was started up and operated constantly at 60 C using magnetic stirrer (DJ-1, Huanyu scientific instrument co., LTD, China) to mix the cultures well. Initially, the reactor was operated in batch mode for 24 h to accumulated biomass for continuous operation. After the batch run, the ASBR was continuously operated based on the feed, react, settle, and decant steps [23]. Digital time controller (HHQ7, Xinling Electronics Co. Ltd, China) was used for automatically controlled the feeding, decanting and settling of the ASBR. The influent and effluent of the reactor were transferred by two peristaltic pumps (BT100-1L, Baoding Longer Precision Pump Co. Ltd, China). The experimental design conditions in the ASBR system were tested according to various HRTs as shown in Table 1. Each condition was operated for several cycles until steady state (constant substrate consumption and H2 production at ±5% variation) [24] was obtained. The volume of gas produced was measured at time intervals by gas collection bag (Dalian Hyde science and technology co. Ltd, China).
Analytical method H2 content of the gas phase was determined by gas chromatography (102 G, Shanghai Analysis Instrument Company, China) using a stainless steel column (Molecular Sieve 5 Å) and thermal conductivity detector with N2 (25 ml/min) as the carrier gas. The injector, detector, and column temperatures
Material and methods Inoculum and culture medium The H2 producer T. thermosaccharolyticum W16 used in this study was isolated from hot spring sediment [22]. The culture medium was prepared as described by Zhao et al. [15] except the sole carbon source was replaced with fungal pretreated
Fig. 1 e Schematic of the ASBR process.
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 4 ) 1 e6
Table 1 e Details of ASBR operation. Parameters
Cycle period (h) Fill period (min) React period (h) Settle period (min) Decanting period (min) Fill &decanting volume (mL)
HRT (h) 6
12
18
24
3 15 2 30 15 250
6 15 5 30 15 250
9 15 8 30 15 250
12 15 11 30 15 250
were 205, 300, and 110 C, respectively [25]. The VFAs and alcohols were determined using a GC (4800, Agilent Technologies, USA) equipped with a hydrogen flame-ionization detector as described by Zhao et al. [26]. The sugars in solution before and after fermentation were separated on an Aminex HPX-87P column (Bio-Rad, Hercules, CA) at 80 C, using 0.02 M H2SO4 as eluent at a flow rate of 0.4 ml/min [27]. A gas chromatograph/mass spectrometer (GCeMS) (6890N-5973, Agilent Cooperation, USA) equipped with a DB column was adopted to detect the existence of furfural, 5-hydroxy-methyl-2-furaldehyde (HMF), and phenols in the fermentation broth [28]. The cell dry weight (CDW) of the effluent was determined from the steady state value of the optical densities at each HRT using the relation CDW (g l1) ¼ (0.258 OD600) þ 0.001 for T. thermosaccharolyticum W16.
Results and discussion
The chemical compositions of fungal pretreated cornstalk hydrolysate were shown in Table 2. Sugars in hydrolysate were oligosaccharides, and the majority was glucose, accounting for 57% of total sugars, whereas pentose (xylose and arabinose) content was a little lower, the value was 43%. No HMF, furfural, and phenols, which are inhibitory to fermenting microorganisms, were detected. Besides, 53 mg/L acetic acid was detected in the cornstalk hydrolysate. Acetic acid is a derivative during hydrolysis of hemicellulose [29], according to Cao et al. [30], acetate was not inhibitory to T. thermosaccharolyticum W16 below 1.0 g/L. As a result, the influence of inhibitors on the subsequent fermentation process could be excluded. In order to improve the H2 production efficiency from fungal pretreated cornstalk hydrolysate, making fully
Table 2 e Characteristics of fungal pretreated cornstalk hydrolysate.
Furfural (mg/L) Phenols (mg/L) HMF (mg/L) Acetate (mg/L) Glucose (g/L) Xylose (g/L) Arabinose (g/L) n.d, not detected.
utilization of both pentose and hexose (C5/C6) contained in hydrolysate was the key issue. Besides development of a suitable bioreactor, selection of appropriate microorganism is also important, T. thermosaccharolyticum W16 applied in this study has been reported owns great potential to ferment both pentose and hexose [31]. We therefore propose that the application of ASBR in continuous H2 production from fungal pretreated cornstalk hydrolysate using T. thermosaccharolyticum W16 would be advantageous to accelerate the rate of H2 production and substrate utilization.
Performance of an ASBR for continuous H2 production from cornstalk hydrolysate Various important parameters including organic loading rate, solid retention time, pH, substrate concentration, cyclic duration and hydraulic retention time (HRT) have been investigated for ASBR operation [32]. Among these, HRT is one of the most important control parameters affecting continuous production of H2 [33], with appropriate HRT, efficient H2 production could be achieved with lower operation cost, which will make the H2 production process more applicable [34]. In this research, continuous H2 production was operated by ASBR with different HRTs of 6, 12, 18, and 24 h respectively by keeping the influent cornstalk hydrolysate consistent at a sugar concentration of 65 mmol/L. Stable condition was lasted for at least 10 cycles and the HRT was then extended.
Effect of HRT on H2 production
Characteristics of fungal pretreated cornstalk hydrolysate
Characteristics
3
Value n.d. n.d. n.d. 53 ± 5 6.1 ± 0.3 3.5 ± 0.2 1.1 ± 0.09
The variation in H2 volume, H2 production rate, and H2 content along with the variation in HRTs were shown in Fig. 2. At an HRT of 12 h, H2 production rate reached peak value of 9.6 mmol H2 L1 h1, which coincided with the maximum H2 volume of 2450 ml H2 L1. A decrease of HRT to 6 h or an increase to 24 h caused H2 production rate decreased by 31% and 60%, respectively. H2 production rate represents the H2 production efficiency of the reactor, the value changed along with the variation of HRT. The marked decrease in H2 production rate at short HRT may be resulted from limited utilization of substrate and washout of H2 producing microorganism according to Chang and Lin [35]. The least biomass concentration experienced at an HRT of 6 h (Table 3) further confirmed this point of view. While at longer HRT, the low H2 production rate might be caused by the depletion of particular nutrients from the feed or the inhibitory intracellular compounds accumulated. During the whole operation process, H2 content in the gas phase was between 55 and 61% (v/v) with only CO2 as the second gas detected, and the maximum value was also achieved at HRT of 12 h (Fig. 2). Therefore, 12 h could be regarded as the optimal operating HRT based on the results obtained. In contrast, H2 yield maintained nearly constant at the HRT ranges of 6e24 h and this value was equal to previous research reported by Zhao et al. [15]. From an engineering point of view, the calculation of H2 yield is meaningful, because it indicates whether the carbohydrates in the reactor are metabolically favourable for H2 production. A constant H2 yield implies a positive correlation between the produced H2 amount and degraded substrate. Result obtained in this research demonstrates the performance of reactor was
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 4 ) 1 e6
steady, and also indicates that the H2 conversion efficiency was independent from HRT variation.
Effect of HRT on substrate utilization An economically feasible H2 production process did not only dependent on the high H2 production efficiency, but also on the efficiency of substrate utilization. Consequently, effect of HRT on substrate utilization from fungal pretreated cornstalk by T. thermosaccharolyticum W16 was investigated. The substrate utilization efficiency and soluble metabolites production under steady-state conditions at each HRT were summarized in Fig. 3 and Table 3. Complete or almost complete glucose utilization was accomplished when HRT was longer than 6 h. While xylose and arabinose utilization efficiency increased from 22% and 41% at HRT 6 h to 87% and 96% at HRT 24 h. Similar to the result observed for pentose utilization, total substrate consumption in hydrolysate increased from 41.6% to 91.5% with the variation of HRT from 6 h to 24 h. These results indicated that glucose was preferentially consumed than pentose. The increase of residual xylose and arabinose in ASBR immediately after lowering the HRT suggests that the system needs some adaptation time to xylose and arabinose loading condition, which may be causeed by “the glucose effect” [36]. Overall, little residual substrate was detected in the effluent stream except at some stages of the fermentation when the HRT was sharply decreased confirmed that the performance of ASBR is of great beneficial for sufficient substrate consumption. Consistent with the utilization of substrate, higher biomass concentrations were acquired with the yield of 0.9 g CDW L1 and 1.0 g CDW L1 at longer HRT of 18 h and 24 h, respectively (Table 3). Although, long HRT (18 and 24 h) benefitted pentose degradation, H2 production was not improved with the increase of HRT (Figs. 2 and 3), this may be attributed to the reasons mentioned above. Under all fermentation conditions, acetate and butyrate were the main fermentation products along with little amount of ethanol, butanol, and propionate. Thus, the bacterial metabolism in ASBR during these periods was following butyrateeacetate type fermentation, which is quite the same to that obtained in batch and continuous cultures [15,31]. The pH value varied between 6.1 and 4.9 with increasing HRT, related to a progressed acid fermentation at long HRT, which caused an accumulation in VFAs concentrations and then a decrease in alkalinity. In conclusion, selection of an HRT which owns high H2 production rate and reasonable substrate utilization efficiency would be very important for the operation of a bioreactor, results obtained in this research indicated the HRT of 12 h was the optimal for ASBR. Interestingly, the maximum H2 production rate of 9.6 mmol H2 L1 h1 achieved at HRT 12 h in this research was higher than values obtained in other studies using UASB of
Fig. 2 e Effect of HRTs on H2 production efficiency.
Table 3 e Fermentation profile at steady state for each HRT. HRT (h)
Acetate (mmol/L)
Butyrate (mmol/L)
6 12 18 24
19.5 ± 0.2 23.1 ± 0.1 35.6 ± 0.7 50.3 ± 0.3
13.8 15.1 19.3 24.5
± 0.1 ± 0.4 ± 0.5 ± 0.3
Ethanol (mmol/L) 6.3 ± 7.2 ± 10.6 ± 15.2 ±
0.2 0.2 0.3 0.3
Butanol (mmol/L) 0.6 ± 0.9 ± 1.2 ± 1.5 ±
0.1 0.09 0.05 0.04
Propionate (mmol/L) 0.3 0.5 0.6 1.0
± 0.01 ± 0.02 ± 0.02 ± 0.01
pH 6.1 ± 0.2 5.8 ± 0.1 5.5 ± 0.1 4.9 ± 0.1
H2 yield (mol H2 mol1substrate) 1.5 ± 1.9 ± 1.8 ± 1.6 ±
0.2 0.2 0.1 0.1
CDW (g L1) 0.4 0.7 0.9 1.0
± 0.09 ± 0.1 ± 0.1 ± 0.1
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
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utilized more sufficient. In addition, the H2 concentrations achieved in this research was slightly higher than values achieved in CSTR. It is apparent that an ASBR developed here would be a promising method for continuous H2 production from lignocellulose hydrolysate. The application of ASBR in continuous H2 production from cornstalk hydrolysate using T. thermosaccharolyticum W16 was successfully carried out in our investigation. Although the use of thermophilic operation conditions could increase the economic burden of the process, it is obvious that such conditions could avoid or minimize microbial contamination, which is a significant problem in many industrial processes [37], and maintain fermentation reactions that are favourable for H2 production [38]. As a result, an ASBR is more suitable and effective for fermentative H2 production from a harsh substrate like lignocellulose hydrolysate.
Conclusion This work demonstrated the feasibility of improving H2 production from cornstalk hydrolysate via an ASBR system using a thermophilic strain, T. thermosaccharolyticum W16. HRT displayed an important role in producing H2 continuously using an ASBR. At HRT 12 h, the H2 production rate reached the maximum of 9.6 mmol H2 L1 h1 with the H2 volume of 2450 ml H2 L1 and substrate utilization of 90.3%. ASBR yielded higher H2 production than that obtained using CSTR. Overall results showed that it is great potential to produce H2 via an ASBR. However, proper HRT control is necessary to obtain efficient H2 production.
Acknowledgements This research was supported by 1) National Natural Science Foundation of China (No. 51178140, No. 30870037, and No. 31100095), China; 2) Shanghai Tongji Gao Tingyao Environmental Science & Development Foundation, China; 3) Academician Workstation Construction in Guangdong Province (2012B090500018), China; 4) China Postdoctoral Science Foundation (20110491053), China; 5) Heilongjiang Postdoctoral Science Foundation (No. LBH-Z11133), China; 6) State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2014TS07), China. Fig. 3 e Effect of HRTs on substrate utilization efficiency.
references 1.52 mmol H2 L1 h1 from wheat straw hydrolysate [14] and semi-continuous reactor of 6.21 mmol H2 L1 h1 from cassava stillage hydrolysate [16]. Moreover, the H2 production rate in the case of ASBR is higher than that reported in our previous study of 8.4 mmol H2 L1 h1 using CSTR under the same condition [15].Compared with the system of CSTR, ASBR is more suitable for H2 production from lignocellulose hydrolysate because this process could retain microorganisms growing faster than the dilution rate, which avoids the main disadvantage of the CSTR system, so higher H2 production rate under lower HRT was achieved, and the substrate could be
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Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/he
An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate Lei Zhao a, Guang-Li Cao a,b,**, Ai-Jie Wang a, Hong-Yu Ren a, Nan-Qi Ren a,* a
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China b School of Life Science and Technology, Harbin Institute of Technology, Harbin 150090, China
abstract Keywords:
Anaerobic sequencing batch reactor (ASBR) process offers great potential for H2 production
Hydrogen production
from wastewaters. In this study, an ASBR was used at first time for enhanced continuous
Thermoanaerobacterium thermo-
H2 production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium
saccharolyticum W16
thermosaccharolyticum W16. The reactor was operated at different hydraulic retention times
Cornstalk hydrolysate
(HRTs) of 6, 12, 18, and 24 h by keeping the influent hydrolysate constant at
Anaerobic sequential batch reactor
65 mmol sugars L1. Results showed that increasing the HRT from 6 to 12 h led to the H2
Hydraulic retention time
production rate increased from 6.7 to the maximum of 9.6 mmol H2 L1 h1 and the substrate conversion reached 90.3%, although the H2 yield remained at the same level of 1.7 mol H2 mol1 substrate. Taking into account both H2 production and substrate utilization efficiencies, the optimum HRT for continuous H2 production via an ASBR was determined at 12 h. Compared with other continuous H2 production processes, ASBR yield higher H2 production at relatively lower HRT. ASBR is shown to be another promising process for continuous fermentative H2 production from lignocellulosic biomass. Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Concerns about the excessive development of fossil fuels, as well as the related environmental problems caused by their usage, have promoted research for the production of alternative fuels [1]. Hydrogen gas (H2) is regarded as a promising alternative because it is an inexhaustible, clean, and renewable energy carrier and owns the potential to eliminate all of the problems that the fossil fuels create [2]. Of all the H2
production ways, biological H2 production is one of the alternative methods which is gaining more and more attention in recent years. Bio-H2 production processes can be classified as photosynthetic bio-H2 production and anaerobic bio-H2 production [3,4]. Compared these two methods, anaerobic bio-H2 process is more favourable owning to its simpler to operate and easy to control system, higher H2 production rates, and broader substrates utilization spectra, such as renewable organic waste [5,6].
* Corresponding author. Fax: þ86 451 86282008. ** Corresponding author. State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China. Fax: þ86 451 86402695. E-mail addresses: [email protected] (G.-L. Cao), [email protected] (N.-Q. Ren). http://dx.doi.org/10.1016/j.ijhydene.2014.05.167 0360-3199/Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
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Lignocellulosic biomass, the underutilized low value renewable resource with the largest abundance on earth, is regarded as one of the most promising substrate for anaerobic bio-H2 production [7,8]. In China, lignocellulosic biomass mainly contains residues after agricultural crop harvest, forestry and related industries, energy crops, as well as municipal waste [9]. Among them, agricultural residues, which is produced at a rate of more than 700 million tons per year accounted for the largest proportion of lignocellulose and applied the greatest potential for bio-H2 production. However, the crystallinity of cellulose and the chemical resistance of lignin gives lignocellulose its recalcitrant nature [1], restricting the efficiency of direct utilization of parent biomass. Thus, to realize the biological conversion of lignocellulosic biomass to H2, lignocellulosic biomass should be hydrolyzed to fermentable sugars (mainly pentose and hexose) by pretreatment and enzymatic hydrolysis processes at first, and then fermented by organisms which could utilize both pentose and hexose [10]. Although several species of anaerobic bacteria or microflora have been reported owns potential to produce H2 from lignocelllulosic hydrolysate, the reported rates and yields of fermentative H2 production are not high enough [10e12]. Currently, most researches focused on developing different operating modes of bioreactors, such as upflow anaerobic sludge blanket bioreactor (UASB), semicontinuous reactor, and continuous stirred tank reactor (CSTR) to cultivate anaerobic bacteria for improving H2 production [13e16]. However, the performance of bioreactors was limited by low substrate utilization, especially pentose utilization, and biomass retention capacity. As a result, for enhanced H2 production from lignocellulosic biomass hydrolysate, development of a bioreactor which could improve H2 production efficiency and make fully use of pentose and hexose contained in the hydrolysate is crucial. Anaerobic sequencing batch reactor (ASBR) holds some advantages over other systems, including relative ease of operation, flexibility, high biomass retaining, low operation costs and solid/liquid separation in a single vessel [17,18]. Consequently, ASBR has been widely used in the H2 production processes from wastewater [19,20]. However, no studies have been demonstrated that ASBR could apply into continuous H2 production process from lignocellulose hydrolysate to enhance H2 production. In addition, in the previous study, an efficient and environmentally friendly biological pretreatment and hydrolysis procedure has been established [21]. Therefore, ASBR process in this research was used to improve H2 production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium thermosaccharolyticum W16. Moreover, different operating strategies were studied in this research to explore optimal operational parameters.
cornstalk hydrolysate [21]. The microorganism was cultivated in 100 ml anaerobic bottles containing 60 ml culture medium at 60 C for 18 h with a rotation speed of 130 rpm, and then the fermentation broth was stored as inoculum.
Reactor setup and operation The schematic of the investigated ASBR unit in this research is shown in Fig. 1. The laboratory-scale ASBR was a sealed glass cylinder with a total volume of 800 ml. The reactor contained 490 ml culture medium was purged with N2 for 30 min to create anaerobic condition, after that, the reactor was airtight and autoclaved at 121 C for 15 min. By inoculating 10 ml of seed inoculum into the ASBR, the reactor was started up and operated constantly at 60 C using magnetic stirrer (DJ-1, Huanyu scientific instrument co., LTD, China) to mix the cultures well. Initially, the reactor was operated in batch mode for 24 h to accumulated biomass for continuous operation. After the batch run, the ASBR was continuously operated based on the feed, react, settle, and decant steps [23]. Digital time controller (HHQ7, Xinling Electronics Co. Ltd, China) was used for automatically controlled the feeding, decanting and settling of the ASBR. The influent and effluent of the reactor were transferred by two peristaltic pumps (BT100-1L, Baoding Longer Precision Pump Co. Ltd, China). The experimental design conditions in the ASBR system were tested according to various HRTs as shown in Table 1. Each condition was operated for several cycles until steady state (constant substrate consumption and H2 production at ±5% variation) [24] was obtained. The volume of gas produced was measured at time intervals by gas collection bag (Dalian Hyde science and technology co. Ltd, China).
Analytical method H2 content of the gas phase was determined by gas chromatography (102 G, Shanghai Analysis Instrument Company, China) using a stainless steel column (Molecular Sieve 5 Å) and thermal conductivity detector with N2 (25 ml/min) as the carrier gas. The injector, detector, and column temperatures
Material and methods Inoculum and culture medium The H2 producer T. thermosaccharolyticum W16 used in this study was isolated from hot spring sediment [22]. The culture medium was prepared as described by Zhao et al. [15] except the sole carbon source was replaced with fungal pretreated
Fig. 1 e Schematic of the ASBR process.
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
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Table 1 e Details of ASBR operation. Parameters
Cycle period (h) Fill period (min) React period (h) Settle period (min) Decanting period (min) Fill &decanting volume (mL)
HRT (h) 6
12
18
24
3 15 2 30 15 250
6 15 5 30 15 250
9 15 8 30 15 250
12 15 11 30 15 250
were 205, 300, and 110 C, respectively [25]. The VFAs and alcohols were determined using a GC (4800, Agilent Technologies, USA) equipped with a hydrogen flame-ionization detector as described by Zhao et al. [26]. The sugars in solution before and after fermentation were separated on an Aminex HPX-87P column (Bio-Rad, Hercules, CA) at 80 C, using 0.02 M H2SO4 as eluent at a flow rate of 0.4 ml/min [27]. A gas chromatograph/mass spectrometer (GCeMS) (6890N-5973, Agilent Cooperation, USA) equipped with a DB column was adopted to detect the existence of furfural, 5-hydroxy-methyl-2-furaldehyde (HMF), and phenols in the fermentation broth [28]. The cell dry weight (CDW) of the effluent was determined from the steady state value of the optical densities at each HRT using the relation CDW (g l1) ¼ (0.258 OD600) þ 0.001 for T. thermosaccharolyticum W16.
Results and discussion
The chemical compositions of fungal pretreated cornstalk hydrolysate were shown in Table 2. Sugars in hydrolysate were oligosaccharides, and the majority was glucose, accounting for 57% of total sugars, whereas pentose (xylose and arabinose) content was a little lower, the value was 43%. No HMF, furfural, and phenols, which are inhibitory to fermenting microorganisms, were detected. Besides, 53 mg/L acetic acid was detected in the cornstalk hydrolysate. Acetic acid is a derivative during hydrolysis of hemicellulose [29], according to Cao et al. [30], acetate was not inhibitory to T. thermosaccharolyticum W16 below 1.0 g/L. As a result, the influence of inhibitors on the subsequent fermentation process could be excluded. In order to improve the H2 production efficiency from fungal pretreated cornstalk hydrolysate, making fully
Table 2 e Characteristics of fungal pretreated cornstalk hydrolysate.
Furfural (mg/L) Phenols (mg/L) HMF (mg/L) Acetate (mg/L) Glucose (g/L) Xylose (g/L) Arabinose (g/L) n.d, not detected.
utilization of both pentose and hexose (C5/C6) contained in hydrolysate was the key issue. Besides development of a suitable bioreactor, selection of appropriate microorganism is also important, T. thermosaccharolyticum W16 applied in this study has been reported owns great potential to ferment both pentose and hexose [31]. We therefore propose that the application of ASBR in continuous H2 production from fungal pretreated cornstalk hydrolysate using T. thermosaccharolyticum W16 would be advantageous to accelerate the rate of H2 production and substrate utilization.
Performance of an ASBR for continuous H2 production from cornstalk hydrolysate Various important parameters including organic loading rate, solid retention time, pH, substrate concentration, cyclic duration and hydraulic retention time (HRT) have been investigated for ASBR operation [32]. Among these, HRT is one of the most important control parameters affecting continuous production of H2 [33], with appropriate HRT, efficient H2 production could be achieved with lower operation cost, which will make the H2 production process more applicable [34]. In this research, continuous H2 production was operated by ASBR with different HRTs of 6, 12, 18, and 24 h respectively by keeping the influent cornstalk hydrolysate consistent at a sugar concentration of 65 mmol/L. Stable condition was lasted for at least 10 cycles and the HRT was then extended.
Effect of HRT on H2 production
Characteristics of fungal pretreated cornstalk hydrolysate
Characteristics
3
Value n.d. n.d. n.d. 53 ± 5 6.1 ± 0.3 3.5 ± 0.2 1.1 ± 0.09
The variation in H2 volume, H2 production rate, and H2 content along with the variation in HRTs were shown in Fig. 2. At an HRT of 12 h, H2 production rate reached peak value of 9.6 mmol H2 L1 h1, which coincided with the maximum H2 volume of 2450 ml H2 L1. A decrease of HRT to 6 h or an increase to 24 h caused H2 production rate decreased by 31% and 60%, respectively. H2 production rate represents the H2 production efficiency of the reactor, the value changed along with the variation of HRT. The marked decrease in H2 production rate at short HRT may be resulted from limited utilization of substrate and washout of H2 producing microorganism according to Chang and Lin [35]. The least biomass concentration experienced at an HRT of 6 h (Table 3) further confirmed this point of view. While at longer HRT, the low H2 production rate might be caused by the depletion of particular nutrients from the feed or the inhibitory intracellular compounds accumulated. During the whole operation process, H2 content in the gas phase was between 55 and 61% (v/v) with only CO2 as the second gas detected, and the maximum value was also achieved at HRT of 12 h (Fig. 2). Therefore, 12 h could be regarded as the optimal operating HRT based on the results obtained. In contrast, H2 yield maintained nearly constant at the HRT ranges of 6e24 h and this value was equal to previous research reported by Zhao et al. [15]. From an engineering point of view, the calculation of H2 yield is meaningful, because it indicates whether the carbohydrates in the reactor are metabolically favourable for H2 production. A constant H2 yield implies a positive correlation between the produced H2 amount and degraded substrate. Result obtained in this research demonstrates the performance of reactor was
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
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steady, and also indicates that the H2 conversion efficiency was independent from HRT variation.
Effect of HRT on substrate utilization An economically feasible H2 production process did not only dependent on the high H2 production efficiency, but also on the efficiency of substrate utilization. Consequently, effect of HRT on substrate utilization from fungal pretreated cornstalk by T. thermosaccharolyticum W16 was investigated. The substrate utilization efficiency and soluble metabolites production under steady-state conditions at each HRT were summarized in Fig. 3 and Table 3. Complete or almost complete glucose utilization was accomplished when HRT was longer than 6 h. While xylose and arabinose utilization efficiency increased from 22% and 41% at HRT 6 h to 87% and 96% at HRT 24 h. Similar to the result observed for pentose utilization, total substrate consumption in hydrolysate increased from 41.6% to 91.5% with the variation of HRT from 6 h to 24 h. These results indicated that glucose was preferentially consumed than pentose. The increase of residual xylose and arabinose in ASBR immediately after lowering the HRT suggests that the system needs some adaptation time to xylose and arabinose loading condition, which may be causeed by “the glucose effect” [36]. Overall, little residual substrate was detected in the effluent stream except at some stages of the fermentation when the HRT was sharply decreased confirmed that the performance of ASBR is of great beneficial for sufficient substrate consumption. Consistent with the utilization of substrate, higher biomass concentrations were acquired with the yield of 0.9 g CDW L1 and 1.0 g CDW L1 at longer HRT of 18 h and 24 h, respectively (Table 3). Although, long HRT (18 and 24 h) benefitted pentose degradation, H2 production was not improved with the increase of HRT (Figs. 2 and 3), this may be attributed to the reasons mentioned above. Under all fermentation conditions, acetate and butyrate were the main fermentation products along with little amount of ethanol, butanol, and propionate. Thus, the bacterial metabolism in ASBR during these periods was following butyrateeacetate type fermentation, which is quite the same to that obtained in batch and continuous cultures [15,31]. The pH value varied between 6.1 and 4.9 with increasing HRT, related to a progressed acid fermentation at long HRT, which caused an accumulation in VFAs concentrations and then a decrease in alkalinity. In conclusion, selection of an HRT which owns high H2 production rate and reasonable substrate utilization efficiency would be very important for the operation of a bioreactor, results obtained in this research indicated the HRT of 12 h was the optimal for ASBR. Interestingly, the maximum H2 production rate of 9.6 mmol H2 L1 h1 achieved at HRT 12 h in this research was higher than values obtained in other studies using UASB of
Fig. 2 e Effect of HRTs on H2 production efficiency.
Table 3 e Fermentation profile at steady state for each HRT. HRT (h)
Acetate (mmol/L)
Butyrate (mmol/L)
6 12 18 24
19.5 ± 0.2 23.1 ± 0.1 35.6 ± 0.7 50.3 ± 0.3
13.8 15.1 19.3 24.5
± 0.1 ± 0.4 ± 0.5 ± 0.3
Ethanol (mmol/L) 6.3 ± 7.2 ± 10.6 ± 15.2 ±
0.2 0.2 0.3 0.3
Butanol (mmol/L) 0.6 ± 0.9 ± 1.2 ± 1.5 ±
0.1 0.09 0.05 0.04
Propionate (mmol/L) 0.3 0.5 0.6 1.0
± 0.01 ± 0.02 ± 0.02 ± 0.01
pH 6.1 ± 0.2 5.8 ± 0.1 5.5 ± 0.1 4.9 ± 0.1
H2 yield (mol H2 mol1substrate) 1.5 ± 1.9 ± 1.8 ± 1.6 ±
0.2 0.2 0.1 0.1
CDW (g L1) 0.4 0.7 0.9 1.0
± 0.09 ± 0.1 ± 0.1 ± 0.1
Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167
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utilized more sufficient. In addition, the H2 concentrations achieved in this research was slightly higher than values achieved in CSTR. It is apparent that an ASBR developed here would be a promising method for continuous H2 production from lignocellulose hydrolysate. The application of ASBR in continuous H2 production from cornstalk hydrolysate using T. thermosaccharolyticum W16 was successfully carried out in our investigation. Although the use of thermophilic operation conditions could increase the economic burden of the process, it is obvious that such conditions could avoid or minimize microbial contamination, which is a significant problem in many industrial processes [37], and maintain fermentation reactions that are favourable for H2 production [38]. As a result, an ASBR is more suitable and effective for fermentative H2 production from a harsh substrate like lignocellulose hydrolysate.
Conclusion This work demonstrated the feasibility of improving H2 production from cornstalk hydrolysate via an ASBR system using a thermophilic strain, T. thermosaccharolyticum W16. HRT displayed an important role in producing H2 continuously using an ASBR. At HRT 12 h, the H2 production rate reached the maximum of 9.6 mmol H2 L1 h1 with the H2 volume of 2450 ml H2 L1 and substrate utilization of 90.3%. ASBR yielded higher H2 production than that obtained using CSTR. Overall results showed that it is great potential to produce H2 via an ASBR. However, proper HRT control is necessary to obtain efficient H2 production.
Acknowledgements This research was supported by 1) National Natural Science Foundation of China (No. 51178140, No. 30870037, and No. 31100095), China; 2) Shanghai Tongji Gao Tingyao Environmental Science & Development Foundation, China; 3) Academician Workstation Construction in Guangdong Province (2012B090500018), China; 4) China Postdoctoral Science Foundation (20110491053), China; 5) Heilongjiang Postdoctoral Science Foundation (No. LBH-Z11133), China; 6) State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2014TS07), China. Fig. 3 e Effect of HRTs on substrate utilization efficiency.
references 1.52 mmol H2 L1 h1 from wheat straw hydrolysate [14] and semi-continuous reactor of 6.21 mmol H2 L1 h1 from cassava stillage hydrolysate [16]. Moreover, the H2 production rate in the case of ASBR is higher than that reported in our previous study of 8.4 mmol H2 L1 h1 using CSTR under the same condition [15].Compared with the system of CSTR, ASBR is more suitable for H2 production from lignocellulose hydrolysate because this process could retain microorganisms growing faster than the dilution rate, which avoids the main disadvantage of the CSTR system, so higher H2 production rate under lower HRT was achieved, and the substrate could be
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Please cite this article in press as: Zhao L, et al., An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate, International Journal of Hydrogen Energy (2014), http://dx.doi.org/ 10.1016/j.ijhydene.2014.05.167