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Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria
international journal of hydrogen energy 35 (2010) 4041–4047
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Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria Maojin Cui, Zhuliang Yuan, Xiaohua Zhi, Liling Wei, Jianquan Shen* Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of New Materials, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun North First Street 2, Beijing 100190, PR China
article info
abstract
Article history:
Leaves are one of the main by-products of forestry. In this study, batch experiments were
Received 14 September 2009
carried out to convert poplar leaves pretreated by different methods into hydrogen using
Received in revised form
anaerobic mixed bacteria at 35 C. The effects of acid (HCl), alkaline (NaOH) and enzymatic
5 February 2010
(Viscozyme L, a mixture of arabanase, cellulase, b-glucanase, hemicellulase and xylanase)
Accepted 6 February 2010
pretreatments on the saccharification of poplar leaves were studied. Furthermore, the
Available online 12 March 2010
effects of acid and enzymatic pretreatment on hydrogen production, together with their corresponding degradation efficiencies for the total reducing sugar (TRS) and metabolites
Keywords:
were compared. A maximum cumulative hydrogen yield of 44.92 mL/g-dry poplar leaves
Poplar leaves
was achieved from substrate pretreated with 2% Vicozyme L, which was approximately
Pretreatment
3-fold greater than that in raw substrate and 1.34-fold greater than that from substrate
Anaerobic fermentation
pretreated with 4% HCl. The results show that enzymatic pretreatment is an effective
Biohydrogen production
method for enhancing the hydrogen yield from poplar leaves. ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Fossil fuels such as oil, coal and natural gas are the main energy sources in current society. However, the inevitable depletion of fossil fuels, combined with concerns about their impact on our environment has led to an interest in nonpolluting and alternative energy sources. In comparison with fossil fuels, hydrogen is a promising alternative fuel of the future as it is a clean energy carrier with a high energy-yield (122 kJ/g) [1,2], the highest of all known fuels, and generates no pollutants producing only water upon combustion. Cellulosic biomass from agricultural and forest residue is an abundantly renewable resource [3]. The annual yield of biomass wastes is more than 0.7 billion tons in China alone [4]. Much cellulosic biomass, such as poplar leaves, however, is burned or discarded as environmental pollutants. Therefore, the production of hydrogen from cellulosic biomass has
attracted increasing attention. Many studies on biohydrogen from cellulosic biomass have been carried out [4–15]. The conversion of raw cellulosic biomass into hydrogen directly by microorganisms, however, is very difficult due to the complicated polymer structure of hemicellulose and cellulose. To enhance hydrogen yield, therefore, it is necessary to pretreat the substrate [5]. Nowadays, the methods of pretreating cellulosic biomass mainly include steam explosion, mechanical, thermal, acid, alkaline, ammonia and oxidative treatment [4–12,16]. Acid hydrolysis is considered to be one of the most effective methods of solubilizing hemicellulose, hydrolysis products contain not only soluble sugars but also a variety of by-products, such as phenol, furan and furfural compounds, adversely inhibiting the capability of hydrogenproducing bacteria to produce hydrogen [10,17]. In addition, acid hydrolysis is commonly carried out at high temperature and corrodes equipment [18]. In contrast, enzymatic
* Corresponding author. Tel.: þ86 10 62620903; fax: þ86 10 62559373. E-mail address: [email protected] (J. Shen). 0360-3199/$ – see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.02.035
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international journal of hydrogen energy 35 (2010) 4041–4047
hydrolysis is considered to be an effective method for pretreating cellulosic materials because it can be carried out under mild conditions without corroding equipment [18]. In this study, we explored the feasibility of converting poplar leaves into hydrogen with acid and enzymatic (Viscozyme L, a mixture of arabanase, cellulase, b-glucanase, hemicellulase and xylanase) pretreatments using anaerobic mixed microbial cultures, aiming at enhancing hydrogen yield by the pretreatment of substrate. For this purpose, the changes in the total reducing sugar (TRS) with different pretreatment methods and the effects of HCl and Viscozyme L concentrations on hydrogen production and liquid metabolites were studied by anaerobic fermentation in batch cultivation. The results show that enzymatic pretreatment is better than acid pretreatment in enhancing hydrogen yield from poplar leaves. To the best of our knowledge, this is the first report of hydrogen production from poplar leaves with pretreatment using anaerobic mixed bacteria.
2.
Materials and methods
2.1.
Materials
The poplar leaves used in this study were collected in autumn from a suburb of Beijing, China. They were dried in sunlight, and were then comminuted to more than 20-mesh using a comminutor and dried again in a thermoelectric oven for 3 h at 105 C. All chemicals were analytical reagent grade except where otherwise specified.
2.2.
Seed microorganisms
The hydrogen-producing mixed cultures were enriched from cracked cereal and identified to be dominated by Clostridium pasteurianum [19]. The cultures were acclimated in a completely stirred tank reactor (CSTR) in a chemostat for approximately one month. The reactor was operated at 35 C, an 8 h hydraulic retention time, and stirred by gas circulation [20]. One liter of culture medium used to ferment contained NH4HCO3, 3770 mg; K2HPO4, 125 mg; NaHCO3, 2000 mg; CuSO4$5H2O, 5 mg; MgCl2$6H2O, 100 mg; MnSO4$4H2O, 15 mg; FeSO4$7H2O, 25 mg; CoCl2$6H2O, 0.125 mg.
2.3.
Pretreatment
2.3.1.
Acid and alkaline pretreatment
1.0 g of dry poplar leaves were mixed with 20 mL of dilute HCl (or NaOH) aqueous solution at different concentrations (0.5%, 1%, 2%, 4% and 8% (w/v), respectively) (5% (w/v) solids loading) and boiled for 30 min in serum vials. The mixture was then neutralized to pH 7.0 by the addition of dilute NaOH (or HCl) aqueous solution at different concentrations (0.5%, 1%, 2%, 4% and 8% (w/v), respectively).
2.3.2.
Enzymatic pretreatment
Viscozyme L (VL) was purchased from Novozymes (China) Biotechnology Co., Ltd. (batch number KTN02161). VL is a mixture of many enzymes, including arabanase, cellulase, b-glucanase,
hemicellulase and xylanase. The optimal working conditions for VL are pH 3.3–5.5 and temperature 25–55 C. 1.0 g of dry poplar leaves were mixed with 20 mL of VL aqueous solution at different concentrations ([VL]), pH, enzymolysis time (t) and temperatures (T ), respectively (5% (w/v) solids loading). Four series of experiments were carried out: Series 1 investigated the effect of VL concentration (0.25–4%, v/v) on the saccharification of poplar leaves; Series 2 investigated the effect of pH (3.0–7.0) on the saccharification of poplar leaves, and a buffer containing 0.1 mol/L citric acid and 0.1 mol/L trisodium citrate was used to adjust the pH; Series 3 investigated the effect of enzymolysis time (1–5 h) on the saccharification of poplar leaves; Series 4 investigated the effect of enzymolysis temperature (35–55 C) on the saccharification of poplar leaves.
2.4.
Experimental procedures
Batch experiments were carried out to produce hydrogen by anaerobic fermentation in 120 mL serum vials using poplar leaves pretreated with HCl and VL as substrate. The total working volume was 80 mL (approximately 1.25% (w/v) solids loading) in each case. (1) Acid pretreatment: approximately 40 mL of mixture after pretreatment (20 mL of dilute HCl used to pretreat substrate and approximately 20 mL of dilute NaOH used to adjust pH), 20 mL inoculum, 10 mL nutrient solution (1 L contained NH4HCO3, 30 160 mg; K2HPO4, 1000 mg; NaHCO3, 16 000 mg; CuSO4$5H2O, 40 mg; MgCl2$6H2O, 800 mg; MnSO4$4H2O, 120 mg; FeSO4$7H2O, 200 mg; CoCl2$6H2O, 1 mg), and approximately 10 mL distilled water; (2) enzymatic pretreatment: 20 mL of mixture after pretreatment, 20 mL inoculum and 40 mL nutrient solution (1 L contained NH4HCO3, 7540 mg; K2HPO4, 250 mg; NaHCO3, 4000 mg; CuSO4$5H2O, 10 mg; MgCl2$6H2O, 200 mg; MnSO4$4H2O, 30 mg; FeSO4$7H2O, 50 mg; CoCl2$6H2O, 0.25 mg). The air was removed from the solution and the headspace by argon gas for 3 min before the vials were capped with rubber septum stoppers and placed in a reciprocal shaker (120 rpm). The batch experiments were performed at 35 C in the dark. Each experimental condition was carried out in triplicate.
2.5.
Analytical methods
The hydrogen content was determined by a gas chromatograph (Techcomp. Co., China, 7890II) equipped with a thermal conductivity detector (TCD) and a 2-m stainless steel column packed with Porapak Q (80–100 mesh). The operating temperatures of the injection port, the oven and the detector were set at 70, 50 and 70 C, respectively. Argon was used as the carrier gas at a flow rate of 30 mL/min. At each time interval, the total volume of biogas production was measured by a plunger displacement method using appropriately sized glass syringes, ranging from 10 to 100 mL [21]. The cumulative hydrogen volume was calculated by Eq. (1) [22], VH;i ¼ VH;i1 þ CH;i VG;i VG;i1 þ VH;0 CH;i CH;i1
(1)
where VH, i and VH, i1 are cumulative hydrogen volumes at the current (i) and previous (i 1) time intervals, VG, i and VG, i1 are the total biogas volumes in the current (i) and previous (i 1) time intervals, CH, i and CH, i1 are the fraction of
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hydrogen in the headspace of the bottle at the current (i) and previous (i 1) time intervals and VH,0 is the total volume of headspace in the bottle. Detection of the alcohols and volatile fatty acids (VFAs, C2–C5) was measured by a gas chromatograph using a flame ionization detector (FID) and a 2-m glass column packed with Unisole F-200 (30–60 mesh). The temperatures of the injection port, the oven and the detector were set at 200, 165 and 200 C, respectively. The carrier gas was also argon at a flow rate of 30 mL/min. The concentration of the total reducing sugar was determined by the phenol– sulfuric acid method using glucose as a standard [23].
2.6.
Model analysis
Content of the total reducing sugar (%)
international journal of hydrogen energy 35 (2010) 4041–4047
HCl NaOH
30
25
20
15
10
5
0
The cumulative hydrogen production during the batch experiments followed the modified Gompertz equation [24,25]: Rm e H ¼ P exp ðl tÞ þ 1 P
(2)
Where H is the cumulative hydrogen production (mL), P is hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL/h), e is 2.72, l is the lag-phase time (h), and t is the incubation time (h). The corresponding values of P, Rm and l for each batch were estimated using Origin 7.5, which is a scientific graphing and data analysis software.
3.
Results and discussion
3.1. Effect of pretreatment on the saccharification of poplar leaves 3.1.1. Effects of HCl and NaOH pretreatment on the saccharification of poplar leaves Acid pretreatment mainly converts hemicellulose in the cellulosic biomass into xylose [26], which is the main component of hemicellulose [27]. HCl was less active than sulfuric acid in the degradation of xylose, which is the feedstock for producing hydrogen by bacteria [28]. Therefore, HCl was used to pretreat the substrate. The TRS contents from 1.0 g of dry poplar leaves after HCl and NaOH pretreatment were analyzed and are depicted in Fig. 1. As shown in Fig. 1, the TRS content continuously increased with increasing HCl and NaOH concentrations. When comparing the contents of the TRS from substrate pretreated with 4% and 8% HCl (or NaOH), the increment was found to be small. The amounts of TRSs with 4% HCl and 4% NaOH pretreatment were approximately 2.33-fold and 1.66-fold greater compared with that in the raw substrate. It was noted that the TRS content with HCl pretreatment was always higher than that with NaOH pretreatment at the same HCl and NaOH concentrations. These results indicate that HCl pretreatment was superior to NaOH pretreatment, consistent with previous studies [4,5].
3.1.2. Effect of enzymatic pretreatment on the saccharification of poplar leaves Enzyme concentration, pH value, enzymolysis time and temperature are the main factors that affect the saccharification
UP
0.5%
1%
2%
4%
8%
HCl and NaOH concentrations (w/v)
Fig. 1 – Effects of HCl and NaOH concentrations on the saccharification of poplar leaves. UP means unpretreatment. of poplar leaves. Therefore, the effects of VL concentration, pH, enzymolysis time and temperature on the contents of the TRS from 1.0 g of dry poplar leaves were studied. The results are shown in Table 1. The TRS content continuously increased when the VL concentration increased from 0.25% to 1% but the increment was small when the VL concentration increased from 1% to 4%. Therefore, 1% VL was used to investigate the effect of pH on the saccharification of poplar leaves. The TRS content continuously decreased with increasing pH. The optimal pH was in the range of 3.0–4.0. Therefore, the effect of enzymolysis time on the saccharification of poplar leaves was studied at pH 4.0.
Table 1 – Effects of VL concentration, pH, enzymolysis time and enzymolysis temperature on the saccharification of poplar leaves. [VL] (v/v)
pH
t (h)
T ( C)
CS (%)
0.25% 0.5% 1% 2% 4%
4.0
3
50
18.51 20.27 25.07 25.33 26.03
1%
3.0 4.0 5.0 6.0 7.0
3
50
25.75 25.07 23.01 22.51 22.39
1%
4.0
1 2 3 4 5
50
22.34 23.92 25.07 25.25 26.00
1%
4.0
3
35 40 45 50 55
18.93 20.03 21.79 25.07 24.63
‘CS’ means the content of the total reducing sugar.
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international journal of hydrogen energy 35 (2010) 4041–4047
The TRS content continuously increased with the increasing enzymolysis time. The increment, however, was small when the enzymolysis time exceeded 3 h. The effect of enzymolysis temperature on the saccharification of poplar leaves was also studied using an enzymolysis time of 3 h. The TRS content initially increased then decreased with increasing enzymolysis temperature, and the optimal temperature was 50 C. According to the above results, the optimal conditions for saccharification of poplar leaves were [VL] ¼ 1%, pH ¼ 3.0–4.0, t ¼ 3 h and T ¼ 50 C. To summarize, the TRS content from pretreated poplar leaves was higher than that from raw poplar leaves, indicating that pretreatment is very necessary for further saccharification of poplar leaves. The TRS contents after enzymatic pretreatment were lower than those after HCl pretreatment but higher than those after NaOH pretreatment. Therefore, poplar leaves pretreated by HCl and VL were used as a substrate in the fermentation to produce hydrogen.
3.2.
Effect of pretreatment on hydrogen production
3.2.1.
Effect of HCl pretreatment on hydrogen production
The effects of HCl concentration on the cumulative hydrogen volumes are presented in Fig. 2. 1.0 g of dry poplar leaves pretreated with different HCl concentrations were used as the substrate to produce hydrogen at an initial pH of 7.0. It can be seen from Fig. 2 that the cumulative hydrogen volume from raw poplar leaves was 15.04 mL but that the cumulative hydrogen volumes continued to increase (27.56–33.45 mL) when the HCl concentration increased (0.5–4%). The maximum hydrogen volume of 33.45 mL was observed at 4% HCl, which was 2.22-fold higher than that from the raw substrate. To further investigate the effect of HCl concentration on hydrogen production, the data in Fig. 2 were simulated using Eq. (2) and the hydrogen production characteristics are shown in Table 2. According to the data in Table 2, all R2 values were more than 0.990, indicating that the fitted curves matched well with the experimental points. The specific hydrogen
Table 2 – Hydrogen production characteristics at different HCl concentrations. HCl (w/v)
UP 0.5% 1% 2% 4%
25
15 10
R2
15.04 27.56 28.44 29.14 33.45
1.25 2.74 1.73 1.53 1.18
3.60 11.90 10.00 9.40 8.76
0.9992 0.9994 0.9993 0.9979 0.9937
production rate continuously decreased with increasing HCl concentration (0.5–4%), possibly because HCl pretreatment produced some inhibitors such as furfural that inhibited the specific hydrogen production rate of hydrogen-producing bacteria [29]. The maximum specific hydrogen production rate of 2.74 mL/h was observed at 0.5% HCl. The shortest duration of 3.60 h occurred with the raw substrate, because the microorganisms used here were acclimated to raw poplar leaves. The duration continuously decreased with increasing HCl concentration (0.5–4%).
3.2.2. Effect of enzymatic pretreatment on hydrogen production 1.0 g of dry poplar leaves pretreated with various VL concentrations at optimal pretreatment conditions (pH ¼ 4.0, t ¼ 3 h, T ¼ 50 C) were used as substrate to produce hydrogen at an initial pH of 7.0. The effects of VL concentration on the cumulative hydrogen volumes are presented in Fig. 3. As shown in Fig. 3, the cumulative hydrogen volumes initially increased then decreased with increasing VL concentration, suggesting that excessive VL was unfavorable to hydrogen production. The maximum hydrogen volume of 44.92 mL was observed at 2%, which was 2.99-fold higher than that from the
UP 0.25% 0.5% 1% 2% 4%
40
20
l (h)
P is hydrogen production potential (mL). Rm is the maximum hydrogen production rate (mL/h). l is the lag-phase time (h). R2 is correlation coefficient.
Cumulative hydrogen (mL)
Cumulative hydrogen (mL)
30
Rm (mL/h)
0 0.5% 1% 2% 4%
50 35
P (mL)
30
20
10
5 0
0 0
20
40
60
80
Time (hour)
Fig. 2 – Cumulative hydrogen volumes from 1.0 g of dry poplar leaves pretreated by different HCl concentrations versus corresponding fermentation time. The operation was at 35 8C and initial pH 7.0.
0
10
20
30
40
50
60
70
Time (hour)
Fig. 3 – Cumulative hydrogen volumes from 1.0 g of dry poplar leaves pretreated by different VL concentrations versus corresponding fermentation time. The operation was at 35 8C and initial pH 7.0.
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international journal of hydrogen energy 35 (2010) 4041–4047
Table 3 – Hydrogen production characteristics at different VL concentrations. VL (v/v) 0 0.25% 0.5% 1% 2% 4%
P (mL)
Rm (mL/h)
l (h)
R2
15.04 27.32 33.13 36.90 44.92 35.47
1.25 1.85 2.14 2.17 1.97 1.32
3.60 8.04 7.71 7.88 7.98 7.13
0.9992 0.9922 0.9938 0.9957 0.9922 0.9863
raw substrate and better than that from pretreated substrate with 4% HCl, indicating that enzymatic pretreatment was better than HCl pretreatment. To further investigate the effect of VL concentration on hydrogen production, the data in Fig. 3 were simulated using Eq. (2) and the hydrogen production characteristics are shown in Table 3. According to the data in Table 3, all R2 values were more than 0.986, indicating that the fitted curves matched well with the experimental points. The specific hydrogen production rate initially increased and then decreased with increasing VL concentration, suggesting that excessive VL was unfavorable to enhance the specific hydrogen production rate. The maximum specific hydrogen production rate of 2.17 mL/h was observed at 1%. The shortest duration of 3.60 h occurred with the raw substrate, possibly due to the same reason given above. The changes in duration with poplar leaves pretreated with different VL concentrations were small with increasing VL concentration (0.25–4%). To summarize, the cumulative hydrogen volume from poplar leaves with pretreatment was higher than that from raw poplar leaves, indicating that pretreatment is necessary for increasing hydrogen production. The result was consistent with previous studies, which are summarized in Table 4. As shown in Table 4, the maximum hydrogen yields from pretreated substrates were much higher than hydrogen yields from raw substrates. The TRS content from the pretreated substrate with 4% HCl was higher than that from the pretreated substrate with 2% VL but the former cumulative hydrogen volume was lower than the latter. This is possibly because HCl pretreatment produced some inhibitors [29], inhibiting the hydrogen-producing bacteria from producing hydrogen using sugar.
3.3.
Effect of pretreatment on metabolites
Gas product analyses showed that only H2 and CO2 were present in the biogas, without any detectable CH4 during
the course of hydrogen production, suggesting that there were no methanogens in the anaerobic mixed bacteria used here. The effects of HCl and VL concentrations on the sugar degradation efficiency, H2 contents, H2 yields, and liquid metabolites are shown in Table 5. The sugar degradation efficiency exceeded 92%, indicating that most of the sugar was consumed by the bacteria, and the changes were small with increasing HCl and VL concentrations. Hydrogen contents in the biogas varied from 28.80% to 32.51% with a change in HCl concentration from 0.5% to 4% and the divergence was also small. The maximum of 32.51% was observed at 1% HCl. Hydrogen contents in the biogas varied from 26.92% to 46.08% with a change in VL concentration from 0.25% to 4% and the maximum of 46.08% was observed at 0.5% VL. The liquid product analysis showed that the metabolites found after fermentation were ethanol, acetic acid, propionic acid and butyric acid. It should be noted that high yields of ethanol and VFAs were obtained from the raw poplar leaves but a relatively low cumulative hydrogen yield (15.04 mL/g-dry poplar leaves) was produced. This is possibly because some of the hemicellulose and cellulose in raw poplar leaves was consumed in the production of ethanol and VFAs [26]. When the HCl concentration increased from 0.5% to 4%, the yields of acetic acid and propionic acid decreased gradually, while ethanol and n-butyric acid initially increased then decreased. A maximum of four liquid metabolites appeared at 2% VL. The possible metabolic pathway of soluble sugar (mainly glucose) to produce hydrogen at the maximum hydrogen yield (2% VL pretreatment) is shown as Eq. (3):
C6H12O6 / 1.315H2 þ 1.873CO2 þ 0.086CH3CH2OH þ 0.552CH3COOH þ 0.125CH3CH2COOH þ 0.074CH3CH2CH2COOH
(3)
As shown in Eq. (3), the metabolic pathway was mainly acetic acid fermentation, and the conversion efficiency of H element in glucose into H2 was 22%. The mechanism of hydrogen production from glucose and xylose by bacterial fermentation has been reported previously [30–32]. Theoretically the metabolic pathways of the four liquid products (ethanol, acetic acid, propionic acid and nbutyric acid) from xylose and glucose are as follows: C5H10O5 / 1.67CH3CH2OH þ 1.67CO2
(4)
C5H10O5 þ 1.67H2O / 1.67CH3COOH þ 3.33H2 þ 1.67CO2
(5)
Table 4 – Biohydrogen production from various cellulosic biomasses with different pretreatment methods. Feedstock Poplar leaves Beer lees Cornstalk Wheat straw Corn stalk
Pretreatment method
H2 yield from raw material
Maximum H2 yield from pretreated substrate
Reference
Viscozyme L HCl HCl HCl-Microwave Biopretreatment
15.04 mL/g 3.16 mL/g 3.16 mL/g-TVS 0.5 mL/g-TVS 20 mL/g-TS
44.92 mL/g 53.03 mL/g 149.69 mL/g-TVS 68.1 mL/g-TVS 176 mL/g-TS
This study [26] [4] [6] [11]
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international journal of hydrogen energy 35 (2010) 4041–4047
Table 5 – Effects of HCl and VL concentrations on the degradation efficiency of total reducing sugar, H2 contents, H2 yields and yields of ethanol and VFAs after fermentation. DESa (%)
H2 contentb (%)
H2 yield (mL/g-S)
Ethanol (mg/g-S)c
Acetic acid (mg/g-S)
Propionic acid (mg/g-S)
n-Butyric acid (mg/g-S)
UP
0
93.28
26.89
15.04
4.20
26.10
9.28
6.06
HCl (w/v)
0.5% 1% 2% 4%
94.98 95.81 94.68 92.44
30.09 32.51 28.80 30.06
27.56 28.44 29.14 33.45
2.27 4.02 5.49 4.87
17.31 16.01 15.96 9.15
5.76 5.16 2.64 2.17
2.21 2.81 1.38 0.94
VL (v/v)
0.25% 0.5% 1% 2% 4%
95.48 96.03 96.03 95.76 95.82
39.25 46.08 40.37 41.25 26.92
27.32 33.13 36.90 44.92 35.47
3.41 4.31 4.30 5.31 4.18
29.72 36.01 34.80 44.68 35.80
5.45 5.30 6.73 12.42 9.63
2.93 3.00 4.40 8.78 5.01
a DES means degradation efficiency of total reducing sugar. b H2 content means percentage of the total cumulative hydrogen produced to total biogas. c (mg/g-S) means (mg/g-dry poplar leaves).
C5H10O5 þ 1.67H2 / 1.67CH3CH2COOH þ 1.67H2O
(6)
C5H10O5 / 0.83CH3(CH2)2COOH þ 1.67H2 þ 1.67CO2
(7)
C6H12O6 / 2CH3CH2OH þ 2CO2
(8)
pretreatment. Therefore, enzymatic pretreatment is an ideal method for increasing the hydrogen yield from poplar leaves by anaerobic fermentation.
Acknowledgments The authors would like to thank the Chinese Academy of Sciences for financial support (Item No. KJCX2-YW-H21). C6H12O6þ2H2O / 2CH3COOH þ 4H2 þ 2CO2
(9)
references C6H12O6 þ 2H2 / 2CH3CH2COOH þ 2H2O
C6H12O6 / CH3(CH2)2COOH þ 2H2 þ 2CO2
(10)
(11)
According to Eqs. (4)–(11), the production of ethanol has no influence on the production of hydrogen whereas the production of acetic and n-butyric acid favors the production of hydrogen. In contrast, the production of propionic acid resulted in less hydrogen production. According to Eq. (3), it was mainly acetic acid fermentation, so it favored the production of hydrogen.
4.
Conclusions
In this work, three different methods, acid (HCl), alkaline (NaOH) and enzymatic treatment, were used to pretreat poplar leaves. The TRS content in pretreated substrate was higher than that in raw substrate, indicating that pretreatment is required for further saccharification of substrate. The TRS contents after enzymatic pretreatment were lower than those after HCl pretreatment but higher than those after NaOH pretreatment. Nevertheless, the maximum cumulative hydrogen yield (44.92 mL/g-dry poplar leaves) from substrate pretreated by 2% VL was higher than that from substrate pretreated by 4% HCl (33.45 mL/g-dry poplar leaves), indicating that enzymatic pretreatment was better than HCl
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[21] Owen WF, Stuckey DC, Healy JB, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 1979;13:485–93. [22] Logan BE, Oh SE, Kim IS, Ginkel SV. Biological hydrogen production measured in batch anaerobic respirometers. Environ Sci Technol 2002;36:2530–5. [23] Dubios M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28:350–6. [24] Zwietering MH, Jongenburger I, Rombouts FM, Riet KV. Modeling of the bacterial-growth curve. Appl Environ Microbiol 1990;56:1875–81. [25] Fang HHP, Li C, Zhang T. Acidophilic biohydrogen production from rice slurry. Int J Hydrogen Energy 2006;31:683–92. [26] Cui M, Yuan Z, Zhi X, Shen J. Optimization of biohydrogen production from beer lees using anaerobic mixed bacteria. Int J Hydrogen Energy 2009;34:7971–8. [27] Thomsen AB, Thygesen A, Bohn V, Nielsen KV, Pallesen B, Jorgensen MS. Effects of chemical–physical pre-treatment processes on hemp fibres for reinforcement of composites and for textiles. Ind Crops Prod 2006;24(2):113–8. [28] Lavarack BP, Griffin GJ, Rodman D. The acid hydrolysis of sugarcane bagasse hemicellulose to produce xylose, arabinose, glucose and other products. Biomass Bioenergy 2002;23:367–80. [29] Laser M, Schulman D, Allen SG, Lichwa J, Antal MJ, Lynd LR. A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol. Bioresour Technol 2002;81:33–44. [30] Lin CY, Lay CH. Carbon/nitrogen-ratio effect on fermentative hydrogen production by mixed microflora. Int J Hydrogen Energy 2004;29:41–5. [31] Zhu Y, Yang ST. Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum. J Biotechnol 2004;110:143–57. [32] Kongjan P, Min B, Angelidaki I. Biohydrogen production from extreme thermophilic temperatures (70 C) by mixed culture fermentation. Water Res 2009;43:1414–24.
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Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria Maojin Cui, Zhuliang Yuan, Xiaohua Zhi, Liling Wei, Jianquan Shen* Beijing National Laboratory for Molecular Sciences (BNLMS), Laboratory of New Materials, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun North First Street 2, Beijing 100190, PR China
article info
abstract
Article history:
Leaves are one of the main by-products of forestry. In this study, batch experiments were
Received 14 September 2009
carried out to convert poplar leaves pretreated by different methods into hydrogen using
Received in revised form
anaerobic mixed bacteria at 35 C. The effects of acid (HCl), alkaline (NaOH) and enzymatic
5 February 2010
(Viscozyme L, a mixture of arabanase, cellulase, b-glucanase, hemicellulase and xylanase)
Accepted 6 February 2010
pretreatments on the saccharification of poplar leaves were studied. Furthermore, the
Available online 12 March 2010
effects of acid and enzymatic pretreatment on hydrogen production, together with their corresponding degradation efficiencies for the total reducing sugar (TRS) and metabolites
Keywords:
were compared. A maximum cumulative hydrogen yield of 44.92 mL/g-dry poplar leaves
Poplar leaves
was achieved from substrate pretreated with 2% Vicozyme L, which was approximately
Pretreatment
3-fold greater than that in raw substrate and 1.34-fold greater than that from substrate
Anaerobic fermentation
pretreated with 4% HCl. The results show that enzymatic pretreatment is an effective
Biohydrogen production
method for enhancing the hydrogen yield from poplar leaves. ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Fossil fuels such as oil, coal and natural gas are the main energy sources in current society. However, the inevitable depletion of fossil fuels, combined with concerns about their impact on our environment has led to an interest in nonpolluting and alternative energy sources. In comparison with fossil fuels, hydrogen is a promising alternative fuel of the future as it is a clean energy carrier with a high energy-yield (122 kJ/g) [1,2], the highest of all known fuels, and generates no pollutants producing only water upon combustion. Cellulosic biomass from agricultural and forest residue is an abundantly renewable resource [3]. The annual yield of biomass wastes is more than 0.7 billion tons in China alone [4]. Much cellulosic biomass, such as poplar leaves, however, is burned or discarded as environmental pollutants. Therefore, the production of hydrogen from cellulosic biomass has
attracted increasing attention. Many studies on biohydrogen from cellulosic biomass have been carried out [4–15]. The conversion of raw cellulosic biomass into hydrogen directly by microorganisms, however, is very difficult due to the complicated polymer structure of hemicellulose and cellulose. To enhance hydrogen yield, therefore, it is necessary to pretreat the substrate [5]. Nowadays, the methods of pretreating cellulosic biomass mainly include steam explosion, mechanical, thermal, acid, alkaline, ammonia and oxidative treatment [4–12,16]. Acid hydrolysis is considered to be one of the most effective methods of solubilizing hemicellulose, hydrolysis products contain not only soluble sugars but also a variety of by-products, such as phenol, furan and furfural compounds, adversely inhibiting the capability of hydrogenproducing bacteria to produce hydrogen [10,17]. In addition, acid hydrolysis is commonly carried out at high temperature and corrodes equipment [18]. In contrast, enzymatic
* Corresponding author. Tel.: þ86 10 62620903; fax: þ86 10 62559373. E-mail address: [email protected] (J. Shen). 0360-3199/$ – see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.02.035
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international journal of hydrogen energy 35 (2010) 4041–4047
hydrolysis is considered to be an effective method for pretreating cellulosic materials because it can be carried out under mild conditions without corroding equipment [18]. In this study, we explored the feasibility of converting poplar leaves into hydrogen with acid and enzymatic (Viscozyme L, a mixture of arabanase, cellulase, b-glucanase, hemicellulase and xylanase) pretreatments using anaerobic mixed microbial cultures, aiming at enhancing hydrogen yield by the pretreatment of substrate. For this purpose, the changes in the total reducing sugar (TRS) with different pretreatment methods and the effects of HCl and Viscozyme L concentrations on hydrogen production and liquid metabolites were studied by anaerobic fermentation in batch cultivation. The results show that enzymatic pretreatment is better than acid pretreatment in enhancing hydrogen yield from poplar leaves. To the best of our knowledge, this is the first report of hydrogen production from poplar leaves with pretreatment using anaerobic mixed bacteria.
2.
Materials and methods
2.1.
Materials
The poplar leaves used in this study were collected in autumn from a suburb of Beijing, China. They were dried in sunlight, and were then comminuted to more than 20-mesh using a comminutor and dried again in a thermoelectric oven for 3 h at 105 C. All chemicals were analytical reagent grade except where otherwise specified.
2.2.
Seed microorganisms
The hydrogen-producing mixed cultures were enriched from cracked cereal and identified to be dominated by Clostridium pasteurianum [19]. The cultures were acclimated in a completely stirred tank reactor (CSTR) in a chemostat for approximately one month. The reactor was operated at 35 C, an 8 h hydraulic retention time, and stirred by gas circulation [20]. One liter of culture medium used to ferment contained NH4HCO3, 3770 mg; K2HPO4, 125 mg; NaHCO3, 2000 mg; CuSO4$5H2O, 5 mg; MgCl2$6H2O, 100 mg; MnSO4$4H2O, 15 mg; FeSO4$7H2O, 25 mg; CoCl2$6H2O, 0.125 mg.
2.3.
Pretreatment
2.3.1.
Acid and alkaline pretreatment
1.0 g of dry poplar leaves were mixed with 20 mL of dilute HCl (or NaOH) aqueous solution at different concentrations (0.5%, 1%, 2%, 4% and 8% (w/v), respectively) (5% (w/v) solids loading) and boiled for 30 min in serum vials. The mixture was then neutralized to pH 7.0 by the addition of dilute NaOH (or HCl) aqueous solution at different concentrations (0.5%, 1%, 2%, 4% and 8% (w/v), respectively).
2.3.2.
Enzymatic pretreatment
Viscozyme L (VL) was purchased from Novozymes (China) Biotechnology Co., Ltd. (batch number KTN02161). VL is a mixture of many enzymes, including arabanase, cellulase, b-glucanase,
hemicellulase and xylanase. The optimal working conditions for VL are pH 3.3–5.5 and temperature 25–55 C. 1.0 g of dry poplar leaves were mixed with 20 mL of VL aqueous solution at different concentrations ([VL]), pH, enzymolysis time (t) and temperatures (T ), respectively (5% (w/v) solids loading). Four series of experiments were carried out: Series 1 investigated the effect of VL concentration (0.25–4%, v/v) on the saccharification of poplar leaves; Series 2 investigated the effect of pH (3.0–7.0) on the saccharification of poplar leaves, and a buffer containing 0.1 mol/L citric acid and 0.1 mol/L trisodium citrate was used to adjust the pH; Series 3 investigated the effect of enzymolysis time (1–5 h) on the saccharification of poplar leaves; Series 4 investigated the effect of enzymolysis temperature (35–55 C) on the saccharification of poplar leaves.
2.4.
Experimental procedures
Batch experiments were carried out to produce hydrogen by anaerobic fermentation in 120 mL serum vials using poplar leaves pretreated with HCl and VL as substrate. The total working volume was 80 mL (approximately 1.25% (w/v) solids loading) in each case. (1) Acid pretreatment: approximately 40 mL of mixture after pretreatment (20 mL of dilute HCl used to pretreat substrate and approximately 20 mL of dilute NaOH used to adjust pH), 20 mL inoculum, 10 mL nutrient solution (1 L contained NH4HCO3, 30 160 mg; K2HPO4, 1000 mg; NaHCO3, 16 000 mg; CuSO4$5H2O, 40 mg; MgCl2$6H2O, 800 mg; MnSO4$4H2O, 120 mg; FeSO4$7H2O, 200 mg; CoCl2$6H2O, 1 mg), and approximately 10 mL distilled water; (2) enzymatic pretreatment: 20 mL of mixture after pretreatment, 20 mL inoculum and 40 mL nutrient solution (1 L contained NH4HCO3, 7540 mg; K2HPO4, 250 mg; NaHCO3, 4000 mg; CuSO4$5H2O, 10 mg; MgCl2$6H2O, 200 mg; MnSO4$4H2O, 30 mg; FeSO4$7H2O, 50 mg; CoCl2$6H2O, 0.25 mg). The air was removed from the solution and the headspace by argon gas for 3 min before the vials were capped with rubber septum stoppers and placed in a reciprocal shaker (120 rpm). The batch experiments were performed at 35 C in the dark. Each experimental condition was carried out in triplicate.
2.5.
Analytical methods
The hydrogen content was determined by a gas chromatograph (Techcomp. Co., China, 7890II) equipped with a thermal conductivity detector (TCD) and a 2-m stainless steel column packed with Porapak Q (80–100 mesh). The operating temperatures of the injection port, the oven and the detector were set at 70, 50 and 70 C, respectively. Argon was used as the carrier gas at a flow rate of 30 mL/min. At each time interval, the total volume of biogas production was measured by a plunger displacement method using appropriately sized glass syringes, ranging from 10 to 100 mL [21]. The cumulative hydrogen volume was calculated by Eq. (1) [22], VH;i ¼ VH;i1 þ CH;i VG;i VG;i1 þ VH;0 CH;i CH;i1
(1)
where VH, i and VH, i1 are cumulative hydrogen volumes at the current (i) and previous (i 1) time intervals, VG, i and VG, i1 are the total biogas volumes in the current (i) and previous (i 1) time intervals, CH, i and CH, i1 are the fraction of
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hydrogen in the headspace of the bottle at the current (i) and previous (i 1) time intervals and VH,0 is the total volume of headspace in the bottle. Detection of the alcohols and volatile fatty acids (VFAs, C2–C5) was measured by a gas chromatograph using a flame ionization detector (FID) and a 2-m glass column packed with Unisole F-200 (30–60 mesh). The temperatures of the injection port, the oven and the detector were set at 200, 165 and 200 C, respectively. The carrier gas was also argon at a flow rate of 30 mL/min. The concentration of the total reducing sugar was determined by the phenol– sulfuric acid method using glucose as a standard [23].
2.6.
Model analysis
Content of the total reducing sugar (%)
international journal of hydrogen energy 35 (2010) 4041–4047
HCl NaOH
30
25
20
15
10
5
0
The cumulative hydrogen production during the batch experiments followed the modified Gompertz equation [24,25]: Rm e H ¼ P exp ðl tÞ þ 1 P
(2)
Where H is the cumulative hydrogen production (mL), P is hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL/h), e is 2.72, l is the lag-phase time (h), and t is the incubation time (h). The corresponding values of P, Rm and l for each batch were estimated using Origin 7.5, which is a scientific graphing and data analysis software.
3.
Results and discussion
3.1. Effect of pretreatment on the saccharification of poplar leaves 3.1.1. Effects of HCl and NaOH pretreatment on the saccharification of poplar leaves Acid pretreatment mainly converts hemicellulose in the cellulosic biomass into xylose [26], which is the main component of hemicellulose [27]. HCl was less active than sulfuric acid in the degradation of xylose, which is the feedstock for producing hydrogen by bacteria [28]. Therefore, HCl was used to pretreat the substrate. The TRS contents from 1.0 g of dry poplar leaves after HCl and NaOH pretreatment were analyzed and are depicted in Fig. 1. As shown in Fig. 1, the TRS content continuously increased with increasing HCl and NaOH concentrations. When comparing the contents of the TRS from substrate pretreated with 4% and 8% HCl (or NaOH), the increment was found to be small. The amounts of TRSs with 4% HCl and 4% NaOH pretreatment were approximately 2.33-fold and 1.66-fold greater compared with that in the raw substrate. It was noted that the TRS content with HCl pretreatment was always higher than that with NaOH pretreatment at the same HCl and NaOH concentrations. These results indicate that HCl pretreatment was superior to NaOH pretreatment, consistent with previous studies [4,5].
3.1.2. Effect of enzymatic pretreatment on the saccharification of poplar leaves Enzyme concentration, pH value, enzymolysis time and temperature are the main factors that affect the saccharification
UP
0.5%
1%
2%
4%
8%
HCl and NaOH concentrations (w/v)
Fig. 1 – Effects of HCl and NaOH concentrations on the saccharification of poplar leaves. UP means unpretreatment. of poplar leaves. Therefore, the effects of VL concentration, pH, enzymolysis time and temperature on the contents of the TRS from 1.0 g of dry poplar leaves were studied. The results are shown in Table 1. The TRS content continuously increased when the VL concentration increased from 0.25% to 1% but the increment was small when the VL concentration increased from 1% to 4%. Therefore, 1% VL was used to investigate the effect of pH on the saccharification of poplar leaves. The TRS content continuously decreased with increasing pH. The optimal pH was in the range of 3.0–4.0. Therefore, the effect of enzymolysis time on the saccharification of poplar leaves was studied at pH 4.0.
Table 1 – Effects of VL concentration, pH, enzymolysis time and enzymolysis temperature on the saccharification of poplar leaves. [VL] (v/v)
pH
t (h)
T ( C)
CS (%)
0.25% 0.5% 1% 2% 4%
4.0
3
50
18.51 20.27 25.07 25.33 26.03
1%
3.0 4.0 5.0 6.0 7.0
3
50
25.75 25.07 23.01 22.51 22.39
1%
4.0
1 2 3 4 5
50
22.34 23.92 25.07 25.25 26.00
1%
4.0
3
35 40 45 50 55
18.93 20.03 21.79 25.07 24.63
‘CS’ means the content of the total reducing sugar.
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international journal of hydrogen energy 35 (2010) 4041–4047
The TRS content continuously increased with the increasing enzymolysis time. The increment, however, was small when the enzymolysis time exceeded 3 h. The effect of enzymolysis temperature on the saccharification of poplar leaves was also studied using an enzymolysis time of 3 h. The TRS content initially increased then decreased with increasing enzymolysis temperature, and the optimal temperature was 50 C. According to the above results, the optimal conditions for saccharification of poplar leaves were [VL] ¼ 1%, pH ¼ 3.0–4.0, t ¼ 3 h and T ¼ 50 C. To summarize, the TRS content from pretreated poplar leaves was higher than that from raw poplar leaves, indicating that pretreatment is very necessary for further saccharification of poplar leaves. The TRS contents after enzymatic pretreatment were lower than those after HCl pretreatment but higher than those after NaOH pretreatment. Therefore, poplar leaves pretreated by HCl and VL were used as a substrate in the fermentation to produce hydrogen.
3.2.
Effect of pretreatment on hydrogen production
3.2.1.
Effect of HCl pretreatment on hydrogen production
The effects of HCl concentration on the cumulative hydrogen volumes are presented in Fig. 2. 1.0 g of dry poplar leaves pretreated with different HCl concentrations were used as the substrate to produce hydrogen at an initial pH of 7.0. It can be seen from Fig. 2 that the cumulative hydrogen volume from raw poplar leaves was 15.04 mL but that the cumulative hydrogen volumes continued to increase (27.56–33.45 mL) when the HCl concentration increased (0.5–4%). The maximum hydrogen volume of 33.45 mL was observed at 4% HCl, which was 2.22-fold higher than that from the raw substrate. To further investigate the effect of HCl concentration on hydrogen production, the data in Fig. 2 were simulated using Eq. (2) and the hydrogen production characteristics are shown in Table 2. According to the data in Table 2, all R2 values were more than 0.990, indicating that the fitted curves matched well with the experimental points. The specific hydrogen
Table 2 – Hydrogen production characteristics at different HCl concentrations. HCl (w/v)
UP 0.5% 1% 2% 4%
25
15 10
R2
15.04 27.56 28.44 29.14 33.45
1.25 2.74 1.73 1.53 1.18
3.60 11.90 10.00 9.40 8.76
0.9992 0.9994 0.9993 0.9979 0.9937
production rate continuously decreased with increasing HCl concentration (0.5–4%), possibly because HCl pretreatment produced some inhibitors such as furfural that inhibited the specific hydrogen production rate of hydrogen-producing bacteria [29]. The maximum specific hydrogen production rate of 2.74 mL/h was observed at 0.5% HCl. The shortest duration of 3.60 h occurred with the raw substrate, because the microorganisms used here were acclimated to raw poplar leaves. The duration continuously decreased with increasing HCl concentration (0.5–4%).
3.2.2. Effect of enzymatic pretreatment on hydrogen production 1.0 g of dry poplar leaves pretreated with various VL concentrations at optimal pretreatment conditions (pH ¼ 4.0, t ¼ 3 h, T ¼ 50 C) were used as substrate to produce hydrogen at an initial pH of 7.0. The effects of VL concentration on the cumulative hydrogen volumes are presented in Fig. 3. As shown in Fig. 3, the cumulative hydrogen volumes initially increased then decreased with increasing VL concentration, suggesting that excessive VL was unfavorable to hydrogen production. The maximum hydrogen volume of 44.92 mL was observed at 2%, which was 2.99-fold higher than that from the
UP 0.25% 0.5% 1% 2% 4%
40
20
l (h)
P is hydrogen production potential (mL). Rm is the maximum hydrogen production rate (mL/h). l is the lag-phase time (h). R2 is correlation coefficient.
Cumulative hydrogen (mL)
Cumulative hydrogen (mL)
30
Rm (mL/h)
0 0.5% 1% 2% 4%
50 35
P (mL)
30
20
10
5 0
0 0
20
40
60
80
Time (hour)
Fig. 2 – Cumulative hydrogen volumes from 1.0 g of dry poplar leaves pretreated by different HCl concentrations versus corresponding fermentation time. The operation was at 35 8C and initial pH 7.0.
0
10
20
30
40
50
60
70
Time (hour)
Fig. 3 – Cumulative hydrogen volumes from 1.0 g of dry poplar leaves pretreated by different VL concentrations versus corresponding fermentation time. The operation was at 35 8C and initial pH 7.0.
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international journal of hydrogen energy 35 (2010) 4041–4047
Table 3 – Hydrogen production characteristics at different VL concentrations. VL (v/v) 0 0.25% 0.5% 1% 2% 4%
P (mL)
Rm (mL/h)
l (h)
R2
15.04 27.32 33.13 36.90 44.92 35.47
1.25 1.85 2.14 2.17 1.97 1.32
3.60 8.04 7.71 7.88 7.98 7.13
0.9992 0.9922 0.9938 0.9957 0.9922 0.9863
raw substrate and better than that from pretreated substrate with 4% HCl, indicating that enzymatic pretreatment was better than HCl pretreatment. To further investigate the effect of VL concentration on hydrogen production, the data in Fig. 3 were simulated using Eq. (2) and the hydrogen production characteristics are shown in Table 3. According to the data in Table 3, all R2 values were more than 0.986, indicating that the fitted curves matched well with the experimental points. The specific hydrogen production rate initially increased and then decreased with increasing VL concentration, suggesting that excessive VL was unfavorable to enhance the specific hydrogen production rate. The maximum specific hydrogen production rate of 2.17 mL/h was observed at 1%. The shortest duration of 3.60 h occurred with the raw substrate, possibly due to the same reason given above. The changes in duration with poplar leaves pretreated with different VL concentrations were small with increasing VL concentration (0.25–4%). To summarize, the cumulative hydrogen volume from poplar leaves with pretreatment was higher than that from raw poplar leaves, indicating that pretreatment is necessary for increasing hydrogen production. The result was consistent with previous studies, which are summarized in Table 4. As shown in Table 4, the maximum hydrogen yields from pretreated substrates were much higher than hydrogen yields from raw substrates. The TRS content from the pretreated substrate with 4% HCl was higher than that from the pretreated substrate with 2% VL but the former cumulative hydrogen volume was lower than the latter. This is possibly because HCl pretreatment produced some inhibitors [29], inhibiting the hydrogen-producing bacteria from producing hydrogen using sugar.
3.3.
Effect of pretreatment on metabolites
Gas product analyses showed that only H2 and CO2 were present in the biogas, without any detectable CH4 during
the course of hydrogen production, suggesting that there were no methanogens in the anaerobic mixed bacteria used here. The effects of HCl and VL concentrations on the sugar degradation efficiency, H2 contents, H2 yields, and liquid metabolites are shown in Table 5. The sugar degradation efficiency exceeded 92%, indicating that most of the sugar was consumed by the bacteria, and the changes were small with increasing HCl and VL concentrations. Hydrogen contents in the biogas varied from 28.80% to 32.51% with a change in HCl concentration from 0.5% to 4% and the divergence was also small. The maximum of 32.51% was observed at 1% HCl. Hydrogen contents in the biogas varied from 26.92% to 46.08% with a change in VL concentration from 0.25% to 4% and the maximum of 46.08% was observed at 0.5% VL. The liquid product analysis showed that the metabolites found after fermentation were ethanol, acetic acid, propionic acid and butyric acid. It should be noted that high yields of ethanol and VFAs were obtained from the raw poplar leaves but a relatively low cumulative hydrogen yield (15.04 mL/g-dry poplar leaves) was produced. This is possibly because some of the hemicellulose and cellulose in raw poplar leaves was consumed in the production of ethanol and VFAs [26]. When the HCl concentration increased from 0.5% to 4%, the yields of acetic acid and propionic acid decreased gradually, while ethanol and n-butyric acid initially increased then decreased. A maximum of four liquid metabolites appeared at 2% VL. The possible metabolic pathway of soluble sugar (mainly glucose) to produce hydrogen at the maximum hydrogen yield (2% VL pretreatment) is shown as Eq. (3):
C6H12O6 / 1.315H2 þ 1.873CO2 þ 0.086CH3CH2OH þ 0.552CH3COOH þ 0.125CH3CH2COOH þ 0.074CH3CH2CH2COOH
(3)
As shown in Eq. (3), the metabolic pathway was mainly acetic acid fermentation, and the conversion efficiency of H element in glucose into H2 was 22%. The mechanism of hydrogen production from glucose and xylose by bacterial fermentation has been reported previously [30–32]. Theoretically the metabolic pathways of the four liquid products (ethanol, acetic acid, propionic acid and nbutyric acid) from xylose and glucose are as follows: C5H10O5 / 1.67CH3CH2OH þ 1.67CO2
(4)
C5H10O5 þ 1.67H2O / 1.67CH3COOH þ 3.33H2 þ 1.67CO2
(5)
Table 4 – Biohydrogen production from various cellulosic biomasses with different pretreatment methods. Feedstock Poplar leaves Beer lees Cornstalk Wheat straw Corn stalk
Pretreatment method
H2 yield from raw material
Maximum H2 yield from pretreated substrate
Reference
Viscozyme L HCl HCl HCl-Microwave Biopretreatment
15.04 mL/g 3.16 mL/g 3.16 mL/g-TVS 0.5 mL/g-TVS 20 mL/g-TS
44.92 mL/g 53.03 mL/g 149.69 mL/g-TVS 68.1 mL/g-TVS 176 mL/g-TS
This study [26] [4] [6] [11]
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international journal of hydrogen energy 35 (2010) 4041–4047
Table 5 – Effects of HCl and VL concentrations on the degradation efficiency of total reducing sugar, H2 contents, H2 yields and yields of ethanol and VFAs after fermentation. DESa (%)
H2 contentb (%)
H2 yield (mL/g-S)
Ethanol (mg/g-S)c
Acetic acid (mg/g-S)
Propionic acid (mg/g-S)
n-Butyric acid (mg/g-S)
UP
0
93.28
26.89
15.04
4.20
26.10
9.28
6.06
HCl (w/v)
0.5% 1% 2% 4%
94.98 95.81 94.68 92.44
30.09 32.51 28.80 30.06
27.56 28.44 29.14 33.45
2.27 4.02 5.49 4.87
17.31 16.01 15.96 9.15
5.76 5.16 2.64 2.17
2.21 2.81 1.38 0.94
VL (v/v)
0.25% 0.5% 1% 2% 4%
95.48 96.03 96.03 95.76 95.82
39.25 46.08 40.37 41.25 26.92
27.32 33.13 36.90 44.92 35.47
3.41 4.31 4.30 5.31 4.18
29.72 36.01 34.80 44.68 35.80
5.45 5.30 6.73 12.42 9.63
2.93 3.00 4.40 8.78 5.01
a DES means degradation efficiency of total reducing sugar. b H2 content means percentage of the total cumulative hydrogen produced to total biogas. c (mg/g-S) means (mg/g-dry poplar leaves).
C5H10O5 þ 1.67H2 / 1.67CH3CH2COOH þ 1.67H2O
(6)
C5H10O5 / 0.83CH3(CH2)2COOH þ 1.67H2 þ 1.67CO2
(7)
C6H12O6 / 2CH3CH2OH þ 2CO2
(8)
pretreatment. Therefore, enzymatic pretreatment is an ideal method for increasing the hydrogen yield from poplar leaves by anaerobic fermentation.
Acknowledgments The authors would like to thank the Chinese Academy of Sciences for financial support (Item No. KJCX2-YW-H21). C6H12O6þ2H2O / 2CH3COOH þ 4H2 þ 2CO2
(9)
references C6H12O6 þ 2H2 / 2CH3CH2COOH þ 2H2O
C6H12O6 / CH3(CH2)2COOH þ 2H2 þ 2CO2
(10)
(11)
According to Eqs. (4)–(11), the production of ethanol has no influence on the production of hydrogen whereas the production of acetic and n-butyric acid favors the production of hydrogen. In contrast, the production of propionic acid resulted in less hydrogen production. According to Eq. (3), it was mainly acetic acid fermentation, so it favored the production of hydrogen.
4.
Conclusions
In this work, three different methods, acid (HCl), alkaline (NaOH) and enzymatic treatment, were used to pretreat poplar leaves. The TRS content in pretreated substrate was higher than that in raw substrate, indicating that pretreatment is required for further saccharification of substrate. The TRS contents after enzymatic pretreatment were lower than those after HCl pretreatment but higher than those after NaOH pretreatment. Nevertheless, the maximum cumulative hydrogen yield (44.92 mL/g-dry poplar leaves) from substrate pretreated by 2% VL was higher than that from substrate pretreated by 4% HCl (33.45 mL/g-dry poplar leaves), indicating that enzymatic pretreatment was better than HCl
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