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Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis
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 5 ) 1 e8
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Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkalicellulase two-step hydrolysis Honghui Yang a,b,*, Bofang Shi a, Hongyu Ma a, Liejin Guo b a b
Department of Environmental Science and Engineering, Xi'an Jiaotong University, Xi'an 710049, PR China State Key Laboratory of Multiphase Flow in Power Engineering, Xi'an Jiaotong University, Xi'an 710049, PR China
article info
abstract
Article history:
The effect of a two-step cornstalk pretreating process, NaOH delignification followed by
Received 8 May 2015
enzymolysis with cellulase and hemicellase, on dark- and photo-fermentative H2 production
Received in revised form
was studied. A five-factor and five-level orthogonal experimental array was designed and
2 July 2015
conducted to study the effect of NaOH concentration (0e1%), hydrolysis time (0e3 h), hydrolysis
Accepted 12 July 2015
temperature (98e126 C), cellulase (0e18 IU/g-cornstalk) and hemicellulase dosage (0e2400 IU/
Available online xxx
g-cornstalk) on pretreatment efficiency determined by H2 production. With NaOH 0.75%, hydrolysis temperature 108 C, hydrolysis time 0.5 h, cellulase dosage 12 IU/g-CS and hemi-
Keywords:
cellulase 2400 IU/g-CS, a maximum reducing sugar yield of 0.56 ± 0.03 g/g-CS and maximum H2
Hydrogen production
yield of 163.1 mL-H2/g-cornstalk were obtained under dark-fermentation, and a maximum H2
Cornstalk
yield of 339.5 mL-H2/g-cornstalk was obtained under photo-fermentation. According to the
NaOH pretreatment
results, the significance of the five parameters on H2 production was listed in high-to-low order
Orthogonal experimental array
as: NaOH concentration, cellulase dosage, hydrolysis temperature, hemicellulase and hydro-
Photo-fermentative hydrogen
lysis time. The effect of the alkaline delignification on pretreatment efficiency and the future
production
efforts on improving H2 production from agricultural wastes were also discussed. Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Dark-fermentative process is accepted as a promising way for sustainable hydrogen production [1]. But it is impractical to use the regular substrates such as glucose, sucrose and starch to produce hydrogen for the high price of these food-based materials and food shortage [2,3]. Cellulosic biomass, such as agricultural straw, is abundant and potential feedstock for fermentative hydrogen production. Although some thermophilic bacteria are able to produce hydrogen directly from
cellulosic biomass [4e7], these organisms showed low hydrogen production rate and most of them need additional vitamins for growth, which makes it uneconomical for industrial application. Cellulase is efficient in enzymolysis of cellulosic biomass specifically without producing inhibitory by-products for fermentation, such as acetate, furan derivates and phenolic monomers [8], which could be produced by chemical hydrolysis. The enzymolysis efficiency was quite low when the cellulosic biomass was directly applied as substrate without chemical pretreatment [9]. Pretreatment is an effective
* Corresponding author. Department of Environmental Science and Engineering, Xi'an Jiaotong University, Xi'an 710049, PR China. Tel./ fax: þ86 29 82664731. E-mail address: [email protected] (H. Yang). http://dx.doi.org/10.1016/j.ijhydene.2015.07.062 0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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process in promoting the digestibility of cellulosic materials [10] by cellulase and increasing its fermentative performance [11,12]. Fan et al. employed cornstalk pretreated by diluted HCl as feedstock and heat-shocked cow dung as inoculum, and a maximum hydrogen yield of 149.69 mL/g-TVS was obtained at initial pH 7.0 and substrate concentration of 15 g/L [13]. Datar et al. [14] found that the optimum parameters for gas explosion of cornstalk were pH 7.0, temperature 220 C and reaction time 3 min, and the maximum hydrogen yield was 25.6 mL/gcornstalk. Ren et al. employed acetic acid-gas explosion and followed by cellulase hydrolysis process to pretreat cornstalk [15]. They obtained a maximum hydrogen yield of 72 mL/gcornstalk when acetic acid concentration was 16% and cellulase dosage was 120 IU and 180 IU, respectively. Wang et al. designed a set of orthogonal experiments to optimize the parameters of cornstalk enzymolysis [16]. It was found that enzymolysis temperature 50 C, enzymolysis time 72 h, initial pH 7.0 and substrate concentration 10 g/L were the optimum parameters, and maximum hydrogen yield of 141.3 mL/gcornstalk was obtained. Cornstalk, as a typical agricultural waste, mainly comprises of cellulose, hemicellulose and lignin. The three components are highly crystallized and convolved with each other to hinder the enzymolysis efficiency. Cellulose is packed into microfibrils by hydrogen bonds and Van der Waals bonds. Hemicellulose and lignin cover the microfibrils [17]. Lignin imparts structural support, and it is impermeable and resistant to microbial attack [18]. Especially since lignin could not be decomposed by cellulase, it retards the enzymolysis of hemicellulose and cellulose [19]. Lignin is dissolvable in alkaline solution, therefore, alkaline pretreatment could be able to decrease the crystallinity degree of cornstalk and ease its enzymolysis process. Compared with acid pretreatment processes, alkaline pretreatment processes utilize low temperature and pressure, and cause less sugar degradation [20]. NaOH is effective for delignification. Alkaline pretreatment increases the sugars yield about three times comparing with untreated and treated wheat straw reported by Carrillo et al. [21]. The enzymatic digestibility of alkaline pretreated barley straw was greatly enhanced [22]. However, the effect of NaOH on pretreating agricultural wastes for hydrogen production [23], especially for photo-fermentative hydrogen production was not well studies. In this study, in order to evaluated the enhancement effect of alkaline pretreatment on enzymolysis and hydrogen production performance of corn straw, a set of orthogonal experiments were conducted to optimize the operational parameters, including NaOH concentration, alkaline hydrolysis temperature, hydrolysis time, cellulase dosage and hemicellulase dosage. The effects of NaOH pretreatment on cornstalk dissolution ratio and enzymolysis efficiency were studied. The feasibility of one-stage photo-fermentation using corn stalk hydrolysate was also demonstrated.
farmland with water content of 3.49%, ash content of 11.90% and volatile solids (VS) content of 84.61%. Cornstalk was shattered into 20e40 meshes and mixed with tap water in a ratio of 1:15. NaOH was used to pretreat the cornstalk before enzymolysis. The parameters set for alkaline hydrolysis and enzymolysis are listed in Table 1. After alkaline hydrolysis, a certain amount of glutamic acid was supplemented into the mixture to make its final concentration 1 g/L in the medium for fermentation. The pH value of the mixture was then adjusted to 4.80 with 1 mol/L HCl. A certain amount of cellulase (Chende Tianfeng Biotechnology Co. LTD, FPU, 100 IU/g) and hemicellulase (KND biotechnology Co. LTD, 20,000 IU/mL) was added into the mixture, and it was kept at 50 C for 10 h. After the enzymolysis, the pH value of the mixture was adjusted to 7.0 with 1 M NaOH solution. All of the hydrolysis and enzymolysis experiments were conducted in triplicate.
Orthogonal experimental array A L25 (55) orthogonal experimental array was designed and conducted to optimize the key parameters affecting hydrogen production including NaOH concentration (0e1%), alkaline hydrolysis time (0e3 h), alkaline hydrolysis temperature (98 C126 C), cellulose (0e12 IU/g-CS) and hemicellulase dosage (0e2400 IU/g-CS) as shown in Table 1. Range analysis was conducted to analyze the significance of these parameters on hydrogen production [25,26]. Each of the experiments listed in Table 1 was conducted in triplicate.
Batch tests In the dark fermentation tests, the batch experiments were performed with 400 mL homemade glass bottles as reactors filled with heat-shocked cow dung of 2 g, a certain amount of cornstalk hydrolysate and mineral nutrient solution of 12.5 mL, and then the mixture was diluted to 250 mL with water. The final concentration of cornstalk hydrolysate was 15 g-CS/L. The components of the mineral nutrient were as follows: (g/L) KH2PO4 5, CaCl2 0.2, FeSO4$7H2O 0.13, MgSO4 1.0, Na2MO4$2H2O 0.06, MnSO4$H2O 0.3. These reactors were filled with pure nitrogen gas for 10 min to remove oxygen to obtain the initial anaerobic environment. The reactors were then set in a water bath shaker at 150 rpm and 35 ± 0.5 C. For photo-fermentation using corn stalk hydrolysate as substrate, the hydrolysate were centrifuged to remove the solid residue, and then diluted with H2O to concentration of 5 g-CS/L, 10 g-CS/L and 15 g-CS/L for photo-fermentation. Rhodobacter sphaeroides HY01 used as inocula was precultured as described in literature [27]. 30 mL syringes were employed as photo-reactor as shown in literature [28]. Each of the experiments in dark-fermentation and photofermentation were conducted in triplicate.
Analytical methods
Materials and methods Inoculum and feedstock The heat-shocked cow dung was employed as inoculum as described previously [24]. Cornstalk (CS) was harvested from
Volumes of the biogas were measured with volumetric bottle as described previously [29], and then calculated into that in standard condition (273.15 K, 1 atm). The components of biogas were analyzed as shown in previous report [27]. Briefly, a gas chromatography (GC, Beifen Co., Ltd. Model, SP-2100)
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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Table 1 e Parameters for orthogonal experiments. Test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
T/ C
NaOH/%
t/h
CD/IU/g-CS
HD/IU/g-CS
98 98 98 98 98 108 108 108 108 108 115 115 115 115 115 121 121 121 121 121 126 126 126 126 126
0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1
0.25 0.5 1 2 3 2 3 0.25 0.5 1 0.5 1 2 3 0.25 3 0.25 0.5 1 2 1 2 3 0.25 0.5
0 3 6 9 12 3 6 9 12 0 6 9 12 0 3 9 12 0 3 6 12 0 3 6 9
0 600 1200 1800 2400 1200 1800 2400 0 600 2400 0 600 1200 1800 600 1200 1800 2400 0 1800 2400 0 600 1200
Pretreatment efficiency % 4.4 ± 12.0 ± 16.3 ± 30.3 ± 41.2 ± 23.2 ± 27.2 ± 17.8 ± 41.0 ± 51.7 ± 16.9 ± 29.3 ± 38.7 ± 52.3 ± 49.2 ± 18.8 ± 30.0 ± 45.4 ± 50.6 ± 49.6 ± 22.8 ± 30.0 ± 45.8 ± 48.0 ± 52.8 ±
Enzymolysis efficiency% 5.2 ± 40.3 ± 66.2 ± 70.6 ± 70.2 ± 32.3 ± 47.5 ± 69.2 ± 73.0 ± 55.6 ± 34.5 ± 50.5 ± 72.0 ± 54.6 ± 66.5 ± 36.8 ± 44.9 ± 46.7 ± 58.6 ± 54.1 ± 28.9 ± 39.3 ± 48.5 ± 51.9 ± 65.1 ±
1.2 1.4 2.1 3.2 2.2 4.3 1.6 3.3 2.1 2.3 1.3 2.7 1.6 1.4 1.3 2.1 2.5 3.3 3.7 4.1 2.0 3.2 4.1 6.0 4.7
1.3 3.1 4.2 5.1 4.7 1.6 2.8 3.7 3.9 2.3 1.8 2.5 4.6 2.5 3.8 2.1 1.2 2.1 3.1 2.9 1.6 1.8 2.3 2.5 3.6
T: temperature; NaOH: NaOH concentration; CD: cellulase dosage; HD, hemicellulase dosage; t, hydrolysis time.
equipped with a thermal conduct detector and a 4 m stainless chromatographic column filled with Hayesep padding was used to determine the gas products. Concentrations of volatile fatty acids (VFAs) in the liquid samples were analyzed with a GC (Varian, CP-3800) equipped with a flame ionization detector and a capillary column coated with terephthalic acid modified PEG (AT.FFAP). The alkaline pretreatment efficiency and enzymolysis efficiency were determined by gravimetric method. The reducing sugar content was determined by anthrone-sulfuric method [30]. Pretreatment efficiency was defined by the percentage of dissolved cornstalk after alkaline hydrolysis, and enzymolysis efficiency stands for the percentage of dissolved cornstalk after enzymolysis. The cumulative hydrogen production data were fitted with modified Gompertz equation [3]. The experimental data points were described as average value plus deviation.
Results and discussion
and so on. And R stands for the largest range of the data in five levels. The largest range of pretreatment efficiency from level 1 to level 5 was 32.68% in terms of NaOH concentration, which means NaOH concentration showed the most significant effect on pretreatment efficiency. The range 19.08% reflected the effect of temperature on pretreatment efficiency. And hydrolysis time, at a range of 6.58%, showed the least significance on pretreatment efficiency. As for the hydrogen production performance, NaOH concentration also showed the largest range 19.18 mL/g-CS among the three factors. Temperature and hydrolysis time showed less significance at a range of 4.44 mL/ g-CS and 5.16 mL/g-CS, respectively. The optimum parameters based on pretreatment efficiency were NaOH 1%, temperature 126 C and hydrolysis time 3 h. The maximum
Table 2 e Range analysis of the effect of temperature (T), NaOH concentration and hydrolysis time (t) on pretreatment efficiency and hydrogen production performance of cornstalk. Pretreatment efficiency/%
Effect of alkaline hydrolysis on cornstalk dissolution and hydrogen yield Table 1 shows the alkaline pretreatment efficiency and enzymolysis efficiency of cornstalk in orthogonal experiments. Table 2 shows the effect of temperature, NaOH concentration and hydrolysis time on pretreatment efficiency and hydrogen production performance of pretreated cornstalk. In Table 2, L1 stands for level 1 in each factor. For instance, (L1, T) means the average datum obtained from the L25 (55) orthogonal experimental array at the temperature level 1 of 98 C,
L1 L2 L3 L4 L5 R Optimal level
T 20.80 30.18 37.28 38.48 39.88 19.08 L5
NaOH 15.22 28.10 32.40 43.24 47.90 32.68 L5
t 30.08 32.62 34.14 32.36 36.66 6.58 L5
Hydrogen yield/mL/g-CS T 18.30 22.74 21.98 21.62 21.52 4.44 L2
NaOH 8.16 19.76 26.60 27.34 26.32 19.18 L4
t 19.76 23.94 18.78 22.66 23.04 5.16 L2
L1-L5: level 1-level 5.
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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hydrogen yield of 34.1 mL H2/g-CS appeared at NaOH of 0.75%, temperature of 108 C and hydrolysis time of 0.5 h for the pretreated cornstalk. Fig. 1 shows the reducing sugar yields and hydrogen yields of NaOH pretreated cornstalks. As shown in Fig. 1, the hydrogen yield increased from 9 mL H2/g-CS in test 1 to 34.1 mL H2/g-CS in test 9. The main products of the effluent from darkfermentation were acetate and butyrate, and the concentrations of acetate and butyrate were around 1 g/L and 0.5 g/L, respectively. The concentration of propionate below 0.2 g/L was detected in most of the tests. The pH values of the effluents were around 5.2e6.0. The corresponding pretreatment efficiency increased by 9 times ranging from 4.4% in test 1 to 41.2% in test 9, suggesting that although 41.2% cornstalk was converted to soluble organic matter, but the reducing sugar content of cornstalk only increased from 0.07 g/g-CS into 0.19 g/gCS in the whole hydrolysis process. The result indicated that the most of hydrolysate could not be utilized by microbes to produce hydrogen except reducing sugar. The NaOH pretreatment dissolved lignin and hemicelluose, but few useful saccharide was produced as shown in Fig. 1 and previous report [14]. Therefore, the NaOH pretreatment was effective on decomposing the cornstalk structure formed by lignin, hemicellulose and cellulose, and another treatment should be applied for increasing the reducing sugar content of the hydrolysate to improve hydrogen production performance. All of these data suggest that these tests showed normal butyric acid metabolic pathway and the reason for the low hydrogen yield was the low amount of reducing sugar content in hydrolysate. Sodium hydroxide and hydrogen peroxide were employed to pretreat soybean straw, and a maximum hydrogen yield of 60.2 mL/g-straw was obtained, which was quite lower than those obtained from dilute acid or enzyme preteated biomass [12]. Therefore, an effective saccharification method is needed for increasing the hydrogen yield.
Effect of alkaline-enzymatic treatment on cornstalk dissolution and hydrogen yield The enzymolysis efficiencies of cornstalk obtained by cellulase and hemicellulase were shown in Table 1. In test 2, the
dissolution ratio of cornstalk increased from 12% to 40.3% when cellulase and hemicellulase dosage was 3 IU/g-CS and 600 IU/g-CS, respectively. From test 3 to test 5, the dissolution ratio of cornstalk increased further to around 70%. In test 10, 14 and 22, in which only hemicellulase was used for enzymolysis, it only increased from 51.7%, 52.3% and 30% into 55.6%, 54.6% and 39.3%, respectively. As mentioned before, part of hemicellulose was dissolved in alkaline hydrolysis; therefore, hemicellulase only increased the dissolution ratio of cornstalk slightly. In test 23, 24 and 25, the dissolution ratio of cornstalk increased from 45.8%, 48% and 52.8% to 48.5%, 51.9% and 65.1%, respectively. Enzymolysis efficiency increased only by 2.7%, 3.9% and 12.1%, which was much less than that of 28.3%, 49.9%, 40.3% and 29% obtained from test 2 to test 5, respectively. The reason was that low alkaline pretreatment efficiency was obtained at lower alkaline hydrolysis temperature, and less hemicellulose was degraded into volatile fatty acids, thus the enzymatic hydrolysis efficiencies were increased dramatically. Many volatile fatty acids are inhibitors and not fermentable for hydrogen production [31]. A considerable amount of acetic acid was detected in the NaOH pretreated mixture in this study. Hence, the more hemicellulose remained in solid after NaOH pretreatment, the higher reducing sugar and the lower inhibitors concentration of the hydrolysate were obtained by enzymolysis. According to the results of range analysis, as shown in Table 3, the significance of these five parameters follows the order: NaOH concentration > cellulase dosage > hydrolysis temperature > hemicellulase dosage > hydrolysis time. The highest enzymolysis efficiency was obtained in the followed treatment condition: NaOH 0.75%, cellulase dosage (CD) 12 IU/ g-CS, temperature (T) 108 C, hemicellulase dosage (HD) 2400 IU/g-CS and alkali hydrolysis time (t) 0.5 h. Fig. 2 shows the hydrogen yields of these 25 tests, and Fig. 1S (Supplementary Information) shows the cumulative hydrogen production profiles of these 25 tests. In test 9, the highest hydrogen yield of 153.9 mL/g-CS and reducing sugar yield of 0.52 ± 0.03 g/g-CS were obtained when the parameters were NaOH 0.75%, T 108 C, t 0.5 h, CD 12 IU/g-CS. Table 4 shows the range analysis results on hydrogen yield according to the mean value of each level. As shown in Table 4, in the control (L1), the hydrogen yield of 37.5 mL/g-CS was obtained. Thereafter the hydrogen yield was gradually increased with the increase of NaOH concentration in the CS pretreatment
Table 3 e Range analysis the effect of temperature (T), NaOH concentration, hydrolysis time (t), cellulase (CD) and hemicellulase dosage (HD) on the dissolution ratio of cornstalk. Pretreatment efficiency/%
Fig. 1 e Reducing sugar yields and dark-fermentative hydrogen production performance of cornstalk pretreated with NaOH hydrolysis.
T1 T2 T3 T4 T5 R Optimal level
T 52.8 57.5 55.0 53.4 44.8 12.7 L2
NaOH 27.4 48.5 59.1 65.1 63.4 37.7 L4
t 47.4 51.4 53.1 56.4 55.1 9.0 L4
CD 37.5 51.0 56.8 58.8 59.4 21.9 L5
HD 46.7 49.3 51.5 56.8 59.1 12.4 L5
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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According to the range analysis, the optimum parameters based on hydrogen yield were listed as follows: NaOH 0.75%, CD 12 IU/g-CS, T 108 C, HD 2400 IU/g-CS and t 0.5 h.
Confirmation experiments
Fig. 2 e Reducing sugar yields and dark-fermentative hydrogen yields of cornstalk after NaOH pretreatment and cellulase-hemicellulase enzymolysis.
from 62.9 mL/g-CS for 0.25% NaOH to maximum value of 103.8 mL/g-CS for 0.75% NaOH (L2~L4), and then the hydrogen yield was decreased with further increase of NaOH concentration(L5). We also found that both cellulase and hemicellulase were employed as the enzymolysis step in the case of without NaOH pretreatment, the hydrogen yield was only similar with that of NaOH pretreatment alone. The results indicated that the NaOH pretreatment significantly affected the hydrogen production performance of the CS. The cellulase dosage showed the second significant influence with a range of 60.1 mL/g-CS. Without cellulase, the average hydrogen yield was 43.3 mL/g-CS; this is close to the mean value that obtained without NaOH pretreatment. The mean value of hydrogen yield increased with the increase of cellulase dosage and maximum yield of 103.4 mL/g-CS was obtained when the cellulase dosage was 12 IU/g-CS. In the range from 98 C to 126 C, the alkaline hydrolysis temperature showed the third significant influence. The mean value reached the maximum, 90 mL/g-CS at 108 C, and then reduced into 68.7 mL/g-CS at 126 C. Hemicellulase showed less effect on hydrogen yield. The mean value of hydrogen yield based on hemicellulase dosage increased from 71.0 mL/ g-CS to 90.8 mL/g-CS when it was increased from 0 to 2400 IU/ g-CS. From 0.25 h to 3 h, hydrolysis time showed the slightest influence on hydrogen yield. And when it was 0.5 h, the highest hydrogen yield of 87.6 mL/g-CS was obtained.
According to the above results of orthogonal experiments, the confirmation experiments were also arranged, and the results were listed in Table 5 and Fig. 2S (Supplementary Information), respectively. As shown in Table 5 and Fig. 2S (Supplementary Information), Y1 listed reducing sugar yield and hydrogen yield to be 0.56 ± 0.03 g/g-CS and 163.1 mL/g-CS (equals 192.9 mL/g-TVS) under the optimum conditions from orthogonal experiment. It was about 6% higher than that obtained in test 9. This slight increase of hydrogen yield was mainly attributed to the function of hemicellulase. In this case, the enzymolysis efficiency of hemicellulose increased slightly. A cellulase dosage of 18 IU/g-CS was conducted in Y2. The hydrogen yield of Y2 was 157.7 mL/g-CS, which was slightly lower than that of Y1, and the reducing sugar yield, 0.56 ± 0.04 g/g-CS, was also close to that obtained in Y1. And the enzymolysis efficiency of cornstalk was similar with that of Y1. This means the increase of cellulase dosage over 12 IU/ g-CS could not increase the hydrogen yield, and this might be caused by some inhibition factors and it is consistent with Carrillo's report [21]. The results of range analysis indicated that the effect of alkaline hydrolysis temperature at 98 C was close to those at 108 C, hence the experimental conditions of Y3 was the same as that of Y1 except for hydrolysis temperature of 98 C, the hydrogen yield of 158.9 mL H2/g-CS was close to that of Y1. In test Y4, the alkaline hydrolysis time was increased from 0.5 h into 1 h, while the hydrogen yield was lower than that of Y1. This result suggests that longer alkaline hydrolysis time could not increase the hydrogen yield. Comparing the hydrogen yield in test Y4 and Y5, it was noticed that hemicellulase played a less important role. The commercial cellulase used in this study was not purified and it showed some hemicellulase activity, thus, when high dosage of cellulase was used, its hemicellulase activity was enough for hemicellose enzymolysis, and the additional hemicellulase could not increase the enzymolysis efficiency clearly. While in low cellulase dosage, additional hemicellulase could increase the hydrogen yield. The results indicate that additional hemicellulase is not needed when the dosage of cellulase was higher than 9 IU/g-CS.
Photosynthetic hydrogen production using corn stalk hydrolysate Table 4 e Range analysis the effect of temperature (T), NaOH concentration, hydrolysis time(t), cellulase (CD) and hemicellulase dosage (HD) on the hydrogen yield. Hydrogen yield/mL/g-CS L1 L2 L3 L4 L5 R Optimal level
T 86.4 90.0 83.5 66.4 68.7 23.6 L2
NaOH 37.5 62.9 93.3 103.8 97.5 66.3 L4
t 76.1 87.6 76.9 78.7 75.7 11.9 L2
CD 43.3 70.1 78.5 99.7 103.4 60.1 L5
HD 71.0 71.5 81.0 80.7 90.8 19.8 L5
As showed in previous reports, two-step process integrated with dark- and photo-fermentation obtained extremely high hydrogen yields [24,32e34], but the rate limiting step of the whole process is the photo-fermentation step [35]. One-step process using reducing sugar as substrate for photofermentation could be an alternative way of the two-step process. As proven by Abo-Hashesh et al., a hydrogen yield of 3.3 mol-H2/mol-glucose and 4.73 mol-H2/mol-glucose could be obtained in batch tests [27,36], and the yield increased dramatically into 9.0 mol-H2/mol-glucose when the pH value of the culture was controlled properly in continuous process
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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Table 5 e Confirmation experiments for orthogonal experiments. No. Y1 Y2 Y3 Y4 Y5
T/ C
NaOH/%
CD/IU/g-CS
HD/IU/g-CS
t/h
Reducing sugar yield/g/g-CS
108 108 98 108 108
0.75 0.75 0.75 0.75 0.75
12 18 12 12 12
2400 2400 2400 2400 /
0.5 0.5 0.5 1 1
0.56 ± 0.03 0.56 ± 0.04 0.52 ± 0.04 0.52 ± 0.02 0.50 ± 0.03
PE/% 67 67 65 68 64
± 3.2 ± 2.1 ± 2.4 ± 3.6 ± 3.3
n/H2max 7.3 6.5 6.4 6.1 4.7
± 0.6 ± 0.4 ± 0.3 ± 0.5 ± 0.6
HY/mL/g-CS 163.1 ± 157.7 ± 158.9 ± 154.1 ± 149.0 ±
5.3 6.1 3.7 2.5 6.3
PE: pretreatment efficiency; HY: hydrogen yield. n/H2max: maximum hydrogen production rate, mL-H2/g-CS/h.
[37]. As shown in our previous report [28], when proper amount of acetate and butyrate were co-fed with glucose to Rubrivivax gelatinosus M002, some synergistic effect showed up and the pH value could be tuned automatically. In this study, the cornstalk hydrolysate was also fed to R. sphaeroides HY01, which is a purple non-sulfur bacterium with high performance on photo-fermentative H2 production [27], for studying its potential on one-step photo-fermentative hydrogen production. Fig. 3 shows the cumulative hydrogen yield of photofermentation using cornstalk hydrolysate prepared by the optimum hydrolysis and enzymolysis parameters obtained above. The hydrogen yields were 339.5 mL/g-CS (4.87 mol-H2/ mol-reducing sugar, 401.5 mL/g-TVS) at 5 g-CS/L, 239.9 mL/gCS at 10 g-CS/L and 43.2 mL/g-CS at 15 g-CS/L. The rapid decrease of hydrogen yield when the substrate concentration increased might be due to the decrease of pH value in the batch test culture since it was 6.56, 5.3 and 4.8 respectively in the final culture, and this phenomenon was also observed in our previously report [27]. When with the substrate of 15 g-CS/ L, the extremely low hydrogen yield may also suggest the inhibition effect of the hydrolyste [27,36]. The maximum hydrogen production rates were 74 mL/(Lh) at 5 g-CS/L, 71 mL/ (Lh) at 10 g-CS/L and 72 mL/(Lh) at 15 g-CS/L. The hydrogen yield increased by 108% compared to that obtained in darkfermentation at 5 g-CS/L. While the maximum hydrogen production rate decreased from about 90 mL/(Lh) to 74 mL/(Lh) in batch tests. In our previous work [27], the hydrogen production rates of R. sphaeroides HY01 with glucose or xylose as
substrates were around 90 mL/(Lh). The color of cornstalk hydrolysate may block part of light and shows some negative effect on hydrogen production compared with those with glucose or xylose as substrate. Therefore, additional efforts should be paid to pretreat the hydrolysate for more efficient photo-fermentative hydrogen production.
Current state of hydrogen yield from agricultural wastes In order to compare the hydrogen yields from pretreated agricultural wastes, a series of reported data are listed in Table 6. Ref. [38] reported that a hydrogen yield of 176 mL/g-TVS was obtained when microbe pretreated cornstalk was used as substrate and lesser panda dung was used as inoculum. The amount of microbe additive used was 7.5 g/kg-cornstalk, and the pretreatment time was extended to 15 days at 25 C for obtaining the high hydrogen yield. The function of this microbe additive is similar with that of cellulase, but the amount used was much less than that of cellulase, hence, it must be more economic than using cellulase if the pretreatment period could be shortened. In Ref. [3], cornstalk was pretreated for 7.5 days by solid state enzymolysis, and 205.5 mL-H2/g-TVS was observed. The hydrogen yield of 192.9 mL/g-TVS obtained in this study was slightly lower than the highest reported datum. Since we know that the cornstalk is composed of around 30% cellulose and 30% hemicellulose [11], the hydrogen yield of around 200 mL/g-TVS might be close to the maximum by using the current mixture culture fermentation. More attention should be paid to develop cheap pretreatment methods and enzymes. Since alkali shows much less intense of corrosion on steel reactors than diluted acid, Using cheaper alkali like Ca(OH)2 and with higher operation temperature might be able to shorten pretreatment time and reduce the enzyme and alkali dosage, and thus reduce the pretreatment cost.
Conclusions
Fig. 3 e Cumulative photo-fermentative hydrogen production profiles using different concentration of corn stalk hydrolysate.
A L25 (55) orthogonal experimental array was designed and conducted to optimize the key parameters affecting hydrogen production including NaOH concentration, alkaline hydrolysis temperature, alkaline hydrolysis temperature, cellulase and hemicellulase dosage. When the cornstalk was only pretreated with NaOH, a hydrogen yield of 34.1 mL/g-cornstalk was obtained. The hydrogen yield increased to 163.1 mL/g-CS in dark-fermentation and 339.5 mL/g-CS in photo-
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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Table 6 e List of hydrogen yield from published papers. Feedstock treatment-biomass Cornstalk treated by microbe Cornstalk by solid state enzymolysis Cornstalk treated by diluted HCl Wheat straw treated by diluted HCl Cornsalk treated by gas explosion Cellulase treated cornstalk Microwave-assisted alkali-cellulase treated Rice straw Microwave-assisted alkali-cellulase treated Rice straw Diluted HCl-Cellulase treated Corncob Ca(OH)2-Accellerase-1500 treated wheat straw Alkali-cellulase corn cob Ozonation-cellulase wheat straw Alkaline-cellulase treated cornstalk Alkaline-cellulase treated cornstalk a b c d
Inoculum Lesser panda dung Lesser panda dung Aerated cow dung Cow dungc Activated sludge Sludgec Activated sludge
Max. H2 rate mL/(L h)
H2 yield mL/g-TVS
18a e 50.7 10.1a e 12.31a 169
176 205.5 149.7 68.1 65b 164.5 155
[38] [39] [13] [40] [14] [16] [41]
Ref.
Two-stage
e
463
[41]
Two-stage Sewage sludge
e e
738 58.8
[24] [20]
100
[23]
80.7 192.9 401.5
[10] This study This study
Clostridium hydrogeniproducens HR-1 Cow manure Cow dungc R. sphaeroides HY01d
52
110 74
mL/(g-TS-h). mL/g-cornstalk or mL/g-corncob. Heat shocked. Operated under photosynthetic condition.
fermentation under the optimum pretreatment conditions with NaOH 0.75%, temperature 108 C, alkali hydrolysis time 0.5 h, cellulase dosage 12 IU/g-CS and hemicellulase dosage 2400 IU/g-CS. The additional hemicellulase could increase the hydrogen yield remarkably at low cellulase dosage, but it only increased the hydrogen yield slightly when the cellulase dosage was higher than 9 IU/g-CS. The overtreatment of cornstalk with NaOH could degrade the hemicellulose, and therefore resulted in the decrease of hydrogen yield.
Acknowledgments The authors thank greatly for the financial support of the National Natural Science Foundation of China for the Youth (No. 51308452) and the National Key Basic Research Program 973 Project founded by MOST of China (No. 2012CB215303). One author (HY) was supported by “the Fundamental Research Funds for the Central University” and Specialized Research Fund for the Doctoral Program of Higher Education (No. 20120201120058).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ijhydene.2015.07.062.
references
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Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkalicellulase two-step hydrolysis Honghui Yang a,b,*, Bofang Shi a, Hongyu Ma a, Liejin Guo b a b
Department of Environmental Science and Engineering, Xi'an Jiaotong University, Xi'an 710049, PR China State Key Laboratory of Multiphase Flow in Power Engineering, Xi'an Jiaotong University, Xi'an 710049, PR China
article info
abstract
Article history:
The effect of a two-step cornstalk pretreating process, NaOH delignification followed by
Received 8 May 2015
enzymolysis with cellulase and hemicellase, on dark- and photo-fermentative H2 production
Received in revised form
was studied. A five-factor and five-level orthogonal experimental array was designed and
2 July 2015
conducted to study the effect of NaOH concentration (0e1%), hydrolysis time (0e3 h), hydrolysis
Accepted 12 July 2015
temperature (98e126 C), cellulase (0e18 IU/g-cornstalk) and hemicellulase dosage (0e2400 IU/
Available online xxx
g-cornstalk) on pretreatment efficiency determined by H2 production. With NaOH 0.75%, hydrolysis temperature 108 C, hydrolysis time 0.5 h, cellulase dosage 12 IU/g-CS and hemi-
Keywords:
cellulase 2400 IU/g-CS, a maximum reducing sugar yield of 0.56 ± 0.03 g/g-CS and maximum H2
Hydrogen production
yield of 163.1 mL-H2/g-cornstalk were obtained under dark-fermentation, and a maximum H2
Cornstalk
yield of 339.5 mL-H2/g-cornstalk was obtained under photo-fermentation. According to the
NaOH pretreatment
results, the significance of the five parameters on H2 production was listed in high-to-low order
Orthogonal experimental array
as: NaOH concentration, cellulase dosage, hydrolysis temperature, hemicellulase and hydro-
Photo-fermentative hydrogen
lysis time. The effect of the alkaline delignification on pretreatment efficiency and the future
production
efforts on improving H2 production from agricultural wastes were also discussed. Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Dark-fermentative process is accepted as a promising way for sustainable hydrogen production [1]. But it is impractical to use the regular substrates such as glucose, sucrose and starch to produce hydrogen for the high price of these food-based materials and food shortage [2,3]. Cellulosic biomass, such as agricultural straw, is abundant and potential feedstock for fermentative hydrogen production. Although some thermophilic bacteria are able to produce hydrogen directly from
cellulosic biomass [4e7], these organisms showed low hydrogen production rate and most of them need additional vitamins for growth, which makes it uneconomical for industrial application. Cellulase is efficient in enzymolysis of cellulosic biomass specifically without producing inhibitory by-products for fermentation, such as acetate, furan derivates and phenolic monomers [8], which could be produced by chemical hydrolysis. The enzymolysis efficiency was quite low when the cellulosic biomass was directly applied as substrate without chemical pretreatment [9]. Pretreatment is an effective
* Corresponding author. Department of Environmental Science and Engineering, Xi'an Jiaotong University, Xi'an 710049, PR China. Tel./ fax: þ86 29 82664731. E-mail address: [email protected] (H. Yang). http://dx.doi.org/10.1016/j.ijhydene.2015.07.062 0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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process in promoting the digestibility of cellulosic materials [10] by cellulase and increasing its fermentative performance [11,12]. Fan et al. employed cornstalk pretreated by diluted HCl as feedstock and heat-shocked cow dung as inoculum, and a maximum hydrogen yield of 149.69 mL/g-TVS was obtained at initial pH 7.0 and substrate concentration of 15 g/L [13]. Datar et al. [14] found that the optimum parameters for gas explosion of cornstalk were pH 7.0, temperature 220 C and reaction time 3 min, and the maximum hydrogen yield was 25.6 mL/gcornstalk. Ren et al. employed acetic acid-gas explosion and followed by cellulase hydrolysis process to pretreat cornstalk [15]. They obtained a maximum hydrogen yield of 72 mL/gcornstalk when acetic acid concentration was 16% and cellulase dosage was 120 IU and 180 IU, respectively. Wang et al. designed a set of orthogonal experiments to optimize the parameters of cornstalk enzymolysis [16]. It was found that enzymolysis temperature 50 C, enzymolysis time 72 h, initial pH 7.0 and substrate concentration 10 g/L were the optimum parameters, and maximum hydrogen yield of 141.3 mL/gcornstalk was obtained. Cornstalk, as a typical agricultural waste, mainly comprises of cellulose, hemicellulose and lignin. The three components are highly crystallized and convolved with each other to hinder the enzymolysis efficiency. Cellulose is packed into microfibrils by hydrogen bonds and Van der Waals bonds. Hemicellulose and lignin cover the microfibrils [17]. Lignin imparts structural support, and it is impermeable and resistant to microbial attack [18]. Especially since lignin could not be decomposed by cellulase, it retards the enzymolysis of hemicellulose and cellulose [19]. Lignin is dissolvable in alkaline solution, therefore, alkaline pretreatment could be able to decrease the crystallinity degree of cornstalk and ease its enzymolysis process. Compared with acid pretreatment processes, alkaline pretreatment processes utilize low temperature and pressure, and cause less sugar degradation [20]. NaOH is effective for delignification. Alkaline pretreatment increases the sugars yield about three times comparing with untreated and treated wheat straw reported by Carrillo et al. [21]. The enzymatic digestibility of alkaline pretreated barley straw was greatly enhanced [22]. However, the effect of NaOH on pretreating agricultural wastes for hydrogen production [23], especially for photo-fermentative hydrogen production was not well studies. In this study, in order to evaluated the enhancement effect of alkaline pretreatment on enzymolysis and hydrogen production performance of corn straw, a set of orthogonal experiments were conducted to optimize the operational parameters, including NaOH concentration, alkaline hydrolysis temperature, hydrolysis time, cellulase dosage and hemicellulase dosage. The effects of NaOH pretreatment on cornstalk dissolution ratio and enzymolysis efficiency were studied. The feasibility of one-stage photo-fermentation using corn stalk hydrolysate was also demonstrated.
farmland with water content of 3.49%, ash content of 11.90% and volatile solids (VS) content of 84.61%. Cornstalk was shattered into 20e40 meshes and mixed with tap water in a ratio of 1:15. NaOH was used to pretreat the cornstalk before enzymolysis. The parameters set for alkaline hydrolysis and enzymolysis are listed in Table 1. After alkaline hydrolysis, a certain amount of glutamic acid was supplemented into the mixture to make its final concentration 1 g/L in the medium for fermentation. The pH value of the mixture was then adjusted to 4.80 with 1 mol/L HCl. A certain amount of cellulase (Chende Tianfeng Biotechnology Co. LTD, FPU, 100 IU/g) and hemicellulase (KND biotechnology Co. LTD, 20,000 IU/mL) was added into the mixture, and it was kept at 50 C for 10 h. After the enzymolysis, the pH value of the mixture was adjusted to 7.0 with 1 M NaOH solution. All of the hydrolysis and enzymolysis experiments were conducted in triplicate.
Orthogonal experimental array A L25 (55) orthogonal experimental array was designed and conducted to optimize the key parameters affecting hydrogen production including NaOH concentration (0e1%), alkaline hydrolysis time (0e3 h), alkaline hydrolysis temperature (98 C126 C), cellulose (0e12 IU/g-CS) and hemicellulase dosage (0e2400 IU/g-CS) as shown in Table 1. Range analysis was conducted to analyze the significance of these parameters on hydrogen production [25,26]. Each of the experiments listed in Table 1 was conducted in triplicate.
Batch tests In the dark fermentation tests, the batch experiments were performed with 400 mL homemade glass bottles as reactors filled with heat-shocked cow dung of 2 g, a certain amount of cornstalk hydrolysate and mineral nutrient solution of 12.5 mL, and then the mixture was diluted to 250 mL with water. The final concentration of cornstalk hydrolysate was 15 g-CS/L. The components of the mineral nutrient were as follows: (g/L) KH2PO4 5, CaCl2 0.2, FeSO4$7H2O 0.13, MgSO4 1.0, Na2MO4$2H2O 0.06, MnSO4$H2O 0.3. These reactors were filled with pure nitrogen gas for 10 min to remove oxygen to obtain the initial anaerobic environment. The reactors were then set in a water bath shaker at 150 rpm and 35 ± 0.5 C. For photo-fermentation using corn stalk hydrolysate as substrate, the hydrolysate were centrifuged to remove the solid residue, and then diluted with H2O to concentration of 5 g-CS/L, 10 g-CS/L and 15 g-CS/L for photo-fermentation. Rhodobacter sphaeroides HY01 used as inocula was precultured as described in literature [27]. 30 mL syringes were employed as photo-reactor as shown in literature [28]. Each of the experiments in dark-fermentation and photofermentation were conducted in triplicate.
Analytical methods
Materials and methods Inoculum and feedstock The heat-shocked cow dung was employed as inoculum as described previously [24]. Cornstalk (CS) was harvested from
Volumes of the biogas were measured with volumetric bottle as described previously [29], and then calculated into that in standard condition (273.15 K, 1 atm). The components of biogas were analyzed as shown in previous report [27]. Briefly, a gas chromatography (GC, Beifen Co., Ltd. Model, SP-2100)
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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Table 1 e Parameters for orthogonal experiments. Test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
T/ C
NaOH/%
t/h
CD/IU/g-CS
HD/IU/g-CS
98 98 98 98 98 108 108 108 108 108 115 115 115 115 115 121 121 121 121 121 126 126 126 126 126
0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1
0.25 0.5 1 2 3 2 3 0.25 0.5 1 0.5 1 2 3 0.25 3 0.25 0.5 1 2 1 2 3 0.25 0.5
0 3 6 9 12 3 6 9 12 0 6 9 12 0 3 9 12 0 3 6 12 0 3 6 9
0 600 1200 1800 2400 1200 1800 2400 0 600 2400 0 600 1200 1800 600 1200 1800 2400 0 1800 2400 0 600 1200
Pretreatment efficiency % 4.4 ± 12.0 ± 16.3 ± 30.3 ± 41.2 ± 23.2 ± 27.2 ± 17.8 ± 41.0 ± 51.7 ± 16.9 ± 29.3 ± 38.7 ± 52.3 ± 49.2 ± 18.8 ± 30.0 ± 45.4 ± 50.6 ± 49.6 ± 22.8 ± 30.0 ± 45.8 ± 48.0 ± 52.8 ±
Enzymolysis efficiency% 5.2 ± 40.3 ± 66.2 ± 70.6 ± 70.2 ± 32.3 ± 47.5 ± 69.2 ± 73.0 ± 55.6 ± 34.5 ± 50.5 ± 72.0 ± 54.6 ± 66.5 ± 36.8 ± 44.9 ± 46.7 ± 58.6 ± 54.1 ± 28.9 ± 39.3 ± 48.5 ± 51.9 ± 65.1 ±
1.2 1.4 2.1 3.2 2.2 4.3 1.6 3.3 2.1 2.3 1.3 2.7 1.6 1.4 1.3 2.1 2.5 3.3 3.7 4.1 2.0 3.2 4.1 6.0 4.7
1.3 3.1 4.2 5.1 4.7 1.6 2.8 3.7 3.9 2.3 1.8 2.5 4.6 2.5 3.8 2.1 1.2 2.1 3.1 2.9 1.6 1.8 2.3 2.5 3.6
T: temperature; NaOH: NaOH concentration; CD: cellulase dosage; HD, hemicellulase dosage; t, hydrolysis time.
equipped with a thermal conduct detector and a 4 m stainless chromatographic column filled with Hayesep padding was used to determine the gas products. Concentrations of volatile fatty acids (VFAs) in the liquid samples were analyzed with a GC (Varian, CP-3800) equipped with a flame ionization detector and a capillary column coated with terephthalic acid modified PEG (AT.FFAP). The alkaline pretreatment efficiency and enzymolysis efficiency were determined by gravimetric method. The reducing sugar content was determined by anthrone-sulfuric method [30]. Pretreatment efficiency was defined by the percentage of dissolved cornstalk after alkaline hydrolysis, and enzymolysis efficiency stands for the percentage of dissolved cornstalk after enzymolysis. The cumulative hydrogen production data were fitted with modified Gompertz equation [3]. The experimental data points were described as average value plus deviation.
Results and discussion
and so on. And R stands for the largest range of the data in five levels. The largest range of pretreatment efficiency from level 1 to level 5 was 32.68% in terms of NaOH concentration, which means NaOH concentration showed the most significant effect on pretreatment efficiency. The range 19.08% reflected the effect of temperature on pretreatment efficiency. And hydrolysis time, at a range of 6.58%, showed the least significance on pretreatment efficiency. As for the hydrogen production performance, NaOH concentration also showed the largest range 19.18 mL/g-CS among the three factors. Temperature and hydrolysis time showed less significance at a range of 4.44 mL/ g-CS and 5.16 mL/g-CS, respectively. The optimum parameters based on pretreatment efficiency were NaOH 1%, temperature 126 C and hydrolysis time 3 h. The maximum
Table 2 e Range analysis of the effect of temperature (T), NaOH concentration and hydrolysis time (t) on pretreatment efficiency and hydrogen production performance of cornstalk. Pretreatment efficiency/%
Effect of alkaline hydrolysis on cornstalk dissolution and hydrogen yield Table 1 shows the alkaline pretreatment efficiency and enzymolysis efficiency of cornstalk in orthogonal experiments. Table 2 shows the effect of temperature, NaOH concentration and hydrolysis time on pretreatment efficiency and hydrogen production performance of pretreated cornstalk. In Table 2, L1 stands for level 1 in each factor. For instance, (L1, T) means the average datum obtained from the L25 (55) orthogonal experimental array at the temperature level 1 of 98 C,
L1 L2 L3 L4 L5 R Optimal level
T 20.80 30.18 37.28 38.48 39.88 19.08 L5
NaOH 15.22 28.10 32.40 43.24 47.90 32.68 L5
t 30.08 32.62 34.14 32.36 36.66 6.58 L5
Hydrogen yield/mL/g-CS T 18.30 22.74 21.98 21.62 21.52 4.44 L2
NaOH 8.16 19.76 26.60 27.34 26.32 19.18 L4
t 19.76 23.94 18.78 22.66 23.04 5.16 L2
L1-L5: level 1-level 5.
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hydrogen yield of 34.1 mL H2/g-CS appeared at NaOH of 0.75%, temperature of 108 C and hydrolysis time of 0.5 h for the pretreated cornstalk. Fig. 1 shows the reducing sugar yields and hydrogen yields of NaOH pretreated cornstalks. As shown in Fig. 1, the hydrogen yield increased from 9 mL H2/g-CS in test 1 to 34.1 mL H2/g-CS in test 9. The main products of the effluent from darkfermentation were acetate and butyrate, and the concentrations of acetate and butyrate were around 1 g/L and 0.5 g/L, respectively. The concentration of propionate below 0.2 g/L was detected in most of the tests. The pH values of the effluents were around 5.2e6.0. The corresponding pretreatment efficiency increased by 9 times ranging from 4.4% in test 1 to 41.2% in test 9, suggesting that although 41.2% cornstalk was converted to soluble organic matter, but the reducing sugar content of cornstalk only increased from 0.07 g/g-CS into 0.19 g/gCS in the whole hydrolysis process. The result indicated that the most of hydrolysate could not be utilized by microbes to produce hydrogen except reducing sugar. The NaOH pretreatment dissolved lignin and hemicelluose, but few useful saccharide was produced as shown in Fig. 1 and previous report [14]. Therefore, the NaOH pretreatment was effective on decomposing the cornstalk structure formed by lignin, hemicellulose and cellulose, and another treatment should be applied for increasing the reducing sugar content of the hydrolysate to improve hydrogen production performance. All of these data suggest that these tests showed normal butyric acid metabolic pathway and the reason for the low hydrogen yield was the low amount of reducing sugar content in hydrolysate. Sodium hydroxide and hydrogen peroxide were employed to pretreat soybean straw, and a maximum hydrogen yield of 60.2 mL/g-straw was obtained, which was quite lower than those obtained from dilute acid or enzyme preteated biomass [12]. Therefore, an effective saccharification method is needed for increasing the hydrogen yield.
Effect of alkaline-enzymatic treatment on cornstalk dissolution and hydrogen yield The enzymolysis efficiencies of cornstalk obtained by cellulase and hemicellulase were shown in Table 1. In test 2, the
dissolution ratio of cornstalk increased from 12% to 40.3% when cellulase and hemicellulase dosage was 3 IU/g-CS and 600 IU/g-CS, respectively. From test 3 to test 5, the dissolution ratio of cornstalk increased further to around 70%. In test 10, 14 and 22, in which only hemicellulase was used for enzymolysis, it only increased from 51.7%, 52.3% and 30% into 55.6%, 54.6% and 39.3%, respectively. As mentioned before, part of hemicellulose was dissolved in alkaline hydrolysis; therefore, hemicellulase only increased the dissolution ratio of cornstalk slightly. In test 23, 24 and 25, the dissolution ratio of cornstalk increased from 45.8%, 48% and 52.8% to 48.5%, 51.9% and 65.1%, respectively. Enzymolysis efficiency increased only by 2.7%, 3.9% and 12.1%, which was much less than that of 28.3%, 49.9%, 40.3% and 29% obtained from test 2 to test 5, respectively. The reason was that low alkaline pretreatment efficiency was obtained at lower alkaline hydrolysis temperature, and less hemicellulose was degraded into volatile fatty acids, thus the enzymatic hydrolysis efficiencies were increased dramatically. Many volatile fatty acids are inhibitors and not fermentable for hydrogen production [31]. A considerable amount of acetic acid was detected in the NaOH pretreated mixture in this study. Hence, the more hemicellulose remained in solid after NaOH pretreatment, the higher reducing sugar and the lower inhibitors concentration of the hydrolysate were obtained by enzymolysis. According to the results of range analysis, as shown in Table 3, the significance of these five parameters follows the order: NaOH concentration > cellulase dosage > hydrolysis temperature > hemicellulase dosage > hydrolysis time. The highest enzymolysis efficiency was obtained in the followed treatment condition: NaOH 0.75%, cellulase dosage (CD) 12 IU/ g-CS, temperature (T) 108 C, hemicellulase dosage (HD) 2400 IU/g-CS and alkali hydrolysis time (t) 0.5 h. Fig. 2 shows the hydrogen yields of these 25 tests, and Fig. 1S (Supplementary Information) shows the cumulative hydrogen production profiles of these 25 tests. In test 9, the highest hydrogen yield of 153.9 mL/g-CS and reducing sugar yield of 0.52 ± 0.03 g/g-CS were obtained when the parameters were NaOH 0.75%, T 108 C, t 0.5 h, CD 12 IU/g-CS. Table 4 shows the range analysis results on hydrogen yield according to the mean value of each level. As shown in Table 4, in the control (L1), the hydrogen yield of 37.5 mL/g-CS was obtained. Thereafter the hydrogen yield was gradually increased with the increase of NaOH concentration in the CS pretreatment
Table 3 e Range analysis the effect of temperature (T), NaOH concentration, hydrolysis time (t), cellulase (CD) and hemicellulase dosage (HD) on the dissolution ratio of cornstalk. Pretreatment efficiency/%
Fig. 1 e Reducing sugar yields and dark-fermentative hydrogen production performance of cornstalk pretreated with NaOH hydrolysis.
T1 T2 T3 T4 T5 R Optimal level
T 52.8 57.5 55.0 53.4 44.8 12.7 L2
NaOH 27.4 48.5 59.1 65.1 63.4 37.7 L4
t 47.4 51.4 53.1 56.4 55.1 9.0 L4
CD 37.5 51.0 56.8 58.8 59.4 21.9 L5
HD 46.7 49.3 51.5 56.8 59.1 12.4 L5
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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5
According to the range analysis, the optimum parameters based on hydrogen yield were listed as follows: NaOH 0.75%, CD 12 IU/g-CS, T 108 C, HD 2400 IU/g-CS and t 0.5 h.
Confirmation experiments
Fig. 2 e Reducing sugar yields and dark-fermentative hydrogen yields of cornstalk after NaOH pretreatment and cellulase-hemicellulase enzymolysis.
from 62.9 mL/g-CS for 0.25% NaOH to maximum value of 103.8 mL/g-CS for 0.75% NaOH (L2~L4), and then the hydrogen yield was decreased with further increase of NaOH concentration(L5). We also found that both cellulase and hemicellulase were employed as the enzymolysis step in the case of without NaOH pretreatment, the hydrogen yield was only similar with that of NaOH pretreatment alone. The results indicated that the NaOH pretreatment significantly affected the hydrogen production performance of the CS. The cellulase dosage showed the second significant influence with a range of 60.1 mL/g-CS. Without cellulase, the average hydrogen yield was 43.3 mL/g-CS; this is close to the mean value that obtained without NaOH pretreatment. The mean value of hydrogen yield increased with the increase of cellulase dosage and maximum yield of 103.4 mL/g-CS was obtained when the cellulase dosage was 12 IU/g-CS. In the range from 98 C to 126 C, the alkaline hydrolysis temperature showed the third significant influence. The mean value reached the maximum, 90 mL/g-CS at 108 C, and then reduced into 68.7 mL/g-CS at 126 C. Hemicellulase showed less effect on hydrogen yield. The mean value of hydrogen yield based on hemicellulase dosage increased from 71.0 mL/ g-CS to 90.8 mL/g-CS when it was increased from 0 to 2400 IU/ g-CS. From 0.25 h to 3 h, hydrolysis time showed the slightest influence on hydrogen yield. And when it was 0.5 h, the highest hydrogen yield of 87.6 mL/g-CS was obtained.
According to the above results of orthogonal experiments, the confirmation experiments were also arranged, and the results were listed in Table 5 and Fig. 2S (Supplementary Information), respectively. As shown in Table 5 and Fig. 2S (Supplementary Information), Y1 listed reducing sugar yield and hydrogen yield to be 0.56 ± 0.03 g/g-CS and 163.1 mL/g-CS (equals 192.9 mL/g-TVS) under the optimum conditions from orthogonal experiment. It was about 6% higher than that obtained in test 9. This slight increase of hydrogen yield was mainly attributed to the function of hemicellulase. In this case, the enzymolysis efficiency of hemicellulose increased slightly. A cellulase dosage of 18 IU/g-CS was conducted in Y2. The hydrogen yield of Y2 was 157.7 mL/g-CS, which was slightly lower than that of Y1, and the reducing sugar yield, 0.56 ± 0.04 g/g-CS, was also close to that obtained in Y1. And the enzymolysis efficiency of cornstalk was similar with that of Y1. This means the increase of cellulase dosage over 12 IU/ g-CS could not increase the hydrogen yield, and this might be caused by some inhibition factors and it is consistent with Carrillo's report [21]. The results of range analysis indicated that the effect of alkaline hydrolysis temperature at 98 C was close to those at 108 C, hence the experimental conditions of Y3 was the same as that of Y1 except for hydrolysis temperature of 98 C, the hydrogen yield of 158.9 mL H2/g-CS was close to that of Y1. In test Y4, the alkaline hydrolysis time was increased from 0.5 h into 1 h, while the hydrogen yield was lower than that of Y1. This result suggests that longer alkaline hydrolysis time could not increase the hydrogen yield. Comparing the hydrogen yield in test Y4 and Y5, it was noticed that hemicellulase played a less important role. The commercial cellulase used in this study was not purified and it showed some hemicellulase activity, thus, when high dosage of cellulase was used, its hemicellulase activity was enough for hemicellose enzymolysis, and the additional hemicellulase could not increase the enzymolysis efficiency clearly. While in low cellulase dosage, additional hemicellulase could increase the hydrogen yield. The results indicate that additional hemicellulase is not needed when the dosage of cellulase was higher than 9 IU/g-CS.
Photosynthetic hydrogen production using corn stalk hydrolysate Table 4 e Range analysis the effect of temperature (T), NaOH concentration, hydrolysis time(t), cellulase (CD) and hemicellulase dosage (HD) on the hydrogen yield. Hydrogen yield/mL/g-CS L1 L2 L3 L4 L5 R Optimal level
T 86.4 90.0 83.5 66.4 68.7 23.6 L2
NaOH 37.5 62.9 93.3 103.8 97.5 66.3 L4
t 76.1 87.6 76.9 78.7 75.7 11.9 L2
CD 43.3 70.1 78.5 99.7 103.4 60.1 L5
HD 71.0 71.5 81.0 80.7 90.8 19.8 L5
As showed in previous reports, two-step process integrated with dark- and photo-fermentation obtained extremely high hydrogen yields [24,32e34], but the rate limiting step of the whole process is the photo-fermentation step [35]. One-step process using reducing sugar as substrate for photofermentation could be an alternative way of the two-step process. As proven by Abo-Hashesh et al., a hydrogen yield of 3.3 mol-H2/mol-glucose and 4.73 mol-H2/mol-glucose could be obtained in batch tests [27,36], and the yield increased dramatically into 9.0 mol-H2/mol-glucose when the pH value of the culture was controlled properly in continuous process
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Table 5 e Confirmation experiments for orthogonal experiments. No. Y1 Y2 Y3 Y4 Y5
T/ C
NaOH/%
CD/IU/g-CS
HD/IU/g-CS
t/h
Reducing sugar yield/g/g-CS
108 108 98 108 108
0.75 0.75 0.75 0.75 0.75
12 18 12 12 12
2400 2400 2400 2400 /
0.5 0.5 0.5 1 1
0.56 ± 0.03 0.56 ± 0.04 0.52 ± 0.04 0.52 ± 0.02 0.50 ± 0.03
PE/% 67 67 65 68 64
± 3.2 ± 2.1 ± 2.4 ± 3.6 ± 3.3
n/H2max 7.3 6.5 6.4 6.1 4.7
± 0.6 ± 0.4 ± 0.3 ± 0.5 ± 0.6
HY/mL/g-CS 163.1 ± 157.7 ± 158.9 ± 154.1 ± 149.0 ±
5.3 6.1 3.7 2.5 6.3
PE: pretreatment efficiency; HY: hydrogen yield. n/H2max: maximum hydrogen production rate, mL-H2/g-CS/h.
[37]. As shown in our previous report [28], when proper amount of acetate and butyrate were co-fed with glucose to Rubrivivax gelatinosus M002, some synergistic effect showed up and the pH value could be tuned automatically. In this study, the cornstalk hydrolysate was also fed to R. sphaeroides HY01, which is a purple non-sulfur bacterium with high performance on photo-fermentative H2 production [27], for studying its potential on one-step photo-fermentative hydrogen production. Fig. 3 shows the cumulative hydrogen yield of photofermentation using cornstalk hydrolysate prepared by the optimum hydrolysis and enzymolysis parameters obtained above. The hydrogen yields were 339.5 mL/g-CS (4.87 mol-H2/ mol-reducing sugar, 401.5 mL/g-TVS) at 5 g-CS/L, 239.9 mL/gCS at 10 g-CS/L and 43.2 mL/g-CS at 15 g-CS/L. The rapid decrease of hydrogen yield when the substrate concentration increased might be due to the decrease of pH value in the batch test culture since it was 6.56, 5.3 and 4.8 respectively in the final culture, and this phenomenon was also observed in our previously report [27]. When with the substrate of 15 g-CS/ L, the extremely low hydrogen yield may also suggest the inhibition effect of the hydrolyste [27,36]. The maximum hydrogen production rates were 74 mL/(Lh) at 5 g-CS/L, 71 mL/ (Lh) at 10 g-CS/L and 72 mL/(Lh) at 15 g-CS/L. The hydrogen yield increased by 108% compared to that obtained in darkfermentation at 5 g-CS/L. While the maximum hydrogen production rate decreased from about 90 mL/(Lh) to 74 mL/(Lh) in batch tests. In our previous work [27], the hydrogen production rates of R. sphaeroides HY01 with glucose or xylose as
substrates were around 90 mL/(Lh). The color of cornstalk hydrolysate may block part of light and shows some negative effect on hydrogen production compared with those with glucose or xylose as substrate. Therefore, additional efforts should be paid to pretreat the hydrolysate for more efficient photo-fermentative hydrogen production.
Current state of hydrogen yield from agricultural wastes In order to compare the hydrogen yields from pretreated agricultural wastes, a series of reported data are listed in Table 6. Ref. [38] reported that a hydrogen yield of 176 mL/g-TVS was obtained when microbe pretreated cornstalk was used as substrate and lesser panda dung was used as inoculum. The amount of microbe additive used was 7.5 g/kg-cornstalk, and the pretreatment time was extended to 15 days at 25 C for obtaining the high hydrogen yield. The function of this microbe additive is similar with that of cellulase, but the amount used was much less than that of cellulase, hence, it must be more economic than using cellulase if the pretreatment period could be shortened. In Ref. [3], cornstalk was pretreated for 7.5 days by solid state enzymolysis, and 205.5 mL-H2/g-TVS was observed. The hydrogen yield of 192.9 mL/g-TVS obtained in this study was slightly lower than the highest reported datum. Since we know that the cornstalk is composed of around 30% cellulose and 30% hemicellulose [11], the hydrogen yield of around 200 mL/g-TVS might be close to the maximum by using the current mixture culture fermentation. More attention should be paid to develop cheap pretreatment methods and enzymes. Since alkali shows much less intense of corrosion on steel reactors than diluted acid, Using cheaper alkali like Ca(OH)2 and with higher operation temperature might be able to shorten pretreatment time and reduce the enzyme and alkali dosage, and thus reduce the pretreatment cost.
Conclusions
Fig. 3 e Cumulative photo-fermentative hydrogen production profiles using different concentration of corn stalk hydrolysate.
A L25 (55) orthogonal experimental array was designed and conducted to optimize the key parameters affecting hydrogen production including NaOH concentration, alkaline hydrolysis temperature, alkaline hydrolysis temperature, cellulase and hemicellulase dosage. When the cornstalk was only pretreated with NaOH, a hydrogen yield of 34.1 mL/g-cornstalk was obtained. The hydrogen yield increased to 163.1 mL/g-CS in dark-fermentation and 339.5 mL/g-CS in photo-
Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062
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Table 6 e List of hydrogen yield from published papers. Feedstock treatment-biomass Cornstalk treated by microbe Cornstalk by solid state enzymolysis Cornstalk treated by diluted HCl Wheat straw treated by diluted HCl Cornsalk treated by gas explosion Cellulase treated cornstalk Microwave-assisted alkali-cellulase treated Rice straw Microwave-assisted alkali-cellulase treated Rice straw Diluted HCl-Cellulase treated Corncob Ca(OH)2-Accellerase-1500 treated wheat straw Alkali-cellulase corn cob Ozonation-cellulase wheat straw Alkaline-cellulase treated cornstalk Alkaline-cellulase treated cornstalk a b c d
Inoculum Lesser panda dung Lesser panda dung Aerated cow dung Cow dungc Activated sludge Sludgec Activated sludge
Max. H2 rate mL/(L h)
H2 yield mL/g-TVS
18a e 50.7 10.1a e 12.31a 169
176 205.5 149.7 68.1 65b 164.5 155
[38] [39] [13] [40] [14] [16] [41]
Ref.
Two-stage
e
463
[41]
Two-stage Sewage sludge
e e
738 58.8
[24] [20]
100
[23]
80.7 192.9 401.5
[10] This study This study
Clostridium hydrogeniproducens HR-1 Cow manure Cow dungc R. sphaeroides HY01d
52
110 74
mL/(g-TS-h). mL/g-cornstalk or mL/g-corncob. Heat shocked. Operated under photosynthetic condition.
fermentation under the optimum pretreatment conditions with NaOH 0.75%, temperature 108 C, alkali hydrolysis time 0.5 h, cellulase dosage 12 IU/g-CS and hemicellulase dosage 2400 IU/g-CS. The additional hemicellulase could increase the hydrogen yield remarkably at low cellulase dosage, but it only increased the hydrogen yield slightly when the cellulase dosage was higher than 9 IU/g-CS. The overtreatment of cornstalk with NaOH could degrade the hemicellulose, and therefore resulted in the decrease of hydrogen yield.
Acknowledgments The authors thank greatly for the financial support of the National Natural Science Foundation of China for the Youth (No. 51308452) and the National Key Basic Research Program 973 Project founded by MOST of China (No. 2012CB215303). One author (HY) was supported by “the Fundamental Research Funds for the Central University” and Specialized Research Fund for the Doctoral Program of Higher Education (No. 20120201120058).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ijhydene.2015.07.062.
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Please cite this article in press as: Yang H, et al., Enhanced hydrogen production from cornstalk by dark- and photo-fermentation with diluted alkali-cellulase two-step hydrolysis, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/ j.ijhydene.2015.07.062