Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder

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Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder Shengnan Zhu, Zhiping Zhang, Yameng Li, Nadeem Tahir, Huiliang Liu, Quanguo Zhang* Key Laboratory of New Materials and Facilities for Rural Renewable Energy, Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China

article info

abstract

Article history:

High solid phase and easily congeal affect the mass transfer in the photo-fermentative

Received 1 June 2018

biohydrogen production when taken straw biomass as substrate. Hence, oscillator was

Received in revised form

adopted to provide the shaking condition to enhance the mass transfer situation in this

8 September 2018

paper. Diverse shaking velocity (0, 80, 120 and 160 rpm) and substrate concentration (0, 2, 4,

Accepted 21 September 2018

6, 8 and 10 g) were studied, to evaluate the influence on the hydrogen yield capacity. The

Available online xxx

results showed that shaking could help to accelerate of gas release, shorten the fermentation time, and improve hydrogen production rate. Hydrogen yield was significantly

Keywords:

enhanced at high substrate concentration under shaking condition. Highest hydrogen yield

Shaking velocity

of 57.08 ± 0.83, 57.62 ± 1.37, 62.28 ± 0.84 mL/g-volatile solids (VS) were observed at shaking

Corn stover powder

velocities of 80, 120 and 160 rpm with 6, 8 and 10 g corn stover powder, respectively. On the

Substrate concentration

contrary, shaking significantly reduced the potential of hydrogen yield at a low substrate

Photo-fermentative hydrogen

concentration, and the lower hydrogen yield obtained at the higher shaking velocity. As the

production

lowest hydrogen yields of 27.68 ± 1.02 and 41.93 ± 0.40 mL/g VS were obtained at shaking velocity of 160 rpm with 2 and 4 g corn stover powder, respectively. © 2018 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC.

Introduction Energy crisis and environmental problems caused by the excessive use of fossil fuels have obtained more and more attention, it is urgent to produce and use renewable energy sources [1e3]. Hydrogen energy, with no greenhouse gas emissions and high energy density (122 kJ/g), is an attractive alternative energy source [4,5]. As one of the various hydrogen production methods, bio-hydrogen production is considered to be a promising approach to produce hydrogen, because that

hydrogen can be produced at room temperature and atmosphere pressure without consuming fossil energy [6e8]. Biohydrogen production mainly includes photo-fermentation and dark-fermentation. Compare with dark-fermentation, photo-fermentation is attracting more attention due to the diversity of the available substrates and high substrate conversion efficiencies [9e11]. It is estimated that about 5.0
109 tons of crop stalks are annually produced in China, and corn stover accounted for the majority. Most of agriculture waste is burned in the open-

* Corresponding author. E-mail address: [email protected] (Q. Zhang). https://doi.org/10.1016/j.ijhydene.2018.09.150 0360-3199/© 2018 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150

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field, which seriously polluted the air [12]. Crop stalk is rich in cellulose and hemicellulose, converting them into valuable energy source, such as methane, ethanol and hydrogen, which can be an effective and sustainable alternative [13e15]. Up to date, there have been many studies on biological hydrogen production using crop stalk as a substrate [16,17]. However, crop stalks have the characteristics of specific physical properties of heterogeneity, low density, and high water-holding capacity, which make it not uniformly distributed inside of reactor, resulting in low hydrogen production potential and substrate conversion efficiency [18,19]. Giving that, mixing methods are proposed due to it can prevent stratification, keep the solids suspension and homogenize the contents of reactors [20e23]. Mixing intensity, as one of the key operational variables, has direct effect on performance of anaerobic fermentation [24e26]. Too high mixing intensity showed a negative effect on substrate-microbe aggregates, resulting in instable performances and low gas production [13,27]. Too low mixing intensity led to sedimentation and floatation of the substrate, gas evolution could not be effectively improved [28]. Optimal mixing intensity is closely related to the substrate concentration, substrate type and mixing mode. Karim et al. [29] showed that yield of biogas can be enhanced by 10%e30% as compare to the unmixed under the cow manure concentration of 100 g/L and 150 g/L, and there was no obvious difference in biogas yield between mixed and unmixed with the substrate concentration 50 g/L. The difference in types of substrate change the rheological properties and thereby affect the optimal operating conditions. When total solid (TS) concentration is relatively high, which will cause high viscosity, and more external forces for mixing to be needed correspondingly [20,30]. Mechanical mixing, hydraulic mixing and pneumatic mixing are the main mixing technology [31e33]. Since mixing plays a key role in exchange of substrate, enhancement of microorganisms growth and gas release, oscillator was adopted to provide the shaking condition to enhance the mass transfer situation in this study. Corn stover powder was taken as substrate, batch experiments were carried out to study the shaking effect on photo-fermentative hydrogen production with different substrate concentrations. A modified Gompertz model was adopted to evaluate the experimental parameter. In addition, the effect of shaking on hydrogen yield, gas quality, pH, reducing sugar and final metabolic by-products were also discussed.

and micronutrient solution (1 mL/L) which contains FeCl3$6H2O（5 mg/L), ZnSO4$7H2O (1 mg/L),CuSO4$5H2O (0.05 mg/L), H3BO4 (1 mg/L), MnCl2$4H2O (0.05 mg/L), and Co(NO3)2$6H2O (0.5 mg/L). The pH value was adjusted to 7 by using 50% (w/w) KOH solution. The consortium was cultured in 500 mL colorless glass bottle for 48 h at 30  C with incandescent lamp provide illumination, light intensity was set 3000 Lux. The hydrogen production medium was supplied through the following chemicals: NH4Cl (0.4 g/L), MgCI2 (0.2 g/ L), yeast extract (0.1 g/L), K2HPO4 (0.5 g/L), NaCl (2 g/L), and sodium glutamate (3.5 g/L).

Raw material Samples of corn stover were collected from an agriculture farm in Zhengzhou, Henan province, China. The raw materials were air-dried, crushed into powder (LG-02, Ruian, China), and screened with 60-mesh sieve. The compositions of corn stover powder were analyzed according to NREL method [35], 39.12 ± 0.68% of cellulose, 30.95 ± 0.54% of hemicellulose, 10.73 ± 0.28% of lignin. Total solids of 95.65%, and volatile solids of 94.21%.

Experimental procedures of photo-fermentative hydrogen production All experiments were carried out in 180 mL conical flasks with corn stover powder, cellulase (35 u/mg), 100 mL citric acid buffer (pH 4.8). The concentrations of corn stover powder in the six groups were 0, 2, 4, 6, 8 and 10 g, respectively, and cellulase was added with enzyme load of 150 mg/g corn stover. The initial pH was adjusted to 6.5 by 50% (w/w) KOH solution. Then, 45 mL photosynthetic consortium in exponential growth phase and 25 mL hydrogen production medium were added into flasks. Argon gas was used to remove oxygen to create the anaerobic condition, and flasks were sealed with rubber stopper. Sealed flasks were placed at constant temperature shaker with shaking velocity of 80, 120 and 160 rpm, respectively. Temperature was adjusted to 30  C and light intensity was set at 6000e7000 Lux. The blank control (0 rpm) was conducted at a constant temperature incubator at temperature of 30  C. The produced gas was collected by gas sampling bags. The volume and composition of gas were measured with 12 h time interval. Each experiment was repeated three times to ensure the accuracy of the data.

Analytical methods

Materials and methods Microorganisms and medium The photosynthetic consortium HAU-M1 were used as the photo-hydrogen producer, which were extracted from mixed substrate of silt sewage, pig manure, and cow dung. It consists of Rhodobacter sphaeroides, Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodopseudomonas palustris [34]. The growth culture medium of photo-fermentation consortium contains NH4Cl (1 g/L), NaHCO3 (2 g/L), K2HPO4 (0.2 g/L), CH3COOHNa (3 g/L), MgSO4$7H2O (0.2 g/L), NaCl (2 g/L), yeast extract (1 g/L),

The composition of produced gas was analyzed by a gas chromatography (6820GC-14B, Agilent, USA) using nitrogen gas as carrier with a flow rate of 45 mL/min. The operational temperature of column oven, injection port, and detector were set to 80  C, 100  C, and 150  C, respectively. The liquid samples were collected at the end of fermentation process. Samples were centrifuged at 10000 rpm for 10 min and filtered with 0.45-mm drainage membrane filer. The filtered supernatant was used for the analysis of Volatile fatty acids (VFAs) and ethanol. The VFAs and ethanol concentrations were measured use the same instrument as measuring gas

Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150

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composition (6820 GC-14B, Agilent, USA), equipped with a capillary column (DB-FFAP) and a flame ionization detector, nitrogen gas was used as carrier with a flow rate of 30 mL/min. The operational temperatures of column oven, injection port, and detector were 100  C, 200  C, 250  C, respectively. Light intensity was measured with a digital light meter (TES-1330, Shanghai, China). pH values were measured with pH meter (PHS-3C, Shanghai, China). The reducing sugar concentrations were calculated by employing the di-nitro salicylic (DNS) colorimetric method [36]. The turbidity of fermented broth was measured by Spectrophotometric [37]. For substrate concentration of 0, 2, 4, 6, 8 and 10 g, the turbidity was 829.09, 3481.82, 4963.64, 7781.82, 9354.55 and 13927.27 NTU, respectively. Fermentation time is defined as a time period, between the starting of inoculating the photosynthetic bacteria into sterilized medium and stopping of no hydrogen generation. Average hydrogen concentration is determined as the ratio of the total hydrogen production to the total gas production during the whole fermentation process.

Results and discussions Effect of shaking velocity on photo-fermentative hydrogen production under different substrate concentrations Cumulative hydrogen production Fig. 1 depicts the change of cumulative hydrogen production at different shaking velocities with different substrate concentrations. There was no hydrogen was detected in fermented bottles at different shaking velocities with none corn stover powder (Fig. 1a), because no organic matters were used by bacteria to hydrogen production. As seen from Fig. 1b, hydrogen production fermentation from 2 g corn stover powder at shaking velocity of 120 rpm, the hydrogen production increased rapidly during 12e48 h and remained stable afterwards. While hydrogen production began to increase in 24 h at shaking velocity of 0 and 80 rpm. This difference might be due to that higher shaking velocity contributes to the higher hydrolysis efficiency of the substrate, as a result, the hydrogen is released earlier. In Fig. 1c, Hydrogen production of higher shaking velocity (120 and 160 rpm) increased sharply during initial 48 h compared with low shaking velocity (0 and 80 rpm). Then, there is almost no hydrogen production under higher shaking velocity, while the production of hydrogen continued for lower shaking velocity until exceed higher shaking velocity, especially when shaking velocity was 0 rpm. A possible reason for this phenomenon was that higher shaking velocity can help fermentation bacteria to utilize substrate rapidly and to release the trapped gas bubbles from the flask, which result more hydrogen in mixed bottles in the early stage of reaction were released [40]. But the total hydrogen yield was much lower than standing-culture due to high shaking velocity break the aggregates of microbe and substrate in lower substrate concentration. The hydrogen

Kinetic analysis The cumulative hydrogen production data from different substrate concentrations with different shaking velocities were fitted by Modified Gompertz model [38,39], which is shown in Eq. (1):    Rmax e ðl  tÞ þ 1 H ¼ Hmax exp  exp Hmax

(1)

where, H is the cumulative hydrogen production (mL), Hmax is the maximum cumulative hydrogen production (mL), Rmax is the maximum hydrogen production rate (mL/h),l is the lag time (h), and e is 2.72.

Cumulative hydrogen production (mL)

Cumulative hydrogen production (mL)

250 0 rpm 80 rpm 120 rpm 160 rpm

200

0 rpm 80 rpm 120 rpm 160 rpm

a

150

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0 rpm 80 rpm 120 rpm 160 rpm

c

e

0 rpm 80 rpm 120 rpm 160 rpm

f

100 50 0 600

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500 400

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100

120

0

20

40

60

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100

120

0

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80

100

120

Fig. 1 e Effect of shaking velocity on cumulative hydrogen production under (a) 0 g corn stover powder, (b) 2 g corn stover powder, (c) 4 g corn stover powder, (d) 6 g corn stover powder, (e) 8 g corn stover powder, (f) 10 g corn stover powder. Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150

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production process with different velocities under substrate concentration of 6 g are shown in Fig. 1d. During the first 60 h, the hydrogen production increased rapidly at shaking velocities of 0 rpm. Then, hydrogen production continued to increase slowly till to the end of 96 h. On the other hand, hydrogen production curves for shaking velocity of 80 and 120 rpm had similar trend, the high-rate hydrogen production after 12 h until the end of hydrogen production. At shaking velocity of 160 rpm, the cumulative hydrogen production in the early stage of the fermentation was ahead of the other velocities, but the hydrogen yield was the lowest in the whole fermentation stage. Seen from Fig. 1d, the shaking velocity of 80 rpm is the most effective shaking velocity to improve hydrogen yield when fermented bottles were loaded 6 g corn stover powder, too high shaking velocity can reduce hydrogen production. With substrate concentration of 8 g (Fig. 1e), at shaking velocity of 120 and 160 rpm, hydrogen production had similar growth pattern, which high-rate hydrogen production lasted for a long time until nearly the end of photofermentation, this result is consistent with literature [28]. Moreover, the production of hydrogen shown almost linear growth during the whole fermentation process. The cumulative hydrogen production at shaking velocity of 160 rpm was higher than that of the shaking velocity of 120 rpm in the initial 60 h. Hydrogen production increased rapidly from 12 to 48 h at shaking velocity of 80 rpm, and then increased slowly. The cumulative hydrogen production at the shaking velocity of 0 rpm was much lower than the other three shaking velocities. The huge difference in values of cumulative hydrogen production may be assign to the shaking velocity, lower shaking velocity is unable to meet the full contact of bacteria and substrate with higher substrate concentration, resulting in incomplete hydrolysis of substrate, thus reduces the hydrogen production yield [41,42]. But too high shaking velocity has also negative effect on bacteria, like shaking velocity of 160 rpm (Fig. 1e). When fermented bottles were loaded 10 g corn stover powder (Fig. 1f), there is a remarkable difference in cumulative hydrogen yield at various shaking velocities. Hydrogen production was observed in the fermented bottle after 12 h and increased rapidly at higher shaking velocity (120 and 160 rpm), while there was an apparent lag for shaking velocity of 80 and 0 rpm and same profile were obtained. At shaking velocity of 160 rpm, hydrogen yield increased rapidly during the initial 48 h, then, hydrogen production rate decreased significantly, and no more hydrogen was detected after 84 h. The hydrogen production at 120 rpm velocity was significantly lower than that of 160 rpm at the later stage, and fermentation stopped at 108 h. At shaking velocity of 0 and 80 rpm, hydrogen yield was almost the same, and the cumulative hydrogen production at 80 rpm was higher than that of 0 rpm during the whole fermentation process.

Kinetic analysis A modified Gompertz model was used to fit the cumulative hydrogen production data for different substrate concentrations under different shaking velocities, and the results of fitting are presented in Table 1. Since fermentation time of hydrogen production with 2 g corn stover powder was short at shaking velocities of 160 rpm, cumulative hydrogen

Table 1 e Kinetic parameters of modified Gompertz model. Substrate concentration (g) 0

2

4

6

8

10

Shaking velocity (rpm)

Hmax (mL)

R2

Rm (mL/h)

l (h)

0 80 120 160 0 80 120 160 0 80 120 160 0 80 120 160 0 80 120 160 0 80 120 160

e e e e 85.29 83.20 67.43 e 226.46 191.26 175.52 159.31 281.11 324.30 272.96 258.67 335.97 369.88 439.07 402.13 353.91 364.77 491.07 579.00

e e e e 0.9925 0.9990 0.9999 e 0.9990 0.9974 0.9989 0.9984 0.9893 0.9971 0.9956 0.9927 0.9940 0.9915 0.9963 0.9978 0.9902 0.9936 0.9844 0.9940

e e e e 2.76 4.38 4.69 e 5.09 4.92 7.62 6.39 4.50 8.91 6.95 11.13 6.30 9.50 10.27 14.51 5.67 7.19 23.44 21.27

e e e e 23.53 25.82 21.19 e 22.73 19.52 16.45 11.41 10.57 13.24 13.19 12.28 11.23 11.29 13.60 13.70 9.80 11.77 10.71 7.72

production data could not be fitted by modified Gompertz model, the fitting data was not shown in Table 1. The R2 value (>0.98) shows that there was a strong connection between the measured cumulative hydrogen production data and the fitted data. With the increase of substrate concentration, the maximum cumulative hydrogen production and maximum hydrogen production rate also show increasing trend. When the fermented bottles loaded 2 g corn stover powder, the maximum cumulative hydrogen production potential decreased from 85.29 to 67.43 mL while maximum hydrogen production rate increased from 2.76 to 4.69 mL/h with the increasing of shaking velocity from 0 to 120 rpm. The highest cumulative hydrogen production was obtained at shaking velocity of 0 rpm (standing-culture). The lag time of 120 rpm was lower as compare to other shaking velocities, which is in agreement with that hydrogen was released earlier at higher velocity. Similar result was also observed at substrate concentration of 4 g corn stover powder. At higher shaking velocities of 120 and 160 rpm, the maximum hydrogen production rates were higher and lag times were shorter than lower shaking velocity but hydrogen potentials are low compared to the lower shaking velocity (0 and 80 rpm). The highest cumulative hydrogen production of 324.30 mL and hydrogen production rate of 11.13 mL/h were achieved at shaking velocity of 80 and 160 rpm with 6 g corn stover powder. Standing-culture had the shortest lag time. When fermented bottles loaded with 8 g corn stover powder, highest cumulative hydrogen production was obtained at shaking velocity of 120 rpm. The hydrogen production rate showed increasing trend with the increase of shaking velocity. The highest hydrogen production rate of 14.51 mL/g was obtained

Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150

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at 160 rpm. Hydrogen production was improved remarkably under the effect of shaking with substrate concentration of 10 g corn stover powder, and the highest cumulative hydrogen production was achieved at shaking velocity of 160 rpm. This might because that higher shaking velocity is required at high substrate concentration, which helps the hydrogen producers to interact more efficiently with substrate and enhances the hydrogen production. The highest hydrogen production rate of 23.44 mL/h was obtained at velocity of 120 rpm, and the lowest lag time of 7.72 h was obtained at velocity 160 rpm.

Hydrogen yield, gas quality and fermentation time Hydrogen yields, gas quality and fermentation time at different shaking velocities with different substrate concentrations are illustrated in Fig. 2. Since there was no hydrogen production using 0 g corn stover powder for photofermentation, its analysis was not included in this study. When fermented bottles were loaded with 2 and 4 g corn

70

Hydrogen yield (mL/g-VS)

a 60 50 40 2g 4g 6g 8g 10 g

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c

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60 40 20 0

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Fig. 2 e Effect of shaking velocity on (a) hydrogen yield, (b) average hydrogen concentration, and (c) fermentation time.

5

stover powder, the hydrogen yield decreased significantly with the increase of shaking velocity. Under higher shaking velocity, lower hydrogen yield was obtained. The hydrogen yields of 0, 80, 120 and 160 rpm were 46.33 ± 1.23 and 58.79 ± 1.14 mL/g VS, 44.35 ± 1.21 and 49.70 ± 1.39 mL/g VS, 35.70 ± 0.80 and 46.59 ± 0.59 mL/g VS, 27.68 ± 1.02 and 41.93 ± 0.40 mL/g VS at corn stover powder of 2 and 4 g, respectively. Apparently, 0 rpm was the optimum condition and shaking was not suitable for hydrogen production by photo-fermentation at lower substrate concentration. In addition, hydrogen yield of 4 g substrate concentration was significantly higher than that of 2 g corn stover powder, especially at higher shaking velocities (120 and 160 rpm). This result suggests that lower substrate concentration is not favorable to hydrogen production despite standing-culture or mixing. When fermented bottles were loaded 6 g corn stover powder, the highest hydrogen yield of 57.08 ± 0.83 mL/g VS were obtained at 80 rpm followed by 0 rpm. With the further increase in shaking velocity (120 and 160 rpm), hydrogen yields dropped remarkably, 47.78 ± 2.04 and 46.21 ± 0.99 mL/g VS were obtained, respectively. The role of mixing became more important when the fermented bottles were loaded with higher concentration of corn stover powder. Hydrogen yield with 8 g corn stover powder showed increasing trend when shaking velocity increased from 0 to 120 rpm, continue to increase the shaking velocity to 160 rpm led to a slight decrease in hydrogen production. At corn stover powder of 10 g, hydrogen yields were only 37.48 ± 1.00 and 39.54 ± 0.81 mL/g VS at velocities of 0 and 80 rpm, respectively. A more significant increase in hydrogen yield was observed with further increase of shaking velocity (120 and 160 rpm), 54.84 ± 1.42 and 62.28 ± 0.84 mL/g VS were achieved, respectively. Furthermore, the hydrogen yield of 160 rpm was higher than that of standing-culture at 4 g corn stover powder, indicating that high-yield photo-fermentative hydrogen production can be obtained under high substrate concentration by adding shaking. In general, it is necessary to add the shaking to improve hydrogen production potential at high substrate concentration, and the optimum shaking velocity increased with the increase of substrate concentration. This phenomenon may be due to the increase in shaking velocity causes the photosynthetic bacteria more collided with the inner surface of bottle at low substrate concentration, which had a negative effect on bacteria. However, at high substrate concentration, more total solid in fermented bottles is distributed, more bacteria are adsorbed on the surface of corn stover powder, the bad effect on bacteria is weakened, leading to the increase of hydrogen production. Higher the concentration of the substrate, the greater the strength of the external force is required to accelerate the mixing process and make hydrogen producers, cellulase and substrate fully contact. The profile of the average hydrogen concentration was similar to hydrogen yield. As shown in Fig. 2b, the highest average hydrogen concentration was almost achieved under highest hydrogen yields with different substrate concentrations. At substrate concentration of 2, 4 and 6 g, the highest average hydrogen concentration of 42.00 ± 0.99%, 49.90 ± 0.68% and 54.49 ± 0.49% were obtained at shaking velocities of 0, 0 and 80 rpm, respectively. At high velocity of 160 rpm with substrate concentration of 2 and 4 g, the lowest

Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150

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values of 34.54 ± 0.41% and 39.21 ± 0.34% were observed respectively. The result indicated that the high shaking velocity had bad effect on gas quality at lower substrate concentration. As for high substrate concentration of 8 and 10 g, the average hydrogen concentrations of 0 and 80 rpm are significantly lower than the shaking velocities of 120 and 160 rpm, 43.10 ± 0.88% and 41.18 ± 0.65%, 45.83 ± 0.78% and 42.68 ± 0.73%, 52.58 ± 0.42% and 54.27 ± 0.39%, 52.64 ± 0.47% and 57.02 ± 0.40% were observed at 0, 80, 120 and 160 rpm, respectively. These results revealed that adding shaking at high substrate concentration increased gas quality compared with standing-culture. In addition, the average hydrogen concentration of high shaking velocities (120 and 160 rpm) increased significantly with the increase of substrate concentration.

Fermentation time decreased with the increase of shaking velocity and increased with the increase of substrate concentration (Fig. 2c). Clearly, the lowest fermentation was obtained at 160 rpm with different substrate concentrations. At high substrate concentration, the addition of shaking not only increased the hydrogen yield but also improved the hydrogen production rate.

Effect of shaking velocity on pH and reducing sugar concentration under different substrate concentrations As shown in Fig. 3, the change of pH value in fermented bottles with none corn stover (Fig. 3a) was different from that of other concentration of corn stover powder at different shaking velocities, which might be caused by the metabolism of bacteria

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Fig. 3 e Effect of shaking velocity on pH and reducing sugar concentration under (a) 0 g corn stover powder, (b) 2 g corn stover powder, (c) 4 g corn stover powder, (d) 6 g corn stover powder, and (e) 8 g corn stover powder, (f) 10 g corn stover powder. Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150

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activity to ensure better adapt to the environment [16]. For other group experiments, pH decreased significantly in the early stage and then gradually increased at different substrate concentrations with different shaking velocities. Similar result was reported by Zhang et al. [43]. The drop of pH was due to the accumulation of VFAs in substrate hydrolysis, and the increase of pH in the later stage of process was attributed to the accumulation of VFAs consumed by photosynthetic bacteria for growth and hydrogen production. The final pH values showed an increasing trend with the increase in shaking velocity at substrate concentration of 2 and 4 g corn stover powder (Fig. 3b and c), which is consistent with low VFAs concentration at the end of fermentation (Fig. 4b and c). With the increase of substrate concentration, low pH values were observed around 24e48 h regardless of the shaking velocity (Fig. 3d, e and f), which was lower as compare to values at 2 and 4 g corn stover, indicating that higher substrate concentration can lead to more VFAs accumulation in the initial fermentation stage, the results consistent with previous literature [44]. Besides, the final pH values of substrate concentration 6, 8 and 10 g was lower than substrate concentration of 2 and 4 g, the reason is that accumulated VFAs not fully utilized by photosynthetic bacteria due to death in the late stage. During the process of hydrogen production by simultaneous saccharification and fermentation, reducing sugars were produced by enzymatic hydrolysis of corn stover powder, there were no reducing sugars were detected in fermented broth with none corn stover (Fig. 3a), for other group experiments, the concentration of reducing sugar increased rapidly in 0e12 h, enzymatic hydrolysis occupied the main position during this period followed with hydrogen production, the rate of sugar production was higher than the rate of consumption. Then, sugar concentration decreased rapidly, which was due to consumed by bacteria for growth and hydrogen production. In the later stage of reaction, the sugar

Liquid metabolites (mg/L)

500

b

Effect of shaking on liquid metabolites under different substrate concentrations The degradation of substrate is accompanied by the formation of VFAs and ethanol. Since none corn stover powder was added to the fermented bottles in the first set of experiments, no metabolites were detected (Fig. 4a). For other group experiments in this study, butyric acid, acetic acid and ethanol were the main metabolites after fermentation finished. As shown in Fig. 4b, the content of butyric acid decreased with the addition of shaking at 2 g corn stover, and ethanol concentration increased from 104.48 ± 12.34 to 151.53 ± 10.21 mg/ L. The similar trends were obtained in 4 g corn stover (Fig. 4c), the concentrations of butyric acid decreased from 247.85 ± 21.52 to 148.38 ± 15.46 mg/L, while the content of ethanol increased from 125.40 ± 13.56 to 269.68 ± 11.45 mg/L. As shown in Fig. 4d, e and f, with the increase of substrate concentration, the concentration of butyric obtained highest at 80, 120 and 160 rpm when fermented bottles were loaded 6, 8, 10 g corn stover, were 403.02 ± 4.51, 848.16 ± 48.69 and 1019.28 ± 96.34 mg/L, respectively. At the same time, the content of ethanol observed lowest at 80 and 120 rpm with 6 and 8 g corn stover. In summary, the content of butyric acid has a direct relationship with the production of hydrogen, high

Butyric acid Acetic acid Ethanol

400

c

300 200 100

0 2100 Liquid metabolites (mg/L)

a

concentration fluctuated up and down and reached a weak dynamic equilibrium. Variation of the sugar concentration at other concentrations of corn stover powder with different shaking velocities were indistinguishable except 2 g corn stover powder (Fig. 3b). As shown in Fig. 3b, when fermented bottles were loaded 2 g corn stover powder, reducing sugar concentration declined with time at 0 rpm. However, sugar concentration varied slightly after 36 h with the other three shaking velocities. The result indicated that the consuming of reducing sugar is coincide with the hydrogen production.

d

Butyric acid Acetic acid Ethanol

1800

f

e

1500 1200 900 600 300 0

0 rpm

80 rpm

120 rpm

160 rpm

0 rpm

80 rpm

120 rpm

160 rpm

0 rpm

80 rpm

120 rpm

160 rpm

Fig. 4 e Effect of shaking velocity on final liquid metabolites under (a) 0 g corn stover powder, (b) 2 g corn stover powder, (c) 4 g corn stover powder, (d) 6 g corn stover powder, (e) 8 g corn stover powder, (f) 10 g corn stover powder. Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150

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concentrations of butyric acid occurred simultaneously with high hydrogen production, while high ethanol content lead to low hydrogen production, indicating that ethanol fermentation was unfavorable for hydrogen production by HAU-M1.

Conclusions This study shown that the addition of shaking could accelerate gas release, shorten the fermentation time and improve hydrogen production rate. Hydrogen yield was significantly enhanced at high substrate concentration under shaking condition, highest hydrogen yield of 57.08 ± 0.83, 57.62 ± 1.37, 62.28 ± 0.84 mL/g VS were obtained at 80, 120 and 160 rpm with 6, 8 and 10 g corn stover powder, respectively. With the increase of substrate concentration, more external force was needed to accelerate the mixing process. However, shaking reduced hydrogen production potential of lower concentration corn stover powder (2, 4 g), and the lower hydrogen yield was obtained at the higher shaking velocity.

Acknowledgements This study was supported by the National Natural Science Foundation of China (51676065,51806061).

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Please cite this article in press as: Zhu S, et al., Analysis of shaking effect on photo-fermentative hydrogen production under different concentrations of corn stover powder, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/j.ijhydene.2018.09.150