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Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor
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 6 ) 1 e7
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Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor Ibdal Satar a, Mostafa Ghasemi b,*, Saad A. Aljlil c, Wan Nor Roslam Wan Isahak d, Abdalla M. Abdalla e, Javed Alam f, Wan Ramli Wan Daud a,d, Mohd Ambar Yarmo g, Omid Akbarzadeh h a
Fuel Cell Institute, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia Petroleum Engineering Department, Universiti Teknologi PETRONAS, Seri Iskandar, 32610, Perak, Malaysia c National Centre for Water Treatment and Desalination Technology, KACST, P.O. Box 6086, Riyadh, 11442, Saudi Arabia d Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia e Faculty of Integrated Technologies, Universiti Brunei Darussalam, JalanTunku Link, Gadong, BE 1410, Brunei Darussalam f King Abdullah Institute for Nanotechnology, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia g School of Chemical Science and Food Technology, Faculty of Science and Technology, Universiti Kebangsaaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia h Chemical Engineering Department, Universiti Teknologi PETRONAS, Seri Iskandar, 32610, Perak, Malaysia b
article info
abstract
Article history:
The production of hydrogen from glucose by using Enterobacter aerogenes ATCC 13048 (E.
Received 6 October 2015
aerogenes) in an immobilized cell reactor (ICR) was investigated. The effect of several fac-
Received in revised form
tors, such as the glucose concentration, feed flow rate, and fermentation time were
20 April 2016
examined. The highest amount of hydrogen (9.44 mmol H2/g glucose) was obtained at a
Accepted 20 April 2016
glucose concentration of 8 g/L, flow rate of 0.5 mL/min, retention time of 24 h and at a
Available online xxx
temperature of 30 C. Meanwhile, the highest amount of carbon dioxide (1.68 mmol CO2/g glucose) was obtained at a glucose concentration of 10 g/L, flow rate of 0.7 mL/min, hy-
Keywords:
draulic retention time of 24 h and at a temperature of 30 C. The hydrogen and carbon
Hydrogen
dioxide production were affected by glucose concentration, hydraulic retention time (HRT)
Carbon dioxide
and fermentation time. This study showed that the ICR was a very efficient method for the
Immobilized cell reactor
production of hydrogen and carbon dioxide gases.
Enterobacter aerogenes
© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Hydrogen (H2) is a promising energy carrier which can play a significant role in the future since it has a high energy content, is a non-pollutant and is clean compared to carbon-based fossil
energy sources [1e3]. One of the scenarios of the current energy sector is the utilization of organic waste as an alternative renewable energy source [4]. Organic waste is most commonly found in the environment, for example, distillery effluent [5,6], molasses, sugary wastewater [7], paper sludge [8], rice winery
* Corresponding author. Tel.: þ60 5 3687375; fax: þ60 5 3687139. E-mail addresses: [email protected], [email protected] (M. Ghasemi). http://dx.doi.org/10.1016/j.ijhydene.2016.04.150 0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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wastewater and corn syrup water [9]. This research is significant from an industrial and commercial point of view due to the abundance of valuable organic waste in waste streams. Hydrogen can be produced by a simple biological method [10]. Immobilized microorganisms can be used for the production of hydrogen [2,11] at a low temperature (30e70 C). Immobilized microorganisms are suitable for a variety of feedstock sources such as organic waste, and this method is less energy intensive compared to other hydrogen production processes [10]. Hydrogen can be produced by means of immobilized microorganisms via anaerobic dark fermentation, whereby organic wastes are converted by anaerobic organisms into hydrogen and carbon dioxide gases [12]. Several factors must be taken into consideration during the process for the production of hydrogen, such as the inoculum and substrate type, and the concentration, culture condition, reactor type, flow rate, fermentation time and temperature [10,11,13]. These factors are essential to understanding the interaction between the factors and their further effects on the production of hydrogen and carbon dioxide [6]. In this research, the immobilized cells of the E. aerogenes were used for hydrogen production. Moreover, several of the main parameters of the hydrogen and carbon dioxide production process, such as the flow rate (mL/min), glucose concentration (g/L), fermentation time (h) and temperature ( C), were evaluated. However the immobilized cell system mostly used for production of materials such as acid, lactic acid etc, but we have used this system for hydrogen production from glucose and simultaneously treatment of wastewaters. Also we have shown that, the effluents of the system can be used as valuable substrate in MET such as microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) for simultaneous generating electricity and hydrogen production as well as wastewater treatment [3].
Materials and methods Bacteria source and culture medium E. aerogenes, a potential hydrogen-producing bacterium, was used in all the experiments. This strain was grown under anaerobic conditions in a medium consisting of 10 g/L glucose, 10 g/L tryptone, 2 g/L K2HPO4, 0.5 g/L MgSO4, 7 g/L H2O and 0.5 g/L yeast extract at a pH of 7.0 and temperature of 30 C inside an incubator shaker at 150 rpm for 24 h.
Immobilization of E. aerogenes The E. aerogenes cells were kept in the refrigerator for about 24 h. Then, the culture was harvested and mixed with 3% (w/v) sodium alginate to make a final concentration of 1.5% (w/w) sodium alginate. The E. aerogenes culture-sodium alginate mixture was dropped into 2% (w/v) calcium chloride solution by using a pipette to form beads with a diameter of 3 mm. The beads were allowed to harden for about 2 h, and were then washed three times with distilled water to remove excess cells and calcium ions. The beads were stored in 0.2% (w/v) glucose and 0.2% (w/v) yeast extract solution [14] for further experimentation.
Bioreactor setup for hydrogen (H2) production The bioreactor or immobilized cell reactor (ICR) is a tubular column constructed with an internal diameter (ID) of 5.0 cm and height (h) of 100 cm (Fig. 1), and having a working volume of 1570 mL. A peristaltic pump was used to pump fresh media (Mettler Toledo DU200) from the media tank to the ICR at a flow rate of 0.5 mL/min. The gas product was collected by using another pump at a flow rate of 0.7 mL/min from the ICR to the gasbag. Two containers (2.5 L) were used as tanks for the fresh media and waste media, respectively. Different concentrations of glucose of 2, 4, 6, 8 and 10 g/L were used to evaluate the effect of glucose concentration on hydrogen and carbon dioxide production. About 80% of the ICR was filled with beads and the remaining volume in the column was left empty for the possible expansion of the beads by the fresh media. The hydrogen and carbon dioxide production yields were monitored after 8, 12, 16, 20, and 24 h of the fermentation process. The dilution rate (D) is the inverse of the hydraulic retention time (HRT), i.e. D ¼ 1/HRT. The HRT (q ¼ V/Q) for feeding in the ICR column was 8, 12, 16, 20, and 24 h corresponding to about 0.125, 0.083, 0.062, 0.05 and 0.042 h1, respectively. As a comparison between continuous and batch mode in ICR operation, the fermentation time was also investigated in this study where gases produced were monitored after 8, 12, 16, 20 and 24 h using 8.0 of glucose at 30 C.
Hydrogen (H2) and carbon dioxide (CO2) production analysis Gas samples were collected by using a gasbag (500 ml). About 1.0 mL of gas samples were analysed by using a Gas Chromatograph (GC) with a thermal conductivity detector (GC-TCD 6890). The retention time was about 15 min, and the column was Porapak Q & Molecular Sieve (6 ft & 10 ft). The temperature of the injector and detector was 150 C and 180 C, respectively. The hydrogen and carbon dioxide peak areas were expressed as a mol percentage (%) of hydrogen production.
Chemical oxygen demand (COD) analysis The organic content of the synthetic waste was determined by using the chemical oxygen demand (COD) method. The chemical oxygen demand (COD), which is expressed in mg COD/L, indicates the mass of oxygen consumed by a litre of solution. In this research, the COD was determined according to APHA standard methods by using a COD measurement instrument set (DRB 200 and DR 2800 spectrophotometer HACH, USA) [6,15]. A total 0.2 ml of the sample was added into a highrange COD reagent vial containing potassium dichromate and was heated to 150 C in the DRB 200 reactor. The vial was then cooled to room temperature and the final COD was recorded using the DR 2800 spectrophotometer HACH at 600 nm.
Organic acid analysis A total of 1.5 mL of effluent was passed through a 0.45 mm membrane filter and analysed by high performance liquid chromatography (HPLC 1100s with UV detector at 220 nm). The mobile phase was a mixture of 95% sulphuric acid (2 mM) and 5% acetonitrile with a flow rate of 0.6 ml/min, oven
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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Fig. 1 e Immobilized Cell Reactor (ICR) schematic (a) Experimental setup of ICR (b).
Scanning electron microscopic (SEM) analysis The scanning electron microscopic (SEM) analysis was performed according to the method described by Kumar and Das [16] and Zao [17]. The bead samples were taken from the ICR column when fresh, and after 10 and 20 days. The dried samples were mounted on SEM stubs coated with gold. Different sections of the solid matrices were examined and photographed by SEM (JEOL JSM 5800) using an accelerated voltage of 20 kV at a distance of 10 mm. The digital images were captured using a resolution of 1280 960 and a dwell time of 160 s.
Results and discussion Hydrogen and carbon dioxide yield The mechanism of the reaction between organic compounds (as a source of carbon) catalysed by microorganisms is quite complicated and unpredictable. This is because the biological reactions depend on the operational conditions such as the pH, temperature and substrates. Therefore, the operational conditions must be controlled during the bio-hydrogen production process. In general, the bio-catalysed reaction in the presence of glucose (hexose) can be described as follows (reactions 1 and/or 2):
C6 H12 O6 þ 6H2 O0 12H2 þ 6CO2
(1)
both photo fermentation and dark fermentation are considered as bio-hydrogen production methods in which microorganisms act as a biocatalyst. The production of hydrogen by using an ICR is a type of dark fermentation process in which no light source is required in the system. Fig. 2 shows the maximum yield of hydrogen and carbon dioxide production with 2 g/L up to 10 g/L of glucose concentration in 0.5 ml/min and 0.7 ml/min of flow rate. The highest yield of hydrogen was 9.44 mmol/g glucose at 8.0 g/l of glucose and 0.5 ml/min of flow rate. The hydrogen production increased with increasing the glucose concentration up to 8.0 g/L, but then the hydrogen yield was decreased at 10.0 g/L of glucose and 0.5 ml/min of flow rate. Its means that the hydrogen yield at 10.0 g/L of glucose was lower than 8.0 g/L of glucose but higher than 6, 4 and 2 g/L of glucose. This might be due to an inhibitory effect which caused by excessive amounts of carbon sources which is glucose here [20,21] moreover immobilized E. aerogenes cells were damaged couldn't consume the nutrient well [1]. Furthermore, the hydrogen production from various (2 g/L to 10 g/L) of glucose concentration and 0.7 ml/min of flow rate were showed a bit different trends, in which the hydrogen yield was increased
10 H Yield at 0.5 ml/min CO Yield at 0.5 ml/min 8
H Yield at 0.7 ml/min CO Yield at 0.7 ml/min
Yield (mmol/g Glucose)
temperature of 40 C, and retention time of 8 min. The stationary phase was a GRACE Genesis C-18 4u column (150 mm 4 mm).
6
4
2
C6 H12 O6 þ 6H2 O0 ð3 4ÞH2 þ xCO2 þ yOrganic acid
(2)
In a photo fermentation process, 1 mol of glucose (e.g. hexose) can produce 12 mol of hydrogen and 6 mol of carbon dioxide (reaction 1), whereas in a dark fermentation process, 1 mol of glucose can only produce about 3e4 mol of hydrogen (or 16.6e22.2 mmol H2/g glucose) (reaction 2) [18,19]. However,
0 0
2
4
6
8
10
12
Glucose concentration (g/l)
Fig. 2 e Effect of glucose concentration and flow rate on H2 and CO2 yields.
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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with increasing glucose concentration. Its mean, the hydrogen yield at 10.0 g/L of glucose was higher than 8, 6, 4, and 2 g/L of glucose. This fact shows that the high flow rate could reduce fermentation time and the amounts of carbon source and nutrient were therefore lower. Hence, the hydrogen yield at 0.7 of flow rate lower than 0.5 ml/min of flow rate. As a whole, the yield of carbon dioxide at a flow rate of 0.5 ml/min was lower than 0.7 ml/min. The highest yield of carbon dioxide was 1.68 mmol per g glucose at 8.0 g/l of glucose and a flow rate of 0.7 ml/min. The yield of carbon dioxide generally increased with elevation of glucose concentration, however suddenly decreased at 10.0 g/l of glucose (1.56 mmol/g glucose). This result shows a same trend, in which the trend of carbon dioxide production at a flow rate of 0.7 ml/min was similar with the trend of hydrogen production at a flow rate of 0.5 ml/min. Based on this fact, the yield of both hydrogen and carbon dioxide production using immobilized E. aerogenes could be controlled by flow rate and substrate concentration.
Hydraulic retention time (HRT) Fig. 3 shows the effect of the hydraulic retention time (HRT) on the production rates and yields of hydrogen and carbon dioxide. The hydrogen and carbon dioxide yields were high at a HRT of 24 h (D ¼ 0.042 h1) owing to the fact that the substrate loading rate was able to increase the capability of the ICR. However, the HRT did not influence the activity of the microbial community in the conversion of the organic source (i.e., glucose) into hydrogen [22,23] and carbon dioxide. The hydrogen yield at a HRT of 24 h (D ¼ 0.05 h1) was 2.37 mmol/g glucose, higher than a HRT of 8 h (0.9 mmol/g glucose), 12 h (1.23 mmol/g glucose), 16 h (1.71 mmol/g glucose) and 20 h (1.45 mmol/g glucose). The low of hydrogen yield at HRT of 8, 12, 16 and 20 h (compare with a HRT of 16 h and a HRT of 24 h) might be caused by the amounts of substrate loaded to ICR was insufficient. Its mean, the hydrogen yield was lead increased when the HRT was decreased. In addition, according to Chang [24], the HRT
has an inhibitory effect on the side products such as organic acids or alcohol, which is inadequately flushed out. Therefore, the combination of product types could force unstable conditions onto the ICR system. Generally, an increase in the HRT resulted in an increase in the hydrogen and carbon dioxide yields.
Effect of fermentation time The maximum yields of H2 and CO2 were 9.44 and 1.68 mmol/g glucose, respectively. The capability of E. aerogenes to produce maximum amount of H2 was at 8.0 g/L of glucose and 0.5 mL/ min of flow rate. Meanwhile, the maximum CO2 yield was obtained at 0.8 g/L of glucose and 0.7 mL/min of flow rate. H2 yield decreased with increasing the glucose composition to 10 g/L. This could because of inhibition effect [20], and the polymer structure of immobilized E. aerogenes cells damage due to excess amount of carbon source [21] and poor nutrient consumption [1]. In the other side, the capability of E. aerogenes to produce H2 was low at lower glucose concentration. This is due to insufficient carbon source. It clearly showed that the glucose and feed flow rate are directly influence on the immobilized E. aerogenes performance in terms of H2 and CO2 production. At the same experimental condition (i.e., concentration of glucose 0.8% w/v, temperature 30 C), the high H2 yield was obtained at fermentation time of 20 h (Fig 4). Moreover, H2 yield at 24 h was lower than the yield at 12 h and 16 h. However, H2 yield was higher compared to the CO2 yield at same condition. This fact indicates that the time of fermentation affect on the immobilized E. aerogenes performance due to possible damage of the cells after 20 h.
Organic acid types Fig. 5 (a) shows the standard chromatogram for acetic acid, in which the acetic acid peak appeared at 3.940 min Fig. 5 (b) is the chromatogram for the effluent, in which the lactic acid and acetic acid peaks appeared at 3.284 min and 3.913 min, respectively. This result indicates that besides acetic acid as
2.5
2.0
H2 CO2 2.0
CO
1.6
Yield (mmol/g Glucose)
Yield (mmol/g Glucose)
H
1.8
1.5
1.0
0.5
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
0.0 HRT=8
HRT=12
HRT=16
HRT=20
HRT=24
HRT (h)
Fig. 3 e Effect of hydraulic retention time on H2 and CO2 yields using 8.0 g/l glucose at 30 C.
6
8
10
12
14
16
18
20
22
24
26
Time(h)
Fig. 4 e Effect of fermentation time on H2 and CO2 yields using 8.0 g/l of glucose at 30 C.
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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 6 ) 1 e7
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Fig. 5 e HPLC chromatograms of: (a) a standard solution of acetic acid, and (b) effluent of fermentation process. Peaks identified: (1) lactic acid and (2) acetic acid. Absorbance time of lactic and acetic acids were represented by x-axes (minute, min). Intensity of absorbance of both lactic and acetic acid were represented by y-axes (mili absorbance unit, mAU).
the main by-product, lactic acid is also produced from synthetic waste fermentation by the use of immobilized E. aerogenes ATCC 13048. Theoretically, glucose can be oxidized completely by bacteria (microorganisms) to produce hydrogen, carbon dioxide, carbon monoxide, methane, acetic acid and/or butyric acid [25], but practically, another organic acid, such as lactic acid, is also produced. This is due to the difficulty in maintaining optimum conditions during the fermentation process. The yield of hydrogen depends on the organic acid type in the end product (effluent). The yield of hydrogen might have been increased if acetic acid, instead of lactic acid, was the main by-product in the effluent [26e28]. These results show that the effluent of ICR fermentation could be used as fuel or feedstock in microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) for generating electricity and hydrogen production, respectively. In both MFCs and MECs system, acetate was oxidized by microorganisms
release electrons and protons at the anodic chamber. At the cathodic chamber, electrons and protons are combined to release water (in MFCs) and hydrogen (MECs).
Scanning electron microscopic (SEM) images A series of SEM images of the beads were taken at the beginning and final days of the ICR process. As shown in Fig. 6 (a), the sodium alginate had a polymeric structure with many pores for the entrapment of the E. aerogenes. Fig. 6 (b) shows the structure of the beads at the end of the experiment. As shown in the figures, the surface of the polymeric structure of the sodium alginate was covered by the cylindrical-shaped E. aerogenes which doing fermentation process and hydrogen production. On the first day, the production of hydrogen was still low due to the poor reproduction rate of the E. aerogenes bacteria
Fig. 6 e (a) SEM image of fresh beads and (b) SEM image of beads after 20 days. Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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Table 1 e The summary of the results in this study. All experiments were run at a temperature of 30 C. Glucose (g/l)
Flow rate (ml/min)
HRT (h)
Fermentation time (h)
H2
CO2
2 4 6 8 10 2 4 6 8 10
0.5 0.5 0.5 0.5 0.5 0.7 0.7 0.7 0.7 0.7
e e e e e e e e e e
e e e e e e e e e e
2.53 4.36 5.01 9.44 9.07 1.97 2.05 2.52 3.85 6.35
0.36 0.62 0.42 1.02 1.06 1.40 0.48 0.88 1.68 1.56
8 8 8 8 8
e e e e e
8 12 16 20 24
e e e e e
0.90 1.23 1.71 1.45 2.37
0.40 0.76 0.90 1.10 1.47
8 8 8 8 8
e e e e e
e e e e e
8 12 16 20 24
0.39 1.19 1.37 1.79 1.13
0.04 0.14 0.15 0.26 0.58
colony. Meanwhile, the hydrogen production increased after 20 days to 9.44 mmol H2/g glucose compared to 2.53 mmol H2/g glucose on the first day. However, the hydrogen production yield decreased after 25 days when the glucose concentration was increased, thus indicating the fact that the glucose concentration is an important factor that affects the capability of the bacteria to produce hydrogen. Table 1 shows the summary of the results in this research. The maximum condition was highlighted by red colour in the table. It has been known that the flow rate and fermentation time were two important operating condition in ICR system, where the flow rate defined as rate of substrate added into the system and products removed continuously from the system in the same rate [29], meanwhile fermentation time was used in batch mode operation for growing up of the bacteria and harvest of any product.
Yield (mmol/g glucose)
electrolysis cells (MECs) for generating electricity and hydrogen gas. The ICR method can be used for the production of hydrogen from industrial wastes and/or agricultural wastes waste that containing glucose.
Acknowledgement The authors gratefully acknowledge financial supports and kind collaboration of King Abdul Aziz City for Science and Technology (KACST) of Saud Arabia through the agreement No.1-2015 KACST-ITM-CNR. The authors extend their appreciations to the fuel cell institute of UKM as well as Universiti Teknologi Petronas of Malaysia for their supports.
references
Conclusion The glucose concentration, feed flow rate, and fermentation time were significant factors in the performance of immobilized E. aerogenes in terms of the hydrogen and carbon dioxide production. The yields of hydrogen and carbon dioxide were increased with increasing glucose concentration, HRT and time fermentation. The highest yield of hydrogen was 9.44 mmol/g glucose using 8 g/L of glucose, a flow rate of 0.5 mL/min and at a temperature of 30 C. Whereas, the highest yield of carbon dioxide was 1.68 mmol/g glucose using 10.0 g/L of glucose, a flow rate of 0.7 ml/min and at a temperature of 30 C. In addition, acetic acid and lactic acid were main organic compound in effluent of the ICR process. For future work, the effluent from the ICR process can also be used as a substrate in both microbial fuel cells (MFCs) and microbial
[1] Sivagurunathan P, Kumar G, Lin CY. Hydrogen and ethanol fermentation of various carbon sources by immobilized Escherichia coli. Int J Hydrogen Energy 2014;39:6881e8. [2] Chookaew T, O-Thong S, Prasertsan P. Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions. Int J Hydrogen Energy 2014;39:9580e7. [3] Wang A, Sun D, Cao G, Wang H, Ren N, Wu WM, et al. Integrated hydrogen production process from cellulose by combining dark fermentation, microbial fuel cells, and a microbial electrolysis cell. Bioresour Technol 2011;102:4137e43. [4] Ghasemi M, Wan Daud WR, Alam J, Ilbeygi H, Sedighi M, Ismail AF, et al. Treatment of two different water resources in desalination and microbial fuel cell processes by poly
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
sulfone/Sulfonated poly ether ether ketone hybrid membrane. Energy 2016;96:303e13. Kamalaskar LB, Dhakephalkar PK, Meher KK, Ranade DR. High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant. Int J Hydrogen Energy 2010;35:10639e44. Mishra M, Das D. Biohydrogen production from Enterobacter cloacae IIT-BT 08 using distillery effluent. Int J Hydrogen Energy 2014;39:7496e507. Ueno Y, Otsuka S, Morimoto M. Hydrogen production from industrial wastewater by anaerobic microflora in chemostat culture. J Ferment Bioeng 1996;82:194e7. Kadar Z, Vrije TD, Noorden G, Budde MA, Szengyel Z, Reczey K. Yields from glucose, xylose and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus. Appl Biochem Biotechnol 2004;113:497e508. Hafez H, Nakhla G, El-Naggar HM. Biological hydrogen production from corn-syrup waste using a novel system. Energies 2009;2:445e55. Elsharnouby O, Hafez H, Nakhla J, El-Naggar HM. A critical literature review on biohydrogen by pure cultures. Int J Hydrogen Energy 2013;38:4945e66. Yokoi H, Maki R, Hirose J, Hayashi S. Microbial production of hydrogen from starch-manufacturing wastes. Biomass Bioenergy 2002;22:389e95. Keskin T, Aksoyek E, Azbar N. Comparative analysis of thermophilic immobilized biohydrogen production using packed materials of ceramic ring and pumice stone. Int J Hydrogen Energy 2011;36:5160e7. Wang J, Wan W. Factors influencing fermentative hydrogen production: a review. Int J Hydrogen Energy 2009;34:799e811. Ghorbani F, Younesi H, Sari AE, Najafpour G. Cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by Saccharomyces. Renew Energy 2011;36(2):503e9. Lay JJ. Modelling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol Bioeng 2000;68:269e78. Kumar N, Das D. Electron microscopy of hydrogen producing immobilized E. cloacae IIT-BT 08 on natural polymers. Int J Hydrogen Energy 2001b;26:1155e63. Zhao L, Cao GL, Wanga AJ, Guo WQ, Liu BF, Ren HY, et al. Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier. Int J Hydrogen Energy 2012;37:162e6.
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[18] Kumar G, Das D. Enhancement of hydrogen production by Enterobacter cloacae IIT-BT 08. Process Biochem 2000;35:589e93. [19] Lima DMF, Moreira WKM, Zaiat M. Comparison of the use of sucrose and glucose as a substrate for hydrogen production in an upflow anaerobic fixed-bed reactor. Int J Hydrogen Energy 2013;38:15074e83. [20] Kumar N, Das D. Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic materials as solid matrices. Enzyme Microb Technol 2001a;29:280e7. [21] Yokoi H, Tokushige T, Hirose J, Hayashi S, Takasaki Y. Hydrogen production by immobilized cells of aciduric Enterobacter aerogenes strain HO-39. J Ferment Bioeng 1997;83:481e3. [22] Zhang Z, Show KY, Tay JH, Liang TD, Lee DJ, Jiang WJ. Effect of hydraulic retention time on biohydrogen production and anaerobic microbial community. Process Biochem 2006b;41:2118e23. [23] Lee DJ, Show KY, Su A. Dark fermentation on biohydrogen production: pure culture. Bioresour Technol 2011;102:8393e402. [24] Chang LS, Lee KS, Lin PJ. Biohydrogen production with fixed-bed bioreactors. Int J Hydrogen Energy 2002;27:1167e74. [25] Logan BE, Call D, Cheng S, Hamelers HVM, Sleutels THJA, Jeremiasse AW, et al. Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environ Sci Technol 2008;42:8630e40. [26] Ghasemi M, Najafpour G, Rahimnejad M, Beigi PA, Sedighi M, Hashemiyeh B. Effect of different media on production of lactic acid from whey by Lactobacillus bulgaricus. Afr J Biotechnol 2009;8(1). [27] Ghasemi M, Najafpour GD, Younesi H, Rahimnejad M, Rezaee PS. Influence of yeast extract concentration on lactic acid production from whey in batch reactor by Lactobacillius bulgaricus. [28] Ghasemi M, Rahimnejad M, Najafpour GD, Sedighi M, Asado M, Hashemiyeh B. Investigation on batch biosorption of lead using Lactobacillius bulgaricus in an aqueous phase system. Biokemistri 2008;20(2). [29] Teo KTK, Tham HJ, Tan MK. Optimization of substrate feed flow rate for fed-batch yeast fermentation process second international conference on computational intelligence. In: Modelling and simulation. Sabah, Malaysia: IEEE computer society; 2010. p. 233e8.
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor Ibdal Satar a, Mostafa Ghasemi b,*, Saad A. Aljlil c, Wan Nor Roslam Wan Isahak d, Abdalla M. Abdalla e, Javed Alam f, Wan Ramli Wan Daud a,d, Mohd Ambar Yarmo g, Omid Akbarzadeh h a
Fuel Cell Institute, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia Petroleum Engineering Department, Universiti Teknologi PETRONAS, Seri Iskandar, 32610, Perak, Malaysia c National Centre for Water Treatment and Desalination Technology, KACST, P.O. Box 6086, Riyadh, 11442, Saudi Arabia d Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia e Faculty of Integrated Technologies, Universiti Brunei Darussalam, JalanTunku Link, Gadong, BE 1410, Brunei Darussalam f King Abdullah Institute for Nanotechnology, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia g School of Chemical Science and Food Technology, Faculty of Science and Technology, Universiti Kebangsaaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia h Chemical Engineering Department, Universiti Teknologi PETRONAS, Seri Iskandar, 32610, Perak, Malaysia b
article info
abstract
Article history:
The production of hydrogen from glucose by using Enterobacter aerogenes ATCC 13048 (E.
Received 6 October 2015
aerogenes) in an immobilized cell reactor (ICR) was investigated. The effect of several fac-
Received in revised form
tors, such as the glucose concentration, feed flow rate, and fermentation time were
20 April 2016
examined. The highest amount of hydrogen (9.44 mmol H2/g glucose) was obtained at a
Accepted 20 April 2016
glucose concentration of 8 g/L, flow rate of 0.5 mL/min, retention time of 24 h and at a
Available online xxx
temperature of 30 C. Meanwhile, the highest amount of carbon dioxide (1.68 mmol CO2/g glucose) was obtained at a glucose concentration of 10 g/L, flow rate of 0.7 mL/min, hy-
Keywords:
draulic retention time of 24 h and at a temperature of 30 C. The hydrogen and carbon
Hydrogen
dioxide production were affected by glucose concentration, hydraulic retention time (HRT)
Carbon dioxide
and fermentation time. This study showed that the ICR was a very efficient method for the
Immobilized cell reactor
production of hydrogen and carbon dioxide gases.
Enterobacter aerogenes
© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Hydrogen (H2) is a promising energy carrier which can play a significant role in the future since it has a high energy content, is a non-pollutant and is clean compared to carbon-based fossil
energy sources [1e3]. One of the scenarios of the current energy sector is the utilization of organic waste as an alternative renewable energy source [4]. Organic waste is most commonly found in the environment, for example, distillery effluent [5,6], molasses, sugary wastewater [7], paper sludge [8], rice winery
* Corresponding author. Tel.: þ60 5 3687375; fax: þ60 5 3687139. E-mail addresses: [email protected], [email protected] (M. Ghasemi). http://dx.doi.org/10.1016/j.ijhydene.2016.04.150 0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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wastewater and corn syrup water [9]. This research is significant from an industrial and commercial point of view due to the abundance of valuable organic waste in waste streams. Hydrogen can be produced by a simple biological method [10]. Immobilized microorganisms can be used for the production of hydrogen [2,11] at a low temperature (30e70 C). Immobilized microorganisms are suitable for a variety of feedstock sources such as organic waste, and this method is less energy intensive compared to other hydrogen production processes [10]. Hydrogen can be produced by means of immobilized microorganisms via anaerobic dark fermentation, whereby organic wastes are converted by anaerobic organisms into hydrogen and carbon dioxide gases [12]. Several factors must be taken into consideration during the process for the production of hydrogen, such as the inoculum and substrate type, and the concentration, culture condition, reactor type, flow rate, fermentation time and temperature [10,11,13]. These factors are essential to understanding the interaction between the factors and their further effects on the production of hydrogen and carbon dioxide [6]. In this research, the immobilized cells of the E. aerogenes were used for hydrogen production. Moreover, several of the main parameters of the hydrogen and carbon dioxide production process, such as the flow rate (mL/min), glucose concentration (g/L), fermentation time (h) and temperature ( C), were evaluated. However the immobilized cell system mostly used for production of materials such as acid, lactic acid etc, but we have used this system for hydrogen production from glucose and simultaneously treatment of wastewaters. Also we have shown that, the effluents of the system can be used as valuable substrate in MET such as microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) for simultaneous generating electricity and hydrogen production as well as wastewater treatment [3].
Materials and methods Bacteria source and culture medium E. aerogenes, a potential hydrogen-producing bacterium, was used in all the experiments. This strain was grown under anaerobic conditions in a medium consisting of 10 g/L glucose, 10 g/L tryptone, 2 g/L K2HPO4, 0.5 g/L MgSO4, 7 g/L H2O and 0.5 g/L yeast extract at a pH of 7.0 and temperature of 30 C inside an incubator shaker at 150 rpm for 24 h.
Immobilization of E. aerogenes The E. aerogenes cells were kept in the refrigerator for about 24 h. Then, the culture was harvested and mixed with 3% (w/v) sodium alginate to make a final concentration of 1.5% (w/w) sodium alginate. The E. aerogenes culture-sodium alginate mixture was dropped into 2% (w/v) calcium chloride solution by using a pipette to form beads with a diameter of 3 mm. The beads were allowed to harden for about 2 h, and were then washed three times with distilled water to remove excess cells and calcium ions. The beads were stored in 0.2% (w/v) glucose and 0.2% (w/v) yeast extract solution [14] for further experimentation.
Bioreactor setup for hydrogen (H2) production The bioreactor or immobilized cell reactor (ICR) is a tubular column constructed with an internal diameter (ID) of 5.0 cm and height (h) of 100 cm (Fig. 1), and having a working volume of 1570 mL. A peristaltic pump was used to pump fresh media (Mettler Toledo DU200) from the media tank to the ICR at a flow rate of 0.5 mL/min. The gas product was collected by using another pump at a flow rate of 0.7 mL/min from the ICR to the gasbag. Two containers (2.5 L) were used as tanks for the fresh media and waste media, respectively. Different concentrations of glucose of 2, 4, 6, 8 and 10 g/L were used to evaluate the effect of glucose concentration on hydrogen and carbon dioxide production. About 80% of the ICR was filled with beads and the remaining volume in the column was left empty for the possible expansion of the beads by the fresh media. The hydrogen and carbon dioxide production yields were monitored after 8, 12, 16, 20, and 24 h of the fermentation process. The dilution rate (D) is the inverse of the hydraulic retention time (HRT), i.e. D ¼ 1/HRT. The HRT (q ¼ V/Q) for feeding in the ICR column was 8, 12, 16, 20, and 24 h corresponding to about 0.125, 0.083, 0.062, 0.05 and 0.042 h1, respectively. As a comparison between continuous and batch mode in ICR operation, the fermentation time was also investigated in this study where gases produced were monitored after 8, 12, 16, 20 and 24 h using 8.0 of glucose at 30 C.
Hydrogen (H2) and carbon dioxide (CO2) production analysis Gas samples were collected by using a gasbag (500 ml). About 1.0 mL of gas samples were analysed by using a Gas Chromatograph (GC) with a thermal conductivity detector (GC-TCD 6890). The retention time was about 15 min, and the column was Porapak Q & Molecular Sieve (6 ft & 10 ft). The temperature of the injector and detector was 150 C and 180 C, respectively. The hydrogen and carbon dioxide peak areas were expressed as a mol percentage (%) of hydrogen production.
Chemical oxygen demand (COD) analysis The organic content of the synthetic waste was determined by using the chemical oxygen demand (COD) method. The chemical oxygen demand (COD), which is expressed in mg COD/L, indicates the mass of oxygen consumed by a litre of solution. In this research, the COD was determined according to APHA standard methods by using a COD measurement instrument set (DRB 200 and DR 2800 spectrophotometer HACH, USA) [6,15]. A total 0.2 ml of the sample was added into a highrange COD reagent vial containing potassium dichromate and was heated to 150 C in the DRB 200 reactor. The vial was then cooled to room temperature and the final COD was recorded using the DR 2800 spectrophotometer HACH at 600 nm.
Organic acid analysis A total of 1.5 mL of effluent was passed through a 0.45 mm membrane filter and analysed by high performance liquid chromatography (HPLC 1100s with UV detector at 220 nm). The mobile phase was a mixture of 95% sulphuric acid (2 mM) and 5% acetonitrile with a flow rate of 0.6 ml/min, oven
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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Fig. 1 e Immobilized Cell Reactor (ICR) schematic (a) Experimental setup of ICR (b).
Scanning electron microscopic (SEM) analysis The scanning electron microscopic (SEM) analysis was performed according to the method described by Kumar and Das [16] and Zao [17]. The bead samples were taken from the ICR column when fresh, and after 10 and 20 days. The dried samples were mounted on SEM stubs coated with gold. Different sections of the solid matrices were examined and photographed by SEM (JEOL JSM 5800) using an accelerated voltage of 20 kV at a distance of 10 mm. The digital images were captured using a resolution of 1280 960 and a dwell time of 160 s.
Results and discussion Hydrogen and carbon dioxide yield The mechanism of the reaction between organic compounds (as a source of carbon) catalysed by microorganisms is quite complicated and unpredictable. This is because the biological reactions depend on the operational conditions such as the pH, temperature and substrates. Therefore, the operational conditions must be controlled during the bio-hydrogen production process. In general, the bio-catalysed reaction in the presence of glucose (hexose) can be described as follows (reactions 1 and/or 2):
C6 H12 O6 þ 6H2 O0 12H2 þ 6CO2
(1)
both photo fermentation and dark fermentation are considered as bio-hydrogen production methods in which microorganisms act as a biocatalyst. The production of hydrogen by using an ICR is a type of dark fermentation process in which no light source is required in the system. Fig. 2 shows the maximum yield of hydrogen and carbon dioxide production with 2 g/L up to 10 g/L of glucose concentration in 0.5 ml/min and 0.7 ml/min of flow rate. The highest yield of hydrogen was 9.44 mmol/g glucose at 8.0 g/l of glucose and 0.5 ml/min of flow rate. The hydrogen production increased with increasing the glucose concentration up to 8.0 g/L, but then the hydrogen yield was decreased at 10.0 g/L of glucose and 0.5 ml/min of flow rate. Its means that the hydrogen yield at 10.0 g/L of glucose was lower than 8.0 g/L of glucose but higher than 6, 4 and 2 g/L of glucose. This might be due to an inhibitory effect which caused by excessive amounts of carbon sources which is glucose here [20,21] moreover immobilized E. aerogenes cells were damaged couldn't consume the nutrient well [1]. Furthermore, the hydrogen production from various (2 g/L to 10 g/L) of glucose concentration and 0.7 ml/min of flow rate were showed a bit different trends, in which the hydrogen yield was increased
10 H Yield at 0.5 ml/min CO Yield at 0.5 ml/min 8
H Yield at 0.7 ml/min CO Yield at 0.7 ml/min
Yield (mmol/g Glucose)
temperature of 40 C, and retention time of 8 min. The stationary phase was a GRACE Genesis C-18 4u column (150 mm 4 mm).
6
4
2
C6 H12 O6 þ 6H2 O0 ð3 4ÞH2 þ xCO2 þ yOrganic acid
(2)
In a photo fermentation process, 1 mol of glucose (e.g. hexose) can produce 12 mol of hydrogen and 6 mol of carbon dioxide (reaction 1), whereas in a dark fermentation process, 1 mol of glucose can only produce about 3e4 mol of hydrogen (or 16.6e22.2 mmol H2/g glucose) (reaction 2) [18,19]. However,
0 0
2
4
6
8
10
12
Glucose concentration (g/l)
Fig. 2 e Effect of glucose concentration and flow rate on H2 and CO2 yields.
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with increasing glucose concentration. Its mean, the hydrogen yield at 10.0 g/L of glucose was higher than 8, 6, 4, and 2 g/L of glucose. This fact shows that the high flow rate could reduce fermentation time and the amounts of carbon source and nutrient were therefore lower. Hence, the hydrogen yield at 0.7 of flow rate lower than 0.5 ml/min of flow rate. As a whole, the yield of carbon dioxide at a flow rate of 0.5 ml/min was lower than 0.7 ml/min. The highest yield of carbon dioxide was 1.68 mmol per g glucose at 8.0 g/l of glucose and a flow rate of 0.7 ml/min. The yield of carbon dioxide generally increased with elevation of glucose concentration, however suddenly decreased at 10.0 g/l of glucose (1.56 mmol/g glucose). This result shows a same trend, in which the trend of carbon dioxide production at a flow rate of 0.7 ml/min was similar with the trend of hydrogen production at a flow rate of 0.5 ml/min. Based on this fact, the yield of both hydrogen and carbon dioxide production using immobilized E. aerogenes could be controlled by flow rate and substrate concentration.
Hydraulic retention time (HRT) Fig. 3 shows the effect of the hydraulic retention time (HRT) on the production rates and yields of hydrogen and carbon dioxide. The hydrogen and carbon dioxide yields were high at a HRT of 24 h (D ¼ 0.042 h1) owing to the fact that the substrate loading rate was able to increase the capability of the ICR. However, the HRT did not influence the activity of the microbial community in the conversion of the organic source (i.e., glucose) into hydrogen [22,23] and carbon dioxide. The hydrogen yield at a HRT of 24 h (D ¼ 0.05 h1) was 2.37 mmol/g glucose, higher than a HRT of 8 h (0.9 mmol/g glucose), 12 h (1.23 mmol/g glucose), 16 h (1.71 mmol/g glucose) and 20 h (1.45 mmol/g glucose). The low of hydrogen yield at HRT of 8, 12, 16 and 20 h (compare with a HRT of 16 h and a HRT of 24 h) might be caused by the amounts of substrate loaded to ICR was insufficient. Its mean, the hydrogen yield was lead increased when the HRT was decreased. In addition, according to Chang [24], the HRT
has an inhibitory effect on the side products such as organic acids or alcohol, which is inadequately flushed out. Therefore, the combination of product types could force unstable conditions onto the ICR system. Generally, an increase in the HRT resulted in an increase in the hydrogen and carbon dioxide yields.
Effect of fermentation time The maximum yields of H2 and CO2 were 9.44 and 1.68 mmol/g glucose, respectively. The capability of E. aerogenes to produce maximum amount of H2 was at 8.0 g/L of glucose and 0.5 mL/ min of flow rate. Meanwhile, the maximum CO2 yield was obtained at 0.8 g/L of glucose and 0.7 mL/min of flow rate. H2 yield decreased with increasing the glucose composition to 10 g/L. This could because of inhibition effect [20], and the polymer structure of immobilized E. aerogenes cells damage due to excess amount of carbon source [21] and poor nutrient consumption [1]. In the other side, the capability of E. aerogenes to produce H2 was low at lower glucose concentration. This is due to insufficient carbon source. It clearly showed that the glucose and feed flow rate are directly influence on the immobilized E. aerogenes performance in terms of H2 and CO2 production. At the same experimental condition (i.e., concentration of glucose 0.8% w/v, temperature 30 C), the high H2 yield was obtained at fermentation time of 20 h (Fig 4). Moreover, H2 yield at 24 h was lower than the yield at 12 h and 16 h. However, H2 yield was higher compared to the CO2 yield at same condition. This fact indicates that the time of fermentation affect on the immobilized E. aerogenes performance due to possible damage of the cells after 20 h.
Organic acid types Fig. 5 (a) shows the standard chromatogram for acetic acid, in which the acetic acid peak appeared at 3.940 min Fig. 5 (b) is the chromatogram for the effluent, in which the lactic acid and acetic acid peaks appeared at 3.284 min and 3.913 min, respectively. This result indicates that besides acetic acid as
2.5
2.0
H2 CO2 2.0
CO
1.6
Yield (mmol/g Glucose)
Yield (mmol/g Glucose)
H
1.8
1.5
1.0
0.5
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
0.0 HRT=8
HRT=12
HRT=16
HRT=20
HRT=24
HRT (h)
Fig. 3 e Effect of hydraulic retention time on H2 and CO2 yields using 8.0 g/l glucose at 30 C.
6
8
10
12
14
16
18
20
22
24
26
Time(h)
Fig. 4 e Effect of fermentation time on H2 and CO2 yields using 8.0 g/l of glucose at 30 C.
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5
Fig. 5 e HPLC chromatograms of: (a) a standard solution of acetic acid, and (b) effluent of fermentation process. Peaks identified: (1) lactic acid and (2) acetic acid. Absorbance time of lactic and acetic acids were represented by x-axes (minute, min). Intensity of absorbance of both lactic and acetic acid were represented by y-axes (mili absorbance unit, mAU).
the main by-product, lactic acid is also produced from synthetic waste fermentation by the use of immobilized E. aerogenes ATCC 13048. Theoretically, glucose can be oxidized completely by bacteria (microorganisms) to produce hydrogen, carbon dioxide, carbon monoxide, methane, acetic acid and/or butyric acid [25], but practically, another organic acid, such as lactic acid, is also produced. This is due to the difficulty in maintaining optimum conditions during the fermentation process. The yield of hydrogen depends on the organic acid type in the end product (effluent). The yield of hydrogen might have been increased if acetic acid, instead of lactic acid, was the main by-product in the effluent [26e28]. These results show that the effluent of ICR fermentation could be used as fuel or feedstock in microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) for generating electricity and hydrogen production, respectively. In both MFCs and MECs system, acetate was oxidized by microorganisms
release electrons and protons at the anodic chamber. At the cathodic chamber, electrons and protons are combined to release water (in MFCs) and hydrogen (MECs).
Scanning electron microscopic (SEM) images A series of SEM images of the beads were taken at the beginning and final days of the ICR process. As shown in Fig. 6 (a), the sodium alginate had a polymeric structure with many pores for the entrapment of the E. aerogenes. Fig. 6 (b) shows the structure of the beads at the end of the experiment. As shown in the figures, the surface of the polymeric structure of the sodium alginate was covered by the cylindrical-shaped E. aerogenes which doing fermentation process and hydrogen production. On the first day, the production of hydrogen was still low due to the poor reproduction rate of the E. aerogenes bacteria
Fig. 6 e (a) SEM image of fresh beads and (b) SEM image of beads after 20 days. Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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Table 1 e The summary of the results in this study. All experiments were run at a temperature of 30 C. Glucose (g/l)
Flow rate (ml/min)
HRT (h)
Fermentation time (h)
H2
CO2
2 4 6 8 10 2 4 6 8 10
0.5 0.5 0.5 0.5 0.5 0.7 0.7 0.7 0.7 0.7
e e e e e e e e e e
e e e e e e e e e e
2.53 4.36 5.01 9.44 9.07 1.97 2.05 2.52 3.85 6.35
0.36 0.62 0.42 1.02 1.06 1.40 0.48 0.88 1.68 1.56
8 8 8 8 8
e e e e e
8 12 16 20 24
e e e e e
0.90 1.23 1.71 1.45 2.37
0.40 0.76 0.90 1.10 1.47
8 8 8 8 8
e e e e e
e e e e e
8 12 16 20 24
0.39 1.19 1.37 1.79 1.13
0.04 0.14 0.15 0.26 0.58
colony. Meanwhile, the hydrogen production increased after 20 days to 9.44 mmol H2/g glucose compared to 2.53 mmol H2/g glucose on the first day. However, the hydrogen production yield decreased after 25 days when the glucose concentration was increased, thus indicating the fact that the glucose concentration is an important factor that affects the capability of the bacteria to produce hydrogen. Table 1 shows the summary of the results in this research. The maximum condition was highlighted by red colour in the table. It has been known that the flow rate and fermentation time were two important operating condition in ICR system, where the flow rate defined as rate of substrate added into the system and products removed continuously from the system in the same rate [29], meanwhile fermentation time was used in batch mode operation for growing up of the bacteria and harvest of any product.
Yield (mmol/g glucose)
electrolysis cells (MECs) for generating electricity and hydrogen gas. The ICR method can be used for the production of hydrogen from industrial wastes and/or agricultural wastes waste that containing glucose.
Acknowledgement The authors gratefully acknowledge financial supports and kind collaboration of King Abdul Aziz City for Science and Technology (KACST) of Saud Arabia through the agreement No.1-2015 KACST-ITM-CNR. The authors extend their appreciations to the fuel cell institute of UKM as well as Universiti Teknologi Petronas of Malaysia for their supports.
references
Conclusion The glucose concentration, feed flow rate, and fermentation time were significant factors in the performance of immobilized E. aerogenes in terms of the hydrogen and carbon dioxide production. The yields of hydrogen and carbon dioxide were increased with increasing glucose concentration, HRT and time fermentation. The highest yield of hydrogen was 9.44 mmol/g glucose using 8 g/L of glucose, a flow rate of 0.5 mL/min and at a temperature of 30 C. Whereas, the highest yield of carbon dioxide was 1.68 mmol/g glucose using 10.0 g/L of glucose, a flow rate of 0.7 ml/min and at a temperature of 30 C. In addition, acetic acid and lactic acid were main organic compound in effluent of the ICR process. For future work, the effluent from the ICR process can also be used as a substrate in both microbial fuel cells (MFCs) and microbial
[1] Sivagurunathan P, Kumar G, Lin CY. Hydrogen and ethanol fermentation of various carbon sources by immobilized Escherichia coli. Int J Hydrogen Energy 2014;39:6881e8. [2] Chookaew T, O-Thong S, Prasertsan P. Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions. Int J Hydrogen Energy 2014;39:9580e7. [3] Wang A, Sun D, Cao G, Wang H, Ren N, Wu WM, et al. Integrated hydrogen production process from cellulose by combining dark fermentation, microbial fuel cells, and a microbial electrolysis cell. Bioresour Technol 2011;102:4137e43. [4] Ghasemi M, Wan Daud WR, Alam J, Ilbeygi H, Sedighi M, Ismail AF, et al. Treatment of two different water resources in desalination and microbial fuel cell processes by poly
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150
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[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
sulfone/Sulfonated poly ether ether ketone hybrid membrane. Energy 2016;96:303e13. Kamalaskar LB, Dhakephalkar PK, Meher KK, Ranade DR. High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant. Int J Hydrogen Energy 2010;35:10639e44. Mishra M, Das D. Biohydrogen production from Enterobacter cloacae IIT-BT 08 using distillery effluent. Int J Hydrogen Energy 2014;39:7496e507. Ueno Y, Otsuka S, Morimoto M. Hydrogen production from industrial wastewater by anaerobic microflora in chemostat culture. J Ferment Bioeng 1996;82:194e7. Kadar Z, Vrije TD, Noorden G, Budde MA, Szengyel Z, Reczey K. Yields from glucose, xylose and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus. Appl Biochem Biotechnol 2004;113:497e508. Hafez H, Nakhla G, El-Naggar HM. Biological hydrogen production from corn-syrup waste using a novel system. Energies 2009;2:445e55. Elsharnouby O, Hafez H, Nakhla J, El-Naggar HM. A critical literature review on biohydrogen by pure cultures. Int J Hydrogen Energy 2013;38:4945e66. Yokoi H, Maki R, Hirose J, Hayashi S. Microbial production of hydrogen from starch-manufacturing wastes. Biomass Bioenergy 2002;22:389e95. Keskin T, Aksoyek E, Azbar N. Comparative analysis of thermophilic immobilized biohydrogen production using packed materials of ceramic ring and pumice stone. Int J Hydrogen Energy 2011;36:5160e7. Wang J, Wan W. Factors influencing fermentative hydrogen production: a review. Int J Hydrogen Energy 2009;34:799e811. Ghorbani F, Younesi H, Sari AE, Najafpour G. Cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by Saccharomyces. Renew Energy 2011;36(2):503e9. Lay JJ. Modelling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol Bioeng 2000;68:269e78. Kumar N, Das D. Electron microscopy of hydrogen producing immobilized E. cloacae IIT-BT 08 on natural polymers. Int J Hydrogen Energy 2001b;26:1155e63. Zhao L, Cao GL, Wanga AJ, Guo WQ, Liu BF, Ren HY, et al. Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier. Int J Hydrogen Energy 2012;37:162e6.
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[18] Kumar G, Das D. Enhancement of hydrogen production by Enterobacter cloacae IIT-BT 08. Process Biochem 2000;35:589e93. [19] Lima DMF, Moreira WKM, Zaiat M. Comparison of the use of sucrose and glucose as a substrate for hydrogen production in an upflow anaerobic fixed-bed reactor. Int J Hydrogen Energy 2013;38:15074e83. [20] Kumar N, Das D. Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic materials as solid matrices. Enzyme Microb Technol 2001a;29:280e7. [21] Yokoi H, Tokushige T, Hirose J, Hayashi S, Takasaki Y. Hydrogen production by immobilized cells of aciduric Enterobacter aerogenes strain HO-39. J Ferment Bioeng 1997;83:481e3. [22] Zhang Z, Show KY, Tay JH, Liang TD, Lee DJ, Jiang WJ. Effect of hydraulic retention time on biohydrogen production and anaerobic microbial community. Process Biochem 2006b;41:2118e23. [23] Lee DJ, Show KY, Su A. Dark fermentation on biohydrogen production: pure culture. Bioresour Technol 2011;102:8393e402. [24] Chang LS, Lee KS, Lin PJ. Biohydrogen production with fixed-bed bioreactors. Int J Hydrogen Energy 2002;27:1167e74. [25] Logan BE, Call D, Cheng S, Hamelers HVM, Sleutels THJA, Jeremiasse AW, et al. Microbial electrolysis cells for high yield hydrogen gas production from organic matter. Environ Sci Technol 2008;42:8630e40. [26] Ghasemi M, Najafpour G, Rahimnejad M, Beigi PA, Sedighi M, Hashemiyeh B. Effect of different media on production of lactic acid from whey by Lactobacillus bulgaricus. Afr J Biotechnol 2009;8(1). [27] Ghasemi M, Najafpour GD, Younesi H, Rahimnejad M, Rezaee PS. Influence of yeast extract concentration on lactic acid production from whey in batch reactor by Lactobacillius bulgaricus. [28] Ghasemi M, Rahimnejad M, Najafpour GD, Sedighi M, Asado M, Hashemiyeh B. Investigation on batch biosorption of lead using Lactobacillius bulgaricus in an aqueous phase system. Biokemistri 2008;20(2). [29] Teo KTK, Tham HJ, Tan MK. Optimization of substrate feed flow rate for fed-batch yeast fermentation process second international conference on computational intelligence. In: Modelling and simulation. Sabah, Malaysia: IEEE computer society; 2010. p. 233e8.
Please cite this article in press as: Satar I, et al., Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.04.150