Effect of calcium ions on biohydrogen production performance in a fluidized bed bioreactor with activated carbon-immobilized cells

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Effect of calcium ions on biohydrogen production performance in a fluidized bed bioreactor with activated carbon-immobilized cells Shu-Yii Wu a,b,c, Chen-Yeon Chu a,b,c,*, Yuan-Chang Shen b a

Green Energy Development Center, Feng Chia University, Taichung 40724, Taiwan Department of Chemical Engineering, Feng Chia University, Taichung 40724, Taiwan c Master’s Program of Green Energy Science and Technology, Feng Chia University, Taichung 40724, Taiwan b

article info

abstract

Article history:

Hydrogen (H2) has become a promising energy source because it is clean and has high-

Received 15 March 2012

energy potential. The aim of this research was to enhance the H2 production efficiency

Received in revised form

under anaerobic condition by addition of calcium ions (Ca2þ) in a fluidized bed reactor (FBR)

23 April 2012

with immobilized cells. Ca2þ ions were added either in the form of Ca(OH)2 or CaCl2.

Accepted 24 April 2012

Immobilized cells were prepared by physical adsorption with activated carbon (AC). The

Available online 31 May 2012

experiments were carried out in a FBR system. The H2 production performance of the FBR fed with sucrose-based synthetic medium, was evaluated under various influent Ca2þ

Keywords:

concentrations [Ca2þ] (50, 100 and 200 ppm) and hydraulic retention times (HRTs) (8, 6, 4

Fluidized bed reactor

and 2 h). The peak value of 1.22 L/h-L was obtained at [Ca2þ] 100 ppm, irrespective of the

Immobilized cells

form of Ca2þ ion added, and at the HRT of 2 h. Although the results were similar for

Anaerobic

different forms of Ca2þ ions, the presence of Ca2þ ions enhanced the H2 producing bio-

Calcium ions

process by 12e18%.

Hydrogen production

Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

The development of renewable energy to reduce the impact on the global environment and climate change, due to the increasing industrial development, has fostered the use of biological processes to produce biofuels like bioH2 [1]. Renewable ways of H2 production, aiming to contribute to “a big future” of low-carbon society, has been already undertaken by some researchers. In the last few decades different methods have been used or invented for H2 production from renewable resources. Among the several methods, biological processes to produce biofuels, in particular bioH2 has higher potential. BioH2 has higher efficiency of conversion to usable

power, generates zero pollutants and has high energy density. Hence, bioH2 has become an attractive energy carrier. Academic institutions in association with industries are attempting to develop newer strategies for H2 production. After the mid 1990s, the focus of bioH2 production research shifted from bio-photolysis of water by algae and cyanobacteria [2] as well as photo-fermentation of organic substrates by photosynthetic bacteria [3] to dark fermentation process. Dark fermentation normally achieves a much higher H2 production rate than water photolysis and photo-fermentation. In addition, dark fermentation also owns the advantage of simultaneous waste reduction and H2 generation [4]. Conventional continuous stirred tank reactor (CSTR) systems have been the

* Corresponding author. Feng Chia University, Green Energy Development Center, No.100, Wenhua Road Seatwen, Taichung 40724, Taiwan. Tel.: þ886 4 24517250x6211/6230; fax: þ886 4 35072114. E-mail address: [email protected] (C.-Y. Chu). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.04.119

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 3 7 ( 2 0 1 2 ) 1 5 4 9 6 e1 5 5 0 2

most frequently used configuration for H2-producing bioreactor [5e8] But CSTR configuration has some disadvantages such as slow-growth of the H2-producing bacteria and washout of cells when operated at high feeding rates leading to reactor instability [9]. In order to overcome these problems addition of immobilized cells into the conventional CSTR for increasing the biomass retention in bioH2 producing fermenters has been attempted by many researchers. BioH2 producing fermenters, such as carrier-induced granular sludge bed reactor (CIGSB) [4,10e12], continuously stirred anaerobic bioreactor (CSABR) with silicone immobilized cells and agitated granular sludge bed reactor (AGSBR) [13e18] have been reported. Attempts to enhance biomass retention by immobilized cells demonstrated better H2 production performance than that of conventional CSTR with H2 production rates ranging from 6 to 360 L/d-L [12e16]. Consequently, immobilized cells created by natural or synthetic matrices [3,13e21] were often used to allow better retention of H2-producing bacterial cells for stable operations at higher feeding rates. Most commonly employed immobilization methods are self-granulation, self-flocculation, gelentrapment, surface attachment or biofilm systems. Cell immobilization from surface attachment [10,20,21] and selfflocculation [16e19,22e26] have demonstrated higher feasibility in practical environmental applications. Activated carbon (AC) is the most common matrix for cell growth and biofilm attachment for H2 production [10,27]. It has been found that addition of Ca2þ ions to a fermentative H2 production reactor enhances the size and growth behavior of the seed sludge granules [28]. The Ca2þ ions might also affect the H2 production performance as it is a key element in the formation of granular bioparticles in a bioreactor [4,29]. In this study, we used a fluidized bed reactor (FBR) for H2 production. Anaerobic sludge immobilized by a physical adsorption with AC was used as the biocatalyst in the FBR. Sucrose was used as the substrate for continuous H2 production by the anaerobic sludge. The FBR was designed and operated under sucrose concentration 20 g COD/l and HRT 2e8 h to assess the H2-producing ability. Reproducibility of continuous H2 operations was also examined to evaluate the

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stability and feasibility of using the AC-immobilized cells in practical H2-producing processes.

2.

Materials and methods

2.1.

Sludge and substrate

The anaerobic seed sludge was obtained from Li-Ming Municipal Sewage Treatment Plant in Taichung, Taiwan. The pH, volatile suspended solids (VSS) and total solids (TS) concentrations of the sludge were found to be 6.8, 33.3 and 65.1 g/L, respectively. The H2 productivity of the seed sludge was enhanced by thermal treatment at 100  C for 1 h to inhibit the methanogenic activity in the sludge. The thermallytreated sludge was used for cell immobilization. The medium used for H2 fermentation consisted of sucrose as the sole carbon substrate at a concentration of 20 g COD/L and sufficient inorganic supplements [7] including (mg/L): NH4HCO3, 5240; NaHCO3, 6720; K2HPO4, 125; MgCl2$6H2O, 100; MnSO4$6H2O, 15; FeSO4$7H2O, 25; CuSO4$5H2O, 5; and CoCl2$5H2O, 0.125.

2.2.

Experimental setup

The schematic representation of experimental setup is shown in Fig. 1. The total working volume of the FBR including the buffer tank and pipe was 2.5 L. The aspect ratio of height (H) to diameter (D) of the fluidized bed bioreactor was 3:1. The operation temperature was 40  C. The recycle rate of the effluent was controlled at 5.5 L/min using a recycle peristaltic pump. The sludge bed was kept up to 10% height of the FBR from the bottom.

2.3.

Analysis

H2 was detected by gas chromatography (Shimadzu GC-14A) using a thermal conductivity detector (TCD). The carrier gas used was argon and the column was packed with Porapak Q (80/100 mesh, Waters Corp., USA). The volatile fatty acids and

Fig. 1 e Schematic representation of the experimental setup.

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ethanol were also detected by gas chromatography (Shimadzu GC-14A) using a flame ionization detector (FID). The temperatures at glass column and injection port were 145 and 175  C, respectively. The carrier gas was nitrogen and the packing material was FON (containing polyethylene glycol and 2nitroterephthalic acid). The COD, pH, TS, VS, were measured according to procedures described in Standard Methods [30]. The sucrose concentration in the effluent was determined according to Anthrone Method [31]. During preparation of samples for Scanning Electron Microscope (SEM) analysis, the immobilized cells were first treated with glutaraldehyde (2.5%) for 3 h followed by dehydration in acetone (50e100%) for 30 min. The pretreated immobilized cell samples were coated with gold via vapor deposition (Model IB-2, Eiko, Japan). The

coated immobilized cell samples were then introduced to the SEM machine (Hitachi S3000, Japan) for observation.

3.

Results and discussion

3.1.

Biohydrogen production performance

Two forms of the Ca2þ ions additives (CaCl2 and Ca(OH)2) were used in the nutrients to compare with the Endo’s nutrient formulation (Fig. 2). Initially, the AC particles were fed into the FBR to adsorb the bacteria in a batch mode for 48 h. The bioreactor was then operated with a low feeding rate for cultivation at HRT of 12 h. When the system attained the

Fig. 2 e Evolutions of bioH2 production rate, H2 content, pH and biomass concentration with respect to different HRTs at substrate concentration of 20 g COD/L in presence of (a) Endo’s nutrient without the Ca2D additives; (b) 100 ppm of Ca(OH)2 as Ca2D additive,; (c)100 ppm of CaCl2 as Ca2D additive (d) 200 ppm of CaCl2 as Ca2D additive and (e)50 ppm CaCl2 as Ca2D additive.

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Table 1 e Effects of HRT and calcium additives on biohydrogen production performance in a fluidized bed reactor. Conditions Endo

100 ppm Ca(OH)2

100 ppm CaCl2

200 ppm CaCl2

50 ppm CaCl2

Sucrose Conversion H2 yield HRT Recycle rate Biomass H2 content H2 production rate pH (%) (%) (L/h-L) (mol H2/mol sucrose) (h) (L/min) (g VSS/L) 8 6 4 8 6 4 2 8 6 4 2 8 6 4 8 6 4 2

5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5

2.05 1.99 1.62 2.48 3.05 2.78 2.76 2.57 2.85 2.58 2.21 2.01 1.78 1.56 2.04 1.85 1.78 1.36

38.8 41.4 41.6 42.1 42.7 42.7 41.5 41.4 42.6 41.0 39.7 43.9 41.8 42.0 42.9 42.5 42.1 42.1

0.43  0.02 0.68  0.04 0.85  0.06 0.48  0.07 0.79  0.02 1.01  0.03 1.22  0.07 0.52  0.02 0.78  0.04 0.93  0.05 1.22  0.08 0.53  0.02 0.69  0.03 0.97  0.01 0.55  0.01 0.71  0.05 0.89  0.03 1.09  0.08

6.45 6.55 6.83 6.34 6.40 6.75 6.91 6.39 6.41 6.65 6.93 6.44 6.44 6.75 6.41 6.42 6.74 7.03

2.62 2.90 2.34 2.87 3.76 3.41 2.07 2.95 3.37 3.01 2.10 3.32 3.31 3.13 3.48 3.37 3.05 1.90

99.1 98.9 91.2 98.6 99.2 92.4 91.1 98.7 97.9 91.3 90.7 99.6 98.2 90.3 98.7 96.4 90.8 89.9

Substrate concentration ¼ 20 g COD/L.

steady state, the feeding rate was varied in the range of HRT 8 to 2 h. As shown in Fig. 2(a), the H2 production rates were 0.43, 0.68 and 0.85 L/h-L at the HRTs 8, 6 and 4 h, respectively. The H2 contents in the biogas ranged from 38.8 to 41.6%. Finally, the system operation failed when the feeding rate was switched to HRT 2 h. This happened due to the biomass washout from the system. Fig. 2(b) shows the evolution of the H2 production performance in presence of 100 ppm Ca(OH)2 as additive. The H2 production rate increased when the feeding rate of the substrate increased, as a result of the lowering of HRT from 12 to 2 h. The H2 production rate increased from 0.29 to 1.22 L/h-L with the H2 content of around 42%. It was also found that the bioreactor operation was stable at the feeding rate of HRT 2 h with the addition of 100 ppm Ca(OH)2. But the system failed, when the feeding rate was further increased (HRT 1 h), due to the biomass washout. Fig. 2(c) shows the evolution of the H2 production performance in presence of 100 ppm CaCl2 as additive. The system took 157 h to attain stability when the FBR was started at HRT of 8 h. As shown in Fig. 2(c), the maximum H2 production rate was 1.23 L/h-L at the HRT of 2 h with H2 content of 39.7%. The effect of different Ca2þ ions in the form of either CaCl2 or Ca(OH)2 on H2 productivity appeared insignificant in this study (Table 1). Fig. 2(d and e) show the evolution of the H2 production performance with addition of 200 and 50 ppm CaCl2, respectively. All the experimental data of the H2 production system mentioned above are shown in Fig. 3 and Table 1. As shown in Fig. 3 and Table 1, it was found that at HRT of 4 h, the H2 production rate (HPR) of 1.01 L/h-L with 100 ppm Ca(OH)2 additive was higher than that of without the Ca2þ ions addition (0.85 L/h-L). At HRT 2 h, the maximum HPR values of 1.22 L/h-L were obtained with 100 ppm Ca2þ ions in the form of CaCl2 and Ca(OH)2, respectively. However, the bioH2 production system failed at the HRT of 2 h in presence (200 ppm Ca2þ) as well as in the absence of Ca2þ ions additives, possibly due to the rapid washout of the biomass. The H2 content in the

biogas, in presence of 100 ppm of CaCl2 or Ca(OH)2 additives, was within the range of 39.7e42.7%, whereas in absence of Ca2þ ions it was found to be within the range of 38.8e41.6%. The maximum H2 yield of 3.76 mol H2/mol sucrose was obtained at 100 ppm of Ca(OH)2 supplementation.

3.2.

Soluble metabolites analysis

The soluble metabolites analysis for H2 production in a fluidized bed bioreactor with and without Ca2þ ion additives is shown in Table 2. The major soluble metabolites detected during H2 fermentation were butyric acid (HBu) and acetic acid (HAc), accounting for 47e58% and 21e25% of total soluble microbial products (SMP), respectively. The ratio of HBu to

Fig. 3 e Hydrogen production rates in fluidized bed bioreactor with respect to different HRTs and Ca2D additives at substrate concentration of 20 g COD/L.

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Table 2 e The soluble metabolites analysis in a hydrogen production fluidized bed reactor with and without calcium additives. Conditions Endo

Ca(OH)2 100 ppm

CaCl2 100 ppm

CaCl2 200 ppm

CaCl2 50 ppm

HRT (h)

Ethnol/SMP

HAc/SMP

HPr/SMP

HBu/SMP

TVFA/SMP

TVFA (mg COD/L)

SMP (mg COD/L)

8 6 4 8 6 4 2 8 6 4 2 8 6 4 8 6 4 2

0.06 0.08 0.06 0.06 0.08 0.06 0.04 0.08 0.07 0.07 0.04 0.08 0.06 0.05 0.06 0.05 0.05 0.04

0.21 0.22 0.24 0.22 0.23 0.23 0.23 0.22 0.21 0.22 0.25 0.22 0.22 0.23 0.22 0.22 0.23 0.24

0.24 0.20 0.20 0.25 0.20 0.19 0.18 0.18 0.23 0.20 0.18 0.19 0.22 0.15 0.16 0.18 0.15 0.14

0.49 0.50 0.49 0.47 0.49 0.52 0.54 0.52 0.49 0.51 0.53 0.52 0.50 0.57 0.56 0.55 0.57 0.58

0.94 0.92 0.94 0.94 0.92 0.94 0.96 0.92 0.93 0.93 0.96 0.92 0.94 0.95 0.94 0.95 0.95 0.96

6574 6165 6385 6223 6103 6963 7542 7103 7873 6569 7731 6575 9507 9550 8673 7551 9507 8887

7011 6707 6826 6631 6624 7444 7885 7684 8510 7086 8089 7113 10,120 10,086 9252 7983 10,040 9217

HAc: acetic acid; HPr: propionic acid; HBu: butyric acid; EtOH: ethanol; TVFA ¼ HAcþHPr-HBu; SMP ¼ TVFA þ EtOH.

HAc (B/A) is often used as a performance indicator for dark H2 fermentation. This ratio was found to be 2.9 in this study and is within the range of favorable B/A ratios as reported by other dark H2 fermentation studies using carbohydrate substrates

[11,28,32]. Inspection of the composition of the soluble metabolites obtained from this work suggests that the cultures carried out metabolic pathways in favor of H2 production, as the soluble metabolites were predominantly

Fig. 4 e (a) Scanning Electron Microscopy (SEM) image of the cross section of carrier particle which was prepared by physical adsorption with activated carbon, (b) SEM image of the bacterial community inside the carrier particle after cultivation.

Fig. 5 e (a) Scanning Electron Microscopy (SEM) image of bacterial suspension on filter paper in presence of Ca2D additive at 2000X magnification, (b) SEM image of bacterial suspension on filter paper in presence of Ca2D additive at 10000X magnification.

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HBu and HAc. This indicates that adding optimum amount of Ca2þ ions in a FBR with immobilized cells can enhance H2producing bioprocess. The SMP was found to increase from 6830 to 10,100 mg COD/L after addition of 200 ppm CaCl2 at HRT of 4 h.

3.3.

SEM observation

Clostridium spp. are well known for their greater ability to produce bioH2. The Clostridium spp. has a shuttle-shaped appearance. They have the advantage of forming spores and enter into a dormant state, reserving their activity when the environmental conditions are unfavorable for their survival and growth. Fig. 4(a and b) show the cross-section of carrier particle prepared by a physical adsorption with AC and the bacterial community inside the carrier particle after cultivation. As shown in Fig. 4(b) it was found that the center of the carrier particles had both forms of the Clostridium spp. i.e., the metabolically active form (shuttle-shaped structure) and the dormant spore (ball-shaped structure). This indicated that the substrate could not enter into the carrier particle, which resulted in the sporulation of the Clostridium spp. However, the spores preserved the activity inside the immobilized cells. Fig. 5(a and b) show the appearance of bacterial suspension under 2000 and 10000X magnification with Ca2þ ions additive on the filter paper. It is evident from the SEM photographs that there is a considerable development of the bacterial community of the Clostridium spp. in the liquid phase.

4.

Conclusions

The optimal H2 production rate of 1.22 L/h-L was obtained with 100 ppm of Ca2þ ions, either CaCl2 or Ca(OH)2, in the immobilized cells-FBR system. The H2 content in the biogas was found to be in the range of 38.8e43.9%. The composition analysis of the soluble metabolites revealed that they were predominantly HBu and HAc. This suggests that the cultures carried out metabolic pathways in favor of H2 production. Thus, adding proper concentration of Ca2þ ions in a FBR system with immobilized cells could be effective in enhancing H2-producing bioprocess by 12e18% when HRT ranged from 4 to 8 h.

Acknowledgments The authors gratefully acknowledge the financial support by National Science Council of Taiwan (grant no. NSC 100-2632E-035-001-MY3), Taiwan’s Bureau of Energy (grant no. 100D0204-3), and Feng Chia University (grant no. FCU-10G27101).

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