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Process Biochemistry 42 (2007) 279–284 www.elsevier.com/locate/procbio

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Batch and continuous fermentative production of hydrogen with anaerobic sludge entrapped in a composite polymeric matrix Ken-Jer Wu a,b, Jo-Shu Chang b,* b

a Department of Biochemical Engineering, Kao Yuan University, Kaohsiung 821, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan

Received 19 December 2005; received in revised form 22 June 2006; accepted 13 July 2006

Abstract Cell immobilization techniques were adopted to biohydrogen production using immobilized anaerobic sludge as the seed culture. Sucrosebased synthetic wastewater was converted to H2 using batch and continuous cultures. A novel composite polymeric material comprising polymethyl methacrylate (PMMA), collagen, and activated carbon was used to entrap biomass for H2 production. Using the PMMA immobilized cells, the favorable conditions for batch H2 fermentation were 35 8C, pH 6.0, and an 20 g COD l1 of sucrose, giving a H2 production rate of 238 ml h1 l1 and a H2 yield of 2.25 mol H2 mol sucrose1. Under these optimal conditions, continuous H2 fermentation was conducted at a hydraulic retention time (HRT) of 4–8 h, giving the best H2-producing rate of 1.8 l h1 l1 (over seven-fold of the best batch result) at a HRT of 6 h and a H2 yield of 2.0 mol H2 mol sucrose1. The sucrose conversion was essentially over 90% in all runs. The biogas consisted of only H2 and CO2. The major soluble metabolites were butyric acid, acetic acid, and 2,3-butandiol, while a small amount of ethanol also detected. The PMMAimmobilized-cell system developed in this work seems to be a promising H2-producing process due to the high stability in continuous operations and the capability of achieving a competitively high H2 production rate under a relatively low organic loading rate. # 2006 Elsevier Ltd. All rights reserved. Keywords: Anaerobic sludge; Biohydrogen production; Continuous culture; Immobilized cell; Polymethyl methacrylate

1. Introduction Hydrogen has been recognized as an ideal energy carrier of the future because it is clean, recyclable, and efficient [1,2]. Being a country importing over 95% of its energy demand, Taiwan has sensed the importance of this new energy carrier and has been devoted to the development of hydrogen energy technology. Production of H2 is one of the vital components in H2 energy platform. Biological production of H2 provides a feasible means for the sustainable supply of H2 with low pollution and high efficiency, thereby being considered a promising way of producing H2 [1,2]. Fermentative H2 production can be achieved by dark fermentation (with obligate or facultative anaerobes) or by photo fermentation (with photoheterotrophic bacteria) [1,2]. Among them, dark fermentation normally achieves a much higher H2 production

* Corresponding author. Tel.: +886 6 2757575x62651; fax: +886 6 2357146/2344496. E-mail address: [email protected] (J.-S. Chang). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.07.021

rate and is considered more applicable for simultaneous waste reduction and H2 generation [1]. Cell immobilization technology has been successfully applied to fermentation and enzymatic transformation [3]. However, the technology has not been widely adopted to H2 production through dark fermentation, whereas there were some examples describing using immobilized cells for phototrophic H2 production [4,5]. Our recent work had developed several immobilized-cell systems for dark H2 fermentation [6,7]. Those cell-entrapment type immobilized cells were effective in H2 production on batch mode but most of them were either infeasible in continuous operations (e.g., calcium alginate (CA)-based and polyurethane (PU) immobilized cells [6]) or have not yet been tested in continuous operations (e.g., immobilized cells created by ethylene-vinyl acetate (EVA) copolymer [7]). In this work, we attempted to create more stable and reliable immobilized cells able to be used on continuous operations. In addition, the effect of the medium and environmental factors was investigated to identify favorable conditions for H2 production with the immobilized-cell system. This work is to our knowledge one

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of the early attempts in using immobilized bacterial microflora for continuous dark H2 production [8]. The outcome of this work is expected to provide useful information for future development of commercial viable bioprocesses for H2 production.

from the culture was measured with a gas meter (Type TG1; Ritter Inc., Germany) with a measuring limit of 10 ml. The gas volumes presented in this work were standardized to 25 8C and 760 mmHg. The time-course H2 evolution data were simulated with modified Gompertz equation (Eq. (1)) [10] to determine the H2 production potential (Hmax), maximum H2 production rate (Rmax), and the lag time (l):

2. Materials and methods

Rmax;H2 e H ¼ H max exp exp ðl tÞ þ 1 H max

2.1. Hydrogen-producing sludge and fermentation medium The seed sludge was collected from the final sedimentation tank of a municipal activated-sludge-based wastewater treatment plant located in central Taiwan. Prior to use, the sludge was subjected to acidic pretreatment [9] and was then acclimated at 35 8C in a continuous-flow reactor operated at a HRT of 6– 12 h to enrich its H2 producing activity. The medium for cell growth and H2 production contained 20 g COD sucrose l1 (adjustable) as the sole carbon source as well as sufficient amounts of inorganic salts [9].

2.2. Immobilization of anaerobic sludge Fifty milliliters (ca. 0.15 g VSS) of acclimated H2-producing sludge was mixed with 25 g of polymethyl methacrylate (PMMA). Supplemental materials (15 g collagen and 10 g activated carbon) were added to modify the physical properties (e.g., density, pore size, mechanical strength, etc.) of the immobilized cells. The core matrix, supplemental materials, and sludge were mixed thoroughly at 45 8C. The colloid mixture was transferred to a syringe, and was then extruded to form disc-like beads with a diameter of 0.5 cm and a volume of ca. 0.25 cm3. No coagulation/curing solution was used after bead extrusion.

2.3. Batch H2 fermentation Twenty-five grams of PMMA-immobilized beads (containing ca. 0.15 g biomass) was inoculated into a 250-ml serum vial containing 100 ml of the aforementioned medium. After inoculation, the vial was sparged thoroughly with argon gas to create an anaerobic condition. The batch operations were conducted at different sucrose concentration (5–30 g COD l1), pH (5.5–7.0), and temperature (30–40 8C) under a fixed agitation rate of 100 rpm. The composition of gaseous (mainly H2 and CO2) and soluble products (volatile fatty acids and alcohols) was monitored with respect to time. The gas produced

(1)

The volumetric H2 production rate and H2 yield were used as major performance indexes assessing the performance of H2 production. The volumetric H2 production rate was determined based on the kinetic constant estimated from modified Gompertz equation (Eq. (1)). The yield was defined as mol of H2 formed per mol of sucrose consumed.

2.4. Continuous H2 fermentation A 2.5-l jar fermentor was used for continuous H2 production using the PMMA-immobilized cells. About 100 g of immobilized beads (containing ca. 0.6 g biomass) were added into 1 l of medium containing 20 g COD l1 of sucrose. The continuous fermentation was operated at 35 8C, pH 6.0, 200 rpm agitation, and a hydraulic retention time (HRT) of 4–8 h. The continuous culture was started up at a HRT of 8 h. While a stable operation was reached, the HRT was progressively decreased from 8 to 6 h, and then finally to 4 h. A gas meter was connected to the gas effluent of the fermentor to measure the amount of biogas produced. Samples were taken from at designated time intervals to detect the gas products and soluble metabolites.

2.5. Analytical methods The gas products were analyzed by gas chromatography (GC) using a thermal conductivity detector (TCD). The volatile fatty acids and ethanol were also detected by GC using a flame ionization detector (FID). The details for GC analysis were described in our recent report [9,11,12]. Standard methods [13] were used to measure the carbohydrate concentration in the effluent and to determine the biomass concentration (in terms of volatile suspended solid; VSS) of the sludge samples. The biomass content in immobilized beads was examined at the end of experiment by mechanical disruption of the beads, followed by similar weighing procedures for VSS measurement.

Table 1 H2 production performance and estimated kinetic parameters with modified Gompertz equation (Eq. (1)) in batch fermentation using PMMA immobilized cells under different combinations of temperature and initial pH Run

1 2 3 4 5 6 7 8 9 10 11 12

Temperature (8C)

Initial pHa

H2 contentb (%)

H2 production rateb,c (ml h1 l1)

H2 yieldb (mol H2 mol sucrose1)

30 30 30 30 35 35 35 35 40 40 40 40

5.5 6.0 6.5 7.0 5.5 6.0 6.5 7.0 5.5 6.0 6.5 7.0

48 2 47 1 27 0 27 1 49 3 43 1 39 1 45 0 50 2 39 0 40 1 40 2

28 2 25 1 33 3 49 2 95 7 238 10 109 4 136 3 129 9 104 8 60 4 44 1

0.31 0.02 0.39 0.03 0.41 0.04 0.30 0.01 1.21 0.07 2.25 0.12 1.42 0.09 1.58 0.17 0.91 0.07 0.80 0.04 1.53 0.11 1.41 0.08

Initial sucrose concentration = 20 g COD l1. a The final pH was within the range of 4.5–4.8. b The data are the mean values of duplicate tests (the ‘‘’’ denotes standard deviation of duplicates). c Maximum volumetric H2 production rate.

Model simulation Total H2 evolution, Hmax (ml)

Rmax (ml h1)

l (h)

R2

34 49 51 36 135 252 158 181 109 96 171 165

2.8 2.5 3.3 4.9 9.5 23.8 10.9 13.6 12.9 10.4 6.0 4.4

8.2 8.5 8.5 7.8 7.7 7.8 8.2 8.1 7.8 7.7 7.7 8.1

0.970 0.998 0.990 0.964 0.986 0.995 0.995 0.998 0.963 0.950 0.964 0.942


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3. Results and discussion 3.1. Batch H2 production with PMMA-immobilized cells To characterize the H2-producing performance of the PMMA immobilized cells, three critical influential factors (temperature, pH, and carbon substrate concentration) on dark H2 fermentation were examined [14]. The kinetic data obtained from batch cultures conducted at various pH and temperatures were simulated by modified Gompertz equation (Eq. (1)). Table 1 shows that the best H2 production rate (Rmax), total H2 evolution (Hmax), and H2 yield (Y H2 ) occurred at pH 6.0 and 35 8C (Table 1). These favorable pH and temperature values are similar to those obtained for suspended or granular sludge systems inoculated with the same seed culture [9,15]. Batch tests were also conducted to identify the best initial sucrose concentration (5–30 g COD l1) when the most favorable pH (6.0) and temperature (35 8C) were used. Table 2 shows that both Rmax and Hmax reached the highest level when the initial sucrose concentration was 20 g COD l1, while both 10 and 20 g COD l1 gave high Y H2 values of 2.68 and 2.25 mol H2 mol sucrose1, respectively (Table 2). Consistently, our previous free-cell work also showed that 20 g COD l1 of sucrose resulted in the best H2 producing performance [9,15,16]. The foregoing results suggest that the cell immobilization procedures did not considerably alter the H2 production properties of the bacterial population in the sludge. 3.2. Continuous H2 production using PMMA-immobilized cells Using the favorable conditions obtained from batch studies (i.e., 35 8C, pH 6.0, 20 g COD l1 of sucrose in feed), continuous cultures were carried out for H2 production under a progressively decreased HRT (from 8 to 4 h). As indicated in Fig. 1, the H2 production rate increased when HRT was decreased from 8 to 6 h, whereas further decrease in HRT to 4 h resulted in a marked decrease in H2 production rate. Further decrease in HRT to 2 h resulted in significant washout of suspended biomass and unstable operation of the system (data not shown). The best H2 production rate in continuous culture

Fig. 1. Time-course profiles of volumetric H2 production rate and H2 content performance in continuous culture containing PMMA immobilized cells under different hydraulic retention time (HRT). (Sucrose concentration in feed = 20 g COD l1, pH 6.0, temperature = 35 8C.)

was ca. 1.8 l h1 l1 (at HRT = 6 h), which is significantly higher than the maximum rate obtained from batch cultures (238 ml h1 l1), indicating a more efficient H2 production performance in continuous fermentation. Table 3 shows that operation at HRT = 6 h also gave a slightly better H2 content in biogas (41%). The H2 yield was similar for HRT = 6 and 8 h with a value of 1.7–2.0 mol H2 mol sucrose1, but dropped considerably to 1.1 mol H2 mol sucrose1 at HRT = 4 h (Table 3). Under identical operation conditions, the continuous culture with suspended cells (SPC) attained similar H2 yield and H2 content in biogas to the immobilized cell (IMC) system at a HRT of 6 and 8 h, while the production rate for SPC was 50–55% lower than that for IMC (Table 3). Meanwhile, the SPC

Table 2 H2 content in biogas and estimated kinetic parameters with modified Gompertz equation (Eq. (1)) in batch fermentation using PMMA immobilized cells under different initial sucrose concentrations Sucrose concentration (g COD l1)

H2 contenta (%)

H2 production ratea,b (ml h1 l1)

H2 yielda (mol H2 mol sucrose1)

Model simulation Total H2 evolution, Hmax (ml)

Rmax (ml h1)

l (h)

R2

5 10 20 30

38 1 41 2 43 1 40 0

57.5 6.3 170 7 238 5 67.5 6.1

1.78 0.11 2.68 0.18 2.25 0.11 0.58 0.05

50 150 252 97

5.75 17 23.8 6.75

14.2 13.5 7.8 16.1

0.912 0.973 0.995 0.948

Initial pH 6.0, temperature = 35 8C. a The data are the mean values of duplicate tests (the ‘‘’’ denotes standard deviation of duplicates). b Maximum volumetric H2 production rate.

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Table 3 H2 production performance in continuous culture containing PMMA immobilized cells and suspended cells (control) under different hydraulic retention time (HRT) HRT (h)

Culture type

H2 content in biogasa (%)

H2 production ratea (l h1 l1)

H2 yielda (mol H2 mol sucrose1)

8

IMC SPC

37 2 38 1

1.42 0.17 0.93 0.07

1.7 .01 1.9 0.3

6

IMC SPC

41 2 40 1

1.80 0.22 1.21 0.10

2.0 0.2 1.9 0.1

4

IMC SPC

39 2 wo

0.83 0.10 wo

1.1 0.2 wo

biocatalysts, as the major H2 producers in fixed-bed and CIGSB system were the surface-attached biofilms [16] and selfflocculated granules [9,12], respectively. In contrast, the H2 producers of this immobilized-cell (IMC) system were entrapped in the porous polymeric materials. Hence, more mass transfer limitations may arise from the IMC system than from biofilm and granular sludge, resulting in the difference in the effect of HRT on H2 production. 3.3. Carbon substrate utilization and soluble metabolites production

Sucrose concentration in feed = 20 g COD l1, pH 6.0, temperature = 35 8C. IMC: immobilized cells, SPC: suspended cells, wo: cell wash-out. a The data are the mean values of duplicate tests (the ‘‘’’ denotes standard deviation of duplicates).

culture was washed out while operating at a HRT of 4 h, at which the IMC system was able to maintain stable, indicating that IMC system was more stable against hydraulic dilution rates. Moreover, compared to our recent continuous H2 producing processes [11,12,16], the present IMC system was much more stable in terms of H2 production rate (Fig. 1) and can be stably operated for over 6 months (data not shown). It is likely that in the immobilized-cell system, most of the bacterial populations were protected inside the polymeric matrix, thereby being able to buffer the impact of variations in environmental factors. However, compared to our recent fixedbed [16] and carrier-induced granular sludge bed (CIGSB) [9] bioreactors, showing an optimal HRT of 0.5–1 h, the best HRT in this system was higher (i.e., HRT = 6 h). This difference could be attributed to different physical characteristics of the

Sucrose conversion in all the batch runs was in general within 83–95% and was as high as 98–99% in continuous runs, indicating a good substrate conversion efficiency of the proposed immobilized-cell system. Accompanying production of biogas (essentially consisting of 30–40% H2 and 60–70% CO2) (Tables 1–3), soluble metabolites (e.g., acids and alcohols) also formed. The major soluble metabolites were butyric acid (HBu), acetic acid (HAc), and 2,3-butandiol (2,3BuOH). These three products accounted for 81–98% of total soluble microbial products (SMP) (Table 4). The only difference compared to our previous H2-producing processes [6–9,12,13,16] was the formation of a considerable amount of 2,3-BuOH, which was absent in our previous studies. This result seems to suggest that some facultative anaerobic H2 producers (e.g., Enterobacter or Klebsiella species) could be present in the immobilized cells, since 2,3-BuOH is one of the major products of those facultative anaerobes while catabolizing carbohydrates [17]. Indeed, one of the pure strain isolated from the immobilized-cell beads was identified as Klebsiella sp. HE1 (NCBI accession no. AY540111) according to 16S

Table 4 Production and composition of soluble metabolites during batch and continuous H2 fermentation with PMMA immobilized cells under different temperature and pH Operation mode

Temperature (8C)

Initial pH

TVFAa (mg COD l1)

SMPa (mg COD l1)

HAc/SMP (%)

HBu/SMP (%)

EtOH/SMP (%)

2,3-BuOH/SMP (%)

TVFA/SMP (%)

Batch

30 30 30 30 35 35 35 35 40 40 40 40

5.5 6.0 6.5 7.0 5.5 6.0 6.5 7.0 5.5 6.0 6.5 7.0

763 68 659 75 739 37 919 18 1040 62 1416 42 983 50 1209 108 1023 71 972 49 941 92 855 61

1519 46 1416 113 1391 97 1761 141 1919 96 1808 72 1877 95 1833 110 1590 48 1691 118 1694 85 1630 65

24 20 23 28 24 29 23 28 25 24 25 23

26 27 30 31 31 49 30 38 39 33 31 29

15 16 15 6 13 2 16 7 8 11 17 19

35 37 38 35 32 20 31 27 28 32 27 29

50 47 53 52 54 78 52 66 64 57 56 52

Operation mode

HRT (h)

TVFAa (mg COD l1)

SMPa (mg COD l1)

HAc/SMP (%)

HBu/SMP (%)

EtOH/SMP (%)

2,3-BuOH/SMP (%)

TVFA/SMP (%)

Continuousb

8 6 4

920 89 2403 144 1981 60

1545 123 3184 159 3905 234

28 33 22

32 42 29

7 3 9

33 22 40

60 75 51

Initial or feeding sucrose concentration = 20 g COD l1. a The data are the mean values of duplicate tests (the ‘‘’’ denotes standard deviation of duplicates). b Conducted at pH 6.0 and temperature = 35 8C; HAc: acetic acid; HBu: normal butyric acid; EtOH: ethanol; 2,3-BuOH: 2,3-butandiol; TVFA (total volatile fatty acid) = HAc + HBu; SMP: soluble microbial products (SMP = TVFA + EtOH + 2,3-BuOH).


rDNA sequence comparison, as the strain has a 99.9% identity to Klebsiella pneumoniae subsp. ozaenae (NCBI accession no. AF228919). This finding may explain why 2,3-BuOH was produced in both batch and continuous cultures. Moreover, there was a general trend that when the H2 production rate was higher, the total volatile fatty acid (TVFA) production was higher, so was the TVFA/SMP ratio (Table 4). This suggests that acid formation was preferable for H2 production, whereas production of alcohols was not favorable. These results are consistent with the common rules that alcohol formation may consume free electrons carried by metabolic reducing power (e.g., NADH), thereby inhibiting H2 production [18]. 3.4. Novelty, significance, and limitation of the proposed immobilized-cell system In our recent work, several cell-entrapment-type immobilized-cell systems were developed [6–8]. However, most of them (e.g., CA-based and PU immobilized cells [6]) have not been successfully utilized in continuous fermentation, due mainly to the insufficient mechanical strength and stability for long-term operation. That was the reason why a stronger polymeric matrix (i.e., PMMA) was used in this study to create a more stable immobilized-cell beads suited for a prolonged continuous operation. It is not easy to use a rigid polymer, like PMMA, as a matrix for cell immobilization. The key technique was the addition of supplemental materials to modify the properties of the matrix and to create appropriate pore size. To our best knowledge, this is the first attempt of using PMMA to immobilize cells for H2 production or other applications. Indeed, the resulting PMMA-immobilized cells displayed a very stable performance in continuous production of H2 from sucrose, allowing stable operation for over 6 months (data not shown). In addition, the PMMA-immobilized cells were able to achieve a high H2-prodcution rate at a relatively low dilution rate (high HRT). Despite operation at a high HRT of 6 h, the PMMA-immobilized cells attained a H2 production rate of 1.8 l h1 l1, which is higher than most of reported values [1,2] and similar to the maximal H2 production rate (1.32 l h1 l1) achieved by our previous fixed-bed process at a much shorter HRT of 1 h [16]. This special feature clearly suggests that using PMMA cells might reduce the operational cost by gaining a comparable H2 producing capacity at a much lower organic load rate (or a much longer HRT). Although the H2 yield (up to 2.68 mol H2 mol sucrose1) obtained from the present study is considerably higher than our recent immobilized-cell systems [7,8], the yield is still lower than that obtained from the suspended-cell systems [9,14–16]. The major cause for the lower yield could be due to the presence of an unfavorable bacterial community structure or the mass transfer limitations arising from cell entrapment. It is likely that the lower pH or higher H2 and CO2 concentration may be present within the entrapped cells due to accumulation of acidic metabolites and gas products, resulting in earlier termination of H2 production or unfavorable H2-producing kinetics. Nevertheless, this limitation might be overcome by an appropriate adjustment of the pore size of the PMMA cells.

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4. Conclusions This work demonstrated a feasible immobilized-cell system for batch and continuous H2 production with a high stability and efficiency. The cells entrapped with novel composite polymeric matrix (PMMA/collagen/activated carbon) displayed good mechanical strength and H2-producing activity. Batch tests were conducted to explore favorable conditions for H2 production with the PMMA immobilized cells. The best sucrose concentration, pH, and temperature obtained from batch fermentation were 20 g COD l1, 6.0, and 35 8C, respectively, giving a H2 production rate of 238 ml h1 l1 and a yield of 2.25 mol H2 mol sucrose1. In continuous culture, the best H2 producing performance occurred when it was carried out at a relatively high HRT of 6 h, attaining an excellent H2 production rate of 1.8 l h1 l1 and a H2 yield of 2.0 mol H2 mol sucrose1. The outcome of this work suggests the potential of using this immobilized-cell system for continuous H2 production in practice. Acknowledgements The authors gratefully acknowledge the financial support of Taiwan’s National Science Council (Grant Nos. 93-2211-E006-040 and NSC 94-2211-E-006-026) and Taiwan’s Bureau of Energy (Grant Nos. NSC93-ET-7-006-001-ET and NSC94-ET7-006-004-ET). References [1] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001;26:13–28. [2] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 2004;29: 173–85. [3] Bickerstaff GF. Immobilization of Enzyme and Cells Totowa, NJ, USA: Humana Press Inc.; 1997. [4] Bagai R, Madamwar D. Prolonged evolution of photohydrogen by intermittent supply of nitrogen using a combined system of Phormidium valderianum, Halobacterium halobium and Escherichia coli. Int J Hydrogen Energy 1998;23:545–50. [5] Zhu H, Suzuki T, Tsygankov AA, Asada Y, Miyake J. Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. Int J Hydrogen Energy 1999;24:305–10. [6] Wu S-Y, Lin C-N, Lee P-J, Chang J-S. Microbial hydrogen production with immobilized anaerobic cultures. Biotechnol Prog 2002;18:921–6. [7] Wu S-Y, Lin C-N, Chang J-S. Biohydrogen production with anaerobic sludge immobilized by ethylene-vinyl acetate copolymer. Int J Hydrogen Energy 2005;30:1375–81. [8] Wu S-Y, Lin C-N, Chang J-S. Hydrogen production with immobilized sewage sludge in three-phase fluidized beds. Biotechnol Prog 2003;19: 828–32. [9] Lee K-S, Wu J-F, Lo Y-S, Lo Y-C, Lin P-J, Chang J-S. Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor. Biotechnol Bioeng 2004;87:648–57. [10] Van Ginkel S, Sung S, Lay J-J. Biohydrogen production as a function of pH and substrate concentration. Environ Sci Technol 2001;35: 4726–30. [11] Lee K-S, Lo Y-S, Lo Y-C, Lin P-J, Chang J-S. H2 production with anaerobic sludge using activated-carbon supported packed-bed bioreactors. Biotechnol Lett 2003;25:133–8.

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