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Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic materials as solid matrices
Enzyme and Microbial Technology 29 (2001) 280 –287
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Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic materials as solid matrices Narendra Kumar, Debabrata Das* Department of Biotechnology, Indian Institute of Technology, Kharagpur, India Received 31 October 2000; received in revised form 23 May 2001; accepted 1 June 2001
Abstract A process was developed for continuous hydrogen production using immobilized Enterobacter cloacae IIT-BT 08 on environmentally friendly lignocellulosic solid matrices. Among the three lignocellulosic carriers used, SM-C (coir) was found to be the best in terms of cell retention (0.44 g dry cell/g dry carrier), packing density (100 g/liter reactor volume), cell loading (44 g dry cell/liter reactor volume) and hydrogen production rate (62 mmol/liter h). Gas hold-up was a major problem in tubular bioreactor with immobilized cells. The tapered and rhomboid bioreactors gave better performance in terms of both the rate of hydrogen production and the gas hold up. The gas hold-up was reduced by 67% using the rhomboid bioreactor as compared to tubular one. The maximum hydrogen production rate achieved was 75.6 mmol/liter h at a dilution rate of 0.93 h⫺1 and recirculation ratio of 6.4. The substrate conversion efficiency was increased by 15% at these conditions as compared to the system with no recycling. There was no substrate inhibition for hydrogen production up to 1.0% glucose concentration. The max and ks for immobilized cells were 1.25 h⫺1 and 9.31 g/liter respectively. The maximum rate of hydrogen production was found to be 2.1 times higher than that of batch system. The process was found more efficient as compared to other continuous hydrogen production processes using packed bed reactor. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Hydrogen; Enterobacter cloacae; Bioreactor configurations; Lignocellulosic materials as solid matrices; Immobilized cells
1. Introduction Hydrogen production is getting importance as an alternative pollution free gaseous fuel for the future. Among different routes of hydrogen production biohydrogen production processes were investigated with both photosynthetic [1,2] and fermentative microorganisms [3– 6]. The rate of fermentative hydrogen production was found faster as compared to photosynthetic processes. These processes still required further improvement for commercial exploitation. The hydrogen production rate using fermentative microorganisms can be enhanced by developing some suitable microbial strain and also by improvement of cell density by whole cell immobilization. One microbial strain with high rate of hydrogen production was isolated and characterized as Enterobacter cloacae IIT-BT 08 [7–12]. Hydrogen production using immobilized whole cells was reported by several researchers [13–15]. The major problems with immobilized hole cells system were low substrate conversion efficiency
and low rate of hydrogen production mainly due to mass transfer resistance in a fixed bed bioreactor. The solid matrices used for the immobilization of the whole cells were mostly either synthetic polymers or inorganic materials. These materials possessed disposal problems. So, environmentally friendly natural polymers such as lignocellulosic materials were chosen as solid matrices in the present studies. The paper deals with the effect of several lignocellulosic materials for hydrogen production. Gas hold-up was found to be a major problem in hydrogen generating system in a packed bed reactor. The effect of different bioreactor configuration was taken into consideration to overcome this problem. Finally attempts were made to increase substrate conversion efficiency and to determine the cell growth kinetic parameters using immobilized Enterobacter cloacae IIT-BT 08.
2. Materials and methods 2.1. Microorganism and culture condition
* Corresponding author. Tel.: ⫹91-3222-82248/78053; fax: ⫹913222-78707. E-mail address: [email protected] (D. Das).
Enterobacter cloacae IIT-BT 08, a locally isolated bacterium was used in all the experiments. The maintenance of
0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 3 9 4 - 5
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
Fig. 1. Schematic diagram of the continuous hydrogen production in packed-bed reactor.
culture, preparation of seed culture and production media were same as reported earlier [10]. The support matrices used for packing the bioreactor were lignocellulosic materials such as rice straw (SM-A), bagasse (SM-B) and coir (SM-C). The pretreatment of the entire solid matrix was done as reported elsewhere [16].
281
Fig. 2. Sketch of different bioreactors configuration.
was followed by passing the production medium at different dilution rates. 2.4. Analysis Sugar, biomass concentration and hydrogen content in the evolved gas were determined by the methods as reported earlier [9].
2.2. Experimental set-up
2.5. Gas hold-up calculation
The schematic diagram of the experimental set-up for the operation of continuous hydrogen production using immobilized whole cell is shown in Fig. 1. A quasi-steady state (5% variation) was confirmed at each dilution rate with respect to constant values of H2 evolution rate, glucose and cell mass concentration in the effluent. The experiments were repeated at different flow rates to get maximum hydrogen production and sugar utilization. Anaerobicity was maintained in all the experiment. All the bioreactors used in the present study were operated continuously for a minimum period of 40 days to find out the operational stability of the system.
The gas hold-up in packed bed reactor was studied by introducing a pulse tracer input to the reactor inlet and its exit concentration profile was measured. The gas hold up was calculated as follows: T ⫽ Vr/f Vr ⫽ Vf ⫹ Vg 1 ⫽ Vf/Vr ⫹ Vg/Vr ⫽ t/T ⫹ Vg/Vr Gas hold-up ⫽ Vg/Vr ⫽ 1 ⫺ t/T where T ⫽ hydraulic retention time, t ⫽ mean residence time calculated by tracer input, F ⫽ flow rate Vr ⫽ reactor volume, Vf ⫽ working volume, Vg ⫽ gas volume inside reactor. 2.6. Generation time of immobilized cells in bioreactor
2.3. Bioreactor system Three bioreactors (Fig. 2) were designed and fabricated. The bioreactors were packed with pretreated solid matrices. To immobilize the whole cells, 35 g/liter of cells suspension in 0.1 M phosphate buffer pH 6 was passed through the packed column at a dilution rate of 0.2 h⫺1 with the help of a peristaltic pump for 8 h. Thereafter, the reactor was kept at rest for 12 h. The reactors were washed with 0.1 M phosphate buffer pH 6.0 at a dilution rate of 0.2 h⫺1. The immobilized cells were immersed in production media (for about 8 –10 h) till the gas production rate reached its maximum and became steady. This
The generation time for immobilized cells was determined according to the method described by Navarro and Table 1 Analysis on loading of E. cloacae on different natural biopolymers Sl. #
Support matrix
Density of packing material (g solid matrix/l or reactor)
Cell loading (g dry cell/g support)
Cell loading (g dry cell/l of reactor)
1 2 3
SM-A SM-B SM-C
54.6 64.7 100
0.32 0.39 0.44
31.73 37.5 44
282
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
Fig. 3. Hydrogen production by immobilized whole cells in a packed bed reactor with SM-A as support matrix (bioreactor configuration–tubular; bioreactor volume–380 ml; temperature–37°C; media used–MYG).
Durand [17]. The bioreactor packed with immobilized cells was continuously fed at the bottom. The flow rate was adjusted such that the residence time of the dislodged cells in the reactor did not exceed the doubling time of the cells. The generation time for freely suspended cells was reported earlier [9]. Multiplication of the free cells in the bioreactor (if any) was prevented because of the high flow rate employed. The optical density of cell suspension leaving the system (effluent) was measured. The peaks indicate the generation time of the cells, which is the difference between any two peaks.
3. Results and discussions 3.1. Retention of E. cloacae IIT-BT 08 on different natural biopolymers Starvation of nutrient enhances the adsorption of microbial cells on solid support matrix [18]. So, in the present studies, adsorption was done in nutrient starved condition using the cells suspension in phosphate buffer. The cell loading in different solid matrices are shown in Table 1. The cell loading both in terms of g cells per g of packing
Fig. 4. Hydrogen production by immobilized whole cells in a packed bed reactor with SM-B as support matrix (bioreactor configuration–tubular; bioreactor volume–380 ml; temperature–37°C; media used–MYG).
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
283
Fig. 5. Hydrogen production by immobilized whole cells in a packed bed reactor with SM-C as support matrix (bioreactor configuration–tubular; bioreactor volume–380 ml; temperature–37°C; media used–MYG).
material and g of cells per volume of reactor was maximum in the case of carrier SM-C and was minimum in the case of carrier SM-A. The packing density was also highest in SM-C. This might be attributed to more surface area due to fibrous and corrugated nature of the same [19]. 3.2. Effect of different carriers on hydrogen production The effect of dilution rate on hydrogen production, substrate conversion efficiency and biomass concentration in the effluent was studied in a packed bed reactor using
different lignocellulosic support matrices. At each dilution rate, the bioreactor was run at least for two days until and unless steady conditions were achieved. The maximum rate of hydrogen production in the case of SM-A was 44 mmol/ liter h (Fig. 3) at a dilution rate of 0.93 h⫺1 with 53% glucose conversion efficiency. The cell concentration in the effluent was almost constant at or above the dilution rate of 0.37 h⫺1. The carrier SM-B showed a better rate of hydrogen production (52 mmol/liter h) and glucose conversion efficiency (61%) than that of SM-A (Fig. 4). This might be due to higher cell density in the case of carrier SM-B.
Fig. 6. Effect of dilution rate on gas hold up using different bioreactor (bioreactor volume–380 ml; support matrix–SM-C; temperature–7°C; media usedMYG).
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N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
Table 2 Performance of different bioreactor using SM-C as solid matrix Type of bioreactor
Dilution rate (h⫺1)
Rate of H2 production (mmol/l h)
Substrate conversion efficiency (%)
Enhancement of H2 production (%)
Tubular Tapered Rhomboid
0.93 0.93 0.93
62.5 65.2 71.0
61 65 69
— 4.3 13.6
Volume of each bioreactor: 380 ml.
However carrier SM-C showed the highest hydrogen production rate (62 mmol/liter h) among all the carriers used (Fig. 5). This might be due to the presence of high concentration of cells adsorbed on SM-C [19]. It is evident from all the three figures that the rate of hydrogen production increased with the increase in dilution rate up to 0.9 h⫺1. However, no significant change in pH was observed in all the three cases (data not shown) [19]. 3.3. Effect of bioreactor configuration Hydrogen production in tubular bioreactor showed that the working volume was decreased with increase in dilution rate (Fig. 6). This could be attributed to the fact that flow of substrate added convective transport contribution to the movement of substrate from the bulk solution to the external surface of the packed matrix where cells were immobilized. It was reported that the mass transfer coefficient increased with the increase of dilution rate in a packed bed bioreactor with immobilized cells [15]. So, mass transfer was more at higher dilution rate, which resulted in production of large volumes of gas and hence higher gas hold-up. The higher gas hold-up resulted in decrease of working volume. To
overcome the gas hold-up problem, different bioreactor configurations were considered. SM-C was chosen as support matrix as it gave the best result in terms of cell density and hydrogen production rate at dilution rate of 0.93 h⫺1. The comparative studies on different bioreactors configuration showed that the rhomboid bioreactor with convergent divergent configuration had maximum hydrogen production rate (Table 2). The enhancement in hydrogen production rate could be attributed to two factors. First due to higher turbulence created by convergent divergent geometry of the reactor (velocity varied from 4 cm h⫺1 to 37 cm h⫺1 in the convergent section and those in the divergent section from 1.3 cm h⫺1 to 12 cm h⫺1) which was responsible for continuous renewal of the surface and thus the substrate had a better interaction with the cells in the bioreactor. Secondly, there was gradual reduction in gas hold-up from tubular to rhomboid bioreactor (Fig. 6). The gas hold up was reduced by 67% by using the rhomboid bioreactor. 3.4. Effect of recycling on hydrogen production The substrate conversion efficiency at maximum hydrogen production rate was found to be low in the case of all the three bioreactors with SM-C as support matrix. Rhomboid bioreactor was chosen for the further studies to increase the substrate conversion efficiency as it gave highest hydrogen production rate with least gas hold up. The effect of recycle ratio on substrate conversion efficiency and hydrogen production rate is shown in Fig. 7. Hydrogen production increased with the recycle ratio. This was mostly due to the reduction of the mass transfer resistance. The maximum hydrogen production rate was 75.6 mmol/liter h at a dilution rate of 0.93 h⫺1 and recirculation ratio of 6.4.
Fig. 7. Effect of recycle ratio on hydrogen production and substrate utilization efficiency in rhomboid bioreactor (bioreactor volume–380 ml; support matrix–SM-C; temperature–37°C; media used–MYG).
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
285
Table 3 Effect of glucose as substrate concentration on hydrogen production Sl. #
Substrate concentration (in feed) (%)
Substrate concentration (in effluent) (%)
Dilution rate (h⫺1)
Cell concentration (g/l)
Conversion efficiency (%)
Rate of hydrogen production (mmol/l h)
1 2 3 4 5 6 7
0.5 1.0 1.5 2.0 2.5 3.0 3.5
0.18 0.25 0.92 1.11 1.50 1.7 2.6
0.93 0.93 0.93 0.93 0.93 0.93 0.93
0.182 0.265 0.279 0.299 0.357 0.413 0.471
64.0 75.0 38.7 44.4 40.0 43.3 25.7
66.7 77.3 72.9 66.8 62.2 62.6 60.8
Bioreactor configuration–rhomboid; total volume of the bioreactor–380 ml, working volume of the bioreactor–250 ml; packing material– coir; density of packing material loaded–76 g/l; dilution rate– 0.9 h⫺1; recirculation ratio– 6.4; temperature–37°C; media used–MY (supplemented with different amount of glucose).
The substrate conversion efficiency was increased by 15% at these conditions as compared to that of without recycling. 3.5. Effect of substrate concentration on hydrogen production The effect of glucose concentration on hydrogen production and growth of E. cloacae IIT-BT 08 is shown in Table 3. The rate of hydrogen production increased with the glucose concentration up to 1% thereafter there was decrease in production rate at 1.5% glucose concentration. There was strong substrate inhibition effect at a concentration of 2% or above and so the rate of hydrogen production was found to decrease. These results were found similar with the batch system where substrate inhibition effect was found at 1% glucose concentration [7]. 3.6. Batch vs continuous system The growth kinetics parameters such as max and ks values for the immobilized cells were calculated by plotting
1/D Vs 1/S. The max and ks for immobilized cells were 1.25h⫺1 and 9.31 g/liter respectively. The max was found little more and there was slight increased in ks value when these values compared with free cells (max ⫽ 1.12 h⫺1 and ks ⫽ 8.89 g/liter) [9]. The immobilized cells system not only maintained a high cell density throughout the process but also it didn’t affect the kinetic parameters to a great extent. The maximum rate of hydrogen production was 2.1 times higher than batch system. 3.7. Generation time of the immobilized cells in bioreactor The generation time for immobilized Enterobacter cloacae IIT-BT 08 was calculated for SM-C as support matrix. The profile of concentration of cells in the effluent is shown in Fig. 8. The growth of cells at different generation time is shown by emergence of corresponding peaks. Daughter cells were reproduced by binary fission [16] of the immobilized cells and thrown out into the outgoing liquid that
Fig. 8. Determination of generation time using immobilized cells (bioreactor configuration–rhomboid; bioreactor volume–380 ml; support matrix–SM-C; temperature ⫺37°C; media used–MYG)
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Table 4 Comparative studies on continuous H2 production in a packed-bed reactor using immobilized whole cells Microorganisms
Support matrix
H2 production rate (mmol/l h)
Reference
Rhodobacter spheroid Rhodospirillum rubrum E. aerogenes NCIMB 10102 E. aerogenes HO-39 E. aerogenes HY-2 E. cloacae IIT-BT 08
Agar gel Ca-alginate Synthetic sponge Porous glass beads Self flocculated cells Lignocellulosic agroresidues
2.1 2.7 10.2 37.9 58.0 75.6
[20] [15] [21] [13] [14] Present work
were rapidly carried out of the reactor due to high dilution rate. This cyclic process led to two conclusions viz. either there was no free sites for attachment on the support (as it had reached its saturation level) or the peaks might have appeared due to interfacial stability. Thus the generation time calculated as the time span between two successive peaks as 1.1 h that was higher than free cells (0.32 h) [9]. 3.8. Comparative studies on continuous hydrogen production processes Several immobilized systems have been reported in the literature on continuous hydrogen production using packed bed reactor [13–15,20,21]. Porous glass-beads and selfflocculated cells have been reported to give better performance as compared to agar gel, Ca-alginate and synthetic sponge. However, the present process showed better rate of hydrogen production (75.6 mmol H2/liter h) as compared to all these systems (Table 4).
4. Conclusion Immobilized Enterobacter cloacae IIT-BT 08 were successfully used for continuous hydrogen production using indigenously available cheap lignocellulosic agroresidue as support matrix. Among the three carriers used, SM-C was found to be the best in terms of cell retention, packing density, concentration of cells and hydrogen production rate. The rhomboid shaped bioreactor with convergent divergent configuration had maximum hydrogen production rate. The gas hold up using this bioreactor was reduced by 67%. The maximum hydrogen production rate achieved was 75.6 mmol/liter h at a dilution rate of 0.93 h⫺1 and recirculation ratio of 6.4. The substrate conversion efficiency was increased by 15% at these conditions. The substrate inhibition both in case of free and immobilized whole cells was found predominant at more than 1.0% glucose concentration. The max and ks for immobilized cells were 1.25 h⫺1 and 9.31 g/liter respectively. The maximum rate of hydrogen production was found to be 2.1 times higher than batch system. The generation time of immobilized cells was 1.1 h, which was higher than free cells. The rate of hydrogen
production achieved in the present system was higher than that of other reported work using different solid matrices and microorganisms.
Acknowledgment This study was supported by the Department of Biotechnology, Government of India.
References [1] Philips EJ, Mitsui A. Role of light intensity and temperature in the regulation of hydrogen photoproduction by the marine cyanobacterium Oscillatoria sp. Miami BG7. Appl Environ Microbiol 1983;45: 1212–20. [2] Das D, Veziroglu NT. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001;26:13–28. [3] Taguchi F, Yamada K, Hasegawa K, Taki-Saito T, Hara K. Continuous hydrogen production by Clostridium sp. strain no.2 from cellulose hydrolysate in an aqueous two phase system. J Ferment Bioeng 1996;82:80 –3. [4] Tanisho S. Feasibility study of biological hydrogen production from sugar cane by fermentation. Proceedings of the 11th world hydrogen energy conference, Stuttgart, Germany. Hydrogen Energy Progress XI 1996;3:2601–2606. [5] Yokoi H, Ohkawara T, Hirose J, Hayashi S, Takasaki Y. Characteristics of H2 production by aciduric Enterobacter aerogenes strain H039. J Ferment Bioeng 1995;80:571– 4. [6] Brosseau JD, Zajic JE. Hydrogen gas production with Citrobacter intermedius and Clostridium pasteurianum. J Chem Tech Biotechnol 1982;32:496 –500. [7] Kumar N, Das D. Production of pollution free gaseous fuel.2000, Patent application # 473/2000/Cal. India. [8] Kumar N, Das D. Studies on molecular hydrogen production by Enterobacter cloacae IIT-BT 08. Paper presented at 9th European Congress on Biotechnology 11–15 July, Brussels, Belgium, 1999. [9] Kumar N, Das D. Enhancement of Hydrogen production by Enterobacter cloacae IIT-BT 08. Process Biochem 2000; 35:589 –593(Erratum,35;9:1074). [10] Kumar N, Das D. Production and purification of ␣-amylase from Hydrogen producing Enterobacter cloacae IIT-BT 08. Bioprocess Engg 2000;23:205– 8. [11] Kumar N, Monga PS, Biswas AK, Das D. Modeling and simulation of cleaner fuel production. Int J Hyd Energy 2000; 25: 945–52. [12] Kumar N, Ghosh A, Das D. Redirection of biochemical pathways for the enhancement of H2 production by Enterobacter cloacae. Biotechnol Letts 2001;23:537– 41.
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287 [13] Yokoi H, Tokushige T, Hirose J, Hayashi S, Takasaki Y. Hydrogen production by immobilised cells of Enterobacter aerogenes strain HO39. J Ferment Bioeng 1997;83:481– 4. [14] Rachman MA, Nkashimada Y, Kakizono T, Nishio N. Hydrogen production with high yield, and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor. Appl Microbiol Biotechnol 1998;49:450 – 4. [15] Seon YH, Lee CG, Park DH, Hwang KY, Joe YI. Hydrogen production by immobilized cells in nozzle loop bioreactor. Biotechnol Letts 1983;15:1275– 80. [16] Ghosh TK, Bandyopadyay K. Rapid ethanol fermentation in yeast cell reactor. Biotechnol Bioeng 1980;12:1489 –96.
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[17] Navarro JM, Durand G. Modification of the growth of Saccharomyces ouvarum by immobilization on a solid carrier. C R Acad Sci, Paris 1980;290:453– 6. [18] Bringi V, Dali BE. Enhanced yeast immobilization by nutrient starvation. Biotechnol Letts 1985;7:905– 8. [19] Kumar N. Hydrogen production by Enterobacter cloacae IIT-BT 08. Ph D Thesis, IIT, Kharagpur, India, 2001. [20] 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. [21] Palazzi E, Fabino B, Perego P. Process development of continuous hydrogen production by Enterobacter aerogenes in a packed column reactor. Bioprocess Engg. 2000;22:205–13.
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Continuous hydrogen production by immobilized Enterobacter cloacae IIT-BT 08 using lignocellulosic materials as solid matrices Narendra Kumar, Debabrata Das* Department of Biotechnology, Indian Institute of Technology, Kharagpur, India Received 31 October 2000; received in revised form 23 May 2001; accepted 1 June 2001
Abstract A process was developed for continuous hydrogen production using immobilized Enterobacter cloacae IIT-BT 08 on environmentally friendly lignocellulosic solid matrices. Among the three lignocellulosic carriers used, SM-C (coir) was found to be the best in terms of cell retention (0.44 g dry cell/g dry carrier), packing density (100 g/liter reactor volume), cell loading (44 g dry cell/liter reactor volume) and hydrogen production rate (62 mmol/liter h). Gas hold-up was a major problem in tubular bioreactor with immobilized cells. The tapered and rhomboid bioreactors gave better performance in terms of both the rate of hydrogen production and the gas hold up. The gas hold-up was reduced by 67% using the rhomboid bioreactor as compared to tubular one. The maximum hydrogen production rate achieved was 75.6 mmol/liter h at a dilution rate of 0.93 h⫺1 and recirculation ratio of 6.4. The substrate conversion efficiency was increased by 15% at these conditions as compared to the system with no recycling. There was no substrate inhibition for hydrogen production up to 1.0% glucose concentration. The max and ks for immobilized cells were 1.25 h⫺1 and 9.31 g/liter respectively. The maximum rate of hydrogen production was found to be 2.1 times higher than that of batch system. The process was found more efficient as compared to other continuous hydrogen production processes using packed bed reactor. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Hydrogen; Enterobacter cloacae; Bioreactor configurations; Lignocellulosic materials as solid matrices; Immobilized cells
1. Introduction Hydrogen production is getting importance as an alternative pollution free gaseous fuel for the future. Among different routes of hydrogen production biohydrogen production processes were investigated with both photosynthetic [1,2] and fermentative microorganisms [3– 6]. The rate of fermentative hydrogen production was found faster as compared to photosynthetic processes. These processes still required further improvement for commercial exploitation. The hydrogen production rate using fermentative microorganisms can be enhanced by developing some suitable microbial strain and also by improvement of cell density by whole cell immobilization. One microbial strain with high rate of hydrogen production was isolated and characterized as Enterobacter cloacae IIT-BT 08 [7–12]. Hydrogen production using immobilized whole cells was reported by several researchers [13–15]. The major problems with immobilized hole cells system were low substrate conversion efficiency
and low rate of hydrogen production mainly due to mass transfer resistance in a fixed bed bioreactor. The solid matrices used for the immobilization of the whole cells were mostly either synthetic polymers or inorganic materials. These materials possessed disposal problems. So, environmentally friendly natural polymers such as lignocellulosic materials were chosen as solid matrices in the present studies. The paper deals with the effect of several lignocellulosic materials for hydrogen production. Gas hold-up was found to be a major problem in hydrogen generating system in a packed bed reactor. The effect of different bioreactor configuration was taken into consideration to overcome this problem. Finally attempts were made to increase substrate conversion efficiency and to determine the cell growth kinetic parameters using immobilized Enterobacter cloacae IIT-BT 08.
2. Materials and methods 2.1. Microorganism and culture condition
* Corresponding author. Tel.: ⫹91-3222-82248/78053; fax: ⫹913222-78707. E-mail address: [email protected] (D. Das).
Enterobacter cloacae IIT-BT 08, a locally isolated bacterium was used in all the experiments. The maintenance of
0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 1 ) 0 0 3 9 4 - 5
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
Fig. 1. Schematic diagram of the continuous hydrogen production in packed-bed reactor.
culture, preparation of seed culture and production media were same as reported earlier [10]. The support matrices used for packing the bioreactor were lignocellulosic materials such as rice straw (SM-A), bagasse (SM-B) and coir (SM-C). The pretreatment of the entire solid matrix was done as reported elsewhere [16].
281
Fig. 2. Sketch of different bioreactors configuration.
was followed by passing the production medium at different dilution rates. 2.4. Analysis Sugar, biomass concentration and hydrogen content in the evolved gas were determined by the methods as reported earlier [9].
2.2. Experimental set-up
2.5. Gas hold-up calculation
The schematic diagram of the experimental set-up for the operation of continuous hydrogen production using immobilized whole cell is shown in Fig. 1. A quasi-steady state (5% variation) was confirmed at each dilution rate with respect to constant values of H2 evolution rate, glucose and cell mass concentration in the effluent. The experiments were repeated at different flow rates to get maximum hydrogen production and sugar utilization. Anaerobicity was maintained in all the experiment. All the bioreactors used in the present study were operated continuously for a minimum period of 40 days to find out the operational stability of the system.
The gas hold-up in packed bed reactor was studied by introducing a pulse tracer input to the reactor inlet and its exit concentration profile was measured. The gas hold up was calculated as follows: T ⫽ Vr/f Vr ⫽ Vf ⫹ Vg 1 ⫽ Vf/Vr ⫹ Vg/Vr ⫽ t/T ⫹ Vg/Vr Gas hold-up ⫽ Vg/Vr ⫽ 1 ⫺ t/T where T ⫽ hydraulic retention time, t ⫽ mean residence time calculated by tracer input, F ⫽ flow rate Vr ⫽ reactor volume, Vf ⫽ working volume, Vg ⫽ gas volume inside reactor. 2.6. Generation time of immobilized cells in bioreactor
2.3. Bioreactor system Three bioreactors (Fig. 2) were designed and fabricated. The bioreactors were packed with pretreated solid matrices. To immobilize the whole cells, 35 g/liter of cells suspension in 0.1 M phosphate buffer pH 6 was passed through the packed column at a dilution rate of 0.2 h⫺1 with the help of a peristaltic pump for 8 h. Thereafter, the reactor was kept at rest for 12 h. The reactors were washed with 0.1 M phosphate buffer pH 6.0 at a dilution rate of 0.2 h⫺1. The immobilized cells were immersed in production media (for about 8 –10 h) till the gas production rate reached its maximum and became steady. This
The generation time for immobilized cells was determined according to the method described by Navarro and Table 1 Analysis on loading of E. cloacae on different natural biopolymers Sl. #
Support matrix
Density of packing material (g solid matrix/l or reactor)
Cell loading (g dry cell/g support)
Cell loading (g dry cell/l of reactor)
1 2 3
SM-A SM-B SM-C
54.6 64.7 100
0.32 0.39 0.44
31.73 37.5 44
282
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
Fig. 3. Hydrogen production by immobilized whole cells in a packed bed reactor with SM-A as support matrix (bioreactor configuration–tubular; bioreactor volume–380 ml; temperature–37°C; media used–MYG).
Durand [17]. The bioreactor packed with immobilized cells was continuously fed at the bottom. The flow rate was adjusted such that the residence time of the dislodged cells in the reactor did not exceed the doubling time of the cells. The generation time for freely suspended cells was reported earlier [9]. Multiplication of the free cells in the bioreactor (if any) was prevented because of the high flow rate employed. The optical density of cell suspension leaving the system (effluent) was measured. The peaks indicate the generation time of the cells, which is the difference between any two peaks.
3. Results and discussions 3.1. Retention of E. cloacae IIT-BT 08 on different natural biopolymers Starvation of nutrient enhances the adsorption of microbial cells on solid support matrix [18]. So, in the present studies, adsorption was done in nutrient starved condition using the cells suspension in phosphate buffer. The cell loading in different solid matrices are shown in Table 1. The cell loading both in terms of g cells per g of packing
Fig. 4. Hydrogen production by immobilized whole cells in a packed bed reactor with SM-B as support matrix (bioreactor configuration–tubular; bioreactor volume–380 ml; temperature–37°C; media used–MYG).
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
283
Fig. 5. Hydrogen production by immobilized whole cells in a packed bed reactor with SM-C as support matrix (bioreactor configuration–tubular; bioreactor volume–380 ml; temperature–37°C; media used–MYG).
material and g of cells per volume of reactor was maximum in the case of carrier SM-C and was minimum in the case of carrier SM-A. The packing density was also highest in SM-C. This might be attributed to more surface area due to fibrous and corrugated nature of the same [19]. 3.2. Effect of different carriers on hydrogen production The effect of dilution rate on hydrogen production, substrate conversion efficiency and biomass concentration in the effluent was studied in a packed bed reactor using
different lignocellulosic support matrices. At each dilution rate, the bioreactor was run at least for two days until and unless steady conditions were achieved. The maximum rate of hydrogen production in the case of SM-A was 44 mmol/ liter h (Fig. 3) at a dilution rate of 0.93 h⫺1 with 53% glucose conversion efficiency. The cell concentration in the effluent was almost constant at or above the dilution rate of 0.37 h⫺1. The carrier SM-B showed a better rate of hydrogen production (52 mmol/liter h) and glucose conversion efficiency (61%) than that of SM-A (Fig. 4). This might be due to higher cell density in the case of carrier SM-B.
Fig. 6. Effect of dilution rate on gas hold up using different bioreactor (bioreactor volume–380 ml; support matrix–SM-C; temperature–7°C; media usedMYG).
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Table 2 Performance of different bioreactor using SM-C as solid matrix Type of bioreactor
Dilution rate (h⫺1)
Rate of H2 production (mmol/l h)
Substrate conversion efficiency (%)
Enhancement of H2 production (%)
Tubular Tapered Rhomboid
0.93 0.93 0.93
62.5 65.2 71.0
61 65 69
— 4.3 13.6
Volume of each bioreactor: 380 ml.
However carrier SM-C showed the highest hydrogen production rate (62 mmol/liter h) among all the carriers used (Fig. 5). This might be due to the presence of high concentration of cells adsorbed on SM-C [19]. It is evident from all the three figures that the rate of hydrogen production increased with the increase in dilution rate up to 0.9 h⫺1. However, no significant change in pH was observed in all the three cases (data not shown) [19]. 3.3. Effect of bioreactor configuration Hydrogen production in tubular bioreactor showed that the working volume was decreased with increase in dilution rate (Fig. 6). This could be attributed to the fact that flow of substrate added convective transport contribution to the movement of substrate from the bulk solution to the external surface of the packed matrix where cells were immobilized. It was reported that the mass transfer coefficient increased with the increase of dilution rate in a packed bed bioreactor with immobilized cells [15]. So, mass transfer was more at higher dilution rate, which resulted in production of large volumes of gas and hence higher gas hold-up. The higher gas hold-up resulted in decrease of working volume. To
overcome the gas hold-up problem, different bioreactor configurations were considered. SM-C was chosen as support matrix as it gave the best result in terms of cell density and hydrogen production rate at dilution rate of 0.93 h⫺1. The comparative studies on different bioreactors configuration showed that the rhomboid bioreactor with convergent divergent configuration had maximum hydrogen production rate (Table 2). The enhancement in hydrogen production rate could be attributed to two factors. First due to higher turbulence created by convergent divergent geometry of the reactor (velocity varied from 4 cm h⫺1 to 37 cm h⫺1 in the convergent section and those in the divergent section from 1.3 cm h⫺1 to 12 cm h⫺1) which was responsible for continuous renewal of the surface and thus the substrate had a better interaction with the cells in the bioreactor. Secondly, there was gradual reduction in gas hold-up from tubular to rhomboid bioreactor (Fig. 6). The gas hold up was reduced by 67% by using the rhomboid bioreactor. 3.4. Effect of recycling on hydrogen production The substrate conversion efficiency at maximum hydrogen production rate was found to be low in the case of all the three bioreactors with SM-C as support matrix. Rhomboid bioreactor was chosen for the further studies to increase the substrate conversion efficiency as it gave highest hydrogen production rate with least gas hold up. The effect of recycle ratio on substrate conversion efficiency and hydrogen production rate is shown in Fig. 7. Hydrogen production increased with the recycle ratio. This was mostly due to the reduction of the mass transfer resistance. The maximum hydrogen production rate was 75.6 mmol/liter h at a dilution rate of 0.93 h⫺1 and recirculation ratio of 6.4.
Fig. 7. Effect of recycle ratio on hydrogen production and substrate utilization efficiency in rhomboid bioreactor (bioreactor volume–380 ml; support matrix–SM-C; temperature–37°C; media used–MYG).
N. Kumar, D. Das / Enzyme and Microbial Technology 29 (2001) 280 –287
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Table 3 Effect of glucose as substrate concentration on hydrogen production Sl. #
Substrate concentration (in feed) (%)
Substrate concentration (in effluent) (%)
Dilution rate (h⫺1)
Cell concentration (g/l)
Conversion efficiency (%)
Rate of hydrogen production (mmol/l h)
1 2 3 4 5 6 7
0.5 1.0 1.5 2.0 2.5 3.0 3.5
0.18 0.25 0.92 1.11 1.50 1.7 2.6
0.93 0.93 0.93 0.93 0.93 0.93 0.93
0.182 0.265 0.279 0.299 0.357 0.413 0.471
64.0 75.0 38.7 44.4 40.0 43.3 25.7
66.7 77.3 72.9 66.8 62.2 62.6 60.8
Bioreactor configuration–rhomboid; total volume of the bioreactor–380 ml, working volume of the bioreactor–250 ml; packing material– coir; density of packing material loaded–76 g/l; dilution rate– 0.9 h⫺1; recirculation ratio– 6.4; temperature–37°C; media used–MY (supplemented with different amount of glucose).
The substrate conversion efficiency was increased by 15% at these conditions as compared to that of without recycling. 3.5. Effect of substrate concentration on hydrogen production The effect of glucose concentration on hydrogen production and growth of E. cloacae IIT-BT 08 is shown in Table 3. The rate of hydrogen production increased with the glucose concentration up to 1% thereafter there was decrease in production rate at 1.5% glucose concentration. There was strong substrate inhibition effect at a concentration of 2% or above and so the rate of hydrogen production was found to decrease. These results were found similar with the batch system where substrate inhibition effect was found at 1% glucose concentration [7]. 3.6. Batch vs continuous system The growth kinetics parameters such as max and ks values for the immobilized cells were calculated by plotting
1/D Vs 1/S. The max and ks for immobilized cells were 1.25h⫺1 and 9.31 g/liter respectively. The max was found little more and there was slight increased in ks value when these values compared with free cells (max ⫽ 1.12 h⫺1 and ks ⫽ 8.89 g/liter) [9]. The immobilized cells system not only maintained a high cell density throughout the process but also it didn’t affect the kinetic parameters to a great extent. The maximum rate of hydrogen production was 2.1 times higher than batch system. 3.7. Generation time of the immobilized cells in bioreactor The generation time for immobilized Enterobacter cloacae IIT-BT 08 was calculated for SM-C as support matrix. The profile of concentration of cells in the effluent is shown in Fig. 8. The growth of cells at different generation time is shown by emergence of corresponding peaks. Daughter cells were reproduced by binary fission [16] of the immobilized cells and thrown out into the outgoing liquid that
Fig. 8. Determination of generation time using immobilized cells (bioreactor configuration–rhomboid; bioreactor volume–380 ml; support matrix–SM-C; temperature ⫺37°C; media used–MYG)
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Table 4 Comparative studies on continuous H2 production in a packed-bed reactor using immobilized whole cells Microorganisms
Support matrix
H2 production rate (mmol/l h)
Reference
Rhodobacter spheroid Rhodospirillum rubrum E. aerogenes NCIMB 10102 E. aerogenes HO-39 E. aerogenes HY-2 E. cloacae IIT-BT 08
Agar gel Ca-alginate Synthetic sponge Porous glass beads Self flocculated cells Lignocellulosic agroresidues
2.1 2.7 10.2 37.9 58.0 75.6
[20] [15] [21] [13] [14] Present work
were rapidly carried out of the reactor due to high dilution rate. This cyclic process led to two conclusions viz. either there was no free sites for attachment on the support (as it had reached its saturation level) or the peaks might have appeared due to interfacial stability. Thus the generation time calculated as the time span between two successive peaks as 1.1 h that was higher than free cells (0.32 h) [9]. 3.8. Comparative studies on continuous hydrogen production processes Several immobilized systems have been reported in the literature on continuous hydrogen production using packed bed reactor [13–15,20,21]. Porous glass-beads and selfflocculated cells have been reported to give better performance as compared to agar gel, Ca-alginate and synthetic sponge. However, the present process showed better rate of hydrogen production (75.6 mmol H2/liter h) as compared to all these systems (Table 4).
4. Conclusion Immobilized Enterobacter cloacae IIT-BT 08 were successfully used for continuous hydrogen production using indigenously available cheap lignocellulosic agroresidue as support matrix. Among the three carriers used, SM-C was found to be the best in terms of cell retention, packing density, concentration of cells and hydrogen production rate. The rhomboid shaped bioreactor with convergent divergent configuration had maximum hydrogen production rate. The gas hold up using this bioreactor was reduced by 67%. The maximum hydrogen production rate achieved was 75.6 mmol/liter h at a dilution rate of 0.93 h⫺1 and recirculation ratio of 6.4. The substrate conversion efficiency was increased by 15% at these conditions. The substrate inhibition both in case of free and immobilized whole cells was found predominant at more than 1.0% glucose concentration. The max and ks for immobilized cells were 1.25 h⫺1 and 9.31 g/liter respectively. The maximum rate of hydrogen production was found to be 2.1 times higher than batch system. The generation time of immobilized cells was 1.1 h, which was higher than free cells. The rate of hydrogen
production achieved in the present system was higher than that of other reported work using different solid matrices and microorganisms.
Acknowledgment This study was supported by the Department of Biotechnology, Government of India.
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