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Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus
international journal of hydrogen energy 34 (2009) 180–185
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus Jamila Obeida, Jean-Pierre Magnina,*, Jean-Marie Flausb, Olivier Adrotb, John C. Willisonc, Roumen Zlatevd a
Grenoble Institute of Technology, LEPMI, UMR 5631 (CNRS-INPG-UJF), BP 75, 38402 St Martin d’He`res, France Grenoble Institute of Technology, Laboratoire des sciences pour la conception, l’optimisation et la production, 46, avenue Fe´lix Viallet, 38031 Grenoble, France c Laboratoire de Chimie et Biologie des Me´taux (UMR 5249 CEA-CNRS-UJF), iRTSV/LCBM, CEA-Grenoble, 38054 Grenoble, France d Autonomous University of Baja California, Institute of Engineering, Mexicali, Baja California, Mexico b
article info
abstract
Article history:
The photosynthetic bacterium, Rhodobacter capsulatus, produces hydrogen under nitrogen-
Received 29 July 2008
limited, anaerobic, photosynthetic culture conditions, using various carbon substrates. In
Received in revised form
the present study, the relationship between light intensity and hydrogen production has
12 September 2008
been modelled in order to predict both the rate of hydrogen production and the amount of
Accepted 12 September 2008
hydrogen produced at a given time during batch cultures of R. capsulatus. The experimental
Available online 28 November 2008
data were obtained by investigating the effect of different light intensities (6000–50,000 lux) on hydrogen-producing cultures of R. capsulatus grown in a batch photobioreactor, using
Keywords:
lactate as carbon and hydrogen source. The rate of hydrogen production increased with
Hydrogen production
increasing light intensity in a manner that was described by a static Baly model, modified
Photobioreactor
to include the square of the light intensity. In agreement with previous studies, the kinetics
Rhodobacter capsulatus
of substrate utilization and growth of R. capsulatus was represented by the classical Monod
Modelling
or Michaelis–Menten model. When combined with a dynamic Leudekong–Piret model, the amount of hydrogen produced as a function of time was effectively predicted. These results will be useful for the automatization and control of bioprocesses for the photoproduction of hydrogen. ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Economic development over the last few decades has been strongly dependent on fossil fuels as sources of energy. These resources are not unlimited in the long term, and environmental concerns have led to the search for clean, renewable energy sources. Hydrogen is a clean source of energy, considered as a potential and more sustainable energy substitute for fossil fuels. It has been predicted that the
contribution of hydrogen to global energy consumption will increase dramatically, to approximately 50% by the end of the 21st century, due to the development of efficient end-use technologies, possibly even becoming the major final energy carrier [1–3]. Hydrogen gas can be produced either by electrolysis, coal gasification, steam methane reforming of natural gas, partial oxidation of fuel oil, solar thermal cracking, biomass gasification, or photobiological synthesis [4–8]. Biological hydrogen
* Corresponding author. Fax: þ33 4 76 82 67 77. E-mail address: [email protected] (J.-P. Magnin). 0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2008.09.081
international journal of hydrogen energy 34 (2009) 180–185
Nomenclature X S VH2 P(I ) Vm, Km m(S ) mmax YxP YxS Te 4(I ) b h M0
bacterial concentration [g L1] substrate concentration [g L1] volume of hydrogen produced [L] hydrogen production rate [h1] two parameters to be identified [h1, lux1] specific bacterial growth rate [h1] maximum substrate degradation rate [h1] yield biomass/product [g L1] substrate utilization yield [g L1] time interval [h] yield of light intensity [lux] non-growth-associated product formation coefficient hydrogen production yield initial lactate concentration [M]
production is carried out by a large number of hydrogenproducing microorganisms, including obligate and facultative anaerobes, aerobes, cyanobacteria, photosynthetic bacteria and algae [9]. Among them, photosynthetic bacteria, such as Rhodobacter capsulatus, are favourable candidates for largescale production due to their high energy-conversion efficiencies and their ability to use a wide variety of substrates for growth and hydrogen production [10–14]. Nevertheless, the rate and yield of hydrogen production vary greatly depending on the carbon sources used and physiological growth conditions, such as light intensity. The conversion efficiency of light energy to hydrogen is a key factor in the development of a biological process devoted to hydrogen production [15–17]. The relationship between light intensity and the metabolic products of photosynthetic microorganisms like microalgae has been described by different mathematical equations [18,19]. With regard to photosynthetic bacteria, Nakada et al. [20] measured light penetration into a four-compartment photobioreactor and its relationship to hydrogen production by Rhodobacter sphaeroides [21,22]. They found that the rate of hydrogen production and light penetration both decreased upon passage through the reactor compartments. The effect of light intensity on nitrogenase synthesis and hydrogen evolution at a constant cell density, in a continuous culture of R. capsulatus, was studied by Jouanneau et al. [15]. Under these conditions, the light absorption by the bacterial culture was constant, so the actual light intensity reaching the bacteria was dependent only on the incident light. These results confirmed those of previous studies on batch cultures [6,22], which showed that increasing the light intensity greatly stimulated the hydrogen production capacity of R. capsulatus. The objective of the present study was to develop a model for hydrogen production by the photosynthetic bacterium, R. capsulatus, in a batch photobioreactor, with particular emphasis on the effect of light intensity. The development of such a model is important for the automatization and control of bioprocesses for the photoproduction of hydrogen from industrial waste.
2.
Materials and methods
2.1.
Bacteria and culture medium
181
Precultures of R. capsulatus strain IR3 [23] were grown anaerobically at pH 6.8 and 30 C in RCV medium (lactate 30 mmol L1, DL-malate 7.5 mmol L1, (NH4)2SO4 7.5 mmol L1). Hydrogenproducing cultures were grown at constant temperature (30 C) in a nitrogen-limiting medium containing Na-lactate (between 30 and 80 mmol L1, depending on the experiment) and Na-glutamate (7 mmol L1) as nitrogen source.
2.2.
Photobioreactor
Two types of photobioreactor were used for hydrogen production experiments with R. capsulatus. The first type consisted of a flat glass bottle, 0.125 L in volume, containing a magnetic stirrer for mixing. The opening was hermetically sealed with a rubber stopper, and the biologically produced hydrogen was evacuated via a needle inserted in the stopper, towards an hydrogen measurement system consisting of an inverted, graduated test-tube filled with KCl (0.1 M). The hydrogen gas produced increased the pressure inside the tube and so proportionally decreased the liquid level. The liquid displacement was directly correlated with the volume of hydrogen produced. The second type was a laboratory-made, rectangular-shaped bioreactor, 1 L in volume, comprising two external plexiglass plates (22.5 12.5 0.4 cm) and a smaller, internal plexiglass plate (19 12.5 0.4 cm). The latter divided the reactor into two compartments: one of 3 cm thickness devoted to bacterial growth, and a second (3 cm thickness) for degassing and circulation of the medium. The reactor was hermetically sealed with a gas-tight plexiglass lid (thickness 2 cm), containing three outlets devoted to the pH electrode, the temperature probe and evacuation of the evolved hydrogen. The culture medium was circulated between the
Fig. 1 – Overview of the experimental set-up for H2 production by R. capsulatus IR3: 1-L plexiglas-reactor (A), lamp (B), ventilator (C), H2 flow-meter (D), H2-measurement system (E), pump (F), pH and temperature measurement apparatus (G), data acquisition card (H), computer (I).
182
international journal of hydrogen energy 34 (2009) 180–185
two compartments by means of a pump (4 W, 12 V, 4.5 L min1). The bioreactor was illuminated from one side by a sodium-vapour lamp (OSRAM, Plantastar, 600 W) (Fig. 1). The light intensity at the surface of the reactor was varied by changing the distance between the light source and the illuminated surface. The light intensity was measured by a digital lux (Meter, RO 1332). The conversion factor between the illumination (in lux, official SI Unit) and the photosynthetic active radiation (PAR) irradiance (in mE m2 s1, non-official SI unit) for the sodium-vapour lamp is 0.0138. Anaerobic conditions were obtained by purging the bioreactor with sterile argon. The hydrogen flow rate was measured by associating a mass flowmeter (0–20 ml H2 h1, MacMillan, model 50D) linked to a data acquisition card (PMD-1208LS, SA Measurement Computing) with a graduated-glass tube (150 4 cm) filled with liquid (KCl 0.1 M) as described above.
2.3.
Analytical methods
Bacterial cell density was estimated spectrophotometrically at 660 nm. The concentration of lactate in the growth medium was determined by HPLC (Waters 510), after previous removal of biomass by centrifugation (3000 rpm, 5 min) using an ICECoregel 87H3 column (Transgenics) with sulphuric acid (0.1 mol L1) as the mobile phase. Acquisition and treatment of data were carried out using Azur software (version 4.5, Datalys Company, France).
3.
Results and discussion
3.1. Effect of light intensity on biomass formation and hydrogen production rate
Fig. 3 – Influence of the light intensity on the specific bacterial growth rate of R. capsulatus IR3 during batch culture on 80 mM lactate (110-ml reactor).
50,000 lux, the final protein concentration increased from 3.1 to 4 g L1. This variation represents a 30% increase in protein concentration, which is correlated with biomass production. The specific-growth rate, determined by Monod’s model, was linearly dependent on light intensity (Fig. 3). A plot of the kinetics of hydrogen production as a function of light intensity revealed that the rate of hydrogen production was 6-fold higher at 50,000 lux than at 6000 lux (Fig. 4). The specific production rate of hydrogen was estimated and modelled according to Baly’s model (Fig. 5). The Baly model, which was originally applied to phytoplankton [24], is a statistically simple model, which correlates the specific production rate, P, to light intensity: P¼
Vm I Km þ I
(1)
The influence of the light intensity on biomass formation and the hydrogen production rate was first investigated using a flat-bottle glass reactor, over a range of light intensities from 6000 to 50,000 lux. The kinetics of biomass production in a photosynthetic hydrogen-producing culture is shown in Fig. 2. As the light intensity was increased from 6000 to
In the case of the photosynthetic bacterium R. capsulatus, a better fit to the data was obtained if the term I was replaced by I2:
Fig. 2 – Influence of light intensity on the bacterial concentration of R. capsulatus IR3 during batch culture on 80 mM lactate (110-ml reactor) (A): 6000 lux; (,) 9000 lux; (:) 11,000; (#) 23,000; (C) 30,000; and (D) 50,000 lux.
Fig. 4 – Influence of the light intensity on the H2 production of R. capsulatus IR3 during batch culture on 80 mM lactate (110-ml reactor) (A): 6000 lux; (,) 9000 lux; (:) 11,000; (#) 23,000; (C) 30,000; and (D) 50,000 lux.
international journal of hydrogen energy 34 (2009) 180–185
183
Therefore, the hydrogen production yield (h) is calculated as a percentage of the complete conversion of lactate in H2 and CO2 from the Eq. (5). h¼
VH2 100 6 22:4 M0
(4)
where VH2 is the volume of hydrogen production in litre and M0, the initial concentration of lactate. The correlation between the hydrogen conversion yield and the light intensity in the range 6000–50,000 lux is shown in Fig. 6. As the light intensity increased from 6000 to 25,000 lux, the hydrogen conversion yield increased accordingly. Above 25,000 lux, the hydrogen production yield stabilized for the strain IR3 with 80 mM lactate. Fig. 5 – Modelling, according to Baly’s model, of the influence of light intensity on H2 production during a batch culture of R. capsulatus IR3 (110-ml glass reactor, 80 mM lactate, 7 mM glutamate, 30 8C).
P¼
Vm I2 Km þ I2
The biomass concentration in the batch hydrogen production experiments depends on the concentration in limiting substrate. Theoretically, the growth rate and the substrate utilization are expressed as: (2)
This can be rationalised, since light is involved twice in hydrogen production by R. capsulatus, firstly in synthesis of the enzyme nitrogenase, and secondly in photosynthetic metabolism required to synthesise ATP for enzyme activity [15,17]. The model parameters of Km and Vm were estimated as 104.9 lux and 2.8 h1, respectively, for bacterial cultures grown under a light intensity range of 6000–50,000 lux. The bacterial hydrogen production yield reflects the capability of a bacterial strain to convert the carbon substrate into biogas. The biogas produced is a mixture of hydrogen and carbon dioxide. For lactate, which was the carbon substrate used in this study, 6 mol of hydrogen are expected to be produced per mole of lactate utilized according to Eq. (4) C3 H6 O3 þ 3H2 O / 6H2 þ 3CO2
3.2. Kinetics of the bacteria growth and substrate utilization
dX ¼ mðSÞX dt dS 1 ¼ mðSÞX dt YxS
)
(5)
where X is the bacteria concentration (g L1), m(S ) the specificgrowth rate (h1), S the substrate concentration (g L1), and YxS the substrate utilization yield. The R. capsulatus growth curves were well fitted by the Monod model, and by its modified versions, the Michaelis– Menten’s model, according to: mðSÞ ¼ mmax
S Ks þ S
(6)
where mmax is the maximum specific substrate degradation rate (h1), Ks the dissociation constant (g L1), and S the substrate concentration (g L1).
(3)
Fig. 6 – Influence of light intensity on H2 conversion rate by R. capsulatus IR3 during a batch culture (110-ml reactor, 80 mM lactate, 7 mM glutamate, 30 8C).
Fig. 7 – Kinetics of the lactate consumption (S, 3) and the biomass production (X, :) during a batch culture of R. capsulatus IR3. Data was fitted using the Monod model (1-L bioreactor, 30 mM lactate, 30,000 lux).
184
international journal of hydrogen energy 34 (2009) 180–185
By integration of Eq. (6) and substitution of the variable of Eq. (7), the following equations are obtained: Si Xi DT þ Xi Ks þ Si 1 Si ¼ mmax Xi DT þ Si Ki þ Si YxS
Xiþ1 ¼ mmax Siþ1
)
(7)
where Xiþ1 is the biomass concentration at time i þ 1 (g L1), Xi the biomass concentration at time i, Siþ1 the substrate concentration at time i þ 1, Si the substrate concentration at time i, DT the time interval (h). Fig. 7 shows the relationship between biomass concentration, substrate utilization and cultivation time, including both experimental data and the fitted curve based on the model. The model described correctly the experimental data. The corresponding model parameters of Ks, mmax, YxS, and DT, were estimated as 10 g L1, 0.3 h1, 0.1 g L1 and 0.5 h, respectively.
3.3.
Kinetics of hydrogen production
The production rate of hydrogen with respect to light intensity irrespective of cell concentration was estimated by Eq. (2). Luedeking–Piret proposed a relationship describing the specific production rate in terms of the cellular concentration [25] according to: dP 1 mðSÞX þ bX ¼ dt YxP
bioreactor and to the light intensity. Therefore, an additional factor was introduced in the Eq. (8) to take into consideration the light intensity effect, as shown in Eq. (9). dVH2 1 ¼ 4ðIÞmðSÞX þ bX dt YxP
Integration of Eq. (9) gives Equation (10): 1 4ðIÞmðSÞ þ b Xi Te þ VH2 i VH2 iþ1 ¼ YxP
(9)
(10)
where (H2)iþ1 is the volume of hydrogen produced at time i þ 1 (ml), (H2)i is the volume of hydrogen produced at time i (ml), X the biomass concentration (g L1), S the substrate concentration (g L1), m(S ) the bacterial specific-growth rate (h1), 4(I ) the yield of light energy (lux), YxP the hydrogen production yield, b the non-growth-associated formation coefficient of product and Te the interval time. The fitted and experimental curves representing the kinetics of hydrogen production during lactate consumption are shown in Fig. 8. A good agreement was obtained between the experimental and the modelled data. The model parameters of Ks, YxP, Te, b, 4(I ), and mmax were estimated as 10 g L1, 0.7 g L1, 0.5 h, 12, 3 lux and 0.3 h1. Thus, the relationship between biomass, product (hydrogen) and light intensity during hydrogen production process by R. capsulatus is well described by the modified Luedeking–Piret model.
(8)
where X is the biomass concentration (g L1), S the substrate concentration (g L1), and m(S ) the bacterial specific-growth rate (h1). The first term, (ð1=YxP ÞmðSÞX), is referred, together, to the microbial growth and the product formation rate. The second term bX is associated with bacterial growth. The dynamic model of hydrogen production reflects the fact that the specific hydrogen production rate (ml/h) is proportional both to the quantity of bacteria present in the
4.
Conclusions
The effect of light intensity on hydrogen production by the photosynthetic bacterium R. capsulatus was investigated and a mathematical model describing substrate utilization, biomass formation and H2 production was obtained, based on experimental results from hydrogen-producing batch cultures. This model allows command variables to be defined, thus enabling automatization of a bioprocess for hydrogen photoproduction with optimization of parameters such as the quantity and quality of the biogas produced.
Acknowledgments This work was supported by grants from the Rhoˆne-Alpes Region (France) and by a doctoral grant awarded to Jamila Obeid by the Syrian Government (The Ministry of Higher Education, Al Baath University, Faculty of Chemical and Petroleum Engineering).
references
Fig. 8 – Modelling, according to a modified Luedeking– Piret’s model, of the kinetics of a batch H2-production by R. capsulatus IR3 (30 mM lactate, 30,000 lux).
[1] Barreto L, Makihira A, Riahi K. The hydrogen economy in the 21st century: a sustainable development scenario. Int J Hydrogen Energy 2003;28:267–84. [2] Barbosa MJ, Rocha JMS, Tramper J, Wijffels RH. Acetate as a carbon source for hydrogen production by photosynthetic bacteria. J Biotechnol 2001;85:25–33. [3] Koku H, Erog˘lu I, Gu¨ndu¨z U, Yu¨cel M, Tu¨rker L. Kinetics of biological hydrogen production by the photosynthetic
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bacterium Rhodobacter sphaeroides O.U. 001. Int J Hydrogen Energy 2003;28:381–8. Cortright RD, Davda RR, Dumesic JA. Hydrogen from catalytic reforming of biomass-derived hydrocarbons in liquid water. Nature 2002;418:964–7. Elliott DC, Sealock LJ, Baker EG. Chemical processing in highpressure aqueous environments 2. Development of catalysts for gasification. Ind Eng Chem Res 1993;32:1542–8. Hillmer P, Gest H. H2 metabolism in photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures. J Bacteriol 1977;129:724–31. Miyake J, Mao XY, Kawamura S. Photoproduction of hydrogen from glucose by a co-culture of a photosynthetic bacterium and Clostridium butyricum. J Ferment Technol 1984; 62:531–5. Monod J. La technique de culture continue. The´orie et application. Ann Inst Pasteur 1950;79:390–410. Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of literature. Int J Hydrogen Energy 2001; 26:13–28. Dierstein R, Drews G. Nitrogen-limited continuous culture of R. capsulata growing photosynthetically or heterotrophically under low oxygen tensions. Arch Microbiol 1974;99:117–28. Eroglu I, Aslan K, Gu¨ndu¨z U, Yu¨cel M, Tu¨rker L. Substrate consumption rates for hydrogen production by Rhodobacter sphaeroides in a column photobioreactor. J Biotechnol 1999; 70:103–13. Sasikala K, Ramana CV, Rao PR. Environmental regulation for optimal biomass yield and photoproduction of hydrogen by Rhodobacter sphaeroides O.U. 001. Int J Hydrogen Energy 1991; 16:597–601. Sistrom WR. Observations on the relationship between the formation of photopigments and the synthesis of protein in Rhodopseudomonas sphaeroides. J Gen Microbiol 1962;28: 599–605. Tsygankov AA, Fedorov AS, Laurinavichene TV, Gogotov IN, Rao KK, Hall DO. Actual and potential rates of hydrogen photoproduction by continuous culture of the purple
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non-sulphur bacterium Rhodobacter capsulatus. Appl Microbiol Biotechnol 1998;49:102–7. Jouanneau Y, Wong B, Vignais PM. Stimulation by light of nitrogenase synthesis in cells of Rhodopseudomonas capsulata growing in N-limited continuous cultures. Biochem Biophys Acta 1985;808:149–55. Katsuda T, Arimoto T, Igarashi K, et al. Light intensity distribution in the externally illuminated cylindrical photobioreactor and its application to hydrogen production by Rhodobacter capsulatus. J Biochem Eng 2000;5:157–64. Meyer J, Kelley BC, Vignais PM. Effect of light on nitrogenase function and synthesis in Rhodopseudomonas capsulata. J Bacteriol 1978;136(1):201–8. Aiba S. Growth kinetics of photosynthetic microorganisms. Adv Biochem Eng 1982;23:86–156. Ragonese FP, Williams JA. A mathematical model for the batch reactor kinetics of algal growth. Biotechnol Bioeng 1968;10:83–8. Nakada E, Asada Y, Arai T, Miyake J. Light penetration into cell suspension of photosynthetic bacteria and relation to hydrogen production. J Ferment Bioeng 1995;80:53–7. Arik T, Gunduz U, Yucel M, Turker L, Sediroglu V, Eroglu I. Photoproduction of hydrogen by Rhodobacter sphaeroides O. U. 001. Proceedings of the Eleventh WHEC, Stuttgart, Germany: International Association for Hydrogen Energy 1996; vol. 3: p. 2407–2416. Hillmer P, Gest H. H2 metabolism in photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by resting cells. J Bacteriol 1977;129:732–9. Willison JC, Madern D, Vignais PM. Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata. J Biochem 1984;219:593–600. Jassby AD, Platt T. Mathematical formulation of the relationship between photosynthesis and light for phytoplankton. Limnol Oceanogr 1976;21:540–7. Luedeking R, Pired EL. Transient and steady states in continuous fermentation, theory and experiment. J Biochem Microbiol Technol Eng 1959;1:431–59.
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Modelling of hydrogen production in batch cultures of the photosynthetic bacterium Rhodobacter capsulatus Jamila Obeida, Jean-Pierre Magnina,*, Jean-Marie Flausb, Olivier Adrotb, John C. Willisonc, Roumen Zlatevd a
Grenoble Institute of Technology, LEPMI, UMR 5631 (CNRS-INPG-UJF), BP 75, 38402 St Martin d’He`res, France Grenoble Institute of Technology, Laboratoire des sciences pour la conception, l’optimisation et la production, 46, avenue Fe´lix Viallet, 38031 Grenoble, France c Laboratoire de Chimie et Biologie des Me´taux (UMR 5249 CEA-CNRS-UJF), iRTSV/LCBM, CEA-Grenoble, 38054 Grenoble, France d Autonomous University of Baja California, Institute of Engineering, Mexicali, Baja California, Mexico b
article info
abstract
Article history:
The photosynthetic bacterium, Rhodobacter capsulatus, produces hydrogen under nitrogen-
Received 29 July 2008
limited, anaerobic, photosynthetic culture conditions, using various carbon substrates. In
Received in revised form
the present study, the relationship between light intensity and hydrogen production has
12 September 2008
been modelled in order to predict both the rate of hydrogen production and the amount of
Accepted 12 September 2008
hydrogen produced at a given time during batch cultures of R. capsulatus. The experimental
Available online 28 November 2008
data were obtained by investigating the effect of different light intensities (6000–50,000 lux) on hydrogen-producing cultures of R. capsulatus grown in a batch photobioreactor, using
Keywords:
lactate as carbon and hydrogen source. The rate of hydrogen production increased with
Hydrogen production
increasing light intensity in a manner that was described by a static Baly model, modified
Photobioreactor
to include the square of the light intensity. In agreement with previous studies, the kinetics
Rhodobacter capsulatus
of substrate utilization and growth of R. capsulatus was represented by the classical Monod
Modelling
or Michaelis–Menten model. When combined with a dynamic Leudekong–Piret model, the amount of hydrogen produced as a function of time was effectively predicted. These results will be useful for the automatization and control of bioprocesses for the photoproduction of hydrogen. ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Economic development over the last few decades has been strongly dependent on fossil fuels as sources of energy. These resources are not unlimited in the long term, and environmental concerns have led to the search for clean, renewable energy sources. Hydrogen is a clean source of energy, considered as a potential and more sustainable energy substitute for fossil fuels. It has been predicted that the
contribution of hydrogen to global energy consumption will increase dramatically, to approximately 50% by the end of the 21st century, due to the development of efficient end-use technologies, possibly even becoming the major final energy carrier [1–3]. Hydrogen gas can be produced either by electrolysis, coal gasification, steam methane reforming of natural gas, partial oxidation of fuel oil, solar thermal cracking, biomass gasification, or photobiological synthesis [4–8]. Biological hydrogen
* Corresponding author. Fax: þ33 4 76 82 67 77. E-mail address: [email protected] (J.-P. Magnin). 0360-3199/$ – see front matter ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2008.09.081
international journal of hydrogen energy 34 (2009) 180–185
Nomenclature X S VH2 P(I ) Vm, Km m(S ) mmax YxP YxS Te 4(I ) b h M0
bacterial concentration [g L1] substrate concentration [g L1] volume of hydrogen produced [L] hydrogen production rate [h1] two parameters to be identified [h1, lux1] specific bacterial growth rate [h1] maximum substrate degradation rate [h1] yield biomass/product [g L1] substrate utilization yield [g L1] time interval [h] yield of light intensity [lux] non-growth-associated product formation coefficient hydrogen production yield initial lactate concentration [M]
production is carried out by a large number of hydrogenproducing microorganisms, including obligate and facultative anaerobes, aerobes, cyanobacteria, photosynthetic bacteria and algae [9]. Among them, photosynthetic bacteria, such as Rhodobacter capsulatus, are favourable candidates for largescale production due to their high energy-conversion efficiencies and their ability to use a wide variety of substrates for growth and hydrogen production [10–14]. Nevertheless, the rate and yield of hydrogen production vary greatly depending on the carbon sources used and physiological growth conditions, such as light intensity. The conversion efficiency of light energy to hydrogen is a key factor in the development of a biological process devoted to hydrogen production [15–17]. The relationship between light intensity and the metabolic products of photosynthetic microorganisms like microalgae has been described by different mathematical equations [18,19]. With regard to photosynthetic bacteria, Nakada et al. [20] measured light penetration into a four-compartment photobioreactor and its relationship to hydrogen production by Rhodobacter sphaeroides [21,22]. They found that the rate of hydrogen production and light penetration both decreased upon passage through the reactor compartments. The effect of light intensity on nitrogenase synthesis and hydrogen evolution at a constant cell density, in a continuous culture of R. capsulatus, was studied by Jouanneau et al. [15]. Under these conditions, the light absorption by the bacterial culture was constant, so the actual light intensity reaching the bacteria was dependent only on the incident light. These results confirmed those of previous studies on batch cultures [6,22], which showed that increasing the light intensity greatly stimulated the hydrogen production capacity of R. capsulatus. The objective of the present study was to develop a model for hydrogen production by the photosynthetic bacterium, R. capsulatus, in a batch photobioreactor, with particular emphasis on the effect of light intensity. The development of such a model is important for the automatization and control of bioprocesses for the photoproduction of hydrogen from industrial waste.
2.
Materials and methods
2.1.
Bacteria and culture medium
181
Precultures of R. capsulatus strain IR3 [23] were grown anaerobically at pH 6.8 and 30 C in RCV medium (lactate 30 mmol L1, DL-malate 7.5 mmol L1, (NH4)2SO4 7.5 mmol L1). Hydrogenproducing cultures were grown at constant temperature (30 C) in a nitrogen-limiting medium containing Na-lactate (between 30 and 80 mmol L1, depending on the experiment) and Na-glutamate (7 mmol L1) as nitrogen source.
2.2.
Photobioreactor
Two types of photobioreactor were used for hydrogen production experiments with R. capsulatus. The first type consisted of a flat glass bottle, 0.125 L in volume, containing a magnetic stirrer for mixing. The opening was hermetically sealed with a rubber stopper, and the biologically produced hydrogen was evacuated via a needle inserted in the stopper, towards an hydrogen measurement system consisting of an inverted, graduated test-tube filled with KCl (0.1 M). The hydrogen gas produced increased the pressure inside the tube and so proportionally decreased the liquid level. The liquid displacement was directly correlated with the volume of hydrogen produced. The second type was a laboratory-made, rectangular-shaped bioreactor, 1 L in volume, comprising two external plexiglass plates (22.5 12.5 0.4 cm) and a smaller, internal plexiglass plate (19 12.5 0.4 cm). The latter divided the reactor into two compartments: one of 3 cm thickness devoted to bacterial growth, and a second (3 cm thickness) for degassing and circulation of the medium. The reactor was hermetically sealed with a gas-tight plexiglass lid (thickness 2 cm), containing three outlets devoted to the pH electrode, the temperature probe and evacuation of the evolved hydrogen. The culture medium was circulated between the
Fig. 1 – Overview of the experimental set-up for H2 production by R. capsulatus IR3: 1-L plexiglas-reactor (A), lamp (B), ventilator (C), H2 flow-meter (D), H2-measurement system (E), pump (F), pH and temperature measurement apparatus (G), data acquisition card (H), computer (I).
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international journal of hydrogen energy 34 (2009) 180–185
two compartments by means of a pump (4 W, 12 V, 4.5 L min1). The bioreactor was illuminated from one side by a sodium-vapour lamp (OSRAM, Plantastar, 600 W) (Fig. 1). The light intensity at the surface of the reactor was varied by changing the distance between the light source and the illuminated surface. The light intensity was measured by a digital lux (Meter, RO 1332). The conversion factor between the illumination (in lux, official SI Unit) and the photosynthetic active radiation (PAR) irradiance (in mE m2 s1, non-official SI unit) for the sodium-vapour lamp is 0.0138. Anaerobic conditions were obtained by purging the bioreactor with sterile argon. The hydrogen flow rate was measured by associating a mass flowmeter (0–20 ml H2 h1, MacMillan, model 50D) linked to a data acquisition card (PMD-1208LS, SA Measurement Computing) with a graduated-glass tube (150 4 cm) filled with liquid (KCl 0.1 M) as described above.
2.3.
Analytical methods
Bacterial cell density was estimated spectrophotometrically at 660 nm. The concentration of lactate in the growth medium was determined by HPLC (Waters 510), after previous removal of biomass by centrifugation (3000 rpm, 5 min) using an ICECoregel 87H3 column (Transgenics) with sulphuric acid (0.1 mol L1) as the mobile phase. Acquisition and treatment of data were carried out using Azur software (version 4.5, Datalys Company, France).
3.
Results and discussion
3.1. Effect of light intensity on biomass formation and hydrogen production rate
Fig. 3 – Influence of the light intensity on the specific bacterial growth rate of R. capsulatus IR3 during batch culture on 80 mM lactate (110-ml reactor).
50,000 lux, the final protein concentration increased from 3.1 to 4 g L1. This variation represents a 30% increase in protein concentration, which is correlated with biomass production. The specific-growth rate, determined by Monod’s model, was linearly dependent on light intensity (Fig. 3). A plot of the kinetics of hydrogen production as a function of light intensity revealed that the rate of hydrogen production was 6-fold higher at 50,000 lux than at 6000 lux (Fig. 4). The specific production rate of hydrogen was estimated and modelled according to Baly’s model (Fig. 5). The Baly model, which was originally applied to phytoplankton [24], is a statistically simple model, which correlates the specific production rate, P, to light intensity: P¼
Vm I Km þ I
(1)
The influence of the light intensity on biomass formation and the hydrogen production rate was first investigated using a flat-bottle glass reactor, over a range of light intensities from 6000 to 50,000 lux. The kinetics of biomass production in a photosynthetic hydrogen-producing culture is shown in Fig. 2. As the light intensity was increased from 6000 to
In the case of the photosynthetic bacterium R. capsulatus, a better fit to the data was obtained if the term I was replaced by I2:
Fig. 2 – Influence of light intensity on the bacterial concentration of R. capsulatus IR3 during batch culture on 80 mM lactate (110-ml reactor) (A): 6000 lux; (,) 9000 lux; (:) 11,000; (#) 23,000; (C) 30,000; and (D) 50,000 lux.
Fig. 4 – Influence of the light intensity on the H2 production of R. capsulatus IR3 during batch culture on 80 mM lactate (110-ml reactor) (A): 6000 lux; (,) 9000 lux; (:) 11,000; (#) 23,000; (C) 30,000; and (D) 50,000 lux.
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Therefore, the hydrogen production yield (h) is calculated as a percentage of the complete conversion of lactate in H2 and CO2 from the Eq. (5). h¼
VH2 100 6 22:4 M0
(4)
where VH2 is the volume of hydrogen production in litre and M0, the initial concentration of lactate. The correlation between the hydrogen conversion yield and the light intensity in the range 6000–50,000 lux is shown in Fig. 6. As the light intensity increased from 6000 to 25,000 lux, the hydrogen conversion yield increased accordingly. Above 25,000 lux, the hydrogen production yield stabilized for the strain IR3 with 80 mM lactate. Fig. 5 – Modelling, according to Baly’s model, of the influence of light intensity on H2 production during a batch culture of R. capsulatus IR3 (110-ml glass reactor, 80 mM lactate, 7 mM glutamate, 30 8C).
P¼
Vm I2 Km þ I2
The biomass concentration in the batch hydrogen production experiments depends on the concentration in limiting substrate. Theoretically, the growth rate and the substrate utilization are expressed as: (2)
This can be rationalised, since light is involved twice in hydrogen production by R. capsulatus, firstly in synthesis of the enzyme nitrogenase, and secondly in photosynthetic metabolism required to synthesise ATP for enzyme activity [15,17]. The model parameters of Km and Vm were estimated as 104.9 lux and 2.8 h1, respectively, for bacterial cultures grown under a light intensity range of 6000–50,000 lux. The bacterial hydrogen production yield reflects the capability of a bacterial strain to convert the carbon substrate into biogas. The biogas produced is a mixture of hydrogen and carbon dioxide. For lactate, which was the carbon substrate used in this study, 6 mol of hydrogen are expected to be produced per mole of lactate utilized according to Eq. (4) C3 H6 O3 þ 3H2 O / 6H2 þ 3CO2
3.2. Kinetics of the bacteria growth and substrate utilization
dX ¼ mðSÞX dt dS 1 ¼ mðSÞX dt YxS
)
(5)
where X is the bacteria concentration (g L1), m(S ) the specificgrowth rate (h1), S the substrate concentration (g L1), and YxS the substrate utilization yield. The R. capsulatus growth curves were well fitted by the Monod model, and by its modified versions, the Michaelis– Menten’s model, according to: mðSÞ ¼ mmax
S Ks þ S
(6)
where mmax is the maximum specific substrate degradation rate (h1), Ks the dissociation constant (g L1), and S the substrate concentration (g L1).
(3)
Fig. 6 – Influence of light intensity on H2 conversion rate by R. capsulatus IR3 during a batch culture (110-ml reactor, 80 mM lactate, 7 mM glutamate, 30 8C).
Fig. 7 – Kinetics of the lactate consumption (S, 3) and the biomass production (X, :) during a batch culture of R. capsulatus IR3. Data was fitted using the Monod model (1-L bioreactor, 30 mM lactate, 30,000 lux).
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By integration of Eq. (6) and substitution of the variable of Eq. (7), the following equations are obtained: Si Xi DT þ Xi Ks þ Si 1 Si ¼ mmax Xi DT þ Si Ki þ Si YxS
Xiþ1 ¼ mmax Siþ1
)
(7)
where Xiþ1 is the biomass concentration at time i þ 1 (g L1), Xi the biomass concentration at time i, Siþ1 the substrate concentration at time i þ 1, Si the substrate concentration at time i, DT the time interval (h). Fig. 7 shows the relationship between biomass concentration, substrate utilization and cultivation time, including both experimental data and the fitted curve based on the model. The model described correctly the experimental data. The corresponding model parameters of Ks, mmax, YxS, and DT, were estimated as 10 g L1, 0.3 h1, 0.1 g L1 and 0.5 h, respectively.
3.3.
Kinetics of hydrogen production
The production rate of hydrogen with respect to light intensity irrespective of cell concentration was estimated by Eq. (2). Luedeking–Piret proposed a relationship describing the specific production rate in terms of the cellular concentration [25] according to: dP 1 mðSÞX þ bX ¼ dt YxP
bioreactor and to the light intensity. Therefore, an additional factor was introduced in the Eq. (8) to take into consideration the light intensity effect, as shown in Eq. (9). dVH2 1 ¼ 4ðIÞmðSÞX þ bX dt YxP
Integration of Eq. (9) gives Equation (10): 1 4ðIÞmðSÞ þ b Xi Te þ VH2 i VH2 iþ1 ¼ YxP
(9)
(10)
where (H2)iþ1 is the volume of hydrogen produced at time i þ 1 (ml), (H2)i is the volume of hydrogen produced at time i (ml), X the biomass concentration (g L1), S the substrate concentration (g L1), m(S ) the bacterial specific-growth rate (h1), 4(I ) the yield of light energy (lux), YxP the hydrogen production yield, b the non-growth-associated formation coefficient of product and Te the interval time. The fitted and experimental curves representing the kinetics of hydrogen production during lactate consumption are shown in Fig. 8. A good agreement was obtained between the experimental and the modelled data. The model parameters of Ks, YxP, Te, b, 4(I ), and mmax were estimated as 10 g L1, 0.7 g L1, 0.5 h, 12, 3 lux and 0.3 h1. Thus, the relationship between biomass, product (hydrogen) and light intensity during hydrogen production process by R. capsulatus is well described by the modified Luedeking–Piret model.
(8)
where X is the biomass concentration (g L1), S the substrate concentration (g L1), and m(S ) the bacterial specific-growth rate (h1). The first term, (ð1=YxP ÞmðSÞX), is referred, together, to the microbial growth and the product formation rate. The second term bX is associated with bacterial growth. The dynamic model of hydrogen production reflects the fact that the specific hydrogen production rate (ml/h) is proportional both to the quantity of bacteria present in the
4.
Conclusions
The effect of light intensity on hydrogen production by the photosynthetic bacterium R. capsulatus was investigated and a mathematical model describing substrate utilization, biomass formation and H2 production was obtained, based on experimental results from hydrogen-producing batch cultures. This model allows command variables to be defined, thus enabling automatization of a bioprocess for hydrogen photoproduction with optimization of parameters such as the quantity and quality of the biogas produced.
Acknowledgments This work was supported by grants from the Rhoˆne-Alpes Region (France) and by a doctoral grant awarded to Jamila Obeid by the Syrian Government (The Ministry of Higher Education, Al Baath University, Faculty of Chemical and Petroleum Engineering).
references
Fig. 8 – Modelling, according to a modified Luedeking– Piret’s model, of the kinetics of a batch H2-production by R. capsulatus IR3 (30 mM lactate, 30,000 lux).
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