international journal of hydrogen energy 34 (2009) 180185

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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 dHe`res, France
Grenoble Institute of Technology, Laboratoire des sciences pour la conception, loptimisation et la production,
46, avenue Felix Viallet, 38031 Grenoble, France
c
Laboratoire de Chimie et Biologie des Metaux (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 (600050,000 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 MichaelisMenten model. When combined with a dynamic LeudekongPiret 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.
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 [13].
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 [48]. Biological hydrogen

* Corresponding author. Fax: 33 4 76 82 67 77.

E-mail address: Jean-Pierre.Magnin@lepmi.inpg.fr (J.-P. Magnin).
0360-3199/$ see front matter 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) 180185

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 [1014]. 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 [1517].
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) 180185

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 (020 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 Monods 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 Balys 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:
P

Vm I
Km 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) 180185

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).
h

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 600050,000 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 Balys 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).

Vm I2
Km 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 600050,000 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 3H2 O / 6H2 3CO2

3.2.
Kinetics of the bacteria growth and substrate
utilization

dX
mSX
dt
dS
1
 mSX
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
Mentens model, according to:
mS mmax

S
Ks 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) 180185

By integration of Eq. (6) and substitution of the variable of

Eq. (7), the following equations are obtained:
Si
Xi DT Xi
Ks Si
1
Si
 mmax
Xi DT Si
Ki Si
YxS

Xi1 mmax
Si1

(7)

where Xi1 is the biomass concentration at time i 1 (g L1), Xi

the biomass concentration at time i, Si1 the substrate
concentration at time i 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).
LuedekingPiret proposed a relationship describing the
specific production rate in terms of the cellular concentration
[25] according to:
dP
1
mSX bX

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

4ImSX bX
dt
YxP

Integration of Eq. (9) gives Equation (10):






1
4ImS b Xi Te VH2 i
VH2 i1
YxP

(9)

(10)

where (H2)i1 is the volume of hydrogen produced at time i 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 LuedekingPiret 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, (1=YxP mSX), 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 Rhone-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).

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Fig. 8 Modelling, according to a modified Luedeking

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R. capsulatus IR3 (30 mM lactate, 30,000 lux).

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