Journal of Biotechnology 162 (2012) 89–96

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Process development for hydrogen production with Chlamydomonas reinhardtii

based on growth and product formation kinetics
Florian Lehr a , Michael Morweiser a , Rosa Rosello Sastre a,∗ , Olaf Kruse b , Clemens Posten a
a
Institute of Engineering in Life Sciences, Department Bioprocess Engineering, Karlsruhe Institute of Technology KIT, Karlsruhe, Germany
b
Department of Biology, Center for Biotechnology, Bielefeld University, Bielefeld, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Certain strains of microalgae are long known to produce hydrogen under anaerobic conditions. In
Received 31 December 2011 Chlamydomonas reinhardtii the oxygen-sensitive hydrogenase enzyme recombines electrons from the
Received in revised form 25 May 2012 chloroplast electron transport chain with protons to form molecular hydrogen directly inside the chloro-
Accepted 1 June 2012
plast. A sustained hydrogen production can be obtained under low sulfur conditions in C. reinhardtii,
Available online 29 June 2012
reducing the net oxygen evolution by reducing the photosystem II activity and thereby overcoming the
inhibition of the hydrogenases. The development of specially adapted hydrogen production strains led
Keywords:
to higher yields and optimized biological process preconditions.
Chlamydomonas reinhardtii
H2 production
So far sustainable hydrogen production required a complete exchange of the growth medium to estab-
Process development lish sulfur-deprived conditions after biomass growth. In this work we demonstrate the transition from
Single batch culture the biomass growth phase to the hydrogen production phase in a single batch culture only by exact dosage
Growth kinetics of sulfur. This eliminates the elaborate and energy intensive solid–liquid separation step and establishes
Product formation kinetics a process strategy to proceed further versus large scale production. This strategy has been applied to
determine light dependent biomass growth and hydrogen production kinetics to assess the potential of
H2 production with C. reinhardtii as a basis for scale up and further process optimization.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction waste of chemical energy by the formation of organic intermediates

Sustainable energy supply is one of the most challenging prob-
lems mankind is facing today. While the solar irradiation energy
of 1.5 × 1018 kWh would theoretically cover the annual global
demand of 1.3 × 1014 kWh more than sufficiently, yet the known
conversion and storage techniques are limiting the use of this enor-
mous potential.
As renewable energy availability is subject to natural fluctu-
ations, not only the generation of energy, but especially energy
storage is becoming an important issue. In contrast to electrical
energy hydrogen can serve directly as a storable energy carrier.
Hydrogen offers the highest mass based chemical energy density
and can be used in fuel cells or combustion engines.
One sustainable hydrogen production process is the natural abil-
ity of certain green algae to produce H2 in a defined environment.
Hydrogen as a biological product is especially interesting since as a
gaseous product, no elaborate downstream processing is necessary.
Moreover, the biochemical pathway of H2 formation in green algae
is connected very closely to the photosynthetic reaction complexes;
∗ Corresponding author.
E-mail address: rosa.rosello@kit.edu (R. Rosello Sastre).
can be largely avoided.
First experimental works on hydrogen production with green
algae have been published in 1942 by Gaffron and Rubin (Gaffron
and Rubin, 1942). Fundamental advances in knowledge of the bio-
chemistry of the process paved the way for protocols that ensured
prolonged light driven H2 production.
The hydrogen production process in Chlamydomonas is located
in the chloroplast. Electrons leave the chloroplast electron trans-
port chain via ferredoxin. In normal oxygenic photosynthesis
mainly the ferredoxin-NADP+ -oxireductase (FNR) enzyme reduces
NADP+ to NADPH. Under anaerobic conditions, however, an
oxygen-sensitive [Fe]-hydrogenase enzyme (Happe et al., 1994;
Happe and Naber, 1993; Stripp et al., 2009) reduces protons in the
chloroplast to molecular hydrogen. Electrons can enter the elec-
tron transport chain by two mechanisms: the photosynthetic water
splitting reaction at photosystem II (PS II) or via the plastoquinone
(PQ) pool by oxidation of reducing equivalents (Godde and Trebst,
1980; Melis and Happe, 2001; Winkler et al., 2009).
The role of intracellular starch for hydrogen production was first
addressed by Gfeller and Gibbs with an analysis of the product of
starch catabolism (Gfeller and Gibbs, 1984). During hydrogen pho-
toproduction a decrease in intracellular starch content is commonly
observed (Chochois et al., 2009; Fouchard et al., 2005; Melis et al.,

0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jbiotec.2012.06.002
90 F. Lehr et al. / Journal of Biotechnology 162 (2012) 89–96
2000; Timmins et al., 2009; Tsygankov et al., 2002) The finding that
Rubisco-deficient mutants, lacking to accumulate starch, feature
a reduced hydrogen production capability (Chochois et al., 2009;
Posewitz et al., 2004) led to the assumption that the PSII indepen-
dent pathway requires starch as a substrate. According to Melis
(Melis, 2007) starch catabolism plays a role in both H2 production
pathways: for the PSII dependent pathway as an electron donor
via PQ and in for both pathways as a substrate for mitochondrial
respiration to maintain anaerobiosis.
For an efficient hydrogen production it is therefore essential to
have an ample flow of electrons from the electron transport chain
directly to the hydrogenase, avoiding competing electron sinks,
one of which would be the cyclic electron flow. This loop pathway
enables the system to redirect the electron flow from ferredoxin
via the cytochrome b6 f complex back to PSI. This mechanism is
driven by light absorption at the PSI and allows producing ATP inde-
pendently from NADPH. Kruse and co-workers have constructed
mutants deficient in cyclic electron transport. By screening these
mutants for high hydrogen production the stm6 mutant has been
found (Kruse et al., 2005), additionally featuring high intracellular
starch reserves which serve as electron donors to the electron trans-
port chain via the PQ pool after glycolysis. Integration of a hexose
symporter in the stm6 mutant led to the stm6 Glc4 strain (Doebbe
et al., 2007) with even higher H2 production capacity. The stm6Glc4
mutant was therefore chosen for the experiments described below.
One major challenge for the photobiological hydrogen produc-
tion with Chlamydomonas is the O2 -sensitivity of the hydrogenase,
which prevents a simultaneous biomass growth together with H2
evolution. Moreover, without special preconditions illuminated
cultures produce oxygen even under externally anaerobic condi-
tions, thereby inhibiting the hydrogenase eventually. To overcome
this problem, PSII activity can be reduced, so that the photo-
synthetic oxygen evolution is balanced by respirational oxygen
consumption. These conditions can be established by sulfur limita-
tion of the culture (Melis and Happe, 2001; Melis et al., 2000). This
necessarily implies a two-stage process: First a biomass growth
phase, in which sulfur is abundant and the environment is aerobic.
Since under these conditions no hydrogen production is possible, a
distinct hydrogen production phase must follow under anaerobio-
sis and sulfur-limiting conditions. The standard procedure (Melis
et al., 2000) features a solid–liquid separation step of biomass
and culture medium after the growth phase, followed by several
washing steps. Finally, the biomass is transferred to a new sulfur-
deficient culture medium. Numerous works have been based on a
similar protocol (Antal et al., 2003; Chochois et al., 2009; Forestier
et al., 2003; Fouchard et al., 2005; Jo et al., 2006; Kim et al., 2010;
Kosourov et al., 2007; Tamburic et al., 2011; Tsygankov et al., 2002;
Zhang et al., 2002).
While this method is very reliable and robust, the solid–liquid
separation step, typically centrifugation, inevitably implies high
energy consumption. With dry biomass concentrations ranging
between 1 and 10 g/L, at least 99% of the kinetic energy demand
is due to water acceleration. At lab scale the total energy demand
is negligible and the accuracy of the above protocol is preferred
over energy savings, at larger scale, however, the energy demand
and expenses for additional periphery must be taken into account,
especially with an energy carrier as the final product. One major
task in this work was therefore to improve the process control
by replacing the culture medium exchange entirely and perform
biomass growth phase and hydrogen production phase in a single
batch culture. This new process strategy together with a custom
illumination system allowed the determination of light dependent
hydrogen production kinetics based on a scalable process manage-
ment. In combination with the presented light dependent biomass
growth kinetics these data can serve as a basis to assess the current
potential for hydrogen production with C. reinhardtii.
2. Material and methods
2.1. Strains and preculture conditions
C. reinhardtii mutants Stm6 (Schonfeld et al., 2004) and Stm6Glc4
(Doebbe et al., 2007) were provided by the Algae Biotech Group
headed by Prof. Olaf Kruse from the University of Bielefeld, as
part of a cooperation work within the Solar Biofuels Consortium
(http://www.solarbiofuels.org). For maintenance and preculturing
the strains were grown axenic under mixotrophic conditions using
TAP (Tris Acetate Phosphate) liquid medium (Gaffron and Rubin,
1942). Therefore 200 mL of an axenic culture were cultivated in
500 mL Erlenmeyer flasks on an orbital shaker incubator at 100 rpm,
25 ◦ C and 100 ␮E/(m2 s).
2.2. Shaking flasks cultivations
The sulphur demand during biomass growth was investigated
in parallelized shaking flask cultivations with varying sulphur con-
centrations in the liquid medium. Therefore special TAP medium
(TAP-S) was used, were the sulphate salts of the original protocol
were replaced by their chloride analogues. The resulting sulphate
concentrations of the flasks were adjusted by adding corresponding
volumes of a 5 g/L MgSO4 stock solution.
After autoclaving the flasks were inoculated with 1 mL of a
mid-log pre-culture of C. reinhardtii Stm6Glc4 and placed on a LED-
orbital shaker for incubation at 25 ◦ C and 100 rpm. Samples were
taken at regular intervals for optical density (OD) measurement and
ion chromatographic (IC) analysis.
2.3. Reactor cultivations
2.3.1. 2 L-model reactor
For the measurement of precise light dependent kinetics, a
commercially available stirred tank reactor (model KLF 2000, Bio-
engineering AG) was modified with a custom lighting device
in order to obtain the needed homogenous illumination pattern
within the algal culture. The reactor consisted of a cylindrical glass
vessel with an inner diameter of 100 mm and a height of 300 mm
(2 L working volume). Agitation and dispersion was realised with
two six-blade Rushton turbines (stirrer/vessel ratio = 0.63) installed
above a membrane equipped aeration tube. The lighting device
has been developed at our institute (Lehr et al., 2007) and con-
sists of two printed circuit board half shells which are assembled
with warm-white SMD-LEDs and additional collimating lenses (6◦
radiation angle) for increased light focusing towards the centre of
the reactor. At low cell densities (<1 g/L), this effect almost com-
pensates the light attenuation caused by the mutual shading of the
cells resulting in an almost homogenous light distribution within
the reactor. Fig. 1 shows the 2 L-modelreactor with installed LED-
lighting system.
2.3.2. Light measurement
The light distribution inside the 2l-model reactor was mea-
sured using a spherical quantum sensor (QLS-2101, Biospherical
Instruments Inc.). The dependence between electric current at the
LED-lighting system and resulting photon flux density (PFD) was
measured inside the reactor filled with algal suspension with an
optical density of OD750nm = 0.46 corresponding to a dry biomass
concentration of 0.2 g/L. At this biomass concentration the reac-
tor volume is homogenously illuminated (results not shown). The
spherical sensor was placed at the middle of the reactor vessel in
axial direction and at a distance of 14.7 mm in radial direction from
the vessel wall, corresponding to the volumetric average of the
illuminated reactor volume.
 F. Lehr et al. / Journal of Biotechnology 162 (2012) 89–96
Fig. 1. Picture showing the 2 L-model reactor with installed LED-lighting device
(LED-half shell opened).
2.3.3. Cultivation conditions
Reactor cultivations for determining the light-dependent
growth kinetics were carried out photoautotrophically in TAP
medium without addition of acetate (TP medium). Temperature
was controlled at 25 ◦ C, pH at 7 and the gassing rate was set at
30 mL/(L min) with 5% (v/v) CO2.
For the determination of the light-dependent hydrogen produc-
tion kinetics, the associated cultivations were performed using TAP
medium with a defined sulphate concentration of 11 mg/L. Initial
gassing during the biomass growth stage was set at 30 mL/(L min)
with 5% (v/v) CO2 . The temperature was kept constant at 25 ◦ C
throughout the process.
2.4. Analytics
2.4.1. Biomass dry weight determination
Biomass dry weight (BDM) was correlated with the optical den-
sity (OD) of the microalgal suspension at a wavelength of 750 nm
against demineralized water using a spectrophotometer (model
T60, PG Instruments Ltd.). At 750 nm no pigment absorption by
C. reinhardtii takes place, thus the extinction almost corresponds to
biomass dependent light scattering. For each Chlamydomonas strain
a correlation curve between OD and biomass dry concentration
was determined. For each correlation, liquid samples at different
biomass densities were taken during cultivations in the 2 L-model
reactor. Besides OD measurements at 750 nm, 30 mL of the sample
were centrifuged (15 min, 15,000 g, 4 ◦ C) and the resulting pellet
was washed twice with ultrapure water, centrifuged again and
dried (24 h, 80 ◦ C) in tared stainless steel centrifuge tubes. After
drying the weight of the pellet was determines on an analytical
balance. Each sample was replicated four times.
91
2.4.2. Starch content
The intracellular starch concentration was determined after
cell disruption and acidic starch hydrolysis by colorimetric quan-
tification of the glucose monomers after reaction with sodium
3,5-dinitrosalicylate (Smith and Cheng, 1978). A sample (2 mL) was
centrifuged (10 min, 15,000 g, 4 ◦ C) and the pellet was resuspended
in 1 mL 0.1 molar sodium acetate buffer (pH 5). 5 stainless steel
grinding balls were added to the resuspended pellet for cell dis-
ruption on a mixer mill (Retsch MM 300, 5 min, 30 Hz). Afterwards
1.5 mL of a 2 molar hydrogen chloride solution was added and the
mixture was boiled for 60 min. Than 2 mL of a 2 molar sodium
hydroxide solution were added and the mixture was centrifuge for
15 min at 20,000 g. 900 ␮L of the resulting interphase were mixed
with 200 ␮L sodium 3,5-dinitrosalicylate solution and boiled again
for 5 min. The absorption of the sample was measured at 546 nm
against a blank sample using a spectrophotometer (T60, PG Instru-
ments Ltd.).
2.4.3. Gas chromatography
During the cultivations in the 2 L-model reactor, the off-gas
composition was measured using a two channel micro gas chro-
matograph (model 3000 Micro GC, Agilent Technologies) which
was directly connected to the headspace of the reactor via a bypass
line. Argon was used as carrier gas. In order to protect the gas
chromatograph analyser against liquid entrained in sample gas, a
gas/liquid membrane separator was installed (Genie® Model 170,
A+ Corporation, LLC). The gas chromatograph was calibrated using
pure H2 , N2 and CO2 standards as well as specially produced calibra-
tion gas mixtures (Air Liquid). Data analysis was performed using
the software EZChrom SI (Agilent, Version 3.2.1).
2.4.4. Ion chromatography
Anion analyses were performed using an ion chromatogra-
phy system (IC-System, model Compact IC Plus 882, Metrohm
GmbH) with auto sampler unit (model Sample Processor 585)
and inline-dialysis cell for fully automated sample processing
including sample dilution, dialysis and chromatographic analy-
sis. 1.5 mL sample (supernatant from microalgal suspension) was
diluted (1:10) and dialyzed (0.2 ␮m cellulose acetate membrane)
against ultra-pure water automated through the IC-System. 20 ␮L
of the dialyzed sample were injected and analyzed using a Met-
rosep A Supp 5 150/4.0 separation column (Metrohm GmbH) and
a carbonate eluent (3.2 mM Na2 CO3 , 1.0 mM NaHCO3 , 12,5% (v/v)
acetonitrile) with a flux rate of 0.7 mL/min.
3. Results and discussion
3.1. Light-dependent biomass growth kinetics
Incident light is one of the major parameters influencing H2 pro-
duction with microalgae, in both biomass growth and hydrogen
production phase. To improve the entire two-stage production pro-
cess the determination of optimum conditions is required for both
phases individually. In a series of growth experiments a defined
constant light intensity was applied to C. reinhardtii Stm6 cultures
in a lab scale bioreactor system under photoautotrophic conditions.
The target biomass concentration was kept intentionally low to
avoid dark zones and light limitation during the experiments. Culti-
vations were performed in batch mode over several days to ensure
full adaption of the culture to the environment. To each cultivation
a different photon flux density (PFD) was applied, ranging from
below 50 to more than 2100 ␮E/(m2 s). For each experiment the
maximum specific growth rate  of the cultivation was determined
and plotted against the PFD, hence resulting in a light dependent
growth kinetics curve (Fig. 2).
92 F. Lehr et al. / Journal of Biotechnology 162 (2012) 89–96

Fig. 2. Light dependent biomass growth kinetics curve with three distinct sections. Fig. 3. Biomass growth under different initial sulfur concentrations.

According to the kinetics, three distinct light intensity sections
can be defined. In section I (0–200 ␮E/(m2 s))  is increasing rapidly
with a higher PFD, indicating a high efficiency of light energy
use. Section II is characterized by a more moderate increase of
 with the incident light intensity, covering a wide PFD range
(200–1700 ␮E/(m2 s)). The maximum  of 1.3 d−1 was measured
at 1700 ␮E/(m2 s). Light intensities above 1700 ␮E/(m2 s) lead to
a drastic decline in the specific growth rate (section III) due to
phototoxic effects.
These data allow the definition of the optimum working PFD
close to 200 ␮E/(m2 s). At this point the efficient light energy use of
section I meets a relatively high specific growth rate. The broad PFD
range of section II provides an ample buffer zone for higher light
intensities, where the efficiency of light use declines without dam-
aging the culture by photoinhibition. Lower PFDs would provide
the same efficiency in light use at the expense of overall production
time. In conclusion, for biomass growth there is a defined optimum
light intensity, but a wide working PFD range in which damage to
the culture can be avoided.
3.2. Light-dependent hydrogen production kinetics
initial medium sulfur content. The decrease in biomass after the
growth phase occurring mainly with low sulfur concentration is
probably due to the effects of sulfur depletion. The most promi-
nent biomass losses occur in cultures with 5–15 mg initial sulfate
concentration. Fig. 4 indicates that in these cultures from t = 75 h
no sulfate was remaining in the medium, whereas the decrease in
biomass did not occur until t = 120 h. Between t = 75 h and t = 100 h
biomass increased in all 3 cultures, probably due to carbohy-
drate accumulation. Successively, the damage in PSII results in a
lower photosynthesis rate and therefore to less conversion of light
energy, which may be compensated by catabolism of carbohydrate
stores. After biomass growth no remaining sulfur could be detected
up to an initial concentration of 30 mg/L, indicating that below
this concentration sulfate is the limiting nutrient during biomass
growth. Initial availability of significantly more than 20 mg/L does
not increase the maximum total biomass, with more than 30 mg/L
excessive sulfur is clearly detectable in the medium at the end of
the biomass growth step. Thus, sulfate concentrations must be kept
below 20 mg/L to avoid remaining sulfate in the medium, conse-
quently allowing the system to enter sulfate depleted conditions
autonomously subsequent to the biomass growth stage.

3.2.1. Determination of limiting sulfur concentration 3.2.2. Balanced batch approach

As already mentioned, in most experiments on hydrogen pro- One major task was the reduction of process complexity by
duction with Chlamydomonas sulfur limitation has been established eliminating the solid–liquid separation step. The determined lim-
by an exchange of the culture medium. This process step involves iting sulfur concentration allowed establishing an exactly balanced
solid–liquid separation, which implies additional equipment and batch system that induces the required sulfur deprived state only
energy expenses. In order to avoid this procedure a self-limiting
system has been established based on an exact dosage of sulfur in
the culture medium.
One important precondition for such an approach is the limit-
ing concentration of sulfur. To this end parallelized experiments on
sulfur nutrient limitation have been conducted with C. reinhardtii
Stm6Glc4 cultures. In 200 mL scale batch experiments the initial sul-
fur concentration in the medium was varied between 5 and 75 mg/L
sulfate. Other medium components were dosed in double excess to
avoid limitation by another nutrient component. The PFD was set
to 350 ␮E/(m2 s). Previously performed experiments revealed that
in a range between 150 and 500 ␮E/(m2 s) the growth response to
sulfur availability is independent from the light intensity (data not
shown). The respective biomass concentrations were determined
and plotted against the process time (Fig. 3). In parallel the remain-
ing sulfur concentration in the growth medium was determined by
IC at each measurement point (Fig. 4).
Both diagrams indicate that the critical sulfur concentration
is above 20 mg/L. Below this value total biomass increased with Fig. 4. remaining sulfur during biomass growth.
 F. Lehr et al. / Journal of Biotechnology 162 (2012) 89–96
Fig. 5. Complete balanced batch process with hydrogen production; transition from
growth phase to production phase at t = 120 h; course of dry biomass (black solid
line), sulfur in medium (purple dot-dash-line), produced hydrogen (green dotted
line) and intracellular starch content (yellow diamonds).
by sulfur consumption. Therefore, a 2 L scale bioreactor experiment
was performed with C. reinhardtii Stm6Glc4 with an initial medium
sulfur content of 11 mg/L. The cultivation was carried out in batch
mode with a PFD of 280 ␮E/(m2 s). The system was equipped with a
pO2 probe and a pressure sensor. Biomass concentration, medium
sulfur content and intracellular starch content were determined by
external measurements.
In the first 40 h of cultivation an exponential growth of the
biomass could be detected, parallel to a decline of the medium
sulfur concentration (Fig. 5). After 50 h the sulfur was totally con-
sumed, biomass growth continued to reach a maximum at about
90 h after inoculation, followed by a slight decrease thereafter. Dur-
ing the entire growth phase the pO2 remained constant.
At 120 h the reactor was sealed with the gassing turned off.
The reactor headspace was minimized by adding 200 mL ultrapure
water in order to increase the analysis sensitivity of newly produced
gas, visible in the sudden down step in the biomass signal. In the
following a steep decline in the pO2 was observed. As soon as the
pO2 value dropped below the detection limit, a pressure increase
inside the reactor could be recorded. Subsequently it remained con-
stant for 230 h, thereafter a constant decrease of biomass over 300 h
could be observed. Gas samples were taken regularly for GC anal-
ysis. Remaining gauge pressure was released to quantify the gas
production. The duration of the production phase was 540 h, the
overall process duration 660 h. Total hydrogen production added
up to 835 mL.
With this experiment it could be proved that it is possible to
realize the transition from growth phase to hydrogen production
phase without solid–liquid separation. With the previously cal-
culated limiting sulfur concentration the system entered a sulfur
deprived state at the end of the biomass growth phase. The res-
pirational activity of the culture was sufficient to consume the
remaining oxygen in the system after the biomass growth. Hydro-
gen evolution started immediately after the total consumption of
oxygen and could be maintained for several hundred hours.
3.2.3. Gas balance
To investigate the gas production in detail, gas chromatogra-
phy samples were taken regularly during the production process.
As stated above, the production process was carried out in a sealed
reactor; consequently, gauge pressure was being built up inside the
reactor headspace, which was released after every GC analysis of
93
Fig. 6. Actual headspace gas composition during H2 production at GC measurement
points.
the gas volume. The measured gas composition does therefore not
represent the total gas content in the whole reactor including gas
and fluid phase, but shows the respective actual headspace gas frac-
tions only. As the physical solubility processes are comparatively
fast in comparison to the biological turnover rates, quasi-stationary
conditions according to Henry’s law can be assumed. Nevertheless,
stationary conditions are not reached with respect to balances of
produced and released gas amounts especially not for CO2 .
As shown in Fig. 6, an oxygen fraction is only detectable until
25 h after the initiation of the production process. This residual oxy-
gen, originating from the headspace when the reactor was sealed,
was consumed by respiration. In contrast, the nitrogen fraction was
diminished only by the successive wash out from the headspace
during pressure releases.
The CO2 fraction slightly decreased for 120 h after the beginning
of the production phase, subsequently a continuous increase could
be observed. This behavior can be explained by the comparatively
high solubility of CO2 and the dissociated species according to the
carbonic acid equilibrium. At the beginning of the production phase
pCO2 was decreasing due to the missing CO2 -enriched gassing. In
the following, more CO2 was produced by respirational activity,
most of which remaining in the liquid phase in the form of solute
CO2 gas and carbonate species. Only minor amounts are visible in
the gas phase according to equilibrium conditions. With more CO2
being produced by the culture, more CO2 gas could accumulate in
the medium leading to an increasing partial pressure and conse-
quently to a higher concentration in the reactor headspace, leading
to a higher wash out rate.
The hydrogen fraction measured in the reactor headspace con-
tinuously increased for approx. 200 h up to 86% and stayed constant
thereafter for another 200 h before a slight decrease could be
recorded. The fact that hydrogen accounts by far for the dominant
gas fraction during the production phase indicates a high percent-
age of hydrogen in the biologically produced gas mixture. However,
these data can only give a rough estimation of the composition of
the actual gas production rates, since back mixing and gas solubility
are not taken into account (see above).
3.2.4. Intracellular starch
The intracellular starch content was followed by analyses of
biomass samples throughout the experiment. During the exponen-
tial growth of the biomass – the first 40 h of the cultivation – the
starch content remained at a comparatively low level of roughly
15% of the biomass dry weight. After full consumption of sulfur
and onset of sulfur limitation, the starch content was remarkably
94 F. Lehr et al. / Journal of Biotechnology 162 (2012) 89–96
Fig. 7. H2 production courses for different PFD values; A: specific H2 production
rates; B: integral H2 production.
increasing within 25 h to reach a maximum of 65% at the point
of maximum biomass concentration, followed by a drop to approx.
45% at the beginning of the H2 production phase. The starch content
remained approximately constant for more than 230 h of hydro-
gen production. Afterwards, together with the observed decline in
overall biomass concentration, the starch fraction drops constantly
towards the end of the experiment.
The observed nearly constant value of biomass and starch con-
tent during the beginning of the production phase indicates a minor
contribution of starch catabolism to the electron flow towards the
hydrogenase. In this case starch would only be necessary for res-
piration directly related to produced and respired oxygen. In the
following 300 h of hydrogen production, however, biomass and
starch content were decreasing. At the same time, the hydrogen
fraction in the reactor headspace increased (Fig. 6, see also Fig. 7A).
These findings indicate that in the first phase of H2 production the
electron flow is based mainly on residual PSII activity, whereas in
the second phase starch catabolism has a major contribution to the
electron flow towards the hydrogenase. However, that has to be
proven be a more detailed model based analysis of CO2 production
rate and of possibly formed fermentative products.
3.2.5. Hydrogen production kinetics
The established balanced batch system allowed for a detailed
investigation of the optimum light conditions for hydrogen produc-
tion. Therefore, a series of cultivations with C. reinhardtii Stm6Glc4
analogous to the previously described were performed with the
PFD as the variable parameter. The light intensity ranged from 80 to
720 ␮E/(m2 s) continuous illumination. Hydrogen production was
analyzed discontinuously by a gravimetric system and gas chro-
matography.
As shown in Fig. 7A the specific hydrogen production rate is
strongly impacted by the applied light intensity. Low PFD val-
ues lead to low production rates, for 80 ␮E/(m2 s) approx. 0.6 mL
H2 per hour and g dry biomass compared to 2.4 mL/(g h) at
720 ␮E/(m2 s). The correlation is not linear and shows satura-
tion above 280 ␮E/(m2 s). In addition, high incident light reduces
the time span between the point of established anaerobiosis and
maximum production rate, but leads also to shorter production
durations. One explanation would be that higher irradiances lead
to quicker irreversible damage in the photosystems, so the capac-
ity for long term production is reduced. A second argument is
that higher production rates result in a quicker depletion of starch
reserves, while this cannot be the single reason, since the total pro-
duction per biomass varies with light intensity. Transcriptional or
metabolomics data would be needed would be needed to give bet-
ter insight on this issue. The appearance of a second peak or at
least an attenuation in the decrease in production rate could be
observed in all cultivations, it is most prominent in the culture
under 80 and 280 ␮E/(m2 s). It could be speculated that differen-
tial expression profiles at various points during sulfur depletion
(Nguyen et al., 2011) lead to changes in hydrogenase abundance
or activity, to shifts in substrate supply for the hydrogen produc-
tion or even a shift between H2 production pathways, but again a
thorough investigation of the cell state would be needed to confirm
these assumptions.
The total hydrogen produced per biomass shows a maximum
at 280 ␮E/(m2 s) of 650 mL H2 per g dry biomass, obtained after
535 h of hydrogen production (Fig. 7B). The plot against the PFD
reveals a narrow maximum, total hydrogen volumes at 240 and
400 ␮E/(m2 s) were 43% and 39% lower respectively (Fig. 8B). The
average specific production rate calculated from total hydrogen
produced over production time and dry biomass (Fig. 8C) increases
with the incident light up to 280 ␮E/(m2 s), above this value no
further increase can be obtained. The productivity calculated by
average production rate per light intensity is again maximal at
280 ␮E/(m2 s) (Fig. 8D), but at lower PFD values it is in a compara-
ble range. Exceeding 280 ␮E/(m2 s) the productivity decreases with
higher illumination.
Production rates and total H2 productions per biomass obtained
are in the same range as data found in literature for stm6 and
stm6Glc4 cultivation (Doebbe et al., 2007; Kruse et al., 2005; Nguyen
et al., 2011; Timmins et al., 2009). However, due to different illu-
mination setups and different measures for biomass concentration
a direct comparison is not possible.
Compared to the light-dependent kinetics for biomass growth
the optimum PFD range for H2 production is very similar. Hydrogen
production rates, however, show a steep decline to both sides of the
optimum light intensity. To reach a high efficiency of light use, for
the overall process a light intensity between 250 and 280 ␮E/(m2 s)
should be applied. If fluctuations in PFD are expected, a devia-
tion towards lower light intensities is preferable with regards to
light efficiency. However, low PFDs lead to longer overall process
durations, which may imply higher costs and energy demands.
In the long run, hydrogen production with microalgae can only
be efficient with sunlight as the light source. The optimum PFD
range of 250–280 ␮E/(m2 s) is comparatively low in contrast to con-
ditions of direct sunlight exposure (>1000 ␮E/(m2 s)). Most current
photobioreactor designs feature the concept of “light dilution”, i.e.
the culture is not exposed to the full sunlight intensity but the light
is distributed over a large reactor surface (Lehr and Posten, 2009).
With the optimum PFD being clearly below midday sunlight inten-
sities, a custom bioreactor design can provide the optimum PFD
for each production site. This requires knowledge of local sunlight
variation and weather conditions, but also the behavior of the algae
culture in fluctuating light conditions. While for short light–dark
cycles frequency-dependent effects for biomass growth could be
 F. Lehr et al. / Journal of Biotechnology 162 (2012) 89–96
Fig. 8. H2 production parameters against light intensity; A maximum specific H2
production rates; B: total H2 production; C: average H2 production rate; D: produc-
tivity of H2 production.
detected (Rosello Sastre et al., 2007), long day-night cycles together
with weather-dependent variation of sunlight intensity may have
a more severe impact on both biomass growth and hydrogen pro-
duction. Since H2 production is light dependent in both pathways
with the given strong variation in productivity, the sustainability of
a large scale outdoor production is dependent on a good adaption
of the production system to local irradiance conditions.
4. Conclusion
In this work we could demonstrate that it is possible to replace
the solid–liquid separation step between the two process phases
95
by a self-inducting system via a precise dosage of sulfur in a single
batch system. We present light dependent biomass growth kinet-
ics based on long-term 2 L batch cultivations similar to conditions
expected for larger production volumes. Kinetic data on a series of
hydrogen production experiments indicate a similar PFD optimum
for both growth and H2 evolution between 250 and 280 ␮E/(m2 s).
The intracellular starch content is being depleted during hydrogen
production, but not linearly during the process. To ensure a high
efficiency in light use, low PFD values are preferable. Due to the
comparatively low optimum PFD these can be provided by appro-
priate reactor design.
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