International Journal of Hydrogen Energy 53 (2024) 760–769

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International Journal of Hydrogen Energy

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Photoautotrophic and sustained H2 production by the pgr5 mutant of

Chlamydomonas reinhardtii in simulated daily light conditions
Valéria Nagy a, Zsombor Dabosi a, b, Soujanya Kuntam a, Krisztián Csankó c, László Kovács a,
Szilvia Z. Tóth a, *
a
Institute of Plant Biology, HUN-REN Biological Research Centre, Szeged, Temesvári krt. 62, H-6726, Szeged, Hungary
b
Institute of Biology, Faculty of Science and Informatics, University of Szeged, Aradi Vértanúk Tere 1, H-6720, Szeged, Hungary
c
CS-Smartlab Devices Ltd., Kübekháza, Fő Utca 86, H-6755, Hungary

A R T I C L E I N F O A B S T R A C T

Handling Editor: Ramazan Solmaz Green algae, such as Chlamydomonas reinhardtii, can produce H2 efficiently using their hydrogenases, but these
enzymes are O2-sensitive and compete with CO2-fixation for electrons. To overcome these limitations, we pre­
Keywords: viously developed an anaerobic, carbon-limited protocol that keeps the Calvin-Benson cycle inactive, and the
Chlamydomonas reinhardtii evolved O2 is scavenged by an absorbent. The PROTON-GRADIENT REGULATION5 (PGR5)-deficient mutant
Biohydrogen
performs better than the wild type in this system, as it produces at least 2.5-fold more H2 and maintains its
Light stress
photosynthetic apparatus and hydrogenase activity even at the intensity of sunlight. However, as the pgr5 mutant
Photosynthesis
PGR5 is known to be sensitive to fluctuating light conditions, it is of paramount interest to determine how it reacts to
Thin cell layer cultures changes in light intensity during H2 production. Therefore, we developed an automated system to monitor H2
production in thin layer cultures under simulated daily light conditions. We found that the pgr5 mutant out­
performed the wild type strain by 100 % when light intensity was varied stepwise between 0 and 1000 μmol
photons m− 2s− 1 during the day. Photosynthetic subunits, including PsbA, PSBO, CP47, PetB and PsaA, were fully
preserved in the pgr5 mutant, and approximately 29 % of its original hydrogenase activity was sustained after 85
h of H2 production in simulated daily light conditions. Hence, the pgr5 mutant is a promising candidate for H2
production under the adverse light conditions that algae may encounter in bioindustry settings.

1. Introduction HydA2), located at the acceptor side of photosystem I (PSI) [4,6,7].

Among the potential renewable energy sources that could ensure
global energy needs and compete with conventional fossil fuel utiliza­
tion, hydrogen (H2) stands out as a promising solution. It has a high
energy content and its combustion releases only water vapor. The pro­
duction of H2 by steam reformation of natural gas requires specific
conditions at high energy inputs and has a large carbon footprint [1]. On
the other hand, the production of biological H2 by microorganisms takes
place at ambient temperature and pressure (reviewed by Refs. [2–5]).
Employing algal cells as whole-cell biocatalysts is a promising strategy
to produce biofuels and high-value products, owing to their easy and
rapid cultivation and remarkable CO2 mitigation capacity. Several
photosynthetic organisms are capable of directly converting light energy
into H2. Green algae produce H2 with a high theoretical energy con­
version efficiency by their [FeFe]-type hydrogenases (HydA1 and
In nature, the primary role of H2 production is to act as a safety valve
protecting the photosynthetic electron transport chain from over-
reduction that may typically occur upon dark-to-light transitions [8].
Once the Calvin-Benson cycle is fully operative, HydA is outcompeted by
the electron acceptor ferredoxin NADP+ reductase (FNR) and electrons
are directed towards CO2 assimilation [9,10]. In addition, O2-dependent
alternative electron transport processes and cyclic electron transfer
around PSI may also compete with HydA for electrons [8,11]. Finally,
molecular oxygen (O2), generated by photosystem II (PSII), inactivates
HydA and H2 production is ceased typically after a few minutes of
continuous illumination [12,13].
H2 production can be prolonged by sulfur deprivation ([14],
reviewed by Ref. [15]), which leads to substantial inhibition of the
oxygen-evolving complex, PSII reaction center damage and finally
degradation of the entire photosynthetic apparatus [16]. Inhibition of

* Corresponding author.
E-mail addresses: nagy.valeria@brc.hu (V. Nagy), dabosi.zsombor@brc.hu (Z. Dabosi), soujanya.kuntam@brc.hu (S. Kuntam), csankok@cs-sld.com (K. Csankó),
kovacs.laszlo@brc.hu (L. Kovács), toth.szilviazita@brc.hu (S.Z. Tóth).

https://doi.org/10.1016/j.ijhydene.2023.12.126
Received 21 September 2023; Received in revised form 21 November 2023; Accepted 13 December 2023
Available online 19 December 2023
0360-3199/© 2023 The Authors. Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
V. Nagy et al.
oxygen-evolving activity results in anoxia, expression of HydA, and H2
production lasting for a few days. Since sulfur deprivation results in
severe cellular damage, and the efficacy of H2 photoproduction is
severely diminished in strong light [17,18], this protocol is unlikely to
be employable in the bioindustry. As the ultimate goal is to exploit algal
H2 production in natural light conditions, it would be highly advanta­
geous to develop robust H2 production systems able to perform in
varying light conditions, from twilight to the intensity of sunlight.
A possible way to sustain H2 production is via limiting the Calvin-
Benson cycle activity, as being a major competitor with HydA for elec­
trons [19]. Employing pulse illumination may prevent activation of the
Calvin-Benson cycle and a build-up of inhibitory O2 concentrations [20,
21]. Using short (1 s) light pulses with dim background light, H2 pro­
duction may last for several days [21]. On the other hand, H2 production
can be initiated by a few hours of anaerobic induction, and the activa­
tion of Calvin-Benson cycle prevented by substrate limitation (i.e., by
the absence of CO2 and acetate [10]).
Low O2 concentration level, essential for maintaining hydrogenase
activity, can be ensured in various ways. In the sulfur deprivation pro­
tocol, this is achieved by diminished PSII activity [14,16,22].
Alga-bacteria consortia are characterized by low O2 levels, due to bac­
terial respiration, enabling H2 production lasting for days [23]. In case
of the anaerobiosis-induced, carbon-limited H2 production, an iron-salt
based O2 absorbent is employed to reduce the O2 level in the headspace
of the culture [10], thereby maintaining HydA activity. Alternatively, a
mixture of ascorbate and copper (Oxysorb) could be added to the alga
culture to reduce O2 [24]. Additionally, engineering O2-tolerant hy­
drogenases could also contribute to sustained H2 production in the
future [25–27].
Various C. reinhardtii mutant strains have been identified with
enhanced H2 production. One of the most promising H2 producing
strains is the PROTON-GRADIENT REGULATION5 (PGR5)-deficient
mutant [28]. PGR5 is involved in the partitioning of linear and cyclic
electron transport, and in its absence PSI cyclic electron flow is impaired
[29]. The pgr5 mutant has been successfully employed in
sulfur-deprivation experiments [30], anaerobiosis-induced, carbon-­
limited conditions [31], in TAP medium with Oxysorb [24], and in
anaerobiosis-induced cultures supplied with acetate [32].
Under all these conditions, the pgr5 mutant produced two to tenfold
more H2 than the control strain. The outstanding performance of the
pgr5 mutant may be due to its following properties: 1) impaired Q-cycle
and diminished PSI cyclic electron flow, thereby 2) compromised lumen
acidification and photosynthetic control, and 3) lower ATP production
in the chloroplast. As an overall result of these properties, the Calvin-
Benson cycle is activated less, thus competing less for the electrons
with HydA. In order to compensate for the lack of ATP, mitochondrial
respiration is increased and thereby a relatively low O2 level is estab­
lished in the culture, diminishing the risk of HydA inactivation by O2
[32,33]. In addition, the association of FNR with the thylakoid mem­
brane is impaired in the absence of PGR5, possibly enhancing the rate of
electron flow towards HydA [29,34].
Under aerobic conditions, the pgr5 mutant is very sensitive to high
light, especially when grown photoautotrophically. This may be due to
the insufficient induction of photosynthetic control mechanisms and
photoprotective non-photochemical quenching (NPQ) [28], and
enhanced electron donation to O2 [34]. Moreover, the H2 production
performance of the pgr5 mutant is strongly compromised at medium
light intensity (approx. 200 μmol photons m− 2s− 1) upon
sulfur-deprivation induced H2 production [30].
On the other hand, we have shown that the pgr5 mutant can with­
stand the intensity of sunlight (approx. 1000 μmol photons m− 2s− 1) for
several days in an anaerobiosis-induced, carbon-limited H2 production
protocol without significant damage to its photosynthetic apparatus
[31]. These data highlighted the potential of using the pgr5 mutant in
future bioindustry applications. Nevertheless, it is unclear whether this
strain can also withstand varying light conditions during H2 production,
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International Journal of Hydrogen Energy 53 (2024) 760–769
including very high light intensities. The effect of varying light is rarely
considered in algal H2 production studies; in a previous study it was
demonstrated that C. reinhardtii subjected to sulfur-deprivation induced
H2 production protocol, cannot withstand natural light conditions and
the cultures rapidly decay [17]. On the other hand, resistance to varying
light conditions, including strong light exposure is particularly relevant
for future outdoor biohydrogen fields.
Here we have tested the H2 production capacity of the pgr5 mutant
under so-called “simulated daily light conditions”, in which the light
intensity was changed every 2 h from 0 to 1000 μmol photons m− 2s− 1
during the day. An automated thin cell layer H2 production system,
capable of continuously monitoring production yield, was built partic­
ularly for this purpose. We found that during the anaerobiosis-induced,
carbon-limited H2 production, the pgr5 mutant responds well to varying
light conditions, and its hydrogenase and photosynthetic apparatus
remain functional with little damage occurring in comparison with the
CC-124 wild-type strain.
2. Materials and Methods
2.1. Algal growth conditions and H2 production
Wild type strain of C. reinhardtii, CC-124 (137c), was obtained from
the Chlamydomonas Resource Center (University of Minnesota, USA).
The pgr5 DNA insertional mutant [28] and the T222 (137c) strain were
provided by Prof. Michael Hippler (University of Münster, Germany).
The strains were grown initially at 23 ◦ C in 500 ml Erlenmeyer flasks
containing 300 ml Tris-acetate-phosphate (TAP) medium [35], shaken
at 120 rpm in under continuous illumination of 80–90 μmol photons
m− 2 s− 1, provided by T8 cool white fluorescent light tubes (Sylvania
luxline plus).
After three days of cultivation, alga cells were transferred to high-salt
(HS) medium (http://www.chlamycollection.org/methods/media-reci
pes/), and the Chl content was set to 50 μg Chl (a+b)/ml, based on
the method by Porra et al. [36].
Thin layer cell cultures were established in modified 1-L Pyrex®
Roux culture bottles with a culture volume of 100 ml, placed horizon­
tally on a shaker. This created a light path of approximately 5 mm and
provided a surface-to-volume ratio of approximately 240 cm2/1000 cm3
(see also in Ref. [31]).
An iron-salt-based, non-cytotoxic O2 absorbent (O2Zero-50 cc loose;
Global Reach Ltd, London, UK) was employed to minimize the O2 con­
centration in the headspace. For this, a holder containing 20 g of O2
absorbent was introduced into Roux culture bottles. The O2 level was
below 0.05 % during the entire experiment, as verified by gas chroma­
tography measurements [31].
Dark anaerobic incubation was performed by flushing the headspace
with N2 gas for 20 min and keeping the cultures in the dark for 4 h. The
cultures were shaken at 120 rpm, and kept at 25 ◦ C for 120 h. The
cultures were 1) illuminated with continuous light of 550 μmol photons
m− 2 s− 1, or 2) subjected to dark-light cycles (8 h darkness/16 h at 550
μmol photons m− 2 s− 1), or 3) subjected to simulated daily light condi­
tions, consisting of 8 h darkness, followed by 2 h of illumination at 100,
250, 500, 750, 1000, 750, 500 and 250 μmol photons m− 2 s− 1. The
protocol was set in a way that the total amount of light energy provided
to the culture was the same as during the dark-light cycles with 550 μmol
photons m− 2s− 1. Light was provided by a warm white LED panel.
2.2. Construction of an automated thin cell layer H2 production system
An automated thin cell layer H2 production device, with pre-
programmed sampling, pressure control, temperature monitoring and
gas rinsing was developed jointly with CS-Smartlab Devices Ltd.
(Szeged).
Thin cell layer photobioreactors (TCL-PBRs) were prepared as
described above. The PBRs were connected to a gas chromatography
V. Nagy et al. International Journal of Hydrogen Energy 53 (2024) 760–769

system through a custom-made control unit. The system enables moni­
toring up to three TCL-PBRs simultaneously (Fig. 1). An inert gas
purging system flushes the headspace of the PBRs with N2 gas. Over­
pressure is prevented by a blow-off device. A sampling manifold is used
to deliver the sample from the headspace into the sample inlet pipe and
then into the loop, from where the sample is injected into the gas
chromatograph (Hewlett Packard 5890) by a 6/2-way sampling valve.
The pressure in the PBR is registered by a pressure gauge. Before sample
injection the sample loop and inlet tube is flushed by N2 via a flushing
valve and pressure release valve. The system also registers the ambient
temperature and pressure for each vessel. The device is controlled by a
custom-made software.
The sample taken from the PBR headspace is introduced into the 100
μl sample loop of the gas chromatograph through a stainless steel
capillary (0.4 mm inner diameter) passively through the valves and the
sample inlet pipes under the influence of the pressure prevailing in the
headspace. The necessary pressure of the headspace was provided by
pressurization to 1300 mbar with compressed N2 prior to each sampling.
The gas compounds of the headspace were separated on an HP-PLOT
Molesieve column (30 m*0.53 mm*0.25 μm) set to 40 ◦ C and connected
to a thermal conductivity detector set at 160 ◦ C. The carrier gas was
argon, linear velocity 115 cm/s.
The headspace was sampled every 2 h in the light periods and PBRs
were flushed with N2 gas for 20 min once a day as indicated in the
figures.
2.3. Chl a fluorescence measurements
Chl a fluorescence measurements were carried out as described
earlier [16]. To measure the photosynthetic activity of C. reinhardtii, we
first dark-adapted the cultures for about 15 min. Then, we transferred
60 μl of cell suspension (50 μg Chl (a+b)/ml) to a Whatman glass
microfibre filter (GF/B). We placed the filter in a leafclip and used a
HandyPEA instrument (Hansatech Instruments Ltd, UK) to record the chl
a fluorescence signals.
2.4. Immunoblot analysis
At each sampling point, 2 ml of culture was collected, spun-down to
remove the supernatant and frozen in liquid nitrogen. The samples were
then solubilized as described in Nagy et al. [37]. For each sample, ex­
tracts equivalent to 1 million cells were mixed with 6 × Laemmli buffer
(375 mM Tris/HCl [pH 6.8], 60 % [v/v] glycerin, 12.6 % [w/v] sodium
dodecyl sulfate, 600 mM dithiothreitol, 0.09 % [w/v] bromophenol
blue) and incubated at 75 ◦ C for 10 min before loading. Protein sepa­
ration and immunoblot analyses were carried out as described in Pod­
maniczki et al. [38]. Specific polyclonal antibodies (produced in rabbit)
against PsbA (N-terminal) (1:2000, AS11-1786), PSBO (1:1000,
AS06-142-33), PetB (1:10000, AS18-4169), CP47 (1:2500, AS04-038),
PsaA (1:2000, AS06-172) and HydA (1:2000, AS09-514) were pur­
chased from Agrisera AB.
2.5. In vitro hydrogenase activity assay
In vitro hydrogenase activity was measured after 3 h of dark anaer­
obic incubation and after 85 h of H2 production in simulated daily light
conditions. The assay was carried out at 37 ◦ C, in darkness in 16-ml
serum bottles. The reaction mixture consisted of 1.9 ml of 100 mM po­
tassium phosphate buffer (pH 6.8), 560 μl of deionized water, 100 μl of
10 % Triton X-100, 40 μl of 1 M methylviologen, 400 μl of anaerobic 1 M
sodium dithionite and 400 μl of algal culture (similarly to Ref. [39]). The
H2 concentration in the headspace was measured by gas chromatog­
raphy every 20 min and fitted with linear regression. The in vitro hy­
drogenase activity was provided as μmole H2/mg chl (a+b) h− 1.
2.6. Statistical analysis
The presented data are based on at least three independent experi­
ments. The exact number of the biological repetitions are indicated in
the figure captions. When applicable, averages and standard errors
(±SE) were calculated. The significance of the mean differences between
the pgr5 mutant and the wild type strain were analyzed by Student’s t-
test or by two-way mixed ANOVA with Tukey post-hoc test at p < 0.1
level using OriginPro 9.5 software.
3. Results
3.1. Construction of an automated thin cell layer H2 production system
Thin cell layer photobioreactors (TCL-PBR) are characterized by low
culture thickness (<10 mm), high surface-to-volume ratios, and high cell
densities (up to 1000 μg Chl (a+b)/ml culture, [40,41]). With turbulent
mixing, high cell densities in TCL-PBR also enable short light/dark cy­
cles (e.g. in the range of 500 ms− 1; [40,42]), preventing over-reduction
of the photosynthetic electron transport [43,44], and the activation of
the Calvin-Benson cycle, which competes with H2 production [9,10,20].
Thin cell layer cultures with a large surface to volume ratio have
improved light utilization efficiency and have been successfully

Fig. 1. Automated thin cell layer H2 production device, with pre-programmed sampling, pressure control, temperature monitoring and gas rinsing. A) Thin layer
C. reinhardtii cell cultures (100 ml) in modified 1-L Pyrex® Roux culture bottles (TCL-PBRs), placed horizontally on a shaker. An iron-salt-based, non-cytotoxic O2
absorbent is placed into the culture bottles to diminish the O2 concentration below 0.05 % in the headspace. The cultures are illuminated from top by a warm-white
LED panel. The TCL-PBRs are connected to a gas chromatography system through a control unit. B) Technical design of the automated thin cell layer H2 production
system. The system is designed to monitor up to three TCL-PBRs (1) simultaneously. The inert gas purging system (11) flushes the air space of the bioreactor with N2
gas (2). The overpressure is prevented by the blow-off device. The manifold (4) is used to deliver the sample from the headspace into the sample inlet pipe and then
into the loop (5), from where the sample is injected into the gas chromatograph (7) by the 6/2-way sampling valve (8). The pressure in the TCL-PBR is registered by
the pressure gauge (3). Before sample injection, the sample loop and the inlet tube are flushed with N2 via a flushing valve (10) and pressure release valve (9). For the
gas chromatography measurements, argon is used as carrier gas (6).

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employed in photoautotrophic conditions to achieve high biomass
accumulation [45,46]. On the other hand, we have shown that the thin
layer approach has a high potential in H2 production as well, due
improved O2 removal from the liquid phase that can be effectively
captured by an O2 absorbent in the gas phase. We have shown that
relative to cultures in serum bottles, thin layer cultures may produce
approx. 3-fold more H2 [31].
The primary goal of this study is to assess the effect of varying light
conditions on the pgr5 mutant’s H2 production. To this end, we have
built an automated thin cell layer H2 production system. The system is
capable of sampling the gas phases of the TCL-PBRs in a pre-
programmed way and inject the samples into a gas chromatograph.
The TCL-PBRs are of 1 L volume, and 100 ml alga culture was placed
inside. Low O2 level (below 0.05 %) was ensured by placing iron-salt
based O2 absorbent in the headspace. The TCL-PBRs are illuminated
with warm-white LEDs from the top (Fig. 1, see Materials and Methods
for more details).
3.2. Photoautotrophic H2 production capacity of the pgr5 mutant in
various light conditions
H2 production was initiated by incubating the cultures in HS medium
(50 μg Chl (a+b)/ml) in dark anaerobic conditions for 4 h, achieved by
N2 flushing. The dark anaerobic conditions enable high expression of
HydA, and when the cultures are placed in continuous light, H2 pro­
duction starts immediately, without any delay [10]. This H2 production
system operates without CO2 or organic carbon source, therefore the
Calvin-Benson cycle remains inactive in the light [10]. Due to the
absence of organic carbon source, it is fully photoautotrophic and most
electrons supporting H2 production originate from water, split by the
oxygen-evolving complex of PSII [10]. Moreover, this protocol enables
the usage of high light intensities (in the range of 1000 μmol photons
m− 2s− 1) and continuous H2 production for at least six days, at a high
yield (approx. 1.2 ml H2/ml culture was produced in a TCL-PBR by the
pgr5 mutant), and without significant damage to the photosynthetic
apparatus [31]. The advantage of omitting acetate as an organic carbon
source from the culture media is multiple: 1) acetate may indirectly feed
the Calvin-Benson cycle, being in competition with H2 production 2)
reducing the costs of H2 production, and 3) reducing the risk of
contamination in bioindustry applications.
Following the dark incubation in N2 atmosphere, continuous illu­
mination was started using white LEDs at an intensity of 550 μmol
photons m− 2s− 1 (Fig. 2A). The gas sampling was performed every 2 h
with the aid of our automated thin cell layer H2 production system. The
CC-124 wild type strain produced approximately 90 μl H2/ml culture
within the first 24 h, whereas the pgr5 mutant produced almost twice as
much. In the CC-124 strain and pgr5 mutant, H2 production increased
almost linearly during the first 24 h at a rate of approximately 3.65 and
6.75 μl H2/ml culture/h, respectively (Fig. 2A). After 24 h, the gas phase
was rinsed with N2, in order to prevent the accumulation of H2 over 5 %,
which may diminish the H2 production efficiency of HydA [47]. On the
second day, both strains produced similar amounts of H2 as on the first
day. On the consecutive days, the H2 production yield diminished
gradually. In total, approx. 291 μl H2/ml culture was produced by the
CC-124 strain in 120 h, and approx. 636 μl H2/ml by the pgr5 mutant
(Fig. 2B).
To confirm the high performance of the thin layer system, we tested
cultures in serum bottles. We found that the CC-124 strain produced
approximately 60 % less H2 in serum bottles than in thin layer cultures
(Suppl. Fig. 1A), in line with our previous results [31]. In this set of
experiments, we also included the direct parental strain of the pgr5
mutant, the T222 line [28,29,32]. Its H2 production yield was slightly
lower than that of the CC-124 strain (Suppl. Fig. 1A). The pgr5 mutant
produced approximately 50 % more H2 than CC-124, meaning that the
enhancement was milder in the serum bottles than in the thin layer
system, where it reached over 100 % increase (c.f. Fig. 2B and Suppl.
Fig. 1A).
Next, the effects of dark-light cycles were studied in the automated
thin cell layer H2 production system. For this, 16 h of continuous illu­
mination at 550 μmol photon m− 2s− 1 was provided followed by 8 h of
darkness (Fig. 3A). At the beginning of the dark period, the headspace
was flushed with N2. As a consequence of diminishing the duration of
illumination by 30 % in comparison with continuous illumination
(Fig. 2), the amount of obtained H2 was reduced by approx. 39 % in the
CC-124 strain (to approx. 176 μl H2/ml culture), and by 28 % in the pgr5
mutant (to approx. 456 μl H2/ml culture, Fig. 3B). Thus, the difference
between the CC-124 strain and the pgr5 mutant became larger.
In a third setting, the effects of varying light conditions, simulating
the changes in light intensities during a day, were assessed. Cultures
were illuminated consecutively at 100, 250, 500, 750 and 1000 μmol
photons m− 2s− 1 light intensities, then in a reverse order, for 2 h at each
intensity (Fig. 4A). These light intensities and durations were set in a
way that the total amount of light energy provided to the culture was the
same as during the dark-light cycles with 550 μmol photons m− 2s− 1 (in

Fig. 2. Anaerobiosis-induced, carbon-limited H2 production by the CC-124 wild type strain and the PSI cyclic electron transport mutant pgr5 in automated TCL-PBR
at continuous white light of 550 μmol photons m− 2s− 1. A) H2 production kinetics B) Total amount of H2 produced in 120 h. The initial Chl concentration was 50 μg
Chl (a+b)/ml. H2 concentration in the headspace was detected every 2 h. The headspace was flushed with N2 at the end of each 24 h-cycle, indicated by arrows in
panel A. The presented data originate from five independent experiments. The * in panel B designates significantly different means, as determined by Student t-test, p
< 0.05.

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Fig. 3. Anaerobiosis-induced, carbon-limited H2 production by the CC-124 strain and the pgr5 mutant in automated TCL-PBR in dark-light cycles of 8 h darkness and
16 h of illumination at 550 μmol photons m− 2s− 1. A) H2 production kinetics B) Total amount of H2 produced in 120 h. The initial Chl concentration was 50 μg Chl
(a+b)/ml. H2 concentration in the headspace was detected every 2 h. The headspace was flushed with N2 at the end of each 24 h-cycle, indicated by arrows in panel
A. The presented data originate from five independent experiments. The * in panel B designates significantly different means, as determined by Student t-test, p
< 0.05.

Fig. 4. Anaerobiosis-induced, carbon-limited H2 production by the CC-124 strain and the pgr5 mutant in automated TCL-PBR in simulated daily light conditions. The
cultures were exposed to 8 h of darkness followed by 2 h of illumination at 100, 250, 500, 750, 1000, 750, 500 and 250 μmol photons m− 2 s− 1. A) H2 production
kinetics B) Total amount of H2 produced in 120 h. The initial Chl concentration was 50 μg Chl (a+b)/ml. H2 concentration in the headspace was detected every 2 h.
The headspace was flushed with N2 at the end of each 24 h-cycle, indicated by arrows in panel A. The red arrow indicates the sampling time for Figs. 6 and 7. The
presented data originate from five independent experiments. The * in panel B designates significantly different means, as determined by Student t-test, p < 0.05. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3). respectively (Fig. 5B). The H2 production yield slowly decreased from
In simulated daily light conditions, the amount of H2 produced was this level during the day, regardless of the light intensity (Fig. 5B).
about 40 % higher in the CC-124 strain (approx. 248 μl H2/ml culture in These results demonstrate that varying illumination conditions,
total), and a slight (approx. 10 %) improvement was observed in case of involving ten-fold intensity changes and very high light intensities,
the pgr5 mutant (502 μl H2/ml culture in total; compare Fig. 3B and 4B). positively affect photoautotrophic H2 production.
We note that the direct parental strain of the pgr5 mutant, the T222
strain performed similarly to the CC-124 strain (Suppl. Fig. 1B).
3.3. Changes in the photosynthetic apparatus and HydA in simulated
The kinetics of H2 gas production in a 2-h time window are shown in
daily light conditions
Fig. 5. We found that in dark-light cycles of 8 h darkness and 16 h of
illumination at 550 μmol photons m− 2s− 1, the amount of H2 produced
Next, we investigated how the photosynthetic apparatus responds to
was rather stable throughout the day in the CC-124 strain and the pgr5
the simulated daily light conditions employed during H2 production
mutant. On day 2, when the maximum H2 production is reached,
(Fig. 4). The investigations were carried out after 85 h of H2 production,
approximately 7 and 17 μl H2/ml culture was produced in 2 h by the CC-
with the sampling performed in the early light period, when H2 pro­
124 strain and the pgr5 mutant, respectively (Fig. 5A). In simulated daily
duction is substantial (Fig. 4A and 5B).
light conditions, the maximum H2 production was observed at the
First, we employed the fast Chl a fluorescence (OJIP) transient,
beginning of each illumination period, at 100 and 250 μmol photons
which is a sensitive and widely used method to detect alterations in the
m− 2s− 1 (Fig. 5B). On day 2, approximately 8 and 21H2/ml culture was
function of the photosynthetic electron transport chain (e.g. Ref. [48]).
produced in the first 2 h in the CC-124 strain and the pgr5 mutant,
Under regular growth conditions, the Chl a fluorescence transients of the

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Fig. 5. H2 production in dark-light cycles (A) and in simulated daily light conditions (B) by the CC-124 strain and the pgr5 mutant in automated TCL-PBR. The data
are based on the experiments presented in Figs. 3 and 4. The amounts of H2 produced in a 2-h window is presented as the function of the actual light intensity. The
duration of the dark period (indicated by 0) was 8 h. The presented data originate from five independent experiments.
CC-124 strain and the pgr5 mutant were very similar and typical of
healthy cultures (Fig. 6A). After 85 h of H2 production, the amplitude of
the Chl a fluorescence diminished and its kinetics changed remarkably
in both strains. However, the diminishment of the maximum fluores­
cence (FM) value was smaller in the pgr5 mutant than in the CC-124
strain (Fig. 6B). The FV/FM value, an indicator of photosynthetic per­
formance [49], decreased from approximately 0.77 to 0.3 in the CC-124
strain and 0.25 in the pgr5 mutant (Fig. 6C).
When the cultures were transferred to normal growth conditions for
3 h of recovery (i.e., to regular Erlenmeyer bottles, moderate light in­
tensity, atmospheric conditions), the Chl a fluorescence kinetics have
partially recovered and the FV/FM also increased to approximately 0.55
in both strains (Fig. 6A,C). These data indicate that the changes in the
fast Chl a fluorescence transient and the diminishment of FV/FM were
mainly caused by a downregulation of the photosynthetic electron
transport via photosynthetic control and various non-photochemical
quenching mechanisms [50–52], instead of irreversible damage. We
have also observed that the chl (a+b) content significantly diminished in
the CC-124 strain during 85 h of H2 production (by about 18 %),
whereas it remained at the same level in the pgr5 mutant (Fig. 6D).
A small aliquot (0.5 ml) of the culture was placed in TAP medium, in
regular growth conditions, in order to assess the viability of the cultures
that had been producing H2 for 85 h. The growth of both genotypes was
vigorous and in three days, the CC-124 strain and the pgr5 mutant
reached a chl (a+b) content of 32 and 27 μg Chl (a+b)/ml, respectively
(Fig. 6E).
Next, immunoblot analysis was used to assess the levels of photo­
synthetic subunits. In the CC-124 strain, the relative amounts of the PSII
subunits, PsbA, PSBO, and CP47 decreased significantly during 85 h of
H2 production, by approximately 25, 77, and 43 %, respectively
(Fig. 7A). The level of the cytb6f subunit, PetB showed a slight decrease,
and the PSI reaction centre subunit, PsaA diminished by approximately
20 % as well (Fig. 7A, representative immunoblots are shown in Fig. 7B).
In contrast to the CC-124 strain, the PSBO and CP47 photosynthetic
subunits were almost completely preserved in the pgr5 mutant (Fig. 7A).
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International Journal of Hydrogen Energy 53 (2024) 760–769
The diminishment in the levels of PsbA, PetB and PsaA was similar as in
the CC-124 strain, i.e., approximately 20 % during 85 h of H2 production
(Fig. 7A).
During a 3-h recovery period in aerobic conditions, the levels of most
photosynthetic subunits did not change substantially (Fig. 7), supporting
that the recovery in photosynthetic activity (as assessed by the FV/FM
value, Fig. 6C) was not due to a resynthesis of photosynthetic subunits,
but to the release of the downregulation of photosynthetic electron
transport.
There was a strong, approximately 60 % diminishment in the HydA
level of the CC-124 strain during 85 h of H2 production (Fig. 7A). On the
other hand, the level of HydA in the pgr5 mutant decreased only by 24 %
(Fig. 7A). The in vitro activity of HydA was also assessed, in the presence
of reduced methylviologen as an electron donor. Following the dark
anaerobic incubation, the in vitro hydrogenase activity was approx. 350
μmole H2/mg chl (a+b) h− 1 in both strains. After 85 h of H2 production,
approximately 17 % of the initial HydA activity was found in the CC-124
strain, whereas 29 % of the original activity could be detected in the pgr5
mutant (Fig. 7C).
4. Discussion
We have designed an automated thin cell layer H2 production system
that may serve as a model for future bio-industial TCL-PBRs for H2
production. It enables regular N2 flushing, pressure adjustment and
automated gas sampling to monitor the yield of H2 production (Fig. 1).
In bioindustry applications, these three elements will be also required
for establishing anaerobic conditions, and collecting the produced H2 on
a regular basis in order to prevent feedback inhibition on HydA [47].
The anoxic environment (i.e., O2 level below 0.05 %) was maintained
here by iron-salt type O2 absorbent in the gas phase. We believe that
there might be other, more economical solutions to keep the O2 level
low, a subject that needs further investigation and optimization for
bioindustry settings.
Our automated thin cell layer H2 production system enabled testing
V. Nagy et al. International Journal of Hydrogen Energy 53 (2024) 760–769

Fig. 6. Characterization of the photosynthetic apparatus of the CC-124 strain and the pgr5 mutant during anaerobiosis-induced, carbon-limited H2 production in
simulated daily light conditions. The measurements were performed at 0 h (control), after 85 h of H2 production, and after 3 h of recovery under standard growth
(oxic) conditions. A) Fast Chl a fluorescence (OJIP) transients; the typical steps (O, J, I, P) are indicated. B) FM values C) FV/FM values derived from the OJIP
transient. D) Chl (a+b) content. E) Photos of the cultures after 85 h of H2 production (diluted by approx. 100-fold), and after an additional three days of growth in
TAP medium and regular conditions. The presented data originate from three independent experiments. The significance of differences between the means was
determined by ANOVA with Tukey post-hoc test. The means with different letters are significantly different (P < 0.1).

and challenging the pgr5 mutant, known for its light-sensitivity in reg­
ular aerobic conditions [28,34], and outstanding H2 production capac­
ities [30,32]. We could confirm that it performs well in
anaerobiosis-induced carbon-limited conditions [31], i.e., it produced
at least two-fold more H2 than the CC-124 strain (Fig. 2).
Considering that it is likely that alga cultures will be subjected to
light-dark cycles in future bioindustrial applications, it was interesting
to determine whether the amount of H2 decreases proportionally with
illumination time. We found that the extent of diminishment was
slightly less in the pgr5 mutant than in the CC-124 strain, due to a more
sustained H2 production yield towards the end of the experiment.
Interestingly, the simulated daily light conditions (between 0 and 1000
μmol photons m− 2s− 1) positively affected the yield, i.e., it was increased
in both genotypes in comparison with the dark-light cycles with the
same average light intensity (Figs. 3 and 4).
The photosynthetic apparatus of the CC-124 strain suffered a more
substantial damage than the pgr5 strain (Fig. 7). Interestingly, the PSII
subunits CP47 and PSBO were the most severely affected, whereas PsbA,
PetB and PsaA diminished by approximately 20 % after 85 h of H2
production. The level of HydA in the CC-124 strain strongly decreased.
On the other hand, the pgr5 mutant was much more robust: all the
photosynthetic subunits and HydA were preserved at a level of 75–100
%. The hydrogenase activity of the pgr5 mutant was also better main­
tained, i.e., 29 % of the original, in vitro activity was preserved, whereas
approximately 17 % of the initial activity was found in the CC-124 strain
after 85 h of H2 production in simulated daily light conditions.
The reason behind the high performance of the pgr5 mutant under
anaerobiosis induced carbon-limited H2 producing conditions may be
due to several factors. First of all, the compromised PSI cyclic electron
transport may result in diminished proton motive force thereby photo­
synthetic control [33], which would otherwise downregulate the
photosynthetic electron transport feeding H2 production. We have
shown previously, that photosynthetic control is a major limitation
regarding H2 production [10], which is at least partially relieved in the
pgr5 mutant.
The compromised PSI cyclic electron transport and proton motive
force in the pgr5 mutant result in a less activated Calvin-Benson cycle,
which contributes to the high H2 production yield of this mutant [32].
This factor, however, is of less importance in the anaerobiosis-induced
carbon-limited H2 production protocol, since the inactive state of the
Calvin-Benson cycle is ensured by the absence of CO2 and other carbon
sources.
The increased respiration of the pgr5 mutant ensures a low O2 level
within the cell, which may help to preserve HydA activity and to miti­
gate oxidative stress, occurring in sulfur-deprivation induced H2 pro­
duction [16,30], and probably in the so-called ambient protocol as well,
induced by anaerobiosis in TAP [32]. In these protocols, an approx.
10-fold difference was observed between the wild type and the pgr5
mutant. In the anaerobiosis-induced carbon-limited H2 production
protocol, a very low O2 level (below 0.05 %) is ensured by placing O2
absorbent in the headspace. As an alternative approach, low O2 con­
centration was achieved by adding Oxysorb to the culture [24]. Upon
the application of O2-absorbing materials, the difference in H2 produc­
tion between the wild type and the pgr5 mutant is less, i.e., 2- to 3-fold,

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Fig. 7. Assessment of the levels of various photosynthetic subunits and HydA, and in vitro hydrogenase activity in the CC-124 wild type strain and the pgr5 mutant
during anaerobiosis-induced, carbon-limited H2 production in simulated daily light conditions. A) Densitometry analysis on PsbA, PSBO, CP47, PetB, PsaA and HydA,
originating from blots as presented in panel B. The data are based on four independent experiments. The significance of differences between means were determined
by ANOVA with Tukey post-hoc test. The means with different letters are significantly different (P < 0.1). B) Representative immunoblots of PsbA, PSBO, CP47, PetB,
PsaA and HydA. The 0 h samples represent the control taken after 4 h of anaerobic induction; the 25 %, 50 %, and 100 % samples were used for the approximate
quantitation of the proteins by densitometry. The 85 h samples were taken after 85 h of H2 production. The recovery samples (rec.) were collected after 3 h of
recovery in regular growth conditions. C) In vitro hydrogenase activity determined on a Chl content basis (in μmole H2/mg chl (a+b) h− 1) in the CC-124 strain and the
pgr5 mutant, after 3 h of anaerobic induction (control) and after 85 h of H2 production carried out in simulated daily light conditions. The data represent means ± SE
(in brackets) of four independent biological replicates (Student t-test, *p < 0.05). The remaining activities after 85 h of H2 production are also indicated (in %).

as the photosynthetic apparatus and HydA activity of the wild type
strains are better preserved.
Iron-salt O2 absorbents establish much lower O2 levels in the head­
space of the culture than those observed in the pgr5 mutant under any
condition (compare [10] with [30,32]. Still, it is conceivable that the O2
produced by PSII has inhibitory effect on HydA and that the presence of
a low amount of O2 in the chloroplast leads to oxidative stress, thereby a
partial degradation of photosynthetic complexes. As a result of the
enhanced respiration in the pgr5 mutant, the intracellular O2 level may
be further diminished, which may contribute to a lesser degree of HydA
inhibition and oxidative stress. On the other hand, it has been shown
that in the absence of PGR5 the association of FNR with the thylakoid
membrane is impaired that may lead to in improved electron transfer
toward HydA [29,34], thereby H2 production.
Upon dark-light conditions, the total H2 yield is diminished relative
to continuous illumination, because the electrons feeding H2 production
originate from photosynthesis. However, employing dark-light cycles
had an advantage in the pgr5 mutant: In continuous light, the amount of
daily H2 production strongly decreased towards the end of the experi­
ment, whereas in dark-light cycles, H2 production was more stable
(Figs. 3 and 4). The reason may be that upon the repetitive dark-light
cycles, HydA may partially regenerate in the dark.
Interestingly, varying the light intensity between 0 and 1000 μmol
photons m− 2s− 1 had a positive effect on H2 production, in both the CC-
124 strain and the pgr5 mutant. This may be explained by the physio­
logical role of HydA in releasing the overpressure on photosynthetic
electron transport. It is therefore likely that electrons are transferred to
HydA more readily when the light intensity is increased.
5. Conclusions
We have developed an automated system to continuously monitor H2

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production in thin layer cultures of green algae under various light re­
gimes. We have demonstrated that the pgr5 C. reinhardtii mutant, which
has impaired PSI cyclic electron transport, outperformed the wild type
strain by at least 100 % in continuous light, dark-light cycles, and
simulated daily light conditions, involving very high light intensities.
The pgr5 mutant also preserved its photosynthetic and hydrogenase
activities for several days, while the wild type strain showed signs of
photodamage. Therefore, the pgr5 mutant is a promising candidate for
H2 production in bioindustry settings, even under adverse conditions.
However, to achieve economic viability, more optimization of the cul­
ture conditions, and the O2 removal method is required. Additionally, it
is conceivable that further genetic engineering of the pgr5 mutant may
also contribute to increased H2 yield in the future.
Funding
This work was supported by the Lendület/Momentum Programme of
the Hungarian Academy of Sciences (LP2014/19), the National
Research, Development, and Innovation Office (K132600, and
FK135633, research grants to SZT and VN), and the Eötvös Loránd
Research Network (SA-109).
Data availability
Data sharing does not apply to this article as all newly created data is
already contained within this article and in its supplementary material.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors thank Prof. Dr. Michael Hippler (University of Münster,
Germany) for providing us with the pgr5 mutant.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijhydene.2023.12.126.
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