ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 921-933 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 1004-1017.
921
Relationship between the Photosystem II
Regulation Mechanisms and Hydrogen Production
in Chlamydomonas reinhardtii under Nitrogen
or Sulfur Deprivation
Vera I. Grechanik
1,a
*, Maksim A. Bol’shakov
1
, and Anatoly A. Tsygankov
1
1
Institute of Basic Biological Problems Russian Academy of Sciences –
Separate Division of Federal Research Center “Pushchino Scientific Center for Biological Research
of the Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia
a
e-mail: vi.semina@gmail.com
Received April 2, 2025
Revised May 19, 2025
Accepted May 21, 2025
Abstract— Some microalgae are capable of light-dependent hydrogen production after a period of anaerobic
adaptation, thus performing biophotolysis of water. The rate of hydrogen production the start of illumination
has the rate equal to the maximum rate of photosynthesis. However, this process is short-lived: oxygen pro-
duced during photosynthesis quickly inactivates the key enzyme of biophotolysis, hydrogenase, and inhibits its
expression. To date, approaches have been developed to achieve sustained hydrogen production by microal-
gae. The most studied are those based on transferring microalgae to nutrient-deficient conditions. However,
it is known that hydrogen production under nutrient deficiency is always accompanied by the decrease in
activity of photosystem II (PSII). Several mechanisms for suppression of PSII activity have been described in
the literature, and there is no consensus on which mechanism is the determining one. The aim of this work
was to test the hypothesis that realization of a particular mechanism of PSII suppression depends not only
on the type of stress but also on the growth conditions. For this purpose, the photoautotrophic culture of the
microalga Chlamydomonas reinhardtii was grown under nitrogen or sulfur deficiency under different light
regimes, and realization of the following mechanisms of PSII activity suppression was analyzed: over-reduction
of the plastoquinone pool (coupled with over-reduction of the entire photosynthetic electron transport chain),
decoupling of PSII (based on the kinetics of ascorbate accumulation and the JIP test) with water-oxidizing
complex, violaxanthin cycle, anaerobic stress associated with the creation of a reducing redox potential of the
culture suspension. It was found that the key mechanism determining hydrogen production is the over-reduc-
tion of the plastoquinone pool. Other mechanisms are also realized under various conditions but do not show
clear correlation with hydrogen production. The obtained results indicate that induction of stress through
starvation of cultures is a convenient approach for studying hydrogen production by microalgae, but due to
the low activity of PSII, it is impractical. New approaches are required to create industrial systems based on
microalgae, allowing full realization of their photosynthetic potential.
DOI: 10.1134/S0006297925600929
Keywords: photohydrogen production by microalgae, photosystem II, photoautotrophic cultures of Chlamydo-
monas reinhardtii, sulfur deprivation, nitrogen deprivation
* To whom correspondence should be addressed.
INTRODUCTION
Environmentally friendly energy production is
a global concern. Despite the significant advances in
“green energy” research, the proposed methods are
still inefficient and economically unfeasible. Molec-
ular hydrogen (H
2
) is considered a promising ener-
gy carrier because it can be produced from water,
and its combustion product is water. Certain groups
of microalgae can produce hydrogen under light
GRECHANIK et al.922
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
after a period of anaerobic adaptation. However, this
process is inhibited by oxygen and is therefore short-
lived. Upon illumination, the accumulated oxygen
inactivates hydrogenase, and hydrogen production
ceases [1-5]. In most studies on hydrogen produc-
tion, the green microalga Chlamydomonas reinhardtii
has been used as a model organism [6]. Long-term
light-dependent hydrogen production by this microal-
ga has been observed under photoheterotrophic con-
ditions with sulfur [7], nitrogen [8], or phosphorus [9]
deprivation.
We previously showed that under nitrogen depri-
vation, the photoautotrophic cultures of C. reinhardtii
did not produce hydrogen, although they had reduced
effective quantum yield of photosystem II, PSII (F
v
/F
m
′)
[10]. For hydrogen production, it was necessary to use
a special light regime (reducing light intensity during
the oxygen absorption phase), which led to over-re-
duction of the plastoquinone pool at the onset of an-
aerobiosis. Under sulfur deprivation, at the onset of
anaerobiosis, the effective quantum yield decreased,
and the plastoquinone pool became over-reduced with
increase in the proportion of “closed” reaction centers
(RC) [11]. It should be noted that in all experiments
describing hydrogen production, there was a decrease
in PSII activity. The literature data imply that several
regulatory mechanisms could be involved in reduc-
ing PSII activity: accumulation of “closed” Q
B
centers
[7, 12], over-reduction of the plastoquinone pool (PQ)
[13], appearance of the stably reduced forms of Q
A
[14], state transitions [12], xanthophyll cycle [12, 15],
degradation of D1 [16, 17], degradation of RbcL and
CP43 [17], decoupling of the water-oxidizing complex
and PSII [11, 18], photoinhibition (oxidative stress)
[17]. At the same time, different authors point to one
or two mechanisms of PSII inhibition [4]. It can be as-
sumed that all these mechanisms are simultaneously
involved in the suppression of PSII activity and are
important for hydrogen production, and the authors
studied different mechanisms separately. It is also
possible that under conditions of sulfur or nitrogen
deprivation (as well as under deprivation of other
nutrients, except carbon), the decrease in PSII activi-
ty could be regulated by various mechanisms due to
additional environmental factors determining differ-
ence in the rates of photosynthesis and respiration
at the moment of transition of cultures to anaerobic
conditions. As indirect confirmation of the latter as-
sumption, it can be noted that photoautotrophic cul-
tures under sulfur deprivation also did not always
produce hydrogen, and at the onset of anaerobiosis,
both preservation of potential PSII activity and sharp
decrease in the effective quantum yield could be
observed [19, 20].
The aim of this work was to examine correlation
between the hydrogen production and the involved
mechanisms of PSII regulation under nitrogen or
sulfur deprivation in photoautotrophic cultures of
C. reinhardtii. For this purpose, we used combina-
tion of methods based on chlorophyll fluorescence of
microalgae cultures both under natural illumination
and after dark adaptation, HPLC analysis of pigments
and ascorbate, as well as gas chromatography to de-
termine the amount of H
2
. The obtained results in-
dicate that inducing stress through starvation of cul-
tures is a convenient approach for studying hydrogen
production by microalgae, and its use reveals new
details of the mechanisms of hydrogen production
regulation.
MATERIALS AND METHODS
Study object and cultivation methods. Initial
cultures of C. reinhardtii Dang, strain CC-124, were
maintained on agar plates with standard Tris-ace-
tate-phosphate (TAP) medium (pH 6.9) [21] at 28°C and
illumination of 36 μmol photons m
−2
∙s
−1
. Single colo-
nies were transferred to 10ml of liquid TAP medium
and incubated for 2 days under the same conditions.
The cultures were then grown photoautotrophically
on High-Salt Medium (HSM) [22] in 500-ml Erlenmey-
er flasks, which were bubbled with a mixture of air
and 2% CO
2
through membrane filters with pore size
of 0.2μm (Acro37 TF, Gelman Sciences, Inc., USA) un-
til the late exponential phase (usually three days un-
der used conditions). For experiments, cultures were
grown under photoautotrophic conditions in a pho-
tobioreactor and next used as inoculum in the same
reactor, which reduced the stress caused by culture
transfer. Control cultures were grown under optimal
illumination without nutrient limitation.
C. reinhardtii was cultivated in a 1.5-liter photo-
bioreactor consisting of coaxial glass cylinders with
an internal stirrer, manufactured at the IBBP RAS
[23]. Light path (thickness of the illuminated suspen-
sion layer) was 22 mm. Temperature (28°C) and pH
(7.4) were automatically controlled by a micropro-
cessor-based system and PC as described previously
[11]. During cultivation, the cultures were aerated
with a gas mixture (98% air + 2% CO
2
or 98% argon +
2% CO
2
, 100 ml/min) through Acro 37 TF membrane
filters with pore size of 0.2 μm (Gelman Sciences,
Inc.). For illumination of cultures in the photobiore-
actor, cold white fluorescent lamps (Navigator NKL-
4U, 30 W, 4000 K, China) were placed at the axis
of glass cylinders. Light intensity on the surface of
cultures was 169 μmol photons m
−2
∙s
−1
of photosyn-
thetically active radiation (PAR) (measured using a
LI-250 recorder (LiCOR, USA) equipped with a quan-
tum light sensor). The photobioreactor was connected
physically and digitally to a JUNIOR-PAM fluorimeter
REGULATION OF PSII AND HYDROGEN PRODUCTION IN C.reinhardtii 923
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
(Walz, Germany), as described previously [11], with
movement of the culture suspension in the recircula-
tion loop carried out by pulsed pneumatic pumps used
in equipment for cultivating microorganisms, such as
ANKUM (SKB BP RAS, USSR). The pulses significantly
slowed down surface overgrowth in the cuvette. Be-
fore measurement, the pumps were turned off for 4 s,
which excluded the movement of liquid in the mea-
suring cuvette.
Obtaining starved cultures. To obtain sulfur-de-
prived cultures of C. reinhardtii, we used the dilution
method: cultures were added to a sulfur-free medi-
um, residual sulfur was consumed during growth, and
the cultures gradually transitioned to sulfur depriva-
tion[24]. To minimize stress during inoculation when
transferring from a glass vessel to a photobioreactor,
C. reinhardtii for inoculum was cultivated in a pho-
tobioreactor using HSM medium with doubled salt
concentration (2HSM) to avoid uncontrolled limita-
tion by any salt. It was previously shown that this
medium did not inhibit growth of microalgae and
allowed achieving higher chlorophyll concentrations.
When the culture accumulated 50 mg/l of chloro-
phyll a and b (Chl (a + b)), part of the suspension
was taken from the photobioreactor and remain-
ing 300 ml of the suspension in the photobioreactor
was diluted with 700 ml of 2HSM medium without
sulfur (2*HSM − S) [11].
To obtain nitrogen-deprived cultures, we used
a modified dilution method [10]. Nitrogen is a vital
component of proteins and nucleic acids, so precise
control of nitrogen deficiency during microalgae cul-
tivation is crucial to prevent cell death. To select a
suitable initial concentration of nitrogen in the medi-
um, we studied dependence of the final concentration
of Chl (a + b) in cultures on the initial concentration
of NH
4
Cl in the 2*HSM medium, as described previ-
ously [10]. Final concentration of Chl (a + b) increased
with increasing initial concentration of NH
4
Cl up to
0.25 g/l and remained constant (40 mg/l) with further
increases in NH
4
Cl concentration. This means that the
initial (0.25 g/l) and higher concentrations of NH
4
Cl
do not lead to transition of cultures to the stationary
phase. At NH
4
Cl concentration of 0.125 g/l, final con-
centration of Chl (a + b) reached 21.5 mg/l, meaning
that it was precisely exhaustion of nitrogen that led
to transition of cultures to the stationary phase. This
concentration of NH
4
Cl in the medium was chosen
for subsequent experiments. Under these conditions,
the cultures began to grow under full nitrogen supply
and gradually transitioned to nitrogen deprivation as
it was consumed.
OJIP kinetic. The JIP test for a dark-adapted
culture is a reliable and highly informative method
reflecting all stages of electron transfer from the re-
action center to photosystem I (PSI) [25, 26]. OJIP ki-
netics measurements were carried out using an Aqua-
Pen 110-C fluorimeter (Photon Systems Instruments,
Czech Republic). Before measurement, the culture was
dark-adapted for 15 min. In the case of an anaerobic
(oxygen-free) culture in the photobioreactor, hermet-
ically sealed tubes filled with argon were used for
sampling. During sampling, the culture did not come
into contact with air. Light pulse had duration of 1 s
and intensity of 3000 μmol photons m
−2
∙s
−1
.
Below are the JIP test parameters determined in
this work:
• F
v
/F
m
and (F
m
− F
0
)/F
m
– maximum photochemi-
cal quantum yield of PSII;
• V
j
= (F
j
− F
0
)/(F
m
− F
0
) – normalized variable fluo-
rescence at the J stage (2 ms after light onset);
• d
V
/d
t0
= 4(F
300
− F
50
)/(F
m
− F
0
) – averaged initial
slope of relative variable fluorescence, measured
from 50 to 300 μs;
• ABS/RC – energy flux absorbed by one active RC
of PSII, proportional to the apparent size of the
antenna of active RCs of PSII capable of reduc-
ing QA;
• PIabs – performance index, an indicator of func-
tional activity of PSII, related to the absorbed
energy.
Ascorbate quantity measurements. To mea-
sure quantity of ascorbate with HPLC, the described
previously protocol for sample preparation and mo-
bile phases for ascorbate elution was employed [27].
For measurements, an Agilent 1100 HPLC system
equipped with a 4.6×250 mm column (Waters Spher-
isorb ODS2, 5 μm, Supelco Inc., USA) was used. A
20-μl sample was loaded onto the column. For ascor-
bate elution, an isocratic mobile phase A was 50 mM
KH
2
PO
4
(pH 2.5, acidified with orthophosphoric acid)
was used for 5min at flow rate of 1ml/min, followed
by a short gradient of acetonitrile (mobile phase B)
from 0% to 30% over the interval from 3.5 to 6 min
to elute less polar components from the column. Per-
centage of mobile phase B was reduced to 0 over time
between minutes 8 and 9, and the column was equili-
brated with a mobile phaseA for an additional 9min.
Total analysis time was 18 min. The analysis was
carried out at room temperature. Signal (absorbance)
was recorded in the range from 190 to 500 nm. The
ascorbate peak appeared at approximately 5.1 min
with maximum absorption at ~244 nm. As a stan-
dard, a serial dilution of ascorbate from 50 to 0.2 μM
in extraction buffer containing tris(2-carboxyethyl)
phosphine hydrochloride (TCEP) was used. TCEP is a
reducing agent that prevents oxidation of ascorbate.
Areas under the ascorbate peaks in the HPLC chro-
matograms were determined using software with
linear approximation for constructing the calibration
curve. Measurements for each sample were carried
out in three biological replicates.
GRECHANIK et al.924
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Carotenoid analysis. Pigment analysis of the
samples was carried out using a Shimadzu HPLC sys-
tem (Shimadzu, Japan) with a reverse-phase Agilent
Zorbax SB-C18 column (4.6×250 mm, particle size
5μm, Agilent, USA). To separate the pigment mixture,
a solution A (23% ethyl acetate, 69% acetonitrile, and
8% water) and solution B (pure ethyl acetate) were
used. The following solvent gradient was applied: for
the first 5 min, solution A was pumped through the
column. Then, from 5 to 25 min, solution A was re-
placed with a linear gradient of solution B (0-25%).
Further, from 25 to 40 min, proportion of solution B
was gradually increased from 25% to 100%. At the
end of analysis, 100% solution B was passed through
the column for 3 min. Flow rate of all solvents was
1 ml/min, and temperature was 25°C. Carotenoids
were identified based on their absorption spectra and
retention times on the column [28].
Results are presented as percentage content of
violaxanthin and zeaxanthin, where 100% is the to-
tal amount of carotenoids (neoxanthin, violaxanthin,
lutein, zeaxanthin, neolutein B, beta-carotene, alpha-
carotene).
Other methods. Content of Chl (a + b) and Chl a/b
ratio were determined spectrophotometrically (spec-
trophotometer U-VIS 1240 Mini, Shimadzu) after ex-
traction with a 95% ethanol solution [29]. Extraction
was carried out in the dark at room temperature for
5min. Amount of starch in the cells was measured as
glucose equivalents after enzymatic hydrolysis using
a Glucose GOD FS kit (DiaSys, Germany) according to
the method of Gfeller and Gibbs [30]. Final data on
starch content are presented as glucose equivalents.
Redox potential (eH) of the medium was measured
as potential of a platinum electrode compared to an
Ag/AgCl electrode (Mettler Toledo, USA). Percentage
of H
2
in the gas from photobioreactor was analyzed
using gas chromatography as described previously
[19]. All experiments on cultivation of C. reinhardtii
were repeated 2-4 times, and the figures show data
from typical growth curves. Amount of H
2
produced
was calculated taking into account gas flow rate
(100 ml/min) and percentage of H
2
at the outlet using
the following formula (1):
V(H
2
) = H
2
,
% × V
gas
(ml/min) × 60/100% × V
culture
.
(1)
Accumulation of H
2
was calculated as the sum
of the corresponding time periods with the measured
rates of H
2
production.
Statistical analysis. Each measurement was
carried out in triplicate. For statistical processing
Excel 2016 was used, and data were visualized with
SigmaPlot12. Data are presented as mean values and
95% confidence intervals.
RESULTS
Cultivation of C. reinhardtii in the atmosphere
of air + CO
2
(control culture). As a control, we used
photoautotrophic cultures of C. reinhardtii in a pho-
tobioreactor with the medium (2*HSM), aerated with
a mixture of air + 2% CO
2
. When cultivating C. rein-
hardtii under these conditions, the concentration of
Chl (a + b) increased during the first 66 h, followed
by the decrease (Fig. 1a). The maximum concentra-
tion of chlorophyll was 40 mg/l. It can be stated that
after 66 h, the culture transitioned to the stationary
phase due to depletion of some mineral component
[11]. At the beginning of cultivation, pO
2
increased
during the first 20h. After 40-60h of cultivation, pO
2
gradually decreased, indicating slowdown in the rate
of photosynthetic oxygen production by the culture,
which occurred before the transition to the station-
ary phase. Starch content in the cells and eH (Fig. 1,a
and b) did not change during the entire cultivation
period. Ascorbate content in the cells changed slightly
and was minimal at the end of the exponential phase.
Effective quantum yield of PSII (Y(II)) in the cultures
initially was 0.73-0.77, gradually decreasing with this
decrease accelerating at the onset of the stationary
phase and reaching 0.5 at the end of cultivation.
These changes are typical for photoautotrophic cul-
tures and, in general, correspond to those described
previously [10, 11, 31].
Analysis of the carotenoid content and of the Chl
a/b ratio (Fig. 1d) was carried out. The obtained data
confirm that in the control cultures, there is no activa-
tion of violaxanthin cycle and additional synthesis of
antenna complexes throughout the entire cultivation
period, including the late exponential phase, in which
the cultures experienced light limitation.
Overall, the growth of C. reinhardtii under aero-
bic conditions represents a typical picture of photo-
autotrophic cultivation of microalgae without stress,
but with transition from unlimited growth to light
limitation followed by the depletion of some nutrient
component in the stationary phase.
Cultivation of C. reinhardtii in the atmosphere
of argon + CO
2
under nitrogen deprivation using
two-stage light protocol. Decrease in light intensi-
ty from 169 to 30 μmol photons m
−2
·s
−1
. Photoau-
totrophic cultures of C. reinhardtii did not produce
hydrogen under nitrogen deprivation in our exper-
iments [10]. For comparison, photoautotrophic cul-
tures of the same microalga, starved for sulfur, pro-
duce a very small amount of hydrogen at constant
light intensity of 110 μmol photons m
−2
∙s
−1
if the in-
oculum was previously cultivated under illumination
of 120 μmol photons m
−2
∙s
−1
[19]. However, if at the
early stage of oxygen consumption, the light intensity
was switched from 110 to 20 μmol photons m
−2
∙s
−1
,
REGULATION OF PSII AND HYDROGEN PRODUCTION IN C.reinhardtii 925
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 1. Cultivation of C.reinhardtii in a photobioreactor in the gas phase of air + 2% CO2 (control). Light intensity 169μmol
photons m
−2
∙s
−1
. a) Chl (a + b) (1); starch (2), ascorbate (3). b) Partial pressure of oxygen, expressed as percentage of
air saturation at 28°C (4); eH against Ag/AgCl electrode (5); H
2
content in the gas phase (6). c) Y(II) (7); F
T
(8); F
m
′ (9).
d)Ratio of Chl a/b (12) and percentage content of violaxanthin (10) and zeaxanthin (11) (100% – total amount of carotenoids).
hydrogen production significantly increased. There-
fore, we applied a two-stage light protocol for the
nitrogen-deprived cultures. For this, the culture of
C. reinhardtii was inoculated as described previous-
ly [10] into the 2*HSM medium with 0.125 g/l NH
4
Cl
(Fig. 1). The culture smoothly transitioned from the
unlimited growth for 43 h, to the stage of limitation
and next to nitrogen deprivation, which generally
corresponds to the data described previously [10].
After 43h, when pO
2
in the culture began to decrease
(early oxygen consumption stage in adaptation to ni-
trogen deprivation), the light intensity was reduced
from 169 to 30μmol photons m
−2
∙s
−1
. pO
2
decreased to
zero within 60 min after changing the light intensity.
When pO
2
reached zero, eH decreased from 300 mV
and reached –60 mV after 30 min (Fig. 2). Two hours
after changing the light intensity, the content of
Chl (a + b) and starch began to decrease, as did the
ascorbate content. Y(II) decreased from 0.74 to 0.4
(Fig. 2c, additional data are provided in the Online
Resource 1), F
v
/F
m
decreased from 0.72 to 0.39. It is
important to note that during these 2 h, the ascor-
bate content significantly decreased. These changes
were accompanied by the decrease in PIabs. ABS/RC
increased moderately, while other JIP test parame-
ters were stable (additional data are provided in the
Online Resource 1). H
2
was not detected at 43.0 and
43.25h but appeared at 44h in the gas phase (Fig.2b).
This indicates that the anaerobic stage of adaptation
to nitrogen deprivation exists but is very short.
During subsequent incubation of the culture at
the reduced light intensity, the content of Chl (a + b)
and starch gradually decreased (Fig. 2a). The ascor-
bate content increased up to 116 h, followed by the
decrease. The H
2
content in the gas phase reached
maximum at 67h. H
2
was produced at a gradually de-
creasing rate until the end of the experiment (Fig.2b).
Total amount of H
2
produced during the entire experi-
ment was 122ml per 1liter of culture. Y(II) and F
v
/F
m
,
after the significant decrease due to changing light
intensity and establishment of anaerobiosis, gradu-
ally increased from 43 h to the end of the experi-
ment. V
j
, d
V
/d
t0
, and ABS/RC doubled after reduction
in light intensity. PIabs reached minimum value at
66 h. After 137 h, there was a recovery of all JIP test
parameters.
Analysis of the carotenoid content and of the
Chl a/b ratio (Fig. 2d) was carried out. The obtained
data confirm that there was no activation of the vi-
olaxanthin cycle and additional synthesis of antenna
complexes in the cultures throughout the entire cul-
tivation period.
GRECHANIK et al.926
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 2. Cultivation of C. reinhardtii in the photobioreactor in the gas phase of argon + 2% CO
2
under nitrogen deprivation
using two-stage light protocol. Light intensity 167μmol photons m
−2
∙s
−1
up to 43.6h, then 30μmol photons m
−2
∙s
−1
(vertical
dotted line – change in light intensity). a) Chl (a + b) (1); starch (2), ascorbate (3). b) Partial pressure of oxygen, expressed
as percentage of air saturation at 28°C (4); eH against Ag/AgCl electrode (5); H
2
content in the gas phase (6). c) Y(II) (7);
F
T
(8); F
m
′ (9). d)Ratio of Chl a/b (12) and percentage content of violaxanthin (10) and zeaxanthin (11) (100% – total amount
of carotenoids). Gap in the measurements of F
T
and F
m
′ is associated with a technical malfunction.
Cultivation of C. reinhardtii in the atmosphere
of argon + CO
2
under nitrogen deprivation using
two-stage light protocol. Increase in the light inten-
sity from 169 to 300 μmol photons m
−2
·s
−1
. In this
experiment, the effect of high light intensity on PSII
activity in the C. reinhardtii cultures under nitrogen
deprivation was studied (Fig. 3). We were unable to
set the light intensity to 300 μmol photons m
−2
∙s
−1
from the beginning of the experiment, as at this light
intensity, the culture at the time of inoculation, even
at a Chl (a + b) concentration of about 12 mg/l, did
not grow. Therefore, the two-stage light protocol was
applied to the cultures: for the first 46 h of cultivation,
the light intensity was 169 μmol photons m
−2
∙s
−1
, and
after 46h of cultivation (period of nitrogen starvation)
it was increased to 300 μmol photons m
−2
∙s
−1
.
The culture smoothly transitioned from unlimited
growth for 23 h, to the limitation stage and next to
nitrogen deprivation. At 46 h of cultivation, the light
intensity was increased to 300 μmol photons m
−2
∙s
−1
;
by this time, nitrogen limitation was already observed.
Chl (a + b), pO
2
, ascorbate, and the effective quantum
yield (Y) decreased. Starch accumulation occurred,
lasting until the end of the experiment and reaching
maximum values compared to all previously conduct-
ed experiments. Increasing the light intensity led to
increase in the amount of ascorbate, with further de-
crease after 60 h. Maximum concentration of ascor-
bate was 5.5 nmol/mg Chl (a + b). eH remained stable
throughout the cultivation, and although pO
2
de-
creased, it was not less than 10% of saturation, mean-
ing that the transition to anaerobic conditions did not
occur. Therefore, we were unable to detect hydrogen
in the gas phase of the photobioreactor at any stage
of the experiment.
All parameters obtained from the JIP test (addi-
tional data are provided in the Online Resource1) did
not differ from those obtained for the cultures grown
under constant light intensity, except for the initial
slope of the fluorescence curve (d
V
/d
t0
(M
0
)), which
showed increase after 65 h and correlated with the
changes in the amount of ascorbate. These changes
indicate decoupling of the water-oxidizing complex
and PSII [31, 32].
Analysis of the carotenoid content and of the
Chl a/b ratio (Fig. 3d) was carried out. The obtained
data confirm that there was no activation of vio-
laxanthin cycle and additional synthesis of antenna
REGULATION OF PSII AND HYDROGEN PRODUCTION IN C.reinhardtii 927
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 3. Cultivation of C. reinhardtii in a photobioreactor in the gas phase of argon + 2% CO
2
under nitrogen deprivation
using two-stage light protocol. Light intensity 167μmol photons m
−2
∙s
−1
up to 46h, then 300μmol photons m
−2
∙s
−1
(vertical
dotted line – change in light intensity). a) Chl (a + b) (1); starch (2), ascorbate (3). b) Partial pressure of oxygen, expressed
as percentage of air saturation at 28°C (4); eH against Ag/AgCl electrode (5); H
2
content in the gas phase (6). c) Y(II) (7);
F
T
(8); F
m
′(9). d)Ratio of Chla/b(12) and percentage content of violaxanthin(10) and zeaxanthin(11) (100% – total amount
of carotenoids).
complexes throughout the entire cultivation period in
the cultures.
Cultivation of C. reinhardtii in the atmosphere
of argon + CO
2
under sulfur deprivation. Light in-
tensity 169 μmol photons m
−2
·s
−1
. When cultivating
C. reinhardtii in the atmosphere of argon + 2% CO
2
,
with the culture inoculated into the sulfur-free medi-
um, concentration of Chl (a + b) increased up to 23 h
(beginning of sulfur starvation) and was stable up to
41 h, after which it began to decrease (Fig. 4).
The culture smoothly transitioned from unlim-
ited growth for 23 h to the stage of limitation and
transition to sulfur deprivation, which generally cor-
responds to the data described previously [11]. pO
2
remained stable up to 23 h, after which it decreased
(oxygen consumption stage), reaching zero (anaer-
obic and hydrogen production stage) by the 65
th
h.
The redox potential was stable when pO
2
was stable,
then decreased simultaneously with pO
2
and sharp-
ly dropped when pO
2
reached zero, followed by the
increase in the late hydrogen production stage, i.e.,
under anaerobic conditions, the redox potential of
the culture liquid was reducing. The ascorbate con-
tent increased throughout the experiment from 10.5
to 31.7 nmol/mg Chl (a + b). Hydrogen was detected
in the gas phase at 66 h. H
2
content in the gas phase
increased up to 93h and then decreased. H
2
in the gas
phase was detected even after 130h of cultivation. To-
tal hydrogen production during the entire incubation
period was 170 ml/l. The effective quantum yield of
PSII, Y(II), began to decrease after 60 h of cultivation,
mainly due to decrease in Fm′.
All parameters obtained from the OJIP test (ad-
ditional data are provided in the Online Resource 2)
23 h after inoculation in the atmosphere of ar-
gon + CO
2
without sulfur were very close to those for
the control culture with similar concentration of Chl
(a + b). Thus, at this time, the culture did not experi-
ence sulfur deprivation. At the 41.5-h point, Y, F
v
/F
m
,
V
j
, Sm, N did not change, while Pabs significantly
decreased.
After 68 h of growth, when H
2
appeared in the
gas phase, Y, F
v
/F
m
, Pabs decreased, while V
j
, S
m
, and
N did not change significantly.
The results of pigment analysis (Fig. 4d) demon-
strate that in this experiment, during adaptation to
high light intensities, there is enzymatic interconver-
sion between violaxanthin and zeaxanthin.
GRECHANIK et al.928
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 4. Cultivation of C. reinhardtii in a photobioreactor in the gas phase of argon + 2% CO
2
under sulfur deprivation. Light
intensity 167 μmol photons m
−2
∙s
−1
. a) Chl (a + b) (1); starch (2), ascorbate (3). b) Partial pressure of oxygen, expressed
as percentage of air saturation at 28°C (4); eH against Ag/AgCl electrode (5); H
2
content in the gas phase (6). c) Y(II) (7);
F
T
(8); F
m
′ (9). d)Ratio of Chla/b (12) and percentage content of violaxanthin (10) and zeaxanthin (11) (100% – total amount
of carotenoids).
Cultivation of C. reinhardtii in the atmosphere
of argon + CO
2
under sulfur deprivation using a
two-stage light protocol. Decrease in light intensity
from 169 to 30 μmol photons m
−2
·s
−1
. When culti-
vating C. reinhardtii in the atmosphere of argon + 2%
CO
2
, with the culture inoculated into the sulfur-free
medium, concentration of Chl (a + b) increased up to
40h, after which it began to decrease (Fig.5). The cul-
ture smoothly transitioned from the unlimited growth
for 40h to the stage of limitation and transition to sul-
fur deprivation. After 40h, the light intensity was re-
duced to 30μmol photons m
−2
∙s
−1
. pO
2
and the redox
potential sharply dropped at the moment of reducing
the light intensity. Sharp drop was also observed in
the effective quantum yield of PSII, mainly due to
the sharp decrease in F
m
′. Starch, ascorbate, and the
Chl a/b ratio remained stable throughout the cultiva-
tion. H
2
content in the gas phase increased up to 89h
and then decreased. Total hydrogen production during
the entire incubation period was 84 ml/l.
Additionally, a pigment analysis was carried out
to assess operation of the violaxanthin cycle. In this
experiment (Fig. 5d), zeaxanthin was absent through-
out the entire cultivation. The percentage content
of violaxanthin decreased.
All parameters obtained from the JIP test (addi-
tional data are provided in the Online Resource 2)
40 h after inoculation in the atmosphere of ar-
gon + CO
2
without sulfur were very close to those for
the cultures grown on complete medium, confirming
the conclusion that, at this time the culture did not
experience sulfur deprivation. After reducing the light
intensity, decrease in F
v
/F
m
and PIabs and increase in
V
j
, d
V
/d
t0
, ABS/RC were observed. After 66h of cultiva-
tion, V
j
, d
V
/d
t0
, ABS/RC doubled, while PIabs decreased
to almost zero.
Cultivation of C. reinhardtii in the atmosphere
of argon + CO
2
under sulfur deprivation using a
three-stage light protocol. Increase in the light in-
tensity from 169 to 300μmol photons m
−2
·s
−1
. In this
experiment, the effect of high light intensity on PSII ac-
tivity in the cultures adapted to low light intensity was
studied (Fig.6). For this, a three-stage transition was ap-
plied, as cultures died when exposed to high light inten-
sity immediately after inoculation at low concentration.
On the first day after inoculation, the light intensity was
40 μmol photons m
−2
∙s
−1
; after 20 h, the light inten-
sity was increased to 167 μmol photons m
−2
∙s
−1
, and
after 46 h of cultivation (period of sulfur starvation),
it was increased to 300 μmol photons m
−2
∙s
−1
.
REGULATION OF PSII AND HYDROGEN PRODUCTION IN C.reinhardtii 929
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 5. Cultivation of C. reinhardtii in a photobioreactor in the gas phase of argon + 2% CO
2
under sulfur deprivation using
two-stage light protocol. Light intensity 167 μmol photons m
−2
∙s
−1
up to 40 h, then 30μmol photons m
−2
∙s
−1
(vertical dotted
line– change in light intensity). a)Chl (a + b) (1); starch(2), ascorbate(3). b)Partial pressure of oxygen, expressed as percent-
age of air saturation at 28°C(4); eH against Ag/AgCl electrode (5); H
2
content in the gas phase (6). c) Y(II) (7); F
T
(8); F
m
′ (9).
d)Ratio of Chl a/b(12) and percentage content of violaxanthin(10) and zeaxanthin(11) (100% – total amount of carotenoids).
On the first day of cultivation, concentration of
Chl (a + b) did not increase, and no culture growth
was observed. At this time, the effective quantum
yield of PSII was 0.750. After increasing the light in-
tensity to 167 μmol photons m
−2
∙s
−1
(21 h), sharp de-
crease in the effective quantum yield was observed,
with rapid recovery to previous values. pO
2
and the
amount of Chl (a + b) increased up to 40h. Increase in
the amount of ascorbate up to 5 nmol/mg Chl (a + b)
was observed. The ratio of Chla/b did not change, and
the amount of starch remained stably low throughout
the experiment.
After 46 h from the start of cultivation, the light
intensity was increased to 300 μmol photons m
−2
∙s
−1
;
at this moment, sharp increase in pO
2
was observed,
followed by sharp decrease to 0. The amount of
ascorbate increased twofold 20 min after increasing
the light intensity and continued to increase through-
out the remaining cultivation period. After the deple-
tion of oxygen in the medium, the redox potential
sharply decreased, indicating synthesis of the re-
dox-active reduced compounds. The H
2
content in the
gas phase increased up to 72 h and then decreased.
Total hydrogen production during the entire incuba-
tion period was 101.6 ml/l.
All parameters obtained from the JIP test (addi-
tional data are provided in the Online Resource 2)
did not change after increasing the light intensity and
up to 46 h were very close to those for the cultures
grown on complete medium, confirming the conclu-
sion that, at this time the culture did not experience
sulfur deprivation. After 70 h of cultivation, at the
onset of anaerobiosis, V
j
, d
V
/d
t0
, ABS/RC doubled,
while PIabs decreased to almost zero.
The results of pigment analysis (Fig. 6d) demon-
strate that in this experiment, during adaptation to
high light intensities, there is an enzymatic inter-
conversion between violaxanthin and zeaxanthin.
Thus, cultures adapted to low light intensities and
transferred to very high light intensities under sul-
fur starvation regulate PSII activity by at least three
mechanisms: over-reduction of the plastoquinone
pool, decoupling of the water-oxidizing complex and
PSII, as well as violaxanthin cycle.
DISCUSSION
In this work, we investigated the mechanisms
of PSII activity regulation in the photoautotrophic
GRECHANIK et al.930
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
Fig. 6. Cultivation of C. reinhardtii in a photobioreactor in the gas phase of argon + 2% CO
2
under sulfur deprivation
using three-stage light protocol. Light intensity 40 μmol photons m
−2
·s
−1
up to 20 h, 167 μmol photons m
−2
·s
−1
up to 46 h,
next 300 μmol photons m
−2
·s
−1
(vertical dotted line – change in light intensity). a) Chl (a + b) (1); starch (2), ascorbate (3).
b) Partial pressure of oxygen, expressed as percentage of air saturation at 28°C (4); eH against Ag/AgCl electrode (5);
H
2
content in the gas phase(6). c)Y(II)(7); F
T
(8); F
m
′(9). d)Ratio of Chl a/b(12) and percentage content of violaxanthin(10)
and zeaxanthin (11) (100% – total amount of carotenoids).
cultures of C. reinhardtii under nitrogen or sulfur
deprivation under various light conditions and their
relationship with hydrogen production.
Control cultures grown in the complete HSM me-
dium at light intensity of 169 μmol photons m
−2
∙s
−1
demonstrated typical photoautotrophic growth with-
out signs of stress. The JIP test parameters indicated
high efficiency of PSII, and the level of reduction of
the plastoquinone pool remained low, as evidenced by
the high ratio of the area above the OJIP curve to the
total area and the stable level of F
0
[26]. Under these
conditions, hydrogen was not produced.
When cultivated in the nitrogen-free medium
with decrease in light intensity from 169 to 30 μmol
photons m
−2
∙s
−1
during the oxygen consumption peri-
od, we observed a significant increase in the reduction
of the plastoquinone pool, previously observed for
the sulfur-deprived cultures [19]. This was accompa-
nied by the decrease in photosynthetic activity (Y(II),
F
v
/F
m
) due to decrease in F
m
′ and increase in F
T
, and
hydrogen production (up to 122 ml/l). At the same
time, as previously noted [13, 33], the increase in FT
is due to the sharp increase in the number of active
centers with reduced QA, caused by the sharp over-re-
duction of the plastoquinone pool. In contrast, in the
case of increasing the light intensity to 300 μmol
photons m
−2
∙s
−1
, over-reduction of the plastoquinone
pool did not occur, despite the decrease in F
v
/F
m
, and
hydrogen was not produced. These results are con-
sistent with the data obtained in photoheterotrophic
cultures [16, 24].
Similar patterns were observed for the culture
grown in the sulfur-free medium. At constant light
intensity of 169μmol photons m
−2
∙s
−1
and when light
intensity increased to 300μmol photons m
−2
∙s
−1
, there
was a moderate over-reduction of the plastoquinone
pool, observed as increase in FT, and hydrogen pro-
duction (170 and 101.6 ml/l). In both cases, there was
enzymatic interconversion between violaxanthin and
zeaxanthin, and decoupling of the water-oxidizing
complex (WOC) and PSII was observed for the cul-
tures only at constant light intensity. Decreasing the
light intensity to 30 μmol photons m
−2
∙s
−1
led only to
over-reduction of the plastoquinone pool and, conse-
quently, to hydrogen production. With the sharp de-
crease in light intensity, the rate of photosynthesis was
lower than the rate of respiration, and cultures very
quickly transitioned to anaerobic conditions (where
REGULATION OF PSII AND HYDROGEN PRODUCTION IN C.reinhardtii 931
BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025
this occurred). Anaerobic conditions led to inabili-
ty to consume NADPH (especially under conditions
where NADPH was not consumed in the Calvin cycle
due to the sulfur or nitrogen starvation). As a result,
the entire photosynthetic electron transport chain
(ETC) became over-reduced, including the plastoqui-
none pool. This phenomenon was first discovered
in 2001 [33].
Comparison of the obtained results with the liter-
ature data shows that the behavior of photoautotro-
phic and photoheterotrophic cultures of C. reinhardtii
under sulfur and nitrogen starvation is largely simi-
lar [16, 24]. In both cases, there is a decrease in PSII
activity manifested by the measured JIP test param-
eters – maximum (F
v
/F
m
) and effective (Y(II)) quan-
tum yield, which is accompanied by accumulation
of the reduced forms of the plastoquinone pool.
CONCLUSION
A key observation of our work is that hydrogen
production was observed only in those cultures where
there was a significant over-reduction of the plas-
toquinone pool, as evidenced by the sharp increase
in FT. This was observed both under sulfur and ni-
trogen deprivation. In the cases where there was no
rapid over-reduction of the plastoquinone pool associ-
ated with rapid transition of the cultures to anaerobic
conditions, hydrogen was not produced, despite the
decrease in photosynthetic activity.
Other mechanisms of PSII regulation studied by
us, such as the violaxanthin cycle or increase in the
proportion of PSII antenna complexes (decrease in the
ratio of Chla/b), apparently play a secondary role and
do not have a determining effect on hydrogen pro-
duction. Accumulation of ascorbate under starvation
also did not correlate unambiguously with hydrogen
production.
Thus, our data allow us to conclude that the key
mechanism determining hydrogen production under
sulfur or nitrogen deprivation in C. reinhardtii is
over-reduction of the plastoquinone pool, which is
achieved under certain light regimes against the back-
ground of suppressed photosynthesis. Other studied
mechanisms play a lesser role. The obtained results
expand our understanding of the regulation of photo-
synthetic apparatus under stress conditions and could
be used to optimize the processes of hydrogen produc-
tion by microalgae.
Abbreviations. PSII, photosystem II; Y(II), effec-
tive quantum yield of photosystemII.
Supplementary information. The online version
contains supplementary material available at https://
doi.org/10.1134/S0006297925600929.
Acknowledgments. The equipment (Agilent HPLC
chromatograph, Tsvet 800 gas chromatograph, and
JUNIOR-PAM and Aquapen PSI fluorimeters) of the
CKP PNCBI RAS (No. 670266, https://www.ckp-rf.ru/
ckp/670266/) was used in the work.
Contributions. V. I. Grechanik – conducting ex-
periments, discussing results, discussing and editing
the manuscript; M. A. Bolshakov – measuring ascor-
bate and analyzing pigment composition, discussing
results; A. A. Tsygankov – concept, discussing results,
writing the manuscript, discussing and editing the ar-
ticle.
Funding. This work was partially carried out
within the framework of the State assignment
125051305944-7 (experiments on microalgae cultiva-
tion under sulfur deprivation under constant light re-
gime) and with the financial support of the Russian
Science Foundation (grant no. 19-14-00255, the re-
maining part).
Ethics approval and consent to participate.
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest. The authors of this work de-
clare that they have no conflicts of interest.
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