Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 7, pp. 934-942 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 7, pp. 1018-1027.

934

Short-Term Photoinhibition Induces Long-Term

Hydrogen Photoproduction in a Phototrophic Culture

of Chlorella sorokiniana on Complete Medium

Alena A. Volgusheva

1,a

* and Taras K. Antal

Department of Biophysics, Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

Laboratory of Integrated Environmental Research, Pskov State University, 180000 Pskov, Russia

e-mail: volgusheva_alena@mail.ru

Received April 5, 2025

Revised May 27, 2025

Accepted May 28, 2025

Abstract— This work demonstrates, for the first time, capacity of the Chlorella sorokiniana immobilized in

alginate to produce hydrogen (H

) over an extended period of time when cultivated under strictly photoauto-

tropic conditions on complete mineral medium. In order to reduce photosynthetic activity, immobilized cells

were subjected to a 30-minute pre-incubation period at high light intensity of 1000  μmol  photons  m

−2

∙s

−1

The ability to produce H

was evaluated under illumination of 40  μmol/(m

∙s). The culture not bubbled with

argon produced H

for 9 days; total gas yield was 0.1  mol  H

. In the culture under argon atmosphere,

the release of H

continued for 51 days, resulting in a total yield of 0.55  mol  H

. The immobilized culture

was capable of H

production at 16% O

in the gas phase, which may be due to the effects of photoinhibition

and activation of oxygen uptake pathways in mitochondria and chloroplast. Analysis of the functioning of

electron-transport chain in the microalgae cells revealed decrease in the rate of electron transport, increase

in the size of the PSII antenna, and development of non-photochemical quenching processes, while activity

of PSII remained moderately high (Fv/Fm  =  0.4-0.6). Inhibitor analysis using 10

−5

  M DCMU demonstrated that

contribution of PSII to hydrogenase reaction increased from 30% on the first day of the experiment to 50%

by the fourth day. Addition of 10

−5

  M DBMIB led to the 90% reduction in the rate of H

formation on both

day 1 and day 4.

DOI: 10.1134/S000629792560098X

Keywords: green microalgae, primary reactions of photosynthesis, hydrogen photoproduction, photoinhibition,

hydrogenase

* To whom correspondence should be addressed.

INTRODUCTION

Molecular hydrogen (H

) has the highest specific

energy capacity among all known types of fuel. Its

combustion does not produce carbon, which makes

hydrogen an ecologically clean source of energy.

Moreover, H

plays a key role in a number of indus-

trial processes such as production of methane, meth-

anol, and ammonia [1].

Use of cyanobacteria and green algae for pro-

duction of H

is considered as one of the areas of

biotechnology aimed at solving global energy crisis

problem. These organisms have unique ability to con-

vert energy of light into H

using photosynthesis. The-

oretically, efficiency of transformation of solar light

into H

could reach 10-13% for algae and 6% for ni-

trogen-fixing cyanobacteria. However, in practice the

levels of efficiency of H

production by photosynthe-

sizing organisms observed under laboratory settings

are significantly lower [2]. This emphasizes the need

to further investigate mechanisms of these processes,

as well as to develop new technologies to improve

efficiency.

Photoproduction of H

by green algae occur with

participation of the enzyme [FeFe]-hydrogenase, which

is synthesized and perform its reaction under anaer-

obic conditions in chloroplast stroma. This enzyme

utilizes electrons generated in the course of water

PHOTOPRODUCTION OF HYDROGEN UPON PHOTOINHIBITION 935

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

photolysis in photosystem II (PSII) and reduces pro-

tons to molecular H

. Activation of hydrogenase play

a crucial role for maintenance of continuous photosyn-

thetic electron flow under anaerobic conditions [2-6].

Two independent studies demonstrated the pos-

sibility of expression of the hydrogenase gene and its

protein synthesis in the green microalgae under an-

aerobic conditions when photosynthetic activity was

reduced[7,8]. It was also shown that the switch from

dark aerobic to light conditions resulted in the de-

crease of H

production mainly due to activation of

Calvin cycle, which effectively competes with hydro-

genase for electrons [9]. In the process, hydrogenase

activity is irreversibly suppressed by O

only 4 min

after cessation of H

release. More over, new strains

of Chlorella vulgaris have been identified that were

capable of H

production under aerobic conditions

and continuous illumination [10]. The presented re-

sults allow considering revision of the existing notions

on fast inhibiting effects of O

on activity of [FeFe]-hy-

drogenase in vivo. The mechanisms of this phenom-

enon are poorly understood at present and require

further investigation. It could be suggested that tol-

erance of hydrogen photoproduction to O

is due to

ability of microalgae to maintain microanaerobic con-

ditions in chloroplast due to partial decrease of the

rate of photosynthesis and simultaneous increase of

the rate of alternative pathways of electron transfer

in chloroplasts, including photorespiration and Meler’s

reaction [7]. Activity of PSII and the rate of mitochon-

drial respiration, which change significantly depending

on cultivation conditions, also play an important role.

It is well known that sulfur starvation efficiently

stimulates long-term H

photoproduction by algae un-

der illumination [11, 12]. However, this method, simi-

lar to other types of biogenic starvation [13-17], has its

limitations: insufficient yield of H

for industrial scale

and short lifespan of algae. Alternative approaches to

stimulate photoproduction of H

by microalgae during

cultivation in completer nutrient medium have been

suggested in the recent decades. Development of

such approaches could facilitate increase of the algae

lifespan and, as a consequence, prolongation of the

period of gas release. In particular, the possibility of

prolonged generation of H

by the C.  reinhardtii cells

through modification of photosynthetic electron trans-

port has been demonstrated[18,19]. Furthermore, an

innovative method of H

photoproduction based on

pulsed illumination regime has suggested [8], howev-

er, its efficiency is limited by the short period of H

release (several days).

Use of immobilized algae cells for production of

under illumination is a promising approach with

several advantages. Immobilization of cell culture

improves their stability and resistance to external

factors, slows down their metabolism (while activity

of the enzymes remains high), and allows reaching

higher cell density, could facilitate more uniform il-

lumination of cells in photobioreactor, and does not

limit gas exchange [20-22]. All these factors taken to-

gether results in the increase of yield and duration

of H

production.

Majority of the studies on production of H

were

conducted with photoheterotrophic cultures, i.e., cul-

tures with added acetate to the nutrient medium.

Acetate serves as an alternative source of carbon,

energy (ATP), and as a reducing agent (NADH), pro-

viding, in particular, a reducing agent for mitochon-

drial respiration stimulating transition of the system

to anaerobic conditions. However, its use increase

significantly costs of H

production. From economical

point of view the use of photoautotrophic cultures

that do not need additional organic substrates seems

more promising, which was successfully demonstrated

with sulfur-starving culture C. reinhardtii as an exam-

ple [23, 24].

In this study we suggest a protocol ensuring pro-

longed photoproduction of H

by the C. sorokiniana

cells. This protocol is the first to combine advantages

of the approaches discussed above; in particular, it in-

volves use of short-term photoinhibition as a stimu-

lating factor, immobilization of microalgae in alginate

films to ensure stability of the process, and photo-

autotrophic cultivation in complete mineral medium.

MATERIALS AND METHODS

Cultivation of algae. C.  sorokiniana (isolated from

waters of the White Sea, Russia, GenBank ID:

KC678067) [25] were cultivated in a HS mineral me-

dium [26] in 300-ml Erlenmeyer flasks at 24°C under

continuous illumination. Photon flux density (PFD)

100 µmol photons∙m

−2

∙s

−1

with stirring rate 120 rpm

on an ELMI shaker (ELMI, Latvia).

Immobilization of algae. Immobilization of al-

gae in alginate films under sterile conditions was

carried out according to the protocol developed by

Kosourov and Seibert [20] with some modifications.

Algae were precipitated by centrifugation (3000 rpm,

10 min) followed by mixing the obtained precipitate

with aqueous solution of sodium alginate (Fluka,

USA) at a ratio of 1 ml of 4% alginate solution per

1g of algae wet weight. The obtained suspension was

spread evenly across a mosquito net. Alginate polym-

erization was induced by spraying films with 50 mM

MgCl

solution. Next films were cut into strips with

size 1×3 cm. Each strip was placed into hermetically

sealed vials with volume 18 or 68ml filled with 10ml

of HS medium.

Photoinhibition and cultivation of immobilized

cultures. Immobilized algae in hermetically sealed

VOLGUSHEVA, ANTAL936

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

vials with volume 18 ml or 68 ml were subjected to

a single 30-min period of illumination with light of

intensity 1000 µmol photons∙m

−2

∙s

−1

. Next 68-ml vol-

ume vials were bubbled with an argon flow 30 min

(at room light). Next, cultures in both types of vials

were incubated from 24 h to 51 days under continu-

ous illumination with PFD 40, 100, or 200  µmol pho-

tons∙m

−2

∙s

−1

depending on experimental conditions at

room temperature. Culture vials of smaller volume not

bubbled with argon flow were used for faster estab-

lishing of anaerobic conditions.

Inhibitory analysis. To investigate impact of

photosynthesis inhibitors on hydrogen production

DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea, di-

uron) and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-

p-benzoquinone, dibromothymoquinone) were used

at final concentration 10

−5

M.

Determination of chlorophyll and starch con-

tent. Immobilized algae were treated with 0.05  M

NaEDTA followed by intensive stirring to complete

destruction of alginate matrix. Next cells were pre-

cipitated with centrifugation (3000 rpm, 10 min), 6 ml

of 90% ethanol was added to the precipitate followed

by incubation for 1 day in a fridge. Quantitative eval-

uation of extracted chlorophyll (Chl) was carried spec-

trophotometrically [27].

After Chl extraction starch content was deter-

mined according to the method described by Gfeller

and Gibbs [28] with some modifications. Cell precip-

itate was washed twice from ethanol by centrifuga-

tion (3000 rpm, 10 min) in 1 ml of sodium acetate

solution (100mM, pH4.5). Next cells were suspended

1 ml of sodium acetate and transferred into sterile

centrifuge tubes, which was followed by treatment in

an autoclave for 10 min at 1 atmosphere. After cool-

ing the samples to room temperature amyloglucosidase

(Sigma, USA) was added to each tube at the amount

corresponding to 2 activity units and incubated in a

thermostat at 55°C for 18-20h. Next fluid volume was

adjusted to 2ml with sodium acetate solution and cen-

trifuged. Glucose content in a supernatant was deter-

mined spectrophotometrically using glucose oxidase –

peroxidase reagent kit (Sigma).

Recording Chl fluorescence. Induction curves

of Chl fluorescence (OJIP transients) were record-

ed with a PEA fluorimeter (Hansatech, United King-

dom). Chl fluorescence was initiated with red light

(λ  =  650  nm), PFD 1500 µmol photons∙m

−2

∙s

−1

. Effi-

ciency of photochemical transformation of energy

in PSII (Fv/Fm) was calculated using the following

equation: Fv/Fm  =  (Fm  −  Fo)/Fm, where Fo and Fm –

minimal and maximal fluorescence values in samples

adapted to dark. OJIP-transients were analyzed with

the help of JIP-test [29,  30] using equation and pa-

rameters presented in Table  1. Immobilized cultures

were dark-adapted for 15min in vials, after which the

sample was removed and placed in the fluorescence

measuring clump.

Gas chromatography. Content of H

was mea-

sured with the help of a LKhM-80 gas chromatograph

(Russia). Argon flow rate used as a carrier gas was

70 ml/min. Column thermostat temperature was set

at 60°C, electric current in katharometer was 70 mA.

Quantitative determination of H

was carried out with

the help of calibration curve constructed based on

analysis of the mixtures of argon (99%) and H

(1%)

in the range of volumes from 50 to 350µl. Calibration

for O

was carried out using air samples with volumes

from 50 to 350 µl.

Statistical analysis. Three biological replicates

were performed for each type of experiment. To

evaluate statistical significance of differences One-

way ANOVA was used together with Tukey test. Dif-

ferences were considered statistically significant

at p < 0.05.

RESULTS

Effect of light intensity on activity of PSII and

yield of H

. In the beginning of the study the effects

of light intensity on activity of PSII were evaluated

in order to determine the level of illumination that

causes significant decrease of the PSII activity but

without its complete inactivation. The obtained data

(Table  2) indicate that the highest decrease of the

Table 1. List of equations used in JIP-test with explanations

Parameters of JIP-test Equation for calculation Interpretation of the parameter

4(F

300µs

 − F

)/(Fm  −  Fo) initial slope of induction curve

2ms

 − Fo)/(F

  −  Fo) relative variable fluorescence at 2 ms

/RC M

∙(1/V

)∙(1  −  V

) electron flow through RC

/RC ABS/RC  −  TR

/RC energy scattered energy by RC

ABS/RC M

∙(1/V

)∙(1/(Fv/Fm)) energy absorbed by RC

PHOTOPRODUCTION OF HYDROGEN UPON PHOTOINHIBITION 937

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Table 2. Effect of light intensity on Fv/Fm and H2 yield in immobilized cells of C.sorokiniana

Intensity of moderate (40)

and photoinhibiting light,

µmol photons∙m

−2

∙s

−1

Fv/Fm

Intensity of illumination after the action

of photoinhibiting light (PFD 1000),

µmol photons∙m

−2

∙s

−1

Yield of H

mmol/m

40 0.704 ± 0.02 40 3.80 ± 0,5

500 0.448* ± 0.03 100 1.14* ± 0.2

1000 0.253* ± 0.02 200 0.76* ± 0.2

Note. Fv/Fm measurement were conducted 30min after incubation of cultures under illumination with light of intensity 40,

500, and 1000µmol photons∙m

−2

∙s

−1

. Prior to measuring Chl fluorescence samples were adapted in the dark for 15min. Yield

of H

was determined after 24-h incubation of the cultures placed in the 18-ml volume vials not bubbled with argon under

light intensity 40, 100, and 200 µmol photons∙m

−2

∙s

−1

. Initial

concentration of Chl ~ 32  µg per strip. *  Significant difference

from the values obtained with illumination of 40 µmol photons∙m

−2

∙s

−1

, p < 0.05.

efficiency ofphotochemical energy conversion in PSII

(parameter Fv/Fm) was recorded at the level of illu-

mination of 1000 µmol photons∙m

−2

∙s

−1

. In this case

the Fv/Fm value decreased by 64% in comparison with

the initial level (from 0.70 to 0.25).

In the second part of the study effects of the light

intensity on efficiency of H

production by the im-

mobilized cultures were examined. First the samples

were subjected to the action of high-intensity light

(1000µmol photons∙m

−2

∙s

−1

) for 30min, and next the

samples were incubated for one day under illumina-

tion 40, 100, and 200 µmol photons∙m

−2

∙s

−1

. Maximal

amount of released H

(3.8 mmol/m

) was recorded

at PFD of 40 µmol photons∙m

−2

∙s

−1

. Increase of illu-

mination to 100 or 200µmol photons∙m

−2

∙s

−1

resulted

in the decrease of H

by 70% and 80%, respectively.

The changes of Fv/Fm and content of O

, H

glucose. In the second stage of the study, duration of

release and its amount during incubation of the

cultures without replacing air with inert gas and in

argon atmosphere were evaluated. As can be seen in

Fig.  1, in both cases the cultures released ~0.1  mol

during 9 days. However, production of H

the cultures not bubbled with argon ceased before this

time point. On the contrary, the immobilized algae cul-

tivated in argon atmosphere continued to release H

for 51 days. Yield of H

was 0.55 mol H

Further study concentrated on the culture not

bubbled with argon, because this approach does not

involve use of expensive argon. Content of O

in the

gas phase of vials decreased gradually from 20% to

9% during the first 8 days of the experiment (Fig.1a).

At the 9th day concentration of O

increased to 14%

and remained at this level until the end of experiment.

To evaluate photosynthetic activity of the algae

the Fv/Fm parameter was used. Value of the Fv/Fm

parameter decreased from 0.7 to 0.25 after initial pho-

toinhibition (Fig. 1a). However, already after one day

the Fv/Fm parameters was recovered to the level of

0.4, and at the 8th day it reached the value of 0.62.

By the 20th day of the experiment the Fv/Fm value

reached control levels (0.7), which indicated complete

recovery of the PSII activity.

Content of glucose in the cells increased by 30%

already after 30min of photoinhibition and continued

to grow increasing more than 5-fold in comparison

with the control level at the 4th day of the experi-

ment. However, the glucose content decreased from

1.13  ng to 0.36  mg on the 5th day of the experiment

followed by the decrease in the next days reaching

minimum value of 0.1  mg at the 13th day of the ex-

periment. This was followed by the gradual increase

of the glucose content up to control levels (0.2  mg).

Analysis of OJIP-transients. Fluorescence induc-

tion curves (OJIP-transients) were recorded and ana-

lyzed to investigate functioning of photosynthetic elec-

tron transport chain (ETC). Typical curves obtained

with the immobilized cultures not bubbled with argon

at the days 1, 4, 8, and 20 of the experiment are pre-

sented in Fig.  2. Typical curve recorded in the control

culture with all phases of fluorescence growth (OJ, JI,

and IP) clearly visible, is shown in the inset. The OJIP-

curves recorded at different days of the experiment

differed significantly in the variable fluorescence (OP).

Minimal OP value was observed at the first day of the

experiment, with amplitude of the OJ phase contrib-

uting significantly to the variable fluorescence, while

the JIP phase was only weakly pronounced. Gradual

increase of OP was observed in the following days,

which indicates growth of the number of active cen-

ters in PSII. The JIP phase became clearly visible on

the 4th day of the experiment.

OJIP-transients were analyzed using JIP-test, which

allows evaluation of the processes of transformation

of light energy in photosynthetic reactions (Table  3).

The Vj parameter, reflecting the portion of closed RC

of PSII uncapable of transfer electron from Q

(prima-

ry quinone acceptor in PSII) to further ETC, was 50%

higher than in the control group during first four

day of the experiment. On the day 8 this parameter

VOLGUSHEVA, ANTAL938

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Fig. 1. Effect of gas phase composition on metabolism and release of hydrogen. Change of content of O

in gas phase,

of glucose in the cells, and of activity of PSII (Fv/Fm) in immobilized algae not bubbled with argon (a). Effect of gas phase

composition on release of H

by C. sorokiniana (b). Algae were cultivated in argon atmosphere (black circles) and with-

out replacing air with inert gas (gray circles). Cultures were incubated in complete HS-medium under illumination with

40 µmol photons∙m

−2

∙s

−1

light. Initial concentration of Chl ~32  µg per strip. Results of a typical experiment are presented

demonstrating average value of H

yield calculated based on the data obtained for 3 vials. Experiments with duration

49 days was repeated twice, and with 21 days – three times.

Fig. 2. Typical OJIP-transients recorded with immobilized

cultures of C. sorokiniana on the 1st, 4th, 8th, and 20th

day after photoinhibition. Algae were cultivated under con-

ditions without air replacement with inert gas in complete

HS medium and illumination with 40 µmol photons∙m

−2

∙s

−1

light. Inset shows a typical curve recorded for immobilized

culture not subjected to photoinhibition, which served as a

control. OJIP transients were recorded one hour after the

end of immobilization and incubation under conditions

described above.

remained higher by 25% than in the control, and by

the day 20 it returned to the control values. The rate

of electron transport to RC (ETo/RC) remained by 60-

70% lower than in the control over the entire dura-

tion of the experiment. The parameter ABS/RC, which

reflects size of antenna, increased more than 6-fold

in the first day. In the following days there was an

insignificant decrease of this parameter, but on the

day 8 it was still 4-fold higher, and at the day 20 –

almost 3-fold higher than the control level. The DIo/

RC parameter characterizing development of non-pho-

tochemical quenching increased more than 9-fold in

the first day of the experiment. This was followed by

its gradual decrease, however, by the day 20 this pa-

rameter was still 2-fold higher than in the control.

Effect of DCMU on H

evolution. Effect of ad-

dition of 10

−5

 M DCMU on the rate of H

produc-

tion (a) and total yield of H

 (b) are shown in Fig. 3.

In the control the rate of H

generation on the days

1 and 2 of the experiment was 12.5 µmol H

−1

Chl h

−1

. Addition of DCMU resulted in the decrease of

the rate of H

production by 30% during the first day.

PHOTOPRODUCTION OF HYDROGEN UPON PHOTOINHIBITION 939

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

Table 3. Parameters of JIP-test calculated based of the fluorescence data obtained from the OJIP-curves

Parameters of JIP-test Control Day 1 Day 4 Day 8 Day 20

Vj 0.52 ± 0.03 0.79* ± 0.02 0.70* ± 0.04 0.65* ± 0.03 0.54 ± 0.05

ABS/RC 0.92 ± 0.21 5.63* ± 0.16 4.69* ± 0.25 3.70* ± 0.34 2.63* ± 0.25

DIo/RC 0.35 ± 0.10 3.35* ± 0.11 2.40* ± 0.18 1.66* ± 0.18 0.71* ± 0.16

ETo/RC 1.61 ± 0.42 0.47* ± 0.51 0.58* ± 0.48 0.63* ± 0.43 0.68* ± 0.45

Note. Mean values based on 3-5 replicated are presented. * Significant difference from the control, p < 0.05.

Fig. 3. Effect of 10

−5

 M DCMU on the rate of H

formation (a) and total yield of H

evolution (b) in the immobilized C.  so-

rokiniana cells cultivated under conditions without replacement of air with inert gas.

After addition of the inhibitor release of H

contin-

ued up to the day 4 of the experiment, however, total

yield of the gas was by 50% lower in comparison with

the control. The rate of H

production in the control

from the day 4 to the day 5 of the experiment was

9.05 µmol H

−1

Chl h

−1

, and addition of DCMU on

the day 4 resulted in the decrease of the rate by 50%.

Accumulation of H

in the gas phase of vials was ob-

served only during the first day after inhibitor addi-

tion, and after that the process completely stopped.

Addition of 10

−5

M DBMIB both in the first and fourth

day of the experiment inhibited formation of H

90% (data not shown).

DISCUSSION

The suggested approach – induction of long-

term photoproduction of H

 – facilitated release of

0.10 mol H

by the immobilized photoautotrophic

C. sorokiniana culture cultivated in the complete me-

dium for 9 days in a natural atmosphere, and 0.55mol

during 51-day cultivation in argon atmosphere.

Reaching these levels of H

production was possible

due to the use of protocol involving initial preillu-

mination of immobilized algae with photoinhibiting

light with intensity 1000 µmol photons∙m

−2

∙s

−1

for

30 min followed by cultivation under light intensity

of 40 µmol photons∙m

−2

∙s

−1

The method of photoinhibition of algae has been

used previously for production of H

. In particular, it

was shown that the phototrophic culture Chlamydomo-

nas reinhardtii, which was pre-treated with nitrogen

bubbling, is capable of long-term (96h) production of

under illumination of 320 µmol photons∙m

−2

∙s

−1

Total yield was 227 µl of H

per ml of culture. To re-

move the released O

a sorbent based on iron salts was

used [31]. An approach has been suggested based on

short-term photoinhibition when the C.reinhardtii cul-

ture grown on the medium with acetate was subjected

to the short-term (15-30 min) illumination with high

light (2000  µmol photons∙m

−2

∙s

−1

). To facilitate anaer-

obic conditions the culture was additionally bubbled

with nitrogen. The following illumination with 15  µmol

photons∙m

−2

∙s

−1

light resulted in production of H

which, however, continued no more than 5  h  [32].

In this study we concentrated on investigation

of photoproduction of H

by the algae cultivated

VOLGUSHEVA, ANTAL940

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

in the air. This approach allows avoiding the use of

costly inert gases, which is significant from econom-

ical point of view. Concentration of O

in the compo-

sition of gas phase in vials decreased from the initial

20.9% to 9% by the day 9 of the experiment (Fig. 1a).

Remarkably, release of H

was observed at O

content

in the gas phase of 16%, which could be explained

by activation of alternative electron transport chains.

Such processes as Meler reaction, photorespiration,

chlororespiration, and reactions with participation of

flavoproteins could results in formation of microan-

aerobic areas in chloroplast, thus protecting hydro-

genase against inactivation by O

 [2,  4,  7,  33]. It must

be mentioned that immobilized cells are arranged

in layers, which could cause differences in physio-

logical states of algae depending on their location.

The surface cells are in contact with external O

, while

the cells in inner layers could be protected form its

effects.

In the time period from day 1 to day 8 of the

experiment the PSII activity (Fv/Fm) was moderate-

ly high (0.4-0.6). However, significant fraction of the

reaction centers in PSII (Vj) were in the closed state

during first 4 days (Table 3). Moreover, electron flow

to RC (ETo/RC) was lower by 60% in comparison with

the control. At the same time, significant increase of

antenna size (ABS/RC) was observed, as well as de-

velopment of the processes of non-photochemical

quenching (DIo/RC). Increase of the antenna size could

be due to the self-shading effect emerging because of

high density of immobilized cells in the alginate layers.

It is known that hydrogenase receives electrons

both from PSII and from reducing equivalents formed

during starch degradation, which are transferred

to ETC via pool of quinones involved in process of

chlororespiration [2-4]. To determine contribution of

PSII-dependent and PSII-independent pathways to pro-

duction of H

, an inhibitory analysis was conducted

on the 1st and 4th days of the experiment. Addition

of 10

−5

  M DCMU that blocks electron flux at the level

of Q

-centers of PSII, decreased the rate of H

release

by 30% in the first day of incubation (Fig. 3a). Hence,

PSII was a minor donor of electrons in comparison

with the electron flux from chloroplast stroma to the

pool of plastoquinones. Addition of DCMU on the 4th

day of experiment, when sharp decrease of glucose

content was observed, resulted in 50% decrease of

the rate of H

production indicate growth of PSII

contribution to hydrogen photoproduction. Addition

of DBMIB that inhibits electron transport at the lev-

el of cytochrome b

f-complex (i.e., blocking electron

flux both from PSII-dependent and PSII-independent

pathways) both on the day 1 and day 4 of the ex-

periment resulted in the significant (90%) decrease

of the rate of H

production. The obtained results

indicate that the electrons coming from PSII and

from the processes of starch degradation play a key

role in generation of H

in C. sorokiniana under the

used experimental conditions.

Differences in contribution of PSII to production

of H

on the first and fourth day of the experiment

could be due to the changes in activity of Calvin cy-

cle. Gradual increase of the glucose level inside the

cells was revealed in the course of our study (Fig.1a).

Nevertheless, significant (68%) decrease of the glucose

level was observed from the day 4 to the day 5 of

the experiment. This fact could be explained by dif-

ferent activity of the reactions of Calvin cycle, which

is the main process competing with hydrogenase re-

action, activity of which could cause termination of

production prior to inactivation of hydrogenase

by oxygen [9].

CONCLUSIONS

Photoproduction of H

by green microalgae is

a promising direction for biotechnology research.

Despite the achieved progress in this area, many issues

remain unresolved. One of the key problems is devel-

opment of economically profitable process capable of

ensuring long-term production of H

by algae cells.

In this study we suggest the method for stimu-

lation of hydrogen photoproduction in the green mi-

croalgae cells based on combination of photoinhibi-

tion induced by short-term illumination of algae cell

with high-intensity light, immobilization of microalgae

in alginate, and photoautotrophic cultivation of cells

in complete mineral medium. This approach allows

achieving long-term production of H

up to 9 day in

the natural air environment and up to 51 days in ar-

gon atmosphere with simultaneous decrease of the

costs of the process. In our experiments total yield

of hydrogen was 0.55  mol  H

in the case of in-

cubation in argon atmosphere, which is equivalent

with regard to Chl to 16.7  mmol H

mg Chl

−1

−2

This result exceeds the previous reported for the free

(non-immobilized) C. reinhardtii cells, which released

for more than 40 days during incubation in the

medium with acetate, but total yield did not exceed

2  mmol  H

  Chl

−1

(0.33  mmol  H

  mg  Chl

−1

) [18].

It is important to note that it is the longest duration

of H

generation reported in the literature.

We also demonstrated in this study that the cul-

ture not-bubbled with argon released 0.1  mol  H

−2

for 9 days. For comparison, the sulfur-starving pho-

toheterotrophic culture of C. reinhardtii in air atmo-

sphere and with similar concentration of Chl releases

0.04  mol  H

 [20]. Under strict photoautotrophic

conditions the non-immobilized sulfur-starving C. re-

inhardtii cells were able to release around 3  mmol

  Chl

−1

  [24].

PHOTOPRODUCTION OF HYDROGEN UPON PHOTOINHIBITION 941

BIOCHEMISTRY (Moscow) Vol. 90 No. 7 2025

The obtained results show that the method has

competitive potential, which could be realized with

optimization of cultivation conditions. However, fur-

ther optimization of the method requires understand-

ing of the mechanisms of the observed glucose con-

tent changes in the microalgae cells during incubation

in air atmosphere and associated changes in activity

of Calvin cycle and respiration, which would require

additional investigations.

Abbreviations. PSII, photosystem II; Chl, chloro-

phyll; DCMU, (3-(3,4-dichlorophenyl)-1,1-dimethylurea)

(diuron); Fv/Fm, efficiency of photochemical energy

transformation in PSII.

Contributions. A. A. Volgusheva – concept of the

study, conducting experiments, and writing text of the

paper; A. A. Volgusheva and T. K. Antal – discussion of

the study results; T. K. Antal– editing text of the paper.

Funding. This study was financially supported by

the State Budget project of the Lomonosov Moscow

State University.

Ethics approval and consent to participate. This

article does not contain any studies with human par-

ticipants or animals performed by any of the authors.

Conflict of interest. The authors of this work de-

clare that they have no conflicts of interest.

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