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. 2022 Jul 15;14(4):893–904. doi: 10.1007/s12551-022-00977-z

The relationship between photosystem II regulation and light-dependent hydrogen production by microalgae

V I Grechanik 1,✉, A A Tsygankov 1
PMCID: PMC9481780  PMID: 36124275

Abstract 

Certain microalgae species are capable of light-dependent hydrogen production under conditions of dark anaerobic incubation or nutrient deprivation. From the biotechnological point of view, this phenomenon is a process of synthesizing the energy carrier H2 while consuming light energy. Here, we overview the functional connection between the photosynthetic machinery and light-dependent hydrogen production and assess the physiological significance of this process. We characterize events involved in PSII downregulation, as well as the relationship between PSII regulation mechanisms and hydrogen generation. We suggest that the light-dependent hydrogen production forms part and parcel of the sophisticated regulatory network ensuring adaptation of microalgae to such environmental stresses as anaerobiosis or nutrient deprivation.

Keywords: Chlamydomonas reinhardtii, Microalgal hydrogen production, Physiological role of hydrogen production, Photosystem II, Nutrient deprivation, Photosystem II downregulation

Introduction 

An environmentally friendly energy production is a pressing issue for sustainable development all over the world. Utilizing fossil fuels proved to be unsustainable due to the limited fossil sources and ultimately inducing the poorly understood global consequences including global warming, greenhouse effects, and pollution. Despite considerable advances in the so-called green energy research, the proposed ways are still far from being reliable or economically valid.

Molecular hydrogen (H2) is commonly considered to be a promising energy carrier since it can be derived from water and produces water upon combustion. Photosynthetic water biophotolysis performed by microalgae is one of the putative mechanisms of hydrogen generation from water (Sakurai and Tsygankov 2019; Petrova et al. 2020; Touloupakis et al. 2021). Most research on hydrogen production used Chlamydomonas reinhardtii as a model organism. This microalga acts as a viable model for research in the assembly and function of the eukaryotic flagellum, chloroplast biogenesis, photosynthesis, nutrient deprivation, dark respiration, and fermentation processes (Harris 2001), as well as in hydrogen production. Thus, the review focuses mostly on the data on this particular organism.

Microalgae under continuous or flashlight with low intensity produce hydrogen with quantum efficiency equal or even higher than the theoretical maximum (Boichenko and Hoffman 1994). Furthermore, the maximum radiant energy-saturated rate of the hydrogen production in C. reinhardtii and Scenedesmus obliquus under optimal conditions reaches the maximum steady-state rate of CO2-dependent O2 evolution (Boichenko et al. 1989). However, implementing microalgal light-dependent H2 production into general practice requires a thorough research into the process as closely tied with other mechanisms of photosynthetic machinery regulation. Although microalgae have potentially high efficiency and rate of hydrogen generation, there are several obstacles for the practical application of the process. They include oxygen toxicity, reduced rates of hydrogen production under stress conditions, and low efficiency of PSII operation under nutrient-deprived conditions. This review describes the modern conceptual landscape in understanding this process focusing on the relationship between the mechanisms involved in the photosystem II downregulation and light-dependent hydrogen production by microalgae.

Photosynthetic apparatus of microalgae

Light is a driving force of hydrogen production by microalgae. Photosynthesis in microalgae resembles photosynthesis in plants though having certain differences. However, these differences are not essential for our considerations. This section shortly describes the photosynthetic machinery in C. reinhardtii without explanation of plant and microalgal differences in photosynthesis.

C. reinhardtii cells contain one chloroplast occupying up to 40% of the cell volume. Chloroplast genome retained core genes inherited from their cyanobacterial ancestor, most of them being required for light reactions of photosynthesis or performing functions related to transcription and translation (Green 2011). Similar to plants and other green microalgae, chloroplast thylakoid membranes contain specialized photosystems, PSI and PSII, both absorbing light energy and converting it into chemical energy of transmembrane proton gradient and NADPH production during the light reactions of photosynthesis, as shown in Fig. 1. ATP (generated by ATP synthase due to proton gradient) and NADPH are further utilized in the Calvin-Benson-Bassham cycle of CO2 fixation and other anabolic processes during the dark reactions of photosynthesis.

Fig. 1.

Fig. 1

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Schematic representation of the photosynthetic electron transfer chain in microalgae. Fd, ferredoxin; FNR, ferredoxin-NADP + reductase; NADP, nicotinamide adenine dinucleotide phosphate; PC, plastocyanine; PQ, plastoquinone; PSI, photosystem I; PS II, photosystem II; LHCII, light-harvesting complex II; NDA2, NADPH dehydrogenase; ATP, adenosine triphosphate

To put it in nutshell, PSII can be functionally divided into a reaction center and the surrounding light-harvesting antennae made up of pigment-protein complexes. The absorption of light photons by the light-harvesting PSII pigment–protein complexes generates the excited singlet electronic state of Chl molecules. This energy is transferred to PS II reaction center (P680). The excited special pair in the reaction center translocates one electron to the pheophytin molecule associated with the D1 protein and then to QA, the plastoquinone molecule harbored by PSII complex (Shevela et al. 2021). When a positively charged reaction center and a negatively charged QA are formed, two chemical events occur. First, oxidation of water to molecular oxygen after four turnovers of PS II and second, reduction of plastoquinone to plastohydroquinone after two turnovers of PS II (Mamedov et al. 2015). Reduced plastoquinone leaves PSII and enters the plastoquinone pool in the membrane with the subsequent electron transfer to the Cyt b6f complex and further to plastocyanine (Fig. 1). Of note, the Cyt b6f complex can be reduced by two electrons from the PQ pool; however, it transfers them to plastocyanine (PC) in one-electron mode.

Similar to PSII, PSI also absorbs photons with the subsequent charge separation in PSI. After that, the PSI reaction center is ready to accept the electron from PC in an one-electron mode. The electron transfer chain of PSI consists of P700 (the primary electron donor), A0 (a pair of Chl molecules), A1 (phylloquinone molecules), and iron–sulfur clusters Fx, FA, and FB (Mamedov et al. 2015). The final step is the electron transfer to ferredoxin (Fd) which serves as a hub that transfers electrons to ferredoxin-NADP + reductase (FNR) in classic photosynthesis or hydrogenase in hydrogen production. Note that thirteen ferredoxin genes have been identified in the C. reinhardtii genome; however, only six (PETF and FDX2-6) have been characterized in any detail (Sawyer and Winkler 2017). PETF can also transfer electrons to a large number of other proteins. One important PETF electron acceptor found in green microalgae is the biologically and biotechnologically important [FeFe]-hydrogenases HYDA1-2, which catalyzes the production of molecular hydrogen (H2) from protons and electrons (Sawyer and Winkler 2017).

Hydrogen production by microalgae

Microalgae are able to produce molecular hydrogen under light after dark anaerobic adaptation. Noteworthy, this anaerobic adaptation is a prerequisite for the hydrogenase expression in microalgae (Forestier et al. 2003; Ghirardi et al. 2005). Although several authors reported about hydrogen production and hydrogenase synthesis under aerobic conditions (Liran et al. 2016; Nikolova et al. 2018), the hydrogenase content was very low and used cultures should be checked for the inhomogeneity. Light absorption activates the photosynthetic ETC and electrons are transferred from water to ferredoxin (Fd) as shown in Fig. 1. Under anaerobic conditions, Fd can transfer electrons to hydrogenases which catalyze the reversible reaction.

2H++2e-↔H2

C. reinhardtii has two hydrogenase genes hydA1 and hydA2 (Forestier et al. 2003; Ghirardi et al. 2007). Both genes encode monomeric FeFe hydrogenases that contain the FeFe active site. Among other FeFe hydrogenases and all NiFe hydrogenases, hydA1 and hydA2 exert highest activity towards hydrogen production (Vignais et al. 2001; Vignais and Billoud 2007). Another specific feature of FeFe hydrogenases is their exceptionally high sensitivity to oxygen toxicity. Oxygen represses the synthesis and inhibits hydrogenase activity in C. reinhardtii (Yanyushin 1982; Ghirardi et al. 1997) and other microalgae.

Figure 1 shows that Fd is the turning point of ETC that switches between ordinary photosynthesis and hydrogen production (Tsygankov and Abdullatypov 2016; Grechanik and Tsygankov 2021). Many species of microalgae have a number of genes encoding ferredoxins. For example, Chlamydomonas reinhardtii expresses at least six different ferredoxins (Winkler et al. 2010). While most of them can interact with hydrogenase, petF ferredoxin has the highest reaction rate (Terauchi et al. 2009).

Upon anaerobic adaptation, the microalgal cells switch to anaerobic energy acquisition via decomposing the accumulated starch when the Calvin cycle is not active and cells are staying in the overreduced state. This explains why the entire photosynthetic electron flow is directed towards a hydrogenase immediately after illumination, and the rate of H2 production reaches the highest photosynthetic rate (Boichenko et al. 1989). During illumination, oxygen concentration grows, while inhibition of hydrogenase activity increases. As a result, the rate of hydrogen production declines. Thus, hydrogen production by microalgae adapted to anaerobiosis is a transient process with the rate decreasing from maximum hydrogen production rate with zero of usual photosynthesis to zero hydrogen production rate and maximum of conventional photosynthesis. Depending on the experimental conditions, this process lasts from several seconds to several minutes (Boichenko and Hoffmann 1994; Boichenko et al. 2004).

High sensitivity of hydrogenase to oxygen hampers implementing this process in biofuel (molecular hydrogen) production.

To overcome the instability of hydrogen production by microalgae, different approaches were applied such as adding DCMU, oxygen consumption with dithionite or other reduced compounds, and oxygen evacuation with inert gas purging through the culture (reviewed in Grechanik and Tsygankov 2021; Touloupakis et al. 2021)). However, these methods did not provide long-term (days) and stable hydrogen production. Furthermore, these methods are not cost-effective.

Spatial and temporal separation of microalgal oxygen and hydrogen production

Temporal and spatial separation of oxygen and hydrogen production while growing microalgae under optimal conditions for starch production with the following fermentation of microalgal biomass is another promising approach aiming to circumvent the difficulties outlined above. This approach was developed in 1980s (Boichenko 1994). The process appears to be stable and provide moderate hydrogen yield however reduced by organic acids, by-products of fermentation. This two-stage approach has been recently redesigned into a three-stage process. The third stage involved the photofermentation of organic acids produced during fermentation stage by purple nonsulfur bacteria (Gavrisheva et al. 2015). Experiments proved the process to be reproducible and stable. Hydrogen was produced during the dark fermentation of microalgal biomass as well as by purple bacteria using the artificial fermentation broth without ammonium. Further detailed analysis showed that the efficiency of light energy bioconversion into an energy carrier (hydrogen) was low (0.55%). Furthermore, successful fermentation required preliminary processing of microalgal biomass increasing the cost of the three-stage process while reducing the efficiency of light energy bioconversion.

Another way to eliminate oxygen from the medium is to co-cultivate hydrogen-producing algae and chemotrophic bacteria (Xu et al. 2016; Ban et al. 2018). Chemotrophic bacteria consume oxygen produced by microalgae, thus maintaining anaerobic conditions. Yet, this method requires the presence of oxidizable organic substrates in the medium complicating it even further.

Temporal separation of microalgal oxygen and hydrogen production by sulfur deprivation

Two decades ago, Melis et al. showed that during adaptation to sulfur deficiency, sulfur-deprived C. reinhardtii cells temporally separated oxygen and hydrogen production in a two-stage process (Melis et al. 2000). During the first stage, cultures accumulated starch followed by a sulfur deficiency-induced decrease in PSII activity. The cultures entered anaerobiosis and produced hydrogen at the second stage which lasted for days (Melis et al. 2000). This work promoted research into hydrogen production by nutrient deficient microalgae all over the world.

Detailed analysis showed that this process, commonly divided into two stages (oxygen production in the first stage and hydrogen production in the second one), in fact involved five steps (Kosourov et al. 2002):

  • The aerobic (oxygen production) stage. At this stage, cells still exert high photosynthetic activity and produce oxygen. Sulfur deficiency is indicated by predominant starch accumulation accompanied by the insignificant growth of biomass and chlorophyll content (unbalanced cell growth). pH is generally elevated; medium redox-potential indicates oxidative conditions while acetate is consumed. Finally, starch is accumulated and the photosynthesis rate decreases.

  • The oxygen consumption stage. At this stage, the photosynthesis rate is lower than the respiration rate reducing oxygen content. The effective quantum yield of PSII (Y(II)) measured by PAM fluorimetry gradually declines (Antal et al. 2003) as well as PSII putative activity (Melis et al., 2000). Redox-potential of the medium decreases but still demonstrates certain oxidative conditions; starch content is usually reduced, while acetate is consumed. Depending on light illumination, temperature, pH, duration of the 1st stage and some other factors, the Chl (a + b) content either is stable or declines.

  • The anaerobic stage. It starts when oxygen content in the medium falls below 1 µM and is accompanied by dramatic changes in the culture. The effective quantum yield Y(II) drops to a very low value in 5–15 min (Antal et al. 2003; Touloupakis et al. 2021); anaerobic metabolism is activated. Redox potential can achieve − 200 ÷  − 500 mV (relative to the Ag/AgCl couple) in 30–90 min. Under anaerobic conditions, this parameter is determined by the presence of reduced compounds derived from fermentation, such as formate (Tsygankov et al., 2002). The expression of the hydrogenase gene is observed. At the end of this stage, Y(II) slightly goes up.

  • Hydrogen production stage. Oxygen is absent in the medium, and redox potential and pH are stable or decline due to the accumulation of fermentation products. Starch content is also reduced being the main source for fermentation. Evidently, at this stage, three different metabolic modes coexist in the culture: photosynthesis indicated by the presence of Y(II); respiration which evidently consumes traces of the oxygen produced by photosynthesis, and fermentation indicated by an increase in the acetate content goes up, and accumulation of ethanol and formate along with the starch degradation (Grechanik and Tsygankov 2021).

  • The termination stage. During this stage, hydrogen is not produced; however, respiratory and photosynthetic activity are still observed.

The duration of each stage depends on the light intensity, cell concentration, phase of cell cycle division, culture prehistory, pH, acetate concentration, sulfur deprivation method, the sulfur concentration at the beginning of the deprivation, and many other factors. For example, the oxygen production stage can vary from 17 to 53 h depending on the cell cycle in the synchronous culture at the beginning of the experiment (Tsygankov et al. 2002).

After the first publication, hydrogen production by sulfur-deprived microalgae was scrutinized by many research groups worldwide. The role of pH (Kosourov et al. 2003), light intensity (Laurinavichene et al. 2004), adding different uncouplers like carbonyl cyanide m-chlorophenylhydrazone (Zhang et al. 2012), and sulfur transport were thoroughly examined (Chen et al. 2005). The profile analysis of the selected photosynthetic proteins showed a drastic decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) during S deprivation, a more gradual decline in the level of PSII and PSI proteins, and an altered composition of the PSII light-harvesting complex (LHC-II) (Zhang et al. 2002). For the sulfur deprivation, a dilution method was developed (Laurinavichene et al. 2002).

The majority of researchers used photoheterotrophic microalgae in their experiments since inducing hydrogen production is rather simple in these cultures. However, hydrogen production requires acetate along with light energy. To develop hydrogen production into a method of acetate-independent light energy bioconversion, researchers designed a protocol for photoautotrophic hydrogen production by microalgae (Tsygankov et al. 2006; Kosourov et al. 2007). The authors showed that S-deprived photoautotrophic cultures passed through the same stages as photoheterotrophic cultures.

Thus, although the sulfur deprivation protocol has not been implemented into a biotechnological process of renewable H2 production, it served as a useful tool for studying microalgal metabolic and photosynthetic flexibility potentially opening up the avenue for future H2 production procedures.

Hydrogen production under limitation by other nutrients

Detecting hydrogen production by S-deprived cultures raises the question of whether microalgae could produce hydrogen under deprivation by other nutrients. Various research groups eliminated different mineral components from the medium and studied hydrogen production by microalgae under deficiency of various nutrients.

Excluding the combined nitrogen from the medium was shown to induce N deprivation of C. reinhardtii (Philipps et al. 2012) and Chlorella protothecoides (He et al. 2012; Zhang et al. 2014). Upon N deprivation, the cultures produced hydrogen in photoheterotrophic culture. Similar to S deprivation, the adaptation of microalgae to N deprivation brought about a decline in the PSII activity, transition to anaerobic conditions, and accumulation of starch and lipids (Philipps et al. 2012). In contrast to sulfur deprivation, the decrease in PSII activity was much slower. Furthermore, the Cyt b6f complex underwent degradation upon N deprivation. As a result, the electron transport to PSI was inhibited leading to a much lower H2 production as compared to S deprivation. In previous studies, in N-deprived photoautotrophic cultures of C. reinhardtii, hydrogen was not detected under high light intensity (Grechanik et al. 2021). Hydrogen production was observed only under a particular light illumination protocol: high light intensity during oxygen production and oxygen consumption stages followed by a low light intensity at the end of the oxygen consumption stage and at the later stages.

P deprivation of microalgae could not be achieved by the simple elimination of phosphates due to the intracellular phosphorus pool. For P deprivation of C. reinhardtii, cells were inoculated in -P medium at low concentrations (Batyrova et al. 2012). After the initial growth period, when cells had utilized intracellular phosphorus, algae established the anaerobic environment followed by the period of H2 photoproduction. Under optimal concentration of the inoculum, H2 production was ~ 70 ml per liter of culture. P-deprived cultures passed similar stages of adaptation to the deprivation as S-deprived cultures. After demonstrating H2 production by P deprived cultures, it was observed in marine microalgae Chlorella sp. upon P deprivation as well (Batyrova et al. 2015). The previous attempts to detect H2 production by marine microalgae under S deprivation were not successful due to the high content of sulfur in seawater.

The processes triggered by excluding other mineral components in the medium were studied by different groups (Papazi et al. 2014; Volgusheva et al. 2015; Touloupakis et al. 2021). However, data analysis is complicated since the authors did not show that exclusion of a particular element led to deprivation and/or limited cell growth.

Physiological importance of light-dependent hydrogen production by microalgae

The physiological significance of light-depended hydrogen evolution in microalgae has been widely discussed since it was initially discovered. Kessler suggested that during the transient period from dark anaerobic to light aerobic conditions, hydrogenases enable the rapid oxidation of reduced PSII (Kessler 1973). It accelerates the transition from a fermentative to a photosynthetic mode of energy metabolism. Later, this suggestion was revisited. The hydrogenases were proposed to serve as important regulatory devices for proper redox poising (Appel and Schulz 1998). Given the accumulated evidence in the last decades, this hypothesis seems fairly reasonable.

The microalgae adapted to anaerobiosis in the darkness do not fix CO2 since substrate phosphorylation cannot support this energy-demanding process (2 ATP molecules are produced during decomposition of one glucose molecule during glycolysis but 30 ATP molecules are consumed in Calvin-Benson-Bassham cycle for one glucose molecule synthesis). Upon illumination after the dark period, photosynthesis is inhibited since all cellular components including ETC are over-reduced. The latter is a common problem of fermentation. To facilitate switching from the dark anaerobic to oxygenic photosynthetic metabolism, the hydrogenases ensure photosynthesis proceeding with a near-maximum rate, accompanied by H2 production in the absence of NADPH formation. Likewise, oxygen is produced at a maximum rate. As a result, cells accumulate oxygen and establish aerobic conditions followed by the ETC oxidation and switching to the photosynthetic mode of energy metabolism.

This hypothesis allows explaining the highest rate of photosynthesis upon transition from dark anaerobic to light conditions. However, while explaining the significant production of H2 under nutrient deprivation, additional considerations should be taken into account. The microalgae deprived of S, N, or P have a low rate of photosynthesis due to the over-reduction of photosynthetic ETC. S-deprived photoheterotrophic anaerobic cultures exhibit that photosynthesis, respiration, and fermentation are going on simultaneously (see Sect. 3.2). In the absence of photosynthesis, no oxygen is produced and respiration is impeded. That is why cells have only substrate phosphorylation for the energy metabolism. Therefore, light-dependent hydrogen evolution oxidizes photosynthetic ETC and simultaneously maintains the transmembrane proton gradient high enough for ATP synthesis and thus generates oxygen required for respiration supplying cells by ATP.

The ratio between photosynthesis, respiration, and fermentation is not constant depending on numerous environmental factors. Therefore, the photosynthetic hydrogen production should be considered as a constituent element of a complex regulatory network maintaining intracellular redox balance and ATP supply through several growth modes.

This hypothesis could be tested by comparing the wild-type strains and mutants lacking the hydrogenases (hyd−). Comparing the viability of S-deprived hyd− mutants with the wild-type strains led to contradictory results. Direct counting of colony-forming units demonstrated that under sulfur deprivation of photoheterotrophic cultures, the viability of the wild-type stains was similar to hyd− mutants (Tolstygina et al. 2006). However, measuring the percentage of dead cells by staining time in a buffer with phenosafranine, methylene blue, and ethanol showed that upon S deprivation under anaerobic conditions, the percentage of dead cells was significantly elevated in hyd− mutants (Antal et al. 2020). However, the hyd− mutants were still alive under anaerobic S-deprived conditions. Probably, hyd− mutants possess different regulating mechanisms that circumvent for hydrogen evolution process.

This suggestion was supported by Ghysels et al. who studied the time required for transferring from anaerobic to aerobic conditions upon illumination in the wild-type strain, hyd− mutant (maturation mutant, hydEF-), and double mutant (hydEF stt7-9) without hydrogenases and locked in the state 1 (Ghysels et al. 2013). The wild strain was shown to produce detectable levels of O2 after 125 s of illumination, more rapidly than the mutant that lack hydrogenases (230 s). However, the double mutant did not yield any detectable levels of O2 even after 600 s. Therefore, hydrogen production seems not to be the single mechanism of regulating the electron flow through ETC. This conclusion is corroborated by the existence of microalgae lacking hydrogenases and their ability for a successful transition from anaerobiosis to conventional photosynthesis.

PSII activity and hydrogen production in microalgae

Upon illumination and after dark anaerobic adaptation, microalgae typically produce hydrogen as described above. Simultaneously, PSII donates electrons to photosynthetic ETC via water oxidation accompanied by O2 production (Fig. 1), thus providing electrons for hydrogen production. In turn, the generated oxygen is toxic to hydrogenase(s) inhibiting hydrogen production. Therefore, PSII exerts dual effects as it provides electrons for hydrogen production, thus being directly involved in it, and at the same time, it generates oxygen, thus providing negative feedback to it. Noteworthy, electron production by PSII is coordinated with the overall activity of the photosynthetic ETC. PSII activity different from the potential rate of ETC is tightly modulated by several regulatory chains. Since stable hydrogen production by microalgae upon nutrient deprivation required a decreased PSII activity, we will shortly describe the regulatory mechanisms of PSII activity.

The downregulation of PSII activity occurs at different levels, including photosynthetic antennas, reaction centers of photosynthesis, ETC modifications, metabolic changes induced by additional protein synthesis, and chloroplast movement.

Non-photochemical quenching (NPQ)

The light energy captured by chlorophyll molecules can have several routes: fluorescence, internal conversion, photochemical reaction, energy transfer to neighboring molecules, intersystem crossing, and excitation quenching by molecular species (Goltsev et al. 2016). Fluorescence yield is affected by the alterations in any of these pathways. This explains the decrease in the yield of chlorophyll fluorescence that is not caused by photosynthetic activity and thus termed as non-photochemical quenching (NPQ), which is attributed to several mechanisms. This network of regulatory pathways is essential for the adaptation of microalgae to changing environment.

Under the excessive light, the most rapid mechanism of NPQ, operating within the range of seconds to minutes, is commonly ascribed to energy dissipation (qE). In C. reinhardtii, the LHCSR1 and LHCSR3 proteins have been reported to be essential for qE induction (Peers et al. 2009; Bonente et al. 2011). Both proteins are located inside the thylakoid membrane and have the acidic residues exposed to the luminal side that are responsible for sensing acidification and triggering heat dissipation (Ballottari et al. 2016; Camargo et al. 2021). LHCSRs in algae are pigment-binding proteins containing 6–7 Chls and 2–3 carotenoids with expression largely conditioned by the environment (Peers et al. 2009; Bonente et al. 2011). LHCSR3 is expressed in the presence of blue light of high intensity (Petroutsos et al. 2016), low CO2 (Miura et al. 2004), and under various stress conditions. LHCSR1 is expressed upon UV-B radiation as well (Allorent et al. 2016). The pH-dependent activation of quenching mechanisms mediated by LHCSR3 depends on acidic residues E221, E224, and D117 involved in the pH-dependent qE activation via lutein radical cation formation (Ballottari et al. 2016), while acidic residues at the C-terminal loop (E231, E233, E237, D239, D240, E242, D244, and D254) mediate the pH-dependent activation of the other LHCSR3 quenching mechanisms which do not depend on the interactions between Chls and Cars and could involve the Chl-Chl interactions (Camargo et al. 2021).

The slower components of NPQ are related to the enhanced energy dissipation due to zeaxanthin (qZ) accumulation through the xanthophyll cycle (Goldschmidt-Clermont and Bassi 2015). De-epoxidation of violaxanthin to zeaxanthin through antheraxanthin occurs in the light and leads to an elevated dissipation of excess excitation energy in the photosystem II (PSII) antenna system preventing the inactivation and damage of the photosynthetic apparatus (Goss and Jakob 2010). Excess excitation energy is dissipated as heat that can be registered through NPQ of chlorophyll fluorescence. The reverse conversion of zeaxanthin to violaxanthin takes place in the dark. In vascular plants and green algae, enhanced thermal dissipation of excessive light energy requires the presence of both a trans-thylakoid proton gradient and high concentrations of zeaxanthin (Goss and Jakob 2010).

The state transition component of NPQ operates within a few minutes and reflects the dynamic allocation of light-harvesting antennae LHCII to the photosystems (qT) (Goldschmidt-Clermont and Bassi 2015). State transitions are regulated by the redox state of the PQ pool. It is activated when plastoquinol occupies the quinol oxidation site of the Cyt b6f complex (Vener et al. 1997). In state 1, upon PQ pool oxidation, the LHCII antenna is connected to PSII. The PQ pool over-reduction induces transition to state 2: a mobile component of the LHCII detaches from PSII and at least partially binds to PSI. The cross-sections of PSII and PSI decreased and increased, respectively, leading to re-balancing the PQ pool redox status. Conversely, the over-oxidized PQ pool induces a transition towards state 1 with the mobile antenna from PSI dissociated and further attached to PSII. Experimental conditions, can manipulate the system transition between to states 1 or 2. However, natural and mild environment tends to maintain the dynamic balance with LHCII being only partially bound to PSII and to PSI (Goldschmidt-Clermont and Bassi 2015).

A key component in state transitions in C. reinhardtii is the chloroplast protein kinase Stt7 (Stn7 in Arabidopsis) that senses the redox state of the plastoquinone pool (Lemeille et al. 2010). When photosystem II is excessively excited, this kinase is activated via the cytochrome b6f complex. This is required for the phosphorylation of the light-harvesting system of photosystem II, which partially migrates to photosystem I (state 2). Excessive excitation of photosystem I inactivates Stt7 causing LHCII dephosphorylation and its translocation back to photosystem II (state 1). Therefore, state transitions can be considered as a regulatory mechanism ensuring a homeostatic regulation of the redox poise in the photosynthetic electron transfer chain.

Linear electron flow depends on both photosystems and generates NADPH as well as ATP, while cyclic electron flow around PSI produces only ATP. State 2, which favors PSI excitation, can enhance the cyclic electron flow rate. Apart from the impact on the Stn7-dependent PSI cross-section, state 2 induce a regulatory switch from the linear electron flow to the cyclic electron flow in C. reinhardtii (Finazzi et al. 2002).

Photoinhibition

Photoinhibition of photosystem II (PSII) is the light-induced loss of oxygen evolution and PSII electron transport activity. Since its discovery, the photoinhibition was thought to occur only in the strong light. It destroys the D1 protein in PSII which does not happen upon a low light intensity. However, further insights into this process implied that the D1 protein degradation takes place under any light intensity and correlates with the loss of PSII activity (Tyystjarvi and Aro 1996). According to the mechanism of photoinhibition suggested in (Nishiyama and Murata 2014), the excess light energy generates reactive oxygen species such as H2O2 and singlet oxygen. Monitoring photodamage and the PSII repair separately revealed that the repair is suppressed by the factors also modulating the photoinhibition. Reactive oxygen species affect PSII by inhibiting protein synthesis rather than directly being essential for the PSII repair. Salt, temperature, and oxidative stress decrease the maximum repair rate thus facilitating the photoinhibition (Allakhverdiev and Murata 2004).

The oxygen-independent mechanisms of the PSII downregulation

Studying chlorophyll fluorescence decay kinetics in picosecond time resolution suggested some oxygen-independent mechanism to inactivate PSII in S-deprived C. reinhardtii (Volgusheva et al. 2007). The authors concluded that a decline in the PSII photochemistry in S-deprived cells resulted from the stably reduced QA forms that in their turn triggered the electrostatic repulsive action on the primary radical pair. The stably reduced QA states can be protonated and dissociate from the D1-protein inducing the protease-dependent PSII disassembly. This process does not require oxygen and might play an important role in the subsequent anaerobic phase of S deprivation (reviewed in (Antal et al. 2011)). Evidently, it is induced by the over-reduced PQ pool, which explains why it can take place even under aerobic conditions.

Another mechanism of the oxygen-evolving activity downregulation was proposed in (Nagy et al. 2016, 2018). In C. reinhardtii, the D1 protein (PsbA) has a significant turnover under S deprivation. However, the other PSII subunits, RbcL and CP43, are degraded more rapidly than D1 protein. GDP-L-galactose phosphorylase significantly contributing to ascorbate biosynthesis is upregulated as well. In millimolar concentrations, ascorbate can inactivate the oxygen-evolving complex and provide electrons to PSII instead of the oxygen-evolving complex. In the absence of a functional donor and sufficient electron transport, the PSII reaction centers are inactivated and degraded.

The PSII downregulation under nutrient deprivation

The PSII activity in microalgae has been consistently reported to decrease upon sulfur, nitrogen, or phosphorus deprivation (Wykoff et al. 1998; Melis et al. 2000; Batyrova et al. 2012; Philipps et al. 2012; Shastik et al. 2020). However, various mechanisms were suggested to mediate the PSII degradation (Table 1).

Table 1.

Mechanisms of PSII downregulation in sulfur deprived hydrogen-producing microalgae 

Mechanisms of PSII downregulation Culture conditions, strain Occurrence under S deprivation Reference
1 Accumulation of QB non-reducing centers S deprivation and P deprivation under aerobic conditions, photoheterotrophic C. reinhardtii Yes (Wykoff et al. 1998; Melis et al. 2000)
2 Over-reduction of PQ pool S deprivation under anaerobic conditions, photoheterotrophic C. reinhardtii Yes (Antal et al. 2003)
3 Appearance of stably reduced forms of QA S deprivation under anaerobic conditions, photoheterotrophic C. reinhardtii Yes (Volgusheva et al., 2007)
4 State transitions S deprivation and P deprivation under aerobic conditions, photoheterotrophic C. reinhardtii Yes (Wykoff et al., 1998)
5 Xanthophyll cycle S deprivation and P deprivation under aerobic conditions, photoheterotrophic C. reinhardtii Yes (Wykoff et al., 1998)
S deprivation and P deprivation under aerobic conditions, photoheterotrophic C. reinhardtii No (Antal et al., 2006)
6 D1 degradation S deprivation under anaerobic conditions, photoheterotrophic C. reinhardtii Yes (Zhang et al., 2002)
S deprivation under anaerobic conditions, photoheterotrophic C. reinhardtii No (Nagy et al., 2018)
7 RbcL and CP43 degradation S deprivation under anaerobic conditions, photoheterotrophic C. reinhardtii Yes (Nagy et al., 2018)
8 Photoinhibition (oxidative stress) Early stage of S deprivation, photoheterotrophic C. reinhardtii Yes (Nagy et al., 2018)
9 Donor side disorder S deprivation under anaerobic conditions, photoheterotrophic C. reinhardtii Yes (Antal et al., 2006)
S deprivation under anaerobic conditions, photoautotrophic C. reinhardtii Yes (Grechanik et al. 2020)
10 Accumulation of ascorbate, Inactivation of an oxygen-evolving complex and substitution of the complex for electron donation to PSII S deprivation under anaerobic conditions, photoheterotrophic C. reinhardtii Yes (Nagy et al., 2018)
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Comparing PSII downregulation variants in nuance indicates several overlapping mechanisms. For example, the over-reduction of the PQ pool (position 2 in Table 1) often leads to the accumulation of stably reduced QB which results in the accumulation of overreduced QA in the light (position 3 in Table 1) and finally in an increased population of QB non-reducing centers (position 1). Stably reduced QA induces the electrostatic repulsive action on the primary radical pair with the following QA disconnection from the D1 protein (Volgusheva et al. 2007; Antal et al. 2011).

So, position 3 describes several consequent steps from PQ over-reduction to the functional inactivation of PSII resulting from the inability of D1 protein to interact with QB.

Another example is photoinhibition (position 8, Table 1), that is the light-induced reduction of the photosynthetic activity in organisms with oxygenic photosynthesis. Photosystem II (PSII) is more sensitive to light than the rest of the photosynthetic machinery. That explains why photoinhibition is commonly believed to be induced by light-induced damage of PSII. In living organisms, photoinhibited PSII centers are continuously repaired via the cleavage of the key D1 protein followed by its de novo synthesis and the PSII photosynthetic reaction center reassembling (Nishiyama and Murata 2014). Thus, position 6 in Table 1 provides a detailed description of position 8 (photoinhibition). Furthermore, all light-induced phenomena that reduce photosynthesis efficiency are often regarded as photoinhibition (Tyystjarvi and Aro 1996). In this term, the degradation of the RbcL and CP43 components of PSII (position 7, Table 1) can be considered as photoinhibition as well.

Noteworthy, there are certain contradictions in the data on downregulation mechanisms. The earliest observations revealed the degradation of the D1 protein (position 6, Table 1) along with the main components of rubisco in S-deprived C. reinhardtii (Zhang et al. 2002). In contrast, a later study reported the degradation of RbcL and CP43 in the absence of a significant loss of the D1 protein (Nagy et al. 2018).

Table 1 lists virtually all known mechanisms of the PSII downregulation which sometimes operate at the same time. For example, in the microalgal cultures adapted to low light intensity, NPQ qE, qZ, and qT are elevated right after being exposed to strong light under nutrient deprivation, similarly to non-deprived cultures (see Sect. 5.1). At the same time, the photoinhibition results from the excess of energy and oxygen as well as reactive oxygen species elevation. This is often observed in the cultures adapted to low light and then exposed to strong light (Nishiyama and Murata 2014).

On the other hand, there is a possibility that different mechanisms of PSII downregulation operate sequentially and can be activated at different stages of S deprivation. The second suggestion seems to be true. This suggestion is based on a number of observations that the PQ pool over-reduction varies at different stages of S deprivation and is further supported by the fact that PSII undergoes significant changes immediately after the switch to anaerobiosis (Antal et al. 2003). The number of closed reaction centers is different at the aerobic and anaerobic stages (Volgusheva et al. 2007).

Along with S deprivation, the particular mechanism of PSII downregulation to be chosen depends on a number of environmental factors such as light intensity, temperature and pH of cultivation, the presence of salt, Chl concentration, and the photobioreactor geometry as well as microalgal culture prehistory. Although it is almost next to impossible to predict the putative mechanism employed, our current understanding allows for making certain assumptions. For example, in photoautotrophic C. reinhardtii cultures, pre-grown under low light and incubated under S-deprivation and low light, the transition to anaerobiosis, required long incubations or was not observed at all (Tsygankov et al. 2006). Under particular conditions, the D1 degradation rate was very slow implying that other mechanisms underlying PSII downregulation are also involved. Conversely, the anaerobic stage starts in 24–25 h after being exposed to strong light during S deprivation in photoautotrophic culture adapted to the moderate light intensity (Tolstygina et al. 2009). Furthermore, the oxygen-consuming stage (the stage with the highest PSII degradation rate) lasted for 2–3 h, which takes less time than the adaptation to low light. Indeed, D1 degradation is observed in many photosynthetic cultures. This mechanism might be utilized in the cultures characterized by PSII degradation induced by the ascorbate accumulation. However, further research is required to validate these assumptions as well as to determine the individual contribution of S, P, or N deprivation and other environmental factors to the PSII downregulation mechanisms in various photosynthetic organisms.

In conclusion, the light-dependent hydrogen production in living photosynthetic microalgae accelerates the transition of cultures from anaerobic to aerobic conditions. Under S deprivation microalgal cultures downregulate PSII activity. The survey of literature shows that different authors observed the incorporation of various mechanisms of PSII downregulation. We suggest that all reported mechanisms can be realized and the particular scenario of downregulation depends on environmental factors including light intensity, temperature, pH of cultivation, the presence of salt, Chl concentration, and the photobioreactor geometry as well as microalgal culture prehistory. Furthermore, under S deprivation, after initial PSII downregulation, the start of hydrogen production upregulates PSII supplying cells with energy for anabolic processes increasing transmembrane proton gradient even if most part of the light energy is wasted. However, this suggestion needs an experimental verification.

So, very intensive research on microalgal hydrogen production for the last two decades does not transfer this process from laboratories to industry. However, the conversion of light energy to hydrogen that serves as an energy carrier appears to be a very useful tool for studying hydrogen metabolism and designing new approaches that could be employed for future artificial photosynthesis.

Author contributions

All authors contributed to the study conception and design. The authors read and approved the final manuscript.

Funding

This work was supported by the Russian Science Foundation grant # 19–14-00255, https://rscf.ru/en/project/19-14-00255/.

Declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Conflict of interests

The authors have no relevant financial or non-financial interests to disclose.

Footnotes

Publisher's Note

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