Transcription of the Hydrogenase Gene during H2 Production in Scenedesmus Obliquus and Chlorella Vulgaris

There is ongoing research related to the production of molecular hydrogen today and algae have proven to be good biological models for producing several compounds of interest. We analyzed how genetic variations in hydrogenase genes (hyd) can affect the production of molecular hydrogen in the algae Chlorella vulgaris and Scenedesmus obliquus. Through isolation and genetic characterization of hyd genes in S. obliquus and C. vulgaris, we made in-silico 3D modeling of the hydrogenase proteins and compared these in 11 algal genera. The 3D structure of hydrogenases indicated its structural conservation in 10 genera of algae, and the results of our grouping according to the aa characteristics of the proteins showed the formation of two groups, which were unrelated to the algae’s phylogenetic classication. By growing C. vulgaris and S. obliquus in anaerobic conditions (in darkness) during 24 h and after exposing the cultures to light, we observed H 2 production values of 9.0 ± 0.40 mL H 2 /L and 16 ± 0.50 mL H 2 /L, respectively. The highest global relative expression of hyd genes was reached during the rst 30 min of exposure to light. The behavior of the expression of the hyd genes in these species of algae proved to be species specic and involved in the production of H 2 . Future identication of isoforms of hyd genes in algae would allow a better understanding of the regulation of the hydrogenase enzyme.


Introduction
The pressure exerted on the environment by the demand for natural resources has promoted proposals for improving and developing new production and biotechnology models compatible with the development of alternative energies (Dincer and Acar 2015; Nikolaidis and Paullikkas 2017). Molecular hydrogen (H 2 ) is a fuel alternative due to its high energy content and its relatively clean combustion (Show et al. 2018; Srivastava et al. 2020). Currently, the global annual hydrogen production is approximately 0.1 GT, which is widely applied for re ning and metal treatment, and a small fraction of it is used as automobile fuel (Nikolaidis and Paullikkas 2017; Sharma and Ghoshal 2015). H 2 is also used as a source of energy, and the generation of electricity by means of fuel cells is already in the expansion phase in the US and the UK, making its use as an energy source to become a reality (Staffell et  Algae are organisms capable of using sunlight for water oxidation through oxygenic photosynthesis. In algae, the light energy absorbed during photosynthesis facilitates the oxidation of water molecules, the release of electrons and protons, and the endergonic transport of these electrons to ferredoxin. Then, the enzyme hydrogenase reduces the protons to hydrogen, oxidizing ferredoxin, which passes from its reduced state to the oxidized state (Rochaix 2020).
The ability of algae to generate molecular hydrogen through oxygenic photosynthesis has led related research to run in two directions: the optimization of abiotic factors (pH, temperature, and light quality, Green algae have demonstrated their ability to decompose water into H 2 and O 2 aided by light energy. Two photosynthetic systems are involved in this process: photosystem I (PSI) and photosystem II (PSII).
In PSII, photons derived from light energy split water into O 2 and electrons. The electrons are then activated in PSI, which will reduce ferredoxin (Fd). By the activity of hydrogenases, Fd (red) can be reoxidized, forming H 2 [FeFe]-hydrogenase mediates this reaction in algae ).
Photobiological production of H 2 seems to be an economically viable alternative; however, at present it has not yet been fully understood how the activity of enzymes involved in the production of molecular hydrogen in algae is regulated. Recently, attention has been given to hydrogenase enzymes. In green microalgae, [FeFe]-hydrogenases are enzymes localized in the chloroplast that release electrons from H 2 and oxidize molecular hydrogen into two protons and two electrons (Carrieri et al. 2011), whose activity has been reported to be induced through anaerobic adaptation in culture supplemented with argon or nitrogen and incubated in the dark (Florin et  However, the induction of hydrogenases has been shown to be transient and regulated by O 2 and CO 2 , making it necessary to regulate the level of photosynthesis and cellular respiration of these algal systems (Carrieri et al. 2011; Kim and Kim 2011).
The literature contains more research reports about algal culture optimization than about genetic manipulation of genes involved in H2 production in algae. Within this context, Chlamydomonas reinhardtii, Chlorella vulgaris, Desmodesmus, and Chlorella have been shown to have good H 2 production in sulphide-free media (Timmins et al. 2009). Furthermore, in C. reinhardtii, the high production of H 2 has been veri ed to be induced by sudden change from dark to light, so that the cells that had become anaerobic in the dark begin to express the hydrogenase gene in the presence of light; however, the effect only lasts a few minutes (Mus et al. 2005). In this system, the hydrogenase accepts electrons produced by photosystem II (PSII) until the activation of the Calvin cycle, and then, the hydrogenase is inhibited by the increasing concentration of O 2 in the medium. Apparently, the regulation of the activity of hydrogenase enzymes is related to three main pathways involving the regulation of photosystems, the respiratory chain, and the Calvin cycle. The nucleotide sequences were aligned (BLASTX) and compared with those in the GenBank database.
DNAMAN version 4.0 was used to translate these sequences and to identify the open reading frame. Predicted amino acid (aa) sequences relative to hyd nucleotide sequences were used in combination with related sequences retrieved from GenBank to build a phylogenetic aligning. Conserved aligned regions (>90%) were selected in all sequences and phylogenetic analysis was performed in software MEGA Each reactor was placed inside the chamber, N 2 gas was purged into the medium for 10 min to remove dissolved oxygen and were kept in the dark for 24 h to achieve anaerobic conditions. Subsequently, the cultures were exposed to light (140 µE m -2 s -1 ) and H 2 was measured at 4 h intervals for one day. . The hyd primers used for relative expression analysis were the same as described above (Table S1) and the 18S rDNA genes were used as reference genes according to Dong et al. (2012).
Ampli cations of the hyd genes were carried out as described above in the same PCR ampli cation conditions used for the phylogenetic analysis. The melt curve analysis and negative controls for the reference and target genes were always included in the experiments in order to eliminate DNA contamination. The relative expression of each gene was determined by the ∆∆Cq method between the target (hyd) and reference (18S rDNA) genes by the equation: Relative expression =

Results
The algal strains of C. vulgaris and S. obliquus were identi ed by our results of sequencing of a partial region of 18S rDNA. The ampli ed 135 bp of 18S rDNA partial regions from C. vulgaris and S. obliquus DNA had 100% identity with Scenedesmus and Chlorella accessions in the NCBI's GenBank (Fig. S1).
We also ampli ed a partial 700 bp (233 aa) region corresponding to the C-terminal domain of the iron hydrogenase large subunit. The identi cation of the hydA gene of C. vulgaris and S. obliquus provided us information about the genetic differences between phylogenetically distant groups of the algal hydrogenases, about non-synonymous changes in algal hydrogenases that could affect the protein's functionality, and about the possibility of differential values in the production of H 2 associated with the regulation in the transcription of the hydA gene in C. vulgaris and S. obliquus.

The hydrogenases of Scenedesmus obliquus and Chlorella vulgaris and their intra and interspeci c evolutionary relationships with other algae
In order to establish genetic differences in the hydrogenase enzyme among algae, and to know if these non-synonymous changes affecting the 3D structure of the protein were related to the evolution of each species in the genus, we made a bioinformatic analysis including dendrogram construction and 3D structure modeling of the enzyme in 24 accessions, including 11 algal genera.
The results from our phylogenetic analysis indicated the formation of a main group including the genera Chlorella, Tetraspora, Scenedesmus, Tetradesmus, Monoraphidium, Raphidocells, Chlamydomonas, Volvox, and Coccomyxa. A second subgroup included the genus Tetraselmis. Some accessions of Chlorella, Raphidocelis, and Nannochloropsis formed a third group (Fig. 1), indicating that perhaps there are variants of hydrogenases in Chlorella and Rhapidocelis.
The dendrograms resulting from grouping algal accessions according to the characteristics of their hydrogenases did not in all cases correspond to the organisms' phylogenetic classi cation. On one side, the genera Chlorella and Coccomyxa belong to the class Trebouxiophyceae but their accessions formed two subgroups within the main branch, similarly to the grouping pattern followed by accessions of Volvox and Tetraspora, both in the order Chlamydomonadales. On the other side, accessions of the genera Scenedesmus and Tetradesmus -belonging to the family Scenedesmaceae-grouped together re ecting a close relationship, and this same grouping pattern occurred between accessions in the genera Monoraphidium and Rhapidocelis both in the family Selenastraceae.
Our results from the 3D modeling of the HYD enzymes from C. vulgaris and S. obliquus showed found a 60% and 55% sequence identity with the [FeFe]-hydrogenase described for Chlamydomonas reinhardtii, respectively (Swanson et al. 2015) (Table S2). Almost all 3D models demonstrated similar structures, except that for Coccomyxa subellipsoidea XP_005643907.1 (Fig. 2, Fig. S2, Fig. S3). In almost all the 3D [FeFe]-hydrogenase structures modeled for the algal genera included in this study, we identi ed the aa residues responsible for the binding of the Iron/sulfur cluster (CPCACGCG; Fig. 2, Fig. S2, Fig. S3), but this sequence of residues was absent in the accessions Coccomyxa subellipsoidea XP_005643907.  (Fig. 3a).
Furthermore, our mutational analysis of [FeFe]-hydrogenase indicated conservation of 120 aa residues among accessions from species of Chlorella (CAC83290.1, ADK77883.1, PRW60372.1, AEA34989.1) and Scenedesmus (AXU2407.1) -including the V3CHLO and V4SCEN sequences we identi ed in this studyand distinguished an interspeci c relationship between the V15CHL (small subunit domain and Cterminal) and V9CHLO (small subunit domain) variants of Chlorella ( Fig. 3a and b) Overall, this analysis demonstrates interspeci c diversity of the [FeFe]-hydrogenase C-terminal and small subunit domains among microalgae taxa (Fig. 3). Interestingly, the evolutionary path of [FeFe]hydrogenase indicated a de nite relationship between species of Chlorella, Scenedesmus, and Tetradesmus in the minimal haplotype network, and the identi cation of eight missing variants and the re ected evolutionary pattern were supported by the analysis of the enzyme's aa similarity in different species of microalgae (Fig. 1). Furthermore, the interspeci c diversity we observed between species of microalgae suggests a divergent evolutionary strategy in the The determination of H 2 in Chlorella vulgaris and Scenedesmus obliquus indicated some points to be highlighted (Table 1 and Fig. 4). When the microalgae were exposed to 1h of white light (140 µE m − 2 s − 1 ),  Fig. 4a).  On the other hand, monitoring the evolution of H 2 during the rst 5 h evaluated, with short intervals of 30 min (Fig. 4b), indicated that it is possible that H 2 is detected in both microalgae from the rst 30 min under white light exposure (Table 1 and Fig. 4b) had previously argued that both algae presented variations in the duration of the lag phase before initiating H 2 production after being exposed to different light intensities, and suggested that the

Regulation of hyd genes in microalgae Scenedesmus obliquus and Chlorella vulgaris
The results of our analysis through qPCR of the relative expression of Hyd in the algae cultured in hydrogenase inducing media -made to know if the hyd gene is differentially regulated in C. vulgaris and S. obliquusindicated that both strains showed higher levels of hyd expression at 1 h of culture (Fig. 5a). C. vulgaris showed 2.3 times more expression of the hyd gene than S. obliquus. Subsequently, in both strains, a decrease in the relative expression values was observed at 5 h of culture, until reaching a basal expression during the following 19 h.
It is known that the expression of the hyd genes is transient, and that their regulation can be inhibited by the products of photosynthesis (Weiner et al. 2018). Studies have shown that hydrogenase accumulates under dark anoxic adaptation conditions. Following such induction, exposure of algae to light supports high rates of H 2 production, but H 2 production ceases within a few minutes of illumination (Ghirardi 2015;Noone et al. 2017). This suggests that the observed increase in the expression of the hyd gene in C. vulgaris and S. obliquus within the rst few hours after the transition from dark anoxia to light is the result of transcriptional regulation of the algal enzymes involved in this sudden change from dark to light, which takes place until the acclimatization of the algae and the activity of the hydrogenase becomes constant and basal (Fig. 5a).
With the aim of deepening the knowledge about regulation of hyd genes in the studied algae, we evaluated the relative expression of hyd genes in a 5 h period at 30 min intervals by qPCR. The results indicated higher expression levels at 30 min after the sudden change from darkness to light, reaching stable levels at 90 min (Fig. 5b).
This behavior pattern of hyd genes transcription in C. vulgaris and S. obliquus suggests that Hyd is involved in the production of H 2 in the rst 90 min, suggesting that possibly after the rst 90 min hydrogenase is regulated by the levels of O 2 as a product of the bio-photolysis of H 2 O that occurred during PSII in microalgae (Antal et al. 2003;Burlacot and Peltier 2018;Ghirardi et al. 2010). If we compare the levels of H 2 production during the rst 30 min in both microalgae, we observe that it is not differential between algae (2 mL H 2 /L) ( Table 1 and Fig. 4b). Suggesting that the elevated expression levels in the rst 30 minutes of light exposure (after a period of darkness) deregulates hydrogenase, enhancing its expression, later as the microalgae acclimatize to the new condition submitted, hydrogenase tends to regulate until to express su cient levels to carry out its activity and contribute to the production of H 2 in both microalgae (Fig. 5b)

Discussion
The exploration of evolutionary trends in microalgae through sequence similarity data allows understanding and establishing families of genes and enzymes with biotechnological potential, such as [FeFe]-hydrogenase (Wang et al. 2020). However, the scarce information available on gene and enzyme sequences is a limiting factor for their potential application. It is of relevance to continue analyzing the coding genes for Hyd in algae in order to better understand the evolution and adaptation of enzymes in microalgae, and even to predict evolutionary trajectories of protein families of interest.
In this study we genetically characterize the hyd gene encoding hydrogenase in two microalgae (C. vulgaris and S. obliquus). According to the primary amino acid characteristics of the enzyme, we model dendrograms, construct a network of haplotypes and compare their three-dimensional structure among 11 genera of algae. The results indicated clustering variations (observed in the dendrogram and in the haplotype network) and in some algal genera the loss of aa residues responsible for the binding of the Iron/sulfur cluster "CPCACGCG" (Coccomyxa subellipsoidea (XP_005643907. 1) and Tetradesmus obliquus (AA65921.1)) was observed. These differences found suggest the presence of isoforms or gene variants that encode hydrogenases in algae (Meuser et , 2015).
From the biological perspective, the presence of Hyd isoforms in algae has a relevance because the hydrogenase enzymes participate in a process that ensures their survival by participating in the vital process of photosynthesis (Petrova et al. 2020), it is therefore not surprising that these enzymes are highly conserved (Fig. 2, Fig. S2, Fig. S3). However, as it was demonstrated, small mutational changes could lead to the inactivation of genes (pseudogenization) or subject them to processes such as neofunctionalization, functionalization, and subfunctionalization, impacting on the process of photosynthesis and the production of H 2 in algae.
The differences in the grouping of hydrogenase enzymes in these algal genera that is observed in our dendrogram ( Fig. 1) (Table 1 and Fig. 4a). The rst point of detection of H 2 in both microalgae was at 30 min, which correlated with the highest levels of hydrogenase expression. It is observed that at the beginning of the kinetics both microalgae show similar trends in H 2 production, but after 5h under continuous light exposure, S. obliquus demonstrated to produce higher H 2 values compared to Chlorella vulgaris (Table 1 and Fig. 5a).
The higher H 2 production values we observed in C. vulgaris compared to S. obliquus may depend on the presence in both algae of different genes coding for HYD and FDX, which results in differences in both μmol H 2 mg − 1 min − 1 with Fdx2, respectively. These results suggest that the a nity to electron donors of HYD isoforms depends on their encoding genes, which can be re ected in the production of H 2 .
On the other hand, Ruiz-Marin et al. (2020), reported that this same Scenedesmus strain turned out to be more H 2 producer compared to Chlorella, indicating that by stimulation with violet light, showed productivities of 128.0 mL H 2 /L and 60.4 mL H 2 /L, respectively. This author highlighted that both algae presented variations in time in the lag phase, as they were exposed to different intensities of light, to later initiate H 2 production, suggesting that the fact that the H2 production kinetics in both microalgae differ after 5h under continuous white light exposure, it will depend on the ability of the microalgae to adapt  used is a determining factor. According to the genetics of the strain, some algae could grow faster and accumulate higher biomass levels in less time, which could cause fast nutrients consumption and inhibit H 2 production. As mentioned above, our results of H 2 production in C. vulgaris are similar to those reported by Sun et al. (2011). In the case of S. obliquus, the values of H 2 production we obtained are different and higher than those reported in the literature, which suggests that the genetic background of this strain, associated with the genes encoding the enzymes involved in H 2 production, is an important factor for H 2 production.
In C. reinhardtii incubated for 1 h in the dark and after exposed to light (370 μEm −2 s − 1 ) for 15 min, the activity of Hyd reached values of 2.3 µg HydA mg Chl -1 during the rst 2 minutes, which was related not to the increased production of H 2 , but of oxygen (Weiner et al. 2018). Also, the accumulation of Hyd proteins in aerobically grown C. reinhardtii after the rst 30 min of anaerobic adaptation in a solution with argon was also reported (Happe and Kaminski 2002) The differential expression of the genes coding for HydA1 and HydA2 was demonstrated by transcriptional analysis of C. reinhardtii cultures incubated in a sealed photobioreactor in sulfur-deprived TAP medium for 1-3 days, in which hydA1 was more expressed than hydA2.
The differential behavior of hyd gene expression in C. reinhardtii was similarly observed by construction of a transcriptomic database in H 2 production conditions. The expression of two contigs, annotated as It is important to highlight, that the search for alternative energy sources that do not involve fossil fuels has been a relevant goal that continues to be of importance today. Algae are organisms that have demonstrated their ability to produce H 2 through photosynthesis involving PSI and PSII. Hydrogenase is a key enzyme with activity related to H 2 yields in algae. In this study, we show that C. vulgaris and S. obliquus produce 9.0 ± 0.40 mL H 2 /L and 16 ± 0.50 mL H 2 /L, respectively, after exposing to light cultures previously grown for 24 h in anaerobic conditions in darkness. Also, the results of our analysis of the relative expression of hyd genes showed their expression 30 min after exposing cultures to light. The differential behavior in the production of H 2 in the analyzed microalgae could be due to each microalgae's capacity to adapt to the culture conditions. In addition, the differential regulation of hyd isoforms in these microalgae could be key to S. obliquus being a better producer of H 2 in comparison to C. vulgaris.Deeper studies allowing us to identify all the Hyd isoforms in algae, would provide a better understanding of the evolution, regulation of transcription, and activity of this enzyme. Future studies of overexpression of this enzyme using different hosts could be relevant to increase H 2 yields in different organisms.

Declarations
Ethics approval and consent to participate Authors declare that they have no con ict of interest, nancial or otherwise.
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Consent to submit has been received explicitly from all co-authors, as well as from the responsible authorities -tacitly or explicitly -at the Autonomous University of Carmen, where the work has been carried out.
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Consent for publication
Not applicable Availability of data and materials Evolutionary relationship of [FeFe]-hydrogenase (Hyd) in algae. Phylogeny was reconstructed by the neighbor-joining method (NJ) and cluster con dence was tested by 1000 bootstrap iterations. The amino acid sequences were aligned with Alignment Explorer/CLUSTALW program and the software Genetic and Molecular Evolution Analyses (MEGA version 6.0).

Figure 2
In silico tertiary structures of [FeFe]-hydrogenase (Hyd) in algae. The prediction of tertiary conformation was performed using the SWISSMODEL program and Chlamydomonas reinharditti was used as a model.  Hydrogen production in Chlorella vulgaris and Scenedesmus Obliquus. The algal biomass was obtained by growth for 7 days and the algae were subsequently exposed under anaerobic conditions in sulfate free medium. The determination of molecular hydrogen production was by HPLC during the time intervals of