Introduction

Biohydrogen is one of the alternative potential energy carriers which can be produced by various kinds of microorganisms via different processes. Cyanobacterial H2 production can be performed by either/both a photosynthetic pathway or/and a dark fermentation from the degradation of accumulated carbohydrate in the cells. There are at least three enzymes involved in H2 metabolism of cyanobacteria. Nitrogenase catalyzes the reduction of atmospheric N2 to ammonia and simultaneously produces H2 as a by-product. This reaction requires numerous ATP. Uptake hydrogenase catalyzes the oxidation of H2, produced by N2 fixation, to protons. These two enzymes are mainly found in heterocysts of N2-fixing cyanobacteria. The last enzyme, bidirectional hydrogenase, catalyzes both consumption and production of H2 and is distributed in both N2-fixing and non-N2-fixing cyanobacteria (Tamagnini et al. 2000, 2002).

Bidirectional hydrogenase is a heterotetrameric subunit consisting of hydrogenase structural complex (HoxY and HoxH encoded by hoxY and hoxH, respectively) and a diaphorase component (HoxF and HoxU encoded by hoxF and hoxU, respectively). Besides HoxF and HoxU, an additional subunit HoxE encoded by hoxE might be found as one of diaphorase components in some cyanobacteria such as Synechocystis sp. PCC 6803 (Appel and Schulz 1996), Synechococcus sp. PCC 6301 (Boison et al. 1998), and Anabaena sp. PCC 7120 (Tamagnini et al. 2002). Nucleotide sequences of these hox genes in several cyanobacteria have been reported. The cyanobacterial hox gene cluster can be organized as an operon (Appel and Schulz 1996; Boison et al. 2000) or separated into two gene clusters (hoxEF and hoxUYH) (Tamagnini et al. 2007). In addition, hox gene expression in cyanobacteria differs among various species depending on the environmental condition and their genetic characteristics. The relative transcript levels of hox genes in most cyanobacterial species increase under nitrogen starvation/limitation condition (Boison et al. 2000; Sheremetieva et al. 2002; Troshina et al. 2002; Antal et al. 2006; Osanai et al. 2006). Furthermore, an absence or a low level of oxygen can induce both the enzyme activity as well as the relative amount of hoxH (Axelsson and Lindblad 2002; Houchins and Burris 1981; Sheremetieva et al. 2002; Schmitz and Bothe 1996).

The unicellular halotolerant cyanobacterium Aphanothece halophytica has been shown to be one of the high potential H2 producers (Taikhao et al. 2013). It is capable of producing high amount of H2 under nitrogen starvation and dark anaerobic condition. In addition, it could grow in seawater supplemented with as little as 1.76 mM NaNO3 and showed a long-term H2 accumulation for at least 14 days (Taikhao et al. 2015). To fully understand the mechanisms and functions of bidirectional hydrogenase in A. halophytica, genetic characterization and transcriptional analysis of bidirectional hydrogenase genes and other related genes under nitrogen and sulfur deprivation condition were investigated.

Materials and methods

Aphanothece halophytica was grown in a 250-mL Erlenmeyer flask containing 100 mL of BG11 medium as previously described with slight modification (Wiangnon et al. 2007). The culture was buffered with 10 mM TES (pH 8.0) and supplemented with Turk Island salt solution (Garlick et al. 1977). Cells were cultivated at 30 °C under a white-light illumination of 30 μmol photons m−2 s−1 on a rotary shaker with a shaking speed of 120 rpm for 7 days. Escherichia coli DH5α used for plasmid DNA manipulation was grown in a 250-mL Erlenmeyer flask containing 100 mL of LB broth (Bertani 1951) at 37 °C with a shaking speed of 250 rpm. For a blue-white screening of correct transformants, E. coli cells were selected on LB agar containing 50 μg mL−1 kanamycin, 50 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 80 μg mL−1 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (X-gal).

DNA and total RNA isolation

Genomic DNA of Aphanothece halophytica was isolated by using Wizard SV genomic DNA purification system kit (Promega, USA). Plasmid DNA isolation was performed by using High-Speed Plasmid DNA Mini Kit (Geneaid, Taiwan). Total RNA was isolated following the protocol of Gutekunst et al. (2005).

DNA amplification by PCR and DNA walking

First, DNA fragments of partial hoxUY and partial hoxH of A. halophytica were amplified by PCR using conserved primers designed from other cyanobacterial genes (Table 1). The PCR reactions and conditions were performed according to Phunpruch et al. (2006) in a Master gradient thermal cycler (Eppendorf, Germany). The PCR products were subsequently cloned and sequenced. The specific primers of hoxY and hoxH designed from the obtained sequences were used for PCR amplification of DNA fragment of hoxYH, and then its PCR product was cloned and sequenced. Finally, the partial sequence of hoxU, the complete sequence of hoxY, and the partial sequence of hoxH were obtained. DNA fragments of the other genes were amplified by three nested PCR reactions with the target specific primers (Table 1) and the supplied DW-ACP primers using DNA walking speedup premix kit (Seegene, Korea). The final PCR products were purified by using QIAquick PCR Purification kit (Qiagen, Germany) before cloning and sequencing.

Table 1 Primers used in PCR, genome walking, co-transcriptional analysis, and transcriptional analysis
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Cloning and nucleotide sequencing

The purified PCR products were cloned into pDrive (Qiagen, Germany). The ligation mixture was incubated at 16 °C for 16 h and then transformed to the competent cells of E. coli DH5α. The recombinant plasmid was isolated by High-Speed Plasmid DNA Mini Kit (Geneaid, Taiwan) and sequenced with Big-Dye terminator cycle sequencing ready reaction kit (Perkin Elmer, USA) using ABI PRISM 3700 DNA analyzer at First BASE Laboratories (Malaysia). Universal primers T7 and SP6 were used for both strand sequencing. The obtained nucleotide sequences were compared with GenBank nucleotide sequence databases.

Co-transcriptional analysis

Contaminating DNA from 1 μg of total RNA was digested with 6 units of RQ1 RNase-free DNase (Promega, USA) in buffer containing 4 units of RNasin plus RNase inhibitor (Promega, USA) at 37 °C for 1 h before inactivating the enzyme by heating at 65 °C for 10 min. The single-stranded RNA was precipitated by phenol-chloroform extraction and suspended in a buffer containing 4 units of RNasin plus RNase inhibitor, 1 mM dNTPs (Promega, USA), and 1 μM RT-primer RhydApha3 (Table 1) which is located at downstream region of hoxH. The mixture was pre-incubated at 70 °C for 10 min and immediately chilled on ice for 10 min before adding 200 units of M-MuLV reverse transcriptase (Thermo Scientific, Lithuania) followed by incubating at 42 °C for 1 h. DI water containing 0.1 % (v/v) DEPC was used instead of M-MuLV reverse transcriptase in a negative control. DNA amplification of hoxEF, hoxFU, hoxUY, and hoxYH fragments by PCR using the obtained complementary DNA as template was performed with four primer pairs, CoFhoxE1Apha-TSP1hoxFApha, CoFhoxF1Apha-TSP3hoxUApha, CoFhoxUApha-CoRhoxYApha, and FhoxY4-RhoxH1Apha, respectively (Table 1).

Transcriptional analysis

In this study, transcriptional analysis of hoxH and other genes involved in H2 metabolism was performed by using QIAGEN OneStep reverse transcription polymerase chain reaction (RT-PCR) kit (Qiagen, Germany). Total RNA was isolated from cells treated under 24-h nitrogen-deprived and sulfur-deprived conditions, and then the contaminating DNA was digested by DNaseI as above described. The expression of a housekeeping gene 16S rRNA was used as a control for each sample. The sequences and origins of primers are shown in Table 1. The RT-PCR condition was performed as follows: RT reaction at 50 °C for 30 s, initial denaturation at 95 °C for 15 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 50–55 °C for 30 s, extension at 72 °C for 90 s, and followed by the final extension at 72 °C for 10 min. The PCR product was analyzed by 1.0 % (w/v) agarose gel electrophoresis. The intensity of DNA bands was analyzed by GeneTools analysis software (version 4.01.02) from SynGene Genius system.

Determination of bidirectional hydrogenase activity, chlorophyll a concentration, glycogen content, and dark respiration rate

Bidirectional hydrogenase activity was determined by measuring H2 production in the presence of dithionite reduced methyl viologen according to Baebprasert et al. (2010). Chlorophyll a was extracted by methanol and determined spectrophotometrically (MacKinney 1941). The glycogen extraction and hydrolysis was performed following the procedure of Ernst and coworkers (1984). Glycogen content was determined as glucose concentration by phenol-sulfuric acid assay (Dubois et al. 1956).The dark respiration rate was measured in a 2-mL cell suspension with a Clark-type oxygen electrode (Oxygraph plus system, Hansatech, UK). All experiments were done in triplicates.

Results

Nucleotide sequences of hox genes in Aphanothece halophytica

The 4960-bp hox gene cluster of A. halophytica consisted of five open reading frames (ORFs), namely, hoxE, hoxF, hoxU, hoxY, and hoxH, without any insertion of ORFs (Fig 1). Their nucleotide sequences were deposited to GenBank under accession number KF885784.1. The upstream gene, hoxE, encoding the bidirectional hydrogenase complex protein HoxE consisted of 516 bp and encoded 171 amino acids with a calculated molecular mass of 19.0 kDa (http://web.expasy.org/compute_pi/). The large diaphorase subunit gene, hoxF, comprised 1617 bp and encoded 538 amino acids with a predicted polypeptide of 57.8 kDa whereas the small diaphorase subunit gene, hoxU, contained 720 bp encoding 239 amino acids with a predicted polypeptide of 26.5 kDa. In addition, the large and small hydrogenase subunit genes, hoxH and hoxY, constituted 1428 bp encoding 475 amino acids, and 549 bp encoding 182 amino acids, respectively. The HoxH and HoxY of A. halophytica showed the estimated molecular masses of 53.1 and 20.1 kDa, respectively. Nucleotide sequences of hox genes in A. halophytica were compared with those from other cyanobacteria reported in GenBank nucleotide sequence database by ClustalW program (Higgins et al. 1994).The hox genes of A. halophytica showed approximately 65–81 % nucleotide identity and 77–91 % amino acid similarity to those reported in other cyanobacteria (excluding those for Halothece sp. PCC 7418 where 99–100 % nucleotide homology and amino acid similarity were detected).

Fig. 1
figure 1

Physical map of hox genes in A. halophytica compared with other cyanobacteria

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Co-transcriptional analysis by RT-PCR

The co-transcriptional analysis of hox gene cluster in A. halophytica was performed by RT-PCR. Figure 2a shows the strategy for the analysis in which the single-stranded mRNA was reverse-transcribed into complementary DNA (cDNA) by M-MuLV reverse transcriptase using hoxH-specific antisense RhydApha3 as a RT-primer. The obtained cDNA was used as a template for amplification of the DNA fragments of hoxEF, hoxFU, hoxUY, and hoxYH using specific primer pairs listed in Table 1. Only one specific RT-PCR product was found in each RT-PCR reaction (Fig. 2b). RT-PCR products of hoxEF, hoxFU, hoxUY, and hoxYH showed similar size to those of the expected PCR products of 1477, 434, 779, and 777 bp, respectively. No RT-PCR products were observed in negative RT-PCR reactions (Fig. 2b). The results indicated that all structural genes encoding bidirectional hydrogenase were transcribed as a single operon in A. halophytica.

Fig. 2
figure 2

Strategy of co-transcriptional analysis of hox gene cluster in A. halophytica. The mRNA was isolated and transcribed into the cDNA using RT-primer RhydApha1. The obtained cDNA was then used as a template for DNA amplification of hoxEF, hoxFU, hoxUY, and hoxYH genes by polymerase chain reaction (a). Agarose gel analysis of RT-PCR products of hoxEF, hoxFU, hoxUY, and hoxYH genes (b). Lane 1, RT-PCR reaction; lane 2, negative RT-PCR reaction without M-MuLV reverse transcriptase; lane 3, positive PCR reaction (using genomic DNA as template); lane 4, negative PCR reaction (without template DNA); lane M, 100-bp DNA ladder

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Transcriptional analysis of large subunit bidirectional hydrogenase gene and other related genes in Aphanothece halophytica

Aphanothece halophytica grown in BG11 supplemented with Turk Island salt solution was harvested and resuspended in three different media; normal medium (BG11 + Turk), sulfur-free medium (BG11 + Turk (-S)), and nitrate-free medium (BG11 + Turk (-N)). Transcriptional analysis of hoxH revealed the upregulation in A. halophytica incubated under nitrate-deprived condition for 24 and 48 h (Fig. 3a, b). This was correlated with the increased bidirectional H2ase activity in cells under nitrate-deprived condition (Fig. 4a). Transcript abundance of two genes involved in glycogen metabolism, glgB encoding 1,4-α-glucan branching enzyme in glycogen synthetic pathway and glgP encoding glycogen phosphorylase in glycogen breakdown pathway, showed the upregulation and downregulation of glgB and glgP, respectively, in cells under nitrate starvation for 24 and 48 h (Fig. 3a, b). Glycogen content was highest, 55.3 ± 9.7 % of dry cell weight, in cells under nitrate-deprived condition for 48 h, a twofold increase compared to cells under normal condition (Fig. 4b). Moreover, the relative transcript levels of coxA and ndhB, encoding respectively subunit I of cytochrome oxidase and subunit B of NADH dehydrogenase in the respiratory electron transport system, were also upregulated in cells under nitrate deprivation for 24 and 48 h (Fig. 3a, b). This upregulation of coxA and ndhB caused the increase in dark respiration rate of cells under nitrate deprivation whereas no apparent changes in dark respiration were observed under sulfur deprivation (Fig. 4c). In contrast, a downregulation of narB, encoding ferredoxin-nitrate reductase, was observed in cells under nitrate deprivation (Fig. 3a, b). No differences of relative transcript level of psbA encoding D1 protein of photosystem II were observed under normal condition. Expression of psbA was decreased and increased respectively under sulfur and nitrate deprivation (Fig. 3a, b). Similar results were observed for the expression of psaA encoding core protein PsaA of photosystem I with the exception of that under sulfur deprivation showing a slightly increased expression.

Fig. 3
figure 3

Relative transcript levels of hox genes and other genes involved in H2 metabolism by OneStep RT-PCR (a). Total RNA was isolated from A. halophytica incubated in BG11 + Turk (control), BG11 + Turk (-S) (without sulfur), and BG11 + Turk (-N) (without nitrate) for 0, 24, and 48 h. RT-PCR products were analyzed by 1.0 % agarose gel electrophoresis. The expression of a housekeeping gene, 16S rRNA gene, was used as a reference for each sample. Relative quantification of transcriptional level of hoxH, glgB, glgP, coxA, ndhB, narB, psbA, and psaA of A. halophytica was obtained by comparison to transcription level of the housekeeping 16S rRNA gene (b)

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Fig. 4
figure 4

Bidirectional H2ase activity (a), glycogen content (b), and dark respiration rate (c) of A. halophytica incubated in normal medium (BG11 + Turk control), sulfur-deprived medium (BG11 + Turk (-S)), and nitrate-deprived medium (BG11 + Turk (-N)) for 0, 24, and 48 h. Data are means ± SD (n = 3)

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Discussion

In this study, we could isolate hox gene cluster with complete nucleotide sequences from A. halophytica. Previously, bidirectional hydrogenase gene was detected in A. halophytica by Southern blot hybridization (Ludwig et al. 2006), and later, Barz and colleagues (2010) reported the partial sequence of a large subunit of bidirectional hydrogenase (hoxH) in A. halophytica. Aphanothece halophytica contains five structural bidirectional hydrogenase genes (hoxE, hoxF, hoxU, hoxY, and hoxH) similar to those normally found in other cyanobacteria (Fig. 1). After homology search of the entire genes, the nucleotide and amino acid sequences of A. halophytica hoxEFUYH showed the highest identity and similarity (99–100 %) to those reported in Halothece sp. PCC 7418 whose genome sequences were launched in GenBank in 2013 (accession number CP003945.1). Nucleotide sequences of hoxF and hoxH in Halothece sp. PCC 7418 showed five and one nucleotide differences, respectively, compared to those of A. halophytica in the present study, i.e., the amino acid sequences of HoxF and HoxH in both organisms differed in three and one amino acids, respectively. In addition, by phylogenetic tree analysis, all hox genes in A. halophytica showed a very close relationship with all hox genes in Halothece sp. PCC 7418 (data not shown). The results suggested that the studied cyanobacterium A. halophytica is in the same genus and species as Halothece sp. PCC7418 but might be different in strain or at subspecies level.

In hox gene cluster of A. halophytica, no other ORFs were observed (Fig. 1). Normally, the orientation of hox gene cluster in several cyanobacteria is similar, but it might have some differences regarding a direction of hox genes or an insertion of ORFs. One or several additional ORFs have been identified between the structural genes in some species of cyanobacteria, for instance Anabaena sp. PCC 7120 (Kaneko et al. 2001), Anabaena variabilis ATCC 29413 (Schmitz and Bothe 1996), Anabaena variabilis IAM M58 (Tamagnini et al. 2002), Arthrospira platensis NIES-39 (Zhang et al. 2005a, b), Lyngbya majuscula CCAP 1446/4 (Ferreira et al. 2009), Synechococcus sp. PCC 6301 (Boison et al. 1998), and Synechocystis sp. PCC 6803 (Appel and Schulz 1996) (Fig. 1). In agreement with our results, no ORFs are found between hox structural genes in Cyanothece sp. ATCC 51142 (Welsh et al. 2008) which showed high potential as H2 producer (Min and Sherman 2010). Nevertheless, the functions of additional ORFs between structural genes in some cyanobacteria need to be further investigated. The hoxEFUYH genes in A. halophytica encode five proteins, HoxE, HoxF, HoxU, HoxY, and HoxH, with calculated molecular masses of 19.0, 57.8, 26.5, 20.1, and 53.1 kDa, respectively. Their molecular masses are in good agreement with the data of five dissimilar subunits of about 19, 58, 26, 20, and 53 kDa reported in the unicellular cyanobacterium Synechocystis sp. PCC 6803 or other cyanobacteria (Appel and Schulz 1996; Ghirardi et al. 2007).

The prediction of conserved motif of hox genes in A. halophytica was performed according to Tamagnini and coworkers (2002). In all Hox subunits of A. halophytica, conserved cysteine motifs, functioning in coordination of iron-sulfur clusters involved in an electron transfer, were found. HoxE in A. halophytica contained four conserved cysteine residues, which were likely to harbor a [2Fe-2S] cluster. The pattern of binding of a putative [2Fe-2S] cluster in HoxE of A. halophytica was C94-x4-C99-x35-C135-x3-C139. A similar result was found in HoxE of Synechocystis sp. PCC 6803 harboring a putative [2Fe-2S] cluster that contained cysteine residues at C96, C101, C137, and C141 (Appel and Schulz 1996). Normally, the HoxE harbors a motif for binding an iron center and is thought to couple with the enzyme in the respiratory and photosynthetic electron transport chain on the thylakoid and also possibly at the cytoplasmic membrane (Bothe et al. 2010). In the middle region of large subunit of diaphorase HoxF in A. halophytica, typical glycine-rich binding sites for oxidized and reduced nicotinamide adenine dinucleotide (NAD+ and NADH) and flavin mononucleotide (FMN and FMNH2) were found between amino acid positions from 171 to 180 (G171-x-G173-x2-G176-x3-G180) and from 183 to 204 (G183-x-G185-x4-G190-x10-G201-x2-G204), respectively, corresponding to the general HoxF glycine-rich binding sites GxGxxGxxxG and GxGxxxxGx10GxxG in other cyanobacteria (Schmitz et al. 1995; Bothe et al. 2010). In addition, HoxF also contained a binding [2Fe-2S] cluster (C25-x4-C30-x30-C61-x3-C65) at the N-terminus and a binding putative [4Fe-4S] cluster (C455-x4-C460-x2-C463-x33-C497) at the C-terminus. These results were in agreement with those from Anabaena variabilis ATCC 29413 (Schmitz et al. 1995) and Synechocystis sp. PCC 6803 (Appel and Schulz 1996). The small subunit of diaphorase moiety, HoxU protein, comprised several conserved cysteine residues involved in the binding of a putative [Fe-S] cluster (Appel and Schulz 1996; Schmitz and Bothe 1996). Two motifs were located at the N-terminal part of the protein; the first motif harbored four cysteine residues probably involved in binding a putative [2Fe-2S] cluster (C36-x10-C47-x2-C50-x13-C64), and the second motif harbored three cysteine residues involved in binding a putative [3Fe-4S] cluster or a putative [4Fe-4S] cluster (C100-x2-C103-x5-C109). Moreover, the C-terminal part of HoxU possessed two typical [4Fe-4S] cluster-binding sites with conserved cysteine residues of C149-x2-C152-x2-C155-x3-C159 and C193-x2-C196-x2-C199-x3-C203. A similar result was reported for HoxU of Anabaena variabilis ATCC 29413 (Schmitz et al. 1995), Synechococcus sp. PCC 6301 (Boison et al. 1996), and Synechocystis sp. PCC 6803 (Appel and Schulz 1996). The small subunit of hydrogenase protein (HoxY) in A. halophytica contained C14, C17, C87, and C151 as conserved cysteine residues, found to be involved in the binding of a putative [4Fe-4S] (Volbeda et al. 1995). The large subunit hydrogenase protein (HoxH) comprised four conserved cysteine residues (C62, C65, C444, C447) involved in the binding of nickel to the active site of hydrogenase (Volbeda et al. 1995; Schmitz and Bothe 1996).

In A. halophytica, five structural genes of bidirectional hydrogenase, hoxEFUYH, were clustered and oriented in the same direction. Furthermore, these five structural genes encoding bidirectional hydrogenase were transcribed as a single operon in A. halophytica (Fig. 2). In cyanobacteria, structural genes encoding bidirectional hydrogenases are differently organized (Tamagnini et al. 2007). The arrangement of hox operon in A. halophytica is very similar to that of hoxEFUYH genes in Halothece sp. PCC 7418 (accession no. CP003945.1) and Cyanothece sp. ATCC 51142 (Welsh et al. 2008). In addition, hox operon in A. halophytica was co-transcribed as a single transcript similar to that reported in Anabaena variabilis (Boison et al. 2000), Lyngbya majuscula CCAP 1446/4 (Ferreira et al. 2009), and Synechocystis sp. PCC 6803 (Gutekunst et al. 2005; Oliveira and Lindblad 2005), whereas hox gene cluster was separated into two different clusters, hoxEF and hoxUYH, in Synechococcus sp. PCC 6301 (Boison et al. 1998) and Anabaena sp. PCC 7120 (Kaneko et al. 2001).

By relative quantification of RT-PCR products, hoxH was upregulated under nitrate starvation (Fig. 3a, b). It has been reported that the relative transcript level of hox genes in most cyanobacterial species increased under nitrogen starvation/limitation condition (Boison et al. 2000; Sheremetieva et al. 2002; Troshina et al. 2002; Antal et al. 2006; Osanai et al. 2006; Baebprasert et al. 2011). An increase of H2ase activity in A. halophytica was also observed after 24 h of N-deprived incubation (Fig. 4a), which was in line with the increased transcript of hoxH (Fig. 3a, b). This was likely due to the nitrate-deficiency-induced increase of both NAD(P)H and electrons for promoting H2 production via bidirectional hydrogenase. Similar observation was reported for Synechocystis sp. PCC 6803 disrupted in nitrate assimilation pathway (Baebprasert et al. 2011). In addition, glgB encoding 1,4-α-glucan branching enzyme involved in glycogen synthesis was upregulated under N-deprived condition (Fig. 3a, b), which was correlated with an increase in glycogen accumulation in A. halophytica (Fig. 4b). In contrast, glgP encoding glycogen phosphorylase involved in glycogen degradation was downregulated under N-deprived condition (Fig. 3a, b). Similar results were observed in a freshwater cyanobacterium Synechocystis sp. PCC 6803 showing the upregulation of glgB after nitrogen starvation for 4 h (Osanai et al. 2006) and for 12 h (Krasikov et al. 2010). The results indicated that cells rather synthesized than degraded glycogen during nitrate starvation. A storage glycogen in the cells might be broken down through glycolysis or other pathways during dark anaerobic fermentation to provide sufficient ATP and NAD(P)H for H2 production via bidirectional hydrogenase.

The two genes involved in the respiratory electron transport, coxA encoding subunit I of cytochrome oxidase and ndhB encoding subunit B of NADH dehydrogenase, in A. halophytica were upregulated under N-starved condition (Fig. 3a, b), which was supported by the increased respiration rate found in N-starved cells (Fig. 4c). The upregulation of coxA and ndhB expression in A. halophytica was in line with the increased expression of all genes encoding terminal respiratory oxidases under N deprivation in Synechocystis sp. PCC 6803 (Krasikov et al. 2010) as well as in Nostoc sp. PCC 7120 showing strong expression of coxA under N2-fixing condition (Wünschiers et al. 2007). The upregulation of terminal respiratory oxidase could dissipate reducing equivalents in the thylakoid membranes to protect photosystem II (PSII) from photoinhibition (Krasikov et al. 2010). The storage carbohydrate content and the respiration rate were increased whereas the rates of O2 evolution and CO2 fixation were decreased in Synechocystis sp. PCC 6308 under nitrogen starvation (Allen et al. 1990). In the N-starved cells of A. halophytica, an increased respiration would help eliminate O2 remaining within the cells, thus providing protection on the enzymes involved in H2 production against inhibition by O2.

The relative transcript level of narB encoding ferredoxin-nitrate reductase was upregulated under both control and S-deprived conditions after 24 h, whereas no changes of narB transcript were detected under N-deprived condition (Fig. 3a, b). This suggested a low nitrate reduction activity in A. halophytica under N-deprived condition. On contrary, narB in Synechocystis sp. PCC 6803 was downregulated under N starvation (Krasikov et al. 2010). Nitrate is transported into the cells by an active transport system and reduced to nitrite by nitrate reductase followed by the action of nitrite reductase (encoded by nirA) to yield ammonium (Incharoensakdi 2006). Nitrate reduction to ammonium through the two sequential reactions of nitrate reductase and nitrite reductase requires two electrons and six electrons, respectively (Flores et al. 2005). Therefore, nitrate reduction competes for electrons that would flow to hydrogenase because nitrate reduction is a better electron sink than hydrogenase (Ananyev et al. 2008). The availability of NaNO3 in the medium would limit H2 production.

The relative transcript level of psbA encoding D1 protein of photosystem II was downregulated under S starvation (Fig. 3a, b) which was in line with a previous study in Synechocystis sp. PCC 6803 (Zhang et al. 2008). The lack of sulfur affected cells by reduction of photosystem II activity, resulting in cessation of O2 evolution and subsequent reactivation of H2ase activity (Melis et al. 2000; Zhang et al. 2002). The expression of psaA encoding core protein PsaA of photosystem I was slightly increased under N starvation (Fig. 3a, b). The increased activity of photosystem I and II under light condition would produce ATP and NADPH which can then provide electron for the reduction of proton to generate H2 in A. halophytica under N starvation. The schematic mechanism of oxygenic photosynthesis and H2 metabolism of A. halophytica under nitrate deprivation is shown in Fig. 5. Under photosynthetic process, electrons from photosystem II (PSII) to photosystem I (PSI) are transferred through plastoquinone (Pq), cytochrome b6f complex (Cyt b6f), plastocyanin (PC), and cytochrome c6 (Cyt c6). From PSI, electrons can be transferred to ferredoxin (Fd) via ferredoxin/NADP+ reductase (FNR) to generate NAD(P)H. Bidirectional hydrogenase (NiFe hydrogenase) can accept electrons from NAD(P)H, but this enzyme is inhibited by O2 produced from the water splitting via PSII. Protons transferred across the thylakoid membrane are used to synthesize ATP by the ATPase. Under N deprivation, A. halophytica converts the energy of photons into energy in the form of ATP and NAD(P)H to promote an increased accumulation of glycogen, via CO2 fixation in Calvin cycle, which is the main storage of carbon and energy in cyanobacterial cells. In addition, nitrate assimilation pathway responsible mainly by the expression of narB was downregulated under N-deprived condition (Fig. 3a, b), thus further enhancing the flow of electrons toward Fd resulting in an increase of the electrons to generate NAD(P)H. Thereafter, H2 is generated under dark anoxic conditions as a result of bidirectional hydrogenase reactivation in the absence of O2 and an over-reduction of NADH during glycogen catabolism. Respiratory electron transfer also includes NADH dehydrogenase (NDH), cytochrome-quinol oxidases (QOX), and cytochrome oxidase (COX), which are involved in the consumption of NADH and O2. Under N deprivation, the increased expression of coxA and ndhB in A. halophytica was observed. This would benefit A. halophytica to maintain the high O2 consumption rate under anaerobic condition resulting in an increased bidirectional hydrogenase activity due to the protection of enzyme against inhibition by O2.

Fig. 5
figure 5

Schematic mechanism of oxygenic photosynthesis and H2 metabolism under N deprivation in A. halophytica. Abbreviations: PSII photosystem II, PSI photosystem I, Pq plastoquinone, Cyt b6f cytochrome b6f complex, PC plastocyanin, Cyt c6 cytochrome c6, Fd ferredoxin, FNR ferredoxin/NADP+ reductase, NiFe hydrogenase bidirectional hydrogenase, NDH NADH dehydrogenase, QOX cytochrome-quinol oxidase, COX cytochrome oxidase

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