Cyanobacteria have different life forms: some species are unicellular, others form colonies and filaments, or live in symbiosis with eukaryotic organisms. Accordingly,
the protection of O2 sensitive enzymes from photosyn — hetically evolved oxygen has evolved through several different strategies.

In the absence of combined nitrogen, many filamen­tous N2-fixing cyanobacteria physically separate oxygenic photosynthesis and N2-fixing enzymes by differentiating specialized heterocyst cells, which are regularly spaced among vegetative cells. Mature hetero­cysts are unique cells providing a microaerobic environ­ment suitable for the enzymes involved in N2 fixation. The microaerobic environment inside of heterocysts is maintained by an elevated rate of respiration, lack of active PSII complexes resulting in an absence of photo­synthetic O2 evolution, and a thick cell wall (Wolk et al., 1994). These two cell types, the vegetative cells and heterocysts, depend on each other. During diazotro­phic growth the vegetative cells perform photosynthetic CO2 fixation and provide the heterocysts with organic carbon intermediates, like sucrose (Lopez-Igual et al.,

2010) , whereas heterocysts provide vegetative cells with fixed nitrogen required for cell growth (Figure 21.2). Since H2 production in heterocysts depends on carbohy­drates produced in vegetative cells, this process of H2 production has been classified as indirect water biophotolysis.

Heterocyst differentiation is tightly regulated by NtcA, a global transcription factor of carbon and nitro­gen metabolism (Zhao et al., 2010). HetR is another essential protein specifically involved in the initial steps of heterocyst development. The patterned differentia­tion of heterocysts is controlled by the ratio of activator, HetR, and suppressor molecules, peptides derived from PetS and HetN (Muro-Pastor and Hess, 2012; Risser and Callahan, 2009). For the heterocystous strain, Anabaena PCC 7120 the frequency of heterocysts is approximately 10% under optimal laboratory growth conditions. Such a low frequency of heterocysts in the filament might result in modest yields of net H2 production. Thus, one possible strategy to improve H2 production is to increase the number of heterocysts in the filaments. However, although the overexpression of HetR resulted in an over­all enhancement of heterocyst frequency up to 29% in

-(oxygenic photosynthesis)

Heterocysts

© [psii PSI

fixatioi

Anabaena PCC 7120 mutant, no increase in the nitroge — nase activity of the filaments took place (Buikema and Haselkorn, 2001). It is possible that the relative decrease in the number of vegetative cells makes them incapable of producing enough reducing power to be transferred to heterocysts for enhanced H2 production.

Unicellular and filamentous nonheterocystous N2-fixing cyanobacteria apply mostly temporal separa­tion mechanism, by performing photosynthesis during the daytime and N2 fixation at night (Compaore and Stal, 2010). The energy generated by photosynthesis is stored in glycogen granules, which are later subjected to oxidative breakdown.

Trichodesmium are unique cyanobacteria, because these filamentous nonheterocystous cyanobacteria are able to fix N2 simultaneously with oxygenic photosynthesis during the photoperiod (Berman-Frank et al., 2001). Nitrogenase is localized in subsets of cells in each trichome, which also contain photosynthetic complexes. During hours of high N2 fixation the cells can turn photo­synthetic activity down within 10 min, which is observed as unequally distributed inactive zones in whole fila­ments. Importantly, the PSII activity was shown to be essential for N2 fixation in Trichodesmium (Berman-Frank et al., 2001). According to the authors, Trichodesmium utilizes photosynthetic electron transport to support N2 fixation and concomitantly enhances the Mehler reaction, which efficiently eliminates the evolved O2. Recently published complete genome sequence of Trichodesmium erythraeum (http://www. ncbi. nlm. nih. gov) shows that the strain indeed possesses the genes encoding the Flv1 and Flv3 proteins, which are involved in the "Mehler — like" reaction in cyanobacteria. Thus, the nitrogenase enzyme in this organism is protected from O2 by a com­bined and modulated temporal and spatial segregation of N2 fixation and oxygenic photosynthesis within indi­vidual cells (Berman-Frank et al., 2001).

The N2-fixing, unicellular cyanobacteria Cyanothece has recently attracted lots of research interest as a highly efficient H2 producer under natural aerobic conditions. Cyanothece sp. ATCC 51142 is the best hydrogen pro­ducer among the known wild-type cyanobacterial strains (Bandyopadhyay et al., 2010). Also, the ability to grow phototrophically, mixotrophically, and hetero- trophically makes this strain an attractive organism for biotechnology. Cyanothece demonstrates temporal sepa­ration of oxygenic photosynthesis and N2 fixation by performing photosynthesis in daytime and N2 fixation at night. Moreover, these alternating processes are regu­lated by an intrinsic circadian rhythm. The genome sequence reveals the presence of the bidirectional [Ni—Fe]-hydrogenase, uptake hydrogenase, and the conventional Mo—Fe nitrogenase. Diazotrophically grown Cyanothece cells entrained in 12-h light/12-h dark cycles exhibit a light-induced H2 production (specific rate >150—300 mmol H2 mg/Chl h) under aero­bic conditions during "subject dark" (Bandyopadhyay et al., 2010). Interestingly, the robust circadian rhythm of Cyanothece allows cells to fix N2 and produce H2 at reasonably high rates even when grown under contin­uous light (Min and Sherman, 2010).

In the presence of combined nitrogen, Cyanothece pro­duces H2 at very low rates, 2—10 mmol H2 mg/Chl h. This H2 production is catalyzed by the bidirectional hydrogenase and is dependent on PSII activity. In diaz — otrophically grown cultures, the production of H2 is driven by the nitrogenase enzyme and the activity of the enzyme is linked to PSI and respiratory electron flow (Min and Sherman, 2010). Moreover, the rates of H2 production in Cyanothece 51142 could be greatly enhanced when cells were grown in the presence of additional carbon sources, as observed in cultures sup­plemented with high concentrations of CO2 or glycerol (Bandyopadhyay et al., 2010). Photoproduction of H2 can be significantly enhanced by increasing reductant availability via dark anaerobic preincubation. This indi­cates the tight coupling of H2 photoproduction to the dark, anaerobic metabolism (Skizim et al., 2012).

Recently, it was reported that Cyanothece can copro­duce H2 and O2 over 100 h under continuous illumina­tion and uninterrupted photosynthetic electron transport (Melnicki et al., 2012). Of course, Cyanothece has a very flexible metabolism and the existence of intra­cellular O2 gradient within the cells cannot be excluded. Despite many interesting papers describing the H2 production in Cyanothece, the molecular mechanisms behind the regulation of the nitrogenase and protection against oxygenic photosynthesis are still under debate.