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Within the cyanobacterial world there are many species adapted to life in hypersaline environments. Some can even grow at salt concentrations approaching NaCl saturation. Halophilic cyanobacteria often form dense mats in salt lakes, and on the bottom of solar saltern ponds, hypersaline lagoons, and saline sulfur springs, and they may be found in evaporite crusts of gypsum and halite. A wide range of species were reported to live at high salinities. These include unicellular types ( Aphanothece halophytica and similar morphotypes described as Euhalothece and Halothece ), as well as non-heterocystous filamentous species ( Coleofasciculus chthonoplastes , species of Phormidium , Halospirulina tapeticola , Halomicronema excentricum , and others). Cyanobacterial diversity in high-salt environments has been explored using both classic, morphology-based taxonomy and molecular, small subunit rRNA sequence-based techniques. This paper reviews the diversity of the cyanobacterial communities in hypersaline environments worldwide, as well as the physiological adaptations that enable these cyanobacteria to grow at high salt concentrations. To withstand the high osmotic pressure of their surrounding medium, halophilic cyanobacteria accumulate organic solutes: glycine betaine is the preferred solute in the most salt-tolerant types; Coleofasciculus produces the heteroside glucosylglycerol, and the less salt-tolerant cyanobacteria generally accumulate the disaccharides sucrose and trehalose under salt stress. Some cyanobacteria growing in benthic mats in hypersaline environments are adapted to life under anoxic conditions and they can use sulfide as an alternative electron donor in an anoxygenic type of photosynthesis through a process which involves photosystem I only.

Unearthing new sustainable and economically viable sources for biofuel production which do not affect the environment is a dire need of the hour. Microalgae is one such promising source due to its high lipid content, productivity, and carbon neutrality. Identification of appropriate strain and process optimization decides the biomass productivity, nutrient value, and oil content which are the major factors for commercialization. In the present work, mass cultivation of halophilic Aphanothece halophytica in raceway ponds was optimized by using organic and inorganic nutrients by using design of experiments. Organic flocculant, neem plus was successfully adapted for harvesting the biomass and oil extraction was done with solvent methodology. A maximum lipid yield of 29.3% was obtained on wet basis, when the reaction temperature, reaction time, biomass-to-solvent ratio and mixing intensity were kept at 68 ºC, 190 min, 9:1, and 300 rpm respectively. Similarly, on dry basis, a lipid yield of 27.5% was reported when the reaction temperature, reaction time, biomass-to-solvent ratio and mixing intensity were maintained at 68 ºC, 190 min, 12:1, and 300 rpm respectively. GC–MS analysis of the lipid was done to appropriate the combination of fatty acid for enhancing the biofuel production.

Cell immobilization is one of the techniques used to improve H2 productivity in cyanobacteria. In this study, H2 production by immobilized cells of unicellular halotolerant cyanobacterium Aphanothece halophytica was investigated and optimized. The results showed that immobilized cells of A. halophytica had higher H2 production than free cells under nitrogen-deprived condition. Among various support material types used, agar-immobilized cells showed the highest H2 production rate. Under nitrogen deprivation, the optimal conditions of cell immobilization for H2 production were 3% (w/v) agar concentration, 0.2 mg dry cell weight per mL of gel solution, and 0.125 cm3 of agar cube. The optimum pH of medium and incubation temperature for H2 production by agar-immobilized cells were pH 7.4 and 40 °C, respectively. Using a large glass vial and headspace volume resulted in enhancement of H2 production by agar-immobilized cells. Finally, H2 production by agar-immobilized cells was analyzed for three consecutive cycles. H2 production could be maintained at the highest level after two cycles when half of immobilized cells were replaced with fresh immobilized cells. These findings indicate that the enhanced H2 production of the unicellular halotolerant cyanobacterium A. halophytica can be achieved by immobilization method, thus providing the possibility to improve H2 production by cyanobacteria in the future.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H 2 ) producer. This study aimed to investigate the enhancement of H 2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H 2 production by A. halophytica , while no differences in H 2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H 2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H 2 production was investigated. The results showed that the highest H 2 accumulation of 1,261.96 ± 96.99 µmol H 2 g dry wt −1 and maximum hydrogenase activity of 179.39 ± 8.18 µmol H 2 g dry wt −1 min −1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG11 0 -K) for 48 h. An increase in hydrogenase activity was attributed to the decreased O 2 concentration in the system, due to a reduction of photosynthetic O 2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H 2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis ( glgA , glgB , and glgP ) were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H 2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.

Aphanothece cells could take up Na super(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na super(+) exhibited Na super(+) extrusion ability upon energizing with glucose. Na super(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na super(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na super(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na super(+) but not with K super(+), Ca super(2+) and Li super(+). The K sub(m) values for ATP and Na super(+) were 1.66 plus or minus 0.12 and 25.0 plus or minus 1.8 mM, respectively, whereas the V sub(max) value was 0.66 plus or minus 0.05 mu mol min super(-1)&thins p; mg super(-1). Mg super(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na super(+)H super(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na super(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.

The halotolerant cyanobacterium Aphanothece halophytica is a potential H 2 producer that induces H 2 evolution under nitrogen deprivation. H 2 is mainly produced via the catabolism of stored glycogen under dark anaerobic condition. H 2 evolution is catalyzed by O 2 -sensitive bidirectional hydrogenase. The aim of this study was to improve H 2 production by A. halophytica using various kinds of inhibitors. Among all types of inhibitors, simazine efficiently promoted the highest H 2 production under dark conditions. High simazine concentration and long-term incubation resulted in a decrease in cell and chlorophyll concentrations. The optimal simazine concentration for H 2 production by A. halophytica was 25 µM. Simazine inhibited photosynthetic O 2 evolution but promoted dark respiration, resulting in a decrease in O 2 level. Hence, the bidirectional hydrogenase activity and H 2 production was increased. A. halophytica showed the highest H 2 production rate at 58.88 ± 0.22 µmol H 2 g −1 dry weight h −1 and H 2 accumulation at 356.21 ± 6.04 μmol H 2 g −1 dry weight after treatment with 25 µM simazine under dark anaerobic condition for 2 and 24 h, respectively. This study demonstrates the potential of simazine for the enhancement of dark fermentative H 2 production by A. halophytica .

A link between carbon and nitrogen metabolism is important for serving as metabolic ancillary reactions. Here, we identified and characterized the alanine dehydrogenase gene in Aphanothece halophytica (ApalaDH) that is involved in alanine assimilation/dissimilation. Functional analysis revealed that ApalaDH encodes a bifunctional protein catalyzing the reversible reaction of pyruvate to l-alanine via its pyruvate reductive aminase (PvRA) activity, the reaction of l-alanine to pyruvate via its alanine oxidative dehydrogenase activity, and the non-reversible reaction of glyoxylate to glycine via its glyoxylate reductive aminase (GxRA) activity. Kinetic analysis showed the lowest affinity for pyruvate followed by l-alanine and glyoxylate with a Km of 0.22 ± 0.02, 0.72 ± 0.04, and 1.91 ± 0.43 mM, respectively. ApalaDH expression was upregulated by salt. Only PvRA and GxRA activities were detected in vivo and both activities increased about 1.2- and 2.7-fold upon salt stress. These features implicate that the assimilatory/dissimilatory roles of ApAlaDH are not only selective for l-alanine and pyruvate, but also, upon salt stress, can catabolize glyoxylate to generate glycine.

Aminotransferases catalyze the reversible pyridoxal phosphate–dependent transfer of amino groups from amino acids to oxo acids and play important roles for the balance between carbon and nitrogen metabolism. In this report, four aminotransferases (Ap1–Ap4) from a halotolerant cyanobacterium Aphanothece halophytica were examined. The results revealed that Ap1 and Ap2 exhibited the aspartate:2-oxoglutarate aminotransferase (AspAT) activity whereas Ap2 catalyzed further aminotransferase activities with alanine (AlaAT) and LL-diaminopimelate (an intermediate for the synthesis of Lys/peptidoglycan) as amino donors. Ap4 exhibited bifunctional aminotransferase with phosphoserine (PSAT) and glycine (GGAT) as amino donors. No activity was observed for Ap3. We identified third gene encoding phosphoserine phosphatase (PSP) in phosphorylate serine biosynthetic pathway. The levels of mRNA for Ap2 and ApMurE encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase were increased after salt stress. These results suggest the link among photorespiratory metabolite (serine, glycine, glyoxylate), phosphorylate serine biosynthetic pathway and aspartate metabolism via aminotransferases for the synthesis of peptidoglycan and betaine under salt stress conditions.

γ-Aminobutyric acid (GABA) is known as an inhibitory neurotransmitter in human, while in plants, GABA is an intermediate for amino acid metabolism and also is accumulated in response to a wide range of environmental stress. In the present study, GABA accumulation in Aphanothece halophytica was increased 2-fold in mid-log phase cells grown under salt stress (2.0 M NaCl). When mid-log phase cells were subjected to changes in NaCl concentrations and pH for 4 h, the highest GABA accumulation was observed in cells adapted in medium that contained 2.0 M NaCl and that was adjusted to pH 4.0, respectively. The increase of GABA accumulation was accompanied by an increased glutamate decarboxylase activity. Addition of glutamate to growth medium stimulated GABA accumulation under acid stress but had no effect under salt stress. However, the highest GABA accumulation was detected in cells exposed to both high salt and acid stresses combined with the 5 mM glutamate supplementation with an approximately 3-fold increase as compared to the control. The unicellular A. halophytica showed a similarly high content of GABA to that of a filamentous Arthrospira platensis suggesting the possibility of genetic manipulation of the genes of A. halophytica involved in GABA synthesis to increase GABA yield.

Biohydrogen is an environmentally friendly alternative energy carrier that can be produced by a number of different microorganisms. The unicellular halotolerant cyanobacterium Aphanothece halophytica is one of the high potential H₂producers. Under dark fermentation, it is capable of producing H₂by the bidirectional hydrogenase activity via the catabolism of glycogen stored during photosynthesis. This work aimed to cultivate A. halophytica in natural seawater containing high salinity and minerals, with an addition of some essential nutrients, and to investigate effects of various nutritional and physical factors on its dark fermentative H₂production. A. halophytica was able to grow in natural seawater added with NaNO₃. Cells grown in seawater supplemented with as little as 1.76 mM NaNO₃showed similar growth to those cultivated in normal BG11 supplemented with Turk Island salt solution. H₂production was the highest when incubating the cells in seawater without any supplementation of NaNO₃. Under this condition, the highest rate of dark fermentative H₂production of 82.79 ± 3.47 nmol H₂ mg⁻¹dry weight h⁻¹was found in cells incubated at 35 °C, pH 6 with the supplementation of 378 mmolC L⁻¹glucose, 0.25 M NaCl, and 0.4 μM Fe³⁺. Long-term H₂accumulation of 1,864 ± 81 nmol H₂ mg⁻¹dry weight was observed after 8 days of dark incubation under anoxic condition, and the high yield of H₂was sustained at least up to 14 days, suggesting the possibility of utilizing natural seawater to grow A. halophytica for long-term production of H₂.