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Dark fermentative hydrogen production and transcriptional analysis of genes involved in the unicellular halotolerant cyanobacterium Aphanothece halophytica under nitrogen and potassium deprivation
(2023-01-06)
Chinchusak, Nattanon
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Incharoensakdi, Aran
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Phunpruch, Saranya
The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H<inf>2</inf>) producer. This study aimed to investigate the enhancement of H<inf>2</inf> production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H<inf>2</inf> production by A. halophytica, while no differences in H<inf>2</inf> production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H<inf>2</inf> production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H<inf>2</inf> production was investigated. The results showed that the highest H<inf>2</inf> accumulation of 1,261.96 ± 96.99 µmol H<inf>2</inf> g dry wt−1 and maximum hydrogenase activity of 179.39 ± 8.18 µmol H<inf>2</inf> 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<inf>0</inf>-K) for 48 h. An increase in hydrogenase activity was attributed to the decreased O<inf>2</inf> concentration in the system, due to a reduction of photosynthetic O<inf>2</inf> 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<inf>2</inf> 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<inf>2</inf> production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.
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Enhancement of dark fermentative hydrogen production in nitrogen-deprived halotolerant unicellular cyanobacterium Aphanothece halophytica by treatment with reducing agents
(2022-12-01)
Chinchusak, Nattanon
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Incharoensakdi, Aran
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Phunpruch, Saranya
To enhance H<inf>2</inf> production by the halotolerant unicellular cyanobacterium Aphanothece halophytica, effect of various kinds of reducing sugar and reducing agent on H<inf>2</inf> production was investigated. The highest H<inf>2</inf> production rate of 55.80 ± 0.50 μmol H<inf>2</inf> g dry weight−1 h−1 was obtained when the cells were incubated in BG11<inf>0</inf> medium containing 0.189 mmol C-atom L−1 glucose under dark anaerobic condition. This rate was 1.5 folds higher than that without glucose. Among ten reducing agents tested, β-mercaptoethanol, dithiothreitol, L-cysteine and sodium sulfide had high potential as an effective reducing agent to increase H<inf>2</inf> production by A. halophytica. Cells treated with 50 mM sodium sulfide showed the highest H<inf>2</inf> accumulation with 4815.59 ± 194.78 μmol H<inf>2</inf> g dry weight−1 after 24 h of dark anaerobic incubation. An increase in H<inf>2</inf> production was ascribed to an increase of hydrogenase activity and a decrease of O<inf>2</inf> production rate. This H<inf>2</inf> production yield was approximately 20 folds higher than that without reducing agent. Furthermore, 50 mM sodium sulfide appeared to be non-toxic to A. halophytica cells, since the IC<inf>50</inf> of sodium sulfide was higher than 100 mM. The reduced ferredoxin at 1.5 μM-1.5 mM, NADH and NADPH at 0.15–1.5 mM could support in vitro [NiFe]-H<inf>2</inf>ase activity, demonstrating the ability of these compounds to provide electrons towards [NiFe]-H<inf>2</inf>ase in A. halophytica.
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Simazine Enhances Dark Fermentative H2 Production by Unicellular Halotolerant Cyanobacterium Aphanothece halophytica
(2022-07-15)
Pansook, Sunisa
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Incharoensakdi, Aran
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Phunpruch, Saranya
The halotolerant cyanobacterium Aphanothece halophytica is a potential H<inf>2</inf> producer that induces H<inf>2</inf> evolution under nitrogen deprivation. H<inf>2</inf> is mainly produced via the catabolism of stored glycogen under dark anaerobic condition. H<inf>2</inf> evolution is catalyzed by O<inf>2</inf>-sensitive bidirectional hydrogenase. The aim of this study was to improve H<inf>2</inf> production by A. halophytica using various kinds of inhibitors. Among all types of inhibitors, simazine efficiently promoted the highest H<inf>2</inf> 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<inf>2</inf> production by A. halophytica was 25 µM. Simazine inhibited photosynthetic O<inf>2</inf> evolution but promoted dark respiration, resulting in a decrease in O<inf>2</inf> level. Hence, the bidirectional hydrogenase activity and H<inf>2</inf> production was increased. A. halophytica showed the highest H<inf>2</inf> production rate at 58.88 ± 0.22 µmol H<inf>2</inf> g−1 dry weight h−1 and H<inf>2</inf> accumulation at 356.21 ± 6.04 μmol H<inf>2</inf> 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<inf>2</inf> production by A. halophytica.
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Enhanced dark fermentative H2 production by agar-immobilized cyanobacterium Aphanothece halophytica
(2019-10-01)
Pansook, Sunisa
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Incharoensakdi, Aran
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Phunpruch, Saranya
Cell immobilization is one of the techniques used to improve H<inf>2</inf> productivity in cyanobacteria. In this study, H<inf>2</inf> 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 H<inf>2</inf> production than free cells under nitrogen-deprived condition. Among various support material types used, agar-immobilized cells showed the highest H<inf>2</inf> production rate. Under nitrogen deprivation, the optimal conditions of cell immobilization for H<inf>2</inf> 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 H<inf>2</inf> 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 H<inf>2</inf> production by agar-immobilized cells. Finally, H<inf>2</inf> production by agar-immobilized cells was analyzed for three consecutive cycles. H<inf>2</inf> 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 H<inf>2</inf> production of the unicellular halotolerant cyanobacterium A. halophytica can be achieved by immobilization method, thus providing the possibility to improve H<inf>2</inf> production by cyanobacteria in the future.
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Identification of bidirectional hydrogenase genes and their co-transcription in unicellular halotolerant cyanobacterium Aphanothece halophytica
(2016-04-01)
Phunpruch, Saranya
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Taikhao, Samart
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Incharoensakdi, Aran
The halotolerant cyanobacterium Aphanothece halophytica has been shown to produce H<inf>2</inf> via dark fermentation of accumulated glycogen under anoxic condition. One set of hox genes encoding a bidirectional hydrogenase is present in A. halophytica. In this study, the nucleotide sequence and the transcriptional analysis of hox genes in A. halophytica were investigated. The results revealed that A. halophytica contained five structural genes, hoxE, hoxF, hoxU, hoxY, and hoxH, without an insertion of other open reading frames (ORFs). The conserved cysteine motifs of iron-sulfur clusters involved in an electron transfer were found in all Hox subunits. The nucleotide and deduced amino acid sequences of hox genes in A. halophytica showed the highest identity and similarity to those of Halothece sp. PCC 7418. By reverse transcription polymerase chain reaction (RT-PCR) analysis, hox genes in A. halophytica were co-transcribed as a single operon. Under nitrogen-deprived condition, the transcripts of hoxH, glgB, coxA, ndhB, and psaA were upregulated whereas those of glgP and narB were downregulated which resulted in an increase of H<inf>2</inf> production, H<inf>2</inf>ase activity, glycogen content, and dark respiration rate.
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Dark fermentative hydrogen production by the unicellular halotolerant cyanobacterium Aphanothece halophytica grown in seawater
(2015-02-01)
Taikhao, Samart
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Incharoensakdi, Aran
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Phunpruch, Saranya
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 H<inf>2</inf> producers. Under dark fermentation, it is capable of producing H<inf>2</inf> by 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 H<inf>2</inf> production. A. halophytica was able to grow in natural seawater added with NaNO<inf>3</inf>. Cells grown in seawater supplemented with as little as 1.76 mM NaNO<inf>3</inf> showed similar growth to those cultivated in normal BG11 supplemented with Turk Island salt solution. H<inf>2</inf> production was the highest when incubating the cells in seawater without any supplementation of NaNO<inf>3</inf>. Under this condition, the highest rate of dark fermentative H<inf>2</inf> production of 82.79 ± 3.47 nmol H<inf>2</inf> mg-1 dry weight h−1 was found in cells incubated at 35 °C, pH 6 with the supplementation of 378 mmolC L−1 glucose, 0.25 M NaCl, and 0.4 μM Fe3+. Long-term H<inf>2</inf> accumulation of 1,864 ± 81 nmol H<inf>2</inf> mg−1 dry weight was observed after 8 days of dark incubation under anoxic condition, and the high yield of H<inf>2</inf> was sustained at least up to 14 days, suggesting the possibility of utilizing natural seawater to grow A. halophytica for long-term production of H<inf>2</inf>.
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Factors affecting biohydrogen production by unicellular halotolerant cyanobacterium Aphanothece halophytica
(2013-04-01)
Taikhao, Samart
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Junyapoon, Suwannee
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Incharoensakdi, Aran
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Phunpruch, Saranya
The effects of several physiological parameters on H<inf>2</inf> production rate in the unicellular halotolerant cyanobacterium Aphanothece halophytica were investigated. Under nitrogen deprivation, the growth of cells was inhibited, but H<inf>2</inf> production rate was enhanced approximately fourfold. Interestingly, cells grown under sulfur deprivation exhibited a decrease in cell growth, H<inf>2</inf> production rate, and bidirectional hydrogenase activity. Glucose was the preferred sugar source for H<inf>2</inf> production by A. halophytica, but H<inf>2</inf> production decreased at high glucose concentrations. H<inf>2</inf> production rate was optimum when cells were grown in the presence of 0. 75 M NaCl, or 0. 4 μM Fe3+, or 1 μM Ni2+. The optimum light intensity and temperature for H<inf>2</inf> production were 30 μmol photons m-2 s-1 and 35 °C, respectively. A two-stage culture of A. halophytica was performed in order to overcome the reduction of cell growth in N-free medium. In the first stage, cells were grown in normal medium to accumulate biomass, and in the second stage, H<inf>2</inf> production by the obtained biomass was induced by growing cells in N-free medium supplemented with various chemicals for 24 h. A. halophytica grown in N-free medium containing various MgSO<inf>4</inf> concentrations had a high H<inf>2</inf> production rate between 11. 432 and 12. 767 μmol H<inf>2</inf> mg chlorophyll a (chl a)-1 h-1, a 30-fold increase compared to cells grown in normal medium. The highest rate of 13. 804 μmol H<inf>2</inf> mg chl a-1 h-1 was obtained when the N-free growth medium contained 0. 4 μM Fe3+. These results suggested the possibility of using A. halophytica and some other halotolerant cyanobacteria thriving under extreme environmental conditions in the sea as potential sources for H<inf>2</inf> production in the future. © 2012 Springer Science+Business Media B.V.

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