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Enhanced Hydrogen Production by the Halotolerant Cyanobacterium Aphanothece halophytica Through Bacterial Co-Cultivation
(2026-05-01)
Somsin, Chutikarn
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Chinchusak, Nattanon
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Incharoensakdi, Aran
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Phunpruch, Saranya
Hydrogen (H<inf>2</inf>) is a promising clean energy carrier with the potential to partially replace fossil fuels. Biological H<inf>2</inf> production using microorganisms offers an environmentally friendly alternative. The halotolerant cyanobacterium Aphanothece halophytica can produce H<inf>2</inf> under nitrogen-deprived and dark anaerobic conditions. In this study, a co-culture strategy was investigated to enhance H<inf>2</inf> production. Five bacterial strains were screened for their ability to improve H<inf>2</inf> production when co-cultivated with A. halophytica. Among them, Staphylococcus aureus significantly enhanced H<inf>2</inf> production, achieving a maximum rate of 11.11 ± 0.18 µmol H<inf>2</inf> g−1 dry weight h−1. Optimization of the bacterial partner revealed that S. aureus cells harvested at 12 h in the mid-logarithmic phase with an OD<inf>600</inf> of 4.0 were the most effective. An inoculum ratio of A. halophytica to S. aureus of 4:1 further enhanced H<inf>2</inf> production, increased bidirectional hydrogenase activity, and reduced O<inf>2</inf> accumulation. Under optimal conditions (0.945 mmol C-atom L−1 glucose, 0.25 M NaCl, pH 7.4, and 35 °C), the maximum H<inf>2</inf> production rate reached 132.49 ± 4.45 µmol H<inf>2</inf> g−1 dry weight h−1, approximately 5.5-fold higher than that under normal conditions. The co-culture achieved a cumulative H<inf>2</inf> yield of 3248.51 ± 88.11 µmol H<inf>2</inf> g−1 dry weight after 48 h.
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Enhanced hydrogen production by green alga Scenedesmus obliquus TISTR 8546 under atmospheric air through potassium deprivation and cysteine supplementation
(2026-03-01)
Sereetrakul, Kodchaporn
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Taikhao, Samart
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Chinchusak, Nattanon
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Phunpruch, Saranya
Hydrogen is a promising alternative energy carrier that can be produced by green microalgae. However, the key enzyme catalyzing this process, [FeFe]‑hydrogenase, is highly sensitive to O<inf>2</inf> generated during photosynthesis, which severely restricts its activity. This study aimed to screen the microalgal strain capable of producing high levels of H<inf>2</inf> in the presence of O<inf>2</inf> and to enhance H<inf>2</inf> production yields using O<inf>2</inf>-scavenging strategies to levels comparable to or exceeding those achieved under anaerobic conditions. Among the 24 strains tested, Scenedesmus obliquus TISTR 8546 demonstrated the highest H<inf>2</inf> production under both aerobic and anaerobic conditions. The O<inf>2</inf>I<inf>50</inf> of S. obliquus TISTR 8546 cells for H<inf>2</inf> evolution was 15.93 ± 0.24%. Potassium deprivation significantly enhanced H<inf>2</inf> production and hydrogenase activity by lowering O<inf>2</inf> levels through reduced photosynthetic O<inf>2</inf> evolution and increased dark respiration. Moreover, potassium deprivation promoted intracellular starch accumulation, providing reducing equivalents for H<inf>2</inf> generation. Cysteine supplementation further stimulated H<inf>2</inf> production by serving as a reducing agent. S. obliquus TISTR 8546 exhibited a maximum H<inf>2</inf> production rate of 22.92 ± 1.05 μmol H<inf>2</inf> mg Chl−1 h−1 and achieved a maximum cumulative H<inf>2</inf> production of 1153.78 ± 52.65 μmol H<inf>2</inf> mg Chl−1 when incubated in potassium-deprived TAP medium supplemented with 0.1 mM cysteine under atmospheric air for 6 days. This rate was 23.6- and 2.7-fold higher than those obtained in TAP and TAP-K media, respectively. These findings demonstrate the potential of S. obliquus TISTR 8546 as a robust microalgal strain for sustainable H<inf>2</inf> production under atmospheric air, highlighting its promise for future industrial applications.
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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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