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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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Biomass Production of Chlorella vulgaris var. vulgaris TISTR 8261 During Cultivation in Modified Food Industry Wastewater
(2025-12-01)
Taikhao, Samart
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Phunpruch, Saranya
Industrial wastewater can serve as a low-cost nutritional source for sustainable microalgal biomass production. This study investigated the biomass of Chlorella vulgaris var. vulgaris TISTR 8261 grown in untreated wastewater collected from four food industry factories in Phra Nakhon Sri Ayutthaya Province, Thailand. Among them, wastewater from a processed food production plant (PFPP) supported the highest algal growth. Supplementation with 17.4 mM sodium acetate significantly improved algal biomass yield. Further optimization with 3.7 mM NH<inf>4</inf>Cl, 1.0 mM KH<inf>2</inf>PO<inf>4</inf>, 0.2 mM MgSO<inf>4</inf>, and a moderate concentration of trace minerals enhanced the specific growth rate and chlorophyll concentration. Scaled-up cultivation in 3.5 L culture bottles in optimized PFPP yielded a maximum biomass yield of 8.436 ± 0.378 g L−1, comparable to 6.498 ± 0.436 g L−1 in standard TAP medium. Biomass composition analysis after 15 days of cultivation revealed 42.70 ± 1.40% protein, 17.10 ± 1.60% carbohydrate, and 1.90 ± 0.10% lipid on a dry weight basis. These findings demonstrate that optimized PFPP wastewater can effectively support high-density cultivation of C. vulgaris var. vulgaris TISTR 8261, yielding nutritionally rich biomass, and offering a cost-effective and environmentally sustainable strategy for industrial-scale microalgal production.
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Biomass and biohydrogen production by unicellular green alga Chlorella vulgaris var. vulgaris TISTR 8261 using frozen food industrial wastewater
(2022-01-01)
Taikhao, Samart
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Phunpruch, Saranya
Biohydrogen production by green algal biomass is promising method for sustainable H<inf>2</inf> production and bioenergy recovery. In this approach, green algae convert organic and inorganic substances (used as the sole source of carbon and electrons) in wastewater into H<inf>2</inf>. In this study, biomass and H<inf>2</inf> production from the unicellular green alga Chlorella vulgaris var. vulgaris TISTR 8261, cultivated in frozen food industrial wastewater, was investigated. The results revealed that growth rate of algal cells cultivated in treated wastewater was significantly higher than that in untreated wastewater but lower than that in the synthetic control Tris acetate phosphate (TAP) medium. In addition, the cells grown in treated wastewater exhibited a high ability to remove nitrate, nitrite, phosphate, and sulfate from the water. Furthermore, algal cells were cultured with various concentrations of sodium acetate (0-17.4 mM); the optical density of the cultures at 750 nm increased with increase in acetate concentration. Cell growth in treated wastewater supplemented with 17.4 mM sodium acetate was similar to that in TAP medium. The highest H<inf>2</inf> production of 12.87 ± 0.58 µmolH<inf>2</inf> mg Chl a-1 was observed in cells incubated in treated wastewater supplemented with 17.4 mM sodium acetate; this yield was higher than that obtained from cells incubated in nitrogen-free TAP medium. The results of this study support the potential use of wastewater for biomass and biohydrogen production by C. vulgaris var. vulgaris TISTR 8261.
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Increasing Hydrogen Production Efficiency of N2-Fixing Cyanobacterium Anabaena siamensis TISTR 8012 by Cell Immobilization
(2017-01-01)
Taikhao, Samart
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Phunpruch, Saranya
H<inf>2</inf> produced by cyanobacteria is one of the interesting alternative energy carriers for the future. The filamentous N<inf>2</inf>-fixing cyanobacterium Anabaena siamensis TISTR 8012 is a potential microorganism for H<inf>2</inf> production. It can produce H<inf>2</inf> via both photosynthesis and nitrogen fixation processes. This study aimed to increase the efficiency of H<inf>2</inf> production by immobilization of A. siamensis cells. The result showed that H<inf>2</inf> production rate by A. siamensis grown in BG11<inf>0</inf> medium was higher than that in BG11 and Allen-Arnon media due to an increase of the heterocyst cells. The sulfur deprivation during adaptation period for 24 hours increased its H<inf>2</inf> production rate. The optimal conditions for H<inf>2</inf> production by immobilized cells were immobilization with alginate and using 150 gel beads in 20 mL glass vial. In immobilized cells, an addition of 0.5% fructose resulted in a 2-fold increase of H<inf>2</inf> production rate. Finally, the reducing agents β-mercaptoethanol and methylviologen enhanced H<inf>2</inf> production rate with 3.092 and 2.426 μmolH<inf>2</inf> mg chl a-1 h-1, respectively, in A. siamensis immobilized cells whereas NADH, dithiothreitol and sodium dithionite were not capable of increasing H<inf>2</inf> production rate.
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Effect of Metal Cofactors of Key Enzymes on Biohydrogen Production by Nitrogen Fixing Cyanobacterium Anabaena siamensis TISIR 8012
(2017-01-01)
Taikhao, Samart
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Phunpruch, Saranya
In N<inf>2</inf>-fixing cyanobacteria, three enzymes are involved in the H<inf>2</inf> metabolism. Nitrogenase catalyzes the N<inf>2</inf> fixation which produces H<inf>2</inf> as a by-product. The produced H<inf>2</inf> is taken up to protons and electrons by an activity of uptake hydrogenase. Reversible enzyme catalyzes both reactions of the H<inf>2</inf> evolution and the H<inf>2</inf> uptake. These enzymes are all metalloenzyme. The cyanobacterial nitrogenase normally requires molybdenum and iron as cofactors; however nitrogenase of few cyanobacterial species is dependent on vanadium. The cyanobacterial uptake and reversible hydrogenase requires nickel and iron as cofactors. This research aimed to investigate the effect of these metal cofactors on H<inf>2</inf> production and hydrogenase activity by N<inf>2</inf>-fixing cyanobacterium Anabaena siamensis TISTR 8012 isolated from rice paddle field in Thailand. The result showed that A. siamensis cells incubated in N-deprived BG11 medium (BG11<inf>0</inf>) gave clearly higher H<inf>2</inf> production rate and hydrogenase activity than those in normal BG11 medium. Under nitrogen deprivation, an increase of iron, nickel, and molybdenum concentrations obviously enhanced H<inf>2</inf> production rate. But only higher iron concentrations increased hydrogenase activity, indicating that the iron metal assisted in the function of reversible hydrogenase activity. In addition, vanadium seemed not to be a metal cofactor of key enzymes involving in H<inf>2</inf> production in A. siamensis. The optimal concentrations of iron, nickel and molybdenum ions for H<inf>2</inf> production rate by A. siamensis were 60 μM, 4 μM and 4 μM, respectively. The highest H<inf>2</inf> production rate of 0.057 μmolH<inf>2</inf> mg chl a-1 h-1 was observed in cells incubated in BG11<inf>0</inf> medium supplemented with 4 μM nickel ion.
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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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