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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
;
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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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
;
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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New batch and continuous systems for converting hydrogen from water hyacinth
(2018-10-03)
Ruen-ngam, Duangkamol
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Jaruyanon, Pongsiri
Water hyacinth was the chosen alternative biomass for H<inf>2</inf> production in this study. A batch and a continuous pilot plant systems are proposed. A batch system of 500 ml capacity was developed first for finding an appropriate biomass ratio which was found to be 2.5%wt. Results from the batch system was used to construct a kinetic model of water hyacinth hydrolysis. The same biomass and water ratio was used in the continuous system. Besides water hyacinth, glucose and cellulose + lignin biomasses were tested. During processing, temperature, pressure, and electric consumption of the system were recorded. The quality and quantity of gas products were analyzed by gas chromatography (GC). The water hyacinth biomass attained the highest rate of H<inf>2</inf> production (providing energy of 11.43 kWh/mole of glucose). Comparing the batch and continuous pilot systems, the continuous system achieved 2.7 times more H<inf>2</inf> mole% than the batch system did.
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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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