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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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Light-Emitting Diode Illumination Enhances Biomass, Pigment, and Lipid Production in Halotolerant Cyanobacterium Aphanothece halophytica
(2025-06-01)
Thongtha, Sitthichai
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Kittiwongwattana, Chokchai
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Incharoensakdi, Aran
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Phunpruch, Saranya
Light characteristics, including spectrum and intensity, significantly impact cyanobacterial biomass production, pigment biosynthesis, and cellular metabolism, influencing the composition of various biochemical compounds. This study aimed to investigate the effects of light-emitting diode (LED) illumination on biomass, pigment, and lipid production in the unicellular halotolerant cyanobacterium Aphanothece halophytica, cultivated in a suitable natural seawater (SNSW) medium. The results revealed that LED light outperformed fluorescent light, with blue LED light, particularly at an intensity of 60 μmol photons m−2 s−1, significantly enhancing growth, pigment synthesis, and lipid accumulation. This resulted in a maximum cell density of 68.96 ± 1.52 × 106 cells mL−1, a specific growth rate of 0.302 ± 0.002 day−1, and a lipid productivity of 56.81 ± 0.75 mg L−1 day−1. White LED light produced lipids suitable for biodiesel, whereas blue, green, and red LEDs promoted the accumulation of polyunsaturated fatty acids (PUFAs), beneficial for food supplements. These findings highlight the potential of LED-based cultivation strategies for optimizing biomass and biochemical compound production in A. halophytica.
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Modified natural seawater as growth medium for the halotolerant cyanobacterium Aphanothece halophytica to increase lipid content for biodiesel production
(2025-02-01)
Thongtha, Sitthichai
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Aryusuk, Kornkanok
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Kittiwongwattana, Chokchai
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Incharoensakdi, Aran
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Phunpruch, Saranya
Biodiesel derived from cyanobacterial oils becomes attractive as an efficient renewable energy. The present study aims to optimize growth and lipid production of the halotolerant unicellular cyanobacterium Aphanothece halophytica cultivated in natural seawater. In this study A. halophytica was able to grow in natural seawater when supplemented with low concentration of NaNO<inf>3</inf>, whereas no growth occurred without supplementation. The specific growth rate of 0.230 day-1 and cell concentration of 25.17 x 106 cells mL-1 were achieved in A. halophytica cultivated in natural seawater supplemented with 17.6 mM NaNO<inf>3</inf> and Turk Island salt solution (suitable natural seawater; SNSW) for 14 days. This growth rate was comparable to that of cells grown in normal BG11 plus Turk Island salt solution. The lipid content and fatty acid profiles of A. halophytica varied with changes in NaCl concentrations. The highest lipid content of 50.47 % and lipid productivity of 48.33 mg L-1 day-1 were obtained in cultures supplemented with 1.89 mmol C-atom L-1 glucose and 0.75 M NaCl. The optimal medium pH and cultivation temperature for lipid production was 7.5 and 25-35 °C, respectively. When cultivating A. halophytica in optimized SNSW with various NaCl concentrations, the highest contents of linoleic and linolenic acids, and the lowest contents of palmitic, stearic, and oleic acids were observed with 0.75 M NaCl. In contrast, cultures grown in optimized SNSW with 0.5 M NaCl showed fatty acid methyl ester profiles rich in monounsaturated fatty acids, which are favorable for high-quality biodiesel production.
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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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Upregulation of Hox-hydrogenase gene expression by nutrient adjustment in the filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005
(2020-12-01)
Raksajit, Wuttinun
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Maneeruttanarungroj, Cherdsak
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Mäenpää, Pirkko
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Lehto, Kirsi
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Incharoensakdi, Aran
Arthrospira sp. PCC 8005 is potentially able to produce hydrogen catalysed by hox gene–encoded bidirectional hydrogenase with the aid of hyp gene–encoded accessory proteins. In the present study, we investigated the physiological factors affecting the hoxE, hoxY, hoxH, and hypF transcription in Arthrospira sp. PCC 8005. About a 4-fold increase of biomass and chlorophyll-a content was observed in cells grown for 7 days in Zarrouk’s medium supplemented with Fe2+. Cells grown in N-deprived medium with added Ni2+ had increased H<inf>2</inf> production and hydrogenase activity with a maximal value of 7.24 ± 0.25 μmol H<inf>2</inf> mg−1 Chla h−1 and 0.61 ± 0.03 μmol H<inf>2</inf> mg−1 Chla h−1, respectively. RT-PCR analysis revealed that cells grown in the N-deprived medium supplemented with Fe2+ or Ni2+ increased hoxE, hoxY, and hoxH transcripts. However, the highest increase of the hoxE, hoxY, hoxH, and hypF transcripts was observed in cells grown in the S-deprived medium supplemented with a combination of Fe2+ and β-mercaptoethanol. These results indicated that the increased expression of hox genes in Arthrospira sp. PCC 8005 can be achieved by proper adjustment of the nutrients in the growth medium. Phylogenetic analysis revealed that the small hydrogenase subunit, HoxY sequence, from Arthrospira sp. PCC 8005 was clustered together along with other cyanobacterial HoxY which is highly related to Arthrospira platensis NIES46.
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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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Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica
(2019-01-01)
Pansook, Sunisa
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Incharoensakdi, Aran
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Phunpruch, Saranya
The unicellular halotolerant cyanobacterium Aphanothece halophytica is a potential dark fermentative producer of molecular hydrogen (H<inf>2</inf>) that produces very little H<inf>2</inf> under illumination. One factor limiting the H<inf>2</inf> photoproduction of this cyanobacterium is an inhibition of bidirectional hydrogenase activity by oxygen (O<inf>2</inf>) obtained from splitting water molecules via photosystem II activity. The present study aimed to investigate the effects of the photosystem II inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on H<inf>2</inf> production of A. halophytica under light and dark conditions and on photosynthetic and respiratory activities. The results showed that A. halophytica treated with CCCP and DCMU produced H<inf>2</inf> at three to five times the rate of untreated cells, when exposed to light. The highest H<inf>2</inf> photoproduction rates, 2. 26 ± 0. 24 and 3. 63 ± 0. 26 μmol H<inf>2</inf>g-1 dry weight h-1, were found in cells treated with 0.5 μM CCCP and 50 μM DCMU, respectively. Without inhibitor treatment, A. halophytica incubated in the dark showed a significant increase in H<inf>2</inf> production compared with cells that were incubated in the light. Only CCCP treatment increased H<inf>2</inf> production of A. halophytica during dark incubation, because CCCP functions as an uncoupling agent of oxidative phosphorylation. The highest dark fermentative H<inf>2</inf> production rate of 39. 50 ± 2. 13 μmol H<inf>2</inf>g-1 dry weight h-1 was found in cells treated with 0.5 μM CCCP after 2 h of dark incubation. Under illumination, CCCP and DCMU inhibited chlorophyll fluorescence, resulting in a low level of O<inf>2</inf>, which promoted bidirectional hydrogenase activity in A. halophytica cells. In addition, only CCCP enhanced the respiration rate, further reducing the O<inf>2</inf> level. In contrast, DCMU reduced the respiration rate in A. halophytica.
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Screening cyanobacteria from marine coastal waters of Thailand for biohydrogen production
(2018-12-01)
Tinpranee, Nichanan
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Incharoensakdi, Aran
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Phunpruch, Saranya
Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. H <inf>2</inf> can be produced by cyanobacterial bidirectional hydrogenase, but mainly during anaerobic dark fermentation. Here, we screened H <inf>2</inf> producing cyanobacteria isolated from marine environments in Thailand and optimized physiological conditions for maximizing H <inf>2</inf> production of the selected isolate. Most of the 54 cyanobacterial strains isolated and purified from samples of seawater, stones, sand, and shells in the Gulf of Thailand and the Andaman Sea, southern part of Thailand produced H <inf>2</inf> when cells were incubated in nitrogen-deprived medium under dark/anaerobic condition. The filamentous non-heterocystous cyanobacterium Geitlerinema sp. RMK-SH10 gave the highest H <inf>2</inf> yield with highest H <inf>2</inf> production rate found in 7-day grown cells. Geitlerinema sp. RMK-SH10 showed maximum H <inf>2</inf> production rate of 0.271 ± 0.013 μmol H <inf>2</inf> mg −1 dry weight h −1 when incubated in NaNO <inf>3</inf> -free ASN III medium containing 0.2 M NaCl, 18.9 mmol C-atom of glucose L −1 , and 0.1 μM Ni 2+ . These results suggest that the marine filamentous cyanobacterium Geitlerinema sp. RMK-SH10 has high potential as a H <inf>2</inf> producer amenable for further improvement by genetic manipulation.

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