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Article

Enhanced Hydrogen Production by the Halotolerant Cyanobacterium Aphanothece halophytica Through Bacterial Co-Cultivation

by
Chutikarn Somsin
1,
Nattanon Chinchusak
1,
Aran Incharoensakdi
2,3 and
Saranya Phunpruch
1,4,*
1
Department of Biology, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
2
Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3
Academy of Science, Royal Society of Thailand, Bangkok 10300, Thailand
4
Bioenergy Research Unit, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand
*
Author to whom correspondence should be addressed.
Fermentation 2026, 12(5), 221; https://doi.org/10.3390/fermentation12050221
Submission received: 20 March 2026 / Revised: 22 April 2026 / Accepted: 25 April 2026 / Published: 29 April 2026
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

Hydrogen (H2) is a promising clean energy carrier with the potential to partially replace fossil fuels. Biological H2 production using microorganisms offers an environmentally friendly alternative. The halotolerant cyanobacterium Aphanothece halophytica can produce H2 under nitrogen-deprived and dark anaerobic conditions. In this study, a co-culture strategy was investigated to enhance H2 production. Five bacterial strains were screened for their ability to improve H2 production when co-cultivated with A. halophytica. Among them, Staphylococcus aureus significantly enhanced H2 production, achieving a maximum rate of 11.11 ± 0.18 µmol H2 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 OD600 of 4.0 were the most effective. An inoculum ratio of A. halophytica to S. aureus of 4:1 further enhanced H2 production, increased bidirectional hydrogenase activity, and reduced O2 accumulation. Under optimal conditions (0.945 mmol C-atom L−1 glucose, 0.25 M NaCl, pH 7.4, and 35 °C), the maximum H2 production rate reached 132.49 ± 4.45 µmol H2 g−1 dry weight h−1, approximately 5.5-fold higher than that under normal conditions. The co-culture achieved a cumulative H2 yield of 3248.51 ± 88.11 µmol H2 g−1 dry weight after 48 h.

1. Introduction

The depletion of fossil fuel reserves and their contribution to CO2 emissions have intensified global energy and climate challenges, underscoring the urgent need for a transition to renewable energy sources [1,2]. Hydrogen (H2) is widely regarded as a promising clean energy carrier due to its high energy density (141.9 MJ kg−1) and its potential applications in heavy transportation and power generation [3,4]. However, most conventional H2 production methods rely on fossil fuels and are associated with high energy consumption and significant carbon emissions [5]. In contrast, biological H2 production offers a sustainable alternative by operating under mild conditions, utilizing renewable substrates, and generating minimal CO2 emissions. Consequently, microbial H2 production using bacteria, green algae, and cyanobacteria has attracted increasing research interest.
Cyanobacteria, also known as blue-green algae, are photosynthetic prokaryotes capable of producing H2 through the activity of three key enzymes: nitrogenase, bidirectional hydrogenase (H2ase), and uptake hydrogenase. Nitrogenase produces H2 as a by-product of nitrogen fixation under nitrogen-deprived and anaerobic conditions, whereas bidirectional H2ase catalyzes H2 evolution using electrons derived from NAD(P)H or reduced ferredoxin. In contrast, uptake H2ase oxidizes H2, thereby decreasing net H2 yields [6]. The efficiency of cyanobacterial H2 production is influenced by multiple biological and environmental factors, including cell age and density, nutrient availability, salinity, carbon source, light intensity, pH, and temperature, all of which affect enzyme activity and cellular metabolism [7,8,9].
Among cyanobacteria, the unicellular halotolerant species Aphanothece halophytica has shown considerable potential for H2 production. Under nitrogen-deprived conditions, it can evolve H2 via bidirectional H2ase in both BG11 medium and seawater [10,11]. Its capacity to thrive in high-salinity environments allows for cultivation in seawater or saline wastewater, reducing freshwater demand and operational costs while supporting sustainable biohydrogen production [12]. However, H2ase activity in A. halophytica is highly sensitive to O2, which remains a major limitation to H2 productivity. To address this challenge, several strategies have been explored, including nitrogen and potassium deprivation [13], supplementation with reducing sugars and reducing agents [14], cell immobilization in agar matrices [15], and inhibition of photosystem II combined with enhanced dark respiration [16].
Among these approaches, co-cultivation has gained increasing attention as an effective strategy to enhance H2 production by alleviating O2 inhibition. In such systems, heterotrophic bacteria consume O2 produced during photosynthesis or respiration, thereby creating more favorable anaerobic conditions for H2ase activity [17,18]. For instance, co-cultivation of Chlamydomonas reinhardtii with bacteria has been shown to reduce O2 accumulation, enabling sustained H2 production even under low light conditions [19,20]. Similarly, co-cultivation of the cyanobacterium Phormidium keutzingianum with activated sludge, in combination with sugar supplementation, enhanced H2 production through bacterial O2 consumption [18]. In addition, co-cultivation systems involving green algae, bacteria, or cyanobacteria have been reported to improve substrate utilization, prolong H2 production, and reduce inhibitory metabolite accumulation, resulting in higher H2 yields than those of monocultures [21,22].
Despite growing interest in microbial co-culture strategies for biohydrogen production in freshwater cyanobacteria, studies focusing on halotolerant cyanobacteria remain limited. Although A. halophytica can tolerate high salinity and produce H2 under dark anaerobic conditions, its performance is still constrained by O2 sensitivity and metabolic limitations. Co-cultivation with heterotrophic bacteria offers a promising solution by promoting O2 consumption, metabolic exchange, and greater environmental stability within the culture system. In this study, five bacterial strains were systematically screened for their ability to enhance H2 production when co-cultivated with A. halophytica. The effects of key operational parameters on H2 production were also investigated. By identifying an effective bacterial partner, this work provides new insights into microbial interactions that can improve biohydrogen production in halotolerant cyanobacterial systems. These findings support the development of more efficient production strategies and highlight the potential of such systems for applications in saline and wastewater-based bioprocesses.

2. Materials and Methods

2.1. Cyanobacterial Cultivation

The unicellular halotolerant cyanobacterium Aphanothece halophytica, originally isolated from Solar Lake, Israel, was kindly provided by Dr. Tetsuko Takabe (Nagoya University, Japan). A. halophytica was cultivated in 250-mL Erlenmeyer flasks containing 100 mL of BG11 medium (pH 7.4) [23] and supplemented with Turks Island salt solution [24]. The initial optical density of the culture was adjusted to approximately 0.1 at 730 nm (OD730). Cultures were incubated at 30 °C under continuous agitation at 120 rpm and illuminated at 30 µmol photons m−2 s−1 with a 16:8 h light–dark photoperiod.

2.2. Bacterial Cultivation

Five bacterial strains, including Bacillus cereus TISTR 1449, Escherichia coli TISTR 074, Micrococcus luteus TISTR 2374, Pseudomonas aeruginosa TISTR 2370, and Staphylococcus aureus TISTR 746, were obtained from the Thailand Institute of Scientific and Technological Research (TISTR), Pathum Thani, Thailand. The strains were cultivated in 250-mL Erlenmeyer flasks containing 100 mL of Luria–Bertani (LB) medium (pH 7.0) (HiMedia, Mumbai, India). The initial optical density was adjusted to approximately 0.1 at 600 nm (OD600). Cultures were incubated at 37 °C with shaking at 120 rpm for 24 h.

2.3. Co-Cultivation of Cyanobacteria with Bacteria

In principle, cyanobacteria are typically grown under light-driven conditions, whereas bacteria are cultivated under dark fermentation. In this study, both microorganisms were co-cultivated under dark anaerobic conditions. Anaerobiosis was established by purging with argon gas, and cultures were incubated in complete darkness to prevent O2 generation from cyanobacterial photosynthesis. A. halophytica was first cultivated in BG11 medium supplemented with Turks Island salt solution under the conditions described above for 7 days. The cells were then harvested by centrifugation at 8000× g for 10 min at 4 °C, washed twice, and resuspended in 100 mL of nitrogen-deprived BG11 medium (BG110) supplemented with Turks Island salt solution. The culture was further incubated for 2 days. Subsequently, the cells were collected, resuspended in fresh medium, and adjusted to a relative OD730 of 20.0. At this density, the cell concentration, determined by direct counting using a hemocytometer (Boeco, Hamburg, Germany) under a light microscope, was approximately 2 × 109 cells mL−1. Bacterial cultures were grown in LB medium for 24 h, harvested by centrifugation at 8000× g for 10 min at 4 °C, and resuspended in BG110 medium supplemented with Turks Island salt solution. The bacterial suspensions were adjusted to an OD600 of approximately 2.0. Equal volumes (2.5 mL each) of A. halophytica and bacterial suspensions were then mixed in 12.5-mL gas-tight glass vials to initiate co-cultivation.

2.4. Screening of Bacterial Partners for Enhancing H2 Production by A. halophytica

Equal volumes (2.5 mL each) of A. halophytica and individual bacterial suspensions were mixed in sealed vials as previously described. The vials were sealed with rubber stoppers and flushed with argon gas for 15 min to establish anaerobic conditions. Co-cultures were incubated at 30 °C with shaking at 120 rpm in the dark for 72 h. H2 and O2 production were quantified using gas chromatography at 24 h intervals.

2.5. Measurement of H2 and O2 Production

A 500 µL sample of headspace gas was withdrawn from each co-culture vial and analyzed for H2 and O2 concentrations using a gas chromatograph (Hewlett-Packard HP5890A, Avondale, PA, USA) equipped with a thermal conductivity detector (TCD) and a molecular sieve 5 Å (60/80 mesh) packed column. High-purity argon gas (99.999%, v/v) was used as the carrier gas. The operating conditions were as follows: injector temperature of 100 °C, column temperature of 50 °C, detector temperature of 100 °C, and carrier gas flow rate of 20 mL min−1 [25]. H2 measurements were conducted at 30 °C and 101.325 kPa. A 4% (v/v) H2 gas mixture in argon (Praxair, Bangkok, Thailand) was used as the calibration standard. The same analytical procedure was applied for O2 quantification. H2 and O2 production rates were expressed as the amount of gas produced per dry cell weight per incubation time (µmol H2 g−1 dry weight h−1 and µmol O2 g−1 dry weight h−1, respectively). For dry cell weight determination, 5 mL of cell suspension was filtered through a glass microfiber filter (GF/C, Whatman, UK). The filters containing cells were dried in a hot-air oven at 70 °C until a constant weight was achieved. Dry cell weight was calculated as the difference between the weight of the filter containing cells and the weight of the empty filter.

2.6. Bidirectional H2ase Activity Measurement

Bidirectional H2ase activity was determined by measuring H2 production in the presence of dithionite-reduced methyl viologen, following the method of Chinchusak et al. [13]. The assays were conducted in sealed glass vials under anaerobic conditions. The reaction mixture contained 1 mL of cell suspension and 1 mL of 25 mM sodium phosphate buffer (pH 7.0) supplemented with 10 mM methyl viologen dichloride hydrate (Sigma, Darmstadt, Germany) and 40 mM sodium dithionite (Sigma, Darmstadt, Germany). The mixture was purged with argon gas to establish an anaerobic atmosphere and incubated at 30 °C in the dark for 30 min. H2 production was quantified using GC–TCD under the same analytical conditions described previously. H2ase activity was expressed as the amount of H2 produced per dry cell weight per incubation time (µmol H2 g−1 dry weight min−1).

2.7. Effect of Bacterial Cell Age, Cell Density, and Inoculum Ratio on H2 Production

Selected bacterial cultures at cell ages of 6, 12, 18, and 24 h were co-cultured with A. halophytica cells. The cultures were suspended in BG110 medium supplemented with Turks Island salt solution. Each 12.5-mL gas-tight vial contained 2.5 mL of A. halophytica at a relative OD730 of 20.0 and 2.5 mL of S. aureus at an OD600 of 2.0, corresponding to a 1:1 inoculum ratio. The vials were sealed and purged with argon gas for 15 min, then incubated under the conditions described above. H2 and O2 production were quantified using gas chromatography. To examine the effect of bacterial cell density, bacterial cultures at 12 h of cell age were adjusted to OD600 values of 0.5, 1.0, 2.0, 4.0, and 6.0. The corresponding cultures were subjected to a 10-fold serial dilution, and 100 µL of each dilution was spread onto LB agar. Cell concentration was determined by the plate count method and expressed as CFU mL−1. Finally, to investigate the effect of inoculum ratio, co-cultivation was performed at different A. halophytica:S. aureus volumetric ratios (5:0, 4:1, 2:1, 1:1, 1:2, and 1:4), with a total working volume of 5 mL.

2.8. Investigation of the Effect of Carbon Sources and Concentration on H2 Production

Two-day nitrogen-deprived A. halophytica cells and bacterial cells at their optimal age and density were co-cultured in BG110 medium supplemented with Turks Island salt solution using the optimal inoculum ratio. Various carbon sources, including acetic acid (Carlo Erba, Milan, Italy), citric acid (Ajax Finechem, Sydney, Australia), fructose (Merck, Darmstadt, Germany), glucose (BiomarkTM laboratories, Pune, India), glycerol (Fisher Scientific, Loughborough, UK), and sucrose (Fisher Scientific, Loughborough, UK), were added to the medium at 0.189 mmol C-atom L−1, matching the carbon concentration of the standard BG110 medium. Co-cultivation was conducted in sealed glass vials flushed with argon gas and incubated at 30 °C under dark conditions with shaking at 120 rpm. H2 and O2 production were analyzed using gas chromatography. To investigate the effect of carbon source concentration on H2 production, the concentration of glucose was varied to 0.189, 0.378, 0.945, 1.89, 3.78, 9.45, and 18.9 mmol C-atom L−1. The glucose supplementation at 0.189 mmol C-atom L−1 was used as a control. H2 and O2 production were measured as described above.

2.9. Investigation of the Effect of Salinity on H2 Production

A. halophytica cells were cultured and adapted to nitrogen deprivation as previously described. The bacterial strain was cultivated in LB medium, then harvested and resuspended in BG110 medium supplemented with Turks Island salt solution. Co-cultivation was carried out by mixing the cell suspensions at the optimal inoculum ratio in BG110 medium containing glucose at a concentration of 0.945 mmol C-atom L−1. NaCl was added to the medium at final concentrations of 0, 0.25, 0.5, 1.0, 2.0, and 3.0 M with 0.5 M NaCl in normal BG110 medium used as a control. Co-cultures were incubated as previously described, and H2 and O2 production were determined using gas chromatography.

2.10. Investigation of the Effect of pH and Temperature on H2 Production

A. halophytica and bacterial cell suspensions were prepared in fresh BG110 medium supplemented with Turks Island salt solution as described previously. Co-cultivation was performed in fresh BG110 containing 0.945 mmol C-atom L−1 glucose and 0.25 M NaCl. To determine the effect of pH, the initial pH of the medium was adjusted to 6.0, 7.0, 7.4, 8.0, and 9.0 prior to incubation. To evaluate the effect of temperature, co-cultures were incubated at 30, 35, 40, 45, and 50 °C. H2 and O2 production were measured using gas chromatography.

2.11. Statistical Analysis

All experiments were conducted independently in triplicate to ensure reliability and reproducibility. Data are presented as the mean ± standard deviation (SD). Statistical differences among treatments were analyzed using one-way analysis of variance (ANOVA) with IBM SPSS Statistics version 25 (IBM Corp., Armonk, NY, USA), followed by Duncan’s multiple range test as a post hoc analysis to identify significant differences among groups at the 95% confidence level (p < 0.05).

3. Results

3.1. Bacterial Screening for Enhancement of H2 Production in Co-Culture

In this study, H2 production was evaluated in monocultures of A. halophytica and five bacterial strains, Staphylococcus aureus TISTR 746, Bacillus cereus TISTR 1449, Escherichia coli TISTR 074, Micrococcus luteus TISTR 2374, and Pseudomonas aeruginosa TISTR 2370, as well as in their respective co-cultures with A. halophytica under dark anaerobic conditions. The monoculture of A. halophytica produced H2 at a maximum rate of 4.87 ± 0.19 µmol H2 g−1 dry weight h−1, reaching a maximum cumulative H2 yield of 116.97 ± 4.50 µmol H2 g−1 dry weight at 24 h of incubation (Table 1). All bacterial monocultures produced only negligible amounts of H2, and no detectable H2 production was observed in the E. coli TISTR 074 monoculture (Table 1). In contrast, co-cultivation of A. halophytica with bacterial partners resulted in significantly higher H2 production than that of the A. halophytica monoculture, except for the co-culture with E. coli TISTR 074. Among the tested combinations, co-culture with S. aureus TISTR 746 exhibited the highest H2 production rate of 11.11 ± 0.18 µmol H2 g−1 dry weight h−1, corresponding to a 2.3-fold increase compared with the A. halophytica monoculture (Table 1). This co-culture reached a maximum cumulative H2 yield of 293.46 ± 5.12 µmol H2 g−1 dry weight at 48 h of incubation (Table 1). Co-cultures with B. cereus, M. luteus, and P. aeruginosa also showed significantly enhanced H2 production, although to a lesser extent than that observed with S. aureus. In addition to enhanced H2 production, the co-culture of A. halophytica with S. aureus TISTR 746 exhibited the lowest O2 production rate among all tested combinations (Table 1). Based on its superior H2 production performance and reduced O2 accumulation, S. aureus TISTR 746 was selected as the bacterial partner for subsequent co-culture optimization experiments.

3.2. Effect of S. aureus TISTR 746 Cell Age on H2 Production

The effect of S. aureus TISTR 746 cell age on H2 production by A. halophytica was investigated under dark anaerobic co-cultivation conditions. The results demonstrated that S. aureus TISTR 746 cells harvested at the mid-logarithmic phase, at a 12 h cell age, yielded the highest H2 production rate of 17.92 ± 0.21 µmol H2 g−1 dry weight h−1 (Table 2). In contrast, co-cultures established with S. aureus cells at 6, 18, and 24 h exhibited significantly lower H2 production rates (Table 2). To investigate long-term H2 production, co-cultures were further incubated in BG110 medium supplemented with Turks Island salt solution for 24, 48, and 72 h before H2 measurement. The maximum cumulative H2 production of 473.74 ± 13.31 µmol H2 g−1 dry weight was found in 12-h-old cells incubated for 48 h (Table 2). Notably, the highest H2 production rate occurred at 24 h of incubation, whereas the maximum cumulative H2 yield was obtained at 48 h. Under most experimental conditions, H2 production rates peaked at 24 h; therefore, this time point was selected for comparative analyses. Based on these findings, S. aureus TISTR 746 cells at the mid-logarithmic phase, at 12 h, were selected for subsequent experiments.

3.3. Effect of S. aureus TISTR 746 Cell Density on H2 Production

The effect of bacterial cell density on H2 production was examined using co-cultures with S. aureus TISTR 746 at OD600 values ranging from 0.5 to 6.0. Among the tested cell densities, the co-culture of A. halophytica with S. aureus TISTR 746 at an OD600 of 4.0 (corresponding to 37.3 ± 3.4 × 108 CFU mL−1) exhibited the highest H2 production rate of 21.33 ± 0.50 µmol H2 g−1 dry weight h−1 and the lowest O2 evolution rate of 7.13 ± 0.20 µmol O2 g−1 dry weight h−1 (Table 3). The enhancement of H2 production at higher bacterial densities is likely attributable to increased respiratory O2 consumption, thereby alleviating O2 inhibition of H2ase activity. However, a further increase in S. aureus TISTR 746 cell density at an OD600 of 6.0 resulted in a significant decline in H2 production, possibly due to excessive competition for substrates or unfavorable metabolic interactions. Consequently, S. aureus density at OD600 of 4.0 was selected as the optimal bacterial density.

3.4. Impact of Inoculum Ratio on H2 Production, O2 Production, and Bidirectional H2ase Activity

Co-cultures of A. halophytica and S. aureus TISTR 746 exhibited enhanced H2 production and bidirectional H2ase activity compared with the A. halophytica monoculture (Table 4). The highest H2 production rate of 23.97 ± 0.54 µmol H2 g−1 dry weight h−1 and the maximum cumulative H2 yield of 771.56 ± 8.20 µmol H2 g−1 dry weight at 48 h of dark anaerobic incubation were observed at an inoculum ratio of 4:1 (A. halophytica/S. aureus). At this ratio, O2 evolution was minimized at 4.61 ± 0.10 µmol O2 g−1 dry weight h−1 (Table 4). This suggested that co-cultivation at a 4:1 inoculum ratio reduces O2 levels, creating more favorable O2 balance for enhancing H2 production. Consistently, the same ratio exhibited the highest bidirectional H2ase activity at 1.55 ± 0.08 µmol H2 g−1 dry weight min−1, approximately twofold higher than that of the monoculture (inoculum ratio of 5:0). Based on these results, a 4:1 inoculum ratio was selected for further studies.

3.5. Effect of Carbon Sources and Concentrations on H2 Production

H2 production by the co-culture at a 4:1 inoculum ratio was evaluated in BG110 medium supplemented with Turks Island salt solution and various carbon sources at an equivalent C-atom concentration of 0.189 mmol L−1. Among the tested carbon sources, glucose supplementation resulted in the highest H2 production rate of 45.09 ± 0.43 µmol H2 g−1 dry weight h−1, approximately twofold higher than the BG110 control (Figure 1A). Other carbon sources, including acetic acid, citric acid, glycerol, and sucrose, also enhanced H2 production, whereas fructose showed the lowest effect. Given its superior performance, glucose was further examined at different concentrations. Increasing glucose levels from 0.189 to 0.945 mmol C-atom L−1 led to a progressive increase in H2 production, reaching a maximum rate of 90.45 ± 2.78 µmol H2 g−1 dry weight h−1 at 0.945 mmol C-atom L−1 (Figure 1B). This value was approximately twofold higher than that at 0.189 mmol C-atom L−1 and threefold higher than that in BG110 without glucose. At higher glucose concentrations (1.89–18.9 mmol C-atom L−1), H2 production declined, likely due to substrate inhibition or metabolic imbalance. Therefore, 0.945 mmol C-atom L−1 glucose was selected for subsequent experiments.

3.6. Effect of Salinity on H2 Production

The influence of salinity on H2 production was assessed by supplementing BG110 medium with Turks Island salt solution at different NaCl concentrations. The highest H2 production rate of 103.31 ± 2.74 µmol H2 g−1 dry weight h−1 was observed at 0.25 M NaCl in the presence of 0.945 mmol C-atom L−1 glucose (Figure 2). This rate was approximately twofold higher than that observed in NaCl-free medium. Further increases in NaCl concentration above 0.5 M caused a sharp decline in H2 production, likely due to osmotic stress adversely affecting both cyanobacterial and bacterial physiology. Accordingly, 0.25 M NaCl was selected as the optimal salinity.

3.7. Effect of pH and Temperature on H2 Production

Among the tested initial pH values (6.0–9.0), the highest H2 production rate of 103.30 ± 1.92 µmol H2 g−1 dry weight h−1 was achieved at pH 7.4, which was significantly higher than those at other pH levels (Figure 3A). Lower or higher pH values than 7.4 led to decreased H2 production, likely reflecting less favorable conditions for H2ase activity. Under the various incubation temperatures, the highest H2 production rate of 132.49 ± 4.45 µmol H2 g−1 dry weight h−1 was found at 35 °C, which was approximately 1.3-fold higher than at the control temperature of 30 °C (Figure 3B). Temperatures above 35 °C led to a decline in H2 production, presumably due to thermal inhibition of enzymatic activity.

3.8. H2 Production Under Optimal Conditions

From the overall results, the optimal conditions for H2 production by A. halophytica co-cultured with S. aureus TISTR 746 were the co-cultivation of both organisms in BG110 medium supplemented with Turks Island salt solution containing 0.945 mmol C-atom L−1 glucose and 0.25 M NaCl, with an initial pH of 7.4 and an incubation temperature of 35 °C under dark conditions. These conditions are hereafter referred to as the “optimal conditions”. Under the optimal conditions, the co-cultures exhibited a maximum H2 production rate of 132.49 ± 4.45 µmol H2 g−1 dry weight h−1, approximately 5.5-fold higher than that under normal conditions (BG110 medium supplemented with Turks Island salt solution, pH 7.4 and 30 °C). The co-culture under optimal conditions also achieved a cumulative H2 yield of 3248.51 ± 88.11 µmol H2 g−1 dry weight after 48 h, representing a fourfold increase compared with the normal conditions (771.56 ± 8.20 µmol H2 g−1 dry weight) (Figure 4). This enhancement corresponded to approximately 25- and 55-fold increases relative to A. halophytica and S. aureus monocultures, respectively. In addition, the co-cultures under optimal conditions exhibited substantially lower O2 evolution (Figure 4), consistent with the highest H2 production observed. These results demonstrate that the optimal co-culture system markedly outperformed the normal conditions, highlighting the effectiveness of co-cultivation and environmental optimization in enhancing biological H2 production.

4. Discussion

The co-cultivation of A. halophytica with nearly all tested bacterial strains resulted in significantly higher H2 yields than those observed in A. halophytica monocultures (Table 1), demonstrating a clear synergistic interaction between cyanobacteria and heterotrophic bacteria. This enhancement is primarily driven by bacterial O2 consumption, which alleviates the inhibitory effect of O2 on [NiFe]-H2ase and enables sustained H2 evolution. O2 sensitivity is a major limitation in cyanobacterial H2 production; the rapid establishment of microoxic conditions through bacterial respiration is therefore critical for maintaining H2ase activity. A comparable mechanism has been reported in co-cultures of C. reinhardtii with activated-sludge bacteria, where rapid O2 depletion supported prolonged H2 production [26].
Beyond O2 removal, bacterial partners likely contribute through metabolic interactions that support electron transfer toward H2ase. Algal-bacterial systems have been shown to release metabolites such as formate, acetate, lactate, and butyrate, which function as electron donors or redox-mediators, thereby maintaining intracellular redox balance and facilitating H2 evolution [27]. In defined consortia, such as Bacillus cereus strain A1 with Brevundimonas naejangsanensis strain B1, exchange of these metabolites enhances interspecies electron transfer and stimulate H2 production [28]. Consistent with this, bacterial monocultures in the present study produced minimal H2, reflecting the absence of fermentable substrates in BG110 medium and indicating that the observed enhancement arises from cooperative interactions rather than intrinsic bacterial productivity.
Among the co-cultures tested, A. halophytica with S. aureus TISTR 746 exhibited the highest H2 production rate, reaching a 2.3-fold increase over that of the A. halophytica monoculture (Table 1). This effect can be interpreted as a combination of efficient O2 depletion and additional biochemical contributions. S. aureus has been reported to produce hydrogen cyanide (HCN) [29], and the predicted reaction (NH2CH2COOH → HCN + CO2 + 4e− + 4H+) suggests a potential influence on the redox environment. Because CN− is an intrinsic ligand in the [NiFe]-H2ase active site, HCN production may be associated with enhanced enzyme functionality, although this relationship remains speculative and warrants further verification. In parallel, the metabolic flexibility of S. aureus, which is capable of aerobic respiration, nitrate respiration, or fermentation depending on O2 availability, supports sustained low-O2 conditions conductive to H2ase activity [30]. While H2 production by S. aureus itself was low, the present results provide preliminary evidence of this capability. Supporting observations from Staphylococcus epidermidis B-6 indicate that members of this genus can produce H2 efficiently under suitable anaerobic and substrate conditions [31]. The use of S. aureus represents a limitation due to its pathogenicity. In this study, the strain was employed exclusively for laboratory-scale screening to identify functional traits, particularly O2 consumption and metabolic exchange, that enhance H2 production. Although unsuitable for industrial application, these findings provide a basis for selecting non-pathogenic strains with comparable physiological roles.
Enhanced H2 production was also observed in co-cultures with B. cereus TISTR 1449, M. luteus TISTR 2374, and P. aeruginosa TISTR 2370 (Table 1). In these systems, improved performance is consistent with the same O2-dependent mechanism described above, with additional strain-specific contributions. For B. cereus, previous studies indicate the ability to produce H2 and to form co-culture-specific exopolysaccharides that promote algal–bacterial aggregation, thereby stabilizing localized microoxic environments favorable for H2ase activity [32,33]. Although H2 production by M. luteus has not been previously reported, the present study demonstrates detectable H2 evolution under both mono- and co-culture conditions. In P. aeruginosa, rapid O2 consumption has been shown to accelerate the transition toward oxygen-limited conditions, and co-cultivation with S. aureus further enhances this effect [34,35]. In contrast, co-culture with E. coli TISTR 074 resulted in the lowest H2 production, with no detectable H2 in monoculture. This behavior can be explained by its hydrogenase system: Hyd-1 and Hyd-2 primarily function in H2 uptake, whereas Hyd-3 and Hyd-4 mediate H2 evolution under fermentative conditions [36]. Under the conditions used here, metabolic activity likely favored H2 oxidation rather than production, consistent with the observed outcome. Hyd-1 and Hyd-2 are known to function as active H2 oxidizers under anaerobic conditions [37]. Notably, enhanced H2 production has been reported in Chlamydomonas–E. coli co-cultures when fermentable substrates are available, highlighting the importance of medium composition [38].
The physiological state and density of S. aureus TISTR 746 strongly influenced co-culture performance. The highest H2 production rate was obtained using mid-logarithmic phase cells (12 h) (Table 2), corresponding to high respiratory activity. Increasing cell density enhanced H2 production up to an optimal OD600 of 4.0 (37.3 ± 3.4 × 108 CFU mL−1), whereas both lower and higher densities reduced yields (Table 3). At densities above OD600 6.0 (53.6 × 108 CFU mL−1), decreased H2 production may be associated with accumulation of electron-rich metabolites and shifts in redox balance. Similarly, an inoculum ratio of 4:1 (A. halophytica/S. aureus) yielded maximal H2 production and H2ase activity (Table 4). Comparable trends have been reported in other algal–bacterial systems, where optimal ratios of 40:1 or 100:1 were required for maximal H2 production [39]. These results indicate that A. halophytica serves as the primary H2 producer, whereas S. aureus modulates the physicochemical environment; excessive bacterial abundance likely introduces competing metabolic effects, including organic acid accumulation.
Carbon source optimization further influenced H2 production. Among the tested substrates, glucose yielded the highest H2 production rate, consistent with its efficient catabolism and the generation of reducing equivalents (NADH and NADPH) via glycolysis and the oxidative pentose phosphate pathway. These reducing equivalents support electron transfer to ferredoxin and H2ase, promoting H2 evolution. Similar trends have been reported in A. halophytica monocultures [10,14]. In contrast, other cyanobacteria preferentially utilize alternative substrates, such as fructose in Anabaena sp. CH3 [40] and Nostoc sp. ARM 411 [41], or glycerol in Dolichospermum sp. [42]. Enhanced H2 production in the co-culture may also reflect glucose utilization by S. aureus [43], which metabolizes glucose via glycolysis and lactic acid fermentation [30] and upregulates fermentative pathways under anaerobic conditions [44]. Increasing glucose concentration up to 0.945 mmol C-atom L−1 improved H2 production, whereas higher concentrations likely caused metabolic imbalance or substrate inhibition [10].
Salinity influenced co-culture performance, with maximal H2 production observed at 0.25 M NaCl. While A. halophytica tolerates higher salinity (optimal growth at 0.5–1.0 M; tolerance up to 3.0 M [45], the lower optimum in co-culture likely reflects the salt sensitivity of S. aureus, which varies among strains [46,47]. At NaCl concentrations above 1.0 M, H2 production declined sharply, likely due to disruption of photosynthetic membranes and PSII, leading to reduced electron transport and limited reductant availability for H2ase [48].
Optimal H2 production was observed at pH 7.4, consistent with previous reports for A. halophytica [11]. Deviations from near-neutral pH adversely affected H2 production. Acidic conditions can disrupt proton homeostasis and enzyme activity, whereas alkaline conditions may destabilize cellular membranes, reduce nutrient uptake efficiency, and inhibit key metabolic pathways. Both extremes can suppress [NiFe]-H2ase activity, leading to reduced H2 evolution. However, contrasting observations have been reported for some microalgae and cyanobacteria that produce H2 from stored carbohydrates such as glycogen or starch. During the metabolism of these reserves, acetate can accumulate, resulting in acidification of the surrounding medium [49]. This suggests that pH dynamics during H2 production may depend on the metabolic pathway and carbon source utilized. Furthermore, in co-culture systems, microenvironmental buffering, interspecies metabolic interactions, and localized pH heterogeneity may mitigate bulk acidification effects, thereby allowing H2ase activity to be maintained despite changes in the overall medium pH. Temperature also affected H2 production, with a maximum at 35 °C, consistent with previous studies [10,11]. Higher temperatures (>40 °C) reduced H2 production, likely due to thermal effects on photosynthetic electron transport and H2ase stability. S. aureus exhibits a broad growth range (7–48 °C; pH 4–10) with optimal growth near 35–37 °C [50]. Detection of H2 at 50 °C likely reflects residual enzymatic activity rather than sustained metabolic function.
During long-term H2 production, the co-culture of A. halophytica with S. aureus TISTR 746 under optimal conditions produced a higher cumulative H2 yield than that observed under normal conditions (Figure 4). This improvement reflects a stable metabolic coupling in which bacterial respiration maintains low O2 levels while cyanobacterial metabolism supplies reducing equivalents, enabling sustained electron flow to H2ase and prolonged H2 production (up to 120 h). From an application perspective, this co-culture strategy enhances H2 productivity without requiring genetic modification or complex process engineering. The use of a halotolerant cyanobacterium also enables potential cultivation in saline or seawater-based systems, reducing freshwater demand and contamination risk. Although a detailed techno-economic analysis was beyond the scope of this study, the observed improvements suggest that microbial co-culture systems may enhance the feasibility of biological H2 production. Future work should focus on scale-up and the identification of non-pathogenic strains with comparable functional roles.

5. Conclusions

Co-cultivation of Aphanothece halophytica with Staphylococcus aureus TISTR 746 significantly enhanced H2 production compared with cyanobacterial monocultures. Optimal enhancement was achieved using S. aureus cells at mid-logarithmic growth (12 h), an OD600 of 4.0, and an inoculum ratio of 4:1 (A. halophytica/S. aureus). Glucose supplementation at 0.945 mmol C-atom L−1, together with controlled salinity (0.25 M NaCl), pH 7.4, and temperature of 35 °C, maximized H2 production and bidirectional H2ase activity while minimizing O2 accumulation. These results indicate that co-cultivation with heterotrophic bacteria can enhance cyanobacterial H2 production by alleviating O2 inhibition and promoting favorable metabolic interactions. This study provides a robust strategy for improving biological hydrogen generation, with potential applications in scalable and sustainable bioenergy production.

Author Contributions

Conceptualization, S.P.; methodology, C.S. and S.P.; validation, C.S., N.C. and S.P.; formal analysis, C.S.; investigation, C.S.; resources, S.P. and A.I.; data curation, C.S.; writing—original draft preparation, C.S. and N.C.; writing—review and editing, N.C., A.I. and S.P.; visualization, C.S.; supervision, S.P.; project administration, S.P.; funding acquisition, S.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by King Mongkut’s Institute of Technology Ladkrabang (Grant No. KREF016621).

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

The data presented in this study are available from the corresponding author upon reasonable request.

Acknowledgments

C.S. thanks the KMITL Research and Innovation Services (KRIS) for her scholarship, and the School of Science at King Mongkut’s Institute of Technology Ladkrabang for providing laboratory facilities and technical support.

Conflicts of Interest

The authors declare no competing interests.

References

  1. Atwoli, L.; Erhabor, G.E.; Gbakima, A.A.; Haileamlak, A.; Ntumba, J.-M.K.; Kigera, J.; Laybourn-Langton, L.; Mash, R.; Muhia, J.; Mulaudzi, F.M.; et al. COP27 Climate Change Conference: Urgent Action Needed for Africa and the World. Lancet Infect. Dis. 2023, 23, 19–21. [Google Scholar] [CrossRef]
  2. Wang, J.; Azam, W. Natural Resource Scarcity, Fossil Fuel Energy Consumption, and Total Greenhouse Gas Emissions in Top Emitting Countries. Geosci. Front. 2024, 15, 101757. [Google Scholar] [CrossRef]
  3. Hassan, Q.; Algburi, S.; Sameen, A.Z.; Jaszczur, M.; Salman, H.M. Hydrogen as an Energy Carrier: Properties, Storage Methods, Challenges, and Future Implications. Environ. Syst. Decis. 2024, 44, 327–350. [Google Scholar] [CrossRef]
  4. Wallington, T.J.; Woody, M.; Lewis, G.M.; Keoleian, G.A.; Adler, E.J.; Martins, J.R.R.A.; Collette, M.D. Hydrogen as a Sustainable Transportation Fuel. Renew. Sustain. Energy Rev. 2025, 217, 115725. [Google Scholar] [CrossRef]
  5. Osman, A.I.; Chen, L.; Yang, M.; Msigwa, G.; Farghali, M.; Fawzy, S.; Rooney, D.W.; Yap, P.-S. Cost, Environmental Impact, and Resilience of Renewable Energy under a Changing Climate: A Review. Environ. Chem. Lett. 2023, 21, 741–764. [Google Scholar] [CrossRef]
  6. Roumezi, B.; Avilan, L.; Risoul, V.; Brugna, M.; Rabouille, S.; Latifi, A. Overproduction of the Flv3B Flavodiiron, Enhances the Photobiological Hydrogen Production by the Nitrogen-Fixing Cyanobacterium Nostoc PCC 7120. Microb. Cell Fact. 2020, 19, 65. [Google Scholar] [CrossRef] [PubMed]
  7. Broussos, P.-I.; Romanos, G.E.; Stamatakis, K. H2 Production by the Unicellular Freshwater Cyanobacterium Synechococcus elongatus PCC7942 PAMCOD Strain. Int. J. Hydrogen Energy 2024, 52, 1298–1303. [Google Scholar] [CrossRef]
  8. Jiao, H.; Tsigkou, K.; Elsamahy, T.; Pispas, K.; Sun, J.; Manthos, G.; Schagerl, M.; Sventzouri, E.; Al-Tohamy, R.; Kornaros, M.; et al. Recent Advances in Sustainable Hydrogen Production from Microalgae: Mechanisms, Challenges, and Future Perspectives. Ecotoxicol. Environ. Saf. 2024, 270, 115908. [Google Scholar] [CrossRef]
  9. Zarei, Z.; Malekshahi, P.; Morowvat, M.H.; Trzcinski, A.P. A Review of Bioreactor Configurations for Hydrogen Production by Cyanobacteria and Microalgae. Int. J. Hydrogen Energy 2024, 49, 472–495. [Google Scholar] [CrossRef]
  10. Taikhao, S.; Junyapoon, S.; Incharoensakdi, A.; Phunpruch, S. Factors Affecting Biohydrogen Production by Unicellular Halotolerant Cyanobacterium Aphanothece halophytica. J. Appl. Phycol. 2013, 25, 575–585. [Google Scholar] [CrossRef]
  11. Taikhao, S.; Incharoensakdi, A.; Phunpruch, S. Dark Fermentative Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica Grown in Seawater. J. Appl. Phycol. 2015, 27, 187–196. [Google Scholar] [CrossRef]
  12. Jaramillo, A.; Satta, A.; Pinto, F.; Faraloni, C.; Zittelli, G.C.; Silva Benavides, A.M.; Torzillo, G.; Schumann, C.; Méndez, J.F.; Berggren, G.; et al. Outlook on Synthetic Biology-Driven Hydrogen Production: Lessons from Algal Photosynthesis Applied to Cyanobacteria. Energy Fuels 2025, 39, 4987–5006. [Google Scholar] [CrossRef]
  13. Chinchusak, N.; Incharoensakdi, A.; Phunpruch, S. Dark Fermentative Hydrogen Production and Transcriptional Analysis of Genes Involved in the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica under Nitrogen and Potassium Deprivation. Front. Bioeng. Biotechnol. 2023, 10, 1028151. [Google Scholar] [CrossRef] [PubMed]
  14. Chinchusak, N.; Incharoensakdi, A.; Phunpruch, S. Enhancement of Dark Fermentative Hydrogen Production in Nitrogen-Deprived Halotolerant Unicellular Cyanobacterium Aphanothece halophytica by Treatment with Reducing Agents. Biomass Bioenergy 2022, 167, 106624. [Google Scholar] [CrossRef]
  15. Pansook, S.; Incharoensakdi, A.; Phunpruch, S. Enhanced Dark Fermentative H2 Production by Agar-Immobilized Cyanobacterium Aphanothece halophytica. J. Appl. Phycol. 2019, 31, 2869–2879. [Google Scholar] [CrossRef]
  16. Pansook, S.; Incharoensakdi, A.; Phunpruch, S. Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica. Sci. World J. 2019, 2019, 1030236. [Google Scholar] [CrossRef] [PubMed]
  17. Javed, M.A.; Zafar, A.M.; Al-Zuhair, S.; El Badawy, A.; Aly Hassan, A. Exogenous Carbon Substrates for Biohydrogen Production and Organics Removal Using Microalgal-Bacterial Co-Culture. ACS Sustain. Chem. Eng. 2022, 10, 15490–15500. [Google Scholar] [CrossRef]
  18. Mohammed Al Nuaimi, M.; Javed, M.A.; El-Tarabily, K.A.; Hyoung Lee, W.; Aly Hassan, A. Biohydrogen Production of a Halophytic Cyanobacteria Phormidium keutzingium and Activated Sludge Co-Culture Using Different Carbon Substrates and Saline Concentrations. Energy Convers. Manag. X 2023, 20, 100487. [Google Scholar] [CrossRef]
  19. Fakhimi, N.; Tavakoli, O. Improving Hydrogen Production Using Co-Cultivation of Bacteria with Chlamydomonas reinhardtii Microalga. Mater. Sci. Energy Technol. 2019, 2, 1–7. [Google Scholar] [CrossRef]
  20. Yu, Q.; He, J.; Zhao, Q.; Wang, X.; Zhi, Y.; Li, X.; Li, X.; Li, L.; Ge, B. Regulation of Nitrogen Source for Enhanced Photobiological H2 Production by Co-Culture of Chlamydomonas reinhardtii and Mesorhizobium sangaii. Algal Res. 2021, 58, 102422. [Google Scholar] [CrossRef]
  21. Fakhimi, N.; Dubini, A.; Tavakoli, O.; González-Ballester, D. Acetic Acid Is Key for Synergetic Hydrogen Production in Chlamydomonas-Bacteria Co-Cultures. Bioresour. Technol. 2019, 289, 121648. [Google Scholar] [CrossRef]
  22. Laurinavichene, T.; Laurinavichius, K.; Shastik, E.; Tsygankov, A. Long-Term H2 Photoproduction from Starch by Co-Culture of Clostridium butyricum and Rhodobacter sphaeroides in a Repeated Batch Process. Biotechnol. Lett. 2018, 40, 309–314. [Google Scholar] [CrossRef] [PubMed]
  23. Rippka, R.; Stanier, R.Y.; Deruelles, J.; Waterbury, J.B. Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. Microbiology 1979, 111, 1–61. [Google Scholar] [CrossRef]
  24. Garlick, S.; Oren, A.; Padan, E. Occurrence of Facultative Anoxygenic Photosynthesis among Filamentous and Unicellular Cyanobacteria. J. Bacteriol. 1977, 129, 623–629. [Google Scholar] [CrossRef]
  25. Sereetrakul, K.; Taikhao, S.; Chinchusak, N.; Phunpruch, S. Enhanced Hydrogen Production by Green Alga Scenedesmus obliquus TISTR 8546 under Atmospheric Air through Potassium Deprivation and Cysteine Supplementation. Algal Res. 2026, 94, 104559. [Google Scholar] [CrossRef]
  26. Zidan, A.; Javed, M.A.; Hassan, A.A. Optimization of Hydrogen Production Using a Coculture of Chlamydomonas reinhardtii and Activated Sludge Bacteria. Chemosphere 2024, 369, 143789. [Google Scholar] [CrossRef] [PubMed]
  27. Fakhimi, N.; Gonzalez-Ballester, D.; Fernández, E.; Galván, A.; Dubini, A. Algae-Bacteria Consortia as a Strategy to Enhance H2 Production. Cells 2020, 9, 1353. [Google Scholar] [CrossRef]
  28. Wang, S.; Tang, H.; Peng, F.; Yu, X.; Su, H.; Xu, P.; Tan, T. Metabolite-Based Mutualism Enhances Hydrogen Production in a Two-Species Microbial Consortium. Commun. Biol. 2019, 2, 82. [Google Scholar] [CrossRef]
  29. Neerincx, A.H.; Linders, Y.A.M.; Vermeulen, L.; Belderbos, R.A.; Mandon, J.; van Mastrigt, E.; Pijnenburg, M.W.; van Ingen, J.; Mouton, J.W.; Kluijtmans, L.A.J.; et al. Hydrogen Cyanide Emission in the Lung by Staphylococcus aureus. Eur. Respir. J. 2016, 48, 577–579. [Google Scholar] [CrossRef]
  30. Troitzsch, A.; Loi, V.V.; Methling, K.; Zühlke, D.; Lalk, M.; Riedel, K.; Bernhardt, J.; Elsayed, E.M.; Bange, G.; Antelmann, H.; et al. Carbon Source-Dependent Reprogramming of Anaerobic Metabolism in Staphylococcus aureus. J. Bacteriol. 2021, 203, e00639-20. [Google Scholar] [CrossRef] [PubMed]
  31. Mazumder, P.; Nath, D.; Manhar, A.K.; Gupta, K.; Saikia, D.; Mandal, M. Dark Fermentative Hydrogen Production from Lignocellulosic Agro-Waste by a Newly Isolated Bacteria Staphylococcus epidermidis B-6. In Resilience, Response, and Risk in Water Systems; Kumar, M., Munoz-Arriola, F., Furumai, H., Chaminda, T., Eds.; Springer Transactions in Civil and Environmental Engineering; Springer: Singapore, 2020; pp. 381–395. [Google Scholar]
  32. Greening, C.; Constant, P.; Hards, K.; Morales, S.E.; Oakeshott, J.G.; Russell, R.J.; Taylor, M.C.; Berney, M.; Conrad, R.; Cook, G.M. Atmospheric Hydrogen Scavenging: From Enzymes to Ecosystems. Appl. Environ. Microbiol. 2015, 81, 1190–1199. [Google Scholar] [CrossRef]
  33. Hupp, B.; Huszár, G.; Farkas, A.; Maróti, G. Algal Hydrogen Production and Exopolysaccharide Patterns in Chlorella–Bacillus Inter-Kingdom Co-Cultures. Fermentation 2023, 9, 424. [Google Scholar] [CrossRef]
  34. Li, Z.; Wang, J.; Feng, K.; Li, Y.; Ding, J.; Liu, B.; Ren, N.; Xing, D. Rapid Recruitment of Hydrogen-Producing Biofilms for Hydrogen Production in a Moving Bed Biofilm Reactor by a Sequential Immobilization and Deoxygenization Approach. Bioresour. Technol. 2020, 317, 123979. [Google Scholar] [CrossRef] [PubMed]
  35. Tognon, M.; Köhler, T.; Luscher, A.; Van Delden, C. Transcriptional Profiling of Pseudomonas aeruginosa and Staphylococcus aureus during in Vitro Co-Culture. BMC Genom. 2019, 20, 30. [Google Scholar] [CrossRef] [PubMed]
  36. Steinhilper, R.; Höff, G.; Heider, J.; Murphy, B.J. Structure of the Membrane-Bound Formate Hydrogenlyase Complex from Escherichia coli. Nat. Commun. 2022, 13, 5395. [Google Scholar] [CrossRef] [PubMed]
  37. Beaton, S.E.; Evans, R.M.; Finney, A.J.; Lamont, C.M.; Armstrong, F.A.; Sargent, F.; Carr, S.B. The Structure of Hydrogenase-2 from Escherichia coli: Implications for H2-Driven Proton Pumping. Biochem. J. 2018, 475, 1353–1370. [Google Scholar] [CrossRef]
  38. Lakatos, G.; Deák, Z.; Vass, I.; Rétfalvi, T.; Rozgonyi, S.; Rákhely, G.; Ördög, V.; Kondorosi, É.; Maróti, G. Bacterial Symbionts Enhance Photo-Fermentative Hydrogen Evolution of Chlamydomonas Algae. Green Chem. 2014, 16, 4716–4727. [Google Scholar] [CrossRef]
  39. Wu, S.; Li, X.; Yu, J.; Wang, Q. Increased Hydrogen Production in Co-Culture of Chlamydomonas reinhardtii and Bradyrhizobium japonicum. Bioresour. Technol. 2012, 123, 184–188. [Google Scholar] [CrossRef]
  40. Chen, P.-C.; Fan, S.-H.; Chiang, C.-L.; Lee, C.-M. Effect of Growth Conditions on the Hydrogen Production with Cyanobacterium Anabaena sp. Strain CH3. Int. J. Hydrogen Energy 2008, 33, 1460–1464. [Google Scholar] [CrossRef]
  41. Sukrachan, T.; Incharoensakdi, A. Enhanced Hydrogen Production by Nostoc sp. CU2561 Immobilized in a Novel Agar Bead. J. Appl. Phycol. 2020, 32, 1103–1115. [Google Scholar] [CrossRef]
  42. Bozieva, A.M.; Khasimov, M.K.; Rao, M.S.; Sinetova, M.A.; Voloshin, R.A.; Dunikov, D.O.; Tsygankov, A.A.; Leong, Y.K.; Chang, J.-S.; Allakhverdiev, S.I.; et al. Optimizing Cyanobacterial Hydrogen Production: Metabolic and Genetic Strategies with Glycerol Supplementation. Front. Energy Res. 2025, 13, 1547215. [Google Scholar] [CrossRef]
  43. Richardson, A.R. Virulence and Metabolism. Microbiol. Spectr. 2019, 7, GPP3-0011-2018. [Google Scholar] [CrossRef] [PubMed]
  44. Vitko, N.P.; Grosser, M.R.; Khatri, D.; Lance, T.R.; Richardson, A.R. Expanded Glucose Import Capability Affords Staphylococcus aureus Optimized Glycolytic Flux during Infection. mBio 2016, 7, e00296-16. [Google Scholar] [CrossRef] [PubMed]
  45. Laloknam, S.; Tanaka, K.; Buaboocha, T.; Waditee, R.; Incharoensakdi, A.; Hibino, T.; Tanaka, Y.; Takabe, T. Halotolerant Cyanobacterium Aphanothece halophytica Contains a Betaine Transporter Active at Alkaline pH and High Salinity. Appl. Environ. Microbiol. 2006, 72, 6018–6026. [Google Scholar] [CrossRef] [PubMed]
  46. Ochiai, T. Salt-Sensitive Growth of Staphylococcus aureus: Stimulation of Salt-Induced Autolysis by Multiple Environmental Factors. Microbiol. Immunol. 1999, 43, 705–709. [Google Scholar] [CrossRef]
  47. Feng, Y.; Ming, T.; Zhou, J.; Lu, C.; Wang, R.; Su, X. The Response and Survival Mechanisms of Staphylococcus aureus under High Salinity Stress in Salted Foods. Foods 2022, 11, 1503. [Google Scholar] [CrossRef]
  48. Yang, W.; Wang, F.; Liu, L.-N.; Sui, N. Responses of Membranes and the Photosynthetic Apparatus to Salt Stress in Cyanobacteria. Front. Plant Sci. 2020, 11, 713. [Google Scholar] [CrossRef]
  49. Rodionova, M.V.; Poudyal, R.S.; Tiwari, I.; Voloshin, R.A.; Zharmukhamedov, S.K.; Nam, H.G.; Zayadan, B.K.; Bruce, B.D.; Hou, H.J.M.; Allakhverdiev, S.I. Biofuel Production: Challenges and Opportunities. Int. J. Hydrogen Energy 2017, 42, 8450–8461. [Google Scholar] [CrossRef]
  50. Lee, H.; Park, J.H.; Park, Y.K.; Kim, H.J. Mathematical Modeling for the Growth of Salmonella spp. and Staphylococcus aureus in Cake at Fluctuating Temperatures. Appl. Sci. 2021, 11, 2475. [Google Scholar] [CrossRef]
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Figure 1. H2 production rate by A. halophytica co-cultivated with S. aureus at 24 h of dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution containing different carbon sources at a concentration of 0.189 mmol C-atom L−1 (A) and glucose at various concentrations. (B) Data are presented as means ± SD (n = 3). Different letters indicate significant differences among samples at the 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
Figure 1. H2 production rate by A. halophytica co-cultivated with S. aureus at 24 h of dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution containing different carbon sources at a concentration of 0.189 mmol C-atom L−1 (A) and glucose at various concentrations. (B) Data are presented as means ± SD (n = 3). Different letters indicate significant differences among samples at the 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
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Figure 2. H2 production rate by A. halophytica co-cultivated with S. aureus at 24 h of dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution containing various NaCl concentrations. Data are presented as means ± SD (n = 3). Different letters indicate significant differences among samples at the 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
Figure 2. H2 production rate by A. halophytica co-cultivated with S. aureus at 24 h of dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution containing various NaCl concentrations. Data are presented as means ± SD (n = 3). Different letters indicate significant differences among samples at the 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
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Figure 3. H2 production rate by A. halophytica co-cultivated with S. aureus at 24 h of dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution. (A) Effect of different pH values at 30 °C; (B) effect of various temperatures at pH 7.4. Data are presented as means ± SD (n = 3). Different letters indicate significant differences among samples at the 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
Figure 3. H2 production rate by A. halophytica co-cultivated with S. aureus at 24 h of dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution. (A) Effect of different pH values at 30 °C; (B) effect of various temperatures at pH 7.4. Data are presented as means ± SD (n = 3). Different letters indicate significant differences among samples at the 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
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Figure 4. Long-term H2 and O2 production by the co-culture of A. halophytica with S. aureus incubated under normal and optimal conditions for 120 h. Data are presented as mean ± SD (n = 3).
Figure 4. Long-term H2 and O2 production by the co-culture of A. halophytica with S. aureus incubated under normal and optimal conditions for 120 h. Data are presented as mean ± SD (n = 3).
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Table 1. Maximum H2 production rate, maximum cumulative H2 production, and O2 production rate by monocultures of the cyanobacterium A. halophytica and five bacterial strains, including B. cereus TISTR 1449, E. coli TISTR 074, M. luteus TISTR 2374, P. aeruginosa TISTR 2370 and S. aureus TISTR 746, as well as co-cultures of A. halophytica with each of these bacteria. All cultures were incubated in BG110 medium supplemented with Turks Island salt solution under dark anaerobic conditions. Data are presented as means ± SD (n = 3). Superscript letters within each column indicate significant differences between samples at a 95% significance level, according to Duncan’s multiple range test (p < 0.05).
Table 1. Maximum H2 production rate, maximum cumulative H2 production, and O2 production rate by monocultures of the cyanobacterium A. halophytica and five bacterial strains, including B. cereus TISTR 1449, E. coli TISTR 074, M. luteus TISTR 2374, P. aeruginosa TISTR 2370 and S. aureus TISTR 746, as well as co-cultures of A. halophytica with each of these bacteria. All cultures were incubated in BG110 medium supplemented with Turks Island salt solution under dark anaerobic conditions. Data are presented as means ± SD (n = 3). Superscript letters within each column indicate significant differences between samples at a 95% significance level, according to Duncan’s multiple range test (p < 0.05).
MicroorganismsMaximum H2 Production Rate
(µmol H2 g−1 Dry Weight h−1)		Maximum Cumulative H2 Production
(µmol H2 g−1 Dry Weight)		O2 Production Rate
(µmol O2 g−1 Dry Weight h−1)
Monocultures
A. halophytica4.87 ± 0.19 c116.97 ± 4.50 d22.27 ± 0.22 d
B. cereus TISTR 14490.20 ± 0.01 e23.41 ± 1.84 g2.77 ± 0.03 a
E. coli TISTR 074nd 1nd 1nd 1
M. luteus TISTR 23740.19 ± 0.01 e23.28 ± 1.64 gnd 1
P. aeruginosa TISTR 23700.50 ± 0.04 d63.72 ± 4.77 e3.11 ± 0.10 a
S. aureus TISTR 7460.66 ± 0.07 d57.19 ± 4.39 f3.47 ± 0.11 a
Co-cultures of A. halophytica with
B. cereus TISTR 14499.50 ± 0.43 b265.47 ± 9.12 b14.67 ± 0.60 c
E. coli TISTR 0740.27 ± 0.08 e6.49 ± 1.81 h21.84 ± 1.12 d
M. luteus TISTR 23749.59 ± 0.36 b230.21 ± 8.58 c14.36 ± 0.79 c
P. aeruginosa TISTR 23709.72 ± 0.30 b233.35 ± 7.31 c14.12 ± 0.60 c
S. aureus TISTR 74611.11 ± 0.18 a293.46 ± 5.12 a10.03 ± 0.28 b
1 nd = not detected.
Table 2. Maximum H2 production rate and cumulative H2 production of A. halophytica co-cultivated with S. aureus at different bacterial cell ages (6, 12, 18, and 24 h) under dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution for 24, 48 and 72 h. Data are presented as means ± SD (n = 3). Different superscript letters within each column indicate significant differences between samples at a 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
Table 2. Maximum H2 production rate and cumulative H2 production of A. halophytica co-cultivated with S. aureus at different bacterial cell ages (6, 12, 18, and 24 h) under dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution for 24, 48 and 72 h. Data are presented as means ± SD (n = 3). Different superscript letters within each column indicate significant differences between samples at a 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
S. aureus
Cell Age (h)		Maximum H2
Production Rate
(µmol H2 g−1 Dry Weight h−1)		Cumulative H2 Production (µmol H2 g−1 Dry Weight)
Incubation Time
24 h		Incubation Time
48 h		Incubation Time
72 h
612.05 ± 0.20 c289.26 ± 4.74 c330.18 ± 11.70 c235.11 ± 4.51 c
1217.92 ± 0.21 a429.99 ± 4.93 a473.74 ± 13.31 a307.31 ± 8.63 a
1815.23 ± 0.21 b365.52 ± 4.95 b373.42 ± 17.03 b253.72 ± 5.38 b
2411.12 ± 0.25 d266.79 ± 5.95 d293.18 ± 8.40 d207.54 ± 8.80 d
Table 3. Maximum H2 production rate, maximum cumulative H2 production, and O2 production rate by A. halophytica (2 × 109 cells mL−1) co-cultivated with S. aureus at various cell concentrations, measured by optical density at 600 nm and colony counting, during dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution. Data are presented as means ± SD (n = 3). Different superscript letters within each column indicate significant differences between samples at a 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
Table 3. Maximum H2 production rate, maximum cumulative H2 production, and O2 production rate by A. halophytica (2 × 109 cells mL−1) co-cultivated with S. aureus at various cell concentrations, measured by optical density at 600 nm and colony counting, during dark anaerobic incubation in BG110 medium supplemented with Turks Island salt solution. Data are presented as means ± SD (n = 3). Different superscript letters within each column indicate significant differences between samples at a 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
OD600Cell
Concentration
(×108 CFU
mL−1)		Maximum H2 Production Rate
(µmol H2 g−1 Dry Weight h−1)		Maximum Cumulative H2 Production
(µmol H2 g−1 Dry Weight)		O2 Production Rate
(µmol O2 g−1 Dry Weight h−1)
0.52.6 ± 0.12.77 ± 0.18 d66.38 ± 4.29 e14.52 ± 0.23 d
1.05.1 ± 0.32.89 ± 0.19 d438.57 ± 3.84 d12.68 ± 0.26 c
2.015.2 ± 0.917.98 ± 0.39 c480.07 ± 7.82 c9.86 ± 0.26 b
4.037.3 ± 3.421.33 ± 0.50 a700.04 ± 6.47 a7.13 ± 0.20 a
6.053.6 ± 6.518.79 ± 0.57 b584.20 ± 8.06 b7.19 ± 0.21 a
Table 4. Maximum H2 production rate, O2 production rate, H2ase activity, and maximum cumulative H2 production by A. halophytica co-cultivated with S. aureus at different inoculum ratios. H2ase activity was measured after mixing the cell suspension with the reaction mixture for 30 min. Data are presented as means ± SD (n = 3). Different superscript letters within each column indicate significant differences between samples at a 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
Table 4. Maximum H2 production rate, O2 production rate, H2ase activity, and maximum cumulative H2 production by A. halophytica co-cultivated with S. aureus at different inoculum ratios. H2ase activity was measured after mixing the cell suspension with the reaction mixture for 30 min. Data are presented as means ± SD (n = 3). Different superscript letters within each column indicate significant differences between samples at a 95% confidence level, according to Duncan’s multiple range test (p < 0.05).
A. halophytica
to S. aureus
(v/v)		Maximum H2 Production Rate
(µmol H2 g−1 Dry Weight h−1)		O2 Production Rate
(µmol O2 g−1 Dry Weight h−1)		H2ase Activity
(µmol H2 g−1 Dry Weight min−1)		Maximum
Cumulative
H2 Production
(µmol H2 g−1 Dry Weight)
5:05.19 ± 0.26 e18.20 ± 0.52 f0.83 ± 0.03 d124.66 ± 6.13 f
4:123.97 ± 0.54 a4.61 ± 0.10 a1.55 ± 0.08 a771.56 ± 8.20 a
2:122.80 ± 0.53 b6.31 ± 0.17 b1.39 ± 0.11 b714.87 ± 9.07 b
1:121.05 ± 0.37 c7.18 ± 0.11 c1.02 ± 0.07 c699.27 ± 7.65 c
1:220.32 ± 0.38 c7.98 ± 0.15 d0.82 ± 0.06 d588.37 ± 7.30 d
1:418.84 ± 0.56 d8.97 ± 0.19 e0.78 ± 0.86 d517.39 ± 8.99 e
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Somsin, C.; Chinchusak, N.; Incharoensakdi, A.; Phunpruch, S. Enhanced Hydrogen Production by the Halotolerant Cyanobacterium Aphanothece halophytica Through Bacterial Co-Cultivation. Fermentation 2026, 12, 221. https://doi.org/10.3390/fermentation12050221

AMA Style

Somsin C, Chinchusak N, Incharoensakdi A, Phunpruch S. Enhanced Hydrogen Production by the Halotolerant Cyanobacterium Aphanothece halophytica Through Bacterial Co-Cultivation. Fermentation. 2026; 12(5):221. https://doi.org/10.3390/fermentation12050221

Chicago/Turabian Style

Somsin, Chutikarn, Nattanon Chinchusak, Aran Incharoensakdi, and Saranya Phunpruch. 2026. "Enhanced Hydrogen Production by the Halotolerant Cyanobacterium Aphanothece halophytica Through Bacterial Co-Cultivation" Fermentation 12, no. 5: 221. https://doi.org/10.3390/fermentation12050221

APA Style

Somsin, C., Chinchusak, N., Incharoensakdi, A., & Phunpruch, S. (2026). Enhanced Hydrogen Production by the Halotolerant Cyanobacterium Aphanothece halophytica Through Bacterial Co-Cultivation. Fermentation, 12(5), 221. https://doi.org/10.3390/fermentation12050221

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