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Article

Biohydrogen Production under Aerial Conditions by a Nitrogen-Fixing Bacterium Isolated from a Steel Signboard

by
Nobuhiro Aburai
*,
Honami Tanaka
,
Hana Kohira
and
Tinami Sekine
Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University, 2665-1 Nakano-machi, Hachioji 192-0015, Tokyo, Japan
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(5), 248; https://doi.org/10.3390/fermentation10050248
Submission received: 17 April 2024 / Revised: 2 May 2024 / Accepted: 7 May 2024 / Published: 10 May 2024
(This article belongs to the Special Issue Microbial Culture and Isolation for the Production of Biofuels)
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Abstract

:
Hydrogen gas is attractive as a clean fuel source if it can be produced efficiently without relying on fossil fuels. Biohydrogen production using photosynthetic bacteria may enable environmentally friendly hydrogen production but is currently limited by factors such as low oxygen tolerance. In this study, we isolate a new strain of bacteria that can produce hydrogen under aerial-phase conditions compared with those under liquid-phase conditions in a nitrogen gas or an argon gas atmosphere. Bacterial strains were cultured from scrapings taken from a steel signboard. Investigation of the hydrogen production of the strains under aerial- and liquid-phase conditions and subsequent DNA sequencing led to identification of the bacterium Cereibacter sp. KGU-NF001. Aerial-phase conditions were achieved by filter membranes with the bacterial strains and placing the membranes on medium-soaked cotton wool. The gas atmosphere affected the behavior of the isolated bacterial strains under both aerial- and liquid-phase conditions. Cereibacter sp. KGU-NF001 showed promising oxygen tolerance and was able to maintain hydrogen production of 1.33 mL/mg/d even when the atmosphere contained 12% oxygen. Our findings illustrate that biohydrogen production may be achieved by photosynthetic bacteria under oxygen-containing aerial-phase conditions, indicating a possible pathway to help lower our reliance on fossil fuels.

1. Introduction

Currently, the use of fossil fuels is causing serious climate change because it is increasing airborne concentrations of greenhouse gases such as carbon dioxide (CO2). There are also concerns about resource depletion because of the mass consumption of fossil fuels. To solve these problems, there is a need for clean energy that can replace fossil fuels and does not burden the global environment [1]. Hydrogen (H2) is attracting attention as a clean energy source. H2 has a very high combustion efficiency—2.75 times that of fossil fuels—and because only water remains after combustion, it does not burden the environment. However, H2 is almost nonexistent in Earth’s atmosphere [2,3]. Currently, the primary method of producing H2 is steam reformation, which uses methane gas as its main reagent; this method relies on fossil fuels and thus does not solve the fundamental problem of fossil fuel use [4,5]. In a bioprocess using photosynthetic bacteria, H2 can be produced by an enzymatic reaction that uses light as an energy source and an organic compound as an electron donor [6,7,8]. As such, this reaction has a low environmental impact. Bioprocess-based H2 production using organic waste has been reported [9,10].
H2 production through bioprocesses can generally be divided into two broad categories. The first involves H2 production from water decomposition by green algae and cyanobacteria under anaerobic conditions, which is accompanied by oxygen (O2) generation [11,12,13]. The second is H2 production by photosynthetic bacteria. Photosynthetic bacteria are bacteria that grow by receiving light energy from the sun; they are classified into green sulfur bacteria, red sulfur bacteria, and red non-sulfur bacteria [6,14]. Among them, red non-sulfur bacteria use light energy to obtain reducing power from carbon sources to produce H2. H2 production by algae uses water, which is an abundant resource on Earth, but the energy required to decompose water and form H2 is large (ΔG0′ = −242 kJ/mol), and it is difficult to maintain anaerobic conditions because O2 is produced by photosynthesis. In contrast, H2 production by non-oxygen-evolving photosynthesis in red non-sulfur bacteria is energetically achievable; for example, ΔG0′ = −25 kJ/mol for glucose [8]. Many bacterial species are also able to use various carbon sources as substrates for H2 production, including starch and aromatic compounds [15].
The enzymes used by red non-sulfur bacteria to generate H2 as a byproduct are nitrogenases and hydrogenases [16]. Nitrogenases contain molybdenum and catalyze adenosine triphosphate (ATP)-dependent and irreversible nitrogen fixation reactions, usually in the presence of molecular nitrogen (N2) [17,18,19]. Nitrogenase receives electrons from nicotinamide adenine dinucleotide and ferredoxin by utilizing ATP produced by ATP synthase and acquired from the photosynthetic apparatus. The protons are subsequently transformed into H2 [6,16]. When nitrogen is plentiful, one mole of H2 is produced, along with ammonia, whereas in the absence of nitrogen, four molecules of H2 are generated. The reactions are represented by Equations (1) and (2).
N2 + 8H+ + 8e− + 16ATP → 2NH3 + H2 + 16ADP + 16Pi,
2H+ + 2e− + 4ATP → H2 + 4ADP + 4Pi,
The reaction involves the reduction of atmospheric N2 to ammonia (NH3), but in the absence of N2, the reaction also involves proton reduction with wide substrate specificity. Although nitrogen fixation and proton reduction reactions compete, H2 production occurs at a ratio of 20%–30% even in the presence of pure N2 [20].
Hydrogenases are enzymes that catalyze the reversible redox reaction of H2 (here, X is an electron carrier):
reduced X + H+ ⇌ oxidized X+ + 1/2H2,
2H+ + 2e− ⇌ H2,
Nitrogenases and hydrogenases are normally inactivated by O2. Although some hydrogenases have been found to be relatively tolerant of O2, H2 production by hydrogenases occurs in the absence of O2, making culture under anaerobic conditions essential for H2 production [21]. Conversely, N2 gas displacement is essential for H2 production using nitrogen-fixing bacteria.
Biofilms in which terrestrial microalgae are attached to a base such as a membrane filter and cultured under aerial conditions have been reported [22,23,24]. In batch culture, aerial conditions allow for more direct light exposure to the bacteria in the culture vessel than is the case under normal liquid-phase conditions, improving light utilization and lowering external energy costs by eliminating the need for agitation of the culture medium, leading to a decrease in total operating costs. Cost is an issue in bioprocess-based H2 production, so H2 formation by biofilm under aerial conditions is an attractive prospect [25,26].
Photosynthetic bacteria are found primarily in the hydrosphere and soil under anaerobic conditions. In a previous study, aerial microalgae were isolated from the surface of a nutrient-poor steel billboard and photosynthetic nitrogen-fixing bacteria were found near the algae bodies [27]. In this study, photosynthetic bacteria are isolated from a wall surface, and suitable strains for H2 production under aerial-phase conditions are selected. H2 production by the isolated bacteria under various O2 concentrations is then examined.

2. Materials and Methods

2.1. Subsection

Bacteria were collected from the surfaces of a steel signboard at the Hachioji campus of Kogakuin University in Japan (35°68′54″ N, 139°31′88″ E) in 2013 by removal and direct deposition into plastic tubes. Then, bacterial samples were placed on SA medium (Medium No. 360) supplemented with 1% w/v agar in a Petri dish. The SA medium consisted of 1 g sodium acetate, 1 g sodium succinate, 0.5 g KH2PO4, 0.6 g K2HPO4, 1 g (NH4)2SO4, 0.2 g MgSO4·7H2O, 0.2 g NaCl, 0.05 g CaCl2·2H2O, 0.1 g Na2S2O3·5H2O, 0.1 g yeast extract, 0.5 mg thiamin-HCl, 0.5 mg niacine, 0.3 mg p-aminobenzoic acid, 0.1 mg pyridoxal-HCl, 0.05 mg biotin, 0.05 mg vitamin B12, 1 mg 2Na-EDTA, 2 mg FeCl3⸱6H2O, 0.1 mg ZnCl2, 0.1 mg MnCl2⸱6H2O, 0.1 mg H3BO3, 0.1 mg CoCl2⸱6H2O, 20 µg Na2MoO4⸱2H2O, 10 µg CuCl2⸱2H2O, 10 µg NiCl2⸱6H2O, and 5 µg Na2SeO3 in 1 L of distilled water. The pH was adjusted to 6.7 with NaOH prior to autoclaving. After more than 1 week of growth, single colonies that formed on the agar-solidified medium were manually isolated under a microscope (BX51; Olympus, Tokyo, Japan). Isolated strains, which were named BRCNF-001 to BRCNF-008, and Rhodobacter sphaeroides (NBRC 12203) were precultured in SA medium and maintained for 7 days at 25 ± 2 °C and 40 μmol photons/m2/s using a cool-white fluorescent lamp under anaerobic conditions in a 200 mL Erlenmeyer flask that was flushed with N2 gas for 1 min and then capped with a lid.

2.2. Liquid and Aerial Culture Conditions

After preculture for 7 days, the cells were incubated for 7 days at 25 °C and 25 μmol photons/m2/s using a cool-white fluorescent lamp under two sets of conditions (see Figure 1 and Figure S1): (1) liquid-phase conditions—where 0.5 mg cells were cultured in 21 mL glass tubes containing 3 mL N-free SA medium with 0.3% glucose as the carbon source, and (2) aerial-phase conditions, which were achieved as follows. Precultured cells (0.5 mg) were layered on 0.20 μm polytetrafluoroethylene membrane filters, which are water permeable under reduced pressure, and then washed with distilled water. The biofilm on the membrane filter was placed on cotton wool wetted with 3 mL N-free SA medium with 0.3% glucose as the carbon source in a 21 mL glass tube. The headspace of each tube was flushed with N2 gas (with 1.5, 2.0, 2.5, 4.0, 6.0, 10.0, 12.0, 13.0, or 15.0% O2 gas), Ar gas, or air (21% O2) for 1 min, and then the tubes were sealed with a rubber cap.

2.3. Hydrogen Analysis

Gas components in the headspace of each glass tube on day 7 of culture were analyzed as described elsewhere [28]. H2 produced during the one-week fermentation was collected with a microsyringe and analyzed using a gas chromatography (GC) system (GC-2014, Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector (GC-TCD, Shimadzu, Kyoto, Japan) under the following conditions. Column: Shincarbon ST column 50–80 (4.0 m × 3.0 mm internal diameter, Shinwa Chemical Industries, Kyoto, Japan), injection volume: 0.5 mL, carrier gas: Ar (43.5 mL/min), and column temperature: 80 °C. The detection limit of the GC-TCD for H2 quantification was 0.02 µL.

2.4. 16S rRNA Analysis and Phylogenetic Study

Crude DNA was extracted from bacterial cells using the bead-beating method [29]. The partial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using universal primers [29]. The PCR products were checked using 1.5% (w/v) agarose-gel electrophoresis and purified using Wizard® SV Gel and PCR Clean-Up System (Promega, Singapore). DNA sequencing was performed using the PCR primers and then determined by Fasmac Co., Ltd. (Atsugi-shi, Japan). BLAST searches were used to compare the sequences of the PCR products with those of known microbial species in the GenBank. A phylogenetic tree was constructed with Cereibacter spp. and closely related bacteria based on 16S rRNA gene sequences. Positions at which the alignment resulted in gaps or ambiguities were omitted from the analysis. The DNA sequence of the 16S rRNA gene for the isolated strain have been deposited in GenBank under accession number LC814985. Gram staining was carried out as described in [30].

2.5. Statistical Analysis

The mean and standard deviation for each treatment were calculated. Data were subjected to analysis of variance. Differences between groups were considered statistically significant at p < 0.05. Statistical analyses were conducted using Microsoft Excel 2019. Statistical analyses of the experimental data were performed using Student’s t-test.

3. Results and Discussion

3.1. Biohydrogen Production under Aerial- and Liquid-Phase Conditions

Eight strains denoted as BRCNF001–008 were isolated from a steel signboard. These eight strains and R. sphaeroides were cultured for 7 days under liquid- or aerial-phase conditions with N2 as the displacement gas. BRCNF001, BRCNF004, BRCNF007, BRCNF008, and R. sphaeroides had significantly lower cell weights under aerial-phase conditions than under liquid-phase conditions (Figure 2a). Only BRCNF003 displayed a larger cell weight under aerial-phase conditions than under liquid-phase ones. Among these strains, the weights of BRCNF004 and BRCNF007 increased about 16-fold from 0.5 mg of bacteria at the beginning of culture under liquid-phase conditions. The weight of BRCNF003 increased about 12-fold under aerial-phase conditions. These results indicate that all strains except BRCNF003 grew with nitrogen fixation under liquid-phase conditions. Compared to the other strains, BRCNF003 grew more easily under aerial-phase conditions.
H2 production rates were greater under aerial-phase conditions than under liquid-phase ones for BRCNF001, BRCNF005, BRCNF007, BRCNF008, and R. sphaeroides (Figure 2b). R. sphaeroides resulted in higher bacterial abundance and lower H2 production rates under liquid-phase conditions than under aerial-phase conditions. The solubility of N2 gas in the liquid medium was only about 0.016 mL mL−1 under standard conditions (0 °C, 1013 hPa), suggesting that the bacteria grew by increasing nitrogenase activity, actively fixing nitrogen, and obtaining a nitrogen source. Conversely, under aerial-phase conditions, the bacteria had easier access to the N2 gas, so the N2 gas utilization efficiency was higher than that in the liquid phase. Due to the fact that the nitrogen source in the cells under liquid-phase conditions was insufficient for growth as a result of low nitrogenase activity, the carbon source in the medium was used for hydrogenase activity, which generated H2 [31]. In general, H2 generation and cell proliferation are reported to be in a competitive relationship, with NH3 generated by nitrogenases and carbon sources in the medium being used for growth [32]. H2 generated by nitrogenases is trapped by hydrogenases in the cell and becomes a source of electrons and protons through oxidative reactions [33]. Strains with more reductively reactive hydrogenases are more promising as biohydrogen-producing bacteria than strains with high nitrogenase activity.
Next, BRCNF001–008 and R. sphaeroides were cultured under liquid- or aerial-phase conditions in an argon (Ar) gas atmosphere for 7 days. Ar is commonly used as a replacement gas in biohydrogen production studies because it is inert and does not react with the bacteria. In an Ar atmosphere, nitrogenases remain inactive because of the lack of a substrate, and biohydrogen is produced by hydrogenases [34]. In an Ar atmosphere, BRCNF001, BRCNF007, and R. sphaeroides had significantly lower cell weights under aerial-phase conditions than under liquid-phase ones, the cell weights of BRCNF002 and BRCNF008 were not significantly different, and the cell weights of BRCNF003, BRCNF004, and BRCNF006 were larger under aerial-phase conditions than under liquid-phase ones (Figure 3a). Cell weights of BRCNF001–004, BRCNF008, and R. sphaeroides increased about 7–8-fold from 0.5 mg of bacteria at the beginning of incubation under liquid-phase conditions in an Ar atmosphere. In contrast, cell weights of BRCNF003 and BRCNF004 increased by about 12 times from the initial bacterial weight under aerial-phase conditions in an Ar atmosphere. The inactivity of nitrogenases under an Ar atmosphere and the lack of a nitrogen source indicate that these strains took up carbon to grow rather than growing by cell division. BRCNF003 grew better under aerial-phase conditions than liquid-phase ones in both N2 and Ar atmospheres.
The H2 production rate in an Ar atmosphere was greater or equal for all strains cultured under aerial-phase conditions than was the case under liquid-phase conditions (Figure 3b). Although nitrogenases did not function in the Ar atmosphere and H2 was produced by hydrogenase activity, the aerial-phase conditions were more efficient than the liquid-phase conditions because of the more uniform light exposure to bacterial cells in the former compared with that in the latter [8]. Comparing the results for N2 and Ar gas, BRCNF002–004 displayed significantly (p < 0.05) greater H2 production rates under both aerial- and liquid-phase conditions compared with those of the other strains. In other words, these strains exhibited low nitrogenase activity and high hydrogenase activity (Figure 2b and Figure 3b). It is believed that N2 gas-fixing nitrogenases produce NH3 simultaneously with H2 and NH3 acts as an inhibitor of nitrogenase activity, so nitrogenase activity gradually weakens as NH3 and H2 are produced [35]. N2 is important as a replacement for air because it makes up 78% of Earth’s atmosphere and is inexpensive. Although Ar is an inert gas that does not affect H2 production, it is much more expensive than N2 and therefore not desirable as an atmosphere for cost-effective biohydrogen production. BRCNF008 showed the highest H2 production rate among the nine strains in both N2 and Ar gas atmospheres under aerial-phase conditions, indicating that it is attractive for H2 production under aerial-phase conditions.

3.2. Phylogenetic Analysis of the Bacterial Strain of BRCNF008

Using PCR amplification and subsequent DNA sequencing, we determined almost the full length of the 16S rRNA gene of the strain BRCNF008, which showed promise for H2 production under aerial-phase conditions. The strain was observed as a Gram-negative bacillus (Figure 4a). The sequence information obtained for BRCNF008 was compared with those of known related species (Figure 4b and Figure S2). The strain was most closely related to Cereibacter species of the class Alphaproteobacteria and named Cereibacter sp. KGU-NF001 (Accession Number: LC814985). The most closely related species, Cereibacter johrii JA192, is a strain discovered in 2010 and recently reanalyzed to be best placed in the genus Cereibacter [36,37]. The nitrogenase and hydrogenase activities (H2 production rate) of Cereibacter are not well understood. Cereibacter sphaeroides (strains IFO12203 and KC2139) has recently undergone a phylogenetic tree reanalysis and been renamed from Rhodobacter sphaeroides [37]. According to the National Institute of Technology and Evaluation’s NBRC database, strain NBRC12203 is derived from IFO12203, so Rhodobacter sphaeroides strain NBRC12203 and BRCNF008 are relatively close species.

3.3. Influence of O2 on H2 Production

The influence of O2 concentration on dry cell weight (Figure 5a) and H2 production rate (Figure 5b) was investigated under aerial-phase conditions in a N2 gas atmosphere. First, when the tubes were filled with air (i.e., 21% O2) instead of N2 gas and incubated for 7 days under otherwise identical aerial-phase conditions, dry cell weight did not increase and H2 was not produced. When the cells were incubated under aerial-phase conditions in a N2 gas atmosphere with an O2 concentration lower than that of air, BRCNF008 and R. sphaeroides showed increases of dry cell weight in the 10–15% O2 concentration range that were similar to those at 0% O2, approximately 10 times higher than at the start of incubation. BRCNF008 and R. sphaeroides produced 1.33 and 1.00 mL/mg/d of H2 under 12% O2, respectively, and did not produce H2 under O2 concentrations above 13.0% (Figure 5b). The H2 production rate of BRCNF008 was similar to that of R. sphaeroides under anaerobic conditions in the O2 concentration range of 6–12%, whereas that of R. sphaeroides was approximately half that under anaerobic conditions in the O2 concentration range of 6–12%. R. capsulatus JP91 was reported to produce H2 under microaerobic dark conditions (2–10% O2) [38]. BRCNF008 showed higher O2 resistance than R. sphaeroides and R. capsulatus JP91. O2 showed 50% inhibitory activity against a nitrogenase in a system with an acetylene concentration of 0.37 µM (e.g., below 0.001% O2) [39]. In comparison, the O2 concentration of 12.0% used here is far greater, indicating that H2 can be generated sufficiently by BRCNF008 in the presence of O2 in the concentration range of 0–12%. Although it is difficult to accurately measure the concentration of CO2 generated by BRCNF008, the rate of CO2 generation was able to be measured by GC (Figure 5c). In an O2 concentration range of 0–21%, CO2 was produced in a concentration range of about 0.15–0.25 mL/mg/d. Because R. sphaeroides is a facultative anaerobe, BRCNF008, which is closely related to Rhodobacter, also respires when O2 is present. Even in the absence of O2, anaerobic respiration and fermentation produce CO2 [40]. Even at an O2 concentration of 13.0%, CO2 was produced and the H2 production rate decreased despite the respiration (Figure 5b,c), suggesting that the O2 concentration remaining after some is consumed by respiration during the 7-day incubation period may affect H2 production. After 7 days of incubation, 9.0% (74.7 μmol) O2 remained in the BRCNF008 culture when the initial O2 concentration was 12.0%, and 10.7% (102.7 μmol) O2 remained when the initial O2 concentration was 13.0%. For R. sphaeroides, 8.0% (84.8 μmol) O2 remained when the initial O2 concentration was 12.0% and 11.0% (100.7 μmol) O2 remained when the initial O2 concentration was 13.0%. The nitrogenase activity of R. capsulatus MNL was sufficiently inhibited by 5%–7.5% O2 [41], indicating that BRCNF008 shows high O2 tolerance. It was shown that H2 production is possible because O2 is consumed to some extent by respiration.
The same culture conditions in air were extended to 14 days, and the rate of H2 production was measured. H2 evolution was observed, albeit weakly, in air, with H2 production rates of 0.239 and 0.157 mL/mg/d for BRCNF008 and R. sphaeroides, respectively. The CO2 production rates for BRCNF008 and R. sphaeroides were 0.493 and 0.359 mL/mg/d, respectively. At the end of incubation (day 14), the residual O2 gas in the systems with BRCNF008 and R. sphaeroides was 15.0% (140.9 μmol) and 12.8% (119.9 μmol), respectively. When incubated in air without replacement gas such as N2 or Ar, H2 was not produced after 7 days, but was produced after 14 days of incubation. It is thought that not only the high tolerance to O2, but also the accumulation of excess ATP by respiration, which was used for hydrogenase activity, resulted in the generation of H2 after 14 days [42].

4. Conclusions

To construct a H2 production system using photosynthetic bacteria isolated from a wall, strains that produced H2 under aerial-phase conditions were identified. The effect of the replacement gas composition in the reaction-vessel headspace on H2 production by the systems was investigated. In both N2 and Ar gas, BRCNF001, BRCNF005, and BRCNF008 showed higher H2 production rates under aerial-phase conditions than under liquid-phase conditions, with BRCNF008 showing the highest H2 production rate of the strains. BRCNF008 was also shown to have relatively high O2 tolerance. Examination of the O2 concentration in the aerial phase showed that BRCNF008 and R. sphaeroides were able to produce H2 at up to 12.0% O2 concentration over 7 days of culture. CO2 was also produced, suggesting that the bacteria were respiring and consuming O2 to produce anaerobic conditions in the aerial phase. When the bacteria were incubated in air for 14 days, H2 production was observed, even though H2 was not produced after 7 days under these conditions. This study demonstrates the possibility of H2 production under aerial-phase conditions using photosynthetic bacteria and the potential for low-cost biohydrogen production in air. In the future, more efficient H2 production will be achieved by optimizing various basic culture conditions such as light, pH, temperature, and scale.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation10050248/s1, Figure S1: Photograph of biofilms seen from an angle; Figure S2: Phylogenetic tree of the isolated bacteria with its known neighbors based on 16S rRNA gene sequences constructed using the bootstrapped neighbor-joining method.

Author Contributions

Conceptualization, N.A.; methodology, N.A., H.T., H.K. and T.S.; validation, N.A.; investigation, N.A., H.T., H.K. and T.S.; writing—original draft preparation, H.T., H.K. and T.S.; writing—review and editing, N.A.; supervision, N.A.; project administration, N.A.; funding acquisition, N.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Strategic Research Foundation Grant-Aided Project for Private Universities from the Ministry of Education, Culture, Sport, Science, and Technology, Japan (S1411005); and by the Research Institute for Science and Technology of Kogakuin University for a special Grant-in-Aid to earn KAKENHI by the Japan Society for the Promotion of Science.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. (a) Images of cultivation under liquid and aerial culture conditions and (b) photograph of biofilms cultivation under aerial culture conditions.
Figure 1. (a) Images of cultivation under liquid and aerial culture conditions and (b) photograph of biofilms cultivation under aerial culture conditions.
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Figure 2. (a) Dry cell weight and (b) H2 production of the isolated bacteria (BRCNF001–008) and Rhodobacter sphaeroides cultured under aerial- or liquid-phase conditions. Dry cell weights are per membrane filter (aerial-phase conditions) or per 3 mL of medium (liquid-phase conditions). The carbon source in the N-free SA medium was glucose. The headspace of each tube was flushed with N2 gas. Data are mean ± standard deviation of three replicate incubations. Asterisks indicate statistically significant differences at p < 0.05.
Figure 2. (a) Dry cell weight and (b) H2 production of the isolated bacteria (BRCNF001–008) and Rhodobacter sphaeroides cultured under aerial- or liquid-phase conditions. Dry cell weights are per membrane filter (aerial-phase conditions) or per 3 mL of medium (liquid-phase conditions). The carbon source in the N-free SA medium was glucose. The headspace of each tube was flushed with N2 gas. Data are mean ± standard deviation of three replicate incubations. Asterisks indicate statistically significant differences at p < 0.05.
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Figure 3. (a) Dry cell weight and (b) H2 production of the isolated bacteria (BRCNF001–008) and Rhodobacter sphaeroides cultured under aerial- or liquid-phase conditions. Dry cell weights are shown per membrane filter (aerial-phase conditions) or per 3 mL of medium (liquid-phase conditions). The carbon source in the N-free SA medium was glucose. The headspace of each tube was flushed with Ar gas. Data are mean ± standard deviation of three replicate incubations. Asterisks indicate statistically significant differences at p < 0.05.
Figure 3. (a) Dry cell weight and (b) H2 production of the isolated bacteria (BRCNF001–008) and Rhodobacter sphaeroides cultured under aerial- or liquid-phase conditions. Dry cell weights are shown per membrane filter (aerial-phase conditions) or per 3 mL of medium (liquid-phase conditions). The carbon source in the N-free SA medium was glucose. The headspace of each tube was flushed with Ar gas. Data are mean ± standard deviation of three replicate incubations. Asterisks indicate statistically significant differences at p < 0.05.
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Figure 4. Micrographs of Gram staining cells of the strain BRCNF008 (a) and phylogenetic tree of the isolated bacteria with its known neighbors based on 16S rRNA gene sequences constructed using the maximum-likelihood method (b). The scale bar in phylogenetic tree represents an evolutionary distance (Knuc) of 0.05. Numbers at branch nodes are bootstrap values (1000 replicates) obtained using the maximum-likelihood method. Accession numbers for DNA sequences are indicated in parentheses. T is type strain.
Figure 4. Micrographs of Gram staining cells of the strain BRCNF008 (a) and phylogenetic tree of the isolated bacteria with its known neighbors based on 16S rRNA gene sequences constructed using the maximum-likelihood method (b). The scale bar in phylogenetic tree represents an evolutionary distance (Knuc) of 0.05. Numbers at branch nodes are bootstrap values (1000 replicates) obtained using the maximum-likelihood method. Accession numbers for DNA sequences are indicated in parentheses. T is type strain.
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Figure 5. Influence of oxygen (O2) concentration on the (a) dry cell weight, (b) H2 production, and (c) CO2 production of Rhodobacter sphaeroides and the strain BRCNF008 cultured under aerial-phase conditions. The carbon source in the N-free SA medium was glucose. The headspace of each tube was flushed with N2 and O2 gas (or air for the 21% O2 conditions). Data are mean ± standard deviation of three replicate incubations.
Figure 5. Influence of oxygen (O2) concentration on the (a) dry cell weight, (b) H2 production, and (c) CO2 production of Rhodobacter sphaeroides and the strain BRCNF008 cultured under aerial-phase conditions. The carbon source in the N-free SA medium was glucose. The headspace of each tube was flushed with N2 and O2 gas (or air for the 21% O2 conditions). Data are mean ± standard deviation of three replicate incubations.
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MDPI and ACS Style

Aburai, N.; Tanaka, H.; Kohira, H.; Sekine, T. Biohydrogen Production under Aerial Conditions by a Nitrogen-Fixing Bacterium Isolated from a Steel Signboard. Fermentation 2024, 10, 248. https://doi.org/10.3390/fermentation10050248

AMA Style

Aburai N, Tanaka H, Kohira H, Sekine T. Biohydrogen Production under Aerial Conditions by a Nitrogen-Fixing Bacterium Isolated from a Steel Signboard. Fermentation. 2024; 10(5):248. https://doi.org/10.3390/fermentation10050248

Chicago/Turabian Style

Aburai, Nobuhiro, Honami Tanaka, Hana Kohira, and Tinami Sekine. 2024. "Biohydrogen Production under Aerial Conditions by a Nitrogen-Fixing Bacterium Isolated from a Steel Signboard" Fermentation 10, no. 5: 248. https://doi.org/10.3390/fermentation10050248

APA Style

Aburai, N., Tanaka, H., Kohira, H., & Sekine, T. (2024). Biohydrogen Production under Aerial Conditions by a Nitrogen-Fixing Bacterium Isolated from a Steel Signboard. Fermentation, 10(5), 248. https://doi.org/10.3390/fermentation10050248

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