The impact of Fe2+ and Na+ concentrations on hydrogen production with three different fermenter bacteria

Abstract

Batch experiments were conducted to investigate the effects of Fe²⁺ and Na⁺ on the hydrogen (H₂) production performance from three different metabolic type hydrogen-producing bacterial strains. The appropriate amount of Fe²⁺ significantly promoted the H₂ production of all three hydrogen-producing bacteria. The combination of H₂ production and liquid products showed that Fe²⁺ was more suitable for the H₂ production and metabolism of E. harbinense ZGX4. When the Fe²⁺ concentration was 0.05 g/L, the H₂ production and liquid products concentrations were 2170 mL/L-medium and 6530 mg/L, respectively. Na⁺ enhanced the H₂ production of E. harbinense ZGX4 and C. butyricum 1.209 but inhibited the H₂ production of E. cloacae 1.2022. Na⁺ made C. butyricum 1.209 exhibit the best H₂ production and metabolic performance when the Na⁺ concentration was 2 g/L, while the H₂ production, and liquid products concentration were 2460 mL/L-medium and 5350 mg/L, respectively. At the end of the experiment, it was found that the addition of Fe²⁺ could change the type of fermentation in C. butyricum 1.209. Therefore, further exploration of the effects of other metal ions on model hydrogen-producing strains has great potential for achieving high hydrogen production rates, among other things.

INTRODUCTION

Biological hydrogen (H₂) production by fermentation is a key technology for H₂ production due to its advantages of relatively high H₂ production capacity, abundant raw material resources, low carbon footprint, and environmental friendliness (Yin and Wang 2017; Łukajtis et al. 2018; Zhang et al. 2021a). Hydrogen-producing microorganisms in the fermentation system are the main actors in obtaining hydrogen, the target product. This is achieved by converting organic substrates into H₂ through the catalytic activity of two key enzymes hydrogenase and nitrogenase (Akhlaghi and Najafpour-Darzi 2020; Baeyens et al. 2020; Sivaramakrishnan et al. 2021). Research in this field focuses on how to effectively utilize hydrogen-producing fermentation bacteria and thereby improve the activity of hydrogen-producing enzymes to increase the efficiency and capacity of the reaction system.

Microorganisms are affected by various factors during hydrogen-producing fermentation, such as substrate, pH, temperature, and metal ions, among which metal ions are the key factors affecting microbial H₂ production (Infantes et al. 2011; Tandon et al. 2018). The type and concentration of metal ions affect the growth of hydrogen-producing bacteria and the structure and function of hydrogen-producing enzymes, which affect the efficiency of H₂ production. Hydrogenase is a type of protein containing iron, nickel, and other metal elements that can catalyze the reversible oxidation of H₂. By adding metal ions as cofactors to the fermentation medium, the catalytic activity of hydrogenase can be effectively enhanced to improve the H₂ production capacity of microbial fermentation and the H₂ production rate of the reaction system (Bao et al. 2013; Srivastava et al. 2019). For example, Ni²⁺ and Fe²⁺ are cofactors of enzymes and necessary factors for H₂ synthesis (Baeyens et al. 2020). Zhao et al. (2017) investigated the effect of Fe²⁺ and Mg²⁺ on the H₂ production of Ethanoligenens harbinense and obtained the maximum H₂ production by adding 600 mg/L MgCl₂·6H₂O and 100 mg/L FeSO₄·7H₂O in the optimal medium. Sekoai and Daramola (2018) evaluated the effect of different concentrations of Fe²⁺, Mg²⁺, Ca²⁺, and Ni²⁺ on H₂ production by fermentation using potato waste as a substrate, and 293 mLH₂/g·TVS (total volatile solids) was obtained. In addition, Mg²⁺, Ca²⁺, and Ni²⁺ also affect H₂ production (Sekoai and Daramola 2018). Boni et al. (2014) investigated the effect of ferrous ion concentration on H₂ production in organic waste fermentation. They found that a suitable ferrous ion concentration would stimulate H₂ production by hydrogen-producing bacteria. Regarding zinc ion, Zhang et al. (2021b) reported that increasing its concentration from 1 mg/L to 5 mg/L can effectively increase hydrogen production. At 2 mg/L Zn²⁺, the maximum hydrogen production was 592 ± 13 mL. Salazar-Batres and Moreno-Andrade (2022) reported that when using glucose as a substrate, 2 mg Ni²⁺/g VS_inoculum generated the highest hydrogen production (774±7.3 mL H₂/L/d). In another study, the authors evaluated the effect of three different iron compounds (Fe₂O₃, FeSO₄, FeCl₃) on hydrogen production from sugar beet pulp. The authors observed that the addition of 0.1 gFe₂O₃/dm³ in an intermittent experiment resulted in a maximum hydrogen production of more than 200 dm³H₂/kgVS, which was twice that of the control (Cieciura-Włoch et al. 2020).

The studies mentioned above demonstrated the existence of a certain promoting effect of metal ions on the fermentation H₂ production of strains, but the effect of Fe²⁺ and Na⁺ on the H₂ production from different types of fermentation strains has rarely been reported under the same cultural conditions. This study reveals the impact of metal ions on the hydrogen production of different fermentation strains. Fe is the main component of hydrogenase, which catalyzes the reduction of protons into hydrogen. Na⁺ promotes H₂ production by promoting the growth of H₂-producing microorganisms. In this study, three different type of fermentation strains, namely Ethanoligenens harbinense ZGX4, Clostridium butyricum 1.209, and Enterobacter cloacae 1.2022, were used to probe into the effects of Fe²⁺ and Na⁺ on H₂ production in the same cultural conditions. Batch experiments were applied to investigate cell growth, H₂ production, and liquid products. Finally, combining H₂ and liquid products production, Fe²⁺ was the most suitable for the H₂ production metabolism of the ethanol-type fermentative hydrogen-producing bacteria E. harbinense ZGX4. At the same time, Na⁺ was found to be more suitable for butyric acid-type fermentative hydrogen-producing bacteria C. butyricum 1.209. This study has the potential to benefit the regulation and optimization of the fermentative process in biohydrogen production.

EXPERIMENTAL

Bacteria and Culture Condition

An ethanol-fermenting hydrogen-producing bacterium, Ethanoligenens harbinense ZGX4, was obtained from the State Key Laboratory of Urban Water Resources and Environment of Harbin Institute of Technology (Xing et al. 2006; Li et al. 2019). Clostridium butyricum 1.209 and Enterobacter cloacae 1.2022 were obtained from the Strain Preservation Center of the Chinese Academy of Sciences. The medium for cell growth and H₂ production was composed of glucose 20 g/L, peptone 4.0 g/L, beef paste 2.0 g/L, yeast juice 1.0 g/L, K₂HPO₄ 1.5 g/L, MgCl₂·6H₂O 0.2 g/L, L-cysteine 0.5 g/L, vitamin solution (Table 1) 10 mL, trace element solution (Table 1) 10 mL, and resazurin (0.2% v/v) 1 mL. The concentrations of Fe²⁺ (from FeSO₄·7H₂O) and Na⁺ (from NaCl) were adjusted according to the fermentative experimental requirements, and the pH of the system was adjusted to 6 to 6.4 by 1 M KOH and 1 M HCl.

Table 1. Composition of Vitamin Solution and Trace Element Solution

Serum bottles with a total volume of 250 mL were used for the batch culture experiment, and the volume of solution was 100 mL. The mouths of the bottles were tightly corked to prevent gas leakage. N₂ was used to purge the whole system for 5 min and then the bottle was sealed quickly to ensure the entire system oxygen-free. Hydrogen was collected continually using a deformable gas bag, and the air pressure of the closed system was the same as atmospheric pressure. The serum bottles were placed in a constant temperature shaker and incubated at a speed of 130 rpm and a temperature of 35 ± 1 ℃. When the activated microbial cells grew to the mid-log stage, they were inoculated into each test vial with the same inoculum amount of 2% (v/v) per vial. Gas samples and liquid fermentation products were taken regularly. A three-way valve was used at the gas sampling port to maintain the sampled gas consistent at the gas concentration of the fermentation flask, and the H₂ content was determined.

Hydrogen Production Experiment Design

The effects of different concentrations of Fe²⁺ and Na⁺ on three model hydrogen-producing strains in their growth and hydrogen production were compared and analyzed by batch culture experiments. The three strains have different optimal concentrations of Fe²⁺ and Na⁺. In previous experiments, a wider range of metal ion concentrations (Fe²⁺ and Na⁺) was selected to investigate their effect on hydrogen production in advance. By this means, a suitable concentration was determined for each metal ion that was selected for different bacteria in this study.

To investigate the effect of metal ions on the H₂ production of the three bacteria, preliminary experiments were carried out to determine the concentration range of Fe²⁺ and Na⁺ by batch culture. For E. harbinense ZGX4, the concentrations of Fe²⁺ in the medium were 0, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5, and 1.0 g/L, according to the experimentally set gradient. For C. butyricum 1.209, Fe²⁺ concentrations were set at 0, 0.05, 0.1, 0.2, 0.25, 0.5, 1, 1.5, 2, and 4 g/L. For E. cloacae 1.2022, Fe²⁺ concentrations were set to 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, and 1.0, 1.5, 2.0, and 4.0 g/L. Na⁺ concentrations were set at 0, 0.5, 1, 1.5, 2, 2.5, and 3 g/L for E. harbinense ZGX4. For C. butyricum 1.209, the Na⁺ concentrations were set at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 10 g/L. For E. cloacae 1.2022, the Na⁺ concentrations were set at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 10.0 g/L. All operations were performed three times to ensure the reproducibility of the data, and the results were summarized as means with standard deviations.

Analysis Methods

Bacteria were diluted appropriately with physiological saline, and the optical density of the culture was measured at 600 nm. The fresh medium with the same dilution times was used as the control, and the dilution times multiplied by the optical density value of the sample was taken to be the true optical density value of the sample. A fermentation solution was taken and centrifuged at 4500 rpm to collect the bacteria. After suspending with normal saline and washing twice, the bacteria were dried at 105 ℃ to constant weight. An analytical balance was used to determine the dry weight of the cells (Song et al. 2013; Trchounian et al. 2016). Excel software was used to obtain the regression equation of absorbance and cell concentration: y=0.4229x-0.008, R²=0.9936, where x is optical density and y is cell concentration. H₂ content was determined using a Shanghai Analytical Instrument Factory SC-II gas chromatograph, thermal conductivity cell detector, stainless steel column, column length and diameter of 2 m×φ5, carrier TDS-01, 60-80 mesh, using high-purity nitrogen as carrier, flow rate 70 mL/min, measured at room temperature, and injection volume 500 μL (Jitrwung and Yargeau 2015; Lamont et al. 2017). The liquid products (ethanol and volatile fatty acids) were determined by GC122 gas chromatograph of Shanghai Analytical Instrument Factory, with a stainless steel column, 2 m column length (5 mm inner diameter), carrier GDX103, 60 to 80 mesh, H₂ flame monitor, evaporation chamber 200 ℃, column temperature 190 ℃, detection chamber temperature 240 ℃, carrier gas nitrogen, flow rate 50 mL/min, H₂ flow rate 50 mL/min, and air flow rate 500 mL/min. Tests were carried out with 1 mL of culture solution, adding 1 to 2 drops of 6 M HCl, centrifuging at 5000 rpm for 15 min, and taking 2 μL of supernatant into the sample for detection (Wang et al. 2017).

RESULTS AND DISCUSSION

Effect of Fe²⁺ on the Growth of Three Hydrogen-Producing Bacteria

Fe²⁺ is one of the most important metal ions for microorganisms’ growth and H₂ production. Duran Padilla et al. (2014) studied the growth of the hydrogen-producing strain Clostridium acetobutylicum ATCC 824 and found that adding Fe²⁺ (20 mg/L) enhanced its growth (Durán-Padilla et al. 2014).

As shown in Fig. 1, the bacterial concentration of E. harbinense ZGX4 was the highest when the Fe²⁺ concentration was 0.05 to 0.1 g/L. The bacterial concentration was significantly lower when the Fe²⁺ concentration was greater than 0.1 g/L or less than 0.05 g/L. These findings showed that both low and high concentrations of Fe²⁺ had a negative effect on the growth of E. harbinense ZGX4, and when the concentration was 0.05 g/L, the maximum bacterial concentration was 0.53 g/L. For C. butyricum 1.209, the bacterial concentration fluctuated less with the change in Fe²⁺ concentration, and the growth was better. When the Fe²⁺ concentration was 0.25 g/L, the bacteria reached the maximum concentration value of 0.52 g/L. For E. cloacae 1.2022, with the increase of Fe²⁺ concentration, the bacterial concentration showed a trend to increase and then decrease and reached the maximum (0.32 g/L) at the Fe²⁺ of 0.25 g/L, and its average biomass was the lowest among the three strains.

Fig. 1. Effect of Fe2+ on the growth of three bacteria

Effect of Fe²⁺ on H₂ Production by Three Hydrogen-Producing Bacteria

Figure 2 shows changes in H₂ content and H₂ production with Fe²⁺ concentration from the three bacteria. For E. harbinense ZGX4, when the Fe²⁺ concentration was less than 0.1 g/L, the H₂ content had little difference and it reached a maximum of 72.7%(v/v) when Fe²⁺ was 0.1 g/L. Its H₂ production fluctuated less with the change in Fe²⁺ concentration. When the Fe²⁺ concentration was at 0.05 g/L, the H₂ production reached the maximum of 2170 mL/L-medium (19500 mL H₂/mol glucose). Below or above this concentration, the H₂ production decreased. The concentration of 0.05 to 0.1 g/L was the most suitable Fe²⁺ concentration range for E. harbinense ZGX4 to produce H₂. Zhang et al. (2015) investigated the effect of temperature, pH, Mg²⁺, and Fe²⁺ on the H₂ production of Ethanoligenens harbinense YUAN-3, and this strain was the same as the one in this study. Moreover, when MgCl₂·6H₂O and FeSO₄·7H₂O were both 300 mg/L and under the optimal other conditions, the maximum H₂ yield of 2140 mL/L could be obtained, which was similar to the results of this study (Zhang et al. 2015). For C. butyricum 1.209, H₂ content showed a trend of increase and then decrease in the process of increasing Fe²⁺ concentration, reaching a maximum value of 64.5% when Fe²⁺ concentration was 0.2 g/L. H₂ production fluctuated greatly with the change in Fe²⁺ concentration. H₂ production was more sensitive to Fe²⁺. When the Fe²⁺ concentration was 0.2 to 0.25 g/L, H₂ production was the highest. When the Fe²⁺ concentration was 0.2 g/L, H₂ production reached a maximum value of 2540 mL/L-medium (22900 mL H₂/mol glucose). Above or below this concentration, H₂ production decreased. When Fe²⁺ concentration exceeded 1.0 g/L, H₂ content and H₂ production were low. H₂ production was completed within 20 hours, indicating that Fe²⁺ at this concentration (1.0 g/L) had an inhibitory effect on the H₂ production of C. butyricum 1.209. The most suitable Fe²⁺ concentration for H₂ production was 0.2 to 0.25 g/L. For E. cloacae 1.2022, when the Fe²⁺ concentration was less than 0.25g/L, H₂ contents showed little difference, fluctuating around 20%. When the Fe²⁺ concentration was 0.5 g/L, H₂ content reached the maximum value of 27.5%. H₂ production reached the maximum value of 785 mL/L- medium (7070 mL H₂/mol glucose) with a Fe²⁺ concentration of 0.75 g/L. Compared with the above two bacteria, the H₂ production of this bacterium was lower, and the fluctuation was the smallest. The Fe²⁺ concentration range corresponding to the optimal H₂ production lagged, which indicated that E. cloacae 1.2022 was less affected by Fe²⁺ than E. harbinense ZGX4 and C. butyricum 1.209, and the suitable Fe²⁺ concentration for H₂ production was 0.5 to 1 g/L.

Except for a few points, the changes in the growth of the three bacteria were consistent with the changes in H₂ production, indicating that within a certain range, H₂ production increased with the growth of bacteria and that bacterial growth was positively correlated with the H₂ production.

Fig. 2. Effect of different Fe2+ concentrations on H2 production. (a) H2 content (b) H2 production

Effect of Fe²⁺ on the Liquid Products of Three Bacteria

With the change in metal ion concentration, the liquid products of the three bacteria were mainly ethanol and volatile fatty acids (VFAs). The VFAs were mainly acetic acid, propionic acid, and butyric acid. Ethanol can be used as a liquid fuel and chemical feedstock, while VFAs have applications in the industrial food and pharmaceutical industries and are considered renewable carbon sources (Agnihotri et al. 2022; Yasser Farouk et al. 2022). Therefore, the hydrogen-producing bacteria can produce valuable liquid products while fermenting and producing H₂.

In terms of liquid products (Fig. 3), the effect of Fe²⁺ concentration on the liquid products of E. harbinense ZGX4 showed good consistency with H₂ production. The amount of total liquid products reflects the bacterial group metabolism of the H₂ production operating system. Its specific composition and proportion will reflect the metabolic type of H₂ production operating system. The main types of liquid products were ethanol and acetic acid, which was a typical ethanol-type fermentation. The overall amount of liquid products reached its maximum when Fe²⁺ was 0.05 g/L, which was 6530 mg/L, the highest among the three bacteria (Fig. 3(a)). The effect of Fe²⁺ on the liquid products of C. butyricum 1.209 was relatively stable with minor fluctuation. The total amount of liquid products reached a maximum value of 4850 mg/L when the concentration was 0.25 g/L. However, with the increase of Fe²⁺, ethanol production increased, and butyric acid fermentation was replaced by mixed acid fermentation. The main liquid products were ethanol, acetic acid, and butyric acid, indicating that adding Fe²⁺ to the fermentation system can alter the fermentation type (Fig. 3(b)). The conversion of bacterial fermentation mechanism to ethanol-type fermentation had better H₂ production capacity (Wang et al. 2018). The main liquid products of E. cloacae 1.2022 were ethanol, acetic acid, and formic acid, which are characteristic of mixed acid fermentation (Chen et al. 2021b). The lowest total amount of liquid products of the three bacteria were obtained, whereas when Fe²⁺ was 0.75 g/L, the highest total amount of liquid products was observed, 2205.1 mg/L.

Fig. 3. Effect of different Fe2+ concentrations on the liquid products for three bacteria. (a) E. harbinense ZGX4 (b) C. butyricum 1.209 (c) E. cloacae 1.2022

In summary, the appropriate concentration of Fe²⁺ promotes H₂ production in the three bacteria, which may be due to the participation of Fe²⁺ in the synthesis of ferredoxin, an essential coenzyme in microbial H₂ production metabolism that activates catalase (Gad El-Rab et al. 2018; Morra et al. 2014). The proper concentration of Fe²⁺ can improve hydrogenase activity in hydrogen production (Zhang et al. 2017). A study on H₂ production from sludge in the presence of Fe²⁺ revealed the mechanism of enhanced H₂ production by Fe²⁺ (Yin and Wang 2021). Compared with E. cloacae 1.2022, E. harbinense ZGX4 and C. butyricum 1.209 had better H₂ production performance with the addition of Fe²⁺. However, Fe²⁺ changed the fermentation type of C. butyricum 1.209. The optimal H₂ production of E. harbinense ZGX4 and C. butyricum 1.209 was similar. When the optimal gas production was reached, E. harbinense ZGX4 required less Fe²⁺ while producing the highest total amount of liquid products. E. harbinense ZGX4 generates more H₂ production and has the highest total amount of liquid products when Fe²⁺ is added.

Effect of Na⁺ on the Growth of Three Hydrogen-Producing Bacteria

Sodium salt is an important medium component in the expanded culture of hydrogen-producing microorganisms. However, sodium salt’s effect on hydrogen-producing fermentation bacteria has been the subject of few studies (Junghare et al. 2012). Therefore, it is necessary to study the effect of sodium salt on hydrogen-producing fermentation bacteria. NaCl addition improves H₂ production and promotes cell growth. This will eliminate sodium ions’ inhibitory effect in the medium preparation process.

As shown in Fig. 4, the growth amount of E. harbinense ZGX4 was the highest when Na⁺ concentration was 1.0 to 1.5 g/L. The growth amount was relatively low when Na⁺ concentration exceeded 1.5 g/L or was less than 1.0 g/L. This showed that both low and high concentrations of Na⁺ inhibited the growth of E. harbinense ZGX4. The maximum increase was 0.485 g/L when the concentration was 1.0 g/L. For C. butyricum 1.209, the growth amount of C. butyricum 1.209 was higher when the Na⁺ concentration was 2.0 to 3.0 g/L, reaching a maximum value of 0.511 g/L when Na⁺ concentration was 3.0 g/L. However, when Na⁺ concentration exceeded 3.0 g/L or was less than 2.0 g/L, the growth amount was lower, indicating that the low and high concentrations of Na⁺ would inhibit the growth of C. butyricum 1.209. For E. cloacae 1.2022, the growth of E. cloacae 1.2022 was higher when Na⁺ concentration was 1.5 to 2.0 g/L. When Na⁺ concentration was 1.5 g/L, the growth amount reached a maximum value of 0.38 g/L (Fig. 4).

Fig. 4. Effect of Na+ on the growth of three bacteria

Effect of Na⁺ on the H₂ Production of Three Bacteria

Figure 5 shows these three hydrogen-producing bacteria were more sensitive to Fe²⁺ than Na⁺. The optimal Na⁺ concentration range for H₂ production lagged behind that of Fe²⁺. For E. harbinense ZGX4, H₂ content ranged between 55% and 65% with a small range, reaching a maximum Na⁺ concentration of 1.5 g/L. When Na⁺ concentration was higher than 1.5 g/L, H₂ production decreased with the increase of Na⁺ concentration. When Na⁺ concentration was higher than 1.5 g/L or lower than 1.0 g/L, H₂ production declined. The fluctuation of H₂ production was slight. When Na⁺ concentration was 1.5 g/L, it reached a maximum value of 1510 mL/L-medium (13600 mL H₂/mol glucose), and 1.0 to 1.5 g/L was the most suitable Na⁺ concentration range of E. harbinense ZGX4 for H₂ production. For C. butyricum 1.209, the change in H₂ content had a similar trend to H₂ production. When Na⁺ concentration was 2.0 g/L, it reached a maximum value of 65.2%. When Na⁺ concentration was lower or higher than 2.0 g/L, H₂ production was low. When Na⁺ concentration was 2.0 g/L, H₂ production reached the maximum value of 2460 mL/L-medium (22100 mL H₂/mol glucose). Among the three bacteria, its H₂ production was the highest. However, its fluctuation was significant and more sensitive to the change in Na⁺ concentration. Its optimal H₂ production threshold was 2.0 to 2.5 g/L. C. butyricum 1.209 was Clostridium, and it has been reported that Clostridium is an ideal H₂ producer (Cai et al. 2021). For E. cloacae 1.2022, H₂ content reached the highest value of 27.0% only when Na⁺ concentration was 1.5 g/L. The other concentrations had little impact, fluctuating between 21.9% and 23.3%. H₂ production decreased as Na⁺ concentration increased. H₂ production in the control group was 72.9 mL/L-medium (656 mL H₂/mol glucose). However, when Na⁺ concentration exceeded 1.5 g/L, H₂ production was significantly reduced, and the lowest was only 3.39 mL/L-medium. This showed that adding Na⁺ did not promote H₂ production but inhibited H₂ production by fermentation of hydrogen-producing bacteria. H₂ production was the lowest among the three bacteria.

Fig. 5. Effects of different Na+ concentrations on H2 production. (a) H2 content (b) H2 production

Table 2.Hydrogen Production of Different Strains

In this study, three model hydrogen-producing strains were used. The effects of Fe²⁺ and Na⁺ on hydrogen production by three strains were investigated, and the highest hydrogen production was obtained. Compared with other strains (Table 2), these three strains also had higher H₂ production efficiency.

Effect of Na⁺ on the Liquid Products of Three Bacteria

Ethanol and acetic acid were the main liquid products for E. harbinense ZGX4 with Na⁺ concentration changes (Fig. 6).

The changing trend was consistent with the changing direction of H₂ production. When the concentration of Na⁺ was 1.5 g/L, total liquid products reached a maximum of 4920 mg/L. For C. butyricum 1.209, the main kinds of liquid products were acetic acid and butyric acid. The change rule of the total amount of liquid products was consistent with the change rule of H₂ production when Na⁺ concentration was higher than 3.0 g/L. It was significantly inhibited and fluctuated greatly due to the change in Na⁺ concentration, reaching a maximum value of 5350 mg/L when the Na⁺ concentration was 2.0 g/L. For E. cloacae 1.2022, combined with its liquid products, the main liquid products were ethanol, acetic acid, formic acid, and a small amount of propionic acid. The law was inconsistent with the changing direction of H₂ production when the concentration of Na⁺ was higher than 2.0 g/L. Obvious inhibition occurred when the concentration of Na⁺ was 2.0 g/L. The total of liquid products was the largest, at 3250 mg/L. The liquid products production was relatively high, but H₂ production was low, probably because the addition of Na⁺ or the output of VFAs inhibited H₂ production of hydrogen-producing bacteria (Boni et al. 2014; Singh and Wahid 2015; Chen et al. 2021a). Average liquid product production was the lowest among the three strains.

Fig. 6. Effect of different Na+ concentrations on the liquid products for three bacteria. (a) E. harbinense ZGX4; (b) C. butyricum 1.209; (c) E. cloacae 1.2022

Overall, it was found that the appropriate Na⁺ concentration could improve H₂ production of E. harbinense ZGX4 and C. butyricum 1.209. On the contrary, Na⁺ could inhibit the hydrogen production of E. cloacae 1.2022, which was different from Fe²⁺. The optimal H₂ production thresholds were relatively close for E. harbinense ZGX4 and C. butyricum 1.209. However, C. butyricum 1.209 had a higher hydrogen production. Comparing the best H₂ production, C. butyricum 1.209 was 38.7% higher than E. harbinense ZGX4, and within the optimal H₂ production range, it had higher liquid product production. Adding Na⁺ would make C. butyricum 1.209 have the most increased H₂ and liquid products production.

CONCLUSIONS

1. The effects of Fe²⁺ and Na⁺ on the H₂ production performance of the strains was investigated using three different fermentation types of hydrogen-producing strains. The results showed that different metal ions had various effects on the H₂ production performance of the strains.

2. Except for Na⁺, which inhibited the H₂ production of E. cloacae 1.2022, Fe²⁺ and Na⁺ positively impacted the growth and H₂ production metabolism of the three different fermentation types of strains.

3. Combining H₂ and liquid product production, Fe²⁺ was the most suitable for H₂ production metabolism of ethanol-type fermentative hydrogen-producing bacteria E. harbinense ZGX4. At the same time, Na⁺ was found to be more suitable for butyric acid-type fermentative hydrogen-producing bacteria C. butyricum 1.209.

4. At the end of the experiment, it was found that adding Fe²⁺ could change the type of fermentation in C. butyricum 1.209.

ACKNOWLEDGMENTS

This study was supported by the Key Research and Development projects of Heilongjiang Province, China (2022ZX02C14) and The Technological Project of Heilongjiang Province, China “the Open Competition Mechanism to Select the Best Candidates” (2022ZXJ05C01).

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