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Biological Hydrogen Energy Production by Novel Strains Bacillus paramycoides and Cereibacter azotoformans through Dark and Photo Fermentation

Department of Chemical Engineering, Faculty of Engineering and Science, Curtin University Malaysia, CDT 250, Miri 98009, Sarawak, Malaysia
Department of Electrical & Computer Engineering, Faculty of Engineering and Science, Curtin University Malaysia, CDT 250, Miri 98009, Sarawak, Malaysia
Mechanical Engineering Department, Abu Dhabi University, Abu Dhabi P.O. Box 59911, United Arab Emirates
Center for Renewable Energy and Energy Efficiency, Abu Dhabi University, Abu Dhabi P.O. Box 59911, United Arab Emirates
Singapore Institute of Food and Biotechnology Innovation, 31 Biopolis Way, #01-02, Nanos, Singapore 138669, Singapore
Author to whom correspondence should be addressed.
Energies 2023, 16(9), 3807;
Submission received: 31 March 2023 / Revised: 23 April 2023 / Accepted: 25 April 2023 / Published: 28 April 2023
(This article belongs to the Special Issue Advances in Renewable Energy Research and Applications)


In daily life, energy plays a critical role. Hydrogen energy is widely recognized as one of the cleanest energy carriers available today. However, hydrogen must be produced as it does not exist freely in nature. Various methods are available for hydrogen production, including electrolysis, thermochemical technology, and biological methods. This study explores the production of biological hydrogen through the degradation of organic substrates by anaerobic microorganisms. Bacillus paramycoides and Cereibacter azotoformans strains were selected as they have not yet been studied for biological hydrogen fermentation. This study investigates the ability of these microorganisms to produce biological hydrogen. Initially, the cells were identified using cell morphology study, gram staining procedure, and 16S ribosomal RNA (rRNA) gene polymerase chain reaction. The cells were revealed as Bacillus paramycoides (MCCC 1A04098) and Cereibacter azotoformans (JCM 9340). Moreover, the growth behaviour and biological hydrogen production of the dark and photo fermentative cells were studied. The inoculum concentrations experimented with were 1% and 10% inoculum size. This study found that Bacillus paramycoides and Cereibacter azotoformans are promising strains for hydrogen production, but further optimization processes should be performed to obtain the highest hydrogen yield.

1. Introduction

Energy is vital in this burgeoning epoch of science and technology. The oil crisis in the 1970s indicated that the recent energy management system is not sustainable over a long period of time [1]. Global reliance on a myriad quantity of fossil fuels has led to excessive greenhouse gas (GHG) emissions. GHG created by the combustion of fossil fuels have caused climate change and global warming which has led to increasing sea levels [2,3]. The use of alternative technologies for energy conversion is necessary to reduce global dependence on non-renewable energy sources. Renewable energy, particularly hydrogen energy, has been proposed due to its high calorific value (140 kJ/g), carbon-neutral nature, and environmentally friendliness [4,5]. The development of hydrogen energy will play a significant role in the future of energy management and contribute to the reduction in environmental pollution.
Hydrogen, although the most abundant element in the universe, cannot be found in pure form on Earth and requires further isolation from various molecules, such as hydrocarbons, water, hydrides, and acids, to obtain it in its gaseous state [6]. The production of hydrogen energy can be classified into three types: grey, blue, and green hydrogen, depending on the feedstocks and production methods used (Figure 1) [7]. Grey hydrogen is produced from hydrocarbons through high-energy-requiring thermochemical conversion technology. Blue hydrogen is produced in the same way but with carbon capture and storage [8]. On the other hand, green hydrogen is obtained from renewable resources such as biomass and water, and metabolic engineering is one of the production technologies for green hydrogen generation. This method is adapted to create a biorefinery sustainable pathway for biomass waste conversion into valuable biochemicals by-products, or namely biohydrogen fuel [9].
Biological hydrogen production through bacteria biodegradation offers several environmental and economic benefits if cost-effective biomass waste is utilized as the feedstock [10]. Lignocellulosic and biomass wastes, such as palm oil mill effluent, winery wastewater, paper waste, post-harvest agricultural waste, and woody biomass, have been shown to significantly contribute to the economic viability of sustainable biopathway hydrogen formation [11,12,13]. Despite the many efforts made to produce biological hydrogen through fermentation technology, the main limitation remains the low hydrogen yield from fermentative bacteria. Furthermore, different microorganism strains may possess varying potentials in hydrogen production. Therefore, to ensure sustainable biofuel generation, the development of biological hydrogen generation through fermentation necessitates further exploration of new hydrogen-producing strains [14].
In recent years, the use of novel microbial strains for biological hydrogen production has gained significant attention. These novel strains have the potential to improve the efficiency and yield of hydrogen production and to overcome the limitations of traditional hydrogen production methods. For instance, some of these strains can tolerate high concentrations of organic matter and produce hydrogen gas through a biodegradation process. A research study conducted by Pu et al. (2019) investigated the effect of substrate concentration on biological hydrogen production by anaerobic seed sludge. The study revealed that the anaerobic seed sludge was able to produce biological hydrogen even at high concentrations of volatile solid substrate, with a hydrogen yield of 5.3 mL per gram of volatile solid substrate. This clarifies that the high substrate concentration did not prevent the production of biological hydrogen from anaerobic degradation [15]. Palafox-Félix et al. (2022) found that the concentration of a carbon source can influence the formation of metabolites in biochemical processes, as indicated by proteomic analysis. Specifically, when glucose is not present, the bacterium shifts its carbon flow towards the production of antifungal substances by utilizing alternative carbon and nitrogen sources. Conversely, when glucose is available, the bacterium prioritizes energy generation and cell growth [16]. Therefore, it is important to explore the metabolic behaviour of this novel strain under different operational conditions, particularly for the generation of biological hydrogen.
Furthermore, some strains can produce hydrogen gas over a wide range of pH and temperature conditions, making the process more flexible and adaptable. For example, Tang et al. (2022) studied the effect of various initial pH levels on hydrogen fermentation by Clostridium sensu stricto 12 sp. The study found that the maximum cumulative hydrogen production occurred at an initial pH of 5, with 70.94 mL of hydrogen gas produced per gram of volatile solid substrate. Additionally, even at a low initial pH of 4, the dark fermentation process still yielded 24.93 mL of hydrogen per gram of volatile solid substrate [17]. Another fermentation study investigated the effect of temperature on anaerobic mixed microflora to produce biological hydrogen. The study examined three temperature conditions: mesophilic (37 °C), thermophilic (55 °C), and hyper-thermophilic (80 °C). The experimental results showed that the thermophilic condition yielded the highest biological hydrogen, with a production rate of 12.28 mmol/g cellulose. At the hyper-thermophilic temperature of 80 °C, a hydrogen yield of 9.72 mmol/g cellulose was obtained [18]. Another study examined the impact of metabolic heat within a biofilm on biological hydrogen production. The researchers employed a fiber Bragg grating (FBG) sensor to measure the temperature of Rhodopseudomonas palustris CQK-01 biofilm and investigate its effect on hydrogen generation. The study found that both the biofilm thickness and the temperature had a significant influence on the efficiency of biological hydrogen production via fermentation [19].
In this regard, this research work is a preliminary trial that explores the potential of two novel strains, the dark fermentative Bacillus paramycoides and the photo fermentative Cereibacter azotoformans, for biological hydrogen production. Bacillus paramycoides and Cereibacter azotoformans are facultative anaerobes, allowing them to produce hydrogen under anaerobic and mild aerobic conditions, providing flexibility in the production process [20,21]. Additionally, Bacillus sp. is widely available and Cereibacter sp. has the potential to reduce the cost of hydrogen production as it requires less nutrition. Thus, they were selected for the experimental study. Moreover, these novel strains have not yet been investigated for biological hydrogen production. According to the literature, Cereibacter sp. could produce additional hydrogen by using the by-products from Bacillus sp. [22]. Therefore, this research explores the potential of the two novel strains for hydrogen production, followed by single strain parametric study and co-culture system for future research and development. In this study, the cells were identified and their growth behaviour study was performed. The preliminary trial showed that Bacillus paramycoides and Cereibacter azotoformans can produce biological hydrogen energy through dark and photo fermentation, respectively. Additionally, these strains demonstrated a fast onset of hydrogen evolution from media inoculation, indicating their potential for efficient biological hydrogen production. Nevertheless, this study is just a basic exploration of cells and hydrogen production, and further optimization processes will be required to maximize the potential of these strains for biological hydrogen production. The results of these optimization processes will be presented as future work, building upon the initial findings presented in this research.

2. Materials and Methods

2.1. Microorganisms and Culture Medium

The freeze-dried cells (DSM14 and DSM5864) were obtained from DSMZ and activated in Pyrex borosilicate conical flasks for 48 h before the experimental study. The photo fermentative microbe was cultivated in a nutrient broth solution consisting of 8 g nutrient broth in 1 L of Milli-Q water, with an initial pH of 6.61. On the other hand, the dark fermentative microbe was grown in a culture media comprising 10 g of glucose, 3 g of peptone, 1 g of yeast extract, 2.8 g of K2HPO4, 3.9 g of KH2PO4, 0.2 g of MgSO47H2O, 0.1 g of NaCl, 0.01 g of CaCl26H2O, 0.05 g of FeSO47H2O, 0.2 g of L-cysteine, and 1 mL of microelements in 1 L of solution [22]. The initial pH of the activation broth for the dark fermentative microbe was 6.68. The microelements solution (1 L) contained 0.07 g of ZnCl2, 0.1 g of MnCl24H2O, 0.06 g of H3BO3, 0.2 g of CoCl26H2O, 0.02 g of CuCl22H2O, 0.02 g of NiCl26H2O, and 0.04 g of NaMoO42H2O. Prior to every experimental study, the conical flasks and cultivating media were autoclaved (HV-110 Hirayama) at 121 °C for 20 min. Carbon sources were autoclaved separately at 110 °C for 20 min before being added to the cultivating media.

2.2. Experimental Setup and Analytical Method

The growth behaviour study was performed based on bacterial concentrations of 1% and 10% in 250 mL Pyrex conical flasks, with a working volume of 200 mL of medium. To generate biological hydrogen, both dark and light fermentative bacteria require anaerobic conditions. To achieve this condition during the experimental study, the conical flask with fermentation medium was first flushed with oxygen-free argon gas for 15 min to remove any remaining oxygen. The flask was then closed with a rubber stopper. Bacteria culturing was performed in a closed desiccator with lit candles to remove any excess oxygen from the environment. Additionally, for the light fermentation process, the setup was constantly supplied with illumination by an Osram 300 W Ultra-Vitalux lamp, with light intensity of 15 klx. For the dark fermentation process, it was performed in a closed incubator to avoid light. Fermentation temperature for both dark and photo fermentation was fixed at 33 °C. A detailed illustration of the experimental setup is presented in Figure 2:
The analytical method is being used to monitor the growth behaviour of bacteria and measure the concentration of biological hydrogen produced by dark and photo fermentation processes. For the growth behaviour study, 2 mL samples of the solution were collected periodically every 4 h and inserted into a 2.5 mL cuvette (Kartell) for analysis. The liquid sample was analysed using a UV-VIS spectrometer (Perkin Elmer) with an optical density (OD) of 600 nm for both dark and photo fermentative cells [23,24]. This method of analysis is known as spectrophotometry and involves measuring the amount of light absorbed by the sample at a specific wavelength [25]. The absorption of light is directly proportional to the concentration of the sample, so by measuring the amount of light absorbed, the concentration of bacteria in the solution can be determined. Additionally, to measure the concentration of biological hydrogen produced by both dark and photo fermentation processes, a portable hydrogen gas detector (ATO) equipped with an electrochemical detector was used. To measure the gas produced from the fermentation process, the inlet and outlet of the hydrogen detector were plugged into the conical flask, creating a loop to detect the real-time concentration in the conical flask. The constant pump rate of 75 mL/min from the detector was utilized. After recording the hydrogen reading from the detector, the outlet was unplugged, and the detector continued to pump to flush away the hydrogen gas present in the conical flask, thus resetting the hydrogen gas concentration to zero. The procedure was repeated every 4 h to measure the hydrogen gas produced periodically. Furthermore, the inoculation, the liquid sample collection, and the hydrogen quantification procedures were performed in a class II biological safety cabinet (Esco Scientific) to prevent contamination.

2.3. Gram Staining Procedure

The gram staining procedure was performed to classify the cells as either gram-positive or gram-negative microorganisms. First, the cells were air-dried and heat-fixed onto a microscope glass slide. Next, the heat-fixed cells were flooded with a crystal violet staining reagent for 60 s. In the following step, the glass slide was washed with a gentle stream of tap water for 2 s, followed by flooding with gram’s iodine for 60 s. Furthermore, the glass slide was washed with ethyl alcohol for 5 to 10 s to remove the iodine reagent. Lastly, safranin was used as a counterstain, which was flooded onto the glass slide for roughly 45 s, followed by flushing with an indirect stream of tap water until no colour appeared in the effluent [26].

3. Results and Discussion

3.1. Morphology and Gram Staining

The morphology of dark and light fermentative cells was observed using a scanning electron microscope (SEM), and the results are presented in Figure 3. The dark fermentative cell, as shown in Figure 3A, was identified as a rod-shaped bacterium with a cell length ranging from 1.8–2.2 µm and a cell width ranging from 0.8–1.2 µm. On the other hand, the photo fermentative cell, as shown in Figure 3B, was identified as an ovoid bacterium with a cell length ranging from 1.5–2 µm and a cell width ranging from 1.2–1.5 µm.
The distinction between a gram-positive and a gram-negative bacterium lies in the thickness of the peptidoglycan layer in the cell walls. Gram-positive bacteria have thick layers of peptidoglycan in their cell walls, while gram-negative bacteria have thin layers of peptidoglycan in their cell walls [27]. The gram staining procedure was employed to differentiate between gram-positive and gram-negative bacteria. Gram-positive organisms retain the primary colour and appear purple under a microscope, whereas gram-negative organisms appear red under a microscope (Figure 4) [26].
According to the gram staining procedure, the cells were classified as gram-positive and gram-negative organisms, displaying purple and red staining under the microscope. This finding suggests that the identity of these cells could be from the Corynebacterium, Clostridium, or Bacillus family for purple staining cells [28,29,30]. Conversely, the red staining observed under the microscope confirmed the cell identity as gram-negative bacteria. These cells could possibly belong to the Neisseria, Pseudomonas, or Cereibacter family [31,32,33]. To further confirm the strains of the microbes, DNA sequencing was performed for the respective strains.

3.2. DNA Sequencing

The DNA was isolated and identified under the 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR). The reaction for PCR is shown in Figure 5. From the total DNA, the 16S rRNA gene from the gDNA of bacterial isolates were PCR amplified using the primer 785F and 907R. The amplified product was run on to a 0.6 agarose. The agarose gel was documented and the PCR amplified product weight showed prominent DNA bands with approximate sizes of 1500 base pairs. PCR amplified products were run on 1% agarose gel. Lane M indicates the DNA ladder (DNA Ladder Mix 250 to 10,000 base pair, catalogue number BIO-5140). Markers with high intensity were indicated by their size. Lanes 1 and 2 indicate the PCR amplified 16S rRNA gene of the respective bacterial isolate. This analysis indicates specific amplification of the 16S rRNA gene. Sequence analysis was performed using both forward and reverse primers and results were edited and assembled into one full-length sequence.
The sizes of the 16S rRNA sequences obtained for each of the bacterial isolates are presented in Table 1. The 16S rRNA sequence of isolate 1 shows 99.93% identity to Bacillus paramycoides. For isolate 2, the 16S rRNA sequence is showing 100% identity to Cereibacter azotoformans.
Additionally, the isolates were subjected to phylogenetic analysis based on their 16S rRNA gene sequences, which were compared to the 16S rRNA gene sequences of hit species. This comparison highlighted the differential alignment of bacterial isolates with various species. The resulting phylogenetic tree (see Figure 6) classifies the dark and photo fermentative bacteria as Bacillus paramycoides (MCCC 1A04098) and Cereibacter azotoformans (JCM 9340).
Once the morphology, the gram staining characteristics, and the identity of both bacteria were determined, a growth behaviour study was conducted. The results of this study will be presented in the following section.

3.3. Growth Behaviour Study and Biological Hydrogen Production with 1% Inoculum

During the growth behaviour study for both strains, simultaneous analysis of biological hydrogen production was performed. Prior to the inoculation process, the initial pH for the Cereibacter sp. culture broth was measured as 6.61, while the initial pH for the Bacillus sp. broth was recorded as 6.68. Figure 7 displays the growth curves for both bacterial strains along with the biological hydrogen production. The biological hydrogen generation and liquid samples were collected and analysed periodically every 4 h for a duration of 4 days.
Figure 7 illustrates that rapid biological hydrogen production occurred during the first 24 h of dark and light biodegradation, and that the hydrogen production rate began to decrease and achieve a steady state from 48 h onwards. The cumulative biological hydrogen production was 5739 ppm and 3654 ppm for dark fermentative Bacillus sp. and light fermentative Cereibacter sp., respectively. Additionally, two growth curves for Cereibacter azotoformans and Bacillus paramycoides were shown based on a 1% inoculum concentration. The biological hydrogen was produced rapidly during the initial phase for both dark and photo fermentative bacteria. Once the bacteria began to grow and increase in cell numbers, the hydrogen production decreased significantly. The growth behaviour of Bacillus sp. and Cereibacter sp. was studied for 96 hours. During 0 to 38 hours, Bacillus sp. experienced a lag phase due to strain adaptation to a new environment. After the lag phase, the exponential phase lasted for 28 hours, from 38 to 66 hours of fermentation time, where maximum growth rate appeared. Lastly, from 66 to 96 hours of growth behaviour study, a stationary phase occurred with no net increase in cell numbers. For Cereibacter sp., the lag phase occurred from 0 to 50 hours fermentation time, after which the exponential phase appeared from 52 to 96 hours of fermentation time. The doubling mechanism of Cereibacter sp. occurred during the log phase, illustrating a proportional growth curve in Figure 7. Nonetheless, the growth curve study for Cereibacter sp. did not reach the stationary phase, so a growth study for both dark and light fermentative bacteria based on a 10% inoculum concentration will be conducted to obtain a full growth curve study and biological hydrogen production analysis. In addition, the biological hydrogen yield can also be compared between small (1%) and large (10%) inoculum sizes.

3.4. Growth Behaviour Study and Biological Hydrogen Production with 10% Inoculum

During the study on growth behaviour at a 1% inoculum concentration, it can be observed that Bacillus sp. underwent lag, log, and stationary phases. However, Cereibacter sp. was only able to achieve lag and part of the exponential phase. As a result, another growth curve study was at a 10% inoculum concentration, which is illustrated in Figure 8.
At a 10% inoculum concentration, Bacillus sp. and Cereibacter sp. were inoculated to study their growth behaviour and biological hydrogen production for 96 h. During this period, simultaneous analysis of biological hydrogen production was carried out for both dark and light fermentation. The results showed that rapid biological hydrogen production occurred during the first 20 h of both dark and light fermentation. However, the biological hydrogen production rate decreased significantly from the 20th to the 44th hour of fermentation time. After the 44th hour, a steady state biological hydrogen production was achieved until the end of the fermentation period. Dark fermentative Bacillus sp. accumulated 4668 ppm of cumulative biological hydrogen production, while photo fermentative Cereibacter sp. produced 2564 ppm after 96 h of fermentation. Both bacterial strains achieved up to the stationary phase in their growth behaviour. The growth curve for Bacillus sp. in Figure 8 demonstrates that the lag phase was from 0 to 20 h, and the log phase occurred from the 20th to 44th hours of fermentation time. The stationary phase for Bacillus sp. began at the 48th hour and lasted until the end of the fermentation period. In contrast, for Cereibacter sp., the lag phase occurred from 0 to 12 h, followed by the exponential phase that lasted up to the 76th hour. The steady phase then appeared from the 76th to the 96th hour of the fermentation period.
In both the 1% and 10% inoculum growth behaviour studies, the lag phase was observed as the initial period in the life of a bacterial population where cells adapted to a new environment. During the lag phase, cells prepared to generate proteins and cellular enzymes, increasing in size but not in cell numbers. The duration of the lag phase could be affected by the inoculum size, physiochemical environment of both origin and new fermentation broth, and physiological history of the bacteria [34]. In both the 1% and 10% inoculum growth studies, rapid biological hydrogen production occurred during the initial lag phase of bacterial growth. Additionally, the hydrogen yield from 1% inoculum was higher than that of the 10% inoculum size. Comparatively, 1% inoculum of dark and photo fermentation showed 22.9% and 42.5% hydrogen yield enhancements, respectively, compared to 10% inoculum size. The phenomenon of a high hydrogen yield resulting from a lower inoculum size could be explained by the density-dependent communication system known as quorum sensing [35]. In the quorum sensing communication system, bacterial cell-to-cell communication involves production, detection, and response to extracellular signaling molecules called autoinducers. During the lag phase of bacterial growth, the cells could send autoinducer signals between the inter-species community to improve the microbial concentration. Furthermore, quorum sensing is involved in regulating enzyme production for microbial growth purposes [36]. The quorum sensing system between bacteria could improve hydrogenase or nitrogenase enzyme activity, regulating a higher hydrogen production rate. Therefore, when the bacterial community achieved significant numbers, or namely the stationary phase, biological hydrogen production decreased and became stable, which could be due to the decreasing rate of quorum sensing. A similar result was obtained by Ulhiza et al. (2018) in an experimental research work to investigate the biological hydrogen yield from different yeast concentrations. They reported that a 5% inoculum resulted in a higher hydrogen yield compared to a 9% inoculum size. The biological hydrogen yields were 52 μmol and 32 μmol for 5% and 9% inoculum sizes, respectively [37].
Palafox-Félix et al. (2022) utilized a label-free quantitative proteomic method to investigate the metabolic control of Amycolatopsis sp. BX17’s metabolism under different glucose concentrations. The findings of their research showed that when glucose was absent from the culture medium, a metabolic shift occurred that favoured the utilization of alternative carbon sources, resulting in a decrease in the bacterial growth rate and an increase in the production of secondary metabolites [16]. In this study, a lower inoculum may have led to higher biological hydrogen production, possibly due to the availability of sufficient glucose for hydrogen metabolism. Under a high inoculum concentration and glucose depletion, the metabolism of microorganisms may shift towards the utilization of alternative carbon sources, resulting in an increased production of secondary metabolites and an inhibition of hydrogen production. Another study enhanced biological hydrogen production by optimizing biofilm growth in a photobioreactor. The study found that a thick biofilm can reduce its porosity, which can result in increased resistance to mass transfer. Additionally, the substrate at the bottom of the biofilm layer may become limited, while the outer layer of the biofilm may experience a lack of light. Consequently, this can cause a decrease in hydrogen production by bacteria [38]. As a result, high inoculum concentration may lead to lower biological hydrogen production due to biofilm inhibition. In summary, biological hydrogen production is significantly affected by the community of inter-bacteria species.
After the lag phase, bacterial growth enters the exponential phase where cell doubling occurs through binary fission, resulting in a periodic doubling of the population [39]. However, during this phase, the rate of biological hydrogen production decreases with increasing cell numbers. This may be due to a reduction in the rate of quorum sensing as the cell population grows, leading to a decrease in enzyme activity responsible for hydrogen production. Furthermore, accumulation of toxic metabolites during cell doubling may inhibit enzyme activity, further decreasing the biological hydrogen production rate. The pH changes observed during the experiment, with a final pH of 6.89 for Cereibacter sp. and 4.81 for Bacillus sp., indicate that increasing cell numbers can alter the fermentation environment, potentially leading to changes in the biological hydrogen metabolism. As a result, higher cell numbers can lead to a lower hydrogen production rate. Nevertheless, with a 1% inoculum concentration of Cereibacter sp., only a portion of the log phase is achieved due to the low cell concentration, resulting in a lower cell division rate. A complete growth curve for Cereibacter sp. can be achieved with a 10% inoculum, as shown in Figure 8. During the steady state, the rate of dividing cells equals the rate of dying cells, resulting in no net increase in the number of viable cells. Moreover, bacteria enter the stationary phase due to various reasons, including limited nutrients, stress factors, such as changes in osmolarity, pH or temperature, and an accumulation of toxic metabolites [40,41]. It is essential to consider all these changing parameters in order to optimize hydrogen production.

4. Conclusions

This journal paper reports the results of a DNA identification study which identified two strains of bacteria as Bacillus paramycoides (MCCC 1A04098) and Cereibacter azotoformans (JCM 9340). This study on the growth behaviour of these strains indicated that both dark and photo fermentative phases, including lag, log, and steady phases, were achieved using a 10% inoculum size. Furthermore, this study aimed to investigate the potential of the novel strains, Bacillus paramycoides and Cereibacter azotoformans, to produce biological hydrogen, making this study a preliminary trial. The experimental results revealed that both strains could carry out hydrogen fermentation and produced the highest yield when a lower bacteria inoculum size was used. This study also highlighted the significant impact of quorum sensing on hydrogen fermentation. Overall, Bacillus paramycoides and Cereibacter azotoformans are considered promising strains for hydrogen fermentation due to their fast onset of hydrogen evolution from media inoculation. Due to their ability to thrive under facultative anaerobic conditions, these strains have shown promise for use in hydrogen fermentation. This paper suggests that future studies should focus on exploring various operating conditions, co-culture symbiotic fermentation, and genetic engineering modifications to enhance biological hydrogen yield through fermentation. This article provides new evidence for the potential of hydrogen production through fermentation and makes a significant contribution towards the development of green energy.

Author Contributions

Methodology, E.C.H.C.; Validation, K.H.L.; Formal analysis, E.C.H.C. and J.K.; Investigation, E.C.H.C., S.K.W., S.Y.L. and A.N.L.; Resources, J.K. and S.S.D.; Writing—original draft, E.C.H.C.; Writing—review & editing, S.K.W., S.Y.L. and S.S.D.; Supervision, S.K.W., J.K., S.Y.L., K.H.L. and S.S.D. All authors have read and agreed to the published version of the manuscript.


This research was conducted at Curtin University Malaysia and was funded by the Ministry of Higher Education Malaysia (MoHE) Fundamental Research Grant Scheme (FRGS/1/2019/TK10/CURTIN/02/1). The authors gratefully acknowledge the financial support provided by MoHE for this research project.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to confidentiality concerns.

Conflicts of Interest

The authors declare no conflict of interest.


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Figure 1. Differentiation of hydrogen production pathways into grey, blue, and green colour code.
Figure 1. Differentiation of hydrogen production pathways into grey, blue, and green colour code.
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Figure 2. Experimental setup for biological hydrogen fermentation.
Figure 2. Experimental setup for biological hydrogen fermentation.
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Figure 3. (A) Dark fermentative cell and (B) photo fermentative cell under SEM.
Figure 3. (A) Dark fermentative cell and (B) photo fermentative cell under SEM.
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Figure 4. (A) Bacillus sp. before and (B) after gram staining, and (C) Cereibacter sp. before and (D) after gram staining.
Figure 4. (A) Bacillus sp. before and (B) after gram staining, and (C) Cereibacter sp. before and (D) after gram staining.
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Figure 5. Agarose gel electrophoresis analysis of 16S rRNA genes amplified from two bacterial isolates.
Figure 5. Agarose gel electrophoresis analysis of 16S rRNA genes amplified from two bacterial isolates.
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Figure 6. Phylogenetic tree for both isolate.
Figure 6. Phylogenetic tree for both isolate.
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Figure 7. Growth curve and simultaneous biological hydrogen production by Cereibacter azotoformans and Bacillus paramycoides (1% inoculum).
Figure 7. Growth curve and simultaneous biological hydrogen production by Cereibacter azotoformans and Bacillus paramycoides (1% inoculum).
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Figure 8. Growth curve and simultaneous biological hydrogen production by Cereibacter azotoformans and Bacillus paramycoides (10% inoculum).
Figure 8. Growth curve and simultaneous biological hydrogen production by Cereibacter azotoformans and Bacillus paramycoides (10% inoculum).
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Table 1. Bacteria isolated identified.
Table 1. Bacteria isolated identified.
Sample16S rRNA Sequenced Gene Size (Base Pair)GenBank Accession Number% Identity% Query CoverScientific Name
11509NR_157734.199.93100Bacillus paramycoides
21418NR_113300.110099Cereibacter azotoformans
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Chung Han Chua, E.; Wee, S.K.; Kansedo, J.; Lau, S.Y.; Lim, K.H.; Dol, S.S.; Lipton, A.N. Biological Hydrogen Energy Production by Novel Strains Bacillus paramycoides and Cereibacter azotoformans through Dark and Photo Fermentation. Energies 2023, 16, 3807.

AMA Style

Chung Han Chua E, Wee SK, Kansedo J, Lau SY, Lim KH, Dol SS, Lipton AN. Biological Hydrogen Energy Production by Novel Strains Bacillus paramycoides and Cereibacter azotoformans through Dark and Photo Fermentation. Energies. 2023; 16(9):3807.

Chicago/Turabian Style

Chung Han Chua, Eldon, Siaw Khur Wee, Jibrail Kansedo, Sie Yon Lau, King Hann Lim, Sharul Sham Dol, and Anuj Nishanth Lipton. 2023. "Biological Hydrogen Energy Production by Novel Strains Bacillus paramycoides and Cereibacter azotoformans through Dark and Photo Fermentation" Energies 16, no. 9: 3807.

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