Growth Study and Biological Hydrogen Production by novel strain Bacillus paramycoides

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hydrogen gas was generated through dark fermentation process. For Bacillus sp. growth study, lag, log, and stationary phase have been achieved in 96 hours. In a summary, metabolic engineering to degrade abundant biomass wastes is a sustainable pathway to produce hydrogen energy, simultaneously resolve waste management issue around the globe.
Keywords: Green energy, Microbes degradation, Biological hydrogen production, Dark Fermentation

Introduction
Energy is essential to fuel up our daily processes. Oil crisis in 1970s indicated that fossil fuel is not sustainable for future energy creation. Industrial revolution has created high dependent on fossil fuels for energy production. However, combustion of oil and gas produced excessive amount of greenhouse gases, hence led to climate change [1], [2] . Therefore, energy transition is crucial to reduce fossil fuels for energy creation.
Renewable energy has been proposed to resolve environmental pollution issues. One of the renewable energies that receive much attention is the hydrogen energy. Properties such as high calorific value (140 kJ/g), carbon neutral, and environmentally friendly could make hydrogen as an important element in the energy sector [3], [4]. Nonetheless to create hydrogen energy, further process is required to isolate hydrogen from molecules such as hydrocarbons, water, and acids [5]. Commercialized electrolysis and thermochemical processes are utilized to create hydrogen element. These processes require fossil fuels or high energy to operate, creating greenhouse gases which making it unsustainable for the future [6]. To overcome these issues, organic substrate degradation by microorganisms could be a better alternative to create green hydrogen energy. The biorefinery pathway is able to degrade biomass into valuable biochemicals by-product, or even biofuel [7]. For instance, energy-efficient and environmentally friendly dark fermentation technology is proposed to create green hydrogen energy [8].
Anaerobic microorganisms are utilized in dark fermentation to transform organic substrates into hydrogen energy. For instance, Bacillus sp., Enterobacter sp., and Clostridium sp. are the most common hydrogen producing strains [9]. Furthermore, agricultural wastes, food wastes, and wastewater can be utilized and treated through dark fermentation process [10]. The phenomenon of dark fermentation can be described as: 6

Bacteria Identification
The cell was observed under scanning electron microscope ( This fits one of the characteristics of Bacillus sp., where it is a rod-shaped growing pattern bacteria [12]. The difference between gram-positive and gram-negative bacteria is grampositive bacteria has thick layers of peptidoglycan in the cell walls. For gram-negative bacteria, it has a thin layer of peptidoglycan in the cell walls [13]. Besides, to differentiate gram-positive and gram-negative bacteria, gram staining procedure can be employed. The organisms that retain the primary colour and appear purple under a microscope is grampositive organisms, whereas organisms that appear red under a microscope are gramnegative organisms [11]. According to literature, Bacillus sp. are gram-positive rods microorganisms [14]. Thus, the gram staining procedure has proved the identity of the After identifying the cell as gram-positive, rod-shaped Bacillus paramycoides, a respective growth behaviour study and the hydrogen producing ability was examined.
The results are included in section 3.2 and 3.3.    Figure 3.5, rapid biological hydrogen production rate appeared during the first 20 hours of fermentation, which is the lag phase of Bacillus sp. growth. This phenomenon can be explained by the density-dependent communication system of microorganisms which also known as quorum sensing [15].

Biological Hydrogen Production by Dark Fermentation
Quorum sensing involves cell-cell communication process which responsible for production, detection, and response towards extracellular signalling molecules named autoinducers. In a result, Bacillus sp. may send autoinducer signals between each other to improve their microbial concentration during lag phase. In addition, quorum sensing regulates enzyme production for microbial growth purpose [16]. Thus, the high-rate communication between cells may increase the enzyme production which in turn increasing the biological hydrogen formation. Lastly, when Bacillus sp. achieved the stationary growth phase, decreasing rate of density-dependent communication system may reduce the rate of enzyme production. This may decrease the biological hydrogen The study showed that pH 6 and 1.5% salinity of swine wastewater was optimum for dark fermentative hydrogen formation. The experimental work also studied that biological hydrogen yield was affected by soluble chemical oxygen demand during alkaline condition and 3-3.5% high salinity solution [18]. Additionally, micronutrients such as Zn, Mn, Ca, Co, Ni, Fe, Cu have significant impact towards the growth and hydrogen production of dark fermentative microbes.
Enzymes such as nitrogenase and hydrogenase produced from fermentative microbes may be enhanced or inhibited by the addition of microelements [19]- [24]. Therefore, Bacillus paramycoides could be further experimented with various operating conditions to enhance its ability for hydrogen production. Different type of biomass wastes can also be experimented with Bacillus paramycoides dark fermentation for useful bioproducts formation.

Conclusion
After the cell identification process, the DNA was proved to be gram-positive, rod-shaped organisms which show purple colour staining under a microscope. The cell was identified as Bacillus paramycoides (MCCC 1A04098). For cell growth behaviour study, lag phase achieved was 20 hours, followed by 28 hours of exponential phase and 48 hours of stationary phase. Moreover, cumulative biological hydrogen produced was 4668 ± 120 ppm from Bacillus sp. It was found that cell number has significant impact on biological hydrogen production rate due to the density-dependent communication system. In a nutshell, the cell identification and basic hydrogen fermentation trial found that Bacillus paramycoides are feasible to produce hydrogen. This article provides new evidence for the potential of hydrogen production from fermentation process. Further experimental work on investigating the effect of various operating conditions towards Bacillus sp.
hydrogen fermentation is crucial. In the meantime, exploring novel strains, genetic engineering, reactor system optimization, and substrates processing methods can also be explored for hydrogen fermentation commercialization. This is to ensure sufficient hydrogen energy to be generated to support the world energy crisis.