# Microbes and Parameters Influencing Dark Fermentation for Hydrogen Production
## 1 Introduction  
The depletion of fossil fuels due to their continuous consumption is attracting attention. These fossil fuels adversely affect the environment due to the release of greenhouse gases. The United Nations Sustainable Development Goals, 7 th and 13 th, aim to develop affordable and clean energy to meet energy needs. Thus, various alternatives are being evaluated to switch to environmentally friendly resources [1], as investing in renewable energy reduces the overall generation of carbon dioxide (CO\({}_{2}\)) [2]. Hydrogen (H\({}_{2}\)) is a clean and environmentally friendly fuel that can replace current fossil fuel resources in the future. It is estimated that the global H\({}_{2}\) market will reach up to USD 1.6 trillion by 2050 [3]. Investment in H\({}_{2}\) production has increased significantly. The United States is expected to become the largest producer of clean H\({}_{2}\) by 2030, accounting for 37% of the global supply. It is estimated that clean H\({}_{2}\) policies in Europe, Japan, and Korea alone can support up to 1.6 Mt of energy production by 2030 [4]. Hydrogen is a high-energy-density gas that does not release greenhouse gases during combustion. Still, conventional methods of H\({}_{2}\) production, such as water electrolysis, require temperatures of around 50-100 \({}^{\circ}\)C, which increases energy demands and relies on freshwater, a resource that is becoming increasingly scarce. Photoelectrolysis, although being presented as an effective approach for H\({}_{2}\) production, has low photolytic efficiency [5], using complexsystems with a short lifetime [6]. The production of H\({}_{2}\) using renewable resources is considered a sustainable and environmentally friendly approach. Organic wastes, such as sewage sludge, organic wastewater, and lignocellulosic, contain a huge amount of energy. It has been observed that each kilogram of chemical oxygen demand (COD) generates about 1.4\(\cdot\)10\({}^{7}\) kJ of metabolic heat [7]. Biological methods of H\({}_{2}\) production are environmentally friendly and theoretically require low energy demands, as they occur under ambient conditions. Biological processes are being scaled up around the world. Cemvita, a startup based in Texas, USA, is pioneering H\({}_{2}\) production through microbial degradation of residual oil hydrocarbons in depleted wells. The company aims to achieve H\({}_{2}\) production at a cost of USD 1 per kg [8]. These methods include photofermentation, biophotolysis, microbial electrohydrogenesis, dark fermentation, and combined system methods. Figure 1 illustrates the various biological methods utilized for H\({}_{2}\) generation. Photodetermination refers to the process through which bacteria, under light exposure, convert organic matter into H\({}_{2}\) gas. Purple non-sulfur bacteria can utilize a variety of substrates, including simple sugars, volatile fatty acids, and industrial and agricultural waste products, in the presence of light and anaerobic conditions. These bacteria have been employed in numerous photobioreactors (PBRs) for the enhancement of H\({}_{2}\) production [9]. The genera _Rlodobacter_, _Rlodobium_, _Rlodoseudomonas_, and _Rlodospirillum_ are among the microbes involved in photofermentation. They convert organic acids into H\({}_{2}\) and CO\({}_{2}\)[10]. Biophotolysis is another biological light-dependent method for H\({}_{2}\) production. It entails capturing and transforming light energy into H\({}_{2}\) via photosynthesis, during which microbes split water molecules. There are two microbial photosynthesis pathways, one comprising microalgae that utilize a hydrogenase enzyme and the other involving cyanobacteria (blue-green algae), which employ a nitrogenase enzyme and sometimes a hydrogenase enzyme. This process creates an anaerobic environment that is necessary to sustain H\({}_{2}\) production [11]. In direct biophotolysis, the natural splitting of water molecules during photosynthesis is facilitated by the action of light.