# Hydrogen from Depleted/Depleting Hydrocarbon Reservoirs: A Reservoir Engineering Perspective
## 3 Microbial H\({}_{2}\)-Bio-H\({}_{2}\) Generation in DORs  
### Concept  
The generation of H\({}_{2}\) from biomass and/or organic wastes through biological and chemical processes is a well-defined, commercially applied process [20, 21, 22]. The energy required for biochemical reactions that generate H\({}_{2}\) can be provided by solar power or by converting fluid carbon sources, or by a combination of both. The conversion of carbon sources involves microbial metabolism, and one of the related technologies is called dark fermentation (DF). DF refers to the degradation of organic substrates by anaerobic bacteria in an environment without a light source or oxygen to produce bio-H\({}_{2}\). The DF is industrially tested in up-scaled bioreactors, creating the required thermodynamic and physiological environments to generate optimum amounts of bio-H\({}_{2}\)[22, 23, 24]. Numerous studies have investigated the microorganisms with their origins and capacities in terms of DF under various conditions [25, 26, 27, 28, 29, 30]. Clostridiaceae, Methylotrophs, Enterobactericeae, and Thermoanaerobacterium are anaerobic or facultative genera of microorganisms commonly utilized for their superior H\({}_{2}\) production capacity [22, 28, 30, 31]. DF bacteria can decompose a variety of substrates (formic acid, lactic acid, cellulose disaccharide, sulfide, etc.) into H\({}_{2}\) under the action of nitrogenase or hydroenzyme. Raw materials such as food and agricultural wastes are said to trigger chemical reactions that produce H\({}_{2}\) at relatively competitive prices. However, the H\({}_{2}\) yield and production rate are low, presenting a significant challenge for large-scale industrial applications. Various publications discuss economic indicators for small DF units, and based on the provided data, the cost of H\({}_{2}\) production shows a wide range from 2.5 to 34 USD\({}_{2023}\) per kg of H\({}_{2}\), with daily production rates of up to 10 tons [21, 32, 33]. This cost range is considerably higher than the actual costs of any conventional H\({}_{2}\) production. Moreover, despite its low global warming potential, DF exhibits very poor energy and exergy efficiencies [33]. The main technical barriers in industrial applications under discussion are the development and operation of functional bioreactors and the thermodynamic limitations on the H\({}_{2}\) yield by microbial fermentation. The availability of raw materials and their costs are cited as the primary economic barriers to the economic potential of large-scale DF applications. The energy produced from glucose (or sucrose) is often cited as one of the primary and most effective examples of DF reactions:  
\[\text{C}_{6}\text{H}_{12}\text{O}_{6}+2\text{H}_{2}\text{O}\to 2\text{CH}_{3} \text{COOH}+4\text{H}_{2}+2\text{CO}_{2} \tag{1}\]  
This reaction involves fermentative microorganisms and produces two useful products: acetic acid (CH\({}_{3}\)COOH) and acetate, as well as H\({}_{2}\), after a certain incubation period. [PERSON] and [PERSON] have conducted an in-depth and critical review of various factors involved in the DF process [34, 35]. These factors include H\({}_{2}\) consumers, bacteria in different media, and inhibitory factors. The authors also provided a summary of the principal biochemical reactions that lead to bio-H\({}_{2}\) generation, and they presented the H\({}_{2}\) yield resulting from this mechanism, as reported in various studies [20; 25; 27; 28; 31; 36]. Table 2 presents the results of selected experimental works including the H\({}_{2}\) yield per type of carbohydrate used, the microorganisms involved, and the experimental conditions. The use of depleted and even depleting hydrocarbon reservoirs for bio-H\({}_{2}\) production is a derivative of the DF efforts discussed earlier at both the laboratory and industrial scales. It is well known that hydrocarbon reservoirs host various types of microorganisms as long as the thermophysical conditions allow this.