**1. Introduction**

Hydrogen technologies, combined with sector coupling, can decarbonize sectors that cannot be electrified (e.g., the chemical industry, steel industry, etc.) [1].

In 2030, hydrogen will account for 0.5% of the electricity mix, according to studies by DNV. In 2050, DNV expects it to be 5%. However, according to the Paris Agreement, at least 15% hydrogen is required in the electricity mix [1]. Yet, expanding renewable energy and electrolysis technologies at the necessary pace will be nearly impossible. Both renewables and electrolysis-ready water are critical resources that could lead to supply insecurities [2].

For this reason, we need new technologies for the production of hydrogen in the course of decarbonization.

#### **1.1 Biological hydrogen as an alternative to electrolysis**

In 2021 bioenergy covered about 55 % of global renewable energy supply (6 % of total energy supply) which makes it the largest source of renewable energy [3]. According to Net Zero Emissions by 2050 the use of bioenergy will increase rapidly by 2030 [4].

The conversion of biomass can take place *via* three routes: thermo-chemical (pyrolysis, gasification), physicochemical (e.g., chemical conversion), or biochemical (anaerobic fermentation, alcoholic fermentation). All three approaches can produce gaseous and liquid fuels, with the first two having high energy requirements. Biochemical transformation, on the other hand, proceeds essentially in a self-determined manner and with a lower external energy requirement. [5, 6]

A general advantage of biomass conversion is the sensible use of biological waste products (e.g., from agriculture) [6]. A significant disadvantage is the current inefficiency of the hydrogen production method [7]. The resulting biogas contains only small amounts of hydrogen since, in most cases, hydrogen is directly decomposed to form methane [8].

This effect can be reduced by so-called dark fermentation, an anaerobic fermentation without light. Methanogenesis is suppressed using bacterial cultures in which few hydrogen-consuming microorganisms (MOs) are present [8]. The process's main advantages are the simple reactor configuration and the ability to produce hydrogen continuously regardless of ambient conditions.

#### **1.2 The efficiency of hydrogen** *via* **dark fermentation**

Dark fermentation has not yet been investigated on a large scale at the current stage. Laboratory studies suggest substantial production variations depending on inoculum, substrate, and substrate pretreatment. Values between 27 L H2/kg TVS (Total Volatile Solids) [9] and 125 L H2/kg TVS [10] were measured for continuously operated biogas plants. This corresponds to a maximum of one-third of converted initial biomass [11].

#### **1.3 Challenges in optimizing biological processes**

Efficiency assessment is essential, especially for sector coupling processes, but it is also difficult. Depending on the (sub)process of the overall process under consideration, there are various methods and key figures for efficiency evaluation. This makes a holistic, objective comparison of the impossible processes. However, this is indispensable if the optimizability of the overall process is to be determined [12]. While the efficiency of purely technical processes can at least be described thoroughly, biological (sub)processes are even more difficult to describe due to their complexity. Biological processes can hardly ever be considered completely isolated since they usually interact with many other processes and are thus dependent on them. On the one hand, this makes it challenging to draw balance boundaries. On the other hand, not all interaction partners are known, so a complete consideration of the isolated process is impossible. Thus, a comprehensive efficiency assessment of these processes is (almost) impossible, even if the state of knowledge yields new information.

Therefore, this chapter develops a model to describe dark fermentation's physical optimum (PhO). Based on this model, the efficiency potentials of the process can be determined, and recommendations for improvement can be derived. An in-depth understanding of the process is required for model development. For this reason, the anaerobic degradation of biomass is described first. The dark fermentation process is then mapped using Petri nets (PNs) to create an idealized comparison process for evaluating existing processes.

## **2. Theoretical basics of dark fermentation**

In biogas plants, biomass is converted into biogas by bacterial decomposition. Primarily methane (60% [13]) and carbon dioxide (35% [13]), but also hydrogen is produced. The biochemical conversion of biomass to biogas is divided into four reaction stages: hydrolysis, acidogenesis, acetogenesis, and acetylation [5, 13].

Dark fermentation, the anaerobic degradation of biomass without light, is the term for hydrogen formation. Dark fermentation is thus identical to anaerobic digestion up to hydrogen formation (acetogenesis). Only methanogenesis is inhibited in dark fermentation [14]. In an actual biogas plant, the degradation process does not simply stop after hydrogen formation. Instead, the resulting H2 is further converted to methane with CO2. Accordingly, this step must be eliminated to increase the hydrogen yield.

#### **2.1 Degradation step 1: Hydrolysis**

During hydrolysis, the complex biomass is enzymatically split into its monomers. This process divides complex carbohydrates into monosaccharides, primarily glucose, by amylases (Eq. 1 [5, 15]).

$$n\left(\mathrm{C}\_{6}H\_{10}O\_{5}\right)\_{n} + nH\_{2}O \to n\mathrm{C}\_{6}H\_{12}O\_{6}\tag{1}$$

Proteins are cleaved by extracellular proteases into shorter peptide chains (di- and tripeptides) and amino acids (AA) (**Figure 1** [15]). Their degradability depends on their AA sequence, hydrophobicity, and the milieu conditions (especially acidity).

The peptides are subsequently cleaved intracellularly into AA. These are used for protein synthesis and energy production of the MO [16]. Lipids are cleaved by lipases into fatty acids and glycerol (**Figure 2** [17, 18]). This degradation is done by anaerobic bacteria such as *Bacteroides*, Clostridia, or Bifidobacteria [19, 20].

#### **2.2 Degradation step 2: Acidogenesis**

The monomers are converted into even simpler components in the second degradation step. Here, AA and monosaccharides are metabolized, and long-chain saturated

**Figure 1.** *Protein hydrolysis reaction scheme [15].*

**Figure 2.** *Reaction scheme of lipid hydrolysis.*

fatty acids (LCFA, e.g. C4 to C18 by *Syntrophomonas* spp. [21]) are converted by βoxidation [17, 22] by facultative and anaerobic acidogenic bacteria such as *Bacteroides*, *Clostridium*, *Butyribacterium*, *Propionibacterium*, *Klebsiella*, *Escherichia, Syntrophomonas,* and *Thermoanaerobacterium* to various organic acids (OAs) such as propionic or butyric acid, but mainly to acetic acid (**Figure 3**) [13, 20, 24, 25]. Carbon dioxide, water, and hydrogen are also formed as waste products [13].

The glycerol is converted in the glycolysis process at which hydrogen is produced [17]. Protein-rich substrates yield exceptionally high levels of butyric and valeric acids [13]. These are released as the carbon residue of the AA after deamination or transamination (loss of the amino group). The amino group is separated as an ammonia molecule at the end of the metabolic pathway. It is, therefore, the main product of protein catabolism [16].

#### **2.3 Degradation step 3: Acetogenesis**

In the third degradation step, the metabolic products of acidogenesis, mainly propionic and butyric acids, but also ethanol (Eq. 2 [19]) and lactate (eq. 3 [19]), are further degraded by anaerobic oxidation.

$$\text{H}\_3\text{C}-\text{CH}\_2-\text{OH} + \text{H}\_2\text{O} \rightarrow \text{H}\_3\text{C}-\text{COOH} + \text{H}\_2\tag{2}$$

$$\text{H}\_3\text{C}-\text{HCOH}-\text{COOH} + \text{H}\_2 \rightarrow \text{H}\_3\text{C}-\text{CH}\_2-\text{COOH} + \text{H}\_2\text{O} \tag{3}$$

Butyric and other longer OAs are degraded according to the same principle during β oxidation (Eq. 3 [26]). Propionic acid is metabolized to acetic acid (Eq. 4) [13, 19].

$$\text{H}\_3\text{C}-\text{CH}\_2-\text{COOH} + \text{3H}\_2\text{O} \rightarrow \text{H}\_3\text{C}-\text{COOH} + \text{CO}\_2 + \text{H}\_2\text{O} + \text{3H}\_2\tag{4}$$

However, the butyrate degradation to acetate and H2 is, under standard conditions, an endergonic reaction [27]. For this reaction to occur, hydrogen elimination is

**Figure 3.**

*Various fermentation reactions with glucose as a reactant and various organic acids as products [23].*

*Optimizability of Biogenic Hydrogen Production DOI: http://dx.doi.org/10.5772/intechopen.111853*

strictly necessary. By forming so-called syntrophic communities consisting of hydrogen producers (e.g., Clostridium spp.) and hydrogen consumers (e.g., Methanoculleus spp.), the metabolization of butyrate to methane can be achieved despite being thermodynamically unfavorable.

Similarly, lactate degradation can be performed by several members of the genus Clostridium, resulting in the formation of acetate, propionate, and carbon dioxide. In sulfate-limited and hydrogen-consuming milieus, Desulvovibrio vulgaris is furthermore able to generate H2, besides acetate and CO2, from lactate, making it a desirable species for dark fermentation [28]. Additionally, lactate seems to be cometabolized by syntrophic acetate oxidizers such as Syntrophaceticus schinkii [29].

#### **2.4 Degradation step 4: Methanogenesis**

In the last fermentation step, methane is formed. There are two possible metabolic pathways.

On the one hand, there are hydrogenotrophic methanogenic MO, such as *Methanoculleus*, which produces methane from hydrogen and carbon dioxide (Eq. 5 [30]). On the other hand, there are acetoclastic methane producers, such as Methanosaeta, which form methane based on acetate (Eq. 6 [20, 30]. The hydrogenotrophic methane formation is energetically preferred.

$$4H\_2 + CO\_2 \rightarrow CH\_4 + CO\_2\tag{5}$$

$$\text{CH}\_3\text{C}-\text{COOH} \rightarrow \text{CH}\_4 + \text{CO}\_2 \tag{6}$$

This changes as soon as the critical hydrogen partial pressure is below a certain threshold. A peculiarity is that the transforming MO is now exclusively Archeans, in contrast to the previous bacteria [19]. These exhibit two temperature optima: at 32– 42°C in the mesophilic [20] and at 48–55°C in the thermophilic range [20], as well as a pH optimum at pH 7 to 7.5 [13, 20]. If the pH value falls below a critical threshold, e.g., to pH 6.5, short OAs and acetate accumulate because methanogenesis is inhibited, further lowering the pH [20].

Archaea activity shows a stronger inhibition by lower pH values than bacteria in the previous degradation steps [13].

#### **2.5 Inhibition of methanogenesis in favor of hydrogen production**

Methanogenesis is the final step in the anaerobic degradation of biomass. In this step, methane is formed from H2 and CO2 on the one hand (hydrogenotrophic) and from acetate on the other (acetoclastic). Since hydrogenotrophic methane formation is H2-consuming, this step must be inhibited for a higher hydrogen yield and the H2 producing reactions must be enabled [20, 30].

Hydrogen partial pressure is the most crucial factor influencing H2 formation and consumption. According to Ahlert, this must be below 50,66 Pa [19] so that H2 can be formed. According to Cazier, a value of 101,33 Pa [31] is sufficient to inhibit acetogenesis. A higher value inhibits production [13, 24]. At the same time, methanogenesis is inhibited by a low H2 partial pressure (below 6,48 Pa [19]). Then, acetoclastic methanogenesis is favored instead of hydrogenotrophic methanogenesis, so no H2 is consumed but acetate.

Another possibility to inhibit hydrogenotrophic methanogenesis is the removal of CO2 from the reactor air [31]. However, this does not solve the problem of H2 formation inhibition when the H2 partial pressure becomes too high, so this method should be used as a supplement, but not alone.

Two other important influencing factors are temperature and pH. Methanogenic MO is archaea, which are more susceptible to stress than bacteria. They can better withstand temperature fluctuations and survive somewhat lower pH values, whereas methanogenic archaea can withstand higher pH values. Methanogenic archaea are strongly inhibited at a pH below seven, already in the slightly acidic range [13, 19, 20]. Therefore, to inhibit methanogenesis, a slightly acidic pH value such as 6.5 would be advantageous for inhibiting methanogenesis.

The low pH must not be caused by LCFA enrichment, as this inhibits hydrolysis and acidogenesis in addition to methanogenesis, which is essential for H2 formation. Methanogenic MO is the first to be inhibited by LCFA accumulation, starting at 1.0– 2.9 kg COD/m3 biomass [32]. Acidogenic MO is inhibited from 2.1–7.9 kg COD/m<sup>3</sup> biomass [32] and hydrolytic ones only from 2.6–9.4 kg COD/m3 biomass [32]. Thus, if the concentration of LCFA is between 1.0–2.1 kgCOD/m3 biomass, methanogenesis should be inhibited, not the other two degradation steps.
