# Sustainable production of biohydrogen: Feedstock, pretreatment methods, production processes, and environmental impact

Aleksandra Modzelewska, Mateusz Jackowski, Panagiotis Boutikos, Magdalena Lech, Maciej Grabowski, Krystian Krochmalny, María González Martínez, Christian Aragón-Briceño, Amit Arora, Hao Luo, et al.

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## ► To cite this version:

Aleksandra Modzelewska, Mateusz Jackowski, Panagiotis Boutikos, Magdalena Lech, Maciej Grabowski, et al. Sustainable production of biohydrogen: Feedstock, pretreatment methods, production processes, and environmental impact. Fuel Processing Technology, 2024, 266, pp.108158. 10.1016/j.fuproc.2024.108158. hal-04805068

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https://imt-mines-albi.hal.science/hal-04805068v1

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# Sustainable production of biohydrogen: Feedstock, pretreatment methods, production processes, and environmental impact

Aleksandra Modzelewska<sup>a</sup>, Mateusz Jackowski<sup>a</sup>, Panagiotis Boutikos<sup>b</sup>, Magdalena Lech<sup>a</sup>, Maciej Grabowski<sup>a</sup>, Krystian Krochmalny<sup>c</sup>, María González Martínez<sup>d</sup>, Christian Aragón-Briceño<sup>e</sup>, Amit Arora<sup>f,g</sup>, Hao Luo<sup>h</sup>, Luca Fiori<sup>i</sup>, Qingang Xiong<sup>j</sup>, Muhammad Yousaf Arshad<sup>k</sup>, Anna Trusek<sup>a</sup>, Halina Pawlak-Kruczek<sup>c</sup>, Lukasz Niedzwiecki<sup>i,l,*</sup>

## ARTICLE INFO

### Keywords:

Biohydrogen

Biomass

Sustainability

LCA

## ABSTRACT

A significant increase in the use of hydrogen, expected to reach between 667 and 4000 TWh, is forecasted for the whole EU in 2050. Electrolysis is believed to be a "silver bullet" due to its synergy with the needs of the grid. However, biohydrogen generation could be complimentary to electrolysis since it does not depend on electricity prices. This review presents a comprehensive picture of the landscape in biohydrogen production, showing state-of-the-art research on different biohydrogen production processes and highlighting potential problems and shortcomings for different processes, including microbial-based production and thermal processes. The work shows that "colour coding" used nowadays is insufficient in terms of providing accurate information regarding the sustainability of particular biohydrogen production technologies. Instead, LCA can provide substantial information for each investigated process. However, there is a need for a wider scope of LCA studies since currently published studies present a syndrome of "carbon tunnel vision", often ignoring impacts other than global warming. Moreover, studies often tend to exclude the impact of capital goods production, which might provide an incomplete overview of such technologies. Moreover, it should not be overlooked that biohydrogen is capable of achieving negative values of CO₂ emissions if CCS is implemented.

## 1. Introduction

One of the challenges for mankind in the upcoming decades will be the decarbonisation of the world's economy and decreasing greenhouse gases emissions, as outlined in the Paris Agreement. In order to achieve

such a goal, it will be necessary to increase the share of renewable energy sources in the energy system [1]. In that context, any fuel that does not emit greenhouse gases during energy conversion becomes increasingly important. Moreover, reducing the use of fossil fuels will require transitions in sectors other than energy generation, such as the synthesis

<footnote><sup>a</sup> Department of Micro, Nano and Bioprocess Engineering, Faculty of Chemistry, Wroclaw University of Science and Technology, 50-373 Wroclaw, Poland</footnote>

<footnote><sup>b</sup> Foundation for Research and Technology Hellas, Institute of Chemical Engineering Sciences (FORTH/ICEHT), Stadiou Str, Platani Rion, 26504 Patras, Greece</footnote>

<footnote><sup>c</sup> Department of Energy Conversion Engineering, Faculty of Mechanical and Power Engineering, Wroclaw University of Science and Technology, Wyb. Wyspiańskiego 27, 50-370 Wroclaw, Poland</footnote>

<footnote><sup>d</sup> Université de Toulouse, Mines Albi, UMR CNRS 5302, Centre RAPSODEE, Campus Jarlard, Route de Teillet, 81000 Albi, France</footnote>

<footnote><sup>e</sup> Industry and Energy Department, CIRCE-Research Centre for Energy Resources and Consumption, Dinamiza Business Park, Ranillas Avenue, Building 3D, 1st Floor, 50018, Zaragoza, Spain</footnote>

<footnote><sup>f</sup> Department of Chemical Engineering, National Institute of Technology Hamirpur, Himachal Pradesh 177005, India</footnote>

<footnote><sup>g</sup> Department of Chemical Engineering, Shaheed Bhagat Singh State University, Punjab, Ferozepur 152004, India</footnote>

<footnote><sup>h</sup> School of Chemistry and Chemical Engineering, Hubei Key Laboratory of Coal Conversion and New Carbon Materials, Wuhan University of Science and Technology, Wuhan, Hubei 430081, China</footnote>

<footnote><sup>i</sup> Department of Civil, Environmental and Mechanical Engineering (DICAM), University of Trento, Via Mesiano 77, 38123 Trento, Italy</footnote>

<footnote><sup>j</sup> State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China</footnote>

<footnote><sup>k</sup> School of Chemical Engineering, University of Adelaide, Adelaide SA 5005, Australia</footnote>

<footnote><sup>l</sup> Energy Research Centre, Centre for Energy and Environmental Technologies, VSB-Technical University of Ostrava, Czechia</footnote>

<footnote>* Corresponding author at: Department of Civil, Environmental and Mechanical Engineering (DICAM), University of Trento, Via Mesiano 77, 38123 Trento, Italy. E-mail address: lukasz.niedzwiecki@unitn.it (L. Niedzwiecki).</footnote>
of chemicals and the production of steel, just to name a few. Hydrogen
can be use used for such purposes.

Currently, the EU uses hydrogen, equivalent to 339 TWh of its chemical energy, with the vast majority generated on-site in “large industry settings” [2]. Reforming (mainly steam reforming and, to some extent, autothermal reforming) is dominant and accounts for 95 % of the hydrogen produced in the EU [2]. A significant increase in the use of hydrogen, expected to reach between 667 and 4000 TWh, is forecasted for the whole EU in 2050 [2]. Internationa Energy Agency (IEA) indicates that global use of hydrogen will rise from 95 Mt. (data from 2022) to approx. 150 Mt. by 2030 [3].

Therefore, it seems plausible to expect the production of hydrogen from biomass to become complementary to hydrogen being produced through electrolysis since biohydrogen production exhibits comparably lower consumption of electricity. This trend could take place in the more general effort required for the substantial advancement of modern bioenergy [4].

Performed studies found that the energy requirement for electric pyrolysis could be as low as 79.37 MJ/kgH₂ [5]. This could be easily replaced with conventional pyrolysis, using other heat sources than electricity. This is a significant improvement in comparison to values reported in the literature for various electrolyzers. Stack energy consumption could range between 170.3 and 239.2 MJ/kgH₂ for alkaline electrolyzers, as reported by Carmo et al. [6], whereas system energy consumption for such solution could reach values between 182.4 and 283.8 MJ/kgH₂ [6]. As for PEM electrolyzers, stack and system energy consumption could range between 170.3 and 227.0 MJ/kgH₂ and 182.4 and 304.1 MJ/kgH₂, respectively [6]. Some LCA analyses for PEM electrolyzers assumed 198.0 MJ/kgH₂ and 162.0 MJ/kgH₂, as values typical nowadays and in 2030 [7], respectively. Energy consumption at the stack for H₂ production was reported by Karayel et al. [8] to be 178.4 MJ/kgH₂, 182.4 MJ/kgH₂, and 133.8 MJ/kgH₂ for alkaline, PEM, and solid oxide electrolyzer, respectively. There are ongoing efforts aiming at decreasing electricity consumption for electrolysis. However, such installations will still use electricity nonetheless, which always brings a penalty related to the efficiency of electricity generation. Thus, direct use of heat for hydrogen production could still be beneficial in comparison to the conversion of such heat to electricity and subsequent electrolysis. One could argue that electricity from intermittent sources, such as solar and wind, could be used for electrolysis, thus preventing

the wasting of energy during times of high supply from wind and solar,
coinciding with low demand from the grid. In such a sense, electrolysis
would become a solution for energy storage, and H₂ would become a
storage medium. However, a simple look at the load of the electric grid
and supply from different energy sources in the EU (Fig. 1), taking into
account both intermittent and controllable sources, reveals that such a
use of a high supply from wind and solar is likely to limit the number of
annual hours of operation of the system, thus heavily influencing the
economics of this solution. Conversely, increasing the operation of
electrolysis to 8000 h per year or more would require the use of energy
currently present in the mix (including both nuclear and fossil).
Furthermore, generating H₂ using excess electricity from intermittent
sources could, in many cases, require reliable long-term H₂ storage. This
is still an issue despite intensive investigations on hydrogen storage [9].

Interestingly, a multitude of different types of waste biomass is available worldwide, including woody biomass [10,11], sewage sludge [12–14], digestates [15,16], organic fraction of municipal solid waste [17], residues from food production, such as meat and bone meal [18], multitude of different energy crops, including those grown for phytor-emediation purposes [19], agricultural residues [20], different types of aquatic biomass [21], to name a few. The most common approach is to use waste biomass as an energy source [22–24]. However, since biomass contains hydrogen as one of its building blocks, it might very well be used as a feedstock for the production of renewable hydrogen. In such a case, the production of hydrogen on-demand is possible, as biomass itself can be considered a form of storage.

Due to these reasons, it seems plausible to suspect that in the fore-
seeable future, hydrogen will not be produced exclusively by electrol-
ysis, but there will be competition between different technologies, with
many different solutions being present on the market, including bio-
hydrogen [3].

Therefore, the scope of this work is a review of different possible
routes of production of biohydrogen presented in the literature so far,
taking into account the complete value chain from feedstock to purified
hydrogen.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Public net electricity generation in the European Union in 2024, with a closeup to week 13, showing the effect of abrupt change in supply from intermittent RES (RES – Renewable Energy Sources) [25]. Note: Pumped storage essentially generates electricity using the same technology as hydro but could be considered renewable only if it stores excess supply from RES.</figcaption></figure>
## 2. Production methods of biohydrogen

### 2.1. Photo-fermentation

Photo fermentation is observed among anoxygenic photosynthetic bacteria (Fig. 2). The principle of photo fermentation is to digest organic substances into H₂. The main advantages of such a process are the achievable purity of the product (see Table 1) and the possibility of using solar energy directly without the need to convert it to electricity. Moreover, a synergy can be found between dark fermentation and photo-fermentation as the post-fermentation broths after the former can be reduced during the latter by as much as 82 % in terms of COD (Chemical Oxygen Demand) [26].

The process is limited by the activity of nitrogenase, the enzyme that catalyzes the reduction of N₂ to ammonia, which is represented by eq. (1):

$$N_2 + 8H^+ + 8e^- + 16ATP \rightarrow 2NH_3 + H_2 + 16ADP + 16Pi \quad (1)$$

During enzyme activity, the processing of 1 mol of N₂ leads to the production of 1 mol of H₂. Under scarcity of nitrogen, the above-mentioned enzyme acts in a different manner. Nitrogenase acts as hydrogenase and catalyzes the reaction described in eq. (2):

$$2H^+ + 2e^- + 4ATP \rightarrow H_2 + 4ADP + 4Pi \quad (2)$$

In that reaction, protons are reduced to achieve one mole of H₂ at the expense of 4 mol of ATP [27].

The main advantages of photofermentation are:

* Utilisation of an easily available source of energy, which is light [28].

* Fixation of atmospheric nitrogen during photofermentation [29].

* Numerous raw materials may be used as feedstock for biohydrogen production [30].

The disadvantages of photofermentation are:

* Oxygen inhibits nitrogenase activity. Thus, the removal of oxygen is necessary for proper photofermentation [29].

* The requirement of light limits production possibilities to daytime, making continuous processes difficult without additional support of energy [28]. However, this could be fixed by using excess electricity from wind farms during nighttime for the generation of artificial light to sustain the process since there is typically an off-peak period in the middle of the night (see Fig. 1).

* The colour and the turbidity of the cultivation medium may negatively affect its penetration by light, therefore lowering the efficiency of the process [31].

Photofermentation was reported among numerous microorganisms. So far, the process has been conducted using Rhodobacter bacteria. For

instance, Rhodobacter sphaeroides reached 2.24 L$_{H_2}$/L when used for fermentation of brewery wastewater [32]. Mixed fermentation using Clostridium butyricum and Rhodopseudomonas palustris resulted in 840 mL$_{H_2}$/g production when using cassava starch and 463 mL$_{H_2}$/g for rice straw [29,33]. Cofermentation using Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodopseudomonas palustris with potato residue as a feedstock resulted in hydrogen production of 77.78 mL$_{H_2}$/L·h [34]. The use of Rhodobacter capsulatus DSM155 and B10 strains resulted in efficiencies of volatile fatty acids conversion ranging between 14.66 and 23.11 %, depending on the strain and composition of the medium [35]. Various values of light conversion efficiency (LCE) have been reported in the literature, depending on different process conditions and microorganisms used. Chookaew et al. [36] demonstrated the effect of glutamate concentration on H₂ production by an anoxygenic photosynthetic bacteria Rhodopseudomonas palustris TN1 using dark fermentation effluent as a feedstock under anaerobic-low light condition (3000 lx) after 192 h of cultivation with maximum LCE of 1.43 % [36]. Rodríguez-Valderrama et al. [35] reported LCE ranging between 2.67 and 7.47 %. Jiang et al. [37] have shown that LCE can be improved by as much as 114.7 % by alternating the light and dark cycles (24 h dark +24 h light) in comparison to LCE obtained during continuous light conditions. Zhang et al. [38] achieved an LCE of 30.67 % using HAU-M1 photosynthetic bacteria in their exponential growth phase, immobilized in a gel containing SnO₂ nanoparticles, using corn cobs as a feedstock. However, the energy recovery efficiency reported by Zhang et al. [38] was 2.44 %.

### 2.2. Biophotolysis

Bio-photolysis, a widely adopted method employing cyanobacteria and blue-green algae, harnesses photon energy to produce biohydrogen. It operates on a principle akin to photosynthesis in plants and can be classified into two main categories: direct bio-photolysis (Fig. 3) and indirect bio-photolysis (Fig. 4).

Direct bio-photolysis involves photosynthesis, in which algae and cyanobacteria break the water into hydrogen and oxygen. Within the chloroplasts of microalgae, Photosystem I (PSI) and Photosystem II (PSII) serve as the primary sites for photosynthetic reactions, absorbing photons to generate a potent oxidant used in the water oxidation process, producing oxygen (O₂), electrons, and protons. The reduction of a proton by an electron, supplied by the reduced ferredoxin (Fd) enzyme within the cells, leads to the formation of hydrogen. In blue-green algae [FeFe], hydrogenase plays a crucial role in H₂ production. The mentioned enzyme is strongly O₂ sensitive, making the process vulnerable to oxygen. Therefore, hydrogen production is limited to the narrow time gap between illumination and accumulation of inhibiting concentration of oxygen [30,39–41].

There are a few methods to overcome such disadvantage:

<figure><img src="image_5.png" /><figcaption>Fig. 2. Simplified scheme of photofermentation process.</figcaption></figure>
<table><caption>**Table 1**
Hydrogen production via biophotolysis and photofermentation for various feedstocks, reactors and organism types.</caption><thead><tr><th>Substrate</th><th>Culture</th><th>Reactor</th><th>Process conditions</th><th>H<sub>2</sub> product.</th><th>% H<sub>2</sub></th><th>Ref.</th></tr></thead><tbody><tr><td>Potato residue (9.48 g/L sugars)</td><td>PSB HAU-M1 (Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palustris)</td><td>Batch Photofermentation</td><td>pH 7<br>30 °C<br>3000 Lux</td><td>77.78 mL H<sub>2</sub> / (L·h)</td><td>37</td><td>[34]</td></tr><tr><td>Glycerol</td><td>Klebsiella sp. TR17 (dark fermentation)<br>Rhodopseudomonas palustris TN1 (photofermentation)</td><td>Batch Coupling of photofermentation and dark fermentation</td><td>40 °C<br>3000 Lux</td><td>6.42 mmol<sub>H2</sub> /g<sub>COD</sub><br>(0.68 mmol<sub>H2</sub> /g<sub>COD</sub> for photofermentation only)</td><td>N.D.</td><td>[36]</td></tr><tr><td>Tequila vinasses (COD 22085 mg/L)</td><td>Rhodopseudomonas pseudopalustris DSM 123</td><td>Batch Photofermentation</td><td>30 °C<br>Light 13.5 W/m<sup>2</sup></td><td>0.65 mmol<sub>H2</sub> /L·h</td><td>Up to 99.1</td><td>[45]</td></tr><tr><td>Beet molasses (51 % sugars)</td><td>Rhodobacter capsulatus JP91</td><td>Batch Photofermentation</td><td>30 °C<br>Light 200 W/m<sup>2</sup></td><td>10.5 mol<sub>H2</sub> /mol<sub>sucrose</sub></td><td>N.D.</td><td>[46]</td></tr><tr><td>Black strap molasses (65 % sugars)</td><td>Rhodobacter capsulatus JP91</td><td>Batch Photofermentation</td><td>30 °C<br>Light 200 W/m<sup>2</sup></td><td>8 mol H<sub>2</sub>/mol sucrose</td><td>N.D.</td><td>[46]</td></tr><tr><td>Distillery wastewater (5.0–5.8 g/L glucose)</td><td>Rhodobacter capsulatus B10 and Rhodobacter sphaeroides B-3059</td><td>Batch photofermentation</td><td>pH > 5.0<br>30 °C<br>Light 30 W/m<sup>2</sup></td><td>Up to 12 mL<sub>H2</sub>/ml of wastewater</td><td>Up to 60</td><td>[47]</td></tr><tr><td>Van Niel's Yeast Broth with 5 M glycerol</td><td>Rhodobacter palustris NCIMB 11774</td><td>Column photobioreactor</td><td>35–38 °C<br>Light 100 W/m<sup>2</sup></td><td>Approx. 2500 mL<sub>H2</sub>/L</td><td>N.D.</td><td>[48]</td></tr><tr><td>Media containing acetic and butyric acids</td><td>Rhodobacter capsulatus DSM155 and B10 strains</td><td>Batch</td><td>25 °C, light intensity 10, 20, and 30 klux</td><td>1.95–2.27 mol<sub>H2</sub> /mol<sub>VFA</sub> consumed</td><td>N.D.</td><td>[35]</td></tr><tr><td>TAP medium</td><td>Chlamydomonas reinhardtii mutants</td><td>Batch biophotolysis</td><td>25 °C<br>Light 15.1 W/m<sup>2</sup></td><td>Up to 13.80 L/kg·h</td><td>N.D.</td><td>[49]</td></tr><tr><td>Crude glycerol (COD 1,884,600 mg/L, total N 1149 mg/L, P 336 mg/L)</td><td>Chlorella vulgaris TISTR 8580<br>Chlorella protothecoides TISTR 8243</td><td>Batch biophotolysis</td><td>pH 6.8<br>PPFD 48 mol<sub>photon</sub> /m<sup>2</sup>·s</td><td>Up to 1.084 mmol/L·h</td><td>20–44 %</td><td>[50]</td></tr></tbody></table>

COD – chemical oxygen demand; N.D. – not defined; PPFD – Photosynthetically active photon flux density: the amount of photosynthetic active photons in µmol striking a surface of 1 m² in 1 s; TAP – Tris-acetate-phosphate.

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Simplified diagram of direct biophotolysis.</figcaption></figure>

* Use of oxygen absorbers in order to bind produced oxygen

* Purging the mixture with an inert gas like N₂

* Reduction of sulfur in the cultivation medium

Indirect bio-photolysis starts with the binding and accumulation of CO₂. This step is done by standard photosynthesis. In the next step, carbohydrates created during photosynthesis are fermented with the release of H₂ and CO₂. Through this mechanism, cyanobacteria and microalgae can produce H₂ from stored glycogen, effectively addressing the challenge of oxygen sensitivity by temporarily separating the H₂ and O₂ evolution processes into two distinct steps, with the involvement of CO₂ evolution or fixation. In the initial phase, microalgae are cultivated under standard aerobic conditions, allowing them to generate lipids,

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Simplified scheme of indirect biophotolysis.</figcaption></figure>

carbohydrates, and other organic compounds through the conversion of solar energy and CO₂ fixation. The transition to the anaerobic phase occurs when O₂ levels are depleted, halting the electron transfer chain. In Photosystem I (PSI), electrons derived from endogenous substrates via catabolic processes in the citric acid cycle and glycolytic pathway are utilized for non-photochemical reduction of the PQ pool through light-dependent ferredoxin reduction. Eventually, this process leads to the production of hydrogen as the electrons are transferred to [Fe-Fe] hydrogenases [42–44]. Table 1 shows hydrogen production by biophotolysis and photofermentation for various feedstocks, reactors and organism types.

## 2.3. Dark fermentation

Dark fermentation (DF) is a form of biological hydrogen production. Dark fermentation is carried out by obligate anaerobic bacteria and facultative anaerobic bacteria in the absence of light and oxygen. In dark
fermentation, bacteria act on the substrate to produce hydrogen. During DF, microorganisms convert organic and inorganic substrates to produce the energy needed for the vital processes of the cells. The main extracellular products of such a process are hydrogen, volatile fatty acids and alcohols that are valuable from the energy point of view [51,52]. Preparing the inoculum helps isolate specific dark fermentative bacteria by removing other microorganisms that could interfere with the fermentation process [53]. If methanogens are not present in the culture, the produced gas consists of hydrogen and carbon dioxide [54,55]. Nevertheless, the ratio between hydrogen and carbon dioxide varies and depends on fermentation conditions, substrate utilized, microorganisms applied, salts supplementation, etc. Compared to methanogenic fermentation, the final yield of gas produced via dark fermentation is lower because carbon atoms that would typically form methane and carbon dioxide instead turn into short-chain carboxylic acids (mainly acetic and butyric acids) in the liquid waste [56].

A multitude of different microorganisms, including mixed cultures, as well as selected species, have been used so far for dark fermentation, including *Bacillus macerans*, *Clostridium butyricum*, *Clostridium beijerinckii*, *Enterobacter asburiae* and *Enterobacter aerogenes*, with positive effects on hydrogen productivity. In terms of enzymes, hydrogenase and nitrogenase are the two enzymes involved in the acetate and butyrate hydrogen-producing pathways. Theoretically, 1 mol of glucose yields 4 mol of hydrogen in the acetate pathway [57]. The butyrate pathway produces 2 mol of hydrogen from 1 mol of glucose [57], as shown by the following reaction eq. (3):

$$C_6H_{12}O_6 + 2H_2O \rightarrow 2CH_3COOH + 2CO_2 + 4H_2 \quad (3)$$

In this reaction, glucose (a common substrate) is converted into $H_2$, $CO_2$, and acetic acid. However, the actual biochemical pathways are more complex and involve multiple intermediate compounds and microbial communities [58]. In many cases, acidogenesis is also taken into account in the modelling of dark fermentation [59].

*Clostridium* is one of the most often mentioned groups of hydrogen-producing bacteria. Its ability to form protective spores seems to be an important advantage of *Clostridium*. Protective spores provide *Clostridia* with substantial chances of survival in harsh conditions, including extreme temperatures, low or high pH, and the presence of detrimental chemicals. Another characteristic of *Clostridia* is the lack of cytochrome. Therefore, inoculum containing *Clostridium* can be pre-treated with heat, pH or chemicals to increase the rate of $H_2$ production and get rid of other unwanted microorganisms. The most efficient hydrogenic bacteria from this genus include *Clostridium kluyveri*, *Clostridium butylicum*, *Clostridium pasteurianum*, and *Clostridium butyricum* [60,61].

Similarly to *Clostridium*, *Bacillus* are gram-positive, rod-shaped bacteria capable of producing $H_2$ by fermentation of organic acids present in a fermentation broth. The species most commonly used for $H_2$ production using the DF process include *Bacillus cloacae*, *Bacillus macerans* (and *acetoethylicus*), *Bacillus polymyxa*, and *Bacillus licheniformis* [62]. The hydrogen yield using *Bacillus licheniformis* can reach 0.37 and 1.1 mol of $H_2$ per 1 mol of glucose in batch and continuous mode, respectively [63].

*Enterobacteriaceae* is a family of bacteria that grow anaerobically or aerobically, depending on the pH. These organisms have high air resistance. Although oxygen blocks the growth of the bacterial biomass, it does not reduce the hydrogen yield. *Enterobacter* are, therefore, often applied to mixed cultures that are more sensitive to oxygen. They are less commonly used in monoculture than *Clostridium* because of the lower hydrogen yield [60]. The most commonly used bacteria from the *Enterobacteriaceae* genus is a facultative anaerobe, *Enterobacter aerogenes*. It is highly adaptable to the environment and can produce hydrogen at high conversion rates under strictly anaerobic conditions. In addition, *E. aerogenes* has the advantage of a wide substrate utilisation range, relatively fast growth, easy cultivation, as well as mature methods of genetic modification. Thus, for large-scale biological hydrogen production, it is considered an ideal strain. Nonetheless, an incomplete

metabolic process results in $H_2$ production much lower than the theoretical 4 mol $H_2$/mol glucose [64].

Mayor pathways for $H_2$ production by DF include acetate-ethanol and acetate-butyrate pathways. Many studies have reported acetate-butyrate as a dominant pathway for $H_2$ production despite the detrimental influence of butyrate-fermenting bacteria such as *Clostridium*, *Butyrviribrio* and *Bacillus*. However, this pathway may be converted to the butanol production pathway, thus consuming $H_2$. In comparison to the acetate-butyrate metabolic pathway, the acetate-ethanol pathway is more stable in terms of $H_2$ production, and it seems that this pathway has been utilized by *Ethanoligenens*, *Acetanaerobacterium*, *Clostridium*, *Rhodopseudomonas*, and *Citrobacter* [65].

Several factors affect the efficiency of hydrogen production in DF. These include pH, temperature, substrate concentration and hydraulic retention time. Optimal conditions vary depending on the microbial community and substrate used.

One of the key parameters influencing the efficiency of hydrogen production is the control of pH in the cultivation media [66,67]. According to studies, as acidity increases, the efficiency of the $H_2$ production process significantly decreases, indicating an inhibitory effect of pH. Therefore, different methods for controlling pH are used to enhance the efficiency of dark fermentation towards $H_2$ production [68–70]. The decrease in hydrogen production is attributed to the mechanism responsible for producing short-chain acids. The aforementioned acetic and butyric acids are commonly detected during DF of various starting materials, especially when *Clostridium* species dominate [71]. Additionally, researchers developed and validated a model based on mass balances of hydrogen ions, which describes the relationship between hydrogen production from dark fermentation and the pH changes over time [71]. Evidence derived from statistical analysis indicates that thermophilic strains exhibit high efficiency in substrate conversion, while mesophilic strains demonstrate superior volumetric productivity [72]. When aiming for a high amount of hydrogen production per unit of substrate consumed, microbes from the *Thermoanaerobacterales* (Family III) should be chosen over those from the *Clostridiaceae* and *Enterobacteriaceae* families, as supported by statistical validation [72]. The yield of 1.33 mol $H_2$/mol glucose equivalent was observed at pH 6.0 [73].

Moreover, fermentation temperature is also an important parameter affecting bacterial growth rate and substrate conversion efficiency to hydrogen. Thermophilic DF has a faster hydrolysis rate than mesophilic dark fermentation, resulting in increased volatile fatty acid production [74]. However, mesophilic DF is also widely used for biohydrogen production due to its advantages of low energy consumption and stable operation. Some works also investigated DF at psychrophilic temperatures, i.e. 21 °C [75]. In general, the temperature affinities of different hydrogen-producing bacteria differ, which is likely to lead to different biohydrogen production pathways and production rates from dark fermentation [76].

Important parameters in the development of the fermentation process are substrate concentration and hydraulic retention time (HRT). HRT corresponds to the time the substrate is in contact with the fermentative microbial population. HRT is a relevant operational parameter that can be used to optimise the $H_2$ yield. As the HRT controls the contact time between the organic substrate and fermentative population, its variation also affects the organic loading rate of bioreactors. Therefore, as the HRT increases, the organic loading rate decreases and vice versa. The optimum HRT value depends on the ability of the fermentative population to degrade the feedstock and grow in the bioreactor, as well as the feedstock’s composition and biodegradability [77,78]. Typical HRT reported by the literature is a couple of hours [62].

Hydrogen production using DF processes exhibits well-known drawbacks, including relatively low $H_2$ yield due to metabolic fundamentals such as stabilisation problems of the oxidative-reductive reaction, weak degradation and hydration capabilities, and rapid uptake by methanogens, homoacetogens and sulfate-reducing bacteria. Fermentation-induced metabolites such as organic acids, additional
microbial contaminants and toxic substances produced by the metabolic pathways also affect H₂ production performance [79]. Other limitations include high operating costs, high pre-treatment requirements for feed and ammonia nitrogen, limited processing capacity, and inhibition by salts. In addition, digestate might be a source of pollution. Moreover, some substrates, particularly lignocellulosic materials, require pre-treatment to improve their biodegradability and hydrogen yield. Pre-treatment methods can add to the overall cost and complexity of the process. Accumulation of metabolic by-products such as butyric acid or acetic acid is also a major problem in DF because it can inhibit microbial activity and reduce hydrogen production efficiency. Also, ammonia in certain concentrations inhibits DF [80,81]. Strategies to mitigate inhibition include the removal or conversion of inhibitory compounds [82,83]. Results presented in Table 2 showcase the potential for sustainable hydrogen generation from diverse feedstocks.

## 2.4. Production of H₂-rich gas in thermochemical processes

Thermochemical routes to produce renewable hydrogen could be considered an interesting alternative to microbial-based processes. Conditions in the reactors are typically more severe in comparison to bioreactors, i.e., much higher temperatures are typically applied. On the one hand, this allows the reactors to be significantly more compact in comparison to bioreactors, which is essential when it comes to the required investment costs. On the other hand, high temperatures create their own drawbacks in terms of requirements for materials, such as additional insulation needed to obtain certain efficiency, etc. Moreover, due to the heat requirements of drying, such processes might not always be well suited for the biomass types characterized by significant moisture content. In general, both pyrolysis (Fig. 5) and gasification (Fig. 6) routes could be used for the production of biohydrogen.

Gasification using oxygen and steam has an important advantage over using air as a gasification agent, as the main gaseous products after the WGS reactor are H₂ and CO₂ (Fig. 6) if impurities are not taken into account. Thus, the separation of CO₂ for subsequent CCS/CCU purposes could be easier. This resembles the difference between oxy-combustion and combustion using air, as in the latter case, the energy cost related to the separation of CO₂ from flue gases is substantial [96]. Gasification

with CO₂ can be considered as a CCU pathway since there is a possibility of reduction to CO during reactions between solid phase and gas (e.g., Boudouard reaction) [97,98].

Another type of gasification process that should not be overlooked in terms of hydrogen production is supercritical water gasification. This particular process is particularly advantageous for wet types of biomass, as water is necessary as a medium, and no prior drying is required [99–101]. Actually, drying wet substrates is energy-intensive, thus making biomass types with high moisture content not attractive for pyrolysis or gasification.

# 3. Suitable feedstocks and pretreatment methods

## 3.1. Sugar-rich feedstocks

Currently, efforts are made to ensure that microbiological (biotechnological) processes are based on renewable raw materials and those that do not compete with the food industry.

The use of food, industrial or even municipal waste for this purpose fits into this assumption. This type of waste contains organic compounds needed by microorganisms to produce biohydrogen. As in every fermentation, a medium rich in carbon sources should be provided. Hence, waste containing glucose, lactose or sucrose is an ideal medium for this purpose [102–104]. This may be waste from the food industry, namely fruit, vegetable, sugar cane, sugar beet or milk processing.

A wide range of organic substrates can be used for dark fermentation, including agricultural residues, food waste and wastewater. The choice of substrate has a significant effect on hydrogen yield and by-product composition. For example, starch-rich substrates tend to produce higher hydrogen yields compared to lignocellulosic materials, which are more recalcitrant to microbial degradation. From the point of view of fermentation processes, monosaccharides, such as hexoses (e.g. glucose) and pentoses (e.g. xylose), and Disaccharides, in particular sucrose and lactose, are substrates that are readily biodegradable. They are currently being used to test new designs as their use in fermentation processes does not require complicated processing, and the results are reproducible. The most recent trends in dark fermentation studies involve technologies capable of continuous production of hydrogen. Simple

<caption>**Table 2**</caption>

Hydrogen production via dark fermentation for various feedstocks, reactors and organism types.

<table><thead><tr><th>Substrate</th><th>Culture</th><th>Reactor</th><th>pH/temp.</th><th>H₂ product.</th><th>% H₂</th><th>Ref.</th></tr></thead><tbody><tr><td>Sucrose; 10 g/L</td><td>E. cloacae IIT-BT 08</td><td>Batch</td><td>pH 6.0<br>T = 36 °C</td><td>660 mL H₂ /<br>(L·h)</td><td>92</td><td>[84]</td></tr><tr><td>Cheese whey +2 M NaOH (to maintain pH 6)</td><td>Mixed culture (indigenous microorganisms in cheese whey)</td><td>CSTR</td><td>pH<br>6.0–6.9<br>T = 38 °C</td><td>65.8 L H₂ /<br>kg TS</td><td>43</td><td>[85]</td></tr><tr><td>Fruit and vegetable waste</td><td>Mixed culture</td><td>Batch</td><td>pH 5.52<br>T = 35 °C</td><td>223.8 mL H₂/gglucose</td><td>50.9</td><td>[86]</td></tr><tr><td>Glucose; 10 g/L</td><td>Flexibacteraceae and Clostridiaceae</td><td>Membrane<br>Continuous</td><td>pH 5.5<br>T = 35 °C</td><td>640 mL H₂ /<br>(L·h)</td><td>60</td><td>[87]</td></tr><tr><td>Organic fraction of municipal solid waste;<br>110 gys/(L·d)</td><td>Mixed cultures</td><td>CSTR Semi-continuous</td><td>pH 5.0<br>T = 50 °C</td><td>5.7 L H₂ /<br>(L·d)</td><td>55</td><td>[88]</td></tr><tr><td>Starch from raw cassava</td><td>Facultative anaerobic</td><td>Batch</td><td>T = 35 °C</td><td>N.D.</td><td>61</td><td>[89]</td></tr><tr><td>Kitchen waste from small gastronomy; 50<br>gCOD/L</td><td>Sludge from anaerobic treatment as a source of mixed culture</td><td>Batch</td><td>pH 5.5<br>T = 55 °C</td><td>79 mmol H₂/<br>(L·medium·d)</td><td>54.4</td><td>[90]</td></tr><tr><td>Swine manure (liquid); 13.94 gCOD/L</td><td>Anaerobic digestion providing mixed cultures</td><td>ASBR Batch</td><td>pH 5.0<br>T = 37 °C</td><td>0.1 L H₂ /<br>(L·h)</td><td>43</td><td>[80]</td></tr><tr><td>Manure from dairy cows; 70 g/L</td><td>Clostridium sp</td><td>CSABR Continuous</td><td>pH 5.0<br>T = 36 °C</td><td>N.D.</td><td>38.6</td><td>[91]</td></tr><tr><td>Cattle wastewater; 1.3 gCOD/L</td><td>Sewage sludge</td><td>Batch</td><td>pH 5.5<br>T = 45 °C</td><td>0.34 L /<br>(L·h)</td><td>32</td><td>[92]</td></tr><tr><td>Jatropha curcas – biodiesel industry residue</td><td>Activated sludge as a source of mixed cultures</td><td>CSTR Continuous</td><td>pH 5.5<br>T = 37 °C</td><td>3.65 L/<br>(L·d)</td><td>58</td><td>[93]</td></tr><tr><td>Citric acid wastewater 18 g COD / dm³</td><td>Mixed cultures</td><td>USAB Continuous</td><td>pH 7.0<br>T = 36 °C</td><td>0.72 L H₂/<br>(L·d)</td><td>67</td><td>[94]</td></tr><tr><td>Palm oil mill effluent 59 g COD / dm³</td><td>Mixed cultures (isolated from cow dung)</td><td>USAB Continuous</td><td>pH 5.0</td><td>73 L/d</td><td>56</td><td>[95]</td></tr></tbody></table>

COD – chemical oxygen demand, TS – total solids, TVS – total volatile solids, VS – volatile solids, N.D. – not defined, MS – model substance, RR – renewable resources, AW – animal waste, PPIW – post-production industrial wastes, CSABR – continuously stirred anaerobic bioreactor, CSTR – continuously stirred tank reactor.
<figure><img src="image_8.png" /><figcaption>Fig. 5. Route of biohydrogen production using pyrolysis (WGS – water gas shift).</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 6. Routes of biohydrogen production using gasification: A – gasifiers using air as gasification agent; B – gasification using agents such as steam, CO₂, or O₂, resulting in gas not diluted by N₂ from air (WGS – water gas shift).</figcaption></figure>

carbohydrates are not suitable for this type of production as their cost is too high, making their use on an industrial scale unprofitable [62]. Sewage sludge is another example of a raw material for dark fermentation. For fermentation processes, literature reports the use of feedstocks such as waste from sugar industry [105,106], wineries, distilleries [107], different types of wastewater [108–110], jatropha waste [111], whey [112], brewer's spent grain [113], sugarcane bagasse [114], wheat straw [115], oat straw [116], or sweet sorghum stover [117] or cassava [118].

An interesting approach is connecting biohydrogen fermentation with biodiesel production since glycerol is formed as a by-product during this process. It is a great source of carbon [119]. However, the most promising feedstock is lignocellulosic waste. This abundant and highly available material can be used as a source of fermentative sugars as well. However, in its basic form, this material is not available to microorganisms used in fermentation. The sugars present in this

material are entrapped in the long biopolymers (cellulose and hemicellulose) [120] and can be released through enzymatic hydrolysis.

## 3.2. Pretreatment methods

Some of the waste, such as the mentioned cellulosic one, requires preliminary treatment in order to make available the carbon sources they contain. These methods include mechanical, e.g. milling [121], thermal [122,123], chemical [124], and enzymatic [124–126] treatment of the material. When it comes to pretreatment of the feedstock for microbial H₂ production routes, pretreatments aim at breaking down the bonds between biopolymer-containing sugars (cellulose and hemicellulose) to make them available to microorganisms. When thermochemical processes for H₂ production are considered, the moisture content of the fuel is extremely important from the point of view of the energy balance of the process. Also the reactivity of the feedstock is
important [127], especially for processes with relatively low residence time, such as entrained flow gasification. Since increased reactivity could be achieved by making more surface area available to the reactants (in case of gasification – gasifying agent) by grinding into finer particles, grindability of the feedstock is important. Oxygen content is also important, e.g., from the point of view of gasification, since primary tars are predominantly oxygenated organic compounds [128,129]. Last, but not the least, since solid fuel is a form of chemical energy storage, aspects relevant to storage of feedstock are crucial, i.e., making the feedstock more hydrophobic decreases the risk of moisture reabsorption by stored biomass, either by reducing water sorption from humid air or from water droplets by changing the contact angle [130].

As far as wet feedstocks are considered from the point of view of increasing the availability of sugars for microbial processes (also performed at relatively high moisture content), enzymatic hydrolysis could be considered effective and non-invasive for the environment. Depending on the material to be decomposed, the enzyme should be selected appropriately. There are various types of cellulases in lignocellulosic materials. Many companies provide ready-made enzyme mixtures that break down cellulosic (and lignocellulosic) waste into the desired fermentation sugars, such as Celluclast® 1.5 L, Novozymes [131], Macerozyme R-10, Gluczyme NL4.2, or Amano Enzyme [125]. However, enzymes are quite sensitive to changes in temperature and pH. They are also quite expensive, so their optimal concentration in the reaction mixture should also be carefully selected. Mussatto et al. [131] delivered data about the hydrolysis by Celluclast: time of reaction - 96 h, temperature - 45 °C, pH 4.8 and enzyme/substrate ratio 45 FPU per 1 g of substrate. Shindo and Tachibana [125] tested Macerozyme R-10 and indicated a time of reaction of 72 h, temperature - 30 °C and enzyme concentration 0.1 % (w/v). It can be seen that they differ slightly, and in order to determine the optimal operating conditions, research should be carried out to ensure the highest possible efficiency of the process. Al-Shorgani et al. [132] used the Celluclast Novo and Novozyme 188 to decompose the lignocellulosic material (rice bran, deoiled rice bran, sago starch) in an anaerobic fermentation to yield biohydrogen. The productivity of biohydrogen reached 3.172 mL H₂/L [132]. Physical pretreatments, such as mechanical milling, ultrasonic treatment, and microwave heating, reduce particle size and cellulose crystallinity, which increases surface area and facilitates enzyme penetration [133,134]. These methods, while effective in enhancing enzyme accessibility, often require high energy inputs, making them more suitable as a complementary approach to other pretreatments [133,134].

Microbial treatment is another form of pretreatment capable of breaking down lignocellulosic biomass [135,136]. During microbial hydrolysis, carbohydrates, proteins, and lipids in the substrate are broken down by extracellular enzymes into low molecular-weight intermediates with molecular weight < 1000 Da [135]. The treatment is similar to enzymatic treatment, with the difference that enzymes are secreted by the microbial community [135], which consists of bacteria or fungi [136]. Strains of *Bacillus*, such as *Bacillus cereus* or *Bacillus lentus*, were successfully used to obtain peptides with antioxidative properties through the hydrolysis of proteins [137]. Moreover, bio-delignification presents an interesting pathway, as microorganisms involved in this process are able to produce hydroxyl radicals through the reaction between iron and hydrogen peroxide [136]. Hydrogen peroxide can be produced by fungi owing to enzymes like aryl-alcohol oxidase, pyranose-2 oxidase, and glyoxaline oxidase [136]. This leads to the degradation of lignin in the oxidative mechanism and the breakdown of glycosidic linkages with carbohydrates [136]. Nonetheless, Wu et al. [138] demonstrated that lignin degradation on its own is not sufficient to increase H₂ production, and higher temperatures of microbial processes (65 °C) are the key component [138].

Chemical pretreatments leverage the reactivity of various chemical agents to disrupt lignin and hemicellulose structures, improving cellulose accessibility. Acid and alkali treatments are among the most commonly used chemical methods. Alkali treatments, such as sodium

hydroxide, are highly effective in breaking down lignin [134]. Acid hydrolysis can significantly change the structural and functional properties of carbohydrates [139]. Among key process parameters of acid hydrolysis temperature, acid concentration, residence time, and solid-to-liquid ratio are mentioned in the literature [140]. The use of strong acids can be advantageous. Sulfuric acid results in relatively high sugar yields and hydrolysate can be used directly after neutralisation with lime [140]. Nitric acid can also solubilise lignin [140]. However, such harsh conditions are related to relatively high investment costs owing to corrosion risk [140]. Weak acids are capable of facilitating the hydrolysis of polysaccharides, e.g., Santoso et al. [141] have shown that hydrolysis with the addition of 5% of acetic acid enabled the production of water-soluble chitosan from shrimp carapaces [141].

Apart from enzymatic and microbial pretreatment, hydrothermal conditions offer interesting possibilities in terms of biomass valorisation, and such an approach has been used so far for the valorisation of biomass as a solid fuel [142,143]. Hydrothermal conditions facilitate breaking down natural polymers, such as cellulose, into sugars [144], which could be subsequently fermented to produce hydrogen.

Hydrothermal carbonisation (HTC) is a thermochemical process that is carried out in a subcritical water reaction environment at temperatures between 180 and 260 °C and at pressures of tens of bar, which ensures the water remains in the liquid state [145,146]. The ionic product of water considerably increases between 200 and 280 °C. Water thus begins to behave as a non-polar solvent [147,148]. When using various types of biomass, the process occurs with a large number of concurrent reactions taking place, which result in a wide variety of products [145,146]. Hydrolysis is the initial step in the HTC process, which is then followed by dehydration and decarboxylation [149]. Hydroxyl groups become fewer as a result of dehydration [145]. CO₂ is formed more quickly as a result of decarboxylation [150,151]. A reduced O/C ratio is a consequence of the reduction in the number of OH groups (due to dehydration) and COOH and C=O groups (due to decarboxylation) [145,152,153]. In series to hydrolysis, dehydration and decarboxylation, aromatization, and polymerization reactions occur during HTC [145,149]. HTC as a pyrolysis pretreatment is advantageous because it reduces the O/C ratio of the treated feedstock [154–156]. This makes HTC an interesting valorisation method prior to pyrolysis, and this is particularly true for wet types of biomass [157–159]. HTC makes the treated feedstock hydrophobic, which positively impacts the mechanical dewatering performances and thus the energy needs of the whole process, considering that high-moisture content biomass should be dewatered/dried before pyrolysis [160–163]. When compared to the raw feedstock, hydrochar has a greater heating value as a result of HTC [164–166]. Additionally, a portion of the biomass’s inorganic fraction is also removed (actually, leached) during the HTC process [167,168]. Furthermore, given that entrained flow gasification relies heavily on particle size, the HTC process’ beneficial effects on biomass grindability should not be disregarded [169–171].

Advanced chemical approaches, such as oxidative pretreatment and organosolv processes, have also shown promise in selectively removing lignin [133,134]. However, these methods can lead to the formation of inhibitory by-products, necessitating additional detoxification steps. Recent innovations in green solvents—such as ionic liquids and deep eutectic solvents (DES)—offer environmentally friendly alternatives with high lignin solubility and reduced toxicity, though scalability remains a challenge due to solvent recovery costs [133].

Regarding dry biomass and thermal biohydrogen production routes that require dry feedstock, torrefaction is one of the processes suitable for the valorisation of relatively dry types of biomass [172]. Typically, the process is performed in an inert environment at elevated temperatures, ranging between 220 and 300 °C. Type of reactor and the feedstocks used are deciding factors in terms of residence time [173–175], although industrial applications tend to favour shorter residence times, due to increased productivity [176,177]. The process takes place in inert conditions or with very small concentrations of oxygen in case of
torrefaction with flue gases [178,179]. Some works also reported using superheated steam for torrefaction [174,180,181].

Torrefaction increases the higher heating value of biomass, thus leading to energy yields higher than one [182–184] and producing solid fuel that resembles low caloric coals often used in power generation units [185] since the first intended application of the product (bio-coal) was co-firing with coal [186–190]. Overall, torrefaction improves the grindability of valorised biomass [191–193]. One of the benefits is also reducing the moisture content and making biomass more hydrophobic [156,194–197], thus providing all the benefits important from the point of view of fuel logistics and storage. Moreover, a decrease in oxygen content could be observed during torrefaction [198–202], which could be beneficial for fast pyrolysis [154,203–205] or gasification [206–209].

In many cases, torrefaction is preceded by drying, which in itself is an important pretreatment process for biomass. Some literature sources make a distinction between non-reactive and reactive drying [210]. The latter takes place at temperatures ranging between 150 and 200 °C for lignocellulosic biomass and leads to an initial breakdown of the structure [210]. This could also be beneficial for subsequent hydrolysis and microbial processes. Hua et al. [211] applied an air frying pretreatment process (200 °C for 30 min) to corn stover, which subsequently increased the saccharification rate and cellulose conversion rate in subsequent enzymatic hydrolysis when compared to samples subjected only to hydrolysis [211].

Apart from torrefaction, using superheated steam, other biomass valorisation processes are performed in atmospheres with significant amounts of steam. Steam explosion is a process in which saturated steam with pressure reaching 2.5 MPa is injected into a pressure-tight reactor [212]. The temperature of the steam explosion process ranges between 180 and 260 °C, with residence time ranging between 2 and 30 min. The steam explosion can improve the reactivity of biomass despite a rather modest influence on its calorific value and carbon content [213]. The difference in reactivity can be attributed mostly to a finer particle size of biomass treated with steam explosion [214], which confirms the influence of the process on biomass structure which results in improved grindability.

Another pretreatment process, similar to a steam explosion, is called Instant Controlled Pressure Drop, with DIC often used as an abbreviation due to the French name of the process (Détente Instantanée Contrôlée) [212]. In DIC, additional vacuum steps are added before and after the treatment with steam that reaches 170 °C and 30 bar [212]. The rapid pressure drop with $\Delta p/\Delta t$ reaching between 2.5 and 5.0 bar/s is a

characteristic feature of this process [212]. The process enhances saccharification. Messaoudi et al. [215] reported an increase in the yield of fermentable sugars for the production of ethanol when DIC pretreatment was applied to eucalyptus chips.

Nonetheless, care should be taken when applying thermal pretreatment processes prior to enzymatic hydrolysis, as such processes could generate pseudo-lignins as by-products [216–218]. In general, the linkage between lignin and polysaccharides in lignocellulosic biomass is an obstacle in terms of the separation of the former [212]. Furthermore, the presence of lignin may cause non-productive adsorption of enzymes like cellulase, thus decreasing the overall performance of the enzymatic hydrolysis process [219]. A qualitative assessment of each pretreatment process for different biohydrogen production routes is summarised in Table 3.

# 4. Types of reactors suitable for hydrogen production

## 4.1. Bioreactors

### 4.1.1. Process parameters that can be controlled in bioreactors

Biohydrogen production yield is affected by a variety of parameters. A number of them can be controlled in state-of-the-art bioreactors. The most important parameters are pH, temperature and hydraulic retention time (HRT).

#### 4.1.1.1. Culture pH.
Culture pH is considered one of the most important factors determining metabolic pathways of the fermentation and, therefore, the biohydrogen yield. The optimal pH depends on the type of the microorganism used. Lu et al. [230] used a mixed culture from a domestic sewage treatment plant in Zhengzhou, China, in a pilot scale unit, and in this case, pH varied between 5.0 and 6.2, depending on the retention time [230]. In photo-fermentation, an alkaline environment at a pH exceeding 6.0 inhibits $H_2$ production by photosynthetic bacteria [231]. In thermophilic dark fermentation, the favourable pH value for anaerobic bacteria appears to be 5.0 [232]. The application of systems with pH control enhances the overall biohydrogen yield and production rate, along with shortening the lag time. pH can be controlled, i.e. by the application of a pH probe connected to an automatic pH titrator, delivering NaOH solution to the bioreactor whenever the low limit is exceeded [233]. Additional $NaHCO_3$ dosing can provide conditions for sulfate removal [234].

<table><caption>**Table 3**

Different pretreatment processes and their suitability for different biohydrogen production routes.</caption><thead><tr><th>Process</th><th>Typical conditions</th><th>Preferred moisture content</th><th>Suitability for thermal production of hydrogen</th><th>Suitability for microbial production of hydrogen</th><th>Ref.</th></tr></thead><tbody><tr><td>Torrefaction</td><td>T: 220–300 °C<br/>t: 20–240 min<br/>Dry or wet (steam), depending on the heat supply design</td><td>Low moisture content feedstock</td><td>Excellent</td><td>Poor</td><td>[220–222]</td></tr><tr><td>HTC</td><td>T: 220–300 °C<br/>t: 30–240 min<br/>Liquid water</td><td>High moisture content feedstock</td><td>Excellent</td><td>Limited</td><td>[223–225]</td></tr><tr><td>Steam explosion</td><td>T: 180–260 °C<br/>p: up to 25 bar<br/>t: 2–30 min<br/>Steam</td><td>Moderate moisture content feedstock</td><td>Limited</td><td>Good</td><td>[212]</td></tr><tr><td>Instant Controlled Pressure Drop</td><td>T: up to 170 °C<br/>p: up to 30 bar<br/>Δp/Δt: 2.5–5.0 bar/s<br/>Steam</td><td>Moderate moisture content feedstock</td><td>No info available</td><td>Excellent</td><td>[212]</td></tr><tr><td>Enzymatic/microbial hydrolysis</td><td>T: 35–70 °C<br/>t: 2–10 days<br/>Liquid water</td><td>High moisture content feedstock</td><td>No info available</td><td>Excellent</td><td>[123,135,137]</td></tr><tr><td>Thermal hydrolysis</td><td>T: 160–190 °C<br/>t: 20–30 min<br/>Liquid water</td><td>High moisture content feedstock</td><td>Good</td><td>Limited</td><td>[226–229]</td></tr></tbody></table>
**4.1.1.2. Temperature.** Biohydrogen production is strongly dependent on the temperature. The optimal temperature range is individual for the following groups of microorganisms: mesophilic, thermophilic, extreme thermophilic or hyperthermophilic. However, most of the laboratory-scale studies on dark fermentation have been conducted using mesophiles [235]. Mesophilic cultures appear to reach their optimal productivity level at temperatures between 30 and 40 °C. Researchers have proved that temperature increase within the given range always improves hydrogen production. However, exceeding the upper value leads to a biohydrogen production decrease, firstly as a result of the denaturation of microbial enzymes responsible for the fermentative hydrogen production process and, secondly, due to the death of microorganisms [236]. Recent studies have considered hyper-thermophilic conditions due to their advantages, such as better pathogenic destruction, less likely methanogenic bacteria contamination, and better conditions for biohydrogen production, resulting in higher yields [236].

**4.1.1.3. Hydraulic retention time (HRT).** HRT is another crucial process parameter that can be controlled in hydrogen-producing bioreactors. HRT is the time period for a specific liquid volume to be retained in the working volume of the reactor. HRT cannot be too short, as the biomass could potentially be washed out from the system. On the other hand, HRT, which is too long, causes a decline in the H₂ production rate. It is also important to consider that the typical specific growth rate of methane-producing bacteria is much lower than that of hydrogen-producing bacteria (0.00162–0.02 h⁻¹ vs 0.0172 h⁻¹). Therefore, carefully regulated HRT could allow the retention of H₂-producing bacteria and get rid of unwanted methane-producing bacteria [230,237–239].

**4.1.1.4. Organic loading rate.** The organic loading rate (OLR), which is defined as the amount of organic waste fed per unit volume of the digester per unit of time [240], has to be optimized in order to obtain a high H₂ yield. According to Khan et al. [238], an initial increase in the OLR aids the production of biohydrogen. However, exceeding 22 gCOD/L-d caused the biohydrogen production to decrease. OLR is closely related to HRT, and these two parameters strongly depend on each other. Therefore, defining the optimal operating conditions should consider both parameters simultaneously [238,241].

**4.1.1.5. Hydrogen partial pressure.** Microbial pathways in fermentative microbial production are strongly dependent on the hydrogen partial pressure. A higher concentration of the dissolved hydrogen in the liquid phase inhibits the hydrogen production process. Therefore, the removal of excess hydrogen from the system is important [230]. However, it is important to point out that in such cases, the direct applicability of Henry's law is often considered, as the concentration of dissolved hydrogen in the liquid phase should be linked at equilibrium to its partial pressure. In fact, due to possible mass transfer limitations encountered by low soluble gases, the system might not be in equilibrium, and Henry's law could not be exactly applicable [242].

**4.1.1.6. Additives.** The conversion of cellulosic biomass to hydrogen can be enhanced by supplementing the nutrients needed for microbial activity [243]. Research has shown that a variety of additives up to a certain concentration can increase the biohydrogen production yield, including metals, metal oxides and other chemicals (enzymes, buffers, vitamins, etc.). For example, the thoughtful addition of metals, metal ions and metal oxides can strengthen the transfer of electrons in bacteria cells, while the addition of immobilization media, such as activated carbon, can mitigate the inhibition of metabolites and increase the biomass density [244]. However, excessive concentrations of additives can have inhibitory effects [245].

**4.1.1.7. Light intensity.** In the case of photofermentation, light intensity

plays a crucial role in determining the hydrogen yield. In hydrogen formation, light saturation requirements are higher (60–100 W/m²) than in cell growth (40–60 W/m²), which is due to higher light intensity requirements for maximum nitrogenase activity [246]. Although the hydrogen production rate increases almost linearly in the range of 10–120 W/m² it is important not to reach photoinhibition levels (for example, 400 W/m² for R. sphaeroides) [247].

**4.1.2. Types of bioreactors**

The type of bioreactor selected for biohydrogen production depends primarily on the type of process carried out, namely, whether it is photofermentation or dark fermentation.

**4.1.2.1. Photoreactors.** The first one uses bioreactors exposed to UV light, i.e., photobioreactors. In the case of this type of bioreactor, the biocatalyst itself is very important (microalgae, diatoms or cyanobacteria). Crucial is also the control of pH, oxygen, sulfur and hydrogen concentrations, and temperature in order to maintain optimal conditions [230]. However, the most important aspect is the amount of light provided. A crucial thing here is the surface-to-volume ratio - it should be as high as possible. As for the shape of the photobioreactor, it can be used as a flat plate, tubular, pond or pool-type. However, it is almost always about access to light. Due to the fact that the efficiency of the process in terms of light conversion is quite low, diluted light should be supplied, and the structure and hydraulics of the bioreactor should ensure very good mixing of the bioreactor content.

**4.1.2.2. Dark fermentation bioreactors.** In the case of dark fermentation, the variety of bioreactor choices is much greater. Due to the fact that this is a process similar to classic fermentation, we can use various types of bioreactors. They are presented in Table 4.

**4.2. Reactors for thermal processes**

**4.2.1. Pyrolysis**

Pyrolysis is a thermal process that causes decomposition to occur in the absence of oxygen [270,271]. As a result, biomass can be transformed into a variety of solid, liquid, and gaseous molecules [272–274]. Solid materials can be put into the soil in the form of biochar or utilized as solid fuel [159,174,275]. On-site process heat can be provided through pyrolysis gaseous products. Products that condense can also be used locally to meet energy needs [276,277]. To obtain pyrolysis oil, which has a relatively high energy density and can be regarded as a superior energy carrier, those components can also be condensed. Pyrolysis is a general term that refers to a large variety of distinct processes. Mild pyrolysis is another name for torrefaction [178,278,279]. Slow pyrolysis intends to produce gases for a variety of uses, including polygeneration, with the production of H₂ [5,177,280] and biochar. The production of bio-oil, sometimes referred to as pyrolysis oil, is increased through quick pyrolysis. With temperatures between 450 and 550 °C and a residence time of 1 s, it uses high heating rates of 100 °C/s [281–283].

From the point of view of hydrogen production, fast pyrolysis is not suitable, as it is designed to maximize the yield of pyrolysis oil, which requires quenching. Apart from having a low yield of gases, there is also a loss of enthalpy during the quenching process, which would cause unnecessary consumption of energy to heat it back to the typical temperature range for reformers. The use of pyrolysis oil is possible, but it would require a pre-reforming/cracking unit operation included. In general, hydrogen content in pyrolysis gas (Table 5) is not enough on its own. Thus, further processing is required. In terms of heating, many pathways could be used, including electric heating [284], which could not only simplify the construction of such reactors and make it relatively cheaper but also serve as a demand-side response. Another interesting possibility is the direct use of solar energy to supply heat necessary for
<table><caption>**Table 4**

Bioreactors suitable for biohydrogen production through microbial processes.</caption><thead><tr><th>Type of bioreactor</th><th>Characteristic</th><th>Results of application</th><th>Ref.</th></tr></thead><tbody><tr><td>Continuous stirred tank reactor (CSTR)</td><td>-The tank reactor with stirring and constant dosing of substrate and collection of products<br/>-Good mixing and, consequently, effective mass transfer<br/>-Low concentration of microorganisms due to the high biomass washout rate<br/>-Formation of granular sludge increased biomass concentration and efficiency of hydrogen formation</td><td>-Hydrogen production rate of up to 0.54 L/ (L·h). Conditions: substrate - synthetic sucrose-base wastewater; 26 °C; pH 5.5; HRT= 6 h;<br/>-Hydrogen production rate of up to 15 L/(L·h). Conditions: silicone-immobilized sludge; 40 °C; pH 6.6 ± 0.2, HRT = 0.5 h;<br/>-Hydrogen production rate of up to 3.2 L/(L·h). Conditions: granular sludge fermenting; glucose wastewater (10 g/L); pH 5.5; 37 °C, HRT = 0.5 h.</td><td>[248–250]</td></tr><tr><td>Aerobic sequencing batch reactor (ASBR)</td><td>-High-rate liquid digestion system that is divided into four separated and sequenced parts: feeding influent chamber, mixing the reactor, settling solids chamber, and decanting liquid chamber</td><td>-Hydrogen production yield from 13.44 mol H<sub>2</sub>/(kgCOD·d). Conditions: COD = 7.9 kgCOD/(m<sup>3</sup>·d); anaerobic mixed microflora; pH 6.0;<br/>-Volumetric hydrogen production rate of 50.5 ml H<sub>2</sub>/(h·L). Conditions: 3 gCOD/L; 35 °C; HRT = 12 h.</td><td>[110,251]</td></tr><tr><td>Membrane bioreactor</td><td>-The CSTR connected with the membrane placed at the outlet stream<br/>-High concentration of biomass<br/>-Membrane fouling decreases the efficiency of biohydrogen production</td><td>-Hydrogen production rate 0.64 L·h. Conditions: HRT = 3.3 h, sludge retention time (SRT) 12 h; substrate 10 g/L glucose;</td><td>[87]</td></tr><tr><td>Fixed-bed bioreactor</td><td>-Operated with support carriers packed in the bioreactor<br/>-Higher mass transfer resistance, lowered rates of substrate conversion and hydrogen production (the flow through the reactor is less turbulent compared with the CSTR)<br/>-pH gradient distribution along the reactor<br/>-Different microbial activity along the reactor<br/>-Recirculation flow should be applied</td><td>-Hydrogen yield (3.5 mol H<sub>2</sub>/mol sucrose); 0.4 L/ (L·h) and 15.8 % of H<sub>2</sub>. Conditions: soft-drink wastewater in up-flow anaerobic packed-bed reactors; pH of 6.5; Recycled low-density polyethylene particles as a support, HRT = 0.5 h<br/>-Hydrogen production 1 h·L and yield 1.1 mol H<sub>2</sub>/mol lactose. Conditions: organic loading rate 37 gCOD/L; pH 5; HRT = 24 h; cheese whey as a substrate (25 gCOD/L)</td><td>[252,253]</td></tr><tr><td>Fluidized-bed bioreactor</td><td>-The column bioreactor with the microorganism immobilized on the bed in a state of fluidization<br/>-The high level of accumulation of a large amount of biomass on the bed</td><td>-Hydrogen production: 1.82 L/(L·h), yield of production: 4.26 mol H<sub>2</sub>/mol sucrose. Conditions: immobilized sewage sludge (ratio of 10 % (v/v)) by ethylene-vinyl acetate copolymer, 3-phase fluidized-bed reactor;</td><td>[254–256]</td></tr></tbody></table>

<table><caption>**Table 4 (continued)**</caption><thead><tr><th>Type of bioreactor</th><th>Characteristic</th><th>Results of application</th><th>Ref.</th></tr></thead><tbody><tr><td></td><td>-High organic loading rate (OLR)<br/>-Short retention time<br/>-Good mixing conditions</td><td>HRT = 6 h; 40 °C, Sucrose (5, 10, 20, and 40 g COD/L) solution as a feedstock<br/>-Hydrogen production rate of 2.36 L/(L·h); hydrogen yield 1.16 mol-H<sub>2</sub>/mol glucose. Conditions: HRT = 4 h; granulated activated carbon; glucose solutions (10–30 g/L) as a feedstock, pH 4.0, temperature 37 °C</td><td>[230,257]</td></tr><tr><td></td><td>-Gas flowing from the bottom (vertically) lifts the bed in the bioreactor volume<br/>-The tank reactor with cross-flow of liquid medium and gas<br/>-Good treatment efficiency and capability in retaining high biomass concentration</td><td>-Hydrogen production rate of 12.3 L/(L·d). Conditions: HRT = 3 h, starchy wastewater supplemented with groundnut de-oiled cake used as a feedstock, COD of medium 26 g/L, total sugars 17 g/L in feedstock; pH 6.75, 37 °C;</td><td>[258]</td></tr><tr><td></td><td>-Good surface to volume ratio<br/>-Relatively easy to scale-up<br/>-High mixing rate<br/>-Low shear<br/>-Temperature control through heat exchanging coils<br/>-Agitation and gas exchange mostly done by bubbling</td><td>1.52 mL/(L·h), with light intensity 12 W/m<sup>2</sup></td><td>[259,260]</td></tr><tr><td></td><td>-Agitation through sea motion<br/>-Low operating cost<br/>-Uses space on the sea surface that is mostly unutilized<br/>—No coling required</td><td>100–300 mL/L·day in early stage reaching up to 500 mL/L·day in later stage</td><td></td></tr><tr><td></td><td>-High S/V due to alveolar panels, -uniform distribution of light<br/>—O2 buildup, high air flow rates required to move across the channels</td><td>-Hydrogen production yield: 57.6 mL/(L·h) accompany with the mean H<sub>2</sub> yield of 1.8 mol H<sub>2</sub>/mol glucose<br/>-Conditions: Rhodopseudomonas palustris anaerobic cultivation for 48 h at pH 7.0, 30 °C; modified RCVBN medium, light irradiation of 50 W/m<sup>2</sup> —H<sub>2</sub> production was conducted by using a basal medium. The initial pH of medium was adjusted 7.0 by 0.1 M NaOH and HCL</td><td>[261,262]</td></tr><tr><td></td><td>-The culture being circulated by the rotation of a marine impeller,<br/>-Combination of an impeller with the loop configuration of the torus geometry allows for a good mixing, without dead volume<br/>-The light supplying device is placed in</td><td>-Hydrogen production yield: 1.9 mL/(L·h) Conditions: Chlamydomonas reinhardtii; pH 7.2; T = 25 °C, batch mode</td><td>[263–265]</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 4 (continued)**</caption><thead><tr><th>Type of bioreactor</th><th>Characteristic</th><th>Results of application</th><th>Ref.</th></tr></thead><tbody><tr><td rowspan="3">Vertical tubular</td><td>front of the photobioreactor enabling a rectangular one-dimensional treatment</td><td></td><td></td></tr><tr><td>- Low surface to volume area</td><td></td><td></td></tr><tr><td>- low material cost for their construction</td><td rowspan="2">-Hydrogen production of 371 mL/L, achieved by Anabaena sp., conditions: gas velocity of 0.524 cm s<sup>1</sup>, light intensity 140 mE m<sup>2</sup>/s, and temperature, 30 °C,</td><td rowspan="2">[266–268]</td></tr><tr><td>- the contact between the bacterial culture and the light source is reduced with the increase of column diameter</td></tr><tr><td rowspan="5">Horizontal tubular</td><td>- usually agitated by sparging of air</td><td></td><td></td></tr><tr><td>- Large surface to volume ratio, which is decreased by scaling up</td><td rowspan="5">-Hydrogen production of 850 mL H<sub>2</sub> (artificial light) and 930 mL (solar light). Conditions: sulfur-deprived cultures of C. reinhardtii, 50 L photobioreactor, 28.0 °C ± 0.5 °C, initial pH of 7.2, culture velocity of 0.36 m s<sup>−1</sup>;</td><td rowspan="5">[267–269]</td></tr><tr><td>-gas directly injected in the tube connections or in the gas exchange unit</td></tr><tr><td>-agitation achieved by mechanical pumps</td></tr><tr><td>-high sunlight absorption and light conversion efficiency</td></tr><tr><td>-Possibility of high biomass density</td></tr></tbody></table>

HRT – hydraulic retention time; COD – chemical oxygen demand

the pyrolysis process [285], which could be performed in a similar manner to solar gasification.

### 4.2.2. Gasification

Solid fuels are converted to gas through a process called gasification [98,294,295]. It was created in the 18th century, with Robert Gardner receiving the first gasification patent in 1788 [97,296]. City lights frequently use so-called “town gas” (also known as producer gas) [97]. Producer gas (that’s what gas from gasification used to be called) was also used by vehicles during periods of severe shortages of liquid fuel for transportation [294]. For instance, in 1945, 54.9 % of vehicles and 77.0 % of buses in Sweden ran on producer gas produced by gasifying wood and charcoal [294]. Types of gasification reactors are shown in Fig. 7, ordered by the size that could be achieved through scaling up.

The gas generated by a gasifier, also known as producer gas or more commonly known as syngas, is mostly composed of CO, H₂, CO₂, H₂O (vapour), and N₂ (from the gasification when using air) [297]. Additionally, the syngas contains hydrocarbons, which include compounds that remain gases in ambient conditions, such as methane [97], as well as more complex substances like phenols, toluene, naphthalene, benzene, and other aromatic chemicals [298,299]. These mostly volatile organic compounds are called tars [300,301]. The most significant obstacles to the subsequent use of gas following gasification are its composition and tar content. Studies on tar concentration in producer gas, modelling of its creation, and strategies for reducing its content in producer gas have been published in large amounts [128,129,298,302–306]. However, the majority of research either focuses on the total tar concentration (all tars) [129,303] or employs model substances such as benzene, toluene, phenol, or naphthalene [129,307–310]. According to a widely accepted definition, organic substances are classified as tars if their molecular weight is greater than that of benzene [302]. The first class of tars is reserved for compounds that cannot be detected by GC, the second class is for heterocyclic

<table><caption>**Table 5**

Hydrogen content – gas from pyrolysis.</caption><thead><tr><th>Feedstock</th><th>Pyrolysis process conditions</th><th>H<sub>2</sub> content</th><th>Ref.</th></tr></thead><tbody><tr><td rowspan="2">Solid mixture of 10 g of lignin with the addition of 1.9 g of FeSO<sub>4</sub></td><td>550 °C; RT 20 min; in N<sub>2</sub></td><td>7.5% vol</td><td rowspan="2">[286]</td></tr><tr><td>800 °C; RT 20 min; in N<sub>2</sub></td><td>33.0% vol</td></tr><tr><td>Food waste</td><td>550 °C; in N<sub>2</sub></td><td>4.2% mass</td><td>[287]</td></tr><tr><td>Pinewood</td><td>1600 °C; HR 50 °C/min; RT 11 min; in N<sub>2</sub></td><td>41.7% vol</td><td rowspan="2">[288]</td></tr><tr><td>Sugarcane bagasse</td><td>680 °C; HR 47.5 °C/min; RT 60 min; in He</td><td>28.8% vol</td></tr><tr><td></td><td>580 °C; HR 47.5 °C/min; RT 60 min; in He</td><td>21.3% vol</td><td></td></tr><tr><td></td><td>480 °C; HR 47.5 °C/min; RT 60 min; in He</td><td>9.6% vol</td><td></td></tr><tr><td></td><td>680 °C; HR 123.5 °C/min; RT 20 min; in He</td><td>45.3% vol</td><td rowspan="2">[289]</td></tr><tr><td></td><td>580 °C; HR 123.5 °C/min; RT 20 min; in He</td><td>15.2% vol</td></tr><tr><td></td><td>480 °C; HR 123.5 °C/min; RT 20 min; in He</td><td>8.7% vol</td><td></td></tr><tr><td rowspan="2">Organic fraction of municipal solid waste</td><td>450 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>47.97% vol</td><td rowspan="2">[290]</td></tr><tr><td>550 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>55.42% vol</td></tr><tr><td></td><td>650 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>61.13% vol</td><td></td></tr><tr><td rowspan="2">dry digestate</td><td>450 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>35.49% vol</td><td rowspan="2">[290]</td></tr><tr><td>550 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>52.01% vol</td></tr><tr><td></td><td>650 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>60.94% vol</td><td></td></tr><tr><td rowspan="2">dried sewage sludge</td><td>450 °C; HR 8 °C/min; RT 60 min; in N<sub>2</sub></td><td>10.41% vol</td><td rowspan="2">[291]</td></tr><tr><td>500 °C; HR 8 °C/min; RT 60 min; in N<sub>2</sub></td><td>12.46% vol</td></tr><tr><td></td><td>550 °C; HR 8 °C/min; RT 60 min; in N<sub>2</sub></td><td>17.93% vol</td><td></td></tr><tr><td></td><td>650 °C; HR 8 °C/min; RT 60 min; in N<sub>2</sub></td><td>26.9% vol</td><td></td></tr><tr><td rowspan="2">Chlorella vulgaris</td><td>450 °C; HR 100 °C/min; RT 60 min; in N<sub>2</sub></td><td>8.96% vol</td><td rowspan="2">[291]</td></tr><tr><td>500 °C; HR 100 °C/min; RT 60 min; in N<sub>2</sub></td><td>18.95% vol</td></tr><tr><td></td><td>550 °C; HR 100 °C/min; RT 60 min; in N<sub>2</sub></td><td>19.16% vol</td><td></td></tr><tr><td></td><td>600 °C; HR 100 °C/min; RT 60 min; in N<sub>2</sub></td><td>17.73% vol</td><td></td></tr><tr><td></td><td>650 °C; HR 100 °C/min; RT 60 min; in N<sub>2</sub></td><td>22.29% vol</td><td></td></tr><tr><td></td><td>600 °C; RT 5.5 s; in N<sub>2</sub></td><td>9% vol</td><td></td></tr><tr><td></td><td>700 °C; RT 5.5 s; in N<sub>2</sub></td><td>27% vol</td><td></td></tr><tr><td></td><td>800 °C; RT 5.5 s; in N<sub>2</sub></td><td>44% vol</td><td></td></tr><tr><td></td><td>900 °C; RT 5.5 s; in N<sub>2</sub></td><td>38% vol</td><td></td></tr><tr><td rowspan="2">Palm kernel shell</td><td>600 °C; RT 5.5 s; in N<sub>2</sub></td><td>19% vol</td><td rowspan="2">[292]</td></tr><tr><td>700 °C; RT 5.5 s; in N<sub>2</sub></td><td>17% vol</td></tr><tr><td></td><td>800 °C; RT 5.5 s; in N<sub>2</sub></td><td>22% vol</td><td></td></tr><tr><td></td><td>900 °C; RT 5.5 s; in N<sub>2</sub></td><td>25% vol</td><td></td></tr><tr><td rowspan="2">Red algae (Furcellaria lumbracilis and Polysiphonia-Ceramium), dried</td><td>600 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>23% vol</td><td rowspan="2">[293]</td></tr><tr><td>400 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>3% vol</td></tr><tr><td rowspan="2">Red algae (Furcellaria lumbracilis and Polysiphonia-Ceramium), washed and dried</td><td>500 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>13% vol</td><td rowspan="2">[293]</td></tr><tr><td>600 °C; HR 15 °C/min; RT 60 min; in N<sub>2</sub></td><td>21% vol</td></tr></tbody></table>
* Concentration read from a figure; HR – heating rate; RT – residence time.

compounds, and the next three classes (from third to fifth) are depen-
dent on the number of aromatic rings [303]. Additionally, as the sig-
nificance of the primary ways of tar reduction cannot be exaggerated
[302], management of the gasification process inside the gasifier's
volume is essential in the case of any measurements of tar content.
Unfortunately, this calls for consistent performance under ideal cir-
cumstances [302]. In general, fixed-bed gasifier technology is widely
known [294,311–315]. However, there is still room for development.
Additionally, fixed bed gasifiers appear promising in terms of balancing
the intermittent sources, as the literature sources mention that the
minimum load (defined as the volume of gas) is as low as 5.5 % for
Zeuch-type gasifiers with changeable cast rings, 33 % for Imbert-type
gasifiers, and 22 % for V-Hearth type gasifiers [294,311].

Among fixed-bed gasification reactors, particular interest is currently
directed towards the direct use of concentrated solar power for
improving gasification since studies reported that biomass conversion
could double under such conditions [316].

Tars remain problematic, especially for fixed-bed gasifiers. Total tar concentration was reported to be 32.3 and 132.4 g/m³ for downdraft and updraft operation of a stratified gasifier, respectively [317]. Downdraft gasifiers were reported to produce 4.9 g of tars per m³ of gas for oil palm fronds [318]. Feedstock such as hazelnut shells achieved tar concentrations ranging between 2.6 and 4.0 g/m³ at the outlet of the downdraft gasifier, with a throat resembling the Imbert-type [319]. Gasification of sewage sludge, depending on pretreatment, resulted in concentrations of phenols (including derivatives) ranging between 100.2 and 792.5 mg/m³ [320].

From the principle point of view, a fluidised bed seems to be pref-
erable due to its superb mixing [321], which in terms of fluidised bed
gasifiers means mixing of bed, fuel particles, gasification agent, and
products. This could help avoid significant temperature gradients within
the reactor, thus making temperature control relatively simple when
compared to fixed bed gasifiers. Depending on the feedstock and oper-
ating parameters, such as temperature or equivalence ratio (ER), flui-
dised bed gasification can also result in a significant concentration of
tars. For example, fluidised bed gasification of woody biomass using a
pilot-scale bubbling fluidised bed gasifier was reported to reach tar

concentrations ranging between 7.5 and 8.0 g/m³ while operating at
800 °C with ER between 0.25 and 0.35 [322].

The literature shows that compounds such as naphthalene, with relatively high melting points, can be especially detrimental to the operation of downstream equipment in the gasification installation [323]. [324] suggested using a tar deposition diagram to predict the potential severity of the tar deposition issues in downstream equipment. An industrial-scale dual fluidized bed biomass steam gasification of woodchips can result in concentrations of naphthalene and acenaphthalene reaching 3822 and 233 mg/m³, respectively [325]. Gasification of *Cynara cardunculus* L. in a bubbling fluidized bed filled with olivine is capable of producing more than 5.0 g/m³ of naphthalene at 800 °C and approximately 3.5 g/m³ of naphthalene at 700 °C [326].

Apart from tars, other pollutants present in the gas, especially com-
pounds containing sulfur, like H₂S, could be detrimental to downstream
unit processes that use catalysts (reforming, WGS). However, such pol-
lutants can be dealt with effectively by caustic scrubbers [327] or even
char from gasification used as a sorbent [328].

Despite problems with tars, the gasification process is capable of
generating a producer gas with a relatively high concentration of
hydrogen. For the fluidized bed gasification of sewage sludge in a lab-
scale gasifier, some investigations observed a relatively high hydrogen
concentration, topping 40 % [329]. Fixed-bed gasification values were
slightly lower but still more than 30 % [330]. By raising the air tem-
perature at a fixed bed gasifier's input, more combustible compounds
can be produced during the gasification of sewage sludge [312].
Nonetheless, hydrogen content in producer gas (Table 6) is insufficient.
Thus, further processing is required (Fig. 6).

4.2.2.1. *Supercritical water gasification.* Supercritical water has been gaining attention lately as a gasification medium [337,338]. In nature, water does not often appear in a supercritical state, apart from primary jets of hydrothermal vents located at large depths in the oceans, where the temperature can locally exceed 390 °C [339,340]. On the industrial scale, supercritical water is commonly used in supercritical and ultra-supercritical water boilers, which offer important benefits in terms of efficiency improvements with increased temperature and pressure in the superheater section [341,342]. In recent years, some attention has also been directed towards the thermal processing of biomass using

<figure><img src="image_10.png" /><figcaption>Fig. 7. Types of gasifiers.</figcaption></figure>
<table><caption>**Table 6**
Hydrogen content – gas from gasification.</caption><thead><tr><th>Feedstock</th><th>Gasifier</th><th>Process conditions</th><th>H<sub>2</sub> content</th><th>Ref.</th></tr></thead><tbody><tr><td>softwood pellets</td><td></td><td>722.5 °C; p 0.2 kPa below ambient; ER = 0.31; air as gasifying agent</td><td>6 %vol</td><td></td></tr><tr><td>solid recovered fuel</td><td>pilot-scale; hybrid updraft/cross-draft; autothermal; continuous</td><td>773.7 °C; p 0.2 kPa below ambient; ER = 0.31; air as gasifying agent</td><td>9 %vol</td><td>[331]</td></tr><tr><td>small wood chips (15 mm part. size)</td><td></td><td>684.5 °C; ER = 0.06; air as gasifying agent</td><td>5 %vol</td><td>[323]</td></tr><tr><td rowspan="3">brewers spent grain</td><td rowspan="3">lab-scale fixed bed; allothermal; batch</td><td>800 °C; CO<sub>2</sub> as gasifying agent</td><td>8.7 % vol</td><td></td></tr><tr><td>900 °C; CO<sub>2</sub> as gasifying agent</td><td>40 %vol</td><td></td></tr><tr><td>1000 °C; CO<sub>2</sub> as gasifying agent</td><td>31 %vol</td><td></td></tr><tr><td rowspan="3">wheat straw</td><td rowspan="3"></td><td>800 °C; CO<sub>2</sub> as gasifying agent</td><td>30 %vol</td><td></td></tr><tr><td>900 °C; CO<sub>2</sub> as gasifying agent</td><td>23 %vol</td><td>[332]</td></tr><tr><td>1000 °C; CO<sub>2</sub> as gasifying agent</td><td>20 %vol</td><td></td></tr><tr><td rowspan="3">pine sawdust</td><td rowspan="3"></td><td>800 °C; CO<sub>2</sub> as gasifying agent</td><td>24 %vol</td><td></td></tr><tr><td>900 °C; CO<sub>2</sub> as gasifying agent</td><td>24 %vol</td><td></td></tr><tr><td>1000 °C; CO<sub>2</sub> as gasifying agent</td><td>28 %vol</td><td></td></tr><tr><td>sewage sludge</td><td>pilot-scale; downdraft; 20 kg/h of feedstock; autothermal with electric air preheating</td><td>725 ± 50 °C; ER = 0.4; air preheated to 400 °C as gasifying agent</td><td>3.18 % vol</td><td>[333]</td></tr><tr><td rowspan="2">wheat straw pellets</td><td rowspan="2">lab-scale; updraft; autothermal; batch</td><td>ER range 0.12-0.28; air as gasifying agent</td><td>4.5* % vol</td><td></td></tr><tr><td>ER range 0.30-0.45; air as gasifying agent</td><td>4.8* % vol</td><td>[334]</td></tr><tr><td rowspan="2">laying hens manure pellets</td><td rowspan="2"></td><td>ER range 0.12-0.28; air as gasifying agent</td><td>3.8* % vol</td><td></td></tr><tr><td>ER range 0.30-0.45; air as gasifying agent</td><td>4.5* % vol</td><td></td></tr><tr><td rowspan="2">wood pellets</td><td rowspan="2">low-tar biomass (LTB) gasifier; autothermal; pilot-scale</td><td>1100 °C* in oxidation zone; ER 0.13; air as gasifying agent</td><td>23.8 % vol</td><td></td></tr><tr><td>950 °C* in oxidation zone; ER 0.27; air as gasifying agent</td><td>20.6 % vol</td><td>[335]</td></tr><tr><td rowspan="2">wood chips</td><td rowspan="2"></td><td>1000 °C* in oxidation zone; ER 0.41; air as gasifying agent</td><td>15.8 % vol</td><td></td></tr><tr><td>750 °C; steam gasification; S/B ratio 0.5</td><td>22.8 ± 0.6 % vol</td><td></td></tr><tr><td rowspan="5">brewers spent grain</td><td rowspan="5">lab-scale; allothermal;</td><td>750 °C; steam gasification; S/B ratio 1.0</td><td>25.3 ± 0.4 % vol</td><td></td></tr><tr><td>800 °C; steam gasification; S/B ratio 0.5</td><td>23.9 ± 0.1 % vol</td><td>[336]</td></tr><tr><td>800 °C; steam gasification; S/B ratio 1.0</td><td>26.6 ± 0.9 % vol</td><td></td></tr><tr><td>850 °C; steam gasification; S/B ratio 0.5</td><td>27.8 ± 0.3 % vol</td><td></td></tr></tbody></table>

<table><caption>**Table 6 (continued)**</caption><thead><tr><th>Feedstock</th><th>Gasifier</th><th>Process conditions</th><th>H<sub>2</sub> content</th><th>Ref.</th></tr></thead><tbody><tr><td></td><td></td><td>850 °C; steam gasification; S/B ratio 1.0</td><td>30.2 ± 2.8 % vol</td><td></td></tr></tbody></table>

1 ER – equivalence ratio; S/B – steam to biomass ratio; * – approximate value, read from a figure.

supercritical water [343,344] since hydrothermal processing is especially suitable for wet types of biomass [345,346]. Moreover, interesting changes in water properties were observed when reaching supercritical parameters, including a significant decrease in dielectric constant, ionic product, density, and viscosity [343]. Due to relatively high temperatures, pressures, and corrosive environment, owing to the use of supercritical water, reactors for supercritical water gasification (SWG) operate in a harsh environment, which requires the use of alloys with high resistance to corrosion [343]. An overview of the reactors for SWG is presented in Fig. 8. In general, the use of such materials combined with the overall mass of the reactors (as a result of temperature and pressure) makes SWG reactors relatively expensive [343].

Some of the reactors shown in Fig. 8 are suitable for fundamental research purposes, which makes their scale limited. Moreover, reactors based on tanks could also be considered limited in terms of their upscaling potential due to comparably higher diameters compared to tube reactors, which require relatively higher thickness. Moreover, tube reactors operating in a continuous manner can get close to plug flow, thus having limited requirements for mixing, whereas the mixing problem in big tank reactors requires customised solutions.

Despite these problems, SWG has been considered by many as an interesting process in terms of biohydrogen production since it is one of few processes suitable for wet biomass [347,348]. A pressurised environment ensures relatively compact size reactors. Residence time is comparably short, in contrast to retention times of microbial-based processes (e.g., dark fermentation), i.e., it is possible for SWG to achieve residence times within the order of magnitude of minutes [347], in contrast to hours in the case of dark fermentation [62]. However, the mass of the reactor and its maintenance becomes an important issue at such temperatures and pressures. Apart from corrosion, deposition problems can also negatively influence the operation of such reactors [343]. Relatively high concentrations of hydrogen can be achieved (Table 7), depending on feedstock and process conditions. Also, tars could potentially be considered a problem, as Zhang et al. [349] reported tar yield as high as 1.1 % for SGW of glucose at 700 °C. Nonetheless, the use of 0.1RuNi/γ-Al<sub>2</sub>O<sub>3</sub> catalyst allowed for decreasing the tar yield to values as low as 0.08 % for SWG of glucose while applying the same process conditions [349]. This was accompanied by a substantial increase in H<sub>2</sub> concentration in the gas from 30.9 to 61.9 % vol (see Table 7). From the point of view of further development of this particular technology for hydrogen production, it seems sensible to advise more work focused on the generation of tars, as well as other deposits, potentially detrimental from the maintenance perspective.

## 5. Upgrading of produced hydrogen

### 5.1. Upgrading via reforming and WGS

It is possible to convert hydrocarbons containing hydrogen atoms to molecular hydrogen. The most common process performing such conversion is steam reforming of methane, described by the reaction shown in eq. (3) [352,353]:

$$CH_4 + H_2O \rightarrow CO + 3H_2 \quad (4)$$

Several different reactions are possible during steam reforming, including those shown by eqs. (4) and (5), depending on steam to carbon
<figure><img src="image_11.png" /><figcaption>Fig. 8. Types of supercritical water reactors suitable to SWG (based on [343], with the addition of ceramic reactors reported by [344]; B – batch, C – continuous, SB – semi-batch).</figcaption></figure>

ratio [352]:

$$C_x H_y + x H_2 O \rightarrow x CO + (x + y/2) H_2 \quad (5)$$

$$C_x H_y + 2x H_2 O \rightarrow x CO_2 + (2x + y/2) H_2 \quad (6)$$

As clearly shown by eqs. (4) and (5), apart from methane, other hydrocarbons formed in the producer gas could react with steam. Steam reforming of toluene is a good example of steam reforming of a compound considered as tar – as shown by the reactions depicted in eqs. (6) and (7) [354,355]:

$$C_7 H_8 + 7 H_2 O \rightarrow 7 CO + 11 H_2 \quad (7)$$

$$C_7 H_8 + 14 H_2 O \rightarrow 7 CO_2 + 18 H_2 \quad (8)$$

Due to these reasons, reforming of hydrocarbons in the presence of steam results in increased formation of CO and CO₂ [354].

Due to the presence of CO₂ in the producer gas, a dry reforming reaction can also take place, as described by eq. (8) [356]:

$$CH_4 + CO_2 \rightarrow 2CO + 2H_2 \quad (9)$$

Dry reforming was first investigated by Fischer and Tropsch as early as 1928, using Ni and Co catalysts [356]. Both steam reforming and dry reforming are endothermic and require heat [356]. Due to these reasons, partial oxidative reforming has been considered [353,356], especially in the context of methane reforming, which is exothermic and occurs as shown in eq. (9) [356]:

$$CH_4 + 1/2 O_2 \rightarrow CO + 2H_2 \quad (10)$$

Combining steam reforming and partial oxidation results in so-called autothermal reforming, which is depicted in eq. (10) [356]:

$$7CH_4 + 3O_2 + H_2O \rightarrow 7CO + 15H_2 + CO_2 \quad (11)$$

This process is mildly exothermic [356]. On the other hand, so-called tri-reforming is mildly endothermic [356] and could be considered an interesting alternative from the energy standpoint. The tri-reforming of methane is shown in eq. (11):

$$20CH_4 + 9O_2 + H_2O + CO_2 \rightarrow 21CO + 41H_2 \quad (12)$$

Typically, reforming is a catalytic process, with the range of

temperatures provided by literature as 700–1000 °C [353]. No matter what type of reforming is performed, substantial amounts of CO are found among the products. Thus, the water gas shift (WGS) reaction is intensively investigated in the context of biohydrogen production [357]. WGS reaction is shown in eq. (12) [353]:

$$CO + H_2O \rightarrow CO_2 + H_2 \quad (13)$$

WGS is one of the reactions that take place during reforming [357]. However, full conversion of CO to CO₂ could be achieved with proper catalysts at temperatures lower than those typical of reforming, with temperatures provided in the literature ranging between 175 and 280 °C [357]. Typical catalysts for WGS are Fe, Ni, Cu, Ru, Rh, Pt and Pd-based catalysts supported in Al₂O₃, TiO₂, CeO₂, or their combinations. Less noble supports, such as MgAl, Mg, Al and CaAl [358] and other innovative supports, such as carbon nanofibers or nanoparticles, are also used [359]. Recent developments in this field proposed the use of bio-sourced catalysts based on biochar rich in less toxic metals, such as iron, for WGS and RWGS reactions. In this case, the role of the catalyst structure and the synergistic effect between the metals present in the bio-sourced material were shown to be crucial for enhancing catalyst activity [360].

Interestingly, the steam reforming of biomass (equivalently: steam gasification of biomass), whether it occurs at atmospheric pressure or at high pressure, downstream of the decomposition of the biomass, can be simplified and studied at thermodynamic equilibrium using only the two reactions (3) and (12) reported above, as demonstrated by Castello and Fiori [361,362]. Reforming catalysts are also based on noble metals. Ni-based catalysts were reported to be the better compromise between efficiency and cost for steam reforming, compared to Ru, Rh and Pt-based catalysts. Alumina (Al₂O₃) is the most usual support for reforming catalysts, especially due to its low cost, neutrality in the process, lifetime and regenerability [363]. The limitation of the use of bio-sourced catalysts in high-temperature thermochemical conversion processes is related to the degradation of the carbon structure, which needs to be previously stabilized.

It should not be overlooked that sulfur content in biomass can be problematic when it comes to reforming of hydrogen reach gas through thermal processes, such as biomass gasification, due to potential issues with catalyst poisoning. In some conditions, coking could also be a problem if deposition takes place at active sites of the catalyst. However,
<table><caption>**Table 7**

Hydrogen content – gas from supercritical water gasification.</caption><thead><tr><th>Feedstock</th><th>Process conditions and reactor</th><th>H<sub>2</sub> content</th><th>Ref.</th></tr></thead><tbody><tr><td rowspan="5">water 95.0%<sub>m</sub>, glucose 5.0%<sub>m</sub><br>water 95.0%<sub>m</sub>, glucose 4.5%<sub>m</sub>, phenol 0.5%<sub>m</sub><br>water 95.0%<sub>m</sub>, glucose 4.0%<sub>m</sub>, phenol 1.0%<sub>m</sub><br>water 95.0%<sub>m</sub>, glucose 3.5%<sub>m</sub>, phenol 1.5%<sub>m</sub><br>Beech sawdust</td><td>T = 400 °C; RT 240 s</td><td>12.0%<sub>vol</sub><br>9.8%<sub>vol</sub></td><td>[350]</td></tr><tr><td>T = 400 °C; RT 16 h; Inconel 625 reactor</td><td>*6%<sub>vol</sub></td><td>[344]</td></tr><tr><td>T = 400 °C; RT 16 h; Stainless steel reactor</td><td>*13%<sub>vol</sub></td><td></td></tr><tr><td>T = 400 °C; RT 16 h; Ceramic reactor</td><td>*7%<sub>vol</sub></td><td></td></tr><tr><td>Municipal waste</td><td>T = 400 °C; RT 16 h; Inconel 625 reactor</td><td>*6%<sub>vol</sub></td><td></td></tr><tr><td rowspan="5">Spent grains</td><td>T = 400 °C; RT 16 h; Stainless steel reactor</td><td>*10%<sub>vol</sub></td><td></td></tr><tr><td>T = 400 °C; RT 16 h; Ceramic reactor</td><td>*8%<sub>vol</sub></td><td></td></tr><tr><td>T = 400 °C; RT 16 h; Inconel 625 reactor</td><td>*4.5%<sub>vol</sub></td><td></td></tr><tr><td>T = 400 °C; RT 16 h; Stainless steel reactor</td><td>*5%<sub>vol</sub></td><td></td></tr><tr><td>T = 400 °C; RT 16 h; Ceramic reactor</td><td>*4%<sub>vol</sub></td><td></td></tr><tr><td>liquid fraction after HTC of digestate from anaerobic digestion of cattle manure at 180 °C; concentr. of HTC liquid in water 6.7%<sub>vol</sub></td><td>T = 500 °C; RT 30 s; Stainless steel reactor</td><td>*73%<sub>vol</sub></td><td>[351]</td></tr><tr><td>liquid fraction after HTC of digestate from anaerobic digestion of cattle manure at 220 °C; concentr. of HTC liquid in water 6.7%<sub>vol</sub></td><td>T = 600 °C; RT 90 s; Stainless steel reactor</td><td>*73%<sub>vol</sub></td><td></td></tr><tr><td>liquid fraction after HTC of digestate from anaerobic digestion of cattle manure at 180 °C; concentr. of HTC liquid in water 20.0%<sub>vol</sub></td><td>T = 600 °C; RT 30 s; Stainless steel reactor</td><td>*58%<sub>vol</sub></td><td></td></tr><tr><td>Glucose, concentration 50 g/L</td><td>T = 700 °C; p = 24 MPa; bench-scale down-flow tubular reactor, made of Inconel 625</td><td>30.9%<sub>vol</sub></td><td>[349]</td></tr><tr><td rowspan="3">Glucose, concentration 50 g/L</td><td>T = 700 °C; p = 24 MPa; bench-scale down-flow tubular reactor, made of Inconel 625; 0.1RuNi/ γ-Al<sub>2</sub>O<sub>3</sub> catalyst; Weight hourly space velocity 6/h</td><td>61.9%<sub>vol</sub></td><td></td></tr><tr><td>T = 700 °C; p = 24 MPa; bench-scale down-flow tubular reactor, made of Inconel 625; 0.1RuNi/ γ-Al<sub>2</sub>O<sub>3</sub> catalyst; Weight hourly space velocity 6/h</td><td>62.8%<sub>vol</sub></td><td></td></tr><tr><td>T = 600 °C; p = 24 MPa; bench-scale down-flow tubular reactor, made of Inconel 625; 0.1RuNi/ γ-Al<sub>2</sub>O<sub>3</sub> catalyst; Weight hourly space velocity 6/h</td><td>39.5%<sub>vol</sub></td><td></td></tr><tr><td>simulated waste: anhydrous D-glucose, acetic acid and guaiacol at a mass ratio of 84:15:1; feedstock concentration 50 g/L</td><td>T = 700 °C; p = 24 MPa; bench-scale down-flow tubular reactor, made of Inconel 625; 0.1RuNi/ γ-Al<sub>2</sub>O<sub>3</sub> catalyst; Weight hourly space velocity 6/h</td><td>41.0%<sub>vol</sub></td><td></td></tr><tr><td>simulated waste: anhydrous D-glucose, acetic acid and guaiacol at a mass ratio of 84:15:1; feedstock concentration 50 g/L</td><td>T = 700 °C; p = 24 MPa; bench-scale down-flow tubular reactor, made of Inconel 625; 0.1RuNi/ γ-Al<sub>2</sub>O<sub>3</sub> catalyst; Weight hourly space velocity 6/h</td><td>41.0%<sub>vol</sub></td><td></td></tr><tr><td>aqueous organic waste fraction from hydrothermal co-liquefaction of a mixture of secondary pulp/paper-mill sludge and waste newspaper at 300 °C; RT 20 min</td><td>T = 700 °C; p = 24 MPa; bench-scale down-flow tubular reactor, made of Inconel 625; 0.1RuNi/ γ-Al<sub>2</sub>O<sub>3</sub> catalyst; Weight hourly space velocity 2.5/h</td><td>41.0%<sub>vol</sub></td><td></td></tr></tbody></table>

this problem can be dealt with by the use of additives, such as hydrogen peroxide, for the steam reforming process [364].

## 5.2. Separation of produced hydrogen

Membrane separation in the context of purification of hydrogen produced by biosynthesis is becoming popular. Among many membrane techniques, those where the separation mechanism occurs by sorption and diffusion are used. Porous membranes are less popular in this case. Two directions are widely tested here: separation of gaseous streams using polymer and ceramic membranes and separation using liquid membranes.

In the case of the separation of gases and their mixtures, two parameters are important: selectivity and permeability. The first parameter should be as high as possible in relation to the desired product - in this case, hydrogen - and the lowest for others, e.g. CO₂. Permeability should be as high as possible - it indicates the amount of permeate – product. Selectivity and permeation depend on many factors: temperature, partial pressure (gas composition) or working pressure, water content and, above all, the type (material, structure, thickness) of the membrane itself [230].

The benefits of membrane applications include lower energy consumption, reduced maintenance requirements, increased selectivity, higher energy efficiency, simplicity in operation and control, and lower cost of installation and maintenance than other separation techniques [230].

Membranes for gas separation can be divided into three major groups: polymeric, dense metal, silica and zeolitic membranes [230]. Often tested are polymeric ones, mainly due to the low cost. The most popular are cellulose acetate, polysulfone, polyethersulfone, polyimide, polybenzimidazole or polyetherimide. However, their biggest disadvantage is their low resistance against high temperatures.

Recently, Shao et al. [365] tested different types of polymeric membranes. Depending on the material, H₂ and CO₂ permeabilities fluctuated between 2.4 and 125 and 0.6–84.6 barrer, respectively. The selectivity of H₂/CO₂ was 1.5–5.9 for the group of H₂-selective membranes [365]. Polfus et al. [366] investigated the dense ceramic composites of La₂₇W₅O₅₅.₅–LaCrO₃ and obtained a hydrogen permeability of 1.1–10.3 ml/(min·cm²) with a wet sweep gas at 700 °C. The membranes remained stable and demonstrated high performance at this high temperature. Furthermore, they improved the process by coating a Pd layer on the membrane, which can lead to a lowering of the process temperature [366]. Significant lower hydrogen permeability was reported by Islam et al. [367], i.e., 0.07 mL/(min·cm²) for Ni–Ba₀.₈Ce₀.₃₅Zr₀.₅Tb₀.₁₅O₃·₆-based ceramic membrane at 900 °C. Another metallic membrane used by Fasolin et al. [368] allowed for a hydrogen flux of 0.26 mol/(m²·s) by vanadium-based membranes at 375 °C – the membranes were synthesized by the supporting dense and crystalline Pd/V93Pd7/Pd multilayers onto the stable and porous alumina. All of the mentioned membranes were stable in these high-temperature conditions. However, the efficiency was unfortunately quite low.

The second type of inorganic membranes are those made from silica material. They have good permeability of hydrogen, and simultaneously, they are cheaper (in comparison to the other types of membranes) and have good scalability. The preparation methods are also easier than metallic or zeolitic ones [369]. Hence, they are promising in terms of hydrogen separation. Kajama et al. [370] tested silica sol-gel membranes and measured a hydrogen gas permeate flux of 0.482 mol/(s·m²) at 1.0 bar feed pressure. Selectivities of H₂ over N₂, CH₄, and Ar were 3.07, 2.23, and 3.75, respectively.

Another type of membrane worth mentioning is the zeolitic membrane. These inorganic membranes can separate liquid and gas species of very similar size and shape, mainly due to their defined pore size at a molecular level and high adsorption properties [371]. Wang et al. [372] used a membrane consisting of a thin ZSM-5 layer on a thick silicalite

<footnote>%m – fraction by mass; %vol – fraction by volume; RT – residence time; * – approximate value, read from a figure.</footnote>
base layer supported on macroporous α-alumina with a yttria-stabilized zirconia intermediate barrier layer. The zeolitic pores of the thin ZSM-5 layer were narrowed by catalytic cracking deposition. They obtained hydrogen permeability of 1.2·10⁻⁷ mol/(m²·s·Pa), along with the 23, 28, and 180 selectivity of H₂ to CO₂, CO, and H₂O vapours at 500 °C, respectively [372].

A completely different type of membrane separation used in bio-
hydrogen treatment is a process using liquid membranes. In the case of
this system, membranes are in the form of (usually) ionic liquids
immobilized on porous membranes (SLM – supported liquid mem-
branes). In the membrane structure, the sorption-diffusion mechanism
occurs. Neves et al. [373] studied the efficiency of this process and ob-
tained quite promising results. Namely, the highest permeability value
(30.70*10⁻¹¹ m²/s) for the PVDF membrane immobilized with the RTIL
[OMIM][PF6] and the high selectivity values towards CO₂, between 22
(for [OMIM][PF6]) and 35 (for [BMIM][BF4]) [373].

6. Sustainability aspects of biohydrogen production

6.1. *The colours of hydrogen concerning pathways towards decarbonisation*

Since the main impulse for the development of hydrogen production technologies is derived from efforts to decrease emissions and achieve a more environmentally friendly industry and society, it seems sensible to mention current initiatives regarding taxonomy for hydrogen produced using different technologies. Currently, one can distinguish the so-called grey hydrogen, blue hydrogen, turquoise hydrogen, green hydrogen, etc., which emerged as a way to differentiate between hydrogen from different sources [7,374]. Originally, it seemed to be a good idea from a regulatory perspective. However, currently recognized colours of

hydrogen do not seem to have a specific label for biohydrogen (see Fig. 9). Since biomass can be considered carbon-neutral in rough estimates, sometimes biohydrogen is considered green hydrogen. However, most often, green hydrogen is associated with electrolysis powered by renewable energy [7,374]. Moreover, biohydrogen has the potential to achieve a negative carbon footprint [375] as an analogy to bioenergy carbon capture and storage (BECCS) in the energy sector.

The use of BECCS has the potential to significantly reduce CO₂ emissions, achieving negative emissions and carbon offsetting [376,377]. There have been some suggested gasification-based BECCS alternatives [378,379]. Some authors have suggested that pre-combustion CO₂ capture might be feasible [155]. Such methods, however, could only be implemented on a large scale. Some studies suggested smaller co-generation plants that might provide 750 kW_e of installed electricity and 600 kW_t of heat while also capturing 1620 t of CO₂ annually [380]. These plants would use 1.5 MW of biomass and 1.4 MW of biogas [380]. However, for such small-scale installations, the logistics problem between the capture site and storage is still open. Biochar is feasible for long-term carbon storage in soil [381–383]. Moreover, it could be beneficial regarding the amendment of soil properties, including the holding capacity of water, organic matter content, and wettability [381–383], thus resulting in improved agricultural yields [384]. This shows that significant synergistic effects could be achieved by using residues from hydrogen production, even if hydrogen is not produced by electrolysis, with electricity from BECCS, but also when it is produced directly as biohydrogen coupled with carbon capture and storage (BCCCS).

Potentially, BCCCS could provide the market with hydrogen associ-
ated with a negative carbon footprint [375]. This would require adding
another colour to the existing palette of colours, which would still serve
in various future regulations as a practical means of differentiation

<figure><img src="image_12.png" /><figcaption>Fig. 9. The palette of colours for hydrogen – based on [7,374,386,387].</figcaption></figure>
between hydrogen from different sources. However, this would undoubtedly blur the overall picture and make it increasingly more difficult to differentiate between different hydrogen sources on the market. Moreover, Howarth and Jacobson have demonstrated that taking into account methane emissions across the value chain of natural gas, including extraction, transportation, and processing, the greenhouse gas footprint of blue hydrogen is not significantly different to grey hydrogen [385].

Therefore, it might be much simpler and more effective to simply drop the existing labels altogether and instead provide a framework for comprehensive analysis of carbon footprint or greenhouse gas footprint for any particular hydrogen source, e.g., using LCA methodology and tools for such purposes. Some works have already attempted this approach, e.g., by providing the so-called hydrogen cleanness index (HCI) [388]. Nonetheless, simplifications such as using CO₂ emission equivalent per kg of hydrogen seem to be even better than unitless coefficients. Such change would provide useful information for potential customers as well as for different market regulatory bodies and institutions responsible for policies.

## 6.2. Environmental sustainability across the life cycle of biohydrogen

Life cycle assessment (LCA) is a tool with a coherent and standardised methodology which enables the evaluation of the environmental impact of products and processes, including innovations [389]. Numerous examples show the possibility of implementing LCA in assessing the sustainability of novel solutions in various distinctly different fields, such as household [390] or textile [391] waste management, recycling of plastics [392], design of municipal infrastructure [390], valorisation of lignocellulosic waste [393], seaweed cultivation [394] or valorisation [293], utilisation of waste from wind turbines [395], production of sorbents [331,396], or bio-based peat alternatives [397], just to name a few. Prospective LCA is deemed a viable tool to assess the environmental implications of technological innovations [398], and it has also been applied to hydrogen production [399]. Such an ex-ante type of LCA is not used to predict the future but rather to explore the range of possible scenarios within the market space where the technology under investigation could operate [398].

LCA has also been used to assess the sustainability of the reforming of natural gas, which is currently a dominant method of hydrogen production worldwide [400]. Reforming can result in direct CO₂ emissions ranging between 8.8 kgCO₂/kgH₂ and 12.9 kgCO₂/kgH₂ [400], depending on the technologies used for reforming and CO₂ capture. However, Khojasteh Salkuyeh et al. [400] determined that across the complete life cycle, the global warming potential of production of hydrogen through steam methane reforming with subsequent carbon capture and storage could be as low as 3.5 kgCO₂eq/kgH₂. On the other hand, Howarth and Jacobson demonstrated that upstream emissions of methane could have detrimental effects in terms of the global warming potential of so-called blue hydrogen [385]. Thus, much depends on the scope of such assessment, as well as on upstream emissions of methane within a particular value chain. Motazedi et al. [401] demonstrated that life cycle GHG emissions for high-temperature steam electrolysis can be as low as 3.0 kgCO₂eq/kgH₂. Although the relatively expensive cost of CO₂, reaching 360 USD/tonneCO₂ is needed to make such technologies competitive with steam reforming in terms of the economics of such systems [401]. Moreover, the life cycle costing analysis performed by Khzouz et al. [402] demonstrated the crucial role of capacity factors in hydrogen nominal cost when the production of hydrogen for vehicles is considered. This might pose some problems in the future if the production of hydrogen by electrolysis is considered a way to prevent curtailment of electricity generation from intermittent renewable energy sources since, by default, the production of hydrogen in such a way could hardly be expected to reach high-capacity factors. On the other hand, novel technologies might benefit from their current position on the learning curve, which could lead to the revision of economic parameters in the

future, at least in terms of the capital costs of such investment. Due to this fact, it seems sensible to investigate different H₂ production routes, including biohydrogen. In such cases, LCA should go hand in hand with economic analyses, or even better, Life Cycle Costing (LCC), since the primary goal of the energy transition is sustainability, and economic analyses can only reveal the most suitable pathways in the foreseeable future.

In terms of biohydrogen production, the sustainability and environmental impact of many different prospective systems have been assessed using LCA (Table 8) and, in many cases, compared to state-of-the-art technologies, like steam methane reforming. Many sources provide similar values of the global warming impact of steam methane reforming without carbon capture and storage, typically ranging between 10 and 12 kgCO₂eq/kgH₂ [400,401,403]. A comparison of global warming impact against steam methane reforming seems to be relatively straightforward since this is a mature technology which could be applied on a large scale. Moreover, in the case of global warming, the impact of fossil-based technologies and capital goods has relatively minor significance [404], which indicates that excluding the construction of the facilities and manufacturing of equipment used in such installations might not lead to significant errors. This could be attributed to the effect of scale, as fossil-based refineries are typically implemented on a large scale [399]. However, this might not be the case for newly developed technologies, which in many cases are still between technology readiness levels (TRL) 4 and 7 [405]. Thus, it should not be discouraging to find a significantly higher impact for novel technologies since they are located relatively low on the learning curve, and much can be improved between laboratory experiments and the operation of pilot plants. This seems to be supported by the data from existing studies (Table 8), as in many cases, laboratory and pilot-scale technologies result in a relatively high global warming impact.

Studies at such an early stage could be used for the identification of hot spots, facilitating the search for areas where the highest improvement of environmental performance could be achieved during the next stages of technology development. Surprisingly, in many cases, sensitivity analysis has not been performed in LCA studies concerning biohydrogen (Table 8). Also, in many cases, LCA studies could have provided much better information if the uncertainty aspect was a subject of consideration. This is especially important, taking into account that error bars can easily be as high as the values for such prospective LCA studies, as demonstrated by LCA studies on electrolysis [401]. Another issue is the tendency to overfocus on the category of global warming impact (Table 8), which is certainly important. However, flagging the potential hot spots and searching for improvements could, in many cases, become a very important outcome of a prospective LCA study [398,399,413]. Thus, it seems sensible to advise reporting results from many different impact categories if applied methods and collected data allow for obtaining such information.

Another important information provided by LCA studies is the fact that it is indeed possible to obtain negative values within the global warming impact category (Table 8). This is important not only in the context of BECCS plants. It also indicates possibilities of decarbonisation in branches of industry other than energy generation. Care should be taken when the substitution of hydrogen from fossil sources is considered since substitution hardly ever could be assumed to be equal to 1 [414]. Nonetheless, considering full product systems, which not only produce a hydrogen-rich gas but are capable of producing high-purity hydrogen (see section 5), gives higher chances of achieving a higher realistic substitution ratio, especially if it could be conclusively proven that produced hydrogen falls within industrial quality specifications.

## 6.3. Economic sustainability

The sustainability of any technology always rests on three pillars, namely ecological, economic, and social sustainability. Thus, bio-hydrogen production processes must be economically feasible in
<table><caption>**Table 8**

Results of LCA of different biohydrogen production technologies per functional unit (* indicates reading values from graphs; ** 90 % confidence interval; COD – chemical oxygen demand; CTU - comparative toxic units; DCB – dichlorobenzene; FU – functional unit; HHV – higher heating value basis; MSW – municipal solid waste; NMVOC – non-methane volatile organic compounds; PSA – pressure swing adsorption).</caption><thead><tr><th rowspan="2">Technology</th><th colspan="9">Selected impact categories</th><th rowspan="2">Functional unit</th><th rowspan="2">Construction of facilities included</th><th rowspan="2">Uncertainty / sensitivity</th><th rowspan="2">Ref</th></tr><tr><th>Global warming, g CO<sub>2</sub>-eq / FU</th><th>Acidification (units below)</th><th>Eutrophication, (units below)</th><th>Ecotoxicity fresh water, (units below)</th><th>Water consumption, m<sup>3</sup> / FU</th><th>Photochemical ozone formation, kg NMVOC<sub>eq</sub></th><th>Ozone layer depletion, kg CFC-11<sub>eq</sub> / FU</th><th>Non-renewable energy, MJ / FU</th></tr></thead><tbody><tr><td>Microbial electrolysis cell; pilot scale; urban wastewater 65 L/d, COD 500 mg/L; hydraulic retention time 2 d; organic loading rate 0.25 gCOD·L<sup>-1</sup>·d<sup>-1</sup>; hydrogen production rate 4.1 L/d</td><td>min: 18.8 max: *430</td><td>min: *1.6 kg SO<sub>2</sub>-eq max: *2.8 kg SO<sub>2</sub>-eq</td><td>-</td><td>-</td><td>-</td><td>-</td><td>min: *-3.5·10<sup>-6</sup> max: *6.5·10<sup>-6</sup></td><td>min: *2500 max: *5300</td><td>1 kg H<sub>2</sub></td><td></td><td>Sensitivity analysis: H<sub>2</sub> recovery ratio, COD removal efficiency, inlet COD, COD volume loading rate, cathodic gas recovery, energy conversion efficiency, applied voltage.</td><td>[403]</td></tr><tr><td>Semi-commercial gasification of municipal solid waste in United Kingdom: fluidised bed gasifier using oxygen and steam, tar reforming, syngas cleaning, water gas shift reactor, CO<sub>2</sub> separation with PSA and subsequent liquefaction (for storage)</td><td>- 183</td><td>0.204 M of H<sub>eq</sub><sup>+</sup></td><td>0.00741 kg P<sub>eq</sub></td><td>2930 CTU</td><td>-</td><td>0.243</td><td>-</td><td>-</td><td>1 MWh<sub>HHV</sub></td><td>No</td><td>Uncertainty analysis performed</td><td>[406]</td></tr><tr><td>Sequential dark fermentation - photofermentation system, using orange peel waste as a feedstock (laboratory scale for identification of hot spots)</td><td>*650</td><td>-</td><td>-</td><td>-</td><td>*8</td><td>-</td><td>-</td><td>-</td><td>1 kg H<sub>2</sub></td><td>No</td><td>No</td><td>[407]</td></tr><tr><td>Dark fermentation, using orange peel waste as a feedstock (laboratory scale for identification of hot spots)</td><td>*350</td><td>-</td><td>-</td><td>-</td><td>*12</td><td>-</td><td>-</td><td>-</td><td></td><td></td><td>No</td><td>[407]</td></tr><tr><td>Photofermentation, using orange peel waste as a feedstock (laboratory scale for identification of hot spots)</td><td>1642.8</td><td>-</td><td>-</td><td>-</td><td>22.3</td><td>-</td><td>-</td><td>-</td><td></td><td></td><td>No</td><td>[407]</td></tr><tr><td>Two fluidised-bed reactors: gasifier and char combustor; tar reformer; H<sub>2</sub> separation with PSA adsorption; CO<sub>2</sub> capture and compression. System simulated in Aspen Plus.</td><td>-14.63</td><td>2.07·10<sup>-2</sup> kg SO<sub>2</sub>-eq</td><td>4.66·10<sup>-3</sup> kg PO<sub>4</sub><sup>3-</sup></td><td>-</td><td>-</td><td>-</td><td>2.85·10<sup>-7</sup></td><td>48.54</td><td>1 kg H<sub>2</sub></td><td>No</td><td>No</td><td>[408]</td></tr><tr><td>Fluidised bed gasification (oxygen as agent),</td><td>-0.5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>280</td><td>1 kg H<sub>2</sub></td><td>No</td><td>No (sensitivity analysis performed)</td><td>[409]</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 8 (continued)</caption><thead><tr><th rowspan="2">Technology</th><th colspan="8">Selected impact categories</th><th rowspan="2">Functional unit</th><th rowspan="2">Construction of facilities included</th><th rowspan="2">Uncertainty / sensitivity</th><th rowspan="2">Ref</th></tr><tr><th>Global warming, g CO<sub>2</sub>-eq / FU</th><th>Acidification (units below)</th><th>Eutrophication, (units below)</th><th>Ecotoxicity fresh water, (units below)</th><th>Water consumption, m<sup>3</sup> / FU</th><th>Photochemical ozone formation, kg NMVOC<sub>eq</sub></th><th>Ozone layer depletion, kg CFC-11<sub>eq</sub> / FU</th><th>Non-renewable energy, MJ / FU</th></tr></thead><tbody><tr><td>including upstream preparation of biomass (pine wood), without CO<sub>2</sub> capture</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>just for economic assessment)</td></tr><tr><td>Fluidised bed gasification (oxygen as agent), including upstream preparation of biomass (pine wood), with CO<sub>2</sub> capture</td><td>-15.8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>310</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>Entrained flow gasification (oxygen as agent), including upstream preparation of biomass (pine wood), without CO<sub>2</sub> capture</td><td>-0.1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td></td><td></td></tr><tr><td></td><td>Entrained flow gasification (oxygen as agent), including upstream preparation of biomass (pine wood), with CO<sub>2</sub> capture</td><td>-21.8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td><td></td><td></td></tr><tr><td>Solar-driven (concentrated solar power) indirect gasification of biomass. Data from modelling and pilot plant.</td><td>1.04 (**2.19-0.61)</td><td>0.0369 (**0.0367-0.0369) kg SO<sub>2</sub>-eq</td><td>0.0009 (**0.0009-0.0009) kg P<sub>eq</sub></td><td>0.0377 (**0.0318-0.0328) kg 1,4-DCB<sub>eq</sub></td><td>4.9425 (** 4.9420-4.9416)</td><td>-</td><td>2.50·10<sup>-5</sup> (**-2.38·10<sup>-5</sup> - 2.45·10<sup>-5</sup>)</td><td>-</td><td>1 kg H<sub>2</sub></td><td>Yes</td><td>Pedigree matrix; Monte Carlo, 10,000 iterations, assumed log-normal distributions; sensitivity analysis for capacity-factor of solar driven operation, feedstock consumption, average biomass transport distance, and flue gas emissions. Monte Carlo, 1000 iterations, triangular distributions. For sensitivity analysis variables such as feedstock conversion rate, [410]</td></tr><tr><td>Comparative analysis for different technologies for production of vehicle-grade hydrogen, using municipal waste: MSW gasification (Scenario 1), waste wood gasification</td><td>min: *4 max: *6.5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>1 kg H<sub>2</sub></td><td>No</td><td>Pedigree matrix; Monte Carlo, 10,000 iterations, assumed log-normal distributions; sensitivity analysis for capacity-factor of solar driven operation, feedstock consumption, average biomass transport distance, and flue gas emissions. Monte Carlo, 1000 iterations, triangular distributions. For sensitivity analysis variables such as feedstock conversion rate, [411]</td></tr></tbody></table>
<table><caption>Table 8 (continued)</caption><thead><tr><th rowspan="2">Technology</th><th colspan="3">Selected impact categories</th><th colspan="3">Functional unit</th><th rowspan="2">Construction of facilities included</th><th rowspan="2">Uncertainty / sensitivity</th><th rowspan="2">Ref</th></tr><tr><th>Global warming, g CO<sub>2-eq</sub> / FU</th><th>Acidification (units below)</th><th>Eutrophication, (units below)</th><th>Ecotoxicity fresh water, (units below)</th><th>Water consumption, m<sup>3</sup> / FU</th><th>Photochemical ozone formation, kg NMVOC<sub>eq</sub></th><th>Ozone layer depletion, kg CFC-11<sub>eq</sub> / FU</th><th>Non-renewable energy, MJ / FU</th><th>capacity factor, and operational time in hours were considered.</th></tr></thead><tbody><tr><td>(Scenario 2), wet waste dark fermentation (Scenario 3) and wet waste combined dark and photo fermentation (Scenario 4). Secondary data used for each scenario.</td><td>min: *100 max: *500</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1 ton of MSW</td><td></td></tr><tr><td>Biohydrogen production from palm oil mill effluent in a pilot-scale up-flow anaerobic sludge blanket fixed-film reactor (treatment of effluent not included)</td><td>818</td><td>2.17 kg SO<sub>2-eq</sub></td><td>3.43-10<sup>-3</sup> kg P<sub>eq</sub></td><td>1.14 kg 1,4-DCB<sub>eq</sub></td><td>2.91</td><td></td><td>1.1-10<sup>-4</sup></td><td>1 kg H<sub>2</sub></td><td>Yes</td><td>No [412]</td></tr></tbody></table>

order to ensure their sustainability. A techno-economic assessment can provide a quantitative and qualitative comparison between the different production pathways to evaluate their feasibility and their future contribution in the development of a sustainable hydrogen economy [415].

Techno-economic assessment (TEA) is a method for estimating the technical and economic performance of a process and/or product. TEA is an important tool for evaluating the profitability of an industrial process and determining the short- and long-term commercialization potential of a new technology [416,417]. In this regard, TEA will be used to assess the technical aspects and analyze the production costs of both thermo-chemical and biological bio-hydrogen conversion routes. Technical and economic parameters of each bio-hydrogen production pathway are obtained from research studies (e.g. systematic reviews, lab-scale experimental data, simulation analysis).

The cost of H₂ varies widely between 0.8 and 4 USD/kg in different regions, mainly due to the technology and raw materials costs [418,419]. The industrial H₂ demand is also anticipated to increase, especially for ammonia production (51 %) and oil refining (31 %), by about 2 EJ/y, while the H₂ demand as a transportation fuel in the most developed countries is estimated to reach 22 EJ/y by 2050 [420]. Capital investment (CAPEX) and Operation costs (OPEX) are the two main economic metrics that determine the bio-hydrogen cost. An optimum case is increasing bio-hydrogen productivity while decreasing CAPEX and OPEX, consequently obtaining a low bio-hydrogen selling price. CAPEX is related to the direct (process equipment, installation, instrumentation, control systems and service facilities) and indirect (construction and engineering) costs. OPEX involves feedstock, labour, safety, maintenance, patents royalties, cooling water and electricity costs [421,422]. The type of biomass feedstock, feedstock availability and transport and the need for the pre-treatment step are also crucial parameters related to the operating costs. Furthermore, variations in fossil fuel prices, different biofuel policies in each country and carbon tax (CO₂ emissions) influence the production costs [418,423]. A significant indicator is the Technology Readiness Level (TRL), which grades the maturity of each technology and its development towards commercialization [416]. To quantitatively evaluate the different technologies for hydrogen production from fossil fuels (natural gas, coal) and biomass, indicators such as type of feedstock, TRL, CAPEX, H₂ yield and H₂ production costs are presented in Table 9.

As shown in Table 9, steam reforming of natural gas (SRNG) is currently the most economically feasible and preferred method for commercial hydrogen production (TRL 8–9), even though it emits a significant amount of CO₂ emissions. The cost of H₂ production from SRNG ranges from 0.77 to 1.22 USD/kg, mainly depending on the feedstock price, global energy demand and H₂ economy [419,428]. Lee and Lee [429] reported that the increasing energy demand can be covered by converting natural gas into H₂ and thereby decreasing CO₂ emissions. Nikolaidis and Poullikkas [415] documented that SRNG, followed by CO₂ capture and sequestration (CS), presented a higher H₂ price (2.27 USD/kg) than SRNG without CS (2.08 USD/kg) correspondingly. The same authors also reported that coal gasification without CS process has the lowest value of production cost per kg H₂ compared to SRNG and CG with CS processes. The coal gasification process demands high CAPEX and OPEX compared to feedstock cost. On the contrary, feedstock cost is much higher than CAPEX during fossil fuel gasification [430].

Although biomass prices are still high, biomass gasification and pyrolysis are economically viable approaches for biohydrogen production. Their maturity reaches high TRL values (6–8), and gasification is anticipated to reach a TRL 9 soon. As discussed by Hoang et al. [431], critical factors affecting hydrogen production yield from biomass gasification are the type of feedstock, type of gasifier, operating conditions and catalysts used in the process. The generation of tar is one of the main technical challenges in the commercialization of biomass gasification. The choice of the gasifier depends on the type of biomass feedstock and
<table><caption>**Table 9**
Technical, Economic and Commercialization parameters of different H₂ production technologies (TRL – Technology Readiness Level).</caption><thead><tr><th>Process</th><th>Feedstock</th><th>TRL</th><th>CAPEX (M€)</th><th>H₂ yield (g/kg feedstock)</th><th>H₂ Production Cost (USD/kg)</th><th>Ref.</th></tr></thead><tbody><tr><td>SRNG</td><td>Natural Gas</td><td>9</td><td>170-240</td><td>40-130</td><td>0.77-1.22</td><td>[418,424,425]</td></tr><tr><td>SRNG with CCS</td><td>Natural Gas</td><td>9</td><td>1183-1571</td><td>-</td><td>0.8-1.66</td><td>[418,424,425]</td></tr><tr><td>Coal Gasification</td><td>Coal</td><td>9</td><td>256-435.9</td><td>-</td><td>0.92-2.83</td><td>[418,424]</td></tr><tr><td>Coal Gasification with CCS</td><td>Coal</td><td>9</td><td>545.6</td><td>-</td><td>1.63</td><td>[424] [415,418]</td></tr><tr><td>Biomass Gasification</td><td>Woody Biomass</td><td>7-8</td><td>6.4-149.3</td><td>50-180</td><td>1.2-3.5</td><td>[418,426]</td></tr><tr><td>Biomass Pyrolysis</td><td>Woody Biomass</td><td>6-7</td><td>3.1-53.4</td><td>25-65</td><td>1.25-2.6</td><td>[418,427]</td></tr><tr><td>Dark Fermentation</td><td>Organic Biomass</td><td>5</td><td>478.2-1615</td><td>4-45</td><td>2.57-2.80</td><td>[418]</td></tr><tr><td>Photo Fermentation</td><td>Biomass</td><td>4</td><td>115.6</td><td>9-45</td><td>2.83-3.5</td><td>[418]</td></tr></tbody></table>

reaction conditions. The development of novel catalysts, such as dolo-mite and olivine, can lead to an increase in H₂ yield and a reduction in operating costs [432]. An average value of H₂ production cost from biomass gasification has been estimated to be within a range of 1.2–3.5 USD/kg [416,418,426]. Many factors, such as the size of the facility and the cost of biomass, affect economics. It was estimated for a plant (biomass gasification-steam reforming-PSA route) with produced H₂ output of 139,700 kg/day and with a biomass cost in the range of 46–80 USD/dry-ton, the resulting H₂ production cost was 1.77–2.05 USD/kg [430]. Salkuyeh et al. [409] carried out a techno-economic analysis and LCA of two reactor configurations for bio-hydrogen production from biomass gasification, a fluidized bed (FB) and an entrained flow (EF) reactor. They also investigated the impact of including a carbon capture and liquefaction unit. Their analysis revealed that the thermal efficiency of the process performed using the EF reactor was 9–11 % higher than the efficiency in the case of the FB, depending upon the carbon capture option. However, the lowest value of the H₂ selling price was 3.1 USD/kg for the FB process. The H₂ price for the EF reactor was 14 % higher, at 3.4 USD/kg, mainly due to the higher capital cost of the EF gasifier. In addition, the minimum H₂ selling price is higher 1.3 to 2.4 USD/kg compared to the price of H₂ generated from SRNG, due to the higher feedstock cost and the pre-treatment unit. The incorporation of a carbon capture and liquefaction system increased the minimum price by 3 % in the EF process and by 11 % in the FB process. In a recent study, Al-Qahtani et al. [433] found that the addition of a CCS unit increased the H₂ cost from 2.2 to 3.4 USD/kg.

The pyrolysis process is similar to gasification, but it takes place at lower temperatures without the requirement of an oxidizing agent. According to its operating temperature (400–800 °C), it is divided into conventional (or slow), fast and flash pyrolysis. Among slow and fast pyrolysis, the latter is frequently used for H₂ production. The product yield from pyrolysis strongly depends on biomass composition, temperature, decomposition time, heating rate, residence time and reactor design [419]. Al Arni [289] compared the fast and slow pyrolysis in terms of H₂ yield at different temperatures. A maximum value of H₂ yield (45 %vol) was obtained at 953 K by fast pyrolysis in a decomposition time of 5–7 min with a heating rate of 120–150 °C/min and residence time of about 20 min. However, in slow pyrolysis, at the same temperature, the H₂ yield was lower (28 %vol) in a decomposition time of 60 min with a rate of 45–50 °C/min and residence time of 1 h. Bartels, Pate, and Olson [430] reported that an increase in biomass cost (0.92 USD/kg biomass) induced a decrease in H₂ price (2.57 USD/kg H₂). Simulation results of fast pyrolysis also revealed that the H₂ production cost from corn waste is strongly affected by the biomass cost [434]. Nikolaidis and Poulikkas [415] and Lepage et al. [418] compared the H₂ production costs for several biomass-based technologies. Steam reforming of natural gas presented the lowest H₂ production cost (0.72 USD/kg), followed by biomass pyrolysis (1.21 to 2.57 USD/kg H₂). Recently, a techno-economic analysis of corn straw pyrolysis for H₂ production showed that biomass costs (68 %) and catalyst costs (12 %) had a significant influence on H₂ and biochar sales [435]. An annual processing of 40,000 tons of corn straw could achieve a maximum return of 6.327 million yuan and an IRR of 22 % in 7.3 years.

Contrary to biomass gasification and pyrolysis, biological methods

for bio-hydrogen production occur at lower temperatures (30–60 °C) and pressure (1 atm), resulting in reduced energy costs. They utilize low-cost feedstock, and they contribute to waste management and recycling as they enable the conversion of various waste materials [418]. However, biological processes provide lower biomass conversion rates and lower H₂ yield, as well as require larger reactor volumes, making them less competitive compared to thermochemical processes and thus are adapted to a small scale. Dark fermentation and photo-fermentation are the two main biological processes for bio-hydrogen production.

Dark fermentation is a widely applied process for bio-hydrogen production from various biomass feedstock (lignocellulose, crop residues, algal) and biological and organic wastes [419]. It takes place when anaerobic microorganisms (e.g. specific bacteria) are sustained in the absence of light at mesophilic (25–45 °C) or thermophilic (45–80 °C) temperatures. The yield of produced H₂ relies on the metabolic pathway, the by-products (e.g. organic acids) of the reaction, the pH and the hydraulic retention time [428]. Some advantages compared to photo-fermentation are the high H₂ yield, low capital and production costs [436]. [437] constructed a business model that converts the costs of procuring biomass into revenue via the production of hydrogen from organic wastewater by biological dark fermentation. In the model, the H₂ production costs also included the income from biomass hydrogen production, as well as the revenue from processing biowaste and reduced fuel source costs. The total annual costs of the system involved the equipment and installation costs, annual depreciation expenses, and annual operational costs. The equipment costs included the cost of the H₂ production unit, H₂ purification unit, compressor, and storage unit. Their analysis showed that the revenue attained by selling the produced H₂ (63,000 m³/y) was 355,827 NTD/yr, while the revenue by selling electricity was 226,800 NTD/yr. A value of 1 USD/m³ for H₂ production cost was calculated. Simulation analysis by [438] revealed that by using wastewater as feedstock for H₂ production by biological fermentation, the revenue generated by selling H₂ and CO₂ was 16,000 USD/y and 4000 USD/y, respectively. However, using agricultural residue as feedstock, the revenue for H₂ and CO₂ sales was 4000 USD/ and 1000 USD/y, respectively. The price of H₂ production was estimated at 2.7 USD/kg. According to [415], the H₂ price by dark fermentation and photo-fermentation was 2.5 USD/kg and 2.8 USD/kg, respectively, mainly due to the low conversion rate, low H₂ yield and the requirement of larger reactors and H₂ separation unit. Photo-fermentation lies on the need for a light source, which increases the process costs.

From a techno-economic point of view, it is viable to combine fermentation technologies in a single process. [439] performed a techno-economic assessment of a combined system of solid-state fermentation and dark fermentation for producing H₂ in a plant with a capacity of 10 t/d of food waste. It was reported that the total capital investment, annual operation cost and annual revenue of the plant were 707,850 USD, 366700 USD/year and 574,800 USD/year, respectively, with an IRR of 20.2 %. The H₂ production cost was 2.29 USD/m³, cheaper than the H₂ market price (2.7 USD/m³), proving the feasibility of this novel bioprocess. In another study, an integrated biological process for H₂ production based on thermophilic and photo-heterotrophic fermentation was techno-economically evaluated [440]. The analysis showed that photo-fermentation is the main contributor to the H₂ production
cost, mainly because the cost of plastic tubing represents 40.5 % of the H₂ price. The CAPEX and the chemicals cost are also notable contribu- tors to the H₂ production cost. Estimating an H₂ production cost of 6.54 USD/kg, major biological improvements should be achieved to make this process economically viable.

In conclusion, as observed in Table 1, biomass gasification is in the commercial stage with an H₂ production lower than pyrolysis and bio- logical processes. Biomass pyrolysis presents H₂ prices close to gasifi- cation with a high potential to be commercialized soon. Dark fermentation and photo-fermentation are on a pilot and lab scale, respectively, with some hurdles to overcome on the way towards com- mercialisation. Future improvements should focus on the reduction of biomass (feedstock) cost, optimization of bioreactors and separation units, and development of small-scale plants. Nonetheless, for the sake of future decision-making, economics should not be considered in complete isolation. To achieve sustainability, all three pillars should be taken into consideration, namely economic, ecological, and social. This is confirmed by works that assessed both economic and ecological impact [422], which clearly demonstrated that biohydrogen could outperform conventional hydrogen when externalities are considered, and both economics and ecological performance are considered from a holistic perspective.

## 6.4. Social sustainability of biohydrogen

Social sustainability is one of the pillars of sustainability that should not be ignored when sustainability is assessed. This is primarily driven by the fact that social acceptance is a *conditio sine qua non* for the suc- cess of any disruptive change in modern societies. On the other hand, the “not in my backyard” syndrome could be especially detrimental for technologies implemented on a small scale. Due to the inherent prop- erties of biomass, such as relatively low bulk density, the achievable scale of biohydrogen production has some natural limitations, especially when considering biobased processes suitable for wet types of biomass.

The work of De-Leon Almaraz et al. [441] identified various social aspects relevant to the hydrogen economy, such as acceptability, accessibility, policies, stakeholders, externalities, as well as other socio- economic factors and technical factors related to safety. Social LCA and social cost-benefit analysis have been determined as key analytical tools for the evaluation of the social sustainability of any particular solution in the hydrogen economy [441]. The trust of stakeholders could be strengthened on a structural level by promoting responsible innovation, which includes a large degree of openness, which can foster trust and promote acceptance among the stakeholders [442]. Such work should be organic and focused on local communities, as suggested by Vallejos- Romero et al. [443]. Social LCA can provide some important insights into social aspects of sustainability for hydrogen production, especially by identifying hot spots [444,445]. A study performed for green hydrogen showed that on-site value chains outperformed off-site value chains [444], which require imported electricity generated in more distant locations. Another social LCA study performed for green hydrogen revealed that working conditions within the green hydrogen value chain are crucial in terms of its social sustainability [445].

Overall, the scarcity of social LCA studies focused on biohydrogen is a significant research gap that does not allow us to assess the sustain- ability of such solutions fully. More studies are needed to fully assess the social aspect of sustainability for biohydrogen production. It should not be overlooked that sustainability has three pillars (see Fig. 10), i.e., it is plausible to state that something is sustainable if sustainability is proven not only from the point of view of the environment, as environmental sustainability is only one of the pillars.

Economic and social sustainability also need to be demonstrated [446]. Social sustainability can be determined using social LCA. There is more than one way to prove economic sustainability (e.g., through different forms of economic or techno-economic analyses). However, life cycle costing (LCC) is capable of demonstrating economic sustainability

<figure><img src="image_13.png" /><figcaption>Fig. 10. Three pillars of sustainability and their relationship with different types of LCA (LCA – Life Cycle Assessment; LCC – Life Cycle Costing; S-LCA – Social LCA).</figcaption></figure>

and determining potential hot spots, which could be helpful in designing a resilient supply chain, thus further strengthening the economic sus- tainability of novel solutions, such as biohydrogen production technologies.

## 7. Conclusions and recommendations

Despite the fact that intensive development in terms of green hydrogen production using electrolysis could be clearly observed, it should not be overlooked that other production routes of green hydrogen are possible and worthy of attention. Since they require different expertise, in comparison to electrolysis, their development might facilitate the overall development of the hydrogen economy by removing bottlenecks and tapping into resources so far underexploited. The following points should be especially taken into consideration.

* Labelling of hydrogen should convey useful information, such as carbon/greenhouse gas footprint, rather than a somewhat useless “palette of colours”.

* Biohydrogen production is complementary to electrolysis, offering a stable and predictable supply since biomass can be stored relatively easily compared to hydrogen storage.

* Biological pathways result in a gas, which requires less extensive treatment in comparison to thermal processes.

* Thermal processes offer additional possibilities, e.g., the use of CO₂ as a gasifying agent, providing opportunities in terms of applications of such processes in CCU.

* Supercritical water gasification offers unique possibilities regarding the use of wet biomass with high moisture content, which would typically require pre-drying prior to pyrolysis or other types of gasification.

* The strength of biohydrogen coupled with CCS is its negative carbon footprint.

Since none of the biohydrogen production processes produce pure hydrogen, each process requires a downstream cascade for gas purifi- cation and treatment. In many cases, impurities (e.g., tars or compounds containing sulfur) might be detrimental to catalysts used downstream. Therefore, it seems sensible to recommend further research on both primary (fuel pretreatment and process conditions optimisation) and secondary measures (gas cleaning) to minimise the production of said impurities.
Any installation for biohydrogen production would consist of many unit operations. Due to their complexity, it might be difficult to compare them directly in terms of their performance. Comprehensive analytical tools, such as life cycle assessment, seem to be the best suited for such purposes. Most of the published LCA studies involve the use of pilot scale and laboratory scale data. Nonetheless, such prospective studies can still serve the purpose of hot spot analysis, which should be an integral part of any technology development. Among the LCA studies, a lack of any kind of uncertainty analysis is often visible. Not many studies include contributions from infrastructure and equipment manufacturing, simply focusing on the processes. Carbon tunnel vision, with an overemphasis on global warming impact, is also a deficiency observed among many LCA studies on biohydrogen. Few studies present data on water consumption and other impact categories. Nonetheless, LCA is a powerful tool for ensuring the sustainability of biohydrogen value chains. Social LCA can be an extremely helpful tool, especially in combination with classical LCA studies focused on environmental impacts, as well as life cycle costing. Social sustainability seems to be the least investigated of all three sustainability pillars, thus suggesting that more social LCA studies are needed to close this knowledge gap.

It cannot be emphasized enough that biohydrogen production could be crucial not only from the energy sector perspective but also from other branches of industry, including the production of fertilisers, other chemicals, or steel, just to name a few. Biohydrogen coupled with carbon capture and storage could further decrease the carbon footprint of these sectors.

## CRediT authorship contribution statement

**Aleksandra Modzelewska:** Writing – original draft, Data curation.
**Mateusz Jackowski:** Writing – original draft, Conceptualization.
**Panagiotis Boutikos:** Writing – original draft, Conceptualization.
**Magdalena Lech:** Writing – original draft, Data curation.
**Maciej Grabowski:** Writing – original draft, Data curation.
**Krystian Krochmalny:** Writing – original draft, Data curation.
**María González Martínez:** Writing – original draft.
**Christian Aragón-Briceno:** Writing – review & editing, Conceptualization.
**Amit Arora:** Writing – review & editing, Data curation.
**Hao Luo:** Writing – review & editing, Data curation.
**Luca Fiori:** Writing – review & editing, Supervision.
**Qingang Xiong:** Writing – review & editing, Supervision.
**Muhammad Yousaf Arshad:** Writing – review & editing, Data curation.
**Anna Trusek:** Validation, Supervision.
**Halina Pawlak-Kruczek:** Validation, Supervision.
**Lukasz Niedźwiecki:** Writing – review & editing, Writing – original draft, Visualization, Data curation, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

Luca Fiori acknowledges the financial support from the project "Produrre Idrogeno in Trentino—H2@TN" (PAT-Trento).

## Data availability

It's a review. Data comes from other publications.

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