9: Biohydrogen - An Introduction

Hydrogen as a fuel

Hydrogen gas would be ideal if it could be produced at scale, easily transported and stored, or produced on-site locally on demand (all of these are long shots, as described at the end of this chapter).   The reaction for the "burning" of hydrogen shows that the only greenhouse gas emitted is H₂O.

\begin{equation}
2 \mathrm{H}_2(\mathrm{~g})+\mathrm{O}_2(\mathrm{~g}) \rightarrow 2 \mathrm{H}_2 \mathrm{O}(\mathrm{g})
\end{equation}

H₂O comes and goes in our atmosphere on short timescales and does not continually build up, as does CO₂ from burning fossil fuels. The standard heat of combustion (in kJ/g or kcal/kg) for H₂ is far higher than any other fuel, as shown in Table  \(\PageIndex{1}\) below, making it an ideal fuel.

Table  \(\PageIndex{1}\):  Energy values for various fuels. Data source:  https://www.engineeringtoolbox.com/s...nt-d_1987.html

We won't discuss large-scale H₂ storage or transport, two fundamental and likely intractable engineering problems. Instead, we will focus on producing "biohydrogen." Of course, the prefix "bio" can mean many things, including the production of H₂ in syngas using cellulose as a feedstock, the electrolysis of water powered by solar/wind energy, and its production by hydrogenases, enzymes found in some microbes.  

The fuel industry uses different colors to describe hydrogen based on how it is produced. They are shown in Table  \(\PageIndex{2}\).

Table  \(\PageIndex{2}\):  Different "colors" of hydrogen based on production methods

Of course, H₂ in syngas can be produced from biomass, as described in Chapter 32.8, but it is unclear if a hydrogen color has been assigned to it.

At present, the important feedstocks for H₂ production worldwide are natural gas (48%), oil (30%), coal (18%), and electrolysis (4%)—mostly fossil fuels.

Methods of Production

H₂ production is also classified based on the chemical processes used to produce it. These processes include

1. Biological: use of live bacteria and algae cells)
2. Thermochemical:  (gas and liquid fuel reforming, coal and biomass gasification)
3. Electrochemical (electrolytic):  (photothermal, photoelectrolytic, and photobiological)

We will organize this chapter section using these three processes. We will start with Biological (1), followed by Electrochemical/Electrolytic (3), and end with Thermochemical (2). They are summarized in Figure \(\PageIndex{1}\). 

Figure \(\PageIndex{1}\):  The main pathways for H₂ production based on biomass. M.G. Eloffy et al., Chemical Engineering Journal Advances, 12 (2022). https://doi.org/10.1016/j.ceja.2022.100410.  Creative Commons license

Biomass can serve as feedstock for all these methods, and the resulting product can be called biohydrogen. Of course, as discussed in the previous chapter, nonbiological feedstocks are also the predominant sources used in thermochemical and electrochemical methods.  

2H⁺ ↔  H₂: An Overview

We will discuss the production of H₂ as an energy source for society. For industrial use, it can be used in fuel cells to power spacecraft and cars, as shown in the reaction below.

\begin{equation}
\begin{aligned}
& \mathrm{O}_2+4 \mathrm{H}^{+}+4 \mathrm{e}^{-} \longrightarrow 2 \mathrm{H}_2 \mathrm{O} \\
& \mathrm{H}_2 \longrightarrow 4 \mathrm{H}^{+}+4 \mathrm{e}^{-}
\end{aligned}
\end{equation}

In the next section, we will discuss the hydrogenases that produce and use H₂ in microbes.  This chapter will treat them very generally. However, we need to review the topic.

Use of H₂ as a source of electrons for reduction reactions.  

Each hydrogen in H₂ has an oxidation number of 0. Each can be oxidized to H⁺ (oxidation number +1), with the 2 electrons transferred to a substrate/cofactor or to a series of substrates with successively higher standard reduction potentials (better oxidizing agents), forming reduced products.  

H₂ + (substrate)_OX  →  2H⁺  +  (product)_RED

This general reaction is analogous to the mitochondrial electron transport chain, in which electrons are passed from a source (NADH) to oxidized acceptor molecules. The general reaction below shows each redox pair in the electron transport chain.

NADH/NAD⁺ →  FAD/FADH₂ →  UQ/UQH2  →  Cyto C_OX/Cyto C_RED  →   O₂/H₂O 

Some organisms have evolved to produce energy by oxidizing H₂. This reaction is analogous to the way photosynthetic organisms obtain energy via water oxidation. In photosystem II, the oxygen-evolving complex oxidizes oxygen in H₂O (oxidation number -2) to O₂ (oxidation number 0). Some redox pairs, starting with H₂O/O₂, are shown below for photosystem II.  

H₂O/O₂ →  P680/P680* →  (Plastoquione)_OX/(Plastoquione)_RED 

The first reaction is endergonic and requires photons as an energy source.

Use of H⁺ as a sink for electrons for oxidation reactions that produce H₂.  

H⁺ has an oxidation number of +1. Hence, it can be reduced to H₂ (oxidation number 0) as it gains electrons from substrates/cofactors that become oxidized. This general reaction is shown below.  

2H⁺ + (substrate/cofactor)RED  →  H₂  + (substrate/cofactor)OX 

Many microorganisms can produce H₂ through variants of photosynthesis or fermentation, both of which provide the two electrons needed. E. coli has four hydrogenases (Hyd 1, 2, 3, and 4). It forms H₂ through two reactions catalyzed by:

• formate (HCO₂^-) dehydrogenase (FDH):  2HCO₂^- ⇌ 2CO₂ + 2H⁺ + 2e^- 
• hydrogenase (H₂ase):  2H⁺ + 2e^- → H₂ 

The C in formate has an oxidation number of +2 and is oxidized to CO_2, in which the C has an oxidation number of +4. 

Biohydrogen from Microalgae 

We will focus most of our attention on the Biological (1) and Electrolytic (3) processes for producing biohydrogen from microalgae. The Biological processes (1) require hydrogenases for H₂ production within cells. The Electrolytic (3) processes use microalgae as a feedstock to provide substrates that other microbes can ferment. These can be combined to increase production. Figure \(\PageIndex{3}\) below summarizes the Biological (1) and Electrolytic (3)  metabolic processes that can be used for microalgae H₂ production.

Figure \(\PageIndex{3}\): Metabolic pathways of biohydrogen production by micro-algal biomass. modified from Ahmed SF et al. Front. Energy Res. 9:753878. doi: 10.3389/fenrg.2021.753878.   Creative Commons Attribution License (CC BY).

These are mainly classified into three categories: i) the photobiological process through which biohydrogen is produced via direct and indirect photolysis in the microalgae, ii) fermentation, and iii) the electrochemical process that comprises photoelectrochemical and electrolytic.

2. Thermochemical from Biomass

We have already explored thermochemical methods to produce syngas (H₂ and CO) and to further use it in the Fischer-Tropsch reaction to make small and large molecules for chemical feedstocks and fuels. We also discussed electrochemical methods for producing syngas and other small organic molecules, such as formate and ethanol, from CO₂. Figure \(\PageIndex{15}\) shows a schematic diagram for thermochemical (gasification) processes to produce H₂.  

Figure \(\PageIndex{15}\):  Schematic diagram for thermochemical (aqueous phase reforming) process. M.G. Eloffy et al.

Summary

(Summary written by Claude, Anthropic)

This chapter evaluates hydrogen as a potential clean fuel — examining its thermodynamic advantages, the biological and electrochemical pathways for its production, and a sobering assessment of the practical and thermodynamic limitations that constrain its role in the energy transition.

Hydrogen's promise and limitations as a fuel. With a combustion enthalpy of 141.58 kJ/g — far exceeding methane (55.51 kJ/g), octane (47.87 kJ/g), and ethanol (29.67 kJ/g) — and producing only water upon combustion, hydrogen is theoretically the ideal fuel. The fossil fuel industry classifies hydrogen by production method (green: electrolysis from renewable energy; blue: steam reforming with carbon capture; grey: steam reforming without carbon capture; black: coal gasification; pink: nuclear electrolysis; turquoise: methane pyrolysis). Currently, 96% of global H₂ production derives from fossil fuels; only 4% from electrolysis. Critically, H₂ is not a direct greenhouse gas (it lacks IR-absorbing bond dipoles) but is an indirect one: atmospheric H₂ reacts with the hydroxyl radical (•OH) — the "detergent of the atmosphere" produced photochemically from ozone and water vapor — competing with •OH's reaction with methane and thereby extending CH₄'s atmospheric lifetime. Atomic hydrogen (H•) produced in this reaction also initiates free-radical chains that generate tropospheric ozone, a health hazard and a greenhouse gas. These indirect warming effects mean H₂ leakage during production, storage, and transport must be minimized.

Hydrogen redox chemistry. H₂ (oxidation state 0) can act as either a reductant (donating 2 electrons to reduce substrates) or as the product of H⁺ reduction (gaining 2 electrons). E. coli produces H₂ through a coupled two-enzyme system: formate dehydrogenase oxidizes formate (C oxidation state +2) to CO₂ (+4), generating H⁺ and electrons, and hydrogenase reduces 2H⁺ to H₂ using those electrons. This electron flow parallels the mitochondrial electron transport chain, with ferredoxin serving an analogous role to the ubiquinone pool.

Direct biophotolysis. In Chlamydomonas reinhardtii and other green microalgae, direct biophotolysis routes electrons from water oxidation at PSII through cytochrome b6f, PSI, and ferredoxin directly to hydrogenase rather than to NADP⁺, producing H₂ as the terminal product. The overall reaction is 2H₂O → 2H₂ + O₂. The major limitation is that O₂ produced by PSII irreversibly damages [Fe-Fe] hydrogenases. Sulfur deprivation selectively suppresses PSII activity, reducing O₂ evolution and enabling sustained H₂ production under illumination. Approximately 90% of C. reinhardtii H₂ production occurs via this direct photolytic route.

Indirect biophotolysis. Under anaerobic conditions in the dark, glucose from prior photosynthesis is oxidized through glycolysis to pyruvate, which is then oxidatively decarboxylated by pyruvate:ferredoxin oxidoreductase (PFOR) — a 267 kDa homodimer with three [Fe₄S₄] clusters per monomer using TPP as cofactor — to acetyl-CoA and CO₂, with electrons transferred to ferredoxin. Reduced ferredoxin then donates electrons via hydrogenase to H⁺, producing H₂. This pathway avoids O₂ exposure of hydrogenase. An alternative route via pyruvate:formate lyase (PFL1) produces formate and acetyl-CoA under prolonged dark anoxia.

Photofermentation. Purple non-sulfur bacteria (PNSB) and some microalgae produce H₂ from organic acids (acetate, butyrate, lactate, succinate) using a single photosystem under strict anaerobic conditions. Electrons from organic acid oxidation flow through the photosystem to ferredoxin and then to nitrogenase — which normally reduces N₂ to NH₃ (consuming 16 ATP per H₂ produced as a byproduct) but, in the absence of N₂, can dedicate all electron flow to H₂ production at a significantly reduced cost of 4 ATP per H₂. The photosystem cannot oxidize water but can oxidize H₂S or organic acids as electron donors.

Dark fermentation. Heterotrophic anaerobic bacteria and microalgae produce H₂ from organic biomass feedstocks — including industrial wastewater — through a four-stage sequence: hydrolysis of complex polymers, acidogenesis (producing short-chain fatty acids and H₂), acetogenesis (producing acetic acid and H₂), and methanogenesis (producing CH₄ consuming H₂). Representative acidogenic reactions include glucose → 2 acetate + 2CO₂ + 4H₂, and acetogenic reactions include lactate + H₂O → acetate + CO₂ + 2H₂. Methanogenic archaea consume H₂ (4H₂ + CO₂ → CH₄ + 2H₂O), representing a competing pathway that diverts electrons from H₂ toward CH₄.

Electrochemical and thermochemical production. Microbial electrolytic cells (MECs) combine microbial oxidation of organic substrates at the anode with proton reduction to H₂ at the cathode, enhanced by an applied external voltage. Photoelectrochemical cells (PECs) use light-sensitive semiconductor electrodes to directly drive water splitting. Thermochemical gasification of biomass produces syngas, whose H₂/CO ratio is adjusted by the water-gas shift reaction for downstream applications.

The honest assessment of hydrogen as an energy carrier. Despite its theoretical appeal, the chapter closes with a critical evaluation that punctures much of the hydrogen hype. Electrolysis captures only ~30% of input electricity in H₂; using renewables directly for electrification is far more efficient. H₂ embrittles steel pipelines, requiring entirely new and enormously expensive infrastructure. Liquid H₂ storage at 20K requires energy equivalent to ~50% of H₂'s combustion value. Producing the current 100 million metric tons per year of industrial H₂ from renewables would consume the entire US electricity grid. The thermodynamic absurdity of capturing dilute, stable CO₂ and using it to make energy-dense H₂ fuels, only to combust them again, is explicit. H₂'s legitimate near-term role is as a chemical feedstock — primarily for NH₃ fertilizer synthesis (the Haber-Bosch process) and for fuel upgrading — not as an energy carrier to power transportation or heating. Direct electrification via electric vehicles and heat pumps is the rational path for most energy applications, making genuine clean hydrogen a complement to, not a substitute for, a renewable electricity grid.