9: Biohydrogen - An Introduction

BIOLOGICAL (1) - Biophotolysis (photosynthesis)

This consists of two processes: Direct and Indirect Photolysis (photosynthesis). Both use light to drive the ultimate reduction of 2H⁺ to H₂ using hydrogenase or nitrogenase. We will explore the details in the next chapter section. The biophotolysis process is divided into indirect (using electrons from substrates) and direct (using electrons from water). These processes are simplified in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Schematic diagram for biological (biophotolysis) process.   M.G. Eloffy et al.

Direct Biophotolysis (photosynthesis)

In direct biophotolysis (photosynthesis), water molecules are oxidized in Photosystem II, which contains the Oxygen Evolving Complex (OEC). This endergonic process is driven by light. The electrons lost from water are passed through Cytochrome b₆f and Photosystem I to ferredoxin, then NADP⁺, which gets reduced to NADPH (as discussed in Chapter 20). These reactions are illustrated in Figure \(\PageIndex{5}\). 

Figure \(\PageIndex{5}\): Light reaction of photosynthesis and associated standard reduction potentials

In direct photolysis, electrons are passed directly from reduced ferredoxin to 2H⁺ in a reaction catalyzed by a hydrogenase, as shown in Figure \(\PageIndex{6}\) below.

Figure \(\PageIndex{6}\): Metabolic hydrogen production pathways used by Chlamydomonas reinhartii.FDX: ferredoxin; H₂ase: hydrogenase; NPQR: NADPH−plastoquinone oxidoreductase; PFR: pyruvate:ferredoxin oxidoreductase; PSI: photosystem I; PSII: photosystem II. Touloupakis, E.; Faraloni, C.; Silva Benavides, A.M.; Torzillo, G. Recent Achievements in Microalgal Photobiological Hydrogen Production. Energies 2021, 14, 7170. https://doi.org/10.3390/en14217170.  Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

 The overall reaction is simplified in the equation below.

\begin{equation}
2 \mathrm{H}_2 \mathrm{O}+\text { Light } \rightarrow 2 \mathrm{H}_2+\mathrm{O}_2
\end{equation}

One problem with direct photolysis is that O2 can damage hydrogenases. Again, we will discuss the biochemistry of hydrogenases in great detail in the next chapter section.

Indirect Biophotolysis

This process bypasses the damaging effects of O₂ on hydrogenase by being carried out in the absence of O₂ using fermentation to provide electrons for the hydrogenase reduction of 2H⁺ to H₂. Photosynthesis is required to make the carbohydrates necessary for fermentation. Glucose can then be oxidized anaerobically (in the dark to avoid O₂ formation from photosynthesis) to form pyruvate through the glycolytic pathway. Pyruvate can then be oxidatively decarboxylated through the pyruvate:ferredoxin oxioreductase (PFR) as ferredoxin gets reduced. It then passes its electrons on through hydrogenase to produce H₂. The pathway is illustrated in the top/right parts of the above figure and the reaction diagram in Figure \(\PageIndex{7}\) below.

Figure \(\PageIndex{7}\): Model of fermentative pathways involved in dark anaerobic H₂ production in C. reinhardtii. Proteins are shown as ovals. Photosynthetic ferredoxin (PETF).   Jens Noth et al., Journal of Biological Chemistry, 288 (2013). https://doi.org/10.1074/jbc.M112.429985.  Creative Commons license.

Glucose and some amino acids can be converted into pyruvate, a substrate for PFR1 in the single-cell algae C. reinhardtii. PFR1 converts pyruvate to acetyl-CoA and CO₂ with the electrons that reduce ferredoxin. The reduced FDX2 passes electrons through hydrogenase (HYDA1) to form H₂. 

Another enzyme used to continue fermentation, pyruvate:formate lyase (PFL1), converts pyruvate to formate and acetyl-CoA, which can be metabolized further to acetate and ethanol. A shift to pyruvate oxidation to PFR1 occurs if PFL1 is mutated or in long-term anoxic conditions.

The key enzyme, pyruvate:ferredoxin oxioreductase(PFR), uses thiamine pyrophosphate (TPP) as a cofactor for the oxidative decarboxylation of the α-keto acid pyruvate, as expected. Figure \(\PageIndex{8}\) shows an interactive iCn3D modelof the pyruvate ferredoxin oxidoreductase (PFOR) from Desulfocurvibacter africanus in anaerobic conditions (7PLM).

Figure \(\PageIndex{8}\): Pyruvate ferredoxin oxidoreductase (PFOR) from Desulfocurvibacter africanus in anaerobic conditions (7PLM). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...XRFMoVhaoRbW86

PFOR, abbreviated here, is a 267 kDa homodimer containing three [Fe₄S₄] clusters (spacefill) per monomer. Only one monomer is shown, and TPP is shown in sticks.

Again, indirect photolysis occurs in the absence of O₂. Light illumination leads to only transient H₂ synthesis. If sulfur is limited in the growth media of the algae, more sustained H₂ production occurs, as the lack of sulfur reduces PSII activity. Hence, H₂ production can be maximized by depleting sulfur and minimizing O_2, even in the presence of light. In the absence of O₂, hydrogenase gene expression increases. Nutrient depletion also produces formate and acetyl-CoA through the enzyme pyruvate:formate lyase (PFL1). This is predominant in Chlamydomonas cells in the dark.

The green microalgae C. reinhardtii makes most of its H₂ (approximately 90%) using direct photolysis.   Commercially, the production of H₂ in indirect photolysis is carried out in a separate sealed bioreactor to avoid  O₂. Indirect photolysis is shown in the above figures.

The reactions to this process are as follows :

\begin{equation}
\begin{gathered}
12 \mathrm{H}_2 \mathrm{O}+6 \mathrm{CO}_2+\text { hν } \rightarrow \mathrm{C}_6 \mathrm{H}_{12} \mathrm{O}_6+6 \mathrm{O}_2 \\
\mathrm{C}_6 \mathrm{H}_{12} \mathrm{O}_6+12 \mathrm{H}_2 \mathrm{O}+\text { hν } \rightarrow 12 \mathrm{H}_2+6 \mathrm{CO}_2
\end{gathered}
\end{equation}

BIOLOGICAL (1) - Fermentation

We have just discussed fermentation processes within living microalgae cells. Now, let's consider fermentation processes using nonliving biomass feedstocks supplied to microbes to produce H₂. This offers a significant way to make biohydrogen. A schematic diagram for Biological (1) fermentation is shown below in Figure \(\PageIndex{9}\):

Figure \(\PageIndex{9}\): Schematic diagram for biological (fermentation) process. M.G. Eloffy et al.

Fermentation involves the decomposition of organic biomass to produce CO₂ and H₂.   The fermentation process can be separated into photofermentation (light fermentation) and dark fermentation.

Photofermentation

Some photosynthetic bacteria and microalgae use Photofermentation to produce H₂ from organic acids like acetic, butyric, lactic, and succinic acids. Oxidation of the acids produces CO₂, along with H⁺s and e^- for H₂ production. Electrons are transferred through photosystem I and eventually, believe it or not, nitrogenase. It is a fermentation process as the process is anoxic. 

Some photosynthetic bacteria, like the purple nonsulfur bacteria, a facultative anoxygenic phototroph, and some microalgae, can produce H₂ using a simplified system that has only one photosystem and uses the enzyme nitrogenase to produce H₂. The photosystem can not generate an oxidizing agent strong enough to oxidize H₂O, but under anaerobic conditions, they can oxidize organic acids and even H₂S to provide electrons from H₂ production. These reactions are shown below in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Photofermentative hydrogen production in PNSB. 

Deo, D., Ozgur, E., Eroglu, I., Gunduz, U., & Yucel, M. (2012). Photofermentative Hydrogen Production in Outdoor Conditions. Hydrogen Energy - Challenges and Perspectives. doi: 10.5772/50390. Creative Commons Attribution 3.0 License, 

We studied nitrogenase in Chapter x.xx. The net reaction for the fixation of nitrogen is shown below.

\begin{equation}
\mathrm{N}_2+8 \mathrm{H}^{+}+8 \mathrm{e}^{-}+16 \mathrm{ATP} \rightarrow 2 \mathrm{NH}_3+\mathrm{H}_2+16 \mathrm{ADP}+16 \mathrm{Pi}
\end{equation}

In this reaction, N₂ is reduced as the N atoms go from a 0 oxidation state to +3 in NH₃. The needed electrons are made from organic acids and fed into the system and eventually go to ferredoxin, which transfers them to protons. The ratio of N₂ to H₂ produced is 1:1, at the expense of 16ATPs per H₂ produced.

The ATP produced by the collapse of the produced proton gradient through FoF1ATPase powers the reaction.

In the absence of N_2, the net reaction becomes

\begin{equation}
2 \mathrm{H}^{+}+2 \mathrm{e}^{-}+4 \mathrm{ATP} \rightarrow \mathrm{H}_2+4 \mathrm{ADP}+4 \mathrm{Pi}
\end{equation}

The electrons are still fed into nitrogenase, but in the absence of the substrate N_2, they are used to reduce 2H⁺ to H₂. Note that only 4 ATPs are required per each H₂ produced, a significant energy gain.

ATP produced during photosynthesis would be used for anabolic biosynthesis contributing to biomass, so extra ATP is needed to support H₂ synthesis past that needed for growth. As anabolism is a reductive process (compared to oxidative catabolism), adequate sources of electrons for reduction are required. Multiple pathways need electrons, including CO₂ fixation, N₂ fixation (with associated H₂ production, and organic acids like polyhydroxbutyrate.  The bacteria use photosynthesis and the Calvin cycle under photoautotrophic conditions to fix CO₂. When external energy supplies from organic acids are present, the bacteria can become photoheterotropic. Under these conditions, the Calvin cycle is used to maintain redox balance.  

Dark Fermentation

We studied dark fermentation indirectly above in our discussion of hydrogenases in microalgae. Hydrogenases are induced in dark conditions, and this pathway involves heterotrophic fermentation (anaerobic) in some bacteria and microalgae. Many microbial species are used. Industrial wastewater enriched in organic material can be used as a feedstock. 

Feedstock materials are hydrolyzed and subjected to fermentation, during which H₂ can be produced. For example, pyruvate produced by glycolytic fermentation can be oxidatively decarboxylated to acetyl-CoA and CO₂ by pyruvate:ferredoxin oxidoreductase with electrons passed on to ferredoxin and even through hydrogenase to form H₂ (as we described above). H₂ is involved in steps after fermentation, including acetogenesis and methanogenesis.    These processes are illustrated in Figure \(\PageIndex{11}\) below.

Figure \(\PageIndex{11}\): The steps involved in anaerobic digestion [9]. Rosa, P. R. F., & Silva, E. L. (2017). Review of Continuous Fermentative Hydrogen-Producing Bioreactors from Complex Wastewater. Frontiers in Bioenergy and Biofuels. doi: 10.5772/65548. Creative Commons Attribution 3.0 License

Examples of acidogenic (formation of short carboxylic/fatty acid), acetogenic (formation of acetic acid), and methanogenic (formation of methane) reactions that produce (and a few that consume) H₂ are shown in Table \(\PageIndex{3}\) below.

Table \(\PageIndex{3}\):  Example of acidogenic, acetogenic, and methanogenic reactions in dark fermentation. Adapted from Rosa, P. R. F., & Silva, E. L., ibid.

We have described a few of the enzymes involved in acidogenic reactions above. Figure \(\PageIndex{12}\) summarizes the steps in acidogenesis.

Figure \(\PageIndex{12}\): An overview of the metabolic pathways of acidogenesis.  Dzulkarnain, E.L.N., Audu, J.O., Wan Dagang, W.R.Z. et al. Microbiomes of biohydrogen production from dark fermentation of industrial wastes: current trends, advanced tools, and future outlook. Bioresour. Bioprocess. 9, 16 (2022). https://doi.org/10.1186/s40643-022-00504-8.  http://creativecommons.org/licenses/by/4.0/.

Figure \(\PageIndex{13}\) below shows a more complex list and summary of dark fermentation reactions.

Figure \(\PageIndex{13}\): Key enzymes and dominant microbial taxa involved during anaerobic digestion of organic matter.  Dzulkarnain, E.L.N.et al. Ibid.

ELECTROCHEMICAL/ELECTROLYTIC (3)

Two primary electrochemical/electrolytic methods for H₂ production are photoelectrochemical and electrolytic, as shown below in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\):  Schematic diagram for the electrochemical process. M.G. Eloffy et al.

Electrolysis

In a microbial electrolytic cell (MEC), microalgae/cyanobacteria use industrially- and metabolically-processed feedstocks to oxidize organic substrates (for example, acetic acid) to CO₂. The released electrons move from the anode (where the oxidation occurs) to the cathode for H⁺ reduction to H₂. An external voltage increases electron flow to the cathode to facilitate the process. This increases the production of H₂ over and above that of just fermentation by microbes in the electrolytic cell. Cyanobacteria and a mix of green microalgae are used, as well as bacteria that can use dark fermentation (i.e., combining the processes described above).  

Photoelectrochemical

Microbial photoelectrochemical cells (PEC) use light-sensitive semiconductor electrodes for water electrolysis. A membrane separates the two electrodes so the protons can be reduced to form H₂.