Hydrogenase: Hydrogen from green algae?
It’s been quiet here on the blog lately. Four years ago, I swapped pipette and lab coat for science management and a mundane desk. For the past 1.5 years, I have been working at The Hydrogen and Fuel Cell Center (ZBT), which researches and develops hydrogen technologies. Hydrogen is a potentially sustainable energy carrier, but here and now I don’t want to get into a debate about its applications. Out of a spontaneous longing for genetics, I instead looked up whether hydrogen could also be produced biotechnologically – and that’s how I came across hydrogenase and the green algae Chlamydomonas reinhardtii.
Hydrogenase?
Hydrogenases are enzymes that combine protons (H+) and electrons (e-) to form molecular hydrogen (H₂). All hydrogenases have a metal cluster in their active center – but different types differ in this cluster. [FeFe]Hydrogenases carry iron atoms in this center and are mainly found in anaerobic organisms, because even the smallest amount of oxygen is enough to shut off this sensitive system.
In photosynthesis, plants and some microorganisms use sunlight to convert water (H₂O) and carbon dioxide (CO₂) into glucose (C₆H₁₂O₆) and oxygen (O₂). This process takes place in two stages: In the light-dependent phase, water is split at photosystem II (PSII), releasing oxygen and releasing electrons. These electrons travel through an electron transport chain, then get “charged” again with light energy at photosystem I (PSI) and are finally used for the synthesis of NADPH. In the light-independent phase, the so-called Calvin cycle, this NADPH supplies the electrons for the fixation of CO₂ in order to ultimately build sugar.
However, instead of building sugars like plants, Chlamydomonas reinhardtii sometimes takes a different path. The [FeFe]hydrogenase can capture the electrons from photosynthesis before they are being fed into the Calvin cycle. Together with protons from water splitting, it then produces H₂. This helps the algae to get rid of excess electrons – effectively acting as a kind of pressure relief valve as long as there is no oxygen nearby.
Chlamydomonas reinhardtii?
Chlamydomonas reinhardtii can be cultivated and genetically modified quite easily. It therefore makes sense to try out whether this metabolic pathway – possibly after a few genetic optimizations – could be used to produce hydrogen.
Left: Chlamydomonas reinhardtii under the scanning electron microscope. Measuring bar: 10µm. Right: Schamtic structure of a single Chlamydomonas reinhardtii alga: 1. flagellum, 2. mitochondrion, 3. contractile vacuole, 4. eyespot, 5. cup-shaped chloroplast, 6. Golgi apparatus, 7. starch granules, 8. pyrenoid, 9. vacuole, 10. nucleus, 11. endoplasmic reticulum, 12. cell membrane, 13. cell wall.
One promising approach is the direct coupling of hydrogenase to PSI: a 2020 study described the genetic fusion of [FeFe]hydrogenase to PsaD, a PSI subunit. The aim: to direct electrons that are normally channeled to the Calvin cycle via ferredoxin directly to the hydrogenase instead. This shortcut in the electron chain increases the efficiency of hydrogen production, as fewer electrons flow into the competing Calvin cycle.
More hydrogen thorugh less PGR5 and PGRL1
An alternative approach to weaken the competing Calvin cycle against the hydrogenase reaction is the pgr5 mutant. PGR5 (Proton Gradient Regulation 5) normally regulates the cyclic electron transport around PSI, which produces ATP. Without PGR5, more electrons reach the hydrogenase, while less ATP slows down the Calvin cycle – which increases hydrogen production.
Until now, pgr5 mutants have mostly been tested under constant lighting. As described in a study published in 2024, researchers have now exposed them to more realistic conditions and simulated daylight conditions with varying light intensity. The results were impressive: the mutant produced more hydrogen than the wild type, kept its hydrogenase and photosynthetic activity stable over several days and was also efficient in fluctuating light.
A highlight of the study was a specially developed thin-film photobioreactor that almost completely removed the oxygen from the environment using an iron-based absorber – perfect for the sensitive hydrogenase. At the same time, the hydrogen produced could be precisely measured.
Another group of researchers used a different mutant in another 2024 study. They had switched off Proton Gradient Regulation Like 1 (PGRL1). Like PGR5, PGRL1 plays a key role in cyclic electron transport around PSI and ensures ATP production. This mutant also leaves more electrons for the hydrogenase – a clear advantage for hydrogen production.
In addition, the authors optimized the culture conditions by avoiding stressful steps such as transferring the algae to new culture media. Instead, they combined a growth phase under oxygen and a production phase in which the oxygen content remained minimal in order to keep the hydrogenase active. Again, a bioreactor was used to continuously collect and analyze the hydrogen produced in order to closely monitor production.
Both studies show how genetic modifications in Chlamydomonas can optimize hydrogen production. Nevertheless, at 0.5 (Nagy et al, 2024) and 0.3 milliliters (Yarkent et al, 2024) of hydrogen per liter of culture solution per hour, we are still a long way from industrially viable standards.
