20 June 2021 ESRFnews

BIOFUELS

FAD stays bent during the process (Science 372 148). This latter discovery is important, because it may explain how the photoenzyme manages to absorb photons in a region of the visible light spectrum that is not used by the organism for photosynthesis. Understanding better how the enzyme works should help [us to] improve its efficiency and stabil- ity, says Beisson. The C. variabilis photoenzyme is not the only way of

exploiting microalgae as an alternative fuel source. For years, academic labs and companies have been culti- vating algal biomass and encouraging it to accumulate intracellular fat, which can be converted either into a biocrude for refinement or into fatty acid methyl esters, which can be used as biofuels. However, harvesting bio- mass at scale and converting it requires a lot of energy, marring its green credentials. The photoenzyme which Beisson and colleagues now believe exists in many species of microalgae is a potentially more attractive option, because it generates ready-to-use volatile hydrocarbons, 15 17 carbon atoms long, like diesel. There is even the possibility of shortening the fatty acids first, so that the reaction directly produces petrol or jet fuel. In fact, Beisson s group already demonstrated that the

process can work in 2019, in a lab-based apparatus that ran non-stop for five days (see Light, algae, CO2: ingre- dients for a hydrocarbon factory , above). Modifying the genome of microalgae to express the necessary high levels of the photoenzyme for an industrial setting will not be so straightforward, nor will scaling up the factory and overcoming the various engineering and economic hurdles. But Beisson believes the challenges are sur- mountable. Another key to success will be to improve the photoenzyme itself so that it is more efficient and stable in the light, he says. This is why it is so important to understand how it works at the molecular level. 

Jon Cartwright

convert fatty acids to hydrocarbons within the microalga. It turned out to be a new photoenzyme an amazing discovery, as, besides those involved with photosynthesis, light-driven enzymes are very rare in living organisms. Beisson and col- leagues sent crystals of it in complex with a fatty acid to the ESRF s highly automated MASSIF-1 macromolecular crystallography beamline. The data revealed the fatty acid to be in a tunnel inside the protein, close to where the light-capturing part of the photoenzyme known as flavin adenine dinucleotide (FAD) is able to be held. It seemed that, under light, FAD steals an electron from the fatty acid, which responds by detaching its carboxylic head to make a hydrocarbon and a CO2 molecule. Thanks to this abil- ity, the researchers called the new photoenzyme fatty acid photodecarboxylase (Science 357 903).

New insights This model left some questions unanswered. For instance, what structural features of the active site in the pho- toenzyme allow it to steal the electron? Is the removal of the carboxylic head instantaneous, or is there a barrier to activation? Beisson and colleagues returned to the ESRF, this time employing a host of techniques: cryo- crystallography at the ID29 and MASSIF-3 beamlines, and ultraviolet-visible absorption and Raman spectroscopy at the ID29 and BM30A beamlines and the ESRF s in crystallo optical spectroscopy (icOS) lab. The work was part of a large international collaboration and was complemented with data taken at other major infrastructures including the SOLEIL synchrotron in France and the Linac Coher- ent Light Source, an X-ray free-electron laser based at the SLAC National Accelerator Laboratory in California, US. From it, the researchers were able to paint a much more detailed picture of the photoenzyme s active site, and the mechanism in general. In their study, published in April this year, they describe how the carboxylic-head removal is near-instantaneous, and that the flavin molecule within

LIGHT, ALGAE, CO2: INGREDIENTS FOR A HYDROCARBON FACTORY

The concept of a photoenzyme-based hydrocarbon cell factory was demonstrated by Fred Beisson and colleagues in the laboratory in 2019. The researchers genetically modified a bacterium to express high levels of the photoenzyme, and sealed a culture of it inside a glass cell though which they bubbled air. Illuminating the cell with LEDs, they could collect 30% of the hydrocarbons emitted in a charcoal trap inside an outlet pipe. The proto-factory ran with a constant production rate for five days (Sci. Rep. 9 13713). According to Beisson, a full-scale hydrocarbon

algal factory might consist of fields of flat-panel photobioreactors (PBRs) bubbled with CO2-enriched air and installed on non-arable land in sunny climes such as those in the Mediterranean close to industries producing CO2, such as cement plants. Elsewhere in Europe, PBRs may still be viable, although productivity would be lower due to the diminished sunlight.

The fatty acids could even be shortened first, so that the reaction directly produces petrol or jet fuel

air input

LED panel

hydrocarbon trap (activated charcoal)

air bubbling system

magnetic stirring bar

microbe culture (300 mL)

air output

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