**1. Introduction**

Microalgae are capable of converting light energy into chemical energy. Biofuels such as biodiesel, biohydrogen, and bioethanol can be derived from microalgae [1]. Photosynthesis in microalgae is coupled to the splitting of water and the evolution of oxygen (O2). This process is catalyzed by the membrane-bound multi-protein complex photosystem II (PSII) [2].

It has has been known since 1942, when Gaffron and co-workers noticed that under anaerobic conditions *Scenedesmus obliquus* cells can transiently produce hydrogen (H2) upon illumination when deprived of oxygen [3]. In microalgae, hydrogenase enzyme catalyzes H2 production in a light-dependent process [4]. Upon illumination, after a dark incubation period, due to light-driven electron transport from ferredoxin to hydrogenase, H2 production is observed. H2 production in microalgae can be divided into direct or indirect processes [5]. A direct process occurs when electrons (e−) from water splitting are transferred via PSII and ferredoxin to hydrogenase. An indirect process occurs when e<sup>−</sup> are derived from the metabolism of carbohydrates, previously accumulated during the (light) aerobic phase, and then utilized for H2 production via both a photo-fermentation

**Citation:** 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

Academic Editor: Wei-Hsin Chen

Received: 6 October 2021 Accepted: 27 October 2021 Published: 1 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

process involving photosystem I (PSI) and in a process in the dark, involving the enzyme pyruvate:ferredoxin oxidoreductase (PFR). In *Chlamydomonas reinhardtii* (hereafter *C. reinhardtii*), PFR enzyme catalyzes the reduction of ferredoxin (Fdx) and the transfer of e<sup>−</sup> to hydrogenase in a similar pathway to that utilized by bacteria (Figure 1) [6]. In *C. reinhardtii,* up to 92% of the final H2 output comes from the direct photolysis coupled to the water oxidation operated by PSII [7]. Contribution of dark fermentation to the overall H2 output is considered negligible (about 4%) in *C. reinhardtii*, but it can be significant in other microalgae such as *Chlorella,* as recently shown [8]. Microalgal hydrogenase enzymes are inactivated by the presence of molecular oxygen, and their expression is induced under anaerobic conditions.

**Figure 1.** 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.

In recent years, energy-related H2 demands have prompted scientists to develop methods that greatly enhance the H2-evolving ability of microalgae. The most promising approach has been the so-called "two-stage process" of photosynthesis (stage 1) and H2 production (stage 2) [9]. In this process, there is a separation of the reactions of oxygen and hydrogen production. This bypasses the sensitivity of the hydrogenase enzyme to oxygen. Under such conditions, it was possible to produce significant volumes of H2 by *C. reinhardtii* in a sustained process.

Several microalgae species have been studied for H2 production, especially *C. reinhardtii*, *Chlorella vulgaris*, and *Chlorella pyrenoidosa* [10–13]. Among them, *C. reinhardtii* is a model microorganism widely recognized as an H2 producer, presenting a hydrogenase with an enzymatic activity 10 to 100 times higher than other species [14]. H2 production requires many optimization steps in order to reach a sustainable process [8,14–17]. Some of these parameters include choosing a proper microalgae strain and selecting appropriate culture conditions (growth media, light, pH, temperature, chlorophyll concentration) and proper photobioreactor (PBR) designs [18–20].

Many works have reported improved H2 production in many microalgal strains by using sulfur, phosphorus, or nitrogen-depleted media [12,21–23]. In such culture conditions, microalgae sustain H2 production only for some days since macro/micronutrient depletion in the culture compromises cell viability. This is the major drawback in microalgal H2 production processes carried out by nutrient deprivation. Microalgaebased H2 production requires anaerobic conditions due to the sensitivity of hydrogenase to O2 [24]. O2 sensitivity of hydrogenase is a major issue for H2 production; therefore, there are many studies on oxygen suppression in order to improve H2 production yield. Genetic and metabolic engineering of microalgae [25,26], nutrient stresses [27,28], light conditions optimization [29], and elimination of competing pathways for electrons [30] are examples of strategies used to improve H2 evolution in microalgae.

This review provides an overview of the most relevant achievements in the photobiological production of H2 by microalgae, and proposes a change of paradigm for the future research in the field.
