Sustained photobiological hydrogen production by Chlorella vulgaris without nutrient starvation
Introduction
Growing concerns about global warming and the limited amount of available fossil energy have increased the need to shift the energy production towards renewable sources. Hydrogen (H2) is extensively proposed as a future source of alternative energy. Some species of microalgae are currently being investigated as potential sources of bioenergy and biofuels such as H2 [[1], [2], [3], [4], [5]]. In microalgae, H2 production is catalyzed by the enzyme hydrogenase in a light-dependent process since hydrogenases are coupled to the photosynthetic electron transport chain via a specific ferredoxin [6,7].
Microalgae and cyanobacteria (blue-green algae) are the only organisms able to combine oxygenic photosynthesis with the production of H2, an attractive pathway for a direct production of H2 from solar energy and water [8]. Following the enthusiasm over the sulfur-deprivation process that greatly stimulates algal H2 production from water in Chlamydomonas reinhardtii, many papers, reviews and book, have been recently published on this subject [5,7,[9], [10], [11], [12]]. Recent reports have also shown that certain new isolates of Chlorella can produce high amounts of hydrogen when suitable organic substrates and reducing agents are supplied to nutrient replete cultures under low irradiance [13]. It has also been found that some isolates of Chlorella vulgaris can produce small amounts of hydrogen even under aerobic conditions, provided that the ratio CO2:O2 is much higher than that normally occurring in the atmosphere [14].
The simplest most sustainable and efficient way to produce H2 with microalgae is the so-called direct biophotolysis, which involves direct transfer of electrons from water to the hydrogenase. However, until to date H2 production of significant amounts of hydrogen from direct biophotolysis is strongly limited by the O2 sensitivity of hydrogenase which is the most challenging barrier to overcome [15,16].
Oxygen acts as a transcriptional repressor, an inhibitor of hydrogenase maturation, and an irreversible inhibitor of hydrogenase catalytic activity [[17], [18], [19]].
The sulfur-starvation technique proposed by Melis et al. [8] is still the preferred way to induce H2 production in Chlamydomonas reinhardtii but it imposes a severe stress leading to the progressive degradation of the photosynthetic apparatus, and as a result light conversion efficiency is very low (0.1% of PAR, photosynthetically active irradiance) [5,20]. Since the water splitting function of photosystem II (PSII) is the main source of electrons for H2 photoproduction, it is important to maintain its activity for as long as possible [1,[21], [22], [23]].
The possibility of improving the biomass and output rates of hydrogen production has been reported in C. reinhardtii, by introducing an alternative route to supply H+ and e− to the hydrogenase enzyme utilizing glucose an alternative source of electrons [24]. In particular, the authors reported an increased H2 production by about 50% in a C. reinhardtii when glucose was added to the sulfur-deprived medium.
Here we report an evidence of sustained photobiological H2 production by Chlorella vulgaris BEIJ, strain G-120 under continuous illumination without applying any nutrient starvation. Its high respiration rate, coupled to a high light compensation point made it possible to efficiently dispose the O2 produced by the water splitting process thus maintaining anaerobiosis, even under relatively high irradiance. This strain is able to grow vigorously in the dark using glucose as a source of carbon and energy, while under illumination it can easily convert its metabolism towards photo autotrophy and produce H2 in a sealed reactor without the need for starvation.
Section snippets
Microalgal strains and culture medium
The green microalga Chlorella vulgaris G-120 (registered as Chlorella vulgaris BEIJ., 1996/H 14, CCALA 30001, Culture Collection of Autotrophic Organisms, Institute of Botany, Třeboň, Czech Republic) is a natural, non-GMO strain (cell size between 3 and 5 μm), which grows fast when cultured heterotrophically (Fig. 1).
The Chlorella G-120 cultures were grown heterotrophically in a medium (hereafter HM) as already described [25,26]. The composition of nutrients solution related to 10 g of glucose:
Photosynthetic rate
The net photosynthetic rates of Chlorella strain G-120 was compared with those of Chlamydomonas reinhardtii strain CC-124 which is commonly used for the hydrogen production experiments (Fig. 3). The important photosynthetic variables estimated from the curves of both the strains are reported in Table 1.
Compared to CC-124, the strain G-120 presents significant differences: a 4-fold higher maximum photosynthesis rate (Pmax), and an 8-fold higher saturation irradiance (Ik) (Table 1). More
Conclusions
This study demonstrated that the microalga Chlorella vulgaris, strain G-120 is capable to produce a sizeable amount of biohydrogen without nutrient starvation, which represents an important step forward in the scale-up process. Indeed, the use of sulfur starvation strongly reduces the economy of the process since it requires to eliminate sulfur residues by repeatedly centrifugate the cells, enhancing the risk of contamination of the culture. Moreover, the strong downregulation of the PSII
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The work was partially supported by the Vicerrectoria de Investigacion, University of Costa Rica, project number C0107: Produccion biológica de hidrógeno. We thank Dr. Bernardo Cicchi for the English revision of the manuscript.
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