Sustained photobiological hydrogen production by Chlorella vulgaris without nutrient starvation

Highlights

• Chlorella produced H₂ by direct photosynthesis without applying sulfur-starvation.

• Chlorella G-120 strain can release H₂ both in the light and in the dark.

• Chlorella G-120 strain can be grown both autotrophically and heterotrophically.

• Photobiological H₂ production by strain G-120 reached 7.7% efficiency (H₂ to light).

• Photosynthetic rate of Chlorella G-120 surpassed that of C. reinhardtii CC-124.

Abstract

This article describes the ability of the Chlorella vulgaris BEIJ strain G-120 to produce hydrogen (H₂) via both direct and indirect pathways without the use of nutrient starvation. Photobiological H₂ production reached a maximum rate of 12 mL H₂ L⁻¹ h⁻¹, corresponding to a light conversion efficiency (light to H₂) of 7.7% (average 3.2%, over the 8-day period) of PAR, (photosynthetically active irradiance). Cells presented a maximum in vivo hydrogenase activity of 25.5 ± 0.2 nmoles H₂ μgChl⁻¹ h⁻¹ and the calculated in vitro hydrogenase activity was 830 ± 61 nmoles H₂ μgChl⁻¹ h⁻¹. The strain is able to grow either heterotrophically or photo autotrophically. The total output of 896 mL of H₂ was attained for illuminated culture and 405 mL for dark cultures. The average H₂ production rate was 4.98 mL L⁻¹ h⁻¹ for the illuminated culture and 2.08 mL L⁻¹ h⁻¹ for the one maintained in the dark.

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.