Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions
Introduction
Prolonged exposure of green algae to anaerobic conditions in the dark leads to the expression of the hydrogenase enzyme and subsequent H2 evolution in the light. This phenomenon was first reported over 60 years ago in the pioneering work of Gaffron and Rubin [1], but until recently algal H2 production remained a laboratory curiosity, despite its fundamental importance and practical potential. Recent studies reported that H2 photoproduction in anaerobically adapted green algae proceeds at higher rates compared to other microalgae [2] and that the light conversion efficiency of the process can be very high [3], [4]. Nevertheless, the generation of bulk quantities of H2 by green algal cultures was considered impractical, due to the fact that hydrogenase in green algae is very sensitive to the O2 co-evolved during photosynthesis. This problem was deemed unsolvable [5]. As a consequence of its sensitivity to O2, H2 photoproduction in anaerobically adapted cells can be sustained for only short periods of time in the absence of O2 scavengers [2], [6]. Recent studies revealed that not only the hydrogenase enzyme per se is irreversibly inhibited by molecular O2 but also the expression of the genes associated with H2 metabolism is down regulated in the presence of O2 [7], [8], [9], [10].
A few years ago, a team at UC Berkeley and NREL devised a system for bulk production of molecular H2 in Chlamydomonas reinhardtii cultures [11], [12]. The system is based on the partial inactivation of photosystem II O2-evolving activity in algal cells in response to sulfur-deprivation stress [13]. The inhibition of photosystem II (PSII) activity results in the transition of the cultures to anaerobic conditions, expression of the hydrogenase enzyme, and H2 gas production in the light for several days [11], [12]. In the first report of the sulfur-deprived H2-producing system [11], it was proposed that protein degradation was the main process feeding electrons to PSI, since starch degradation during H2 production stage was not significant in the algal strain used. Later, it was shown that electrons for H2 evolution were derived mostly from residual PSII activity [14], [15], [16], and that the relative contribution of PSII to H2 photoproduction depends on the stage of sulfur deprivation [17]. It is now accepted that both water oxidation and the endogenous catabolism of starch and/or protein contribute electrons to H2 production [16], [18]. Moreover, organic substrate degradation fuels the respiratory consumption of O2 produced by residual PSII activity during the H2-production stage and is responsible for maintaining culture anaerobicity [12], [16], [19], [20]. Finally, substrate degradation during the H2-production stage is also required to maintain the appropriate intracellular redox potential to control the expression of the hydrogenase gene in C. reinhardtii [10].
All experiments on H2 production by sulfur-deprived cultures have been done so far with photoheterotrophic cultures using TRIS-acetate-phosphate (TAP) medium. When photoheterotrophic cultures are sulfur-deprived, acetate is consumed during the O2-producing and O2-consuming, aerobic stages, but not during the H2-production anaerobic stage [11]. Indeed, some acetate is even produced during H2-production stage [16]. The use of acetate in the growth medium increases the expense associated with maintenance of the system and, as a consequence, the cost of the H2 gas produced [21]. Indeed, the molar ratio of H2 produced per mole of acetate consumed during the aerobic phase is 1.0 at an initial pH of 7.7 and lower at other pH values [16]. In contrast, purple bacteria use light in combination with organics to produce H2 much more efficiently [22], and are able to achieve molar ratios of 2.67 H2/acetate [23] compared to a maximum theoretical ratio of 4 [24]. These observations raised the question of the feasibility of using photoautotrophic instead of photoheterotrophic sulfur-deprived green algae for H2 production.
The present study demonstrates that sustained H2 photoproduction by the sulfur-deprived green alga, C. reinhardtii, is possible under strictly photoautotrophic conditions, in the absence of acetate or any other organic substrate in the medium. We accomplished this by pre-cultivating cells under a special light regime and CO2 supplementation during S-deprivation of the culture.
Section snippets
Cell growth conditions and sulfur-deprivation procedure
Wild-type C. reinhardtii Dang 137C was grown photoautotrophically in flat glass bottles containing 1.5 l of high salt (HS) medium, pH 7.0 [25], at . Algal cultures were bubbled continuously with 2% CO2 in air. During growth, the algae were illuminated from two sides with cool-white fluorescence lamps providing an average incident light intensity of about PAR (low light conditions, LL) or PAR (high light conditions, HL) on each surface of the culture bottles. If
Demonstration of photoautotrophic H2 production
H2 production by sulfur-deprived C. reinhardtii cultures under photoautotrophic conditions can indeed be demonstrated in HS medium with added bicarbonate. Initially, photoautotrophic cultures, pre-grown under HL conditions ( PAR), were sulfur-deprived and incubated under PAR in a non-automated PhBR system. At the beginning of S-deprivation, NaHCO3 was introduced into each PhBR as the sole carbon source, and the PhBRs were sealed on the second day of sulfur
Discussion
Our first attempt to induce photoautotrophic production of H2 in sulfur-deprived cultures of C. reinhardtii entailed simply excluding acetate from both the pre-growth and the sulfur-deprivation media [29]. However, in the absence of acetate, the algal cultures did not transition to anaerobiosis and, hence, they were unable to induce hydrogenase activity or photoproduce molecular H2. In addition, those cultures did not accumulate starch during the O2-producing stage of sulfur deprivation. Since
Acknowledgments
This work was supported in part by the Hydrogen, Fuel Cells, and Infrastructure Technologies Program, EERE, US Department of Energy, and by the Russian Federation of Basic Research (Grant # 04-04-97205).
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