Effects of Light Intensity and Oxidized Nitrogen Sources on Hydrogen Production by Chlamydomonas reinhardii

Abstract

Chlamydomonas reinhardii cells, after a period of dark anaerobic adaptation, evolve H2 not only in the dark but also in the light. Our results show that high irradiances impair prolonged H2 evolution, while under low irradiances or darkness H2 evolution proceeds for more than 50 hours. NO3-and NO2-suppress H2 evolution both in the dark or under low irradiance. Apparently the cells prefer these oxidized nitrogen sources to protons as electron acceptors, since both N03-and NOTbecome reduced to NHW+, which is excreted to the culture medium in high amounts. H2 evolution started once these oxidized anions were largely depleted from the medium. Moreover, H2 evolution was consistently associated with NH4I excretion even if NH4' was already present in high amounts in the medium. This observation indicates that the cells utilize not only their carbohydrate but also their protein reserves as sources of reducing power for H2 evolution. This conclusion was supported by the observation that when nitrogen-starved cells were made anaerobic in a nitrogen-free medium, they not only evolved H2 at very high rates but excreted concomitantly NH4W up to concentrations in the millimolar range.After some controversy following the pioneering work of Gaffron and Rubin (1 1) on H2 evolution by green algae, it is now widely accepted that two different pathways exist in these organisms for H2 photoproduction with either water or carbohydrates as electron donors (1). Direct coupling between oxygenic photosynthetic activity and the H2 evolving system has been demonstrated to take place preferentially during initial periods of light exposure (7,12,22). Alternatively, simultaneous evolution of CO2 and H2 have been observed both in the dark (15) and in a light-dependent process involving solely PSI (18). In these two latter cases, cellular carbon reserves provide ultimately the reducing equivalents for H2 evolution, with H+ acting as final electron acceptors. Since H2 evolution implies such a simple redox reaction, it should provide an effective pathway for the disposal of excess internal reducing power, especially under low 02 tensions (18,26). A similar relief valve operates in anaerobic bacteria (1).In photosynthetic eukaryotes, the reductive utilization ofNO3-is carried out by two different enzymes: NAD(P)H-nitrate reductase which reduces N03 to N02 , and reduced ferredoxin-nitrite reductase which reduces N02-to NH4' (14 to the medium, most probably to unload excess photosynthetically generated reducing power (2, 3). Moreover, it was found that in this organism inorganic nitrogen metabolism was modulated by blue light (4).We report here the effect ofthe physiological electron acceptors N03 and N02 on H2 production by anaerobically adapted cells of C. reinhardii. When either of these oxidized nitrogen compounds were present, H2 production was suppressed, while, depending on the oxidized nitrogen source, NO2-and/or NH4' were released into the medium. However, with NH4' as the only nitrogen source, H2 evolution was enhanced. Furthermo...

Optimization of Metabolic Capacity and Flux through Environmental Cues To Maximize Hydrogen Production by the Cyanobacterium “ Arthrospira ( Spirulina ) maxima ”

“…The competition with nitrate for hydrogen production has been previously reported for Synechocystis sp. strain PCC 6803 (24) and in the green alga Chlamydomonas reinhartii (5). We expect that older cultures grown in batch mode will have naturally depleted nitrate from cellular uptake.…”

COPPER RESPONSE REGULATOR1–Dependent and –Independent Responses of theChlamydomonas reinhardtiiTranscriptome to Dark Anoxia

“…It might be proposed that the glyoxylate cycle was employed to convert amino acids to fermentable substrates also after the onset of anaerobiosis. The cycle is active in anaerobic C. reinhardtii cells (Gibbs et al, 1986;Willeford and Gibbs, 1989) and dark-anoxic algae excrete ammonium (Aparicio et al, 1985). The products of amino acid degradation might be used for gluconeogenesis, whose induction was indicated by increased amounts of the pyruvate:phosphate dikinase transcript PPD1 and MME2, a transcript encoding an NADP-dependent malic enzyme.…”

Relevance of nutrient media composition for hydrogen production in Chlamydomonas

“…According to Philipps et al (2012), the H 2 production rates are also lower than those obtained under S deficiency. Intriguingly, Aparicio et al (1985) reported no advantage in H 2 production when comparing N-depleted vs ammonium-containing cultures. Hence, H 2 production under N deficiency in Chlamydomonas needs to be better studied.…”

“…Thus, it is proposed that fermentative H 2 production plays an important role in N-depleted cells. As suggested by Aparicio et al (1985), protein degradation could be the major source or reductive equivalents under N deficiency; this suggestion is based on the observation that cells excrete ammonium during H 2 production even in N-depleted cultures. Hence, the pyruvate resulting from protein degradation could contribute to H 2 production via PFR in N-depleted cultures.…”

“…Finally, prolonged nutrient stress can cause the mobilization of internal reserves as sources of ATP with the concomitant generation of reduced equivalents (predominantly NADH and NAD(P)H). Indeed, the mobilization phase of internal metabolites (e.g., starch and proteins) is often reported to be coordinated with the H 2 -production phase Aparicio et al 1985;Batyrova et al 2012;Volgusheva et al 2014). …”