CMldmyuOnas rihardii cells, growning photoautotropbical under air, excreted to the culture medium much higher amounts of N02 and NH4 I under ble than under red fight. Under shmilar conditions, but with N02-as the only nitrogen source, the cells consumed NO2 -and excreted NH4, at shilar rates under blue and red light. In the presence of N03-and air with 2% CO2 (v/v) incorporate al the pbotogenerated NH4 +. Because these cells should have high levels of reducing power, they might use N03-or, in its absence, NO,-as terminal electron acceptors. The excretion of the products of N02-and NH4, to the medium may provide a mechanism to control reductant level in the cells. Blue lght is suggested as an important regulatory factor of this photorespiratory conmption of N03-and possibly of the whole nitrogen metabolism in green algae.Nitrate assimilation in green algae and higher plants is a basic metabolic process because it uses more than 20o of the reducing power generated by their photosynthetic apparatus (11). Among the different steps involved in this metabolic pathway, reduction of NO3 to N02 catalyzed by nitrate reductase has become particularly relevant due to its regulatory features on nitrogen metabolism (5,12,36).NADH-nitrate reductase from green algae is a multimeric enzyme of high mol wt with several electron transport components such as flavin adenine dinucleotide, protoheme b557, and molybdenum (8). Recently, it has been reported that molybdenum is held in a special cofactor that contains an unidentified pterin (13).

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...

The utilization of N03-by green algae growing photoautotrophically under air, which are growth conditions close to their more habitual situations in nature, is associated with the excretion of N02-and NHW to the culture medium. The entire process is promoted by blue light and depends on photosynthetically active radiation for the required reducing equivalents. The (18).Although in green algae the reduction of nitrate has been currently considered mainly an assimilatory process, recent studies have shown that these organisms growing under the prevailing air-CO2 tensions excrete high amounts of NO2-and NH4' to the culture medium in the light. Under these conditions, the cells utilize NO3-and NO2-as electron sinks probably to unload photosynthetically generated reducing power. Recently, it was also shown that this process was sustained much more efficiently by blue than by red light (4, 6). When the CO2 tension in the gas phase was increased to 2%, NH4' and NO2-excretions ceased and previously excreted NO2-and NHI4 became rapidly assimilated, most probably as a consequence of the increase in the availability of carbon skeletons for incorporating NH4' (7).As shown in this paper, to achieve high rates of NO3-utilization and N02 and NH4' excretions, either high irradiance of blue light or combined high irradiance of red light with small quantum fluxes of blue light are required. High irradiances of PAR radiations would generate the necessary reducing equivalents, while blue light has the additional role of activating in situ nitrate reductase.A close correlation was found between the blue light-induced increase in nitrate reductase activity and the blue light stimulation of the NO2-and NH4-excretion rates.In eukaryotic photosynthetic organisms, the reduction ofNO3-to NH4' takes place in two well defined steps: (a) the reduction of NO3-to NO2-catalyzed by the NAD(P)H-nitrate reductase wherein 2e-are involved and (b) the reduction of NO2-to NH4' catalyzed by the Fd-nitrite reductase wherein 6e-are involved (13).

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The blue light dependent utilization of nitrate by green algae under common air and high irradiances, besides its assimilatory nature, is associated with the release of NO2 (-) and NH4 (+) to the culture medium. If the CO2 content of the sparging air was increased up to 2%, previously excreted NO2 (-) and NH4 (+) were rapidly assimilated. When under air and high irradiances the cell density in the culture reached values corresponding to 25 μg Ch 1.ml(-1), no further growth was observed and the highest values of NO3 (-) consumption and NO2 (-) and NH4 (+) release were attained. Besides low CO2 tensions, increasing NO3 (-) concentrations in the medium stimulated the release of NO3 (-) and NH4 (+). Under CO2-free air the consumption of NO3 (-) and the release of NO2 (-) and NH4 (+) on a total N bases were almost stoichiometric and their rates saturated at much lower irradiances than under air. Under CO2-free air high rates of NO2 (-) release were only observed under the blue radiations that were effectively absorbed by photosynthetically active pigments, i.e. 460 nm, but not under 404 and 630 nm radiations. However, the simultaneous illumination of the cells with 404 and 630 nm monochromatic light showed a remarkable synergistic effect on NO2 (-) release.The results are discussed in terms of the close relationship between C and N metabolism, the photosynthetic reducing power required to convert NO inf3 (sup±) -N into R - NH2-N and the blue light activation of nitrate reductase.