Cecilia Faraloni

Sustained H2 production in a Chlamydomonas reinhardtii D1 protein mutant

In the present investigation, a detailed biochemical analysis of the high H₂ producer D1 protein mutant strain L159I-N230Y of Chlamydomonas reinhardtii, carrying a double amino acid substitution, was made. The leucine residue L159 was replaced by isoleucine, and the N230 asparagine was replaced by tyrosine. The performance of this strain was compared to that of the cc124 strain. The mutant showed a sustained capacity to donate electrons by means of direct biophotolysis for H₂ production, as demonstrated by the higher efficiency of utilization of the hydrogenase enzyme when carried out under anaerobic conditions. The latter property was maintained also under sulfur deprivation. Furthermore, when compared to the cc124, the mutant showed a higher amount of D1 protein content, a higher carbohydrate storage capacity and a sustained PSII direct contribution to the H₂ production during sulfur deprivation. The addition of DCMU to the cells showed that as much as 7.0 mL H₂ liter of culture h⁻¹ were produced by means of direct biophotolysis. The maximum apparent light-to-hydrogen conversion efficiency expressed on PAR (photosynthetically active radiation) reached 3.22%, while PSII efficiency to perform direct biophotolysis was calculated to be 2.03%. These values are significantly higher than what has been reported in the literature.

Interplay between photochemical activities and pigment composition in an outdoor culture of Haematococcus pluvialis during the shift from the green to red stage

The transfer of laboratory cultures of H. pluvialis to high irradiance outdoors caused a substantial decline in the maximum quantum yield of photosystem II (PSII), from 0.65 in the morning to 0.45 at midday, as measured by the ratio of variable to maximum fluorescence yields (Fv/Fm), and a steep rise in non-photochemical quenching (NPQ). Chlorophyll fluorescence induction curves of morning samples showed a clear I-step, reflecting a certain PSII heterogeneity. Single turnover flash measurements on samples taken from the outdoor photobioreactor in the middle of day showed an increase in the reoxidation time constant of the reduced plastoquinone QA−, i.e., the time required for electron transfer from the primary plastoquinone acceptor of PSII QA− to the secondary plastoquinone acceptor QB. Photosynthesis rates were almost constant during the day. Along with the increase in non-photochemical quenching, there was a slight increase in zeaxanthin and antheraxanthin contents and decrease in violaxanthin, showing the presence of an operative xanthophyll cycle in this microalga. A marked increase of secondary carotenoids was found at the end of the first day of exposure to sunlight, mainly astaxanthin monoester, which reached 15.5% of the total carotenoid content. Though cells turned reddish during the second day, the decline in the fluorescence parameter Fv/Fm in the middle of the day was less than during the first day, and there was no further increase in the value for NPQ. Similar behaviour was observed during the third day when the culture was fully red. After four days of exposure to sunlight, the dry weight reached 800 mg L−1 and the concentration of secondary carotenoids (81% astaxanthin monoester) reached 4.4% dry weight.

Outdoor H2 production in a 50-L tubular photobioreactor by means of a sulfur-deprived culture of the microalga Chlamydomonas reinhardtii

In the past decade, H₂ production using the green microalga Chlamydomonas reinhardtii has been extensively studied under laboratory-scale photobioreactors, while information on outdoor cultures is still lacking. In this paper, the results of experiments conducted with sulfur-deprived cultures of C. reinhardtii carried out in a 50-L horizontal tubular photobioreactor are presented. Hydrogen production experiments were carried out under both artificial and direct solar light. In both cases, the H₂ output attained was 18-20% of what obtained in the laboratory. However, no significant changes in the H₂ production were observed when cells grown outdoors were tested under laboratory conditions. Chlorophyll fluorescence measurements showed that outdoor cultures were subjected to strong photo-inhibition, due to the combination of high solar light intensity and sulfur-deprivation. Indeed, H₂ production was only achieved outdoors when cultures were previously acclimated to sunlight, a condition that caused a number of physiological changes, namely: (i) a decrease in the chlorophyll content per unit of dry weight; (ii) an increase in the photosynthesis and respiration rates, and (iii) a higher induction of the xanthophyll cycle pigments as compared to non-acclimated cultures. It was concluded that the reduced H₂ output achieved in the 50-L photobioreactor was due to the different illumination pattern to which the cultures were exposed (one-sided vs. two-sided illumination provided in the laboratory), as well as to the great difference in the mixing times (60 min vs. 15.5s achieved in the lab-scale photobioreactor). To the very best of our knowledge this is the first time that H₂ production with green algae has been achieved by means of solar light.

PHENOTYPIC CHARACTERIZATION AND HYDROGEN PRODUCTION IN CHLAMYDOMONAS REINHARDTII QB-BINDING D1-PROTEIN MUTANTS UNDER SULFUR STARVATION: CHANGES IN CHL FLUORESCENCE AND PIGMENT COMPOSITION1

The effects of QB‐binding D1‐protein mutations on the phenotypic characteristics and on hydrogen production of sulfur‐deprived Chlamydomonas reinhardtii P. A. Dang. cultures were investigated. The mutation involved one (D240) or double (D239–40) amino‐acid deletions at positions 240 and 239–240, respectively, in the loop connecting helices D and E of the D1 protein. Phenotypic characterization of the mutants showed the following peculiarities as compared to the wildtype (WT): (i) a higher sensitivity to photoinhibition, (ii) a reduced amount of chl per dry weight and per cell, (iii) a higher respiration‐to‐photosynthesis ratio, (iv) a higher carbohydrate accumulation during the aerobic phase, and (v) a higher synthesis of xanthophyll‐cycle pigments. These differences were translated into a 12‐ to 18‐fold higher hydrogen biogas production.

Ionizing radiation impacts photochemical quantum yield and oxygen evolution activity of Photosystem II in photosynthetic microorganisms.

Purpose: Long-term space exploration requires biological life support systems capable of coping with the deleterious space environment. The use of oxygenic photosynthetic microorganisms represents an intriguing topic in this context, mainly from the point of view of food and O2 production. The aim of the present study was to assess the effects of space ionizing radiation exposure on the photosynthetic activity of various microorganisms. Materials and methods: Ground-based irradiation experiments were performed using fast neutrons and gamma rays on microorganisms maintained at various light conditions. A stratospheric balloon and a European Space Agency (ESA) flight facility were used to deliver organisms to space at the altitude of 38 and 300 km, respectively. During the balloon flight, the fluorescence activity of the organisms was real-time monitored by means of a special biosensor. Results: The quantum yield of Photosystem II (PSII), measured directly in flight, varied among the microorganisms depending on the light conditions. Darkness and irradiation of cells at 120 and 180 μmol m−2 s−1 enhanced the radiation-induced inhibition of photosynthetic activity, while exposure to weaker light irradiance of 20 and 70 μmol m−2 s−1 protected the cells against damage. Cell permanence in space reduced the photosynthetic growth while the oxygen evolution capacity of the cells after the flight was enhanced. Conclusions: A potential role of PSII in capturing and utilizing ionizing radiation energy is postulated.

Morphological and biochemical characterization of the exocellular investments of polysaccharide-producing Nostoc strains from the Pasteur Culture Collection

A total of 40 Nostoc strains, belonging to the Pasteur Culture Collection and originally isolated from different habitats, were photoautotrophically grown in liquid cultures and tested for the presence of exocellular polysaccharidic investments surrounding the trichomes. However, 25 of them showed a significant presence of these structures, coupled with the release of polysaccharidic material (RPS) into the medium. A rather large number of different morphological forms was observed in the cultures during growth, but at the time of harvesting the predominant morphological form was, in most cases, the vegetative trichome. With regard to the exocellular mucilaginous investments, three main types of morphologies were observed: (i) capsules surrounded by an external pellicle, (ii) capsules with sharp outlines but without an external pellicle, (iii) slimy investments that either loosely surrounded the trichomes without following their shape or were organized in large globular lumps. Among the twenty-five strains that released polysaccharides into the culture medium, three showed mean daily productivities ranging from 30 to 50 mg (RPS) l−1 d−1, values comparable with those of the most productive cyanobacterial strains so far described. The morphological characteristics of the polysaccharidic investments produced by the Nostoc strains seem not to be related to their original habitats. Furthermore, the differences in RPS productivities observed among the strains seem not to be related to the shape of the mucilaginous exocellular investments. Chemical analysis of purified samples of the polysaccharides demonstrated that all the polymers possess an acidic nature, due to the presence of uronic acids, and that they are characterized by the presence of a peptidic moiety and of amino sugars.

Photoacclimation of Phaeodactylum tricornutum (Bacillariophyceae) cultures grown outdoors in photobioreactors and open ponds

The acclimation to high light of Phaeodactylum tricornutum cultures grown outdoors both in an open pond and in tubular photobioreactors (PBRs) was studied by means of chlorophyll fluorescence, pigment analysis and growth. Cultures grown in PBRs (5-cm tube diameter) at two biomass concentrations (0.3 and 0.6 g l−1) were compared with a culture grown in a 10-cm deep open pond (0.3 g l−1). Therefore, the performance of the cultures was compared on the basis of both the same biomass concentration and areal density. Cultures grown at 0.3 g l−1 in PBRs experienced the highest light stress, which resulted in dramatic changes in both chlorophyll fluorescence and photosynthesis parameters, and in low areal productivity. In this culture, the Fv/Fm ratio was 0.5 in the morning and dropped to about 0.1 within 1 h of exposure to bright sunlight. Similar behaviour was observed with the measurements of rETR, and the initial slope (αETR) of the photosynthesis curve, while the saturation irradiance parameter (Ik) increased about four-fold compared to the morning value. These changes were accompanied by a higher induction of the diadinoxanthin-cycle pigments, evidenced by an increasing ratio of diatoxanthin to diadinoxanthin during the day (up to 104% at 1400 h), and a higher value of non-photochemical quenching (NPQ) that rose to up to 1.2 in the high stress culture. The culture grown in the open pond at the same biomass concentration (0.3 g l−1) did not show sizeable changes either in pigments or in chlorophyll fluorescence parameters. Denser cultures in PBRs (0.6 g l−1) showed less dramatic changes in the photochemical parameters. Changes in the areal productivity of the cultures correlated well with changes in the photochemical parameters. Areal productivity was about 27% higher in the open pond than in PBRs when cultivated at the same concentration of 0.3 g l−1.

The effect of ionising radiation on photosynthetic oxygenic microorganisms for survival in space flight revealed by automatic photosystem II-based biosensors

Photosynthetic microorganisms are expected to be useful to maintain an oxygenic atmosphere and to provide biomass for astronauts in the International Space Station as well as in future long-term space flights. However, fluxes of complex ionizing radiation of various intensities and energies make space an extreme environment for the microorganisms, affecting their photosynthetic efficiency. To automatically monitor the photosynthetic Photosystem II (PSII) activity of microorganisms under space conditions an optical biosensor, which utilizes chlorophyll fluorescence as biological transduction system, was built; the PSII activity was monitored by the biosensor during balloon flights at stratospheric altitudes of about 40 km. The effect of space stress on quantum yield of PSII varied among the tested species depending on the growth light conditions at which they were exposed during the flights.

Carbohydrate synthesis by two Navicula strains isolated from benthic and pelagic mucilages in the Tyrrhenian Sea (Tuscan Archipelago)

Two exopolysaccharide (EPS)-producing strains of the diatom Navicula, were isolated from benthic and pelagic mucilaginous aggregates sampled in the Tyrrhenian Sea and cultured under laboratory conditions. The amount of carbohydrate formed over the growth period and on a per cell basis was quite similar. However, the benthic strain showed a preferential synthesis of the bound (i.e., cellular and capsular) carbohydrate fraction, whereas the pelagic strain preferentially synthesised soluble, polymeric carbohydrates. The polysaccharides released into the medium by the two strains showed the same qualitative monosaccharidic composition, being constituted by two acidic and six neutral sugars. It is suggested that the difference between the benthic and the pelagic strain in the synthesis of bound or soluble carbohydrates may be related to the different role of these compounds in the particular habitats of the strains.

H2 production in Rhodopseudomonas palustris as a way to cope with high light intensities

The ability of coping with the damaging effects of high light intensity represents an essential issue when purple non-sulfur bacteria (PNSB) are grown under direct sunlight for photobiological hydrogen production. This study was aimed at investigating whether H2 photo-evolution could represent, for Rhodopseudomonas palustris 42OL, a safety valve to dissipate an excess of reducing power generated under high light intensities. The physiological status of this strain was assessed under anaerobic (AnG) and aerobic (AG) growing conditions and under H2-producing (HP) conditions at low and high light intensities. The results obtained clearly showed that Fv/Fm ratio was significantly affected by the light intensity under which R. palustris 42OL cells were grown, under either AnG or AG conditions, while, under HP, it constantly remained at its highest value. The increase in light intensity significantly increased the H2 production rate, which showed a positive correlation with the maximum electron transfer rate (rETRmax). These findings are important for optimization of hydrogen production by PNSB under solar light.