Giuseppe Torzillo

ON-LINE MONITORING OF CHLOROPHYLL FLUORESCENCE TO ASSESS THE EXTENT OF PHOTOINHIBITION OF PHOTOSYNTHESIS INDUCED BY HIGH OXYGEN CONCENTRATION AND LOW TEMPERATURE AND ITS EFFECT ON THE PRODUCTIVITY OF OUTDOOR CULTURES OF SPIRULINA PLATENSIS (CYANOBACTERIA)

The saturating pulse fluorescence technique was applied to study photoinhibition of photosynthesis in outdoor cultures of the cyanobacterium Spirulina platensis (Nordstedt) Geitler strain M2 grown under high oxygen and low temperature stress. Diurnal changes in maximum photochemical yield (Fv/Fm), photon yield of PSII (ΔF/F′m), and nonphotochemical quenching (qN) were measured using a portable, pulse‐amplitude–modulated fluorometer. When solar irradiance reached the maximum value, the Fv/Fm and ΔF/F′m ratios of theSpirulina cultures grown under high oxygen stress decreased by 35% and 60%, respectively, as compared with morning values. The depression of the Fv/Fm and ΔF/F′m ratios reached 55% and 84%, respectively, when high oxygen stress was combined with low temperature (i.e. 10° C below the optimal value for growth). Photoinhibition reduced the daily productivity of the culture grown under high oxygen stress by 33% and that of the culture grown under high oxygen–low temperature stress by 60%. Changes in the biomass yield of the cultures correlated well with changes in the daily integrated value of the estimated electron transport rate through the PSII (ΔF/F′m × photon flux density). The results indicate that on‐line chlorophyll fluorescence measurement is a powerful tool for assessing the photosynthetic performance of outdoorSpirulina cultures.

Effect of temperature on yield and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors

Outdoor experiments carried out in Florence, Italy (latitude 43.8° N, longitude 11.3° E), using tubular photobioreactors have shown that in summer the average net productivity of a Spirulina platensis culture grown at the optimal temperature of 35 °C was superior by 23% to that observed in a culture grown at 25 °C. The rates of night biomass loss were higher in the culture grown at 25 °C (average 7.6% of total dry weight) than in the one grown at 35 °C (average 5%). Night biomass loss depended on the temperature and light irradiance at which the cultures were grown, since these factors influenced the biomass composition. A net increase in carbohydrate synthesis occurred when the culture was grown at a low biomass concentration under high light irradiance or at the suboptimal temperature of 25 °C. Excess carbohydrate synthesized during the day was only partially utilized for night protein synthesis.

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.

Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and the xanthophyll cycle

Abstract. The role of the xanthophyll cycle in the adaptation of two chlorococcal algae Scenedesmus quadricauda and Chlorella sorokiniana to high irradiance was studied under laboratory and outdoor conditions. We wished to elucidate whether the xanthophyll cycle plays a key role in dissipating the excesses of absorbed light, as in higher plants, and to characterise the relationship between chlorophyll fluorescence parameters and the content of xanthophyll-cycle pigments. The xanthophyll cycle was found to be operative in both species; however, its contribution to overall non-photochemical quenching (NPQ) could only be distinguished in Scenedesmus (15–20% of total NPQ). The Scenedesmus cultures showed a larger pool of xanthophyll-cycle pigments than Chlorella, and lower sensitivity to photoinhibition as judged from the reduction of maximum quantum yield of photosystem II. In general, both algae had a larger xanthophyll-cycle pool when grown outdoors than in laboratory cultures. Comparing the two species, Scenedesmus exhibited a higher capacity to adapt to high irradiance, due to an effective quenching mechanism and high photosynthetic capacity; in contrast, Chlorella represents a species with a larger antennae system, less-efficient quenching and lower photosynthetic performance. Non-photochemical quenching (NPQ) induced through the xanthophyll cycle can, to a limited extent, represent a regulatory factor in diluted algal cultures grown in outdoor solar photobioreactors, as well as in natural algal phytoplankton populations exposed transiently to high irradiance. However, it does not play an appreciable role in dense, well-mixed microalgal suspensions.

Interplay between light intensity, chlorophyll concentration and culture mixing on the hydrogen production in sulfur‐deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors

Relationships between light intensity and chlorophyll concentration on hydrogen production were investigated in a sulfur‐deprived Chlamydomonas reinhardtii culture in a laboratory scale photobioreactor (PBR) equipped with two different stirring devices. In the first case, the culture was mixed using a conventional magnetic stir bar, while in the second it was mixed using an impeller equipped with five turbines. Experiments were carried out at 70 and 140 µmol photons m−2 s−1 in combination with chlorophyll concentrations of 12 and 24 mg L−1. A high light intensity (140 µmol photons m−2 s−1, supplied on both sides of the PBR) in combination with a low chlorophyll concentration (12 mg L−1) inhibited the production of hydrogen, in particular in the culture mixed with the stir bar. An optimal combination for hydrogen production was found when the cultures were exposed to 140 µmol photons m−2 s−1 (on both sides) and 24 mg L−1 of chlorophyll. Under these conditions, the hydrogen production output rate reached about 120 mL L−1 in the culture mixed with the stir bar, and rose to about 170 mL L−1 in the one mixed with the impeller. These outputs corresponded to a mean light conversion efficiency of 0.56% and 0.81%, respectively. However, the efficiency increased to 1.08% and 1.64%, respectively, when maximum hydrogen rates were considered. The better performance of the dense cultures mixed with an impeller was mainly attributed to an intermittent illumination pattern to which the cells were subjected (time cycles within 50–100 ms) which influenced the hydrogen production (1) directly, by providing the PSII with a higher production of electrons for the hydrogenase and (2) indirectly, through a higher synthesis of carbohydrates. The fluid dynamics in the PBR equipped with the impeller was characterized. The better mixing state achieved in the PBR of the new configuration makes it a useful tool for studying the hydrogen production process involving photosynthetic microorganisms, and provides a better insight into the physiology of the process. Biotechnol. Bioeng. 2009; 104: 76–90

Biological constraints in algal biotechnology

In the past decade, considerable progress has been made in developing the appropriate biotechnology for microalgal mass cultivation aimed at establishing a new agro-industry. This review points out the main biological constraints affecting algal biotechnology outdoors and the requirements for making this biotechnology economically viable. One of them is the availability of a wide variety of algal species and improved strains that favorably respond to varying environmental conditions existing outdoors. It is thus just a matter of time and effort before a new methodology like genetic engineering can and will be applied in this field as well. The study of stress physiology and adaptation of microalgae has also an important application in further development of the biotechnology for mass culturing of microalgae. In outdoor cultures, cells are exposed to severe changes in light and temperature much faster than the time scale required for the cells to acclimate. A better understanding of those parameters and the ability to rapidly monitor those conditions will provide the growers with a better knowledge on how to optimize growth and productivity. Induction of accumulation of high value products is associated with stress conditions. Understanding the physiological response may help in providing a better production system for the desired product and, at a later stage, give an insight of the potential for genetic modification of desired strains. The potential use of microalgae as part of a biological system for bioremediation/detoxification and wastewater treatment is also associated with growing the cells under stress conditions. Important developments in monitoring and feedback control of the culture behavior through application of on-line chlorophyll fluorescence technique are in progress. Understanding the process associated with those unique environmental conditions may help in choosing the right culture conditions as well as selecting strains in order to improve the efficiency of the biological process.

Temperature as an important factor affecting productivity and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors

Outdoor experiments using tubular photobioreactors have shown that in summer the average net productivity of a Spirulina platensis culture grown at the optimal temperature of 35°C was superior by 23% to that observed in another culture grown at 25°C. The rates of night biomass loss were higher in the culture grown at 25°C (average 7·6% of dry weight) than in the one grown at 35°C (average 5% of dry weight). We found that the night biomass loss was dependent on the temperature and light irradiance at which the cells were grown, since these factors influence the biomass composition. A net increase in carbohydrate synthesis was observed when the cells were grown under high light irradiance or at the suboptimal temperature of 25°C. The excess of carbohydrate synthesized during the day was only partially utilized for night protein synthesis.

Microbial biomass recovery using a synthetic cationic polymer

Abstract A synthetic cationic polymer (Praestol) for biomass recovery by flocculation of Tetraselmis suecica, Spirulina platensis and Rhodopseudomonas palustris was tested both in the laboratory and outdoors. The flocculating efficiency of Praestol increased with the dose used up to 1 mg litre−1 for all microorganisms tested. With this flocculant dose, the percentage of biomass separated was 86% for Rhodopseudomonas and 70% for Tetraselmis and Spirulina. The maximum flocculation efficiency was observed when the flocculant: biomass ratio was 1:1000 (by weight). Moreover, satisfactory flocculation efficiency was maintained when Praestol was used on cultures grown in sea- or brackish-water media. No inhibitory effect on the growth of the cultures was noted when the medium was recycled in the pond after flocculation.

In situ monitoring of chlorophyll fluorescence to assess the synergistic effect of low temperature and high irradiance stresses inSpirulina cultures grown outdoors in photobioreactors

A chlorophyll fluorescence technique was applied to anin situ study on the effects of low temperature and high light stresses onSpirulina cultures grown outdoors in controlled tubular photobioreactors at high (1.1 g L−1) and low (0.44 g L−1) biomass concentrations. Diurnal changes in PSII photochemistry (Fv/Fm) after 15 min of darkness, or in the light (dF/F′m), and non-photochemical (qN) quenching were measured using a portable, pulse-amplitude-modulated fluorometer. The depression of theFv/Fm ratio ofSpirulina cultures grown outdoors at 25°C (i.e. 10°C below optimum for growth) and 0.44 g L−1, reached 30% at the middle of the day. At the same time of the day thedF/F′m ratio showed a reduction of up to 52%. The depression of bothFv/Fm anddF/F′m was lower in the cultures grown at 1.1 g L−1. Photoinhibition reduced the daily productivity of the culture grown at 0.44 g L−1 and 25°C by 33% with respect to that grown at 35°C. Changes in the growth yields of the cultures grown under different temperatures and growth rates correlate well with analogous changes in photon yield (dF/F′m). Simple measurements of photochemical yield (Fv/Fm) can be used to test the physiological status ofSpirulina cultures. The results indicate that the saturating pulse fluorescence technique, when usedin situ, is a powerful tool for assessment of the photosynthetic characteristics of outdoor cultures ofSpirulina.

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.