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Carbon dioxide (CO2) and light are essential for high photosynthetic rates of microalgal cultures. Microalgal photosynthetic behavior at low CO2 concentrations has not been revealed yet at the same level of detail as leaf photosynthesis. In the present study, we investigated the short-term photosynthetic response of suspended Chlorella sorokiniana to limiting light intensity and CO2 concentration. We used a novel CO2-based photosynthesis monitor originating from leaf research but equipped with an aquatic chamber sparged with CO2 enriched air. Photosynthesis was measured by employing a steady-state CO2 mass balance over the chamber. Light and carbon response curves were determined under constant pH, temperature, dissolved oxygen, light intensity or dissolved carbon dioxide. We determined the volumetric mass transfer coefficient of the aquatic chamber to accurately convert gaseous CO2 partial pressure into aqueous CO2 concentration to evaluate the CO2 response measurements. Light response measurements on dilute algal cultures revealed a high photosynthetic capacity on a time scale of minutes which by far exceeded the average CO2 uptake on a time scale of hours (cell growth). Light response measurements with dense and fully absorbing cultures provided accurate insight into the response of microalgal mass culture to changing incident irradiance, including the efficiency of photosynthesis of the algal culture as a whole. CO2 response measurements demonstrated severe CO2 limitation at dissolved CO2 levels of

In reef-building corals (order Scleractinia) and giant clams (phylum Molluca), V-type H + -ATPase (VHA) in host cells is part of a carbon concentrating mechanism (CCM) that regulates photosynthetic rates of their symbiotic algae. Here, we show that VHA plays a similar role in the sea anemone Anemonia majano , a member of the order Actinaria and sister group to the Scleractinia, which in contrast to their colonial calcifying coral relatives is a solitary, soft-bodied taxa. Western blotting and immunofluorescence revealed that VHA was abundantly present in the host-derived symbiosome membrane surrounding the photosymbionts. Pharmacological inhibition of VHA activity in individual anemones resulted in an approximately 80% decrease of photosynthetic O 2 production. These results extend the presence of a host-controlled VHA-dependent CCM to non-calcifying cnidarians of the order Actiniaria, suggesting it is widespread among photosymbiosis between aquatic invertebrates and Symbiodiniaceae algae.

In reef-building corals (order Scleractinia) and giant clams (phylum Molluca), V-type H+-ATPase (VHA) in host cells is part of a carbon concentrating mechanism (CCM) that regulates photosynthetic rates of their symbiotic algae. Here, we show that VHA plays a similar role in the sea anemone Anemonia majano, a member of the order Actinaria and sister group to the Scleractinia, which in contrast to their colonial calcifying coral relatives is a solitary, soft-bodied taxa. Western blotting and immunofluorescence revealed that VHA was abundantly present in the host-derived symbiosome membrane surrounding the photosymbionts. Pharmacological inhibition of VHA activity in individual anemones resulted in an approximately 80% decrease of photosynthetic O2 production. These results extend the presence of a host-controlled VHA-dependent CCM to non-calcifying cnidarians of the order Actiniaria, suggesting it is widespread among photosymbiosis between aquatic invertebrates and Symbiodiniaceae algae.

In algae, it is well established that the pyrenoid, a component of the carbon-concentrating mechanism (CCM), is essential for efficient photosynthesis at low CO2. However, the signal that triggers the formation of the pyrenoid has remained elusive. Here, we show that, in Chlamydomonas reinhardtii, the pyrenoid is strongly induced by hyperoxia, even at high CO2 or bicarbonate levels. These results suggest that the pyrenoid can be induced by a common product of photosynthesis specific to low CO2 or hyperoxia. Consistent with this view, the photorespiratory by-product, H2O2, induced the pyrenoid, suggesting that it acts as a signal. Finally, we show evidence for linkages between genetic variations in hyperoxia tolerance, H2O2 signaling, and pyrenoid morphologies.

In algae, it is well established that the pyrenoid, a component of the carbon-concentrating mechanism (CCM), is essential for efficient photosynthesis at low CO2. However, the signal that triggers the formation of the pyrenoid has remained elusive. Here, we show that, in Chlamydomonas reinhardtii, the pyrenoid is strongly induced by hyperoxia, even at high CO2 or bicarbonate levels. These results suggest that the pyrenoid can be induced by a common product of photosynthesis specific to low CO2 or hyperoxia. Consistent with this view, the photorespiratory by-product, H2O2, induced the pyrenoid, suggesting that it acts as a signal. Finally, we show evidence for linkages between genetic variations in hyperoxia tolerance, H2O2 signaling, and pyrenoid morphologies.

In reef-building corals (order Scleractinia) and giant clams (phylum Molluca), V-type H+-ATPase (VHA) in host cells is part of a carbon concentrating mechanism (CCM) that regulates photosynthetic rates of their symbiotic algae. Here, we show that VHA plays a similar role in the sea anemone Anemonia majano, a member of the order Actinaria and sister group to the Scleractinia, which in contrast to their colonial calcifying coral relatives is a solitary, soft-bodied taxa. Western blotting and immunofluorescence revealed that VHA was abundantly present in the host-derived symbiosome membrane surrounding the photosymbionts. Pharmacological inhibition of VHA activity in individual anemones resulted in an approximately 80% decrease of photosynthetic O2 production. These results extend the presence of a host-controlled VHA-dependent CCM to non-calcifying cnidarians of the order Actiniaria, suggesting it is widespread among photosymbiosis between aquatic invertebrates and Symbiodiniaceae algae.

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) is the carbon-fixing enzyme present in most photosynthetic organisms, converting CO2 into organic matter. Globally, photosynthetic efficiency in terrestrial plants has become increasingly challenged in recent decades due to a rapid increase in atmospheric CO2 and associated changes toward warmer and dryer environments. Well adapted for these new climatic conditions, the C4 photosynthetic pathway utilizes carbon concentrating mechanisms to increase CO2 concentrations surrounding RuBisCO, suppressing photorespiration from the oxygenase catalyzed reaction with O2. The energy efficiency of C3 photosynthesis, from which the C4 pathway evolved, is thought to rely critically on an uninterrupted supply of chloroplast CO2. Part of the homeostatic mechanism that maintains this constancy of supply involves the CO2 produced as a byproduct of photorespiration in a negative feedback loop. Analyzing the database of RuBisCO kinetic parameters, we suggest that in genera (Flaveria and Panicum) for which both C3 and C4 examples are available, the C4 pathway evolved only from C3 ancestors possessing much lower than the average carboxylase specificity relative to that of the oxygenase reaction (SC/O=SC/SO), and hence, the higher CO2 levels required for development of the photorespiratory CO2 pump (C2 photosynthesis) essential in the initial stages of C4 evolution, while in the later stage (final optimization phase in the Flaveria model) increased CO2 turnover may have occurred, which would have been supported by the higher CO2 levels. Otherwise, C4 RuBisCO kinetic traits remain little changed from the ancestral C3 species. At the opposite end of the spectrum, C3 plants (from Limonium) with higher than average SC/O, which may be associated with the ability of increased CO2, relative to O2, affinity to offset reduced photorespiration and chloroplast CO2 levels, can tolerate high stress environments. It is suggested that, instead of inherently constrained by its kinetic mechanism, RuBisCO possesses the extensive kinetic plasticity necessary for adaptation to changes in photorespiration that occur in the homeostatic regulation of CO2 supply under a broad range of abiotic environmental conditions.

Hygrophila polysperma is a heterophyllous amphibious plant. The growth of H. polysperma in submerged conditions is challenging due to the low CO2 environment, increased resistance to gas diffusion, and bicarbonate ion (HCO3–) being the dominant dissolved inorganic carbon source. The submerged leaves of H. polysperma have significantly higher rates of underwater photosynthesis compared with the terrestrial leaves. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), an anion exchanger protein inhibitor, and ethoxyzolamide (EZ), an inhibitor of internal carbonic anhydrase, repressed underwater photosynthesis by the submerged leaves. These results suggested that H. polysperma acclimates to the submerged condition by using HCO3– for photosynthesis. H. polysperma transports HCO3– into the leaf by a DIDS-sensitive HCO3– transporter and converted to CO2 by carbonic anhydrase. Additionally, proteome analysis revealed that submerged leaves accumulated fewer proteins associated with C4 photosynthesis compared with terrestrial leaves. This finding suggested that H. polysperma is capable of C4 and C3 photosynthesis in the terrestrial and submerged leaves, respectively. The ratio of phosphoenol pyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the submerged leaves was less than that in the terrestrial leaves. Upon anatomical observation, the terrestrial leaves exhibited a phenotype similar to the Kranz anatomy found among C4 plants; however, chloroplasts in the bundle sheath cells were not located adjacent to the vascular bundles, and the typical Kranz anatomy was absent in submerged leaves. These results suggest that H. polysperma performs proto-Kranz type photosynthesis in a terrestrial environment and shifts from a proto-Kranz type in terrestrial leaves to a HCO3– use photosynthesis in the submerged environments.

Hygrophila polysperma is a heterophyllous amphibious plant. The growth of H. polysperma in submerged conditions is challenging due to the low CO2 environment, increased resistance to gas diffusion, and bicarbonate ion (HCO3–) being the dominant dissolved inorganic carbon source. The submerged leaves of H. polysperma have significantly higher rates of underwater photosynthesis compared with the terrestrial leaves. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonate (DIDS), an anion exchanger protein inhibitor, and ethoxyzolamide (EZ), an inhibitor of internal carbonic anhydrase, repressed underwater photosynthesis by the submerged leaves. These results suggested that H. polysperma acclimates to the submerged condition by using HCO3– for photosynthesis. H. polysperma transports HCO3– into the leaf by a DIDS-sensitive HCO3– transporter and converted to CO2 by carbonic anhydrase. Additionally, proteome analysis revealed that submerged leaves accumulated fewer proteins associated with C4 photosynthesis compared with terrestrial leaves. This finding suggested that H. polysperma is capable of C4 and C3 photosynthesis in the terrestrial and submerged leaves, respectively. The ratio of phosphoenol pyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the submerged leaves was less than that in the terrestrial leaves. Upon anatomical observation, the terrestrial leaves exhibited a phenotype similar to the Kranz anatomy found among C4 plants; however, chloroplasts in the bundle sheath cells were not located adjacent to the vascular bundles, and the typical Kranz anatomy was absent in submerged leaves. These results suggest that H. polysperma performs proto-Kranz type photosynthesis in a terrestrial environment and shifts from a proto-Kranz type in terrestrial leaves to a HCO3– use photosynthesis in the submerged environments.

A field dataset detailing the abundance and carbon use strategies (obtained with C13 isotopes) of marine macroalgae within 3 wave-exposed and 4 wave-sheltered coastal kelp forests in Otago, New Zealand, along with site information such as temperature, light, nutrients of each site. Additionally, a dataset of the results from a laboratory experiment measuring the photosynthetic response of Xiphophora gladiata and Hymenena palmata to increasing DIC concentrations and under high or low water motion and high (8.1) or low (7.6) pH.

Membrane permeabilities to CO2 and HCO3− constrain the function of CO2 concentrating mechanisms that algae use to supply inorganic carbon for photosynthesis. In diatoms and green algae, plasma membranes are moderately to highly permeable to CO2 but effectively impermeable to HCO3−. Here, CO2 and HCO3− membrane permeabilities were measured using an 18O‐exchange technique on two species of haptophyte algae, Emiliania huxleyi and Calcidiscus leptoporus , which showed that the plasma membranes of these species are also highly permeable to CO2 (0.006–0.02 cm · s−1) but minimally permeable to HCO3−. Increased temperature and CO2 generally increased CO2 membrane permeabilities in both species, possibly due to changes in lipid composition or CO2 channel proteins. Changes in CO2 membrane permeabilities showed no association with the density of calcium carbonate coccoliths surrounding the cell, which could potentially impede passage of compounds. Haptophyte plasma‐membrane permeabilities to CO2 were somewhat lower than those of diatoms but generally higher than membrane permeabilities of green algae. One caveat of these measurements is that the model used to interpret 18O‐exchange data assumes that carbonic anhydrase, which catalyzes 18O‐exchange, is homogeneously distributed in the cell. The implications of this assumption were tested using a two‐compartment model with an inhomogeneous distribution of carbonic anhydrase to simulate 18O‐exchange data and then inferring plasma‐membrane CO2 permeabilities from the simulated data. This analysis showed that the inferred plasma‐membrane CO2 permeabilities are minimal estimates but should be quite accurate under most conditions.(© 2020 Phycological Society of America). ISSN:0022-3646 ISSN:1529-8817

Photoautotrophic organisms globally fix 111-117x1015 grams of carbon per year and around half of this global net primary production is aquatic (Behrenfeld et al., 2001; Field et al., 1998), with green algae a major contributor to this global carbon fixation. However, aquatic environments have some limitations The concentration of CO2 is often 2,200 times lower in water than in air, and diffusion is also 8,000 times slower. In addition, Rubisco, which catalyses the first major step of carbon fixation, converting atmospheric CO2 into precursors of energy-rich molecules, exhibits slow catalytic rates, low affinity for CO2 and competition from O2 for the active sites. Therefore, most green algae have developed a Carbon Concentrating Mechanism (CCM). In eukaryotic micro-algae, the Rubisco micro-compartment is called the pyrenoid and together with active inorganic carbon transporters and strategically located carbonic anhydrases, elevated CO2 within the pyrenoid improves photosynthetic efficiency. Most photosynthetic organisms have an hexadecameric Rubisco holoenzyme (L8S8), composed of eight ~55-kDa large subunit (LSU), encoded by a chloroplast gene (rbcL) and eight ~15-kDa small subunit (SSU), encoded by a nuclear gene family (RbcS) in Form I Rubisco. The CCM has been particularly well-defined in the model unicellular chlorophyte Chlamydomonas reinhardtii and recent studies showed that for full CCM induction, a key protein linker EPYC1 and its interaction with Rubisco SSU were necessary. The overall goal of this study was to use a phylogenetic approach, firstly to investigate SSU structure across the green algal phylogeny, and also to explore CCM diversity in two specific groups of species. This study used a variety of methodologies combining physiological experiments, biochemistry, imaging and bioinformatic analyses. The results firstly showed the presence of two different Rubisco SSU structures within the green algae. Secondly, the Rubisco catalytic properties found in streptophyte algae closely related to land plants (streptophytes) reflect the strength of any CCM and pyrenoid leakiness, whereas Rubisco in extant land plants reflects more recent selective pressures associated with the terrestrial atmospheric environment. This research also provides evidence for diversity of CCM expression in two closely related genera (Chlamydomonas and Chloromonas), ranging from species expressing a CCM and pyrenoid, or a CCM without a pyrenoid, to neither pyrenoid or CCM. This study provides the first preliminary analyses of five different genomes confirming multiple independent origins of the pyrenoid in green algae but has also allowed an initial comparison of the molecular components essential for pyrenoid formation across these species., Natural Environment Research Council (grant number NE/L002507/1 to Howard Griffiths) and resources associated with BBSRC-BB/M007693/1, BB/I024518/1 as part of the Combining Algal and Plant Photosynthesis (CAPP), supported by BBSRC and NSF. We are grateful for a Cambridge Trust Vice Chancellor’s award and Lucy Cavendish College, Cambridge, for supporting the PhD scholarship of Myriam Goudet. Doug Orr and Elizabete Carmo-Silva acknowledge support from the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant number BB/I024488/1).

Background Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations. Results Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low. Conclusion We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.

The signaling transduction proteins of PII superfamily are one of the ancient and highly conserved protein families in nature and widely distributed in all domains of life. Canonical PII exerts it’s signaling function via the binding of small effector molecules, including ATP, ADP, and 2-oxoglutarate (2-OG). All cyanobacteria contain a PII homologue of the GlnB-type, which is involved in the regulation of nitrogen assimilation by binding to N-acetyl-L-glutamate kinase (NAGK), the key enzyme of arginine biosynthesis, and to PII-interacting protein X (PipX), the co-activator of the global nitrogen-trascription factor NtcA. The binding of PII to NAGK enhances NAGK activity and prevents arginine (Arg) feedback inhibition, thereby increasing the flux into the arginine pathway. Formation of the PII-PipX complex causes a deactivation of the NtcA regulon. In this study, we identified Lys58 to be a key residue for proper sensing/signaling function of cyanobacterial PII protein. Furthermore, this study provided the first biochemical characterization of red algal PII protein from Porphyra purpurea. Its properties represent an intermediary state between PII properties in Cyanobacteria and Chlorophyta. A comparison to Chlorophyta PII proteins showed that during later stages of evolution of Chlorophyta, the PII proteins diverged in their properties, becoming very heterogeneous with respect to ADP and to 2-OG binding, while only the binding of ATP in presence of 2-OG has been conserved. Further, we characterized the first metabolic adaptation strategies of colorless-nonphotosynthetic alga Polytomella parva, in comparison to closely related green-photosynthetic alga Chlamydomonas reinhardtii, in response to nitrogen starvation. Metabolome analysis revealed that P. parva accumulates higher amounts of TCA intermediates as well as arginine, glutamate and aspartate. Strikingly, the PII and NAGK proteins in P. parva have coevolved into a stable hetero-oligomeric complex, irrespective of effector molecules such that PII evolved into a subunit of NAGK. The PII-like proteins lack the canonical PII signature sequences but are structurally clearly related to classical PII proteins. Their functions, targets and regulatory responses are unknown. A close examination of available cyanobacteria genomes revealed several different PII-like proteins. In this study, we focused on two PII-like proteins in cyanobacteria namely: 1) SbtB protein, which is located in one operon next to sodium dependent bicarbonate transporter (SbtA) and 2) the divalent ion tolerance protein CutA. Biochemical and physiological studies showed that the SbtB protein is an important component of the cyanobacterial carbon concentration mechanism (CCM) for sensing the fluctuation levels of inorganic carbon (Ci) via binding to the secondary messenger cAMP. Also, we demonstrated that cyanobacterial CutA protein is not involved directly in heavy metal sensing. SbtB was revealed as the first protein of the PII superfamily, which specifically binds cAMP in addition to ATP, ADP and AMP. However, for CutA, no binding of effector molecules could be identified. I resolved the crystal structures of CutA (in apo form) and SbtB (in apo status and in complex with AMP and cAMP) proteins. The nucleotide-binding pocket of SbtB is located between the lateral cleft of two subunits, as in canonical PII proteins. Apparently, the trimeric core architectural principle of PII-like proteins of CutA and SbtB are widely distributed similar to the canonical PII core architecture. This clearly indicates that the proteins of the PII superfamily arose evolutionary from a common trimeric ancestor protein. Die Signaltransduktionsproteine der PII-Superfamilie stellen eine der ältesten hochkonservierten Proteinfamilien dar und finden sich in allen Domänen des Lebens. Kanonische PII-Signalproteine üben ihre Funktion dadurch aus, dass sie als Effektormoleküle ATP, ADP und 2-Oxoglutarat (2-OG) binden. Alle bislang untersuchten Cyanobakterien enthalten PII-Homologe des GlnB-Typs. Es reguliert den zellulären Stickstoff-Stoffwechsel, indem es an die N-Acetyl-L-Glutamat-Kinase (NAGK, katalysiert den geschwindigkeitsbestimmenden Schritt der Arginin-Biosynthese) sowie an das PII-Interacting Protein X (PipX, Co-Aktivator des globalen Stickstoff-Transkriptionsfaktors NtcA) bindet. Die Interaktion von PII mit NAGK führt zur Erhöhung der NAGK-Aktivität und unterbindet die hemmende Wirkung von Arginin auf NAGK, was letztlich zu einer verstärkten Produktion von Arginin führt. Die Bildung eines PII-PipX-Komplexes wiederum führt zur Deaktivierung des NtcA-Regulons. In dieser Arbeit konnte gezeigt werden, dass der Lys58-Rest eine Schlüsselrolle bei der Signaltransduktion des cyanobakteriellen PII spielt. Das PII-Protein aus der Rotalge Porphyra purpurea wurde erstmals biochemisch charakterisiert. Es stellt einen Übergangszustand zwischen den PII-Proteinen aus Cyanobakterien und Chlorophyta dar. Durch den Vergleich der sensorischen Eigenschaften des PII-Proteins aus Rotalgen mit PII-Proteinen aus verschiedenen Phyla oxygener phototropher Organismen (Cyanobakterien, Grünalgen und Moose) zeigte sich, dass die PII-Signaltransduktion in Rotalgen weitgehend der in Cyanobakterien gleicht, wohingegen sich die Eigenschaften der PII-Proteine im Laufe der Entwicklung der Chlorophyta verändert haben. So verhalten sie sich sehr heterogen bezüglich der Bindung von ADP und 2-OG, nur die Bindung von ATP in Gegenwart von 2-OG ist konserviert. Eine weitere Studie befasste sich mit der PII-Signaltransduktion in der nicht photosynthetisch aktiven Alge Polytomella parva im Vergleich zur nahe verwandten, Photosynthese betreibenden Alge Chlamydomonas reinhardtii. Metabolom-Daten zufolge akkumuliert P. parva TCA-Intermediate sowie Arginin, Glutamat und Aspartat. Ferner zeigte diese Studie dass sich im Lauf der P. parva Evolution PII und NAGK zu einem stabilen, heterooligomeren Komplex entwickelt haben hat, der nicht mehr durch Effektormoleküle dissoziiert wird. Dementsprechend kann PII in diesem Fall als Untereinheit von NAGK angesehen werden. PII-ähnliche Proteine ähneln strukturell den klassischen PII-Proteinen, besitzen aber nicht die kanonischen PII Signaturmotive. Bislang war nichts über ihre Funktionen, Interaktionspartner und regulatorischen Reaktionen bekannt. Eine in-silico Analyse der Genome von Cyanobakterien zeigte das Vorhandensein verschiedener PII-ähnlicher Proteine (PII-like proteins). Im Rahmen dieser Arbeit lag der Fokus auf zwei PII-ähnlichen Proteinen in Cyanobakterien: 1) SbtB, dessen Gen in einem Operon in direkter Nachbarschaft zum Natriumabhängigen Bicarbonat-Transporter (SbtA) liegt und 2) CutA (divalent ion tolerance protein). Es konnte sowohl biochemisch als auch physiologisch gezeigt werden, dass SbtB eine wichtige Komponente des Kohlenstoffanreichungsmechanismus (carbon concentration mechanism, CCM) ist, indem es Änderungen der intrazellulären Konzentration von anorganischem Kohlenstoff (Ci) durch Bindung des sekundären Messengers cAMP verarbeitet. Darüber hinaus konnten wir zeigen, dass CutA in Cyanobakterien nicht direkt an der Erkennung von Schwermetallionen beteiligt ist. Weiterhin konnten wir für SbtB als erstes Mitglied der PII-Superfamilie zeigen, dass neben den bekannten Effektoren ATP, ADP und AMP auch cAMP spezifisch gebunden wird. Jedoch blieben die sensorischen Eigenschaften von CutA unklar. Die Kristallstrukturen von CutA (Apo-Form) und SbtB (Apo-Form und im Komplex mit AMP und cAMP) konnte aufgeklärt werden. Dadurch konnte die Nukleotid-Bindetasche von SbtB in der lateralen Falte zweier Untereinheiten lokalisiert werden. Offenbar ist die trimere Grundstruktur der PII-ähnlichen Proteine SbtB und CutA ähnlich wie die der kanonischen PII-Proteine weit verbreitet. Dies zeigt eindeutig, dass die Proteine der PII-Superfamilie evolutionär aus einem gemeinsamen Vorfahren hervorgegangen sind.

Red coralline algae are projected to be sensitive to ocean acidification, particularly in polar oceans. As important ecosystem engineers, their potential sensitivity has broad implications, and understanding their carbon acquisition mechanisms is necessary for making reliable predictions. Therefore, we investigated the localized carbonate chemistry at the surface of Arctic coralline algae using microsensors. We report for the first time carbonate ion concentration and pH measurements ([CO3 2-]) at and above the algal surface in the microenvironment. We show that surface pH and [CO3 2-] are higher than the bulk seawater in the light, and even after hours of darkness. We further show that three species of Arctic coralline algae have efficient carbon concentrating mechanisms including direct bicarbonate uptake and indirect bicarbonate use via a carbonic anhydrase enzyme. Our results suggest that Arctic corallines have strong biological control over their surface chemistry, where active calcification occurs, and that net dissolution in the dark does not occur. We suggest that the elevated pH and [CO3 2-] in the dark could be explained by a high rate of light independent carbon fixation that reduces respiratory CO2 release. This mechanism could provide a potential adaptation to ocean acidification in Arctic coralline algae, which has important implications for future Arctic marine ecosystems.

Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid., BBSRC/NSF Combining Algal and Plant Photosynthesis (CAPP), Cambridge-IDB International Scholarship, Cambridge Trust PhD Overrun Fund, Cambridge Philosophical Society Research Studentship, Lundgren Research Award, Houston Putnam Lowry Fund - Fitzwilliam College

We explored the metabolic pathways in two industrially relevant marine microorganisms to understand their core metabolic capabilities. It is necessary to track how an organism distributes organic building blocks throughout its metabolic pathways so that we can devise strategies to alter its metabolism and reroute substantial metabolic flux towards target compound(s). Though we cannot measure intracellular metabolic fluxes directly, we can retro-biosynthetically calculate them by supplying substrates labeled with non-radioactive isotopes to an organism. We then measure the resulting isotope labeling patterns of metabolites and calculate the fluxes that produced them. We addressed three goals with our research, (i) resolving questions surrounding organic carbon metabolism in the diatom Phaeodactylum tricornutum (P. tricornutum), (ii) identifying reactions in a putative photosynthetic carbon concentrating mechanism in P. tricornutum and (iii) mapping central carbon metabolism of the cellulolytic aerobe Saccharophagus degradans (S. degradans). Towards goal (i) we show that P. tricornutum predominantly consumes glucose, as opposed to atmospheric CO2, under mixotrophic conditions using the Entner-Doudoroff (ED) glycolytic pathway instead of the more common Embden-Meyerhof-Parnas pathway (EMP). We utilized metabolic flux analysis (MFA) to discover that acetate is metabolized for energy production instead of for biomass formation during mixotrophic growth on CO2 and acetate. Finally, we developed a method for measuring isotopic labeling in polyunsaturated fatty acids via gas chromatography-mass spectrometry (GC-MS), and demonstrated its utility in resolving outstanding questions about glucose metabolism by P. tricornutum. Towards goal (ii) we utilized isotope labeling and gene silencing in combination to identify pyruvate carboxylase as a key enzyme in a C4 carbon concentrating mechanism in P. tricornutum, while also ruling out phosphoenolpyruvate carboxylase as a key enzyme in the pathway. Towards goal (iii) we present 13C-MFA of aerobic consumption of glucose, xylose, and cellobiose by S. degradans. This is the first reported MFA of cellobiose metabolism and one of only a handful analyzing xylose metabolism in an aerobic microorganism.

Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO2 retro diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements which are thought be essential for recruitment of Rubisco to the pyrenoid, viz the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing “pyrenoid-competent” chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing the Chlamydomonas SSU differed from wild type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.

Marine diatoms play a critical role in the global carbon cycle, where they are responsible for 20% of all primary production. RubisCO, the rate limiting enzyme in carbon fixation, has a half-saturation constant many times higher than oceanic CO2 concentrations. In order to overcome this limitation, diatom species have evolved a diverse array of highly efficient carbon concentrating mechanisms. Increasing our understanding of these mechanisms provides a foundation to improve genetic engineering of these organisms for biofuel production and increased carbon sequestration, as well as a basis to potentially improve the efficiency of photosynthesis in terrestrial plants.In this study, the carbon concentrating mechanism of the diatom species Phaeodactylum tricornutum is investigated through kinetic modeling of potential pathways and analysis of differential gene expression of CCM-related genes. An existing kinetic model is rebuilt and expanded to explore proposed carbon-concentrating mechanisms and provide predictive values of carbon fluxes through the system at varying external conditions. The feasibility of each of three potential mechanisms is evaluated. Through gene expression analysis, potential major regulators of the carbon concentrating mechanism are identified.This investigation finds that the presence of bicarbonate transporters on the plasmalemma, CER, and chloroplast membranes is needed for the CCM to achieve expected carbon uptake and photosynthetic fluxes, but a bicarbonate transporter on the PPC membrane is not necessary. Additionally, the down-regulation of a chloroplast-membrane targeted bicarbonate transporter at sub-atmospheric CO2 indicates that a secondary CCM pathway takes over under low carbon stress.

The activities of extra and intracellular carbonic anhydrases (CA) were studied in the microalgae Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) growing in laboratory cultivation. During ten days of batch cultivation, daily determinations of pH, cell number, enzymatic activity, and total dissolved inorganic carbon (DIC), as well as its main species, CO2 and HCO3-, were performed. Enzymatic activity increased as the growing cell population depleted inorganic carbon from the medium. Carbon dioxide concentration decreased quickly, especially in the third day of cultivation, when a significant increase of the intracellular enzymatic activity was observed. Bicarbonate concentration had its largest decrease in the cultivation medium in the fourth day, when the activity of the extracellular enzyme had its largest increase, suggesting its use by the alga through CA activity. After the fourth cultivation day, half of the cultures were aerated with CO2-free atmospheric air, which caused an increase in the total and external activity of the enzyme, although, in this condition, the stationary growth phase began earlier than in cultures aerated with atmospheric air. The pH of the media was measured daily, increasing from the first to the fourth day, and remaining almost constant until the end of the cultivation. Algal material transferred to the dark lost all enzymatic activity.As atividades da Anidrase Carbônica (AC) extra e intracelular foram estudadas na microalga marinha Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) crescendo em cultivos laboratoriais. Durante dez dias de cultivo, determinações diárias do pH, número de células, atividades enzimáticas, carbono inorgânico total dissolvido (CID) e suas principais espécies CO2 e HCO3- foram feitas. A atividade enzimática aumentou na medida em que a população celular em crescimento retirava carbono inorgânico do meio de cultivo. A concentração de dióxido de carbono decresceu rapidamente, especialmente no terceiro dia do cultivo, quando um significante aumento na atividade enzimática intracelular foi observado. A concentração de bicarbonato teve seu maior decréscimo no meio de cultivo no quarto dia, quando a atividade da enzima extracelular teve seu maior aumento, sugerindo seu uso pela alga através da atividade da AC. Após o quarto dia de cultivo, metade das culturas passou a ser aerada com ar atmosférico sem CO2, o que causou um aumento na atividade total e externa da enzima, fazendo com que esses cultivos entrassem na fase estacionária do crescimento antes que aqueles aerados com ar atmosférico normal. O pH do meio foi medido diariamente, aumentando desde o primeiro até o quarto dia e permanecendo quase constante até o fim do cultivo. Material algal transferido para o escuro perdeu toda a atividade enzimática.

Symbiotic dinoflagellate algae residing inside coral tissues supply the host with the majority of their energy requirements through the translocation of photosynthetically fixed carbon. The algae, in turn, rely on the host for the supply of inorganic carbon. Carbon must be concentrated as CO2 in order for photosynthesis to proceed, and here we show that the coral host plays an active role in this process. The host-derived symbiosome membrane surrounding the algae abundantly expresses vacuolar H(+)-ATPase (VHA), which acidifies the symbiosome space down to pH ∼ 4. Inhibition of VHA results in a significant decrease in average H(+) activity in the symbiosome of up to 75% and a significant reduction in O2 production rate, a measure of photosynthetic activity. These results suggest that host VHA is part of a previously unidentified carbon concentrating mechanism for algal photosynthesis and provide mechanistic evidence that coral host cells can actively modulate the physiology of their symbionts.

Photosynthesis is crucial for life but is a slow process because the CO2 concentration near the principal carbon-assimilation enzyme RuBisCO is extremely low. Very few plants and algae perform a carbon-concentrating mechanism (CCM) to overcome the insufficiency, which are classified into biophysical and biochemical (C4) mechanism. The enzyme CA catalyzes the reversible dehydration of HCO3- to CO2 in biophysical CCMs and its active site contains a Zn2+. In this study, we hypothesized that Zn2+ availability can impact CCMs and therefore investigated the effect of Zn2+ availability on photosynthetic metabolism in a unicellular marine diatom Phaeodactylum tricornutum. P. tricornutum has a sequenced genome and can conduct both biophysical and C4 CCMs. We observed that Zn2+ has a significant effect on cell growth rate but no significant interference on intracellular metabolism, suggesting no essential compensation of C4 CCMs for biophysical CCMs even at low CA activity anticipated at low Zn2+ concentration.

We investigated the catalytic activity and inhibition of the beta-class carbonic anhydrase (CA, EC 4.2.1.1) CahB1, from the relict cyanobacterium Coleofasciculus chthonoplastes (previously denominated Microcoleus chthonoplastes). The enzyme showed good activity as a catalyst for the CO2 hydration, with a k(cat) of 2.4 x 10(5) s (1) and a k(cat)/K-m of 6.3 x 10(7) M (1) s (1). A range of inorganic anions and small molecules were investigated as inhibitors of CahB1. Perchlorate and tetrafluoroborate did not inhibit the enzyme (K(I)s >200 mM) whereas selenate and selenocyanide were ineffective inhibitors too, with K(I)s of 29.9-48.61 mM. The halides, pseudohalides, carbonate, bicarbonate, trithiocarbonate and a range of heavy metal ions-containing anions were submillimolar-millimolar inhibitors (K(I)s in the range of 0.15-0.90 mM). The best CahB1 inhibitors were N,N-diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K(I)s in the range of 8-75 mu M, whereas acetazolamide inhibited the enzyme with a K-I of 76 nM. This is the first kinetic and inhibition study of a cyanobacterial CA. As these enzymes are widespread in many cyanobacteria, being crucial for the carbon concentrating mechanism which assures substrate to RubisCO for the CO2 fixation by these organisms, a detailed kinetic/inhibition study may be essential for a better understanding of this superfamily of metalloenzymes and for potential biotechnological applications in biomimetic CO2 capture processes. (C) 2014 Elsevier Ltd. All rights reserved.

International audience; Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites.

Many photosynthetic organisms have developed inorganic carbon (Ci) concentrating mechanisms (CCMs) that increase the CO2 concentration within the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Several CCMs, such as four carbon (C4) and crassulacean acid metabolism (CAM), bicarbonate accumulation systems and capsular structures around RubisCO have been described in great detail. These systems are believed to have evolved several times as mechanisms that acclimate organisms to unfavourable growth conditions. Based on recent experimental evidence we propose the occurrence of another more general CCM system present in all plants. This basal CCM (bCCM) is supposed to be composed of mitochondrial carbonic anhydrases (a β-type carbonic anhydrase and the γ-type carbonic anhydrase domain of the mitochondrial NADH dehydrogenase complex) and probably further unknown components. The bCCM is proposed to reduce leakage of CO2 from plant cells and allow efficient recycling of mitochondrial CO2 for carbon fixation in chloroplasts. Fil: Zabaleta, Eduardo Julian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Martin, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Braun, Hans Peter. Leibniz Universität Hannover; Alemania

The fluxes of CO 2 and oxygen during photosynthesis by cell suspensions of Tessellaria volvocina and Mallomonas papillosa were monitored mass spectrometrically. There was no rapid uptake of CO 2, only a slow drawdown to compensation concentrations of 26 lM for T. volvocina and 18 lM for M. papillosa, when O 2 evolution ceased, indicating a lack of active bicarbonate uptake by the cells. Darkening of the cells after a period of photosynthesis did not cause rapid release of CO 2, indicating the absence of an intracellular inorganic carbon pool. However, upon darkening a brief burst of CO 2 was observed similar to the post-illumination burst characteristic of C3 higher plants. Treatment of the cells of both species with the membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide had no adverse effect on photosynthetic rate, but stimulated the dark CO 2 burst indicating the dark oxidation of a compound formed in the light. In the absence of any active accumulation of inorganic carbon photosynthesis in these species should be inhibited by O 2. This was investigated in four synurophyte species T. volvocina, M. papillosa, Synura petersenii, and Synura uvella: Photosynthetic O 2 evolution rates in all four algae, measured by O 2 electrode, were significantly higher (40-50%) in media at low O 2 (4%) than in airequilibrated (21% O 2) media, indicating an O 2 inhibition of photosynthesis (Warburg effect) and thus the occurrence of photorespiration in these species. © Springer Science+Business Media B.V. 2011.

Thesis is under embargo due to publisher requirements., Aquatic photosynthesis accounts for 50% of the global annual net primary production (NPP), despite frequent low availability and limited diffusion of CO2 in the aquatic milieu, and low affinity for CO2 by the primary carboxylating enzyme, Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Many eukaryotic algae, and a single group of land plants, the hornworts, have an inducible carbon concentrating mechanism (CCM), to overcome these limitations. The efficiency of the CCM is improved when RuBisCO is localised to a subcellular compartment, the pyrenoid, which is hypothesised to act as a diffusion barrier for CO2 . Although the pyrenoid is a major player in global carbon balance (we estimate 10-15% of NPP), it is one of the few remaining prominent cellular features without a precise molecular or physiological definition. Under ambient CO2 , at least 90% of the cellular RuBisCO is packed into a dense matrix, together with the chaperone RuBisCO activase. Thylakoid membranes usually traverse the pyrenoid matrix, and the carboxylating substrate is thought to be delivered to the active sites of the enzyme via a carbonic anhydrase located in the lumen of these thylakoids. The mechanism of aggregation of constituents within the pyrenoid, however, still remains largely unknown. Comprehensive mutant screens have yet to reveal mutants incapable of forming pyrenoids other than those mutants with a defective RuBisCO holoenzyme, whereas DNA microarray studies uncovered little with reference to pyrenoid ultrastructure or aggregation. Taken together, this evidence raises the possibility that the basis of pyrenoid ultrastructure and aggregation lies entirely in sequence variations of RuBisCO itself. This work explored, firstly, the advantages conferred by an active CCM in hornworts and in unicellular algae, compared with the passive CO2 acquisition in most terrestrial plants. A physiological framework to CCM and pyrenoid-based photosynthesis, and isotopic discrimination, was provided by comparing the photosynthetic characteristics of selected bryophytes and algae, differing in chloroplast morphology and degrees of internalisation of gas exchanges. The results showed that on-line, carbon isotope discrimination values were a good indicator of CCM occurrence, as well as liquid-phase diffusion limitation, and biochemical limitations resulting from declining RuBisCO activity and electron transport. The methodology was used to diagnose the presence of an active CCM, and the extent of CO2 leakage. Secondly, the effect of RuBisCO sequence variations on the pyrenoid, and associated CCM, was studied using the model alga Chlamydomonas reinhardtii. The starting premise was the report by Nozaki et al. (2002) that, in some species of the family Chlamydomonaceae, a few amino acid residues within the RuBisCO large subunit (LSU) correlated strongly with pyrenoid formation. The specific roles of seven LSU residues were studied by site-directed mutagenesis. Whilst the mutations reduced the affinity of RuBisCO for CO2 and increased CO2 leakage, compared to wild-type Chlamydomonas, there was no effect on the pyrenoid phenotype. Informed by observations that Chlamydomonas mutants with a hybrid RuBisCO, composed of a native LSU, and higher plant small subunit (SSU), lacked a pyrenoid (Genkov et al., 2010), and that defined SSU alterations were neutral with respect to the pyrenoid (Genkov and Spreitzer, 2006), hitherto unexplored SSU domains were modified. A pyrenoid was successfully restored by replacing jointly the two solvent-exposed α-helices, whereas single α-helix replacements had no effect. However, leakage values indicated that the associated CCM was not fully operative, suggesting important correlates between the RuBisCO SSU and the CCM, besides the conditioning of pyrenoid formation. If the pyrenoid is partly defined by simple sequence variations in the RuBisCO SSU, as suggested by the evidence outlined in this thesis, there is the tantalising possibility that transformation of a biophysical CCM into crop plants could be a tractable approach for the future., This work was supported by the Fonds National de la Recherche, Luxembourg [grant AFR 06/030], and sponsored by Dr. Christian Ries, curator at the National Museum of Natural History, Luxembourg, and president of the Luxembourg Naturalist Society

The activities of extra and intracellular carbonic anhydrases (CA) were studied in the microalgae Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) growing in laboratory cultivation. During ten days of batch cultivation, daily determinations of pH, cell number, enzymatic activity, and total dissolved inorganic carbon (DIC), as well as its main species, CO2 and HCO3-, were performed. Enzymatic activity increased as the growing cell population depleted inorganic carbon from the medium. Carbon dioxide concentration decreased quickly, especially in the third day of cultivation, when a significant increase of the intracellular enzymatic activity was observed. Bicarbonate concentration had its largest decrease in the cultivation medium in the fourth day, when the activity of the extracellular enzyme had its largest increase, suggesting its use by the alga through CA activity. After the fourth cultivation day, half of the cultures were aerated with CO2-free atmospheric air, which caused an increase in the total and external activity of the enzyme, although, in this condition, the stationary growth phase began earlier than in cultures aerated with atmospheric air. The pH of the media was measured daily, increasing from the first to the fourth day, and remaining almost constant until the end of the cultivation. Algal material transferred to the dark lost all enzymatic activity. As atividades da Anidrase Carbônica (AC) extra e intracelular foram estudadas na microalga marinha Tetraselmis gracilis (Kylin) Butcher (Chlorophyta, Prasinophyceae) crescendo em cultivos laboratoriais. Durante dez dias de cultivo, determinações diárias do pH, número de células, atividades enzimáticas, carbono inorgânico total dissolvido (CID) e suas principais espécies CO2 e HCO3- foram feitas. A atividade enzimática aumentou na medida em que a população celular em crescimento retirava carbono inorgânico do meio de cultivo. A concentração de dióxido de carbono decresceu rapidamente, especialmente no terceiro dia do cultivo, quando um significante aumento na atividade enzimática intracelular foi observado. A concentração de bicarbonato teve seu maior decréscimo no meio de cultivo no quarto dia, quando a atividade da enzima extracelular teve seu maior aumento, sugerindo seu uso pela alga através da atividade da AC. Após o quarto dia de cultivo, metade das culturas passou a ser aerada com ar atmosférico sem CO2, o que causou um aumento na atividade total e externa da enzima, fazendo com que esses cultivos entrassem na fase estacionária do crescimento antes que aqueles aerados com ar atmosférico normal. O pH do meio foi medido diariamente, aumentando desde o primeiro até o quarto dia e permanecendo quase constante até o fim do cultivo. Material algal transferido para o escuro perdeu toda a atividade enzimática.

Inorganic carbon (Ci) is the major sink for photosynthetic reductant in organisms capable of oxygenic photosynthesis. In the absence of abundant Ci, the cyanobacterium Synechocystis sp. strain PCC6803 expresses a high affinity Ci acquisition system, the CO2-concentrating mechanisms (CCM), controlled by the transcriptional regulator CcmR and the metabolites NADP+ and α-ketoglutarate, which act as co-repressors of CcmR by modulating its DNA binding. The CCM thus responds to internal cellular redox changes during the transition from Ci-replete to Ci-limited conditions. However, the actual changes in the metabolic state of the NADPH/NADP+ system that occur during the transition to Ci-limited conditions remain ill-defined. Analysis of changes in the redox state of cells experiencing Ci limitation reveals systematic changes associated with physiological adjustments and a trend towards the quinone and NADP pools becoming highly reduced. A rapid and persistent increase in F0 was observed in cells reaching the Ci-limited state, as was the induction of photoprotective fluorescence quenching. Systematic changes in the fluorescence induction transients were also observed. As with Chl fluorescence, a transient reduction of the NADPH pool (‘M’ peak), is assigned to State 2→State 1 transition associated with increased electron flow to NADP+. This was followed by a characteristic decline, which was abolished by Ci limitation or inhibition of the Calvin–Benson–Bassham (CBB) cycle and is thus assigned to the activation of the CBB cycle. The results are consistent with the proposed regulation of the CCM and provide new information on the nature of the Chl and NADPH fluorescence induction curves.

Bicarbonate species in the aqueous phase is the primary source for CO2 for the growth of microalgae. The potential of carbon dioxide (CO2) fixation by Chlorella pyrenoidosa in enriched bicarbonate medium was evaluated. In the present study, effects of parameters such as pH, sodium bicarbonate concentration and inoculum size were assessed for the removal of CO2 by C. pyrenoidosa under mixotrophic condition. Central composite design tool from response surface methodology was used to validate statistical methods in order to study the influence of these parameters. The obtained results reveal that the maximum removal of CO2 was attained at pH 8 with sodium bicarbonate concentration of 3.33 g/l, and inoculum size of 30 %. The experimental results were statistically significant with R 2 value of 0.9527 and 0.960 for CO2 removal and accumulation of chlorophyll content, respectively. Among the various interactions, interactive effects between the parameters pH and inoculum size was statistically significant (P