Carbon fixation in marine phytoplankton: carboxylase activities and stable carbon-isotope ratios; physiological and paleoclimatological aspects

We measured the activity of three carboxylases: RuBP carboxylase, PEP carboxylase and PEP carboxykinase of marine phytoplankton species in culture and in natural communities. Activities of the three carboxylases were measured simultaneously with stable carbon-isotope ratios. The enzymatic activities have been used to estimate the importance of carboxylation and its impact on the ¹³C:¹²C ratio (expressed as ¹³C). The marine phytoplankton species in culture were Fragilariopsis kerguelensis, Nitzschia turgiduloides, Skeletonema costatum, Phaeodactylum tricornutum, Isochrysis galbana, Dunaliella marina, and Prorocentrum micans, and the field samples were collected from different depths off the coast of Portugal (August/September, 1981). Our results indicate that, as in terrestrial plants, the ¹³C value is a good indicator of the extent of carboxylation. RuBP carboxylase activity was always predominant, whereas the ¹³C value never reached values typical of the C₄ pathway. The carboxylases could be PEP carboxylase (in dinoflagellates) or PEP carboxykinase (in diatoms). carboxylation increased at the end of the exponential growth phase in a diatom culture and with increased biomass in natural samples. We interpret these increases as an adaptative response mechanism to poor environmental conditions, especially to low light intensity.     

Growth,photosynthesis and carbon metabolism in the temperate marine diatom Skeletonema costatum adapted to low temperature and low photon-flux density

The temperate diatom Skeletonema costatum (Grev.) Cleve was grown in low temperature and/or low light conditions. The cultures were acclimatized for at least three months before experiments were begun. Our data indicate that the initial slope of the photosynthesis vs irradiance curve () is controlled predominantly by light history and the light-saturated photosynthesis (P _max) by temperature. The number of divisions per day decreased with decreasing light intensity, but was identical for cultures grown at 3° or 18°C. The metabolic pathways of inorganic carbon fixation were not fundamentally affected by low temperature or low light intensity, but both these factors increased labelling of C₃ compounds, synthesized by the Calvin-Benson cycle, and decreased that of phosphoenolpyruvate (PEP) and other metabolites. This indicates an enhancement of ribulose-1,5-bisphosphate (RuBP) carboxylase activity, which is the first step in the C₃ pathway (3-phosphoglycerate and sugar phosphate synthesis); this may optimize cell functions. At low temperatures, a seven-fold increase in RuBP carboxylase activity per cell was observed. S. costatum is able to adapt to low irradiance by increasing and decreasing I _k (the ratio of P _max:, light intensity at onset of light saturation), and to low temperature by increasing its cellular chlorophyll a and RuBP carboxylase content. However, in the latter case, adaptation is not optimal. This study revealed two main features: (1) there is evidence that RuBP carboxylase has a key function in adjustment to high rates of photosynthesis at suboptimal temperatures or irradiances; (2) adaptive mechanisms are dynamic processes and the role of the time scale in physiological adaptation should be considered.     

Photosynthetic carbon acquisition in the red alga Gracilaria conferta

In this continuing study on photosynthesis of the marine red alga Gracilaria conferta, it was found that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in crude extracts had a K _m (CO₂) of 85 M. Since seawater contains only ca. 10 M CO₂, it appears that this alga must possess a CO₂ concetrating system in order to supply sufficient CO₂ to the vicinity of the enzyme. Because this species is a C₃ plant (and thus lacks the C₄ system for concentrating CO₂), but can utilize HCO₃ ^- as an exogenous carbon source, we examined whether HCO₃ ^- uptake could be the initial step of such a CO₂ concetrating system. The surface pH of G. conferta thalli was 9.4 during photosynthesis. At this pH, estimated maximal uncatalyzed HCO₃ ^- dehydration (CO₂ formation) within the unstirred layer was too slow to account for measured phostosynthetic rates, even in the presence of an external carbonic anhydrase inhibitor. This observation, and the marked pH increase in the unstirred layer following the onset of light, suggests that a HCO₃ ^- transport system (probably coupled to transmembrane H⁺/OH^- fluxes) operates at the plasmalemma level. The involvement of surface-bound carbonic anhydrase in such a system remains, however, obscure. The apparent need of marine macroalgae such as G. conferta for CO₂ concentrating mechanisms is discussed with regard to their low affinity of Rubisco to CO₂ and the low rate of CO₂ supply in water. The close similarity between rates of Rubisco carboxylation and measured photosynthesis further suggests that the carboxylase activity, rather than inorganic carbon transport and intercoversion events, could be an internal limiting factor for photosynthetic rates of G. conferta.     

Physiological and immunological evidence for two distinct C1-utilizing pathways in Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), a dual endosymbiotic mussel from the Mid-Atlantic Ridge

The existence of endosymbiotic sulfur-oxidizing chemoautotrophic and methanotrophic bacteria associating with marine mytilid mussels has previously been inferred by 16S rDNA analysis in Bathymodiolus puteoserpentis Von Cosel et al. 1994, a hydrothermal vent mussel from a site on the Mid-Atlantic Ridge. In mussels collected in June 1993, we found evidence of enzymes diagnostic of two distinct C₁ assimilation pathways in this symbiosis. Assays for the utilization of radiolabelled methane and for immunodetection of methanol dehydrogenase were positive, indicating that oxidation and incorporation of this substrate are occurring in this symbiosis. Sulfide or thiosulfate had no detectable stimulatory effect on CO₂ incorporation, and assays for the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), an enzyme diagnostic for the Calvin–Benson cycle, were negative. RubisCO was detected in all samples examined by immunoblot analysis, indicating this enzyme is expressed in the B. puteoserpentis symbiosis. Stable isotope data showed that carbon isotope values were in agreement with previously reported values, and nitrogen isotope values were among the most depleted ever reported for bivalve symbioses. The carbon isotope values do not preclude the utilization of vent-derived methane. These data could be explained by the presence of two metabolically distinct bacterial symbionts or a Type X methanotrophic symbiont. Received: 3 October 1997 / Accepted: 23 July 1998     

Antioxidant defense properties of Arctic amphipods: comparison between deep-, sublittoral and surface-water species

The toxic reactive oxygen species (ROS), produced naturally in an oxygenated environment, led to the development of antioxidant defenses by aerobic organisms to prevent tissue damage. Polar marine waters are regarded as a strongly prooxidant ecosystem, due to the high dissolved oxygen level and intense UV radiation during summer, while the deep waters are considered refuges against oxidative stress. In order to further elucidate the prooxidant and antioxidant forces associated with cold-water ecosystems and the implications of UV radiation, we sampled three amphipod species living at three different depths in the same water mass of the Arctic Ocean, characterized by low temperature (ca. 0°C), high oxygen level, and high ROS concentration in the surface water. The three species were the deep-sea amphipod Eurythenes gryllus, the benthic sublittoral amphipod Anonyx nugax and the surface-water species Gammarus wilkitzkii inhabiting the ice pack. The total oxyradical scavenging capacity (TOSC) was measured in the cytosolic fraction of the digestive gland and in the cell-free hemolymph of the three amphipod species. A significantly low TOSC toward peroxyl and peroxynitrite radicals (P<0.05) in E. gryllus compared with the shallow-water species (A. nugax) can be explained by the low metabolism of the deep-sea species. In the cell-free hemolymph, TOSC values are similar between E. gryllus and A. nugax, while in G. wilkitzkii a lower and higher TOSC toward hydroxyl and peroxynitrite, respectively, indicate specific adaptation to oxidative stress. Experimental exposure of A. nugax and G. wilkitzkii to H₂O₂ resulted in a significant change in TOSC (P<0.05) measured in the digestive gland and in the cell-free hemolymph of A. nugax, while no change (P>0.05) was noted in G. wilkitzkii. These data suggest that A. nugax is highly susceptible to oxidative stress and that G. wilkitzkii is characterized by a mechanism that prevents the diffusion of exogenous ROS through the gills or allows excretion of internal H₂O₂ through the gills to the environment.Communicated by M. Kühl, Helsingør     

Localization of RubisCO and sulfur in endosymbiotic bacteria of the gutless marine oligochaete Inanidrilus leukodermatus (Annelida)

 The chemoautotrophic potential of the two co-occurring larger and smaller bacterial endosymbionts of the gutless marine oligochaete Inanidrilus leukodermatus was determined using immunocytochemistry. An antibody directed against the Form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key CO₂-fixing enzyme of the Calvin–Benson cycle, consistently labeled the larger symbionts. Electron microscopic spectroscopy showed that the larger symbionts contained sulfur in intracellular globules and to a lesser degree in the cytoplasm. The presence of RubisCO and sulfur indicates that the larger endosymbionts of I. leukodermatus are chemoautotrophic sulfur-oxidizing bacteria. In contrast, no RubisCO or sulfur was detected in the smaller endosymbionts of this host. Received: 28 September 1999 / Accepted: 29 May 2000     

The pattern of carbon fixation in the marine unicellular alga Phaeodactylum tricornutum

The short- and long-term fixation of ¹⁴CO₂ by Phaeodactylum tricornutum was studied using methods of fractionation that allowed examination of all the products labelled with ¹⁴C. There was no doubt that the major pathway of CO₂ fixation was into 3-phosphoglycerate, but there was also significant incorporation by -carboxylation by means of phosphoenol-pyruvate carboxylase and transamination into aspartate. At short time intervals (10 sec), 90% of the radioactive products found were accounted for by 3-phosphoglycerate and aspartate. The lipids associated with the photosynthetic apparatus contained a high proportion of the ¹⁴C fixed, which at 60 sec was located mainly in the carbohydrate portion of the lipids. At 30 and 300 min, the chlorophylls, carotenoids and the long-chain fatty acids were heavily labelled with ¹⁴C. The monogalacto-diglycerides, the digalacto-diglycerides and the sulpholipids each had a characteristic long-chain fatty acid composition. The cell proteins and a reserve polysaccharide were also labelled with ¹⁴C at short time intervals and increased their radioactivity in a linear fashion up to the longest period studied, 300 min. The activity of the enzymes ribulose diphosphate carboxylase and phosphoenolpyruvate carboxylase was sufficient to account for the pattern of fixation found.     

Cloning and expression of high choriolytic enzyme,a component of the hatching enzyme system,during embryonic development of the marine ornamental fish<Emphasis Type="Italic"> Chrysiptera parasema</Emphasis>

In the present study, a cDNA for the hatching enzyme of a marine tropical fish, Chysiptera parasema, was cloned. This is the first demonstration of hatching enzyme cDNA from a marine tropical fish. The amino acid (aa) sequence deduced from the cDNA consisted of an 18-aa signal sequence, a 53-aa propeptide sequence and a 196-aa mature enzyme portion, having a consensus active site sequence for astacin family proteases. Phylogenetic analysis showed that the C. parasema enzyme was included in the clade of HCEs (high choriolytic enzymes), one of the hatching enzymes of freshwater fishes such as medaka (Oryzias latipes), masu salmon (Oncorhynchus masou) and zebrafish (Danio rerio), but not in the group of LCEs (low choriolytic enzymes), another type of hatching enzymes identified in the medaka. The developmental expression patterns of the C. parasema HCE gene were highly similar to that of the medaka HCE gene. The results suggested that the hatching enzyme system is highly conserved between these marine and freshwater fish species.Communicated by R. Cattaneo-Vietti, Genova     

Acute and chronic toxic effect of lead (Pb) and zinc (Zn) on biomarker response in post larvae of Penaeus monodon (Fabricus, 1798)

Metal pollution produces damage to marine organisms at the cellular level possibly leading to ecological imbalance. The present investigation focused on the acute and chronic toxicity of lead (Pb) and zinc (Zn) by examining the effects of biomarker enzymes in post-larvae of Penaeus monodon (Tiger prawn). Antioxidant biomarker responses such as lipid peroxidation (LPO) and catalase (CAT) activity for Pb and Zn were determined following chronic exposure. Acute Lethal Concentration ₅₀ (LC₅₀) values observed in the study at 96?h for Pb and Zn at 5.77?±?0.32?mg?L^?1 and 3.02?±?0.82?mg?L^?1, respectively. The estimated No Observed Effect Concentration and Lowest Observed Effect Concentration values for Pb were 0.014 and 0.029?mg?L^?1 and that recorded for Zn was 0.011 and 0.022?mg?L^?1, respectively. Among the two metals studied, toxicity of Zn was found to be greater to P. monodon than Pb. The activities of antioxidant defense enzymes and total protein content differed significantly from control following exposure to both metals. Overall, the biomarker studies demonstrated that alterations in antioxidant enzymes and induction of LPO reflect the consequences of heavy metal exposure in P. monodon.     

The concept of light intensity adaptation in marine phytoplankton: Some experiments with Phaeodactylum tricornutum

The historical background on adaptation of algae to various light intensities is analysed. It is argued that there is little evidence to suggest that previous growth at low light intensities enhances the ability of an alga to utilize these low light levels. Rather, the published evidence suggests that the most general response to growth at sub-optimal light intensities is a reduced ability to utilize saturating levels. The present experiments with Phaeodactylum tricornutum Bohlin have tested this concept of light intensity adaptation. Changing photosynthetic abilities during batch growth depended on the light intensity used for growth and these changes affected interpretations of the data. When measurements were made intensities appeared to photosynthesize (at all intensities) better than did those grown at higher light levels. When the changes during batch growth were taken into account, or when the alga was grown in turbidostat cultures, a different picture was obtained. Growth at reduced light intensities was accompanied by (a) increased chlorophyll content, (b) decreased rate of light-saturated photosynthesis expressed on a chlorophyll, cell number or cell protein basis, and (c) decreased activity of RuDP carboxylase. One result suggested that growth at a suboptimal light intensity did enhance the ability to utilize lower light levels. The light-saturation curve of cells grown in batch culture at 0.7 klux showed higher slopes at the low light intensities than did those grown at 12 klux. This was most marked when photosynthesis was expressed per cell, but was also apparent when it was put on a per chlorophyll basis.     

The gill symbiont of the hydrothermal vent mussel Bathymodiolus thermophilus is a psychrophilic,chemoautotrophic, sulfur bacterium

Certain hydrothermal vent invertebrates, e.g. Riftia pachyptila and Calyptogena magnifica, are clearly established as harboring dense populations of chemoautotrophic sulfur bacteria in specialized tissues. By contrast, the physiological characteristics of the abundant intracellular gill symbiont of the vent mussel Bathymodiolus thermophilus have been questioned. The low activities of enzymes diagnostic for CO₂ fixation (Calvin cycle) and for sulfur-driven energy generation, as measured by other investigators, have been attributed to bacterial contamination of the gill surface. Based on research at the Galápagos Rift hydrothermal vents in 1988 and subsequent laboratory experiments, the current study confirms that the B. thermophilus symbiont is a psychrophile for which thiosulfate and sulfide stimulate CO₂ fixation. It strongly indicates that the symbiont is a chemoautotroph by establishing the following: (1) Sulfide and thiosulfate can stimulate CO₂ fixation by partially purified symbionts by up to 43-fold and 120-fold, respectively; (2) the ribulose-1,5-bisphosphate carboxylase/oxygenase activity of the symbiont is sufficient to account for its sulfide- or thiosulfate-stimulated CO₂ incorporation; (3) the symbiont's molar growth yield on thiosulfate, as judged by CO₂ incorporation, is indistinguishable from that of free-living chemoautotrophs. Due to the high protein-degrading activity of B. thermophilus gill lysate, it is also suggested that host lysis of symbionts plays a more important role in the nutrition of the vent mussel than in R. pachyptila or C. magnifica, for which no comparable protein-degrading activity was found.     

Studies on 14CO2-assimilation in marine rhodophyceae

Experiments on enzymatic (in vitro) and in vivo CO₂-fixation using a variety of marine Rhodophyceae such as Porphyra umbilicalis (L.) J.Ag., Rhodomela confervoides (Huds.) Silva, Corallina officinalis L. and Chondrus crispus Stackh. revealed that carbon assimilation in the Rhodophyceae is almost exclusively performed by photosynthesis via ribulose-1, 5-di-phosphate carboxylase, whereas light-independent CO₂-fixation via -carboxylation by phosphoenolpyruvate carboxykinase scarcely exceeds 1% of the total carbon fixation potential of the plants. Activity of phosphoenolpyruvate carboxylase could not be detected. With respect to the main accumulation products of photosynthetic CO₂-fixation, the Rhodophyceae investigated are not uniform: Corallina officinalis L., Rhodymenia palmata (L.) Grev., and Gigartina stellata (Stackh.) Batt. have been found to accumulate ¹⁴C in the neutral compound floridoside (=2-O-glycerol--D-galactopyranose), whereas Delesseria sanguinea (Huds.) Lamour., Ceramium cubrum (Huds,) C.Ag., and Rhodomela confervoides (Huds.) Silva, representing members of the Delesseriaceae, Ceramiaceae and Rhodomelaceae, respectively, do not photosynthesize floridoside, but show intense ¹⁴C-labelling in an acidic constituent, mannosidoglycerate (= digeneaside). This compound is reported for the first time as a rapidly ¹⁴C-labelled and accumulated photosynthate in a variety of red algal species exclusively belonging to the Ceramiales.     

Activities of sulfur-oxidizing bacteria at the 21°N East Pacific Rise vent site

The incorporation of inorganic and organic carbon into cell material, as well as the activities of carboxylating enzymes (ribulose bisphosphate carboxylase and phosphoenol pyruvate carboxylase), were measured in waters emitted from warm vents at the 21°N ocean spreading site (depth 2 600 m) of the East Pacific Rise. Both obligately and facultatively chemoautotrophic bacteria were present and comprised a significant but variable portion of the total microbial population as indicated by comparisons of microscopic cell counts with liquid enrichments and colony counts on media containing reduced sulfur compounds. The proportion of chemoautotrophic, sulfur-oxidizing bacteria maximally reached 79% of the total microbial population based on ribulose bisphosphate carboxylase activity. Variability of chemoautotrophic activity occurred between vents at different locations, but was also observed in one individual vent. Maximum rates of CO₂ incorporation in warm vent water were similar to levels measured previously in the O₂/H₂S interface of the Black Sea. In shipboard experiments, these rates were virtually unaffected by in-situ pressures (ca 260 atm). Rate measurements at various temperatures, as well as the observed mesophilic characteristics of all isolates obtained, suggest that the microbial, chemoautotrophic activity decreases rapidly as freshly emitted vent water is diluted with cold, ambient, deep-sea water.Contribution No. 6071 of the Woods Hole Oceanographic InstitutionContribution No. 1708 of the Center for Environmental and Estuarine Studies of the University of Maryland     

Effects of O2, pH and DIC on photosynthetic net-O2 evolution by marine macroalgae

Net photosynthetic O₂ evolution by five marine macroalgae:Ulva lactuca L.,Enteromorpha sp.,Ceramium strictum Harvey,Fucus serratus L., andF. vesiculosus L., collected from Danish waters in the summer of 1983 was followed at increasing O₂ and with pH either fixed close to pH 7, 8 or 9, or drifting upwards during photosynthesis in a closed chamber to determine the effects of changing O₂, pH and DIC (dissolved inorganic carbon) on photosynthesis. Increasing O₂, increasing pH and decreasing DIC together limited O₂ evolution. Raising the O₂ concentration with pH and DIC held constant resulted in less inhibition of net-O₂ evolution than when all three factors acted together. The O₂ inhibition of photosynthesis was similar to the reported O₂ inhibition of ribulose 1,5-bisphosphate carboxylase isolated from lower and higher plants. Net-O₂ evolution as a function of the molar ratio of O₂ to HCO^– ₃ + CO₂ in solution provided a general, linear relationship (r ² = 0.72 to 0.84), predicting inhibition of photosynthesis based on O₂ pH and DIC changing together. Slopes of this relationship, representing competition between O₂ and carbon based on external concentrations, were similar for the five taxonomically different algae, suggesting that similar processes act to reduce net-O₂ evolution.     

Chemoautotrophic symbionts and translocation of fixed carbon from bacteria to host tissues in the littoral bivalve Loripes lucinalis (Lucinidae)

Specimens of Loripes lucinalis (Lucinidae) living in reducing sediments were collected near a sewage outfall at low tide on the Moulin Blanc beach, Brest, France, from January to March 1987. Electron microscope studies revealed numerous Gram-negative-type bacteria in the gill cells. Ribulosebiphosphate carboxylase, a diagnostic enzyme of the Calvin-Benson cycle of CO₂-fixation was measured only in the gill extracts. Various tissues of L. lucinalis were examined for activity of APS reductase, (EC 1.8.99.2), ATP sulphurylase (EC 2.7.7.4) and rhodanese (EC 2.8.1.1), enzymes involved in sulphide oxidation. APS reductase was only found in symbiont-containing tissues, i.e., gills. These enzymatic studies characterise the symbionts as chemoautotrophic sulphide-oxidizing bacteria. Histoautoradiography demonstrated that part of the carbon dioxide fixed by symbiotic bacteria in the gills is translocated to symbiont-free tissues of the bivalve. The ultrastructure of the gill is detailed and a nomenclature based on established and new terminology is proposed to describe the various cellular types comprising the gill filament.     

Effect of carbon dioxide concentration on calcification in the red coralline alga Bossiella orbigniana

The relationship between various experimental concentrations of CO₂ and calcification in Bossiella orbigniana (Decaisne) was studied by measuring Ca-45 incorporation into the crystalline matrix. Air containing CO₂ at partial pressures (P_{CO 2}) of 0.04 to 5.5% was bubbled through synthetic seawater in incubation vessels. The resultant pH values in the presence of plants ranged from 6.5 to 8.7. The maximum calcification rate appears to lie between 0.11 and 1.05% P_{CO 2}. The data suggest that calcification is controlled by a biological process that may be sensitive to pH and/or to the relative bicarbonate concentration. The data also suggest that a severalfold increase in CO₂ over the present atmospheric level might lead to increased calcification in this marine alga.     

Regulation of fructose-2,6-bisphosphate content in mantle tissue of the sea musselMytilus galloprovincialis

6-phosphofructo-2-kinase (PFK-2) from the mantle of the sea musselMytilus galloprovincialis Lmk, collected from the Ría de Arosa (NW Spain) in 1990, was purified 550-fold by extraction and sequential affinity chromatography on Affi-gel Blue and ATP-agarose columns. The enzyme was a dimer with a native molecular weight of 100 kilodaltons (KDa) and a subunitM _r of 53 KDa. PFK-2 activity is dependent on the presence of P_i. At physiological P_i concentrations, the enzyme exhibits hyperbolic kinetics with both ATP and Fru-6-P, withK _m values of 0.62 and 0.37 mM respectively. In vivo, PFK-2 activity is limited by the concentration of Fru-6-P which is low in comparison with theK _m for this substrate. Citrate and PEP inhibited PFK-2 activity.     

Study on phosphate uptake of the marine cyanophyte Synechococcus sp. NIBB 1071 in relation to oligotrophic environments in the open ocean

Inorganic phosphate (Pi) uptake by the marine cyanophyte Synechococcus sp. NIBB 1071 was studied using cells grown in an artificial seawater medium. The phosphate uptake was markedly enhanced in cells grown in the medium of low phosphate concentrations (phosphate-limited cells) than in cells grown in the phosphate-rich medium (phosphate-replete cells). The diagnosis of kinetics of instantaneous phosphate-uptake showed that V _max of the former was more than two orders of magnitude greater than that of the latter, and the k _m of the former was about 1/20 of that of the latter. The enhancement of the phosphate uptake was completed after a 40-h incubation of phosphate-replete cells in the phosphate-free medium. The activation was suppressed by chloramphenicol, an inhibitor of protein synthesis. The uptake developed in phosphate-limited cells was energy dependent and susceptive to osmotic shock, which suggests the involvement of a periplasmic phosphate-binding protein, analogous to that found in heterotrophic gram-negative eubacterial cells. The relationship between phosphate quota and growth rate, together with the kinetical data for phosphate uptake, predicted that ambient phosphate as low as 0.5 nM could support cell growth at a rate of one division per day. Results indicate that cells can grow rapidly even at phosphate concentrations as low as nanomolar levels. A possible regulatory mechanism of phosphate uptake in marine Synechococcus spp. is discussed in relation to a wide distribution of this picophytoplankton in the ocean environment. Received: 19 March 1997 / Accepted: 2 April 1997     

Photosynthetic carbon acquisition in the red algaGracilaria conferta

Photosynthetic properties of the common red algaGracilaria conferta, collected from the eastern Mediterranean Sea were investigated in 1989, in order to begin evaluating its adaptative strategies with regard to the inorganic carbon composition of seawater, and to test whether the alleged C₄ photosynthesis of anotherGracilaria species is common within the genus. Net photosynthetic rates ofG. conferta were, under ambient conditions of inorganic carbon (ca. 10µM, CO₂ and 2.2 mM HCO ₃ ^- ), not sensitive to O₂ over the range 10 to 300µM, and the CO₂ compensation point was low (ca. 0.005µM). Ribulose-1,5-bisphosphate carboxylase/oxygenase was the major carboxylating enzyme, with a crude extract activity of 175µmol CO₂ g^–1 fresh wt h^–1 while phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase were present at 70 and 20%, respectively, of that activity. No activities of the decarboxylases NAD-and NADP-malic enzyme could be detected. The¹⁴C pulse-chase incorporation pattern showed thatG. conferta fixes inorganic carbon via the photosynthetic carbon reduction cycle only, with no evidence for photosynthetic C₄ acid metabolism. Photosynthesis at the natural seawater pH of 8.2 was, at 25°C and saturating light, saturated at the ambient inorganic carbon concentration of 2.5 mM. It is proposed that, under ambient inorganic carbon conditions, a CO₂ concentrating system other than C₄ metabolism provides an internal CO₂ concentration sufficient to suppress the O₂ effect on ribulose-1,5-bisphosphate carboxylase/oxygenase and, thus, on photorespiration, in a medium where the external free CO₂ concentration is lower than theK _m(CO₂) of the carboxylating enzyme. Since inorganic carbon, under natural saturating light conditions, seems not to be a limiting factor for photosynthesis ofG. conferta, it likely follows that other nutrients limit the growth of this alga in nature.     

Products of denitrification by a marine bacterium as revealed by gas chromatography

Gas chromatography was used to analyse the gases released by growing cultures of the denitrifying marine bacterium Pseudomonas perjectomarinus. When nitrate was provided as final oxidant, CO₂ was the first detectable gas released. Following the peak rate of release of CO₂ at approximately 16 h, N₂ liheration began and peaked at 30 h. When N₂O was provided as terminal oxidant, CO₂ and N₂ release began within a few hours and the rate of liberation of both increased for 45 to 50 h before leveling. Cell-free extracts of bacteria from denitrifying cultures of P. perfectomarinus were incubated anaerobically with nitrate, malic acid and electron transfer cofactors provided, and the gases in the atmosphere above the reaction mixtures were analysed. NO was found to be present transiently, and N₂O was discerned after prolonged incubation. The applicability of gas chromatography to analyses of marine ecosystems and the need for better means of sampling the atmosphere above enzymatic reaction mixtures were disoussed.