An investigation of glycolate excretion in two species of blue-green algae

Summary The amount of ¹⁴C-glycolate excreted by Oscillatoria sp. and Anabaena flos-aquae is less than 1% of the ¹⁴C fixed by the algae during photosynthesis. Transfer of cells grown on 5% CO₂ in air to a medium of low bicarbonate concentration or treatment of the cells with isonicotinyl hydrazide (INH) during photosynthesis, caused little increase in glycolate excretion. -Hydroxysulfonates failed to stimulate massive excretion of glycolate. Although these blue-green algae excreted little glycolate, a significant proportion of the photosynthetically fixed carbon was excreted in the form of basic, neutral and acidic compounds, and such excretion was greater in 5% CO₂-grown cells than in air-grown cells.     

THE DISTRIBUTION OF PHOTOSYNTHATE IN ASCOPHYLLUM NODOSUM AS IT RELATES TO EPIPHYTIC POLYSIPHONIA LANOSA12

The brown alga Ascophyllum nodosum is not a major source of organic carbon for its epiphytic red alga Polysiphonia lanosa. Plants pulse-labeled for 24–25 hr with NaH¹⁴CO₃ were examined for exudation and translocation. The maximum amount of radioactive carbon compounds lost from A. nodosum during this experimental period was less than 0.3% of the total ¹⁴C fixed by the alga, and of this amount, only 5% could, have moved through the frond. The remaining fraction of the ¹⁴C lost from the thallus was released into the water. The total exudate from A. nodosum was collected for 1 week in a series of flasks of filtered seawater changed at 12-hr intervals corresponding with the beginning of the light and dark periods, respectively. During 7 days at 15 C only 1.5% of the total ¹⁴C originally fixed had been released as radioactive organic carbon, whereas at 5 C, 0.6% of the total ¹⁴C fixed was found in the medium. No significant difference in the rate of exudation of organic ¹⁴C was observed in light or darkness. After fractionation of the exudate it was found that only 10% of the radioactivity in these exudates was composed of organic acids or amino acids. P. lanosa, on the other hand, is perfectly capable of fixing its own carbon. The photosynthetic rates measured by H¹⁴CO₃ uptake confirm the observations of Bidwell: 3.96 mg CO₂/g/hr (0.09 m mole/g/hr).     

Excretion of Glycolate, Mesotartrate and Isocitrate Lactone by Synchronized Cultures of Ankistrodesmus braunii

Fixation of ¹⁴CO₂ by synchronized cultures of Ankistrodesmus braunii was highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of ¹⁴C excreted during photosynthesis followed the same trend. Cells at the end of the growing phase, after 10 hours of a 16-hour light phase, excreted nearly 35% of the total ¹⁴C fixed as one product, glycolate. Dividing cells from the dark phase, when tested in the light, excreted only 4% as much glycolate-¹⁴C as the young growing cells. Dividing cells also excreted as much mesotartrate as glycolate and also some isocitrate lactone and an unidentified acid. None of these excreted acids were found inside the cells in significant amounts. Methods for isolation and identification of the excreted acids are present. With ¹⁴C-labeled algae, it was shown that the excretion of glycolate was light-dependent and inhibited by 1,1-dimethyl-3-(p-chlorophenyl) urea. The excretion of labeled mesotartrate, isocitrate lactone, and an unknown acid, but not glycolate, also occurred in the dark. The excreted mesotartrate was predominantly carboxyl-labeled even after long periods of ¹⁴CO₂ fixation. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of the carboxyl-labeled mesotartrate. The reason for the specific excretion of glycolate, mesotartrate, and isocitrate lactone is not known, but the metabolism of all three acids by the algae may be limited and each can form dilactides or lactones by dehydration. In this context isocitrate lactone was excreted rather than the free acid.     

CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

The role of carbon dioxide in the formation of end-products by Hymenolepis diminuta

The hypothesis that ambient CO₂ levels determine the end-products of energy metabolism excreted by Hymenolepis diminuta was tested by incubating the parasite in a range of CO₂ concentrations and measuring internal concentrations of adenine nucleotides and the excretion of organic acids. The strain of H. diminuta used was found to excrete mainly lactic acid and acetic acid. Succinic acid production was generally less than 5–10% of the total. At high CO₂ concentrations, the rate of excretion of lactic acid decreased while that of succinic acid increased, which conforms with the hypothesis. Acetic acid excretion did not vary significantly over the range of CO₂ concentrations used. Other results did not support the hypothesis. High CO₂ levels reduced the total amounts of acids excreted and the rate of succinic acid excretion was so small as to be ineffective in preventing the accumulation of H⁺ ions. When present in the incubation medium, succinic acid was taken up by H. diminuta. Lactic and acetic acid excretion was always sufficient to limit the accumulation of H⁺ ions. The conditions of incubation were shown not to be responsible for the low rates of succinic acid excreted. Incubation conditions and metabolic end-products were found to affect the rates of excretion of organic acids. There is thus a need, in work of this nature, to regulate and specify experimental conditions and to stipulate the strain of parasite used. The hypothesis was rejected and it was suggested that the energy metabolism of parasitic helminths is adapted to fluctuating O₂ and CO₂ tensions.     

ALGAL EXCRETION AND BACTERIAL ASSIMILATION IN HOT SPRING ALGAL MATS1

Benthic algal-bacterial mats are present in the effluents of alkaline hot springs at temperatures between 50 and 73 C. The thin surface layer is composed of the unicellular blue-green alga Synechococcus lividus. Also present in the surface layer and forming thick, orange mats beneath it, are filamentous, phototrophic, gliding bacteria of the genus Chloroflexis, also capable of heterotrophic growth. The very low species diversity and the constancy of the hot spring environment, make these mats a good ecosystem for studying the transfer of nutrients from the algae to the bacteria. To determine whether the alga might supply organic materials to the bacterium, excretion by natural populations of S. lividus was studied in the field by means of short-term radioisotope experiments. Under optimal conditions for photosynthesis, between 3 and 12% of the total ¹⁴C fixed was excreted as ¹⁴C-labeled organic compounds. Variations in cell density at concentrations of S. lividus approximating those found in the mat had no effect on the percentage excretion. However, at cell densities below a threshold, level, the percentage excretion increased with diminishing cell density. Except at very low light intensities the percentage of fixed carbon excreted, was very similar for all light intensities tested. Excretion at temperatures approaching the upper limit for growth was not significantly different from the percentage excretion values observed at lower temperatures. ¹⁴C-labeled organic compounds excreted during algal photosynthesis could be subsequently assimilated by natural populations of the bacteria present in the mat.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas

Aminooxyacetate (1 millimolar) did not inhibit photosynthetic ¹⁴CO₂ fixation by Chlamydomonas reinhardtii Dangeard, (−) strain (N.90) but greatly stimulated the biosynthesis and excretion of glycolate. Similar results were obtained from cells grown with 5% CO₂ or low CO₂ (air). After 2 minutes with air-grown cells, [¹⁴C]glycolate increased from 0.3% of the total ¹⁴C fixed by the control to 11.7% in the presence of aminooxyacetate and after 10 minutes from 3.8% to 41.1%. Ammonium nitrate (0.2 millimolar) in the media blocked the aminooxyacetate stimulation of glycolate excretion. Chromatographic analyses of the labeled products in the cells and supernatant media indicated that aminooxyacetate also completely inhibited the labeling of alanine while some pyruvate accumulated and was excreted. A high percentage (35%) of initial ¹⁴CO₂ fixation was into C₄ acids. Initial products of ¹⁴CO₂ fixation included phosphate esters as well as malate, aspartate, and glutamate in treated or untreated cells. Lactate was also a major early product of photosynthesis, and its labeling was reduced by aminooxyacetate. Inasmuch as lactate was not excreted, glycolate excretion seemed to be specific. When photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, labeled organic and amino acids but not phosphate esters were lost from the cells. Aminooxyacetate did not inhibit the enzymes associated with glycolate synthesis from ribulose bisphosphate.     

TRANSFER OF N2 AND CO2 FIXATION PRODUCTS FROM ANABAENA OSCILLARIOIDES TO ASSOCIATED BACTERIA DURING INORGANIC CARBON SUFFICIENCY AND DEFICIENCY1

Transfer of N₂ and CO₂ fixation products from the bloom forming blue-green alga, Anabaena oscillarioides Bory, to attached and free swimming bacteria is common during active growth of the former. Incubation with ¹⁵N₂ and ¹⁴CO₂ followed by size fractionation filtration reveals that: i) magnitudes of fixed N and C excretion, relative to N₂ and CO₂ fixation, are dictated by dissolved inorganic carbon (DIC) availability for A. oscillarioides photosynthetic production, ii) associated bacteria exhibit preferences for recently fixed excreted N compounds, iii) bacterial utilization of excreted N is independent of ambient light conditions, and iv) lag times between N₂ fixation and detectable bacterial assimilation of excreted fixed N compounds are ca. 1–2 h. Both ¹⁴NH₄Cl dilution and Hg(NH₃)₂ Cl₂ precipitation techniques indicate that NH₃ is a major excretion product from A. oscillarioides, particularly during DIC limited growth. Active N and C excretion and transfer to associated bacteria are features of viable A. oscillarioides filaments. Hence, transfer of these metabolites reflects complex mutualistic, and possibly symbiotic associations rather than solely signaling senescence.     

Relationship between leaf development and primary photosynthetic products in the C4 plant Portulaca oleracea L.

Summary The photosynthetic products of Portulaca oleracea differ greatly depending on leaf age and length of exposure to ¹⁴CO₂. Mature leaves of P. oleracea fix ¹⁴CO₂ primarily into organic and amino acids during a 10-s exposure period. Less than 2% of the ¹⁴CO₂ fixed appears in phosphorylated compounds. In contrast, incorporation into amino acids can account for over 60% of the total ¹⁴CO₂ fixed by young leaves in an equal time period, and incorporation into alanine alone can account for up to one half of this amount. Senescent leaves display a quantitative shift of primary products toward phosphorylated compounds with a concomitant reduction of the label residing in malate and asparate. About 8 times more phosphoglyceric acid is produced in senescent leaves than in mature leaves. The aspartate/ malate ratio is not constant and depends on the length of time the leaves are exposed to ¹⁴CO₂ and the age of the leaves under study. It appears as if the stage of leaf development is one of the most important factors determining the operation of a particular enzyme system in C₄ plants.     

Metabolic Pathway of alpha-Ketoglutarate in Citrus Leaves as Affected by Phosphorus Nutrition

The uptake and metabolism of α-[5-¹⁴C]ketoglutarate by phosphorus-deficient and full nutrient (control) lemon (Citrus limon) leaves were studied over various time intervals. After 45 minutes in P-deficient leaves, the bulk of incorporated ¹⁴C appeared in organic acids and much less in amino acids, while in the control leaves, the ¹⁴C contents of organic and amino acids were equal. In P-deficient leaves, after longer incubation times the ¹⁴C content of organic acids and amino acids increased, while that of CO₂ and residue fractions remained low. In full nutrient leaves the ¹⁴C content of amino acids and organic acids decreased after longer incubation time and increased in the insoluble residue and CO₂. In full nutrient leaves the organic and amino acid metabolism were closely related and accompanied by protein synthesis and CO₂ release, while in P-deficient leaves an accelerating accumulation of arginine and citric acid was linked together with inhibition of protein synthesis and CO₂ liberation.     

Temperature-Enhanced Osmotic Growth Requirement of Crithidia*†

SYNOPSIS. An osmotic growth requirement for the trypanosomatid Crithidia fasciculata became conspicuous at 32 C. Osmo-supportive compounds were surveyed at 28 vs. 33 C, in “low-osmotic” defined medium. Effective osmotic support was provided by many compounds, e.g., glycerophosphate, sorbitol, mannitol, glycerol, Na isethionate, glycine, arginine, KCl, NaCl, NH₄Cl, and K₂SO₄. The nonspecificity of the requirement was thus evident, but inactivity of the presumably poorly adsorbable pentaerythritol indicated that osmotic pressure was a likely but insufficient condition for satisfying the temperature-enhanced growth requirement most clearly expressed as a need for osmotic support. Fortification of the medium with a combination of glycerophosphate, glycine, glycerol, and Tween 80 permitted good growth at 35 C. Possible relations between the temperature-enhanced osmotic requirement cell membrane damage, and morphological phases of Trypanosoma and Leishmania are discussed.     

Halotolerance studies onChlamydomonas reinhardtii: glycerol excretion by free and immobilized cells

The freshwater green algaChlamydomonas reinhardtii can tolerate a maximum saline concentration of 200 mM NaCl. In response to this osmotic shock, the cells accumulated during the first 24 h 15% of the total glycerol synthesized as osmoregulatory metabolite, to provide the corresponding osmotic balance. After this period all glycerol synthesized was excreted to the medium, 4 g L^-1 at 120 h in optimal conditions, before cell degradation occurred. This excretion was about 2-fold higher in Ca-alginate entrapped cells in the presence of 250 mM NaCl. It was concluded that immobilized cells may be of biotechnological interest for continuous glycerol photoproduction in air-lift bioreactors.     

Influence of gibberellic acid on <Superscript>14</Superscript>CO<Subscript>2</Subscript> metabolism,growth, and production of alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Changes in growth parameters, carbon assimilation efficiency, and utilization of ¹⁴CO₂ assimilate into alkaloids in plant parts were investigated at whole plant level by treatment of Catharanthus roseus with gibberellic acid (GA). Application of GA (1 000 g m⁻³) resulted in changes in leaf morphology, increase in stem elongation, leaf and internode length, plant height, and decrease in biomass content. Phenotypic changes were accompanied by decrease in contents of chlorophylls and in photosynthetic capacity. GA application resulted in higher % of total alkaloids accumulated in leaf, stem, and root. GA treatment produced negative phenotypic response in total biomass production but positive response in content of total alkaloids in leaf, stem, and roots. ¹⁴C assimilate partitioning revealed that ¹⁴C distribution in leaf, stem, and root of treated plants was higher than in untreated and variations were observed in contents of metabolites as sugars, amino acids, and organic acids. Capacity to utilize current fixed ¹⁴C derived assimilates for alkaloid production was high in leaves but low in roots of treated plants despite higher content of ¹⁴C metabolites such as sugars, amino acids, and organic acids. In spite of higher availability of metabolites, their utilization into alkaloid production is low in GA-treated roots.     

Transient excretion of succinate from <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> submerged mycelia reveals the complex movements and metabolism of carboxylates

Submerged growth of Trichoderma atroviride CCM F 534 on glucose-containing medium was accompanied by the excretion of organic acids (succinate, citrate, alpha-ketoglutarate, fumarate, aconitate). The excretion of succinate was transient. After 48–72 h cultivation, millimolar amounts of succinate disappeared from the medium. We studied the mechanism of the removal of succinate from the medium and demonstrated the activation of the inward transport of succinate by submerged mycelia. This transport was carrier-mediated, had a low solute specificity, and was driven by proton-motive force. The last aspect was provided by the activation of the H⁺-ATPase, as documented by measurements of ATPase activity and expression of the pma gene. The disruption of the pma gene abolished the capacity of the mycelia to re-uptake succinate but not its production. Results show that excreted carboxylates could serve as alternative nutrients in the late phase(s) of submerged growth, explain why inward transport system(s) for carboxylates are induced, and indicate that the inward-directed transport could interfere with the production of carboxylic acids by fungi.     

Inhibierungserscheinungen bei der alkoholishchen Gärung III. Der Einfluß des osmotischen Druckes auf die Lebensfähigkeit von Saccharomyces cerevisiae

The influence of the osmotic pressure on the viability of yeast cells was studied in batch processes. It could be found, that the viability is a function of the total osmotic pressure obtained by adding the partial osmotic values of the principal ingredients saccharose, ethanol and salts dissolved in the medium. At optimum process conditions (pH, T, etc.) the cells can tolerate the osmotic pressure up to fixed value π₁. Above π₁ the viability decreases linearly and upward of a second threshold value π₂ nonlinearly. In the case of the used strain Saccharomyces cerevisiae Hansen Sc 5 the values of the both points are π₁ = 25 atm and π₂ = 47 atm.     

Synthesis of metabolism-resistant substrates for the transport system for cationic amino acids; their stimulation of the release of insulin and glucagon, and of the urinary loss of amino acids related to cystinuria

Several amino acids have been synthesized as model transport substrates building on the piperidine and cyclohexane rings. Only when the distal N atom is part of an unambiguously cationic structure are these compounds transported predominantly by the cationic amino acid system. These amino acids in labeled form are excreted rather slowly in unmodified state, very little ¹⁴CO₂ being released. Those which are unambiguously cationic (including also homoarginine) led to a greatly increased excretion of arginine, lysine, ornithine and citrulline. Those which might be expected to act as lysine analogs had little effect on the excretion of the basic amino acids, although the excretion of citrulline and the sum of glutamine plus asparagine was accelerated. Certain of the analogs intensified the excretion of citrulline in dissociation from effects on resorption of the basic amino acids, also in dissociation from effects on cystine resorption. These results indicate citrulline resorption does not occur principally by the same agency serving for the basic amino acids, nor by the agency serving for cystine, despite the observed interactions for resorption. The injection of either of three transport analogs for arginine into the rat leads to early increases in the circulating levels of immunologically reactive insulin and glucagon.     

ALGAL EXCRETION OF 14C-LABELED COMPOUNDS AND MICROBIAL INTERACTIONS IN CYANIDIUM CALDARIUM MATS1

Acid spring effluents are often covered with mats of the eucaryotic phycocyanin-containing alga. Cyanidium caldarium. The primary bacterial component of such mats is an acidophilic strain of Bacillus coagulans, and the primary fungal component is Dactylaria gallopava. Because of the limited species diversity, C. caldarium mats appeared to be an excellent system for studying algal excretion and various microbial interactions in nature. From 2 to 6% of the NaH¹⁴CO₃ taken up by natural or laboratory populations of the alga was excreted as ¹⁴C-labeled materials. The maximum excretion occurred at temperature, light, and pH values optimum for NaH¹⁴CO₃ uptake. However, when excretion was expressed as a percentage of NaH14 CO₃ uptake, a higher percentage of the radioactivity was excreted at nonoptimal conditions for NaH¹⁴CO₃ uptake. Fungal biomass was directly proportional to algal density, but bacterial numbers varied widely and did not correlate with algal numbers. The bacterial and fungal components could be grown in mixed culture with either growing C. caldarium cultures or in an extract prepared, by healing algal cells.     

Metabolic Study of Douglas-fir Pollen Germination in vitro

Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] pollen was germinated and grown in mass in a sterile mineral medium supplemented with 0.3 M mannitol as osmotic stabilizer. During the 4-day period, pollen elongated threefold; free sugar and amino acid contents did not change significantly; soluble protein, insoluble protein, and RNA decreased; starch reduced to 30%; and DNA doubled in quantity. Respiration rate remained high and constant during the first 36 h and then increased, but the respiratory quotient averaged about 1.0 throughout the period. The pool size of adenine nucleotides remained constant, whereas ATP content and energy charge increased rapidly during the first 8 h and remained high for the remainder of the period. ¹⁴C-glucose was rapidly metabolized within 2 h at 25°C to amino acids (33%), organic acids (22%), sugars (20%), CO₂ (15%), lipids (5%), and insoluble components (3%). Experimental data indicated that the cultural conditions provided a suitable environment for rapid germination and active metabolism of Douglas-fir pollen.     

ORGANIC COMPOUNDS RELEASED FROM A NATURAL POPULATION OF EPIBENTHIC BLUEGREEN ALGAE1

Extracellular release of dissolved organic compounds by the bluegreen algal community of a brackish marsh was studied using ¹⁴C techniques. Mannitol and trehalose were identified as the most commonly released compounds. The proportions of these two extracellular compounds varied in response to light intensity and the water potential of the environment. The presence of mannitol, in particular, suggests that excretion of organic compounds in natural situations is a function of osmotic adjustment.