Growth Delay and Intracellular Changes in Chlorella ellipsoidea C-27 as a Result of Deuteration

The effects of deuterium (D) on Chlorella ellipsoidea C-27 wereinvestigated. Cells grown in a medium prepared with deuteriumoxide (D₂O) showed pronounced delays in cell growth and division;the length of a cell cycle in medium with 100 mol% D₂O was morethan 5 times longer than that in medium prepared in H₂O Thedelay caused by D₂O was not overcome by either indoleaceticacid or kinetin. The biological and ultrastractural characteristicsof deuterated .Chlorella (D-Chlorella) cells were examined.The responses of D-Chlorella to cell wall-digesting enzymesdid not differ from those of normal (H-Chlorella) cells. D-Chlorellacells were enlarged, and cellular components, such as proteins,nucleic acids, lipids and ATP, were present in larger quantitiesthan those in H-cells. The chloroplast of D-Chlorella was enlarged,but the levels of component photosynthetic pigments were significantlyreduced. By contrast, mitochondria of D-Chlorella were smallerthan those of H-cells. These changes in levels of cellular componentsand in the sizes of organelles seem to be unique to deuteration. (Received May 13, 1992; Accepted July 28, 1992)     

Metabolism of urea in Chlorella ellipsoidea

The effect of cyanide on ammonia and urea metabolism was studiedwith intact cells of Chlorella ellipsoidea Gerneck, a greenalga which apparently lacks urease. Ammonia uptake was inhibited more readily by cyanide than wasurea uptake. Urea uptake was stimulated by lower concentrationsof cyanide. The addition of cyanide caused the formation ofammonia from some cellular nitrogenous compounds. In the presenceof exogenously added urea, the molar ratio of ammonia accumulatedin the medium to urea taken up exceeded 2.0 as the cyanide concentrationincreased. However, the molar ratio of ammonia actually producedfrom urea nitrogen to urea taken up was less than 1.35 at anyconcentration of cyanide tested. In the presence of higher concentrationsof cyanide, the rate of incorporation of ¹⁵N into amino acidsfrom ¹⁵N-urea was higher than that from ¹⁵N-ammonium sulfate. The results suggest that Chlorella ellipsoidea possesses a pathwaythrough which urea nitrogen is assimilated directly withouta preliminary breakdown to ammonia. (Received October 18, 1976; )     

Studies on frost hardiness in Chlorella ellipsoidea I. Development of frost hardiness of Chlorella ellipsoidea in synchronous culture

Cells of Chlorella ellipsoidea Gerneck (IAM C-27) were synchronouslygrown under a 28-hr light-14-hr dark regime at 25°C. Thealgal cells at different stages during the cell cycle were hardenedat 3°C for 48 hr. The survival rate of hardened cells wasmaximum (70%) at the L₂ stage(ripening phase) in the life cycle.The average cell volume of L₂ cells increased during hardening,but the process of nuclear division scarcely advanced. The hardinessof L₂ cells increasedwith prolongation of hardening time upto 48 hr. Their viability decreased upon increasing the ratof cooling and lowering the final freezing temperature. Butthe hardened cells, which had been prefrozen stepwise, showeda survival rate above 50% even at –196°C when thawedrapidlyin a bath at 25°C. Although L₂ cells were somewhathardened in the dark, illumination was the more effective whenused with bubbling gas. Under illumination, bubbling of 1% CO₂-airincreased the hardiness more than CO₂-free air, but in the dark,this relation was reversed. The hardiness was lowest with nitrogengas bubbling under both conditions. (Received December 3, 1975; )     

Triparanol Inhibition of Sterol Biosynthesis in Chlorella ellipsoidea

The sterol composition of C. ellipsoidea was markedly changed when this alga was grown in the presence of 1 μg/g triparanol. Triparanol appears to inhibit the removal of 14α-methyl group, the second alkylation at C-24, Δ⁷-reductase, and Δ⁸ → Δ⁷-isomerase. The effect of triparanol in Chlorella is much more diversified than the specific effect originally assigned to it in animals.     

Action Spectra of Photosynthetic Activities of Chlorella ellipsoidea

• 1) Suspensions of Chlorella show an even stronger light scattering than suspensions of chloroplasts of spinach. The bands of absorption are thus broadened and, at higher concentrations, moved to lower wave-lengths. The intensity of the photosynthesis closely follows the curves of light scattering, a fact partly explaining the high efficiency of green light. Calculated per unit thermoelectrically measured incident energy the action spectrum shows bands at 660–670 nm and c. 500 nm and a comparatively high level of the whole region 500–560 nm.
• 2) Flash experiments show the existence of a steady state carotene/xanthophyll that is moved to reduction (c/x > 1) in blue and green light and to oxidation (c/x < 1) in red light. All experiments point to the existence of two light reactions, the first one involving excitation of carotenoids, with ferredoxin-TPN as acceptor, the second one involving excitation of chlorophyll, with the cytochrome system of the chloroplasts acting as donors of electrons and thus completing an energy converting circulation between pigments and enzyme systems.
• 3) The operation of combined light reactions appears also from experiments with simultaneous or succedaneous illumination with monochromatic light of different wave-lengths. Some effects may be explained from separate excitations of carotenoids and chlorophylls, others may depend on still unknown photic reactions.
• 4) The action spectrum in ultrared shows a positive band at c. 900 nm but no or only very small effects in the region 950–1400 nm. Ultrared radiation has on the other hand an enhancing effect on the light excitation in the visible spectrum. A combination of infrared and visible radiation shows a roughly linear relation between incident energy and photosynthetic effect.
• 5) All experiments were performed in the region of linear relation between intensity of incident light and O₂-production. Induced effects of combined monochromatic regions show a very rapid initial change in the steady states that in one or two minutes simmers down to a balanced state of continued photosynthesis. No change was observed in the total quantity of the pigments.

     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

Superoxide dismutase and chilling injury in Chlorella ellipsoidea

The relationship between superoxide dismutase (SOD) and chilling injury was examined in chilling-sensitive and chilling-resistant strains of Chlorella ellipsoidea. The sensitive strain contained less SOD than the resistant strain. Moreover, all of the SOD in the sensitive strain was the H2O2-sensitive, iron-containing SOD, whereas most of the SOD in the resistant strain was the H2O2-resistant, manganese-containing SOD. Illumination further enhanced the disparity in SOD content between the sensitive and resistant strains since the SOD in the former declined during illumination, whereas the SOD in the latter strain did not. It was possible to elevate the SOD content of the sensitive strain and to increase the proportion of MnSOD by prior growth in the presence of 50 microM paraquat. The SOD content of the cultures after 5 h of illumination at 4 degrees C fell in the order sensitive strain less than paraquat-induced sensitive strain less than resistant strain. The resistance of these cultures to chilling injury was related to SOD content. This was the case whether resistance was assessed in terms of growth rate after chilling, bleaching of chlorophyll during chilling, or loss of viability during chilling. It thus appears likely that O2- is an agent of chilling injury.     

Deuteration Causes the Decreased Induction of Heat-Shock Proteins and Increased Sensitivity to Heat Denaturation of Proteins in Chlorella

Deuterated Chlorella (D-Chlorella) cells were obtained by cultivationin a medium that contained D₂O. The modification of cell componentsby deuterium (D) increased the heat sensitivity of cells, whichdepended on the extent of deuteration. To elucidate the mechanismof the D-induced increase in the heat sensitivity, the effectof incorporation of D on the denaturation of proteins was investigated.Proteins obtained from D-Chlorella cells, when preheated at37°C, were aggregated to a greater extent than those fromH-Chlorella against the heating at 45°C. The rate of synthesisof heat-shock proteins (hsps) in D-Chlorella was consistentlylower than that in Hcells. Furthermore, an experiment in vitroindicated that deuterated proteins denatured more rapidly thannormal proteins upon heating at 60°C. (Received August 12, 1993; Accepted November 30, 1993)     

Isolation and Characterization of Chloroplast DNA from Chlorella ellipsoidea

A circular DNA molecule was isolated from chloroplasts of Chorella ellipsoidea. The DNA had a buoyant density of 1.695 grams per cubic centimeter (36% GC) and a contour length of 56 micrometers (175 kilobase pairs). The restriction endonuclease analysis gave the same size. Agarose gel electrophoretic patterns of chloroplast DNA digested by several restriction endonucleases were also presented. The digestion by the restriction enzymes, HpaII, MspI, SmaI, and XmaI revealed no appreciable methylation at CG sites in chloroplast DNA.     

Metabolism of 24-dihydrolanosterol in Ochromonas malhamensis and Chlorella ellipsoidea

24-Dihydrolanosterol-[2-³H] was converted to cholesterol in Chlorella ellipsoidea but ergost-5-enol, poriferasterol, clionasterol were not labelled. The absence of the necessary 24(25) double bond precursor eliminates the possibility of C₂₈ and C₂₉ sterol synthesis. However, it was confirmed that 24-dihydrolanosterol was metabolized by Ochromonas malhamensis to give cholesterol, brassicasterol, and poriferasterol.     

Uptake of Inorganic Carbon by Isolated Chloroplasts of the Unicellular Green Alga Chlorella ellipsoidea

Chloroplasts, isolated from protoplasts of the green alga, Chlorella ellipsoidea, were estimated to be 99% intact by the ferricyanide-reduction assay, and gave CO₂ and PGA-dependent rates of O₂ evolution of 64.5 to 150 micromoles per milligram of chlorophyll per hour, that is 30 to 70% of the photosynthetic activity of the parent cells. Intact chloroplasts showed no carbonic anhydrase activity, but it was detected in preparations of ruptured organelles. Rates of photosynthesis, measured in a closed system at pH 7.5, were twice the calculated rate of CO₂ supply from the uncatalyzed dehydration of HCO₃⁻ indicating a direct uptake of bicarbonate by the intact chloroplasts. Mass spectrometric measurements of CO₂ depletion from the medium on the illumination of chloroplasts indicate the lack of an active CO₂ transport across the chloroplast envelope.     

The acquisition and accumulation of inorganic carbon by the unicellular green alga Chlorella ellipsoidea

Abstract. The uptake and accumulation of inorganic carbon has been investigated in Chlorella ellipsoidea cells grown at acid or alkaline pH. Carbonic anhydrase (CA) was detected in ceil extracts but not in intact cells and CA activity in acid-grown cells was considerably less than that in alkali-grown cells. Both cell types demonstrates low K_1/2 (CO₂) values in the range pH 7.0–8.0 and these were unaffected by O₂ concentration. The CO₂ compensation concentrations of acid- and alkali-grown cells suspended in aqueous media were not significantly different in the range of pH 6.0–8.0, but at pH 5.0, the CO₂ compensation concentrations of acid-grown cells (57.4cm³ m⁻³) were lower than those of alkali-grown cells (79.2cm³ m⁻³). The rate of photo-synthetic O₂ evolution in the range pH 7.5–8.0 exceeded the calculated rate of CO₂ supply two- to three-fold, in both acid- and alkali-grown cells, indicating that HCO₃⁻ was taken up by the cells. Accumulation of inorganic carbon was measured at pH 7.5 by silicone-oil centri-fugation, and the concentration of unfixed inorganic carbon was found to be 5.1 mol m⁻³ in acid-grown and 6.4mol m⁻³ in alkali-grown cells. These concentrations were 4.6- and 5.9-fold greater than in the external medium. These results indicate that photorespiration is suppressed in both acid- and alkali-grown cells by an intracellular accumulation of inorganic carbon due, in part, to an active uptake of bicarbonate.     

Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga

Eight bacterial strains identified as P1, P2, Y1, Y2, W1, W2, G, and R were isolated from a long-term laboratory culture of the green alga Chlorella ellipsoidea. Although it is unknown how these bacterial strains have been maintained with the C. ellipsoidea culture, all appeared to promote the growth of C. ellipsoidea. Co-inoculation of each bacterial strain with C. ellipsoidea resulted in 0.5–3 times greater algal growth than that of C. ellipsoidea alone. The most effective bacterium (i.e., strain P1) was selected and further characterized. Biochemical analysis and transmission electron microscopy revealed that strain P1 is closely related to the genus Brevundimonas. Sequence analysis of the 16S rRNA of strain P1 showed 99.9 and 99.4% nucleotide sequence identity to that of B. nasdae and B. vesicularis, respectively. In addition to the growth promotion of C. ellipsoidea by strain P1, the growth of strain P1 was also significantly enhanced by co-culturing with C. ellipsoidea, indicating a symbiotic relationship between the bacterium and alga. Scanning electron microscopy showed the direct adhesion of strain P1 cells to the surface of C. ellipsoidea cells, as well as the development of abundant crinkles on the surface of co-cultured C. ellipsoidea cells. Handling editor: J. Padisak     

Optimization of pressurized liquid extraction of zeaxanthin from Chlorella ellipsoidea

The total zeaxanthin level in Chlorella ellipsoidea, a green microalga, was more than nine times that of red pepper, a plant source of zeaxanthin. Additionally, the zeaxanthin in C. ellipsoidea consisted of the free form, while those in other plants exist as zeaxanthin mono- and diesters. Pressurized liquid extraction (PLE) was used to extract zeaxanthin from C. ellipsoidea. Both the extraction temperature and extraction time, the two main factors in PLE, were optimized with a central composite design to obtain the highest extraction efficiency of zeaxanthin. Hexane, ethanol, and isopropanol were used as PLE extraction solvents. Ethanol extracted zeaxanthin most efficiently from C. ellipsoidea. Temperature was the parameter with the strongest influence on the extraction of zeaxanthin. The optimum extraction temperature and time for zeaxanthin were 115.4°C and 23.3?min, respectively. The maximum predicted value of 4.28?mg?g^?1 agreed with the experimental value of 4.26?mg?g^?1, supporting the quality of the fitted model. These results indicate that PLE using ethanol may be a useful method for extracting zeaxanthin from C. ellipsoidea.     

Characteristics of the Photosynthetic Metabolism of Carbon in Deuterated Chlorella ellipsoidea C-27

The photosynthetic metabolism of carbon in fully deuteratedcells of Chlorella ellipsoidea C-27 (D-Chlorella), obtainedby culture in medium prepared with 100 mol% D₂O, was characterizedby examining the activities of several enzymes and the levelsof metabolic regulators in a comparison with those of ordinarycells (H-Chlorella). The cellular content of starch in D-Chlorellawas more than twice that in H-Chlorella, whereas those of sucroseand glucose were significantly lower in D-Chlorella. Deuterationof Chlorella caused marked alterations in the activities ofenzymes involved in starch metabolism. There was a significantdecrease in the activity of phosphorylase, a catabolic enzyme,and a significant increase in the activity of starch synthase,an anabolic enzyme. These alterations are probably responsiblefor the increase in the amount of starch in cells. By contrast,no marked changes were observed in the activities of enzymesand the levels of metabolic inhibitors that are involved inthe synthesis of sucrose. It seems likely, therefore, that thedecrease in the amount of sucrose in D-Chlorella was causedmainly by a deficiency in sources of carbon in the cytoplasm,as a consequence of an increase in levels of starch in chloroplasts. (Received May 13, 1992; Accepted December 1, 1992)     

Accumulation of Free Fatty Acids during Hardening of Chlorella ellipsoidea

Chlorella ellipsoidea Gerneck (IAM C-27) was synchronously grown, and cells at an intermediate stage in the ripening phase of the cell cycle were hardened at 3°C for 48 hours. A nonpolar lipid which increased greatly during hardening was analyzed by gas-liquid chromatography. Palmitic, oleic, linoleic, and linolenic acids were the main components of the lipid. Electron micrographs revealed the appearance of lipid bodies in hardened cells. When formation of free fatty acids and lipid bodies was inhibited with cycloheximide, oligomycin, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the development of a high level of hardiness was always inhibited. However, the converse results were not always realized. Cells hardened in the dark in the absence of glucose developed a measurable hardiness in spite of their failure to form free fatty acids. The appearance of lipid bodies was invariably accompanied by the formation of the fatty acids. In pulse-labeling with [¹⁴C]NaHCO₃ for 4 minutes at zero time and at the 12th hour of hardening, initial incorporation rates of ¹⁴C into total lipids of whole cells and the cellular membrane fraction were significantly higher than that into free fatty acids. These results suggest that, although fatty acids are inserted into membrane lipids during hardening, the accumulation of free fatty acids and the appearance of lipid bodies per se are not involved in the development of frost hardiness.