The effects of some inhibitors on the rates of photosynthesis and respiration by Chlorella

The effects of several inhibitors on the rates of photosynthesis and of respiration by Chlorella pyrenoidosa have been studied. The inhibitors used in this study include potassium cyanide, hydroxylamine hydrogen chloride, dinitrophenol,and sodium azide. Each inhibitor seems to have its own characteristic action in photosynthesis and in respiration. With the exception of hydroxylamine, all the inhibitors show a stimulation of respiration at low concentrations of the inhibitors. Only dinitrophenol inhibits respiration to a marked extent. Potassium cyanide, hydroxylamine, and dinitrophenol are about equally effective in inhibiting photosynthesis, but sodium azide is nearly ten times as effective.     

Effects of Environmental pH on the Internal pH of Chlorella pyrenoidosa, Scenedesmus quadricauda, and Euglena mutabilis

The effect of external pH on two laboratory-cultured acid-intolerant species (Chlorella pyrenoidosa Chick and Scenedesmus quadricauda Turp. Bréb.) and one acid-tolerant species from a natural population (Euglena mutabilis Schmitz) was examined by measuring internal pH. These measurements were made with the weak acid ¹⁴C-dimethyloxazolidine-2,4-dione after cells had been incubated for 2 and 6 hours at external pH levels from 3.0 to 8.0. Photosynthetic and respiration rates of the three species were also measured over the range of external pH levels.     

RuBP Limitation of Photosynthetic Carbon Fixation during NH(3) Assimilation : Interactions between Photosynthesis, Respiration, and Ammonium Assimilation in N-Limited Green Algae

The effects of ammonium assimilation on photosynthetic carbon fixation and O₂ exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net O₂ evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light.     

Isolation and Characterization of a Novel Antialgal Allelochemical from Phragmites communis

Antialgal allelochemicals were isolated from Phragmites communis Tris. The isolated allelopathic fraction showed strong inhibition activity on the growth of Chlorella pyrenoidosa and Microcystis aeruginosa but had no inhibition on Chlorella vulgaris. The 50% effective concentrations (EC₅₀) of the allelopathic fractions on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.79 mg/liter, respectively. The allelopathic activity of the fraction was species-specific. The isolated allelopathic fraction caused metal ion leakage from algal cells. The fraction decreased the activities of antioxidant enzymes, such as superoxide dismutase and peroxidase. The addition of the isolated fraction increased the concentration of unsaturated lipid fatty acids in cell membrane of C. pyrenoidosa and M. aeruginosa. This caused a change in plasma membrane integrity and the leakage of ions in the protoplast. The allelopathic compound was identified by nuclear magnetic resonance and gas chromatography-mass spectrometry as ethyl 2-methylacetoacetate. Synthesized ethyl 2-methylacetoacetate also showed allelopathic activity on C. pyrenoidosa and M. aeruginosa. The EC₅₀ of synthesized ethyl 2-methylacetoacetate on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.65 mg/liter, respectively.     

Synchronization of cell division ofChlorella pyrenoidosa

A modification of the synchronization method forChlorella pyrenoidosa was suggested, based on the mechanical separation of small and large cells by differential centrifugation and dilution of the population after division to a constant density (maximally 3×10⁶ cells/ml.). Using this system, with a 16-hour day and eight hours' darkness, the ontogenetic cycle ofChlorella pyrenoidosa takes 24 hours and synchronous growth and development can be maintained for eight generations.     

Sorption of some ions by algae related to their trophic conditions

The unicellular algaeScenedesmus obliquus (125),Chlorella pyrenoidosa (82) andCoccomyxa solorinae saccatae (111) were studied with respect to the form of uptake of potassium, phosphate, calcium and zinc ions and to the energy sources involved: light under autotrophic conditions, glucose under mixotrophic or heterotrophic conditions (in light and in darkness or together with yeast extract as an auxotrophic substrate). We respected the trophic conditions of algae when preparing the experimental material (precultivation). The following conditions were reached:

1. (1)
   The three algae grow faster in a glucose medium under mixotrophic conditions and are capable of growing on it also heterotrophically:Ch. pyrenoidosa andSc. obliquus grow substantially better thanC. solorinae saccatae. The first two algae grow more intensively in a glucose medium containing yeast extract whileCoccomyxa does not. After cultivation under mixotrophic conditions the first two diminish endogenous respiration, the third raises it. Glucose stimulates respiration in the first two when grown autotrophically, while after mixotrophic cultivation the effect of glucose is suppressed inCh. pyrenoidosa and in the other two only after growth on glucose with yeast extract.     
   
   

自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。     

Specific factor inducing cell division of the algaChlorella pyrenoidosa and of the yeastCandida utilis

From cells of the green algaChlorella pyrenoidosa and of the yeastCandida utilis a substance was isolated which stimulated cell division in these microorganisms. The synthesis of this division-inducing factor (DIF) takes place inChlorella pyrenoidosa during the first half of the development cycle. During the second half of the ontogenetic development it is not possible to restore the inhibited division processes by its application. DIF increases aerobic respiration and aerobic and anaerobic glycolysis. Under aerobic conditions, in the presence of a source of energy and of DIF, cell division takes place without simultaneous synthesis of cell mass. Consequently, the substance can separate the processes of growth and division in that division can take place without growth.     

TEMPERATURE CHARACTERISTICS FOR METABOLISM OF CHLORELLA : I. THE RATE OF O2 UTILIZATION OF CHLORELLA PYRENOIDOSA WITH ADDED DEXTROSE

The temperature characteristic for the rate of O₂ consumption by Chlorella pyrenoidosa suspended in Knop solution containing 1 per cent glucose was studied between 1° and 27°C. with the Warburg technic. The value of µ was found to be about 19,000 ±1,000 cal. There is some indication of a critical temperature at 20°C., with shift to a lower µ above this temperature. The effect of sudden changes in temperature on the rate of respiration and the variation of the latter with time at constant temperatures are discussed. It is concluded that the "normal" respiration (in absence of external glucose) does not appear in the determination of this temperature characteristic.     

The course of respiration during the life cycle of Chlorella cells

Endogenous and glucose respiration were studied during the life history of Chlorella pyrenoidosa. A generalized picture of the course of respiration during the life cycle is suggested. At the liberation of daughter cells from the wall of the mother cell, or soon after, the respiration rate reaches its lowest level. If the daughter cells are placed in light the respiration rate rapidly increases with time, soon reaches a maximum, and then declines slowly. Two factors are important in the initial increase—the early developmental stage of the cells and the influence of light. In autotrophically developing algae the parts played by developmental processes and by light have not been separated. Direct activation of respiratory enzymes by light, in addition to the level of respiratory substrate, cannot be excluded. The decline of respiration rate over most of the cell history seems to have no connection with light and is probably bound to the developmental processes per se. Darkening the suspension interrupts growth and induces liberation of daughter cells, with concomitant faster decrease in respiration rate. The rate of respiration of small daughter cells decreases in darkness only slowly with time. Illumination seems necessary to bring these cells back to a high level of respiratory activity.     

The role of sugars in the respiration of differently cultivated green algae

A study of the effect of exogenous sugars (fructose, glucose, arabinose and xylose) on Qo₂ inChioreila pyrenoidosa (A 82) and inScenedesmus obliquus (A 125) showed that the effect of sugars on respiration depends on previous cultivation of the alga. In autotrophically cultivated algae all sugars contribute to the increase of Qo₂, in those cultivated rnixotrophically (with the addition of glucose or together with yeast extract, and with xylose) this occurs only in Scenedesmus when glucose or xylose are applied. The effect of cultivation was apparent in that mixotrophically cultivated algae had larger endogenous reserves of substrates than the autotrophic ones. The exogenous substrate is not preferentially respired and does not affect respiration solely as its substrate. In Chlorella, the effect of different cultivation conditions is more pronounced, the QO₂ being usually higher than inScenedesmes.     

Efficacy of Chlorella pyrenoidosa and Scenedesmus abundans for Nutrient Removal in Rice Mill Effluent (Paddy Soaked Water)

Microalgae are product of sustainable development owing to its ability to treat variety of wastewater effluents and thus produced biomass can serve as value added product for various commercial applications. This paper deals with the cultivation of microalgae species namely Chlorella pyrenoidosa and Scenedesmus abundans in rice mill effluent (i.e., paddy soaked water) for nutrient removal. In order to investigate the nutrient removal capability, microalgae are subjected to cultivation in both raw and autoclaved samples. The maximum phosphate removal by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 98.3% and 97.6%, respectively, whereas, the removal of ammoniacal nitrogen by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 92% and 90.3%, respectively. The growth (measured in terms of chlorophyll content) of Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 3.88 mg/l and 5.55 mg/l, respectively. The results indicate the suitability of microalgae cultivation in rice mill effluent treatment for nutrient removal.     

Sustained photo-hydrogen production by <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> without sulfur depletion

The sustained production of H₂ by Chlorella pyrenoidosa was achieved without sulfur deficiency or PSII inhibition. C. pyrenoidosa preserved hydrogenase activity for several hours in the dark. Hydrogenase activity in vitro is O₂ sensitive, which indicates that respiration may play an important role in H₂ production. A sustainable production of H₂ was obtained for 200 h under illumination. Further, by using 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a PSII inhibitor, at least 70% electrons for H₂ production were generated from PSII-catalyzed-H₂O oxidation. The remaining electrons were probably from endogenous substrate degradation.     

TEMPERATURE CHARACTERISTICS FOR THE METABOLISM OF CHLORELLA : II. THE RATE OF RESPIRATION OF CULTURES OF CHLORELLA PYRENOIDOSA AS A FUNCTION OF TIME AND OF TEMPERATURE

The respiration of the green alga Chlorella pyrenoidosa, suspended in Knop''s solution, has been studied in the dark as a function of time and of temperature. The rates of oxygen consumption and of carbon dioxide production (at constant temperature) decline for about 25 hours to a low, constant level. From an analysis of the curves it is suggested that two substances, A and B, are utilized, whose respiratory quotients are 1 and 0.65 respectively. The values of the temperature characteristics were found to be: for oxidation of A, 19,500 (0.6 to 11.5°C.) and 3,500 (11.5 to 28°C.); for oxidation of B, 5,600 (23.4 to 0.6°C.).     

Studies of sulfate utilization by algae: 8. The ubiquity of sulfate reduction to thiosulfate

Cell-free extracts from several microorganisms, when prepared by methods originally devised for Chlorella pyrenoidosa (Emerson strain 3) and incubated anaerobically with ATP, Mg²⁺, and 2, 3-dimercaptopropan-1-ol, are capable of reducing sulfate-³⁵S to thiosulfate. These microorganisms include, in addition to C. pyrenoidosa (Emerson strain 3), several other strains of C. pyrenoidosa, Chlorella protothecoides, Chlorella vulgaris, Anacystis sp., Chlamydomonas reinhardi, Escherichia coli, Salmonella typhimurium, and baker's yeast. Three of these organisms, E. coli, S. typhimurium, and baker's yeast, were previously reported by others to reduce sulfate to sulfite. Moreover, three mutant strains of S. typhimurium (Ba-25, Ce-363, and Bc-482) previously reported by other workers to be unable to reduce sulfate to sulfite also cannot form thiosulfate, and one mutant strain (Cd-68) reportedly able to form sulfite can also form thiosulfate. Taken together, this suggests that thiosulfate-forming activity may be a common feature of sulfate-reducing systems, and it may be present in enzymatic systems previously thought to be forming sulfite. Reasonably conclusive identification of thiosulfate is provided by ion exchange chromatography and by paper electrophoresis; the ambiguities associated with other analytical methods are discussed.     

Early effect of sodium pentachlorophenate on photosynthetic activity of the alga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> Chick. S-39

Early effects of 0.0001–10 mM sodium pentachlorophenate (PCP-Na) on the green alga Chlorella pyrenoidosa Chick. S-39 involves a rapid (within 1–2 min) decrease in the light-induced oxygen evolution by algal cells. The suppressed relative yield of variable chlorophyll fluorescence in C. pyrenoidosa in the presence of high PCP-Na concentrations and its dynamics provide evidence for rapid inactivation of photosystem 2, which is not observed at low concentrations of the toxicant. An analysis of the induction curve of delayed chlorophyll fluorescence in algal cells suggests that PCP-Na at low concentrations disturbs the coupling of electron transport and phosphorylation, whereas at high concentrations it inhibits electron transport and decreases the energy potential of photosynthetic membranes. The early toxic effect of PCP-Na is responsible for subsequent impairment of C. pyrenoidosa productivity.     

防己抑藻效应及其化感物质的HPLC分析

通过常温水浸提方法获得12种药用植物浸提液,并分别研究其抑藻效应。结果显示：防己水浸提物(相对浓度1 g·L^-1)抑制蛋白核小球藻的效应最强,最大比生长率为-0.28 d^-1。防己甲醇浸提物对蛋白核小球藻的抑藻效应显著,且持续时间长,最低有效抑藻浓度为30 mg·L^-1。HPLC鉴定结果显示：防己水浸提液和甲醇浸提液中主要含有防己诺林碱、粉防己碱等生物碱类化学成分,防己对治理水华发生有较大的应用前景。     

Inhibition of oxygen evolution following illumination of Chlorella cells with far-red light

The effect of brief far-red illumination on Chlorella pyrenoidosa cells has been investigated.An inhibition of oxygen evolution occurs 20 to 30 s after the end of the far-red illumination. This inhibition occurs in a step following the initial charge separation process by the System II centers. It is reversible in the light through a purely photochemical process.     

Countercurrent distribution and partition of Chlorella-sensitive and -resistant strain to the lytic agent in two-phase system. I

The influence of various buffering ions and NaCl concentrations on the distribution of Chlorella cells of two strains having differing surface and size were studied in an aqueous dextran-polyethylene glycol phase system using the one-step partition and CCD method.As models for these experiments we used an artificial mixture of Chlorella pyrenoidosa A 24 or Chlorella sp. 366 (strains sensitive to the infective lytic agent) and cells of Chlorella vulgaris B. resistant to this agent. The average size of Chlorella pyrenoidosa A 24 cells was 5.3 ± 0.53 μm and of Chlorella vulgaris B. 6.8 ± 0.55 μm.The condition of complete distribution of this algal mixture in 0.01 M sodium and potassium phosphate buffer is fulfilled when the concentration of NaCl is equal to or greater than a critical concentration of 15 mM of NaCl in the first, and 10 mM of NaCl in the second buffer.In buffers containing—COOH groups at pH 7.3 and low molarity a complete separation of mixture components was obtained. The addition of NaCl to maleate- and fumarate-NaOH buffers of pH 7.3 slightly worsened the separation effect which was visualized when decreasing the r_max difference value of both algal components on comparison with the separation obtained in a system without NaCl.At pH 6.0 in various buffers containing —COOH groups a complete separation of the algal components of the mixture was observed only when NaCl was absent. The addition of NaCl to some buffers of pH 6.0, e.g. maleate, malonate, and succinate, destroyed completely the successful separation and gave only a common peak of small r_max value.The fumarate buffer of pH 6.0, in the absence of NaCl, was an exception and gave only one common peak of small r_max value in contrast to its stereo-isomer maleic acid where two peak separation was obtained.     

Photosynthetic enhancement at high light intensities

Enhancement of photosynthetic oxygen evolution by Chlorella pyrenoidosa (211-8b) decreased almost linearly as the rate of photosynthesis was increased. The observed decrease was predicted most accurately by a model assuming exponential rate versus intensity curves as opposed to a model assuming hyperbolic light curves. The decrease in enhancement was not changed significantly upon changing from 0.03% to 5% carbon dioxide.