The Influence of CO2 Concentration and pH on Two Chlorella Species Grown in Continuous Light

Both Chlorella pyrenoidosa and Chlorella vulgaris grow equally well at 20°C aerated with ordinary air or mixtures of air with 5 or 12 per cent CO₂ (5 klux continuous light). Whereas C. vulgaris relatively rapidly adapts to a higher CO₂ tension, adaptation takes about 24 hours for C. pyrenoidosa. In Chlorella vulgaris pH in the range 3.6–7.6 has no apparent influence on the rate of photosynthesis in experiments having a duration of two hours. This is true both for algae grown aerated by ordinary air and for algae grown with a mixture of 5 per cent CO₂ in air. The adaptation time must be short. In Chlorella pyrenoidosa the same is found for algae in ordinary air, whereas an influence of pH is seen in some experiments where the aeration was by 5 per cent CO₂ in air. As is to be expected, the rate of photosynthesis in C. pyrenoidosa during the first two hours is very much influenced by the concentration of free CO₂. The highest rate is found at the CO₂ concentration at which the algae had been growing previously. The influence on the rate of photosynthesis in C. vulgaris is very much less, although in principle the same. The investigation of the corresponding influence on the rate of respiration is complicated by considerable variation from one series to another. In C. vulgaris this is particularly of importance. In C. pyrenoidosa, the highest rate of respiration is generally found at the CO₂-concentration at which the alga had been growing before the experiment. It seems probable that variations between similar series is due to the fact that the algae were grown in continuous light but with dilution with fresh culture medium when the optical density had reached a certain magnitude. Algae grown in this way are neither synchronized nor non-synchronized.Our thanks are due to the Danish State Research Foundation for financial support.     

EFFECTS OF CO2 CONCENTRATION DURING GROWTH ON THE INTRACELLULAR STRUCTURE OF CHLORELLA AND SCENEDESMUS (CHLOROPHYTA)1

Effects of CO₂ concentration during growth on intracellular structure were studied with ftve species of Chlorella and Scenedesmus obliquus. Cells grown under ordinary air conditions (low-CO₂ cells) had a well developed pyrenoid surrounded by starch, while those grown under high CO₂ conditions (high-CO₂ cells) had a less developed pyrenoid or no detectable pyrenoid. Two mitochondria, one at each side of the neck of the projection of the chloroplast close to the pyrenoid, were found in low CO₂ cells of C. vulgaris 11h. Usually, lamellar stacks extended in parallel in the chloroplast of low-CO₂ cells of C. vulgaris 11h, while a grana-like structure was found in high-CO₂ cells. However, in C. pyrenoidosa, grana like structures were found more commonly in low-CO₂ cells than in high-CO₂ cells. These results suggest that development of pyrenoid starch is generally correlated with growth under low CO₂ conditions, whereas CO₂-effects on lamellar stacking are species dependent.     

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.     

Effects of culture temperature shift and light-dark time cycle on the cellular sugar accumulation of Chlorella pyrenoidosa

We investigated the effect of culture temperature on the maximum specific growth rate and the cellular sugar accumulation, and the effect of a temperature shift on the sugar accumulation of Chlorella pyrenoidosa cells in a batch culture system. Increase in temperature below 30?°C appeared to correlate with increase in the maximum specific growth rate, on the contrary the cellular sugar content showed a reverse tendency against temperature. We attempted to utilize this tendency for improving sugar productivity in Chlorella. First, we cultured Chlorella at 28?°C during the logarithmic growth phase to obtain a high specific growth rate. The culture temperature was then shifted from 28?°C to 22?°C at the late logarithmic growth phase in order to reduce the specific growth rate and enhance the cellular sugar accumulation. As a result, we obtained a 15% increase in sugar production over that obtained by cultivation at 28?°C throughout the culture. We also investigated the effect of light-dark time cycle on the sugar productivity and found that this operating variable did not affect the cellular sugar content but influenced the final cell concentration. Among the examined light-dark time cycles, maximum sugar productivity was obtained in the case of 12?h light and 12?h dark period.     

Antimicrobial effects of esters and amides of 3-(5-nitro-2-furyl)acrylic acid

The effect of 18 newly synthesized esters and amides of 3-(5-nitro-2-furyl)acrylic acid on bacteria (Escherichia coli, Staphylococcus aureus), yeasts (Saccharomyces cerevisiae, Candida albicans), molds (Aspergillus niger, Penicillium cyclopium, Rhizopus oryzae) and algae (Chlorella pyrenoidosa, Euglena gracilis, Scenedesmus obliquus) was investigated. The MIC values revealed antimycotic, antialgal and antibacterial activity of the studied derivates. The antimycotic activity was found to decrease with increasing the length of the alkyl chain of esters and after introduction of amino nitrogen into the furylethylene backbone. The inhibitory effect on growth is caused by blocking bioenergetic processes, glycolysis in particular.     

Regulation of polyamine synthesis in physarum polycephalum during growth and differentiation

The levels and synthesis of polyamines were investigated in Physarum polycephalum to obtain information about their regulation during growth and differentiation in a lower eukaryote. Putrescine pools rapidly increased 4–5 fold during the change from dormant spherules to growing plasmodia. The activity of ornithine decarboxylase (EC 4.1.1.17), which converts ornithine to putrescine, reflected this rapid change in the level of putrescine. Spermidine levels were closely correlated with protein concentrations during differentiation due to variations in the activity of S-adenosyl-l-methionine decarboxylase which is involved in the conversion of putrescine to spermidine This enzyme was not stimulated by putrescine, unlike the similar enzyme in other eukaryotes, thereby permitting independent regulation of putrescine and spermidine levels. The high levels of both putrescine and spermidine suggest separate functions for these polyamines in Physarum.The half-lives of ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase were 14 and 21.5 min, respectively. These short half-lives keep the polyamine metabolism under a very tight control as illustrated by the rapid fluctuations in enzyme activity during differentiation and the synchronous mitotic cycle. The step patterns of these unstable enzymes during the mitotic cycle suggest that these enzyme levels are limited by gene dosage.     

O-Dealkylation of coumarin and resorufin ethers by unicellular green algae: kinetic properties of Chlorella fusca and Chlorella sorokiniana

O-Dealkylations of resorufin and coumarin ethers, mediated by microsomal cytochrome P450 mono-oxygenases from animals, plants and microorganisms, are shown here to be performed also by intact cells of the unicellular green algaeChlorella fusca andChlorella sorokiniana. The activity of theO-dealkylation of these ethers was up to tenfold higher withChlorella sorokiniana. Both algae dealkylated methyl-, ethyl-, and pentylethers of resorufin and coumarin. Dealkylation in vivo indicated efficient absorption of methoxy- and ethoxyresorufin, confirmed by the respective absorption kinetics. Piperonylbutoxide and 1-aminobenzotriazole, known inhibitors of plant and mammalian cytochrome P450s, significantly inhibited theO-dealkylase activity of both algal strains. The use of synchronized cultures of both algae revealed that efficiency ofO-dealkylation depends on the stage of the cell cycle: during the growth phase, theO-dealkylase activities increased more than proportional, and the distinct drop in activity during the last hours of the light period indicated the appearance of an endogenous substrate.     

Dynamic aspects of radiorespirometry during the cell cycle of Candida utilis: Some observations in phased culture under carbon-, nitrogen-, and phosphorus-limited growth

Many changes that occur in a cell during the cell cycle can be demonstrated in synchronous cultures and can reveal dimensions of cell metabolism not attainable by the study of balanced growth of asynchronous populations in batch cultures or the steady state in chemostat cultures. The release of ¹⁴CO₂ from specifically labeled glucose by phased (continuously synchronized) cultures follows a characteristic pattern (profile) that depends upon the stage in the cell cycle and the period of labeling used. Successive profiles throughout a cycle showed differences that were altered under different nutrient-limiting growth conditions. Profiles obtained with glucose-1-¹⁴C, glucose-2-¹⁴C, glucose-3,4-¹⁴C, and glucose-6-¹⁴C and phased cells of Candida utilis under N-, P-, and C-limited growth demonstrated the variable character of the metabolic activity that occurred in the cells while contour changes within the profiles across the cycle indicated possible correlations with activities of the hexose monophosphate, Embden-Meyerhof-Parnas, and tricarboxylic acid cycle pathways during the cell cycle. The basis of these changes and their use as elementary parameters for study of problems of physiological changes in vivo are considered.     

Isolation and characterization of a starchless mutant of Chlorella pyrenoidosa STL-PI with a high growth rate, and high protein and polyunsaturated fatty acid content

The growth characteristics, biochemical composition and ultrastructure of a novel starchless mutant of Chlorella pyrenoidosa, designated as STL‐PI, are compared to the same characteristics of its parental strain, C. pyrenoidosa 82T. The STL‐PI mutant had a 22 ± 5% higher growth rate, and 24.5 ± 4.2% more protein than the parental strain, 82T. Furthermore, the STL‐PI mutant accumulated 20.4% more polyunsaturated fatty acids and 18% less saturated fatty acids than the parental 82T. When the parental 82T was cultured in a nitrogen‐free media, their starch content increased from 6.8 ± 2.8% to 22.5 ± 3.1%. In contrast, the STL‐PI mutant produced no starch, regardless of the growth conditions. Instead, the mutant cells responded to nitrogen limitation by further increasing their lipid content from 25.2 ± 1.2% to 38.0 ± 2.3% per dry weight. Transmission electron micrography revealed that nitrogen limitation typically stimulates the formation of starch granules in the chloroplast of 82T cells. Yet no starch granules were observed in the STL‐PI cells. Instead, only the formation of large lipid globules was observed in the mutant cells. These results demonstrate that the starchless mutant STL‐P1 possesses novel physiological and phytochemical properties distinct from the 82T cells: their cells were deficient in starch synthesis and showed higher growth rates and productivity than 82T cells.     

Impacts of Elevated CO2 Concentration on Biochemical Composition,Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations,Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2concentrations within the range 3-186 μmol/L and the biochemical composition, carbonic anhydrase (CA),and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate,and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186μmol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 μmol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.     

Separation,antitumor activities,and encapsulation of polypeptide from Chlorella pyrenoidosa

Chlorella pyrenoidosa is a unicellular green algae and has been a popular foodstuff worldwide. However, no reports on the antitumor peptides from such a microalgae are available in the literature. In this study, using low‐temperature high‐pressure extraction, enzymatic hydrolysis, ion exchange, and gel filtration chromatography, we separated a polypeptide that exhibited inhibitory activity on human liver cancer HepG2 cells, and named the polypeptide CPAP (C. pyrenoidosa antitumor polypeptide). Furthermore, the micro‐ and nanoencapsulation of CPAP were investigated by using two methods: complex coacervation and ionotropic gelation. The in vitro release tests revealed that CPAP was well preserved against gastric enzymatic degradation after micro/nanoencapsulation and the slowly controlled release in the intestine could be potentially achieved. These results suggest that CPAP may be a useful ingredient in food, nutraceutical, and pharmaceutical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:681–687, 2013     

Ultraviolet Inactivation and Photoreactivation in Synchronized Chlorella

Synchronous cultures of Chlorella pyrenoidosa have been used in studies of the action of UV-irradiation during different stages in the life cycle on cell division capacity. These experiments show that there is a considerably higher sensitivity to UV-irradiation during the first hours of the life cycle. The variations in photoreactivating capacity of UV-damaged cells during a life cycle have been investigated. The results from these investigations show that the photoreactivating capacity varies and that it is higher in young daughter cells (autospores) and 12–15 hour cells. These variations can be due to variations in the activity of the photoreactivating enzyme during the life cycle.     

Effects of Indole-3-acetic Acid and Gibberellin on Synchronous Cultures of Chlorella fusca

The applicability of synchronous cultures of Chlorella fusca as a reproducible experimental system for the study of growth regulators has been investigated using indole-3-acetic acid (IAA) and gibberellin.

• 1 None of these compounds stimulated growth or sporulation.
• 2 IAA inhibited growth and sporulation at concentrations higher than 6 × 10^?5M, the effect increasing with increasing acidity. Gibberellin had no effect.

     

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana

By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions.     

Inhibition of Chlorella growth by degradation and related products of linoleic and linolenic acids and the possible significance of polyunsaturated fatty acids in phytoplankton ecology

The activity of the degradation products of linoleic and linolenic acids in inhibiting the growth of the green alga Chlorella pyrenoidosa was determined using the paper disk-agar plate method. Although hydroperoxides derived from these acids and aldehyde biodegradation products and other related aldehydes and alcohols showed inhibitory activity, it is concluded from the weakness of the activities that the inhibitory effects of polyunsaturated fatty acids(PUFAs) on Chlorella growth is due mainly to the acids themselves and not their degradation products. The evidence for the possible ecological role of PUFAs as toxic agents against phytoplankton is presented. This revised version was published online in August 2006 with corrections to the Cover Date.     

THE UPTAKE OF MANNITOL AND SORBITOL BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

Chlorella saccharophila (Krüger) Nadson takes up mannitol and sorbitol in the light and the dark. The rate of uptake is concentration dependent. is not affected by pH in the range pH 6.0 to 8.0 and ii not stimulated by light. Uptake is inhibited by the respiration inhibitor sodium azide (10^-2 M) but not by 3-(3,4-dichlorophenyl)-1,1-di-methyl urea (10^-6 M), an inhibitor of photosynthesis. Sorbitol. but not mannitol, stimulates the rate of dark respiration but both support the heterotrophic growth of the alga. Both compounds permeate the cells of C. miniata. and two strains of C. pyrenoidosa but do not support the heterotrophic growth of these algae. The cells of C. vulgaris are impermeable to both compounds.     

Activities of mitochondrial enzymes during aerobic synchronous growth of aerobically and anaerobically grownSaccharomyces cerevisiae

The mitochondrial enzymes eytoehrome-c: O₂-oxidoreductase (E.C.1.9.3.1), NADH: cytochrome-c-oxidoreductase (E.C.1.6.2.1), NADH: ferrioyanide oxidoreductase (E.C.1.6.2.99),l-malate hydrolyase (E.C.4.2.1.2) andl-malate: NADH-oxidoreductase (E.C.1.1.3.7) increase their activities during the aerobic synchronous growth of aerobically grownSaccharomyces cerevisiae in discrete steps and only once during the cell cycle. An identical phenomenon was observed during the aerobic synchronous growth of anaerobically grown yeast. The mechanism of completization of mitochondrial membranes is thus likely to be discontinuous and the same during both mitochondrial multiplication and the conversion of promitochondria to fully functioning mitochondria.     

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。     

Effect of light fluctuations on photosynthesis and metabolic flux in Synechocystis sp. PCC 6803

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds. In the present study, we investigated the metabolic response of Synechocystis sp. PCC 6803 to light fluctuations using ¹³C-metabolic flux analysis. The light intensity and duty ratio were adjusted such that the total number of photons or the light intensity during the low-light phase was equal. Light fluctuations affected cell growth and photosynthetic activity under the experimental conditions. However, metabolic flux distributions and cofactor production rates were not affected by the light fluctuations. Furthermore, the estimated ATP and NADPH production rates in the photosystems suggest that NADPH-consuming electron dissipation occurs under fluctuating light conditions. Although we focused on the water–water cycle as the electron dissipation path, no growth effect was observed in an flv3-disrupted strain under fluctuating light, suggesting that another path contributes to electron dissipation under these conditions.     

棕鞭藻对蛋白核小球藻的摄食及其营养策略

在研究稻草秸秆对蛋白核小球藻(Chlorella pyrenoidosa)的化感作用时,偶然发现一株对蛋白核小球藻具有强烈的取食作用的鞭毛虫,能有效抑制小球藻的大量繁殖。经形态学观察,该种鞭毛虫具有2片淡黄色色素体,片状、周生;虫体呈球形、椭圆形或卵形,能变形,具2根不等长的鞭毛,从细胞顶部伸出;长鞭毛约为虫体的1.5倍,短鞭毛约为虫体的0.5倍;对蛋白核小球藻的吞噬能力极强,每个虫体可以吞噬3~4个小球藻,虫体大小因吞噬物的多少变化较大。初步确定这种鞭毛虫为棕鞭藻(Ochromonas sp.)。在SE、稻草以及麦粒3种培养基中的种群生长表明,这种棕鞭藻在麦粒培养基最易培养,生长速度最快。结果表明,这种棕鞭藻为混合营养类型,但以吞噬营养为主。