Studies on Scenedesmus acutus growth. I effect of autotrophic and mixotrophic conditions on the growth of Scenedesmus acutus

Among sugars, glucose and mannose were found to be the most suitable substrates for mixotrophic growth, uptake of galactose and its influence on growth was negligible, and sucrose and fructose occupied intermediary positions. The optimum temperature for sugar uptake was 30 degrees C, both under light and in darkness. Enhancement in the photosynthetic oxygen-evolution rate, based on the utilization of substrates, was foremost in the presence of glucose, followed by mannose, sucrose, and fructose. Industrial by-products such as sugarcane molasses also were utilized to increase the algal growth under mixotrophic conditions. A maximum yield in biomass was obtained subsequent to the combined supply of sugarcane molasses with carbon dioxide to indoor as well as outdoor mixotrophic cultures. Doubling the carbon dioxide supply alone above a certain level, under autotrophic and mixotrophic outdoor conditions, did not produce a pronounced increase in the algal growth rate. The results on autotrophic and mixotrophic growth variations are discussed in the article.     

Glutamate dehydrogenase of the unicellular green alga Scenedesmus acutus

The coenzyme-non-specific glutamate dehydrogenase (EC 1.4.1.3) from Scenedesmus acutus in inhibited by p-hydroxymercuribenzoate only in the deamination reaction. From this result and from its stability in the presence of urea it is concluded that this enzyme exhibits and equilibrium between three conformations: aminating and deaminating conformations induced by NADH-2-oxoglutarate and NAD⁺-glutamate, respectively, and the “native” conformation in the absence of substrates.     

Studies on Scenedesmus acutus growth. The incorporation of 14C-glucose by Scenedesmus acutus under light and dark periods

Qualitative and quantitative estimations of alcohol-extractable compounds from (14)C-glucose-incorporated Scendesmus acutus cells were performed at various intervals of time. After two hours of incubation with (14)C-glucose in light, amino acids were synthesized at 65% and sucrose at 26% of the total input label. In the dark incorporation, 30% of the total radioactivity was found in amino acids and 46% in sucrose. Leucine and valine were detected only during the oxidation of glucose in light. The concentration of serine increased more in the presence of light, as compared to dark. These results on the oxidation of glucose are discussed in this article.     

Influence of light on plastocyanin formation in the alga Scenedesmus acutus

Scenedesmus acutus, pregrown autotrophically, forms high levels of plastocyanin during an initiation period of 24 hr in the light, after cupric ions have been added to depleted cells. In the dark, no plastocyanin formation is observed. This is in contrast to heterotrophic cells which, over the same period and under identical conditions, yield about half the plastocyanin level regardless of whether they are incubated in the dark or in the light. The ATP level is high in all cases when substantial formation of plastocyanin takes place, while the ³⁵S-sulfolipid level, the marker of thylakoid formation, is at variance. After the 24-hr plastocyanin formation period, the ³⁵S-sulfolipid level is high in illuminated autotrophic cells as well as in dark-incubated heterotrophic ones, but low in illuminated heterotrophic cells, all of them having substantial plastocyanin contents. Additional experiments on light dependence of copper uptake, influence of light quality, occurrence of apoplastocyanins, provide a set of data to assume that the role of light in plastocyanin formation is primarily through photophosphorylation, the ATP presumably being necessary for processing of plastocyanin precursors to holoprotein.     

Studies on Scenedesmus acutus growth II. Effect of autotrophic and mixotrophic growth on the amino acid and the carbohydrate composition of Scenedesmus acutus

As a general rule an increase in carbohydrates occurs during the light phase of the cell cycle and that of protein during phase, although variations were found in these components under autotrophic and mixotrophic growth conditions. The results are based on the quantitative determination of carvohydrates as trimethylsilyl (TMS) derivatives and amino acids as N-trifluoroacetyl-n-butyl (TAB) esters in algal cells cultured in light and dark periods by gas-liquid chromatography (LC). Cells harvested during the dark period contained more amino acids as compared to similar cultures harvested during the light phase. In light, the production of amino acids of the aspartate family increased in cells cultivated with glucose and carbon dioxide. With glucose as sole carbon source, the carbohydrate content was higher in the dark than in the light period. Under continuous light conditions, in the presence of carbon dioxide, there was a decrease in the carbohydrate content also. Gas-liquid chromatography analysis of the extract of the purified cell walls showed that they are made up of 0.076% carbohydrates and 0.28% amino acids on the dry weight (DW) basis of whole cells. The results on the metabolism of cells, under autotrophic and mixotrophic conditions, are discussed in this article.     

Bioenergetic analysis of mixotrophic growth in Chlorella vulgaris and Scenedesmus acutus

The specific growth rate of Chlorella vulgaris in the presence of glucose (mixotroph) was always larger than that in autotroph when the light intensity was less than 10 klux. However, Scenedesmus acutus behaved differently, i.e., when the light intensity was more than 6 klux, the specific growth rate in mixotroph became smaller than that in autotroph. Cellular contents of chlorophyll a and b in Scenedesmus acutus that deteriorated more markedly in mixotroph than those of Chlorella vulgaris could account for these different growth behaviors. In other words, mechanisms relevant to the photosynthesis and the oxidative phosphorylation of glucose seem to function independently with respect to Chlorella Vulugaris.     

Electron-dense Regions in Dividing Mitochondria of a Green Alga, Scenedesmus acutus

The dividing and the constricted regions of mitochondria ina green alga, Scenedesmus acutus , were studied by electronmicroscopy. Electron-dense substances were always visible inthese specific regions of mitochondria that had been fixed byfreeze-substitution. Thin fibres, which seemed to surround theconstricted regions, were also seen. The possibility that theelectron-dense regions constitute part of the dividing apparatusof the mitochondria is discussed.Copyright 1993, 1999 AcademicPress Division apparatus, division of mitochondria, electron microscopy, mitochondria, Scenedesmus     

Triacylglycerol content,productivity and fatty acid profile in Scenedesmus acutus PVUW12

A detailed lipid characterization of Scenedesmus acutus PVUW12, with emphasis on the evaluation of triacylglycerols (TAGs) as a biodiesel feedstock, is presented. When algal cells were grown in nitrogen-free medium (N stress), a lipid increase was detected that was mainly due to TAG accumulation. In situ fluorescence measurements allowed the kinetics and extent of neutral lipid accumulation to be followed. Under N stress, the productivity of total lipids and TAGs increased significantly (80.99 and 63.74 mg L^?1 day^?1, respectively) compared with controls (29.51 and 16.23 mg L^?1 day^?1, respectively). Monounsaturated fatty acids were the major fraction and increased further (49.74 %) in stressed cells, with oleic acid as the most abundant compound (46.97 %). The polyunsaturated fatty acid composition of this algal oil appears to meet the European Standard EN 14214. These results indicate that S. acutus oil meets the requirements for its use as a biodiesel feedstock. Since this strain was also proposed for wastewater bioremediation, this opens up the possibility of its use in an integrated system combined with biofuel production.     

Zooplankton-induced unicell-colony transformation in Scenedesmus acutus and its effect on growth of herbivore Daphnia

The effects of colonial ecomorphs of the green alga Scenedesmus acutus on growth of Daphnia cucullata and D. pulex were examined. In ecologically relevant densities (up to 200 animals l^-1) the relatively small D. cucullata did not induce colony formation in Scenedesmus acutus, whereas the larger congener D. pulex significantly promoted colony formation. Both clearance rate and population growth rate (r) were significantly lower in D. cucullata when fed colonial Scenedesmus then when fed unicellular food. However, for D. pulex no effects of food type were observed. These results show that large Daphnia may influence its food in such a way that smaller congeners and competitors are negatively affected.     

Cytoskeleton mediating transport between the ER system and the Golgi apparatus in the green alga Scenedesmus acutus

In the green alga Scenedesmus acutus, Golgi bodies are located near the nucleus and supplied with transition vesicles that bud from the outer nuclear envelope membrane. Using this alga, we have shown previously that thiamine pyrophosphatase (TPPase), a marker enzyme of Golgi bodies, migrates in vesicles from the Golgi bodies to the ER via the nuclear envelope in the presence of BFA (Noguchi et al., Protoplasma 201, 202-212, 1998). In this study we demonstrate that both cytochalasin B and oryzalin (microtubule-disrupting agent) inhibit the BFA-induced migration of TPPase from Golgi bodies to the nuclear envelope. However, only actin filaments--not microtubules--can be detected between the nuclear envelope and the Golgi bodies in both BFA-treated and untreated cells. These observations suggest that actin filaments mediate the BFA-induced retrograde transport of vesicles. This mechanism differs from that found in mammalian cells, in which microtubules mediate BFA-induced retrograde transport by the elongation of membrane tubules from the Golgi cisternae. We also discuss the non-participation of the cytoskeleton in anterograde transport from the nuclear envelope to the Golgi bodies.     

Stimulation of lactobacilli by an aqueous extract of the green alga Scenedesmus acutus 276-3a

Summary The hot-water extract of Scenedesmus acutus 276-3a enhances acid formation by Lactobacillus casei var. sbirota, Streptococcus lactis and Streptococcus thermophilus appreciably. At least in S. lactis the stimulation is not caused by mineral constituents such as Mn²⁺. In order to facilitate screening, a quick test in tubes was used. Stimulation of lactobacilli by algal extract is also demonstrated in tests on agar plates containing TTC. Bioautography of algal extract fractions on TTC plates enabled adenine and hypoxanthine to be identified as growth factors in algal extract which contributed towards the stimulation of S. lactis.Dedicated to Professor Dr. Wilhelm Halbsgut on the occasion of his 65th birthday     

Isolation and characterization of a Scenedesmus acutus strain to be used for bioremediation of urban wastewater

Green microalgae, due to their short growth cycle and to their ability to photosynthetically fix carbon dioxide producing an oil-rich biomass, have been proposed as an attractive alternative feedstock for the production of “second generation” biofuels. However, it has been anticipated that owing to their ability to colonize very different environments characterized by high levels of nitrogen, they can also be good candidates for bioremediation, thus integrating environmental protection with sustainable biomass production. We have isolated a strain belonging to Scenedesmus genus from urban wastewater. This isolate, Scenedesmus acutus PVUW12, was tested for its ability to grow and actively deplete eutrophicating inorganic molecules present in wastewater. In order to test its biomass productivity, the PVUW12 strain was grown in a vertical-column photobioreactor using standard growth medium obtaining a maximal productivity of 0.3?g dry weight L^?1?d. When the same strain was grown in the photobioreactor filled with wastewater collected from the final step of the local urban purifier plant containing 18.8?mg?L^?1 nitrate, we observed complete nitrogen removal coupled with a biomass production of about 0.74?g dry weight L^?1 within 3 days. After 10?days, the recovered biomass was analyzed for triglyceride content which was found to be 9.3% of the dry biomass. However, when algal cells were left for additional 10?days in static conditions the triglyceride content increased to 28.8%. These data show that this Scenedesmus strain can be used for wastewater bioremediation producing a biomass suitable for energy production.     

Morpho-physiological aspects of Scenedesmus acutus PVUW12 cultivated with a dairy industry waste and after starvation

Among green microalgae, Scenedesmus sp. is known for its potential in wastewater remediation and lipid production, especially under starvation. Moreover, it is often characterised by a mixotrophic metabolism. In this work, we cultivated S. acutus PVUW12 in the presence of a liquid fraction of scotta (LFS), a cheese whey by-product, as source of nutrients. Subsequently, cultures were starved to evaluate lipid production. Cells were analysed to obtain information about growth, nutrient consumption during LFS cultivation, morphology and photosynthetic efficiency. We found that the alga boosted its growth when cultured in presence of LFS. Production of stromatic starch grains, polyphosphate granules, cell wall enlargement and reduction of the photosynthetic efficiency were also induced. Massive lipid accumulation was observed only during starvation, which also induced a strong slowdown of growth, loss of polyphosphate grains and further decrease in photosynthetic efficiency. This study demonstrates that S. acutus PVUW12 can be involved in a two-step cultivation, first by promoting growth using a by-product from cheese industry and second by transferring the microalgae on starvation to induce lipid accumulation for bioenergetics purposes.     

Isolation and characterization of soluble cytochrome c-553 and membrane-bound cytochrome f-553 from thylakoids of the green alga Scenedesmus acutus

Soluble cytochrome c-553 and membrane-bound cytochrome f-553 from the alga Scenedesmus acutus were purified to apparent homogeneity. The properties of cytochrome c-553 are comparable to preparations obtained from other eukaryotic algae, whereas the thylakoid-bound species resembles higher plant cytochrome f. Common characteristics are: 1. An asymmetrical alpha-band at 553 nm. 2. A midpoint redox potential of +38 MV (pH 7.0), with a pH dependency above pH 8.0 of -60mV/pH unit. 3. Formation of a pyridine hemochromogen with a maximum at 550 nm; no adducts with CN- or CO are observed. Distinguishing features are: 1. Cytochrome f-553 has a more complicated beta-band, with maxima at 531.5 and 524 nm, and hence a more complex low-temperature spectrum. Also the positions of the gamma- and delta-bank at 421.5 and 331 nm, respectively, distinguish cytochrome f-553 from cytochrome c-553, with gamma- and delta-bands at 416 and 318 nm. 2. The ferricytochrome c-553 spectrum exhibits a weak band at 692 nm, which is not observed with cytochrome f.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Role of oxidative stress and thiol antioxidant enzymes in nickel toxicity and resistance in strains of the green alga Scenedesmus acutus f. alternans.

Treatment with Ni(NO3)2 leads to the formation of reactive oxygen species (ROS) in the green alga Scenedesmus acutus f. alternans, causing lipid peroxidation. This effect was stronger in a Ni-sensitive strain, UTEX72, than in a Ni-resistant strain, B4. In the resistant strain, Ni induced an increased ratio of reduced to oxidized glutathione (GSH:GSSG), whereas it caused a lowered ratio in the sensitive strain. Enzymes involved in the control of ROS were studied in these strains as well as two others that have shown different degrees of nickel resistance. The resistant strain, B4, which grows while containing large amounts of internal Ni, had much higher levels of glutathione reductase and catalase than the other strains. The sensitive strain, UTEX72, had higher levels of glutathione peroxidase, superoxide dismutase, and glucose-6-phosphate dehydrogenase than did strain B4. The resistant strains, Ni-Tol and Cu-Tol, derived from strain UTEX72, which are partly able to exclude Ni, had enzyme profiles that resembled that of UTEX72 more closely than that of B4. Treatment with 10 and 100 microM Ni for 4 or 22 h had complex effects on enzyme levels in all four strains. Ni decreased glutathione reductase in B4, slightly increased it in Ni-Tol and Cu-Tol, and did not affect the low levels of this enzyme in UTEX72. Ni lowered glutathione peroxidase in B4 and either did not affect it or slightly raised it in the other strains. Ni lowered catalase in B4 and did not affect the other strains. Superoxide dismutase was raised in B4 and Ni-Tol and lowered in Cu-Tol and UTEX72, and glucose-6-phosphate dehydrogenase was lowered in all four strains. These results suggest that one major mechanism of Ni resistance, especially in strain B4, may be the ability to combat the formation of ROS when exposed to this metal, likely by maintaining a high GSH:GSSG ratio.     

Comparative study of nickel toxicity to growth and photosynthesis in nickel-resistant and -sensitive strains of Scenedesmus acutus f. alternans (Chlorophyceae)

Nickel (Ni) toxicity to growth and photosynthesis was studied in four strains of Scenedesmus acutus f. alternans. Effects of Ni dosage and duration of exposure on growth and photosynthesis were strain specific. Large differences in responses of both growth and photosynthesis to Ni were detected between three resistant strains (B4, Cu-Tol, and Ni-Tol) and one sensitive strain (UTEX 72). Growth of UTEX 72 was 18 times more sensitive to Ni than those of the three resistant strains. The order of Ni dosages (fmol Ni/pg cell dry weight) causing 50% inhibition (D₁₅₀) of growth rates in the four strains was Ni-Tol (10.5) > B4 (8.19) > Cu-Tol (4.60) > UTEX 72 (0.25). The effect of Ni dosage on photosynthetic rate as percentage of control corresponded to a saturation curve and was a strong function of duration of exposure. The DI₅₀s of photosynthetic rates were 3.5 times lower in UTEX 72 than in the three resistant strains, and in all four strains they decreased sharply with the increase in duration of exposure. The order of the four strains in DI₅₀s of photosynthetic rate was B4 (58.2) > Cu-Tol (38.0) > Ni-Tol (28.9) > UTEX 72 (8.24) for 6-h exposure and Ni-Tol (2.88) > Cu-Tol (1.30) > B4 (1.01) > UTEX 72 (0.15) for 24-h exposure. The DI₅₀s of photosynthetic rate for 6-h exposure were higher than those of growth rate in all four strains, and for 24-h exposure they were lower, except in UTEX 72. Thus, the relative Ni sensitivity of growth and photosynthesis of the four strains depends on the duration of exposure. The results of factorial analysis of variance suggested that Ni toxicity to photosynthesis is a consequence of a strong interaction among strain, Ni dosage, and duration of exposure.