Biostimulant activity of humic-like substances from agro-industrial waste on Chlorella vulgaris and Scenedesmus quadricauda

The use of microalgae in a number of sectors, including biodiesel, feed and food production, is proving to be of great interest. An evaluation was made of the possible biostimulant effects on Chlorella vulgaris and Scenedesmus quadricauda of humic-like substances (HLs) extracted from agro-industrial wastes. These included digestate from the waste of an agro-livestock farm (D-HL), oil extraction residues from rape (B-HL, Brassica napus L.) and tomato residues (T-HL). The microalgal growth medium (BG11) was supplemented with HLs to evaluate their effect on biomass yield as well as carbohydrate, chlorophylls a and b, lipid and fatty acid contents. Our results showed that the HLs used in the test are effective biostimulants of C. vulgaris and S. quadricauda. The biostimulant effect seems to depend on the type of extract used for cultivating the microalgae, the concentration and the species treated. Among the extracts applied to the growth medium, D-HL and T-HL seem to have a significant effect on microalgal biomass and lipid production. Although B-HL showed no significant effect on the biomass and lipid content of C. vulgaris and S. quadricauda, its presence in the growth medium increased the saturated:unsaturated fatty acid ratio (SFA/UFA) and stimulated the sugar metabolism of the microalgae by increasing their carbohydrate and chlorophyll content.     

Variations temporelles de l'ultrastructure de Scenedesmus quadricauda exposée au cuivre lors d'une expérience à long terme

An experiment was conducted with a continuous culture of Scenedesmus quadricauda for 50 days in a copper concentration of 250 μg l⁻¹. Ultrastructural examinations were carried out at different times to obtain the sequence of events. After 1.5 h of exposure, the ultrastructural effects were obvious: the chloroplasts were retracted and all cell structures appeared diffuse. The same observations were reported after 3, 6, 12 and 24 hours. Between the 3rd and 15th days of the experiment, the cells showed major accumulations of starch, lipid and electron-dense inclusions in the vacuoles, and the cell walls became thicker. After 22,32 and 50 days the cells appeared normal, but the cytoplasm was densely packed with ribosomes and the endoplasmic reticulum was evident in most cells. However, atomic absorption analysis for copper showed that there was no bio-accumulation after 50 days of exposure. These findings suggest the implication of a second tolerance mechanism to copper (possibly extracellular complexation or removal) that is activated after long exposure, in contrast to electron-dense inclusions, which appear after a relatively short time (2 days).     

Bioassays using Stigeoclonium tenue Kütz. and Scenedesmus quadricauda (Turp.) Bréb. as testorganisms; a comparative study

Three different bioassays, two culture tube test methods with respectively Stigeoclonium tenue or Scenedesmus quadricauda and one bottle test with S. quadricauda, were compared. The yields obtained in the various tests were linearily correlated (r = 0.86, P < 0.001). The same primary limiting nutrient was indicated by the bioassays in most cases. However the algal growth in the tube test using Stigeoclonium was more often P-limited. In the case of S. quadricauda both test methods (tube and bottle) were nearly equally effective. The yields of N-limited samples were significantly correlated with the inorganic-N as well as total-N concentration of the water sample. A significant correlation of the ortho-P as well as total-P concentration with the yield of the P-limited assays was only found for Stigeoclonium tenue. The critical total N/P ratio (by weight) for N or P limitation was approximately 17: 1 for Stigeoclonium tenue and 22: 1 for Scenedesmus quadricauda.     

Effects of phenolic compounds on growth and metabolic activities of Chlorella vulgaris and Scenedesmus bijugatus isolated from soil

Phenol and three nitrophenols (o-nitrophenol, m-nitrophenol, p-nitrophenol), commonly occurring pollutants in natural eco-systems, were tested for their toxic effects on soil isolates of Chlorella vulgaris and Scenedesmus bijugatus, growing under phototrophic, photoheterotrophic and heterotrophic conditions. The toxicity criteria included cell number, chlorophyll, total protein and carbohydrate content, ¹⁴CO₂ uptake and in vivo nitrate reductase activity. Both C. vulgaris and S. bijugatus were sensitive to the pollutants when the cultures were grown under phototrophic or heterotrophic conditions. However, the toxicity was found reversed or alleviated upon photoheterotrophic growth of the cultures. Transmission electron microscope studies revealed various cytological abnormalities in C. vulgaris in the presence of phenolics at algistatic levels.     

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.     

The effects of 5‐azacytidine and cadmium on global 5‐methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S‐adenosyl‐L‐methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5‐azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S‐adenosylhomocysteine, 5′‐methylthioadenosine and global 5‐methylcytosine (5‐mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5‐mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5‐azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5‐mC content of C. reinhardtii was 1%–1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S‐adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.     

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.     

Regiospecific reduction of adrenosterone to 11-ketotestosterone by microalga T76 Scenedesmus quadricauda

Summary Adrenosterone is reduced to 11-ketotestosterone by cultures of microalga Scenedesmus quadricauda. The best bioconversion yields (62%) were obtained in cultures containing 3×10⁶ cells/mL of alga and 100 mg of adrenosterone in 400 mL of medium after 10 days at 24°C with a photoperiod light - dark 16:8 h.     

Increase of Copper Toxicity to Growth of Chlorella vulgaris with Increase of Light Intensity

Abstract In the absence of inhibitory concentrations of copper, the photoautotrophic growth of Chlorella vulgaris INETI58C at 27°C exhibited a higher specific growth rate and reached a higher maximal concentration of biomass, under irradiance of 150 W m⁻², compared with 100 W m⁻². However, when the mineral growth medium was supplemented with CuSO₄ (range 40–80 μM), algal growth was significantly affected at the higher light intensity. In the presence of Cu²⁺, the increase in dry biomass was uncoupled from the increase in cell concentration since more than 16 autospores gathered together, inside the enlarged mother cell, suggesting that copper arrested the normal bursting of the mother cell wall. At the higher irradiance, growth medium supplementation with 80 μM of CuSO₄ led to bleaching of photosynthetic pigments. No growth was observed, while, under the lower irradiance, growth was only slightly inhibited. Results clearly showed that copper toxicity to growth of C. vulgaris was strongly influenced by light intensity. Higher light intensity elicits lethal or sublethal Cu²⁺ damage at concentrations lower than the threshold level for damage at lower light intensities. Cu²⁺ may elicit lethal or sublethal light damage at irradiances lower than the threshold level for unpolluted aquatic systems. Received: 17 January 1997; Accepted 15 April 1997     

Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l⁻¹ day⁻¹. We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l⁻¹ day⁻¹, biomass density was 5.7 g l⁻¹ dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.     

The Effects of Some Chelating Agents and Their Copper Complexes on Photosynthesis in Scenedesmus quadricauda

The effects on apparent photosynthesis in the green alga Scenedesmus quadricauda of four chelating substances, 8-hydroxyquinoline (= oxine), β-isopropyltropolone (=β-thujaplicin), sodium dimethyldithiocarbamate and 3-(β-hydroxyethyl)-C-pentamethylene-3-aza-dithiocarbamate have been investigated. The first three compounds mentioned had all an inhibitory effect on photosynthesis, measured as oxygen production with a manometric, a volumetric or a polarographic method. In combination with certain concentrations of CuSO₄ bimodal dose response curves were observed, similar to those obtained on the growth of different microorganisms. Calculations of the amounts of different complexes that appeared in the nutrient solutions during the experiments are compared with the recorded degrees of inhibition. The results strongly indicate that the 1: 1-complexes between Cu²⁺ and the chelating substances are the most toxic agents at low concentrations of these compounds.     

Antioxidant responses of microalgal species to pyrene

The antioxidant response of four freshwater microalgal species, Chlorella vulgaris Beij., Scenedesmus platydiscus (G. M. Smith) Chod., Scenedesmus quadricauda(Turp.) Bréb., and Selenastrum capricornutum Printz without pyrene addition (control) and at two pyrene concentrations (0.1 and 1.0 mgL⁻¹) were investigated. Under the control condition, the values of the antioxidant parameters differed significantly among species and the difference was seemed not to be related to their susceptibility to pyrene. The antioxidant response to pyrene treatments also varied from species to species. Pyrene led to a significant increase in total glutathione (GSH) content in all species except C. vulgaris, a species did not exhibit any ability to metabolize pyrene. The glutathione-S-transferase (GST) activities also remained unchanged in pyrene treated C. vulgaris, increased greatly in S. platydiscus and Se. capricornutum (the two species with higher pyrene metabolism ability), but inhibited remarkably in S. quadricauda (the only species sensitive to pyrene toxicity). On the other hand, the glutathione reductase (GR) activities increased in C. vulgaris but remained at a similar level as the control in the other three species. The malondialdehyde (MDA) content, an indicator of lipid peroxidation, declined in S. quadricauda but showed no significant change in the other three species. The activities of glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) in pyrene treated cells remained almost the same as the controls for all microalgal species. Results suggested that the alterations of antioxidant systems in microalgae might not be useful indicators of pyrene exposure but pyrene-enhanced GSH metabolism might be important in pyrene biotransformation.     

Influence of mixing and shear stress on Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii

Photosynthetic activity (PA) and growth of different microalgae species (Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii) depends in addition to other factors on mixing (tip speed) and shear stress (friction velocity) and was studied in a stirring tank (microcosm). In order to detect cause–effect relationships for an increase in photosynthetic activity, experiments were conducted under different pH values (6.0–8.5) and CO₂ concentrations (0.038 and 4 % (v/v)). The PA was determined as the effective quantum yield by pulse amplitude modulation during a stepwise increase of the tip speed from 0 to 589 cm s^?1 (friction velocity: 0–6.05 cm s^?1) in short-term experiments. The increase caused a distinctive pattern of PA of each species. Compared to 0 cm s^?1, C. vulgaris and S. obliquus showed a 4.0 and 4.8 % higher PA at the optimum tip speed of 126 cm s^?1 (friction velocity of 2.09 cm s^?1) and a 48 and 71 % higher growth, respectively. At 203 cm s^?1, the PA dropped to the value of the unstirred control, while at 589 cm s^?1, the PA decreased of up to 7 and 8 %. In contrast, C. reinhardtii showed 7 % stronger growth at 126 cm s^?1, while the PA decreased about 15 % at an increase of tip speed to 589 cm s^?1. For all investigated microalgae, the pattern of PA and higher growth was not only explained by the main contributing factors like light supply, nutrient supply, and overcoming diffusion gradients. The results indicate that hydrodynamic forces have a stimulating effect on the physiological processes within the cells.     

Growth of Chlorella vulgaris and associated bacteria in photobioreactors

The aim of this study was to test three flat plate photobioreactor configurations for growth of Chlorella vulgaris under non‐axenic conditions and to characterize and quantify associated bacterial communities. The photobioreactor cultivations were conducted using tap water‐based media to introduce background bacterial population. Growth of algae was monitored over time with three independent methods. Additionally, the quantity and quality of eukaryotes and bacteria were analysed using culture‐independent molecular tools based on denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative polymerase chain reaction (QPCR). Static mixers used in the flat plate photobioreactors did not generally enhance the growth at the low light intensities used. The maximum biomass concentration and maximum specific growth rate were 1.0 g l^?1 and 2.0 day^?1 respectively. Bacterial growth as determined by QPCR was associated with the growth of C. vulgaris. Based on PCR‐DGGE, bacteria in the cultures mainly originated from the tap water. Bacterial community profiles were diverse but reproducible in all flat plate cultures. Most prominent bacteria in the C. vulgaris cultures belonged to the class Alphaproteobacteria and especially to the genus Sphingomonas. Analysis of the diversity of non‐photosynthetic microorganisms in algal mass cultures can provide useful information on the public health aspects and unravel community interactions.     

Tolerance for Prolonged Darkness of Three Phytoplankton Species, Microcystis aeruginosa (Cyanophyceae), Scenedesmus quadricauda (Chlorophyceae), and Melosira ambigua (Bacillariophyceae)

Phytoplankton community dynamics are affected not only by atural events such as overwintering but also by artificial events such as artificial circulation and related darkness conditions. In order to clarify the effect of tolerance for prolonged darkness on community succession, laboratory cultures of three phytoplankton taxa, Microcystis aeruginosa(Cyanophyceae), Scenedesmus quadricauda(Chlorophyceae), and Melosira ambigua(Bacillariophyceae) were carried out in darkness. The period of darkness was varied: 5, 10, 15, 20 days, and the control. Thereafter, all samples were reilluminated. After more than 10 days of darkness, M. aeruginosa decreased markedly with the length of the darkness period and was reduced to only 1% of the initial cell number after 20 days darkness. In contrast, S. quadricauda and M. ambigua retained their biomass even after 20 days of darkness. After restarting the light–dark cycle, however, all three species similarly increased exponentially and reached their maximum biomass levels. These results suggest that differences in tolerance for prolonged darkness may cause the succession of the phytoplankton under certain conditions.     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998     

Blockade of heavy metals accumulation in Chlorella vulgaris cells by 24-epibrassinolide

This study was conducted on the influence of 24-epibrassinolide (24-epiBL) mixed with varying concentrations of heavy metals (copper, lead, cadmium, zinc) upon the growth and accumulation of these heavy metals in the cell of the alga Chlorella vulgaris Beijerinck (Chlorophyceae). Heavy metals at the concentration of 10^–3 M, alone or mixed with 24-epiBL, showed a lethal effect on C. vulgaris. At metal concentrations of 10^–6–10^–4 M, a combination with 24-epiBL appeared to have a stronger stimulatory effect on a number of cells than a single metal (a stronger inhibitory effect). 24-EpiBL at the concentration of 10^–8 M in combination with heavy metals (in the range 10^–6–10^–4 M) blocked metal accumulation in algal cells. 24-EpiBL has an anti-stress effect on C. vulgaris contaminated by heavy metals. The inhibitory effect on metal accumulation of 24-epiBL mixed with different heavy metals was arranged in the following order: zinc > cadmium > lead > copper. This process is correlated with the stimulation of growth of C. vulgaris. The stimulatory effect of 24-epiBL mixed with heavy metals leading to an increased pH in the medium (5.28–6.20) was significantly higher than the impact due to the increased acidity in the medium due to metals alone (pH 3.10–5.85). Lower pH increased the toxicity of heavy metals in C. vulgaris cells.     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Carbon dioxide biofixation by Chlorella vulgaris at different CO2 concentrations and light intensities

In order to develop an effective CO₂ mitigation process using microalgae for potential industrial application, the growth and physiological activity of Chlorella vulgaris in photobioreactor cultures were studied. C. vulgaris was grown at two CO₂ concentrations (2 and 13% of CO₂ v/v) and at three incident light intensities (50, 120 and 180 μmol m^?2 s^?1) for 9 days. The measured specific growth rate was similar under all conditions tested but an increase in light intensity and CO₂ concentration affected the biomass and cell concentrations. Although carbon limitation was observed at 2% CO₂, similar cellular composition was measured in both conditions. Light limitation induced a net change in the growth behavior of C. vulgaris. Nitrogen limitation seemed to decrease the nitrogen quota of the cells and rise the intracellular carbon:nitrogen ratio. Exopolysaccharide production per cell appeared to be affected by light intensity. In order to avoid underestimation of the CO₂ biofixation rate of the microalgae, exopolysaccharide production was taken into account. The maximum CO₂ removal rate (0.98 g CO₂ L^?1 d^?1) and the highest biomass concentration (4.14 g DW L^?1) were determined at 13% (v/v) CO₂ and 180 μmol m^?2 s^?1. Our results show that C. vulgaris has a real potential for industrial CO₂ remediation.     

Adsorption of Zinc Ions by Scenedesmus Obliquus and S. Quadricauda and its Effect on Growth and Metabolism

Zinc adsorption by two green algae, Scenedesmus obliquus and Scenedesmus quadricauda, was investigated. The maximum adsorption capability of zinc ion obtained from the Langmuir adsorption isotherms was higher for S. obliquus (6.67) than for S. quadricauda (5.03), and S. obliquus was more tolerant to zinc phytotoxicity than S. quadricauda. Lower concentrations of zinc increased dry mass, chlorophylls a and b, carotenoids, and total amino acid contents in both algae. On the other hand, higher concentrations of zinc were inhibitory for growth and the other metabolic activities in a concentration dependent manner.