Effects of Oil Refinery Effluents on Selenastrum capricornutum PRINTZ

Laboratory-based bioassay experiments using Selenastrum capricornutum PRINTZ as the test organism were conducted to evaluate the potentiality of refinery effluents to sustain algal growth. The raw effluents were remarkably toxic to the test alga, but satisfactory algal growth occurred in (diluted) 0.01 % effluents. The effluents from subsequent treatment stages required much less dilution to eliminate the toxicity. This study implicates oil and phenol in the reduction of algal growth in refinery effluents. A highly significant negative correlation was found between the final yield of the test alga and the concentration of oil or phenol in the culture suspension. The bioassay experiments therefore point to the toxic nature of refinery effluents. On the basis of these findings it is recommended that proper dilution of refinery effluents is necessary before they are discharged into any body of water.     

Photosynthetic Effect in Selenastrum capricornutum Progeny after Carbon-Ion Irradiation

A large proportion of mutants with altered pigment features have been obtained via exposure to heavy-ion beams, a technique that is efficient for trait improvement in the breeding of plants and algae. However, little is known about the underlying mechanisms by which the photosynthetic pigments are altered by heavy-ion irradiation. In our study, the photosynthetic characteristics of progenies from carbon-ion irradiated Selenastrum capricornutum were investigated. Five progenies deficient in chlorophyll a were isolated after carbon-ion exposure. Photosynthetic characteristics, photoprotection capacity and gene expression of the light-harvesting complex in these progenies were further characterized by the measurement of chlorophyll fluorescence parameters (Fv/Fm, ФPSII, NPQ, ETR), the de-epoxidation state of the xanthophyll cycle, the amount of lutein and quantitative real-time PCR. High maximum quantum yield of photosystem II at day 10 and high thermal dissipation ability were observed in progenies #23 and #37 under normal culture condition. Progenies #18, #19 and #20 showed stronger resistance against high levels of light steps than the control group (612–1077 μmol photons m ^-2 s ^-1, p< 0.05). The progenies #20 and #23 exhibited strong photoprotection by thermal dissipation and quenching of ³Chl* after 24 h of high light treatment. The mRNA levels of Lhcb5, Lhcbm5 and Lhcbm1 of the light-harvesting complex revealed markedly differential expression in the five progenies irradiated by carbon-ion beams. This work indicates that photosynthetic efficiency, photoprotection ability and the expression of light-harvesting antennae in unicellular green algae can be markedly influenced by irradiation. To our knowledge, this is the first report on changes in the photosynthetic pigments of green algae after treatment with carbon-ion beams.     

Round Robin testing with the Selenastrum capricornutum microplate toxicity assay

Three Quebec-based ecotoxicological laboratories participated in an intercalibration exercise to assess the performance of a recently-published cost-efficient algal microplate toxicity assay. Three test series were carried out with six operators (2 from each laboratory) and two reference toxicants (Cd²⁺ as CdCl₂ and phenol). Variables included algal cultivation technique (series 1), presence or absence of Na₂EDTA in the growth medium (series 2), and passive or active gas exchange during incubation (series 3). Control growth variability conferred an overall test precision of 8.7% (coefficient of variation obtained for 204 microplate tests). Cadmium (96 h EC50 = 56 µg · l^–1) and phenol (96 h EC50 = 69.7 mg · l^–1) toxicity test reproducibility was reflected by coefficients of variation of 24.3% and 34.9%, respectively. Algal cultivation technique, whether standardized or in house, had no effect on toxicity results. Na₂EDTA, as part of the growth medium, significantly ameliorated algal growth and toxicity. While active gas exchange during microplate incubation significantly improved growth, toxicity results were unaffected. Phenol volatility was found to have a marked influence on algal growth. This effect can be offset, however, by providing appropriate modifications to better seal individual wells and to improve experimental design.     

Small-scale fluid motion mediates growth and nutrient uptake of Selenastrum capricornutum

1. A fluid‐flow reactor using submersible speakers was constructed to generate small‐scale fluid motion similar to conditions measured in open water environments; flow was quantified by particle image velocimetry. Additionally a Couette‐type rotating cylinder was used to generate shear flows; flow was quantified using an optical hotwire probe and torque measurements. Growth rates of the green alga Selenastrum capricornutum were determined from changes in cell counts and viability was tested using the fluorogenic probe fluoresceine diacetate. 2. Evidence that fluid motion directly affects growth rates was obtained as a significant difference between growth in a moving versus non‐moving fluid. A near 2‐fold increase in growth rate was achieved for an energy dissipation rate of ? = 10^?7 m² s^?3; a rate common in lakes and oceans. The onset of the viability equilibrium, identified as the day of the test period when the number of active cells equalled non‐active cells, was delayed by 2 days for moving fluid conditions as compared with a non‐moving fluid. 3. Nutrient uptake was determined by a decrease in the bulk fluid concentration and cellular phosphorus concentration was also estimated. The thickness of the diffusive sublayer surrounding a cell, a zone dominated by molecular diffusion, was estimated. Increasing fluid motion was found to decrease the thickness of this layer. The Sherwood number (ratio of total mass flux to molecular mass flux) showed that advective flux surrounding cells dominated molecular diffusion flux with regard to Péclet numbers (ratio of advective transport to molecular diffusion transport). Fluid motion facilitated uptake rates and resulted in increased growth rates, compared with no‐flow conditions. The rate‐of‐rotation and the rate‐of‐strain in a moving fluid equally mediated the diffusive sublayer thickness surrounding the cells. Our study demonstrates that small‐scale fluid motion mediates algal growth kinetics and therefore should be included in predictive models for algal blooms.     

Adenosine Triphosphate Pools in Methanobacterium

Certain aspects of adenosine triphosphate (ATP) metabolism in the strict anaerobe Methanobacterium strain M.o.H. have been investigated. Results of growth yield studies suggest that ATP conservation is very inefficient (0.06 mole of ATP per mole of hydrogen) under the conditions used to grow the bacterium in a fermentor. Experiments designed to demonstrate net ATP formation in cell-free extracts were negative. In whole-cell studies, substances which decreased ATP pool levels and increased adenosine monophosphate (AMP) pool levels were air, chloroform, 2,4-dinitrophenol, carbonylcyanide-m-chlorophenylhydrazone, and pentachlorophenol. The results suggest that the latter compounds act either as inhibitors of electron transport or as uncouplers of an energy-linked process. All the above compounds also inhibit methane formation in cell-free extracts, an ATP-requiring process. Methods are described for estimation of ATP, adenosine diphosphate (ADP), and AMP in whole cells, with a sensitivity in the range of 10 to 200 pmoles. An apparatus for quick sampling from an anaerobic suspension of whole cells also is described.     

Inhibition of Glycolysis by Adenosine Triphosphate

The effect of Adenosine triphosphate in inhibiting glycolysisin mitochondrial extracts from pea cotyledons was studied. Itwas possible to show a direct inhibition of adenosine triphosphateon glyceraldehyde phosphate dehydrogenase and on alcohol dehydrogenase.This inhibition was sufficient to explain the inhibition ofglycolytic phosphorlation observed previously.     

Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum

Extracts from the unicellular green alga Selenastrum capricornutum exhibit high superoxide dismutase activity, but only traces of catalase activity. The excess hydrogen peroxide (HO) generated by the superoxide dismutase in S. capricornutum may be degraded by a unique peroxidase. This peroxidase has a high specificity for ascorbate as its electron donor. The enzyme has an optimum pH at 8, is insensitive to cyanide and is inhibited by oxine. Addition of low concentrations of copper to algal cultures stimulates the peroxidase activity threefold. This enzymatic system could be used as a sensitive bioindicator for copper in fresh water.     

Quantitative Extraction of Adenosine Triphosphate from Cultivable and Host-Grown Microbes: Calculation of Adenosine Triphosphate Pools

Existing data on adenosine triphosphate (ATP) pools in microbes are deficient for two reasons: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei, and Mycobacterium lepraemurium. Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time (E. coli, 3 min; M. phlei, 5 min; M. lepraemurium, 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.     

Adenosine Triphosphate Inhibition of Yeast Trehalase

Yeast trehalase has been found to be inhibited non-competitively by adenosine triphosphate. Such a biological control could explain the accumulation of trehalose during the stationary phase of the growth curve.     

Impact of light quality and quantity on growth rate kinetics of Selenastrum capricornutum

Microalgal biomass produced in indoor photobioreactors can be used as inoculum for large‐scale outdoor cultures or directly for the production of high‐value bioproducts due to the higher control of these cultures compared with outdoor systems. One of the main costs of indoor microalgal cultures is the illumination. This work can be used as a basis for the optimization of the light source for indoor microalgal biomass production, based on the light source type, irradiance, productivity, growth rate, attenuation coefficients, and contaminant growth on the reactor's side‐walls. Four commercially available near 400‐W artificial light sources for microalgal cultures (metal halide (MH), high‐pressure sodium (HPS), Son Agro^®, and fluorescent) were compared. The light elevation and the surface scalar irradiance were shown to have a linear relationship. The attenuation coefficient in air (k_a) was highest with Son Agro^®. A linear partition of the attenuation coefficient between the water and biomass and an exponential relationship between average scalar irradiance and depth were found. An empirical overall scalar attenuation coefficient for each light source was obtained. The lowest maximum observed growth rate was obtained with fluorescent light (0.98 d^?1) and the highest with Son Agro^® (2.39 d^?1). The highest growth on the reactor's wall was obtained with Son Agro^®. Further studies resulted in a higher maximum specific growth rate and optimum irradiance for HPS (2.37 d^?1 and 460 μmol s^?1 m^?2) compared with those observed with MH (1.73 d^?1 and 391 μmol s^?1 m^?2).     

Kinetics of Phosphate Incorporation into Adenosine Triphosphate and Guanosine Triphosphate in Bacteria

Escherichia coli cells were labeled with (33)PO(4) for several generations and then (32)PO(4) was added, samples were taken at various times, and adenosine triphosphate (ATP) and guanosine triphosphate (GTP) were purified from an acid extract. The ratio of (32)P/(33)P in each of the six phosphate species was determined and compared to the ratio expected at isotopic steady state. The rate constants of the simplest network of pools which could explain the data, and were consistent with metabolic pathways, were then calculated. In the range of times studied (30 to 3,000 s at 21.5 C) the following generalizations can be made. (i) The gamma-phosphate at ATP requires 2,200 s to achieve 50% of its final specific activity and behaves as if it were exchanging with another species 4.4 times as large, with this other species turning over in 540 s. (ii) The beta-phosphate of ATP and the gamma-phosphate of GTP are in equilibrium with the gamma-phosphate of ATP. (iii) The specific activity of the gamma-phosphate of GTP lags 66 s behind the gamma-phosphate of ATP. (iv) The alpha-phosphates of ATP and GTP have equal specific activities which lag far behind the other four phosphates. A single precursor pool with a turnover time of 416s fits the data best, although a fair fit can be obtained assuming several sequential precursor pools with much shorter turnover times. These findings are consistent with known biosynthetic pathways and the probable flows through them, including the relationship of nucleotide biosynthesis to ribonucleic acid synthesis and turnover.     

The Effect of Some Inhibitory Sugars upon the Content of Adenosine Triphosphate in Wheat Roots

The content of adenosine triphosphate (ATP) in roots of -wheat (Triticum aestivum L.) was determined with the fire-fly-luciferase method. The content is decreased by D-mannose, which inhibits root growth, respiration and chloride uptake. In intact seedlings the inhibition of root growth is relieved by other sugars and also by the flavanone naringenin and by 2,4-dinitrophenol. This reversal is combined with an increased content of ATP. The inhibition of chloride uptake by mannose in excised roots is reversed by some other sugars (including D-galactose which is in itself inhibitory to root growth), and also in this case the ATP content is increased. Naringenin and dinitrophenol do not relieve the inhibition of chloride uptake caused by mannose. Nor do they increase the content of ATP in this case. The primary effect of mannose seems to be inhibition of glycolysis whereas the effect upon root growth is secondary. Galactose, which also inhibits root growth, does not inhibit respiration or reduce the ATP content and the primary effect of galactose (and also of 2-deoxy-D-glucose and 2-deoxy-D-galactose) seems to be on the synthesis of cell wall substances.     

Conjugation of benzo[a]pyrene metabolites by freshwater green alga Selenastrum capricornutum

Benzo[a]pyrene (BaP) undergoes metabolic transformation in mammals via oxidative, hydrolytic, and conjugative processes; however, little is known concerning BaP conjugation in freshwater algae. It has been shown in this laboratory that BaP is metabolized by Selenastrum capricornutum via a dioxygenase pathway. This study describes the conjugation of BaP metabolites by a green alga, Selenastrum capricornutum. Cultures were exposed to 1160 micrograms/l [14C]BaP for 4 days at 23 degrees C under gold fluorescent lights on a diurnal cycle of 16 h light, 8 h dark. Of the total metabolites in the algal culture, 89% were present in media. BaP and non-conjugated metabolites were separated from conjugated metabolites by chromatography on neutral alumina columns using solvents of increasing polarity. Seventy-one percent of the BaP metabolites were conjugates of which 12.2%, 12.0% and 12.4% were sulfate ester and alpha- and beta-glucose conjugates, respectively. Conjugates that coeluted with sulfate esters were hydrolyzed with arylsulfatase, alpha- or beta-glucosidase; high performance liquid chromatography (HPLC) analysis indicated that the major product of each enzymatic hydrolysis was the 4,5-dihydrodiol (87.2, 69 and 53%, respectively). Eighty-six percent of the conjugates were acid labile following incubation for 2 h in 4 N HCl at 37 degrees C. To our knowledge this is the first demonstration of the metabolism of a polynuclear aromatic hydrocarbon by a freshwater green alga through a dioxygenase pathway and subsequent conjugation and excretion.     

Variation in cell composition and utilization of N and P for growth of Selenastrum capricornutum

Cell material from different phases of growth for the green alga Selenastrum capricornutum Printz was analysed for major elements and chlorophyll a content. The ranges in percentage of the dry weight were 50–80%, 4.4–8.8%, 0.24–3.0%, and 0.20–3.23% for C, N, P and chlorophyll a, respectively. The dry weight per cell varied 2.3 times between different growth phases. A N/P-quotient of about 10 (by weight) was found to be a critical level for Selenastrum when these elements were the most limiting nutrients for growth. 31 different organic N-compounds were tested as single N sources in growth experiments with Selenastrum. At least 19 of them could be utilized to varying extent for algal growth. Three organic P-compounds of six tested, were utilized for algal growth. Yield coefficients (mm³ cell volume/mg limiting nutrient element) for Selenastrum were determined with NO₃, -N (y_N= 134). NH₄-N (Y_N= 111) and PO₄-P (Y_P= 2580).     

Comparison of Reductions in Adenosine Triphosphate Content, Plasma Membrane-associated Adenosine Triphosphatase Activity, and Potassium Absorption in Oat Roots by Diethylstilbestrol

The possibility was investigated that diethylstilbestrol (DES) inhibits potassium absorption in oat (Avena sativa L. cv. Goodfield) roots by inhibiting mitochondrial functions in addition to inhibiting the plasma membrane ATPase. DES at 10⁻⁶ molar stimulated the mitochondrial ATPase slightly, but higher concentrations had no effect. Oxidative phosphorylation by isolated mitochondria was inhibited 50% by 2.6 × 10⁻⁵ molar DES; concentrations of 10⁻⁴ molar or greater were completely inhibitory. After a lag of about 2 minutes, 10⁻⁴ molar DES produced a linear decrease in ATP content of excised roots. After 20 minutes, the ATP content of the tissue was about 50% of the control and remained at that level after 30 minutes in DES.     

Adenosine Triphosphate and Synchronous Mitosis in Physarum polycephalum

Synchronous mitoses occur in Physarum polycephalum in the absence of cell division. Nucleoside and nucleotide profiles were prepared from synchronously growing P. polycephalum at intervals throughout the growth cycle. Comparison of these profiles demonstrates that the pool of adenosine triphosphate decreases from a high level at prophase to a minimum through mitosis and increases again in the postmitotic period. These events appear to coincide with changes in the pools of adenosine diphosphate and adenosine but not with that of adenosine monophosphate. This observed decrease in the pool of adenosine triphosphate during mitosis was confirmed by direct enzymatic assay. These results presumably reflect the energy demands of the cell during mitosis.     

Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity

Background

Human PMS2 (hPMS2) homologues act to nick 5′ and 3′ to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn⁺⁺ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)₂E(X)₄E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.

Methodologies/Principal Findings

We examined the effect ATP had on the Mn⁺⁺ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6±0.08×10⁻⁵ s⁻¹ and 4.2±0.3×10⁻⁵ s⁻¹ in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)₂E(X)₄E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.

Conclusions

ATP stimulated the Mn⁺⁺ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)₂E(X)₄E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn⁺⁺ induced nicking activity.     

Respiration in Relation to Adenosine Triphosphate Content during Desiccation and Rehydration of a Desiccation-tolerant and a Desiccation-intolerant Moss

O₂ consumption by the desiccation-tolerant moss Tortula ruralis and the desiccation-intolerant Cratoneuron filicinum increased markedly during the latter stages of desiccation. ATP content of the mosses during desiccation was not correlated with O₂ consumption, but was influenced by the rate at which the mosses lost water. The more rapid the water loss, the more ATP that was present in the dry mosses. The pattern of O₂ consumption on rehydration also was influenced by the previous rate of desiccation. After rapid desiccation of T. ruralis O₂ consumption upon rehydration was considerably elevated, and for up to 24 hours. After very slow desiccation the elevation was small and brief. Normal O₂ consumption did not occur in C. filicinum after rapid desiccation, but did so within a few hours of rehydration after slower speeds of drying. ATP levels in T. ruralis returned to normal within 5 to 10 minutes of rehydration. In C. filicinum, increases in ATP were closely correlated with O₂ consumption. These observations are considered to be related to differential damage caused to mitochondria and to cellular integrity by different speeds of water loss. The desiccation-tolerant moss appears to be able to repair the severe damage imposed by rapid desiccation whereas the desiccation-intolerant moss cannot.     

Antioxidant responses of two microalgae, Selenastrum capricornutum and Chlorella sp., to estradiol and ethinylestradiol

The cellular characteristics and antioxidant responses including reactive oxygen species (ROS), esterase activity, superoxide dismutase, peroxidase (POD), glutathione (GSH), and malondialdehyde (MDA) content of two microalgal species, Selenastrum capricornutum and Chlorella sp., exposed to single and mixed estradiol (E2) and ethinlyestradiol (EE2) at a final concentration of 200 μg L^?1 were investigated. The growth, autofluorescence, cell complexity, and cell size of S. capricornutum and Chlorella sp. were not affected by single and mixed estrogen treatments, but the temporal trends of these two species were different. The temporal changes of the esterase activity and ROS level in these two species were also very different, with a continuous increase with time in Chlorella but peak on Day 2 in S. capricornutum. The esterase activity of S. capricornutum was not affected by estrogen treatments; however, the activity in treated Chlorella decreased significantly from Day 2 onwards. The single EE2 and mixed E2 and EE2 treatments induced more ROS of S. capricornutum than the single E2 treatment and control on Day 4, but no other difference was found between treatments and control of Chlorella on that day. The other oxidative damage responses of S. capricornutum were not affected by estrogen treatments, except MDA increased in EE2 treatment, on the other hand, POD and GSH of Chlorella increased in both single and mixed estrogen treatments on Day 4. These results suggested that microalgae species were tolerant to E2 and EE2, but some species-specific changes occurred to combat the oxidative stress posed by estrogens.     

Respiration Rate and Adenosine Triphosphate Formation in Tissues4

When leaves of Nicotiana tabacum L. var. Xanthi were inoculatedwith TMV, local lesion formation gave enhanced ATP concentrationaccompanied by increased respiration. Phosphorus metabolismin healthy and infected leaves was investigated either by floatingleaf tissue on ³²P₁-phosphate buffer or by allowing intact leavesto take up ³²P₁-phosphate buffer through the petioles. Bothmethods gave increased ³²P₁ incorporation into inorganic andorganic acid-soluble phosphorus fractions of the infected leaf.Comparison of the specific activity of ³²P₁-phosphate buffersupplied with the specific activity of the -phosphate groupof ATP demonstrated that 50 per cent of the ATP in the healthyleaf compared with over 78 per cent of the ATP in the infectedleaf was metabolically active in respiration. As the ATP concentrationin the infected leaf is much greater than that in the healthyleaf, this means that the amount of ATP being utilized and resynthesizedas a result of respiratory metabolism in the infected leaf ismore than twice that in the healthy leaf. Pulse-labelling experimentsdemonstrated that the rate of ATP turnover was very similarin healthy and infected leaves; therefore the increased respirationin the infected leaf results from the larger ATP pool with aturnover rate of ATP similar to that in the healthy leaf. Withtobacco callus tissue cultures infected by TMV, where infectiondoes not result in local-lesion formation, phosphorus metabolismwas unaltered in the infected tissue. It is concluded that necroticlocal-lesion formation results from increased availability ofATP at sites of ATP utilization, and this is aggravated by over-productionof ATP in the infected leaf.