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.     

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P_s) of 7.784 mL and production yield of (P_r) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R² 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H₂ evolution rate and higher butyrate concentration in the fermentate.     

Action Spectra for Photosystems I and II in Formaldehyde Fixed Algae

Action spectra were obtained for photosystems I and II in chemically fixed algal cells and for photosystem I in unfixed lysozyme treated cells. Untreated algal cells yielded neither of the 2 light reactions with the reaction mixtures used. The action spectra for photosystem I in the blue-green alga Anacystis nidulans and red alga Porphyridium cruentum follow the absorption spectrum of chlorophyll a with a small peak in the region of the accessory pigments. In the green alga Chlorella pyrenoidosa the photosystem I action spectrum follows the absorption spectrum of chlorophyll a. Photosystem II action spectra in A. nidulans and P. cruentum follow the absorption spectra of the accessory pigments while that in C. pyrenoidosa is shifted slightly toward the blue spectral region. These results provide additional evidence that formaldehyde fixed cells are valid models for studying the light reactions of photosynthesis.     

Quantum Requirements of Photosynthetic Oxygen Evolution and 77 K Fluorescence Emission Spectra in Unicellular Green Algae Grown Under Low- and High-CO2-Conditions

Quantum requirements of photosynthetic oxygen evolution at 682 nm and fluorescence spectra at liquid nitrogen temperature (77 K), were investigated in Dunaliella tertiolecta, Chlamydomonas reinhardtii C-9, Chlorella vulgaris 11g, Chlorella vulgaris C3, and Chlorella pyrenoidosa 8b grown under low- and high-CO₂ conditions. Dunaliella, Chlamydomonas and C. vulgaris 11g show higher quantum requirements and a higher ratio of F_710–740/F_680–695 fluorescence when grown under low-CO₂ conditions, indicating a change in excitation energy distribution towards PS I. In C. pyrenoidosa the quantum requirement for low-CO₂ grown cells is higher than in high-CO₂ grown cells, but there was practically no change in the fluorescence ratio. In C. vulgaris C3, the quantum requirements of low- and high-CO₂ grown cells are the same, but the fluorescence ratio is higher in high-CO₂ grown cells than in low-CO₂ grown cells. These results indicate that most of the low-CO₂ grown cells require more PS I light than high-CO₂ grown cells. It is possible that this energy is used for cyclic electron flow. In C. vulgaris C 3, a mechanism may exist for excitation energy distribution which leads to the same quantum requirements under low- and high-CO₂ conditions.     

HYDROGEN ION BUFFERING OF CULTURE MEDIA FOR ALGAE FROM MODERATELY ACIDIC,OLIGOTROPHIC WATERS 1

Five hydrogen ion buffers were compared for their usefulness in regulating pH in a model oligotrophic, moderately acidic (pH 6.0) algal growth medium. These were 3,3-dimethylglutaric acid (DMGA), tricarbaliylic acid (TCA), trans-aconitic acid (tAA), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 2-(N-morpholino) ethanesulfonic acid (MES). All buffers (2.5 mM) except HEPES limited the reduction of pH in a NH₄⁺-based medium during growth of Chrysochromulina breviturrita Nich. to less than 0.12 units, compared with more than 2 units in an unbuffered medium. Long term growth of C. breviturrita in these media was significantly inhibited (P < 0.05) by TCA and tAA. MES was able to control pH with the minimum amount of NaOH (1.0 mM) added to the medium to adjust to pH 6.0. Four of five bacterial isolates were capable of utilizing tAA as a sole organic-C source, and no isolate could metabolize HEPES or MES. No significant differences (P > 0.05) were found in the maximum growth rates of six algal species (from five classes) in a medium with or without MES buffer, although significantly greater cell yields of Ochromonas danica Prings. were obtained in the buffered medium. MES (pK₄=6.15) was considered to be the most useful buffer in the pH range 5.0–6.5, due to its biological inertness, buffering capacity, the minimal requirement for excess base to adjust pH and its minimal metal complexing ability.     

Algicidal effect of bromine and chlorine on Chlorella pyrenoidosa

Chlorella pyrenoidosa was found to grow rapidly in tap water. Peak growth was reached after 2 to 3 days. Chlorine and bromine, added to such water, were shown to be effective inhibitors of algal growth. Bromine and bromamine were primarily algicidal, whereas chlorine and chloramines were mainly algistatic. It is assumed that the mechanisms of action of these halogens on Chlorella are not the same.     

Occurrence of 2-hydroxy acids in microalgae

2-Hydroxy acids were believed to be absent in algae until this study, in which the analysis of microalgae belonging to Chlorophyta (Chlamydomonas reinhardtii and Chlorella pyrenoidosa), Rhodophyta (Cyanidium caldarium M-8 and Cyanidium caldarium RK-1) and Cyanophyta (Anbaena variabilis, Anacystis nidulans, Oscillatoria species and Phormidium foveolarum) is reported. 2-Hydroxy adds with carbon chain lengths of C₁₆-C₂₆, were found in all the algal samples studied, ranging in concentrations from 4.0 to 320μg/g dry alga. The dominant constituents are 2-hydroxyhexadecanoic, 2-hydroxynonadecanoic, 2-hydroxyhexacosanoic and a branched 2-hydroxy-C₁₉ acid. The distribution patterns of the acids differed significantly among the algal samples. Hence 2-hydroxy acids may be useful for the classification of algal species as well as being an important source of 2-hydroxy acids in the natural environment.     

Harvesting Chlorella species using magnetic iron oxide nanoparticles

Microalgae are very useful organisms as it provides many beneficial products for human use. For large scale cultivation and further applications, its harvesting procedure needs to be enhanced to make the production process of the end product highly affordable. Magnetic nanoparticles have great potential to harvest microalgae as it can easily attract and attach to the algal cell surface forming a layer, which can be harvested quickly under the influence of a magnetic field. Our work on Chlorella pyrenoidosa and Chlorella minutissima shows, 500 mg of the synthesized bare iron oxide nanoparticles, harvests 90% of Chlorella pyrenoidosa (1 g L^?1), in 60 s at pH 3 and 600 mg iron oxide nanoparticles, harvests 85% of Chlorella minutissima (1 g L^?1) in 60 s at pH 5, which can decrease the amount of time and energy consumed in the overall production costs.     

Influence of endophytic strains of the bacterium Bacillus subtilis on cell number in monocultures of green algae

Effect of several endophytic bacterial strains of Bacillus subtilis on growth of Scenedesmus quadricauda and Chlorella vulgaris algae was investigated. Introduction of bacillus strains into the algal cultures diminished the cell number of microalgae. The cells of C. vulgaris exhibited equal sensitivity to all the endophyte strains examined. The number of S. quadricaudas cells reduced to larger extent by the 7th day of algal culturing in the presence of B. subtilis 11V or 11VM strains than in the presence of B. subtilis 26D strain, the latter strain being the main component of the commercial biofungicide Fitosporin-M. Washed bacterial cells inactivated by autoclaving did not reduce the number of C. vulgaris cells. The bacterial cells that were autoclaved without separation from the culture liquid, as well as the autoclaved supernatant of cell suspension exerted the same inhibitory effect as living bacterial cells. The decrease in the algal cell number under the action of endophytic bacillus strains is presumably due to thermostable biologically active substances synthesized and secreted by the bacillus strains.     

Effect of pH on Inorganic Carbon Uptake in Algal Cultures

Biomass production by the green algae Scenedesmus obliquus and Chlorella vulgaris in intensive laboratory continuous cultures was considerably affected by the pH at which the cultures were maintained. Carbon photoassimilation experiments revealed that pH values in the range of 8 to 9 were important for determining the free CO₂ concentrations in the medium. With higher pH values, additional pH effects were observed involving a decrease in the relative high affinity of low CO₂-adapted algae to free CO₂. The carbon uptake rate by high CO₂-adapted algae after transfer to low free CO₂ medium was characterized by a lag period of about 30 min, after which the affinity of the algae to CO₂ increased considerably. Both continuous growth and carbon uptake experiments indicated that artificially maintained high free CO₂ concentrations are recommended for maximal production in intensive outdoor algal cultures.     

Synchronization of cell division ofChlorella pyrenoidosa

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

Steady-state rotifer growth in a two-stage, computer-controlled turbidostat

Steady-state populations of rotifers (Brachionus calyciflorus)were maintained in twostage, continuous-flow turbidostatic cultureon the green alga Chlorella pyrenoidosa. In this system, themaximum specific growth rate,µ^max of the rotifers wasmaintained by using a computer to control the concentrationof algae, as rotifer food, in the rotifer culture. As rotifersconsumed algae, the turbidity decreased until a set-point wasreached. Then fresh algal suspension (supplied from a steady-statealgal chemostat) was metered into the rotifer culture, whichwas held in the dark. Rotifer and algal populations, as wellas rotifer µ^max entered steady states. These steady-stateresults were consistent with previous data from chemostat studies,but growth transients indicated that the of the µ^maxrotifersmay be subject to selection. The system is unique in providinga means to explore population dynamics of a metazoan maintainednear its µ^max.     

Chronic toxicity of methylparathion to the rotifer Brachionus calyciflorus fed on Nannochloris oculata and Chlorella pyrenoidosa

The effect of sublethal levels of methylparathion (0, 1, 3, 5, 7 mg l^–1) on the freshwater rotifer, Brachionus calyciflorus, during their entire life cycle was studied. Rotifers were fed on two species of unicellular algae: Nannochloris oculata and Chlorella pyrenoidosa; both algal concentrations were 5 × 10⁵ cell ml^–1.The parameters used to determine the toxicity of this compound were survival, fecundity, net reproductive rate (R)_o, generation time (T), intrinsic rate of natural increase (r), reproductive value (V _x/V_o) and life expectancy at hatching (eo). All the demographic parameters studied were affected by methyl-parathion exposure on rotifers fed on both species of algae, but the toxic effect was larger when animals were fed on Chlorella pyrenoidosa; in this case, animals showed a decreased in fertility and also a delayed first reproduction. Sublethal methylparathion levels produced a reduction in most of the parameters selected, especially after exposure to 7 mg l^–1, where the animals died before reproducing.     

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

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

Zur Glykolatoxydation einzelliger Grünalgen

Summary O₂-uptake and CO₂-release by a chlorophyll-free, carotenoid-containing mutant of Chlorella vulgaris increase on addition of Na-glycolate by factors of 4–5 and 5–6, respectively (Fig. 1). In an enzyme preparation of that alga (sonification, centrifugation, precipitation with 0–30% (NH₄)₂SO₄, dialysis) activity of glycolate oxidase can be demonstrated by O₂-uptake (Fig. 2a) as well as by reduction of the artificial electron acceptor DCPIP (Fig. 2b). The same holds true for whole cells as well as equally prepared enzyme preparations of heterotrophically or autotrophically grown wildtype Chlorella vulgaris, provided the cells are cracked by a French press instead of a sonicator (Figs. 3a-c and 4a-c). Glyoxylate is the main reaction product (Table). Oxidation of exogenous glycolate is rapidly performed by whole cells of Scenedesmus quadricauda and of Ankistrodesmus convolutus, too, but hardly or not at all by Chlorella pyrenoidosa and Ankistrodesmus braunii. No definite influence of the level of CO₂ applied during growth is found: Chlorella vulgaris and Ankistrodesmus convolutus show a rapid oxidation of glycolate after growth under 0,03 and 1,5% CO₂ in air, whereas Chlorella pyrenoidosa and Ankistrodesmus braunii do not show an enhanced O₂-uptake on addition of glycolate after either condition (Fig. 5). Various developmental stages of Chlorella pyrenoidosa respond differently to addition of glycolate, the extra O₂-consumption varying between about 25% (mature cells) and 50–60% (young cells) of the endogenous rate (Fig. 6). It thus appears that species of unicellular green algae within the same genus have strong or weak glycolate oxidase activity and that several external factors have only a modifying effect on that enzyme.     

Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State 1H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state ¹H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative ¹H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r ² = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.     

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.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4 A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO₄ concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5–150 ng/ml concentration range [r²=0.999 for (+)- and (−)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (−)-tramadol, respectively.     

Toxic and non-toxic strains of Microcystis aeruginosa induce temperature dependent allelopathy toward growth and photosynthesis of Chlorella vulgaris

Global warming was believed to accelerate the expansion of cyanobacterial blooms. However, the impact of changes due to the allelopathic effects of cyanobacterial blooms with or without algal toxin production on the ecophysiology of its coexisting phytoplankton species arising from global warming were unknown until recently. In this study, the allelopathic effects of toxic and non-toxic Microcystis aeruginosa strains on the growth of green alga Chlorella vulgaris and photosynthesis of the co-cultivations of C. vulgaris and toxic M. aeruginosa FACHB-905 or non-toxic M. aeruginosa FACHB-469 were investigated at different temperatures. The growth of C. vulgaris, co-cultured with the toxic or non-toxic M. aeruginosa strains, was promoted at 20 °C but inhibited at temperatures ≥25 °C. The inhibitory effects of the toxic and non-toxic M. aeruginosa strains on of the co-cultivations (C. vulgaris and non-toxic M. aeruginosa FACHB-469 or toxic M. aeruginosa FACHB-905) also linearly increased with elevated temperatures. Furthermore, toxic M. aeruginosa FACHB-905 induced more inhibition toward growth of C. vulgaris or P_max and R_d of the mixtures than non-toxic M. aeruginosa FACHB-469. C. vulgaris dominated over non-toxic M. aeruginosa FACHB-469 but toxic M. aeruginosa FACHB-905 overcame C. vulgaris when they were co-cultured in mesocosms in water temperatures from 20 to 25 °C. The results indicate that allelopathic effects of M. aeruginosa strains on C. vulgaris are both temperature- and species-dependent: it was stimulative for C. vulgaris at low temperatures such as 20 °C, but inhibitory at high temperatures (≥25 °C); the toxic strain was determined to be more harmful to C. vulgaris than the non-toxic one. This suggests that global warming may aggravate the ecological risk of cyanobacteria blooms, especially those with toxic species as the main contributors.     

Biodegradation of p-nitrophenol by microalgae

A study was made on the use of a mixed microalgal consortium to degrade p-nitrophenol. The consortium was obtained from a microbial community in a waste container fed with the remains and by-products of medium culture containing substituted aromatic pollutants (nitrophenols, chlorophenols, fluorobenzene). After selective enrichment with p-nitrophenol (p-NP), followed by an antibiotic treatment, an axenic microalgal consortium was recovered, which was able to degrade p-nitrophenol. At a concentration of 50 mg L^–1, total degradation occurred within 5 days. Two species, Chlorella vulgaris var. vulgaris f. minuscula and Coenochloris pyrenoidosa, were isolated from the microalgal consortium. The species were able to accomplish p-NP biodegradation when cultured separately, although Coenochloris pyrenoidosa was more efficient, achieving the same degradation rate as the original axenic microalgal consortium. When Coenochloris pyrenoidosa was associated with Chlorella vulgaris in a 3:1 ratio, complete removal of the nitro-aromatic compound occurred within three days. This is apparently the first report on the degradation of a nitro-aromatic compound by microalgae.