Single-Cell Mass Spectrometry Analysis of Metabolites Facilitated by Cell Electro-Migration and Electroporation.

Single-cell metabolite analysis plays an important role in biological study. While mass spectrometry is a powerful tool for identification and quantitation of metabolites, the low absolute analyte amounts in single cell and difficulty in sampling represent significant challenges in single cell analysis. In this study, we developed an effective method with a simple sampling procedure for analyzing single cells. A single cell was driven to a capillary tip through electro-migration, followed by releasing the cell contents through electroporation, into a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent mass spectrometry analysis was performed directly with nanoelectrospray ionization. This method was applied for analyzing a variety of cells and monitoring the metabolic changes in response to perturbed cell culturing conditions. This method opens a new avenue for easy and rapid analysis of single cells with high sensitivity.

A study into the species sensitivity of green algae towards imidazolium-based ionic liquids using flow cytometry.

The sensitivity of individual organisms towards toxic agents is an important indicator of environmental pollution. However, organism-specific quantification of sensitivity towards pollutants remains a challenge. In this study, we determined the sensitivity of Chlorella vulgaris (C. vulgaris) and Scenedesmus quadricauda (S. quadricauda) towards three ionic liquids (ILs), 1-alkyl-3-methyl-imidazolium chlorides [Cnmim][Cl] (n = 4,6,8). We kept all external parameters constant to identify the biotic parameters responsible for discrepancies in species sensitivity, and used flow cytometry to determine four conventional endpoints to characterise cell viability and cell vitality. Our results demonstrate that after exposure to the ILs, cell proliferation was inhibited in both species. At the same time, the cell size, complexity and membrane permeability of both algae also increased. However, while Chl a synthesis by S. quadricauda was inhibited, that of C. vulgaris was enhanced. S. quadricauda has evolved a metabolic defense that can counteract the decreased esterase activity that has been shown to occur in the presence of ILs. While it is likely that S. quadricauda was less sensitive than C. vulgaris to the ILs because of this metabolic defense, this alga may also exhibit better membrane resistance towards ILs.

Combined Confocal Laser Scanning Microscopy Techniques for A Rapid Assessment of the Effect and Cell Viability of Scenedesmus sp. DE2009 Under Metal Stress.

Phototrophic microorganisms are the dominant populations in microbial mats, which play an important role in stabilizing sediments, such as happens in the Ebro Delta. These microorganisms are exposed to low metal concentrations over a long period of time. Distinct methods have been used to evaluate their toxic effect on the preservation of these ecosystems. Nevertheless, most of these techniques are difficult to apply in isolated phototrophs because (i) they usually form consortia with heterotrophic bacteria, (ii) are difficult to obtain in axenic cultures, and (iii) do not grow on solid media.In this study, and for the first time, a combination of fast, non-invasive, and in vivo Confocal Laser Scanning Microscopy (CLSM) techniques were applied in a consortium of Scenedesmus sp. DE2009 to analyze its physiological state and viability under metal stress conditions. Microalga was more resistant to Pb followed by Cr and Cu. However, in multimetal combinations, the presence of Cu negatively affected microalga growth. Additionally, the inhibitory concentration (IC) values were also calculated by CLSM pigment analysis. The result determines a higher degree of toxicity for Cu and Cr in comparison to Pb. The high sensitivity of these CLSM-methods to detect low concentrations allows consideration of Scenedesmus sp. DE2009 as a good bioindicator of metal pollution in natural environments.

Effect of algal surface area and species interactions in toxicity testing bioassays.

Single and multispecies algal bioassays were assessed using copper toxicity with three green algae (Scenedesmus subspicatus, Scenedesmus quadricauda and Ankistrodesmus angustus) and one blue-green algae species (Oscillatoria prolifera). Single and multispecies toxicity tests were conducted based on cell density as per standard toxicity testing, and on equivalent surface area. A higher copper sulfate toxicity was registered for O. prolifera, followed by S. subspicatus, S. quadricauda, and A. angustus in single-species toxicity tests based on cell density. Single species toxicity tests based on surface area showed increased copper toxicity with increasing algal surface area except for A. angustus. In multispecies control bioassays, the growth of A. angustus was inhibited in the presence of other species in surface area-based tests. As compared to single species bioassays, O. prolifera, and S. quadricauda showed a decreased sensitivity to copper sulfate in both cell density and surface area based multispecies tests. However, for the algae species with the smallest surface area, S. subspicatus, 96h-EC50 value decreased in multispecies bioassays based on surface area as compared to the single species test, while it increased in multispecies bioassays based on cell density. The difference in S. subspicatus sensitivity to copper between tests based on cell density and surface area supports the need to adopt multispecies toxicity testing based on surface area to avoid the confounding effect on copper toxicity of increased biomass for metal binding. 96h-EC50 values for all species combined in the multispecies test based on cell density and on surface area were significantly different from 96h-EC50 values obtained in single species bioassays. These results demonstrate that single-species bioassays may over- or underestimate metal toxicity in natural waters.

Development of a helical coagulation reactor for harvesting microalgae.

In this study, an innovative helical coagulation reactor (HCR) was developed for harvesting microalgae by sedimentation with polyaluminium chloride (PAC). The effects of construction and hydrodynamic characteristics on harvesting performance were investigated. Results showed that a higher harvesting efficiency, 96.37%, was achieved for the large and compact flocs generated by the HCR, and the settling rate of flocs was substantially influenced by the velocity gradient (G) and the Reynolds number (Re). When the Reynolds number closed to the transition between laminar and turbulent flow (4000), the flocs settled faster (20.51 m h-1), although settling slowed as the Reynolds number increased further because of ruptured flocs. The settling rate of flocs could be further improved to 23.27 m h-1 by a pulse flow field, mainly due to larger and more compact flocs forming in the plug pipe flow. Furthermore, a comparative investigation of a mechanically agitated vessel and the HCR with the same Camp number (Gt) showed that the HCR achieved higher settling rates and a shorter residence time than those with a mechanical agitator. The HCR provided a uniform dissipation of energy and high velocity gradient while avoiding electrical and mechanical energy consumption, suggesting this reactor is an efficient and economic option for microalgae harvesting.

Effect of ionic liquids with different cations and anions on photosystem and cell structure of Scenedesmus obliquus.

The rapid increase in the production and practical application of ionic liquids (ILs) could pose potential threats to aquatic systems. In this study, we investigated the effects of four ILs with different cations and anions, including 1-hexyl-3-methylimidazolium nitrate ([HMIM]NO3), 1-hexyl-3-methylimidazolium chloride ([HMIM]Cl), N-hexyl-3-metylpyridinium chloride ([HMPy]Cl), and N-hexyl-3-metylpyridinium bromide ([HMPy]Br), on photosystem and cellular structure of Scenedesmus obliquus. The results indicated that ILs are phytotoxic to S. obliquus. The contents of chlorophyll a, chlorophyll b and total chlorophyll decreased with increasing ILs concentrations. The chlorophyll fluorescence parameters of photosynthetic system II (PSII), including minimal fluorescence yield (F0), potential efficiency of PSII (Fv/Fo), maximum quantum efficiency of PSII photochemistry (Fv/Fm), yield of photochemical quantum [Y(II)], and non-photochemical quenching coefficient without measuring F0' (NPQ), were all affected. This indicates that ILs could damage PSII, inhibit the primary reaction of photosynthesis, interdict the process of electron-transfer and lead to loss of heat-dissipating ability. ILs also increased cell membrane permeability of S. obliquus, influenced the cellular ultrastructure, changed the morphology of algae cells and destroyed the cell wall, cell membrane and organelles. The results indicated that imidazolium ILs had greater effect than pyridinium ILs, NO3--IL and Br--IL had greater effect than Cl--IL. To minimize threats to the environment, the structure of ILs should be taken into consideration.

Long-term toxicity of ZnO nanoparticles to Scenedesmus rubescens cultivated in different media.

The aim of this work was to investigate the long-term toxic effect of zinc oxide (ZnO) nanoparticles (NPs) on freshwater microalgae, combined with the nutrient consumption in the culture. For this purpose, two common microalgae media (Blue-Green 11, BG-11, and Bold's Basal Medium, BBM) were used. Scenedesmus rubescens was used as freshwater microalgae model species and was exposed to ZnO NPs at different concentrations (0.081 to 810 mg/L) for a period up to 28 d. The experimental results revealed that microalgae growth was affected by the time of exposure and the NPs concentrations, but mainly the culture medium used. Differences in microalgae growth rates were observed and attributed to the selected culture medium. The toxic effect of ZnO NPs was higher on microalgae cultured in modified BG-11 compared to BBM, despite the fact that S. rubescens exhibited higher growth rate in modified BG-11 without the exposure of ZnO NPs.

A novel method to analyse in vivo the physiological state and cell viability of phototrophic microorganisms by confocal laser scanning microscopy using a dual laser.

Phototrophic microorganisms are very abundant in extreme environments, where are subjected to frequent and strong changes in environmental parameters. Nevertheless, little is known about the physiological effects of these changing environmental conditions on viability of these microorganisms, which are difficult to grow in solid media and have the tendency to form aggregates. For that reason, it is essential to develop methodologies that provide data in short time consuming, in vivo and with minimal manipulating the samples, in response to distinct stress conditions. In this paper, we present a novel method using Confocal Laser Scanning Microscopy and a Dual Laser (CLSM-DL) for determining the cell viability of phototrophic microorganisms without the need of either staining or additional use of image treating software. In order to differentiate viable and nonviable Scenedesmus sp. DE2009 cells, a sequential scan in two different channels was carried out from each same xyz optical section. On the one hand, photosynthetic pigments fluorescence signal (living cells) was recorded at the red channel (625- to 785-nm fluorescence emission) exciting the samples with a 561-nm laser diode, and an acousto-optic tunable filter (AOTF) of 20%. On the other hand, nonphotosynthetic autofluorescence signal (dead cells) was recorded at the green channel (500- to 585-nm fluorescence emission) using a 405-nm UV laser, an AOTF of 15%. Both types of fluorescence signatures were captured with a hybrid detector. The validation of the CLSM-DL method was performed with SYTOX green fluorochrome and electron microscopic techniques, and it was also applied for studying the response of distinct light intensities, salinity doses and exposure times on a consortium of Scenedesmus sp. DE2009.

Boosting TAG Accumulation with Improved Biodiesel Production from Novel Oleaginous Microalgae Scenedesmus sp. IITRIND2 Utilizing Waste Sugarcane Bagasse Aqueous Extract (SBAE).

This investigation utilized sugarcane bagasse aqueous extract (SBAE), a nontoxic, cost-effective medium to boost triacylglycerol (TAG) accumulation in novel fresh water microalgal isolate Scenedesmus sp. IITRIND2. Maximum lipid productivity of 112 ± 5.2 mg/L/day was recorded in microalgae grown in SBAE compared to modified BBM (26 ± 3 %). Carotenoid to chlorophyll ratio was 12.5 ± 2 % higher than in photoautotrophic control, indicating an increase in photosystem II activity, thereby increasing growth rate. Fatty acid methyl ester (FAME) profile revealed presence of C14:0 (2.29 %), C16:0 (15.99 %), C16:2 (4.05 %), C18:0 (3.41 %), C18:1 (41.55 %), C18:2 (12.41), and C20:0 (1.21 %) as the major fatty acids. Cetane number (64.03), cold filter plugging property (-1.05 °C), and oxidative stability (12.03 h) indicated quality biodiesel abiding by ASTM D6751 and EN 14214 fuel standards. Results consolidate the candidature of novel freshwater microalgal isolate Scenedesmus sp. IITRIND2 cultivated in SBAE, aqueous extract made from copious, agricultural waste sugarcane bagasse to increase the lipid productivity, and could further be utilized for cost-effective biodiesel production.

Effect of subcellular distribution on nC60 uptake and transfer efficiency from Scenedesmus obliquus to Daphnia magna.

The potential uptake and trophic transfer ability of nanoparticles (NPs) in aquatic organisms have not been well understood yet. There has been an increasing awareness of the subcellular fate of NPs in organisms, but how the subcellular distribution of NPs subsequently affects the trophic transfer to predator remains to be answered. In the present study, the food chain from Scenedesmus obliquus to Daphnia magna was established to simulate the trophic transfer of fullerene aqueous suspension (nC60). The nC60 contaminated algae were separated into three fractions: cell wall (CW), cell organelle (CO), and cell membrane (CM) fractions, and we investigated the nC60 uptake amounts and trophic transfer efficiency to the predator through dietary exposure to algae or algal subcellular fractions. The nC60 distribution in CW fraction of S. obliquus was the highest, following by CO and CM fractions. nC60 uptake amounts in D. magna were found to be mainly relative to the NPs' distribution in CW fraction and daphnia uptake ability from CW fraction, whereas the nC60 trophic transfer efficiency (TE) were mainly in accordance with the transfer ability of NPs from the CO fraction. CW fed group possessed the highest uptake amount, followed by CO and CM fed groups, but the presence of humic acid (HA) significantly decreased the nC60 uptake from CW fed group. The CO fed groups acquired high TE values for nC60, while CM fed groups had low TE values. Moreover, even though CW fed group had a high TE value; it decreased significantly with the presence of HA. This study contributes to the understanding of fullerene NPs' dietary exposure to aquatic organisms, suggesting that NPs in different food forms are not necessarily equally trophically available to the predator.

Microalgal growth with intracellular phosphorus for achieving high biomass growth rate and high lipid/triacylglycerol content simultaneously.

Nutrient deprivation is a commonly-used trigger for microalgal lipid accumulation, but its adverse impact on microalgal growth seems to be inevitable. In this study, Scenedesmus sp. LX1 was found to show similar physiological and biochemical variation under oligotrophic and eutrophic conditions during growth with intracellular phosphorus. Under both conditions microalgal chlorophyll content and photosynthesis activity was stable during this growth process, leading to significant increase of single cell weight and size. Therefore, while algal density growth rate dropped significantly to below 1.0 × 10(5)cells mL(-1) d(-1) under oligotrophic condition, the biomass dry weight growth rate still maintained about 40 mg L(-1) d(-1). Meanwhile, the lipid content in biomass and triacylglycerols (TAGs) content in lipids increased significantly to about 35% and 65%, respectively. Thus, high biomass growth rate and high lipid/TAG content were achieved simultaneously at the late growth phase with intracellular phosphorus. Besides, microalgal biomass produced was rich in carbohydrate with low protein content.

Validation of algal viability treated with total residual oxidant and organic matter by flow cytometry.

Algal cell growth after starch and oxidant treatments in seawater species (Isochrysis galbana and Phaeodactylum tricornutum) and freshwater species (Selenastrum capricornutum and Scenedesmus obliquus) were evaluated by flow cytometry with fluorescein diacetate (FDA) staining to determine algal viability. Growth of algal cell was found to be significantly different among groups treated with NaOCl, starch and/or sodium thiosulfate, which are active substance (Total Residual Oxidant; TRO as Cl2), organic compound to meet efficacy testing standard and neutralizer of TRO by Ballast Water Management Convention of International Maritime Organization, respectively. The viability of algal cell treated with TRO in starch-add culture of 5days after treatment and neutralization was decreased significantly. ATP contents of the treated algal cells corresponded to the FL1 fluorescent signal of flow cytometry with FDA staining. I. galbana was the most sensitive to TRO-neutralized cultures during viability analysis.

Using carbon dioxide to maintain an elevated oleaginous microalga concentration in mixed-culture photo-bioreactors.

Microbial contamination of growth reactors is a major concern for microalgal biofuel production. In this study, the oleaginous, CO2-tolerant microalga Scenedesmus dimorphus was combined with a wastewater-derived microbial community and grown in replicated sequencing batch photobioreactors. The reactors were sparged with either ambient air or 20% v/v CO2. In the initial growth cycles, air and the 20% CO2 reactors were similar in terms of growth and microbial community structure. Beyond the fourth growth cycle, however, the ambient air reactors had larger decreases in cell density and growth rate, and increases in species richness and non-algal microorganisms compared to the 20% CO2 reactors. Both qPCR and rDNA sequence analyses demonstrated a greater loss in S. dimorphus enrichment in the ambient-air reactors compared to the 20% CO2 reactors. These results demonstrate that environmental parameters can be used to delay the adverse impacts of microbial contamination in open, mixed-culture microalgae bioreactors.

Microalgal cell disruption via ultrasonic nozzle spraying.

The objective of this study was to understand the effect of operating parameters, including ultrasound amplitude, spraying pressure, nozzle orifice diameter, and initial cell concentration on microalgal cell disruption and lipid extraction in an ultrasonic nozzle spraying system (UNSS). Two algal species including Scenedesmus dimorphus and Nannochloropsis oculata were evaluated. Experimental results demonstrated that the UNSS was effective in the disruption of microalgal cells indicated by significant changes in cell concentration and Nile red-stained lipid fluorescence density between all treatments and the control. It was found that increasing ultrasound amplitude generally enhanced cell disruption and lipid recovery although excessive input energy was not necessary for best results. The effect of spraying pressure and nozzle orifice diameter on cell disruption and lipid recovery was believed to be dependent on the competition between ultrasound-induced cavitation and spraying-generated shear forces. Optimal cell disruption was not always achieved at the highest spraying pressure or biggest nozzle orifice diameter; instead, they appeared at moderate levels depending on the algal strain and specific settings. Increasing initial algal cell concentration significantly reduced cell disruption efficiency. In all UNSS treatments, the effectiveness of cell disruption and lipid recovery was found to be dependent on the algal species treated.

Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3.

In this study, the effects of the type and concentration of nitrogen sources on the cell growth and lutein content of an isolated microalga Scenedesmus obliquus FSP-3 were investigated. With batch culture, the highest lutein content (4.61 mg/g) and lutein productivity (4.35 mg/L/day) were obtained when using 8.0 mM calcium nitrate as the nitrogen source. With this best nitrogen source condition, the microalgae cultivation was performed using two bioreactor strategies (namely, semi-continuous and two-stage operations) to further enhance the lutein content and productivity. Using semi-continuous operation with a 10% medium replacement ratio could obtain the highest biomass productivity (1304.8 mg/L/day) and lutein productivity (6.01 mg/L/day). This performance is better than most related studies.

Utilization of xylose as a carbon source for mixotrophic growth of Scenedesmus obliquus.

Mixotrophic cultivation is one potential mode for microalgae production, and an economically acceptable and environmentally sustainable organic carbon source is essential. The potential use of xylose for culturing Scenedesmus obliquus in a mixotrophic mode and physiological features of xylose-grown S. obliquus were studied. S. obliquus had a certain xylose tolerance, and was capable of utilizing xylose for growth. At a xylose concentration of 4gL(-1), the maximal cell density was 2.2gL(-1), being 2.9-fold of that under photoautotrophic condition and arriving to the level of mixotrophic growth using 4gL(-1) glucose. No changes in cellular morphology of the cells grown with or without xylose were detected. Fluorescence emission from photosystem II (PS II) relative to photosystem I (PS I) was decreased in mixotrophic cells, implying that the PSII activity was decreased. The biomass lipid content was enhanced and carbohydrate concentration was decreased, in relation to photoautotrophic controls.

Fixed-bed biosorption of cadmium using immobilized Scenedesmus obliquus CNW-N cells on loofa (Luffa cylindrica) sponge.

A continuous fixed-bed biosorption process was established for cadmium (Cd) removal by Scenedesmus obliquus CNW-N (isolated from southern Taiwan) cells immobilized onto loofa sponge. This immobilized-cell biosorption process allows better recovery and reusability of the microalgal biomass. The growth of microalgae on the matrix support with appropriate nutrient supplementation could enhance the overall metal removal activity. Major operating parameters (e.g., feeding flow rate, cycle number of medium replacement, and particle diameter of the sponge) were studied for treatability evaluation. The most promising cell growth on the sponge support was obtained at a flow rate of 0.284 bed volume (BV)/min, sponge particle diameter of 1 cm, and with one cycle of medium replacement. The performance of fixed-bed biosorption (adsorption capacity of 38.4 mg, breakthrough time at 15.5 h) was achieved at a flow rate of 5 ml/min with an influent concentration of 7.5 mg Cd/l.

Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077.

The aim of present study was to investigate the effects of nitrogen limitation as well as sequential nitrogen starvation on morphological and biochemical changes in Scenedesmus sp. CCNM 1077. The results revealed that the nitrogen limitation and sequential nitrogen starvation conditions significantly decreases the photosynthetic activity as well as crude protein content in the organism, while dry cell weight and biomass productivity are largely unaffected up to nitrate concentration of about 30.87mg/L and 3 days nitrate limitation condition. Nitrate stress was found to have a significant effect on cell morphology of Scenedesmus sp. CCNM 1077. Total removal of nitrate from the growth medium resulted in highest lipid (27.93%) and carbohydrate content (45.74%), making it a potential feed stock for biodiesel and bio-ethanol production. This is a unique approach to understand morphological and biochemical changes in freshwater microalgae under nitrate limitation as well as sequential nitrate removal conditions.

Selection and evaluation of CO2 tolerant indigenous microalga Scenedesmus dimorphus for unsaturated fatty acid rich lipid production under different culture conditions.

Five indigenous microalgal strains of Scenedesmus, Chlorococcum, Coelastrum, and Ankistrodesmus genera, isolated from Indian fresh water habitats, were studied for carbon-dioxide tolerance and its effect on growth, lipid and fatty acid profile. Scenedesmus dimorphus strain showed maximum growth (1.5 g/L) and lipid content (17.83% w/w) under CO2 supplementation, hence selected for detailed evaluation. The selected strain was alkaline adapted but tolerated (i) wide range of pH (5-11); (ii) elevated salinity levels (up to 100 mM, NaCl) with low biomass yields and increased carotenoids (19.34 mg/g biomass); (iii) elevated CO2 levels up to 15% v/v with enhancement in specific growth rate (0.137 d(-1)), biomass yield (1.57 g/L), lipid content (19.6% w/w) and CO2 biofixation rate (0.174 g L(-1) d(-1)). Unsaturated fatty acid content (alpha linolenic acid) increased with CO2 supplementation in the strain.

The influence of ultrasound frequency and power, on the algal species Microcystis aeruginosa, Aphanizomenon flos-aquae, Scenedesmus subspicatus and Melosira sp.

We report on the effectiveness of sonication on controlling the growth of four problematic algal species which are morphologically different and from three algal divisions. Two cyanobacterial species Microcystis aeruginosa (unicellular) and Aphanizomenon flos-aquae (filamentous), one green alga Scenedesmus subspicatus (colonial) and lastly a diatom species Melosira sp. (filamentous) were subjected to ultrasound of selected low to high frequencies ranging from 20 to 1144 kHz. Microcystis aeruginosa and Scenedesmus subspicatus highest cell removal rates were 16 +/- 2% and 20 +/- 3% when treated with the same ultrasound frequency of 862 kHz but differing energy levels of 133 and 67 kWh m(-3), respectively. Aphanizomenon flos-aquae best removal rate was 99 +/- 1% after 862 kHz and 133 kWh m(-3) of energy, with Melosira sp. achieving its highest cell removal at 83% subsequent to ultrasound of 20 kHz and 19 kWh m(-3). Microcystis aeruginosa and Scenedesmus subspicatus are considered non-susceptible species to ultrasound treatment from a water treatment perspective due to their low cell removal rates; however, photosynthetic activity reduction of 65% for Microcystis aeruginosa does indicate the possible utilization of ultrasound to control bloom growth, rather than bloom elimination. Conversely, Aphanizomenon flos-aquae and Melosira sp. are deemed species highly susceptible to ultrasound. Morphological differences in shape (filamentous/non-filamentous) and cell wall structure (silica/peptidoglycan), and presence of gas vacuoles are probable reasons for these differing levels of susceptibility to ultrasound.