Characterization of Particle Movement and High-Resolution Separation of Microalgal Cells via Induced-Charge Electroosmotic Advective Spiral Flow.

Microalgae are renewable, sustainable, and economical sources of biofuels and are capable of addressing pressing global demand for energy security. However, two challenging issues to produce high-level biofuels are to separate promising algal strains and protect biofuels from contamination of undesired bacteria, which rely on an economical and high-resolution separation technology. Separation technology based on induced-charge electroosmotic (ICEO) vortices offers excellent promise in economical microalga separation for producing biofuels because of its reconfigurable and flexible profiles and sensitive and precise selectivity. In this work, a practical ICEO vortex device is developed to facilitate high-resolution isolation of rich-lipid microalgae for the first time. We investigate electrokinetic equilibrium states of particles and particle-fluid ICEO effect in binary-particle manipulation. Nanoparticle separation is performed to demonstrate the feasibility and resolution of this device, yielding clear separation. Afterward, we leverage this technology in isolation of Chlorella vulgaris from heterogeneous microalgae with the purity exceeding 96.4%. Besides, this platform is successfully engineered for the extraction of single-cell Oocystis sp., obtaining the purity surpassing 95.2%. Moreover, with modulating parameters, we isolate desired-cell-number Oocystis sp. enabling us to investigate proliferation mode and carry out transcriptome analyses of Oocystis sp. for high-quality neutral lipids. This platform can be extended directly to economically separate other biological micro/nanosamples to address pressing issues, involving energy security, environmental monitoring, and disease diagnosis.

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.

Effects of mesotrione on oxidative stress, subcellular structure, and membrane integrity in Chlorella vulgaris.

Mesotrione is a selective herbicide used to prevent weed attack of corn. It is extensively used, and hence, is being increasingly detected in aquatic ecosystems and may exert adverse effects on aquatic organisms. To evaluate the effects of mesotrione on photosynthesis-related gene expression, antioxidant enzyme activities, subcellular structure, and membrane integrity in algal cells, a comprehensive study was conducted using the green alga, Chlorella vulgaris. Exposure to 4-50 mg/L mesotrione resulted in a progressive inhibition of cell growth, with a 96-h median inhibition concentration (96 h- ErC50) value of 18.8 mg/L. Further, 18 and 37.5 mg/L mesotrione affected the algal photosynthetic capacity by decreasing the cell pigment content and reducing transcript abundance of photosynthesis-related genes. Mesotrione induced oxidative stress, as confirmed by increased cellular levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and altered antioxidant enzyme activities. It also damaged the algal cellular structure, observed as plasmolysis, blurred organelle shape, and disruption of the chloroplast structure. Flow cytometry analysis revealed that mesotrione exposure led to uneven cell growth and interior irregularities in the algal cell. The apparent propidium iodide (PI) influx also confirmed that the herbicide induced damage of the cell membrane integrity. This study will facilitate the understanding of the physiological and morphological changes induced by mesotrione in C. vulgaris cells, and provide basic information for understanding the biological mechanisms of mesotrione-induced algal toxicity.

Aged microplastics polyvinyl chloride interact with copper and cause oxidative stress towards microalgae Chlorella vulgaris.

Microplastics (MPs) could pose potential risks to microalgae, the primary producer of marine ecosystems. Currently, few studies focus on the interaction of aged MPs with other pollutants and their toxic effects to microalgae. Therefore, the present study aimed to investigate i) the aging of microplastics polyvinyl chloride (mPVC) in simulated seawater and the changes in physical and chemical properties; ii) the effects of single mPVC (virgin and aged) and copper on microalgae Chlorella vulgaris; and iii) the interaction of aged mPVC and copper and the oxidative stress towards C. vulgaris. In this study, some wrinkles, rough and fractured surface textures can be observed on the aged mPVC, accompanying with increased hydroxyl groups and aromatic carbon-carbon double bond but decreased carbon hydrogen bond. It was found that single virgin or aged mPVC at low concentration (10 mg/L) had significant inhibition on the growth of C. vulgaris but no inhibition at higher concentration (100, 1,000 mg/L), which can be reasonably explained by the aggregation and precipitation of mPVC at high concentration. The aging of mPVC inhibited the growth of C. vulgaris with the maximum growth inhibition ratio (IR) of 35.26% as compared with that of virgin mPVC (IR = 28.5%). However, the single copper could significantly inhibit the growth of C. vulgaris and the inhibitory effects increased with concentration (0.2, 0.5, 1.0 mg/L). Furthermore, both the single aged mPVC (10 mg/L) and copper (0.5 mg/L) caused serious cell damage, although the concentration of superoxide dismutase (SOD) and the intracellular malonaldehyde (MDA) increased. In contrast to single treatment, the growth of C. vulgaris can be enhanced by the combined group with copper (0.5 mg/L) and aged mPVC (10 mg/L).

Effect of light on the transformation of BDE-47 by living and autoclaved cultures of Microcystis flos-aquae and Chlorella vulgaris.

Polybrominated diphenyl ethers (PBDEs) are ubiquitous and toxic contaminants found in high concentrations in watercourses, and are not well removed by conventional wastewater treatment facilities. This study aimed to evaluate the removal and transformation of BDE-47, one of the environmentally predominant PBDE congener, by a green alga (Chlorella vulgaris) and a cyanobacterium (Microcystis flos-aquae) under different light conditions. Living and autoclaved cultures were exposed to BDE-47 at a concentration of 10 µg L-1 for 7 days. Both species removed >90% of BDE-47 very shortly after spiking. Light intensity affected the transformation of BDE-47 in living cultures of both species, since 5 to 11 times more debromination products were measured at a light intensity of 100 µmol photons m-2 s-1 than at 20 µmol photons m-2 s-1. Living cultures of M. flos-aquae transformed BDE-47 at a rate of 0.22 day-1 while no transformation was observed in the respective autoclaved cultures. On the contrary, both living and autoclaved cultures of C. vulgaris had similar BDE-47 transformation rates of 0.05-0.06 day-1. Debromination of BDE-47 was a predominant transformation pathway in cultures of C. vulgaris, with two times higher BDE-28 concentrations measured than in M. flos-aquae, while hydroxylation was more dominant with the cyanobacterium. Most BDE-47 and its debromination product BDE-28 were found on the cell surface of both species. These results reveal that different transformation mechanisms were involved in C. vulgaris and M. flos-aquae cultures and confirm the importance of species selection for the removal of PBDEs from contaminated environments.

Quantifying silver nanoparticle association and elemental content in single cells using dual mass mode in quadrupole-based inductively coupled plasma-mass spectrometry.

In this study, a method of simultaneous dual mass detection for single cell analysis by quadrupole-based ICP-MS (ICP-QMS) is proposed. The method shows potential for use in quantitative investigations of nanoparticle association and elemental composition of cells. Dual mass detection had been attempted in the analysis of two-element core-shell nanoparticles and in isotope dilution analysis. In this method the detector switches between two selected masses during the analysis. Dual mass mode eliminates the discrepancies in signal that can occur due to sample instability or fluctuation in sample uptake when two masses are analysed sequentially by conventional single cell analysis (SP mode). Preliminary tests showed that using an Mg spike as marker of cells in dual mass mode was feasible for the quantification of cells. The method showed good linearity and a reproducible detection rate, and the results were comparable to the SP mode. The approach was then employed with algal cells exposed to silver nanoparticles (AgNP), to study on the Ag-associated cells and AgNP by monitoring the Ag and Mg signal in one analytical run. Finally, Mg and Mn were detected, and then quantified using the same approach to evaluate the elemental composition and correlation between different elements of the exposed cells. It is believed that this dual mass approach can extend the capability of ICP-QMS for multi-elemental detection at the single cell level, representing an enormous potential for size characterization, quantification and elemental composition evaluation in single cell (particle) analysis.

Growth and secretome analysis of possible synergistic interaction between green algae and cyanobacteria.

Synergistic coexistence of nitrogen fixing cyanobacteria such as Anabaena variabilis, Nostoc muscorum and Westiellopsis prolifica with green algae namely Scenedesmus obliquus, Chlorella vulgaris and Botryococcus braunii was studied under nitrogen deficient conditions. The effect of these interactions was investigated on growth, fixed nitrogen content, lipid content and their secretomes in individual cultures and cocultures. Based on the cocultivation studies, it was found that out of the nine interactions studied, B. braunii-N. muscorum synergism was best established. This interaction resulted in a maximum of 50% enhancement in nitrogen fixation in B. braunii-N. muscorum co-culture leading to 27% enhancement in lipid content (membrane and neutral lipid). In general, B. braunii co-cultures showed an enhancement in biomass content of up to 38%. Secretome analysis showed presence of new and modified secondary metabolites having roles in quorum sensing/quenching, interspecies signaling, N-fixation, carbon metabolism, lipid metabolism, antimicrobial activity. Compounds such as trichloroacetic acid and hexadecane were identified that are known to have roles in nitrogen assimilation and carbon metabolism, respectively, were present in some of the co-culture secretomes. The combination of B. braunii-N. muscorum led to the formation of new compounds such as triacontanol which have role in improvement of glucose-lipid metabolism and 9-octadecenamide that is known to be a phytohormone.

Auto-flocculation through cultivation of Chlorella vulgaris in seafood wastewater discharge: Influence of culture conditions on microalgae growth and nutrient removal.

Nowadays, the pretreatment of wastewater prior to discharge is very important in various industries as the wastewater without any treatment contains high organic pollution loads that would pollute the receiving waterbody and potentially cause eutrophication and oxygen depletion to aquatic life. The reuse of seafood wastewater discharge in microalgae cultivation offers beneficial purposes such as reduced processing cost for wastewater treatment, replenishing ground water basin as well as financial savings for microalgae cultivation. In this paper, the cultivation of Chlorella vulgaris with an initial concentration of 0.01 ± 0.001 g⋅L-1 using seafood sewage discharge under sunlight and fluorescent illumination was investigated in laboratory-scale without adjusting mineral nutrients and pH. The ability of nutrient removal under different lighting conditions, the metabolism of C. vulgaris and new medium as well as the occurrence of auto-flocculation of microalgae biomass were evaluated for 14 days. The results showed that different illumination sources did not influence the microalgae growth, chemical oxygen demand (COD) and biochemical oxygen demand (BOD) significantly. However, the total nitrogen (total-N) and total phosphorus (total-P) contents of microalgae were sensitive to the illumination mode. The amount of COD, BOD, total-N and total-P were decreased by 88%, 81%, 95%, and 83% under sunlight mode and 81%, 74%, 79%, and 72% under fluorescent illumination, respectively. Furthermore, microalgae were auto-flocculated at the final days of cultivation with maximum biomass concentration of 0.49 ± 0.01 g⋅L-1, and the pH value had increased to pH 9.8 ± 0.1 under sunlight illumination.

Effect of metformin exposure on growth and photosynthetic performance in the unicellular freshwater chlorophyte, Chlorella vulgaris.

Many pharmaceuticals have negative effects on biota when released into the environment. For example, recent work has shown that the commonly prescribed antidiabetic drug, metformin (N,N-dimethylbiguanide), has endocrine disrupting effects on fish. However, effects of metformin on aquatic primary producers are poorly known. We exposed cultured isolates of a freshwater chlorophyte, Chlorella vulgaris, to a range of metformin concentrations (0-767.9 mg L-1) to test the hypothesis that exposure negatively affects photosynthesis and growth. A cessation of growth, increase in non-photochemical quenching (NPQ, NPQmax), and reduced electron transport rate (ETR) were observed 24 h after exposure to a metformin concentration of 767.8 mg L-1 (4.6 mM). By 48 h, photosynthetic efficiency of photosystem II (Fv/Fm), α, the initial slope of the ETR-irradiance curve, and Ek (minimum irradiance required to saturate photosynthesis) were reduced. At a lower concentration (76.8 mg L-1), negative effects on photosynthesis (increase in NPQ, decrease in ETR) were delayed, occurring between 72 and 96 h. No negative effects on photosynthesis were observed at an exposure concentration of 1.5 mg L-1. It is likely that metformin impairs photosynthesis either through downstream effects from inhibition of complex I of the electron transport chain or via activation of the enzyme, SnRK1 (sucrose non-fermenting-related kinase 1), which acts as a cellular energy regulator in plants and algae and is an ortholog of the mammalian target of metformin, AMPK (5' adenosine monophosphate-activated protein kinase).

Antagonistic effect of nano-ZnO and cetyltrimethyl ammonium chloride on the growth of Chlorella vulgaris: Dissolution and accumulation of nano-ZnO.

The interaction of nanoparticles with coexisting chemicals affects the fate and transport of nanoparticles, as well as their combined effects on aquatic organisms. Here, we evaluated the joint effect of ZnO nanoparticle (nano-ZnO) and cetyltrimethyl ammonium chloride (CTAC) on the growth of Chlorella vulgaris and explored the possible mechanism. Results showed that an antagonistic effect of nano-ZnO and CTAC (0.1, 0.2 and 0.3 mg L-1) was found because CTAC stop nano-ZnO being broken down into solution zinc ions (Zn2+). In the presence of CTAC, the zinc (including nano-ZnO and released Zn2+) showed a higher adsorption on bound extracellular polymeric substances (B-EPS) but lower accumulation in the algal cells. Moreover, we directly demonstrated that nano-ZnO was adsorbed on the algal B-EPS and entered into the algal cells by transmission electron microscope coupled with energy dispersive X-ray (TEM-EDX). Hence, these results suggested that the combined system of nano-ZnO and CTAC exhibited an antagonistic effect due to the inhibition of CTAC on dissolution of nano-ZnO and accumulation of the zinc in the algal cells.

Comparison of Chlorella vulgaris biomass productivity cultivated in biofilm and suspension from the aspect of light transmission and microalgae affinity to carbon dioxide.

To investigate light transmission and cells affinity to CO2, Chlorellavulgaris was attached to microfiltration membrane that laid on the solidified BG11 medium compared to that in suspended cultivation mode in this study. The results showed that C. vulgaris showed a 30.4% higher biomass production (103gm-2) in attached than in suspend system. The upper layer of biofilm with a thickness of 41.31µm (the corresponding areal density of 40gm-2) was effectively illuminated under light intensity of 120µmolm-2s-1 and more than 40% of the microalgal cells were in light even the areal density was high to 100gm-2. While only 2.5% of the cells were effectively illuminated in the suspended cultivation system. Furthermore, microalgae cells in biofilm showed a higher affinity to CO2 compared with that in suspension, and CO2 saturation point of microalgae cells in biofilm was 1.5% but 4.5% in suspension.

Co-cultivation of microalgae and nitrifiers for higher biomass production and better carbon capture.

The aim of this work was to study co-cultivation of nitrifiers with microalgae as a non-intrusive technique for selective removal of oxygen generated by microalgae. Biomass concentration was, at least, 23% higher in mixed-cultures where nitrifiers kept the dissolved oxygen concentration below 9.0µLL(-1) than in control Chlorella vulgaris axenic-cultures where the concentration of dissolved oxygen was higher than 10.0µLL(-1). This approach to eliminating oxygen inhibition of microalgal growth could become the basis for the development of advanced microalgae reactors for removal of CO2 from the atmosphere, and concentrated CO2 streams. CO2 sequestration would become a chemically and geologically safer and environmentally more sound technology provided it uses microalgal, or other biomass, instead of CO2, for carbon storage.

Can spherical eukaryotic microalgae cells be treated as optically homogeneous?

This study aims to answer the question of whether spherical unicellular photoautotrophic eukaryotic microalgae cells, consisting of various intracellular compartments with their respective optical properties, can be modeled as homogeneous spheres with some effective complex index of refraction. The spectral radiation characteristics in the photosynthetically active region of a spherical heterogeneous microalgae cell, representative of Chlamydomonas reinhardtii and consisting of spherical compartments corresponding to the cell wall, cytoplasm, chloroplast, nucleus, and mitochondria, were estimated using the superposition T-matrix method. The effects of the presence of intracellular lipids and/or starch accumulation caused by stresses, such as nitrogen limitation, were explored. Predictions by the T-matrix method were qualitatively and quantitatively consistent with experimental measurements for various microalgae species. The volume-equivalent homogeneous sphere approximation with volume-averaged effective complex index of refraction gave accurate estimates of the spectral (i) absorption and (ii) scattering cross sections of the heterogeneous cells under both nitrogen-replete and nitrogen-limited conditions. In addition, the effect of a strongly refracting cell wall, representative of Chlorella vulgaris, was investigated. In this case, for the purpose of predicting their integral radiation characteristics, the microalgae should be represented as a coated sphere with a coating corresponding to the cell wall and a homogeneous core with volume-averaged complex index of refraction for the rest of the cell. However, both homogeneous sphere and coated sphere approximations predicted strong resonances in the scattering phase function and spectral backscattering cross section that were not observed in that of the heterogeneous cells.

Effect of Ethephon as an Ethylene-Releasing Compound on the Metabolic Profile of Chlorella vulgaris.

In this study, Chlorella vulgaris (C. vulgaris) was treated with ethephon at low (50 µM) and high (200 µM) concentrations in medium and harvested at 0, 7, and 14 days, respectively. The presence of ethephon led to significant metabolic changes in C. vulgaris, with significantly higher levels of α-tocopherol, γ-aminobutyric acid (GABA), asparagine, and proline, but lower levels of glycine, citrate, and galactose relative to control. Ethephon induced increases in saturated fatty acids but decreases in unsaturated fatty acids. The levels of highly saturated sulfoquinovosyldiacylglycerol species and palmitic acid bound phospholipids were increased on day 7 of ethephon treatment. Among the metabolites, the productivities of α-tocopherol (0.70 µg/L/day) and GABA (1.90 µg/L/day) were highest for 50 and 200 µM ethephon on day 7, respectively. We propose that ethephon treatment involves various metabolic processes in C. vulgaris and can be an efficient way to enrich the contents of α-tocopherol and GABA.

Enhanced Harvesting of Chlorella vulgaris Using Combined Flocculants.

In this study, a novel flocculation strategy for harvesting Chlorella vulgaris with combined flocculants, poly (γ-glutamic acid) (γ-PGA) and calcium oxide (CaO), has been developed. The effect of flocculant dosage, the order of flocculant addition, mixing speed, and growth stage on the harvesting efficiency was evaluated. Results showed that the flocculation using combined flocculants significantly decreases the flocculant dosage and settling time compared with control. It was also found that CaO and γ-PGA influenced microalgal flocculation by changing the zeta potential of cells and pH of microalgal suspension. The most suitable order of flocculant addition was CaO first and then γ-PGA. The optimal mixing speed was 200 rpm for 0.5 min, followed by 50 rpm for another 4.5 min for CaO and γ-PGA with the highest flocculation efficiency of 95 % and a concentration factor of 35.5. The biomass concentration and lipid yield of the culture reusing the flocculated medium were similar to those when a fresh medium was used. Overall, the proposed method requires low energy input, alleviates biomass and water contamination, and reduces utilization of water resources and is feasible for harvesting C. vulgaris for biofuel and other bio-based chemical production.

Chlorella vulgaris as a lipid source: Cultivation on air and seawater-simulating medium in a helicoidal photobioreactor.

The freshwater microalga Chlorella vulgaris was cultured batchwise on the seawater-simulating Schlösser medium either in a 1.1-L-working volume helicoidal photobioreactor (HeP) or Erlenmeyer flask (EF) as control and continuously supplying air as CO2 source. In these systems, maximum biomass concentration reached 1.65 ± 0.17 g L(-1) and 1.25 ± 0.06 g L(-1) , and maximum cell productivity 197.6 ± 20.4 mg L(-1)  day(-1) and 160.8 ± 12.2 mg L(-1)  day(-1) , respectively. Compared to the Bold's Basal medium, commonly employed to cultivate this microorganism on a bench-scale, the Schlösser medium ensured significant increases in all the growth parameters, namely maximum cell concentration (268% in EF and 126% in HeP), maximum biomass productivity (554% in EF and 72% in HeP), average specific growth rate (67% in EF and 42% in HeP), and maximum specific growth rate (233% in EF and 22% in HeP). The lipid fraction of biomass collected at the end of runs was analyzed in terms of both lipid content and fatty acid profile. It was found that the seawater-simulating medium, despite of a 56-63% reduction of the overall biomass lipid content compared to the Bold's Basal one, led in HeP to significant increases in both the glycerides-to-total lipid ratio and polyunsaturated fatty acid content compared to the other conditions taken as an average. These results as a whole suggest that the HeP configuration could be a successful alternative to the present means to cultivate C. vulgaris as a lipid source. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:279-284, 2016.

Aggregate formation affects ultrasonic disruption of microalgal cells.

Ultrasonication is a cell disruption process of low energy efficiency. This study dosed K(+), Ca(2+) and Al(3+) to Chlorella vulgaris cultured in Bold's Basal Medium at 25°C and measured the degree of cell disruption under ultrasonication. Adding these metal ions yielded less negatively charged surfaces of cells, while with the latter two ions large and compact cell aggregates were formed. The degree of cell disruption followed: control=K(+)>Ca(2+)>Al(3+) samples. Surface charges of cells and microbubbles have minimal effects on the microbubble number in the proximity of the microalgal cells. Conversely, cell aggregates with large size and compact interior resist cell disruption under ultrasonication. Staining tests revealed high diffusional resistance of stains over the aggregate interior. Microbubbles may not be effective generated and collapsed inside the compact aggregates, hence leading to low cell disruption efficiencies. Effective coagulation/flocculation in cell harvesting may lead to adverse effect on subsequent cell disruption efficiency.

Envelopment-Internalization Synergistic Effects and Metabolic Mechanisms of Graphene Oxide on Single-Cell Chlorella vulgaris Are Dependent on the Nanomaterial Particle Size.

The interactions between nanomaterials and cells are fundamental in biological responses to nanomaterials. However, the size-dependent synergistic effects of envelopment and internalization as well as the metabolic mechanisms of nanomaterials have remained unknown. The nanomaterials tested here were larger graphene oxide nanosheets (GONS) and small graphene oxide quantum dots (GOQD). GONS intensively entrapped single-celled Chlorella vulgaris, and envelopment by GONS reduced the cell permeability. In contrast, GOQD-induced remarkable shrinkage of the plasma membrane and then enhanced cell permeability through strong internalization effects such as plasmolysis, uptake of nanomaterials, an oxidative stress increase, and inhibition of cell division and chlorophyll biosynthesis. Metabolomics analysis showed that amino acid metabolism was sensitive to nanomaterial exposure. Shrinkage of the plasma membrane is proposed to be linked to increases in the isoleucine levels. The inhibition of cell division and chlorophyll a biosynthesis was associated with decreases in aspartic acid and serine, the precursors of chlorophyll a. The increases in mitochondrial membrane potential loss and oxidative stress were correlated with an increase in linolenic acid. The above metabolites can be used as indicators of the corresponding biological responses. These results enhance our systemic understanding of the size-dependent biological effects of nanomaterials.

Combined nitrogen limitation and cadmium stress stimulate total carbohydrates, lipids, protein and amino acid accumulation in Chlorella vulgaris (Trebouxiophyceae).

Metals have interactive effects on the uptake and metabolism of nutrients in microalgae. However, the effect of trace metal toxicity on amino acid composition of Chlorella vulgaris as a function of varying nitrogen concentrations is not known. In this research, C. vulgaris was used to investigate the influence of cadmium (10(-7) and 2.0×10(-8)molL(-1) Cd) under varying nitrogen (2.9×10(-6), 1.1×10(-5) and 1.1×10(-3)molL(-1)N) concentrations on its growth rate, biomass and biochemical composition. Total carbohydrates, total proteins, total lipids, as well as individual amino acid proportions were determined. The combination of Cd stress and N limitation significantly inhibited growth rate and cell density of C. vulgaris. However, increasing N limitation and Cd stress stimulated higher dry weight and chlorophyll a production per cell. Furthermore, biomolecules like total proteins, carbohydrates and lipids increased with increasing N limitation and Cd stress. Ketogenic and glucogenic amino acids were accumulated under the stress conditions investigated in the present study. Amino acids involved in metal chelation like proline, histidine and glutamine were significantly increased after exposure to combined Cd stress and N limitation. We conclude that N limitation and Cd stress affects the physiology of C. vulgaris by not only decreasing its growth but also stimulating biomolecule production.

Rapid one-step purification of single-cells encapsulated in alginate microcapsules from oil to aqueous phase using a hydrophobic filter paper: implications for single-cell experiments.

By virtue of the biocompatibility and physical properties of hydrogel, picoliter-sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one-step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil-suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF-7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (~10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long-term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single-cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single-cell experiments.