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.

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.

Sensing of phosphates by using luminescent Eu(III) and Tb(III) complexes: application to the microalgal cell Chlorella vulgaris.

Phenanthroline-based chiral ligands L(1) and L(2) as well as the corresponding Eu(III) and Tb(III) complexes were synthesized and characterized. The coordination compounds show red and green emission, which was explored for the sensing of a series of anions such as F(-), Cl(-), Br(-), I(-), NO3(-), NO2(-), HPO4(2-), HSO4(-), CH3COO(-), and HCO3(-). Among the anions, HPO4(2-) exhibited a strong response in the emission property of both europium(III) and terbium(III) complexes. The complexes showed interactions with the nucleoside phosphates adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). Owing to this recognition, these complexes have been applied as staining agents in the microalgal cell Chlorella vulgaris. The stained microalgal cells were monitored through fluorescence microscopy and scanning electron microscopy. Initially, the complexes bind to the outer cell wall and then enter the cell wall through holes in which they probably bind to phospholipids. This leads to a quenching of the luminescence properties.

Trapping of redox-mediators at the surface of Chlorella vulgaris leads to error in measurements of cell reducing power.

The reduction of the redox mediator ferricyanide, [Fe(CN)6](3-), by a range of algal and bacterial species, is frequently measured to probe plasma membrane ferrireductase activity or to quantify the reducing power of algal/bacterial biofilms and suspensions. In this study we have used rotating disk electrochemistry (RDE) to investigate the reduction of ferricyanide by the model organism Chlorella vulgaris. Importantly, we have seen that the diffusion limited current due to the oxidation of ferrocyanide, [Fe(CN)6](4-), at the electrode decreased linearly as C. vulgaris was added to the solution, even though in a pure ferrocyanide solution the algae are not able to reduce the mediator further and are simply spectator 'particles'. We attribute this effect to trapping of ferrocyanide at the cell surface, with up to 14% of the ferrocyanide missing from the solution at the highest cell concentration. The result has important implications for all techniques that use electrochemistry and other concentration dependent assays (e.g. fluorescence and colourimetry) to monitor ferrocyanide concentrations in the presence of both biofilms and cell suspensions. Analyte trapping could lead to a substantial underestimation of the concentration of reduced product.

Reduction in microparticle adsorption using a lateral interconnection method in a PDMS-based microfluidic device.

Microparticle adsorption on microchannel walls occurs frequently due to nonspecific interactions, decreasing operational performance in pressure-driven microfluidic systems. However, it is essential for delicate manipulation of microparticles or cells to maintain smooth fluid traffic. Here, we report a novel microparticle injection technique, which prevents particle loss, assisted by sample injection along the direction of fluid flow. Sample fluids, including microparticles, mammalian (U937), and green algae (Chlorella vulgaris) cells, were injected directly via a through hole drilled in the lateral direction, resulting in a significant reduction in microparticle attachment. For digital microfluidic application, the proposed regime achieved a twofold enhancement of single-cell encapsulation compared to the conventional encapsulation rate, based on a Poisson distribution, by reducing the number of empty droplets. This novel interconnection method can be straightforwardly integrated as a microparticle or cell injection component in integrated microfluidic systems.

Adhesion of algal cells to surfaces.

This paper reports the cell-substratum interactions of planktonic (Chlorella vulgaris) and benthic (Botryococcus sudeticus) freshwater green algae with hydrophilic (glass) and hydrophobic (indium tin oxide) substrata to determine the critical parameters controlling the adhesion of algal cells to surfaces. The surface properties of the algae and substrata were quantified by measuring contact angle, electrophoretic mobility, and streaming potential. Using these data, the cell-substratum interactions were modeled using thermodynamic, DLVO, and XDLVO approaches. Finally, the rate of attachment and the strength of adhesion of the algal cells were quantified using a parallel-plate flow chamber. The results indicated that (1) acid-base interactions played a critical role in the adhesion of algae, (2) the hydrophobic alga attached at a higher density and with a higher strength of adhesion on both substrata, and (3) the XDLVO model was the most accurate in predicting the density of cells and their strength of adhesion. These results can be used to select substrata to promote/inhibit the adhesion of algal cells to surfaces.

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.

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria.

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.

Synchronous-scan fluorescence of algal cells for toxicity assessment of heavy metals and herbicides.

Synchronous-scan spectrofluorometry was applied to Chlorella vulgaris cells to assess the toxicity of heavy metals and herbicides in water. Simultaneous scan of both the excitation and emission spectra was done at a constant wavelength difference Deltalambda (20-140 nm) between the emission and excitation wavelengths in the range of 420-700 nm emission, where a peak of fluorescence was observed. Its position depends on Deltalambda. Fluorescence measurements were conducted with algal cells in suspension in water and immobilized in a translucent silica matrix. The influence of toxic chemicals was tested with cadmium as a heavy metal and with atrazine, diuron, DNOC and paraquat as herbicides. The toxic effect of those chemicals mainly results in a quenching of algal cells fluorescence by reducing their photosynthetic activity.

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.

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 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.

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.

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).

Arsenic (V) induces a fluidization of algal cell and liposome membranes.

Arsenate is one of the most poisonous elements for living cells. When cells are exposed to arsenate, their life activities are immediately affected by various biochemical reactions, such as the binding of arsenic to membranes and the substitution of arsenic for phosphate or the choline head of phospholipids in the biological membranes. The effects of arsenate on the life activities of algae Chlorella vulgaris were investigated at various concentrations and exposure times. The results demonstrated that the living activities of algal cells (10(10)cells/L) were seriously affected by arsenate at a concentration of more than 7.5mg As/L within 24h. Algal cells and the artificial membranes (liposomes) were exposed to arsenate to evaluate its effects on the membrane fluidization. In the presence of arsenate, the membranes were fluidized due to the binding and substitution of arsenate groups for phosphates or the choline head on the their membrane surface. This fluidization of the biological membranes was considered to enhance the transport of toxicants across the membrane of algal cells.

Effect of iron on growth and lipid accumulation in Chlorella vulgaris.

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L(-1) FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed.

Effect of L-glutamic acid on the growth and ammonium removal from ammonium solution and natural wastewater by Chlorella vulgaris NTM06.

The main objective of this laboratory scale experiment was to study the effect of l-glutamic acid on the growth in media and removal of ammonium from ammonium solution and natural wastewater by Chlorella vulgaris NTM06. It was observed that higher levels (1.0% and 1.5%) of l-glutamic acid compared to control (0% l-glutamic acid) negatively affected growth of C. vulgaris NTM06 and enhanced removal of ammonium from ammonium solution as well as natural wastewater. After 24h of incubation, 99% of 169.3mg NH(4)(+)-N/l was removed from ammonium solution by 1.5% l-glutamic acid treated C. vulgairs NTM06 cultures; removal in case of control was 70%. In case of natural wastewaters with initial ammonium concentrations of 1550, 775, 310 and 155 mg NH(4)(+)-N/l, removal after 48 h of incubation were 60%, 88%, 61% and 55% respectively. Ammonium removals from ammonium solutions of pH 4.0-8.0 were similar, whereas adsorption of ammonium ions on to the surface of dead C. vulgaris NTM06 cells was around 11%. Compared to dark, cultures incubated under the light showed higher initial removal of ammonium, however, after 24h, differences were not significant. Further research on the role of l-glutamic acid in micro-algal treatment of wastewater and its combination with other approaches such as co-immobilization of micro-algae with other organisms, starvation of micro-algal cells and the use of polymers is recommended.

Effect of 2,4-dichlorophenoxyacetic acid on growth, protein and chlorophyll-a content of Chlorella vulgaris and Spirulina platensis cells.

In this study, effect of different 2,4 -dichlorophenoxyacetic acid (2,4-D) concentrations (0.0, 9.10(-5), 9.10(-4), 9.10(-3) and 9.10(-2) mM) on growth rate, content of protein and chlorophyll-a in Chlorella vulgaris and Spirulina platensis cells was investigated. The most stimulatory effect on growth rate, protein and pigment ratio of C. vulgaris and S. platensis was observed at 9.10(-4) mM concentrations of 2,4-D. The results show that low concentrations of 2,4-D have hormonal effect due to being a synthetic auxin. Cell number protein and pigment rates were inhibited at 9.10(-2) mM concentration in C. vulgaris. Such parameters were inhibited in S. platensis, both at 9.10(-3) and 9.10(-2) mM 2,4-D concentrations. This is due to herbicidal effect of high concentrations of 2,4-D. S. platensis was found to be more sensitive than S. vulgaris to 2,4-D applications. The use of algae as bio-indicators in herbicide contaminated fresh water habitats, was discussed.

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.