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Biochemistry & Analytical Biochemistry
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Quantifying Uptake and Retention of Copper Ions in Silica-Encrusted Chlamydomonas reinhardtii

Xiaohui Li1, Shanying Gui2, Mohammed Bhuiyan2, Weiqiao Zeng2, Yagya Subedi2, Rong Wang1 and Liaohai Chen2*

1Department of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA

2Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA

*Corresponding Author:
Dr. Liaohai Chen
Department of Chemistry and Biochemistry
Utah State University, Logan
UT, 84322, USA
Tel: (435) 797-8626
E-mail: [email protected]

Received Date: October 30, 2015; Accepted Date: November 26, 2015; Published Date: November 30, 2015

Citation: Li X, Gui S, Bhuiyan M, Zeng W, Subedi Y, et al. (2015) Quantifying Uptake and Retention of Copper Ions in Silica-Encrusted Chlamydomonas reinhardtii. Biochem Anal Biochem 4:228. doi:10.4172/2161-1009.1000228

Copyright: © 2015 Li X, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Using copper (II) ion as a model pollutant, we report a new bioremediation concept, which involves the use of green algae Chlamydomonas reinhardtii to efficiently collect copper ions from the solution, followed by encapsulating copper loaded algae with silica, thus reducing the bioavailability of copper ions in the solution. Specifically, the potential of Chlamydomonas reinhardtii as an active copper (II) absorbent was demonstrated by quantifying and characterizing the copper uptake rate, capacity, efficacy, as well as copper retention from C. reinhardtii. Subsequently, a method of encrusting copper loaded C. reinhardtii with silica was developed, taking the advantage that the presences of high abundant polysaccharides and glycoproteins on the cell walls, as well as the presence of (3-amino-propyl) trimethoxysilane (APS) can function as nucleation center for silicification process of tetramethyl orthosilicate (TMOS). Both fluorescence imaging and scanning electron microscope (SEM) imaging confirmed the silica encrustation. It is expected that silica encrustation of algae has the potential for in situ remediation of various contaminants in a wide range of environments while providing long-term stabilization and diminishing the bioavailability of the contaminants.


In vivo copper quantification; Chlamydomonas reinhardtii; Bioremediation; Silica encrustation


Effective management of contaminated marine and freshwater is a challenging problem with far-reaching economic and ecological consequences. Current technologies for remediating contaminated environmental water include both ex situ and in situ remediation. In ex situ remediation, water is forced to pass through immobilized absorbents and the contents are extracted from water and immobilized in the absorbents. ex situ remediation are expensive, as the need for large-scale water and absorption material handling. On the other hand, in situ remediation methods are less energy intensive and less expensive. Thus, it has the potential to remediate contaminated water in large scales.

in situ remediation applies absorbing materials such as activated charcoal or microbes to absorb contaminants [1-3]. Activated charcoal can strongly collect and concentrate various metal ions and organic compounds due to their porous structures. Yet, its unspecific absorbance leads to various organic and inorganic compounds competing for sorption sites, facilitating the release of contaminants to the environment. On the other hand, in situ bioremediation, using microbes to degrade or transform contaminants to less toxic and nontoxic forms, is being used to remediate certain contaminants, including heavy metal ions of uranium and cadmium, organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), organohalides, etc. [4,5]. However, the key drawbacks for in situ bioremediation are the limited availability of organisms to effectively metabolize/detoxify the contaminants as well as slow remediation rate due to the combination of respiratory and co-metabolic processes. Our research aims at tackling these challenges by exploring a new approach of not only efficiently absorbing contaminants, but also encrusting the absorbed contaminants to reduce their bioavailability in the aqueous environment.

Many algae/microbe species are capable of aggressively uptaking and accumulating various contaminants, including organic compounds, metal ions, and metalloids [6-9]. However, the use of marine and freshwater algae for conventional in situ bioremediation is limited because of the ineffective metabolism/detoxification process. We proposed a bold approach to develop a new remediation method by sealing the pregnant algae, thus dramatically reducing the bioavailability of the contaminants to the environment, instead of replying on alga’s metabolic detoxification process. The use of living algae as sorbents for both organic and metal (especially free metal ion and metalloid [8] contaminants could address complex contaminant mixtures in contaminated waters. More importantly, it is environment friendly. In this study, we used a well studied Chlamydomonas reinhardtii (C. reinhardtii) as a model microbe to demonstrate the proof-of-concept.

As one of the major biomineralization processes in nature, silicon mineralization is a widespread biological phenomenon observed in a large number of organisms. Silicon (Si), the second most abundant element on Earth, represents about one-fourth of the Earth’s crust mass in the form of silicon dioxide (or silica) and various silicates. Biospecies face a high concentration of free Si as silicic acid (70 μM in the oceans on average, and ~5 μM in surface waters) [10]. The interaction between algae cell wall and silicic acid in the environment leads to precipitation of minerals and formation of silica crust over living organisms, such as diatoms. The nucleation process occurs directly on the cell wall, resulting in biominerals firmly attaching to and encrusting the cells. The gravitation of encrustation and subsequent mineral growth can overcome buoyancy. Eventually mineral “rocks” of the encrusted cells are settled in water. In this paper, we reported such a new remediation process of using alga to actively uptake and collect metal copper ions, followed by the addition of silicification enhancer molecules and silicic acids to encrust copper-loaded algae. The “mineralized” copper-loaded algae can consequently form large insoluble silica “rocks” and settle in the water, thus leading to the reduction of bioavailability of copper ions in water.

Materials and Methods


C. reinhardtii 1 wild type (ATCC-18798TM) is used throughout the experiments. Copper (II) sulfate is used as copper source for all experiments. Hydroxylamine-hydrochloride (99.999% NH2OH·HCl), copper (II) sulfate pentahydrate (CuSO4·5H2O), disodium bathocuproine disulfonate (2,9-dimethyl-4,7-diphenyl- 1,10-phenanthrolinedisulfonic acid disodium salt, C12H4N2 (CH3)2(C6H4)2(SO3Na)2), 2-(N-morpholino)-ethanesulfonic acid (MES, pKa=6.1), Sodium dodecyl sulfate (SDS), Tetramethyl orthosilicate (≥99%, TMOS), (3-amino-propyl) trimethoxysilane (90%, APS), and fluorescein isothiocyanate isomer 1 (F7250) were purchased from Sigma-Aldrich and used as received.

Hewlett-Packard 8453 UV-Vis spectrophotometer was used for absorbance determination. Olympus IX70 fluorescence microscope was used for fluorescence imaging. High resolution FEI thermal field emission scanning electron microscope (SEM)-Nova NanoSEM 200 was used for SEM imaging.

Culturing C. reinhardtii

C. reinhardtii cells were grown in the modified Gorman and Levine (1965) TAP medium [11] adjusted to pH 7.2, and maintained in Erlenmeyer flasks with a 14:10 hr light–dark cycle using cool-white fluorescent light and a shake speed of 110 rpm at room temperature. All glassware used for culturing and testing was sterilized before using. The density of C. reinhardtii cells was determined by the optical density measurement of cell density at 550 nm. The cell suspension was diluted into the linear response region (absorbance < 1.5 OD), and the cell density for 1.0 OD was 5 ×106 cells/mL

Measurement of the copper (II) ion concentration

The copper (II) ion concentration in aqueous solution was measured according to the modified APHA standards 2005 [12]. Typically, in a 10 mL tube, 0.2 mL of 0.11 g/L hydroxylamine-hydrochiloride solution and 0.2 mL of 0.01 g/L disodium bathocuproine disulfonate solution were added into 2.0 mL of MES buffer (0.2 mol/L, pH=6.5). The absorbance was measured at 484 nm. All the samples tested were diluted to the linear response region of 0.02-4 ppm of copper (II) ion concentration. Since copper ion tends to be adsorbed on the surface of sample containers, samples were analyzed as soon as possible after collection.

Visualization of copper uptake using x-ray-fluorescence

X-ray optics at third-generation synchrotron sources is capable of visualizing and measuring intracellular metals in biological samples with a high sensitivity. In brief, C. reinhardtii cells were incubated with 5 μM copper (CuSO4.5H2O) at room temperature. After 5, 30, and 60 min, the cells were washed with 100 μM EDTA to remove any copper ions that were outside the cells. Copper-loaded cells were then fixed by glutaraldehyde and mounted on silicon nitride windows. Another algae sample without exposed to copper solution was also prepared as an endogenous copper control sample. All the samples were plunge frozen and X-ray fluorescence imaging was conducted in beamline 2-ID-E at the Advanced Photon Source (Argonne, IL).

Characterization of the copper (II) uptake and release process for C. reinhardtii

The copper (II) uptake tests were accomplished in the following manner. The pre-cultured algal cells in the middle of the logarithmic phase were collected by centrifugation at 600 × g for 6 min, and washed thoroughly with the uptake solution and then resuspended in a 250 ml Erlenmeyer flask with 25 mL solution containing copper (II), and then the cells were incubated under the regular cell culturing condition. After the desired incubation period (0, 15, 30, 60, 120, and 180 min), the aqueous solution were separated from the algae by centrifugation at 600 × g for 6 min, and the concentration of copper (II) ion in the supernatant was measured by the colorimetric method described above. The uptake cell density was determined by measuring the optical density at 550 nm. The uptake process was monitored for 3 hours unless specified.

The amount of copper (II) ions taken up per C. reinhardtii cell, q (μg copper /cell), was obtained by using the following expression:

q=[(C0-C)·Vcu ]/( d·Vcell ) ≈ (C0-C)/d (1)

where C0 and C are the concentrations of the heavy metal in the initial solution and after uptake, respectively (μg/mL, ppm); Vcu is the volume of the cell contained copper (II) solution (mL); Vcell is the volume of the copper (II) cell suspension (mL) and d is the density of C. reinhardtii cell (cell/mL). Since the sample for Cu2+ concentration measurement was from the same solution as samples for the cell density measurement, the value of Vcu is approximately equal to that of Vcel. Thus, copper (II) ion is independent of the volume change of the uptake solution. Each experiment was repeated three times and the results given were the average values with error bars indicating the standard deviation.

For the copper (II) release process, the algal cells incubated in the copper (II) solutions for 3 hours were collected by centrifugation at 600 × g for 6 min, and washed thoroughly with water or release solutions and then resuspended with 25 mL of water or release solutions without copper (II) ions. After the desired incubation period (0, 15, 30, 60, 120, and 180 min), the concentration of copper (II) ion and the cell density in this supernatant were measured as stated above. The release test was carried out in sterilized deionized H2O and the MES buffer (0.2 M, pH 6.5), respectively. Different chemical treatments for copper (II) release conditions included: 1) washing the cells with different buffer solution, 2) heating the cells up to 75°C for 1 hours, and 3) treating the cells with chemicals such as 1% SDS for 1 hour, and 70% HNO3 for 3 hours.

Encapsulation of algal cells with silica shells

The FITC-silanes were prepared by mixing 0.265 ml of APS and 5 μmol of fluorescein isothiocyanate (FITC) diluted in 5 ml anhydrous ethanol as a co-solvent [13]. The reaction mixture was stirred at ambient temperature for 3 hours. Water is excluded to prevent hydrolysis and condensation of APS molecules. And storage and synthesis with FITC are performed in the dark to avoid bleaching [14].

Silicic acid solution was freshly prepared by hydrolyzing 1 M TMOS in 1 mM HCl for 15 min at room temperature. The algal cells were suspended in 1 mL of phosphate buffer saline (PBS), followed by addition of 30 μL of pre-hydrolyzed TMOS for SiO2 shell formation [15]. 2 μL of APS was added to catalyze/initialize silica polymerization, and the sample was incubated for 5 min at ambient temperatures. Samples were washed 3 times with PBS buffer to remove free silicic acid, then resuspended in 1 mL PBS buffer followed by adding 10 μL of APS-FITC to coat the silica surface. Samples were washed 3 times with PBS buffer to remove free APS-FITC, and the washed pellets were resuspended in 0.5 mL of PBS buffer for direct visualization of silica shells on algae surface.

Image acquisition and processing

Fluorescence images were obtained using an Olympus IX70 Microscope. Transmitted illumination was provided by a tungstenhalogen lamphouse, and fluorescence illumination was provided by a mercury lamphouse. SEM images were obtained by firstly preparing the sample of silica encapsulated algal cells through fixation and dehydration steps. Algae samples were fixed with formaldehyde (final concentration 2.5%) for 2 hours at room temperature and then washed 3 times with distilled water, followed by washing with ethanol series of 50%, 70%, 80%, 90%, 95%, and 100%. Finally, the samples were coated with gold and analyzed on Nova NanoSEM 200.


Copper (II) uptake by C. reinhardtii

We first examined how different concentrations of Cu2+ ions could influence the growth of C. reinhardtii by monitoring the algae growth rate as a function of copper concentrations (data showed in Supplementary Materials). It was found that Cu2+ ion in the concentration range from 0 to 7.6 ppm is not likely to affect the growth of C. reinhardtii while significant inhibition appeared around 14.2 ppm of copper (II). At 56.9 ppm of copper (II), the algal growth was considerably prohibited (Figure S1). Accordingly, uptake studies were carried out using three different Cu2+ concentrations of 1.62 ppm (no influence), 15.6 ppm (inhibition influence), and 49.6 ppm (toxic influence) to evaluate the uptake efficiencies of C. reinhardtii against Cu2+ ions.

After incubating C. reinhardtii in copper (II) solutions, both concentrations of copper (II) that remained in the solution and the copper (II) concentration accumulated in the algae cells were measured using a Bathocuproine Method. When the Cu2+ concentration was 1.62 ppm, the copper (II) absorbed by C. reinhardtii rapidly increased during the first 30 min and then plateaued gradually within 60 min (Figure 1A). The copper (II) concentration in solution was reduced to 0.25 ppm, indicating that 84% of copper (II) ions were removed from the solution by C. reinhardtii cells (remediation efficiency, Figure 1A inset).


Figure 1: Uptake of Copper (II) on C. reinhardtii. (A) Concentrations of Cu (II) within the C. reinhardtii cells during uptake with the original Cu (II) concentration of 1.62 ppm. The insert graph illustrates the concentrations of Cu (II) in solution during the algae uptake process. (B) X-ray fluorescence images of C. reinhardtii cells with endogenous copper (left) and uptaken copper (right).

The amount of copper (II) absorbed within each cell was calculated based on Equation [1] with a cell density of approximately 7.3×106 cell/mL. The amount of copper (II) absorbed by each cell increased significantly from an undetectable level to 1.86×10-7 μg/cell (about 1.76×109 copper (II) ion/cell) during the uptake period. After this uptake period, the amount of biosorbed copper (II) ions on C. reinhardtii did not change significantly over time.

Copper ion uptake was also visualized directly by using synchrotron x-ray fluorescence. Recent developments in x-ray optics in third-generation synchrotron sources enable highly sensitive visualization and quantification of intracellular metals in biological samples. Accordingly, we applied x-ray fluorescence microscopy (XRF) to visualize and compare both endogenous cellular copper and endocytosed copper in algae. As shown in Figure 1B, algae cells exhibited maximum endogenous copper density of 0.0967 μg/cm2 (Figure 1B, left). However, copper loaded cells had maximum copper density of 9.11 μg/cm2 (Figure 1B, right) —100 times greater than the endogenous value. Since the cells were incubated and washed with EDTA solution multiple times, nonspecific absorption of copper ions on the surface of algae cells had been removed. Thus, the resulting XRF images illustrated the endocytosed coppers within the cells.

Uptake efficiencies were further studied at the copper concentrations of 1.62, 15.6, and 49.6 ppm in deionized H2O. As shown in Figure 2A, the threshold of copper (II) uptake occurred after 30 minutes, regardless of the concentration difference. With a higher initial Cu2+ concentration, more Cu2+ ions were absorbed by each algal cell (Table 1), indicating an increased uptake capacity (Figure 2B). However, with a higher Cu2+ concentration, the remediation efficiency decreased (Table 1), which was possibly due to the uptake capacity gradually saturated when the copper (II) concentration continually increased. The results suggested that C. reinhardtii cells have the ability to uptake large amount of copper (II) ions from aqueous systems, and the remediation efficiency displays a copper (II) concentration dependence.


Figure 2: (A) Effect of the initial concentrations of Cu (II) on the uptake rates by C. reinhardtii. (B) The uptake capacity of algae as a function of the original concentration of Cu (II) ions.

Initial [Cu2+] (ppm) [Cu2+] After Uptake (ppm) Cu2+Removal Efficiency Cell Density (cell/mL) Uptake Capacity (copper/cell)
1.62 0.25 84.5% 7.4×106 1.8×109
15.6 12.4 20.8% 4.3×106 7.3×109
49.6 42.2 14.8% 5.9×106 1.2×1010

Table 1: Effect of the initial concentration of Copper (II) on the uptake rates.

Copper release from copper-loaded C. reinhardtii

To evaluate the retention of copper ions in copper-loaded C. reinhardtii, we quantified the release of copper (II) associated with C. reinhardtii. Briefly, after isolating copper-loaded algae, the cells were incubated in pure water. The changes of Cu2+ concentrations in water were monitored, and the percentage of Cu2+ released to water was calculated. As shown in Figure 3A, within three-hour incubation, the Cu2+ concentration in solution remained lower than 0.1 ppm, and the amount of absorbed Cu2+ ions on C. reinhardtii did not change significantly with time. The release percentages under different original copper concentrations were calculated and listed in Table 2. Less than 5% of Cu2+ was released from C. reinhardtii at copper concentrations of 1.62 and 15.64 ppm, indicating strong binding between copper (II) and cell components of C. reinhardtii.


Figure 3: Characterization of the Copper (II) release process with different original Cu2+ concentrations (A) and different release conditions (B).

Initial [Cu2+] (ppm) [Cu2+] after Uptake (ppm) Cu2+Taken-Up (mg) Cu2+Released (mg) Release Percentage
1.62 0.25 34.2 1.6 4.69%
15.64 12.38 81.4 3.9 4.74%

Table 2: Percentage of Copper (II) released in water.

Furthermore, we studied the Cu2+ release from copper-loaded C. reinhardtii under the influence of chemical treatments. As shown in Figure 3B, less than 5% of copper (II) was released when copper-loaded cells were incubated with MES buffer containing 10% of EDTA. Only 9% of Cu2+ ions were released when the copper-loaded algal cells were washed 3 times with the MES buffer containing 10% of EDTA. Even when the solution of copper-loaded algal cells was heated for 2 hours, the amount of copper (II) released was negligible. This suggests that most of absorbed copper (II) ions are stably associated with algal cells either through cell wall conjugations or intracellular interactions.

When using much harsher conditions like SDS (to dissociate the algal cell) or concentrated HNO3 (to digest cells) treatment, significantly higher amounts of copper (II) were released into solution (24% and 60%, respectively), as shown in Figure 3B. Theoretically, HNO3 digestion could completely lyse algae cells and release 100% of the intracellular copper ions. However, the strong acidic condition could dramatically influence the absorbance of copper-bathocuproine complex in measurement; thus, 60% of releasing efficiency calculated for the HNO3 treatment may not reflect a true number. Nevertheless, these results suggested that C. reinhardtii is an excellent absorbent with great stability under mild conditions.

Silica encapsulation of copper loaded Chlamydomonas reinhardtii

To use algae as a bioremediation platform, the bioavailability of copper ions associated with copper-loaded C. reinhardtii needs to be reduced significantly in water. This was achieved by encrusting algae through biomineralization. The formation of inorganic crust over a living organism is a widespread biological phenomenon, and one of the major biomineralization processes is the deposition of silica in biologic systems. The chemical structure of cell wall of C. reinhardtii, which contains high abundance of hydroxyl (-OH) groups on the sugar/ hydroproline units and amino (-NH2) and carboxyl (-COOH) groups on the protein, can be advantage for silica deposition. We hypothesized that the cell walls of algae can serve as causal agents for nucleation of silica, polymerization of silicic acid, and adhesion to other silica materials for subsequent silica encapsulation, as shown in Figure 4A.


Figure 4: (a) Schematic illustration of silica encapsulation outside algal cells. (b) Chemical reaction of labeling APS with FITC. (c). Schematic representation of the silica crust surface coated with FITC-APS.

In brief, copper loaded algae cells were treated with APS and TMOS for silica condensation on the cell surface. To observe the silica encrustation under microscope, we also coated the algal cells with the fluorescent-labeled silane (APS-FITC) outside the SiO2 layer (Figure 4B and 4C). After silica encapsulation, the size and shape of C. reinhardtii cells ([email protected]2) remained similar (Figure 5a-5c). Due to the broad absorbance of the visible light region in C. reinhardtii, both fluorescence of APS-FITC and autofluorescence of algae can be observed when excited at 480 nm (Figure 5d and 5g). We also chose 535 nm as a control excitation wavelength, where APS-FITC shows no fluorescence (Figure 5d-5f). To demonstrate the green fluorescence from the silicified shell, we incubated the same amount of algae and [email protected]2 with the same amount of APS-FITC. The control group of algae treated with APSFITC only showed only weak fluorescence (Figure 5h) indicating that APS-FITC does not interact strongly with algal cells. On the other hand, most of the [email protected]2 cells exhibit strong fluorescence (Figure 5i) in a ring structure on their surface, indicating the presence of a shell structure after silica encapsulation.


Figure 5: Microscopic images of silica encapsulation of C. reinhardtii. Algal cells, algal cells+APS-FITC, and algal [email protected]2-APS-FITC: a-c, bright field images; d-f, fluorescence images using 535 nm excitation wavelength; and g-i, fluorescence images using 480 nm excitation wavelength.

The structure of encapsulated algae cells were further examined using scanning electron microscope (SEM). As shown in Figure 6, although [email protected]2 maintained its original shape, the surface became rougher after biomimetic silicification (Figure 6a and 6b) compared to the uncoated algae cells (Figure 6c). Moreover, the surface of algae@ SiO2 is composed of silica nanoparticles (Figure 6d). The SEM images confirmed the encapsulation of algal cells within silica shells.


Figure 6: Surface characteristics of [email protected]2 at different magnifications (a,b), free algae (c), and silica nanoparticles alone (d). The scale bars are 10 μm (a) and (c), 2 μm (b), and 500 nm (d). Inset figure in (b) shows the silica nanoparticles on the cell surface.

We further discovered that APS could function as an anchor molecule for silane condensation on the algal cell wall, thus enhancing the silica encapsulation process. Without APS added to the silane precursor (TMOS), the formed SiO2 did not nucleate on the cell surface (Figure 7a and 7c); instead, it self-nucleated in solution and condensed into irregularly shaped aggregates, leaving the cells uncovered with silica. In contrast, when APS was added to TMOS, it led to the nucleation of SiO2 around the algal cells and formed a silica shell to encapsulate algal cells (Figure 7b and 7d). Thus, APS facilitates the formation of silica crust around algal cells.


Figure 7: APS facilitates the silica encapsulation process. Low (a-b) and high (c-d) magnification fluorescence images excited at 480 nm of algal cell encapsulation without addition of APS after TMOS (a,c), and algal cell encapsulation with addition of APS after TMOS (b,d).

Silica encapsulation assisting copper removal from algae

We further tested whether silicification facilitates the copper (II) bioremediation. We quantified and compared the copper concentrations in solution before and after C. reinhardtii treatment as well as after silicification process. With an original Cu2+ concentration of 7.5 ppm, 2.1 ppm copper (II) remained in solution after incubation with algal cells (Figure 8). Furthermore, the amount of copper (II) that remained in solution was reduced to less than 1.0 ppm after silica encapsulation (Figure 8). This indicates that the silica condensation process could further trap copper ions within the silica shells. Overall, the combination of algae treatment and silicification process has the capability to remove 70%-80% of the copper (II) ions from water within 3 hours.


Figure 8: Efficiency of copper (II) removal by the [email protected]2 system before and after algae treatment and silica encapsulation.


Since C. reinhardtii can absorb an array of trace metals, including cobalt, nickel, lead, cadmium, and mercury [16-20], it is not surprising that we observed great copper uptake capability of C. reinhardtii. Although the growth of C. reinhardtii cells is sensitive to copper (Figure S1) [21-23], they exhibit an exceptional capacity for copper uptake (more than 109 copper ions per cell), and they can remove more than 80% of copper (II) in solution at a low copper (II) concentration (Table 1). At a high copper (II) concentration (49.6 ppm), the amount of copper (II) up-taken by C. reinhardtii increased by an order of magnitude, although with a lower remediation efficiency (~15%, Table 1). The low remediation efficiency probably resulted from the saturated absorbance of C. reinhardtii as well as the impaired growth of C. reinhardtii under such a high copper (II) concentration. Moreover, C. reinhardtii showed a remarkably low release percentage of the absorbed copper ions, unless treated with harsh chemical conditions, such as incubation with SDS or HNO3 (Figure 3). These results make C. reinhardtii a great choice for bioremediation.

To reduce the bioavailability of the absorbed copper (II) in algae, we performed biomimetic silicification under mild conditions (room temperature, normal pressure, and neutral pH) to encapsulate and encrust the algal cells with silica. Algal cells were successfully encapsulated with silica shells when APS was used. A plausible explanation for the function of APS is that, as a cation in neutral pH, it could create an anchor point for silane condensation between the hydrolyzed silane anions [24] and the negatively charged carboxyl and hydroxyl groups on the cell surface. At the same time, APS could provide a basic environment for the silicification process and be a good catalyst for the silica encapsulation by offering silicic acid a template for further crosslink. On the other hand, while other organo-alkoxysilanes rapidly condensate to insoluble precipitates, APS remains stable in solution because hydrolyzed APS forms a six- or five- membered chelate ring and sterically hinders condensation [14]. Thus, APS probably acts as a template and catalyst for the silicification process around the cells. With APS added, algal cells were encapsulated with silica shells, so that the algae could be easily removed from the environment due to the “precipitation”. Moreover, copper ions were further removed during silica encapsulation process (Figure 8). Because of the dense deposition of silica over the contaminant–loaded cells, we anticipate little, perhaps no bioavailability of contaminants, which prohibits the heavy metal contamination from re-entering the food chain.

Overall, the potential bioremediation method descried herein is different from “physical” remediation methods such as extraction or filtration, where infrastructure (filtration stations) and (electric) energy need to be involved. It is also different from the conventional bioremediation methods, which rely on the metabolism of microbes to remediation the contaminants. For algae, although they can very aggressively uptake/absorb metal and other contaminants, they lack the capability to convert/metabolize these contaminants. Thus contaminant-impregnated algae need to be separated/removed from water to reduce the contaminant’s bioavailability. While a filtration method could be used, it will be difficult, considering the size of algae unless they can be selectively bound to larger particles (such as charcoal). Precipitation of contaminant-impregnated algae via natural biomineralization/biosilicification processes offers a simple, economic and natural way to reduce the bioavailability of contaminants. Silicic acid is a “natural product” (70 μM in the oceans on average), a minimal environment impact by using silicic acid for sedimentation is expected. Given the fact that algae are very robust microbes that can live in hot, toxic metal-rich, acidic environments [25], and biomineralization/ biosilicification happen in both fresh and marine water, we anticipate the proposed approach has the potential to be extended to remediate real contaminated marine and freshwater.

We demonstrated that the copper level in a water samples can be reduced to less than 1 ppm from an initial concentration of ~50 ppm via alga treatment and silica-encrustation process. Considering the fact that typical copper concentrations in moderately polluted water/ sediment are in the range of 25-50 ppm, while sediments with copper concentrations less than 25 ppm are considered as non-polluted [26], the proposed bioremediation method has a potential capacity to deal with real contaminated samples.

In summary, by using C. reinhardtii to treat a metal ion solution, followed by silicification around the cells, we are able to remove 70%- 80% of the copper (II) ions from water within 3 hours. As a new strategy for bioremediation, which is still in an early, conceptual develop stage, additional studies need to be conducted with other pollutants to remediate pollution in different water systems.


We thank Dr. Lydia Finlay at Argonne National Laboratory for the help with x-ray fluorescence imaging.


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