The Effects of CdSe/ZnS Quantum Dots on the Photosynthesis Rate of the Chlorella Vulgaris Beads

Photosynthesizing microalgae produce more than 50% of oxygen in the atmosphere and are crucial for the survival of many living systems such as coral reefs. To address the declining of coral reefs, artificial reefs have been introduced to encapsulate the aglae cells in a polymer matrix but the effects of nanoscale pollutants on these engineered systems have not been fully understood. In this work, quantum dots with a size smaller than 10 nm are being used to elucidate the photosynthesis performance of the sodium alginate beads encapsulated with Chlorella vulgaris (C. vulgaris). The fluorescent quantum dots can move into the alginate matrix and the fluorescence intensity in the algae beads is correlated with the quantum dot concentration. We further show that the photosynthesis of the algae beads are sensitive to the quantum dot concentration and are also time sensitive. In the first 48 min of quantum dot exposure, both carbon dioxide absorption and oxygen production are low, suggesting limited photosynthesis. After the initial incubation, the photosynthesis rate quickly increases even though more inhibition is still observed with higher concentration of the quantum dots.


Introduction
Green algae, such as Chlorella vulgaris (C. vulgaris), has shown great potential to be used in pollution control 1 , biofuel 2 , and dietary supplement 3 . Containing chloroplasts, algae can photosynthesize and produce half of the atmospheric oxygen 4 . They are also crucial to the marine systems via the formation of symbiotic relationships with other organisms, particularly coral reefs. Coral reefs play an essential role in shoreline protection by reducing the wave energy by 97% 5 . However, the reefs have decreased by ~50% since 1950 due to rapid global warming, thus increasing the risks of global natural hazards 6 . A recent study has shown that heat-evolved algae can endure elevated temperatures and enhance coral bleaching tolerance to marine heat waves 7 . To further enhance the photosynthetic efficiency of coral reefs and potentially create artificial living reefs to combat climate change, 3-D printed structures containing algae cells have been introduced to mimic the morphological features of coral reefs 8,9 . Alginate hydrogel shows great promise as the scaffold material, in which the cells are viable for several days without nutrition 10 .
Another emerging challenge for the coral reefs and future engineering living reefs is the continuous exposure to chemical waste, especially at nanoscale 11,12 . Early results reported decreases in cell growth rate and chlorophyll content when the cell cultures were exposed to nanomaterials such as quantum dots 13 , nanoplastics 14 , and oxide nanoparticles 15 . On the other hand, gold nanoparticles can enhance photosynthesis in microalgae by transferring light into photogenerated electrons. A 42.7% increase in the carotenoids has been reported with this method 16 . To date, most of the research on nanomaterials/microalgae interactions has focused on cell cultures in solution, and little has been investigated with the immobilized microalgae cells that can be constructed as engineering living systems.
Here, we use quantum dots as a model to study the interaction of nanostructures with engineering living systems by encapsulating C. vulgaris in sodium alginate beads (Figure 1a). We show that more quantum dots are presented in the algae beads as the quantum dot concentration in the solution increases. We apply the bicarbonate indicator and gas chromatography-mass spectrometry to study the effect of quantum dots on algae photosynthesis. In the first 48 min after incubation, the photosynthesis is significantly inhibited by the presence of quantum dots. However, the photosynthesis rate increases after the initial incubation, suggesting that some of the quantum dots might be released or digested by the algae cells. At 120 min, algae beads incubated with quantum dots show less oxygen production than the samples without quantum dots, and the oxygen production is inversely correlated with the quantum dot concentration. The results indicate that the microalgae-based living systems are sensitive to the environment with nanoscale pollutants.  Bright-field and fluorescence imaging: The microalgae bead was dissected into hemispheres and prepared on a wet glass slide with a cover. The slide was then viewed using the Bright Field mode of an Amscope XD-RFL microscope. Bright-field imaging was used to identify clusters of the C. vulgaris cells and to quantify the relative concentration of the cells. The Amscope XD-RFL was also used to image the slides fluorescently.

Materials
TEM imaging: Intact microalgae beads were first fixed with 2% glutaraldehyde in 0.2 M cacodylate buffer for 2 hr. They were then rinsed three times (15 min each) in 0.2 M cacodylate buffer. They were then postfixed in 1% OsO4 in 0.2 M cacodylate buffer for two hours, followed by three rinses (15 min each) in 0.2 M cacodylate buffer. The beads were then dehydrated in a series of increasing concentrations (25%, 50%, 75%, and 95%) of ethanol for 10 min at each concentration. Finally, the beads were immersed in 100% acetone for 10 min. The dehydrated beads were immersed in 3/1 acetone/Embed 812 (Electron Microscopy Sciences) for 12 hr, followed by 1/1 acetone/Embed 812 for four hr, followed by 3/1 acetone/Embed 812 for four hr. The beads were then embedded in Embed 812 for 12 hr, followed by polymerization at 60 o C for 12 hr.
Thin sections (70 nm) for imaging were made with a Leica Artos 3D ultramicrotome. The thin sections were floated on water and transferred to 150 mesh copper grids. Staining was performed with UranyLess EM stain (Electron Microscopy Sciences) for two min, followed by two 15 s rinses in distilled water.
Imaging was performed with a JEOL 2010 transmission electron microscope operating at 200 kV. Images were captured with an AMT digital camera.

Quantification of fluorescence:
The fluorescence images were quantified using Fiji ImageJ. Several areas of cells containing quantum dot fluorescence were analyzed to find integrated density. Several areas of background were analyzed to find mean background fluorescence. Thereby the ImageJ program could calculate a baseline for fluorescence which could be used to correct the average fluorescence found from the selected areas of note, using the equation: The area of fluorescence was identified by using threshold values of 83 and 87 HSG, the values most closely matched the emission of the quantum dots, to isolate the fluorescence of the quantum dots. After isolation, the area of these regions was integrated to obtain a total area of fluorescence.
Production of CO2: Stored CO2 was generated by the chemical reaction: Two 4-liter sealable glass containers were connected by Teflon tubing. We added 22 mL of HCl to one of the containers and followed by another 47g of CaCO3. The glass was then promptly sealed, only allowing gas to pass through the Teflon tubing into the second container, separating CO2 from other products. Using a thin 100 µL Hamilton syringe, 3.6 L of CO2 gas was extracted from the second container for later use.

Results and Discussions
The microalgae beads are large in size (diameter: 1.5 mm) and tend to sediment to the bottom of the microtube. We exploited a short and low-speed centrifuge process to facilitate the interaction between quantum dots and algae beads. As shown in Figure 2,  24 . This mechanism would decrease photosynthesis, but not cellular respiration, unlike the inhibition of the cell walls.
We found that the photosynthetic rate increases over time for the algae beads incubated with the quantum dots, regardless of the quantum dot concentration. For example, at 72 min, the O2 level is only 0.3% for the 0.1 µL sample, compared to 0.8% without quantum dots. On the other hand, at 120 min, the difference between the two samples decreases to ~0.3%. As the pore size on the cell wall is 5-20 nm, the quantum dots can enter the cell wall through endocytosis to damage the cell structure, thus lowering the photosynthetic efficiency. However, the ubiquitous rate increase for all the samples indicates that the cells can adapt to the hazardous environment and still perform photosynthesis by either exocytosis or quantum dot digestion 25,26 .