Heavy metal contamination presents a constant threat to biological systems. Simultaneously, heavy metals have become one of the major contaminants in the aquatic ecosystem. In this regard, the investigation of heavy metal-tolerance genes in algae is relevant. Chlamydomonas reinhardtii is a unicellular green alga, and an excellent model organism used in heavy metal studies. In C. reinhardtii, a novel gene designated as Cia7, was hypothesized to play a role in heavy metal homeostasis due to CIA7’s conserved cysteine-residue motif. This study compared two strains of C. reinhardtii, cc4425, the wild-type possessing the functional CIA7 protein and cc5013, the mutant strain possessing the disrupted cia7- gene. The hypothesis was that the expression of Cia7 contributes to an increased cadmium (Cd)-tolerance in C. reinhardtii. The Cd-tolerance would be described by physiological markers of microalgae health, and by intracellular accumulation of the metal. Methods: The objectives of this study were (1) to compare chlorophyll fluorescence and cell size in cc4425 and cc5013 exposed to Cd²⁺, and (2) to compare Cd²⁺ bioaccumulation in cc4425 and cc5013 strains in different growth media. Flow cytometry, and inductively coupled plasma optical emission spectrometry (ICP-OES) analysis were performed. Results: There was no significant statistical difference in Cd²⁺ bioaccumulation between the two strains, cc4425 and cc5013, regardless of growth media. However, a statistically significant difference in Cd²⁺ bioaccumulation (p<0.0001) was determined between the media (with acetate and without acetate). The cia7- mutant, cc5013 was found to be more susceptible to a Cd²⁺-induced decrease in chlorophyll fluorescence and had a reduced cell size compared to cc4425, the wild-type strain. Conclusions: These observed differences between the strains suggest that CIA7’s biological activity could play a direct or indirect role in increasing Cd tolerance in C. reinhardtii.

Cell growth: Liquid cultures were initiated in 50 mL of TAP medium by using two loopfuls (4 mm spherical scraping) of cells for inoculation. Culture flasks were placed in an orbital shaker (VWR DS2-500-1) at 130 rpm under normal fluorescent room lighting until cultures reached optical density (OD650) of 0.800 +/- 0.050, measured using a Spectronic Genesys 8 spectrophotometer. This OD650 of 0.800 has been calibrated to be equal to 1.0-3.0 x 10⁶ cells/mL which is log phase in C. reinhardtii. The data were generated from a 2 x 2 x 2 factorial experiment in randomized complete block design.  The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), two levels of Cd concentration (0 µM, 10 µM), and two levels of media (1/4X TAP, Min). In this experiment there were 3 blocks.

Flow cytometry: Three replicates (in this experiment blocks are synonymous to replicates) with three trials were performed per concentration and per strain. Chlorophyll intensity and cell size were taken using a Cytek DxP8 FACSCalibur Flow Cytometer acquiring 10,000 events. Chlorophyll intensity was determined by utilizing laser BluFL3 with a wavelength filter 695/40 nm. Cell size was determined as a function of the forward scatter signal (FSC). Cell cultures cc4425 and cc5013 were treated with 0, 5, and 10 µM CdCl2 at midlog phase (OD650 = 0.400). Samples were analyzed at 24-hour time points for a duration of 96 hours via flow cytometry.

The data were generated from a 2 x 3 x 4 factorial experiment in randomized complete block design. The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), three levels of Cd concentration (0 µM, 5 µM, and 10 µM), and four levels of time (1, 2, 3, and 4 days).

ICP-OES: The treatment period was 4 days, after which cells reached the logarithmic phase of growth. On the 4th day, cells were equilibrated at OD650 = 0.800 (an absorbance at log phase) and harvested by centrifugation at 3,000 rpm for 10 minutes. The pellets were washed twice with millipore H2O, then resuspended, followed by centrifugation at 3,000 rpm for 5 minutes to rinse pellets of extracellular metals. Washed pellets were desiccated at 100°C, and either frozen at -80°C or used for downstream analysis. Dry pellets were acidified with 5 mL of HNO3, incubated for 20 minutes, and digested in a MARS 6 microwave following the machine’s internal protocol for plant material. Following digestion, samples were diluted 1:10, and analyzed for cadmium, by ICP-OES (Agilent Varian 720-ES). The data were generated from a 2 x 2 x 2 factorial experiment in randomized complete block design. The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), two levels of media (¼X TAP and Min), and two levels of Cd concentration (0 ppb [0 μM] and 1830 ppb [10 μM]).

Cell growth: The data were generated from a 2 x 2 x 2 factorial experiment in randomized complete block design.  The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), two levels of Cd concentration (0 µM, 10 µM), and two levels of media (1/4X TAP, Min). In this experiment there were 3 blocks.

Flow cytometry: Three replicates (in this experiment blocks are synonymous to replicates) with three trials were performed per concentration and per strain. The data were generated from a 2 x 3 x 4 factorial experiment in randomized complete block design. The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), three levels of Cd concentration (0 µM, 5 µM, and 10 µM), and four levels of time (1, 2, 3, and 4 days).

ICP-OES: The data were generated from a 2 x 2 x 2 factorial experiment in randomized complete block design. The factorial arrangement of treatments was the result of two levels of strain (cc5013, cc4425), two levels of media (¼X TAP and Min), and two levels of Cd concentration (0 ppb [0 μM] and 1830 ppb [10 μM]).