Effect of carbon dots supplementation in Chlorella vulgaris biomass production and its composition

CDs nanoparticles were synthesized using a simple carbonization procedure adapted from Chatzimitakos et al (2017). Briefly, orange juice extracted from fresh oranges was centrifuged at 4,000 rpm for 15 min to further filtration under vacuum. The purified juice was mixed with urea –used as a nitrogen source– in a ratio of 1 g and 2 g of urea per 10 ml of juice, respectively. The samples were placed in crucibles and heated for 3 h in a furnace at 220 °C. After cooling to room temperature, the homogenous brown-black residues were ground into a fine powder. Then, the powder was dispersed into 40 ml double distilled water (DDW) under ultrasonication for 10 min. Thereafter, the dispersion was centrifuged at 4,000 rpm for 15 min and the supernatant was filtered using a 0.20 μm syringe filter. CDs were freeze-dried to obtain a light-brown powder, which was stored in dark until its use (figure 1).

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Four samples of CDs were obtained varying the temperature (180 and 220 °C) and the dosage of urea (1 g and 2 g). According to the temperature and urea concentration, samples were identified as 'CDs-180-1gU', 'CDs-180-2gU', 'CDs-220-1gU', and 'CDs-220-2gU'. The CDs sample identified as CDs-220-2gU was selected for further experimentation in microalgae because it exhibited the best results during characterization.

CD samples were analyzed using different characterization techniques to evaluate specific properties. For instance, the Fourier-transform infrared spectroscopy (FTIR) technique was used to identify the functional groups on the surface of CDs. FTIR spectra were obtained using an FT-IR Spectrometer (PerkinElmer, Waltham, MA, USA) with the Universal ATR sampling accessory. The analysis was performed in a transmission range of 400 cm⁻¹ to 4000 cm⁻¹ with 64 scans. On the other hand, the optical properties of CDs were examined using a UV–visible spectroscopy equipment, which revealed the absorption region of CDs. Measurements were performed with a PerkinElmer UV/vis Lambda 365 in the wavelength range of 200–800 nm using dilutions of all CDs' samples. The fluorescence properties of CDs were evaluated as well; the fluorescence intensity and the steady-state emission spectra of CDs were obtained by an F-4600 spectrofluorometer. Finally, the ζ potential analysis was employed to analyze the stability and surface charge of CDs. The technique used was Phase-analysis light scattering (PALS) with a NanoBrook 90Plus PALS Brookhaven, which performed ten measurements for each sample using the Smoluchowski equation.

Chlorella vulgaris from UTEX (UTEX, Austin, TX, USA) was used for further experimentation. Microalgae culture stock was placed with a brand OPTIMA 4.5-Watt pump with an air filter of 0.20 μm, 1.5 l m⁻² min⁻¹ of aeration, and a photoperiod of 12 h:12 h and 16 h:8 h (light:dark) (Hydro grow extreme LED lamps). The temperature was kept at 21 °C. The culture medium used for the microalgae growth was BG11, which contains NaNO₃ 1.5 g l⁻¹, K₂HPO₄ 40 mg l⁻¹, CaCl₂ · 2H₂O 36 mg l⁻¹, MgSO₄ · 7H₂O 75 mg l⁻¹, Citric Acid · H₂O 6 mg l⁻¹, Ferric Ammonium Citrate 6 mg l⁻¹, Na₂EDTA · 2H₂O 1 mg l⁻¹, Na₂CO₃ 20 mg l⁻¹, H₃BO₃ 2.86 mg l⁻¹, MnCl₂ · 4H₂O 1.81 mg l⁻¹, ZnSO₄ · 7H₂O 0.22 mg l⁻¹, Na₂MoO₄ · 2H₂O 0.39 mg l⁻¹, CuSO₄ · 5H₂O 0.079 mg l⁻¹, and Co(NO₃)₂ · 6H₂O 0.04 mg l⁻¹.

For the experiments, 20 ml of C. vulgaris culture at a concentration of 1 g l⁻¹ were inoculated into 180 ml of BG11 medium, using 250 ml Erlenmeyer flasks. To evaluate the photoperiod effect in CDs-microalgae systems, it was used two photoperiod cycles of 12 h:12 h and 16 h:8 h (light:dark) for ten days. The CDs sample identified as 'CDs-220-2gU' was added into the microalgae cultures on days 0 and 5 at different dosages including 0 (control), 0.5, 1.0, and 2.0 mg l⁻¹; all experiments were conducted in triplicate (figure 2).

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The biomass growth of C. vulgaris under different CDs dosages was evaluated every 24 h by measuring the optical density of the cultures at 750 nm using a spectrophotometer (Fluorstar omega, 415-0470). Previously, a standard curve was performed to relate the optical density of the inoculum with its dry mass. The dry weight (g/L) was determined by filtering 10 ml of algal culture using pre-weighed Whatman GF/C glass microfiber filters. The filter with the algal biomass held was dried at 100 °C for 2 h. Then, filters with the algal biomass were cooled at room temperature inside a vacuum desiccator and weighed gravimetrically.

C. vulgaris cultures were sampled by taking 50 ml of the culture on days 5 and 10. These samples were stored at 4 °C until their use. To determine the protein, carbohydrate, and lipid production, biomass was washed with bi-distilled water and centrifuged at 4,000 rpm for 10 min at 10 °C. Then, the supernatant was discharged and the biomass was washed twice and resuspended in distilled water.

2.4.1. Carbohydrate production of C. vulgaris under different concentrations of carbon dots and photoperiods

2.4.2. Protein production of C. vulgaris under different concentrations of carbon dots and photoperiods

2.4.3. Lipid production of C. vulgaris under different concentrations of carbon dots and photoperiods

The statistical analysis was performed using Minitab 21 (Minitab Inc.). A multi-factor analysis of variance (ANOVA) with Tukey's post-hoc tests was employed for the entire experiment, considering both photoperiods and all conditions related to carbon dots. The significance was set at α = 0.05.

The CDs were synthesized via carbonization in an open environment using orange juice and urea as the carbon and nitrogen source, respectively. All CDs' samples exhibited good fluorescence activity under UV light irradiation (figure 3(a)). The surface chemical states and elemental characterization of the CDs were determined by FTIR analysis (figure 3(b)). The peaks identified at 1660,1600, 1400, 1349, 1070, 1000, and 910 cm^–1 confirmed the presence of C=C, C=C, O–H/S=O, C–N, C–O, C=C, and C=C bonds, respectively (Chen et al 2018). Specifically, the samples processed at 180 °C presented a peak at 2920 cm^–1, this peak is attributed to a C–H bond presence, which is formed for a hydrogen atom on the periphery of a benzene structure (Hu et al 2021). The results suggest that the surface of CDs nanoparticles is mainly composed of functional groups containing C, O, and N elements. The N-containing groups are produced because of the addition of urea during the synthesis. The O–H (alcohol) groups detected on the CDs' surface explain the high-water solubility of the synthesized CDs, as a result of their polar molecular bond.

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The UV–vis spectra showed the optical properties of CDs. As shown in figure 3(c), all the samples presented a peak at 260 nm, which reflects a conjugation for the CDs' structure. The specific peak at 260 nm is related to the ${n}{-}{\pi }{* }$ transition of the chromophore C=C. The peak at 285 nm was attributed to the ${n}{-}{\pi }{* }$ transition of C=N (González-González et al 2022b). Finally, the peak presented at 410 nm was related to the ${n}{-}{\pi }{* }$ transition associated with functional groups attached to the surface of CDs (Nie et al 2014).

On the other hand, diluted samples were analyzed in a fluorescence spectrophotometer to determine the CDs emission intensity under an excitation wavelength of 380 nm (figure 3(d)). The CDs-180-2gU sample showed the highest emission intensity, followed by CDs-220-1gU, CDs-180-1gU, and CDs-220-2gU. Yang et al (2018) stated that the temperature of the process altered the degree of carbonization of the carbon core and the compounds attached to the surface of CDs. In this manner, the fluorescence intensity is highly dependent on the carbon core and the surface states (Hu et al 2021). The highest emission intensity obtained with sample CDs-180-2gU might be attributed to the temperature and nitrogen content. During the synthesis, the energy from the spontaneous reaction broke the molecule into smaller pieces, after carbonization, and then different chemical groups are formed over their surface (Yu et al 2012). Contrarily, excess energy during high temperature-synthesis procedures might carbonize the chemical groups formed over the CDs surface (Yu et al 2012), producing a significant decrease in the fluorescence intensity of CDs (Yang et al 2018). As shown in figure 3(d), the maximum emission intensity of CDs samples was presented at a wavelength of approximately 500 nm.

The ζ potential was measured to determine the surface charge due to the presence of functional groups on the surface of CDs nanoparticles and the stability. All samples presented a negative charge (table 1) suggesting that there is an electron concentration over the CDs' surface (Long et al 2022). This negative charge might be associated with carboxyl, hydroxyl, and carbonyl groups, placed over the surface of CDs (Chen et al 2016). The CDs- 220-1gU sample showed the lowest absolute value, which indicates low physical stability and agglomeration tendency produced by cohesion forces (Vieira et al 2015). The other three samples presented a higher value, which is associated with repulsive forces and good electrical stability (Joseph and Singhvi 2019). The selected sample to use in microalgae culture (CDs-220-2gU) presented a value of −26.3 mV, which indicates the presence of functional groups and excellent stability (Shnoudeh et al 2019).

The selection of CDs samples for further addition to microalgae culture was based on the stability, fluorescence intensity, and content of functional groups. The selected sample (CDs-220-2gU) showed excellent stability with a ζ potential value of –26.3 mV, suggesting a negative charge and good stability in the media (González-González et al 2020). The FTIR results showed that the selected sample possesses C–O, C–N, and O–H groups on the CDs' surface, which might be beneficial for further experimentation due to increased water solubility and N-containing groups associated with enhanced optical properties. Finally, the results from fluorescence analyses showed that the selected CDs sample has a maximum peak at 400 and 500 nm (red region), which might shift the light absorption spectra and capture more photons, which in turn might enhance the light absorption and increase microalgae growth (Zhao et al 2021).

The biomass growth of C. vulgaris under different CDs concentration and photoperiod cycles was analyzed. Overall, the addition of CDs increased microalgae biomass production in the first five days of culture. For instance, higher biomass production of 1 g l⁻¹ and 0.96 g l⁻¹ was obtained with the addition of 1.0 and 2.0 mg l⁻¹ of CDs compared with the control under the 12 h:12 h (light:dark) photoperiod, respectively (figure 4(a)). At day 10, C. vulgaris supplemented with 1.0 mg l⁻¹ of CDs showed the highest biomass production (1.04 g l⁻¹) in this photoperiod, followed by the addition of 2.0 mg CDs/L (1.01 g l⁻¹). The results are similar to those reported by Anyanwu et al (2022); in their work, C. vulgaris was cultivated under the same 12 h:12 h photoperiod obtaining a biomass production of 1.482 g l⁻¹ after 16 days of cultivation.

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It should be noted that the cell growth decreased after 5 days, which might be related to the addition of CDs. This phenomenon can be shown in all the treatments, but also, it can be observed that after 8 days, the microalgae started to grow again (also, this can be observed in the 16 h:8 h photoperiod). It is supposed that the exponential phase of cells was affected by the CDs supplementation, is important to note that 1.0 and 2.0 mg l⁻¹ of CDs treatments were less affected in comparison to 0.5 mg l⁻¹ of CDs. This can be associated with the potential of CDs to increase light capture, which is critical during the photosynthesis process for cell growth and biocompounds production (Yan and Zheng 2013). CDs nanoparticles can increment the red light (650–700 nm) absorption by shifting the photoluminescence promoting cell growth and light capture, which has been also demonstrated by other authors (Xue et al 2020). In addition, CDs attached to the cell wall of microalgae can increase the light absorption of blue photons (400–525 nm) (Yang et al 2022).

On the other hand, microalgae cultures under the 16 h:8 h (light:dark) photoperiod presented higher biomass production in comparison to the equivalent samples at the 12 h:12 h photoperiod (figure 4(b)). This demonstrates the strong influence of the light exposure both on the photosynthetic activity of microalgae and on the role of CDs during light capture. Microalgae with no supplementation of CDs presented a biomass production of 2.12 g l⁻¹, which is a higher production compared to 12 h:12 h (light/dark) photoperiod (1 g l⁻¹). Also, biomass production was higher in comparison to the reported by Deng et al (2019), which cultivated Chlorella kessleri under mixotrophic conditions (2 gL^–1 glucose and 20 h:4 h light/dark photoperiod) and obtained a maximum biomass production of 1.17 g l⁻¹ after 7 days of cultivation.

Under 16 h:8 h (light/dark) photoperiod, the supplementation of 0.5 mg l⁻¹ of CDs increased microalgae growth to 2.4 g l⁻¹. Interestingly, higher concentrations of CDs (1.0 and 2.0 mg l⁻¹) decreased biomass production. This result suggests a biomass growth inhibition caused by cellular stress, which is produced by high dosages of CDs under longer periods of light. Excess light produces cell damage and oxidative stress, which might reduce biomass production in those samples (Minhas et al 2023). Therefore, the optimum dosage of CDs was 0.5 mg l⁻¹ under the 16 h:8 h (light/dark) photoperiod. This may imply that at a lower light photoperiod (12 h:12 h light/dark) it is recommended to use concentrations greater than 1.0 mg l⁻¹ of CDs, on the contrary, if there is an extended exposure of light (16 h:8 h light/dark) it is advisable to use lower concentrations of CDs (less than 1.0 mg l⁻¹). These results can be very useful for the growth of microalgae in outdoor conditions, where the light depends on the geographical location. In the statistical analysis (ANOVA-Tukey's test) of cell growth, it was observed that a higher microalgae biomass was obtained under a photoperiod of 16 h:8 h light/dark across all conditions compared to the alternative photoperiod. In addition, the treatment exhibiting the highest biomass production was observed to be the one administrated with 0.5 mg l⁻¹ of CDs after 10 days of culture under a 16 h:8 h light/dark photoperiod, yielding 2.34 g l⁻¹.

The effect of both the addition of CDs and photoperiod cycles on the carbohydrate production was determined after days 5 and 10 of culture (figure 5). Under the photoperiod of 12 h:12 h (light:dark), C. vulgaris (with no addition of CDs) produced 173 mg g⁻¹ dry weight of carbohydrates after 10 days. Hamidian and Zamani (2022) reported a similar value (163 mg g⁻¹ dry weight) of carbohydrates content during Chlorella sp. cultivation without any nanomaterial addition. In our study, the carbohydrate production was considerably increased after the addition of CDs (figure 5(a)). The highest carbohydrate production was achieved with the addition of 1.0 mg l⁻¹ of CDs, which reached 211 mg g⁻¹ dry weight and 217 mg g⁻¹ dry weight on day 5 and 10, respectively, which represents an enhancement of 44 mg g⁻¹ dry weight in carbohydrate production (25% increase in comparison to the control).

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These results do not follow the same trend under the 16 h:8 h (light/dark) photoperiod (figure 5(b)); the maximum carbohydrate production reached 194 mg g⁻¹ dry weight after 5 days with no addition of CDs. This showed that a longer light period contributes to reducing carbohydrate content. Interestingly, the addition of CDs under these conditions had negative effects, which might be attributed to cell damage produced by an excess of light and a modification of the photosynthetic pathways caused by the nanoparticles (Priyadharsini et al 2022). During the photosynthesis process, light is captured and turned into biomolecules; in the first stage, light energy is directed to synthesize pigments, in this manner, a major quantity of energy could have been designated to produce more pigments and only a lower adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) could be able for carbohydrate production (de Carvalho Silvello et al 2022). Xue et al (2020) reported a higher carbohydrate production of 110 mg l⁻¹ (250 mg g⁻¹ dry wt) cultivating Chlorella regularis (FACHB-729). Their culture was supplemented with CDs synthesized from citric acid as the precursor. Their work reported 28% more carbohydrate production than the present study; such differences can be attributed to the precursor nature and microalgae strains. In this respect, the economic feasibility of using pure precursors or natural/waste precursors for the synthesis of CDs should be considered as well. Regarding the statistical analysis (ANOVA-Tukey's test) of carbohydrates, it was observed that a higher amount was synthesized under a photoperiod of 12 h:12 h light/dark throughout the entire experiment. The treatments exhibiting the highest carbohydrate production were those with 1 mg l⁻¹ and 2 mg l⁻¹ of CDs at 10 days of culture under 12 h:12 h light/dark photoperiod, where 217.5 mg g⁻¹ and 216.6 mg g⁻¹ were synthesized, respectively.

The protein production of C. vulgaris under different photoperiod cycles and CDs dosages was determined as well. As presented in figure 6(a), after 10 days, the addition of 2 mg l⁻¹ of CDs showed a maximum protein production (203 mg g⁻¹ dry wt) in a 12 h:12 h (light/dark) photoperiod. In contrast, microalgae without the addition of CDs presented a protein production of 180 mg g⁻¹ dry wt. On the other hand, microalgae cultures under the photoperiod of 16 h:8 h (light/dark) presented higher protein production (figure 6(b)). At day 5, microalgae cultures with 1.0 mg l⁻¹ and 2.0 mg l⁻¹ of CDs presented a protein production of approximately 200 mg g⁻¹ dry wt, which was significantly higher in comparison to the control samples (with no CDs' addition), which presented only 170 mg g⁻¹ dry wt. At day 10, the maximum protein production reached 285 mg g⁻¹ dry wt with the addition of 2.0 mg l⁻¹ of CDs, which represents an enhancement of 74 mg g⁻¹ dry wt in carbohydrate production (35% more compared with the control). It is interesting to observe, that the best concentration of CDs is 0.5 mg l⁻¹ for protein production, since it reached 542 mg of proteins per liter of microalgae culture. In contrast, the protein production obtained by the control and microalgae supplemented with 2.0 mg l⁻¹ of CDs was 430 and 396 mg of proteins per liter, respectively.

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These results showed protein productivity aligned with the work reported by İnan et al (2023); they cultivated Chlorella variabilis and harvested a maximum protein production of 330 mg g⁻¹ dry wt under 24 h of light exposure. The protein production presented in our study is higher than that obtained by Xue et al (2020), who cultivated Chlorella regularis and reported a maximum protein production of 84 mg l⁻¹ (136 mg g⁻¹ dry wt) after 12 days and the addition of 1 mg l⁻¹ of CDs. The significant increment in protein production achieved in this study is attributed to the effect produced by the addition of CDs and their interaction with microalgae cells. In the statistical analysis (ANOVA-Tukey's test) of proteins production, it was determined that a greater quantity was synthesized under a photoperiod of 16 h:8 h light/dark throughout the experiment. The treatment yielding the highest protein production was with 2 mg l⁻¹ of CDs at 10 days of culture under the 16 h:8 h light/dark photoperiod, where 285 mg g⁻¹ of proteins were generated.

The lipid production of C. vulgaris under two different photoperiod conditions and the addition of CDs at different dosages were determined. At day 5 under the photoperiod of 12 h:12 h, the maximum lipid production (260 mg g⁻¹ dry wt) was reached with the addition of CDs at 2.0 mg l⁻¹ (figure 7(a)). In contrast, microalgae with the addition of 1.0 mg l⁻¹ of CDs showed a lower value of 178 mg g⁻¹ dry wt. On the other way, at day 10 of the microalgae culture, the maximum lipid production (298 mg g⁻¹ dry wt) was reached with the addition of CDs at 1.0 mg l⁻¹, which represented an enhancement of 114 mg g⁻¹ dry wt in lipid production (>50% compared with the control).

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An improvement in lipid production under the photoperiod of 16 h:8 h (light/dark) was observed (figure 7(b)). At day 5, the maximum lipid production (173 mg g⁻¹ dry wt) was reached with the addition of CDs at 2.0 mg l⁻¹. On the other hand, the control sample (with no addition of CDs) presented the lowest lipid productivity (99 mg g⁻¹ dry wt). Also, the maximum lipid production (249 mg g⁻¹ dry wt) was obtained with the addition of 2.0 mg l⁻¹ of CDs after 10 days.

According to the literature, Xue et al (2020) cultivated C. regularis with the addition of CDs; they reported a maximum lipid production of 260 mg l⁻¹ (333 mg g⁻¹ dry wt) with a CDs dosage of 1 mg l⁻¹. These results are aligned with the obtained in this work; the addition of 0.5 mg l⁻¹ and 2.0 mg l⁻¹ of CDs with a photoperiod of 16 h:8 h (light/dark) resulted in 175 mg g⁻¹ dry wt and 249 mg g⁻¹ dry wt of lipids, respectively. Under the photoperiod of 12 h:12 h (light/dark) it was obtained 298 mg g⁻¹ dry wt of lipids, after the addition of 1 mg CDs/L. In the statistical analysis (ANOVA-Tukey's test) of lipid production, it was observed that a higher amount was produced under a photoperiod of 12 h:12 h light/dark throughout the experiment. The treatment yielding the highest lipid production occurred with 1 mg l⁻¹ of CDs at 10 days of culture under the 12 h:12 h light/dark photoperiod, resulting in the synthesis of 298 mg g⁻¹ of lipids.

Wahidin et al (2013) studied the effect of the photoperiod cycles on Nannochloropsis sp. cultures; they found that the maximum lipid content was obtained with a longer light period (18:6 h light/dark); however, a complete light period (24 h:0 h light/dark) produced a negative effect on the lipid content. In this sense, the higher lipid production of C. vulgaris cultures under the 12 h:12 h (light/dark) photoperiod can be attributed to the fact that under this photoperiod the cells received the optimum amount of energy to store and use during lipid synthesis. On the other hand, the longer light period (16 h:8 h light/dark) could have produced cellular stress, which decreased lipid synthesis.