Pristine Carbon Dots Boost the Growth of Chlorella vulgaris by

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Pristine carbon dots boost the growth of Chlorella vulgaris by enhancing photosynthesis Mengling Zhang, Huibo Wang, Yuxiang Song, Hui Huang, Mingwang Shao, Yang Liu, Hao Li, and Zhenhui Kang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00319 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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ACS Applied Bio Materials

Pristine carbon dots boost the growth of Chlorella vulgaris by enhancing photosynthesis Mengling Zhang, Huibo Wang, Yuxiang Song, Hui Huang, Mingwang Shao, Yang Liu*, Hao Li* and Zhenhui Kang* Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, China. E-mail: [email protected]; [email protected]; [email protected] KEYWORDS: Carbon dots, Chlorella, Photosynthesis, Antioxidant

ACS Paragon Plus Environment

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ABSTRACT: Microalgae has been identified as a promising sources for biofuels production due to its superior characteristics. Carbon dots (CDs) with low toxicity and appreciable biocompatibility have been widely applied in bio-application. In this work, Chlorella vulgaris is adopted as a model microalgae to study the impacts of CDs on it. The CDs are low toxicity and biodegradable and they can increase the biomass and growth rate by 17% and 21%, respectively. That is due to the CDs could be degraded by Chlorella vulgaris and producing numerous CO2, which could be immobilized by chloroplast and enhanced photosynthesis. And the CDs can improve the activity of Rubisco in vivo by 34%. Furthermore, these CDs as an anti-oxidants can protect Chlorella vulgaris cells against the damage of UV radiation and enhance the specific growth rate and the biomass by 17% and 18% under UV radiation.

1. INTRODUCTION Microalgae has been identified as a promising sources to produce biofuels due to its superior characteristics, for instance, affluent nutrient content, easy to cultivate and valuable by-products13

It has get tons of attention in boosting their nutrient content, like lipid, protein and carbohydrate,

or by-products, like astaxanthin and chlorophyll, which have been widely exploited and applied in industrialization.4-6 Along with the development of nano-technology, the relationship of nanomaterials with microalgae have been exploited and apply in agriculture and environment.7-11 It's worth noting that some metal,12, 13 metal oxides14-16 nanoparticles and some carbon-based nanomaterials (e.g. CNT17, CNF18 and graphene oxide19, 20) have been reported that they have toxicity on microalgae. More importantly, the most of nanoparticles are hardly biodegraded in the natural environment. So, we need to seek a kind of non-toxic and biodegradable material.


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CDs (carbon dots) is a new member of carbon-based nanomaterial family and attracts lots of attention in sensing, bio-imaging, photocatalysis and so on due to their advantages in well water solubility, strong fluorescent, easy modification and low toxicity.21-23 In addition, different with the metallic nanoparticles and other carbon-based nanomaterials, CDs are low toxicity (or nontoxicity) and biodegradable.24, 25 Due to this benign nature, CDs could be absorbed easily by organism.26, 27 However, little attention have been focused on the effect of CDs on microalgae. Previous research, reported by Xiao et al,28 contrasted the toxicity of metal nanoparticles with CDs on Chlorella vulgaris. They discovered the toxicity of CDs is smaller than that of metal nanoparticles on the growth of Chlorella vulgaris, but they ignore the dose-response of CDs during their growth and not study how affect the growth of Chlorella vulgaris treated with CDs. Thus, Uncertainties still exist. Herein, we demonstrate that CDs can increase the growth of Chlorella vulgaris by enhancing their photosynthesis and they as an anti-oxidants can protect Chlorella vulgaris cells against the damage of UV radiation. CDs used as a supplement in the growth medium can increase their specific growth rate and the production of stored energy. The obtained CDs could improve the Rubisco activity of Chlorella vulgaris, which activity directly affects the photosynthetic rate. Furthermore, series of measurements were implemented for simulating the biodegradation process of CDs. The CDs are biodegradable and a large amount of CO2 are generated during the degradation process. The more available CO2 could utilized by Chlorella vulgaris for photosynthesis. 2. EXPERIMENTAL SECTION 2.1. Chemicals and characterization methods All the chemicals were purchased from Beijing Chemical Reagent (Beijing, China) and Sigmaaldrich (Beijing, China), which were used without any further purification. The graphite rod


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(99.99%) was purchased from Alfa Aesar Co. Ltd. The Rubisco ELISA test kit was provided by Comin Biotechnology Co. Ltd (Suzhou, China). Bicinchoninic acid (BCA) protein concentration test kit was purchased from Solarbio Science & Technology Co. Ltd (Beijing, China). The Chlorella vulgaris strain were purchased from Ivy Biotech Co. Ltd (Tianjin, China). The X-ray photoelectron spectroscopy (XPS) spectrum were measured using an Axis Ultra DLD X-ray photoelectron spectroscope. A FEI/Philips Tecnai G2 F20 transmission electron microscope was adopted for obtaining Transmission electron microscopy (TEM) images. Scanning electron microscopy (SEM) images were obtained on FEI-quanta 200 scanning electron microscope with an acceleration voltage of 20 kV equipped. Fourier Transform Infrared (FT-IR) spectra of CDs were acquireed by a Perkin Elmer FT-IR spectrometer. Photoluminescence (PL) and ultravioletvisible (UV-vis) absorption spectrum was obtained with Fluromax-4 (France Jobin Yvon company) and Perkin Elmer UV–vis spectrophotometer (Lambda 750), respectively. 2.2. Synthesis of CDs. The CDs were synthesized by a typical electrochemical etching method developed by our group.29 Briefly, Two graphite rods as anode and cathode were inserted into the Milli-Q water. 30 V static potential was employed to the anode and cathode. The anode graphite rod gradually corroded during with electrolysis and the solution turn to black. The CDs solution was obtained after filtered with filter paper by two times. 2.3. Chlorella vulgaris culture Chlorella vulgaris were cultured with F/2 medium30 (the chemical ingredients of F/2 were shown in supporting information) in the illumination incubator (temperature: 25 ±1 °C, light intensity: 5000 lux, light: 24 h) for 9 days and artificial shaking 3 times per day. The cell concentration of Chlorella vulgaris was determined regularly by UV-Vis spectrophotometer at 690 nm. The optical


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density values located in 690 nm (OD690) were converted into dry cell weight via standard curve (Fig. S1a). The growth curve was obtained on the basis of the dry weigh of Chlorella vulgaris. The growth rate was calculated as following equation: μ=

dlnX dt

|max

(1)

X means biomass concentration (mg/L) and t is cultivation time (day). 2.4. UV exposure experiment. Chlorella vulgaris were cultured with F/2 medium in the illumination incubator (temperature: 25 ±1 °C, light intensity: 5000 lux, light: 24 h) for 9 days. Artificial intermittent shaking three times per day. The day after cultivation, the Chlorella vulgaris were exposed by UV radiation of 254 nm (8W) for 15 min every day. 2.5 Determination of nutritional components 2.5.1 Measurement of chlorophyll content Chlorophyll content was detected with colorimetric method.31 Chlorella vulgaris suspension sample (4 mL) was centrifuged (5000 r/min for 10 min) and the algae cells were collected. Then 8 mL of ethyl alcohol was added for extracting till the Chlorella vulgaris cells change to white. After 48h, the sample treated with centrifugation (5000 r/min, 10 min) for removing residue. The supernatant is chlorophyll solution and monitored OD649 and OD665 by UV-Vis spectrophotometer (Fig. S2). Three independent experiments were measured and the Chlorophyll contents (CT) were determined using the following equation: Chla (mg/L) = 13.95 × 𝑂𝐷665 − 6.88 × 𝑂𝐷649

(2)

Chlb (mg/L) = 24.96 × 𝑂𝐷649 − 7.32 × 𝑂𝐷665

(3)

𝐶𝑇 (mg/L) = (𝐶ℎ𝑙𝑎 + 𝐶ℎ𝑙𝑏) × 𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑖𝑜

(4)


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2.5.2 Measurement of carbohydrate content Anthrone was used for detecting the carbohydrate content of Chlorella vulgaris.32 30 mL of Chlorella vulgaris suspension sample was harvested by centrifugation and washed with deionized water for removing culture solution. The supernatant was discarded and added into distilled water (5 mL). The mixture was heated at boiling-water for extracting carbohydrate. After 20 min, the extraction was treated with centrifugation (5000 r/min, 10 min) to remove residue. The above extraction (1 mL) was added into of anthrone solution (5 mL, 0.1 g anthrone powder dissolved in 100 mL 80 % H2SO4). Then take the mixture solution reacted in boiling-water for 10 min. The optical density values located in 620 nm of mixture solution was detected. Triplicate actions were performed for statistical analysis. The carbohydrate content was obtained via Fig. S1b (difference concentration of glucose solution used the same treatment to measure OD620 values). 2.5.3 Measurement of protein content BCA protein concentration test kit was adopted for measuring the protein content of Chlorella vulgaris, which includes two kinds of Reagents (Reagent BCA and Reagent Cu). First, 50 mL Reagent BCA mixt mixed with 1 mL Reagent Cu and the working solution was obtained. The extraction procedure as follows: Chlorella vulgaris cells were collected by centrifugation (5000 r/min for 10 min). Then, this cells were crushed by ultrasonic. The homogenates was treated with centrifugation (5000 r/min, 10 min) and the protein extract was collected. Then 200 μL of sample extract was added into working solution (2 mL) and this mixture was incubated in 30 oC. After 15 min, the optical density values located in 562 nm of mixture solution was detected by UV/Vis spectrophotometer. The protein content was obtained via Fig. S1c (Using the same test procedure for measuring OD562 values of difference concentration of BSA protein marker). 2.5.4 Determination of the lipid content


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The Bligh and Dyer method was employed for detecting the lipid content of Chlorella vulgaris.33 The sample cells was collected by centrifugation (5000 r/min, 10 min) then lyophilized by freeze dryer. This dried cells were crushed by mortar with liquid N2. Chloroform and methanol mixture (volume ratio, 2:1) was added for extracting lipid at 25 °C for 24 h (total volume: 18 mL). The lipid extract was collected by centrifugation (5000 r/min for 10 min). A second Chloroform and methanol mixture were added for re-extracting. All lipid extract were transferred into round bottomed flask and evaporated by rotary evaporator for weigh. The lipid content was calculated according to microalgae biomass. 2.6 CDs degradation by HRP 1 mg of CDs was transferred into a vial and dissolved in PBS (4 mL, 0.01 mol/L, pH = 7.0). Then, 4 mL 0.385 mg/mL Horseradish Peroxidase (HRP) solution was added. After 24 h incubation at 25 °C away from light. The sample was placed on a magnetic stirrer with 220 rpm constant shaking. 8 mL H2O2 (800 μmol/L) was added to start the degradation process. The vial was sealed by rubber stoppers and parafilm. During this process, 250 μL H2O2 (800 μmol/L) was added in order to compensate for H2O2 consumption each day. 2.7 In vivo effects of CDs on Rubisco activity The sample extraction of Chlorella vulgaris was extracted by ultrasonication. And the Rubisco (ribulose bisphosphate carboxylase oxygenase) activity was measured by Rubisco ELISA test kit, which provided by Comin Biotechnology Co. Ltd. The detection procedure are referenced with previous report.8 2.8 The antioxidant activity of carbon dots 2.8.1 DPPH∙ scavenging assay The DPPH∙ free radical assay was determined by Fukumoto and Mazza reported.34 Purple


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DPPH∙ change to yellow in the presence of antioxidants and its concentration was monitored by UV-Vis spectrophotometer. 20 μL of 10 mmol/L DPPH∙ in absolute methanol was mixed with different concentration of CDs in absolute methanol (0-500 nmol/L, the calculation of molecular weight of CDs detailed in supporting information) and the mixture (2 mL) was incubated in the dark for 60 min. AA (Ascorbic acid) as standard was adopted. The decrease of OD515 was obtained. These measurements were repeated three times and the radical scavenging activity (RSA) of CDs with different concentration was calculated by following: RSA (%) =

𝐴𝑐− 𝐴𝐶𝐷𝑠 𝐴𝑐

(6)

Where Ac and ACDs are OD515 of control (without CDs) and CDs-DPPH mixture solution. 2.8.2 KMnO4 reduction assay The KMnO4 reduction assay was carried out for assessing the antioxidant activity of the obtained CDs as reported by Das et al.35 20 μL of 10 mmol/L acidified KMnO4 solution (pH = 2.0) was mixed with CDs in different concentrations (0-200 nmol/L) and the mixture (total volume, 2 mL) was incubated in the dark for 60 min. OD626 was measured and the percentage of the decrease of which represent the RSA of CDs. The experiment were repeated three times and the RSA of CDs with different concentration was computed with equation (6). The Ac and ACDs are the OD526 of control (without CDs) and CDs- KMnO4 mixture solution. AA was used as standard. 3. RESULTS AND DISCUSSION 3.1. Characterization of CDs Fig. 1a is the typical TEM image of the obtained CDs, demonstrating that CDs are well dispersed in water and their diamater was approximately 5 nm. Their diameter distribution was obtained by counting 200 particles in one image. HRTEM image of CDs is shown in the inset of Fig. 1a, revealing the lattice spacing of CDs is about 0.21 nm,


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which according with the (100) facet of graphite. The FT-IR spectrum is mesured for analyzing surface functional groups and shown in Fig. 1b (black line). The peak loacted at 3450 cm-1 corresponds to the of O-H stretch. The peak loacted at 2918 cm-1 attributed to the stretch of C-H. The two peaks located at 1720 cm-1 and 1388 cm-1 are ascribe to the stretching vibration of C=O and C-O, respectively.29, 36 These hydrophilic functional groups (hydroxyl and carboxyl groups) result in the excellent water solubility of CDs and significantly extend their applications in the water system. Fig. 1b(red line) is the Raman spectrum of CDs. There are two peaks at 1341 cm-1 and 1609 cm-1, corresponding with D band and G band, which ascribe to vibrations of sp3 carbon in disordered graphite and sp2 carbon in a two dimensional hexagonal lattice, respectively. 29, 37 The ratio of sp3/sp2 of CDs is 1.43. XPS spectrum was obtained for analyzing the composition of CDs. As shown in Fig. S3a, there are two signals at 284.80 and 532.88 eV in the full-scan XPS spectrum of CDs, corresponding to C 1s and O 1s. It contains 74.27 at.% of carbon and 25.73 at.% of oxygen. Fig. 1c is the partial XPS spectrum of C 1s. Three component peaks, located at 284.80, 286.00 and 289.80 eV, can be resolved and these peaks correspond to C-C, C-O and O-C=O, respectively.38, 39 The high resolution scan XPS spectrum of O 1s is shown in Fig. S3b, the peak located 532.38 eV represents O=C and another peak located at 533.70 eV attributes to O-C. Fig. 1d (black line) displays the UV-vis absorption spectrum of CDs. There is a characteristic peak located at 230 nm, attributing to π-π* transition of C=C units. It is similar to the other carbon dots reported earlier.23, 40 The PL and PLE spectra of CDs are shown in Fig. 1d. The PL of CDs has the strongest emission located at 457 nm in the 330


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nm excitation. In addition, the cytotoxicity of CDs in vitro was detected with 293T cells (shown in Fig. 1e). These cells were incubated with increasing concentrations of CDs (0500 µg/mL). Even at ultrachigh concentraction of CDs (500 µg/mL), their is not significant decrease in 293T cell viability. Which demonstrates the obtained CDs have good biocompatibility for the organism.

Figure 1. (a) The TEM image (the HRTEM image and diameter distribution are shown in insets), (b) FT-IR (black trace) and Raman spectra (red trace), (c) Partial XPS spectrum of C 1s, (d) PL spectrum (red line), PLE spectrum (red line) and UV-vis absorption spectrum (black line) of CDs. (e) Viability of 293T cells after 48 h incubation with different


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concentrations of CDs. The mean and standard deviation are represented by column and error bar. 3.2 The effects of CDs on Chlorella vulgaris In the following work, Chlorella vulgaris was adopted as a model microalgae for studing the impacts of CDs on it. Fig. 2a is the photo of Chlorella vulgaris incubated by F/2 medium (control) and CDs treated F/2 medium (CDs) for 8 days. The Chlorella vulgaris cells treated with CDs are more viridity than control group. The UV-Vis absorption spectrum of Chlorella vulgaris solution was measured for monitoring the biomass of it (Fig. S4). To further investigate the dose-response on the phytotoxicity of CDs during Chlorella vulgaris growth period, the control group (without CDs) and CDs treated group (treated with 1, 5, 10, 20 µg/mL CDs) were cultivated and the growth curves are displayed in Fig. S5a. The biomass and growth rate are presented in Fig. 2b. It turned out that a concentration threshold (10 µg/mL) is presented and the biomass and of growth rate of Chlorella vulgaris treated with 10 µg/mL CDs increased by 17% and 21% than the control group, respectively. Below 10 µg/mL CDs, the biomass and growth rate of Chlorella vulgaris gradually improve with the increasing concentration of CDs. However, at the high concentrations of CDs (20 µg/mL), the presence of CDs inhibited the growth of Chlorella vulgaris . Moreover, the content of main components of Chlorella vulgaris was detected. Fig. 2c is the contents of chlorophyll (Chl), carbohydrate ((CH2O)n), lipid (Lip) and protein (Pr) of control group (without CDs) and CDs treated group (treated with 10 µg/mL CDs), respectively. In control group, the contents of Chl, (CH2O)n, Lip and Pr are 9.92, 262.51, 136.00 and 320.56 mg/L, respectively. And the CDs treated group are 12.60, 321.64, 163.20 and 410.31 mg/L, respectively. Compared with control group, the contents of Chl, (CH2O)n, Lip and Pr


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increased by 27%, 23%, 20% and 28%, respectively. The low concentration of CDs do not have toxicity and they could promote the the growth of Chlorella vulgaris. In other words, the addition of CDs could increase the nutritive contents of Chlorella vulgaris.

Figure 2. (a) The photo of Chlorella vulgaris incubated by F/2 medium (control) and 10 µg/mL CDs - F/2 medium (CDs) for 8 days (scale bar:1 cm). (b) The biomass and growth rate of Chlorella vulgaris treated with 1, 5, 10 and 20 µg/mL CDs. (c) The content of chlorophyll (Chl), carbohydrate ((CH2O)n), lipid (Lip) and protein (Pr) of control group and CDs treated group (10 µg/mL CDs), respectively, * P < 0.05. The mean and standard deviation are represented by column and error bar. 3.3 CDs protect Chlorella vulgaris cells against the damage of UV radiation The UV radiation has to be considered unfavorably to living matter and many


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protecting strategies have been developed to cope with these negative impacts. 41,

42

Therefore, the effects of CDs on Chlorella vulgaris exposed by UV light of 254 nm were researched. Fig. 3a exhibits the photo of Chlorella vulgaris incubated by F/2 medium in fluorescent lamp light (control+Vis), F/2 medium in UV exposure (control+UV) and 10 µg/mL CDs - F/2 medium in UV exposure (CDs+UV) for 8 days, respectively (the growth curve shown in Fig. S5b). After the UV radiation, the color of Chlorella vulgaris is went from the emerald green to yellow green. However, the Chlorella vulgaris treated with CDs was still emerald. In the UV radiation, the biomass and growth rate of Chlorella vulgaris incubated with and without CDs are displayed in Fig. 3b, respectively. After the radiation of UV, the biomass and growth rate of CDs treated Chlorella vulgaris increased by 18% and 17% than control group, respectively. The UV radiation can inhibit the growth of Chlorella vulgaris, but the density of CDs treated Chlorella vulgaris is higher, which illustrate the addition of CDs could protect Chlorella vulgaris cells against the UV radiation so that the Chlorella vulgaris cells could grow faster. Moreover, the content of main components of Chlorella vulgaris incubated with different culture conditions was detected (shown in Fig. 3c). The contents of Chl, (CH2O)n, Lip and Pr of Chlorella vulgaris exposed by UV are 6.63, 152.30, 112.88 and 97.38 mg/L, respectively, while the Chlorella vulgaris treated with CDs exposed by UV are 7.90, 190.76, 130.00 and 158.54 mg/L, respectively. In the present of UV radiation, the content of nutritional components was markedly decrease for Chlorella vulgaris. But the CDs (10 µg/mL) treated Chlorella vulgaris has superior nutritional components than that of Chlorella vulgaris (without CDs) exposed by UV and the contents of Chl, (CH2O)n, Lip and Pr of Chlorella vulgaris treated with CDs enhanced 19%, 25%, 15% and 32% under the UV


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radiation. This data demonstrate the obtained CDs could oppose the damage of the UV radiation on Chlorella vulgaris.

Figure 3. (a) The photo of Chlorella vulgaris incubated by F/2 medium in fluorescent lamp light (control), F/2 medium in UV exposure (control+UV) and 10 µg/mL CDs - F/2 medium in UV exposure (CDs+UV) for 8 days, respectively (scale bar:1 cm). (b) The specific growth rate, biomass and (c) content of Chl, (CH2O)n, Lip and Pr of control group and CDs treated group (10 µg/mL CDs) after UV exposure, respectively, * P < 0.05. The mean and standard deviation are represented by column and error bar. 3.4 TEM and SEM images of Chlorella vulgaris cells For understanding if CDs could be uptaked by Chlorella vulgaris during their growth, TEM images of ultrathin section of Chlorella vulgaris treated with CDs (Fig. 4a-4c) were acquired after 5 days incubation. In HRTEM images of ultrathin section (Fig. 4d), 0.21 nm


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lattice spacing from CDs was found in Chlorella vulgaris cells. It is demonstrated the CDs could be absorbed by Chlorella vulgaris. Next, the SEM images of Chlorella vulgaris were obtained for observing the impacts of CDs on the chlorella cell wall. Fig. 4e and Fig. 4f shows the SEM images of Chlorella vulgaris cells incubated with and without CDs in 5000 lux fluorescent lamp light, respectively. There is no difference in chlorella cells treated with and without CDs, which represent CDs have not destroy the cell wall of Chlorella vulgaris. Furthermore, the Chlorella vulgaris was incubated by UV exposure. The control group was incubated by F/2 medium and expose by UV radiation for 15 min every day after the second day of growth. The CDs treated group was added 10 µg/mL CDs in the F/2 medium. After UV radiation for 4 days, the SEM images of chlorella treated without and with CDs are shown in Fig. 4e and Fig. 4f. In control group, most of the cytoderm of Chlorella vulgaris cells were destroyed under UV radiation. Interestingly, only little small part of Chlorella vulgaris cells treated with CDs were flawed. Therefore, the as-prepare CDs could protect Chlorella vulgaris cells far from the destruction of UV radiation.


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Figure 4. (a-c) TEM and (d) HRTEM images of ultrathin section of Chlorella vulgaris treated with CDs. SEM images of Chlorella vulgaris incubated by F/2 medium and 10 µg/mL CDs-F/2 medium in (e/f) 5000 lux fluorescent lamp light or (g/h) UV light of 254 nm, respectively. 3.5 Effects of CDs on photosynthesis Different with the metallic nanoparticles and other carbon nanomaterials, CDs are low toxicity (or non-toxicity) and biodegradable. Therefore, series of measurements were implemented for simulating the biodegradation process of CDs in vivo. Horseradish Peroxidase (HRP), an important peroxidase enzyme, was used for enzymatically degrading CDs in the present of H2O2.43 Fig. S6a displays the photos of CDs in predegradation (0 day) and after 20 days degradation with HRP and H2O2. There is an obvious fading after degradation process. TEM images

was used to track the

morphological variation during the process of enzymatic degradation (Fig. S6b). Before degradation process, the diameter of CDs is about 5 nm. After 20 days, it was seen that the particle size of CDs decreases to 3 nm (Fig. S6c). In addition, the degradation process was also monitored by UV-Vis absorption spectrum (Fig. S6d). After 20 days of degradation, the absorption of CDs was obviously weakened and the peak located at 230 nm (π-π* transition) disappeared. The degradation of CDs is an oxidation process and CO2 as a final oxidation product would present. To prove this point, GC-MS was used for dectecting the gas in the top of vial. The experimental data indicate that the degradation of the gas product is CO2.The insert of Fig. S6d shows a large amount of CO2 were produced after 20 days of degradation. The equation of degradation of CDs is shown in Fig. 5a. Due to this results of the degradation process of CDs, and the HRP and


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H2O2 widely exist in the biological body. There is a assumption that the CDs could be degraded by Chlorella vulgaris cells and produce a lot of CO2, which could utilized by Chlorella vulgaris for photosynthesis. Based above discussions, the degradation product, CO2, is the main reason why CDs could enhance the photosynthesis. In addation, Rubisco is one of the key enzyme during photosynthesis. Its activity directly affects the photosynthetic rate.44,

45

The effects of CDs on Rubisco activity were detected for

exploring the role of CDs on photosynthesis. Fig. 5b is the effect of CDs on Rubisco activity in vitro. The addition of CDs could enhance the Rubisco activity by 38.22%. This phenomenon was also found in vivo experiment. Such as Fig. 5c, the Rubisco activity of chlorella treated without and with CDs are 0.37 and 0.50 nmol/mL/min. Compared with control group, the rubisco activity (CDs treated group)increased 34.48%. In the other words, CDs could observably enhance the photosynthesis of Chlorella vulgaris. The increase of Rubisco activity is the other reason that is CDs could enhance the photosynthesis.


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Figure 5. (a) The equation of degradation of CDs. (b) In vitro and (c) in vivo Rubisco activity of control and CDs treated,** P < 0.01. The mean and standard deviation are represented by column and error bar. 3.6 The antioxidant activity of CDs In the process of UV irradation, Chlorella vulgaris would sharply generate more reactive oxygen species (ROS), such as OH∙, HO2∙ and so on.46 This ROS could destroy the structure of Chlorella vulgaris cells, so that the UV irradation would inhibit the growth of Chlorella vulgaris. However, the Chlorella vulgaris incubated by CDs relieves the repressive effect from UV irradation. In the above discussion, CDs could absorb the ultraviolet light (200400 nm) (Fig. 1d),. So, CDs can reduce ultraviolet radiation damage and protect Chlorella vulgaris.In addition, the good free radical scavengers always have long conjugated C=C chains. 47 In the following, the antioxidant activity of the as-prepare CDs has been assessed and compared with AA. The RSA of the CDs was assessed using DPPH∙, which is a model radical and commonly adopted for evaluating the antioxidant activity of materials. Fig. 6a shows the absorbance of DPPH∙ methanol solutions incubated with a series of CDs (0-500 nmol/L). The characteristic DPPH∙ absorbance peak gradually disappeared with increasing CDs. The RSA of CDs and AA with different concentrations are contrasted in Fig. 6b. The RSA of CDs as well as AA. The superior RSA of CDs attribute to the large amount -OH group and conjugate sp2 carbon for delocalizing and stabilizing free electron.48 Furthermore, KMnO4 reduction assay was carried out for evaluating the antioxidant activity of CDs. Fig. 6c is the absorption spectrum of KMnO4 mixed with increasing concentrations of CDs (0200 nmol/L) after reaction. The characteristic peaks of KMnO4 solution are gradually bleached during with the increase of CDs, which illustrate the reduction of KMnO4 by CDs.


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The RSA of CDs and AA were calculated and shown in Fig. 6d. The data of RSA reveal that CDs have the highest antioxidant activity than that of AA. These results demonstrate the obtained CDs as an anti-oxidants can protect Chlorella vulgaris cells against oxidative damage.

Figure 6. (a) The absorption spectrum of DPPH∙ (100 μmol/L) with the increasing molarities of CDs (0-500 nmol/L) after incubation of 60 min; (b) DPPH∙ scavenging assay: RSA vs. Concentration of CDs. (c) The absorption spectrum of KMnO4 (100 μmol/L) with the increasing molarities of CDs (0-200 nmol/L) after incubation of 60 min; (d) KMnO4 reduction assay: RSA vs. Concentration of CDs (mean ±standard deviation). From the above discussion, the obtained CDs have positively effects on Chlorella vulgaris. Two effects of CDs are sorted out in Fig. 7. One is the obtained CDs could improve the growth of Chlorella vulgaris with high quality. That is because the CDs could enhance the activity of Rubisco, which directly affects the photosynthetic rate. In addition,


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the CDs could be degraded by Chlorella vulgaris and produced a number of CO2 and some small organic molecules. The producted CO2 could be immobilized by chloroplast. Due to the increase of Rubisco activity and more available CO2, the photosynthesis of Chlorella vulgaris observably increase. The specific growth rate and the biomass of CDs (10 µg/mL) treated Chlorella vulgaris increased 21% and 17% than that of the control group. The accelerated photosynthesis make the biomass of Chlorella vulgaris to improve and the content of nutritional components increased by 20%~32%. The other is that they can protect Chlorella vulgaris cells against the damage of UV radiation on account of their optical properties and high antioxidant activity. Furthermore, the obtained CDs have benign free radical scavenging activity, which ascribe that CDs is good electron acceptor due to the abundant -OH group and conjugate sp2 carbon for delocalizing and stabilizing free electron.48 They as an anti-oxidants can protect Chlorella vulgaris cells against the damage of UV radiation. These property of CDs render the biomass and growth rate of Chlorella vulgaris treated with CDs increased 18% and 17% compared with the control exposed by UV radiation.

Figure 7. The participation of CDs in the growth of Chlorella vulgaris.


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4. CONCLUSION We have studied the impacts of CDs on the growth of Chlorella vulgaris, which treated with CDs could grow faster and the biomass and growth rate of Chlorella vulgaris treated with CDs increased by 17% and 21%, respectively. That is because the CDs could be degraded by Chlorella vulgaris and produced a number of CO2, which could be immobilized by photosynthesis. In addition, the CDs can also increase the in vitro and in vivo activity of Rubisco by 38% and 34%, respectively. That could accelerate the photosynthesis of Chlorella vulgaris as well. Moreover, the CDs could absorb ultraviolet radiation and have the highest antioxidant activity than that of AA. They as an anti-oxidants can protect Chlorella vulgaris cells against the damage of UV radiation and enhance the specific growth rate and the biomass by 17% and 18% under the UV radiation. In a word, the low toxicity and biodegradation of CDs have positive effects on the growth of Chlorella vulgaris, by which they not only improve the growth of Chlorella vulgaris with high quality and can protect Chlorella vulgaris cells against the damage of UV radiation. This study may offer a practical way to improve the growth of Chlorella vulgaris and could be deepen to understand the relationship of carbon-based nanomaterials to microalgae. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the Internet at http://pubs.acs.org. TEM images, XPS spectra and UV spectra of CDs. The chemical ingredients of F/2. Detail procedures of experimentation. The UV-Vis spectrum of Chlorella vulgaris. Standard curve of carbohydrate content and protein content. The growth curve of chlorella and the digital photos of CDs during degradation process.


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AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]; [email protected] NOTES The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENT This work is supported by the Collaborative Innovation Center of Suzhou Nano Science and Technology, the National Natural Science Foundation of China (51725204, 51572179, 21471106, 21771132, 21501126), the Natural Science Foundation of Jiangsu Province (BK20161216), the China Postdoctoral Science Foundation (2017M611902) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). REFERENCES (1) Chisti, Y. Biodiesel from Microalgae. Biotechnol. Adv. 2007, 25 (3), 294-306. (2) Mata, T. M.; Martins, A. A.; Caetano, N. S. Microalgae for Biodiesel Production and other Applications: A review. Renewable Sustainable Energy Rev. 2010, 14 (1), 217-232. (3) Safi, C.; Zebib, B.; Merah, O.; Pontalier, P. Y.; Vaca-Garcia, C. Morphology, Composition, Production, Processing and Applications of Chlorella vulgaris: A review. Renewable Sustainable Energy Rev. 2014, 35, 265-278. (4) Lee, Y. C.; Lee, K.; Oh, Y. K. Recent Nanoparticle Engineering Advances in Microalgal Cultivation and Harvesting Processes of Biodiesel Production: A review. Bioresour. Technol. 2015, 184, 63-72.


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Graphical Abstract


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Pristine Carbon Dots Boost the Growth of Chlorella vulgaris by

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