Protection of Photosynthetic Algae against Ultraviolet Radiation by

Feb 15, 2017 - Blachon, Harb, Munteanu, Piednoir, Fulcrand, Charitat, Fragneto, Pierre-Louis, Tinland, and Rieu. 2017 33 (9), pp 2444–2453. Abstract...
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Protection of Photosynthetic Algae against Ultraviolet Radiation by One-step CeO Shellization 2

Pengqiang Duan, Tingting Huang, Wei Xiong, Lei Shu, Yuling Yang, Changyu Shao, Xurong Xu, Weimin Ma, and Ruikang Tang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04421 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

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Protection of Photosynthetic Algae against Ultraviolet Radiation by One-step CeO2 Shellization Pengqiang Duan,† Tingting Huang,‡ Wei Xiong,† Lei Shu,† Yuling Yang§,† Changyu Shao,† Xurong Xu,*,†,# Weimin Ma*,‡ and Ruikang Tang*,†,# †

Center for Biomaterials and Biopathways, Department of Chemistry, Zhejiang

University, Hangzhou, Zhejiang 310027, China. ‡

College of Life and Environmental Science, Shanghai Normal University, Shanghai

200234, China. §

Electric Power Science and Research Institute, Yunnan Power Grid, Kunming,

Yunnan 650217, China. #

Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, Zhejiang

310027, China.

ABSTRACT: Photosynthetic microalgae play an important role in solar-to-chemical energy conversion on Earth but the increasing solar ultraviolet (UV) radiation seriously reduces the biological photosynthesis. Here we developed a one-step approach to construct cell-in-shell hybrid structure by using direct adsorption of CeO2 nanoparticles onto cells. The engineered CeO2 nanoshell can efficiently protect the 1 ACS Paragon Plus Environment

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enclosed Chlorella cell due to its excellent UV filter property, which can also eliminate UV-induced oxidative stress. The experiments demonstrate that the resulted algae-CeO2 composites can guarantee their biological photosynthetic process and efficiency even under UV. This study follows a feasible strategy to protect living organisms by using functional nanomaterials to improve their biological functions.

INTRODUCTION Algae are the most widespread and fastest-growing photosynthetic organisms in nature and their photo-chemical efficiency is 7-31 times greater than the land-based plants. Therefore, algae are considered as the most promising long-term, sustainable sources of biomass,1 with an indication of that algae will play an important role in overcoming the increasing energy demands and environmental concerns in the near future.2-4 However, the efficiency of photosynthesis of algae is extremely sensitive to environmental stresses.5,6 Ultraviolet radiation (UV) in solar spectrum, a permanently existing environmental factor, can result in a significant inhibitory effect on the photosynthetic microalgae.7,8 Generally, the biological effects of UV radiation involve photosensitization and formation of toxic reactive oxygen species (ROS),9,10 which are fatal to microalgae. Previous studies have evidenced that the natural levels of UV radiation can significantly affect activities of relevant enzymes,11 damage DNA,12,13 reduce photosynthetic pigments and even destroy PSII structure of protein–pigment complex in algae,14 which may finally lead to the decrease of photosynthesis. Currently, there is a great challenge to eliminate the biological damages caused by UV radiation. 2 ACS Paragon Plus Environment

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During biological evolution, some organisms have developed biomineralization strategy to create exquisite inorganic compounds with extraordinary properties in optics,15,16 photonics,17 magnetics,18,19 and mechanics etc.20 Recently, scientists have successfully protected several living cells from harsh environmental conditions by using biomineralization-inspired material shells.21-24 These studies highlight the important

role

of

biomimetic

shellization

in

organism

protections

and

improvements.25-28 Furthermore, it has been demonstrated that the artificially conferred silica shell can alleviate high light-induced photoinhibition on microalgae.23 However, these modified microalgae cells must receive extra layer-by-layer or polyelectrolyte treatments to enhance their biomineralization abilities to receive the shell protections. Unfortunately, these treatments are relatively complicated and time-consuming.

Beyond

the

above-mentioned

attempts,

other

biomimetic

shellization methods such as ion binding and heterogeneous nucleation, etc. require cell surfaces with relatively high density of receptors for ion accumlations.29 However, it has been noticed that the excessive ion treatment can cause damage to algae due to the salt stress.30,31 Alternatively, it would be of importance to minimize the encapsulation steps for the cells, which not only reduces the possible damage but also saves the treatment cost. With the aim of protecting algae from harmful UV, a biocompatible shell with UV filter property should be rationally designed and constructed. Besides, the fabrication process should be under physiological conditions and biologically friendly. We noted that CeO2 nanoparticles could be used as UV filter materials and they were 3 ACS Paragon Plus Environment

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biocompatible for a wide range of microorganisms.32-35 Chlorella pyrenoidosa (C. pyrenoidosa) is a genus of single-cell green algae and in nature, its surface is a negatively charged cell wall. In this case, CeO2 nanoparticles, whose surface are positively charged, could be introduced to the cell wall electrostatically without any modification. Herein, we report an one-step method to construct functional alga-CeO2 composites, which can guarantee the cell photosynthesis under UV radiation.

EXPERIMENTAL SECTION Cells Culture Conditions. Chlorella pyrenoidosa cells were cultured in Tris-Acetate-Phosphate (TAP) medium. The medium was autoclaved at 121 °C for 20 min and its pH was adjusted by using 0.1 M acetic acid. The Chlorella cells were cultured in the TAP medium bubbled with sterile air at 25°C with continuous illumination of 40 µmol m-2 s-1 by the fluorescent lamps for 24 h (A750=0.8-1.0). Encapsulation of Chlorella Cells with CeO2 Nanoparticles. Chlorella cells were harvested by centrifugation (5,000g) for 5 min at room temperature and suspended in tphosphate buffer saline (PBS) solution (10 mM, pH 7.0). The cell density was adjusted

to

6×106

cells/mL,

and

then

CeO2

nanoparticles

(nanopowder,

Sigma-Aldrich, USA, morphology shown in Figure S1, size distribution was provided in Figure S2) were added to the solution and the final concentration of CeO2 in the PBS was 0.05 mg/mL. The mixture was stirred for 60 min and the resulted suspensions were centrifuged and re-suspended to TAP for further investigation.

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Material Characterizations. Scanning electron microscope studies (SEM) were conducted with a S-4800 (HITACHI, Japan) equipped with an energy-dispersive spectroscope (EDAX, USA). Samples of Chlorella and Chlorella@CeO2 were fixed by another PBS solution (10 mM) containing 2.5 wt.% glutaraldehyde. The fixed ones were washed by 0.01 M NaCl solution and dehydrated using ethanol. The samples were transferred on silicon chips and air-dried overnight at room temperature. Transmission electron microscopy (TEM) was performed on a HT-7700 (HITACHI, Japan). The biological specimens were fixed by using glutaraldehyde, OsO4, and K2Cr2O7 and the cells were dehydrated in ethanol/acetone and embedded in Epon 812/Araldite M resin. Thin sections were cut by using a Reichert ultratome (Zeiss, Germany) and the cells were stained with uranyl acetate and lead citrate. Zeta Potential Measurement. The zeta potentials of the native Chlorella, CeO2 nanoparticles and Chlorella@CeO2 were determined by a Zetasizer Nano S (Malvern, UK). In this examination, all sample densities were about 6×106 cells/mL in PBS buffer and the CeO2 concentrations were 0.05 mg/mL. Photosynthesis Measurement and UV Exposure Investigation. The yields of chlorophyll fluorescence at steady-state of electron transport were measured by using a Dual-PAM-100 monitoring system (Walz, Effeltrich, Germany) equipped with an ED-101US/MD unit at room temperature.36 Minimal fluorescence at open PSII centers in the dark-adapted state (Fo) was excited by a weak measuring light (650 nm) at a PFD of 0.05 to 0.15 µmol m-2 s-1. A saturating pulse of red light (600 ms, 10,000 µmol m-2 s-1) was applied to determine the maximal fluorescence at the closed PSII 5 ACS Paragon Plus Environment

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centers in the dark-adapted state (Fm). The maximal quantum yield of PSII (Fv/Fm) was evaluated by using an equation of (Fm - Fo)/Fm.37 The electron transfer rate of PSII (ETR(II)) was measured under 200 µmol m-2 s-1 photosynthetic active radiation (PAR). The photosynthetic O2 production in native Chlorella and Chlorella@CeO2 cells were measured at 25°C by monitoring the evolution of O2 with a Clark-type oxygen electrode (Hansatech Instruments, Kings Lynn, UK). Oxygen evolution was measured in the presence of 10 mM NaHCO3 under a light intensity of 500 µmol m-2 s-1. The UV-Vis spectra were obtained with a PGENERAL T6-New Century spectrometer in the transmission mode. All absorption spectra were determined in the aqueous state of the samples. In the UV exposure test, both the native Chlorella cells and Chlorella@CeO2 cells were exposed under an artificial UVB (280-320 nm) irradiation system equipped with a 1.2 W ultraviolet lamp (Philips, Netherlands). The actual UVB radiation power on the algae was adjusted by the distance and measured by a UV radiometer. An additional 40 µmol m-2 s-1 photosynthetic active radiation was also illuminated during the examinations to provide the growth light for the algae cells. The cell suspensions were filled within quartz cuvettes (width of 1 cm) and then used for the exposure tests. Detection of UV-induced H2O2 Levels. The measured Chlorella cells were broken by using a 5-min ultrasonic treatment and then the samples were obtained by using centrifugation (10000 g for 3 min) and the H2O2 levels in the supernatants were detected using a commercial examination kit (A064-1, Nanjing Jiancheng Bioengineering Institute, China). 6 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION The zeta potentials (Figure 1) of the CeO2 nanoparticles and the native Chlorella cells in the 10 mM PBS were 20.70 ± 6.57 and -19.10 ± 8.64 mV, respectively. Theoretically, the material particles could be absorbed onto the cell surface due to the electrostatic interaction. And as the expectation, the cell-material composites were resulted spontaneously by mixing the algae and CeO2 nanoparticles. We also found that the Chlorella cells slightly aggregated in the beginning upon the addition of the nanoparticles but after 1 h stirring, the cells dispersed well in the solution and the zeta potential of the engineered Chlorella cells was reduced to -4.20 ± 3.09 mV, which implied the incorporation of the positively charged CeO2 materials.

Figure 1. Zeta potentials of the native Chlorella cells, CeO2 nanoparticles and Chlorella@CeO2. The data were obtained in their 10 mM PBS buffer solutions.

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Figure 2a shows native Chlorella cells under SEM, which were typical spheres. The cell surfaces were relatively smooth. After the material treatment, the incorporated CeO2 nanoparticles could be observed onto the cells (Figure 2b) and they resulted in the thin layers around the cells, forming shell-like structures. TEM of the microtome-sliced Chlorella@CeO2 showed that the layer thickness was 40-50 nm (Figure 2d). The composite structure of the Chlorella core and the CeO2 shell was further confirmed by EDAX mapping examination (Figure 2f).

Figure 2. SEM micrographs of (a) native Chlorella and (b) Chlorella@CeO2. TEM micrographs of microtome-sliced (c) native Chlorella and (d) Chlorella@CeO2.

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EDAX elemental mapping of (e) native Chlorella and (f) Chlorella@CeO2. The red, green and yellow represent C, O and Ce, respectively.

Photosystem II (PSII), an oxygen involved process, is mainly dependent on the highly structured photosynthetic machinery in organisms. Thus, the maximum quantum yield of PSII (Fv/Fm) is closely related to the cell integrity and cell viability. At the early state after the material treatment, the photosynthetic activity of PSII was around 85% (=0.63/0.74) in comparison with the native ones (Figure 3a). But it could recover to the normal level within 6 h, which implied that the engineered cells compromised to the new CeO2 shell at the early stage of the material incorporation but could adapt the shell structure gradually. This phenomenon might be attributed to several factors. First, the cell walls of algae consist of cellulose, polysaccharides, and glycoproteins and they could provide enough binding sites for CeO2.38,39 Since the nanoparticles were bonded to the cell surface by nonspecific (electrostatic, hydrogen bonding, and hydrophobic) interactions, the cell viability was reduced by these newly formed surface bonds at the early stage. As the binding energy was relatively weak, the engineered cells could keep their integrity inside the nanoshell and the photosynthetic activities could recover to the normal level with time. Another factor that would influence the cell viability was the cerium ions release. In the experiment, we found that the cerium ions reached the equilibrium concentration in 48 h (Figure S3). The final concentration of cerium ions in the medium was always < 0.2 mg/L, which were reported to be safe to the green algae.40,41 Besides, it has been reported 9 ACS Paragon Plus Environment

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that CeO2 nanoparticles with different surface properties might have different effects on the cell growth.35 Thus, we measured the growth curves of both native and engineered cells (Figure S4). The experimental data revealed that the CeO2 shells could inhibit cell division in the early stage. But for 84 h cell culture, we found that the coated Chlorella could proliferate and reach the same cell density just with a time delay of 12 hours. This delay was attributed to the shell formation, which partially limited the cell activity, especially at the beginning of the material treatment. These results showed that the CeO2 nanoparticles were non-toxic and the nanoshell was biocompatible to the enclosed Chlorella cell.

Figure 3. Comparison of several parameters in native Chlorella (black lines) and Chlorella@CeO2 (red lines). (a) Photosynthetic activity (Fv/Fm) variation with time after CeO2 treatment (n≥5). (b) UV-vis absorption spectra of native Chlorella,

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Chlorella@CeO2 and CeO2 nanoparticles (blue line). (c) The maximum quantum yield (Fv/Fm) of PSII after exposure under 0.3 mW/cm2 UVB radiation (n≥5).

The UV-Vis spectrum of the engineered Chlorella cell was examined to confirm the expected property from the shell structure (Figure 3b). The native Chlorella cells could absorb UV light due to the biomacromolecules within the organisms.42 Noticeably, the CeO2 nanoparticles had a broad and strong absorption band at the UV range (250–400 nm) but the visible light (400-760 nm) used for photosynthesis was not absorbed. After the coating using CeO2 nanoparticles, a similar adsorption band in the UV range was detected and the peak was analogous to that of the material phase. Since the material encapsulated the cells to result in core-shell structure, any light must pass through the outmost CeO2 shells before reach to the inside cells. Therefore, the functional shells efficiently filtered UV radiation so that the enclosed Chlorella cells could be protected, guarding their biosystem under UV radiations. To further investigate the effect of the CeO2 nanoshells on the UV protection, both the engineered Chlorella cells and native Chlorella cells were exposed to an artificial UVB irradiation system as UVB can cause much more biological damages than the other UV on the earth ground. The maximum quantum yield of PSII was measured to evaluate UV induced damage to the Chlorella cells (Figure 3c). For a long-term exposure under the continuous UVB radiation with a dose of 0.3 mW/cm2, we found that the native Chlorella cells were inactivated and the photosynthetic activity decreased dramatically (0.74 to 0.16) in the first 12 h and turned to 0.10 after 24 h. 11 ACS Paragon Plus Environment

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However, the CeO2 engineered cells showed a significantly higher survival ability. Different from the native ones, their Fv/Fm kept at 76.39% (=0.55/0.72) as the beginning and this value remained even after 24 h radiation. The effective quantum yield of PSII , Y(II), (Figure S5) demonstrated the similar tendency about the improvement . These results indicated that the artificial CeO2 shell could protect the Chlorella from UV radiation and the cell-in-shell structure could provide the photosynthetic organism a more favorable and stable environment. The well-established Z-scheme photosynthetic electron transport mechanism suggests that the efficient photosynthesis requires continuous photoelectrons transportation with a high and constant rate.43 Once linear electron transport chains are damaged by some environmental factors, the photoelectrons cannot transfer from PSII to the site for carbon reduction so that the subsequent carbon assimilation will be suppressed. The linear photosynthetic electron transport rate (ETR(II)) is a key photosynthetic factor in the photosynthesis process. Figure 4a shows that ETR(II) of the engineered Chlorella cell always kept at a high level (57.97 to 45.93 µmol/m2s) in the whole exposure experiment test but that of the native ones turned to zero after the 12 h treatment. It followed that the photosynthetic electron transport chain can be completely blocked by UV, but fortunately this negative effect can be reduced by a rationally designed material shell. Besides, we found that the electron transport rate decreased much more obviously than the activity of PSII in native Chlorella cells in the early stage. For example, Fv/Fm reduced 16.46% but ETR(II) reduced 60.55% after 3 h treatment. Thus, the 12 ACS Paragon Plus Environment

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photosynthetic electrons generated on PSII could not be fully used for subsequent carbon reduction and then O2 would play as electron acceptors to counteract the excessive photosynthetic electrons.44 But this would lead to the formation of reactive oxygen species (ROS). ROS, such as hydrogen peroxide (H2O2), hydroxyl radical (HO·) or hydroperoxyl (HO2·), could destroy macromolecules essential for the integrity of the cell and even lead to cell death.45 Fortunately, the CeO2 nanoparticles, which have an auto regenerative surface redox reaction cycle (Ce3+↔Ce4+) (confirmed by X-ray photoelectron spectroscopy in Figure S6), were reported to have an unprecedented antioxidant activity.46,47 In this experiment, the antioxidant properties were investigated by detecting the concentration of H2O2 because it had a relatively long life time and was readily for detection compared to the other free radicals. After 24 h exposure to the UV radiation (0.3 mW/m2), the ROS level (Figure 4b) of native Chlorella cell suspension surged to as high as 1.09 µmol/µg chlorophyll. However, for the CeO2 engineered cells with the same initial cell density, the ROS level was almost flat and near zero. Two possible reasons might account for this experimental phenomenon: 1) most of the UV light was filtered by the CeO2 nanoshell so that there was less possibility to generate massive ROS; 2) even a small amount of UV light passed through the shell, the ROS formed in the engineered Chlorella cells would be scavenged due to the surface redox reaction of the CeO2 nanoshell. Since the UV-induced ROS could be eliminated, the cell-material composites could be guarantee the photosynthetic system to operate normally under UV radiations. 13 ACS Paragon Plus Environment

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Figure 4. (a) Photosynthetic electron transfer rate (ETR) of PSII after exposure under 0.3mW/cm2 UVB radiation (n≥5). (b) The toxic ROS (H2O2) induced by the UVB radiation (n=3). (c) Photosynthetic electron transfer rate after treated with H2O2 (1%) (n≥5). (d) photosynthetic production of O2 with 10 mM NaHCO3 as the artificial electron acceptor (n≥5).

Further experiments were conducted to examine whether the fashionable shell could defend against exogenous oxidative stress. A higher concentration of H2O2 (1%) was added to the Chlorella cell suspension (without UV radiation). After 2 h treatment, the ETR(II) (Figure 4c) of Chlorella@CeO2 decreased from 58.52 to 45.98 µmol/m2s but that for native Chlorella, from 57.84 to 27.36 µmol/m2s. Since the ETR(II) value of the native Chlorella decayed 2.6 folds in comparison with the 14 ACS Paragon Plus Environment

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engineered ones, the data suggested an excellent protection of the material shell against the exogenous oxidative stress. The rate of O2 evolution from the algae is the most important indicator in the photosynthesis system. Accordingly, we measured the photosynthetic evolution of O2 of the native and engineered algae cells after 6 h exposure (Figure 4d) under UV. In these experiments, NaHCO3 (10 mM) was used as an artificial electron acceptor. We found that the rate of O2 evolution decreased rapidly with the increasing of UVB intensity (from 0.1 to 0.3 mW/cm2) in the native ones and the O2 evolution even stopped at the intensity of 1 mW/cm2. This phenomenon followed that the photosynthesis system was irreversibly damaged by the radiation. In contrast, the O2 evolution of the engineered ones kept the original ability of 45.53% even if they were exposed under UVB radiation of 1 mW/cm2 after 6 h. The UVB dose at ground level is related to ozone concentration, weather, altitude and the time in a day. Generally, it reaches the maximum value at noon and the maximum radiation power of UVB is different from place to place. It should be mentioned that at ground level the maximum radiation power of UVB is no more than 0.3 mw/cm2.48,49 So these results indicated the engineered Chlorella cells could survive in the high UVB region successfully. It also follows that the rationally engineered Chlorella cells could produce more biomass because the biomass was positively related O2 evolution in the photosynthetic system.

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Our present work has successfully fabricated CeO2 nanoshell on the surface of Chlorella pyrenoidosa through a one-step method (Scheme 1). The engineered shells endow the Chlorella cell with ability to protect them from UV radiation due to their excellent UV filter property as well as the elimination of UV-induced oxidative stresses. Therefore, the engineered Chlorella cells can survive even under UV radiations. This strategy of constructing biotic-abiotic hybrid using functional nanoparticles would improve microalgae to survive under a harsher environment and ensure their photo-chemical efficiency. It is believed that such hybrid organism has potential as an important tool to solve the environmental problem and the green energy production.

Scheme 1. Scheme of one-step CeO2 nanoshell formation on the Chlorella cell and the mechanism of UV protection.

ASSOCIATED CONTENT

Supporting Information.

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Additional information is available free of charge on the ACS Publications website. The file includes SEM micrographs of CeO2 nanoparticles (Figure S1), TEM micrographs of CeO2 nanoparticles and their size distribution (Figure S2), inductively coupled plasma mass spectrometry (ICP-MS) measurement of cerium ions release in different culture time (Figure S3), the growth curves of native Chlorella cells and CeO2 coated cells (Figure S4), the effective yield of PSII (Y(II)) of native Chlorella and Chlorella@CeO2 in different exposure time (Figure S5) and XPS spectrum of the CeO2 nanoparticles (Figure S6). AUTHOR INFORMATION Corresponding Author * [email protected].

* [email protected].

* [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank Mengjie Yu and her colleagues for their assistance in biological specimen preparation. This research was supported by the National Natural Science Foundation of China (21625105 and 21471129), the Zhejiang Provincial Natural

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Science Foundation of China (LY17B010001) and the Fundamental Research Funds for Central Universities (2016QN81020). ABBREVIATIONS UV, ultraviolet; UVB, ultraviolet B; ETR, electron transport rate; PS, photosystem; ROS, reactive oxygen species; TAP, Tris-Acetate-Phosphate; PBS, phosphate buffer saline;

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One-step approach to construct cell-in-shell hybrid structure and the resulted algae-CeO2 complex can guarantee its photosynthetic efficiency even under high ultraviolet conditions.

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