Using N-Doped Carbon Nanosheets - ACS Publications - American

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Sustainable growth and lipid production from Chlorella pyrenoidosa using N-doped carbon nanosheets: Unravelling the role of Graphitic Nitrogen Anwesha Khanra, Sujata Sangam, Adeeba Shakeel, Deepa Suhag, Subhradeep Mistry, Monika Prakash Rai, Sandip Chakrabarti, and Monalisa Mukherjee ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03103 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 8, 2017

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ACS Sustainable Chemistry & Engineering

Sustainable growth and lipid production from Chlorella pyrenoidosa using N-doped carbon nanosheets: Unravelling the role of Graphitic Nitrogen Anwesha Khanra‡1, Sujata Sangam‡1, Adeeba Shakeel‡1, Deepa Suhag‡2, Subhradeep Mistry3, Monika Prakash Rai*1, Sandip Chakrabarti*4, Monalisa Mukherjee*1,2

1

Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector-125, Noida – 201313

(UP), India. Tel: +91(0)-120-4392194. *E-mail: [email protected]; [email protected] 2

Amity Institute of Click Chemistry Research and Studies, Amity University Uttar Pradesh,

Sector-125,

Noida

–

201313

(UP),

India.

Tel:

+91(0)-120-4586945.

*E-mail:

[email protected] 3

Framework Solid Laboratory, Solid State and Structural Chemistry Unit, Indian Institute of

Science, Bangalore, 560012, India. Tel: +91(0)-80-22932551. 4

Amity Institute of Nanotechnology, Amity University Uttar Pradesh, Sector-125, Noida –

201313 (UP), India. Tel: +91(0)-120-4392130. *E-mail: [email protected] ‡ These authors contributed equally to this work.

KEYWORDS: Bioenergy, Carbonaceous nanomaterials, Microalgae, N-doped graphene nanosheets, Lipid enhancement, Pharmaceuticals, Nutraceuticals.

ACS Paragon Plus Environment

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ABSTRACT The advent of novel carbonaceous nanomaterials (CMs) associated with microalgae paved an alternate way for the bioeconomic production of biofuels as well as high value added compounds. Herein, we for the first time, present a holistic approach for sustainable biomass and lipid production from Chlorella pyrenoidosa, wherein, CMs, namely N-doped carbon nanosheets (CNS) and N-doped graphene nanosheets (NGS) were used as one of the algal growth supporting factors. Doping carbon nanomaterials with nitrogen can effectively tune its electronic structure and other intrinsic properties for efficient photocatalysis. The utilization of CNS and NGS in this process lead to rapid, environment friendly, and facile assimilation of biomass and lipids for the development of nutraceuticals, pharmaceuticals, and other bioenergy associated applications. Employing a suite of characterization methods, the intrinsic structural and morphological properties of CMs were revealed. Compared with control, the lipid content obtained in the presence of undoped carbonized carbon materials (CCM), CNS, and NGS were found to be around 1.5, 2 and 6 folds higher, respectively, at similar growth conditions. We, therefore, envisage that graphitic nitrogen rich NGS plays a pivotal role in enhancing the lipid production from algae. This finding therefore, exhibits a promising potential to bring about a paradigm shift in the field of bioenergy frameworks. INTRODUCTION The growing interest in utilizing microalgal biomass and lipids for the production of biofuels, nutraceuticals, and pharmaceuticals have spurred the researchers to explore the innovative and economically-viable approach using carbonaceous nanomaterials.1,2 Oxygenic photosynthetic microalgae have drawn significant interest due to their ability for rapid growth, accumulation of storage molecules, facile cultivation, biodegradable fuel production, and synthesis of value added


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co-products such as long chain Poly unsaturated fatty acids (PUFA) and carotenoids in a diverse manner.3 Harvesting solar energy by photosynthetic machinery of microalgae is a facile and economic process for the translation of sunlight, water, and CO2 for the production of biomass and triacylglycerols (TAG).4,5 India is very fortunate to have ample source of solar energy, thereby making the photoautotrophic cultivation of the chosen algal strain technically feasible for the fixation of large amount of CO2, without the involvement of any expensive organic supplements.4 The selection of a prospective microalgal strain and mode of cultivation are the major bottlenecks for commercialization. To overcome these limitations related to the synthesis of bio-products from microalgae, nanomaterials have been introduced into the cultivation medium.6,7 Microalgae, Chlorella pyrenoidosa is a robust, fresh water, unicellular chlorophycean, known for their ability to grow even under environmental stress conditions and have already been reported for their remarkable bioremediation properties from industrial as well as municipal waste water.8,9 Therefore, C. pyrenoidosa has been largely investigated for their potential in carbon dioxide sequestration, phycoremediation, and large scale production of nontoxic biodiesel and several other biomolecules.5,10,11 A number of literatures depict the utilization of nanomaterials wherein, carbon nanotubes (CNT) are shown to optimize microalgal fitness by enhancing the production yield, however the challenges lie in their inherently toxic nature.12,13 In addition to this, there are few reports demonstrating the enhanced lipid extraction efficiency from the microalgal cells using carbon nanomaterials with adverse effects on cell density.6,14


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In a recent report, carbon nanomaterials were conjugated with the plant cells and their photosynthetic activity was noted to rise by 30 %. This observation could be accredited to the ability of the carbon nanomaterial to share electrons through the thylakoid of chloroplasts, thereby, allowing the chloroplasts to capture a broader range of visible light.15 Hence, the idea for this novel nanobionic approach is to incorporate synthetic photocatalyst (CMs) to impart microalgae with augmented photosynthetic activity for efficient lipid synthesis and biomass production. Cultivation of microalgae using nitrogen doped carbon nanosheets is in its embryonic stage. Band gap engineering is key to developing new artificial photocatalysts (via doping) for effective conversion of solar energy into chemical energy in the field of biomimetic carbon materials.16-19 The unique properties of CNS and NGS such as large surface-to-volume ratio, electrochemical stability, immobilizing ability, catalytic activity, high chemical tunability, prolonged visible-light absorption, extended π-conjugated system, and sharp edge planes render them suited as a growth substrate for algae. Moreover, the improved catalytic activity and dispersibility of NGS over CNS and CCM would present them as suitable substrates for algal culturing for enhanced lipid production. Plausibly, the sharp edge planes may also create stress by proposing the chance to modify the characteristics of feedstock materials for pilot scale cultivation.20,21 In order to explore this field further, we have prepared doped and undoped CMs, which were synthesized in bulk from common and inexpensive chemical reagents. These materials were investigated for sustainable growth and lipid production from C. pyrenoidosa. Additionally, nitrogen doping (Ndoping) is expected to introduce additional n-type carriers in carbon system resulting into highly porous architecture, enhanced catalysis and biocompatibility.19,22,23 These properties further facilitate the adherence of nitrogen doped CMs to cellular surfaces, thereby enhancing the yield


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of metabolic products.14 Oleaginous microalgae possess a thick cell wall composed of complex carbohydrates and glycoproteins, manifesting chemical resistance and high mechanical strength leading to an expensive and energy-intensive process of lipid extraction.4 Moreover, it is very challenging to remove the capping agents like polysaccharides, proteins, and lipids.6 Immobilization of functional groups of NGS with C. pyrenoidosa possibly weakens the cell-wall that would lead to easy cell-disruption for facile lipid extraction.10 Furthermore, as reported earlier by our group, the sharp edges of N-doped CMs can be an effective tool to overcome this barrier, eliminating the need of any toxic solvents and chemicals.21,24 Subsequently, additional advantage of employing these CMs is their reusability which is attributed to their easy recovery from organic phase via centrifugation after lipid extraction. Incorporation of nitrogen atoms into CCM could be responsible for physio-chemical changes depending on doping configuration.22 there is a tendency of enrichment of graphitic nitrogen after the reduction of CNS.20 Therefore, the present study is the first report of the plausible role of surface area, hierarchical micro-macroporous organization, and graphitic nitrogen available in NGS for obtaining higher lipid as well as biomass production, consequently opening up the field of graphitic sp2 nitrogen catalysis for numerous biotechnological and biomedical applications. EXPERIMENTAL SECTION Synthesis of CMs CMs were synthesized using solvothermal techniques. CNS was synthesized with some modification in the literature procedure.20 The ratio of melamine: glycerol: H2SO4 was kept as 0.4 g : 10 ml : 10 ml to influence the incorporation of appropriate ‘N’ configuration into the carbon framework of CNS. In comparison with our earlier reports the amount of melamine was reduced to 0.4 g.


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Scheme 1 Hydrothermal synthesis of CNS and NGS NGS was obtained by reduction of CNS as reported earlier by our group using de-ionized (DI) water as a solvent (Scheme 1).20 Undoped carbonaceous material CCM was synthesized using similar technique in absence of dopants.21 Characterization Chemical Composition Raman Spectra was performed on confocal micro-Raman LabRam HR spectrometer (Horiba Scientific) by using Backscattering geometry with a CCD detector at 633 nm Ar laser and 100 X magnification. Calibration of the Raman spectrometer was done using an internal silicon reference at 520 cm-1, resulting into a peak position resolution of less than 1 cm-1. X-ray Photoelectron Spectroscopy were recorded using a Kratos Axis Ultra Photoelectron Spectrometer that uses Al Kα (1253.6 eV) X-rays. Casa XPS version 2.2.73 software was used for curve fitting and background subtraction. The powder X-Ray diffraction (XRD) pattern was obtained from PANalytical EMPYREAN diffractometer using CuKα radiation in the 2θ range at room temperature. The N2 adsorption experiments were performed on the Belsorp max instrument at 77 K. Morphology The surface morphology of CNS, CCM and NGS were observed using Scanning Electron Microscopy (SEM) Zeiss EVO40. Transmission Electron Microscopy (TEM) and High Resolution Transmission Electron Microscopy (HRTEM) were carried out on a JEOL, JEM2100F electron microscope at an acceleration voltage of 200 kV. Samples were prepared by drop


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casting the carbonaceous nanomaterials dispersion onto a carbon coated copper grid followed by drying at room temperature. Microalgal strain and media preparation The fresh water green microalgae C. pyrenoidosa NCIM 2738, obtained from National Collection of Industrial Micro-organism, National Chemical Laboratory, Pune, India, has been grown photoautotrophically in Fogg’s media.8 The microalga was cultivated in 1 L Erlenmeyer flask having 500 ml autoclaved media. The algal cultures were maintained in a temperature controlled incubator at 28 ± 2 °C, providing continuous light (24 h) illumination (40 Watt, white tube light) of 100 µmol m-2s-1 and shaken intermittently. Microalgae were initially cultured photoautotropically on atmospheric CO2 and pH of the medium was maintained at 7.5 by adding 0.1 mol L-1 HCl to the culture medium. Inoculation was done using exponentially growing cells to maintain the initial optical density (O.D) at 0.1. Experiments were performed in triplicates and the average data were plotted in the form of graphs and tables. Growth Analysis The microalgal cells were cultivated in 100 ml Erlenmeyer flask, containing 50 ml autoclaved medium in the presence or absence of different concentrations (0, 1, 2, 3, 4, 5 mg/50 ml) of Ndoped CMs (NGS and CNS) and undoped CMs (CCM). Growth of the microalgal strain was measured at an optical density of 665 nm spectrophotometrically (UV/Vis. Schimadzu) after every 24 h of inoculation. After 25 days, photoautotrophic cultures with NGS, CNS, and CCM were harvested by using centrifugation (Eppendorf 5810R) at 960 x g for 15 mins. The algalCMs conjugates were seen to be insoluble in the culture medium. Therefore, they could be easily harvested followed by decantation with further removal of water via centrifugation. The pellet


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was then washed twice by distilled water to remove the cell debris and dried in oven at 60 oC to obtain the dry cell mass. Biomass concentration was calculated by using the following formulae

(1)

Lipid analysis Lipid biomolecules were extracted from dried microalgal biomass by using Bligh & Dyer method.25 The lipid production and lipid content were calculated as ! !"

! %

#$#% &

(#)* #$#% (#)*

+ 100

(2)

(3)

RESULTS AND DISCUSSION Surface and Morphological characterization of CMs

Figure 1 CM structural morphology without microalgae; (a) SEM image of CNS; (b) HRTEM image of CNS; (c) SEM image of NGS, and (d) HRTEM image of NGS.


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SEM images of NGS (Figure 1c) exhibited thin graphitic flakes, however CNS (Figure 1a) was found to be thin, folded and wrinkled flower petal like structure. The HRTEM image of NGS (Figure1d) exhibited parallel fringes indicating lattice lines confirming its crystalline nature. The SAED pattern of CNS (inset of Figure 1b) displayed diffused ring pattern, suggesting its amorphous nature which upon reduction shows progressive ordering of the nanosheets for NGS due to self healing.20 Furthermore, recently reported AFM images confirm the height profile of the as-synthesized CNS and NGS to lie in the nanometer range.24,26 The presence of grooves and folded regions of NGS along with their highly porous nature20 enable NGS to enact as a stable platform for algal growth. The sharp edges present in the 2D NGS results in creating stress, which eventually yields high lipid content. The interlayer lattice spacing of NGS is around 0.35 nm (Figure 1d), which is consistent with reported literatures.20 The morphology of C. pyrenoidosa cells after the exposure to NGS, CNS, and undoped CCM have been analysed by SEM (Figure 2). In order to get an insight on the reproduction pattern of this microalga, we have captured the SEM images during the early exponential phase in the presence and absence of CMs. The C. pyrenoidosa cells are noted to be very small in size as these are newly divided daughter cells emerged from multiple fission27 depending upon the genetic potential, culture compositions, and growth conditions. The green microalgal cells displayed unicellular, spherical shaped morphology, containing smooth cell wall with the cell sizes ranging from 90 nm to 130 nm in both control (Figure 2c) and undoped CCM (Figure 2d). In case of CNS (Figure 2a), a dense and uniform growth of the algal cells was observed (like lamellar wafers in appearance) on the surface of carbon nanosheets due to continuous multiplication of microalgae that ultimately leads to cracking of the sheets. These cracks may be due to high pressure exerted by dividing microalgal cells on the CNS. It was determined from


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SEM image that the shape remained similar in control as well as in the sample treated with CNS (Figure 2c and 2a); however, the size varies. In control, the typical diameter of the C. pyrenoidosa was seen to be around 0.1 µm, whereas, the diameter of microalgae with NGS was found to be in the range of 0.2-0.3 µm (Figure 2b). To corroborate the effect of nitrogen doping on the growth of algal cells, we analyzed the morphology of the cells in presence of undoped material CCM (Figure 2d). Under these circumstances, the cells exhibited moderate growth as compared to the control. In presence of CNS, the cell growth was found to be more condensed; however, the cell size was noted to be smaller in comparison with NGS mediated cells. The increased cell size in the presence of NGS may be ascribed to higher cellular lipid accumulation, thereby, establishing the supremacy of NGS for lipid production in C. pyrenoidosa. The gravimetric analysis of lipid production was further supported by SEM results.

Figure 2 SEM micrographs of (a) CNS with microalgae (b) NGS with microalgae; (c) C. Pyrenoidosa (control); and (d) CCM with microalgae


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Chemical and compositional characterization of CMs Further characterization of CMs has been performed using powder XRD. XRD patterns of NGS, CNS, and CCM are reported in previous literatures.20,21,24 No diffraction pattern was seen for CNS, indicating that CNS produced through solvothermal method is amorphous in nature. The as-synthesized NGS displayed a broad peak (002) at 22.678° (d = 0.35nm) after being treated hydrothermally for 10 h, thereby demonstrating that the exfoliation resulted in the expansion of NGS nanostructure along with partial restoration of the graphitic crystal structure. The specific surface area of the as-prepared CNS and NGS were estimated by the Brunauer– Emmett–Teller (BET) method by analyzing the N2 adsorption/desorption isotherm (Figure 3). The nitrogen doped graphene nanosheets exhibited type-IV characteristics. Subsequently, the isotherm corresponding to NGS displayed a H3 hysteresis loop at the relative pressure (P/P0) ̴ 0.3–1.0, which is symptomatic of a framework-like structure. Moreover, the considerable volumes adsorbed at lower P/P0 = 0–0.3 indicates the presence of microporosity along with displaying an irreversible desorption phenomenon which is characteristic of mesoporous frameworks.28

Figure 3 Nitrogen adsorption/desorption isotherms of NGS and CNS


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The SBET for NGS was calculated to be 152 m2g-1 with a pore volume of 1.3 cm3g-1. In contrast to NGS, CNS displayed a H4 hysteresis loop which is conformed to insubstantial amounts of mesopores restrained by the presence of micropores. The SBET for CNS was found to be 78 m2g-1 with a pore volume of 0.87 cm3g-1. X-ray photoelectron spectroscopy was used to investigate the surface composition and bonding states of elements present within the CMs.29 Survey spectra (Figure 4a) taken over a wide range of binding energies (0-1200 eV) displayed a pre-dominant narrow O1s peak at 531.5 eV and C1s peak at 284.5 eV for both CNS and CCM, respectively. N1s peak obtained at 400.5 eV in case of CNS and NGS proves the incorporation of nitrogen in its structure. The decrease in melamine concentration in CNS resulted in increased pyrrolic nitrogen domain which is evident from its deconvoluted N1s spectrum (Figure S1). The ratio of pyridinic ‘N’ to pyrrolic ‘N’ is seen to decrease as compared to the previously synthesized CNS.24 The results further proved that CNS has been deoxygenated to form NGS due to partial reduction which is evident from the decrease in O1s peak in NGS compared to CNS.20 In all the carbon samples, main peak of C1s can be seen at 282 eV, which correspond to sp2 carbon. The deconvulation of the N1s spectrum in CNS shows a peak at 398.6 eV and 400.1 eV attributed to pyridinic and pyrrolic nitrogen, respectively. The increased pyrrolic ‘N’ domain of CNS when exposed to longer reaction time under hydrothermal condition resulted in re-orientation and rearrangement of the ‘N’ functionalities.30 This results in a surge in the graphitic ‘N’ domains (401.1 eV) of NGS (Figure 4b). Pyridinic and graphitic nitrogen exert a minimal impact on the graphene structure, because of similar bond lengths of C–N (1.41 Å) and C–C (1.42 Å), whereas sp3 hybridized pyrrolic nitrogen disturbs the planar structure of graphene. Graphitic nitrogen refers to a nitrogen atom at the junction of three hexagonal lattices. In case of


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graphitic nitrogen, three valence electrons of nitrogen form three σ-bonds with the neighboring carbon atoms, wherein one electron is engaged in π- bond formation, and fifth electron is partially involved in the π* state of the conduction band. Each graphitic nitrogen results into ndoping effect due to the contribution of ~0.5 electron to the π- system of graphene whereas pdoping affects the defect sites by withdrawing electron from the graphene sheet by pyridinic and pyrrolic nitrogen.31 Catalytic activity is not directly related to the surface area of catalysts and higher content of nitrogen.32 Catalytic activity can be enhanced even at relatively low N content and a high ratio of graphitic-N/pyridinic-N in nitrogen doped graphene. It was reported earlier that more graphitic nitrogen sites in catalyst is critical for higher catalytic activities.33,34 NGS is rich in graphitic nitrogen which is a kind of bound nitrogen and not easily available for algae, resulting into lipid production. Structure of native carbon matrix driving defects or impurities in graphitic carbon have been excellently determined using a versatile, rapid, and non-destructive method of Raman spectroscopy.35,36 Under doping conditions, peak positions of the G and D band always shift due to electron-phonon coupling.35 In graphene, Raman D-peak is proportional to the excitation laser energy, but Raman G peak is not sensitive to the excitation energy.37 Undoped CCM exhibited very low density of states at the Fermi level similar to graphene’s unique band structure around the K point in the Brillouin zone.38 Graphene nanosheets underwent charge transfer interaction with electron-donor and acceptor molecules resulting in marked changes in Raman and electronic spectra.


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Figure 4 (a) Full scan XPS spectra of CCM, CNS, and NGS; (b) The high-resolution N1s spectrum NGS Raman spectra of CNS show a red shift/downfield shift of both G and D band exhibiting a broad peak at 1565 cm-1 and 1346.8 cm-1, respectively, as compared to CCM with G and D band occurring at 1574.5 cm-1 and 1355.8 cm-1, respectively (Figure 5). This broadened red shift in the spectrum of CNS can possibly be due to the electron doping available in amino group of melamine that disturbs the aromatization. This thereby, shifts the peak towards lower wavenumber, indicating an increase in Fermi level of graphene due to the presence of N dopants, consequently establishing the relationship between Fermi energy and the Raman peak positions.26,38 Although, G mode intensity increase is an additional and independent measurement of Fermi level shift, however G peak changes in absolute intensity as well as position when graphene is highly doped.38 The frequency of G band is red shifted with an increase in uniaxial strain, demonstrating a correlation between weakened carbon bonds and lower vibrational frequency due to raised distance between carbon atoms.35 In contrast with CCM (ID/IG = 0.87), CNS displayed an ID/IG ratio of 0.99, thereby hinting at higher number of boundaries, growth nucleation sites and disorderliness in N-doped CNS further validating the presence of the heteroatom in its carbon structure.21,22 Moreover, in case of NGS, the G and D bands are blue-shifted to 1580 cm-1 and 1355.8 cm-1, respectively, with a ID/IG ratio of 0.82 (Figure 5).


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Figure 5 Raman spectra of CCM, CNS, and NGS (at 633nm excitation) showing ID/IG ratio of 0.87, 0.99, and 0.82, respectively. This decrease in the ID/IG ratio is indicative of the defect healing phenomenon and restoration of the sp2 carbon domains resulting in an increase in the π-π delocalization leading to its stronger interaction and increase in the graphitic nitrogen. It has already been proven that the ID/IG ratio is inversely proportional to the crystalline dimension. The increase in the G band intensity and corresponding decrease in the D band shows that NGS has crystalline structure which is in consistency with the TEM and XRD studies.

Response of growth and lipid production by C. pyrenoidosa through CMs The major goal of present research work was to determine the effect of variety of CMs and concentration on the physiological and biochemical behavior of the chosen microalgal species, namely C. pyrenoidosa. The growth of algae is highly influenced by environmental factors and nutrients availability. Plausibly, CMs adsorbed on the algal cell surface, may aid in the release of digestive enzymes for nutrients acquisition, which are required for cell growth. In presence of CMs, the nutrient acquisition is more compared to the control, which is reflected in the biomass


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density. Furthermore, the two factors which govern the cell growth in presence of CMs are first the adsorption capacity, and second the type of nitrogen on CMs. We have recently reported that CNS is an excellent absorbent material compared to other carbon based nanomaterials.24 Furthermore, it is composed of pyrrolic and pyridinic nitrogen which are free N compared to NGS having higher number of graphitic bound nitrogen. Hence, CNS can provide better platform for release of digestive enzyme which is indispensable for nutrient acquisition. In presence of NGS, the bound graphitic nitrogen partly perturbs the release of enzymes, causing early stationary phase, eventually leading to highest lipid production. Interestingly, we for the first time have established the influence of graphitic nitrogen present in NGS for enhanced lipid production. This was further validated by the absence of rise in lipid production by C. pyrenoidosa in the presence of CCM and CNS, which were devoid of any graphitic nitrogen. Similar observations were made for biomass production at all concentrations of CNS as predicted from the growth curve (Figure 6b). The result depicts that CNS provides favorable condition for the growth of C. pyrenoidosa. On the 7th day of cell cultivation, maximum biomass was achieved using 3 mg of CNS (Figure S2). In case of CCM, stressed condition was seen at relatively higher concentration (5 mg), whereas, other concentrations displayed good results with the control for higher biomass production (Figure 6a). In case of higher concentration of NGS i.e. 4 mg and 5 mg, the cell division gets restricted therefore resulting in decreased biomass assimilation (Figure 6c). In the presence of NGS, the highest concentration i.e. 5 mg was found to be inhibitory for algal growth. Additionally, similar result was also obtained with CCM, however higher concentration of CNS showed moderate growth.


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Figure 6 Growth patterns of C. pyrenoidosain photoautotrophic cultivation in presence of CCM (a), CNS (b) and NGS (c). This may be due to increase in oxidative stress of the cell in presence of CMs.14 Maximum lipid efficiency of 18.02 ± 5 (%) was achieved in presence of NGS with biomass 0.340 (g/L) (Figure 7). Possibly, NGS provides optimal amount of available nitrogen which is responsible for higher lipid content along with sustainable biomass production. Moderate lipid production of 5.7 ± 2 (%) was observed with CNS with a biomass culture density of 0.536 (g/L). From these growth curves, we can also justify our proposed theory that optimum nitrogen concentration and carbon source play a major role in the lipid production with higher nitrogen content as is the case of NGS. Pyridinic and graphitic nitrogen play an important role in increasing the lipid content from algae when grown on N-doped graphene nanosheets.


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Figure 7 Response of NGS, CNS, and CCM with C. Pyrenoidosa on biomass concentration (red), lipid production (green) and lipid content (Inset Fig 7). It is known that increasing pyridinic and pyrrolic N-doping induces a large amount of disorder mostly due to bonding disorder and vacancies. Pyridinic and pyrrolic nitrogen atoms are different in view of their bonding configurations with carbon atoms. They play a similar role since introduction of either pyridinic or pyrrolic nitrogen atoms would induce a large number of structural defects. Hydrothermal reduction of amorphous CNS sheets decreases the defects by self-repairing and changing the orientation of N to graphitic one and imparting crystallinity to the sheets. This packed crystal not only increases the stress for lipid production but also acts as catalyst for their production in a specified time. Presence of nitrogen is necessary for cell growth and division of microalgae. However, in the presence of sufficient amount of carbon and lower amount of nitrogen, cell division would be forced to cease. The deprivation of nitrogen would inhibit the protein formation and trigger lipid accumulation inside the cells.39 This result is in accordance with earlier reports showing lower lipid content for higher nitrogen supplementation.11,40


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Light adsorption and scattering at specific wavelength can be amplified by resonant interactions between light (photons) and suspended CMs that result in a significant increase in the accumulation of both chlorophyll and carotenoid pigments, which further improves light uptake by C. pyrenoidosa. Furthermore, these carbon nanomaterial suspensions could be effectively recycled without any toxicological contamination issues. CMs might be generating various reactive oxygen species (ROS) that could lead to oxidative stress to the microalgae thus inducing triacylglycerol synthesis and lipid accumulation.14 The obtained results indicate that NGS acts as promising candidate for lipid production after interacting with C. pyrenoidosa. This barrier can be overcome using sharp edges of NGS followed by CNS, eliminating the need of any toxic solvents and chemicals. Herein, our work highlights hitherto unexplored algal-CMs platforms featuring microalgae C. pyrenoidosa which offer a high degree of lipid yield with NGS in contrast to inherent unaided C. pyrenoidosa’s low lipid production ability. This study convincingly demonstrated the NGS, CNS, and CCM incorporated approach for lipid synthesis. This synergistic approach for lipid production could offer a sustainable alterative for current methods for the production of nutraceuticals, pharmaceuticals, and replacement of petroleum-based fuels for the sustainable development of bio-based economy in the near future. The significant role of graphitic nitrogen has been emphasized for obtaining higher lipid as well as biomass production. The obtained results, thereby, validate that N-doped CMs could be established as an engineering marvel for enhancing photosynthesis artificially. These N-doped carbon nanomaterial substrates have carved a niche for themselves in the field of metal free and visible light responsive photocatalysts for solar energy conversion that leads to the enhancement of cell growth and/or pigments.


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Our results suggest that NGS is one of the suitable carbon nanomaterials for lipid production without any lethal effect on algal growth. The blend of photocatalytic and antimicrobial properties coupled with their intrinsic biocompatibility and eco-friendliness make these nanoparticles particularly attractive over other conventional techniques.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXX. Graphical representation of carbon nanomaterials, SEM images, additional figures of lipid and algae, plots of biomass and lipid production. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT We thank Amity University Uttar Pradesh, Noida, Uttar Pradesh, India for providing us with research facilities and laboratory space. REFERENCES (1) Dineshkumar, R.; Paul, A.; Gangopadhyay, M.; Singh, P.D.N.; Sen, R. Smart and Reusable Biopolymer Nanocomposite for Simultaneous Microalgal Biomass Harvesting and Disruption: Integrated Downstream Processing for a Sustainable Biorefinery. ACS Sustain Chem. Eng. 2017, 5 (1), 852–861.


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For Table of Contents Use Only

Herein, we report the investigation of sustainable as well as bio-economic biomass and lipid production from Chlorella pyrenoidosa associated with as-synthesized carbon nanosheets particularly N-doped graphene sheets (for high value biological products).


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Herein, we report the investigation of sustainable as well as bioeconomic biomass and lipid production from Chlorella pyrenoidosa associated with as-synthesized carbon nanosheets particularly N-doped graphene sheets (for high value biological products). 30x22mm (300 x 300 DPI)


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Using N-Doped Carbon Nanosheets - ACS Publications - American

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