Sustainable Changes in the Contents of Metallic Micronutrients in First

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Sustainable changes in the contents of metallic micronutrients in first generation gram seeds imposed by carbon nano-onions: A life cycle seed to seed study Kumud Malika Tripathi, Anshu Bhati, Anupriya Singh, Amit Kumar Sonker, Sabyasachi Sarkar, and Sumit Kumar Sonkar ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01937 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 16, 2017

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ACS Sustainable Chemistry & Engineering

Sustainable changes in the contents of metallic micronutrients in first generation gram seeds imposed by carbon nano-onions: A life cycle seed to seed study Kumud Malika Tripathi,‡† Anshu Bhati,§‡ Anupriya Singh,§ Amit Kumar Sonker,ɸ Sabyasachi Sarkar,†,₸ and Sumit Kumar Sonkar*§ †

Department of Chemistry, Indian Institute of Technology, Kanpur, Kanpur-208016, India. Department of Chemistry, Malaviya National Institute of Technology, Jaipur, Jaipur-302017, India. ɸ Department of Materials Science & Engineering, Indian Institute of Technology, Kanpur, Kanpur-208016, India. ₸ Department of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah-711103, India. E-mail: [email protected] §

‡

K.M.T. and A.B. Contributed equally to this work

Abstract: Plant-nano-carbons interaction have been mostly explored for enhanced germination, cell growth, plant growth, with a limited study on the productivity of seeds under controlled conditions. The present finding reports the sustainable impacts of bio-waste (wood wool) derived nano-carbons as carbon nano-onions (CNOs). On the entire life cycle of gram plants to obtain the “seed to seed” as first generation seeds (FGSs). Water soluble version of CNOs as water soluble carbon nano-onions (wsCNOs) at 0 (control), 10, 20 and 30 µg mL-1 were used for the germination of gram seeds, for the initial 10 days only. Followed by the transferring of 10 days old baby plants into the soil to complete their natural life cycle (~ 4 months). FGSs harvested from the wsCNOs treated plants showed a significant increase in their yield and health with respect to their individual weight, overall dimensions, enhanced protein, stored electrolytes and other micronutrient contents. The protein content increased from 96 µg mL-1 to 170 µg mL-1 and the level of electrolytic conductivity increased from 2.2 mS to 3.4 mS in the FGSs, harvested from the plants treated with 0 µg mL-1 (control) to 30 µg mL-1 of wsCNOs respectively. 1 ACS Paragon Plus Environment


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wsCNOs used here were presumably acting as a stimulant to increase the contents of metallic micronutrients (Mn, Mo, Cu, Zn, Fe, and Ni) in FGSs without showing its inside accumulations as a contaminant examined by transmission electron microscope (TEM) and Raman spectral analysis. In future, a sustainable approach for the utilization of wood waste as nano-fertilizer could provide a possible approach in agricultural science to overcome the shortage of stored nutrients inside the seeds and also to limit the excessive use of fertilizers. Keywords:

Waste

wood

wool,

Water

soluble

carbon

nano-onions,

nano-fertilizer,

micronutrients, protein content, conductivity, enhanced productivity. 1. Introduction The sustainable demand for food with increase in overall global population is a serious concern in current time, worldwide.1,2 Presently, agricultural sciences need to adopt strategies for boost up crop productivity to manage the continuously increasing food demand along with maintaining its nutritional values.1,3-5 As a possible measure to increase the productivity of plants, exploration of diverse nanoparticles as nano-fertilizer to stimulate physiological and biochemical changes in plants have increased in recent years.6-11 Related to using nanoparticles in agriculture the adaptation of sustainable strategies are crucial. Precisely related to increased in productivity3,12 that directly depends on the nutrition provided and can resolve the increased food demands.7,13,14 However, the accumulations of nanoparticles mainly in edible part (seeds) of plants is a major concern and need in-depth and detailed analysis.15 Stimulating the healthier contents of micronutrient contents in plants in an environmental friendly approach can holds great future promises because of its significant roles in all the metabolic and cellular functions.16 Such as in plant growth, production of chlorophyll, synthesis of growth hormones, gene expressions, cell division, photosynthetic activities, root development, N2 and CO2 fixation, etc.17,18 Deficiency of 2 ACS Paragon Plus Environment

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micronutrients in plant lead to the retarded growth, deferred flowering, chloris of matured leaves, reduction in protein expressions, total protein synthesis and consequently, decrease in productivity.17-20 To be prepared in advance the present need is to explore and optimized the use of advanced organic based fertilizers compared to synthetic chemical fertilizers. Like the sustainable nano-carbons,3,12,21-23 for increasing the productivity and nutrient contents in plants. Why nano-carbons? It's completely based on age-old practice to explore the scientific concern in detail besides charring the crop wastes for the fabrications of biochar,24,25 just after harvesting and before sowing the next crop.21 Our approach in the present finding is very basic and straight forward for the utilization of bio-waste derived nano-carbons as nano-fertilizer (like biochars) to boost the stored nutrient contents of plants. Biochar are scientifically explored carbon rich substances with porous structures, fabricated with bio-waste provided the sustainable benefits as fertilizer. Possessing the high-level of chemical and biological activity, capable enough to exchange various cations in the form of nutrients. Additionaly high surface porosity and larger active surface area lead to increased adsorption capacity of bio-chars.26-30 Similarly, nanocarbons like carbon nano-onions (CNOs) are having very high aspect ratio, tunable chemical and physical characteristic based on the size and surface properties31 that makes them very significant material that can be explored as nano-fertilizers like biochars. Sarkar and co-workers reported the positive impacts of biochar to increases the growth of wheat plants and control the flow of nutrients.21 Several other reports are available to explore the positive prospects of plant-nanocarbons interactions.3,9,12,15,21-23,32-52 Single walled carbon nanotubes (SWCNTs)15,47 and multiwalled carbon nanotubes (MWCNTs)3,15,51 stimulated the overall growth of tomato plants by penetrating inside the thick seed coat in tomato seeds. Water soluble carbon nanotubes (wsCNTs) showed the growth stimulating effects in gram22 and wheat plants.44 Cup stacked

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carbon nanotubes (CSCNTs) were equated with the bio-synthesis of lignin in Arabidopsis thaliana.49 Recently, the SWCNTs in plant nano-bionics is reported which increase the total efficiency of photosynthetic machinery of chloroplasts in spinach.50 Other reports for the use of nano-carbons associated varied plants such as mustard,32 bitter melon,46 alfa-alfa,45 barley,39 cotton,34 maize,41 cucumber,48 onions,37 rice,36 rape,48 and soyabean39 are documented. The aforementioned studies have focused on the short term effects of nano-carbons in plant from the stage of seedlings to matured plants. Considering the numerous applications of nano-carbons in plant and lack of study on their impacts on the stored nutrients of next generation (first) seeds. Herein, we investigated the potential variations in micronutrients, protein and electrolytic contents of first generation seeds (FGSs) obtained from wsCNOs treated and control gram plants. Significant differences were observed in the stored contents of protein, electrolyte, and micronutrient in FGSs obtained from wsCNOs treated plants compared to the control plants. We described a simple finding, focused on the simple conversion of wood waste as wood wool to wsCNOs for their long term sustainable applications in agricultural sciences. 2. Results and discussion 2.1. Absorbance/FTIR/Raman/TGA and XPS analysis of wsCNOs A long known conventional oxidative treatment method was used for the water solubilization of Soxhlet purified soot under refluxing conditions (~ 12 hours), that led to the introduction of negative functionalities like carboxylic and hydroxyl groups on CNOs surface.5356

The aqueous solution of wsCNOs was quite stable against an extended period even after the ~

10 months from its initial solubility. Figure 1 (a) shows the absorbance spectrum of wsCNOs, with a band ~ 270 nm, that was attributed because of the involvements of π-π* transitions. A comparative FTIR, thermogravimetric analysis (TGA), Raman and X-ray photoelectron


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spectroscopy (XPS) analysis were carried out to investigate the surface functionalities of wsCNOs and soxhlet purified soot. Figure 1 and Figure 2 shows the detailed comparative spectroscopic analysis regarding nature and degree of functionalization of wsCNOs. FTIR spectrum of Soxhlet purified soot (blue line-Figure 1 (b)) shows the presence of vibrational bands associated to sp2 and sp3 carbon atoms and the emergence of additional negative surface functionalities in the case of wsCNOs (black line).57,58 The Soxhlet purified soot (Figure 1(b) blue line) shows the stretching bands at 2924 cm-1 and 2853 cm-1 (weak, sp3 –C-H), 1593 cm1

(strong, C=C), 1384 cm-1 (medium. phenyl –C-H ) and 1280 cm-1 (medium,-CH2 bending).

Oxidative treatment of the Soxhlet purified soot shows the additions of few new stretching bands ((black line-Figure 1 (b)), at 3428 cm-1 (broad, O-H), 2925 cm-1 (weak, C-H), 1721 cm-1 (strong C=O), 1606 cm-1 (strong, C=C), 1339 cm-1 (medium, phenyl C-H), 1242 cm-1 (medium,O-H bending) and 1130 cm-1(weak, C-O). a)

b)

c)

d)



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Figure 1: (a) Absorption spectrum of wsCNOs; (b) FTIR spectra; (c) Raman and (d) TGA spectra of Soxhlet purified soot (blue line) and wsCNOs (black line) respectively. Raman spectroscopy is quite significant to reveal the electronic arrangement of nanoparticles and applied for the characterization of graphitic (G band) and disordered (D band) carbon structures in nano-carbons. G band attributed due to the first-order scattering of E2g phonons by sp2 carbon and D band results from the breathing mode of j-point phonons of A1g symmetry.

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Significant differences concerning peak positions and intensities were observed

between Soxhlet purified soot (blue line) and wsCNOs (black line) as illustrated in Figure 1 (c). The shifting of both D (1346 cm-1 from 1357 cm-1) and G bands (1582 cm-1 from 1588 cm-1) toward lower wavenumber, in wsCNOs (in comparison with a Soxhlet purified soot) confirm the weakening of the carbon framework during the oxidation process. The increase in surface defects concerning the number of sp3 carbons during oxidation led to the increased D-band area as compared with G-band. The degree of functionalization can be quantified by a relative IG/ID intensity ratio. Decrease in IG/ID ratio from 0.52 (Soxhlet purified soot) to 0.44 (for wsCNOs) was attributed due to considerable augment in the active area of D-band (from the destruction of sp2 carbon clusters during oxidation).60,61 Surface defects in forms of sp3 carbons in sp2 islands of wsCNOs were crucial regarding functionalization and further their water solubilization. Further, the Zeta potential measurements were performed to analyze the surface charge of wsCNOs. The high negative zeta potential value ~ -71 mV (for the most soluble fraction achieved by gel separation)62 in comparison with as prepared wsCNOs (~ - 56 mV) supporting the high degree of negative surface functionalization.63 The extent of thermal stability of wsCNOs comparing with Soxhlet purified soot was analyzed with TGA measurements. Figure 1 (d) shows the comparative weight loss versus temperature plots of Soxhlet purified soot and wsCNOs. It is evident that


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wsCNOs exhibited less thermal stability with a significant weight loss of ~ 67 % in contrast to Soxhlet purified soot, which showed only ~ 32% weight loss at 900 ˚C. Weight loss in the case of wsCNOs was more prominent due to the decomposition of surfacial oxygenous groups incorporated during oxidation along with weakening and destruction of surfacial graphitic framework.61 wsCNOs showed 35 % of more weight loss than Soxhlet purified soot, that justifies the surfacial functionalization of thermally labile groups as evidenced by FTIR and Raman spectroscopy. XPS analysis was performed to get into detailed analysis of wsCNOs composition and chemical nature of negative surface functionalities of wsCNOs. Figure 2 (a) shows the full XPS survey scan of the wsCNOs. Quantification of carbon (C1s) and oxygen (O1s) by XPS as illustrated in Figure 2 (a) shows ~ 45 % of oxygenous group over surface. The detailed analysis shows the presence of C1s at 285.6 eV and O1s at 534.5eV in wsCNOs. Over the deconvolution of C1s short scan, we can easily differentiate the different modes of carbon binding with oxygen and carbon in four different ways as –C-C- (284.4eV), –C=C- (286.5 eV), -C-O- (287.2eV) and – C=O (288.8eV). Similarly, for O1s deconvolution short scan shows the differential binding of oxygen with carbon as –C-O- (531.1eV) and -C=O (533.1 eV) and COO- (534.2eV).61,64,65

Figure 2: (a) XPS full scan of wsCNOs and (b) its C1s short scan; (c) O1s short scan. 2.2. Microscopic studies: 7 ACS Paragon Plus Environment


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Morphological and microstructural characterization of wsCNOs was carried out with field emission scanning electron microscope (FESEM), Atomic force microscope (AFM), TEM and high-resolution transmission electron microscope (HRTEM) analysis. FESEM and AFM (Figure 3 (a,b)) images illustrate the spherical morphology of wsCNOs, along with the AFM height profile (inset of Figure 3 (b)) of wsCNOs. Low-resolution TEM image (Figure 4 (a)) confirms the spherical and homogeneous distribution of wsCNOs without accompanying any other morphological impurities. The diameter distributions of wsCNOs mostly range from 20 to 40 nm and were analyzed statistically as illustrated in Figure 4 (b).

Figure 3: (a) FESEM image of wsCNOs; (b) AFM image of wsCNOs (inset- height profile analysis of wsCNOs). High-resolution TEM images displayed in Figure 4 (c-e) reveal the spherical onion like arrangements of concentric graphitic rings. Extensive surface derivatization via the impregnation of negative surface functionalities render the defective outer surface as shown in Figure 4 (d) marked by black arrows. A red box in Figure 4 (f) shows the missing graphitic planes in wsCNOs and marked white arrows demonstrate the distance between interlayer graphitic planes (0.26 nm).


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Figure 4: (a) TEM image of wsCNOs corresponding to its (b) size distribution histogram. (c-e) HRTEM images of wsCNOs (f) HRTEM images of wsCNOs showing interlayer distance of 0.26 nm in graphitic plane. 2.3 Effect of wsCNOs on the growth of FGSs In contrast to our previous study that addressed the impact of wood wool derived wsCNOs only on the phenotype of gram plants starting from the germination of seeds to harvesting of FGSs, i.e. “seed to seed cycle”. The positive impact gained in our former work was observed here, in terms of the improved in stored micronutrients, protein and the electrolytic contents of FGSs harvested from wsCNOs treated plants, compared to control plants. Compared to the previous reports showing the accumulations of nano-carbons inside the fruits/flowers.3 At present, we are concerned about the contamination of fresh fruits/seeds by the used nano9 ACS Paragon Plus Environment


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carbons. As the healthier seeds are the only medium that can stored a sustainable future in the form of food. In this quest to deliberately restrict the accumulation of wsCNOs inside the FGSs. At the preliminary phase of our experiments we exposed the seeds to wsCNOs for initial 10 days only, in the laboratory conditions in DI water (without using any growth medium).12,22. Subsequently, we placed the same 10 day old baby plants in soil under optimal conditions in a greenhouse to complete their life cycle12,22 from "source to seed" to yield FGSs. The wsCNOs internalized into the plants were mostly working within water transport channels (xylem vessels)12,22,40 to enhance the overall productivity of plants. 2.3.1. Phenotypical Analysis of FGSs obtained from wsCNOs exposed and control plants The phenotype of FGSs was significantly affected by the interactions of wsCNOs. Figure 5 (ad) illustrated the comparative digital camera images of FGSs (20 in number) obtained from control (0 µg mL-1), 10 µg mL-1, 20 µg mL-1 and 30 µg mL-1 wsCNOs treated plants, just after the 15 days of harvesting. The weight and dimensions (length, width and height) of FGSs obtained from control and treated plants were measured with microbalance and with a standard micrometer. It was observed that weight of FGSs was remarkably increased by using wsCNOs concentrations of 10 µg mL-1, 20 µg mL-1 and 30 µg mL-1 which were significantly higher in comparison to control FGSs (p

Sustainable Changes in the Contents of Metallic Micronutrients in First

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