Gaurav Srivastava*a,
Chinmaya Kumar Dasa,
Anubhav Dasa,
Satish Kumar Singhb,
Manas Royc,
Hansung Kimd,
Niroj Sethye,
Ashok Kumarf,
Raj Kishore Sharmag,
Sushil Kumar Singhh,
Deepu Philipij and
Mainak Das*aj
aBioelectricity, Green Energy, Physiology & Sensor Group, Laboratory XII, Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India. E-mail: mainakd@iitk.ac.in
bInstitute Nursery, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India
cDepartment of Chemistry, Lovely Professional University, Phagwara, Punjab 144411, India
dDepartment of Chemical & Biomolecular Engineering, Yonsei University, 134 Sinchon-dong, Seodaemon-gu, Seoul, 120-749, South Korea
eDefense Institute of Physiology & Allied Sciences, Defense Research Development Organization, Government of India, Timarpur, Delhi 110054, India
fDepartment of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India
gElectrochemical Materials Research Group, Division of Physical Chemistry, Department of Chemistry, Delhi University, Delhi, 110007, India
hFunctional Materials Division, Solid State Physics Laboratory, Defense Research Development Organization, Government of India, Timarpur, Delhi 110054, India
iIndustrial & Management Engineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India
jDesign Program, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India
First published on 29th October 2014
Certain nano-materials are known to have plant growth promoting effects, which could find applications in agriculture. We drew inspiration from the nano-factories of deep-sea hydrothermal vents; where iron pyrite nanoparticles serve as fertilizer to sustain chemoautotrophic life forms. We synthesized such iron pyrite nanoparticles in a controlled environment and used them as seed treatment agent (Pro-fertilizer). For us, the term ‘pro-fertilizers’ represents those materials that cause enhanced plant growth with minimum interference to the soil ecosystem when used for seed treatment. We conducted multi-location field trials on spinach crops, since it is a globally popular crop, consumed as both fresh (salads) and processed food. The spinach seeds were treated for 14 hours in an aqueous suspension of iron pyrite nanoparticle (FeS2 + H2O) and thereafter directly sown in the field setup for the experiment. The control seeds were only treated in water for the same duration and sown directly in the field. After 50 days, the crop yields from iron-pyrite nanoparticle treated seeds and control seeds were evaluated. The plants developed from iron pyrite nanoparticle treated seeds exhibited significantly broader leaf morphology, larger leaf numbers, increased biomass; along with higher concentration of calcium, manganese and zinc in the leaves when compared to the plants developed from control seeds. We further investigated the possible mechanism resulting in the biomass enhancement following seed-treatment. Our results indicate that there is an enhanced breakdown of stored starch in the iron pyrite treated seeds resulting in significantly better growth. This raises the possibility of developing iron pyrite nanoparticles as a commercial seed-treatment agent (pro-fertilizer) for spinach crops.
Agricultural productivity directly depends on an optimal plant nutrient management system. The past few decades of intensive crop production strategies have resulted in excessive use of chemical fertilizers, which in turn resulted in deteriorating soil health and increasing water pollution.7 Strategists, planners and thinkers of modern agricultural practices are pondering the question of ‘How to reduce the use of chemical fertilizers without compromising production?’ In other words, how to develop a sustainable strategy for fertilizer usage.
One approach could be to reduce the size of fertilizers to nano-dimensions so that high surface area to volume ratio can be achieved; where altered surface properties will reduce the dose requirements. This makes nano-fertilizers more advantageous over conventional fertilizers.5,6 In addition, this will reduce the cost of fertilizers and will significantly reduce soil–water pollution. Effectiveness of nano-strategy in developing slow release of fertilizer is reported in case of potash fertilizers8 and in urea-modified hydroxyapatite nanoparticles for sustained release of nitrogen in the soil.9 Carbon nano-structures are also used to increase plant growth.10,11 Most of these nanoparticles that are being tested for their plant growth promoting effects; are of anthropogenic origin (do not exist in nature) and get directly applied in the growth substrate (soil or water). There is no study till date, which attempted to exploit the potency of any kind of nanoparticle as a seed-treatment agent. Although a wide variety of physical and chemical approaches for seed treatment are documented in the literature; however, the nanomaterial as a seed treatment agent is a comparatively novel approach (ESI: Section S1: Table S1.† Detailed list of different seed treatment agents). If such an approach is successful in increasing the yield, then we can reduce the consumption of fertilizers and reap rewards mentioned earlier. We speculate that during the onset of germination, when the seeds are experiencing an extremely fertile metabolic phase, certain nanoparticles may significantly influence the physiology of plant growth; which essentially becomes the premise of this work. In this work, we develop and demonstrate a seed treatment strategy for spinach crop using iron pyrite nanoparticles, which resulted in significantly higher yield.
The most obvious question is ‘why iron pyrite nanoparticles were chosen for our study?’ We drew inspiration from the hydrothermal vents of nature. In late 1970s, several deep-sea expeditions resulted in the discovery of hydrothermal vents and the amazing life forms (giant tubeworms, shrimp, clams and limpets) surrounding these vents. The ecosystem of hydrothermal vent is devoid of sunlight, has high pressure and temperature, and also is rich in iron pyrite nanoparticles and sulfides. These nanoparticles are naturally synthesized in abundance in the nano-factories of the hydrothermal vents. One of the major question arise out of this discovery was ‘how in such an extreme environment, devoid of light, the life form thrives?’ On further probing, it was discovered that the giant tubeworms, which accounts mostly for the very high population in such vents, symbiotically harbors wide range of chemo-autotrophic microbes in their body. The symbiotic microbes are reported to house proteins that are involved in energy coupling by oxidation of the available metal sulfides; primarily FeS2.12 Thus FeS2 nanoparticle synthesized in the hydrothermal vents act as an energy source for chemoautotrophic life forms.13–15 It is through this oxidation of sulfides and energy coupling, the higher symbiotic organism i.e. tubeworms obtain its energy for survival. Iron pyrite is also known to be linked with other chemoautotrophic organisms16 and has great evolutionary significance.17 Researchers also showed interactions between microbes such as Acidothiobaccilus feroxidans and pyrite surface.18 Thus FeS2 nanoparticle function as a fertilizer for sustaining life forms in deep ocean floor and in oxygen deficient environments. These findings inspired us to look for any relationship between the FeS2 and higher autotrophic life forms.
In order to address this problem, we needed pure iron pyrite nanoparticles. Iron pyrite is abundantly present in the nature, but it is contaminated with arsenic19 and other heavy metals. Therefore, we developed a simple strategy to synthesize pure FeS2 nanoparticles for our studies. Keeping in mind that in future, we need to reduce the consumption of commercial fertilizers without compromising on the productivity; we decided to use FeS2 nano-particles as a seed treatment agent. In one of our recently concluded studies, we have shown that in acute and sterile laboratory conditions; when Cicer arietinum seeds are treated with iron pyrite nanoparticles, and grown for 7 days in pure, sterile water; significantly healthy plants with increased dry-weight were observed.20 Though our preliminary results were promising; but, from the perspective of a farmer, the major question is: ‘will this novel approach of seed treatment with iron pyrite nano-particles will be effective in the field trials?’ Moreover, ‘will this nano-particle based strategy of crop production is economically effective?’
To address these questions, we evaluated the agricultural production of spinach crop (Spinacia oleracea) after seed treatment with FeS2 nanoparticles. Spinach is popular and global vegetable, which is rich in iron,21 calcium22 and many vitamins, mainly vitamin A.23 Further, spinach leaf extracts has demonstrated anti-proliferative, anti-aging, anti-inflammatory, and anti-oxidant properties in different experimental models.24 We conducted multiple location field trials on this short duration leafy vegetable crop; where harvesting happens after 60 days of sowing. The FeS2 nanoparticle treated spinach seeds are sown in the fields. After 50 days, we observed that the FeS2 treated seeds resulted in significantly higher: number of leaves per plant, fresh and dry weight per plant, leaf and leaf area index per plant and higher concentration of calcium, manganese and zinc in the leaves; when compared to control seeds. Further, we proposed a possible mechanism for the observed effects of FeS2 nanoparticles on the seed physiology. Overall schematic of the study is shown in Fig. 1.
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Fig. 1 The outline of the study involving synthesis and characterization of nanoparticles, and using them as seed treatment agent, followed by monitoring of the crop yield. |
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Fig. 2 Characterization of synthesized iron pyrite nanoparticle. (a) XRD (b and c) SEM images of the particles showing pitcher like morphology. |
Total leaf area was calculated (Fig. 5a) and found to be significantly higher for the test samples. Next, we calculated the ‘Leaf Area Index (LAI)’, which is a measure of the total photosynthetic area available to the plant. Test samples demonstrated significantly higher ‘LAI’ with values of 1.53 ± 0.07, when compared to the control value of 0.94 ± 0.02 (Fig. 5b). Representative pictures of the leaves obtained from the test and the control plants are shown in Fig. 5c. High leaf area index values also hints at high biomass content of the test samples in comparison to the control.
Next we evaluated the fresh and dry weight of control and test plants. Test samples showed tremendous increase in both fresh and dry weight, which is indicative of significant increase in biomass of the test sample upon FeS2 seed treatment (Fig. 6a and b).
We conducted six sets of experiments, with each replicated six times (n = 6) to confirm each of our findings. In the first five cases, the control experiment was seeds treated with double distilled water. For the comparative experiments, the seeds were treated with FeS2, water, and specific chemicals in certain cases to modulate certain starch breakdown pathways. The experimental layout is given in Fig. 8.
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Fig. 8 The overall experimental layout to dissect the mechanism of action of FeS2 on the germination of seed. |
In the first experiment, we collected the leachate after 14 hours of seeds treatment from both control and test group. It was analyzed for the presence of reducing sugar. It was found that the test group leachate was 40% more rich in reducing sugars when compared with the control (Fig. 9a); allowing us to postulate that higher amylase like activity is promoted by FeS2 treatment of seeds. Next, we tested for amylase activity in control and treated seeds for durations of 14 hours after treatment and fully germinated seeds at day 7. The amylase activity was found to be 10% and 30% more in both cases respectively (Fig. 9b and c). The generally accepted fact is that vigorous amylase activity occurs after 72–96 hours of water imbibition during germination, and hence, the higher amylase activity is observed in the later case (Fig. 9c). To quantify the influence of FeS2 on amylase activity, we conducted another set of experiments; where the intrinsic activator of amylase in seeds, viz., Gibberellin was blocked using abscisic acid (ABA). After the treatment with ABA, the amylase activity in the germinated seeds was significantly reduced as per the accepted theory. However, we observed that fully germinated seeds treated with ABA and FeS2 demonstrated slight increase (∼5%) in the amylase activity at day 7 (Fig. 9d). Continuing our investigation, we conducted another set of experiments, where the intrinsic amylase activity was blocked using amylase inhibitor. Interestingly, we found that the fully germinated seeds treated with amylase inhibitor and FeS2 exhibited ∼20% more amylase activity when compared with fully germinated seeds treated with just amylase inhibitor (Fig. 9e). These results provoked our curiosity, and we conducted a simple starch breakdown assay in a controlled setting to investigate the capability of FeS2 alone in breaking down starch molecules in the presence of water. In this experiment, the control was starch plus double distilled water; whereas, the test setup contained starch with double distilled water along with FeS2. Both setups were kept aside for 14 hours at room temperature; after which the percentage of reducing sugars were estimated in both cases. To our surprise, it was found that the test case demonstrated ∼40% more reducing sugar content than the control (Fig. 9f).
The hydrolysis of starch to reducing sugars by FeS2 in the presence of water, and the continuing starch breakdown to reducing sugar even in the presence of amylase inhibitor suggest that FeS2 alone can breakdown starch in the presence of water. Thus one can say that FeS2 could mimic the enzymatic activity of amylase enzyme. So the next pertinent question is ‘How starch could be hydrolyzed by FeS2 nanoparticles?’ The most plausible reason lies in the intricate surface chemistry of iron pyrite molecule. Iron pyrite surface has ‘iron defect’ sites.29–31 Further our XPS results highlighted the presence of such surface defects on the iron pyrite nanoparticles (Fig. 3). A surface mediated reaction on these defect sites between pyrite and water (either in the absence or presence of oxygen) leads to the production of significant amount of hydrogen peroxide (H2O2).29–31 The amount of H2O2 liberated in such reactions is a direct function of defect site density and available surface area of the particles.29–31 So unlike the ‘classical Fenton reagent’, which is a mixture of hydrogen peroxide and ferrous salts, there is an in situ production of H2O2 on the surface of iron pyrite in the presence of water.29–31 This attribute of FeS2 has led scientists to coin the term the ‘pyrite-only Fenton-like’ (PF) reagent.32 Both Fenton and PF reagent are effective oxidants by virtue of their ability to generate highly reactive hydroxyl radicals. PF has been shown to oxidize wide range of organic compounds including lactate,32 carbon tetrachloride,33 chlorinated ethylenes,34–37 aromatic nitro compounds38 and copper phthalocyanine.39 Earlier it has been demonstrated that starch could be hydrolyzed by Fenton reagent.40 Here we are demonstrating that PF also has the ability to hydrolyze starch. So we could consider iron pyrite nanoparticle system (FeS2 + H2O + starch) as the ‘Artificial Enzyme System’ mimicking amylase activity in hydrolyzing starch. Thus when seeds are treated with FeS2, they have more amylase activity as compared to the control seeds. This enhanced amylase activity and pronounced breakdown of stored starch in the seeds act as a strong growth booster in future development of the plant, as we observe in the field trial.
Next we asked ourselves another question. Is the enhanced biomass of adult plant exclusively due to enhanced amylase activity of the germinated seeds or during that germination phase some other growth promoting pathway might have got triggered? Here we speculate that H2O2 generated by FeS2 + H2O + seed could act as a chemical messenger. Earlier research has reported that H2O2 is involved as a secondary messenger in stimulating brassinosteroids mediated CO2 assimilation, redox signaling and carbohydrate metabolism.41,42 Since, FeS2 can generate hydrogen peroxide in aqueous environment; the brassinosteroid mediated pathway of CO2 assimilation and carbohydrate metabolism can be enhanced by increased supply of hydrogen peroxide by pyrite. The overall proposed mechanism is outlined in Fig. 10. Thus, the increase in biomass and plant size upon seed treatment with FeS2, can be attributed to the modulation and increased activity of the aforementioned pathway by H2O2 generated by FeS2.
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Fig. 10 Proposed outline of the mechanism of action of FeS2 on spinach seed in enhancing germination and plant growth. |
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Fig. 11 Representative scheme of iron pyrite nanoparticle synthesis. TSC = Tri-sodium citrate; Na2Sx = sodium polysulfide. |
1. Biomass and number of leaves per plant: the seeds were allowed to germinate and grow in the fields for 50 days and then harvested manually. Total produce was cleared of any mud attached in the roots by washing in running water and weighed to obtain the fresh weight of the samples. Number of leaves in each plant was counted manually and the samples were further subjected to dry heat in an oven at 75 °C for 48 hours. The dried samples were again weighed to get the total dry weight of the produce. This whole procedure was carried out for all the three plots.
2. Leaf area per plant: ten random fresh leaves were taken from each group i.e. control and test, thickness of each leaf in the middle portion was measured with a micrometer, and no significant difference was found. This data was further corroborated with specific leaf area measurement. One square inch area from 10 random leaves was carefully marked and cut with a scalpel. This area was weighed and total plant area was measured by multiplying the fresh weight of one square inch leaf to the total fresh weight of the produce. The leaf area per plant is calculated by dividing the total leaf area by the total number of plants. Photosynthesis is a function of total leaf area and signifies the amount of biomass produced.
3. Leaf area index: leaf area index signifies the total photosynthetic area available to the plant and is calculated as the ratio of total leaf area to the total field area. The formula for calculating ‘Leaf area index’ is [Leaf area index = total leaf area/total field area]. Leaf area index was calculated for all three trials and mean was calculated.
4. Specific leaf area: specific leaf area is calculated as the ratio of leaf area to dry mass. It signifies the thickness of the leaves. The formula for calculating ‘Specific leaf area’ is [Specific leaf area = Leaf area/Dry mass]. The data obtained was found in corroboration with leaf thickness measurements with the help of micrometer.
2. Crude seed extract: crude seed extract was prepared by grinding the germinated seedlings using a pestle mortar, in 10 ml distilled water along with small amount of silicate. The extract thus obtained was filtered and volume was made upto 50 ml.
3. Amylase activity test: amylase test was performed as reported in previous literature with slight modification.44 1 ml Crude amylase extract was mixed with 1 ml citrate buffer (pH 5.6) and incubated at 40 °C in a water bath for 10 minutes. To this solution, 2 ml of 1% starch solution was added and again kept for incubation at 40 °C for 10 minutes. Further to stop the amylase activity, 4 ml of 0.4 M NaOH was added. 1 ml of this final solution was used for DNSA assay for reducing sugars.
4. ABA (Gibberellin inhibitor): abscisic acid (ABA) is a dormancy inducer and an inhibitor of gibberellin. We used 50 μM of ABA independently and along with FeS2 during the time of seed treatment. In the control, no ABA was used. ABA was obtained from commercial vendor.
5. Amylase inhibitor extraction from Ragi seeds: amylase inhibitor extraction was done as described by Kumar et al. from ragi seeds.44 80 gram of seeds were grounded in a pestle mortar to fine powder. 240 ml of 0.15 M NaCl was added and stirred for 3 hours at room temperature. The slurry obtained was filtered under vacuum. 52 grams of ammonium sulfate was added to 150 ml of filtrate and was mixed thoroughly. The solution was allowed to stand overnight at 4 °C. This was further centrifuged at 24000g for 20 minutes and the precipitate was dissolved in 50 ml distilled water. The solution was properly dialyzed against distilled water. Dialyzed solution was centrifuged for 20 minutes at 24
000g. Further 40 ml of clear supernatant was heated at 70 °C for 30 minutes to deactivate amylase and to remove heat labile proteins. The solution was again centrifuged at 24
000g for 20 minutes. The supernatant was carefully taken and stored at 4 °C for further use.
6. Effect of iron pyrite on amylase inhibitor: 2 ml of extracted amylase inhibitor was incubated with 100 μl of iron pyrite suspension (10 mg ml−1) for 3 hours. This was then centrifuged at 5000 rpm for 20 minutes to get a clear supernatant of amylase inhibitor. Similar procedure was done for control with 100 μl distilled water. 1 ml of supernatant was carefully taken in a test tube. 1 ml amylase (1 mg ml−1) and 1 ml citrate buffer (pH 5.6) were added. This mixture was incubated in a water bath at 40 °C for 30 minutes. 2 ml of 1% starch solution was added and incubated at same temperature for 10 minutes. This solution was used to carry out DNSA assay for reducing sugars.
7. Iron pyrite and starch incubation: 2 ml of 1% starch solution was mixed with 1 ml citrate buffer (pH 5.6). 100 μl of iron pyrite suspension was added to the test solution and volume of control solution was made up with 100 μl distilled water (1 mg FeS2 nanoparticle). This was kept overnight (12 hours) in the dark. After incubation the solution was centrifuged at 5000 rpm for 30 minutes and 2 ml of the supernatant was carefully taken for DNSA assay.
8. DNSA reagent: 1 gram of di-nitro salicylic acid was added to 50 ml water. To this solution 30 grams of sodium–potassium tartrate was added in small amounts. Milky yellow color is observed, which turns to transparent yellow upon addition of 20 ml of 2 M NaOH. Final volume is made to 100 ml and the reagent is kept at 4 °C protected from light. We prepared fresh solution for every individual assay.
Footnote |
† Electronic supplementary information (ESI) available: The detailed data, synthesis and characterization of the cerium and graphene oxide nanoparticles are given in section S2. See DOI: 10.1039/c4ra06861k |
This journal is © The Royal Society of Chemistry 2014 |