Carbon Nanotubes as Plant Growth Regulators - Wiley Online Library

Carbon Nanotubes

Carbon Nanotubes as Plant Growth Regulators: Effects on Tomato Growth, Reproductive System, and Soil Microbial Community Mariya V. Khodakovskaya,* Bong-Soo Kim, Jong Nam Kim, Mohammad Alimohammadi, Enkeleda Dervishi, Thikra Mustafa, and Carl E. Cernigla

Multi-walled carbon nanotubes (CNTs) can affect plant phenotype and the composition of soil microbiota. Tomato plants grown in soil supplemented with CNTs produce two times more flowers and fruit compared to plants grown in control soil. The effect of carbon nanotubes on microbial community of CNT-treated soil is determined by denaturing gradient gel electrophoresis and pyrosequencing analysis. Phylogenetic analysis indicates that Proteobacteria and Bacteroidetes are the most dominant groups in the microbial community of soil. The relative abundances of Bacteroidetes and Firmicutes are found to increase, whereas Proteobacteria and Verrucomicorbia decrease with increasing concentration of CNTs. The results of comparing diversity indices and species level phylotypes (OTUs) between samples showed that there is not a significant affect on bacterial diversity.

1. Introduction New discoveries in nanotechnology provided knowledge and technological platforms for a number of applications in medical science, aerospace, electronics and defense industries.[1–3] Success of application of nanotechnology to these areas has generated interest in introduction of nanotechnological approaches in agricultural and food systems.[4] It has been experimentally observed that specific types of

Prof. M. V. Khodakovskaya, M. Alimohammadi Department of Applied Science University of Arkansas at Little Rock Little Rock, AR, 72204, USA E-mail: [email protected] Dr. B.-S. Kim, Dr. J. N. Kim, Prof. C. E. Cernigla National Center for Toxicological Research Jefferson, AR, 72079, USA Prof. E. Dervishi, T. Mustafa Center for Integrative Nanotechnology Sciences University of Arkansas at Little Rock Little Rock, AR, 72204, USA DOI: 10.1002/smll.201201225 small 2013, 9, No. 1, 115–123

nanoparticles in low doses are able to activate physiological processes in plants. For example, TiO2 nanoparticles at an optimal concentration were able to improve the growth of spinach plants through activation of photosynthesis.[5,6] The positive effects of carbon nanotubes on plant growth and development has been described by number of research groups. Thus, increase of root growth in response to carbon nanotubes was documented for onion, cucumber[7] and ryegrass.[8] We recently demonstrated that multi-walled carbon nanotubes (MWCNTs) can activate growth of tomato plants[9] and affect the expression of genes that are essential for cell division and plant development.[9,10] Liu et al. demonstrated that single walled nanotubes (SWCNTs) can penetrate the walls and membranes of tobacco cells.[11] The ability of nanoparticles to penetrate plant cells has generated interest in the possibility of using nanoparticles as smart treatment-delivery systems in plants.[12] Torney et al. have reported that goldcapped mesoporous silica nanoparticles (MSNs) are able to penetrate cell walls and deliver DNA into plant cells by using a bombardment method.[13] Nanocapsules can be used to deliver herbicide to plants. This approach has the potential to provide better penetration through plant tissues and allow slow and constant release of herbicides.[14] There are a few interesting reports about possibility to use nanomaterials as

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component or enhancer of fertilizers. Hossail et al. success- to tomato plants through watering of the soil mixture can fully used nanoporous silica with inserted urease enzymes to increase production of flowers and fruits of compared to the better control the ammonia release from urea fertilizer.[15] plants that were unexposed to CNTs. We also explored the In another study, zinc-aluminum-layered double-hydroxide effect of carbon nanoparticles applied as growth regulators nanocomposites were used for controlled release of a plant on the composition of microbiota in the plant soil habitat. growth regulator (NAA).[16] Soil is one of the major recipients of carbon nanomaterials The emergence of recent discoveries of the unique effects released into the environment. To investigate effects of CNTs of nano-sized materials on plant cell and whole plant organism on the soil microbiota, we used denaturing gradient gel elechas promoted an increase for the use of nanomaterials in plant trophoresis (DGGE) and pyrosequencing techniques with biotechnology, crop management, plant production for non- bacterial 16S rRNA genes. The resulting analysis showed food use, and the biofuel industry. However, one of the crucial the effect of CNT treatment on the composition of the soil issues that should be addressed to guide studies on successful microbiota. Taken together, the data in this report provides applications of nanotechnology is the toxicity of nanomaterials valuable information in determining the potential environto humans and their impact on the environment.[17] According mental effects associated with the use of carbon nanotubes to recent predictions, the production of significant amounts of as regulators of plant growth. Although all described experiengineered nano-materials for different applications may lead ments were carried out in plant growth chambers in a laborato the contamination of terrestrial and aquatic ecosystems.[18] tory setting, the experiments were designed in such a way as The changes in physical and structural properties and signifi- to mimic events that could occur in a natural environment cant decrease in size of man-made nanomaterials could lead contaminated with carbon nanotubes. to unpredictable effects on plants, animals, and humans.[19,20] In the case of nano-contamination of soil, surface or ground water, plants could easily absorb and accumulate man-made 2. Results and Discussion nanoparticles. A study by Zhu et al. revealed that pumpkin plants, grown in an aqueous medium containing nano-Fe3O4 2.1. Phenotypical Analysis of Tomato Plants Grown in Soil particles, can absorb, translocate and accumulate the particles Supplemented with Multi-Walled Carbon Nanotubes in the plant tissue.[21] The dynamic uptake and distribution of carbon nanoparticles in rice plants exposed to C70 (fullerene) Previously, we demonstrated that introduction multi-walled has been reported by Lin et al.[22] Recently we showed that carbon nanotubes or single-walled carbon nanotubes in agar multi-walled carbon nanotubes can be absorbed from medium MS medium resulted in activation of growth of young tomato or soil by the root system of tomato plants and subsequently seedlings grown in nanotube supplemented medium in vitro.[9] distribute inside the tomato plants and reach the leaves and To further assess the potential of carbon nanotubes in activathe fruits. Thus, carbon nanotubes were detected by Raman tion of plant growth, we investigated the effects multi-walled spectroscopy in the fruits of tomato plants grown in soil sup- carbon nanotubes (CNTs) introduced in soil on phenotype of plied with multi-walled carbon nanotubes during regular tomato plants grown in CNT-treated soil mix from juvenile to watering.[9] It is possible that the extensive use of nanomate- mature stages of plant development (Figure 1, Figure 2). The rials as plant growth regulators can lead to their accumulation CNTs were dispersed in water (50 mL) through sonication in landfills and as a result can affect the soil microbial commu- and added to the soil mix once per week in concentrations nity. The alteration of the microbial community could cause an imbalance or a shift in the bacterial diversity with impact on their functional role in the established soil habitats. However, few studies are available on the toxicity of nanoparticles against soil microorganisms. Gajjar et al. reported that man-made nanoparticles of Ag, CuO and ZnO composition had toxic effects on a soil bacteria isolate, Pseudomonas putida KT2440.[23] High concentration of multiwalled carbon nanotubes; (≥500 μg/g of soil) could affect the microbial activity and biomass in soils.[24] In contrast, other investigators found that the carbon nanomaterial fullerene (C60)[25,26] introduced in soil had little impact on the structure and funcFigure 1. Effect of carbon nanotubes (CNT) supplied with watering (50 and 200 μg/mL) tion of the soil microbial community. on phenotype of tomato plants. Average plant height (A), number of leaves (C), number of In this study, the effects of CNTs, deliv- flowers (B,D) were measured for CNT-exposed plants (CNT 50 and CNT 200) plants exposed ered during watering, on phenotype of to activated carbon (AC) and unexposed tomato plants (control) at flowering stage. Each mature tomato plants were investigated. data point is the average of 20 individual measurements. Thus, vertical bars indicate ±SE We determined that application of CNTs (n = 20).

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Carbon Nanotubes as Plant Growth Regulators

Figure 2. Effect of carbon nanotubes (CNT) supplied with watering (50 and 200 μg/mL) on development of tomato fruits. Average number of fruits (A), number of seeds per fruit (B) and size of fruits (C) were measured for CNT-exposed plants (CNT 50 and CNT 200), plants exposed to activated carbon (AC) and unexposed tomato plants (control) at stage of mature (red) fruits. Each data point is the average of 20 individual measurements. Thus, vertical bars indicate ±SE (n = 20).

50 and 200 μg/mL to tomato plants (10 plants for each CNT concentration). Another 10 plants (control) received only regular watering. A third group of plants (AC) was watered with solution containing activated carbon in concentration 50 μg/mL. At flowering stage the overall growth response of all 3 experimental groups was evaluated. Out of the growth parameters observed (plant height, number of leaves and number of flowers) two parameters were affected by supply of CNTs to the soil. Thus, the height was slightly but statistically significantly higher for plants receiving CNTs compared with plants exposed to activated carbon or control plants (Figure 1A). No differences in number of developed leaves were observed between all experimental groups (Figure 1C). However, CNT-treated tomato plants produced two times more flowers at both tested nanotube concentrations (50 and 200 μg/mL) compared with control plants or plants treated small 2013, 9, No. 1, 115–123

with activated carbon (Figure 1B,D). The increase of flower number for CNT-treated plants resulted in correspondent increase of production of tomato fruits (Figure 2A). Thus, tomato plants receiving CNT solution during watering produced two-times more fruit per plant compared with control plants and plants receiving activated carbon. The number of seeds and size of fruits per plant were not affected by application of CNTs to tomato plants and were similar between all experimental groups (Figure 2B,C). Our experiments indicated that delivery of carbon nanotubes to the plants through watering can be efficient for activation of the reproductive system of plants and lead to increase of the production of fruits. The mechanism of observed effects of carbon nanotubes on plant reproductive system will require future detailed investigation. However, most likely this mechanism is associated to the already documented ability of carbon nanotubes to activate expression of genes/proteins essential for plant growth and development. Previously we demonstrated that CNTs in low doses (100–200 μg/mL) can activate expression of water channel protein (aquaporin) in tomato seedlings grown on sterile agar medium supplemented with CNT.[10] It is well known that aquaporins are key regulators of plant growth and development.[27] Thus, it was demonstrated that overexpression of Arabidopsis aquaporin in tobacco plants that lead to increase in plant growth and photosynthetic efficiency. We observed recently that CNTs can stimulate growth and activate gene and protein expression of aquaporin in tobacco cells.[28] Additionally, marker genes for cell division and cell extension were up-regulated in tobacco cells exposed to CNTs compared to unexposed cells.[28] These gene expression studies provided evidence that carbon nanotubes have an ability to affect physiology of plants through up-regulation genes essential for plant development. However, the mechanism of how the nanotubes can trigger the expression of specific genes is still an open question and requires further experimentations. Differences in the physiological response of tomato plants exposed to carbon nanomaterials is raising a question about the importance of their size, shape and other properties for bioeffects that such materials can cause. We believe that the properties differences in between CNTs and activated carbon might have a strong effect on the plant growth. We have previously reported that the surface charge of CNTs has an effect on the growth and expression of water channel protein in tomato plants.[10] We found that the level of aggregation, type and charge of the functional group in surface of applied CNTs is critical for specific effects of CNTs on germination and growth of tomato seedlings.[10] Moreover, the highest increase of tomato growth was achieved when tomato seedlings were exposed to well-dispersed functionalized CNTs with high negative surface charge. Furthermore, the large differences in size, shape and crystallinity between the nano-materials might be one of the major factors contributing to the differences seen in plants, when exposed to these various carbon nanomaterials. More specifically the tubular structure of crystalline CNTs, with outer diameters only in the range of 25 nm, may allow and enhance their further uptake and interaction with biological system, when compared to the non-crystalline relatively larger activated carbon materials. Similarly with the results presented here (Figure 2),


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conclude that the uptake of CNTs occurs relatively easily and distributed inside tomato plants when exposed to nanotubes. Such observations raise an important question about the safety of the use of carbon nanomaterials as regulators of growth for food crops. The use of carbon nanomaterials for activation of biomass production or regulation of reproductive development stage could be more appropriate for non-food sectors of agriculture and horticulture. It could be suggested that carbon nanotubes can have a potential for regulation of growth of ornamental plants, biofuel crops, plants produced specifically for extraction of metabolites and proteins. However, even non-food plants exposed to CNTs can serve as a potential pathway for nanomaterials to enter the food chain and affect soil ecosystems including the microbiota. A comprehensive assessment of the toxicity of nano-exposed plant organs Figure 3. Raman-scattering detection of CNT aggregates in tomato flowers (A,B) based on the (leaves, roots, fruits, seeds) to animals and spectroscopic properties of the individual CNTs. It can be clearly observed the presence of a the effects of such nano-materials residues 1587 cm−1 peak in several spots along the surface of the surface of the flowers from the CNTs- on soil microbial community should be exposed plants (C). This peak corresponds to the G band of the multiwalled carbon nanotubes considered for human and environmental and it was not detected at all in the control samples (flowers of untreated plants). A 633 nm health. The development of new analytical laser excitation with a power of 5 mW was used for these studies. techniques for quantitative measurements of nanomaterials uptake and distribution application of activated carbon did not cause an increase in in different plant organs is another important avenue for plant biomass whereas application multi-walled carbon nano- future investigation. tubes or single-walled carbon nanotubes resulted in the activation of biomass production of tomato seedlings.[9] 2.3. DGGE Proﬁles of Soil Microbiota from CNT-Treated Soils 2.2. Detection of CNT in the Reproductive System of Tomato Plants Grown in CNT-Containing Soil In order to assess if effect of increase of number of reproductive organs is associated with accumulation of CNTs by reproductive organs, Raman spectroscopy was used to detect the presence of the nanotubes in the flowers of the plants grown on CNT-treated soil (Figure 3). Given the unique spectroscopic signature of the carbon nanotubes, Raman spectroscopy is one of the most sensitive methods, that can non-destructively analyze the presence of these nanomaterials in plant organs and plant cells.[9,10] Raman analysis clearly indicated the presence of clustered CNTs in the flower structures (Figure 3). The analysis was done on the flowers collected from the plants exposed to CNTs and we demonstrated the presence and the intensity of the G band, which is characteristic to the nanotubes. No CNTcharacteristic G peak (1587 cm−1) was detected in the control samples, clearly indicating that the CNTs distribute within the plant systems and reach the flowers. These results are in excellent agreement with our previous studies, which clearly showed by Raman, photothermal and photoacoustic spectroscopy, that CNTs reach the leaves and fruits of the tomato plants that were watered with nanotubes.[9] Thus, we can


The addition of carbon nanotubes to soil through watering practice can affect microorganisms that interact with soil environment. To assess environmental influences associated with CNT contamination of soil, we analyzed soil samples originating from phenotypical experiment for comprehensive analysis of the soil microbial community. The denaturing gradient gel electrophoresis (DGGE) method was used to initially screen the differences of microbial community in soil samples. DGGE profiles were conducted to compare bacterial communities between soils with different treatments of CNTs (Figure 4A). Although some variation was observed in triplicate soil samples from each test group, we could statistically compare the DGGE profiles of bacterial communities in control soil samples with those from each CNT treatment group. These variations and differences of community compositions were analyzed by clustering analysis of the DGGE gel profiles. Figure 4B shows that the DGGE profiles clustered as patterns with each concentration of carbon nanotube treated soil. CNT 50 samples (50 μg/mL of CNT) clustered together with an average of 89.6% similarity between triplicate samples. One profile of CNT 0 (r9) had 81.4% similarity with the other two CNT 0 samples (control), and had 83.6% of similarity (average value) with all of CNT 50 samples. CNT 200 (r7) profiles had a 69.5% of similarity with the other two CNT


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(accounting for 86.2% of the total obtained reads) were analyzed after trimming process (discarded reads containing low quality score, short read length, chimera sequences) and 19 130 reads were randomly subtracted from each concentration of pooled triplicates for comparison of pyrosequencing reads between samples (Table 1). Their average length was 450.8 bp and coverage of sample was 80.3%. Although the Shannon diversity index of control sample (CNT 0) was higher than those of treated samples (CNT 50 and 200), the values of Shannon diversity index were similar each other (6.58 to 6.84). The number of observed phylotypes ranged from 5227 to 5490 and estimated phylotypes from 15 414 to 17 073 were similar among three different CNT concentration soils. These results indiFigure 4. DGGE gel profiles obtained from triplicate CNT-treated and control soil samples cated that the diversities of microbial com(A). Clustering analysis of DGGE profiles using Dice coefficient (B). Dendrograms were generated munity were similar between control and with normalized gel profiles from the BioNumerics program. ‘M’ indicates the marker and the CNT treated soils. The richness of bactenumber followed by ‘CNT’ represent the concentration of treated CNTs (50 or 200 μg/mL). rial community in samples and the depth of sequencing were observed in rarefaction 200 samples (200 μg/mL of CNT), but had over 76.7% simi- curves (Supporting Information (SI), Figure S1). The slopes of larity with the CNT 50 samples. These similarities indicated plots were not gradual over 19 000 reads sequencing, and rarthat one profile of triplicate CNT 0 and 200 samples clustered efaction curves of three samples were similar with increased with CNT 50 samples group. Similarities of clustered profiles number of analyzed reads. The increasing rarefaction curves within CNT 0 and CNT 200 samples were 94.3% and 83.02%, over 19,000 reads were consistent with coverage values (80.3% respectively. The similarities of CNT 50 profiles with cluster of Good’s coverage). This result indicated that the diversity of of CNT 0 profiles were higher (78.6% of average similarity) microbial communities was not significantly affected by the than those of CNT 200 (74.2%). These similarities between treatment of the soil with CNTs. However, there was impact on the composition of the bactesamples represented the homologies of bacterial communities between soil samples with treated different concentra- rial community caused by the exposure to different concentrations of CNTs. Higher similarities between CNT 0 (control) tions of CNT treatments at the each phylogenetic level. Phyla and CNT 50 than CNT 50 and CNT 200 indicated that the of Bacteroidetes and Proteobacteria were dominant groups differences in the bacterial communities were related to the in three different soils (Figure 5A). Relative abundances of concentration of CNTs. Therefore, the microbial communities Bacteroidetes (33.1% in CNT 0 shifted to 57.7% in CNT 200) could be affected by introduction of high concentrations of and Firmicutes (1.9% to 3.1%) were increased with increasing concentration of carbon nanotubes, whereas Proteobacteria CNTs compared to the low exposure levels in soil. (50.3% to 28.3%) and Verrucomicrobia (3.5% to 2%) were reduced. At the class level, Sphingobacteria within the phylum 2.4. Pyrosequencing Analysis of CNT-Treated Soils of Bacteroidetes was significantly increased with treatment of carbon nanotube (8.2% in CNT 0 shifted to 24.7% in CNT 200), We further analyzed the control and CNT treated soil by pyro- while Alphaproteobacteia within Proteobacteria was the most sequencing analyses of samples from triplicates of each test decreased member (39.3% to 22.7%) in samples (Figure 5B). group. A total of 81,788 sequence reads were obtained from The other members of Bacteroidetes were also increased control and MWCNT treated soil samples and 70 525 reads and those of Proteobacteria were decreased due to exposure Table 1. Summary of pyrosequencing results obtained from control and CNT-treated soils. Pyrosequencing reads were pooled with triplicate samples of each soil. Observed

Estimated values

Total

Analyzed

Normalized

Mean

phylotypes

Chao1

Shannon

Good’s

reads

reads

reads

length [bp]

[OTUs]

[OTUs]

diversity

coverage

CNT 0

29 483

24 787

19 130

444.8

5490

17 073.8

6.84

79.6

CNT 50

21 624

19 139

19 130

452.9

5227

15 414.4

6.81

80.9

CNT 200

30 681

26 599

19 130

454.6

5273

16 478.0

6.58

80.4

Samples

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the three different soil samples, and their reads numbers were 41 723 (72.7% of total sequences). This indicated that dominant OTUs were not affected by treatment of carbon nanotube materials. Approximately 2505 OTUs (3342 sequences) declined with exposure to carbon nanotubes with Proteobacteria (1423 OTUs) as the major member that was effected. Approximately 420 OTUs (1,867 sequences) declined for the CNT 200 samples and 368 OTUs (1697 reads with Bacteroidetes as major member) were commonly found only in carbon nanotube treated samples. 2428 OTUs (3,101 sequences) were present only in CNT 200 soil sample. These results suggest that the impacted bacterial species were replaced by other species and they are relatively Figure 5. Compositions of bacterial community in control soils compared with those of minor members in samples. Uncultured CNT-treated soils. Relative abundances of phyla (A) and classes (B) were calculated from Flavobacterium was the dominant spepyrosequencing data. The phyla and classes were selected by the abundance greater than cies in all samples and their abundances 0.5% in any of three soil samples. increased with the increased concentrations of carbon nanotube materials i.e., of carbon nanotube materials. This indicated that the CNT approximately 15.2% at the control (CNT 0) soil shifted to treatment influenced the relative abundances of each bacte- 18.9% at CNT 200. Uncultured Chitinophaga within the class rial group. The sequence information also showed that within of Sphingobacteria was the most increased species from 1.9% Sphingobacteria there was a increased of uncultured Chiti- in control soil (CNT 0) to 9.9% at CNT 200, whereas Masnophaga and uncultured clone EF612370, while the most silia aerilata within class of Betaproteobacteria was the most reduced species within Alphaproteobacteria was uncultured decreased species with nanotube treatments. Our results shed light on understanding the interactions clone EU881088. Taxonomic analysis of pyrosequencing reads indicated that most of species detected in all samples were of carbon nano-materials with soil microbiota. We noticed uncultured bacteria. Therefore, it was difficult to survey their that diversity of microbial community was not affected sigrelated ecological roles in the environment. The shared Oper- nificantly in CNT treated soil compared to control soil. Nevational Taxonomic Units (OTUs) were obtained by compara- ertheless, some shift in bacterial community was detected in tive analysis of OTUs between nanotube treated and control nanotube treated soil. The results of this investigation are in samples (Figure 6). A total of 994 OTUs were common among agreement with other studies that did not observe significant impact of carbon nano-materials applied at a wide range of concentrations on indigenous soil microorganisms.[24–26] However, instead of using only traditional approaches to monitor microbial diversity, we used advanced molecular methods including pyrosequencing to conduct a comprehensive evaluation of impact of CNTs on soil microbiota.

3. Conclusion

Figure 6. The analysis of common and different OTUs among control and CNT-treated samples obtained from pyrosequencing data. The upper numbers indicate the number of OTUs and numbers in the blank are total read numbers of each OTU. Most of the sequences were common among the three CNT-treated soil samples.


In summary, we have demonstrated that CNTs introduced in soil mix through watering can affect the phenotype of tomato plants. Tomato plants grown on soil supplemented with CNTs produced the same amount of leaves but two-times more flowers and fruits than plants grown in regular soil. This observation opens new perspectives on technological applications for the introduction of CNTs as growth regulators in agricultural practices. Thus, accelerating plant growth by application of carbon nanotubes can open new perspectives for a number of avenues ranging from biofuel crops to spacegrown plants. However, the documented ability of plants to uptake carbon nanotubes from soil and accumulate these nano-particles in reproductive organs also raises questions


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about potential hazard to the environment. In this study, as a first step in the evaluation of influences associated with carbon nano-materials used as plant growth regulators, we performed analysis of bacterial diversity and distribution of microorganisms in the control and CNT-treated soils. Comparative metagenomic analysis of microbial communities revealed that the diversity and richness of microbial communities was not affected by multi-walled carbon nanotubes, while the abundance of each bacterial group was influenced by treatment of CNTs. Although the profiles of community composition shifted by exposed carbon nanotubes, major phylotypes (OTUs) were maintained with treatments. This result indicated that the microbial community in soil shifted their composition mainly minor phylotypes after introduction of carbon nanotubes to the soil used for tomato cultivation. To evaluate the change of ecosystem in CNT-treated soil, more detailed studies using functional genes are necessary. Therefore, comprehensive investigation of the impact and evaluation of toxicity of carbon nanotubes used as plant growth regulators is recommended at all levels of ecosystem including microbiota, animals and humans.

4. Experimental Section 4.1. Synthesis and Characterization of Carbonaceous Materials The carbon nanotube samples were grown and processed as previously reported in our earlier manuscripts.[10,28] More specifically, multi-walled carbon nanotubes (CNTs) were synthesized at 720 °C, on a Fe:Co:CaCO3 catalyst system with a stoichiometric composition of 2.5:2.5:95 wt%, using a the radio frequency chemical vapor deposition technique (RFCVD).[29] High and low resolution TEM images of the CNTs are shown in the SI, Figure S2C,D. TEM analysis indicated that CNTs have an inner and outer diameter of 10 nm and 25 nm, respectively. The length of the nanotubes was found to be in the range of few microns. We have described the properties of CNTs such as length, size and surface charge in more details in our previous publications.[10,28] At the end of the synthesis, CNTs were acid treated until a purity of over 98% was reached. For a better dispersion in water, the nanotubes were sliglty oxidized and were found to have a negative surface charge.[10] Thorough microscopy analysis were also performed on the as-purchased activated carbon and images are presented in the SI, Figure S2A,B. TEM images of the activated carbon indicated the presence of various noncrystalline carbon structures with irregular shapes and nonhomogeneous sizes varying between 125–250 nm. 4.2. Plant Growth, Phenotypical and Statistical Analysis The tomato seeds (cv. Micro-Tom) were sterilized and placed on sterile agar MS medium as described early.[9] Three types of MS medium were used for experiments: standard MS medium (control), MS medium supplemented with 50 μg/mL of activated carbon and MS supplemented with 50 μg/mL or 200 μg/mL of highly purified multi-walled carbon nanotubes. small 2013, 9, No. 1, 115–123

Seedlings germinated on four described types of medium were transferred in pots containing Sun Gro Redi-earth Plug and Seedling Mix (Sun Gro Horticulture, Inc.). Four experimental groups of tomato plants grown in soil conditions were selected. Each group contained ten tomato plants. Seedlings germinated on standard MS medium were selected as CNT 0 group (control). Seedlings germinated on standard MS medium supplemented with activated carbon were selected as AC group. Seedlings germinated on standard MS medium supplemented with 50 μg/mL or 200 μg/mL of multi-walled carbon nanotubes were selected as CNT 50 and CNT 200 groups respectively. Tomato plants from all groups were grown in a growth chamber under 9 h light (26 °C) and 15 h dark (22 °C), 45% humidity and 500 μmol m−2 s−1 light intensity. The supply of multi-walled carbon nanotubes to pots was carried out through regular watering with tap water. Plants of CNT 0 group were watered once at day with tap water. The plants from AC, CNT 50 and CNT 200 groups were additionally watered with tap water containing 50 μg/mL of activated carbon (group AC), CNTs in concentration of 50 μg/mL (group CNT 50) or CNTs in concentration of 200 μg/mL (group CNT 200) once a week. To achieve this task, CNTwater solution (50 mL for each used concentration) was added inside of soil mix into each experimental pot. During other days of week, these plants of AC, CNT 50 and CNT 200 groups were watered only with regular water. The morphological studies were performed when plants were in flowering stage (6-week-old plants) and when all plants had mature (red) fruits (9-week-old plants). The plant height, number of leaves, number of flowers, number of fruits, size of fruits, number of seeds in fruits were measured for all plants of all experimental groups. Whole experiment was repeated twice; therefore, each data point is the average of 20 individual measurements. Thus, vertical bars indicate ±SE (n = 20).

4.3. Detection of CNTs in Flowers by Raman Spectroscopy The flowers from mature plant grown in regular soil mix and soil mix supplemented with CNTs through watering were collected and stabilized in a small amount of buffer (RLT, buffer, Qiagen, Inc) supplemented with β-mercaproethanol. Flowers spread in thin films were analyzed by Raman spectroscopy at room temperature. Raman scattering spectra were recorded using a Horiba Jobin Yvon LabRam HR800 equipped with a CCD, and a grating of 600 lines/mm. For excitation a He-Ne laser (633 nm) was used as the excitation source. The laser beam intensity measured at the sample was 5 mW, and Raman shifts were calibrated with a silicon wafer at a peak of 521 cm−1.

4.4. DNA Extraction and DGGE Analysis of CNT-Treated and Control Samples After final phenotypical measurements and harvesting of tomato plants, pots with control soil mixed and those with carbon nanotubes (CNT 0, CNT 50 and CNT 200 groups)



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were used for analysis of the soil microbial community. Genomic DNA was extracted from 0.5 g (dry weight) of each soil (CNT 0, 50, and 200 experimental conditions) using DNA elution accessory kit with RNA Powersoil total RNA extraction kit (MoBio Laboratories, Carlsbad, CA, USA). To reduce bias of extraction, extractions of genomic DNA in triplicate from each soil were pooled and used for further analysis.[30] 16S rRNA genes (V3 region) were amplified for conducting DGGE analysis using primers GC-clamp-340f (5′-TCC TAC GGG AGG CAG CAG-3′) and 518r (5′-ATT ACC GCG GCT GCT GG-3′).[31,32] The amplification was performed using a Mastercycler gradient instrument (Eppendorf, Hauppauge, NY, USA), in a final volume of 50 μL with 10X Taq buffer, dNTP mixture (Takara, Shiga, Japan), 10 μM of each primer (MWG-Biotech, Ebersberg, Germany), 2 U of Taq polymerase (Ex Taq; Takara). The condition of amplification was initial denaturation at 94 °C for 5 min, and then 30 cycles of denaturation (30 s, 94 °C), primer annealing (30 s, 55 °C), and extension (30 s, 72 °C), with a final extension step of 7 min at 72 °C. The amplified product was confirmed by 2% agarose gel electrophoresis and visualized with a Gel Doc system (BioRad, Hercules, CA, USA). The extraction and amplification of genomic DNA from triplicate soil samples were conducted independently. Purified amplicons were obtained using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA), and concentrations were determined on a NanoDrop 1000 instrument (NanoDrop Technologies, Wilmington, DE, USA). Equal amounts of amplified products from each sample were loaded on the DGGE gel with denaturing gradient ranged from 40% to 65% and the run was at 70 V for 16 h 30 min at 60 °C, using the Dcode system (BioRad). DGGE gel profiles were visualized by ethidium bromide staining and photographed using the Gel Doc system (BioRad). Normalization and clustering analysis of gel profiles were conducted using the BioNumerics program version 6.0 (Applied Maths, St.-Martens-Latem, Belgium).

4.5. Pyrosequencing Analysis of CNT-Treated and Control Samples After DGGE analysis for preliminary sample comparisons of microbial community from CNT treated and control samples, we obtained detailed information of microbial community composition using high-throughput pyrosequencing. 16S rRNA genes (ranged from V1 to V3) amplified from genomic DNA of each sample using barcoded primers. The amplification, sequencing and analysis were followed by previous descriptions[33,34] using a 454 GS Junior Sequencing system (Roche, Branford, CT, USA). Briefly, filtering process were conducted (any reads containing two or more ambiguous nucleotides, average quality score