Abstract
Several soil microbes are present in the rhizosphere zone, especially plant growth promoting rhizobacteria (PGPR), which are best known for their plant growth promoting activities. The present study reflects the effect of gold nanoparticles (GNPs) at various concentrations on the growth of PGPR. GNPs were synthesized chemically, by reduction of HAuCl4, and further characterized by UV-Vis spectroscopy, X-ray diffraction technique (XRD), and transmission electron microscopy (TEM), etc. The impact of GNPs on PGPR was investigated by Clinical Laboratory Standards Institute (CLSI) recommended Broth-Microdilution technique against four selected PGPR viz., Pseudomonasfluorescens, Bacillussubtilis, Paenibacillus elgii, and Pseudomonas putida. Neither accelerating nor reducing impact was observed in P. putida due to GNPs. On the contrary, significant increase was observed in the case of P.fluorescens, P. elgii, and B.subtilis, and hence, GNPs can be exploited as nano-biofertilizers.
1 Introduction
In view of the escalating global population, ample crop yield is one of the main objectives of agricultural scientists. The arable land getting quickly reduced and inadequate energy resources provoked the problem; synthetic fertilizers are one of the responsible factors for the present situation. Synthetic fertilizers, though playing an important role in the current agricultural scenario, are adversely affecting the health of human beings as well as soil because of their indiscriminate and insane usage [1]. Biofertilizers from a wide range of lifeforms, like cyano-algae, rhizobacteria, etc., have shown a promising hope in overcoming the problem of land deterioration and soil health [2, 3].
Soil being one of the most complex and rich nutritional resources present in nature is under constant threat of becoming barren. In particular, the role of soil microbes grouped under plant growth promoting rhizobacteria (PGPR) is very significant in current agricultural practices. PGPR, a group of beneficial and heterogeneous bacterial forms found in soil are being widely used in agriculture [1]. These microorganisms promote plant growth by several mechanisms, such as better nutritional attainment, hormone production, induced systemic resistance (ISR), systemic acquired resistance (SAR), production of pneumatophores and siderophores. Agricultural scientists have shown their interest in biodiversity of PGPR, such as climate specificity and crop specificity.
In hunt of more food production with eco-friendly approach in limited resources, novel technologies are habitually employed to achieve the required crop yields. To go ahead with accomplishing the aforementioned investigation on interactive studies of PGPR with gold nanoparticles (GNPs) with a cost-effective and result-oriented approach, prediction-based validative method using biotechnological and bio-informatical tools have been taken into consideration for reducing the cost of experimentation [4].
The dawn of nanoparticles like GNPs in biological sciences has been noteworthy for delivering desired results such as they have made their presence in the remediation of environment as well as in soil transport system. GNPs are known for their specific size, shape, inertness, and surface coating materials. They are less toxic in nature under certain conditions and provide strong resistance against surface oxidation.
Moreover, the nanoparticles are transported into the cell by different methods depending upon their size. PGPR are soil microorganisms that are considered as the building blocks of the ecosystem are susceptible to nanoparticles [5], but their mechanisms of action are not well understood to date. This might be due to different types and nature of nanoparticles, and there is no exact model that can interpret the interaction of nanoparticles with PGPR.
2 Materials and methods
2.1 Synthesis of gold nanoparticles
Trisodium citrate-stabilized GNPs have been synthesized as per the protocol [6]. In brief, a composition of trisodium citrate (3.4 ×10-2M, 4.0 ml), tannic acid (10 g/dl, 1.0 ml), and potassium carbonate (0.025 m, 1.0 ml) was diluted with deionized water (14.0 ml) and gradually heated up to 60±1°C. These contents were added to a solution of 1.176 g/dl, 80 ml, and chloroauric acid (HAuCl4) at the rate of 1 ml/min at 60±1°C with continuous stirring for 30 min.
2.2 Characterizations of GNPs
Characterizations of GNPs were carried out by the aid of UV-visible (UV-Vis) absorption spectroscopy, XRD, TEM, and cyclic voltammetry.
2.2.1 UV-Vis spectra
Characterization of GNPs were done using UV-Vis spectrophotometer and recorded on Genesis 10 Thermospectronic spectrophotometer (USA) using quartz cuvette at a wavelength range of 200–700/800 nm.
2.2.2 X-ray diffraction (XRD)
XRD was performed on air-dried GNPs with a 40-kV voltage, 30-mA current at a wavelength of 1.54 Å on copper grids using Rigaku Smartlab X-ray diffractometer, Japan. Spectra were obtained in the 2θ range of 10°–90°.
2.2.3 Transmission electron microscopy (TEM)
TEM analysis of GNPs was done by preparing a sample of 50 μg/ml in ethanol followed by applying a drop of the suspension over the copper grid. The grid was dried at room temperature (23±1°C). TEM (JEOL 1011 [Tokyo, Japan]) was operated with a primary beam voltage of 80 kV, and data were recorded at 1.7 s exposure time.
2.2.4 Cyclic voltammetry
The cyclic voltammetry was done to see the binding of GNPs with experimented PGPR by its electrochemical behavior. The electrochemical characterization of analytes (100 μl) in medium (10 ml) was conducted using cyclic voltammogram over IVIUM Potentiostat Galvanostat at a scan rate of 0.05 V/s at -1.1 V using a three-electrode cell (20 ml), namely, Ag/AgCl (3 m NaCl) as reference, platinum wire (1 mm diameter) as counter, and glassy carbon (2 mm diameter) as working electrodes. Prior to the electrochemical analysis, the working electrode was polished with 3 μm and 0.05 μm alumina slurries, rinsed with water, followed by 3 min of sonication time. This mechanical cleaning procedure was performed once, before a series of runs. The electrochemical data were analyzed using IVIUMSOFT VS.1.821 software.
2.2.5 GNPs-PGPR cell interactions
Morphological observations of PGPR cells along with GNP attachment onto the membrane were done under scanning electron microscope (SEM). Precipitates formed in experimental PGPR culture were separated from liquid medium (ratio 1:10) by brief centrifugation, mixed with GNPs for 1 h, according to the 0.5 McFarland turbidity [7] centrifuged again. The samples were progressively dehydrated for 24 h dried at room temperature followed by gold coating during desiccation period of 24–48 h. Dried samples were placed on a silicon grid at 5.0 current, 8.6 WD with 100.00KX magnification, and examined with Carl Zeiss ultra™55 SEM.
2.3 Growth promotion assay
2.3.1 Collection of bacterial cultures
The dominance of Pseudomonas fluorescens and Bacillus subtilis in the rhizosphere soil ecology prompted us to investigate the growth promoting characteristics with both Gram-positive and Gram-negative cultures and select the suitable cultures for the present investigation. The standard cultures of Pseudomonas fluorescens (MTCC-9768), Bacillus subtilis (MTCC-2274), and Pseudomonas putida (MTCC-1259) were procured from the Microbial Type Culture Collection (MTCC) Chandigarh, India, and Paenibacillus elgii (ARP-1) was received from Prof. Appa Rao Podile’s Laboratory at University of Hyderabad, India.
2.3.2 Selection of medium
The selected medium for experiment was Mueller Hinton broth (MHB) with the following composition: beef extract 2.0 g/l, starch 1.5 g/l, casein hydrolysate 17.5 g/l; at pH 7.4±0.1 recommended by the FDA, World Health Organization, and Clinical Laboratory Standards Institute (CLSI).
2.3.3 Inoculum preparation
Inocula were prepared according to the 0.5 McFarland standards containing 1–5×106 cells/ml at 625 nm absorbance [7].
2.3.4 Growth experiments
The present experiment was designed to observe the effect of GNPs (Figure 1C) on P. fluorescens and B. subtilis (Figure 1A, B) using the broth microdilution method recommended by CLSI [8]. Sterile 96-well microtiter plates with lids (SPL) were used for the assay of rhizobacterial culture, cultured overnight at 37°C in a selected medium (MHB). Initial dispensing of 100 μl medium (MHB) in all wells of 96-well microtiter plates was followed by the addition of 90 μl and 80 μl of MHB in columns 3 and 4. Then, 10-μl and 20-μl samples, i.e. GNPs at column 3 (sample control) and column 4 (dilution well) were added; the former was vertically diluted from 1 to 8 followed by serial dilution from the 4th to 11th row, and after dilution, excess broth was discarded from the last row. Inocula, 100 μl, was added to make a final volume up to 200 μl. Column 12 was taken as the positive control (O. D. Control), which contains 100 μl medium (MHB) and 100 μl inocula. After an incubation of 24 h at 35±2°C, bacterial growth was measured by absorbance due to turbidity, i.e. optical density (O. D.), as a measure of turbidity recorded using SpectramaxPlus384 (Molecular Devices Corporation, USA).
Selected standard cultures of PGPR viz. (A) P. fluorescens-MTCC-9768; (B) B. subtilis- MTCC-2274; and (C) fold nanoparticles (GNPs).
Comparative growth percent of PGPR in media supplemented with GNPs was calculated by using the following formula.
The observations were recorded as mean value of three replicates.
3 Predictive method
Growth promotion in P. fluorescens and B. subtilis with GNPs forced us to think that a similar result can also be expected in other strains, too. For validation and reducing the cost of the experiment, a predictive method using bioinformatics in which five strains each of Pseudomonas and Bacillus were taken.
3.1 Phylogenetic analysis
Th 16S rRNA gene was selected for phylogenetic study of Pseudomonas and Bacillus. The gene sequences of five homologous strains obtained by the blastx run of both selected cultures were procured from the Gen Bank NCBI database. The alignments of gene sequences were done by Clustal W analysis, and further phylogeny was constructed in the form of N-J bootstrapped phylogenetic tree [10–12] by MEGA 4.0 [13].
4 Results and discussion
4.1 Characterizations of GNPs
In the present study, GNPs have been synthesized through citrate reduction method and characterized by UV-Vis spectra, XRD, and TEM [6]. UV-Vis spectra of HAuCl4 and respective GNPs are given in Figure 2A, which shows the absorption spectra of HAuCl4 at 221 nm. Reduction of HAuCl4 under trisodium citrate, tannic acid system showed GNPs with plasmonic absorption spectra at 536 nm [6]. The absorption spectra obtained from the spectrophotometer of gold nanoparticles corresponding to 536 nm refer to the particle size to be around 45 nm (Figure 2A). This was further confirmed by transmission electron micrographs of GNPs, which confirmed the average particle size varying at a range of 15–45 nm (Figure 3A, B) [14]. The X-ray diffraction spectra confirmed the crystalline structure of the synthesized particles. The 2θ obtained at 38°–40° peak (111) confirmed it (Figure 2B). This made the experimented GNPs more significant because of their size-dependent activity, such as that the 13-nm GNPs have lethal effect on cell viability [15], while the 12-nm GNPs show more toxicity [16]. Smaller nanoparticles are more reactive than bigger ones; they can disrupt cell membranes as well as membrane transport, as the experimented GNPs were bigger in size, i.e. 45 nm, and bacterial cells are usually about 1–2 μm in size, which will not permit the penetration. This might be a reason for aggregation on the cell surface and indirectly promotes the bacterial growth [17].
(A) UV-Vis spectra of chloroauric acid (a), absorption of gold nanoparticles (b) and characteristic plasmonic peak of gold nanoparticles (c). (B) X-ray diffraction spectra of gold nanoparticles confirm crystalline nature.
(A, B) Transmission electron micrograph (at 1.37 KX) representing the formation of spherical gold nanoparticles within size ranges of 15–45 nm. (C) Cyclic voltammogram of gold nanoparticles (a), media with PGPR containing gold nanoparticles (b) and media without gold nanoparticles (c).
The presence of GNPs in the medium in testing PGPR has clearly been reflected through cyclic voltammogram. The voltammogram of GNPs shows an oxidation peak current of 23.5 μA at -0.46 mV [Figure 3C (a)]. The medium with the tested PGPR containing GNPs exhibit oxidation peak currents of 12.12 μA at -0.46 mV [Figure 3C (b)], whereas the voltammogram of the medium without gold nanoparticles (200 mm, pH 7.0) does not indicate any electrochemical behavior [Figure 3C (c)].
4.2 Effect of GNPs on P. fluorescens and B. subtilis
The initial concentrations of GNPs were not found effective as clearly shown in Figure 4A and B. But as the GNP concentration increased, a noteworthy growth promotion was observed in P. fluorescens and B. subtilis. The significant R2-value, i.e. 0.945 and 0.804, for the aforesaid PGPR also supported the growth promotion and established the proper relationship between the growth percentage and the concentration of GNPs.
Average growth percentages of P. fluorescens (A) and B. subtilis (B) with respect to the concentration of gold nanoparticles.
4.3 Phylogeny of P. fluorescens and B. subtilis
Molecular phylogeny was done to obtain the homologous strains of P. fluorescens and B. subtilis. The accession nos. and gene sequences were procured from the NCBI gene bank and further blasted these sequences in blastx. After plotting a phylogenetic tree, it was found that P. elgii, a Gram-positive strain lies in between P. fluorescens and B. subtilis. So, for checking the physiological similarity to one much closer strain, i.e. P. elgii and another strain from another branch P. putida, a Gram-negative strain, which was located far from these strains, was taken (Figure 5). After plotting the phylogenetic tree, literatures showed that several strains of trees were found as growth promoting agents [18], but experimentation could not be carried out due to cost expenses. Therefore, the representative strains of Gram-positive P. elgii and Gram-negative P. putida with plant growth promoting character have been selected for prediction and validation through in vitro investigations.
Phylogenetic neighbor-joining tree based on the 16S rRNA gene sequences. The number of branch points represents the percentage of 1000 bootstrapped datasets showing specific internal branches. Bar: 2 nucleotide substitution of 16S rRNA gene sequence (sequences of the strains were obtained from the NCBI database–accession no. given). (Construct by MEGA software version 4.0.)
Molecular phylogeny was used earlier for phosphate solubilization [19], separation of type strains [20], and for analyzing the variability in MIC against Malassezia [4]. However, in the present paper, molecular phylogeny was used with the objective of tracking the accelerating effect of GNPs on the growth of PGPR, which showed nearness in the N-J tree based on 16S rRNA gene sequences. Thus, the closely related P. elgii exhibited accelerated growth due to the nearness with P. fluorescens and B. subtilis. On the contrary, P. putida, which was distantly located in the N-J tree did not show any acceleration in growth; rather, it exhibited inhibition in vitro during the validative study (Figure 6).
Cultures of selected PGPR strains for validation: (A) P. elgii, (B) P. putida, (C) graph represents the average growth % of P. elgii along with GNP Conc.
4.4 Validation study
4.4.1 Effect of GNPs on P. elgii and P. putida
The same protocol was followed to perform the growth experiment of P. elgii and P. putida as was in the case of P. fluorescens and B. subtilis. The result was shown in the form of a graph depicting the relationship between the average growth percent of GNP concentration with the noteworthy R2-value. The R2-values for P. elgii and P. putida were 0.944 and 0.145, respectively, pointing out the fact that P. elgii shows very good growth in the presence of GNPs. The growth of P. elgii increases as the concentration of GNPs increases. On the other hand, P. putida did not show any significant effect and, hence, was not considered for further study, as the growth percentage of P. fluorescens and B. subtilis originated from -10, but P. elgii in comparison to the former two rises from the base. It starts from 0, and as the concentration increases gradually, its growth flows parallel.
The GNPs were used with the aforesaid PGPR to avoid any adverse effect by nanoparticles. The direct growth promotion potentiality of GNPs and silver nanoparticles for enhancing the growth of Brassica juncea, a higher plant, has been reported earlier [21, 22]. But these nanoparticles when administered directly in the soil have been reported to have a toxic effect on the environment as well as on the soil [23]. Thus, with an objective to utilize the growth-accelerating activity of GNPs and minimizing its nano-toxicity, the soil can be subjected to treatment with PGPR, which are already used as bio-fertilizers leading toward economic and less time-consuming agricultural practices. Hence, to the best of our knowledge, the impact of GNPs on the experimented PGPR has been investigated for the first time. Growth promotion can be observed by morphological parameters like length of the root, shoot, pod size, and weight [15, 16] in response to higher plants, whereas the population density of PGPR in broth is measured by turbidity through qualitative method, indicating positive or negative responses. In the present paper, we have used the quantitative detection of growth promotion in broth applying 96-well microtiter plates through the CLSI-recommended technique of broth microdilution [8]. This technique is rapid, economical, and accurate. As large numbers of strains of PGPR are available, to reduce the cost of experimentation, a predictive method using the bioinformatics tool has been performed. To validate the predicted result, the growth-promotion assay has been done.
5 Interaction of GNPs with used PGPR
Interaction of GNPs with PGPR has been always a matter of interest, but still lacks a solid basis for its mechanism of action. The most apparent method of interaction is the direct attachment of nanoparticles to the cell membrane of organisms [24]. GNPs get completely aggregated, whereas few nanoparticles are found shrunken on the cell surface of P. fluorescens (Figure 7A), having similar results obtained for in B. subtilis (Figure 7B). GNPs are found deeply shrunken in P. elgii (Figure 7C) as contrary to its presence on P. putida (Figure 7D), which are not much visible on the cell surface and are supposed to penetrate in the cell resulting in the production of reactive oxygen species, which might have blocked the cell communication pathways of plasma membrane leading to cell death.
Scanning electron micrographs (SEM) of GNPs with P. fluorescens (A), B. subtilis (B), P. elgii (C), and P. putida (D). The black arrows are showing the attached GNPs.
Some broken cells are visible, which could have resulted from the sample preparation. GNPs are found aggregated because of their specific size; due to this, they could not penetrate the cell surface. An interactive study of the used PGPR with GNPs is a step toward the mechanism of action of nanoparticles. Attachments of nanoparticles to the bacterial surface may change the shape and size of the bacterial cell resulting in accelerating growth [25] or nano-toxic effect, which causes the death of cells.
6 Conclusions
The qualitative and quantitative effects of GNPs (6.25 μg/ml) on accelerating the growth of P. fluorescens and B. subtilis prompted the performing of the predictive analysis of related cultures, with which P. elgii and P. putida emerged. Subsequent in vitro validation exhibited no acceleration in P. putida contrary to 63% acceleration in P. elgii, 57% in P. fluorescens, and 33% in B. subtilis (Table 1). Thus, P. elgii along with GNPs has a lot of potential to be used for the development of nano-biofertilizer after undergoing ecotoxicological investigations. The interactive study of the aforesaid PGPR with GNPs is a step toward the mode of action of GNPs on rhizosphere genera.
Effect of GNPs (6.25 μg/ml) on population density of tested strains of PGPR after 24 h of incubation.
| S. n. | Test strains | Qualitative by visualizing | Quantitative growth (%) |
|---|---|---|---|
| 1 | P. fluorescens | ++ | 57 |
| 2 | B. subtilis | + | 33 |
| 3 | P. elgii | +++ | 63 |
| 4 | P. putida | – | Not checked |
+++, excellent growth; ++, moderate growth; +, poor growth; –, no growth.
About the authors
Shashi Kant Shukla is currently perusing his doctoral research under the guidance of Professor Anupam Dikshit FNASc, Biological Product Laboratory, Head, Department of Botany, University of Allahabad, Allahabad, India. His research interest includes the different aspects of PGPR and synthesis, fundamental properties, and interactive study of nanomaterials with PGPR. He has been awarded thrice in national/international presentations for his publications.
Rajesh Kumar is currently working as Senior Research Fellow (upgraded from CSIR-JRF). He has expertise in culturing and isolation of genetic material of microbes and in handling of instruments such as Rotary Evaporator, Clevenger Apparatus, Spectramax (384 plus), RT PCR, UV-Vis Spectrophotometer, Shaker Incubator, Ultrasonicator, etc. He has three publications in his credit including one book, one book chapter, and one journal paper.
MGH Zaidi earned his PhD degree in Chemistry from Lucknow University, India, in 1992. He is currently a Professor at the Department of Chemistry, G. B. Pant University of Agriculture and Technology, Pantnagar Uttarakhand, India. His area of interest is in the development of environmentally benign methods of processing of technologically important polymers, composites, nanomaterials for structural, electrochemical and biomedical applications.
Sanjeev Kr. Srivastava obtained his PhD in the Polymer Nanocomposites from Instrument Design Development Centre, IIT Delhi. He has been the Technology Manager at the Centre for Nano Science and Engineering, IISc Bangalore, since March 2012. He has published 15 research papers in international and national journals. He is the coordinator for Indian Nanoelectronics User Program (INUP) of MCIT Government of India.
Anupam Dikshit, FNASc, is the Head of the Department of Botany, and the Coordinator for the Enviornmental Sciences, University of Allahabad, India, He has a number of international patents and awards for developing various biological products for agricultural and human health after visiting UK, USA, and Germany. Professor Dikshit has more than 100 publications in national and international journals. He has guided one DSc and 19 DPhil students.
Acknowledgments
We acknowledge the support from the Head of the Department of Botany, University of Allahabad, for providing the facilities; Prof. Appa Rao Podile for the culture; Dr. Sai Mutthukumar, SSSIHL, Prashanthinilayam, A.P., and Prof. Appa Rao M. Rao, Clemson University, USA, for the valuable discussion; to Dr. Amarnath Dwivedi, Retd., Professor, English Department, University of Allahabad, for proof reading and language improvement of the manuscript; Mr. M.P. Singh, Department of Vetenary Sciences, G. B. Pantnagar University, Uttarakhand, for the TEM analysis; Mr. Varadwaj, Mr. Pradeep, IISc., Bangalore, for the SEM and XRD; and UGC for the financial support.
Conflict of interest statement: The authors have no conflict of interests.
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