- Email: [email protected]
The effect of nanoparticles on soil and rhizosphere bacteria and plant growth in lettuce seedlings
Chemosphere 221 (2019) 703e707
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
The effect of nanoparticles on soil and rhizosphere bacteria and plant growth in lettuce seedlings Tohren C.G. Kibbey*, Keith A. Strevett School of Civil Engineering and Environmental Science, University of Oklahoma, Norman, OK 73019, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
TiO2 and positive polystyrene nanoparticles reduced rhizosphere bacteria. Low rhizosphere bacteria correlated with inhibited root and stem length. Nanoparticle attachment to root surfaces was not required for growth inhibition. Negative polystyrene nanoparticles increased bacteria but not growth.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2018 Received in revised form 8 January 2019 Accepted 15 January 2019 Available online 16 January 2019
Nanomaterials are increasingly being considered for use in agricultural applications, where they have been suggested for a range of uses including fertilizer and pesticide applications. Among nanomaterial applications, agricultural use has a particularly high likelihood of introducing significant quantities of nanomaterials to the environment. The focus of this work was on conducting preliminary experiments examining how nanomaterials might influence rhizosphere bacteria, and in turn influence plant growth. For this work, buttercrunch lettuce seeds were grown in the presence of suspensions of three different nanoparticles. Two of the studied nanomaterials, amine-modified polystyrene nanospheres and titanium dioxide nanoparticles, caused significant decreases in both rhizosphere bacterial counts and plant root and stem growth. In contrast, sulfate-modified polystyrene nanospheres actually increased rhizosphere bacterial counts, but had no significant impact on growth. Only the amine-modified polystyrene nanospheres were found to attach to root surfaces, suggesting that nanomaterial attachment to root surfaces is not a requirement for hindered plant growth. It was hypothesized that attachment of amine-modified polystyrene and TiO2 nanomaterials to bacteria themselves could be changing the bacteria surface properties, and ultimately reducing bacterial affinity for root surfaces. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: Tamara S. Galloway Keywords: Nanoparticles Titanium dioxide Rhizosphere Soil bacteria Plant growth
1. Introduction Over the past two decades, manufactured nanomaterials have been the subject of considerable interest due to their superior
* Corresponding author. E-mail address: [email protected] (T.C.G. Kibbey). https://doi.org/10.1016/j.chemosphere.2019.01.091 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
properties for applications spanning a wide range of industries. Although the potential benefits of nanotechnology are widely recognized, the increased use of nanomaterials may also lead to new concerns for environmental pollution and risks to human health. Among proposed applications of nanomaterials, those with perhaps the greatest potential environmental exposure include terrestrial agricultural applications. In agriculture, nanomaterials
704
T.C.G. Kibbey, K.A. Strevett / Chemosphere 221 (2019) 703e707
have been considered for applications ranging from nano-fertilizers to nano-pesticides, as well as other applications related to enhancing seed germination or growth (e.g., (Khot et al., 2012; Rai et al., 2015)). A common feature among agricultural applications of nanomaterials is the potential for direct introduction of large quantities of nanomaterial into the environment. One of the most significant sources of ecological risk from terrestrial nanomaterial applications is the potential for long-term harm to plant life and soil productivity. Specifically, if nanomaterials are introduced into the subsurface to produce a shortterm benefit, the potential exists for them to accumulate and produce negative impacts over longer time-scales. While much is known about the transport of nanomaterials, particularly in saturated porous media, significantly less is known about their interactions with bacteria and plants in the rhizosphere. The rhizosphere is a region defined by the thin layer of attached soil surrounding plant root surfaces in the unsaturated zone of the subsurface. The rhizosphere is a highly biologically-active region, containing orders of magnitude higher bacterial concentrations than surrounding bulk soil. Synergistic interactions between bacteria and root surfaces that occur in the rhizosphere are a critical part of the nitrogen and carbon cycles, and are essential for plant growth. Anything that disrupts the synergistic interactions between bacteria and roots within the rhizosphere will ultimately disrupt nutrient exchange, and hinder plant growth. Studies examining the interactions of nanoparticles with plants have covered a range of types of nanomaterials. Many studies have emphasized phytotoxicity, uptake and accumulation (e.g., (Ma et al., 2010a; Rico et al., 2011; Jacob et al., 2013)). Transmission and translocation studies have shown the importance of nanoparticle translocation processes as they relate to biomagnification. However, potentially more interesting is the impact of nanoparticles on root elongation and seed germination (e.g., (Yang and Watts, 2005; Lin and Xing, 2007, 2008; Ma et al., 2010b)). While some nanoparticles have been reported to have a positive impact on phosphorus uptake, many have led to significant root truncation, have yielded low above-ground biomass, shrinking root tips or vacuolated root epidermal cells (e.g., (Lin and Xing, 2008; Ma et al., 2010a)). Work examining behaviors of nanomaterials in the rhizosphere has highlighted the importance of interfacial phenomena on rhizosphere interactions (e.g., (Huang, 2008)). Because of the low pore-water content of the rhizosphere, and the presence of multiple participating interfaces, physicochemical properties in the rhizosphere are more complex than in bulk soil. Studies have observed that some metal nanoparticles can increase the biota activity, while other metal nanoparticles have no impact (e.g., (Mishra and Kumar, 2009; Taran et al., 2014)). Molybdenum nanoparticles have been observed to increase physiological processes in plants, while silver nanoparticles have been observed to not create the same cellular damage as in bulk studies (Mirzajani et al., 2013). Mirzajani et al. (2013) showed a shift in biota dominance based on the addition of silver nanoparticles and some enhancement of phosphorous uptake. TiO2 and Fe3O4 nanoparticles were also observed to impact phosphorus uptake (Zahra et al., 2015), although the role of bacteria in the process was not evaluated. While the importance of bacterial interactions in the rhizosphere are increasingly being recognized (e.g., (Sillen et al., 2015)), fundamental information about the interfacial mechanisms that govern nanomaterial-bacteria interactions in the rhizosphere is lacking. The objective of the work described here was to conduct a preliminary investigation of how nanomaterial interactions with bacteria and root surfaces in the rhizosphere could potentially impact plant growth. Specifically, the work used plant growth
experiments to explore how nanomaterials impacts on soil bacteria in the rhizosphere would be reflected in the early stages of plant growth, both above and below ground.
2. Materials and methods 2.1. Materials Three different nanomaterials were selected for the work, covering a range of surface properties. Titanium dioxide (TiO2) nanopowder (P25 Aeroxide) was obtained from Degussa Corporation (Düsseldorf, Germany). Fluorescent red polystyrene latex nanoparticles were purchased from Sigma-Aldrich with both sulfate (negative) and amine (positive) surface functional groups. All nanomaterials were used as received. Nanomaterial suspensions were created in groundwater from Norman, Oklahoma, USA. The groundwater and all suspensions had measured pH values of 9.0. Suspensions were shaken daily, but not sonicated or aggressively mixed, to mimic realistic application conditions; observed sedimentation was minimal in all cases. The suspension of TiO2 was created at a concentration of 100 mg/mL, while the suspensions of the latex materials were created at a concentration of 50 mg/mL; the different concentrations were used to partially compensate for the different solid densities of the materials. The nano-TiO2 used for the work has a reported primary particle size of 21 nm; however, as is the case with many nanomaterials, TiO2 nanoparticles do not exist as individual primary particles in aqueous suspension, but rather as stable aggregates (Wiesner et al., 2006; Chen et al., 2008, 2010). Previous measurements of aggregate size of the material were found to be between approx. 85 and 125 nm at pH values above or below the point of zero charge (PZC, pH 6.2 for this material), and as high as 1700 nm within one pH unit of the PZC (Chen et al., 2008, 2010). The zeta potential of the TiO2 at pH 9, 0.2 mM ionic strength is approx. 27 mV, as determined from interpolation from values reported at pH 7 and 10 by Chen et al. (2010). The sulfate-modified (negative) polystyrene nanospheres have a manufacturer-reported size of 100 nm, and a zeta potential of 20.3 mV (Sun and Hu, 2004). The amine-modified (positive) polystyrene nanospheres have a manufacturer-reported size of 1000 nm, and a likely zeta potential of approx. þ60 mV, based on measurements for different sized nanospheres from the same source (Bihari et al., 2008). Note that while the size of the aminemodified nanospheres used in this work is larger than the upper end of commonly-recognized nanomaterial size range (e.g., 100 nm), it is not outside of the typical size range of nanomaterial aggregates in aqueous suspension. Buttercrunch lettuce seeds were used for all growth experiments. Seeds were purchased from BWI Companies, Inc. (Nash, TX), and were used as received.
2.2. Growth experiments Growth experiments were conducted in 50 mL plastic centrifuge tubes (Fig. 1). Centrifuge tubes were perforated for drainage, with pea-gravel added for soil support. Three buttercrunch lettuce seeds were planted per tube, and three tubes were used per set of samples. Plants were grown in organic potting mix (Espoma, Millville, NJ). Plants were watered twice daily, once in the morning with either groundwater (for the control) or nanoparticle suspension (for all other conditions), and once in the afternoon with groundwater. The volume of each addition was 1 mL per tube. Tubes were located just inside a North-facing laboratory window.
T.C.G. Kibbey, K.A. Strevett / Chemosphere 221 (2019) 703e707
Fig. 1. Images of growing buttercrunch lettuce plants taken just after germination (A), and later in the growth process (B).
2.3. Analyses Germination was observed four days after seeds were planted. Plants were then allowed to grow for three weeks postgermination, and then analyzed on the same day. Stem lengths were measured with a ruler. A sterile laboratory spatula was used to loosen the soil around the edge of the centrifuge tube. The soil plug was then transferred to a sterile 144 cm2 glass pate. The soil plug was dissected to expose roots with bound soil. The roots were gently separated from the soil and measured with a ruler. Soil remaining in each core was considered to be bulk soil, while soil that remained attached to roots was considered to be rhizosphere soil. Bacteria were enumerated using viable plate count. Specifically, 5 g of bulk soil was aseptically transferred to 95 mL of sterile buffer. For rhizosphere soil, stalks were aseptically removed from the soil-bound roots and 1 g of soil-bound root was aseptically transferred to 9 mL of sterile buffer. Suspension was agitated and serially diluted via 1 mLe9 mL. Serial dilutions were used to plate 0.1 mL of suspension on 5 replica plates containing Nutrient Agar (BD Difco). Colony counts were completed at 24, 48 and 72 h of incubation (room temperature), and final stable counts were recorded. Confocal microscopy was used to determine whether fluorescent polystyrene nanospheres were associated with root surfaces. Analyses were conducted with a Leica SP8 confocal microscope. Root samples were placed on a microscope slide in groundwater and covered with a cover slip, and then imaged using a 10 dry objective. An excitation wavelength of 561 nm was used for analyses of nanospheres, while a second channel of transmitted light was also collected to identify root outlines. Scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS) was used to determine whether TiO2 nanoparticles were associated with root surfaces. Analyses were conducted using a Zeiss NEON 40 EsB high-resolution SEM with an EDS attachment used for elemental mapping. Root samples were sputtered with Au/Pd alloy prior to analyses, but were not otherwise processed. EDS mapping was used to search for the element titanium.
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Fig. 2. Measured stem and root lengths for the four systems studied.
all measurements. The data in Fig. 2 illustrate that both stem and root length were strongly impacted by the nanoparticle suspensions, with both the amine-modified polystyrene nanospheres and TiO2 nanoparticles significantly impairing both stem and root growth compared with the control. In contrast, the sulfatemodified polystyrene nanosphere suspension exhibited no significant impact on growth. Fig. 3 shows bacterial counts in the bulk soil and rhizosphere soil for each of the systems, normalized to the counts in the control. Of particular interest, note that the two systems with the greatest impairment in growth (Fig. 2; amine-modified nanospheres and TiO2 nanoparticles) also exhibit significantly decreased rhizosphere bacterial counts in Fig. 3. This result suggests that the nanomaterials may be hindering plant growth in these systems by interfering with growth or attachment of rhizosphere bacteria. It is interesting to note that the TiO2 suspension does not appear to have as substantial an impact on bulk soil bacteria as the rhizosphere bacteria (Fig. 3), in that the bulk soil counts are closer to those of the control, ~83% for the bulk counts compared with ~42% for the rhizosphere counts. (In contrast, the bacterial counts for the aminemodified nanospheres are depleted to ~47% for both the bulk soil and rhizosphere bacteria.) That suggests that the TiO2 in this system is not simply creating toxicity to all soil bacteria, but rather is somehow inhibiting the function or attachment of bacteria in the rhizosphere. Furthermore, it is interesting to note that the bacteria
3. Results and discussion Fig. 2 shows measured stem and root lengths for the four conditions studied, as analyzed 21 days after germination. Error bars in this and all subsequent figures correspond to standard deviations of
Fig. 3. Bacterial counts in the bulk soil and rhizosphere for the four systems studied, normalized to counts in the control system.
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Fig. 4. Relationship between root length and rhizosphere bacterial count for the four systems studied.
counts for the negatively-charged sulfate-modified polystyrene nanospheres are actually higher in both the bulk soil and the rhizosphere compared with the control. The reason for this result is unknown. However, it is interesting to note that despite the increased bacteria compared with the control, no enhancement in plant growth is observed (Fig. 2). This result suggests that while effects that reduce rhizosphere bacteria are able to hinder plant growth, increasing rhizosphere bacteria beyond baseline levels does not necessarily further enhance plant growth. Additional information on the composition of rhizosphere bacteria would be useful to understand these behavior, in that it is likely the sulfatemodified nanospheres are enhancing the growth of bacteria not associated with plant growth. Fig. 4 further illustrates the relationship between the observations in Figs. 2 and 3. Because root and stem length appear to exhibit very similar trends, Fig. 4 only examines root length. The vertical axis in Fig. 4 shows root length for each system studied, while the horizontal axis shows bacterial counts in the rhizosphere. It is clear from the figure that both the amine-modified (positive) polystyrene system and the TiO2 system exhibit both significantly decreased root lengths, and significantly decreased rhizosphere soil bacteria counts compared with the control. In contrast, the sulfatemodified (negative) polystyrene system exhibits significantly higher rhizosphere bacteria counts compared with the control, but no significant difference in root length. In order to further understand the observations in Figs. 2e4,
imaging was used to evaluate the extent to which nanoparticles attached to root surfaces. For this purpose, confocal microscopy was used to image the fluorescent polystyrene nanospheres (Fig. 5A and B), while SEM/EDS was used to look for attached TiO2 nanoparticles (Fig. 5C). Fig. 5A shows a root sample from a lettuce plant grown in the presence of amine-modified (positive) polystyrene nanospheres. The image is a superposition of fluorescence from the nanoparticles, shown in orange, and transmitted light to show the outline of the root. It is apparent from the image that the root is covered with nanoparticles; that is, the positively charged nanospheres have attached strongly to the root. All images of roots that had been grown in the presence of amine-modified (positive) polystyrene nanospheres showed significant nanoparticle coverage. This result is not unexpected, because the strong positive charge of the nanospheres and the typical negative charge of root surfaces produces a significant affinity of the nanospheres for the root surfaces. Fig. 5B shows a root sample from a plant grown in the presence of sulfate-modified (negative) polystyrene nanospheres. Note that the image was collected with the same imaging settings as those used for Fig. 5A. It is apparent from Fig. 5B that the sulfate-modified (negative) nanospheres are not associated with the root surface. Note that a small cluster of suspended nanospheres is evident in the lower right corner of the image, verifying that the confocal imaging is able to detect the sulfate-modified nanospheres. The lack of attachment in this system is not unexpected, because the strong negative charge of the nanospheres and the typical negative charge of root surfaces reduces the affinity of the nanospheres for root surfaces. Fig. 5C shows a SEM/EDS map of a root sample in the system grown with the nano-TiO2 suspension. Spectral analysis of the image found that, as might be expected, carbon and oxygen are the most prevalent, at more than 93 weight percent of the detected elements. For this and all other root images examined, all other detected elements were in the single digit percentages or lower, with titanium either not detected, or detected at 0.1 weight percent (the lowest amount reported by the software, and likely within the noise of the method). No attached TiO2 nanoparticles could be identified in any of the images studied. As was the case with the negatively-charged polystyrene nanospheres, this result is not unexpected. Comparing the results in Fig. 5 with those in Figs. 2e4, it is apparent that while attachment of nanoparticles to root surfaces may inhibit plant growth and deplete rhizosphere bacteria, it is not a necessary condition for those effects to occur. Note that while both the amine-modified polystyrene nanospheres and the nanoTiO2 systems exhibited both depleted rhizosphere bacterial counts
Fig. 5. Confocal (A,B) and EDS (C) images of root surfaces grown in the presence of A: Amine-modified (positive) polystyrene nanospheres, B: Sulfate-modified (negative) polystyrene nanospheres, and C: TiO2 nanoparticles.
T.C.G. Kibbey, K.A. Strevett / Chemosphere 221 (2019) 703e707
and inhibited plant growth, only the amine-modified polystyrene exhibited attachment to root surfaces. While positively charged nanoparticles such as the aminemodified polystyrene nanospheres used here have been shown to cause toxicity to some bacteria (e.g., (Jacobson et al., 2015)), the effect of nano-TiO2 has typically been found to be more subtle, reducing the activity of some bacteria under some conditions (e.g., (Ge et al., 2012)). It is important to note that the bulk soil bacterial counts in the TiO2 system are much closer to those in the control than is the case for the rhizosphere counts (~83% bulk vs. ~42% rhizosphere, Fig. 3), so it is likely that direct toxicity is not the cause of the depleted rhizosphere bacteria in the TiO2 system. One possible explanation for the depleted rhizosphere bacteria in both cases may be that nanoparticles are attaching to bacteria surfaces, and ultimately influencing the attachment of the bacteria to root surfaces by changing bacterial surface properties. Note that although bacteria and TiO2 are both negatively charged at the pH of this work, electrostatic interactions are only one mechanism for attachment. Preliminary XDLVO calculations using bacteria parameter values from Chen and Strevett (2003) and TiO2 parameter values from Huang et al. (2015) show that under a wide range of ionic strengths, attachment of nano-TiO2 to many different bacteria at the secondary minimum should be thermodynamically favorable. (Note also that others have reported attachment of nanoTiO2 to bacteria surfaces (e.g. (Huang et al., 2015)).) The positivelycharged amine-modified polystyrene nanospheres would also be expected to attach strongly to bacteria, just as they do to root surfaces (Fig. 5C). It is possible that attachment of nanoparticles to soil bacteria in both cases reduce the favorability of their attachment to root surfaces. Additional studies of simultaneous nanoparticle and bacterial attachment would be needed to evaluate this hypothesis. 4. Conclusions This work examined how three different nanoparticle suspensions impacted both soil and rhizosphere bacteria, and early plant growth in buttercrunch lettuce plants. The results of the work found that both positively-charged amine-modified polystyrene nanospheres and nano-TiO2 significantly inhibited both rhizosphere bacterial counts and plant growth in the system studied. In contrast, negatively-charged sulfate-modified polystyrene nanospheres appeared to enhance rhizosphere bacterial counts, but did not have a significant impact on plant growth. These results suggest that one potentially important mechanism through which nanoparticles could hinder plant growth could be by interfering with the growth, attachment or function of rhizosphere bacteria. The work found significant attachment of positively-charged amine-modified polystyrene nanospheres to root surfaces, but no detectable attachment of the other two nanoparticles studied. It is hypothesized that the similar impact of both amine-modified polystyrene and nano-TiO2 on both rhizosphere bacteria and plant growth may result from attachment of the nanoparticles to the bacteria themselves, and the effect of the attachment on bacterial affinity for root surfaces. Acknowledgments The authors thank Aderonke Adegbule for her assistance with
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the SEM imaging conducted for this work, and Dr. Tingting Gu for her assistance with the confocal imaging conducted for this work.
References Bihari, P., Vippola, M., Schultes, S., Praetner, M., Khandoga, A.G., Reichel, C.A., Coester, C., Tuomi, T., Rehberg, M., Krombach, F., 2008. Optimized dispersion of nanoparticles for biological in vitro and in vivo studies. Part. Fibre Toxicol. 5. Chen, G., Strevett, K.A., 2003. Microbial surface thermodynamics and interactions in aqueous media. J. Colloid Interface Sci. 261, 283e290. Chen, L.X., Sabatini, D.A., Kibbey, T.C.G., 2008. Role of the air-water interface in the retention of TiO(2) nanoparticles in porous media during primary drainage. Environ. Sci. Technol. 42, 1916e1921. Chen, L.X., Sabatini, D.A., Kibbey, T.C.G., 2010. Retention and release of TiO2 nanoparticles in unsaturated porous media during dynamic saturation change. J. Contam. Hydrol. 118, 199e207. Ge, Y., Schimel, J.P., Holden, P.A., 2012. Identification of soil bacteria susceptible to TiO2 and ZnO nanoparticles. Appl. Environ. Microbiol. 78, 6749e6758. Huang, G.C., Xia, D.H., An, T.C., Ng, T.W., Yip, H.Y., Li, G.Y., Zhao, H.J., Wong, P.K., 2015. Dual roles of capsular extracellular polymeric substances in photocatalytic inactivation of Escherichia coli: comparison of E. coli BW25113 and isogenic mutants. Appl. Environ. Microbiol. 81, 5174e5183. Huang, P.M., 2008. Soil physicochemical and biological interfacial interactions in the rhizosphere: impacts on food security and ecosystem integrity. Rev. Cienc Suelo Nutr. 8. Jacob, D.L., Borchardt, J.D., Navaratnam, L., Otte, M.L., Bezbaruah, A.N., 2013. Uptake and translocation of Ti from nanoparticles in crops and wetland plants. Int. J. Phytoremediat. 15, 142e153. Jacobson, K.H., Gunsolus, I.L., Kuech, T.R., Troiano, J.M., Melby, E.S., Lohse, S.E., Hu, D., Chrisler, W.B., Murphy, C.J., Orr, G., Geiger, F.M., Haynes, C.L., Pedersen, J.A., 2015. Lipopolysaccharide density and structure govern the extent and distance of nanoparticle interaction with actual and model bacterial outer membranes. Environ. Sci. Technol. 49, 10642e10650. Khot, L.R., Sankaran, S., Maja, J.M., Ehsani, R., Schuster, E.W., 2012. Applications of nanomaterials in agricultural production and crop protection: a review. Crop Protect. 35, 64e70. Lin, D.H., Xing, B.S., 2007. Phytotoxicity of nanoparticles: inhibition of seed germination and root growth. Environ. Pollut. 150, 243e250. Lin, D.H., Xing, B.S., 2008. Root uptake and phytotoxicity of ZnO nanoparticles. Environ. Sci. Technol. 42, 5580e5585. Ma, X.M., Geiser-Lee, J., Deng, Y., Kolmakov, A., 2010a. Interactions between engineered nanoparticles (ENPs) and plants: phytotoxicity, uptake and accumulation. Sci. Total Environ. 408, 3053e3061. Ma, Y.H., Kuang, L.L., He, X., Bai, W., Ding, Y.Y., Zhang, Z.Y., Zhao, Y.L., Chai, Z.F., 2010b. Effects of rare earth oxide nanoparticles on root elongation of plants. Chemosphere 78, 273e279. Mirzajani, F., Askari, H., Hamzelou, S., Farzaneh, M., Ghassempour, A., 2013. Effect of silver nanoparticles on Oryza sativa L. and its rhizosphere bacteria. Ecotoxicol. Environ. Saf. 88, 48e54. Mishra, V.K., Kumar, A., 2009. Impact of metal nanoparticles on the plant growth promoting rhizobacteria. Dig. J. Nanomater. Bios. 4, 587e592. Rai, M., Ribeiro, C., Mattoso, L., Duran, N., 2015. Nanotechnologies in Food and Agriculture. Springer, Cham. Rico, C.M., Majumdar, S., Duarte-Gardea, M., Peralta-Videa, J.R., GardeaTorresdey, J.L., 2011. Interaction of nanoparticles with edible plants and their possible implications in the food chain. J. Agric. Food Chem. 59, 3485e3498. Sillen, W.M., Thijs, S., Abbamondi, G.R., Janssen, J., Weyens, N., White, J.C., Vangronsveld, J., 2015. Effects of silver nanoparticles on soil microorganisms and maize biomass are linked in the rhizosphere. Soil Biol. Biochem. 91, 14e22. Sun, H., Hu, N.F., 2004. Voltammetric studies of hemoglobin-coated polystyrene latex bead films on pyrolytic graphite electrodes. Biophys. Chem. 110, 297e308. Taran, N.Y., Gonchar, O.M., Lopatko, K.G., Batsmanova, L.M., Patyka, M.V., Volkogon, M.V., 2014. The effect of colloidal solution of molybdenum nanoparticles on the microbial composition in rhizosphere of Cicer arietinum L. Nanoscale Res. Lett. 9, 1e8. Wiesner, M.R., Lowry, G.V., Alvarez, P., Dionysiou, D., Biswas, P., 2006. Assessing the risks of manufactured nanomaterials. Environ. Sci. Technol. 40, 4336e4345. Yang, L., Watts, D.J., 2005. Particle surface characteristics may play an important role in phytotoxicity of alumina nanoparticles. Toxicol. Lett. 158, 122e132. Zahra, Z., Arshad, M., Rafique, R., Mahmood, A., Habib, A., Qazi, I.A., Khan, S.A., 2015. Metallic nanoparticle (TiO2 and Fe3O4) application modifies rhizosphere phosphorus availability and uptake by Lactuca sativa. J. Agric. Food Chem. 63, 6876e6882.
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
The effect of nanoparticles on soil and rhizosphere bacteria and plant growth in lettuce seedlings Tohren C.G. Kibbey*, Keith A. Strevett School of Civil Engineering and Environmental Science, University of Oklahoma, Norman, OK 73019, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
TiO2 and positive polystyrene nanoparticles reduced rhizosphere bacteria. Low rhizosphere bacteria correlated with inhibited root and stem length. Nanoparticle attachment to root surfaces was not required for growth inhibition. Negative polystyrene nanoparticles increased bacteria but not growth.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2018 Received in revised form 8 January 2019 Accepted 15 January 2019 Available online 16 January 2019
Nanomaterials are increasingly being considered for use in agricultural applications, where they have been suggested for a range of uses including fertilizer and pesticide applications. Among nanomaterial applications, agricultural use has a particularly high likelihood of introducing significant quantities of nanomaterials to the environment. The focus of this work was on conducting preliminary experiments examining how nanomaterials might influence rhizosphere bacteria, and in turn influence plant growth. For this work, buttercrunch lettuce seeds were grown in the presence of suspensions of three different nanoparticles. Two of the studied nanomaterials, amine-modified polystyrene nanospheres and titanium dioxide nanoparticles, caused significant decreases in both rhizosphere bacterial counts and plant root and stem growth. In contrast, sulfate-modified polystyrene nanospheres actually increased rhizosphere bacterial counts, but had no significant impact on growth. Only the amine-modified polystyrene nanospheres were found to attach to root surfaces, suggesting that nanomaterial attachment to root surfaces is not a requirement for hindered plant growth. It was hypothesized that attachment of amine-modified polystyrene and TiO2 nanomaterials to bacteria themselves could be changing the bacteria surface properties, and ultimately reducing bacterial affinity for root surfaces. © 2019 Elsevier Ltd. All rights reserved.
Handling Editor: Tamara S. Galloway Keywords: Nanoparticles Titanium dioxide Rhizosphere Soil bacteria Plant growth
1. Introduction Over the past two decades, manufactured nanomaterials have been the subject of considerable interest due to their superior
* Corresponding author. E-mail address: [email protected] (T.C.G. Kibbey). https://doi.org/10.1016/j.chemosphere.2019.01.091 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
properties for applications spanning a wide range of industries. Although the potential benefits of nanotechnology are widely recognized, the increased use of nanomaterials may also lead to new concerns for environmental pollution and risks to human health. Among proposed applications of nanomaterials, those with perhaps the greatest potential environmental exposure include terrestrial agricultural applications. In agriculture, nanomaterials
704
T.C.G. Kibbey, K.A. Strevett / Chemosphere 221 (2019) 703e707
have been considered for applications ranging from nano-fertilizers to nano-pesticides, as well as other applications related to enhancing seed germination or growth (e.g., (Khot et al., 2012; Rai et al., 2015)). A common feature among agricultural applications of nanomaterials is the potential for direct introduction of large quantities of nanomaterial into the environment. One of the most significant sources of ecological risk from terrestrial nanomaterial applications is the potential for long-term harm to plant life and soil productivity. Specifically, if nanomaterials are introduced into the subsurface to produce a shortterm benefit, the potential exists for them to accumulate and produce negative impacts over longer time-scales. While much is known about the transport of nanomaterials, particularly in saturated porous media, significantly less is known about their interactions with bacteria and plants in the rhizosphere. The rhizosphere is a region defined by the thin layer of attached soil surrounding plant root surfaces in the unsaturated zone of the subsurface. The rhizosphere is a highly biologically-active region, containing orders of magnitude higher bacterial concentrations than surrounding bulk soil. Synergistic interactions between bacteria and root surfaces that occur in the rhizosphere are a critical part of the nitrogen and carbon cycles, and are essential for plant growth. Anything that disrupts the synergistic interactions between bacteria and roots within the rhizosphere will ultimately disrupt nutrient exchange, and hinder plant growth. Studies examining the interactions of nanoparticles with plants have covered a range of types of nanomaterials. Many studies have emphasized phytotoxicity, uptake and accumulation (e.g., (Ma et al., 2010a; Rico et al., 2011; Jacob et al., 2013)). Transmission and translocation studies have shown the importance of nanoparticle translocation processes as they relate to biomagnification. However, potentially more interesting is the impact of nanoparticles on root elongation and seed germination (e.g., (Yang and Watts, 2005; Lin and Xing, 2007, 2008; Ma et al., 2010b)). While some nanoparticles have been reported to have a positive impact on phosphorus uptake, many have led to significant root truncation, have yielded low above-ground biomass, shrinking root tips or vacuolated root epidermal cells (e.g., (Lin and Xing, 2008; Ma et al., 2010a)). Work examining behaviors of nanomaterials in the rhizosphere has highlighted the importance of interfacial phenomena on rhizosphere interactions (e.g., (Huang, 2008)). Because of the low pore-water content of the rhizosphere, and the presence of multiple participating interfaces, physicochemical properties in the rhizosphere are more complex than in bulk soil. Studies have observed that some metal nanoparticles can increase the biota activity, while other metal nanoparticles have no impact (e.g., (Mishra and Kumar, 2009; Taran et al., 2014)). Molybdenum nanoparticles have been observed to increase physiological processes in plants, while silver nanoparticles have been observed to not create the same cellular damage as in bulk studies (Mirzajani et al., 2013). Mirzajani et al. (2013) showed a shift in biota dominance based on the addition of silver nanoparticles and some enhancement of phosphorous uptake. TiO2 and Fe3O4 nanoparticles were also observed to impact phosphorus uptake (Zahra et al., 2015), although the role of bacteria in the process was not evaluated. While the importance of bacterial interactions in the rhizosphere are increasingly being recognized (e.g., (Sillen et al., 2015)), fundamental information about the interfacial mechanisms that govern nanomaterial-bacteria interactions in the rhizosphere is lacking. The objective of the work described here was to conduct a preliminary investigation of how nanomaterial interactions with bacteria and root surfaces in the rhizosphere could potentially impact plant growth. Specifically, the work used plant growth
experiments to explore how nanomaterials impacts on soil bacteria in the rhizosphere would be reflected in the early stages of plant growth, both above and below ground.
2. Materials and methods 2.1. Materials Three different nanomaterials were selected for the work, covering a range of surface properties. Titanium dioxide (TiO2) nanopowder (P25 Aeroxide) was obtained from Degussa Corporation (Düsseldorf, Germany). Fluorescent red polystyrene latex nanoparticles were purchased from Sigma-Aldrich with both sulfate (negative) and amine (positive) surface functional groups. All nanomaterials were used as received. Nanomaterial suspensions were created in groundwater from Norman, Oklahoma, USA. The groundwater and all suspensions had measured pH values of 9.0. Suspensions were shaken daily, but not sonicated or aggressively mixed, to mimic realistic application conditions; observed sedimentation was minimal in all cases. The suspension of TiO2 was created at a concentration of 100 mg/mL, while the suspensions of the latex materials were created at a concentration of 50 mg/mL; the different concentrations were used to partially compensate for the different solid densities of the materials. The nano-TiO2 used for the work has a reported primary particle size of 21 nm; however, as is the case with many nanomaterials, TiO2 nanoparticles do not exist as individual primary particles in aqueous suspension, but rather as stable aggregates (Wiesner et al., 2006; Chen et al., 2008, 2010). Previous measurements of aggregate size of the material were found to be between approx. 85 and 125 nm at pH values above or below the point of zero charge (PZC, pH 6.2 for this material), and as high as 1700 nm within one pH unit of the PZC (Chen et al., 2008, 2010). The zeta potential of the TiO2 at pH 9, 0.2 mM ionic strength is approx. 27 mV, as determined from interpolation from values reported at pH 7 and 10 by Chen et al. (2010). The sulfate-modified (negative) polystyrene nanospheres have a manufacturer-reported size of 100 nm, and a zeta potential of 20.3 mV (Sun and Hu, 2004). The amine-modified (positive) polystyrene nanospheres have a manufacturer-reported size of 1000 nm, and a likely zeta potential of approx. þ60 mV, based on measurements for different sized nanospheres from the same source (Bihari et al., 2008). Note that while the size of the aminemodified nanospheres used in this work is larger than the upper end of commonly-recognized nanomaterial size range (e.g., 100 nm), it is not outside of the typical size range of nanomaterial aggregates in aqueous suspension. Buttercrunch lettuce seeds were used for all growth experiments. Seeds were purchased from BWI Companies, Inc. (Nash, TX), and were used as received.
2.2. Growth experiments Growth experiments were conducted in 50 mL plastic centrifuge tubes (Fig. 1). Centrifuge tubes were perforated for drainage, with pea-gravel added for soil support. Three buttercrunch lettuce seeds were planted per tube, and three tubes were used per set of samples. Plants were grown in organic potting mix (Espoma, Millville, NJ). Plants were watered twice daily, once in the morning with either groundwater (for the control) or nanoparticle suspension (for all other conditions), and once in the afternoon with groundwater. The volume of each addition was 1 mL per tube. Tubes were located just inside a North-facing laboratory window.
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Fig. 1. Images of growing buttercrunch lettuce plants taken just after germination (A), and later in the growth process (B).
2.3. Analyses Germination was observed four days after seeds were planted. Plants were then allowed to grow for three weeks postgermination, and then analyzed on the same day. Stem lengths were measured with a ruler. A sterile laboratory spatula was used to loosen the soil around the edge of the centrifuge tube. The soil plug was then transferred to a sterile 144 cm2 glass pate. The soil plug was dissected to expose roots with bound soil. The roots were gently separated from the soil and measured with a ruler. Soil remaining in each core was considered to be bulk soil, while soil that remained attached to roots was considered to be rhizosphere soil. Bacteria were enumerated using viable plate count. Specifically, 5 g of bulk soil was aseptically transferred to 95 mL of sterile buffer. For rhizosphere soil, stalks were aseptically removed from the soil-bound roots and 1 g of soil-bound root was aseptically transferred to 9 mL of sterile buffer. Suspension was agitated and serially diluted via 1 mLe9 mL. Serial dilutions were used to plate 0.1 mL of suspension on 5 replica plates containing Nutrient Agar (BD Difco). Colony counts were completed at 24, 48 and 72 h of incubation (room temperature), and final stable counts were recorded. Confocal microscopy was used to determine whether fluorescent polystyrene nanospheres were associated with root surfaces. Analyses were conducted with a Leica SP8 confocal microscope. Root samples were placed on a microscope slide in groundwater and covered with a cover slip, and then imaged using a 10 dry objective. An excitation wavelength of 561 nm was used for analyses of nanospheres, while a second channel of transmitted light was also collected to identify root outlines. Scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS) was used to determine whether TiO2 nanoparticles were associated with root surfaces. Analyses were conducted using a Zeiss NEON 40 EsB high-resolution SEM with an EDS attachment used for elemental mapping. Root samples were sputtered with Au/Pd alloy prior to analyses, but were not otherwise processed. EDS mapping was used to search for the element titanium.
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Fig. 2. Measured stem and root lengths for the four systems studied.
all measurements. The data in Fig. 2 illustrate that both stem and root length were strongly impacted by the nanoparticle suspensions, with both the amine-modified polystyrene nanospheres and TiO2 nanoparticles significantly impairing both stem and root growth compared with the control. In contrast, the sulfatemodified polystyrene nanosphere suspension exhibited no significant impact on growth. Fig. 3 shows bacterial counts in the bulk soil and rhizosphere soil for each of the systems, normalized to the counts in the control. Of particular interest, note that the two systems with the greatest impairment in growth (Fig. 2; amine-modified nanospheres and TiO2 nanoparticles) also exhibit significantly decreased rhizosphere bacterial counts in Fig. 3. This result suggests that the nanomaterials may be hindering plant growth in these systems by interfering with growth or attachment of rhizosphere bacteria. It is interesting to note that the TiO2 suspension does not appear to have as substantial an impact on bulk soil bacteria as the rhizosphere bacteria (Fig. 3), in that the bulk soil counts are closer to those of the control, ~83% for the bulk counts compared with ~42% for the rhizosphere counts. (In contrast, the bacterial counts for the aminemodified nanospheres are depleted to ~47% for both the bulk soil and rhizosphere bacteria.) That suggests that the TiO2 in this system is not simply creating toxicity to all soil bacteria, but rather is somehow inhibiting the function or attachment of bacteria in the rhizosphere. Furthermore, it is interesting to note that the bacteria
3. Results and discussion Fig. 2 shows measured stem and root lengths for the four conditions studied, as analyzed 21 days after germination. Error bars in this and all subsequent figures correspond to standard deviations of
Fig. 3. Bacterial counts in the bulk soil and rhizosphere for the four systems studied, normalized to counts in the control system.
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Fig. 4. Relationship between root length and rhizosphere bacterial count for the four systems studied.
counts for the negatively-charged sulfate-modified polystyrene nanospheres are actually higher in both the bulk soil and the rhizosphere compared with the control. The reason for this result is unknown. However, it is interesting to note that despite the increased bacteria compared with the control, no enhancement in plant growth is observed (Fig. 2). This result suggests that while effects that reduce rhizosphere bacteria are able to hinder plant growth, increasing rhizosphere bacteria beyond baseline levels does not necessarily further enhance plant growth. Additional information on the composition of rhizosphere bacteria would be useful to understand these behavior, in that it is likely the sulfatemodified nanospheres are enhancing the growth of bacteria not associated with plant growth. Fig. 4 further illustrates the relationship between the observations in Figs. 2 and 3. Because root and stem length appear to exhibit very similar trends, Fig. 4 only examines root length. The vertical axis in Fig. 4 shows root length for each system studied, while the horizontal axis shows bacterial counts in the rhizosphere. It is clear from the figure that both the amine-modified (positive) polystyrene system and the TiO2 system exhibit both significantly decreased root lengths, and significantly decreased rhizosphere soil bacteria counts compared with the control. In contrast, the sulfatemodified (negative) polystyrene system exhibits significantly higher rhizosphere bacteria counts compared with the control, but no significant difference in root length. In order to further understand the observations in Figs. 2e4,
imaging was used to evaluate the extent to which nanoparticles attached to root surfaces. For this purpose, confocal microscopy was used to image the fluorescent polystyrene nanospheres (Fig. 5A and B), while SEM/EDS was used to look for attached TiO2 nanoparticles (Fig. 5C). Fig. 5A shows a root sample from a lettuce plant grown in the presence of amine-modified (positive) polystyrene nanospheres. The image is a superposition of fluorescence from the nanoparticles, shown in orange, and transmitted light to show the outline of the root. It is apparent from the image that the root is covered with nanoparticles; that is, the positively charged nanospheres have attached strongly to the root. All images of roots that had been grown in the presence of amine-modified (positive) polystyrene nanospheres showed significant nanoparticle coverage. This result is not unexpected, because the strong positive charge of the nanospheres and the typical negative charge of root surfaces produces a significant affinity of the nanospheres for the root surfaces. Fig. 5B shows a root sample from a plant grown in the presence of sulfate-modified (negative) polystyrene nanospheres. Note that the image was collected with the same imaging settings as those used for Fig. 5A. It is apparent from Fig. 5B that the sulfate-modified (negative) nanospheres are not associated with the root surface. Note that a small cluster of suspended nanospheres is evident in the lower right corner of the image, verifying that the confocal imaging is able to detect the sulfate-modified nanospheres. The lack of attachment in this system is not unexpected, because the strong negative charge of the nanospheres and the typical negative charge of root surfaces reduces the affinity of the nanospheres for root surfaces. Fig. 5C shows a SEM/EDS map of a root sample in the system grown with the nano-TiO2 suspension. Spectral analysis of the image found that, as might be expected, carbon and oxygen are the most prevalent, at more than 93 weight percent of the detected elements. For this and all other root images examined, all other detected elements were in the single digit percentages or lower, with titanium either not detected, or detected at 0.1 weight percent (the lowest amount reported by the software, and likely within the noise of the method). No attached TiO2 nanoparticles could be identified in any of the images studied. As was the case with the negatively-charged polystyrene nanospheres, this result is not unexpected. Comparing the results in Fig. 5 with those in Figs. 2e4, it is apparent that while attachment of nanoparticles to root surfaces may inhibit plant growth and deplete rhizosphere bacteria, it is not a necessary condition for those effects to occur. Note that while both the amine-modified polystyrene nanospheres and the nanoTiO2 systems exhibited both depleted rhizosphere bacterial counts
Fig. 5. Confocal (A,B) and EDS (C) images of root surfaces grown in the presence of A: Amine-modified (positive) polystyrene nanospheres, B: Sulfate-modified (negative) polystyrene nanospheres, and C: TiO2 nanoparticles.
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and inhibited plant growth, only the amine-modified polystyrene exhibited attachment to root surfaces. While positively charged nanoparticles such as the aminemodified polystyrene nanospheres used here have been shown to cause toxicity to some bacteria (e.g., (Jacobson et al., 2015)), the effect of nano-TiO2 has typically been found to be more subtle, reducing the activity of some bacteria under some conditions (e.g., (Ge et al., 2012)). It is important to note that the bulk soil bacterial counts in the TiO2 system are much closer to those in the control than is the case for the rhizosphere counts (~83% bulk vs. ~42% rhizosphere, Fig. 3), so it is likely that direct toxicity is not the cause of the depleted rhizosphere bacteria in the TiO2 system. One possible explanation for the depleted rhizosphere bacteria in both cases may be that nanoparticles are attaching to bacteria surfaces, and ultimately influencing the attachment of the bacteria to root surfaces by changing bacterial surface properties. Note that although bacteria and TiO2 are both negatively charged at the pH of this work, electrostatic interactions are only one mechanism for attachment. Preliminary XDLVO calculations using bacteria parameter values from Chen and Strevett (2003) and TiO2 parameter values from Huang et al. (2015) show that under a wide range of ionic strengths, attachment of nano-TiO2 to many different bacteria at the secondary minimum should be thermodynamically favorable. (Note also that others have reported attachment of nanoTiO2 to bacteria surfaces (e.g. (Huang et al., 2015)).) The positivelycharged amine-modified polystyrene nanospheres would also be expected to attach strongly to bacteria, just as they do to root surfaces (Fig. 5C). It is possible that attachment of nanoparticles to soil bacteria in both cases reduce the favorability of their attachment to root surfaces. Additional studies of simultaneous nanoparticle and bacterial attachment would be needed to evaluate this hypothesis. 4. Conclusions This work examined how three different nanoparticle suspensions impacted both soil and rhizosphere bacteria, and early plant growth in buttercrunch lettuce plants. The results of the work found that both positively-charged amine-modified polystyrene nanospheres and nano-TiO2 significantly inhibited both rhizosphere bacterial counts and plant growth in the system studied. In contrast, negatively-charged sulfate-modified polystyrene nanospheres appeared to enhance rhizosphere bacterial counts, but did not have a significant impact on plant growth. These results suggest that one potentially important mechanism through which nanoparticles could hinder plant growth could be by interfering with the growth, attachment or function of rhizosphere bacteria. The work found significant attachment of positively-charged amine-modified polystyrene nanospheres to root surfaces, but no detectable attachment of the other two nanoparticles studied. It is hypothesized that the similar impact of both amine-modified polystyrene and nano-TiO2 on both rhizosphere bacteria and plant growth may result from attachment of the nanoparticles to the bacteria themselves, and the effect of the attachment on bacterial affinity for root surfaces. Acknowledgments The authors thank Aderonke Adegbule for her assistance with
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the SEM imaging conducted for this work, and Dr. Tingting Gu for her assistance with the confocal imaging conducted for this work.
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