Initial Sterilization of Soil Affected the Interactions of Cerium Oxide

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Initial Sterilization of Soil Affected the Interactions of Cerium Oxide Nanoparticles and Soybean Seedlings (Glycine max (L.) Merr.) in a Greenhouse Study Cheyenne Stowers, Maria King, Lorenzo Rossi, Weilan Zhang, Aishwarya Arya, and Xingmao Ma ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01654 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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ACS Sustainable Chemistry & Engineering

Initial Sterilization of Soil Affected the Interactions of Cerium Oxide Nanoparticles and Soybean Seedlings (Glycine max (L.) Merr.) in a Greenhouse Study Cheyenne Stowers1, Maria King2, Lorenzo Rossi3, Weilan Zhang1,a, Aishwarya Arya2, Xingmao Ma1,* 1

Zachry Department of Civil Engineering, Texas A&M University, TAMU 3136, College

Station, TX 77843-3136, USA 2

Department of Biological and Agricultural Engineering, Texas A&M University, TAMU 2474,

College Station, TX 77843-2474, USA 3

Department of Horticultural Sciences, University of Florida, IFAS, Indian River Research and

Education Center, Fort Pierce, FL 34945-3138, USA

*For correspondence: Dr. Xingmao Ma Zachry Department of Civil Engineering Texas A&M University 199 Spence Street, TAMU 3136 College Station, TX 77843-3136 USA

a

Current Address: Dept. of Civil and Environmental Engineering, The Hong Kong University of

Science & Technology, Clear Water Bay, Hong Kong, China

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Abstract Cerium oxide nanoparticles (CeO2NPs) have exhibited strong impacts on many physiological and biochemical processes in agricultural crops. However, almost all previous studies directly linked the responses of plants to the impact of ENPs, ignoring the potential role of local organisms in the rhizosphere. The primary goal of this study was to assess the effects of the rhizosphere bio-community on the interactions of CeO2NPs with soybeans, including CeO2NPs transformation in the rhizosphere and in plant tissues. Soybean seedlings grown in initially sterilized soil or unsterilized soil were exposed to different concentrations of CeO2NPs (0, 100 and 500 mg/kg) in a growth chamber for 27 days. Initial sterilization significantly affected the interactions of CeO2NPs and soybeans, compared with the unsterilized soil (p≤0.05). For example, the net photosynthesis rate of soybean seedlings exposed to 100 mg/kg of CeO2NPs for 25 days was 122% higher in sterilized soil than in unsterilized soil. However, at 500 mg/kg, the net photosynthesis rate was 67.2% lower in sterilized soil than in unsterilized soil. Cerium accumulation in soybean tissues was affected by the exposure concentration and soil type, although strong interaction between the two factors was not observed. The presence of 100 mg/kg of CeO2NPs significantly increased the nodule numbers on soybean roots to about 12 in sterilized soil while only one or two nodules were observed in unsterilized soil at the same concentration, indicating significant impact of soil treatment (p≤0.05). Higher CeO2NPs concentrations and soil treatment also influenced the number of soil nematodes and their viability. In summary, the soil bio-community strongly affected the physiological impact of CeO2NPs on soybean seedlings, the nematode population and the Ce accumulation in soybean tissues. Keywords: cerium oxide nanoparticles, soybean, rhizosphere, nematodes


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Introduction Engineered nanoparticles (ENPs) have been increasingly used in many industries such as manufacturing and energy production due to their high surface-to-volume ratio and abundance of reactive sites.1,2 With the wide usage of ENPs, their environmental fate and potential impact on the human food supply is a growing concern. These ENPs can enter an agricultural system through the land application of bio-solids or the use of pesticides and fertilizers containing ENPs.3 A range of studies have been conducted to determine the impact of ENPs on various agricultural crops, with both positive and negative results reported depending upon the ENPs properties, plant species, dosing concentrations as well as exposure pathways.4-6 Cerium oxide nanoparticles (CeO2NPs) are one of the most commonly used ENPs. There is an estimated global production of 10,000 tons of CeO2NPs per year that can be found in a variety of industrial products such as diesel fuel additives, petroleum refining catalysts, electronics, and automobile catalytic convertors.7 Their interactions with different plant species have been widely studied. For example, CeO2NPs at 500 mg/kg were shown to improve wheat biomass by 12.7% and grain yield by 36.6%.6 When dosed by 100 mg/kg CeO2NPs, the net photosynthesis rate of soybeans increased by 25-50%. However, 500 mg/kg of CeO2NPs decreased the photosynthesis rate by 25-30%.8 These studies showed that CeO2NPs affected plant physiological processes. However, almost all previous studies interpreted the plants’ responses to CeO2NPs as the direct impact of these nanoparticles on plants, ignoring the essential role of rhizosphere biological ecosystem on mediating the impact of ENPs on plants. The rhizosphere is the region around the roots that extends around 2-80 mm depending upon the plant species.9 Soil surrounding plant roots contains a variety of nutrients, microorganisms, and enzymes.10 For example, microfauna (nematodes and protozoa) in this 3 ACS Paragon Plus Environment


region can influence nutrient availability by regulating the size and composition of the microbial community, excreting nutrients themselves, or accelerating the turnover rate of biomass and soil organic matter.11 The microbial diversity and activity in this region is also significantly higher than in the bulk soil.9 Root exudates, including secreted ions, free oxygen and water, enzymes, mucilage, and various carbon-containing primary and secondary metabolites, have been suggested to have a significant role in eliciting the outcomes of such interactions and therefore regulating the plant-rhizosphere dynamics.10 The bacteria in this region of soil are important to plant health, and leguminous plants, such as soybeans, often create symbiotic relationships with nitrogen-fixing bacteria in the soil. Previous studies indicate that the nitrogen-fixing bacteria are particularly sensitive to ENP exposure.3 Another study specifically showed that CeO2NPs significantly decreased the soil microbial carbon and nitrogen contents, after a seven day exposure.12 If ENPs are able to impact rhizosphere bacteria and other organisms such as nematodes, this could impact plants themselves. This in turn, could affect the plant’s response to ENPs such as the production of exudates, which then influences the speciation of ENPs (e.g. particulate ENPs vs. dissolved ions for metallic ENPs) in plant rhizosphere.13 The speciation of CeO2NPs directly determines their accumulation and bioavailability to plants.14 As a result, the degree to which ENPs, rhizosphere organisms, and plants interact is expected to have a profound impact on the environmental consequences of ENPs and require more investigation.13 The primary objectives of this investigation were: (1) to assess the effects of the initial microbial community on the interactions of CeO2NPs with soybeans, and (2) to understand how the microbiological community and wider bio-fauna in the rhizosphere affect the CeO2NPs speciation and uptake by plants.


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Materials and Methods Cerium Oxide Nanoparticle Polyvinylpyrrolidone (PVP)-coated CeO2NPs were purchased from US Research Nanomaterials, Inc (Houston, TX, USA). Characterization of the NPs was reported in a previous publication which used CeO2NPs from the same batch as this investigation.8 The NPs were roughly spherical and the diameter of the particles ranged from 6 to 24 nm, with an average diameter of 10 nm. Zeta potential of the NPs in 500 mg/L of solution was around -51.6 mV. Xray photoelectron spectroscopy (XPS) analysis indicated that the amount of Ce3+ was approximately 12% of the total Ce on nanoparticle surface. Soil Preparation Scotts Topsoil packaged in Marysville, OH was purchased from a commercial outlet. The soil contained a combination of peat, composted forest products, and sphagnum peat moss and was adequately fertilized. Soil was prepared in two conditions: sterilized and unsterilized. Sterilized soil was obtained by placing it in an autoclave (Panasonic MLS – 3781L) for 25 minutes at 121˚C. The soil was sterilized to eradicate the original bio-community. The soil was not kept in a state of sterilization during the experiment. Therefore, it is assumed that bacteria from the air and water would reenter the soil and establish a new microbial community. Nematodes were tested after sterilization and they were able to survive sterilization as used in this study (Details below). Soybean Preparation Soybean seeds (Glycine max (L.) Merr.) were purchased from Johnny’s Selected Seeds (Winslow, Maine). Seeds were sterilized using 1.25% sodium hypochlorite solution and then 5 ACS Paragon Plus Environment


rinsed with deionized (DI) water three times.15 The seeds were germinated in two separate containers with sterilized or unsterilized saturated soil of about 2 inch depth. After 4 days they were transplanted into containers containing 150 grams of the relevant (sterilized or unsterilized) topsoil. Before seeds were transplanted into the soil, a freshly-made dispersion of DI water and CeO2NPs were added to the soil at concentrations of 0 (control), 100, and 500 mg CeO2/kg of dry soil. Amount of DI water needed was determined to be 120 mL per container in order to maintain saturation. In total, each group (sterile and unsterilized) had three treatments with 5 replicates per treatment; for a total of 30 soybean seedlings. They were placed in a growth chamber with a controlled light/dark cycling of 16 hours on and 8 hours off. DI water was given daily to maintain saturation and plants were kept at constant room temperature of 25˚C. Photosynthesis Rate & Stomatal Conductance Analysis Net photosynthesis rate and stomatal conductance were measured with a Licor-6400XT (Lincoln, NE) in conjunction with an infrared gas exchanger at day 11, day 18, and day 25 after CeO2 exposure. This equipment utilizes a lamp that keeps a constant quantum flux of 1000 µmol/m2/s and a constant CO2 exchange rate of 400 µmol/mol between two reference sensors. In addition, a flow rate of 500 µmol/s is utilized to control humidity within the chamber. Chlorophyll Fluorescence Chlorophyll fluorescence values (Fv/Fm) were measured with a continuous excitation chlorophyll fluorescence analyzer (OS1p, Opti-Sciences, Hudson, NH). Measurements took place on the same days the net photosynthesis rate was measured. Leaf clips were placed on the 6 ACS Paragon Plus Environment

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topmost fully expanded leaves of the soybeans and remained there for 30 minutes.16 These clips allowed the leaves to adjust to the darkness and then be excited for measurement purposes. Plant Harvest After 27 days of growth, the plants were pulled from the soil gently and rinsed with DI water to remove soil particles. The roots were separated from the shoots and weighed separately to obtain their fresh weight. Nodules on the fresh soybean roots were then counted and inside color of the nodules, which indicates the activity of nodules, inspected by cutting them open after counting. Some fresh leaves were used for chlorophyll content analysis. Chlorophyll Content Analysis Chlorophyll content of a and b were determined following the methods of Moran (1982)17. Fresh leaf tissues in the amount of 50 mg were weighed from each replicate and placed in a centrifuge tube with 12 mL of dimethyl formamide (DMF). The samples were vortexed for a minute and then kept in the dark for 24 hours. Afterwards, the chlorophyll analysis was conducted using a Lambda 35 UV-Vis spectrophotometer (Perkin Elmer, Waltham, MA). Light absorbance was measured at wavelengths of 664 nm and 647 nm and were then used to calculate chlorophyll a and b values. Ce Analysis in Plant Tissues The total element of cerium in plant tissues (including Ce attached to root surface) was determined by strong acid digestion, following the procedure reported earlier.8 First, the tissues were dehydrated in an oven at 70˚C for 7 days. After fully dried to constant weight, 0.5 grams of tissue material were added into a 4 mL solution of nitric acid (70% by volume) and incubated



overnight at room temperature. They were further digested in a DigiPREP MS hot block digester (SCP science, Clark Graham, Canada) at 95˚C until any remaining residual tissue was fully dissolved. The digestate was then cooled to room temperature, at which point it was further mixed with a 2 mL solution of H2O2 (30% by volume) and heated at 95˚C for two hours. After cooling down to room temperature, this solution was analyzed by inductively coupled plasma mass spectrometry (ICP-MS, Perkin Elmer mod. DRCII, Waltham, MA). An internal standard containing 5 µg/L of rhodium was used for all measurements and instrumental fluctuations were corrected according to the internal standard density variation. Calibration curves were acquired with six concentrations of analytical-grade ICP standards of Ce and a blank and one standard solution was run for every 15 samples to ensure consistency. The plasma Ar flow was 19 L/min. The sample uptake rate is 1 mL/min and the dwell time is set as 50 ms. Ce Speciation Fresh root tissues rinsed with DI water three times were further rinsed with 5 mM CaCl2 washing solution five times.18 This washing solution was used to collect cerium attached on the surface of roots. A 4 mL aliquot of this washing solution was then centrifuged through 10kDa Amicon Ultra-4 Centrifugal Filter (EMD Millipore, Billerica, MA, USA) units to separate the dissolved cerium from particulate cerium. Ce in the filtrate was then quantified by ICP-MS, and this fraction of Ce was considered as the dissolved Ce on the root surface. Another 4 mL of the same washing solution was nitric acid (1% HNO3) digested and quantified by ICP-MS to determine total cerium on root surface. The difference was calculated as Ce in particulate form on root surface. After CaCl2 washing, enzymatic digestion was performed on fresh root and shoot tissues to distinguish the particulate and dissolved cerium in soybean tissues by a recently reported 8 ACS Paragon Plus Environment

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method (Zhang et al., 2017). Fresh root and shoot tissues were cut into small sections with a blade. 0.5 grams of this tissue were mixed into a 9 mL solution of 20 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH=5, adjusted by NaOH). This mixture was homogenized in a centrifuge tube using a handheld homogenizer (150 W, Fisher Scientific). After the mixture was thoroughly homogenized, one mL of 30 mg/mL Macerozyme R-10 enzyme (bioWORLD, Ohio, USA) (prepared in 20 mM MES) was added to the mixture. This mixture, now at 10 mL, was mixed on a shaker for 24 hrs at 37oC. The dissolved and total Ce was then analyzed similarly as described above for the washing solution. Nematode Plating Five grams of each soil sample were resuspended in 1 mL of Phosphate Buffer Saline (PBS) buffer at pH 7.4, and vortexed vigorously for two consecutive 30 sec periods. Aliquots of 100 µL soil extracts were plated on nematode growth medium (NGM)19 and incubated at 30oC for four weeks. The nematode growth on the plates was monitored by microscopy at 1000x magnification. Images were taken within a ten day period starting at week 5 to record changes in the nematode colonies. Statistical Analysis Minitab was used to perform t-tests, one-way analysis of variance (ANOVA), and twoway ANOVA on the data obtained. Two-way ANOVA was used to test the interaction of the independent factors (concentration and soil condition) on the dependent factor (any parameter tested). The one-way ANOVA was used to determine statistical differences exhibited by the CeO2 NP concentration level (0, 100 mg/kg, and 500 mg/kg) on each parameter tested. The t-



test was used to determine the significance of the two soil conditions (unsterilized and sterilized) at a particular concentration level. Results Fresh Biomass No significant differences were found in the fresh biomass of shoots across different CeO2NPs concentrations or soil conditions, Figure S1A. A two-way ANOVA analysis showed no interactions between soil conditions and concentration levels. In contrast, the fresh root biomass was significantly increased by 36% and 40% after exposure to 100 and 500 mg/kg of CeO2NPs in the unsterilized soil compared to its control group, Figure S1B. However, in the sterilized soil, treatment with 100 and 500 mg/kg of CeO2NPs led to approximately 28 and 32% root biomass reduction compared to the controls in the sterilized soil, albeit insignificantly. A comparison of the fresh root biomass between different soil conditions but at the same CeO2NPs exposure showed that only the control groups displayed a significant difference, with the sterilized group being 89% higher than the unsterilized group. Two-way ANOVA analysis indicated that root biomass was impacted by the interaction between CeO2NPs concentrations and soil condition. Nodule Counts The number of nodules on soybean roots was counted at harvest and the nodule count from different treatments is shown in Figure 1. The inside of nodules was inspected, and a reddish color prevailed, indicating that the bacteria present were actively fixing nitrogen.20 In the control group of both soil types, the nodule count was zero. As the dosing level of CeO2NPs increased, the number of nodules present significantly increased. There was only a significant 10 ACS Paragon Plus Environment

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difference between soil conditions at 100 mg/kg CeO2NPs, where plants in the sterilized group had greater than 10 nodules while the unsterilized group had only 1 or 2 present on a root sample. Two-way ANOVA indicated that the nodule count was independently affected by either CeO2NPs or soil condition, but that these two factors did not interact. Chlorophyll Content Chlorophyll a and chlorophyll b contents from different treatments are shown in Figure 2. Significance between soil conditions was seen for both chlorophyll a and b in the control group and at the 100 mg/kg group, with sterilized soil having significantly higher levels. However, at 500 mg/kg, the chlorophyll levels exhibited opposite trends, even though the differences were insignificant. Chlorophyll a was significantly increased by 89% at 500 mg/kg CeO2NPs in the unsterilized soil, compared to the unsterilized control. In the sterilized soil, however, chlorophyll a content decreased by 19% (although not significantly) at the same concentration. Chlorophyll b in 500 mg/kg CeO2NPs treated soybeans significantly decreased by 20% from the control in sterilized soil. Interestingly, the ratio of chlorophyll a and b remained at about 2.55 in sterilized soil, despite the CeO2NPs treatment. On the contrary, 500 mg/kg of CeO2NPs significantly increased the chlorophyll a and b ratio in unsterilized soil from 1.94 to 3.05. Interaction between the two factors was seen for both chlorophyll a and b. Chlorophyll Fluorescence Chlorophyll fluorescence was measured at three different days and the results are shown in Figure S2. 100 mg/kg of CeO2NPs did not affect soybean chlorophyll fluorescence, regardless of the soil condition. In the sterilized soil, the enhancement of chlorophyll fluorescence by 500 mg/kg of CeO2NPs was observed on day 11. However, the enhancement disappeared afterwards. 11 ACS Paragon Plus Environment


In the unsterilized soil, the improvement of the FV/FM ratio was not observed until day 25 at 500 mg/kg. Significant differences between different soil conditions at the 500 mg/kg treatment was noticed on day 18 and 25. Interaction between soil conditions and concentration levels was only significant at day 25.

Stomatal Conductance Three measurements were taken during the growing process and are shown in Figure S3. CeO2NPs had a significant impact on the stomatal conductance at both day 11 and day 25 for both soils. However, the extent of impact varied between the soils. For example, both 100 and 500 mg/kg of CeO2NPs significantly increased the stomatal conductance in unsterilized soil at day 11, but only 500 mg/kg CeO2NPs displayed significant impact in sterilized soil on the same day. At day 18, stomatal conductance was not significantly affected by CeO2NPs and the effect of soil condition was only significant at 500 mg/kg. On day 25, treatment with CeO2NPs again demonstrated significant effects on stomatal conductance in both soils. In addition, the soil condition became a significant factor affecting the stomatal conductance of soybean leaves in the presence of 100 mg/kg and 500 mg/kg CeO2NPs, but not in the control group. Interaction between these two factors was significant on day 11 and day 25. Net Photosynthesis Rate Plant net photosynthesis rate at different treatment conditions are shown in Figure 3. At day 18, neither the soil condition nor the CeO2NPs affect plant photosynthesis efficiency compared to their controls. On day 11 and day 25, CeO2NPs at the tested concentrations had a significant impact on plant photosynthesis rate, and the net effect of CeO2NPs depended upon the soil conditions. The net photosynthesis rate of soybean was significantly increased by


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CeO2NPs at both 100 and 500 mg/kg in unsterilized soil on day 11, however the net photosynthesis rate was significantly reduced by 100 mg/kg of CeO2NPs in sterilized soil on the same day. The photosynthesis rate was 38% higher in the sterilized soil than in the unsterilized group in the control group on day 11. On day 25 the control group was no longer significantly affected by the soil condition, but the other two treated groups were still significantly affected by soil condition, with the unsterilized group being 59% lower than the sterilized group at 100 mg/kg and then the sterilized group being 67% lower than the unsterilized group at 500 mg/kg. When assessing the photosynthesis rates over time, it can be seen how the control group was impacted by soil condition initially, but then showed no impact by day 25, and the 500 mg/kg group having the opposite results. Significant interactions were detected between CeO2NPs treatment and soil conditions on day 11 and day 25. Cerium Accumulation in Plant Tissues Figure S4 shows total Ce in different plant tissues. Total Ce associated with roots was impacted significantly by dosing concentrations, increasing in accumulation as the dosing concentration increased in both soils. No significant differences were noticed for Ce associated with soybean roots at different soil conditions. Neither soil treatment nor CeO2NPs at applied concentrations made a significant difference on Ce in soybean shoots. Although the average value of accumulation for the shoots was higher at 500 mg/kg compared to the other two treatments, this was not statistically significant. In addition to the total Ce, Ce speciation in soybean roots was examined following the enzyme digestion method, Figure 4. Only plants from the control and 500 mg/kg treated group were analyzed. Significantly less Ce (58%) was attached to soybean roots grown in sterilized soil than those in the unsterilized soil. No dissolved Ce was found on the root surface in both soil 13 ACS Paragon Plus Environment


conditions. The Ce concentration in roots in the sterilized soil was 45% lower than those grown in the unsterilized soil at 500 mg/kg. Dissolved cerium was found in soybean roots at 500 mg/kg, but significance between soil conditions could not be established due to high standard deviations. Nematode Growth Different levels of nematode activity were detected on the the agar surfaces plated with the soil extracts, Figure 5. Nematode population was negatively affected but not fully inhibited by sterilization. After four weeks of incubation (Day 27 after seedling transfer), high numbers of nematode larvae were visible all over the plates with non-sterilized soil, while the sterilized soil contained mostly single, immotile (dead) nematodes. Over the additional 10-day period after intial observation, the non-sterilized samples continued to develop mildly dense larval colonies while the sterile samples remained the same, maybe due to the absence of live bacteria that the nematodes would feed on. Plates with non-sterile soil containing 100 mg/kg CeO2NPs exhibited dense colonies that continued to grow into denser populations. The samples with sterile soil also showed very few small colonies, indicating that 100 mg/kg CeO2NPs may potentially enhance nematode growth as the sterilized soil without NPs showed clusters of dead nematodes only. Interestingly, samples with 500 mg/kg CeO2NPs showed very few, scattered colonies in the nonsterilized soil, and even smaller, more scattered colonies in the sterilized samples, indicating that CeO2NPs at high concentratinos adversely affected nematode growth. This may be explained by their ability to induce oxidative damage in the nematodes.21 In addition, the ENPs toxic effect on bacteria that play key roles for the bacterial-feeding nematode life cylce has been known.22 Nematodes in the original soil without plants were also examined in sterilized and unsterilized conditions and the results are shown in Figure S5. After the initial weak growth the nematodes 14 ACS Paragon Plus Environment

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started to develop more colonies in the non-sterile soil. The sterilized soil initially exhibited dead nematodes that continued to grow into weak, immotile colonies. Discussion A majority of the measurements carried out in this study, including the physiological parameters of soybeans as well as the accumulation and speciation of Ce in plant tissues, indicated a strong role of the rhizosphere bio-community in the interactions of CeO2NPs and plants. After four weeks of exposure to CeO2NPs, the net photosynthesis rate, and stomatal conductance as well as the FV/FM ratio were all significantly affected by the soil condition. While chlorophyll a and b were not significantly affected by either the CeO2NPs treatment or the soil condition alone, those factors interacted and led to significant differences in these pigments in plant leaves. At the highest concentration of CeO2NPs used in this study (500 mg/kg), a clear trend could be seen for all measured physiological parameters that they were more negatively impacted in the sterilized treatments than in the unsterilized treatments. As indicated above, after the initial sterilization, the soil was open to the air while the plants were growing. Therefore, it was expected that a new biological community would develop in the sterilized soil, but the biocommunity was probably less diverse and have a smaller metabolic capability than the indigenous community in the unsterilized soil. Our results clearly showed that the composition of the local community in plant rhizosphere is an important factor in the interactions of ENPs and plants, which has been ignored in most previous studies. The change in soil biological composition between treatments had several consequences. One such consequence was the different nodulation rate of soybean roots in the presence of CeO2NPs. There was a drastic increase in the nodule production on soybean roots treated with 100 and 500 mg/kg of CeO2NPs in the sterilized soil compared with the unsterilized soil. In a 15 ACS Paragon Plus Environment


previous study using soybeans and CeO2NPs (concentrations at 0, 0.1, 0.5 and 1 g/kg), the soybean plants produced consistent nodulation (mean = 39 ± 3) for all treatments.23 In that study, the plants were grown to the seed production stage, whereas in this investigation the soybeans were grown for only 27 days. The previous study reported that high concentrations of CeO2NPs (0.5 and 1g/kg) resulted in many dry nodules and significantly reduced nitrogen fixation (80%), indicating that the nodules present were influenced by CeO2NPs and could not fix nitrogen from the atmosphere as effectively as the unexposed nodules. The control in this study showed no sign of nodulation for both sterilized and unsterilized soils until day 27. However, the treatment with CeO2NPs significantly increased the nodulation rate in both soils, in particular the sterilized ones. We did not observe many dried nodules as reported previously, possibly because the observation was conducted toward the end of the soybean life cycle in the previous study while the observation was performed on day 27, when the nodules were probably still fresh and did not have enough time to respond to the impact of CeO2NPs. It is interesting to notice that CeO2NPs stimulated the development of nodules in our study compared with the controls, possibly because that CeO2NPs exerted some stress on plants and limited their capability to directly take up nitrogen from the soil (through possible interference with the nitrogen cycle). Previous research has reported the ability of reduced ceria to cause dissociation of nitrite (NO2-)24 and to catalyze the reduction of NO to N2.25 Plants obtain the majority of their nitrogen through nitrate (NO3-) and ammonium (NH4+). NO3- is produced through the oxidation of NO2- by nitrifying bacteria, so it is possible that two things could have occurred: CeO2NPs might have negatively affected the nitrifying bacteria, therefore impacting their ability to produce nitrate in the soil, or directly reacted with the nitrite, limiting the amount of nitrite available for the nitrification process. We did not investigate the nitrogen fixation efficiency of the nodules except for a visual observation


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of the color, further efforts are required to understand whether the nodules are still efficiently fixing nitrogen after CeO2NPs exposure. One phenomenon particularly interesting to us is that CeO2NPs led to a greater increase of nodules in the sterilized soil than the unsterilized soil. The reasons leading to higher nodulation in sterilized soil are not clear. However, we attribute this partially to the reduced nematode growth in the sterilized soil resulting in less competition between the plant and nematodes for the nitrogen fixing bacteria, Figure 5 .26 Due to the soil treatment, sometimes an opposite effect of CeO2NPs on plants was observed. For example, chlorophyll a levels dropped from control to the 500 mg/kg CeO2NPs treated plants in the sterilized soil, while in the unsterilized soil, chlorophyll a increased. This was also seen in the net photosynthesis rate. For instance, a strong increase in the photosynthesis efficiency was seen in the sterilized group with 100 mg/kg CeO2NPs on day 25, but soybean seedlings had the lowest photosynthesis rate in the unsterilized soil with the same CeO2NPs treatment. Similarly, when the soybeans were exposed to CeO2NPs, their root biomass decreased in the sterilized soil, but increased for plants in the unsterilized group compared with their relative controls. In addition, the different levels of CeO2NPs seem to affect nematode growth and viability differently, potentially affecting the plant-bacterium interactions in the root rhizosphere. The results showcased how significant a role the bio-community can have on the way CeO2NPs impact a plant system. The soil treatment also led to significant differences in Ce accumulation in plant tissues. The enzymatic analysis indicated that plants grown in sterilized soil tended to have lower Ce associated with their root tissues. It is believed that CeO2NPs can be transported from roots to shoots both as particles and as Ce ions. Direct transport of CeO2NPs alone is unlikely the reason for higher upward transfer of CeO2NPs in sterilized soil because upward transport of CeO2NPs is



closely related to the water transpiration and the biomass in the sterilized soil was actually slightly smaller than plants in unsterilized soil. While ionic Ce was detected in soybean roots in both soil conditions, it was not detected in soybean shoots in either soil, suggesting that they might have transformed to Ce containing particles following their root uptake. Previous study has reported the formation of cerium phosphate nanoparticles in plant roots following Ce root uptake and the soil used in this study was fully fertilized.27 How the different biological communities in plant rhizosphere affect Ce distribution and transformation in different soils remains unknown. However, the results highlight the important role of local biological community in the interactions of plants and ENPs. While we did not perform extensive characterization of the biological community in plant rhizosphere, the insight provided by the study is significant and the results may partially explain the inconsistent and sometimes contradictory results concerning the plant responses to ENPs in different studies. Future studies should include the bulk and ionic counterparts as comparisons to see whether similar microbial effects can also be observed when cerium is provided in different forms. In addition, the plants were only grown for 27 days in this study. Future investigations should extend the growth period to maturity so that the role of the rhizosphere bio-community can be further illustrated in the long term impact of ENPs on crop physiological processes, including their impact on crop yield.

Supplementary Information: The fresh weight of plants, the chlorophyll fluorescence, the stomatal conductance of plants as well as the total Ce accumulation in plant tissues after soybean seedlings were exposed to three different concentrations of CeO2NPs in two types of soil for 27 days. The nematode counting in the sterilized and unsterilized soil and the development in 10 days after the first examination.


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Acknowledgments We thank Dr. Leonardo Lombardini (Dept. of Horticultural Sciences, Texas A&M University, College Station, TX) for assistance with the LI-COR 6400xt photosynthetic measurements.

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Figure 1: (A): Nodule counts on soybean roots exposed to three different concentrations of CeO2NPs in two soil conditions (unsterilized and sterilized). Values represent mean ± SD (n=3), with the different letters indicating significant differences (p ≤ 0.05) according to one-way ANOVA for the same soil condition. Asterisks (*) indicate significant differences between soil conditions at a particular concentration, according to t-test. (B): Pictures of nodules found on soybean roots and (C) a typical nodule cut in half.



Figure 2: Chlorophyll a (A) and chlorophyll b (B) content of soybean plants exposed to CeO2NPs. Soybeans were grown in two soil conditions (unsterilized and sterilized) and exposed to three different concentrations of CeO2NPs (0,100 or 500 mg/kg). Reported values represent mean ± SD (n=3), with different letters indicating significant differences (p ≤ 0.05) among concentrations at the same soil condition, according to one-way ANOVA. Asterisks (*) indicate significant differences between soil conditions at a particular concentration, according to t-test.


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Figure 3: Net photosynthesis rate of soybean leaves at day 11 (A), day 18 (B) and day 25 (C) after the seedlings were treated with three different concentrations of CeO2NPs (0, 100 or 500 mg/kg) in two soil conditions (unsterilized and sterilized). Reported values represent mean ± SD (n=3), with different letters indicating significant differences (p ≤ 0.05) according to one-way ANOVA. Asterisks (*) indicate significant differences between soil conditions at a specific concentration, according to t-test.




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Figure 4: (A) Total cerium attached to root surface of soybeans, (B) Total cerium within the root tissues, and (C) Dissolved Ce in soybean roots after the plants were exposed to 0 and 500 mg/kg of CeO2NPs. Soybeans were grown in two soil conditions (unsterilized and sterilized). Reported values represent mean ± SD (n=3), with different letters indicating significant differences (p ≤ 0.05) according t-tests between concentration levels. Asterisks (*) indicate significant differences between soil conditions at a particular concentration.



Figure 5: Representative images of nematode development in soils treated with three different concentrations of CeO2NPs (0, 100 and 500 mg/kg) in two different soil conditions (sterilized and non-sterilized). A-D are soil samples collected from pots treated with 0 mg/kg of CeO2NPs, E-H are soil samples collected from pots treated with 100 mg/kg of CeO2NPs and I-L are soil samples collected from pots treated with 500 mg/kg of CeO2NPs. A-B, E-F and I-J are unsterilized soils and the rest are sterilized soils. A,C, E, G, I, K were observed on the day of harvest (Day 1) and B,D, F, H, J and L were observed 10 days after the first observation (Day 10). Clusters of nematode larvae are shown on the images, in varying sizes, from dense colonies (A, B, E, F) to small numbers (G, H) and barely visible presence (K, L). Immotile (dead) nematodes with straight bodies are shown in both sterilized (I) and non-sterilized soil (K). Bar corresponds to 1 mm.


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TOC

Rhizosphere biological community affects the impact of cerium oxide nanoparticles on soybean physiological processes


Initial Sterilization of Soil Affected the Interactions of Cerium Oxide

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