Synergistic Effects of Zinc Oxide Nanoparticles and Bacteria Reduce Heavy Metals Toxicity in Rice (Oryza sativa L.) Plant

Heavy metals (HMs) are toxic elements which contaminate the water bodies in developing countries because of their excessive discharge from industrial zones. Rice (Oryza sativa L) crops are submerged for a longer period of time in water, so irrigation with HMs polluted water possesses toxic effects on plant growth. This study was initiated to observe the synergistic effect of bacteria (Bacillus cereus and Lysinibacillus macroides) and zinc oxide nanoparticles (ZnO NPs) (5, 10, 15, 20 and 25 mg/L) on the rice that were grown in HMs contaminated water. Current findings have revealed that bacteria, along with ZnO NPs at lower concentration, showed maximum removal of HMs from polluted water at pH 8 (90 min) as compared with higher concentrations. Seeds primed with bacteria grown in HM polluted water containing ZnO NPs (5 mg/L) showed reduced uptake of HMs in root, shoot and leaf, thus resulting in increased plant growth. Furthermore, their combined effects also reduced the bioaccumulation index and metallothionine (MTs) content and enhanced the tolerance index of plants. This study suggested that synergistic treatment of bacteria with lower concentrations of ZnO NPs helped plants to reduce heavy metal toxicity, especially Pb and Cu, and enhanced plant growth.


Introduction
Rapid increase in industrialization and anthropogenic activities has greatly contributed towards Heavy Metals (HMs) pollution in water which affects all aspects of the ecosystem [1]. Heavy metals are metals with relatively high density that are toxic in small (e.g., Cd, Pb, Hg, As) or higher (e.g., Cu, Zn, Co) concentrations [2]. In Pakistan, no sewage treatment plants are available, so crop irrigation with HM contaminated water results in metal accumulation in grains and other tissues of the plant [3]. It is evident from the literature [4] that water samples obtained from Hayatabad Industrial Estate (HIE), Peshawar, Pakistan, showed a higher level of HM contamination than quality standards should allow. Rice is the main staple food used by 2.7 billion people globally as a daily energy requirement. In Pakistan, its production is badly affected due to excesses of HM contamination in irrigated water [5]. Basically, HMs affect the biological systems of rice plants by altering several processes such as water, nutrients and oxygen uptake, and damage the chlorophyll synthesis pathway [6]. HMs damage the structure and quantity of seeds, the area of sugars grains, the cellular organelles, and also break the mitochondria, the nuclear envelope and damage the wall thickness of the plant cell [7]. Toxic metals disturb osmoregulation during the development process in plants by increasing osmolarity, reducing water potential, affecting the leaf area and transpiration level, and creating

Bacterial Strains Collection and Cultivation
The two HMs-resistant bacteria species (Bacillus cereus (PMBL-3) and Lysinibacillus macroides (PMBL-7), isolated from the industrial estate's effluent at Gadoon, were identified by Khattak et al. [25]. These two strains were obtained from the Plant and Microbial Biotechnology Laboratory, Kohat University of Science and Technology, Kohat, Pakistan. Bacterial strains were grown on nutrient broth (1% tryptone (Sigma Aldrich, St. Louis, CA, USA) 1% sodium chloride (Sigma Aldrich, St. Louis, CA, USA), and 0.5% yeast extract (Sigma Aldrich, St. Louis, CA, USA) at pH 7.4-7.6. Fresh culture of the bacteria was inoculated in autoclaved media for 24 h. Afterwards, the culture broth containing bacteria was centrifuged at 3500× g for 10 min, and then the pellet was properly washed with 1 mL of 5% NaCl solution (Sigma Aldrich, St. Louis, CA, USA). The bacterial cells were suspended in sterilized saline water for further experiments.

Heavy Metals Analysis in Polluted Water Samples
Heavy metals (Pb, Cd, Cr, and Cu) content in polluted water was determined by following the methodology of Radulescu et al. [26]. HMs content was analyzed by adding 2 mL of 70% concentrated nitric acid (HNO 3 ) (Sigma Aldrich, St. Louis, CA, USA) and 5 mL of 50% concentrated hydrochloric acid (HCl) (Sigma Aldrich, St. Louis, CA, USA) in (100 mL) water samples boiled at 95 • C on a hot plate (Ceramic hot plate C-MAG HP 4, Rawalpindi, Pakistan). Water was heated until the volume reduced to 15-20 mL. HMs content was than analyzed by atomic absorption spectroscopy (Perkin Elmer, Analyst 4000, Waltham, MA, USA).

Heavy Metals Remediation in Artificially Polluted Water
HMs (Pb, Cd, Cr, and Cu) remediation of artificially polluted water (100 mg/L of each metal) was carried out following the methodology of Bestawy [27]. Erlenmeyer flasks (250 mL) were filled with 100 mL of fresh bacteria strains culture (B. cereus and L. macroides), HMs (Pb, Cd, Cr, and Cu) solution and different concentrations of ZnO NPs (5,10,15,20 and 25 mg/L). Flasks were regularly agitated at 200 rpm for 90 min in the shaker incubator until reaching equilibrium. Flasks were then centrifuged at 4000× g to separate the solid-liquid phase and the samples were filtered through a 0.22 µm filter membrane. The experiment consisted of one positive control (bacteria culture without ZnO NPs) and one negative control (only ZnO NPs). All the experiments were performed in triplicate. HM concentrations in filtered water samples were determined by atomic absorption spectroscopy. HM removal efficiency (%) was determined by the given formula [28]: C 0 is the initial while C e is the final concentration of HMs in solution. The bio-nano remediation experiment further analyzed a polluted water sample designed in batch culture flasks by following the protocol of Bestawy [27] with slight modification. In this method, a series of 500 mL Erlenmeyer flasks were filled with 100 mL of polluted water with an equal volume (100 mL) of different concentrations of ZnO NPs (0, 5, 10, 15, 20 and 25 mg/L). Then 100 mL of fresh culture broth of B. cereus and L. macroides was added and agitated in incubator shaker at 200 rpm (90 min) until equilibrium was reached. The solution was centrifuged and the supernatant was used to investigate the HM (Pb, Cd, Cr and Cu) contents by atomic absorption spectroscopy.

Heavy Metals Remediation at Different pH and Contact Time
Effect of pH and contact time on the removal efficiency of HMs ions were determined by the following methodology [29]. Effect of pH was determined by mixing 100 mL of fresh cultured broth of bacteria strains (B. cereus and L. macroides) with 100 mL of metal ions (Sigma Aldrich, St. Louis, CA, USA) (Pb, Cd, Cr and Cu) (100 mg/L) solution and 100 mL of different concentrations of ZnO NPs (5, 10, 15, 20 and 25 mg/L) solutions. All the flasks were agitated in a shaking incubator for 90 min at different pH (4, 5, 6. 7 and 8) conditions. The pH of each solution was maintained by adding 1 mL of 5 M hydrochloric acid (HCl) and 1 mL of 5 M sodium hydroxide (Sigma Aldrich, St. Louis, CA, USA) (NaOH). The pH of each flask was determined by pH meter (OAKIAN digital pH meter, Waltham, MA, USA). Further experiments were carried out at different time intervals (30,60,90, and 120 min) while pH remained the same. Time at each interval was determined by Time clock (Time clipart digital clock, Waltham, MA, USA).

Heavy Metals Remediation in Plants
The remediation potential of the combined treatment of bacteria and ZnO NPs was further observed in the hydroponic culture experiment. Seeds were primed with bacterial strains (B. cereus and L. macroides) and grown in distilled water for 10 days. After 10 days, young seedlings were immediately transplanted in trays (3 L) containing one quarter strength Hoagland solution. The Hoagland medium composition includes 2.5 mL of 2 M KNO 3 , 2 mL of 2 M Ca(NO 3 ) 2 × 4 H 2 O, 1.5 mL of Iron oxide, 1 mL of 2 M MgSO 4 × 7 H 2 O, 1 mL of 1 M KH 2 PO 4, and 1 mL micronutrients. Plants were grown in the green house at 30 ± 2 • C in 16 h light with 60% humidity. After 21 days of cultivation, seedlings were transferred from the Hoagland solution to the solutions containing 5 and 10 mg/L ZnO NPs in (1 L) and HM contaminated wastewater for 1 week (7 days). The hydroponic system was used to inhibit the sorption of ZnO NPs to the soil surface and ensure that NPs and HMs were fully available [21]. Three replicates for each treatment and control blank were grown. During the exposure time, plants were randomly rotated and relocated to ensure equal light exposure. After HM polluted water and ZnO NPs exposure, the pH of the growth medium was maintained with a pH meter every daily.

Heavy Metals in Water and Plant Tissue (Root, Shoot and Leaf) after Remediation
HM content in different tissues of the plant was investigated by following the protocol of Retka [30]. Plant tissues (root, shoot and leaves) were dried at 80 • C in a dry oven (Dry heat Oven) for 2 days. Dried plant tissues (0.5 g) were taken and crushed with mortar and pestle separately. Crushed root, shoot, and leaf were digested with 3 mL of 70% (v/v) nitric acid and incubated overnight at room temperature. The next day, samples were heated at 95 • C for 4 h. After cooling, 2 mL of 30% (v/v) H 2 O 2 was added in the beaker and reheated at 95 • C until samples were fully digested. HM contents in different plant tissues were analyzed by atomic absorption spectroscopy (Perkin, ELMER, and Analyst 4000).

Heavy Metals Analysis in Polluted Water after Remediation Experiment
Heavy metals (Pb, Cd, Cr and Cu) content in polluted water was again analyzed by using the protocol of Radulescu [26]. To determine the HM content in treated water samples, 2 mL of concentrated nitric acid (HNO 3 ) and 5 mL of hydrochloric acid (HCl) were added to the beaker. The solution was boiled on a hot plate (Ceramic hot plate C-MAG HP 4, Rawalpindi, Punjab, Pakistan) at 95 • C until volume decreased to 15-20 mL. HMs were determined by atomic absorption spectroscopy (Perkin Elmer, Analyst 4000, Waltham, MA, USA).

Quantification of Low Molecular Weight Polypeptide Metallothioneins (MTs)
Metallothionein content was quantified through spectroscopy by following the protocol as described previously by Palmiter et al. [31]. . The solution was maintained at pH 7.4 and placed for 5-10 min. Protein was estimated through Bradford's method by following the protocol as described previously by Laemmli [33]. The low molecular weight proteins of MTs were separated on a 17% SDS twin mini gel electrophoresis unit (Bio-Rad, Hercules, CA, USA) at 80 V for 2.5 h. The gel was stained for 20 min by adding Coomassie blue dye (R-250, Sigma-Aldrich, St. Louis, CA, USA) and 20% methanol. The sample was de-stained by adding 5% acetic acid (Sigma-Aldrich, St. Louis, CA, USA), and then bands were compared with standard protein markers (Benchmark Protein Ladder, Thermo Fisher Scientific Inc. Waltham, MA USA) in the electro photogram (Bio-Rad, Hercules, CA, USA).

Bioaccumulation Index and Tolerance Index (TI) Determination
The bioaccumulation index was analyzed by measuring the number of heavy metals accumulated in grown plants following the previous method [34] with slight modification. The bioaccumulation index was calculated by using the following formula: C plant = Concentration of HMs in rice plant C water = Concentration of HMs in water The tolerance index of plants was measured by using the protocol of Wu et al. [35] with slight modification. The following formula was used to estimate the tolerance index (%) with slight modification. TI (%) = (Dry weight of treated plants/Dry weight of control plants × 100) (4)

Statistical Analysis
The data were subjected to one-way ANOVA by using statistix 9 software (v.10, Informer Technologies, Inc., Los Angeles, CA, USA). Means were separated by following the least significance difference (LSD) at p ≤ 0.05.

ZnO NPs Interaction with Bacteria Improved the Removal Efficiency of HMs
In order to check the synergistic effect of ZnO NPs along with bacterial strains (B. cereus and L. macroides) in remediating HMs, we first wanted to determine the potential effect(s) of ZnO NPs on bacteria at different pH (4, 5, 6, 7, 8, 9 and 10)       x FOR PEER REVIEW 8 of 18

ZnO NPs Interaction with Bacteria Improved the Removal Efficiency of HMs from Polluted Water
We had observed that ZnO NPs at lower concentrations, along with bacteria, efficiently remediate HMs from the media amended with heavy metals at neutral pH (90 min). We further determined the effect of ZnO NPs at lower concentrations along with bacteria on polluted water. For this purpose, both bacterial strains (B. cereus and L. ma-

ZnO NPs Interaction with Bacteria Improved the Removal Efficiency of HMs from Polluted Water
We had observed that ZnO NPs at lower concentrations, along with bacteria, efficiently remediate HMs from the media amended with heavy metals at neutral pH (90 min). We further determined the effect of ZnO NPs at lower concentrations along with bacteria on polluted water. For this purpose, both bacterial strains (B. cereus and L. macroides) along with NPs were applied on HM contaminated water. Results revealed that maximum HMs  (Table 1).

Total Heavy Metal Uptake, and Remediation Percentage in Polluted Water
Plants raised from bacterial primed seeds grown at the lower concentration of ZnO NPs revealed a lower uptake of HM content as compared with control treatments ( Table 2).  Table 2). Table 2. Synergistic effects of the bacterial strains (Bacillus cereus and Lysinibacillus macroides) and ZnO NPs (5 and 10 mg/L) on heavy metal uptake (mg/L) and reduction (%) of rice seedling grown in HM polluted water.  Water analysis revealed that bacteria-NP combined treatments significantly decreased the HM content in polluted water as compared with individual treatments (Table 2) Table 2).

Concentrationof Low Molecular Weight Polypeptide Metallothioneins (MTs)
It is evident from the earlier section that ZnO NPs and bacteria combined treatments, at lower concentrations, improved plant growth under HM stress. In order to prove their role in proteomic level, we determined metallothioneins (MTs) content, as mentioned in materials and methods. It is clear from Figure 6  with plants raised from primed seeds with B. cereus and L. macroides (0.1150 and 0.1350 µ mol) ( Figure 6A). MT concentration was also tested by using SDS PAGE. The synergistic treatment of B.cereus and L.macroides along with ZnO NPs at 5 mg/L in the presence of HMs showed a smaller number of bands at 7 kDa as compared with B.cereus and L.macroides primed seeds without ZnO NPs treatments and individually grown plants at 5 mg/L ZnO NPs treatments, respectively ( Figure 6B).  showing statistical significance at 5% probability level (ANOVA). Different alphabets appeared in superscript on each number showed statistically significant at 5% probability level.

Discussion
Pakistan has an abundance of surface and groundwater resources, but due to industrial discharge, these resources are polluted with HMs, used by farmers for irrigation [36]. HMs also affect plant growth by inhibiting the cellular process, causing low pigmentation and crop production [37]. The individual effect of NPs and bacteria was observed for the remediation of HM contaminated water, but no work so far has been reported on the combined effect of Bacillus spp. and ZnO NPs on the remediation of HMs in rice plants. ZnO NPs have received great attention worldwide and caused detoxification and transformation of metals more efficiently in plants [38]. They remain suspended in water without electrostatic forces, so they provide significant tolerance against HMs [2].
It was observed from the current findings that the removal efficiency of combined treatment of bacteria and ZnO NPs (5 mg/L) was maximum at neutral pH (Figures 1 and  2).This is in agreement with previous observations, where it has been shown that neutral pH condition made the surface of ZnO NPs more negative; thus electrostatic interactions between NPs and metal cations increased, which caused higher removal efficiency, while at low pH, HMs precipitate in the form of hydroxides where hydrogen ions compete for binding with adsorbents. These results were confirmed by the findings of Xie [39] that the hydroxide ions on the functional group of NPs react with hydrous oxide at higher pH to produce deprotonated oxide (MO − ), while at lower pH, the hydrous surface will be completely covered with hydrogen ions [14]. The synergistic effect is more helpful in remediating the Pb and Cu metals from polluted water. It was reported by Karn [38] that ZnO nanoparticles showed 85% removal efficiency of Pb and Cu metal ions in the form of metal reduction/oxidation or adsorption mechanism. The removal efficiency of HMs was also observed at different time intervals (0, 30, 60, 90 and 120 min). Maximum removal Waste water, B.c + w.w (B. cereus + waste water)., L.m + w.w (L. macroides + waste water)., 5 ZnO NPs + w.w (5 mg/L ZnO NPs + waste water), 10 ZnO NPs + w.w (10 mg/L ZnO NPs + waste water), B. c. + 5 + w.w (B. cereus + 5 mg/L ZnO NPs + waste water)., L.m + 5 + w.w (L. macroides + 5 mg/L ZnONPs + waste water), B.c. + 10 + w.w (B. cereus + 10 mg/L ZnO NPs + waste water), L.m. + 10 + w.w (L. macroides + 10 mg/L ZnO NPs + waste water).

Discussion
Pakistan has an abundance of surface and groundwater resources, but due to industrial discharge, these resources are polluted with HMs, used by farmers for irrigation [36]. HMs also affect plant growth by inhibiting the cellular process, causing low pigmentation and crop production [37]. The individual effect of NPs and bacteria was observed for the remediation of HM contaminated water, but no work so far has been reported on the combined effect of Bacillus spp. and ZnO NPs on the remediation of HMs in rice plants. ZnO NPs have received great attention worldwide and caused detoxification and transformation of metals more efficiently in plants [38]. They remain suspended in water without electrostatic forces, so they provide significant tolerance against HMs [2].
It was observed from the current findings that the removal efficiency of combined treatment of bacteria and ZnO NPs (5 mg/L) was maximum at neutral pH (Figures 1 and  2). This is in agreement with previous observations, where it has been shown that neutral pH condition made the surface of ZnO NPs more negative; thus electrostatic interactions between NPs and metal cations increased, which caused higher removal efficiency, while at low pH, HMs precipitate in the form of hydroxides where hydrogen ions compete for binding with adsorbents. These results were confirmed by the findings of Xie [39] that the hydroxide ions on the functional group of NPs react with hydrous oxide at higher pH to produce deprotonated oxide (MO − ), while at lower pH, the hydrous surface will be completely covered with hydrogen ions [14]. The synergistic effect is more helpful in remediating the Pb and Cu metals from polluted water. It was reported by Karn [38] that ZnO nanoparticles showed 85% removal efficiency of Pb and Cu metal ions in the form of metal reduction/oxidation or adsorption mechanism. The removal efficiency of HMs was also observed at different time intervals (0, 30, 60, 90 and 120 min). Maximum removal efficiency of HMs was observed after a 90 min time interval (Figures 3 and 4). It can be observed from the current findings that bacteria inoculation reduced the passage of metals in water, which results in low reactive oxygen species (ROS) in less time. These results were confirmed by the findings of Wang [40] that after 90 min maximum binding sites on NPs' surface were exposed, which effectively bind with metals [41].
The batch culture experiment showed that lower concentrations (5 and 10 mg/L) of ZnO NPs improved the tolerance capacity of bacteria against heavy metals as compared with higher concentrations (Table 1). ZnO NPs at lower concentration (5 and 10 mg/L) help bacteria in remediating HMs in polluted water. Bacillus spp. are actively involved in the biotransformation of metals and use Zn +2 ions in the range of (0.01-1 mM) as micronutrients for their growth, stabilizing the membrane, macromolecules, different steroid receptors and carbohydrate metabolism under stress conditions. These findings revealed that zinc ions are beneficial for Zn regulatory proteins in bacteria, so increasing the tolerance capacity of bacteria against HMs [42]. It was reported by Ashraf [43] that the bacteria strain Klebsiella variicola isolated from industrial effluents has high bio-sorption ability and maximum tolerance against HMs. A recent report has been published by Wątły [44], in which it was observed that the application of titanium oxide NPs and ZnO NPs provided more active sites for HMs and increased remediation of metals.
In addition, it was observed that lower concentrations of ZnO NPs reduced the passage of HMs into different tissues of rice plants ( Figure 5) in the presence of bacteria. Bio-priming of seeds with Bacillus species has been investigated to increase the nutritional pathway of rice under HM stress conditions [45]. Bacteria secreting plant growth-promoting metabolites extract nutrients from water by fixing nitrogen [46]. Bacillus spp. is the most notable genus used in abiotic stress tolerance in potato radish, rice, mung bean and chickpea [47]. The recent Amiard report [48] revealed that a significant reduction of HMs occurred in plants in two ways; the higher secretion of root exudates leads to higher adsorption of HMs on ZnO NPs; surface so reducing the bioavailability of metals. Low pH (5.4-5.8) is associated with retaining most ions at a free-standing position, while higher pH lowers the nutrient bioavailability. It was reported by Sharifan [49] that ZnO NPs act as bio-sorbent for the remediation of cobalt from water, used for the irrigation of crops.
Metallothionein (MTs) content in plants under HM stress is a well-known phenomenon [50]. Current research demonstrated that plants grown under HM stress had high MTs contents as compared with control plants ( Figure 6). Our results showed resemblance to the findings of Khati et al. [51], who revealed that MTs content increased in plants under cadmium (Cd) stress. B. cereus and L. macroides primed seeds with ZnO NPs reduced HMs stress and lowered MT content. HM-resistant bacteria strains prevent the plant from a toxic effect and restrict their inflow in the plant. Earlier studies reported maximum MT contents in lupinus luteus L. plant under HMs stress [52].
Current findings also revealed that the number of HMs was reduced in water by bacteria-nanoparticles interaction ( Table 2). It was also observed in the current study that bioaccumulation index HMs (Pb, Cd, Cr, and Cu) in the plant decreased and tolerance index increased in plants raised from bacteria primed seeds after application of NPs, as compared with HM polluted water (Figure 7). Metal reduction may be due to the ability of heavy metal-resistant bacteria to immobilize the HMs in the root by different mechanisms [52]. According to Abraham [46], seed inoculation with Cr resistance strain P. aeruginosa reduces the amount of chromium and increases plant growth. It was reported by Wu [35] that seed priming with bacteria increased the germination and tolerance capacity of crops by adhering to the seeds and decreased the passage of HMs to plants. It was also reported that ZnO NPs have a high surface to volume ratio in reacting with metals in contaminated media and also triggers the movement of metals in biochemical pathways in plants [44]. It was investigated by Dubchak [53] that metal oxide NPs such as Fe 3 O 4 , ZnO and CuO remove the metals from aqueous solution. These results were further confirmed by Kummar et al. [29], who observed that metal oxide NPs such as Fe 3 O 4 , ZnO and CuO removed the metals from aqueous solution and increased the germination of Mung (Vignaradiata) and gram (Cicer aretinium) plants.

Conclusions
It was concluded from the current study that ZnO NPs (at lower concentrations), along with bacteria, more efficiently bind with HMs ions and remediate metals from polluted water, as compared to their individual effect. The combined effect also showed low bioaccumulation index, metallothionine (MTs) content and inhibited the passage of HMs to plant tissues by improving the plant tolerance index. The combined treatments of ZnO NPs and bacteria play a significant role in plant tolerance level and the removal of HMs from water.