Date of Award

2013-01-01

Degree Name

Doctor of Philosophy

Department

Environmental Science and Engineering

Advisor(s)

Jorge L. Gardea-Torresdey

Abstract

The production of engineered nanoparticles (ENPs) has rapidly increased due to their wide range of applications in the field of electronics, medicine, chemistry and biology. Consequently, concerns have risen about the environmental release and potential negative impact of NPs. A few reports have described the effects of TiO2 NPs and quantum dots upon nitrogen fixing bacteria. However, the understanding of the interaction of NPs in plant-microbe interface (symbiotic association), like alfalfa (Medicago sativa L.)-Sinorhizobium meliloti, is still in its infancy. Alfalfa is the world's most important forage crop. It grows in association with S. meliloti, which is very important in terms of nitrogen fixation and, hence, global nitrogen cycling. This research project was aimed at understanding the effect of two ENPs (CeO2 and ZnO) on S. meliloti and alfalfa, separately, and on their symbiosis. This investigation was completed in three phases. Initially, the associated bacterium was treated, separately, with CeO2 and ZnO NPs. Ten nm CeO2 and ZnO NPs were exposed towards S. meliloti at 10, 31, 62.5,125, and 250 mg/l in liquid Yeast Mannitol Broth (YMB). Toxicological parameters evaluated included UV/Vis measurement of minimum inhibitory concentration, disk diffusion tests, and dynamic growth. Advanced scanning transmission electron microscope (STEM) and infrared spectroscopy (FTIR) were utilized to determine the spatial distribution of NPs and macromolecule changes in bacterial cells, respectively. Results indicate that ZnO NPs were more toxic than CeO2 NPs in terms of inhibition of dynamic growth and viable cells counts. STEM images revealed that CeO2 and ZnO NPs were found on bacterial cell surfaces and ZnO NPs were internalized into the periplasmic space of the cells. FTIR spectra showed changes in protein and polysaccharide structures of extra cellular polymeric substances present in bacterial cell walls treated with both NPs. The growth data showed a bacteriostatic effect of CeO2 NPs, whereas ZnO NPs was bactericidal to S. meliloti. Overall, ZnO NPs were found to be more toxic than CeO2 NPs to S. meliloti. In phase II, the alfalfa plant in association with S. meliloti, were cultivated for 30 d in soil treated with ZnO NPs, ionic (ZnCl2) and bulk ZnO, concentrations ranging from 0 (control)-750 mg kg-1. Plant growth, Zn bioaccumulation, dry biomass, leaf area, total protein, and catalase (CAT) activity were measured. Results showed 50% germination reduction by bulk ZnO at 500 and 750 mg/kg and all ZnCl2 concentrations. ZnO NPs and ionic Zn reduced root and shoot biomass by 80% and 25%, respectively. Conversely, bulk ZnO at 750 mg/kg increased shoot and root biomass by 225% and 10%, respectively, compared to control. At 500 and 750 mg/kg, ZnCl2 reduced CAT activity in stems and leaves. Total leaf protein significantly decreased as external ZnCl2 concentrations increased. STEM analysis revealed the presence of ZnO particles in tissues, suggesting the uptake of NPs. However, ZnO NPs showed less toxicity compared to ZnCl2 on measured traits. Phase III was performed in soil treated with either ZnO or CeO2 NPs at 0, 250, 500, and 750mg/kg for 30 days to study the toxicity effect of ZnO and CeO2 towards alfalfa's secondary metabolites and antioxidative properties. The toxicity was evaluated for leaf using chlorophyll (a & b), carotenoids, phenolic, and flavonoid contents. Results showed that, compared to control, leaf chlorophyll a content reduced to 60% and 40% at 750 mg/kg of CeO2 and ZnO NP treatments, respectively. Chlorophyll b reduced by 64%, 48%, and 60% at 250, 500 and 750 mg/kg CeO2 NP treatments, while chlorophyll b remained the same except 40% reduction at 750 mg/kg bulk ZnO treatment. CeO2 NPs enhanced the root flavonoids content by 34% at 750 mg/kg treatment and 86% in shoot at 500 mg/kg treatment, compared to control. Total root flavonoids decreased by 49% in plants treated with 750 mg/kg of ZnO NP treatment, whereas total flavonoids in shoots remained similar to control in all treatments, except 250 mg/kg ZnO NP treatment, where the flavonoid content decreased to 77%. This is the first complete study in a symbiotic system in terms of nano, bulk and ionic Zn species comparison in soil matrix. In addition, to the best of the authors' knowledge, this is the first report on the effects ZnO and CeO2 on alfalfa's secondary metabolites and chlorophyll content. Our results will help to reveal the toxicity of CeO2 and ZnO NPs on alfalfa and S. meliloti species, as well as to understand the eco-toxicity of NPs in plant-microbe symbiosis.

Language

en

Provenance

Received from ProQuest

File Size

118 pages

File Format

application/pdf

Rights Holder

Susmita Bandyopadhyay

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