Source: UNIVERSITY OF TEXAS AT SAN ANTONIO submitted to NRP
INTERACTION OF METAL OXIDE NANOPARTICLES WITH NITRIFYING BACTERIA IN SOIL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030972
Grant No.
2023-67018-40682
Cumulative Award Amt.
$544,943.00
Proposal No.
2022-08577
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
Aug 31, 2026
Grant Year
2023
Program Code
[A1511]- Agriculture Systems and Technology: Nanotechnology for Agricultural and Food Systems
Recipient Organization
UNIVERSITY OF TEXAS AT SAN ANTONIO
1 UTSA CIRCLE
SAN ANTONIO,TX 78249
Performing Department
(N/A)
Non Technical Summary
The use of metal oxide nanoparticles (MeO NPs) in a wide range of commercial products has grown exponentially in recent years. Consequently, these NPs are indirectly discharged in agricultural soils through irrigation or sewage-sludge application and directly as nanofertilizers or nanopesticides. The introduction of MeO NPs into agricultural soils can be toxic to microorganisms carrying out important biogeochemical processes within the soil ecosystem, including nitrification. Our current understanding of the inhibitory effects of MeO NPs and the mechanism of toxicity towards nitrifying bacteria is limited. To address these research gaps and to better understand the impact of MeO NPs on soil nitrification processes, we will investigate the effects of ZnO NPs and CuO NPs, along with bulk ZnO and CuO, on the physiological, transcriptional and metabolic responses of nitrifying bacteria in agricultural soils. Subsequently, we will develop a path analysis to integrate the different variables measured in our study to assess the multifaceted consequences of MeO NPs on nitrifying communities. We will study the interactions of nitrifying bacteria, in lab-scale soil microcosms, with MeO NPs for environmentally relevant concentrations and transformations of these nanoparticles in soils. We will also compare how the nature (bulk versus particle) of MeO affects the nitrifying communities. The coupling of advanced molecular biology and conventional biochemical tools to develop causal models represents a potentially transformative step forward in evaluating the effects of nanoparticles on microbial processes in soils.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110104050%
1020110202030%
1020110200020%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
0110 - Soil;

Field Of Science
1040 - Molecular biology; 2020 - Engineering; 2000 - Chemistry;
Goals / Objectives
The use of metal oxide nanoparticles (MeO NPs) in a wide range of commercial products has grown exponentially in recent years.The introduction of MeO NPs into agricultural soils can be toxic to microorganisms carrying out important biogeochemical processes within the soil ecosystem. Among these processes, nitrogen (N) cycle is an essential and critical process, in which nitrification and denitrification processes control soil inorganic N availability and subsequent soil fertility. The strong coupling between nitrification and denitrification makes the N cycle an ideal model to study the impacts of environmental disturbances on microbial functioning.we will investigate the effects of ZnO NPs and CuO NPs, along with bulk ZnO and CuO, on the physiological, transcriptional and metabolic responses of nitrifying bacteria in agricultural soils. Subsequently, we will develop a path analysis to integrate the different variables measured in our study to assess the multifaceted consequences of MeO NPs on nitrifying microorganisms in soil ecosystems. We will study the interactions of nitrifying bacteria, in lab-scale soil microcosms, with MeO NPs for environmentally relevant concentrations and transformations of these nanoparticles in soils. We will also compare how the nature (bulk versus particle) of MeO affects the nitrifying communities typically present in soil systems. Integrating the use of molecular biology tools with conventional biochemical methods to describe the causal model of MeO NPs interaction with nitrifying communities in soil is the overall goal of this research plan. There are three specific objectives of this project: (1)Determine the impact of MeO NPs (ZnO NPs and CuO NPs) and bulk material (ZnO and CuO) on the functional gene expression and microbial diversity of soil bacterial communities and evaluate how MeO NPs inhibit nitrification at the molecular level, (2)Determine the effects of MeO NPs and bulk-MeO on the physiological and metabolic responses of nitrifying bacteria in soil, which in turn impacts the N cycle, and (3)Integrate the results of molecular biology tools with biochemical and analytical methods to elucidate causal links between MeO NP concentration and nitrification in soil.
Project Methods
1) Determine the impact of MeO NPs (ZnO NPs and CuO NPs) and bulk material (ZnO and CuO) on the functional gene expression and microbial diversity of soil bacterial communities and evaluate how MeO NPs inhibit nitrification at the molecular level. We will employ reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assays to measure the fold changes in transcript levels of key functional genes involved in N transformations in soil nitrifying communities exposed to different concentrations of MeO NPs and bulk-MeO. For RT-qPCR assays, we will use a novel microfluidics PCR - droplet digital PCR (ddPCR) for gene expression measurements to overcome PCR inhibition associated with complex soil samples. This approach will be able to identify and quantify the effect of nanoparticles at a molecular level. Furthermore, we will identify supplementary functional genes, in addition to those involved in nitrification, which could act as potentially even more sensitive indicators of nitrification inhibition or alternately specific indicators of inhibition by MeO NPs. We will also evaluate the changes in microbial diversity of soil communities exposed to MeO NPs using 16S rRNA sequencing.2) Determine the effects of MeO NPs and bulk-MeO on the physiological and metabolic responses of nitrifying bacteria in soil, which in turn impacts the N cycle. Standard physiological assays such as soil respiration and enzymatic activity will be used to confirm the results of gene expression. Additionally, cellular internalization and/or attachment of MeO NPs to cell surface may lead to alterations in the cells' morphology. Therefore, cell morphology, membrane integrity and the production of intracellular Reactive Oxygen Species (ROS) will also be examined in response to MeO NP induced-cytotoxicity. Toxicological assessments will be accompanied with dissolution and aging measurements of the relevant NPs.3) Integrate the results of molecular biology tools with biochemical and analytical methods to elucidate causal links between MeO NP concentration and nitrification in soil. We will develop causal models using path analysis to describe the effects of MeO NPs on soil nitrifying bacteria. We will determine the link between the expression of known functional genes such as amoA and the kinetics of nitrification during the imposition of MeO NP mediated inhibition. We will compare the results of nitrification inhibition by ZnO NPs and CuO NPs to that of bulk material to understand how the nature of metal affects the same process.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The audience reached during this reporting period included students (graduate and undergraduate students), postdocs, peer researchers, scientists, and professionals from environmental and chemical engineering. The research outcomes of this project were shared at multiple conferences including theNanoscale Science and Engineering for Agriculture and Food Systems Gordon Research Conference (GRC),McNair Baylor Undergraduate Conference and UTSA Undergraduate Research Summer Showcase. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided an opportunity for the professional development and training of one postdoctoral scholar, one graduate student and two undergraduate students recruited during the past year. The postdoctoral scholar was trained in the synthesis and characterization of nanoparticles. The graduate student received hands-on-training in various aspects of molecular biology research including DNA extraction and analysis, enzyme assays, and PCR/qPCR as well as academic abstract and technical report writing. The undergraduate students were mentored by the postdoc and graduate student and assisted in laboratory work. One undergraduate student working on the project was selected as McNair Scholar and presented their work at the McNair Baylor Undergraduate Conference and UTSA Undergraduate Research Summer Showcase. How have the results been disseminated to communities of interest?The results of this research were presented at the Nanoscale Science and Engineering for Agriculture and Food Systems Gordon Research Conference (GRC), Manchester, NH, U.S.A. The undergraduate student working on the project presented the results at various undergraduate conferences including the McNair Baylor Undergraduate Conference and UTSA Undergraduate Research Summer Showcase. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, qPCRs will be conducted to examine functional gene abundance and expression. Sequencing will be performed to analyze the composition of soil microbial communities. The manuscript for the impact of ZnO NPs will be prepared and finalized.

Impacts
What was accomplished under these goals? Zinc oxide (ZnO) nanoparticles (NPs) were synthesized in-house via the hydrolysis of Zinc (II) acetylacetonate [Zn(acac)?] in 1,4-butanediol at 140°C. Briefly, 9.885 g (0.25 mol/L) of Zn(acac)? was dissolved in 150 mL of 1,4-butanediol. The solution was heated to 140°C while stirring with a magnetic stirrer at 800 rpm in a round-bottom (RB) flask equipped with a condenser for reflux. The reaction was allowed to proceed for 5 hours, with ZnO precipitation occurring after approximately 30 minutes. After cooling to room temperature, the reaction mixture was centrifuged at 10,000 rpm for 1 hour to collect the solid particles. The solid phase was washed first with ethanol and centrifuged again under the same conditions. The wash was then repeated with water, followed by another round of centrifugation. Finally, the washed solid particles were redispersed in water, sonicated for 5 minutes using a probe sonicator, and stored in a refrigerator at 4°C in a storage bottle. Similarly, citrate-coated ZnO NPs were synthesized at citrate/ZnO molar ratios of 0.25, 0.5, and 0.8 using 2.757 g, 5.514 g, and 8.823 g of sodium citrate dihydrate, respectively, during the reaction. The rest of the procedure remained unchanged. Microscopic images of the NPs were captured using a JEOL JEM-2010F High-Resolution Transmission Electron Microscope (Nanolab Technology, Milpitas, CA). Raman spectra of freeze-dried ZnO and citrate-coated ZnO NPs powder were obtained using a Horiba LabRam HR-Evolution (Piscataway, NJ, USA), equipped with a 600 grooves/mm grating, 532 nm laser excitation, CCD camera detection, and an MPlan N 100×/0.9 objective from Olympus (Waltham, MA, USA). Powder X-ray diffractograms (XRD) were collected using Malvern Panalytical Empyrean Nano Edition (Westborough, MA, USA). The zeta potential and mean hydrodynamic diameter of ZnO and citrate-coated ZnO NPs dispersions in water at various pH levels were measured using a Malvern Zetasizer Nano ZS (Westborough, MA, USA). Soil microcosms were setup using soil samples containing nitrifying bacteria. These microcosms were exposed to varying concentrations of ZnO NPs: low concentration (LC) at 10 mg/kg soil to represent potential environmental exposure levels; medium concentration (MC) at 100 mg/kg soil for moderate exposure under controlled conditions; and high concentration (HC) at 500 mg/kg soil, where stronger effects, including toxicity, were expected. The experimental design included a positive control, which consisted of untreated natural soil assumed to contain nitrifying bacteria, and a negative control using sterile, autoclaved soil to ensure no bacterial activity. The study included five treatment conditions (positive control, negative control, and low, medium, and high concentrations of ZnO nanoparticles), along with three additional citrate treatments at low, medium, and high concentrations. Each treatment was replicated three times, and samples were collected at five different time points (21, 42, 63, 84, and 105 days), yielding a total of 120 samples for analysis. The experiments ran for over 100 days, with samples taken at regular intervals of 21 days for subsequent analysis. We conducted a series of assays to assess the impact of NPs on nitrifying bacteria. Protein assays were performed to quantify protein content as an indicator of cell viability. Enzyme assays focused on measuring the activities of key enzymes, including AMO, NXR, NIR, and NAR, which play important roles in the nitrification process. Additionally, nucleic acid extractions were performed to analyze microbial community composition and gene expression.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Hussain, A., Hassan, M. J., Rivera, M., Moreno, M., Ure�a-Benavides, E. E., & Kapoor, V. (2024) Interaction of Metal Oxide Nanoparticles with Nitrifying Bacteria in Soil. Poster presentation at the Nanoscale Science and Engineering for Agriculture and Food Systems Gordon Research Conference (GRC), Manchester, NH, U.S.A.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rivera, M., Hussain, A.& Kapoor, V. (2024) Interaction of Metal Oxide Nanoparticles with Nitrifying Bacteria in Soil. Poster presentation at the USDA Eco JEDI Mini Research Symposium, San Antonio, TX, U.S.A.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rivera, M., Hussain, A.& Kapoor, V. (2024) Interaction of Metal Oxide Nanoparticles with Nitrifying Bacteria in Soil. Poster presentation at the McNair Baylor Undergraduate Conference, Waco, TX, U.S.A.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rivera, M., Hussain, A.& Kapoor, V. (2024) Interaction of Metal Oxide Nanoparticles with Nitrifying Bacteria in Soil. Poster presentation at the UTSA Undergraduate Research Summer Showcase, San Antonio, TX, U.S.A.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rivera, M., Hussain, A.& Kapoor, V. (2024) Interaction of Metal Oxide Nanoparticles with Nitrifying Bacteria in Soil. Poster presentation at the 4. Lone Star Sustainability Forum, San Antonio, TX, U.S.A.