Recipient Organization
ARKANSAS STATE UNIVERSITY
(N/A)
STATE UNIVERSITY,AR 72467
Performing Department
Agriculture
Non Technical Summary
In the USA and worldwide, manufacturing of goods that are utilized in all aspects of life can have major negative impacts on the environment. The chemicals released from these industries as well as agricultural chemicals and chemical weapon residuals have negative effects on human, animal and plant health. Methods are needed that allow clean-up of these waste streams in an environmentally friendly manner such that the products of clean-up restore utility of the soil and water resources that were polluted. Some of the chemicals of note are atrazine, a common herbicide for weed control in the corn belt, aniline dyes used for paper and fabric coloration, and complex POPs (persistant organic pollutants) from many sources. These pollutants are found in both water and soil milieus. Thus, a clean-up strategy should be developed that can address the degradation of the chemicals in either environment.This project utilizes a new technology that is biologically based so that the outcomes can be good for the environment. We will use enzymes--protein molecules that catalyze breakdown of specific types of molecules, to break down these pollutants. Our enzyme of choice is called manganese peroxidase--a powerful enzyme that can react with many types of molecules. Because of its versatility, it can be potentially used on a variety of chemicals, even mixed groups of pollutants. What we don't know is how effective it will be in water and soil from polluted sites and how to deliver the necessary added reagents to make the enzyme do its work.The enzyme to be used in the project is produced in corn kernels. This is a very convenient source of the enzyme, as we can just grind the corn into flour and deliver the enzyme directly to the soil sample. Additionally, for treatment of water samples, we may be able to create a filter from ground corn through which the polluted water can be channeled, cleaning it up in the process of passing through the filter. This would be similar to using a filter for your tap water at home, except rather than just removing the pollutants, it changes their nasty structures into environmentally friendly structures.If we are successful, we will have a way to address pollution in our drinking water that will allow it to be safer. Ultimately it is the water that is most affected, as it dissolves and collects the pollutants from soil, so if they are removed from soil, the water running through the soil will be cleaner. The Environmental Protection Agency should be quite interested in this work and we intend to work with the agency to implement application of our research.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Goals / Objectives
The overall dual goal of this project is to strengthen A-State's College of Agriculture and Technology (CoAT) in research in the area of biobased products and to enhance minority student participation in STEM disciplines and careers. The project addresses the following specific questions: 1) Does the plant-produced enzyme degrade or precipitate an aniline dye, atrazine, or a phenol derivative? 2) What type of formulation of the enzyme is most effective? 3) Does ground corn germ deliver enzyme effectively? 4) Does germ trap the products of the reaction? 5) What are the products of the enzyme reactions in the corn formulations? 6) Can corn-derived MnP solve the problem of bioremediation delivery cost effectively?Atrazine is the most common herbicide for agriculture and home use, and can be used to treat weeds in corn, sorghum, sugar cane and lawns. It has been reported that 34.5 million pounds of atrazine are applied to agricultural soils in the US each year. The Natural Resources Defense Council (NRDC) puts that number at 60-80 million pounds. Run-off from these uses allows it to be present in groundwater and soils above the recommended EPA levels of 3 ppb in drinking water. The bioavailability affects bioaccumulation to toxic levels. A remediation method for this chemical in US soil and water is warranted.Dyes used for paper printing, color photography, textile dyeing and as an additive in petroleum products have a synthetic origin and complex aromatic molecular structure; about 15% of those made are discharged in industrial effluents. Current treatment methods are physicochemical and expensive. Some evidence as well as our own data indicate that these dyes may be decolorized and detoxified using peroxidases. What is not known is the corn-derived enzymes' ability to work in a soil/waste water milieu.Aromatic compounds, which include phenols and aromatic amines, anilines, hydroxyquinoline, benzidines, and naphthylamines are contaminants widely found in industry waste waters of coal conversion, oil refining, resin and plastic manufacturing, textiles, and mining, in addition to dye manufacturing; textile and leather treatment; chemical and pharmaceutical industries, foundries, and pulp and paper plants. The POPs comprise some of the most toxic of all environmental hazards (USEPA). These carbon compounds are often chlorinated, have low solubility in water, higher solubility in lipid and as such tend to bioaccumulate. POPs were used extensively for industrial production post World War II when thousands of synthetic chemicals came into common use. Although their properties were useful for many applications, they had the unforeseen consequence of being harmful to humans, animals and the environment. POPs include intentionally produced chemicals used in agriculture and manufacturing as well as unintentionally produced chemicals such as dioxins, that come from incineration of industrial and medical waste. Many remediation strategies have been tested with variable results that often leave additional chemical residues.1. Research Objective: To determine the efficacy of using recombinant manganese peroxidase (MnP) from corn seed for the degradation of environmental pollutants in water and soil.2. Education Objective: To reach our goal of improving the representation of minorities and women in STEM disciplines, we will recruit and train a minority graduate student and two minority undergraduate students for this project, mentoring them into STEM careers.
Project Methods
Objective 1: To determine the efficacy of using recombinant MnP from corn seed for degradation of environmental pollutants in water and soil.Approach: Our research objective will be accomplished through four specific tasks.Specific task 1: Generate three formulations of recombinant manganese peroxidase (MnP) from corn seed. Rationale: Purified enzyme is the ideal formulation to determine the actual reaction and products of incubation with substrate. However, the formulation that will be the product that is a cost-effective application will not be purified enzyme. We anticipate that the enzyme will either be in a concentrated extract or a formulation of defatted germ fraction of the corn kernels that is much more economical to produce and a much more sensible inoculant into a bioremediation milieu.Outcome: Three formulations of MnP for use in Tasks 2-4: a) purified enzyme; b) concentrated extract; c) defatted germ.Specific task 2: Test formulations on three classes of environmental pollutants. Rationale: The literature suggests that MnP will be effective to degrade or polymerize (for precipitation) a number of pollutants that are found in the environment. Most of these experiments have been done with a whole fungus, or with a purified enzyme from the fungus. However, the particular isozyme that is over expressed in corn seed has not been tested. Moreover, recombinant MnP in a corn matrix will have different impurities that may influence the reaction in contrast to enzymes purified or formulated from fungal cultures. It is therefore critical that we test the maize recombinant enzyme on the various chemicals. In addition, for broadest utility, we will test three categories of environmental pollutants--azo dye, atrazine, and phenol. We will continue work with the azo dye, Acid Blue 25, to determine its product and to determine its fate using different formulations of the enzyme. Atrazine is the most commonly used chemical on corn for weed control in the Midwest corn-belt and is often found in drinking water. Phenol is a model compound for the highly toxic derivatives containing chlorine, such as 2,4-Dichlorophenol (DCP).Outcomes: We will understand the amount of enzyme in its different formulations that is effective on the pollutant in aqueous solution. The products of the reactions will be analyzed and identified to molecular group or specific molecule.Specific task 3: Test atrazine degradation in soil. Rationale: Many pollutants are found in solution in waterways, often washed out of contaminated soils. Because of the complexity of extracting and analyzing soil samples, this task will focus only on atrazine. Atrazine is the most common contaminant in Mid-western soils in the corn-belt and is often found in drinking water. The chemical binds to soil particles and is released only when pH and moisture content change (Arias-Estévez et al., 2008). Thus, we will test the efficacy of MnP in degrading atrazine in soil and releasing products of lesser or no toxicity as a means to prevent water contamination.Outcomes: We will understand the conditions necessary for MnP in various formulations to remove atrazine from soils with limited water content.Specific task 4: Analyze products of the reactions. In collaboration with Dr. Stephen Grace, University of Arkansas Little Rock and Dr. Fabricio Medina-Bolivar, A-State, we will analyze the products of the reactions using high-performance thin layer chromatography (HPTLC), Gas Chromatography followed by Mass Spectrometry (GCMS) and/or High-Performance Liquid Chromatography (HPLC-PDA) or Liquid Chromatography Mass Spectrometry (LCMS). Rationale: Analysis of the products of the enzyme reactions is key to understanding the efficacy of using MnP for remediation.Outcomes: Identification of the compounds that result from the enzyme reactions with these pollutants in aqueous and soil based media.Objective 2: Recruit and train a minority graduate and two undergraduate students for this project to increase underrepresented minorities in STEM research.Specific task 1: Recruit a graduate student through the various networks that have been set up in Arkansas (see paragraph above) for minority students and train the student in bench science, as well as mentor him/her in a STEM career or continued graduate training.Specific task 2: Recruit minority undergraduates through this same network or the Honors College at A-State for hands-on experience in a research laboratory, mentoring him/her into a graduate program or STEM career.Evaluation plan: The evaluation of the current project will focus on its two primary objectives: (1) to determine the efficacy of using recombinant MnPOX from corn seed for degradation of environmental pollutants in water and soil, and (2) recruit minority graduate and undergraduate students for this project to improve underrepresented minorities in science. The evaluation will be both formative (focusing on the completion of activities and feedback to guide the efforts) and summative (evaluating the products and successes in achieving the project's goals). The methods to be used in the evaluation will largely fall into qualitative assessments of progress by interviews and quantitative counts of products (e.g., generation of formulations, development of papers and publications), along with examination of discrepancies in timing of milestones. Specific evaluation tasks for each of the program objectives are outlined below.Research Evaluation: The formative evaluation for the Research component of the proposed project will follow the timeline for this proposal to determine whether the project is on track. The timeline will be monitored on a monthly basis using interviews and will focus on the specific tasks of the project:Generation of three formulations of recombinant manganese peroxidase (MnP) from corn seed.Testing of formulations on three classes of environmental pollutants in waterTesting of atrazine in soil.The summative evaluation for this component will be primarily at the end of the grant period and will focus on counts of the products and results of the project:Submission of publications on the use of corn-produced MnPox for treatment of environmental pollutants.The number of tools available for treatment of environmental pollutants, including cost-effective solutions to treating soil and water pollution.Extent to which the project staff have engaged with the EPA and other remediation organizations to offer the technology resulting from the project's experiments.Education Evaluation: The formative evaluation for the Education component of this project will examine the timing of the accomplishments listed in the timetable, monitored on a quarterly basis. Specifically, we will monitor the following:Advertisements for minority graduate studentsAdvertisements for minority undergraduate studentsTraining (type and frequency) provided to undergraduate student involved in the projectThe summative evaluation for this component will focus on the successes of the educational program (using both quantitative and qualitative data):Academic progress of the minority students involved.Extent to which an undergraduate minority student is trained in laboratory science.Undergraduate student application to graduate school.Number of student publications.Reported skills that will allow them to work in the scientific community.Plans and expectations of minority students to become role models for other students.