Source: ARKANSAS STATE UNIVERSITY submitted to NRP
PLANT-PRODUCED MANGANESE PEROXIDASE AS A BIOREMEDIATION AGENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1010673
Grant No.
2017-70001-25987
Cumulative Award Amt.
$149,016.00
Proposal No.
2016-06448
Multistate No.
(N/A)
Project Start Date
Dec 15, 2016
Project End Date
Dec 14, 2019
Grant Year
2017
Program Code
[NLGCA]- Capacity Building Grants for Non Land Grant Colleges of Agriculture
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%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1330110100020%
1330210100060%
9030210100020%
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.

Progress 12/15/18 to 12/14/19

Outputs
Target Audience:The industrial community has numerous issues in the area of mitigation of pollutants that come from their manufacturing processes. In the textiles, printing and leather industries, these pollutants mainly comprise synthetic dyes. This project was designed to find ways to mitigate those dyes through application of enzymes produced recombinantly in corn seed. This low-cost, high volume production system can provide a mechanism of remediation that will be accessible to producers from both a cost-effective and volumetric stand point. Thus, one target audience of this project is the industrial community of users of synthetic dyes to produce colored products. Additional audiences would include the makers and users of persistent organic pollutants, such as 2,4-dichlorophenol, and herbicides, such as atrazine, that are persistent in the environment at sources of manufacture and use. Changes/Problems:I decided not to do experiments on 2,4-dichlorophenol because of its toxicity. We may do something with this in the future. Instead, we expanded the number of dyes and the number of enzymes for treatment. What opportunities for training and professional development has the project provided?For the education objective, I recruited an African American graduate student from an HBCU for this project. He successfully completed his master's degree. We also trained two female undergraduates using these funds in laboratory experimental procedures. In addition, the post doc on the project was an Hispanic female. I supported the graduate student and the post doc to attend professional meetings and present their work. Each has found employment in the scientific field How have the results been disseminated to communities of interest?Joshua's thesis is currently in process at ProQuest. The post doc and I are collaborating with the student to put together at least 2 publications from this work. They are in preparation. What do you plan to do during the next reporting period to accomplish the goals?The project is done and this is the final report.

Impacts
What was accomplished under these goals? For the research objective, my graduate student showed that MnP and laccase were able to degrade 5 different synthetic dyes that are used in the textile and printing industries. Each of the dyes had a different reaction profile to the enzymes. However, the most effective method for degrading the dyes was using MnP first followed by a laccase reaction. One of the dyes, methyl orange (MO), was also used in toxicity assays before and after degradation. Unfortunately, MO was not very toxic prior to degradation, and thus was not reduced in toxicity after enzyme treatment. HPLC was performed to identify the products of the degradation. The enzymes were also applied to atrazine alone and in combination. They had no degradation effects on atrazine. For the education objective, I recruited an African American graduate student from an HBCU for this project. He successfully completed his master's degree. We also trained two female undergraduates using these funds in laboratory experimental procedures. In addition, the post doc on the project was an Hispanic female.

Publications

  • Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2020 Citation: Byrd, Joshua. 2020. PLANT-PRODUCED OXIDATION/REDUCTION ENZYMES AS BIOREMEDIATION AGENTS. A thesis presented in partial fulfillment of the requirements for the Master of Science in Agriculture.


Progress 12/15/16 to 12/14/19

Outputs
Target Audience:The industrial community has numerous issues in the area of mitigation of pollutants that come from their manufacturing processes. In the textiles, printing and leather industries, these pollutants mainly comprise synthetic dyes. This project was designed to find ways to mitigate those dyes through application of enzymes produced recombinantly in corn seed. This low-cost, high volume production system can provide a mechanism of remediation that will be accessible to producers from botha cost-effective and volumetric stand point. Thus, one target audience of this project is the industrial community of users of synthetic dyes to produce colored products. Additional audiences would include the makers and users of persistent organic pollutants, such as 2,4-dichlorophenol, and herbicides, such as atrazine, that are persistent in the environment at sources of manufacture and use. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. The graduate student was recruited from an HBCU. His training was not rigorous and he has faced a number of challengesas a graduate student at A-State. He has taken classes in writing and critical thinking skills that have been very difficult forhim. He has also had content classes that should be follow-ons to previous classes for which he has not enough basicinformation. He is progressing.2. The two undergraduates on the project made great progress. The educational outreach student found a great potentialvocation through her work with our outreach coordinator. The woman in the research lab said it was her first "real" job doingresearch and she absolutely loved it. She did not know what she wanted to do with her education, but has now decided thatgraduate school is her next goal. How have the results been disseminated to communities of interest?Results have been reported to research communities. The patent application was abandoned, and publications will be forthcoming. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Research: This was a very productive year for showing the degradation of pollutants. The graduate student on the project showed significant degradation of five different synthetic dyes using manganese peroxidase. An undergraduate researcher who was also working on the project was using another redox enzyme, laccase, to degrade these same dyes. Because they both showed effects, although not the same patterns, the graduate student used the two redox enzymes in sequential degradation experiments and showed that the enzymes had great synergy. Products of the enzyme reactions were run on HPLC to determine the breakdown compounds which were then also analyzed by LC/MS. With respect to atrazine, our efforts to degrade this molecule were not successful. He wrote his thesis and defended it with great success. We are in the process of writing two publications based on this work. Education: I recruited a minority (African American) graduate student on the project who began in June, 2017. I also recruited a female undergraduate student who worked a few months until she graduated. She worked a few more months before starting graduate school. A second undergraduate student on the project was performing outreach to our local public school community with the outreach coordinator in STEM at A-State. • She provided support and presented to an estimated 300 students in other ABI related events throughout the school year. Examples include a visit of 200 sixth graders from Valley View Elementary and the Arkansas Science Festival located at Little Rock Air Force Base. • Designed and presented "Pollinators in Arkansas" lesson plan @ East Poinsett County SD Warrior Summer camp.Attended by 15 K-4th grade students; • Hands-on activity introduced the importance pollinators to local agricultural and horticulture life cycles. Studentsexplored how bees make honey • Designed and presented "Edible Plant Cell Wall" activity for El Centro Hispano community center in Jonesboro, AR-Summer Camp attended by 35 K-12 Hispanic/Latino students • Hands-on activity introduced students to biological molecules such as enzymes and their uses in agricultural and industrial applications • Designed and presented "Microbes in my food!" activity to 102 6th grade students at Jonesboro Public School (International Studies). • In this hands-on activity the students became forensic epidemiologist and investigated a food-borne pathogen make people sick at their school.

Publications

  • Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2020 Citation: Byrd, Joshua, PLANT-PRODUCED OXIDATION/REDUCTION ENZYMES AS BIOREMEDIATION AGENTS; in partial fulfillment of the Master of Science in Agriculture at Arkansas State University.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Byrd, Joshua and Hood, EE; 2019, Plant-Produced Manganese Peroxidase as a Bioremediation Agent. Oral presentation at Create@state, Arkansas State University.


Progress 12/15/17 to 12/14/18

Outputs
Target Audience:The industrial community has numerous issues in the area of mitigation of pollutants that come from their manufacturing processes. In the textiles, printing and leather industries, these pollutants mainly comprise synthetic dyes. This project is designed to find ways to mitigate those dyes through application of enzymes produced recombinantly in corn seed. This low-cost, high volume production system can provide a mechanism of remediation that will be accessible to producers from both a cost-effective and volumetric stand point. Thus, one target audience of this project is the industrial community of users of synthetic dyes to produce colored products. Additional audiences would include the makers and users of persistent organic pollutants, such as 2,4-dichlorophenol, and herbicides, such as atrazine, that are persistent in the environment at sources of manufacture and use. Changes/Problems:Originally, the plan was to use one enzyme with one dye, one herbicide, and one POP. We expanded the number of dyes to five, each having a unique structure, and treating them with two different redox enzymes. This was fortuitious since the atrazine did not digest under any conditions tested, so we had to drop this molecule from our study. What opportunities for training and professional development has the project provided?1. The graduate student was recruited from an HBCU. His training was not rigorous and he has faced a number of challenges as a graduate student at A-State. He has taken classes in writing and critical thinking skills that have been very difficult for him. He has also had content classes that should be follow-ons to previous classes for which he has not enough basic information. He is progressing. 2. The two undergraduates on the project made great progress. The educational outreach student found a great potential vocation through her work with our outreach coordinator. The woman in the research lab said it was her first "real" job doing research and she absolutely loved it. She did not know what she wanted to do with her education, but has now decided that graduate school is her next goal. How have the results been disseminated to communities of interest?To date, we have not reported results to target audiences, only research communities. Once the patent application is filed, we will contact potential customers of the technology. What do you plan to do during the next reporting period to accomplish the goals?We will finish up the dye degradation experiments--looking at HPLC of degradation products as well as toxicity of the same. We plan to graduate the MS student and publish at least two manuscripts based on his work. We will also look at degradation of the POP, 2,4-dichlorophenol.

Impacts
What was accomplished under these goals? Research: This has been a very productive year for showing the degradation of pollutants. The graduate student on the project has shown significant degradation of five different synthetic dyes using manganese peroxidase. An undergraduate researcher who was also working on the project was using another redox enzyme, laccase, degrade these same dyes. Because they both showed effects, although not the same patterns, the graduate student began using the two redox enzymes in sequential degradation experiments and showed that the enzymes had great synergy. With respect to atrazine, our efforts to degrade this molecule have not been successful. Education: I have recruited a minority (African American) graduate student on the project who began in June, 2017. I also recruited a female undergraduate student who worked a few months until she graduated. She is going to work a few more months before starting graduate school. A second undergraduate student on the project was performing outreach to our local public school community with the outreach coordinator in STEM at A-State. She provided support and presented to an estimated 300 students in other ABI related events throughout the school year. Examples include a visit of 200 sixth graders from Valley View Elementary and the Arkansas Science Festival located at Little Rock Air Force Base. Designed and presented "Pollinators in Arkansas" lesson plan @ East Poinsett County SD Warrior Summer camp. Attended by 15 K-4th grade students; Hands-on activity introduced the importance pollinators to local agricultural and horticulture life cycles. Students explored how bees make honey Designed and presented "Edible Plant Cell Wall" activity for El Centro Hispano community center in Jonesboro, AR- Summer Camp attended by 35 K-12 Hispanic/Latino students Hands-on activity introduced students to biological molecules such as enzymes and their uses in agricultural and industrial applications Designed and presented "Microbes in my food!" activity to 102 6th grade students at Jonesboro Public School (International Studies). In this hands-on activity the students became forensic epidemiologist and investigated a food-borne pathogen make people sick at their school.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Byrd, Joshua, Uyen Tran, Kendall R Hood, Elizabeth E Hood; Plant-Produced Manganese Peroxidase as a Bioremediation Agent; ABI Annual Symposium, September 25, 2018, Little Rock, AR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hood, EE, Byrd, JD, Tran, U. Redox enzymes from the corn grain production system remediate azo-dyes; IAPB Quadrennial meeting, August 22, 2018; Dublin Ireland.


Progress 12/15/16 to 12/14/17

Outputs
Target Audience:I have recruited a minority (African American) graduate student on the project who began in June. We have not made enough progress yet on the project to report any results outside of the lab. Other lab members are hearing about his results. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training a new graduate student recruited from an HBCU. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue to work with various pollutant chemicals that we have in-house to learn about their degradation with the peroxidase enzyme. As we perform these studies, we will report results at regional and national conferences through student poster presentations. I plan to hire an undergraduate student for training on this project as well.

Impacts
What was accomplished under these goals? A minority graduate student began work on the project in June. He has learned several new techniques and is making progress on doing enzyme reactions and readings. After the first of the year, we will recruit an undergraduate to participate in the project. Joshua Byrd (my grad student) has purified the peroxidase from corn seed and made a concentrated extract. He has worked at length with the dyes and shown degradation. He kept the resulting samples in the freezer for eventual analysis at the University of Arkansas Little Rock.

Publications