Source: UNIVERSITY OF DELAWARE submitted to NRP
EVALUATION AND OPTIMIZATION OF INNOVATIVE, MYCOREMEDIATION TECHNOLOGIES TO REMOVE PATHOGENS AND ANTIBIOTICS FROM DAIRY MANURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1015444
Grant No.
2018-67017-27882
Cumulative Award Amt.
$469,999.00
Proposal No.
2017-08055
Multistate No.
(N/A)
Project Start Date
Jun 1, 2018
Project End Date
Sep 30, 2023
Grant Year
2018
Program Code
[A1331]- Improving Food Safety
Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
Entomology & Wildlife Ecology
Non Technical Summary
The long-term goals of this research are to develop and implement sustainable, mycoremediation technologies capable of reducing pathogens and contaminants present in agricultural manure waste streams, which have become a considerable threat to public health and overall food safety. Improper handling of manure has resulted in direct contamination of produce, water, animals and humans. Zoonotic pathogens such as Salmonella, pathogenic and non-pathogenic Escherichia coli, Cryptosporidium, and Listeria are found in animal manure. Manure application can also introduce antibiotic residues into the environment. Antibiotics, used in animal production, and their metabolites are excreted in manure and persist in stored manure, soil, and water. Plants grown in soil containing antibiotic residues uptake and translocate them to the roots, stems, leaves and fruits, which is problematic if ingested as raw produce by humans. Ingestion of these raw plants can cause extraneous exposure to antibiotics. Composting of solid manure at high temperatures can reduce certain pathogens, but pathogen regrowth can occur once temperatures decrease. The white rot fungus (WRF), Pleurotus ostreatus, in bioreactors can reduce E.coli in diary manure. The WRF, Phanerochaete chrysosporium, can degrade oxytetracycline and tetracycline in a crude enzyme extract. The objective of this research is to optimize fungal bioreactor properties, evaluate the predation/degradation behavior of the WRF, and efficiently degrade contaminants in dairy manure. In this project, we will assess the ability of white rot fungi to degrade bacteria and antibiotics in manure. Using bioreactors, we will assess the effect of nutrients and other compounds present in the manure on the degradation ability of the fungi. We will also evaluate how the type of flow within the bioreactor will affect fungal degradation of pathogens and antibiotics. We will also assess the potential of composting manures with fungi to degrade bacteria and antibiotics in solid manure. We will assess ways to improve pathogen removal by manipulating the ratio of manure to fungus.From this, we will improve the understanding of fungal bioremediation and develop methods and technology for treatment of animal manures. We will apply this new knowledge to develop fungal bioreactors that are low cost alternatives for manure management. By improving the ability of composting methods to reduce bacteria in animal manure, we will improve food safety and decrease the risk of human exposure to contaminated food resulting from manure application. By developing Best Management Practices for safe and efficient composting, we will help set safety rules for the application of biological soil amendments. We will improve the sustainability of animal production and increase food safety. This work should help increase the value of biological soil amendments as well.
Animal Health Component
75%
Research Effort Categories
Basic
10%
Applied
75%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
4027410202025%
4034020110225%
7124010110025%
7110110200025%
Knowledge Area
403 - Waste Disposal, Recycling, and Reuse; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 402 - Engineering Systems and Equipment;

Subject Of Investigation
0110 - Soil; 4010 - Bacteria; 4020 - Fungi; 7410 - General technology;

Field Of Science
2020 - Engineering; 1100 - Bacteriology; 1102 - Mycology; 2000 - Chemistry;
Goals / Objectives
1. IntroductionSoil amendment handling practices can reduce food safety risks to fruits and vegetables and to other crops. Bacterial pathogens can be present in biological soil amendments of animal origin and can be transferred to crops via direct application, wind, or rain. The FDA Food Safety Modernization Act of 2011 (FSMA) mandated Produce Safety Rule Standards for the Growing, Harvesting, Packing, and Holding of Produce for Human Consumption. FDA has deferred its decision on an appropriate time interval between the application of untreated biological soil amendments (including raw manure) and crop harvesting until it conducts a risk assessment and extensive research to strengthen scientific support for any future proposal. Data suggest that bacteria that may contaminate raw agricultural commodities (RACs) may persist in manure for hundreds of days (Sharma and Reynnells, 2017). There is a data gap regarding potential for biocontrol of bacteria in manure. While proper composting can inactivate bacterial pathogens, small-to-mid-sized growers may be reluctant to properly compost biological soil amendments due to lack of space and potential issues with adherence to time and temperature conditions and necessary record keeping. Bacterial pathogens may survive or undergo regrowth in finished compost due to incomplete thermal inactivation during composting or recontamination that can occur (Reynnells et al., 2014). These proposed novel studies will provide a means of treatment for raw manure and the potential for use of manure on land used to grow RACs. Bacterial pathogens including Salmonella and Escherichia coli O157:H7 may exist in raw manure; and irrigation water exposed to contaminated surface water runoff from nearby animal production farms has been linked to foodborne outbreaks associated with contaminated leafy greens (Crohn and Bianchi, 2008). Better methods are needed to ensure that the use of animal manures in agricultural production does not impose a risk to food safety nor contribute to environmental contamination. Recent work in the area of mycoremediation has shown that certain white rot fungi (WRF) are capable of killing bacteria present in manure waste streams (Chirnside, 2016). In addition, these WRF are able to degrade recalcitrant contaminants, like antibiotics and their residues, and have been used to remediate contaminated soils (Golan-Rozen et al., 2015). Therefore, it is feasible that technologies utilizing the WRF can be developed to remove pathogenic bacteria and antibiotics from manure before it is exposed to the environment. This proposed project will provide as stated in Program Area Priority A1331, an economical and adoptable control strategy aimed at reducing the incidence of foodborne hazards during the critical pre-harvest production time.1.1 Long-term Goals and Supporting ObjectivesThe long-term goal of this research project is to develop and implement sustainable, low cost, mycoremediation technologies that can reduce pathogens and contaminants present in agricultural manure waste streams, which have become a considerable threat to public health and overall food safety. This will also allow for the use of biological soil amendments of animal origin to be used in crop production. We have preliminary data to support that the WRF, Pleurotus ostreatus, in bioreactors can reduce E.coli in dairy manure (Chirnside, 2016; Chirnside et al., 2013). As discussed in the following Body of Knowledge section, small bench scale fungal bioreactors were able to reduce the number of E. coli naturally present in aqueous dairy manure. Crude extract from the WRF, Phanerochaete chrysosporium, containing LiP enzyme was able to degrade tetracycline and oxytetracycline, two antibiotics, during in vitro incubation (Wen et al., 2009).The goals of this research are to optimize fungal bioreactor properties, to evaluate the predation/degradation behavior of the WRF, and efficiently degrade pathogenic bacteria and antibiotic contaminants in dairy manure. We propose to achieve these goals by evaluating fungal bioreactor properties in laboratory experiments and coordinating these results with composting experiments utilizing dairy manure inoculated with the fungi under study. We have specifically chosen strains of E. coli and E. coli O157:H7 that are well studied in the literature (Sharma et al., 2016; Reynnells et al., 2014; Tomas-Callejas et al., 2011) and have been shown to survive in biological soil amendments; thereby we will be able to directly correlate our studies to a reduction in food safety risks. Specifically these objectives include:To determine the efficacy of two different white rot fungi, Phanerochaete chrysosporium and Pleurotus ostreatus, grown within fungal bioreactors to remove E. coli (strain TVS 355) from aqueous dairy manure through the assessment of different growth support materials and flow regimes.To evaluate the ability of the white rot fungus grown within a packed-bed fungal bioreactor to remove several pathogenic bacteria present in a buffered growth solution and in aqueous dairy manure.To evaluate the ability of the white rot fungus grown within a packed-bed fungal bioreactor to remove antibiotic residues present in water and in aqueous dairy manure.To determine the fate of E. coli TVS 355 and E. coli O157:H7 and antibiotic residues in dairy manure during composting with two different white rot fungi, Phanerochaete chrysosporium and Pleurotus ostreatus. A third inoculant treatment will consist of a mixture of P. chrysosporium and P. ostreatus.
Project Methods
OBJ. 1. Bioreactors will be used to assess the degradative ability of two different white rot fungi (WRF), Phanerochaete chrysosporium (PC) and Pleurotus ostreatus (PO). In order to evaluate the effect of nutrients on fungal predation, three different materials with varying carbon to nitrogen ratios (C:N) will be used as the fungal growth support material; reticulated polyurethane foam (RPF), wood chips (WC) and the Spent Mushroom Compost (SMC). Two types of controls for each support material will be run to allow for the measurement of reduction in E. coli (TVS 355) due to adsorption to the support and/or to the support containing dead mycelia. A robust strain of generic E. coli (TVS 355) will be used in the bioreactors.PC cultures (ATCC #34541) will be maintained on 2% malt agar. Inoculum production will follow typical protocols for fungal inoculum development. For PO, prepared inoculum on sterilized rye berries will be used. The prepared fungal inocula will be used to colonize the different support materials and the support materials will be incubated until fully colonized. The colonized material will be transferred to the bioreactors, allowed to recolonize for 2 weeks to ensure adequate mycelia growth within the reactors. Ligninolytic activity will be induced by pumping a nitrogen-limited media through the bioreactors 2 weeks. We will assess the effect of batch and continuous flow regime hydraulics on fungal degradation ability. Two batch time periods will be evaluated: 60 and 480 minutes. For the different batch periods, the bioreactors will be loaded with approximately 500 mL of aqueous dairy manure inoculated with E. coli strain TVS 355. After the treatment time, the effluent will be collected for E. coli analysis. Additional effluent will be collected for nutrient and pH analysis. Influent samples will be collected at initiation of the test. Samples of the support material for each reactor will be sampled before and after the batch test for C:N. Liquid samples will be analyzed for E. coli TVS 355 survival. Samples will be analyzed for nutrient and carbon content using standard methods.Continuous Flow Loading of BioreactorsAs described above, fungal bioreactors will be tested under a continuous flow regime. New inoculum will be prepared as described above for all of the bioreactors. The same treatments will be used. Aqueous dairy manure inoculated with E. coli strain TVS 355 will be pumped through the bioreactors at the continuous rate of 2.8 mL min-1. Influent and effluent samples will be collected at 4 time intervals. Sampling and analysis will be the same as the batch test. Biomass samples will also be collected.Results will be assessed for design characteristics that resulted in the best degradative performance of the fungi, which will be used in Obj. 2 & 3.Obj.2: Pathogen Removal, Batch and Continuous Flow Study with Pathogens in Buffer and Aqueous Dairy Manure. The fungus that performed the best will be used. The supports that will be evaluated will be the inert RPF and the optimum biomass from Objective 1. The flow regimes will consist of one batch time-period analysis using the most efficient batch time evaluated in Objective 1 and continuous flow. For the continuous flow study, the effluent will be sampled at four time points. Sample collection for both bacteria and for nutrient analysis will occur as described above.Two strains of attenuated E. coli O157:H7 (ATCC 700728 and ATCC 43888), both lacking stx1 and stx2 will be used. Salmonella enterica serovar Typhimurium strain ATCC SL1344 will also be used. Manure samples and subsamples will be collected and analyzed for bacteria content using standard procedures developed by Dr. Kneil.This experiment will be run with the pathogens in a buffer and with the pathogens in aqueous dairy manure. Influent preparation and bacterial analysis will be same as above. Separate samples will be collected for nitrogen, carbon and pH analysis as described above. Samples of the bioreactor biomass will be collected at each time sample point and be analyzed for C:N as described above.Objective 3: Antibiotic Removal, Batch and Continuous Flow Study with Antibiotics in Buffer and in Aqueous Dairy Manure. This study will be set up as described in Objective 2. However, the buffer and aqueous manure will be spiked with the antibiotics in place of the pathogens. Two antibiotics, sulfamethazine and tetracycline, will be tested in the batch and continuous flow studies. The antibiotics will be added to the liquid at a rate of 10 mg L-1. Antibiotic analysis will be performed by Dr. Kang's Lab using the Enzyme-Linked Immunosorbent Assay (ELISA) kits for all antibiotics. Some of the positive samples from ELISA tests will be further tested with GC-MS to confirm the presence of these antibiotics. Influent preparation will be as stated previously. Separate samples will be collected for nitrogen, carbon and pH analysis as described in Objective 1. Antibiotic analysis will be performed as stated above in Experiment 3A. One batch flow and continuous flow regime will be assessed.Results from objectives 2 & 3 will be evaluated in order to assess the effect of C:N on the degradation ability of the fungus. These results will be used to improve bioreactor design characteristics, which can be assessed at field scale and developed into commercial applications.Objective 4: Fungal - Manure CompostingEvaluation We will evaluate the degradation of pathogens and antibiotics in composted dairy manure inoculated with white rot fungi. Three fungal inoculants will be evaluated: a) PC, b) PO, and c) PO and PC combined. Three ratios of inoculum:manure will be evaluated. A fourth treatment of the manure without fungal inoculation will be included. The prepared fungal inoculum will be used to colonize the composted dairy manure at the ratios described above. Growth of the WRF will be monitored daily.Piles will be maintained in controlled biochambers in the greenhouse located at the College of Agriculture and Natural Resources. Piles will be inoculated with E. coli TVS 355 and the two strains of E. coli O157:H7 propagated in a manure slurry using backpack sprayers at 6 log CFU/ml. The piles will be spiked with the antibiotics at a rate of 10 mg kg-1 applied to the manure using a backpack sprayer. The piles will be mixed thoroughly after addition of the antibiotics. The piles will be turned weekly. Composited samples from each pile will be collected on day 0, 30, 45, 60, 75 and 90. The samples will be analyzed using standard methods. Bacterial analysis will be performed as outlined above. The manure samples will be extracted for antibiotic analysis and analyzed as outlined above. Some of the positive samples from ELISA tests will be further tested with GC-MS to confirm the presence of these antibiotics.Efforts: Improved understanding of optimum fungal bioreactor design characteristics will be used to develop field scale experiments in order to develop marketable treatment systems.Extension workshops will be used to promote best practices for composting of animal wastes to increase food safety and improve the sustainability of animal production and agronomic processes. Evaluation: The reduction in pathogens and antibiotics will be compared to the bioreactor characteristics using standard analytical calculations. The new knowledge will used to develop optimized bioreactors to test at field scale in order to evaluate low cost methods for operation of the bioreactors. Ultimately, a commercial bioreactor that can be used on the farm will be developed.The successful composting methods will be developed into management practices that can be demonstrated to growers at workshops and extension activities. These best management practices can help set safety rules for application of biological soil amendments.

Progress 06/01/18 to 09/30/23

Outputs
Target Audience:The target audience for this project includes scientists and researchers interested in food safety and enhancing agricultural practices to mitigate control of pathogens and antimicrobials, as well as growers and land management practitioners. The research conducted in this project and the outputs will be of interest to soil scientists as well as researchers interested in pre-harvest risk reduction strategies. This project includes development of science-based knowledge through research presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Throughout this project undergraduate researchers received training and mentoring in science. These students were successful at completing research experiments and were trained in the scientific method and in advancements in agriculture. There were at least fifteen undergraduate students trained over the course of this project, and approximately 85% of these were women. A PhD student working on this project, Alexis Omar, received several opportunities for professional development by participation in conferences through presentation of her research. Within these same conferences, Alexis participated in workshops on leadership, and diversity equity and inclusion. This was an outstanding opportunity to provide professional development to a highly motivated and passionate young scientist. How have the results been disseminated to communities of interest?Results for this project have been and will be shared in oral talks, poster presentations, and in peer-review journal articles. Several manuscripts are currently being developed that will highlight the chemical analysis in terms of the impact on biological remediation by the fungi. Presentations have been given both orally and in poster-format to the soil science, analytical chemistry, and food safety communities. There has continuously been a great interest in the findings of these projects as presented by the students and co-PIs of theproject. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Overall this project resulted in a change in knowledeg related to mycoremediation of white rot fungi for use as a biocontrol agent for bacterial pathogens. Objective 1.To determine the efficacy of two different white rot fungi,Phanerochaete chrysosporium and Pleurotus ostreatus, grown within fungal bioreactors to removeE. coli(strain TVS 355) from aqueous dairy manure through the assessment of different growth support materials and flow regimes. 1) Major activities completed / experiments conducted: Fungal bioreactors were created using varying matrices (spent mushroom compost, reticulated polyurethan foam, and woodchips) and both Phanerochaete chrysosporium and Pleurotus ostreatus were pumped through the bioreactors in liquid dairy manure and also in phosphate buffered saline. 2) Data collected: The data collected shows the inactivation of E. coli over time in the presence of the fungi. Chemical analysis provided data regarding enzyme amounts indicating fungal growth. 3) Summary statistics and discussion of results: The SMC had higher concentrations of ergosterol compared to the other substrates. Ergosterol was found in the autoclaved fungal treatments indicting that ergosterol has a slow degradation rate within the substrate, suggesting that a longer wait period should occur before starting the experiments to allow for any lingering ergosterol to degrade. Overall, the live fungal treatments had the highest ergosterol concentrations in the separate treatments for each of the substrates. Visible inspection also verified fungal growth within the treatment 1 bioreactors. Concentrations of E. coli varied overtime, with about one log change. 4) Key outcomes or other accomplishments realized: This initial series of experiments led to the further study of woodchips. Other factors would also be optimized following this initial analysis. Objective 2.To evaluate the ability of the white rot fungus grown within a packed-bed fungal bioreactor to remove several pathogenic bacteria present in a buffered growth solution and in aqueous dairy manure. 1) Major activities completed / experiments conducted: Pleurotus ostreatus was grown on varying matrices (spent mushroom compost, reticulated polyurethan foam, and woodchips) in bath and continuous bioreactors. Phanerochaete chrysosporium was evaluated using only woodchips in an optimized batch and continuous flow system. An outbreak strain of E. coli O157:H7 was studied in this system. Other microbial pathogens (Salmonella, virus, and protozoa) were assessed in subsequent experiments. 2) Data collected: Data collected included enumeration of bacteria present and calculation of the reductions of bacteria over time. Chemical analysis indicated the presence of enzymatic activity by the fungi. 3) Summary statistics and discussion of results: Aqueous dairy manure has a basic pH between the range of 8-9. The different fungal treatments did impact the pH of the manure over time. Carbon and nitrogen levels were measured, and no statistical significance was observed at P<0.05 for tests of within treatment-effects for time and the interaction between treatment and time. The ortho phosphorus increased in the bioreactors over time. However, in the treatment with the live fungus the concentration of OP decreased after day 7. This indicated that the fungus was utilizing the nutrients available in the manure and that which leached from the compost support. The fungal growth was able to reduce the levels of E. coli O157:H7 over normal decay levels occurring over time. 4) Key outcomes or other accomplishments realized: This work resulted in a change in knowledge regarding the reduction of pathogenic E. coli. Objective 3.To evaluate the ability of the white rot fungus grown within a packed-bed fungal bioreactor to remove antibiotic residues present in water and in aqueous dairy manure. 1)Major activities completed / experiments conducted: Phanerochaete chrysosporium and Pleurotus ostreatus were grown on two different types of spent mushroom compost, a synthetic foam, and on wood chips and analyzed for their ability to degrade sulfamethazine. Concentrations of the antibiotic were provided at 500 mg/L. 2) Data collected: The data showed the sulfamethazine removal by percentage over time (up to 200 hours). 3) Summary statistics and discussion of results: Sulfamethazine removal varied by the matrix including removal of 80-90% by fungi grown on wood chips and mushroom compost. Fungi grown on synthetic foam failed to remove more than 50% by 200 hours of analysis. 4) Key outcomes or other accomplishments realized: This work resulted in a change in knowledge as sulamethazine was removed by the presence of Phanerochaete chrysosporium and Pleurotus ostreatus. Objective 4. To determine the fate ofE. coliTVS 355 andE. coliO157:H7 and antibiotic residues in dairy manure during composting with two different white rot fungi,Phanerochaete chrysosporiumandPleurotus ostreatus. A third inoculant treatment will consist of a mixture ofP. chrysosporiumandP. ostreatus. 1) Major activities completed / experiments conducted: This objective was modified based on the findings of the previous three objectives. To increase the potential utilization of the bioreactors, the matrices were combined with novel filtration media (zero-valent iron and biochar)to create filters for use in on-farm pre-harvest food safety applications. 2) Data collected: Inoculated artificial ground water was prepared with bacteria, virus, or protozoa and applied to filters built from wood chips containing Phanerochaete chrysosporium layered with novel filtration media. 3) Summary statistics and discussion of results: Chemical analysis of hydrogen peroxide was indicative of fungal ligninolytic activity. Microbial reductions were compared to other controls. 4) Key outcomes or other accomplishments realized: This objective has resulted in a change in knowledge. Incorporation of Phanerochaete chrysosporium into a filter significantly increased removal of E. coli and Salmonella from water, creating an advantageous filtration system for irrigation water.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lopez, S., & Kang, D. H. Adsorption of Sulfamethazine to Biochar. In World Environmental and Water Resources Congress 2023 (pp. 74-79).
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Oral Presentation World Environmental & Water Resources Congress 2023 Batch Study with White Rot Fungi for Antibiotics Degradation Author: Dong hee Kang  Morgan State University Co-Author: Anastasia M. Chirnside, PhD, M.ASCE  University of Delaware
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Oral Presentation World Environmental & Water Resources Congress 2023 Adsorption of Sulfamethazine to Biochar Author: Sebastian Lopes  Morgan State University Co-Author: Dong hee Kang  Morgan State University
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: A Continuous Flow-through System Utilizing White-Rot Fungi, Pleurotus ostreatus, and Its Effects on the Inhibition on Escherichia coli. Alexis N. Omar, Aubrey Inkster, Anastasia E. M. Chirnside and Kalmia Kniel. 2023. CANR Student Research Symposium. November 16, Newark, DE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Novel Mycoremediation Technique Utilizing White-Rot Fungi, Pleurotus ostreatus and Phanerochaete chrysosporium to Inhibit Pathogenic E. coli for Pre-Harvest Food Safety. Alexis N. Omar, Anastasia E. M. Chirnside, and Kalmia E. Kniel. 2023. International Association of Food Protection (IAFP) European Symposium, May 3-5, Aberdeen, Scotland.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: A Continuous Flow-through System Utilizing White-Rot Fungi, Pleurotus ostreatus, and Its Effects on the Inhibition on Escherichia coli. Alexis N. Omar, Anastasia E. M. Chirnside and Kalmia Kniel. 2023. University of Delaware Microbiology Symposium, March 10th, Newark, DE.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Evaluation of White Rot Fungus to Control Growth of Escherichia coli in Cattle Manure. Alexis N. Omar, Anastasia Chirnside, Kalmia E. Kniel. 2024. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2023.100206
  • Type: Conference Papers and Presentations Status: Under Review Year Published: 2024 Citation: Alexis N. Omar, Kyle McCaughan, Anastasia E. M. Chirnside, Manan Sharma, and Kalmia Kniel. Novel ZVI-Biochar-Mycoremediation Filtration Utilizing White-Rot Fungi Phanerochaete chrysosporium, to Inhibit Pathogenic Bacteria inAgricultural Water.


Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Research was presented and shared at scientific conferences with the target audience being largely researchers and scientists. Changes/Problems:A no-cost extension was requested to complete the planned experiments for objective 4. The previous grant director, Dr. Stacey Chirnside, retired during this time period. What opportunities for training and professional development has the project provided?A graduate student continues to be trained in mycology and several undergraduates have been trained in mycology. This is a wonderful opportunity for students to learn how to work with fungi, an understudied microbe. How have the results been disseminated to communities of interest?Results were presented at national scientific conferences. There was great interest from other scientists. What do you plan to do during the next reporting period to accomplish the goals?The next report will be the final report following a request for a no-cost extension in order to work on objective 4.

Impacts
What was accomplished under these goals? This period of time focused on completion of activities related to objective 2 and 3. The major goal of objective 2 is to assess teh ability of white rot fungi to reduce the bacterial load of pathogens, like pathogenic E. coli O157:H7. The major goal of objective 3 is the removal of sulamethazine, an antimicrobial, by the activity of white-rot fungal species.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Dorbert, A., A. Inkster, S. Palani, A. N. Omar, A. E. M. Chirnside, and K. E. Kniel. 2022. Determining the efficacy of different carbon/nitrogen ratios of growth medias for optimal fungal growth. Northeast Agricultural and Biological Engineering Conference. Bel Air, Maryland, July 31-August 3, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Henry, N. L., A. N. Omar, A. E. M. Chirnside, and K. E. Kniel. 2022. Monitoring Fungal Enzyme Activity During Mycoremediation of Bacteria in Animal Waste Streams. Northeast Biological and Agricultural Conference. Bel Air, Maryland, July 31-August 3, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Maddox, P., A. N. Omar, A. E. M. Chirnside, and K. E. Kniel. 2022. Monitoring Soluble Phosphate Activity During Mycoremediation of Bacteria in Animal Waste Streams. UD Envision Symposium. DE State Fair, Harrington, DE. August 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: A Continuous Flow-through System Utilizing White-Rot Fungi, Pleurotus ostreatus, and Its Effects on the Inhibition on Escherichia coli. Alexis N. Omar, Aubrey Inkster, Anastasia E. M. Chirnside and Kalmia Kniel. 2022. Annual Meeting of International Association of Food Protection (IAFP), July 31- Aug, Pittsburg, PA.


Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Other researchers, undergraduate students and graduate students attending the symposiums, thesis defense and conferences. The information will be of interest to practitioners and farmers looking for other technology to reduce pathogens in manure. Changes/Problems:At this time, we are on track and accomplishing the work without interuption. We don't forsee any difficulties in completing the project. What opportunities for training and professional development has the project provided?1. Seven undergraduate students worked on the project and learned microbiological techniques and analytical methods for ergosterol, nitrogen, COD, pH, H2O2, and phosphorus analysis. Two graduate students gained research experience in microbiology and analytical chemistry. Several students did special projects related to the experiments. 2. One undergraduate student participated in a senior research project and wrote a thesis on her work with ergosterol analysis on the project samples. The title of the project was "Quantifying fungal growth in nutrient limited systems for bioremediation purposes." Alayna will present a poster at meeting in the summer of 2022. 3. An undergraduate student intern from Millersville University participated in a summer internship in my lab. She performed the enzyme analyses on the samples from the bioreactor experiments.The title of her work was "Monitoring Fungal Enzyme Activity During Mycoremediation of Bacteria in Animal Waste Streams." Nikki Henry is presented her work as a poster at meetings in summer 2022. 4. Two graduate students participated in the funded research and presented seminars on their work. Alexis Omar has been working with Dr. Kniel monitoring the bacteria degradation within the manure and the bioreactors. Aubrey Inkster has been working with me, Dr. Chirnside, on the fungal bioreactors and has conducted the flow through hydraulic bioreactors. He also conducted following experiments with both fungi grown on the wood chips. How have the results been disseminated to communities of interest?1. Aubrey Inkster presented his Master of Science thesis proposal to his thesis committee in the Microbiology program in the Graduate College at UD. His proposal was entitled:"Determination of the efficacy of fungal bioreactors to remove E. coli from aqueous dairy manure." 2. Aubrey Inkster gave an oral presentation at the 2021 Northeast Agricultural and Biological Engineers Congress. His talk was entitled: "Determination of the efficacy of fungal bioreactors to remove E. coli from aqueous dairy manure." 3. Alexis N. Omar presented a poster at the 2021 IAFP Annual Meeting entitled "Woodchips Increase the Inhibitory Abilities of White-Rot Fungi, Pleurotus Ostreatus, in Manure Inoculated with Escherichia coli ." 4. Alexis N. Omar presented a poster at the University of Delaware's College of Agriculture and Natural Resources' 2021 Fall Research Symposium. 5. Two undergraduate students participated in funded research internships sponsored by the University of Delaware. The projects were related to this research andthey will present their work and write a paper. They gave video presentations of their work to the general public at the Delaware State Fair in July 2021. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we plan onaccomplishing the following: analysis of all the samples, preparing the data, applying statistics and presenting the results in journal articles and oral presentations. If time alows, we may treat solid manure with the white rot fungi to determine the efficicacy of bacteria reduction by the fungi.

Impacts
What was accomplished under these goals? Progress Summary: The following experiments were accomplished: Evaluation of E. Coli Reduction in Batch Bioreactors with Pleurotus Ostreatus (PO) on Reticulated Polyurethane Foam (RPF). The batch experiment consisted of 4 different treatments (Figure 1). Each treatment was done in triplicate. Each Treatment was run separately with one replication of the uninoculated control (T4). For Treatment 1, the bioreactors contained Spent Mushroom Compost (SMC) inoculated with live PO. For Treatment 2, the bioreactors contained inoculated SMC that were autoclaved. Treatment 3 was the negative control consisting of the sterilized SMC. Treatment 4 was the un-inoculated control, which had the sterilized support and was treated with un-inoculated aqueous dairy manure that contained no E. coli TVS355. Aqueous dairy manure inoculated with E. coli TVS355 was applied to all bioreactors with a multichannel peristatic pump at a rate of 35 ml/min. Approximately 1 liter of manure was applied to the reactors (¾ bioreactor volume). The batch experiment was run for 10 days with the following sampling times: Day 0, 1,3,5,7 and 10. Samples were collected from the effluent, the support biomass, the inoculated influent, and the non-inoculated influent. The following analyses were performed on the samples. 1. E. coli TVS 355 : Enumerated on MacConkey agar with Rifampacin or Nalidixic acid 2. Ergosterol : Solid Phase Extraction & HPLC 3. COD : SMWW 5220 A. Acid digestion/ colorimetric 4. pH : pH strips 5. LiP and MnP enzyme assays 6. OP 7. TKN Evaluation of E. Coli Reduction in Packed-bed Flow Through Bioreactors with Pleurotus Ostreatus (PO) on Wood Chips (WC), Spent Mushroom Compost (SMC) and Reticulated Polyurethane Foam (RPF). The batch experiment consisted of 4 different treatments. Each treatment was done in triplicate. Each Treatment was run separately with one replication of the uninoculated control (T4). For Treatment 1, the bioreactors contained Reticulated Polyurethane Foam (RPF) inoculated with live PO. For Treatment 2, the bioreactors contained inoculated RPF that were autoclaved. Treatment 3 was the negative control consisting of the sterilized RPF. Treatment 4 was the un-inoculated control, which had the sterilized support and was treated with un-inoculated aqueous dairy manure that contained no E. coli TVS355. Aqueous dairy manure inoculated with E. coli TVS355 was applied to all bioreactors with a multichannel peristatic pump at a rate of 35 ml/min. Approximately 1 liter of manure was applied to the reactors (¾ bioreactor volume). The batch experiment was run for 10 days with the following sampling times: Day 0, 1,3,5,7 and 10. Samples were collected from the effluent, the support biomass, the inoculated influent, and the non-inoculated influent. The same parameters listed above were monitored throughout the experiment. Evaluation of E. Coli Reduction in Flow-through Bioreactors with Pleurotus Ostreatus (PO) and Phanerochaete chrysosporium (PC) on oak wood chips (WC). The flow-through experiment consisted of 4 different treatments. Each treatment was done in duplicate. For Treatment 1, the bioreactors contained oak wood chips (WC) inoculated with live PC. For Treatment 2, the bioreactors contained WC inoculated with PO that were autoclaved. For Treatment 3, the bioreactors contained PC inoculated WC that were autoclaved. For Treatment 4, the bioreactors contained PO inoculated WC that were autoclaved. Treatment 5 was the un-inoculated control, which had the sterilized support and was treated with un-inoculated aqueous dairy manure that contained no E. coli TVS355. Phosphate buffer solution (pH= 6.5) inoculated with E. coli TVS355 was applied to all bioreactors with a multichannel peristatic pump at a rate of 0.5 ml/min. The experiment was run for 50 days with the following sampling times: Day 0, 5,10,15,20, 25, 30, 35, 40 and 50. Samples were collected from the effluent, the support biomass, the inoculated influent, and the non-inoculated influent. The following analyses were performed on the samples. 1. E. coli TVS 355 : Enumerated on MacConkey agar with Rifampacin or Nalidixic acid 2. Ergosterol : Solid Phase Extraction & HPLC 3. COD : SMWW 5220 A. Acid digestion/ colorimetric 4. pH : pH strips 5. LiP and MnP enzyme assays 6. OP 7. TKN Evaluation of E. Coli Reduction in Batch Bioreactors with Pleurotus Ostreatus (PO) and Phanerochaete chrysosporium (PC) on oak wood chips (WC). The batch experiment consisted of 4 different treatments. Each treatment was done in duplicate. For Treatment 1, the bioreactors contained oak wood chips (WC) inoculated with live PC. For Treatment 2, the bioreactors contained WC inoculated with PO that were autoclaved. Treatment 3 was the un-inoculated control, which had the sterilized support and was treated with un-inoculated aqueous dairy manure that contained no E. coli TVS355. Each reactor was filled with 1.8 L of phosphate buffer solution (pH= 6.5) inoculated with E. coli TVS355 Treatment 3 received the un-inoculated buffer solution. The experiment was run for 50 days with the following sampling times: Day 0, 5,10,15,20, 25, 30, 35, 40 and 50. Samples were collected from the effluent, the support biomass, the inoculated influent, and the non-inoculated influent. The same parameters listed above were monitored throughout the experiment.

Publications

  • Type: Theses/Dissertations Status: Accepted Year Published: 2022 Citation: Dorbert, Alayna. 2022. Quantifying fungal growth in nutrient limited systems for bioremediation purposes. Senior Thesis. University of Delaware 2022-5. Newark, DE
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Omar, A., A. Inkster, A. E. M. Chirnside, and K. Kniel. 2021. Woodchips Increase the Inhibitory Abilities of White-Rot Fungi, Pleurotus ostreatus, in Manure Inoculated with Escherichia coli. International Association for Food Protection Conference Phoenix AZ, July 18-21, 2021 P2-113
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Omar, A., and K. Kniel. 2021. Differential interactions of Pleurotus ostreatus with Escherichia coli TV355 and Escherichia coli O157:H7. International Association for Food Protection Conference Phoenix AZ, July 18-21, 2021 P2-114
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Inkster, A. and A. E. M. Chirnside. 2021. Determination of the efficacy of fungal bioreactors to remove E. coli from aqueous dairy manure. NABEC 2021 Virtual conference July 30-31, 2021.


Progress 06/01/20 to 05/31/21

Outputs
Target Audience: Other researchers and graduate students attending the symposiums and conferences. The information will be of interest to practitioners and farmers looking for other technology to reduce pathogens in manure Changes/Problems:The building where my research laboratory was located was renovated beginning in July 2019. Due to building renovations, my lab has been in transition. We moved into our new lab in April 2021 and had the hood and digestor installed in late May 2021. We were unable to run several different analyses on our samples from the experiments during the transition period. We are currently working on completing all samples. We should be starting the next experiments with the bioreactors in July of 2021. We continue to be limited by the pandemic on the number of students allowed in the lab at the same time. Hopefully, these restrictions will be reduced as we move forward. Although these interruptions have delayed the progress of the bioreactor experiments and has delayed our timeline for accomplishing the research work, we have resumed our research. What opportunities for training and professional development has the project provided? Six undergraduate students worked on the project and learned microbiological techniques and analytical methods for ergosterol, nitrogen, COD, pH, H2O2, and phosphorus analysis. Two graduate students gained research experience in microbiology and analytical chemistry. One undergraduate student participated in a funded research (Delaware Water Resources Agency) project related to this research. Patrick McGay's project was to modify the peroxidase enzyme assays so that they would be accurate for dark colored complex media, which interfered in the standard assays that used changes in absorption to determine activity. The title of the project was 'Modification of Peroxidases Enzyme Analytical Methods for Complex Media from Solid State Bioreactors use to Reduce Pathogens in Dairy Manure.' He presented a poster prestation and will write a paper on the work. Three graduate students participated in the funded research and presented seminars on their work. Sivaranjani Palani graduated in July 2020 with her Master of Science in Environmental Engineering. She is currently a PhD student at North Carolina State University in Environmental engineering with Dr. Francis de los Reyes III. Her project is the Thermodynamic Modeling of Anaerobic Systems. Alexis Omar has been working with Dr. Kniel monitoring the bacteria degradation within the manure and the bioreactors. Aubrey Inkster has been working with me, Dr. Chirnside, on the fungal bioreactors and will concentrate on the flow through hydraulic bioreactors. How have the results been disseminated to communities of interest? Sivaranjani Palani presented her Master of Science thesis in the Department of Civil and Environmental Engineering's Graduate seminar. "Determination of the efficacy of fungal bioreactors to remove E. coli from aqueous dairy manure." Sivaranjani Palani completed and published her Master of Science thesis in the Department of Civil and Environmental Engineering's Graduate seminar. "Determination of the efficacy of fungal bioreactors to remove E. coli from aqueous dairy manure." Alexis N. Omar presented a poster at the 2020 IAFP AnnualMeeting entitled "White-rot Fungi Species used as a Biocontrol Method in Bioreactors to Inhibit Escherichia coli for Pre-Harvest Food Safety. Alexis N. Omar presented a poster at the University of Delaware's College of Agriculture and Natural Resources' 2020 Fall Research Symposium. Patrick McGay, an undergraduate student, gave oral presentations at the annual symposium for the Delaware Water Resources Center's Scholars on May 13, 2021. Pat will also complete a written report on his research. Two undergraduate students participated in funded research (Delaware Water Resources Agency) projects related to this research and wrote a paper submitted to the agency. Sicily Broderick's wrote a paper for the Delaware Water Resources Agency's Internship Program entitled: "Analysis of Hydrogen Peroxide Concentration in Effluent from Fungal Bioreactors." Zachary Burcham's paper was entitled: "Optimization of HPLC analysis of ergosterol to quantify the fungal biomass growth within bioreactors." What do you plan to do during the next reporting period to accomplish the goals?Continue experiments to complete objective 1. We will be evaluating the flow through hydraulics within the bioreactors. We will calculate actual and theoretical hydraulic retention time for the bioreactors using a Mn tracer test. We will continue to work with Dr. Kang who will conduct column tests with the fungi on the support materials to assess the fungi's ability to degrade the antibiotics. We will work with the E. coli in a buffer system instead of the manure to elucidate the mechanism the fungi use to degrade the bacteria. The three support materials will be evaluated for their ability to support and aid the fungi in the reduction of the bacteria.

Impacts
What was accomplished under these goals? Progress Summary: Evaluation of E. Coli Reduction in Batch Bioreactors with Pleurotus Ostreatus (PO) on Spent Mushroom Compost (SMC) The batch experiment consisted of 4 different treatments (Figure 1). Each treatment was done in triplicate. Each Treatment was run separately with one replication of the uninoculated control (T4). For Treatment 1, the bioreactors contained Spent Mushroom Compost (SMC) inoculated with live PO. For Treatment 2, the bioreactors contained inoculated SMC that were autoclaved. Treatment 3 was the negative control consisting of the sterilized SMC. Treatment 4 was the un-inoculated control, which had the sterilized support and was treated with un-inoculated aqueous dairy manure that contained no E. coli TVS355. Aqueous dairy manure inoculated with E. coli TVS355 was applied to all bioreactors with a multichannel peristatic pump at a rate of 35 ml/min. Approximately 1 liter of manure was applied to the reactors (¾ bioreactor volume). The batch experiment was run for 10 days with the following sampling times: Day 0, 1,3,5,7 and 10. Samples were collected from the effluent, the support biomass, the inoculated influent, and the non-inoculated influent. The following analyses were performed on the samples. E. coli TVS 355 : Enumerated on MacConkey agar with Rifampacin or Nalidixic acid Ergosterol : Solid Phase Extraction & HPLC COD : SMWW 5220 A. Acid digestion/ colorimetric pH : pH strips LiP and MnP enzyme assays OP TKN The ortho phosphorus increased in the bioreactors over time (Figure 2). However, in the treatment with the live fungus the concentration of OP decreased after day 7. This indicated that the fungus was utilizing the nuetrients available in the manure and that which leached from the compost support. Figure 1. SMC experimental set up. . Figure 2. Change in Ortho Phosporus over time in Bioreactors with SMC and Pleurotus ostreatus. Modification of Peroxidases Enzyme Analytical Methods for Complex Media from Solid State Bioreactors use to Reduce Pathogens in Dairy Manure (Undergraduate Student Project) The objective of this research was to monitor the fungal bioreactors during treatment of dairy manure containing E. coli for both Lignin Peroxidase (LiP) and Manganese Peroxidase (MnP). The standard enzyme assays used to monitor the ligninolytic enzymes use colorimetric methods. The complex media from the bioreactors interferes with the standard enzyme assays. Therefore, a review of the literature was done to modify the methods so the assays will be effective for the dark-colored media from the bioreactors. The tests were run on the clear growth solution containing the fungus to confirm the success of the analyses. Once the tests were confirmed successful, the assays will be performed on samples taken from the bioreactors during the E. coli degradation experiments. The new procedures were documented in the laboratory protocols and used to assay the enzyme activity during the bioreactor experiments Tables 1 and 2). The highest days of LiP activity occurred on Day 3 (Figure 3) and then again on Day 13 while MnP showed maximum activity on Day 13 (Figure 4). Figure 3. LiP Activity over time in liguid growth cultures Figure 4. MnP Activity over time in liguid growth cultures Table 1. Modified Lignin Peroxide Assay Modified Lignin Peroxide Assay Reagents: 0.5 mL 0.160 mM azure B 1.0 mL 125 mM sodium tartrate buffer, pH 3.0 0.5 mL sample 0.5 mL 2 mM H2O2, prepared daily. Add the H2O2 only when ready to make readings. H2O2 initiates the reaction. Final concentrations for 2.5 mL of reaction mixture: 50 mM sodium tartrate buffer, pH 3.0 0.320 mM azure B 0.1 mM H2O2 Spectrophotometer Use: Mix sample well. Place Sample in Spectrophotometer Add the H2O2 mixing well when adding. Push RUN on Spec. Record the reaction (after adding H2O2) at 5 second intervals for at least 2.5 minutes at 651 nm on a continuous spectrophotometric assay. Make sure the spectrophotometer has ample time to warm up. Data Analysis Find the largest drop in absorbance over the time to calculate enzyme activity, U. U LiP = (Δabs/min * 1000mL/L)/(48.8 cm2 * mmole-1 * 1cm * ml of sample) Table 2. Modified Manganese Peroxidase Assay Modified Manganese Peroxidase Assay Reagents: 300 μL 20 mM sodium succinate buffer, pH 4.5 with 5 mL 2 mM H2O2 per 100 mL (for stabilization) 200 μL 250 mM sodium lactate 200 μL 1 mM MnSO4 200 μL 1 g/L phenol red solution 100 μL 1 g/L Bovine serum albumin 1 mL sample Final concentrations in 2 mL of reaction o 2 mM sodium succinate buffer, pH 4.5 o 25 mM sodium lactate o 0.1 mM MnSO4 o 0.1 mM phenol red o 0.1 mM H2O2 o 1 g/L Bovine serum albumin Spectrophotometer Use: Mix sample well. Place Sample in Spectrophotometer Add the H2O2 mixing well when adding. Push RUN on Spec. Record the reaction (after adding H2O2 in sodium succinate) at 5 second intervals for at least 2.5 minutes at 610 nm on a continuous spectrophotometric assay. Make sure the spectrophotometer has ample time to warm up. Data Analysis Find the largest rise in absorbance over the time to calculate enzyme activity, U. U MnP = (Δabs/min * 1000mL/L)/(22 cm2 * mmole-1 * 1cm * ml of sample)

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Omar, A. S. Palani, P. K. Litt, K. McCaughan, A. E. M. Chirnside and K. Kniel. 2020. White-rot Fungi Species used as a Biocontrol Method in Bioreactors to Inhibit Escherichia coli for Pre-Harvest Food Safety. IAFP Annual Meeting October 26-29, 2020 P3-85.
  • Type: Other Status: Accepted Year Published: 2021 Citation: McGay, P. A. Inkster,and A E M Chirnside. 2021. Modification of Peroxidases Enzyme Analytical Methods for Complex Media from Solid State Bioreactors use to Reduce Pathogens in Dairy Manure.Delaware Water Resource Center's Scholars Annual Symposium May 13, 2021.


Progress 06/01/19 to 05/31/20

Outputs
Target Audience:Other graduate students and researchers attending the University's graduate research symposium. Changes/Problems:The building where my research laboratory is located is being renovated beginning July 2019. Due to building renovations, I have moved to a temporary location in August of 2019. I am still waiting for a chemical hood to be install, which has prevented the analysis of the samples for nitrogen. Due to the pandemic, we have not had access to the lab starting on March 19, 2020. We should have access to the lab sometime in late June or July 2020. These interruptions have delayed the progress of the bioreactor experiments and has delayed our timeline for accomplishing the research work. What opportunities for training and professional development has the project provided? Four undergraduate students worked on the project and learned microbiological techniques and analytical methods for ergosterol, nitrogen, COD, pH, H2O2, and phosphorus analysis. The two graduate students gained research experience in microbiology and analytical chemistry. Two undergraduate students participated in funded research (Delaware Water Resources Agency) projects related to this research. Sicily Bordrick's project was the determination of the mode of action that the White Rot Fungi within Solid State Bioreactors use to Reduce Pathogens in Dairy Manure. Zachary Burcham's project was the optimization of HPLC analysis of ergosterol to quantify the fungal biomass within the bioreactors. How have the results been disseminated to communities of interest? Sivaranjani Palani and Aubrey Inkster presented a poster at the University of Delaware Environmental Institute Graduate Student Symposium, March 5, 2020 entitled "Determination of the efficacy of fungal bioreactors to remove E. coli from aqueous dairy manure through assessment of growth support materials and flow regimes- woodchips and batch flow." Alexis N. Omar presented a poster at the 2019 IAFP AnnualMeeting entitled "Establishing Baseline Inhibition of Escherichia coli in Aqueous Dairy Manure Prior to Treatment by a Fungal Biocontrol Agent" Undergraduate students gave oral presentations at the annual symposium for the Delaware Water Resources Center's Scholars on May 14, 2020. They will also complete a written report on their research. What do you plan to do during the next reporting period to accomplish the goals? Continue experiments to complete objective 1 and move forward to the next 2 objectives. Continue work with Dr. Kang to evaluate extraction and detection methods for antibiotic analysis of the different support materials. Work with Dr. Kniel to move forward to objective 4 in coordination with the other objectives.

Impacts
What was accomplished under these goals? Progress Summary: Evaluation of E. Coli Reduction in Batch Bioreactors with Pleurotus Ostreatus (PO) on Wood Chips The batch experiment consisted of 4 different treatments (Figure 1). Each treatment was done in triplicate except for treatment 4, resulting in 10 total bioreactors. For Treatment 1, the bioreactors contained wood chips inoculated with live PO. For Treatment 2, the bioreactor contained inoculated wood chips that were autoclaved. Treatment 3 was the negative control consisting of the sterilized wood chips. Treatment 4 was the un-inoculated control, which had the sterilized support and was treated with un-inoculated aqueous dairy manure that contained no E. coli TVS355. Aqueous dairy manure inoculated with E. coli TVS355 was applied to all bioreactors with a multichannel peristatic pump at a rate of 35 ml/min. Approximately 1 liter of manure was applied to the reactors (¾ bioreactor volume). The batch experiment was run for 10 days with the following sampling times: Day 0, 1,3,5,7 and 10. Samples were collected from the effluent, the support biomass and the influent. The following analyses were peformed on the samples. E. coli TVS 355 : Enumerated on MacConkey agar with Rifampacin or Nalidixic acid Ergosterol : Solid Phase Extraction & HPLC COD : SMWW 5220 A. Acid digestion/ colorimetric pH : pH strips Figure 1. Batch Flow Experiment 1: Pleurotus ostreatus on wood chips. Treatment 1: live fungus, Treatment 2 autoclaved fungus, Treatment 3 autoclave woodchips, Treatment 4 autoclaved woodchips without E. coli TVS 355. Optimization of Ergosterol Analysis with Quantification to Fungal Growth (Undergraduate Student Project) The objective of this research was to develop an accurate extraction and clean-up procedure to monitor the concentration of ergosterol within fungal biomass growing on complex support media. Specifically, we modified a cleanup procedure to increase the efficiency of ergosterol extraction. Extraction efficiencies were calculated using spiked media samples. Ergosterol concentrations were measured using High Pressure Liquid Chromatography (HPLC). A standard curve was developed relating ergosterol concentration to peak area. The quality control measurements were used to validate the modified procedure developed in this project and to establish method detection limits. The developed ergosterol concentrations were correlated to the actual weight of fungal biomass for each fungus. Production of ergosterol may not be similar with the two different fungi. Development of a Test for Hydrogen Peroxide Concentration within the Bioreactors (Undergraduate Student Project) The objective of this research was to monitor the fungal bioreactors during treatment of dairy manure containing E. coli for both Lignin Peroxidase, Manganese Peroxidase, and hydrogen peroxide in order to ascertain the mode of action the fungi use to degrade the E. coli. The procedures needed to be modified in order to work with the dark colored manure. This work concentrated on the measurement of hydrogen peroxide, H2O2, by a colorimetric procedure that used ferrous ammonium sulfate and xylenol orange to produce an orange color when reacted with hydrogen peroxide. A standard curve was developed, and extraction efficiency was evaluated.

Publications

  • Type: Other Status: Submitted Year Published: 2020 Citation: Burcham, Z., S. Palani, and A. E. M. Chirnside. 2020. Optimization of HPLC Analysis of Ergosterol to Quantify Fungal Biomass Growth within Bioreactors. Delaware Water Resources Center Annual Symposium Report. University of DE. May 16, 2020 Bordrick, S., S. Palani, and A E M Chirnside. 2020. Optimization of Hydrogen Peroxide Analysis to Quantify Enzyme Activity within Bioreactors. Delaware Water Resources Center Annual Symposium Report. University of DE. May 16, 2020


Progress 06/01/18 to 05/31/19

Outputs
Target Audience: Nothing Reported Changes/Problems:The building where my research laboratory is located is being renovated beginning July 2019. Due to the renovations I have to move to a temporary location for 2-3 months and then move a second time to the permanent location. This move schedule has delayed the start of the bioreactor experiments and has delayed our timeline for accomplishing the research work. Once we move to the final location, we should be able to proceed without further delay. What opportunities for training and professional development has the project provided? Undergraduate students worked on the project and learned microbiological techniques and analytical methods for nitrogen analysis. The two graduate students gained research experience in microbiology and analytical chemistry. They also prepared research posters of their work. How have the results been disseminated to communities of interest? Graduate students presented a poster at the University of Delaware Environmental Institute Graduate Student Symposium, March 7, 2019. Poster Title-"Determination of the efficacy of fungal bioreactors to remove E. coli from aqueous dairy manure through assessment of growth support materials and flow regimes." What do you plan to do during the next reporting period to accomplish the goals?Finish Objective 1 and begin Objectives 2 and 3 with the optimized design. Meet with Dr. Kang from Morgan State University to update him on the Objective 1 progress and work with Dr. Kang and Dr. Kniel to accomplish the final objectives.

Impacts
What was accomplished under these goals? Progress Summary: Bioreactor Construction: During the past year, we bought the materials needed to build the bioreactors. The inert support, reticulated polyurethane foam, was purchased from Merryweather Foam, Inc. The other support materials, wood chips (WC) and spent mushroom compost (SMC), were also obtained. The wood chips were purchased from a local supplier. The SMC was donated to the project by Phillips Mushroom Farms, of Kennett Square, PA. The three support materials were analyzed for the following parameters: water holding capacity, bulk density, porosity, and pH. Total carbon and total nitrogen of the organic support materials were measured. The bioreactors were made of rectangular plastic containers of volume 1.89 L (19cm x 10cm x 19cm). A hole was drilled at the bottom of the container for effluent collection. The drain hole was fitted with 1/4" PVC ball valve and fastened with a fitting pipe (1/4" x 1/4"). The fitting pipe was covered with a fine mesh fiberglass screen secured by a rubber band to prevent clogging of the valves. The entire set up was made watertight with a heavy-duty marine water sealant. Inoculum Development: The wood chips were chopped and homogenized by passing through a 5.08 cm sieve. 100 ml deionized distilled water (DDW) was added to each 500 g of woodchips and they were autoclaved twice for 45 minutes with an interval of 24 hours. Pleurotus ostreatus spawn grown on sterilized rye berry seeds were used for inoculating the wood chips. The P.ostreatus inoculum was added in the ratio 1:1 by weight. 100 ml of DDW was added for every 500 g of autoclaved woodchips at the time of inoculation. Two days later 100 ml of DDW was again added to the P.ostreatus inoculated wood chips. Fungal growth solution was added to the woodchips after 4 weeks. The woodchips were inoculated in two batches. The woodchips inoculated jars were closed loosely and the lid was covered with parafilm to avoid contamination. They were frequently mixed for aeration. Once fungal growth was established, the wood ships were added to the bioreactors to initiate the experiments. Evaluation of the persistence of E. coli in the liquid dairy manure Methods: E. coli TVS355 and O157:H7 (4407) were inoculated into liquid dairy manure, obtained locally, at 4 and 8 log-cfu/mL, incubated at 37°C or 22°C, and sampled at 0, 24, 48 and 72hrs. Bacteria were enumerated on MacConkey agar with Rifampicin (MACR) or Nalidixic Acid (MACN). Bacterial survival was evaluated using a one-way Anova across 10 trials, with n=4 per treatment. P. chrysosporium, obtained locally, was maintained on 2% malt agar at 39°C. Results: Liquid manure remained at pH 8.0-8.2 across the sampling time, with a consistent water activity of 0.99. In all studies E. coli did not grow in manure; however, greater die off was observed by E. coli O157:H7 compared to E. coli TVS355 (p>0.05). Regardless of inoculum level, E. coli O157:H7 decreased by 0.71 log-cfu/mL from 0 to 24hrs and by 1.46-1.57 log-cfu/mL from 24 to 48hrs. Comparatively, E. coli TVS355 decreased by 0.21-0.19 log-cfu/mL from 0 to 24hrs and by 0.46-1.11 log-cfu/mL from 24 to 48 hours. E. coli O157:H7 was not detected at 72hrs, but E. coli TVS355 survived in liquid manure at 72hrs at 6.6 log-cfu/mL for higher inoculum and 2.3 log-cfu/mL for the lower inoculum. E. coli inhibition was further assessed in the presence of P. chrysosporium.

Publications