PHOTOCATALYTIC DISINFECTION OF AGRICULTURALLY IMPACTED WATERS: CHARACTERIZATION OF SOLUTION CHEMISTRY ON BACTERIAL ADHESION AND DISINFECTION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1001293
• Grant No.: 2013-67012-21277
• Project No.: CALW-2013-03306
• Proposal No.: 2013-03306
• Program Code: A7201
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2015
• Grant Year: 2013

Project Director
Kinsinger, N.

Recipient Organization
The Regents of University of California
200 University Office Building
Riverside,CA 92521

Performing Department
Chemical and Environmental Eng

Non Technical Summary
My aim as a post-doctoral researcher is to use my background in engineering and science to conduct research and develop new technologies for environmental remediation and protection of public health. Dr. Walker's expertise in bacterial adhesion and water quality will allow me to take my work in new directions, focusing on the application of TiO2 photocatalytic treatment for water and food safety. This USDA Fellowship Grant will enable me to build the skills necessary for a career in environmental research and college-level teaching. This goal is a product of a longstanding enthusiasm for academic pursuits combined with the desire to protect our natural resources. My doctoral training and research interests provide me with the multidisciplinary skills that make me ideally suited to complete the proposed post-doctoral study, which merges material science and environmental engineering. Water is an essential resource for life, yet each year Escherichia coli infects 75,000 people and caused 61 deaths within the United States from contaminated food and drinking water Drinking water sources are frequently contaminated by agricultural run-off or soil penetration from animal waste water lagoons. Many pathogenic viruses and bacteria are known to be resistant to chlorine disinfection. While increasing chlorine concentration will increase its efficacy, it will also lead to an increase in carcinogenic trihalomethanes (THMs) causing increased concern for public health and safety. Disinfection via oxidation of organics by hydroxyl radicals (OH•) is an alternative and supplemental technology for water treatment that does not require significant capital investment compared with processes such as reverse osmosis. It also eliminates the concern of producing secondary byproducts such as THMs due to complete mineralization. Various methods are used to produce OH• including degradation of H2O2, O3, and H2O2/O3, using ultra-violet radiation (UV). Adding a photocatalytic material can significantly accelerate the production of these radicals that degrade organics non-selectively. Photocatalysts work by absorbing sunlight and, in the presence of water, generates OH• radicals. TiO2 is an inexpensive example of a photocatalytic material that can be easily integrated into an existing treatment system and isolated from the effluent liquid stream. Typical TiO2 photocatalytic systems are optimized under ideal slurry reactor conditions based on photocatalyst material properties such as particle size, surface area, phase, and surface charge. However not only the photocatalyst material properties are important as it has been shown that reduced interaction distances between the target (organic or bacteria) and photocatalyst surface enhance the efficiency activity since to the short lifespan of the reactive oxygen species. Photocatalytic technology has the potential to be applied a wide variety of water treatment applications such alternative agriculture runoff treatment, dairy water recycling, and produce rinse water disinfection. However there are limitations to overcome before such technology is deployable for water treatment applications. The proposed work seeks to evaluate the effectiveness of TiO2, in the degradation of a model bacteria and pesticide, and with the mechanistic understanding of these processes, optimize a reactor for water treatment. The specific objectives of this work are as follows: Research Objectives Quantification of bacterial adhesion under flow to immobilized food grade TiO2 nanoparticles surfaces under simulated conditions of agriculturally impacted surface waters. Quantification of batch photocatalytic generation of reactive oxygen species by immobilized food grade TiO2 nanoparticles surfaces under simulated conditions of agriculturally impacted waters. Quantification of batch photocatalytic degradation of model organic pollutants (i.e. pesticides) using immobilized food grade TiO2 nanoparticles surfaces under simulated conditions of agriculturally impacted waters. Evaluate different photocatalytic disinfection reactor designs to optimize cell adhesion, organic degradation, and ROS formation (based upon mechanistic understanding gained from objectives 1-3)

Animal Health Component

0%

Research Effort Categories

Basic

80%

Applied

0%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	4010	2020	70%
712	0210	2020	30%

Knowledge Area
133 - Pollution Prevention and Mitigation; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
0210 - Water resources; 4010 - Bacteria;

Field Of Science
2020 - Engineering;

Keywords

photocatalytic disinfection

bacterial adhesion

titanium dioxide

Goals / Objectives
My goal as a USDA Fellow and post-doctoral researcher is to continue my education and development of necessary skills to be on the cutting edge of environmental and water quality research and continued development of my mentoring skills to be an effective leader in the scientific community. I aim to use my background in engineering and material science to conduct research and develop new technologies for environmental remediation and protection of public health. Dr. Walker's expertise in bacterial adhesion and water quality will allow me to take my expertise in new directions, focusing on the application of TiO2 photocatalytic disinfection for water and food safety. The proposed work seeks to evaluate the effectiveness of TiO2, in the degradation of a model bacteria and pesticide, and with the mechanistic understanding of these processes, optimize a reactor for water treatment. This training, and my research interests, provides me with the multidisciplinary skills that make me ideally suited to complete the proposed post-doctoral study merging material science and environmental engineering. Rationale and Significance With increasing awareness of water conservation and protection, photocatalytic technology has the potential to be applied to a wide variety of water treatment applications such alternatives for agriculture runoff treatment, dairy water recycling, produce rinse water disinfection to protect the food supply from potential bacterial outbreaks while continuing to protect our water supply. Outbreaks observed in produce are becoming increasing common possibly due to contaminated irrigation waters or contaminated waters used during processing. These waters may become contaminated animal manure from runoff or leached into surface and ground waters supplies during rain events 1. This becomes specifically problematic is more rural farming communities that may rely solely on well water which may become contaminated and is of key concern in developing countries. Typical TiO2 photocatalytic systems are optimized under ideal slurry reactor conditions based on photocatalyst material properties such as particle size, surface area, phase, and surface charge. However, not only the photocatalyst material properties are important, since adhesion to the catalyst surface significantly effects disinfection due to the short lifespan of the reactive radical species. There are limitations to overcome before such technology is deployable for water treatment applications. The photocatalytic activity also has the versatility for antimicrobial surface applications such as self-cleaning coatings for use as food preparation surfaces that will also reduce outbreaks and spread of bacteria such as E. coli. This research plan responds to the USDA Program Area Priority #2, Food Safety, Nutrition, and Health; by ensuring that water onsite is free from microbial contamination this will be meeting the USDA's goal of ensuring food safety and security. Research Objectives Quantification of bacterial adhesion under flow to immobilized food grade TiO2 nanoparticles surfaces under simulated conditions of agriculturally impacted surface waters. Quantification of batch photocatalytic generation of reactive oxygen species by immobilized food grade TiO2 nanoparticles surfaces under simulated conditions of agriculturally impacted waters. Quantification of batch photocatalytic degradation of model organic pollutants (i.e. pesticides) using immobilized food grade TiO2 nanoparticles surfaces under simulated conditions of agriculturally impacted waters. Evaluate different photocatalytic disinfection reactor designs to optimize cell adhesion, organic degradation, and ROS formation (based upon mechanistic understanding gained from objectives 1-3)

Project Methods
Objective 1: Ideal and Simulated Bacterial Adhesion Operating parameters such as pH have been reported to greatly affect heterogeneous photocatalytic performance due to particle aggregation which will reduce accessible surface active sites and block excitation events within the catalyst. The system pH also affects the ionization state of catalyst surface and the organic compounds which has been shown to affect the sorbate-surface interactions which impact performance since OH• radicals are easily scavenged. Commercial food grade TiO2 (E171) materials will be selected to vary surface area, particle size, and phase (anatase/brookite/rutile) which are all know to affect photocatalyst performance. These TiO2 materials will be immobilized on a glass annular photoreactor and glass surfaces (to be used in a parallel plate (PP) flow chamber) utilizing a dip-coating procedure outlined by van Grieken et al. The clean glass reactor will be dip-coated using a 150g/L suspension of TiO2 in deionized water adjusted to pH1.5 using nitric acid. The reactor will be dried at 110°C for 24 hours following each coating cycle and then calcined at 500°C for 2 hours heating at a rate of 5°C per minute. Catalyst Material Characterization: TiO2 materials immobilized on the glass surfaceswill be characterized using methods Scanning Electron Microscopy, Bright-field Transmission Electron Microscopy, and X-Ray diffraction will be used to investigate the size, morphology, phase, and thickness of the TiO2 films both prior to and following calcination. The Brunauer, Emmett, and Teller method will be used to determine surface area and pore size distributions. The zeta potential will be determined for each sample surface by measuring the streaming potential. A streaming potential analyzer (EKA, Anton Paar, Graz, Austria) with an asymmetric clamping cell will be used to determine the electrokinetic properties of the sample surfaces. Roughness will be measured by atomic force microscopy to provide a measure of the surface roughness and uniformity of the surface. Bacterial Adhesion Characterization: Escherichia coli O157:H7 will be used as a model pathogen, to characterize effect of TiO2 properties on adhesion under various simulated conditions of agriculturally impacted waters (artificial surface water spiked with model pesticides). Individual solution chemistry and environmental parameters including pH, ionic strength, natural organic matter, water temperature, and the effects of photocatalyst material and morphology and morphology will be investigated. Adhesion studies will be conducted by recycling a stock suspension of E. coli (approximate concentration of 108 cells mL−1) for 6 cycles at various flow rates to account for the effect of shear. Following adhesion the influent solution will be switch to a bacteria-free suspension at the same chemical composition. The effluent will be measured using a UV-visible spectrophotometer at a fixed wavelength of 546nm to characterize the bacteria breakthrough curves. Parallel Plate Bacterial Adhesion Characterization: The mass transfer of bacteria onto the solid-water interface under range of solution chemistry will be measured in a parallel plate (PP) flow chamber system available at UCR. This flow chamber is installed on the stage of an inverted fluorescent microscope (BX-52 Olympus) allowing for the quantification of bacterial adhesion under flowing conditions. The bottom of the flow cell is a microscope slide, which is typically used as the test surface in PP studies. However, the flow cell has been modified to insert a test surface on the upper surface (installed into a grove in the upper acrylic flow deck) such that deposition of cells onto a non-transparent test surface - in this case a immobilized TiO2 film on glass) - will be investigated. Bacterial samples will be suspended in solutions (described above) and injected into a parallel flow chamber system at flow rates within the PP system will be tested across a range of diffusion dominated to turbulent conditions, mimicking the type of flows experienced in various stages within the photocatalytic reactor. We will apply different shear rates in close proximity to the test membrane surface (where shear rate is maximum) in the PP channel that are between 1 to 103 sec-1. Objective 2: Photocatalytic Generation of Reactive Oxygen Species (ROS) Measurement of steady-state hydroxyl radical (OH•) concentrations will be performed using a batch reactor illuminated by a 450W O3-free xenon arc lamp to produce a collimated beam with a water filter to remove infrared radiation. A 305nm long pass filter will be used to produce light in the UV and visible range. Steady-state concentrations of OH• will be determined using phenol as a probe. Prepared sample of immobilized TiO2 on the glass substrate will be immersed in know starting concentrations of phenol (100μM). These reactors will be exposed to the light over several hours will 1mL aliquots taken every hour and analyzed using High Performance Liquid Chromotagraphy. Objective 3: Degradation of Model Organic Pollutants Photocatalytic degradation of organic pollutants (such as herbicide: Atraezine, or insecticide: Diazinon) will be conducted to characterize the effect of immobilized TiO2 properties on degradation rates of such pollutants. During the photocatalytic degradation studies system parameters investigated in Objectives 1-2 will be adjusted individually to identify the effect of individual constituents within agriculture impacted waters such as ionic strength and natural organic material. Subsequently photocatalytic degradation studies using agriculturally impacted water with the cocktail of constituents will be performed. Objective 4: Photocatalytic Disinfection Based on results from Objectives 1-3, studies will be conducted to characterize the effect of bacterial adhesion to the TiO2 photoreactor on disinfection rates. E. coli will be used again as the model pathogen and the same annular photoreactor with immobilized TiO2 (prepared as described above) will be used to conduct the disinfection studies. The disinfection rates will be measured by quantifying the concentration of viable bacteria at predetermined times via serial dilution procedures. During the photocatalytic disinfection studies system parameters investigated in Objective 1 will be adjusted individually to identify the effect of individual constituents within agriculturally impact waters on disinfection. Subsequently photocatalytic disinfection studies using simulated agriculturally impacted water with the combination of constituents will be performed. These detailed studies will develop understanding necessary for real applications of this technology and will guide photocatalytic reactor and system designs to account for natural fluctuations within the influent from various sources and seasonal variations. There are some limitations and issues that might be encountered during this project. Such issues may include low reactive oxygen generation by certain nanoparticle/surface combinations resulting in immeasurable disinfection rates or pesticide degradation, due to material properties or insufficient UV exposure of TiO2 in the reactor geometry. In these cases, different reactor geometries, photocatalyst design (nanoparticle type and coating), and enhanced retention time in reactors will be considered. Annual Milestones: Year 1 Completion of the preparation and characterization of the photocatalytic reactors to be used for bacterial adhesion Completion of bacterial adhesion studies Conference attendance and presentation on bacterial adhesions studies Year 2 Completion of photocatalytic ROS generation studies Completion of pesticide photocatalytic degradation and disinfection studies. Conference attendance and presentation on correlation of adhesion to disinfection rate Manuscript submitted for publication on resulting studies

Progress 09/01/13 to 08/31/15

Outputs
Target Audience:This USDA Fellowship Grant aided my development of skills to be a future leader in environmental research and college level teaching. I was able to be lead my own unique research project as well as mentor and guide several undergraduate and graduate students in their research which has aided me in building my research portfolio and expanding my capabilities as an educator. In addition, I was able to pursue an additional project related to this subject to fully explore the environmental issues related to food safety that I independently developed further preparing me for my future career. Additionally, through Dr. Walker's (faculty mentor) active outreach through a USDA-funded Hispanic Serving Institution (HSI) project, I mentored several Riverside Community College undergraduate students who are considering studies and careers in engineering as part of Dr. Walker's USDA HSI grant. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Throughout this project I had several opportunities for training and professional development that I participated in. These activities included conference attendances, water quality and resource workshops and mentoring undergraduate students (both at University of California, Riverside and the local community college) and graduate students. I was also able to participate in several collaborations further expanding my knowledge and research portfolio. Additionally my adviser included me in several grant application to develop my grant and scientific writing. In addition to formal professional development this project provided the opportunity to expand in new research directions both through the mentoring graduate students on a variety projects, and by pursuing my research interests. In contrast to my graduate work, this fostered an interest in pursuing applied research specifically in food safety which has direct implications in public policy. How have the results been disseminated to communities of interest?The results have been (and will soon be disseminated) through conference presentations, papers (soon to be published) and press releases. While academic research is typically disseminated to communities of interest through conferences and papers, these modes only reach a limited audiences. Therefore a press release was published to reach a broader community since our work is addressing issues of our food safety. Additionally I presented in several undergraduate courses to not only reach a broader community but also to speak of undergraduate research opportunities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact The US Center for Disease Control (CDC) estimates that 1 in 6 Americans becomes ill, 3,000 die and the industry loses over $75 billion annually from foodborne diseases. In 20 years, 350 outbreaks were associated with Escherichia coli O157:H7 and 52% were foodborne. Contamination may occur at any step from farm to table and survive during cleaning, packaging, and storage. While chlorine disinfection can be effective, photocatalytic disinfection is an alternative and supplemental technology that focuses on the prevention of the critical contamination stage - initial adhesion by killing bacteria in the water or on equipment surfaces. A reactor can be installed to disinfect rinse water (frequently recycled) from bacteria that have detached from leaves and have been suspended within the water thus spreading the contamination throughout the crop. There are also many surfaces that the leaves contact during processing and are difficult to clean. These surfaces may be coated with titanium dioxide to create an antimicrobial surface to prevent further contamination as well. Titanium dioxide surfaces with varying roughness, phase, and grain sizes were prepared to evaluate bacterial attachment and photocatalytic activity. Roughness was identified as a key parameter to enhance bacterial surface attachment, however due to processing conditions surfaces with increased roughness also were observed to reduce photocatalytic activity which will reduce the antimicrobial effectiveness. Therefore we identified a processing condition which optimizes bacterial attachment and photocatalytic activity ideal for implementation within a reactor or antimicrobial surface to reduce food-borne outbreaks. Accomplishments Goal #1: Quantification of bacterial adhesion under flow to immobilized food grade TiO2nanoparticles surfaces under simulated conditions of agriculturally impacted surface waters. Titanium dioxide (TiO2) thin films were prepared by dip coating glass coupons. Suspensions were prepared using commercially available TiO2 suspended at varying pH conditions to control the resulting thin film roughness. Subsequently the samples were fired to anneal the TiO2 thin film to the glass and control grain size and phase. These coupons were mounted within a parallel plate flow chamber to quantify bacterial attachment under flow conditions. A simple potassium chloride solution was used to investigate the impact of agriculturally relative ionic strength on the bacteria attachment. Increasing ionic strength increases the rate of bacterial attachment indicating that the attachment mechanism was dominated by electrostatic interactions for all TiO2 samples predominately composed of anatase. However bacterial attachment to TiO2 samples composed of rutile was not observed even at significantly high ionic strengths. This indicates that rutile surface energies are unfavorable for bacterial attachment. For the anatase samples two preparation conditions were relative, suspension pH 1.5 and pH 3. Samples prepared at pH1.5 resulted in weakly attached films and smoother surfaces. Samples prepared at pH 3 resulted in more durable thin films and rougher surfaces. The films were subsequently fired at 3 temperatures: 400C, 600C, and 700C. All samples prepared at pH3 resulted in more than 3 times bacterial attachment compared to films prepared at pH1.5. The increasing firing temperature also resulted in increasing bacterial attachment. This indicates that the increased surface roughness increase bacterial attachment. Goal #2: Quantification of batch photocatalytic generation of reactive oxygen species by immobilized food grade TiO2nanoparticles surfaces under simulated conditions of agriculturally impacted waters. Due to time limitations and equipment availability this segment of the project was delayed until much later into the project than initially anticipated. However to continue to evaluate the potential application of this technology for agricultural disinfection we preceded to address the next objective and this work is still ongoing. Goal #3: Quantification of batch photocatalytic degradation of model organic pollutants using immobilized food grade TiO2nanoparticles surfaces under simulated conditions of agriculturally impacted waters. The films prepared and evaluated in Goal #1 were used to evaluate the impact of the film properties on the photocatalytic activity. A batch reactor was irradiated with UV light (centered around 365nm) over several hours and the process of removing 1mL aliquots was used to evaluated the photocatalytic activity. Since the samples were thin films, a 1 in square coupon was placed within the reactor. The photocatalytic activity, similar to the bacterial attachment, was dependent on both the processing and firing conditions. All samples prepared at pH1.5 resulted in nearly 2 times the activity than the samples prepared at pH3 (the inverse of bacterial attachment). This effect was is attributed to the reduction of surface area of the samples prepared at pH3 due to larger aggregates present in the suspension. In addition, by increasing the firing temperature there was a significant decrease in bacterial attachment due to crystal growth and densification of the films. Goal #4: Evaluate different photocatalytic disinfection reactor designs to optimize cell adhesion, organic degradation, and ROS formation (based upon mechanistic understanding gained from objectives 1-3). Based on the results observed within Goals #1 and #3 the optimal material to incorporate within a reactor or an antimicrobial surface would be the thin film prepared at pH3 and fired at 400C. This is associated with several parameters: bacterial attachment, photocatalytic activity, and durability. The photocatalytic activity of this sample was not the highest amongst all samples however this sample significantly increased the bacterial attachment. Disinfection is significantly dependent on proximity to the catalytic surface due to the short lifespan of radical species. Therefore increasing the number of bacteria directly attached to the catalyst surface would significantly increase the disinfection efficacy despite the reduced photocatalytic activity. Therefore increasing the surface roughness, while minimizing the reduction in surface area associated with thin film processing is vital.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kinsinger, N. M., Honda, R.; Keene, V., Walker, S. L.; Titanium Dioxide Nanoparticle Removal in Primary Stages of Water Treatment: Role of Coating, Natural Organic Matter, Source Water, and Solution Chemistry. Environmental Engineering Science 2015
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Flores, Jessamine, Joung, Young Soo, Kinsinger, Nichola, Lu, Xinglin; Buie, Cullen, Walker, Sharon; Antimicrobial Behavior of Novel Surfaces Generated by Electrophoretic Deposition and Breakdown Anodization. Colloids and Surfaces: B, 2015, in press
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Lin, S., A.A. Taylor, Z. Ji, C.H. Chang, N.M. Kinsinger, W. Ueng, S.L. Walker, and A.E. Nel. Understanding the partitioning, transformation, speciation, and hazard potential of copper-based particles through integrating a model septic tank and zebrafish embryo high-throughput screening ACS Nano, 2015, 9 (2), pp 20382048
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Rapicavoli, J.N., Kinsinger, N.M., Perring, T.M., Backus, E.A., Shugart, H.., Walker, S.L., Roper, M.C.; O-antigen Modulates Insect Vector Acquisition of the Bacterial Plant Pathogen, Xylella fastidiosa. 2015, PLOS Pathogens, in review
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Kinsinger, N. M., Luth, M., Walker, S. L. Efficacy of Rinsing to Detach and Disinfect E. coli O157:H7 on Baby Spinach 2016, in preparation
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Kinsinger, N. M., Luth, M., Walker, S. L., "Agricultural Application Photocatalytic disinfection of E. coli O157:H7" 2016, in preparation
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Conference Presentation Nichola Kinsinger and Sharon Walker, "Is our salad safe? Efficacy of current disinfection rinses and potential photocatalytic technology to prevent foodborne outbreaks" presented at ACS Fall Meeting, Boston, MA, August 2015
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Conference Presentation Nichola Kinsinger and Sharon Walker, Efficacy of Rinsing to Detach and Disinfect E. coli on Baby Spinach poster presented at Gordon Research Conference  Environmental Sciences: Water, Holderness, NH, June 2014