Source: OHIO STATE UNIVERSITY submitted to NRP
BIOLOGICAL APPROACHES TO MITIGATE BIOFILM-ASSOCIATED FOOD SAFETY RISKS IN INDOOR LEAFY GREENS PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1029776
Grant No.
2023-70019-39364
Cumulative Award Amt.
$996,516.00
Proposal No.
2022-10566
Multistate No.
(N/A)
Project Start Date
Mar 15, 2023
Project End Date
Aug 31, 2027
Grant Year
2024
Program Code
[UIE]- Urban, Indoor and Emerging Agriculture Initiative
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
(N/A)
Non Technical Summary
The market share of hydroponic vegetables is rapidly increasing in the US and globally.? Food safety is critical to the hydroponic fresh produce industry. In addition to being a public health concern, food safety issues have had an adverse economic impact on growers and packers. While the FSMA regulations have been implemented, there is a lack of understanding how to mitigate food safety hazards in hydroponic both among growers and regulators. Novel solutions for producing safe hydroponic crops, and particularly meeting the sanitation standards by mitigating biofilms, are urgently needed. In this project, we will characterize Salmonella spp. and Listeria monocytogenes biofilms, determine the role of algae in the formation of biofilms and identify novel bioremediation procedures to co-mitigation of biofilms and environmental algal mats in two types of hydroponic leafy green production systems (nutrient film technique (NFT) and deep-water culture (DWC)). We will translate the finding into the risk communication materials describing biofilm and algae management best practices in commercial NFT and DWC leafy green indoor production systems.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71214301100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The overall goal of this project is to ensure the food safety of indoor grown leafy greens by characterizing biofilms and their management, including determining their contributions to foodborne pathogen contamination. Based on our preliminary data and previous research, we hypothesize that the risk of human pathogen contamination of hydroponic leafy greens grown indoor can be reduced through the mitigation of biofilms using novel bioremediation approaches and protective cultures, without negatively impacting the plant health, its quality, and nutritional attributes.To this end we will complete the following objectives:Characterize Salmonella and Listeria monocytogenes biofilms in commercial NFT (Nutrient Flow Technology) and DWC (Deep Water Culture) leafy green indoor production systems.Evaluate the influence of algae on the establishment ofSalmonellaandListeriamonocytogeneson leafy green crops and environmental surfaces.Establishment of bioremediation procedures and alternative approaches to co-mitigation of biofilms and environmental algal mats in leafy green indoor production systems.Development of risk communication materials describing biofilm and algae management best practices in commercial NFT and DWC leafy green indoor production systems.Upon the completion of the proposed studies, we will have developed evidence-based effective food safety intervention strategies that will enable hydroponic indoor growers to ensure food safety and meet regulatory food safety requirements (FDA FSMA, Produce Safety Rule). We anticipate that much of the proposed research will have substantial near-term impacts (see Logic Model).Our work determining effective biofilm mitigation parameters will be translated into standard operating procedures usable at all spatial scales and types of growing systems in the industry. Co-mitigation practices for algae biofilms will be of huge importance to industry, and the first of its kind. We will work directly with hydroponic growers to translate our research into procedures. We will pre-test developed standard operating procedures in commercial indoor hydroponic settings.Our work will ensure that adequate sanitation practices are used by the industry, thus improving food safety of fresh produce. This is particularly important in the drive to increase consumption of nutrient dense fresh vegetables by the American public, reduce foodborne illnesses while improving nutrition and overall health. Developing new strategies to mitigate the food safety risks in hydroponics will lead to long term public health improvement and enhance the growth and sustainability of the fresh produce industry and US agriculture.
Project Methods
SalmonellaTyphimurium orL.monocytogenespreviously used in our labwill beused for inoculum. Commercial NFT and DWC systems (CropKing, Lodi OH) will be used in this study. One cropping cycle (~4 weeks) of lettuce 'Rex' will be used to establish native biofilms.Biofilms will bemonitored andcharacterizedusingimmersed substratesthat mimic surfaces found in commercial hydroponic production. Totrackbiofilm production in situ for NFT systems,coupons of tested PVC materialswill be inserted inside individual channels invariouslocationsatknowndistances from the point of inoculation (nutrient solution tank). Immersed materialcouponswillbecollected throughout the cycle, andfinallyrecoveredat the end of the crop cycle.Theviable cellswill be enumeratedusingthedrop plate methodandpaired withcharacterization ofbiofilm community using amplicon metabarcoding targeting bacterial and microalgal ribosomal markers.Briefly, at recovery,thebiofilmsformed onimmersedsurfaceswillbecollectedusing sterile sponge-stickswabs, followed by sonication and centrifugation.Each suspension will be independently used forbothDNA extractionand drop plate estimation of bacterial abundance per substrate. Total bacterial counts will be estimated using general R2Amedium, while enumeration of human pathogenswill be determinedby culturing on LB agar amended with 100 µg/mL nalidixic acid forL. monocytogenesor xylose lysine deoxycholate amended with 75 µg/mL kanamycin forSalmonellaTyphimurium.The amount of exopolysaccharide in biofilms will bemeasured using standard crystal violetstaining.Themicrobial community composition of the sampled biofilms will be characterizedthroughamplicon metabarcoding using bacterial and microalgal ribosomal markers for Illumina sequencing.In addition, immersed substrates will be used to capture biofilms for visualization ofspatialstructureof collected biofilmsusing confocalmicroscopycombined withfluorescence in situ hybridization (FISH).The effects of surface type, substrate location (in relation to the point of inoculation)and time of samplingon total bacterial abundance and abundance ofL. monocytogenesandS. typhimuriumon immersed substrates biofilm will be analyzed.The groups ofalgaepresent in hydroponic systemscan vary depending on the type of system, nutrient concentration,temperature,andwater source.Three groups ofalgaeidentifiedinour research DWC systems- blue-green, green andflagellates. Photosynthetic light (12/12hrphotoperiod)will be provided by growth lampsproviding an average light intensity of 31 500 lux, set0.3 m above each culture.Subsamples (250uL) of each of the microcosmsat 24hrwill be tested and visualized.Biocontrol strategies including the use of living bacterial species (protective cultures) with antimicrobial attributes, enzymes targeting EPS, and bacteriophage will be tested to determine their efficacy in preventing or remediating L. monocytegenes, Salmonella Typhimurium, and/or algal biofilms on representative surfaces in NFT and DWC systems as described in Obj. 1. The biofilm remediation treatments will include 1) Nisaplin, a bacteriocin-containing commercial product, 2) Biofilm Buster Pro, a "lactic acid bacteria" enzymatic product, and 3) PhageGuard LISTEX, a commercial product developed from the bacteriophage P100 that induces cell lysis and disintegration of EPS produced by L. monocytogenes. The biocontrol preventive treatments will include 1) a consortium of native hydroponic bacteria, 2) a consortium of commercially available biological control products (Bacillus amyloliquefaciens (DefGard) and Pseudomonas chlororaphis (Howler)) for control of plant pathogens, 3) a consortium of probiotic Lactobacillus spp. A consortium of native hydroponic bacteria comes from Co-PI Benitez-Ponce's collection of 188 hydroponic bacterial strains and represents the top six dominant genera of bacteria according to community profiling. The biological control products represent two commonly applied plant growth promoting microbial inoculants in hydroponic production. A consortium of five probiotic strains of Lactobacillus spp. will be obtained from the USDA-ARS culture collection. We will prescreen treatments individually in model NFT and DWC systems before selecting a subset of treatments to test in combination in commercial scale hydroponic systems.All treatments will be introduced individually to tabletop NFT channels and DWC reservoirs. Human pathogens and algae will be inoculated into hydroponic systems. The algae type that harbors the highest population of human pathogens as determined in Objective 2 will be used in these experiments. Lettuce 'Rex' will be produced in the systems and assessments taken at the end of cropping as described in previous objectives. Each remediation and prevention treatment will be repeated three times and the experiment will be run twice. Non-treated systems will serve as controls. Six coupons of each surface type will be placed in the corresponding hydroponic system. The two best candidates for treatments effective against L. monocytogenes and Salmonella Typhimurium biofilms in each hydroponic system will be scaled-up using commercial-like scale NFT and DWC systems in our BSL2 facilities.Risk communication materials will be developed to raise awareness of food risks associated with biofilm formation and algae in indoor hydroponics. We will use photographs and animations to visually present the biofilm formation process, on hydroponic surfaces. We will develop control and mitigation programs that will include standard operating procedures for the prevention of biofilms using protective cultures identified as effective. All developed materials will be integrated into the following courses / guides.

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

Outputs
Target Audience: Hydroponic crop producers Greenhouse farmers Indoor/Vertical farmers Urban farmers Container farmers High tunnels/hoophouse farmers Aquaponic farming Researchers in academia, industry, and regularity agencies Hydroponic food safety policy makers in industry and government Equipment suppliers Food safety consultants and inspectors Changes/Problems:The activities have been mostly smooth so far, allowing us to advance the experiments as planned. However, we encountered several challenges. The hiring process for a post-doc took longer than expected. We posted the position early in 2024 and identified an excellent candidate from Brazil, but we are still waiting for the paperwork to clear so they can start. Although this has caused minor delays, we expect the candidate to begin theposition in January 2025. What opportunities for training and professional development has the project provided?One MS student graduated, One PhD student graduated. One PhD student and one MS student are currently working on this project. 55 participants from industry took online Food Safety in Hydroponic class in English How have the results been disseminated to communities of interest?Target audience for the work under this award are any stakeholders in hydroponic and indoor crop production. We have delivered evidence collected in this the work under this grant to people through formal or informal educational programs: We have developed a curriculum and integrated the findings into the online asynchronous course targeting hydroponic crop producers. We have delivered instruction to 55 participants in English. We have developed and launched the Spanish version of the training to reach Spanish speaking hydroponic indoor crop producers and greenhouse/indoor farmers and workers. We have published one manuscript reporting the evidence on food safety risks in hydroponic crop production in open access journal, reaching research community We presented four posters describing the findings of our studies in 2024 annual International Association of Food protection conference in Long Beach, CA (attended by over 3,500 food safety professionals from industry, academia, and regulatory agencies from six continents. We presented two posters at European IAFP Symposium 2024 in Geneva Switzerland including the participants from industry, academia, and regulatory agencies. We have presented two talks describing the finding from the studies and their implication to practice to over 200 industry participants representing greenhouse growers and consultants, indoor/vertical farmers, urban and container farmers, aquaponic growers, greenhouse and indoor farming equipment designers. What do you plan to do during the next reporting period to accomplish the goals?Objective 2. Evaluate the influence of algae on the establishment ofSalmonellaandListeriamonocytogeneson leafy green crops and environmental surfaces. We will continue the studies to evaluate the influence of algae on the establishment ofSalmonellaandListeriamonocytogeneson leafy green crops and environmental surfaces. This will include the co-culture of the pathogens and the two algae species and establishment of protocols toevaluate the mixed-culture biofilms first in laboratory conditions and then upscaling to the near-commercial setting. Objective 3.Establishbioremediation procedures and alternative approaches to co-mitigation of biofilms and environmental algal mats in leafy green indoor production systems. We plan to commence the studies with bacterial isolates previously isolated from hydroponic environments and determine the effect of the established cultures on mono- and mixed- culture biofilms. Objective 4.Development of risk communication materials describing biofilm and algae management best practices in commercial NFT and DWC leafy green indoor production systems. We will develop infographics explaining the sanitation protocols to remove the biofilms from surfaces and approaches to prevent biofilm formation. Training and professional development: We are in the process of hiring a post-doc who will have an opportunity to be trained through this project. Two graduate students will present their work at International Association of Food Protection 2025 annual meeting.

Impacts
What was accomplished under these goals? Objective 1. CharacterizeSalmonellaandListeria monocytogenesbiofilms in commercial NFT (Nutrient Flow Technology) and DWC (Deep Water Culture) leafy green indoor production systems. We have characterized biofilms produced by 11 different strains ofSalmonellaspp. isolated from human, environmental, and food samples (list: We have determined What is the ability of different Salmonella serovars in nutrient solution at greenhouse temperature ranges (25°C, 28°C and fluctuating greenhouse conditions). Compared to the control optimal temperature for Salmonella spp. (37°C), most tested strains exhibited optimal biofilm formation capabilities at 28°Cwas the optimal temperature and to a lesser extent fluctuating temperatures in commercial-like greenhouse conditions. Salmonella serotypes that consistently formed moderate-strong biofilms in all media solutions were Anatum, Montevideo, Albany, Newport, and kanamycin resistant Typhimurium. Comparing the media, and the conditions when plant exudates circulate in the systems, glucose was the source of carbohydrates in M9 media while in NPE (nutrient solution + exudate), different sugars were present. This affected the characteristics and the strength of formed biofilms (8 strain formed biofilms in M9 vs 9 in NPE (nutrient solution + exudate)) compared to just nutrient solution (3 biofilm formers). Javiana, Montivideo, Albany showed an RDAR morphology, while Anatum was PDAR,andSalmonellaTyphimurium resistant to kanamycin, and Newport was SAW morphology.In conclusion, the majority of Salmonella strains can form strong biofilms in hydroponic conditions, especially I presence of sugars coming from plant exudates. We have evaluated the following methods for biofilm characterization from coupons: total protein, culturable bacterial counts and bacterial activity based on tetrazolium reduction assay. We have determined the effect of materials (e.g. PVC, high density polystyrene) on biofilm accumulation, including the biofilm formation of an inoculated Bacillus velezensis isolate. We have conducted experiments with L. mocytogenes to better understand its ability to form biofilms on surfaces used deep water culture hydroponics. We demonstrated that L. monocytogenes readily forms biofilms on PVC surfaces used in trays, piping, floaters, and substrates like rockwool and oasis. We showed that sodium hypochlorite, the commonly used sanitizer in the fresh produce industry, is an ineffective treatment against L. monocytogenes and its biofilms formed at 22°C on hydroponic surfaces. Also, once L. monocytogenes biofilms are formed, they reduce the effectiveness of other sanitizers. The findings from this study provide hydroponic growers with information about effectiveness of sanitizers that can help them chose appropriate treatments and improve food safety of hydroponic produce. When used in commercial hydroponic systems. Objective 2. Evaluate the influence of algae on the establishment ofSalmonellaandListeriamonocytogeneson leafy green crops and environmental surfaces. We have established algae culturing lab protocols and optimized the approaches for enumeration of algae in monoculture biofilms in hydroponic nutrient solution. We determined the optimal time and temperature for establishment and maintenance of two common algae (Anabaena sp. and Chlorella vulgaris) and established protocols for their culture and quantification in BG11 algae media and LG (leafy green nutrient solution). We then proceeded to determine the best approaches for simultaneous quantification of two species of algae, Salmonella spp. and Listeria monocytogens on XLD and LB (Nal) culture media. These experiments are ongoing. Objective 4. Development of risk communication materials describing biofilm and algae management best practices in commercial NFT and DWC leafy green indoor production systems. We have integrated the findings from the biofilm and sanitizer efficacy studies into the training and recommendations for greenhouse growers. The training is available in person, and as a asyncronous online course in English in Spanish.

Publications

  • Type: Other Status: Published Year Published: 2024 Citation: Mensah, A.A., Lewis Ivey, M.L., Moodispaw, M.R. and Ilic, S., 2024. Effectiveness of Chemical Sanitizers against Salmonella Typhimurium in Nutrient Film Technique (NFT) Hydroponic Systems: Implications for Food Safety, Crop Quality, and Nutrient Content in Leafy Greens. Foods, 13(12), p.1929.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mensah, A. A., Lewis Ivey, M.L., Diekmann, F., Bang, C. M., Rivas, G., Huagu, P. K., & Ilic, S. (2024). Interventions to control human pathogens in hydroponic crop production: Scoping review. International Associations of Food Protection Annual Conference, Long Beach, CA, July 2024
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Garay G. Aeration, water source, and surface materials influence hydroponic lettuce production. Department of Plant Pathology, The Ohio State University. PhD Dissertation 2024
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Garay G. Aeration, water source, and surface materials influence hydroponic lettuce production. Deparment of Plant Pathology, The Ohio State University, Exit seminar. July 3 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Huagu, P. K., Lewis Ivey, M.L. & Ilic, S. (2024)Effectiveness of Sanitizers Commonly Used by the Greenhouse Industry to Eliminate L. monocytogenes from Hydroponic Surfaces Used in Deep Water Culture Systems. International Associations of Food Protection Annual Conference, Long Beach, CA, July 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Ilic, S., (2024) Interventions to Control Human Pathogen Bacteria in Controlled Environments, International Associations of Food Protection Annual Conference, Long Beach, CA, July 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Ilic, S. (2024) Ensuring Food Safety and Nutrition in CEA, CEAg World Conference and Expo, Raleigh, NC, Aug 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Lewis Ivey, M.L. (2024) Green Guardians: Tackling Algae, Biofilm, and Plant Pathogens in Hydroponic Havens, CEAg World Conference and Expo, Raleigh, NC, Aug 2024
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Huagu, P. K., (2024) Assessing the Effectiveness of Sanitizers against Listeria monocytogenes and its Biofilms on Deep-Water Culture Hydroponic Surfaces, Human Nutrition, Department of Human Sciences, MS Thesis 2024