IMPROVING THE SAFETY OF IRRIGATION WATER USED FOR PRODUCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1008762
• Project Start Date: Dec 15, 2015
• Project End Date: Mar 31, 2020
• Program Code: [(N/A)]- (N/A)

Project Director
Janes, M.

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
Food Science

Non Technical Summary
Produce farmers utilizing open surface irrigation water are at a higher risk for pathogen contamination of crops and are in need of an inexpensive and non-chemical way to remove pathogenic bacteria from irrigation water. We plan on developing a deep-bed zeolite filtration device capable of reducing microbial contamination of irrigation water used for high risk vegetables. Zeolite is a mineral rock that can be modified to enhance the removal of pathogenic bacteria from irrigation water. The objectives of the studyare to optimize a deep-bed filter using modified zeolite capable of reducing E. coli O157:H7 and Salmonella levels from irrigation water and to test the effectiveness of the filtration system in an actual farming environment. Generic E. coli which is non-pathogenic will be used as an indicator to evaluate the performance of the deep-bed zeolite filter on reducing food safety risks associated with surface irrigation water used for irrigating strawberries. The deep-bed zeolite filter system will provide growers with a method to reduce pathogens in irrigation water and comply with the proposed Food Safety Modernization Act (FSMA) produce safety rule, agricultural water requirements.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1122	1100	100%

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

Subject Of Investigation
1122 - Strawberry;

Field Of Science
1100 - Bacteriology;

Keywords

zeolite

salmonella

e. coli

irrigation water

Goals / Objectives
The long term goal of this research project is to develop sustainable practices to mitigate on-farm food safety risks. Agriculture water is one of the important routes of human pathogen food contamination, and growers must take steps to minimize the risk of produce contamination with source water. The purpose of this project is to develop a non-chemical water treatment system that would not adversely affect the quality and productivity of the crops. The beneficiaries of this project are fruit and vegetable growers utilizing open surface irrigation waterwith a higher risk for pathogen contamination of crops. For example, strawberry farmers in Louisiana pump ground water into ponds on the surface of their farms then use this water for irrigation of strawberries. This project is important and timely as the Food and Drug Administration (FDA) Food Safety Modernization Act (FSMA) requires fresh produce growers to meet certain microbial levels depending on the risk associated with the water application. The outcome of this project will help develop a water treatment system useful for fresh produce growers to reduce risk associated with agricultural water and to meet the buyers', state, and federal food safety requirements. Specific objectives are as follows:1. Chemically modified zeolite for removing E. coli O157:H7 and Salmonella Thypimurium from inoculated pond water.2. Develop a deep-bed filter capable of removing E. coli O157:H7, and Salmonella Typhimurium from irrigation water using surfactant modified zeolite.3. Use of a deep-bed zeolite filter system to control generic E. coli in irrigation water applied to produce in a field setting.

Project Methods
Objective 1:The zeolite will be modified by two different hexadecyl(trimethyl)ammonium (HDMTA) aqueous solutions, using either Chloride (Cl) or Bromide (Br) as the mineral (Margeta, 2013). For each gram of zeolite, 600 µl of an aqueous solution of HDMTA Cl or Br at (v/v) concentration 0, 3, 6, 9, 12, 15, or 18% will be sprayed on the surface of the zeolite and then mixed for 10 min. The aqueous solutions of zeolite with HDTMA Cl or Br will be dried in a conventional oven at 150ºC for 10 min then five grams of the surfactant modified zeolite (SMZ) will be placed into an ENASCO water filtration mount (a laboratory scale water filtration system). Sterile deionized water (250 ml) will be run through the ENASCO filtration mount to compact the SMZ.E .coliO157:H7 (ATCC 43895) andSalmonellaTyphimurium will be used in this study. The bacterial cultures will be stored in 30% (wt/wt) glycerol (20% water v/v) at -80 °C in Tryptic soy broth (TSB). Frozen cultures will be activated by three successive passages in TSB. After incubation for 18 h in 100 ml of TSB at 37°C, the cultures will be centrifuged; and the pellet will be suspended into equal volumes of PBS buffer. Then 50 ml of the re-suspended overnight cultures will be added to 1 L of deionized water or sterilized farm pond water for a final concentration of 106CFU/ml. One liter of inoculated water will be passed through the ENASCO SMZ filtration system. Triplicate samples for each concentration and water source will be collected pre and post filtration and tested forE. coliO157:H7, andSalmonellaTyphimurium levels.E. coliO157:H7 will be plated on Sorbitol MacConkey Agar (SMAC) supplemented with Cefixime-Tellurite and incubated at 37ºC for 24±2 hours (US FDA-BAM, 2011).SalmonellaTyphimurium will be plated on Xylose Lysine Deoxycholate Agar (XLD) and incubated at 35ºC for 24±2 hr (US FDA-BAM, 2007). All experiments will be repeated three times.Objective 2:Commercial HydrAid Biosand Filters (Cascade Engineering Grand Rapids, MI, USA) will be used to make the deep-bed zeolite filter system. The filtration column contains underdrain gravel, filtration gravel and 45.4 kg of filtration sand in a 20 L water column. The dimension of the filtration columns are 0.77 m high with a diameter of 0.14 m. The filtration column used for the deep-bed zeolite filter will have the filtration sand replaced with the SMZ. A scale-up bench top experiment will be conducted with the deep-bed zeolite filter system. Pond water (20 L dosages) inoculated withE. coliO157:H7, andSalmonellaTyphimurium,(6 Log CFU/ml) will be passed through the deep-bed filtration system containing either sand or SMZ using a pump. Water samples will be collected to determine how effective the deep-bed zeolite filter works with large volumes of water. Bacterial counts will be enumerated as described above. Modification of the SMZ deep-bed filtration will be done if needed before starting the on-farm experiments.Objective 3:Irrigation water source for this study will be the water from a pond located at the LSU AgCenter Burden research station. Pond water (250 liter) will be collected in a 100-gallon (around 378 liters) water tank (food-grade plastic). Water will be stirred and pH and turbidity will be recorded.This project will use a cocktail of three genericE. colistrains from a lab (TVS 353, 354, 355) that have been used as surrogates for foodborne pathogens in field trials (Suslow et al., 2011). The bacterial cultures will be stored in 30% (wt/wt) glycerol (20% water v/v) at -80 °C in TSB. Frozen cultures will be activated by three successive passages in TSB. For cocktail preparation, 100 ml of each of the three strains will be mixed into an empty sterile l liter bottle through a sterilized glass funnel. The cocktail strain will be stored in an ice chest and transferred to the field. The 250 ml stationary phase cocktail strain will be added to 250 liters water with final bacterial counts of 105- 106CFU/liter. After addition of genericE. colicultures, a vigorous mixing will be donewith the help of a cleaned sanitized paddle.Pond water inoculated with genericE. colifrom the tank will be filtered through the deep-bed sand and deep-bed zeolite filter system that will be used to irrigate strawberries. An overhead irrigation system which is known to deliver water to the harvestable portion of the crops will be used to irrigate strawberries plants. This will let us understand the fate of our research strain on the surface of the fruits. To access the microbial quality of the pond water, water samples will be collected just before adding genericE. colicultures (as described below). Water will be sampled pre- and post-filtration and at the final point of discharge from the sprinkles once a month during the growing season for the specific crop. Three replicate samples will be taken at each sampling point, and samples will be collected monthly until the end of each growing season. Water samples will be collected in sterile 100 ml bottles and transported to the lab on ice and kept at 4°C until processed within 18 h. A standard membrane filter method will be used to analyze treated irrigation waters for genericE. coli (Anonymous, 2009. EPA, Method 1603). Briefly, modified mTEC agar will be prepared, sterilized, and aseptically poured into 60 mm dishes. Water samples will be filtered through 47-mm mixed cellulose ester membrane filters with a pore size of 0.45 µm. Following filtration, the membrane filters will be removed from the filtering apparatus and rolled onto the surface of the modified mTEC agar and incubated inverted at 37°C. Five plates (100 ml sample volumes) will be assayed. For each assay, colonies will be enumerated and recorded for each plate; and microbial concentrations will be calculated based on the number of colonies per 100 mL sample volume. Strawberries will be planted using sustainable growing practices at the LSU AgCenter Burden Center (Baton Rouge, LA). We will plant the strawberries during the early fall before the first hard frost of winter. The pH of the soil will be tested and adjusted to a pH of 5.8 to 6.5 (pH strawberries grow) with either peat moss if pH is above 6.5 or lime if pH is below 5.8. Soil samples will be tested for genericE. colibefore planting as described above. Field measuring of 50 x 60 ft will be divided into nine soil plots having individual plots measuring 5 x 10 ft. Plots will be randomly assigned for each treatment (control, no treatment, sand filter and deep-bed zeolite filter). Each plot will be separated by a minimum of 10 ft to minimize cross contamination between the treatments and for ease of harvesting.We will introducegenericE. coli(105- 106CFU/liter) into the control, deep-bed sand and deep-bed zeolite filtration systems through overhead irrigation once a month during the growing season. Once the strawberries have reached maturity, which will occur in the spring of 2017, we will start harvesting strawberries randomly once a month and test for genericE. coli(n=10 from each plot). After collecting the strawberries, they will be transported to the lab on ice and stored at 4°C for no longer than 18 hour before being tested. Strawberries (100 g) will be placed into 100 ml of DE broth and stomached for 1 min. GenericE. colisamples will be plated on Violet Red Bile Agar using the spread plate method. Duplicate plates will be incubated at 37°C for 24h.Statistical analysis: Data will be analyzed using mixed model procedure of SAS ®. A randomized complete block design with fixed effects of irrigation system and treatment will be used to determine differences in genericE. colilevels of control, deep-bed sand and deep-bed zeolite filter system treatments. For strawberry data, harvest will also be considered as a fixed effect. Individual plot will be designated as blocks (random factor).

Progress 12/15/15 to 03/31/20

Outputs
Target Audience:Water quality has become a principal focus of food safety control for produce farmers with the Food Safety Modernization Act (FSMA). Some produce farmers use surface water for irrigation that may have a higher bacterial content than allowed by the rule. The target audience was produce farmers and regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Publication in Lousiana Agriculture. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This studyevaluated the effectiveness of hexadecyltrimethylammonium bromide (HDTMABr) to develop a SMZ filtration system capable of removing Escherichia coli O157:H7, Salmonella serotypes, and Listeria monocytogenes from irrigation water. A liter of inoculated water with Escherichia coli O157:H7, Salmonella typhimurium, or Listeria monocytogenes at counts of 6 log CFU/ml was filtered through a 20g column of SMZ. The SMZ at a concentration higher than 20% w/w of HDTMABr removed > 6 log CFU/ml of Escherichia coli O157:H7 and Listeria monocytogenesand > 2 log Salmonella sp. The SMZ was tested in a strawberry field using a filtration system with regular sand and with the SMZ operating at 25 GPM. Two controls were used to prove the effectivity of the SMZ-- a positive control of the pond water and a filter system with only regular sand. An Escherichia coli non-pathogenic surrogate was used to inoculate pond water, which naturally contains fecal material, to concentrations >6 log CFU/ml. The SMZ filtration system had a removal capacity of 99.99% of the Escherichia coli in comparison to the two controls that did not remove thebacteria from the system. SMZ modified with HDTMABr could be a viable solution for farmers to comply with new FSMA regulations and provide a way to reduce foodborne outbreaks.

Publications

• Type: Other Status: Published Year Published: 2019 Citation: Louisiana Agriculture. Improving Irrigation Water Safety for Strawberries Using Surfactant-Modified Zeolite Filtration Vol.62, No.4, Fall 2019 Pp.32-33.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Water quality has become a principal focus for produce farmers with the Food Safety Modernization Act (FSMA). Some produce farmers use surface water for irrigation that could have a higher bacterial content than allowed by the rule. The target audience would be produce farmers and regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One internworked on the project and was trained in microbiological techniques and experimental design. How have the results been disseminated to communities of interest?The study was presented at a seminar to graduate students and faculty. What do you plan to do during the next reporting period to accomplish the goals?We plan on preparing the manuscript for publication in a refereed journal and agricultural magazine.

Impacts
What was accomplished under these goals? We have finished statistically analyzing the date for Objective 3 (Use of a deep-bed zeolite filter system to control genericE. coliin irrigation water applied to produce in a field setting).The Surfactant Modified Zeolite (SMZ) filtration system significantly reduced theE. coliand coliform counts in the pond irrigation water compared to the control and sand treatment in the strawberry field. The initial genericE. colicounts inoculated into the pond water before irrigation of the strawberries was about 5.0 Log CFU/ml; and after filtration through the SMZ filtration system, theE. colicounts ranged from 1.31 Log CFU/ml to non-detectable levels over the two-year growing season. After irrigation with the SMZ treated pond water, theE. colicounts on the strawberries were not significantly different from the control.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Jose L. Brandao Delgado, Liliana Avaroma, Gustavo Lira, Andrea Muela, Achyut Adhikari, Kathryn K. Fontenot, and Marlene Janes. 2019.Removal of Nine Salmonella enterica Serotypes from Water by Surfactant Modified Zeolite with Hexadecyltrimethylammonium Bromide, ASM meeting in San Francisco, June 20  24, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Jose Fuentes and Marlene Janes. 2019. Microbiological Quality and Prevalence of pathogens in Strawberries (Fragaria x ananassa) in the United States. IFT meeting in New Orleans, LA, June 2 - 5, 2019.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Water quality has become one of the principal focus for produce farmers with the Food Safety Modernization Act (FSMA). Some produce farmers use surface water for irrigation that could have a higher bacterial content than allowed by the rule. The target audience would be produce farmers and regulatory agencies. Changes/Problems:The strawberry plants were infected with red stele root rot that lowered the amount of strawberries available for sampling in the fall 2018. The strawberry plants were treated with a fungicide to kill the red stele root rot and we plan to complete the sampling of strawberriesin the spring of 2019. What opportunities for training and professional development has the project provided?Two interns Liliana Gutierrez (Bolivia) and Alvaro Garcia (Honduras) who worked on the project were trained in microbiological techniques and experimental design. How have the results been disseminated to communities of interest?The project was presented to produce farmers at the LSU AgCenter Good Agricultural Practice workshop in Alexandria LA March 2018. What do you plan to do during the next reporting period to accomplish the goals?We plan to finish testing the zeolite deep bed filters with pond water inoculated with generic E. coli applied to strawberry plants in a field setting, at the Burden Research Station, Baton Rouge, LA.

Impacts
What was accomplished under these goals? The 20 % hexadecyltrimethylammonium bromide (HDTMABr) surfactant modified zeolite (SMZ) filtration system was tested in a strawberry field located at Burden Research Station, Baton Rouge, LA. Strawberry plants were planted 0.6 m apart in a field measuring 50 x 60 ft. that was divided into nine soil plots having individual plots measuring 5 x 10 ft. The strawberry plot was maintained during this quarter by applying mulch around the strawberry plants with a 76 mm layer of pine mulch. Strawberry plants were watered by overhead irrigation system once a week using pond water located on the Burden Research Station. Application of fertilizers, herbicides, and insecticides were applied as needed. The HDTMABR-SMZ filtration system (operating at 25 gallons per minute), was compared to two controls one containing only agricultural sand in the filtration system and one with no filtration system (control). An Escherichia coli non-pathogenic surrogate was used to spike pond water that naturally contain fecal material to 5 log CFU/ml of E. coli. Irrigation water samples (100 ml) were collected after the inoculated pond water was filtered through the 20 % HDTMABR SMZ and two controls. After irrigation, a composite sample (~300g) of strawberries were collected once a month for three months. Bacterial counts were determined using E. coli/coliform petri-film. The 20 % HDTMABR-SMZ filtration system had a removal capacity of more than 99.99% of the Escherichia coli compared to the two controls. Sand filtration of irrigation water did not remove Escherichia coli from the pond water. The Escherichia coli/coliform counts for the strawberries were not significance different between treatments. The HDTMABR-SMZ filtration system could be a viable solution for farmers to comply with new FSMA regulations and provide a way to reduce foodborne outbreaks.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Jose Brandao Delgado, Jose Fuentes, Kathryn Fontenot, Achyut Adhikari and Marlene Janes 2018. IAFP meeting in Salt Lake City, UT. ⿿Improving the Safety of Strawberry Irrigation Water Using a Hexadecyltrimethylammonium Bromide Modified Zeolite Filtration System⿝

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Water quality has become one of the principal foci of food safety control for produce farmers with the Food Safety Modernization Act (FSMA). Some produce farmers use surface water for irrigation that could have a higher bacterial content than allowed by the rule. The target audience would be produce farmers and regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A summer intern Ligia Fragallo from the Brazilian Scientific Mobility Program (BSMP) was trained in microbiological techniques and experimental design. Her research was presented at the IAFP 2017 meeting in Tampa Fl. One graduate student was supported by this project and was trained in experimental design and data analysis. How have the results been disseminated to communities of interest?The project was presented to produce farmers at the 2017 LSU AgCenter Vegetable Field day at the Burden Research Station in Baton Rouge, LA, Wednesday, May10, 2017. What do you plan to do during the next reporting period to accomplish the goals?We plan to start testing the zeolite deep bed filters with pond water inoculated with generic E. coli applied to strawberry plants in a field setting, at the Burden Research Station.

Impacts
What was accomplished under these goals? We determined if surfactant-modified zeolite (SMZ) was able to removeListeria monocytogenesandSalmonellaTyphimurium from inoculated water.The SMZ was produced by treating zeolite with a solution of hexadecyltrimethylammonium bromide (HDTMA-Br) at 0, 10, 20, 30% (w/w) and drying at 125°C for 30 minutes. The testing of the SMZ was done using a liter of inoculated water withListeria monocytogenesorSalmonellaTyphimurium,at a concentration of 5 log CFU/ml, which was passed through a column with 20 g of SMZ. Bacterial counts were determined before and after filtration. All analysis were based on three separate experiments with each mean + standard deviation being average of six determinations.The SMZ at concentrations of 10, 20, and 30% HDTMA-BR was able to remove 5 log CFU/ml ofListeria monocytogenesfrom the water. The control treatment SMZ 0% HDTMA-BR did not removeListeria monocytogenesfrom the water. The SMZ at concentration of 0% and 10% HDTMA-BR did not remove Salmonella from the water. The SMZ at 20% and 30% HDTMA-BR was able to filter out 0.4 ± 0.02 log CFU/ml and 1.2 ± 0.10 log CFU/ml of Salmonella from the water,respectively.The SMZ was more effective at removingListeria monocytogenesthanSalmonellaTyphimurium from water. We have plantedstrawberry plants in a plot located at Burden Research Station, Baton Rouge, LA. Strawberry plants were planted 0.6 m apart in a field measuring50 x 60 ft that were divided into nine soil plots having individual plots measuring 5 x 10 ft.. Mulch was placed around the strawberry seedlings with a 76mm layer of pine mulch. Strawberry plants were watered by overhead irrigation system once a week using pond water located on the Burden Research Station. Application of fertilizers, herbicides, and insecticides were applied as need.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mohammed Alhejaili, Dorra Djebbi-Simmons, Achyut Adhikari, and Marlene Janes. 2017. IAFP meeting in Tampa Fl. Correlation of Salmonella spp. to generic E. coli in irrigation water.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Jose L. Brandao Delgado, Ligia Fragallo and Marlene Janes. 2017. IAFP meeting in Tampa Fl. Removal of Listeria monocytogenes and Salmonella typhimurium from water using a filtration system with Surfactated modified Zeolite.

Progress 12/15/15 to 09/30/16

Outputs
Target Audience:The target audience includes the farmers that grow produce and the produce industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The next step will be to determinehow effective zeolite modified with hexadecyltrimethylammonium bromide (HDTMA-Br) at 0, 20, 40, 60, 80, 100 mM will beat removing Salmonellafrom water.

Impacts
What was accomplished under these goals? We determined how effective zeolite modified with hexadecyltrimethylammonium bromide (HDTMA-Br) at 0, 20, 40, 60, 80, 100 mM was at removing E. coli O157:H7 from water. This experiment was conducted in a column filled with 20 g of SMZ. One liter of inoculated water (106 CFU/ml) with Escherichia coli O157:H7 (ATCC® 43895™) was filtered through the system. The SMZ was able to remove up to removed 6 log CFU/ml of E. coli O157:H7 from the water. Our results indicated that Zeolite modified with HDTMA-Br could be used to develop a low cost filtration system.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: J. Cormier and M. Janes. 2016. Concentration and detection of hepatitis A virus and its indicator from artificial seawater using zeolite. Journal of Virological Methods, 235:1-8.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Olubode Adeniyi, Amy Hernandez, Mark LeBlanc, Joan King, and Marlene Janes. Quantitation of pesticide residue in water and food in Louisiana, USA. J. Water Resource Protection, (Accepted, 2016).