Progress 07/01/19 to 02/28/21

Outputs
Target Audience:During the side-by-side comparison studies conducted at UC Davis, Fluidion has given instrumentation training to several extension specialists and students at UC Davis (Dr. Trevor Suslow's group) for the operation of the ALERT-AG to perform E. Coli measurements of agricultural water samples. As part of the outreach effort during this project, through our collaborator Dr. Phil Long of Lake Chelan Research Institute (LCRI) we have worked with the Lake Chelan Watershed Planning Unit and contacted a large local fruit cooperative (Chelan Fruit), a major irrigation water provider (Lake Chelan Reclamation District), and a major agricultural supply company (Wilbur-Ellis). Changes/Problems:Due to the Covid-19 pandemic and statewide lockdowns, the ALERT studies with Dr. Suslow's group at UC Davis as well as Dr. Phil Long at Lake Chelan Research Institute (and Chelan Fruit) have been postponed until water sampling activities and laboratory testing can be resumed. Dr. Trevor Suslow has agreed to connect us with local growers to test the ALERT-AG so we can get additional feedback from field operations. We hope to resume these activities in the coming months now that CA has reopened in mid-June and WA is expected to reopen by the end of June this year. What opportunities for training and professional development has the project provided?Several stakeholders were trained in the use of the ALERT-Ag instrument, and operated it autonomously thereafter. How have the results been disseminated to communities of interest?We have provided updates of the ALERT technology and have discussions of the study results from this USDA NIFA project with Dr. Trevor Suslow, Vice President of Food Safety at the Produce Marketing Association (PMA). Dr. Suslow is well recognized in the produce community, and he has been sharing our technology through his network whenever applicable. In addition, we have been working in collaboration with Dr. Phil Long at the Lake Chelan Research Institute (LCRI) to reach out to grower communities in the Washington area. Fluidion has provided LCRI with an ALERT-Ag unit for use in hands-on testing. Dr. Long has worked with an individual orchardist with extensive acreage in cherries, apples and pears (Al Robison) to conduct initial testing in locations irrigated by water from both Lake Chelan and the Columbia River. This outreach effort to communities of interest will continue beyond the project award period. In the past, we have also presented the ALERT technology at produce conferences, such as United Fresh and PMA Tech Knowledge. ALERT Technology was also presented online at the 2020 NEMC conference. Most conferences have been canceled during the Covid pandemic, however, we plan to participate in similar types of events in the coming year to disseminate the latest results of the ALERT-Ag technology to communities of interest. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1)A prototype of a disposable cartridge was designed, initially using 3-D printed technology and then using plastic injection molding. Particular attention was paid to design for manufacturing, and a specialized consultant (Grant Innovation/Actualized Technology) was contracted to help with design and prototyping work. The cartridge prototype was integrated in a test prototype product and tested. Two manufacturing runs have been performed, which has allowed ironing out several assembly issues and injection molded part tolerances. The results using the disposable cartridge show good accuracy and correlation with laboratory measurements. Numerous side-by-side measurements against the approved Colilert Quantitray method have enabled drafting a calibration curve for E.coli, whereas repeatability tests have allowed measuring the precision of the device. The device shows much better usability by untrained personnel. Specifically, maintenance duration has been reduced from 45 minutes (initially) to less than 5 minutes by a person with no prior training, which is extremely promising in terms deploying this technology as an on-farm analyzer. A new hardware modem, capable of operating on the latest Nb-IOT and LTE-M networks, has been integrated in both the Alert-Ag portable device and the ALERT in-situ system. This enables the device to operate and communicated data from areas where previously there wasn't GPRS coverage (which is being shut down in the US). The new modem also allows larger amounts of data to be transmitted faster, and the device firmware was upgraded to implement a "data download" function, allowing recovery of data that was not sent due to poor network coverage. A new user interface was designed as well, simplifying operation, improving the available information and rendering the unit much more user-friendly. A prototype of the new interface is currently operational. 2)To evaluate the usability of the ALERT-AG for agricultural applications, we provided Dr. Trevor Suslow's group (UC Davis) and Dr. Phil Long (Lake Chelan Research Institute, LCRI) with ALERT-AG units. Extension specialists in Dr. Suslow's group were trained to use the ALERT-AG for performing E. Coli measurements in our study. Based on their feedback, the foremost advantage of the ALERT-AG compared to other EPA approved methods (including the IDEXX Colilert) is the ability to measure a large range of E. Coli concentrations (up to 1,000,000 per 100mL) without the need for any dilution of the sample prior to measurements. This provided a significant saving in personnel time and effort, as well as elimination of errors during multiple dilutions in sample preparation using other techniques such as IDEXX Colilert. Other positive factors include: significantly reduced time-to-result, instrument portability, ease of use, wireless data communication and automated data reporting. As for the ALERT-Ag unit with Dr. Long, he worked with an orchardist from Chelan Fruit to conduct initial testing in locations irrigated by water from both Lake Chelan and the Columbia River. 3) Agricultural water samples used for irrigating nuts, tomatoes and leafy greens were collected from canals and reservoirs around Davis (CA), Modesto (CA), and Yuma (AZ) by extension specialists from Dr. Suslow's group. Measurements in Fluidion's ALERT-AG and the Colilert-18 were conducted in parallel using filtered and unfiltered samples. The focus of this study was to assess the ability of the ALERT-Ag to quantify bacterial charge on particles present in suspension, which currently eludes the standard measurement protocols (but membrane filtration and the MPN method such as Colilert Quantitray count each particle as a single bacterium). The particulate bacterial count is extremely important from a risk-assessment perspective, since particles can shield bacteria from UV light as well as disinfecting agents, thus allowing them to possibly reach the final customer. Data analysis of over 100 processed samples indicated that the Colilert tends to measure higher than the ALERT-Ag for reservoir samples, whereas the trend is opposite for canal samples. When comparing the results of filtered vs unfiltered samples in the ALERT Ag, the unfiltered samples consistently measured higher than the filtered samples, thus proving the ability of the ALERT technology to measure bacterial particle content. Another observation drawn from our study is that bacteria bound to particulate matter tend to be predominant in irrigation canals, reservoir samples being generally less affected by fecal particle presence. In addition to UC Davis, we also reached out to growers in the Washington State region through the help of our collaborator, Dr. Phil Long (LCRI), who worked with a local fruit cooperative (Chelan Fruit) in using the Fluidion Alert Ag to conduct initial testing in locations irrigated by water from both Lake Chelan and the Columbia River. 4) To prepare for the EPA approval process, we have drafted an initial study plan for the EPA Alternative Testing Procedure (ATP) and selected a certified water quality testing laboratory, Weck Laboratories. Quantitative measurements of E. Coli concentrations on more than 80 water samples from a variety of water matrices have been conducted in the IDEXX Colilert and Fluidion ALERT-Ag in parallel. The data shows excellent correlation for different water matrices, with Pearson coefficient of rww=0.940 for wastewater, rsw=0.927 for stormwater, and rgw=0.802 for groundwater. The use of wastewater in our study has allowed us to assess whether ALERT-Ag is capable to measure the bacterial abatement factor from industrial tertiary disinfection processes, and to perform a comparison against an EPA-approved method in a water matrix relevant for water reuse. The effects of chlorination on the ALERT-AG meausurement are evaluated through the use of chlorinated tertiary effluent. The data, though limited, indicated that the ALERT-AG is effective in measuring disinfected samples as we can see the E. Coli concentration values drop significantly following the disinfection process. The capability to measure at various stages of the disinfection process throughout wastewater treatment is one of the advantages of the ALERT technology, whereas this would be not possible using other methods which do not include a bacterial culture step. The results from filtered vs unfiltered samples indicated there is better correlation between Colilert and ALERT data for unfiltered samples. This is as expected since the initial calibration for the ALERT was done using unfiltered samples. For unfiltered samples, we have also observed that the Colilert was measuring higher than the ALERT for E. Coli concentrations below 10^4 whereas it was measuring lower for E. Coli concentrations above 10^5. This crossover trend was corrected following recalibration. For Ag samples, the effect of particles on the E. Coli concentration as measured by different methods is of great interest. Based on the ALERT data, an average log reduction from filtration (5mm pore size) is calculated to be 0.35 log10 units. It is important to note that based on our results, standard methods such as the Colilert do not see the effects of particles which can have significant impacts on the effectiveness of UV treatment of irrigation water. The precision of the instrument was evaluated via repeated measurements. Overall, the precison of the ALERT Ag is 0.18 log10 units (after recalibration). For comparison, the same methodology was applied to the Colilert measurements, and the precision of the laboratory measurements was 0.09 log10 units. The level of precision provided by the ALERT is very good, for a portable instrument that does not require any dilutions (by comparison, the laboratory protocol required up to 5 dilution steps per measurement, and multiple measurements performed at different dilutions to ensure that the reading is within range).

Publications

Progress 07/01/19 to 06/30/20

Outputs
Target Audience: Nothing Reported Changes/Problems:Due to the Covid-19 pandemic and statewide lockdowns, the ALERT studies with Dr. Suslow's group at UC Davis as well as Dr. Phil Long at Lake Chelan Research Institute (and Chelan Fruit) have been postponed until water sampling activities and laboratory testing can be resumed. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?We provide periodic updates of the ALERT technology and have discussions of the study results from this USDA NIFA project with Dr. Trevor Suslow, Vice President of Food Safety at the Produce Marketing Association (PMA). Dr. Suslow is well recognized in the produce community, and he has been sharing our technology through his network whenever applicable. In addition, through Dr. Phil Long at the Lake Chelan Research Institute in Washington, we have been able to reach out to grower communities in the area who have expressed interest in the ALERT technology. We will continue with this outreach in the coming months. In the past, we have also presented the ALERT technology at produce conferences, such as United Fresh and PMA Tech Knowledge. ALERT Technology was also presented online at the 2020 NEMC conference. We will continue to participate in these types of events to disseminate the latest results to communities of interest. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, provided that water sampling and laboratory testing can resume following ease of Covid-19 lock-down, we plan to pursue the following tasks: Finalize ALERT V2 development and testing (Objective1) Connect with local growers in CA to get feedback on the use of ALERT LAB in field operations (Objective2) Initiate second ALERT LAB study with Dr. Phil Long and Chelan Fruit in WA (Objectives 2 & 3) Collect and analyze additional samples for the side-by-side study conducted in Dr. Trevor Suslow's group at UC Davis (Objective3) Continue with the side-by-side study at Weck Laboratories, and expand the sample types to include additional agricultural water samples (Objective4)

Impacts
What was accomplished under these goals? Milestone 1 A prototype of a disposable cartridge was designed, initially using 3-Dprinted technology and then using plastic injection molding. Particular attention was paid to design for manufacturing, and a specialized consultant (Grant Innovation/Actualized Technology) was contracted to help with design and prototyping work. The cartridge prototype was integrated in a test prototype product and tested. The results using the disposable cartridge show good accuracy and correlation with laboratory measurements, with much better usability by untrained personnel. Specifically, maintenance duration has been reduced from 45 minutes (initially) to less than 5 minutes by a person with no prior training, which is extremely promising in terms deploying this technology as an on-farm analyzer. Milestone 2 During our current and previous side-by-side comparison studies conducted at UC Davis, several extension specialists in Dr. Trevor Suslow's group have used the ALERT LAB for performing E. Coli measurements of agricultural water samples. Based on their feedback, one main advantage of the ALERT LAB compared to other E. Coli measurement techniques is the ability to measure a large range of E. Coli concentrations (up to 100,000 per 100mL) without the need for any dilution of the sample prior to measurements. Other positive factors include: instrument portability, ease of use with minimal training, wireless data communication and automated reporting of data. Dr. Trevor Suslow has agreed to connect us with local growers to test the ALERT LAB so we can get additional feedback from field operations. Unfortunately, this has been delayed due to Covid-19 lockdowns since mid-March 2020. Milestone 3 Several discussions have been conducted with Dr. Trevor Suslow (Vice President of Food Safety, Produce Marketing Association; UC Davis, Postharvest Technology Center) in the planning of a new round of side-by-side comparison study between Fluidion's ALERT LAB and the Colilert-18 to be carried out in his laboratory at UC Davis. During the months from November 2019 to March 2020, agricultural water samples used for irrigating nuts, tomatoes and leafy greens were collected from canals and reservoirs around Davis (CA), Modesto (CA), and Yuma (AZ). We have provided an ALERT LAB and reagents to Dr. Suslow's laboratory for use in this study, and have trained Dr. Suslow's staff in the operations of the ALERT LAB prior to the study. We have also established a protocol for the measurements to be performed using the ALERT LAB and Colilert-18. For each agricultural water sample collected by Dr. Suslow's staff, four measurements are performed in parallel: 1) ALERT LAB: 25mL unfiltered sample with no dilution, 2) ALERT LAB: 25mL filtered sample (using syringe filters with 5µmpore size) with no dilution, 3) Colilert-18: 100mL unfiltered sample with no dilution, and 4) Colilert-18: 100mL unfiltered sample with 100X dilution. Multiple samples from different physical locations at the sampling site are measured. Data from the ALERT LAB istransmitted wirelessly and quantitative E. Coli concentrations are automatically caluclated and reported on the secure cloud platform, while results from the Colilert-18 are read by Dr. Suslow's staff and recorded on a shared Excel sheet. In this study so far, more than 50 samples have been collected, mostly in the areas around Davis, and some in Modesta and Yuma, reuslting in over 100 samples processed by Dr. Suslow's staff. Side-by-side comparison of the ALERT LAB and Colilert data show good correlation. However, more sample points will be needed to draw more concrete comparisons between different sample types and locations. Unfortunately, due to the Covid-19 pandemic and statewide lock-downs since mid-March 2020, the study has been interrupted. The study will resume as soon as sampling is possible and laboratory measurements in Dr. Suslow's research lab can be conducted again. In addition to UC Davis, we have also started reaching out to growers in WA. Through our collaborator, Dr. Phil Long at the Lake Chelan Research Institute, we plan to initiate a second study of the ALERT LAB using agricultural water in collaboration with Chelan Fruit. We have provided an ALERT LAB unit to Dr. Phil Long for the study, discussions are in progress to establish measurement protocols and define parameters for the study. Milestone 4 To prepare for the EPA approval process for Fluidion's ALERT technology, we have selected a water quality testing laboratory, Weck Laboratories, to perform the EPA approval study. We have initiated side-by-side experiments on an initial set of samples from a variety of water matrixes, including wastewater, stormwater, groundwater, and agricultural water. This initial study will allow us to verify the testing protocol and eliminate possible protocol non-compliance issues prior to the official EPA study. We have provided an ALERT LAB and reagents to Weck Laboratories for use in this study, and have trained their microbiology staff in the operations of the ALERT LAB prior to the study. We have also established a protocol for the measurements to be performed using the ALERT LAB and Colilert-18. For each water sample, six measurements are performed in parallel: 1) ALERT LAB: unfiltered sample with 1:4 dilution in sterilized water, 2-3) ALERT LAB: filtered sample with 1:4 dilution in sterilized water, 4-6) Colilert-18: unfiltered sample with 1-100,000 dilution depending on type of water. Cooled samples are transported to Weck Laboratories directly from the water sources in CA and MA shortly before each measurement. Data from the ALERT LAB is transmitted wirelessly to the secure cloud platform where quantitative E. Coli concentrations are automatically reported, while results from the Colilert-18 are read by Weck Laboratoriesstaff and recorded on a shared Excel sheet. To date, more than 70samples have been collected and processed by Weck Laboratories for this study. Side-by-side comparison of the ALERT and Colilert data show excellent correlation for different water matrices, with Pearson coefficient of rww=0.952 for wastewater, rsw=0.924 for stormwater, and and rgw=0.802 for groundwater. Most of the samples tested so far have been from wastewater sources. However, additional samples from agricultural sources will be included in the study in the coming months.

Publications