Progress 10/01/09 to 09/30/13
Outputs
Target Audience: Fruit and vegetable producers and packers, researchers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Work on this project was the basis for the PhD project of a graduate student. How have the results been disseminated to communities of interest? Results were reported at the 2009 and 2010 Annual Meetings of the American Society of Agricultural and Biological Engineers, and at the 2009 Annual Conference of the Institute of Biological Engineering. These results were also published in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Consumption of fresh fruits and vegetables contaminated with human pathogenic bacteria has caused numerous outbreaks of food-borne disease in recent years. Understanding the nature of bacterial colonization on plant surfaces could lead to more effective practices to prevent contamination or minimize the risk. The goal of this research was to determine the effect of plant surface microstructure on bacterial attachment. The specific objectives were to (1) determine the effect of microstructure on bacterial attachment, (2) evaluate the effect of surface hydrophobicity on bacterial attachment to microstructures, and (3) examine the effect of culture flow on bacterial attachment to microstructures. Plant microstructures come in a wide range of sizes and shapes, causing difficulty in isolating and understanding individual effects. Photolithography was selected as a microfabrication method to build surface structures on silicon to mimic stomata, trichomes, and grooves between plant epidermal cells. These structures were subjected to a culture of Escherichia coli tagged with green fluorescent protein at 30 deg C for 48 h. Bacterial attachment characteristics were determined by observation under a confocal laser scanning microscope. Arrays of stomata and grooves had bacterial attachment levels 2-3 times as large as that of an array of trichomes. The trichome base had attachment levels 3-5 times as large as those of the more distant areas. The stoma opening had the nearby attachment level higher than the more distant areas by 35-97 %. Areas around a groove showed no significant effects on the attachment. To simulate the hydrophobic nature of natural plant surfaces, the microstructures were treated with a chemical vapor deposition process called silanization, using the long-chain hydrocarbon compound octadecyltrichlorosilane. The results indicated that hydrophilic and hydrophobic microstructures had similar attachment levels. Bacteria often encounter a dynamic environment of flow before and during colonization on real plant surfaces. The silanized microstructures were subjected to a continuous flow of the E. coli culture for 48 h at three different flow rates (20, 100, and 500 ml/min). Shear stresses estimated by computational fluid dynamics were in a range of 0.2-24 dyne/cm^2, with a higher shear stress at a faster flow. Arrays of stomata and grooves had bacterial attachment levels 2-4 times as large as that of an array of trichomes, but the difference in attachment level at 500 ml/min was smaller than that at 20 and 100 ml/min. The left and right locations of trichome bases with respect to flow direction had higher bacterial attachment levels and shear stresses than the up-stream and down-stream locations. The down-stream location of stomata at 500 ml/min had an attachment level higher than the other locations, and higher than the down-stream location at 20 and 100 ml/min by 70 and 33 %, respectively. Microstructure geometry clearly affected bacterial attachment in the model system. Culture flow rate and attachment location around microstructures with respect to flow direction also influenced bacteria attachment to a certain degree.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sirinutsomboon, B. and M. J. Delwiche. Effect of Fluid Flow on Attachment of Escherichia coli O137:H41 to Plant Surface Structure Analogs Built by Microfabrication. Biological Engineering Transactions 6(2): 83-104.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2011
Citation:
Citation: Sirinutsomboon, B. 2011. Attachment of Escherichia coli on plant surface structures built by microfabrication. Ph.D. Thesis. University of California, Davis, California. (not previously listed)
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Higher demand for fresh produce in the US and abroad in recent years is associated with the greater number of food-borne outbreaks caused by human pathogenic bacteria. To improve our understanding of bacterial colonization on plant surfaces, we studied the effects of plant surface microstructure on bacterial attachment. The specific objectives were to (1) determine the effect of microstructure on bacterial attachment, (2) evaluate the effect of surface hydrophobicity on bacterial attachment to microstructures, and (3) examine the effect of culture flow on bacterial attachment to microstructures. Arrays of microstructures on silicon surfaces were built to mimic stomata, trichomes, and grooves between plant epidermal cells. We formed a hydrophobic surface on the silicon by silanization (chemical vapor deposition of octadecyltrichlorosilane), resulting in hydrophobic microstructures similar to those on natural plant surfaces. The dynamic environment of flow that bacteria could encounter before and during colonization of plant surfaces was simulated. The structures were subjected to flows of culture medium (20, 100, and 500 ml/min) with green florescent protein-tagged Escherichia coli O137:H41 for 48 h at 30 deg C. Attachment characteristics were determined by observation under a confocal laser scanning microscope. Shear stresses at these flow rates were calculated with simple fluid models and estimated by computational fluid dynamics. A manuscript summarizing the results from the flow cultures is currently under review for publication. PARTICIPANTS: Michael J. Delwiche, professor, principal investigator, mjdelwiche@ucdavis.edu Bunpot Sirinutsomboon, former graduate student, bsirinut@ucdavis.edu Department of Biological and Agricultural Engineering, University of California at Davis, Davis, CA 95616. Phone: (530)752-6731. Fax: (530)752-2640 TARGET AUDIENCES: Food safety specialists, microbiologists, plant pathologists, food engineers, biological engineers, agricultural engineers, food scientists, post-harvest biologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Shear stresses at the three flow rates estimated by computational fluid dynamics were in the range of 0.2-24 dyne/cm^2, with higher shear stress at the greater flow. Bacteria attached to the control areas at cell densities ranging from 2 to 19 cells per 100 um^2, with most of the control areas around 4 cells per 100 um^2. At higher flow rates, the etched control area had increased densities compared with the un-etched control areas. Arrays of stomata and grooves had attachment levels 2 to 4 times as large as for arrays of trichomes. Within an array of trichomes, the area around the base had a higher normalized density than in the more distant areas. Within an array of stomata, the area near the opening had a higher normalized density than further away. These differences tended to be smaller at the higher flows. Within an array of grooves, there were no significant location effects on normalized density at any of the flow rates. The effects of location around each microstructure type with respect to flow direction were evaluated. For trichomes, attachment on the left and right of the structure appeared to be higher than up-stream and down-stream, although the differences were not significant. Flow simulations indicated that the shear stresses in the left and right locations were higher than those in the up-stream and down-stream locations. For stomata, attachment at the down-stream location was larger for the higher flow rate. The flow simulations indicated larger shear stresses at higher flow rates for all the locations around a stoma, suggesting an interesting tendency of higher attachment at higher shear stress. Overall, the results showed that bacterial attachment was influenced by culture flow rate and location around the microstructures with respect to flow direction. This information could be useful for improving current practices of irrigation or washing harvested produce to reduce bacterial attachment.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Consumption of fresh fruits and vegetables contaminated with human pathogenic bacteria has caused numerous outbreaks of food-borne disease in recent years. Understanding the nature of bacterial colonization on plant surfaces could lead to more effective practices to prevent contamination or minimize the risk. The goal of this research is to determine the effect of plant surface microstructure on bacterial attachment. The specific objectives are to (1) determine the effect of microstructure on bacterial attachment, (2) evaluate the effect of surface hydrophobicity on bacterial attachment to microstructures, and (3) examine the effect of culture flow on bacterial attachment to microstructures. Plant microstructures come in a wide range of sizes and shapes, causing difficulty in isolating and understanding individual effects. Photolithography was selected as a microfabrication method to build surface structures on silicon to mimic stomata, trichomes, and grooves between plant epidermal cells. These structures were subjected to a culture of Escherichia coli tagged with green fluorescent protein and attachment characteristics were determined by observation under a confocal laser scanning microscope. The results have been presented at professional conferences and published in an academic journal. Several more manuscripts are being prepared. PARTICIPANTS: Michael J. Delwiche, professor, principal investigator, mjdelwiche@ucdavis.edu Bunpot Sirinutsomboon, graduate student, bsirinut@ucdavis.edu Department of Biological and Agricultural Engineering, University of California at Davis, Davis, CA 95616. Phone: (530)752-6731. Fax: (530)752-2640 TARGET AUDIENCES: Food safety specialists, microbiologists, plant pathologists, food engineers, biological engineers, agricultural engineers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Arrays of stomata and grooves had bacterial attachment levels 2-3 times as large as that of an array of trichomes. The trichome base had attachment levels 3-5 times as large as those of the more distant areas. The stoma opening had the nearby attachment level higher than the more distant areas by 35-97 %. Areas around a groove showed no significant effects on the attachment. To simulate the hydrophobic nature of natural plant surfaces, the microstructures were treated with a chemical vapor deposition process called silanization, using a long-chain hydrocarbon compound, octadecyltrichlorosilane. The results indicated that hydrophilic and hydrophobic microstructures had similar attachment levels. Bacteria often encounter a dynamic environment of flow before and during colonization on real plant surfaces. The silanized microstructures were subjected to a continuous flow of the E. coli culture for 48 h at three different flow rates (20, 100, and 500 ml/min). Estimated by a fluid dynamics program, the shear stresses were in a range of 0.2-24 dyne/cm^2, with a higher shear stress at a faster flow. Arrays of stomata and grooves had bacterial attachment levels 2-4 times as large as that of an array of trichomes, but the difference in attachment level at 500 ml/min was smaller than that at 20 and 100 ml/min. With respect to flow direction, the left and right locations of trichome bases had higher bacterial attachment levels and shear stresses than the up-stream and down-stream locations. The down-stream location of stomata at 500 ml/min had an attachment level higher than the other locations, and higher than the down-stream location at 20 and 100 ml/min by 70 and 33 %, respectively. Microstructure geometry clearly affected bacterial attachment in the model system. Culture flow rate and attachment location around microstructures with respect to flow direction also influenced bacteria attachment.
Publications
- Sirinutsomboon, B. 2011. Attachment of Escherichia coli on plant surface structures built by microfabrication. PhD Thesis, University of California, Davis, California. 207 pp.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In recent years, outbreaks of foodborne disease from produce contaminated with human pathogens have been increasing. A better understanding of bacterial attachment on plant surfaces is required. We microfabricated structures on silicon to simulate natural plant structures (trichomes, stomata, grooves between cells) and incubated them with a culture of Escherichia coli. Size, shape, and distribution of the fabricated microstructures were varied to better understand their effect on bacterial attachment. The results have been presented at professional conferences and published in an academic journal. PARTICIPANTS: Michael J. Delwiche (a), professor, principal investigator, mjdelwiche@ucdavis.edu Bunpot Sirinutsomboon (a), graduate student, bsirinut@ucdavis.edu (a) Department of Biological and Agricultural Engineering, University of California at Davis, Davis, CA 95616. Phone: (530)752-6731. Fax: (530)752-2640 TARGET AUDIENCES: Food safety specialists, microbiologists, plant pathologists, food engineers, biological engineers, agricultural engineers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Bacteria attached to flat (i.e., control) areas on the silicon pieces at a density of 1 to 2 cells per 100 um^2 and there were no differences between etched and un-etched control areas. The level of bacteria in an area with array of trichomes was about half of those in areas with array of stomata or grooves. Within an array of trichomes, the level of bacteria attaching around the base of the trichome was about 4.7 times higher than areas further away. Within an array of stomata, the level of bacteria attaching to the area 2.5-5.0 um away from the pore opening was about 35-59 percent higher compared with the area next to the pore or areas further away. Within an array of grooves, there were no significant location effects. Overall, surface microstructure geometry and position clearly affected bacterial attachment in our model system of etched silicon. These findings may explain why a plant species with certain surface characteristics attracts more or less bacteria. The knowledge could lead to improved means of preventing bacterial attachment. We are also studying the hydrophobicity effect on bacterial attachment. Microstructures are covered with a hydrophobic layer, similar to natural plant surface, by a silanization process. Microstructures will also be under continuous flow of liquid culture of bacteria. The result will show how well the bacteria react to the flow and successfully attach to the microstructures.
Publications
- Sirinutsomboon, B., Delwiche, M.J., and Young, G.M. 2011. Attachment of Escherichia coli on plant surface structures build by microfabrication. Biosystems Engineering (in press).
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