To assess the survival of E. coli in sediment, a lab study will be carried out. Prior to lab study, both water and sediment samples will be collected from two different monitoring sites in Skunk Creek, is a tributary of Big Sioux River. The way study sites are chosen is one site has direct access to cattle and another site is under seasonal area riparian management (SRAM).Experimental setup: For survival study, six glass tanks will be used to act as flow through compartment. The glass tanks dimension is LengthX WidthX Height: 20″X 12″X18″. The top of the tank is removable for sample collection purpose. This setup is a close conduit system maintaining 20 cm of water depth from bed sediment. Total six tanks with two different sites sediment samples and three different temperatures have been prepared for survival study.
For simulating stream condition, the flow in the tank will be generated by using a submersible aquarium pump. The flow rate will be maintained in between 3E-4 to 4E-4 cubic ft per second in all chamber. Two pumps place inside two water tanks where natural water reserved. The water collected from two different study sites. Each water tank connects with three glass tanks, so it will be easy to maintain uniform distribution of water in each tank. The sediment samples will be collected from two locations mentioned above. In each tank, 5cm sediment bed will be seeded by collecting sediment from the study site. The reason of choosing 5cm is past researchers observed the presence of E. coli is highest in top 3cm of streambed sediment. Three temperatures in the tank will be maintained 4⁰C, 16⁰C and 30⁰C. The temperature selected by reviewing literatures. Literature shows under 5⁰C E. coli survival is higher and above 28⁰C survival of E. coli decreased. Therefore, total six tanks with two different sites samples and three different temperatures will be prepared for survival study. Before initiation of the survival study in the flow chamber, sediments from two different sites should be incubated at above three temperatures (4⁰C, 16⁰C and 30⁰C) to adapt with the environment of the flow chamber.
Sample Collection and Analysis
Both water and sediment samples will be collected daily basis for first 7 days and weekly for following six weeks. 500 ml of water samples will be collected from each six tanks. For sample analysis, membrane filtration technique will be used. For sediment sample analysis, samples collection and procedure method carry out by the methods I am following in my project. Samples will be collected in 500ml volume of sterile wide mouth bottle. For sample processing, 1:10 ratio of sample: phosphate buffer solution is used. After sample mixing, samples are shaking in 150 rpm in an orbital shaker for 45 minutes. 50 ml supernatant collects from the mixer and use for sample analysis. Membrane filtration method use for E. coli determination. Both water and sediment samples will be analyzed for two dilutions (100 and 101) in triplicate.
In addition of E. coli enumeration, different other variables will be monitored to identify potential factors that impact on survivability in E. coli in sediment. For each water samples including field samples, pH, electrical conductivity (EC), dissolved oxygen (DO), turbidity will be measured. For measuring pH and EC, AB15, Fisher scientific, Hampton, NH will be used. For measuring DO and turbidity, hand-held DO meter (model HI98193, Hanna Instruments, Smithfield, R.I.) and a field colorimeter kit (DR 890, Cole-Parmer., Vernon Hills, IL.) will be used, respectively. For each location, sediment characterization (i.e. particle size analysis) and organic matter will be analyzed including the field samples. For particle size analysis, American Society of Testing Materials (ASTM) D422 (sieve analysis) method will be followed. For organic matter determination, Loss on ignition method will be used.
In data analysis, at first step it is important to know the distribution of the data. For checking normality of the data, “Shapiro-Wilk” test will be used. For non-normally distributed data, 1-sample Wilcoxon test is suitable for comparing the data where one variable is discrete(X) and another variable is continuous(Y). 1-sample Wilcoxon test can identify is there any significant difference of the median of a population. But to identify the differences among the data itself, a post hoc test “Bonferroni” will be used. If the distribution of the data is normal, then 1-sample t test followed by Duncan’s multiple range test will be used. To determine the correlation between E. coli levels and different sediment sample sizes or, organic matter; correlation factor will be calculated. For determination of correlation factor, spearman rank correlation coefficient test will be used. All the above test will be done in R software. For better representation of the data, different curves for the parameters and percentage reduction of E. coli levels will be plotted and calculated, respectively.
This study focuses on the survival of E. coli in freshwater sediment only- The study based on lab scale experiment, so it will be differing from natural stream such as sunlight, wind or weed in the sediment bed or algal growth, pollutants or nutrients from upstream etc.- Some literatures found E. coli can survive in the sediment bed for 90 days to 120 days, in that case, few E. coli survival time will be neglected in this study. – E. coli survival rate differ from different pollutant source. As the sources of the study site is mainly livestock (animal grazing or feeding operations), manure application in the agricultural field so agricultural runoff, wildlife etc. Therefore, this study result would not represent the survival of E. coli in sediment for the stream which pollution source is septic tank or municipal waste.