September 1 - 1139 - Hao Wang
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11th International Drainage Symposium August 30-September 2, 2022 Des Moines, Iowa
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September 1 - 1139 - Hao Wang
- 1. Characterization and Identification of Microbial Communities in Agricultural Drainage Ditches and Bioaugmentation Potentials for Enhanced Denitrification 11th International Drainage Symposium August 30 โ September 2, 2022 This work is supported by the University of Minnesota, MN Drive Program Hao Wang1, Satoshi Ishii1,2, Andry Ranaivoson1, Jeffrey Strock1 1 Department of Soil, Water, Climate 2 Biotechnology Institute
- 2. Research Site & Background โข University of Minnesota, Southwest Research Outreach Center (SWROC, Lamberton, MN) โข Using low-grade weirs to change the hydraulic characteristics of drainage ditch in order to increase Hydraulic retention time (HRT) and promote nitrogen removal โข Average HRT of treatment ditch during Flow season โ 12-14hrs (Control 30-60 minutes) Control no weir Treatment with weir
- 3. Research Site & Background โข Flow Season: Spring (March-May), Summer (June-August) and Fall (September-November) โข Low air and water temperatures in early spring (1-14o C) โข Drainage flow is seasonal, generally higher in spring and lower in summer - (44551.44 m3 /month vs 22842.25 m3 /month) โข High Nitrate content in tile-drained water (10 and 30mg NO3-N/L)
- 4. Discharge & Nutrient Reductions
- 5. Objectives 1. Identify and characterize the microbial properties of the drainage ditch โ Culture-independent microbial community analysis 2. Use the power of microorganism to enhance the nitrogen removal rate โ Isolation of denitrifying bacteria โ Bioaugmentation/biostimulation
- 6. Microbial Community Analysis โข Objective 1: Identify and characterize the microbial properties of the drainage ditch a) Samples were collected from Inlet, Midpoint, and Outlet for both the Control and the treatment Ditch throughout 2020 and 2021 b) DNA extracted from sediment for downstream analysis
- 7. Microbial Community Analysis โข 16S rRNA gene amplicon sequencing for bacterial community
- 8. Alpha Diversity Analysis (2020) - The diversity is influenced by the location (Statistically significant: (Pr: <0.001) C1:Control Inlet C2: Control Midpoint C3: Control Outlet T1: Treatment Inlet T2: Treatment Midpoint T3: Treatment Outlet Control no weir Treatment with weir
- 9. Alpha Diversity Analysis (2021) Statistically significant: (Pr: <0.001) Control no weir Treatment with weir
- 10. Beta Diversity Analysis (2020) Inlet Outlet - Shift in microbial community from inlet to outlet - Potential factors: DO, DOC, Nitrate availability? Inlet Outlet Midpoint 100m 100m
- 11. Beta Diversity Analysis (2021) Inlet Outlet - Shift in microbial community from inlet to outlet - Potential factors: DO, DOC, Nitrate availability?
- 12. Beta Diversity Analysis Inlet Outlet - Combining two years of data - Control: convergent - Treatment: divergent
- 13. Bioaugmentation โข Objective 2: Use the power of microorganism to enhance the nitrogen removal rate a) Sediment were used to isolate denitrifers
- 14. Denitrifer Isolation & Identification โข Isolation condition: 10 o C (for potential cold-adapted denitrifer) โข 300+ strains isolated โข Among the strains identified (~120 strains) - 57.1% Clostridium sp.; 13.2% Pseudomonas sp; 11% Cryobacterium sp; 6.6% Rahnella sp.
- 15. Denitrifier Verification โข 40 strains tested for denitrification performance โข Confirmed denitrifier strains (N2 producing): 6 - Pure culture N2 production test - Measured by GC-MS - All 6 strains are Pseudomonas sp. - Also found 14 N2O producing strains
- 16. Future Direction of Study Bioaugmentation โข Innovative method: Gel encapsulation โข Enrich pure culture โ encapsulate them into hydrogel beads Microbiome analysis โข Nitrogen Cycle Functional Gene quantification using Nitrogen Cycle Evaluation (NiCE) chip โข Create models to analyze the correlation between microbiome and environmental variables including temperature, nitrate, DOC, DO, etc.
- 17. Acknowledgement โข MnDRIVE โข Dr. Zhiyue Wang โข SWROC, UMN โข Ishii Lab members โข Strock Lab members
Editor's Notes
- Hello everyone, I am Hao Wang and I am a Ph.D. student in the LAAS program, department of Soil, Water, and Climate. Today I would like to present the research project for my Ph.D. Study, Characterization and Identification of Microbial Communities in Agricultural Drainage Ditches and Bioaugmentation Potentials for Enhanced Denitrification. I am co-advised by Prof. Strock and Prof. Ishii for this project.
- A little bit background information. This project was conducted in the University of Minnesota, Southwest Research Outreach Center located at Lamberton, MN. We have two drainage ditches for water removed by subsurface drainage system from a cornfield. The ditches were set up and installed in 2002 and the operation began in 2003. One drainage ditch was unmanaged, and it just like all the other drainage ditches across the upper midwest. And for the other ditch, we are using low-grade weirs to change its hydraulic characteristics in order to increase HRT and to promote nitrogen removal. So by installing the low-grade weirs, it adds some wetland features to a flow-through ditch. The average HRT of treatment ditch during flow season (Spring for snow melt, fall for precipitation) is about 12-14 hours and in comparison, the HRT in the control ditch is about 30-60 minutes.
- Some major characteristics โ Specific values โ temperature & flow rate โ more details presented by Andry/Jeff
- Our water measuring and testing results indicate that there is a 56% cumulative discharge reduction in the treatment ditch compared to the control ditch. And 64% nitrate-nitrogen load reduction. And a 50% flow-weighted nitrate-nitrogen concentration reduction in the treatment ditch. While other researchers in our team are focusing on examining the water quality improvement and the hydraulic characteristics of the experimental ditches, I am working on figuring out the microbiology aspect of the ditches and examining the difference between the control and the treatment ditch in terms of the microbial community composition. -Per cu meter/meter, concentration-based data
- There are two main objectives for this study. For the phase one, we are identifying and characterizing the microbial properties of the drainage ditches and isolate potential denitrifiers from the ditch. The ultimate question that we are trying to answer is if we can use the power of microorganism through bioaugmentation and/or biostimulation to increase the denitrification rate and further improve the output water quality.
- For the microbiome structure analysis, we have 16s rRNA gene amplicon sequencing for bacterial community. Future work: Nitrogen Cycle Functional Gene quantification
- Through statistically analysis of the 2020 samples, we found that the diversity of microbial community is impacted by its location in the ditch channels. It seems like there is an increase in the diversity of microbial community in the treatment ditch form inlet to outlet. And at the same time this trend was not found in the control ditch.
- When looking at the 2021 samples, it shows that the increasing trends was observed in both the control and the treatment ditch. One possible explanation of this is the draught. Even though year 2020 was dry, 2021 was even drier. In 2020, there was still some water discharge from the control outlet โ not in 2021. Still have discharge from control outlet during summer when the flow rate was low. Cross analysis with the precipitation patten, and hydrology data.
- This PCoA graph of 16s Amplicon sequencing results visualizes the dissimilarities in microbial community between samples collected from each location throughout the year. To briefly explain this PCoA graph is that the distance between each dot represents their similarity. The closer two dots are to each other, the more similar they are in terms of microbiome composition. And on the other hand, the further two dots are to each other, the more different they are in terms of microbiome composition. As you can see from the graph, there are some cluster formed. For examples, the red cycle cluster on the bottom right side of the graph indicates that the microbial community at inlets is stable and didnโt change much throughout the entire year or between the control and treatment ditch. We can also pick up some interested trends from the graph, for example, the graph shows that there is a shift in the composition of microbial community from the inlet to the outlet. There are many potential factors that contribute to this change. For example, the nitrate and dissolved oxygen concentration. As well as the organic matter content we will later create a model to predict and understand which factors are affecting the microbiome composition and which factors have a bigger impacts than the other.
- Similar trend in 2021 Coordinate system โ different, it is just how the graph was generated. matrix calculate the dissimilarity between each samples and generate the graph automatically to visually the difference. Not too much meaning to the axises.
- Treatment more divergent: meaning even though the dots are clustered together. They tend to be more spread out than the control ones. -33%
- Currently, more than 300 strains were isolated from the ditch sediment with selecting media. Some common genera of the strains identified include Pseudomonas, Clostridium, Cryobacterium;
- N2 production: inoculate strain with nitrate, and measure n2 production overtime 15N2 โ substrate
- NiCE chip is a high-throughput qPCR system that can quantitatively measure >30 nitrogen cycle related genes for more than 120 samples at the same time. Old days โ a few function genes. woodchipsamples Target genes include key genes for denitrification (nirK, nirS, norB, nosZ) Gel encapsulation โ buy sometime
- Thank you, and I would like to thank the funding source for this project, University of Minnesota MNDrive program. I would also like to thank the Ishii lab, Strock lab and other SWROC members for providing valuable comments and supports to this project.
- Thank you so much for your time and I would like to take questions or comments regarding this project.