Unlocking the bioreactor microbiome for nutrient management and water quality
Issue
Currently, the bioreactor microbial community is a black box of uncertainty in terms of what is responsible for the denitrification and pollution swapping and where the community is originating from. Preliminary data from researchers’ prior work compared woodchips from nine field bioreactors operating for two years at different flow conditions and identified consistent microbial membership between the installed bioreactors as well as consistent load reduction across the range of controlled hydraulic retention times. These observations, that predictable microbes were associated with bioreactors with varied engineered controls, are the rationale for the hypothesis that bioreactor microbiomes can be selected and may also be managed. Researchers believe that understanding how the environmental conditions affect both nutrient removal and the microbiome will benefit bioreactor performance and ensure the sustainability of their operations.
Objective
In this proposal, researchers seek to identify and manipulate microbial communities in corncob and woodchip bioreactors that mediate complete denitrification of nitrate to dinitrogen gas, with minimal release of nitrous oxide, methane or methylmercury to expand the full potential of this conservation practice. Further, treating bioreactor microbial communities as manageable resources, they will evaluate the feasibility of managing and manipulating bioreactor microbiomes to benefit bioreactor performance.
Approach
The researchers will use the data collected to develop a model, which will be the first to link operation, microbiome and function, and will support understanding of the underlying biological and chemical controls for integrating this conservation practice in agroecosystems.
Project Updates
Note: Project reports published on the INRC website are often revised from researchers' original reports to increase consistency.
December 2023
The six upflow columns constructed as lab-scale representation bioreactors are continuing to run. They have completed three months at 8 h HRT (ideal flow rate) and have moved on to the second treatment of an increased 16 h HRT flow rate. In early October, three columns (all corncob) were not operating correctly. After troubleshooting, we decided to replace corncobs, tubing and pumps. Fresh cobs were collected and were inoculated in the columns with fresh tile water for a week. These fresh corncobs were also used for DNA extraction. Samples of used corncobs were taken for potential future analysis. Since tubing, pump, and corncob replacement, no further issues have occurred. In order to prevent future issues, monthly maintenance of tubing is now conducted.
During the replacement period, the three woodchip columns were sampled on a weekly basis as usual. As of late December, there are 30 weeks of collected data: water samples, greenhouse gas samples, water DNA and monthly media DNA. As of December 1, DNA is collected weekly (rather than monthly). Water and media DNA samples are being extracted and diluted in preparation for qPCR analysis to test for microbial processes of denitrification. The three woodchip columns are approaching their three months at 16 h HRT and will be switched to 2 h HRT within the next month. Because of the delay with the three corncob columns, they will not be switched to 2 h HRT until early March.
Photos attached show corncobs before replacement. There is a distinguishable color difference between the three, namely that column 3 (farthest to the right) is a darker color. This column did not remove nitrate as well as columns 1 and 2 (farthest left and middle). The second photo includes the used corncobs inside a tub. We suspect that fungus visibly present in columns 1 and 2 (cobs from left to right: column 2, column 1, column 3) clogged the columns and prevented water from flowing, therefore overworking the pump.
Corn Cob Columns
July 2023
(1) Bioreactor columns are constructed and denitrifying in our laboratory. Nitrate removal was used to compare the performance of fresh media in tile water to fresh media, mixed with active field media, in a lab-created nutrient solution (similar to field conditions). This test was important to determine that bioreactor construction was successful. The columns were started April 27, 2023.
Weekly greenhouse gas and water samples have been taken since, along with initial DNA extraction. We had a challenge after one month when the corncob bioreactors were producing an unprecedented amount of gas. The high concentration of gas was creating a buildup in the system, and the water was not able to escape. This led to leaks, requiring us to stop the columns and adapt. In response, we have developed a method of venting excess gas while ensuring gas samples taken were still accurate. This method has so far been effective, and we are currently implementing it on the woodchip columns. Initial data analysis suggests the corncobs produce more CO2 than the woodchips, showing that the excess gas is a result of an increased decomposition rate. We also see a greater flush of total organic carbon in the corncob columns compared to the woodchip columns. The system is maintained on a daily basis by refilling the influent of nutrient solution, ensuring no leaks are occurring, monitoring flow rate for proper HRT and checking pressure buildup. Dissolved oxygen is measured every sampling day to ensure anoxic conditions are achieved within the system.
(2) From the corn cob and woodchip bioreactors, we have isolated bacteria which are nitrate-reducing and are characterizing their ability to denitrify. We have isolated a total of 26 bacteria, and 13 of them have been shown to reduce nitrate. These microbes are potential inputs into our objective that relates to 'inoculating' bioreactors with optimal microbiomes to help stabilize their performance.
Other activities
The graduate student working on the project, Taylor, will present on this work at the INRC Fall seminar series "Focus on the Future."
December 2022
Researchers recruited a graduate student to help lead this research. An in-lab-scale representation of bioreactors has been constructed. Fresh woodchips have been collected to be placed inside the three designed up-flow columns. The microbiome associated with these woodchips will be characterized through DNA extraction of woodchip surfaces.Quality control is completed, and the bioreactor laboratory design is now operational. Standard procedures are being developed for water sample collection, greenhouse gas sample collection and DNA sample collection and extraction techniques.
Experiments are scheduled to begin in mid-January.