Final report for GNC17-235
In recent years, Minnesota has begun to tackle its significant nitrate pollution, brought to attention in particular by the Dead Zone in the Gulf of Mexico, to which Minnesota is the sixth largest contributor. The greatest input to nitrate pollution is agriculture, which accounts for an estimated 89-95% of the nitrate loads found in the Minnesota, Missouri, and Cedar Rivers, and Lower Mississippi River basins (MPCA 2013). Woodchip bioreactors, which provide the means for the biological reduction of nitrate to inert dinitrogen gas (i.e. denitrification), are a feasible means for reducing nitrate leaching. However, there is a need to enhance bioreactor efficiency in the spring when runoff and nutrient leaching are at their highest in Minnesota. Cold temperatures in early spring most likely limit the activity of denitrifying bacteria in woodchip bioreactors.
This study will employ two methods for enhancing an established woodchip bioreactor located in Willmar, Minnesota: (1) bioaugmentation, in which cold-adapted denitrifying bacteria are introduced to the bioreactor, and
(2) biostimulation, in which an additional carbon source is added to the bioreactor to stimulate microbial activity and denitrification. Bacteria will be isolated from the bioreactor itself and tested for denitrification abilities. The bacteria with the greatest denitrification rates at cold temperatures will be grown in the lab and subsequently reintroduced to the bioreactor. By measuring nitrate reduction over time at this bioreactor, we will be able to determine the optimal microbial community and additional carbon that gives the largest nitrate removal.
This study can be applied to other bioreactors in Minnesota and the Midwest to create the most efficient bioreactors. Enhancement of these bioreactors will contribute significantly to nitrate load reductions from agriculture currently polluting bodies of water. Going forward, this information will be used to establish best management practices to enhance bioreactor in a cost effective way. The overall goal of this research will particularly benefit small-scale farmers as it provides a cost-effective, edge of field option for reducing nitrate pollution.
Having an understanding of the microbial communities naturally present in woodchip bioreactors will allow researchers to optimize the woodchip bioreactors using specific strains of known denitrifying bacteria. Denitrifying bacteria from woodchip bioreactors have not been well characterized, so this study will pave the way for this research and provide impetus for further study of the microbial communities in woodchip bioreactors. Learning about the denitrifying bacterial communities will allow researchers to target those bacteria that are most active at lower temperatures and inoculate the woodchip bioreactors with these strains. Inoculating the woodchip bioreactor with the most efficient strains will contribute significantly to nitrate load reductions from agriculture currently polluting bodies of water, which can be quantified by measuring nitrate load reductions in the wastewater before and after entering the woodchip bioreactor, and it will be invaluable in the execution of the Minnesota Nutrient Reduction Strategy.
Developing a procedure for optimal denitrification will promote the establishment of woodchip bioreactors across the region. Throughout the course of this study, researchers will strive to engage with the Willmar community by informing farmers and community members of the mechanisms behind biological nitrate reduction and sharing progress on the enhancement of this woodchip bioreactor. This will occur through increased presence at community events, such as having a table and poster at a local farmer’s market. Learning about the economic feasibility and effectiveness of these systems will incentivize farmers to establish woodchip bioreactors on their own fields.
For this project, a woodchip bioreactor had already been established and running for several years, however our goal was to enhance this woodchip bioreactor using biostimulation and bioaugmentation techniques. During year one, we spoke with the local farmers to discuss what was needed - what their priorities were and their concerns with the woodchip bioreactor. The idea of the woodchip bioreactor, which requires no loss of farmland and can safely remove nutrients from wastewater, was well-received, but there were concerns about their effectiveness. During the fall, we took samples of the woodchips and wastewater to measure N. With the woodchips, I isolated denitrifying-microorganisms under cold conditions, tested their denitrification abilities, and identified the most efficient strains. We grew those particular strains in large cultures and, during the following spring, brought them to the field for inoculation. We recruited many volunteers (undergraduate/graduate students in various fields of study, technicians, interested professors, USDA employees, one local farmer) for this field campaign - it involved inoculating 3 of the 8 bioreactors in the field with this efficient denitrifier every two weeks for 6 weeks. At the same time, another 3 of the 8 bioreactors were subjected to biostimulation - this involved a programmed pump that would deliver an optimal concentration of acetate to the bioreactors so that the denitrifying microorganisms would thrive. We sampled the water from each of the 8 bioreactors at each trip to the field and then processed the samples in the laboratory on campus, exposing many of the volunteers to new lab techniques.
Currently, the water samples that will tell us how biostimulation and bioaugmentation affected N loading are being analyzed. These results are expected to be part of a published journal article next year. However, the process of identifying and characterizing efficient denitrifiers for bioaugmentation has been submitted to Applied and Environmental Microbiology for publication. Several of the potential denitrifiers were subjected to whole genome sequencing, which revealed that one of the microorganisms has the complete denitrification gene pathway - we are hopeful that by inoculating this strain to the woodchip bioreactor, nitrate reduction will increase significantly, even under cold conditions.
Educational & Outreach Activities
Journal articles: Currently there is one article in review for Environmental Microbiology titled "Cold-Adapted Denitrifying Bacteria in Woodchip Bioreactors". There are also two additional articles that will soon be sent for review to Soil Biology and Biochemistry Journal and Genome Announcement.
Talks and Presentations: I spoke about the woodchip bioreactor research at the Water Resources Symposium at the University of Minnesota in March 2018. I also presented a poster in Atlanta at the American Society for Microbiology (ASM) June 2018 conference about woodchip bioreactors. Additionally, I presented my defense seminar about the woodchip bioreactor findings in August 2018. In attendance were 2-3 farmers interested in the study, as well as technicians, University faculty and students interested in bioremedation. And finally, at the end of August, we will have a stand at the Minnesota State Fair where we will show a mini woodchip bioreactor and talk with the public about water quality and nitrate remediation.
Workshop/Field days: This spring we held a number of field days learning about the bioreactor, with each field day corresponding to a lab day the following day. We had 5-6 volunteers each field day that included U of MN graduate and undergraduate students interested in learning more about nitrate bioremediation, technicians, professors and engineers. The following lab days involved processing water and woodchip samples collected the previous day and included a larger group of students and faculty learning about sample collection and new lab techniques.
Both researchers and members of the public became excited when hearing about the promising new technology of woodchip bioreactors and our plans to enhance them through bioaugmentation. Woodchip bioreactors require no loss of farmland, are simple to install and consistently remove nitrate from wastewater. While woodchip bioreactors have been successful, our findings proposed a combination of inoculating two efficient denitrifiers to a woodchip bioreactor. This was carried out in the spring, and results are still being analyzed. Adding our denitrifying microorgansims should enhance a woodchip bioreactor during cold temperatures, particularly. We have found a cost-effective, environmentally-safe method for enhancing the woodchip bioreactors. In talking to broad groups of people at university-wide events and public outreach events such as the State Fair, there seems to be a growing interest in installing woodchip bioreactors, especially since they could be a more targeted alternative to buffer strips.
Throughout the last year, I have appreciated the opportunity to work with many people that were previously unaware of woodchip bioreactors or nutrient management strategies in general. I have shared the microbiological findings with farmers, technicians and engineers that have not had any experience with microbiology and we have also shared our research with a broad group of University researchers, students that have now expressed interest in sustainable agriculture, and members of the public that were eager to learn more about this research. Sharing our findings with people and witnessing their changing attitudes has made me excited about sustainable agriculture.