Investigating the Ability of Mushroom Mycelium to Reduce Fecal Coliform Bacteria Contamination in Surface Water

Posted 9 months ago by Rob Turner

School(s) : Interdisciplinary Arts and Sciences, STEM
Primary PI Name : Rob Turner
Email : Rturner1@uw.edu
Phone : 425-352-3616
Project/Faculty Website : http://faculty.washington.edu/rturner1
Research Location : UW Bothell
Project Goals : Abstract: Our research project is investigating the ability of mushroom mycelium-inoculated straw wattles to reduce fecal coliform bacteria contamination in small streams of the constructed floodplain wetland of the University of Washington Bothell/Cascadia College campus. The goal is to greatly reduce the concentrated discharges of E. coli bacteria to North Creek, which in turn is advected to Lake Washington via the Sammamish River. Preliminary laboratory tests found reductions of E. coli to be as much as 99% when contaminated water was passed through woodchip cylinders inoculated with King Stropharia (S. rugosoannulata) mycelium, relative to controls without mycelium. Lab experiments conducted with Blue Oyster (P. ostreatus) mushroom mycelium have not shown the same success. Observed variability in the ability of mycelium-inoculated substrates to reduce E. coli counts of contaminated water appears to be dependent on factors such as the retention time of water in the substrate, species of mushroom, and maturity of the mycelium. Experiments with mycelium-inoculated straw wattles placed in contaminated streams have so far yielded inconclusive results, with only 2 of 8 trials demonstrating decreased mean E. coli counts downstream of the wattles. Previous Work: Mycoremediation is the practice of using mushroom mycelium to remove contaminants from soil and surface water. This is a relatively new area of research, combining ecological restoration, environmental chemistry, and microbiology, with the potential to dramatically improve water quality in surface water runoff, and thus in the lakes that receive those remediated discharges. Laboratory and field research conducted by Paul Stamets and others has demonstrated that mushroom inoculated substrates can reduce Escherichia coliform (E. coli) bacteria counts in water that has passed through the substrates (Stamets et al., 2013). Some species of E. coli are pathogenic and their presence indicates contamination via fecal matter and the likely presence of other intestinal pathogens. Site Characteristics: The UWB/Cascadia College campus was established in 2000 on the site of a cattle ranch. The 59 acre floodplain wetland restoration project is the largest such project in Washington State. Rapid growth of the student population campus has fostered nearly continuous building, increased impervious surface, and increased runoff into the wetland. Meanwhile, the successful wetland restoration has attracted a large population of crows that use it as a nightly roost site. The crow population swells to as many as 15,000 birds each night in the winter months (Wacker, 2015). This results in the deposition of a massive amount of fecal matter each day, making the campus and wetland an uncharacteristically large source of pathogen and nutrient loading to North Creek, which flows through the wetland and discharges to the Sammamish River just south of campus (Figure 4). The two sites chosen for field testing were SW8 and SW3 (Figure 5). Both are small tributaries which run through the crow roost area and discharge into North Creek. At SW8, wattles were placed in line with the stream in August, while at SW3 wattles were placed perpendicular across the channel in September (Figures 6 and 7). Field Results: Wattles inoculated with Blue Oyster mycelium were placed in two wetland tributaries and evaluated for their effectiveness at removing E.coli. In all trials, stream water passed through a maximum of two mycelium inoculated wattles (~1ft thickness each). Only 2 of the 8 trials showed reductions in E. coli counts downstream of the wattles (Figure 10) and those observed reductions may just be artifacts of the insufficient precision of the m-ColiBlue method when diluting samples. Laboratory Results: In Winter of 2016, King Stropharia (S. rugosoannulata) was screened for it’s potential to remove E. coli from North Creek water samples at different residence times. Water from North Creek was brought to the lab and poured through cylinders of wood chips inoculated with King Stropharia mycelium (Figure 2), as well as through control cylinders with wood chips and no mycelium. Three residence time treatments were evaluated. Treatment one allowed flow through the cylinder. Treatment two and three retained water in the cylinders for 1 hour and 24 hours, respectively. As residence time increased in treatments, E. coli counts were reduced in output samples and eventually reached almost 100% reduction (Figures 8 and 9). However, similar experiments conducted by running contaminated water through straw inoculated with Blue Oyster mycelium showed no significant reduction in E. coli.Conclusions Laboratory experiments demonstrated that substrates inoculated with King Stropharia mycelium can reduce E. coli counts in water passing through them and that reductions improve with contact time. However, substrates inoculated with Blue Oyster mycelium have had minimal impact on E. coli counts in the lab or in the field. Next steps will be to make wattles with King Stropharia mycelium, install more of them in a stream to increase contact time of water in the wattles, and regularly swap out wattles so they can recover from saturation.
Student Outcomes : Methods: The data set of greatest interest in this study are counts of E. coli bacteria from samples of contaminated water that have passed through inoculated straw or wood chips. In all experiments, samples were collected in triplicates, then filtered, plated, incubated, and counted following the protocols of EPA-approved method 10029 (Hach m-ColiBlue24). Figure 1 shows what filter papers look like using this method. E. coli and Other Coliform bacteria colonies are counted in all replicates, averaged, and expressed as colony forming units per 100 ml. Deionized water blanks are analyzed following the same protocols to test for contamination in our method. Investigation of the ability of mushroom mycelium to reduce E. coli counts in water was conducted by running contaminated water through either straw or wood chips inoculated with King Stropharia or Blue Oyster mycelium (Figures 2 and 3). Creating mycelium inoculated wattles like the one pictured at the top left of this poster involves collecting and sanitizing straw, seeding it with fungi, wrapping it up with burlap, storing it in the dark for a few weeks, and watering intermittently.
Additional information : Figure 1. Photograph of E. coli (blue) colonies and other coliform bacteria (red) colonies on filter paper incubated on mColi Blue broth. Figure 2 (left). Cylinder with inoculated wood chips used in the “Runthrough” and residence time experiments. Figure 3 (right). Filter tower used to test the capabilities of mycelium inoculated straw used in wattles. Figure 4. Map of the Lake Washington area with a box showing the location of the study site. North Creek discharges into the Sammamish River, which then flows into Lake Washington. Map from Kiyohara and Zimmerman, 2009. Figure 5. Surface runoff in the UWB/Cascadia College wetland and our water quality sampling sites. The orange circles mark the locations where we have placed mycelium inoculated wattles. The crow roost zone covers most of the wooded area east of the bike path. Map by Anfal Alwheid. Figure 6. Two wattles installed parallel to stream flow at site SW8. The stream is only about 1 foot wide here. Figure 7 (right). Two wattles installed perpendicular to stream flow at site SW3. The blue arrow shows direction of flow. Wattles were placed here when a rain event blew out the wattles at SW8 and prompted the resumption of flow between the pond at SW2 and North Creek. Figure 8. E. coli counts of North Creek samples were quantified (Input). Then sample water was poured into cylinders with wood chips inoculated with King Stropharia mycelium. Mean E. coli counts fell by almost 50% just by flowing through the cylinders. Increasing the residence time of water in the cylinders dramatically reduced the E. coli counts. Impacts of these treatments on Other Coliform counts were inconclusive. Figure 9. Mean E. coli counts increased as the sample water passed through cylinders that held wood chips with no mushroom mycelium. E. coli counts increased dramatically in sample water that was retained in the cylinders that held wood chips without mushroom mycelium. Figure 10. Two trials showing decreases in mean E. coli counts downstream of wattles. Decreases were modest and the high variation of counts among triplicate samples render the difference in the means of the 8/25/16 trial insignificant. References Cited: Kiyohara, K and Zimmerman, M (2009). Evaluation of Downstream Migrant Salmon Production in 2008 from the Cedar River and Bear Creek. Washington State Department of Fish and Wildlife. http://wdfw.wa.gov/publications/00091/wdfw00091.pdf Stamets, P, Beutel, M, Taylor, A, Flatt, A, Wolff, M, and Brownson, K (2013). Comprehensive Assessment of Mycofiltration Biotechnology to Remove Pathogens from Urban Stormwater. EPA SBIR Phase I Research Results. Fungi Perfecti. http://fungi.com/pdf/articles/Fungi_Perfecti_Phase_I_Report.pdf Wacker, D (2015). Personal communication.

  • School(s) : Interdisciplinary Arts and Sciences, STEM
  • Primary PI Name : Rob Turner
  • Interested? Contact Faculty Researcher by Email : Rturner1@uw.edu
  • Phone : 425-352-3616
  • Project/Faculty Website : http://faculty.washington.edu/rturner1
  • Research Location : UW Bothell
  • Project Goals : Abstract: Our research project is investigating the ability of mushroom mycelium-inoculated straw wattles to reduce fecal coliform bacteria contamination in small streams of the constructed floodplain wetland of the University of Washington Bothell/Cascadia College campus. The goal is to greatly reduce the concentrated discharges of E. coli bacteria to North Creek, which in turn is advected to Lake Washington via the Sammamish River. Preliminary laboratory tests found reductions of E. coli to be as much as 99% when contaminated water was passed through woodchip cylinders inoculated with King Stropharia (S. rugosoannulata) mycelium, relative to controls without mycelium. Lab experiments conducted with Blue Oyster (P. ostreatus) mushroom mycelium have not shown the same success. Observed variability in the ability of mycelium-inoculated substrates to reduce E. coli counts of contaminated water appears to be dependent on factors such as the retention time of water in the substrate, species of mushroom, and maturity of the mycelium. Experiments with mycelium-inoculated straw wattles placed in contaminated streams have so far yielded inconclusive results, with only 2 of 8 trials demonstrating decreased mean E. coli counts downstream of the wattles. Previous Work: Mycoremediation is the practice of using mushroom mycelium to remove contaminants from soil and surface water. This is a relatively new area of research, combining ecological restoration, environmental chemistry, and microbiology, with the potential to dramatically improve water quality in surface water runoff, and thus in the lakes that receive those remediated discharges. Laboratory and field research conducted by Paul Stamets and others has demonstrated that mushroom inoculated substrates can reduce Escherichia coliform (E. coli) bacteria counts in water that has passed through the substrates (Stamets et al., 2013). Some species of E. coli are pathogenic and their presence indicates contamination via fecal matter and the likely presence of other intestinal pathogens. Site Characteristics: The UWB/Cascadia College campus was established in 2000 on the site of a cattle ranch.  The 59 acre floodplain wetland restoration project is the largest such project in Washington State.  Rapid growth of the student population campus has fostered nearly continuous building, increased impervious surface, and increased runoff into the wetland.  Meanwhile, the successful wetland restoration has attracted a large population of crows that use it as a nightly roost site.  The crow population swells to as many as 15,000 birds each night in the winter months (Wacker, 2015).  This results in the deposition of a massive amount of fecal matter each day, making the campus and wetland an uncharacteristically large source of pathogen and nutrient loading to North Creek, which flows through the wetland and discharges to the Sammamish River just south of campus (Figure 4). The two sites chosen for field testing were SW8 and SW3 (Figure 5). Both are small tributaries which run through the crow roost area and discharge into North Creek. At SW8, wattles were placed in line with the stream in August, while at SW3 wattles were placed perpendicular across the channel in September (Figures 6 and 7). Field Results: Wattles inoculated with Blue Oyster mycelium were placed in two wetland tributaries and evaluated for their effectiveness at removing E.coli. In all trials, stream water passed through a maximum of two mycelium inoculated wattles (~1ft thickness each). Only 2 of the 8 trials showed reductions in E. coli counts downstream of the wattles (Figure 10) and those observed reductions may just be artifacts of the insufficient precision of the m-ColiBlue method when diluting samples. Laboratory Results: In Winter of 2016, King Stropharia (S. rugosoannulata) was screened for it’s potential to remove E. coli from North Creek water samples at different residence times. Water from North Creek was brought to the lab and poured through cylinders of wood chips inoculated with King Stropharia mycelium (Figure 2), as well as through control cylinders with wood chips and no mycelium. Three residence time treatments were evaluated. Treatment one allowed flow through the cylinder. Treatment two and three retained water in the cylinders for 1 hour and 24 hours, respectively.  As residence time increased in treatments, E. coli counts were reduced in output samples and eventually reached almost 100% reduction (Figures 8 and 9). However, similar experiments conducted by running contaminated water through straw inoculated with Blue Oyster mycelium showed no significant reduction in E. coli.Conclusions Laboratory experiments demonstrated that substrates inoculated with King Stropharia mycelium can reduce E. coli counts in water passing through them and that reductions improve with contact time. However, substrates inoculated with Blue Oyster mycelium have had minimal impact on E. coli counts in the lab or in the field. Next steps will be to make wattles with King Stropharia mycelium, install more of them in a stream to increase contact time of water in the wattles, and regularly swap out wattles so they can recover from saturation.
  • Student Outcomes : Methods: The data set of greatest interest in this study are counts of E. coli bacteria from samples of contaminated water that have passed through inoculated straw or wood chips. In all experiments, samples were collected in triplicates, then filtered, plated, incubated, and counted following the protocols of EPA-approved method 10029 (Hach m-ColiBlue24). Figure 1 shows what filter papers look like using this method. E. coli and Other Coliform bacteria colonies are counted in all replicates, averaged, and expressed as colony forming units per 100 ml. Deionized water blanks are analyzed following the same protocols to test for contamination in our method. Investigation of the ability of mushroom mycelium to reduce E. coli counts in water was conducted by running contaminated water through either straw or wood chips inoculated with King Stropharia or Blue Oyster mycelium (Figures 2 and 3). Creating mycelium inoculated wattles like the one pictured at the top left of this poster involves collecting and sanitizing straw, seeding it with fungi, wrapping it up with burlap, storing it in the dark for a few weeks, and watering intermittently.
  • Number of Student Positions Available : 2
  • Additional information : Figure 1. Photograph of E. coli (blue) colonies and other coliform bacteria (red) colonies on filter paper incubated on mColi Blue broth. Figure 2 (left). Cylinder with inoculated wood chips used in the “Runthrough” and residence time experiments. Figure 3 (right). Filter tower used to test the capabilities of mycelium inoculated straw used in wattles. Figure 4. Map of the Lake Washington area with a box showing the location of the study site. North Creek discharges into the Sammamish River, which then flows into Lake Washington. Map from Kiyohara and Zimmerman, 2009. Figure 5. Surface runoff in the UWB/Cascadia College wetland and our water quality sampling sites. The orange circles mark the locations where we have placed mycelium inoculated wattles. The crow roost zone covers most of the wooded area east of the bike path. Map by Anfal Alwheid. Figure 6. Two wattles installed parallel to stream flow at site SW8. The stream is only about 1 foot wide here. Figure 7 (right). Two wattles installed perpendicular to stream flow at site SW3. The blue arrow shows direction of flow. Wattles were placed here when a rain event blew out the wattles at SW8 and prompted the resumption of flow between the pond at SW2 and North Creek. Figure 8. E. coli counts of North Creek samples were quantified (Input). Then sample water was poured into cylinders with wood chips inoculated with King Stropharia mycelium. Mean E. coli counts fell by almost 50% just by flowing through the cylinders. Increasing the residence time of water in the cylinders dramatically reduced the E. coli counts. Impacts of these treatments on Other Coliform counts were inconclusive. Figure 9. Mean E. coli counts increased as the sample water passed through cylinders that held wood chips with no mushroom mycelium. E. coli counts increased dramatically in sample water that was retained in the cylinders that held wood chips without mushroom mycelium. Figure 10. Two trials showing decreases in mean E. coli counts downstream of wattles. Decreases were modest and the high variation of counts among triplicate samples render the difference in the means of the 8/25/16 trial insignificant. References Cited: Kiyohara, K and Zimmerman, M (2009). Evaluation of Downstream Migrant Salmon Production in 2008 from the Cedar River and Bear Creek. Washington State Department of Fish and Wildlife. http://wdfw.wa.gov/publications/00091/wdfw00091.pdf Stamets, P, Beutel, M, Taylor, A, Flatt, A, Wolff, M, and Brownson, K (2013). Comprehensive Assessment of Mycofiltration Biotechnology to Remove Pathogens from Urban Stormwater. EPA SBIR Phase I Research Results. Fungi Perfecti. http://fungi.com/pdf/articles/Fungi_Perfecti_Phase_I_Report.pdf Wacker, D (2015). Personal communication.