MYCOFILTRATION Using Fungi To Improve Water Quality Tradd Cotter Mushroom Mountain LLC

PESTICIDES (Herbicides, insecticides) CHEMICAL (PCB’s, Aromatic Hydrocarbons, Dioxins) BIOLOGICAL (Coliforms, parasites) HEAVY METALS (Lead, mercury, arsenic) MYCOREMEDIATION Applications

Total Maximum Daily Loads (TMDLs) Sediment Pathogens Nutrients Metals Dissolved Oxygen Temperature pH Pesticides Mercury Organics

(TMDLs) Standards for Determining Loading Capacity – The greatest amount of a pollutant that a water can assimilate and still meeet water quality standards Identify contamination limits of pathogen in colony forming units per milliliter(CFU/mL) Fecal coliforms - 40FC/100mL Chemical Pollutants – ppm/ppm dependent on toxicity and mortality of organisms

Total Maximum Daily Loads Survey Wildlife and Aquatic Biology Inventory and evidence of biological disruption Determine life cycles of biology and seasonal inputs to identify weaknesses and resilience of systems Identify Short Term to Long Term goals, targets, and indicators

Quality of Media Should provide adequate passage of water for ideal contact time and rate of flow Needs to be composed of a durable matrix that can withstand rate of flow and saturation (wheat straw vs wood chips) Preferably composed of native debris matched with local or regional strains trained to recognize debris

Testing Solutions Herbicides Bean tests (Can also use tomatoe seeds, and other listed on EPA website for threshold toxicity) Test exposures for all 12 dilutions (up to parts per billion, ppb, in replicates of 5) prior to running tabletop units to note damage to assess a physical spectrum of noticeable damage

Testing Solutions Biological Pathogens STREAK ISOLATION AND PURIFICATION Identification of pathogens, SERIAL DILUTIONS and EASYGEL plate pours for population counts – colony forming units (CFU/mL) Test behavioral galleries using any number of orientations Use metabolites formed by CONTACT STIMULATION in agar media or in wells formed in agar to determine rates of inhibition, cell lyses, or inactivation. Bacterial growth curves, controls and dosed with metabolites

ARENA ANALYSIS Harvest cells at critical intervals to determine metabolic responses/variance compared to controls Variances can be determined and quantified for both beneficial and detrimental responses Intracellular metabolites can be analyzed for gene expression when exposed to different combinations of mixed species

Once biomass is fully colonized, even while fruiting mushrooms, water percolation can commence Water can be poured or mechanically pumped to circulate a given volume on a small or large scale Water also contains trace metabolites that are known for their antiviral and immune enhancing properties MYCOFILTRATION

Filling Unit Unit can be filled with precolonized media and allowed to bind, or filled with substrate and spawn and allowed to colonize BEFORE running unit. Make sure the unit is clean and wiped clean of all residual pesticide residues, pump must also be cleaned and run, remove magnetic rotor and clean with a q-tip and alcohol. Once the media is colonized, the bottom tray can be filled with a predetermined volume of water and dosed with contaminant of interest (biological or chemical) calculated at CFU/ml or ppm/ppb.