Georgia State University

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Geosciences Theses Department of Geosciences

Spring 5-14-2021

Examining Mycofiltration Efficacy in a First Order Stream

Lacey J. Davis
Georgia State University

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Davis, Lacey J., "Examining Mycofiltration Efficacy in a First Order Stream." Thesis, Georgia State
University, 2021.
doi: https://doi.org/10.57709/22653283

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 EXAMINING MYCOFILTRATION EFFICACY IN A FIRST ORDER STREAM

by

LACEY J. DAVIS

Under the Direction of Sarah H. Ledford, PhD

ABSTRACT

Bacterial contamination from sanitary and combined sewer overflows, leaking sewer

infrastructure, and stormwater runoff decreases urban surface water quality. This research

investigates a bioremediation technique, mycofiltration, to mitigate episodic bacterial

contamination in first-order urban streams, which has previously been demonstrated to work in

lab experiments. The objectives are: (1) establish the spatial distribution of E. coli in the Upper

South River watershed, and (2) evaluate the potential for Trametes versicolor fungal spawn to

decrease E. coli concentrations when accounting for short hydrologic retention and surface

water-groundwater interactions inherent in streams via a stream table experiment. The Trametes

versicolor mycofilter overall reduced concentrations of E. coli, but no more than was reduced by

stream sediments alone. These findings suggest the usefulness of mycofiltration may be limited

by decreased contact time or hyporheic flow paths that bypass the mycelium installation.

INDEX WORDS: Mycoremediation, Mycofiltration, Urban hydrology, South River, Water

quality, Bacterial contamination
EXAMINING MYCOFILTRATION EFFICACY IN A FIRST ORDER STREAM

by

LACEY J. DAVIS

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science

in the College of Arts and Sciences

Georgia State University

2021
 Copyright by
Lacey Jon Davis
2021
 EXAMINING MYCOFILTRATION EFFICACY IN A FIRST ORDER STREAM

by

LACEY J. DAVIS

Committee Chair: Sarah H. Ledford

Committee: Richard Milligan

Christine Stauber

Electronic Version Approved:

Office of Graduate Services

College of Arts and Sciences

Georgia State University

May 2021
 iv

DEDICATION

I dedicate this thesis to my grandparents, Sherry and Larry Binenfeld, for their

unconditional love, and confidence in me. I would like to also dedicate this work to my mother,

Laura Elaine Binenfeld Negelow Davis Aven, and my sister, Laina Rachel Davis, who are my

greatest teachers and unwavering touchstones.

 v

ACKNOWLEDGEMENTS

I would like to first acknowledge my thesis advisor, Dr. Sarah Ledford, for her consistent

support, guidance, and confidence in me. I would also like to acknowledge my committee, Dr.

Richard Milligan and Dr. Christine Stauber, for their contributions to the expansion and

refinement of this thesis. Additionally, I would like to thank Dr. Christy Visaggi for her

mentorship throughout my academic career. Finally, I would like to acknowledge my formidable

friends: Maggie, Eleanor, Margaret, Sara Ann, Natalie, and my sweetheart, Mara, whose

enduring love provides me the bearings to navigate this ever-changing world. I love you all very

much and am forever grateful for your commitment and care.

 vi

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................................ V

LIST OF TABLES ................................................................................................................... VIII

LIST OF FIGURES .................................................................................................................... IX

1 INTRODUCTION............................................................................................................. 1

1.1 South River Watershed ............................................................................................... 3

1.1.1 Overview of SSOs and CSOs in the Upper South River ......................................... 4

1.1.2 Current E. coli contamination in the Upper South River ....................................... 8

1.1.3 Human health impacts ........................................................................................... 11

1.1.4 What is mycofiltration? .......................................................................................... 12

1.2 Study objectives .......................................................................................................... 13

2 BACKGROUND ............................................................................................................. 15

2.1 Evidence of mycofiltration efficacy .......................................................................... 15

2.1.1 Mycelium behavior ................................................................................................. 16

2.2 Heavy metal and chemical mycoremediation .......................................................... 17

2.3 Bacterial mycoremediation ....................................................................................... 19

3 METHODS ...................................................................................................................... 21

3.1 South River Watershed Alliance sampling procedures .......................................... 21

3.2 Experiment design...................................................................................................... 23

3.2.1 Stream table design and sediment installation ...................................................... 23

 vii

3.2.2 In-situ sample collection ........................................................................................ 25

3.2.3 E. coli isolation and growth ................................................................................... 25

3.2.4 Mycelium cultivation and deployment ................................................................... 28

3.3 Dilution calculations and procedure......................................................................... 31

3.3.1 Analyzing E. coli concentrations ........................................................................... 32

4 RESULTS ........................................................................................................................ 33

4.1 Upper South River E. coli concentrations................................................................ 33

4.2 Lab experiments ......................................................................................................... 37

5 DISCUSSION AND CONCLUSION ............................................................................ 41

5.1 Discussion .................................................................................................................... 41

5.2 Conclusion................................................................................................................... 44

REFERENCES ....................................................................................................................... 46
 viii

LIST OF TABLES

Table 1 Impaired Stream Segments in HUC 0301070301 (Scott, 2014). ....................................... 9

Table 2 Bacterial culture tracking sheet........................................................................................ 26

Table 3 Determining E.coli titer. .................................................................................................. 27

Table 4 Determining E. coli titer continued.................................................................................. 27

Table 5 Calculations for determining E. coli titer......................................................................... 27

Table 6 Procedure for E. coli dilution.......................................................................................... 31

Table 7 E. coli and Total Coliform data for each sample site....................................................... 35

Table 8 Concentration decrease of E. coli for each experiment. ................................................. 40

 ix

LIST OF FIGURES

Figure 1 Impervious surfaces and stormwater runoff (EPA, 2003). ............................................... 2

Figure 2. Headwaters of the Upper South River are a milky white color due to leachate from a

historical cotton processing plant and is designated a class 1 hazardous waste site........... 4

Figure 3 GA EPD's Watershed Protections Branch Monitoring Stations on the Upper South River

(EPD, 2021) ........................................................................................................................ 9

Figure 4 Sample sites along the Upper South River ..................................................................... 22

Figure 5 Stream table filled with Ripplewater Creek sediment. ................................................... 24

Figure 6 Stream table effluent....................................................................................................... 25

Figure 7 E. coli bacterial titer. ...................................................................................................... 28

Figure 8 Isolated wild E. coli type. ............................................................................................... 28

Figure 9 Trametes versicolor mycelium. ..................................................................................... 30

Figure 10 Trametes versicolor burlap mycofilter deployed in stream table. ............................... 30

Figure 11 Sterilized Quercus alba sawdust. ................................................................................. 30

Figure 12 E. coli concentrations in the Upper South River (Note: Because the Tift Site was

below the detection limit, it is not included in this figure.) .............................................. 34

Figure 13 E. coli concentrations every hour for the experiment with bleached sediment and E.

coli-free inflow. Error bars indicate the standard deviation of three outflow samples taken

at each timestep. ................................................................................................................ 37

Figure 14 E. coli concentrations every hour for the experiment with bleached sediment and E.

coli-free inflow on a Log10 scale. .................................................................................... 37

 x

Figure 15 E. coli concentrations every hour during the experiment with E. coli-spiked input

water but no mycelium. Triplicate samples were collected of the initial inflow

concentrations, which is shown at time 0 for each experiment. ....................................... 38

Figure 16 E. coli concentrations every hour during the experiment with E. coli-spiked input

water but no mycelium on a Log10 scale. Triplicate samples were collected of the initial

inflow concentrations, which is shown at time 0 for each experiment. ............................ 39

Figure 17 With Turkey Tail (Trametes versicolor) mycelium. Triplicates were collected of the

input concentrations, shown at time 0, with the standard deviation shown in the error

bars. ................................................................................................................................... 40

Figure 18 With Turkey Tail (Trametes versicolor) mycelium on a Log10 scale. Triplicates were

collected of the input concentrations, shown at time 0. .................................................... 40

 1

1 INTRODUCTION

Urbanization poses threats to the integrity of water quality throughout the United States

primarily due to increased impervious surfaces and associated stormwater runoff, decrease in

groundwater recharge, stream bank destabilization, and increased contaminant transmission

(Alder, 2013; Burns et al., 2005; Gaffield et al., 2003). Various research has shown that

impervious surfaces not only increase flood peaks and runoff volume, but also transmit bacterial

and chemical pollutants into surface waters at rapid rates (Fig. 1; DeWalle and Swistock, 2000;

Gaffield et al., 2003).

Coupling climate change issues with continued population growth exacerbates already

mounting urban water quality issues through the United States, and in particular, in the rapidly

expanding metro Atlanta area (DeWalle and Swistock, 2000; EPD, 2008; Gaffield et al., 2003).

By 2050, population growth in the city of Atlanta is anticipated to almost double (Jeong et al.

2018). Dekalb County, in southeastern metro Atlanta is expecting a population increase of 22%

by the year 2040 (ARC, 2015). This escalated population growth will add strain to an already

aging water infrastructure system and be exacerbated by increased frequency and intensity of

precipitation events likely contributing to increased stormwater runoff, sanitary sewer overflows

(SSOs) and decreased water quality in the coming years (EPD, 2008). Conservative climate

change predictions expect an overall increase of precipitation in certain areas across the United

States (Jeong et al., 2018). Evidence is already being seen, with Dekalb County experiencing its

the third wettest year in 30 years in 2020, with over 60 inches of precipitation (Champion, 2020).

More than 6,000 streams across Georgia are considered impaired and do not meet current

water quality standards (EPD, 2008). Numerous contaminants ranging from heavy metals,

increased sediment loads, and pesticides and fertilizers threaten water quality in the Atlanta area,
 2

but bacterial contamination in surface water is the largest water quality issue to manage in

Georgia (EPA, 2004; GAAS, 2014). According to the Georgia Environmental Protection

Division, over half of stream impairments in Georgia’s surface waters are caused by bacterial

contamination caused by Escherichia coli and Total Coliform (EPD, 2008). These bacteria are a

naturally occurring part of human and animal digestive systems and found in both human and

animal waste (EPA, 2004). They enter urban waterways through leaking sewer infrastructure,

SSOs, combined sewer overflows (CSOs), and stormwater runoff (EPA, 2004; GAAS, 2014).

Although the consequences of bacterial contamination on water quality are well researched, they

remain a significant source of water quality degradation throughout the United States and the

metro Atlanta area and continue to pose numerous human health risks (EPA, 2016; Walsh et al.,

2005). Understanding ways to remove bacterial contamination from urban streams is required to

improve stream health and access to recreation for citizens. This thesis presents the results of an

investigation into the effectiveness of mycofiltration for E. coli removal in a first-order Piedmont

stream as well as the past and current E. coli contamination in the Upper South River.

Figure 1 Impervious surfaces and stormwater runoff (EPA, 2003).

 3

1.1 South River Watershed

The South River is located on Muscogee Creek Indigenous Peoples land (NLD, 2020)

and is part of the Upper Ocmulgee River Basin. Today, the South River provides drinking water

supplies to Clayton, Henry and Rockdale Counties (SRWA, 2020; USGS, 2020). It originates in

highly urbanized South Atlanta (Figure 2) and flows southeast through DeKalb, Fulton, Jasper,

Newton, Rockdale, and Clayton counties until it converges with the Alcovy River and the

Yellow River draining into Lake Jackson (ARK, 2020; SRWA, 2020; USGS, 2020). The outlet

of Lake Jackson marks the beginning of the Ocmulgee River which continues to flow southeast

until it merges into the Oconee River (ARK, 2020; SRWA, 2020; USGS, 2020). Finally, the

Oconee River joins the Altamaha River which continues eastward ultimately draining into the

Atlantic Ocean (ARK, 2020; SRWA, 2020; USGS, 2020). Overall, the South River watershed

contains about 60 tributaries and is comprised of land use ranging from agricultural, residential,

commercial, and industrial (Scott, 2014; USGS, 2020). The Upper South River has an extensive

history and continued issues with bacterial contamination as a result of failed regulatory

enforcement and failing infrastructure (Mitchell, 2019; SRWA, 2020; USACE, 2012). This

section aims to (1) provide an overview of Combined Sewer Overflows (CSOs) and Sanitary

Sewer Overflows (SSOs) in Dekalb County, and (2) frame the following mycofiltration lab

research as a scalable prototype for future field deployment.

 4

Figure 2. Headwaters of the Upper South River are a milky white color due to leachate
from a historical cotton processing plant and is designated a class 1 hazardous waste site.

1.1.1 Overview of SSOs and CSOs in the Upper South River

In urban areas, storm sewers and sanitary sewers are the primary categories of sewer

systems (EPA, 2004; EPA, 2016). Storm sewer systems carry stormwater runoff directly into

surface waters, while sanitary sewer systems are responsible for transporting untreated sewage

(domestic, commercial and industrial) to wastewater treatment facilities for treatment (EPA,

2004; EPA, 2016; USGS, 2020). Sanitary sewers can be further categorized as separate or

combined. The City of Atlanta has historically operated with numerous combined sewer systems

(CSS) which- unlike separate sewer systems (SSS) are constructed to carry stormwater runoff

and sewage in one combined piping system, and are designed to overflow during the highest

precipitation events as a preventative measure against pipe breakage (Borden, 2015; SRWA,
 5

2020). During high precipitation events (and subsequent high stormwater collection) CSS can

result in CSOs and subsequent high concentration of E. coli may enter the surface water (EPA,

2004). Due to the 1,925 CSOs that occurred between 1988-1991 in the South River and

Chattahoochee River, the City of Atlanta was required to eliminate CSOs through the Georgia

Water Control Act (Borden, 2015). However, the Custer Ave CSO facility and the Intrenchment

Creek Water Quality Control Facility remain on the South River and continue to threaten water

quality (Scott, 2014; SRWA, 2020).

In contrast, Sanitary Sewer Overflows (SSOs) generally result from fats, oil, and grease

clogs, aging Wastewater Collection Transmission System (WCTS) and resulting high stormwater

intrusion (the infiltration of precipitation through damaged sewer infrastructure), root intrusion,

lack of maintenance, and population growth (USDC, 2010; CRK, 2014; DWM, 2015; EPA,

2016). Unlike the City of Atlanta, Dekalb County only has SSS and therefore only experiences

SSOs. Between the years 2006 and 2010, Dekalb County experienced at least 2,846 SSOs

(USDC, 2010) and 836 raw sewage spills directly into Snapfinger Creek, and other tributaries to

the Upper Ocmulgee (USDC, 2010).

Both CSOs and SSOs result in the discharge of untreated sewage, wastewater, and

stormwater directly into surface water (Borden, 2015; CRK, 2014; EPA, 2004; EPA, 2016).

Because of this, CSOs and SSOs pose risks to public health and water quality, and both are

considered point source discharges regulated by the EPA and EPD through the Clean Water Act

(CWA) and associated National Pollutant Discharge Elimination Permits (NPDES) permits

(EPA, 2004; EPA, 2016). SSOs and CSOs cause numerous and varied environmental impacts

such as decreases in dissolved oxygen and resulting decrease in aquatic abundance and diversity,
 6

excessive nutrient loads and resulting eutrophic conditions, and entanglement or digestion of

floatables by wildlife (EPA, 2004).

Water quality in the United States is primarily regulated by the CWA (Borden, 2015;

EPA, 2016; Pennington and Cech, 2010). The CWA was signed into law in 1972 to provide a

regulation of pollutant discharges in surface and groundwater throughout the United States

(Borden, 2015; Pennington and Cech, 2010). Essentially, the CWA states that a point source

pollutant can only be discharged into a waterway with the use of permit, and this is enforced by

the EPA and EPD through the use of NPDES permits (Borden, 2015; Doyle, 2012). Although

NPDES permits specify the quantity and type of pollutants allowed for discharge in surface and

groundwater, (Doyle, 2012; Martinez, 2016; Pennington and Cech, 2010), Dekalb County has

continuously failed to meet these requirements and thus violated the CWA (USDC, 2010).

As a result of years of noncompliance with the federal CWA and the GA Water Quality

Control Act, primarily due to excessive sewage spills, the EPA filed a lawsuit against Dekalb

County in 2010 (USDC, 2010). This lawsuit resulted in the agreement of a settlement by way of

a consent decree between Dekalb County, the EPA, and EPD (EPA, 2015; USDC, 2010).

Specific violations charged to Dekalb County include the “illegal discharges of untreated

sewage” and failure “to operate and maintain the collection and transmission systems of its

treatment works” (EPA, 2017). The resulting 2011 consent decree demanded that Dekalb County

repair and update its WCTS in the South River, Snapfinger Creek and Peachtree Creek in order

to reduce its annual SSOs and CSOs within nine years as well as pay almost $900,000 in civil

penalties (USDC, 2010).

According to the consent decree, 64% of the Dekalb County’s WCTS infrastructure is

between 25-50 years old and in need of replacement (USCD, 2010). Dekalb County Department
 7

of Watershed Management (DWM) has developed a rehabilitation plan to limit SSOs (DWM,

2015). Intended rehabilitation methods include techniques such as “cured-in-place line pipe, pipe

bursting, manhole lining, manhole replacement, manhole height adjustment, manhole ring and

cover replacement, manhole frame sealing, open cut pipe replacement, point repairs, manhole

raising, manhole ring and cover replacement, and service lateral rehabilitation” (DWM, 2015).

These rehabilitation methods are intended to reduce pollutants as required by the consent decree

yearly as follows: “Total Suspended Solids (TSS) reduced by 9,743 pounds, Biological Oxygen

Demand (BOD) reduced by 9,424 pounds, Chemical Oxygen Demand (COD) reduced by 22,133

pounds, Total Nitrogen reduced by 1,437 pounds, Total Phosphorous reduced by 272 pounds”

(EPA, 2015). These improvements to Dekalb County’s WCTS are estimated to cost over $1

billion (News Release, 2020). Although the consent decree requires repairment of one-third of

Dekalb County’s sewer infrastructure, it does stipulate repair for the remaining sections despite

continued SSOs throughout the entire system (Mindock, 2020).

The consent decree expired on June 20th, 2020; however, Dekalb County had failed to

meet the requirements of the consent decree by this date (SRWA, 2020). In fact, in 2017, 6.4

million gallons of untreated sewage spilled into Snapfinger Creek, the largest SSO in 11 years,

and within the first 9 months of 2020, Dekalb County SSOs had already resulted in over 2

million gallons of untreated sewage pouring into the Upper South River (DWM, 2020; Niesse,

2017). Due to an inability to meet the initial deadline, in October 2020, the EPA, EPD, and US

Department of Justice extended the consent decree (News Release, 2020). The new, extended

consent decree prioritizes about 100 locations in the WCTS, requires Dekalb County to increase

its reporting to the EPD and EPA, and to pay over $1 million in civil penalties to the United

States and the State of Georgia (News Release, 2020). In response to this inability to make
 8

substantial change in decreasing SSOs since 2011, the SRWA filed a citizen lawsuit stating that

Dekalb County remains in violation of the CWA and NPDES permits (Mindock, 2020).

However, the lawsuit was dismissed in court by U.S. District Judge Steven D. Grimberg because

according to the court, the EPA and EPD extension of the consent decree settles the case

(Mindock, 2020).

1.1.2 Current E. coli contamination in the Upper South River

Although the Georgia Environmental Protection Division (EPD) has monitoring locations

on the Upper South River and its tributaries (Figure 3), consistent Total Coliform monitoring is

lacking. The most recent report (2014) on the Upper South River highlights segments that do not

meet Total Coliform bacteria total maximum daily load (TMDL). TMDL is defined as the

amount of pollutant a body of water can legally contain according to the CWA and is calculated

using the formula below:

TMDL = ∑WLAs + ∑LAs +MOS

where WLAs are the total amount of point source waste allocated, LAs are the total

amount of nonpoint source waste load allocated, and MOS refers to the margin of safety (Scott,

2014). Table 1 lists stream segments in the Upper South River that exceed the TMDL and thus

are considered impaired.

 9

Figure 3 GA EPD's Watershed Protections Branch Monitoring Stations on the Upper

South River (EPD, 2021)

Table 1 Impaired Stream Segments in HUC 0301070301 (Scott, 2014).

 10

Specific causes of stream impairment in these areas are attributed to stormwater runoff,

animal waste, SSOs and CSOs, Wastewater Treatment Facilities, and septic tank failures (Scott,

2014). Unfortunately, the steps towards improving water quality in the South River by the Metro

North Georgia Water Planning District (MNGWPD) such as the creation of a Stormwater

Management Plan (SWMP), public education programs, streambank stabilization and restoration,

and sewer infrastructure repair, have not been sufficient and current SSOs and CSOs remain a

significant and escalating problem for the South River (MNGWPD, 2009; Scott, 2014; SRWA,

2020). To supplement these findings and provide a more current picture of the issue, the South

River Watershed Alliance (SRWA) began a water quality monitoring program in 2019 with the

objective of obtaining consistent water quality data such as Total Coliform and E. coli.

According to the EPA, E. coli concentrations are used as standards for determining water

quality in regard to bacterial contamination and to protect the public from contact with harmful

bacteria during recreation and/or swimming (EPA, 2016; GAAS, 2014). E. coli standards for

Recreational Waters indicate that a designated swimming area must contain <235 (cfu/100mL),

<298 (cfu/100mL) in a moderate swimming area, <410 (cfu/100mL) in a light swimming area,

and <576 (cfu/100mL) in an infrequent swimming area (GAAS, 2014). Because the South River

is not designated for recreational use, the CWA does not necessitate the above E. coli

concentration standards to be met. The South River’s designated use classification is currently

“Fishing” which is the lowest classification for surface water and affords the lowest protection in

terms of pollutant levels (SRWA, 2020). The SRWA has been working to shift the South River’s

designation to “Recreation” in order to force the EPD to implement improved water quality

standards (SRWA, 2020). And although, the state of Georgia has recognized that the need to

redraft the categories of surface water designated uses stating that “the designations for fishing
 11

are not stringent enough for certain sensitive ecosystems” (EPD, 2004) no progress has been

made to execute these changes. Because the South River is unofficially utilized by residents for

recreation and fishing, higher water quality is imperative to minimize public health threats.

1.1.3 Human health impacts

Although policies such as the CWA coupled with modern water treatment technologies

have enabled the majority of the U.S. population access to pathogen free water for consumption

and food production, Total Coliform and E. coli concentrations in surface water regularly exceed

safe standards (Gaffield et al., 2003). CSOs and SSOs in particular threaten public health by

exposure to untreated sewage and subsequent intestinal parasites, viruses and bacteria (EPA,

2004). Additionally, CSO and SSO consequences are exacerbated during precipitation events by

an influx of other pollutants via stormwater runoff into surface water (EPA, 2004). As discussed

preciously, bacterial contamination is the leading cause of stream impairment in the United

States and impaired segments may pose significant public health concerns (EPA, 2004).

Research indicates that nonpoint source pollution, and stormwater runoff in particular,

increases the public’s exposure to bacterial contamination and propensity for acute or chronic

illness and are directly related to increased concentrations of E. coli, Total Coliform, Giardia,

and Cryptospondium pathogens in surface water (Gaffield et al., 2003). E. coli in particular poses

problematic public health risks due to antibiotic resistance of numerous strains (Pini and Geddes,

2020). Accurate data regarding the quantity of illness related to waterborne diseases remains

elusive due to the fact that the majority of cases are unreported due to the difficulty in accurate

diagnosis; however, acute cases of waterborne illness caused by the recreational use of

bacterially contaminated water include “ear and eye discharges, skin rashes, and gastrointestinal

problems” (Gaffield et al., 2003). Immunocompromised people, children, people who are
 12

pregnant, and elderly people remain the most vulnerable to serious illness as a result of such

pathogens (Gaffield et al., 2003). Additionally, the increased temperatures and increased

precipitation rates associated with climate change are likely to increase surface waterborne

illnesses which result from the recreational use of bacterially contaminated water (Gaffield et al.,

2003).

1.1.4 What is mycofiltration?

Mycofiltration is a specific type of mycoremediation and refers primarily to the use of

fungi for contaminant removal from water. Mycoremediation is a technique used to remove a

range of pollutants from soil and/or water (Cotter, 2014; Kulshreshtha et al., 2014). The prefix

‘myco’ refers to fungi and ‘-remediation’, in general, refers to the process of cleaning or

correcting (Cotter, 2014). Research has shown mycelium are capable of removing a variety of

chemical, heavy metal, and bacterial contaminants from terrestrial and aquatic environments

(Taylor and Stamets, 2014). Although it is a relatively new area of research, mycofiltration has

been used for remediation of polluted water due to industrial agricultural practices, animal

husbandry, and certain industrial manufacturing sites, as well as from stormwater runoff and

failing sewer systems (Cotter, 2014; Pini and Geddes, 2020; Singh, 2006; Stamets et al., 2013;

Taylor et al., 2015). Mycofiltration installations vary in scale, medium, and contaminant removal

and although mycofiltration is typically used post contamination, it can be used preventatively in

areas where pollutant contamination is inevitable (Cotter, 2014).

Best management practices (BMP) and green infrastructure may reduce the quantity of

stormwater pollutants such as E. coli after precipitation events by increasing stormwater

infiltration and groundwater recharge; however, they may not completely remove the

contamination (Gaffield et al., 2003; Martinez, 2016). BMPs range in scale from high impact
 13

infrastructures such as sand filtration, wetlands, and retention ponds, to low impact practices

such as household rainwater collection units, swales, and green roofs (DWM, 2018).

Mycoremediation may be a useful addition to the above BMPs due to its relative low cost and

minimal impact. Additionally, research has estimated that mycoremediation may be a more

affordable alternative to current wastewater treatment operations that utilize bacteria and/or

sterilization because mycoremediation (excluding biosorption) does not create byproducts (Molla

and Fakhru’l-Razi, 2012). Coupling various green infrastructure techniques, stormwater

management programs, and mycofiltration installations may be key towards successful

management of non-point source pollution.

1.2 Study objectives

Mycofiltration may be a useful addition to innovative community led responses in lieu of

failed government regulatory enforcement and failure to improve Dekalb County’s sewer

infrastructure, but there has been limited research that considers the impact of hydrology on its

effect. As has been well observed in remediation of nutrient contamination by streams (Craig et

al., 2008; Hall et al., 2009; Bernhardt et al., 2017), the time-scale of interaction between the

remediation media and contaminants may be key in understanding the efficacy of the approach.

However, when this is accounted for, mycofiltration may be an appropriate approach for small

order urban streams. Small scale stream restoration techniques founded in environmental equity

such as mycofiltration may provide an affordable alternative while mitigating environmental

injustice-the unequal distribution of environmental amenities or hazards (Gould and Lewis,

2016). Because environmental restoration can lead to environmental injustice, in particular green

gentrification which results in increased property values and subsequent displacement or

exclusion of economically vulnerable and marginalized residents (Gould and Lewis, 2016), it is
 14

imperative that mycoremediation research balances attainable restoration goals with community

needs while centering community involvement (Doyle and Shields, 2012; Walsh et al., 2005). In

particular, mycoremediation may contribute to increased visibility of the water quality issues

facing the Upper South River and bolster the SRWA’s argument for improved water quality

designation.

This research will specifically answer the questions (1) How effective is Escherichia coli

(E. coli) removal from surface water when interacting with Trametes vericolor fungal spawn for

short periods of time in a laboratory representation of a first-order stream? and (2) Will

statistically significant decreases in E. coli concentrations occur when accounting for hydrologic

interactions? Initially, I hypothesized that the presence of Trametes vericolor would reduce E.

coli contamination linearly, even when the natural flowpaths and retention times of streams were

considered.

This research consisted of a lab-based experiment and field based research which

combined aim to provide an understanding of the current distribution of E. coli contamination in

the Upper South River and evaluate the feasibility of a prototype for future mycofiltration

installations within the Upper South River Watershed. The lab-based experiment utilized a

stream table filled with sediment from Ripplewater Creek (a tributary of the Upper South River)

to simulate a flowing first-order stream and deployed a mycofilter containing Trametes

versicolor mycelium to mitigate E. coli concentrations in the surface water. Naturally occurring

E. coli from Ripplewater Creek was isolated, cultured, and routed through a flow rate variable

pump with deionized water at a constant rate through the stream table for 5 hours. A mycofilter

of Trametes vericolor fungal spawn and Quercus alba sawdust trimmings were deployed at one

section across the stream table, and samples were taken and analyzed with an IDEXX Quanti-
 15

Tray System hourly. These results were compared to experiments where the E. coli-laced water

was run without the mycofilter. Additionally, I analyzed stream samples along the Upper South

River with the IDEXX Quanti-Tray System in order to determine spatial and temporal

distribution of E. coli concentrations. Prototypes are especially imperative for mycofiltration

installations because they help determine the best suited fungal species and substrate for the

contaminant and location, the quantity of mycofilters necessary for substantial contaminant

removal rates in relation to rate of discharge (Cotter, 2014). This study aims to provide data

applicable for future mycofiltration installations within in the Upper South River.

2 BACKGROUND

2.1 Evidence of mycofiltration efficacy

Bioremediation is an umbrella term which describes the use of microorganisms to reduce

contaminants levels (Kapahi and Sachdeva, 2017; Rhodes, 2014); mycoremediation and

mycofiltration are methods of bioremediation that involve fungi. Bioremediation can be

accomplished through a variety of mechanisms such as biosorption, where heavy metals get

adsorbed on the surface of the biosorbent, bioaccumulation, wherein microorganisms bind to

heavy metals and concentrate them (Velásquez and Dussan, 2009), and biodegradation, the

transformation/breaking down of inorganic compounds into usable organic compounds

(Kulshreshtha et al., 2014). It is important to note, that although biosorption (also known as

hyperacuumulation) has been shown to be an effective bioremediation tool, it does not degrade

the contaminant but merely transfers the contaminate to the fungi which then needs to be

properly disposed (Cotter, 2014; Kulshreshtha et. al, 2014). Research demonstrates that

mycelium are capable of biosorption, bioaccumulation, and biodegradation. However,

biodegradation is the proposed mechanism for E. coli reduction through mycofiltration and this
 16

is particularly helpful because biodegradation specifically transforms the contaminant into a

benign substance and does not produce contaminated waste products (Kulshreshtha et al., 2014).

Additionally, although this research focuses on the use of mycelium to remove contamination,

past research has shown that live or dead mushrooms (the fruiting body of fungi) and even the

spent mushroom substrate can be used as for bioremediation (Kapahi and Sachdeva, 2017;

Kulshreshtha et al., 2014).

In a mycoremediation installation, the mycelium removes contamination in a variety of

ways (Bhadouria et al., 2019; Cotter, 2014; Stamets, 2005). Mycelium have shown the ability to

interrupt cellular replication and degrade cell membranes (Cotter, 2014). Interestingly, they can

also change the pH of the surrounding environment thus making the area inhospitable to

microorganisms (Bhadouria et al., 2019; Cotter, 2014; Singh, 2006). Various chemical reactions

such as oxidation, reduction, biological degradation, and co-metabolic reactions are also

pathways towards degradation of contaminants (Bhadouria et al., 2019). Lastly, they may also

limit microbial growth by reducing nutrient availability in surrounding areas through species

exclusion, the taking up of physical space thus preventing growth of other organisms (Cotter,

2014; Singh, 2006). Mycelium are able to do all of the above through enzyme secretion, and they

can be highly effective because (unlike bacteria) they are not bound by cells and therefore they

do not require direct contact “with a compound in order to begin to degrade it” (Cotter, 2014).

This may be particularly useful in mycofiltration projects because the mycelium need only to be

in close (not direct) contact with the pathogen in order to digest it (Cotter, 2014).

2.1.1 Mycelium behavior

Mycelium are the rooting body of fungi and are decomposers by their very nature (Cotter,

2014; Singh, 2006; Kulshreshtha, et al., 2008; Stamets, 2005). In natural systems, they secrete
 17

enzymes which metabolize materials for consumption (Singh, 2006; Stamets, 2005).

Specifically, they manufacture peroxidase, cellulose, ligninase, and laccase enzymes which break

down the lignins and cellulose in woody material (Cotter, 2014; Singh, 2006; Rhodes, 2014;

Stamets, 2005). This decomposition results in humus which is a material full of more accessible

nutrients for other organisms to consume and provides an imperative role in the nutrient cycling

(Rhodes, 2013).

Mycelium can be broken down into two main groups: white rot and brown rot (Cotter,

2014; Stamets, 2005). The majority of literature regarding mycoremediation uses white rot fungi

and this is mainly due to their predication towards consuming lignin whereas brown rot fungi

generally break down cellulose (Cotter, 2014; Rhodes, 2014). White rot fungi use a variety of

enzymes and thus are able to break down and/or transform a variety of organic molecules. This

non-specificity garners them an advantage in the field of mycoremediation (Mir-Tutusaus et al.,

2014). Notable white rot fungal species include Pleurotus spp, Trametes versicolor, Ganoderma

lucidum (Cotter, 2014; Mir-Tutusaus et al., 2014). Certain fungi species have affinities with

particular contaminants and species vary in their ability to bioremediate. Additionally,

bioremediation effectiveness is highly dependent on numerous factors such as contact time,

temperature, substrate, biomass, and pH (Cotter, 2014; Kapahi and Sachdeva, 2017;

Kulshreshtha et al., 2014).

2.2 Heavy metal and chemical mycoremediation

Various mushroom species and genera have been shown to sequester (biosorb) high

concentration of numerous heavy metals, in their mycelium as well as their fruiting bodies while

others have shown the ability to biodegrade heavy metals, hydrocarbons, and other chemical

pollutants (Kapahi and Sachdeva, 2017; Kulshreshtha et al., 2014). For example, the
 18

extracellular enzymes that Pleurotus spp. (oyster mushroom) secrete have shown the ability to

degrade polymers (da Luz et al, 2013), crude oil (Olusola and Anslem, 2010) and sorb heavy

metals such as cadmium, copper, zinc, iron, lead, and nickel (Lamrood and Ralegankar, 2013;

Oyetayo et al., 2012; Tay et al., 2011). More specifically, certain species within the genus show

an affinity for certain heavy metal biosorption; for example, in previous research higher

cadmium uptake was exhibited in P. ostreatus versus higher mercury uptake by P. sajor-caju

(Kapahi and Sachdeva, 2017). A variety of other fungi species such as Lentinula edodes, Corolus

versicolor, Agaricus bisporus, Lactarius piperatus, Trametes versicolor and Fomes fasciatus

have shown degradation and biosorption of a variety of complex pollutants ranging from

Polycyclic aromatic hydrocarbons (PAHs) to a variety of heavy metals (Manna and Amutha,

2017; Kulshreshtha et al., 2014).

Recent research has indicated that white rot fungi may be able to remediate

agrochemicals, organochlorine pesticides, organophosphonate agrochemicals such as

dichlorodiphenyltrichlor oethane (DDT) and pentachlorophenol (PCP) in soil and water

(Bhadouria et al., 2019; Hu et al., 2020). Trametes versicolor in particular was shown to degrade

agrochemicals ranging from carbofuran, oxytetracyclin, imiprothrin, and cypermethrin (Mir-

Tutusaus et al., 2014). Additional research has shown that Trametes versicolor and other white

rot fungi have the ability to remediate certain micro pollutants which are not removed by current

WWTP operations (Álamo et al., 2018; Beltrán-Flores et al., 2020; Mir-Tutusaus et al, 2018).

Micro pollutants including pharmaceutically active compounds (such as acetaminophen,

ibuprofen, naproxen, salicylic acid), various antibiotics, psychiatric drugs, and endocrine

disruptors have been effectively removed or transformed from wastewater using T. versicolor

(Mir-Tutusaus et al., 2018). However, numerous limitations remain which prevent the use of
 19

mycofiltration for wastewater treatment such as the need for nutrient addition to the system,

microorganism competition, and changing pH to optimize growth of fungi (Mir-Tutusaus et al.,

2018).

2.3 Bacterial mycoremediation

Research on mycoremediation for bacterial contamination ranges from use in wastewater

treatment plants, agricultural runoff, and stormwater retention ponds and runoff. Regardless of

the scale of the project, it is imperative to utilize the best fungal species for the contamination

(Cotter, 2014). Numerous species of fungi have shown antimicrobial properties; specifically,

Trametes versicolor (Cotter, 2014), Calvatia spp, Ganoderma spp., and Pleurotus spp (Cotter,

2014; Singh, 2006). Because this field of study is burgeoning, research remains limited;

however, field and lab studies which are foundational to the study of mycofiltration and show

promise for future research will be explored below.

Stamets (2005) details field and lab mycoremediation research in his book Mycelium

Running. He specifically used mycofiltration as a best management practice (BMP) for

controlling bacteria contamination in surface water from agricultural runoff (Stamets, 2005). In

his field experiment, he inoculated Storpharia rugoso annulata mycelium into woodchip swales

downstream from his livestock pen and documented a 100% decrease in Total Coliform

concentrations in the effluent after one year despite an increase in livestock population (Stamets,

2005). His subsequent laboratory research (Stamets et al., 2013) with Washington State

University identified fungal species for mycofiltration, cultivation methods such as the

MycoFilterTM, and demonstrated the potential effectiveness of E. coli removal through

mycofiltration. This research identified Storpharia spp. as the preferred mycelium for

mycofiltration due to its rate of initial colonization and resilience under various environmental
 20

stressors such as temperature and dehydration, and propensity at E. coli concentration reduction

(Stamets et al., 2013). Stamets et al. performed a series of single bucket tests wherein an E. coli

concentration of about 800 cfu/100mL per 30 L bucket, was percolated through a Storpharia spp.

mycelium mycofilter at a flow rate of 0.5L/min and 2.2 L/min (Stamets et al., 2013). The

mycofilters were filled with a 1:1 ratio of mycelium to large and small wood chips. Their results

indicated an E. coli reduction by about 20% at 0.5L/min flow rate and about a 14% reduction of

E. coli at the 2.2 L/min rate (Stamets et al., 2013) indicating that longer contact time between E.

coli and mycelium is necessary for efficacy. Overall, this research concluded that mycofiltration

(unlike other stormwater BMP) are capable of removing free-floating (not sediment-bound)

bacteria (Stamets et al., 2013). It is important to note, that while this research did percolate water

through the filter, it did not account for natural hydrologic conditions including hyporheic flow

through the streambed.

Similarly, Martinez (2016) utilized synthetic stormwater in a pond setting to investigate

the efficacy of Pleurotus ostreaus mycelium for E. coli reduction. Their research showed a 98%

reduction in E. coli concentrations after a three-week period, and importantly, indicated that

reduction increased after each subsequent week (Martinez, 2016). Thomas et al. (2015) also used

Storpharia rugoso annulata coupled with Pleurotus ostreatus, and Pleurotus ulmarius mycelium

inoculated alder mulch in bioretention cells in a human made wetland and compared those

bioretention cells with ones that only had alder mulch without mycelium inoculation. After a six-

month period, the control cells showed an E. coli reduction of 66%, and the mycelia-inoculated

cells produced an E. coli reduction of 90% (Thomas et al., 2015). The most recent research on

mycofiltration for bacterial removal focused on contact time related to E. coli removal and found

that mycelium are capable of E. coli removal up to 96 h after inoculation in a sterile lab
 21

environment (Pini and Geddes, 2020). However, they note, that the efficacy of short term contact

and longer than 96 h contact is unknown (Pini and Geddes, 2020). Again, this research did not

account for hydrology; the lab inoculated and natural stream water were contained in receptacles

and did not flow as would occur in a natural stream environment (Pini and Geddes, 2020).

Despite multiple studies that purport the effectiveness of Storpharia spp in

mycofiltration, Cotter (2014) found that while Storpharia spp may initially reduce bacterial

concentrations, long-term it may ultimately increase the bacterial concertation (Cotter, 2014).

Additionally, although numerous studies have focused on Pleurotus spp usefulness in

mycoremediation in soil, Gulis and Suberkropp (2003)’s research has indicated that the enzymes

secreted by Pleurotus spp may be diluted in aquatic environments and could have limited

efficacy in mycofiltration applications. Trametes vericolor mycelia have been shown to have

antimicrobial activity in isolated laboratory studies against various bacteria including E. coli,

Staphylococcus aureaus, Pseudomonas aeruginosa, and Methicillin-Resistant Staphylococcus

aureus (MRSA) (Gebreyohannes et al., 2019; Hleba et al., 2014).

3 METHODS

3.1 South River Watershed Alliance sampling procedures

To understand the potential applicability of this restoration technique, samples were

collected to quantify the extent of E. coli contamination in the Upper South River. Sampling

locations on the Upper South River headwater, tributaries, and main stem stream segments were

identified, and all samples were obtained utilizing the Adopt a Stream Quality Assurance/Quality

Control (QA/QC) chemical and biological protocols. Each stream sample was taken at base flow,

at roughly the same time of day, and at the exact same location. Samples were analyzed at the

Chattahoochee Riverkeeper’s Neighborhood Water Watch water quality lab (CRK, 2014). Nine
 22

total sample sites were located on the main stem of the Upper South River and its tributaries

(Figure 11).

Figure 4 Sample sites along the Upper South River

Each site was chosen due to accessibility and relation to CSO and WWTP facilities. The

Tift site is located at the southern headwaters of the Upper South River. McDaniel is a tributary

to the South River and is located below the McDaniel Branch constructed wetlands near Arthur

Langford Park in SW Atlanta. SR-1 is located on the main stem of the Upper South River

adjacent to the Brownsmill Golf Course. CSO-1 is a tributary to the Upper South River and is

located upstream from Custer Combined Sewer Overflow Facility. CSO-2 is a tributary of the

Upper South River and is located downstream from Custer Combined Sewer Outflow Facility.

WWTF-1 is located on the main stem of the Upper South River upstream from the Snapfinger

Wastewater Treatment Facility. WWTF-2 is located on the main stem of the Upper South River

downstream from Snapfinger Treatment Facility. SR-2 is on the main stem of the Upper South
 23

River as well at the Panola Shoals Trailhead. Lastly, Ripplewater is a tributary to the Upper

South River and is located south of Brownwood Park.

3.2 Experiment design

Overall, three experiments were performed in order to determine the efficacy of T.

versicolor mycelium in E. coli removal from a first-order stream. A 2.5’ X 6’ stream table was

utilized to simulate a flowing stream. Sediment from Ripplewater Creek (tributary to the Upper

South River) was collected, placed in the bottom of the stream table at a 2-inch depth, and

bleached. A Geotech GeopumpTM Peristaltic DC pump was used to pump water at a constant rate

(5.2 +/- 0.28 mL/s) through the stream table. The first experiment was performed with only

deionized (DI) water to determine the baseline E. coli concentrations after sediment bleaching.

The second experiment ran DI water containing 615.85 +/- 154.72 MPN/100 mL E. coli. The

final experiment was performed with DI water containing 642.08 +/- 211.31 MPN/100mL E. coli

and one cross vein deployment of a mycofilter containing Trametes versicolor (T. versicolor)

mycelium. Each experimental setup was run three times and the system was sterilized with 10%

chlorinated DI water between runs to remove residual E. coli and subsequently flushed with 24 L

DI water to remove residual chlorine.

3.2.1 Stream table design and sediment installation

A 2.5’ X 6’ stream table was sterilized and placed on a worktable in Dr. Sarah H.

Ledford’s urban hydrology laboratory. A hydroponic filter was installed at the outflow end of the

stream table covered with landscaping fabric to mitigate sediment build up and prevent clogging

of the outflow. Ripplewater Creek sediment was collected from the streambed at 33°73'62.6" N,

-84°34'66.1" W and placed inside the steam table at a depth of 2 inches (Figure 5). Sediment was

typical of an urban piedmont streambed and consisted of a mixture of fine clay, sand, and silt
 24

sediment, as well as gravel and pebbles. Pieces of broken glass, trash, and small decomposing

invertebrates were also present in the sediment. A 5-gallon bucket was placed at the head of the

stream table; a steady flow of DI water was pumped into the stream table with a Geotech

GeopumpTM Peristaltic DC pump and MasterflexTM ¼ inch ID tubing. A second 5-gallon bucket

was placed at the tail of the stream table to collect the outflow water (Figure 6). With time, a

channel formed in the sediment between the inflow and outflow points.

Sediment sterilization was achieved before each test by adding 10% bleach to 22L of DI

water and running through the system. This procedure was repeated until an initial sediment

sterilization was obtained and outflow water contained 0-3 MPN/100mL E. coli. After each

experiment, the sediment was re-sterilized until E. coli concentrations returned to low

concentrations (below 10 MPN/100mL).

Figure 5 Stream table filled with Ripplewater Creek sediment.

 25

Figure 6 Stream table effluent

3.2.2 In-situ sample collection

Samples for E. coli isolation from Ripplewater Creek were collected in-situ and stored

according to Georgia Adopt-A-Stream QA/QC Bacterial and Chemical Monitoring Certification

procedures. Procedures included 1) labeling the Nasco Whirl-Pak sample bag with the current

time, date, and experiment, 2) wearing unpowered latex disposable gloves and taking samples

without touching the inside of the sample bag, 3) obtaining a minimum sample volume of

100mL, 4) placing the sample immediately in a refrigerator, 5) collecting the samples at mid-

stream, and 6) analyzing the sample within 6 hours of collection while maintaining a stable

sample temperature between 1-4°C (GAAS, 2014).

3.2.3 E. coli isolation and growth

E. coli from the sample water was isolated and cultured at GSU’s School of Public Health

in Dr. Lisa Casanova’s laboratory (Table 2, 3, 4, 5; Figure 7, 8). Cultured E. coli was stored at -
 26

20 degrees C. After use, the E. coli contaminated water was neutralized with a 10% bleach

solution, and then disposed.

Table 2 Bacterial culture tracking sheet.

 27

Table 3 Determining E.coli titer.

Table 4 Determining E. coli titer continued.

Table 5 Calculations for determining E. coli titer.

 28

Figure 7 E. coli bacterial titer.

Figure 8 Isolated wild E. coli type.

3.2.4 Mycelium cultivation and deployment

As discussed earlier, other species of mycelium such as Pleurotus spp. and Stropharia

rugoso-annulata have shown to be bacteraphagues (Cotter, 2014; Singh, 2006); however, T.

versicolor mycelium was chosen for the experiments due to its ability to survive amid high

discharge rates (Cotter, 2014), which may be useful for in future field deployment. T. versicolor
 29

cultivation is relatively simple because it colonizes on a variety of hardwood mediums and has

shown the ability to outcompete other fungi in its growth rate (Cotter, 2014). Untreated Quercus

alba sawdust trimmings were obtained from a local cabinet shop and stored in a cool, dry place

until needed. T. versicolor sawdust spawn (Cotter, 2014) was purchased from Mushroom

Mountain and kept refrigerated (38°F) until needed for incubation. The spawn is naturally white

in color (Figure 9) and has an acidic odor; no changes in coloration or odor were observed

throughout the experiment.

Untreated burlap mycofilters were constructed (2” W X 14” L) based on Stamets’ (2005)

mycofilter design (Figure 10). The mycofilters were initially sterilized for 10 minutes in de-

chlorinated boiling water and then filled with a 2:1 ratio containing T. vericolor sawdust spawn

and sterilized Quercus alba sawdust weighing a total of 330.0 grams. Sawdust sterilization was

obtained by first vacuum sealing the sawdust and then placing in a 1100 W Sous Vide cooker at

178°F (Figure 11). The mycelium was then incubated at 75-85°F for 2-5 weeks in a sterilized 18-

gallon plastic bin. A heating lamp was used to guarantee consistent temperature and the

inoculated burlap mycofilters were placed in a lattice formation to allow for adequate air

circulation. Suggested incubation time for optimal mycelia efficacy is 2-5 weeks (Cotter, 2014;

Pini and Geddes, 2020). Daily misting with de-chlorinated water prevented the mycelium from

drying out. All equipment was sterilized beforehand with 190 proof Everclear. Latex gloves and

a face mask were worn while handling the mycelium, sawdust, and mycofilters.
 30

Figure 9 Trametes versicolor mycelium.

Figure 10 Trametes versicolor burlap mycofilter deployed in stream table.

Figure 11 Sterilized Quercus alba sawdust.

 31

3.3 Dilution calculations and procedure

A “spike” of E. coli was put into a bucket containing 12L DI water and stirred for 5

minutes to fully incorporate the E. coli, with a goal concentration of 1000MPN/100 mL. The

water in the bucket was then pumped through the stream table with the flow rate variable pump

for 5 hours, requiring 8 input buckets. Details of the dilution process and ratios displayed in

Table 6 and a sample was taken from each bucket to measure inflow E. coli concertation. Sample

water was pumped through the table at a rate of 5.2 +/- 0.28 mL/s, with the pump rate measured

at the beginning of each experiment. Water continuously flowed into 5 gallon buckets at the end

of the stream table. A sample was collected from outflow of the stream table before it entered the

lower buckets each hour for the length of the experiment. Buckets were sterilized with a 10%

bleach solution after use.

Table 6 Procedure for E. coli dilution.

 32

3.3.1 Analyzing E. coli concentrations

Samples of the outflow were collected in Nasco Whirl-Pak and immediately placed in a

refrigerator at 4°C. Samples were prepared for the IDEXX Colilert-18 Quanti-tray system

analyzation by transferring 100mL of the sample into a 120mL sample vessels using a pipette.

Next, the IDEXX Colilert reagent powder was added to each sample and dissolved. This mixture

was then poured into Quanti-Tray/2000 and bubbles were allowed to settle. Finally, the Quanti-

Tray was sealed with the Quanti-Tray Sealer. Each sample was labeled with the date, sample

name, and dilution (no dilution was used). The sealed samples were then placed in an incubator

at 35°C for 18-22 hours. After incubation, the samples were removed and individually inspected

in natural light and using a UV-light. Results were read according to the IDEXX Coliler-18 test

Procedure (2017) and recorded on the back of the Quanti-Tray, in the lab notebook, and

ultimately onto a Microsoft Excel spreadsheet. The IDEXX Coliler-18 test is approved by the

EPA to monitor drinking water, surface water, ground water, and wastewater (CRK 2014,

IDEXX, 2017). The IDEXX Quanti-Tray using the Most Probable Number (MPN) statistical

method to determine bacterial concentrations (IDEXX, 2017). Disinfection was prioritized

throughout the experiment. Sterile latex gloves were worn at all times. Disinfecting wipes were

used to wipe down all surfaces. All materials that were used were disinfected with bleach and

wastewater from the experiment and sterilized with a 10% bleach solution before transmitting it

into the sewer system.

An ANOVA (analysis of variance) statistical analysis was used to determine the

statistical significance of the results on the null hypothesis that the means of the data sets (E. coli

concentrations at the outflow with and without the T. versicolor) are the same and the alternative

hypothesis that they are different. If p<0.05 then the null hypothesis is rejected in favor of the
 33

alternative, while if p>0.05, the null hypothesis is accepted (Helsel et al., 2020). These results

were calculated in Matlab using the anova1() function. In addition, the slope of the linear

regression of outflow E. coli concentrations through each experiment was tested for significance

using ANOVA in Excel. This was to test if E. coli concentrations decreased during each

experiment. Percent retention was calculated utilizing the following equation:

Average Inflow- Average Outflow

Average Inflow

4 RESULTS

4.1 Upper South River E. coli concentrations

Significantly high E. Coli levels were found in all sample sites, with samples collected on

nine dates. On average, 6.4 sites of the nine sampled were over the EPA limit for swimming of

235 cfu/100mL on any one sampling date. The McDaniel, CSO-2, and the Ripplewater sites

seem to have chronic contamination problems due to their consistently high E. coli

concentrations (Figure 12; Table 7). The highest concentrations were seen across all sites on

3/5/2020, where it had rained 1.52 in over 24 hours before sampling and 3.07 inches over 72

hours. This clearly indicates SSO spills and CSOs were likely prevalent during this large rain

event. Atlanta receives approximately 50 inches of rain per year, so this event represents 6% of

annual rain in three days. Interestingly, on this date, CSO-2, below the Custer Avenue CSO, was

the only site below the EPA limit likely due to primary treatment. This impact of stormflow on

E. coli contamination is supported by the samples from 10/17/19, which was after 0.52 inches of

rain over the prior 72 hours. On this date, all sites but CSO-1 were above the limit. CSO-1 is the

only site that routinely fell below the EPA limits. Even at baseflow, when 72-hour antecedent
 34

precipitation was 0 in, any sites were above the standards but there was wide variability in the

absolute concentrations of E. coli at baseflow.

Figure 12 E. coli concentrations in the Upper South River (Note: Because the Tift Site
was below the detection limit, it is not included in this figure.)
 35

Table 7 E. coli and Total Coliform data for each sample site.
36
 37

4.2 Lab experiments

The initial series of experiments (WOEC-1,2,3) determined base line concentrations of E.

coli present in the Ripplewater creek sediment after sterilization of the sediment. As shown in

Figure 13 and 14, temporal variance occurred throughout each experiment (WOEC-1, WOEC-2,

and WOEC-3), but there were no statistically significant temporal trends. During this

experiment, three samples were taken at each timestep to calculate variability.

Figure 13 E. coli concentrations every hour for the experiment with bleached sediment
and E. coli-free inflow. Error bars indicate the standard deviation of three outflow samples taken
at each timestep.

Figure 14 E. coli concentrations every hour for the experiment with bleached sediment
and E. coli-free inflow on a Log10 scale.
 38

The second set of experiments (WEC-1,2,3), determined the input concentrations of E.

coli as well as observed natural changes in the E. coli concentrations due to metabolic processes

in the sediment alone, despite sediment bleaching. Figures 15 and 16 displays the results of the

second set of experiments wherein inputs of DI water spiked with E. coli (MPNs ranging from

(307.6 to 866.4) were pumped into the stream table system after which hourly samples were

obtained from the stream table output. Temporal variance was also observed throughout each

experiment in this subset. However, despite temporal variance, E. coli concentrations nominally

decreased compared to input concentrations by the end of each experiment. Variations through

time in E. coli concentrations here may be due in part by human error and/or IDEXX error.

Possible human error could have occurred during the determination of the titer concentration, in

handling the E. coli during transport and storage, and/or during the performance of the E. coli

dilutions.

Figure 15 E. coli concentrations every hour during the experiment with E. coli-spiked
input water but no mycelium. Triplicate samples were collected of the initial inflow
concentrations, which is shown at time 0 for each experiment.
 39

Figure 16 E. coli concentrations every hour during the experiment with E. coli-spiked
input water but no mycelium on a Log10 scale. Triplicate samples were collected of the initial
inflow concentrations, which is shown at time 0 for each experiment.

In the final series of experiments (WTT-1,2,3), the Turkey Tail (T. versicolor) mycelium

was deployed in one cross section across the stream table (Figures 17, 18). Although overall the

output samples resulted in lower E. coli concentrations than the input, there was not a

statistically significant linear decline in concentration during any experiments. Calculating the

overall decrease in E. coli concentration from input to the end of the experiment shows no

difference in retention with the presence of the mycelium (Table 8, p=0.65). Although E. coli

removal was observed throughout various experiments overall, the removal of E. coli cannot be

contributed to the T. versicolor fungal spawn. However due to budget and time constraints, the

experiment was not performed more than 6 times total, thus limiting the statistical strengths of

these tests.
 40

Figure 17 With Turkey Tail (Trametes versicolor) mycelium. Triplicates were collected
of the input concentrations, shown at time 0, with the standard deviation shown in the error bars.

Figure 18 With Turkey Tail (Trametes versicolor) mycelium on a Log10 scale. Triplicates
were collected of the input concentrations, shown at time 0.

Table 8 Concentration decrease of E. coli for each experiment.

*ANOVA analysis does not indicate a difference between average retention with mycelium and average
retention without mycelium. P=0.6
 41

5 DISCUSSION AND CONCLUSION

5.1 Discussion

The first objective of this study was to explore the effectiveness of mycofiltration in

decreasing E. coli concentrations during a short span of time (5 hours). Previous research

demonstrated E. coli removal success varying from 99.25% to 99.74% from Pleurotus ostreatus

(oyster mushroom) mycelium deployment for 48 to 96 hour durations (Pini and Geddes, 2020);

however, that research occurred on a shaker table where mycelium and contaminants could

interact continuously. In contrast, for actual field applications and to understand real-world

implications, hydrologic flow must be considered. Data produced from this research and

accounting for hyporheic flow paths, suggests that 4.5% more E. coli was removed with the T.

versicolor mycofilter than without the mycofilter.

While these results are discouraging, they do not negate the potential for mycofiltration

efforts in longer term projects but merely suggest the limitations of mycofiltration for rapid

response solutions to bacterial contamination in surface water. Previous research demonstrates

the efficacy of mycoremediation in field and lab applications (Kapahi and Sachdeva, 2017;

Kulshreshtha et al., 2014; Pini and Geddes, 2020; Stamets, 2005; Singh, 2006) and specifically

the use of T. versicolor as a good fit for mycoremediation (Álamo et al., 2018; Beltrán-Flores et

al., 2020; Cotter, 2014; Gebreyohannes et al., 2019; Hleba et al., 2014; Mir-Tutusaus et al.,

2014). Furthermore, this study was limited by the use of only one species of mycelium, only one

row of mycelium installation, and limited trials due to budget and time concerns. Additionally,

due to MPN statistical calculation methodology, the IDEXX Colilert-18 presents a potentially

large margin of error when considering short term E. coli reductions.

 42

The next objective of this study was to investigate effects of hydrology, specifically

discharge rate, on mycofiltration effectiveness and determine the significance of contact time

between E. coli contaminated water and the mycofilter. Research regarding contact time is

limited but thus far, overall data suggests that mycofiltration efficacy increases with an increase

in contact time. For example, E. coli removal increased after each subsequent week to 98% after

a total of three weeks immersed in a lab based pond setting (Martinez, 2016); however,

additional research has indicated a lower rate of reduction after 96 h immersed in a shaker table

with the highest rates at 48 h (Pini and Geddes, 2020). Lastly, increased discharge rate (likely

due to decreased contact time) has been shown to reduce mycofiltration efficacy (Stamets et al.,

2013). For example, research which did not account for hyporheic flow (but instead forced water

through the mycofilter) found a 20% reduction at 8.3 mL/s flow rate and a 14% reduction of E.

coli at the 36.7 mL/s rate with the use of Storpharia spp. mycofilter (Stamets et al., 2013). This

experiment follows that trend, showing a 28.8% reduction of E. coli at 5.2mL/s, although we

cannot differentiate the removal from natural sediment metabolism compared to the removal

from mycofiltration.

In a hypothetical field environment of a small order urban Piedmont stream, water could

be passing through the mycofilters at around 23 L/s discharge (average daily baseflow discharge

at USGS gage 02336030, North Fork of Peachtree, from July 1, 2019 to April 1, 2020) and after

high precipitation events the discharge rate, particularly in urban areas, could increase to 1773

L/s (highest mean daily discharge at that same gage for the same period). These considerations

are imperative to investigate in the laboratory before deployment in the field, and remain in

contrast to previous laboratory research which does not account for discharge (Pini and Geddes,

2020). While E. coli concentrations did decrease throughout the experiment when interacting
 43

with T. versicolor mycelium at an average discharge rate of 5.2 +/- 0.28 mL/s, the results of this

study do not show statistically significant decreases that can be attributed to the mycofilter at

much lower discharges than would be seen in first-order urban streams.

Differences in the response seen in these lab experiments to other published work highlight

multiple issues to be considered and addressed in future work. First, this experiment allowed for

hyporheic flow paths to form around the mycelium structure, a hydrologic flow path that is well-

known but has not been considered by other experiments. These hyporheic flow paths could

allow for water to move around the mycelium structure, depending on how they system is

installed, and decrease the interactions between contaminant and mycelium, especially

considering highly permeable systems. While hyporheic flow is well known for removing

contaminants, such as nitrogen, by driving interactions between streambed microbes, anoxic

zones, and increased residence times (Passeport et al.,2013), without the presences of mycelium

in the hyporheic zone, there is no benefit for E. coli remediation. Consideration of how to

address this issue will greatly improve the potential impact of future restoration approaches. This

includes considerations such as the potential effects of increasing the burlap barrier width or

including multiple burlap barriers along the stream table as well as the potential use of different

species of fungal spawn in future research. Secondly, mycelium deployment duration also plays

a pivotal role in E. coli reduction (Cotter, 2014). Highest rates of E. coli reduction have been

shown within a 48-hour period, but significant reduction rates continue up to 96 hours after

deployment (Pini and Geddes, 2020. This research stands in contrast to previous research

because the time frame of each experiment was significantly shorter (5 hours total). This

research has demonstrated that although E. coli reduction does begin within shorter time frames,

mycelium efficacy may not be significant for episodic bacterial contamination. While contact
 44

time has been explored in past research, variation in E. coli concentrations remains largely

unexamined; however, E. coli input concentrations may also affect the efficacy of mycofiltration.

Of the six experiments during this research, E. coli inputs ranged in average from 472.95 to

873.3 MPN/100mL, other research utilized 800 cfu/100mL (Stamets et al., 2013). Additionally,

this research’s field data shows that E. coli concentrations in the Upper South River are

significantly higher and often exceed 1000 MPN/100mL (with a maximum concentration of

120,980 MPN/100mL observed) which may affect future field applications. Research utilizing

extremely high E. coli concentrations has yet to be explored, but may provide insight into future

mycofiltration efficacy.

5.2 Conclusion

This research examined the efficacy of T. versicolor mycelium in reducing concentrations

of E. coli in flowing waters during short periods of time laboratory-scaled environment. The

findings of this study are that the T. versicolor mycofilter reduced concentrations of E. coli with

temporal variance throughout the experiments; however, results do not indicate any more

removal of E. coli from the water column compared to flow without the mycelium. These

findings suggest the usefulness of mycofiltration as a water quality restoration technique in

streams may be limited by decreased contact time between contaminant and mycelium or

hyporheic flow paths that bypass the mycelium installation. According to this research, more

laboratory and field-scale research is needed to determine the effectiveness of Trametes

versicolor mycelium as well as to determine contact time. However, it must be noted that

mycoremediation is a burgeoning remediation method and has proven effective for heavy metal,

chemical, and bacterial removal from soils (Kapahi and Sachdeva, 2017; Kulshreshtha et al.,

2014; Singh, 2006). Additionally, it shows promise as a viable method for remediation of water
 45

reservoirs, waste water treatment plants, and small scale surface water remediation installations

(Cotter, 2014; Martinez, 2016; Molla and Fakhru’l-Razi, 2012; Pini and Geddes, 2020; Singh,

2006). The usefulness of this technique once refined may be helpful in mitigating high bacterial

concentrations in small order urban streams resulting from increased precipitation events due to

climate change and continued coverage of impervious surfaces. This is important in light of the

lack of policy change and implementation to protect small order streams from consistent

degradation and limit human health exposure to high bacterial loads (Walsh and Ward, 2019).
 46

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