final presentation

1. FILTRATION OF
CONTAMINATED
GREYWATER USING
MYCOREMEDIATION
BIOSYSTEMS CAPSTONE DESIGN PRESENTATION
DECEMBER 6TH , 2022
NATALIE WHITAKER, MARGUERITE AZZARA, FREDDY NOCELLA, ISAIAH DEL CAMPBELL, DAVID
TRAYNHAM, ALLY BARRERA

2. OUTLINE
Background
01
Literature
Review
02
Rationale
03
Objective
04
Tasks
05
Materials
and Methods
06
Results and
Discussion
07
Conclusion
08

3. BACKGROUND

4. WHERE IS DOMINICA

5. 1400 MILES SOUTHEAST OF MIAMI

6. SOUFRIERE, DOMINICA

7. BACKGROUND
–
HURRICANES
IN DOMINICA
• In 2017, the island of Dominica
was devastated by two category
five hurricanes. The first was
Hurricane Irma on September
6th followed closely by
Hurricane Maria on September
18th.
• Landslides caused by the
hurricanes led to heavy
amounts of debris and
contaminants flowing through
the land into the ocean causing
significant damage to the coral
reefs. The people of the island

8. LITERATURE REVIEW

9. LITERATURE REVIEW – WHAT IS
MYCOREMEDIATION AND HOW DOES IT
RELATE TO GREYWATER?
• Mycoremediation is the use of fungus to remove contaminants
from the environment. Greywater is household wastewater that
originates from anywhere except a toilet, like shower water.
Common constituents that are tested for in household greywater
include nitrate, phosphorus, BOD5 and suspended solids (Abed
2016).
• Many fungi absorb and retain specific elements, namely heavy
metals, in their biomass through a process called
hyperaccumulation. (Cotter 2014)

10. LITERATURE REVIEW - NITRATE
REMOVAL USING MUSHROOMS
• Nitrates and nitrites are a very common pollutant in many
streams and runoffs. Using mushrooms to intercept the
pollutants can mitigate the issue before the pollutants enter
oceans or rivers. Mycelium and mushrooms have a sponge like
matrix which can hold onto pollutants, and they can also uptake
them from soils as they grow. Differing species have different
capacities for the uptake, but all can retain some levels of
nitrates and nitrites (Agoroaei 2008).

11. LITERATURE REVIEW – CORAL BLEACHING
AND HURRICANE MARIA IN DOMINICA
• Coral bleaching is one of the negative consequences caused by excess
runoff and greywater inputs. Coral bleaching is caused by a variety
factors, most notably a warming climate. Despite this, there is emerging
research that contaminated water also plays a role. Excess nitrogen
interrupts the symbiosis between the corals and their algae partners,
since cell division of the algae is limited by nitrogen levels. If too much
nitrogen is present, the algae reproduce too much and is the symbiosis
is thrown off (Pogoruetz 2016).

12. RATIONALE

13. RATIONALE
• Coral reefs in Dominica have faced a
large amount of stress from
wastewater runoff and storm runoff
due to recent hurricanes in the last
few years that have led to coral
bleaching. By filtering this wastewater
and stormwater runoff before it
enters the sea, the coral will be
exposed to less harmful
containments that will help
slow/eliminate coral bleaching.

14. MEDIA FROM ISLAND

15. UNITED NATIONS SUSTAINABILITY GOALS
In 2015 the United Nations created the 17 Sustainable Development Goals. The primary
purpose of these goals is to create a blueprint to improve human health and education,
reduce inequality, and spark economic prosperity. Three of these goals relate directly to
our project. By removing nitrate, sulfate, and phosphorus from the wastewater and
runoff, there will be cleaner water, protect the coastal reef population and create a more
sustainable future for Dominica.
Related Goals:
1. Clean Water and Sanitation
2. Sustainable Communities and Cities
3. Life Below Water
https://sdgs.un.org/goa

16. OBJECTIVE

17. OBJECTIVE
• The objective of this project is to engineer and design a
biological treatment system to remove contaminants from waste
streams entering the ocean.

18. VENN DIAGRAM
Sustainable
Communitie
s
Environmental
Solutions
Resiliency
Our
Project
Socio-
economic
Factors
Science/
Biology
Engineering
Design

19. MUSHROOM MOUNTAIN SITE
VISIT

20. MUSHROOM MOUNTAIN
• On October 11th, our group attended a tour of mushroom mountain in
Easley, SC.
• We learned about various types of mushrooms as well as how to grow
them and what conditions/food sources would work best as we grow
Tiger Sawgill mycelium for our project.

21. MUSHROOM MOUNTAIN TRAIL
PICTURES

22. MUSHROOM MOUNTAIN WAREHOUSE
PICTURES

23. LABORATORY PROCEDURE

24. MATERIALS
• Tiger Sawgill mycelium from Mushroom Mountain
• Sand – specify type
• Hickory sawdust (5 pounds)
• Plastic columns
• Peristaltic pump – Cole Parmer Masterflex L/S
• Tubing – Masterflex Pump Precision Pump Silicone Tubing
• Hach DR900
• Nitrate, sulfate, phosphate standards from RICCA chemical
• Nitrate, sulfate, phosphate test reagents from Hach

25. METHODS
1. A 50/50 mixture of sand and sawdust will be created and used in addition to the
mushrooms. This mixture will then be packed into the columns with the mushrooms
in different ratios.
1. Columns A and B will have no mushrooms.
2. Columns C and D will be 90% sand and sawdust and 10% mushrooms.
3. Columns E and F will be 75% sand and sawdust and 25% mushroom.
4. Columns G and H will be 50% sand and sawdust and 50% mushrooms.
2. A nutrient solution will be created with DI water, nitrate standard, phosphate
standard, and sulfate standard.
1. This solution will then be pumped through the column at a rate of 4 mL/min.
2. Three, 200 mL pour volumes will be run through each column.
3. Five, 40 mL samples will be collected from each pore volume.
3. After the samples are collected, they will be analyzed using the DR900 for nitrate,
phosphate, and sulfate.

26. TASKS

27. GANTT CHART

28. EXPERIMENTAL PROCEDURE:
A small portion of medical gauze
was used as a filter to keep
substrate from exiting the
effluent tube.
Columns being packed with
sawdust/sand mixture and
mycelium in the desired
ratios.

29. EXPERIMENTAL PROCEDURES – NIGHT 1:
•The lab setup was effective, and
the first experimental run was
a success.​
•What we noticed could
be improved​:
•The tubes in the peristaltic
were being pulled​.
•Reactors will be placed in the
back while the sample collection
tubes will be moved in front of
the reactor vessels.​
•Mild leakage was observed. The
observed amount is thought to
be negligible.

30. EXPERIMENTAL PROCEDURES – NIGHT 2:
•We took what we learned on day 1
and made a few minor adjustments
for the lab to run more smoothly.
•Peristaltic tubes were cleaned
which helped them not get
pulled in the peristaltic pump.
•Columns were now placed in
the back so that sample
collection tubes could easily be
collected without the risk of
knocking over the lab setup.
•Silicon was applied to the
reactors where the PVC met the
clear plastic tube, and there was
nearly no water leakage.

31. EXPERIMENTAL
PROCEDURES – SAMPLE
ANALYSIS
• The samples coming out of the column
turned out to be colored.
• We think this is from the soil the
mycelium was spawned in since the
columns with more mycelium
produced samples with more color.
• Since the DR900 cannot give accurate
readings with colored samples, the
samples were diluted 1:3 or 1:1 with DI
water depending on how much color
was present.
• Nitrate and phosphate samples were
analyzed first because they can only be
held for 48 hours.
• The readings were recorded in excel.
This is a picture of sample tubes. All together, we took
163 samples between our 8 columns and analyzed for
both nitrate and phosphate.
This picture shows the setup for phosphate analysis. The
more yellow color in the sample after reagents are added,
the more phosphate that is present.

32. EXPERIMENTAL
PROGRESS – SAMPLE
ANALYSIS
• After Fall Break, our plan was to
analyze for sulfate, since its hold
time is 28 days. However, when we
began analyzing, we realized the
samples had become turbid due to
the growth of bacteria and mold.
• This meant our samples were
contaminated and could no longer
be analyzed accurately.
These images show the mold growth (white fuzzy
patches) in the tubes after Fall Break.

33. RESULTS
• The first step in our analysis was determining the actual concentrations of
nitrate and phosphate based on the dilutions we did.
• Sample calculation:

34. RESULTS
• The next step in our analysis was to use the flowrate and the time needed
to saturate each column to find the pore volume.
• The time needed to saturate the column is the time from when water
begins flowing in to when water begins to flow out of the top of the
column.
• Sample calculation:

35. RESULTS
• We then used the calculated pore volumes of each column and the actual
concentrations to generate breakthrough curves, which is a plot C/C0 vs. pore
volumes through the column.
• C/C0 is the concentration of the sample (C) divided by the initial concentration
of the nutrient solution. Theoretically C/C0 should be less than 1.
• C0 nitrate = 30 mg/L
• C0 phosphate = 45 mg/L
• Sample calculation for C/C0 for one of the nitrate samples:

36. RESULTS – BREAKTHROUGH CURVES FOR COLUMNS
A AND E
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.900
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
C/C0
(nitrate)
PV
Column A
Column E
Figure 1. This graph compares the breakthrough curves for
column A (no mycelium) and column E (25% mycelium)
• Based off this graph, column
A has lower C/C0 values,
which implies more nitrate
was retained in the column
without any mycelium

37. RESULTS – BREAKTHROUGH CURVES FOR COLUMNS
A AND C
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.00 0.50 1.00 1.50 2.00 2.50 3.00
C/C0
(nitrate)
PV
Column A
Column C
Figure 2. This graph compares the breakthrough curves for
column A (no mycelium) and column C (10% mycelium)
• In figure 2, column C starts
out with higher C/C0 values
but begins to have lower C/C0
values at around 0.75 pore
volumes.

38. RESULTS – BREAKTHROUGH CURVES FOR COLUMNS
C AND E
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.900
0 0.5 1 1.5 2 2.5 3 3.5
C/C0
(nitrate)
PV
Column C
Column E
Figure 3. This graph compares the breakthrough curves for
column C (10% mycelium) and column E (25% mycelium)
• The C/C0 values for column C
are lower than those for column
E.

39. RESULTS – MASS OF NITRATE REMOVED
• The final step in our analysis was to look at the mass of nitrate each column retained.
To do this, we multiplied the concentration of each sample by the sample volume to get
the mass of nitrate in each sample.
• We then summed all the masses for each column to show how much nitrate was leaving
the column.
• In order to get the mass of nitrate that went into the column, we determined the volume
of nutrient solution that went into each column and then multiplied the volume by the
initial concentration, which was 30 mg/L.
• The mass of nitrate that left the column was then subtracted from the initial mass of
nitrate in the column to get the nitrate retained.

40. RESULTS – MASS OF NITRATE
REMOVED
Column Mass Entering the Column (mg) Mass Leaving the Column (mg) Mass Retained by the Column (mg)
A (0% mycelium) 13.92 5.72 8.2
B (0% mycelium) - - -
C (10% mycelium) 14.19 7.74 6.45
D (10% mycelium) 13.83 8.65 5.18
E (25% mycelium) 12.57 10.74 1.83
F (25% mycelium) 12.84 11.47 1.37
Table 1. This table gives a summary of the mass retained by each column using the
equations previously described

41. RESULTS - PHOSPHATE
0
0.5
1
1.5
2
2.5
0.000 0.500 1.000 1.500 2.000 2.500 3.000 3.500
C/C0
PV
Table 4. This graph shows the breakthrough
curve for phosphate for column E (25%
mycelium). Most of the values on this graph
have C/C0 values >1.
• Our phosphate breakthrough
curves all had C/C0 values
greater than 1. This means there
was more phosphate coming out
of the column than was put in.

42. DISCUSSION - NITRATE
• Each breakthrough curve begins with a spike in the nitrate concentration. This
represents the injection of the nutrient solution. As the solution flows through the
column, the concentration decreases as nitrate is taken up, until it finally drops even
lower once DI water is injected to purge the column.
• The breakthrough curves for the columns with mycelium all exhibit similar behavior in
that they begin with a larger spike in nitrate concentration if they had more mycelium.
A possible explanation for this could be from nitrates in the soil the mycelium is
spawned in, as they could have been washed out of the column in the early samples.
• Since columns C and E had the most similar colors, these are the only two columns that
can be accurately compared for nitrate retention results.
• From table 1, column C retained 6.45 mg/L nitrate and column E retained 1.83
mg/L nitrate.
• Therefore, based off our experiment, a 10% mycelium addition was more effective
at nitrate removal than a 25% mycelium addition.

43. • A possible explanation for why 10% mycelium
was more effective could be because the fungal
cells were better distributed throughout the
column.
• The 25% column could have had larger
aggregations of fungi, rather than
dispersed cells, which would have resulted
in less surface area for nitrate uptake.
• The image to the left gives a visual of this
idea
DISCUSSION - NITRATE

44. DISCUSSION -
PHOSPHATE
• As mentioned previously, all the phosphate graphs resulted in C0 values greater than
1, which means more phosphate came out than went in.
• While we are unsure as to why this is, we think this is from the soil the mycelium was
grown in.
• Another possible reason for this is due to the color of the samples, since the DR900
method for phosphate uses a DI blank rather than a sample blank. A DI blank makes
this method more sensitive to color since the DI water is clear.

45. Option 2: A bed-style filter where water
flows in, moves through the filter, and
out the bottom through a series of
perforated tubes.
CONCLUSION
We believe there are two potential designs that could be implemented in Dominica. Both of these
would be a form of filtration for the greywater leaving the houses, before it reaches the ocean.
Option 1: A tube that would be filled with a
mixture of mycelium, sawdust, and sand. This
would be attached to the pipe leaving the
house.

46. CONCLUSION, CONTINUED
• Important factors to consider whenever picking an application:
• Cost and who would pay
• How often each method would need to be changed out
• Ease of operation and implementation
• These applications have the possibility of being a low-cost solution to help in a
country that does not have the resources available for centralized water
treatment.

47. ACKNOWLEDGEMEN
TS
• We would like to thank Dr. Darnault for his
guidance in the design and execution of our lab
and dr. Dodd for his ongoing support and
knowledge.
• Dr. Ogle for providing project scope and
equipment.
• Brandon at Mushroom Mountain for sharing his
expertise in the area of mushroom research.

48. THANK
YOU