detox :: mycoremediation

d e¡t o x Mycoremediation

Technical Dissertation CARC 7102

Master of Architecture ARB/RIBA Part 2

Ting Yan Yeo 1407327

de¡tox

Mycoremediation

01

gloss a r y

02

ex pe r i m e nt

01

03

ex pe r i m e nt

02

04

ex pe r i m e nt

03

09

> home growing kit // yellow oyster mushroom

> mycoremediation // part I

> mycoremediation // part II

soil t e s t > oil extraction + pH evaluation

o v er a l l a na l y s i s ex pe r i m e nt

04

> mycelium bricks

c o n ten ts

in t r od uct i o n

15

17

[a p p e nd i x]

c on cl us i o n bib lio g r a p hy + we b o g ra p h y

18

19

19

Master of Architecture ARB/RIBA Part II CARC7102 Technical Dissertation

c ol o p ho n

19

Supervisor: Gabor Stark 2015/16

intr o du c t io n >> From the mid of 18th century, Industrial Revolution has been the key success to the global economic growth. Its involvement in vast exploitation of nonrenewable natural resources and emission of unwanted chemical substances into the environment has however led to the chief worry of many on the planet: pollution.

This Detox Project was initiated from the research of my final year thesis project where its topic was at first revolving around Malta’s infrastructural development with its social, political as well as environmental impact and later on narrowed down to Marsa Power Station[MPS] - the main source of electricity to the island since 1953 that has been officially decommissioned early 2015 due to many concerns on its negative impacts and the support of technological advance on renewable energy today. It is planned to be completely demolished by 2018 for future development.

There are many precedents of successful adaptive reuse on post-industrial landscape around the world such as Gas Work Park in Washington, Duisburg Nord in Germany and Tate Modern in London. Clearly, from decommissioning the power plant to being able to reuse the land, it is essential for the remediation works to be done proficiently to prepare the ground condition from an unknown state in terms of risk and hazards to a known and safe state due to its exposure to all sorts of chemical contamination during its working life.

operation SHUTDOWN

decommission COLD STANDBY

DECONTAMINATION

DISMANTLING

WASTE DISPOSAL

R E M E D I A T I O N

reuse LAND REUSE

Bioremediation is one of the waste management techniques that is commonly used to clean up a contaminated site through naturally occurring or deliberately introduced microorganisms. Mycoremediation is a form of bioremediation, a process that breaks down contaminants using fungi, or more commonly known as mushroom. It is the network of white and fuzzy filaments called mycelium that fruits mushroom. Technically, mycelium produces enzymes that break down long-chained toxins into simpler and less toxic molecules. This technique was founded by an American mycologist, Paul Stamets who has spent nearly 3 decades researching on mushroom and his book Mycelium Running: How Mushroom Can Help Save The World has been a huge reference in this technical project.

“Life springs from mycelium. Fungi control the flow of nutrients, and as a consequence they are the primary governors of ecological equilibrium. As ecosystems change, fungi adapt to steer the course of nutrient cycles. The strength and health of any ecosystem is a direct measure of its diverse fungal populations and their interplay with plants, insects, bacteria and other organism.”

[Stamets, 2005:89]

The objectives of this project are: 01 02 03 04

to understand the growth pattern of mycelium to evaluate the capability of mycelium in breaking down the toxin in the soil to study how extreme the condition in which mycoremediation can still happen to analyse the soil condition after mycoremediation has taken place

In order to achieve greater results and to obtain a better understanding on mycoremediation, a number of experiments from a simple home growing mushroom kit to more intricate growing process were later established.

01 / 19

g lo s s ar y

BI·O·RE·ME·DI·A·TION A waste management technique that involves the use of either naturally occurring or deliberately introduced microorganisms or other forms of life to consume and break down environmental pollutants, in order to clean up a contaminated site.

MY·CO·RE·ME·DI·A·TION It is a form of bioremediation, using fungi to degrade the contaminants in soil or water. Myco means fungus and remediation means remedy.

m yc elium is the vegetative part of a fungus, consisting of a network of fine white filaments. Mycelium produces enzymes that are superb at breaking apart hydrocarbons, the base structure common to oils, petroleum products, pesticides, PCBs, and many other pollutants.

g lo s s ar y

C contaminant cultivate D decommission

E environmental control I inoculation

inert L litmus paper

M microbe metabolize

P pasteurisation petroleum hydrocarbons

- the substance[gear oil] that makes something[substrate] impure or polluted - grow

- the preparation of plant, equipment and structures by taking them from an ‘unknown state’ in terms of risk and hazards, to a ‘known state’ ready for dismantling.

- to limit the condition of the room where the experiment takes place such as temperature, moisture and light.

- introduce or insert or vaccinate an infective agent[spawn] into an organism [substrate] - absent from the strength or ability to grow or move

- a strip of paper that is stained with litmus and used to test the alkalinity or acidity of a substance

- microorganism, bacteria or germ - a living organism[mycelium] process a substance[microbe] to maintain life

- a process of partial sterilization to get rid of bacteria - the long-chained compound that is the base structure of pollutants like pesticides, oil and petroleum products - scientific term for oyster mushroom

pleurotus - the earliest stage of fruiting, also known as pinning stage primodia S spawn spawn run

substrate

- the mycelium of fungus [a cultivated mushroom, seed] - the stage where mycelia will start to colonize the substrate and absorb its energy - a substance or material on which an organism grows or an enzyme acts [hay, coffee ground, woodshavings, barleys, hops]

02 / 19

ex pe r i me n t

01

> home growing kit // yellow oyster mushroom

environm ent a l c ont rol 14 - 22 ยบC shaded windowsill spray twice daily [to keep the compost moist]

d ay 0 1 -1 0

d ay 1 1

>> The project started with a home-growing kit that contains prespawned mycelium for yellow oyster mushroom. The purpose of this exercise was to get a better understanding on the growth pattern of mycelium.

da y 12

da y 13

day 14

d ay 15

da y 16

ha r vest x 2 c rop s

>>The whole process took place in 17 days from spawn run to the first crop. The harvest of the second crop happened two weeks after.

da y 17 03 / 19

ex pe r i me n t

02

> mycoremediation // part I

>> After the first exercise, I learned that it is difficult to observe the spawn run stage as the substrate was wrapped in black polyethylene bag and opaque container. On top of that, there is no control over the type of substrates with the prespawned kit.

01 02

03

In order to achieve a clearer observation over each stage of the process in different substrates, it requires starting the growing process from spawn inoculation.

04

eq uip m ent 01. 250ml glass jar [with labels]

05

02. polyethylene containers [as moulds] 03. straining bag 04. syringe 05. rubbing alcohol [Isopropanol Alcohol 70%]

08

06. vinyl disposable gloves

06

07. zip lock // freezer bag 08. gear oil [as toxin] 09. spoon

07

09

10. kettle

11. tub

12. scale

sp a wn

Spawn Run > 20ºC-30ºC // 90%-100% humidity // 7-10 days Primordia > 18ºC-24ºC // 95%-100% humidity // fresh air 3 times a day // full light // 2-4 days Fruiting > 18ºC-27ºC // 85%-95% humidity // fresh air 3 times a day // full light // 3-5 days

ide al c o n dit io n

> pink oyster mushroom [pleurotus djamor] > purchased from mushroombox.co.uk

s u b s tr ate A. waste coffee grounds

A.

D.

B.

E.

B. hay C. woodshavings D. barley E. hops

C.

04 / 19

s tep 01

> boil the water

> fill the straining bag with substrate and place it in a clean container > pour boiling water over the substrate until it is completely soaked

st ep 0 2

> leave the substrate to soak for 30 minutes

> spray all work surfaces, glass jars, moulds and spoon with alcohol and dry. The alcohol has to be completely evaporated to avoid killing the spawn.

+ > wash hands and arms with soap, wear gloves for the next steps.

> mix substrate with spawn in ratio of 20% [ spawn:substrate = 1:5 ] > in this case, 250 grams of substrate + 50 grams of spawn

sub s t r a t e p r ep a r a t ion Pasteurisation is preferable to sterilization as it reduces the competing microbes in the substrate and sets aside ‘good bacteria’ to grow alongside mycelium.

A. waste coffee ground > A bagful was collected fresh from a local coffee house. Since it was already pasteurised during the brewing process, it 35ºC

could be ready for inoculation process.

B. hay

> after 30 minutes, drain off the water > spread the substrate on a tray to cool it down to at least 35ºC before proceed to inoculation [note: temperature higher than 40ºC will kill the mushroom spawn]

C. woodshavings > Both hay and woodshavings were purchased as raw substrate from a pet store.

Hence, they have to undergo pasteurisation process as shown in the diagram.

01

02

03

inoc ula t ion It is important to keep everything as clean as possible during this step to keep down the

B.

+ 8ml gear oil

contamination. + 2ml gear oil

A.

In order to study mycoremediation process, gear oil which contains petroleum hydrocarbons was added onto inoculated substrates.

The experiment is to see if mycelium would be undeterred.

C.

05 / 19

Mycelia will start to colonize the substrate and absorb its energy during spawn-run.

dark [ in an airy box ]

Once the substrate is fully covered by mycelia, it will be ready for pinning. This takes about 2 weeks.

A.

day 01

day 03

day 05

day 07

coffee grounds

20 ยบC - 30 ยบC [ warm room ]

B.

hay

cover each jar loosely with zip lock to maintain high humidity

C.

s tep 03

s paw n ru n

woodshavings

Mycelia started spreading out on day 03.

In the coffee ground substrate, green

They look white and fuzzy. The substrates

mould was observed on day 07. However,

were fully colonized by day 13 and

mycelia managed to outnumber the

tiny pins [baby mushroom] started to

contaminants. By day 13, all the green

develope.

mould was metabolized by mycelia.

A.

B.

o b s er v a tio n

Spawn-run was documented every 2 days.

C.

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day 09

day 11

day 13

All the green mould was

Tiny pins [baby mushroom]

Tiny pins [baby mushroom]

metabolized by mycelia.

started growing out of

started growing out of

mycelia. This means

mycelia. This means

pinning process has started.

pinning process has started.

07 / 19

During pinning[primordia] stage, an increase in fresh air

18 ยบC - 24 ยบC

and light will induce fruiting. It is also essential to keep the

full light + fresh air

content moist.

spray twice daily to maintain moisture content

All the jars were moved to a well-lit and airy room which

s tep 04

p in n in g - fru itin g

maintains the temperature at 23ยบC.

01

02

03

01

A.

A.

B.

B.

C.

C.

d ay 1 7

02

da y 19

03

01

02

03

A.

m yc or em ed ia t ion Mycelium fruits faster and bigger on hay

B.

with 8ml of gear oil compared to the one

Mycelium digests the gear oil by breaking C.

down the petroleum hydrocarbons into non-toxic components [H2O and CO2 ] as

t heor y

without any gear oil.

its nutrient.

d ay 2 1

B.

hay

03

+ 8ml gear oil

A.

coffee ground

02

+ 2ml gear oil

01

C.

woodshavings

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e x t ra ct i o n

03

> mycoremediation // part II

about

ex pe r i me n t

>> Mycoremediation project was carried on by using bigger amount of gear oil to analyse the capacity of mycelium in digesting the contaminants.

With 1:5 ratio of mushroom spawn to hay substrate as the fixed substance, the variable amount of gear oil from 0ml to 160ml established a set of 8 jars

f i xe d

experiment.

s u b s tr ate

sp a wn

hay

pink oyster mushroom [pleurotus djamor]

250 grams

+ 50 grams

v a ri ab le

g ear o il

+ 0 ml

+ 2.5 ml

+ 5ml

+ 10ml

+ 20ml

+ 40ml

+ 80ml

+ 160ml

sub s t r a t e p r ep a r a t ion preparation. The hay underwent pasteurisation process as previously shown in experiment 02 before it was inoculated with mushroom spawn.

s tep 01

Experiment 03 started with substrate

inoc ula t ion substrate with spawn in ratio of 20% was used.

300 grams fixed substance

[ spawn:substrate = 1:5 ] The variable amount of gear oil was

step 02

As the fixed substance, a mixture of

doubled up each time to obtain a greater difference between each result or a pattern later in the process.

01

02

03

04

05

06

07

08

09 / 19

Mycelia start to colonize the substrate and absorb its

20 ยบC - 30 ยบC [ warm room ]

energy during spawn-run.

dark [ in an airy box ] cover each jar loosely with zip lock to maintain high humidity

It takes about 2 weeks for the substrate to be fully covered by mycelia before it is ready for pinning 01

day 01

day 03

day 05

day 07

02

03

04

05

06

07

08

s tep 03

s paw n ru n

Just like in Part I, mycelia started

Mycelia spread evenly throughout hay

spreading out on day 03 in all jars. They

subtrates, but stopped at the oil line.

look white and fuzzy. The substrates were fully colonized by day 15. On day 19, tiny pins started to develop.

No green mould developed in any jar.

o b s er v a tio n

Spawn-run was documented every 2 days.

10 / 19

day 09

day 11

day 13

day 15

Substrates in all the jars were fully colonized by the white and fuzzy mycelia and ready for pinning and fruiting process.

11 / 19

All the jars were moved to a well-lit and airy room which

18 ยบC - 24 ยบC

maintains at 23ยบC to induce fruiting process.

full light + fresh air spray twice daily to maintain moisture content

d ay 1 9

s tep 04

p in n in g - fru itin g I

day

ob s er va t ion The fruiting rate and size have minimal noticable variation regardless of the amount of gear oil except jar 08 which has less fruit compared to the rest.

1s t c rop

day

21

da y 23

25 01 + 0ml gear oil

05 + 20ml gear oil

02 + 2.5ml gear oil

06 + 40ml gear oil

03 + 5.0ml gear oil

07 + 80ml gear oil

04 + 10ml gear oil

08 + 160ml gear oil

05

06

07

08

01

02

03

04

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The mushrooms were harvested from the first crop.

18 ยบC - 24 ยบC

All the jars were remained

full light + fresh air

under the same conditions and

spray twice daily to maintain moisture content

allowed to continue fruiting.

2 nd c ro p

s tep 05

fru itin g II

d ay 2 7

day

d ay 3 7

day

o bs er v atio n After 12 days of 2nd fruiting, Jar 02, 07 and 08 have less and smaller fruit compared to the rest. Their fruiting process is slower.

29

da y 31

35

da y 33 01 + 0ml gear oil

05 + 20ml gear oil

02 + 2.5ml gear oil

06 + 40ml gear oil

03 + 5.0ml gear oil

07 + 80ml gear oil

04 + 10ml gear oil

08 + 160ml gear oil

05

06

07

08

01

02

03

04

13 / 19

All the jars were remained under the same conditions again to continue observing the 3rd fruiting process before emptying out the contents to assess their conditions as

18 ยบC - 24 ยบC full light + fresh air spray twice daily to maintain moisture content

final results.

d ay 4 6

s tep 06

f ru it in g III

day

obse r vation

All jars took nearly twice as long to fru this time. On day 70, mould could be observed on fruits and the surface of

substrates in all jars. They also manag to attract flies.

51

da y 62

uit

ged Jar 02

3 rd c rop

da y 70

01 + 0ml gear oil

05 + 20ml gear oil

02 + 2.5ml gear oil

06 + 40ml gear oil

03 + 5.0ml gear oil

07 + 80ml gear oil

04 + 10ml gear oil

08 + 160ml gear oil

05

06

07

08

01

02

03

04

14 / 19

about

soil test

> oil extraction + pH evaluation

>> After 70 days of mycoremediation in experiment 03, a soil test was conducted to analyse how much gear oil has been digested by mycelium throughout the process as well as how it has affected the pH level of the substrate.

In order to achieve an accurate precision in the results by using proper scientific equipment, a few attempts have made to approach a number of proper laboratories such as School of Biosciences and School of Physical Sciences in University of Kent, as well as Canterbury College. Unfortunately, none of them were able to assist the project.

In the end, I consulted Professor Fran Kerton from University of York who provided me an advice on an oil extraction exercise which I could be able to carry out without a scientific laboratory to evaluate the amount of gear oil left in the substrate.

On top of that, soil tester and litmus paper were also being used to examine the condition of the substrate.

jar

01

02

03

04

05

06

p H l e v el

7.5

7.0

7.0

7.5

8.0

>8.0

mo i s t u re level

3.0

6.0

8.0

2.5

2.5

1.0

eq uip m ent

02 01

01. metal sieve

03

02. electronic scale 04

06

03. litmus paper test strips 04. spoon

05

05. soil tester [moisture + pH level] 06. rubbing alcohol [Isopropanol Alcohol 70%]

s ub s t r a t e c ond it ion 08

The substrate in each jar was first tested with a soil tester to measure its moisture level and pH level. The tip was inserted to the hay substrate above the oil level at the bottom of jar. The results were then

t est 0 1

07

recorded.

8.0

>8.0 substrate in jars with higher amount of gear oil seem to have higher pH and lower moisture level tip level

1.5

<1.0

15 / 19

lit m us p ape r

tes t 02

Before the oil was extracted out from the jar, the liquid mixture[gear oil + water] at the bottom of each jar was tested with litmus paper to find out whether it is acidic or alkaline.

tested liquid

o il ex tr ac tio n

test 03

Extraction test was advised by Professor Fran Kerton as mentioned before.

Step 01:

Step 03:

Step 04:

Identify a type of solvent that the gear

Add 50ml of solvent in the jar,

Leave the extraction in a

oil dissolves in it. Professor Fran Keton

stir it around and then filter it

well-ventilated location for the

suggested nail varnish which is mainly

through a sieve.

solvent to evaporate. Visually

acetone or methylated spirits which is

make notes on the extraction,

mainly methanol/ethanol.

whether or not it looks oily.

Rubbing alcohol [Isopropanol Alcohol 70%] was used in this case.

Step 02: Weigh the container that will be used to have the solvent evaporate in it.

Step 05: Re-weigh the container again to see how much oil if any is left in the substrate and was extractable in 50ml of solvent.

Deduct the weight of the container - 22g to achieve the final results

re s u l t s 01

02

03

04

05

06

07

08

7

7

7

7

7

6

5

4

pH scale 1 2 3 4 5 6 7 8 9 10 11 12 13 14

acid i c

n

a l ka l i ne

neutral

acidic

the liquid mixture in jars with higher amount of gear oil have lower pH [more acidic]

note: The amount extracted will not be 01

16g

05

38g

100% of the oil due to partition coefficients.

Theoretically, jar 01 should not have anything left since no oil was added in the experiment. However, since

02

24g

06

64g

all jars were being watered daily to keep the content moist, hence it is suspected there should be water in the extracted solute.

It was aimed to obtain a set of

03

30g

07

117g

results which will be able to give an overall trend and suggest whether the mushrooms are able to digest the oil and remove it from contaminated substrate.

04

54g

08

188g

16 / 19

01

02

03

+ 0ml gear oil

+ 2.5ml gear oil

+ 5ml gear oil

pH

7.5

7.0

7.0

mois t ure

3.0

6.0

8.0

7

7

fru itin g I

day 2 5

fru itin g II

day 3 7

f ru itin g III

day 7 0

test 01

s u b s tr ate c o n d it io n

test 02

l i t m u s p aper

test 03

o i l ex t r ac t io n

pH of liquid m ix ture

lef tov er liquid m ix ture

7

16g

24g

30g

05

06

07

08

+ 10ml gear oil

+ 20ml gear oil

+ 40ml gear oil

+ 80ml gear oil

+ 160ml gear oil

7.5

8.0

>8.0

8.0

>8.0

2.5

2.5

1.0

1.5

<1.0

7

7

6

5

4

54g

38g

64g

117g

o v er a l l a n a ly s is

l

04

188g

17 / 19

[ a p p p e n d i x]

obser v a t i o n The process of mycelium bricks was started off by creating the substrate in

A.

enclosed condition [as previously shown in experiment 02] and cultivating the spawn in various substrates: A. waste coffee grounds

B.

B. hay C. woodshavings D. barley E. hops

C.

The process was documented every two days. On day 13, only 3 of the substrates [A. B. & C.] have developed the required density level of mycelium. All spawncolonized substrates were removed from

D.

the mould to let it air dry for 3 days before baking in the oven for an hour to make the cultivation inert. This experiment is however failed to achieve a solid mass because of the relatively low density between mycelium and substrate.

After further research, there was evidence that the substrate needs to be compacted into a smaller mould prior to mycelium colonisation, which is during day 03 to day 06.

> mycelium bricks

E.

d a y 05

04

d a y 01

ex pe r i me n t

d a y 13

d a y 11

d a y 09

d a y 07

>> Mycelium brick was conducted as a side experiment to see if thereâ&#x20AC;&#x2122;d be any variation in the density of different substrates after spawn run which may become useful at a later date. However, the core area of the research has later been decided to focus on mycoremediation.

18 / 19

conc lu s ion >> Due to the lacking of proper scientific equipment and access to laboratory, it has led to several constraints in testing the final results of the experiment. Even though the oil extraction test that was established in the end failed to prove whether or not mycelium has digested the gear oil in the substrate, however it was learnt that certain chemical reactions could be achieved with tools like everyday objects.

A lot scientific knowledge was surely gained in the process.

Nonetheless, the surprising finding in Experiment 03 which mycelium was able to undergo spawn-run and even lasted through 3 times of fruiting in the most contaminated jar [+160ml of gear oil] has somehow become a success to accurately prove its ability to survive in the varying amount of contaminated substrate. On top of that, the increasing fruiting rate and size along with the increasing amount of gear oil[contaminant] in Experiment 02 was another favourable outcome.

The speed of its growth and its capacity in metabolizing microbes show beyond doubt its role as the primary governors of ecological equilibrium. The power of mycelium to trigger a sequence of reaction by other organisms in a lifeless landscape as well as its ability as a detoxifier in mycoremediation is definitely an asset to the health of environment.

bib liogr a phy Stamets, Paul (2005) Mycelium Running: How Mushroom Can Help Save The World. New York: Ten Speed Press.

webogr a phy in general: http://www.fungi.com/blog/items/helping-the-ecosystemthrough-mushroom-cultivation.html http://www.ted.com/talks/paul_stamets_on_6_ways_ mushrooms_can_save_the_world#t-286765 http://www.ted.com/talks/eben_bayer_are_mushrooms_ the_new_plastic#t-319581 http://blog.ted.com/9-ways-mushrooms-could-drasticallyimprove-the-world/ experiment 01 > home growing kit: http://www.nutleyskitchengardens.co.uk/merryhill-freshmushroom-kit.html experiment 02 > mycoremediation // part I + experiment 03 > mycoremediation // part II: http://mushroombox.co.uk http://www.sciencestuff.com/playground/agar_powder. shtml https://www.growveg.co.uk/guides/growing-gourmetmushrooms-at-home-from-waste-coffee-grounds/ http://www.instructables.com/id/Oyster-Mushrooms-in-aLaundry-Basket/step4/Load-the-Basket/ https://issuu.com/mycofarmx/docs/mycofarmx

Col op hon Master of Architecture ARB/RIBA Part II CARC7102 Technical Dissertation Ting Yan Yeo tingyan.yeo@gmail.com Supervisor: Gabor Stark Canterbury, UK 2016

experiment 04 > mycelium bricks: http://www.iaacblog.com/programs/mycotecture-growinginto-form-2/ http://www.o-matic.com/blog/?p=3873 http://www.mycoworks.com http://www.terreform.org/projects_habitat_mycoform.html Canterbury School of Architecture New Dover Road Canterbury Kent CT1 3AN United Kingdom www.ucreative.ac.uk www.cantarch.org

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