Design by Degradation - fungal bioremediation of plastic waste in the new construction paradigm

DESIGN BY DEGRADATION DEGRADATION BY DESIGN Fungal bioremediation of plastic waste in the new construction paradigm Master thesis by

Lennert Van Rompaey 2019 - 2020

Master thesis submitted under the supervision of Prof. Dr. Ir. Arch Lars de Laet and co-supervision of Elise Elsacker in order to be awarded the masters degree in Architectural Engineering

WORD OF GRATITUDE Never had I thought that I would one day find myself mixing straw, mushrooms and cat food or sorting trash cans of fast-food chains when I started my curriculum as an architectural engineer. The realisation of this master thesis has been quite the journey, in which I have come to question our current business-as-usual practices within the building industry, but also beyond the building industry, and in which I have dipped my toes into the field of microbiology to search for solutions. Now here I stand, knee-deep in municipal waste, with changed perspective and great gratitude for it.

I would especially like to thank Elise Elsacker for introducing me to the fascinating world of mycology, which is not standard in the curriculum, though I now think that it should be. It has truly opened my eyes and your guidance, patience and trust have been vital whilst developing my ideas and my passion for them. As important in this process was the insight and supervision of Lars de Laet; thank you for the freedom, encouragement and occasional sense of realism you have given me throughout.

It is safe to say that I would have not been as invested in this project if it weren’t for joining the Critical Concrete Summer School; thank you for the hands-on experience and for planting a seed for social and ecological values within architecture. Moreover I would like to thank the department of microbiology who has allowed me to work in their laboratory during the next phase of my thesis. I have entered as a bull in a china shop but have quickly learned the ropes; thanks for that, kind matadors Eveline, Karl and Simon. A big thank you also goes out to the Dopper Changemaker Challenge for funding a part of my expenses and for believing in my wreckless enthusiasm and ideas.

One man’s trash is another man’s treasure they say, hence I would like to thank the local Starbucks stores for letting me plunder their trash cans and for making the building project at the end of this thesis possible.

Last but not least a heartfelt thanks to everyone around me. To my parents, thank you for letting me turn your home into a laboratory and construction site. To my friends, you have all been unconditionally optimistic and loving - it has made all the difference.

ABSTRACT For over thirty years, the biological degradation of plastics by microorganisms has been promoted through microbiological publications as an eco-friendly alternative for landfilling and incinerating – a clarion call for change – yet today these are still the most economically viable ways of discarding plastics. Therefore, this thesis has set out to investigate whether mycelium composites could valourise this waste stream and inject the bioremediation process of plastics into an economically viable material for an industry that in itself is in dire need of sustainable material innovation: the building industry.

Through an experiment-driven approach infused with literature research, three fungal species that are currently used for the production of mycelium composites are tested for their bioremediation potential. Ganoderma Lucidum has thereby come to the fore as a promising candidate to degrade polyethylene as well as PLA, while resorting to the polymer as the only carbon source to sustain its growth. The findings give way to an explorative research and small scale prototyping that puts into perspective the possibilities, limitations and areas for further research of the architectural implementation of the biological degradation process of plastic waste. The thesis concludes in the form of a small scale building project in which 3000 disposable coffee cups were resorted to as a resource to construct a living wall segment that clearly narrates the process of biodegradation, evoking a critical thinking about waste management and starting a debate that transcends disciplines on how architecture could evolve from its current shortcomings in sustainability.

LIST OF TABLES Table 1 Absorption value (abs) at 666nm, final concentration (c) and

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percentage dye degradation (DC) of methylene blue in the liquid culture media 23 days post inoculation.

Table 2 Initial mass; mass after 50 days of colonisation by Ganoderma

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L.; total mass loss and degradation percentage of five equiform LDPE samples.

Table 3 Initial mass; mass after 50 days of colonisation by Ganoderma

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L.; total mass loss and degradation percentage of five equiform HDPE samples.

Table 4 Initial mass; mass after 50 days of colonisation by Ganoderma L.;

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total mass loss and degradation percentage of five equiform PLA samples.

Table 5 Initial mass; mass after 50 days of colonisation by Ganoderma L.;

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total mass loss and degradation percentage of five equiform samples of a wood/PLA composite.

Table 6 Initial mass; mass after 50 days of colonisation by Ganoderma L.;

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total mass loss and degradation percentage of five equiform LDPE samples on a lignin medium.

Table 7 Initial mass; mass after 50 days of colonisation by Ganoderma L.; total mass loss and degradation percentage of five equiform PLA samples on a lignin medium.

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LIST OF FIGURES Figure 1 Hyfi Tower by The Living (2014): a cluster of 13-meter-tall towers

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built using biodegradable bricks made from agricultural byproducts and mycelium Figure 2 mycelium + timber by Sebastian Cox and Ninela Ivanova (2017):

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furniture collection made from mycelium and wood Figure 3 Mycelium Ware by craft combine (2019): homeware collection

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made from agricultural byproducts and mycelium Figure 4 Discarded plastic bottles and other garbage blocks the Vacha

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Dam, near the Bulgarian town of Krichim, on April 25, 2009. Single-use plastic containers like bottles and plastic bags are “the biggest source of trash� found near waterways and beaches, according to the nonprofit Ocean Conservancy.

Figure 5 Mycelium research at the production centre of Critical Concrete

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in Porto (Critical Concrete - Summerschool 2018) Figure 6 Autoclave setup at MICR laboratory - Vrije Universiteit Brussel

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Figure 7 A. Generative hyphae. B. Binding hyphae, the arrow shows the

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origin from a generative hypha. C. Skeletal hyphae, the arrow shows the origin from a generative hypha (Jones et al., 2017).

Figure 8 Maria Lourdes in her uninsulated living space prior to the

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refurbishment (Critical Concrete - Summerschool 2018) Figure 9 Cutting straw into pieces of 5 to 10 cm (Critical Concrete -

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Summerschool 2018) Figure 10 Pasteurisation setup (Critical Concrete - Summerschool 2018)

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Figure 11 Weighing grain spawn while wearing latex gloves and regularly

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cleaning the metal workbench (Critical Concrete - Summerschool 2018) Figure 12 Squeezing the pasteurised substrate and transferring it to

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closed containers (Critical Concrete - Summerschool 2018) Figure 13 Filling the mould with the substrate (Critical Concrete Summerschool 2018)

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Figure 14 Mushrooms sprouting during the growth period (Critical

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Concrete - Summerschool 2018) Figure 15 Building an improvised rocket oven with loosely stacked bricks

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(Critical Concrete - Summerschool 2018) Figure 16 Discolouration of Methylene Blue by four fungal species

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Figure 17 Absorption spectra (400 - 800 nm) of the liquid culture media

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after 23 days the maximum absorption value of the dye was collected at a wavelength of 666nm Figure 18 Agar media with PE or PLA as the sole carbon source,

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inoculated by Ganoderma L. (left page) or Trametes V. (right page).

Clusters of polymer granules can be noticed after agar solidification. No visible growth two weeks post inoculation.

Figure 19 Agar media with PE or PLA as the sole carbon source, inoculated

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by Ganoderma L. (left page) or Trametes V. (right page). Mycelial growth on day 10 post inoculation.

Figure 20 Growth rate of Trametes V. measured over 10 days as surface

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colonised (%) of a 100mm diameter petri plate containing a 2% malt extract agar (MEA-), PLA- or PE-medium Figure 21 Growth rate of Ganoderma L. measured over 10 days as surface

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colonised (%) of a 100mm diameter petri plate containing a 2% malt extract agar (MEA-), PLA- or PE-medium Figure 22 Polyethtylene and PLA samples after fifty days of colonisation

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by Ganoderma L. (from the left column: LDPE on MEA, LDPE on lignin, HDPE on MEA, PLA on MEA, PLA on lignin, wood-PLA on MEA) Figure 23 Degradation percentage of five equiform LDPE samples after

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50 days of colonisation by Ganoderma L. on a 2% malt extract agar medium Figure 24 Scanning electron microscopy images of an LDPE sample prior

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to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom) Figure 25 Degradation percentage of five equiform HDPE samples after 50 days of colonisation by Ganoderma L. on a 2% malt extract agar medium

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Figure 26 Scanning electron microscopy images of an HDPE sample prior

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to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom) Figure 27 Degradation percentage of five equiform PLA samples after 50

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days of colonisation by Ganoderma L. on a 2% malt extract agar medium Figure 28 Scanning electron microscopy images of a PLA sample prior

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to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom) Figure 29 Degradation percentage of five equiform samples of a wood/

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PLA composite after 50 days of colonisation by Ganoderma L. on a 2% malt extract agar medium Figure 30 Scanning electron microscopy images of a wood/PLA sample

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prior to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom) Figure 31 Degradation percentage of five equiform LDPE samples after

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50 days of colonisation by Ganoderma L. on a lignin medium Figure 32 Degradation percentage of five equiform PLA samples after 50

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days of colonisation by Ganoderma L. on a lignin medium Figure 33 Malt extract media supplemented with a range of pigments

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Figure 34 Follow up every two days post inoculation of the pigmented

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growth media - bottom of petri dishes Figure 35 Follow up every two days post inoculation of the pigmented

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growth media - topview of petri dishes Figure 36 Follow up every two days post inoculation of the pigmented

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growth media - bottom of petri dishes Figure 37 Malt extract medium supplemented with PB29 and PR83

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displays the differing discolouration rate by Ganoderma L., undergoing a metamorphosis from purple to red and eventually white Figure 38 Processing HDPE granulates in a beechwood substrate

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Figure 39 Material samples of mycelium composites that contain HDPE

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granulates within the substrate matrix (left) or attached to the surface

Figure 40 LDPE-coated cardboard waste colonised by Ganoderma L.

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Figure 41 Shredded LDPE-coated cardboard (top); material sample of a

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mycelium composite with LDPE-coated cardboard as substrate (bottom) Figure 42 Prototype of a mycelium composite building block, grown on

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a substrate containing 300 disposable coffee cups and beechwood chips Figure 43 woodfibre-PLA cylindric mould with a wall thickness of 0,4 mm,

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two weeks post inoculation by Ganoderma L. Figure 44 standard PLA cylindric mould with a wall thickness of 0,4 mm,

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two weeks post inoculation by Ganoderma L. Figure 45 standard PLA cylindric mould with a wall thickness of 0,4 mm

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and a gradual filament inflow decrease along its height (from 100% at the bottom to 45% at the top. At a filament inflow of 60%, growth on the exterior of the mould can be observed.

Figure 46 standard PLA cylindric mould with a wall thickness of 0,4 mm

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and a filament inflow of 60%, two weeks post inoculation by Ganoderma L. Figure 47 standard PLA mould that explores the possible convergence

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of digital and biological manufacturing, two weeks post inoculation by Ganoderma L. (no established growth due to contamination).

Figure 48 Wall segment resulting from the protocol described on page

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89-100 at the day of unmoulding the building blocks Figure 49 Wall segment resulting from the protocol described on page 89-100, one week after unmoulding the building blocks

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Table of contents

INTRODUCTION.......................................................................................

1 “IT’S ONLY ONE STRAW,” SAID 8 BILLION PEOPLE...................................

1.1 Problem statement: the plastic waste management system....................

1.2 Towards a new plastic waste management system................................

2 OBJECTIVES AND DELIMITATIONS...........................................................

3 A THUMBNAIL OF WHAT’S AHEAD: RESEARCH APPROACH.......................

4 MYCELIUM COMPOSITES AND MATERIAL-MINDFULNESS: CRASH COURSE 4.1 Production process of mycelium-composites in the lab.........................

4.1.1 Strain selection: an introduction to fungi............................................

4.1.2 Substrate selection..........................................................................

4.1.3 Production protocol.........................................................................

4.2 Production process of mycelium-composites in a DIY-environment.........

4.2.1 Production protocol.........................................................................

4.2.2 Scouting for local substrate alternatives..............................................

4.3 Material mindfulness - a critical reflection...........................................

5 PRELIMINARY TESTING AND STRAIN SELECTION.....................................

5.1 Bioremediation potential: discolouration of methylene blue..................

5.2 Plastic as sole carbon source............................................................

5.3 Strain selection...............................................................................

6 DEGRADATION OF PLASTICS....................................................................

6.1 50 days of degradation...................................................................

6.1.1 Low-density polyethylene - LDPE........................................................

6.1.2 High-density polyethylene - HDPE.....................................................

6.1.3 Standard PLA filament.....................................................................

6.1.4 Composite wood-PLA filament..........................................................

6.2 Lignin as a catalyst...........................................................................

6.3 Optimisation of the degradation rate..................................................

6.4 Polymer pigment degradation - a study of colour.................................

7 DEGRADATION MATERIALISED: SMALL SCALE PROTOTYPING...................

7.1 Plastic waste as a substrate...............................................................

7.1.1 HDPE granulates.............................................................................

7.1.2 LDPE-coated cardboard as a substrate................................................

7.1.3 PLA as biodegradable mould for the production of mycelium-composites 8 PRACTICE WHAT YOU PREACH.................................................................

9 CONCLUSION..........................................................................................

10 ANNEXES................................................................................................

11 BIBLIOGRAPHY........................................................................................

Table of contents

Word of gratitude Abstract List of tables List of figures Table of contents

1 7 7 9 11 13 17 17 18 19 20 22 24 31 32 33 34 38 45 47 48 53 55 57 59 60 61 63 71 72 72 76 80 86 104 109 113

Introduction

Scientists, fungi and a whole lot of trash talk in the new construction paradigm INTRODUCTION As we turn a blind eye to the unsustainable nature of today’s building practices, cities thrive and urbanisation sparks growth of urban tissue at unprecedented rates.

Like Ziya Tong puts it: in the twentyfirst century, there are cameras everywhere, except where our food comes from, where our energy comes from, and where our waste goes (Tong, 2019). We are living and producing at a pace that is too demanding for the earth’s metabolism to keep track. Unfortunately, the ecosystem does not offer discounts for negligence. For whilst we trade critical self-reflection of established practices for the glorification of a fast-paced lifestyle, natural ecological systems suffer great losses.

Current business-as-usual practices in the building sector are characterised by a linear use and disposal of materials, making that the building industry alone is responsible for 35% of the total waste generation in the European Union and 40% of the global energy usage (Osmani et al., 2019; European Commission, 2010)

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A raised awareness of the depletion of non-regenerative material sources and the production of non-renewable composites with hardly any end-of-life possibilities, have made it a necessity to take a step back and reassess our current building traditions. To do so, it is an imperative to briefly reflect upon where we come from as an industry and how we got here.

Before the coming of the Industrial Revolution, which imposed the rigors of manufacturing and mass production onto the world of design and architecture, the dialogue between material and architect had been rather straightforward.

The master builder, who assumed the role of architect, engineer, scientist and manufacturer; established an understanding of materials and their properties through an empirical process of observation and documentation. Of trial and error (Addington et al., 2005).

Generating knowledge, experience and an overall affinity with the material, implied that the design process and the creation of architecture inevitably became

part of a very integrated approach.

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However with globalisation, mass production and standardisation of materials taking the wheel, the course of architecture would be steered in a drastically different direction. While resource allocation used to be somewhat inherently regulated by local availability and thus directly reflected the demand of the local population - given that the master builders of the pre-industrial era would have no other choice than to work within the restrictions of transport and scale -, the architects of today are not confronted with such limitations. Today however, the global use of resources adds up to ninety gigatons of raw material per year, which is being transported all over the globe (OECD, 2019). And as modern economy requires constant and indefinite growth in order to survive, standardisation and mass production have made it incredibly difficult to hit the brakes.

Technological advancements and a growing dependency on digital representational tools since the mideighties have thereby completely shifted the relationship between medium and designer (Addington et al., 2005).

One could say that in the digital age, a proverbial firewall between the architect and the manufacturing process has emerged: the design process does no longer necessarily rely on a developed sensibility for handling materials, neither does it depend on pushing material innovation. The 20th century separation of the disciplines has on the one hand brought an end to the romantic vision of the master builder, a homo universalis able to one-handedly push the boundaries of material innovation, to design and

construct his creations. Nevertheless, it has allowed for incredible accomplishments and rapid knowledge acquisition across different fields, giving way to innovative practices combining cross-disciplinary expertise.

Facing a crucial paradigm shift in the way we build and manufacture building components, it is now more than ever the architect’s responsibility to put up for question how we approach architecture; to establish an open conversation that surpasses the separation of academic disciplines and to give voice to new developments. In the up-and-coming pursuit of a circular approach to compete with the tradition of linear resource extraction and disposal for the production of building components, the introduction of biological principles as inoculation, growth and decay have come to the fore. Through a handful of realisations in the field of architecture (Figure 1) and design (Figure 2, Figure 3) that recognise the need for material innovation, fungal mycelium has proven itself as a promising candidate for the cultivation of materials and objects. Through biological growth rather than energy demanding manufacturing processes, organic waste streams (straw, flax, sawdust, etc.) can be resorted to as a valuable resource to produce a self-assembled biomaterial that can eventually return to the earth’s carbon cycle at the end of its useful lifespan (Elsacker et al., 2019).

The research ahead picks up on the existing knowledge and experience regarding mycelium composites in architecture and attempts to introduce new developments from the field of microbiology, which have shown that

Introduction

Figure 1 Hyfi Tower by The Living (2014): a cluster of 13-meter-tall towers built using biodegradable bricks made from agricultural byproducts and mycelium

Figure 2 mycelium + timber by Sebastian Cox and Ninela Ivanova (2017): furniture collection made from mycelium and wood

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Figure 3 Mycelium Ware by craft combine (2019): homeware collection made from agricultural byproducts and mycelium

fungi are not only capable of feeding on organic substrates but might also have the capacity to decompose synthetic compounds.

Plastics.

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Could we valourise the most renowned waste stream of the Anthropocene Epoch and transform it into biomass, for the production of a carbon negative construction material? A concept that could restructure not just our waste management system, but also our approach to resource allocation in architecture and our very way of designing. The capacity of materials to change over time, to wear and deteriorate is seen as something to be avoided in architecture, and rarely perceived as a design opportunity. However, in the pursuit of a circular metabolism in architecture, degradation should come to the fore as an intrinsic design parameter, allowing for the exploration of design by degradation or degradation by design.

Through an explorative process this microbiological research which has not yet found its way to practical applications will be tested both for its feasibility as for its design opportunities. Given that working with living organisms or navigating on the intersection between architecture and microbiology is all but standard in an academic discourse in architectural engineering, the used approach is reminiscent of the spirit of the master builders and aims to stimulate DIY biofabrication as an accessible and ecological action. After a hands-on crash

course in the DIY production of mycelium composites, the research will be taken to the lab. Are the fungal strains used in an architectural framework capable of the same bioremediating properties of strains analysed in microbiological literature? At what rate could we decompose plastic waste with these strains? An empirical process guided by prototyping will provide a better understanding of the working principles and allow for a practical exploration of the architectural potential regarding form, aesthetic, structural relevance and temporality. These findings will then pave the way for a design-andbuild project that showcases the potential of the material: a living wall segment expressing the sustainable ideology of a closed-loop system overarching multiple industries.

Introduction 5

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1.1

Problem Statement

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“It’s only one straw,” said 8 billion people

PROBLEM STATEMENT : THE PLASTIC WASTE MANAGEMENT SYSTEM

Out of sight, out of mind. An aphorism our current waste management system dictates to live by as 99% of consumer goods are trashed within six months after purchase (Hawken et al., 1999). A sheer volume of waste that poses a major threat to the environment when considered to be the dead-end result of a linear production process - an issue that clearly surpasses the confinement of industries. Especially when man develops materials characterised by their inertness and intentionally slow degradation process, such as plastics, this results in an indivertible accumulation of waste. The most widely produced plastics contributing to the current buildup of waste are respectively low-density polyethylene (LDPE), high-density polyethylene (HDPE), polyvinyl chloride (PVC), polystyrene (PS) and polypropylene (PP) (Pathak et al., 2017).

While in 1950 the global production of plastic polymers comprised 1.5 million tons, plastic demand and the ease of manufacturing have increased enormously since. Today, an amount of approximately

381 million tons is produced on a yearly basis and is still increasing, yet most of it retires at an early age with a single ticket to the landfill (Geyer et al., 2015).

The current disposal of plastic waste knows three approaches: storing in landfills, incineration and recycling. The latter involves categorizing and sorting plastic waste into different types, washing, drying, grinding and finally reprocessing the material; making recycling an expensive process (da Luz et al., 2013).

In other words, current economic systems determine that producing new plastic is cheaper than recycling used plastic.

Hence, dictated by a competitive market system, polymer product designers typically choose not to consider what happens after the lifespan of their products but instead opt for the production of new material. Choices that sustain our fast way of living and producing which we have gladly adopted as the reality we live in, but still choices we make as a human collective.

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As such, the current lie of the land shows that approximately only 9% of generated plastic waste is being recycled and that the remaining two approaches are highly favoured; 12% is being incinerated and 79% gets accumulated in landfills or the natural environment (UN Environment report, 2018). Given the constant growth of population going hand in hand with an increasing waste production and the engineered resistance of plastics to degradation, storing plastic waste in landfills only postpones the consequences of our inactions to future generations. In landfill-conditions plastics are thereby reported to partially anaerobically degrade at very slow rates, causing the emission of large volumes of toxic gasses which induce infertility of soil, prevent the degradation of otherwise harmless substances and endanger natural eco-systems (Ebnesajjad, 2002; Ohja et al., 2016). Incineration on the other hand, implies the immediate release of polyaromatic hydrocarbons, carbon dioxide, carbon monoxide, hydrogen chloride, hydrogen cyanide and phosgene into the environment (Peacock, 2000).

Environmental concerns associated with plastics have been around for a while and are ever emerging, leading to some measures such as bans on single use plastic bags, that have been introduced and are now in effect in 74 countries (Nielsen et al., 2019). And already since the 1980s, the development and research of biodegradable plastics has gained interest and has led to the successful commercial development of some biodegradable plastics such as polyhydroxyalkanoates (PHA) and polylactic acid (PLA) which are likely to replace currently used plastics at least in

Figure 4 Discarded plastic bottles and other garbage blocks the Vacha Dam, near the Bulgarian town of Krichim, on April 25, 2009. Single-use plastic containers like bottles and plastic bags are “the biggest source of trash� found near waterways and beaches, according to the nonprofit Ocean Conservancy.

some fields. These new alternatives alternatives opened the way for new considerations of waste management strategies since these materials are designed to degrade under environmental conditions or in municipal and industrial biological waste treatment facilities (Shah et al., 2008). However, none of them are efficiently biodegradable in landfills and a study of 2017 indicated that at 25 °C in seawater, PLA showed no degradation over the span of a year (Bagheri et al., 2017). This confirms that while researching new alternatives and slowing down the plastic waste flow at its source is from the utmost importance, we also need to improve the way we manage our plastic waste.

TOWARDS A NEW PLASTIC WASTE MANAGEMENT SYSTEM

Worldwide research for the last three decades has focused on the rapid biodegradation of plastics by natural decomposers, which is the only ecofriendly process that can solve the problems faced by current plastic waste management methods (Ojha et al., 2017; Shimao, 2001). As a result, numerous studies have shown that some heterotrophic microorganisms, such as bacteria and fungi, which play a significant role in the processing of natural polymer compounds also show significant potential to biologically degrade man-made polymers as a source of carbon to sustain their growth (Shah et al., 2008; Bhardwaj et al., 2012; Restrepo-Florez et al., 2014; Pathak et al., 2017). This biodegradation process by microorganisms takes place in a sequence of steps and is only possible due to the microorganism’s capacity to synthesise specific extracellular oxidative enzymes

“Wood-decaying fungi are to

major the

contributors

earth’s

carbon

cycle and can be seen as the most promising decomposers for tackling the plastic pollution issue” Hassinger (2018)

that facilitate polymer chain cleavage (Shah et al., 2008; Pathak et al., 2017).

The by-products of biodegradation (hummus, carbon dioxide and water) are eco-friendly and are able to be readily assimilated by living organisms (Premraj et al., 2005).

In a review on the current status of polymer degradation, Pathak et al. (2017) catalogues all reported microorganisms associated with the biological degradation of different plastic types (Annex 10.1).

Among the list is a whole range of endophytic fungi such as Pestalotiopsis Microspora, belonging to the taxonomic rank of Ascomycota, which was experimentally demonstrated by Russell et al. (2011) to be capable of decomposing polyester polyurethane (PUR). Most of the focus has been on Ascomycota biodegrading either polyurethane or polyethylene, but a study of 1992 (Milstein et al., 1992) and a more recent study of Hassinger (2018) which were not included in the catalogue also show that fungal strains of the Basidiomycota phylum were capable of biodegrading a range of plastic types. Hassinger (2018) furthermore states that wood-decaying fungi are major contributors to the earth’s carbon cycle and can be seen as the most promising decomposers for tackling the plastic pollution issue, given that these fungi are the only fungi capable of breaking down lignin; a highly complex natural polymer that varies in size and arrangement depending on plant species.

Problem Statement

1.2

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30 years of published microbiological papers promoting the bioremediation of plastics as a viable alternative for landfilling and incinerating should have been a clarion call for change – yet today these are still the most economically viable ways of discarding plastics.

What’s the price of money, right?

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Hassinger (2018) therefore proposes the use of Basidiomycota such as Pleurotus Ostreatus as a more practical way of implementing the bioremediation of plastics, since these fungi are already being commercially cultivated for human consumption. Instead of wood-based resources to grow edible fungi, plastic waste could be used to grow them instead. In like manner, Basidiomycota are currently resorted to for the production of mycelium composites in an architectural and design framework. Hence, the question emerges whether mycelium composites could valourise plastic waste and inject the bioremediation process of plastics into an economically viable material for an industry in dire need of sustainable material innovation.

Objectives and Delimitations

The master thesis aims to investigate the architectural implementation of the biological degradation process of plastic waste by studying plastic as a substrate for the production of mycelium composites through a practical and experiment-driven approach. As such, a stepping stone for theoretical findings in the field of microbiology towards a practical application in architecture and design is pursued. The intent of this research is not to produce new findings in the field of microbiology (I will gladly leave that up to experts in the field). However based on literature research of established microbiological protocols, plastic degradation is pursued by means of simple test setups for a targeted selection of fungal strains that are relevant for the production of mycelium composites.

Hereby, the analysed plastic types will be limited to polyethylene (PE), the most widely used and produced plastic, and polylactic acid (PLA), the most applied material as filament for 3D-printing; which will make for an interesting comparison between the bioremediation of a fossil-based plastic and a bioplastic. The objectives of the master thesis are summarised below in the form of a set of research questions: RQ1: Do the fungal species that are used for the production of mycelium composites have bioremediation potential? If so, how could we select a fungal species that is most suited for the task.

RQ2: What are the rates of plastic degradation that can be achieved in a simple test setup? What rates of degradation are documented in literature?

RQ3: How could the bioremediation of plastic waste be injected in the production process of mycelium composites? What design opportunities and limitations does this entail?

This research does not offer a finished product, nor should it be read as a manifesto with a clear conclusion of how architecture should evolve from its current shortcomings in sustainability. Rather, it serves as an inspiring attempt of a holistic approach towards material innovation, an exploration of new alternatives to put our current business-asusual practices up for question and an open call for other disciplines to join the debate.

Objectives and Delimitations

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Figure 5 Mycelium research at the production centre of Critical Concrete in Porto (Critical Concrete - Summerschool 2018)

Research approach

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A thumbnail of what’s ahead RESEARCH APPROACH

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Finding ourselves at the intersection of architecture and biology, the research ahead is inevitably twofold. Can plastic waste serve as a viable substrate for the production of mycelium composites? If so, what design opportunities does this entail? To formulate an answer to these questions, both practical experience with the manufacturing of mycelium composites and a theoretical backbone in

microbiological principles are required.

Given its very practical objective, the master thesis kicks off with a brief literature study on the production methods of mycelium composites parallel to gaining direct hands-on experience with the material. To do so, I started by joining a one month design-and-build project at Critical Concrete, a collective that emerged from a dissensus with the

prevailing building industry practice and now follows its own understanding of sustainable architecture: long lasting and easily repairable structures, made of locally sourced materials and upcycled trash. Here, I was given the opportunity to be actively involved in their pragmatic research on creating mycelium insulation panels from local agricultural waste streams which showed the potential of mycelium to establish a local, communitybased approach to cultivating materials at a human-scale, not at global industrial scale.

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With a know-how of the DIY-production of mycelium composites for architectural purpose, a new perspective on academic research regarding mycoremediation of plastic waste emerges. Guided by extensive literature analysis, the possibility to combine both is then put up for experimental testing. In this stage, experiments are conducted under laboratory conditions at VUB (Research Group of Microbiology MICR), where accurate measurements are possible. A preliminary investigation of the bioremediating potential of three architecturally relevant fungal species, Pleurotus Ostreatus, Trametes Versicolor and Ganoderma Lucidum, will allow for the selection of one. This microorganism will be subjected to further analysis in which the degradation process of plastic waste streams will be quantified and visualised.

From here, the thesis will focus on how to put theory into practice; taking the findings from the experimentation stage and using them in small-scale prototyping. What design opportunities

does the bioremediation of plastic waste bring to the designer and architect? The production of prototypes that explore this query will provide insight on how plastic remediation could be applied on a bigger scale. However, tangible insight is only taught by real-world execution and the encounter of practical complications.

Hence, the master thesis concludes with a modest design-and-build project; wrapping up in the same fashion it was kicked off.

Research approach 15

16 Figure 6 Autoclave setup at MICR laboratory - Vrije Universiteit Brussel

Crash course

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Mycelium-composites & Material-mindfulness A CRASH COURSE

The production of mycelium composites is a process that is still in search of standardisation and commercialisation. As such, a multitude of academics but also self-proclaimed biohackers have developed a range of experimental approaches and non-standardized procedures, resulting in a very extensive but fragmented knowledgeavailability (Elsacker et al., 2020). A significant foundation in the pursuit of establishing a comprehensive framework for the production process of mycelium composites has nonetheless been laid out by Elsacker et al. (2020). This work will provide the basis for an elementary understanding of fungi and the growing principles of fungal mycelium which will encourage the learning of current production techniques. The theoretical introduction of laboratory manufacturing methods will be followed by a hands-on immersion in the DIY production process at Critical Concrete, encouraging a perspective on both the global and local pursuit of change.

4.1

PRODUCTION PROCESS OF MYCELIUM-COMPOSITES IN THE LAB

Mycelium composites consist of a three-dimensional network of fungal mycelium grown on organic substrate that provides nutrients to the fungus. Serving as a natural glue, the root structure of the fungus binds the feedstock as if it were reinforcement fibres to form a lightweight, cultivated composite. A broad variety of fungi and substrates can be resorted to in the production phase, directly influencing the properties of the resulting material. Hence the importance of the selection of a suitable strain and substrate as an initial step of the production.

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18

STRAIN SELECTION : AN INTRODUCTION TO FUNGI

Though invisible to the eye when not fruiting above the surface in the form of mushrooms, fungi serve an indispensable role in the ecosystem as primary decomposers of dead organic matter.

Through the excretion of digestive enzymes in their environment, they are capable of processing complex organic compounds into soluble nutrients for other organisms.

Germinating spores produce filamentous cells with distinct chitin-based cell walls and thread-like branching structure, the hyphae. Hyphal growth serving as its mode of mobility, the organism explores a substrate for carbon and nitrogen-based nutrients, forming an interwoven network of hyphae that becomes an integral part of its substrate: mycelium. The fungal kingdom is largely unexplored and out of an estimated 2,2 - 3,8 millions of species only 36, all of the Basydiomycota phylum, have been mentioned for the application in mycelium composites (Elsacker et al., 2020). Hyphal architecture, growth rate and the ability to degrade lignin seem to be the performance predictors assisting in species selection, greatly influencing the density and complexity of the mycelium network (Jones et al., 2018; Elsacker et al., 2020).

Hyphal architecture as a first parameter, divides fungal species according to the number of hyphal types it exhibits, described by using the mitic system.

Three principal hyphal systems can be distinguished: generative hyphae, binding hyphae and skeletal hyphae, which are respectively very sparsely branched, moderately branched and highly branched. The number of hyphal types present in a species categorises

them as monomitic (only generative hyphae), dimitic (comprising two hyphal types) or timitic (comprising all three hyphal types). Due to the complexity of its structure, the latter category shows advanced performance for the production of mycelium composites since the direct correlation with its mechanical properties (Webster et al., 2007).

Figure 7 A. Generative hyphae. B. Binding hyphae, the arrow shows the origin from a generative hypha. C. Skeletal hyphae, the arrow shows the origin from a generative hypha (Jones et al., 2017).

4.1.1

The capacity to degrade lignin on the other hand serves as another important parameter for the selection of a fungal strain, allowing to effectively glue lignocellulosic fibres. A trait that is specific for wood-decaying species, which are categorised as white, brown or soft rot fungi along their effectiveness of wood decay by secreting ligninolytic enzymes. White rot fungi are most applied in mycelium composites as they are capable of complete lignin degradation and hence demonstrate good substrate colonisation (Elsacker et al.). Lignin being a highly complex natural polymer, these

In the research ahead, the selection of fungal strains will hence be limited to three trimitic white-rot Basidiomycetes, already extensively used for the production of mycelium composites: Trametes Versicolor (Jones et al., 2017; Lelivelt et al., 2015), Pleurotus Ostreatus (Haneef et al., 2017; Lelivelt et al., 2015; Moser et al., 2017; He et al., 2014) and Ganoderma Lucidum (Haneef et al., 2017; Holt et al., 2012; Travaglini et al., 2016) (Elsacker et al., 2020). The applicability of other fungal strains or targeted genetic modification of strains is however a promising subject for further investigation.

4.1.2

Crash course

wood-decaying fungi are also the most promising decomposers of man-made polymers (Russell et al., 2011).

SUBSTRATE SELECTION

Mycelium composites are typically cultivated from lignocellulosic biomass, the most abundantly available raw material on earth. Lignocellulose, which comprises cellulose, hemicellulose and lignin is the main structural component that provides strength and stability to plant cell walls, making most agricultural waste streams a suitable substrate for production (Van Wylick, 2018; Elsacker et al., 2020). The substrate choice for mycelium-based composites has a direct implication on the material properties. For example, the compressive strength of a Ganoderma based mycelium composite with a cotton plant filler for example, reaches values up to 72 kPa while red oak fibres create a material with a compressive strength of 490 kPa (Jones et al., 2017); which can be attributed to the relative proportions of cellulose, hemicellulose and lignin relative to one another, distinct for each type of vegetation. The supplementation of additives such as sand, gravel, silica or glycerine in the substrate can moreover highly influence growth rates of the fungus and material properties such as compressive strength and flammability of the final product (Jones et al., 2017; Elsacker et al., 2020).

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4.1.3

PRODUCTION PROTOCOL

A seven-step protocol for the production of mycelium composites in laboratory conditions was set up by Elsacker et al. (2020) on the basis of a wide range of open source manuals, research papers and patents:

1

The mycelium is initially grown on agar plates, ingrain-substrate, in a liquid nutrient solution, or in the pre-grown homogenised substrate;

2

The substrate is autoclaved (Figure 6) or pasteurised to eliminate any type of already present microorganisms on the substrate and thereby preventing contamination during the growth and incubation process;

3

A specific amount of the mycelium tissue is added to the substrate. If the substrate was not humidified before autoclaving (step 2) an amount of sterile water is added. To improve growth, a sterile solution of nutrients can also be added;

4

The mycelium grows through the substrate in a controlled environment. The material can be grown in two phases, first in a mould to bind the fibres, and secondly outside the mould to solidify the outer skin of the material during a period;

5

The inoculated substrate is hand-packed in a sterilised mould which has the desired shape. The mould is sealed with a filtered air-permeable cover to maintain a micro-climate;

6

The grown material is heat-treated at a specific temperature for several hours to end the growth process and dehydrate the material;

7

A coating or post-processing can be applied to the material to improve its properties.

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Crash course 21

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4.2

PRODUCTION PROCESS OF MYCELIUM-COMPOSITES IN A DIY-ENVIRONMENT

Despite the added value of the regulated conditions in a laboratory environment, ensuring low risk of contamination and optimal growing conditions; the low-cost and high accessibility of biofabrication allows the production of mycelium composites to be taken up by anyone disposing over a minimal equipment and an ecological mindset. In the context of a design-and-build project in the Islas of Porto, I had the opportunity to actively participate in the pragmatic research undertaken by Critical Concrete focussing on the social and ecological potential of mycelium composites production on a local, community-based scale using available resources and working within financial constraints, not at global industrial scale. With the objective of refurbishing the poor living conditions of a family in financial need, where overheating and coldness turned out to be one of the biggest issues of housing and health, a DIY protocol for the production of cheap, performant and ecological mycelium insulation panels was established (Figure 8).

Following a workshop and keynote lecture by Maurizio Montalti (Officina Corpuscoli/Mogu), 27 mycelium insulation panels were produced from straw parallel to the experimental investigation of a range of locally available substrates for future projects.

Crash course 23

Figure 8 Maria Lourdes in her uninsulated living space prior to the refurbishment (Critical Concrete - Summerschool 2018)

4.2.1

PRODUCTION PROTOCOL

Resources for the production of mycelium composites were obtained from a local mushroom farmer who provided a large amount of straw and a mother culture of Pleurotus Ostreatus. Answering to available equipment and constricted financial limits, a seven-step protocol for the production of mycelium composites in DIY conditions was set up by Critical Concrete (2019):

24

Figure 9 Cutting straw into pieces of 5 to 10 cm (Critical Concrete - Summerschool 2018)

Preparation of the substrate: The substrate (i.e. straw) is cut into pieces of 5 to 10 cm (Figure 9) and supplemented by 10% of the wet substrate weight of an additive of choice (i.e. flour), which will enhance the initial growth rate. Ensuing, the substrate is sterilised by means of pasteurisation (Figure 10), meaning that batch by batch the substrate is boiled in a large pot of water for one and a half hour.

Crash course

1

25

Figure 10 Pasteurisation setup (Critical Concrete Summerschool 2018)

26

Figure 11 Weighing grain spawn while wearing latex gloves and regularly cleaning the metal workbench (Critical Concrete - Summerschool 2018)

Preparation of a sterile workbench and mould: post-pasteurisation working conditions need to be as clean as possible to prevent contamination during the growth period. Hence, in the following steps reusable latex gloves are worn and the gloves, tools and surfaces in contact with the substrate will be regularly cleaned with 70% alcohol. As such, a metal workbench and a mould (here: an aluminium window frame) is sterilised while the substrate is pasteurised (Figure 11).

3

Cooling and squeezing: The substrate is taken out of the pot with sterilised spoons and spread over the workbench. During pasteurisation, the substrate has absorbed a large amount of water. In order to provide suitable growing conditions, the water content and temperature of the straw need to be reduced. The substrate is therefore squeezed and then transferred to sterile closed boxes where it can cool down until reaching room temperature (Figure 12).

Crash course

2

27

Figure 12 Squeezing the pasteurised substrate and transferring it to closed containers (Critical Concrete - Summerschool 2018)

28

4

Inoculation: After sterilising the mould, a homogeneous distribution of substrate and grain spawn is achieved through a process of layering and manual mixing: half of the substrate and half of the quantity of spawn is put into the mould, mixed, then the other half of the substrate and spawn is added and mixed again (Figure 13). The mixture is pressed by hand to obtain a higher density, after which the mould is sealed with a plastic film.

During its growth the fungus needs oxygen and releases CO2; hence a couple of holes are made in the plastic film with a sterile needle.

5

Growing period: the panels are now left to grow in the dark.

Growth rates are variable given the unattainability of a constant temperature and relative humidity in a DIY environment. The panels were exposed to temperature varying between 20°C and 28°C during the day, falling between 15 and 20°C during the night. Close monitoring during the growth period is therefore recommendable, adding blankets to increase the temperature when necessary. When the top layer of the panel has developed a thick white skin, the panel is removed from the mould, flipped and left in the dark for a second growth period (Figure 14).

Figure 13 Filling the mould with the substrate (Critical Concrete - Summerschool 2018)

Figure 14 Mushrooms sprouting during the growth period (Critical Concrete Summerschool 2018)

Crash course 29

Figure 15 Building an improvised rocket oven with loosely stacked bricks (Critical Concrete Summerschool 2018)

30

6

Terminating the growth: After full colonisation of the substrate and a visible formation of a thick white skin wrapping the entire panel, the panel is air-dried in the sun with a temperature varying between 18°C and 28°C for three days.

The microorganism is then killed, ensuring a stable end product, by heating the panel at 80°C until completely dehumidified (one hour for a panel with a thickness of 5cm). To do so, an improvised outdoor rocket oven was constructed using loose bricks and two metal plates (Figure 15).

7

Post-processing: the panel is now finished and has a distinct texture, that depends on the type of substrate and mushroom that is used, which can be left untreated but can also be covered to form a sandwich wall with wood panels, or can be painted with a natural and transpiring paint.

SCOUTING FOR LOCAL SUBSTRATE ALLTERNATIVES

Parallel to the production of mycelium insulation panels, we set up an experiment to investigate locally available resources that could serve as substrates and additives for the production of mycelium composites. Following the DIY protocol established in chapter 4.2.1, a total of nineteen bricks were produced respecting the same proportions of substrate, spawn and additives. Cotton, cardboard, sawdust, cork, coffee ground, line fibre and straw by means of a reference substrate were collected from waste streams in the vicinity of the work centre and were mixed with additives such as calcium, gypsum, grape seeds, corn starch, sugar and cat food. The growth period was closely monitored and visualised in annex 10.2.

All resources up for investigation showed to be effective substrates for the production of mycelium composites, reaching a full colonisation after a period of 19 days on average.

Cardboard seemed to be the most favourable substrate, reaching full colonisation after only 10 days in a mix with corn starch or after 14 days when supplemented with sugar or calcium and gypsum. The supplement of growth rate enhancing additives such as grape seed, corn starch, sugar or cat food seemed to highly increase the risk of contamination for DIY production methods. This was to be expected as they enhance the initial growth rate of the fungus by providing fast, easily soluble nutrients to the microorganism, however also allowing unfavourable moulds and bacteria that are not necessarily capable of producing ligninolytic enzymes to use the mixture as a breeding ground. Calcium or gypsum, additives that are resorted to for optimising the pH of the substrate, therefore do not show this phenomenon.

Crash course

4.2.2

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4.3

MATERIAL MINDFULNESS - A CRITICAL REFLECTION

With the production process of mycelium composites in a regulated laboratory setting as an indispensable knowledge-foundation, a direct involvement in the manufacturing and application of the material in a small-scale, do-it-yourself environment offers new insight. The design-and-build project at Critical Concrete questions what architecture can be and puts decentralised biofabrication forward as a promising alternative for environmentally harmful business-as-usual practices. A project that harnesses the belief that a direct social and ecological impact on the local level might serve as a catalyst for change on a global and industrial level.

32

It is however important to remain very critical when putting forward innovative approaches and a material mindfulness should be employed throughout the whole production process. Hands-on experience learns that growing mycelium requires rigour on cleanliness. Metal and polypropylene, a polymer that is extremely resistant for biological attack, are easily sterilised materials and therefore meticulously resorted to as moulds for mycelium production. While during the project at Critical Concrete a sustainable solution was found by using an aluminium window frame, production in the lab typically resorts to polypropylene containers and growth-bags.

By first-handed screening of waste-streams for locally available substrate alternatives and testing their feasibility for manufacturing mycelium composites, a straightforward dialogue between the architect and material is set up that allows for an unambiguous development of a circular system. Cardboard came to the fore as a promising substrate, opening doors towards local management of cardboard waste in an architectural framework. In the following chapters we investigate whether the same could be achieved with plastics, which would facilitate the management of plastic waste streams by resorting to them as a valuable resource and could allow for investigating a symbiotic relationship between the material and the mould it is created in, making the production process fully sustainable.

Knowledge without know-how is sterile. We use the word academic in a pejorative sense to identify this limitation.

Myron Tribus

Preliminary Experiments

5 Preliminary testing and strain selection PRELIMINARY EXPERIMENTS With a know-how of the DIY-production of mycelium composites for architectural purpose, a new perspective on academic research regarding mycoremediation of plastic waste emerges. Guided by extensive literature analysis, the possibility to combine both is put up for experimental testing. In this stage, experiments are conducted under laboratory conditions where accurate measurements are possible.

As mentioned in chapter 4, fungi classified in the phylum Basidiomycota with a trimitic hyphal architecture and capable of lignin degradation are favoured for manufacturing mycelium-based composites. Wood-decaying fungi are thereby also mentioned as the most promising decomposers of man-made polymers by Russell et al. (2011). Hence, a targeted selection of three trimitic white-rot Basidiomycetes, already extensively used for the production of mycelium composites: Trametes Versicolor (Jones et al., 2017; Lelivelt et al., 2015), Pleurotus Ostreatus (Haneef et al., 2017; Lelivelt et al., 2015; Moser et al., 2017; He et al., 2014) and Ganoderma Lucidum (Haneef et al., 2017; Holt et al., 2012; Travaglini et al., 2016) (Elsacker et al., 2020) will now undergo a preliminary screening for their bioremediating capacity, leading up to the selection of one fungal strain for further analysis and prototyping.

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5.1

BIOREMEDIATION POTENTIAL: DISCOLOURATION OF METHYLENE BLUE IN LIQUID MEDIUM

The use of synthetic dyes as a method to screen microorganisms for their bioremediation potential is an efficient method, that allows both visual and quantitative comparison (Perovano Filho et al., 2011; Menezes et al., 2017). In the experiment at hand we will therefore cultivate three fungal strains suitable for the production of mycelium composites: Pleurotus Ostreatus (Pl.O.), Trametes Versicolor (Tr.V.) and Ganoderma Lucidum (G.L.) side by side with Pestalotiopsis Microspora (P.M.); a fungal strain of the phylum Ascomycota, recorded to have the ability to grow on polyurethane (PUR) as the sole carbon source by producing the enzyme serine hydrolase under both aerobic and anaerobic conditions (Russell et al., 2011). By monitoring the growth of the selected strains in a liquid culture medium containing a synthetic dye, such as methylene blue, we will be able to establish a first indication of each strain’s capacity to degrade plastics.

34

Methylene blue is a heterocyclic aromatic compound that colours deep blue when dissolved in water. Hence, the solution will visually display a degree of discolouration when the chemical compound is degraded. At this point, methylene blue is serving as an indicator for the synthesis of extracellular oxidative enzymes by the growing fungus.

These enzymes show the potential of the fungal isolate to adapt its metabolism to alternative carbon sources by breaking down stable C - C bonds. A feature that might indicate the fungus’ capacity to use synthetic polymers as a carbon source to sustain its growth (Menezes et al., 2017).

Material prep_ Pestalotiopsis Microspora Pleurotus Ostreatus Trametes Versicolor Ganoderma Lucidum Methylene blue 50 Malt extract 17 Distilled water 1

mg/L g/L L

35

The medium is now sterilised by autoclaving at 121°C and 15psi for twenty minutes.

Transfer the flask to a laminar flow cabinet where it can cool down until reaching room temperature. Eight Erlenmeyer flasks are now filled with 100 mL of the prepared medium, in order to produce a duplicate of the experiment for each fungal isolate. A ninth is filled for reference.

Beside the reference medium, each Erlenmeyer flask is then inoculated by adding 5 cm2 of fungal mycelium, which is extracted from the pregrown culture plates. Aluminium foil is used to cover the flasks before removing them from the laminar flow cabinet and maintain a sterile environment throughout the growth period. The reference medium

Figure 16 Discolouration of Methylene Blue by four fungal species Reference sample

Pestalotiopsis M.

Pleurotus O.

Preliminary Experiments

Method_ Following protocols established by Kundjadia et al.

(2016) and Menezes et al. (2017), the experiment is set up for the analysis of Pestalotiopsis M., Pleurotus O., Trametes V. and Ganoderma L. Prior to cultivation in liquid medium containing methylene blue, the four strains are pregrown on a 2% malt agar plate for a week.

Ensuing, a liquid malt extract-based culture medium is prepared in a flask by adding 17 g of malt extract to 1 L of distilled water. After supplementing the medium with 50 mg of methylene blue, the flask is sealed with aluminium foil and placed in an autoclave. Note that in this step the initial concentration of methylene blue is determined as 50 mg/L, which will later on serve as the point of reference to determine the degradation percentage.

Ganoderma L.

Trametes V.

is kept in a cold room to prevent any growth of possible contamination. In a shaking incubator, the cultures are now left to grow for a period of 23 days at 150 rotations per minute and a constant temperature of 26°C.

Results and discussion_ After 23 days of growth, a first visual inspection of the flasks indicates that all tested fungal strains were able to induce a certain degree of discolouration in the medium (Figure 16). Therefore it can be stated that Pestalotiopsis M., Pleurotus O., Trametes V. as well as Ganoderma L. are all capable of synthesising extracellular oxidative enzymes that cause the disintegration of methylene blue. The four fungal strains thus show potential for the bioremediation of recalcitrant compounds, like plastics. However, with the naked eye we can already tell that there is a clear distinction in the effectiveness of degradation across the different strains. Pestalotiopsis M., known for its PUR-degrading capacity, thereby seems to display a much lower efficiency than the Basidiomycetes.

36

The parameters that are resorted to in order to establish a numerical comparison between the tested isolates, are the final concentration of methylene blue and the percentage of dye degradation after 23 days of culture growth. Both can be calculated from the absorption spectra obtained by means of spectrophotometry for which we fill eight cuvettes with a small sample of the inoculated flasks; two extra cuvettes are filled with clear distilled water as blanks in order to calibrate the machine and one more is filled with a sample of the reference medium.

Conform Menezes et al. (2017), the maximum absorption value of the dye was collected at a wavelength of 666nm in the reference medium (Figure 17). Hence, the absorption value of all inoculated samples at 666nm were measured (Table 1). This allows for the calculation of the final concentration of methylene blue and the percentage of dye degradation (DC) by means of the following formula: DC(%) = [(AC - AF)/AC] x 100 where AC is the absorption value of the reference medium and AF is the absorption value of the sample. Pleurotus O., Pestalotiopsis M., Trametes V. and Ganoderma L. respectively showed maximum values of 65,5%; 75,0%; 97,2% and 81,0% degradation.

Menezes et al. (2017) reports 53,3% of dye degradation for Pycnoporus Sanguineus, Fusidium Sphaceliae and Pleurotus Ostreatus, without a significant difference between the isolates. In this study cultures were allowed to grow for a period of 19 days, while here the spectrophotometric analysis was executed after 23 days of growth. The different degradation value for the common denominator Pleurotus Ostreatus could therefore be attributed to the longer cultivation period and shows the time dependency of the experiment. All samples were eventually rendered completely colourless. That is why the obtained degradation percentages should not be interpreted as an absolute

absorbance

3

Preliminary Experiments

value of the total quantity of the dye that can be degraded by the fungus, but rather as an indication of the efficiency of the degradation process. It can be concluded that all tested strains display the capacity to produce oxidative enzymes that break down stable C - C bonds of methylene blue, and that differences in efficiency between the strains can most likely be attributed to varying growth rates or the enzyme-specificity.

Reference Pestalotiopsis M. Pleurotus O. Ganoderma L. Trametes V.

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2

1

0 400

500

600

666

700

800 wavelength Îť [nm]

Figure 17 Absorption spectra (400 - 800 nm) of the liquid culture media after 23 days the maximum absorption value of the dye was collected at a wavelength of 666nm

Reference

Pl.O. 1

Pl.O. 2

P.M. 1

P.M. 2

G.L. 1

G.L. 2

T.V. 1

T.V. 2

abs

2,683

0,925

0,799

0,670

0,630

0,612

0,508

0,076

0,089

c (mg/L)

50

17,2

14,9

12,5

11,7

11,4

9,5

1,4

1,6

DC (%)

0

65,5

70,2

75,0

76,5

77,2

81,0

97,2

96,7

Table 1 Absorption value (abs) at 666nm, final concentration (c) and percentage dye degradation (DC) of methylene blue in the liquid culture media 23 days post inoculation.

5.2

PLASTIC AS SOLE CARBON SOURCE

In the preceding experiment Pl.O., Tr.V. and G.L. have all proven their ability to produce oxidative enzymes that break down stable C-C bonds of methylene blue. A possible indication of the capacity to use plastics as a primary food source to sustain their growth. The two fungal strains that showed the highest degradation rate, Trametes Versicolor and Ganoderma Lucidum, will now be subjected to a synthetic growth medium containing polyethyene (PE) or polylactic acid (PLA) as the sole carbon source.

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Brunner et al. (2018) screens a range of fungi isolated from plastic debris floating in the shoreline of a lake just as Yamada-Onodera et al. (2001) reports Penicillium Simplicissimum, for having the capacity to utilise polyethylene as the only carbon source to facilitate growth. This was examined by observing growth of the fungi in petri dishes on an agar medium containing a range of nutrients (3 g L-1 NH4NO3, 5 g L-1 K2HPO4, 1 g L-1 NaCl, 0.2 g L-1 MgSO4.7H2O, 0.25 ml L-1 Tweed 20, and 15 g L-1 agar) supplemented with 10 g L-1 polyethylene powder immediately after autoclaving. Thus, besides containing nitrogen, phosphorus, sulphur, potassium, magnesium, sodium and chlorine, the only source of carbon in the medium was polyethylene. Fungi capable of degrading the plastic polymer would grow on the plastic granules (Brunner et al., 2018).

Following the train of thought of Yamada-Onodera et al. (2001) and Brunner et al.

(2018), the growth of Trametes Versicolor and Ganoderma Lucidum will now be observed in petri dishes on an agar medium with PE or PLA as the sole carbon source.

PE is a fossil-based polymer obtained by the polymerisation of ethylene monomers, which results in long chains of covalently linked carbon atoms with a pair of hydrogen atoms attached to each carbon atom (-CH2-), terminated by methyl groups (-CH3) at each end (Peacock, 2000). The polymer chains are synthesised with different molecular weights and chain architectures leading to the classification of the resulting material as LDPE (low density polyethylene and molecular weight below 50.000 g/mol ) or HDPE (high density and molecular weight up till 200,000 g/mol). PE plastic is characterised by its toughness, low moisture absorption, good chemical resistance, good electrical resistance, a low coefficient of friction and ease of processing (Rosato et al., 2004).

PLA is a renewable and biodegradable polymer obtained by condensation of lactic acid monomers, naturally processed from fermented plant starch such as corn, cassava, sugarcane or sugar beet pulp (SÜdergard et al., 2010). It is a thermoplastic polyester (C3H4O2)n, designed with an end-of-life scenario in mind where the polymer is returned to earth’s carbon cycle through an industrial composting process. PLA plastic displays mechanical returned to earth’s carbon cycle through an industrial composting process. PLA plastic displays mechanical properties comparable to traditional polymers and is already being applied as filament for 3D-printing or as a decomposable packaging material (Oz et al., 2017).

n

O O

n

Distilled water 1 L agar 15 g/L PE/PLA 20 g/L

Method_ In preparation of the experiment at hand, Trametes V. and Ganoderma L. are pregrown on a 2% malt agar plate for one week. Polyethylene powder (0,92 g/cm3) and PLA powder (1,24 g/cm3) were obtained from Resinex Benelux in order to prepare a growth medium with a homogenous distribution of PE or PLA as only source of carbon for the cultivation of Trametes V. and Ganoderma L. Considering that the melt temperature of polyethylene and the glass transition temperature of PLA would be far exceeded in the process of sterilising with an autoclave, the powders are submerged in 70% ethanol and left to dry overnight in a laminar flow cabinet.

The following day, both the PLA- and PE-media are prepared in an analogous manner. 15 g of agar is added to a flask filled with one litre of distilled water. The flask is now sealed with aluminium foil and placed in an autoclave.

In order to render the medium inert, it is then heated under pressure at 121 °C and 15 psi in the autoclave for twenty minutes. Immediately after autoclaving, the flask is transferred to the laminar flow cabinet. Here, 20 g of the sterilised powder is supplemented to the medium and thoroughly stirred. The medium can now be distributed over petri plates, which are left uncovered in the laminar flow cabinet until agar solidification. Half of the petri dishes are inoculated with a plug of fungal mycelium of the pregrown Tr.V. strain, the other half with G.L., after which the plates are covered and finally sealed with plastic paraffin film. The cultures are incubated in the dark at a constant temperature of 26 °C and visually inspected every few days.

Preliminary Experiments

Material prep_ Trametes Versicolor Ganoderma Lucidum

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SAMPLE GLPLA Ganoderma L. PLA powder

SAMPLE GLPE Ganoderma L. PE powder

40

Results and discussion_ Optical evaluation of the PE- and PLAmedia demonstrates that the powder is not homogenously distributed along the medium as clusters of granules can be noticed after agar solidification. A follow up every few days thereby showed no visible growth of the fungi even after two weeks upon the date of inoculation (Figure 18). This raises the question whether Trametes V. and Ganoderma L. are either incapable of reaching the clusters of PE- and PLA-granules across the medium, or that the fungi are only capable of establishing a co-metabolism of synthetic polymers, where the fungi are coaxed to produce oxidative enzymes as a reaction to carbon sources that are simpler to break down.

Therefore the test was re-executed, this time supplementing the media with 17 g L-1 malt extract. In doing so, the growth medium would

certainly comprise the necessary feedstock to ensure the growth of the cultures. However, after two weeks still no visible growth had been established.

The failed control test made clear that the absence of growth could not be the result of a lack of available nutrients in the media, but rather that the growth of the fungi had been inhibited by a constituent of the medium itself.

This could have happened due to the used sterilisation technique, meaning that the ethanol used to decontaminate the PE- and PLA-powder might not have been sufficiently evaporated during one night of drying in the laminar flow cabinet. When adding the powder to the medium, this would imply that a certain amount of the 70% ethanol was mixed into the medium

SAMPLE TVPE Trametes V. PE powder

Preliminary Experiments

SAMPLE TVPLA Trametes V. PLA powder

41

as well, inhibiting all growth of Trametes V. and Ganoderma L. It is therefore hypothesised that a revision of the used sterilisation technique might bring about a different result of the experiment.

The experiment is executed once more, amping up the concentration of PE and PLA in order to ensure a more even distribution of the granules along the medium. Furthermore, it is decided not to sterilise the powders in a 70% ethanol submersion but merely to rinse the powders with sterile water, significantly increasing the risk of contamination.

Figure 18 Agar media with PE or PLA as the sole carbon source, inoculated by Ganoderma L. (left page) or Trametes V. (right page). Clusters of polymer granules can be noticed after agar solidification. No visible growth two weeks post inoculation.

SAMPLE GLPLA Ganoderma L. PLA powder

SAMPLE GLPE Ganoderma L. PE powder

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Material prep_ Trametes Versicolor Ganoderma Lucidum Distilled water 1 L agar 15 g/L PE/PLA 100 g/L

Revised method_ A flask containing one litre of distilled water is sterilised by means of autoclaving for twenty minutes. Meanwhile, 100 g of PLA powder and 100 g of PE powder are measured in the laminar flow cabinet. Once the sterile water is cooled down till room temperature, both batches of powder are rinsed and left uncovered in the laminar flow cabinet until completely dry. This method does not ensure a sterile powder and a heightened risk on contamination of the medium should be anticipated.

Both the PLA- and PE-medium are now prepared in an analogous manner. Fifteen grams of agar is added to a flask filled with one litre of distilled water. The flask is sealed with aluminium foil and placed in an autoclave, where it is heated under pressure at 121°C and 15psi for twenty minutes. Immediately after autoclaving, the flask is transferred to the laminar flow cabinet. Here 100g of powder, rinsed in sterile water, is supplemented to the medium and thoroughly stirred. The medium can now be distributed over petri plates, which are left uncovered in the laminar flow cabinet until agar solidification. Half of the petri dishes are inoculated with a plug of fungal mycelium of the pregrown Trametes V. strain and the other half with Ganoderma L., after which the plates are covered and finally sealed with plastic paraffin film. The cultures are incubated in the dark at a constant temperature of 26°C and visually inspected every few days.

SAMPLE TVPE Trametes V. PE powder

Preliminary Experiments

SAMPLE TVPLA Trametes V. PLA powder

43

Figure 19 Agar media with PE or PLA as the sole carbon source, inoculated by Ganoderma L. (left page) or Trametes V. (right page). Mycelial growth on day 10 post inoculation.

As such, we are now able to conclude that both Trametes V. and Ganoderma L. can biologically degrade the synthetic polymers polyethylene and polylactic acid in the process of resorting to them as a primary food source to sustain their growth. Full colonisation of the medium does however not imply full degradation of the plastic present within the medium.

Polyethylene or PLA being the sole carbon source in the medium does nonetheless show the fungi’s ability to produce oxidative enzymes that break down stable C - C bonds, leading to polymer chain scission and the production of low molecular weight compounds such as oligomers, monomers and dimers, which can be assimilated later on by the fungus itself or by other microorganisms (da Luz et al., 2013).

surface colonisation [%]

Figure 20 Growth rate of Trametes V. measured over 10 days as surface colonised (%) of a 100mm diameter petri plate containing a 2% malt extract agar (MEA-), PLA- or PE-medium 100

80

60

40

20

T.V. on MEA T.V. on PLA T.V. on PE

0 0

2

4

6

8 10 time [days]

Figure 21 Growth rate of Ganoderma L. measured over 10 days as surface colonised (%) of a 100mm diameter petri plate containing a 2% malt extract agar (MEA-), PLA- or PE-medium surface colonisation [%]

44

Results and discussion_ Due to the increased concentration of PLA- and PE-powder, attaining a fairly homogenous medium has been accomplished. Both the PLA- and PEmedium now have a milky appearance due to an even distribution of granules as described in Brunner et al. (2018).

Optical evaluation every few days now does show a steady growth pattern of both Trametes V. and Ganoderma L. on the PLA medium as well as the PE medium. Eight days after inoculation, the full petri plates are covered in a white blanket of mycelium for samples TVPLA and TVPE (Figure 20). After ten days of growth, full colonisation of the plate is established by GLPLA as well and a radial thickening of the mycelium can be observed in samples TVPLA and TVPE. Sample GLPE displays a slightly slower growth rate, as after ten days of growth only 67% of the petri plate was colonised (Figure 21).

100

80

60

40

20

G.L. on MEA G.L. on PLA G.L. on PE

0 0

2

4

6

8 10 time [days]

5.3

Preliminary Experiments

It should however be noted that deciding not to sterilise the PLA- and PE-powder resulted in a considerable amount of samples that were subjected to contamination.

Samples inoculated with Trametes V. strikingly more so than samples inoculated with Ganoderma L. Other methods of sterilisation, such as gamma irradiation sterilisation, should therefore be considered but will not be explored within the scope of this research.

STRAIN SELECTION

Trametes Versicolor and Ganoderma Lucidum have both displayed significant potential in the preliminary screening for fungal strains with both architectural relevance and bioremediating capacity. In the methylene blue test, Ganoderma L. disintegrated 81,0% of the synthetic dye in a total of 23 days while Trametes V. succeeded in breaking down no less than 97,2%. Moreover, both fungal strains have shown their capability to biologically degrade polyethylene, the most widely used and produced plastic, as well as polylactic acid, the most widely used bioplastic as filament for 3D printing, while resorting to the polymer as the only carbon source to sustain their growth. The slight distinction in degradation rate that came to the fore in the methylene blue test, resurfaces in analysing the rate at which Trametes V. and Ganoderma L. are able to colonise a medium containing polyethylene as the primary food source. While Trametes V. was able to fully colonise the medium in a period of eight days, Ganoderma L. needed ten days.

With the objective of using waste as a resource in mind, and the pragmatic complications of sterilising plastics without the use of growth inhibiting chemicals, a higher resistance of the fungal strain towards contamination by other microorganisms might however be a valuable asset. In the last test setup, the specific strain of Trametes V. that was used appeared to be more vulnerable for contamination in a medium containing unsterilized plastics. In the selection of a fungal strain for further analysis and prototyping, the seemingly higher resilience to contamination of Ganoderma Lucidum therefore outweighs the slightly higher degradation rate of Trametes Versicolor.

45

46

Degradation of Plastics

The previous chapter has indicated that when provided the right conditions, some fungi are all but picky eaters. Through a preliminary screening of four fungal strains, Ganoderma Lucidum has come to the fore as a promising microorganism for combining the biodegradation of plastic waste with the production of mycelium composites. In a medium with a homogenous distribution of polyethylene or polylactic acid particles in water, the fungus has shown its capacity to utilise recalcitrant polymers as a primary food source.

However, a homogenised provision of plastic particles in a water based medium, is not representative for the growth conditions that can be provided when processing plastic waste into mycelium composites. Therefore, the selected fungal strain will now be subjected to a more pragmatic approach of experimenting. In order to create realistic growth conditions, Ganoderma L. will have to feed on plastic in the form of consumer products as they are found in the waste stream of the plastic industry: most often singleuse bags or non-degradable packaging. Is Ganoderma L. able to degrade plastic in its end-of-life appearance and if so, at what rate? These questions lie at the core of the following experiments where the biodegradation of two plastic types, PE and PLA in their consumer product forms, is analysed.

Degradation of Plastics

6

47

6.1

50 DAYS OF DEGRADATION

To test the feasibility and quantify the efficiency of plastic waste management by Ganoderma L., the weight losses of equiform plastic samples were measured after fifty days of incubation. Firstly, we will take a look at polyethylene, processed in either low-density (LDPE) or high-density (HDPE) materials. Ensuing, the degradation of polylactic acid will be analogously observed, both as a standard PLA material and as a bamboofibre/PLA composite. This will make for an interesting comparison between the rate at which Ganoderma L. is able to remediate a bioplastic and a fossil-fuel plastic.

48

Since the biological degradation process of solid polymers can take on two faces, namely through bulk degradation or surface degradation (Ratner et al., 2012), visualising the surface changes of the samples is a useful tool for increasing our understanding of the obtained results. Therefore the surface of each plastic type will be characterised prior to the experiment by means of scanning electron microscopy (SEM), providing a reference for comparison with the surface characteristics after fifty days of colonisation.

If degradation occurs by surface erosion, the process takes place on the exterior which leads to the thinning of the sample with time. In this type of biodegradation, the core of the material remains untouched until the surrounding material has been degraded and the molecular weight of the polymer remains constant. The rate of surface biodegradation is therefore directly proportional to the available surface area of the polymer and remains constant for thin materials (Kumbar et al. 2014; Ulery et al., 2011).

In bulk erosion, polymer biodegradation simultaneously occurs throughout the polymer matrix. The molecular weight decreases throughout the process while the matrix dimensions remain constant until total mechanical failure. In this type of biodegradation, the polymer typically allows penetration of water into the bulk of the material (Ratner et al. 2012).

The determination of whether a material undergoes surface erosion or bulk erosion depends on various factors, such as the linkage between monomers, method of chain scission, mechanism of hydrolysis of monomer units, glass transition temperature, surface-to-volume ratio and porosity of the polymer (Domb et al., 2011). Primarily, it depends on the hydrophobic nature of the polymer matrix. Therefore, it is expected that surface erosion will be the prevailing mechanism of degradation for both PE and PLA.

Distilled water 1 L Agar 15 g/L Malt extract 10 g/L

Method_ Equiform PE- and PLA-samples of two by four centimetres were collected in preparation of the test. To generate an estimation of the degradation rate, a fivefold replication of the test set up will be produced for each plastic type. In order to induce the search for alternative carbon sources within the polymer samples, a sheer 1% malt extract agar medium was resorted to for the cultivation of pure Ganoderma L. cultures for five days before adding the samples to the medium. This 1% malt extract agar (MEA) is prepared by adding 10g of malt extract and 15g of agar to one litre of distilled water. The medium is now sterilised in an autoclave for twenty minutes and inoculated in petri plates with a plug of Ganoderma L.

Plastic samples of two by four centimetres are now rinsed in sterilised water and airdried before listing the initial weight of the samples by means of an analytical scale.

Accurate measurement of the initial weight to a minimum of four decimals is crucial in this stage in order to calculate an estimation of the degradation percentage later on.

Five days after inoculation, the plastic samples are added to the petri dishes. The plates are sealed with paraffin tape to maintain a constant humidity and then placed in an incubator for fifty days at a constant temperature of 26°C. Fifty days after the date of inoculation, all samples are retrieved from the petri plates.

To remove any residual organic matter, the samples are thoroughly rinsed; first with 70% ethanol, then with distilled water and finally air-dried before measuring the final weight. An estimation of the degradation percentage after fifty days can now be made by means of the percent mass loss given by the following formula: ((massinitial – mass50 days )/massinitial) x 100% Samples displaying the highest degree of degradation were then taken for a final observation under the scanning electron microscope, visualising the surface changes that were induced by colonisation of the microorganism.

Degradation of Plastics

Material prep_ Ganoderma Lucidum Plastic samples 2x4 cm

49

50

GLM#1

GLL#1

GHM#1

GLM#2

GLL#2

GHM#2

GLM#3

GLM#3

GHM#3

GLM#4

GLL#4

GHM#4

GLM#5

GLL#5

GHM#5

GPL#1

GWM#1

GPM#2

GPL#2

GWM#2

GPM#3

GPL#3

GWM#3

GPM#4

GPL#4

GWM#4

GPM#5

GPL#5

GWM#5

Figure 22 Polyethtylene and PLA samples after fifty days of colonisation

by Ganoderma L. (from the left column: LDPE on MEA, LDPE on lignin, HDPE on MEA, PLA on MEA, PLA on lignin, wood-PLA on MEA)

Degradation of Plastics

GPM#1

51

˿ ͢

500 µm

post biodegradation

˿ ͢

500 µm

prior to biodegradation

˿ ͢

275 µm

post biodegradation

˿ ͢

275 µm

prior to biodegradation

˿ ͢

100 µm

post biodegradation

˿ ͢

100 µm

prior to biodegradation

52

Low-density polyethylene samples were collected from municipal waste in the form of single-use plastic bags and observation of the petri plates after fifty days of growth showed that two out of five samples had achieved full colonisation of the polymer surface by Ganoderma L., while the remaining three had only been partially colonised.

All samples clearly displayed zones of discolouration, macroscopically manifesting as either faded red areas on red LDPE samples or yellowed spots on white LDPE samples.

10 9 8 7 6 5

2,94 2,62

2,68

2,76

GLM#5

3

GLM#4

4

2,37

2

GLM#3

GLM#2

1

GLM#1

degradation percentage [%]

Figure 24 Scanning electron microscopy images of an LDPE sample prior to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom)

LOW-DENSITY POLYETHYLENE - LDPE

Figure 23 Degradation percentage of five equiform LDPE samples after 50 days of colonisation by Ganoderma L. on a 2% malt extract agar medium

A mean percent mass loss of 2,67% was recorded after the incubation period, ranging from a minimum of 2,37% up till 2,94% respectively in sample GLM#3 and GLM#2. This shows that Ganoderma L. has not only resorted to the malt extract growth medium but also to the polymer chains of the LDPE samples as a carbon source for sustaining its growth. Electron microscopy thereby visualises clear changes in the surface topography of the LDPE samples after colonisation. Besides residues of hyphal growth, it is possible to observe cracking in the polymer surface as a result of biodegradation by means of surface erosion. The pattern of surface cracks is remarkably irregular, which can likely be attributed to the semi-crystalline nature of polyethylene, comprising of crystalline micro-structures, that are surrounded by amorphous regions. It has been experimentally corroborated that amorphous regions are consumed first because they are more accessible to microorganisms (Raghavan and Torma, 1992; Albertsson et al., 1995; Sudhakar et al., 2008; Restrepo-Flòrez et al., 2013).

Table 2 Initial mass; mass after 50 days of colonisation by Ganoderma L.; total mass loss and degradation percentage of five equiform LDPE samples sample

massinitial [mg]

mass50days [mg]

mass loss [mg]

degradation percentage [%]

GLM#1

38,2

37,2

1,0

2,62

GLM#2

40,8

39,6

1,2

2,94

GLM#3

42,2

41,2

1,0

2,37

GLM#4

37,3

36,3

1,0

2,68

GLM#5

36,3

35,2

1,0

2,76

Degradation of Plastics

6.1.1

53

˿ ͢

500 µm

post biodegradation

˿ ͢

500 µm

prior to biodegradation

˿ ͢

275 µm

post biodegradation

˿ ͢

275 µm

prior to biodegradation

˿ ͢

100 µm

post biodegradation

˿ ͢

100 µm

prior to biodegradation

54

Though its chemical structure is similar to that of LDPE, unlike it, HDPE possesses low frequency of chain branching which allows the close approach of polymer molecules and results in a dense, highly crystalline material (Peacock, 2000; Carraher, 2003).

As HDPE exhibits low swelling characteristics it is commonly used to pack juices and other liquids. Samples for the experiments were therefore obtained from shampoo bottles. Full colonisation of the samples was established, except for GHM#1 where a mild contamination of the medium had partially prevented the growth of Ganoderma L.

0,6 0,5 0,4 0,3 0,2 0,1

GHM#5

0,7

GHM#4

0,8

GHM#3

0,9

GHM#2

1

Hadad et al. (2005) reports that the biodegradability of polyethylene is inversely proportional to its molecular weight. Given the higher crystallinity and molecular weight of HDPE compared to LDPE, a lower degradation rate could therefore be expected for HDPE samples. A mean percent mass loss of 0,25% was recorded after a fifty-day degradation period, ranging from a minimum of 0,20% up till 0,34% respectively in sample GHM#1 and GHM#3. However, when comparing the total mass loss of both the LDPE and HDPE samples, it seems that the actual amount of polyethylene degraded is comparable. It becomes clear that the degradation rate of samples does not unambiguously reflect the biodegradability of the material. Chamas et al.

(2020) states that the rate of degradation depends not only on the intrinsic properties of the plastics (polymer type, molecular weight, etc.) but also on the extrinsic properties such as the size and shape of the material. Hence, samples with the same composition and mass but different surface areas can show very different rates of degradation 0,34 (Chamas et al., 2020). Visualisation of the HDPE 0,28 0,24 surface again shows surface cracking, albeit of a 0,22 0,20 much smaller magnitude than recorded for LDPE samples. This reinstates that the biodegradation of polyethylene takes place on the exposed surface of the polymer and that the rate of biodegradation is functionally dependent on the surface area of the plastic. Hence, when exposing HDPE to Figure 25 Degradation percentage of five biological degradation a maximum surface area equiform HDPE samples after 50 days of colonisation by Ganoderma L. on a 2% malt to volume ratio should be pursued.

GHM#1

degradation percentage [%]

Figure 26 Scanning electron microscopy images of an HDPE sample prior to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom)

HIGH-DENSITY POLYETHYLENE - HDPE

extract agar medium

Table 3 Initial mass; mass after 50 days of colonisation by Ganoderma L.; total mass loss and degradation percentage of five equiform HDPE samples sample

massinitial [mg]

mass50days [mg]

mass loss [mg]

degradation percentage [%]

GHM#1

550,2

549,1

1,1

0,20

GHM#2

689,4

687,9

1,5

0,22

GHM#3

591,5

589,5

2,0

0,34

GHM#4

532,9

531,4

1,5

0,28

GHM#5

673,0

671,4

1,6

0,24

Degradation of Plastics

6.1.2

55

˿ ͢

500 µm

post biodegradation

˿ ͢

500 µm

prior to biodegradation

˿ ͢

275 µm

post biodegradation

˿ ͢

275 µm

prior to biodegradation

˿ ͢

100 µm

post biodegradation

˿ ͢

100 µm

prior to biodegradation

56

PLA samples were printed with a standard transparent 3D-printing filament with a minimum layer height of 0,2 millimetre. All five samples showed to have reached full colonisation of the polymer surface by Ganoderma L. during the fifty-day incubation period. After retrieving the samples from the petri plates and thoroughly rinsing them, the structural integrity of the prints seems to be altered as parallel print lines are now only held together by the transverse print line that forms the perimeter of the PLA print.

Moreover, a visual surface change from transparent to opaque can be observed.

10 9 8 7 6 5

4,95 4,54 4,15

4,32

4,20

4 3 2

GPM#5

GPM#4

GPM#3

GPM#2

1

GPM#1

degradation percentage [%]

Figure 28 Scanning electron microscopy images of a PLA sample prior to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom)

STANDARD PLA FILAMENT

Figure 27 Degradation percentage of five equiform PLA samples after 50 days of colonisation by Ganoderma L. on a 2% malt extract agar medium

A mean percent mass loss of 4,43% was recorded after the incubation period, ranging from a minimum of 4,15% up till 4,95% in sample GPM#2 and GPM#1 respectively. As could be expected, the biodegradation rate of the PLA samples seems significantly higher than the rate of biodegradation of polyethylene samples. Despite thorough rinsing of the samples, visualisation by means of scanning electron microscopy shows that a large amount of biomass is still present in between print lines, which suggests that the total mass loss after fifty days would be even higher. The hyphal growth in these gaps have ripped the printed samples apart at the seams which highly increases the polymer surface that is under biological attack. Due to the hydrophobic nature of PLA, the method of biodegradation is nonetheless still surface erosion, which manifests in brittle cracking of the polymer surface.

Table 4 Initial mass; mass after 50 days of colonisation by Ganoderma L.; total mass loss and degradation percentage of five equiform PLA samples sample

massinitial [mg]

mass50days [mg]

mass loss [mg]

degradation percentage [%]

GPM#1

186,0

176,8

9,2

4,95

GPM#2

185,6

177,9

7,7

4,15

GPM#3

185,0

176,6

8,4

4,54

GPM#4

164,4

157,3

7,1

4,32

GPM#5

171,5

164,3

7,2

4,20

Degradation of Plastics

6.1.3

57

˿ ͢

500 µm

post biodegradation

˿ ͢

500 µm

prior to biodegradation

˿ ͢

275 µm

post biodegradation

˿ ͢

275 µm

prior to biodegradation

˿ ͢

100 µm

post biodegradation

˿ ͢

100 µm

prior to biodegradation

58

PLA samples were again printed at the minimum layer height of 0,2 millimetre with a composite filament, commercially going under the name of Bamboofill, containing 80% PLA and 20% recycled bamboo fibres. All five samples were fully colonised within the fifty-day period but unlike the standard PLA samples, the printed wood/PLA-pieces showed no disintegration between print lines.

Fully separating the biomass of the sample could not be achieved as the microorganism had become an integral part of the sample itself. Therefore it should be noted that an underestimation of the degradation percentage was recorded with the mean percent mass loss of 6,78%, ranging from a minimum of 5,61% up till 7,64% respectively in sample GWM#4 and GWM#1. However, we are unable to conclude what ratio of the mass loss is due to lignin degradation and which part due to actual PLA degradation.

10 9 8

7,64 7,23

7,24

7 6,18

6

5,61

5 4 3 2

GWM#5

GWM#4

GWM#3

GWM#2

1

GWM#1

degradation percentage [%]

Figure 30 Scanning electron microscopy images of a wood/PLA sample prior to (left) and post (right) 50 days of colonisation by Ganoderma L. with a width of 500 µm (top), 275 µm (middle) and 100 µm (bottom)

COMPOSITE WOOD-PLA FILAMENT

Figure 29 Degradation percentage of five equiform samples of a wood/PLA composite after 50 days of colonisation by Ganoderma L. on a 2% malt extract agar medium

As a result of the wood/PLA-blend, the susceptibility of the material for fungal growth is highly augmented. The polymer allows penetration of water into the bulk of the material and biodegradation occurs throughout the whole polymer matrix. Contrasting with the images of the surface erosion process of polyethylene and standard PLA, SEM-visualisation now clearly shows the biodegradation of an already porous reference sample through bulk erosion. Residues of hyphal growth can be observed within the pores of the material, which have been enlarged and hollowed out in the process.

Table 5 Initial mass; mass after 50 days of colonisation by Ganoderma L.; total mass loss and degradation percentage of five equiform samples of a wood/PLA composite sample

massinitial [mg]

mass50days [mg]

mass loss [mg]

degradation percentage [%]

GWM#1

179,4

165,7

13,7

7,64

GWM#2

141,1

130,9

10,2

7,23

GWM#3

149,2

138,4

10,8

7,24

GWM#4

160,5

151,5

9,0

5,61

GWM#5

143,9

135,0

8,9

6,18

Degradation of Plastics

6.1.4

59

6.2

60

LIGNIN AS A CATALYST

Hassinger (2018) reports that ligninolytic enzymes produced by fungi as a response to the presence of lignin in the growth medium could function as a catalyst for the biodegradation of plastics. To compare the influence of the growth medium in which the biodegradation of plastic takes place, the degradation assay for LDPE and standard PLA was also executed in a lignin medium. Alongside the MEA medium, a lignin medium was therefore prepared by grinding 200g of beechwood fibres in a blender for ten minutes after which 15g of agar was added to the mixture. The rest of the experiment was continued in an analogous manner.

mean percent mass loss of LDPE samples in a lignin medium was 5,51% while the degradation percentage in a malt extract agar medium was only 2,67%; more than double. The degradation percentage of PLA samples also showed an increase, rising from 4,43% in the malt extract medium to a mean value of 5,90% in the lignin medium.

Conform the findings of Hassinger (2018), Ganoderma L. performed significantly better in the lignin media than in the malt extract agar medium, showing an increase in degradation rate for both the LDPE and PLA samples. The recorded

Table 6 Initial mass; mass after 50 days of colonisation by Ganoderma L.; total mass loss and degradation percentage of five equiform LDPE samples on a lignin medium sample

massinitial [mg]

mass50days [mg]

mass loss [mg]

degradation percentage [%] 6,67

GLL#1

39,0

36,4

2,6

GLL#2

39,0

37,0

2,0

5,13

GLL#3

38,8

36,8

2,0

5,15

GLL#4

40,0

37,8

2,2

5,50

GLL#5

41,1

39,0

2,1

5,11

Table 7 Initial mass; mass after 50 days of colonisation by Ganoderma L.; total mass loss and degradation percentage of five equiform PLA samples on a lignin medium sample

massinitial [mg]

mass50days [mg]

mass loss [mg]

degradation percentage [%]

GPL#1

186,5

175,3

11,2

6,01

GPL#2

181,3

170,5

10,8

5,96

GPL#3

181,9

170,1

11,8

6,49

GPL#4

177,3

167,9

9,4

5,30

GPL#5

184,7

174,1

10,6

5,74

6,67

2

1

1

GPL#1

2

GLL#4

3

GLL#3

3

GLL#2

4

GLL#1

61

5

4

Figure 31 Degradation percentage of five equiform LDPE samples after 50 days of colonisation by Ganoderma L. on a lignin medium

6.3

5,96 5,30

5,11

5

5,74

6,49 6,01

GPL#4

5,15

7 6

5,50 5,13

8

GPL#3

6

9

GPL#2

7

GPL#5

degradation percentage [%]

8

GLL#5

degradation percentage [%]

9

10

Degradation of Plastics

10

Figure 32 Degradation percentage of five equiform PLA samples after 50 days of colonisation

by Ganoderma L. on a lignin medium

OPTIMISATION OF THE DEGRADATION RATE

In the previous paragraphs, an indication of the degradation percentage of polyethylene and PLA by Ganoderma L. was experimentally determined. The importance of pursuing a maximum surface area to volume ratio when exposing hydrophobic polymers to biological degradation came to the fore. Of course this maximum is already achieved for thin LDPE films, but when processing bulky PLA or HDPE in mycelium composites, this parameter will highly influence the rate of degradation and should be well weighed up against the growth period of the fungus. Moreover, the presence of lignin displayed to have a positive effect on the biodegradability of plastics; serving as a catalyst for the production of oxidative enzymes when present in the growth medium and allowing bulk

degradation when present in the polymer matrix.

In a simple test setup, the maximum recording for the percentage of the total weight loss of the HDPE and LDPE samples after fifty days of incubation were found to be 0,34% and 6,67% respectively.

Whereas for PLA, a maximum percentage of the total weight loss of 6,49% was found. These results are promising and reassure the potential of targeted biodegradation of plastics by isolated fungi, having in mind that Otake et al. (1995) reports similar findings of LDPE surface changes after soil burial for over 32 years and that PLA shows no degradation over the span of a year in a marine environment (Bagheri et al., 2017).

However, the findings in this writing only serve as an explorative indication and conceptual confirmation for the feasibility of implementing the biodegradation of plastics in the production process of mycelium composites, which will be further tested through prototyping in Chapter 7. Optimisation of the degradation rate of plastics by Ganoderma L. is beyond the scope of this research but is undoubtedly possible and demands both longer observation of the degradation process and close transdisciplinary collaboration. The achieved degradation percentages in microbiological research are therefore of a different order of magnitude. Weight loss percentages of 58,6% and 34,4% were reported by Ojha et al. (2017) for HDPE and LDPE sheets respectively after ninety days of fungal biodegradation and according to Hidayat et al. (2012) 12, 21, 30 and 48% of polylactic acid samples were degraded in 1, 2, 3 and 6 months.

62

Optimising the biodegradation process could encompass a pre-treatment of the polymer, an adjustment of the growth conditions such as temperature and pH, or an alteration in the composition of the growth medium. As such, physical and chemical treatments of the polymer before biodegradation, including UV irradiation, photooxidation, thermal treatment and oxidation with nitric acid have proven to increase its effectiveness (Pathak et al., 2017). Moreover, Copinet et al. (2004) reports that the degradation rate of PLA can be enhanced by increasing temperature and relative humidity and Jarerat et al. (2001) makes the surprising observation that Tritirachium A. shows no PLA film degradation in liquid culture using basal medium, but displays 76% degradation after only 14 days when supplemented with 0,1% of gelatine.

POLYMER PIGMENT DEGRADATION - A STUDY OF COLOUR

The choice of a substrate for mycelium composites always has an influence on both mechanical and aesthetic qualities of the material. Plastic waste comes in all shapes, strengths and colours; opening doors for further research concerning achievable visual and structural material properties. In this chapter a study of colour is initiated, in order to investigate whether the pigments that give a specific colour to the plastic, have an influence on the growth of Ganoderma L. and could visually offer the possibility of implementing either temporal or permanent colour in the material.

Pigments are insoluble organic or inorganic compounds added to the polymer matrix. Compounds that are organic in nature are hard to disperse and tend to form agglomerates which can cause pigmented spots in the plastic but are commonly resorted to for applications needing high tinting strength and brilliant shades. It could be hypothesised that organic colourants will be used as a source of carbon by Ganoderma L.; will biodegrade and will be rendered colourless over time. Inorganic pigments such as metal oxides, sulphides, etc. are more easily dispersed in the resin and are mainly used when high opacity is needed.

The use of white-rot fungi Phanerochaete C. and Tinctoporia Sp. to decolourise lignincontaining pulp and paper wastewater has already been reported in 1980 (Eaton et al., 1980; Fukuzumi, 1980). Since then, Phanerochaete C. has been studied for the decolourisation of various dyes (Bilgic et al., 1997; Cammarota and Sant’Anna, 1992; Lankinen et al., 1991; Tatarko and Bumpus, 1998; Young and Yu, 1997; Ollikka et al.,1993; Pasti-Grigsby et al., 1992; Spadaro et al., 1992; Glenn and Gold, 1983).

Other white-rot fungi also capable of decolourising colourants, include Trametes V. (Wong and Yu, 1999; Young and Yu, 1997), Coriolus V. (Knapp and Newby, 1999; Knapp et al., 1995) and Funalia T. (Yesilada et al., 1995) (Fu and Viraraghavan, 2001).

In the following experiment, the growth of Ganoderma L. in the presence of a selection of three organic pigments (PG7 green (Phthalocyanine), PB15 azur blue (Phthalocyanine) and PR83 red (Anthraquinone)) and three inorganic pigments (PB29 ultramarine blue (silico aluminate of sodium polysulfides), PR101 red (iron-oxide) and PY1 yellow (mononitrogenous azo compound)) used in plastics, is put up for analysis.

PB29 silico aluminate blue

PB15 phthalocyanine blue

PG7 phthalocyanine green

PY1 azo yellow

PR83 iron-oxide red

PR101 anthraquinone red

Figure 33 Malt extract media supplemented with a range of pigments

Degradation of Plastics

6.4

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Material prep_ Ganoderma Lucidum Distilled water 1 L Peptone 1 g/L Dry yeast 2 g/L Malt extract 20 g/L Agar 15 g/L Arabic gum 120 mL/L Pigment 4 g/L

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Method_ In preparation of the experiment, a pure culture of Ganoderma L. is pregrown on a 2% malt agar plate for one week. Pigments PG7, PB15, PR83, PB29, PR101 and PY1 are obtained in powder form in order to prepare a growth medium containing a homogenous distribution of the dye. To prepare one litre of growth medium, an amount of 4 grams of pigment is placed on a glass surface and crushed with a spatula while adding 120 millilitres of Arabic gum binder. This natural binder allows for the pigments to be dissolved in one litre of distilled water, after which 1 gram of peptone, 2 grams of dry yeast, 20 grams of malt extract and 15 grams of agar are added to the medium. A reference medium without pigments is prepared for comparison.

The medium is now stirred, sealed with aluminium foil and placed in an autoclave. In order to render the medium inert, it is then heated under pressure at 121°C and 15psi in the autoclave for twenty minutes. Immediately after autoclaving, the flask is transferred to the laminar flow cabinet. The medium can now be distributed over petri plates, which are left uncovered in the laminar flow cabinet until agar solidification. The petri dishes are inoculated with a plug of fungal mycelium of the pregrown Ganoderma L. strain, after which the plates are covered and finally sealed with plastic paraffin film.

The cultures are incubated in the dark at a constant temperature of 26°C and visually inspected every few days.

PR101 - day 4

PY1 - day 6

PR83 - day 6

PR101 - day 6

PY1 - day 8

PR83 - day 8

PR101 - day 8

PY1 - day 10

PR83 - day 10

PR101 - day 10

PY1 - day 12

PR83 - day 12

PR101 - day 12

Degradation of Plastics

PR83 - day 4

Figure 34 Follow up every two days post inoculation of the pigmented growth media - bottom of petri dishes

PY1 - day 4

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PR101- day 4

PR83 - day 4

PY1 - day 4

PR101 - day 6

PR83 - day 6

PY1 - day 6

PR101 - day 8

PR83 - day 8

PY1 - day 8

PR101 - day 10

PR83 - day 10

PY1 - day 10

PR101 - day 12

PR83 - day 12

PY1 - day 12

PB29 - day 4

PG7 - day 6

PB15 - day 6

PB29 - day 6

PG7 - day 8

PB15 - day 8

PB29 - day 8

PG7 - day 10

PB15 - day 10

PB29 - day 10

PG7 - day 12

PB15 - day 12

PB29 - day 12

Degradation of Plastics

PB15 - day 4

Figure 35 Follow up every two days post inoculation of the pigmented growth media - topview of petri dishes

PG7 - day 4

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Figure 36 Follow up every two days post inoculation of the pigmented growth media - bottom of petri dishes

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PB29 - day 4

PB15 - day 4

PG7 - day 4

PB29 - day 6

PB15 - day 6

PG7 - day 6

PB29 - day 8

PB15 - day 8

PG7 - day 8

PB29 - day 10

PB15 - day 10

PG7 - day 10

PB29 - day 12

PB15 - day 12

PG7 - day 12

Organic anthraquinone-based dyes for example are more resistant to degradation due to their fused aromatic structures and thus remain their colour for a longer time (Fu and Viraraghavan, 2001), while the inorganic ultramarine blue pigment displays a very high degradation rate by Ganoderma L. and is even decolourised by the fungus’ enzymatic activity before the medium is overgrown by mycelium (Figure 35 and Figure 36).

Hence, in a medium containing both these pigments, the differentiation of degradation rate between pigments facilitates a fast colour-shift from purple to red followed by a slow-paced decolouration of the red pigment (Figure 37).

Figure 37 Malt extract medium supplemented with PB29 and PR83 displays the differing discolouration rate by Ganoderma L., undergoing a metamorphosis from purple to red and eventually white

Degradation of Plastics

Results and discussion_ Optical evaluation every few days shows that the growth rate of Ganoderma L. is not significantly influenced by the presence of pigments in the medium. A steady growth pattern is registered in all petri plates, achieving full colonisation after ten to twelve days following the inoculation date. The hypothesis that organic colourants would be biodegraded at a faster rate, as they could be used as a source of carbon for the microorganism, does not seem to be fulfilled. While all samples display a certain degree of decolourisation, the results of the experiment are very pigment-specific. Most dyes have a synthetic origin and complex aromatic molecular structures which make them very stable and difficult to be biodegraded (Fewson, 1988; Seshadri et al., 1994).

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Degradation materialised

Small scale prototyping

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SMALL SCALE PROTOTYPING While scientists and reason pierce the reality bubble and might pave the way for a new paradigm of construction, the architect and designer should come to the fore as an interpreter; navigating on the intersection between architecture and science and as ever, searching for the equilibrium between purpose and aesthetic. Finding architectural value in the biological principles of inoculation, growth, degradation and regrowth questions our very way of designing and implies giving some control out of hands by setting up a collaboration with the material itself. Up till now, the confinement of petri plates and flasks have formed the invisible borders of our construction site. Here, Ganoderma L. has proven its bioremediating capacities aside from its already established potential to produce mycelium composites.

What design opportunities are brought about by the ability to degrade plastics?

In the previous chapter the degradation process took place both concealed under a white blanket, cleaving polymer chains on the microscopic level, but also with strong aesthetic presence, extravagantly altering colours from purple to red to white. The acquired knowledge can now be materialised

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in a multi-stage exploration by small scale prototyping, in which we investigate how mycelium composites could be a silent vessel for responsible waste management or how the degradation process could be visualised for the creation of materials with a clear narrative and time-bound aesthetic. Hence, in this chapter a series of objects is produced as a means of generating knowledge, experience and an overall affinity with the material; focussing on plastic waste as a substrate choice on the one hand and plastic as a temporary mould for the production of mycelium composites on the other hand.

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PLASTIC WASTE AS A SUBSTRATE

The capacity of Ganoderma L. to degrade polyethylene waste streams allows for investigating methods to incorporate plastic waste management in the production of mycelium composites. The feasibility of plastic degradation by Ganoderma L. has been displayed in previous chapters, where the fungus was able to use polyethylene as the sole carbon source to sustain its growth and induce weight loss of both LDPE and HDPE samples by means of surface erosion. However, whereas the lignocellulosic substrate for standard production of mycelium composites serves as a source of nutrients; as importantly, it serves as a moist absorber and retainer to provide the growing fungus of a constant water supply throughout the medium. A hydrophobic substrate material such as polyethylene therefore requires a different approach.

Notwithstanding the possibility of researching LDPE or HDPE as sole substrate component in laboratory conditions where the relative humidity can be closely monitored, the practicality of the production process and the structural implications on the material itself are questionable. Therefore, two methods of plastic waste management incorporation are tried out where plastics are used as substrate in combination with a moist retaining substance: HDPE waste distributed as small granulates in a lignocellulosic matrix and unsustainable material-composites such as LDPE coated cardboard allocated as substrate.

7.1.1

HDPE GRANULATES

Introducing plastic waste management in the production of mycelium composites should not be overcomplicated. Mycelium composite production is already in the process of becoming standardised and industrialised, and the implementation could be as simple as adding plastic waste to the lignocellulosic substrate. The presence of lignin thereby displayed to have a positive effect on the biodegradability of plastics, serving as a catalyst for the production of oxidative enzymes, which actually makes this method even more interesting. Pursuing a maximum surface area to volume ratio when exposing hydrophobic polymers to biological degradation is crucial for achieving fastpaced degradation since the rate of biodegradation is functionally dependent on the

The production of this plastic-mycelium composite was put to the test by shredding HDPE products, retrieved from municipal waste, into pieces of five to seven millimetres and evenly distributing them in a beechwood substrate. A ratio of 50 grams HDPE granulates to 200 grams of beechwood fibres was used and humidified by 250 millilitres of distilled water. Beyond that, the production process did not have to deviate further from the established standard. Heating polyethylene can release toxic by-products when reaching temperatures at which the polymer decomposes. The decomposition range, which lies at 335°C – 450°C, is however far above the temperature reached by autoclaving (121°C) at which mycelium composite substrates are sterilised (Hilado, 1998). The mixture can thus be autoclaved as a whole, after which it is inoculated with 75 grams of Ganoderma L. grain spawn.

A preliminary growth phase of one week (in the dark at 26°C) in a reusable polypropylene growth bag that is frequently kneaded, ensures full colonisation of the substrate. In a laminar flow cabinet, the substrate is then transferred to a mould in which it will undergo a second growth phase of another two weeks. Hereafter, observation of the material already showed a thick white skin on the outer surface of the object. A growth phase outside the mould was therefore not required, but could take place if not all surfaces show equal colonisation.

Figure 38 Processing HDPE granulates in a beechwood substrate

The resulting material is a sturdy, lightweight composite, by no means differing from standard mycelium composites. When cutting the material in half, the granulates are of course still present but are encapsulated in

Small scale prototyping

surface area of the plastic. With the exception of film materials, plastic waste should therefore be grinded or shredded into granulates of comparable size.

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a biofilm of filamentous cells. Based on the preliminary research we can therefore assume that the biodegradation process has been initiated on the surface of granulates.

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The production of mycelium composites seems to provide the perfect environment for structurally implementing plastic waste management. After a heat-treatment for several hours to dehydrate the material, the growth process and thus the degradation process is terminated, delivering a material ready for application in an architectural or design framework.

Given the differences in degradation rate, accurate sorting of plastic waste in types seems crucial however and further research over longer periods in time are of the essence to investigate

Figure 39 Material samples of mycelium composites that contain HDPE granulates within the substrate matrix (left) or attached to the surface

when specific plastic types might be considered completely degraded and ready for composting. Thereby, tests on plastic degradation in multiple cycles of growth, dehydration, use and regrowth of the material might acclimate this process to our fast paced way of living and producing.

Hereafter, the HDPE granulates were loosely strewn on the mycelium surface after which the samples were placed back in the incubator. After a period of five days, Ganoderma L. had attached itself to all loose granulates and the material samples could now be placed vertically without plastic pieces falling off. In the following period, the growing fungus will peak through the gaps between plastic pieces and further colonise the surfaces of the HDPE granulates with the charismatic unpredictability of its organic growth patterns. As such, the fungal degradation of plastics enables time to become a design parameter, gradually turning a richly coloured and textured object into a soft and white one.

Small scale prototyping

In an attempt to explore plastic degradation as a visible expression and fully exploit the time-bound aesthetic of a living material, a series of four mycelium composites was produced in which the degradation process of HDPE granulates is showcased rather than concealed. Analogous to the aforementioned production method, HDPE waste was shredded into pieces of five to seven millimetres and 200 grams of beechwood was hydrated by 200 millilitres of distilled water. Instead of mixing the plastic pieces among the beechwood fibres prior to sterilisation, both were autoclaved in separate containers. The beechwood substrate was inoculated with 60 grams of Ganoderma L. grain spawn and left in the dark at 26°C to grow for a week, then transferred to a mould and left to grow for another week.

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7.1.2

LDPE-COATED CARDBOARD AS A SUBSTRATE

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Figure 40 LDPE-coated cardboard waste colonised by Ganoderma L.

Whereas HDPE waste still has the potential to be melted and remoulded to form new recycled products, this is not at all the case where product designers use plastic as a coating in multi-layered materials where plastic is often used in combination with other plastic types or other materials such as cardboard and aluminium. To enable recycling of this type of composites, the different materials would first have to be separated and then processed separately too. While efforts are being made in researching end-oflife protocols for this, to date most multi-layers are by default headed to the landfill or incineration plant but nonetheless increasingly used in different applications (Kaiser et al., 2018; European Commission, 2020).

The experiment is now reissued using Ganoderma L. in a LDPE coated cardboard substrate, supplemented with corn starch respecting the same proportions of substrate, spawn and additives. Therefore, LDPE coated cardboard was obtained from disposable coffee cups, which were shredded into pieces of maximum four by four centimetres. After a single growth phase of ten days in a polypropylene mould, the sample reached full colonisation of the substrate (Figure 41).

Figure 41 Shredded LDPE-coated cardboard (top); material sample of a mycelium composite with LDPEcoated cardboard as substrate (bottom)

Small scale prototyping

In this paragraph we investigate whether one of these unsustainable composites, LDPE coated cardboard, could qualify as a valuable resource for the production of mycelium composites. Whereas the separation of material layers poses the main problem in the search for end-of-life possibilities, it is exactly the combined layout of this waste stream that could favour fungal bioremediation and makes it a promising substrate alternative. In chapter 4.2.2, the potential of cardboard waste as a breeding ground for fungi has already been experimentally corroborated, reaching full colonisation of Pleurotus O. after only ten days in a mix with corn starch or after fourteen days when supplemented with sugar or calcium and gypsum.

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Given the successful production of a small scale material sample, a prototype of a building block (60x40x20 cm) was fabricated using three hundred disposable coffee cups, which were shredded by means of a garden chipper and mixed with beechwood chips in a 2:3 volumetric ratio. After two weeks of growth in the mould, full substrate colonisation was established. A one-week growth period outside the mould thereby ensured the formation of a thick skin on the surface, resulting in a sturdy and lightweight building block with an appealing irregularity of colour and texture.

Small scale prototyping

Figure 42 Prototype of a mycelium composite building block, grown on a substrate containing 300 disposable coffee cups and beechwood chips

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7.1.3

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PLA AS A BIODEGRADABLE MOULD FOR THE PRODUCTION OF MYCELIUM COMPOSITES

During the production process in the lab, polypropylene containers and growthbags were typically resorted to as moulds, defining the final shape of the material and providing an ideal microclimate with a constant humidity and filtered air-flow.

The polypropylene moulds are of course reusable, but only to some extent and limit the exploration of free form. Geometrically, mycelium composites are incredibly versatile however and only limited in form by the shape of the mould in which they are created. Therefore, the bioremediating potential of Ganoderma L. is now exerted to, not with the purpose of managing plastic waste streams, but to facilitate the investigation of establishing a symbiotic relationship between the material and the mould itself. That is to say that an inherently biodegradable plastic such as PLA, created from renewable resources, could serve as a temporary form and structure provider during the time mycelium gains its own structural relevance, eventually degrading the PLA mould and thus making the production process wasteless.

In a first attempt, two cylindric moulds with a diameter of 7 centimetres and height of 25 centimetres were printed using standard PLA 3D-printing filament on the one hand and the composite woodfibre-PLA, studied in chapter 6.1, on the other hand. Thereby, the wall thickness of the print was set to a minimum value of 0,4 millimetres. The cylindric envelopes were filled with a beechwood fibre substrate and inoculated by means of Ganoderma L. grain spawn.

Figure 43 woodfibre-PLA cylindric mould with a wall thickness of 0,4 mm, two weeks post inoculation by Ganoderma L.

Small scale prototyping

Two weeks post inoculation date, the woodfibre-PLA cylinder had started to exhibit mycelium growth on the exterior of the mould and a full substrate colonisation could therefore be assumed (Figure 43).

Bulk erosion as the occurring degradation method of the mould, allows Ganoderma L. to grow through the form-defining envelope, favouring its biodegradation.

The standard PLA cylinder also displays full substrate colonisation at this point and is causing some print-line fractures along the cylinder surface (Figure 44). However, growth on the exterior surface of the print is not as easily established given that surface erosion is the occurring method of degradation for standard PLA filament.

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Figure 44 standard PLA cylindric mould with a wall thickness of 0,4 mm, two weeks post inoculation by Ganoderma L.

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Colonisation of both the interior and exterior of the mould should however be pursued to increase the degradation rate of the temporary envelope. The minimum wall thickness that can be printed by a standard 3D-printer is 0,4 millimetres, which corresponds to the width of one print line. A further reduction of this parameter requires advanced equipment.

Nonetheless, the printing process can be tweaked by reducing the inflow of PLAfilament relative to the printing speed. In doing so, a lesser amount of material is resorted to while printing the same distance on a print line and the porosity of the print can be increased. This does however have direct implications on the structural integrity of the print. To find the desired equilibrium, a cylinder with the same dimensions was printed with a gradual filament inflow decrease along the height of the print; starting at 100% at the bottom decreasing in steps of 5% to reach 45% filament inflow at the top.

A clear distinction along the height of the cylinder can be observed at which point the porosity of the mould allows Ganoderma L. to grow through the PLA shell and colonise its outer surface as well. (Figure 45) This happens where the filaments inflow parameter reaches 60%, which will be the preferred setting for further testing as the structural integrity of the print only decreases at lower values.

Figure 45 standard PLA cylindric mould with a wall thickness of 0,4 mm and a gradual filament inflow decrease along its height (from 100% at the bottom to 45% at the top. At a filament inflow of 60%, growth on the exterior of the mould can be observed.

Small scale prototyping

Hereupon, a full standard PLA cylindric mould was printed at 60% filament inflow. The porosity of the mould attained by these settings permits oxygen to reach the growing fungus along the entire envelope, highly favouring the colonisation of both the substrate on the interior as well as the PLA surface forming the exterior of the mould. The degradation of the mould is thereby augmented, but one loses somewhat control over the shape of the final result as the fungus starts to use the predefined shape as a guideline for its growth rather than as a strict form-defining boundary.

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Figure 46 standard PLA cylindric mould with a wall thickness of 0,4 mm and a filament inflow of 60%, two weeks post inoculation by Ganoderma L.

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The affirmed viability of printing biodegradable PLA moulds, supports a possible convergence between digital and biological manufacturing as a multi-scalar construction practice. Despite growth being prevented by contamination; as an impetus for further research on parametric architecture that combines additive manufacturing with biological processes of growth, a final mould was created with an algorithm based on recursive systems defined by the input of an initial generation and its mechanism of explorative growth (Figure 47).

Figure 47 standard PLA mould that explores the possible convergence of digital and biological manufacturing, two weeks post inoculation by Ganoderma L. (no established growth due to contamination).

Small scale prototyping

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8

Practice what you preach A DIY PROTOCOL FOR LOCAL PLASTIC WASTE MANAGEMENT

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As a conclusion of the material research and a start of an open dialogue for contemplation on possible applications, this chapter documents the process of tackling a local waste stream and formulates a DIY production protocol for its bioremediation and valourisation. The research thus far has been explorative.

More methods of how to incorporate plastic bioremediation in the production of mycelium composites are to be examined and a profound study is of the essence to investigate when specific plastic types might be considered completely degraded and the material is ready for composting. However, the indicated potential of creating low-cost building materials through local waste management as a social and ecological act that can be picked up by anyone and that might serve as a catalyst for change on a global and industrial level, is promising and encouraged me to return to a DIYenvironment and build a full scale wall segment.

The aim is to build a walll segment that – not unequivocally – resorts to degradation as construction method, and on the other hand resorts to design as a tool for clearly narrating the process of degradation, evoking a critical thinking and a revolutionised awareness about waste management and the way it is practiced. As such, the wall segment will be constructed from a local plastic waste stream, using mycelium composites as a vessel for its bioremediation, as well as a canvas that showcases the plastic degradation process. The production protocol was documented in the following pages, providing tangible insight on realworld execution of the proposed material concept and its adjoining practical complications.

Selecting a local waste stream. Based on the successful small-scale production of material samples in the previous chapter, LDPE-coated cardboard waste was selected as substrate given its lack of recycling options and its local availability in the form of disposable cups: abundantly used in the city of Brussels by all fast-food and fast-coffee chains.

However, the collection of used disposable cups turned out to be quite the undertaking.

Given its lack of recycling options, disposable cups end up in the residual waste bin after their very short lifespan. Gathering this waste stream for allocation as a resource thus implies getting your hands dirty by manual sorting of trash, for which I was granted permission to do at local Starbucks affiliates. A labour intensive process that nonetheless quickly delivers results. After a few days of sorting through the trash and with a helping hand from ZNA Middelheim and GO! Campus Wemmel, a total amount of 3000 used coffee cups were gathered

Practice what you preach

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2

Shredding waste stream into pulp or granulates.

The 3000 used disposable cups were shredded into a pulp by means of a garden shredder. Since LDPE is present in the form of a film, a maximum surface area to volume ratio is self-evident and the shredding process only facilitates the ease of handling and the density of the substrate.

Since the rate of biodegradation is functionally dependent on the surface area of the plastic, the shredding process gains importance when selecting a bulky plastic waste stream and small granulates of comparable size should be pursued. This would however require an industrial shredder.

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Preparation of the substrate. It has been experimentally corroborated that the presence of lignin serves as a catalyst for bioremediation of plastics (chapter 6.2). The pulp obtained in step 2 is therefore mixed with beechwood chips in a 2:3 volumetric ratio and collected in reusable laundry nets. Growth rate enhancing additives such as grape seed, corn starch, sugar or cat food can be added in this stage but seemed to highly increase the risk of contamination for DIY production methods (chapter 4.2.2). Hence, no additives were supplemented.

Practice what you preach

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Pasteurisation of the substrate. The substrate is now sterilised by means of pasteurisation, meaning that the laundry nets, containing the substrate, are boiled one by one in a large pot of water for one and a half hour. Ensuing, the nets are taken out of the pot and hung up until they no longer leak water. The substrate is then transferred to sterile closed boxes where it can cool down until reaching room temperature.

Construction of moulds. Despite the promosing prototypes of biodegradable PLA moulds in chapter 7.1.3, the time consuming nature of 3D-printing and its limitations of scale were an obstacle and questions the application of this technique on a larger scale. Hence, a series of nine reusable moulds with a dimension of 60x40x20 cm was constructed from laminated chipboard.

Practice what you preach

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Inoculation. To minimise the use of plastic, the preliminary growth phase in polypropylene growth bags is left out. After sterilising the mould and workspace, the mould is directly filled with the pasteurised substrate and supplemented with 10% of the substrate weight of Ganoderma L. grain spawn. A homogeneous distribution of substrate and grain spawn is achieved through a process of layering and manual mixing. The mixture is then pressed by hand to obtain a higher density, after which the mould is sealed with a single growth bag that was cut open. The air filters in the growth bag facilitate the inflow of oxygen and release of CO2 during the growth of the fungus, without increasing the risk of contamination.

First period of growth. The building blocks are now left to grow in a small dark room, where the temperature can be easily regulated. Growth rates depend on the size of the object and are of course variable given the unattainability of a constant temperature and relative humidity in a DIY environment. However, a fairly constant temperature of 26°C was pursued. After one week, the substrate was again manually mixed to ensure a homogenous colonisation throughout the whole substrate. The moulds were then sealed again and left to grow in the dark for another week.

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Adding HDPE granulates on the top surface (optional).

To showcase the degradation of plastic waste as an aesthetic feature, one side of the building blocks is now covered with a layer of colourful HDPE granulates. Shredded HDPE waste was provided by the Antwerp-based non-profit organisation Sheep On Wheels, that is committed to collecting local plastic waste and shredding it for reuse in the design of recycled products. The granulates were pasteurised and subsequently loosely strewn on the mycelium surface after which the moulds were sealed again.

Second period of growth. A second growth phase is now initiated until Ganoderma L. attaches itself to all loose granulates and the building blocks can be placed vertically without plastic pieces falling off. With this objective, the building blocks are left in the mould for another week.

If granulates were not added on the mycelium surface in step 8, a second growth phase outside the mould is recommended to solidify the outer skin of the material.

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Terminating the growth: After full colonisation of the substrate and a visible formation of a thick white skin wrapping the entire building block, the growth process and thus the degradation process is terminated by heating the panel at 80°C until completely dehumidified, ensuring a stable end product.

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For the purpose of creating a living canvas for the degradation process of plastics, the growth was not terminated in this project, allowing the fungus to further colonise the surfaces of the HDPE granulates with the charismatic unpredictability of its organic growth patterns.

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Practice what you preach

Figure 48 Wall segment resulting from the protocol described on page 89-100 at the day of unmoulding the building blocks

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9 Conclusion 104

The pursuit of a circular approach to compete with the tradition of linear resource extraction and disposal, is a phenomenon that surpasses industries and that is highly compromised by economic principles and established practices that put mass production on a pedestal. Valourising waste as a resource and acknowledging the importance of biological remediation, might bring us one step closer to coming full circle in being sustained by the constant transformation of matter. Therefore, this thesis has set out to investigate whether the current material practice of mycelium composites could valourise plastic waste and inject the bioremediation process of plastics into an economically viable material with architectural purpose.

Through a preliminary screening of three fungal species that are currently used in the production of mycelium composites, all three have shown potential for the bioremediation of recalcitrant compounds, like plastics. Thereby, Ganoderma Lucidum has come to the fore as the most promising microorganism (i.e. from the limited selection) for combining the biodegradation of plastic waste with the production of mycelium composites and has shown its capacity to utilise recalcitrant polymers as a primary food source.

An indication of the degradation rate of polyethylene and PLA by Ganoderma L. was experimentally determined. In a simple test setup, the maximum recording for the percentage of the total weight loss of the HDPE and LDPE samples after fifty days of incubation were found to be 0,34% and 6,67% respectively. Whereas for PLA, a maximum percentage of the total weight loss of 6,49% was found. These results are promising and reassure the potential of targeted biodegradation of plastics by isolated fungi, having in mind that Otake et al. (1995) reports similar findings of LDPE surface changes after soil burial for over 32 years and that PLA shows no degradation over the span of a year in a marine environment (Bagheri et al., 2017).

Conclusion

Figure 49 Wall segment resulting from the protocol described on page 89-100, one week after unmoulding the building blocks

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Optimisation of the degradation rate of plastics by Ganoderma L. is beyond the scope of this research but is undoubtedly possible and demands both longer observation of the degradation process and close transdisciplinary collaboration. The achieved degradation percentages in microbiological research are therefore of a different order of magnitude. Weight loss percentages of 58,6% and 34,4% were reported

by Ojha et al. (2017) for HDPE and LDPE sheets respectively after ninety days of fungal biodegradation and according to Hidayat et al. (2012) 12, 21, 30 and 48% of polylactic acid samples were degraded in 1, 2, 3 and 6 months.

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The importance of pursuing a maximum surface area to volume ratio when exposing hydrophobic polymers to biological degradation came to the fore. Moreover, the presence of lignin displayed to have a positive effect on the biodegradability of plastics; serving as a catalyst for the production of oxidative enzymes when present in the growth medium and allowing bulk degradation when present in the polymer matrix (chapter 6). However, the findings in this writing only serve as an explorative indication and conceptual confirmation for the feasibility of implementing the biodegradation of plastics in the production process of mycelium composites.

Optimising the biodegradation process could encompass a pre-treatment of the polymer, an adjustment of the growth conditions such as temperature and pH, or an alteration in the composition of the growth medium. As such, physical and chemical treatments of the polymer before biodegradation, including UV irradiation, photooxidation, thermal treatment and oxidation with nitric acid have proven to increase its effectiveness (Pathak et al., 2017). Moreover, Copinet et al. (2004) reports that the degradation rate of PLA can be enhanced by increasing temperature and relative humidity and Jarerat et al. (2001) makes the surprising observation that Tritirachium A. shows no PLA film degradation in liquid culture using basal medium, but displays 76% degradation after only 14 days when supplemented with 0,1% of gelatine.

Although preliminary, the explorative research and small scale prototyping with plastic-mycelium composite materials have put into perspective the possibilities, limitations and areas for further research of implementing plastic degradation within a material practice, and more broadly within the realm of architecture and design.

In an effort to reduce the dependency on polypropylene moulds in the production process of mycelium composites, the investigation of utilising temporary PLA moulds was successful in the way that an inherently biodegradable plastic is exploited for its malleable qualities, with a clear vision on its end-of-life destination: directly employed in an environment in which it can biologically degrade. Its success is however limited to production in laboratory conditions where the relative humidity and cleanliness of air flow can be closely monitored and restricted in scale by the dimensions of available 3D-printing equipment. Furthermore, successful attempts were made in resorting to HDPE waste streams with limited end-of-life prospects or LDPE-coated multilayers that currently have none, to produce material samples that show an architectural purpose.

The findings were put into practice in the form a small scale building project in which 3000 disposable coffee cups were resorted to as a resource to construct a living wall segment that clearly narrates the process of biodegradation, evoking a critical thinking about waste management and the way it is practised. A physical representation that promotes awareness of the material practice and opens the dialogue for a transdisciplinary and holistic approach towards material innovation on a very direct and tactical level. The established production protocol in a DIY environment thereby stresses the potential of mycelium composites to establish a local, community-based approach to cultivating low-cost materials and manage local waste streams that can be picked up by anyone and might serve as a catalyst for change on a global and industrial level.

Conclusion

The importance was mentioned of further experimentation over longer periods in time, which are of the essence to investigate when specific plastic types might be considered completely degraded and the material is ready for composting.

Hereby, tests on plastic degradation in multiple cycles of growth, dehydration, use and regrowth of the material might acclimate this process to our fast paced way of living and producing.

107

108

Annexes

10 Annexes 10.1 Phatak et al. (2017): List of microorganisms reported to the degrade different types of plastics Type of polymer

Microorganisms

References

Polyethylene

Brevibacillus borstelensis, Comamonas acidovorans TB-35, Pseudomonas chlororaphis, P. aeruginosa, P. fluorescens, Rhodococcus erythropolis, R. rubber, R. rhodochrous, Staphylococcus cohnii, S. epidermidis, S. xylosus, Streptomyces badius, S. setonii, S. viridosporus, Bacillus amyloliquefaciens, B. brevis, B. cereus, B. circulans, B. circulans, B. halodenitrificans, B. mycoides, B. pumilus, B. sphaericus, B. thuringiensis, Arthrobacter paraffineus, A. viscosus, Acinetobacter baumannii, Microbacterium paraoxydans, Nocardia aster- oides, Micrococcus luteus, M. lylae, Lysinibacillus xylanilyticus, Aspergillus niger, A. versicolor, A. flavus, Cladosporium cladosporioides, Fusarium redo- lens, Fusarium spp. AF4, Penicillium simplicissimum YK, P. simplicissimum, P. pinophilum, P. frequentans, Phanerochaete chrysosporium, Verticillium lecanii, Glioclodium virens, Mucor circinelloides, Acremonium Kiliense, Phanerochaete chrysosporium

(Dussud and Ghiglione 2014; Shah et al. 2008a; Kale et al. 2015a, b; Grover et al. 2015; Restrepo-Flรณrez et al. 2014; Bhardwaj et al. 2012a)

Polyvinyl chloride

Pseudomonas fluorescens B-22, P. putida AJ, P. chlororaphis, Ochrobactrum TD, Aspergillus niger

(Dussud and Ghiglione 2014; Shah et al. 2008a; Shah et al. 2008a; Kale et al. 2015a, b; Bhardwaj et al.

2012a)

Polyurethane

Comamonas acidovorans TB-35, Curvularia senegalensis, Fusarium solani, Aureobasidium pullulans, Cladosporium sp., Trichoderma DIA-T spp., Trichoderma sp., Pestalotiopsis microspora

(Dussud and Ghiglione 2014; Shah et al. 2008a; Shah et al. 2008a; Kale et al. 2015a, b; Bhardwaj et al.

2012a)

Poly(3-hydroxybutyrate)

Pseudomonas lemoignei, Alcaligenes faecalis, Schlegelella thermodepolymer- ans, Aspergillus fumigatus, Penicillium spp., Penicillium funiculosum

(Dussud and Ghiglione 2014; Shah et al. 2008a; Kale et al. 2015a, b; Bhardwaj et al. 2012a)

Poly(3-hydroxybutyrate- co-3-hydroxyvalerate)

Clostridium botulinum, C. acetobutylicum, Streptomyces sp. SNG9

(Dussud and Ghiglione 2014; Dussud and Ghiglione 2014; Shah et al.

2008a; Bhardwaj et al. 2012a)

Polycaprolactone

Bacillus brevis, Clostridium botulinum, C. acetobutylicum, Amycolatopsis sp., Fusarium solani, Aspergillus flavus

(Dussud and Ghiglione 2014; Dussud and Ghiglione 2014; Shah et al.

2008a; Bhardwaj et al. 2012a)

Polylactic acid

Penicillium roquefort, Amycolatopsis sp., Bacillus brevis, Rhizopus delemar

(Shah et al. 2008a)

109

10.2 Critical Concrete Summer School (2018): investigation of locally available resources that could serve as substrates and additives for the production of mycelium composites: a recording of the development of material samples over the course of the growth period of Pleurotus O.

110

Annexes

Ca + G

Gs + M + G

S

S + Cf

Additive

150

100

50

130

130

Wet substrate weight

12,5(S) + 12,5(Cf)

30(M)

2(Ca) + 4(G)

5(Gs) + 2,5(M) + 2,5(G)

26(S)

13(S) + 13(Cf)

Additives: 20% of the wet substrate weight

20

12,5

15

10

5

13

13

Quantities (g)

M 125 20(S) + 20 (Cf)

20

Components

S + Cf 200 40(S)

5

brick 3

brick 6 brick 7 brick 8

Cotton

Cardboard

Gs + M + G

S

S + Cf

S + Cf

155

50

155

155

130

32(M)

3(Ca) + 6(G)

5(Gs) + 2,5(M) + 2,5(G)

31(S)

15,5(S) + 15,5(Cf)

13(S) + 13(Cf)

12,5

16

15,5

5

15,5

15,5

13

brick 9

Substrate

S + Cf 200

5(Gs) + 2,5(M) + 2,5(G)

17

20

Grain spawn: 10% of the wet substrate weight

S 50

34(M)

4(Ca) + 8(G)

brick 1

brick 4

Gs + M + G

170

200

brick 2

brick 5

M

Ca + G

Straw

brick 11

brick 10

Ca + G

160

12,5(S) + 12,5(Cf)

Sawdust

M

125

brick 12

brick 16

S + Cf

brick 15

brick 14

brick 13

brick 17 Coffee ground

9

Cork

brick 18

9(S) + 9(Cf) Gs: Grape seed

90

M: Maïzena

S + Cf Ca: Calcium

S: Sugar

Flax fibre

Cf: Cat food

brick 19

G: Gypsum

•

•

•

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•

21°C

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Day 12

21°C

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Day 13

25°C

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Day 14

27°C

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Day 15

27°C

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Day 16

21°C

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Day 17

21°C

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Day 18

20°C

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Day 19

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Cooking time

Drying time

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Inoculation period

•

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Day 11

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21°C

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22°C

Day 10

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Day 9

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20°C

Day 7

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Day 6

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23°C Day 5

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Day 3

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25°C

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21°C Day 2

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Day 1

111

112

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