Applied Science, Faculty of
Chemical and Biological Engineering, Department of
DSpace
UBCV
French, Victoria Marie
Doctor of Philosophy - PhD
University of British Columbia
This thesis explores the engineering of pure mycelium materials as sustainable, functional alternatives to conventional synthetic materials for pollution control. By systematically investigating the influence of fungal species and bioprocessing methods, this work demonstrates that mycelium networks can be tailored to yield materials with tunable network morphology, mechanical strength, and surface chemistry. Compared to liquid state fermentation, solid-state fermentation produced dense mycelial mats with high chitin content and superior mechanical properties. Submerged fermentation methods enabled the formation of porous sheets with high strength values relative to static fermentation methods and a high mannoprotein content. These findings reveal the tunability of fungal material properties and underscore the importance of bioprocess control in designing materials for specific applications. Building on these insights, mycelium-derived materials were applied to address pressing environmental challenges in air and water remediation. Mycelium-modified mask layers achieved high particulate filtration efficiency (up to 97% for PM2.5) with breathability comparable to commercial masks and exhibited asymmetric hydrophobicity for improved moisture management. In water treatment, mycelium membranes demonstrated high adsorption capacities for heavy metals (up to 73.2 mg/g for Cu(II)), outperforming other low cost, biobased adsorbents, and retained performance over multiple regeneration cycles. Hybrid membranes incorporating cellulose nanofibrils further balanced water flux and metal rejection. Additionally, mycelium-based membranes with immobilized laccase were produced for the effective degradation of phenolic pollutants, maintaining high catalytic activity and stability over repeated use. Application of the enzyme-mycelium membrane to water contaminated with azo dye Congo Red resulted in a reduction of 92% of dye using optimal immobilization conditions. By bridging fungal biology, materials science, and environmental engineering, this thesis highlights mycelium as a next-generation sustainable technology. This work demonstrates that materials can be grown, not manufactured, providing solutions for pollution mitigation while reducing reliance on petrochemicals.
Mycelium as an Advanced Functional Material by Victoria Marie French B.S., Virginia Polytechnic Institute and State University, 2020 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2025 © Victoria Marie French, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Mycelium as an Advanced Functional Material submitted by Victoria Marie French in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemical and Biological Engineering Examining Committee: E. Johan Foster, Professor, Chemical and Biological Engineering, UBC Supervisor Scott Renneckar, Professor, Forestry, UBC Supervisory Committee Member Charles Haynes, Professor, Chemical and Biological Engineering, UBC Supervisory Committee Member Susan Baldwin, Professor, Chemical and Biological Engineering, UBC University Examiner Steven Hallam, Professor, Microbiology, UBC University Examiner Kevin Edgar, Professor, Sustainable Biomaterials, Virginia Tech External Examiner iii Abstract This thesis explores the engineering of pure mycelium materials as sustainable, functional alternatives to conventional synthetic materials for pollution control. By systematically investigating the influence of fungal species and bioprocessing methods, this work demonstrates that mycelium networks can be tailored to yield materials with tunable network morphology, mechanical strength, and surface chemistry. Compared to liquid state fermentation, solid-state fermentation produced dense mycelial mats with high chitin content and superior mechanical properties. Submerged fermentation methods enabled the formation of porous sheets with high strength values relative to static fermentation methods and a high mannoprotein content. These findings reveal the tunability of fungal material properties and underscore the importance of bioprocess control in designing materials for specific applications. Building on these insights, mycelium-derived materials were applied to address pressing environmental challenges in air and water remediation. Mycelium-modified mask layers achieved high particulate filtration efficiency (up to 97% for PM2.5) with breathability comparable to commercial masks and exhibited asymmetric hydrophobicity for improved moisture management. In water treatment, mycelium membranes demonstrated high adsorption capacities for heavy metals (up to 73.2 mg/g for Cu(II)), outperforming other low cost, biobased adsorbents, and retained performance over multiple regeneration cycles. Hybrid membranes incorporating cellulose nanofibrils further balanced water flux and metal rejection. Additionally, mycelium-based membranes with immobilized laccase were produced for the effective degradation of phenolic pollutants, maintaining high catalytic activity and stability over repeated use. Application of the enzyme-mycelium membrane to water iv contaminated with azo dye Congo Red resulted in a reduction of 92% of dye using optimal immobilization conditions. By bridging fungal biology, materials science, and environmental engineering, this thesis highlights mycelium as a next-generation sustainable technology. This work demonstrates that materials can be grown, not manufactured, providing solutions for pollution mitigation while reducing reliance on petrochemicals. v Lay Summary This research explores how fungi, specifically their root-like networks called mycelium, can be grown and processed to create new, sustainable materials for cleaning air and water. By adjusting how the mycelium is grown, materials were generated with selectable properties such as strength, flexibility, water-resistance, and chemical composition. These mycelium materials were used to improve face masks, making them better at filtering air pollution while remaining comfortable and compostable. Mycelium membranes were also developed that can remove harmful metals and chemicals from water and showed they can be reused multiple times. Compared to traditional materials made from plastics or other chemicals, mycelium-based materials are cheaper to produce, can be made from agricultural waste, and break down naturally, reducing environmental impact. This work demonstrates that fungi can help us create greener solutions for pollution and move toward a more sustainable future. vi Preface This dissertation consists of original, independent, unpublished work (Chapters 1, 2, and 5) by author V. French as well as collaborative and published works (Chapters 3 and 4). Details for chapters 3 and 4 can be found below. Chapter 3 consists of work conducted at the University of British Columbia by Victoria French, Chuanshen Du, and E. Johan Foster. Victoria French, Chuanshen Du, E. Johan Foster, Mycelium as a self-growing biobased material for the fabrication of single-layer masks, Journal of Bioresources and Bioproducts, Volume 8, Issue 4, 2023, Pages 399-407, ISSN 2369-9698, https://doi.org/10.1016/j.jobab.2023.07.001. V. French was responsible for conceptualization, sample preparation, data analysis, and manuscript preparation. C. Du contributed to manuscript revisions. Chapter 4 consists of work conducted at the University of British Columbia by Victoria French, Maya Kleiman, Kabir Bhalla, Raphaell Moriera, Adel Jalaee, James W Kronstad, and E. Johan Foster. Victoria French, Maya Kleiman, Kabir Bhalla, Raphaell Moreira, Adel Jalaee, James W. Kronstad, E. Johan Foster, Fungal-derived adsorption membrane to capture potentially toxic elements, Chemical Engineering Journal, Volume 488, 2024, 151028, ISSN 1385-8947, https://doi.org/10.1016/j.cej.2024.151028. V. French was responsible for conceptualization, methodology, investigation, data curation, formal analysis, and writing (original draft). M. Kleiman was responsible for conceptualization, data curation, and writing (review and editing). Kabir Bhalla contributed to data curation (culturing and cell plate analysis), formal analysis, and writing (review and editing). Raphaell Moreira contributed to data curation (XPS), formal analysis, and writing (review and editing). Adel vii Jalaee contributed to data curation (DMA), formal analysis, visualization, and writing (review and editing). The use of Generative AI (Perplexity) was utilized in this thesis to aid in grammar, style, and clarity of writing. Prompts were specified to ensure that only language and structure, and not ideas or concepts, were changed. viii Table of contents Abstract ................................................................................................................................... iii Lay Summary ............................................................................................................................ v Preface ..................................................................................................................................... vi Table of contents .................................................................................................................... viii List of tables ........................................................................................................................... xii List of figures ......................................................................................................................... xiii Acknowledgements .................................................................................................................. xx Chapter 1: Introduction ............................................................................................................ 1 1.1 Research motivations ................................................................................................ 1 1.2 Background ............................................................................................................... 3 1.2.1 The need for clean natural resources .................................................................... 3 1.2.2 Biologically inspired solutions for pollution control ............................................. 6 1.2.3 Filamentous fungi ................................................................................................11 1.2.4 The use of mycelium as a material ......................................................................18 1.3 Research aim and objectives ....................................................................................24 1.4 Research questions ...................................................................................................24 1.5 Research road map ...................................................................................................26 1.6 Scope and guiding principles ...................................................................................28 Chapter 2: Preparation and Characterization of Pure Mycelium Materials .........................30 2.1 Introduction ..............................................................................................................31 2.2 Experimental methods and materials .....................................................................33 2.3 Results and discussion .............................................................................................37 2.3.1 Bioprocessing design and production of pure mycelium materials .....................37 2.3.2 Structural differences of pure mycelium materials .............................................42 ix 2.3.3 Cell wall analysis of pure mycelium materials ...................................................47 2.3.4 Material properties of PMMs ...............................................................................50 2.3.5 Selection of applied mycelial mats .......................................................................53 2.3.6 Environmental and economic considerations ......................................................55 2.4 Conclusions ..............................................................................................................56 Chapter 3: Mycelium-modified masks for air pollution control .............................................59 3.1 Introduction ..............................................................................................................59 3.2 Experimental methods .............................................................................................64 3.3 Results and discussion .............................................................................................68 3.3.1 Morphology and composition ...............................................................................68 3.3.2 Filtration efficiency and breathability .................................................................72 3.3.3 Mechanical properties ..........................................................................................77 3.3.4 Conclusions ...........................................................................................................78 Chapter 4: Mycelium membranes for the capture of potentially toxic elements ...................81 4.1 Introduction ..............................................................................................................81 4.2 Results ......................................................................................................................85 4.2.1 Membrane synthesis and characterization..........................................................85 4.2.2 Elucidating the mechanisms of PTE adsorption .................................................90 4.2.3 Performance as an adsorption membrane for PTE removal ...............................94 4.3 Discussion .................................................................................................................97 4.4 Conclusions ............................................................................................................ 101 4.5 Methods & Materials ............................................................................................. 102 4.5.1 Mycelium pellet cultivation & processing.......................................................... 102 4.5.2 Membrane formation .......................................................................................... 102 x 4.5.3 Characterization ................................................................................................. 103 4.5.4 Batch adsorption ................................................................................................ 104 4.5.5 Regeneration and recycling studies ................................................................... 105 4.5.6 Water flux and metal rejection .......................................................................... 106 Chapter 5: Mycelium membranes as enzyme immobilization platforms for treatment of azo dyes ........................................................................................................................................ 108 5.1 Introduction ............................................................................................................ 108 5.2 Methods & materials .............................................................................................. 111 5.2.1 Preparation of mycelial support ......................................................................... 111 5.2.2 Characterization of mycelial support................................................................. 112 5.2.3 Immobilization of laccase ................................................................................... 113 5.2.4 Effect of immobilization on stability, reusability, and kinetics: ....................... 115 5.2.5 Decolorization and degradation of Congo Red: .................................................. 116 5.3 Results & discussion .............................................................................................. 117 5.3.1 Development of a deacetylated mycelial support .............................................. 117 5.3.2 Optimization of immobilization parameters ...................................................... 121 5.3.3 Comparison of the free and immobilized enzyme operational stability and kinetics ............................................................................................................................ 125 5.3.4 Application of the immobilized enzyme system for dye degradation ................ 129 5.4 Conclusions ............................................................................................................ 133 Chapter 6: Conclusions .......................................................................................................... 136 References .............................................................................................................................. 139 Appendix A – Chapter 2 supplementary information .......................................................... 183 xi Appendix B – Chapter 3 supplementary information .......................................................... 190 Appendix C – Chapter 4 supplementary information .......................................................... 192 Appendix D – Chapter 5 supplementary information .......................................................... 203 xii List of tables Table 1.1. Classifications of hyphal types and relevant species ...........................................15 Table 3.1. Fiber/hypha diameters in mask layers and mycelium-modified mask layers .....69 Table 4.1. Parameter and fit values for kinetic models ........................................................94 Table 4.2. Parameter and fit values for isotherm models .....................................................94 Table 4.3. Comparison of low-cost and biobased adsorbents for Cu(II) ................................99 Table 5.1. Calculated kinetic parameters for free and immobilized laccase ...................... 128 xiii List of figures Figure 1.1. Diverse fungal fruiting bodies of British Columbia. Digital images of mushrooms from Pacific Spirit Park, British Columbia, situated in the ancient, unceded xʷməθkʷəy̓əm (Musqueam) First Nation land. ................................................................................................ 2 Figure 1.2. Evolutionary association and timeline of Fungi, Animals, Plants, and Choanoflagellates. Phylogenetic tree of the Kingdom Fungi is shown in blue further specifying the relationship between 6 contemporarily recognized phyla (solid line) and 2 emerging phyla (dashed line). .................................................................................................13 Figure 1.3. General mycelium and fungal cell wall structures. Graphical depiction of mycelium growing through a substrate (vegetative hyphae) and into the air (aerial hyphae). The bubble on the top left shows the coating of hydrophobin proteins on the outer surface of aerial hyphae arranged as rod-like structures, with hydrophobic residues oriented to the air. The arrangement of major cell wall components, including chitin, glucans, and glycoproteins, is shown on the top right. ........................................................................................................18 Figure 1.4 Publication and patent trends for mycelium materials. (a) Trends in global publications on mycelium materials (Web of Science Core collection database; search term: “Mycelium AND material*”) and patents on mycelium materials (Google Patents; search term: “Mycelium AND material*”). (b) Number of patents on mycelium materials filed by countries. Data extracted from Onorato et al. (96). ................................................................19 Figure 1.5 Road map depicting the progression of ideas and research within this thesis, starting from the development of PMMs to application of PMMs as mask materials, adsorption membranes, and towards phenolics degradation. ................................................27 Figure 2.1. Overview of research for the development of pure mycelium materials (PMMs) .................................................................................................................................................30 xiv Figure 2.2. (a) Graphical depiction of static fermentation methods, solid state fermentation (SSF) and liquid state fermentation (LSF), and agitated fermentation methods, submerged fermentation (SmF) and the resulting fungal material after growth time and processing. (b) Digital images of mycelial mats derived from SSF, LSF, and SmF methods using the species Ganoderma lucidum. ...............................................................................................................38 Figure 2.3. (a) Digital images of selected white-rot fungal species after growth for 7 days on malt extract agar plates with activated charcoal (images depict mycelial growth patterns, not actual SSF, LSF, or SmF samples). Mycelial network structure is shown as white filamentous mass. (b) Map of successful, non-homogenous, and unsuccessful mat formation using different fungal species and fermentation methods. ..............................................................41 Figure 2.4. (a) Scanning electron microscope image of the cross section of Ganoderma lucidum produced from solid state fermentation (GL SSF) showing the full vegetative layer. (b) Scanning electron microscope image of the cross section of Ganoderma lucidum produced from liquid state fermentation (GL LSF) showing distinct aerial, vegetative, and submerged hyphal layers. (c) Methodology for hyphal diameter estimation using ImageJ software showing (i) a representative image of vegetative hyphae captured by scanning electron microscopy, (ii) image adjustment with background subtraction, gaussian 3D blur, and thresholding, and (iii) generated local thickness map with circles applied across hyphal widths. Hyphal diameter distribution of (d) GL SSF and (e) GL LSF derived from ImageJ analysis. ...................................................................................................................................43 Figure 2.5. (a) Scanning electron microscope image of the cross section of pellets produced from submerged fermentation (SmF) using (i) Ganoderma lucidum (GL SmF), (ii) Pleurotus ostreatus (PO SmF), and (iii) Trametes versicolor (TV SmF). (b) Hyphal diameter distribution of (i) GL SmF, (ii) PO SmF, and (iii) TV SmF derived from ImageJ analysis. ......................46 xv Figure 2.6. Structures of monosaccharides from fungal cell walls, including glucose (a), mannose (b), and N-acetyl-D-glucosamine (c). (d) The concentration of monosaccharides relative to the total amount of monosaccharides calculated via HPLC analysis. (e) The concentration of glucosamine relative to the total amount of monosaccharides shown on a different scale for clarity. ........................................................................................................49 Figure 2.7. (a) Stress-strain curves from tensile mode using a Dynamic Mechanical Analyzer (DMA) for static and agitated fermentation methods and different species. (b) Ashby plot of Young’s modulus vs. density showing general material categories as envelopes with the materials generated from this study as stars. (c) Contact angle measurements at t=0s and t=60s for generated mycelial materials with aerial side and vegetative side exposed for static fermentation samples. .............................................................................................................51 Figure 2.8. Radar chart depicting key structure and property values of produced mycelial mats. Values are shown as relative values based on the highest numerical value in each variable. ...................................................................................................................................54 Figure 3.1. Scheme of project methods and representative samples (a) Image of a 3-layer commercial non-surgical face mask and the primary functions of each layer. P1 and P2 refer to different layers of the polypropylene mask; (b) Graphical depiction of the method for production of mycelium-modified masks and the selection of P1 and P2 to be inoculated with fungal mycelium; (c) Graphic of the proposed functionality of a mycelium-modified mask; (d) Optical images of a representative mycelium-modified mask using digital camera (i) and the scanning electron microscope (ii). .....................................................................................62 Figure 3.2. Morphology of mask layers before and after mycelium modification: polypropylene fibers in native mask layers for (a) the innermost layer (P1), and (d) the middle layer (P2), the association of mycelial hyphae and polypropylene face masks in the modified material for (b) P1-M and (e) P2-M and at the cross section for (c) P1-M and (f) P2-M. .......69 xvi Figure 3.3. Chemical and thermal analysis of mask layers and mycelium-modified mask layers: (a) ATR-FTIR analysis of mask layers and mycelium-modified mask layers; (b) TGA analysis of mask layers and mycelium-modified mask layers. ..............................................72 Figure 3.4. Filtration efficiency (FE) and breathability of mask-mycelium composites. Filtration efficiency of original mask layers P1, P2, and a commercial mask (all three original mask layers) and mask layers grown with mycelium P1-M, P2-M over the course of 10, 15, and 20 d. GX refers to the time (X) of the sample growth. No time label indicates the sample was grown for only 10 d. ..........................................................................................................74 Figure 3.5. Contact angle tensiometer potential mechanism for utilization of asymmetric hydrophobicity. (a) Contact angle of P1, P2, P1-M, and P2-M (the dashed line represents a contact angle value of 150°.); (b) Graphic illustrating the top and bottom sides of the mycelium-modified mask layers and the potential dual mechanism for water repellency and water uptake (top refers to the composite side contacting the agar plate and bottom refers to the side interfacing with the air; (c) Representative contact angle images selected for P1, P2, P1-M, and P2-M. ......................................................................................................................76 Figure 4.1. Biobased membrane formation and design. (a) Method for preparing biobased membranes from mycelium and TCNF with growth, blending/homogenizing, and vacuum filtration steps (b) Graphical depiction of the adsorption membrane function, the proposed hierarchical porosity of membrane derived from fungal mycelium with pores generated by the fungal filaments as well as pores on the fungal cell wall, and proposed tunability of mycelial membranes with the addition of TCNFs in terms of flux and, consequently, contact time with pollutants. Image made with BioRender. ..............................................................86 Figure 4.2. Structure of Mycelium Membranes and Modification with Tempo-Oxidized CNFs. (a) Images of membranes without TCNF (i) and with 5wt% TCNF (ii) using a digital camera and x-sectional morphology of membranes using scanning electron microscopy. (b) xvii proposed interaction of TCNFs and mycelial hyphae. Image made with BioRender. (c) Average pore size and calculated BET surface area of membranes 0, 1, and 5wt% TCNF using a meso-scale and micro-scale BET instruments (d) Mesopore size distribution from BET of membranes with 0, 1, and 5wt% TCNF. (e) Filtration of pure water over time for mycelium membranes with 0, 1 and 5wt% TCNF. (f) Swelling and storage modulus at 50°C of membranes with 0, 1 and 5wt% TCNF. ..................................................................................89 Figure 4.3. Batch adsorption kinetic, isotherm and chemical analysis. (a) Adsorption efficiency (qe) over time for M, M-1TCNF, and M-5TCNF and fits of the pseudo-second order model (b) Dependence of adsorption efficiency (qe) on the equilibrium concentration of Cu(II) (Ce) and fits of the Langmuir isotherm model (c) Proposed mechanisms for the adsorption of Cu onto the various functional groups associated with fungal cell wall components. Image made with BioRender. (d) Chemical structures of major fungal cell wall components. Structures created with ChemDraw. (e) high-resolution nitrogen (N) and carbon (C) scans of M membrane before and after adsorption. .............................................................................93 Figure 4.4. Adsorption membrane performance for Cu(II) and other PTE removal. (a) Graphical depiction of the colorimetric assay used to detect Cu(II) concentration before adsorption and after adsorption using the M-1TCNF membrane in a dead-end cell apparatus. Graphic created with BioRender. (b) Images of the M-1TCNF membrane before adsorption, after adsorption, and after regeneration in 0.1M HCl. (c) EDX map of the surface and cross section of the membrane after adsorption. (d) Flux and Cu(II) rejection percentages for M, M-1TCNF, and M-5TCNF. (e) Amount of Cu adsorbed by the M, M-1TCNF, and M-5TCNF membranes over 3 cycles of adsorption and desorption. (f) PTE rejection percent of M-1TCNF for Cu, Zn, and Cd. ..................................................................................................................96 Figure 5.1. Effect of NaOH treatment on cell wall chemistry and structure. (a) Generalized overview of the fungal cell wall and depiction of the deacetylation of the cell wall and removal xviii of glycoproteins. Structures of chitin and chitosan drawn with ChemDraw. (b) Representative FTIR-ATR spectra of original mycelia (OG), pretreated mycelia (PT), and mycelia treated with 30, 45, and 60% NaOH (DA 30, DA 45, and DA 60 respectively). The baseline chosen for calculation of the degree of deacetylation is shown by the grey line and the pink highlight represents the characteristic peak chosen while the grey highlight represents the reference peak chosen. (c) The calculated degree of deacetylation for mycelial samples. (d) relative activity (%) of laccase oxidation of ABTS measured by UV-Vis spectrophotometry for the mycelial samples formed into mats. (e) morphology of NaOH-treated mycelial samples visualized by scanning electron microscopy (SEM). ............................................................. 118 Figure 5.2. (a) Schematic representing the immobilization process with the addition of glutaraldehyde (GA) followed by addition of laccase (lacc). (b) Graphic illustrating the points chosen for a Box-Behnken design of experiments and selected parameter ranges. (c) 3D response surface models and (d) contour plots showing the effect of two variables with the third variable set to the median value. ................................................................................. 122 Figure 5.3. Effect of immobilization on thermal stability, pH stability, storage stability, recyclability, and enzyme kinetics. (a) Effect of temperature on the relative activity of free and immobilized enzymes (b) effect of pH on the relative activity of free and immobilized enzymes (c) effect of storage time on the relative activity of free and immobilized enzymes (d) reusability of immobilized enzymes across 5 cycles (e) effect of substrate concentration on the reaction velocity for free enzymes and (f) immobilized enzyme on mycelial support. ......... 126 Figure 5.4. Degradation of Congo Red by different enzyme systems. (a) Image of Congo Red (CR) solutions after treatment with free laccase (F-Lacc), Immobilized laccase on mycelial support (I-Lacc), immobilized laccase on mycelial support with syringaldehyde in solution (I-Lacc_SA), co-immobilized laccase and syringaldehyde on mycelial mat (I-Lacc_I-SA). (b) Decolorization percent of Congo Red with various enzymatic treatments over time (c) xix proposed mechanism of mediator-assisted decolorization of Congo Red with the possible structures resulting from the oxidation of Congo Red. Structures were generated using ChemDraw. ............................................................................................................................ 131 xx Acknowledgements I would like to express my gratitude to Dr. Johan Foster for his support and belief in my vision to explore mycelium, a material new to our laboratory. As an undergraduate at Virginia Tech, working under his mentorship provided both the perspective and encouragement I needed to pursue my interests with confidence, for which I am sincerely grateful. I hope the mycelial network colonized in our group will be strong enough to thrive for many years to come. I also would like to extend my sincere thanks to my committee members, Dr. Scott Renneckar and Dr. Charles Haynes, for their ongoing guidance and thoughtful encouragement throughout this journey. To Dr. Ruby Osei-Bonsu, my valued friend, colleague, and Vancouver family: we began this path together as uncertain first-year students, and it is a privilege to complete it alongside you. The transition to a new country and the early days in the temporary Westbrook laboratory were challenging, yet those experiences marked the beginning of something remarkable. I continue to be inspired by your endless confidence, persistence, and bright smile in the face of hardship! My dear friend and respected mentor, Dr. Jongjit Chalitangkoon, I can’t thank you enough for the endless support and brainstorming sessions, even when they came as late-night research questions on Whatsapp. Your intelligence and love for the scientific process motivated me deeply during our years working together. Outside of the lab, you are a stellar person and friend. Fighting! I would also like to thank the entire Foster Advanced Materials Group (FAMG). The camaraderie, collaboration, and energy within our group have made this experience truly xxi memorable. Sreenath, Adel, Akash, Fernanda, Shaun, Sara, Philip, Pani, and Samin: your individuality and enthusiasm have enriched my academic and personal life. I look forward to seeing your continued success. My appreciation extends to my collaborators across various departments and schools, including Dr. Maya Klein, Dr. Raphael Moriera, Dr. Joice Kaschuk, and Dr. Jim Kronstad, whose expertise and perspectives have greatly contributed to this work. Thank you especially to collaborator Kabir Bhalla, who provided me with great motivation and a shared love for fungi. Finally, I am deeply thankful for the unwavering support of my family in Virginia: my mother, father, and sister, Christina. Your belief in me sustained me through moments of doubt, and your encouragement was felt across every distance. Mom, your constant reminders that I am “good enough, smart enough, and people like me” have shaped my self-belief. Dad, your support in various roles (technical support, crisis manager, etc.) has been indispensable. Christina, your creativity and wit continue to inspire me. I am profoundly grateful for your love and support, which made this achievement possible. xxii 1 Chapter 1: Introduction 1.1 Research motivations The present research is driven by two overarching motivations that converge to form the central goal of this thesis. The first motivation stems from the escalating global pollution crisis, manifested in both visible and invisible contaminants. Visible contaminants, such as floating debris in our oceans, oil spills, and urban smog, serve as stark reminders of the environmental challenges we face. Equally concerning are invisible pollutants, like endocrine-disrupting pharmaceuticals in our water systems and micro- or nano-plastics, which are equally harmful to land and aquatic systems. These pervasive contaminants are particularly formidable due to limited regulatory frameworks and a lack of widespread strategies to address them. As pollution concerns intensify alongside an increasing global population, addressing these challenges demands innovative and sustainable approaches. In this context, it is our responsibility as scientists and engineers to develop responsible and balanced solutions for the management of natural resources to ensure long-term ecological stability and human wellbeing. The second motivation arises from the fascinating properties of fungi and their intricate interactions in natural ecosystems, an area largely overlooked within the lens of materials science and engineering. Fungi are integral to complex natural systems, a reality evident even during casual mushroom foraging in woodland environments and reinforced through my own exploration of local ecosystems (Figure 1.1). The unique shapes and forms of fungal fruiting bodies are on full display across nature's canvas, evolved to enable survival. While plants and trees are often considered the lungs of the earth, fungi and mushrooms act as the stomach and kidneys of the planet, digesting organic matter and 2 recycling nutrients back into the environment. Their ubiquity and functional diversity in nature suggest untapped potential for novel applications, serving as both a material and source of bioinspiration in this work. This Ph.D. research bridges the gap between these two motivations by investigating fungi for pollution control and environmental remediation while exploring the engineering possibilities of mycelium-based materials. By harnessing the unique properties of fungi, this work aims to address pressing environmental issues while advancing our understanding of unconventional bio-based materials. _____________________________________________________________________________________ Figure 1.1. Diverse fungal fruiting bodies of British Columbia. Digital images of mushrooms from Pacific Spirit Park, British Columbia, situated in the ancient, unceded xʷməθkʷəy̓əm (Musqueam) First Nation land. _____________________________________________________________________________________ "In all things of nature there is something of the marvelous" - Aristotle 3 1.2 Background 1.2.1 The need for clean natural resources The preservation of clean air and water is globally recognized as a paramount issue for sustaining human and environmental health (1,2). Pollution of air, water, and workplace is responsible for 9 million deaths each year globally, representing a 66% increase since the year 2000 (3). Despite advancements in technologies for the treatment and production of these critical natural resources, current methods often exhibit limitations in sustainability and functionality, requiring further innovation. Among the wide array of pollutants, this work focuses on three that have significant environmental and health impacts: particulate matter, potentially toxic elements (commonly referred to as heavy metals), and phenolic contaminants. 1.2.1.1 Particulate matter Particulate matter (PM) is a major global concern preventing clean breathing air, derived from anthropogenic and natural sources, such as wildfire smoke and road dust (4). PM2.5 represents those particles with diameters under 2.5µm and are of particular concern because of their unique ability to travel distances of hundreds of miles, as well as penetrate human lungs and bloodstream. The UN Sustainable Resources Outlook report states that 120 million life years are lost every year to outdoor PM2.5 pollution and 80 million life years due to indoor PM2.5 pollution (5). Strategies to reduce the impact of PM2.5 often focus on mitigation, necessitating behavioral or structural changes at the citizen, business, and government level (6,7). These strategies have the potential to be effective if implemented, as shown by emission decline during COVID-19 pandemic. Reductions in traffic and industrial activities during lockdowns are thought to have resulted in the significant decrease of PM2.5 and other air 4 pollutants in 70% of cities across the globe (8). However, these impacts were only temporary as regulations and activity increased to pre-COVID levels. As a result, other methods are needed to deal with current health impacts of PM2.5 pollution, such as the use of face masks or air purification systems (9,10). However, modern facemasks and air filtration systems utilize fossil fuel-based materials, leading to increased strain on the environment and secondary pollutant generation in the form of micro- and nano-plastics (11). 1.2.1.2 Potentially toxic elements Potentially toxic elements (PTEs) are another form of pollutant that has shown increasing concern over recent years. Lead (Pb), cadmium (Cd), zinc (Zn), copper (Cu), arsenic (As), chromium (Cr), nickel (Ni), mercury (Hg), manganese (Mn), and iron (Fe) represent this category of pollutant (12). These elements can exist in the earth naturally at trace levels and some also support life as components of metabolic processes or as cofactors. However, at high enough concentrations, all of these elements can become toxic to animals, plants, and humans, causing protein denaturation and eventually cell death (13). PTEs can leach into waterways by natural (volcanic incidents, weathering) or anthropogenic (industrial, domestic, agricultural, medicinal, technological sectors) means, often resulting in an initial contamination of soil (14). Methods for removing PTEs, such as coagulation, flocculation, precipitation, ion-exchange membranes, and adsorption, each have notable limitations (15–18). While coagulation, flocculation, and precipitation are cost-effective, they generate large volumes of sludge that require further treatment (19,20). Ion-exchange membranes offer rapid removal but suffer from fouling and limited operational conditions. Although ion-exchange resins do not produce sludge, large volumes of acid and/or base are generated during regeneration (21). Adsorption is widely used due to its simplicity and material versatility, yet challenges with adsorbent regeneration and scalability hinder its industrial 5 application (22). These limitations have hindered the implementation of PTE remediation at large scales, underscoring the need for new approaches (23,24). 1.2.1.3 Phenolic contaminants Along with several PTEs, phenol and phenol derivatives, largely derived from industrial processes, make up a significant portion of the priority pollutant list generated by the US Environmental Protection Agency (25). This is because many phenolic compounds can readily absorb into human skin and gastrointestinal tract, resulting in the formation of reactive intermediates that can interact with proteins. This results in both short- and long-term toxicity, such as carcinogenic and endocrine-disruption effects (26). Physical adsorption, ozonation, electrochemical treatments, and microbial degradation techniques have been studies for the removal of phenolic pollutants, but are often limited by low efficiency, limited operational stability, and high cost (27). The growing challenges posed by PM, PTEs, and phenolic pollutants highlight the limitations of conventional treatment methods, including secondary waste generation and prohibitive costs. Biological systems, such as fungi, plants, and microbes, can offer inherently sustainable alternatives to combat these limitations while offering novel functionalities due to their evolved metabolic adaptations for pollutant degradation. By examining these natural mechanisms, from enzymatic pathways to symbiotic relationships, we can identify eco-friendly strategies to combat pollution and protect health. Section 1.2.2 will introduce some of the current literature on bio-based strategies for pollution control. 6 1.2.2 Biologically inspired solutions for pollution control Nature offers a wealth of strategies for environmental regulation, with organisms evolving complex systems to interact with gases, water, contaminants, and competitors. By understanding, using, and/or emulating components of these biological systems, novel sustainable methods have been researched to produce clean air and water. Several of these strategies will be outlined below with particular attention to methods using fungi, although this review is by no means exhaustive. 1.2.2.1 Bioremediation Numerous strategies to remove pollutants rely on living organisms by harnessing metabolic and/or enzymatic processes. Microbial bioremediation uses microorganisms to break down or transform pollutants into smaller and/or detoxified compounds. Yeasts, bacteria, fungi, and algae are commonly used groups of microbes used for bioremediation and can be applied in situ, such as composting, or ex situ, such as in a bioreactor (28). Bioremediation has been studied for a wide variety of pollutants, including PTEs, organic pollutants (hydrocarbons, pesticides, dyes, microplastics, etc.), and emerging contaminants (pharmaceuticals, per- and poly-fluoroalkyl compounds) (29,30). Emerging scientific work has widened bioremediation applications and improved efficacy by using genetically engineered microorganisms and a microbial consortia (29). 1.2.2.2 Bio-oxidation Bio-oxidation is a targeted bioremediation strategy that uses oxidative enzymes from bacteria, fungi, and algae to degrade organic pollutants (31). These bio-oxidant enzymes, such as laccases, tyrosinase, peroxidases, and cytochrome P450, can be secreted directly by microorganisms or extracted from microbes and plants for indirect use (32). Oxidoreductases 7 are particularly valuable due to the broad substrate specificity of some enzymes within this group (33). Particularly, laccase enzymes, involved in lignin degradation, show a broad substrate specificity, with the ability to degrade several aromatic and multi-substituted compounds such as phenols, chlorophenols, nitrophenols, and bisphenols (33,34). While both bacteria and fungi produce lignin-modifying enzymes, fungal enzymes, especially those from white rot fungi, are more effective because of their higher redox potential and robust lignin-degrading systems (35). This makes them especially useful for breaking down persistent pollutants like azo dyes and pharmaceutical compounds, achieving high removal rates (36,37). Compared to traditional chemical oxidation, bio-oxidation offers similar efficacy at a lower cost (32). 1.2.2.3 Biosorption and bioaccumulation Biological systems interact directly with aqueous pollutants through biosorption (surface binding) and bioaccumulation (intracellular uptake), mechanisms critical for contaminant sensing, enzymatic breakdown, and metabolic assimilation (38). Biosorption is a passive process requiring nonliving biomass or microorganisms and dominates recent research due to its cost-effectiveness and operational advantages. In contrast, bioaccumulation relies on living organisms, combining passive biosorption with energy-intensive active transport (39). Although living microbes can be used for biosorption and may allow for higher pollutant removal rates, non-living biomass is favored due to reduced toxicity constraints, minimal maintenance, long-term storage stability, and adaptability to diverse environmental (40). Scalable applications prioritize materials like polysaccharides, plant-derived residues, and biopolymers for their high binding selectivity toward PTEs (Cu, Cd, Cr, Pb, and Zn) (41). Efficiency hinges on optimizing pH, biosorbent dose, pollutant concentration, contact time, and temperature (38). Fungi have been extensively studied in recent years as biosorbents for 8 PTEs, using both living and non-living fungal biomass (42–44). Biosorption to cell wall components is considered to be the predominant mechanism for removal, which includes physical adsorption, ion exchange, electrostatic interactions, and complexation (45). 1.2.2.4 Microbe-assisted phytoremediation Remediation using microbes (bioremediation) is categorically separated from remediation using plants (phytoremediation), but the combination of these biological organisms has been shown to yield synergistic results (46). Mycorrhizal fungi, for example, can be used in conjunction with trees to remove soil contaminants. Plants provide mycorrhizal fungi with nutrients (carbohydrates and lipids) necessary to complete the asexual life cycle, while mycorrhizal fungi extend through the soil to supply the host plant with water and nutrients that are difficult to access (47). This tactic results in the improved growth of 90% of angiosperms in unfavorable conditions. In polluted environments, mycorrhizal fungi aid the plant in remediation by releasing metal-immobilizing compounds (glomalin-related soil proteins), capturing pollutants inside the fungal cell structure, and enabling the host plant to continue growing in toxic environments to further metabolize contaminants (47,48). A recent student found that mycorrhizal treatment of basket willow shrub for phytoremediation of toxic polycyclic aromatic hydrocarbons (PAHs) released organic acids that acted as co-metabolic substrates, increasing removal efficiency of several PAH compounds (49). 1.2.2.5 Biofiltration Current remediation strategies have primarily addressed soil and water contamination, but biological approaches for air pollution also exist. Biofiltration, a well-established method, uses living organisms (typically bacteria or fungi) in fixed-bed reactors to capture and 9 degrade airborne pollutants (50). First applied nearly a century ago to treat odors from wastewater plants and livestock facilities, biofilters operate without liquid phases, making them ideal for poorly water-soluble compounds (51). For highly soluble pollutants, bioscrubbers absorb gases into liquid before microbial treatment in bioreactors, enabling precise control of microbial growth and processing (50). While biofilters primarily target odorous gases and volatile organic compounds (VOCs), emerging research highlights their potential for particulate matter removal. Using plants, researchers have shown PM2.5 removal in urban areas through optimized plant configurations, such as strategically planted trees and shrubs (52). In contrast to the metabolic processes described for microbial biofiltering, plant biofiltering of PM uses physical means, where PM is deposited on the plant leaf surface or encapsulated in the cuticle via wet or dry deposition (53). 1.2.2.6 Mycoremediation Among the diverse organisms in nature, fungi stand out as complex entities, genetically more akin to humans than plants as determined by genomic and protein sequence analysis. Their sophisticated responses to environmental stimuli have long attracted scientific interest, particularly in the context of fungal bioremediation, or mycoremediation, which leverages their intricate enzymatic systems comprised of coordinated intracellular and extracellular enzymes. Compared to bacterial bioremediation, fungal bioremediation has been shown to have several distinct advantages, including degradation of recalcitrant compounds (in particular, more robust capability to degrade chlorinated aromatics, high molecular weight polyaromatic hydrocarbons, and synthetic dyes compared to bacteria), better tolerance to lower temperatures and salinity fluctuations, and lowered risk of by-product formation with complete mineralization capabilities (54–56). Looking at the case of copper bioremediation, marine fungi show double the copper removal compared to 10 autotrophic bacteria, which has been attributed to mobilization of otherwise immobile copper fragments via organic acid secretion (57). Despite these recognized benefits, fungal bioremediation remains a largely untapped field. While an estimated 2–3 million fungal species are known, recent techniques suggest the true number may be as high as 12 million (58). This vast, unexplored fungal diversity holds immense promise for the development of novel remediation strategies. Investigating how fungi respond to environmental cues thus presents an exciting opportunity for advancing sustainable technologies. Filamentous fungi, in particular, offer unique possibilities for creating novel functional materials. Recent research has explored the structural properties of fungal cells to develop self-supporting composite and pure materials, a topic discussed in greater detail in section 1.2.4. Scientific literature also provides inspiration for how these self-structured materials can be harnessed for advanced environmental remediation applications. Mycoremediation studies have targeted the remediation of petroleum-contaminated soils, heavy metals, and hydrocarbons (59). Key species commonly used in filamentous mycoremediation include Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor, and Lentinula edodes (60–63). White rot fungi are utilized far more frequently than brown rot fungi (96% versus 3%), likely due to their native lignin-degrading capabilities (60). Additionally, most studies favor the use of free fungal cells over immobilized cells for bioremediation purposes (60). While the pollutant-degrading abilities of filamentous fungi are well established, the use of their self-structuring mycelial networks is a relatively recent development. One of the earliest applications of self-structured mycelium composites for mycoremediation involved S. rugoso-annulata-bound wood chips and straw to remove E. coli from stormwater (64). As contaminated water passed through the dense mycelial network, the fungi were theorized to 11 produce antibiotics that reduced bacterial loads, resulting in over 90% reduction in E. coli compared to uninoculated controls. Using the same fungal species and substrate, mycelium biofilters were subsequently applied to remediate a broader range of antibiotic-resistant bacteria-S. aureus, E. faecium, C. jejuni, P. aeruginosa, E. coli, and S. enteritidis-achieving removal efficiencies of 100%, 97%, 59%, 54%, 65%, and 27%, respectively, after 24 hours of incubation (65). Two primary mechanisms have been proposed for bacterial remediation in this system: (1) biodegradation via metabolites, such as bacteriolytic enzymes, and (2) non-metabolic association with the fungal cell wall (biosportion or entrapment within growing hyphal fibers). These developments highlight the dual potential of filamentous fungi as both a structural and functional material for bioremediation. Nevertheless, significant research gaps remain. To fully realize the potential of filamentous fungal materials, it is essential to understand the underlying biology and bioprocessing that shape their material properties and functions. The following sections will provide a concise overview of filamentous fungi and summarize the current literature on their use as biobased materials, forming the foundation for the research presented in this thesis. 1.2.3 Filamentous fungi The Kingdom Fungi is a group of eukaryotic organisms that play important and varied ecological roles as saprobes, symbiotes, or parasites. Fungi can exist in a unicellular form (yeast), filamentous form (molds), or some combination of the two (dimorphic fungi) (66). In addition to morphological classification, fungi are more systematically categorized by phylogenetic analysis. Contemporary view of the phylogenetic classification of fungi includes 6 distinct phyla: Phylum Basidiomycota, Phylum Ascomycota, Phylum Glomeromycota, Phylum Blastocladiomycota, Phylum Chytridiomycota, and Phylum Neocallimastigomycota 12 (Figure 1.2) (67). Zygomycetes, sometimes classified as Phylum Zygomycota, represent over 9000 species of fungi that do not currently fit neatly into one of the 6 contemporary Phyla, highlighting the difficult job of mycologists to categorize such a diverse group of organisms (67). This work focuses on filamentous fungi in the Phylum Basidiomycota, which are known for their high ecological importance as contributors to the carbon cycle and symbiotic partners to plants (68). Basidiomycota and Ascomycota combine to make up the sub-kingdom Dikarya, otherwise referred to as ‘higher fungi’. Basidiomycetes are comprised almost exclusively of filamentous fungi, which are capable of forming fruiting bodies, or mushrooms, supported by carefully organized filaments (69). Several of the most important edible mushrooms are basidiomycetes, including Agaricus bisporus (button mushroom) and Pleurotus ostreatus (oyster mushroom). Additionally, wood-decay basidiomycete fungi have industrial and biotechnological relevance due to their production of ligninolytic enzymes (70). 13 _____________________________________________________________________________________ Figure 1.2. Evolutionary association and timeline of Fungi, Animals, Plants, and Choanoflagellates. Phylogenetic tree of the Kingdom Fungi is shown in blue further specifying the relationship between 6 contemporarily recognized phyla (solid line) and 2 emerging phyla (dashed line). _____________________________________________________________________________________ 1.2.3.1 Filamentous fungi life cycle The filamentous fungus begins its lifecycle as a single spore, whereby it finds a substrate with suitable conditions and nutrients to begin germination. In basidiomycetes, germination results in the basidiospore elongating into a germ tube, a singular hypha that can begin the colonization of the substrate and penetration into the substratum. The germ tube forms homokaryotic hyphae (primary mycelium) which fuse with compatible mating strains via plasmogamy to form a heterokaryotic hypha (secondary mycelium), a dominant 14 phase where each cell contains two genetically distinct nuclei (71). This dikaryon grows through clamp connections, maintaining nuclear pairing during mitosis. Under favorable conditions, the dikaryotic mycelium produces a basidiocarp (mushroom) where specialized cells grow into basidia, producing basidiospores to complete the lifecycle. Growth of the hypha is localized at the growing tip where cell wall material is excreted, and direction of the growth is dictated by polarization at the cell tip. Polarization is controlled by Ca2+ gradients, where calcium concentration is highest at the hyphal apex and rapidly declines 10-20 μm behind the tip (72). Similar calcium gradients have been observed in other tip-growing cells like pollen tubes, suggesting a conserved mechanism across different biological systems (73). Hyphae branch sub-apically to form a fractal pattern growing radially on homogeneous substrates to optimize resource scavenging, forming the network of branched filaments called the mycelium (74). The mycelial network allows fungi to build large resource supply systems that can take advantage of spatiotemporally uncoupled carbon and nitrogen in their environment (74). 1.2.3.2 Mycelium morphology Mycelial growth can exhibit two primary forms in dikarya: tomentose (diffuse) and rhizomorphic (cord-like), with specific morphology dictated by environmental factors. Tomentose mycelia form loose, cottony networks of hyphae that spread diffusely across substrates, while rhizomorphic mycelia organize into dense, root-like structures that efficiently transport nutrients over long distances. The differentiation of mycelia into rhizomorphs involves coordinated hyphal aggregation and changes in cell wall composition (75). Poor nutrient availability, increased oxygen availability, and environmental stressors can trigger the growth of rhizomorphic mycelia (75). 15 Hyphae also display structural diversity. Generative hyphae are thin-walled, branched, and capable of forming reproductive structures; ligative (binding) hyphae are thick-walled and highly branched, providing mechanical strength by binding other hyphae together; and skeletal hyphae are long, thick-walled, and unbranched or sparsely branched, contributing rigidity and support to the fungal structure (76,77). Depending on the species, mycelia can contain all or select types of hyphae. Species are referred to as mono-, di-, and tri-mitic based on the number of and type of hyphae present (78). Description of hyphal classifications and classifications of species relevant to this thesis work are shown in Table 1.1. Table 1.1. Classifications of hyphal types and relevant species Hyphal system classification Types of hyphae Species Monomitic Only generative hyphae Hericium spp. (79,80) Dimitic Generative and skeletal hyphae, or generative and binding hyphae. Pleurotus spp. (with limited skeletal hyphae) (81) Trimitic Generative, skeletal, and binding hyphae. Ganoderma lucidum (bovista type) (82), Trametes s.str (coralloid type) (83) 1.2.3.3 Filamentous fungi cell wall structure The fungal cell wall is essential for maintaining cellular shape and integrity, providing mechanical protection against osmotic and environmental stresses (84). It also enables fungi to interact with their environment and is crucial for growth, viability, and 16 adaptation. Therefore, the cell wall is critical to understand and develop functional fungal materials. The cell wall consists of two general layers: 1) an amorphous layer, allowing for cell sensing and environmental interactions, and 2) a structural layer, giving strength and shape to the cell. A depiction of a generalized filamentous fungus with major cell wall components is shown in Figure 1.3. The structural layer of the cell wall primarily consists of chitin microfibrils and glucans. In fungi, glucans can have several arrangements and linkages. One of the common arrangements unique to the fungal cell wall is a β-(1,3) glucan backbone with β-(1,6) branches (85). β-(1,3)-glucans form coiled, spring-like chains that confer tensile strength and elasticity, constituting 30–60% of the cell wall's dry weight (86). These polymers are cross-linked to other cell wall components via the β-(1,6)-glucan branches, which includes chitin, proteins, and other polysaccharides (86). Chitin (1,4-linked N-acetylglucosamine) exists as hydrogen-bonded microfibrils in the cell wall and are complexed to β-glucans, contributing to cell wall rigidity. It comprises ~2% of yeast walls and 10–20% in filamentous fungi with an α-chitin crystalline structure being predominant (87). Chitin deacetylases convert chitin into chitosan in the fungal cell wall, helping fungal pathogens and mutualists evade host immune responses (88). The relative concentration of chitosan to the total amount of chitosan and chitin in some fungal species has been found to be 19–28 wt%, although characterization of the amount of chitosan specifically in the fungal cell wall is relatively unstudied (89). The amorphous layer consists of other structural polysaccharides with an outer layer of glycosylated proteins, often containing mannose residues. Mannan chains, composed of mannose linked in various ways, are found either attached to glucans or covalently bound to proteins as N-linked or O-linked glycoproteins (86). Mannans make up 10–20% of the fungal cell wall’s dry weight, while polypeptide content is around 4% (86). Certain fungal cell wall 17 proteins are essential for the biosynthesis, cross-linking, and remodeling of cell wall polysaccharides, processes that are crucial for dynamic cell wall growth and structural flexibility (90). Cell wall proteins also play a role in environmental sensing and regulation of molecules that can enter the plasma membrane (91). Hydrophobins are an interesting type of cell wall protein unique to filamentous fungi (92). Structurally, hydrophobins are characterized by eight conserved cysteine residues forming four disulfide bonds and are divided into two classes based on differences in sequence and assembly properties. Class I hydrophobins form robust, amyloid-like rodlets, while class II hydrophobins form flower-like structures (93). By self-assembling at hydrophilic–hydrophobic interfaces, hydrophobins reduce surface tension, enabling hyphae to grow out of moist substrates and into the air, and coating these structures with a hydrophobic layer that aids in spore dispersal and prevents waterlogging (94). 18 _____________________________________________________________________________________ Figure 1.3. General mycelium and fungal cell wall structures. Graphical depiction of mycelium growing through a substrate (vegetative hyphae) and into the air (aerial hyphae). The bubble on the top left shows the coating of hydrophobin proteins on the outer surface of aerial hyphae arranged as rod-like structures, with hydrophobic residues oriented to the air. The arrangement of major cell wall components, including chitin, glucans, and glycoproteins, is shown on the top right. _____________________________________________________________________________________ 1.2.4 The use of mycelium as a material Interest in using mycelium as a material has been increasing exponentially over the past decade, as is shown in Figure 1.4 with both publication and patents on mycelium materials exhibiting upward trends since the year 2000. While global science and engineering 19 scientific output grew by 59% over 10 years (2012-2022), mycelium materials research grew by 160% (95). Patent filings for mycelium technologies, particularly leather substitutes, now outpace traditional biomaterials, reflecting an overall shift and improved attitude toward fungal materials (96). Key countries involved in the mycelium material innovation space include China and the US, countries with large and expanding bio-economies. However, a significant number of patents filed in the past 5 years to the European Patent Office (EPO) and World Intellectual Property Organization (WIPO) signify a more global interest in mycelium as a material (96). _____________________________________________________________________________________ Figure 1.4 Publication and patent trends for mycelium materials. (a) Trends in global publications on mycelium materials (Web of Science Core collection database; search term: “Mycelium AND material*”) and patents on mycelium materials (Google Patents; search term: “Mycelium AND material*”). (b) Number of patents on mycelium materials filed by countries. Data extracted from Onorato et al. (96). _____________________________________________________________________________________ While interest in using fungi for materials has surged in recent years, the practice itself has ancient roots. The species F. fomentarius is a key example of the use of fungal materials throughout history. The use of this species dates back at least 5,300 years, with 20 the earliest documented evidence traced to Ötzi, the Neolithic Iceman (3300 BCE), who carried processed F. fomentarius (with common names the Hoof fungus and Tinder fungus) as part of a fire-starting kit (97). While Ötzi's use focused on fire-making, the development of fungi into textiles and functional materials (e.g., hats, bags) emerged later, flourishing in pre-industrial Europe as a specialized craft called Amadou across Central and Eastern regions (97). Amadou is made by trimming, stretching, and dying the F. fomentarius fungal body to transform the hard polypore into smooth and flexible materials. By the 18th–19th centuries, Amadou became concentrated in regions like Corund, Transylvania where it persists today (98). 1.2.4.1 Composite mycelium materials Modern mycelium materials are formed as composite mycelium materials (CMM) or pure mycelium materials (PMMs). Composite mycelium materials are formed by the partial degradation of lignocellulosic material, whereby the mycelial network colonizes and binds together the discrete lignocellulosic segments (99). The type and processing of substrates for CMMs have a large impact on the growth of the CMM. For example, P. ostreatus grown on potato dextrose broth forms thinner, collapsed hyphae with lower chitin content, resulting in materials that absorb more water, are less stiff, and are more stretchable compared to those grown on cellulose (100). Although the nutritional content of the lignocellulosic material has been shown to have an impact on growth and mechanical properties of CMMs, other studies suggest that fiber processing and size plays a larger role in these parameters (101). CMMs have been explored in a variety of applications, including construction, textiles, and insulation material (76,102–105). Utilizing waste streams as the substrate material, such as straw, sawdust, wood chips, rice husks, banana leaves, coffee grounds, coconut coir, 21 and even paper and cardboard waste, CMMs have been heralded as sustainable materials for a circular economy (106–108). Moisture resistance remains a critical area for improvement, particularly toward packaging and structural applications, as mycelium-lignocellulose composites generally exhibit an affinity for water absorption (109). Additionally, there is some difficulty in comparing the effect of variables used across publications because of the lack of uniformity in experimental procedures and data collection. 1.2.4.2 Pure mycelium materials A more recent development in the mycelium material space is the production of pure mycelium materials (PMMs), whereby the mycelium is removed entirely from the substrate, resulting in largely a retention of the aerial hyphae. Over the past decade, PMMs have reached commercial markets as sustainable alternatives to animal leather and synthetic foams, with applications in fashion, packaging, and interior design (110–112). Companies such as Ecovative, MycoWorks, and Bolt Threads have pioneered large-scale production, using proprietary fermentation and post-processing techniques to fine-tune the material properties for specific end uses. Taking mycelium-based leather (MBL) as a model for how mycelium materials function industrially, we find that mycelium can compete well with conventional materials (113). A 2022 pilot study demonstrated that mycelium-based leather (MBL) has a carbon footprint of 6.2 kg CO₂/m², far below bovine leather’s 32.97 kg CO₂/m² (114). While scaling production may raise MBL emissions to an approximated 13.88 kg CO₂/m², adopting bio-gas-free workflows could reduce this to 2.76 kg CO₂/m² (114). Cost analyses further highlight MBL’s advantage, with production costs at $0.18–0.28/m² compared to raw hides’ $5.38–6.24/m², underscoring its environmental and economic potential (115). 22 The structure and properties of PMMs are shaped by both the fungal species and the fermentation methods. Solid-state fermentation (SSF) is widely used for producing foams and leather-like sheets, where aerial hyphae grow densely on the surface of a solid substrate and are then harvested as a pure layer (113). Liquid-state fermentation (LSF), a relatively unstudied technique, allows mycelium mats to form at the air-liquid interface, enabling rapid, scalable production but with increased risk of contamination (116). Recently, VTT demonstrated the production of pure mycelium material from submerged fermentation (SmF), a technique used already in fermentation industries for the production of secondary metabolites (117). This enabled the continuous production of mycelial sheets; however, the properties of the material are not yet disclosed. A variety of fungal species are used in PMM production, with Ganoderma lucidum, Pleurotus ostreatus, and Trametes versicolor among the most common due to their robust aerial mycelium formation and adaptability to different substrates (118). In mycelium materials, species selection was found to significantly impact the growth performance in terms of hyphal extension rate and growth density (119,120). The choice of growth media ranges from agricultural by-products (like corn husks or sawdust) to defined nutrient solutions, with substrate composition and environmental conditions (CO₂ concentration, humidity, temperature) carefully controlled to optimize yield and material characteristics (100,111,112). As the field advances, research is increasingly focused on understanding how fermentation dynamics, feedstock selection, and fungal genetics interact to produce PMMs with tunable properties for diverse applications. At the cutting-edge of PMM research are chemical, genetic, and co-cultivation strategies to enhance mechanical and functional properties. Appels et al. demonstrated that treating mycelium films with glycerol significantly alters their mechanical behavior: at 32% 23 glycerol, Young’s modulus drops to 0.003 GPa (from 0.47 GPa untreated), while strain at failure increases to 29.6%, shifting the material from natural to elastomer-like properties (121). Genetic engineering has also proven effective. Deleting the sc3 hydrophobin gene in Schizophyllum commune increased mycelium density by 1.4–3 fold, yielding a thermoplastic-like material with a Young’s modulus up to 2.7 GPa and tensile strength of 40.4 MPa (122). Co-cultivation approaches, such as combining Ganoderma lucidum with Sporosarcina pasteurii, using bacterial-induced calcium carbonate mineralization to reinforce mycelium networks, enhancing compressive strength (123). 1.2.4.3 Literature gaps Despite growing interest in mycelium-based materials, several significant gaps remain in the current literature. There is a notable lack of research focused on pure mycelium materials, which limits our understanding of mycelium’s intrinsic properties and its specific contributions within composite structures. Additionally, bioprocess control remains underexplored, with few studies addressing how different growth methods influence the structure and functional properties of the resulting mycelial materials. The potential of mycelium as a functional membrane is also poorly understood, particularly regarding its filtration and selective permeability capabilities. Finally, there is little research on the use of pure, non-living mycelial structures for air and water treatment, leaving their potential for environmental remediation largely untapped. Addressing these gaps is essential for advancing both the scientific understanding and practical applications of mycelium-based technologies. 24 1.3 Research aim and objectives This research sets out to develop functional membranes from mycelial biomass toward pollutant remediation by exploring the relationship of mycelium with various contaminants. To meet the core intention of this research, the following objectives were proposed. Objectives: 1. Develop a framework for producing materials from pure mycelia and understand the bioprocess-structure-property relationships. 2. Develop systems for incorporating mycelial biomass into pollutant treatment systems. 3. Assess and optimize the performance of mycelial membranes for the treatment of polluting materials. 4. Understand the mechanisms of removal of polluting substances by mycelial membranes. 1.4 Research questions To meet the objectives proposed in section 1.3, the following research questions and sub-questions were presented. 1. What is the impact of bioprocessing changes on the structure and properties of pure mycelial materials? a. How does the fermentation method impact the chemical composition and morphology of pure mycelial materials? 25 b. How does the species of fungus used to produce pure mycelial materials impact the chemical composition and morphology? c. How do the structure and properties of mycelial materials produced by varying bioprocessing methods inform the material properties? 2. How can mycelial structures be engineered to produce a membrane for the treatment of air pollution? a. What is the impact of mycelia on filtration and water resistance of mask materials? 3. How can mycelial structures be engineered to produce a membrane for treatment of water pollution? a. What is the adsorbance of heavy metals onto a mycelial membranes? i. What is the mechanism of removal of heavy metals by the mycelial membrane? ii. How can we control the adsorbance of heavy metals in a flow reactor using mycelial membranes? b. What is the performance of a mycelial membrane with immobilized enzymes for treatment of phenolic compounds? i. What are the optimal conditions for immobilization of enzymes on the mycelial membrane? ii. How does immobilization impact the properties and function of the enzyme? iii. What is the amount and final degradation products of the phenolic compounds? 26 1.5 Research road map This thesis contains 4 research chapters, with one broad, fundamental chapter and 3 applied chapters. Many of the chapters are derived from published works, which are stated clearly in the Preface. Modifications and expansions of the research articles in the chapters were done to provide detail and aid in the overall clarity of the thesis. Figure 1.5 depicts the overall structure of the thesis. Chapter 2 will provide some of the guiding criteria and selection for later chapters based on research question 1. This chapter provides an overview of fungal types and processing methods and attempts to relate these parameters to the material structure and properties. The results from Chapter 1 can serve as a guiding principle for future work looking to develop and apply pure mycelium materials to a wide array of different applications, not just environmental remediation. Chapters 3, 4, and 5 take insights from the conclusions of this chapter to inform material selection and application selection. Chapter 3 set out to address research question 2. This chapter assesses the structural elements of mycelia and applies it to air filtration applications, as the structure can mimic non-woven filtration layers. This chapter will provide a first step into the application of self-growing, structured mask materials for air pollution control. Chapter 4, based on research question 3(a), will analyze the chemical composition of mycelia as well as the structural elements of the mycelia to develop adsorption materials for PTEs inspired by the interaction of fungi with metals in their environment. This chapter will demonstrate the applicability of native and unmodified mycelia in water remediation applications. 27 Chapter 5, based on research question 3(b), will analyze modifications to the mycelia to provide further avenues for water pollution treatment. Chapter 3 investigates adsorption, whereas chapter 4 investigates degradation of pollutants using the mechanisms of fungal enzymes as the chief motivation. _____________________________________________________________________________________ Figure 1.5 Road map depicting the progression of ideas and research within this thesis, starting from the development of PMMs to application of PMMs as mask materials, adsorption membranes, and towards phenolics degradation. _____________________________________________________________________________________ 28 1.6 Scope and guiding principles This thesis focuses on treatment (not mitigation) of environmental pollutants using filamentous fungi, prioritizing their unique structural advantages over other fungal types. The work centers on pure mycelium materials isolated from substrates to ensure consistency and scalability. In Chapter 3 of this thesis, a non-edible matrix is modified by the mycelium material, which could be considered a composite structure. However, in current terminology related to mycelium materials, this would not fall into the label of “composite mycelium material” that traditionally utilizes a lignocellulosic substrate as the secondary material in the composite. In this instance, we view the mycelium simply as a modifying surface treatment. Sustainability serves as the core framework, with emphasis on triple-bottom-line metrics: environmental benefits (reduced pollution/toxicity), economic viability (cost-effective production), and equitable access (affordability for marginalized communities). The focus of this thesis is on improving and investigating environmental benefits; however, economic viability and equitable access are considered throughout. By integrating bioengineering (mycelium cultivation), materials science (hybrid material design), and environmental science (pollution remediation), this research aims to develop solutions that align with sustainable development goals, particularly targeting the needs of underserved populations while advancing circular economy principles. 29 30 Chapter 2: Preparation and Characterization of Pure Mycelium Materials This chapter sets the stage for the rest of the research presented in this thesis, focusing on the engineering of pure mycelial materials with controlled properties. Currently, most production methods for mycelium materials are not well understood, with much of the available knowledge confined to patent literature and lacking in mechanistic detail. Here, we examine how bioprocessing conditions influence mycelial structure and, in turn, material properties. This approach is intended to give researchers clearer guidance when selecting production parameters for mycelium material development. In subsequent chapters, we refer to the produced mycelium material as a “membrane,” referencing its application to selectively remove pollutants; however, throughout this chapter we use the term “mats” to reflect the potential broader applicability of these materials. Nevertheless, the material properties discussed here are particularly relevant to membrane-based applications. _____________________________________________________________________________________ Figure 2.1. Overview of research for the development of pure mycelium materials (PMMs) _____________________________________________________________________________________ 31 2.1 Introduction The production and consumption of materials shapes global communities and economic landscapes. The exchange of materials between countries drives significant economic transactions, and high consumption of materials and generation of solid waste correlates to high GDPs (124). In 2017, materials consumption was estimated at 90 million tons, a figure expected to double by 2050 (125). Since the 1950s, there has been a remarkable surge in the use of non-renewable, petrochemical-based materials, which has been accompanied by a host of environmental problems. Notably, nearly 79% of petrochemical-based plastic production between 1950 and 2015 is now found in landfills, underscoring the serious challenges associated with the disposal of these materials and the environmental consequences thereof (124). It is evident that the structure of our society relies on materials, with increasing reliance on non-renewables. To reach sustainable goal targets outlined in the United Nations Sustainable Development Goals (SDGs), particularly toward SDG 12 that calls for responsible consumption and production, it is imperative that research and development focuses on the advancement of sustainable materials (126,127). Bio-based materials, derived from terrestrial/marine biomass or biogenic waste, offer a promising pathway toward circular economies (128). Traditional materials like wood and paper now share space with advanced biopolymers such as polylactic acid (PLA) and polyhydroxyalkanoates (PHAs). However, limitations, including moisture sensitivity, low mechanical strength compared to conventional materials, and susceptibility to microbial degradation, limit their widespread adoption (129,130). While life cycle assessments indicate that each metric ton of bio-based material saves 55 gigajoules of energy and 3 tons of CO₂ 32 equivalents compared to conventional counterparts, performance gaps relative to synthetic materials underscore the need for further innovation (128). Microbial materials represent a promising area of research within bio-based materials (131). These include bacterial cellulose, microalgae-derived products, and fungal mycelium. For example, bacterial cellulose produced by certain bacteria (e.g. Gluconacetobacter xylinus) forms strong fibrous mats with high tensile strength (200-2000 MPa) and crystallinity that can be harvested and processed into various materials (132). Microalgae also offer a sustainable source of bioproducts. They can range from pigments, bioplastics, fatty acids used in bioremediation and biorefineries (133). Compared to conventional lignocellulosic feedstock in biorefineries, microalgae are advantageous in that they do not require agricultural land space and clean water (134). Fungal mycelium, the network of hyphae that forms the vegetative part of fungi, can be produced as self-structured materials, whereby the fungal cell becomes the structural element. Mycelium has been used to bind agricultural waste particles like sugarcane bagasse and rice husks into solid bio-composites (CMMs) and removed as a foamy top-layer from the substrate to form pure mycelium biomaterials (PMMs) (135). Fungal mycelium has emerged as a promising sustainable material, yet the relationship between bioprocessing parameters and material properties remains poorly understood, especially to produce PMMs. Numerous factors have been shown to result in distinct material properties, including species and strain selection, concentration of CO2 and O2, direction and strength of airflow, light, humidity, temperature, and substrate composition (136). However, a systematic approach to understanding how these parameters mold the resulting material is limited. 33 In this study, we investigate how fungal species and fermentation methods influence the structure and properties of pure mycelium mats. Four white-rot fungi, Pleurotus ostreatus, Ganoderma lucidum, Trametes versicolor, and Hericium erinaceous were selected based on their non-toxicity, non-parasitic nature, and global availability. Mycelium mats were produced using three fermentation approaches: solid-state fermentation (SSF), liquid-state fermentation (LSF), and submerged fermentation (SmF), while other environmental variables were held constant. We hypothesize that both the fungal species and the fermentation method will significantly affect hyphal morphology, cell wall composition, and, consequently, the mechanical and surface properties of the resulting mycelium mats. By analyzing cell wall chemistry, hyphal structure, mechanical performance, and wettability, this work aims to establish clear relationships between bioprocessing conditions and material outcomes. The results are intended to provide a framework for selecting species and fermentation systems to engineer mycelium materials with properties tailored for specific applications, such as filtration membranes or biodegradable packaging. 2.2 Experimental methods and materials Growth and processing of mycelial samples SSF samples were cultivated in 250 mL beakers containing 50 mL of potato dextrose agar (PDA) medium, which was sterilized via autoclaving at 121°C for 20 minutes. LSF samples were grown in 250 mL Erlenmeyer flasks with 50 mL of potato dextrose broth (PDB), also autoclaved under the same conditions. Inoculation for both SSF and SmF samples was carried out by introducing a drop of mycelial spore culture onto or into the respective media. In the case of SSF, the drop was placed at the center of the agar to allow for even radial growth outward. The mycelium was allowed to grow at 28°C for 15 days, after which the 34 complete mat was harvested. For SSF samples, the mats were carefully peeled off the solid media, whereas for LSF samples, the mats were simply extracted from the liquid media. All harvested mats were washed three times with deionized water and stored in a −30°C freezer overnight before undergoing freeze-drying for 24 hours. Cultures of Pleurotus ostreatus, Ganoderma lucidum, and Trametes versicolor were maintained on PDA plates at 30°C and sub-cultured every three weeks. To produce SmF samples, sterile yeast extract peptone (YEP) broth was prepared by autoclaving a solution containing 1% yeast extract and 2% peptone at 121°C for 20 minutes. A 1 cm × 1 cm agar plug from each fungal culture was used to inoculate 1 L of YEP broth in 2 L Erlenmeyer flasks. Cultures were incubated at 25°C in a shaking incubator set to 220 rpm for 21 days. Mycelial biomass was separated from the liquid medium using vacuum filtration with a nylon cloth, washed three times with distilled water, and freeze-dried for subsequent processing. A suspension of processed mycelial biomass (1% w/v) was prepared in deionized water and homogenized at 10,000 rpm for 10 minutes using an IKA Ultra-Turrax homogenizer. The homogenized suspension was vacuum filtered through a nylon cloth with a pore size of 40 nm until all water was removed. A brief pause of five seconds post-filtration ensured uniform compression across all samples. The resulting wet mats were frozen overnight at −80°C and then freeze-dried to produce self-supporting sheets. All fungal spore cultures used in this study were sourced from Grow Mushrooms Canada. Chemical analysis The fungal cell wall composition of SSF, LSF, and SmF samples was analyzed following the method outlined by François (2007) (137), with a brief summary provided here. Approximately 50 mg of dried mycelial biomass was collected for each sample. To facilitate processing, SSF and LSF samples were blended for one minute to generate smaller hyphal 35 fragments. The cell wall material was isolated by suspending the mycelial biomass in 1 mL of TE buffer within 2 mL Eppendorf tubes and vortexing with acid-washed glass beads. This process involved seven 20-second vortexing intervals, each followed by 30 seconds on ice to minimize thermal degradation. After removing the glass beads, the isolated cell wall material was dried overnight in an oven set to 110°C. To prepare the cell wall material for compositional analysis, acid hydrolysis was performed by adding 75 µL of sulfuric acid to wet the biomass, which was then left at room temperature for three hours. The mixture was subsequently diluted with 0.95 mL of Milli-Q water and heated in a sand bath at 100°C for four hours. The reaction was terminated by neutralizing the solution with barium hydroxide. To determine sugar composition (glucose, mannose, and N-acetylglucosamine), hydrolyzed cell wall material was prepared for HPLC analysis by filtering using 0.22 µm PTFE filters. HPLC methodology followed the protocol by Wong and Guttierez, with the following parameters: instrument: Agilent 1260 HPLC-RID; column: Nucleogel Ion 300 OA column (300 mm × 7.8 mm, 10µm id); flow: 0.45 ml/min; mobile phase: 5 mM H2SO4; column temperature: 71°C; RID temperature: 55°C; and run time: 40 min. Standards were run with solutions of D-(+)-Glucose (Bio-Reagent), D-(+)-Mannose (Bio-Reagent), and HPLC-grade N-Acetyl-D-glucosamine (SigmaAldrich). Morphological analysis The morphology of the mycelial samples was examined using scanning electron microscopy (SEM) and image analysis. Sample preparation involved mounting the biomass onto metal stubs with carbon tape. The samples were then coated with a 10 µm layer of iridium using a Leica EM MED020 coater (Wetzlar, Germany) to enhance conductivity. 36 Imaging was performed with a Helios NanoLab 650 SEM system (Oregon, USA), equipped with an Everhart-Thornley detector. The microscope was operated at an accelerating voltage of 3 kV, with the working distance precisely set to 4 mm to ensure consistency across samples. The vegetative hyphae from various fungal species and fermentation methods were analyzed using Fiji image processing software. Image preparation followed a systematic workflow to ensure accurate measurements, including the application of a Gaussian 3D blur to reduce noise, thresholding, binarization, and despeckling to remove artifacts. Hyphal diameter was quantified using the local thickness function, providing estimates of structural dimensions across samples. Mechanical analysis Dynamic mechanical analysis (DMA) was conducted in both tensile and compression modes using a TA Instruments DMA 850. For compression testing, LSF and SSF samples were prepared by cutting them into circular specimens with a diameter of 1 cm. The tests were performed over a temperature range of 25 to 80°C with a heating rate of 3°C/min, at a frequency of 1 Hz and an oscillation amplitude of 0.1%. Tensile tests were carried out on SSF, LSF, and SmF samples, which were cut into rectangular shapes. These samples were analyzed with a frequency of 1 Hz and an oscillation amplitude of 0.1%. Contact angle analysis Static water contact angles were determined using the sessile drop method with an optical tensiometer (Theta Flex, Biolin Scientific Oy, Finland). Mycelial mats were adhered to glass slides using double-sided tape. A 5 µL droplet was carefully deposited onto the material's surface, and the instrument automatically measured the geometry of the droplet. 37 Contact angle values were recorded over 60 seconds. Triplicates (biological for SSF and LSF) were measured across 2 points each, resulting in 6 total points measured. 2.3 Results and discussion 2.3.1 Bioprocessing design and production of pure mycelium materials This study utilized three distinct fermentation methods: solid-state fermentation (SSF), liquid-state fermentation (LSF), and submerged fermentation (SmF). A graphical overview of these fermentation approaches is provided in Figure 2.2 (a). Static fermentations, encompassing SSF and LSF, require no additional processing, whereas agitated fermentations, represented by SmF, involve further steps to refine the mycelial material. SmF processing includes blending and homogenizing to separate hyphae from pellets, followed by vacuum filtration to form a cohesive mycelial mat. In SSF, fungal growth occurs both within the substrate as vegetative hyphae and outward from the substrate surface, producing aerial hyphae. During the harvesting process, peeling of the mycelial mat from the surface isolates the aerial hyphae while the vegetative hyphae remain embedded in the solid phase. In LSF, fungal growth is concentrated at the air-substrate interface due to static conditions, forming a thick sheet composed of both vegetative and aerial hyphae. The mycelial sheet is harvested by simply pouring off the liquid media, resulting in retention of both layers. Representative samples of SSF-, LSF-, and SmF-grown mats produced from Ganoderma lucidum are displayed in Figure 2.2 (b). 38 _____________________________________________________________________________________ Figure 2.2. (a) Graphical depiction of static fermentation methods, solid state fermentation (SSF) and liquid state fermentation (LSF), and agitated fermentation methods, submerged fermentation (SmF) and the resulting fungal material after growth time and processing. (b) Digital images of mycelial mats derived from SSF, LSF, and SmF methods using the species Ganoderma lucidum. _____________________________________________________________________________________ 39 To ensure fair comparison across all fungal species and fermentation methods, it was necessary to optimize several key parameters at the outset of the study. Media selection was found to be a critical parameter. Potato dextrose (PD) outperformed malt extract (ME) and yeast peptone dextrose (YPD) in terms of yield (mass of biomass per liter of media) for SSF and LSF samples, while YPD broth was optimal for SmF, particularly for Ganoderma lucidum. Therefore, potato dextrose agar (PDA) and potato dextrose broth (PDB) were used for SSF and LSF experiments, respectively, while YPD was employed for SmF experiments. These findings established a standardized growth environment for subsequent experiments. In addition to media selection, gas exchange was found to play a critical role in mycelial growth and mat formation. For SSF and LSF methods, the optimal ratio of media to headspace was established through preliminary experiments. A ratio of 0.2 media volume to total reactor volume yielded the thickest mycelial sheets. Starting colonization with limited gas exchange (by capping the beaker or flask with aluminum foil) and transitioning to increased gas exchange (by replacing the foil with parafilm) on day 5 stimulated the development of a thicker layer of aerial hyphae. Processing conditions for SmF-derived mycelial sheets were also systematically optimized, as described in further detail in Chapter 4. The most effective protocol involved blending SmF pellets for one minute at low speed to break up larger aggregates, followed by homogenization at 10,000 rpm for 10 minutes to gently separate hyphal filaments. Deviations from this protocol often resulted in either complete destruction of hyphal structures or the formation of large, inconsistent chunks, emphasizing the need for precise control during processing. Additionally, controlling the target grammage of mycelium during mat formation was critical as insufficient mass led to uneven coverage, while excessive mass produced overly 40 thick, difficult-to-handle mats. The optimal target grammage was determined to be 0.012 g/cm2 following this experimental protocol. Finally, the choice of drying method significantly influenced the quality of the resulting mycelium materials. Preliminary studies revealed that freeze-drying preserved the material’s shape and porous structure better than air-drying or oven-drying, which often caused non-uniform shrinkage and brittleness. This finding is particularly relevant for applications such as insulation and filtration, where structural integrity and porosity are essential. With an established protocol to develop pure mycelium materials, the key parameters of this research, species and fermentation type, were varied. A survey of 4 species (Ganoderma lucidum, Pleurotus ostreatus, Trametes versicolor, and Hericium erinaceous) were tested for growth in the three fermentation methods as described earlier. The initial colonization of the species on agar plates with activated charcoal for visualization purposes, shown in Figure 2.3 (a), showcases the general mycelial network formation for these different species. The success of the growth of the samples is depicted in Figure 2.3 (b), and is categorized as either successful mat formation, non-homogenous mat formation, or unsuccessful mat formation. 41 _____________________________________________________________________________________ Figure 2.3. (a) Digital images of selected white-rot fungal species after growth for 7 days on malt extract agar plates with activated charcoal (images depict mycelial growth patterns, not actual SSF, LSF, or SmF samples). Mycelial network structure is shown as white filamentous mass. (b) Map of successful, non-homogenous, and unsuccessful mat formation using different fungal species and fermentation methods. _____________________________________________________________________________________ Ganoderma lucidum (GL) produced dense, homogenous mycelial mats in both static fermentation methods, SSF and LSF. Pleurotus ostreatus (PO) generated homogenous mats in LSF; however, in SSF, the aerial hyphae were too fluffy and soft, making consistent and uniform removal from the solid substrate difficult. Trametes versicolor (TV) failed to form homogenous mats in both LSF and SSF, exhibiting central growth inhibition. Growth inhibition in the central region of the static fermentation vessels may be a result of a localized accumulation of inhibitory compounds triggered in the oxygen-limited region (138). For 42 example, TV produces ethanol under hypoxic conditions, which may result in decreased mycelial biomass (139). Utilizing a fermentation set up with homogenous gas and oxygen availability may help produce uniform mats from this species. Evocative patents also show that controlling the direction of airflow in mycelium tray bioreactors can force directional growth of the aerial hyphae, allowing for formation of a cross-hatched material (112). Under static conditions, GL, PO, and TV all displayed tomentose growth, while Hericium erinaceus (HE) showed rhizomorphic growth, suggesting that HE experienced environmental stress or suboptimal conditions (75). Potato dextrose has been shown to be a suitable substrate for HE growth in previous studies, signifying that other conditions (temperature, gas availability, moisture) are responsible for limited growth (140). As a result of limited growth, HE was no longer investigated to produce PMMs. Compared to static fermentation methods, SmF showed successful growth of TV, PO, and GL, producing mycelial pellets. Given its broad success with multiple species, SmF was subsequently used to further investigate species-dependent differences. Notably, only GL was able to form successful mats across all three fermentation methods. As a result, GL was selected for detailed analysis of the effects of different fermentation methods. 2.3.2 Structural differences of pure mycelium materials To compare the structure of filamentous hyphae grown in different reactors and with different species, quantitative measurements were taken of density and hyphal diameter, along with analysis of hyphal shape and branching derived from electron microscopy images. Figure 2.4 compares static fermentation conditions, grown from GL, because these 43 conditions can be directly compared as they both retain the native self-structured mycelial architectures. _____________________________________________________________________________________ Figure 2.4. (a) Scanning electron microscope image of the cross section of Ganoderma lucidum produced from solid state fermentation (GL SSF) showing the full vegetative layer. (b) Scanning electron microscope image of the cross section of Ganoderma lucidum produced from liquid state fermentation (GL LSF) showing distinct aerial, vegetative, and submerged hyphal layers. (c) Methodology for hyphal diameter estimation using ImageJ software showing (i) a representative image of vegetative hyphae captured by scanning electron microscopy, (ii) image adjustment with background subtraction, gaussian 3D blur, and 44 thresholding, and (iii) generated local thickness map with circles applied across hyphal widths. Hyphal diameter distribution of (d) GL SSF and (e) GL LSF derived from ImageJ analysis. _____________________________________________________________________________________ As shown in Figure 2.4 (a), the SSF mat in cross-section shows a comparatively homogenous distribution of hyphae from the vegetative side (contacting the substrate surface) and aerial side (growing into the air). In the removal process of SSF, the hyphae growing through the substrate cannot be retained, resulting in a material composed mostly of aerial hyphae. By comparison, the LSF mat, shown in Figure 2.4 (b) has distinct hyphal layers. A submerged vegetative layer of hyphae is retained because the whole mycelial mat is removed in this method, which contains both hyphae and extracellular polysaccharides. These extracellular polysaccharides show a sheet-like appearance between hyphae upon drying (141). Above the submerged layer is a layer of vegetative and aerial hyphae existing at and above the air-substrate interface. Interestingly, a dense hyphal layer is shown at the top of the LSF mat, possibly a result of a larger amounts of aerial hyphae that collapse into itself upon drying. Despite this, the overall density of LSF (88 kg/m3) was lower than SSF (163 kg/m3), resulting in a more foam-like material. The diameters of vegetative hyphae of SSF and LSF samples were analyzed by image processing software. Previous work has shown hyphal diameter estimation by utilizing the BoneJ plugin in ImageJ; however, this technique requires z-stacked images (142). In this work, we propose a method to use a single-plane image to perform a similar function, creating a method to utilize widely available microscopy images such as electron microscope images. To do this, processed SEM images of the samples were input in ImageJ local thickness analyzer, which fits spheres across the hyphae lengths. Images at various stages of 45 processing are shown in Figure 2.4 (c) with the original image (i), binarized image (ii), and image after local thickness generator with heat map showing the variation of fitted sphere volumes (iii). The volume of the spheres was converted to diameter, and a distribution was generated as shown in Figure 2.4 (d, e) for SSF and LSF respectively. To understand differences in the distribution of hyphal diameters, it is necessary to distinguish the types of hyphae found in filamentous fungi. The main hyphal types include generative hyphae, which are responsible for growth and reproduction; skeletal hyphae, which provide rigidity and support; and ligative (binding) hyphae, which interconnect and reinforce the mycelial network (76). The presence and proportion of these hyphal types contribute to the overall architecture and adaptability of the fungus. The histogram for SSF vegetative hyphal diameters shows a greater spread than the histogram for LSF vegetative hyphal diameters despite similar mean values (3.5 and 3.4 for GL SSF and GL LSF, respectively. SEM images comparing the two structures reveal that GL SSF and GL LSF have similar hyphal morphologies, with a large number of generative hyphae (Appendix A.1). However, SSF appears to have more instances of smaller ligative hyphae and larger skeletal hyphae, contributing to the greater distribution of hyphal diameters from the mean. Studies show that the presence of skeletal and ligative hyphae as found in trimitic species correlates with increased compression modulus and strength in fungal tissues, indicating these hyphal types are produced in response to the need for enhanced mechanical integrity in solid environments (143). In LSF, the mycelium is suspended in liquid, facing less mechanical stress and not requiring as much structural reinforcement. 46 _____________________________________________________________________________________ Figure 2.5. (a) Scanning electron microscope image of the cross section of pellets produced from submerged fermentation (SmF) using (i) Ganoderma lucidum (GL SmF), (ii) Pleurotus ostreatus (PO SmF), and (iii) Trametes versicolor (TV SmF). (b) Hyphal diameter distribution of (i) GL SmF, (ii) PO SmF, and (iii) TV SmF derived from ImageJ analysis. _____________________________________________________________________________________ Fungal hyphal morphology varies significantly between species, with each exhibiting distinct combinations of hyphal types that influence their structural and functional properties. Differences in hyphal composition are reflected in the structure and hyphal diameter distribution observed for each species grown in SmF (Figure 2.5). For example, Ganoderma lucidum (GL) is a trimitic species, meaning it possesses all three hyphal types (82). This results in a wide hyphal diameter distribution, with small-diameter ligative hyphae and large-diameter skeletal hyphae present. However, the structure of GL in SmF is 47 not particularly dense and overall yield was low compared to other species (Appendix A.2), and its growth in submerged fermentation conditions is less successful compared to static fermentation, likely due to less favorable physical support for the development of its full hyphal structure. In contrast, PO typically contains only generative hyphae, with occasional presence of ligative hyphae (81). As a result, the hyphal diameter distribution for PO is narrow, dominated by the uniform size of generative hyphae. Under electron microscopy, PO hyphae appear consistent in size and morphology, with limited branching, reflecting a simpler and more homogeneous mycelial structure. Trametes versicolor (TV), also a trimitic species, theoretically contains all three hyphal types (83). However, in this analysis, TV appears to be composed mostly of generative hyphae, resulting in a diameter distribution that is narrow, though not as restricted as that of PO. The TV mycelium is characterized by a high degree of branching, with shorter hyphal lengths between branches, which may contribute to its adaptability and ability to quickly produce a greater yield of fungal pellets than the other species. Commercial enzyme-producing fungal strains, such as TV, have been described to preferentially display highly-branched macromorphologies as it enables better processing and increased protein production (144). 2.3.3 Cell wall analysis of pure mycelium materials The morphological analysis of PMMs provides a foundation for understanding their structural properties, but their functional performance is equally dependent on chemical composition. Fungal cell walls, which comprise a significant portion of fungal dry mass (20-40%), are critical for mechanical integrity and environmental interactions (84,145). To isolate the contribution of cell wall polymers, PMM samples were subjected to purification, removing cytoplasmic contents and extracellular polysaccharides. The isolated cell walls were then 48 hydrolyzed with sulfuric acid to depolymerize structural polysaccharides into monosaccharides, which were quantified via high-performance liquid chromatography (HPLC). Representative HPLC spectra can be found in Appendix A.3. The three dominant monosaccharides identified are glucose, mannose, and N-acetylglucosamine (chemical structures in Figure 2.6 a–c). These monosaccharides correspond to the primary fungal cell wall polymers: glucans (glucose), mannoproteins (mannose), and chitin (N-acetylglucosamine). Relative concentrations varied across PMM types (Figure 2.6 (d)). Glucose dominates in all samples, consistent with glucans forming the bulk of fungal cell walls. Mannose levels were intermediate, reflecting mannoprotein content, while N-acetylglucosamine, a marker of chitin, was present in minor amounts (Figure 2.6 (e)). Chitin content in filamentous fungi is typically low (~10% of the dry cell wall), which aligns with chitin’s role as a structural reinforcement polymer rather than a bulk component (144). The lower amount of chitin exhibited in this study is likely a result of simultaneous deacetylation of the N-Acetylglucosamine. Additional peaks in the HPLC spectra may be attributed to deacetylated glucosamine residues. 49 _____________________________________________________________________________________ Figure 2.6. Structures of monosaccharides from fungal cell walls, including glucose (a), mannose (b), and N-acetyl-D-glucosamine (c). (d) The concentration of monosaccharides relative to the total amount of monosaccharides calculated via HPLC analysis. (e) The concentration of glucosamine relative to the total amount of monosaccharides shown on a different scale for clarity. _____________________________________________________________________________________ SSF samples exhibited a higher relative concentration of glucosamine compared to all other fermentation methods, indicating elevated chitin content in the cell wall. Chitin, a polymer of N-acetylglucosamine, is critical for mechanical reinforcement in heterogeneous solid substrates, where hyphae face structural stresses during colonization (146). This aligns 50 with the dynamic nature of fungal cell walls, which modulate chitin synthesis in response to mechanical challenges or competitive pressures (e.g., chitinase activity converting chitin to chitosan for pathogen defense) (147). The increased chitin content likely enhances hyphal rigidity and substrate adhesion, traits essential for SSF growth. In contrast, SmF samples showed lower proportions of glucosamine and higher proportions of mannose, suggesting increased mannosylated proteins compared to SSF and LSF. This shift may reflect reduced mechanical demands in liquid environments, where buoyancy mitigates structural stress, allowing metabolic resources to prioritize glycoproteins. Specifically, GL SmF displayed the highest mannose levels, potentially due to environmental stress (such as shear forces from mixing or contamination) upregulating mannoprotein production (148). 2.3.4 Material properties of PMMs Analysis of the mechanical and surface properties of mycelium materials revealed notable differences between SSF, liquid-state fermentation LSF, and SmF methods across different fungal species, as shown in Figure 2.7. 51 _____________________________________________________________________________________ Figure 2.7. (a) Stress-strain curves from tensile mode using a Dynamic Mechanical Analyzer (DMA) for static and agitated fermentation methods and different species. (b) Ashby plot of Young’s modulus vs. density showing general material categories as envelopes with the materials generated from this study as stars. (c) Contact angle measurements at t=0s and t=60s for generated mycelial materials with aerial side and vegetative side exposed for static fermentation samples. _____________________________________________________________________________________ Stress-strain curves shown in Figure 2.7 (a) demonstrate that GL produced via SSF exhibits higher stiffness and strength compared to LSF, while LSF samples are capable of greater elongation before failure. SmF samples generally display high stiffness and strength, except for GL SmF, which likely reflects the species’ limited growth and network formation 52 under these conditions. A table of specific mechanical properties (Young’s modulus, ultimate tensile strength, and elongation) of PMMs is shown in Appendix A.4. Further, GL SSF samples show a higher storage modulus under compression across the tested temperature range (35–75°C) than LSF samples (Appendix A.5), indicating superior resistance to deformation and suggesting a denser, more robust mycelial network. This can be attributed to both the dense micromorphology seen in SEM analysis along with the high chitin content found in GL SSF cell walls. When mapped on an Ashby plot of Young’s modulus versus density, both LSF and SSF materials fall within the foam material category (Figure 2.7 (b)). However, with additional processing using SmF, mycelium materials can approach the property range of natural materials, likely due to the increased density from compressing the mycelial structure during processing. Among SmF samples, GL showed higher elongation which may be a result of higher mannoproteins in the cell wall relative to other structural polysaccharides. Surface property analysis via contact angle measurements reveals that LSF samples exhibit a greater difference in wettability between the aerial and vegetative sides (Figure 2.7 (c)). The aerial side of GL LSF displayed a high water contact angle (153°C), which remained stable over the testing period. This may be attributed to the higher water activity of the substrate in LSF, which could promote increased production of hydrophobins and thus enhance surface hydrophobicity. However, studies on the production of Pleurotus ostreatus exhibit the opposite trend, whereby SSF showed a higher production of hydrophobins, although this was not directly correlated to surface hydrophobicity (149). This indicates that the production of hydrophobins within different environmental conditions is also dependent on species. It was thought that surface roughness differences between SSF and LSF samples may also contribute to differences in surface properties, especially considering the dense top 53 layer of aerial hyphae shown in SEM analysis if GL LSF; however, roughness measurements determined that the surface roughness of GL SSF and GL LSF were similar (Appendix A.6). In contrast to static fermentation samples, SmF samples display a progressive wetting behavior over time, likely due to their distinct porous structure and the absence of aerial hyphae. Notably, PO SmF samples do not exhibit significant wetting over time, possibly because their more homogeneous structure results in less surface topographical variation, reducing pathways for water infiltration. 2.3.5 Selection of applied mycelial mats The future use of mycelial materials will depend on the structure and properties of produced mats shown in Figure 2.8. This radar chart helps to visualize the trade-offs between variables needed for specific applications, especially in air and water treatment. 54 _____________________________________________________________________________________ Figure 2.8. Radar chart depicting key structure and property values of produced mycelial mats. Values are shown as relative values based on the highest numerical value in each variable. _____________________________________________________________________________________ In terms of functionality, mycelial membranes will require different structures and properties dependent on the application. For example, in applications concerning air treatment, high hydrophobicity may be required to mitigate wetting of the mat which would reduce its ability to capture particulate matter. High hydrophobicity can also enable features of the mycelial membrane in air treatment applications such as self-cleaning (150). In this instance, utilizing static fermentation methods where hydrophobic aerial hyphae are retained would be preferred. Specifically, LSF samples which show contact angles above 150° can be used to produce high water resistant and potentially self-cleaning membranes. For water treatment applications, however, wettability of the membrane is imperative to allow for an adequate flux through the membrane and good association of aqueous contaminants to the membrane surface (151). In this case, sheets produced from submerged fermentation, specifically from Trametes or Ganoderma species may provide optimal results in terms of low 55 flux and high contaminant rejection. Mechanical properties of mycelial mats can also help inform its possible applications. Air treatment membranes such as face masks will require higher flexibility to aid in the curved fit to the face. Meanwhile, water treatment membranes may require higher compressive and ultimate strength to achieve a good resistance to pore collapse and deformation. The structure is equally as important as material properties in determining the use case of mycelial mats from different species and fermentation methods. The porous structure, which can be understood in part by the measured density and hyphal diameters and features, is critical to control features such as breathability, flux, and particle size rejection. Additionally, chemical composition of the cell wall can determine the types of contaminants that can adsorb to the membrane surface. For example, mycelia with higher mannoprotein content may be more suitable for applications involving heavy metal adsorption as these components contain necessary functional groups for attachment (152). 2.3.6 Environmental and economic considerations Along with the technical features discussed earlier, environmental and economic considerations will play an important role in the selection of bioprocess and fermentation set ups for mycelium materials in the future. A key advantage of SSF is the ability to utilize agricultural or industrial waste as growth media, enhancing the sustainability and circularity of the process. For example, agricultural residues, such as sawdust (153) or wood pellets (154), are frequently employed in SSF to support fungal growth, thereby enhancing the circularity and sustainability of the process. SmF also has potential for utilization of a waste stream. SmF employs agitation methods such as shaking, bubbling, or stirring to produce mycelial biomass (118,155). This approach parallels processes in the fermentation industry, where mycelia are often treated as byproducts while secondary metabolites are 56 extracted (156). Collaborations with fermentation industries hold significant potential for repurposing waste mycelia directly into value-added materials, further advancing sustainable practices. 2.4 Conclusions In this study, we systematically investigated the relationship between fermentation method, fungal species, and the resulting morphological, chemical, and material properties of PMMs. By comparing static solid-state, liquid-state, and submerged fermentation conditions across multiple species, we demonstrated that both the physical environment and the inherent biology of the fungus drive significant differences in hyphal architecture and cell wall composition. Solid-state fermentation produced denser networks of skeletal and ligative hyphae, as well as higher chitin content, leading to materials with superior stiffness, strength, and storage modulus compared to mycelia produced by liquid-state fermentation. Submerged fermentation generally favored the development of more dense hyphal mats with increased mannoprotein content, resulting in materials with high stiffness and the potential for greater functionality. Differences in fermentation method also resulted in differences in surface properties, with liquid-state fermentation samples producing materials with the highest aerial side hydrophobicity and stability over time, a property that could be valuable in a range of material applications. These findings highlight the dynamic nature of fungal cell walls, which adjust their structure and composition in response to environmental cues. The observed correlations between hyphal morphology, cell wall chemistry, and mechanical performance speak to the importance of controlling fermentation parameters to tailor PMM properties for specific applications. For instance, optimizing for higher chitin content and higher relative amounts 57 of skeletal hyphae may be desirable for structural uses, while promoting generative hyphae and mannoproteins could enhance flexibility and surface functionality. Overall, these results provide a framework for the rational design of mycelium-based materials, leveraging the interplay between fungal biology and processing conditions to engineer desired material characteristics. This work advances the understanding of structure–function relationships in fungal biomaterials and supports the development of sustainable, tunable alternatives to conventional fossil fuel-based materials. 58 59 Chapter 3: Mycelium-modified masks for air pollution control Building on the strategies developed for engineering mycelium materials, this chapter shifts focus to their application as membranes for pollution control. Here, we investigate the potential of mycelium to address air pollution, using its naturally branched, interconnected macrostructure, a feature that resembles the fibrous networks found in conventional synthetic mask materials. Pleurotus was selected for these experiments based on preliminary results highlighting its “fluffy” mycelial network, which was anticipated to enhance breathability, as well as its resilience and adaptability across diverse substrates and conditions. This chapter marks the first exploration into utilizing the network architecture of mycelium for pollutant capture, offering insight into how these biological materials can physically interact with contaminants. 3.1 Introduction Protective face masks have been a crucial measure facing air hazards ever since its first appearance in late 1800s (157,158). Covid-19 has seen a large spike in demand for disposable face masks, especially as personal protective equipment for healthcare workers (159). The rising need for face masks drives concerns about environmental impact and sustainability. Currently, disposable face masks are manufactured from fossil-fuel based materials such as polypropylene, polyethylene, and polyesters (160). Researchers found that these face masks release nano- and micro-scale plastic fibers, silica grains, along with other heavy metal containing pollutants, into the environment (161). The extent of such negative impacts is still not fully understood; however, the increased prevalence of face masks will only serve to heighten these impacts. 60 Recent years have shown increased efforts to generate mask filters from bio-based materials such as cellulose (162), chitin (163,164), polylactic acid (165), and polyhydroxyalkanoates (166). These bio-based polymers are often processed into fibers for the production of masks through electrospinning, wet spinning, and melt spinning (167). Masks derived from these materials often necessitate multiple layers or additional materials to meet the functional requirements of a face mask (168–170). Bacterial cellulose, a material directly produced from acetic acid bacteria, has recently been explored as a mask filtration material (171). The self-structured material shows high tensile strength (~1200-2250 MPa) and high particulate matter removal efficiency (>99%); however, high hydrophilicity and swelling property create challenges in using this material to replace commercial face masks (171). Mycelium, a bio-based material derived from fungi, has never been studied explicitly as a mask filtration material. In comparison to the afore-mentioned bio-based materials, the self-growing nature and tunable hydrophobicity of mycelium makes it a unique source (172). Based on these properties, mycelium-derived materials could be another potential candidate as a replacement material for the fossil-fuel based plastics in current commercial face masks. Commercial face masks are composed of three layers, each serving a unique purpose. The primary functions of these layers are graphically depicted in Figure 3.1 (a) for a face mask derived from polypropylene. Polypropylene mask filter 1 (P1) is the filtration layer in the inside of the mask contacting the face. As such, it is mainly responsible for wicking moisture away from the face and providing comfort to the wearer (173). There is little to no significant filtration performed by this layer. Polypropylene mask filter 2 (P2) is the key filtration layer within the mask. Compared to P1, P2 has a smaller fiber diameter and, subsequently, smaller pore sizes. Tuning of this layer can have fundamental impacts on the 61 usability and rating of the overall mask. The final layer, displayed in Figure 3.1 (a) as the darkest outer layer, functions primarily to provide a hydrophobic coating to protect the mask from wetting and external contaminants (173). Creating a hydrophobic and non-absorbing outer layer is critical as the filtration ability of the inner layer can be dampened by moisture. 62 _____________________________________________________________________________________ Figure 3.1. Scheme of project methods and representative samples (a) Image of a 3-layer commercial non-surgical face mask and the primary functions of each layer. P1 and P2 refer to different layers of the polypropylene mask; (b) Graphical depiction of the method for production of mycelium-modified masks and the selection of P1 and P2 to be inoculated with fungal mycelium; (c) Graphic of the proposed functionality of a mycelium-modified mask; (d) Optical images of a representative mycelium-modified mask using digital camera (i) and the scanning electron microscope (ii). _____________________________________________________________________________________ 63 In this work, a novel method is presented that utilizes the native, self-growing assembly of mycelium to produce a single-layer modified mask with the functionality of a traditional three-layer mask. The production method used in this work is depicted in Figure 3.1 (b). Fungus from the species Pleurotus ostreatus is inoculated on top of a mask layer, forcing the growth of mycelium through the filter template to reach the malt extract agar plate. Pleurotus ostreatus was selected as the fungal species because it is considered non-pathogenic and exhibits good biocompatibility (174). The fungus is incubated between 10–20 days, at which point a network of mycelium is visible throughout the samples. By tuning the growth and processing conditions, the mycelium-modified mask is hypothesized to create a mask material with good filtration efficiency, breathability, and hydrophobicity, as illustrated in Figure 3.1 (c). This work aims to explore the potential applications and functionality of mycelium-modified masks as an alternative to disposable face masks made from synthetic materials and will investigate whether the modified mask can meet the functional requirements of the three-layered commercial non-surgical face masks. Key properties of the mycelium-modified masks discussed in this report are filtration efficiency, breathability, and water resistance, as they are essential parameters for producing filtration materials. Filtration efficiency and breathability will be explored as a function of the fungal incubation period, ranging from 10–20 days. It is expected that increasing the incubation time will lead to a higher density of mycelial filaments, allowing for tailoring of these parameters to fit the needs of the filtration material. Water resistance of the mycelium-modified mask is thought to improve through the introduction of hydrophobic proteins on the hyphal surface, which was not considered a function of incubation time in this report. Two layers of the mask are inoculated with mycelium and referred to as P1-M and P2-M, corresponding to the original mask layer that 64 was inoculated and modified. The template structure is thought to impact the morphology of the grown mycelium, leading to a difference in properties. Furthermore, investigating the difference between P1 and P2 templates can indicate the applicability of mycelium to improve an existing filter layer, P2, and create a primary filter layer from a template with much lower filtration ability, P1. Exploration of other species of non-pathogenic fungi and media are not included here. However, the results of this work help establish a baseline for working with these materials. 3.2 Experimental methods Materials Sterile liquid mycelium culture of Pleurotus ostreatus was purchased from GrowMushroomsCanada (Toronto, ON). Malt extract agar (catalog #: 70145), activated charcoal (catalog #: 05105), hexamethyldisilazane (catalog #: 440191), and glycerol (catalog #: G5516) were purchased from Sigma-Aldrich. 100mm x 15mm sterile polypropylene petri dishes (catalog #: FB0875712) were obtained from Fisher Scientific. 2% glutaraldehyde fixative in 0.1M Cacodylate Buffer, pH 7.4 was purchased from BioPLUS. 3-layer disposable and non-surgical face masks were purchased from JianshiBang (product model: 202). Substrate preparation Agar substrate was prepared by adding 12 g malt extract agar and 1 g activated charcoal to 250 mL deionized water. The solution was boiled for 10 minutes on a heated stir plate and cooled before autoclaving at 121°C for 20 minutes. Approximately 25 mL of agar was poured into each petri dish and allowed to set for 2 hours before storing in the fridge. 65 Mask preparation and growth Cellophane sheets were cut to fit the diameter of the agar plates and polypropylene mask filters were autoclaved at 121°C for 20 minutes. The cellophane sheets were placed over the agar and the mask filters placed on top. 1 drop of liquid mycelium inoculant was carefully deposited over each filter. Parafilm was used to wrap the perimeter of the agar plates. Inoculated plates were placed in an incubator at 27°C and allowed to grow for 10, 15, or 20 days. To terminate fungal growth, samples were placed in an autoclave at 121°C for 15 minutes and then allowed to air dry overnight. Scanning electron microscopy Samples were prepared for Scanning Electron Microscopy (SEM) imaging by soaking in 2% glutaraldehyde solution for 20 minutes followed by washing several times with deionized water (DI). Samples were coated with 10µm iridium with a Leica EM MED020 coater and subsequently imaged using a Helios NanoLab 650 scanning electron microscope with an Everhart-Thornley detector. Fiber and hyphal diameter were estimated using ImageJ software. At least 10 measurements were taken to generate a representative average. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectra were taken using the mask layers as films. A Perkin Elmer Frontier UATR equipped with a ZnSe-Diamond ATR crystal was used with a scan speed of 0.5 cm-1 and resolution of 4 cm-1. Spectra were collected at three distinct points on each sample. Thermogravimetric analysis 66 Thermal analysis was performed using a Thermogravimetric Analyzer TGA 5500. 6 mg samples were weighed and placed in the instrument pan. A 20°C/min ramp was used up to a final temperature of 600°C. Water resistance Static water contact angle was measured using the sessile drop method with a Theta Flex 300-Pulsating Drop 200 Tensiometer. A 5 µL drop was placed on the surface of the material and automatic measurement of the drop geometry was performed by the instrument. Contact angle measurements were calculated over a 5 second time-period. The average contact angle over this time-period was calculated for each sample and three measurements at different points were recorded. Swelling measurements were performed by placing small amounts (approximately 1cm x 1cm square samples) of each sample in DI water and measuring the mass change with respect to initial mass over 5 minutes, 10 minutes, 30 minutes, 1 hour, and 24 hours. 5 replicates of each sample were recorded. Water uptake measurements were also performed by placing similar sized samples in a Tenney Environmental Chamber for 24 hours at 85% relative humidity and 20°C and measuring the mass change with 3 samples each. Mechanical strength Tensile tests were performed using a universal Mechanical Tester (6800, Instron, U.S.). 15 mm × 5 mm samples were cut, and a testing length of 6 mm was used. Tape was folded over the top and bottom edges of the samples to reduce slippage on the grips. A rate of 10 mm/min and grip strength of 60 N was used for each sample. Ultimate tensile strength, elongation at tensile strength, and the Young's modulus were calculated from generated stress-strain curves. 5 replicates of each sample were recorded. 67 Filtration efficiency and breathability A filtration efficiency test was set up by using a particulate aerator to generate NaCl particles with a median diameter of 300 nm in a semi-enclosed chamber. Measurements of the filtration efficiency were performed by a respirator fit tester (Portacount PRO 8030, TSI, USA) attached to a chamber holding the filtering sample cut to a 3.7 cm diameter circle. A personal air sampling pump (GilAir Plus, Gilian, USA) forced NaCl particles through the chamber at a consistent face velocity. Filtration efficiency was generated by the respirator fit tester from the change in the number of particles detected in air without the filter (C0) and with the filter (Ci) (175). 10 readings of filtration efficiency were recorded for each sample. The filtration efficiency (FE) could be calculated by Equation 3.1: (Equation 3.1) 𝐹𝐸 = (1 −𝐶𝑖𝐶0) ∙ 100 Breathability was analyzed by finding the pressure differential (pressure drop) between two sides of the filter. Pressure drop was measured using a pressure gauge attached to the filtration efficiency instrument. Post-processing treatments In some instances, the mycelium-modified masks were treated following autoclave growth termination. Samples that received treatment were soaked in 8% (V/V) glycerol for 1 h. After each treatment, the samples were washed 5 times with deionized water and allowed to air-dry. 68 3.3 Results and discussion 3.3.1 Morphology and composition The association of mycelium within a non-biodegradable matrix has not been visualized prior for material applications. Previous works typically show mycelium growing through, or on, lignocellulosic substrates that the fungus can degrade. Figure 3.2 depicts the morphology of the inedible mask template used in this study. Microscopy reveals that the polypropylene mask fibers do not inhibit the growth of the mycelium, even allowing the mycelial hyphae to grow directly on top of the polypropylene. The mycelium is then able to bridge gaps between the fibers and generate a highly branched network as shown in the top view of samples P1-M and P2-M (Figure 3.2 (b) and (e), respectively) Extensive branching of the mycelium is thought to allow for better entrapment of particulate matter, enabling higher performance as a filtration material. Cross-section images of the mycelium-modified masks (Figure 3.2 (c, f)) show the mycelium was able to penetrate through the height of the mask layer. The mycelium in the mycelium-modified masks greatly reduce the overall fiber/hyphae size. Hyphae diameter is smaller than the original mask fiber diameter for both P1 and P2 as indicated in Table 3.1. The small diameter of the mycelium can allow for the creation of a high number of smaller pores, allowing for improvement of physical filtration properties. _____________________________________________________________________________________ 69 Figure 3.2. Morphology of mask layers before and after mycelium modification: polypropylene fibers in native mask layers for (a) the innermost layer (P1), and (d) the middle layer (P2), the association of mycelial hyphae and polypropylene face masks in the modified material for (b) P1-M and (e) P2-M and at the cross section for (c) P1-M and (f) P2-M. _____________________________________________________________________________________ Table 3.1. Fiber/hypha diameters in mask layers and mycelium-modified mask layers Sample Fiber/Hypha diameter (µm) P1 22.6 ± 1.4 P2 6.81 ± 2.7 P1-M 2.06 ± 0.8 P2-M 2.22 ± 0.8 70 The unique growth design is thought to contribute to the ability of the fungus to associate with the polypropylene. This design is comprised of a bottom layer of nutrient-rich agar, followed by a sheet of cellophane topped with the mask layers. Pleurotus ostreatus was inoculated on top of the mask layers, forcing the fungus to grow down through the mask to reach nutrients in the agar and creating a mass of vegetative hyphae. At the same time, the fungus at the air interface generated a rich layer of aerial hyphae. The cellophane was placed between the mask and agar to aid in clean removal after heat deactivation. Chemical and thermal characteristics of mask layers are shown in Figure 3.3. The ATR-FTIR was performed to confirm the presence and composition of the Pleurotus ostreatus mycelium in the modified material. Spectra of pure mycelium (P1, P2), and modified layers (P1-M and P2-M) are shown in Figure 3.3 (a). Mycelial spectra are in line with previously reported literature data, indicating similar composition of chitin, β-glucans, and glycoproteins (100,176). Additionally, mycelium-modified mask spectra indicates that the mycelial structure is not significantly changed by the growth through polypropylene layer. A broad peak from 3000 to 3600 cm−1 corresponds to OH stretching. Sharpening of the broad OH band around 3270 cm−1 is related to secondary amine and amide groups. Peaks between 2800 and 3000 cm−1 are related to CH stretching. The sharp peaks at 1455 and 1375 cm−1 are associated with CH2 and symmetric CH3 deformation, respectively, specific to polypropylene. Peaks around 2920 cm−1 are referenced as stretching from CH bonds in aldehydes, primarily associated in fungal cell walls with chitin. Peaks around 1630 and 1545 cm−1 are also related to amide I band and amide Ⅱ band, respectively. These peaks can be associated with both chitin and proteins in the fungal mass. Finally, peaks around 1000 cm−1 are associated with C-OH stretching in a variety of polysaccharides in the fungal cell wall, such as chitin, glucan, and mannans. The mask layers were tested as films directly in the ATR-FTIR 71 instrument. As such, the thick layer of mycelia on the outside of P1-M resulted in little recognition of the polypropylene mask portion. However, P2-M shows some of the characteristic CH2 and CH3 peaks related to polypropylene. TGA analysis, shown in Figure 3.3 (b), also confirms both the presence of mycelia and polypropylene, and shows that the mass percent of mycelium in the modified layers is approximately 7% for both P1-M and P2-M. Other interesting information gained from the TGA spectra is the shift of the onset of thermal degradation by about 20°C for mycelium-modified masks. This suggests some thermal insulation effect of the mycelium on the polypropylene. Fire resistance is another consideration for development of commercial masks, as they can also be utilized by individuals in contact with smoke (177). Fire resistance is a parameter that can be explored in more depth in future studies. 72 _____________________________________________________________________________________ Figure 3.3. Chemical and thermal analysis of mask layers and mycelium-modified mask layers: (a) ATR-FTIR analysis of mask layers and mycelium-modified mask layers; (b) TGA analysis of mask layers and mycelium-modified mask layers. _____________________________________________________________________________________ 3.3.2 Filtration efficiency and breathability Figure 3.4 (a) illustrates that the filtration efficiency for P1 is significantly improved by growth of mycelium even after only 10 days, going from an efficiency of 43% to an efficiency near 80%. Filtration efficiency of P2 is slightly improved, with a smaller range of filtration efficiencies detected, for 10-day grown sample as shown in Figure 3.4 (b). Unmodified P2 demonstrates a filtration efficiency of 94%. After modification, the filtration efficiency rises to 97%. As depicted in Figure 3.4 (c), the filtration efficiency of this single layer matches closely with a 3-layer commercial mask. Furthermore, the breathability of both 10-day samples (P1-M and P2-M) is similar to the commercial 3-layer mask. Interestingly, filtration efficiency for samples grown for 15 days and 20 days started to decrease for both P2 and P1. 73 It is unclear what is responsible for the lower filtration efficiency, but it can be theorized that potential changes in composition and morphology as the mycelial mass continues developing could contribute to this observation. However, as expected, there was generally a decrease in pressure drop compared to 10-day samples possibly related to increased mycelial density in the samples. 74 _____________________________________________________________________________________ Figure 3.4. Filtration efficiency (FE) and breathability of mask-mycelium composites. Filtration efficiency of original mask layers P1, P2, and a commercial mask (all three original mask layers) and mask layers grown with mycelium P1-M, P2-M over the course of 10, 15, and 20 d. GX refers to the time (X) of the sample growth. No time label indicates the sample was grown for only 10 d. _____________________________________________________________________________________ Mycelium imparts strong hydrophobicity to the mask filter layers on the top side due to the unique growth method. Figure 3.5 (a) shows contact angle of samples on the top and 75 bottom sides. P1 and P2 are hydrophobic, with contact angles of 144° and 145°, respectively. However, modification of the layers with mycelium creates a potentially superhydrophobic material, with contact angles above 150°. The contact angle for P1-M and P2-M are 157° and 155° on the top side, respectively. The strong hydrophobicity of the top side is potentially due to the dense layer of aerial hyphae that forms a thick skin on the top of the composite. Aerial hyphae tips are known to be coated by hydrophobic proteins that help the fungus modulate environmental interactions (178). Pleurotus species, in particular, are known to possess several types of hydrophobic moieties (179). In dry environments, hydrophobins conform to create a strong hydrophobic interaction. However, in wet conditions, the proteins are able to rearrange to create a hydrophilic interaction (180). The contact angle of samples after reswelling in water for 24 h was around 120°, suggesting this conformational change of the hydrophobic constituents. Lipid residues could also play a role in the observed hydrophobicity of the mycelium. Interestingly, the bottom side of the modified mask layers, grown contacting the cellophane, displayed lower contact angles (70° for P1-M and 110° for P2-M). This may be due to a differing composition of the vegetative hyphae or the lack of hydrophobic protein coating. Relative to P2, P1 had more space between the polypropylene mask fibers which may have allowed for deeper and thicker growth of vegetative mycelium. This could help explain the larger difference in contact angles of P1 on the top and bottom sides. To this author's knowledge, this is the first time this asymmetric hydrophobicity of mycelium composites has been explored. Typically, mycelium is grown through lignocellulosic materials, which have been thought to be responsible for the modified material's hydrophilicity. In this research, however, the modified material is hydrophobic, showing especially in the case of P1 that the mycelium itself is contributing to lower contact angles and increased hydrophilicity on the bottom side. 76 _____________________________________________________________________________________ Figure 3.5. Contact angle tensiometer potential mechanism for utilization of asymmetric hydrophobicity. (a) Contact angle of P1, P2, P1-M, and P2-M (the dashed line represents a contact angle value of 150°.); (b) Graphic illustrating the top and bottom sides of the mycelium-modified mask layers and the potential dual mechanism for water repellency and water uptake (top refers to the composite side contacting the agar plate and bottom refers to the side interfacing with the air; (c) Representative contact angle images selected for P1, P2, P1-M, and P2-M. _____________________________________________________________________________________ 77 Higher swelling of mask layers with mycelium is reported in Appendix B-1. Variation observed in mycelium samples may be a result of pieces of the mycelium breaking off during handling. This result has promising implications for the wicking effects that would be necessary for the inner mask portion. In particular, the contact angle gradient from the bottom side could help drive these wicking effects. It should be noted, however, that no discernable water uptake was measured over 24 h at 85% relative humidity which was chosen to represent the humidity of breath contacting the inner layer of the mask. More studies should be performed to investigate the wicking effects of the mycelium. 3.3.3 Mechanical properties Mechanical testing of the mask layers was performed to ensure the usability of the material. Stress-strain plots of the layers are included in Appendix B-2. A table with mechanical data, including Young's modulus, elongation at break, and ultimate tensile strength is also provided in Appendix B-2. The original mask layers (P1) display higher elongation at tensile strength and lower Young's modulus, owing to higher elasticity. A nearly 10 × increase in Young's modulus of layers impregnated with mycelium suggests a material with significantly improved strength. However, the mycelium imparts some stiffness to the composites, potentially a result of stiff chitosan chains that constitute the cell wall. Mask material must be able to withstand mechanical force and movement while maintaining elasticity, another important metric to create a comfortable material. A post-processing treatment was briefly explored to provide a range of mechanical properties that could give rise to fine-tuning of the material properties and find adequate balance between strength and elasticity. Glycerol, a common plasticizer, was utilized for treatment of fungal materials with improved strength and flexibility (181). Glycerol-treated masks show slight improvements in elongation at tensile strength. The low treatment time (1 h) could have 78 resulted in low amounts of glycerol impregnation in the samples. Further optimization using these treatments should be performed in future studies. 3.3.4 Conclusions The results of this study demonstrate the potential of mycelium to act as a crucial filtration component in mask applications: a) Filtration efficiency and breathability were tuned by the number of days the mycelium was allowed to grow, with 10 days providing a sufficient compromise between the two factors. Filtration efficiency of P1 was improved by approximately 37%, modifying this layer such that it can be utilized as a primary filtration layer. A roughly 3% increase in filtration efficiency of layer P2 allowed the filtration efficiency of this single layer to reach the filtration efficiency of all three layers of a commercial mask; b) A hydrodynamic gradient was established between the top and bottom of the mycelium material to assist in providing a water-barrier needed for the outside of the mask component while potentially allowing for water-wicking with the area in contact with the face; c) Through the stacked growth design, mycelium able to grow through an inedible matrix. The morphology and composition of the mycelium-modified mask layers was explored. Other critical parameters for masks, such as flame resistance, bacterial filtration efficiency, and synthetic blood penetration should be tested in future studies. However, this research underscores the exciting potential of mycelium as self-growing filaments for mask material and highlights the need for further research in this area. In particular, additional development of post-processing treatments and investigations of wicking properties of the vegetative hyphae is needed. Future development of this technology can be envisioned to reduce polypropylene content in disposable face masks by 66%, reducing environmental 79 impacts dramatically. Furthermore, this method can be utilized to recycle disposable face mask layers that would otherwise be subjected to a landfill. 80 81 Chapter 4: Mycelium membranes for the capture of potentially toxic elements Transitioning from the air filtration applications explored in Chapter 3, this chapter shifts focus to water pollution remediation, leveraging mycelium’s intrinsic interactions with metals. Building on the bioprocessing insights from Chapter 2, submerged fermentation (SmF) was selected to produce mycelial membranes for water treatment due to its superior mechanical properties, critical for maintaining structural integrity in flow reactors, and elevated mannoprotein content, which was thought to enhance surface functionality for pollutant binding. The creation and exploration of mycelial membranes for PTE capture in this chapter highlights mycelium’s versatility in addressing diverse pollution streams. Additionally, the development of the membrane for integration in flow reactors highlights the potential of this technology to be applied in larger-scale settings. 4.1 Introduction Hundreds of millions of people across the globe suffer from harmful levels of contaminated or chemically polluted drinking water as a result of improper wastewater management (182). Studies show that marginalized communities are more vulnerable to the exposure and negative effects of pollution, further driving social inequalities (3,183,184). As a result, the World Health Organization underscores the importance of water affordability and accessibility in sustainable development target 6.1 (2). Potentially toxic elements (PTEs) are one type of pollutant that is a growing environmental concern resulting from the release of effluents from a variety of industrial processes (12,185). Excess concentrations of PTEs, such as copper (Cu), mercury (Hg), lead (Pb), cadmium (Cd), arsenic (As), and nickel (Ni), can cause extensive damage to living organisms(185). Even small quantities of PTEs released in 82 the environment pose serious threats to human health through bioaccumulation and biomagnification (186). Rural areas near mining sites or industrial plants, in particular, can be highly impacted by PTE contamination leaching through soil into local waterways or wells (187). Furthermore, marginalized communities in rural areas are historically underfunded in terms of water infrastructure development across ethnic, racial, and class lines (188). In addition to developing social strategies that urge municipalities to provide more equitable resources and funding allocation, it is important to improve engineering strategies that allow for successful implementation. This entails the development of materials for membrane adsorption that will meet the needs of marginalized communities in terms of cost, performance, and sustainability. Well-known strategies to remove PTEs include coagulation, flocculation, ion-exchange membranes, precipitation, and adsorption (15–18). Precipitation, coagulation, and flocculation are considered simple and economical PTE removal techniques (22). However, these strategies suffer from slow operational times and secondary pollutant generation in the form of sludge that requires additional treatments(19,20). Ion-exchange membranes offer fast removal and operation but are restricted by fouling and limited operational parameters. Considered the most prevalent removal technique, adsorption is both simple and able to use a wide variety of starting materials, but the long treatment times relative to other techniques can make translation to industrial scale difficult (22). Membrane adsorption is a promising technology that couples the filtration ability of porous membranes with targeted capturing of pollutants by functional groups on the membrane surface. Compared to the aforementioned traditional strategies, this hybrid system can allow for higher efficiency removal of pollutants and facile integration into industrial processes and/or point-of-use devices (189). A material for membrane adsorption is typically produced by functionalizing or grafting on the surface 83 of polymer membranes (190), exemplified by the work of Hao et. al. in grafting caffeic acid onto the surface of PVDF membranes (191). Current challenges to implementing this technology include limited adsorbate residence time (the amount of time the adsorbate interacts with the adsorbent material before desorbing or passing through the membrane), complex processing, and high production costs (192). Limited research has focused on using biobased or biological materials to generate adsorption membranes, creating a knowledge gap in the literature with regards to these materials despite their large potential (193). Filamentous fungi present an interesting case for development of an adsorption membrane as this biomass has been studied for its adsorbent properties and has been utilized in recent years to develop biobased materials (136,194,195). In nature, fungi are required to sequester small amounts of essential metals from their environment to support metabolism (196). However, high concentrations of metals can be deleterious to the growth and enzymatic function of fungal cells (197). Therefore, complex mechanisms are responsible for mediating interactions between fungi and metals. These interactions include biosorption to cell wall components, bioaccumulation by precipitation in organelles, and chelation mediated by the release of metal-chelating agents (198,199). Biosorption of metals onto living and non-living fungal biomass has become a growing field of interest (42–44). Biosorption functions at the cell wall surface by physical adsorption, ion exchange, electrostatic interactions, and complexation (45). Typically, in living systems, fungi are inoculated in liquid media containing the pollutant and function by bioremediation through the growth period (200–202). In non-living or inactivated systems, the fungal biomass is typically harvested after growth, dried, and added to the pollutant solution as an adsorbent material (203–205). Non-living fungi are considered superior for PTE remediation due to higher resistance to operational parameters, lack of necessity for sterilization or additional nutrients, and 84 reusability (40). Furthermore, non-living fungal biomass has shown higher PTE removal than living counterparts (206). Despite the large body of work using fungi to remove PTEs, there are no studies that exploit the self-structured mycelium, derived from filamentous fungi, as a low-cost and sustainable bio-based membrane for PTE remediation. This work introduces a novel technique to fabricate adsorption membranes from mycelium toward the cleaning of mining-related contaminants by investigating the removal of a model PTE via kinetic, isotherm, and chemical analysis. Control over the porous structure, and therefore the membrane flux, is achieved by modification with a small amount (≤5wt% of the total structure) of an additional biobased material: tempo-oxidized cellulose nanofibers (TCNFs). Cu(II) was selected as a model divalent cation to represent PTEs for batch studies. Zn(II) and Cd(II) were also tested to highlight the applicability of the adsorption membrane for different types of PTEs that are important for communities near mining sites (207). Pleurotus ostreatus, a species of the white-rot fungi, was utilized for this study as it has previously shown an ability to remove a variety of pollutants in aqueous systems, including several metals, as well as produce large amounts of hyphae (208,209). Furthermore, the inherent non-toxic nature of this species with respect to the environment and human health allows for its use in water treatment applications. P. ostreatus was grown in submerged fermentation vessels, a common reactor type for many industrial systems producing fungal mycelium and by-products. Often, these industrial plants discard the mycelium biomass as waste as the secondary metabolites (enzymes, citric acid) are extracted for commercial use (210). As such, the research design presented here encourages the utilization and valorization of a waste stream from fermentation industries, which allows for the envisaged product to be considered low-cost (211). This work represents the next 85 generation of materials toward adsorption membranes that considers the intersectionality of social and engineering challenges. 4.2 Results 4.2.1 Membrane synthesis and characterization Membranes were prepared by processing filamentous biomass from liquid cultures of P. ostreatus as shown in Figure 4.1 (a). An appropriate nutrient media was found to be yeast extract peptone to achieve high growth levels and non-lysed hyphae (Appendix C-1 (a-b)). Biomass collected from shaken liquid state fermentation reactors had a pellet shape, resulting from the entanglement of hyphae under the shaking conditions (Appendix C-1 (c)). After blending and homogenizing, the mycelial network becomes broken into hyphal fragments that can be formed into a membrane via vacuum filtration. Blending and homogenizing times were selected as 1 minute and 10 minutes, respectively, based upon processing optimization (Appendix C-2). Tempo-oxidized cellulose nanofibers (TCNFs) were added during the homogenizing phase at concentrations of 1wt% (M-1TCNF) and 5wt% (M-5TCNF). The membrane mode of action is shown in Figure 4.1 (b). From the unique processing method, a hierarchically porous membrane was generated to attach PTEs to adsorption sites on the membrane surface. TCNF content from 0-5wt% regulated the rate of flow of liquid through the membranes surface area, otherwise known as flux, which in turn controlled the contact time of adsorbates with the membrane. 86 _____________________________________________________________________________________ Figure 4.1. Biobased membrane formation and design. (a) Method for preparing biobased membranes from mycelium and TCNF with growth, blending/homogenizing, and vacuum filtration steps (b) Graphical depiction of the adsorption membrane function, the proposed hierarchical porosity of membrane derived from fungal mycelium with pores generated by the fungal filaments as well as pores on the fungal cell wall, and proposed tunability of mycelial membranes with the addition of TCNFs in terms of flux and, consequently, contact time with pollutants. Image made with BioRender. _____________________________________________________________________________________ The membrane structures for neat mycelium (M) and M-5TCNF are shown in Figure 4.2 (ai-ii). Under the scanning electron microscope, the hyphal structure is shown interweaving to generate a porous material on the surface and in the cross section. Attachment of the TCNFs to an individual hypha can be visualized in Figure 4.2 (aii) and 87 is shown to act as a bridge between hyphae, as graphically illustrated in Figure 4.2 (b). Electrostatic interactions via the partially negative carboxyl units on the TCNF and partially positive acetamide and amine units of the mycelia can mediate this attachment (212). Additionally, non-modified CNFs showed significant aggregation compared to their oxidized counterparts, supporting the need for CNF modification (Appendix C-3). The prepared membranes were porous due to the filamentous structure of the mycelium. Pores were present in both the macro-structure of the membrane and within the fungal cell wall, forming a hierarchically porous material, as can be seen in Figure 4.2 (ai-ii). It was hypothesized that the addition of TCNFs in varying amounts could control the porosity and water flux of the membrane. To verify this hypothesis, analyses of porosity and filtration/flux were subsequently performed. Porosity was calculated through BET using a micro-porous and meso-porous range instrument, which was necessary as a result of the different scales of porosity. Average pore sizes in terms of pore diameter (Pd, avg) and BET surface area (SA) are shown in Figure 4.2 (c). Pores mainly arising from the preparation of the membrane are referred to as processed pores, while cell wall pores refer to pores considered to be associated with the native cell wall architecture. Pores in the range of 2-3 nm are common in the fungal cell wall as they assist in nutrient uptake and cytoplasmic release, which explains the Pd, avg of ~4 nm for the pure mycelium membrane found with micro-porous instrument (213). The increase in the Pd, avg in M-1TCNF is a result of larger pores generated by TCNF bridging. Notably, a ~10x decrease in surface area with the addition of 1wt% TCNF was determined by the micro-porous BET, which can be rationalized by the coverage of the cell wall pores by TCNFs similar to observations seen in membrane fouling (214). Further increase of TCNF 88 content to 5wt% resulted in a decrease in the Pd, avg relative to 1wt% as a higher density of TCNF creates smaller pores. Processed pores calculated from the mesopore-range BET instrument show a Pd, avg between 20 and 30 nm and a greater surface area aligning with increasing TCNF content. Pore size distribution of the membranes is shown in Figure 4.2 (d), as measured with a mesoporous-range BET instrument. The pore area (Parea) was found to greatly increase for M-1TCNF and M-5TCNF relative to the M membrane. The filtration ability and corresponding flux, shown in Figure 4.2 (e), decreased for M-1TCNF and M-5TCNF relative to M. With the addition of 1 and 5wt% TCNF, bridging of the hyphae resulted in a higher number of mesopores and a reduction of macropores, supporting the mesopore size analysis. Pure mycelium membranes contain larger macropores that create a faster filtration rate not captured by BET, as shown in SEM. Other physical characteristics impacted by the addition of TCNF included swelling and mechanical strength, which are reported in Figure 4.2 (f). These criteria are important in assessing the usability of the membranes in real-world systems. A high storage modulus and low swelling are favorable so as not to induce conformational changes to the membrane during its use. Stiffness of the membranes was assessed through dynamic-mechanical analysis (DMA) using sinusoidal tensile deformation. DMA data, in particular, can aid in enhancing our comprehension of the microstructural attributes of the materials used in membrane fabrication. The stiffness of membranes is ultimately influenced by the materials employed for their fabrication and the interplay between the membrane and humidity. Figure 4.2 (f) presents the storage modulus (E') of membranes at 50°C, concurrently demonstrating swelling percentages. Swelling was found to remain similar for membranes 89 with 0 and 1wt% addition of TCNF at 6.73 and 5.96%. However, for the addition of 5wt% TCNF, swelling rose to 9.13%. _____________________________________________________________________________________ Figure 4.2. Structure of Mycelium Membranes and Modification with Tempo-Oxidized CNFs. (a) Images of membranes without TCNF (i) and with 5wt% TCNF (ii) using a digital camera and x-sectional morphology of membranes using scanning electron microscopy. (b) proposed interaction of TCNFs and mycelial hyphae. Image made with BioRender. (c) Average pore size and calculated BET surface area of membranes 0, 1, and 5wt% TCNF using a meso-scale and micro-scale BET instruments (d) Mesopore size distribution from BET of 90 membranes with 0, 1, and 5wt% TCNF. (e) Filtration of pure water over time for mycelium membranes with 0, 1 and 5wt% TCNF. (f) Swelling and storage modulus at 50°C of membranes with 0, 1 and 5wt% TCNF. _____________________________________________________________________________________ As illustrated in Figure 4.2 (f), the incorporation of 1wt% TCNF in the mycelium membrane facilitated an optimal interfacial interaction, leading to strong adhesion between TCNF and mycelium fibers. This resulted in the highest storage modulus and efficient stress transfer (140 MPa). However, introducing higher quantities of TCNFs (5wt%) led to a reduction in the storage modulus. This decline can be attributed to the potential aggregation of TCNFs, which could detrimentally impact the membrane's mechanical characteristics. Imaging of the samples with scanning electron microscopy displayed cases of large aggregates on the surface of the membrane (Appendix C-4(b)). Overall, these results demonstrate that it is crucial to maintain the concentration within the percolation threshold, a range dictated by the aspect ratio and chemical properties of TCNFs. This ensures that the desired mechanical performance is retained in the membrane. 4.2.2 Elucidating the mechanisms of PTE adsorption To elucidate the mechanism underlying the mycelial membrane’s ability to adsorb PTEs, we conducted a comprehensive analysis encompassing kinetic, isotherm, and chemical analyses using Cu (II) as a model divalent cation. These experiments were performed in a batch system, facilitating direct comparability with existing studies in literature. Optimal conditions for maximal adsorption were determined by initially adjusting pH and time (Appendix C-5 (a)). Adsorption efficiency (qe) was measured as the amount of Cu(II) (mg) adsorbed per mass of membrane adsorbent (g) using a colorimetric assay of the Cu(II) solutions. It was found that qe increased from pH 3.5 to 5.5, a result of decreasing competition 91 with hydronium ions at adsorption sites. This result aligns with results from other fungal adsorbent materials (215). However, above a pH of 5.5, precipitation of Cu(II) to Cu(OH) resulted in lower amounts of soluble ions available for adsorption. An equilibrium reaction time of approximately 12 hours was determined for all membranes, past which adsorption remained stable up to 24 hours (Appendix C-5 (b)). Therefore, subsequent experiments used an adsorption time of 24 hours to ensure equilibrium had been reached, and the optimal pH of 5.5. A dose of 1 mg adsorbent/mL adsorbate was used although adsorption was observed to increase with higher dosages (Appendix C-5 (c)). The data from the kinetic analysis was fitted to pseudo-first and pseudo-second order models, with relevant parameters for each detailed in Table 4.1. For all membranes, a pseudo-second order model provided the best fit as depicted in Figure 4.3 (b). A pseudo-second order model assumes chemisorption is the main adsorption process, contrasting with the pseudo-first order model that posits physisorption as the primary mechanism. Isotherm data was fitted to common adsorption isotherm models, Langmuir and Freundlich, and the parameters are noted in Table 4.2. The Langmuir model was found to provide the best fit to the data, implying a monolayer adsorption process, as shown in Figure 4.3 (c). From the Langmuir fit, a maximum theoretical adsorption value, qm, was calculated. For M, M-1TCNF, and M-5TCNF, qm was found to be 72.3, 73.2, and 62.4 mg Cu adsorbed/g mycelium adsorbent respectively. The fungal cell wall has a complex chemical make-up that can contribute to its unique adsorption as shown in Figure 4.3 (c,d). Within the cell wall, a complex of covalently bonded chitin/chitosan and β-glucans form the major structural component. A variety of mannoproteins and α-glucans are also found in the cell wall (216). The polysaccharide and protein constituents of the fungal cell wall create a plethora of functional sites for the 92 adsorption process which include acetamide, amine, hydroxyl, and carboxylic acid moieties. Several mechanisms, including chelation, ion exchange, and electrostatic interactions, are thought to play a role in PTE adsorption by these groups (217). A pseudo-second order fit helps validate this assumption as it implies that chemisorption, rather than physisorption, controls the adsorption process. The adsorption capacity of the material decreases with 5% addition of TCNF, indicating that the mycelium is the major adsorbing material. Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) serve as analytical techniques vital for identifying the specific functional groups and chemical environments in the mycelial membrane that play a crucial role in adsorption. FTIR analysis showed that peaks related to C=O stretching, N-H bending, and C-N stretching exhibit a blue shift after adsorption of Cu (Appendix C-6 (a)). The blue shift is due to π-back donations between the ligand (C=O, N-H, C-N) and metal (Cu(II)) that indicates the involvement of these functional groups in the adsorption of Cu(II). To corroborate these findings, XPS was employed to obtain information about the mycelium membrane’s chemical environments before and after copper adsorption. As evident from Appendix C-6 (b), XPS data comparing the mycelium membrane before and after adsorption demonstrates the retention of adsorbed Cu(II) and confirms no reduction of Cu2+. Notably, the post-adsorption spectrum lacks calcium signatures, hinting at ion-exchange as a key adsorption mechanism. Observations of shifted binding energies in high-resolution nitrogen peaks imply copper’s coordination with mycelium’s amine and amide groups (Figure 4.3 (e)). Similarly, altered peaks in the high-resolution C and O spectra, shown in Figure 4.3 (e), suggests Cu association with carboxylic acid and carbonyl regions. Shifts in these peaks demonstrate the involvement of fungal cell wall constituents containing these functional units, including mannoproteins, chitin, and chitosan. 93 _____________________________________________________________________________________ Figure 4.3. Batch adsorption kinetic, isotherm and chemical analysis. (a) Adsorption efficiency (qe) over time for M, M-1TCNF, and M-5TCNF and fits of the pseudo-second order model (b) Dependence of adsorption efficiency (qe) on the equilibrium concentration of Cu(II) (Ce) and fits of the Langmuir isotherm model (c) Proposed mechanisms for the adsorption of Cu onto the various functional groups associated with fungal cell wall components. Image made with BioRender. (d) Chemical structures of major fungal cell wall components. 94 Structures created with ChemDraw. (e) high-resolution nitrogen (N) and carbon (C) scans of M membrane before and after adsorption. _____________________________________________________________________________________ Table 4.1. Parameter and fit values for kinetic models Table 4.2. Parameter and fit values for isotherm models 4.2.3 Performance as an adsorption membrane for PTE removal The adsorption performance of the membranes was evaluated to understand the effectiveness of the material in a dead-end cell system Figure 4.4. A colorimetric analysis of Adsorbent Pseudo 1st order Pseudo 2nd order k1 qe,calc R² k2 qe,calc R² M 1.049 46.0 0.8851 0.03215 49.6 0.9745 M-1TCNF 0.887 45.1 0.9630 0.03082 49.2 0.9942 M-5TCNF 0.838 35.4 0.9804 0.02823 38.4 0.9975 Adsorbent Langmuir Freundlich KL qm R² KF 1/n R² M 0.0287 72.3 0.980 8.22 0.394 0.925 M-1TCNF 0.0183 73.2 0.931 6.11 0.424 0.877 M-5TCNF 0.0245 62.4 0.921 8.85 0.336 0.868 95 Cu(II) was performed and the indicator color became invisible to the naked eye after membrane adsorption (Figure 4.4 (a)). Digital images of the membrane before adsorption, after adsorption, and after regeneration are shown in Figure 4.4 (b). The blue color on the membrane after adsorption is visual result of Cu attachment to the surface. Cu was not only detected on the surface of the membrane, but through the cross-section as highlighted by Energy-dispersive X-ray spectroscopy (EDX) (Figure 4.4 (c)). After regeneration, the membrane returned to its original color, indicating the release of Cu from the material. As discussed previously, flux of liquid through the membrane can be controlled through the addition of TCNFs. Flux can have a large impact on the residence time of the adsorbate, allowing for potentially higher levels of attachment. Figure 4.4 (d) depicts the flux and metal rejection percentage of M, M-1TCNF, and M-5TCNF. Compared to M, M-1TCNF displayed a lower flux and higher rejection percentage. The higher contact time for Cu(II) ions through M-1TCNF allows metal ions to attach to the adsorption sites. Although M-5TCNF has a similar flux to M-1TCNF, the lower rejection percentage is a result of the lower adsorption capacity and fewer binding sites available as a result of cross-linked mycelium and TCNF. Figure 4.4 (e) depicts the reusability of membranes across three adsorption/desportion cycles using 0.1M hydrichloric acid (HCl) as the desorption solution. After the first desorption cycle, a marked decrease was observed in adsorption. Acid-soluble components of the mycelium may be stripped during the desorption process, although further investigation of this process is needed to understand this anomaly. Using the M-1TCNF as the optimal membrane, the system was tested for removal of additional PTEs: Zn and Cd shown in Figure 4.4 (f). Compared to the optimized removal of Cu(II) at 97.4%, Zn and Cd had lower removal percentages of 49.7% and 68,2%. However, optimization of the system for these metals, particularly in terms of pH, has the potential to improve the values. 96 _____________________________________________________________________________________ Figure 4.4. Adsorption membrane performance for Cu(II) and other PTE removal. (a) Graphical depiction of the colorimetric assay used to detect Cu(II) concentration before adsorption and after adsorption using the M-1TCNF membrane in a dead-end cell apparatus. Graphic created with BioRender. (b) Images of the M-1TCNF membrane before adsorption, after adsorption, and after regeneration in 0.1M HCl. (c) EDX map of the surface and cross section of the membrane after adsorption. (d) Flux and Cu(II) rejection percentages for M, M-1TCNF, and M-5TCNF. (e) Amount of Cu adsorbed by the M, M-1TCNF, and M-5TCNF membranes over 3 cycles of adsorption and desorption. (f) PTE rejection percent of M-1TCNF for Cu, Zn, and Cd. _____________________________________________________________________________________ 97 4.3 Discussion Previous works using mycelium as a material often use solid-state fermentation reactors (100,174,218). This retains the native morphology of the mycelium, but also retains the spatial heterogeneity of the network(219). For functional applications, a controlled morphology may be favorable. VTT Technical Research Centre of Finland shows a large-scale production method using shaken liquid state fermentation followed by the creation of a mycelium slurry(105). The slurry can then be processed via vacuum filtration or lyophilization to produce a fungal sheet. In this work, a similar method of blending, homogenizing, and vacuum filtration was used to develop a porous membrane derived from mycelia. Through this simple processing method, a homogenous mycelial sheet can be derived that can enable a higher level of control in filtration and adsorption. Optimization of the critical production areas for the method introduced in this work, blending and homogenizing times, to properly fibrillate the mycelium is proposed as a baseline for further development of this system. With mycelium alone, it is difficult to control the porosity of the membrane. To overcome this challenge, TCNFs were selected as an additional biobased component to regulate this parameter. In prior works, mycelium has been combined with cellulose nanocrystals and carboxymethylated cellulose nanofibers during the pellet growing process (220). However, this does not allow for precise control over the amount of material attached to the mycelia. Another study describes the blending of larger Kraft fibers and hemp fibers with fungal fibers, resulting in a microporous paper with enhanced air permeability and virus reduction capacity compared to neat cellulose fibers (221). In contrast, this work used the addition of smaller cellulose nanofibers to mycelia after growth and processing to modulate membrane properties. Surface area, porosity, and, thereby, water flux, were able to be 98 tailored by the addition of TCNFs into the system with different concentrations. As well, addition of 1wt% TCNFs served to enhance the mechanical properties of the membrane with little change in swelling, allowing for improved utility. The mechanical reinforcement capabilities of TCNFs have been similarly shown in prior work within a variety of bio-based and petrochemical-based matrices (222,223). However, these beneficial properties, including higher mechanical strength and minimal swelling, appear to be diminished at ≥5wt% TCNF addition. The reduction in mechanical properties can be attributed to aggregation of TCNFs within the membrane, creating void spaces that can act as crack propagation sites (224). Higher concentrations of TCNF also reduce the adsorption capacity of the membrane, resulting from the interaction mycelium and TCNF, a phenomenon seen for other cross-linked adsorption systems (225). Therefore, additions of only small amounts of TCNF (1wt%) are beneficial for the membrane adsorption system to regulate water flux, enhance contact time, improve strength, and maintain adsorption capabilities. The investigation into the mechanism of PTE adsorption enhances our fundamental understanding of mycelial adsorption. Prior works that utilized mycelial pellets have shown that the adsorption of metals follows Langmuir adsorption and pseudo-second order kinetics (215), which was also supported with our experiments. This conclusion is verified by the XPS data showing coordination of Cu(II) onto amine, amide, carbonyl, and carboxylic acid groups present in the fungal cell wall and ion-exchange with Ca2+. Compared to other low-cost and biobased materials, the mycelial membrane, regardless of TCNF concentration, has much higher static adsorption capacities (226–232) (Table 4.3). Additionally, compared to a study using extracted fungal chitin-glucan, the static adsorption capacity of our system is nearly doubled (72.3 mg/g for pure mycelial membrane versus 43.4mg/g (233)). This suggests that the native structure of the mycelia contributes significantly to its adsorption performance. In 99 particular, mannoproteins in the cell wall that are stripped during the isolation of the chitin-glucan complex may be more important to the adsorption of PTEs by the fungus than previously considered. Interestingly, heat treated and powdered P. ostreatus biomass described by Javaid et al. also displayed a significantly lower adsorption capacity for Cu(II) of 8.06mg/g (234). The difference in the adsorption capacities between the current study and previous works insinuates that retention of both the native chemical structure, with the inclusion of cell-wall proteins, and the native filamentous structure, that facilitates the interaction of fungi and PTEs in their natural environment, can promote higher adsorption capacities. Table 4.3. Comparison of low-cost and biobased adsorbents for Cu(II) Adsorbent pH Adsorption capacity (mg/g) Reference Cashew nut shell 5 20 (226,227) Persimmon leaf 6 19.42 (226,229) Capsicum annuum seeds 5 11.53 (226,230) Bagasse fly ash 4 2.36 (226,231) Fish waste 4 2.1 (226,232) S. cerevisiae biomass 4.5 19.53 (226,228) Fungal-derived chitin/glucan nanopapers -- 43.4 (233) Powdered P. ostreatus biomass 4.5 8.06 (234) P. ostreatus mycelial membranes 5.5 62.4-73.2 Current work 100 Metal rejection percentage can be used to evaluate the performance of the membrane in the flow system. A synthetic membrane developed by Mojdehi et al. from polyethersulfone/polyaniline-modified TiO2 demonstrated a rejection percentage of 84% for copper, while Hamid et al. achieved up to 100% copper rejection using a polyether/zeolite membrane (235,236). Biobased chitosan/cellulose-acetate membrane exhibited a rejection percentage of 81.06%, which is significantly lower than the rejection percentage of the biobased mycelial membranes, regardless of TCNF content, shown in the current work (237). P. ostreatus, the fungal species utilized in this study, is a non-pathogenic, saprotrophic species that is cultivated around the world (238). Therefore, the ability of this material to be cultivated and used is not limited to specific geographic regions and is not limited by negative consequences to human or environmental health. The creation of this material as an adsorption membrane for PTE removal facilitates its practical use on an individual and industrial level. For individuals, an adsorption membrane can be utilized as a gravity filter placed in a bottle or tap at the point-of-use. Point-of-use water treatment systems are especially useful for rural communities without access to water treatment facilities. This system could also be envisaged and adapted at an industrial level to mitigate contamination from industrial waste. This initial foray into the exploitation of mycelium for adsorption membranes yields several further areas of interest. The low-cost and sustainable nature of this work is not explicitly analyzed in this report, and future studies should perform a life cycle assessment and technical economic analysis in which the lifespan during industrial implementation of the membrane is considered. Additionally, this work does not investigate different fungal species, which may display interesting and novel interactions between species composition and function. Importantly, species that are commonly grown in bioreactors for fermentation 101 industries can provide a beneficial value-added product to the mycelium waste stream. However, toxicity and availability should be considered when selecting species. Bioengineering specific fungal strains to maximize adsorption performance is an intriguing prospect for future research but may be cost restrictive. Utilization of other non-traditional media such as agricultural waste can be explored as a means of improving the sustainability of the production process. In future works, application of this technology to real industrial wastewater containing mixed metals and salts should be analyzed. The goal of this study was to overcome the limitations of current PTE-removal techniques for marginalized communities by generating a low-cost, sustainable, and effective adsorption membrane. To this end, a framework for generating mycelial membranes and a greater understanding of the mechanisms by which mycelia adsorb PTEs was produced. Furthermore, this study highlights the unique abilities and high performance of filamentous fungi with the hope of encouraging further exploration of this material to meet environmental challenges. 4.4 Conclusions Here, we prepared a fully biobased filtration membrane using P. ostreatus biomass as the main structural component. Modification of the pore size and physical properties was attained with small additions of TCNFs, with optimal properties using 1wt% TCNF. Adsorption capacities reached 73.2 mg/g for M-1TCNF membranes following the Langmuir adsorption isotherm model and pseudo-second order kinetics. Furthermore, the adsorption mechanism was found to be chemisorption on fungal cell wall components. Membranes were utilized in a flow system and achieved up to 97% removal of Cu(II) with contact time, and hence removal efficiency, controlled by the addition of TCNF. The membranes exhibit versatility in PTE removal by eliminating 49.7 and 68.2 % of Zn and Cd, respectively. As 102 such, this technology has the potential to reach the needs of marginalized communities for PTE contamination in terms of cost, performance, and sustainability. This system represents a promising development toward the remediation of contaminated waters using green materials and sustainable technologies. 4.5 Methods & Materials 4.5.1 Mycelium pellet cultivation & processing P. ostreatus cultures were maintained on agar plates at room temperature and sub-cultured every 3 weeks. 12 1x1cm agar plugs were used to inoculate 1L of autoclaved (121°C for 20 minutes) yeast extract peptone dextrose (YPD) broth containing 1% yeast extract, 2% peptone and 2% dextrose. Cultures were grown for 21 days in a shaking incubator at 220 rpm and room temperature (~22°C). Light microscopy of the culture was used to regularly monitor pellet formation. Pellets were isolated with a ceramic filter, washed with deionized water (DI) water 3 times, and freeze-dried. After freeze-drying, pellets were inoculated on agar medium to ensure their non-viability. The non-viable pellets were then processed by blending for 1.5 minutes in DI water and freeze-dried. 4.5.2 Membrane formation TCNFs (Cellulose lab) were added to water and processed pellets and stirred together for 24 hours. The mixture was then homogenized for 10 minutes at 10,000 rpm. The resultant suspension was vacuum filtered using a 40 µm pore size nylon filter to produce a membrane. The time of vacuum filtration was controlled to ensure even compression across samples. Membranes were freeze dried and carefully removed from the nylon filter to produce a self-standing membrane. 103 4.5.3 Characterization Scanning electron microscopy Coating was performed with a Leica EM MED020 coater using 10 µm iridium. Images were taken with a Helios NanoLab 650 scanning electron microscope with an Everhart-Thornley detector at a voltage between 5-15kV and working distance of 4mm. Pore size and surface area Surface area and pore size measurements were conducted using a TriStar II Plus BET Gas Adsorption Analyzer for meso-pore size analysis and Micromeritics 3Flex Surface Area & Pore Size Analyzer for micro-pore size analysis. Both instruments used the Brunauer-Emmett-Teller (BET) method. Membranes were cut into ~0.5cm2 pieces and degassed at 100°C for 24 hours under vacuum. Chemical characterization XPS analyses were carried out with a Kratos AXIS Supra X-ray photoelectron spectrometer using a monochromatic Al K(alpha) source (15mA, 15kV). The instrument work function was calibrated to give a binding energy (BE) of 83.96 eV for the Au 4f7/2 line for metallic gold and the spectrometer dispersion was adjusted to give a BE of 932.62 eV for the Cu 2p3/2 line of metallic copper. The Kratos charge neutralizer system was used on all specimens. Survey scan analyses were carried out with an analysis area of 300 x 700 microns and a pass energy of 160 eV. High resolution analyses were carried out with an analysis area of 300 x 700 microns and a pass energy of 20 eV. Spectra have been charge-corrected to the C-C, C-H (aliphatic) line carbon 1s spectrum set to 284.8 eV. Spectra were analyzed using CasaXPS software (version 2.3.26). Mechanical strength 104 Dynamic mechanical tests were performed in tensile mode in the temperature range from 25 to 80°C and heating rate of 3°C/min, at a frequency of 1 Hz with an oscillation amplitude of 0.1%, which is within the linear viscoelastic region of the specimens were cut in rectangular shapes in a TA Instruments DMA 850. The storage modulus was reported at 50°C, at which the temperature-modulus relationship was linear. Swelling Swelling was measured by cutting 1 cm square pieces of membrane and submerging them in DI water for 24 hours. The mass of the membrane pieces was measured before and after submersion. Wet membranes were shaken three times to remove excess water before recording the mass. 4.5.4 Batch adsorption A specified weight of adsorbent material was added to a specified volume of adsorbate and the solutions were agitated on a shaker plate at 120rpm and 25°C. For kinetic experiments, aliquots of 100 µL were taken at varying time points (20, 40, 60, 80, 160, 320, 720, and 1440 mins) and centrifuged for 10 mins at 10,000 rpm. Otherwise, aliquots were taken at 1440 mins to ensure equilibrium had been reached. A stock solution of 5% Tween 80 in pH 6.5 buffer of potassium phosphate monobasic and NaOH with PAN at a concentration of 2x10-4 M was used as an indicator for Cu following the protocol in Shar et. al. (239). A small amount of Cu(II) containing samples were diluted in the indicator solution and absorbance was measured at 555nm in a microplate reader. Zn was measured with a similar indicator at pH 9 (240). Adsorption efficiency, q, was calculated using equation 4.1. (Equation 4.1) 𝑞 = (𝐶𝑂 − 𝐶𝑓)𝑉/𝑚 105 Where q is the adsorption efficiency, 𝐶𝑂 is the initial metal concentration, 𝐶𝑓 is the final metal concentration, V is the metal solution volume, and m is the mass of adsorbent. Kinetic and isotherm analysis using batch adsorption utilized pseudo-first order, pseudo-second order, Langmuir and Freundlich models(241,242). The non-linear forms of the models are shown in equations 4.2-4.5. (Equation 4.2) Pseudo-first order 𝐹(𝑡) = 1 − 𝑒−𝑘1𝑡 (Equation 4.3) Pseudo-second order 𝐹(𝑡) = (𝑘2∙𝑞𝑒)1+ (𝑘2∙𝑞𝑒)∙𝑡 Where t is time, qe is the equilibrium adsorption capacity, and k1 and k2 are first and second order rate constants, respectively. (Equation 4.4) Langmuir 𝑞𝑒 =𝑞𝑚∙𝐾𝐿∙𝐶𝑒1+ 𝐾𝐿∙𝐶𝑒 Where KL, or the Langmuir constant, is a ratio of the adsorption and desorption rate, qm is the maximum theoretical adsorption capacity, and Ce is the concentration of adsorbate at equilibrium. (Equation 4.5) Freundlich 𝑞𝑒 = 𝐾𝐹 ∙ 𝐶𝑒1𝑛⁄ Where KF, the distribution coefficient, and n, a correction factor, are constants. 4.5.5 Regeneration and recycling studies Cu-loaded membranes were removed from the adsorption liquid and washed with Milli-Q (MQ) water (pH 5.5) three times. Desorption was carried out at 25°C and 120rpm in 0.1M HCl solution for 24 hours. Cu(II) concentration was measured via absorbance at 555nm with the indicator solution described in the previous section. 106 4.5.6 Water flux and metal rejection Water flux measurements were performed using a 50 mL Amicon dead end cell. Flux was calculated using equation 4.6. For measurement of metal rejection using the dead-end filtration system, copper solution at a concentration of 100 mg/L was filled into the cell (or other PTEs) and the concentration of the filtrate and permeate were measured to calculate the PTE rejection rate as shown in equation 4.7. (Equation 4.6) 𝐽 = 𝑉𝐴𝑡 J is the water flux, V is the permeate volume, A is the membrane area, and t is the time for a specific volume of permeate to pass. (Equation 4.7) 𝑅𝑃𝑇𝐸 (%) = (1 − (𝑐𝑝𝑐𝑓)) ∙ 100 𝑅𝑃𝑇𝐸 is the percent rejection of PTEs, Cp is the concentration of metal in the permeate, Cf is the concentration of metal in the filtrate. 107 108 Chapter 5: Mycelium membranes as enzyme immobilization platforms for treatment of azo dyes Expanding on the advances made in Chapter 4, this final research section continues to employ submerged fermentation-derived mycelial membranes for water treatment, but shifts the focus from pollutant capture to active degradation. Inspired by the natural ability of fungi to break down complex contaminants, this chapter explores how integrating fungal enzymes can enhance the remediation process. In particular, we draw on the capabilities of white-rot fungi, which produce enzymes such as laccase known for degrading phenolic and aromatic pollutants. For this purpose, Trametes versicolor was chosen due to its established use in commercial enzyme production. This chapter demonstrates how fungal materials can move beyond adsorption to actively transform and detoxify pollutants, further expanding the role of mycelium-based systems in sustainable water treatment. 5.1 Introduction Laccase, a versatile oxidoreductase enzyme widely applied in industries such as textile, food, and pulp and paper processing, has gained significant attention for its ability to degrade a broad spectrum of environmental pollutants, including synthetic dyes, pharmaceutical residues, and endocrine-disrupting compounds (243). Despite its potential, the broader application of laccase, like many commercial enzymes, is constrained by several limitations. High production costs, coupled with low operational stability and limited reusability, reduce economic feasibility (244). These challenges require innovative strategies to enhance the enzyme's stability, longevity, and cost-effectiveness. Enzyme immobilization is a growing field of research aimed at addressing the aforementioned issues. Enzyme immobilization can improve stability, longevity, substrate 109 selectivity, and activity while facilitating product separation (245). Common methods used for enzyme immobilization include coupling to a pre-existing support matrix (by means of physical binding, covalent binding, or entrapment) or without a support (enzyme crosslinking) (246). Covalent binding has been shown in particular to result in improved longevity and operational stability of the enzyme; however, care must be taken to ensure that the binding does not result in a large loss of catalytic activity due to conformational changes or activation from improper orientation (247,248). Carriers for immobilization are categorized as inorganic and organic (naturally or synthetically derived). Among the inorganic supports are silicas, ceramics, and metal oxides (249). Synthetically derived organic polymers constitute a large share of commercial immobilization matrices, which includes synthetic polymers and resins (250). However, naturally occurring polymer matrices such as chitosan, agarose, and protein biopolymers have also been increasingly studied for enzyme immobilization applications (249). Building on these immobilization techniques, this study proposes a novel approach using mycelial biomass, a by-product of commercial enzyme production, as an immobilization platform for laccase. Mycelium is the filamentous network of branched hyphae that forms the main structure of fungi and has garnered increasing interest as a sustainable biomaterial. Its unique properties have enabled its development into structural and packaging materials, while recent advances have explored its potential as a functional, bio-based resource (218,251,252). The fungal cell wall, composed predominantly of chitin, glucans, and proteins, offers a versatile platform for modification and functionalization (216). Approximately 60% of all industrial enzymes are produced by fungi (244). In liquid fermentation processes used to produce fungal enzymes, vast quantities of mycelial biomass from filamentous fungi are generated, with thousands of tons discarded or incinerated annually as waste (253). This 110 creates a large opportunity to generate a circular economy with established industrial fermentation industries. Past research on the immobilization of living fungi onto support material for whole cell enzyme immobilization has been applied to the removal of organochlorines, industrial wastewater treatment, and biosorption and extraction of heavy metals (254–257). Recently, the work by Xu et al. proposed the use of dispersed waste mycelium as a lipase immobilization support through adsorption to cell wall components (253). This study proposes a novel approach to immobilize laccase on mats derived from Trametes versicolor mycelium by covalent attachment, transforming an underutilized waste stream into a functional material that enhances enzyme reusability and operational efficiency. Unlike previous studies, this work takes advantage of the filamentous and structural nature of mycelia to produce sheets that can be used in batch or flow processes for water treatment. We hypothesize that treating the Trametes versicolor biomass with sodium hydroxide (NaOH) will deacetylate the chitin component of the fungal cell wall, increasing the availability of amine groups for enzyme attachment via glutaraldehyde. To investigate the development of an immobilized membrane system from this bioresource, the effect of NaOH treatment on the biomass is investigated, along with an optimization of several immobilization conditions to reach a high enzymatic activity. The immobilized membrane system is then evaluated for the degradation of an azo dye contaminant, Congo Red. The approach presented here leverages the intrinsic properties of fungal biomass to improve enzyme usability, contributing to the development of sustainable biocatalytic systems. 111 5.2 Methods & materials 5.2.1 Preparation of mycelial support Growth of Trametes versicolor biomass Cultures of Trametes versicolor were maintained on potato dextrose agar (PDA) plates at 30 °C and sub-cultured every three weeks. 1 cm × 1 cm agar plugs were used to inoculate 1 L of sterile yeast extract peptone (YEP) broth in 2L Erlenmeyer flasks. The YEP broth was autoclaved at 121 °C for 20 minutes and consisted of 1% yeast extract and 2% peptone. Cultures were incubated at 25 °C in a shaking incubator at 220 rpm for 21 days. The resulting mycelial biomass was separated from the liquid medium using vacuum filtration with a nylon cloth, washed three times with distilled water, and freeze-dried for further processing. Deacetylation of mycelial biomass The freeze-dried mycelial biomass underwent a two-step pretreatment process. First, the biomass was mixed with 0.5 M HCl for 30 minutes at 25 °C to remove soluble wall components and lipids. The treated biomass was washed three times with deionized (DI) water. Second, the biomass was treated with 1 M NaOH for 1 hour at 90 °C to eliminate acid-soluble proteins. This was followed by sequential washing with DI water, 75% isopropanol (IPA), and 100% IPA. The pretreated biomass was further processed by submerging it in 400 mL of 30%, 45%, or 60% (w/v%) NaOH solution. The reaction was conducted at 80 °C for 4 hours under constant stirring and halted by the addition of acetic acid until the solution reached a neutral pH. The biomass was washed five times with DI water to remove residual reagents. To remove unbound chitosan, the treated biomass was immersed in 1 M acetic acid for 4 hours 112 at 50 °C. The final material was washed three times with DI water and freeze-dried prior to mat formation. Support formation The formation of a mycelial mat followed a procedure outlined in our previous work and is briefly overviewed below (251). A 1% (w/v) suspension of the processed mycelial biomass was prepared in DI water and homogenized at 10,000 rpm for 10 minutes using an IKA Ultra-Turrax homogenizer. The homogenized suspension was vacuum filtered through a nylon cloth with 40 nm pore size until all water was removed. A 5-second pause was observed after filtration to ensure even compression across all samples. The resulting wet mats were frozen overnight at −80 °C and then freeze-dried to produce a self-supporting sheet. 5.2.2 Characterization of mycelial support Determination of the degree of deacetylation Fourier-transform infrared spectroscopy (FTIR) was used to determine the degree of deacetylation (DD%) of the mycelial biomass using a Bruker FTIR equipped with an ATR accessory. Triplicate measurements were recorded for unique sections of each mycelial sample with 64 scans and a resolution of 4cm-1. The peak height of the absorption band at 1630 cm-1 (C=O bending) and the reference band at 3283 cm-1 (O-H stretching) were analyzed using Origin software. DD% was calculated using the following equation based on literature (258,259). (Equation 5.1) 𝐷𝐷 = 97.67 − 𝑏[𝐴1630𝐴3283] 113 Where A1630 and A3283 are the peak intensities at 1630 and 3283 nm respectively, and 𝑏 = 26.486, based on the baseline methodology used. Scanning electron microscopy Mycelial supports were coated with a 10 µm iridium layer using a Leica EM MED020 coater (Wetzlar, Germany). Imaging was conducted with a Helios NanoLab 650 scanning electron microscope (Oregon, USA) equipped with an Everhart-Thornley detector. Operating voltage was 3 kV and the working distance was set to 4 mm. 5.2.3 Immobilization of laccase Support activation and optimization of immobilization parameters Activation of the support was performed by submerging the mycelial mat in glutaraldehyde solution at a ratio of 1mg membrane:1mL glutaraldehyde solution at 30°C and 300rpm in an incubating microplate shaker (Fisher). After activation, the mat was washed three times in excess DI water to remove residual glutaraldehyde. Laccase immobilization was carried out by submerging the activated mat in fresh laccase solution at a ratio of 1 mg membrane:1 mL laccase solution (0.5-1.5 mg/mL) for 12 hours at 4°C. Laccase immobilization was optimized using a Box-Behnken response surface methodology. Independent variables included activation time (tGA), activator concentration [GA], and enzyme concentration [Lacc]. The enzyme activity (μmol min− 1 L− 1) was used as the response variable. Optimal immobilization conditions to maximize enzyme activity were determined by regression analysis. Statistical significance of the estimated parameters was performed by one-way analysis of variance (ANOVA) using Origin software for design of experiments. Statistical significance* was defined as ρ<α where α=0.05. 114 Laccase activity assay Laccase activity (free and immobilized) was assessed by monitoring its reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in 0.1 M citrate-phosphate buffer (pH 4) at 25 °C. Enzyme activity was calculated based on the change in absorbance (ΔA) using a Shimadzu UV-2600 UV-vis spectrophotometer (Kyoto, Japan) over time using the following equation: (Equation 5.2) Enzyme activity 𝑈 = (∆𝐴)(𝑉𝑡)(106)(𝑡)(𝜀)(𝑑) Where U is the non-specific enzyme activity (μmol min− 1), ∆A is the measured change in absorbance, 𝑉𝑡 is the total reaction volume (L), t is the reaction time, ε is the molar extinction coefficient (M− 1 cm− 1 or L mol− 1 cm− 1) (36,000 M-1cm-1), and d is the optical path length (cm) (1 cm). The amount of enzyme attached to the support was determined by measuring the protein content of the enzyme solution before and after immobilization. Protein content was analyzed using the corrected Bradford Assay with BSA as a standard (260). The calibration curve used is shown in Appendix D-1. Using the protein estimation, specific enzymatic activity could be calculated from the following equation: (Equation 5.3) Specific enzyme activity Us = (∆A)(Vt)(106)(t)(ε)(d)(mp) 115 Where 𝑚𝑝 is the mass of protein calculated from the Bradford Assay. 5.2.4 Effect of immobilization on stability, reusability, and kinetics: Assessing reusability and pH, temperature, and storage stability Temperature stability of the enzyme (free and immobilized) was evaluated over a range of 20-80°C using 0.1M citrate-phosphate buffer at pH 4. Samples were incubated at 20, 30, 40, 50, 60, 70, and 80°C using a dry bath for 45 minutes prior to the activity assay measurement. Relative activity was measured by setting the temperature with the highest activity value to 100%. pH stability of free and immobilized enzymes was evaluated over a pH range of 3–7 using 0.1 M citrate-phosphate buffer at a temperature of 20°C. Samples were incubated at 4 °C for 72 hours at pH levels of 3, 4, 5, 6, and 7. Relative activity was measured by setting the pH with the highest activity value to 100%. Storage stability was probed by incubating the enzyme (free and immobilized) for up to 15 days (0, 5, 10, and 15 days) and then measuring activity at pH 4 and 20°C. The reusability of the immobilized laccase measured over 5 cycles, washing with water and pH 4 buffer between cycles and measured at pH 4 and 20°C. Determination of kinetic parameters Kinetic parameters of the enzyme (free and immobilized) were determined by measuring enzymatic activity over a range of ABTS concentrations (0.5-2.1 mM) at pH 4 and 20°C. The Michaelis-Menten equation (equation 4) was linearized by the Lineweaver-Burke method (equation 5) and the kinetic parameters were found by taking the slope and y-intercept of a linear fit to the data. (Equation 5.4) 𝑣 =𝑣𝑚𝑎𝑥[𝑆]𝐾𝑀 + [𝑆] 116 (Equation 5.5) 1𝑣= 𝐾𝑀𝑣𝑚𝑎𝑥(1[𝑆]) + 1[𝑣𝑚𝑎𝑥] 𝑣 is the reaction velocity, [S] is the substrate concentration, 𝐾𝑀 is the Michaelis constant, and 𝑣𝑚𝑎𝑥 is the maximum reaction velocity. 5.2.5 Decolorization and degradation of Congo Red: Congo Red solutions were prepared at a concentration of 300 mg/L in pH 5.6 citrate-phosphate buffer. Solutions were treated at a ratio of 1mL:0.5mg Congo Red solution to mycelial support prior to immobilization. 100µL of laccase solution (at a concentration of 1mg/mL) was added to 1mL Congo Red solutions to probe the effect of the free enzyme. Decolorization of Congo Red was monitored over 96 hours using a UV-vis spectrophotometer and calculated by the following equation: (Equation 5.6) %𝐷𝑒𝑐𝑜𝑙𝑜𝑟𝑖𝑧𝑎𝑡𝑖𝑜𝑛 = (𝐴495,𝑓𝐴495,𝑖) × 100 Where 𝐴495,𝑖 is the absorbance of the initial Congo Red solution at 495 nm and 𝐴495,𝑓 is the absorbance of the treated Congo Red solution at 495 nm. Mass spectroscopy was employed to evaluate of the degradation of Congo Red by analyzing the spectra before and after treatment with the immobilized enzyme. Mass spectrometric analysis was conducted in negative ion mode using a Bruker HCTultra PTM Discovery System with electrospray ionization and ion trap mass analyzer. Optimized parameters used were based on the work of Lark et al. (261): capillary voltage: -400 to -500 V, capillary temperature: 150-200 °C, nebulizer gas flow: 180 μL min−1, and nebulizer pressure: 1 bar. 117 5.3 Results & discussion 5.3.1 Development of a deacetylated mycelial support The production and characterization of the deacetylated mycelial support material is illustrated in Figure 5.1. Fungal biomass was harvested, washed, and subjected to pretreatment with 0.5M HCl and 1M NaOH, resulting in a 55% yield. Subsequent deacetylation was performed using 30, 45, and 60% (wt/vol) NaOH for 4 hours at 80°C, with an average yield of 23% across treatments. 118 _____________________________________________________________________________________ Figure 5.1. Effect of NaOH treatment on cell wall chemistry and structure. (a) Generalized overview of the fungal cell wall and depiction of the deacetylation of the cell wall and removal of glycoproteins. Structures of chitin and chitosan drawn with ChemDraw. (b) Representative FTIR-ATR spectra of original mycelia (OG), pretreated mycelia (PT), and mycelia treated with 30, 45, and 60% NaOH (DA 30, DA 45, and DA 60 respectively). The baseline chosen for calculation of the degree of deacetylation is shown by the grey line and the pink highlight represents the characteristic peak chosen while the grey highlight represents the reference 119 peak chosen. (c) The calculated degree of deacetylation for mycelial samples. (d) relative activity (%) of laccase oxidation of ABTS measured by UV-Vis spectrophotometry for the mycelial samples formed into mats. (e) morphology of NaOH-treated mycelial samples visualized by scanning electron microscopy (SEM). _____________________________________________________________________________________ The hypothesized chemical transformation of chitin to chitosan within the fungal cell wall is depicted in Figure 5.1 (a). FTIR analysis revealed a decrease in the acetyl group peak intensity (1632 cm^-1^) relative to the OH stretching peak intensity (3283 cm^-1^) after pretreatment and NaOH treatment (Figure 5.1 (b)). Derived from the ratio of these peak intensities, a value for the degree of deacetylation was calculated. The degree of deacetylation increased from 64.5% to 73.8% after pretreatment and further to 82.9%, 84.1%, and 83.7% for 30%, 45%, and 60% NaOH treatments, respectively (Figure 5.1(c)). Compared to previous work, the effect of NaOH concentration on the degree of deacetylation was markedly decreased (258). This may be due to the higher concentration range (7.5-15M) and longer reaction time (4 hours) used in this study, resulting in a plateau of the reaction. Additionally, the original Trametes versicolor biomass appears to have a high degree of deacetylation compared to previous studies that measured the degree of deacetylation of mycelia, reaching the classification of chitosan with >50% degree of deacetylation. We hypothesize that this may be a result of 1) the liquid state fermentation method used to produce the mycelia, which would not require a highly rigid cell wall which would be normally used to penetrate a solid substrate and 2) remaining proteins in the mycelial biomass that were not fully removed by the pretreatment steps. The change in the degree of deacetylation observed after treatment, however, is a good indicator that the proposed reaction was successful. 120 The impact of NaOH treatment on the effectiveness of the mycelial biomass as an enzyme immobilization support was screened by measuring the activity of immobilized enzymes on mats formed from each material (Figure 5.1 (d)). Relative enzyme activity decreased slightly from 73.8% to 71.1% after pretreatment. Treatment with 30% and 45% NaOH increased activity to 85.3% and 100%, respectively, while 60% NaOH treatment resulted in a decrease to 84%. The observed changes in relative enzyme activity did not directly correlate with the degree of deacetylation, suggesting that other factors influenced enzyme immobilization effectiveness. To understand the difference in activity between treated samples, SEM microscopy was employed to uncover morphological changes that may have occurred during treatment. SEM images (Figure 5.1 (e)) revealed that NaOH treatment caused fusion of the hyphal structure compared to original and pretreated samples. Images of original and pretreated samples can be found in Appendix D-2. This occurrence has been noted in literature previously (258). At 45% NaOH content, increased breakage of hyphal structures created a highly textured surface on larger sheet structures. However, 60% NaOH treatment led to further fusion and a smoother surface. The surface detail is shown in Appendix D-3. The morphological differences observed in the SEM images, particularly the changes in surface texture and hyphal structure, may contribute to the variations in relative activity. The highly textured surface created by 45% NaOH treatment likely provided an optimal substrate for enzyme immobilization, resulting in higher enzyme loading and the highest relative activity for treated samples. In contrast, the smoother surface produced by 60% NaOH treatment may have reduced the available surface area for enzyme attachment, leading to decreased activity. Samples treated with 45% NaOH were used for all subsequent experiments. 121 5.3.2 Optimization of immobilization parameters Using the optimally treated membrane sample (45DA), the impact of immobilization conditions was analyzed using glutaraldehyde as an activator (Figure 5.2). 122 _____________________________________________________________________________________ Figure 5.2. (a) Schematic representing the immobilization process with the addition of glutaraldehyde (GA) followed by addition of laccase (lacc). (b) Graphic illustrating the points chosen for a Box-Behnken design of experiments and selected parameter ranges. (c) 3D response surface models and (d) contour plots showing the effect of two variables with the third variable set to the median value. _____________________________________________________________________________________ 123 Enzyme immobilization was conducted in a two-step process, as illustrated in Figure 5.2 (a). Initially, the membranes were activated by immersion in a glutaraldehyde solution, followed by the removal of unbound reagent. Subsequently, the activated membranes were immersed in a laccase solution and incubated overnight at 4°C. While the exact mechanism of enzyme immobilization on aminated supports is not yet fully understood, possible mechanisms include ion exchange, hydrophobic interactions, and covalent attachment (262). Covalent attachment is thought to occur via Schiff base, Michael addition, or other pathways dependent on the immobilization conditions and will occur more slowly than the other mechanisms (263). To optimize the immobilization process, a Box-Behnken response surface methodology was employed to evaluate the effects of three independent variables: crosslinking time (t_GA), crosslinker concentration [GA], and enzyme concentration [Lacc] on enzymatic activity (μmol min− 1 L− 1). Figure 5.2 (b) presents the 13 experimental points for the Box-Behnken design, along with the range of values selected for each independent variable. A second-order polynomial regression was applied to the data, yielding an R2 value of 0.97 and an adjusted R2 of 0.90, confirming the model's good fit. All linear and quadratic coefficients were found to be statistically significant, except for the linear [Lacc] term and the quadratic (t_GA) term. Most interaction terms were insignificant, with high P-values, except for the (t_GA)([GA]) interaction. t_GA was identified as the most significant factor affecting activity. The term coefficients and P-Values are listed in Appendix D-4. The predictive equation for laccase activity, excluding highly insignificant terms (P>0.1), is presented as Equation 7. 124 (Equation 5.7) 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑈) = −0.0144 + 0.0954(𝑡𝐺𝐴) − 0.0748([𝐺𝐴]) − 0.0574(𝑡𝐺𝐴)([𝐺𝐴]) + 0.135([GA][GA]) + 0.113([Lacc])([Lacc]) Figure 5.2 (c, d) displays 3-D response surface plots and contour plots, illustrating the relationship between the independent variables based on the model. Optimal conditions within the tested ranges were determined through regression analysis, aiming for maximal enzymatic activity. For subsequent experiments, the local optimum conditions of t_GA = 4 hours, [GA] = 0.5 (wt%), and [Lacc] = 1.5 mg/mL were utilized. In this study, immobilization was conducted at pH 7, where only the terminal amine on the enzyme is believed to be activated, facilitating a direct covalent interaction between glutaraldehyde and the enzyme rather than promoting an intense multipoint covalent interaction (263). Further lines of evidence to suggest covalent attachment were found by analyzing ATR-FTIR spectra of the membrane before and after immobilization of the enzyme (spectra can be found in Appendix D-5). The formation of these direct covalent interactions may require extended periods for immobilization, potentially explaining the positive linear effect of glutaraldehyde activation time (t_GA) on enzyme activity. A longer time range (12-16 hours) may have shown a true optimal value, although this range may reduce practical applicability of this technology to industry (264). In the absence of spacers, lower glutaraldehyde concentrations ([GA]) exhibited a positive effect on activity, likely due to support overcrowding and overheating at higher concentrations. Furthermore, steric hindrance when activation of the membrane is 125 high using a high concentration of glutaraldehyde may impede access to the enzyme's active site. The negative interaction term between activation time and activator concentration highlights the necessity for balanced parameters to achieve optimal results and adequate support activation without overcrowding. Laccase concentration ([Lacc]) displayed a local minimum at 1 mg/mL, possibly due to inter-protein interactions causing enzyme aggregation at this concentration, with higher values reaching saturation (265). 5.3.3 Comparison of the free and immobilized enzyme operational stability and kinetics To assess the stability and usability of the immobilized enzyme to the free enzyme, a series of sensitivity and stability tests were performed. Kinetic analysis was also performed to understand the impact of immobilization on kinetic parameters. 126 _____________________________________________________________________________________ Figure 5.3. Effect of immobilization on thermal stability, pH stability, storage stability, recyclability, and enzyme kinetics. (a) Effect of temperature on the relative activity of free and immobilized enzymes (b) effect of pH on the relative activity of free and immobilized enzymes (c) effect of storage time on the relative activity of free and immobilized enzymes (d) reusability of immobilized enzymes across 5 cycles (e) effect of substrate concentration on the reaction velocity for free enzymes and (f) immobilized enzyme on mycelial support. 127 _____________________________________________________________________________________ The stability of the free and immobilized laccase enzyme to temperatures between 20 and 80°C is shown in Figure 5.3 (a). Enzymes were incubated for 45 minutes prior to performing the activity assay. At lower temperatures (20 and 30°C) the free enzyme exhibited higher activity compared to the immobilized enzyme, while temperatures above 30°C showed higher relative activity (%) for the immobilized enzyme. The optimal temperature was shifted for the immobilized enzyme from 30°C to 40°C. pH stability of the free and immobilized laccase enzyme was assessed over a range of 3-7 using 0.1M citrate-phosphate buffer as shown in Figure 5.3 (b). The immobilized enzyme displayed a broadening of the range of pH values with a higher amount of retained activity. Storage stability of the enzyme, shown in Figure 5.3(c), was also improved by immobilization, with the free enzyme showing only 15% of the initial activity after 5 days whereas the immobilized enzyme still retained 50% of the initial activity. The reusability of the immobilized laccase enzyme over 5 cycles was assessed by measuring activity at optimal conditions and is shown in Figure 5.3 (d). After each cycle, the mat with immobilized enzyme was removed and washed thoroughly with water and buffer. After 5 cycles, the immobilized membrane retained 48% of the initial activity. Kinetic analysis of the free and immobilized enzyme was conducted by measuring the reaction velocity for the enzymes at optimal conditions across a range of substrate concentrations ([ABTS] = 0.5- 2.1µM.) The effect of substrate concentration [S] on reaction velocity (ѵ) are displayed as Lineweaver-Burke plots in Figure 5.3 (e) and Figure 5.3 (f) for the free and immobilized laccase, respectively. Calculated kinetic parameters derived from 128 the Lineweaver-Burke plots, including the Michaelis Constant, KM, and maximum rate velocity, ѵmax, are listed for the free and immobilized laccase in Table 5.1. Table 5.1. Calculated kinetic parameters for free and immobilized laccase Temperature and pH optima shifts after immobilization have been observed for laccase immobilized on chitosan-containing supports previously (266). After immobilization, free movement of the enzyme is reduced by the covalent attachment. Thus, the ability of the enzyme to unfold and denature at elevated temperatures is reduced. At higher temperatures, the substrate has more kinetic energy to reach the enzyme active site quicker, leading to an upward shift in optimal temperature. Changes in the microenvironment and conformation of the enzyme after immobilization can contribute to a shift in pH optimum (5-7) (267). Broadening in the usable pH range of the enzyme has been observed in previous literature on chitosan-based supports (268). In addition to increasing the effectiveness of the enzyme at a larger range of pH and temperature conditions, the ability to reuse the immobilized enzyme helps improve its potential in industry. Enzymes are often expensive to produce, and soluble enzymes can be challenging to remove from the reaction media. Immobilizing the enzyme allows for easy separation of the products and enzyme after reaction and permits the reuse of the enzyme, lowering the cost of using this biocatalyst. The activity retention of <50% after 5 cycles is Maximum rate velocity ѵmax (µmol/min) Michaelis constant KM (µmol) Free laccase 381.68 0.241 Immobilized laccase 123.3 3.030 129 slightly lower than what has been reported in several studies on laccase immobilization with chitosan-based supports, which may be a result of the different immobilization conditions utilized (266,268,269). Further optimization of the immobilization conditions to favor a multi-point covalent attachment during immobilization could help improve the reusability of the enzyme but could have deleterious effects on enzyme activity due to rigidification of the enzyme and reduced substrate affinity (270). Immobilization has been observed to have a strong effect on the kinetic parameters of enzymes and can result in either increased or decreased enzymatic activity based on the specific alterations to the active site after immobilization (271). The 3D structure of the enzyme is fundamental to the configuration of the active and allosteric sites, directly impacting the interaction between the enzyme and substrate. An abnormal conformation of the enzyme from immobilization can cause a reduction in the maximum reaction velocity (ѵmax) and increase in the Michaelis constant (KM), indicating lower substrate affinity. Partition effects and diffusional resistance after immobilization can also play a role in the change of kinetic behavior after immobilization, often resulting in a reduction in ѵmax (272,273). This research shows an approximate 70% decrease in the observed ѵmax and a 10-fold increase in KM, which is in alignment with other studies on similar immobilization (274,275). The kinetic behavior change observed can be attributed to a combination of several effects, with conformational changes from the covalent linkage and complex diffusion effects from the porous structure of the support. 5.3.4 Application of the immobilized enzyme system for dye degradation 130 The effectiveness of the immobilized enzyme on mycelial supports was assessed by using the biocatalyst system to degrade Congo Red. Four treatment conditions were compared: free laccase (F-Lacc), immobilized laccase (I-Lacc), immobilized laccase with syringaldehyde mediator in solution (I-Lacc_SA), and co-immobilized laccase and syringaldehyde mediator (I-lacc_I-SA). Substrate degradation by laccase occurs at the mononuclear T1 copper center of the enzyme, with the redox potential of the T1 center largely determining the ability of the enzyme to oxidize a particular substrate (276). For bulky substrates or substrates with higher redox potential than the enzyme, mediators can be used to shuttle electrons from the active site to the enzyme (277). The use of natural mediators, such as syringaldehyde and vanillin, have been shown to increase the ability of laccase to degrade complex aromatic structures, like synthetic dyes and pharmaceutical compounds (278). From a preliminary survey, it was determined that syringaldehyde was a successful mediator for the degradation of Congo Red compared to vanillin, which prompted the selection of syringaldehyde as the mediator in this study. The decolorization of Congo Red was monitored by measuring the change in absorbance at 495 nm. Breakage of the azo bond, which results in a decrease in the extended conjugation of the structure, results in a decrease of intensity at this wavelength and, thereby, a reduction of the color of the dye solution (279,280). Visual representations of the solution colors before and after treatment and decolorization percentages over 96 hours are presented in Figure 5.4 (a) and Figure 5.4 (b), respectively. 131 _____________________________________________________________________________________ Figure 5.4. Degradation of Congo Red by different enzyme systems. (a) Image of Congo Red (CR) solutions after treatment with free laccase (F-Lacc), Immobilized laccase on mycelial support (I-Lacc), immobilized laccase on mycelial support with syringaldehyde in solution (I-Lacc_SA), co-immobilized laccase and syringaldehyde on mycelial mat (I-Lacc_I-SA). (b) Decolorization percent of Congo Red with various enzymatic treatments over time (c) proposed mechanism of mediator-assisted decolorization of Congo Red with the possible 132 structures resulting from the oxidation of Congo Red. Structures were generated using ChemDraw. _____________________________________________________________________________________ The co-immobilized enzyme and syringaldehyde (I-Lacc_I-SA) demonstrated superior performance, achieving 92% decolorization after 96 hours. The system with syringaldehyde mediator in solution (I-Lacc_SA) exhibited rapid initial decolorization within the first 24 hours but yielded lower overall results compared to I-Lacc_I-SA. This is due to the produced color of the oxidized syringaldehyde in solution over time. The free enzyme (F-Lacc) showed the poorest performance, with the slowest rate and only 45% decolorization after 96 hours. All immobilized enzyme systems naturally demonstrate a dual function to reduce Congo Red colorization: adsorption and enzymatic degradation. The red coloration of the immobilized mats after treatment suggests a significant proportion of the decolorization mechanism is due to adsorption. However, LC-MS analysis of the Congo Red metabolites after treatment with I-Lacc_I-SA evidences the production of degradation products, confirming the contribution of the enzyme (Appendix D-6). The proposed mechanism for the decolorization using this immobilized membrane system is depicted in Figure 5.4 (c), as also suggested in previous literature. Tuning the immobilization conditions further could allow for tailoring of the system to favor enzymatic degradation during future studies. The findings in the current work corroborate previous studies, showing that the addition of a mediator such as syringaldehyde, both to the solution and co-immobilized with the enzyme, can improve the reaction rate and overall decolorization of a synthetic dye (281,282). This work highlights that the co-immobilization of the mediator, creating an enzyme-mediator system, allows for optimal final results and facile separation of the reaction 133 products. Additionally, there is no need to further purify the solution by removing the mediator. These findings showcase the enhanced efficiency of the co-immobilized enzyme and mediator system in Congo Red degradation, suggesting its potential for practical applications in dye removal processes. Still, there are several areas for further development of this system. Optimization of the loading and immobilization conditions of the enzyme-mediator system has the potential to yield superior results in future works. Additionally, bioengineering the mycelium to produce higher amounts of chitosan or using chitin deacetylases during treatment. 5.4 Conclusions This study demonstrates the successful development of a novel laccase immobilization platform using mycelial biomass derived from Trametes versicolor. The deacetylation process of the mycelial support, optimized through various NaOH treatments, resulted in improved enzyme activity. The 45% NaOH treatment yielded the highest relative enzyme activity, a result of both an increase in the degree of deacetylation and change in morphological changes to the hyphae after treatment. Optimization of laccase immobilization to the mycelial mat was performed and conditions for local maximal enzyme activity were found to be [GA]=0.5wt%, tGA=4 hrs, and [Lacc]=1.5mg/mL. The immobilized laccase system using optimal immobilization conditions exhibited enhanced pH and temperature stability, as well as improved reusability, addressing key challenges in enzyme applications. Furthermore, the effective degradation of Congo Red dye highlights the practical applicability of this immobilized enzyme system in environmental remediation. This research not only presents 134 an innovative solution for enzyme immobilization but also contributes to the circular economy by repurposing mycelial waste from industrial enzyme production. Future studies should focus on improvement and optimization of the laccase-mediator system, paving the way for more sustainable and efficient biocatalytic systems. 135 136 Chapter 6: Conclusions This thesis has established a comprehensive framework for understanding and engineering mycelium-based materials, with a particular focus on their application in pollution control and environmental remediation. The research began by systematically investigating how fermentation methods and fungal species selection influence the structural, chemical, and functional properties of pure mycelium materials. Solid-state fermentation (SSF) was shown to produce dense mycelial networks with higher chitin content, with up to 0.84% glucosamine relative to total monosaccharides. Compared to liquid-state fermentation (LSF), SSF exhibits superior mechanical properties, as evidenced by a Young’s modulus of 0.47 GPa for Ganoderma lucidum SSF samples. However, LSF materials showed exceptional surface hydrophobicity, with contact angles reaching 153° on the aerial side. In contrast, submerged fermentation (SmF) enabled the formation of sheets that shifted the PMM material into a different material class (‘foams’ for SSF and LSF and ‘natural materials’ for SmF). SmF offers scalability and the potential to utilize fermentation waste as the PMM material feedstock, though it required additional processing compared to the self-grown SSF and LSF methods. The work highlighted the plasticity of fungal cell walls and the importance of bioprocess control in tailoring material performance for specific applications. Building on these foundational insights, the thesis demonstrated the practical utility of mycelium in three key pollutant remediation contexts. First, mycelium-modified mask layers for air pollution control were shown to significantly enhance filtration efficiency: the inner polypropylene mask layer (P1) improved from 43% to 80% efficiency, while the middle layer (P2) reached 97%, matching the performance of commercial three-layer masks. Notably, these improvements were achieved without compromising breathability (pressure drop: ~0.75 mm H₂O/cm²), and the materials exhibited asymmetric hydrophobicity (157° contact 137 angle on the aerial side versus 70° on the vegetative side), creating an environment to potentially control moisture and manage comfort. Second, mycelium-based membranes applied to potentially toxic element adsorption achieved a Cu(II) adsorption capacity of 73.2 mg/g, outperforming other low-cost, biobased materials. Hybrid membranes containing 1% TEMPO-oxidized cellulose nanofibrils (TCNF) balanced high water flux (1.5-2.5 L/m²s) with metal rejection rates of 97% for copper, 68% for zinc and 50% for cadmium. These membranes retained >30% of their adsorption capacity after three regeneration cycles with 0.1M HCl, demonstrating their reusability and practical potential. Third, the development of enzymatic membrane system using laccase immobilized on deacetylated mycelial supports resulted in robust systems for phenolic pollutant degradation. These immobilized enzyme systems retained 48% enzyme activity after 5 cycles and achieved 92% degradation of Congo Red dye within 96 hours. Collectively, these results advance scientific understanding in several important ways. The research clarifies how the interplay between fungal biology and bioprocess engineering shapes the structure–function relationships of mycelium-based materials, highlighting the importance of a systematic approach to fungal material design. It demonstrates the multi-functionality of mycelium, rooted in its unique biological features such as hydrophobin coatings and the chitin-glucan matrix, and shows mycelium as a viable and sustainable alternative to synthetic materials in diverse environmental applications. Furthermore, the work provides a roadmap for replacing petrochemical-derived products with self-assembling, tunable fungal systems, thus contributing to the broader shift toward a circular bioeconomy. While the technical aspects of mycelium-based materials were the primary focus of this thesis, economic and environmental considerations are equally significant. The 138 cultivation of mycelium relies on low-cost substrates, often agricultural waste, which should reduce the costs of material production of membranes compared to traditional resins. Importantly, mycelium materials are fully compostable, addressing the end-of-life challenges associated with plastic waste and toxic adsorbents, and reinforcing their suitability for a circular economy. Looking forward, several avenues for future research are evident. Scaling up production will require the development of continuous fermentation systems and industry standards for material performance. Advanced functionalization, including genetic engineering to enhance chitin or hydrophobin production, and the creation of hybrid materials with biobased polymers, could further expand the utility and durability of fungal materials. Real-world validation through field testing in industrial and urban environments, coupled with comprehensive life-cycle assessments and techno-economic analyses, will be essential to fully quantify the environmental and economic benefits. Socioeconomic integration, such as decentralized production models utilizing local fungal strains and waste streams, and policy engagement to support green procurement, are also critical for widespread adoption. In summary, this thesis positions mycelium as a cornerstone of next-generation sustainable materials, bridging the gap between biological systems and engineered solutions for environmental challenges. By exploring the relationships between fungal biology, material properties, and application performance, this work not only shows advances in pollution control but also contributes to a fundamental reimagining of how materials can be grown in service of both people and the planet. 139 References 1. Rafaj P, Kiesewetter G, Gül T, Schöpp W, Cofala J, Klimont Z, et al. Outlook for clean air in the context of sustainable development goals. Glob Environ Chang. 2018 Nov 1;53:1–11. 2. SDG Targets 6.1/6.2/6.a Drinking water, sanitation and related official development assistance [Internet]. [cited 2023 Sep 28]. Available from: https://www.who.int/data/gho/data/themes/topics/sdg-target-6-ensure-availability-and-sustainable-management-of-water-and-sanitation-for-all 3. Landrigan PJ, Fuller R, Acosta NJR, Adeyi O, Arnold R, Basu N (Nil), et al. The Lancet Commission on pollution and health. Lancet [Internet]. 2018 Feb 3 [cited 2023 Sep 20];391(10119):462–512. Available from: http://www.thelancet.com/article/S0140673617323450/fulltext 4. Reader GT. Clean Air, Clean Water, Clear Conscience. Sustain Eng Technol Archit [Internet]. 2021 Nov 29 [cited 2024 Jun 14];1-1-1–34. Available from: https://pubs.aip.org/books/monograph/3/chapter/7030/Clean-Air-Clean-Water-Clear-Conscience 5. Global Resources Outlook 2024 | UNEP - UN Environment Programme [Internet]. [cited 2025 Apr 23]. Available from: https://www.unep.org/resources/Global-Resource-Outlook-2024 6. Sofia D, Gioiella F, Lotrecchiano N, Giuliano A. Mitigation strategies for reducing air pollution. Environ Sci Pollut Res [Internet]. 2020 Jun 1 [cited 2025 Apr 23];27(16):19226–35. Available from: https://link.springer.com/article/10.1007/s11356- 140 020-08647-x 7. Tucker WG. An overview of PM2.5 sources and control strategies. Fuel Process Technol. 2000 Jun 1;65–66:379–92. 8. He C, Hong S, Zhang L, Mu H, Xin A, Zhou Y, et al. Global, continental, and national variation in PM2.5, O3, and NO2 concentrations during the early 2020 COVID-19 lockdown. Atmos Pollut Res. 2021 Mar 1;12(3):136–45. 9. Huang G, Zhou W, Qian Y, Fisher B. Breathing the same air? Socioeconomic disparities in PM2.5 exposure and the potential benefits from air filtration. Sci Total Environ. 2019 Mar 20;657:619–26. 10. Zhang J, Mu Q. Air pollution and defensive expenditures: Evidence from particulate-filtering facemasks. J Environ Econ Manage. 2018 Nov 1;92:517–36. 11. Shirvanimoghaddam K, Czech B, Yadav R, Gokce C, Fusco L, Delogu LG, et al. Facemask Global Challenges: The Case of Effective Synthesis, Utilization, and Environmental Sustainability. Sustain 2022, Vol 14, Page 737 [Internet]. 2022 Jan 10 [cited 2025 Apr 23];14(2):737. Available from: https://www.mdpi.com/2071-1050/14/2/737/htm 12. Pourret O, Hursthouse A. It’s Time to Replace the Term “Heavy Metals” with “Potentially Toxic Elements” When Reporting Environmental Research. Int J Environ Res Public Health [Internet]. 2019 Nov 2 [cited 2023 Jul 11];16(22). Available from: https://pubmed.ncbi.nlm.nih.gov/31766104/ 13. Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med. 1995 Feb 1;18(2):321–36. 141 14. Kiran, Bharti R, Sharma R. Effect of heavy metals: An overview. Mater Today Proc. 2022 Jan 1;51:880–5. 15. Xu T. Ion exchange membranes: State of their development and perspective. J Memb Sci. 2005 Oct 15;263(1–2):1–29. 16. Matlock MM, Howerton BS, Atwood DA. Chemical precipitation of heavy metals from acid mine drainage. Water Res. 2002 Nov 1;36(19):4757–64. 17. Shrestha R, Ban S, Devkota S, Sharma S, Joshi R, Tiwari AP, et al. Technological trends in heavy metals removal from industrial wastewater: A review. J Environ Chem Eng. 2021 Aug 1;9(4):105688. 18. Fei Y, Hu YH. Design, synthesis, and performance of adsorbents for heavy metal removal from wastewater: a review. J Mater Chem A [Internet]. 2022 Jan 18 [cited 2023 Aug 3];10(3):1047–85. Available from: https://pubs.rsc.org/en/content/articlehtml/2022/ta/d1ta06612a 19. Kurniawan TA, Chan GYS, Lo WH, Babel S. Physico–chemical treatment techniques for wastewater laden with heavy metals. Chem Eng J. 2006 May 1;118(1–2):83–98. 20. Barakat MA. New trends in removing heavy metals from industrial wastewater. Arab J Chem. 2011 Oct 1;4(4):361–77. 21. Chandrasekara NPGN, Pashley RM. Study of a new process for the efficient regeneration of ion exchange resins. Desalination [Internet]. 2015 Feb 2 [cited 2025 Oct 28];357:131–9. Available from: https://www.sciencedirect.com/science/article/pii/S0011916414006274 22. Abdullah N, Yusof N, Lau WJ, Jaafar J, Ismail AF. Recent trends of heavy metal 142 removal from water/wastewater by membrane technologies. J Ind Eng Chem. 2019 Aug 25;76:17–38. 23. Selvi A, Rajasekar A, Theerthagiri J, Ananthaselvam A, Sathishkumar K, Madhavan J, et al. Integrated remediation processes toward heavy metal removal/recovery from various environments-A review. Front Environ Sci [Internet]. 2019 May 22 [cited 2025 Apr 23];7(May):439466. Available from: www.frontiersin.org 24. Abd Elnabi MK, Elkaliny NE, Elyazied MM, Azab SH, Elkhalifa SA, Elmasry S, et al. Toxicity of Heavy Metals and Recent Advances in Their Removal: A Review. Toxics 2023, Vol 11, Page 580 [Internet]. 2023 Jul 3 [cited 2025 Apr 23];11(7):580. Available from: https://www.mdpi.com/2305-6304/11/7/580/htm 25. Toxic and Priority Pollutants Under the Clean Water Act | US EPA [Internet]. [cited 2025 Apr 23]. Available from: https://www.epa.gov/eg/toxic-and-priority-pollutants-under-clean-water-act 26. Chowdhury P. Degradation of Phenolic Compounds Through Uv and Visible- Light-Driven Photocatalysis Technical and Economic Aspects. 2017 [cited 2025 Apr 23]; Available from: https://books.google.com/books/about/Phenolic_Compounds.html?id=ahCQDwAAQBAJ 27. Jun LY, Yon LS, Mubarak NM, Bing CH, Pan S, Danquah MK, et al. An overview of immobilized enzyme technologies for dye and phenolic removal from wastewater. J Environ Chem Eng. 2019 Apr 1;7(2):102961. 28. Boopathy R. Factors limiting bioremediation technologies. Bioresour Technol. 2000 Aug 1;74(1):63–7. 143 29. Kuppan N, Padman M, Mahadeva M, Srinivasan S, Devarajan R. A comprehensive review of sustainable bioremediation techniques: Eco friendly solutions for waste and pollution management. Waste Manag Bull. 2024 Sep 1;2(3):154–71. 30. Sharma I, Sharma I. Bioremediation Techniques for Polluted Environment: Concept, Advantages, Limitations, and Prospects. Trace Met Environ - New Approaches Recent Adv [Internet]. 2020 Dec 7 [cited 2025 Apr 30]; Available from: https://www.intechopen.com/chapters/70661 31. Mohammadi SA, Najafi H, Zolgharnian S, Sharifian S, Asasian-Kolur N. Biological oxidation methods for the removal of organic and inorganic contaminants from wastewater: A comprehensive review. Sci Total Environ [Internet]. 2022 Oct 15 [cited 2025 Apr 30];843:157026. Available from: https://www.sciencedirect.com/science/article/pii/S0048969722041237 32. Patel AK, Singhania RR, Pandey A. Production, Purification, and Application of Microbial Enzymes. Biotechnol Microb Enzym Prod Biocatal Ind Appl [Internet]. 2017 Jan 1 [cited 2025 Apr 30];13–41. Available from: https://www.sciencedirect.com/science/article/pii/B9780128037256000029 33. Cárdenas-Moreno Y, González-Bacerio J, García Arellano H, del Monte-Martínez A. Oxidoreductase enzymes: Characteristics, applications, and challenges as a biocatalyst. Biotechnol Appl Biochem. 2023 Dec 1;70(6):2108–35. 34. Arregui L, Ayala M, Gómez-Gil X, Gutiérrez-Soto G, Hernández-Luna CE, Herrera De Los Santos M, et al. Laccases: structure, function, and potential application in water bioremediation. Microb Cell Fact [Internet]. 2019 Nov 14 [cited 2025 Apr 30];18(1). Available from: https://pubmed.ncbi.nlm.nih.gov/31727078/ 144 35. Pérez-Boada M, Ruiz-Dueñas FJ, Pogni R, Basosi R, Choinowski T, Martínez MJ, et al. Versatile Peroxidase Oxidation of High Redox Potential Aromatic Compounds: Site-directed Mutagenesis, Spectroscopic and Crystallographic Investigation of Three Long-range Electron Transfer Pathways. J Mol Biol [Internet]. 2005 Nov 25 [cited 2025 Apr 30];354(2):385–402. Available from: https://www.sciencedirect.com/science/article/pii/S0022283605011137 36. Pecková V, Legerská B, Chmelová D, Horník M, Ondrejovič M. Comparison of efficiency for monoazo dye removal by different species of white-rot fungi. Int J Environ Sci Technol [Internet]. 2020 Jun 20 [cited 2025 Apr 30];18(1):21–32. Available from: https://link.springer.com/article/10.1007/s13762-020-02806-w 37. Marco-Urrea E, Pérez-Trujillo M, Vicent T, Caminal G. Ability of white-rot fungi to remove selected pharmaceuticals and identification of degradation products of ibuprofen by Trametes versicolor. Chemosphere [Internet]. 2009 Feb 1 [cited 2025 Apr 30];74(6):765–72. Available from: https://www.sciencedirect.com/science/article/pii/S0045653508013520 38. Hlihor RM, Apostol LC, Gavrilescu M. Environmental bioremediation by biosorption and bioaccumulation: Principles and applications. Enhancing Cleanup Environ Pollut [Internet]. 2017 May 10 [cited 2025 Apr 30];1:289–315. Available from: https://link.springer.com/chapter/10.1007/978-3-319-55426-6_14 39. Chojnacka K. Biosorption and bioaccumulation in practice. 2009;137. 40. Ayele A, Haile S, Alemu D, Kamaraj M. Comparative Utilization of Dead and Live Fungal Biomass for the Removal of Heavy Metal: A Concise Review. Sci World J. 2021;2021. 145 41. Torres E. Biosorption: A Review of the Latest Advances. Process 2020, Vol 8, Page 1584 [Internet]. 2020 Dec 1 [cited 2025 Apr 30];8(12):1584. Available from: https://www.mdpi.com/2227-9717/8/12/1584/htm 42. Dhankhar R, Hooda A. Fungal biosorption – an alternative to meet the challenges of heavy metal pollution in aqueous solutions. http://dx.doi.org/101080/095933302011572922 [Internet]. 2011 [cited 2023 Aug 8];32(5):467–91. Available from: https://www.tandfonline.com/doi/abs/10.1080/09593330.2011.572922 43. Mohamadhasani F, Rahimi M. Growth response and mycoremediation of heavy metals by fungus Pleurotus sp. Sci Reports 2022 121 [Internet]. 2022 Nov 19 [cited 2023 Sep 18];12(1):1–6. Available from: https://www.nature.com/articles/s41598-022-24349-5 44. Alothman ZA, Bahkali AH, Khiyami MA, Alfadul SM, Wabaidur SM, Alam M, et al. Low cost biosorbents from fungi for heavy metals removal from wastewater. https://doi.org/101080/0149639520191608242 [Internet]. 2019 Jul 2 [cited 2023 Aug 9];55(10):1766–75. Available from: https://www.tandfonline.com/doi/abs/10.1080/01496395.2019.1608242 45. Sharma S, Rana S, Thakkar A. Chemical and Phytoremediation Technique for Removal of Heavy Metals. J Heavy Met Toxic Dis [Internet]. 2016 [cited 2023 Aug 10];1(2). Available from: http://www.imedpub.com/ 46. Ramos JL, Marqués S, van Dillewijn P, Espinosa-Urgel M, Segura A, Duque E, et al. Laboratory research aimed at closing the gaps in microbial bioremediation. Trends Biotechnol [Internet]. 2011 Dec 1 [cited 2025 Apr 29];29(12):641–7. Available from: 146 https://www.cell.com/action/showFullText?pii=S0167779911001119 47. Zhang X, Wang Z, Lu Y, Wei J, Qi S, Wu B, et al. Sustainable Remediation of Soil and Water Utilizing Arbuscular Mycorrhizal Fungi: A Review. Microorg 2024, Vol 12, Page 1255 [Internet]. 2024 Jun 21 [cited 2025 Apr 29];12(7):1255. Available from: https://www.mdpi.com/2076-2607/12/7/1255/htm 48. Singh AK, Zhu X, Chen C, Wu J, Yang B, Zakari S, et al. The role of glomalin in mitigation of multiple soil degradation problems. Crit Rev Environ Sci Technol [Internet]. 2022 [cited 2025 Apr 29];52(9):1604–38. Available from: https://www.tandfonline.com/doi/abs/10.1080/10643389.2020.1862561 49. Li X, Kang X, Zou J, Yin J, Wang Y, Li A, et al. Allochthonous arbuscular mycorrhizal fungi promote Salix viminalis L.–mediated phytoremediation of polycyclic aromatic hydrocarbons characterized by increasing the release of organic acids and enzymes in soils. Ecotoxicol Environ Saf. 2023 Jan 1;249:114461. 50. Delhoménie MC, Heitz M. Biofiltration of air: A review. Crit Rev Biotechnol [Internet]. 2005 [cited 2025 Apr 30];25(1–2):53–72. Available from: https://www.tandfonline.com/doi/pdf/10.1080/07388550590935814 51. Malakar S, Saha P Das, Baskaran D, Rajamanickam R. Comparative study of biofiltration process for treatment of VOCs emission from petroleum refinery wastewater—A review. Environ Technol Innov [Internet]. 2017 Nov 1 [cited 2025 Apr 30];8:441–61. Available from: https://www.sciencedirect.com/science/article/pii/S235218641730161X 52. Chen L, Liu C, Zou R, Yang M, Zhang Z. Experimental examination of effectiveness of vegetation as bio-filter of particulate matters in the urban environment. Environ 147 Pollut [Internet]. 2016 Jan 1 [cited 2025 Apr 30];208:198–208. Available from: https://www.sciencedirect.com/science/article/pii/S0269749115300518 53. Wróblewska K, Jeong BR. Effectiveness of plants and green infrastructure utilization in ambient particulate matter removal. Environ Sci Eur 2021 331 [Internet]. 2021 Sep 25 [cited 2025 Apr 30];33(1):1–24. Available from: https://link.springer.com/article/10.1186/s12302-021-00547-2 54. Dinakarkumar Y, Ramakrishnan G, Gujjula KR, Vasu V, Balamurugan P, Murali G. Fungal bioremediation: An overview of the mechanisms, applications and future perspectives. Environ Chem Ecotoxicol [Internet]. 2024 Jan 1 [cited 2025 Apr 30];6:293–302. Available from: https://www.sciencedirect.com/science/article/pii/S2590182624000237 55. Thirumalaivasan N, Gnanasekaran L, Kumar S, Durvasulu R, Sundaram T, Rajendran S, et al. Utilization of fungal and bacterial bioremediation techniques for the treatment of toxic waste and biowaste. Front Mater. 2024 Jul 15;11:1416445. 56. Dinakarkumar Y, Ramakrishnan G, Gujjula KR, Vasu V, Balamurugan P, Murali G. Fungal bioremediation: An overview of the mechanisms, applications and future perspectives. Environ Chem Ecotoxicol [Internet]. 2024 Jan 1 [cited 2025 Nov 6];6:293–302. Available from: https://www.sciencedirect.com/science/article/pii/S2590182624000237 57. Dell’anno F, Rastelli E, Buschi E, Barone G, Beolchini F, Dell’anno A. Fungi Can Be More Effective Than Bacteria for the Bioremediation of Marine Sediments Highly Contaminated with Heavy Metals. Microorganisms [Internet]. 2022 May 1 [cited 2025 Nov 6];10(5):993. Available from: 148 https://pmc.ncbi.nlm.nih.gov/articles/PMC9145406/ 58. Wu B, Hussain M, Zhang W, Stadler M, Liu X, Xiang M. Current insights into fungal species diversity and perspective on naming the environmental DNA sequences of fungi. Mycology [Internet]. 2019 Jul 3 [cited 2025 Apr 4];10(3):127–40. Available from: https://www.tandfonline.com/doi/abs/10.1080/21501203.2019.1614106 59. Akpasi SO, Anekwe IMS, Tetteh EK, Amune UO, Shoyiga HO, Mahlangu TP, et al. Mycoremediation as a Potentially Promising Technology: Current Status and Prospects—A Review. Appl Sci 2023, Vol 13, Page 4978 [Internet]. 2023 Apr 15 [cited 2025 Nov 6];13(8):4978. Available from: https://www.mdpi.com/2076-3417/13/8/4978/htm 60. Rosa FM, Mota TFM, Busso C, Arruda PV de, Brito PEM, Miranda JPM, et al. Filamentous Fungi as Bioremediation Agents of Industrial Effluents: A Systematic Review. Fermentation [Internet]. 2024 Mar 1 [cited 2025 Apr 30];10(3):143. Available from: https://www.mdpi.com/2311-5637/10/3/143/htm 61. K S, T M, Biswas A, T MS. Remediation potential of mushrooms and their spent substrate against environmental contaminants: An overview. Biocatal Agric Biotechnol [Internet]. 2022 Jul 1 [cited 2025 Apr 30];42:102323. Available from: https://www.sciencedirect.com/science/article/pii/S1878818122000500 62. El-Ramady H, Abdalla N, Fawzy Z, Badgar K, Llanaj X, Törős G, et al. Green Biotechnology of Oyster Mushroom (Pleurotus ostreatus L.): A Sustainable Strategy for Myco-Remediation and Bio-Fermentation. Sustain 2022, Vol 14, Page 3667 [Internet]. 2022 Mar 21 [cited 2025 Apr 30];14(6):3667. Available from: https://www.mdpi.com/2071-1050/14/6/3667/htm 149 63. P.M. Tavares A, R. Pereira S, M.R.B. Xavier A. Biotechnological Applications of Trametes versicolor and their Enzymes. Curr Biotechnol. 2016 May 17;6(2):78–88. 64. Taylor A, Flatt A, Beutel M, Wolff M, Brownson K, Stamets P. Removal of Escherichia coli from synthetic stormwater using mycofiltration. Ecol Eng. 2015 May 1;78:79–86. 65. Sen K, Llewellyn M, Taheri B, Turner RJ, Berglund T, Maloney K. Mechanism of fungal remediation of wetland water: Stropharia rugosoannulata as promising fungal species for the development of biofilters to remove clinically important pathogenic and antibiotic resistant bacteria in contaminated water. Front Microbiol [Internet]. 2023 [cited 2025 May 1];14:1234586. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC10642173/ 66. Walker GM, White NA. Introduction to Fungal Physiology. Fungi [Internet]. 2017 Nov 22 [cited 2024 Jan 19];1–35. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/9781119374312.ch1 67. Money NP. Fungal Diversity. Fungi Third Ed. 2016 Jan 1;1–36. 68. De Mattos-Shipley KMJ, Ford KL, Alberti F, Banks AM, Bailey AM, Foster GD. The good, the bad and the tasty: The many roles of mushrooms. Stud Mycol [Internet]. 2016 [cited 2025 Apr 4];85:125–57. Available from: http://dx.doi.org/10.1016/j.simyco.2016.11.002. 69. Oberwinkler F, Bauer R. Ultrastructure in basidiomycetes-requirement for function *1. 70. Maciel MJM, Castro e Silva A, Ribeiro HCT. Industrial and biotechnological 150 applications of ligninolytic enzymes of the basidiomycota: A review. Electron J Biotechnol. 2010;13(6). 71. Coelho MA, Bakkeren G, Sun S, Hood ME, Giraud T. Fungal Sex: The Basidiomycota 7. 2018 [cited 2025 May 1]; Available from: https://onlinelibrary.wiley.com/doi/10.1128/9781555819583.ch7 72. Jackson SL, Heath IB. Roles of calcium ions in hyphal tip growth. Microbiol Rev [Internet]. 1993 [cited 2025 May 1];57(2):367–82. Available from: /doi/pdf/10.1128/mr.57.2.367-382.1993?download=true 73. Scheible N, McCubbin A. Signaling in Pollen Tube Growth: Beyond the Tip of the Polarity Iceberg. Plants [Internet]. 2019 Jun 1 [cited 2025 May 1];8(6):156. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC6630365/ 74. Tlalka M, Bebber D, Darrah PR, Watkinson SC. Chapter 3 Mycelial networks: Nutrient uptake, translocation and role in ecosystems. Br Mycol Soc Symp Ser [Internet]. 2008 Jan 1 [cited 2025 May 1];28(C):43–62. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0275028708800057 75. Yafetto L. The structure of mycelial cords and rhizomorphs of fungi: A mini-review. Mycosphere. 2018;9(5):984–98. 76. Jones M, Mautner A, Luenco S, Bismarck A, John S. Engineered mycelium composite construction materials from fungal biorefineries: A critical review. Mater Des. 2020 Feb 1;187:108397. 77. Introduction to Fungi - John Webster, Roland Weber - Google Books [Internet]. [cited 2025 May 1]. Available from: https://books.google.ca/books?hl=en&lr=&id=HZLXFi- 151 om-0C&oi=fnd&pg=PP6&ots=MA9rcWA8QY&sig=s0EE5Mi7s2mbyZJ40k8zXlVIJrY&redir_esc=y#v=onepage&q&f=false 78. O’brien RW, Ralph BJ. The Cell Wall Composition and Taxonomy of Some Basidiomycetes and Ascomycetes. Ann Bot [Internet]. 1966 Oct 1 [cited 2025 May 2];30(4):831–43. Available from: https://dx.doi.org/10.1093/oxfordjournals.aob.a084117 79. Cunningham GH. Hydnaceae of New Zealand. Part I.-The pileate genera Beenakia, Dentinum, Hericium, Hydnum, Phellodon and Steccherinum. Trans R Soc New Zeal [Internet]. 1958 [cited 2025 May 2];85(4). Available from: https://www.cabidigitallibrary.org/doi/full/10.5555/19591101868 80. Pegler DN. Hyphal analysis of basidiomata. Mycol Res [Internet]. 1996 Feb 1 [cited 2025 May 2];100(2):129–42. Available from: https://www.sciencedirect.com/science/article/pii/S0953756296801110 81. Corner EJH. The agaric genera Lentinus, Panus, and Pleurotus, with particular reference to Malaysian species. 1981;169. 82. Corner EJH. Ad Polyporaceas 1. Amauroderma and Ganoderma. [Internet]. Koeltz Botanical Books; 1983 [cited 2025 May 2]. Available from: https://www.koeltz.com/en/heft-075-corner-ejh-ad-polyporaceas-1amauroderma-and-ganoderma-1983-reprint-2009-31-figs-182-p-gr8vohardcover 83. Corner EJH. Ad Polyporaceas VI. 1990 Feb 5;197. 84. Gow NAR, Latge J-P, Munro CA. The Fungal Cell Wall: Structure, Biosynthesis, and 152 Function. Microbiol Spectr [Internet]. 2017 May 19 [cited 2025 May 2];5(3). Available from: /doi/pdf/10.1128/microbiolspec.funk-0035-2016?download=true 85. Osherov N, Yarden O, Borkovich A, Ebbole DJ. Cellular and Molecular Biology of Filamentous Fungi Edited by K The Cell Wall of Filamentous Fungi. [cited 2025 May 2]; Available from: https://onlinelibrary.wiley.com/doi/10.1128/9781555816636.ch17 86. Lesage G, Bussey H. Cell Wall Assembly in Saccharomyces cerevisiae . Microbiol Mol Biol Rev [Internet]. 2006 Jun [cited 2025 May 2];70(2):317–43. Available from: /doi/pdf/10.1128/mmbr.00038-05?download=true 87. Gong Z, Zhang S, Liu J. Recent Advances in Chitin Biosynthesis Associated with the Morphology and Secondary Metabolite Synthesis of Filamentous Fungi in Submerged Fermentation. J Fungi [Internet]. 2023 Feb 1 [cited 2025 May 2];9(2):205. Available from: https://www.mdpi.com/2309-608X/9/2/205/htm 88. Aranda-Martinez A, Lopez-Moya F, Lopez-Llorca LV. Cell wall composition plays a key role on sensitivity of filamentous fungi to chitosan. J Basic Microbiol [Internet]. 2016 Oct 1 [cited 2025 May 2];56(10):1059–70. Available from: /doi/pdf/10.1002/jobm.201500775 89. Barroso-Solares S, Lopez-Moya F, Fraile T, Prieto ÁC, Lopez-Llorca L, Pinto J. Chitin and chitosan quantification in fungal cell wall via Raman spectroscopy. Spectrochim Acta Part A Mol Biomol Spectrosc [Internet]. 2025 Jun 5 [cited 2025 May 2];334:125928. Available from: https://www.sciencedirect.com/science/article/pii/S1386142525002343 90. Urbar-Ulloa J, Montaño-Silva P, Ramírez-Pelayo AS, Fernández-Castillo E, Amaya-Delgado L, Rodríguez-Garay B, et al. Cell surface display of proteins on filamentous 153 fungi. Appl Microbiol Biotechnol 2019 10317 [Internet]. 2019 Jul 29 [cited 2025 May 2];103(17):6949–72. Available from: https://link.springer.com/article/10.1007/s00253-019-10026-7 91. Zlotnik H, Pilar Fernandez M, Bowers B, Cabib E. Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. J Bacteriol [Internet]. 1984 [cited 2025 May 2];159(3):1018–26. Available from: /doi/pdf/10.1128/jb.159.3.1018-1026.1984?download=true 92. Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latgé JP. Hydrophobins—Unique Fungal Proteins. PLOS Pathog [Internet]. 2012 May [cited 2024 Jan 19];8(5):e1002700. Available from: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002700 93. Sunde M, Kwan AHY, Templeton MD, Beever RE, Mackay JP. Structural analysis of hydrophobins. Micron [Internet]. 2008 Oct 1 [cited 2025 May 2];39(7):773–84. Available from: https://www.sciencedirect.com/science/article/pii/S0968432807001230 94. Wessels JGH. Fungal hydrophobins: Proteins that function at an interface. Trends Plant Sci [Internet]. 1996 Jan 1 [cited 2024 Jan 19];1(1):9–15. Available from: http://www.cell.com/article/S1360138596800173/fulltext 95. (NCSES) NC for S and ES. Publications Output: U.S. Trends and International Comparisons. 2023. 96. Onorato C, Madeu F, Tsakalova M, Deligkiozi I, Zoikis Karathanasis A. Navigating the mycelium patent maze: A holistic approach to patent mapping in production technologies. World Pat Inf. 2024 Mar 1;76:102265. 154 97. Stametes P. Mycelium Running: How Mushrooms Can Help Save the World [Internet]. Ten Speed Press. 2005 [cited 2022 Jun 29]. Available from: https://books.google.ca/books?hl=en&lr=&id=NPI8_-omzvsC&oi=fnd&pg=PP16&dq=mycofiltration+stametes&ots=3coB5_eh1F&sig=Gk0k8glFN8pwpAe6LGr6FI55f3w#v=onepage&q=mycofiltration stametes&f=false 98. Papp N, Rudolf K, Bencsik T, Czégényi D. Ethnomycological use of Fomes fomentarius (L.) Fr. and Piptoporus betulinus (Bull.) P. Karst. in Transylvania, Romania. Genet Resour Crop Evol [Internet]. 2017 Jan 1 [cited 2025 Apr 27];64(1):101–11. Available from: https://link.springer.com/article/10.1007/s10722-015-0335-2 99. Peng L, Yi J, Yang X, Xie J, Chen C. Development and characterization of mycelium bio-composites by utilization of different agricultural residual byproducts. J Bioresour Bioprod [Internet]. 2023 [cited 2023 May 22];8:78–89. Available from: https://doi.org/10.1016/j.jobab.2022.11.005 100. Haneef M, Ceseracciu L, Canale C, Bayer IS, Heredia-Guerrero JA, Athanassiou A. Advanced Materials From Fungal Mycelium: Fabrication and Tuning of Physical Properties. Sci Reports 2017 71 [Internet]. 2017 Jan 24 [cited 2022 Mar 1];7(1):1–11. Available from: https://www.nature.com/articles/srep41292 101. Elsacker E, Vandelook S, Brancart J, Peeters E, De Laet L. Mechanical, physical and chemical characterisation of mycelium-based composites with different types of lignocellulosic substrates. PLoS One [Internet]. 2019 Jun 1 [cited 2025 May 2];14(7):e0213954. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213954 155 102. Pelletier MG, Holt GA, Wanjura JD, Greetham L, McIntyre G, Bayer E, et al. Acoustic evaluation of mycological biopolymer, an all-natural closed cell foam alternative. Ind Crops Prod. 2019 Nov 1;139:111533. 103. Bentangan M, Nugroho. Adi Reza, Hartantyo R. Mycelium Material, its Method to Produce and Usage as a Leather Substitute. WO 2020/136448 A1, 2020. 104. Characterization of mycelium-based composites as foam-like wall insulation material — Eindhoven University of Technology research portal [Internet]. [cited 2022 Mar 2]. Available from: https://research.tue.nl/en/studentTheses/characterization-of-mycelium-based-composites-as-foam-like-wall-i 105. WO2021136883A1 - Methods of making non-woven materials from mycelium - Google Patents [Internet]. [cited 2023 Nov 18]. Available from: https://patents.google.com/patent/WO2021136883A1/en?oq=WO2021136883A1 106. Grimm D, Wösten HAB. Mushroom cultivation in the circular economy. [cited 2021 May 1]; Available from: https://doi.org/10.1007/s00253-018-9226-8 107. Biby SR, Surendran V, Kundanati L. Mycelium Biocomposites from Agricultural and Paper waste: Sustainable alternative to plastic foam based secondary packaging. [cited 2025 May 2]; Available from: https://doi.org/10.1101/2025.02.18.638821 108. Akromah S, Chandarana N, Eichhorn SJ. Mycelium Composites for Sustainable Development in Developing Countries: The Case for Africa. Adv Sustain Syst [Internet]. 2024 Jan 1 [cited 2025 May 2];8(1):2300305. Available from: /doi/pdf/10.1002/adsu.202300305 109. Girometta C, Picco AM, Baiguera RM, Dondi D, Babbini S, Cartabia M, et al. 156 Physico-Mechanical and Thermodynamic Properties of Mycelium-Based Biocomposites: A Review. Sustainability [Internet]. 2019 Jan 8 [cited 2021 May 1];11(1):281. Available from: https://www.mdpi.com/2071-1050/11/1/281 110. Elsacker E, Vandelook S, Peeters E. Recent technological innovations in mycelium materials as leather substitutes: a patent review. Front Bioeng Biotechnol [Internet]. 2023 [cited 2025 May 5];11:1204861. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC10441217/ 111. Method of producing fungal materials and objects made therefrom. 2017 Jul 14; 112. Increased homogeneity of mycological biopolymer grown into void space. 2018 Nov 14; 113. Whabi V, Yu B, Xu J. From Nature to Design: Tailoring Pure Mycelial Materials for the Needs of Tomorrow. J Fungi 2024, Vol 10, Page 183 [Internet]. 2024 Feb 28 [cited 2025 May 5];10(3):183. Available from: https://www.mdpi.com/2309-608X/10/3/183/htm 114. Williams E, Cenian K, Golsteijn L, Morris B, Scullin ML. Life cycle assessment of MycoWorks’ ReishiTM: the first low-carbon and biodegradable alternative leather. Environ Sci Eur [Internet]. 2022 Dec 1 [cited 2025 May 5];34(1):1–18. Available from: https://enveurope.springeropen.com/articles/10.1186/s12302-022-00689-x 115. Jones M, Gandia A, John S, Bismarck A. Leather-like material biofabrication using fungi. Nat Sustain 2020 41 [Internet]. 2020 Sep 7 [cited 2022 Feb 22];4(1):9–16. Available from: https://www.nature.com/articles/s41893-020-00606-1 116. Gandia A, van den Brandhof JG, Appels FVW, Jones MP. Flexible Fungal Materials: 157 Shaping the Future. Trends Biotechnol [Internet]. 2021 Dec 1 [cited 2025 May 5];39(12):1321–31. Available from: https://www.cell.com/action/showFullText?pii=S0167779921000603 117. WO2021136883A1 - Methods of making non-woven materials from mycelium - Google Patents [Internet]. [cited 2025 May 5]. Available from: https://patents.google.com/patent/WO2021136883A1/en 118. Elsacker E, Vandelook S, Peeters E. Recent technological innovations in mycelium materials as leather substitutes: a patent review. Front Bioeng Biotechnol. 2023;11. 119. Jones M, Huynh T, John S. Inherent species characteristic influence and growth performance assessment for mycelium composite applications. Res Artic [Internet]. 2018 [cited 2025 May 5];2018(1):71–80. Available from: www.vbripress.com/aml 120. Cartabia M, Girometta CE, Milanese C, Baiguera RM, Buratti S, Branciforti DS, et al. Collection and characterization of wood decay fungal strains for developing pure mycelium mats. J Fungi [Internet]. 2021 Dec 1 [cited 2025 May 5];7(12):1008. Available from: https://www.mdpi.com/2309-608X/7/12/1008/htm 121. Appels FVW, van den Brandhof JG, Dijksterhuis J, de Kort GW, Wösten HAB. Fungal mycelium classified in different material families based on glycerol treatment. Commun Biol 2020 31 [Internet]. 2020 Jun 26 [cited 2023 Feb 17];3(1):1–5. Available from: https://www.nature.com/articles/s42003-020-1064-4 122. Appels FVW, Dijksterhuis J, Lukasiewicz CE, Jansen KMB, Wösten HAB, Krijgsheld P. Hydrophobin gene deletion and environmental growth conditions impact mechanical properties of mycelium by affecting the density of the material. Sci Reports 2018 81 [Internet]. 2018 Mar 16 [cited 2023 May 22];8(1):1–7. Available 158 from: https://www.nature.com/articles/s41598-018-23171-2 123. US20210317433A9 - Bio-Manufacturing Process - Google Patents [Internet]. [cited 2025 May 5]. Available from: https://patents.google.com/?assignee=ecovative&oq=ecovative 124. Geyer R, Jambeck JR, Law KL. Production, use, and fate of all plastics ever made. Sci Adv [Internet]. 2017 Jul 5 [cited 2023 Dec 5];3(7). Available from: https://www.science.org/doi/10.1126/sciadv.1700782 125. Olivetti EA, Cullen JM. Toward a sustainable materials system. Science (80- ) [Internet]. 2018 Jun 29 [cited 2023 Dec 5];360(6396):1396–8. Available from: https://www.science.org/doi/10.1126/science.aat6821 126. Ali SH. The materials science imperative in meeting the Sustainable Development Goals. Nat Mater 2018 1712 [Internet]. 2018 Nov 23 [cited 2023 Dec 5];17(12):1052–3. Available from: https://www.nature.com/articles/s41563-018-0228-9 127. THE 17 GOALS | Sustainable Development [Internet]. [cited 2023 Dec 5]. Available from: https://sdgs.un.org/goals 128. Weiss M, Haufe J, Carus M, Brandão M, Bringezu S, Hermann B, et al. A Review of the Environmental Impacts of Biobased Materials. J Ind Ecol [Internet]. 2012 Apr [cited 2025 May 12];16(SUPPL.1):S169–81. Available from: /doi/pdf/10.1111/j.1530-9290.2012.00468.x 129. Fratzl P. Introduction: Sustainable Materials. Chem Rev [Internet]. 2023 Mar 8 [cited 2025 May 12];123(5):1841–2. Available from: /doi/pdf/10.1021/acs.chemrev.3c00091 159 130. Vinod A, Sanjay MR, Suchart S, Jyotishkumar P. Renewable and sustainable biobased materials: An assessment on biofibers, biofilms, biopolymers and biocomposites. J Clean Prod [Internet]. 2020 Jun 10 [cited 2025 May 12];258:120978. Available from: https://www.sciencedirect.com/science/article/pii/S0959652620310258 131. Armstrong R. Towards the microbial home: An overview of developments in next-generation sustainable architecture. Microb Biotechnol [Internet]. 2023 Jun 1 [cited 2025 May 12];16(6):1112–30. Available from: /doi/pdf/10.1111/1751-7915.14256 132. Campano C, Balea A, Blanco A, Negro C. Enhancement of the fermentation process and properties of bacterial cellulose: a review. Cellul 2015 231 [Internet]. 2015 Nov 18 [cited 2025 May 12];23(1):57–91. Available from: https://link.springer.com/article/10.1007/s10570-015-0802-0 133. Calijuri ML, Silva TA, Magalhães IB, Pereira ASA de P, Marangon BB, Assis LR de, et al. Bioproducts from microalgae biomass: Technology, sustainability, challenges and opportunities. Chemosphere [Internet]. 2022 Oct 1 [cited 2025 May 12];305:135508. Available from: https://www.sciencedirect.com/science/article/pii/S004565352202001X 134. Javed F, Aslam M, Rashid N, Shamair Z, Khan AL, Yasin M, et al. Microalgae-based biofuels, resource recovery and wastewater treatment: A pathway towards sustainable biorefinery. Fuel [Internet]. 2019 Nov 1 [cited 2025 May 12];255:115826. Available from: https://www.sciencedirect.com/science/article/pii/S0016236119311780 135. Alemu D, Tafesse M, Mondal AK. Mycelium-Based Composite: The Future Sustainable Biomaterial. Int J Biomater [Internet]. 2022 Jan 1 [cited 2025 May 13];2022(1):8401528. Available from: /doi/pdf/10.1155/2022/8401528 160 136. Elsacker E, Vandelook S, Van Wylick A, Ruytinx J, De Laet L, Peeters E. A comprehensive framework for the production of mycelium-based lignocellulosic composites. Sci Total Environ. 2020 Jul 10;725:138431. 137. François JM. A simple method for quantitative determination of polysaccharides in fungal cell walls. Nat Protoc 2007 16 [Internet]. 2007 Jan 25 [cited 2025 Apr 8];1(6):2995–3000. Available from: https://www.nature.com/articles/nprot.2006.457 138. Kudahettige RL, Holmgren M, Imerzeel P, Sellstedt A. Characterization of bioethanol production from hexoses and xylose by the white rot fungus Trametes versicolor. Bioenergy Res [Internet]. 2012 Mar 12 [cited 2025 Jun 1];5(2):277–85. Available from: https://link.springer.com/article/10.1007/s12155-011-9119-5 139. Paschos T, Xiros C, Christakopoulos P. Ethanol effect on metabolic activity of the ethalogenic fungus Fusarium oxysporum. BMC Biotechnol [Internet]. 2015 Mar 12 [cited 2025 Jun 1];15(1):15. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4417268/ 140. Imtiaj A, Jayasinghe C, Lee GW, Shim MJ, Rho H-S, Lee HS, et al. Vegetative Growth of Four Strains of Hericium erinaceus Collected from Different Habitats. Mycobiology [Internet]. 2008 [cited 2025 Jun 1];36(2):88. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC3755240/ 141. Mahapatra S, Banerjee D. Fungal Exopolysaccharide: Production, Composition and Applications. Microbiol Insights [Internet]. 2013 Jan [cited 2025 Jun 1];6:MBI.S10957. Available from: https://scholar.google.com/scholar_url?url=https://journals.sagepub.com/doi/pdf/10.4137/MBI.S10957&hl=en&sa=T&oi=ucasa&ct=ufr&ei=PDQ9aLeHDam7ieoP1JuLsA8& 161 scisig=AAZF9b8eygCePQbYQJzcMjOIvQXr 142. Islam MR, Tudryn G, Bucinell R, Schadler L, Picu RC. Morphology and mechanics of fungal mycelium. Sci Reports 2017 71 [Internet]. 2017 Oct 12 [cited 2022 Mar 2];7(1):1–12. Available from: https://www.nature.com/articles/s41598-017-13295-2 143. Porter DL, Naleway SE. Hyphal systems and their effect on the mechanical properties of fungal sporocarps. Acta Biomater [Internet]. 2022 Jun 1 [cited 2025 Jun 2];145:272–82. Available from: https://www.sciencedirect.com/science/article/pii/S1742706122002161 144. Ost KJ, Student M, Cord-Landwehr S, Moerschbacher BM, Ram AFJ, Dirks-Hofmeister ME. Cell walls of filamentous fungi – challenges and opportunities for biotechnology. Appl Microbiol Biotechnol 2025 1091 [Internet]. 2025 May 24 [cited 2025 Jun 2];109(1):1–22. Available from: https://link.springer.com/article/10.1007/s00253-025-13512-3 145. Lamon G, Lends A, Valsecchi I, Wong SSW, Duprès V, Lafont F, et al. Solid-state NMR molecular snapshots of Aspergillus fumigatus cell wall architecture during a conidial morphotype transition. Proc Natl Acad Sci U S A [Internet]. 2023 Feb 7 [cited 2025 Jun 2];120(6):e2212003120. Available from: /doi/pdf/10.1073/pnas.2212003120?download=true 146. Bowman SM, Free SJ. The structure and synthesis of the fungal cell wall. BioEssays [Internet]. 2006 Aug [cited 2025 Jun 2];28(8):799–808. Available from: https://pubmed.ncbi.nlm.nih.gov/16927300/ 147. Gow NAR, Lenardon MD. Architecture of the dynamic fungal cell wall. Nat Rev Microbiol 2022 214 [Internet]. 2022 Oct 20 [cited 2025 Jun 2];21(4):248–59. Available 162 from: https://www.nature.com/articles/s41579-022-00796-9 148. Ibe C, Munro CA. Fungal Cell Wall Proteins and Signaling Pathways Form a Cytoprotective Network to Combat Stresses. J Fungi 2021, Vol 7, Page 739 [Internet]. 2021 Sep 8 [cited 2025 Jun 2];7(9):739. Available from: https://www.mdpi.com/2309-608X/7/9/739/htm 149. kulkarni SS, Nene SN, Joshi KS. A comparative study of production of hydrophobin like proteins (HYD-LPs) in submerged liquid and solid state fermentation from white rot fungus Pleurotus ostreatus. Biocatal Agric Biotechnol [Internet]. 2020 Jan 1 [cited 2025 Jun 2];23:101440. Available from: https://www.sciencedirect.com/science/article/pii/S187881811931117X 150. Ji X, Huang J, Teng L, Li S, Li X, Cai W, et al. Advances in particulate matter filtration: Materials, performance, and application. Green Energy Environ [Internet]. 2023 Jun 1 [cited 2025 Jul 10];8(3):673–97. Available from: https://www.sciencedirect.com/science/article/pii/S2468025722000632 151. Mccutcheon JR, Elimelech M. Influence of membrane support layer hydrophobicity on water flux in osmotically driven membrane processes. J Memb Sci [Internet]. 2008 [cited 2025 Jul 10];318:458–66. Available from: www.elsevier.com/locate/memsci 152. Wang Y, Selvamani V, Yoo IK, Kim TW, Hong SH. A Novel Strategy for the Microbial Removal of Heavy Metals: Cell-surface Display of Peptides. Biotechnol Bioprocess Eng [Internet]. 2021 Jan 1 [cited 2025 Jul 10];26(1):1–9. Available from: https://link.springer.com/article/10.1007/s12257-020-0218-z 153. Raman J, Kim DS, Kim HS, Oh DS, Shin HJ. Mycofabrication of Mycelium-Based Leather from Brown-Rot Fungi. J Fungi [Internet]. 2022 Mar 1 [cited 2025 Apr 163 8];8(3):317. Available from: https://www.mdpi.com/2309-608X/8/3/317/htm 154. Baars JJP, Mishra P, Hendrickx P, Van Der Horst C, Van Peer A. Development of a circular sustainable culturing process for natural leather-like materials based on fungal mycelium. 2024 [cited 2025 Apr 8]; Available from: https://doi.org/10.18174/676633 155. A. Pilot scale of continuous mycelium leather production at VTT... | Download Scientific Diagram [Internet]. [cited 2025 Apr 8]. Available from: https://www.researchgate.net/figure/A-Pilot-scale-of-continuous-mycelium-leather-production-at-VTT-bioreactor-fermentation_fig3_357190129 156. Fourest E, Roux JC. Heavy metal biosorption by fungal mycelial by-products: mechanisms and influence of pH. Appl Microbiol Biotechnol [Internet]. 1992 Jun [cited 2023 Aug 11];37(3):399–403. Available from: https://link.springer.com/article/10.1007/BF00211001 157. MacIntyre CR, Seale H, Dung TC, Hien NT, Nga PT, Chughtai AA, et al. A cluster randomised trial of cloth masks compared with medical masks in healthcare workers. BMJ Open [Internet]. 2015 Apr 1 [cited 2022 Feb 18];5(4):e006577. Available from: https://bmjopen.bmj.com/content/5/4/e006577 158. Strasser BJ, Schlich T. A history of the medical mask and the rise of throwaway culture. Lancet [Internet]. 2020 Jul 4 [cited 2022 Feb 18];396(10243):19–20. Available from: http://www.thelancet.com/article/S0140673620312071/fulltext 159. Aragaw TA. Surgical face masks as a potential source for microplastic pollution in the COVID-19 scenario. Mar Pollut Bull. 2020 Oct 1;159:111517. 164 160. Fadare OO, Okoffo ED. Covid-19 face masks: A potential source of microplastic fibers in the environment. Sci Total Environ [Internet]. 2020 Oct 1 [cited 2022 Feb 18];737:140279. Available from: /pmc/articles/PMC7297173/ 161. Sullivan GL, Delgado-Gallardo J, Watson TM, Sarp S. An investigation into the leaching of micro and nano particles and chemical pollutants from disposable face masks - linked to the COVID-19 pandemic. Water Res. 2021 May 15;196:117033. 162. Sangeetha K, Pragadheeswari R, Arunkumar K V, Rajakumar R, Professor A, Professor A. Nano Membrane Fibrous on lyocell non-woven fabric for bacterial filtration efficiency in surgical face mask CLOTHING PREFERENCE View project MEDICAL TEXTILES View project Nano Membrane Fibrous on Lyocell Non-Woven Fabric for Bacterial Filitration Efficiency in Surgical Face Mask. 2014 [cited 2022 Feb 18];2:598–9. Available from: www.indiasciencetech.com 163. Abbas WA, Shaheen BS, Ghanem LG, Badawy IM, Abodouh MM, Abdou SM, et al. Cost-Effective Face Mask Filter Based on Hybrid Composite Nanofibrous Layers with High Filtration Efficiency. Langmuir [Internet]. 2021 Jun 22 [cited 2022 Feb 18];37(24):7492–502. Available from: https://pubs.acs.org/doi/abs/10.1021/acs.langmuir.1c00926 164. Choi S, Jeon H, Jang M, Kim H, Shin G, Mo Koo J, et al. Biodegradable, Efficient, and Breathable Multi-Use Face Mask Filter. Adv Sci [Internet]. 2021 Mar 1 [cited 2022 Feb 18];8(6):2003155. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/advs.202003155 165. He H, Gao M, Illés B, Molnar K. 3D Printed and Electrospun, Transparent, Hierarchical Polylactic Acid Mask Nanoporous Filter. Int J Bioprinting [Internet]. 165 2020 [cited 2022 Feb 18];6(4). Available from: /pmc/articles/PMC7557519/ 166. Al-Hazeem NZ. Manufacture of fibroustructure facemask to protect against coronavirus using electrospinning. Med Res Chronicles [Internet]. 2021 Apr 28 [cited 2022 Feb 18];8(2):103–10. Available from: http://medrech.com/index.php/medrech/article/view/480 167. Babaahmadi V, Amid H, Naeimirad M, Ramakrishna S. Biodegradable and multifunctional surgical face masks: A brief review on demands during COVID-19 pandemic, recent developments, and future perspectives. Sci Total Environ. 2021 Dec 1;798:149233. 168. Liu H, Huang J, Mao J, Chen Z, Chen G, Lai Y. Transparent Antibacterial Nanofiber Air Filters with Highly Efficient Moisture Resistance for Sustainable Particulate Matter Capture. iScience [Internet]. 2019 Sep [cited 2023 May 22];19:214–23. Available from: https://pubmed.ncbi.nlm.nih.gov/31377666/ 169. Wang L, Gao Y, Xiong J, Shao W, Cui C, Sun N, et al. Biodegradable and high-performance multiscale structured nanofiber membrane as mask filter media via poly(lactic acid) electrospinning. 2021 [cited 2023 May 22]; Available from: https://doi.org/10.1016/j.jcis.2021.08.079 170. Athukoralalage SSA, Bell CA, Gemmell AC, Rowan AE, Amiralian N. Recent advances and future perspectives in engineering biodegradable face masks. J Mater Chem A [Internet]. 2023 Jan 24 [cited 2023 May 22];11(4):1575–92. Available from: https://pubs.rsc.org/en/content/articlehtml/2023/ta/d2ta08019b 171. Jonsirivilai B, Torgbo S, Sukyai P. Multifunctional filter membrane for face mask using bacterial cellulose for highly efficient particulate matter removal. Cellulose 166 [Internet]. 2022 Jul 1 [cited 2023 May 22];29(11):6205–18. Available from: https://link.springer.com/article/10.1007/s10570-022-04641-3 172. Sun W, Tajvidi M, Hunt CG, Howell C. All-Natural Smart Mycelium Surface with Tunable Wettability. 2021 [cited 2023 May 22]; Available from: https://dx.doi.org/10.1021/acsabm.0c01449 173. Garcia RA, Stevanovic T, Berthier J, Njamen G, Tolnai B, Achim A. Cellulose, nanocellulose, and antimicrobial materials for the manufacture of disposable face masks: A review. BioResources. 2021 Feb 25;16(2):4321–53. 174. Antinori ME, Contardi M, Suarato G, Armirotti A, Bertorelli R, Mancini G, et al. Advanced mycelium materials as potential self-growing biomedical scaffolds. Sci Reports 2021 111 [Internet]. 2021 Jun 16 [cited 2022 Dec 9];11(1):1–14. Available from: https://www.nature.com/articles/s41598-021-91572-x 175. Kwong LH, Wilson R, Kumar S, Crider YS, Reyes Sanchez Y, Rempel D, et al. Review of the Breathability and Filtration Efficiency of Common Household Materials for Face Masks. ACS Nano [Internet]. 2021 Apr 27 [cited 2022 Dec 1];15(4):5904–24. Available from: https://pubs.acs.org/doi/full/10.1021/acsnano.0c10146 176. Sayfutdinova A, Samofalova I, Barkov A, Cherednichenko K, Rimashevskiy D. Structure and Properties of Cellulose/Mycelium Biocomposites. Polymers (Basel) [Internet]. 2022 Apr 1 [cited 2023 Feb 17];14(8):1519. Available from: https://www.mdpi.com/2073-4360/14/8/1519/htm 177. O’Dowd K, Nair KM, Forouzandeh P, Mathew S, Grant J, Moran R, et al. Face Masks and Respirators in the Fight Against the COVID-19 Pandemic: A Review of Current Materials, Advances and Future Perspectives. Mater 2020, Vol 13, Page 3363 167 [Internet]. 2020 Jul 29 [cited 2023 Feb 17];13(15):3363. Available from: https://www.mdpi.com/1996-1944/13/15/3363/htm 178. Elliot MA, Talbot NJ. Building filaments in the air: aerial morphogenesis in bacteria and fungi. Curr Opin Microbiol. 2004 Dec 1;7(6):594–601. 179. Han J, Kawauchi M, Schiphof K, Terauchi Y, Yoshimi A, Tanaka C, et al. Features of disruption mutants of genes encoding for hydrophobin Vmh2 and Vmh3 in mycelial formation and resistance to environmental stress in Pleurotus ostreatus. FEMS Microbiol Lett [Internet]. 2023 Jan 17 [cited 2023 May 22];370. Available from: https://academic.oup.com/femsle/article/doi/10.1093/femsle/fnad036/7134070 180. Berger BW, Sallada ND. Hydrophobins: multifunctional biosurfactants for interface engineering. J Biol Eng [Internet]. 2019 Jan 23 [cited 2025 May 2];13(1):10. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC6343262/ 181. Appels FVW, van den Brandhof JG, Dijksterhuis J, de Kort GW, Wösten HAB. Fungal mycelium classified in different material families based on glycerol treatment. Commun Biol [Internet]. 2020 Dec 1 [cited 2021 May 1];3(1). Available from: https://pubmed.ncbi.nlm.nih.gov/32591629/ 182. Drinking-water [Internet]. [cited 2023 Sep 20]. Available from: https://www.who.int/news-room/fact-sheets/detail/drinking-water 183. Jones DH, Yu X, Guo Q, Duan X, Jia C. Racial Disparities in the Heavy Metal Contamination of Urban Soil in the Southeastern United States. Int J Environ Res Public Health [Internet]. 2022 Feb 1 [cited 2023 Sep 20];19(3). Available from: https://pubmed.ncbi.nlm.nih.gov/35162130/ 168 184. Gochfeld M, Burger J. Disproportionate Exposures in Environmental Justice and Other Populations: The Importance of Outliers. Am J Public Health [Internet]. 2011 Dec 1 [cited 2023 Sep 20];101(Suppl 1):S53. Available from: /pmc/articles/PMC3222496/ 185. Sharma S. Heavy Metals In Water: Presence, Removal and Safety - Google Books [Internet]. Royal Society of Chemistry. 2014 [cited 2023 Jul 11]. Available from: https://books.google.ca/books?hl=en&lr=&id=e2ooDwAAQBAJ&oi=fnd&pg=PR13&dq=heavy+metals+in+water&ots=z4pi47Wbwg&sig=MIfr05pCr4tG4djD5swYgSz5TRw#v=onepage&q=heavy metals in water&f=false 186. Szynkowska MI, Pawlaczyk A, Maćkiewicz E. Bioaccumulation and Biomagnification of Trace Elements in the Environment. Recent Adv Trace Elem [Internet]. 2017 Dec 18 [cited 2023 Jul 11];251–76. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/9781119133780.ch13 187. Zhao H, Xia B, Fan C, Zhao P, Shen S. Human health risk from soil heavy metal contamination under different land uses near Dabaoshan Mine, Southern China. Sci Total Environ. 2012 Feb 15;417–418:45–54. 188. Mueller JT, Gasteyer S. The ethnically and racially uneven role of water infrastructure spending in rural economic development. Nat Water 2023 11 [Internet]. 2023 Jan 19 [cited 2023 Sep 28];1(1):74–82. Available from: https://www.nature.com/articles/s44221-022-00007-y 189. Khulbe KC, Matsuura T. Removal of heavy metals and pollutants by membrane adsorption techniques. Appl Water Sci 2018 81 [Internet]. 2018 Jan 24 [cited 2023 Aug 3];8(1):1–30. Available from: https://link.springer.com/article/10.1007/s13201- 169 018-0661-6 190. Kim S, Nam SN, Jang A, Jang M, Park CM, Son A, et al. Review of adsorption–membrane hybrid systems for water and wastewater treatment. Chemosphere. 2022 Jan 1;286:131916. 191. Hao S, Geng Y, Jia Z. UV pre-activation/thermal initiated grafting of caffeic acid on PVDF for preparation of adsorptive membranes for cesium. React Funct Polym. 2018 Nov 1;132:120–6. 192. Hao S, Jia Z, Wen J, Li S, Peng W, Huang R, et al. Progress in adsorptive membranes for separation – A review. Sep Purif Technol. 2021 Jan 15;255:117772. 193. Park D, Yun YS, Park JM. The past, present, and future trends of biosorption. Biotechnol Bioprocess Eng [Internet]. 2010 Feb 18 [cited 2023 Aug 8];15(1):86–102. Available from: https://link.springer.com/article/10.1007/s12257-009-0199-4 194. Desai K, Kit K, Li J, Michael Davidson P, Zivanovic S, Meyer H. Nanofibrous chitosan non-wovens for filtration applications. Polymer (Guildf). 2009 Jul 17;50(15):3661–9. 195. Yang L, Park D, Qin Z. Material Function of Mycelium-Based Bio-Composite: A Review. Front Mater. 2021 Sep 30;8:737377. 196. Baldrian P. Interactions of heavy metals with white-rot fungi. Enzyme Microb Technol. 2003 Jan 2;32(1):78–91. 197. Baldrian P, Baldrian P. Effect of Heavy Metals on Saprotrophic Soil Fungi. Soil Biol [Internet]. 2010 [cited 2023 Aug 9];19:263–79. Available from: https://link.springer.com/chapter/10.1007/978-3-642-02436-8_12 170 198. Priyadarshini E, Priyadarshini SS, Cousins BG, Pradhan N. Metal-Fungus interaction: Review on cellular processes underlying heavy metal detoxification and synthesis of metal nanoparticles. Chemosphere. 2021 Jul 1;274:129976. 199. Priyanka, Dwivedi SK. Fungi mediated detoxification of heavy metals: Insights on mechanisms, influencing factors and recent developments. J Water Process Eng. 2023 Jul 1;53:103800. 200. Liapun V, Motola M. Current overview and future perspective in fungal biorecovery of metals from secondary sources. J Environ Manage. 2023 Apr 15;332:117345. 201. Mohanty S, Ghosh S, Nayak S, Das AP. Bioleaching of manganese by Aspergillus sp. isolated from mining deposits. Chemosphere. 2017 Apr 1;172:302–9. 202. Patel F, Lakshmi B. Bioleaching of copper and nickel from mobile phone printed circuit board using Aspergillus fumigatus A2DS. Brazilian J Microbiol [Internet]. 2021 Sep 1 [cited 2023 Aug 10];52(3):1475–87. Available from: https://link.springer.com/article/10.1007/s42770-021-00526-y 203. Pang C, Liu YH, Cao XH, Li M, Huang GL, Hua R, et al. Biosorption of uranium(VI) from aqueous solution by dead fungal biomass of Penicillium citrinum. Chem Eng J. 2011 May 15;170(1):1–6. 204. Yang SK, Tan N, Yan XM, Chen F, Lin YC. Adsorption of thorium(IV) from aqueous solution by non-living biomass of mangrove endophytic fungus Fusarium sp. #ZZF51. J Radioanal Nucl Chem [Internet]. 2013 Nov 5 [cited 2023 Aug 10];298(2):827–33. Available from: https://link.springer.com/article/10.1007/s10967-013-2567-y 205. Sen M. Adsorption-Desorption Studies on Cr(VI) Using Non-Living Fungal Biomass. 171 Artic Asian J Chem [Internet]. 2010 [cited 2023 Aug 10]; Available from: https://www.researchgate.net/publication/279548837 206. (PDF) Removal of lead from aqueous solution by filamentous fungi [Internet]. [cited 2023 Aug 10]. Available from: https://www.researchgate.net/publication/288448199_Removal_of_lead_from_aqueous_solution_by_filamentous_fungi 207. Shi J, Zhao D, Ren F, Huang L. Spatiotemporal variation of soil heavy metals in China: The pollution status and risk assessment. Sci Total Environ. 2023 May 1;871:161768. 208. Tay CC, Liew HH, Yin CY, Abdul-Talib S, Surif S, Suhaimi AA, et al. Biosorption of cadmium ions using Pleurotus ostreatus: Growth kinetics, isotherm study and biosorption mechanism. Korean J Chem Eng [Internet]. 2011 Mar 16 [cited 2023 Aug 11];28(3):825–30. Available from: https://link.springer.com/article/10.1007/s11814-010-0435-9 209. Xiangliang P, Jianlong W, Daoyong Z. Biosorption of Pb(II) by Pleurotus ostreatus immobilized in calcium alginate gel. Process Biochem. 2005 Jul 1;40(8):2799–803. 210. Fourest E, Roux JC. Heavy metal biosorption by fungal mycelial by-products: mechanisms and influence of pH. Appl Microbiol Biotechnol [Internet]. 1992 Jun [cited 2023 Jun 7];37(3):399–403. Available from: https://link.springer.com/article/10.1007/BF00211001 211. Joseph L, Jun BM, Flora JRV, Park CM, Yoon Y. Removal of heavy metals from water sources in the developing world using low-cost materials: A review. Chemosphere. 2019 Aug 1;229:142–59. 172 212. Tang R, Yu Z, Renneckar S, Zhang Y. Coupling chitosan and TEMPO-oxidized nanofibrilliated cellulose by electrostatic attraction and chemical reaction. Carbohydr Polym. 2018 Dec 15;202:84–90. 213. Money NP. Measurement of pore size in the hyphal cell wall ofAchlya bisexualis. Exp Mycol. 1990 Sep 1;14(3):234–42. 214. Xiong J, Fu D, Singh RP, Ducoste JJ. Structural characteristics and development of the cake layer in a dynamic membrane bioreactor. Sep Purif Technol. 2016 Jul 14;167:88–96. 215. Li X, Zhang D, Sheng F, Qing H. Adsorption characteristics of Copper (Ⅱ), Zinc (Ⅱ) and Mercury (Ⅱ) by four kinds of immobilized fungi residues. Ecotoxicol Environ Saf. 2018 Jan 1;147:357–66. 216. Feofilova EP. The fungal cell wall: Modern concepts of its composition and biological function. Microbiology [Internet]. 2010 Dec 7 [cited 2023 Sep 7];79(6):711–20. Available from: https://link.springer.com/article/10.1134/S0026261710060019 217. Veglio’ F, Beolchini F. Removal of metals by biosorption: a review. Hydrometallurgy. 1997 Mar 1;44(3):301–16. 218. Vandelook S, Elsacker E, Van Wylick A, De Laet L, Peeters E. Current state and future prospects of pure mycelium materials. Fungal Biol Biotechnol [Internet]. 2021 Dec 1 [cited 2022 Feb 22];8(1):1–10. Available from: https://fungalbiolbiotech.biomedcentral.com/articles/10.1186/s40694-021-00128-1 219. Regalado CM, Crawford JW, Ritz K, Sleeman BD. The origins of spatial heterogeneity in vegetative mycelia: a reaction-diffusion model. Mycol Res. 1996 Jan 173 1;100(12):1473–80. 220. Attias N, Reid M, Mijowska SC, Dobryden I, Isaksson M, Pokroy B, et al. Biofabrication of Nanocellulose–Mycelium Hybrid Materials. Adv Sustain Syst [Internet]. 2021 Feb 1 [cited 2023 Oct 25];5(2):2000196. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/adsu.202000196 221. Irbe I, Filipova I, Skute M, Zajakina A, Spunde K, Juhna T. Characterization of Novel Biopolymer Blend Mycocel from Plant Cellulose and Fungal Fibers. Polym 2021, Vol 13, Page 1086 [Internet]. 2021 Mar 30 [cited 2023 Oct 25];13(7):1086. Available from: https://www.mdpi.com/2073-4360/13/7/1086/htm 222. Jalaee A, Kamkar M, French V, Rojas OJ, Foster EJ. Direct milling: Efficient, facile, and green method for processing fibrillated cellulose/polymeric nanocomposites with boosted thermomechanical and rheological performance. Carbohydr Polym. 2023 Aug 15;314:120932. 223. Saba N, Mohammad F, Pervaiz M, Jawaid M, Alothman OY, Sain M. Mechanical, morphological and structural properties of cellulose nanofibers reinforced epoxy composites. Int J Biol Macromol. 2017 Apr 1;97:190–200. 224. Gan PG, Sam ST, Abdullah MF bin, Omar MF. Thermal properties of nanocellulose-reinforced composites: A review. J Appl Polym Sci [Internet]. 2020 Mar 15 [cited 2023 Nov 29];137(11):48544. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/app.48544 225. Huang C, Chung YC, Liou MR. Adsorption of Cu(II) and Ni(II) by pelletized biopolymer. J Hazard Mater. 1996 Feb 1;45(2–3):265–77. 174 226. Bilal M, Ihsanullah I, Younas M, Ul Hassan Shah M. Recent advances in applications of low-cost adsorbents for the removal of heavy metals from water: A critical review. Sep Purif Technol. 2021 Dec 1;278:119510. 227. SenthilKumar P, Ramalingam S, Sathyaselvabala V, Kirupha SD, Sivanesan S. Removal of copper(II) ions from aqueous solution by adsorption using cashew nut shell. Desalination. 2011 Jan 31;266(1–3):63–71. 228. Stanescu AM, Stoica L, Constantin C, Lacatusu I, Oprea O, Miculescu F. Physicochemical Characterization and Use of Heat Pretreated Commercial Instant Dry Baker’s Yeast as a Potential Biosorbent for Cu(II) Removal. CLEAN – Soil, Air, Water [Internet]. 2014 Nov 1 [cited 2023 Nov 29];42(11):1632–41. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/clen.201300484 229. Lee SY, Choi HJ. Persimmon leaf bio-waste for adsorptive removal of heavy metals from aqueous solution. J Environ Manage. 2018 Mar 1;209:382–92. 230. Özcan A, Özcan AS, Tunali S, Akar T, Kiran I. Determination of the equilibrium, kinetic and thermodynamic parameters of adsorption of copper(II) ions onto seeds of Capsicum annuum. J Hazard Mater. 2005 Sep 30;124(1–3):200–8. 231. Gupta VK, Ali I. Utilisation of bagasse fly ash (a sugar industry waste) for the removal of copper and zinc from wastewater. Sep Purif Technol. 2000 Mar 6;18(2):131–40. 232. El Haouti R, Anfar Z, Chennah A, Amaterz E, Zbair M, El Alem N, et al. Synthesis of sustainable mesoporous treated fish waste as adsorbent for copper removal. Groundw Sustain Dev. 2019 Apr 1;8:1–9. 175 233. Yousefi N, Jones M, Bismarck A, Mautner A. Fungal chitin-glucan nanopapers with heavy metal adsorption properties for ultrafiltration of organic solvents and water. Carbohydr Polym. 2021 Feb 1;253:117273. 234. Javaid A, Bajwa R, Shafique U, Anwar J. Removal of heavy metals by adsorption on Pleurotus ostreatus. Biomass and Bioenergy. 2011 May 1;35(5):1675–82. 235. Hamid SA, Azha SF, Sellaoui L, Bonilla-Petriciolet A, Ismail S. Adsorption of copper (II) cation on polysulfone/zeolite blend sheet membrane: Synthesis, characterization, experiments and adsorption modelling. Colloids Surfaces A Physicochem Eng Asp. 2020 Sep 20;601:124980. 236. Pakdel Mojdehi A, Pourafshari Chenar M, Namvar-Mahboub M, Eftekhari M. Development of PES/polyaniline-modified TiO2 adsorptive membrane for copper removal. Colloids Surfaces A Physicochem Eng Asp. 2019 Dec 20;583:123931. 237. Ghaee A, Shariaty-Niassar M, Barzin J, Matsuura T, Fauzi Ismail A. Preparation of chitosan/cellulose acetate composite nanofiltration membrane for wastewater treatment. Desalin Water Treat [Internet]. 2016 Jul 2 [cited 2024 Mar 6];57(31):14453–60. Available from: https://www.tandfonline.com/doi/abs/10.1080/19443994.2015.1068228 238. Törős GH, El-Ramady H, Prokisch J. Edible Mushroom of Pleurotus spp.:A Case Study of Oyster Mushroom (Pleurotus ostreatus L.). Environ Biodivers Soil Secur [Internet]. 2022 Feb 1 [cited 2023 Oct 13];6(2022):51–9. Available from: https://jenvbs.journals.ekb.eg/article_231627.html 239. Shar GA, Soomro GA. SPECTROPHOTOMETRIC DETERMINATION OF COBALT(II), NICKEL(II) AND COPPER (II) WITH 1-(2 PYRIDYLAZO)-2- 176 NAPHTHOL IN MICELLAR MEDIUM. Nucl [Internet]. 2004 [cited 2023 Jul 27];41(1–4):77–82. Available from: http://www.thenucleuspak.org.pk/index.php/Nucleus/article/view/1068 240. Terletskaya A V., Ievleva OS, Bogoslovskaya TA, Goncharuk V V. Selective photometric determination of nickel, cobal, and zinc in drinking and natural waters using 1-(2-pyridylazo)-2-naphthol and surfactants (SAS). J Water Chem Technol [Internet]. 2013 Nov 15 [cited 2023 Jul 27];35(6):246–52. Available from: https://link.springer.com/article/10.3103/S1063455X13060027 241. Revellame ED, Fortela DL, Sharp W, Hernandez R, Zappi ME. Adsorption kinetic modeling using pseudo-first order and pseudo-second order rate laws: A review. Clean Eng Technol. 2020 Dec 1;1:100032. 242. Wang J, Guo X. Adsorption isotherm models: Classification, physical meaning, application and solving method. Chemosphere. 2020 Nov 1;258:127279. 243. Bhardwaj P, Kaur N, Selvaraj M, Ghramh HA, Al-Shehri BM, Singh G, et al. Laccase-assisted degradation of emerging recalcitrant compounds – A review. Bioresour Technol. 2022 Nov 1;364:128031. 244. Loi M, Glazunova O, Fedorova T, Logrieco AF, Mulè G. Fungal Laccases: The Forefront of Enzymes for Sustainability. J Fungi 2021, Vol 7, Page 1048 [Internet]. 2021 Dec 7 [cited 2024 Dec 9];7(12):1048. Available from: https://www.mdpi.com/2309-608X/7/12/1048/htm 245. Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol. 2007 May 2;40(6):1451–63. 177 246. Garcia-Galan C, Berenguer-Murcia Á, Fernandez-Lafuente R, Rodrigues RC. Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance. Adv Synth Catal [Internet]. 2011 Nov 1 [cited 2024 Dec 11];353(16):2885–904. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/adsc.201100534 247. Datta S, Christena LR, Rajaram YRS. Enzyme immobilization: An overview on techniques and support materials. 3 Biotech [Internet]. 2013 Jun 6 [cited 2024 Dec 11];3(1):1–9. Available from: https://link.springer.com/article/10.1007/s13205-012-0071-7 248. Maghraby YR, El-Shabasy RM, Ibrahim AH, Azzazy HMES. Enzyme Immobilization Technologies and Industrial Applications. ACS Omega [Internet]. 2023 Feb 14 [cited 2024 Dec 12];8(6):5184–96. Available from: https://pubs.acs.org/doi/full/10.1021/acsomega.2c07560 249. Aggarwal S, Chakravarty A, Ikram S. A comprehensive review on incredible renewable carriers as promising platforms for enzyme immobilization & thereof strategies. Int J Biol Macromol. 2021 Jan 15;167:962–86. 250. Ansorge-Schumacher MB. Enzyme Immobilization. 2024 [cited 2024 Dec 11];219–41. Available from: https://link.springer.com/chapter/10.1007/978-3-031-42999-6_11 251. French V, Kleiman M, Bhalla K, Moreira R, Jalaee A, Kronstad JW, et al. Fungal-derived adsorption membrane to capture potentially toxic elements. Chem Eng J. 2024 May 15;488:151028. 252. Manan S, Ullah MW, Ul-Islam M, Atta OM, Yang G. Synthesis and applications of fungal mycelium-based advanced functional materials. J Bioresour Bioprod. 2021 Feb 178 1;6(1):1–10. 253. Xu J, Zhang R, Han Z, Wang Z, Wang F, Deng L, et al. The highly-stable immobilization of enzymes on a waste mycelium carrier. J Environ Manage. 2020 Oct 1;271:111032. 254. Pereira JCV, Serbent MP, Skoronski E. Application of immobilized mycelium-based pellets for the removal of organochlorine compounds: a review. Water Sci Technol [Internet]. 2021 Apr 15 [cited 2025 Apr 10];83(8):1781–96. Available from: http://creativecommons.org/licenses/by/4.0/ 255. Zahmatkesh M, Spanjers H, van Lier JB. A novel approach for application of white rot fungi in wastewater treatment under non-sterile conditions: immobilization of fungi on sorghum. Environ Technol [Internet]. 2018 Aug 18 [cited 2025 Apr 10];39(16):2030–40. Available from: https://www.tandfonline.com/doi/abs/10.1080/09593330.2017.1347718 256. Chen HY, Guan YX, Yao SJ. A novel two-species whole-cell immobilization system composed of marine-derived fungi and its application in wastewater treatment. J Chem Technol Biotechnol [Internet]. 2014 Nov 1 [cited 2025 Apr 10];89(11):1733–40. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/jctb.4253 257. Mahmoud ME, Yakout AA, Abdel-Aal H, Osman MM. Immobilization of Fusarium verticillioides fungus on nano-silica (NSi–Fus): A novel and efficient biosorbent for water treatment and solid phase extraction of Mg(II) and Ca(II). Bioresour Technol. 2013 Apr 1;134:324–30. 258. Chulikavit N, Huynh T, Wang C, Yuen ACY, Khatibi A, Mouritz A, et al. Engineering mycelium fungi into an effective char-forming thermal protection material via 179 alkaline deacetylation. Polym Degrad Stab. 2023 Jun 1;212:110355. 259. Sabnis S, Block LH. Improved infrared spectroscopic method for the analysis of degree of N-deacetylation of chitosan. Polym Bull [Internet]. 1997 Jul 1 [cited 2024 Nov 24];39(1):67–71. Available from: https://link.springer.com/article/10.1007/s002890050121 260. Zor T, Selinger Z. Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies. Anal Biochem. 1996 May 1;236(2):302–8. 261. Iark D, Buzzo AJ dos R, Garcia JAA, Côrrea VG, Helm CV, Corrêa RCG, et al. Enzymatic degradation and detoxification of azo dye Congo red by a new laccase from Oudemansiella canarii. Bioresour Technol. 2019 Oct 1;289:121655. 262. Boudrant J, Woodley JM, Fernandez-Lafuente R. Parameters necessary to define an immobilized enzyme preparation. Process Biochem. 2020 Mar 1;90:66–80. 263. Barbosa O, Ortiz C, Berenguer-Murcia Á, Torres R, Rodrigues RC, Fernandez-Lafuente R. Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv [Internet]. 2013 Dec 2 [cited 2024 Dec 5];4(4):1583–600. Available from: https://pubs.rsc.org/en/content/articlehtml/2014/ra/c3ra45991h 264. Betancor L, López-Gallego F, Hidalgo A, Alonso-Morales N, Dellamora-Ortiz G, CesarMateo GDO, et al. Different mechanisms of protein immobilization on glutaraldehyde activated supports: Effect of support activation and immobilization conditions. Enzyme Microb Technol. 2006 Aug 2;39(4):877–82. 180 265. Bes MT, Gomez-Moreno C, Guisan JM, Fernandez-Lafuente R. Selective oxidation: stabilisation by multipoint attachment of ferredoxin NADP+ reductase, an interesting cofactor recycling enzyme. J Mol Catal A Chem. 1995 May 25;98(3):161–9. 266. Zheng F, Cui BK, Wu XJ, Meng G, Liu HX, Si J. Immobilization of laccase onto chitosan beads to enhance its capability to degrade synthetic dyes. Int Biodeterior Biodegradation. 2016 May 1;110:69–78. 267. Dwevedi A. Basics of Enzyme Immobilization. Enzym Immobil [Internet]. 2016 [cited 2024 Dec 5];21–44. Available from: https://link.springer.com/chapter/10.1007/978-3-319-41418-8_2 268. Xu R, Zhou Q, Li F, Zhang B. Laccase immobilization on chitosan/poly(vinyl alcohol) composite nanofibrous membranes for 2,4-dichlorophenol removal. Chem Eng J. 2013 Apr 15;222:321–9. 269. Bilal M, Jing Z, Zhao Y, Iqbal HMN. Immobilization of fungal laccase on glutaraldehyde cross-linked chitosan beads and its bio-catalytic potential to degrade bisphenol A. Biocatal Agric Biotechnol. 2019 May 1;19:101174. 270. Othman AM, Sanromán Á, Moldes D. Laccase multi-point covalent immobilization: characterization, kinetics, and its hydrophobicity applications. Appl Microbiol Biotechnol [Internet]. 2023 Feb 1 [cited 2024 Dec 12];107(2–3):719–33. Available from: https://link.springer.com/article/10.1007/s00253-022-12352-9 271. Almeida FLC, Prata AS, Forte MBS. Enzyme immobilization: what have we learned in the past five years? Biofuels, Bioprod Biorefining [Internet]. 2022 Mar 1 [cited 2024 Dec 12];16(2):587–608. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/bbb.2313 181 272. Engasser J-M, Horvath C. Diffusion and Kinetics with Immobilized Enzymes. 1976 Jan 1;1:127–220. 273. Rees DC. A general solution for the steady-state kinetics of immobilized enzyme systems. Bull Math Biol [Internet]. 1984 Mar [cited 2024 Dec 12];46(2):229–34. Available from: https://link.springer.com/article/10.1007/BF02460071 274. Jiang DS, Long SY, Huang J, Xiao HY, Zhou JY. Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres. Biochem Eng J. 2005 Aug 1;25(1):15–23. 275. Fang H, Huang J, Ding L, Li M, Chen Z. Preparation of magnetic chitosan nanoparticles and immobilization of laccase. J Wuhan Univ Technol Mater Sci Ed [Internet]. 2009 Feb 20 [cited 2024 Dec 12];24(1):42–7. Available from: https://link.springer.com/article/10.1007/s11595-009-1042-7 276. Loi M, Glazunova O, Fedorova T, Logrieco AF, Mulè G. Fungal Laccases: The Forefront of Enzymes for Sustainability. J Fungi 2021, Vol 7, Page 1048 [Internet]. 2021 Dec 7 [cited 2024 Dec 11];7(12):1048. Available from: https://www.mdpi.com/2309-608X/7/12/1048/htm 277. Jeyabalan J, Veluchamy A, V VP, Kumar A, Chandrasekar R, Narayanasamy S. A review on the laccase assisted decolourization of dyes: Recent trends and research progress. J Taiwan Inst Chem Eng. 2023 Oct 1;151:105081. 278. Cañas AI, Camarero S. Laccases and their natural mediators: Biotechnological tools for sustainable eco-friendly processes. Biotechnol Adv. 2010 Nov 1;28(6):694–705. 279. Stolz A. Basic and applied aspects in the microbial degradation of azo dyes. Appl 182 Microbiol Biotechnol [Internet]. 2001 [cited 2025 Apr 7];56(1–2):69–80. Available from: https://pubmed.ncbi.nlm.nih.gov/11499949/ 280. Silva ER, Dall’oglio EL, Vasconcelos LG, Morais EB. Decolorization of the benzidine-based azo dye Congo red by the new strain Shewanella xiamenensis G5-03. Braz J Biol [Internet]. 2021 [cited 2025 Apr 7];82. Available from: https://pubmed.ncbi.nlm.nih.gov/33978076/ 281. Gu Y, Yuan L, Jia L, Xue P, Yao H. Recent developments of a co-immobilized laccase–mediator system: a review. RSC Adv [Internet]. 2021 Sep 2 [cited 2025 Apr 8];11(47):29498–506. Available from: https://pubs.rsc.org/en/content/articlehtml/2021/ra/d1ra05104k 282. Liu R, Wang S, Han M, Zhang W, Xu H, Hu Y. Co-immobilization of electron mediator and laccase onto dialdehyde starch cross-linked magnetic chitosan nanomaterials for organic pollutants’ removal. Bioprocess Biosyst Eng [Internet]. 2022 Dec 1 [cited 2025 Apr 8];45(12):1955–66. Available from: https://link.springer.com/article/10.1007/s00449-022-02799-5 183 Appendix A – Chapter 2 supplementary information A.1 Morphology of static fermentation samples SEM images of GL SSF (a) and GL LSF (b) are shown. Hyphal types appear similar, with most hyphae as generative (the filled structure of hyphae in cross-section indicates the presence of intracellular material, indicative of generative hyphae). However, SSF shows a larger range of hyphal diameters, with several larger structures present (skeletal hyphae) and several thinner segments of hyphae branching from skeletal and generative hyphae (ligative hyphae). A.2 Yield of SmF pellets The yield of SmF pellets for GL, PO, and TV species is shown in the table below as the dry weight of sample after washing and freeze-drying. 184 GL SmF 0.56 g TV SmF 2.21 g PO SmF 1.89 g A.3 Representative HPLC spectra for PMM monosaccharide analysis HPLC spectra for quantitative determination of glucose, mannose, and glucosamine for GL SSF (a), GL LSF (b), GL SmF (c), PO SmF (d), and TV SmF (e). Spectra show peaks for monosaccharides based on analysis of standards and determination of their residence time, which was 15.2 min for glucose, 16.3 min for mannose, and 20.5 mins for glucosamine. The appearance of other small peaks around 18-19 mins in samples indicates the presence of another monosaccharide not analyzed here. 185 186 A.4 Mechanical properties of PMMs in tensile mode Young’s modulus, UTS, and elongation values for samples of PMMs. Biological triplicates were taken for GL SSF and GL LSF whereas duplicates were measured for SmF samples across fungal species. 187 Sample Young’s modulus (MPa) Ultimate tensile strength (MPa) Elongation (%) GL SSF 1 130 0.8 0.8 GL SSF 2 125 0.75 2 GL SSF 3 100 0.65 1.6 GL LSF 1 75 0.55 2.7 GL LSF 2 20 0.2 1.4 GL LSF 3 31 0.4 3 TV SmF 1 375 1.10 0.3 TV SmF 2 231 0.8 0.4 PO SmF 1 506 1.4 0.3 PO SmF 2 365 0.87 0.2 GL SmF 1 78 0.21 0.5 GL SmF 2 41 0.11 0.3 A.5 Compression testing of static fermentations PMMs Storage modulus vs. temperature (35-75°C) for Ganoderma lucidum mycelium produced by static fermentation methods. 188 A.6 Surface roughness of PMMs Surface roughness of static fermentation PMMs were calculated by using the 3D Topography module of the Theta Flex 300-Pulsating Drop 200 Tensiometer. Surfaces were analyzed by the instrument and a roughness value (r) is recorded in the table below. Sample r GL SSF aerial 1.09 ± 0.04 GL LSF aerial 1.17 ± 0.12 A sample of the image processing performed by the 3D topography module is shown below for GL LSF. 189 190 Appendix B – Chapter 3 supplementary information B.1 Swelling of mycelium-modified masks Swelling properties of mask layers and mycelium-modified mask layers (triplicate swelling measurements were taken over the course of 1 440 min, and the swelling results were normalized by the samples’ initial weight and displayed as a function of time.) B-2 Mechanical data for mycelium-modified masks Representative stress-strain curves for P1, P1-M, P2, P2-M, and samples with glycerol treatment, P1-M-G, and P2-M-G 191 Table of calculated mechanical properties for air-dried samples with various post-processing treatments Sample Ultimate tensile strength (MPa) Elongation at tensile strength (%) Young’s modulus (MPa) P1 1.37 ± 0.40 184.470 ± 85.21 0.37 ± 0.02 P1-M 85.82 ± 3.19 1.403 ± 0.21 9.17 ± 0.03 P1-M-G 84.78 ± 11.04 1.208 ± 0.13 6.46 ± 0.02 P2 0.43 ± 0.10 17.210 ± 5.11 9.54 ± 0.00 P2-M 9.07 ± 0.40 0.592 ± 0.03 16.25 ± 0.05 P2-M-G 7.07 ± 1.12 1.228 ± 0.09 9.21 ± 0.02 192 Appendix C – Chapter 4 supplementary information C-1 Media selection and pellet structure of mycelium (a) Image of Pleurotus ostreatus culture on agar plates with the following media: yeast extract peptone dextrose (YPD), yeast nitrogen media (YNM), Sabouraud agar (SDA), and potato dextrose agar (PDA) with the most growth is shown on the YPD and PDB plate. (b) DIC images of liquid cultures in YPD and PDB are shown to show the health of the hyphae in YPD versus PDB after 14 days of growth. Hyphal lysis is evident in PDB media. (c) Images of pellet growth in liquid media after shaking for 7 days and the freeze-dried pellet structure. C-2. Table of media, pH, and C/N sources 193 Media Type pH Carbohydrate source Nitrogen source PDA (potato dextrose agar/broth) Complex 5.6 Dextrose Potato infusion YPD (yeast peptone dextrose) Complex 6.5 Dextrose Peptone, yeast extract SDA (Sauboraud agar) Complex 5.6 Dextrose Mycological peptone YNB (Yeast nitrogen base) Defined 4.5 D-glucose Ammonium sulphate C-3. Process optimization of blending and homogenizing mycelium Optical microscopy of a 1% dispersion of mycelium pellets in water after processing by blending and homogenizing. Blending for more than 1 min results in broken hyphal filaments. For dispersions blended for 1 min 10 min versus 30 min homogenizing times does not significantly improve dispersions. 194 C-4. Influence of CNF type and quantity on composite properties (a) Scanning electron microscopy images of Pleurotus ostreatus pellets neat (i) or with the addition of 2wt% unmodified cellulose nanofiber (ii) or tempo-oxidized cellulose nanofiber (iii). The highly aggregated appearance of CNF relative to TCNF (marked by blue arrows) informed the decision to utilize TCNFs in the membrane construction. (b) A scanning electron microscopy image depicting a visible instance aggregation of TCNFs at 5wt% addition. 195 C-5. Batch adsorption parameters (a) pH dependence of the adsorption process for mycelium membrane (M) with an adsorption time of 3 hours and a graphical depiction of the competition for adsorption sites with hydronium ions at varying pHs. (b) Batch adsorption experiment of Cu(II) with a starting concentration of 100 mg/L at pH 5.5 and shaking speed of 220 rpm. Complete adsorption for all membranes was found at t=12 hours. All subsequent experiments used an adsorption time of 24 hours to ensure this equilibrium had been reached based on this experiment. (c) Percent Cu(II) adsorbed by mycelium membrane at different dosages (0.5, 1, 2, 4, and 8 mg adsorbent/mL adsorbate solution.) Percent adsorption follows an increasing trend with dosage. Systems can be tailored using this information to remove specific quantities of contaminant. 196 C-6. Validation of adsorption mechanism using FTIR and XPS (a) FTIR spectra of the M membrane before adsorption (M) and after adsorption (M+Cu). Peak values are shown with the associated bonds. (b) XPS survey scan of M membrane before and after adsorption of Cu(II). 197 C-7. Morphology of M-1TCNF membrane Images of M-1TCNF using a digital camera and x-sectional morphology of membranes using scanning electron microscopy. Association of TCNF and mycelium is shown in the scanning electron microscopy, where smaller fibers are shown to generate small pores over the mycelia. Surface area and pore size analysis C-8. Micrometrics surface area and pore size curves 198 Micromeritics 3Flex Surface Area & Pore Size Analyzer BET adsorption isotherm curves for M (a), M-1TCNF (b), M-5TCNF (c) and BJH adsorption incremental pore area distribution for M (d), M-1TCNF (e), M-5TCNF (f). C-9. Tristar II plus surface area and pore size curves TriStar II Plus BET Gas Adsorption Analyzer BET adsorption isotherm curves for M (a), M-1TCNF (b), M-5TCNF (c) and BJH adsorption incremental pore area distribution for M (d), M-1TCNF (e), M-5TCNF (f). 199 Colorimetric determination of Cu concentration C-10. Copper calibration curves Cu(II) calibration curves for UV-vis spectrometer and microplate reader. The difference in slope between the two instruments is related to pathlengths differences based on Beer-Lamberts law. These equations were utilized to calculate Cu(II) concentration in samples. 200 C-11. Parameters for the linear fit to UV-vis and microplate Cu(II) calibration curves UV Vis spectrophotometer Microplate reader Slope 0.11564 0.06057 Intersect Set @ 0 Set @ 0 Pearsons R 0.99968 0.99646 R square 0.99936 0.99293 Adj. R square 0.9993 0.99222 Kinetic and isotherm models C-12. Pseudo-first order kinetic model fits Non-linear fit of kinetic data for M, M-1TCNF, and M-5TCNF to pseudo-1st order model. 201 C-13. Freundlich model fits Non-linear fit of isotherm data for M, M-1TCNF, and M-5TCNF to Freundlich model. 202 XPS analysis C-14. XPS survey scans XPS survey scans for M-1TCNF before (a) and after Cu adsorption (b) and M-5TCNF before (c) and after Cu adsorption (d). 203 Appendix D – Chapter 5 supplementary information D-1. Table listing the degree of deacetylation (DD%), and relative activity values for untreated and treated mycelial samples Samples treated with 30, 45, and 60% NaOH. The highest relative activity is shown for mycelial samples treated with 45% NaOH, which is also the sample with the highest degree of deacetylation. D-2. Calibration curve for protein estimation using Bradford Assay correction UV-vis spectra (left) of samples with 0-25mg/mL BSA. Corrected calibration curve (left) using the ratio of the absorbance at 590nm to the absorbance at 450nm. The selected range was the linear portion of the previous range. DD (%) Relative activity Untreated 64.5 ± 4.1 73.8% Pretreated 73.8 ± 0.9 71.1% Treated 30 82.9 ± 0.4 85.3% Treated 45 84.1 ± 1.1 100% Treated 60 83.7 ± 1.5 84.0% 204 D-3. Morphology of original (OG) and pre-treated (PT) mycelial biomass. Original biomass shows typical TV hyphal morphology. Pretreated biomass shows flattening and welding of hyphal structures. D-4. Surface of 45% and 60% NaOH treated biomass 45% treated mycelia show increased surface structures and small pores generated on the surface of the sheet. 60% treated mycelia show a smooth sheet with minimal surface structures. D-5. Immobilization optimization terms, coefficients, and significance 205 Coefficient estimate P-value t_GA 0.0954 0.00762 ** [GA] -0.0748 0.01503 * [Lacc] 0.01518 0.3814 (t_GA)2 -0.01665 0.03622 * [GA]2 0.12741 0.04659 * [Lacc]2 0.10572 0.03178 * t_GA * [GA] -0.05379 0.08287 t_GA * [Lacc] -0.00517 0.82135 [GA] * [Lacc] -0.0299 0.24921 *= significant results (P< 0.05) **= highly significant results (P<0.01) D-6. FTIR spectra of the mycelial mat before (M) and after (M IE) enzyme immobilization Increased peak shoulder at ~1650 cm-1 indicates the presence of an imine peak related to a Schiff-base reaction mechanism. 206 D-7. HPLC analysis of degradation products Congo Red solutions before (top) and after (bottom) treatment with I-Lacc & I-SA enzyme-mediator system on mycelial support.
Thesis/Dissertation
eng
Chemical and Biological Engineering
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Foster, E. Johan
Mycelium as an advanced functional material
Text