Campbell et al. PDF 2023 Annual Review of Materials Research Progress in Sustainable Polymers from Biological Matter

Annual Review of Materials Research Progress in Sustainable Polymers from Biological...

Annual Review of Materials Research Progress in Sustainable Polymers from Biological Matter Ian R. Campbell,∗ Meng-Yen Lin,∗ Hareesh Iyer, Mallory Parker, Jeremy L. Fredricks, Kuotian Liao, Andrew M. Jimenez, Paul Grandgeorge, and Eleftheria Roumeli Department of Materials Science and Engineering, University of Washington, Seattle, Washington, USA; email: [email protected] Annu. Rev. Mater. Res. 2023. 53:81–104 Keywords First published as a Review in Advance on sustainability, biological matter, biopolymers, biocomposites, cementitious March 31, 2023 materials, renewable resources The Annual Review of Materials Research is online at matsci.annualreviews.org Abstract https://doi.org/10.1146/annurev-matsci-080921- The increasing consumption of nonrenewable materials urgently calls for 083655 the design and fabrication of sustainable alternatives. New generations of Copyright © 2023 by the author(s). This work is materials should be derived from renewable sources, processed using en- licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted vironmentally friendly methods, and designed considering their full life use, distribution, and reproduction in any medium, cycle, especially their end-of-life fate. Here, we review recent advances in provided the original author and source are credited. developing sustainable polymers from biological matter (biomatter), includ- See credit lines of images or other third-party material in this article for license information. ing progress in the extraction and utilization of bioderived monomers and ∗ polymers, as well as the emergence of polymers produced directly from These authors contributed equally to this article unprocessed biomatter (entire cells or tissues). We also discuss applica- tions of sustainable polymers in bioplastics, biocomposites, and cementitious biomaterials, with emphasis on relating their performance to underlying fundamental mechanisms. Finally, we provide a future outlook for sus- tainable material development, highlighting the need for more accurate and accessible tools for assessing life-cycle impacts and socioeconomic challenges as this field advances. 81  1. INTRODUCTION Since the beginning of large-scale production and use of plastics in the 1950s, approximately Biomass: renewable 8.3 billion tonnes (Bt) of pure plastics (not including fillers and additives) have been produced, organic material from from which 6.3 Bt are generated waste (1). Of this plastic waste, less than 10% has been recycled, plants or animals, approximately 12% has been incinerated, and almost 80% (∼5 Bt) has accumulated in landfills or including individual the natural environment. Plastics accumulated in the environment will represent 12 Bt by 2050 extracted components thereof if current production and waste management trends continue unaltered (1), at which point the weight of plastic waste in the ocean will equal that of fish (2). The negative impacts of the prolifer- Biopolymer: polymer ation of plastic waste in the natural environment are multifold, including acute trauma to animals synthesized from biological organisms upon ingestion as well as long-term hazards created by concentrating organic pollutants in the environment (2). These alarming data, along with the irreplaceable role played by plastics in our Self-bonded society and economy, motivate the development of sustainable polymers. This broad term includes polymeric material: polymer that does not new and existing macromolecules that are derived entirely or partially from renewable (nonfossil) require an external feedstocks for the production of biodegradable or recyclable polymers (3). To supplant commod- binder to form a ity polymers, sustainable polymers must combine performance and scalability (including source self-standing matrix feedstock availability, cost efficiency, and ability to be processed with the existing plastics manu- Biomatter: biomass facturing infrastructure). In terms of mechanical properties, which are the primary performance synthesized by any metrics we focus on in this review, commodity plastics have an elastic modulus between 0.1 and living organism (plant, 1 GPa, strength between 1 and 50 MPa, and elongation to break between 1% and 1,000%, while animal, bacterium, engineering and high-performance plastics have higher modulus and strength values, 1–10 GPa alga, fungus) that retains a degree of and 30–200 MPa, respectively, but are brittle, with elongation to break values below 10%. native, hierarchical Here, we provide an overview of current research efforts in creating polymers from renewable organization feedstocks, offering promising solutions to the ever-increasing pollution problem. We outline three distinct tracks, introduced in Figure 1, and present exciting findings that highlight the po- tential of each approach in providing sustainable polymer solutions addressing the sourcing and end-of-life issues simultaneously. First, we describe monomers derived from biomass or waste to produce existing or entirely novel polymers. Using well-developed polymerization reactions en- ables seamless integration of bioderived monomers in existing infrastructures, while the derived monomers can also serve as functional organic molecules for other synthetic materials. Next, we present advances in extracting and utilizing biopolymers as self-bonded polymeric materials or as components for synthetic polymers or inorganic structural materials. This approach utilizes the high-molecular-weight biopolymers as produced from organisms and often maintains their secondary or tertiary structure as a template for forming hierarchical materials. Finally, we re- view recent advances in using entire organisms in the form of cells or tissues as building blocks to create either self-bonded materials or novel composites. This approach can potentially lead to wasteless manufacturing (i.e., where no biomass is wasted) and can be built around local resource utilization and existing processing infrastructures. An example of this emerging approach is de- veloping cell cultures locally as tunable material platforms that eliminate transportation needs, bypass nonsustainable supply chains, and are independent of climate and land fertility. 2. FROM BIOMATTER-DERIVED MONOMERS TO SUSTAINABLE POLYMERS Recent innovations in the chemical and enzymatic extraction and polymerization of monomers derived from biological matter (biomatter) and, specifically, from vegetal biomass, organic waste, and microorganisms have augmented the potential for biobased monomers to supplant petroleum-based equivalents (2) (Figure 2a). In this section, we discuss examples of abundant and versatile macromolecules of plant biomass such as carbohydrates, fatty acids, terpenes, 82 Campbell et al.  Extraction Assembly Product a Biomatter Extraction b HO OH purification R' O O Monomer HO OH O R C O R' C R n 5 cm Fungi Curli fiber Protein c E. coli Polymer 2 cm Algae Protein Biofilm network Aquaplastic house d Bacteria Organisms 2 cm 100 μm and tissues Hypha Mycelium Mycelium composite network Direct 10 μm 1 cm Spider silk valorization Plant cell Self-bonded plant cells Plant cell bioplastic beam Figure 1 Research tracks to produce sustainable materials from renewable sources. (a) Examples of biomatter resources. (b) Extracting monomers to synthesize existing or entirely novel polymers. (c) Extracting biopolymers to create self-bonded or composite materials. (d) Using the entire organism as a polymer composite building block. The extraction needs and generated waste increase from bottom to top. Image of flip-flops in panel b adapted from Reference 4. Illustrations of extractions in panels b and c and image of protein structure in panel c adapted from images created with BioRender.com. Network illustration and image of the 3D aquaplastic house in panel c adapted with permission from Reference 5. Upper scanning electron microscopy snapshot in panel d adapted with permission from Reference 6. Image of plant cell bioplastic beam in panel d adapted with permission from Reference 7. polyphenols, organic acids, and their derivatives, which are used as the principal renewable sources of high-value monomers. One of the most important monomers for commodity plastics, ethylene, has been derived through the fermentation and dehydration of glucose, extracted from agricultural products such as sugarcane, and used commercially to produce bio-polyethylene (Bio-PE) since approximately 2010 (10). Other industrially significant olefin monomers have also been derived from fatty acids via the decarbonylation of carboxylic acids (11). By using common nickel salts to catalyze deoxy- genation, John et al. (11) achieved up to 82% yield of olefins from fatty acids while reducing the number and rarity of reactants and eliminating the need for an acid-activating species. Abdelrah- Polyphenol: man et al. (8) similarly improved the yield of monomer extraction by hydrogenation and dehydra- biopolymer formed from aromatic decyclization of itaconic acid to isolate dienes and isoprene with a 72% selectivity (Figure 2b). phenolic networks While these techniques efficiently transform biosourced monomers into traditionally petroleum- derived polymers, they do not improve upon detrimental end-of-life environmental persistence. Bioplastic: polymer derived from biomass Arguably one of the most industrially significant bioplastics (12) in the world, polylactic that, at some stage in acid (PLA), is obtained by the ring-opening polymerization of lactic acid monomers, which are its processing, can be produced through the fermentation of starch-rich biomass. Unlike commodity plastics such as shaped by flow Bio-PE, PLA can be degraded, albeit slowly, via synthetic hydrolysis of backbone ester groups www.annualreviews.org Biomatter for Sustainable Materials 83  WoodI WoodII Cellulose Elastic modulus (GPa) Natural source Monomer Polymer 102 a Natural Enzymatic 100 materials Crystalline Monomer cellulose extraction 10–2 Chemical Synthetic 1 process Foams polymers 2 10–4 n 3 Extraction Polymerization 10–2 10–1 100 101 102 103 104 Strength (MPa) b Commodity plastics c Novel polymers O O O 0 O One-pot Gas-phase dehydra- O O decyclization O + O O O −10 O n O HO O O 3-MTHF Isoprene −20 ΔGrxn (Kcal/mol) 400°C O −30 15 30% O Stress (MPa) 200°C 49% PLLA ΔGvap −40 25°C 10 23% −50 γMCL polymer backbone 5 15% O O O O −60 OH OH Liquid HO 0 −70 HO cascade 0 500 1,000 1,500 2,000 O PLLA grafts reactions Strain (%) Itaconic acid MGBL MBDO 3-MTHF −80 Bottlebrush copolymers enable Hydrogenation and dehydra-decyclization to isolate monomer mechanical property tuning Figure 2 Monomer extraction and polymerization. (a) Visualization of chemical or enzymatic extraction of monomers from biomass to synthesize polymers and a graphical representation of their mechanical performance from the literature (triangles are from Reference 14, squares are from Reference 9, and circles are from Reference 13). Illustration of extraction adapted from BioRender.com. Ashby plot adapted with permission from Reference 7. (b) The energetic reaction pathway for the isolation of isoprene from itaconic acid and the 3-MTHF intermediate. Panel adapted with permission from Reference 8. (c) The variation in mechanical responses enabled by differing degrees of copolymerization of (red) poly(γMCL) and (blue) PLLA. Panel adapted with permission from Reference 9. Abbreviations: 3-MTHF, 3-methyl-tetrahydrofuran; γMCL, γ-methyl-ε-caprolactone; MBDO, 2-methyl-1,4-butanediol; MGBL, methyl-γ-butyrolactone; PLLA, poly(l-lactide). (2). Other monomers have been isolated from biomass specifically to facilitate degradation, such as β-methyl-δ-valerolactone (MVL), which can be derived from sugar to produce ther- moplastic polyurethane (PU), PU foams (13), and poly(MVL), which is depolymerizable at low temperatures, enabling the recovery and reuse of approximately 97% of the MVL monomer. Other recent examples include work from Manker et al. (14), who synthesized thermoplastic poly(alkylene xylosediglyoxylates) from lignocellulosic saccharides that are chemically recyclable and degradable via hydrolysis. Their results show that the degree of degradation depends on the diol molecule used in the polymerization. Shen et al. (15) promoted degradation by including acetal groups in the backbone of lignin-derived monomers. The resulting polycycloacetals are semicrystalline thermoplastics and can be degraded via the cleavage of acetal groups by hydrolysis in acidic organic solvents. Novel monomers can also be used to create block copolymers that diversify polymer mechanical performance. The terpene pinene, a product of conifer sap, was used to synthe- size thermoplastic elastomers (TPEs) from myrcene and α-methyl-p-methylstyrene (AMMS) monomers (16). When polymerized into an ABA triblock copolymer, the poly(myrcene)- poly(AMMS) copolymers have elongations and tensile strengths comparable to petroleum-based 84 Campbell et al. styrenic TPEs. Fournier et al. (9) synthesized a bottlebrush copolymer elastomer with tunable mechanical properties from poly(l-lactide) (PLLA) and lignin-derived γ-methyl-ε-caprolactone (γMCL). By varying the density and length of PLLA grafts, the copolymer mechanical properties Polysaccharide: can be tuned significantly (Figure 2c). linear or branched Microbial cultures have also been used as a sustainable route for the isolation of monomers. carbohydrate polymer Liang et al. (17) utilized genetically modified Escherichia coli to improve the extraction of styrene of monosaccharides by a factor of 3.45, although the resulting polystyrene had a low molecular weight. Microalgal oils (e.g., glucose) bound together through have been used to isolate polyols, which serve as polyester monomers, with examples including glycosidic linkages PU foams that meet the requirements for commercial footwear while demonstrating excellent (covalent bonds) compostability in soil (4). Nanopaper: sheet composed of 3. BIOPOLYMERS FROM BIOMATTER nanofibers (with at least one dimension Biological organisms synthesize and utilize hierarchical biopolymers, often of high molecular below 100 nm) weight, as structural and functional building blocks. Leveraging the diversity of biopolymers and developing methods for their extraction constitute a promising emerging field within sustainable Cellulose nanofibril (CNF): cellulose fibril polymers. In this section, we present advances in sustainable materials utilizing polysaccharides, comprising both polyphenols, and proteins. amorphous and crystalline regions 3.1. Cellulose (700% strain to break for a graphene concentration of 10 wt%. However, this large extensibil- ity comes at the cost of reduced Young’s modulus (4 MPa) and strength (0.5 MPa). Finally, an unprecedented approach for introducing nanocarbon compounds in silks was adopted by Wang et al. (40) and Lepore et al. (57), who fed nanocarbon materials directly to silkworms and spi- ders, respectively. The silkworms fed with a 0.2 wt% solution of single-walled carbon nanotubes www.annualreviews.org Biomatter for Sustainable Materials 91  (CNTs) produced a silk that was 639% stiffer and 113% stronger (40). Spiders fed with CNTs produced fibers 1,084% stiffer and 631% stronger than the control (57). Cellulose 3.6.2. α-helix-dominant proteins. Proteins forming predominantly α-helix secondary struc- nanocrystal (CNC): tures have also been used for sustainable materials. The Nelson group (41) developed a resin purely crystalline for stereolithography (SLA) by adding comonomers and a photoinitiator to a solution of a glob- whisker of cellulose (3–20 nm in width and ular α-helix-forming protein, bovine serum albumin (BSA). Their strategy capitalized on the 50–2,000 nm in stimuli sensitivity of the BSA molecular conformation to develop hydrogels with shape memory length) behaviors. While printed structures retained the native globular BSA conformation, postprint- ing thermal curing caused β-sheet formation and, subsequently, material stiffening. Moreover, the BSA globules in the printed structures exhibited plasticity during deformation due to pro- tein unfolding, which can be recovered via heating or swelling in water (Figure 4d) (41). Smith et al. (58) further enhanced SLA BSA materials by soaking printed structures in tannic acid (TA), a natural polyphenol. Infusing biodegradable methacrylated BSA structures with TA formed an intermolecular H-bonding network that improved toughness almost 10-fold. 3.6.3. Hybrid α–β protein structures. Sustainable materials developed from proteins with quaternary organization and mixed secondary structures have also been reported. Lysozyme, for instance, conforms in both α-helix and β-sheet arrangements. Recently, De France et al. (59) added lysozyme to cellulose nanocrystal (CNC) films and studied the effects of sonication as a means to change the lysozyme conformation from globules to fibers. The α/β ratio of lysozyme re- mained unchanged and independent of the altered conformation achieved through sonication. α/β ratio stability and strong electrostatic interactions enabled reinforcement of the CNC network by protein fibrils and improved its toughness by 84% (at 10 wt% protein). Casein, a micellar protein found in milk, has been combined with additives such as glycerol and pectin to prepare films with properties suitable for edible food packaging (60). As discussed previ- ously, the final properties are defined by the interplay between the structural function of protein, its plastic deformability, the interactions with polysaccharides, and the ordered domain disruption facilitated from plasticizers. Building on the anticipated enhanced interactions between lignocel- lulosic wood particles and casein, durable wood panels were prepared without requiring the use of formaldehyde or other petrochemical binders (61). Gluten, like casein, is a complex mixture of distinct proteins: polymeric glutenins and monomeric gliadins. The network of proteins grants gluten viscoelastic properties that enable thermomechanical processing. By exploiting gluten vis- coelasticity and utilizing glycerol as a plasticizer, Jiménez-Rosado et al. (62) produced bioplastics via extrusion and injection molding. 4. BIOMATTER AS A MATERIAL BUILDING BLOCK: USING ENTIRE ORGANISMS AND TISSUES The extraction of biopolymers or monomers, as described in the previous sections, is a costly, energy-intensive, and wasteful process. Recent advances in circumventing extraction processes rely on using raw or minimally preprocessed biomatter. In this section, we discuss innovative utilizations of biomatter as a filler or a matrix material with little or no preprocessing and present emerging approaches such as material farming as viable implementations of sustainable polymer design. 4.1. Bacteria Cultured bacteria have recently emerged as alternative polymer materials. Manjula-Basavanna et al. (63) demonstrated that bacteria, E. coli and Lactobacillus rhamnosus, or yeast, Saccharomyces 92 Campbell et al. cerevisiae, could be cultured, harvested, and air-casted into self-bonded films showing indentation moduli of 5–42 GPa for E. coli, 5–30 GPa for L. rhamnosus, and 1–30 GPa for S. cerevisiae. Rather than culturing a single strain, Das et al. (64) utilized the symbiotic relationship between Biofilm: consortium Acetobacter aceti bacteria and Chlamydomonas reinhardtii microalgae to create integrated compos- of microorganisms ite biofilms. The by-products of microalgal metabolism were used in the production of BC from where cells adhere to bacteria, and the by-products of BC synthesis acted as nutrients for the microalgae. BC was dis- each other and often tributed throughout the hybrid material instead of concentrating at the air–medium interface, to a surface while varying the component ratio influenced the microalgae distribution. Similarly, Birnbaum Biomineralization: et al. (65) designed a bacterial coculture of a BC-producing strain, Gluconacetobacter hansenii, and mineral precipitation, genetically engineered E. coli to produce unique composite capsules. By using droplets of growth particularly of CaCO3 , by living cells media, a BC matrix was formed on the droplet surface while intertwined bacteria cells and curli fibers remained at the core of the produced spheres. Further property tuning can occur in this versatile system as a result of bacterial biomineralization; CaCO3 mineral deposits increase the hardness and stiffness of the composite spheres. An emerging theme in sustainable materials is capitalizing on bacterial biomineralization, or microbially induced calcium carbonate precipitation (MICP), to develop self-healing construction materials, primarily concrete. When a crack is formed in concrete that contains bacterial spores, air and water come in contact with those spores. Bacterial metabolic processes then alter the pH and carbonate concentration of the surrounding matrix, causing MICP to accumulate and fill the cracks (66). Biomineralization mechanisms include urea hydrolysis performed by alkali-tolerant ureolytic bacteria such as Bacillus sp. strains (66) and NO3 reduction performed by denitrifying bacteria (67). Alternatively, photosynthetic cyanobacteria in mortar induce MICP by metaboliz- ing CO2 , releasing OH− , and increasing the surrounding pH. The associated carbon fixation was reported to promote CaCO3 precipitation and facilitated 35% strength recovery after micro- cracking for mortars containing 12 wt% Spirulina sp. (68) (the mechanism and self-healing re- sults are shown in Figure 5a). Commercialization efforts have been reported, such as BioLITH tiles [from Biomason (https://biomason.com)], that show mechanical properties acceptable for structural applications despite consuming only a fraction (namely 3.5%) of the manufacturing energy required for conventional competing materials. In addition to their use as fillers for crack remediation, living microorganisms have been uti- lized as binders and compatibilizers in building materials. Raut et al. (73) utilized Bacillus pasteurii biomineralization to bind clay and sand and formed bricks with 8-MPa compressive strength af- ter 28 days, which is 30–50% of the strength of conventional bricks. Similarly, Heveran et al. (74) combined Spirulina, gelatin, and a sand scaffold to create a living building material (LBM) with 9% cell viability at high humidity (>50%) and low temperature (4°C). Cell viability was further increased to 37% by tuning the component concentrations (69) (Figure 5b). The cell viability in LBMs is higher than that of encapsulated microorganisms in cement due to the absence of an alkaline environment, demonstrating the outstanding potential of LBMs as sustainable build- ing materials. Lastly, MICP was used to improve compatibility between 5 wt% recycled plastic filler and mortar and to elicit compressive strengths of approximately 55 MPa (75), exemplifying another strategy to reduce the environmental footprint of cementitious materials. 4.2. Fungi Fungi colonize substrates with filamentous cells called hyphae (2–10 μm in diameter), which form an extended network called mycelium. Mycelia naturally adopt the shape of the substrate as they grow, enabling straightforward shaping of the resulting mycelium/substrate composites. These attractive biomatter-based materials offer diverse mechanical properties and densities, fast growth times, low fabrication costs, and biodegradability. www.annualreviews.org Biomatter for Sustainable Materials 93  Bacterial biomineralization Fungal applications a Carbonic anhydrase c Bending stress (MPa) i interconversion of ii 0.8 i ii a CO2 b HCO3– b CaCO3 0.6 Hot pressed HCO3– OH– c 0.4 Bacterial Cold pressed HCO3– CO2 cell 0.2 c Not pressed 100 μm 50 μm CaCO3 1 μm 0 Rubisco 0 1.0 2.0 3.0 4.0 5.0 a Aerial hyphae b Mycelium c Substrate Cyanobacteria Strain (%) iii Crack healing d i ii iii Hypha Crack 14 days Biomass 20 mm 20 mm substrate b Gel Extracellular i Sand ii RH%+ 20 mm polymeric 100 40x substances Viability [%, cfu/(g/s)] Viability 80 Gel/sand = 0.13 e Gel/sand = 0.3 CaCO3 60 Hypha 1mm 40 Strength 20 50 μm Living building 0 material E. coli Spirulina RH%– Figure 5 Bacteria- and fungi-based materials. (a) Cyanobacteria carry out (i) carbonic anhydrase interconversion of HCO− 3 , promoting (ii) CaCO3 precipitation that (iii) seals cracks. (b, i) Microbially induced calcium carbonate precipitation combined with sand and gelatin to form a living building material. Higher humidity increases viability while decreasing strength of the living building material. (b, ii) Spirulina was found to be more viable than Escherichia coli in living building materials. (c, i) Mycelia grown in different substrates were hot pressed at 150°C, cold pressed at 20°C, and not pressed, showing up to eightfold increases in flexural strength depending on fungal species, substrate, and processing conditions. (c, ii) Microscopy images of unpressed Trametes multicolor mycelium on rapeseed straw. (d, i) 3D-printed mycelium structure with (ii) microscope image. (d, iii) Illustration showing bonding between mycelium and the biomass substrate assisted via the secretion of extracellular polymeric substances consisting of polysaccharides and proteins. (e) Cement-free sandstone bonded by fungi-induced CaCO3. Abbreviation: RH, relative humidity. Panel a, subpanels ii and iii adapted with permission from Reference 68. Panel b adapted with permission from Reference 69. Panel c adapted with permission from Reference 70. Panel d adapted (i,ii) with permission from Reference 71 and (iii) from images created with BioRender.com. Panel e adapted with permission from Reference 72. The principal structural components of hyphal cell walls are chitin, β-glucans, and proteins. Tuning the relative concentration of the cell wall components by altering growth conditions [e.g., by changing media composition and morphology, incubation temperature, lighting, or humidity (6, 76)] or by genetic engineering (77) allows for the bottom-up control of the macroscopic me- chanical properties of the produced material. In addition, the choice of feedstock can influence the composite properties if nondegraded feedstock remains within the final composite (70, 78). Finally, the processing method used to form mycelium-based materials enables further property tuning (70). 4.2.1. Fungal foams. Joshi et al. (6) tuned the properties of composite fungi foams by changing the feedstock material on which mycelia of Pleurotus ostreatus were grown. Their results showed that pure sawdust and mixed sugarcane–sawdust substrates lead to foams with the highest den- sity and strength, 0.3 g/cm3 and 6.7–7.5 MPa, respectively. The high lignin content of sawdust, especially in comparison with other commonly used substrates such as rapeseed straw (79) and 94 Campbell et al. sugarcane bagasse (80), may cause the higher performance of the composite foams. Elsacker et al. (78) examined the effects of varying the feedstock form (loose, chopped, dust, precom- pressed, or tow forms were tested) on the mechanical properties of Tinea versicolor composite Organomineraliz- foams. Precompressed substrates lead to significantly higher compressive moduli (ranging from ation: the 1.1 to 1.4 MPa), compared with loose natural fibers, which consistently lead to softer foams (com- precipitation of pressive modulus from 0.1 to 0.6 MPa) for all types of substrates. In composites of Basidiomycota minerals, particularly sp. mycelium with wood particles, Sun et al. (81) showed that when the mycelium is first grown CaCO3 , facilitated by dead biological matter in culture media and subsequently mixed with wood particles, the produced composite is softer rather than living cells (Young’s modulus decreases from 225 to 40 MPa) and weaker (strength decreases from 1.20 to (cf. biomineralization) 0.5 MPa) compared with the same species grown directly on wood particles. Moreover, the use of CNFs at low concentrations (2.5 wt%) substantially improves the mechanical performance (mod- ulus increases from 225 to 660 MPa, and strength increases from 1.2 to 3.5 MPa) by enhancing the bonding between the wood particles and mycelium via extensive H-bonding. Highlighting the influence of the processing method, Appels et al. (70) showed that the elastic modulus and flexural strength for hot-pressed composites of P. ostreatus and Trametes ochracea/multicolor are 5–35 and 3–8 times higher than unpressed and cold-pressed foams, respec- tively, under all tested conditions (Figure 5c). Alternatively, Haneef et al. (76) investigated the influence of the culture medium on the mechanical performance of pure mycelium, without a feedstock residing within the final product. They found that adding potato dextrose broth to a pure cellulose substrate altered the macromolecular makeup of hyphal cell walls. These composi- tional changes caused the Young’s modulus and extensibility of Ganoderma lucidum films to change from 12 to 4 MPa and 14% to 33%, respectively, owing to the plasticizing effects of lipids. Appels et al. (77) demonstrated a drastic improvement on the mechanical performance of genetically al- tered Schizophyllum commune mycelium foams by modifying light and CO2 levels during growth. The genetically modified foams (sc3) cultured in high light and high CO2 flow reached an elastic modulus of 2.73 GPa and a strength of 40.4 MPa (compared with 0.91 GPa and 9.5 MPa, respec- tively, for unaltered foams). The sc3 gene deletion in S. commune resulted in the highest reported density and mechanical properties among all pure mycelium films. The higher thermal stability results for sc3 suggest that the comprising biopolymers are either more crosslinked or of higher molecular weight in the case of the mutant. These and other recent successes have encouraged rapid commercialization of mycelium-based materials as demonstrated by 47 patents filed from 2009 to 2018 (82). 4.2.2. Fungal inks. Additive manufacturing (AM) can further expand the use of mycelium- based composites (83). Successful demonstrations of 3D-printed fungal materials rely on printing a mixture of growth media and cells as shown in Figure 5d. The same constraints of traditional AM are present in mycelium printing, although the need to preserve cell viability through the control of feedstock type, morphology, distribution, and porosity adds an extra layer of difficulty (84, 85). Burry et al. (71) showed that inter- and intralayer adhesion in mycelium prints can be improved by a cell-secreted extracellular polymeric substance that bonds and physically interlocks with the lignocellulosic substrate, as mentioned in the previous sections. 4.2.3. Fungi in cementitious materials. Despite bacterial biomineralization being primar- ily utilized in self-healing concrete, challenges such as the limited lifetime of bacterial spores (