*1.1. Honeybee/Hive as a Model Organism/Habitat*

Honeybees are a semi-domesticated animal species: there still are wild colonies thriving in forests or urban areas without human interference [15]. Their natural nests are mostly found in tree cavities that differ from the artificially made habitats concerning their microclimates and microbiomes. Cavities that are large enough for honeybee colony preference (25 L to 40 L), have potentially been formed, thus being occupied by a variety of species for hundreds of years. Such cavities are incrementally formed by various types of other organisms. Mostly initiated with wood rot fungi, other micro and macro-organisms such as invertebrates continue colonising depending on the microclimatic conditions established as a result of the location and sizes of the cavities [16]. On top of pre-existing multispecies communities in these cavities, honeybee colonies continually add diverse types of organic and inorganic materials by collecting particles. These particles range from the size of fungal spores, pollen grains, road and coal dust, and sawdust, up to dead wood [17]. Chlorella alga is one example of an organic material beneficial to honeybees, which they bring to their nest, and which provides nutritional benefits. Researchers discovered that when colonies had access to foraging lands with this alga, they produced more honey than when they moved to a location without it [18]. Forager bees have also been found digging in soil and cattle dung [19] but the reason—if there is one—has not been identified yet.

Honeybee colonies exhibit community-level immunity that also includes the nest material which is essentially a microhabitat for their beneficial symbionts. Resin use and propolis provide self-medication for honeybee colonies with their antiviral, antibacterial, and antifungal properties [20]. Propolis is collected plant resins, mixed with saliva, and wax that the bees use to coat the inner nest surfaces [21]. Beeswax in honeybee nests does not support microbial growth. However, it can be a bioindicator of environmental toxins and colony health: particulates such as larval faeces, shed exuviae, lipophilic chemicals and environmental toxins have been found in beeswax [22]. Pollen is generally the medium in which foragers bring nutrition but also environmental hazards (chemical and biological) to their nests. Additionally, studies have shown that mycotoxin-producing moulds and

yeasts that ferment pollen into digestible bee bread, thrive in the conditions in which pollen is stored in the hive [23,24]. Findings on such microbial dependencies advise against the indiscriminate use of pesticides in agriculture. In the case of honeybee colonies, even if the substances do not affect bees directly, they still may be harmful to the microorganisms they essentially need to thrive. It is important to note that determining the microbial communities and their interactions with other organisms or other communities is a complex ecological and technological study. moulds and yeasts that ferment pollen into digestible bee bread, thrive in the conditions in which pollen is stored in the hive [23,24]. Findings on such microbial dependencies advise against the indiscriminate use of pesticides in agriculture. In the case of honeybee colonies, even if the substances do not affect bees directly, they still may be harmful to the microorganisms they essentially need to thrive. It is important to note that determining the microbial communities and their interactions with other organisms or other communities is a complex ecological and technological study. Honeybee nests differ significantly from other social insect nests, as the only adapta-

medium in which foragers bring nutrition but also environmental hazards (chemical and biological) to their nests. Additionally, studies have shown that mycotoxin-producing

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Honeybee nests differ significantly from other social insect nests, as the only adaptability found in bees' nest building is the way they fill pre-given cavities with their combs. They cannot (re-)shape the cavity itself. In contrast to that, termites and ants can dynamically alter their nest enclosures. Still, honeybees can actively regulate the climate and maintain homeostasis within their "prefabricated" nest enclosures. They fill the cavity adaptively as an efficient movement and storage platform for specific behaviour such as fanning, heating, clustering, etc. [25]. Inhabiting a structured but rather static nest topology requires high connectivity between different functional areas, for example efficiently connecting transport paths between the nectar handover area—in the front the near the entrance—and the honey storage area in the back of the hive. The duration of nectar-storing trips between these areas is one of the key regulatory types of feedback of honeybee foraging, ultimately affecting the colony's pollination activity [26–28]. This is just one out of many prerequisites to be considered in a colony's "hive architecture". The ventilation and gas exchange should support a fluent transition from a colony's foraging season mode to the colony's winter mode and vice versa. However, again, honeybees are highly adaptive and resilient, they flourish in a variety of habitats given the warmth and darkness. Nature-inspired designs imitating or literally being tree trunks have been adopted by beekeepers and designers throughout history, employing a broad range of regionally and organically derived materials (Figure 1a–e). Belgian artist Annemarie Maes uses microorganisms, digital design, and fabrication to connect advanced technology with a living biosystem such as a beehive [29]. There are also projects that use mycelium-grown materials as part of beehive enclosures, but these ideas have never been tested against the risks of long-term colony habitation [30,31]. bility found in bees' nest building is the way they fill pre-given cavities with their combs. They cannot (re-)shape the cavity itself. In contrast to that, termites and ants can dynamically alter their nest enclosures. Still, honeybees can actively regulate the climate and maintain homeostasis within their "prefabricated" nest enclosures. They fill the cavity adaptively as an efficient movement and storage platform for specific behaviour such as fanning, heating, clustering, etc. [25]. Inhabiting a structured but rather static nest topology requires high connectivity between different functional areas, for example efficiently connecting transport paths between the nectar handover area—in the front the near the entrance—and the honey storage area in the back of the hive. The duration of nectar-storing trips between these areas is one of the key regulatory types of feedback of honeybee foraging, ultimately affecting the colony's pollination activity [26–28]. This is just one out of many prerequisites to be considered in a colony's "hive architecture". The ventilation and gas exchange should support a fluent transition from a colony's foraging season mode to the colony's winter mode and vice versa. However, again, honeybees are highly adaptive and resilient, they flourish in a variety of habitats given the warmth and darkness. Nature-inspired designs imitating or literally being tree trunks have been adopted by beekeepers and designers throughout history, employing a broad range of regionally and organically derived materials (Figure 1a–e). Belgian artist Annemarie Maes uses microorganisms, digital design, and fabrication to connect advanced technology with a living biosystem such as a beehive [29]. There are also projects that use mycelium-grown materials as part of beehive enclosures, but these ideas have never been tested against the risks of long-term colony habitation [30,31].

**Figure 1.** Diversity of man-made beehives and a natural honeybee nest. (**a**) Mediaeval bee haven found in Rosslyn Chapel, Scotland. The north-facing side of the pinnacle when bees are returning to their haven in 2015. (**b**) Fossilised bees' nest within the pinnacle of Rosslyn Chapel. Photo Credit: Rosslyn Chapel Trust. (**c**) An apiary of stacked mud hives in central Egypt. Photo Credit: Gene Kritsky. (**d**) Bee bole embedded in a historic cottage, UK. cc-by-sa/2.0 by Oast House Archive. Source: https://www.geograph.org.uk/photo/1296874 (accessed on 8 February 2022). (**e**) A handwoven basket hive, coated with ash and cobb, photo taken Cine Beekeeping Museum, Aydin, Turkey. (**f**) Urban beehives near an industrial area, 2021, Graz, Austria. (**g**) A feral honeybee nests. **Figure 1.** Diversity of man-made beehives and a natural honeybee nest. (**a**) Mediaeval bee haven found in Rosslyn Chapel, Scotland. The north-facing side of the pinnacle when bees are returning to their haven in 2015. (**b**) Fossilised bees' nest within the pinnacle of Rosslyn Chapel. Photo Credit: Rosslyn Chapel Trust. (**c**) An apiary of stacked mud hives in central Egypt. Photo Credit: Gene Kritsky. (**d**) Bee bole embedded in a historic cottage, UK. cc-by-sa/2.0 by Oast House Archive. Source: https://www.geograph.org.uk/photo/1296874 (accessed on 8 February 2022). (**e**) A handwoven basket hive, coated with ash and cobb, photo taken Cine Beekeeping Museum, Aydin, Turkey. (**f**) Urban beehives near an industrial area, 2021, Graz, Austria. (**g**) A feral honeybee nests. Source: https://forum.canberrabees.com/t/mount-taylor-act-wild-feral-bee-hive-in-a-tree/309 (accessed on 8 February 2022).

### *1.2. Fungal Biofabrication*

In nature, fungi take the role of primary microbial decomposers, meaning they decompose material in the world's ecosystems. However, fungi can also be used not only to decompose but also to compose new structures. Mycelium is the vegetative growth of filamentous fungi that bonds organic matter through a network of hyphal microfilaments and it is currently a competitor of several synthetic materials [32]. There are two main types of mycelium-based materials: pure mycelium materials and mycelium-based composites. Pure mycelium materials are generally used and studied for smaller-scale applications such as paper or textile making [33,34] and biomedical applications such as wound healing [35] and tissue engineering [36]. Mycelium-based composites are made by growing the mycelium homogeneously in and around organic waste materials and are generally used for mesoscale modular applications such as bricks [37], thermal insulation, or acoustic panels [38,39], and low-value materials such as packaging [40]. Mycelium materials can be compared to one of the oldest types of composite materials, cob. Cob has organic fibrous material reinforcement, such as straw, which is bound by subsoil. In mycelium composites, instead of the extracted soil, mycelial hyphae are the fully organic and naturally grown binders. This means, if not treated additionally, the whole yielding material is organic. One can also grow these materials with zero waste and tune them to be mechanically intact, thermally insulative and 100% biodegradable. These properties highly depend on the growth factors including the type of lignocellulosic substrate base selected, fungal strains, and climatic control of the growth medium [41]. On the downside, an increased natural degradability means faster degradation or decay, a feature that is usually avoided in traditional building materials. Thus, the use of these materials demands solving additional challenges, e.g., repair or replacement regimes, or finding specific use cases, where such dynamics are not detrimental or even desired.

During the degradation processes, mycelial fungi's metabolic activities lead to selfhealing [42], beneficial volatile production, and detoxification. However, especially inside buildings with no sufficient natural air ventilation and humidity control, competing bacteria and fungi can harm the mechanical structure of the mycelium. Additionally, mycelium can produce mycotoxins and sporulate which can harm its co-inhabitants. As a result, prior to use, mycelium biofabrication methods and design application scenarios include desiccation of the mycelium biomass.

To date, several biofabrication technologies have been developed to achieve desired shapes and functionalities in mycelium materials. To visually measure growth, the simplest, least complicated, and most generally used approach is to cast a mixture of organic substrate and mycelium inoculum into premade moulds, usually translucent plastic enclosures [43]. This method works effectively for specific design scales where the end product can be grown uniformly. Biofabrication techniques, on the other hand, that specify and generate mycelia's growth boundaries using computational design and digital fabrication tools, can allow for local variation in material qualities and result in more complex geometries. Textiles can be used to define stay-in scaffolds for mycelium-based composites [44]. Textile logics can be translated into the filament scale, such as the structural stay-in scaffolds produced using the Kagome weaving method in the FUNGAR Project's building elements [45]. In another recent work, computationally generated scaffold morphologies have been 3D printed and inoculated via a robot arm equipped with sensors [46]. Furthermore, researchers and designers have been successful in 3D printing pre-inoculated viscous materials directly [47,48].

## *1.3. A Therapeutic Design Problem*

In this article, we propose a hybrid construction method for building more bioreceptive and bioactive beehives using living fungal mycelia formed via 3D printed stay-in scaffolds. Our main goal is to combine the thermally insulative properties of mycelia with its medicinal, potentially microbiome modulating properties. We use parametric design tools and fused deposition manufacturing to produce these mycelium scaffolds. Our hive

morphologies are designed aiming at honeybee colonies that self-organise similar to how they reside in hollow trees (Section 3.3) while reducing the energy loss of the hive. We used the quantitative and qualitative aspects of tall and narrow tree cavities as a design reference since the community-level immunity of honeybees has evolved in such environments. The main function of the overall hive morphology is to be durable supporting a full bee colony, living mycelium body and against changing weather conditions. This "therapeutic design problem" is a challenging task in creating and testing artificial habitats. It requires setting up empirical experiments to study one-to-one scale hive designs, to compare morphologies that are successfully occupied long-term (minimum one year) and both by honeybees and mycelia.

The design of therapeutic inner nest environments starts with a bioreceptive strategy for the overall morphology. There are two layers of bioreceptivity to be considered in fungal architectures. First is the receptivity (to mycelia) strategy used in designing the overall morphologies. The overall morphology of the fungal construct is primarily the morphology of the reusable or sacrificial formworks in/on which the mycelia grow. This first layer affects the second layer, which is the receptivity (to any other microorganisms and insects) of the pre-established mycelium arising from variation in surface qualities, or density differentiation throughout mycelial volumes. For functional applications, the second layer of bioreceptivity is minimised when the mycelium is heated and desiccated after its dense network formation. However, our goal with therapeutic mycelial beehives includes both layers of bioreceptive design. First is to enable the release of beneficial fungal compounds towards the hive interior. This is only possible with a living—not necessarily growing—mycelium structurally supported by 3D printed stay-in scaffolds. This approach can be a counteraction to the modern beekeeping sector. The modern beehives fall behind in terms of design characteristics that affect the climate conditions that are most relevant for honeybees and their symbionts. For example, one study shows that tree cavities provide better humidity levels compared to traditional modular box hives [48].

A targeted approach to therapeutic design problems can be about designing with specific and known medicinal properties of some fungi. Recent research shows that fungus species with antiviral and antibacterial compounds modulating microbial communities that are beneficial to humans can also boost the immune system of honeybees against specific viruses and bacteria when fed to the bees [49–51]. For embedding such properties in the enclosure material by growing specific fungal strains into hive morphologies the mycelia need to be kept alive during the honeybee colony inhabitation. In a targeted case like this, the challenge is to find a way to match the environmental conditions—temperatures, humidity, pH and oxygen levels—of the fungal habitats with those of the harmful organisms. For instance, a directed evolution strategy is used to breed an entomopathogenic fungus *Metarhizium brunneum.* This fungus is known to inhibit the growth of a honeybee pathogen Varroa Destructor, but naturally lives in lower temperatures, so it is bred to thrive in mostly affected brood areas in the hive (avg. 35 ◦C) [52]. Genetic modification or breeding of mycelial fungal species are interesting for these applications. In addition to what mycelia can do for honeybees, one example of how honeybee activities might support mycelial life can be the propolis enrichment of habitats. It has been shown that propolis can be used as a growth supplement for decreasing contamination risk in the mushroom production industry [53].

Moreover, novel therapeutic properties of mycelial habitats for honeybees might lay hidden in plain sight, as microorganisms embedded in the construction material may be able to perform certain beneficial support functions for the honeybee collective. However, these properties and abilities may well depend on the environmental conditions they are growing in the microclimate of the hive, which is a special ecological niche for microorganisms, precisely controlled by the bees but still affected by the environmental conditions of the hive surrounding. This creates the main aspect of complexity that apply to the case of fungal biodesign for social animals. A circular feedback loop is in effect here, as the bees control the inner nest climate to a high extent, affecting the microbial communities, which

in turn can affect the bees in return. The mycelium hive body with a large number of fungal cells also has an impact on the inner climate when they respire, degrade and regenerate. Even though this might not alter the inner hive climate as much as or as rapidly as the honeybee colony can, it would trigger the highly sensitive bees to more actively regulate the inhive climate in a homeostatic state. This would in turn affect the mycelium's morphology, molecular composition, and survival.

### **2. Methods**

### *2.1. Bio (Material) Coupling*

To investigate the interactions between honeybee nest-related materials, honeybee pathogens, and mycelial fungi, we employ methods from classical microbiology in-lab and honeybee behavioural biology on-field. For measuring the therapeutic properties of mycelia, we set up microbiological assays commonly used in insect immunity studies such as the lytic zone assay. These assays are used to measure the ability of any substance to break apart bacterial cell walls. Bioassays are controlled experiments that are commonly used to assess the potency of a bioactive agent—in our case, living or inactive mycelium—as inhibitors of pathogenic microorganism growth in comparison to standard measures. These experiments are typically designed in such a way that the environmental conditions are suitable to the pathogenic matter whose growth the test matter is hypothesised to inhibit. In addition to the inhibition assays, we make Petri dish experiments where a selection of organic nest matter—propolis, wax, pollen, honey, bee bread, etc. —are placed next to a mycelium patch and incubated at temperatures in which the nest materials would exist naturally. For coupling the living bees and our hive material designs, we make field experiments with full sized bee colonies. In these experiments, the prior criterion for the mycelium material is the selected fungus species being non-pathogenic to humans, bees, and plants, as well as to the local ecosystem.

### *2.2. Bio (Scaffolding) Design*

We developed a design-to-fabrication methodology (digital design tailored for additive manufacturing) to make structural and nutritional scaffolds in/on which mycelia attach and grow. This method promotes a homogenous and fast growth of the selected fungal matrix (mycelium of fungus species and strain). In our case, we used fused deposition modelling, a sub-caste in the 3D printing family holds forth the promise of "digital craft" [54]: a set of topological and geometrical operations to produce patterns as continuous extrusion paths and overall morphologies which at the end are represented as a set of instructions for the 3D printer.

**Topological Operations:** Our current toolpath drawing pattern is based on radial or mesh hexagonal grid topologies. Following a continuous weaving sequence of hexagonal cell control points, one polyline is formed. We call it "continuous weaving" because the toolpath is not interrupted, extrusion is continuous. The deposited material is like a continuous thread (Figure 2).

**Geometrical Operations:** In the first digital method, which we call the "variable-offset (VO)" method, multiple values are used to offset the contour lines of the user-specified geometry (distances), defining the outer boundary as well as the inner material density with consideration of the spaces for mycelium inoculate. The spaces between the offset lines are then intertwined using hexagonal weaving (Figure 2A). The initial geometries can be created with the intuitive top-down control of parameters. From there on a clusteroriented Genetic Algorithm, using the Biomorpher [55] plug-in for Grasshopper3D [56] rapidly explores a confined design space inside a parametric design model and provides interim 3D representations of design varieties. With an interactive evolution design tool, it is possible to rapidly generate morphological varieties while dealing with competing quantitative factors, such as the sizes of spaces required for the occupation of different scales of organisms and the time/material needed for 3D printing [57,58].

method.

**Figure 2.** Topological operations for continuous toolpath drawing. (**A**) Hexagonal weaving. (a) Point bonding areas. (b) Linear bonding area. (c) Drape zone. (**B**) Density differentiation via the VO method. **Figure 2.** Topological operations for continuous toolpath drawing. (**A**) Hexagonal weaving. (a) Point bonding areas. (b) Linear bonding area. (c) Drape zone. (**B**) Density differentiation via the VO method. tive optimisation process using finite element functions. Density distribution throughout this voxelised space is defined in accordance with benchmarks, which in our fungal hive

(**A**) (**B**)

**Figure 2.** Topological operations for continuous toolpath drawing. (**A**) Hexagonal weaving. (a) Point bonding areas. (b) Linear bonding area. (c) Drape zone. (**B**) Density differentiation via the VO

**Geometrical Operations:** In the first digital method, which we call the "variable-off-

set (VO)" method, multiple values are used to offset the contour lines of the user-specified geometry (distances), defining the outer boundary as well as the inner material density

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**Geometrical Operations:** In the first digital method, which we call the "variable-offset (VO)" method, multiple values are used to offset the contour lines of the user-specified geometry (distances), defining the outer boundary as well as the inner material density with consideration of the spaces for mycelium inoculate. The spaces between the offset lines are then intertwined using hexagonal weaving (Figure 2A). The initial geometries can be created with the intuitive top-down control of parameters. From there on a clusteroriented Genetic Algorithm, using the Biomorpher [55] plug-in for Grasshopper3D [56] rapidly explores a confined design space inside a parametric design model and provides interim 3D representations of design varieties. With an interactive evolution design tool, it is possible to rapidly generate morphological varieties while dealing with competing quantitative factors, such as the sizes of spaces required for the occupation of different scales of organisms and the time/material needed for 3D printing [57,58]. The second digital method, "iterative-subtraction (IS)", allows for the distribution of voxels in 3D space according to specified load and support requirements. This is an iterative optimisation process using finite element functions. Density distribution throughout this voxelised space is defined in accordance with benchmarks, which in our fungal hive case are drawn in consideration of honeybee occupancy loads, spatial layouts for its landing zone, protected spaces or dark zones, and minimisation of material used for production [59]. The voxels are then replaced with hexcells and translated into a toolpath with the hexagonal weaving drawing method (Figure 3). Numerous toolkits can significantly assist in the topology design process in Grasshopper3D, we used Millipede [60]. This mode of operation yields a material microstructure with uniform porosity. So, if needed, geometrical attractors—lines or points—in the parametric hive model can be used to achieve density gradients. We previously presented this application of topology optimisation in a speculative design and construction scenario for multispecies architectural boundaries, the *Co-occupied Boundaries* Project [61]. case are drawn in consideration of honeybee occupancy loads, spatial layouts for its landing zone, protected spaces or dark zones, and minimisation of material used for production [59]. The voxels are then replaced with hexcells and translated into a toolpath with the hexagonal weaving drawing method (Figure 3). Numerous toolkits can significantly assist in the topology design process in Grasshopper3D, we used Millipede [60]. This mode of operation yields a material microstructure with uniform porosity. So, if needed, geometrical attractors—lines or points—in the parametric hive model can be used to achieve density gradients. We previously presented this application of topology optimisation in a speculative design and construction scenario for multispecies architectural boundaries, the *Co-occupied Boundaries* Project [61].

assist in the topology design process in Grasshopper3D, we used Millipede [60]. This mode of operation yields a material microstructure with uniform porosity. So, if needed, **Figure 3.** IS method. From left to right: density distribution in voxel space using Millipede, isomesh visualisation, and hexagonal weaving for voxel definition. **Figure 3.** IS method. From left to right: density distribution in voxel space using Millipede, isomesh visualisation, and hexagonal weaving for voxel definition.

the hexagonal weaving drawing method (Figure 3). Numerous toolkits can significantly

**Figure 3.** IS method. From left to right: density distribution in voxel space using Millipede, isomesh

geometrical attractors—lines or points—in the parametric hive model can be used to achieve density gradients. We previously presented this application of topology optimisation in a speculative design and construction scenario for multispecies architectural boundaries, the *Co-occupied Boundaries* Project [61]. Using our design approach, we are able to provide more direct lines of communication between the digital geometries, machine parameters and physical model. Finally, we may also adjust both surface and inner porosities of the scaffold based on performance parameters for temperature and humidity control. Because it enables structural design with physics calculation, the IS technique may be more beneficial for designing overall structures capable of housing a beehive colony in an elevated position above the ground-similar to forest habitats in large trees.

### *2.3. Biofabrication 2.3. Biofabrication*

similar to forest habitats in large trees.

Depending on the material and microstructure resolution defining the overall morphology, we use different fungal inoculation methods. In what we call the "infill-feed" method, we manually fill the vertical tubular cells with mycelium inoculated solid substrates (Figure 4a). In the second method, the "self-feed" method, the liquid fungal culture is poured directly onto the printed scaffold. In this case, the printed scaffolds should have higher grid resolution and therefore must provide enough nutrition for the mycelia, nutrition providing surface area for mycelium growth (Figure 4b). Depending on the material and microstructure resolution defining the overall morphology, we use different fungal inoculation methods. In what we call the "infill-feed" method, we manually fill the vertical tubular cells with mycelium inoculated solid substrates (Figure 4a). In the second method, the "self-feed" method, the liquid fungal culture is poured directly onto the printed scaffold. In this case, the printed scaffolds should have higher grid resolution and therefore must provide enough nutrition for the mycelia, nutrition providing surface area for mycelium growth (Figure 4b).

Using our design approach, we are able to provide more direct lines of communication between the digital geometries, machine parameters and physical model. Finally, we may also adjust both surface and inner porosities of the scaffold based on performance parameters for temperature and humidity control. Because it enables structural design with physics calculation, the IS technique may be more beneficial for designing overall structures capable of housing a beehive colony in an elevated position above the ground-

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**Figure 4.** Artefacts produced with different methods. (**a**) "Infill-feed". (**b**) "Self-feed". **Figure 4.** Artefacts produced with different methods. (**a**) "Infill-feed". (**b**) "Self-feed".

### **3. Experiments 3. Experiments**

### *3.1. Materials*

*3.1. Materials*  The surface area of the nutritional matter that a fungus can attach to is an important factor that influences hyphae breakdown of specified material. It can be increased, or controlled, by using extrusion of lignocellulose rich substances. First, we took a preliminary investigation into a wide range of manufacturing parameters that affect the crucial geometrical attributes necessary for the growth of mycelium. In a continuous deposition, variables like print head speed and distance from the previously printed layer greatly impact the thickness of extruded lines, thus the surface area of the organic molecules on which fungal cells can attach. An experimental composite filament (GrowLay™) [62] proved to be the best candidate in our commercially available polymeric material assortment. GrowLay™ is made of cellulose particles, polyvinyl alcohol or PVA which is a water-soluble synthetic polymer and another backbone polymer –that the producer does not prefer sharing. Once the PVA is removed, the printed structures retain microcapillaries and increased surface area of the cellulose. Using the "self-feed" method, we prepared samples with TV hyphae growing on GrowLay™ and *T. Versicolor* (TV) hyphae growing on lignin infused polylactic acid (lignin PLA). In Figure 5, we demonstrated our observation of TV hyphae growth on these two 3D printing materials using microscopic scanning technology. As foreseen, hyphae could grow across the GrowLay™ material (Figure 5), which remains with microcapillaries after the removal of the water-soluble polymer. We also explored paper clay as a stay-in scaffold material [63] and clay extrusion to build. When compared to synthetic and engineered bioplastics, clay printing comes with its own set of challenges due to its organic nature. Therefore, we first experimented with the toolpaths and the 3D printing parameters in order to establish porous boundaries which are able to The surface area of the nutritional matter that a fungus can attach to is an important factor that influences hyphae breakdown of specified material. It can be increased, or controlled, by using extrusion of lignocellulose rich substances. First, we took a preliminary investigation into a wide range of manufacturing parameters that affect the crucial geometrical attributes necessary for the growth of mycelium. In a continuous deposition, variables like print head speed and distance from the previously printed layer greatly impact the thickness of extruded lines, thus the surface area of the organic molecules on which fungal cells can attach. An experimental composite filament (GrowLay™) [62] proved to be the best candidate in our commercially available polymeric material assortment. GrowLay™ is made of cellulose particles, polyvinyl alcohol or PVA which is a water-soluble synthetic polymer and another backbone polymer –that the producer does not prefer sharing. Once the PVA is removed, the printed structures retain microcapillaries and increased surface area of the cellulose. Using the "self-feed" method, we prepared samples with TV hyphae growing on GrowLay™ and *T. Versicolor* (TV) hyphae growing on lignin infused polylactic acid (lignin PLA). In Figure 5, we demonstrated our observation of TV hyphae growth on these two 3D printing materials using microscopic scanning technology. As foreseen, hyphae could grow across the GrowLay™ material (Figure 5), which remains with microcapillaries after the removal of the water-soluble polymer. We also explored paper clay as a stay-in scaffold material [63] and clay extrusion to build. When compared to synthetic and engineered bioplastics, clay printing comes with its own set of challenges due to its organic nature. Therefore, we first experimented with the toolpaths and the 3D printing parameters in order to establish porous boundaries which are able to hold the mycelium substrates and avoid layer collapses (Figure 6). However, we did not make a microscopic scan of clay mycelium samples.

make a microscopic scan of clay mycelium samples.

make a microscopic scan of clay mycelium samples.

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hold the mycelium substrates and avoid layer collapses (Figure 6). However, we did not

hold the mycelium substrates and avoid layer collapses (Figure 6). However, we did not

**Figure 5.** Autofluorescence of fungal hyphae (white) was captured with confocal laser-scanning microscopy and used for visualising the virtual slices of material. Left: TV mycelium and lignin PLA: Right: TV mycelium and GrowLay™. **Figure 5.** Autofluorescence of fungal hyphae (white) was captured with confocal laser-scanning microscopy and used for visualising the virtual slices of material. Left: TV mycelium and lignin PLA: Right: TV mycelium and GrowLay™. **Figure 5.** Autofluorescence of fungal hyphae (white) was captured with confocal laser-scanning microscopy and used for visualising the virtual slices of material. Left: TV mycelium and lignin PLA: Right: TV mycelium and GrowLay™.

**Figure 6.** Clay extrusion toolpath tests to find the hex-weaving parameters and overall geometry. The darker red colour shows the final iteration which we used to 3D print the whole clay scaffold. **Figure 6.** Clay extrusion toolpath tests to find the hex-weaving parameters and overall geometry. The darker red colour shows the final iteration which we used to 3D print the whole clay scaffold. **Figure 6.** Clay extrusion toolpath tests to find the hex-weaving parameters and overall geometry. The darker red colour shows the final iteration which we used to 3D print the whole clay scaffold.

For the large-scale prototypes, we used a large thermoplastic 3D printer, Reprap BIG, using the GrowLay™ filament described above. We refer to them as GrowLay Hive-1 and GrowLay Hive-2. The third and the most recent one was printed with clay, with a liquid deposition modelling printer, Delta WASP 40 100 Clay, and we refer to it as the Mycelial Clay Hive. Both printers were placed in room conditions in springtime without indoor For the large-scale prototypes, we used a large thermoplastic 3D printer, Reprap BIG, using the GrowLay™ filament described above. We refer to them as GrowLay Hive-1 and GrowLay Hive-2. The third and the most recent one was printed with clay, with a liquid deposition modelling printer, Delta WASP 40 100 Clay, and we refer to it as the Mycelial Clay Hive. Both printers were placed in room conditions in springtime without indoor For the large-scale prototypes, we used a large thermoplastic 3D printer, Reprap BIG, using the GrowLay™ filament described above. We refer to them as GrowLay Hive-1 and GrowLay Hive-2. The third and the most recent one was printed with clay, with a liquid deposition modelling printer, Delta WASP 40 100 Clay, and we refer to it as the Mycelial Clay Hive. Both printers were placed in room conditions in springtime without indoor climate control. These hives are described in more detail in Section 3.4.

climate control. These hives are described in more detail in Section 3.4. For measuring the fungal mycelia's potency as bioactive agents against potential honeybee pathogens, we used two bacterial players. *Micrococcus luteus* is a gram-positive bacterium commonly used in the initial stages of inhibition zone assays in insect immunology studies. For more targeted studies we used Paenibacillus larvae cells—the causative bacterium of deadly American Foulbrood (AFB) disease in honeybee colonies. Both agents were pre-initiated and grown to an active stage in agar Petri dishes. As fungal players in these assays, we used mycelia of five different species: *Trametes versicolor* (TV), *Pleurotus*  climate control. These hives are described in more detail in Section 3.4. For measuring the fungal mycelia's potency as bioactive agents against potential honeybee pathogens, we used two bacterial players. *Micrococcus luteus* is a gram-positive bacterium commonly used in the initial stages of inhibition zone assays in insect immunology studies. For more targeted studies we used Paenibacillus larvae cells—the causative bacterium of deadly American Foulbrood (AFB) disease in honeybee colonies. Both agents were pre-initiated and grown to an active stage in agar Petri dishes. As fungal players in these assays, we used mycelia of five different species: *Trametes versicolor* (TV), *Pleurotus*  For measuring the fungal mycelia's potency as bioactive agents against potential honeybee pathogens, we used two bacterial players. *Micrococcus luteus* is a gram-positive bacterium commonly used in the initial stages of inhibition zone assays in insect immunology studies. For more targeted studies we used Paenibacillus larvae cells—the causative bacterium of deadly American Foulbrood (AFB) disease in honeybee colonies. Both agents were pre-initiated and grown to an active stage in agar Petri dishes. As fungal players in these assays, we used mycelia of five different species: *Trametes versicolor* (TV), *Pleurotus ostreatus* (PO), *Ganoderma lucidum* (GL), *Hericium erinaceus* (HE), and *Grifola frondosa* (GF). However, for the targeted assays we mainly focused on the mycelium

*ostreatus* (PO), *Ganoderma lucidum* (GL), *Hericium erinaceus* (HE), and *Grifola frondosa* (GF).

*ostreatus* (PO), *Ganoderma lucidum* (GL), *Hericium erinaceus* (HE), and *Grifola frondosa* (GF). However, for the targeted assays we mainly focused on the mycelium of *Trametes versi-*

known for its anticancer ingredient Krestin (PSK, a protein-bound polysaccharide). It has

known for its anticancer ingredient Krestin (PSK, a protein-bound polysaccharide). It has

of *Trametes versicolor* (TV) which is a common mushroom producing polypore fungus and is commercially known for its anticancer ingredient Krestin (PSK, a protein-bound polysaccharide). It has also been found that Krestin is also a strong antibiotic against microbes pathogenic to humans. This and other medicinal compounds are present in the mycelium of TV. For the large-scale design prototypes, we used PO, GL, and TV mycelia. PO, also known as oyster mushroom, is a widely grown edible mushroom with a rapidly spreading mycelium that efficiently utilises substrate resources, making it a good material maker [64]. GL belongs to the *Ganoderma* species which is broadly studied and showed antiviral properties effective against honeybee deformed wing virus [49]. In general, our biological organism selection criteria were mostly about the honeybee related properties.
