**1. Introduction**

There is a growing interest in harnessing living systems in the fabrication of materials and structures. Biological systems are capable of self- assembling complex materials and composites in highly energy efficient ways. While we make use of the materials provided by nature after the organism that created them is dead, utilizing living process may offer new methods of material assembly. These methods, however, will also require novel design tools and a new understanding of the relationship between the designer and their materials.

A promising group of organisms for biological material fabrication is fungi. For example, fungi can be manipulated at various scales for different purposes, such as in leather form with similar texture to animal leather and as a binder for bulk material (as mycelium composites). It also can act as a functional material when it is still alive, to form networks for microorganisms (see Fiber Highways Project [1]), or as a sensor (see Fungal Architecture project [2]). Most of these projects utilize fungal mycelium; however, few design projects address the fruiting body. Unlike mycelium, the fruiting bodies of many fungus species exhibit complex morphologies and self-assemble without the 'scaffold' of a substrate or aggregate. While we tend to harvest the fruiting bodies as food, the morphological complexity and their sensitivity to environmental conditions, as well as their speed of growth make them especially suitable for studies on how biological systems fabricate complex forms and materials. To this end we provide an early study in which the fruiting bodies of a well-studied fungal species (*Pleurotus ostreatus*) are shown to be somewhat controllable given their sensitivity to key environmental parameters. They were used as a biomaterial probe to test the concept of biological parametrics [3].

Biomaterial probes are defined as experiments that are carried out on biological materials or fabrication strategies without designed goals, but which are used to understand the factors influencing a biological system [4]. As Ramirez-Figueroa explains, it focuses on design explorations which show how the practice of design is transformed and redefined by using living systems. Although mycelium was used as a material probe in the preliminary experiments, the main design experiments were conducted here using fruiting bodies [5].

**Citation:** Ozkan, D.; Morrow, R.; Zhang, M.; Dade-Robertson, M. Are Mushrooms Parametric? *Biomimetics* **2022**, *7*, 60. https://doi.org/10.3390/ biomimetics7020060

Academic Editors: Andrew Adamatzky, Han A. B. Wösten and Phil Ayres

Received: 15 March 2022 Accepted: 29 April 2022 Published: 10 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The goal of the design experiments was to intervene in the fruiting body formation of oyster mushrooms by altering the environmental factors for growth.

### *1.1. Biological Parametrics*

Parametric design is a broad concept that connects data to the design of form and structure. Often synonymous with generative design, the role of the designer in a parametric design process is not to design the form of the object or system directly but rather to define the key controlling parameters and their relationships [6]. In Architecture, parametric design is often associated with the development of complex organic forms derived from initial conditions created by, for example, site mappings or simulations of use and function [7,8].

There is an analogy between parametric and biological processes in that in many examples of biological growth, especially in plants and fungi, the form of the organism is often, in part, derived from an interaction with environmental factors, including, access to sunlight and nutrients, physical constraints and barriers and interaction with other organisms. To some extent we already intervene in these biological processes in agriculture. A tomato, cultivated in the highly controlled, nutrient rich environment of a greenhouse, for example, could be described as 'parametrically designed'. Refined crafts such as bonsai tree growing are also examples of intervening in biological growth with specific forms in mind. The Bonsai tree is produced through direct and 'coercive' control through the 'directing' of branches and the severe limitations of nutrients to keep the trees in dwarf form. In design terms this cultivation approach is more akin to direct control than parametric design, which implies a separation between the intervention (through data) and the generated design outcome.

Our research into fruiting bodies (of *P. ostreatus* known as *oyster mushrooms*) has, however, suggested that, for certain biological systems, a parametric approach to their 'design' and cultivation may be possible. To this end the paper will introduce the concept of biological parametric design as a fabrication strategy through design experiments which investigate the relationship between environmental parameters and fungal fruiting body morphology.

### *1.2. Plasticity*

While it is often stated that DNA is the 'blueprint of life', biological systems are only partially shaped by the information contained in genes. Biological systems are subject to epigenetic influences i.e., environmental conditions which will cause genes to activate or not [9]. This relationship between phenotype and environment is sometimes referred to as plasticity and can be measured in terms of the degree of variation between organisms given the same genome [10]. Plasticity is exhibited at different stages of an organism's life. Here, however, we will focus on developmental plasticity of mushroom fruiting bodies which lead to a variation in morphogenesis and final form. The concept of plasticity implies a pliability of developmental processes which may, we suggest, enable human intervention in direct parametric control.

As Dade-Robertson discusses, these indirect methods of affecting a living material through environmental parameters use "nature's own agencies" without human imposition through "forcible constraints" such as cutting and molding the organism, or genetic manipulation [10]. A question remains, however, as to what degree this plasticity is amenable to a parametric approach. Biological systems and processes often exhibit non-linear behavior with, for example small changes in environmental conditions creating tipping points and leading to developmental outcomes that are not easy to attribute to single or limited sets of parameters and/or where the same effect does not always cause the same results [11]. An organism growing under exactly the same environmental conditions can form different morphologies. It is the non-linear behavioral pattern of the living materials that leads to an abundance of variations in the final product. Biological systems are also subject to noise and exhibit cell-to-cell variation and emergence where outcomes are not easily reducible

to the behavior of parts. This biological complexity, therefore, challenges a parametric approach, and at the same time requires designers to have deep knowledge about the biological materials and bioprocess for the fabrication of the materials. Designers need to explore the value ranges and tipping points where the organism presents a linear change, so (if applicable) they can apply parametric design principles.

## *1.3. Prior Work*

There are a number of notable precedents for a parametric approach to fabricating with biological systems outside the context of fungus. The works of Jiwei Zhou et al., Thora Arnardottir et al., and Neri Oxman et al., using plant roots, bacteria, and silkworms, respectively, show approaches to influence the environmental conditions of living organisms to achieve a desired material [9–11]. For instance, Arnodottir uses urease producing bacteria to calcify sand, creating cemented columns of material, without including digital tools to control the parameters [12]. By altering the cast sizes, inlet positions for nutrients and reactants, she shows that parameters which affect biological growth can be influenced. The influence of the parameters can be predicted while creating cast materials and the final form of the cemented columns does not have to be dictated by the shape of the cast. More complex forms emerge because of the interaction of these biological and environmental factors. In the case of the Silkworm Pavilion-II project by Oxman et al., they guide silkworms to cover the woven surface of the pavilion [13]. The distribution of the silkworms was controlled by heat, gravity, and light as variables. Since environmental conditions were directly linked to silk production, they could spread the fibers homogenously as they intended. Zhou et al., uses plant roots to test digital biofabrication strategies for product design purposes [14]. They fabricate self-supported 3D structures by altering the growth media, direction of gravity and porosity of their digitally fabricated mold. These variables allowed them to manipulate plant roots, since the nutritional richness and the force of gravity have an impact on the root growth [14].

In each example above, designers initially define the environmental factors (in a parametric manner) as variables they can work with to manipulate the final outcome. In each case they have shown that, to some extent (within a value range) there is a somewhat predictable relationship between environmental parameters and specific material outcomes. The outcomes of these processes also exhibit variations, however, and this challenges notions of fabrication tolerances.

### *1.4. Focus*

This paper extends these works on biological parametric design by reporting four design experiments using fungal fruiting bodies. In each case the objectives are to find the environmental parameters responsible for different fruiting body morphologies and to see whether such morphologies can be predicted. The fruiting bodies of the selected fungus have the benefit of being complex, in terms of morphology but also plastic, in that they exhibit significant phenotypic variation given the same genetic information. They can also be grown quickly. These experiments seek to answer the question: To what extent is mushroom growth parametric?

### **2. Materials**

Oyster mushrooms (*P. ostreatus*) were used in this study because of their fast growth (compare to other species used in the design field such as *ganoderma resinaceum* and *trametes versicolor*) and the wide variety of known fruiting body morphologies due to their gastronomic use, indicating a high level of developmental plasticity [15]. In addition to the rapid growth rate and plasticity, fungal fruiting bodies possess totipotency. Totipotency describes the ability of a cell to divide and produce all the differentiated cells of an organism autonomously [16]. This means if even a tiny amount of mushroom tissue is transplanted onto a nutrient medium, it can initiate new growth [15]. Totipotency enables the harvested cells to be used as the basis for a new experiment.

In each experiment the mushroom growth followed a common and well described developmental pathway starting with the vegetative phase (hypha growth), which continued to the reproductive phase (fruiting body formation) (Figure 1) [17]. Hypha filaments transform to the fertile tissue of a fruiting body under suitable conditions. The organization of hyphae significantly changes while creating fruiting bodies. Normally, the filaments show positive autotropism by growing in an upwards direction; however while forming a fruiting body structure, they start to grow inwards and show negative autotropism [18]. This is due to hyphae forming a three-dimensional compound complex by interlocking with other hyphae structures, instead of simply forming an unconstrained mesh. The initial development of the fruiting body begins with a hyphal knot, which can be triggered by a disturbance such as an injury, edge encounter or changes in nutrient levels, temperature, or light exposure [18]. In the formation of *Basidiomycota* fungi, hyphae form knots by reducing their level of chitin and the knots become mushrooms by expansion and inflation of pre-existing hyphae. Depending on the species' phototropic requirements, the progress can proceed with the introduction of light that leads to cellular differentiation [17]. The formation of stipe (stalk), cap (pileus), and gill cells occur during this process, during which the mushroom takes on its characteristic appearance [15]. The spores are discharged from the surface of gills. Therefore, gills increase their surface area by folding, to allow the production of more spores. ganization of hyphae significantly changes while creating fruiting bodies. Normally, the filaments show positive autotropism by growing in an upwards direction; however while forming a fruiting body structure, they start to grow inwards and show negative autotropism [18]. This is due to hyphae forming a three-dimensional compound complex by interlocking with other hyphae structures, instead of simply forming an unconstrained mesh. The initial development of the fruiting body begins with a hyphal knot, which can be triggered by a disturbance such as an injury, edge encounter or changes in nutrient levels, temperature, or light exposure [18]. In the formation of *Basidiomycota* fungi, hyphae form knots by reducing their level of chitin and the knots become mushrooms by expansion and inflation of pre-existing hyphae. Depending on the species' phototropic requirements, the progress can proceed with the introduction of light that leads to cellular differentiation [17]. The formation of stipe (stalk), cap (pileus), and gill cells occur during this process, during which the mushroom takes on its characteristic appearance [15]. The spores are discharged from the surface of gills. Therefore, gills increase their surface area by folding, to allow the production of more spores.

**Figure 1.** The developmental path of the fungal reproductive phase. **Figure 1.** The developmental path of the fungal reproductive phase.

### **3. Methods**

**3. Methods** 

gravity [19].

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 4 of 18

the harvested cells to be used as the basis for a new experiment.

the rapid growth rate and plasticity, fungal fruiting bodies possess totipotency. Totipotency describes the ability of a cell to divide and produce all the differentiated cells of an organism autonomously [16]. This means if even a tiny amount of mushroom tissue is transplanted onto a nutrient medium, it can initiate new growth [15]. Totipotency enables

In each experiment the mushroom growth followed a common and well described

tinued to the reproductive phase (fruiting body formation) (Figure 1) [17]. Hypha filaments transform to the fertile tissue of a fruiting body under suitable conditions. The or-

### *3.1. Factors in the Morphogenesis of Mushrooms*

*3.1. Factors in the Morphogenesis of Mushrooms*  In previous literature it has been shown that different mushroom species adapt to the environment they occupy to maintain their life and chances of reproducing [19]. Mushroom morphology is connected to the transportation of spores, where the fungi adopt forms that optimize the diffusing of spores [15]. For instance, the umbrella shape of mushrooms comes from the upward development of stipes under the influence of light, In previous literature it has been shown that different mushroom species adapt to the environment they occupy to maintain their life and chances of reproducing [19]. Mushroom morphology is connected to the transportation of spores, where the fungi adopt forms that optimize the diffusing of spores [15]. For instance, the umbrella shape of mushrooms comes from the upward development of stipes under the influence of light, whereas the gills that diffuse the spores develop downward and are affected by gravity [19]. However, the umbrella shape can be changed by altering the direction of light and gravity [19].

whereas the gills that diffuse the spores develop downward and are affected by gravity [19]. However, the umbrella shape can be changed by altering the direction of light and The major factors that affect the form of mushrooms depend on the species. Bellettini et al. has conducted experiments which show the key parameters affecting the mushroom morphology of oyster mushrooms: air temperature, light, humidity, CO2 levels, gravity, substrate amount and size [20]. These factors influence the cap and stalk's shape, size, and surface finish of oyster mushrooms [21]. Therefore, in this study humidity, CO2 level, The major factors that affect the form of mushrooms depend on the species. Bellettini et al. has conducted experiments which show the key parameters affecting the mushroom morphology of oyster mushrooms: air temperature, light, humidity, CO<sup>2</sup> levels, gravity, substrate amount and size [20]. These factors influence the cap and stalk's shape, size, and surface finish of oyster mushrooms [21]. Therefore, in this study humidity, CO<sup>2</sup> level, gravitational force and substrate amount are used as variables to test the parametric qualities of mushrooms. Light duration and temperature are kept as constant values since we found across our interaction with fungi that they are more effective in initiating the mushroom formation rather than affecting mushroom morphology. High humidity environments provide favorable conditions for mushrooms to thrive in and bear fruit [18]. Different sources state that using 90–95% humidity or using 80–85% humidified culture room as well as spraying their fungi three times a day helps to achieve the optimal mushroom yield [20,22]. Stalk thickness tends to decrease

with the decrease in the level of humidity, since there is not enough water for mushroom development [18,23].

A change in CO<sup>2</sup> concentration also triggers different stages of the fungal life cycle and affects the morphology of mushrooms. During the development of mushrooms, respiration activity increases, so the preferred CO<sup>2</sup> level decreases. While the preferred CO<sup>2</sup> concentration is 2000–2500 mg/L for mycelium growth, it decreases to 1500–2000 mg/L for fruiting body development. If the CO<sup>2</sup> level remains high, the cap formation may not occur [20]. High CO<sup>2</sup> concentration blocks pileus formation while boosting stalk elongation because the cell wall is affected by elevated CO<sup>2</sup> levels [19].

Many mushroom stalks possess negative gravitropism [24] as the fruiting bodies grow in the opposite direction of gravity and bending of the stalk occurs at the upper region closest to the cap [19]. In the literature, the substrate mass has often been studied as it affects the size and number of mushroom blooming because of the impact on nutrient availability.

### *3.2. The Experimental Design*

From the literature above the effects of humidity, CO<sup>2</sup> levels, gravitational force and substrate amount were chosen as variables as these had the potential to have the most significant impact on mushroom morphology. To validate this decision, a series of experiments were conducted testing the effect of different conditions in isolation. The experiments were carried out during the COVID-19 period and hence some of the experimental setups were improvised around the available equipment and facilities.

Humidity and CO<sup>2</sup> levels in the experiments were controlled by a growth chamber that consists of an Arduino UNO (connected to a laptop), Arduino sensors (DHT11 air humidity and temperature sensor, SEN0219 infrared CO<sup>2</sup> sensor, V1.0 soil moisture sensor and HC-SP04 ultrasonic distance sensor) and devices (12V DC fan, humidifier, 450 nm LED blue light source and 75 watt heat bulb) [3]. The chamber also helped to keep temperature and light exposure stable. Only one variable was changed at a time and the others were kept constant for each experiment (Table 1).

**Table 1.** The variables used for four experiments.


Each set started with the same substrate ratios with 25% of strawbale, 25% of wood shavings, and 25% of coffee grounds. Straw was blended in a Nutri Ninja Blender & Smoothie Maker 900 W for 5 s to a homogeneous mixture. The wood shavings and coffee grounds were not blended since they already had uniform size. The substrates were prepared and sterilized in an autoclave at 121 ◦C for 15 min. This mixture was then seeded with 25% of oyster mushroom spawn (*P. ostreatus*) from GroCycle-UK, and sealed in (10 × 10 × 3 cm) plastic boxes, in the dark, at ambient temperature. The experiments ran for 29 days. After an initial three weeks of growth the samples were exposed to different environmental conditions for eight days in the growth chambers. All experiments were conducted in triplicate.

By altering the parameters incrementally across different experiments, as seen in Table 1, we were able to measure the scale effect of different environmental conditions and relate specific parameters with mushroom dimensions.

Variables

### 3.2.1. The Humidity Experiment 3.2.1. The Humidity Experiment

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 6 of 18

Humidity (%) 95 85 80 75 80 75 80 CO2 (ppm) 2000 1000 3000 5000 5000 300

Gravity (degree) 90° 90° 90° 135° 180° 90° Light (nm) 4 h, 450 nm 4 h, 450 nm 4 h, 450 nm 4 h, 450 nm Temp. (°C) 20–22 °C 20–22 °C 20–22 °C 20–22 °C

relate specific parameters with mushroom dimensions.

Sub. Amount (g) 55 55 55 40 80 120 160

**Table 1.** The variables used for four experiments.

The variable of humidity level was set to four different levels as discussed in our previous paper and seen in Table 1 [3]. The variable of humidity level was set to four different levels as discussed in our previous paper and seen in Table 1 [3].

By altering the parameters incrementally across different experiments, as seen in Table 1, we were able to measure the scale effect of different environmental conditions and

**Experiments [1] Humidity [2] CO2 [3] Gravity [4] Sub. Amount** 

### 3.2.2. The CO<sup>2</sup> Experiment 3.2.2. The CO2 Experiment

The variable of CO<sup>2</sup> level was set to three different levels as explained in our previous paper and seen in Table 1 [3]. The variable of CO2 level was set to three different levels as explained in our previous paper and seen in Table 1 [3].

### 3.2.3. The Gravity Experiment 3.2.3. The Gravity Experiment

In this experiment the angle of growth was tested. The effect of gravity upon the growing mushroom was adjusted as a means of support by using the aforementioned plastic containers. After being removed from the containers, the mycelium tiles were kept in 90◦ , 135◦ and 180◦ angles, as seen in Figure 2. The samples with 180◦ angles were positioned on a box. Lifting them prevented moistening and mushroom growth on the contact surface. In this experiment the angle of growth was tested. The effect of gravity upon the growing mushroom was adjusted as a means of support by using the aforementioned plastic containers. After being removed from the containers, the mycelium tiles were kept in 90°, 135° and 180° angles, as seen in Figure 2. The samples with 180° angles were positioned on a box. Lifting them prevented moistening and mushroom growth on the contact surface.

**Figure 2.** The positioning of the mushrooms in the gravity experiment. **Figure 2.** The positioning of the mushrooms in the gravity experiment.

The experiment was repeated under 2000 ppm CO2 level. In this way, it was possible to see the effect of gravity on caps in different sizes. The experiment was repeated under 2000 ppm CO<sup>2</sup> level. In this way, it was possible to see the effect of gravity on caps in different sizes.

### 3.2.4. The Substrate Amount Experiment 3.2.4. The Substrate Amount Experiment

In this set of experiments, the effect of substrate amount on mushroom size was tested. 40 g, 80 g, 120 g and 160 g mycelium and various substrates were mixed in the ratio of 25% of strawbale, 25% of wood shavings, 25% of coffee grounds, and 25% of mushroom spawn, as mentioned before. All mixtures were kept in (10 × 10 × 3 cm) plastic box and covered with aluminum foil with a 4 × 4 cm hole in the middle of one of the widest surfaces, as seen in Figure 3. The aim of guiding the mushroom growth from a single opening In this set of experiments, the effect of substrate amount on mushroom size was tested. 40 g, 80 g, 120 g and 160 g mycelium and various substrates were mixed in the ratio of 25% of strawbale, 25% of wood shavings, 25% of coffee grounds, and 25% of mushroom spawn, as mentioned before. All mixtures were kept in (10 × 10 × 3 cm) plastic box and covered with aluminum foil with a 4 × 4 cm hole in the middle of one of the widest surfaces, as seen in Figure 3. The aim of guiding the mushroom growth from a single opening was to limit the number of fruiting bodies, thus, to prevent overcrowding, to focus on the size of the mushrooms. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 7 of 18 was to limit the number of fruiting bodies, thus, to prevent overcrowding, to focus on the size of the mushrooms.

The mushroom morphology was documented at the end of day 27, through photog-

The biggest mushroom from each replicate was selected as the most mature specimen (Figure 4). Measurements were made digitally using Rhinoceros 3D due to the difficulty

The location of the measurement points for each specimen were standardized as fol-

• Capsize and stalk length are measured using curved lines. To measure the capsize |AB|, point-A is selected arbitrary on the cap edge, and point-B is located on the opposite side of the edge/point-A. To measure the stalk length |EF|, point-F is selected as the bottom of the stalk and point-E is selected as the lowest mid-point of the

2. Measuring the angle between this line and the x-axis (parallel to the ground). • The stalk curvature angle (G°) is measured by drawing two lines parallel to the stalk (one from underneath the cap, the other from the base of the stalk) and measuring

1. Drawing a line between the lowest and highest point on the cap edge.

alyze the overall mushroom forms by allowing for the digital measurement of dimensions

**Figure 3.** The preparation of the samples for the substrate amount experiment. **Figure 3.** The preparation of the samples for the substrate amount experiment.

in measuring delicate mushrooms of a small size.

• The angle of the cap curvature (D°) is measured by:

the angle between these two lines.

*3.3. Measuring the Results* 

of the caps and stalks [3].

lows:

cap.
