**1. Introduction**

The growing interest in the use of living materials in the design field is motivated by the desire to create a renewable resource of sustainable materials that is biodegradable and grown using waste materials. This approach offers the potential to produce a lowcost alternative to commercial synthetic materials whilst developing a new spectrum of functional properties [1]. For instance, while fungi as mycelium composites can be used as a structural bulk material as in the MycoTree, MycoCreate-2, and El Monolito Micelio projects [2–5], bacterial cellulose shares many properties with plant cellulose and is already successfully used in the medical field [6] and in fashion design projects [7,8].

Whilst current research has focused on optimising the growth and performance of individual microorganisms for design applications, our research considers the potential to combine different microbial systems in order to achieve new functional possibilities. For example, can multi-microbial systems produce stronger, more durable biomaterials whilst transforming the look and feel of materials for architecture? Using biological materials at different stages of their lifecycle for different purposes requires developing new methods of fabrication that allow the designers to reveal their various features. *BioKnit* is a prototype designed and built in the Hub for Biotechnology in the Built Environment (HBBE) under the Living Construction theme [9]. It focuses on bringing mycelium and bacterial cellulose together with textiles using knitting technologies. Knitted fabric, the first component of the prototype, acts as a scaffold and guides, enhances, and restricts the organism as it grows or attaches to it. Mycelium in a composite form, the second component, acts as a bulk material that gives sufficient compressive strength to the knitted structure to enable the production of a 1.8 m high, free-standing vault. Bacterial cellulose (BC) is the third component and adds a layer of complexity to the system by adding a new optical and

**Citation:** Hoenerloh, A.; Ozkan, D.; Scott, J. Multi-Organism Composites: Combined Growth Potential of Mycelium and Bacterial Cellulose. *Biomimetics* **2022**, *7*, 55. https:// doi.org/10.3390/biomimetics7020055

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

Received: 15 March 2022 Accepted: 29 April 2022 Published: 3 May 2022

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**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/).

tactile quality. BC is used as a surface treatment to coat the knit/mycelium composite and as a tactile skin, self-adhered to the mycelium/knit composite. Mycelium and BC, as two commonly used biomaterials in the design field, were chosen as organisms since cellulose can be used to cultivate mycelium [9]. It proves that they can establish a relationship, and one of the organisms could be used to support the other.

Fungi mainly consist of two parts: the fruiting body/mushroom and the mycelium/roots. The mycelium is of general interest as a biomaterial due to its ability to rapidly grow on various forms of waste as a composite, creating a bulk building material [5]. There are numerous studies on the properties of mycelium composite materials grown on agricultural waste, such as the works of Appels et al. [10] and Jones et al. [5]. Another approach was taken by Elsacker et al. by growing BC to be utilized as a nutritious substrate additive with the focus being on improving the mycelium growth and incorporating the bio-organism in a dried form [11].

BC is a biopolymer that can be grown as a pure culture from a single bacterial strain in sterile lab conditions or with a symbiotic culture of bacteria and yeast (SCOBY), which is commonly used to create fermented kombucha tea in household kitchens. Properties such as fine crystalline structures, biodegradability and biocompatibility, good water-holding capacity, and chemical stability can explain the growing interest in BC as a biomaterial [6]. Due to the large-scale nature of the *BioKnit* prototype, the more resilient kombucha method was chosen for these experiments.

This paper introduces the initial design experiments that were conducted during the prototyping process and asks the following questions: (1) how two organisms grow together and how they influence each other, (2) what is the potential of a knit scaffold as a technique to assemble multiple living materials through growth, and (3) how can the challenge of contamination due to varying growth requirements be addressed? These questions are tested to develop protocols for multi-kingdom textile composites through a methodology based on explorative experimentation using material tinkering [12] and based within established biomaterial protocols rather than a biomimetic investigation translating functional models from nature. Each organism's behavior was observed in an iterative array of experiments.

### **2. Materials and Methods**

### *2.1. Mycelium Composite Preparation*

Mycelium grown in the experiments was inoculated with a mixed substrate (10 g of strawbale, 10 g of wood shavings, 10 g of coffee grounds), which was sterilized in an autoclave at 121 ◦C for 15 min. The sterile mixture was seeded with 10 g of oyster mushroom spawn from GroCycle, UK and kept in sealed plastic boxes (100 × 100 × 30 mm), in the dark, at ambient temperature. After three weeks, the samples were taken out of the boxes and kept in three different forms: oven-dried, air-dried, and living. The first set was air-dried for 8 days and then oven-dried (for 2 h under 60 ◦C). The second set was air-dried for 2 weeks at room temperature. Finally, the third set of tiles were kept alive in a closed container. These three sets of tiles were then integrated in the bacterial cellulose experiments.
