*3.2. In Vitro Coupling*

We had a series of laboratory experiments to characterise the antibacterial activity of specific fungal species. First assays showed us the ability of TV mycelium grown on GrowLay™ to lyse bacterial cell walls, based on the lytic plate assay, using freeze-dried cells of *M. luteus*. Following this, we carried out the inhibition assay with living *P. larvae* cells. This is different from lytic activity, in that it shows the ability of the samples to inhibit the proliferation of bacterial cells, leading to the death of the bacterium. However, we had yeast contamination in all our plates potentially due to the incubation temperatures (35 ◦C), a much higher temperature than what living mycelium needs to grow. As the third testing method, we homogenised the patches of mycelia of different fungal strains. This process breaks down the cell walls of fungi and frees compounds which might be bioactive and antibacterial, and it potentially kills all living agents in the solution. We first cultured the mycelia of TV, PO, GL, HE, and GF. After we scraped 1 cm × 1 cm mycelium patches from the agar cultures, we diluted them in 1 mL PSB (phosphate solubilising bacteria) water. Then, we used a TissueLyser II to break down the fungal cells, for 15 min and shake with maximum frequency. Since the scraped mycelium culture was mixed with the substrate, the solutions were too thick for the micro filtering process which we needed for the last phase. Therefore, we used a centrifugal shaker to have clearer liquids at the end. After we filtered the solutions, we incubated the *M. luteus* cultured media with these solutions in conditions where the *M. luteus* thrive (35 ◦C). However, this assay did not show us any positive results as opposed to the first assay where we have used a grown and living patch of TV mycelium.

In unsterile conditions, we placed living TV mycelium and propolis in six malt extract agar dishes. The propolis was collected directly from our hives at the HIVEOPOLIS Honeybee Research Field Laboratory at the Botanical Gardens in Graz, Austria and we did not pre-process it to pasteurise or similar. We did not have any control samples, yet from our experience, agar cultures should be prepared in highly sterile environments to avoid contamination. Yet, the mycelium grew fast and healthy in our dishes with propolis. This can be in favour of the mycelium survival in a living fungal honeybee hive. Then, in a closed plastic container, we placed living TV mycelium grown on a solid substrate together with a wax comb piece, collected from one of our hives and not pre-processed to sterilise. We observed a superficial mycelium coverage on beeswax without degradation of the wax, potentially caused by the fattiness of the wax. We have not performed any coupling assays with pollen, honey, or other substances such as bee bread or royal jelly. Among these, we think that the pollen storage area is a good candidate for the fungal mycelia that we introduced to find nutrition and water. Thus, if our mycelial fungi can survive on pollen as a bio-fungicide, the mycotoxin release can be inhibited in favour of the honeybee colony. This also opens mutualism, in terms of an organic coupling towards therapeutic hives indoors. In terms of specific honeybee bacterial pathogens, we concentrated on AFB disease, which can be detrimental to any colony as fast as three weeks. AFB only attacks the honeybee larvae, located in the brood comb area within the nest with stable temperatures between 32–36 ◦C. The conditions that AFB and larvae live in, especially the temperature, are different from the conditions of basidiomycetes fungi habitats. However, in the longer term, as directed evolution techniques become more common, we can improve our materialmaker fungi to survive in higher temperatures and train them to inhibit the growth of pathogenic bacteria. *3.3. First field Experiment with Living Bees: "Beeocompatibility"*  To test if honeybees are tolerant to mycelium materials, we set up a controlled experiment with living bees. We adapted the natural beekeeping friendly and open-source BCN Wárre Hive design and fabrication models provided by the OSBeehives Project [65]. We

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 11 of 21

can improve our material-maker fungi to survive in higher temperatures and train them

### *3.3. First field Experiment with Living Bees: "Beeocompatibility"* selected the mycelium of GL. As growth substrates, we used coconut fibre mats since co-

to inhibit the growth of pathogenic bacteria.

To test if honeybees are tolerant to mycelium materials, we set up a controlled experiment with living bees. We adapted the natural beekeeping friendly and open-source BCN Wárre Hive design and fabrication models provided by the OSBeehives Project [65]. We selected the mycelium of GL. As growth substrates, we used coconut fibre mats since coconut is hostile to bacterial and yeast contamination and has acidity levels suitable for mycelium (5.5–6.5). The cut-out coconut mats were soaked in water for 12 h, hand pressed, and autoclaved at 121 ◦C for 20 min. We laid the pieces from a pregrown GL mycelium on the fibre mats, placed them in shallow plastic boxes without lids, enclosed in large plastic bags and then incubated them for 14 days at 23 ◦C. When we were satisfied with the mycelial colonisation, we dried the pieces in a kitchen oven at 50 ◦C for 2 h each. We used the mycelial side oriented towards the inside of the hive. We stapled the leatherlike edges on the wooden panels and placed a metal mesh to protect the fibres from being eaten by other animals (Figure 7). conut is hostile to bacterial and yeast contamination and has acidity levels suitable for mycelium (5.5–6.5). The cut-out coconut mats were soaked in water for 12 h, hand pressed, and autoclaved at 121 °C for 20 min. We laid the pieces from a pregrown GL mycelium on the fibre mats, placed them in shallow plastic boxes without lids, enclosed in large plastic bags and then incubated them for 14 days at 23 °C. When we were satisfied with the mycelial colonisation, we dried the pieces in a kitchen oven at 50 °C for 2 h each. We used the mycelial side oriented towards the inside of the hive. We stapled the leatherlike edges on the wooden panels and placed a metal mesh to protect the fibres from being eaten by other animals (Figure 7).

**Figure 7.** BCN Wárre Hive with mycelium retrofit. (**a**) Retrofit hive wall with GL mycelium grown on coconut fibre mat. (**b**) Fixing details. (**c**) One of the three mycelium retrofit hives. **Figure 7.** BCN Wárre Hive with mycelium retrofit. (**a**) Retrofit hive wall with GL mycelium grown on coconut fibre mat. (**b**) Fixing details. (**c**) One of the three mycelium retrofit hives.

On 20 July 2019, we introduced six small-sized bee colonies (3000–4000 bees each) into each hive, three of them with mycelial walls and three with regular 18 mm thick wooden walls. We monitored the climate within the hives for two months from July 26th to September 6th, with combined temperature and relative humidity sensors, Beebots, provided by one of the HIVEOPOLIS research groups, Pollenity. The ambient temperature and relative humidity measurements were from near mycelium walls in three of the On 20 July 2019, we introduced six small-sized bee colonies (3000–4000 bees each) into each hive, three of them with mycelial walls and three with regular 18 mm thick wooden walls. We monitored the climate within the hives for two months from 26 July 26 to 6 September, with combined temperature and relative humidity sensors, Beebots, provided by one of the HIVEOPOLIS research groups, Pollenity. The ambient temperature and relative humidity measurements were from near mycelium walls in three of the hives, and from the same locations in the control hives. These measurements were essential for tracking the microclimatic differences that occurred near two materials, as well as for our long-term hive design programme where the mycelium remains alive.

hives, and from the same locations in the control hives. These measurements were essential for tracking the microclimatic differences that occurred near two materials, as well as This experiment gave us several insights into the honeybee and mycelium material coupling and what to consider in experiment set-ups with hives accommodating full colonies.

for our long-term hive design programme where the mycelium remains alive.

This experiment gave us several insights into the honeybee and mycelium material

learned that the contamination can be avoided only with premeditated clean lab protocols, especially in such experimental scenarios in which the materials must be ready to be tested on-site with strict deadlines. The preparation labour and errors cost us a

• Bees chewed out the mycelium from areas that are softer than others. We can only

• Regarding the hive set-up, we realised that the bees should have been blocked from

for the colony given the nutritional and therapeutic benefits of mycelium.

hypothesise about what they did with the mycelium: they might have moved it out from the hive or consumed it. We think that the second possibility could be beneficial

the empty quilt box (a shallow empty volume below the feeder designed to be filled with humidity capturing material). As this area gets warmer and nearer to the feeder,

coupling and what to consider in experiment set-ups with hives accommodating full col-

late start of the whole field experiment.

onies.


Only one of the six hives survived until the following summer season. This survivor hive was one with mycelium attached. We think that the reason is that we populated the hives late in the season, not giving bees enough time to reproduce, collect pollen and nectar, and build wax combs to store enough honey. Additionally, the weather conditions were particularly challenging that year, with heavy rainfall leading to floodings in many adjacent buildings. The colonies and their wax combs were so small that there was too much empty space in the hives before the winter and not enough insulation on the walls.
