HARDWARE

Inspired by solid-state fermentation and mycofiltration, the MycoFlux apparatus combines the cultivation of filamentous fungi and their deployment as a sustainable filter matrix. The fungi is grown on packages of solid substrate and - once a desired amount of biomaterial is achieved - an integrated liquid distribution system circulates a solution to be filtered.

In the case of Rare-Earth recovery, one would deploy our genetically engineered fungus that expresses metal-binding proteins on its surface. The recombinant fungus can be grown on the substrate within the apparatus and a bioleached solution of e-waste circulated until the Rare-Earth Elements are adsorbed onto the biomaterial. Then, a desorption agent could redissolve the ions into a concentrated solution.

Due to its design, the MycoFlux offers a high degree of modularity, which can be adapted to the specific conditions of a project, i.e., the used fungus, the desired filtrate and retentate. In addition, the handling of the apparatus is designed for convenience, which allows, for example, a quick and easy exchange of old substrate against new one.

A suite of sensors is integrated directly into the box, so it is possible to control the growth parameters of the fungi. Automated data processing and visualization enable the user to monitor the MycoFlux easily.

One particularly interesting aspect is the use of separate trays for the cultivation of fungi to achieve an optimized heat and mass distribution, which is not guaranteed in a solid block of a mycofilter. 5 It is also our goal to introduce technology that is modular and can be adapted for different purposes. In the context of Rare-Earth recycling this could mean the selective separation of different (groups of) Rare-Earth Elements.

By using trays, scale-up is also more straightforward and it could be possible to accommodate different trays over the height of the apparatus with fungus that exhibits selectivity for different Rare-Earth Elements, thereby realizing multiple separations in a single setup.

Therefore, we decided to incorporate all the above features in a tray-based solid-state bioreactor, which is an interesting approach for fungus cultivation as well as beneficial for the eventual extraction process of Rare-Earth Elements.

The solution that we want to process in the MycoFlux is filled into a tub at the bottom of the apparatus. Through a pump and hose, it is transported to the top of the apparatus and distributed evenly over the filter medium through a nozzle.

Through the choice of the nozzle and the geometry of the apparatus, we ensured sufficient coverage of the filter with liquid medium while considering the ease of building and maintaining the apparatus. The nozzle used is a stainless-steel spiral nozzle with a spray pattern of multiple concentric cones and is especially well suited against blockage by solid residues in the liquid medium. As we cannot fully prevent the washing out of biomass or filter matrix, this was an important design choice to ensure long-term usage.

We also opted for a square layout of the device instead of a circular one, as this is easier to construct with the low-barrier materials we chose because of our overarching goals. The liquid passes through the filter medium and exits each filtering layer via a metallic mesh at the bottom of every tray. After the filter trays, it trickles into the tub again and can be continuously recirculated until a desired Rare-Earth recovery is achieved.

Our key takeaways from the interviews are as follows:

The overall reception was very positive, and many potential partners worldwide are excited to utilize our hardware solution. We received user feedback regarding the handling of our apparatus: as we want to place our system in community workshops and community laboratories, it is important for our future users that the apparatus can be moved and have parts of it manipulated as needed in restricted spaces. To cater to that need, we installed transport rollers at the bottom of the apparatus so it can be moved more flexibly even in smaller areas. We also installed handles to the top lid and mounting for the hose as quality-of-use improvements. We realized that the deployment of our apparatus in community working spaces with genetically modified fungi inside is not in the scope of current legislation. Therefore, we consulted security experts on potential prospects in this field and how we can prepare our technology to be ready to go once the legislative framework is set. The main adjustment to our current build was adding polytetrafluoroethylene (PTFE) lining to each wood surface that touches the fungus to minimize the chance of the fungus growing through gaps in the wood. We also plan to add additional filters to the air and liquid outlets to prevent fungal spores from exiting the apparatus.

We gathered more user feedback by inviting interested students from our university to visit our hardware in person and experience working with it first-hand. From their feedback, we designed an improved bottom drain. In the future, we would also improve the handling of the trays by using a different material for the tray guideways.

Early on, we also consulted woodworking specialists from our university’s community workshop. They advised us to impregnate our wood against humidity and biocontamination by using wood glaze and putty. We are extremely satisfied with the results of these safeguards as our constructions have survived heavy exposure to the elements outside as well as the growing fungus inside.

In order to be able to monitor the growth conditions of the mycelium and to maintain a permanently optimal environment, we decided to integrate sensor technology into the MycoFlux.

For our current field-test, we chose an array of sensors that measures temperature, air humidity and CO₂ and O₂ concentrations. We also created a custom-made sensor data visualization program with Python to get easy access to the generated data. These information sources enable the user to introduce control measures to ensure optimal fungal growth.

When selecting the sensors, there are some points you should consider. First, you must consider which parameters you want to measure at all. We chose temperature, humidity, O₂ and CO₂ sensors for this purpose because these parameters are important in general mushroom cultivation 6 and our mushroom has no other special requirements for mycelium formation. This can be adapted and extended as desired, depending on the mushroom. When buying, you should also make sure that the sensors have a tolerant working range in terms of temperature fluctuations and humidity so that they do not break. This information can be found in the data sheets of the sensors before purchasing.

Because of our focus on flexibility and customization we decided on the combination of an Arduino as a microcontroller and Python as the programming language for the visualization program. They provide a plethora of tutorials and resources and are very beginner-friendly, allowing the user to adapt the system according to their requirements. For the implementation, we decided on a modular system using plug-in boards, whereby no soldering is necessary in order to make subsequent changes quickly and to maintain the system easily.

The sensors are located inside the MycoFlux between the individual trays and are protected from direct contact with liquid by a small case made of acrylic glass. The rest of the hardware, such as the microcontroller and the display, is located on the opposite side outside the chassis in a waterproof plastic box. The design allows the user to gain access to the microcontroller quickly and easily, e.g., for testing, making changes to the hardware, changing the battery or reading out the data.

Due to our requirements regarding flexibility and expandability, we decided on the Arduino Mega 2560 as a microcontroller. Compared to the much more common Arduino Uno, this unit provides a larger memory and more I/O Pins, making it possible to easily adapt or extend the current configuration for a specific use case.

The Arduino reads the sensor data in a given frequency, displaying them on the connected display for easy access. Furthermore, it collects the data, internally storing it until being connected via USB to our custom-made data visualization program, through which the data is archived and subsequently deleted from the measurement unit to make room for more data. The visualization program also provides an interface to the unit, which can be expanded according to the user's needs. This includes setting a new time interval and registering a new Arduino unit. The tool also offers a visualization of the stored data, giving the user the ability to monitor and interpret the measurements, and an option to export and import data to enable access from multiple devices.

We used the substrate from one bag to mix it with the mycelium in the MycoFlux drawers, which provide a non-sterile environment and directly inoculated the other bag under sterile conditions. We simultaneously inoculated unsterilized straw in a plastic box.

While the mycelium in the Mycoflux drawers took around two weeks to overgrow the new substrate, the mycelium in the autoclaving bag took around five weeks. This difference in growth speed can likely be explained by the larger quantities of already-existing mycelium in the drawers compared to the relatively small quantities of mycelium used for the autoclaving bag.

In contrast, the mycelium in the unsterilized substrate did not show any signs of growth and died after around four weeks.

It has been concluded that the sterility of the substrate plays a major role in fungal proliferation. However, the environment in which the fungi are cultivated does not need to be completely sterile and can most likely be counteracted by larger amounts of inoculation mycelium.

However, further modifications are necessary to safely grow and accommodate genetically modified fungi that are suitable for the actual extraction and recycling of the REEs. For one, the liquid distribution system could be further optimized by adding improved drainage from the bottom while ensuring that the pump is always below the liquid level.

In our current prototype, the guiding rails for the drawers are uniform so differently sized drawers from both sides can be inserted. While a nice idea in theory, this makes taking the drawers out relatively sluggish. In the future, we would recommend installing a drawer system like those found in cabinets and probably forgo the added modulary for easier handling. A specialized system that allows for both could be designed, however it would come with increased complexity.

When developing MycoFlux, we came across more possible implementations of this technology in various fields beyond our original intent. As mycofiltration is already a well-researched concept for water treatment (e.g., rain water), integrating this approach in our course should not be far from reach.

We believe, the MycoFlux is an exciting opportunity for communities to unleash the bioremediation powers of fungi within their immediate surroundings, be it cleaning water or extracting valuable resources. Through our documentation and continued development, we want to bring synthetic biology into the daily lives of communities everywhere on earth!

Thinking outside the box, the potential of MycoFlux is not just limited in the field of bioremediation. With a bit of finetuning, MycoFlux could play a key role in developing novel mycelial-based applications. For instance, it could be used as a powerful tool in the optimization of fungal farming of medicinal and edible mushrooms, as the growth and fruiting phases can be optimized by changing the growth environment of the fungus.

By sharing our knowledge in building and adapting the functionalities of MycoFlux with the iGEM communities and interested parties, we look forward to expanding the possibilities of MycoFlux as a multipurpose tool not only for the recycling of REEs, but also for a variety of needs and applications. To achieve this, it is essential to establish a network of users who share their experiences or problems and work together towards solutions. Here, we can count on the network our Human Practices and Sustainable Development Impact have built!