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\begin{document}

\title{3D printed cellulose-based fungal battery}



\author[1]{Carolina Reyes}%
\author[1]{Erika Fivaz}%
\author[1]{Zsofia Sajo}%
\author[1]{Aaron Schneider}%
\author[1]{Gilberto Siqueira}%
\author[1]{Javier Ribera}%
\author[1]{Alexandre Poulin}%
\author[1]{Francis Schwarze}%
\author[1]{Gustav  Nyström}%
\affil[1]{Empa Materials Science and Technology}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
Growing e-waste and the dwindling of non-renewable materials underscores
the urgency to develop electronics based on renewable natural resources.
Using cellu-lose as a material for 3D printing living fungal electrodes,
is a novel way to harness their metabolic activity for potential use in
electrochemical devices. This study shows that the yeast Saccharomyces
cerevisiae and the white-rot fungus Trametes pubescens in combination
with cellulose nanocrystals (CNC) and cellulose nano-fibrils can be 3D
printed and that both fungi grow inside the inks. Adding carbon black
and graphite flakes to the inks makes them electronically conductive for
po-tential use as electrodes in fungal batteries, specifically microbial
fuel cells (MFC). A single battery produces a maximum power density of
12.5 \selectlanguage{greek}μ\selectlanguage{english}W/cm2 and a maxi-mum current density of 49.2 \selectlanguage{greek}μ\selectlanguage{english}A/cm2 (22 k\selectlanguage{greek}Ω\selectlanguage{english}). These
fungal biobatteries can produce between 300-600 mV for several days,
delivering 3-20 \selectlanguage{greek}μ\selectlanguage{english}A for external loads be-tween 10 and 100 k\selectlanguage{greek}Ω. \selectlanguage{english}Attaching
four batteries in parallel can power a small sensor for 65 hours. Also,
a fully biodegradable fungal MFC can be constructed with bees-wax and a
customized cellulose proton exchange membrane. This is the first study
that reports on 3D printed cellulose-based fungal electrodes in an MFC.%
\end{abstract}%



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\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

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\textbf{1. Introduction}

It is estimated that 74.7 Mt of e-waste will be reached by 2030 due to
the disposal of electronics with components that are not readily
recyclable or that easily degrade\textsuperscript{{[}1{]}}. Thus, there
is a rapid need for developing green electronics that use more
environmentally friendly, non-toxic and renewable materials in their
components.

One type of potential green electronics are microbial fuel cells or
biobatteries. Microbial fuel cells (MFCs) use the redox metabolism of
microorganisms to produce energy. Microorganisms used in MFCs either
metabolize organic matter, such as wastewater or sugars or use light
energy and photosynthesis in the absence of organic matter. An MFC
consists of an anode and a cathode and they are either separated by a
proton exchange membrane (PEM), or left open in a single compartment
device \textsuperscript{{[}2{]}{[}3{]}}.

MFCs are of interest for applications such as biomedical diagnostic
devices\textsuperscript{{[}4{]}{[}5{]}}, wearable
electronics\textsuperscript{{[}6{]}}, remote sensing and environmental
monitoring devices\textsuperscript{{[}7{]}}. They are especially
beneficial for stand-alone applications that do not need access to the
main energy grid. There are many different types of microbial fuel cells
including
bacterial\textsuperscript{{[}8{]}{[}9{]}{[}10{]}{[}11{]}{[}12{]}},
archaeal\textsuperscript{{[}13{]}},
algal\textsuperscript{{[}14{]}{[}15{]}{[}16{]}} and mixed consortia
MFCs\textsuperscript{{[}17{]}}. Various MFCs have been used for
wastewater treatment\textsuperscript{{[}18{]}}, to power low power
devices \textsuperscript{{[}7{]}{[}17{]}{[}19{]}}, to power a
microprocessor\textsuperscript{{[}19{]}} and to bio-remediate
dyes\textsuperscript{{[}20{]}{[}21{]}}. Fungal MFCs have also been
studied, including MFCs with yeast\textsuperscript{{[}22{]}{[}23{]}} and
recently with white-rot fungi that decompose
wood\textsuperscript{{[}21{]}{[}20{]}}.However, studies combining both
yeast and white-rot fungi as anode and cathode in MFCs have not been
made. Combining both types of fungi in an MFC system offered the
opportunity to use their different metabolic capabilities, including
cellulose degradation and enzyme production, for enhanced MFC
performance.

Several different pathways of electron transport are possible in an MFC.
When a microorganism oxidizes a substrate in the anode, it produces
electrons. Due to the potential difference between the substrate and the
anode surface, electrons travel to its surface by way of enzymes and
molecules that traverse the cell. The cathode receives the electrons
generated in the anode through a connecting wire and they can travel
either directly to a terminal electron acceptor (TEA), such as oxygen,
or indirectly by way of a series of molecules and proteins. A PEM can
also be used to physically separate the anode and cathode chambers and
has the benefit that it allows protons to enter the cathode chamber
while keeping oxygen out of the anode chamber.

Studies with the yeast \emph{Saccharomyces cerevisiae}
and\emph{Hansenula anamola} have hypothesized dehydrogenases as possible
enzymes involved in shuttling electrons from yeast cells to the surface
of the anode either with or without the help of a redox
mediator\textsuperscript{{[}24{]}{[}22{]}}. Redox active molecules
observed in the supernatant of some yeast MFCs such as \emph{Arxula
adeninivorans}and \emph{Candida melebiosica,} suggest that these yeasts
can secrete their own mediator\textsuperscript{{[}25{]}{[}26{]}}. Other
studies involving white-rot fungi show that the enzyme cellobiose
dehydrogenase can directly transfer electrons to the anode
surface\textsuperscript{{[}27{]}}. Recent studies involving white-rot
fungi in the cathode have used the enzyme laccase, with and without a
mediator, to reduce oxygen in wood and dye based
MFCs\textsuperscript{{[}20{]}{[}21{]}}.

Just as there are various types of microorganisms for use in MFC
studies, there are also many different types of materials to make the
electrodes, PEM and the MFC itself including the use of glass or acrylic
polymer chambers with Nafion membrane and graphite felt or carbon paper
materials\textsuperscript{{[}28{]}{[}29{]}{[}2{]}{[}3{]}}. Recently, MFC
reactors and reactor components such as electrodes, have started to be
3D printed from various materials including: polylactic acid, UV curable
resin, stainless steel and aluminum
alloy\textsuperscript{{[}30{]}{[}31{]}}. Concurrently, microorganisms
have been incorporated into inks that were 3D printed into objects and
structures. Bacteria were mixed into alginate\textsuperscript{{[}32{]}},
hyaluronic \textsuperscript{{[}33{]}}, or graphene based
inks\textsuperscript{{[}34{]}}, and yeast were mixed into cellulose
based ink\textsuperscript{{[}35{]}} for various projects. One of the
first groups to 3D print microbial hydrogels was a paper-based
photovoltaic study\textsuperscript{{[}36{]}}. They used extrusion-based
printing to print a cyanobacteria-alginate-hydrogel on top of an anode
containing \emph{Shewanella onedensis} bacteria. Simultaneously, other
groups started to 3D print electrodes, made from carbon black mixed with
organic and inorganic polymers for use in
MFCs\textsuperscript{{[}37{]}{[}38{]}}. 3D bio-printing have also been
used to make a bacterial hydrogel anode for application in a Plexiglass
MFC\textsuperscript{{[}39{]}}. The use of 3D bioprinting for printing
bioelectrodes was seen as an advantage because it would allow one to
more easily control the geometry, porosity, dimensions and efficient
mass transport of culture medium, electrolyte or synthesized products of
the bioelectrode. The bacterium \emph{S.} \emph{onedensis} MR-1 was
combined with microcrystalline cellulose, alginate and carbon black to
make a cured 3D printed lattice structure anode with a Pt wire attached
to it and a carbon cloth cathode. Ferricyanide was used as a TEA and a
polystyrene cation exchange membrane was used as a separator.
Nevertheless, the use of organic and biodegradative materials in the
overall construction of MFCs remains to be improved.

Previously, our team developed materials, inks and devices for a new
branch of green electronics including
batteries\textsuperscript{{[}40{]}},
supercapacitors\textsuperscript{{[}41{]}},
sensors\textsuperscript{{[}42{]}} and
displays\textsuperscript{{[}43{]}}. As part of these devices, cellulose,
an abundant, renewable and biodegradable polymer derived from wood, was
used as a main component. The goal of this study is to expand the
possibilities demonstrated in our previous work by providing devices
that can not only store electric energy but also generate energy in a
system which is completely safe for the environment. To realize this, we
developed hydrogel fungal inks that were compatible with 3D printing.
This printing technique allows for full geometric freedom to print
devices of arbitrary form and shape that can be seamlessly integrated
with other electronic components. For the hydrogel inks, nanocellulose
was used as a matrix, which when combined with carbon particles and
graphite flakes, enabled tunable viscosities and electronic
conductivities for the fungal containing inks. The
yeast\emph{Saccharomyces cerevisiae} (DSMZ 1333) and the white-rot
fungus\emph{Trametes pubescens} (Empa 220) were used as active
microorganisms in the MFC. Neither fungal strain has been previously
used in an MFC. As proof of concept, we developed a fully biodegradable
device prototype based on beeswax enclosure, shellac resin and a
customized cellulose-based PEM. To our knowledge, this is the first
study to produce a 3D printed cellulose fungal based MFC.

\textbf{2. Results and Discussion}

\textbf{2.1 Biodegradable 3D printed fungi battery concept}

Living fungal inks were prepared by combining a sterile cellulose ink
containing carbon black, graphite flakes, glycerol and nutrients
together with either yeast cells or white-rot fungi mycelia and
mediators (\textbf{Figure 1A} ) (see SI and methods section for
details). Fungal biobatteries were assembled by printing a previously
developed carbon based current collector (CC) \textsuperscript{{[}41{]}}
directly onto the Plexiglass used for the MFC chamber (see SI for
details). Mediators were absorbed to the CC as a way to deliver it to
the fungi\textsuperscript{{[}44{]}}. The fungi inks were thereafter
printed using direct ink writing/robocasting extrusion-based 3D printing
directly onto the CC (\textbf{Figure 1B} ). The schematic
(\textbf{Figure 1C)} , depicts how the different fungi could be using
nutrients and mediators to donate and accept electrons in the assembled
MFC. The anode chamber was filled with 2\% Malt Extract (ME) as a
nutrient source while the cathode chamber was filled with a 4\% ME
filtered supernatant containing laccase, copper and mediator.

\textbf{2.2 Preparation and properties of living fungi electrode inks}

To tune the properties of the hydrogel-based inks for fungal
encapsulation, rheological characterization was performed on 10 wt\%
cellulose nanocrystals (CNC) and 1.2 wt\% TEMPO
(2,2,6,6-tetramethylpiperidine-1-oxyl radical) oxidized cellulose
nanofibrils (TEMPO-oxidized CNF) inks that were autoclaved for
sterilization and subsequently loaded with \emph{T. pubescens} Empa 220
cells. To enable 3D printing, the gel inks\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

shall exhibit strong shear thinning behavior to allow extrusion through
the printing nozzle. Based on previously developed 3D printing of
hydrogel inks and our experimental
setup\textsuperscript{{[}45{]}{[}46{]}{[}41{]}}, shape retention after
printing is made possible by a storage (G`) modulus higher than a few
kPa and significantly higher than the loss modulus (G'') as well as a
dynamic yield shear stress (\selectlanguage{greek}τ\selectlanguage{english}\textsubscript{y}) of 100
Pa\textsuperscript{{[}47{]}{[}45{]}{[}48{]}}. Oscillatory rheological
results show that the dynamic yield shear stress (\selectlanguage{greek}τ\selectlanguage{english}\textsubscript{y})
was 161 Pa \selectlanguage{ngerman}± 69 and 152 Pa ± 17 for the 10\% inks with and without cells
respectively.

Adding \emph{T. pubescens} Empa 220 cells thus increased the dynamic
yield shear stress of the ink (\textbf{Figure 2A} ), but only
marginally. The autoclaved ink with 20 mg of added mycelial cells was
slightly stiffer (higher G`) compared to the autoclaved ink without any
cells at low shear rates (\textbf{Figure 2A} ). Rotational rheology
results showed that the viscosity at rotational shear rates between 0.01
and 1000 s\textsuperscript{-1} was similar between both inks, which both
showed a strong shear thinning behavior (\textbf{Figure 2B} ). In
combination, these results showed that both inks satisfied the printing
requirements in that they were shear thinning viscoelastic materials
which flow during printing and are capable of shape retention.

We next determined the rheological behavior of 15 wt\% CNC and 1.2 wt\%
TEMPO-oxidized CNF inks that were autoclaved, and to which carbon black,
graphite flakes, glycerol and mediators were added. Results showed that
the dynamic yield shear stress of both inks was 1288 Pa ± 276 (anode
inks) and 1390 Pa ± 234 (cathode inks). Also, both inks had a G` (1175
Pa ± 392; anode inks, 3972 Pa ± 1674 cathode inks) and G`` (2273 Pa ±
349; anode inks, 5576 Pa ± 1909 cathode inks) higher than
10\textsuperscript{3} Pa (\textbf{Figure 2C and 2D} ). Thus, these inks
also satisfied the shear thinning and viscoelastic properties required
for 3D printing. Examples of 3D printed anode and cathode inks are shown
in \textbf{Figure 2E} and \textbf{2H} displaying excellent shape
fidelity.

\textbf{2.3 Viability of Cells in the 3D printed Conductive Inks}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

After establishing the rheological properties of the inks, we next
tested the viability of \emph{S. cerevisiae} and \emph{T. pubescens}Empa
220 in the electrode ink formulations following the ink preparation
steps and 3D printing. Their viability was tested by combining the cells
with the inks using an orbital mixer (see methods section for details),
then 3D printing the inks, using sterile techniques, on top of Petri
dishes containing 2\% Malt Extract Agar (MEA). The prints were incubated
at 28 \selectlanguage{ngerman}°C in the dark and monitored for growth. White-rot fungi will
produce filamentous ''hair-like'' mycelia when grown on MEA and yeast
will form a creamy-like smooth structure on MEA. Results showed that
\emph{S. cerevisi} ae and \emph{T. pubescens} Empa 220 were able to grow
in printed inks (\textbf{Figure 2F-G and I-J} ). In addition, we
determined the time needed to grow \emph{T. pubescens} inside printed
inks in order for the fungi to produce laccase. Laccase is an enzyme
secreted by white-rot fungi in the presence of
cellulose\textsuperscript{{[}49{]}}and lignin. When combined with copper
and 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS),
laccase is an efficient electron
transporter\textsuperscript{{[}50{]}{[}20{]}{[}13{]}}. Laccase
production was tested in the presence of copper (0.1 mM
CuSO\textsubscript{4}) as copper has been shown to induce laccase
production in white-rot fungi\textsuperscript{{[}51{]}}. Copper
concentrations added to the inks were within the range of Cu
concentrations found in various soils in the environment and were only
small catalytical amounts\textsuperscript{{[}52{]}{[}53{]}}. Laccase
production was assessed using a laccase enzyme
assay\textsuperscript{{[}49{]}} and visually using a colorimetric
Petri-dish assay\textsuperscript{{[}54{]}{[}55{]}}. After 4-5 days of
growth, a purple ring around the printed ink and the presence of mycelia
in and around the printed inks were visible indicative of laccase
production (\textbf{Figure 2G and Figure S2A} ). Also, laccase enzyme
activities were highest in inks when a suspension of filtered media
(from a 2-week grown \emph{T. pubescens} 220 culture) was used for
growth (\textbf{Figure S2B} ). It is possible that sugars, such as
glucose, present in the 2\% ME repress laccase production compared to
the 2-week filtered suspension. A study found that laccase production
only increased in \emph{T. pubescens} cultures when glucose was depleted
in the culture media over time\textsuperscript{{[}53{]}}.Together these
results helped us to determine the length of time required to
grow\emph{T. pubescens} Empa 220 in the printed inks and also which
media type to use for optimal laccase production in the cathode. To
visualize the fungal cells within the hydrogel inks, the printed anode
was also analyzed by scanning electron microscopy (SEM). Inks containing
carbon black and graphite flakes without \emph{S. cerevisiae} showed
small globular grains \textless{} 5 \selectlanguage{greek}μ\selectlanguage{english}m (\textbf{Figure S3A)} . In
contrast, a control sample of \emph{S. cerevisiae} cells clearly showed
the structure of the yeast cells being \textasciitilde{}5 \selectlanguage{greek}μ\selectlanguage{english}m in size
(\textbf{Figure S3B} ). After identifying the ink material and yeast, we
could next study how \emph{S. cerevisiae} was embedded in the ink
material where the yellow arrows are pointing to yeast cells
homogenously embedded in the conductive hydrogel matrix (\textbf{Figure
3A and B} ). Next, the printed cathode was analyzed. When magnified to
50 \selectlanguage{greek}μ\selectlanguage{english}m, the carbon black ink (no cells) appeared as a flaky ink.
(\textbf{Figure S3C} ). A control sample of \emph{T. pubescens} Empa.
220 in contrast, appeared as filamentous rod like structures with
lengths \textgreater{} 30 \selectlanguage{greek}μ\selectlanguage{english}m (\textbf{Figure S3D} ). When examining the
cell containing cathode inks, single rod-like filaments of \emph{T.
pubescens} Empa 220 were observed embedded in the matrix (\textbf{Figure
3C and D} ). These combined results show that the two fungi are both
well embedded in the layers of the printed inks. Having established that
the fungi were viable in the printed inks, we next proceeded to
characterize their electrochemical activity.

\textbf{2.4 Cells in the 3D printed conductive inks are viable} After
printing the \emph{S. cerevisiae} yeast anode on the surface of the CC,
we compared its electrochemical activity to a control printed anode with
no yeast. The mediator thionine was used based on previous studies with
yeast anodes\textsuperscript{{[}56{]}}. Cyclic voltammetry (CV)
experiments of the \emph{S. cerevisiae} anode produced an oxidation peak
(-0.06 V) and a reduction peak (-0.29 V) that was absent from the
control ink with no yeast (\textbf{Figure 4A} ). A potential sweep
window of -0.6 to 0.6 V was selected to include the expected thionine
redox peaks which should appear within this
range\textsuperscript{{[}57{]}{[}58{]}}. Based on these results, it
shows that the yeast anode printed ink was electrochemically active.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Next, we tested the electrochemical activity of the yeast anode in a
standardized MFC setup using commercial Nafion 117 membrane and a
commercial Pt coated graphite paper cathode. A small volume of 2\% ME (3
mL) was added to the anode chamber as a nutrient source for the yeast.
Results showed that the anode MFC had an average open circuit voltage
(OCV) of \textasciitilde{} 400 mV and that the voltage output was in the
range of 250-350 mV with a 10 k\selectlanguage{greek}Ω \selectlanguage{english}load (\textbf{Figure 4B} ). This
voltage output was stable between 3 and 5 days in different experiments
before decreasing steadily. The anodes with no yeast showed a voltage
output in the range of 100-180 mV with a 10 k\selectlanguage{greek}Ω \selectlanguage{english}load and that the voltage
output lasted less than one day before decreasing (\textbf{Figure 4B} ).
These results confirmed that the \emph{S. cerevisiae} anode was
electrochemically active. We hypothesized the variation in voltage
output between different experiments could be due to the differences in
cell number used in each experiment. Differences in cell number have
been shown to affect voltage output in bacterial
MFCs\textsuperscript{{[}59{]}{[}60{]}}. To address this hypothesis
different cell numbers were added to the printed inks and the MFC
voltage output measured. When cell numbers were 5.2 x
10\textsuperscript{7} cells to 1 x 10\textsuperscript{8} cells, the
voltage output was 250-300 mV with a 10 k\selectlanguage{greek}Ω \selectlanguage{english}resistor (\textbf{Figure S4}
). When the cell numbers increased between 8.4 x 10\textsuperscript{8}
and 1.4 x 10\textsuperscript{9} cells, the voltage output decreased
below 100 mV in less than 1 day (\textbf{Figure S4} ). Overall, these
results show that the anode produces the highest power for the longest
amount of time when cell numbers are low at the start of the experiment.
A possible explanation for this observation is that at high cell number,
the yeast consumes nutrients in the hydrogel ink at a faster rate than
at a lower cell number.

To demonstrate the concept of using our fungal batteries in self-powered
biodegradable environmental sensing units, we tested if the power
generated by the anode in a single MFC could be used to power a
temperature and voltage sensor. For the test, we used an IoT bluetooth
AP4470 sensor (AshaiKasei) that reports temperature and voltage
requiring a nominal minimum 290 mV voltage and 10-15 \selectlanguage{greek}μ\selectlanguage{english}A to power the
sensor. In our experiments, we found that an output of 270 mV was enough
to trigger and start the sensor. Results show that the anode could power
the sensor for over 2.5 hours (\textbf{Figure S5} ). Based on these
results, we concluded that the yeast anode is compatible with low power
sensing applications and could be used in further tests with the
cathode.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

The electrochemical activity of the cathode was also tested individually
in a three-electrode setup. Results show that addition of 10 mM and 20
mM ABTS to the electrolyte produced an oxidation peak at +0.55 V and a
reduction peak at +0.45 V as previously
reported\textsuperscript{{[}61{]}} (\textbf{Figure S3B} ). Previous
studies show that ABTS can be used as a mediator with white-rot fungi in
the cathode\textsuperscript{{[}62{]}}. Furthermore, increasing the ABTS
concentration led to increasing ABTS oxidation and reduction peaks as
expected. Next, we performed CV on the \emph{T. pubescens} Empa 220
printed inks by adding filtered supernatant with high laccase activity
to the inks. We found that the redox activity of the printed inks was
lower compared to ABTS alone (no inks) with the supernatant addition
(\textbf{Figure 4C} ). Thus, the cathode printed ink appeared not to
contribute to redox activity of the ABTS. Perhaps the presence of
the\emph{T. pubescens} Empa 220 increases the resistance and lowers the
transport of electrons across the gel and to the surface of the cathode
current collector. In an MFC study where the white-rot
fungus\emph{Trametes versicolor} was used, voltage output was lower
compared to an MFC containing only laccase and ABTS. The authors
hypothesized that perhaps adhesion of the fungus to the carbon fiber
cathode hindered electron transfer from ABTS to the cathode
surface\textsuperscript{{[}62{]}}. As a result of this finding, we
proceeded with \emph{T. pubescens} Empa 220 added directly to the MFC
without dispersing it in a gel matrix.

To determine the electrochemical activity of the cathode, we compared
voltage output from an MFC with and without \emph{T. pubescens} Empa 220
in the cathode compartment. Several pieces of mycelia were introduced
into the cathode compartment and the laccase containing supernatant was
used as the catalyst. The MFC with \emph{T. pubescens} Empa 220 and 20
mM ABTS had an OCV of 600 mV (\textbf{Figure 4D} ). The internal
resistance of the MFC was 17k\selectlanguage{greek}Ω. \selectlanguage{english}When a 100k\selectlanguage{greek}Ω \selectlanguage{english}load was attached, 300-500
mV was generated for several days. On day 5 of the experiment the 100k\selectlanguage{greek}Ω
\selectlanguage{english}load was replaced with a 10k\selectlanguage{greek}Ω \selectlanguage{english}load so that the external load could be
closer to the internal resistance. It is known that a higher voltage
output can be obtained the closer the external load is to the internal
resistance of an MFC\textsuperscript{{[}63{]}}. Switching the load to
10k\selectlanguage{greek}Ω \selectlanguage{english}dropped the voltage to 30 mV on day 6 (\textbf{Figure 4D} ).
Additionally, the internal resistance appeared to increase to 80k\selectlanguage{greek}Ω \selectlanguage{english}due
to drying out of the anode chamber. Refilling the anode compartment and
reattaching the 10k\selectlanguage{greek}Ω \selectlanguage{english}load increased the voltage up to 200 mV on day 7.
These results show that voltage and power output rely on maintaining
adequate growth conditions in the anode and, importantly, that the
fungal battery can be reactivated by rehydration.

To study the effect of ABTS on the voltage and power output, the
experiment was repeated again but this time with 10 mM ABTS. Adding 10
mM ABTS and \emph{T. pubescens} Empa 220 resulted in a lower OCV of 400
mV, and when a 100k\selectlanguage{greek}Ω \selectlanguage{english}load was attached, \textgreater{} 200 mV was
produced for two days (\textbf{Figure 4D} ). Hence, using a lower ABTS
concentration lowers the power output. Thus, a concentration of 20 mM
ABTS was selected for further tests. Next, we performed a control
experiment where the fungus was left out of the cathode compartment and
20 mM ABTS was used. The OCV was 160 mV and after attaching a 100k\selectlanguage{greek}Ω \selectlanguage{english}on
day 3, the voltage dropped drastically to 13 mV (\textbf{Figure 4D} ).
Together these results confirmed that the presence of \emph{T.
pubescens} Empa 220 cells were needed for maximum power output in the
MFC.

\textbf{2.5 Fungal MFC can produce energy} Having identified the ideal
parameters to use for both anode (7-layer print, 1 x
10\textsuperscript{8} yeast cells, 3 mL of 2\% ME) and cathode (4 mL
laccase supernatant, white-rot fragments, 20 mM ABTS), we proceeded to
test the maximum power and current output of the MFC. For a single MFC,
a maximum power of 12.54 \selectlanguage{greek}μ\selectlanguage{english}W/cm\textsuperscript{2} was obtained at a
current density of 49.2 \selectlanguage{greek}μ\selectlanguage{english}A/cm\textsuperscript{2} with a 22 k\selectlanguage{greek}Ω \selectlanguage{english}load
attached to the MFC (\textbf{Figure 5A} ). Attaching four MFCs in
parallel to the sensor without any load could power it for 65 hours
(\textbf{Figure 5B} ).

\textbf{2.6 Beeswax MFC and Cellulose PEM.} To build a fully
biodegradable MFC prototype we used natural beeswax without any
additives to cast an MFC circular enclosure. This type of casted beeswax
enclosure has been used to make an edible non-microbial
battery\textsuperscript{{[}64{]}}. A paper was adhered to the two pieces
of wax with a shellac resin to test the possibility of using a cellulose
PEM (\textbf{Figure 5C} ). After filling the enclosure with water, the
paper could prevent water from crossing over into one of the empty
chambers. Furthermore, there was no leakage of water from the assembled
enclosure.

To optimize ionic conductivity several cellulose based PEMs were
developed (\textbf{Table S3} ). Through-plane conductivity measurements
showed that certain cellulose PEMs could be made more conductive than
Nafion 117 (\textbf{Figure S7A} ). This was especially true when the PEM
was tested with a yeast minimal growth medium (YMM) as electrolyte (See
supporting methods). At least one other study involving acetate-based
PEMs has shown that cellulose-based PEMs can produce high
conductivities\textsuperscript{{[}65{]}}. However, this study did not
proceed with testing the membrane in an MFC. With the developed
membranes at hand, we first tested cellulose PEMs with high
conductivities in an air cathode MFC. In the air cathode test, results
showed that the MFCs with cellulose PEMs produced lower voltage output
compared to when Nafion 117 was used instead (\textbf{Figure S7B} ).
These results show that despite through-plane conductivity tests showing
high conductivity of cellulose-based PEMs in different electrolytes,
additional resistances are introduced in an assembled MFC that can
result in low voltage output.

As a final step, we proceeded with testing the best performing cellulose
PEM (scaa27) and inks in a fully assembled wax enclosure. Either YMM or
a 2\% ME media was used in the anode compartment in separate tests.
Results using YMM in the anode compartment with \emph{T. pubescens} Empa
220 in the cathode compartment showed that the OCV was 176 mV for a few
hours before decreasing, then increased and remained at 120 mV for
several days (Figure S8A). The internal resistance was 11 k\selectlanguage{greek}Ω. \selectlanguage{english}The low
voltage output coincides with the results of the test enclosure using
the cellulose-based PEM instead of Nafion 117 (Figure S6B). At the end
of a week-long experiment, the beeswax enclosure was opened and, in some
enclosures, fungal growth on the surface of the PEM was observed in the
cathode compartment (Figure S8B). This indicates that \emph{T.
pubescens} Empa 220 can decompose the cellulose PEM, an advantage for
building a biodegradable prototype. In addition, the yeast anode ink
showed signs of yeast growth on the surface of the ink (Figure S8C) and
dark white-rot mycelial pellets were present on the cathode surface
(Figure S8D). These results further show that the beeswax does not
inhibit fungal growth in either compartment. However, binding of the
white-rot to the cathode surface may have prevented electron transfer to
the CC.

We next made MFC tests using 2\% ME media in the anode compartment.
Several modifications were made to further improve the MFCs including:
attaching white-rot mycelial pellets around the CC instead of its
surface, printing 5 layers instead of 4 layers of anode ink and adding
more laccase supernatant to the cathode chamber. Results, showed that
the fungal MFCs had OCVs between 400-500 mV for a few hours on day one
before dropping steadily over time (\textbf{Figure 5E} ). When compared
to the fungal MFCs, the OCV of the non-fungal MFC dropped in voltage
faster over time. Attaching a 50 k\selectlanguage{greek}Ω \selectlanguage{english}load to the MFCs allowed for testing
the internal resistance of the MFCs. The internal resistance of the
fungal MFCs were higher (139 k\selectlanguage{greek}Ω \selectlanguage{english}and 330 k\selectlanguage{greek}Ω\selectlanguage{english}) than the non-fungal MFC (100
k\selectlanguage{greek}Ω\selectlanguage{english}), possibly due to the presence of the fungus. Switching the load to
loads matching the internal resistance of each MFC showed the fungal
MFCs produced voltages between 100 mV and 200 mV, while the non-fungal
MFC produced between 75 and 50 mV (Figure 5F).

Finally, we also made tests to determine if the fungal biobatteries
would disintegrate in artificial compost. We observed continuous
disintegration and mass loss of the assembled MFC (\textbf{Figure S6} ).
After 3 weeks, most of the wax had disintegrated into fragments visible
by eye (\textbf{Figure 5D} ).

Overall, these results show that it is possible to assemble working
fungal MFC prototypes that can be designed for autonomous operation and
to degrade at their end of use without any harm for the environment.
Future optimization could be envisioned to increase the power output of
the MFCs, by e.g., higher performance PEMs and current collectors, as
well as to increase customizability by 3D printing, thereby enabling
more uniform fungal cell and electrode integration. The presented
technology integrating metabolic energy generation by fungi with
additive 3D printing into biodegradable devices, has the potential to
open new avenues within sustainable electronic applications and use
fungal batteries as power sources for environmental monitoring, remote
sensing and smart agriculture.

\textbf{3. Conclusions}

In this study we developed a cellulose-based MFC with a 3D printed yeast
anode and a white-rot fungus present in the cathode. These MFCs could be
assembled in parallel to power a temperature sensor for several days. By
using a synergistic approach, it was possible to harness the breakdown
of sugars by yeast in a 3D printed anode with the laccase enzyme
activity of the white-rot in the cathode for enhanced energy production.
These findings are a step forward in the field of microbial
electrochemistry and material science because it creates opportunities
for 3D printing different types of cellulose-based fungal electrodes for
use in MFCs, and using organic and environmentally friendly materials in
their construction. Future research could focus on optimizing the device
by making it fully 3D printable and ready for use in practical
applications in the field, particularly in conjunction with
environmental sensors.

\textbf{4. Methods}

\emph{\textbf{Electrode Ink Preparation}:} Anode and cathode inks were
prepared based on methods previously described in (Aeby et al. 2021,
Reyes et al. 2022). Sulfuric acid hydrolyzed CNC (15\% wt) and
TEMPO-oxidized CNF (1.2\% wt) was prepared by combining 6 g of CNC, 0.48
g of TEMPO-oxidized CNF, and 34 g of water with a resistivity of [?]18
M\selectlanguage{greek}Ω\selectlanguage{english}cm\textsuperscript{-2} in a 125 mL SpeedMixer container. The ink was
first mixed with a spatula and then using an orbital mixer (Hauschild
and Co. KG, SpeedMixer DAC 600) at 2300 rpm for 1 min before storing it
at 4\selectlanguage{ngerman}°C for 1-2 days to allow it to swell. Next, 7-10 \selectlanguage{greek}μ\selectlanguage{english}m
high-aspect-ratio graphite flakes (99\% metals basis, Thermo Scientific,
Switzerland) and carbon-black particles (carbon ECP, LION) were added to
the ink as electrically conductive material. 6 g of glycerol was added
to the ink to prevent the ink from drying after printing. The inks were
mixed again by orbital mixing at 1500 rpm for 1 min and 2300 rpm for 4
min. A 3-roll mill (EXAKT, Switzerland) was used to homogenize the inks
with the following settings: first pass (level 6, back rolls 14, front
roll 7), second pass (level 6, back rolls 4, front roll 1). 0.8 g of
malt extract was added to the inks and mixed by orbital mixing at 2300
rpm for 1 min before autoclaving the inks for 15 min at 100\selectlanguage{ngerman}°C (Systec
VX-121). Inks were stored at 4°C until ready for cell addition and
printing.

\emph{\textbf{3D printing of electrodes}:} The anode and cathode were
printed using direct ink writing (DIW) Ultimaker 2+ instrument
(my3DWorld GmbH, Switzerland) enclosed in a Plexiglass enclosure to
maintain sterile conditions during printing steps. The printing platform
and the Plexiglass enclosure were sterilized with 70\% ethanol before
printing. Next, the cellulose ink was loaded into a sterile 20 mL
Luer-lock syringe inside a laminar flow-hood using a sterile spatula.
The syringe was capped with an ethanol sterilized syringe barrel end cap
(S\&P Shop, H. Sigrist \& Partner AG, Switzerland) and then centrifuged
for 10 min at 3,000 rpm (Rotina 380, Hettich AG, Switzerland) before
printing. At the printing step, the end cap was replaced with an
ethanol-sterilized, 0.84 mm diameter, tapered dispensing tip. Printing
was done at room temperature. The prints were 30 mm x 25 mm x 5 mm. The
anode was printed as a line pattern and the cathode as a grid pattern to
allow for air to come in contact with the white-rot ink.

\emph{\textbf{Anode Ink}:} The anode was prepared by adding 100-200 \selectlanguage{greek}μ\selectlanguage{english}L
of a resuspension of yeast (\emph{Saccharomyces cerevisiae} ) grown
overnight in 2\% ME (30\selectlanguage{ngerman}°C, 150 rpm, 40\% RH) to 15 g of the above
electrode ink. In addition to the yeast, 900 \selectlanguage{greek}μ\selectlanguage{english}L of thionine (5 mM) was
added to the ink. The cells and thionine were first mixed with a spatula
into the ink and then the ink mixed by orbital mixing at 1500 rpm for 1
min and 2300 rpm for 4 min.

\emph{\textbf{Cathode Ink}:} The cathode inks used in the CV experiment
were prepared the following way: 15 g of the above electrode ink was
combined with 0.1 g of \emph{T. pubescens} Empa 220 biomass. To produce
the biomass, \emph{T. pubescens} Empa 220 was grown for 4-5 days in
50-75 mL of 4\% ME with 0.1 mM CuSO\textsubscript{4} in a 500 mL
Erlenmeyer flask. Cultures were grown at ambient temperature with
shaking at 120 rpm. The biomass was prepared by first centrifuging 40 mL
of the culture in a 50 mL conical tube at 3K rpm for 20 minutes to
collect the biomass. Supernatant was removed after centrifugation and
the biomass was torn apart into smaller pieces with a lancet. Following
addition of the biomass, 450 \selectlanguage{greek}μ\selectlanguage{english}L ABTS was always added to the ink either
from a 10 mM or 20 mM stock solution. The ABTS stock solution was
prepared using 18\selectlanguage{greek}Ω \selectlanguage{english}MilliQ water and filter sterilized using a 0.2 \selectlanguage{greek}μ\selectlanguage{english}m
PVDF filter. The ink syringe was then prepared as described above for
the yeast anode.

Cellulose Ink for Rheology Measurements: To study the rheological
properties of cellulose inks with and without cells, the cellulose inks
were prepared as follows: sulfuric acid hydrolyzed CNC (10 wt\%) and
TEMPO-oxidized CNF (1.2 wt\%) were prepared by combining 4 g of CNC,
0.48 g of TEMPO-oxidized CNF, and 36 g of water with a resistivity of
[?]18 M\selectlanguage{greek}Ω\selectlanguage{english}cm\textsuperscript{-2} in a 125 mL SpeedMixer container. The ink
was first mixed with a spatula and then using an orbital mixer
(Hauschild and Co. KG, SpeedMixer DAC 600) at 1500 rpm for 1 min and
2300 rpm for 4 min before storing it at 4\selectlanguage{ngerman}°C for 1-2 days to allow it to
swell. 0.8 g of malt extract was added to the inks and mixed by orbital
mixing as described above before autoclaving the inks for 15 min at
100°C (Systec VX-121). \emph{T. pubescens} Empa 220 was transferred to
inks by scraping off 0.02 g of mycelium from a 2\% MEA plate. The sample
was transferred to a 50 mL sterile Falcon tube and 5 mL of 1X PBS added
along with sterile glass beads (Sigma, 2 mm diameter). The suspension
was mixed (Vortex Genie 2, Scientific Industries, 2 min, level 10) until
the fibers broke apart. 2 mL of this suspension was added to the ink and
orbital mixed as above before storing the ink at 4°C in the dark. For
the rheology measurements, three replicates per gel were measured (from
the same batch of gel). The rheology of gels was characterized at 20°C
with an MCR 302 Anton Paar Rheometer. A plate geometry of 25 mm diameter
and 0.5 mm gap was used. The storage modulus \emph{G`} and the loss
modulus \emph{G``} were obtained using a logarithmic sweep of
oscillatory strain from 0.01 \% to 1000 \%. The apparent yield stress
\selectlanguage{greek}τ\selectlanguage{english}\textsubscript{y} is the stress at which \emph{G`} and \emph{G``}
intersect. Flow viscosity was measured using a logarithmic sweep of
rotational shear rate from 0.01 to 1000\textsuperscript{s-1}.

To study the rheology of cellulose inks with carbon black, graphite
flakes, and mediators (no cells added), inks were prepared as described
under electrode ink, anode and cathode inks. Similar rheological
conditions were used as described above, except that a 1 mm gap was used
instead due to the increased viscosity of the ink. Oscillatory
measurements were determined as described above.

\emph{\textbf{Current collector ink}:} The current collector ink was
prepared as previously described\textsuperscript{{[}41{]}}. Briefly, the
ink was composed of 26.3 wt\% of graphite flakes (7-10 \selectlanguage{greek}μ\selectlanguage{english}m
high-aspect-ratio graphite flakes) and 6.56 wt\% of carbon-black
particles. The mixture was ball-milled (Pulverisette 7 from Fritsch,
Germany) at 5 x 2 min, 800 rpm with a 1 min pause in between (revers
on). Next, 50 wt\% of shellac at 50 wt\% in ethanol and 15.7 wt\% of
penthanol was added and the mixture milled again as described above. The
shellac ethanol mixture was prepared by mixing 50 g of shellac flakes
(Blattershellack rubin wachsfrei, Kremer Pigmente) with 50 g of ethanol
(98\%) and letting the mixture stir for 2 hr at 90\selectlanguage{ngerman}°C until complete
dissolution of the flakes. The mixture was ball-milled again with the
same parameters. Following milling, the current collector ink was stored
at 4°C until use.

\emph{\textbf{Casting of current collector}:} The CC was cast by using a
laser-cut PTE film stencil. The stencil was glued to the surface of a
PMME laser cut part using non-permanent spray glue. A spatula was used
to apply the current collector ink to the surface and a razor blade was
used to spread the ink over the stencil. Afterwards, the ink was dried
for at least 24 hr at 60°C. 150 \selectlanguage{greek}μ\selectlanguage{english}L of thionine (5 mM) or ABTS (20 mM)
was pipetted onto the surface of the CC to temporarily adsorb the
mediator to the surface of the electrodes. This was done to ensure that
mediator was available to the fungi for electron transport at the
surface of the electrode\textsuperscript{{[}44{]}}. Following addition
of the mediator, the dye was air dried and then the surface sterilized
by UV light for 1 hr.

\emph{\textbf{Scanning electron microscopy imaging of electrodes and
control cells}:} Anode or cathode inks, with or without cells, were 3D
printed on top of Plexiglas and immediately frozen at -80 \selectlanguage{ngerman}°C. The inks
were lyophilized until dry (1-2 days). A small part of the printed ink
was broken and placed on top of an SEM holder. The sample was coated
with a 7 nm layer of platinum (Bal-Tec MED 020 Modular High Vacuum
Coating Systems, Bal-Tec AG, Liechtenstein), and assessed via SEM
(Quanta 650 FEG ESEM, FEI, Hillsboro, Oregon, USA). For imaging
of\emph{S. cerevisiae} and \emph{T. pubescens} Empa 220 cells, the cells
were scraped off directly from an actively growing agar culture and
placed on top of the SEM holder before coating with platinum as
described above.

\emph{\textbf{Cyclic voltammetry experiments}:} Cyclic voltammetry (CH
Instruments, 660E) was used to characterize the oxidation-reduction
reactions of the electrodes. The anode or cathode was the working
electrode, a Ag/AgCl reference electrode prepared in
house\textsuperscript{{[}66{]}} and a platinum counter electrode were
also used. For the anode CV experiments 7 layers were printed ontop of
the CC PMAA plate. The anode print containing yeast was incubated for
3-4 days at 28°C, under static conditions, to ensure growth of the yeast
in the ink. 5-10 drops of 1X PBS were added on top of the prints prior
to measurements to hydrate the ink due to dryness. Scan rates of 10 and
100 mV/s were used and voltage window of -0.6 and + 0.6 V. For the
cathode CV experiments, 12 layers of ink were printed as this thickness
was found to be most stable for inserting the electrodes into these
prints. The inks were printed as described above. Cathode prints were
incubated for 4 days at 28°C under static conditions to ensure growth
and laccase production. 5 drops of 1X PBS were added on top of prints
prior to measurements as with the anode. A 10 mV/s scan rate was used
and a -1 to +1 V window.

MFC experiments : The anode was characterized by using a one-chamber
MFC. A Pt carbon cathode (40\% Pt 0.5 mg/cm\textsuperscript{2} on carbon
paper, Fuel Cell Earth) and Nafion 117 PME were used for testing the
voltage output of the anode. A 10K Ohm resistor was selected following
tests with several different resistors. The anode was printed as 7
layers and the compartment was filled with 3 mL of 2\% ME and sealed
with a putty adhesive (Tesa Tack). The cathode was characterized using a
two-chamber MFC. The anode printed ink and Nafion 117 PME were used for
testing. When a cathode ink was present in the cathode chamber, it was
filled with 2 mL of supernatant containing laccase. To obtain the
laccase containing-supernatant, a \emph{T. pubescencs} Empa 220 culture
was collected after 2 weeks of growth in liquid 4\% ME containing copper
(as described above for inks). The biomass was removed using a bottle
top vacuum 0.2 \selectlanguage{greek}μ\selectlanguage{english}m PVDF filter (VWR). In the MFC experiments without the
printed cathode, the cathode chamber was prepared by filling it with 4
mL of laccase containing supernatant and 150 \selectlanguage{greek}μ\selectlanguage{english}L of 10 or 20 mM ABTS
stock solution. 7-8 fungal pieces were added into the chamber through a
drilled port at the top of the PMMA using a sterile 18 G needle and
sterile forceps. The biomass was prepared as described above in the
cathode ink section. Copper tape was used to connect the anode and
cathode electrodes with the load. The MFCs were placed inside a plastic
container with a beaker of DI water to increase the humidity to
\textgreater{} 80\% during the experiment. This was necessary to prevent
drying out of the inks during the experiment.

\emph{\textbf{Use of the MFC to power sensors}:} The MFC was connected
to an IoT Bluetooth AP4470 demo sensor node (AshaiKasei) that reports
temperature (TMP117 sensor) and voltage using a Bluetooth BL9 receiver.
The sensor requires a minimum 290 mV input and draws 12-13 \selectlanguage{greek}μ\selectlanguage{english}A away from
the MFC to power the sensor (Kobayashi Makato, personal communication).
For tests with the anode, the MFC anode and cathode ends were attached
to the Bluetooth sensor using an RND Components Experiment development
board (RND 255-00004) and circuit wires. Readings were recorded using an
Arduino M5 stack core development kit and Arduino IDE version 2.0.3. A
script was provided by AshaiKasei for the AP4470 sensor node to collect
the readings using the Arduino M5 stack.

\emph{\textbf{Disintegration Experiments}:} Disintegration experiments
were carried using a previously described method used to study the
disintegration of a supercapacitor under simulated composting
conditions\textsuperscript{{[}41{]}}. This method is based on the
International Organization of Standardization 20200 for plastics
disintegration using sawdust, rabbit feed, cornstarch, sugar, corn oil,
urea and compost. The casted cylinders were made using white
pharmaceutical grade beeswax (Rayher). The CC was cast on top of the wax
cylinders using a silicone spatula and allowed to air dry at 4-10\% RH
for 5-7 days before mediators were added as described above. The anode
ink was prepared as described above and 3D printed as layers on top of
the CC and wax casted cylinders. Ports and openings for the Cu wire were
introduced prior to assembly using 19G needles and an X-Acto knife.
Also, several pieces of white-rot fungi were added to the surface of the
cathode CC before assembly. The white-rot fungi culture was prepared as
described above for the MFC experiments. Both chambers were attached to
the cellulose PEM with shellac resin and the assembled enclosure was
allowed to dry overnight with plastic clamps holding the cylinder pieces
in place. The next day, hot wax was applied to the middle of the
cylinder pieces, sealing the enclosure. The wax MFCs were incubated at
58\selectlanguage{ngerman}°C and mass loss measured weekly for up to 7 weeks as previously
described\textsuperscript{{[}41{]}}.

Beeswax MFC Assembly: The beeswax MFCs were prepared as described for
the disintegration experiment with the following modifications: The MFC
with 2\% ME was assembled using a 5-layer printed yeast anode. The
cellulose PEM scaa 27 was used in all MFCs. Copper tape was adhered to
the CC using shellac resin. 5-7 pieces of the white-rot fungus was
attached around the CC prior to the assembly step, using sterile
forceps. Following assembly, 6 mL of 2\% ME was added to the anode
compartment and 4 mL of laccase supernatant was added to the cathode
compartment with 150 \selectlanguage{greek}μ\selectlanguage{english}L of 20 mM ABTS. Beeswax was adhered around the
assembled MFC prior to the addition of media.

\emph{Wax Based Enclosure and Shellac Resin}\textbf{:} A silicone
circular casting mold (OD \selectlanguage{ngerman}Ø 5.5 cm, ID Ø 4 cm, 1.5 cm H) was used to
prepare the beeswax enclosure. White colored 100\% beeswax was used to
cast the enclosure by heating the wax to 60 °C and pouring this into the
enclosure. Two circular wax molds were prepared for anode and cathode
chamber. A shellac resin was prepared to glue the cellulose PEM to the
beeswax, and to glue the copper tape to the CC, by grinding 2 g of
orange shellac flakes (Blättershellack orange aus Indien, Kremer
Pigmente) with a mortar/pestal and then mixed with 4 mL of ethanol
(98\%). The mixture was capped and parafilmed overnight to avoid
evaporation and to allow the shellac to dissolve completely before use.
The final solution was stored in a glass vial with parafilm around the
cap.

\textbf{Supporting Information}

\begin{itemize}
\tightlist
\item
  MFC and ink schematic (Figure S1)
\item
  Laccase activity experiments (Figure S2)
\item
  SEM images of controls (Figure S3)
\item
  Anode cell number experiments (Figure S4)
\item
  Anode powers sensor (Figure S5)
\item
  Disintegration of wax MFC enclosures (Figure S6)
\item
  Cellulose membranes (Figure S7)
\item
  MFC voltage outputs of wax MFC enclosure (Figure S8)
\end{itemize}

\textbf{Acknowledgements}

The authors would like to thank Dr. Xavier Aeby, Sefora Riilo and Dr.
Fabian Wiesemüller for technical support, Dr. Xiaomae Yan for help with
preparation of the silver wire electrode for the CV measurements, Anja
Huch for SEM analysis, Luis Rozas for helping to prepare the conductive
ink using the 3-roll mill, Dr. Silvia Campioni for advice regarding the
use of laccase supernatant in the cathode compartment, Yannick Nagel for
help with the assembly and design of the 3D printer custom extruder and
Dr. Luca Müller for rheology support and input on assembling the MFC
laser-cut testing enclosure. Dr. Luiz Garcia Greca for help measuring
and recording disintegration of samples. Funding for the project was
provided by Gebert Rüf Stiftung Microbials Initiative (Project no.
GRS-08/19).

\textbf{Conflicts of Interest} : The authors declare no conflicts of
interest.

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