Intrinsic electrical properties of cable bacteria reveal an Arrhenius temperature dependence

Abstract

Abstract Filamentous cable bacteria exhibit long-range electron transport over centimetre-scale distances, which takes place in a parallel fibre structure with high electrical conductivity. Still, the underlying electron transport mechanism remains undisclosed. Here we determine the intrinsic electrical properties of the conductive fibres in cable bacteria from a material science perspective. Impedance spectroscopy provides an equivalent electrical circuit model, which demonstrates that dry cable bacteria filaments function as resistive biological wires. Temperature-dependent electrical characterization reveals that the conductivity can be described with an Arrhenius-type relation over a broad temperature range (− 195 °C to + 50 °C), demonstrating that charge transport is thermally activated with a low activation energy of 40–50 meV. Furthermore, when cable bacterium filaments are utilized as the channel in a field-effect transistor, they show n-type transport suggesting that electrons are the charge carriers. Electron mobility values are ~ 0.1 cm2/Vs at room temperature and display a similar Arrhenius temperature dependence as conductivity. Overall, our results demonstrate that the intrinsic electrical properties of the conductive fibres in cable bacteria are comparable to synthetic organic semiconductor materials, and so they offer promising perspectives for both fundamental studies of biological electron transport as well as applications in microbial electrochemical technologies and bioelectronics.

Full text

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Intrinsic electrical properties
of cable bacteria reveal
an Arrhenius temperature
dependence
Robin Bonné1, Ji‑Ling Hou1, Jeroen Hustings1, Koen Wouters1, Mathijs Meert1,
Silvia Hidalgo‑Martinez2, Rob Cornelissen1, Filippo Morini1, Soe Thijs3, Jaco Vangronsveld3,4,
Roland Valcke5, Bart Cleuren6, Filip J. R. Meysman2,7 & Jean V. Manca1*
Filamentous cable bacteria exhibit long‑range electron transport over centimetre‑scale distances,
which takes place in a parallel bre structure with high electrical conductivity. Still, the underlying
electron transport mechanism remains undisclosed. Here we determine the intrinsic electrical
properties of the conductive bres in cable bacteria from a material science perspective. Impedance
spectroscopy provides an equivalent electrical circuit model, which demonstrates that dry cable
bacteria laments function as resistive biological wires. Temperature‑dependent electrical
characterization reveals that the conductivity can be described with an Arrhenius‑type relation over
a broad temperature range (− 195 °C to + 50 °C), demonstrating that charge transport is thermally
activated with a low activation energy of 40–50 meV. Furthermore, when cable bacterium laments
are utilized as the channel in a eld‑eect transistor, they show n‑type transport suggesting that
electrons are the charge carriers. Electron mobility values are ~ 0.1 cm2/Vs at room temperature
and display a similar Arrhenius temperature dependence as conductivity. Overall, our results
demonstrate that the intrinsic electrical properties of the conductive bres in cable bacteria are
comparable to synthetic organic semiconductor materials, and so they oer promising perspectives
for both fundamental studies of biological electron transport as well as applications in microbial
electrochemical technologies and bioelectronics.
In 2012, a novel group of filamentous bacteria was discovered1, which thrive in marine and freshwater
sediments2,3. From the analysis of the sediment chemistry, it was proposed that they can transport electrical
currents over centimetre distances1,4. ese so-called cable bacteria form unbranched chains of over 10,000 cells
that vertically orient in the sediment to take advantage of the redox gradients that occur in natural sediment
(Fig.1A,B)2. Metabolic oxidation and reduction reactions occur in dierent parts of the lament, and to ensure
the electrical coupling of these redox half-reactions, electrons are transported over centimetre-scale distances
along the lament1.
Direct electrode measurements reveal that individual cable bacterium laments can guide electrical currents
over distances up to 1cm under an externally applied potential5. is length scale of conduction for a single
organism surpasses greatly that of other known current-producing bacteria, such as Geobacter sulfurreducens and
Shewanella oneidensis MR-1. ese organisms form conductive nanowires that are a few micrometres long, and
act as model organisms in the eld of electromicrobiology6,7. e conductive structures that enable the long-range
transport in cable bacteria have recently been disclosed. Microscopy investigations reveal that all cells within a
cable bacterium lament share a common space within the cell envelope, and that a network of parallel bres
run within this periplasmic space along the whole lament8,9. ese periplasmic bres comprise the primary
OPEN
1X-LAB, Hasselt University, Agoralaan D, 3590 Diepenbeek, Belgium. 2Department of Biology, University of
Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. 3Centre for Environmental Sciences, Hasselt University,
Agoralaan D, 3590 Diepenbeek, Belgium. 4Department of Plant Physiology, Faculty of Biology and Biotechnology,
Maria Curie-Sklodowska University, Plac Marii Skłodowskiej-Curie 5, 20-400 Lublin, Poland. 5Molecular and
Physical Plant Physiology, Hasselt University, Agoralaan D, 3590 Diepenbeek, Belgium. 6Theory Laboratory,
Hasselt University, Agoralaan D, 3590 Diepenbeek, Belgium. 7Department of Biotechnology, Delft University of
Technology, Van der Maasweg 9, 2629HZ Delft, The Netherlands. *email: jean.manca@uhasselt.be
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conductive structures of cable bacteria, forming an ordered and fail-safe network10 with conductivities up to
79 S/cm5 (Fig.1A). Nevertheless, the underlying electron transport mechanism remains currently undisclosed.
To gain fundamental insight into the long-range electron transport of cable bacteria, we have investigated
the intrinsic electrical properties of individual laments that were isolated from sediment enrichments. Using
a variety of electrical characterization techniques, we studied dried intact laments as well as so-called “bre
sheaths”, i.e. laments from which the lipid membranes and internal cytoplasm are removed by chemical extrac-
tion, thus retaining a sheath structure that embeds the conductive bres5,9(Fig.1A). tted to an (RC) circuit, the
parallel resistance (DC) and alternating current (AC) measurements to determine the intrinsic conductivity and
the inuence of contact resistances. Furthermore, the tunability of the transport was examined in a eld-eect
transistor set-up, which enables us to determine the charge carrier mobility. Finally, we employed the same
techniques in a cryostat set-up to study the conductivity and mobility as a function of temperature.
Results
Cable bacteria act as resistive biological wires. In order to study the intrinsic electrical properties of
cable bacteria, it is crucial to unravel the electrical equivalent circuit and the inuence of contacts on the overall
electrical response. Previous measurements5 have produced linear current/voltage (IV) curves for both indi-
vidual intact laments and bre sheaths. Since these experiments were performed for a restricted voltage range
(− 0.1 to 0.1V), in this work we repeated them for larger voltage ranges (− 1 to 1V and − 10 to 10V). Measure-
ments were conducted in a probe stage set-up with gold, silver and carbon electrodes, and to minimize degrada-
tion of the conductive structures under the inuence of oxygen5, measurements were always performed under a
nitrogen atmosphere. Regardless of the voltage range, we consistently observed the same straight IV behaviour
(Fig.S1), which excludes that Schottky barriers are present at the lament/electrode interface and proving the
ohmic nature of these contacts.
To obtain a representative equivalent electrical circuit for cable bacteria, we performed electrical impedance
spectroscopy, where an AC voltage with varying frequency (range from 1Hz to 1MHz; amplitude 0.1V) is
applied to a single lament in the probe stage conguration. Individual laments were isolated from sediment
enrichments and used either as an intact lament (number of samples n = 6) or as a bre sheath (n = 4). Filaments
were positioned between two gold electrodes on glass or SiO2 substrates with a non-conductive interspacing
(100 to 500µm) (Fig.1C). Carbon paste was added at both ends to ensure a good electrical connection between
laments and gold electrodes (Fig. S2). All samples showed a similar response to the impedance measure-
ments, providing a semicircle in the complex impedance plane (Fig.2A,B). is behaviour can be described by
an equivalent electrical circuit that contains two serial resistors (
Rs
and
Rp
) of which one is in parallel with a
capacitor11 (Fig.2C). From a reference measurement where no lament was placed between the electrodes, an
equivalent circuit is obtained that does not include the resistance
Rp
(i.e.
Rp→∞
), showing that the components
Rs
and
Cp
are inherent to the measurement setup, while
Rp
is attributed to the lament. e equivalent electri-
cal circuit is hence interpreted as follows: the series resistance
Rs
represents the combination of the resistance
of the measurement system wires and the resistance of the probe-electrode interface, while the capacitance
Cp
is attributed to the capacitance of the electrodes and the measurement system. e parallel resistance
Rp
then
comprises both the bulk resistance of the cable bacterium lament
RBulk
and the contact resistance between the
electrodes and the lament
RContact
(Fig.2C).
Values for
Rp
range from 0.8 MΩ to 3.6 GΩ (TableS1), corresponding with previously reported conduc-
tivity values5. Moreover, as expected,
Rp
is equal in value to the total resistance of the sample measured in a
Figure1. Electrical measurement set-ups for cable bacterium laments. (A) A graphical representation
of a cable bacterium with a set of parallel conductive bres in the cell envelope. Filaments for electrical
measurements are prepared either as intact laments or as a bre sheath aer removal of the cytoplasm
and membranes. (B) A SEM image of an intact cable bacterium shows the cells and ridges going along the
lament. (C) e lament is positioned between two electrodes on a non-conductive substrate for DC or AC
measurements. (D) In the FET measurements, two gold electrodes act as source S and drain D, while the highly
n-doped silicon gate electrode G imposes the eld eect at the bottom.
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subsequently performed DC measurement. Values for the other parameters were found to be
Rs=0.8 ±1.0
kΩ
and
Cp=34 ±15
pF. e ratio
R
s
/Rp=0.004 ±0.008%
is consistently small, which aligns with the expected low
resistance of the measurement system connections. Over the broad range of experimental conditions examined,
which include dierent lament types (intact laments and bre sheaths), dierent lament lengths, a range
of lament conductivities as well as dierent electrode substrates, impedance results always showed a single
semicircle with no distinguishable other components in parallel with the system capacitance
Cp
. In order to
determine
RContact
, an additional DC measurement as a function of distance was performed (see Fig.S2 and
TableS2), yielding a signicantly smaller value compared to
RBulk
.
Overall, the obtained equivalent electrical circuit thus demonstrates that cable bacterium laments can be
considered as biological resistive wires with purely ohmic behaviour.
Transistor measurements show n‑type charge transport with high mobility. To determine the
magnitude of the charge carrier mobility, we examined laments in a eld-eect transistor (FET) conguration
(Fig.1D), where the inuence of an externally applied electric eld on the conduction is evaluated. In a bottom-
gate/bottom-contact FET conguration—as typically used to investigate the electrical properties of (in)organic
semiconductor lms—a single lament is placed across the source (S) and drain (D) electrodes separated by
various channel lengths (100 to 300µm) on top of a silicon dioxide/n-doped silicon gate (G) substrate. Since the
eld-eect is typically only present in a thin layer of the sample (~ 10–100nm) near the dielectric substrate, we
opted to work with bre sheaths, for which the distance between conductive bres and substrate is smaller than
for intact bacterial laments.
Transfer curves for a bre sheath are shown in Fig.3A. Here,
ID
,
VGS
, and
VDS
represent the drain current,
gate-to-source voltage, and drain-to-source voltage, respectively. At zero gate bias (
VGS
= 0) and
VDS
= 0.1V,
the sample shows a high o-state
ID
, which will be further discussed later on. With increasing positive gate bias
(
VGS
> 0) at 1V/s (other scan rates in Fig.S3),
ID
slightly increases (about 9% at
VGS
= + 80V). In contrast, at
VGS
= − 80V,
ID
decreases with 9%. is indicates that the charge density at the interface between bre sheath
and dielectric increases with increasing gate voltage, consistent with n-type semiconductor behaviour where
electrons are the maincharge carriers. To verify this, the leakage current
IG
was monitored for all measurements
(n = 4), which was always more than two orders of magnitude smaller(1–10 pA) than the change in
ID
(Fig.S4).
Additionally, the output characteristics (
ID
versus
VDS
) were determined for
VGS
varying from − 50V to + 50V.
A typical graph is given in Fig.3B, where the gate bias modulates the linear slope (
∂ID/∂VDS
) of the IV curve.
e conductivity linearly increases with gate bias
VGS
, yielding a modulation rate of 3 mS/cm per volt (Fig.S5).
Given the bias condition (
VDS ≪VGS
), the transistor response is found to be linear over the gate voltage
domain, as shown in Fig.3A. An estimate for the mobility of the electrons can be obtained by using the formula
µ=(∂ID/∂ VGS )·l/(w·VDS ·Ci)
in the linear bias mode condition at positive gate voltage, where
l
= 0.1–1mm
is the channel length and
w
= 4µm is a conservative estimate of the channel width12 since it corresponds to the
Figure2. e equivalent electrical circuit for an individual cable bacterium lament probed by impedance
spectroscopy. Nyquist plots of (A) intact laments and (B) bre sheaths show a similar single semicircle in the
complex plane. (C) e data were described as an equivalent electrical circuit consisting of a resistor Rs in series
with a parallel stack of a capacitor Cp and resistor Rp, comprising the cable bacterium RBulk and its contact with
the electrode RContact.
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width of the total bre sheath.
Ci
is the gate capacitance per unit area and can be calculated as
Ci
=
εr
·
ε0/d
, with
d
the substrate oxide thickness,
ε0
the vacuum permittivity and
εr
the relative dielectric permittivity of the gate
insulator. For the four bre sheaths examined, the electron mobility was found to be in the range of 0.09–0.27
cm2/Vs (TableS3), which is in the same order of magnitude as many organic semiconductors13.
Conduction is thermally activated with low activation energy over a wide temperature
range. To further understand the charge transport mechanism14–16 in cable bacteria, we studied the con-
ductivity at dierent temperatures for a broad temperature range in a helium atmosphere (see “Methods” sec-
tion). Figure4A shows the conductivity
σ
(see “Materials and Methods”) as a function of the inverted thermal
energy
1/kT
, for both an intact lament and a bre sheath, when cooled down in discrete steps from + 50°C
Figure3. FET measurements reveal an n-type semiconductor behaviour when bre sheaths are used as the
channel. (A) Transfer characteristics of a bre sheath measured at a constant
VDS
=0.05V (20°C) show a
modulation of the drain current
ID
when the gate bias
VGS
is changed from 0 to 80V to − 80V and back to 0V.
e inset shows a bre sheath to be a attened ~ 150nm double stack of bres contained in a thin sheath. (B)
Output characteristics of a bre sheath under a constant gate voltage varying from − 50 to + 50V in steps of 20V
show the slope of the current–voltage curve to change as a function of gate bias
VGS
.
Figure4. Temperature-dependent electrical characterization shows thermally activated charge transport.
(A) e conductivity
σ
of intact laments and bre sheaths show a linear relation with the inverted thermal
energy
1/kT
, thus following an Arrhenius behaviour with activation energy in the range of 40–50meV. (B)
Independent measurements of the impedance response as a function of temperature conrm this result. e
similarity in the semicircle for every temperature implies the thermal activation only to be present in the
(bulk) parallel resistance. (C) When tted to an (RC) circuit, the parallel resistance
Rp
shows a similar thermal
activation as found in (A), while the capacitance
Cp
remained constant as a function of temperature.
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to − 195°C. Both lament types demonstrate a similar behaviour; the conductance decreases with decreasing
temperature, thereby excluding the possibility of metal-like conduction. e activation energy
Ea
is determined
by tting the data with the Arrhenius function
σ=σ0exp(−Ea/kT)
17 (Fig.4A). e tted curves show similar
slopes, indicating comparable activation energies; the dierences in osets indicate a dierent room temperature
conductivity as observed before. Heating the samples back from − 195 to + 50°C resulted in similar activation
energy, thereby demonstrating any lament decay to be small (Fig. S6). An average of the activation energy for
all samples (TableS4) results in 42.3 ± 6.5meV for intact laments (n = 8) and 48.4 ± 7.4meV for bre sheaths
(n = 10)—very close to the room temperature
kT
value of 25meV and low compared to typical activation ener-
gies in the order of 500meV for biological conductors16 like S. oneidensis nanowires18. is result demonstrates
that electron transport in cable bacteria is thermally activated, and laments remain conductive far beyond the
natural physiological temperature range of living cable bacteria.
To verify whether the low activation energy is intrinsic, the impedance response was measured as a func-
tion of temperature in a new set of experiments. Figure4B shows a complex plane plot for an intact lament for
temperatures ranging from − 175 to + 50°C (n = 3). Again we deduce for all temperatures a similar semicircle as
shown in Fig.4B, showing a negligible system resistance
Rs
. is time, the equivalent circuit (
CpRp
) was tted to
the data (TableS5). e
Rp
value agrees well with the corresponding DC value, and a similar Arrhenius behaviour
is found with a corresponding activation energy
Ea
= 40.1 ± 5.3meV, while
Cp
was found to be constant over the
whole temperature range (Fig.4C).
Temperature‑dependent FET measurements show a similar thermally activated mobility. In
order to further unravel the electron transport mechanism, the FET characteristics are likewise studied as a
function of temperature for the same range of − 195°C to + 50°C, with smaller increments. Fibre sheath samples
were prepared as before and laid on interdigitated gold electrodes (10 lines, interspacing 20µm) to enhance the
current signal at low temperatures. For a series of 30 dierent temperatures, a transfer curve is made. As shown
in Fig.5A, the transfer curves at low temperature more resemble a classical n-type FET behaviour as compared
to room temperature (Fig.3A), with a higher change in
ID
at positive gate voltages and almost no eect at nega-
tive gate voltages.
e mobility was calculated from a t over the positive gate voltages, again using the linear mode bias condi-
tion. In Fig.5B the calculated mobility is plotted as a function of temperature. At temperatures above − 100°C
(I.E. the le part of the graph), the retrieved mobility values show more variation, which is attributed to a less pro-
nounced transistor response at those temperatures (see also the “Discussion”). An Arrhenius behaviour becomes
apparent over the temperature range of − 195°C to − 100°C. When tting the data to the Arrhenius relationship
µ=µ0exp(Ea/kT)
, similar activation energy for the mobility as for the conductivity can be determined. Aver-
aged over n = 3 measurements (TableS6), the activation energy for the electron mobility is 36 ± 5meV, compared
to a value for the activation energy of conductivity of 50 ± 2meV, measured on the same samples.
Discussion
In this work, we report the intrinsic electrical properties of dry cable bacterium laments with dierent char-
acterization techniques. Using electrical impedance spectroscopy, we found a single semicircle in the complex
plane, indicating that cable bacteria can be considered as biological electrical wires with ohmic contacts. ese
results are in line with a theoretical impedance analysis for transport in stochastic systems19, but also correspond
toGeobacter nanowires20,21 with the exclusion of an ionic component to the overall conductivity.
Alongside a high electrical conductivity (> 10 S/cm;6 and this work), our results demonstrate that the con-
ductive bres in the cell envelope of cable bacteria display high electron mobility (10–1 cm2/Vs) for a biological
material. ese values are similar in magnitude to organic semiconducting nanowires13,22,23 like P3HT24 and
PEDOT:PSS25 and close to the charge carrier mobility in amorphous silicon (1 cm2/Vs)22. Furthermore, the
Figure5. e electron mobility of the conductive structures in cable bacteria is thermally activated. (A) A
transfer characteristic at lower temperature (at a constant
VDS
=0.5V) indicates the n-type eect is more
prominent at lower temperatures. Calculated from transfer curves at dierent temperatures, (B) the mobility
is plotted as a function of temperature to reveal that the electron mobility is thermally activated, following an
Arrhenius relationship for temperatures below -100°C.
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estimated charge carrier mobility is higher than that of nanowires from G. sulfurreducens (10–4 to 10–2 cm2/Vs)26,27
and comparable to the hole mobility of the conductive structures of S. oneidensis (10–1 cm2/Vs)28. e promis-
ing values for the mobility for the conductive structures in cable bacteria make them an interesting candidate
material in the search for organic and biological alternatives to classical semiconductors. Furthermore, cable
bacteria show a transistor response that is detectable over a broad voltage range, though with a smaller eect
as compared to Geobacter20 and Shewanella nanowires28, but becoming more apparent at lower temperatures.
is limited tunability may be at least in part due to the particular geometric conguration of the bre sheaths
examined here. e conductive bres are embedded in a non-conductive matrix, and only the bottom layer of
the ~ 120nm double stacked bres is expected to be inuenced by the gate electric eld9, while the upper layer
will act as a temperature dependent conductive pathway, which will disturb the transistor behaviour at higher
temperatures (Fig.4A, inset). For individually isolated bres, we expect a higher tunability.
Our temperature-dependent experiments reveal that the conductivity and electron mobility is thermally
activated and can be described by an Arrhenius relation with an activation energy of around 45meV. Arrhenius
behaviour has also been found for thin lms of proteins and peptides placed between planar electrodes15,16,
as well as in inorganic nanowires with surface defects29,30, and is commonly attributed to (multistep) hopping
transport. However, where a multistep hopping transport is proposed for S. oneidensis nanowires6, the charge
transport mechanism in G. sulfurreducens nanowires remains unclear and under debate21,31–33. Future structural
and electrical studies are needed to elucidate the electrical transport mechanism inside the periplasmic bres
of cable bacteria and distinguish between multistep hopping26,33, variable range hopping34, coherence assistant
hopping, or other mechanisms30,35.
e electrical properties of cable bacteria described here oer new perspectives not only for fundamental
studies, but also for technological applications. Our observations that cable bacteria can function as electrical
interconnections with low contact resistance, as well as active electrical channels in FETs, show that they can be
envisioned as suitable future materials for the emerging eld of bioelectronics36, including visionary technolo-
gies such as biodegradable electronics. e reported intrinsic electrical properties, together with the long-range
electron transport and the wide temperature range of operation, are unique assets to envisage cable bacteria for
these future electronic applications.
Materials and methods
Sample preparation. Cable bacteria were enriched in natural sediment cores incubated in oxygenated
seawater, as described previously3. Sediment was collected at Rattekaai (Oosterschelde, e Netherlands). Single
laments of cable bacteria were picked from the sediment enrichment, as described previously9. Filaments were
washed at least six times in MilliQ water to remove sediment debris, thus providing so-called “intact laments”.
Any excess of water was removed with a pipette and the sample was le to dry. Overall, about 5min passed
between the picking of a lament and the start of the current measurement. Alternatively, aer washes with Mil-
liQ, laments were exposed to a sequential extraction procedure, thus removing the cytoplasm and membranes,
as described previously9. is provided so-called bre sheaths. Aer about 45min of preparation time, the sam-
ple was transferred onto the electrode substrate.
AC/DC electrical measurements. For all electrical measurements, the substrate was placed at a probe
station with two needle probes connecting to the two electrodes. e probe stage is housed in a nitrogen glove-
box to prevent sample decay. In the DC measurements, the probe station was connected to a Keithley 2450A
sourcemeter (Keithley, USA) with triax cables, driven by the multi-tool control soware SweepMe, as described
earlier5. For AC impedance measurements, the sample is probed with a VersaSTAT3F potentiostat (Ametek,
USA), allowing impedance measurements in the range 1MHz to 1Hz or 100mHz at bias voltage 0.1V. ese
results were veried with a MFIA impedance analyser (Zurich Instruments, Switzerland) for frequencies in the
range of 5MHz to 1Hz. Data tting was done with both the ZSimpWin and ZView soware packages (Solartron,
USA). Conductivity
σ
was calculated for all samples using
σ=Gl/A
, with
l
the conduction length,
A
= 0.12 µm2
the conductive area (about 60 bres of 50nm diameter)9 and
G=�I/�V
the conductance calculated with a
linear t to the IV-diagrams.
Field‑eect transistor measurements. Field-eect measurements were done on a highly n-doped sili-
con wafer in a bottom-gate bottom-contact FET conguration. A 150nm thick thermally grown silicon oxide
layer served as a dielectric layer, and the bottom drain and source gold electrodes with a thickness of 50nm of
the coplanar FET were dened by optical lithography to yield a channel length of 100µm. Aer the washing
and extraction treatments, as mentioned above, an individual lament was placed across the source and drain
contacts. Gate-source and drain-source voltages were applied by two separate Keithley 2450A sourcemeters, for
which the current response was continuously monitored.
e time response of the drain current upon applying a positive gate bias depends on the time to build up the
conductive channel. erefore, in order to measure consistent transfer curves, a proper gate sweeping speed was
crucial. Fig.S3 shows the transfer curves measured at a gate sweeping speed varying from 10, 2, 1, and 0.5V/s.
Using a fast scanning speed of 10V/s, the signal of
ID
shows a large hysteresis. Stable
ID
is obtained by reducing
the gate sweeping speed down to 2V/s and 1V/s.
Temperature‑dependent measurements. Temperature measurements were performed in two dier-
ent cryostats. Initial DC measurements were performed with a commercial model OptistatDN by Oxford Instru-
ments. e cryostat is liquid nitrogen-based, allowing the sample to be cooled down from ambient temperature
to − 195°C. Cable bacterium laments were dropcasted on glass substrates, and carbon paste was applied to both
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Scientic Reports | (2020) 10:19798 | https://doi.org/10.1038/s41598-020-76671-5
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ends to form electrical contact points. ese were mounted in a double-walled cryostat using spring contacts.
e inner vessel was lled with helium as exchange gas, the outer vessel by a high vacuum, maintaining a pres-
sure around 10–9bar for thermal insulation. e sample was heated to 50°C and then cooled down to − 195°C in
steps of 25°C. For each step, current–voltage curves were measured to obtain the conductivity
σ
. Cooling down
was achieved by adjustment of a needle valve for the liquid nitrogen ow towards the heat-exchanger. A steady-
state temperature is established by the combination of the heat exchanger and an adjacent heating element. e
latter was coupled in a feedback loop to a temperature control unit, model ITC 502 by Oxford Instruments. Aer
stabilization of the current (about 15min), a current–voltage measurement was performed at each temperature.
For temperature-dependent AC and FET measurements, and as verication experiments of the previous
set-up, a cryostat probe stage HFS350EV-PB4 with liquid nitrogen pump LNP96-S and LINK soware was used
(Linkam, UK). Coax outlets were coupled to the MFIA or adapted to triax cables to connect to the Keithley
2450A sourcemeters. e prepared sample is loaded on the stage and purged with nitrogen gas for 3min. It
was then cooled down to − 195°C with liquid nitrogen in the disk underneath the sample. In steps of 25°C
or smaller, the sample is heated to 50°C. e current was monitored to stabilize (aer 1 to 5min) before the
characterization measurement was performed. For AC measurements, a bias voltage of 300mV was applied;
for FET measurements
VDS =5V
was chosen to enhance the current output signal. Aer the measurements, all
samples were studied with an optical microscope to verify if there was no degradation or damage due to cooling
to low temperatures.
Statistics. All measurements were at least performed in triplicates. e value “n” in the text symbolizes the
number of samples, and averages are given
±
the standard deviation.
Received: 28 August 2020; Accepted: 30 October 2020
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Acknowledgements
e authors thank the colleagues from X-LAB from Hasselt University and the Microbial Electricity team from
the University of Antwerp for discussions and feedback. Special thanks to K. Ceyssens and T. Custers for the
graphics in Fig.1A; R. Lempens and M. De Roeve for help with the experimental set-up; J. D’Haen for SEM
imaging (Fig.1B). Graphics for Fig.1C,D and Fig.3A were made by RB with Adobe illustrator. is research was
nancially supported by the Research Foundation—Flanders (FWO project grant G031416N to FJRM and JM
and FWO aspirant grant 1180517N to RB). FJRM was additionally supported by the Netherlands Organization
for Scientic Research (VICI grant 016.VICI.170.072).
Author contributions
Impedance and contact resistance measurements were performed by R.B.; transistor measurements by R.B.,
J.L.H. and M.M.; cryostat measurements by R.B., J.L.H., K.W. and J.H.; SEM measurements by J.D. Cable bacte-
ria cultivation and sample preparation were done by S.H.M., R.B., J.L.H. and F.J.R.M. e study was conceived
by J.M. Furthermore, R.C., S.T., F.M., J.V., R.V., F.J.R.M. and B.C. contributed to discussions and preparation of
the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https:// doi. org/ 10. 1038/ s41598- 020- 76671-5.
Correspondence and requests for materials should be addressed to J.V.M.
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